Method for obtaining a plant-based yoghurt analog

Abstract

The present disclosure generally relates to the field of starter cultures for dairy alternatives. In particular, a method for producing a fermented plant-based composition, a fermented plant-based composition, and a use of a Ligilactobacillus salivarius strain to ferment a plant base. 5

Description

[0001] P8194EP00

[0002] 1

[0003] TITLE

[0004] Method for obtaining a plant-based yoghurt analog

[0005] FIELD

[0006] The present disclosure generally relates to the field of starter cultures for dairy alternatives. In particular, a method for producing a fermented plant-based composition, a fermented plant-based composition, and a use of a Ligilactobacillus salivarius strain to ferment a plant base.

[0007] BACKGROUND

[0008] In the manufacture of yoghurt analogs, plant bases are fermented by lactic acid bacteria (LABs) to provide flavor, texture, and acidification. From a food safety point of view, it is desirable to ferment plant bases to a pH of 4.6 or lower within 10 hours, however, these acidification targets cannot be met for some plant bases with currently used starter cultures, such as cultures containing Streptococcus thermophilus and Lactobacillus bulgaricus strains, without the addition of sugar to the base. The necessity to add sugar adds to the costs of manufacturing yoghurt analogs and conflicts with manufacturers wish to produce healthier products that contain less sugar.

[0009] Yoghurt analogs may be produced from a plant base, or a mixture of a plant base and milk obtained from mammals, such as cow's milk. Analogs manufactured from mixtures of plant bases and milk obtained from mammals are often referred to as hybrid products. Various plant bases are commercially available, such as almond, rice, pea, fava, oat, and soy bases. Conventionally, combinations of one or more Streptococcus thermophilus (S. thermophilus') strains and one or more Lactobacillus delbrueckii subsp. bulgaricus Lb. bulgaricus) strains are inoculated into the plant base and allowed to ferment the base at a suitable temperature to provide the yoghurt analog. The strains used to provide yoghurt analogs should provide a good texture, which typically has a high mouth thickness and viscosity (measured as high shear stress using a rheometer) and high gel firmness.

[0010] Soy bases are currently the most popular plant base used in the manufacture of yoghurt analogs. The main fermentable sugar in this base is sucrose, which means that Lb. bulgaricus, which is known to not ferment on sucrose, essentially does not contribute to the acidification. Thus, when using conventional S. thermophilus and Lb. bulgaricus strains in the fermentation of soy bases, it is essentially only S. thermophilus that provides acidification. Typically, soy bases contain in the range of 0.6-0.9 wt% sucrose, however, this concentration of sucrose is insufficient for reaching the acidification targetP8194EP00

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[0012] (pH 4.6 within 10 hours) when using conventional S. thermophilus. Examples of insufficient acidifications of soy bases with conventional cultures are shown in figure 1. Here, acidification curves of two commercially available soy bases, which were fermented with a commercially available starter culture containing S. thermophilus and Lb. bulgaricus, are shown. The first soy base is acidified from a pH of 7.2 to a pH of 5.3 at 10 hours, whereas the second soy base is acidified from a pH of about 7 to a pH of 4.7 at 10 hours. To aid the fermentation such that the acidification target can be reached, additional sucrose is commonly added to the soy base, the specific amount of sucrose depending on the soy base's natural sucrose content, start pH, and buffering capacity. Manufacturers, however, wish to reduce the amount of added sugars to meet consumer's demand for healthier products and to reduce costs.

[0013] Soy bases naturally contain high levels of protein, often in the range of 3 to 6 wt%, which adds to the base' buffering capacity, hampering acidification. It is desirable from a nutritional perspective, to have such high amounts of protein.

[0014] Hence, there remains a need for a method for fermenting plant bases to food safety acidification targets, using lower amounts of added sugar, and without compromising on other key product qualities such as protein content and texture.

