Process for manufacturing a meat substitute composition and related products

Abstract

A process (100) for manufacturing a meat substitute composition from leguminous substrates and a meat substitute composition obtainable by said process are provided. The process comprises fermenting leguminous substrates, such as dehulled faba beans, using a starter composition comprising a co-culture of Rhizopus oligosporus and Propionibacterium freudenreichii, in solid-state fermentation conditions. A process for manufacturing edible meat substitute items for human and animal consumption, and use of a co-culture of R. oligosporus and P. freudenreichii as a starter culture in manufacturing fermented meat substitutes from dehulled faba beans are further provided.

Description

[0001] PROCESS FOR MANUFACTURING A MEAT SUBSTITUTE

[0002] COMPOSITION AND RELATED PRODUCTS

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the processes of manufacturing fermented leguminous plant-based meat substitute compositions and foodstuffs obtained therefrom. The invention further relates to preparing a microbiological starter to be used in manufacturing of the meat substitute composition.

[0005] BACKGROUND

[0006] Growing numbers of people choose vegetarian diet due to economical and ethical considerations, as well as for health reasons. Indeed, there is an increased consumer demand for alternative protein-rich foods that are not derived from animals and hence essentially void of saturated fatty acids cholesterol, but that have flavor, taste and texture similar to animal-derived meat products.

[0007] Leguminous plants or legumes are well-acknowledged as valuable alternatives to animal-based proteins. The term “legume” encompasses the plants in the Fabaceae (or Leguminosae) botanical family, as well as seeds of these plants. When used as dry grains, edible seeds of certain leguminous plants are known as “pulses”. Most common pulses include for example dried beans, chickpeas, lentils, peas and soybeans.

[0008] Nutritional properties, as well as vitamin and mineral composition and content of plant-based foods can be further enhanced using fermentation of plant-based substrates with various microorganisms, such as bacteria or filamentous fungi.

[0009] Vitamins are important components of balanced nutrition. Vitamin B 12 (cobalamin) is an essential vitamin for humans as being a cofactor of enzymes which are involved in the synthesis of DNA, fatty acids and myelin. Vitamin B12 is involved in development of red blood cells, nerve cells and brain function; its deficiency can cause a variety of symptoms including fatigue, headache and depression, and lead to more severe conditions, such as megaloblastic anemia, peripheral arterial diseases and various neurological disorders. Humans cannot synthesize vitamin B12, and hence it must be received from external sources. Moreover, to be effective in humans, vitamin B 12 must be provided in a metabolically active form. In nature this vitamin is synthesized by few bacteria, and it is concentrated mainly in the bodies of ruminants and higher predatory organisms in the natural food chain system. Hence, major natural dietary sources of vitamin B 12 are represented with animal foods (meat, fish, milk, etc.) but not plant foods.

[0010] Although few bacteria produce active vitamin B12 de novo, a majority of B12- producing microorganisms generate a so-called pseudoform which is inactive. Active vitamin B12 is distinguished from the pseudovitamin by the presence of 5,6- dimethylbenzimidazole (DMBI) as a lower ligand coordinated to a central atom of cobalt (in the pseudoform the lower ligand is adenine). DMBI as the lower ligand is crucial for binding of cobalamin to a glycoprotein responsible for its transport in the human gastrointestinal tract; therefore, cobalamins comprising DMBI as lower ligands are of special human nutritional value. Hence, the processes of natural fortification of foods by microbial fermentation aim at production of active vitamin B12.

[0011] Propionibacterium freudenreichii has been utilized to fortify some plant-based foods, such as cereal and cereal bran with vitamin B12. Anaerobic, aerotolerant P. freudenreichii produces notable amounts of active vitamin B12 and can be used as such in food production as it is generally recognized as safe (GRAS). However, production of vitamin B12 by fermentation of plant-based substrates with this propionibacterium is limited because of low cobalt content in plants.

[0012] This problem can be tackled by using legumes instead of cereals, because cobalt is essential for bacteria on root nodules of legumes, and it is typically supplied to legume crops during cultivation.

