EP4658093A1 - Food composition comprising chlorophyll-deficient chlorella biomass with high protein content >50wt% - Google Patents

Abstract

The present invention relates to a food composition comprising Chlorella biomass material, wherein the biomass material is sourced from a chlorophyll-deficient strain of Chlorella microalgae and wherein the Chlorella biomass material comprises at least 50 wt% of Chlorella protein by dry weight of the Chlorella biomass and wherein the food composition is in the form of a meat analogue or a dry savoury snack.

Description

FOOD COMPOSITION COMPRISING CHLOROPHYLL-DEFICIENT CHLORELLA BIOMASS WITH HIGH PROTEIN CONTENT >50WT%

Field of the Invention

This invention relates to a food composition comprising Chlorella biomass material. In particular, it relates to a food product in the form of a meat analogue or in the form of a dry savoury snack.

Background of the Invention

Protein is an important element of human and animal nutrition. Therefore, both the quantitative and qualitative protein content is an important property of food products.

The widespread, ongoing adoption and resulting market growth of plant-based or flexitarian diets, as a result of increasing consumer awareness of the environmental and health consequences of a diet rich in animal protein, is often referred to as a "disruptive shift in consumption habits". The retail plant-based protein market has achieved a global double-digit growth (14% CAGR) of worth 18.5 billion USD in 2020-21 and expected to reach >40 billion USD by 2026 with meat alternatives, plant-based milk and dairy and egg replacements representing >80% share that have a broad demographic appeal, including those who do not necessarily identify as vegetarian or vegan.

Soy and pea protein (concentrates or isolates) currently dominate the global market for plant-based protein ingredients despite challenges around environmental impact, allergenicity and poor taste associated therewith. Therefore, food manufacturers are actively seeking alternative options; recognising the competitive advantage of plant-based proteins, such as oats, potato and chickpea, in addition to mycoprotein, insect protein and cultured or synthetic proteins, which have improved taste, nutrition, sustainability or allergenicity, consistent with evolving consumer preference. However, there is a growing urgent need for new sources of plant-based protein, beyond soy and pea, to meet consumer demand and expectations. First and foremost, these ingredients should deliver high nutritive value at an attractive cost and low environmental impact, while being readily scalable. Ingredients that can also provide functional, bioavailable protein with neutral taste and colour are even more desirable to the food and beverage manufacturer. Lastly, ingredients that can deliver on all of the above while delivering added health benefits to the consumer and potentially be produced without reliance on specific geographies, fitting strongly within a distributed robust food system have further value as we appreciate a world where external factors including global pandemics and conflicts can radically disrupt global supply chains and population health. All of these attributes align strongly to global priorities, for instance, within the UN's Sustainable Development Goals.

In this regard, algae in general have been identified as potential sources of vegetarian and/or vegan foods. While microalgae in particular, such as Chlorella sp. have been traditionally used as a food source for both human and animal consumption, recent trends in nutraceuticals and food industries have identified microalgae as a potential source of essential nutrients that provide several other benefits. For at least these reasons, Chlorella microalgae has a growing market opportunity as a food ingredient, largely owing to its high protein & fibre content and economical, heterotrophic production method.

For example, the green microalga, Chlorella vulgaris, has been produced commercially as a food and dietary supplement for at least the last 50 years. Moreover, Chlorella vulgaris is exempted from EU Novel Food Regulation (EU) 2015/2283 - being as it was "on the market as a food or food ingredient and consumed to a significant degree (within the EU) before 15 May 1997". In addition to being safe to eat for both humans and animals, both as a whole food and as an ingredient, Chlorella vulgaris is also present on the Cl RS China List of approved cosmetic ingredients both as whole cell and as extract, as well as being included on the European Cosmetics Ingredients list.

Besides Chlorella vulgaris, other species of Chlorella such as Chlorella sorokiniana, as well as other microalgae related to the Chlorella genus especially those selected from the family Chlorellaceae, may be exploited commercially for various applications for example in food, nutraceuticals, cosmetics, and so on. For example, it will be appreciated that Chlorella sorokiniana UTEX 1230 and its equivalent strains (SAG 211-8k and CCAP 211/8k) represented in other culture collections, has an established history of consumption within the EU (and globally) before 15 May 1997, meaning that it is does not fall under the scope of Regulation (EU) 2015/2283 of the European Parliament and of the Council of 25 November 2015 on novel foods, (as clarified by the Czech Republic, Ministry of Agriculture in their Consultation of 4 March 2022). Chlorella sorokiniana will be likely to emerge as a preferred species for food applications as the market for microalgae-based plant protein continues to grow, owing to its productive growth rate when cultivated heterotrophically on glucose, favourable regulatory status in major markets and higher average protein content.

However, as a microalgae, a single-cell aquatic plant, Chlorella naturally contains chlorophyll, which limits the application of the whole-cell ingredient in food formulations, owing to its poor organoleptic properties (colour, taste and smell). To reduce such undesirable sensory properties, microalgae variants with reduced levels of chlorophyll have been developed by a number of producers. However, current market microalgae with reduced chlorophyll also exhibit a reduction in their protein content as a consequence of the strain development process. For example, wild-type (green) Chlorella sorokiniana currently on the market typically has a protein content of 63.1 %, 64.4% or 65.0% of biomass dry weight (GRAS Notice No. 986; December 17, 2020) However, a typical market chlorophyll-reduced, (white) variant of Chlorella sorokiniana produced by Aliga Microalgae of Denmark has a protein content of circa 45-50% (https://www.foodnavigator.eom/Article/2022/07/20/lntroducing-neutral-tasting-white-Chlorella- This-enables-application-in-formulations-that-previously-weren-t-possible, accessed

16/09/2022).

Similarly, wild-type (green) Chlorella vulgaris typically exhibits a protein content of 42- 58% of biomass dry weight, depending on growth conditions (Safi et al. 2014; DOI: 10.1016/j.rser.2014.04.007), but white and yellow, chlorophyll-reduced Chlorella variants produced by Allmicroalgae (Portugal) have 30% and 32.5% (w/w) protein, respectively (allmashop.com; accessed 24/05/2022). The problem is that, although the organoleptic properties of the Chlorella microalgae are improved, and enable a higher-incorporation rate as a plant-based protein and food ingredient, in doing so, the existing solution results in a concurrent reduction in overall protein content of the microalgae. This is likely to happen as a direct consequence of the absence of proteinaceous photosynthetic pigments, with the outcome being to reduce the market applicability and potential desirability of the product as a plant-based protein ingredient. For instance, a reduced total protein content may require manufacturers to incorporate other sources of protein in the food compositions to achieve an overall target protein threshold enabling a "source of plant-based protein" label, or to achieve a desired function within the formulation, which is only achieved as a reflection of the overall protein content. While increases in the protein content relative to wild-type Chlorella vulgaris of green or reduced- chlorophyll Chlorella vulgaris variants have been reported following random mutagenesis (from 30.5 ± 0.8 % w/w, to 39.5 ± 0.9%and 48.7 ± 1.3% (w/w), respectively, when cultivated heterotrophically, in the dark, in Erlenmeyer flasks; Schuler et al. (2020; DOI: 10.3389/fbioe.2020.00469) and Gabriel da Cunha Bombo (2021 ; thesis), this does not represent an increase in protein content above 50% (w/w), and is not even beyond a typical level of protein observed in wild-type Chlorella vulgaris, as described hereinabove. Accordingly, although random mutagenesis is a technique that would be known to the skilled person for improvement of microalgal traits (Trovao et al. 2022; DOI: 10.3390/md20070440), its application for improving protein content of microalgae beyond 50% (w/w) does not form part of the current state-of-the art (Trovao et al. 2022; Table 1 and Supplementary Table S1 : “Targets” column). Furthermore, the concept of using random mutagenesis to stack multiple improved traits in microalgae; such as reduced chlorophyll content (in a range of 0.001-0.5 mg/g dry cell weight), high protein content (at least 50% w/w) and a modified cell wall also does not form part of the current state-of-the-art. It is an object of the invention to provide a food composition comprising microalgal biomass with a relatively high amount of protein.

It is another object of the invention to provide such a food composition in the form of a meat analogue or a dry savoury snack.

It is a further object of the invention to provide a foodstuff comprising microalgal biomass with a consumer-acceptable sensory profile.

It is another object of the invention to provide a food composition comprising biomass obtained from a genetically stable, non-recombinant variant strain of Chlorella microalgae with improved organoleptic properties and protein content and suitable for use in consumer products.

Summary of the Invention

It has now been found that one or more of the above objects are achieved by a composition according to the invention. In particular, it was found that - whereas Chlorella biomass is usually associated with off-flavour and off-odour - by including specific Chlorella biomass material in a food composition, its protein content can be increased with no or only a minimal effect on the sensory/organoleptic properties of the food product.

Therefore, according to a first aspect, the invention relates to a food composition comprising Chlorella biomass material, wherein the biomass material is sourced from a chlorophyll-deficient strain of Chlorella microalgae and wherein the Chlorella biomass material comprises at least 50 wt% of Chlorella protein by dry weight of the Chlorella biomass and wherein the food composition is in the form of a meat analogue or a dry savoury snack.

Detailed Description of the Invention

The word ‘comprising’ as used herein is intended to mean ‘including’ but not necessarily ‘consisting of’ or ‘composed of’. In other words, the listed steps or options need not be exhaustive.

Unless specified otherwise, numerical ranges expressed in the format ‘from x to y’ or ‘x-y’ are understood to include x and y. When for a specific feature multiple preferred ranges are described in the format ‘from x to y’ or ‘x-y’, it is understood that all ranges combining the different endpoints are also contemplated. For the purpose of the invention ambient temperature is defined as a temperature of about 20°C. Except in the examples and comparative experiments, or where otherwise explicitly indicated, all numbers are to be understood as modified by the word “about”.

Unless indicated otherwise, weight percentages (wt.%) are based on the total weight of the composition.

The terms "a" and "an" and "the" and similar referents as used herein refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The term "mouth feel" as used herein refers to the overall appeal of a food product, which stems from the combination of characteristics such as aroma, moistness, chewiness, bite force, degradation, and fattiness that together provide a satisfactory sensory experience.

The term “protein isolate” as used herein refers to material having at least 90 wt% of protein based on dry matter. Examples include soy protein isolate, pea protein isolate, potato protein isolate and Chlorella protein isolate. Thus, a protein isolate is a refined form (typically the most highly refined form) of protein product that is separated from other biomass components by physical or chemical means. It typically contains substantially no dietary fibre.

The term “protein concentrate” as used herein refers to material having at least 60 wt% protein based on dry matter. A protein concentrate is typically obtained from a natural source (such as for example Chlorella biomass or soy meal) by removing at least part of the non- proteinaceous constituents, thereby ending up with a relatively higher protein content., Thus a protein concentrate is a refined protein product that is less concentrated than protein isolates, as it may contain residual carbohydrate and dietary fibre. Accordingly, protein concentrates typically comprise 80% protein by dry weight.

The food composition of the present invention comprises Chlorella biomass material, wherein the biomass material is sourced from a chlorophyll-deficient strain of Chlorella microalgae and wherein the Chlorella biomass material comprises at least 50 wt% of Chlorella protein by dry weight of the Chlorella biomass.

The Chlorella biomass material is preferably present in the food composition in an amount of from 0.1 to 60% by dry weight of the food composition, more preferably from 0.5 to 50%, even more preferably from 1 to 40% and still more preferably from 1.5 to 10%. It will be readily understood by the skilled person that a suitable inclusion level may for instance be chosen based on the desired amount of protein (in particular the desired amount of Chlorella protein in the food product) and the level of protein in the Chlorella biomass.

In the context of the present invention, “Chlorella biomass” refers to the biomass as it is grown upon cultivation or fermentation or other means of growing it, whether in unrefined form or after downstream processing. Thus, the term encompasses both wet and dry biomass, concentrated and purified biomass, protein concentrates and protein isolates.

Thus, for example, a Chlorella protein concentrate or Chlorella protein isolate that is sourced from a Chlorella strain that is contemplated herein for use in the food composition of the present invention is also encompassed in the term “Chlorella biomass”. Chlorophyll-deficient Chlorella strains

The present invention overcomes the drawbacks mentioned above by using a whole algal cell, selected from Chlorella microalgae having a reduced chlorophyll content optionally less than 0.5 mg/g dry cell weight, and high protein content of at least 50% w/w. The algae strain of the invention is preferably produced by a non-recombinant method, and the invention is, therefore, preferably a non-genetically modified whole algal cell that is genetically stable. In particular, the chlorophyll-deficient strain (or hereafter referred to as the "variant(s)" or "variant strain(s)") of Chlorella microalgae having reduced chlorophyll content and high protein content enable improved organoleptic properties as well as a wide range of applications thereof, such as wholecell algal ingredients and their applications. Beneficially, by combining low chlorophyll, while maintaining a comparative or greater level of protein content as compared to the wild-type, the variant strains of Chlorella microalgae are a more commercially competitive plant-based protein ingredient than current market microalgae. Moreover, the low chlorophyll, high protein variant strains of Chlorella microalgae of the invention may also have improved digestibility though a modification of the cell wall. Beneficially, this may eliminate the need to mechanically "crack" the ingredient to enhance the nutritional properties thereof, which simplifies production and lowers cost by the elimination of this need to carry out a mechanical “cracking” step. Alternatively, the energy required to crack or mechanically lyse the Chlorella microalgae of the invention is reduced when compared to the wild-type as a result of a modification of the cell wall. Furthermore, the functional properties of the Chlorella microalgae of the invention (with or without modification of the cell wall) may be favourably enhanced in a food composition as a result of cracking or lysis with respect to emulsion capacity, foaming capacity, gelling capacity, viscosity and the like.

It is understood that the invention, methods and approach described herein also apply to the identification and isolation of chlorophyll-deficient variant strains of other algal biomass (such as green algae) that are haploid genetically. The term "algal biomass" (or “algae biomass") as used herein refers to a biomass derived from algae, such as Chlorella microalgae. It will be appreciated that the algal biomass may typically be selected from the Chlorellaceae taxonomic family of green algae of which notable genera include the true Chlorella species, such as but not limited to Chlorella sorokiniana or Chlorella vulgaris in addition to other species that have historically been identified, consumed or commercially sold as genus Chlorella including, but not limited to Parachlorella kessleri, Auxenochlorella protothecoides, Auxenochlorella pyrenoidosa, or Heterochlorella luteoviridis.

The term "chlorophyll" as used herein refers to a group of green pigments contained in cells of green plants. Chlorophyll is essential for photosynthesis and allows photosynthetic organisms to absorb energy from sunlight (absorbing blue and red lights and reflecting green light from the visible region of the electromagnetic spectrum). It will be appreciated that the chlorophyll content is associated with at least one of: chlorophyll a (a-chlorophyll or Chl-a) and/or chlorophyll b (P-chlorophyll or Chl-b). Chlorophyll a is a primary photosynthetic pigment, which participates directly in the light-driven reactions of photosynthesis, while chlorophyll b is an accessory pigment operable to collect energy primarily from blue wavelengths of sunlight and pass it on to chlorophyll а. Moreover, the chlorophyll content can be influenced strongly by cultivation conditions, in particular the absence or presence of light. In the dark, chlorophyll content can be reduced or even significantly suppressed.

Moreover, the protein content of the Chlorella microalgae may be identified as the protein concentrate therein. The term "protein concentrate" refers to a certain threshold level of protein content, typically produced using aqueous or mild alkali extraction (pH 7-10) of proteins and soluble carbohydrates. The insoluble residue, mostly carbohydrate, is thus removed by centrifugation, followed by precipitation of protein at its isoelectric point (pH ~ 4.5). The precipitated protein is separated by mechanical decanting, washed, and neutralized to a pH of б.8 and then spray-dried. In the chlorophyll-deficient strain of Chlorella microalgae of the present invention, the protein content is at least 50% w/w (protein content compared to the dry cell weight of the algal biomass).

Moreover, a reduced chlorophyll content of the variant strain of Chlorella microalgae beneficially affects its organoleptic properties (for example, taste and smell). When combined with increased protein content, the variant strain of Chlorella microalgae further exhibits improved formulation and nutritional properties. Moreover, all modifications, independently or combined, have a positive impact on the overall consumer acceptance of the variant strain of Chlorella microalgae.

It will be appreciated that the Chlorella microalgae of the invention are not capable of photoautotrophic growth, being that they have a chlorophyll content in a range of 0.001-0.5 mg/g dry cell weight.

Without wishing to be bound by theory, it will be understood that this low chlorophyll content can suitably be achieved as a result of the mutations in genes which encode for phytoene desaturase (or a subunit thereof) and which encode for magnesium chelatase (or a subunit thereof), preferably subunit Chll or ChIH of magnesium chelatase and more preferably subunit Chll of magnesium chelatase as hereinbelow described.

Beneficially, the colour of the Chlorella protein material and/or the Chlorella biomass material which contains the protein material or from which it is sourced is such that it does not lead to off-colour in the food composition of the invention. Therefore, the colour is preferably white or close to white. Therefore, the invention also provides, according to one preference a Chlorella biomass material, that can suitably be used in the food composition of the invention, wherein said biomass has a L* value in an L* a* b* colour space of greater than about 78.

According to a preferred aspect, the present invention involves use of an algae biomass that has an L* value, in terms of Cl ELAB L* a* b* colour space values, that is higher than that of other known algae biomass materials. This is particularly advantageous as such an algae biomass therefore has improved properties that relate to consumer acceptance of consumer goods (e.g. food products) that contain the algae biomass, and in particular improved visual properties, improved taste properties, and improved smell properties, which are desirable in particular in combination with the relatively high protein content of the Chlorella biomass used in the present invention.

As described above, the L* range in the Cl ELAB colour space has a range of 0 to 100. The algae biomass of the present invention has an L* value of greater than about 78. Preferably, the algae biomass has an L* value of greater than about 79, about 80, about 81 , about 82, about 83, about 84, or about 85. Particularly preferably, the algae biomass has an L* value of greater than about 81.0, about 81 .5, about 82.0, about 82.5, about 83.0, about 83.5, about 84.0, or about 84.5.

The algae biomass may have an L* value in the range of from between 78 and 90. Preferably, the algae biomass has an L* value in the range of from 79 to 85. Particularly preferably, the algae biomass has an L* value in the range of from 81 to 85.

It has, surprisingly, been found that L* values of above about 81 , such as in the range of from between 81 and 85, correspond to algae biomass that has significantly improved properties, particularly visual properties and/or organoleptic properties (i.e. properties relating to smell and taste) that are relevant to consumer acceptance when the algae biomass is incorporated into consumer goods, such as food products. This particularly beneficial value, or range, of L* values, has not previously been described in the art in relation to algae biomass of this type.

The algae biomass preferably has an a* value in an L* a* b* colour space in the range of between 0.1 and 4.0. Particularly preferably, the algae biomass has an a* value in the range of between 1.5 and 3.5. Still more preferably, the algae biomass has an a* value in the range of between 1.8 and 3.1.

The algae biomass preferably has a b* value in an L* a* b* colour space in the range of between 10 and 27. Particularly preferably, the algae biomass has a b* value in the range of between 15 and 25. Still more preferably, the algae biomass has a b* value in the range of between 15 and 20, such as between 16 and 19.

In one preferred embodiment, the algae biomass has a L* value in an L* a* b* colour space in the range of between 81.0 and 85.0; an a* value of in the range of between 1.8 and 3.1 ; and a b* value in the range of between 16.0 and 19.0. In one preferred embodiment, the algae biomass has a L* value in an L* a* b* colour space in the range of between 82.0 and 89.0; an a* value of in the range of between 0.4 and 2.2; and a b* value in the range of between 13.0 and 18.0.

In one preferred embodiment, the algae biomass has a L* value in an L* a* b* colour space in the range of between 80.0 and 87.0; an a* value of in the range of between 0.7 and 2.0; and a b* value in the range of between 15.0 and 19.0.

In one preferred embodiment, the algae biomass has a L* value in an L* a* b* colour space in the range of between 85.0 and 90.0; an a* value of in the range of between 0.3 and 1 .2; and a b* value in the range of between 9.0 and 13.0.

In one preferred embodiment, the algae biomass has a L* value in an L* a* b* colour space in the range of between 87.0 and 89.0; an a* value of in the range of between 0.5 and 0.6; and a b* value in the range of between 11.0 and 12.0.

Preferably, the algae biomass has a ratio of the b* value to the a* value of at least 5:1 , preferably of at least 10:1 , more preferably of at least 20: 1 , still more preferably of at least 30: 1.

Preferably, the algae biomass has a ratio of the b* value to the a* value of less than 40:1 , preferably of less than 30:1 , more preferably of less than 20:1 , still more preferably of less than 10:1.

It has surprisingly been found that L* a* b* values in these combinations of ranges correspond to algae biomass that has significantly improved visual properties, particularly visual properties that are relevant to consumer acceptance when the algae biomass is incorporated into consumer goods, such as food products. This particularly beneficial value, or range, of L* values, has not previously been described in the art in relation to algae biomass of this type.

In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae is a modified strain of a Chlorella microalgae species. Typically, the modified strain (namely, 'variant strain') of Chlorella microalgae is a modification of the progenitor cells of the Chlorella microalgae. Herein, the term "progenitor" refers to a wild-type or parental strain of the Chlorella microalgae.

The term "wild-type strain" (or wild-type) refers to a typical form of an organism as it occurs in nature. Specifically, the wild-type is a typical form of an organism of a species comprising a set of genes characteristic to a naturally existing organism of that species, i.e. comprising normal occurrence of a gene at a locus, and exhibiting the associated phenotypes thereof. The wild-type strain of Chlorella microalgae can be obtained from its usual dwelling sites such as land, rivers, ponds, lakes, brackish water, wastewater and the like. The naturally existing wild-type strain of Chlorella microalgae is able to grow autotrophically by performing photosynthesis (producing a biomass of alga by utilizing sunlight, carbon dioxide, water and a few nutrients). However, the wild-type strain of Chlorella microalgae can also be cultivated using heterotrophic and/or mixotrophic growth modes. Wild-type strains of Chlorella microalgae are haploid in their normal growth phase, i.e. have only one copy of the genome, thereby making Chlorella microalgae particularly amenable to a phenotypic trait improvement approach using genetics as, for some traits, a single genetic change could yield the desired phenotype. Furthermore, being haploid, these variant or improved strains are likely to be genetically stable as there is essentially no capacity of the mutant strain to easily correct or revert to the wild-type state; moreover, there is no other genetic copy of the DNA that can act as a correction template to facilitate this process. Here, comparative taxonomic analysis by alignment of ITS2 genetic sequences using ClustaW2 software (Madeira et al. 2019; DOI: 0.1093/nar/gkz268) was used to establish that proprietary Chlorella vulgaris strain 4TC3/16 (4TC3) is a wild-type strain of Chlorella vulgaris’, being that is taxonomically identical to the culture collection type strain of Chlorella vulgaris 211/11b.

The term "parent strain" as used herein, refers to a progenitor organism that, during the process of division, replicates its DNA, which is then inherited by an offspring or daughter cell thereof. Specifically, Chlorella microalgae reproduce asexually by multiple fission, with the basic rule that one mother cell reproduces its DNA synchronously to produce at least two daughter cells per division event (or burst). Occasionally, a division burst may comprise four, eight and rarely, sixteen daughter cells (Mandalam and Palsson 1997; DOI 10.1023/A: 1018310008826). The number of Chlorella microalgae daughter cells produced per division burst is thought to be modifiable by environmental factors such as light and temperature - being as they directly affect growth rate, and consequently, the coordination between DNA replication and division events in the cell cycle (Bisova and Zachleder 2014; DOI: 10.1093/jxb/ert466). Given this asexual method of whole genome reproduction and inheritance, it can be understood that Chlorella microalgae strains exhibit an extremely high degree of genetic stability between generations. Further, in the context of a mutagenesis campaign, the parent strain may be a wild-type strain of Chlorella microalgae or a variation (i.e. a genetic variant) of the wild-type strain of Chlorella microalgae. The term "parental strain", therefore, may also refer to a genetic variant or subtype of Chlorella microalgae, preferably a previous generation.

It will be appreciated that a variation of the wild-type strain of Chlorella microalgae differs from the parent strain (namely, the wild-type strain) only by the mutated gene(s) (and in some cases closely linked genes). Such variant strains of Chlorella microalgae are valuable in understanding the effect of a single or multiple gene mutations in the organism. The variation of the wild-type strain of Chlorella microalgae may be a genetic mutant.

Therefore, the invention also provides a Chlorella microalgae strain having a mutation in at least one gene that encodes for phytoene desaturase or a subunit thereof. The invention also provides a Chlorella microalgae strain having a mutation in at least one gene that encodes for magnesium chelatase or a subunit thereof. The invention furthermore provides a Chlorella microalgae strain having a first mutation in at least one gene that encodes for phytoene desaturase or a subunit thereof, and wherein said Chlorella microalgae has a second mutation in at least one gene that encodes for magnesium chelatase or a subunit thereof.

Algae produce a number of pigments including chlorophylls and carotenoids, which capture energy from light as part of the process of photosynthesis. These pigments are critical for photosynthesis and, as such, their production is tightly controlled by a number of enzymes as part of either the chlorophyll or carotenoid synthesis pathways.

Accordingly, “magnesium chelatase" (EC 6.6.1.1) is an enzyme that catalyses the first committed step of the chlorophyll synthesis pathway; being the insertion of Mg²⁺ into protoporphyrin IX.

“Phytoene desaturase" (EC 1.3.5.5) is an enzyme essential to the carotenoid biosynthesis pathway and controls the conversion phytoene into lycopene. Within the carotenoid synthesis pathway, phytoene desaturase converts phytoene into zeta-carotene, which forms the basis of all other plant carotenoids.

“Geranylgeranyl diphosphate synthase" (EC 2.5.1.29) is an enzyme required for the synthesis of geranylgeranyl diphosphate (GGPP), which is the precursor for the biosynthesis of carotenoids and chlorophylls.

It has surprisingly been found that Chlorella microalgae that have a first mutation in a gene that encodes for phytoene desaturase, or a subunit thereof, and additionally have a second mutation in a gene that encodes for magnesium chelatase, or a subunit thereof, have improved properties relating to the use of such microalgae and algae biomass derivatives thereof in consumer goods (e.g. food products). In particular, this combination of mutations has surprisingly been found to result in Chlorella microalgae from which algal biomass may be derived that has an L* value, in terms of Cl ELAB L* a* b* colour space values, that is higher than that of other known algae biomass materials, as described above. It has also surprisingly been found that this combination of mutations results in Chlorella microalgae from which algal biomass may be derived that has L* a* b* values, in terms of CIELAB L* a* b* colour space values, that is particularly beneficial.

In preferred Chlorella microalgae, as hereinbefore described, the second mutation is in a gene that encodes for subunit Chll of magnesium chelatase or subunit ChIH of magnesium chelatase. In particularly preferred Chlorella microalgae, as hereinbefore described, wherein the second mutation is in a gene that encodes for subunit Chll of magnesium chelatase.

In some preferred Chlorella microalgae as hereinbefore described, a mutation is present in a gene that encodes for Geranylgeranyl diphosphate synthase (GGPP), or a subunit thereof. Thus, some preferred Chlorella microalgae of the invention have a first mutation in a gene that encodes for phytoene desaturase, or a subunit thereof, and additionally have a second mutation in a gene that encodes for magnesium chelatase, or a subunit thereof, and additionally have a third mutation in a gene that encodes for Geranylgeranyl diphosphate synthase, or a subunit thereof.

It has surprisingly been found that Chlorella microalgae having a first mutation in a gene that encodes for phytoene desaturase, or a subunit thereof, and additionally having a second mutation in a gene that encodes for subunit Chll of magnesium chelatase have particularly improved properties relating to the use of such microalgae and algae biomass derivatives thereof in consumer goods (e.g. food products). In particular, this combination of mutations has surprisingly been found to result in Chlorella microalgae from which algal biomass may be derived that has an L* value, in terms of Cl ELAB L* a* b* colour space values, that is higher than that of other known algae biomass materials, as described above. It has also surprisingly been found that this combination of mutations results in Chlorella microalgae from which algal biomass may be derived that has L* a* b* values, in terms of CIELAB L* a* b* colour space values, that is highly beneficial.

It has surprisingly been found that Chlorella microalgae that have a first mutation in a gene that encodes for phytoene desaturase, or a subunit thereof, and additionally have a second mutation in a gene that encodes for magnesium chelatase, or a subunit thereof, and additionally have a third mutation in a gene that encodes for Geranylgeranyl diphosphate synthase, or a subunit thereof, have even further improved properties relating to the use of such microalgae and algae biomass derivatives thereof in consumer goods (e.g. food products). In particular, this combination of mutations has surprisingly been found to result in Chlorella microalgae from which algal biomass may be derived that has an L* value, in terms of CIELAB L* a* b* colour space values, that is higher than that of other known algae biomass materials, as described above. It has also surprisingly been found that this combination of mutations results in Chlorella microalgae from which algal biomass may be derived that has L* a* b* values, in terms of CIELAB L* a* b* colour space values, that is highly beneficial.

Preferably, in the Chlorella microalgae as hereinbefore described, the second mutation results in a loss of function, but could also result in a reduction in function. This is more likely to be caused by a frameshift mutation as a result of an IN DEL, but could also be caused by a SNP resulting in a nonsynonymous mutation with either the protein rendered nonfunctional due to a change in the amino acid structure, or in a premature stop codon resulting in a truncated protein.

In an embodiment, the Chlorella microalgae species is selected from Chlorella sorokiniana or Chlorella vulgaris. The term "Chlorella vulgaris" as used herein, refers to a species of singlecell aquatic plant, termed microalgae, falling under Division "Chlorophyta" within the plant taxonomic Kingdom. The full taxonomic assignment is: Biota Plantae (Kingdom) Viridiplantae (Subkingdom) Chlorophyta (Phylum (Division)) Chlorophytina (Subphylum (Subdivision)) Trebouxiophyceae (Class) Chlorellales (Order) Chlorellaceae (Family) Chlorella (Genus) Chlorella vulgaris (Species). The microalgae are photosynthetic organisms that grow in diverse habitats ranging from regions of varying hardness of growth medium (such as soil or water), humidity, salinity, light-access, and temperature conditions, such as land, rivers, ponds, lakes, sea, brackish water, wastewater and the like. Typically, the wild-type strains of Chlorella vulgaris are associated with a dark-green colour, a specific smell (such as aquatic, fish-like, earthy or mouldy smell), an unpleasant taste, in addition to a cell wall; which has glucosamine as its main component, and generally comprises an alkali soluble hemicellulose fraction, and a residue fraction, the rigid wall.

The term "Chlorella sorokiniana" as used herein, refers to a species of green microalgae, that can grow in freshwater and consumes both organic and inorganic carbon, falling under Division "Chlorophyta" within the plant taxonomic Kingdom. The full taxonomic assignment is: Biota Plantae (Kingdom) Viridiplantae (Subkingdom) Chlorophyta (Phylum (Division)) Trebouxiophyceae (Class) Chlorellales (Order) Chlorellaceae (Family) Chlorella (Genus) Chlorella sorokiniana (Species). Typically, the wild-type strains of Chlorella sorokiniana are associated with a characteristic emerald-green colour and pleasant grass odour, in addition to a cell wall which has glucosamine as its main component, and generally comprises an alkali soluble hemicellulose fraction, and a residue fraction, the rigid wall. The hemicellulose fraction of the Chlorella sorokiniana cell wall may contain 50% higher proportion of rhamnose, compared to Chlorella vulgaris. In addition, Algenan (previously “sporopollenin”), a highly-resistant biopolymer, is a long-suspected component of the Chlorella sorokiniana UTEX1230 cell wall (e.g. Rosen et al. 1985, DOI: 10.1016/0168-9452(85)90061-5; Kodner et al. 2009, 10.1016/j.orggeochem.2009.05.003). Further, the highly-conserved N-terminal glycan structures of the cell walls within genus Chlorella are sufficiently diverse that they can be used to differentiate between species (Mocsai et al 2020; DOI: 10.1111/tpj.14718).

In addition to the composition of cell wall carbohydrates and general chemotaxonomy, Chlorella sorokiniana (such as type-strain LITEX 1230) may be further distinguished from Chlorella vulgaris on the basis that the former tolerates a higher cultivation temperature; reportedly up-to 39 °C for LITEX 1230 (Sorokin & Myers 1953; DOI: 10.1126/science.117.3039.330), whereas the latter typically exhibits an upper temperature tolerance of 28-30 °C (Kessler 1985; DOI: 10.1007.bf02418020).

In addition to being phototrophs, both Chlorella vulgaris and Chlorella sorokiniana exhibit the ability to grow heterotrophically, on glucose (or other suitable organic carbon source), in the absence of light. Further, both Chlorella vulgaris and Chlorella sorokiniana are cultivable in mixotrophic growth mode, using a mixture of light and glucose, or other suitable organic carbon source. The wild-type or parent strain of Chlorella microalgae may be obtained from their natural habitats or from laboratory cultures. The obtained strains of Chlorella microalgae are genetically defined as Chlorella microalgae using PCR amplification, sequencing and alignment of the genetic material with a reference sequence. Examples of useful genetic sequencing targets for the purpose of taxonomic identification of Chlorella microalgae include, but are not limited to: 18S rRNA gene sequence, the internally transcribed spacer (ITS) regions between the 18S rRNA gene, 5.8S rRNA gene and the 28S rRNA gene sequence. Such regions have been used extensively for intra and inter genus phylogenetic analysis of the Chlorellaceae (green algae) family (Huss et al. 1999; DOI: 10.1046/j.1529-8817.1999.3530587.x, Krienitz et al., 2015; DOI: 10.1016/j.tplants.2014.11.005, Darienko and Prdschold 2015; DOI: 10.1111/jpy.12279, and Heeg and Wolf 2015; DOI: 10.1016/j. plgene.2015.08.001). Other statistics and additional sequences derived from whole genome sequencing are another method for strain identification.

For example, phylogenetic tree construction from the ITS2 sequences demonstrated the evolutionary relationship between Parachlorella kessleri, Chlamydomonas reinhardtii, and members of the genus Chlorella. The Neighbour-joining tree indicated that Parachlorella kessleri and Chlamydomonas reinhardtii are significantly different to the species within the Chlorella genus as shown by the longer branch lengths due to earlier genetic divergence. Strains of Chlorella vulgaris including 4TC3 formed a distinct clade demonstrating the similarity between these isolates and confirming the designation of strain 4TC3 as Chlorella vulgaris.

