WO2024218330A1 - Production of fermented plant-based products - Google Patents

Abstract

The present invention relates to the field of food technology, in particular to the preparations plant- based products by fermentation. The plant-based products may be legume based and a selection of suitable strains provide improved flavor profiles in the fermented product.

Description

PRODUCTION OF FERMENTED PLANT-BASED PRODUCTS

FIELD

The present invention relates to the field of food technology, in particular to the preparations plantbased products by fermentation.

BACKGROUND

Many food consumers request plant-based alternatives to conventional dairy and meat products for reasons such as sustainability, lifestyle, and dietary restrictions. However, dairy- and meatalternative products are often nutritionally unbalanced, and their flavor profiles have limited their general consumer acceptance. Additionally, the texture of fermented milk or plant-based alternatives is an important quality parameter, affecting consumer acceptance. Fermentation can aid in improving the sensory profiles, nutritional properties, texture, and microbial safety of plant-based dairy and meat alternatives whereby possibly eliminating the use of flavor masking and texturing ingredients.

However, many fermented plant-based dairy and meat alternatives are still rejected by some consumers due to issues with flatulence. A major cause of flatulence is the presence of raffinose family oligosaccharides (RFOs), which are widespread across the plant kingdom, and ubiquitous in legume seeds (Minorsky 2003. The Hot and the Classic. Plant Physiol. 131 25-26.). Many RFOs are composed of a-(1 ,6)-galactosides linked to a sucrose unity (Cardoso et al 2021 . Novel and emerging prebiotics: advances and opportunities. Adv. Food Nutr. Res. 95 41-95). Humans and animals do not produce an a-galactosidase enzyme to synthesize and digest the RFOs in the intestine (Minorsky, 2003; Mao et al. (2014). In Vitro Fermentation of Lactulose by Human Gut Bacteria. J. Agric. Food Chem. 62 10970-10977.). As a result, RFOs are instead metabolized by gut microbes (bacteria) to synthesize flatulence causing by-products like hydrogen (H2), carbon dioxide (CO2), and methane (CH4). In fact, flatulence is the single most important factor that deters consumption and utilization of legumes in human and animal diets (Elango et al 2022. Raffinose Family Oligosaccharides: Friend or Foe for Human and Plant Health? Front Plant Sci. 13:829118).

Today, commercial supplement products are available which supplies a- galactosidase and sucrase enzymes in the human gut to hydrolyze RFOs counter the flatulence problem. Recently, the a-galactosidase gene (galC) was cloned from Aspergillus oryzae (YZ1) and expressed in Pichia pastoris for protein production, and galC effectively degraded the RFOs (primarily raffinose and stachyose) in soymilk (Wang 2020. Characterization of a protease-resistant a-galactosidase from Aspergillus oryzae YZ1 and its application in hydrolysis of raffinose family oligosaccharides from soymilk. Int. J. Biol. Macromol. 158 708-720). Moreover, stachyose, raffinose, verbascose can be effectively digested by certain microbes (Heravi et al 2019. The melREDCA Operon Encodes a Utilization System for the Raffinose Family of Oligosaccharides in Bacillus subtilis. Journal of Bacteriology, vol 201 , no. 15).

Nevertheless, there remains a need for a cost-effective way to produce well-tasting, texturized plantbased dairy and meat alternatives that are free from or have reduced levels of RFOs.

SUMMARY

Accordingly, the present invention seeks to mitigate the shortcomings of existing processes, by providing a combination of strains for fermenting a plant base, which reduces the amount of RFOs (raffinose, stachyose, and verbascose) in the plant base while also providing improved flavor and texture to the plant-based products.

As shown in the Figs 14-17, the inventors of the present invention have found that although Bacillus subtilis strains degrade RFOs raffinose, stachyose, and verbascose by itself in different plant bases, the reduction in RFOs is hampered where the Bacillus subtilis strains are combined with lactic acid bacteria starter cultures such as Leuconostoc mesenteroides strains, Lactiplantibacillus plantarum/pentosus/paraplantarum strains, and Lacticaseibacillus paracasei strains. However, the inventors have surprisingly found that combinations of Bacillus subtilis strains with Streptococcus thermophilus strains do substantially not lessen the reduction of RFOs, and in some cases improves the reduction of stachyose and verbascose.

Thus, by combining a Streptococcus thermophilus strain with a Bacillus subtilis strain, a surprising synergistic effect is achieved where:

1) RFOs are significantly degraded,

2) an improved flavor is achieved through the production of diacetyl (desired dairy flavor) and removal of hexanal (undesirable beany off-flavor)

3) an improved texture is achieved from using Streptococcus thermophilus, which acidifies the plant base, thereby forming gels, and

4) an improved microbial safety is achieved as a result of the relatively quick pH drop produced by Streptococcus thermophilus.

Taken together, the fermentation with a combination of a Streptococcus thermophilus strain with a Bacillus subtilis strain can be used to produce plant-based dairy and meat alternatives that are more acceptable to consumers.

According to a first aspect, a process for producing a fermented plant-based product with a reduced stachyose and/or verbascose content is provided, the method comprising the steps of: a) providing a substrate comprising a plant base, b) adding to the substrate at least one Streptococcus thermophilus strain and at least one Bacillus subtilis strain, c) fermenting the substrate until a stop criterion is reached, and d) obtaining fermented plant-based product.

In a preferred embodiment, the plant base is a legume substrate.

In an even more preferred embodiment, the legume substrate is faba bean or chickpea substrate.

In one embodiment, the at least one Streptococcus thermophilus is Streptococcus thermophilus (DSM 17876).

In one embodiment, the Bacillus subtilis strain is Bacillus subtilis (DSM 33181) or Bacillus subtilis (DSM 33182).

In one embodiment, the fermented plant-based product is a dairy analogue product.

In one embodiment, the fermented plant-based product is a meat analogue product.

In one embodiment, the stop criteria is a predetermined fermentation time, such as 8 hours, 12 hours, 18 hours, 24 hours or 36 hours.

In one embodiment, the stop criteria is a predetermined pH level, such as when pH is below 4.6, below 4.7, below 4.8, below 4.9 or below 5.0.

According to a second aspect, a product obtainable by the process according to the first aspect is provided.

According to a third aspect, a starter culture composition comprising at least one Streptococcus thermophilus strain and at least one Bacillus subtilis strain is provided.

In an embodiment, the at least one Streptococcus thermophilus is Streptococcus thermophilus (DSM 17876).

In an embodiment, the Bacillus subtilis strain is Bacillus subtilis (DSM 33181) or Bacillus subtilis (DSM 33182).