[0015] SUMMARY

[0016] The inventors of the present invention have surprisingly found that by using a Ligilactobacillus salivarius Lb. salivarius) strain in the fermentation of a soy base, said soy base can be fermented to a pH of 4.6 or below within 10 hours without the addition of sugars. However, it is contemplated that a Ligilactobacillus salivarius strain may be used in the fermentation of any plant base, using only low amounts of added sugar or no added sugar at all.

[0017] Accordingly, the present disclosure provides in a first aspect a method for producing a fermented plant-based product, the method comprising the steps of:

[0018] i) providing a plant base,

[0019] ii) inoculating a Ligilactobacillus salivarius strain into the plant base, and iii) allowing the Ligilactobacillus salivarius strain to acidify the plant base to a pH of 4.6 or lower, thereby obtaining the fermented plant-based composition.

[0020] Advantageously, this fermentation can be achieved using only a Ligilactobacillus salivarius, i.e., without the addition of other bacterial cultures, simplifying the manufacturing process, and lowering production costs. Also, since lower amounts ofP8194EP00

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[0022] sugars are required, as compared to when using other LABs for the fermentation, the ingredient costs are even further reduced.

[0023] The Lb. salivarius strain of the method can be any Lb. salivarius strain, as evident from example 1 and figure 1-3, which show five Lb. salivarius strains used to ferment a soy base to a pH of 4.6. The Lb. salivarius strain is preferably a strain safe to use in the production of food grade products. Preferably, the Lb. salivarius strain is provided as a starter culture.

[0024] Definitions

[0025] As used herein, the term "wt%" means weight percent, i.e., "g per 100 g".

[0026] As used herein, the terms such as "2 % sucrose" and "2 % fructose" refer to 2% weight / volume solution, i.e., "g per 100 ml_".

[0027] The terms "yoghurt analog" or "plant-based composition" used herein are meant to refer to dairy-like products, which are products used as culinary replacements for dairy products, prepared where one or more mammalian milk constituents have been replaced with plant material and the resulting food resembles the original product. The mammalian milk constituents are replaced completely or at least 30% by dry weight with plant material, for example, using planted-based milks derived from legumes (such as soybeans, pea, lentils or chickpeas), nuts (such as coconut), cereals (such as oat).

[0028] As used herein, the adjective "dairy" shall be taken to mean a composition or product comprises or consists of mammalian milk matter, i.e., the lacteal secretion obtainable by milking.

[0029] As used herein, the term "free from" should be understood as a composition or product which does not contain a given substance but where trace amounts or contaminants thereof may be present.

[0030] As used herein, the term "added sugar" shall refer to sugars that are added during the processing of foods (e.g. refined sugars that may be added to a plant base of processed plant matter) as opposed to sugars naturally occurring in said foods. Added sugars include sugars (free, mono- and disaccharides), sugars from syrups and honey, and sugars from concentrated fruit or vegetable juices that are in excess of whatP8194EP00

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[0032] would be expected from the same volume of 100 percent fruit or vegetable juice of the same type.

[0033] As used herein, the term "fermentation" or "fermenting" refers to a process wherein carbohydrates are transformed into a range of metabolites through chemical reactions carried out by one or more microorganisms.

[0034] The term "lactic acid bacteria" ("LAB") designates 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, low-GC, acid tolerant, non-sporulating, non-respiring, rodshaped bacilli or cocci.

[0035] 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.

[0036] The term "plant base", is used to describe the plant material used as a base for fermentation. As an example, the plant base may be a soy base. Such a soy base may e.g., be obtained by soaking and grinding soybeans, boiling the mixture, and filtering out remaining particulates. Soy base can, e.g., be a soy milk, which is a plant-based drink.

[0037] The relatedness between two amino acid sequences or between two nucleotide sequences is described herein by the parameter "sequence identity".

[0038] For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

[0039] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)P8194EP00

[0040] 5

[0041] As used herein, the term "alpha-galactosidase activity" refers to the hydrolyzation of the a-l,6-galactosidic bonds present in saccharides such as melibiose, raffinose, stachyose, and verbascose, i.e., the removal of galactose residues from a saccharide backbone. This activity is characteristic of the enzyme alpha-galactosidase (a-gal) (EC.