Furthermore, a comparison of complete chloroplast sequences of Chlorella vulgaris 4TC3, Chlorella vulgaris C27, Chlorella sorokiniana 1230, Parachlorella kessleri 211-11g and Chlamydomonas reinhardtii Its1-3O demonstrated that the intraspecies similarity between the Chlorella vulgaris strains was 98.7%, and this high similarity of the chloroplast genome further confirms the identity of strain 4TC3 (Table 1). When compared with another member of the genus Chlorella, Chlorella sorokiniana 1230, strains Chlorella vulgaris 4TC3 and C27 were 84.1% and 82.8% similar respectively, highlighting the considerable genetic diversity contained within this genus. A greater level of dissimilarity was determined between Chlorella vulgaris 4TC3 in comparison with Parachlorella kessleri 211-11g (81.2%) and Chlamydomonas reinhardtii Its 1 -30 (80.1%) which is to be expected due to these organisms belonging to three different genera (Table 1). Table 1 - Pairwise Average Nucleotide Identity calculated from chloroplast sequences, numbers denote percentage similarity

Typically, Chlorella sorokiniana or Chlorella vulgaris are robust species with a high consumer interest owing to their biotechnological and economical potential including, but not limited to, a wide variety of primary biomolecules (such as proteins, carbohydrates and lipids) and several intermediate compounds, nutritional value, and so forth.

In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae has a chlorophyll content in a range of 0.001-0.5 mg/g dry cell weight. The chlorophyll-deficient strain of Chlorella microalgae has a chlorophyll content in a range of 0.001 to 0.5 mg/g dry cell weight (DCW), preferably 0.01 to 0.25 mg/g dry cell weight, or 0.02 to 0.1 mg/g DCW. For example, the chlorophyll content of the chlorophyll-deficient strain of Chlorella microalgae may be 0.001 , 0.002, 0.003, 0.004, 0.005, 0.01 , 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 or 0.45 mg/g DCW up to 0.002, 0.003, 0.004, 0.005, 0.01 , 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.50 mg/g DCW, preferably

0.25, 0.3, 0.35, 0.4 or 0.45 mg/g DCW up to 0.3, 0.35, 0.4, 0.45 or 0.50 mg/g DCW, 0.1 , 0.15 or 0.2 mg/g DCW up to 0.15, 0.2 or 0.25 mg/g DCW, or 0.001 , 0.002, 0.003, 0.004, 0.005, 0.01 , 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045 or 0.05 mg/g DCW up to 0.002, 0.003, 0.004, 0.005, 0.01 , 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05 or 0.1 mg/g DCW.

In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae has a protein content in a range of 50-85% w/w. For example, the protein content of the chlorophyll-deficient strain of Chlorella microalgae may be 50, 55, 60, 65, 70, 75 or 80% w/w up to 55, 60, 65, 70, 75, 80 or 85% w/w. Optionally, the chlorophyll-deficient strain of Chlorella microalgae has a protein content in a range of 50-75% w/w, preferably 50-70% w/w, more preferably 50-60% w/w. For example, the protein content of the chlorophyll-deficient strain of Chlorella microalgae may be 50, 55, 60, 65 or 70% w/w up to 55, 60, 65, 70 or 75% w/w, preferably 50, 55, 60 or 65% w/w up to 55, 60, 65 or 70% w/w.

Beneficially, a higher protein content enhances nutritional value of the variant strain of Chlorella microalgae, that may be used to replace currently available animal-based protein diets. Additionally, beneficially, the variant strain of Chlorella microalgae can displace pea protein and other new plant-based ingredients by outcompeting on costs, features and benefits.

In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae has a starch content of less than 25 % w/w. The term "starch" as used herein refers to a type of carbohydrate that accumulates in cells of a biological organism, such as Chlorella, under specific conditions such as for example heterotrophic, mixotrophic, phototrophic, high CO2 concentration, high light intensity, limited nitrogen concentration, and so on. Optionally, the chlorophyll-deficient strain of Chlorella microalgae has a starch content in a range of 0.1 to 25% w/w, 0.2 to 20% w/w, 0.5 to 15% w/w, 1 to 10% w/w, 2 to 5% w/w, or about 3% w/w. For example, the starch content of the chlorophyll-deficient strain of Chlorella microalgae may be O.1 , 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, 16.0, 17.0, 18.0, 19.0 20.0, 21.0, 22.0, 23.0 or 24.0% w/w up to 25% w/w, 0.2, 0.5, 1.0, 5.0, 10.0 or 15.0% w/w up to 0.5, 1.0, 5.0, 10.0, 15.0 or 20% w/w, 0.5, 1.0, 5.0 or 10.0% w/w up to 1.0, 5.0, 10.0 or 15.0% w/w, 1.0, 2.5, 5.0 or 7.5% w/w up to 2.5, 5.0, 7.5 or 10.0% w/w, 2.0, 2.5, 3.0, 3.5, 4.0 or 4.5% w/w up to 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0% w/w, about 3% w/w.

In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae has a 50% or greater reduction in starch content as compared to a starch content of a parent strain of Chlorella microalgae, grown under the same conditions. Specifically, the variant strains of Chlorella microalgae records approximately 50% to 100%, preferably, 50 to 90%, or 60 to 87% (e.g. about 86%) reduction in the starch content as compared to a parent (or wild-type) strain of Chlorella microalgae. For example, the reduction in starch content of the chlorophyll-deficient strain of Chlorella microalgae may be 50, 60, 70, 80 or 90% up to 60, 70, 80, 90 or 100%, preferably, 50, 60, 70 or 80% up to 60, 70, 80 or 90%, or 60, 70 or 80% up to 70, 80 or 87%. A greater than 50% reduction in starch content of the variant strain of Chlorella microalgae compared to the starch content of the parent strain of Chlorella microalgae is associated with an improved protein content in the variant strain of Chlorella microalgae compared to the protein content in the parent strain of Chlorella microalgae.

In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae preferably has a chitin content in a range of 0.001 to 4.0 mg/g dry cell weight. The term "chitin" as used herein refers to a structural polysaccharide containing nitrogen and glucosamine and includes chitin, chitin-like polysaccharide and chitosan. Chitin is synthesized from monomer units N-acetyl-D- glucosamine linked by a |3-(1 ,4) covalent linkage, by a chitin synthase enzyme. Chitin is insoluble in most organic solvents, water and dilute acids and, as a result, imparts rigidity to the cell walls. Chitin is a primary component of cell walls of a varied group of organisms, such as yeast, fungi, molluscs, arthropods (for example insects and crustaceans), nematodes, and marine animals. Interestingly, chitin is not a common component of the cell walls in green algae and the presence of chitin is a genus-defining characteristic of Chlorella species.

It will be appreciated that besides the preferred low chitin content, a preferred chlorophylldeficient strain of Chlorella microalgae has a low assayable chitosan content. The term "chitosan" as used herein refers to a linear polysaccharide composed of p-(1 — >4)-linked D- glucosamine (deacetylated unit) and /V-acetyl-D-glucosamine (acetylated unit). Typically, chitosan is a copolymer of 2-amino-2-deoxy-p-d-glucopyranose (glucosamine) and 2-acetamide- 2-deoxy-p-d-glucopyranose (N-acetylglucosamine). Moreover, chitosan is obtained by deacetylation of chitin. Chitosan is soluble in organic acids unlike chitin that is insoluble in water and most organic solvents. It will be appreciated that calcofluor white staining may be used for flow cytometry isolation of chitin mutants as well as chitosan.

Optionally, the variant strain of Chlorella microalgae has a chitin content in a range of 0.001 to 4.0 milligram per gram of dry cell weight, preferably 0.1 to 4.0 mg/g DCW, 0.4 to 2.0 mg/g DCW. For example, the chitin content of the variant strain of Chlorella microalgae may be in a range of 0.001 , 0.003, 0.006, 0.009, 0.012, 0.015, 0.018, 0.021 , 0.024, 0.027, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.50, 1.00 mg/g DCW up to 1.01 , 1.50, 2.00, 2.50, 3.00, 3.50, 3.60, 3.70, 3.80, 3.90, or 4.00 mg/g DCW, preferably 0.10, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00 or

3.50 mg/g DCW up to 1.50, 2.00, 2.50, 3.00, 3.50, or 4.00 mg/g DCW, or 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.50, 1.00 or 1.50 mg/g DCW up to 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.50, 1.00,

1.50 mg/g or 2.00 DCW. In an example, the variant strain of Chlorella microalgae may contain a chitin content of 2.36 mg/g DCW up to 3.09 mg/g DCW as compared to a chitin content 4.94 mg/g DCW in the parent (or wild-type) strain of Chlorella microalgae. The reduction in chitin content results in higher overall digestibility of the variant strain of Chlorella microalgae, which is beneficial when the algae are used as food ingredients. In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae has a 25% or greater reduction in chitin content as compared to a chitin content of a parent strain of Chlorella microalgae, grown under the same conditions. A greater than 25% reduction in chitin content of the variant strain of Chlorella microalgae compared to the chitin content of the parent strain of Chlorella microalgae is associated with an improved protein digestibility-corrected amino acid score (PDCAAS) score. Specifically, the variant strain of Chlorella microalgae records approximately 37.40% up to 52.30%%, preferably, 40-51%, or 45-50% reduction in the chitin content as compared to a parent (or wild-type) strain of Chlorella microalgae. For example, the reduction in chitin content of the chlorophyll-deficient strain of Chlorella microalgae may be 37.40, 40.40, 44.40 or 48.40% up to 40.40, 44.40, 48.40 or 51.30%, preferably 40, 44 or 48% up to 44, 48 or 51 %, or 45, 47 or 49% up to 47, 49 or 50%.

In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae has a protein digestibility-corrected amino acid score (PDCAAS) in a range of 0.75 to 1. The term "Protein digestibility-corrected amino acid score (PDCAAS)" refers to a standard method used to quantify the nutritional quality of a given protein source. PDCAAS rating has been adopted by the Food and Drug Administration (FDA, US) and the Food and Agricultural Organization (FAO, WHO) as "the preferred 'best'" assay for determining protein quality. Specifically, the PDCAAS assay combines a protein digestibility score with a protein quality score based on the amino acid requirements of humans and expresses the result as a percentage score of overall protein quality. It is well-understood in the art that the calculated value PDCAAS can exceed 100% (equivalent to a ratio of 1.0) by this method, so calculated PDCAAS values that exceed 100% may, by convention, be optionally truncated to 100% (or expressed as a ratio of 1.0). To arrive at the final PDCAAS score, the protein digestibility is determined by the difference in protein of input and output material of a digestion model. Beneficially, the present disclosure uses the Protein Digestibility Assay KitTM (K-PDCAASTM) (using the Animal-Safe Accurate Protein Quality Score (ASAP-Quality Score Method) developed under U.S. Patent No. 9,738,920 by Medallion Labs), manufactured by Megazyme, Ireland to determine the protein digestibility score. Further, the protein quality or amino acid score is calculated by identifying the limiting amino acid in the sample (which has the lowest ratio against recommended values); therefore, it follows that amino acid compositions which are more similar to the recommended values will have higher amino acid scores. The essential amino acids include histidine, isoleucine, leucine, lysine, methionine, cystine, phenylalanine, tyrosine, threonine, tryptophan and valine. The total protein content, which is determined from the total nitrogen content (via DUMAS analysis), is used to convert the mass of amino acids from a proportion of total mass to a proportion of protein. Therefore, the amino acid score is impacted by the nitrogen-to-protein (NtP) coefficient used to convert elemental nitrogen into protein content. In the present disclosure, total protein content is calculated using the Dumas method for protein assay, and an NtP conversion factor of 6.25 (Nx6.25) to normalize the calculated protein content to other protein sources for comparison. Nx6.25 is the established, standard NtP conversion factor that used to calculate Dumas protein content for most foods including microalgae (McCance and Widdowson’s The Composition of Foods; ISBN 978-1- 84973-636-7). The typical PDCAAS of the variant strain of Chlorella microalgae may be for example in a range from 0.75, 0.80, 0.85, 0.90 or 0.95 up to 0.80, 0.85, 0.90, 0.95 or 1.0.

In addition to the low chlorophyll content, high protein content, and preferably also the low chitin content, which is linked to strain digestibility and nutrient bioavailability, the chlorophylldeficient strain of Chlorella microalgae of the invention may have one or more additional desirable phenotypes as a result of further stable genetic mutations. In an embodiment, the one or more additional desirable phenotypes is selected from a group comprising: a colour, a pigment content, a smell, a taste, a texture, a biochemical composition and improved tolerance to process conditions. Such desirable phenotypes may be, for example, associated with organoleptic properties of the strain (e.g. colour, flavour, smell), content of other components such as, but not limited to, proteins, pigments (such as lutein, carotenoids and chlorophyll), micro- and macronutrients (for example potassium, sodium and nitrogen), minerals, vitamins (such as B and E vitamins), carbohydrates, fatty acids, antioxidants, glycoproteins, glycerols, phytochemicals (such as flavonoids and tannins), biochemical composition, dietary fibre content, and improved tolerance to process conditions.

Optionally, the chlorophyll-deficient strain of Chlorella microalgae is incapable of producing, or has substantially reduced ability to produce chlorophyll pigments (chlorophyll a and/or chlorophyll b), however possesses a variable, but genetically-determined ability to produce other pigments, such as for example lutein, xanthophylls other carotenoids and tetrapyrroles. The term “lutein" refers to a primary xanthophyll (carotenoid) in green microalgae that enables the microalgae to absorb blue light and reflect yellow or orange-red light from the visible region of the electromagnetic spectrum. Lutein functions as a light energy modulator in the microalgae and serves as a non-photochemical quenching agent that protects cells of the microalgae from photochemical damage caused by high intensity of light during photosynthesis. Moreover, the lutein content in an organism is typically genetically determined and regulated by growth conditions, including but not limited to temperature, pH of growth medium, exposure to light, nitrogen content in the growth medium or atmosphere, salinity of growth medium, rate of growth and so forth. Optionally, the variant strain of Chlorella microalgae has a lutein content in a range of 3 to 10 mg/g DOW, preferably 5 to 8, 4 to 7, or 3 to 4.5. For example, the lutein content of the parent strain of Chlorella microalgae may be 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or 9.5 mg/g DOW up to 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg/g DOW, preferably 5, 5.5, 6, 6.5, 7 or 7.5 mg/g DOW up to 5.5, 6, 6.5, 7, 7.5 or 8 mg/g DOW, 4, 4.5, 5, 5.5, 6 or 6.5 mg/g DCW up to 4.5, 5, 5.5, 6, 6.5 or 7 mg/g DCW, or 3, 3.5 or 4 mg/g DCW up to 3.5, 4 or 4.5 mg/g DCW. Optionally, the Chlorella microalgae has a lutein content below 9 mg/g DCW, more preferably below 8 mg/g DCW, yet more preferably below 7 mg/g DCW, yet more preferably still below 6 mg/g DCW, yet more preferably still below 5 mg/g DCW, yet more preferably below 4 mg/g DCW, yet more preferably still below 3 mg/g DCW, yet more preferably still below 2 mg/g DCW, yet more preferably still below 1 mg/g DCW, and yet more preferably up to 0.1 mg/g DCW of the lutein content of a parent strain of Chlorella microalgae. Typically, the average normal amount of lutein in the parent strain of Chlorella microalgae is 5 mg/g DCW.

Moreover, the content of chlorophyll a, chlorophyll b and/or lutein and/or other pigments in the chlorophyll-deficient strain of Chlorella microalgae can be determined using analytical methods known to the skilled person, for example chromatographic or spectrophotometric techniques.

In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae has a colour selected from at least one of white, cream, pale yellow, yellow, lime, pale green, golden, caramel, orange, pink, red, deep-red, red-brown or brown. Optionally, the said colours may also be associated strongly with a change in smell and taste of the chlorophyll-deficient strain of Chlorella microalgae as compared to its wild-type or parental strain. Notably, as a result of the presence of lutein, xanthophylls other carotenoids and tetrapyrroles, the colour of such chlorophyll-deficient strains of Chlorella microalgae is one of: pink, red, deep-red, red-brown, brown or yellow-brown colour. Typically, the colour may be determined by visual inspection of the strains, however, several other analytical methods (such as those stated herein, including L*a*b Cl ELAB colour values) may also be used to determine and measure the colour of the chlorophyll-deficient strain of Chlorella microalgae.

In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae is obtained from a parent strain of Chlorella microalgae, by performing mutagenesis of the parent strain of Chlorella microalgae. The term "mutagenesis" as used herein, relates to a technique of inducing mutations by artificially exposing the organism to mutagens using laboratory procedures. Mutagens have the effect of increasing the frequency of genetic mutation over and above the natural frequency of spontaneously occurring mutations. The variation of the wild-type or parental strain may be a genetic mutant.

In an embodiment, mutagenesis is performed by exposure of the parent strain of Chlorella microalgae to a mutagenic chemical. It will be appreciated that chemical mutagenesis is not considered to produce Genetically Modified Organisms (GMOs) as defined by the current Ell legislation; European Union Directive 2001/18/EC (Annex 1 B).

In an embodiment, the mutagenic chemical is an alkylating agent. The term "alkylating agent' as used herein, refers to one or more classes of alkylating agents functioning as mutagens. Generally, the alkylating agents transfer alkyl groups (such as methyl or ethyl group) to macromolecules (such as bases, or the backbone phosphate groups of the nucleic acids) under physiological conditions. Typically, the alkyl group acts on nucleophilic sites of the macromolecule, for example, nitrogen or oxygen nucleophiles in DNA (as described by Gates 2009; DOI: 10.1021/tx900242k). Such transfers result in alkylation of bases (for example guanine) and subsequent mispairing of said base during DNA replication (with for example, thymine instead of cytosine). Normally, the alkylating agents that function as mutagens include but are not limited to: sulphur mustards, nitrogen mustards, epoxides, ethylene imines, alkyl alkanesulphonates, dialkyl sulphates, beta-lactones, diazo compounds and nitroso compounds. Examples of such alkylating agents from each of these respective classes include: mustard gas, nitrogen mustard (HN2), ethylene oxide (EG), diepoxybutane (DEB), ethyleneimine (El), triethylenemelamine (TEM), ethyl methanesulphonate (EMS) and methyl methansulphonate (MMS), diethylsulphate (DES), beta-propiolactone, diazomethane, N-Nitroso-N-methylurea (NMU) and N-methyl-N’-nitro-N-nitrosoguanidine (NG or NTG or MNNG) (as described by Auerbach 1976; DOI: 10.1007/978-1-4899-3103-0_16).

In an embodiment, the concentration of the mutagenic chemical is in a range from 0.1 to 2.0 M. Typically, mutagenesis is performed by exposure of the parent strain of Chlorella microalgae to a sub-lethal quantity of the mutagenic chemical. The sub-lethal quantity of the mutagenic chemical is defined as the amount or quantity of the mutagenic chemical that results in less than 100% kill of the parent strain of Chlorella microalgae in a given time. The concentration of the mutagenic chemical may be 0.1 to 2.0 M, 0.2 to 2.0 M, 0.5 to 2.0 M, 0.7 to 1.0 M. The concentration of the mutagenic chemical may be for example from 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 M up to 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 M, preferably 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 M up to 0.3, 0.4, 0.5, 0.6, 0.7,

0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 M, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2,

1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 M up to 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,

1.9 or 2.0 M, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 .8 or 1 .9 M up to 0.8, 0.9, 1.0, 1.1 ,

1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 M, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 M up to 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0. In an example, the concentration of the mutagenic chemical is 0.2 M of EMS that is non-lethal to the Chlorella microalgae species. In another example, the sub-lethal quantity is 0.2 M of MMS that produces a 20% lethality to the Chlorella microalgae species (referred to as "mutagen kil hereafter). In yet another example, the sub-lethal quantity is 0.8 M of EMS that produces a 40% mutagen kill. In still another example, the sub-lethal quantity is 0.8 M of MMS that produces a 60% mutagen kill. In case of EMS, repeated replication of such mispaired DNA can result in a transition mutation, wherein original G:C base pairs change to A:T base pairs, thereby changing the genetic makeup of the organism. In such case, the replication of such mutated DNA may create heritable missense mutations or nonsense mutations within coding sequences or impacting gene expression or gene function by compromising regulatory sequence functionality including RNA splice-site mutations or promoter or other regulatory sequence mutations. Beneficially, the use of alkylating agents as mutagenic chemicals for plant breeding, for human consumption, is not considered to produce Genetically Modified Organism (GMOs) as defined by the current Ell legislation; European Union Directive 2001/18/EC (Annex 1 B), and is therefore acceptable for further applications in various industries, such as food, health, biotechnology and biofuels.

The mutagenesis may be performed by exposure of the parent strain of Chlorella microalgae to a mutagenic chemical for a specific time. The exposure time to a given concentration of the mutagenic chemical also influences its lethality. In other words, the concentration (and quantity) of the mutagenic chemical used for performing the mutagenesis, combined with the exposure time, can determine the amount of mutation undergone by the organism. Optionally, the specific time for treatment with the mutagenic chemical is 1 to 120 minutes. More optionally, the quantity of the mutagen (or mutagen dose) is defined as a concentration of the mutagen multiplied by an exposure time. Specifically, the sub-lethal quantity of the mutagen is obtained by altering the mutagen concentration, the exposure time, or a combination of both, for example.

It will be appreciated that heavily mutagenized cells of the organism accumulate multiple mutations of genetic material, which are often deleterious to the viability or overall fitness of the mutagenized strain as assessed using standard growth performance assays, for example. This is of particular importance in a haploid organism such as Chlorella microalgae, where only a single copy of each gene is present. It is common that multiple mutations occur within the genomes of mutagenized strains. Use of a high quantity of alkylating agent for performing the mutagenesis may result in point mutations that create aberrations in the mutated strain of Chlorella microalgae as compared to the parent strain of Chlorella microalgae, or may result in death of the mutated strain of Chlorella microalgae. Optionally, the degree of mutagen kill may be measured by determining cell viability using a conventional quantification technique (for example, viable counts, viability staining, flow cytometry, and the like) known in the art.

Therefore, using a sub-lethal or non-lethal quantity (0.1 to 2.0 M) of the mutagenic chemical, such as alkylating agents, for a specific time, enables generation of desired phenotypes while preventing or minimising accumulation of undesirable traits that might reduce overall strain fitness, hamper growth, or result in death of the organism. Typically, optimal mutagen dose is determined empirically for a specific species of an organism, and varies from organism to organism. Similarly, different mutagens have different mechanisms of action, and an optimal dosing strategy (i.e. relative concentration multiplied by time) using a mutagen for the target organism can be determined for each. Therefore, besides determining the dosing strategy, identification of a suitable mutagen is equally essential as each mutagen class has a specific mechanism of action that directly affects the diversity of mutations generated in the target organism which, thereby, also impacts the utility of the resulting mutant library.

In an example, the mutagenesis of the parent strain of Chlorella microalgae may be performed by exposure of the parent strain of Chlorella microalgae to a 1.0 M dose of EMS for an exposure time of 1 minute or a dose of EMS above 1 .0 M for exposure time of a shorter period, for example 30 seconds, to produce a 50% lethality to the Chlorella microalgae species, for example. This process, i.e. combining mutagenic chemical concentration and exposure time to said mutagen, results in a kill rate which acts as a proxy for mutation frequency. Consequently, surviving cells of said process have one or more mutations within their genomes. Collectively, such cells comprise a pool (or library) of mutations from which can be selected desirable variant strains using a suitable method.

Alternatively, mutagenesis is performed by exposure of the parent strain of Chlorella microalgae to a physical mutagen, optionally wherein the physical mutagen comprises at least one of UV light, gamma rays, X-rays. These mutagens cause changes in the genotype of the parent strain of Chlorella microalgae to result in the mutated strain of Chlorella microalgae. In such an instance, as an alternative to performing the mutagenesis of the parent strain of Chlorella microalgae by exposure to the mutagenic chemicals, mutagenesis by exposure to physical mutagens can be performed to obtain the mutated strain of Chlorella microalgae.

Beneficially, mutagenesis and, in particular, the use of a mutagenic chemical, preferably sub-lethal quantities thereof, according to the invention, results in genetic variant strains of Chlorella microalgae in which the overall chlorophyll content, chitin content, protein content and high digestibility thereof as disclosed of the strain is the result of a stable genetic mutation.

In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae is genetically stable. The term "genetically stable" as used herein, refers to a characteristic of a species or a strain/isolate to resist changes and maintain its genotype over multiple generations or cell divisions, ideally hundreds to several thousand generations, in non-selective conditions.

Notably, the parent strains of Chlorella microalgae are haploid. A haploid parent strain prevents the variant strains thereof from reverting back from a desired genotype to the genotype commonly associated with the parent strain of Chlorella microalgae over successive generations of cultivation, beneficially exhibiting relative stability of the desired phenotype in such strains. The chlorophyll-deficient strain of Chlorella microalgae (i.e. the variant strain of Chlorella microalgae) is genetically stable. Notably, the quantitative analysis, including flow cytometry or, optionally, qualitatively, confocal microscopy of variant strains of Chlorella microalgae maintained both on agar and in liquid culture is sufficient to conclude that the phenotype, such as reduced chlorophyll content, is genetically stable in the variant strain of Chlorella microalgae. Further, the stability of genetic mutations can also be confirmed by direct genetic sequencing.

Optionally, the variant strain of Chlorella microalgae is genetically stable and is electrocompetent or has improved genetic transformation capacity to take up exogenous DNA, RNA, protein, polypeptides or complexes derived therefrom as compared to its parent strain. For example, a reduced chitin content of a variant strain of Chlorella microalgae improves the genetic transformation efficiency of the variant strain of Chlorella microalgae. The genetic transformation of a variant strain of Chlorella microalgae with heterologous DNA may be achieved using conventional techniques, such as for example nanoparticle-based gene gun ("biolistics") and electroporation.

In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae is cultivated in a heterotrophic growth mode. Notably, algae such as Chlorella microalgae can grow in conditions ranging from optimal to extreme and in varied habitats. The variant strains of the invention can be mixotrophs or heterotrophs. In a preferred embodiment, the variant strains of the invention are cultivated in the heterotrophic growth mode (i.e. cultivatable solely on an organic carbon energy source, such as glucose, in the absence of light). Beneficially, such heterotrophic growth allows large scale economical production of the variant strains as a result of the superior growth rate and biomass yield that can be produced in proven existing plant designs, when compared to phototrophic or mixotrophic methods of microalgal cultivation.

Preferably, Chlorella microalgae of the invention is produced by a food-grade process to deliver a food-grade product. The Chlorella microalgae are cultivated in fermentation medium using a heterotrophic production process; in which an organic carbon energy source, preferably glucose, is used as feedstock and is supplied either in batch mode or preferably, fed-batch mode. Beneficially, the fed-batch process can deliver a higher final DCW and normally results in higher biomass productivities and consequently, a faster process. Optionally, the glucose feeding process could be continuous, providing an accurate feeding profile can be achieved. Such a feeding profile takes into account a feed rate, a current growth rate of the culture and a target media glucose concentration to achieve optimal growth of the low chlorophyll, high protein Chlorella variants. Moreover, calculations for the rate of feed of glucose are standard and known in the art. Optionally, the fermentation can also operate in semi-continuous mode with several draws, providing that nutrients are added to compensate for the broth removal rate. The process will start in batch mode (“the seed train”); being fed one or multiple glucose boluses as required to achieve the desired biomass density that is required to inoculate the main fed-batch production process. Specifically, the process will start with a 1mL vial of Chlorella microalgal biomass frozen at -80 °C that, when thawed, inoculates a seed train that takes place in two phases (P1 and P2) in sterile baffled and ventilated Erlenmeyer flasks. After completion of the seed train, the production fermenter is inoculated with the required volume to produce an initial concentration of at least 0.8 grams per litre, or, in an alternative embodiment, of at least 3 grams per litre.

In an embodiment, the chlorophyll-deficient strain of Chlorella microalgae is cultivated:

- at a specific temperature;

- for a predefined period of time;

- without the presence of light; and

- in the presence of an organic carbon energy source.

In an embodiment, the specific temperature is in a range of 20 to 35 °C, optionally in the range of 26 to 29 °C, e.g. about 28 °C. These temperatures are particularly preferred in case the Chlorella microalgae are Chlorella vulgaris microalgae.

In an embodiment, the specific temperature is in a range of 20 to 35 °C, optionally in the range of 25 to 30 °C, optionally in the range 28 °C to 30 °C, e.g. about 29 °C. These temperatures are particularly preferred in case the Chlorella microalgae are Chlorella sorokiniana microalgae.

In an embodiment, the predefined period of time is in a range of 1 to 5 weeks, optionally in the range of 1 to 3 weeks. Alternatively, the predefined period of time is in the range of 1 to 7 days, optionally in the range of 1 to 5 days.

In an embodiment, the organic carbon energy source is glucose and/or acetate, preferably glucose. Optionally, the organic carbon energy source is derived from invert sugar, where the enzyme invertase has been used to hydrolyse sucrose into glucose and fructose.

With the process as hereinbefore described, biomass densities that are typically greater than 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 100, 115, 120, 125, 130, 135, 140, 145, 150, 155, or 160 g/L can be achieved. A higher final biomass density could be achieved by running a longer fermentation process, or using a continuous feed profile, for example. After the fermentation process, the biomass is optionally washed with water and/or concentrated to, for example, 200 g/L by centrifugation. Additionally, optionally, the biomass is then lysed by mechanical means to crack or break open the cells. The processed biomass is then spray dried. After spray drying, the powder is packed promptly to avoid moisture increase and oxidative phenomena.

In an embodiment, the organic carbon energy source is glucose having a glucose to biomass conversion ratio of more than 0.45. It will be appreciated that the high protein (and optionally white and/or low starch) variant strains of Chlorella microalgae require a controlled concentration of glucose in fermentation broth to maintain optimal (or economical) growth, thereof. The conversion of glucose to biomass as a result of cultivating Chlorella microalgae in the fermentation broth is typically measured as a glucose to biomass conversion ratio or glucose to biomass yield coefficient. Moreover, said variants of Chlorella microalgae require a continuous feeding throughout the fermentation run, in order to sustain optimal growth by maintaining the optimal glucose level in the fermentation broth (as described above). In this regard, a glucose to biomass conversion ratio of >0.45, such as 0.55, is preferred. To achieve this, an automated feed profile may be developed to maintain a target of 20 g/L optimal glucose concentration in the fermentation broth for the said strain. It will be appreciated that the optimal glucose level can vary for different species of Chlorella microalgae. For example, variants of Chlorella sorokiniana with a low starch phenotype may exhibit optimal growth and glucose to biomass conversion ratio at circa 10 g/L glucose, but exhibit growth inhibition at circa 30 g/L glucose concentration in the fermentation broth. By contrast, a low starch variant of Chlorella vulgaris does not show decline in growth until a concentration of 60-70 g/L glucose in the fermentation broth. Sub-optimal growth of Chlorella microalgae resulting from the non-optimised feeding or concentration of glucose typically also results in a reduction in the glucose to biomass yield coefficient. Therefore, it will be appreciated that an optimal glucose feed regime for the particular Chlorella microalgae is required throughout the fermentation to achieve efficient, economic and timely conversion of glucose to biomass.

Typically, the variant strain of Chlorella sorokiniana is cultivated at a specific temperature, optionally ranging from 20 to 35 °C and more optionally in a range from 28 to 30 °C, for a predefined period of time, such as in a range of 1 to 5 weeks, optionally in a range of 1 to 3 weeks, more optionally less than 7 days, optionally without the presence of light, i.e. in the dark or absence of light, and in the presence of an organic carbon energy source such as for example glucose (heterotrophic growth mode) and/or acetate (mixotrophic growth mode).

Alternatively, the mutated strain of Chlorella microalgae is cultivated under mixotrophic growth mode with partial presence of light, such as by exposure of the mutated strain of Chlorella microalgae to light for a limited time per day or at a minimally set light intensity. In such an example, the mixotrophic growth is performed by employing simultaneous use of different sources of energy for cultivating the mutated strain of Chlorella microalgae. Alternatively, the mutated strain of Chlorella microalgae is cultivated under phototrophic growth mode in the light with supply of air or a specific CO2 supply to facilitate photosynthetic growth.

Optionally, the characteristics of the light used (e.g. intensity of light, wavelength (colour) of light and so forth) during mutagenesis, are defined. More optionally, the light intensity range, wavelength and quality may be, e.g. white LED, white fluorescent, daylight fluorescent, red LED, or mix of white and red or other LED, with light intensity values ranging from 5 micromoles/m2/s to 300 micromoles/m2/s, most preferably low light conditions comprise 2 to 25 micromoles/m2/s of white LED light.

According to a preference, the invention provides a strain of Chlorella microalgae comprising a genomic DNA sequence that is at least 50% identical to Sequence 2 (SEQ ID NO: 2). The genomic DNA sequence Sequence 2 (SEQ ID NO: 2) encodes for a Chll subunit of magnesium chelatase. Preferably the genomic DNA sequence is at least 60% identical to Sequence 2 (SEQ ID NO: 2), more preferably at least 70% identical to Sequence 2 (SEQ ID NO: 2), still more preferably at least 80% identical to Sequence 2 (SEQ ID NO: 2). In some particularly preferred embodiments, the genomic DNA sequence is at least 85% identical to Sequence 2 (SEQ ID NO: 2), such as at least 80% identical to Sequence 2 (SEQ ID NO: 2), at least 85% identical to Sequence 2 (SEQ ID NO: 2), at least 90% identical to Sequence 2 (SEQ ID NO: 2), or at least 95% identical to Sequence 2 (SEQ ID NO: 2), more preferably at least 99% identical to Sequence 2 (SEQ ID NO: 2) and still more preferably it is identical to Sequence 2 (SEQ ID NO: 2).

According to a preference, the invention provides a strain of Chlorella microalgae comprising a genomic DNA sequence that is at least 50% identical to Sequence 4 (SEQ ID NO: 4). The genomic DNA sequence Sequence 4 (SEQ ID NO: 4) encodes for a phytoene desaturase. Preferably the genomic DNA sequence is at least 60% identical to Sequence 4 (SEQ ID NO: 4), more preferably at least 70% identical to Sequence 4 (SEQ ID NO: 4), still more preferably at least 80% identical to Sequence 4 (SEQ ID NO: 4). In some particularly preferred embodiments, the genomic DNA sequence is at least 85% identical to Sequence 4 (SEQ ID NO: 4), such as at least 80% identical to Sequence 4 (SEQ ID NO: 4), at least 85% identical to Sequence 4 (SEQ ID NO: 4), at least 90% identical to Sequence 4 (SEQ ID NO: 4), or at least 95% identical to Sequence 4 (SEQ ID NO: 4), more preferably at least 99% identical to Sequence 4 (SEQ ID NO:

4) and still more preferably it is identical to Sequence 4 (SEQ ID NO: 4).

