EP3035807A2

EP3035807A2 - Method for preparing lipid-rich compositions of microalga flour with optimised organoleptic properties

Info

Publication number: EP3035807A2
Application number: EP14789310.1A
Authority: EP
Inventors: Amandine DRUON; Marie LE RUYET; Laurent Segueilha
Original assignee: Roquette Freres SA
Current assignee: Corbion Biotech Inc
Priority date: 2013-08-13
Filing date: 2014-08-12
Publication date: 2016-06-29
Also published as: WO2015022469A3; US20160192691A1; US20200046002A1; WO2015022469A2; JP2016527898A; KR20160041927A; MX2016001917A; CN105451575A

Abstract

The present invention relates to a method for preparing, by fermentation, lipid-rich compositions of microalga flour having optimised organoleptic properties, characterised in that the microalgae are cultured in a minimal culture medium.

Description

PROCESS FOR THE PREPARATION OF FLOUR COMPOSITIONS OF MICROALGUES RICH IN LIPIDS OF OPTIMIZED ORGANOLEPTIC QUALITY

The present invention relates to a novel process for the fermentative preparation of flour compositions of lipid-rich Chlorella microalgae having an optimized sensory profile, which allows them to be incorporated into food formulations without generation of flavors. undesirable.

Presentation of the state of the art

Historically requiring "only water and sunlight" to grow, algae have long been considered a source of food.

There are several species of algae that can be used for food, most of which are "macro-algae" such as kelp, sea lettuce (Ulva lactuca) and red algae Porphyra (cultivated in Japan) or "dulse" (Palmaria palmata).

However, in addition to these macroalgae, there are also other sources of algae represented by "microalgae", ie photosynthetic or unicellular microscopic algae algae, of marine origin or not, grown for their applications in the biofuels or food.

For example, spirulina (Arthrospira platensis) is grown in open lagoons (by phototrophy) for use as a dietary supplement or incorporated in small amounts in confectionery or beverages (usually less than 0.5% w / w).

Other microalgae rich in lipids, including some species of Chlorella type, are also very popular in Asian countries as food supplements (mention is made of microalgae of the genus Crypthecodinium or Schizochytrium producers of omega 3 fatty acids).

The production and use of Chlorella microalgae flour are for example described in WO 2010/120923 and WO 2010/045368.

The oil fraction of microalgae flour, which can be composed mainly of monounsaturated oils, can offer nutritional and health benefits over saturated, hydrogenated and polyunsaturated oils often found in conventional food products.

When it is desired to industrially manufacture microalgae flour powders from their biomass, significant difficulties remain, not only from the technological point of view, but also from the point of view of the sensory profile of the compositions produced. Indeed, if algae powders, for example manufactured with photosynthetically grown algae in outdoor ponds or by photobioreactors, are commercially available, they have a dark green color (bound to chlorophyll) and a strong, unpleasant taste. .

Even formulated in food products or as nutritional supplements, these algae powders always communicate this green color visually unattractive to the food product or nutritional supplement and have an unpleasant taste of fish or seaweed flavor.

Moreover, it is known that certain species of blue-green algae naturally produce odorous chemical molecules such as geosmin (trans-1, 10-dimethyl-trans-9-decalol) or MIB (2-methylisoborneol), generating odors earthy or moldy.

As for chlorella, the descriptor commonly accepted in this field is the taste of "green tea", a little similar to other green vegetable powders such as green barley powder or green wheat powder, taste attributed to its strong chlorophyll content.

Their flavor is usually masked only when mixed with strong vegetables or citrus juices.

There is therefore still an unmet need for microorganic flour compositions of the Chlorella genus of suitable organoleptic quality allowing the use thereof in more numerous and diversified food products.

Summary of the invention

The applicant company has found that this need could be met by proposing a new process for the fermentative preparation of flour compositions of Chlorella microalgae rich in lipids.

Thus, the present invention relates to a fermentation process in which the microalgae are cultured in a so-called "minimum" culture medium.

Preferably, the minimum medium:

contains a reduced amount of sulfur source and / or

- is deprived of one or more vitamins of group B, in particular vitamins of the choline and inositol type.