[0013] R freudenreichii used together with a mold fungus Rhizopus oligosporus in cofermentation of soybean has been reported to increase the vitamin B 12 content in an Indonesian food tempeh (He & Howell, 2022 [1]), usually produced from soybeans fermented with R. oligosporus. Similar study on fortifying a legume lupin-based tempeh analog with vitamin B12 as a result of cofermentation of lupin (namely, Lupinus albus, L. angustifolius and L. mulabilis) seeds with R. oligosporus and P freudenreichii has been reported by Signorini et al., 2018 [2],

[0014] However, one of the major problems encountered so far with the most dietary legumes is their allergenicity. Amongst those, soybeans, peanuts and lupin represent the top allergens. Because of associated allergies, as well as cross-reactivity observed therebetween, consumption of these pulses has been naturally limited. In this regard, it would be desirable to update the field of technology related to production of meat substitutes from leguminous feedstocks, in view of exploiting an entire range of nutritive components included in pulses and / or avoiding health risks potentially associated with (over)consumption of animal protein and fats obtained mainly from red meat, as well as those associated with food allergies. Hence, further advances in development of meat substitutes suitable for a broader range of consumer groups is still highly looked for. It is further desirable that the resulted meat substitute product would be appealing to the customer in terms of aroma, taste and appearance.

[0015] SUMMARY OF THE INVENTION

[0016] An objective of the present invention is to solve or to at least mitigate at least some of the problems arising from the limitations and disadvantages of the related art. One or more objectives are achieved by various embodiments of a process for manufacturing a meat substitute composition from leguminous substrates, a process for manufacturing a meat substitute product for human or animal consumption, related products and uses as defined herein. Thereby, in one aspect of the invention the manufacturing process is provided, according to what is defined in the independent claim 1.

[0017] In an aspect, a process is provided for manufacturing a meat substitute composition from leguminous substrates, in accordance with what is defined in the independent claim 1.

[0018] In an embodiment, the process comprises: (z) preparing a starter culture comprising a co-culture of Rhizopus oligosporus and Propionibacterium freudenreichii in a cultivation medium; (zz) obtaining a leguminous substrate and inoculating thereof with the starter culture obtained at step (i), and (zzz) fermenting the leguminous substrate in solid-state fermentation conditions, wherein the leguminous substrate is dehulled faba beans.

[0019] In an embodiment, in said process, the starter culture is added to the leguminous substrate to a concentration within a range of about 0.7 wt-% to about 1.5 wt-%.

[0020] In an embodiment, the process further comprises admixing of at least one enzyme with the leguminous substrate to a concentration of about 2 wt-%, wherein said at least one enzyme is preferably transglutaminase.

[0021] In an embodiment, in said process, preparation of the starter culture comprises incubation of a co-culture of R. oligosporus and R freudenreichii with the cultivation medium at a temperature within a range of about 28-38 °C for a period of about 72 hours to about 96 hours, wherein the co-culture is provided in the cultivation medium in a concentration of about 1 wt-%. In some exemplary embodiment, the cultivation medium is rice.

[0022] In an embodiment, in said process, prior to inoculation with the starter culture the leguminous substrate is pretreated by soaking in water for a period of about 6 hours to about 12 hours followed with boiling and drying.

[0023] In an embodiment, in said process, fermentation of the leguminous substrate using the starter culture is implemented in a batch reactor of a packed-bed type, wherein a ratio between a bed volume occupied by the substrate and a headspace volume is about 30 vol-% to 70 vol-%. In an embodiment, said fermentation procedure is conducted at a temperature within a range of about 28-33 °C during a period of about 18 hours to about 24 hours.

[0024] In an embodiment, the process further comprises: (zv) homogenization of a fermented leguminous substrate obtainable at step (zzz), followed with cooling and drying to obtain a homogenized product with a moisture content within a range of about 65% to about 70%.

[0025] In another aspect, a meat substitute composition is provided, in accordance with what is defined in the independent claim 10.

[0026] In another aspect, a process for manufacturing a meat substitute product for human or animal consumption is provided, in accordance with what is defined in the independent claim 11. In an embodiment, manufacturing of the meat substitute product comprises a process for manufacturing the meat substitute composition according to some previously defined aspects and embodiments, and further comprises post-treatment of said meat substitute composition to form an edible meat substitute item. In an embodiment, the meat substitute product comprises the meat substitute composition obtainable according to some previously defined aspects and embodiments.

