WO2024008866A1 - Method for the production of a protein matrix composition having a textured structure - Google Patents

Method for the production of a protein matrix composition having a textured structure Download PDF

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Publication number
WO2024008866A1
WO2024008866A1 PCT/EP2023/068708 EP2023068708W WO2024008866A1 WO 2024008866 A1 WO2024008866 A1 WO 2024008866A1 EP 2023068708 W EP2023068708 W EP 2023068708W WO 2024008866 A1 WO2024008866 A1 WO 2024008866A1
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WIPO (PCT)
Prior art keywords
matrix composition
protein matrix
biomass substrate
substrate
fungal
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PCT/EP2023/068708
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French (fr)
Inventor
Paul DEN DULK
Sabine Emma VERSTEEGE
Nicolaas Adriaan Van Biezen
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Proteins Of Tomorrow B.V.
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Publication of WO2024008866A1 publication Critical patent/WO2024008866A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/18—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from yeasts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/008—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/12—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from cereals, wheat, bran, or molasses
    • A23J1/125—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from cereals, wheat, bran, or molasses by treatment involving enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
    • A23J1/148—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds by treatment involving enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/22—Working-up of proteins for foodstuffs by texturising
    • A23J3/225—Texturised simulated foods with high protein content
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/22—Working-up of proteins for foodstuffs by texturising
    • A23J3/225—Texturised simulated foods with high protein content
    • A23J3/227—Meat-like textured foods
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/16—Solid state fermenters, e.g. for koji production

Definitions

  • the present invention relates to a method for the production of a protein matrix composition having a textured structure from solid-state fermentation of food grade and/or agricultural biomass substrate, wherein the protein matrix composition is suitable for human consumption. Furthermore, the present invention relates to a protein matrix composition obtained via the method based on solid state fermentation, as well as to the use thereof as a meat substitute composition.
  • a demand for edible products having a high protein content that are of plant based origin (or non-animal) is increasing.
  • An increasing awareness of personal health in combination with an increasing demand for a green footprint leads to increased demand for edible products that include non-animal, plant sourced components such as proteins and fibers, including a growing demand for edible meat substitutes that mimic meat in its composition and texture but are composed of non- animal components.
  • vegetarian food is gaining acceptance due to health concerns and environmental issues related with the production of meat. For example, with the production of beef meat large agricultural areas are involved in order to provide sufficient feed for the animals.
  • meat substitutes which aim to provide the proteins which are recommended for daily intake as well as having an appealing texture.
  • meat substitutes are tofu, tempeh and mycoproteins known under the name Quorn.
  • the mycoprotein which is used in Quorn is refined from Fusarium venenatum.
  • the fungus is grown in continually oxygenated water in large fermentation tanks: so called liquid fermentation.
  • the mycoprotein derived is washed and further texturized by using a binder in order to provide a meat like texture.
  • the process involved to provide a palatable product requires considerable inputs in the form of energy and materials and is thus not efficient.
  • Tofu is produced by using soybean derived proteins in a relatively simple and efficient process.
  • present meat analogues are mostly comprised of texturized vegetable proteins that have been obtained via costly texturization processes and suffer from a limited supply of raw material and low manufacturing capacity. Furthermore, most meat analogues products need multiple processing steps to provide a suitable end product for human consumption, making the production process time consuming and costly.
  • the above object is met, according to a first aspect, by the present invention by a method for the production of a protein matrix composition having a textured structure from solid-state fermentation of food grade and/or agricultural biomass substrate, wherein the protein matrix composition is suitable for human consumption, wherein the method comprises the steps of:
  • solid state fermentation means fermentation under controlled circumstances with solid nutrients, as opposed to liquid fermentation which takes place in an oxygenated broth in large vessels. In contrast to liquid fermentation, with solid state fermentation there is no need for expensive soluble nutrients. Materials with low value (like sugar cane or beet waste stream or Asparagus peelings) can thus be upgraded to a valuable food product.
  • solid state fermentation produces a food product that does not require further processing: it can be grown in the desired shape and size, the fully grown substrate can be sold as such, or cut in slices and sold in separate packages, it can be dried or stored in a freezer. There is no need for refining the fibers and extruding them to obtain a reasonable texture.
  • the resulting protein matrix composition obtained via the method of present invention can be used as such, comparable to other solid state processes that produce protein compositions where fungi is harvested from a substrate which is not food-grade, i.e wood- (wood fibre), gras- or strawtype substrates, wool, or manure substrates. Further processing steps, such as clean up and removal of the growth substrate, or dewatering steps are not needed to make the substrate ready for use.
  • the technical ingredient or semi-finished product can be used as is, or in, or part of vegan, vegetarian, hybrid or conventional animal based products and may for example be processed by increasing the heat kernel temperature of the protein matrix composition of 72 °C to enable human consumption.
