WO2025247765A1 - Non-heated fermented meat analogue product prepared with vegetable protein transglutaminase and pediococcus strains - Google Patents

Abstract

The present invention relates to a method for providing a fermented food using a transglutaminase and culture, and a fermented food product obtained by this method.

Description

TITLE

NON-HEATED FERMENTED MEAT ANALOGUE PRODUCT PREPARED WITH VEGETABLE PROTEIN TRANSGLUTAMINASE AND PEDIOCOCCUS STRAINS

FIELD

The present disclosure generally relates to a prepared food. In particular, it relates to a method for obtaining a plant based fermented food, and a fermented food obtained by this method.

BACKGROUND

The demand for meat alternatives is growing due to ethical, health, and environmental concerns of conventional meat production and consumption. Meat alternatives are food products free from meat, which approximates qualities of specific types of meat, such as mouthfeel, flavor, appearance, or chemical characteristics. To approximate these qualities, most of the meat alternatives available today are made from a range of different ingredients such as water, non-textured/textured protein, flavor enhancing additives, fats or oils, colorants, and binders followed by heat treatment (Kyriakopoulou et al. Functionality of ingredients and additives in plant-based meat analogues. Foods 2021 , 10(3):600). Often, hydrocolloids are used as a binder to improve the texture, and in particular, the firmness of meat alternatives. However, hydrocolloids are often perceived as a non-desirable ingredient by consumers. Furthermore, the addition of hydrocolloids is undesired since it dilutes the product and adds to the overall costs of manufacture.

Hence, there is a need for a meat alternative, which has a suitable firmness and more closely approximates the texture of meats without the use of hydrocolloids.

SUMMARY

The inventors of the present invention have found that by using a combination of a transglutaminase (TG) and a starter culture for processing a substrate comprising a legume protein isolate, a firm food may be obtained without the use of hydrocolloids. As is evident from figures 10-13, the firmness obtained with the combination of TG and cultures is greater than the sum of the firmness of the food obtained with TG and the firmness of the food obtained with the same cultures.

Adding a functional legume protein to water provides an aqueous solution and/or suspension of legume protein, which upon fermentation with cultures and/or cross-linking with transglutaminase provides a gel. When producing such a gel, it is preferable that the gel loses water during processing, since this contributes to a mouthfeel desired by consumers. More specifically, processes for obtaining a dry sausage alternative should preferably have a water loss in the range of 17-20 wt% for an optimal texture, and for food safety reasons, the water activity should preferably be 0.94 or lower. When the gel is formed, water is shed from the gel in a process known as syneresis. When syneresis occurs, water is able to migrate to the surface of the food, such as just under the surface of a casing of the food. This facilitates drying, as the water at the surface evaporates more easily off of the food. It is advantageous that water loss is facilitated at least partially by syneresis, such that drying by means of energy intensive climate control can be minimized (climate control may be, e.g., heating, low humidity, and/or blowing). Instead of water loss, one might refer to the yield, which is the weight of the gel divided by the sum of the weight of the water and the weight of the gel. Greater gel yield means higher water retention, which involves more protein-water interactions. A sufficient yield is crucial to guarantee a high gelling capacity that enhances the functional properties of the gel, such as its elasticity and firmness. Le., although water loss from syneresis is desired, too much water loss will compromise the textural properties of the gel. As can be seen from figures 22-24, varying the legume protein content in the substrates results in distinct patterns of yields depending on whether the substrate was processed with TG, cultures, or both. When the substrate is processed with TG, as seen in fig. 22, the yield is around 93-99% regardless of the protein content. On the other hand, fig. 23 shows that the yield increases with increasing protein content when the substrate is fermented with cultures. The inventors found that processing the substrates with both cultures and TG results in a yield pattern, which could not have been anticipated from the yield patterns achieved with either TG or culture. As can be seen in fig. 24, the yield is around 52% when the protein content is in the range of 11.3-13.6 wt%, but the yield decreases sharply to around 35% when the protein content drops to 9.0 wt%. At 35%, the yield is very low, which is known to lead to a less firm and less elastic gel. As such, the inventors have found that it is preferable for the substrate to have a functional protein content of above 9 wt% to obtain a gel with a suitably high yield.

Generally, it is desirable to obtain a suitable gel strength with as little protein as possible due to the high cost of protein. The inventors have found that when fermenting substrates with cultures, the gel strength does not develop further once the protein content is about 18 wt%, indicating that the solution/suspension is saturated with functional protein. This is evident from the fig. 27 of the present disclosure which shows work value and peak load to increase linearly until a protein content of 18 wt%. Furthermore, fig. 27 shows that at 21 wt% protein, the gel strength becomes more variable, indicating that the suspension is completely saturated with protein. Since the cross-linking reaction of TG is dependent on protein in solution, increasing the protein content beyond 18% is thus not expected to provide any further increase in gel strength when the gel is prepared with TG and cultures. Hence, it is preferable to have a protein content of 18 wt% or less to not waste expensive protein.

In addition, the inventors found that a high legume protein content such as 18 wt% and above results in a grainy texture, which can be seen in fig. 28. It is more desirable to obtain a smooth texture, which is achieved at a lower protein content, preferably the texture observed for the 13.5 wt% soy protein in fig. 28. As such, the inventors have found that it is preferable for the substrate to have a protein content of less than 18 wt% to obtain a desired texture. Accordingly, the present disclosure provides in a first aspect a method for preparing a fermented food, the method comprising the steps of: i) providing a substrate comprising an aqueous phase, the aqueous phase having a legume protein content in the range of at least 10 wt% and below 18 wt% of the total weight of the aqueous phase, ii) inoculating a starter culture comprising a bacterial strain in the substrate, iii) adding transglutaminase to the substrate, wherein steps ii) and iii) are performed in any order or simultaneously, iv) allowing the transglutaminase to catalyze cross-linking of the legume protein, wherein step ii) is performed before step iv) or during step iv), and v) allowing the starter culture to ferment the substrate until a pH of the substrate is in the range of 4-5.

DEFINITIONS

As used herein, "starter culture" should be understood to be a composition comprising live microorganisms that are capable of initiating or effecting fermentation of organic material after being cultivated in a separate starter medium. Starter cultures may contain one or more microorganism species.

