Method of preparing fermented plant-based products with umami taste and products thereof

Abstract

The present invention relates to a method of preparing a fermented plant-based product having a umami taste. The method comprises the step of providing a plant protein substrate and the step of fermenting the plant protein substrate with a starter culture comprising Lactobacillus johnsonii. The present invention also relates to such a fermented plant-based product and to Lactobacillus johnsonii strains suitable for use in the present invention. The present invention also relates to peptides that impart and / or enhance umami taste, to methods for preparing such peptides, to compositions comprising such peptides, and to the use of such peptides as flavoring agents.

Description

Technical Field

[0001] This invention relates generally to the field of fermentation of plant materials. Specifically, this invention relates to a method for preparing fermented plant-based products with umami flavor, and the fermented plant-based products thereof. Background Technology

[0002] Along with sweet, bitter, salty, and sour, umami is one of the five basic tastes. Umami is a taste that spreads and covers the tongue, lingers, and brings a mouthwatering and pleasantly delicious sensation.

[0003] Umami-imparting and umami-enhancing substances, including peptides, have been found in many animal-based foods such as ham and cheese, as well as plant-based foods such as soy sauce and sourdough bread.

[0004] Umami-imparting and umami-enhancing substances, especially peptides, stimulate saliva production, appetite, and increase the palatability of food. Therefore, they are valuable for improving the sensory quality of food.

[0005] Plant-based products are gaining increasing attention. Therefore, there is a desire to generate umami-enhancing and flavor-improving substances, particularly peptides, in plant-based products to provide an enhanced sensory experience.

[0006] There are two methods to generate umami-imparting and umami-enhancing peptides.

[0007] On the one hand, these peptides can be produced through enzymatic hydrolysis. Several commercially available peptidase preparations have been studied for the hydrolysis of plant proteins. Examples include flavor enzymes from *Aspergillus niger* (Novozyme), alkaline proteases from *Bacillus subtilis*, ProteAXK from *Rhizopus oryzae* (AMANO), and Umamyzyme from *Aspergillus oryzae*. However, the resulting hydrolysates typically exhibit an unpleasant bitter taste.

[0008] On the other hand, these peptides can be produced through fermentation. Such peptides are typically produced via processes involving more than one step or more strains of bacteria. For example, the production of soy sauce requires Aspergillus and lactic acid bacteria, while sourdough bread requires fungi or maltase and lactic acid bacteria.

[0009] Therefore, it is desirable to provide a method for generating or enhancing umami flavor in plant-based products. The method is desirable to be natural and involve a limited number of steps and / or a limited number of ingredients. It is also desirable that the method produces plant-based products with a limited degree of bitterness.

[0010] It is also desirable to provide molecules and compositions that can be used as flavoring agents, particularly to impart or enhance umami flavor. Such molecules and compositions are expected to be plant-derived and not artificial, i.e., not produced through chemical synthesis.

[0011] Any references to prior art documents in this specification should not be construed as an admission that such prior art is well-known or part of common knowledge in the field. Summary of the Invention

[0012] The object of the present invention is to improve the prior art, and in particular to provide a method, fermentation of plant-based products, Lactobacillus johnsonii strain, uses, compositions and peptides that overcome the problems of the prior art and solve the above-mentioned needs, or at least to provide a useful alternative.

[0013] The inventors were surprised to find that the objective of the invention could be achieved through the subject matter of the independent claims. The dependent claims further expand the conception of the invention.

[0014] Therefore, a first aspect of the present invention provides a method for preparing a fermented plant-based product, the method comprising providing a plant protein substrate and fermenting the plant protein substrate with a starter culture containing Lactobacillus johnsonii.

[0015] It has been found that fermenting plant proteins with Lactobacillus johnsonii effectively imparts and / or enhances umami flavor in plant-based products while limiting the generation of bitterness. The method of the present invention is simple and involves a single fermentation step.

[0016] A second aspect of the invention provides a fermented plant-based product comprising plant proteins fermented with Lactobacillus johnsonii.

[0017] The fermented plant-based products of this invention have a pleasant, pronounced, and enhanced umami flavor, while exhibiting limited bitterness.

[0018] A third aspect of the invention proposes Lactobacillus johnsonii deposited in CNCM with accession number CNCM I-5910.

[0019] A fourth aspect of the invention provides for the use of *Lactobacillus johnsonii* in the production of fermented plant-based products, wherein *Lactobacillus johnsonii* is selected from the list consisting of: *Lactobacillus johnsonii* deposited in CNCM with accession number CNCM I-5910, *Lactobacillus johnsonii* deposited in CNCM with accession number CNCM I-1225, *Lactobacillus johnsonii* deposited in CNCM with accession number CNCM I-2474, or combinations thereof.

[0020] It has been found that the aforementioned Lactobacillus johnsonii strain effectively provides / enhances the umami flavor in plant-based products through the fermentation of plant proteins.

[0021] The fifth aspect of the invention provides a peptide having an amino acid sequence of SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11.

[0022] A sixth aspect of the present invention provides a method for preparing one or more peptides, the method comprising the following steps:

[0023] Provides plant protein substrates,

[0024] Fermentation of plant protein substrates using starter cultures containing Lactobacillus johnsonii.