According to a preference, the invention provides a strain of Chlorella microalgae comprising a genomic DNA sequence that is at least 50% identical to Sequence 5 (SEQ ID NO:

5). The genomic DNA sequence Sequence 5 (SEQ ID NO: 5) encodes for a phytoene desaturase. Preferably the genomic DNA sequence is at least 60% identical to Sequence 5 (SEQ ID NO: 5), more preferably at least 70% identical to Sequence 5 (SEQ ID NO: 5), still more preferably at least 80% identical to Sequence 5 (SEQ ID NO: 5). In some particularly preferred embodiments, the genomic DNA sequence is at least 85% identical to Sequence 5 (SEQ ID NO: 5), such as at least 80% identical to Sequence 5 (SEQ ID NO: 5), at least 85% identical to Sequence 5 (SEQ ID NO: 5), at least 90% identical to Sequence 5 (SEQ ID NO: 5), or at least 95% identical to Sequence 5 (SEQ ID NO: 5), more preferably at least 99% identical to Sequence 4 (SEQ ID NO: 4) and still more preferably it is identical to Sequence 5 (SEQ ID NO: 5).

According to a preference, the invention provides a strain of Chlorella microalgae comprising a genomic DNA sequence that is at least 50% identical to Sequence 7 (SEQ ID NO: 7). The genomic DNA sequence Sequence 7 (SEQ ID NO: 7) encodes for a ChIH subunit of a magnesium chelatase. Preferably the genomic DNA sequence is at least 60% identical to Sequence 7 (SEQ ID NO: 7), more preferably at least 70% identical to Sequence 7 (SEQ ID NO: 7), still more preferably at least 80% identical to Sequence 7 (SEQ ID NO: 7). In some particularly preferred embodiments, the genomic DNA sequence is at least 85% identical to Sequence 7 (SEQ ID NO: 7), such as at least 80% identical to Sequence 7 (SEQ ID NO: 7), at least 85% identical to Sequence 7 (SEQ ID NO: 7), at least 90% identical to Sequence 7 (SEQ ID NO: 7), or at least 95% identical to Sequence 7 (SEQ ID NO: 7), more preferably at least 99% identical to Sequence 7 (SEQ ID NO: 7) and still more preferably it is identical to Sequence 7 (SEQ ID NO: 7).

According to a preference, the invention provides a strain of Chlorella microalgae comprising a genomic DNA sequence that is at least 50% identical to Sequence 25 (SEQ ID NO: 25). The genomic DNA sequence Sequence 25 (SEQ ID NO: 25) encodes for a ChIH subunit of a magnesium chelatase. Preferably the genomic DNA sequence is at least 60% identical to Sequence 25 (SEQ ID NO: 25), more preferably at least 70% identical to Sequence 25 (SEQ ID NO: 25), still more preferably at least 80% identical to Sequence 25 (SEQ ID NO: 25). In some particularly preferred embodiments, the genomic DNA sequence is at least 85% identical to Sequence 25 (SEQ ID NO: 25), such as at least 80% identical to Sequence 25 (SEQ ID NO: 25), at least 85% identical to Sequence 25 (SEQ ID NO: 25), at least 90% identical to Sequence 25 (SEQ ID NO: 25), or at least 95% identical to Sequence 25 (SEQ ID NO: 25), more preferably at least 99% identical to Sequence 25 (SEQ ID NO: 25) and still more preferably it is identical to Sequence 25 (SEQ ID NO: 25).

According to a preference, the invention provides a strain of Chlorella microalgae comprising a genomic DNA sequence that is at least 50% identical to Sequence 66 (SEQ ID NO: 66). The genomic DNA sequence Sequence 66 (SEQ ID NO: 66) encodes for a Chll subunit of a magnesium chelatase. Preferably the genomic DNA sequence is at least 60% identical to Sequence 66 (SEQ ID NO: 66), more preferably at least 70% identical to Sequence 66 (SEQ ID NO: 66), still more preferably at least 80% identical to Sequence 66 (SEQ ID NO: 66). In some particularly preferred embodiments, the genomic DNA sequence is at least 85% identical to Sequence 66 (SEQ ID NO: 66), such as at least 80% identical to Sequence 66 (SEQ ID NO: 66), at least 85% identical to Sequence 66 (SEQ ID NO: 66), at least 90% identical to Sequence 66 (SEQ ID NO: 66), or at least 95% identical to Sequence 66 (SEQ ID NO: 66), more preferably at least 99% identical to Sequence 66 (SEQ ID NO: 66) and still more preferably it is identical to Sequence 66 (SEQ ID NO: 66).

According to a preference, the invention provides a strain of Chlorella microalgae comprising a genomic DNA sequence that is at least 50% identical to Sequence 27 (SEQ ID NO: 27). The genomic DNA sequence Sequence 27 (SEQ ID NO: 27) encodes for a phytoene desaturase. Preferably the genomic DNA sequence is at least 60% identical to Sequence 27 (SEQ ID NO: 27), more preferably at least 70% identical to Sequence 66 (SEQ ID NO: 27), still more preferably at least 80% identical to Sequence 27 (SEQ ID NO: 27). In some particularly preferred embodiments, the genomic DNA sequence is at least 85% identical to Sequence 27 (SEQ ID NO: 27), such as at least 80% identical to Sequence 27 (SEQ ID NO: 27), at least 85% identical to Sequence 27 (SEQ ID NO: 27), at least 90% identical to Sequence 27 (SEQ ID NO: 27), or at least 95% identical to Sequence 27 (SEQ ID NO: 27), more preferably at least 99% identical to Sequence 27 (SEQ ID NO: 27) and still more preferably it is identical to Sequence 27 (SEQ ID NO: 27).

According to a particularly preferred aspect, the invention provides a Chlorella vulgaris strain designated WC03, deposited on December 14, 2023 at the Culture Collection of Algae and Protozoa (CCAP), SAMS Ltd., Scottish Marine Institute, OBAN, Argyll, PA37 1QA, United Kingdom, in accordance with the Budapest Treaty, with a Patent Deposit Designation of CCAP 211/143.

According to a particularly preferred aspect, the invention also provides a Chlorella sorokiniana strain designated CS174, deposited on December 14, 2023 at the Culture Collection of Algae and Protozoa (CCAP), SAMS Ltd., Scottish Marine Institute, OBAN, Argyll, PA37 1QA, United Kingdom, in accordance with the Budapest Treaty, with a Patent Deposit Designation of CCAP 211/142.

The Chlorella biomass used in the present invention is preferably derived from one or more of the above identified preferred strains.

The invention also provides a method of producing a chlorophyll-deficient strain of Chlorella microalgae having a protein content of at least 50% w/w, the method comprising: a) obtaining a parent strain of Chlorella microalgae; b) performing mutagenesis of the parent strain of Chlorella microalgae; c) cultivating the mutated strain of Chlorella microalgae at a specific temperature, for a predefined period of time, and in the presence of an organic carbon source; and d) identifying and isolating chlorophyll-deficient mutants of the parent strain of Chlorella microalgae having a protein content of at least 50% w/w.

The modified strains of Chlorella may be subjected to additional rounds of mutagenesis, both before and after the steps in which mutants with a protein content of at least 50% w/w is isolated. Optionally, the method further comprises performing steps (b) to (d) repeatedly for selecting healthy colonies of the modified strain of Chlorella based on desired traits, wherein the desired traits comprise a colour, a pigment content, a protein content, cell wall modification and improved tolerance to process conditions selected from a group of temperature, pH, sheer stress and osmolality. Furthermore, the Chlorella strains are preferably selected to be stable through generations.

Thus, the identification and isolation of mutant strains in one or more of these rounds of mutagenesis, may suitably also involve involve identifying and isolating mutants of the parent strain of Chlorella microalgae having a mutation in at least one gene that encodes for phytoene desaturase or a subunit thereof, and further having a mutation in at least one gene that encodes for magnesium chelatase or a subunit thereof. The method may also involve two rounds of mutagenesis, wherein the two identifying and isolating steps of the mutants each involve one of identifying and isolating mutants having a mutation in at least one gene that encodes for phytoene desaturase or a subunit thereof and of having a mutation in at least one gene that encodes for magnesium chelatase or a subunit thereof.

Thus, it is conceived that for instance a first one or more rounds of isolating and cultivating mutants are directed at improving their colour (improved whiteness, reduced chlorophyll levels and/or reduced carotenoid levels) and subsequently, a further round of mutagenesis is directed towards isolating mutants with a higher level of protein in the corresponding Chlorella biomass.

Various embodiments and variants disclosed above apply mutatis mutandis to the method.

In this regard, after mutagenesis, chlorophyll-deficient strain of Chlorella microalgae are isolated after cultivation under suitable growth condition, preferably cultivated in a heterotrophic growth mode at a specific temperature ranging from 20 to 35 °C, optionally, in a range from 25 to 28 °C, for a predefined period of time ranging from 1 to 5 weeks, optionally in a range of 1 to 3 weeks, more optionally less than 7 days, and in the presence of an organic carbon source such as for example glucose and/or acetate, without the presence of light, i.e. in the dark or in the absence of light. Isolation of suitable variants may be performed by any means known to the skilled person.

Flow cytometry (FCM) is a technique for detecting and measuring physical and chemical characteristics of a sample containing cells or particles. The sample containing cells or particles are often labelled with fluorescent markers for analysing cells and components. Flow cytometry is based upon analysis of the relative signal strength of fluorescence of a sample (autofluorescence) or fluorescent marker bound to a sample containing cells or particles. Optionally, flow cytometry serves as an enrichment step of physically sorting (namely, separating and isolating) desired cells away from cells with a parental phenotype and thereby purifying cells of interest based on their specific optical properties, referred to as fluorescence-activated cell sorting or cell sorting by flow cytometry. Optionally, such isolated cells are expanded by cultivation and re-sorted through one or more additional rounds of flow cytometry to confirm the stability of the phenotype or to enrich for a secondary mutant phenotype, for example a chlorophyll-deficient phenotype, or a colour phenotype, according to the fluorescence parameters chosen. They can then be further expanded in liquid culture or plated onto agar plus glucose plates for scoring of colours with respect to other mutations. Optionally, the identification of the variant strain of Chlorella microalgae comprises calcofluor white staining of the cells and sorting of the cells with flow cytometry. Calcofluor white staining along with flow cytometry is a well-known technique for rapid detection of the cell wall of various organisms, such as yeast and fungi. Calcofluor white (CFW) is a fluorescent blue dye or stain that binds to |3-(1-3)- and |3-(1 -4) polysaccharides, such as chitin in the cell wall. Moreover, CFW stained samples can be analysed using epifluorescence microscopy or flow cytometry for diagnosing, identifying, and counting the cells containing varying levels of chitin in cell walls. CFW has an absorption spectrum ranging from 300 to 412 nanometres (nm) with a peak at 347 nm. The CFW dye fluoresces when exposed to ultraviolet light, violet light or blue-violet light.

In an embodiment, the identification of the chlorophyll-deficient strain of Chlorella microalgae of the invention comprises sorting or screening the cells by any suitable technique, such as by using flow cytometry. The variant strain of Chlorella microalgae may be further selected for example based on a desirable pigment or protein content, wherein the desirable pigment or protein content is based upon a relative signal obtained on cell sorting by flow cytometry. The use of flow cytometry provides the advantages of examining thousands of cells per second and in real time and processing quantifiable data over a computer coupled to a flow cytometer. Furthermore, flow cytometry helps in cell counting, cell sorting, determining cell characteristics and function and detecting microorganisms.

The method further comprises selecting healthy (or viable) cells or filtering out unhealthy cells of the chlorophyll-deficient strain of Chlorella microalgae, preferably by cultivation under non-permissive or stressful conditions. It will be appreciated that during mutagenesis of the parent strain of Chlorella microalgae, cells of the Chlorella microalgae may acquire mutations at multiple sites within the genome, including a mutation or mutations that are causative for the desired phenotype. However, some mutated cells (strains) of Chlorella microalgae may additionally acquire deleterious mutations as a consequence of exposure to the mutagenic agent, resulting in one or more undesired mutations, for instance in essential genes. In such an instance, it is essential to filter out these unhealthy cells of the Chlorella microalgae associated with the deleterious mutations, to ensure selection of only those cells which are robust and able to grow well under desired cultivation conditions. This can be achieved by cultivation of the mutated strains during the period immediately following exposure to the chemical or physical mutagen under stressed or less permissive (or non-permissive) conditions, for instance at the limit of, or slightly above the normal upper temperature for cultivation and in the absence of light but in the presence of glucose. Optionally, mutated strains are cultivated under phototrophic conditions, more optionally, mutated strains are cultivated under mixotrophic conditions. Only robust strains are able to proliferate under stressful conditions. This approach enriches for strains that are not compromised in their general growth characteristics. Furthermore, after cultivation, the desired phenotypes related to reduced chlorophyll or chitin content can be scored. Undesired phenotypes, including chlorophyll or chitin content at levels associated with the parent strain or the wild-type strains of Chlorella microalgae, are not selected. In other words, they are filtered out. Optionally, cells of Chlorella microalgae that exhibit the desired phenotype across a series of generations are selected as healthy cells. More optionally, the mutated strain of Chlorella microalgae is cultivated at a temperature that is slightly higher than an ideal temperature for cultivation of the microalgal strain, to select only healthy cells of the Chlorella microalgae.

Optionally, the method comprises recovering the mutant strains of Chlorella microalgae on a solid agar plate. Recovering the mutant strains of Chlorella microalgae on the solid agar plate ensures isolation of only the viable cells for use in later steps of isolation of variant strains of Chlorella microalgae. Preferably, the mutant strains are sub-cultured several times on the solid agar plates to ensure they are free from a potential contamination from bacteria or fungi. Flow cytometry can be used to determine the chitin content of the variant strains in a quantitative manner, as described herein above.

Optionally, the method further comprises repeating, several times, mutagenesis and strain selection of the parent strain of Chlorella microalgae. The said repetition of mutagenesis, cultivation and isolation steps enables selecting healthy cells of the variant strains of Chlorella microalgae based on desired phenotypes (or traits) such as reduced chlorophyll content, preferably a combination of such phenotypes for example reduced chitin content, desirable colours, a pigment content, a high protein content or improved tolerance to process conditions. Incubating the library for a number of generations following mutagenesis is a useful strategy for removing viable, but undesirable genetic mutations which adversely affect overall cell performance, or "fitness". Such a method permits the “stacking” of desirable traits in a Chlorella microalgae in a controlled manner. This is a significant improvement over prior art methods involving random mutagenesis followed by selection of desired mutants (e.g. desired phenotypes) as it is a directed process that does not rely on a random occurrence of a desired combination of mutations in a Chlorella microalgae.

Preferably, the Chlorella biomass used in the present invention is an algae biomass derived from the aforementioned chlorophyll-deficient strain of Chlorella microalgae , or obtained by performing the aforementioned method.

Various embodiments and variants disclosed above apply mutatis mutandis to the composition.

The Chlorella biomass as used herein is preferably derived from algae (microalgae or macroalgae) that is cultivated heterotrophically. Optionally, the algae biomass can be obtained from the variant strain of Chlorella microalgae under current good manufacturing practice (cGMP) conditions. Notably, lower chlorophyll content of the variant strain of Chlorella microalgae renders the Chlorella microalgae more commercially acceptable. For example, a variant strain of Chlorella microalgae with chlorophyll content of 0.001 mg/g DCWwill be more commercially acceptable in industries that require no colour in their final manufactured products, as compared to the variant strain of Chlorella microalgae with chlorophyll content of 0.10 mg/g DCW. Similarly, lower lutein content of the variant strain of Chlorella microalgae renders the Chlorella microalgae more commercially acceptable. For example, a variant strain of Chlorella microalgae with a lutein content of 0.01 mg/g DCW will be more commercially acceptable for certain applications, as compared to the variant strain of Chlorella microalgae with lutein content of 1 mg/g DCW.

Beneficially, the variant strain of Chlorella microalgae having the reduced chlorophyll content is a potential ingredient in various food and personal care applications. Furthermore, the reduced chlorophyll content of the variant strain of Chlorella microalgae is also associated with reduction in the unpleasant colour, smell and taste (organoleptics) associated with the wild-type strain of Chlorella microalgae, when used in the food and personal care applications. Additionally, beneficially, the variant strain of Chlorella microalgae having the reduced chlorophyll content can be incorporated at a higher percentage as an ingredient in food compositions, compared with the wild-type, as a result of such improvements in the organoleptic properties which gives the chlorophyll-reduced Chlorella a neutral flavour.

In an embodiment, the composition may be employed in at least one of: human foods, human nutraceutical preparations or formulations, animal feeds, pharmaceutical compositions including vaccines, cosmetics, personal care compositions, personal care devices.

The term "food' refers to an edible product that can be directly or indirectly (such as, subsequent to preparation) consumed by humans and/or animals. The term "food ingredient" refers to a substance incorporated into food during one of: production, processing, treatment, packaging, transportation, distribution, preservation, storage and so forth of food. Optionally, the food ingredients are incorporated into the food to improve and/or maintain freshness, nutritional value, appearance, texture, taste and safety of the food.

The non-genetically modified and non-transgenic Chlorella microalgae biomass is suitable for direct incorporation into food compositions and products according to the present invention, whole or as an ingredient. Food products also include, but are not limited to, bakery products, microalgae flour, pasta, rice, breakfast cereals, cereal bars, confections, sauces, soups, dairy substitutes, frozen desserts, ice creams, yoghurts, smoothies, creams, spreads, salad dressings, mayonnaises, food garnishing and seasoning, candies, gums, jellies, beverages, snacks.

The term "microalgae flour¹' (used interchangeably herein with the term “algae flour¹’, “algal flour¹’, “algae powder”, or “algal powder”) is used to refer to an edible composition comprising a plurality of particles of algae biomass. Thus, Chlorella flour, or Chlorella powder are examples of a microalgae flour. Optionally, the plurality of particles of algae biomass is any one of: whole cells, lysed cells or a mixture thereof. More optionally, the microalgae flour comprises one or more of significant digestible proteins, dietary fibre content, associated water binding attributes, healthy oil delivering attributes, spices, herbs, a flow agent, an antioxidant and so forth. It may be appreciated that the microalgae flour lacks visible oil and is preferably in a powdered form. The microalgae flour can be produced under current Good Manufacturing Practice (cGMP) conditions using any method known in the art.

According to a particular preference, the Chlorella biomass used in the present invention is a protein isolate or concentrate derived from an algae biomass, wherein the algae biomass is derived from one or more of the chlorophyll-deficient strains of Chlorella microalgae as hereinbefore described.

Protein isolates and protein concentrates are produced by such methods as: wet extraction (alkali extraction/isoelectric precipitation), dry fractionation (air classification), salt extraction, micellization and mild fractionation. The efficiency of the extraction process depends on the physiochemical properties of the starting material, in addition to the method and conditions (such as pH, temperature, time of treatment etc) applied. Furthermore, it is known in the art that the physiochemical and functional properties of protein extracts; such as: emulsifying, foaming and gelling properties, in addition to solubility, water holding capacity, oil holding capacity, flavour, texture, digestibility, hydrophobicity and the like, can be modified or enhanced by physical, chemical or biological processes to improve their function and application as a food ingredient. Examples of such physical modification processes include: high-pressure treatment, heat with sheer treatment (extrusion), cold atmospheric pressure plasma treatment and ultrasonic treatment. Examples of such chemical modification processes include: glycation, acylation and deamidation. Examples of such biological modification processes include: fermentation and enzymatic modification (Shanthakumar et al. 2022; DOI: 10.3390/molecules27165354)

Advantageously, the Chlorella algal biomass of the invention is suitable as a food ingredient in vegan products, dairy and/or egg-free products. Another advantage of the algal biomass of the invention is that, due to the low chlorophyll, and high protein content, the optional whiter appearance, and the preferably low chitin content and, therefore, improved digestibility, they may be incorporated into food products as whole or as an ingredient without the need for physical downstream processes such as pulverizing, milling, breaking, grinding, cracking. This reduces the energy intensity of the manufacturing process, thereby lowering the overall carbon footprint, creating a more sustainable, lower environmental impact route to the desirable ingredient. The Chlorella microalgae biomass of the invention is a suitable ingredient in the production of texturized vegetable protein (TVP) or similar meat analogue or meat extender (products typically produced by extrusion) owing to its improved processability characteristics.

Other uses include nutraceuticals, nutritional supplements (for example, nutritional supplements, hormone tablets, digestive capsules, tablets, powders, oils and the like) and animal feed. Additionally, the other uses of the algal biomass include cosmetics (for example, in lipsticks, powders, creams, exfoliants, facial packs, and so forth), personal care compositions and personal care devices (for example toothpastes, mouthwash, hand-wash, body-wash, body soaps, shampoos, oils, sun-creams, after-sun creams, sunblock and so forth), colourants. Further uses include pharmaceuticals (such as vaccines, various bioactives and delivery routes for other recombinant proteins and enzymes).

It is further understood that reduced chitin content, beneficially enables improvements in the overall economic return of investment related to biofuels production from the said organism.

Optionally, in an embodiment, provided is a method of using the aforementioned composition as an ingredient in at least one of: human foods, human nutraceutical preparations or formulations, animal feeds, pharmaceutical compositions including vaccines, cosmetics, personal care compositions, supplements, personal care devices or textiles, dyes, inks or for the production of fuels. The method of use comprises using the algae biomass ingredient comprising the variant strain of Chlorella microalgae as any one of: a dried powder, dried flakes, a frozen paste, an extract (protein isolate or protein concentrate), solutions, suspensions, solution preconcentrates, emulsions, emulsion pre-concentrates, a concoction, tablets, pills, pellets, capsules, caplet, concentrates, granules, and so forth. Furthermore, a dried, fresh, or frozen part of the Chlorella microalgae, oil derived from the Chlorella microalgae, a homogenate, whole cell, lysed cell and so forth can be used in preparation of human foods, human nutraceutical preparations or formulations, animal feeds, pharmaceutical compositions, cosmetics, personal care compositions, personal care devices and fuels. Moreover, the Chlorella microalgae can be used to prepare compositions in any way known to the skilled person.

Method of producing Chlorella microalgae strain

The Chlorella strain used to prepare the Chlorella biomass that is applied in the food compositions of the present invention can suitably be produced using the following method.

Referring to Figure 1 , shown is a flowchart 100 of steps of a method of producing a chlorophyll-deficient strain of Chlorella microalgae having a protein content of at least 50% w/w. At step 102, a parent strain of Chlorella microalgae is obtained, such as from its natural habitat or a laboratory culture. At step 104, mutagenesis of the parent strain of Chlorella microalgae is performed. Herein, a mutagenic chemical such as an alkylating agent in its sublethal quantity and for a specific duration of time is used for mutagenesis of the obtained parent strain of Chlorella microalgae. The parent strain of Chlorella microalgae is subjected to mutagenesis in order to produce mutated, variant strains of Chlorella microalgae exhibiting a different phenotype, such as reduced chlorophyll content, high protein content, and so on, from that exhibited by the parent strain of Chlorella microalgae. Herein, mutagenesis is performed by exposing the obtained parent strain of Chlorella microalgae to EMS having a concentration in a range from 0.1 to 2.0 M for 1 to 120 minutes.

At step 106, the mutated strain of Chlorella microalgae is cultivated at a specific temperature, for a specific time, and in the presence of an organic carbon source. For example, the mutated strain of Chlorella microalgae is cultivated under heterotrophic growth mode using a source of carbon and energy, such as glucose, without any presence of light (i.e. in the dark or in the absence of light). In such case, as an example, the petri dishes containing the sample of Chlorella microalgae may be wrapped individually in a substantially opaque sheet, such as a foil, and then the wrapped-up petri dishes may be placed inside a cardboard box in the incubator. Other suitable ways of cultivating in the dark or without the presence of light can be used. Moreover, the heterotrophic growth of the mutated strain of Chlorella microalgae is achieved under suitable aseptic conditions.

Optionally, the mutated strain of Chlorella microalgae is obtained from a parent strain of Chlorella microalgae, cultivated using one or more of: a liquid or solid growth medium, including a fermentation medium containing an added carbon source such as glucose, or a mixotrophic growth medium containing acetate or a heterotrophic growth medium or a phototrophic growth medium whereby CO2 is used as the carbon source via a photosynthetic route and growth in the light. In an example, the mutated strain of Chlorella microalgae is obtained from a parent strain of Chlorella microalgae, cultivated using a solid medium. Such a solid medium can be a regular agar plate. In such an instance, cells of the mutated strain of Chlorella microalgae are inoculated on agar plates at an appropriate cell density to achieve a dense biomass growth on the surface of the agar plates. The solid medium can be a high salt medium-glucose agar plate, wherein the high salt medium-glucose agar plate comprises: a growth medium such as High Salt Medium (HSM), glucose (for example, 1% w/v) and agar.

In another example, the mutated strain of Chlorella microalgae is cultivated using a liquid medium. Such a liquid medium can be at least one of TAP (Tris-Acetate-Phosphate), High Salt Medium (HSM), glucose (for example, having consistency of 1 % w/v) and so forth. The fermentation medium comprises a source of nitrogen (such as proteins or nitrate or, more usually, ammonium), minerals (including magnesium, phosphorus, potassium, sulphur, calcium, and iron), trace elements (zinc, cobalt, copper, boron, manganese, molybdenum), an optional pH buffer, a source of carbon and energy (such as glucose, acetate) and so forth. Optionally, the parent strain of Chlorella microalgae is cultivated in a fermenter.

Furthermore, cultivation of the cells that have been exposed to mutagenesis at a higher than optimal cultivation temperature acts as a ‘stress’ filter such that only the more robust strains - where accumulated mutations have not produced a weakened or crippled organism can produce colonies on agar or viable daughter cells identified through a screen such as flow cytometry. As a result of cultivation of mutagenized cells at such elevated temperature or temperatures, fewer overall cells grow but those that do grow are more biologically and genetically fit with regard to growth and/or biomass production. Hence, those strains with reduced chlorophyll content that grow under these conditions and are scored based upon initial chlorophyll content should also be expected to be more robust with regard to application within an ultimate scalable commercially-relevant bioprocess. The repeated cultivation of the strains in the same growth conditions, i.e. heterotrophic growth conditions, produces generations of the variant strain of Chlorella microalgae with the desired phenotype compared to the starting mutant strain thereof.

At step 108, chlorophyll-deficient mutants of the parent strain of Chlorella microalgae are identified and isolated. Cells of the mutated strain of Chlorella microalgae having a phenotype different from the parent strain of Chlorella microalgae, are identified as the variant strain of Chlorella microalgae, and subsequently isolated for further application thereof. For example, when the mutated strain of Chlorella microalgae is cultivated using agar plates, colonies of the mutated strain of Chlorella microalgae on the agar plates that exhibit a different phenotype than the parent strain of Chlorella microalgae are identified as the variant strain of Chlorella microalgae. It will be appreciated that the mutated strains (or variants) are then selected based on one or more additional desirable phenotype, preferably reduced chlorophyll content, after growth on solid or liquid medium. Optionally, the phenotype is a scorable phenotype, wherein such phenotypes may be identifiable by various methods for such identification known to a person skilled in the art. In an example, white colour variant strains of Chlorella microalgae may be identified using L*a*b* CIELAB colour values.

Therefore, the Chlorella microalgae strain is preferably capable of yielding upon cultivation a Chlorella biomass material with an L* value within an L*a*b* colour space of greater than about 78, more preferably greater than about 79, about 80, about 81, about 82, about 83, about 84, or about 85 and preferably an a* value in the range of between 0.1 and 4.0, more preferably between 1.5 and 3.5 and even more preferably between 1.8 and 3.1 , and preferably a b* value in the range of between 10 and 27, more preferably between 15 and 25, even more preferably between 15 and 20 and still more preferably between 16 and 19. Thus, the Chlorella microalgae strain is preferably capable of yielding upon cultivation a Chlorella biomass material with an L* value in an L* a* b* colour space in the range of between 81.0 and 85.0; an a* value of in the range of between 1.8 and 3.1 ; and a b* value in the range of between 16.0 and 19.0. These values correspond to a colour which is generally perceived as white, in the context of algae biomass materials.

At step 110, chlorophyll-deficient mutants of the parent strain of Chlorella microalgae having a protein content of at least 50% w/w are identified and isolated. Cells of the mutated strain of Chlorella microalgae having a phenotype different from the parent strain of Chlorella microalgae, are identified as the variant strain of Chlorella microalgae, and subsequently isolated for further application thereof. For example, when the mutated strain of Chlorella microalgae is cultivated using agar plates, colonies of the mutated strain of Chlorella microalgae on the agar plates that exhibit a different phenotype than the parent strain of Chlorella microalgae are identified as the variant strain of Chlorella microalgae. It will be appreciated that the mutated strains (or variants) are then selected based on one or more additional desirable phenotype, i.e. increased protein content, after growth on solid or liquid medium. Optionally, the phenotype is a scorable phenotype, wherein such phenotypes may be identifiable by various methods for such identification known to a person skilled in the art. In an example, the Chlorella microalgae is selected based on a desirable protein content, wherein the desirable protein content is based upon a relative signal obtained on cell sorting by flow cytometry or iodine staining, preferably by iodine staining.

The steps 102, 104, 106, 108, and 110 are only illustrative and other alternatives can also be provided where one or more steps are added, one or more steps are removed, or one or more steps are provided in a different sequence without departing from the scope of the claims herein. The method may, for example, further comprise repeating, several times, mutagenesis and strain selection of the parent strain of Chlorella microalgae. The said repetition of mutagenesis, cultivation and isolation steps enables selecting healthy cells of the variant strains of Chlorella microalgae based on desired phenotypes (or traits) such as reduced chlorophyll content, preferably a combination of such phenotypes for example reduced chitin content, desirable colours, a pigment content, a high protein content or improved tolerance to process conditions. Incubating the library for a number of generations following mutagenesis is a useful strategy for removing viable, but undesirable genetic mutations which adversely affect overall cell performance, or "fitness". Such a method permits the “stacking” of desirable traits in a Chlorella microalgae in a controlled manner.

Chlorella biomass can straightforwardly be obtained from the Chlorella strains described herein, using well-known fermentation and/or cultivation methods suitable to grow quantitative amounts of it. As described herein, the resulting biomass may be washed, concentrated and/or dried before use, for example by spray-drying. Thus, for instance, the Chlorella biomass is preferably obtainable from a strain of Chlorella microalgae according to one of the following clauses:

1. A chlorophyll-deficient strain of Chlorella microalgae having a protein content of at least 50% w/w.

2. The chlorophyll-deficient strain of Chlorella microalgae of clause 1 , being a modified strain of a Chlorella microalgae species.

3. The chlorophyll-deficient strain of Chlorella microalgae of clause 2, wherein the Chlorella microalgae species is selected from Parachlorella kessleri, Auxenochlorella protothecoides, Auxenochlorella pyrenoidosa, or Heterochlorella luteoviridis, Chlorella sorokiniana or Chlorella vulgaris.

4. The chlorophyll-deficient strain of Chlorella microalgae of any one of clauses 1 to 3, having a chlorophyll content in a range of 0.001-0.5 mg/g dry cell weight.

5. The chlorophyll-deficient strain of Chlorella microalgae of any one of clauses 1 to 4, having a protein content in a range of 50-85% w/w.

6. A chlorophyll-deficient strain of Chlorella microalgae of any one of clauses 1 to 5, having a starch content of less than 25% w/w.

7. The chlorophyll-deficient strain of Chlorella microalgae of clause 6, having a 50% or greater reduction in starch content as compared to a starch content of a parent strain of Chlorella microalgae, grown under the same conditions.

8. The chlorophyll-deficient strain of Chlorella microalgae of any one of clauses 1 to 7, having a chitin and/or chitosan, poly-d-glucosamine and poly-acetyl-D-glucosamine content in a range of 0.001 to 4.8 mg/g dry cell weight.

9. The chlorophyll-deficient strain of Chlorella microalgae of clause 8, having a 25% or greater reduction in chitin content as compared to a chitin content of a parent strain of Chlorella microalgae, grown under the same conditions.

10. The chlorophyll-deficient strain of Chlorella microalgae of any one of clauses 1 to 9, having a protein digestibility-corrected amino acid score (PDCAAS) in a range of 0.75 to 1.

11. The chlorophyll-deficient strain of Chlorella microalgae of any one of clauses 1 to 10, having one or more additional desirable phenotypes, wherein the one or more additional desirable phenotypes is selected from a group comprising: a colour, a pigment content, a smell, a taste, a texture, a biochemical composition and improved tolerance to process conditions.

12. The chlorophyll-deficient strain of Chlorella microalgae of any one of clauses 1 to 11, having a colour selected from at least one of white, cream, pale yellow, yellow, lime, pale green, golden, caramel, orange, pink, red, deep-red, red-brown or brown. 13. The chlorophyll-deficient strain of Chlorella microalgae of any one of clauses 1 to 12, obtained from a parent strain of Chlorella microalgae, by performing mutagenesis of the parent strain of Chlorella microalgae.

14. The chlorophyll-deficient strain of Chlorella microalgae of clause 13, wherein mutagenesis is performed by exposure of the parent strain of Chlorella microalgae to a mutagenic chemical.

15. The chlorophyll-deficient strain of Chlorella microalgae of clause 14, wherein the mutagenic chemical is an alkylating agent.

16. The chlorophyll-deficient strain of Chlorella microalgae of clause 14 or clause 15, wherein the concentration of the mutagenic chemical is in a range from 0.1 to 2.0 M.

17. The chlorophyll-deficient strain of Chlorella microalgae of clause 15, wherein mutagenesis is performed by exposure of the parent strain of Chlorella microalgae to a physical mutagen, optionally wherein the physical mutagen comprises at least one of UV light, gamma rays, X-rays.

18. The chlorophyll-deficient strain of Chlorella microalgae of any one of clauses 1 to 17, cultivated in a heterotrophic growth mode.

19. The chlorophyll-deficient strain of Chlorella microalgae of any any one of clauses 1 to 18, cultivated:

- at a specific temperature;

- for a predefined period of time;

- without the presence of light; and

- in the presence of an organic carbon energy source.