As used herein, “non-heated” should be understood as not heated to a temperature at or above 42°C, such as above 45°C, such as above 50°C, such as above 60°C. Conversely, “heated” or “heat- treated” should be understood as the food having been subjected to a temperature at or above 42°C, such as above 45°C, such as above 50°C, such as above 60°C. In general, temperature driven coagulation does not occur below 42°C for most legume proteins. In some meat alternative products such as salami alternatives, avoiding coagulation produces a product mimicking the raw fermented character of traditional meat-based salami.

As used herein, a food that is “plant-based” should be understood as when the food comprises at least 90% plant or plant-derived ingredients by dry weight.

As used herein, a “meat alternative” should be understood as a food product that is free from meat, and which approximates qualities of specific types of meat, such as mouthfeel, flavor, appearance, or chemical characteristics. For instance, a sliced salami alternative may have a color contrast between fat replacers and other ingredients to emulate the overall appearance of a sliced meatbased salami. A meat alternative may sometimes be referred to as a meat substitute or a meat analogue. As used herein, a “hybrid product” should be understood as a food product that comprises both plant and meat sources of protein, and which approximates qualities of specific types of meat, such as mouthfeel, flavor, appearance, or chemical characteristics. For instance, a sliced salami hybrid product may have a color contrast between fat replacers and other ingredients to emulate the overall appearance of a sliced meat-based salami.

As used herein, “protein isolate”, “protein concentrate”, or "protein flours" are interchangeable and synonymous and should be understood as a granular substance (such as a powder) prepared from legume matrices which naturally contain protein, such as soy, pea, and faba. The functional protein content of protein isolate is at least 40 wt%.

As used herein, “protein isolate”, “protein concentrate” or "protein flours" are interchangeable and synonyms and should be understood as a functional protein powder prepared from vegetable matrices which naturally contain protein, such as legumes. The protein content of these functional protein powders is at least 40 wt%

As an example, soy protein isolates are traditionally prepared from defatted soy meal using aqueous or mild alkali extraction (pH 7-10) of proteins and soluble carbohydrates. As another example, pea protein isolates are usually obtained by removing the pea's outer shell, milling it into a flour, followed by removal of fiber and starch through a filtration process.

As used herein, “functional protein” should be understood to be the water-soluble protein part of the protein isolate, protein concentrate, or protein flour. E.g., a protein isolate may have a functional protein content of 85 wt%. In the context of the present invention, functional protein of legume origin is simply referred to as “legume protein”, and functional protein which is of a specific legume is simply referred to as the name of the specific legume followed by “protein”, e.g., “pea protein”.

As used herein, the terms “transglutaminase”, “microbial transglutaminase”, “MTGase”, or “TG” are synonymous and interchangeable, and they mean a mature polypeptide having transglutaminase activity (EC 2.3.2.13) that catalyzes the formation of a covalent bond between the y-carboxamide group of protein- or peptide-bound glutamine (acyl donors) and the free amine group of protein- or peptide-bound lysine (acyl acceptors), which is microbially produced and derived from a microbial source or donor if recombinantly produced. A mature polypeptide should be understood as the transglutaminase being in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art, that a host cell may produce a mixture of two of more different mature polypeptides (/.e., with different C-terminal and/or N-terminal amino acid residues) expressed from the same polynucleotide. The transglutaminase in SEQ ID NO:1 is produced in vivo in at least 3 different mature forms shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, wherein the N-terminal amino acid in the mature polypeptide differs by one residue in each sequence, respectively.

For purposes of the present invention, microbial transglutaminase activity is determined according to the procedure described in the Examples. As used herein, the term “expression” includes any step involved in the production of a transglutaminase polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

As used herein, the term “isolated” means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).

It is also known in the art that different host cells process polypeptides differently, and also that the pro-peptide may influence the N-terminal of the mature transglutaminase and, consequently, one host cell expressing a polynucleotide encoding a full-length polypeptide incl. signal- and pro-peptide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide. The inventors expect that any number of differently processed mature transglutaminase polypeptides may be effective in the present invention; it is entirely trivial to test the suitability of any mature transglutaminase or the different mature forms thereof to identify one or more effective enzyme.

The relatedness between two amino acid sequences or between two nucleotide sequences is described herein by the parameter “sequence identity”.

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

As used herein, the term “variant” means a polypeptide having transglutaminase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. As used herein, the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

As used herein, a gel should be understood as a substantially dilute cross-linked system of proteins, which exhibits no flow when in the steady state, although the liquid phase may still diffuse through this system. Proteins in a gel may be cross-linked through one or more types of intermolecular interactions or bonds, such as covalent bonds or aggregation.

Pediococcus pentosaceus is interchangeable and synonymous with P. pentosaceus. Likewise, Pediococcus acidilactici interchangeable and synonymous with P. acidilactici. Furthermore, “L.” in L. plantarum is short for Lactiplantibacillus or the legacy naming Lactobacillus and in and L. sakei, it is short for Latilactobacillus (legacy naming Lactobacillus).

As used herein, the term “fermentation” or “fermenting” refers to a process wherein carbohydrates are transformed into a range of metabolites through chemical reactions carried out by microorganisms, such as a bacterial culture. Besides lactic acid, metabolites produced during fermentation include also volatile organic compound that can affect the flavor of the fermentation product.

BRIEF DESCRIPTION OF THE FIGURES

Fig 1 shows a box plot of peak loads of two food samples: a non-heated fermented food according to an embodiment of the invention and a food with a composition similar to the embodiment’s composition, but which food was heat-treated and comprises a hydrocolloid.

Fig 2 shows a picture of a non-heated fermented food according to an embodiment of the invention (right) and a picture of a food with a composition similar to the embodiment’s composition, but which similar food is heated and comprises a hydrocolloid (left).

Fig 3 shows a bar plot of fermentations times to a pH of 5 for twenty different non-heated food compositions with varying pea protein content and functional ingredients, wherein some of the compositions are according to embodiments of the present invention.

Fig 4 shows 10 images of different non-heated food compositions with varying soy protein content and functional ingredients. A = P. acidilactici and 9wt% soy protein content, B = P. acidilactici and 11.3wt% soy protein content, C = P. acidilactici and 13.6wt% soy protein content, D = microbial transglutaminase and 4.6wt% soy protein content, E = microbial transglutaminase 6.8wt% soy protein content, F = microbial transglutaminase and 9wt% soy protein content, G = microbial transglutaminase and 11.3wt% soy protein content, H = microbial transglutaminase and 13.6wt% soy protein content, I = microbial transglutaminase, P. acidilactici and 11.3wt% soy protein content, and J = microbial transglutaminase, P. acidilactici and 13.6wt% soy protein content. Fig 5 shows a line chart of the pH value during fermentation of four different non-heated foods with a soy protein content of 4.6wt% on a wet weight basis: a soy protein suspension inoculated with P. acidilactici and P. pentosaceus; a soy protein suspension with MTGase; a composition according to an embodiment of the invention, the composition being a soy protein suspension which was inoculated with P. acidilactici and P. pentosaceus and which contains MTGase; and a soy protein suspension inoculated with Streptomyces .