[0027] In an embodiment, post-treatment of the meat substitute composition comprises a procedure selected from the group consisting of: thermal processing, non-thermal processing, shaping into mold, extrusion, pressing, drying, freezing, and any combination thereof. In an embodiment, post-treatment of the meat substitute composition comprises at least one procedure configured to yield the meat substitute product in the form of: a solid food item, a semisolid food item, or a dried food item in the form of powder, a granule, or a pellet.

[0028] In another aspect, a meat substitute product for human or animal consumption is provided, in accordance with what is defined in the independent claim 14.

[0029] In another aspect, use of the process according to previously defined embodiments is provided in manufacturing of meat substitute products for human or animal consumption.

[0030] The co-culture of Rhizopus oligosporus and Propionibacterium freudenreichii obtainable as described in the present disclosure can be used as a starter culture in manufacturing fermented meat substitutes from leguminous substrates, preferably, from dehulled faba beans.

[0031] The utility of the present invention arises from a variety of reasons depending on each particular embodiment thereof. At first, the invention generally aims at solving the problem of providing a method for manufacturing a fermented, high-protein meat substitute composition which can be used per se in various recipes available for consumer and further process to form an edible meat substitute products for human or animal consumption. The disclosed method enables manufacturing of a meat-like ingredient from improved in terms of its nutritional properties (high-protein, low-fat and naturally gluten free) and further provided as non-GMO and not a regulated allergen. This is achieved by using novel leguminous substrates, such as faba beans that undergo fermentation in specially developed process conditions.

[0032] In the present disclosure, the term “co-fermentation” generally relates to fermentation of a substrate with at least two different microorganisms. Accordingly, the term “costarter” generally relates to an inoculum comprising a combination of at least two different microorganisms.

[0033] Within the present disclosure the term “comprises” is intended to be construed as “includes, among other things”; whereas the term “consists of’ is intended to be construed as “consists of only”.

[0034] Different embodiments of the present invention will become apparent by consideration of the detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Fig. 1 is a flow chart that illustrates, at 100, a process for manufacturing an edible meat substitute composition, according to the embodiments.

[0036] Fig. 2 is a flow chart that illustrates, at 200, a process for manufacturing a meat substitute product for human and animal consumption, according to the embodiments.

[0037] Fig. 3 is a flow chart that illustrates, at 300, a process for preparing a (co)starter culture used in the processes 100 and 200.

[0038] Figs. 4A-4C show the results of experimentally assessing the ability of Propionibacterium freudenreichii to produce vitamin B 12 in solid state fermentation conditions.

[0039] DETAILED DESCRIPTION OF THE DRAWINGS

[0040] Fig. 1 illustrates, at 100, a concept underlying various embodiments of a process for manufacturing a meat substitute composition from leguminous substrates. The leguminous substrates preferably comprise pulse substrates. Still preferably, the leguminous substrate comprises dehulled faba beans (Vicia faba Lf

[0041] Along with soybeans, the faba bean has a highest protein content amongst pulses. Amount of lean protein in the faba bean is thus about 25-35%. However, compared to soy the faba bean is not genetically modified (non-GMO) and not a regulated allergen. In the other hand, in comparison to cereals (e.g. wheat), faba bean substrates contain twice as much protein. Additionally, faba bean is naturally gluten-free and offers a rich selection of vitamins, minerals and bioactive compounds. In comparison to soybeans, for example, commonly cultivated in North and South Americas, the faba bean can be grown worldwide and further - some varieties of the faba bean are capable of surviving cold winters (down to about -15 to -17 °C) common in Nordic countries and not uncommon on the European continent.

[0042] The process 100 enables manufacturing of the meat substitute composition which can be further used as an ingredient for production of meat substitute products (meat substitute food items suitable for human and animal consumption).