20. The chlorophyll-deficient strain of Chlorella microalgae of clause 19, wherein the specific temperature is in a range of 20 to 35 °C, optionally in the range of 25 to 28 °C.

21. The chlorophyll-deficient strain of Chlorella microalgae of clause 19 or clause 20, wherein the predefined period of time is in a range of 1 to 5 weeks, optionally in the range of 1 to 3 weeks.

22. The chlorophyll-deficient strain of Chlorella microalgae of any one of clauses 19 to 21 , wherein the organic carbon energy source is glucose and/or acetate.

23. The chlorophyll-deficient strain of Chlorella microalgae of clause 22, wherein the organic carbon energy source is glucose having a glucose to biomass conversion ratio of more than 0.45.

24. The chlorophyll-deficient strain of Chlorella microalgae of any one of clauses 1 to 23, being genetically stable.

25. A strain of Chlorella microalgae having a first mutation in at least one gene that encodes for phytoene desaturase or a subunit thereof, and wherein said Chlorella microalgae has a second mutation in at least one gene that encodes for magnesium chelatase or a subunit thereof.

26. The strain of Chlorella microalgae of clause 25, wherein the second mutation is in a gene that encodes for subunit Chll of magnesium chelatase or subunit ChIH of magnesium chelatase.

27. The strain of Chlorella microalgae of clause 26, wherein the second mutation is in a gene that encodes for subunit Chll of magnesium chelatase.

28. The strain of Chlorella microalgae of any one of clauses 25 to 27, wherein the second mutation is a Single Nucleotide Polymorphism (SNP) or Insertion/deletion (INDEL).

29. A strain of Chlorella microalgae comprising a genomic DNA sequence that is at least 50% identical to Sequence 2 (SEQ ID NO: 2).

30. A strain of Chlorella microalgae according to any one of clauses 25 to 29 which also is a chlorophyll-deficient strain of Chlorella microalgae according to any one of clause 1 to 24.

31. A Chlorella vulgaris strain designated CCAP 211/143 deposited under provisions of the Budapest Treaty at the Culture Collection of Algae and Protozoa (CCAP, SAMS Ltd., Scottish Marine Institute, OBAN, Argyll, PA37 1QA, United Kingdom) on December 14, 2023 (Patent Deposit Designation of CCAP 211/143).

32. A Chlorella sorokiniana strain designated CCAP 211/142 deposited under provisions of the Budapest Treaty at the Culture Collection of Algae and Protozoa (CCAP, SAMS Ltd., Scottish Marine Institute, OBAN, Argyll, PA37 1QA, United Kingdom) on December 14, 2023 (Patent Deposit Designation of CCAP 211/142).

Thus, the Chlorella biomass is preferably according to one of the following clauses:

33. An algae biomass, wherein said biomass has a L* value in an L* a* b* colour space of greater than about 78.

34. The algae biomass of clause 33, having an a* value in an L* a* b* colour space in the range of between 0.1 and 4.0.

35. The algae biomass of clause 33 or clause 34, having a b* value in an L* a* b* colour space in the range of between 10 and 27.

36. The algae biomass of any one of clauses 33 to 35, wherein said biomass has a L* value in an L* a* b* colour space in the range of between 81.0 and 85.0; an a* value in the range of between 1.8 and 3.1; and a b* value in the range of between 16.0 and 19.0.

37. The algae biomass of any one of clauses 33 to 36, wherein said biomass has a protein content of at least 50% w/w.

38. The algae biomass of any one of clauses 33 to 37 in the form of a powder or flour. Food Chlorella biomass

The Chlorella biomass contained in the food product of the invention comprises at least 50 wt% of Chlorella protein by dry weight of the Chlorella biomass. More preferably, the Chlorella biomass material has a protein content in a range of 50-85% w/w.

In this context, protein content is preferably determined using the Dumas method as described herein, using an NtP factor of 6.25.

Thus, based on the protein content of the Chlorella biomass, the amount of Chlorella protein in the food product can easily be set and/or determined.

Preferably, the Chlorella protein material is present in the food composition of the invention in an amount of from 0.1 to 60% by dry weight of the food composition, more preferably from 0.2 to 45%, even more preferably from 0.3 to 30%, still more preferably from 0.4 to 25%, yet more preferably from 0.5 to 20%, even still more preferably from 1 to 10% and still more preferably from 1.5 to 5% by dry weight of the food composition. As is clear to the skilled person, preferred amounts of protein may further depend on the type of food composition. For example, a meat analogue composition will typically have a higher overall protein content than for instance an instant soup composition (by dry weight of the composition). The preferred amount of Chlorella protein in a product with a higher overall protein content can feasibly be higher than in a product in which the desired total amount of protein is lower.

Beneficially, the Chlorella biomass is very low in chlorophyll. Chlorophyll is believed to play a role in the off-flavour, off-odour and/or off-colour of food products. This is particularly true for C/i/ore/Za-originating chlorophyll. Therefore, the food composition preferably comprises less than 0.05 wt% of Chlorella chlorophyll by dry weight of the product, more preferably less than 0.02 wt% of Chlorella chlorophyll, and even more preferably less than 0.01 wt%. Still more preferably, the food composition is substantially free from Chlorella chlorophyll.

The beneficial sensory properties of the present food composition are more pronounced if also the overall level of chlorophyll in the product is relatively limited. Thus, the food composition comprises less than 0.05 wt%, more preferably less than 0.02 wt% and even more preferably less than 0.01 wt% of chlorophyll by dry weight of the food product. Still more preferably, the food composition is substantially free from chlorophyll.

Likewise, the beneficial sensory properties of the present food composition are more pronounced if the Chlorella biomass material is relatively white, more preferably if it is white.

Therefore, according to one preference, the invention also provides a food composition, wherein the food composition comprises Chlorella biomass material comprising the Chlorella protein material, and wherein the Chlorella biomass is sourced from a chlorophyl-deficient strain of Chlorella microalgae, wherein the said strain is capable of yielding upon cultivation a Chlorella biomass material with an L* value within an L*a*b* colour space of greater than about 78, more preferably greater than about 79, about 80, about 81 , about 82, about 83, about 84, or about 85.

Preferably, the said strain is capable of yielding upon cultivation a Chlorella biomass material with an L* value in the range of from between 78 and 90, more with an L* value in the range of from 79 to 85, and even more preferably, with an L* value in the range of from 81 to 85.

Preferably, the said strain is capable of yielding upon cultivation a Chlorella biomass material with an a* value in an L* a* b* colour space in the range of between 0.1 and 4.0, and more preferably, with an a* value in the range of between 1.5 and 3.5; and even more preferably, with an a* value in the range of between 1.8 and 3.1.

Preferably, the said strain is capable of yielding upon cultivation a Chlorella biomass material with a b* value in an L* a* b* colour space in the range of between 10 and 27, more preferably with a b* value in the range of between 15 and 25; and even more preferably with a b* value in the range of between 15 and 20, such as between 16 and 19.

Preferably, the said strain is capable of yielding upon cultivation a Chlorella biomass material with an L* value in an L* a* b* colour space in the range of between 81 .0 and 85.0; an a* value of in the range of between 1.8 and 3.1 ; and a b* value in the range of between 16.0 and 19.0. It has surprisingly been found that L* a* b* values in this combination of ranges correspond to Chlorella biomass that has significantly improved visual properties, particularly visual properties that are relevant to consumer acceptance when the algae biomass is incorporated into consumer goods, such as food products.

Several species of Chlorella are suitable to yield biomass for use in the food composition of the invention. Preferably, the strain of Chlorella microalgae is a strain of Chlorella vulgaris or a strain of Chlorella sorokiniana microalgae, and more preferably one of the strains identified herein.

In some embodiments of the food composition it is preferred that the strain of Chlorella microalgae is a strain of Chlorella vulgaris microalgae. In other embodiments of the food composition, it is preferred that the strain of Chlorella microalgae is a strain of Chlorella sorokiniana microalgae.

Without wishing to be limited by theory, it is believed that the superior sensory properties of the Chlorella biomass are at least in part related to certain mutations.

Thus, it is preferred that the genome of the strain of Chlorella microalgae includes an effective mutation in a magnesium chelatase gene and/or an effective mutation in a phytoene desaturase gene.

Magnesium chelatase (EC 6.6.1.1) is an enzyme that catalyses the first committed step of the chlorophyll synthesis pathway; being the insertion of Mg²⁺ into protoporphyrin IX. Magnesium chelatase is a highly-conserved enzyme composed of three subunits: Chll, ChlD, and ChlH. The subunits are postulated to have distinct roles in forming the catalytically-active holoenzyme that, ultimately, performs the magnesium chelation reaction; broadly, Chll and ChlD are thought to form an ATP-associated complex, while ChIH binds to the magnesium ion, leading to the formation of the active Mg chelatase holoenzyme (Xhang et al. 2018; DOI: 10.3389/fpls.2018.00720).

Thus, the strain of Chlorella microalgae is preferably characterised by the presence of an effective mutation in a magnesium chelatase gene, more preferably in a magnesium chelatase subunit ChIH and or Chll gene, and even more preferably in a magnesium chelatase subunit Chll gene.

It is even more preferred that the genome of the strain of Chlorella microalgae includes an effective mutation in a magnesium chelatase gene and an effective mutation in a phytoene desaturase gene.

In the context of the present application, an effective mutation refers to a mutation such that less, or no active enzyme is formed (e.g. less or no active Mg chelatase). This may for instance be due to the fact that no Mg chelatase is formed at all by the strain, or because the protein is modified such that it can no longer function as an effective (subunit of) Mg chelatase.

Typical effective mutations include a stop gained mutation, a frameshift deletion, (high impact) and missense mutation and an in-frame deletion (with moderate impact), an exon variant, or a downstream gene variant (modifier impact).

Preferably, the strain of Chlorella microalgae includes one or more of the effective mutations specified in Table 4 or in Table 9. Thus, the strain of Chlorella microalgae preferably comprises a genomic DNA sequence that is at least 50% identical to Sequence 2 (SEQ ID NO: 2). The genomic DNA sequence Sequence 2 (SEQ ID NO: 2) encodes for a Chll subunit of magnesium chelatase. Preferably the genomic DNA sequence is at least 60% identical to Sequence 2 (SEQ ID NO: 2), more preferably at least 70% identical to Sequence 2 (SEQ ID NO: 2), still more preferably at least 80% identical to Sequence 2 (SEQ ID NO: 2). In some particularly preferred embodiments, the genomic DNA sequence is at least 85% identical to Sequence 2 (SEQ ID NO: 2), such as at least 80% identical to Sequence 2 (SEQ ID NO: 2), at least 85% identical to Sequence 2 (SEQ ID NO: 2), at least 90% identical to Sequence 2 (SEQ ID NO: 2), or at least 95% identical to Sequence 2 (SEQ ID NO: 2), more preferably at least 99% identical to Sequence 2 (SEQ ID NO: 2) and still more preferably it is identical to Sequence 2 (SEQ ID NO: 2).

The strain of Chlorella microalgae preferably comprises a genomic DNA sequence that is at least 50% identical to Sequence 4 (SEQ ID NO: 4). The genomic DNA sequence Sequence 4 (SEQ ID NO: 4) encodes for a phytoene desaturase. Preferably the genomic DNA sequence is at least 60% identical to Sequence 4 (SEQ ID NO: 4), more preferably at least 70% identical to Sequence 4 (SEQ ID NO: 4), still more preferably at least 80% identical to Sequence 4 (SEQ ID NO: 4). In some particularly preferred embodiments, the genomic DNA sequence is at least 85% identical to Sequence 4 (SEQ ID NO: 4), such as at least 80% identical to Sequence 4 (SEQ ID NO: 4), at least 85% identical to Sequence 4 (SEQ ID NO: 4), at least 90% identical to Sequence

4 (SEQ ID NO: 4), or at least 95% identical to Sequence 4 (SEQ ID NO: 4), more preferably at least 99% identical to Sequence 4 (SEQ ID NO: 4) and still more preferably it is identical to Sequence 4 (SEQ ID NO: 4).

The strain of Chlorella microalgae preferably comprises a genomic DNA sequence that is at least 50% identical to Sequence 5 (SEQ ID NO: 5). The genomic DNA sequence Sequence 5 (SEQ ID NO: 5) encodes for a phytoene desaturase. Preferably the genomic DNA sequence is at least 60% identical to Sequence 5 (SEQ ID NO: 5), more preferably at least 70% identical to Sequence 5 (SEQ ID NO: 5), still more preferably at least 80% identical to Sequence 5 (SEQ ID NO: 5). In some particularly preferred embodiments, the genomic DNA sequence is at least 85% identical to Sequence 5 (SEQ ID NO: 5), such as at least 80% identical to Sequence 5 (SEQ ID NO: 5), at least 85% identical to Sequence 5 (SEQ ID NO: 5), at least 90% identical to Sequence

5 (SEQ ID NO: 5), or at least 95% identical to Sequence 5 (SEQ ID NO: 5), more preferably at least 99% identical to Sequence 4 (SEQ ID NO: 4) and still more preferably it is identical to Sequence 5 (SEQ ID NO: 5).

The strain of Chlorella microalgae preferably comprises a genomic DNA sequence that is at least 50% identical to Sequence 7 (SEQ ID NO: 7). The genomic DNA sequence Sequence 7 (SEQ ID NO: 7) encodes for a ChIH subunit of a magnesium chelatase. Preferably the genomic DNA sequence is at least 60% identical to Sequence 7 (SEQ ID NO: 7), more preferably at least 70% identical to Sequence 7 (SEQ ID NO: 7), still more preferably at least 80% identical to Sequence 7 (SEQ ID NO: 7). In some particularly preferred embodiments, the genomic DNA sequence is at least 85% identical to Sequence 7 (SEQ ID NO: 7), such as at least 80% identical to Sequence 7 (SEQ ID NO: 7), at least 85% identical to Sequence 7 (SEQ ID NO: 7), at least 90% identical to Sequence 7 (SEQ ID NO: 7), or at least 95% identical to Sequence 7 (SEQ ID NO: 7), more preferably at least 99% identical to Sequence 7 (SEQ ID NO: 7) and still more preferably it is identical to Sequence 7 (SEQ ID NO: 7).

In order to arrive at the desired mutations, one or more cycles of mutagenesis and desirable trait selection may be performed, typically starting from a wildtype Chlorella or from a variant which already contains one or more desirable traits.

In some cases, a desirable trait or combination of traits may already be arrived at after one round of mutagenesis. Moreover, a preferred first round of mutagenesis preferably involves selection of a mutated strain of Chlorella microalgae which is chlorophyll-deficient. Thus, the food composition preferably comprises Chlorella biomass material that is obtainable by a process including the step of fermentation of the chlorophyll-deficient strain of Chlorella microalgae and wherein the chlorophyll-deficient strain of Chlorella microalgae is obtainable by a method of producing the chlorophyll-deficient strain of Chlorella microalgae comprising the steps of: a) obtaining a parent strain of Chlorella microalgae; b) performing mutagenesis of the parent strain of Chlorella microalgae; c) cultivating the mutated Chlorella microalgae at a specific temperature, for a predefined period of time, and in the presence of an organic carbon source; and d) identifying and isolating chlorophyll-deficient mutants of the parent strain of Chlorella microalgae.

Here, the parent strain of Chlorella microalgae is preferably a wildtype strain, for example wildtype Chlorella vulgaris or wildtype Chlorella sorokiniana. The preferred way of performing mutagenesis is as described hereinabove, for example by means of an alkylating agent, preferably ethyl methanesulphonate or methyl methansulphonate, more preferably ethyl methanesulphonate.

In this context, Chlorella microalgae are identified as chlorophyll-deficient when they preferably have a chlorophyll content of up to 0.5 mg/g by dry cell weight (DCW), more preferably up to 0.25 mg/g by dry cell weight, even more preferably of up to 0.1 mg/g by dry cell weight and still more preferably of up to 0.05 mg/g by dry cell weight. Thus, the chlorophyll content preferably is in the range of 0.001 to 0.5 mg/g by dry cell weight (DCW), more preferably 0.01 to 0.25 mg/g dry cell weight, and even more preferably 0.02 to 0.1 mg/g DCW.

The identified and isolated chlorophyll-deficient mutants preferably comprise a mutation that affects the capability of the mutants to produce chlorophyll. For example, it is preferred that the genome of the chlorophyll-deficient Chlorella microalgae includes an effective mutation in a magnesium chelatase gene as described hereinabove.

The chlorophyl-deficient strain of Chlorella microalgae obtained from the steps of the method of producing described above, can suitably be used as the starting point for a further round of mutagenesis and trait selection. Thus, the method of producing the chlorophylldeficient strain of Chlorella microalgae preferably comprises the further steps of e) performing mutagenesis of the chlorophyll-deficient strain of Chlorella microalgae; f) cultivating the further-mutated Chlorella microalgae at a specific temperature, for a predefined period of time, and in the presence of an organic carbon source; and g) identifying and isolating chlorophyll-deficient and whiter mutants of the parent strain of Chlorella microalgae. The preferred way of performing mutagenesis is as described hereinabove, for example by means of an alkylating agent, preferably ethyl methanesulphonate or methyl methansulphonate, more preferably methyl methanesulphonate.

The whiteness of the mutants can be assessed by any suitable means, for example by the colorimetric methods as described herein. For example, when the colours of microalgae are expressed by means of CIE-lab L*a*b* parameters, a mutant is considered whiter when it is capable of yielding upon cultivation a Chlorella biomass material with an L* value, and/or an a* value and/or a b* value within an L*a*b* colour space that is closer to the desired range of L* values, and/or a* values and/or b* values, respectively, than that of the strain from which the mutant was obtained. Here, the desired ranges are for L*, and L* value of greater than about 78, more preferably greater than about 79, about 80, about 81 , about 82, about 83, about 84, or about 85, the desired ranges for the value of a* are in the range of between 0.1 and 4.0, more preferably between 1.5 and 3.5 and even more preferably between 1.8 and 3.1 , and the desired ranges for the desired value of b* are in the range of between 10 and 27, more preferably between 15 and 25, even more preferably between 15 and 20 and still more preferably between 16 and 19. It is particularly preferred that the identified and isolated chlorophyll-deficient and whiter mutants have an L* value within an L*a*b* colour space of greater than about 78, more preferably greater than about 79, about 80, about 81, about 82, about 83, about 84, or about 85 and preferably an a* value in the range of between 0.1 and 4.0, more preferably between 1.5 and 3.5 and even more preferably between 1.8 and 3.1 , and preferably a b* value in the range of between 10 and 27, more preferably between 15 and 25, even more preferably between 15 and 20 and still more preferably between 16 and 19. Thus, the whiter mutant is preferably capable of yielding upon cultivation a Chlorella biomass material with an L* value in an L* a* b* colour space in the range of between 81.0 and 85.0; an a* value of in the range of between 1.8 and 3.1; and a b* value in the range of between 16.0 and 19.0. The chlorella-deficient mutant is preferably white.

Without wishing to be limited by theory, it is hypothesised that the genome of a whiter mutant suitably involves a mutation affecting the capability of the Chlorella microalgae to produce coloured compounds such as coloured carotenoids. Thus, it is for instance preferred that the genome of the chlorophyll-deficient and whiter Chlorella microalgae includes an effective mutation in a phytoene desaturase gene as described herein.

The chlorophyl-deficient and whiter strain of Chlorella microalgae obtained from the steps of the method of producing described above, can suitably be used as the starting point for an even further round of mutagenesis and trait selection. Thus, the method of producing the chlorophyll-deficient strain of Chlorella microalgae preferably comprises the further steps of h) performing mutagenesis of the chlorophyll-deficient and whiter strain of Chlorella microalgae; i) cultivating the further-mutated Chlorella microalgae at a specific temperature, for a predefined period of time, and in the presence of an organic carbon source; and j) identifying and isolating chlorophyll-deficient and whiter mutants of the parent strain of Chlorella microalgae capable of yielding upon fermentation a protein content of at least 50% w/w by dry cell weight.

The protein content is preferably determined by the Dumas method as described herein, using an NtP factor of 6.25. Preferably, the protein content is at least 50 wt% by dry cell weight, more preferably it is in the range of 50 to 85 % by dry cell weight.

A relatively high protein content can in part be achieved by ensuring a relatively low starch content, as explained hereinbefore. Therefore, the identification and isolation step j) may suitably also include the identification of mutants that, upon cultivation, are relatively low in starch, for instance having a starch content of less than 25 wt%, more preferably less than 15 wt%, even more preferably less than 10 wt% by dry cell weight.

Performing three or more rounds of mutagenesis and trait selection is particularly preferred. Therefore, it is preferred that the food composition comprises Chlorella biomass material obtainable by a process including the step of fermentation of the chlorophyll-deficient strain of Chlorella microalgae and wherein the chlorophyll-deficient strain of Chlorella microalgae is obtainable by a method of producing a chlorophyll-deficient strain of Chlorella microalgae, that is capable of being grown to yield Chlorella biomass comprising at least 50% by dry weight of protein and not more than 10 wt% by dry weight of starch, wherein the method comprises the steps of: a) obtaining a parent strain of Chlorella microalgae, the strain preferably being a wildtype strain; b) performing mutagenesis of the parent strain of Chlorella microalgae; c) cultivating the mutated Chlorella microalgae at a specific temperature, for a predefined period of time, and in the presence of an organic carbon source; and d) identifying and isolating mutants of the parent strain of Chlorella microalgae that exhibit a first trait, e) performing mutagenesis on a strain of Chlorella microalgae that stably exhibit the first trait; f) cultivating the further-mutated Chlorella microalgae at a specific temperature, for a predefined period of time, and in the presence of an organic carbon source; and g) identifying and isolating mutants of the parent strain of Chlorella microalgae that exhibit the first and a second trait; h) performing mutagenesis on a strain of Chlorella microalgae that stably exhibit the first and second trait; i) cultivating the further-mutated Chlorella microalgae at a specific temperature, for a predefined period of time, and in the presence of an organic carbon source; and j) identifying and isolating mutants of the parent strain of Chlorella microalgae that exhibit the first, the second and a third trait, wherein the first, second and third trait each are one of chlorophyll deficiency, whiteness and capability of yielding upon fermentation a protein content of at least 50 wt% by dry weight of the Chlorella biomass.

In this method, the order in which the desired traits are used for selection and further mutagenesis may suitably be selected by the skilled person, depending for instance on the phenotypes observed after a particular round of mutagenesis. In addition to specified traits, mutants may also suitably be selected based on other traits, such as the suppression of the formation of starch or chitin deficiency.

One factor that may contribute to obtaining Chlorella biomass with a relatively high protein is the suppression of the formation of starch by the microalgae during their cultivation. Therefore, the Chlorella biomass material preferably has a starch content of less than 25 wt% by dry weight of the Chlorella biomass material, more preferably less than 20 wt%, even more preferably less than 15 wt%, still more preferably less than 10 wt%, yet more preferably less than 5 wt%, and even still more preferably less than 3 wt% by dry weight of the Chlorella biomass. Thus, the Chlorella biomass material preferably has a starch content of 0.1 wt% to 25 wt%, more preferably 0.2 to 20 wt%, even more preferably 0.5 to 15 wt%, still more preferably 1 to 10 wt%, yet more preferably 2 to 5 wt% by dry weight of the Chlorella biomass material.

Another contributor to a relatively high protein content may be the over-expression of certain proteins. Thus, genome of the strain of Chlorella microalgae preferably contains one or more mutations leading to the over-expression of protein-encoding genes.

Both lysed and non-lysed Chlorella biomass material can have desirable properties, depending on the type of application. Therefore, preferably at least 60 wt%, preferably at least 70 wt%, more preferably at least 80 wt% and even more preferably at least 90 wt% of the Chlorella biomass material by dry weight of the food product is present in the form of lysed Chlorella biomass material.

Conversely, in other embodiments of the food composition, preferably at least 60 wt%, preferably at least 70 wt%, more preferably at least 80 wt% and even more preferably at least 90 wt% of the Chlorella biomass material by dry weight of the food product is present in the form of non-lysed Chlorella biomass material.

The Chlorella biomass material contained in the food product of the present invention is preferably obtainable by a process including the step of heterotrophic fermentation of the desired strain of Chlorella microalgae, wherein the desired strain preferably is one of the strains described herein.

Methods to work up or fractionate the Chlorella biomass material are well known. Thus, the Chlorella biomass material may for instance be in the form of a protein isolate or a protein concentrate.

It is particularly preferred that the Chlorella biomass material is spray-dried.

Food composition

The food composition of the present invention is a food composition in the form of a meat analogue or a dry savoury snack.

Meat analogues

According to one preference, the food composition is in the form of a meat analogue.

Thus, the present invention provides a food composition comprising Chlorella biomass material, wherein the biomass material is sourced from a chlorophyll-deficient strain of Chlorella microalgae and wherein the Chlorella biomass material comprises at least 50 wt% of Chlorella protein by dry weight of the Chlorella biomass and wherein the food composition is in the form of a meat analogue.

It is particularly preferred that the meat analogue is a vegan meat analogue or a vegetarian meat analogue.

The Chlorella protein material may be included in the meat analogue for any reason for which proteins would usually be included. Thus it may add to the desired nutritional profile of the meat analogue. According to one preference, the Chlorella protein material is present as a binder.

The term “meat analogue” is understood to refer to a food product resembling a real- meat product (in particular in appearance, mouthfeel and/or use) whereby at least 90 wt% of the protein is of non-animal origin. Preferably, a meat analogue is substantially free of egg white protein and dairy protein. Even more preferably a meat analogue is substantially free of animal protein. Even more preferably a meat analogue is substantially free of animal fat. Even more preferably a meat analogue comprises less than 0.1 wt% of ingredients derived from animals, more preferably less than 0.01wt%. Preferably the meat analogue according to the invention is free from haem-containing protein. “Substantially free from” as used herein means that such ingredients are not added as such for a specific functionality but can be present in trace amounts as part of a non-animal derived ingredient.

The term “minced meat analogue” as used herein refers to a vegetarian product that has an appearance and structure similar to that of minced meat. More particularly, like minced meat, the minced meat analogue of the present invention is largely (> 40 wt.%) composed of small pieces of elastic hydrated material that are wetted on the outside by an aqueous liquid. The term “minced meat analogue” also encompasses products shaped from such minced meat analogue, such as patties, sausages, and meat balls.

The term "non-animal" as used herein refers to a plant, algae, fungus, or microbe. The term "texturized protein" or TP as used herein refers to solid fibrous particles, which are produced by cooking the starting materials in an extruder cooker and extruding them to form a texturized protein.

The term "texturized vegetable protein" or TVP as used herein refers to TP based on plant protein such as soy protein, pea protein, sunflower protein, mung bean protein. TVP is often made from protein concentrates. For example, soy protein concentrates are prepared by removing soluble carbohydrate fraction as well as some flavour compounds from defatted soy meal.

The meat analogue according to the invention preferably has a moisture content of 45 to 69 wt%, more preferably 56 to 65 wt%, more preferably 60 to 65 wt% by weight of the total composition.

Non-animal protein

The meat analogue according to the invention preferably comprises from 4 to 60 wt%, preferably from 5 to 35 wt% of non-animal protein, more preferably 6 to 29 wt%, even more preferably 7 to 24 wt%, even more preferably 8 to 19 wt%, even more preferably 10 to 15 wt% by total weight of the meat analogue. Preferably the non-animal protein is selected from plant protein, algal protein (including Chlorella protein), fungal protein, or microbial protein. Plant protein is preferably selected from legume protein. In particular, the meat analogue according to the invention preferably comprises - at levels according to each of the ranges recited abovesoy protein, pea protein, fungal protein, mung bean protein, algal protein, wheat protein, oat protein, lentil protein, faba bean protein, lupin protein and combination thereof.

Preferably, at least 70 wt%, more preferably at least 80 wt%, even more preferably at least 90 wt%, even more preferably at least 95 wt% of the total amount of protein of the meat analogue is plant protein selected from legume protein, wheat protein, rice protein and combinations thereof. Examples of legume proteins that can be used include soy protein, lentil protein, pea protein, faba bean protein, lupin protein and combinations thereof.

According to another preference, a substantial proportion of the protein is Chlorella protein.

Thus, depending on the desired function in the meat analogue, the Chlorella protein may be present in relatively high amounts, for instance from 10 to 60 wt%, more preferably from 20 to 45 wt% by dry weight of the composition, or it may be present at more modest levels, for instance from 0.1 to 10wt%, more preferably from 0.5 to 8 wt%, even more preferably from 1 to 5 wt% by dry weight of the composition.

Texturized Protein (TP) and TVP

At least part of the non-animal protein may be present in the meat analogue in the form of a texturized protein (TP). Preferably, at least 90 wt% of the non-animal protein in the meat analogue according to the invention is in the form of TP.

Typically, TP particles are purchased as dry fibrous particles. TP particles are hydrated with sufficient hydration solution to obtain hydrated TP particles. When TP is made from plant protein, it is also referred to as textured vegetable protein (TVP). Preferred TVP is made from legume protein containing material, in particular from soy and pea sometimes in combination with wheat protein or oat protein. TVP particles can for instance be obtained from suppliers like Roquette™, ADM™ and Solae™. Preferably the meat analogue according to the invention comprises 30 to 70 wt% of hydrated TP particles, more preferably 40 to 65 wt% by weight of the total composition. Preferably the meat analogue according to the invention comprises 30 to 70 wt% of hydrated TVP particles, more preferably 40 to 65 wt% by weight of the total composition. Preferably the meat analogue according to the invention comprises 30 to 70 wt% of hydrated legume TVP particles, more preferably 40 to 65 wt% by weight of the total composition whereby the TVP particles comprise soy protein, pea protein and combinations thereof. Preferably, the meat analogue is free from spun protein fibres such as solution-spun protein fibres. These protein fibres are typically prepared from a spinning dope of protein which is forced through a porous membrane such as a spinneret to form fibres which are coagulated in an acid salt bath and oriented by suitable means, such as by a series of rolls revolving at increasing speeds like described in US 2682466 (Boyer) and US 3314356.

Binding agents

The meat analogue according to the invention preferably comprises a binding agent to bind other ingredients of the meat analogue, e.g., to bind textured vegetable protein particles. A suitable binding agent can be identified by titrating different binding agents against the cohesiveness and fracturability of the meat analogue. The binding agent preferably includes a gel forming agent. Preferably, the binding agent comprises a combination of gel forming protein (preferably gel-forming plant protein), a gel forming polysaccharide and combinations thereof. The meat analogue according to the invention preferably comprises 0.1 to 25 wt% of binding agent, preferably 0.1 to 15 wt% more preferably 0.2 to 11 wt%, even more preferably 0.3 to 7 wt% by weight of the total composition. The binding agent preferably comprises the combination of 0.3 to 7 wt% of a gel forming protein and 0.1 to 5 wt% of a gel forming polysaccharide like methylcellulose.

The binder may also include Chlorella protein or Chlorella biomass of the present invention.

Gel forming agent

The meat analogue according to the invention preferably comprises a ungelatinized gel forming agent, preferably in an amount of from 0.1 to 25 wt%, preferably 0.1 to 15 wt% more preferably 0.2 to 11 wt%, even more preferably 0.3 to 7 wt%, more preferably 0.1 to 9 wt%, more preferably 0.2 to 7 wt%, even more preferably 0.3 to 5 wt% even more preferably 0.5 to 5 wt% by weight of the total composition. The term “gel forming agent” as used herein refers to a compound which is able to form a gel at the concentration, pH and salt level used in the meat analogue when heat is applied, preferably to a temperature of at least 40°C, more preferably at least 45°C, even more preferably at least 50°C, even more preferably at least 60°C, even more preferably at least 70°C, even more preferably at least 80°C. Such gel forming agent may also be referred to as a “heat-inducible gel forming agent”. Particularly preferred, is a heat-inducible gel forming agent which is able to form a gel when heat is applied, preferably to a temperature at least 60°C whereby the heat-inducible gel forming agent comprises at least one protein. When heat is applied to a solution of a gel forming agent it forms a gel when subsequent to the heating it is cooled. The gel forming agent is thought to form a gel by creating a network of gel forming agents holding the water phase. The term “ungelatinized gel forming agent” as used herein refers to the gel forming agent when it is not a gel. This is the case when it has not been heated.

The term “gelatinized gel forming agent” as used herein refers to the gel forming agent when it is a gel after heating - as described above and subsequent cooling. Preferably, it is cooled to less than 60°C, more preferably less than 50°C, more preferably less than 40°C. One exception is methyl cellulose which forms gels when heated (typically to at least about 60°C) but a methylcellulose gel becomes liquid when cooled.

The gel forming agent may preferably be present in the meat analogue as an ungelatinized gel forming agent. The advantage thereof is that the meat analogue gets firmer during cooking just like real meat thereby providing the consumer with an even better meat analogue experience. This is for instance preferred when the meat analogue is intended as an analogue of raw meat.

The gel forming agent may be a non-animal protein isolate or non-animal protein concentrate or a non-animal polysaccharide. Preferably the non-animal gel forming agent is derived from a plant, algae, microbe. If the gel forming agent is a gel forming plant protein, preferred examples include but are not limited legume protein isolates like soy protein isolate, pea protein isolate, mung bean protein isolate and other plant proteins like potato protein, RuBisCo, moong 8S globulin, a pea globulin, a pea albumin, a lentil protein, zein, or an oleosin and combinations thereof. The gel forming agent can also be a gel forming Chlorella protein. The meat analogue according to the invention preferably comprises an ungelatinized gel forming protein (preferably a plant protein), preferably in an amount of from 0.1 to 25 wt%, preferably 0.1 to 15 wt%, more preferably 0.2 to 11 wt%, even more preferably 0.3 to 7 wt%, more preferably 0.1 to 9 wt%, more preferably 0.2 to 7 wt%, even more preferably 0.3 to 5 wt%, even more preferably 0.5 to 5 wt% by weight of the total composition. The meat analogue according to the invention preferably comprises an ungelatinized gel forming protein selected from potato protein, soy protein isolate, pea protein isolate or Chlorella protein, preferably in an amount of from 0.1 to 25 wt%, preferably 0.1 to 15 wt%, more preferably 0.2 to 11 wt%, even more preferably 0.3 to 7 wt%, more preferably 0.1 to 9 wt%, more preferably 0.2 to 7 wt%, even more preferably 0.3 to 5 wt%, even more preferably 0.5 to 5 wt% by weight of the total composition.