Fig 6 shows a line chart of the pH value during fermentation of four different non-heated foods with a soy protein content of 6.8wt% on a wet weight basis: a soy protein suspension inoculated with P. acidilactici and P. pentosaceus; a soy protein suspension with MTGase; a composition according to an embodiment of the invention, the composition being a soy protein suspension which was inoculated with P. acidilactici and P. pentosaceus and which contains MTGase; and a soy protein suspension inoculated with Streptomyces.

Fig 7 shows a line chart of the pH value during fermentation of four different non-heated food compositions with a soy protein content of 9wt% on a wet weight basis: a soy protein suspension inoculated with P. acidilactici and P. pentosaceus; a soy protein suspension with MTGase; a composition according to an embodiment of the invention, the composition being a soy protein suspension which was inoculated with P. acidilactici and P. pentosaceus and which contains MTGase; and a soy protein suspension inoculated with Streptomyces.

Fig 8 shows a line chart of the pH value during fermentation of four different non-heated food compositions with a soy protein content of 11 .3wt% on a wet weight basis: a soy protein suspension inoculated with P. acidilactici and P. pentosaceus; a soy protein suspension with MTGase; a composition according to an embodiment of the invention, the composition being a soy protein suspension which was inoculated with P. acidilactici and P. pentosaceus and which contains MTGase; and a soy protein suspension inoculated with Streptomyces.

Fig 9 shows a line chart of the pH value during fermentation of four different non-heated food compositions with a soy protein content of 13.6wt% on a wet weight basis: a soy protein suspension inoculated with P. acidilactici and P. pentosaceus; a soy protein suspension with MTGase; a composition according to an embodiment of the invention, the composition being a soy protein suspension which was inoculated with P. acidilactici and P. pentosaceus and which contains MTGase; and a soy protein suspension inoculated with Streptomyces.

Figs 10-13 show the peak load for different non-heated food compositions with varying functional pea protein content and varying functional ingredients (with or without TG and with different starter cultures).

Fig 14-21 show images of different non-heated food compositions with varying functional protein content, varying functional ingredients (different legume protein isolates, with or without TG, and with different starter cultures). Fig 22 shows that for soy and pea-based gels, the yield obtained when using only TG is the same regardless of the protein content and which type of legume protein is used.

Fig 23 shows that for soy-based gels, the yield obtained when using only a starter culture (P. acidilactici DSM 28308 + P. pentosaceus DSM 35016) increases with the concentration of functional protein.

Fig 24 shows that for pea-based gels, the yield obtained when using a starter culture and transglutaminase is stable from 11 .3% functional protein and above, and that the yield is lower at 9%.

Fig 25 shows that the effect on yield observed in fig 23 is also observed when the soy base gel is obtained with different starter cultures and TG.

Fig 26 shows that the effect observed in fig 24 is also observed in faba-based gels.

Fig 27 shows that for soy-based gels, the work value and the peak load increases with the functional protein content when the functional protein content is in the range of 9-18%. At 21 % functional protein content, the work value and peak load does not increase compared to 18%.

Fig 28 shows images of soy protein suspensions with different levels of protein content.

DETAILED DESCRIPTION OF THE INVENTION

The starter culture is allowed to ferment the substrate to a pH in the range of 4-5, however, it is preferred that the fermentation is to a pH in the range of 4.7-4.9. The maximum gel formation is obtained at the isoelectric point of the functional protein, which is usually a pH in the range of 4.2 to 4.3. However, such low pH values can be perceived as too sour by consumers. Hence, a pH in the range of 4.7-4.9 is preferred, as it is a compromise between flavor profile (less sour than pH 4.2-4.3) and adequate gel formation.

Any starter culture producing lactic acid as the major metabolic end-product of carbohydrate fermentation, and which ferments the substrate to the target pH range of 4-5 may be used. This is evident from the examples and the figures of the present disclosure, which shows that foods obtained with either Pediococcus acidilactici DSM 28307, Lactiplantibacillus plantarum DSM 35015, or Latilactobacillus sakei DSM 14022 all provided suitable firmness (peak load) and about the same yields.

In an embodiment, the starter culture comprises a Pediococcus strain. The Pediococcus strain may be any Pediococcus species producing lactic acid as the major metabolic end-product of carbohydrate fermentation. In a preferred embodiment, the Pediococcus strain is a Pediococcus acidilactici strain. In an even more preferred embodiment, the Pediococcus strain is Pediococcus acidilactici DSM 28307. During the fermentation stage, the consumption of carbohydrate by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum.

In an embodiment, the starter culture comprises an auxiliary Pediococcus strain, which auxiliary Pediococcus strain is a Pediococcus pentosaceus strain. In a preferred version of this embodiment, the Pediococcus strain is a Pediococcus acidilactici strain and the auxiliary Pediococcus strain is a Pediococcus pentosaceus strain. In an even more preferred version of this embodiment the Pediococcus strain is Pediococcus acidilactici strain DSM 28307 and the auxiliary Pediococcus strain is Pediococcus pentosaceus strain DSM 35016.

In another embodiment, the fermented food is a salami hybrid product. In a preferred version of this embodiment, the fermented food furthermore comprises an auxiliary Pediococcus strain, which auxiliary Pediococcus strain is a Pediococcus pentosaceus strain. In an even more preferred version of this embodiment, the Pediococcus strain is a Pediococcus acidilactici strain and the auxiliary Pediococcus strain is a Pediococcus pentosaceus strain. In an even more preferred version of this embodiment the Pediococcus strain is Pediococcus acidilactici strain DSM 28307 and the auxiliary Pediococcus strain is Pediococcus pentosaceus strain DSM 35016.

In an embodiment, the starter culture comprises a Pediococcus acidilactici strain, preferably Pediococcus acidilactici DSM 28307.

In an embodiment, the starter culture comprises a Lactiplantibacillus plantarum strain, preferably Lactiplantibacillus plantarum DSM 35015.