[0042] 3.2.1.22). For purposes of the present invention, alpha-galactosidase activity activity is determined according to the procedure described in the Examples. In the context of the present invention, the polypeptides mentioned as having alpha-galactosidase activity should be understood as mature polypeptides. A mature polypeptide should be understood as the polypeptide being in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art, that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acid residues) expressed from the same polynucleotide.

[0043] As used herein, S.t. or S. thermophilus is short for Streptococcus thermophilus and Lb.s. or Lb. salivarius is short for Ligilactobacillus salivarius (also known as Lactobacillus salivarius according to legacy naming).

[0044] BRIEF DESCRIPTION OF THE FIGURES

[0045] Figures 1 shows in a) acidification curves for milk and soy base fermentations prepared with and without a strain according to the prior art, i.e., an Lb. delbrucki bulgaricus strain, and shows in b) fermentation curves for milk and soy base fermentations conducted with and without Lb. salivarius strains cultures.

[0046] Figures 2 and 3 show acidification curves for milk and soy base fermentations conducted with and without Lb. salivarius strains.

[0047] Figure 4 shows acidification curves for milk and soy base fermentations conducted with and without different cultures, wherein the different cultures each comprises a different S. thermophilus strain and Lb. salivarius DSM 35221.

[0048] Figure 5 shows in a) that a soy base fermented with an Lb. salivarius strains has a high shear stress when compared to soy base fermented with a prior art Lb. delbrucki bulgaricus strain. Figure 5 furthermore shows in b) that milk supplied with 1 % sucrose and fermented with Lb. salivarius strains have a suitable shear rate, comparable to a prior art Lb. paracasei strain, albeit lower than a prior art Lb. delbrucki bulgaricus strain.P8194EP00

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[0050] Figure 6 shows acidification curves of soy bases containing different protein levels, fermented with different cultures comprising an Lb. salivarius strain.

[0051] Detailed description

[0052] In an embodiment of the method, the Ligilactobacillus salivarius strain is selected from Ligilactobacillus salivarius DSM 35221, Ligilactobacillus salivarius DSM 35243, Ligilactobacillus salivarius DSM 35244, Ligilactobacillus salivarius DSM 35245, and Ligilactobacillus salivarius DSM 35246. Preferably, the Ligilactobacillus salivarius strain is Ligilactobacillus salivarius DSM 35221.

[0053] In an embodiment, the step iii) is carried out within 10 hours.

[0054] In an embodiment, the plant base is kept at a temperature in the range of 37-44 °C during the step of iii).

[0055] In an embodiment, the plant base is substantially free from added sugars.

[0056] In an embodiment, the plant base has a natural sucrose content of at most 1.5 wt%, such as 0.3 to 1.5 wt%, preferably at most 1.0 wt%, such as 0.3 to 1.0 wt%, more preferably at most 0.9 wt%, such as 0.3 to 0.9 wt%.

[0057] In an embodiment, wherein the plant base is selected from almond base, rice base, and soy base. Preferably, the plant base is a soy base.

[0058] In an embodiment, the plant base has a pH in the range of 6.5 to 7.3, such as in the range of 6.7 to 7.2.

[0059] In an embodiment, the plant base has a protein content in the range of 3.6 to 6.2 wt%, such as in the range of 3.8 to 5.5 wt%, such as in the range of 3.9 to 5.1 wt%.

[0060] In an embodiment, the plant base has a pH in the range of 6.5 to 7.3, and a protein content in the range of 3.6 to 6.2 wt%. In another version of this embodiment, the plant base has a pH in the range of 6.5 to 7.3, and a protein content in the range of 4 to 5 wt%.

