Enzymatic treatment to improve texture of fermented plant-based cheese-analogue

Abstract

The present invention relates to a method for improving organoleptic properties of a plant-based cheese-analogue, and the resulting products as such. In particular, the combination of fermentation and enzymatic treatment yields a plant-based matrix with improved gel properties, such as increased hardness and springiness, to provide a product with a sensory feel that mimic dairy-based cheese. In addition, the treatment also reduces the number of bitterness-associated peptides found in the product.

Description

[0001] ENZYMATIC TREATMENT TO IMPROVE TEXTURE OF FERMENTED PLANT-BASED CHEESE-ANALOGUE

[0002] Technical field of the invention

[0003] The present invention relates to a method for improving organoleptic properties of a plant-based cheese-analogue, and the resulting products as such. In particular, the combination of fermentation and enzymatic treatment yields a plant-based matrix with improved gel properties, such as increased hardness and springiness, to provide a product with a sensory feel that mimic dairy-based cheese. In addition, the treatment also reduces the number of bitterness-associated peptides found in the product.

[0004] Background of the invention

[0005] The production of dairy cheese is an ancient practice that started as a way of preserving milk and turned into the development of fermented products with a broad range of flavors and interesting textures that are nowadays widely consumed on a regular basis. The characteristic organoleptic properties of cheese are directly linked to the production processes, the performance of different microorganisms, and most importantly, the nature of dairy milk. This colloidal dispersion of fat globules stabilized by casein micelles presents extremely particular behaviour upon heat treatment or acidification, and its replication with plant raw materials a difficult challenge to overcome.

[0006] In the production of cheese, milk is heat-treated to ensure safety and inactivate endogenous microorganisms that could be present in the raw product. During this process, proteins denature and unfold. Microorganisms, e.g., lactic acid bacteria, are then inoculated and start fermenting the milk, namely transforming lactose into lactic acid, and thus acidifying the media. This pH drop complements the action of rennet, encouraging proteins to interact with each other and start forming a protein network known as curd, which entraps fat globules in its pores and conforms the firm texture of cheese. Milk casein is responsible for the formation of this three-dimensional network, and its characteristic molecular structure is responsible for its versatility to confer liquid but also gel-like textures.

[0007] The demand for plant-based alternatives to dairy cheese is exponentially growing, mostly due to an increasing awareness of sustainability and the large amount of resources that animal protein production requires, and consumers are looking for products that can provide a similar experience to eating dairy cheese but are based on plant-based raw materials. One of the most important challenges in plant-based cheese-analogue production is texture, with producers trying to replicate the curd formed from milk casein mostly relying on functional ingredients such as texturizers for the development of this attribute. Accordingly, current plant-based cheese-analogues comprise additives, including oils, such as coconut oil, hydrocolloids, such as agar or carrageenans, or gums, such as guar, xanthan, or Arabic gum, to obtain a suitable firm texture.

[0008] However, despite recent efforts, currently commercialized plant-based cheeseanalogues do not meet consumer satisfaction with regards to texture. One of the issues is that the gel strength is generally weaker in plant protein systems than in dairy curds, and therefore the textural and rheological properties of these plant protein gels remain far from those of dairy curds and cheeses.

[0009] Plant protein gels are three-dimensional protein networks that may be heat-induced, acid-induced, or fermentation-induced. All three methods transform a liquid matrix into a gel structure through protein aggregation that is characterized as soft matter, since it is composed of protein molecules that are dispersed in a liquid matrix, giving them a semi-solid consistency. The textural and rheological properties of these gels can vary with pH, ionic strength, reducing agents, or protein concentration, among other factors. Moreover, the gelation dynamics of plant protein gels are dependent on the molecular structure of the proteins present in the gel. The protein-protein interaction conditions gel strength, but also other factors such as the size of the protein aggregates, if the protein network is fine or coarse and how well-structured it is, and the degree of crosslinking between the proteins.

[0010] For these reasons, knowledge on plant protein behaviour under different processing conditions is required to develop high quality products, and new strategies to improve plant protein gelation for the purpose of producing plant-based cheese-analogues are therefore at a premium.

[0011] Accordingly, there is clear need for development of plant-based systems that contain a suitable texture to provide the consumers with an organoleptic feeling similar to that experienced with dairy cheeses.

[0012] Thus, it would be advantageous to provide a method for improving the gel properties of a plant-base, such as a pea-base, for implementation in plant-based cheese-analogues. In particular, it would be advantageous to provide a simple method for increasing the hardness and springiness of a plant-based matrix to more accurately mimic a dairybased cheese.

[0013] Summary of the invention

[0014] The development of plant-based alternatives to dairy-based cheese is driven mainly by the sustainability agenda and the awareness of reducing the resource burden required by animal protein production. However, the plant-based cheese-analogue options available on the market today fail to meet the organoleptic expectations of the consumers. In particular, the texture of the plant-based cheese-analogues are too squishy, contain excessive texturizing agents, and nevertheless lack the firmness of dairy-based cheeses.

[0015] The present invention relates to a method for treatment of a plant-base, such as a peabase, to supply a plant-based matrix with improved texture similar to dairy-based cheeses. The method includes the combination of fermentation and enzymatic treatment of the plant-base. The enzymatic treatment with transglutaminase strengthens proteinprotein interactions to yield a self-supporting three-dimensional gel with a texture alike a dairy cheese and obviates the need to include any texturizing agents. The present invention also provides plant-based cheese-analogues with improved firmness and organoleptic experience.

[0016] Thus, an object of the present invention relates to the provision of a simple method for preparing a plant-based cheese-analogue with a texture resembling that of a dairybased cheese.

[0017] Another object of the present invention relates to the provision of a method for preparing a plant-based cheese-analogue without the need of any texturizing agents.

[0018] Thus, an aspect of the present invention relates to a method for producing a fermented pea-based product, the method comprising the following steps:

[0019] (i) providing a pea-base composition,

[0020] (ii) adding a bacterial culture comprising one or more lactic acid bacteria to said pea-base composition,

[0021] (iii) adding transglutaminase to said pea-base composition, and

[0022] (iv) fermenting the pea-base composition for a period of time until a predetermined pH is reached, thereby producing a fermented pea-based product. Another aspect of the present invention relates to a fermented pea-based product obtainable by the method as described herein.

[0023] Yet another aspect of the present invention relates to a bacterial blend comprising:

[0024] - one or more lactic acid bacteria, and

[0025] - transglutaminase.

[0026] A further aspect of the present invention relates to a fermented pea-based cheeseanalogue comprising:

[0027] - a fermented pea-base composition,

[0028] - one or more lactic acid bacteria, and

[0029] - transglutaminase.

[0030] A still further aspect of the present invention relates to use of transglutaminase in the production of a fermented pea-based cheese-analogue.

[0031] Brief description of the figures

[0032] Figure 1 shows (A) storage modulus G' (filled squares) and loss modulus G" (unfilled circles) as a frequency sweep of fermentation-induced pea protein gels with (light gray) and without (dark gray) TG, i.e. The dark gray unfilled circles (lowest data points) correspond to the loss modulus G" of a fermentation-induced pea protein gel without TG. The sweep was performed at 0.1 strain. (B) Storage modulus (white) and loss modulus (cross-hatched) of fermentation-induced pea protein gels with (right, TG) and without (left, none) TG at a frequency of 1 Hz.

[0033] Figure 2 shows (A) fermentation-induced pea protein gels without TG (left) and with TG (right). The top image shows the gels standing prior to manual pressing, and the lower image shows the gels after manual pressing. (B) Hardness and springiness of fermentation-induced pea protein gels with (right, TG) and without TG (left, none).

[0034] Figure 3 shows a bar graph displaying the number of bitterness-associated and nonbitter peptides of different pea protein gels obtained through fermentation with select combinations of lactic acid bacteria and with and without TG. Detailed description of the invention

[0035] Definitions

[0036] Prior to outlining the present invention in more details, a set of terms and conventions is first defined:

[0037] Dairy

[0038] In the present context, the term "dairy" refers to the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes, or camels.

[0039] The terms "dairy" and "milk" may be used interchangeably herein. Thus, for most practical purposes, the term "dairy-based" products (or cheese) refers to products (or cheese) obtained from cow's milk.

[0040] Plant-based cheese-analogues

[0041] In the present context, the term "plant-based cheese-analogue" 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, such as a pea-base comprising pea protein.

[0042] Pea -base composition

[0043] In the present context, the term "pea-base composition" refers to the plant-based material used as a base for fermentation. The pea-base composition comprises a peaprotein component, which can be, but is not limited to, a pea protein isolate, a pea protein concentrate, pea protein powder, or a pea protein flour. The pea-base composition may comprise additional ingredients beneficial for the fermentation, such as sugars.

[0044] Lactic acid bacteria

[0045] In the present context, the term "lactic acid bacteria" refers to food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram positive, catalase negative, acid tolerant, non-motile, non- sporulating, microaerophilic or anaerobic bacteria.