In an embodiment, the starter culture comprises a Latilactobacillus sakei strain, preferably Latilactobacillus sakei DSM 14022.

As mentioned in WO2016128508A1 , the strain Pediococcus acidilactici DSM 28307 was deposited by Chr. Hansen A/S at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

As mentioned in W02019043055A1 , the strain Latilactobacillus sakei DSM 14022 was deposited by Chr. Hansen A/S at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The starter culture should preferably be inoculated in the substrate before the transglutaminase reaction turns the substrate into a gel. This is because inoculating the culture in the substrate will shear the cross-linked protein network thereby destroying the gel structure. Also, further incubation with transglutaminase will not sufficiently re-establish the protein network. Generally, the transglutaminase reaction results in gel formation within 0.5-12 hours depending on temperature, pH, and other factors known in the art to affect transglutaminase activity.

In a preferred embodiment, step ii) is performed before step iv) or during step iv), such that the starter culture is inoculated in the substrate before gel formation occurs due to cross-linking of legume protein.

In a preferred embodiment, step ii) is performed at most 0.5 hours after the start of step iv).

The optimal pH for transglutaminase is in the range of 6-8. The substrate is generally in this range before fermentation and during the lag phase of the culture. Therefore, to obtain the highest degree of protein cross-linking, the step of allowing transglutaminase activity should preferably be performed before the substrate is acidified by fermentation.

In a preferred embodiment, the step of iv) is performed at a pH in the range of 5-8.5, preferably in the range of 6-8.

In a preferred embodiment, the step of iv) is completed during a lag phase of the starter culture.

In an embodiment, step iv) is performed in less than less than 3 hours, preferably in less than 1.5 hours.

In an embodiment, step v) is performed in less than 11 hours, preferably in less than 6 hours.

The substrate may comprise a liquid fat source, such as an oil. Legume protein in the substrate may act like an emulsifier between the oil and aqueous phase, which alters the way cross-linking occurs in the substrate. Without being bound by theory, it is believed that when most of the cross-linking of legume proteins occurs between proteins in the aqueous phase, and not between proteins bound on oil surfaces, a preferred firm texture is obtained. Since the proteins in the aqueous phase have more sites available for the cross-linking reaction than proteins bound on oil surfaces, it is believed that a greater firmness may be achieved when the substrate has a suitably low oil content.

In an embodiment of the method, the substrate furthermore comprises an oil-in-water emulsion, the oil-in-water emulsion comprising the aqueous phase and an oil phase, wherein the oil phase comprises legume protein, and wherein oil of the oil phase constitutes in the range of 1 to 15 wt% of the total weight of the oil-in-water emulsion. In a preferred version of this embodiment, the oil of the oil phase constitutes in the range of 5 to 14.5 wt% of the total weight of the oil-in-water emulsion, more preferably 7 to 14 wt% of the total weight of the oil-in-water emulsion.

In an embodiment, the oil-in-water emulsion has an oil to protein weight ratio of less than 1.25, preferably in the range of 0.5-1 .2, most preferably in the range of 0.9-1 .1 , wherein the oil to protein weight ratio is calculated as: (weight of the oil of the oil phase)/(weight of the legume protein in the oil phase + weight of the legume protein in the aqueous phase).

In a preferred embodiment, the method comprises the step of:

- heating the substrate such that the functional protein unfolds. By unfolding the functional protein, additional exposition of hydrophobic regions takes place, which further enhances hydrophobic clustering. Consequently, the gel network becomes stronger and more cohesive as the proteins aggregate further. It is preferable that the heating to unfold the functional protein is gentle, such that the cross-linking (isopeptide bonds) and disulfide bonds of the functional proteins are kept intact. The skilled person knows how to provide a suitable heating step for unfolding the functional protein, also such that cross-linking and disulfide bonds are substantially not disrupted. Generally, the heating step involves a temperature in the range of 65 to 100°C and lasts for less than 12 min. As an example, a substrate containing pea protein or soy protein may be heated to a temperature in the range of 68-74°C for 0.5-4 minutes, preferably 71-73°C for 1-4 minutes. In one embodiment, the heating step is carried out after the step of iv) and v). In another embodiment, the heating step is carried out before the steps of ii) and iii).

In an embodiment, the method furthermore comprises a step of shaping the substrate into an elongated shape. In a preferred version of this embodiment, the substrate is shaped by stuffing the substrate into a casing, preferably a cellulose or alginate casing.

In an embodiment of the method, the method furthermore comprises a step of drying the fermented substrate obtained in step v). In a specific version of this embodiment, the fermented substrate is dried to a moisture content in the range of 20wt% to 40wt% and/or to a water activity below 0.95.

Advantageously, the suitable firmness provided by the combination of the starter culture and the transglutaminase is achievable without heating and/or the use of hydrocolloids. Without being bound by theory, it is believed that not heating the food in the process of manufacture leads to lesser denaturation, caramelization, melting, and/or disintegration of fat replacer or pieces of fat present in food, and/or lesser convection and/or diffusing of fat replacer or pieces of fat into the surrounding material of the food. As a result, the boundaries between fat replacer or fat pieces and the surrounding food material are clearly discernable, which more closely resembles the appearance of traditionally non-heated meat-based sausages. The difference between a non-heated fermented food according to an embodiment of the invention and a similar composition, but which is heated and comprises a hydrocolloid, is shown in fig. 2. It is evident from fig. 2 that use of hydrocolloids and heat-treatment results in significantly less discernable contrast and boundaries between fat replacer or fat pieces and surrounding material of the food, whereas the non-heated fermented food has clearly discernable boundaries.

In an embodiment, the method does not comprise a step of heating the substrate or the fermented substrate obtained in step v). Preferably, the substrate or the fermented substrate is not heated to a temperature at or above 42°C, such as above 45°C, such as above 50°C, such as above 60°C.

In an embodiment, the substrate furthermore comprises a texturized vegetable protein. In a preferred version of this embodiment, the texturized vegetable protein is soy based, pea based, soy and wheat based, or pea and wheat based. Texturized vegetable proteins (TVPs) are derived from protein concentrates or isolates that are extruded and texturized. During this process, the proteins are stretched into an extended shape. TVPs are commonly formulated as dry bites and can be produced in different granulometries, depending on the use. In some meat alternative applications, TVPs are hydrated either before addition to other components of the meat alternative or hydrated upon mixing with other components of the meat alternative. TVPs have high water absorbency and, once cooked, have a texture similar to meat and fish. They also have a fairly neutral taste and odor and are therefore suitable for flavoring.