  • Another major advantage of solid-state fermentation is that, because dewatering is not needed, also much less energy is used and less waste is produced.
  • the moisture content of the biomass substrate is at least 50 wt %, such as from 60 to 80 wt %. Dry matter of the biomass substrate should be between 15 to 40wt%. Above 40 wt% dry matter the biomass substrate mixture will become too dry resulting in suboptimal growth conditions in support of the cultivation and development of the mycelium in order to form the protein matrix composition having the desired textured structure. In case the biomass substrate mixture comprises too little dry mass, i.e. a dry matter content of below 15 wt%, the textured structure will not be obtained and since there is more moist available in the biomass substrate mixture there is a higher chance of obtaining unwanted fungal infections or growth of microbes.
  • Mycelium is preferably done in growing conditions which are favourable for the used fungal- or yeast inoculum. Different strains each have their own preferences, however, most of the edible fungus have good mycelial growth at temperatures in the substrate of 18 to 35 C, more notably 25 C.
  • the mycelium is allowed to grow in said substrate for between 24 to 96 hours, preferably 36 to 72 hours, more preferably 40 to 52 hours, most preferably 48 to 50 hours, at a temperature of between 20 to 30 °C, providing the protein matrix composition having a textured structure.
  • the fungal- or yeast inoculum should be edible, i.e. suitable for human consumption and derived from Ascomycetes and/or Zygomycota. Furthermore, fungal- or yeast mycelium that are non-sporulating, and have a high reproduction cycle and mainly produce white mycelium are preferred in the method of present invention. As such, Ascomycetes are used in the method of present invention since these groups of fungi do not produce external spore formations as seen for example with mushroom type fungi. In contrast Ascomycetes produce microscopic spores inside special, elongated cells or sacs, known as 'asci', which are responsible for the rapid spread of these fungi.
  • Ascomycytes have a more rapid growth rate than for example fungi that produce external spore formations or structures such as Basidiomycota fungi. Also Zygomycota do not form external spore formations for reproduction and may therefore also be suitable for use in the method of present invention.
  • the present invention relates to the method, wherein the edible fungal- or yeast inoculum from the group of Ascomycetes or Zygomycota is one or more selected from the group consisting of Rhizopus oligosporus, Rhiz,opus oryzae, Aspergillus oryzae, Aspergillus tamarii, Trichoderma viride, Trichoderma reesi, Aspergillus nidulans, Aspergillus niger, Penicillium, Mucorales, preferably Rhizopus oligosporus .
  • fungal- or yeast inoculum provide a particular firm mycelium through the present substrate, therewith providing for example a suitable meat substitute composition having an increased firmness.
  • suitable meat substitute composition having an increased firmness.
  • no known allergies have been detected and no levels of mycotoxins are produced.
  • multiple strains can be cultivated at once to tune the protein, amino acid, mineral, texture, and flavour profiles of the final biomass.
  • the present invention relates to the method, wherein the biomass substrate comprises at least between 15 to 35 wt%, preferably 20 to 30 wt% of a plant derived ingredient or product selected from the group consisting of pea fibre, beet pulp, potatoes, soy, faba bean, corn, sugar cane, cellulose and bamboo, barley, brewers’ spent grain, rice, oat, wheat, preferably pea fibre and/or beet pulp.
  • a plant derived ingredient or product selected from the group consisting of pea fibre, beet pulp, potatoes, soy, faba bean, corn, sugar cane, cellulose and bamboo, barley, brewers’ spent grain, rice, oat, wheat, preferably pea fibre and/or beet pulp.
  • the inclusion of the edible fibrous plant derived ingredients provide a firm texture to the protein matrix composition.
  • the present invention relates to the method, wherein the biomass substrate further comprises at least between 0.5 to 10 wt%, preferably 1 to 5 wt% of a cereal derived ingredient or product selected from the group consisting of rice, including rice flour, brewer’s spent grain, oats, wheat, maize, barley, rye, preferably rice.
  • a cereal derived ingredient is preferred to provide the fungal- or yeast inoculum an optimal start of growth due to the increased presence of complex sugars such as starch.
  • rice flour has shown to provide fast production of high quality protein matrix composition using the method of present invention.
  • the present invention relates to the method, wherein said biomass substrate is inoculated with fungal- or yeast inoculum at a concentration of between 100000 to 500000 cfu’s/kg, preferably 200000 to 400000 cfu’s/kg, most preferably 250000 to 300000 cfu’s/kg. If the fungal- or yeast inoculum concentration is too low (i.e. below 100000 cfu’s/kg), there is an increased chance of that an infection will take place by another microorganism, which will get the upper hand to grow further in the culture.
  • the present invention relates to the method, wherein the heat treatment comprises sterilizing or pasteurization of the present biomass substrate before introducing the fungal- or yeast inoculum.