[0046] 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. These bacteria are thus generally responsible for the acidification of milk and for the texture of various dairy products. Beyond production of lactic acid, also acetic acid, formic acid, and propionic acid are generated by lactic acid bacteria.

[0047] The industrially most useful lactic acid bacteria include, but are not limited to, Lactococcus species (spp.), Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacterium spp, Enterococcus spp. and Propionibacterium spp. Additionally, lactic acid producing bacteria belonging to the group of the strict anaerobic bacteria, Bifidobacteria, i.e. Bifidobacterium spp. which are frequently used as food starter cultures alone or in combination with lactic acid bacteria, are generally included in the group of lactic acid bacteria.

[0048] Lactic acid bacteria may also be referred to by the abbreviation LAB.

[0049] Fermentation

[0050] In the present context, 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. Herein, the carbohydrates are provided in a pea-base composition and the bacterial culture comprises one or more lactic acid bacteria. Besides lactic acid, metabolites produced during fermentation include also volatile organic compound that can affect the flavour of the fermentation product.

[0051] Storage modulus

[0052] In the present context, the term "storage modulus" refers to the storage modulus G' in Pascal (Pa) determined from shear experiments, i.e., the ability of a material to store energy elastically when it is subjected to deformation. At low frequency, the rate of shear is low, and therefore the capacity of retaining the original strength of media is high. When the frequency increases, the rate of shear also increases resulting in an increased amount of energy put into the material. Accordingly, the storage modulus increases with frequency.

[0053] Texture profile analysis (TP A)

[0054] In the present context, the term "texture profile analysis (TPA)" refers to double compression test for determining the textural properties of foods. During texture profile analysis samples are compressed over two deformation cycles separated by a period of sample resting time. The TPA test is performed on a texture analyzer which generates a force-time graph from which mechanical textural attributes, such as hardness and springiness, can be extracted.

[0055] TPA may be measured according to ISO 11036:2020 Sensory analysis - Methodology - Texture profile. The texture analyzer may be a TA-XT2 (Stable Micro Systems, Godaiming, England).

[0056] Hardness

[0057] In the present context, the term "hardness" refers to the maximum force (or load) reached during the first deformation cycle resulting from texture profile analysis (TPA) of a material. The hardness is a measure of the firmness of the material. Hardness is measured in units of gravitational force equivalents (g).

[0058] Hardness may be measured according to ISO 11036:2020 Sensory analysis - Methodology - Texture profile. The texture analyzer may be a TA-XT2 (Stable Micro Systems, Godaiming, England).

[0059] Springiness

[0060] In the present context, the term "springiness" refers to the ratio calculated as the detected height during the second compression divided by the original compression distance resulting from texture profile analysis (TPA) of a material. The springiness reflects the ability of a sample to recover its structure after being compressed. Springiness is measured in percentage (%).

[0061] Springiness may be measured according to ISO 11036:2020 Sensory analysis - Methodology - Texture profile. The texture analyzer may be a TA-XT2 (Stable Micro Systems, Godaiming, England).

[0062] About

[0063] Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, weights, concentrations, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ±10% about the number 10, which is anything between 9% and 11%). Number of bitterness-associated peptides

[0064] The skilled person knows how to identify protein and peptide sequences of a pea base composition. An example of a method to determine peptide sequences in such samples is described in example 4, however, an array of other methods to achieve the same are available to the skilled person. Bitterness-associated peptides should in the context of the present disclosure be understood as a peptide which has been predicted to be bitter by the sequence-based predictor, iBitter-Fuse, developed by Charoenkwan et al. 2021. The predictor is available online through the following hyperlink: https: / / camt.pythonanywhere.com / bittersarpredict. Instructions of how to use the predictor is described by Charoenkwan et al 2021. Non-bitter peptides should in the context of the present disclosure be understood as a peptide which has been predicted to be non-bitter by the sequence-based predictor, iBitter-Fuse.

[0065] Improved plant-based cheese-analooues with texture mimickino dairy cheese

[0066] One of the main hurdles in manufacture of plant-based cheese-analogues is to produce a product with a texture resembling that of a corresponding dairy cheese. Current efforts typically include addition of texturizers, but the resulting mechanical textural attributes does not meet the expectations of the consumers. Thus, a sustainable and improved organoleptic offering to the consumers is needed to attain a more sizable commitment to plant-based cheese-analogues.

[0067] Therefore, there is an increasing interest in new ways of producing gels where the plant proteins are the main structural units of the gel structure. Pea protein is an exceptional plant-base as a dairy alternative because it is easily digestible, rich in iron, and largely hypoallergenic. Fermentation of the pea base has great potential to modify and improve the physicochemical and sensory properties of proteins, thus creating pleasant textures and flavors that might improve the quality of plant-based cheese-analogues.

[0068] When lactic acid bacteria are grown in a plant protein matrix, they produce lactic acid, thus decreasing the pH to values near the isoelectric point of the plant proteins. The protein net charges are then close to zero and they start aggregating and forming a curd that retains water and oil inside its pores. Some lactic acid bacteria contribute to an improved texture due to their ability to produce exo- (or extracellular) polysaccharides (EPS), which can be capsular (remain attached to the cell in the form of capsules) or secreted into the media. EPS consists of either a single type of sugar (homo-exopolysaccharides) or repeating units made of different sugars (hetero- exopolysaccharides). EPS-producing LAB are of interest since EPS act as natural viscosifiers and texture enhancers of fermented foods. Furthermore, EPS from food- grade LAB with defined rheological properties have potential for development and exploitation as food additives.

[0069] However, the three-dimensional matrix- or gel-like structure obtained from fermentation of the pea base does still not adequately mimic the texture of a corresponding dairy cheese, with the primary challenge being that the mechanical properties are weaker.

[0070] Herein are described a method for producing a pea-based product with excellent texture. The method is based on the combination of fermentation and enzymatic treatment of a pea-base composition to achieve a product with a matrix- or gel-like structure of great mechanical textural attributes.

[0071] The inventors have found that enzymatic treatment of the pea-base composition with transglutaminase (TG) was very effective in promoting texture building of the fermented product. Transglutaminases are a group of enzymes which catalyse the formation of an isopeptide bond between y-carboxamide groups of glutamine residue side chains and the E-amino groups of lysine residue side chains. While TG has been used in food processing previously as a means for bonding proteins together, e.g., in imitated meats, the efficiency with which TG improved texture while enduring the acidic environment of fermentation was a highly surprising finding.

[0072] In particular, the results obtained by the inventors indicate that TG under suboptimal pH conditions is capable of establishing covalent bonds between the pea proteins to provide a fermented pea-based product with excellent mechanical textural attributes.

[0073] Thus, an aspect of the present invention relates to a method for producing a fermented pea-based product, the method comprising the following steps:

[0074] (i) providing a pea-base composition,

[0075] (ii) adding a bacterial culture comprising one or more lactic acid bacteria to said pea-base composition,

[0076] (iii) adding transglutaminase to said pea-base composition, and

[0077] (iv) fermenting the pea-base composition for a period of time until a predetermined pH is reached, thereby producing a fermented pea-based product.

[0078] Improved flavor of plant-based cheese analogues Another important issue in plant-based cheese analogues is the oral perception of bitterness. Unfortunately, plant-based cheese analogues, such as analogues based on pea compositions, are often perceived as too bitter by consumers. Besides volatile compounds, peptides present in the analogues contribute to the perception bitterness. Hydrophobic peptides have previously been associated with the oral perception of bitterness, and it has been shown that TG can alter the peptide composition of pea protein isolate hydrolysates to affect a lower bitterness perception (Cosson et al., 2022; Su et al., 2023). The inventors have surprisingly shown that by processing a pea-based composition with TG and fermentation with select lactic acid bacteria, the number of bitterness-associated peptides is reduced even further than what can be achieved with TG alone. In other words, the results obtained by the inventors indicate a synergistic effect of using TG in combination with lactic acid bacteria for obtaining plant-based cheese analogues with improved flavor. It has in particular been shown that by using a Streptococcus thermophilus strain and either a Lacticaseibacillus rhamnosus strain or a Lentilactobacillus kefiri strain in combination with TG, an improved reduction in the number of bitterness-associated peptides is achieved.

[0079] Hence, an embodiment of the present invention relates to the method as described herein, wherein the one or more lactic acid bacteria comprises a Streptococcus thermophilus strain and either a Lacticaseibacillus rhamnosus strain or a Lentilactobacillus kefiri strain. In a specific version of this embodiment, the one or more lactic acid bacteria comprises Streptococcus thermophilus and either Lacticaseibacillus rhamnosus DSM33870 or Lentilactobacillus kefiri DSM34723. In another specific version of this embodiment, the one or more lactic acid bacteria comprises Streptococcus thermophilus DSM34727 and either Lacticaseibacillus rhamnosus DSM33870 or Lentilactobacillus kefiri DSM34723. In another specific version of this embodiment, the one or more lactic acid bacteria comprises Streptococcus thermophilus DSM34727 and / or DSM 19242, and Lacticaseibacillus rhamnosus DSM33870. In another specific version of this embodiment, the one or more lactic acid bacteria comprises Streptococcus thermophilus STH79, DSM34726, DSM34727, and / or DSM 19242, and Lentilactobacillus kefiri DSM34723. In another specific version of this embodiment, the one or more lactic acid bacteria comprises Streptococcus thermophilus DSM34725 and Lacticaseibacillus rhamnosus DSM33870. Preferably, the one or more lactic acid bacteria does not comprise Lactobacillus bulgaricus DSM28910.