[0061] In an embodiment of the method, the plant base has a pH in the range of 6.5 to 7.3, a protein content in the range of 3.6 to 6.2 wt%, and is substantially free from added sugar.P8194EP00

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[0063] In an embodiment of the method, the Ligilactobacillus salivarius is allowed in the step of iii) to acidify the plant base to a pH in the range of 4.5 to 4.6.

[0064] According to carbohydrate-active enzymes database (CAZy), there are two

[0065] a-galactosidase genes in L. salivarius Ren, GalAl and GalA2 (Wang W, Sudun, Hu H, et al. Unraveling the mechanism of raffinose utilization in Ligilactobacillus salivarius Ren by transcriptomic analysis. 3 Biotech. 2022;12(9):229.). Both GalAl and GalA2 belong to the glycosyl hydrolases family 36 and might catalyze the hydrolysis of a-1,6-galactoside links present in raffinose, resulting in D-galactose and sucrose. Amino acid sequence alignments showed that GalAl shares 100% sequence identity with the a-galactosidase in L. salivarius DSM 20492. Analysis of the regions flanking galAl in L. salivarius Ren genome revealed a 9.32 kb region encoding enzymes required for raffinose and galactose metabolism. The galA2 gene encodes a putative a-galactosidase. Gene galA2 was predicted to be co-transcribed with lsr_RS05020 which encodes a putative membrane protein YitT with unknown function. Both

[0066] galAl and galA2 were up-regulated during growth on raffinose in L. salivarius Ren.

[0067] Sugar analysis of soy bases fermented with L. salivarius were carried out as mentioned in the Examples. The analysis showed that all raffinose was consumed at end of fermentation, see table 1.

[0068] Table 1: Sugar analysis of UHT Provamel Soy base at end of fermentation

[0069]

[0070] Without being bound by theory, it is believed that the consumption of raffinose by L. salivarius is at least partly responsible for the improved acidification provided by L. salivarius, and that GalAl is a key enzyme in L. salivarius' metabolism of raffinose.P8194EP00

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[0072] Accordingly, in an embodiment the Ligilactobacillus salivarius strain comprises a first nucleotide encoding a first polypeptide with alpha-galactosidase activity, said first polypeptide having at least 96%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% amino acid identity with any one of the polypeptides of SEQ ID NO:9 or SEQ ID NO: 15-17. The polypeptides of SEQ ID NO:9 and SEQ ID NO:15-17 are GalAl.

[0073] Furthermore, without being bound by theory, it is believed that GalA2 is another key enzyme in L. salivarius' metabolism of raffinose.

[0074] Accordingly, in an embodiment the Ligilactobacillus salivarius strain comprises a second nucleotide encoding a second polypeptide with alpha-galactosidase activity, the second polypeptide having at least 96%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% amino acid identity with any one of the polypeptides of SEQ ID NO: 12-14. The polypeptide of SEQ ID NO: 12-14 are GalA2.

[0075] In some strains a putative GalA2 is encoded by two nucleic acids. Accordingly, in an embodiment the Ligilactobacillus salivarius strain comprises a third nucleotide encoding a polypeptide having at least 96%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% amino acid identity with the polypeptide of SEQ ID NO: 10, and a fourth nucleotide encoding a polypeptide having at least 96%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% amino acid identity with the polypeptide of SEQ ID NO: 11.

[0076] The inventors of the present invention have furthermore surprisingly found that when an Lb. salivarius strain is used in combination with a Streptococcus thermophilus (S. thermophilus') strain to ferment a soy base without added sugar, the time to reach the target pH of 4.6 may be further reduced. This is evident from example 1 and figure 4, and example 2 and figure 6 of the present application. The compatibility of Lb. salivarius and S. thermophilus strains is a surprising finding, since Lb. salivarius is a known to produce bacteriocins.