  • the sterilization is carried out by pasteurization, steam sterilization, gamma radiation, dry heat sterilization or chemical sterilization.
  • the present invention relates to the method, wherein in step iv) the solid-state fermentation and said fungal- or yeast inoculum and mycelium growth in said biomass occurs in filter bags or vacuum sealed bags. Also, plastic containers or larger metal containers may be used. Filter bags provide sufficient aeration for a time period which is sufficient to saturate the substrate with mycelium. Saturation of the substrate is usually obtained before the moment when spore formation of the mycelium begins.
  • the present invention relates to the method, wherein pH regulators, nitrogen sources, such as amino acids and/or urea derivates, enzymes, preservatives, colorants, flavouring agents, binders, hydrocolloids, starches, gums, fats, and/or salt are added to the mixture of step i).
  • Additive such as colorants and/or flavourings should be mixed in with the substrate in step i, since after step iv, solid state fermentation, the mixing would result in a break up the mycelium structure of the end product.
  • the present invention relates to the method, wherein the food grade and/or agricultural biomass substrate is a residual or waste substrate from the food and/or agricultural industry.
  • the food and agricultural industries produce high volume food grade waste streams containing quality nutrients, which often goes to feed animals at an inefficient conversion rate or is treated as a complete waste.
  • Such food grade waste streams provide a valuable food grade source of nutrients that is very suitable for use in the method of present invention.
  • the present invention relates to a protein matrix composition having a textured structure obtainable by the method of present invention as disclosed above, wherein said protein matrix composition has a firmness of between 6 to 8 Newton, as determined by a peak force at first compression by a texture profile analysis (TPA) according to example 3.
  • TPA texture profile analysis
  • the protein matrix composition of present invention has a textured structure and a firmness that is comparable to that of meat, cheese or milk product, seafood, and known me at and seafood vegetarian substitute products, preferably meat or seafood.
  • the firmness is determined as described in example 3 using the TPA method.
  • the product according to the method of present invention remained its hardness between 6 to 8 Newton, also after frying and is within the range of the other meat/vegetarian products.
  • the present invention relates to a meat substitute composition comprised of the protein matrix composition or to the use of the protein matrix composition for the provision of an edible consumer product such as a meat- or seafood analogues or hybrid thereof, a cheese product, or a vegetarian product.
  • Meat-, seafood substitutes or hybrid food products thereof, or a cheese product, or a vegetarian product may be comprised of the protein matrix composition obtained via the method of present invention.
  • Figure 1 Shows the harness of various edible products including a protein matrix composition (PMC) having a textured structure obtained via the method of present invention, and different meat and vegetarian products.
  • PMC protein matrix composition
  • the firmness of the products was determined by TPA, before (raw product) and after frying the products in a pan.
  • the product according to the method of present invention remained its harness also after frying and is within the range of the other meat/vegetarian products.
  • Example 1 agricultural side-streams as substrate
  • Beet pulp is the portion of sugar beet that remains after the sugar has been removed from the beet pulp at a processing plant.
  • the pulp was washed, strained, and put through a de -juicer to provide a moisture content of 88 wt%.
  • 4% w/w of rice flour was added in a vertical mixer.
  • the substrate mixture comprised of 16 wt% dry matter was put in vacuum sealed bags and pasteurized in a water bath at 72-82 °C for about 1-2 hours. After pasteurization, the mixture was allowed to cool to room temperature, about 18 to 25°C.
  • the mixture was inoculated with a strain of Rhizopus oligosporus at a concentration of 2g/kg in a vertical mixer disinfected with 70% ethanol.
  • the inoculated substrate placed in a vacuum bag and formed in a rectangular shape. Air holes were created by puncturing the vacuum bag with a metal skewer.
  • the bag was placed on a wired shelf in an incubator at 25-30 °C for 40- 60 hours to undergo solid-state fermentation.
  • the product after fermentation had a water content of about 70 wt%
  • BSG potato pulp or brewer’s spent grain
  • a substrate mixture comprised of 20-30% pea fibre, 4% rice flour , 0-5% protein isolate, 0-5% starch and 0-5% fibre from other origin were mixed with drinking-quality tap water in a vertical mixer, to obtain a total dry matter content of 30 wt%.
  • the mixture was put in vacuum sealed bags and pasteurized in a water bath at 72-82 °C for 1-2 hours. After pasteurization, the mixture was allowed to cool to room temperature.
  • the mixture was inoculated with a strain of Rhizopus oligosporus at a concentration of 2g/kg in a vertical mixer disinfected with 70% ethanol.
  • the inoculated substrate was placed in a vacuum bag and formed in a rectangular shape. Air holes were created by puncturing the vacuum bag with a metal skewer.
  • the bag was placed on a wired shelf in an incubator at 25-30 °C for 40- 60 hours.