In an embodiment, the composition has a metabolic capacity to consume stachyose in separate fermentations of a faba bean isolate and a chickpea isolate, and wherein the metabolic capacity to consume stachyose is at least 80%, such as at least 85%, preferably at least 90% of the least one Bacillus subtilis strain’s metabolic capacity to consume stachyose in separate fermentations of the faba bean isolate and the chickpea isolate.

In an embodiment, the composition has a metabolic capacity to consume verbascose in a fermentation of a faba bean isolate, and wherein the metabolic capacity to consume verbascose is at least 80%, such as at least 85%, preferably at least 90% of the least one Bacillus subtilis strain’s metabolic capacity to consume verbascose in a fermentation of the faba bean isolate.

According to a fourth aspect, a kit-of-parts comprising at least one Streptococcus thermophilus strain and at least one Bacillus subtilis strain is provided.

According to a fifth aspect, use of a composition of the third aspect or a kit-of-parts of the fourth aspect for fermenting a plant-based product is provided. In an embodiment, the use is furthermore for reducing a stachyose and/or a verbascose content in the plant-based product.

In an embodiment, the use is furthermore for acidifying the plant-based product.

In an embodiment, the plant-based product is a dairy analogue product.

In an embodiment, the plant-based product is a meat analogue product.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows graphs displaying texturizing data, i.e. texture data, for plant bases according to embodiments.

Figure 2 is a graph displaying off-flavor associated compounds for a plant base according to an embodiment.

Figure 3 is a graph displaying off-flavor associated compounds for a plant base according to an embodiment.

Figure 4 is a graph displaying off-flavor associated compounds for a plant base according to an embodiment.

Figure 5 is a graph displaying off-flavor associated compounds for a plant base according to an embodiment.

Figure 6 is a graph displaying dairy-associated flavor compounds for a plant base according to an embodiment.

Figure 7 is a graph displaying dairy-associated flavor compounds for a plant base according to an embodiment.

Figure 8 is a graph displaying dairy-associated flavor compounds for a plant base according to an embodiment.

Figure 9 is a graph displaying dairy-associated flavor compounds for a plant base according to an embodiment.

Figure 10 is a graph displaying production of ethanol and organic acids in a plant base according to an embodiment.

Figure 11 is a graph displaying production of ethanol and organic acids in a plant base according to an embodiment.

Figure 12 is a graph displaying production of ethanol and organic acids in a plant base according to an embodiment.

Figure 13 is a graph displaying production of ethanol and organic acids in a plant base according to an embodiment. Figure 14 shows graphs displaying data regarding degradation of sugars for plant bases according to embodiments.

Figure 15 shows graphs displaying data regarding degradation of sugars for plant bases according to embodiments.

Figure 16 shows graphs displaying data regarding degradation of sugars for plant bases according to embodiments.

Figure 17 shows graphs displaying data regarding degradation of sugars for plant bases according to embodiments.

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 forthe Purposes of Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

The strain Lactocaseibacillus rhamnosus, in the figures referred to as DSMZ33156, is a commercially available deposit with the American Type Culture Collection (ATCC), with accession number ATCC 53103. DETAILED DESCRIPTION

Further embodiments are described in the following detailed description.

Probiotic strains

The term “probiotic bacteria” refers to viable bacteria which are administered in adequate amounts to a consumer for the purpose of achieving a health-promoting effect in the consumer. Probiotic bacteria are capable of surviving the conditions of the gastrointestinal tract after ingestion and colonize the intestine of the consumer. Probiotic bacterial strains may be added before or after fermentation. If added before fermentation the probiotic bacterial strain also act as a fermentative bacteria.

Among the lactic acid bacteria used in the food industry, Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and Bifidobacterium are predominantly applied. The lactic acid bacteria of the species Streptococcus thermophilus (S. thermophilus) are used extensively alone or in combination with other bacteria such as Lactobacillus for the production of food products, in particular fermented food products.

It will be appreciated that the Lactobacillus genus taxonomy was updated in 2020. The new taxonomy is disclosed in Zheng et al. 2020 and will be cohered to herein if not otherwise indicated. For the purpose of the present invention, the table below presents a list of new and old names of some Lactobacillus species relevant to the present invention.

Table 2. New and old names of some Lactobacillus species relevant to the present invention.

In a particular embodiment the probiotic strain according to the present invention is selected from the group consisting of bacteria of the genus Lactobacillus, such as Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lacticaseibacillus easel, Lactobacillus delbrueckii, Lactobacillus lactis, Lactiplantibacillus plantarum, Lactiplantibacillus pentosus, Lactiplantibacillus paraplantarum, Limosilactobacillus reuteri and Lactobacillus johnsonii, the genus Bifidobacterium, such as the Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum and Bifidobacterium infantis, and the like.

In a particular embodiment, the probiotic Lactobacillus strain is selected from the group consisting of Lactiplantibacillus plantarum, Lactiplantibacillus pentosus, Lactiplantibacillus paraplantarum, Lacticaseibacillus paracasei and Lacticaseibacillus rhamnosus.

In a particular embodiment, the probiotic Lactobacillus strain is selected from the group consisting of a Lacticaseibacillus rhamnosus strain and a Lacticaseibacillus paracasei strain.

In a particular embodiment, the probiotic strain is Streptococcus thermophilus (DSM 17876).

In a particular embodiment, the probiotic strain is Leuconostoc mesenteroides (DSM 32865).

In a particular embodiment, the probiotic strain is Lactiplantibacillus plantarum/pentosus/paraplantarum (DSM 34551).

In a particular embodiment, the probiotic strain is Lacticaseibacillus paracasei (DSM 34552).

In a particular embodiment, the probiotic strain is Lacticaseibacillus paracasei (DSM 34553).

In a particular embodiment, the probiotic strain is Lactocaseibacillus rhamnosus (ATCC 53103).

In a particular embodiment, the probiotic strain is Bacillus subtilis (DSM 33181).

In a particular embodiment, the probiotic strain is Bacillus subtilis (DSM 33182).

The above probiotic strains may be further combined with each other, or with other lactic acid bacteria.

In one embodiment, the probiotic strains are combined in a mixture.

In one embodiment, the probiotic strains are combined as a kit-of-parts.

In one embodiment the composition and/or mixture or kit-of-parts may comprise S. thermophilus strain DSM 33118 or DSM 33182, and one or more of Streptococcus thermophilus (DSM 17876), Leuconostoc mesenteroides (DSM 32865), Lactiplantibacillus plantarum/pentosus/paraplantarum (DSM 34551), Lacticaseibacillus paracasei (DSM 34552), Lacticaseibacillus paracasei (DSM 34553), Lactocaseibacillus rhamnosus (ATCC 53103, in the figures also referred to as DSMZ33156).

