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WO2025120178A1 - Souches de streptococcus thermophilus à acidifiacation rapide dans le soja - Google Patents

Souches de streptococcus thermophilus à acidifiacation rapide dans le soja Download PDF

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Publication number
WO2025120178A1
WO2025120178A1 PCT/EP2024/085118 EP2024085118W WO2025120178A1 WO 2025120178 A1 WO2025120178 A1 WO 2025120178A1 EP 2024085118 W EP2024085118 W EP 2024085118W WO 2025120178 A1 WO2025120178 A1 WO 2025120178A1
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Prior art keywords
plant
fermented
strain
dsm
prts
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Elise Manoury
Armelle COCHU-BLACHÈRE
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International N&H Denmark ApS
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International N&H Denmark ApS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
    • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
    • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
    • A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
    • A23C11/106—Addition of, or treatment with, microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C20/00—Cheese substitutes
    • A23C20/02—Cheese substitutes containing neither milk components, nor caseinate, nor lactose, as sources of fats, proteins or carbohydrates
    • A23C20/025—Cheese substitutes containing neither milk components, nor caseinate, nor lactose, as sources of fats, proteins or carbohydrates mainly containing proteins from pulses or oilseeds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS
    • A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
    • A23D7/005—Edible oil or fat compositions containing an aqueous phase, e.g. margarines characterised by ingredients other than fatty acid triglycerides
    • A23D7/0053—Compositions other than spreads
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS
    • A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
    • A23D7/005—Edible oil or fat compositions containing an aqueous phase, e.g. margarines characterised by ingredients other than fatty acid triglycerides
    • A23D7/0056—Spread compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L9/00—Puddings; Cream substitutes; Preparation or treatment thereof
    • A23L9/20—Cream substitutes
    • A23L9/24—Cream substitutes containing non-milk fats and non-milk proteins, e.g. eggs or soybeans

Definitions

  • he present invention relates to strains of Streptococcus thermophilus in which the cell- envelope proteinase (PrtS) or a homologue thereof is functionally inactive or absent, the use of these PrtS-negative strains for the fermentation of preparations derived from a plant, in particu- lar fermented food or feed products based on a plant, as well as methods for the preparation of fermented products based on plants.
  • PrtS cell- envelope proteinase
  • BACKGROUND OF THE INVENTION In soy yogurt-type technology, customers are continuously looking for fast-acidifying starter cultures for the purpose of improved productivity.
  • C urrent starter cultures used in the industry mainly comprise Streptococcus thermophilus (ST) strains that are cell-envelope proteinase (PrtS) positive.
  • ST Streptococcus thermophilus
  • PrtS cell-envelope proteinase
  • Fig. 2 ABSCIA method for characterizing strain acidification kinetic descriptors.
  • Fig. 3 Preparation of soy model prior to pH monitoring with HydroPlate®.
  • Fig. 4 HydroPlate® method for characterizing strain acidification kinetic descriptors.
  • Fig. 5 Preparation of soy model prior to pH measurement with microprobes.
  • Fig. 6 Microprobe method for characterizing pH24h in soy model.
  • Fig. 7 Acidification kinetics of soy model fermented 24 hours at 37°C with S.
  • I t is an object of embodiments of the invention to provide strains of Streptococcus ther- mophilus that are suitable for the fermentation of plant-based products.
  • the present invention relates in a broad aspect to a Streptococcus thermophilus strain in which the cell-envelope proteinase (PrtS) or a homologue thereof is functionally inactive or absent.
  • the present invention relates to a Streptococcus thermophi- lus strain in which the cell-envelope proteinase (PrtS) or a homologue thereof is functionally in- active or absent, and wherein the acidification kinetics of said strain in soy model is character- ized by: - an average slope between pH 6.0 and 5.3 at least about 100 (x104 pHU/min) measured as de- scribed in Assay 3, and - an average pH after 24 hours of fermentation measured as described in Assay 4 of at least about 4.20 , and not higher than pH 5.5.
  • PrtS cell-envelope proteinase
  • the present invention relates to a Streptococcus thermophilus strain according to the present invention, which is selected from the group consisting of: (1) the DSM 21892 bacterial strain deposited under the Budapest Treaty on 7 October 2008 in the name of Danisco GmbH, GmbH at the Leibniz Institute DSMZ-German Col- lection of Microorganisms and Cell Cultures GmbH; (2) the DSM 34703 bacterial strain deposited under the Budapest Treaty on 19 July 2023 in the name of DuPont Nutrition Biosciences ApS, Denmark at the Leibniz Institute DSMZ-German Col- lection of Microorganisms and Cell Cultures GmbH; (3) the DSM 28128 bacterial strain deposited under the Budapest Treaty on 4 December 2013 in the name of Danisco GmbH, Germany at the Leibniz Institute DSMZ-German Col- lection of Microorganisms and Cell Cultures GmbH; and (4) the DSM 34705 bacterial strain deposited under the Budapest Treaty on 19 July 2023 in the name of Du
  • the present invention relates to a composition, such as a composition of a starter culture comprising or consisting of one or more culture of a Streptococcus thermophilus strain as defined in the present disclosure, optionally further comprising at least one other micro- organism, such as at least one other lactic acid bacterium and/or at least one Propionibacterium.
  • the present invention relates to the use of a culture of a Streptococcus thermophilus strain as defined in the present disclosure or of a composition as defined in the present disclosure, such as a starter culture, for the fermentation of an aqueous preparation de- rived from a plant, in particular a fermented food or feed product based on a plant.
  • the present invention relates to a method for preparing a fermented product, in particular a fermented food or a feed product based on a plant, such as pea, soy, and oat, wherein the method comprises putting into contact a plant-based substrate, with or in the presence of a culture of a bacterial strain as defined in the present disclosure or a composition as defined in the present disclosure, and obtaining said fermented product.
  • the present invention relates to a method for acidifying an aqueous preparation derived from a plant, such as pea, soy, and oat, the method comprising putting into contact a plant-based substrate, with or in the presence of a culture of a bacterial strain as de- fined in the present disclosure or a composition as defined in the present disclosure.
  • the present invention relates to a fermented product, in particular a fermented food or a fermented feed product, in particular a fermented food or feed product based on a plant, such as pea, soy, and oat, obtainable by the methods as defined in the present disclosure.
  • PrtS cell-envelope proteinase
  • CEP cell-envelope pro- teinase
  • prtS gene refers to a gene encoding the “cell-envelope proteinase (PrtS)”.
  • prtS genes vary among the many known strains of S. thermophilus. However, they show more than 90% of sequence identity between the different PrtS protein sequences of the strains.
  • One suitable sequence of a prtS gene within the present definition is that of strain CNRZ385 available under the accession number AF243528 (SEQ ID NO:1).
  • One suitable example of a cell-envelope proteinase (PrtS) or a homologue thereof within the present invention is that of strain CNRZ385 available under the accession number AF243528 (SEQ ID NO:2).
  • PrtS-positive or PrtS+ ST strain refers to a strain Streptococcus ther- mophilus that contains a functional cell-envelope proteinase (PrtS) encoded by a functional prtS gene.
  • a PrtS-negative or PrtS- ST strain refers to a strain Streptococcus ther- mophilus that contains a functionally inactive or absent cell-envelope proteinase (PrtS).
  • the PrtS-negative or PrtS- ST strain has a functionally inactive or absent cell-enve- lope proteinase (PrtS) due to a mutation, disruption or deletion of the prtS gene.
  • the present invention may encompass variants, homologues, derivatives and fragments thereof.
  • variant is used to mean a naturally occurring nucleotide sequence or amino acid sequence which differs from a wild-type sequence.
  • homologue means an entity having a certain homology with the subject nu- cleotide sequences or amino acid sequence.