If the gel forming agent is a gel forming polysaccharide, preferred examples include but are not limited to carob bean gum, tara gum, cassia gum, gum arabic, konjac mannan gum, carrageenan, methylcellulose, xanthan gum, pectin, starch and combinations thereof. The meat analogue according to the invention preferably comprises an ungelatinized gel forming polysaccharide, preferably in an amount of from 0.1 to 5 wt% even more preferably 0.2 to 3 wt% by weight of the total composition. Preferably, the ungelatinized gel forming agent comprises a combination of 1 to 5 wt% methylcellulose and 0.5 to 7 wt% potato protein by weight of the total composition. Preferably, the meat analogue according to the invention is substantially free from carob bean gum, tara gum, cassia gum, gum arabic, konjac mannan gum, carrageenan, xanthan gum and combinations thereof, if such ingredients are undesired.

Fibre

The meat analogue according to the invention preferably comprises 0.1 to 9 wt% of fibre, more preferably 0.2 to 7 wt%, even more preferably 0.3 to 6 wt% by weight of the total composition. Preferred examples of suitable edible fibre include but are not limited to psyllium fibre, citrus fibre, potato fibre, bamboo fibre, barley bran, carrot fibre, corn bran, soluble dietary fibre, insoluble dietary fibre, pea fibre, rice bran, head husks, soy fibre, soy polysaccharide, wheat bran, wood pulp cellulose and combinations thereof. The Chlorella biomass of the present invention also provides a suitable source of fibre. Preferably, the meat analogue according to the invention comprises 0.3 to 6 wt% of fibre selected from psyllium fibre, citrus fibre, potato fibre bamboo fibre and combinations thereof.

The meat analogue according to the invention preferably comprises lipid. The term “lipid” as used herein refers to a glyceride component that contains at least 80 wt% of glycerides selected from triglycerides, diglycerides and combinations thereof. Any food grade lipid may be used. Lipid may be a liquid oil or a solid fat or preferably both. Preferably the lipid comprises both liquid oil droplets and solid fat particles.

The term “liquid oil” as used herein refers to an oil that contains no solid at 20°C (N20 = 0%). The solid fat content at 20°C can be determined using ISO method ISO 8292-2:2008. The term “solid fat” as used herein refers to a fat that contains at least 20% solid fat at 20°C (N20 >20%). Preferably, the meat analogue according to the invention comprises 1 to 25 wt% of lipid, more preferably 5 to 22 wt%, even more preferably 8 to 20wt% by weight of the total composition. Preferably, the meat analogue according to the invention comprises 1 to 20 wt% of liquid oil, more preferably 2 to 15 wt%, even more preferably 3 to 10 wt% by weight of the total composition. Preferably, the meat analogue according to the invention comprises 1 to 20 wt% of solid fat, more preferably 2 to 15 wt%, even more preferably 3 to 10 wt% by weight of the total composition. Preferably, the meat analogue according to the invention comprises 1 to 25 wt% of lipid, more preferably 5 to 22 wt%, even more preferably 8 to 20wt% by weight of the total composition, whereby the meat analogue comprises both liquid oil droplet and solid fat particles, preferably whereby the meat analogue comprises

• 1 to 20 wt% of liquid oil, more preferably 2 to 15 wt%, even more preferably 3 to 10 wt% in the form of liquid oil droplets, and

• 1 to 20 wt% of solid fat, more preferably 2 to 15 wt%, even more preferably 3 to 10 wt% in the form of solid fat particles.

Any food grade lipid may be used. Preferred liquid oils include one or more of vegetable oil, an algal oil, sunflower oil, corn oil, soybean oil, palm fruit oil, palm kernel oil, safflower oil, flaxseed oil, rice bran oil, cottonseed oil, olive oil, canola oil, coconut oil, and mango oil.

Preferred solid fat particles include shea fat, coconut fat and palm fat. Preferably, solid fat is added as flakes. Preferably the solid fat particles in the meat analogue have size from 0.2 to 10 mm, preferably 0.5 to 9 mm, more preferably from 2 to 8 mm. The term size in this context refers to the longest dimension of a solid fat particle measured from one end through the center to the other end of the particle.

Preferably, meat analogue according to the invention comprises 1 to 25 wt% of lipid comprising at least sunflower oil droplets and coconut fat particles.

If the lipid comprises both liquid oil droplets and solid fat particles, the liquid oil and solid fat are mixed as individual ingredients with the other ingredients of the meat analogue. If the solid fat is first melted and then mixed with liquid oil this will result in a new triglyceride composition which is either liquid or solid but not both.

Flavouring agent

The meat analogue according to the invention preferably comprises flavouring agent, preferably 0.1 to 20 wt% of flavouring agent, more preferably 0.5 to 10 wt%, even more preferably 1 to 10 wt% by weight of the total composition. Any food grade flavouring agent to provide the desired flavour may be used. Examples include, beef flavour, pork flavour, meat flavour, fish flavour, taste enhancer, yeast extract, spices, herbs, and combinations thereof.

Colouring agent

The meat analogue according to the invention preferably comprises coloring agent, preferably 0.01 to 10 wt% of colouring agent, more preferably 0.05 to 5 wt%, even more preferably 0.1 to 3 wt% by weight of the meat analogue. Any food grade coloring agent to provide the desired colour may be used. Preferred are non-animal derived coloring agents like extracts, juices, powders from red or orange coloured fruit, vegetable, root. Examples include beet, bell pepper, pomegranate, mandarin, carrot, barley malt and combinations thereof.

Salt

The meat analogue according to the invention preferably comprises NaCI, preferably from 0.01 to 5 wt% of NaCI, more preferably 0.05 to 3 wt% of NaCI more preferably 0.1 to 2 wt% of NaCI, more preferably 0.3 to 1.7wt%, more preferably 0.5 to 1.7wt%, more preferably 1.1 to 1.5 wt% by weight of the total composition. In addition, part of the NaCI may be replaced by KCI.

The invention further provides a meat analogue comprising a) non-animal protein; b) a binding agent; and c) preferably 0.01 to 5 wt% of NaCI and wherein the meat analogue comprises from 0.1 to 30 wt%, more preferably from 0.5 to 20 wt%, even more preferably from 1 to 10 wt% and still more preferably from 2 to 5 wt% of Chlorella protein of the present invention by dry weight of the meat analogue. Here, the Chlorella of the present invention is preferably derived from or part of Chlorella vulgaris and/or Chlorella sorokiniana biomass. It is especially preferred that the Chlorella protein is introduced in the form of a spray-dried powder. Any further preferences expressed hereinabove with regard to the Chlorella protein and/or biomass of the invention also apply to its use in the meat analogue.

Here, the Chlorella protein may suitably form part of the non-animal protein base of the meat analogue. Alternatively, it may also serve as a component of the binding agent.

The food composition in the form of a meat analogue can be prepared by usual means. During preparation of the meat analogue, the Chlorella biomass can be introduced at any suitable stage of the production process, for instance together with (or instead of) any other non-animal protein. In case the biomass is in powder form, it can also conveniently be dosed or mixed in at any other stage at which powders can suitably be mixed into the meat analogue composition.

Savoury snacks

According to one preference, the food composition is in the form of a dry savoury snack.

A dry savoury snack is generally understood to be a food composition with a savoury sensory character. Thus, a savoury snack typically has a savoury flavour. Savoury flavour is a well-known sensory attribute. Usually, but not necessarily, it associated with a taste and aroma profile in which salty, umami, and/or spicy elements provide a dominant contribution. Conversely, though sweet notes may be present in a savoury flavour, they are usually not dominant.

Similarly, food components that are intended to be eaten with condiments that have a savoury character are also considered savoury by themselves, for example non-sweet cerealbased products (pasta/noodles or baked snacks), potato-based products (instant mash, crisps), etcetera.

Examples of typical food products regarded as savoury snacks are detailed hereinbelow.

According to a preference, the food composition in the form of a dry savoury snack has a water content of up to 25 wt% by weight of the composition. According to some preferences the water content is up to 20 wt%, more preferably up to 16 wt%, and even more preferably up to 12 wt% by weight of the composition. It is well-known that a suitable water content may depend on the exact product format of the dry savoury snack.

According to a preference, the food composition in the form of a dry savoury snack has a water activity A_w of up to 0.65, more preferably up to 0.60 and even more preferably up to 0.55. Thus, it preferably has a water activity A_w of between 0.10 and 0.65, more preferably between 0.20 and 0.60, even more preferably between 0.25 and 0.55 and still more preferably between 0.30 and 0.50.

The savoury snack may be in a ready-to-eat format or may be suitable for further preparation steps before consumption. For instance, the palatability of a savoury snack may be improved by or even require warming. According to a particular preference, the dry savoury snack is a reconstitutable savoury snack. That is, a food product which is intended for reconstitution with a liquid, typically a water-based liquid and, in particular, a warm or hot (up to boiling) liquid. For example, dry noodles and dry soup concentrates or sauce concentrates are or form part of such reconstitutable snacks.

Dry concentrate

A preferred type of dry savoury snack is a dry concentrate.

Dry concentrates, in particular savoury concentrates are product formats in which the present invention can suitably be applied. Therefore, the composition of the invention preferably is a savoury concentrate. Such concentrates typically serve to prepare ready-to-eat compositions. Thus, savoury concentrates include for instance dry soups, dry sauces, seasonings, bouillon powders, and meal-makers. It is preferred that the savoury concentrate has a portion size suitable for use as a snack. For example, the savoury concentrate may be packed in a portion pack, for example in a portion suitable for providing a single serving of a soup or a sauce. Alternatively, the savoury concentrate may be used as a seasoning mix, for instance one that is used in a product that also includes a carbohydrate component.

When the food product of the invention is in the form of a savoury concentrate, it preferably comprises from 0.5 to 20 wt-%, more preferably from 1 to 10 wt-% and even more preferably from 2 to 5 wt-% of Chlorella biomass by dry weight of the composition.

The savoury concentrate preferably comprises from 0.1 to 15 wt%, more preferably, from 0.2 to 10 wt% and even more preferably from 0.5 to 5 wt% of Chlorella protein by dry weight of the composition.

The savoury concentrate preferably comprises a) 3 to 85 wt-% of inorganic salt; b) 0.5 to 60 wt-% of fat; c) 1 to 10 wt-%, more preferably 2 to 5 wt-% of Chlorella biomass d) optional further components; wherein the wt-% is by weight of dry matter of the total composition.

More preferably, the savoury concentrate comprises a) 3 to 85 wt-% of inorganic salt; b) 0.5 to 60 wt-% of fat; c) 2 to 5 wt-% of Chlorella biomass; d) 0 to 50 wt-% of savoury taste-giving ingredients selected from glutamate, 5’- ribonucleotides, sucrose, glucose, fructose, lactic acid, citric acid and combinations thereof; e) 0 to 25 wt-% of starch component selected from native starch, pregelatinised starch, maltodextrin, modified starch and combinations thereof; f) 0 to 45 wt-% of vegetable matter other than (c), selected from vegetables, herbs, spices and combinations thereof; g) 0 to 10 wt-% of water; wherein the wt-% is by weight of dry matter of the total composition.

Here, the components a) to e) together preferably constitute at least 55 wt.% of the savoury concentrate and the components a) to g) together preferably constitute at least 75 wt.% of the savoury concentrate.

The dry concentrate, in particular the savoury concentrate, can come in several forms or shapes: typical forms are free-flowing powders, granulates, shaped concentrates and pastes.

Savoury concentrate in particulate or powder form

The composition of the invention preferably is a dry concentrate in powder form, comprising a) 3 to 85 wt-% of inorganic salt; b) 0.5 to 2 wt-%, more preferably 1 to 1.5 wt-% of of edible oil; c) 2 to 5 wt-% of Chlorella biomass; and d) optional further components; wherein the wt-% is by weight of dry matter of the total composition.

More preferred oils in this product format are soybean oil, sunflower oil, rapeseed oil, corn oil (maize oil), olive oil, linseed oil, palm olein and fractions and combinations thereof, and even more preferably oils are sunflower oil, rapeseed oil, olive oil and linseed oil.

Alternatively, the food composition can be in the form of particulate savoury composition comprising: a) 1-80 wt.%, by weight of the composition, of an edible salt selected from sodium chloride, potassium chloride and combinations thereof; b) 1-30 wt.%, by weight of the composition, of savoury taste giving ingredients selected from glutamate, 5’-ribonucleotides, sucrose, glucose, fructose, lactic acid, citric acid and combinations thereof; c) up to 10 wt.%, by weight of the composition, of water; and d) from 1 to 10 wt%, more preferably from 2 to 5 wt% of Chlorella biomass by weight of the composition, wherein the sum of a) and b) is at least 20 wt.%, of the total weight of the composition.

This type of formulation is especially preferred if the composition serves as a seasoning composition.

Granulated savoury concentrate

In one preferred embodiment, the savoury concentrate is a granulate having a mass weighted average diameter in the range of 0.1-5 mm, said granulate comprising the following components: a) 1-85 wt%, more preferably 35-85 wt.%, preferably 40-75 wt.% of inorganic salt; b) 3-20 wt.%, preferably 4-15 wt.% fat; c) 2 to 5 wt-% of Chlorella biomass; d) 2-20 wt.%, preferably 5-15 wt.% of the savoury taste-giving ingredients; wherein the wt-% is by weight of dry matter of the total composition.

The granulate preferably has a mass weighted average diameter in the range of 0.2-2 mm, most preferably in the range of 0.25-1.5 mm.

Shaped savoury concentrate

A savoury concentrate may suitably be shaped. For example, a composition comprising mainly powderous ingredients can be shaped into a desirable shape by known means, including for instance mould-casting, sintering, freeze-drying, etc.

In accordance with another preferred embodiment of the invention, the savoury concentrate is a shaped article having a weight of 2-50 g, said shaped article comprising the following components: a) 35-70 wt.%, preferably 40-60 wt.% of the inorganic salt; b) 5-30 wt.%, preferably 15-25 wt.% of the fat, said fat having a solid fat content at 20°C (N20) of at least 5%; c) 2 to 5 wt-% of Chlorella biomass; d) 0-20 wt.%, preferably 2-18 wt.% of the savoury taste-giving ingredients; wherein the wt-% is by weight of dry matter of the total composition.

The shaped article preferably has a weight in the range of 2.5-30 g, more preferably in the range of 3.0-28 g and most preferably of 3.2-24 g. The shaped concentrate article can suitably be provided in different forms.

Savoury concentrate in paste form

In yet another embodiment the savoury concentrate is in the form of a paste. Such a paste preferably comprises a) 3 to 30 wt-% by dry weight of the total composition of the inorganic salt; b) at least 30 wt-% by dry weight of the total composition of an oil phase comprising liquid oil in an amount of at least 30 wt-% by weight of the oil phase; c) 2 to 5 wt-% of Chlorella biomass; d) 1 to 50 wt-% by dry weight of the total composition of the savoury taste giving ingredients.

The term ‘oil phase’ as used herein refers to a distinct lipid phase within the savoury concentrate that contains oil and optionally other lipids. Non-lipid components that are dispersed in the oil phase are not part of the oil phase. The concentration of liquid oil in the oil phase of a savoury concentrate equals 100% - N₂₀. Thus, a savoury concentrate containing 48 wt.% oil phase having a N₂₀ of 5 wt.%, has a liquid oil content of 0.48 x 95 = 45.6 wt.%. Preferably, the oil phase contains at least 50 wt.% of vegetable oil, more preferably at least 70 wt.% and even more preferably the oil phase contains at least 90 wt.% of vegetable oil.

Preferred components of the savoury concentrate

The savoury concentrate preferably comprises inorganic salt. The inorganic salt is added to provide a salty taste. The salt preferably comprises NaCI, KCI and mixtures thereof. The high level of inorganic salt is predominantly present to provide the desired salty taste impact after dissolution in a relatively high volume. Preferably, the amount of inorganic salt in the food concentrate is at least 3 wt%, more preferably at least 5 wt%, even more preferably at least 8 wt%, still more preferably at least 10 wt%, yet more preferably at least 15 wt%, and even still more preferably at least 20 wt% by dry weight of the composition. Preferably, the amount of inorganic salt is at most 70 wt%, more preferably at most 60 wt%, even more preferably at most 50 wt%, and still more preferably at most 40 wt%, by dry weight of the composition. Preferably, the amount of NaCI in the savoury concentrate is at least 3 wt%, more preferably at least 5 wt%, even more preferably at least 10 wt%, still more preferably at least 15 wt% and preferably at most 60 wt%, more preferably at most 55 wt%, and still more preferably at most 50 wt%, by dry weight of the total composition.

In addition to the preferences expressed above, for the fat contained in the food composition, the fat contained in the savoury concentrate preferably has a N₂₀ of 0-60%, more preferably of 5-40% and most preferably of 10-30%.

The savoury concentrate is for example for preparing a bouillon, a soup, a sauce, a gravy or a seasoned dish. To contribute to the savoury taste, the savoury concentrate may further comprise savoury taste-giving ingredients selected from the group consisting of glutamate, 5’-ribonucleotides, sucrose, glucose, fructose, lactic acid, citric acid and mixtures thereof. The term savoury taste-giving ingredients used in the plural may refer to a single compound or a mixture of more than one taste-giving compounds. The amount of savoury taste-giving ingredients present in the savoury concentrate is preferably an effective amount to obtain the desired level in the ready-to-eat product that is prepared from the concentrate. The effective amount depends on the desired dilution rate and amount in the ready-to-eat product. The savoury taste-giving ingredient in the concentrate is preferably present in an amount of at most 40 wt%, more preferably of at most 30 wt%, more preferably in an amount of at most 25 wt%, most preferably in an amount of at most 15 wt%, and preferably at least 0.1 wt%, more preferably at least 0.5 wt%, more preferably at least 1 wt%, more preferably at least 5 wt%, based on the dry weight of the total savoury concentrate. It is understood that any savoury taste-giving compound can be added as such or as part of more complex food ingredients like yeast extract; hydrolyzed proteins of vegetables-, soy-, fish-, or meat-origin, malt extract, beef flavourings, onion flavouring, liquid or dissolvable extracts or concentrates selected from the group consisting of meat, fish, crustaceans, herbs, fruit, vegetable and mixtures thereof.

The savoury concentrate preferably contains a starch component selected from native starch, pregelatinised starch, maltodextrin, modified starch and combinations thereof. The starch component is preferably present in the savoury concentrate in a concentration of 3-20 wt.%, more preferably of 4-18 wt.% and most preferably of 5-15 wt.%. The starch component is preferably selected from native starch, maltodextrin, pregelatinised starch and combinations thereof. Even more preferably, the starch is selected from native starch, pregelatinised starch and combinations thereof. Most preferably, the starch component is native starch. The starch component typically has a mass weighted mean diameter in the range of 5-200 pm, more preferably of 10-100 pm, most preferably of 12-60 pm.

The savoury concentrate preferably comprises vegetable matter. This vegetable matter is preferably applied in the form of leafs, slices, florets, dices or other pieces. Thus, the savoury concentrate preferably comprises 0 to 30 wt.%, more preferably 0 to 20 wt-% and even more preferably 1 to 10 wt% by dry weight of the total composition of such vegetable matter (other than the specified plant powders) selected from vegetables, herbs, spices and combinations thereof. Examples of sources of vegetable matter include parsley, dill, basil, chives, sage, rosemary, thyme, oregano, leek, onion, mushrooms, broccoli, cauliflower, tomato, courgette, asparagus, bell pepper, egg plant, cucumber, carrot and coconut flesh.

According to another preferred embodiment, the savoury concentrate contains 0-10 wt.%, more preferably 0.5-8 wt.% and most preferably 1-5 wt.% of gelatine component, said gelatine component being selected from gelatine, hydrolysed gelatine and combinations thereof.

The savoury concentrate typically contains less than 9 wt.% water. More preferably, the concentrate contains 1-8 wt.% water. Most preferably, the concentrate contains 2-7 wt.% water. The water content in the food concentrate can be measured by any standard method including drying the food concentrate and comparing the weight before and after drying.

The food concentrate according to the invention preferably has a water activity of less than 0.65, more preferably less than 0.5, even more preferably less than 0.4, more preferably less than 0.3 and preferably more than 0.15.

The savoury concentrate of the present invention preferably is a packaged savoury concentrate. The portion of the concentrate as packaged preferably has a weight (excluding packaging) of 1 g to 1 kg, preferably 2-250 g, more preferably 5-50 g. The packaging can be e.g. a container, a pouch or a wrapper.

Pasta

According to another preference, the food product in the form of a dry savoury snack is a dried pasta. Such pasta can conveniently be used as a savoury snack or as part of a savoury snack. Thus, the food product preferably is a dried savoury snack in the form of pasta. The pasta preferably comprises from 1 to 25 wt%, more preferably from 2 to 20 wt% and even more preferably from 5 to 15 wt% of Chlorella biomass by dry weight of the composition. It is particularly preferred that the Chlorella biomass is used in the form of a dry powder, especially a spray-dried powder.

In addition, the pasta can include any conventional pasta ingredients, and can for example be in the form of cereal pasta or rice noodles. Thus, the pasta preferably comprises at least 50 wt%, more preferably at least 75 wt% of farinaceous material, which is preferably selected from wheat, corn, soy flour, semolina, farina, rice flour, pulse flour or combinations thereof.

The pasta may be prepared by any usual production process. Typically, a dough (which may conveniently include the Chlorella biomass) is formed first, e.g. by kneading. The dough is then shaped and optionally cooked or fried. The pasta is dried until the water level is sufficiently low. The dried pasta preferably comprises between 1 and 20 wt% of water by weight of the composition, more preferably between 5 and 15 wt% and even more preferably between 10 and 14 wt%. Many standard dried pasta products have a water content of about 12 wt%.

The pasta product may include any other conventional pasta ingredients in the usual amounts. Usual ingredients include egg or egg components, starches, salt, flavouring, colourants and the like.

Instant meal snack

A very convenient product format for a dried savoury snack is an instant meal snack. Typically, at least one of the components of the instant meal snack is reconstitutable with liquid, especially with hot water. Typically such products include a carbohydrate base, a dry seasoning or sauce mix, and in some cases a dressing for flavour additions. The carbohydrates can be pre-cooked (e.g., by steaming, frying, pre-gelling) for quick re-hydration, and could be, for example: pasta (fusilli, vermicelli, penne formats), noodles (fried noodles, air-dried rice noodles), rice (long, medium, short grains), or wheat (semolina, barley). Other suitable carbohydrate bases are legumes, including for instance lentils, especially legumes that have been pre-treated to make them instant-reconstitutable. Another suitable carbohydrate base is dehydrated potato, especially dehydrated mashed potato. Dehydrated mashed potato is suitably prepared in the form of granules or flakes. Well-known manufacturing processes for dehydrated mashed potato are described for instance by D. Hadziyev and L. Steele, Dehydrated mashed potatoes - chemical and biochemical aspects, Advances in Food Research. Vol. 25, pages 55-136 (1979), and M. Willard, Potato Processing: Past, Present and Future, American Potato Journal, vol 170, pages 406 to 418. Another suitable carbohydrate base is a reconstitutable porridge base, comprising for instance durum wheat semolina, wheat flakes, oats and the like.

The instant meal suitably also comprises dry seasoning components. These can be mixed in with the carbohydrate base, or be available separately, in the sense that they have to be added or dosed into the carbohydrate base (e.g. after rehydration) by the consumer. Common seasoning mixes could include one or more of on top of flavour components (flavours, herbs, spices, salt), taste enhancer, fat, garnish and thickeners, dry vegetables, dairy, vegetable extracts, tomato powder. Specific seasoning mixes can aim toward a bouillon type of seasoning or specific types of sauces (e.g., bolognese, carbonara, Alfredo, creamy chicken mushroom), where different ingredients are brought together, including dehydrated/powder tomato, dried dairy components (e.g., whey, cream, butter, cheese powder), meat (e.g., freeze dried bacon, ham, chicken), binders (e.g., rice/wheat flour, corn starch), dehydrated vegetables (e.g., broccoli, mushrooms, carrots, chives, parsley). Dressing could include, for example, flavoured oil and soy sauce.

Both the carbohydrate base and the seasoning may suitably comprise the Chlorella biomass, though in many cases it will suffice if only one of the components comprises the Chlorella biomass. Thus, the carbohydrate base of the instant meal is preferably a pasta as described hereinbefore and/or the seasoning is preferably in the form of a savoury concentrate as described hereinbefore, and even more preferably a savoury concentrate in powder or granulate form.

The instant meal-type snack is suitably packed in a sachet, a container or similar type of packaging. A preferred type of packaging is a cup (esp. a “snack cup”). Typically such cups are designed such that - after removal of the lid - the contents (including at least the carbohydrate base) can easily be reconstituted by pouring in liquid (esp. hot water). Thus, according to one aspect, the invention relates to a container, preferably in the form of a sachet or a snack cup, more preferably a snack cup, wherein the container comprises a food product in the form of a reconstitutable dry savoury snack, said food product comprising a) at least 45 wt% of a carbohydrate base component by weight of the dry product b) at least 1 wt%, more preferably at least 2 wt% and even more preferably at least 5wt% of a dry seasoning component; wherein the food product comprises from 0.1 to 20 wt%, more preferably from 0.2 to 15 wt%, even more preferably from 0.5 to 10 wt% and still more preferably from 1 to 5 wt% of Chlorella biomass by dry weight of the food product and wherein the Chlorella biomass is preferably incorporated (upon preparation) in the form of a dry powder, more preferably a spray-dried powder. Here, depending e.g. on the desired amount of Chlorella biomass and the sensory or organoleptic properties of the carbohydrate base, the Chlorella biomass may be part of the carbohydrate base component or of the dry seasoning component or it may be incorporated in both.

Savoury bites

According to another preference, the food composition in the form of a dry savoury snack is in the form of a savoury bite. In this context, a savoury bite denotes a food product in biteable form or in the form of bitesize elements. Thus, such a dry savoury snack can be a savoury snack bar, a granola bar, a crisp, a puff, a crouton, krupuk, baked savoury bites, finger food and similar product formats. The savoury bites may be packed individually, in portion packs or in larger packs suitable for snacking by multiple consumers. The savoury bite preferably comprises from 0.1 to 20 wt%, more preferably from 0.2 to 15 wt%, even more preferably from 0.5 to 10 wt% and still more preferably from 1 to 5 wt% of Chlorella biomass by dry weight of the food product.

FIG. 1 is an illustration of steps of a method of producing a chlorophyll-deficient strain of Chlorella microalgae having a protein content of at least 50% w/w.

FIG. 2 shows the iteration of new Chlorella vulgaris variants, achieved through successive rounds of chemical mutagenesis, originating from wild-type 4TC3/16.

FIG. 3 shows the iteration of new Chlorella sorokiniana variants, achieved through successive rounds of chemical mutagenesis, originating from wild-type UTEX1230.

FIG. 4 shows the iteration of new Chlorella vulgaris variant WC03, achieved through successive rounds of chemical mutagenesis, originating from wild-type 4TC3/16. FIG. 5 shows the iteration of new Chlorella sorokiniana variants, achieved through successive rounds of chemical mutagenesis, originating from wild-type UTEX1230.

Examples

Part I - Chlorella biomass preparation

Example 1 - Production of Chlorella strains

Genetically defining Chlorella microalgae strains using PCR amplification:

Chlorella microalgae was genetically defined by 18S and ITS2 seguencing as described above. In particular, it was confirmed that the strain 4TC3/16 was a wildtype Chlorella vulgaris strain.

FERM media described herein, denotes growth media with the following composition: glucose (111 mM), (NH₄)₂SO₄ (47.7 mM), MgSO₄.7H₂O (2.8 mM), CaCI₂.2H₂O (204 pM), K₂HPO₄ (51.7 mM), NaH₂PO₄.H₂O (63.3 mM), KOH (40 mM), citric acid (8.8 mM), H3BO3 (1.1 mM), Na₂MoO₄ (32 pM), ZnSO₄.7H₂O (974 pM), MnSO₄.H₂O (958 pM), NiCI₂.6H₂O (11 pM), FeSO₄.7H₂O (79.1 pM), CuSO₄.5H₂O (8 pM), Thiamine hydrochloride (5.65 pM), Biotin (92.1 nM), Cyanocobalamin (13.3 nM), D-Pantothenic acid (205.3 nM), 4-Aminobenzoic acid (656.3 nM).

The high salt medium (HSM) described herein comprised: NH₄CI (7 mM), MgSO₄.7H₂O (400 pM), CaCI₂.2H₂O (340 pM), K₂HPO₄ (4.13 mM), KH₂PO₄ (2.67 mM), Na₂-EDTA (57.75 pM), (NH₄)₆MO₇O₂₄.4H₂O (28.5 nM), Na₂SeO₃ (100 nM), ZnSO₄.7H₂O (2.5 pM), MnCI₂.4H₂O (6 pM), Na₂CO₃ (21.9 pM), FeCI₃.6H₂O (20 pM), CuCI₂.2H₂O (2 pM). Glucose is added at 1% (w/v) or 2% (w/v) or 3% (w/v) where indicated, in addition to:Thiamine hydrochloride (5.65 pM), Biotin (92.1 nM), Cyanocobalamin (13.3 nM), D-Pantothenic acid (205.3 nM), 4-Aminobenzoic acid (656.3 nM) to produce HSM 1 GV or HSM2GV or HSM3GV, respectively.

Cultivating Chlorella microalgae strains under heterotrophic growth mode: Chlorella microalgae strains were grown in 20 millilitres (ml) of liguid medium containing glucose at a starting cell density of 2x10® cells/ml. Cells were grown in the dark at 26°C for 6 days. A 10 ml aliguot was removed and centrifuged (4500 x g, 10 minutes) to collect the cells; the pellets were washed in 1 ml double-distilled (dd) H2O and centrifuged again (4500 x g, 10 minutes). The resulting biomass pellets were dried by lyophilisation in pre-weighed tubes. Once dry, the dry cell weight (DCW) was determined before carrying out the extraction. Selecting viable cells of the Chlorella microalqae Cells growing as colonies of the variant strains of Chlorella microalgae associated with the desired phenotypes were isolated and streaked sequentially and iteratively on a solid medium to obtain axenic, isogenic strains as well as to assess the stability of the chlorophyll-reduction phenotype under conditions more approximating a commercial cultivation scheme. An axenic culture is a microbial culture in which only a single strain of one organism is present and which is entirely free of all other strains or other contaminating microorganisms. The colonies were further inoculated using a liquid media. The liquid media comprising TAP (Tris-Acetate-Phosphate), High Salt Medium (HSM) plus glucose (for example, having 1 to 3% w/v glucose). The colonies were cultivated in dark conditions at the specific temperature of 15 to 30 °C for 3 weeks, and monitored over multiple successive generations for stable phenotypes.

Detailed description of preferred mutagenesis method

Chlorella microalgae strains were grown in 100 millilitres (ml) of nutrient rich liquid medium containing glucose or acetate (such as FERM or HSM as described herein) at a starting cell density of 2x10⁶ cells/ml. Cells were grown in the dark at 28 °C for 3-6 days, with 120 rpm agitation. Cell number was recorded during the incubation period using a haemocytometer and a light microscope. When cells are still in exponential phase of growth, reaching 1x10⁷ to 1x10⁸ cells/ml, an aliquot containing 1x10⁹ cells was harvested by centrifugation (4500g for 10 minutes).

The resulting cell pellet was resuspended in 1 ml HSM media + 1 % glucose. An aliquot of ethyl methanesulphonate (EMS), with a resulting final concentration of 0.5M was added to the resuspended cells. The cells were incubated in the dark at 25 °C for 30-120 minutes. EMS is known to produce random mutations, such as nucleotide substitution, transition mutation, single nucleotide polymorphisms (SNPs) and the like, in the genetic makeup of the organism exposed thereto. The use of EMS may result in a mutated ethylguanine base in the DNA as a result of guanine alkylation. Repeated replication of such mutated DNA can result in a transition mutation, wherein original G:C base pairs change to A:T base pairs, thereby significantly changing the genetic makeup of the organism. In such case, the replication of such mutated DNA may create missense mutations or nonsense mutations within coding sequences or impacting gene expression or gene function by compromising regulatory sequence functionality including splicesite mutations.

After the mutation incubation period an aliquot of 30% sodium thiosulphate, in HSM + 1 % glucose media was added to the cell solution, with a final concentration of 5% sodium thiosulphate. The cells were incubated in the dark for a further 10 minutes. The sodium thiosulphate inactivates the alkylating agent and slows the rate of mutagenesis. After the inactivation incubation the cell solution was diluted in 25 ml of HSM 1 % glucose media, centrifuged, and the supernatant discarded (into a 30% sodium thiosulphate solution). This wash step was repeated 2 more times to eliminate the residual EMS and stop the mutagenesis process.

After washing, the cells were resuspended in 50 ml of HSM + 1 % Glucose, TAP, or other nutrient rich media with a glucose or acetate carbon source. The cells were incubated in the dark, at 28 °C, with 120 rpm agitation, for at least 24 hours. The cells were then plated on solid agar media (HSM + 1% glucose or TAP) to isolate viable cell mutants derived from single cells. After 2-4 weeks the colonies were ready for phenotype selection. Alternatively, after 24 hours the mutant pool can be screened with flow activated cell sorting (flow cytometry), to isolate single cells with a desired phenotype, or a mutant pool enriched with the desired phenotype.

Identifying and isolating variant strains of Chlorella microalgae with reduced chlorophyll content

Chlorella microalgae strains were grown in 20 millilitres (ml) of liquid medium containing glucose or acetate at a starting cell density of 2x10⁶ cells/ml. Cells were grown in the dark at 26 °C for 6 days. A 10 ml aliquot was removed and centrifuged (4500 x g, 10 minutes) to collect the cells; the pellets were washed in 1 ml double-distilled (dd) H2O and centrifuged again (4500 x g, 10 minutes). The resulting biomass pellets were dried by lyophilisation in pre-weighed tubes. Once dry, the dry cell weight (DCW) was determined before carrying out the extraction. To extract the chlorophyll, 1 ml of methanokacetone (1 : 1) was added to each sample, samples were then mixed by vortexing and pelleted (4500 x g, 5 minutes). The supernatants were collected into separate tubes. This was repeated 4 times for each sample with each supernatant pooled with the previous until 5 ml was collected for each sample. To complete the extraction, 1 ml of dichloromethane: methanol (1 :3) was added to the pellet and the previous step repeated and the 1 ml supernatant added to the previous 5 ml. This was followed by 1 ml dichloromethane to yield a total of 7 ml total supernatant. To extract any residual pigment that was remaining in biomass pellets after all the above extraction steps had been performed, 1 ml dichloromethane: methanol (1 : 1) was added to the samples with 500 micrometre (pm) glass beads and sonicated for 10 minutes and the supernatant was again added to the previous 7 ml. The extractions were carried out in low light conditions - samples were wrapped in foil between processing steps to protect any pigments from degradation by light or chemical reactions catalysed by light.