The expression “mixture” means that the strain(s) are physically mixed together. In an embodiment, the S. thermophilus strain(s) and the Lactobacillus strain(s) such as e.g. Lactobacillus delbrueckii subsp bulgaricus, Lactobacillus acidophilus, Lactobacillus easel, Lactobacillus paracasei, and/or Lactobacillus rhamnosus are in the same box or in the same pouch.

In contrast, the expression “A kit-of-part” comprising strain(s) means that strains or culture of strain(s) are physically separated but intended to be used together. Thus, the strains or culture of S. thermophilus strain(s) and Lactobacillus strain(s) are in different boxes or sachets. In an embodiment, the S. thermophilus strain(s) and the Lactobacillus such as e.g. Lactobacillus delbrueckii subsp bulgaricus, Lactobacillus acidophilus, Lactobacillus easel, Lactobacillus paracasei, and/or Lactobacillus rhamnosus strain(s) are under the same format, i.e., are in a frozen format, in the form of pellets or frozen pellets, a powder form, such as a dried or freeze-dried powder.

In a particular embodiment of the present invention, the composition comprises from 10⁴ to 10¹² CFU (colony forming units)/g of the S. thermophilus strain, such as from 10⁵ to 10¹¹ CFU/g, from 10⁶ to

10¹⁰ CFU/g, or from 10⁷ to 10⁹ CFU/g of the S. thermophilus strain.

In a particular embodiment the composition further comprises from 10⁴ to 10¹² CFU/g of the Lactobacillus strain, such as from 10⁵ to 10¹¹ CFU/g, such as from 10⁶ to 1 O¹⁰ CFU/g, or such as from 10⁷ to 10⁹ CFU/g of the Lactobacillus strain.

In a particular embodiment the composition comprises from 10⁴ to 10¹² CFU/g, such as from 10⁵ to

10¹¹ CFU/g, from 10⁶ to 1 O¹⁰ CFU/g, or from 10⁷ to 10⁹ CFU/g of each of the Lactobacillus delbrueckii subsp bulgaricus, Lactobacillus acidophilus, Lactobacillus easel, Lactobacillus paracasei, and/or Lactobacillus rhamnosus strain(s).

S. thermophilus and Lactobacillus such as L. bulgaricus, L. acidophilus, L. easel, L. paracasei, and/or L. rhamnosus and other lactic acid bacteria are commonly used as starter cultures serving a technological purpose in the production of various foods, such as in the dairy industry, such as for fermented milk products. Thus, in another preferred embodiment the composition is suitable as a starter culture.

The composition may be a starter culture such as a yoghurt, vegurt, creme fraTche (which may be a plant-based creme fraTche), sour cream (which may be a plant-based sour cream), or meat analogue starter culture.

The composition and/or starter culture may be frozen, spray-dried, freeze-dried, vacuum-dried, air dried, tray dried or in liquid form. Typically, the storage stability of the composition and/or starter culture can be extended by formulating the product with low water activity. By controlling the water activity (Aw), it is possible to predict and regulate the effect of moisture migration on the product. Therefore, it may be preferred that the water activity (Aw) of the dried compositions herein is in the range from 0.01-0.8, preferably in the range from 0.05-0.4.

As used herein, the term "dairy analogue" is meant to refers to dairy-like products, which are products used as culinary replacements for dairy products, prepared where one or more milk constituents have been replaced with other ingredients and the resulting food resembles the original product. The milk constituents are replaced completely or substantially with plant material, for example, using planted-based milks derived from legumes (such as soybeans), nuts (such as almonds cashews, coconuts), cereals such as (oat, rice, corn, or wheat). Such plant-based milk, prepared from plant material, is referred to herein as “plant milk” or “plant milk base”.

It should be noted that “dairy analogue”, “plant milk” or “plant milk base” used herein does not refer to alcoholic beverages, or fruit and vegetable juices in general, since such beverages are generally not considered as culinary replacements for dairy products. As used herein, the term "meat analogue" is meant to refers to meat-like products, which are products used as culinary replacements for meat products, prepared where one or more animal tissue constituents (such as skin, muscles, fats, etc.) have been replaced with other ingredients and the resulting food resembles the original product. The animal tissue constituents are replaced completely or substantially with plant material, for example, using planted bases or plant-based matrices derived from legumes (such as soybeans, fava, and pea), nuts (such as almonds cashews, coconuts), root fruits (such as beetroot), and/or cereals (such as oat, rice, corn, or wheat).

The term "legume" refers to any plant belonging to the family Fabaceae. Fabaceae is a large and economically important family of flowering plants, which is commonly known as the legume family, pea family, bean family or pulse family. A variety of different legumes can be consumed. Legumes typically have a pod or hull that opens along two sutures when the seeds of the legume are ripe. The Fabaceae family includes over 750 genera and 16,000 to 19,000 species.

Examples of “legumes” include peanuts (Arachis hypogaea), pigeon peas (Cajanus cajan), chickpea (Cicerarietinum), soy bean (Glycine max), lentils (Lens culinaris), lupins (Lupinus spp.), peas (Pisum sativum), field peas (Pisum arvense), beans (Phaseolus spp.), common beans (Phaseolus vulgaris) and its various cultivars and varieties, vetches (Vicia spp.), faba beans (Vicia faba), beans (Vigna spp.), cow peas (Vigna unguiculata), azuki beans (Vigna angularis) and bambara beans (Voandzeia subterranea).

The term “improving the flavor” of a product means to making the product more palatable. This can be determined for example by sensory assessment known to a skilled person in the art.

The composition of the present invention may additionally comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorants or mixtures thereof. The composition preferably comprises one or more of cryoprotectants, lyoprotectants, antioxidants and/or nutrients, more preferably cryoprotectants, lyoprotectants and/or antioxidants and most preferably cryoprotectants or lyoprotectants, or both. Use of protectants such as cryoprotectants and lyoprotectantare known to a skilled person in the art. Suitable cryoprotectants or lyoprotectants include mono-, di-, tri-and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia) and the like), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol and the like), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptones, gelatin, yeast extract) and inorganic compounds (such as sodium tripolyphosphate).

In one embodiment, the composition according to the present invention may comprise one or more cryoprotective agent(s) selected from the group consisting of inosine-5’-monophosphate (IMP), adenosine -5’-monophosphate (AMP), guanosine-5’-monophosphate (GMP), uranosine-5’- monophosphate (UMP), cytidine-5’-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine and a derivative of any such compounds. Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B-family, vitamin C). The composition may optionally comprise further substances including fillers (such as lactose, maltodextrin, cyclodextrin) and/or flavorants.