  • the product after fermentation had a water content of about 75 wt%.
  • TPA Texture Profile Analysis
  • Example 4 agricultural side-streams in combination with refined food ingredients as substrate
  • beet pulp is the portion of sugar beet that remains after the sugar has been removed from the beet pulp at a processing plant.
  • the pulp was washed, strained and put through a de-juicer to provide a moisture content of 88 wt%.
  • 4% w/w of rice flour and 15 % of pea fibre was added in a vertical mixer.
  • the substrate mixture comprised of 26 wt% dry matter was put in vacuum sealed bags and pasteurized in a water bath at 72-82 °C for about 1,5 hours. After pasteurization, the mixture was allowed to cool to room temperature, about 22 °C.
  • the mixture was inoculated with a strain of Rhizopus oligo poru at a concentration of 2g/kg in a vertical mixer disinfected with 70% ethanol.
  • the inoculated substrate placed in a closable stainless-steel container and formed in a rectangular shape. Air holes were created by puncturing the vacuum bag with a metal skewer.
  • the bag was placed on a wired shelf in an incubator at 28 °C for about 1 hour to undergo solid-state fermentation.
  • the product after fermentation had a water content of about 70 wt%.

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Abstract

The present invention relates to a method for the production of a protein matrix composition having a textured structure from solid-state fermentation of food grade and/or agricultural biomass substrate, wherein the protein matrix composition is suitable for human consumption. Furthermore, the present invention relates to a protein matrix composition obtained via the method based on solid state fermentation, as well as to the use thereof as a meat- or seafood substitute composition.

Description

METHOD FOR THE PRODUCTION OF A PROTEIN MATRIX COMPOSITION
HAVING A TEXTURED STRUCTURE
Description
The present invention relates to a method for the production of a protein matrix composition having a textured structure from solid-state fermentation of food grade and/or agricultural biomass substrate, wherein the protein matrix composition is suitable for human consumption. Furthermore, the present invention relates to a protein matrix composition obtained via the method based on solid state fermentation, as well as to the use thereof as a meat substitute composition.
A demand for edible products having a high protein content that are of plant based origin (or non-animal) is increasing. An increasing awareness of personal health in combination with an increasing demand for a green footprint, leads to increased demand for edible products that include non-animal, plant sourced components such as proteins and fibers, including a growing demand for edible meat substitutes that mimic meat in its composition and texture but are composed of non- animal components. Furthermore, vegetarian food is gaining acceptance due to health concerns and environmental issues related with the production of meat. For example, with the production of beef meat large agricultural areas are involved in order to provide sufficient feed for the animals.
Within the vegetarian food sector there are relatively few meat substitutes which aim to provide the proteins which are recommended for daily intake as well as having an appealing texture. Examples of known meat substitutes are tofu, tempeh and mycoproteins known under the name Quorn. The mycoprotein which is used in Quorn is refined from Fusarium venenatum. The fungus is grown in continually oxygenated water in large fermentation tanks: so called liquid fermentation. The mycoprotein derived is washed and further texturized by using a binder in order to provide a meat like texture. The process involved to provide a palatable product requires considerable inputs in the form of energy and materials and is thus not efficient. Tofu is produced by using soybean derived proteins in a relatively simple and efficient process. However, tofu lacks taste and texture. Furthermore, present meat analogues are mostly comprised of texturized vegetable proteins that have been obtained via costly texturization processes and suffer from a limited supply of raw material and low manufacturing capacity. Furthermore, most meat analogues products need multiple processing steps to provide a suitable end product for human consumption, making the production process time consuming and costly.
In view of the above there is a need in the art for a cost effective method that enables the provision of a biobased (non-animal) protein source. The method should facilitate large scale production and adoption of biobased edible products, including a meat substitute composition by an efficient process. In addition there is a need in the art for a meat substitute having a texture which is meat like, comprises essential amino acids and provides a desired taste profile.
It is an object of the present invention, amongst other objects, to address the above need in the art. The object of present invention, amongst other objects, is met by the present invention as outlined in the appended claims.