[0080] An embodiment of the present invention relates to the method as described herein, wherein the period of time is at least 1 hour, preferably at least 1.2 hours. An embodiment of the present invention relates to the method as described herein, wherein the content of transglutaminase is in the range of about 0.01 % (w / w) to about 10 % (w / w), such as about 0.1 % (w / w) to about 5 % (w / w), such as about 0.2 % (w / w) to about 1 % (w / w), with respect to the total weight of the pea-base composition.

[0081] The pea-base composition comprises pea proteins which during fermentation and enzymatic treatment are crosslinked to provide a matrix- or gel-like structure. The pea protein is typically extracted from yellow split peas and may be provided in several different forms. One preferred form is pea protein isolate in which the outer shell of the peas is removed, and the remainder is milled into a flour. The flour then undergoes separation of fibers and starch in a filtration process and is finally distilled into a white precipitate known as pea protein isolate or analogously as pea protein powder. The proteins in the pea protein component are mostly denatured during the purification process. The amount of pea protein may be adjusted in accordance with the desired properties of the final fermented pea-based product.

[0082] Thus, an embodiment of the present invention relates to the method as described herein, wherein the pea-base composition comprises a pea protein component in a form selected from the group consisting of a pea protein isolate, a pea protein concentrate, pea protein powder, and a pea protein flour, preferably a pea protein isolate.

[0083] Another embodiment of the present invention relates to the method as described herein, wherein the pea protein component comprises at least about 50% (w / w) protein, such as at least about 60% (w / w) protein, such as at least about 70% (w / w) protein, preferably at least about 80% (w / w) protein, with respect to the total weight of the pea protein component.

[0084] A further embodiment of the present invention relates to the method as described herein, wherein the pea-base composition has a content of pea protein component in the range of about 2 % (w / w) to about 20% (w / w), such as about 5 % (w / w) to about 15 % (w / w), such as about 8 % (w / w) to about 12 % (w / w), with respect to the total weight of the pea-base composition.

[0085] A still further embodiment of the present invention relates to the method as described herein, wherein the pea-base composition has a content of pea protein component of about 10 % (w / w), with respect to the total weight of the pea-base composition. The pea-base composition is a viscoelastic material, preferably in liquid form, and may comprise additional ingredients in addition to the pea protein component. Oil may be included in the pea-base composition to mimic the fat contained in dairy cheese, thereby improving the mouthfeel of the final product. The oil may be in liquid form at room temperature, i.e., have a melting temperature below room temperature, such as below 0 °C. Examples of such oils include, but are not limited to, sunflower oil and rapeseed oil.

[0086] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the pea-base composition is a viscoelastic material.

[0087] Another embodiment of the present invention relates to the method as described herein, wherein the pea-base composition is in liquid form.

[0088] Yet another embodiment of the present invention relates to the method as described herein, wherein the pea-base composition is in the form of a colloidal suspension.

[0089] Still another embodiment of the present invention relates to the method as described herein, wherein the colloidal suspension is an emulsion or sol, preferably an emulsion.

[0090] A further embodiment of the present invention relates to the method as described herein, wherein the pea-base composition comprises one or more oils or fats.

[0091] An even further embodiment of the present invention relates to the method as described herein, wherein the one or more oils or fats are liquid at room temperature and / or have a melting temperature of less than 0°C.

[0092] Sugars can be added to the pea-base composition to induce fermentation. They act as carbohydrate substrates in the fermentation reaction and as nutrient for increased microbial proliferation. The method is not limited to any specific sugars or amount thereof.

[0093] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the pea-base composition comprises one or more sugars.

[0094] Another embodiment of the present invention relates to the method as described herein, wherein the content of said one or more sugars is in the range of about 0.5 % (w / w) to about 5 % (w / w), such as about 1 % (w / w) to about 3 % (w / w), with respect to the total weight of the pea-base composition.

[0095] A further embodiment of the present invention relates to the method as described herein, wherein the one or more sugars are monosaccharides and / or disaccharides.

[0096] A still further embodiment of the present invention relates to the method as described herein, wherein the one or more sugars are sucrose and / or glucose.

[0097] Pea globulins, such as vicilin, convicilin, and legumin, in their native state has a tightly folded structure which makes them less susceptible to crosslinking through the action of transglutaminase. Legumin and vicilin, in particular, present hexameric and trimeric structures, respectively, where TG could have difficulty accessing glutamine and lysine. Thus, to maximise inter- and intra-molecular crosslinking induced by TG, the pea-base composition may be heat treated prior to inoculation with bacterial culture and addition of TG to unfold and expose the hydrophobic sites of the pea proteins, including glutamine and lysine residues.

[0098] Thus, an embodiment of the present invention relates to the method as described herein, wherein the method comprises a step of subjecting the pea-base composition to heat treatment prior to adding the bacterial culture and transglutaminase.

[0099] Another embodiment of the present invention relates to the method as described herein, wherein the pea-base composition is subjected to heat treatment at a temperature sufficient for denaturing the pea proteins.

[0100] Still another embodiment of the present invention relates to the method as described herein, wherein said heat treatment is performed at a temperature of at least about 80°C, such as at least about 85°C, preferably about 90°C.

[0101] An even further embodiment of the present invention relates to the method as described herein, wherein the heat treatment is performed for at least about 5 min, such as at least about 10 min, such as at least about 15 min, preferably at least about 20 min.

[0102] The method described herein involves fermentation of a pea-base composition utilizing lactic acid bacteria. The method is not limited to any particular combination of lactic acid bacteria, and it is contemplated that the method will find utility with lactic acid bacteria usually utilised for food processing. These include, but are not limited to, lactic acid bacteria traditionally used for fermentation of dairy products.

[0103] Thus, an embodiment of the present invention relates to the method as described herein, wherein the one or more lactic acid bacteria are of a genus selected from the group consisting of Streptococcus, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Fructilactobacillus, Lactococcus, Bifidobacterium, and Pediococcus.

[0104] 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 nothing else is noticed. For the purpose of the present invention, table 1 presents a list of new and old names of some Lactobacillus species relevant to the present invention.

[0105] Table 1. New and old names of some Lactobacillus species relevant to the present invention.

[0106] Another embodiment of the present invention relates to the method as described herein, wherein the one or more lactic acid bacteria are selected from the group consisting of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lacticaseibacillus paracasei, Bifidobacterium, Bifidobacterium lactis, Lactobacillus helveticus, Lacticaseibacillus casei, Pediococcus, Lactiplantibacillus plantarum, Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, Lactococcus lactis and Lacticaseibacillus rhamnosus.

[0107] A further embodiment of the present invention relates to the method as described herein, wherein the one or more lactic acid bacteria comprises Streptococcus thermophilus and Lactobacillus bulgaricus. A preferred embodiment of the present invention relates to the method as described herein, wherein the one or more lactic acid bacteria consists of:

[0108] - DSM28910,

[0109] - DSM22589, and / or

[0110] - DSM15957.

[0111] Another preferred embodiment of the present invention relates to the method as described herein, wherein the one or more lactic acid bacteria consists of:

[0112] - DSM28910,

[0113] - DSM22589, and

[0114] - DSM15957.

[0115] Yet another embodiment of the present invention relates to the method as described herein, wherein the bacterial culture comprises Lactiplantibacillus plantarum, S. thermophilus and / or Lacticaseibacillus rhamnosus, and the bacterial culture further comprises an auxiliary culture, the auxiliary culture comprising one or more lactic acid bacteria selected from the group consisting of Lactobacillus bulgaricus, Lactobacillus helveticus, Pediococcus acidilactici, and Lacticaseibacillus casei. In one version of this embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus. In another version of this embodiment, the auxiliary culture comprises a Lactobacillus helveticus. In another version of this embodiment, the auxiliary culture comprises a Pediococcus acidilactici. In one version of this embodiment, the auxiliary culture comprises a Lacticaseibacillus casei. In a preferred version of this embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus and a Lactobacillus helveticus. In another preferred version of this embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus and a Pediococcus acidilactici. In another preferred version of this embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus and a Lacticaseibacillus casei. In a preferred version of this embodiment, the auxiliary culture comprises a Lactobacillus helveticus and a Pediococcus acidilactici. In another preferred version of this embodiment, the auxiliary culture comprises a Lactobacillus helveticus and a Lacticaseibacillus casei. In a preferred version of this embodiment, the auxiliary culture comprises a Pediococcus acidilactici and a Lactobacillus helveticus. In a preferred version of this embodiment, the auxiliary culture comprises a Pediococcus acidilactici and a Lacticaseibacillus casei. In a preferred embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, and a Pediococcus acidilactici. In another preferred embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, and a Lacticaseibacillus casei. In another preferred embodiment, the auxiliary culture comprises a Lactobacillus bulgaricus, a Lacticaseibacillus casei, and a Pediococcus acidilactici. In another preferred embodiment, the auxiliary culture comprises a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei.