In one embodiment the cryoprotective agent is an agent or mixture of agents, which in addition to its cryoprotectivity has a booster effect.

The expression "booster effect" is used to describe the situation wherein the cryoprotective agent confers an increased metabolic activity (booster effect) on to the thawed or reconstituted culture when it is inoculated into the medium to be fermented or converted. Viability and metabolic activity are not synonymous concepts. Commercial frozen or freeze-dried cultures may retain their viability, although they may have lost a significant portion of their metabolic activity e.g. cultures may lose their acid-producing (acidification) activity when kept stored even for shorter periods of time. Thus viability and booster effect has to be evaluated by different assays. Whereas viability is assessed by viability assays such as the determination of colony forming units, booster effect is assessed by quantifying the relevant metabolic activity of the thawed or reconstituted culture relative to the viability of the culture. The term "metabolic activity" refers to the oxygen removal activity of the cultures, its acid-producing activity, i. e. the production of e. g. lactic acid, acetic acid, formic acid and/or propionic acid, or its metabolite producing activity such as the production of aroma compounds such as acetaldehyde, (a-acetolactate, acetoin, diacetyl and 2,3-butylene glycol (butanediol)).

In one embodiment the composition contains or comprises from 0.2% to 20% of the cryoprotective agent or mixture of agents measured as % w/w of the material. It is, however, preferable to add the cryoprotective agent or mixture of agents at an amount which is in the range from 0.2% to 15%, from 0.2% to 10%, from 0.5% to 7%, and from 1 % to 6% by weight, including within the range from 2% to 5% of the cryoprotective agent or mixture of agents measured as % w/w of the frozen material by weight. In a preferred embodiment the culture comprises approximately 3% of the cryoprotective agent or mixture of agents measured as % w/w of the material by weight. The amount of approximately 3% of the cryoprotective agent corresponds to concentrations in the 100 mM range. It should be recognized that for each aspect of embodiment the ranges may be increments of the described ranges.

In a further embodiment, the composition of the present invention contains or comprises an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) as a booster (e.g. growth booster or acidification booster) for bacterial cells, such as cells belonging to the species S. thermophilus, e.g. (substantial) urease negative bacterial cells. The term "ammonium salt", "ammonium formate", etc., should be understood as a source of the salt or a combination of the ions. The term "source" of e.g. "ammonium formate" or "ammonium salt" refers to a compound or mix of compounds that when added to a culture of cells, provides ammonium formate or an ammonium salt. In some embodiments, the source of ammonium releases ammonium into a growth medium, while in other embodiments, the ammonium source is metabolized to produce ammonium. In some preferred embodiments, the ammonium source is exogenous. In some particularly preferred embodiments, ammonium is not provided by the dairy substrate. It should of course be understood that ammonia may be added instead of ammonium salt. Thus, the term ammonium salt comprises ammonia (NH3), NH4OH, NH⁴⁺, and the like.

In one embodiment the composition may comprise thickener and/or stabilizer, such as pectin (e.g. HM pectin, LM pectin), gelatin, CMC, Soya Bean Fiber/Soya Bean Polymer, starch, modified starch, carrageenan, alginate, and guar gum.

In the context of the present disclosure, “isolate”, such as a faba bean or a chickpea isolate, should be understood as plant material that has been processed to isolate or concentrate desired substances of the plant material. E.g., in a plant protein isolate, a plant material has been processed to obtain an isolate with an increased protein content.

In an embodiment, the faba bean isolate is a faba bean protein isolate.

In an embodiment, the chickpea isolate is a chickea protein isolate.

In the context of the present disclosure, “metabolic capacity” should be understood as the difference in concentration of a compound that a bacterial strain or a composition of bacterial strains can produce in one or more substrates by fermenting the one or more substrates. Where the metabolic capacity refers to the concentration difference produced in two or more substrates, the metabolic capacity is the total of the concentration differences produced in each of the substrates. As an example, a strain’s metabolic capacity to consume stachyose in separate fermentations of a faba bean isolate and a chickpea isolate is calculated as follows: faba + ^chickpea

Where

MC is the metabolic capacity, c_faba is the stachyose concentration difference produced by fermentation with the strain in the faba been isolate,

^chickpea is the stachyose concentration difference produced by fermentation with the strain in the chickpea isolate.

A general formula for calculating the metabolic capacity can therefore be formulated as:

Where n is the number of substrates.

Methods for determining the concentration of compounds such as the RFOs raffinose, stachyose and verbascose are well known in the art and are available to the skilled person.

Methods to ferment a plant base, such as a faba bean isolate or a chickpea isolate with a lactic acid bacteria strain or a Bacillus subtilis strain are available to the skilled person. In an embodiment, the stachyose and/or the verbascose concentration in a faba bean isolate or a chickpea isolate is determined by high performance an-ion exchange with pulsed amperometry detection (HPAE-PAD) on a high pressure ion chromatography (HPIC), using a 250x1 mm hydrophobic, polymeric, pellicular anion-exchange resin column, a gradient elution of 4 min 10 mM KOH, followed by 1.5 min 100 mM KOH, followed by 8.5 min 200 mM KOH, followed by 16 min 10m M KOH, with an elution flow rate of 63 pL min^-1 , using conductivity detection, and peak area relative to stachyose and/or the verbascose standards of 2.5pg mL^-1 , 50pg mL^-1, 1 mg mL^-1 , 20mg mL^-1 , and 400 mg L^-1 .

In an embodiment, the fermentation of a faba bean isolate and/or a chickpea isolate is performed by addition of an inoculum of a strain of the disclosure to a homogenized, pasteurized aqueous solution of the faba bean and/or chickpea isolate (7% w/v), glucose (1 % w/v), and sucrose (1 % w/v), followed by incubation at either 30 or 37°C for 12 hours to produce a fermentate, followed by addition of 60 % v/v ethanol to a final ratio of 2:1 v/v (60 % v/v ethanol to the fermentate).

EXAMPLES

Specific strains were tested fortheir ability to ferment four different legume bases, i.e., protein bases, such as pea from two different suppliers, chickpea and faba bean and improve its flavor by reducing levels of compounds associated with beany off-flavor (such as hexanal) and producing “good flavor” metabolites associated with umami taste and “dairy notes” (dairy flavor associated) metabolites such as diacetyl.