Specifically, the above object, amongst other objects, is met, according to a first aspect, by the present invention by a method for the production of a protein matrix composition having a textured structure from solid-state fermentation of food grade and/or agricultural biomass substrate, wherein the protein matrix composition is suitable for human consumption, wherein the method comprises the steps of:
(i) providing and mixing of the food grade and/or agricultural biomass substrate comprising at least two different ingredients, wherein at least one of said ingredients comprises a fibrous texture, wherein the biomass substrate comprises a dry matter content of between 15 to 40 wt%, preferably between 20 to 35 wt%, and a moisture content of at least 50 wt%, based on the weight of the biomass substrate,
(ii) heat treatment of said biomass substrate until a core temperature of at least 72 °C, preferably at least 75 °C, more preferably at least 85 °C, most preferably at least 100 °C is achieved, for example by pasteurization or sterilization, followed by allowing the biomass substrate cool to room temperature, for example between 18 to 35 °C, preferably 20 to 25 °C,
(iii) inoculating said biomass substrate with an edible fungal- or yeast inoculum (spores/cells/mycelium) from the group of Ascomycetes and/or Zygomycoia: and
(iv) allow solid state fermentation to occur and said fungal- or yeast inoculum to grow in said biomass substrate for a period of between 24 to 96 hours, preferably 36 to 72 hours, to saturate the biomass substrate with mycelium and to provide the protein matrix composition having a textured structure comprised of at least 5 to 35 wt% protein, preferably 8 to 30 wt%, more preferably 10 to 25 wt%, most preferably 15 to 20 wt%, based on the total weight of the protein matrix composition.
By fermentation, it is possible to transform the substrate from a loose, granular structure, to a strong, cohesive but also flexible structure. This is due to the filamentous fungi which grow around and between the substrate, thereby forming a network. By using the present method, it becomes possible to produce a protein matrix composition having a texture that is meat-like with regard to the firmness. The term solid state fermentation as used in the present context means fermentation under controlled circumstances with solid nutrients, as opposed to liquid fermentation which takes place in an oxygenated broth in large vessels. In contrast to liquid fermentation, with solid state fermentation there is no need for expensive soluble nutrients. Materials with low value (like sugar cane or beet waste stream or Asparagus peelings) can thus be upgraded to a valuable food product. Another advantage of solid state fermentation is that it produces a food product that does not require further processing: it can be grown in the desired shape and size, the fully grown substrate can be sold as such, or cut in slices and sold in separate packages, it can be dried or stored in a freezer. There is no need for refining the fibers and extruding them to obtain a reasonable texture. The resulting protein matrix composition obtained via the method of present invention can be used as such, comparable to other solid state processes that produce protein compositions where fungi is harvested from a substrate which is not food-grade, i.e wood- (wood fibre), gras- or strawtype substrates, wool, or manure substrates. Further processing steps, such as clean up and removal of the growth substrate, or dewatering steps are not needed to make the substrate ready for use. The technical ingredient or semi-finished product can be used as is, or in, or part of vegan, vegetarian, hybrid or conventional animal based products and may for example be processed by increasing the heat kernel temperature of the protein matrix composition of 72 °C to enable human consumption. Another major advantage of solid-state fermentation is that, because dewatering is not needed, also much less energy is used and less waste is produced.
The moisture content of the biomass substrate is at least 50 wt %, such as from 60 to 80 wt %. Dry matter of the biomass substrate should be between 15 to 40wt%. Above 40 wt% dry matter the biomass substrate mixture will become too dry resulting in suboptimal growth conditions in support of the cultivation and development of the mycelium in order to form the protein matrix composition having the desired textured structure. In case the biomass substrate mixture comprises too little dry mass, i.e. a dry matter content of below 15 wt%, the textured structure will not be obtained and since there is more moist available in the biomass substrate mixture there is a higher chance of obtaining unwanted fungal infections or growth of microbes.
Growing of the present fungal- or yeast inoculum into mycelium is preferably done in growing conditions which are favourable for the used fungal- or yeast inoculum. Different strains each have their own preferences, however, most of the edible fungus have good mycelial growth at temperatures in the substrate of 18 to 35 C, more notably 25 C. The mycelium is allowed to grow in said substrate for between 24 to 96 hours, preferably 36 to 72 hours, more preferably 40 to 52 hours, most preferably 48 to 50 hours, at a temperature of between 20 to 30 °C, providing the protein matrix composition having a textured structure.
The fungal- or yeast inoculum should be edible, i.e. suitable for human consumption and derived from Ascomycetes and/or Zygomycota. Furthermore, fungal- or yeast mycelium that are non-sporulating, and have a high reproduction cycle and mainly produce white mycelium are preferred in the method of present invention. As such, Ascomycetes are used in the method of present invention since these groups of fungi do not produce external spore formations as seen for example with mushroom type fungi. In contrast Ascomycetes produce microscopic spores inside special, elongated cells or sacs, known as 'asci', which are responsible for the rapid spread of these fungi. Therefore, Ascomycytes have a more rapid growth rate than for example fungi that produce external spore formations or structures such as Basidiomycota fungi. Also Zygomycota do not form external spore formations for reproduction and may therefore also be suitable for use in the method of present invention.