In an embodiment, the substrate comprises a fat replacer or pieces of fat. The pieces of fat may for example be based on plant-based fats such as coconut or palm fat. Also, the pieces of fat may be fat emulsions stabilized with proteins, hydrocolloids, and/or transglutaminase.

Fat replacers are subcategorized into fat substitutes and fat mimetics. Fat substitutes are molecules that possess the physical and functional characteristics of conventional fat molecules (e.g., triglycerides). Fat substitutes can directly replace conventional fat molecules in foods on a weight- for-weight basis. They are typically synthetic molecules which provide no energy calories or structured lipid molecules which provide reduced energy calories. Fat substitutes can successfully maintain the palatability of foods as they can reproduce the texture and mouthfeel of fat. They may not reproduce the taste properties of fat as fat itself provides flavor to foods and is a carrier of other fat-soluble flavor compounds in foods.

Fat mimetics are substances which can mimic some of the organoleptic and physical properties of conventional fat molecules. However, they cannot replace fat molecules in food on a weight-for- weight basis. Fat mimetics are typically protein- or carbohydrate-based molecules that may be modified to mimic some of the properties of conventional fats. Fat mimetics are generally not suitable for high temperature applications, such as frying, as they are susceptible to denaturation or caramelization. Fat mimetics are generally polar, water-soluble compounds. Thus, they cannot replace some of the non-polar functional characteristics of fats, such as lipid-soluble flavor carrying capacity. However, their polar nature facilitates water binding which helps generate a sense of creaminess and lubricity in foods similar to that found in full-fat products.

In an embodiment, the substrate is substantially free from hydrocolloids or free from hydrocolloids. Hydrocolloids are a class of food ingredients (mainly polysaccharides and some proteins) that are widely applied in various food products. Examples of hydrocolloids are alginate, pectin, carrageenan, gelatin, gellan, and agar.

In an embodiment, the substrate furthermore comprises a carbohydrate source, preferably a monosaccharide or a disaccharide preferably dextrin or glucose.

As is evident from the examples and the figures disclosed herein, the substrate may comprise any type of legume protein. The legume protein should be soluble and/or suspendable in water at least up to a protein content of 17 wt%. Preferably, the legume protein should be suspendable at least up to a protein content of 17 wt% without forming aggregates, i.e., without forming a grainy texture. In an embodiment, the legume protein is soy protein, pea protein, and/or faba protein. In a preferred embodiment, the legume protein is pea protein. In another preferred embodiment, the legume protein is soy protein.

In a preferred embodiment, the source of the legume protein is a legume protein isolate, preferably a soy protein isolate, pea protein isolate, and/or faba protein isolate, more preferably a pea isolate or a soy protein isolate, most preferably a pea protein isolate.

In a preferred embodiment, the aqueous phase comprises legume protein in the range of 11 to 17 wt% of the total weight of the aqueous phase, more preferably in the range of 12 to 16.5 wt% of the total weight of the aqueous phase, most preferably in the range of 15 to 16 wt% of the total weight of the aqueous phase.

In the context of the present disclosure, the functional protein content in an aqueous phase is calculated as follows: weight of functional protein 100 weight of water in the aqueous phase + weight of functional protein

An example of how to obtain the values for the formula (a) is provided below the ingredients of the substrate contributing water to the aqueous phase are identified, e.g., hydrated texturized vegetable protein (HTVP), ice, culture suspension, etc. the water content of each of the identified ingredients is determined. E.g., the water content may be 75 wt% for HTVP, 99 wt% for the culture suspension, and 100 wt% for the ice. the weight of water in the aqueous phase is then calculated as: weight of HTVP x 75% + the weight of the culture suspension x 99% + weight of the ice the total weight of the functional protein content of the protein containing ingredients is then determined. For instance, if the substrate comprises a soy protein isolate with 85 wt% functional protein and a pea protein isolate with 80 wt% functional protein, the total weight of functional protein is calculated as the sum of the weight of the soy protein isolate times 85% and the weight of the pea protein isolate times 80%. the functional protein content in the aqueous phase is then calculated by using the above stated formula (a).

Where the fermented food also contains an oil, the functional protein content of the aqueous phase is lower than the value calculated in the formula (a), since some of the functional protein will be bound on oil surfaces.

Also, not all water containing ingredients contribute with water to the aqueous phase. For instance, a gelled ingredient, such as a fat mimetic, which traps water in its gel structure, does not contribute with water to the aqueous phase of the food. The skilled person knows which ingredients contribute with water to the aqueous phase and by how much. In a preferred embodiment, the transglutaminase has a sequence identity to the mature polypeptide of any of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In one embodiment, the polypeptides differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

In another preferred embodiment, the transglutaminase has been isolated. A transglutaminase of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or an allelic variant thereof; or is a fragment thereof having transglutaminase activity.

Yet another preferred embodiment relates to transglutaminase variants of the SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the transglutaminase of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino or carboxyl-terminal extensions, such as an aminoterminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physio-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for transglutaminase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al.,

1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al. , 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

The transglutaminase may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

The transglutaminase may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin etal., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al.,

1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

The transglutaminase may be obtained from a microorganism of any genus. For purposes of the present invention, the terms “obtained from” or “derived from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one embodiment, the transglutaminase obtained from a given source is secreted extracellularly.

The transglutaminase may be a bacterial transglutaminase. For example, the transglutaminase may be a Gram-positive bacterial transglutaminase such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces polypeptide having transglutaminase activity, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma transglutaminase.

In one embodiment, the transglutaminase is selected from Bacillus alkalophilus, Bacillus altitudinis, Bacillus amyloliquefaciens, B. amyloliquefaciens subsp. plantarum, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus safensis, Bacillus stearothermophilus , Bacillus subtilis, and Bacillus thuringiensis transglutaminases.

In another embodiment, the transglutaminase is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus transglutaminase.

In another embodiment, the transglutaminase is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, Streptomyces mobaraensis, or Streptomyces lividans transglutaminase.

In yet another embodiment, the transglutaminase comprises or consists of a transglutaminase derived from Streptomyces mobaraensis, Streptomyces caniferus or Streptoverticillium ladakanum, preferably the transglutaminase comprises or consists of a transglutaminase derived from Streptomyces mobaraensis.

The transglutaminase may be a fungal transglutaminase. For example, the transglutaminase may be a yeast transglutaminase such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xy/ar/a transglutaminase.