According to one embodiment, the minimum medium contains a reduced amount of sulfur source, preferably a reduced amount of MgSO 4 and / or (NhU 3 SCU), In particular, the minimum medium may not contain more than 1 g / MgSC> 4 or may contain about 1 g / L MgSC> 4. It may also contain no more than 0.2 g / L of (NhU ^ SCU or may contain about 0.2 g / L of (NhU ^ UCS. According to another embodiment, the minimum medium is deprived of one or more vitamins of group B. In particular, the minimum medium may not contain choline and / or inositol. Preferably, it contains neither choline nor inositol.

The organoleptic qualities of the flour compositions can be evaluated by means of a tasting composition prepared by mixing 5-10% of microalgae flour composition, 0.5-2% sugar, 0.1-0.5% d vanilla aroma and skim milk; the percentages being expressed by weight of the tasting composition, homogenizing the composition prior to heating at 60-85 ° C for 2-10 minutes.

The microalgae may be selected from Chlorella protothecoides, Chlorella kessleri, Chlorella minutissima, Chlorella sp., Chlorella sorokiniama, Chlorella luteoviridis, Chlorella vulgaris, Chlorella reisiglii, Chlorella ellipsoidea, Chlorella saccarophila, Parachlorella kessleri, Parachlorella beijerinkii, Prototheca stagnora and Prototheca moriformis.

Preferably, the microalgae belong to the genus Chlorella, and most preferably are Chlorella protothecoides.

According to one embodiment, the microalgae are deprived of chlorophyll pigments. These microalgae can especially be grown in the dark or are unable to produce, or have a reduced production capacity, chlorophyll pigments.

The present invention also relates to a lipid-rich microalgae flour composition obtained by the process according to the invention, as well as the use of this flour composition for the preparation of a food composition.

Detailed description of the invention

Within the meaning of the invention, a microalgae flour composition has an "optimized sensory profile" or "optimized organoleptic quality", when its evaluation by a sensory panel in food formulation (for example in an ice cream or in a composition of tasting as described below) concludes that there are no defects that alter the organoleptic quality of said food formulations containing these microalgae flour compositions.

By "organoleptic quality" is meant the property of a food in terms of taste, smell, appearance, color and consistency.

These defects (Anglo-Saxon term "off-notes") are associated with the presence of odor-specific and / or aromatic undesirable molecules which are characterized by a threshold of perception corresponding to the minimum value of the sensory stimulus necessary for the awakening of 'a feeling. The "optimized sensory profile" or "optimized organoleptic quality" is then translated by a sensory panel by obtaining the best scores on a scale of evaluation of the 4 sensory criteria (appearance, texture, flavors and flavors).

By "total content" is meant the sum of the contents for each of the volatile organic compounds in the list.

As used herein, the term "about" refers to a value of +/- 20%, 10%, 5% or

2%.

For the purposes of the present invention, the term "microalgae flour" should be understood in its broadest interpretation and as designating, for example, a composition comprising a plurality of microalgae biomass particles. The microalgae biomass is derived from microalgae cells, which may be whole or broken, or a mixture of whole and broken cells.

A number of state-of-the-art documents, such as the international patent application WO 2010/120923, describe methods for the preparation and use in feeding the biomass of Chlorella microalgae.

The microalgae that are the subject of the present invention are therefore preferably microalgae of the genus Chlorella, more particularly Chlorella protothecoides, more particularly Chlorella deprived of chlorophyllian pigments, by any method known per se to those skilled in the art (either by the fact that the culture is carried out in the dark, or because the strain has been mutated so as to no longer produce these pigments). In particular, the lipid-rich microalgae may be chosen, in a non-exhaustive manner, from Chlorella protothecoides, Chlorella kessieri, Chlorella minutissima, Chlorella sp., Chlorella sorokiniama, Chlorella luteoviridis, Chlorella vulgaris, Chlorella reisiglii, Chlorella ellipsoidea, Chlorella saccarophila, Parachlorella kessieri, Parachlorella beijerinkii, Prototheca stagnora and Prototheca moriformis. Thus, in a very particular embodiment, the microalgae flour composition is a composition of Chlorella flour, and in particular Chlorella protothecoides.