[0116] In a preferred embodiment, the bacterial culture comprises a Lactiplantibacillus plantarum, a S. thermophilus, and a Lacticaseibacillus rhamnosus, and the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei. In a version of this embodiment, the S. thermophilus is S. thermophilus DSM34725. In another version of this embodiment, the Lacticaseibacillus rhamnosus is Lacticaseibacillus rhamnosus DSM33870.

[0117] In an embodiment, the Lactobacillus bulgaricus is Lactobacillus bulgaricus DSM28910. In an embodiment, the Lactobacillus helveticus is Lactobacillus helveticus DSM 19499. In an embodiment, the Pediococcus acidilactici is Pediococcus acidilactici DSM28307. In an embodiment the Lacticaseibacillus casei is Lacticaseibacillus casei ATCC55544.

[0118] In a preferred embodiment, the bacterial culture comprises Lactiplantibacillus plantarum DSM35094, S. thermophilus DSM34725, and Lacticaseibacillus rhamnosus DSM33870, and the auxiliary culture comprises Lactobacillus bulgaricus DSM28910, Lactobacillus helveticus DSM 19499, Pediococcus acidilactici DSM28307, and Lacticaseibacillus casei ATCC55544.

[0119] In a preferred embodiment, the bacterial culture comprises Lactiplantibacillus plantarum, S. thermophilus DSM34725, and Lacticaseibacillus rhamnosus DSM33870, and the auxiliary culture comprises Lactobacillus bulgaricus DSM28910, Lactobacillus helveticus DSM 19499, Pediococcus acidilactici DSM28307, and Lacticaseibacillus casei ATCC55544.

[0120] In an embodiment, the bacterial culture comprises a Lactiplantibacillus plantarum, S. thermophilus DSM34725, and a Lacticaseibacillus rhamnosus, and the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei. In another version of the preferred embodiment, the bacterial culture comprises a Lactiplantibacillus plantarum, a S. thermophilus and Lacticaseibacillus rhamnosus DSM33870, and the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei. In another version of the preferred embodiment, the bacterial culture comprises a Lactiplantibacillus plantarum, a S. thermophilus, and a Lacticaseibacillus rhamnosus, and the auxiliary culture comprises Lactobacillus bulgaricus DSM 28910, a Lactobacillus helveticus, a Pediococcus acidilactici, and a Lacticaseibacillus casei. In another version of the preferred embodiment, the bacterial culture comprises a Lactiplantibacillus plantarum, a S. thermophilus, and a Lacticaseibacillus rhamnosus, and the auxiliary culture comprises a Lactobacillus bulgaricus, Lactobacillus helveticus DSM 19499, a Pediococcus acidilactici, and a Lacticaseibacillus casei. In another version of the preferred embodiment, the bacterial culture comprises a Lactiplantibacillus plantarum, a S. thermophilus, and a Lacticaseibacillus rhamnosus, and the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, Pediococcus acidilactici DSM28307, and a Lacticaseibacillus casei. In another version of the preferred embodiment, the bacterial culture comprises a Lactiplantibacillus plantarum, a S. thermophilus, and a Lacticaseibacillus rhamnosus, and the auxiliary culture comprises a Lactobacillus bulgaricus, a Lactobacillus helveticus, a Pediococcus acidilactici, and Lacticaseibacillus casei ATCC55544.

[0121] Another embodiment of the present invention relates to the method as described herein, wherein the bacterial culture comprises Lentilactobacillus kefiri, Streptococcus thermophilus, and Lactococcus lactis. In a preferred version of this embodiment, the bacterial culture comprises Lentilactobacillus kefiri, Streptococcus thermophilus, and Lactococcus lactis. In a specific version of this embodiment, the bacterial culture comprises Lentilactobacillus kefiri DSM34723, Streptococcus thermophilus DSM34727, Streptococcus thermophilus DSM 19242, and Lacticaseibacillus rhamnosus DSM33870, and Lactococcus lactis DSM34729. In another version of this embodiment, the bacterial culture comprises Lentilactobacillus kefiri DSM34723, a Streptococcus thermophilus, and a Lacticaseibacillus rhamnosus, and a Lactococcus lactis. In another version of this embodiment, the bacterial culture comprises a Lentilactobacillus kefiri, Streptococcus thermophilus DSM 34727, and a Lacticaseibacillus rhamnosus, and a Lactococcus lactis. In another version of this embodiment, the bacterial culture comprises Lentilactobacillus kefiri, Streptococcus thermophilus DSM 19242, and a Lacticaseibacillus rhamnosus, and a Lactococcus lactis. In another version of this embodiment, the bacterial culture comprises a Lentilactobacillus kefiri, a Streptococcus thermophilus and Lacticaseibacillus rhamnosus DSM33870, and a Lactococcus lactis. In yet another version of this embodiment, the bacterial culture comprises a Lentilactobacillus kefiri, a Streptococcus thermophilus and a Lacticaseibacillus rhamnosus, and Lactococcus lactis DSM34729.

[0122] Another embodiment of the present invention relates to the method as described herein, wherein the bacterial culture comprises Levilactobacillus brevis, Lentilactobacillus kefiri, and Streptococcus thermophilus. In a preferred version of this embodiment, Levilactobacillus brevis is Levilactobacillus brevis DSM34744,

[0123] In a preferred version of this embodiment, Lentilactobacillus kefiri is Lentilactobacillus kefiri DSM 34723.

[0124] In a preferred version of this embodiment, Streptococcus thermophilus is Streptococcus thermophilus DSM34726 and / or Streptococcus thermophilus DSM34727.

[0125] Another embodiment of the present invention relates to the method as described herein, wherein the bacterial culture comprises Lentilactobacillus kefiri, Streptococcus thermophilus, Lacticaseibacillus rhamnosus, and Lactococcus lactis.

[0126] In a preferred version of this embodiment, Lentilactobacillus kefiri is Lentilactobacillus kefiri DSM 34723.

[0127] In a preferred version of this embodiment, Streptococcus thermophilus is Streptococcus thermophilus DSM34727 and / or Streptococcus thermophilus DSM19242. In a preferred version of this embodiment Lacticaseibacillus rhamnosus is Lacticaseibacillus rhamnosus DSM33870. In a preferred version of this embodiment Lactococcus lactis is Lactococcus lactis DSM34729.

[0128] In a preferred version of the above embodiments that comprise Lactiplantibacillus plantarum, the Lactiplantibacillus plantarum is DSM35094.

[0129] Yet another embodiment of the present invention relates to the method as described herein, wherein the content of bacterial culture is in the range of about 0.01 % (w / w) to about 0.1 % (w / w), with respect to the total weight of the pea-base composition.

[0130] The pea-base composition is acidified during fermentation as the lactic acid bacteria converts carbohydrates into lactic acid and other metabolites. Lactic acid bacteria which induce fast acidification are preferred for food safety reasons. Fermentation of the peabase composition is continued until a predetermined pH is reached. This target pH value can be set, amongst others, based on the desired texture of the fermented pea-based product. Typically, the predetermined pH value will be less than pH 5 to realise the full benefits of the fermentation process where texture as well as flavour is developed.

[0131] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the predetermined pH is less than about pH 5, such as less than about pH 4.9, such as less than about pH 4.8, such as less than about pH 4.7 such as less than about pH 4.6, preferably about pH 4.5. Another embodiment of the present invention relates to the method as described herein, wherein the predetermined pH is in the range of about pH 4 to about pH 5, such as about pH 4.3 to about pH 4.7.

[0132] At the end of the fermentation the pea-base composition has been transformed into a matrix or gel with mechanical textural attributes similar to a dairy cheese. This include a certain gel strength and firmness that keeps the product from collapsing under pressure, but also some elasticity as would be expected from a dairy cheese.

[0133] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product is in the form of a matrix or gel.

[0134] Another embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product is a fermented pea-based cheese-analogue.

[0135] The form and texture of the fermented pea-based product obtained by the method described herein is achieved without additives such as texturizers or thickening agents. Producers of plant-based products, such as cheese-analogues, would like to avoid use of these additives because it would reduce cost and give a cleaner label.

[0136] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product does not contain any texturizers.

[0137] Another embodiment of the present invention relates to the method as described herein, wherein the texturizers are selected from the group consisting of coconut oil, hydrocolloids, and gums.