  • identity can be equated with "identity”.
  • a homologous sequence is taken to include an amino acid or a nu- cleotide sequence which may be at least 80, 85 or 90% identical, preferably at least 95%, 96%, 97%, 98 % or 99% identical to the subject sequence.
  • the homologues will comprise the same active sites etc. as the subject amino acid sequence for instance.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • a homologous sequence is taken to include an amino acid sequence or nu- cleotide sequence which has one or several additions, deletions and/or substitutions compared with the subject sequence.
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the cor- responding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short num- ber of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed.
  • the present invention defines suitable Streptococcus thermophilus strains ac- cording to the present invention to have an average slope between pH 6.0 and 5.3 at least about 100 (x104 pHU/min) measured as described in Assay 3 as described herein (sometimes referred to as a fast acidifying Streptococcus thermophilus strain); and having an average pH after 24 hours of fermentation measured as described in Assay 4 as described herein of at least about 4.20, and not higher than pH 5.5.
  • the Streptococcus thermophilus strains according to the present in- vention have an average slope between pH 6.0 and 5.3 at least about 110 (x104 pHU/min) measured as described in Assay 3, such as at least about 115, 120, 125, 130, 135, 140, 145, or 150 or 160 (x10 4 pHU/min).
  • the Streptococcus thermophilus strains in which the cell-envelope proteinase (PrtS) or a homologue thereof is functionally inactive or absent of the invention is characterized by the fact that the strain leads to a fermented plant-based product, not undergo- ing significant post-acidification for at least about 24h at 37°C.
  • T he expressions “not undergoing significant post-acidification” or “less post-acidifying” as used herein refers to a Streptococcus thermophilus strain, which when fermented in soy model described herein provided for an average pH after 24 hours of fermentation measured as de- scribed in Assay 4 of at least about 4.20, and not higher than pH 5.5.
  • soy model refers to the French commercial soy Bjorg beverage (“Bjorg soja sanster BIO”) supplemented with 2% filtrated (0.2 ⁇ m) glucose as described in assays 1, 3 and 4 disclosed herein.
  • Aqueous preparation derived from a plant One aspect of the present invention relates to the use of a culture of a Streptococcus thermophilus strain for the fermentation of an aqueous preparation derived from a plant, i.e. plant-based fermentation in order to produce plant-based compositions.
  • the fermented plant-based composition is produced by inoculat- ing, e.g., with bacteria or a bacterial composition described herein, and fermenting a vegetal base.
  • a vegetal base such as an aqueous preparation derived from a plant for producing the fermented plant-based composition may be any raw and/or pro- Switchd plant-based material, e.g., plant matter, that can be subjected to fermentation according to the methods provided herein.
  • the vegetal base is or includes fully or partially hydrolysed plant matter, for example cereal.
  • the cereal is wheat, rye, spelt, barley, oat, millet, sorghum, rice, teff, or combinations thereof.
  • the vegetal base is or includes plant matter from legumes.
  • the vegetal base is or includes pulses, for example split peas, field peas, dry peas, lentil, chickpeas, garbanzo bean, konda, navy bean, white navy bean, white pea bean, pea bean, cow pea, horse bean, haricot, pinot bean, mottled bean, small red bean, red Mexican bean, kidney bean, black bean, black turtle bean, cranberry bean, roman bean, speckled sugar bean, lima bean, haba bean, Madagascar bean, green gram, mung bean, green bean, black gram, urad dal, soy and/or lupin.
  • pulses for example split peas, field peas, dry peas, lentil, chickpeas, garbanzo bean, konda, navy bean, white navy bean, white pea bean, pea bean, cow pea, horse bean, haricot, pinot bean, mottled bean, small red bean, red Mexican bean, kidney bean, black bean, black turtle bean, cra
  • the vegetal base such as an aqueous preparation derived from a plant is or includes soy (e.g., soy plant matter).
  • the vegetal base is or includes plant matter from nuts.
  • nuts contemplated for use herein include almonds, cashews, pecans, macada- mias, hazelnuts, pistachio, walnuts, or combinations thereof.
  • the vegetal base is or includes seeds.
  • seeds contemplated for use herein include hemp, pumpkin, quinoa, sesame, tiger nut, flax, chia, sunflower, coconut, or combinations thereof.
  • the vegetal base is a substrate of vegetal origin, such as a water- based plant extract.
  • the vegetal base is or includes a water-based plant extract.
  • the vegetal base is or includes a water-based plant extract of any of the plant matter described herein.
  • the vegetal base is a wa- ter-based plant extract of a cereal, a legume, a nut, a seed, or a combination thereof.
  • the water-based plant extract is from a nut.
  • the water- based plant extract is from a cereal.
  • the water-based plant extract is from a legume.
  • the water-based plant extract is from a plant seed. In some embodiments, the water-based plant extract is from soy. In some embodiments, the wa- ter-based plant extract is from soy.
  • plant-based substrate and “water-based plant extract” or “vegetal base drink” may be used interchangeably.
  • water-based plant extract may be used inter- changeably with a “plant-milk” or “plant-based milk” or “plant-based drink”, or “plant-based dairy alternative product”.
  • the vegetal base is a suspension. In some embodiments, the veg- etal base is an aqueous suspension containing water and plant matter as described herein.
  • Meth- ods of preparing a suspension of plant matter suitable for vegetal bases for fermentation as de- scribed herein are known in the art.
  • Exemplary processes for preparing plant matter include me- chanical and/or enzymatic disruption of the plant matter, optionally in combination with a solu- tion or other form of hydration to produce an aqueous fraction which may be separate, e.g., decanted, centrifuged, and/or filtered, from the remaining plant matter, e.g., starchy, fibrous matter.
  • the vegetal base is a butter alternative or beverage, such as a nut, seed, or pulse butter alternative or beverage. Methods for preparing butter alternatives for use as described herein are known in the art.
  • Exemplary methods of preparing a butter alterna- tive include wet or dry grinding roasted or unroasted e.g., nuts, seeds, pulses, to a paste.
  • the size of the particles of the paste are suitable for the preparation of bev- erages.
  • the vegetal base does not include added sugars or proteins.
  • the carbohydrate of the vegetal base is supplied only from the plant matter.
  • the vegetal base includes proteins.
  • the proteins are supplied only from the plant matter.
  • the amount of protein is in the range of or of about 1 to 5% (% weight) of the total vegetal base.
  • the amount of protein is in the range of or of about 2 to 5% (% weight) of the total vegetal base. In some embodiments, the amount of protein is in the range of or of about 3 to 5% (% weight) of the total vegetal base. In some embodiments, the amount of protein is in the range of or of about 4 to 5% (% weight) of the total vegetal base. In some embodiments, the amount of protein is in the range of or of about 1 to 4% (% weight) of the total vegetal base. In some em- bodiments, the amount of protein is in the range of or of about 1 to 3% (% weight) of the total vegetal base.
  • the amount of protein is in the range of or of about 1 to 2% (% weight) of the total vegetal base.
  • sugars may be added to the vegetal base. In some embodiments, the sugars are added in the range of or of about 0.5 to 5% (% weight) of the total vegetal base. In some embodiments, the sugars are added in the range of or of about 1 to 5% (% weight) of the total vegetal base. In some embodiments, the sugars are added in the range of or of about 2 to 5% (% weight) of the total vegetal base. In some embodiments, the sugars are added in the range of or of about 2 to 4% (% weight) of the total vegetal base.
  • the one or more bacterial strains are contained in a bacterial composition, such as a starter culture. In some embodiments, the one or more bacterial strains are contained in a starter culture. In some embodiments, the starter culture is a preparation of living bacteria able to assist in fermentation. In some embodiments, the vegetal base is inocu- lated with a starter culture. In some embodiments, the vegetal base is inoculated with a starter culture and a protective culture. In some embodiments, a protective culture is a culture able to reduce or prevent the growth of biological contaminants, such as yeast and mold. In some em- bodiments, the protective culture may include one or more bacterial strains described herein, e.g., L.