[0025] Separate one or more peptides,

[0026] One or more of the peptides are selected from the list consisting of: a peptide having the amino acid sequence of SEQ ID NO: 4, a peptide having the amino acid sequence of SEQ ID NO: 5, a peptide having the amino acid sequence of SEQ ID NO: 6, a peptide having the amino acid sequence of SEQ ID NO: 7, a peptide having the amino acid sequence of SEQ ID NO: 8, a peptide having the amino acid sequence of SEQ ID NO: 9, a peptide having the amino acid sequence of SEQ ID NO: 10, a peptide having the amino acid sequence of SEQ ID NO: 11, or a combination thereof.

[0027] A seventh aspect of the invention provides a composition comprising one or more peptides, wherein the one or more peptides are selected from the list consisting of: a peptide having the amino acid sequence of SEQ ID NO: 4, a peptide having the amino acid sequence of SEQ ID NO: 5, a peptide having the amino acid sequence of SEQ ID NO: 6, a peptide having the amino acid sequence of SEQ ID NO: 7, a peptide having the amino acid sequence of SEQ ID NO: 8, a peptide having the amino acid sequence of SEQ ID NO: 9, a peptide having the amino acid sequence of SEQ ID NO: 10, a peptide having the amino acid sequence of SEQ ID NO: 11, or combinations thereof.

[0028] The eighth aspect of the invention provides for the use of one or more peptides as flavoring agents, wherein the one or more peptides are selected from the list consisting of: a peptide having the amino acid sequence of SEQ ID NO: 4, a peptide having the amino acid sequence of SEQ ID NO: 5, a peptide having the amino acid sequence of SEQ ID NO: 6, a peptide having the amino acid sequence of SEQ ID NO: 7, a peptide having the amino acid sequence of SEQ ID NO: 8, a peptide having the amino acid sequence of SEQ ID NO: 9, a peptide having the amino acid sequence of SEQ ID NO: 10, a peptide having the amino acid sequence of SEQ ID NO: 11, or combinations thereof.

[0029] The ninth aspect of the present invention discloses the use of the composition of the seventh aspect of the present invention as a flavoring agent.

[0030] It has been found that the aforementioned peptides and compositions containing one or more of these peptides effectively impart and / or enhance umami flavor in products. These peptides or compositions can be used as umami flavoring agents, umami flavor enhancers, and / or flavor masking agents.

[0031] Those skilled in the art will gain a clearer understanding of these and other aspects, features, and advantages of the present invention after reading the detailed description of the embodiments of the present invention in conjunction with the accompanying drawings. Attached Figure Description

[0032] Figure 1 The sensory scores of umami from fermented plant-based milk prepared with pea protein and fermented with different starter cultures are shown to be compared with the sensory scores of umami from reference unfermented plant-based milk prepared with pea protein (labeled unfermented pea protein isolate).

[0033] Figure 2 The table shows the umami peptide counts of a reference unfermented plant-based milk prepared with pea protein (labeled unfermented pea protein isolate) and the umami peptide counts of fermented plant-based milk prepared with pea protein and fermented with different strains of Lactobacillus johnsonii. Detailed Implementation

[0034] As used in this specification, the terms "including" and "containing" should be interpreted as having a inclusive meaning, the opposite of exclusive or exhaustive, that is, meaning "including but not limited to".

[0035] All numerical ranges should be understood to include every integer within that range.

[0036] As used in this specification, the singular forms “a,” “an,” and “the” include multiple referents unless the context clearly indicates otherwise.

[0037] As used in the specification, the terms "substantially free" or "substantially absent" mean the presence of no more than 10% by weight, preferably no more than 5% by weight, and more preferably no more than 1% by weight of the excluded material. In a preferred embodiment, "substantially free" or "substantially absent" means that the remaining excluded material is no more than 0.1% by weight. "Completely free" or "completely absent" generally means that at most only trace amounts of the excluded material are present, and preferably there are no detectable amounts of the excluded material.

[0038] Unless otherwise stated, all percentages in this specification refer to weight percentages where applicable.

[0039] Unless otherwise defined, all technical terms have and should be given the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] As used herein, the term "aqueous liquid" refers to a liquid containing water. Preferably, it refers to a liquid containing at least 50%, more preferably at least 70% by weight of water, more preferably at least 80% by weight of water, or even more preferably at least 90% by weight of water. Most preferably, it refers to a liquid composed entirely of water.

[0041] As used herein, the term "vegan" refers to an edible composition or product that contains no animal products or products of animal origin.

[0042] As used herein, the term "vegetarian" refers to edible compositions or products that do not contain meat (including fish).

[0043] As used herein, the term "plant powder" refers to a plant composition containing 5% to 49.9% protein.

[0044] As used herein, the term "plant protein concentrate" refers to a plant composition containing 50% to 79.9% plant protein.

[0045] As used herein, the term "plant protein isolate" refers to a composition containing 80.0% to 99.0% plant protein.

[0046] As used herein, the term "argininoyl peptide" refers to a peptide having an amino acid sequence containing at least one arginine residue. In particular, when the peptide is in its three-dimensional structure, at least one arginine residue can be accessed by umami taste receptors in the mouth, such as GluR4, mGluR1, and type 1 taste receptors (TAS1R1+TAS1R3).