According to a preferred embodiment, the present invention relates to the method, wherein the edible fungal- or yeast inoculum from the group of Ascomycetes or Zygomycota is one or more selected from the group consisting of Rhizopus oligosporus, Rhiz,opus oryzae, Aspergillus oryzae, Aspergillus tamarii, Trichoderma viride, Trichoderma reesi, Aspergillus nidulans, Aspergillus niger, Penicillium, Mucorales, preferably Rhizopus oligosporus . These fungal- or yeast inoculum provide a particular firm mycelium through the present substrate, therewith providing for example a suitable meat substitute composition having an increased firmness. For certain species, no known allergies have been detected and no levels of mycotoxins are produced. In addition to monocultures of filamentous fungi, multiple strains can be cultivated at once to tune the protein, amino acid, mineral, texture, and flavour profiles of the final biomass.
According to another preferred embodiment, the present invention relates to the method, wherein the biomass substrate comprises at least between 15 to 35 wt%, preferably 20 to 30 wt% of a plant derived ingredient or product selected from the group consisting of pea fibre, beet pulp, potatoes, soy, faba bean, corn, sugar cane, cellulose and bamboo, barley, brewers’ spent grain, rice, oat, wheat, preferably pea fibre and/or beet pulp. The inclusion of the edible fibrous plant derived ingredients provide a firm texture to the protein matrix composition.
According to yet another preferred embodiment, the present invention relates to the method, wherein the biomass substrate further comprises at least between 0.5 to 10 wt%, preferably 1 to 5 wt% of a cereal derived ingredient or product selected from the group consisting of rice, including rice flour, brewer’s spent grain, oats, wheat, maize, barley, rye, preferably rice. Inclusion of a cereal derived ingredient is preferred to provide the fungal- or yeast inoculum an optimal start of growth due to the increased presence of complex sugars such as starch. Furthermore, including for example rice flour has shown to provide fast production of high quality protein matrix composition using the method of present invention.
According to a preferred embodiment, the present invention relates to the method, wherein said biomass substrate is inoculated with fungal- or yeast inoculum at a concentration of between 100000 to 500000 cfu’s/kg, preferably 200000 to 400000 cfu’s/kg, most preferably 250000 to 300000 cfu’s/kg. If the fungal- or yeast inoculum concentration is too low (i.e. below 100000 cfu’s/kg), there is an increased chance of that an infection will take place by another microorganism, which will get the upper hand to grow further in the culture. However, when the inoculum concentration is too high, the mycelium will grow faster or too fast, which is economically less interesting and allows the mycelium to sporulate which is less attractive to the end product, providing black spores. It was found that 250000 to 300000 cfu’s/kg is most optimal.
According to a preferred embodiment, the present invention relates to the method, wherein the heat treatment comprises sterilizing or pasteurization of the present biomass substrate before introducing the fungal- or yeast inoculum. Preferably the sterilization is carried out by pasteurization, steam sterilization, gamma radiation, dry heat sterilization or chemical sterilization.
According to a preferred embodiment, the present invention relates to the method, wherein in step iv) the solid-state fermentation and said fungal- or yeast inoculum and mycelium growth in said biomass occurs in filter bags or vacuum sealed bags. Also, plastic containers or larger metal containers may be used. Filter bags provide sufficient aeration for a time period which is sufficient to saturate the substrate with mycelium. Saturation of the substrate is usually obtained before the moment when spore formation of the mycelium begins.
According to another preferred embodiment, the present invention relates to the method, wherein pH regulators, nitrogen sources, such as amino acids and/or urea derivates, enzymes, preservatives, colorants, flavouring agents, binders, hydrocolloids, starches, gums, fats, and/or salt are added to the mixture of step i). Additive such as colorants and/or flavourings should be mixed in with the substrate in step i, since after step iv, solid state fermentation, the mixing would result in a break up the mycelium structure of the end product.
According to another preferred embodiment, the present invention relates to the method, wherein the food grade and/or agricultural biomass substrate is a residual or waste substrate from the food and/or agricultural industry. The food and agricultural industries produce high volume food grade waste streams containing quality nutrients, which often goes to feed animals at an inefficient conversion rate or is treated as a complete waste. However, such food grade waste streams provide a valuable food grade source of nutrients that is very suitable for use in the method of present invention.
The present invention, according to a second aspect, relates to a protein matrix composition having a textured structure obtainable by the method of present invention as disclosed above, wherein said protein matrix composition has a firmness of between 6 to 8 Newton, as determined by a peak force at first compression by a texture profile analysis (TPA) according to example 3. The protein matrix composition of present invention has a textured structure and a firmness that is comparable to that of meat, cheese or milk product, seafood, and known me at and seafood vegetarian substitute products, preferably meat or seafood. The firmness is determined as described in example 3 using the TPA method. The product according to the method of present invention remained its hardness between 6 to 8 Newton, also after frying and is within the range of the other meat/vegetarian products. The present invention, according to a further aspect, relates to a meat substitute composition comprised of the protein matrix composition or to the use of the protein matrix composition for the provision of an edible consumer product such as a meat- or seafood analogues or hybrid thereof, a cheese product, or a vegetarian product. Meat-, seafood substitutes or hybrid food products thereof, or a cheese product, or a vegetarian product may be comprised of the protein matrix composition obtained via the method of present invention.