Bacillus are known for their production of y-PGA, which is a naturally occurring biopolymer made from repeating units of l-glutamic acid, d-glutamic acid, or both. y-PGA hydrolysis by an exohydrolase results in a release of glutamate, which might enhance umami taste desirable to have in meat alternative plant-based products. Because of its ability to produce glutamate and glutamate polymers (y-PGA), Bacillus was in focus in our work. It has a known history of safe use in foods in fermented plant-based foods in Africa and Asia and is essential for the alkaline fermentation of various soy products, e.g. Bacillus subtilis and Bacillus licheniformis are the dominant fermentative organisms in doenjang - a traditional, fermented soybean food product. The Japanese appreciate the activity of Bacillus subtilis on soybean in the form of the fermented product Natto. DSM 33181 and DSM 33182 are two Bacillus Natto spore(-) strains. We have investigated Bacillus for their ability to ferment four different legume bases such as pea from two different suppliers, chickpea and faba bean and improve flavor of the bases. Both the strains DSM 33181 and DSM 33182 had the ability to reduce the amount of hexanal, which is considered as the major volatile responsible for beany/rancid/off-flavor (i.e., rancid off-flavour) in legumes after an overnight incubation at 30°C or at 37°C.

Fermentation Four legume matrices, i.e., legume protein powders, (pea from two different suppliers, chickpea and faba bean) were prepared by the addition of distilled water to protein concentrates, homogenization, and pasteurization at 90°C for 20 min while stirring. The matrices were left at room temperature overnight, and the pasteurization step was repeated. When the matrices were cooled down to the incubation temperature, sugar solution (1 % sucrose and 1 % glucose) and 1 % overnight inoculum of Bacillus and I or 1 % overnight inoculum of LAB were added to the matrices, and the matrices were fermented overnight at either 30 or 37°C.

Bacillus were incubated in LB medium overnight at 37°C while shaking at 250 rpm. LAB were incubated in MRS-Difco (in the case of Lactobacillus and Leuconostoc) or M17 + 1 % sucrose and 1 % glucose (in the case of S. thermophilus) at 30°C (in the case of Lactobacillus, Leuconostoc and L. lactis strains) or 37°C (in the case of S. thermophilus). A full list of the strains used, together with deposit receipts, is found in Table 1.

VITESSENCE™ 1803 Pea Protein (isolate pea protein) containing a minimum of 80 % protein content on dry matter basis was used to prepare a 4 % solution (4 g powder + 96 ml ddbW). ADM ProFam™ Pea Protein 580 (isolate pea protein) with 80 % protein content was used to prepare a 4 % solution (4 g powder + 96 ml ddFLO). VITESSENCE® Prista P 360 faba bean protein (concentrate faba bean protein), which is 60% protein concentrate (on a dry basis), was used to prepare 8 % solution (8 g powder + 92 ml ddFLO). CP-PRO70® Chickpea Protein Concentrate from Innovapro containing at least 68 % protein was used to prepare a 7 % solution (7 g powder + 96 ml ddH₂O).

The fermentation was performed in four different formats: 2 ml samples in 20 ml headspace vials for volatile compound analysis, 0.2 ml samples in an MTP for sugar analysis, 1 ml samples in 1 ml 96- well MTP for texture measurements (compression test), and 2 ml samples in 2 ml 96-well MTP for texture measurements (TADM). Before subjected to the volatile compound analysis, the fermentation was stopped by adding saturated sulfuric acid (0.4 ml of 2 M FLSC to each vial containing 2 ml sample) and freezing the samples to -20°C. For sugar measurements, 2 volumes of 60 % ethanol were added (0.4 ml to 0.2 ml samples) after the fermentation.

Texture measurements

For fermented milk and plant-based products, the texture is an important quality parameter and depends on both the microbes driving the fermentation as well as the process conditions. Traditionally, rheometer and texture analyzer are used to assess texture in fermented milk products. Large samples (30-100 mL), high workload, rather low throughput, and relatively long test time are required for the traditional texture methods using rheometer and texture analyzer. TADM (total aspiration and dispense monitoring) method as described in (Poulsen et al. 2019. High-throughput screening for texturing Lactococcus strains. FEMS Microbiology Letters, Vol 366, Issue 2) has been applied to measure relative shear stress in micro-titer plate (MTP) format (Fig. 1). A high-throughput (HTP) compression test in 1 ml MTP scale resembling compression tests by Texture Analyzer (TA- TX) in 100 ml cups was used to evaluate the stiffness of the samples (Fig. 1). TA-TX records the force of resistance of the instrument probe as it penetrates the sample. The measurement consists in a back extrusion test, from which the following parameters can be extrapolated: Firmness (max. positive force), Consistency (positive compression area), Cohesiveness (max. negative force), and Viscosity (negative area). Hamilton Start liquid handling robot equipped with custom-made metal micro-tools applied a compression on 1 ml samples situated in a 96-well 1 ml MTP, where the MTP is placed on a precision balance from Mettler Toledo. A distribution plate on the balance assures equal responses along the different positions. The force resistance is recorded, while the micro-tools penetrate one sample at a time. The precision balance carrying the samples records the force resistance when this is applied every 10 msec, and reports in an csv file. Data acquisition is automatic, and results are contained in unique file names arranged by origin (design), sample and time. The output obtained resembles the compression curves obtained from texture analyzer TA-TX, with Time (ms) on the x axis and force (g) on the y-axis. The method speed is faster than the reference method, requiring <1 min per sample. It is fully automatic for the run of a 96-well MTP. The HTP method uses a plunger that has a 4/10 plunger/sample surface ratio with individual probes of approximately 4 mm. The results are expected in the range 2-20 g. TA-TX uses a plunger that has approximately 3/4 plunger/sample surface ratio and reuses the same probe of 30 mm for all samples (needs cleaning every time); results are within the range of 200-1000 g.

Figure 1 shows the results of the compression test (left panel) and TADM (right panel) for the four matrices fermented at two different temperatures, 30 or 37°C. “Control” represents matrices fermented using Bacillus alone: either the strain DSMZ33181 or DSMZ33182. While fermentations using Bacillus alone did not result in a gel formation, a texture enhancement caused by the acidification by LAB resulting in a gel formation occurred. Moreover, some LAB strains are known for their exo-cellular polysaccharide production, which can enhance the texture of milk and plant-based matrices (Poulsen et al, 2022. Versatile Lactococcus lactis strains improve texture in both fermented milk and soybean matrices. FEMS Microbiology Letters, Vol 369, Issue 1). Some strains resulted in higher texture at one of the temperatures used, e.g. DSM 17876 resulted in a higher texture at 37°C, which is expected for a S. thermophilus strain, as 30°C is not an optimal temperature for S. thermophilus. DSM 32865 resulted in a higher texture at 30°C, which is expected for a Leuconostoc strain, as Leuconostoc prefer 20-30°C and not 37°C.