[0047] As used herein, the term "prolyl peptide" refers to a peptide having an amino acid sequence containing at least one proline residue. In particular, when the peptide is in its three-dimensional structure, at least one proline residue can be accessed by umami taste receptors in the mouth, such as GluR4, mGluR1, and type 1 taste receptors (TAS1R1+TAS1R3).

[0048] As used herein, the term "glutamine peptide" refers to a peptide having an amino acid sequence containing at least one glutamine residue. In particular, when the peptide is in its three-dimensional structure, at least one glutamine residue can be accessed by umami taste receptors in the mouth, such as GluR4, mGluR1, and type 1 taste receptors (TAS1R1+TAS1R3).

[0049] For clarity, in the context of this invention, "argininoyl peptide," "prolyl peptide," and / or "glutamyl peptide" are not Maillard reaction products, particularly Maillard reaction peptides. Similarly, for clarity, in any relevant aspect of this invention (i.e., all aspects of the invention except the third aspect), any peptide having the amino acid sequence SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, and 11 is not a Maillard reaction product, particularly a Maillard reaction peptide.

[0050] As used herein, the term "added reducing sugar" refers to reducing sugars that are added to and are not inherently present in the compositions of the present invention and in the components of fermented plant-based products (e.g., plant protein substrates, plant proteins).

[0051] As used herein, the term "high-methoxyl pectin" refers to pectin with a degree of esterification (DE) of at least 50%, preferably 55% to 75%. The degree of esterification (DE) is defined as the number of methylated galacturonic acid units, expressed as a percentage of total galacturonic acid units in the pectin molecule.

[0052] In a first aspect, the present invention relates to a method for preparing a fermented plant-based product. The fermented plant-based product may be a fermented plant-based food or beverage. The fermented plant-based food or beverage may be selected from a list consisting of: sauces, broths, dips, seasonings, fermented dairy analogues, meat analogues, pet food, confectionery, snacks, or soups. Preferably, the fermented plant-based food or beverage is vegetarian or vegan.

[0053] In a preferred embodiment, the fermented plant-based food or beverage is a fermented plant-based dairy or meat analogue. In a more preferred embodiment, the fermented plant-based food or beverage is a fermented plant-based dairy analogue. The fermented plant-based dairy analogue can be selected from a list consisting of: cheese analogues, fresh cheese analogues, cooking cream analogues, farm cheese analogues, yogurt analogues, kefir analogues, fermented milk analogues, or combinations thereof. In the most preferred embodiment, the fermented plant-based food or beverage is a fermented plant-based beverage, particularly a fermented plant-based dairy beverage analogue.

[0054] In some implementations, the fermented plant-based products are free of pectin, especially high-methoxyl pectin.

[0055] The method includes the step of providing a plant protein substrate. Preferably, the plant protein substrate is a plant protein suspension. The plant protein suspension is prepared by suspending the plant protein in an aqueous liquid. In a preferred embodiment, the aqueous liquid is water.

[0056] In the embodiments, the plant protein substrate, preferably the plant protein substrate, contains at least 0.5% by weight of plant protein, preferably 0.5% to 18% by weight, more preferably 1.5% to 18% by weight, more preferably 2% to 12% by weight, and even more preferably 5% to 12% by weight of plant protein.

[0057] In the implementation plan, plant protein is different from soybean protein and / or cereal protein.

[0058] Advantageously, the plant protein includes, and preferably consists of, legume proteins. Legume proteins are selected from the list consisting of: pea protein, mung bean protein, northern bean protein, white kidney bean protein, flavours bean protein, navy bean protein, red bean protein, black bean protein, kidney bean protein, lima bean protein, mung bean protein, chickpea protein, broad bean protein, lentil protein, or combinations thereof. More preferably, the plant protein includes, and preferably consists of, pea protein.

[0059] Plant proteins can be in the form of plant powder, plant protein concentrate, plant protein isolate, or combinations thereof. Preferably, the plant protein is in the form of a plant protein isolate, more preferably a legume protein isolate, and more preferably a pea protein isolate.

[0060] The method also includes the step of fermenting a plant protein substrate with a starter culture containing Lactobacillus johnsonii.

[0061] It has been found that fermenting plant protein substrates with *Lactobacillus johnsonii* effectively imparts and / or enhances umami flavor in plant-based products while limiting the formation of bitterness. The method of the present invention is simple and involves a single fermentation step.

[0062] Advantageously, the starter culture consists solely of *Lactobacillus johnsonii*. It has been observed that the umami flavor generation / enhancement effects provided by the method of the present invention can be achieved by fermenting plant protein substrates with a single strain, particularly with only *Lactobacillus johnsonii*. In other words, the fermented plant-based product contains no bacteria other than *Lactobacillus johnsonii*, particularly other than *Lactobacillus johnsonii* as described below.

[0063] In a preferred embodiment, *Lactobacillus johnsonii* expresses protease PrtP and / or protease PrtM and / or oligopeptide permease OppA. Proteases PrtP and PrtM are cell wall-bound proteases.

[0064] The protease PrtP has an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably 99% identical to that of SEQ ID NO: 1. Most preferably, the protease PrtP has the amino acid sequence of SEQ ID NO: 1.

[0065] The protease PrtM has an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably 99% identical to that of SEQ ID NO: 2. Most preferably, the protease PrtM has the amino acid sequence of SEQ ID NO: 2.