The extracted pigments were dried at 60 °C by evaporation and dried pellets were resuspended in 80% acetone. Absorbance was measured spectrophotometrically at 647, 664, and 750 nanometers (nm).

The following formulas were used to calculate chlorophyll content as described in Porra et al. (1989; DOI: 10.1016/S0005-2728(89)80347-0).

Chlorophyll a = (12.25 x (A664-A750)) - (2.55 *(A647-A750))

Chlorophyll b = (20.31 x (A647-A750)) - (4.91 *(A664-A750)) Total chlorophyll = (17.76 x (A647-A750)) + (7.43 *(A664-A750))

All strains were analysed in biological triplicate.

Thin layer chromatography can be used to separate and visualise the pigment composition in different strains. For example, Chlorella vulgaris strains were grown in 20 ml of liquid growth medium containing 1% glucose. Cells were grown in the dark at 26°C for 6 days. A 10 ml aliquot was removed and the cells were collected by centrifugation (4500 x g, 10 minutes).

To extract the pigments, 0.5 ml of dichloromethane: methanol (1 : 1) was added to each sample, samples were then mixed by vortexing and centrifuged again (21000 x g, 5 minutes). The supernatants, containing the extracted pigments, were collected in separate collection tubes. This extraction was repeated on the pellet 2 times and pooled into the same collection tube each time for each sample. The entire extraction process was carried out in low light laboratory conditions (< 50 pmol rm² s^-1), samples were wrapped in aluminium foil between processing steps. Small quantities of the samples were deposited on a Silica gel on TLC Alu foil plate (91835-50EA, Sigma-Aldrich (RTM)) and developed with a solvent solution of 5:3:2 Hexane: Ethyl Acetate: acetone. Resultant plates were imaged to record the separation and relative composition of pigments for each sample.

Cell sorting by flow cytometry can be used as an enrichment step to sort chlorophylldeficient cells away from wild-type cells based upon the relative signal strength of autofluorescence. For example, a sample containing cells was suspended in a fluid and injected into a flow cytometer instrument, wherein the flow of the sample was set at one cell at a time. The flow rate of the flow cytometer instrument may be any suitable rate. Optionally, the flow rate of the flow cytometer instrument was 60.000 to 500.000 events per minute. The flow rate may also be lower than 60.000 or higher than 500.000 events per minute. The flow cytometer employs lasers of various wavelengths for multi-parametric analysis of the cells in a heterogenous cell population. The light scattered and fluoresced by the cell is a characteristic of the cell and components therein. A 488 or 561 nm laser was used to elicit strong chlorophyll autofluorescence from a mixture of live cells. The population of cells that exhibit strong autofluorescence was sorted away from those cells that have null or significantly reduced signal as an enrichment step to enrich for those cells within the total population that have accumulated mutations that knock down or abolish the chlorophyll signal. This step can be applied optionally between 2-7 days postexposure to mutagen and is applied in liquid culture. As a control to calibrate the cytometry, wildtype cells were extracted using 90% acetone to remove chlorophyll and were then photobleached using strong light for 20-30 minutes. These chlorophyll null cells were then used to calibrate the sorter with regard to chlorophyll deficient particles. Further, flow cytometry enables cell counting, cell sorting, determining cell characteristics and functions, detecting microorganisms, biomarker detection, protein engineering detection, and the like. Null cells including those desired cells with reduced chlorophyll content and expanded and resorted through one additional round to confirm the stability of the chlorophyll deficient phenotype. These cells can be further expanded in liquid culture or plated onto agar plus glucose plates for scoring of colour with respect to other mutants. One such scoring method that can be used to quantify and thereby directly compare the colour of chlorophyll-deficient Chlorella variants is the use of L*a*b* Cl ELAB colour values as enumerated, for example using the PCE-CSM 1 colourimeter (https://www.pce-instruments.com/) and the accompanying manufacturer’s protocol.

Once expanded, the chlorophyll deficient cell population that is actively growing can be sorted into sub-populations or single cells using the application of different lasers exciting at specific wavelengths and concurrent detection of deflection of the laser beam and specific fluorescence emissions of higher wavelength photons from cellular compounds, which can be used to differentiate specific pigment combinations that would ultimately influence the resultant stable biomass colour for a given biomass that is derived from a particular population of cells or single cells carrying specific genotype. Unhealthy colonies of the modified strain of Chlorella (e.g. Chlorella vulgaris) can be removed by filtration. It will be appreciated that during mutagenesis of the wild-type strain of Chlorella, cells of the modified strain of Chlorella may acquire mutations at multiple sites within the genome, including a mutation or mutations that are causative for the desired phenotype. However, some colonies of the modified strain of Chlorella may additionally acquire deleterious mutations corresponding to one or more undesired phenotypes, for instance in essential genes. In such an instance, it is essential to filter out these unhealthy colonies of the modified strain of Chlorella associated with the deleterious mutations, to ensure selection of only those colonies that are robust and able to grow well under desired cultivation conditions. This can be achieved by cultivation of the organisms during the period immediately following exposure to the chemical or physical mutagen under stressed or less permissive (or non-permissive) conditions, for instance at the limit of or slightly above the normal upper temperature for cultivation and in the absence of light but in the presence of glucose. Only robust strains are able to proliferate under these conditions. This approach enriches for strains that are not compromised in their replication capacity. Furthermore, after cultivation in the dark, the desired phenotypes related to colour can be scored using techniques described herein above. In one such example, the desired phenotype of the modified strain of Chlorella vulgaris is associated with white, cream, pale yellow, yellow, pale green, golden, caramel, orange, red or lime colour. Undesired colonies will be associated with other colours including the wild-type, dark green colour and are not selected. In other words, they are filtered out. Colonies of modified strain of Chlorella vulgaris that exhibit the desired phenotype across a series of generations are selected as healthy colonies. The mutated strain of Chlorella vulgaris is cultivated at a temperature that is slightly higher than an ideal temperature (such as, above 28 °C) for cultivation of the microalgal strain, to select only healthy colonies of the modified strain of Chlorella vulgaris.

Colonies of the modified strain of Chlorella (e.g. Chlorella vulgaris) associated with the desired phenotypes are isolated and streaked sequentially and iteratively on a solid medium to obtain pure colonies as well as to assess the stability of the colour phenotype under conditions more approximating a commercial cultivation scheme. The pure colonies are further inoculated using a liquid media. Optionally, the liquid media may be at least one of TAP (Tris-Acetate- Phosphate), High Salt Medium (HSM) plus glucose (for example, having 1 % w/v glucose). More optionally, the pure colonies are cultivated in dark conditions at the specific temperature of 25 °C (or between 20 and 35 °C) for 1-3 weeks and monitored over multiple successive generations for stable phenotypes. Such stable phenotypes may be associated with a lack of green colour within the pure colonies of the modified strain of Chlorella vulgaris and/or the presence of white, cream, pale yellow, yellow, pale green, golden, caramel, orange, red or lime colour phenotypes.

Single chlorophyll-deficient Chlorella microalgae (with a chlorophyll content below 0.5 mg/g dry cell weight) can be isolated from a mixed mutant pool using flow cytometry. When light energy is absorbed by chlorophyll, part of the energy is used to drive photosynthesis via photochemical energy conversion, the remaining energy is lost as heat or emitted as fluorescence radiation. This fluorescence is also called chlorophyll autofluorescence.

When carrying out automated cell sorting, cells in solution are drawn into a flow cytometer and manipulated by fluidics into a separated single file cell stream (hydrodynamic focusing). Cells pass through the laser, where natural (such as chlorophyll) or artificial fluorophores are excited by this light and emit fluorescence with a specific wavelength spectrum. The fluorescent emission is detected by photomultiplier tubes (PMTs) or photodiodes. A voltage pulse (an event) is created when a change in the number of photons is detected by a PMT. The area of this pulse correlates to the fluorescence intensity (Fl) of the fluorophore. This information is automatically collected by the machine and is displayed live on flow cytometry software.

The combination of mirrors, filters and detectors allows the machine to detect fluorescence at specific bands of wavelength. Commercially available lasers suitable for optimal excitation of chlorophyll are either 488 and 561 nm. The chlorophyll emission from this excitation ranges from 640 to 850 nm. Preferably the emission is monitored using a 695 ± 40 nm dichromatic filter. Chlorophyll and additional pigments within the cell can also be excited by other lasers, including 349 nm, 355 nm, 405nm, 445 nm, 532 nm, 594nm, 640nm, 740 nm, with emissions ranging from 350-850 nm.

The 695 ± 40 nm Fl of individual Chlorella cells is correlated to the chlorophyll content of the cells. Software controlling a flow cytometer allows one or a series of custom gates to be created containing cells with specific fluorescent properties at different excitation and emission combinations. A sorting flow cytometer, often referred to as fluorescence-activated cell sorters, can deflect the stream of cells to isolate single cells that have a specific fluorescence fingerprint that falls within the selected gates (sorting gates). Cells that are not deflected are discarded.

Chlorophyll-deficient Chlorella microalgae strains having a chlorophyll content below 0.5 mg/g DCW were therefore identified by their Fl and deflected into a single tube, creating an enriched pool of genetically unique mutants with similar fluorescence phenotypes. Alternatively, they were sorted into individual tubes, or wells within a microplate to isolate single mutant lines.

A positive control was used to calibrate or specify the sorting gates for selecting cells with the desired fluorescent properties. This positive control can be wild-type cells which have had their chlorophyll extracted using 90% acetone and photobleached for 20-30 minutes, or existing mutant strains that have the desired chlorophyll content. A negative control is also used to calibrate the sorting gates. The negative control can be the parent strain of the mutant pool, which has above, at most, 0.5 mg/g DCW chlorophyll. The positive controls should have a low Fl signal at 495 nm excitation, 695 ± 40 nm emission channel, 561 excitation, 695 ± 40 nm emission channel. The negative control exhibits a high signal in the same channels. Fluorescent properties associated with low chlorophyll, of all cells (within a sampled pool) within the negative control should fall outside the sorting gate, to avoid sorting false positives. Once the gates are calibrated the mutant pool of cells can be sorted, isolating cell lines with below 0.5 mg/g DCW chlorophyll content. Both the positive and negative control can be used to gate to exclude unhealthy or dead cells using forward and side scatter of the 488nm laser.

Before sorting the cells are grown to mid exponential phase in HSM media with glucose. The cells were sorted into the same media (either a single tube or microplate), and incubated for 1-4 weeks in the dark at 28°C. After 1-4 weeks the cells were either scaled up to assess the phenotype, or plated to isolate single cell lines if sorted into an enriched mutant pool.

The isolation of chlorophyll-deficient Chlorella microalgae may for instance be achieved using fluorescence-activated cell sorting (FACS).

The sequential rounds of mutagenesis of Chlorella vulgaris were followed by cultivating the mutated strains of Chlorella vulgaris at a specific temperature, for a predefined period of time, without presence of light and in the presence of an organic carbon energy source, and identifying colonies of the mutated strain of Chlorella vulgaris having a phenotype (or desired trait) different from the parental strain of Chlorella vulgaris. The method can use cell sorting by flow cytometry to sort cells based on the desired traits. However, the selection for desired traits may be achieved using any of the disclosed methods or methods available to those skilled in the art. Desired traits to include, but not limited to, pigment content, colour, protein content and improved tolerance to process conditions including but not limited to cultivation temperature, pH, sheer stress and osmolality. Identifying and isolating variant strains of Chlorella microalgae with an increased protein content and reduced starch content

Isolation of suitable variants may be performed by any means known to the skilled person. The use of a staining or indicator agents, such as iodine, including iodine vapor or solution, preferably iodine vapour, to stain the intracellular starch present within the cell, specifically within the chloroplast, flow cytometry or a combination thereof are preferred.

The identification of a modified strain of Chlorella microalgae comprises sorting or screening the cells by any suitable technique, such as by using flow cytometry. The protein and starch modified strain of Chlorella microalgae may, be selected based upon the degree of staining by iodine vapour, detectable by visual inspection or intensity of starch-iodine fluorescence signal obtained on cell sorting by flow cytometry. The modified strain of Chlorella microalgae may be further selected, based on a desirable pigment or cell wall composition, wherein the desirable pigment or cell wall composition is based upon a relative signal obtained on cell sorting by flow cytometry. The use of flow cytometry provides the advantages of examining thousands of cells per second and in real time and processing quantifiable data over a computer coupled to a flow cytometer. Furthermore, flow cytometry helps in cell counting, cell sorting, determining cell characteristics and function and detecting microorganisms.

Isolated mutant strains of Chlorella microalgae, derived from a chlorophyll deficient parent mutant strain, grown on solid nutrient replete media, were stained in a sealed glass container saturated with iodine vapor. Typically, 5g of iodine granules were placed on the clean lid of a petri dish, located within a wide 1 L glass beaker. The corresponding bottom of the second petri dish, holding the Chlorella colonies, is then positioned above the iodine granules, with colonies facing downwards, and the beaker is sealed with a lid for 1.5 minutes to enable staining. After staining, colonies with higher starch content appear darker and can be selected for resuspension in 20pL of HSM +3% glucose medium and subsequently spotted onto HSM +3% glucose plates. Alternative chemicals can be bound to intracellular starch or protein, with different spectral properties. The mutant strains were stained for 1 minute and 30 seconds. Iodine vapor permeates through the cell wall and cell membrane. Polyiodide ions (l_n ^_) form a complex with the amylose fraction of starch; this complex has specific light absorption spectra, appearing dark blue. When cells with the wild-type phenotype (i.e. high starch) are stained, a dark blue colour results, corresponding to a high concentration of starch, including amylose. Mutant strains of Chlorella microalgae with a different, lighter or undetectable colour change compared to the parent strain (dark blue) were selected. Strains with mutations in starch biosynthesis, can exhibit higher protein content.

Alternatively, the mutant strains of Chlorella microalgae were stained with iodine solution (2% KI w/v and 1 % I2 w/v) for 1-60 minutes. Samples were washed with phosphate-buffered saline (PBS) or liquid media to remove the excess iodine. Resuspended, stained cells were sorted by flow cytometry, with 488 nm excitation, according to their fluorescence emission shift at 515 nm (or between 500-530 nm) and compared to the non-mutated parent strain of Chlorella microalgae (namely, control). A single suspension of cells was prepared, effectively stained, and allowed to flow through the flow cytometer in a single flow through the light beam for sensing. The laser was used to elicit strong fluorophore fluorescence from intracellular starch-iodine complexes in viable cells. The dye-specific fluorescence signals were analysed by a computer physically connected to the flow cytometer. The population of cells that exhibited strong fluorescence was sorted away from those cells that had null or significantly reduced signal as an enrichment step to enrich for those cells within the total population that had accumulated mutations that knockdown or abolish the starch content signal. This step was applied between 2-7 days post-exposure to mutagen and in liquid culture.

The screening step was repeated up to 5 times in order to isolate cells with a stable genetic trait, rather than strains with an unstable and variable trait due, for instance, to natural phenotypic plasticity (i.e. not due to stable, inherited genetic mutation). Furthermore, both the mutagenesis and screening step can be repeated on the same strain lineage to isolate mutants with still lower starch content, thereby selecting strains with multiple knock-out or knock-down mutations in different genes or associated genetic regulatory elements involved in the starch synthesis or protein synthesis pathway.

The identification of starch deficient strains from chlorophyll-deficient parents (due to stable, inherited genetic traits) was possible under nutrient replete conditions (i.e. without nitrogen deficiency induced chlorosis), demonstrating that this method enables isolation of mutants of Chlorella microalgae that have a genetically-determined, constitutively lower starch content, rather than those which exhibit starch accumulation in response to, for example nutrient stress. Parent strain selection is important for the strain development process; parent strains of Chlorella microalgae with a desired phenotype are screened for high protein, and high growth rate prior to mutagenesis and screening of starch deficiency (i.e. resulting in higher protein levels). The starch content was verified using the Total Starch Assay Kit Assay Kit™ (K-TSTA; Megazyme, Ireland). Further, the manufacturer’s “total starch content of samples containing resistant starch (RTS- NaOH)” procedure was followed as described in https://doi.org/10.1002/star.201800146.

Selected Chlorella microalgae mutants, characterised by starch deficiency, were further screened to ensure a higher protein content was maintained in various, controlled, growth conditions. Protein quantification was carried out using several methods, including the Biuret, Bradford, BCA, Lowry, Fluorescent, Pierce, Kjeldahl, Dumas methods, and amino acid quantification. The Dumas method is preferred due to its suitability in determining total protein content. This method involves combusting a small, known mass of sample (100 mg-1 g) at high temperatures (800-900 °C) in the presence of oxygen. This combustion process leads to the release of gases, including nitrogen. The nitrogen gas is then separated and quantified using a thermal conductivity detector, providing an estimation of crude protein content. This nitrogen value is converted to a protein value using the established nitrogen to protein (NtP) conversion factor of 6.25 (Nx6.25), as per McCance and Widdowson’s "The Composition of Foods" (ISBN 978-1-84973-636-7). This factor is widely accepted for various foods, including microalgae, and it normalises the protein content to make it comparable with other protein sources.

For the Dumas analysis, Chlorella microalgae cultures were grown in glass Erlenmeyer flasks using nutrient-rich media supplemented with glucose. The cultures were maintained in the dark at 28 °C with 120 rpm agitation for 3-7 days until a biomass density of 4-8 g/litre was achieved. Sampling occurred during late exponential growth phase to avoid stress-induced variations. Alternatively, samples were also obtained from cells cultured in controlled batch-fed fermenters, harvested during exponential growth at densities ranging from 4-100 g/l. Prior to Dumas analysis, the samples were washed with distilled water and freeze-dried. This method does not account for variation in non-protein nitrogen.

Alternatively, the protein content was measured by amino acid content. In this method, the dry biomass (50g) produced by the same method as above is processed by a suitable method to break down the proteins into separate amino acids (hydrolysis). The amino acids were derivatised to aid detection, then separated with ion exchange chromatography, liquid chromatography (LC), high pressure liquid chromatography (HPLC), other similar chromatography, gas chromatography, and detected with UV, fluorescence, pulse amperometry, flame ionisation detection (FID), mass spectrophotometry (MS) or nuclear magnetic resonance (NMR).

Strains with protein contents higher than 50% (Dumas 6.25 NtP) were selected. This method resulted in a systematic, consistent, directed increase in the desired phenotype, i.e. increased relative protein concentration in a chlorophyll-deficient Chlorella.

Preferred method for mutagenesis and flow cytometry:

Herein follows a description of the preferred method for isolating chlorophyll deficient Chlorella microalgae strains using flow cytometry cell sorting. The described method is suitable to isolate chlorophyll deficient mutants of Chlorella microalgae in general.

Exponential phase wild type (4TC3/16, chlorophyll replete) cells were concentrated to 1x10⁹ cells per ml in HSM +3% glucose media. An aliquot of methyl methanesulphonate (MMS) was added to the cell solution at a final concentration of 0.12M (10 pl per 1 ml). The cell mixture was incubated for 1 hour in the dark. A 30% sodium thiosulphate solution was added to the cell mixture to a final concentration of 5% sodium thiosulphate. The culture was incubated for 10 minutes. The cells were washed 3 times in 25 ml of HSM + 1% glucose. The cells were resuspended in 25 ml HSM + 3% glucose and left to recover for 144 hours in the dark, at 28°C and 120 rpm agitation.

After a period of recovery, the cells were sub-cultured into fresh media, further incubated, and sampled during mid-exponential phase growth (5x10⁶ cells ml’¹ to 5x10⁷). 100,000 cells were initially analysed with a BD FACSAria Fusion (Becton Dickinson, USA) or Bigfoot Spectral Cell Sorter (Thermo Fisher Scientific, USA) in order to detect their fluorescence properties. In addition to the mutant pool, wild type cells and chlorophyll deficient mutants (isolated by alternative methods or previous flow cytometry isolation) were analysed. These single strain cells were used as controls to design gates to sort populations.

First, using the forward and side scatter (FSC and SSC parameters) of the excitation laser, healthy single cells were gated, excluding cell debris and clumped or dividing cells. The chlorophyll content of cells was analysed by measuring the emission fluorescence intensity at 695±40 (or 670±30) after excitation ftom 488nm and 561 nm lasers. Gates were designed based on the fluorescence properties of the control cells. Gates were drawn to instruct the flow cytometry sorter to isolate cells that have fluorescence properties within the range of fluorescent intensities which match the chlorophyll deficient control population, excluding to the wild type chlorophyll replete control cell population.

Using live data and prepared gates, cells with the desired chlorophyll deficient fluorescent properties were sorted away from a stream of a mixed mutant population into a tube or microplate well. The nozzle tip size used for sorting was 100 pm. The sorted cells were sorted into 100 pl HSM + 3% glucose media with 300 pg/ml carbenicil lin and 85 pg/ml cefotaxime. After 2 weeks of growth, single cells multiplied into large populations. The chlorophyll content was screened by visual colour. False positive mutant populations were discarded, chlorophyll deficient mutant populations were scaled up to a larger cell culture and validated by chlorophyll analysis.

Experimental protocol for mutant YC27:

Mutant YC27 was isolated by mutating 4TC3/16 and screening via flow cytometry (see Figure 2). 4TC3/16 was grown to exponential phase in FERM media in the dark, at 28°C and with 120 rpm agitation. Cells were concentrated to 1x10⁹ cells per ml, in 5ml of HSM +1% glucose media. A 50 pl aliquot of MMS was added and the culture was incubated for 1 hour. After quenching the mutagenesis with the addition of 30% (final concentration 5%) sodium thiosulphate, the cells were washed 3 times and left to recover in HSM +3% glucose media for 6 days and sub-cultured. After 4 days, when the culture had reached mid-exponential phase growth, cells were sorted by flow cytometry sorting (florescence activated cell sorting, FACS), using 488 nm and 561 nm lasers and 670±30 nm emission as previously described herein. Cells with low florescence intensity matching chlorophyll deficient control cell population were gated away from the main population and sorting into 96-well microplates, with 100 pl HSM + 3% glucose media with 300 pg/ml carbenicillin and 85 pg/ml cefotaxime. The cells were incubated in the same conditions described above. After 2 weeks chlorophyll deficient colonies, including YC27 were sub cultured into 25ml HSM + 3% glucose media for further validation.

YC27 isolated in this manner was yellow in colour, had a chlorophyll content of 0.15 mg/g, and a protein content of 45.9 % w/w.

Experimental protocol for mutant WC12

Mutant WC12 was isolated by mutating YC27 as previously described herein and screening via visual plate screening (see also Figure 2). YC27 was grown to exponential phase in FERM media, in the dark at 28°C and 120 rpm agitation. Cells were concentrated to 1x10⁹ cells per ml, in 1 ml of HSM +1 % glucose media. A 51 pl aliquot of EMS was added (0.5M final concentration) and the culture was incubated for 2 hours. After quenching the mutagenesis with the addition of 30% (final concentration 5%) sodium thiosulphate, the cells were washed 3 times and left to recover in HSM +3% glucose media. After 24 hours, the cells were plated on HSM + 3% glucose agar plates. An aliquot of 5000 cells per plated on each plate. After 3 weeks plates were visually screened, colonies with low levels of carotenoid, including WC12 were isolated and sub cultured into 25 ml HSM + 3% glucose for further validation of chlorophyll and carotenoid content. Cultures were incubated in the same conditions as described above.

WC12 isolated in this manner was white in colour, had a chlorophyll content of 0.04 mg/g, and a protein content of 45.2 % w/w.

Experimental protocol for mutants WCLS04, WCLS05 and WCLS06

Mutants WCLS04, WCLS05 and WCLS06 were isolated by mutating WC12 and screening via starch staining plate screening as follows (see also Figure 2). WC12 was grown to exponential phase in FERM media, in the dark at 28°C and 120 rpm agitation. Cells were concentrated to 1x10⁹ cells per ml, in 1 ml of HSM +1 % glucose media. A 51 pl aliquot of EMS was added (0.5M final concentration) and the culture was incubated for 1 hours. After quenching the mutagenesis with the addition of 30% (final concentration 5%) sodium thiosulphate, the cells were washed 3 times and left to recover in HSM +3% glucose media. After 24 hours, the cells were plated on HSM + 3% glucose agar plates. An aliquot of 5000 cells per plated on each plate. After 4 weeks plates were stained with iodine vapour for 1 .5 minutes. Colonies with low levels of stain compared to non-mutated controls and the majority of the mutant population were isolated and restreaked onto HSM + 3% glucose agar plates. After 2 weeks, the iodine staining was repeated to ensure consistent low staining. Single colonies were isolated and scaled up for starch and protein analysis. From these colonies WCLS04, WCLS05, WCLS06 were isolated.

WCLS04 isolated in this manner was white in colour, had a chlorophyll content of <0.03 mg/g, and a protein content of 56.2 % w/w.

WCLS05 isolated in this manner was white in colour, had a chlorophyll content of <0.03 mg/g, and a protein content of 54.1 % w/w.

WCLS06 isolated in this manner was white in colour, had a chlorophyll content of <0.03 mg/g, and a protein content of 58.7 % w/w.

Experimental protocol for isolation of mutant YC03:

First, mutant YC03 was isolated by mutating 4TC3/16 and screening via visual plate screening. 4TC3/16 was grown to exponential phase in HSM +1% glucose media in the light, at 25°C and with 120 rpm agitation. Cells were concentrated to 1x10⁹ cells per ml, in 1ml of HSM +1 % glucose media. A 51 pl aliquot of EMS was added (0.5M final concentration) and the culture was incubated for 2 hours. The cells were washed 3 times and left to recover in HSM +1 % glucose media in the dark, at 25°C and 120 rpm agitation. After 24 hours, the cells were plated on HSM + 1% glucose agar plates in aliquots of 5000 cells per plate. Plates were stored in the dark at 28°C. After 3 weeks plates were visually screened, colonies with low levels of chlorophyll, including YC03 were isolated and sub cultured into 25 ml HSM + 3% glucose for further validation of chlorophyll and carotenoid content. Cultures were maintained in the same conditions as described above.

YC03 isolated in this manner was yellow in colour, had a chlorophyll content of 0.05 mg/g, and a protein content of 35.95% w/w.

Experimental protocol for isolation of mutant WC03:

Mutant WC03 was isolated by mutating YC03 and screening via visual plate screening. YC03 was grown to exponential phase in FERM media, in the dark at 28°C and 120 rpm agitation. Cells were concentrated to 1x10⁹ cells per ml, in 1ml of HSM +1 % glucose media. A 10 pl aliquot of MMS was added and the culture was incubated for 1 hours. After quenching the mutagen with the addition of 30% (final concentration 5%) sodium thiosulphate, the cells were washed 3 times and left to recover in HSM +3% glucose media in the dark, at 25°C and 120 rpm agitation. After 24 hours, the cells were plated on HSM + 3% glucose agar plates in aliquots of 5000 cells per plate. After 3 weeks plates were visually screened, colonies with low levels of carotenoid and chlorophyll, including WC03 were isolated and sub cultured into 25 ml HSM + 3% glucose for further validation of chlorophyll and carotenoid content. Cultures were maintained in the same conditions as described above. WC03 isolated in this manner was white in colour, had a chlorophyll content of 0.001 mg/g, and a protein content of 34.5% w/w.

Chlorella vulgaris WC03 genome sequence analysis

There follows a description of the preferred method for genome sequencing and annotation of Chlorella microalgae. Optionally, the Chlorella microalgae is Chlorella vulgaris mutant strain WC03. However, the described method is suitable to identify genetic mutations in Chlorella microalgae in general. Genome sequencing of Chlorella vulgaris WC03 was performed using Illumina sequencing, resulting in a final genome assembly of 38.1 Mbp with an average GC content of 61.5%. A total of 10,542 genes were predicted and annotated via InterProScan and KEGG, with variant analysis identifying 25 mutations in WC03 compared to Chlorella vulgaris 4TC3, consisting of 11 SNPs and 14 INDELs.

An axenic culture of Chlorella vulgaris WC03 was cultivated from a thawed cryostock in FERM complete media comprising: glucose (111 mM), (NH4)2SO4 (47.7 mM), MgSO4.7H2O (2.8 mM), CaCI₂.2H₂O (204 pM), K₂HPO₄ (51.7 mM), NaH₂PO₄.H₂O (63.3 mM), KOH (40 mM), citric acid (8.8 mM), H3BO3 (1.1 mM), Na₂MoO₄ (32 pM), ZnSO₄.7H₂O (974 pM), MnSO₄.H₂O (958 pM), NiCI₂.6H₂O (11 pM), FeSO₄.7H₂O (79.1 pM), CuSO₄.5H₂O (8 pM), Thiamine hydrochloride (5.65 pM), Biotin (92.1 nM), Cyanocobalamin (13.3 nM), D-Pantothenic acid (205.3 nM), 4-Aminobenzoic acid (656.3 nM) at 28°C under heterotrophic conditions with shaking at 130 rpm. Cells were harvested after seven days by centrifugation at 13300 g for 5 minutes. DNA was extracted from Chlorella vulgaris WC03 biomass by mechanical bead-beating and TRIzol™ reagent. Extracted DNA was quantified via Nanodrop™ and DNA integrity was assessed by gel electrophoresis on a 1% agarose gel. Library preparation, including DNA fragmentation, adapter ligation, amplification and size selection was performed by Eurofins Genomics using proprietary methods. The quality of the final library was assessed by determination of size distribution and quantification, prior to sequencing on the Illumina NovaSeq 6000 platform using 2x150 sequence mode. Quality filtering of genetic data was performed by Eurofins Genomics using Illumina CASAVA software (95% of the bases with a quality of PH RED score 28 or better, no adapter trimming) prior to the reporting of raw reads. Adaptor sequences were removed with T rimmomatic (v0.38.0, Bolger et al., 2014; DOI: 10.1093/bioinformatics/btu170) and quality was checked after trimming using FastQC (vO.11.8, Andrews, 2010; online). Reference-based contig assembly was performed using SPAdes (v 3.12.0, Bankevich et al., 2012; DOI: 10.1089/cmb.2012.0021) with careful correction and automatic k-mer value detection, with the complete genome sequence of Chlorella vulgaris 4TC3 used as the reference. Coverage was assessed via Qualimap 2 (v 2.2.2, Okonechnikov et al., 2015; DOI: 10.1093/bioinformatics/btv566) and determined to be approximately 186 x coverage with 99.7% of reads mapping to the 4TC3 reference genome. The statistics of the final assembly are reported in Table 2.

Table 2: Features of the Chlorella vulgaris 4TC3 and WC03 genome assemblies. 4TC3 is a complete genome sequence, WC03 is a draft genome sequence; accounting for the difference in total length.

(*) WGS = whole genome sequence

The completeness of the genome assembly was further assessed by the single copy orthologs (BUSCO, v 5.2.2, Manni et a!., 2021 ; DOI: 10.1093/molbev/msab199) with WC03 being 95.7% complete and 1.4% partial genes of the 1519 belonging to the Chlorophyta dataset identified in WC03. Gene prediction was carried out by alignment of gene-models from Chlorella vulgaris 4TC3 with the WC03 genome assembly using Exonerate included in MAKER (v 2.31.11 , Cantarel et al., 2008; DOI: 10.1101/gr.6743907). The ab initio gene predictor Augustus (v 3.4.0, Stanke et al., 2004; DOI: 10.1093/nar/gkh379) was trained and a second round of gene prediction in the soft-masked genome was performed using the MAKER pipeline combining the homology-based predictions and ab initio gene prediction, with repeats identified via RepeatMasker (v 4.0.9, Smit et al., 2013; online) and Dfam (v 3.5, Storer et al., 2021 ; DOI: 10.1186/s13100-020-00230-y). A total of 10,542 genes were identified with BUSCO analysis (v 5.2.2, Manni et al., 2021) identifying 95.1 % complete and 2.4% partial genes of the 1519 belonging to the Chlorophyta dataset. For gene function annotation, protein-coding genes were translated into amino acid sequences via the MAKER-P pipeline and annotated using InterProScan (v 5.0.0, Blum et al., 2021 ; DOI: 10.1093/nar/gkaa977) against the TIGRFAM, Panther and PfamA databases. Proteins were also mapped against the KEGG database (v 101.0, Kanehisa et al., 2022; DOI: 10.1002/pro.4172). A total of 1571 genes were assigned to a metabolic pathway via KEGG analysis. To identify genetic variations in Chlorella vulgaris WC03, the assembled, annotated genome sequence was aligned to the reference genome of 4TC3 using BWA-MEM (v 0.7.17.2, Li and Durbin, 2009; DOI: 10.1093/bioinformatics/btp324). Sorting and dereplication were performed using Picard (v 2.26.10, Broad Institute, 2019) prior to base recalibration and variant calling using GATK BQSR and Haplotype Caller (v 4.1.3.0, Poplin et al., 2017; DOI: 10.1101/201178) with ploidy set to 1. Variant filtering was performed using GATK Select Variants and Variant Filtration tools. All variants were filtered with the Quality by Depth filter of < 2.0 and Quality < 30.0. Additional filtering was performed on SNPs with FisherStrand > 60.0 and RMSMappingQuality < 40.0, and on INDELs with FisherStrand > 200.0. SnpEff (v4.3, Cingolani et al., 2012; DOI: 10.4161 /fly.19695) was used to annotate and predict the effect of the variants on gene function. Functional information for the identified genes was obtained by InterProScan (v 5.0.0, Blum et al., 2021). A total of 25 mutations were identified, comprising 11 SNPs and 14 INDELs. Genetic variations in Chlorella vulgaris strains due to mutations identified herein are summarised in Table 3. The likelihood of a variant impacting the protein sequence and ascribed function of the protein and, therefore, potentially resulting in a phenotypic change is summarised in Table 4.

Table 3 - Genetic variations in Chlorella vulgaris strains due to mutations

1 . Single Nucleotide Polymorphism (SNP) or Insertion/deletion (INDEL).