[0043] The process starts at 102, with preparation of a starter culture composition further referred to as a “starter culture”. The starter culture is preferably provided as a so- called co-starter comprising a co-culture (mixed culture) of at least two different microorganisms cultivated together on same cultivation medium. The starter culture composition preferably comprises a co-culture of the fungus Rhizopus oligosporus and the bacterium Propionibacterium freudenreichii inoculated on a cultivation medium. In embodiments, the cultivation medium comprises or consists of rice grains and / or rice flour; however, any other appropriate medium is not excluded.

[0044] In the process 100, any suitable strain of R. oligosporus and of P. freudenreichii can be utilized.

[0045] In a non-limiting example, the process 100 utilized propionibacterium P freudenreichii ssp. freudenreichii, in particular, P freudenreichii ssp. freudenreichii, strain DSM 20271 purchased from the Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures and the spores of mold fungus R. oligosporus available as a commercial starter from the TopCultures.

[0046] The above indicated combination of microorganisms selected for producing the (co)starter culture for co-fermentation of faba beans is substantiated with the fact that P freudenreichii is capable of naturally producing vitamin B12 in its metabolically active form, hence having a special nutritional value for humans. On the other hand, R. oligosporus hydrolyzes the faba bean storage proteins releasing essential amino acids and peptides, therefore nutritional value of the fermented product is improved. Ability of P. freudenreichii to produce vitamin B 12 was experimentally evaluated, as described hereinbelow with reference to Example 1.

[0047] The process may include pretreatment of the cultivation medium (not shown). In an event dry (rice) grains are used as cultivation medium, pretreatment may include soaking those in water or in appropriate aqueous solution(s) followed with heat treatment (e.g. boiling, steaming or the like), drying and cooling. Exemplary pretreatment procedures include soaking during a time period within a range of 1-2 hours, followed with draining and boiling for about 5-10 minutes. Thermally treated cultivated medium is then cooled down to about 33-38 degrees Celsius (°C).

[0048] Fig. 3 illustrates, at 300, a process of preparation of the starter culture composition at stage 102. Stage 102 of the process 100 (Fig. 1) thus comprises or consists of the process 300 of Fig. 3.

[0049] In an exemplary process 300, rice grains used as cultivation medium are pretreated by procedure(s) as described herein above. A co-culture of R. oligosporus and P freudenreichii obtained at 302 is combined with the cultivation medium (step 304) to a final concentration of 1% (w / w) (or 1 percent by weight, wt-%). In embodiments, isolated spores of R. oligosporus (104spores / g) and a concentrated bacterial culture of R freudenreichii (104CFU / g) are combined with rice flour and further admixed with the cultivation medium, such as pretreated rice grains, to a final concentration of about 1% (w / w) (or 1 percent by weight, wt-%). In some other embodiments, the above-mentioned microorganisms are combined with the cultivation medium comprising a mixture of rice grains (pretreated as above) and rice flour, to a final concentration of about 1 wt-%. An amount of rice flour in a mixture of microorganisms with the cultivation medium is about 10 wt-%. Using rice flour or similar powder-textured material is advantageous as it prevents clumping of the cultivation medium. Alternatively, a co-culture of microorganisms as above can be added directly to pretreated rice grains. The co-culture of R. oligosporus and P freudenreichii is further incubated (306, Fig. 3) with the cultivation medium at a temperature within a range of about 28-38 °C for a predetermined period of time. The incubation period is provided within a range of about 72 hours to about 96 hours. Thereafter, the starter culture composition is obtained, and it can used in the process 100.

[0050] With reference back to Fig. 1, at 102, a (co)starter culture composition is thus obtained, comprising R. oligosporus and P freudenreichii in a suitable cultivation medium. This (co)starter composition is advantageously configured to perform (co)fermentation of leguminous substrates, as described further below.

[0051] The process 100 (Fig. 1) continues at 104, wherein the leguminous substrate comprising dehulled faba beans is inoculated with the starter culture prepared at 102 (through the process 300). The starter culture is thus added to the leguminous substrate to achieve final concentration within a range of about 0.7 wt-% to about 1.5 wt-%.