[0138] A further embodiment of the present invention relates to the method as described herein, wherein the texturizers are selected from the group consisting of coconut oil, agar, carrageenans, guar gum, xanthan gum and Arabic gum.

[0139] The present method produces fermented pea-based products with excellent mechanical textural attributes despite the absence of texturizers or thickening agents. Important parameters include storage modulus, hardness, and springiness. The storage modulus is a measure of the ability of the fermented pea-based product to store energy elastically when it is subjected to deformation. The springiness reflects the ability of the fermented pea-based product to recover its structure after being compressed. Together, these two parameters contribute significantly to the chewiness of the product, i.e., the mouthfeel sensation experienced by the consumer during chewing due to sustained and elastic resistance from the food. Hardness is a measure of the firmness of the product and relates to the force applied by the molar teeth to compress the food. Accordingly, all three parameters are important characteristic of cheese and will influence how the food is perceived by the consumer.

[0140] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the fermented pea-based product has a storage modulus (G') of at least about 10,000 Pa, such as at least about 12,000 Pa, at a frequency of 1 Hz at 0.1% strain at room temperature.

[0141] Another embodiment of the present invention relates to the method as described herein, wherein fermented pea-based product has a hardness of at least about 1,000 g, such as at least about 1,100 g.

[0142] A further embodiment of the present invention relates to the method as described herein, wherein fermented pea-based product has a springiness of at least about 70%, such as at least about 75%, preferably at least about 80%.

[0143] A still further embodiment of the present invention relates to the method as described herein, wherein the hardness and / or springiness are measured according to ISO 11036:2020 Sensory analysis - Methodology - Texture profile.

[0144] The present method may include an additional step following fermentation of inactivating the transglutaminase. This step may be performed by heating the fermented pea-based product to a temperature of approx. 70°C. The properties induced by transglutaminase is retained after heat treatment.

[0145] Thus, an embodiment of the present invention relates to the method as described herein further comprising a heat treatment step following step (iv) of heat treatment of the fermented pea-based product.

[0146] Another embodiment of the present invention relates to the method as described herein, wherein the heat treatment step is performed at about 70°C.

[0147] The fermented pea-based product obtained from the present method has all of the above advantageous mechanical textural attributes. Provided herein is therefore a fermented pea-based product, such as a cheese-analogue, which have mechanical properties that resembles those of a dairy cheese.

[0148] Accordingly, an aspect of the present invention relates to a fermented pea-based product obtainable by the method as described herein.

[0149] In an embodiment, the fermented pea-based product is obtained by a method comprising the steps of:

[0150] (i) providing a pea-base composition,

[0151] (ii) adding a bacterial culture comprising one or more lactic acid bacteria to said pea-base composition,

[0152] (iii) adding transglutaminase to said pea-base composition, and

[0153] (iv) fermenting the pea-base composition for a period of time until a predetermined pH is reached, wherein the one or more lactic acid bacteria comprises a Streptococcus thermophilus strain and either a Lacticaseibacillus rhamnosus strain or a Lentilactobacillus kefiri strain. In a preferred version of this embodiment, the fermented pea-based product has a lower number of bitterness-associated peptides as compared to an auxiliary fermented pea-based product obtained by a method comprising the following steps:

[0154] (a) providing the same pea-base composition used to produce the fermented peabased product,

[0155] (b) adding the same bacterial culture used to produce the fermented pea-based product to said pea-base composition, and

[0156] (c) fermenting the pea-base composition to the predetermined pH.

[0157] A bacterial blend comprising transglutaminase may be used as ingredient in the process of preparing a fermented pea-based cheese-analogue.

[0158] Thus, an aspect of the present invention relates to a kit comprising transglutaminase and a bacterial culture comprising Lactobacillus delbrueckii subsp. bulgaricus and / or Streptococcus thermophilus. In a preferred embodiment, the kit comprising transglutaminase and a bacterial culture comprising DSM28910, DSM22589, and / or- DSM15957.

[0159] Another aspect of the present invention relates to a fermented pea-based cheeseanalogue comprising:

[0160] - a fermented pea-base composition,

[0161] - one or more lactic acid bacteria, and transglutaminase.

[0162] In an embodiment, the one or more lactic acid bacteria comprises a Streptococcus thermophilus strain and either a Lacticaseibacillus rhamnosus strain or a Lentilactobacillus kefiri strain.

[0163] Yet another aspect of the present invention relates to a fermented pea-based cheeseanalogue comprising:

[0164] - a fermented pea-based composition,

[0165] - one or more lactic acid bacteria, and

[0166] - optionally, transglutaminase.

[0167] An embodiment of the present invention relates to the fermented pea-based cheeseanalogue as described herein, wherein the transglutaminase has been inactivated.

[0168] A further aspect of the present invention relates to use of transglutaminase in the production of a fermented pea-based cheese-analogue.

[0169] In an embodiment of the use of transglutaminase in the production of a fermented peabased cheese-analogue, the fermented pea-based cheese analogue is fermented by using one or more lactic acid bacteria comprising a Streptococcus thermophilus strain and either a Lacticaseibacillus rhamnosus strain or a Lentilactobacillus kefiri strain.

[0170] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0171] Preferences, options, and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options, and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the method for producing the fermented pea-based product and all its features, which may readily be part of the resulting fermented pea-based product, such as a fermented pea-based cheese-analogue. Moreover, said features may also be transferred to the use of transglutaminase (TG) for production of a fermented pea-based cheese-analogue. Embodiments and features of the present invention are also outlined in the following items. Items

[0172] XI. A method for producing a fermented pea-based product, the method comprising the following steps:

[0173] (i) providing a pea-base composition,

[0174] (ii) adding a bacterial culture comprising one or more lactic acid bacteria to said pea-base composition,

[0175] (iii) adding transglutaminase to said pea-base composition, and

[0176] (iv) fermenting the pea-base composition for a period of time until a predetermined pH is reached, thereby producing a fermented pea-based product.

[0177] X2. The method according to item XI, wherein the content of transglutaminase is in the range of about 0.01 % (w / w) to about 10 % (w / w), such as about 0.1 % (w / w) to about 5 % (w / w), such as about 0.2 % (w / w) to about 1 % (w / w), with respect to the total weight of the pea-base composition.

[0178] X3. The method according to any one of items XI or X2, wherein the pea-base composition comprises a pea protein component in a form selected from the group consisting of a pea protein isolate, a pea protein concentrate, pea protein powder, and a pea protein flour, preferably a pea protein isolate.

[0179] X4. The method according to item X3, wherein the pea protein component comprises at least about 50% (w / w) protein, such as at least about 60% (w / w) protein, such as at least about 70% (w / w) protein, preferably at least about 80% (w / w) protein, with respect to the total weight of the pea protein component.

[0180] X5. The method according to any one of items X3 or X4, wherein the pea-base composition has a content of pea protein component in the range of about 2 % (w / w) to about 20% (w / w), such as about 5 % (w / w) to about 15 % (w / w), such as about 8 % (w / w) to about 12 % (w / w), with respect to the total weight of the pea-base composition.

[0181] X6. The method according to any one of items X3-X5, wherein the pea-base composition has a content of pea protein component of about 10 % (w / w), with respect to the total weight of the pea-base composition.

[0182] X7. The method according to any one of the preceding items, wherein the pea-base composition is in liquid form. X8. The method according to any one of the preceding items, wherein the pea-base composition is in the form of a colloidal suspension.

[0183] X9. The method according to item X8, wherein the colloidal suspension is an emulsion or sol, preferably an emulsion.

[0184] X10. The method according to any one of the preceding items, wherein the pea-base composition comprises one or more oils or fats.

[0185] XI 1. The method according to any one of the preceding items, wherein the pea-base composition comprises one or more sugars.

[0186] X12. The method according to item Xll, wherein the content of said one or more sugars is in the range of about 0.5 % (w / w) to about 5 % (w / w), such as about 1 % (w / w) to about 3 % (w / w), with respect to the total weight of the pea-base composition.

[0187] X13. The method according to any one of items Xll or X12, wherein the one or more sugars are monosaccharides and / or disaccharides.

[0188] X14. The method according to any one of items X11-X13, wherein the one or more sugars are sucrose and / or glucose.

[0189] X15. The method according to any one of the preceding items, wherein the method comprises a step of subjecting the pea-base composition to heat treatment prior to adding the bacterial culture and transglutaminase.

[0190] X16. The method according to item X15, wherein said heat treatment is performed at a temperature of at least about 80°C, such as at least about 85°C, preferably about 90°C.

[0191] X17. The method according to any one of the preceding items, wherein the one or more lactic acid bacteria are of a genus selected from the group consisting of Streptococcus, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Fructilactobacillus, Lactococcus, Bifidobacterium, and Pediococcus.