  • inoculating and adding may be used in- terchangeably to refer to contacting a vegetal base with one or more bacteria, for example as contained in a bacterial composition, such as a starter culture or a protective culture.
  • the vegetal base is inoculated with one or more bacterial strains separately.
  • the bacterial strains are not mixed together prior to being added to the vegetal base.
  • the bacterial strains are mixed together prior to being added to the vegetal base.
  • the strain or mixtures of strains used for inoculation may be referred to as or included in a starter culture or protective culture.
  • the starter culture is a thermophilic starter culture, and the composition of the starter culture is suitable for thermophilic fermentation as described herein.
  • the starter culture includes a combination of bacteria suitable for thermophilic and mesophilic fermentation.
  • the starter culture includes thermophilic and mesophilic bacteria, and the composition of the starter culture is suita- ble for thermophilic and mesophilic fermentation, e.g., as described herein.
  • thermophilic microorganisms such as thermophilic bacteria, refer to microorganisms that function preferentially at temperatures above 37°C, e.g., 37 to 50°C.
  • mesophilic microorganisms such as mesophilic bacteria, refer to microor- ganisms that function preferentially at temperatures at or below 37°C, e.g., 25 to 37°C.
  • the starter culture is a pure culture, i.e., comprises or consists of a single bacterial strain being a Streptococcus thermophilus strain in which the cell-envelope pro- teinase (PrtS) or a homologue thereof is functionally inactive or absent.
  • the starter culture is a mixed culture, i.e. comprises or consists of at least one bacterial strain of the invention as described herein and at least one other bacterial strain.
  • the starter culture contains one or more lactic acid bacteria.
  • the starter culture contains at least two lactic acid bacteria.
  • the composition may in some embodiments include a multiplicity of strains either belonging to the same species or belonging to different species.
  • the lactic acid bacte- rium in the starter culture is or includes a mixture of a Lactobacillus delbrueckii subsp bulgaricus strain, a Streptococcus thermophilus strain at including a Streptococcus thermophilus strain of the present invention, and an L. rhamnosus strain.
  • the starter culture includes at least a bacterial strain from the genera Streptococcus and Lacticaseibacillus.
  • the starter culture includes or consists of bacteria from the genus Lactococcus, Lactobacillus, Streptococcus, Lacticaseibacil- lus, Leuconostoc, Pediococcus, Enterococcus, Bifidobacterium, Paralactobacillus, Acetilactobacil- lus, Agrilactobacillus, Amylolactobacillus, Apilactobacillus, Bombilactobacillus, Companilactobacil- lus, Dellaglioa, Fructilactobacillus, Furfurilactobacillus, Holzapfelia, Lacticaseibacillus, Lactiplanti- bacillus, Lapidilactobacillus, Latilactobacillus, Lentilactobacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus, Liquorilactobacillus, Loigolacto
  • the starter culture includes or consists of bacteria from the genus Lactococcus, Lactobacillus, Streptococcus, Lacti- caseibacillus, Leuconostoc, Pediococcus, Enterococcus or Bifidobacterium, or any combination thereof.
  • the starter culture includes or consists of bacteria from the ge- nus Lactococcus, Lactobacillus, Streptococcus, Lacticaseibacillus, Leuconostoc, Pediococcus, or Bifidobacterium, or any combination thereof.
  • the starter culture includes or consists of bacteria from the genus Lactococcus, Lactobacillus, Streptococcus, Lacticaseibacil- lus, or any combination thereof. In some embodiments, the starter culture includes or consists of bacteria from the genus Lactobacillus, Streptococcus, Lacticaseibacillus, or any combination thereof. In some embodiments, the starter culture includes or consists of bacteria from the ge- nus Streptococcus and Lactobacillus. In some embodiments, the starter culture includes at least a Streptococcus thermophilus strain and a Lactobacillus delbrueckii subsp bulgaricus strain.
  • the starter culture includes one or more of a Streptococcus thermophilus strain, a Lacticaseibacillus rhamno- sus strain, a Lactobacillus acidophilus strain, a Bifidobacterium lactis strain, a Limosilactobacillus fermentum strain, a Lacticaseibacillus paracasei strain, a Lactiplantibacillus plantarum strain, a Lactobacillus delbrueckii subsp bulgaricus strain, a Propionibacteria freudenreichii strain, a Pedio- coccus acidilactici strain, an Enterococcus faecium strain, a Lactococcus lactis strain, Lactococcus cremoris strain, or any combination of the foregoing.
  • the starter culture includes one or more of a Streptococcus thermophilus strain, a Lactobacillus acidophilus strain, a Lacticaseibacillus rhamnosus strain, a Bifidobacterium lactis strain, a Limosilactobacillus fermen- tum strain, a Lacticaseibacillus paracasei strain, a Lactiplantibacillus plantarum strain, a Lactoba- cillus delbrueckii subsp bulgaricus strain, a Propionibacteria freudenreichii strain, a Pediococcus acidilactici strain, a Lactococcus lactis strain, a Lactococcus cremoris strain, or any combination of the foregoing.
  • the starter culture includes one or more of a Strepto- coccus thermophilus strain, a Lacticaseibacillus rhamnosus strain, a Lactobacillus delbrueckii subsp bulgaricus strain, or any combination of the foregoing.
  • the starter culture includes a Streptococcus thermophilus strain and a Lacticaseibacillus rhamnosus strain.
  • the starter culture includes more than one strain of a species.
  • the starter culture includes at least a Streptococcus thermophilus strain and one Lactococcus strain.
  • the starter culture may include one or more of a Lactococcus strain known in the art, such as a strain of Lactococcus cremoris subsp cremoris (previously referred to as Lactococcus lactis subsp. cremoris), Lactococcus lactis subsp hordniae, or Lactococcus lactis subsp lactis.
  • the starter culture includes a Lactococ- cus cremoris subsp cremoris and/or a Lactococcus lactis subsp lactis strain.
  • the starter culture includes one or more Lacticaseibacillus rham- nosus strains.
  • the starter culture includes one or more Lacticaseibacillus rhamnosus strains of vegetal origin.
  • Lacticaseibacillus rhamnosus strain is derived, selected, or isolated from plant material, e.g., fermented plant material.
  • the starter culture either as a pure or mixed culture as defined above, is in frozen, dried, freeze-dried, liquid or solid format, in the form of pellets or frozen pel- lets, or in a powder or dried powder.
  • the starter culture is in a frozen for- mat or in the form of pellets or frozen pellets, in particular contained in one or more boxes or sa- chets.
  • the starter cultures as defined herein are in a powder form, such as a dried or freeze-dried powder, in particular contained in one or more boxes or sachets.
  • the liquid format is a bulk starter, such as a bacterial strain or mixture of bacterial strains previously propagated in a growth medium to obtain the required concentration for inoculation.
  • the starter culture either as a pure culture or mixed culture as defined above, and whatever the format (frozen, dried, freeze-dried, liquid or solid format, in the form of pellets or frozen pellets, or in a powder or dried powder) includes the bacterial strain(s) described herein in a concentration in the range of or of about 10 5 to 10 13 cfu (colony forming units) per gram of the starter culture.
  • the concentration of the bacterial strain(s) described herein within the starter culture of the invention is in the range of or of about 10 5 to 10 13 cfu per gram of the starter culture, and in particular at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 or at least 10 11 CFU/g of the starter culture.
  • the concentration of bacterial strain(s) described herein – as a pure culture or as a mixed culture - within the starter culture is in the range of or of about 10 6 to 10 13 cfu/g of frozen concentrate or dried concentrate, and more pref- erably at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , or at least 10 13 cfu/g of frozen concentrate or dried concentrate.