[0052] In embodiments, the process 100 further comprises subjecting the leguminous substrate comprising dehulled faba beans to enzymatic treatment. At 114, at least one enzyme is thus admixed with the substrate to achieve final concentration of about 2 wt-%. In embodiment, said at least one enzyme is admixed with the substate essentially simultaneously with the starter culture. Alternatively, the at least one enzyme can be added to the substrate pre-inoculated with the starter culture. In some instances, the starter culture may be added to the substrate pre-supplied with the at least one enzyme.

[0053] In embodiment, the at least one enzyme is a protein crosslinking enzyme, preferably, transglutaminase. Transglutaminases (TG, EC 2.3.2.13, gamma-glutamyl-peptide, amine-gamma-glutamyl-transferase) belong to a group of acyltransferases that catalyze formation of an isopeptide bond between glutamine and lysine residues and amine incorporation into specific glutamine residues. As a results, new macromolecular clusters are created having physicochemical properties different from those of a native protein. Treatment with protein crosslinking enzyme(s), such as transglutaminases, enhance nutritional properties of faba bean substrates; therefore, improved functional ingredients, such as meat substitute composition, described herewith, can be formulated. Additionally, transglutaminase improves gelling properties of faba bean substrates. Increased gelling can be attributed to enhanced protein-protein interactions and improvement in the mechanical and rheological properties such as the gel strength, structural, and conformational flexibility.

[0054] Enzymatic modification of leguminous substrates may include, in addition to or instead of crosslinking, proteolytic treatment with various proteases (e.g. pepsin, trypsin, etc.). Enzymatic treatment of faba beans with proteases may significantly increase protein solubility and oil-holding capacities.

[0055] Prior to being inoculated, the leguminous substrate is preferably pretreated (not shown). Pretreatment includes washing and dehulling faba bean feedstocks, soaking thus prepared substrate medium (dehulled faba beans) in water or appropriate aqueous solution(s) followed with heat treatment (e.g. boiling, steaming or the like) and cooling. During the pretreatment stage, the faba bean substrate is preferably size- reduced through grinding and / or shear mixing, for example.

[0056] Pretreatment of an exemplary 200 kg batch of dehulled faba bean substrate thus includes soaking in 300 liters of water for a time period of about 6-12 hours. After draining, soaked faba beans are further heat-treated for about 15-20 min followed with drying to a temperature of about 40 °C or less at room temperature. Exemplary heat treatment includes boiling (350 L water with 0.3 % acetic acid). Moisture content in pretreated substrate is within a range of about 60-70 % (to compare, freshly harvested faba bean biomass has moisture content of about 10%).

[0057] The starter culture and the at least one enzyme may be mixed with the (pretreated) substrate in a fermentation reactor or in a separate vessel. Mixing of the (co)starter microorganisms and the enzyme(s) may be performed at the same time or in separated steps. Size-reduction of the substrate may be performed simultaneously with the mixing procedure. The process 100 continues at 106, comprising fermenting of the leguminous substrates in solid-state fermentation conditions. Solid-state fermentation (SSF) systems are generally used for fermentation of moist solid substrates with microorganisms in an absence of free-flowing water. In embodiments, the SSF process 106 is implemented in a batch reactor, preferably of a “packed-bed” type. The packed-bed (bio)reactor may be implemented with a static bed and essentially no aeration or the bed may be mixed very infrequently, such as once per day, for example. In some instances, the SSF process may include blowing air through the bed of substrate.

[0058] Trial batch included 200 kg of the (pretreated) substrate; however, the process is fully scalable to operate with larger (or smaller) batches. In the SSF reactor, a bed volume occupied by the substrate constitutes about 25-40 percent by volume (vol-%) of the volume of the reactor; while a headspace volume constitutes about 60-75 vol-%, respectively. In an embodiment, this ratio (bed volume occupied by the substrate vs headspace volume) is about 30 vol-% to about 70 vol-%. Humidity level (defined hereby as a percentage of liquid / water vapor in the air) in the reactor thus constitutes 60-70%. Solid-state fermentation 106 is conducted at a temperature within a range of about 28-33 °C during a period of about 18 hours to about 24 hours.