[0192] X18. The method according to any one of the preceding items, wherein the one or more lactic acid bacteria are selected from the group consisting of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lacticaseibacillus paracasei, Bifidobacterium, Bifidobacterium lactis, Lactobacillus helveticus, Lacticaseibacillus casei, Pediococcus, Lactiplantibacillus plantarum, Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, Lactococcus lactis and Lacticaseibacillus rhamnosus.

[0193] X19. The method according to any one of the preceding items, wherein the one or more lactic acid bacteria comprises Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0194] X20. The method according to any one of the preceding items, wherein the one or more lactic acid bacteria consists of:

[0195] - DSM28910

[0196] DSM22589, and / or

[0197] - DSM15957.

[0198] X21. The method according to any one of the preceding items, wherein the content of bacterial culture is in the range of about 0.01 % (w / w) to about 0.1 % (w / w), with respect to the total weight of the pea-base composition.

[0199] X22. The method according to any one of the preceding items, wherein the predetermined pH is less than about pH 5, such as less than about pH 4.9, such as less than about pH 4.8, such as less than about pH 4.7 such as less than about pH 4.6, preferably about pH 4.5.

[0200] X23. The method according to any one of the preceding items, wherein the predetermined pH is in the range of about pH 4 to about pH 5, such as about pH 4.3 to about pH 4.7.

[0201] X24. The method according to any one of the preceding items, wherein the fermented pea-based product is in the form of a matrix or gel.

[0202] X25. The method according to any one of the preceding items, wherein the fermented pea-based product is a fermented pea-based cheese-analogue.

[0203] X26. The method according to any one of the preceding items, wherein the fermented pea-based product has a storage modulus (G') of at least about 10,000 Pa, such as at least about 12,000 Pa, at a frequency of 1 Hz at 0.1% strain at room temperature. X27. The method according to any one of the preceding items, wherein fermented peabased product has a hardness of at least about 1,000 g, such as at least about 1,100 g.

[0204] X28. The method according to any one of the preceding items, wherein fermented peabased product has a springiness of at least about 70%, such as at least about 75%, preferably at least about 80%.

[0205] Yl. A fermented pea-based product obtainable by the method according to any one of the preceding items.

[0206] Ul. A kit comprising transglutaminase and a bacterial culture comprising Lactobacillus delbrueckii subsp. bulgaricus and / or Streptococcus thermophilus.

[0207] U2. A kit comprising transglutaminase and a bacterial culture comprising

[0208] - DSM28910,

[0209] - DSM22589, or

[0210] - DSM15957.

[0211] U3. A kit comprising transglutaminase and a bacterial culture comprising

[0212] - DSM28910

[0213] - DSM22589, and

[0214] - DSM15957.

[0215] Zl. A fermented pea-based cheese-analogue comprising:

[0216] - a fermented pea-base composition,

[0217] - one or more lactic acid bacteria, and

[0218] - transglutaminase.

[0219] Z2. The fermented pea-based cheese-analogue according to item Zl, wherein the content of transglutaminase is in the range of about 0.01 % (w / w) to about 10 % (w / w), such as about 0.1 % (w / w) to about 5 % (w / w), such as about 0.2 % (w / w) to about 1 % (w / w), with respect to the total weight of the fermented pea-base composition.

[0220] Z3. The fermented pea-based cheese-analogue according to any one of items Zl or Z2, wherein the fermented pea-base composition has a content of pea protein in the range of about 2 % (w / w) to about 20% (w / w), such as about 4 % (w / w) to about 12% (w / w), such as about 6 % (w / w) to about 10 % (w / w), with respect to the total weight of the fermented pea-base composition. Z4. The fermented pea-based cheese-analogue according to any one of items Z1-Z3, wherein the one or more lactic acid bacteria are of a genus selected from the group consisting of Streptococcus, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Fructilactobacillus, Lactococcus, Bifidobacterium, and Pediococcus.

[0221] Z5. The fermented pea-based cheese-analogue according to any one of items Z1-Z4, wherein the one or more lactic acid bacteria are selected from the group consisting of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lacticaseibacillus paracasei, Bifidobacterium, Bifidobacterium lactis, Lactobacillus helveticus, Lacticaseibacillus casei, Pediococcus, Lactiplantibacillus plantarum, Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, Lactococcus lactis and Lacticaseibacillus rhamnosus.

[0222] Z6. The fermented pea-based cheese-analogue according to any one of items Z1-Z5, wherein the one or more lactic acid bacteria comprises Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0223] 71. The fermented pea-based cheese-analogue according to any one of items Z1-Z6, wherein the one or more lactic acid bacteria consists of:

[0224] - DSM28910,

[0225] - DSM22589, and / or

[0226] - DSM15957.

[0227] Z8. The fermented pea-based cheese-analogue according to any one of items Z1-Z7, wherein the content of bacterial culture is in the range of about 0.01 % (w / w) to about 0.1 % (w / w), with respect to the total weight of the fermented pea-base composition.

[0228] Z9. The fermented pea-based cheese-analogue according to any one of items Z1-Z8, wherein the fermented pea-based cheese-analogue has a storage modulus (G') of at least about 10,000 Pa, such as at least about 12,000 Pa, at a frequency of 1 Hz at 0.1% strain at room temperature.

[0229] Z10. The fermented pea-based cheese-analogue according to any one of items Z1-Z9, wherein fermented pea-based cheese-analogue has a hardness of at least about 1,000 g, such as at least about 1,100 g. Zll. The fermented pea-based cheese-analogue according to any one of items Z1-Z10, wherein fermented pea-based cheese-analogue has a springiness of at least about 70%, such as at least about 75%, preferably at least about 80%.

[0230] Wl. Use of transglutaminase in the production of a fermented pea-based cheeseanalogue.

[0231] W2. The use according to item Wl, wherein the transglutaminase is added during fermentation with one or more lactic acid bacteria.

[0232] W3. The use according to any one of items Wl or W2, wherein the transglutaminase improves the texture of the fermented pea-based cheese-analogue compared to a fermented pea-based cheese-analogue produced without transglutaminase.

[0233] W4. The use according to item W3, wherein improved texture is defined as an increase in storage modulus, hardness and / or springiness of the fermented pea-based cheeseanalogue.

[0234] W5. The use according to any one of items W1-W4, wherein the fermented pea-based cheese-analogue comprises a fermented pea-base composition with a content of pea protein in the range of about 2 % (w / w) to about 20% (w / w), such as about 4 % (w / w) to about 12% (w / w), such as about 6 % (w / w) to about 10 % (w / w), with respect to the total weight of the fermented pea-base composition.

[0235] The invention will now be described in further details in the following non-limiting examples.

[0236] Examples

[0237] Example 1: Preparation of fermented pea-based product

[0238] This example describes how the fermented pea-based product can be produced by a combination of fermentation and enzymatic treatment.

[0239] Method

[0240] Preparation of pea-base composition

[0241] Pea protein isolate (ADM, Chicago, IL, USA) with a composition of 81.3% protein, 7% fat, and 9% fiber was suspended at 10% w / w in a water solution of 1% w / w glucose- and 1% w / w sucrose (Sigma Aldrich, Soborg, Denmark) and blended with a triple-blade hand blender (Kenwood Limited, Woking, United Kingdom) until dispersed. The suspension was emulsified with 10% sunflower oil (Woolworths, Auckland, New Zealand) with the same blender and was homogenized in a Twin Panda 400 homogenizer (GEA, Dusseldorf, Germany) using two stages (150; 50 bars) in a single pass. Homogenized emulsions were then heat-treated at 90 °C for 20 min while stirring in a thermoregulated mixer (Bellini Supercook, New York, USA) and cooled down to room temperature prior to microbial inoculation.

[0242] Fermentation and enzymatic treatment

[0243] The bacterial culture used in this example are from Chr. Hansen A / S (Horsholm, Denmark) and is commercial starter culture comprising two strains of S. thermophilus (DSM22589 and DSM15957) and a single strain of L. delbrueckii subsp. bulgaricus (DSM28910).

[0244] A volume of 0.02 % w / w inoculum of the bacterial culture was inoculated in the prepared pea-base composition. 0.5% w / w transglutaminase (TG) (Ajinomoto, Chuo City, Tokyo, Japan) was added to half of the samples. Inoculated samples were incubated at 43°C for 8 hours until the pH reached 4.5. Samples were then stored either at 4°C or at 22°C. After 7 days of storage, all samples were drained to avoid the expelled water maintaining continuous contact with the gel during storage.

[0245] The resulting fermented pea-based product was further analyzed for rheological properties (example 2) and mechanical textural attributes (example 3).

[0246] Results

[0247] The fermented pea-based product prepared by the present method, with and without TG, had a three-dimensional gel-like structure fully self-supported by the proteinprotein interactions of the protein network (figure 2A, top).

[0248] Conclusion

[0249] The described method successfully produced a fermented pea-based product with a structure that resembles a plant-based cheese-analogue.

[0250] Example 2: Rheological measurements of the fermented pea-based product

[0251] This example describes the measurements performed to determine the rheological properties of the fermented pea-based product, hereunder the storage modulus (G').