The fermentative process described in patent application WO 2010/120923 allows the production of a certain number of microalgae flour compositions of variable organoleptic quality.

The culture medium recommended in this patent application is a complex fermentation medium, comprising:

a carbon source (glucose, fructose, sucrose, galactose, xylose mannose, rhamnose, arabinose ...),

a source of nitrogen (such as proteins, soy flour, yeast extract, corn quenching water, etc.), elements in trace (zinc, boron, cobalt, copper, manganese and molybdate, made in the form of ZnC, H3BO4, C0CI2.6H2O, CUCI2.2H2O, MnCl ₂ .4H ₂ 0 (ΝΗ ₄₎ 6Μθ7θ24 .4Η ₂ 0),

optionally a pH buffer and

- Phosphate (as a source of phosphorus, phosphate salts can be used).

For strains maintained alive by the University of Texas at Austin for its microalgae collection (UTEX), different media are available (see their website http://www.utew.org/).

Furthermore, in the patent application WO 2010/120923, it is specified that a high oil content can be produced by the supply of excess carbon source and under nitrogen limitation.

But nowhere in this document is it suggested or suggested to use a minimum medium, whether to grow the microalgae, or to promote the production of flour compositions having an optimized sensory profile.

A minimal medium is conventionally defined as a medium containing only the chemical elements strictly necessary for the growth of the microalgae, in a form usable by microalgae having no particular requirement.

The minimum medium then contains:

a source of carbon and energy: generally glucose;

a source of potassium and phosphorus: for example K2HPO4; a source of nitrogen and sulfur: for example (NH ₄ ) 2SO _4;

a source of magnesium and sulfur: for example MgSCUTHbO; a source of calcium: for example CaCl2.2H20;

a source of iron: for example FeSCMJhbO;

sources of trace elements: salts of Cu, Zn, Co, B, Mn, Mo; and

vitamins (thiamine, biotin, vitamin B12 ...).

However, as will be exemplified below, the Applicant Company has found that if we further reduce the sulfur intake of this minimum medium and / or eliminates certain vitamins (especially choline and inositol, assimilated to vitamins group B), flour compositions of these microalgae having an optimized sensory profile were then obtained.

To determine this sensory profile, the Applicant company has defined a very simple tasting matrix that nevertheless makes it possible to make an organoleptic evaluation similar to that obtained with much more complex and very different recipes such as an ice cream or a brioche.

This tasting matrix of microalgae flour compositions includes: 5-10% of microalgae flour composition, preferably about 7%; 0.5-2% sugar, preferably about 1%;

0.1 -0.5% vanilla flavor, preferably about 0.25%; and

the remainder in skim milk, preferably about 91.75%;

the percentages being expressed by weight of the tasting composition.

The preparation of a tasting composition as described above is homogenized, heated to 60-85 ° C, preferably about 75 ° C, for 2-10 minutes, preferably about 5 minutes.

To test and evaluate the organoleptic qualities of a microalgae flour composition included in the tasting composition as described above, a sensory panel is defined.

This sensory panel is formed to evaluate the sensory properties of different batches of microalgal flour compositions, particularly Chlorella protothecoides biomass flour.

A set of individuals, at least 10, 20 or 30, in particular about 15, are pooled to evaluate descriptors of several microalgal flour compositions, preferably in comparison with a reference microalgae flour sample identified as consistent, that is to say of acceptable organoleptic quality (lot reference No. 1) and another sample of organoleptic quality very unacceptable (lot referenced No. 2).

Preferably, the microalgal flour compositions are tested in the form of a tasting composition according to the present document. Alternatively, the compositions may be tested in any other form desired by those skilled in the art, for example in the form of ice cream or bread-making product such as a brioche.

Preferably, the reference products as presented in the following table are associated with each descriptor:

Reference descriptors

Color aspect (from light to dark)

Texture Nappant whole milk + 5% cream

Flavors Sweet Sucrose 1%

100 g mushrooms in 100 ml cold water

Mushroom

/ dilution X 4

Ebly Solution Cereals 10%

Flavors Butter / dairy product

Rancid oil Oxidized oil 1, 5%

Very microalgae flour composition

Back vegetable taste

unacceptable Of course, those skilled in the art can define other reference products if they wish.