[0059] Fermented substrate is further subjected, at 108, to mechanical processing implemented through mixing and / or homogenization. During homogenization, the fermented faba bean is disintegrated into smaller particles to create a stable, preferably homogenous (uniform) dispersion. Homogenization is advantageously performed in a separate homogenizer apparatus configured as a shear-mixer or a high- pressure homogenizer, for example, at a temperature within a range of about 80 °C to about 95 °C for 20-30 minutes. In some instances, homogenization time is 25 minutes. At 108, the substrate can be supplied with salt (NaCl) added to a final concentration of about 0.5 % (w / w).

[0060] Alternatively, homogenization may be conducted during the SSF process, whether the latter is implemented a bioreactor equipped with agitation means.

[0061] At 110, fermented and homogenized faba bean is drained (mostly aiming at removal of surface moisture) and cooled to a temperature of about 6 °C or less, such as to about 4-6 °C. Draining is typically realized by letting the liquid flow away when the substrate is taken off the (bio)reactor / homogenizer. In some instances, draining and / or cooling processes may be optionally accompanied with drying, such as airdrying, for example, whereupon a fermented faba bean material having moisture content of about 60-75%, preferably 65-70%, further referred to as a meat substitute composition is obtained (see step 112). It is preferred that the moisture context of the meat substitute composition is adjusted to correspond to the moisture context in a nature analog (beef, lamb, poultry, etc.).

[0062] Meat substitute composition was analyzed for the content of faba bean protein and vitamin B 12. To measure protein content, the Kjeldahl method was utilized. Content of B12 vitamin was analyzed using Ultra-High-Performance Liquid Chromatography (UHPLC) and common microbiological assays. In embodiments, the meat substitute composition comprises faba bean protein in an amount of at least 16 gram per 100 gram of the composition and B12 vitamin in an amount of at least 0,009 pg / g (0,9 microgram per 100 gram of the composition). Overall, the content of the faba bean protein may vary in said composition within a range of about 16-30 g per 100 g of the composition, and the content of vitamin B 12 may vary within a range of about 0,009-0,015 pg / g. To compare, 100 g of boiled beef contains approximately 1,3 pg of B12 vitamin. Thus obtained composition can be further used as an ingredient in manufacturing of a variety of edible meat substitute (or meat replacement) products, according to a process described herein below.

[0063] The invention further concerns use of the process 100 and / or the composition manufacturing by the process 100 in manufacturing of edible meat substitute products for human or animal consumption.

[0064] Fig. 2 illustrates, at 200, a process for manufacturing a meat substitute product for human or animal consumption. The process 200 is implemented based on the process 100 according to the embodiments described above and it comprises steps 102-112, with or without 114. Additionally, the process 200 comprises a post-treatment 116 of a meat substitute composition obtained by the process 100, whereby an edible meat substitute item is formed.

[0065] Post-treatment 116 generally involves conversion of the meat substitute composition (a foodstuff ingredient) into an edible food product suitable for consumption by humans or animals. The meat substitute product is thus formulated as an essentially complete meal which can be consumed as such or after minor processing, such as heating in a microwave, baking, frying or grilling. Post-treatment 116 of the meat substitute composition may be implemented through at least procedure including: thermal processing (e.g. thermal pasteurization), non-thermal processing (e.g. ultrasonication, UV treatment, etc.), shaping into mold, extrusion, pressing, drying, freezing (including dry-freezing), or any combination thereof. In embodiment, post-treatment 116 of the meat substitute composition comprises at least one procedure configured to yield the meat substitute product in the form of: a solid food item, a semisolid food item, or a dried food item in the form of powder, a granule, or a pellet.

[0066] The invention further provides for an edible meat substitute product for human or animal consumption obtainable by the manufacturing process 200 and / or comprising or consisting of a fermented leguminous substrate based composition obtainable in the process 100 according to the embodiments described hereinabove. The meat substitute product is thus provided in the form of any one of: a solid food item, a semisolid food item, and a dried food item in the form of powder, a granule, or a pellet.

[0067] A variety of meat substitute products include for example burger-type steaks, fillets, chops, chunks, slices, minced meat-like products, sausages, and the like, shaped items such as meatballs, nuggets and sticks, various ragout-type foods, etc. A variety of pet foods can be produced accordingly, formulated as dry or wet foodstuff products. Diversity of these products may vary with consistency, texture, taste, aroma, and flavor.