  • the concentration of the bacterial strain(s) described herein within the starter culture of the invention is the range of or of about 10 5 to 10 9 CFU/g of the starter culture.
  • the concentration of the bacterial strain(s) described herein within the starter culture of the invention is the range of or of about 10 6 to 10 9 CFU/g of the starter culture. In some embodiments, the concentration of the bacterial strain(s) described herein within the starter culture of the invention is the range of or of about 10 6 to 10 8 CFU/g of the starter culture. In some embodiments, the concentration of the bacterial strain(s) described herein within the starter culture of the invention is the range of or of about 10 6 to 10 7 CFU/g of the starter culture. In some embodiments, the concentration of the bacterial strain(s) described herein within the starter culture of the invention is, is at least, or is about 10 6 CFU/g of the starter culture.
  • the concentration of the bac- terial strain(s) described herein within the starter culture of the invention is, is at least, or is about 10 7 CFU/g of the starter culture.
  • Starter cultures may be prepared by techniques well known in the art such as those dis- closed in US 4,621,058.
  • starter cultures may be prepared by the introduction of an inoculum, for example a bacterium, to a growth medium to produce an inoculated medium and ripening the inoculated medium to produce a starter culture.
  • Dried starter cultures may be prepared by techniques well known in the art, such as those discussed in US 4,423,079 and US 4,140,800.
  • the starter culture is in the form of concentrated frozen pel- lets.
  • the addition of the starter culture to the vegetal base is direct, e.g., as a direct vat inoculate, direct vat starter, direct vat set culture.
  • the vegetal base may be inoculated with the starter culture prior to initiating fermentation.
  • the vegetal base is inoculated with the starter culture following, e.g., at a time point after, fermentation is initiated.
  • the vegetal base is inoculated with the starter culture during fermentation. For example, in some cases where strains of the starter culture are added separately, e.g., not in a mixture, the strains may be added at different times before or during fermentation.
  • microorganisms and/or additives may be added to the vegetal base before, dur- ing or after fermentation of the vegetal base.
  • boosters such as yeast extracts and/or amino acid containing compositions may be added to the vegetal base in addition to a starter culture, to support or promote microorganism function and facilitate successful production of the fermented plant-based compositions.
  • Microorganisms that may be added to the vegetal base include those that will contribute in an advantageous manner to the properties of the fer- mented plant-based compositions, e.g., fermented plant-based food products.
  • added microorganisms, yeast extracts, or amino acid compositions may improve or support diac- etyl production, acetaldehyde conversion and breakdown, the viscosity, gel stiffness, mouth coating, flavor, post acidification, and/or acidification speed in the fermented plant-based compo- sitions.
  • other ingredients may be added to the vegetal base, such as colors, stabilizers, e.g., pectin, starch, modified starch, CMC, etc.; or polyunsaturated fatty acids, e.g. omega-3 fatty acids.
  • Such ingredients may be added at any point during the production process, e.g. before or after fermentation.
  • cryoprotectants and/or conventional additives including nutrients such as yeast extracts, sugars and vitamins, e.g., vitamin A, C, D, K or vitamins of the vitamin B family may also be added to the vegetal base either with or separate from the starter culture or other additives.
  • Suitable cryoprotectants that may be added include components that improve the cold tolerance of the microorganisms, such as mannitol, sorbitol, sodium tripolyphosphate, xylitol, glycerol, raffinose, maltodextrin, erythritol, threitol, trehalose, glucose and fructose.
  • additives may include carbohydrates, flavors, minerals, enzymes (e.g., rennet, lactase and/or phospholipase).
  • additives, microorganisms, or other components as described in the preceding paragraphs are added simultaneously or nearly simultaneously with the starter culture to the vegetal base, e.g., at the same time as inoculation of the vegetal base with the starter culture.
  • additives, microorganisms, or other components as de- scribed in the preceding paragraphs are added to the vegetal base before, e.g., temporally prior to, inoculation of the vegetal base with the starter culture.
  • additives, mi- croorganisms, or other components as described in the preceding paragraphs are added after, e.g., temporally following, inoculation of the vegetal base with the starter culture.
  • additives, microorganisms, or other components as described in the preceding para- graphs may be added to the vegetal base before, during, or after fermentation, for example in- dependent of the inoculation with the starter culture.
  • the vegetal base is inoculated with the starter culture, and op- tionally additives, microorganisms, or other components as described in the preceding para- graphs above, by any suitable method.
  • the vegetal base may be inoculated by di- rect inoculation into a fermentation vessel.
  • Fermentation The methods for producing a fermented plant-based composition include fermentation, e.g., thermophilic fermentation, of a vegetal base, for example as described herein, inoculated with a starter culture, and optionally other additives, microorganisms, and components, as de- scribed herein. Fermentation according to the methods provided herein facilitates the conversion of car- bohydrates into alcohols or acids through the action of a microorganism, e.g., bacteria present, for example, in a starter culture. Fermentation processes may be selected to optimize parameters such as temperature, oxygen, process time to achieve a suitable fermented plant-based product.
  • the inoculated vegetal base is fermented at a temperature at or above about 25°C, 30°C, 35°C, 40°C, or 50°C. In some embodiments, the inoculated vegetal base is fermented at a temperature of between or between about 20 to 50°C, 30 to 50°C, or 40 to 50°C. In some embodiments, the inoculated vegetal base is fermented at a temperature of above or of about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, or 50°C.
  • the inoculated vegetal base is fer- mented at a temperature of between or between about 35 to 47°C, 35 to 46°C, 35 to 45°C, 35 to 44°C, 35 to 43°C, 36 to 43°C, 37 to 43°C, 38 to 43°C, 39 to 43°C, 40 to 43°C, 41 to 43°C, or 42 to 43°C.
  • the inoculated vegetal base is fermented at a temperature of or of about 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, or 44°C.
  • the inoculated vegetal base is fermented at a temperature of or of about 37°C, e.g., 37 ⁇ 1°C. In some embodiments, fermentation of the inoculated vegetal base is carried out for at least 4 hours, e.g., 5, 6, 7, 8, 9, 10, 15, 20, or 24 hours. In some embodiments, fermentation of the inoculated vegetal base is carried out for or for about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, fermentation of the inocu- lated vegetal base is carried out for at most 24 hours.
  • fermentation of the inoculated vegetal base is carried out for between or between about 4 to 24, 4 to 23, 4 to 22, 4 to 21, 4 to 20, 4 to 19, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 hours.
  • fermentation of the inoculated vegetal base is carried out for or for about 10 hours.
  • fermen- tation of the inoculated vegetal base is carried out for or for about 9 hours.
  • fermentation of the inoculated vegetal base is carried out for or for about 8 hours.
  • fermentation of the inoculated vegetal base is carried out for or for about 7 hours. In some embodiments, fermentation of the inoculated vegetal base is carried out for or for about 6 hours. In some embodiments, fermentation of the inoculated vegetal base is carried out for or for about 5 hours. In some embodiments, fermentation of the inoculated vegetal base is carried out for or for about 4 hours. In some embodiments, fermentation of the inoculated vegetal base is carried out for an amount of time needed for the vegetal base to reach a target pH. In some embodiments, the target pH is a suitable pH for producing the fermented plant-based composition that are food products, e.g., as described herein.
  • the target pH is in a range of or of about 3.4 to 5. In some embodiments, the target pH is in a range of or of about 3.8 to 5. In some embodiments, the target pH is in a range of or of about 4 to 5, such as in the range of 4.5 to 5. In some embodiments, the target pH is in a range of or of about 4.2 to 4.7, such as 4.5 to 4.7.