The invention will be further elucidated in the following figure and examples, showing non limitative embodiments of the present invention.
Figure 1: Shows the harness of various edible products including a protein matrix composition (PMC) having a textured structure obtained via the method of present invention, and different meat and vegetarian products. The firmness of the products was determined by TPA, before (raw product) and after frying the products in a pan. The product according to the method of present invention remained its harness also after frying and is within the range of the other meat/vegetarian products.
Examples
Example 1: agricultural side-streams as substrate
As agricultural side-stream sugar beet pulp was used. Beet pulp is the portion of sugar beet that remains after the sugar has been removed from the beet pulp at a processing plant. The pulp was washed, strained, and put through a de -juicer to provide a moisture content of 88 wt%. 4% w/w of rice flour was added in a vertical mixer. The substrate mixture comprised of 16 wt% dry matter was put in vacuum sealed bags and pasteurized in a water bath at 72-82 °C for about 1-2 hours. After pasteurization, the mixture was allowed to cool to room temperature, about 18 to 25°C.
The mixture was inoculated with a strain of Rhizopus oligosporus at a concentration of 2g/kg in a vertical mixer disinfected with 70% ethanol. The inoculated substrate placed in a vacuum bag and formed in a rectangular shape. Air holes were created by puncturing the vacuum bag with a metal skewer. The bag was placed on a wired shelf in an incubator at 25-30 °C for 40- 60 hours to undergo solid-state fermentation. The product after fermentation had a water content of about 70 wt%
The same process was also applied to other agricultural side-streams including potato pulp or brewer’s spent grain (BSG). BSG is obtained as a mostly solid residue after wort production in the beer brewing process. Example 2: refined ingredients as substrate
As refined food ingredient, commercial pea fibre was used. A substrate mixture comprised of 20-30% pea fibre, 4% rice flour , 0-5% protein isolate, 0-5% starch and 0-5% fibre from other origin were mixed with drinking-quality tap water in a vertical mixer, to obtain a total dry matter content of 30 wt%. The mixture was put in vacuum sealed bags and pasteurized in a water bath at 72-82 °C for 1-2 hours. After pasteurization, the mixture was allowed to cool to room temperature.
The mixture was inoculated with a strain of Rhizopus oligosporus at a concentration of 2g/kg in a vertical mixer disinfected with 70% ethanol. The inoculated substrate was placed in a vacuum bag and formed in a rectangular shape. Air holes were created by puncturing the vacuum bag with a metal skewer. The bag was placed on a wired shelf in an incubator at 25-30 °C for 40- 60 hours. The product after fermentation had a water content of about 75 wt%.
By fermentation, it is possible to transform the substrate from a loose, granular structure, to a strong, cohesive but also flexible structure. This is due to the filamentous fungi which grow around and between the substrate, thereby forming a network.
Example 3: Texture profile analysis (TPA)
The texture and hardness or firmness of different types of meat and meat substitutes and the protein matrix composition having a textured structure obtained via the method of present invention, using Texture Profile Analysis (TPA) was determined, which provides a measure of the force required to compress the product. Briefly, samples of 30 mm thickness were measured at a “peak force at first compression” (= Fmaxl), using TEXTURE ANALYZER TX-700 (Lamy Rheology), compression until 5 mm, compression speed 1.5 mm/s, time between first and second compression 5 seconds, and measured at room temperature. Results were analysed using Rheotex software.
It was determined that for meat the Fmaxl was between 4.5-20 N (N=Newton), with majority of meats tested were on the higher end of this range. For vegetarian product the Fmaxl was between 3.0 - 20 N, but here majority of vegetarian burgers were on the low end of this range. In general, vegetarian products are therefore on the soft side (the exception was vegetarian chicken, which was very firm) and meat is firmer. Next, a product obtained via the method of present invention, comprised of 25% pea fiber and 4% rice flour was determined to have a Fmaxl of between 6-8 N. Therefore, the protein matrix composition having a textured structure is therefore somewhat between the Fmaxl values of meat and vegetarian products.
Next, TPA measurements were performed with the products after frying the products in a pan. The product according to the method of present invention remained its hardness also after
Figure imgf000009_0001
frying and is within the range of the other meat/vegetarian products. Figure 1 summarizes the results of the TPA experiment.
Example 4: agricultural side-streams in combination with refined food ingredients as substrate
As agricultural side-stream sugar beet pulp was used in combination with pea fibre. Beet pulp is the portion of sugar beet that remains after the sugar has been removed from the beet pulp at a processing plant. The pulp was washed, strained and put through a de-juicer to provide a moisture content of 88 wt%. 4% w/w of rice flour and 15 % of pea fibre was added in a vertical mixer. The substrate mixture comprised of 26 wt% dry matter was put in vacuum sealed bags and pasteurized in a water bath at 72-82 °C for about 1,5 hours. After pasteurization, the mixture was allowed to cool to room temperature, about 22 °C.