In another embodiment, the transglutaminase is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis transglutaminase.

In another embodiment, the transglutaminase is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride transglutaminase.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The transglutaminase may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.). Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the transglutaminase may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a transglutaminase has been detected by probes well known in the art, the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Methods of producing a transglutaminase are well-known to the skilled person, typically, comprising (a) cultivating a cell, which in its wild-type form produces the transglutaminase, under conditions conducive for production of the transglutaminase; and optionally, (b) recovering the transglutaminase, OR comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the transglutaminase; and optionally, (b) recovering the transglutaminase.

The host cells are cultivated in a nutrient medium suitable for production of the transglutaminase using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the transglutaminase to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the transglutaminase is secreted into the nutrient medium, the transglutaminase can be recovered directly from the medium. If the transglutaminase is not secreted, it can be recovered from cell lysates.

The transglutaminase may be detected using methods known in the art that are specific for the transglutaminases. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the transglutaminase.

The transglutaminase may be recovered using methods known in the art. For example, the transglutaminase may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

The transglutaminase may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDSPAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure transglutaminases.

In an alternative embodiment, the transglutaminase is not recovered, but rather a host cell of the present invention expressing the transglutaminase is used as a source of the transglutaminase. The term transglutaminase includes whatever auxiliary compounds may be necessary for the enzyme's catalytic activity, such as, e.g., an appropriate acceptor or cofactor, which may or may not be naturally present in the substrate.

The transglutaminase may be in any form suited for the use in question, such as, e.g., in the form of a dry powder or granulate, a non-dusting granulates, a liquid, a stabilized liquid, or a protected enzyme.

In a second aspect, the present disclosure provides a fermented food obtained by the method according to the first aspect.

In an embodiment, the fermented food has a protein content in the range of 10.5-12 wt% and a firmness measured by peak load of at least 100 g.

In an embodiment, the fermented food has a protein content in the range of 13.5-14.5 wt% and a firmness measured by peak load of at least 200 g.

In an embodiment, the fermented food has a protein content in the range of 14.6-15.5 wt% and a firmness measured by peak load of at least 300 g.

In an embodiment, the fermented food is a meat alternative. In a specific version of this embodiment, the fermented food is a plant-based meat alternative, such as a plant-based sausage alternative. In an even more preferred version of this embodiment, the fermented food is a salami alternative.

In a preferred embodiment, the fermented food is a plant-based sausage alternative comprising a fat replacer or pieces of fat.

In an embodiment, the fermented food is a hybrid product, such as a hybrid product having a plant protein to meat protein ratio in the range of 0.01 to 0.99, such as a ratio in the range of 0.05 to 0.95, such as a ratio in the range of 0.15 to 0.85. In a specific version of this embodiment the hybrid product is a sausage hybrid product. In another version of this embodiment, the hybrid product is a salami hybrid product. In an embodiment, the fermented food is a salami alternative.

DEPOSIT AND EXPERT SOLUTION

The applicant requests that a sample of the deposited microorganisms stated below may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted. Table 1 : The applicant has made the following deposits at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

EXAMPLES

MTGase activity assay:

Transglutaminase activity may be determined by any method known in the art. For example, analysis of transglutaminase activity may be done by quantitation of the released ammonia resulting from the formation of an isopeptide bond between a free amino group (6-aminohexanoic acid) and an acyl group from a glutamine (Z-GLN-GLY) like described below. Chemicals and enzymes used: 10 Z- GLN-GLY. E.g., Sigma C6154 6-aminohexanoic acid. Eg. Sigma 07260 L-Gluthatione reduced. Eg. Sigma G4251 a-Ketoglutarate. Eg. Sigma K3752 NADH 15 L-GLDH. Eg. Roche 107735 MOPS. Eg. Sigma M-1254.Transglutaminase standard Method: To 75 microliter of an enzyme solution, dissolved in 0.1 M MOPS/5 mM L-Gluthatione reduced pH 7.0, is added 50 microliter of 1 % 6-aminohexanoic acid, and 75 microliter of 1 % Z-GLN-GLY, and 75 microliter of (0.44 g/L NADH, 2.5 g/L o- Ketoglutarate in 0.1 M MOPS pH 7.0). The absorbance at 340 nm is followed by kinetic measurement for 5 min at 30 °C.

The enzyme activity is determined similar to a transglutaminase standard that has been aligned to be the transglutaminase Unit Definition (Folk, J. E. and Cole, P. W. (1966) Biochim. Biophys. Acta.241 , 5518-5525). MTGase activity expressed as TGHU(A).

The present invention has been described with reference to various embodiments, aspects, examples, or the like. It is not intended that these elements be read in isolation from one another. Thus, the present disclosure provides for the combination of two or more of the embodiments, aspects, examples, or the like.

All embodiments described herein are intended to be within the scope of the invention disclosed. These and other embodiments of the present invention will become readily apparent to those skilled in the art from the following detailed description of the preferred embodiments having reference to the whole description, the invention not being limited to any particular preferred embodiment(s) disclosed.

Water activity

Water activity (a_w) was measured with an Aqua Lab 4TE. Samples with a temperature of 25°C +/- 4°C are measured.

Firmness analysis

The peak load (firmness) analysis was performed with a Texture Analyzer device CT3. Samples were prepared by slicing a thickness of 1.5 cm and storing at room temperature (approx. 19-21 °C) for at least 4 hours. The Texture Analyzer device CT3 is configured to “Normal” mode and set to trigger: 100 g; deformation: 3.0 mm; and speed: 0.5 mm/s. The Texture Analyzer device CT3 is fitted with a Texture Analyzer Dough Extensibility Jig (TA-DE) and a spherical probe (TA18, 12.7mm). Peak load in gram is the maximum measured load during the test. Work value is defined as the energy required to deform a sample. Work value was calculated as the integral of load over the time to the first fracture. Example 1

Fermented non-heated food peak load

A non-heated salami alternative was prepared from the components of table 2:

The non-heated salami alternative was prepared using the following process: a) The non-heated salami alternative was obtained through the following process: b) mixing the suspension of Pediococcus acidilactici strain DSM 28307 and Pediococcus pentosaceus strain DSM 35016 with the water and ice, c) adding colorings and salt (sodium chloride) and mixing, d) adding functional protein and enzyme (transglutaminase), and mixing, e) adding spices, flavorings, and dextrose, and mixing, f) adding TVP and fat replacer and blending at a temperature in the range of 13-15°C, g) stuffing in a plant-based casing (viscofan veggie provided by viscofan, s.a.), h) fermenting at a temperature of 37°C, a room humidity of 99%, until a pH of the fermentate is in the range of 4.8-5.0, i) drying at 14°C, at a room humidity of 83%, until a_w = 0.94 whereby the non-heated salami alternative is obtained. For comparison purposes, a heated salami alternative was prepared from the components of table 3: | Fat replacer | | 151