[0066] The oligopeptide permease OppA has an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably 99% identical to that of SEQ ID NO: 3. Most preferably, the oligopeptide permease OppA has the amino acid sequence of SEQ ID NO: 3.

[0067] It has been found that Lactobacillus strains expressing protease PrtP and / or protease PrtM and / or oligopeptide permease OPPA effectively produce peptides that provide and / or enhance umami flavor after fermentation.

[0068] Specifically, Lactobacillus johnsonii can be selected from a list consisting of the following: Lactobacillus johnsonii deposited in CNCM with accession number CNCM I-5910, Lactobacillus johnsonii deposited in CNCM with accession number CNCM I-1225, Lactobacillus johnsonii deposited in CNCM with accession number CNCM I-2474, or a combination thereof.

[0069] These Lactobacillus johnsonii strains effectively deliver / enhance umami flavor in plant-based products through the fermentation of plant proteins. In particular, they are believed to hydrolyze plant proteins into peptides that impart and / or enhance umami flavor during fermentation.

[0070] Lactobacillus johnsonii, deposited at CNCM with accession number CNCM I-5910, was deposited by Société des Produits Nestlé SA on October 5, 2022, at the French National Center for Microbial Collections (CNCM), Pasteur Institute, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, under the Budapest Treaty.

[0071] Lactobacillus johnsonii, deposited at CNCM with accession number CNCM I-1225, was deposited by Neste SA on June 30, 1992, under the Budapest Treaty, at the French National Center for Microbial Collections (CNCM), Pasteur Institute, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15.

[0072] Lactobacillus johnsonii, deposited at CNCM with accession number CNCM I-2474, was deposited by Nestlé on May 9, 2000, at the French National Center for Microbial Collection (CNCM), Pasteur Institute, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, under the Budapest Treaty.

[0073] The applicant, Nestlé (SPN), is the successor to the depositor, Neste SA.

[0074] In a preferred embodiment, the fermentation step of the plant protein substrate with a starter culture lasts for at least 16 hours, preferably at least 18 hours, more preferably at least 24 hours, even more preferably at least 35 hours, and most preferably at least 40 hours. Without being bound by theory, it is believed that this minimum fermentation time ensures that the proteolytic mechanism of *Lactobacillus johnsonii* has sufficient time to initiate and produce a sufficient amount of peptides during fermentation, which impart and / or enhance the umami flavor of the plant-based product. In another embodiment, the fermentation step of the plant protein substrate with a starter culture lasts for up to 50 hours, more preferably up to 48 hours.

[0075] In the implementation plan, the fermentation step is carried out at a temperature of 35°C to 45°C.

[0076] In another embodiment, the fermentation step is carried out under stirring, preferably at a stirring speed of 200 rpm to 1200 rpm.

[0077] The fermentation step can be carried out under anaerobic or aerobic conditions, preferably under aerobic conditions.

[0078] The method of this invention allows for the production of argininoyl peptides, prolyl peptides, and / or glutamyl peptides that impart and / or enhance umami flavor. In particular, fermented plant-based products contain argininoyl peptides, prolyl peptides, and / or glutamyl peptides.

[0079] In a preferred embodiment, the argininoyl peptide, prolyl peptide, and / or glutamyl peptide consists of 2 to 20 amino acids, preferably 2 to 15 amino acids. Argininoyl peptides, prolyl peptides, and / or glutamyl peptides with limited amino acid sequence lengths are more likely to impart and / or enhance umami flavor.

[0080] Fermented dairy products contain elevated levels of these peptides. In a preferred embodiment, the concentration of argininoyl peptide, prolyl peptide, and / or glutamyl peptide in the fermented plant-based product is at least 0.2 µmol per liter of fermented plant-based product, preferably at least 0.5 µmol, more preferably at least 1.0 µmol, even more preferably at least 1.5 µmol, and most preferably 2.0 µmol.

[0081] In a preferred embodiment, the concentration of argininosine, prolyl peptide and / or glutamyl peptide in the fermented plant-based product is at most 100 µmol per liter of fermented plant-based product, preferably at most 50 µmol, more preferably at most 30 µmol, even more preferably at most 20 µmol, and most preferably at most 19 µmol.

[0082] In some embodiments, argininosine, prolyl peptide and / or glutamyl peptide may be derived from plants, preferably from plants other than soybeans and / or cereals.

[0083] Advantageously, argininoyl peptides, proline peptides, and / or glutamyl peptides may be derived from legumes. Legumes are selected from the list consisting of: peas, adzuki beans, broad beans, white kidney beans, flavour beans, navy beans, red beans, black beans, pinto beans, kidney beans, lima beans, mung beans, chickpeas, broad beans, lentils, or combinations thereof. More preferably, the legume is pea.

[0084] It has been discovered that the method of the present invention allows for the production of novel peptides that effectively impart or enhance umami flavor. Fermented plant-based products comprise peptides selected from the list consisting of: peptides having the amino acid sequence of SEQ ID NO: 4, peptides having the amino acid sequence of SEQ ID NO: 5, peptides having the amino acid sequence of SEQ ID NO: 6, peptides having the amino acid sequence of SEQ ID NO: 7, peptides having the amino acid sequence of SEQ ID NO: 8, peptides having the amino acid sequence of SEQ ID NO: 9, peptides having the amino acid sequence of SEQ ID NO: 10, peptides having the amino acid sequence of SEQ ID NO: 11, or combinations thereof.