  • the fermented plant-based composi- tion has a pH in the range of or of about 3.4 to 5. In some embodiments, the fermented plant- based composition has a pH in the range of or of about 3.8 to 5 at the end of fermentation.
  • the fermented plant-based composition has a pH in the range of or of about 4 to 5 at the end of fermentation. In some embodiments, the fermented plant-based composition has a pH of or of about 4.1, 4.2, 4.3, 4.4., 4.5, 4.6, or 4.7 at the end of fermentation. In some embodiments, the fermented plant-based composition has a pH in the range of or of about 4.2 to 4.7 at the end of fermentation. In some embodiments, the fermented plant-based composition has a pH of or of about 4.6 at the end of fermentation. In some embodiments, the pH of the inoculated vegetal base is measured, e.g., moni- tored, during fermentation.
  • the pH of the inoculated vegetal base is measured, e.g., monitored, at specific time points during fermentation. For example, a pH meas- urement may be taken every 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, or every hour, 2 hours, 3 hours, 4 hours, or 5 hours.
  • the pH of the inoculated veg- etal base is measured, e.g., monitored, continuously during fermentation.
  • the fermented plant-based composition produced according to the methods provided herein is stored.
  • the fermented plant-based composition is stored to produce a stored fermented plant-based composition.
  • the fermented plant-based composition is stored in one or more containers. Suitable containers for storage included, but are not limited to, containers capable of securing and protecting the fermented plant-based composition, e.g., protecting the fermented plant-based composition from contamination.
  • the fermented plant-based composition is packaged in a sealed container.
  • the fermented plant- based composition is packaged in a container after fermentation has occurred. In some embodi- ments, the fermentation occurs in a container that can be sealed.
  • a vegetal base e.g., inoculated vegetal base
  • a container such that at the end of fermentation the container contains a plant-based fermented composition and the con- tainer is then sealed.
  • the vegetal base is inoculated prior to placement in the container for fermentation.
  • the vegetal base is inoculated after to placement in the container for fermentation.
  • the one or more containers are useful for storing the fermented plant-based composition at a cool temperature, e.g., a cooler temperature than used for fermen- tation, such as at or at about 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C or 26°C.
  • a cool temperature e.g., a cooler temperature than used for fermen- tation, such as at or at about 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°
  • the fermented plant-based composition is stored at a temperature in the range of or of about 1 to 26°C, 1 to 24°C, 1 to 22°C, 1 to 20°C, 1 to 18°C, 1 to 16°C, 1 to 14°C, 1 to 12°C, 1 to 10°C, 1 to 8°C, 1 to 6°C, 1 to 4°C, or 1 to 2°C.
  • the fer- mented plant-based composition is stored at a temperature in the range of or of about 1 to 14°C, 1 to 12°C, 1 to 10°C, 1 to 8°C, 1 to 6°C, or 1 to 4°C.
  • the fer- mented plant-based composition is stored at a temperature in the range of or of about 4 to 8°C.
  • the fermented plant-based composition is stored at or at about a temper- ature of 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C or 26°C.
  • the fermented plant-based composition is stored at or at about a temperature of 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C or 14°C.
  • the fer- mented plant-based composition is stored at or at about a temperature of 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C.
  • the fermented plant-based composition is stored at or at about a temperature of 6°C, e.g., 6 ⁇ 1°C.
  • stored fermented plant-based composition maintains a target pH achieved during fermentation.
  • the target pH is a suitable pH for produc- ing the fermented plant-based composition that is a food product, e.g., as described herein.
  • the target pH is in a range of or of about 3.8 to 5.
  • the target pH is in a range of or of about to 5.
  • the target pH is or is about 4.1, 4.2, 4.3, 4.4., 4.5, 4.6, or 4.7.
  • the target pH is in a range of or of about 4.2 to 4.7.
  • the target pH is or is about 4.6.
  • the stored fermented plant-based composition has a pH in the range of or of about 3.4 to 4.5. In some embodiments, the stored fermented plant-based compo- sition has a pH in the range of or of about 3.8 to 4.2. In some embodiments, the stored fer- mented plant-based composition has a pH of or of about 3.8, 3.9, 4.0, 4.1, 4.2, or 4.3. In some embodiments, the stored fermented plant-based composition has a pH in the range of or of about 3.9 to 4.1. In some embodiments, the stored fermented plant-based composition has a pH of or of about 4.0.
  • the pH of the stored fermented plant-based composition is meas- ured, e.g., monitored, during storage.
  • the pH of the stored fermented plant-based composition is measured, e.g., monitored, at specific time points during storage. For example, a pH measurement may be taken every or every other day, every third, every fourth, every fifth, every sixth, every seventh, every eighth, every ninth, every tenth, every eleventh, or every twelfth, every thirteenth, or every fourteenth day in storage for the duration of storage.
  • the pH of the stored fermented plant-based composition is measured, e.g., monitored at about every 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 hours, 60 hours, or every 72 for the duration of time in storage.
  • the pH of the stored fermented plant-based composition is measured, e.g., monitored, continuously during storage.
  • the pH of the stored fermented plant-based composition is measured at a temperature of or of about 20°C.
  • the stored fermented plant-based composition may be removed from storage for a period of time to increase the temperature of the stored fermented plant-based composition prior to measuring the pH.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition, is stored for at least or at least about 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for at least or at least about 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, or 90 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for at least or at least about 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
  • the fermented plant-based com- position e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for at least or at least about 120 days.
  • the fermented plant-based composition e.g., stored fermented plant-based com- position
  • the fermented plant-based composition is stored for at least or at least about 90 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition e.g., stored fermented plant-based composition, is stored for at least or at least about 28 days.
  • the fer- mented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composi- tion e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for at least or at least about 7 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for at most or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition, is stored for at most or at most about 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for at most or at most about 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 40, 50, 60, 70, 80, or 90 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for at most or at most about 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
  • the fermented plant-based com- position e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for at most or at most about 90 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composi- tion
  • the fermented plant-based composition is stored for at most or at most about 60 days.
  • the fermented plant- based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition e.g., stored fermented plant-based composi- tion
  • the fermented plant- based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for at most or at most about 14 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fer- mented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for a dura- tion of or of about 7 to 120 days.
  • the fermented plant-based composi- tion e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for a duration of or of about 7 to 90 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant- based composition e.g., stored fermented plant-based composition, is stored for a duration of or of about 7 to 21 or 7 to 14 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for a duration of or of about 120 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based compo- sition e.g., stored fermented plant-based composition, is stored for a duration of or of about 30 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition is stored for a duration of or of about 28 days.
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition e.g., stored fermented plant-based composition
  • the fermented plant-based composition e.g., stored fer- mented plant-based composition, is stored for a duration of or of about 7 days.
  • a fer- mented plant-based composition is a fermented plant-based food product.
  • the fermented plant-based food product is a soy-based composition.
  • the fermented plant-based food product is a fermented soy-based food product.
  • Fermented plant-based food products contemplated herein include, but are not limited to, a food such as, but not limited to, fermented dairy alternative food products.
  • the fermented plant-based food product is a fermented dairy alternative food product.
  • the term "food” is used in a broad sense and includes feeds, foodstuffs, food ingredients, food supplements, and functional foods.
  • the term “food” is used in a broad sense - and co- vers food for humans as well as food for animals (i.e. a feed).
  • the food is for human consumption.
  • the fermented plant-based composition produced according to the methods described herein produces a fermented plant-based food product that is a functional food.
  • the term “functional food” means a food which is capable of providing not only a nutritional effect and/or a taste satisfaction but is also capable of delivering a further ben- eficial effect to consumer.
  • the bacterial strains or bacterial compositions, e.g., of the starter culture, described herein may be - or may be added to - a food ingredient, a food supplement, or a functional food.
  • the food may be in the form of a solution or as a solid - depend- ing on the use and/or the mode of application and/or the mode of administration.