The heated salami alternative was prepared using the following process: a) mixing the suspension of Pediococcus acidilactici strain DSM 28307 and Pediococcus pentosaceus strain DSM 35016 with the water and ice, b) adding colorings, potassium chloride and sodium chloride, k-Carrageenan LBG, and mixing, c) adding functional protein, and mixing, d) adding spices, flavorings, and dextrose, and mixing, e) adding TVP and fat replacer and blending at a temperature in the range of 13-15°C, f) stuffing in a plant-based casing (viscofan veggie provided by viscofan, s.a.), g) fermenting at a temperature of 37°C, a room humidity of 98%, until a pH of the fermentate is in the range of 4.6-4.8, h) cooking at 87°C, room humidity 99%, until a core temperature of the fermentate is 87°C, i) cooling at 7°C, until a core temperature of the fermentate is 10-20°C, j) drying at 14°C, at a room humidity of 85%, until a_w = 0.94 whereby the heated salami alternative is obtained.

The non-heated salami alternative and the heated salami alternative’s peak load were measured according to the firmness analysis described above. The peak loads of each salami alternative is shown in fig. 1 , wherein the bar titled “fermented heated food with hydrocolloids” shows the heated salami alternative’s peak load and the bar titled “Fermented non-heated food” shows the non. heated salami alternative’s peak load.

Example 2

Appearance of salami alternatives

A non-heated salami alternative was prepared using the ingredients of table 4:

The non-heated salami alternative was prepared using the following process: k) combining and mixing water, red beet, the suspension of Pediococcus acidilactici strain DSM 28307 and Pediococcus pentosaceus strain DSM 35016, and microbial transglutaminase, l) adding herbs and spices and mixing, m) adding soy protein isolate and mixing, n) adding red wine and mixing, o) adding gluten network and mixing, p) adding hydrated plums and mixing, q) adding fermented pea TVP and mixing, r) adding fermented wheat TVP and mixing, s) adding fat replacer and sundried tomatoes and mixing, t) vacuumize, u) stuffing in a plant-based casing (viscofan veggie provided by viscofan, s.a.), v) fermenting at a temperature of 37°C, a room humidity of 95 %, for 10-15 hours until a pH of the fermentate is in the range of 4.8-5.0, w) drying at 12°C, at a room humidity of 85 %, until a_w = 0.94 (30-35 % weight loss), and x) smoking at for 5 min at a temperature of 12°C and a room humidity of 85%, whereby the non-heated salami alternative is obtained.

A heated salami alternative was prepared using the ingredients of table 5:

The heated salami alternative was prepared using the following process: a) combining and mixing water, red beet, and the suspension of Pediococcus acidilactici strain DSM 28307 and Pediococcus pentosaceus strain DSM 35016, b) adding herbs and spices and mixing, c) adding soy protein isolate and mixing, d) adding red wine and mixing, e) adding gluten network and mixing, f) adding hydrated plums and mixing, g) adding fermented pea TVP and mixing, h) adding fermented wheat TVP and mixing, i) adding fat replacer and sundried tomatoes and mixing, j) vacuumize, k) stuffing in a plant-based casing (viscofan veggie provided by viscofan, s.a.), l) fermenting at a temperature of 37°C, a room humidity of 95 %, for 10-15 hours until a pH of the fermentate is in the range of 4.8-5.0, m) cooking at a temperature of 85°C, room humidity of 99 %, until a core temperature of the fermentate is 82°C, n) cooling at 0-7°C, until a core temperature of the fermentate is less than 12°C o) drying at 12°C, at a room humidity of 85 %, until a_w = 0.94 (30-35 % weight loss), and p) smoking at for 5 min at a temperature of 12°C and a room humidity of 85%, whereby the heated salami alternative is obtained.

The heated and non-heated salami alternatives where sliced and a picture of each salami alternative was taken, as seen in fig. 2. In fig 2, the picture of the heated salami alternative is shown above the text “Fermented heated food with hydrocolloids” and the non-heated salami alternative is shown above the text “Fermented non-heated food".

Example 3

Firmness, acidification, yield, and appearance of fermented legume protein-based foods

Twenty non-heated foods were prepared in four groups, l)-IV), each group having a different combination of functional ingredients and five protein content levels:

I) Pediococcus acidilactici strain DSM 28307 and Pediococcus pentosaceus strain DSM 35016, and with a soy protein content of 4.6wt%, 6.8wt%, 9wt%, 11 .3wt%, and 13,6%

II) Microbial transglutaminase (MTGase) and with a soy protein content of 4.6wt%, 6.8wt%, 9wt%, 11.3wt%, and 13,6%

III) Pediococcus acidilactici strain DSM 28307 and Pediococcus pentosaceus strain DSM 35016 and microbial transglutaminase (MTGase), and with a soy protein content of 4.6wt%, 6.8wt%, 9wt%, 11.3wt%, and 13,6%

IV) Streptomyces and with a soy protein content of 4.6wt%, 6.8wt%, 9wt%, 11 .3wt%, and 13,6%

The compositions for the five different protein contents were as in table 6:

The soy protein isolate being Vittesence 1803, with a 80wt% protein content and the culture suspension being either P. acidilactici strain DSM 28307 and P. pentosaceus strain DSM 35016 suspended in water (1 :39, culture:water) or a Streptomyces species in water. In group II) compositions, i.e. compositions without a culture suspension, 10 g of additional water was added instead of a culture suspension.

The twenty non-heated foods were prepared as follows: a) functional ingredients are added to the water and mixed, thereby obtaining a suspension, b) soy protein isolate is mixed with dextrose, thereby obtaining a powder, c) the powder and the suspension are mixed to a mixture, d) the mixture is added into cups at 10Og/cup e) the mixture is allowed to be fermented at 37°C until pH is in the range of 4.3 to 4.4 thereby obtaining the non-heated foods.

Time to reach pH 5 for each of the twenty gels are shown in fig. 3.