[0085] In some implementations, the peptides listed above may be derived from plants, preferably from plants other than soybeans and / or cereals.

[0086] Advantageously, the peptides listed above can be derived from legumes. Legumes are selected from the list consisting of: peas, adzuki beans, broad beans, white kidney beans, flavour beans, navy beans, red beans, black beans, pinto beans, kidney beans, lima beans, mung beans, chickpeas, fava beans, lentils, or combinations thereof. More preferably, peas are a preferred legume.

[0087] In the embodiments, the fermented plant-based product may comprise at least 0.2 µmol, preferably at least 0.5 µmol, more preferably at least 1.0 µmol, even more preferably at least 1.5 µmol, and most preferably 2.0 µmol of peptide per liter of fermented plant-based product, wherein the peptide is selected from the list consisting of: peptides having the amino acid sequence of SEQ ID NO: 4, peptides having the amino acid sequence of SEQ ID NO: 5, peptides having the amino acid sequence of SEQ ID NO: 6, peptides having the amino acid sequence of SEQ ID NO: 7, peptides having the amino acid sequence of SEQ ID NO: 8, peptides having the amino acid sequence of SEQ ID NO: 9, peptides having the amino acid sequence of SEQ ID NO: 10, peptides having the amino acid sequence of SEQ ID NO: 11, or combinations thereof.

[0088] In the embodiments, the fermented plant-based product may contain up to 100 µmol, preferably up to 50 µmol, more preferably up to 30 µmol, even more preferably up to 20 µmol, and most preferably up to 19 µmol of peptide per liter of fermented plant-based product, wherein the peptide is selected from the list consisting of: peptides having the amino acid sequence of SEQ ID NO: 4, peptides having the amino acid sequence of SEQ ID NO: 5, peptides having the amino acid sequence of SEQ ID NO: 6, peptides having the amino acid sequence of SEQ ID NO: 7, peptides having the amino acid sequence of SEQ ID NO: 8, peptides having the amino acid sequence of SEQ ID NO: 9, peptides having the amino acid sequence of SEQ ID NO: 10, peptides having the amino acid sequence of SEQ ID NO: 11, or combinations thereof.

[0089] Fermented plant-based products possess umami flavor. Umami flavor can be assessed by a trained panel of evaluators who assess the umami flavor of food products, using a rating scale ranging from 0 (undetectable) to 5 (strongly detectable). An example of a method for assessing umami flavor is provided in this sample.

[0090] Plant-based products are substantially free of, and preferably free of, added sweetening ingredients. The term "added sweetening ingredients" refers to exogenous components that impart a sweet taste when added to a composition or product, such as sucrose, honey, or fruit. The term "exogenous" does not include sweetening ingredients inherently present in plant protein substrates, and preferably in aqueous liquids of plant proteins and plant protein suspensions. In particular, plant-based products are substantially free of, and preferably free of, added sucrose and / or added fruit. This avoids the presence of a primary sweet taste that could overshadow the umami flavor imparted and / or enhanced by the methods of the present invention.

[0091] In the implementation scheme, the fermented plant-based product has an acidic pH, particularly less than 5.5, preferably less than 5.0, more preferably less than 4.6, and most preferably 3.0 to 4.6.

[0092] In another embodiment, the fermented plant-based product may also contain ingredients selected from a list of, for example, vitamins, minerals, pigments, fiber, prebiotics, hydrocolloids, salts, buffers, fats, oils, emulsifiers, plant-based milk substitutes, plant-based butter substitutes, solid food particles, or mixtures thereof.

[0093] In some implementations, the method does not include any step of the Maillard reaction. Therefore, the method does not include any step of the Maillard reaction consisting of:

[0094] The fermentation of plant-based products obtained by heat treatment at a temperature above 100°C for at least 30 minutes, preferably at least 1 hour, or

[0095] One or more reducing sugars are added to the obtained fermented plant-based product, and then the fermented plant-based product in which one or more reducing sugars have been added is heat-treated at a temperature above 100°C for at least 30 minutes, preferably at least 1 hour.

[0096] For example, the added reducing sugar can be selected from xylose, arabinose, ribose, glucose, galactose, and mixtures thereof.

[0097] In some implementations, the fermented plant-based product may be free of one or more added reducing sugars. For example, one or more added reducing sugars may be selected from xylose, arabinose, ribose, glucose, galactose, and mixtures thereof.

[0098] In the context of this invention, the Maillard reaction is undesirable because it requires the addition of additional reducing sugars and a prolonged, harsh heat treatment at high temperatures, which can generate undesirable flavor compounds in fermented plant-based products. Furthermore, this harsh heat treatment can have other undesirable consequences for fermented plant-based products: loss of compounds of interest (including macronutrients and micronutrients), protein aggregation, protein denaturation, etc. Moreover, the specific implementation of the Maillard reaction introduces further complexity to the process and indicates higher energy consumption.

[0099] In a second aspect, the present invention relates to a fermented plant-based product comprising plant proteins fermented with Lactobacillus johnsonii.

[0100] Plant proteins and Lactobacillus johnsonii can be as described in the first aspect of the invention.