[0252] Method The fermented pea-based product was prepared according to Example 1.

[0253] Rheoloqical analysis

[0254] The rheological properties of the gels were analyzed with a rheometer (Advanced Rheometric Expansion System (ARES), TA Instruments, New Castle, USA) equipped with a plate-to-plate geometry (flat parallel plate diameter: 40 mm). Slices of each sample of fermented pea-based product were placed between the plates at a 1 mm gap and subjected to a frequency sweep from 0.01 to 10 Hz at 0.1 strain at room temperature.

[0255] The storage modulus (G') and loss modulus (G") of each sample was obtained to evaluate their viscoelastic properties. Rheological tests were performed in technical duplicates and biological triplicates, obtaining a total of 6 readings for each time point, as well as for the enzymatically treated (TG sample), and non-TG treated samples. The

[0256] Storage modulus changes during gelation were followed by fermenting the pea-base composition with and without TG in a viscoelasticity testing platform (ElastoSens™Bio, Rheolution, Montreal, Canada) at 43 °C for 8 h. G' was measured every minute at 0.1 strain and a fixed frequency of 0.8 Hz, and acidification was simultaneously followed in a parallel setting with a pH meter.

[0257] Statistical Analysis

[0258] All samples were compared using analysis of variance with a factorial design, where the effect of the presence of TG was evaluated. Tukey tests were performed in cases where there were more than two levels per factor, and t-student tests in the case of two levels per factor, to assess significance. The software used for the statistical analysis was JMP Pro 16 (SAS Institute, Cary, North Carolina, United States), and p values of less than 0.05 were interpreted as significant differences. All analyses were performed in biological triplicates and technical duplicates.

[0259] Results

[0260] When investigating the gelation dynamics of pea-base compositions into fermentation- induced gels, two gels were subjected to fermentation under the same incubation conditions (43 °C until reaching a pH of 4.5). It was observed that in the gel without TG, the storage modulus began to increase after approximately 0.5 hours of fermentation and at a pH of around 6.8. In contrast, the gel with TG had a delayed onset of the gelation process with the storage modulus starting to increase after approximately 1.2 hours of incubation and at a pH of around 6.7.

[0261] Without being bound by theory, it is contemplated that the delayed onset of gelation is caused by an increased temporal requirement for TG to establish sufficient intermolecular connections. The crosslinking between proteins facilitated by TG leads to formation of more stable and structured networks within the gel matrix. This could potentially be explained by a higher crosslinking density in samples with TG.

[0262] Indeed, the rheological data supported that the storage modulus is significantly increased for samples fermented in the presence of TG compared to samples prepared in the absence of TG (Figures 1A-B). The sample without enzymatic treatment presented a G' of 7850 Pa, whereas the sample with TG had a G' of 12051 Pa (Figure IB). These results reflect that the elastic component of the sample with TG was significantly higher, and therefore, firmer, and more resistant to deformation than the one without TG.

[0263] Both samples prepared with and without TG showed higher storage moduli (G') than loss moduli (G") indicating gel-like behaviour in both cases (Figures 1A-B).

[0264] Conclusion

[0265] Rheological analysis of the fermented pea-based product revealed that the crosslinking induced by transglutaminase (TG) significantly increased the elastic response to produce a gel which more closely resembled dairy cheese than the sample without addition of TG.

[0266] Example 3: Texture profile analysis (TPA) of the fermented pea-based product This example describes the measurements performed to determine the mechanical textural attributes, hardness, and springiness, of the fermented pea-based product.

[0267] Method

[0268] The fermented pea-based product was prepared according to Example 1.

[0269] Texture profile analysis (TPA)

[0270] Texture profile analysis (TPA) of the fermented pea-based product was performed for samples with and without transglutaminase.

[0271] Samples were subjected to double compression at room temperature in a texture analyzer (TA-XT2, Stable Micro Systems, Godaiming, England). Cylinders of 2.5 cm x 2.5 cm were cut with a stainless-steel cylinder from the central part of the samples, and a sample compression of 40% was applied with a 50mm diameter cylindrical aluminum probe using two compression cycles at a constant crosshead speed of 2 mm / sec using a trigger force of 3 g. The resulting texture profile analysis (TPA) reported the hardness and springiness of the fermented samples. Compression tests were performed in technical duplicates and biological triplicates, obtaining a total of six readings for each storage time point, as well as for the samples treated with and without TG.

[0272] Hardness was calculated as the maximum force of the first compression, and springiness was calculated as a ratio between the detected height during the second compression divided by the original compression distance.

[0273] Statistical

[0274] All samples were compared using analysis of variance with a factorial design, where the effect of the presence of TG was evaluated. Tukey tests were performed in cases where there were more than two levels per factor, and t-student tests in the case of two levels per factor, to assess significance. The software used for the statistical analysis was JMP Pro 16 (SAS Institute, Cary, North Carolina, United States), and p values of less than 0.05 were interpreted as significant differences. All analyses were performed in biological triplicates and technical duplicates.

[0275] Results

[0276] All gels were subjected to a double compression test, and their textural properties were evaluated by their hardness and springiness. Hardness values provide information about the firmness of the gels, whereas springiness values reflect the ability of a sample to recover its structure after being compressed.

[0277] Prior to the compression test, it was already possible to detect differences when the samples were pressed by hand (Figure 2A). Samples without TG (left) readily collapsed upon gentle pressure, whereas samples with TG (right) withstood pressure and retained their shape.

[0278] TPA testing revealed that samples prepared with TG resulted in significantly firmer gels than samples prepared in absence of TG, with hardness values of 1153 g and 456 g, respectively (Figure 2B). Moreover, samples containing TG were able to recover 86% of their structure, whereas those without TG were fractured after the first compression and only recovered 43% of their structure (Figure 2B). Without being bound by theory, it is contemplated that this markedly difference in mechanical textural attributes is due to the covalent bonds between lysine and glutamine residues facilitated by the presence of TG. In comparison, fermentation-induced gels prepared in the absence of TG are supported by hydrophobic interactions and hydrogen bonds, which supposedly are weaker in nature. Conclusion

[0279] The TPA results conclude that TG treatment leads to significantly higher gel hardness and springiness compared to the samples without TG treatment. Surprisingly, both hardness and springiness values were doubled resulting in a fermented pea-based product with textural attributes similar to those of a dairy-based cheese.

[0280] Example 4: Peptide profiles of the fermented pea-based product

[0281] Peptides significantly influence flavor and peptidomics analyses were thus performed for the different fermented pea-based product.

[0282] Fermented pea-based products were prepared according to Example 1, but with the bacterial blends as shown in table 2 below. The fermented pea-based products were then stored for 16 weeks under refrigeration before being subjected to peptidomics analyses.

[0283] Peptide sample preparation

[0284] Water-soluble peptides were extracted from the samples that were stored for 16 weeks under refrigeration, by mixing the samples and water (ratio 1:2). The mixture was allowed to stand at 40°C for 30 min before the water-insoluble large peptides and intact proteins were removed by centrifugation (10,000 x g for 20 min at 4°C) and clear supernatant was stored at -20°C. The tubes were then lyophilized, and the dried pellets were reconstituted with water (MilliQ purified) and filtered through 0.45 pm centrifugal filters (Millipore, Australia). The collected eluents were subsequently lyophilized. The lyophilized pellets were resuspended in 100 zL of 1% formic acid to provide peptide samples.

[0285] LC-MS analysis of peptide samples

[0286] The peptides samples were chromatographically separated (5 pL) on an a liquid chromatograph (Ekspert nanoLC415, Eksigent, Dublin, CA, USA) coupled to a mass spectrometer (Triple TOF 6600 MS, SCIEX, Redwood City, CA, USA) as described previously (Bose et al., 2019). In brief, the peptides were desalted for 5 min on a C18 trap column (ChromXP C18, 3 pm, 120 A, 10 x 0.3mm) at a flow rate of 10 pL / min solvent A and separated on another C18 column (ChromXP, 3 pm, 120 A, 150 mmx0.3mm) at a flow rate of 5pL / min. The solvents used were, solvent A: 5% DMSO, 0.1% formic acid, 94.9% water; solvent B: 5% DMSO, 0.1% formic acid, 90% acetonitrile, 4.9% water. A linear gradient from 5 to 45% solvent B over 40 min was employed, followed by 45-90% B over 5 min, a 5 min hold at 90% B, a return to 5% B over 1 min, and 14 min of re-equilibration. The eluent from the HPLC was directly coupled to the ion source (DuoSpray) of the Triple TOF 6600 MS. The ion spray voltage was set to 5500 V; the curtain gas was set to 138 kPa (20 psi), and the ion source gas 1 and 2 (GS1 and GS2) were set to 103 kPa and 138 kPa (15 psi and 20 psi). The heated interface was set to 100°C. Raw spectral data files were processed using Protein-Pilot™ 5.0 software (AB SCIEX) with integrated false discovery rate (FDR) analysis, and the Paragon Algorithm was used for peptide identification.