2. Effect of the mutation on the protein sequence derived from the translation of this gene. Missense variants result in a change to a single amino acid, frameshift variants result in a disruption of the reading frame and the subsequent translation, intron variants affect the intron regions. 3. Likelihood of this variant impacting the protein sequence and therefore potentially resulting in a phenotypic change, see Table 4 for more details

4. Contig names and position numbers relate to the location on the WT 4TC3 genome assembly.

Table 4: Putative variant impact as identified via SnpEFF (Cingolani et al., 2012)

Where there is reference to specific mutations, the positions are described in reference to the Wild Type genome sequence. To identify these positions the mutant genome sequence is mapped against the relevant reference genome. The term “Contigs” refer to specific contigs of the reference genome (4TC3 for C. vulgaris, UTEX1230 for C. sorokiniana) and the “variant position” is specific to this contig (position numbers restart from 1 at each new contig). For example, a variant in C. vulgaris at Contig 11 position 69376 would be found in the variant sequences that align to Contig 11 of 4TC3, at the position located 69376 bases from the start of the sequence.

Genetic description of strains

There follows a genetic description of Chlorella vulgaris WC03 and various Chlorella vulgaris strains that form embodiments of the invention; having a chlorophyll content in a range 0.001-0.5 mg/g dry cell weight and a protein content of at least 50% w/w determined using this method. It will be appreciated that the skilled person could use this information to reproduce such strains and thereby, various embodiments of the invention without undue experimentation, by using direct gene-editing methods, (in addition to the mutagenesis methods described herein). Such suitable gene editing tools or methods that would be known to the person skilled in the art include, but are not limited to: genetic recombination, zinc finger nucleases, transcription activator-like effector nucleases (TALENS), CRISPR-Cas9 gene editing, base editing (e.g. using dCas9), prime editing (e.g. using pegRNA) or Programmable Addition via Site-specific Targeting Elements (PASTE). YC03 is characterised by mutations in the gene encoding magnesium chelatase, subunit I (Chi I) . It exhibits a reduction in chlorophyll in comparison to the WT strain 4TC3.

WC03 is characterised by mutations in genes encoding magnesium chelatase, subunit I (Chll; Sequence 2 (SEQ ID NO: 2)) and phytoene desaturase (Sequence 4 (SEQ ID NO: 4), EC: 1.3.5.5). It exhibits a reduction in chlorophyll in comparison to the WT strain 4TC3.

YC27 is characterised by mutations in the gene encoding magnesium-chelatase, subunit H (ChIH). It exhibits a reduction in chlorophyll in comparison to the WT strain 4TC3.

WC12 is characterised by mutations in the genes encoding magnesium-chelatase, subunit H (ChIH; Sequence 7 (SEQ ID NO: 7)), phytoene desaturase (Sequence 5 (SEQ ID NO: 5), EC:1.3.5.5), alcohol dehydrogenase (Sequence 9 (SEQ ID NO: 9)), and starch binding domain (Sequence 11 (SEQ ID NO: 11)). It exhibits a reduction in chlorophyll in comparison to the WT strain 4TC3. It also exhibits an increase in protein content and decrease in starch content compared to WT strain, 4TC3 and WC03.

WCLS04 is characterised by mutations in the genes encoding magnesium-chelatase, subunit ChIH (Sequence 7 (SEQ ID NO: 7)), phytoene desaturase (Sequence 5 (SEQ ID NO: 5), EC: 1.3.5.5), alcohol dehydrogenase (Sequence 9 (SEQ ID NO: 9)) starch binding domain gene (Sequence 11 (SEQ ID NO: 11)), glycogen phosphorylase (Sequence 13 (SEQ ID NO: 13), EC:2.4.1.1) and cellulose synthase (UDP-forming) (Sequence 15 (SEQ ID NO: 15), EC:2.4.1.12). It exhibits a reduction in chlorophyll in comparison to the WT strain 4TC3. It also exhibits an increase in protein and decrease in starch compared to 4TC3, and WC12.

WCLS05 is characterised by mutations in the genes encoding magnesium-chelatase, subunit ChIH (Sequence 7 (SEQ ID NO: 7)), phytoene desaturase (Sequence 5 (SEQ ID NO: 5), EC: 1.3.5.5), alcohol dehydrogenase (Sequence 9 (SEQ ID NO: 9)) starch binding domain gene (Sequence 11 (SEQ ID NO: 11)), isoamylase (Sequence 17 (SEQ ID NO: 17), EC:3.2.1.68) and glucose-6-phosphate isomerase (Sequence 19 (SEQ ID NO: 19)). It exhibits a reduction in chlorophyll in comparison to the WT strain 4TC3. It also exhibits an increase in protein and decrease in starch compared to 4TC3 and WC12.

WCLS06 is characterised by mutations in the genes encoding magnesium-chelatase, subunit ChIH (Sequence 7 (SEQ ID NO: 7)), phytoene desaturase (Sequence 5 (SEQ ID NO: 5), EC: 1.3.5.5), alcohol dehydrogenase (Sequence 9 (SEQ ID NO: 9)) starch binding domain gene (Sequence 11 (SEQ ID NO: 11)), glucose-6-phosphate isomerase (Sequence 21 (SEQ ID NO: 21)), and trehalose 6-phosphate synthase (Sequence 23 (SEQ ID NO: 23), EC 3.1.3.12). It also exhibits an increase in protein and decrease in starch compared to 4TC3 and WC12. The whole genome sequence of WC06 is Sequence 63 (SEQ ID NO: 63). Chlorophyll, protein and starch content

Table 5 shows chlorophyll content in chlorophyll deficient colour variants of Chlorella vulgaris as compared to the chlorophyll content produced in wild-type cells for the parental strain (4TC3/16) and a comparative, well characterised culture collection strain of Chlorella vulgaris (CCAP 211/11b) cultivated under the same conditions. The relative amounts of chlorophyll a, chlorophyll b and total chlorophyll calculated are represented in mg/g DCW when grown under heterotrophic conditions using glucose as the primary carbon source, in accordance with various embodiments of the present disclosure.

Table 5: Chlorophyll content (chlorophyll a + chlorophyll b = total chlorophyll; mg/g DCW) in chlorophylldeficient colour variants of Chlorella vulgaris, compared to WT 211/11 b and 4TC3/16

Table 6 shows protein content in chlorophyll deficient colour variants of Chlorella vulgaris as compared to the protein content produced in wild-type cells for the parental strain (4TC3/16) cultivated under the same conditions. The relative amounts of protein calculated (N • 6.25) are represented in g/100g DCW when grown under heterotrophic conditions using glucose as the primary carbon source, in accordance with various embodiments of the present disclosure.

Table 6. Protein content (g/100g DCW; DUMAS N • 6.25) in chlorophyll deficient colour variants of Chlorella vulgaris as compared to the protein content produced in wild-type cells for the parent, WT strain (4TC3/16)

Table 7 shows starch content in chlorophyll deficient colour variants of Chlorella vulgaris having a protein content of more-than or equal-to 50% (w/w) as compared to the starch content produced in wild-type cells for the parental strain (4TC3/16) and a comparative, well characterised culture collection strain of Chlorella vulgaris (CCAP 211/11 b) cultivated under the same conditions. The relative amounts of starch calculated are represented in g/100g DCW when grown under heterotrophic conditions using glucose as the primary carbon source, in accordance with various embodiments of the present disclosure.

Table 7: Starch content (g/100g DCW) in chlorophyll deficient colour variants of Chlorella vulgaris as compared to the starch content produced in wild-type cells for the parent, WT strain (4TC3/16)

Chlorella sorokiniana:

Genetically identifying Chlorella sorokiniana using PCR amplification

Wild-type Chlorella sorokiniana LITEX 1230 was obtained from LITEX Culture Collection of Algae at UT-Austin, Texas, USA. It was genetically verified in-house by 18S and ITS2 sequencing as described above.

Experimental protocol for isolation of Chlorella sorokiniana CS04 (YSK04):

Strain CS04 was isolated by mutating Chlorella sorokiniana UTEX1230 and screening via visual plate screening. UTEX1230 was grown to exponential phase in FERM media. The cell concentration was adjusted to 0.5x10⁹ cells per ml in a 0.5 mL volume of their respective growth media. Mutagenesis was performed using 0.5M EMS and incubating for 1 hour. After quenching the mutagenesis with the addition of 30% (final concentration 5%) sodium thiosulphate, the cells were washed 3 times and left to recover in FERM media for 2 days.

After the recovery period, the cells were plated on HSM media enriched with 1% glucose and vitamins (HSM 1 GV) agar plates in aliquots of 500 cells per plate and incubated at 25 °C in the dark. After 1-2 weeks plates were visually screened, and colonies with a yellow phenotype, including CS04, were isolated. From these plates, yellow UTEX1230 mutants were isolated, initially cultured in 500 pL snap cap tubes, and later transferred to 20 mL FERM media in 50 mL flasks for further growth. Over a period of 2-3 weeks, these mutants predominantly maintained a yellow coloration. One particular yellow mutant was identified and named CS04.

CS04 isolated in this manner was yellow in colour, had a chlorophyll content of 0.47 mg/g, and a protein content of 33.8 % w/w.

Experimental protocol for isolation of Chlorella sorokiniana strains CS10 (WSK04) and CS11 (WSK05), OS 12 (WSK06):

Mutants CS10, CS11 and CS12 were isolated by mutating CS04 and screening via starch- stained plate screening, as follows. CS04 was grown to exponential phase in FERM media, in the dark at 28°C and 120 rpm agitation. Cells were concentrated to 1x10⁹ cells per ml, in 1 ml of HSM +1% glucose media. A 51 pl aliquot of EMS was added (0.5M final concentration) and the culture was incubated for 1 hours. After quenching the mutagenesis with the addition of 30% (final concentration 5%) sodium thiosulphate, the cells were washed 3 times and left to recover in HSM +3% glucose media. After 24 hours, the cells were plated on HSM + 3% glucose agar plates. An aliquot of 5000 cells per plated on each plate. After 4 weeks plates were stained with iodine vapour for 1.5 minutes (using the iodine granule method; as described herein above). Colonies with low levels of stain compared to non-mutated controls and the majority of the mutant population were isolated and restreaked onto HSM + 3% glucose agar plates. After 2 weeks, the iodine staining was repeated to ensure consistent low staining. Single colonies were isolated and scaled up for starch and protein analysis. From these colonies CS10, CS11 and CS12 was isolated.

CS10 isolated in this manner was white in colour, had a chlorophyll content of <0.07 mg/g, and a protein content of 51.32 % w/w.

CS11 isolated in this manner was white in colour, had a chlorophyll content of <0.07 mg/g, and a protein content of 50.10 % w/w.

CS12 isolated in this manner was white in colour, had a chlorophyll content of <0.07 mg/g, and a protein content of 51 .67 % w/w. Experimental protocol for isolation of Chlorella sorokiniana CS107:

Strain CS107 was isolated by mutating CS04 and screening via visual plate screening. CS04 was grown to exponential phase in FERM media, in the dark at 28 °C and 120 rpm agitation. Cells were concentrated to 1x10⁹ cells per mL, in FERM media and incubated in the presence of 0.5M EMS for 1 hour. After quenching the mutagen with the addition of 30% (final concentration 5%) sodium thiosulphate, the cells were washed 3 times and left to recover in % FERM media in the dark, at 25 °C and 120 rpm agitation. After 24 hours, the cells were plated on FERM agar plates in aliquots of 1000 cells per plate and incubated at 25 °C in the dark. After 2 weeks plates were visually screened, and colonies with a white phenotype, including CS107, were isolated and sub cultured into 25 mL FERM for further validation of chlorophyll and carotenoid content. Cultures were maintained in the same conditions as described above.

CS107 isolated in this manner was white in colour, had a chlorophyll content of 0.074 mg/g, and a protein content of 43 % w/w.

Experimental protocol for isolation of Chlorella sorokiniana CS172:

Strain CS172 was isolated by mutating CS107 and screening via visual plate screening. CS107 was grown to exponential phase in FERM media, in the dark at 28 °C and 120 rpm agitation. Cells were concentrated to 1x10⁹ cells per mL, in FERM media and incubated in the presence of 0.5M EMS for 1 hour. After quenching the mutagen with the addition of 30% (final concentration 5%) sodium thiosulphate, the cells were washed 3 times and left to recover in % FERM media in the dark, at 32 °C and 120 rpm agitation. After 24 hours, the cells were plated on FERM agar plates in aliquots of 700 cells per plate and incubated at 32 °C in the presence of 20% CO2, in the dark. After 3 weeks plates were visually screened and colonies with a white phenotype, including CS172, were isolated and sub cultured into 25 mL FERM for further validation of chlorophyll and carotenoid content. Cultures were maintained in the same conditions as described above.

CS172 isolated in this manner was white in colour, had a chlorophyll content of 0.054 mg/g, and a protein content of 31 .9 % w/w.

Experimental protocol for isolation of Chlorella sorokiniana CS174:

Strain CS174 was isolated by mutating CS172 and screening via starch staining plate screening as previously described herein. CS172 was grown to exponential phase in FERM media, in the dark at 28 °C and 120 rpm agitation. Cells were concentrated to 1x10⁹ cells per ml, in FERM media and incubated in the presence of 0.5M EMS for 1 hour. After quenching the mutagen with the addition of 30% (final concentration 5%) sodium thiosulphate, the cells were washed 3 times and left to recover in % FERM media in the dark, at 32 °C and 120 rpm agitation. After 24 hours, the cells were plated on FERM agar plates in aliquots of 1000 cells per plate and incubated at 28 °C in the dark. After 1 week, plates were visually screened and colonies with a white phenotype, including CS174, were isolated and sub cultured into snap cap tubes containing 10 mL 1/4 FERM. Colonies were incubated in the dark, at 32 °C and 120 rpm agitation for 72 hours. 1 OD (750 nm) of cells from each colony was resuspended and then replica-plated (in 100 uL spots) onto FERM agar plates. Spots were directly stained with iodine to identify mutant strains exhibiting “low starch” phenotypes; such strains were isolated and scaled up for starch and protein analysis. One such strain was CS174.

CS174 isolated in this manner was white in colour, had a chlorophyll content of 0.052 mg/g, and a protein content of 59 % w/w.

Experimental protocol for isolation of Chlorella sorokiniana CS09 (WSK03):

Mutant CS04 was isolated through mutagenesis of Chlorella sorokiniana LITEX 1230 and subsequent flow cytometry-based selection. Herein follows a description of the preferred method for isolating chlorophyll-deficient Chlorella microalgae strains using flow cytometry cell sorting. In this example, the chlorophyll-deficient Chlorella strain is CS04, a mutant of Chlorella sorokiniana LITEX 1230. However, the described method is suitable for isolating chlorophyll-deficient mutants of Chlorella microalgae in general.

Exponential phase LITEX 1230 cells were concentrated to 1x10^A9 cells per ml in HSM3GV + 3% glucose media. An aliquot of 0.5M Ethyl Methanesulfonate (EMS) was added to the cell solution at a final concentration of 0.12M (10 pl per 1 ml). The cell mixture was incubated for 1 hour in the dark. A 30% sodium thiosulphate solution was added to the cell mixture to a final concentration of 5% sodium thiosulphate. The culture was incubated for 10 minutes. The cells were washed three times in 25 ml of HSM + 1% glucose. The cells were resuspended in 25 ml HSM + 3% glucose and left to recover for 24 hours in the dark, at 28°C, with agitation at 120 rpm.

After the recovery period, the cells were sub-cultured into fresh media, further incubated, and sampled during mid-exponential phase growth (between 5x10^A6 cells/ml and 5x10^A7 cells/ml). An initial analysis of 100,000 cells was conducted using a BD FACSAria Fusion (Becton Dickinson, USA) or Bigfoot Spectral Cell Sorter (Thermo Fisher Scientific, USA) to detect their fluorescence properties. In addition to the mutant pool, wild-type cells and chlorophyll-deficient mutants (isolated by alternative methods or previous flow cytometry isolation) were analysed. These single-strain cells were used as controls to design gates to sort populations.

First, using the forward and side scatter (FSC and SSC parameters) of the excitation laser, healthy single cells were gated, excluding cell debris and clumped or dividing cells. The chlorophyll content of cells was analysed by measuring the emission fluorescence intensity at 695±40 (or 670±30) after excitation from 488nm and 561 nm lasers. Gates were designed based on the fluorescence properties of the control cells. The gates were drawn to instruct the flow cytometry sorter to isolate cells that have fluorescence properties within the range of fluorescent intensities which match the chlorophyll-deficient control population, excluding the wild-type chlorophyll-replete control cell population.

Using live data and prepared gates, cells with the desired chlorophyll-deficient fluorescent properties were sorted away from a stream of a mixed mutant population into a tube or microplate well. The nozzle tip size used for sorting was 100 pm. The sorted cells were sorted into 100 pl HSM + 3% glucose media with 300 pg/ml carbenicillin and 85 pg/ml cefotaxime, then plated onto HSM + 3% glucose plates on the same day. After 2 weeks chlorophyll deficient colonies, including YC27 were sub cultured into 25ml HSM + 3% glucose media for further validation. One particular mint-white mutant was identified and named CS09.

CS09 isolated in this manner was mint-white in colour, had a chlorophyll content of 0.11 mg/g, and a protein content of 36.7 % w/w.

Experimental protocol for isolation of Chlorella sorokiniana strains WSK03/VLS7 (CSV), WSK03/VLS19 (CS20), WSK06/VW19 (CS23) and WSK06/VW17 (CS24)

Mutants CSV, CS20, CS23 and CS24 were isolated by mutating CS09 and screening via starch- stained plate screening, as follows. Cs09 was grown to exponential phase in FERM media, in the dark at 28°C and 120 rpm agitation. Cells were concentrated to 1x10⁹ cells per ml, in 1ml of HSM +1 % glucose media. A 51 pl aliquot of EMS was added (0.5M final concentration) and the culture was incubated for 1 hours. After quenching the mutagenesis with the addition of 30% (final concentration 5%) sodium thiosulphate, the cells were washed 3 times and left to recover in HSM +3% glucose media. After 24 hours, the cells were plated on HSM + 3% glucose agar plates. An aliquot of 5000 cells per plated on each plate. After 4 weeks plates were stained with iodine vapour for 1.5 minutes (using the iodine granule method; as described herein above). Colonies with low levels of stain compared to non-mutated controls and the majority of the mutant population were isolated and restreaked onto HSM + 3% glucose agar plates. After 2 weeks, the iodine staining was repeated to ensure consistent low staining. Single colonies were isolated and scaled up for starch and protein analysis. From these colonies CSV, CS20, CS23 and CS24 were isolated.

CSV isolated in this manner was white in colour, had a chlorophyll content of <0.17 mg/g, and a protein content of 56.37 % w/w.

CS20 isolated in this manner was white in colour, had a chlorophyll content of <0.17 mg/g, and a protein content of 55.50 % w/w. CS23 isolated in this manner was white in colour, had a chlorophyll content of <0.17 mg/g, and a protein content of 58.50 % w/w.

CS24 isolated in this manner was white in colour, had a chlorophyll content of <0.17 mg/g, and a protein content of 59.93 % w/w.

Experimental protocol for isolation of Chlorella sorokiniana CS73:

Strain CS73 was isolated by mutating Chlorella sorokiniana UTEX1230 and screening via flow cytometry. UTEX1230 was grown to exponential phase in FERM media. The cell concentration was adjusted to 1x10⁹ cells per mL in a 1 mL volume of their respective growth media. Mutagenesis was performed using 0.5M EMS and incubating for 1 hour. After quenching the mutagenesis with the addition of 30% (final concentration 5%) sodium thiosulphate, the cells were washed 3 times and left to recover in HSM1GV for 3 days and sub-cultured. After multiple subcultures, 13 days, when the culture had reached mid-exponential phase growth, cells were sorted by flow cytometry sorting florescence activated cell sorting (FACS), using 488 nm and 561 nm lasers and 670±30 nm emission as previously described herein.

Sorting gates were set using the distinction in autofluorescence between wild type and mutant yellow control cells, allowing for precise gating of chlorophyll-deficient mutants. New mutant yellow cells of UTEX1230 were sorted into HSM1GV. These cells were then incubated under the same conditions described above. After a recovery period, the cells were plated on HSM + 1% glucose media plates. From these plates, yellow UTEX1230 mutants were isolated, initially cultured in 500 pL snap cap tubes, and later transferred to 20 mL FERM media in 50 mL flasks for further growth. Over a period of 2-3 weeks, these mutants predominantly maintained a yellow coloration. One particular yellow mutant was identified and named CS73.

Experimental protocol isolation of Chlorella sorokiniana CS120:

Strain CS120 was isolated by mutating CS73 and screening via visual plate screening. CS73 was grown to exponential phase in FERM media, in the dark at 28 °C and 120 rpm agitation. Cells were concentrated to 1x10⁹ cells per mL, in 1 mL of HSM1GV and incubated in the presence of 0.5M EMS for 1 hour. After quenching the mutagen with the addition of 30% (final concentration 5%) sodium thiosulphate, the cells were washed 3 times and left to recover in HSM 1 GV in the dark, at 25 °C and 120 rpm agitation. After 24 hours, the cells were plated on HSM1GV agar plates in aliquots of 1000 cells per plate and incubated at 25 °C in the dark. After 3 weeks plates were visually screened and colonies with a white phenotype, including CS120, were isolated and sub cultured into 25 mL HSM 1 GV for further validation of chlorophyll and carotenoid content. Cultures were maintained in the same conditions as described above. Genomic sequence analysis of Chlorella sorokiniana strains

There follows a description of the preferred method for genome sequencing and annotation of Chlorella microalgae. Genome sequencing of Chlorella sorokiniana LITEX 1230 was performed by (Hovde etal., 2018; DOI: 10.1016/j. algal.2018.09.012) using Illumina and PacBio sequencing, resulting in a final genome assembly of 58.5 Mbp with an average GC content of 63.8%. The genome sequence of C. sorokiniana LITEX 1230 is publicly available to download from the National Center for Biotechnology Information database under bioproject PRJNA422912, genome assembly ASM313072v1.

The statistics of the final genome assemblies are reported in Table 2 and Table 8 for the wild-type strains of C. vulgaris and C. sorokiniana, respectively.

Gene prediction was carried out by the inventors via alignment of gene-models from C. sorokiniana LITEX 1230 (Blake et al., 2018; DOI: 10.1016/j. algal.2018.09.012) using Exonerate included in MAKER (v 2.31.11, Cantarel et al., 2008; DOI: 10.1101/gr.6743907).

The ab initio gene predictors Augustus (v 3.4.0, Stanke et al., 2004; DOI: 10.1093/nar/gkh379) and Snap (v 2013_11_29, http://korflab.ucdavis.edu/software.html) were trained and a second round of gene prediction in the soft-masked genome was performed using the MAKER pipeline combining the homology-based predictions and ab initio gene prediction, with repeats identified via RepeatMasker (v 4.0.9, Smit et al., 2013; online) and Dfam (v 3.5, Storer et al., 2021 ; DOI: 10.1186/s13100-020-00230- y). A total of 13,021 genes were identified with BLISCO analysis (v 5.2.2, Manni et al., 2021) identifying 96% complete and 1.9 % partial genes of the 1519 belonging to the Chlorophyta dataset. For gene function annotation, proteincoding genes were translated into amino acid sequences via the MAKER-P pipeline and annotated using InterProScan (v 5.0.0, Blum etal., 2021; DOI: 10.1093/nar/gkaa977) against the TIGRFAM, Panther and PfamA databases. Proteins were also mapped against the KEGG database (v 101.0, Kanehisa et al., 2022; DOI: 10.1002/pro.4172). A total of 1822 genes were assigned to a metabolic pathway via KEGG analysis.

There follows a description of the preferred method to identify genetic variations in Chlorella sorokiniana mutant strains by genome sequencing and annotation. Optionally, the mutant strain of Chlorella sorokiniana microalgae is Chlorella sorokiniana CS172. However, the described method is suitable to identify genetic mutations in Chlorella microalgae in general.

Genome sequencing of Chlorella sorokiniana CS172 was performed using Nanopore sequencing, resulting in a final genome assembly of 57.4 Mbp with an average GC content of 63.85%.

An axenic culture of C. sorokiniana CS172 was cultivated from a thawed cryostock in FERM complete media at 28°C under heterotrophic conditions with shaking at 130 rpm. Cells were harvested after four days by centrifugation at 13300 x g for 5 minutes. DNA was extracted from C. sorokiniana CS172 biomass using the Quick-DNA Plant/Seed Miniprep Kit (Zymo).

Extracted DNA was quantified via Nanodrop(RTM) and DNA integrity was assessed by gel electrophoresis on a 1 % agarose gel. Library preparation was performed using the Ligation Sequencing Kit V14 (SQK-LSK114, Oxford Nanopore) according to the manufacturer’s instructions and sequenced on the MinlON using a R10.4.1 flow cell. Raw fast5 files were basecalled using Guppy (v 6.5.7, 400bps) and sequences with a Q score >9 and longer than 200bp were used for analysis. Assembly was performed using Flye (v 2.9.1 , Lin et al., 2016; DOI :10.1073/pnas.1604560113) with one round of polishing. The statistics of the final assembly are reported in Table 8.

Table 8: Features of the C. sorokiniana UTEX1230 and CS172 genome assemblies.

(*) WGS = whole genome sequence

The completeness of the genome assembly was further assessed by the single copy orthologs (BUSCO, v 5.2.2, Manni et al., 2021 ; DOI: 10.1093/molbev/msab199) with CS172 containing 99.2% complete and 0.3% partial genes of the 1519 belonging to the Chlorophyta dataset.

Sequences were mapped against the published UTEX 1230 genome using Minimap2 (v 2.26, -ax map-ont -L), read groups added using Picard (v 2.26.10), reads sorted and indexed using samtools (v 1.18) and variants called using Medaka (v 1.7.2, model r1041_e82_400bps_fast_g615). Variants were filtered using SnpSift (v 4.3, QUAL > 30, DP > 2, MQ > 40) and SnpEff (v4.3, Cingolani et al., 2012) was used to annotate and predict the effect of the variants on gene function. Functional information for the identified genes was obtained by InterProScan (v 5.0.0, Blum et al., 2021).

Where there is reference to specific mutations, the positions are described in reference to the Wild Type genome sequence. To identify these positions the mutant genome sequence is mapped against the relevant reference genome. The term “Contigs” refer to specific contigs of the reference genome (4TC3 for C. vulgaris, UTEX1230 for C. sorokiniana) and the “variant position” is specific to this contig (position numbers restart from 1 at each new contig). For example, a variant in C. vulgaris at Contig 11 position 69376 would be found in the variant sequences that align to Contig 11 of 4TC3, at the position located 69376 bases from the start of the sequence.

Genetic variations in Chlorella sorokiniana strains due to mutations identified herein are summarised in Table 9. The likelihood of a variant impacting the protein sequence and ascribed function of the protein and, therefore, potentially resulting in a phenotypic change is summarised in Table 4.

Table 9 - Genetic variations in Chlorella sorokiniana strains due to mutations

1. Single Nucleotide Polymorphism (SNP) or Insertion/deletion ( NDEL).

2. Effect of the mutation on the protein sequence derived from the translation of this gene. Missense variants result in a change to a single amino acid, frameshift variants result in a disruption of the reading frame and the subsequent translation, intron variants affect the intron regions. 3. Likelihood of this variant impacting the protein sequence and therefore potentially resulting in a phenotypic change, see Table 4 for more details

4. Contig names and position numbers relate to the location on the LITEX 1230 genome assembly.

Genetic description of Chlorella sorokiniana strains CS04, CS10, CS12, CS120, CS107, CS172 and CS174

There follows a genetic description of Chlorella sorokiniana CS172, CS174, and various Chlorella sorokiniana strains described herein. It will be appreciated that the skilled person could use this information to reproduce such strains and thereby, various embodiments of the invention without undue experimentation, by using direct gene-editing methods, (in addition to the mutagenesis methods described herein). Such suitable gene editing tools or methods that would be known to the person skilled in the art include, but are not limited to: genetic recombination, zinc finger nucleases, transcription activator-like effector nucleases (TALENS), CRISPR-Cas9 gene editing, base editing (e.g. using dCas9), prime editing (e.g. using pegRNA) or Programmable Addition via Site-specific Targeting Elements (PASTE).

Geranylgeranyl diphosphate synthase (EC 2.5.1.29) is an enzyme required to the synthesis of Geranylgeranyl diphosphate (GGPP), which is the precursor for the biosynthesis of carotenoids and chlorophylls. Phytoene desaturase (EC 1.3.5.5) is an enzyme essential to the carotenoid biosynthesis pathway and controls the conversion phytoene into lycopene. Magnesium chelatase (EC 6.6.1.1) is an enzyme that catalyses the first committed step of the chlorophyll synthesis pathway; being the insertion of Mg²⁺ into protoporphyrin IX. Magnesium chelatase is a highly-conserved enzyme composed of three subunits: Chll, ChlD, and ChlH. The subunits are postulated to have distinct roles in forming the catalytical ly-active holoenzyme that, ultimately, performs the magnesium chelation reaction; broadly, Chll and ChlD are thought to form an ATP- associated complex, while ChlH binds to the magnesium ion, leading to the formation of the active Mg chelatase holoenzyme (Xhang et al. 2018; DOI: 10.3389/fpls.2018.00720).

CS04 (YSK04) is characterised by mutations in the genes encoding magnesium- chelatase, subunit H (ChlH; Sequence 24 (SEQ ID NO: 24)). It exhibits a reduction in chlorophyll in comparison to the WT strain LITEX 1230.

CS107 is characterised by mutations in the genes encoding magnesium-chelatase, subunit H (ChlH; Sequence 25 (SEQ ID NO: 25)) and geranylgeranyl diphosphate synthase (Sequence 26 (SEQ ID NO: 26)). It exhibits a reduction in chlorophyll in comparison to the WT strain UTEX 1230.

CS172 (whole genome: Sequence 64 (SEQ ID NO: 64)) is characterised by mutations in the genes encoding magnesium-chelatase, subunit H (ChlH; Sequence 25 (SEQ ID NO: 25)), geranylgeranyl diphosphate synthase (Sequence 26 (SEQ ID NO: 26)), and phytoene desaturase (Sequence 27 (SEQ ID NO: 27)). It exhibits a reduction in chlorophyll in comparison to the WT strain UTEX 1230. CS174 is characterised by mutations in the genes encoding magnesium-chelatase, subunit H (ChIH; Sequence 25 (SEQ ID NO: 25)), geranylgeranyl diphosphate synthase (Sequence 26 (SEQ ID NO: 26)), and phytoene desaturase (Sequence 27 (SEQ ID NO: 27)). It exhibits a reduction in chlorophyll in comparison to the WT strain LITEX 1230.

CS10 (WSK04) is characterised by mutations in genes encoding magnesium chelatase, subunit H (ChIH; Sequence 24 (SEQ ID NO: 24)) and phytoene desaturase (EC:1.3.5.5). It exhibits a reduction in chlorophyll in comparison to the WT strain LITEX 1230.

CS12 (WSK06) is characterised by mutations in the genes encoding magnesium- chelatase, subunit H (ChIH; Sequence 24 (SEQ ID NO: 24)) and phytoene desaturase (Sequence 28 (SEQ ID NO: 28), EC: 1.3.5.5). It exhibits a reduction in chlorophyll in comparison to the WT strain UTEX 1230.

CS120 is characterised by mutations in the genes encoding magnesium-chelatase, subunit I (Chll; Sequence 66 (SEQ ID NO: 66)) and phytoene desaturase (Sequence 65 (SEQ ID NO: 65), EC: 1.3.5.5). It exhibits a reduction in chlorophyll in comparison to the WT strain UTEX 1230.

Properties of Chlorella sorokiniana strains

Table 10 shows chlorophyll content in chlorophyll deficient colour variants of Chlorella sorokiniana as compared to the chlorophyll content produced in wild-type cells for the parental strain (UTEX1230), which is a comparative, well characterised culture collection strain of Chlorella sorokiniana cultivated under the same conditions. The relative amounts of chlorophyll a, chlorophyll b and total chlorophyll calculated are represented in mg/g DCW when grown under heterotrophic conditions using glucose as the primary carbon source, in accordance with various embodiments of the present disclosure.

Table 10: Chlorophyll content (chi a + chi b = total chi; mg/g DCW) in chlorophyll-deficient colour variants of Chlorella sorokiniana, compared to WT UTEX1230

Table 11 shows protein content in chlorophyll deficient colour variants of Chlorella sorokiniana as compared to the protein content produced in wild-type cells for the parental strain (UTEX1230), which is a comparative, well characterised culture collection strain of Chlorella sorokiniana cultivated under the same conditions. The relative amounts of protein calculated (N • 6.25) are represented in g/100g DCW when grown under heterotrophic conditions using glucose as the primary carbon source, in accordance with various embodiments of the present disclosure.

Table 11 : Protein content (g/100g DCW; DUMAS N • 6.25) in chlorophyll deficient colour variants of Chlorella sorokiniana as compared to the protein content produced in wild-type cells for the parent, WT strain (UTEX1230)

Table 12: Starch content (g/100g DCW) in chlorophyll deficient colour variants of Chlorella sorokiniana as compared to the starch content produced in wild-type cells for the parent, WT strain (UTEX 1230)

Table 12 shows starch content in chlorophyll deficient colour variants of Chlorella sorokiniana having a protein content of more-than or equal-to 50% (w/w) as compared to the starch content produced in wild-type cells for the parental strain (UTEX1230) cultivated under the same conditions. The relative amounts of starch calculated are represented in g/100g DCW when grown under heterotrophic conditions using glucose as the primary carbon source, in accordance with various embodiments of the present disclosure.

Identifying and isolating variant strains of Chlorella microalgae with reduced chitin content: The recovered mutant strains were resuspended in a phosphate-buffered saline, stained with calcofluor white (CFW) fluorescent dye and sorted by using flow cytometry according to their fluorescence shift at 380 nm and compared to the parent strain of microalgae (namely, control). A single suspension of cells was prepared, effectively stained and allowed to flow through the flow cytometer in a single flow through the light beam for sensing. The laser was used to elicit strong chitin autofluorescence from a mixture of viable cells. The dye-specific autofluorescence signals were analysed by a computer physically connected to the flow cytometer. The population of cells that exhibited strong autofluorescence was sorted away from those cells that had null or significantly reduced signal as an enrichment step to enrich for those cells within the total population that had accumulated mutations that knocked down or abolished the chitin content signal. This step was applied between 2-7 days post-exposure to mutagen and in liquid culture.