[0077] Any Streptococcus thermophilus which is suitable for use as a starter culture may be used. In an embodiment of the method, the Streptococcus thermophilus strain is selected from Streptococcus thermophilus DSM 35268, Streptococcus thermophilus DSM 35267, Streptococcus thermophilus DSM 34854, Streptococcus thermophilus DSM 24023, and Streptococcus thermophilus DSM 34853. The StreptococcusP8194EP00

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[0079] thermophilus have previously been deposited at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

[0080] In an embodiment of the method, in the step of iib) a plurality of Streptococcus thermophilus strains are inoculated into the plant base. The strains are added before the step of iii), however, the sequence of adding the Streptococcus thermophilus strains to the plant base is not of importance and can be carried out in any order or simultaneously. In a preferred version of this embodiment, the plurality of Streptococcus thermophilus strains are Streptococcus thermophilus DSM 24023 and Streptococcus thermophilus DSM 33745. In another preferred version of this embodiment, the plurality of Streptococcus thermophilus strains are Streptococcus thermophilus DSM 24023, Streptococcus thermophilus DSM 33745, and Streptococcus thermophilus DSM 34853.

[0081] In an embodiment, the method does not comprise adding a bacterial strain to the plant base that is not selected from at least one Ligilactobacillus salivarius or at least one Ligilactobacillus salivarius strain and at least one Streptococcus thermophilus strains.

[0082] In a second aspect, the present invention relates to a fermented plant-based composition obtained from a method according to any one of the above embodiments.

[0083] In third aspect, the present invention relates to a fermented plant-based composition comprising a Ligilactobacillus salivarius strain, said fermented plant-based composition having a pH of 4.6 or lower.

[0084] In an embodiment, the fermented plant-based composition has a protein content in the range of 3.6 to 6.2 wt%, and wherein at least 95 wt% of the protein content of the plant-based composition is protein from a fermented plant base.

[0085] In an embodiment, the fermented plant-based composition is a soy-based composition.

[0086] In an embodiment, the fermented plant-based composition is a soy-based

[0087] composition.P8194EP00

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[0089] In an embodiment, the fermented plant-based composition furthermore comprises a Streptococcus thermophilus strain.

[0090] In an embodiment, the fermented plant-based composition is substantially free from added sugars. In a specific version of this embodiment, the fermented plant-based composition is free from sugars.

[0091] In a fourth aspect, the present invention relates to a use of a Ligilactobacillus salivarius strain for fermenting a plant base with a pH of 4.6 or lower.

[0092] In an embodiment of the use, the plant base has a protein content in the range of 3.6 to 6.2 wt%.

[0093] In an embodiment of the use, the plant base is a soy-based composition.

[0094] In a fifth aspect, the present invention relates to a container, the container comprising a Ligilactobacillus salivarius strain.

[0095] In an embodiment, the container comprises a Ligilactobacillus salivarius strain and a Streptococcus thermophilus strain.

[0096] In an embodiment, the container comprises the Ligilactobacillus salivarius as a dried or frozen pellet, preferably freeze-dried, or spray-dried pellet.

[0097] DEPOSIT AND EXPERT SOLUTION

[0098] 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.

[0099] Table 2: The applicant has made the following deposits at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

[0100]

[0101] P8194EP00

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[0103]

[0104] EXAMPLES

[0105] Example 1: assessment of strain's soy base and milk acidification and texturing properties

[0106] Soy base and milk acidification data were obtained in 200 ml format by inoculating strains in either soy base containing no added sugar (Provamel Organic Soya Drink unsweetened, Alpro C.V.A., Vlamingstraat 28, BE-8560 Wevelgem), B-milk added 1 wt% sucrose, or B-milk added 2 wt% sucrose (2 g or 4 g sucrose in 200 ml milk) and incubating the samples at 43°C, while recording the pH using a multichannel pH meter system (iCinac, AMS Alliance, France).