The mixture was inoculated with a strain of Rhizopus oligo poru at a concentration of 2g/kg in a vertical mixer disinfected with 70% ethanol. The inoculated substrate placed in a closable stainless-steel container and formed in a rectangular shape. Air holes were created by puncturing the vacuum bag with a metal skewer. The bag was placed on a wired shelf in an incubator at 28 °C for about 1 hour to undergo solid-state fermentation. The product after fermentation had a water content of about 70 wt%.
SUBSTITUTE SHEET (RULE 26)

Claims

1. Method for the production of a protein matrix composition having a textured structure from solid-state fermentation of food grade and/or agricultural biomass substrate, wherein the protein matrix composition is suitable for human consumption, wherein the method comprises the steps of:
(i) providing and mixing of the food grade and/or agricultural biomass substrate comprising at least two different solid ingredients, wherein at least one of said ingredients one comprises a fibrous texture, wherein the biomass substrate comprises a dry matter content of between 15 to 40 wt%, and a moisture content of at least 50 wt%,
(ii) heat treatment of said biomass substrate until a core temperature of at least 72 °C is achieved, followed by allowing the biomass substrate cool to room temperature, for example between 18 to 35 °C,
(iii) inoculating said biomass substrate with an edible fungal- or yeast inoculum from the group of Ascomycetes or Zygomycota,' and
(iv) allow solid state fermentation to occur and said fungal- or yeast inoculum to grow in said biomass substrate for a period of between 24 to 96 hours, preferably 36 to 72 hours, to saturate the biomass substrate with mycelium and to provide the protein matrix composition having a textured structure comprised of at least 5 to 35 wt% protein, based on the total weight of the protein matrix composition.
2. Method according to claim 1, wherein the edible fungal- or yeast inoculum from the group of Ascomycetes or Zygomycota is one or more selected from the group consisting of Rhizopus oligosporus, Rhizopus oryzae, Aspergillus oryzae, Aspergillus tamarii, Trichoderma viride, Trichoderma reesi, Aspergillus nidulans, Aspergillus niger, Penicillium, Mucorales, preferably Rhizopus oligosporus.
3. Method according to claim 1 or 2, wherein the biomass substrate comprises at least between 15 to 35 wt%, preferably 20 to 30 wt% of a plant derived ingredient or product selected from the group consisting of pea fibre, beet pulp, potatoes, soy, faba bean, corn, sugar cane, cellulose, bamboo, barley, brewers’ spent grain, rice, oat, wheat, preferably pea fibre and/or beet pulp.
4. Method according to any of the claims 1 to 3, wherein the biomass substrate further comprises at least between 0.5 to 10 wt%, preferably 1 to 5 wt% of a cereal derived ingredient or product selected from the group consisting of rice, including rice meal or rice flour, brewer’s spent grain, oats, wheat, maize, barley, rye, preferably rice.
5. Method according to any of the claims 1 to 4, wherein said biomass substrate is inoculated with fungal- or yeast inoculum at a concentration of between 100000 to 500000 cfu’s/kg, preferably 200000 to 400000 cfu’s/kg, most preferably 250000 to 300000 cfu’s/kg.
6. Method according to any of the claims 1 to 5, wherein the heat treatment comprises sterilizing or pasteurization of the present biomass substrate before introducing the fungal- or yeast inoculum, preferably the sterilization is carried out by pasteurization, steam sterilization, gamma radiation, dry heat sterilization or chemical sterilization.
7. Method according to any of the claims 1 to 6, wherein in step iv) the solid state fermentation and said mycelium growth in said biomass occurs in filter bags or vacuum sealed bags.
8. Method according to any of the claims 1 to 7, wherein pH regulators, nitrogen sources, such as amino acids and/or urea derivates, enzymes, preservatives, colorants, flavouring agents, binders, hydrocolloids, starches, gums, fats, and/or salt are added to the mixture of step i).
9. Method according to any of the claims 1 to 8, wherein the food grade and/or agricultural biomass substrate is a residual or waste substrate from the food and/or agricultural industry.
10. A protein matrix composition having a textured structure obtainable by the method according to any of the claims 1 to 8, wherein said protein matrix composition has a firmness of between 6 to 8 Newton, as determined by a peak force at first compression by a texture profile analysis (TP A) as disclosed in example 3.
11. Meat substitute composition comprised of a protein matrix composition obtainable by the method according to any of the claims 1 to 9.
12. Use of a protein matrix composition according to claim 10, for the provision of an edible consumer product such as a meat- or seafood analogues or hybrid thereof, a cheese product, or a vegetarian product.
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