[0068] The meat substitute product may further comprise supplementary components, such as stabilizers, preservatives, and / or flavor-enhancing agents.

[0069] The meat substitute product can be further finished and packed depending on the nature thereof. Suitable packing includes, but is not limited with boxes, bags or cans. In some instances, the end product may comprise a properly packed meat substitute composition for use as a meat replacement ingredient in various recipes.

[0070] The meat substitute product is advantageously rendered with texture, flavor and taste of meat (e.g. beef, lamb or pork). Alternatively, the meat substitute product may be rendered with texture, flavor and taste of poultry birds (e.g. chicken or turkey), fish or shellfish.

[0071] The invention further concerns use of a co-culture of Rhizopus oligosporus and Propionibacterium freudenreichii as a starter culture in manufacturing fermented meat substitutes from leguminous substrates, preferably, from dehulled faba beans.

[0072] Example 1. Evaluation of P freudenreichii ability to produce vitamin B 12 in solid state fermentation conditions. In order to assess ability of P freudenreichii to produce vitamin B12, bacterial population densities (CFU / g) were evaluated in faba bean substrates before and after fermentation, and B12-vitamin content was quantified. Faba bean substrates were pretreated as described hereinabove.

[0073] In experimental trials, a starter culture was produced as described hereinabove, but as a monoculture of R. oligosporus. Concentration of R. oligosporus in the starter culture was about 3.5E7. This starter was added to faba beans in an amount of about 1 wt-%. R freudenreichii was cultured separately in liquid cultures to achieve needed cell densities, thereafter bacterial cells were washed in 0.9 % NaCl. Concentrated cells were then added to faba beans pre-inoculated with R. oligosporus so that P freudenreichii cell densities in pretreated faba bean (already containing R. oligosporus) were about 1.85E9.

[0074] Four (4) solid-state fermentation trials designated in Figs. 4A-4C with roman numerals IV, V, VI and VII were conducted, and cell densities of P freudenreichii per a gram of pretreated faba bean were analyzed by quantifying colony forming units (CFU) prior to fermentation (in an inoculate designated as FB for Faba Bean) and in the fermented faba bean (FFB for Fermented Faba Bean) using a plate count method. Three repetitions were conducted for each trial (Replicates I, II, and III). The results are presented in tables shown on Figs. 4A and 4B, respectively.

[0075] Fermentation trials IV- VII were conducted in laboratory scale, hence, a total amount of faba bean subjected to fermentation was about 410 g. A series of nine dilutions were prepared for colony counting (1.00E-1 to 1.00E-9, see columns designated as “Dilutions” in Figs. 4A and 4B). Empty cells in these columns indicate that corresponding samples produced a lawn of bacteria too dense to be countable.

[0076] Experimentally obtained values for colony forming units (CFU) per a gram of sample (CFU / g) for the inoculate (CFU / g FB, Fig. 4A) and the fermented faba bean (CFU / g FFB, Fig. 4B) are shown in the column designated as “Sample (g)”. Average (avg) and standard (std) values were calculated, respectively. Fig. 4C summarizes the results of average and standard value calculations shown on Figs. 4A and 4B for trials IV- VII before and after fermentation (designated with signs (*) and (**), respectively). A graph shown on Fig. 4C visualizes the results shown in a corresponding table.

[0077] Vitamin B12 content in the fermented faba bean substrate obtained in fermentation trial VII was analyzed using a method SLMB 62 / 9.2.1: 2002-05. The content of vitamin B12 for trial VII was determined as 0.38 pg / g (Table 4B). After subsequent pasteurization of fermented faba bean substrate, the content of vitamin B12 content was about 0.26 pg / g. Results indicate that R freudenreichii preserves its viability in proposed solid-state fermentation conditions and is capable of vitamin B12 in required amounts even with lower cell concentrations.

[0078] R. Oligosporus grew as expected in all fermentation trials (IV- VII) with no inhibition occurred (data not shown).