  • the bacterial strains or bacterial compositions, e.g., start cultures, described herein can be used in the preparation of food products such as one or more of fer- mented plant-based food products.
  • the bacterial strain or bacterial composition can be used as an ingredient to prepare milk-type drinks, lactic acid bacteria drinks, yoghurt alterna- tives, and/or drinking yoghurt alternatives that are made from plant matter (e.g., a vegetal base) and referred to as a dairy alternative food product, e.g., plant-based yoghurt alternative.
  • a dairy alternative food product e.g., plant-based yoghurt alternative.
  • the fermented plant-based food product is a stirred-type dairy al- ternative food product.
  • the fermented plant-based food product is a set- type dairy alternative food product.
  • the fermented pant-based food prod- uct is a plant-based yogurt alternative, a plant-based cream alternative, a plant-based matured cream alternative, a plant-based butter alternative, a plant-based fat spread, a plant-based cheese alternative, a plant-based fromage frais alternative, a plant-based product beverage al- ternative, a plant-based processed cheese alternative, a plant-based cream dessert alternative, a plant-based cottage cheese alternative, or a plant-based kefir alternative.
  • the Streptococcus thermophilus strain according to embodiments 1 or 2, wherein the aver- age pH after 24 hours of fermentation measured as described in Assay 4 is at least about 4.21 such as at least about 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.30, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, 4.40, 4.41, 4.42, 4.43, 4.44, 4.45, 4.46, 4.47, 4.48, 4.49, 4.50, 4.51, 4.52, 4.53, 4.54, 4.55, 4.56, 4.57, 4.58, 4.59, or 4.60, and not higher than pH 5.5.
  • the Streptococcus thermophilus strain according to any one of embodiments 1-3, wherein the average slope between pH 6.0 and 5.3 measured as described in Assay 3 is increased relative to the same value for DSM 33651 with at least about 10%, such as 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, or 42%.
  • the Streptococcus thermophilus strain according to any one of embodiments 1-4, wherein the PrtS protein or a homologue thereof is functionally inactive or absent due to a modifica- tion introduced into the prtS gene, such as a deletion, a mutation, or an insertion, such as an insertion that causes a frame shift, a deletion or a mutation causing a premature stop codon, or any other insertion such as of a gene or transposable element disrupting the prtS gene, or complete removal or absence of the prtS gene or replacement with a non-func- tional prtS gene.
  • a modifica- tion introduced into the prtS gene such as a deletion, a mutation, or an insertion, such as an insertion that causes a frame shift, a deletion or a mutation causing a premature stop codon, or any other insertion such as of a gene or transposable element disrupting the prtS gene, or complete removal or absence of the prtS gene or replacement with a
  • the Streptococcus thermophilus strain according to any one of embodiments 1-5, which is selected from the group consisting of: (1) the DSM 21892 bacterial strain deposited under the Budapest Treaty on 7 October 2008 in the name of Danisco GmbH, GmbH at the Leibniz Institute DSMZ-Ger- man Collection of Microorganisms and Cell Cultures; (2) the DSM 34703 bacterial strain deposited under the Budapest Treaty on 19 July 2023 in the name of DuPont Nutrition Biosciences ApS, Denmark at the Leibniz Institute DSMZ- German Collection of Microorganisms and Cell Cultures; (3) the DSM 28128 bacterial strain deposited under the Budapest Treaty on 4 December 2 013 in the name of Danisco GmbH, Germany at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures; and (4) the DSM 34705 bacterial strain deposited under the Budapest Treaty on 19 July 2023 in the name of DuPont Nutrition Biosciences ApS, Denmark at the Leibniz Institute
  • a composition such as a composition of a starter culture comprising or consisting of a one or more culture of a Streptococcus thermophilus strain as defined in any one of embodi- ments 1-6, and optionally further comprising at least one other microorganism, such as at least one other lactic acid bacterium and/or at least one propionic bacterium.
  • composition according to embodiment 7, which composition comprises or consists of one or more, such as two, three or more cultures of a Streptococcus thermophilus strain as defined in any one of embodiments 1-6 and optionally one or more Streptococcus ther- m ophilus strain in which the cell-envelope proteinase (PrtS) or a homologue thereof is func- tionally active.
  • the composition according to embodiment 7, which composition comprises or consists of one or more culture of a Streptococcus thermophilus strain as defined in any one of embod- iments 1-6 and no other Streptococcus thermophilus strain. 10.
  • said plant is selected from legumes, such as the seeds of legumes including beans, such as soybeans, peas, favabeans, chickpea, lentils, mung bean, and any isolate thereof; nuts such as almond, coconut, cashew nut, Brazil nut, hazelnut, macadamia nut, pecan nut, pistachio and walnut; cereals and pseudo cereals such as wheat, corn/maize, oats, sorghum, rice, barley, millet, triticale, buckwheat, rye, teff; tuber such as cassava, potato, tapioca, arrowroot; oleaginous plants such as hemp, c anola, rapeseed, and sunflowers, and any isolate thereof.
  • legumes such as the seeds of legumes including beans, such as soybeans, peas, favabeans, chickpea, lentils, mung bean, and any isolate thereof.
  • nuts such as almond, coconut,
  • a method for acidifying an aqueous preparation derived from a plant, such as pea, soy, and oat comprising putting into contact a plant-based substrate, with or in the presence of a culture of a bacterial strain as defined in any one of embodiments 1-6 or a composition as defined in any one of embodiments 7-9. 15.
  • said plant is selected from legumes, such as the seeds of legumes including beans, such as soybeans, peas, favabeans, chickpea, lentils, mung bean, and any isolate thereof; nuts such as almond, co- conut, cashew nut, Brazil nut, hazelnut, macadamia nut, pecan nut, pistachio and walnut; cereals and pseudo cereals such as wheat, corn/maize, oats, sorghum, rice, barley, millet, triticale, buckwheat, rye, teff; tuber such as cassava, potato, tapioca, arrowroot; oleagi- nous plants such as hemp, canola, rapeseed, and sunflowers, and any isolate thereof.
  • legumes such as the seeds of legumes including beans, such as soybeans, peas, favabeans, chickpea, lentils, mung bean, and any isolate thereof.
  • nuts such as almond,
  • a fermented product in particular a fermented food or a fermented feed product, in partic- ular a fermented food or feed product based on a plant, such as a plant-based substrate, such as from pea, soy, and oat, obtainable by the method of embodiment 13-15. 17.
  • a fermented product in particular a fermented food or a fermented feed product, in partic- ular a fermented food or feed product based on a plant, such as a plant-based substrate, s uch as from pea, soy, and oat, comprising a culture of a Streptococcus thermophilus strain as defined in any one of embodiments 1-6 or a composition as defined in any one of em- bodiments 7-9. 18.
  • a plant-based dairy al- ternative product in particular a product based on a plant, such as a plant-based substrate, such as from pea, soy, and oat, such as a plant-based yoghurt, plant-based cheese, plant
  • a method for selecting a strain of Streptococcus thermophilus suitable for fermentation of a plant-based product comprising the steps of a) selecting a strain in which the cell-envelope proteinase (PrtS) or a homologue thereof is functionally inactive or absent; s electing a strain wherein the acidification kinetics of said strain in soy model is character- ized by: - an average slope between pH 6.0 and 5.3 at least about 100 (x104 pHU/min) measured as described in Assay 3, and - an average pH after 24 hours of fermentation measured as described in Assay 4 of at least about 4.20, and not higher than pH 5.5. 20.