[0101] The features of the fermented plant-based products disclosed in the first aspect of the invention are applicable to the fermented plant-based products of the second aspect of the invention, and vice versa.

[0102] In the implementation scheme, the fermented plant-based product can be obtained or is available through the method of the first aspect of the invention.

[0103] The fermented plant-based products of this invention have a pleasant, pronounced, and enhanced umami flavor, while exhibiting limited bitterness.

[0104] In some embodiments, the fermented plant-based products do not undergo the Maillard reaction, particularly the Maillard reaction as described in the first aspect of the invention.

[0105] In some implementations, the fermented plant-based product may be free of one or more added reducing sugars. For example, one or more added reducing sugars may be selected from xylose, arabinose, ribose, glucose, galactose, and mixtures thereof.

[0106] In the context of this invention, the Maillard reaction is undesirable because it requires the addition of additional reducing sugars and necessitates prolonged and harsh heat treatment at high temperatures, which can generate undesirable flavor compounds in fermented plant-based products. Furthermore, this harsh heat treatment can have other undesirable consequences for fermented plant-based products: loss of compounds of interest (including macronutrients and micronutrients), protein aggregation, protein denaturation, etc. Moreover, the specific implementation of the Maillard reaction introduces further complexity and indicates higher energy consumption.

[0107] In a third aspect, the present invention relates to Lactobacillus johnsonii deposited in CNCM with accession number CNCM I-5910.

[0108] This strain of *Lactobacillus johnsonii* effectively generates / enhances umami flavor in plant-based products through the fermentation of plant proteins, while producing limited bitterness. It is believed that during fermentation, the strain hydrolyzes plant proteins into peptides that impart and / or enhance umami flavor.

[0109] In a fourth aspect, the present invention relates to the use of *Lactobacillus johnsonii* in the production of fermented plant-based products, wherein *Lactobacillus johnsonii* is selected from the list consisting of: *Lactobacillus johnsonii* deposited in CNCM with accession number CNCM I-5910, *Lactobacillus johnsonii* deposited in CNCM with accession number CNCM I-1225, *Lactobacillus johnsonii* deposited in CNCM with accession number CNCM I-2474, or combinations thereof.

[0110] The fermented plant-based product can be a fermented plant-based product as described in the first and second aspects of the present invention.

[0111] The benefits of such strains are described in the first and third aspects of the present invention.

[0112] In a fifth aspect, the present invention relates to peptides having an amino acid sequence of SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11.

[0113] These peptides effectively impart and / or enhance the umami flavor in products.

[0114] In some preferred embodiments, the peptide having the amino acid sequence of SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11 is isolated and / or purified. In some preferred embodiments, the peptide is not combined with one or more reducing sugars, particularly one or more added reducing sugars. For example, one or more reducing sugars, particularly one or more added reducing sugars, may be selected from xylose, arabinose, ribose, glucose, galactose, and mixtures thereof. In some embodiments, the peptide has not undergone a Maillard reaction, particularly the Maillard reaction as described in the first aspect of the invention.

[0115] In the context of this invention, the Maillard reaction is undesirable because it requires the addition of additional reducing sugars and a prolonged, harsh heat treatment at high temperatures, which may generate undesirable flavor compounds. Furthermore, this harsh heat treatment can produce other undesirable consequences: loss of compounds of interest, protein aggregation, protein denaturation, etc. Moreover, the specific implementation of the Maillard reaction introduces further complexity and indicates higher energy consumption.

[0116] In a sixth aspect, the present invention relates to a method for preparing one or more peptides, the method comprising the following steps:

[0117] Provides plant protein substrates,

[0118] Fermentation of plant protein substrates using starter cultures containing Lactobacillus johnsonii.

[0119] Separate one or more peptides,

[0120] Optionally, one or more peptides are purified.

[0121] One or more of the peptides are selected from the list consisting of: a peptide having the amino acid sequence of SEQ ID NO: 4, a peptide having the amino acid sequence of SEQ ID NO: 5, a peptide having the amino acid sequence of SEQ ID NO: 6, a peptide having the amino acid sequence of SEQ ID NO: 7, a peptide having the amino acid sequence of SEQ ID NO: 8, a peptide having the amino acid sequence of SEQ ID NO: 9, a peptide having the amino acid sequence of SEQ ID NO: 10, a peptide having the amino acid sequence of SEQ ID NO: 11, or a combination thereof.

[0122] The plant protein substrate, plant protein, starter culture, Lactobacillus johnsonii, and fermentation steps are as provided in the first aspect of the invention.

[0123] In some implementations, the peptides listed above may be derived from plants, preferably from plants other than soybeans and / or cereals.

[0124] Advantageously, the peptides listed above can be derived from legumes. Legumes are selected from the list consisting of: peas, adzuki beans, broad beans, white kidney beans, flavour beans, navy beans, red beans, black beans, pinto beans, kidney beans, lima beans, mung beans, chickpeas, fava beans, lentils, or combinations thereof. More preferably, peas are a preferred legume.

[0125] The separation and purification of peptides can be accomplished using methods well known to those skilled in the art.