Degradation of off-flavor related compounds and production of desirable flavor compounds

The selected strains were grown in the different legume protein emulsions as described above. Two grams of the different emulsions were aliquoted directly in a 20ml headspace vials. Uninoculated emulsions were used as negative controls and were not incubated. All samples were prepared in duplicates. After incubation, 400pl of sulfuric acid 2M was added to the headspace vials and samples were stored at -18°C until analysis. Volatile organic compounds produced during fermentation were determined by head space solid phase microextraction gas chromatography coupled to mass spectrometry (HS-SPME-GC-MS). The instrument was a Multi Purpose Sampler (Gerstel, MSCI, Skovlunde, Denmark), with a 7890B GC (Agilent Technologies, Denmark) and a 5977A MS (Agilent Technologies, Denmark). VOCs were extracted by SPME using a DVB/Car/PDMS-fiber (Supelco#57299, VWR, Denmark) for 20 min. at 60°C, desorbed splitless at 270°C onto a TenaxTA-filled liner (Gerstel#012438, MSCI, Skovlunde, Denmark) kept at -30°C. After fiber desorption, the TenaxTA-filled liner were heated to 300°C and the trapped VOCs transferred splitless and separated on a DB-5MS Ul column 30m x 0.25mm x 1 pm (Agilent#122-5533UI, Agilent Technologies, Denmark) at 170 kPa constant pressure using helium as carrier gas. Oven temperature program was as follows: starting at 32°C/2min - increased to 102°C@10°C/min - further increased to 145°C@5°C/min - further increased to 200°C@15°C/min - further increased to 200°C@15°C/min - further increased to 280°C@20°C/min - hold at 280°C for 5 min. The mass spectrometer operated in electron impact mode at -70eV and the analyzer was scanning from 29-209 amu.

NIST 17 library search and Retention Indexes were used for identification of VOCs. Feature extraction was done using MassHunter Quantitative Analysis (Version 10.2, Build 10.2.733.8, Agilent Technologies, Denmark) and results calculated as peak height divided by baseline noise (signal-to- noise, S/N). Removal ratio for the beany off-flavors and the enhancement formation ratio for dairy notes compounds were calculated comparing the detected S/N values of those in the fermented samples to those in the respective uninoculated matrix.

Figures 2 to 5 show the degradation (level of decrease compared to the unfermented sample, in signal to noise) of different off-flavor associated compounds in legume bases. More specifically, Figure 2 shows the degradation of off-flavor associated compounds as a result of fermentation of a faba bean protein concentrate (VITESSENCE® Prista P 360, Ingredion, USA) using strains or strain combinations, each of said strains or strain combinations corresponding to an embodiment. Figure 3 shows the degradation of off-flavor associated compounds as a result of fermentation of chickpea protein concentrate (CP-PRO70® concentrate, InnovoPro) using strains or strain combinations, each of said strains or strain combinations corresponding to an embodiment. Figure 4 shows the degradation of off-flavor associated compounds as a result of fermentation of isolate pea protein (ProFam™ 580, ADM) using strains or strain combinations, each of said strains or strain combinations corresponding to an embodiment. Figure 5 shows the degradation of off-flavor associated compounds as a result of fermentation of isolate pea protein (VITESSENCE™ 1803, Ingredion, USA) using strains or strain combinations, each of said strains or strain combinations corresponding to an embodiment.

Figures 6 to 9 show the production (level of increase compared to the unfermented sample, in signal to noise) of desirable dairy-associated flavor compounds in legume bases. More specifically, figure 6 shows the production of dairy associated compounds as a result of fermentation of faba bean protein concentrate (VITESSENCE® Prista P 360, Ingredion, USA) using strains or strain combinations, each of which strains or strain combinations corresponding to an embodiment. Figure 7 shows the production of dairy associated compounds as a result of fermentation of chickpea protein concentrate (CP-PRO70® concentrate, InnovoPro) using strains or strain combinations, each of which strains or strain combinations corresponding to an embodiment. Figure 8 shows the production of dairy associated compounds as a result of fermentation of isolate pea protein (ProFam™ 580, ADM) using strains or strain combinations, each of which strains or strain combinations corresponding to an embodiment. Figure 9 shows the production of dairy associated compounds as a result of fermentation of isolate pea protein (VITESSENCE™ 1803, Ingredion, USA) using strains or strain combinations, each of which strains or strain combinations corresponding to an embodiment.

Figures 10 to 13 show the production (level of increase compared to the unfermented sample, in signal to noise) of ethanol and esters by the heterofermentative Leuconostoc in legume bases. Figure 10 shows the production of ethanol and esters as a result of fermentation of faba bean protein concentrate (VITESSENCE® Prista P 360, Ingredion, USA) by the heterofermentative Leuconostoc. Figure 11 shows the production of ethanol and esters as a result of fermentation of chickpea protein concentrate (CP-PRO70® concentrate, InnovoPro) by the heterofermentative Leuconostoc. Figure 12 shows the production of ethanol and esters as a result of fermentation of isolate pea protein (ProFam™ 580, ADM) by the heterofermentative Leuconostoc. Figure 13 shows the production ethanol and esters as a result of fermentation of isolate pea protein (VITESSENCE™ 1803, Ingredion, USA) by the heterofermentative Leuconostoc.

Raffinose family type, alpha-galactooligosaccharide analysis

A set of experiments with the aim to quantify sucrose-based alpha-galactooligosaccharides was conducted. More precisely, sucrose, melibiose, raffinose, stachyose and verbascose from plantbased fermented products were characterized. In addition, the method also quantifies Glucose and Fructose typically also present in the samples.

These analyses were performed by HPAE-PAD (High Performance An-ion Exchange with Pulsed amperometry detection) on a Dionex ICS-6000 system (ThermoFisher, Sunnyvale, CA, USA). The alpha-galactooligosaccharides were separated with an 250x1 mm PA-200 column plus guard column and quantified by conductivity detection. The gradient elution were from the generated potassium hydroxide (A). 0-4min 10mM KOH, 4-4.5min. 100mM KOH, 4-5-13min 200mM KOH, 13-13.5min 10mM KOH, 13.5min-30min 10mM KOH.

The flow rate of the gradient elution was set to 63uL min^-1 with a total run time of 30 min. Retention time and peak area were used to quantify the sugars in the samples. Standards from 2.5ug mL^-1 to 500 mg L^-1 were used for calibration curves.