[0107] Inoculation material of S. thermophilus strains was prepared by incubating single strains in 10 ml M17 broth containing 2% sucrose as C-source and incubated overnight at 37°C. Inoculation material of lactobacilli was prepared by adding incubating single strains in 10 ml MRS-Difco broth and incubated overnight at 37°C. A volume of 2 ml of each strain was transferred to the 200 ml soy base, B-milk containing 1 wt% sucrose, or B-milk containing 2 wt% sucrose. The following strains and strain combinations were inoculated in the 200 ml format: Ligilactobacillus salivarius DSM 35221, Ligilactobacillus salivarius DSM 35243, Ligilactobacillus salivarius DSM 35244, Ligilactobacillus salivarius DSM 35245, Ligilactobacillus salivarius DSM 35246, Ligilactobacillus salivarius DSM 35221 and Streptococcus thermophilus CHCC21564, Ligilactobacillus salivarius DSM 35221 and Streptococcus thermophilus DSM 35268, Ligilactobacillus salivarius DSM 35221 and Streptococcus thermophilus DSM 35267, and Lactobacillus delbrueckii subsp. bulgaricus DSM 19252.

[0108] B-milk was prepared by reconstituting low fat skim milk powder to a level of dry matter of 9.5% and pasteurized at 99°C for 30 min, followed by cooling to the fermentation temperature. For texture measurements, fermentations were stopped at approx. pH 4.6, as monitored by pH electrodes, followed by cooling to 4°C and overnight storage at 4°C. After storage, the fermented milk was manually stirred gently by means of aP8194EP00

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[0110] stick fitted with a bored disc until homogeneity of the sample. Shear stress of the samples was assessed at 13°C using HTR 302 compact rheometer (HTR1) (Anton Paar GmbH, Graz, Austria). Shear stress measurements [Pa] at different shear rates [1 / s] were obtained and used to assess the texturing capabilities of the strains under investigation.

[0111] Example 2: assessment of strain acidification properties in different soy media with different protein content

[0112] 200 mL sterile plastic bottles were filled up in duplicates with Provamel UHT treated unsweetened soy milk (3.7% protein) or Olga soy milk (5.0% protein, Olga, 2 rue Julien, Neveu, 35531 Noyal-sur-Vilaine, France) and placed closed with their lids in a waterbath filled with water at 43°C. The temperature of the soy milk was monitored with a sterile thermometer and when temperature reached 43°C, all bottles were inoculated with the cultures at an inoculating rate of 0.025%. The fermentation time and pH were monitored with the use of an AXONE System. Acidification curves resultant of the fermentations are shown in figure 6.

[0113] Sugars present in the soy base at end of fermentation were determined LC-MS. Fermented fermented Provamel UHT treated unsweetened soy milk was filtered and the filtrate was analyzed on LC-MS using standard curves of pure sugars. Results are provided in table 1.

[0114] Example 3: alpha-galactosidase activity assay

[0115] Alpha-galactosidase activity may be determined by any method known in the art. For example, analysis of alpha-galactosidase activity may be done by quantitation of the release of p-nitrophenol resulting from the cleavage of the substrate p-nitrophenyl-a-galactopyranoside into free galactose and p-nitrophenol, like described below.

[0116] Samples comprising alpha-galactosidase may be obtained by any method known in the art. The skilled person knows how to produce and / or isolate alpha-galactosidase from cultures. 20 pL of samples comprising alpha-galactosidase were transferred into a 384-well plate containing in each well 20 pL of 4-nitrophenyl-a-galactopyranoside (pNP-a-Gal) dissolved in the filter-sterilized Mcllvaine buffer (40 mM citric acid, 120 mM disodium hydrogen phosphate, pH 5.8) at the concentration of 0.45 mg / mL (1.5 mM). The mixing of the supernatant with the substrate solution was followed by a short trituration sequence. The reaction with the substrate was allowed to proceed for 5 hours at 37 °C without agitation. After the assay time had elapsed, 20 pL of the stopping reagent (0.5M sodium carbonate) was added to the reaction mixture in orderP8194EP00

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[0118] to deprotonate the liberated 4-nitrophenol into the 4-nitrophenolate form which in alkaline conditions absorbs at 400 nm.