[0126] In some preferred embodiments, after separation, the one or more peptides are not combined with one or more reducing sugars, particularly one or more added reducing sugars. For example, the one or more reducing sugars, particularly one or more added reducing sugars, may be selected from xylose, arabinose, ribose, glucose, galactose, and mixtures thereof. In some embodiments, the one or more peptides do not undergo the Maillard reaction. In the context of this invention, the Maillard reaction is undesirable because it requires the addition of reducing sugars and necessitates harsh heat treatment at high temperatures for extended periods, which may generate undesirable flavor compounds as reaction products. Furthermore, such harsh heat treatment may lead to other undesirable consequences: loss of compounds of interest, protein aggregation, protein denaturation, etc.

[0127] In a seventh aspect, the present invention relates to a composition comprising one or more peptides, wherein the one or more peptides are selected from the list consisting of: a peptide having the amino acid sequence of SEQ ID NO: 4, a peptide having the amino acid sequence of SEQ ID NO: 5, a peptide having the amino acid sequence of SEQ ID NO: 6, a peptide having the amino acid sequence of SEQ ID NO: 7, a peptide having the amino acid sequence of SEQ ID NO: 8, a peptide having the amino acid sequence of SEQ ID NO: 9, a peptide having the amino acid sequence of SEQ ID NO: 10, a peptide having the amino acid sequence of SEQ ID NO: 11, or combinations thereof.

[0128] This composition has a pleasant, significant, and enhanced umami flavor, while exhibiting limited bitterness. The composition can also be added to products, particularly food products, to impart and / or enhance umami flavor.

[0129] In some implementations, the peptides listed above may be derived from plants, preferably from plants other than soybeans and / or cereals.

[0130] Advantageously, the peptides listed above can be derived from legumes. Legumes are selected from the list consisting of: peas, adzuki beans, broad beans, white kidney beans, flavour beans, navy beans, red beans, black beans, pinto beans, kidney beans, lima beans, mung beans, chickpeas, fava beans, lentils, or combinations thereof. More preferably, peas are a preferred legume.

[0131] In particular, one or more peptides are added peptides, meaning they are added to the composition and are not inherently present in the composition.

[0132] In some embodiments, one or more peptides, particularly one or more added peptides, are isolated and / or purified, i.e., they are added in isolated and / or purified form.

[0133] In some embodiments, the composition is an oral composition, preferably a food composition, a beverage composition, or a nutritional composition.

[0134] In some implementations, the composition is not fermented.

[0135] In some embodiments, the composition is pectin-free, particularly high-methoxyl pectin.

[0136] In some preferred embodiments, the composition does not contain one or more added reducing sugars. For example, one or more added reducing sugars may be selected from xylose, arabinose, ribose, glucose, galactose, and mixtures thereof. In some embodiments, one or more peptides of the composition have not undergone the Maillard reaction. In the context of this invention, the Maillard reaction is undesirable because it requires the addition of additional reducing sugars and necessitates harsh heat treatment at high temperatures for extended periods, which may generate undesirable flavor compounds as reaction products. Furthermore, such harsh heat treatment may lead to other undesirable consequences: loss of compounds of interest, protein aggregation, protein denaturation, etc.

[0137] In an eighth aspect, the present invention relates to the use of one or more peptides as flavoring agents, wherein the one or more peptides are selected from the list consisting of: peptides having the amino acid sequence of SEQ ID NO: 4, peptides having the amino acid sequence of SEQ ID NO: 5, peptides having the amino acid sequence of SEQ ID NO: 6, peptides having the amino acid sequence of SEQ ID NO: 7, peptides having the amino acid sequence of SEQ ID NO: 8, peptides having the amino acid sequence of SEQ ID NO: 9, peptides having the amino acid sequence of SEQ ID NO: 10, peptides having the amino acid sequence of SEQ ID NO: 11, or combinations thereof.

[0138] In some implementations, the peptides listed above may be derived from plants, preferably from plants other than soybeans and / or cereals.

[0139] Advantageously, the peptides listed above can be derived from legumes. Legumes are selected from the list consisting of: peas, adzuki beans, broad beans, white kidney beans, flavour beans, navy beans, red beans, black beans, pinto beans, kidney beans, lima beans, mung beans, chickpeas, fava beans, lentils, or combinations thereof. More preferably, peas are a preferred legume.

[0140] In particular, these peptides can be used as umami flavoring agents, umami flavor enhancers, and / or flavor masking agents.

[0141] In some preferred embodiments, the one or more peptides are not combined with one or more reducing sugars, particularly one or more added reducing sugars. For example, the one or more reducing sugars, particularly one or more added reducing sugars, may be selected from xylose, arabinose, ribose, glucose, galactose, and mixtures thereof. In some embodiments, the one or more peptides of the composition have not undergone the Maillard reaction. In the context of this invention, the Maillard reaction is undesirable because it requires the addition of additional reducing sugars and necessitates harsh heat treatment at high temperatures for extended periods, which may generate undesirable flavor compounds as reaction products. Furthermore, such harsh heat treatment may lead to other undesirable consequences: loss of compounds of interest, protein aggregation, protein denaturation, etc.

[0142] In a ninth aspect, the present invention relates to the use of the composition of the seventh aspect of the invention as a flavoring agent. In particular, it can be used as a umami flavoring agent, a umami flavor enhancer, and / or a flavor masking agent.