[0287] Identification of peptides and prediction of bitterness-associated peptides

[0288] Tandem mass spectrometry data was searched against a custom-built database comprising the pea reference genome Pisum sativum via (urgi. versailles.inra.fr / Species / Pisum / Pea-Genome-project) appended to the UniProt Pisum sativum database (version 20220406) and a database of contaminant proteins known as the standard repository of adventitious proteins (cRAP) and the Biognosys-11 iRT_C18 peptide standards ("UP291121_

[0289] Pisum_sativum_Genome_vla_cRAP_iRTl.new.fasta"). The total number of proteins in the custom database was 62,311 proteins. The individual data files were searched using the 'no enzyme' criteria. The database search results were curated to yield the protein identifications using a 1% global FDR determined by the built-in FDR tool within ProteinPilot software. Peptide lists were curated for the proteins detected in 1% FDR and peptides with 95% confidence. The different sequences were associated with bitter and non-bitter human flavor perception through the predictor iBitter-Fuse developed by Charoenkwan et al. 2021.

[0290] Results

[0291] Hydrophobic peptides have previously been associated with the oral perception of bitterness (Cosson et al., 2022; Su et al., 2023). While non-bitter peptides dominated the peptide profile of all samples (Figure 3), the analysis of peptidomic data allowed the identification of specific peptides believed to impart bitterness in pea protein. In samples fermented by B6, B64, and A2, more bitterness-associated peptides were detected in the absence of TG (Su et al., 2023). In contrast, in samples fermented by Al, the presence of TG caused a higher number of bitter peptides.

[0292] Conclusion

[0293] Pea protein fermented gels prepared using transglutaminase and the B6, B64, and A2 blends have a lower level (number) of bitterness-associated peptides than the gels prepared only with the B6, B64 and A2 blends. Therefore, consumers may prefer gels prepared with these cultures and transglutaminase over gels only prepared with the culture blends. Deposits and Expert Solution

[0294] The applicant requests that a sample of the deposited microorganism stated in table 3 below may only be made available to an expert, until the date on which the patent is granted. The applicant requests that the availability of the deposited microorganism referred to in Rule 33 EPC shall be effected only by the issue of a sample to an independent expert nominated by the requester (Rule 32(1) EPC). If an expert solution has been requested, restrictions concerning the furnishing of samples apply.

[0295] The deposit was made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D- 38124 Braunschweig, Germany.

[0296] The Budapest Treaty provides that any restriction of public access to samples of deposited biological material must be irrevocably removed as of the date of grant of the relevant patent.

[0297] Table 3. Deposited strains The strain Lactobacillus paracasei (also referred to as Lacticaseibacillus paracasei,

[0298] CRL431, and L. casei 431®) was deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Tissue type Collection Center, 10801 University Blvd, Manassas, VA 20110, USA on 24 January 1994 under accession number ATCC 55544. The well-known probiotic bacterium is commercially available from Chr. Hansen A / S, 10-12 Boege Alle, DK-2970 Hoersholm, Denmark, under the product name Probio-Tec® F-DVS L. casei-431®, Item number 501749, and under the product name Probio-Tec® C-Powder-30, Item number 687018.

[0299] References

[0300] Bose, U., Broadbent, J. A., Byrne, K., Hasan, S., Howitt, C. A., 8<. Colgrave, M. L. (2019). Optimisation of protein extraction for in-depth profiling of the cereal grain proteome. Journal of Proteomics, 197, 23-33. https: / / doi.org / 10.1016 / jjprot.2019.02.009

[0301] Charoenkwan P, Nantasenamat C, Hasan MM, Moni MA, Lio' P, Shoombuatong W. iBitter- Fuse: A Novel Sequence-Based Bitter Peptide Predictor by Fusing Multi-View Features. Int J Mol Sci. 2021 Aug 19;22(16):8958. doi: 10.3390 / ijms22168958. PMID: 34445663; PMCID: PMC8396555.

[0302] Cosson, A., Oliveira Correia, L., Descamps, N., Saint-Eve, A., & Souchon, I. (2022). Identification and characterization of the main peptides in pea protein isolates using ultra high-performance liquid chromatography coupled with mass spectrometry and bioinformatics tools. Food Chemistry, 367, 130747. https: / / doi.Org / 10.1016 / j.foodchem.2021.130747

[0303] Su, G., Xie, Y., Liu, R., Cui, G., Zhao, M., & Zhang, J. (2023). Effect of transglutaminase on taste characteristics of pea protein hydrolysates through altering the composition of amino acids and peptides. Food Bioscience, 56, 103261. https: / / doi.Org / 10.1016 / j.fbio.2023.103261

[0304] Zheng et al. (2020), Int. J. Syst. Evol. Microbiol., 70, 2782-2858

Claims

Claims1. A method for producing a fermented pea-based product, the method comprising the following steps:(i) providing a pea-base composition,(ii) adding a bacterial culture comprising one or more lactic acid bacteria to said pea-base composition,(iii) adding transglutaminase to said pea-base composition, and(iv) fermenting the pea-base composition for a period of time until a predetermined pH is reached, thereby producing a fermented pea-based product.

2. The method according to claim 1, wherein the content of transglutaminase is in the range of about 0.01 % (w / w) to about 10 % (w / w), such as about 0.1 % (w / w) to about 5 % (w / w), such as about 0.2 % (w / w) to about 1 % (w / w), with respect to the total weight of the pea-base composition.

3. The method according to any one of claims 1 or 2, wherein the pea-base composition comprises a pea protein component in a form selected from the group consisting of a pea protein isolate, a pea protein concentrate, pea protein powder, and a pea protein flour, preferably a pea protein isolate.

4. The method according to claim 3, wherein the pea-base composition has a content of pea protein component in the range of about 2 % (w / w) to about 20% (w / w), such as about 5 % (w / w) to about 15 % (w / w), such as about 8 % (w / w) to about 12 % (w / w), with respect to the total weight of the pea-base composition.

5. The method according to any one of the preceding claims, wherein the method comprises a step of subjecting the pea-base composition to heat treatment prior to adding the bacterial culture and transglutaminase.

6. The method according to any one of the preceding claims, wherein the one or more lactic acid bacteria are of a genus selected from the group consisting of Streptococcus, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Fructilactobacillus, Lactococcus, Bifidobacterium, and Pediococcus.

7. The method according to any one of the preceding claims, wherein the one or more lactic acid bacteria comprises a Streptococcus thermophilus strain and either a Lacticaseibacillus rhamnosus strain or a Lentilactobacillus kefiri strain.

8. The method according to any one of the preceding claims, wherein the predetermined pH is less than about pH 5, such as less than about pH 4.9, such as less than about pH 4.8, such as less than about pH 4.7 such as less than about pH 4.6, preferably about pH 4.5.

9. The method according to any one of the preceding claims, wherein the fermented pea-based product is in the form of a matrix or gel.

10. The method according to any one of the preceding claims, wherein the fermented pea-based product is a fermented pea-based cheese-analogue.

11. The method according to any one of the preceding claims, wherein fermented peabased product has a hardness of at least about 1,000 g, such as at least about 1,100 g.

12. The method according to any one of the preceding claims, wherein fermented peabased product has a springiness of at least about 70%, such as at least about 75%, preferably at least about 80%.

13. A fermented pea-based product obtainable by the method according to any one of the preceding claims.

14. The fermented pea-based product of claim 13, wherein the fermented pea-based product is obtained by the method of claim 7.

15. The fermented pea-based product of claim 14, wherein the fermented pea-based product has a lower number of bitterness-associated peptides as compared to an auxiliary fermented pea-based product obtained by a method comprising the following steps:(a) providing the same pea-base composition used to produce the fermented peabased product,(b) adding the same bacterial culture used to produce the fermented pea-based product to said pea-base composition, and(c) fermenting the pea-base composition to the predetermined pH.

16. A kit comprising transglutaminase and a bacterial culture comprising Lactobacillus delbrueckii subsp. bulgaricus and / or Streptococcus thermophilus, wherein theLactobacillus delbrueckii subsp. bulgaricus is preferably DSM28910, and wherein the Streptococcus thermophilus is DSM22589 and / or DSM 15957.

17. A fermented pea-based cheese-analogue comprising: - a fermented pea-base composition,- one or more lactic acid bacteria, and- transglutaminase.

18. The fermented pea-based cheese-analogue of claim 17, wherein the one or more lactic acid bacteria comprises a Streptococcus thermophilus strain and either a Lacticaseibacillus rhamnosus strain or a Lentilactobacillus kefiri strain.

19. Use of transglutaminase in the production of a fermented pea-based cheeseanalogue.

20. The use according to claim 19, wherein the fermented pea-based cheese analogue is fermented by one or more lactic acid bacteria comprising a Streptococcus thermophilus strain and either a Lacticaseibacillus rhamnosus strain or a Lentilactobacillus kefiri strain.

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