  • PrtS cell-envelope proteinase
  • the method according to embodiments 19 or 20, wherein the average pH after 24 hours of fermentation measured as described in Assay 4 is at least about 4.21 such as at least about 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.30, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, 4.40, 4.41, 4.42, 4.43, 4.44, 4.45, 4.46, 4.47, 4.48, 4.49, 4.50, 4.51, 4.52, 4.53, 4.54, 4.55, 4.56, 4.57, 4.58, 4.59, or 4.60, and not higher than pH 5.5. 22.
  • the PrtS protein or a homologue thereof is functionally inactive or absent due to a modification introduced into t he prtS gene, such as a deletion, a mutation, or an insertion, such as an insertion that causes a frame shift, a deletion or a mutation causing a premature stop codon, or any other i nsertion such as of a gene or transposable element disrupting the prtS gene, or complete removal or absence of the prtS gene or replacement with a non-functional prtS gene.
  • a modification introduced into t he prtS gene such as a deletion, a mutation, or an insertion, such as an insertion that causes a frame shift, a deletion or a mutation causing a premature stop codon, or any other i nsertion such as of a gene or transposable element disrupting the prtS gene, or complete removal or absence of the prtS gene or replacement with a non-functional prtS gene.
  • a Streptococcus thermophilus obtained by a method according to any one of embodiments 19-23 such as any one selected from the group consisting of: (1) the DSM 34703 bacterial strain deposited under the Budapest Treaty on 19 July 2023 in the name of DuPont Nutrition Biosciences ApS, Denmark at the Leibniz Institute DSMZ-Ger- man Collection of Microorganisms and Cell Cultures; (2) the DSM 34705 bacterial strain deposited under the Budapest Treaty on 19 July 2023 in the name of DuPont Nutrition Biosciences ApS, Denmark at the Leibniz Institute DSMZ-Ger- man Collection of Microorganisms and Cell Cultures; and (3) the DSM 34704 bacterial strain deposited under the Budapest Treaty on 19 July 2023 in the name of DuPont Nutrition Biosciences ApS, Denmark at the Leibniz Institute DSMZ-Ger- man Collection of Microorganisms and Cell Cultures.
  • a composition such as a composition of a starter culture comprising or consisting of a one or more culture of a Streptococcus thermophilus strain as defined in embodiment 24, and optionally further comprising at least one other microorganism, such as at least one other lactic acid bacterium and/or at least one propionic bacterium.
  • PrtS cell-envelope proteinase
  • composition according to embodiments 25 or 26, which composition comprises or con- sists of one or more culture of a Streptococcus thermophilus strain as defined in embodi- ment 24 and no other Streptococcus thermophilus strain.
  • said plant is selected from legumes, such as the seeds of legumes including beans, such as soybeans, peas, favabeans, chickpea, lentils, mung bean, and any isolate thereof; nuts such as almond, coconut, cashew nut, Brazil nut, hazelnut, macadamia nut, pecan nut, pistachio and walnut; cereals and pseudo cereals such as wheat, corn/maize, oats, sorghum, rice, barley, millet, triticale, buckwheat, rye, teff; tuber such as cassava, potato, tapioca, arrowroot; oleaginous plants such as hemp, canola, rapeseed, and sunflowers, and any isolate thereof.
  • legumes such as the seeds of legumes including beans, such as soybeans, peas, favabeans, chickpea, lentils, mung bean, and any isolate thereof.
  • nuts such as almond, coconut, cashe
  • a method for preparing a fermented product, in particular a fermented food or a feed prod- uct based on a plant, such as pea, soy, and oat comprising putting into contact a plant-based substrate, with or in the presence of a culture of a bacterial strain as defined in embodiment 24; or selected from (1) the DSM 21892 bacterial strain deposited under the Budapest Treaty on 7 October 2008 in the name of Danisco GmbH, GmbH at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures and (2) the DSM 28128 bacterial strain deposited under the Budapest Treaty on 4 December 2013 in the name of Danisco GmbH, GmbH at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures; or a composition as defined in any one of embodiments 25-27, and obtaining said fermented product.
  • a method for acidifying an aqueous preparation derived from a plant, such as pea, soy, and oat comprising putting into contact a plant-based substrate, with or in the presence of a culture of a bacterial strain as defined in embodiment 24 or a composition as defined in any one of embodiments 25-27.
  • said plant is selected from legumes, such as the seeds of legumes including beans, such as soybeans, peas, favabeans, chickpea, lentils, mung bean, and any isolate thereof; nuts such as almond, co- conut, cashew nut, Brazil nut, hazelnut, macadamia nut, pecan nut, pistachio and walnut; cereals and pseudo cereals such as wheat, corn/maize, oats, sorghum, rice, barley, millet, triticale, buckwheat, rye, teff; tuber such as cassava, potato, tapioca, arrowroot; oleagi- nous plants such as hemp, canola, rapeseed, and sunflowers, and any isolate thereof.
  • legumes such as the seeds of legumes including beans, such as soybeans, peas, favabeans, chickpea, lentils, mung bean, and any isolate thereof.
  • nuts such as almond,
  • a fermented product in particular a fermented food or a fermented feed product, in partic- ular a fermented food or feed product based on a plant, such as a plant-based substrate, such as from pea, soy, and oat, obtainable by the method of embodiment 31-33.
  • a fermented product in particular a fermented food or a fermented feed product, in partic- ular a fermented food or feed product based on a plant, such as a plant-based substrate, such as from pea, soy, and oat, comprising a culture of a Streptococcus thermophilus strain as defined in embodiment 24; or selected from (1) the DSM 21892 bacterial strain depos- ited under the Budapest Treaty on 7 October 2008 in the name of Danisco GmbH, GmbH at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures and (2) the DSM 28128 bacterial strain deposited under the Budapest Treaty on 4 December 2013 in the name of Danisco GmbH, GmbH at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures; or a composition as defined in any one of embodiments 25-27.
  • a product according to any one of embodiments 34 or 35 which is a plant-based dairy al- ternative product, in particular a product based on a plant, such as a plant-based substrate, such as from pea, soy, and oat, such as a plant-based yoghurt, plant-based cheese, plant- based quark, a plant-based sour cream, plant-based kefir, a plant-based koumiss, plant- based butter, a plant-based beverage, a yoghurt-alternative plant drink, a fermented plant- based product, a matured cream-alternative, a fromage frais-alternative, a cottage cheese- alternative, or a cream dessert-alternative.
  • a plant-based dairy al- ternative product in particular a product based on a plant, such as a plant-based substrate, such as from pea, soy, and oat, such as a plant-based yoghurt, plant-based cheese, plant
  • DSMZ referring to Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany. It is requested that the biological material shall be made available only by the issue of a sample to an expert nominated by the requester.
  • Assays used herein Assay 1 The soy model is prepared as following: add 100 mL of filtrated (0.2 ⁇ m) glucose solution 200 g/L to 900 mL of French commercial soy Bjorg beverage (“Bjorg soja sanster BIO”) under sterile conditions and homogenize manually. Distribute soy model in 50 mL Schott flasks under sterile conditions. Let it warm at 37°C for at least 30 min before inoculation ( Figure 1).
  • the ABSCIA automatically calculates several descriptive descriptors: - Slope between pH 6 to pH 5.3 (*104 pHU/min): slope between the pH values 6.00 and 5.30, reflecting the acidification speed in a relevant and a comparable way from one strain to another. - Time to pH 4.6 (min): time when the pH 4.60 is reached. It’s the technological time usually used by soy yogurt-type producers. - pH24h: pH value after 24 hours of fermentation. The curves are usually recorded for 24 hours, so it gives the final picture of the acidification.
  • Assay 2 Defrozen pre-culture (in reconstituted milk containing 10% (w/v) skim milk powder pasteurized 20 min at 120°C and incubated 16h at 37°C) of the strains to be tested was used to inoculate at 2% (v/v) 100 mL of plain milk (“Candia Grand Lait” pasteurized 10 min at 90 °C diluted with water at 93% (v/v)) and 100 mL of sweet milk (“Candia Grand Lait” pasteurized 10 min at 90 °C sup- plemented with sucrose at 6% (w/v)). The inoculated milks were statically incubated in a water- bath at 43°C for 24h.