Figure 14 shows the degradation (mg/g, level of decrease compared to the unfermented matrix) of undesirable sugars causing discomfort in three legume bases as a result of fermentation at 30°C by the 6 LAB strains in the presence of Bacillus DSM 33182. Figure 15 shows the degradation (mg/g, level of decrease compared to the unfermented matrix) of undesirable sugars causing discomfort in three legume bases as a result of fermentation at 37°C by the 6 LAB strains in the presence of Bacillus DSM 33182.

Figure 16 shows the degradation (mg/g, level of decrease compared to the unfermented matrix) of undesirable sugars causing discomfort in three legume bases as a result of fermentation at 30°C by the 6 LAB strains in the presence of Bacillus DSM 33181 .

Figure 17 shows the degradation (mg/g, level of decrease compared to the unfermented matrix) of undesirable sugars causing discomfort in three legume bases as a result of fermentation at 37°C by the 6 LAB strains in the presence of Bacillus DSM 33181 .

Discussion

The reduction of off-flavors and formation of desirable compounds for the different plant bases is summarized below.

Reduction of off-flavors

Four different compounds are reported here as representative for the off-flavor such as 3-methyl- butanal, pentanal, hexanal and 1-penten-3-ol. Pentanal and hexanal are selected due to their reported involvement in the green and beany off-flavor (Engels et al., “Metabolic Conversions by Lactic acid bacteria during Plant Protein Fermentations”. Foods 11 , 1005 (2022); Fisher et al., “Impact of Ageing on Pea Protein Volatile Compounds and Correlation with Odor”, Molecules 27, 852 (2022)). 3-Methyl-butanal has a malty flavor (Wang et al., “Flavor challenges in extruded plantbased meat alternatives: A review”. Compr Rev Food Sci Food Saf. 21 (2022)). 1-Penten-3-ol is described also as with a green, vegetable perception in legume protein matrices (Xu et al. “HS- SPME-GC-MS/olfactometry combined with chemometrics to assess the impact of germination on flavor attributes of chickpea, lentil, and yellow pea flours”, Food Chemistry 280, 83-9584 (2019); Youseff et al., “Sensory Improvement of a Pea Protein-Based Product Using Microbial Co-Cultures of Lactic Acid Bacteria and Yeasts”, Foods 9, 349 (2022)).

In general terms, those compounds are reduced in all fermented samples by Bacillus or Lactic acid bacteria strains alone and in their combinations at the two different temperatures tested. In general, pentanal and hexanal are reduced more when Bacillus is combined with Leuconostoc, Lb. Plantarum, Rhamnosus and Paracasei than Bacillus alone. Each of the bases show slightly different behaviour for those compounds. For example:

In Faba, it is observed that 1-penten-3-ol is not founded in any of the conditions. Besides, pentanal and hexanal are degraded more when Bacillus and Leuconostoc, Bacillus and Lb. Plantarum, Bacillus and Streptococcus (only for pentanal). However, it is possible to see an increase of those two compounds when Bacillus and Paracasei (DSM 34552) are combined and fermented at 37°C. It is observed also that hexanal is increasing when Bacillus (DSM 33182) at both temperatures and when Bacillus (DSM 33181 and DSM 33182) are combined with Paracasei. 3-Methyl-butanal is reduced in all conditions tested.

In Chickpea, it is observed that 1-penten-3-ol is degraded for all combinations except for Bacillus subtilis (DSM 33182) at 30°C, Bacillus subtilis (DSM 33181) with Paracasei (DSM 34553) at 37°C, and Bacillus subtilis (DSM 33181 and DSM 33182) with Lb. Plantarum (DSM 34551). Pentanal and hexanal are degraded in a slightly higher degree when Bacillus is combined with Leuconostoc. 3- Methylbutanal is reduced to a large extent when Bacillus and Streptococcus combination is used.

In ADM Pea, it is observed that 1-penten-3-ol is degraded in a higher degree when Bacillus is combined with the other strains. The highest reduction is observed when Bacillus is combined with Leuconostoc. Pentanal and hexanal are degraded in all combinations tested and in a higher degree when Bacillus is combined with Leuconostoc, with Rhamnosus and Lacticaseibacillus paracasei at 37°C. 3-Methylbutanal shows than the Bacillus alone can reduce this compound to a larger extent.

In V. Pea, no degradation is observed for any of the above-mentioned compounds when Bacillus is fermented at 30°C. 1-Penten-3-ol is better reduced when Bacillus is combined with Leuconostoc. Pentanal and hexanal are degraded in a higher degree when Bacillus is combined with Leuconostoc, Paracasei and Rhamnosus. 3-Methyl-butanal is more degraded when Bacillus is combined with Rhamnosus.

Formation of desirable dairy note compounds

Four compounds are selected to describe the formation of dairy notes in the fermented samples. Those compounds are diacetyl, acetoin, 2,3-pentadione and 2-nonanone. Diacetyl and acetoin are important aroma compound in dairy products due to their buttery flavor (Macciola et al., “Rapid gas- chromatographic method for the determination of diacetyl in milk, fermented milk and butter”. Food Control 19 (9) (2008)). 2,3-Pentadione and 2-nonanone are also related to buttery, creamy, and sweet flavor (Zhao et al., “Variation of Aroma Components of Pasteurized Yogurt with Different Process Combination before and after Aging by DHS/GC-O-MS”. Molecules 28 (4) (2023)).

In general, all Bacillus and Lactic acid bacteria combinations (except Leuconostoc and Plantarum) result in more diacetyl production compared to Bacillus alone. Each of the bases show slightly different behavior for those compounds. For example:

In Faba, it is observed that diacetyl and acetoin produced for all Bacillus and Lactic acid bacteria combinations, except for Bacillus with Leuconostoc and with Plantarum. 2,3-Pentadione is produced more when Bacillus is combined with Streptococcus. 2-Nonanone is increased for all combinations of Bacillus and Lactic acid bacteria.

In Chickpea, diacetyl is formed for all combination except for Bacillus combined with Leuconostoc and Plantarum. Acetoin is not formed at all for any combinations. 2,3-Pentadione is produced more when Bacillus is combined with Streptococcus. 2-Nonanone is produced to a large extent when Bacillus and Rhamnosus and it shows a degradation when Bacillus and Leuconostoc is combined. In ADM pea, it is observed that diacetyl and acetoin are formed for all combinations except for Leuconostoc. 2,3-Pentadione is formed more when Bacillus is combined with Streptococcus. 2- Nonanone is formed more when Bacillus is combined with Paracasei (at 37°C) and Rhamnosus.