At each tasting session, several products, for example 4 to 5, are evaluated on each descriptor in comparison with a batch or reference sample, preferably a batch or reference sample considered as acceptable organoleptic quality.

All products are evaluated one after the other, on scales ranging for example from 1 to 9 as follows:

Value of 1: the evaluated descriptor is not present in the product;

Value of 5: the evaluated descriptor is present in the product in exactly the same way as the acceptable organoleptic reference product;

Value of 9: the evaluated descriptor is very present in the product.

The reference batch of acceptable organoleptic quality is a composition of microalgae flour in the sense that it has the "satisfactory" sensory profile of all these descriptors. The reference batch of organoleptic quality very unacceptable is a lot not satisfying the descriptors relating to aromatic notes, that is to say the descriptors flavors and flavors, because it has for example a significant vegetable aftertaste.

It is important to note that the reference batch of acceptable organoleptic quality is not necessarily the microalgae flour composition having the optimal sensory profile: it is preferably a microalgae meal composition perceived by the sensory panel. as "satisfactory", in particular with a score of 5, on all the descriptors tested. In this embodiment, the microalgae flour tested compositions are classified by the sensory panel on either side of this reference batch of acceptable organoleptic quality.

In general, the compositions tested are classified by the sensory panel relative to the reference batch (s) of acceptable or unacceptable organoleptic quality, preferably relative to the reference batch (s) of acceptable organoleptic quality. .

Thus, the first step results in the classification of the various flour compositions of microalgae tested according to their organoleptic quality.

In particular, analysis of variances (ANOVA) are performed to evaluate the discriminating power of the descriptors (descriptors whose p-value associated with the Fisher - ANOVA type 3 test) is less than 0.20 for the Composition effect in the model. descriptor ~ Composition + judge). The Composition effect is interpreted as the discriminating power of the descriptors: if there is no effect (Critical Probability> 0.20), the compositions have not been discriminated according to this criterion. The smaller the critical probability, the more discriminating the descriptor. A Principal Component Analysis (PCA) is then performed to obtain a sensory map of the compositions, and a simultaneous representation of all the compositions on all the descriptors.

Thus, the present invention relates to a process for the preparation, by fermentation, of flour compositions of microalgae rich in lipids of optimized organoleptic quality.

In the process according to the invention, the microalgae, preferably microalgae of the genus Chlorella and more particularly Chlorella protothecoides, are cultivated in a minimum medium. The biomass thus obtained can then be transformed into microalgae flour.

The minimum medium as used in the present invention comprises a source of carbon, potassium, phosphorus, nitrogen, magnesium, calcium, iron, trace elements and vitamins.

According to one particular embodiment, the minimum medium comprises the following compounds: Glucose, KH ₂ PO ₄ , NaH ₂ PO ₄ , MgSO ₄ , (NH ₄ ) ₂ SO ₄ , CaCl ₂ , FeSO ₄ , MnSO ₄ , CoSO ₄ , CuSO ₄ , ZnSO ₄ , H ₃ BO 3, Na ₂ MoO ₄ , Thiamine, Biotin, Vitamin B 12, Calcium pantotenate and p-Aminobenzoic acid, and optionally Inositol and / or Choline chloride.

Preferably, this minimum medium comprises a reduced amount of sulfur source and / or is deprived of one or more vitamins of group B.

According to one embodiment, the minimum medium comprises a reduced amount of sulfur source.

As used herein, the term "reduced amount" preferably means that the amount in the medium is less than the amount known to support the strain. Preferably this term means less than 20, 25, 30, 35, 40, 45 or 50% to the amount known to support the strain. Alternatively, a reduced amount may mean that the amount contained in the medium meets only 50, 55, 60, 65, 70, 75 or 80% of the requirements of the cultivated strain.

The sulfur contained in the minimum medium comes essentially from the macro-elements containing this element. Indeed, the contribution of micronutrients is usually negligible. Thus, according to one embodiment, the sulfur contained in the minimum medium comes essentially from MgSO ₄ and / or (NH ₄ ) ₂ SO ₄ .