[0119] Measurement

[0120] Pure 4-nitrophenol (pNP) dissolved in the stopping reagent (0.5M sodium carbonate) was used as an internal calibrant with 8 calibrations points between 174 pg / mL and 13 pg / mL. All samples and internal calibrant were measured after 300 minutes at 400 nm.

[0121] Quantification of a-galactosidase activity

[0122] The activity was calculated according to the following equation:

[0123] a-galactosidase activity (pIU / mL,(pmol / mL) / min) = 109(AU(400nm) / (slope(pNP standard mg / mL)) / (MW(pNP,g / mol))) / 300min

[0124] The calculation procedure is based on the definition of 1 International Unit of activity as 1 pmol of pNP released per milliliter per minute. Briefly, the absorbance at 400 nm (quantification of released pNA amount), is divided by the slope of the pNP standard curve to calculate the mass equivalents of pNP. This value is then divided by the molecular weight of pNP to calculate the molar equivalents of pNP. This value is then divided by minutes to calculate activity in kilo-international Units per milliliter (klU / mL). Finally, this value is multiplied by lOgto calculate micro-IU per milliliter (pIU / mL).

Claims

1. P8194EP0014CLAIMS1. A method for producing a fermented plant-based composition, the method comprising the steps of:i) providing a plant base,ii) inoculating a Ligilactobacillus salivarius strain into the plant base, and iii) allowing the Ligilactobacillus salivarius strain to acidify the plant base to a pH of 4.6 or lower, thereby obtaining the fermented plant-based composition.

2. The method of claim 1, wherein step iii) is carried out within 10 hours.

3. The method of claim 1 or 2, further comprising the step of:iib) inoculating a Streptococcus thermophilus strain into the plant base, wherein the step of iib) is carried out before the step of iii), and wherein the steps of ii) and iib) can be carried out in any order or simultaneously.

4. The method according to any one of the preceding claims, wherein the plant base has a protein content in the range of 3.6 to 6.2 wt%.

5. The method according to any one of the preceding claims, wherein the plant base is substantially free from added sugars.

6. The method according to any one of the preceding claims, wherein the plant base is a soy base.

7. The method according to any one of the preceding claims, wherein the Ligilactobacillus salivarius strain comprisesa) a first nucleotide encoding a first polypeptide with alpha-galactosidase activity, said first polypeptide having at least 96%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% amino acid identity with any one of the polypeptides of SEQ ID NO:9 or SEQ ID NO: 15- 17.

8. The method according to any one of the preceding claims, wherein the Ligilactobacillus salivarius strain comprisesbi) a second nucleotide encoding a second polypeptide with alpha-galactosidase activity, the second polypeptide having at least 96%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% amino acid identity with any one of the polypeptides of SEQ ID NO: 12-14.P8194EP0015orbii) a third nucleotide encoding a polypeptide having at least 96%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% amino acid identity with the polypeptide of SEQ ID NO: 10, and a fourth nucleotide encoding a polypeptide having at least 96%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% amino acid identity with the polypeptide of SEQ ID NO:11.

9. A fermented plant-based composition comprising a Ligilactobacillus salivarius strain, said fermented plant-based composition having a pH of 4.6 or lower.

10. The fermented plant-based composition of claim 8, wherein the fermented plant-based composition has a protein content in the range of 3.6 to 6.2 wt%, and wherein at least 95 wt% of the protein content of the plant-based composition is protein from a fermented plant base.

11. The fermented plant-based composition of claim 8 or 9, wherein the fermented plant-based composition is a soy-based composition.

12. The fermented plant-based composition of any one of the claims 8 to 10, wherein the fermented plant-based composition furthermore comprises a Streptococcus thermophilus strain.

13. The fermented plant-based composition of any one of the claims 8 to 11, wherein the fermented plant-based composition is substantially free from added sugars.

14. Use of a Ligilactobacillus salivarius strain for fermenting a plant base with a pH of 4.6 or lower.

15. The use according to claim 13 or 14, wherein the plant base is a soy base.

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