  • the acidifying properties of the strains were evaluated by recording the pH during milk fermentation.
  • the pH was monitored for 24 hours using the CINAC system (Alliance Instruments, France; pH electrode Mettler 405 DPAS SC, Toledo, Spain). The pH was recorded every 5 minutes.
  • the CINAC automatically calculates several descriptive descriptors: - Slope between pH 6 to pH 5.3 (*104 pHU/min): slope between the pH values 6.00 and 5.30, reflecting the acidification speed in a relevant and a comparable way from one strain to another.
  • - Time to pH 4.6 (min) time when the pH 4.60 is reached. It’s the technological time usually used by milk yogurt producers.
  • - pH24h pH value after 24 hours of fermentation.
  • the soy model is prepared as following: add 100 mL of filtrated (0.2 ⁇ m) glucose solution 200 g/L to 900 mL of French commercial soy Bjorg beverage (“Bjorg soja sansender BIO”) under sterile conditions and homogenize manually. Distribute soy model in 10 mL tubes under sterile conditions and store them in a waterbath at 6°C ( Figure 3).
  • Assay 4 The soy model is prepared as following: add 100 mL of filtrated (0.2 ⁇ m) glucose solution 200 g/L to 900 mL of French commercial soy Bjorg beverage (“Bjorg soja sansender BIO”) under sterile conditions and homogenize manually. Distribute soy model in 10 mL tubes under sterile conditions and store them in a waterbath at 6°C ( Figure 5). These tubes stored at 6°C will allow to prepare intermediate dilutions (4.106 cfu/mL) of the Streptococcus thermophilus strain being tested in order to have the same volume to add in the HydroPlate® (PreSens, Precision Sensing GmbH, Germany) whatever the strain.
  • HydroPlate® PreSens, Precision Sensing GmbH, Germany
  • refrigerated tubes will allow not to start the fermenta- tion yet in these tubes.
  • the aim is to try to better control the conditions before the inoculation of HydroPlate®.
  • Take the required volume in order to reach an inoculation rate of 4.106 cfu/mL in 10 mL soy model tubes.
  • thermophilus strains carrying the prtS gene were shown to be of interest in milk since they are faster in acidification than strains without this gene (Boulay et al., 2020, International Journal of Food Microbiology, Volume 335, 16 December 2020).
  • derivatives of S. thermophilus 34703-1 strain, selected from parental strain DSM 34703, were constructed into which the prtS gene has been introduced using natural competence as shown in Table 1.
  • S. thermophilus LMD-9 strain was cho- sen as reference strain bearing the prtS gene (Boulay et al., 2020).
  • Figure 7 presents acidification kinetics in soy model, measured in the conditions of Assay 1 and Table 2 summarizes the acidification parameters in both soy and milk models, measured in the conditions of Assay 1 and Assay 2.
  • strains 34703- 1.1 and 34703-1.2 have a low effect on these descriptors in soy model, whereas it has a strong effect in milk model by highly reducing both pH 24h and time to reach pH4.6, and deeply increasing the slope between pH6 to pH5.3. This indicates that the presence of the prtS gene does not seem to systematically improve the acidification speed in soy model (as in the current experimental condi- tions), contrary to what has been described in Boulay et al. (2020). Moreover, in this example, strains DSM 34703 and 34703-1, which are not bearing the prtS gene, display the highest slope as compared to the reference PrtS-positive S.
  • thermophilus LMD-9 strain in soy model This raises the question about the importance of the prtS gene in S. thermophilus for fast soy fermentation, contrary to milk fermentation.
  • Table 1 Strains used in the example 1 PrtS S trains Mother Presence (+) Absence (-) D SM 34703 - - 34703-1 DSM 34703 - 34703-1.1 34703-1 + 34703-1.2 34703-1 + LMD-9 - + Table 2. Descriptors from ABSCIA of soy (Assay 1) and milk (Assay 2) models fermented 24 hours at 37°C with S.
  • thermophilus LMD-9 DSM 34703 strain and its derivatives 34703-1 (Prts-nega- tive), 34703-1.1 (Prts-positive) and 34703-1.2 (Prts-positive) strains.
  • Soy model 1 Milk model 2 Slope Slope Strains pH 6.0 to 5.3 Time to pH 6.0 to 5 Time to pH4.6 pH 24h .3 pH4.6 pH (*10 4 ( 4 24h (min) *10 (min) pHU/min) pHU/min) DSM 34703 98 602 4.25 27 not reached 4.64 34703-1 117 532 4.22 33 not reached 4.67 34703-1.1 91 571 4.17 145 290 4.18 34703-1.2 106 569 4.16 134 310 4.29 LMD-9 82 571 4.19 nd* nd* *nd: not determined.
  • ST PrtS-negative strains (more than half of the tested ST PrtS- negative strains) are among those fastest 20 ST strains, DSM 21892, DSM 34703, DSM 28128, DSM 34705, DSM 34704, ST-9, ST-12 and ST-13.
  • five strains (DSM 21892, DSM 34703, DSM 28128, DSM 34705, DSM 34704) in the top-6 of strains displaying the highest slope ( ⁇ 132 * 104 pHU/min) in soy model are prtS-negative.
  • thermophilus PrtS-negative strains (DSM 21892 and DSM 34703) display a higher slope ( ⁇ 151 * 104 pHU/min) than the best PrtS-positive strain (ST-1, ⁇ 144 * 104 pHU/min). These results confirm that the prtS gene is not mandatory for increasing the acidification kinetics in soy model. O n top and advantageously, among the 20 fastest strains, the average pH24h of the eight ST PrtS-negative strains is 4.43 while the one of the 12 PrtS-positive strains is 4.17, making those PrtS-negative strains even more interesting to produce milder fermented soy products.

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Abstract

La présente invention concerne des souches de Streptococcus thermophilus dans lesquelles la protéinase de cellule-enveloppe (PrtS) ou son homologue est fonctionnellement inactif ou absent, l'utilisation de ces souches à PrtS négative pour la fermentation de préparations dérivées d'une plante, en particulier des produits alimentaires ou des aliments pour animaux fermentés à base d'une plante, ainsi que des procédés de préparation de produits fermentés à base de plantes.
PCT/EP2024/085118 2023-12-08 2024-12-06 Souches de streptococcus thermophilus à acidifiacation rapide dans le soja Pending WO2025120178A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010066907A1 (fr) * 2008-12-12 2010-06-17 Danisco A/S Agrégat génétique de souches de streptococcus thermophilus ayant des propriétés rhéologiques uniques pour la fermentation laitière
WO2015140211A1 (fr) * 2014-03-19 2015-09-24 Dupont Nutrition Biosciences Aps Amélioration de la fabrication de viande cuite

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010066907A1 (fr) * 2008-12-12 2010-06-17 Danisco A/S Agrégat génétique de souches de streptococcus thermophilus ayant des propriétés rhéologiques uniques pour la fermentation laitière
WO2015140211A1 (fr) * 2014-03-19 2015-09-24 Dupont Nutrition Biosciences Aps Amélioration de la fabrication de viande cuite

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BOULAY MYLÈNE ET AL: "Streptococcus thermophilus growth in soya milk: Sucrose consumption, nitrogen metabolism, soya protein hydrolysis and role of the cell-wall protease PrtS", INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, ELSEVIER BV, NL, vol. 335, 8 October 2020 (2020-10-08), XP086329179, ISSN: 0168-1605, [retrieved on 20201008], DOI: 10.1016/J.IJFOODMICRO.2020.108903 *

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