Pictures taken of some of the non-heated foods are shown in fig. 4. A = P. acidilactici and 9wt% soy protein content, B = P. acidilactici and 11.3wt% soy protein content, C = P. acidilactici and 13.6wt% soy protein content, D = microbial transglutaminase and 4.6wt% soy protein content, E = microbial transglutaminase 6.8wt% soy protein content, F = microbial transglutaminase and 9wt% soy protein content, G = microbial transglutaminase and 11.3wt% soy protein content, H = microbial transglutaminase and 13.6wt% soy protein content, I = microbial transglutaminase, P. acidilactici and 11.3wt% soy protein content, and J = microbial transglutaminase, P. acidilactici and 13.6wt% soy protein content.

The acidification during fermentation of the non-heated food products were measured. The figs. 5 to 9 shows line plots of the measured pH values of each of the non-heated food products. pH was measured with a multi-channel pH meter.

Using the same method as for the twenty foods mentioned above, four new groups of foods were prepared, V)-XVI), each group having a different combination of functional ingredients, i.e., different types of legume proteins, different cultures, with and without TG, and different protein levels:

V) Pea protein and TG

VI) Pea protein and Pediococcus acidilactici strain DSM 28307 and Pediococcus pentosaceus strain DSM 35016

VII) Pea protein, TG, and Pediococcus acidilactici strain DSM 28307 and Pediococcus pentosaceus strain DSM 35016

VIII) Pea protein, TG, and Pediococcus acidilactici strain DSM 28307

IX) Pea protein, TG, and L. plantarum DSM 35015

X) Pea protein, TG, and L. sake/ DSM 14022

XI) Soy protein and TG

XII) Soy protein and Pediococcus acidilactici strain DSM 28307 and Pediococcus pentosaceus strain DSM 35016 XIII) Faba protein, TG, and Pediococcus acidilactici strain DSM 28307 and Pediococcus pentosaceus strain DSM 35016

XIV) Faba protein and TG

XV) Faba protein and Pediococcus acidilactici strain DSM 28307 and Pediococcus pentosaceus strain DSM 35016

XVI) Faba protein, TG, and Pediococcus acidilactici strain DSM 28307 and Pediococcus pentosaceus strain DSM 35016

Vittesence 1803 pea protein isolate (80% pea protein, Ingredion USA) was used for preparing the foods containing pea protein, Unisol NRG soy protein isolate (90% soy protein, Vitablend, The Netherlands) was used for preparing the foods containing soy protein, and Tendra faba protein isolate (85% faba protein, Cosun Protein, The Netherlands) was used for the foods containing faba protein.

Pictures taken of the foods are shown in figs. 14-21.

The firmness (peak load) was measured for each of the foods according to the firmness analysis described above. The peak loads are shown in figs. 10-13.

Yields were obtained by weighing the foods and the water shed from the foods. The yields were calculated as (the weight of the food)/(weight of the water + the weight of the food). The yields are shown in figs. 22-26.

Example 4

Texture of high protein content fermented soy-based foods

An additional set of soy-based foods were prepared as before, with Unisol NRG soy protein isolate and P. acidilactici strain DSM 28307 and P. pentosaceus strain DSM 35016.

Firmness of the foods was measured (work value and peak loads) as described above, and are shown in fig. 27.

Example 5

A set of non-fermented aqueous soy and pea protein suspensions were prepared. Pictures of the suspension are shown in fig. 28. The suspension with 17.5 wt% pea protein and 18 wt% soy protein shows grainy texture, while the suspension with 13.5 wt% soy protein shows a smooth texture. The suspension with 4.5 wt% and 9 wt% soy protein shows suspension having a wet consistency. The consistency of the suspension carries over into the final fermented product, and as such a protein content of 13.5 wt% is preferred over the other protein concentrations.

Claims

1 . A method for preparing a fermented food, the method comprising the steps of: i) providing a substrate comprising an aqueous phase, the aqueous phase having a legume protein content of at least 10 wt% and below 18 wt% ofthe total weight of the aqueous phase, ii) inoculating a starter culture comprising a bacterial strain in the substrate, iii) adding transglutaminase to the substrate, wherein steps ii) and iii) are performed in any order or simultaneously, iv) allowing the transglutaminase to catalyze cross-linking of the legume protein, wherein step ii) is performed before step iv) or during step iv), and v) allowing the starter culture to ferment the substrate until a pH of the substrate is in the range of 4-5.

2. The method of claim 1 , wherein the substrate furthermore comprises an oil-in-water emulsion, the oil-in-water emulsion comprising the aqueous phase and an oil phase, wherein the oil phase comprises legume protein, and wherein oil of the oil phase constitutes in the range of 1 to 15 wt% of the total weight of the oil-in-water emulsion.

3. The method of claim 2, wherein the oil-in-water emulsion has an oil to protein weight ratio of less than 1.25, preferably in the range of 0.5-1 .2, wherein the oil to protein weight ratio is calculated as: (weight ofthe oil ofthe oil phase)/(weight ofthe legume protein in the oil phase + weight of the legume protein in the aqueous phase).

4. The method according to any one of the preceding claims, wherein the legume protein is soy protein, pea protein, and/or faba protein.

5. The method according to any one of the preceding claims, wherein the bacterial strain is selected from a Pediococcus acidilactici strain, a Latilactobacillus sakei strain, or a Lactiplantibacillus plantarum strain.

6. The method according to any one of the preceding claims, wherein the method further comprises the step of:

- heating the substrate such that the functional protein unfolds.

7. The method according to any one ofthe preceding claims, wherein the substrate furthermore comprises fat replacer or pieces of fat.

8. The method according to any one of the preceding claims, wherein the substrate is substantially free from hydrocolloids.

9. The method according to any one of the preceding claims, wherein the method furthermore comprises the step of: vi) drying the fermented substrate obtained in step v).

10. The method according to any one of the preceding claims, wherein the method furthermore comprises a step of shaping the substrate into an elongated shape.

11. The method according to any one of the preceding claims, wherein step iv) is performed in less than 3 hours, preferably in less than 1 .5 hours.

12. The method according to any one of the preceding claims, wherein step v) is performed in less than 11 hours, preferably in less than 6 hours.

13. The method according to any one of the preceding claims, wherein the transglutaminase has a sequence identity to the mature polypeptide of any of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 of at least 60%.

14. The method according to any one of the preceding claims, wherein the method does not comprise a step of heating the substrate or the fermented substrate obtained in step v).

15. A product obtained by a process according to any one of the claims 1-14.