Thus, according to a particular embodiment, the minimum medium used comprises at most the equivalent of about 1 g / L of MgSO ₄ and about 0.2 g / L of (NH ₄ ) ₂ SO ₄ as a source of sulfur. Preferably, the minimum medium used comprises at most the equivalent of 1 g / L of MgSO ₄ and 0.2 g / L of (NH ₄ ) ₂ SO ₄ as a source of sulfur. According to a preferred embodiment, the minimum medium used comprises the equivalent of about 1 g / L MgSC and about 0.2 g / L of (NhU ^ SC source of sulfur, and more preferably, the equivalent of 1 g / L MgSC and 0.2 g / L of (NhU ^ SC.

According to one embodiment, the minimum medium is deprived of one or more vitamins of group B.

Preferably, the minimum medium does not contain choline and / or inositol. More particularly preferably, the minimum medium contains neither choline nor inositol.

Preferably, the microalgae are cultured in the minimum medium throughout the duration of the fermentation, that is to say until a biomass sufficient to be converted into flour. Typically, the fermentation has a duration of more than 50 hours, preferably between 50 and 150 hours. The biomass produced is preferably greater than 100 or 150 g / L medium, and very particularly preferably between 100 and 250 g / L medium.

As used herein, the term "lipid-rich microalgae" refers more particularly to microalgae producing a biomass comprising more than 30, 35, 40 or 45% lipid by dry weight of biomass.

Microalgae are preferably deprived of chlorophyll pigments. In particular, the fermentation can be conducted in the dark. Alternatively, microalgae may also be unable to produce, or have reduced production capacity, chlorophyll pigments.

Thus, according to a preferred embodiment, the microalgae are cultured under heterotrophic conditions, that is to say without light by using a carbon substrate (preferably glucose) as a source of carbon and energy.

According to a preferred embodiment, the biomass obtained contains little or no organoleptically undesirable compounds such as the oxidative degradation products of monounsaturated fatty acids. In particular, the biomass contains little or no oxidative degradation products of oleic acid. Preferably, the content of linoleic acid (oxidative degradation product of oleic acid) is less than 18, 15, 10, 8 or 7% by weight relative to the total weight of fatty acids in the dry biomass.

Although usable on a smaller scale, the process is preferably carried out on an industrial scale, that is to say on fermenters of average (from about 1 to 100 m ³ ) and large capacity (from more than 100 m ³ ). According to one embodiment, the process is carried out on fermentors with a capacity of at least 1, 10, 25, 50, 75, 100, 500 or 1000 m ³ .

The process according to the invention may also comprise a step of transforming the biomass obtained into microalgae flour. This step may involve any method known to those skilled in the art for obtaining microalgae flours. The present invention also relates to a lipid rich microalgae flour composition obtained according to the method of the present invention.

It also relates to the use of this flour composition for the preparation of food compositions such as food products, nutritional supplements, confectionery or beverages.

The invention will be better understood with the aid of the examples which follow, which are intended to be illustrative and not limiting.

EXAMPLES

Example 1: Production of Chlorella protothecoides rich in lipids - Reduction of the MgSQ4 or vitamins intake

The strain used is Chlorella protothecoides UTEX 250 Preculture:

500 mL of medium in a 2L Erlenmeyer flask;

Composition of the medium (in g / L or mg / L):

The incubation takes place under the following conditions: duration: 72 h; agitation temperature: 1 10 rpm (Infors Multitron Incubator).

The preculture is then transferred to a 30L fermentor of the Sartorius type.

Culture for biomass production:

The basic medium is:

The supply of MgSO4 is reduced to 1 g / L in test 2 and Choline chloride and Nnositol are eliminated in test 3.

The initial volume (Vi) of the fermenter is adjusted to 7 L after seeding. It is brought to 15 - 20 L in final.

The driving parameters of fermentation are as follows:

Temperature 28 ° C

pH 6.8 by NH ₃ 28% w / w then KOH 5N

p0 ₂ > 20% (maintained by stirring) Agitation 300 RPM mini

Air flow 15 L / min

When the residual glucose concentration falls below 10 g / L, a glucose supply in the form of a concentrated solution at 700 g / L is continuously carried out so as to maintain the glucose content between 0 and 20 g / L. in the fermenter.