Calcofluor white (CFW) staining: Typically, before staining, a stock solution of CFW was prepared by adding 35 milligram (mg) of CFW to 7 millilitre (ml) of sterile distilled water, and a few drops of 10 Normal (N) sodium Hydroxide (NaOH) was added to increase the pH of said stock solution to 10 to 11 and increase solubility of CFW in sterile distilled water. A final stock solution (adjusted to 10 ml with addition of sterile distilled water) was divided into small aliquots (for example, of 150 microlitre (pl), and stored in the dark at a temperature of -20°C until use. Alternatively, the CFW stock solution was obtained from the manufacturer at a premade concentration of 0.1 % w/v and diluted to a final staining concentration of 25pg/mL before use. Rapid staining with one or two drops of 0.1% CFW can be used to stain a sample, such as the microalgae strains, for detection of chitin in the cell wall. Samples are incubated for a time period of, for example, 1 minute or less before performing analysis.

Cell sorting using Flow Cytometry for the isolation of Chlorella microalgae mutants reduced chitin content: The sample containing cells or particles was suspended in a fluid and injected into a flow cytometer instrument. The flow rate was controlled to allow one cell at a time to flow through a laser beam (355 nm excitation and either 447_60 nm or 450_50 emission filter) where the light that is scattered or emitted as fluorescence is characteristic of cells and components thereof.

The flow cytometry gates were set to capture 'events' that lay outside the variance of the parent strain of Chlorella microalgae, i.e. the control (no events in control). Three gates were captured (P4, P5, P6), with increasing distance from the parent strain of Chlorella microalgae population (less fluorescence). Further, single cells from gates P5 and P6 were sorted into standard 24-well cell-culture plates containing mixotrophic growth medium containing glucose as the carbon source, and incubated further. The incubation period was typically around 1-3 weeks. At the end of said incubation period, viable isolated strains were obtained, as indicated by for example green-coloured or turbid cell cultures.

Sequence listings index

Sequences 1-28 (SEQ ID NO: 1-28), 65 (SEQ ID NO: 65) and 66 (SEQ ID NO: 66) are individual genes.

Sequences 29-62 (SEQ ID NO: 29-62) are the whole genome sequence of C. vulgaris 4TC3, split into the contigs that are used to describe the position of variants.

Sequences 63 (SEQ ID NO: 63) and 64 (SEQ ID NO: 64) are the whole genome sequences of individual mutant strains.

Example 2: Production of microalgal biomass powder

To produce Chlorella biomass, microalgal strains of Example 1 were cultivated at 100 I scale in a liquid fermentation medium. The fermentation medium (FERM as described herein) comprised: glucose (111 mM), (NH₄)₂SO₄ (47.7 mM), MgSO₄.7H₂O (2.8 mM), CaCI₂.2H₂O (204 pM), K₂HPO₄ (51.7 mM), NaH₂PO₄.H₂O (63.3 mM), KOH (40 mM), citric acid (8.8 mM), H3BO3 (1.1 mM), Na₂MoO₄ (32 pM), ZnSO₄.7H₂O (974 pM), MnSO₄.H₂O (958 pM), NiCI₂.6H₂O (11 pM), FeSO₄.7H₂O (79.1 pM), CuSO₄.5H₂O (8 pM), Thiamine hydrochloride (5.65 pM), Biotin (92.1 nM), Cyanocobalamin (13.3 nM), D-Pantothenic acid (205.3 nM), 4-Aminobenzoic acid (656.3 nM) and the microalgae were grown at 29°C to a cell density of between 50-100 g/l.

The microalgae biomass (e.g. flour) can be produced under current Good Manufacturing Practice (cGMP) conditions using any method known in the art. Typically, the procedure begins with the seed train phase, where a cryovial containing the microalgae culture is used to inoculate a series of increasing media volumes, culminating in a prepared inoculum for the fermentation phase. Fermentation is conducted in bioreactors with controlled environmental conditions.

Chlorella vulgaris algae biomass production

In a first example of the production of algae biomass, Chlorella vulgaris microalgae strain WC03 was cultivated at 100 L scale in a liquid fermentation medium (FERM as described herein), beginning with a 1.5 mL cryovial that was used to inoculate a 50 mL flask, incubated for 7 days. The culture was then expanded in volume to 500 mL for 5 days, followed by a 5 L vessel for 5 to 6 days of either batch or fed-batch fermentation. The target initial density of biomass for the 100 L fermenter, referred to as the initial after inoculation concentration (AIC), was equal to or greater than 3 g/L. The final 100L fermentation was closely controlled; pH and nitrogen requirements were managed by adding a 25% to 30% ammonia solution via the fermenter's pH control loop. The pH was maintained at 6.5. Glucose, the primary carbon source, was administered to maintain concentrations between 10 to 20 g/L, utilising either continuous feed or bolus additions to maintain this range. Temperature was maintained at 28 °C, airflow rates were set between 0.5 to 1.0 vvm to facilitate oxygen transfer, with a fixed stirrer speed for consistent mixing. The fermentation process was completed with the separation of the biomass, followed by drying and packaging.

Chlorella sorokiniana algae biomass production

In a second example of the production of algae biomass, Chlorella sorokiniana microalgae strain CS172 was cultivated at 100 L scale in a liquid fermentation medium (FERM as described herein), beginning with a 1 .5 mL cryovial inoculating a 500 mL flask for 3 days. This was followed by a 2 to 3-day batch or fed-batch fermentation in a 5 L vessel. The mature culture was then inoculated in a 100 L fermenter for a 1 .7 to 3-day fed-batch fermentation process, with a target AIC equal to or greater than 3 g/L. Glucose concentration in the fermentation broth was controlled within a 10 to 20 g/L range, and an ammonia solution at a 25% to 30% concentration was used to maintain pH at 6.5 and as a nitrogen feed. Temperature was maintained at 28 °C, airflow rates were set between 0.5 to 1.0 vvm to facilitate oxygen transfer, with a fixed stirrer speed for consistent mixing. Following fermentation, the biomass underwent the downstream process of separation, drying, and packaging as described above, to obtain the final product.

Downstream processing

For each of the described strains, microalgae powder was produced by a three-step downstream process (DSP) from algae biomass, which is the product of the heterotrophic fermentation process described herein above. The three-step DSP process comprised the following steps: washing, concentration and drying. The purpose of the washing and concentration step is to reduce the spent medium carryover to a value that does not impact the organoleptic characteristics of the dried product and to achieve a dry cell weight concentration that favours the performance of the drying unit operation. Specifically, the biomass was harvested, washed once using an equivalent volume of city water and concentrated using a nozzle centrifuge to increase the biomass concentration up to 200g/L. The biomass was then spray-dried using a stage spray drying system with external vibrating fluid bed in 15s @ 80°C, yielding the Chlorella biomass as a free-flowing bright white powder.

Quantification of the colour (CIELAB) of algal biomass (e.q. flour) and algal biomass (e.q flour)

Method

The colour of algal biomass (e.g. flour) and algal biomass (e.g. flour) resuspended in a specific volume of liquid at a specific solid %, can be quantified by a tristimulus colorimeter. The colorimeter works by quantifying the change in the intensity of electromagnetic radiation (within the visible wavelength spectrum 400 to 700 nm) after transmitting or reflectance; the absorbency of light waves, i.e the colour, can be measured. In a tristimulus, radiant power from a light source illuminates an object. The reflected or transmitted radiant power from the object channels is channelled through three independent tristimulus filters reaching a photo-detector. The response from the photodetector, is proportional to the corresponding tristimulus value of the object-source combination. This raw data is processed by a microprocessor for the computation of the absolute CIE tristimulus values. The values can be given as CIE LAB, XYZ, Lch, RGB and LUV.

In this instance the colour of Chlorella powder samples is measured using the PCE- CSM 2 colourimeter (https://www.pce-instruments.com/) according to the manufacturer's instructions. The device is calibrated only when first used, after significant environmental changes, after prolonged use or if results are inaccurate compared to a reference sample. To obtain the colour measurement, the powder compartment on the measuring plate is filled with Chlorella powder; overfilling without compacting, and the powder test box is assembled, ensuring a straight, snug screw joint and no air gaps under the glass in order to compact the powder and obtain an accurate measurement. The colorimeter is connected to a laptop using a USB cable and operated as per the manufacturer’s instructions, using the supplied software which records CIEXYZ and CIELAB values. The colorimeter's measuring hole is placed over the test box and the measurement taken by pressing the test button. Three measurements are recorded per sample to obtain an average measurement. Between samples, the powder test box assembly is disassembled, wiped with a dry cloth and a fresh sample loaded as described above. Post-use, the test box assembly is cleaned with 70% (v/v) ethanol, dried and stored appropriately.

For liquid samples, a 5% solution is prepared by mixing 0.5 g of powder in 10 mL of deionised water. The mixture is allowed to hydrate at room temperature for 10 minutes, using a homogeniser for 5 seconds at maximum speed if necessary to produce a uniform, hydrated suspension. Immediately before the measurement is taken, the solution is remixed, before transferring to the powder compartment of the measuring plate using a Pasteur pipette; overfilling as before and removing any foam or bubbles by pipette aspiration. The test box is then assembled, avoiding bubbles and ensuring a straight, snug screw joint, and any overspill is dried to ensure it does not come into contact with the colourimeter. The colorimeter is operated as instructed by the manufacturer; following the same procedure as described above for powder samples to obtain triplicate measurements measurement. In between liquid samples, the test box is disassembled, rinsed with tap water and dried. After use, the test box is cleaned with 70% (v/v) ethanol, dried and stored appropriately.

Results

A comparison table showing the L*a*b* values of dry, powdered biomass obtained from variants with a first mutation in at least one gene that encodes for phytoene desaturase or a subunit thereof, and i) a second mutation in at least one gene that encodes for subunit Chll of magnesium chelatase (Samples 2:1 , 2:2 and 2:3 - obtained from Chlorella vulgaris strain WC03); and ii) a second mutation in at least one gene that encodes for subunit ChIH of magnesium chelatase (Samples 2:4, 2:5 and 2:6 - obtained from Chlorella vulgaris strains WC12, WCLS04 and WCLS06, respectively); is shown below in Table 13 . Also included are the values for Comparative Sample 2:A, which is a commercially available C/7/ore//a-derived algae biomass (ex Alima, Portugal). There is a statistically-significant difference in AE values between strains with a MgCI subunit Chll and MgCI subunit ChIH mutation (two-tailed t.test, p<0.05, t= t = 3.89, d.f = 4). Samples 2:1 , 2:2, 2:3, 2:4, 2:5, and 2:6 were obtained by the methodology described above. Comparative Sample 2:A does not form part of the invention and was obtained commercially from a standard supplier.

Table 13 provides the L*a*b* values of the dried powder samples and their AE (a measure of the change in visual perception) relative to a pure white reference (L* 100, a* 0, b* 0). A two tail t-test was performed on the AE values comparing samples with the mutation in MgCI subunit Chll (group 1) against samples with the mutation in MgCI subunit ChIH (group 2), yielding a significant difference (p<0.05, t= t = 3.89, d.f = 4). Table 13: Colorimeter values of dried powder samples. AE is the measure of change in visual perception of two given colours, in this case against pure white reference L* 100, a* 0, b* 0.

A further comparison table showing the L*a*b* values of algae biomass according to the present invention is shown below in Table 14.

Table 14: Colorimeter values of dried powder samples.

Functional properties of Chlorella biomass

Modifying the ratio of protein and starch content in microalgae enabled the production of a range of microalgal biomass ingredients with different functional properties. For example, algal biomass with an increased protein and lower starch content (when compared to algae biomass produced from a strain with wild type genotype) resulted in a significant increase in apparent viscosity of an algae flour resuspended solution at a range of shear rates (Table 15). Furthermore, algal biomass with a high protein content has a higher emulsification capacity compared to microalgal biomass with a lower protein content (Table 15). For viscosity analysis, cracked algae biomass was resuspended in water (10% solids). Viscosity was analysed at a range of shear rates (within measurable torque range), using a rotatory viscosity meter (NDJ-9S) and reporting the mean (apparent) viscosities obtained over the measurable torque ranges. Viscosity was measured at 25°C. The biomass solution was heated to 85°C for 10 minutes, then left to cool to 25°C before measuring viscosity (heated treatment). For emulsion capacity analysis, cracked algae biomass was resuspended in water normalised to a 3% protein content, oil (Canola) was added at variable ratios, and the mixture was homogenised at 2000 rpm for 2 minutes. A positive conductivity measure indicated that the emulsions were oil in water (rather than water in oil) in a continuous phase. Values of 0 indicated the emulsion had collapsed. All emulsions were stable for over 5 days.

Table 15: Functional properties of microalgae biomass with different protein and starch content. Units in brackets represent standard error of the mean (SEM). Samples of whole cell (non-lysed) Chlorella biomass showed the same trends in viscosity and apparent viscosity as observed for the above samples of lysed biomass.

Part II - Food compositions

Method 1 : Structured Team Assessment

The sensory properties of food compositions were determined using a structured team assessment. The panellists were instructed to score the products on various attributes. The scoring system used to assess the differences is shown below:

The sensory scoring system

4 = same as the reference

5 = slightly higher than reference

6 = higher than reference

7 = significantly higher than reference

3 = slightly lower than reference

2 = lower than reference

1= significantly lower than reference

Sensory attributes are described in Table 16. Sensory scores which are good when higher than the reference are the flavour scores of dairy, milk, cheese, butter, cream flavour and creamy texture mouthfeel and in the taste score for mushroom soup the mushroom flavour and mushroom odour. Sensory scores which are better when lower than the reference are off- flavour odour and off-flavour taste, astringency and bitter taste.

Table 16

Method 2. Colour measurement of food compositions.

Digital images were made under controlled lighting using a DigiEye imaging system (VeriVide Ltd) that allows documenting the appearance by making colorimetrically accurate images, which are suitable for the measurement of colour uniformity, size, and shape.

The DigiEye lamps (D65) were switched on in angled mode, at least for 10 minutes before start of the calibration using the White Uniformity board and for the colour reference the DigiTizer calibration chart, both from VeriVide Ltd.

The colour is expressed using the CIELAB (L*a*b*) colour scale (colour space or model). This scale consists of a luminance or intensity component L* ranging from 0 (black) to 100 (white), along with two chromatic components a* (degree of redness if values are positive or greenness in negative values) and b* (degree of yellowness for positive values or blueness if values are negative). The difference between two Colours can be expressed as AE, the distance between two points in L*a*b* space and is calculated as follows:

AE = { (AL*)²+ (Aa*)²+ (Ab*)²} where:

AL* is calculated as L*_Sam_Pie - L* _REF,

Aa* is calculated as a*sample ^— a*REF,

Ab* is calculated as b*_sampie - b*_REF,

The AE value of 2.3 is generally considered to be just visible for the human eye (for homogeneously distributed colours).

Materials:

Microalgae biomass batches used are summarised in Table 17: Table 17

Each microalgae biomass material was used in the form of a free-flowing powder. Example 3 Carbonara sauce

As benchmark, a dairy containing dry mix for carbonara sauce was made and 2 non-dairy carbonara-like sauce powder mixes containing microalgae powder were prepared using the recipes listed in Table 18. To prepare the non-dairy sauces, 68.5 g of each dry preparation was added to 450g just boiled water in a pan, then mixed using a whisk and boiled for 2 min for a final product of approximately 500g (as about 20g evaporated during boiling). The sauce was transported in preheated thermos flasks and stored (0.5-1 hour) prior to tasting. For preparation of the reference, 129g dairy-based sauce-powder was added to 900g just boiled water, mixed using a whisk while boiling the sauce for 2 min and then stored in a thermos flask. The sauces were tasted without pasta. The taste results are given in Table 19.

Table 18 Formulations.

Table 19 The mean taste scores of 6 panellists.

The taste scores show that the best scored sample is example 3:1 , the non-dairy sauce with White Chlorella vulgaris WCLS06, which scored close to the benchmark (score 4). The appearance of example 3:1 was also whiter than the comparative non-dairy sample. Therefore, the examples according to the invention were closest to the benchmark not only in flavour but also in colour. The comparative sample 3:A, the non-dairy sauces with White Chlorella CsWC1 had high off-flavour which made this samples less acceptable. In detail the off-flavour was described in the sample with 5% White Chlorella CsWC1 as a green - grass flavour, e.g. like eating spinach.

Example 4 Pasta

Compositions shown in Table 20 were prepared by mixing wheat flour with Chlorella powder: white Chlorella WCLS06 or White Chlorella CsWC1 or Golden Chlorella CsGC1. Next, fresh whole egg was added and slowly worked into the flour mixes with a fork. The reference sample was prepared using only wheat flour and egg. Next, the pasta dough was mixed by hand, followed by kneading for ~5 minutes until it became cohesive and smooth. The dough was flattened into a thin sheet of approximately 3 mm using a rolling pin and then cut into pasta noodles with a width of around 3mm. Table 20

Next, the pasta was dried in a kitchen oven at approximately 70 °C for approximately 4 hours to reach moisture level below 12 wt%.

Moisture calculation after drying the pasta

The moisture level in the dried pasta is calculated using the following formula: Here, Msolids, is the mass of the dry solids of component / in the dough and the sum is over all n dough components and Ma is the weight of the pasta after drying.

Pasta cooking and assessment

Dry pasta was put into ~500ml of boiling water and then cooked for 15 min. After cooking it was cooled down and served to the panellists for organoleptic assessment. Four panellists tasted all samples and then agreed on attributes to be scored according to scale described in Method 1. For each sample, the score was agreed per attribute. The results are presented in Table 21.

Table 21

The tasting results (Table 21) showed that the pasta with 10%wt. of White Chlorella vulgaris WCLS06 powder (example 4:1) had the best taste result, being close to the reference in comparison to other microalgae biomass powders tested.

In detail: the sample prepared with White Chlorella WCLS06 scored slightly higher on the off-odour than the reference, namely 5 and not 4 as the reference. The off-odour for samples of example 4:1 (with White Chlorella WCLS06) was surprisingly described as musty, earthy, yeastlike, sour dough and not fishy, green, seaweed as typically defined for macro and microalgae biomass powders). The comparative sample containing White Chlorella CsWC1 (comparative 4:A) scored higher (6) on fishy, green off-odour than the reference or examples 4:1, whilst pasta with Golden Chlorella CsGC1 (comparative sample 4:B) scored highest, namely 7. In the case of samples prepared with White Chlorella CsWC1 and Golden Chlorella CsGC1 , the off-odour was described as fishy, green and seaweed, in general not pleasant and typical for micro- and macroalgae biomass powders

The pasta with White Chlorella WCLS06 (example 4:1) had a slight increase in the off- taste, scoring 5. The direction of the off-taste was described, similarly to the off-odour, as musty, earthy, yeast-like, green, sour dough. For pasta samples prepared with White Chlorella CsWC1 (comparative 4:A) and with Golden Chlorella CsGC1 (comparative 4:B) a slight increase in astringency/dry aftertaste was noted. No astringency was noted for samples with White Chlorella WCLS06, both scoring 4, same as the reference.

Pasta prepared with Golden Chlorella CsGC1 (comparative sample 4:B) was concluded to be most prone towards disintegration in the mouth upon chewing. It fell apart easier, giving an astringent maybe even powdery perception. For pasta prepared with White Chlorella WCLS06 (example 4:1) or White Chlorella CsWC1 (comparative 4:A) were somewhere in between, with a score of 5.

Table 22 - Colour measurements and colour difference calculations for cooked pasta

Results of colorimetric analysis of the pastas with the DigiEye are provided in Table 22. Pasta prepared with White Chlorella CsWC1 (Comparative 4:A) is greener and darker than the reference and products prepared with White Chlorella WCLS06 (examples 4:1). This colour of comparative sample 4:A is not appealing and not appetizing from the consumer perspective. Pasta /noodles prepared with Golden Chlorella CsGC1 are even darker than the reference and all pastas prepared with White Chlorella (WCLS06 and CsWC1). They are also stronger in yellow and red colour directions, as measured with L*a*b* . The AE calculations reveal that pasta prepared with White Chlorella WCLS06 where the AE is ~4, the colour of the pasta is so similar to the regular colour of that reference pasta, that it can only be recognized as slightly different when placed directly next to the reference. The AEs for pasta made with White Chlorella CsWC1 (Comparative 4:A) and with Golden Chlorella CsGC1 (Comparative 4:B) are much larger and their colours can be easily recognized to be different from regular pasta, even without proximal comparison.

Example 5 Instant tomato soup

Compositions shown in Table 23 were prepared by mixing typical ingredients for instant tomato soup, in which savoury creamer and native starch were partially replaced by Chlorella flour in amounts that enable ‘high in protein’ claims (according to Ell regulations) by increasing the protein content in the dry mix from 12 wt.% (as in REFERENCE) to at least 17 wt.% (as in Comparative 5:A or Example 5:1). 17% wt.% of protein in the dry mix corresponds to 20 cal.% from protein per serving. To realize protein enrichment, microalgae biomass powder was used: White Chlorella WCLS06 (Example 5:1) or White Chlorella CsWC1 (Comparative 5:A). To achieve the target protein concentration, (Table 23) 20 wt% of White Chlorella CsWC1 (containing 30% protein) had to be used in the dry mix, whereas only 12 wt.% of White Chlorella WCLS06 (containing 50.3% protein) was needed to reach the same protein level, needed for high protein claims. Table 23

Instant soup dry mix powders were combined with freshly boiled water (in the weight ratio of 1 :9) and then tasted by 2 panellists. The results are shown in Table 24

Table 24

The taste scores clearly indicate that the soup prepared with 12 wt.% of White Chlorella WCLS06 (example 5:1) in dry mix scored closest to the reference. In detail, off-odour was scored 4, whilst for the White Chlorella CsWC1 containing sample (comparative 5:A) it was 7. In the case of off- taste, still some was detected for example 5:1 , as it scored 5 on the off-taste scale, surprisingly, it was not in the direction of fish, seaweed or ocean-like as typical for microalgae biomass powder off-taste. Instead, it was described as musky, earthy, yeast-like or sour dough-like. This direction fitted well with tomato taste direction of the soup. Addition of WCLS06 (example 5:1) did not supress the tomato odour and tomato flavour to the same extent as the White Chlorella CsWC1. Both for tomato odour and tomato taste the scores were 3 and not 1 as in the case of comparative 5:A White Chlorella CsWC1.

Example 6 Savoury bites

Savoury bites or balls preparations were made using ingredients listed in Table 25 Rolled oats, almonds, flaxseed, toasted onion, garlic paste, salt and microalgae powder ingredients were combined and processed using thermomixer for 2 minutes at maximum speed in such all ingredients formed uniformed dry powder mix. Next, puffed buckwheat was added and mixed for 10 s, assuring it was mixed into the dry powder but not mashed. The dry mix was stored in its dry format till the savoury bites had to be prepared for the tasting. Table 25 The savoury bites were prepared by combining 90g of the dry mix with 40g of cold water and 5 g of sunflower oil in the thermomixer and mixed at room temperature at medium speed for 2 minutes assuring formation of pasty, dough-like mixture. Immediately after that, savoury bites were shaped by hand in a form of 5 cm diameter spheres/balls and served for sensory assessment.

To achieve the protein enrichment, microalgae powder, namely high protein White Chlorella WCLS06 (Example 6:1 and example 6:2) or White Chlorella CsWC1 (Comparative 6:A) were used. Forthat (table 21) 15 wt% of White Chlorella CsWC1 powder (containing 30% protein) had to be used where only 7 wt.% of White Chlorella WCLS06 (containing 50.3% protein) was needed to reach the same protein target (Example 6:2).

Three panellists tasted all samples and then agreed on attributes to be scored according to scale described in Method 1 . For each sample, the score was agreed per attribute. The results are presented in Table 26.

The results clearly indicate that savoury bites can be enriched with proteins without compromise on the taste and odour when using White Chlorella WCLS06 at the inclusion level of around 5 wt.% (Example 6:2) in the final product. Products assessed in Example 6:2 scored 4 both on off-odour and off-taste, indicating no difference from the reference i.e., from bites prepared without White Chlorella. Comparative 6:A to have the same protein enrichment as example 6:2 had to comprise 11 wt.% of White Chlorella CsWC1 in the final product and scored significantly worse both on off-odour and off-flavour (score of 7 for both attributes). Moreover, bites prepared with the same amount of microalgae powder, i.e., 11 wt.% of White Chlorella WCLS06 (Example 6.1) scored still lower on the off-odour and off-taste than Comparative 6:A (6 and 5 respectively and not 7). The slightly higher off-odour of sample 6:1 was surprisingly described as musty, earthy, yeast-like, sour dough and not fishy, green, seaweed as typically defined for macro and microalgae powders. In contrast, comparative sample 6:A comprising CsWC1 displayed that typical off-taste and off-odour.

In conclusion, when using the same amount of protein powder with higher protein concentration (example 6:1) savoury bite products could be enriched further with proteins with less compromise on flavour than the comparative sample 6:A.

Table 26

Example 7 - Alfredo pasta snack

Material:

Microalgae biomass batches used are:

• White Chlorella sorokiniana, CS172, spray dried powder, obtained from Example 2

• White Chlorella CsWC1 purchased from Allmicroalgae (Alima) Portugal, dried powder,

As a benchmark, a cream containing dry mix for instant Alfredo pasta snack was made and 2 Alfredo dry mixes containing microalgae powder instead of cream powder were prepared using the recipes listed in Table 27. To prepare the final pasta snack, 50 g of each dry preparation was mixed into 150g just boiled water, using a spoon, and left for 5 min (occasionally stirred), until pasta was rehydrated, and the snack was ready for consumption. The averages of the sensory scores given by 8 panellists are reported in Table 28.

Table 27 Dry mixes compositions for Alfredo pasta snacks

Taste evaluation was carried out as detailed in Method 1 above (see Table 16). The taste evaluation shows that the sample that scored most close to the benchmark is example 7:1 , the pasta snack with White Chlorella CS172. It scored 3 (slightly less) on dairy flavour and cheese flavour and 5 (slightly higher) on the fishy off-taste. For comparison the sample with white Chlorella CsWC1 (Comparative 7:A) scored 6 (higher) on fishy off-flavour and 2 (lower) on dairy flavour. The cheese flavour, though, was scored 3 in both cases, indicating the similar impact on this attribute of both chlorella. Colour measurements are in Table 29. The colour of Example 7:1 was also whiter, scoring 3 (slightly green). In detail, Example 7:1 with white Chlorella CS172 was closer to the benchmark not only in respect to flavour but also in respect to dairy-like appearance. The comparative sample 7:A, i.e. , the pasta pot snack with white Chlorella CsWC1 had high fishy off-flavour (scored as 6) and high green colour (scoring 2) which made this samples less acceptable. In detail, the sample with 6.5% white Chlorella CsWC1, was more fishy in taste and more green in colour than sample with 6.5% white Chlorella CS172. Furthermore, Chlorella CsWC1 supressed the dairy flavour more than white Chlorella CS172.

Table 28 - Tasting scores for samples prepared with dry mixes made according to formulations in Table 27

Table 29 - Colour measurements and colour difference calculations for Alfredo pasta snacks (pasta+sauce) (as prepared)

Example 8: Savoury model formulation

A taste session with 5 panellists was performed with model savoury dispersions. Dry mixes were prepared by mixing sodium chloride, maltodextrin, and two batches of spray dried microalgae in amounts as given in Table 30. For the preparation of the model savoury dispersion 1/10 of the dry mix was added to 9/10 wt. of just boiled tap water (final dosage 1.5% wt. microalgae powder in a model savoury dispersion). For the reference I benchmark model savoury dispersion, the % ingredients are given in the same table and this dispersion does not contain microalgae powder. Table 30 - Dry mix compositions

Table 31 -Taste scores

The tasting results (Table 31) showed that the model savoury dispersion with 1.5 %wt. white C. sorokiniana (Example 8:1) had the best taste results and is the closest to the reference. This model savoury dispersion had only a slight fishy off-taste and slight other off-taste. The latter was described as musty, green. Some cardboard was detected and was higher than in the reference, scoring 5, whilst 4 is scored for the reference. The comparative White Chlorella CsWC1 microalgae tasted in the savoury model dispersions had a clearly discernible higher fishy off- flavour and higher other off flavour, scoring 6. It was commented to taste unpleasant, cardboard, fishy and had a wet cardboard odour.

The savoury model dispersions were also assessed for the whiteness colour. The scores higher than 4 indicated the samples were whiter, whilst scores below 4 were less white and indicating towards green/grey. Example 8:1 scored in white direction (on par with the reference), whilst Comparative 8:A was assessed to be greener and scoring 3.

Example 9: Tomato Cup a Soup

A taste session with 5 panellists was performed with Tomato Cup-a-Soup samples. Dry mixes were prepared by mixing a commercially available tomato soup powder mix (“llnox Cup-a-Soup Tomaat”, obtained from a local supplier) with two batches of spray dried microalgae in amounts as given in Table 32. For the preparation of the soup snack, 15 gram of the dry mix was added to 150 gram just boiled tap water (final dosage 1.4% wt. microalgae powder in wet soup). For the reference I benchmark 1.4% maltodextrin powder was dosed instead of microalgae. The % ingredients of the tomato commercial soup powder mix are tomato powder 38%, potato starch, croutons 11% (flour, salt, yeast, rapeseed oil), palm oil, antioxidant: rosemary extract), sugar, pasta 8%, yeast extract, iodised salt, dextrose, salt, onion powder, corn oil, mineral salt (potassium), glucose syrup, spices (garlic, pepper), parsley 0,5%, concentrated vegetable extract (celeriac, carrot, leek, onion).

Table 32 - Compositions of soup powder samples

Table 33 - Taste score in average values

The tasting results (Table 33) showed that the soup with 1.4 %wt. white C. sorokiniana CS172 (Example 9:1) had both best taste results close to the reference in comparison to the comparative sample tested. The soup with C. sorokiniana CS172 had a slight other off-taste described as green and cardboard that was higher than in the reference, scoring 5, whilst 4 is scored for the reference. The comparative example tomato soup snack containing White Chlorella CsWC1 microalgae had the highest other off-flavour, scoring 6, and was commented to have a strong aftertaste of cardboard, a green aftertaste and had an unpleasant cardboard odour.

Claims

Claims

1. Food composition comprising Chlorella biomass material, wherein the biomass material is sourced from a chlorophyll-deficient strain of Chlorella microalgae and wherein the Chlorella biomass material comprises at least 50 wt% of Chlorella protein by dry weight of the Chlorella biomass and wherein the food composition is in the form of a meat analogue or a dry savoury snack.

2. Food composition according to claim 1, wherein the Chlorella biomass material is present in an amount of from 0.1 to 75% by dry weight of the food composition, more preferably from 0.5 to 50%, even more preferably from 1 to 40% and still more preferably from 1.5 to 10%.

3. Food composition according to claim 1 or 2, wherein the Chlorella biomass material comprises having a protein content in a range of 50-85% w/w.

4. Food composition according to any one of claims 1 to 3, wherein the Chlorella protein material is present in an amount of from 0.1 to 60% by dry weight of the food composition, more preferably from 0.2 to 45%, even more preferably from 0.3 to 30%, still more preferably from 0.4 to 25%, yet more preferably from 0.5 to 20%, even still more preferably from 1 to 10% and still more preferably from 1.5 to 5% by dry weight of the food composition.

5. Food composition according to any one of claims 1 to 4, wherein the food composition comprises less than 0.05 wt%, more preferably less than 0.02 wt% and even more preferably less than 0.01 wt% of chlorophyll by dry weight of the food composition.

6. Food composition according to any one of claims 1 to 5, wherein the strain of Chlorella microalgae is a strain of Chlorella vulgaris or a strain of Chlorella sorokiniana microalgae.

7. Food composition according to claim 6, wherein the strain of Chlorella microalgae is a strain of Chlorella vulgaris microalgae.

8. Food composition according to any one of claims 1 to 7, wherein the genome of the strain of Chlorella microalgae includes an effective mutation in a magnesium chelatase gene and/or an effective mutation in a phytoene desaturase gene.

9. Food composition according to any one of claims 1 to 8, wherein the Chlorella biomass material has a starch content of less than 10 wt%, more preferably less than 5 wt% by dry weight of the Chlorella biomass material.

10. Food composition according to any one of claims 1 to 9, wherein at least 60 wt%, preferably at least 70 wt%, more preferably at least 80 wt% and even more preferably at least 90 wt% of the biomass material by dry weight of the food composition is present in the form of lysed biomass material.

11. Food composition according to any one of claims 1 to 10 wherein at least 60 wt%, preferably at least 70 wt%, more preferably at least 80 wt% and even more preferably at least 90 wt% of the biomass material by dry weight of the food composition is present in the form of non-lysed biomass material.

12. Food composition according to any one of claims 1 to 11 , wherein the Chlorella biomass material is obtainable by a process including the step of heterotrophic fermentation of the chlorophyll-deficient strain of Chlorella microalgae.

13. Food composition according to any one of claims 1 to 12, wherein the Chlorella biomass material is obtainable by a process including the step of fermentation of the chlorophylldeficient strain of Chlorella microalgae and wherein the chlorophyll-deficient strain of Chlorella microalgae is obtainable by a method of producing a chlorophyll-deficient strain of Chlorella microalgae, that is capable of being grown to yield Chlorella biomass comprising at least 50% by dry weight of protein and not more than 10 wt% by dry weight of starch, wherein the method comprises the steps of: a) obtaining a parent strain of Chlorella microalgae, the strain preferably being a wildtype strain; b) performing mutagenesis of the parent strain of Chlorella microalgae; c) cultivating the mutated Chlorella microalgae at a specific temperature, for a predefined period of time, and in the presence of an organic carbon source; and d) identifying and isolating mutants of the parent strain of Chlorella microalgae that exhibit a first trait, e) performing mutagenesis on a strain of Chlorella microalgae that stably exhibit the first trait; f) cultivating the further-mutated Chlorella microalgae at a specific temperature, for a predefined period of time, and in the presence of an organic carbon source; and g) identifying and isolating mutants of the parent strain of Chlorella microalgae that exhibit the first and a second trait; h) performing mutagenesis on a strain of Chlorella microalgae that stably exhibit the first and second trait; i) cultivating the further-mutated Chlorella microalgae at a specific temperature, for a predefined period of time, and in the presence of an organic carbon source; and j) identifying and isolating mutants of the parent strain of Chlorella microalgae that exhibit the first, the second and a third trait, wherein the first, second and third trait each are one of chlorophyll deficiency, whiteness and capability of yielding upon fermentation a protein content of at least 50 wt% by dry weight of the Chlorella biomass.

14. Food composition according to any one of claims 1 to 13, wherein the food composition is in the form of a meat analogue.

15. Food composition according to any one of claims 1 to 14, wherein the food composition is in the form of a savoury snack.