[0079] It is clear to a person skilled in the art that with the advancement of technology the basic ideas of the present invention may be implemented in various ways. The invention and its embodiments may generally vary within the scope of the appended claims.

[0080] REFERENCES

[0081] 1. He & Howell. Vitamin-B12 enrichment in tempeh by co-culture with Propionibacterium freudenreichii during fermentation. BioRxiv preprint version posted November 7, 2022. DOI: 10.1101 / 2022.11.06.515253.

[0082] 2. Signorini et al. Enhanced vitamin B12 production in an innovative lupin tempeh is due to synergic effects of Rhizopus and Propionibacterium in cofermentation. International Journal of Food Sciences and Nutrition (2018): Vol. 69, No. 4, 451-457, DOI: 10.1080 / 09637486.2017.1386627.

Claims

Claims1. A process (100) for manufacturing a meat substitute composition from dehulled faba beans, the process comprising:(z) preparing (102) a starter culture comprising a co-culture of Rhizopus oligosporus and Propionibacterium freudenreichii in a cultivation medium;(zz) obtaining a dehulled faba bean substrate and inoculating (104) thereof with the starter culture, and(zzz) fermenting (106) the faba bean substrate in solid-state fermentation conditions.

2. The process of claim 1, wherein the starter culture is added to the faba bean substrate to a concentration within a range of about 0.7 wt-% to about 1.5 wt- %.

3. The process of any one of claims 1 or 2, further comprising admixing (114) of at least one enzyme with the faba bean substrate to a concentration of about 2 wt-%, wherein said at least one enzyme is transglutaminase.

4. The process of claim 1, wherein preparation (102) of the starter culture comprises incubation of a co-culture of R. oligosporus and R freudenreichii with the cultivation medium at a temperature within a range of about 28-38 °C for a period of about 72 hours to about 96 hours, wherein the co-culture is provided in the cultivation medium in a concentration of about 1 wt-%.

5. The process of claim 4, wherein the cultivation medium is rice.

6. The process of claim 1, wherein prior to inoculation with the starter culture the faba bean substrate is pretreated by soaking in water for a period of about 6 hours to about 12 hours followed with boiling and drying.

7. The process of claim 1, wherein fermentation (106) of the faba bean substrate using the starter culture is implemented in a batch reactor of a packed-bed type, wherein a ratio between a bed volume occupied by the substrate and a headspace volume is about 30 vol-% to 70 vol-%.

8. The process of claim 7, wherein fermentation (106) of the faba bean substrate with the starter culture is conducted at a temperature within a range of about 28-33 °C during a period of about 18 hours to about 24 hours.

9. The process of claim 1, further comprising:(zv) homogenization (108) of a fermented faba bean substrate obtainable at fermentation step (106), followed with cooling and drying (110) to obtain a homogenized product with a moisture content within a range of about 65% to about 70%.

10. A meat substitute composition obtainable by the process (100) according to any one of claims 1-9, and comprising a faba bean protein in an amount of at least 16 gram per 100 gram of the composition and B 12 vitamin in an amount of at least 0,9 microgram per 100 gram of the composition.

11. A process (200) for manufacturing a meat substitute product for human or animal consumption, comprising a process (100) according to any one of claims 1-9, and further comprising post-treatment (116) of a meat substitute composition obtained by the process (100) to form an edible meat substitute item.

12. The process of claim 11, wherein post-treatment (116) of the meat substitute composition comprises a procedure selected from the group consisting of: thermal processing, non-thermal processing, shaping into mold, extrusion, pressing, drying, freezing, and any combination thereof.

13. The process of any one of claims 11 or 12, wherein post-treatment (116) of the meat substitute composition comprises at least one procedure configured to yield the meat substitute product in the form of: a solid food item, a semisolid food item, or a dried food item in the form of powder, a granule, or a pellet.

14. A meat substitute product for human or animal consumption comprising or consisting of the meat substitute composition according to claim 10 posttreated to form any one of: a solid food item, a semisolid food item, or a dried food item in the form of powder, a granule, or a pellet.

15. Use of the process according to any one of claims 1-9 in manufacturing of meat substitute products for human or animal consumption.

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