When 1000 g of glucose were consumed and the biomass reached a concentration of 70 g / L, ammonia is replaced by potash for pH regulation. This allows the biomass to accumulate lipids

Results:

The higher the score given by the sensory panel, the more the composition is characterized by the presence of "off-notes", that is to say organoleptic defects.

The flour compositions obtained with tests 2 and 3 obtain a grade of less than 5, that is to say a score lower than the reference composition of satisfactory organoleptic quality. The compositions of tests 2 and 3 therefore have better organoleptic properties than those of the reference composition.

These results therefore show that limiting the medium to sulfur, in particular by reducing the concentration of MgSC, which is the main source of sulfur, or the absence of certain vitamins, in particular inositol and / or choline, leads to a reduction significant level of off-notes and thus an improvement of the organoleptic quality of the composition.

Claims

1. Process for the fermentative preparation of lipid-rich microalgae flour compositions of optimized organoleptic quality, characterized in that the microalgae are cultured in a minimum culture medium.

2. Method according to claim 1, characterized in that the minimum medium: contains a reduced amount of sulfur source and / or

is deprived of one or more vitamins of group B.

3. Method according to any one of the preceding claims characterized in that the minimum medium contains a reduced amount of sulfur source.

4. Method according to any one of the preceding claims, characterized in that the minimum medium contains a reduced amount of MgSC and / or (NhU ^ SC.

5. Method according to any one of the preceding claims, characterized in that the minimum medium does not contain more than 1 g / L of MgSC.

6. Method according to any one of the preceding claims, characterized in that the minimum medium contains about 1 g / l of MgSC.

7. Method according to any one of the preceding claims, characterized in that the minimum medium contains not more than 0.2 g / L of (NhU ^ SC.

8. Method according to any one of the preceding claims, characterized in that the minimum medium contains about 0.2 g / L of (NhU ^ SC.

9. Method according to any one of the preceding claims, characterized in that the minimum medium comprises at most the equivalent of about 1 g / L of MgSC and about 0.2 g / L of (NhU ^ SC source of sulfur.

10. Method according to any one of the preceding claims, characterized in that the minimum medium is deprived of one or more vitamins of group B.

1 1. Process according to any one of the preceding claims, characterized in that the minimum medium does not contain choline.

12. Method according to any one of the preceding claims, characterized in that the minimum medium does not contain inositol.

13. Process according to any one of the preceding claims, characterized in that the minimum medium does not contain either choline or inositol.

14. Method according to any one of the preceding claims, characterized in that the organoleptic qualities of the flour compositions are evaluated by means of a tasting composition prepared by mixing 5-10% of microalgae flour composition, 0.5- 2% sugar, 0.1-0.5% vanilla flavor and skimmed milk; the percentages being expressed by weight of the tasting composition, homogenizing the composition prior to heating at 60-85 ° C for 2-10 minutes.

15. Process according to any one of the preceding claims, characterized in that the microalgae are selected from Chlorella protothecoides, Chlorella kessleri, Chlorella minutissima, Chlorella sp., Chlorella sorokiniama, Chlorella luteoviridis, Chlorella vulgaris, Chlorella reisiglii, Chlorella ellipsoidea, Chlorella saccarophila, Parachlorella kessleri, Parachlorella beijerinkii, Prototheca stagnora and Prototheca moriformis.

16. Process according to any one of the preceding claims, characterized in that the microalgae belong to the genus Chlorella.

17. Method according to any one of the preceding claims, characterized in that the microalgae are Chlorella protothecoides.

18. Process according to any one of the preceding claims, characterized in that the microalgae are deprived of chlorophyll pigments.

19. The method of claim 18 characterized in that the microalgae are grown in the dark.

20. The method of claim 18 or 19 characterized in that the microalgae are unable to produce, or have a reduced production capacity, chlorophyll pigments.

21. Process according to any one of the preceding claims, characterized in that it further comprises a stage of transformation of microalgae into microalgae flour.

22. Lipid-rich microalgae flour composition obtained by the process according to claims 1 to 21.

23. Use of the lipid-rich microalgae flour composition of claim 22 for the preparation of a food composition.