In V. Pea, diacetyl and acetoin increase for all combinations except for Bacillus at 37°C. Those compounds are former more than Bacillus alone when Bacillus and Paracasei (at 37°C), Bacillus and Rhamnosus and Bacillus and Streptococcus. 2,3-Pentadione and 2-nonanone are not formed in any of the conditions.

Ethanol and esters formation

The formation of ethyl esters is an important contributor for cheese flavor. This reaction is dependent on available ethanol and other factors such as esterase activity and free fatty acids (Pedersen et al., “Potential impact on cheese flavour of heterofermentative bacteria from starter cultures”. International Dairy Journal 33 (2) (2013)). Here, ethyl-acetate and ethyl-hexanoate are selected due to their impact on the fruity flavor of fermented products (Rajendran et al., “Flavour Volatiles of Fermented Vegetable and fruit substrates: a Review”, Molecules 28 (7) (2023)).

It is possible to see a formation for all those compounds for all the bases when fermented with Bacillus and the heterofermentative Leuconostoc.

In summary, the results show that the strains in Table 1 were able to reduce the amount of the beany- associated off-flavor compound hexanal, while producing several metabolites associated with pleasant flavor with dairy and umami notes. Spore negative phenotype is important, if the strains are to be used for food applications.

Oligosaccharides (Raffinose family type)

The reduction of raffinose family oligosaccharides (RFO) for the different plant bases is summarized in the following.

ADM pea 30° C - reduction RFO family oligosaccharides

Stachyose and Verbascose are reduced in all fermented samples, by Bacillus or Lactic acid bacteria strains alone & in their combinations. Raffinose is reduced with DSMZ33182 alone and in combination with DSM17876 and DSM32865 respectively.

Chickpea 30° C - reduction RFO family oligosaccharides

Verbascose is not present in the chickpea so no reduction is seen. Stachyose are reduced in all fermented samples, by Bacillus or Lactic acid bacteria strains alone & in their combinations. Raffinose is reduced with DSMZ33182 alone and in combination with DSM17876 and DSM32865 respectively and in the other combinations to a lesser degree.

Faba 30° C - reduction RFO family oligosaccharides Stachyose and Verbascose are reduced with DSMZ33182 alone and in combination with DSM17876 and DSM32865 respectively and Stachyose is reduced in the other combinations to a lesser degree. Verbascose is not reduced in other combinations.

ADM pea 37° C - reduction RFO family oligosaccharides

Stachyose and Verbascose are reduced in all fermented samples, by Bacillus or Lactic acid bacteria strains alone & in their combinations. Raffinose is reduced the most with DSMZ33182 alone but also a little in all combinations.

Chickpea 37° C - reduction RFO family oligosaccharides

Verbascose is not present in the chickpea so no reduction is seen. Stachyose are reduced in all fermented samples, by Bacillus or Lactic acid bacteria strains alone & in their combinations. Raffinose is reduced with DSMZ33182 alone and in combination with DSM17876 and in the other combinations to a lesser degree.

Faba 37° C - reduction RFO family oligosaccharides

Stachyose and Verbascose are reduced with DSMZ33182 alone and in combination with DSM17876 and DSM34553 respectively and with combination with ATCC53103 to a lesser degree. Verbascose is not reduced in other combinations.

Conclusion, Oligosaccharides (Raffinose family type)

From figures 14-17 it is clear that although the Bacillus subtilis strains degrade RFOs raffinose, stachyose, and verbascose by themselves in different plant bases, the reduction in RFOs is hampered where the Bacillus subtilis strains are combined with lactic acid bacteria starter cultures such as Leuconostoc mesenteroides strains, Lactiplantibacillus plantarum/pentosus/paraplantarum strains, and Lacticaseibacillus paracasei strains. The exception being that combinations of Bacillus subtilis strains with Streptococcus thermophilus strain DSM17876 do substantially not lessen the reduction of RFOs, and in some cases improves the reduction of stachyose and verbascose.

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 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 preferred embodiment(s) disclosed.

Claims

1 . A process for producing a fermented plant-based product with a reduced stachyose and/or verbascose content, the method comprising the steps of: a) providing a substrate comprising a plant base, b) adding to the substrate at least one Streptococcus thermophilus strain and at least one Bacillus subtilis strain, c) fermenting the substrate until a stop criterion is reached, and d) obtaining fermented plant-based product.

2. The process according to claim 1 , wherein the plant base is a legume substrate.

3. The process according to claim 2, wherein the legume substrate is a faba bean or chickpea substrate.

4. The process according to any one of the preceding claims, wherein the at least one Streptococcus thermophilus strain is Streptococcus thermophilus DSM 17876.

5. The process according to any one of the preceding claims, wherein the at least one Bacillus subtilis strain is Bacillus subtilis (DSM 33181) or Bacillus subtilis (DSM 33182).

6. A product obtainable by the process according to any one of the claims 1 to 5.

7. A starter culture composition comprising at least one Streptococcus thermophilus strain and at least one Bacillus subtilis strain.

8. The starter culture composition according to claim 7, wherein the at least one Streptococcus thermophilus is Streptococcus thermophilus (DSM 17876).

9. The starter culture composition according to claim 7 or 8, wherein the Bacillus subtilis strain is Bacillus subtilis (DSM 33181) or Bacillus subtilis (DSM 33182).

10. The starter culture composition according to claims 8 or 9, wherein the composition has a metabolic capacity to consume stachyose in separate fermentations of a faba bean isolate and a chickpea isolate, and wherein the metabolic capacity to consume stachyose is at least 80% of the least one Bacillus subtilis strain’s metabolic capacity to consume stachyose in separate fermentations of the faba bean isolate and the chickpea isolate.

1 1. The starter culture composition according to any one of the claims 8 to 10, wherein the composition has a metabolic capacity to consume verbascose in a fermentation of a faba bean isolate, and wherein the metabolic capacity to consume verbascose is at least 80% of the least one Bacillus subtilis strain’s metabolic capacity to consume verbascose in a fermentation of the faba bean isolate.

12. A kit-of-parts comprising at least one Streptococcus thermophilus strain and at least one Bacillus subtilis strain.

13. Use of a composition according to any one of the claims 8 to 11 or a kit-of-parts according to claim 12 for fermenting a plant-based product.

14. The use according to claim 13, wherein the use is furthermore for reducing a stachyose and/or a verbascose content in the plant-based product.

15. The use according to claim 13 or 14, wherein the use is furthermore for acidifying the plantbased product.

16. The use according to any one of the claims 13 to 15, wherein the plant-based product is a dairy analogue product.

17. The use according to any one of the claims 13 to 16, wherein the plant-based product is a meat analogue product.