[0143] protein sequence

[0144] •SEQ ID NO: 1 (Length: 298)

[0145] MKRIIKVGALAASSFSLLFLAGCSSNNKEVAQYGDKKISQEQFYQELNSSPTSKTILANMLIYGALKEAYGSKLNKDQVEADYNSYKKRYGSQFDSFLKENSYTKKSFKQMIELNHLSKIALKKQMKPTTSQLKEEWKDYQPKITVQHISTTSRDTANTVIEKLNNGESFASLASKYSVDSLTSSNGGKLPAFDMQNRRYDSTFKKGAYKLKNDEYTKEPIKVTNGYEVIKMINHPKKGNFADKQKELTEAIYNKWASNSRIMNNVISQVLKEQHVSIKDKDLKSALDMYKGSNKANF

[0146] ·SEQ ID NO: 2 (Length: 2209)

[0147]

[0148] •SEQ ID NO: 3 (Length: 584)

[0149] MKMSKSKLLYVTLASSLTIFATACGAKKTDSSNQTIKFSQNVPKKAIKKGGTLTYALENESPFTGIFLSEIADTSPDVEAAAPGDESLFSINDQYQITDKGAATLKLNHHDNTAKIKVKDKVRWSDGKPVVAKDLEYSYEILANPK VQTTQYTSSLENIKGMAEYHQGKAKEISGIEMPDGPNGKTLILHFKELKPAMLNAASGFYWEHAAPYHYLKKVPFEKLVSSDQVRKHPLYFGPYKMDKTVQGQSTSWSRNPYYWRGKPNFAHIYMSNITNSNVSQAIKSHKFDVAS VLDSQWLQVKNTKGVNFVGKKTLSYNYLAFKVGKWDQKLGKNVENPHAKMNNPALRKAMAYAMNVDVINNRFYHGLKFRVNSLIPSQFTPYYDKNIPTYSYNLNKANELLDKAGYKKAPGALYRRQPNGKPLVINIAVRGSGENSE AIWRNYIQQWKKAGLNVKFLGGRPMEFNRWVAAVKSSDPKIDVLEGSWGAAGDPSPSVFYGEKMPYNFARFVSPTNTKLLDEIDSAKSFDKNYRVQKFHEWQRWMYNKAYVVPTSGAYSVTAVNSKVTGWSLKPSANVWYEAGFSK

[0150] peptide sequence

[0151]

[0152] Those skilled in the art will understand that they are free to combine all the features of the invention disclosed herein. In particular, features described for products of the invention can be combined with uses or methods of the invention, and vice versa. Furthermore, features described for different embodiments of the invention can be combined.

[0153] Furthermore, if known equivalents exist for a specific feature, such equivalents should be incorporated as expressly mentioned in this specification. Further advantages and features of the invention will become apparent upon reference to the accompanying drawings and non-limiting embodiments.

[0154] Example

[0155] Example 1: Materials and Methods

[0156] Materials and Methods 1: Preparation of Fermented Plant-Based Milk

[0157] Preparation of fermented plant-based milk substitutes.

[0158] To this end, a 200ml emulsion of 10% pea protein was first prepared by mixing pea protein isolate (85% pea protein) with water.

[0159] Different emulsions were inoculated with a starter culture containing one of the strains listed in Table 1, and fermented with the starter culture for 48 hours under the conditions provided in Table 1 and with a stirring speed of 120 rpm to obtain fermented plant-based milk substitutes. All fermentations were carried out in 500 ml sterile Erlenmeyer flasks using laminar flow and sterile pipettes. For Bifidobacterium, an anaerobic incubator was used for the fermentation step, and the oxygen level was set to 0.5%.

[0160]

[0161] Table 1

[0162] Materials and Methods 2: Preparation of Reference Unfermented Plant-Based Milk Substitutes

[0163] The following is a reference unfermented plant-based milk substitute prepared as follows: Pea protein isolate (85% pea protein) was mixed with water to obtain 200 ml of an emulsion containing 10% pea protein. This emulsion corresponds to the reference unfermented plant-based milk substitute.

[0164] Materials and Methods 3: Sensory Analysis

[0165] The sensory evaluation of different samples was conducted by a trained panel of experts who assess the taste of plant-based milk.

[0166] Sensory evaluations were conducted on different days and through different sensory stages to avoid sensory fatigue. Additionally, bottled water was provided during sensory analysis to serve as mouthwash at the start of sensory evaluations and between test samples.

[0167] Specifically, 5 ml aliquots of the unfermented plant-based milk substitute from Material and Method 2 were presented to the panel along with the same aliquots of the fermented plant-based milk substitute from Material and Method 1. One unfermented sample (reference) and two fermented samples were evaluated at each sensory stage, for a total of three samples.

[0168] Throughout the testing period, both unfermented and fermented plant-based milk substitutes were stirred to prevent sedimentation.

[0169] For sensory evaluation, the panel was asked to assess the perception of umami or bitterness by providing a score (= sensory score) on a scale of 0 to 5 for each sample.

[0170] Materials and Methods 4: Solid-phase extraction fractionation of plant-based milk substitutes to obtain SPE fractions containing metabolites, including umami-contributing and / or enhancing peptides.

[0171] Solvent extraction was performed on both fermented plant-based milk substitutes prepared with different strains from Material and Method 1 and a reference unfermented plant-based milk substitute from Material and Method 2. The extraction protocol involved stirring 340 g of dried protein three times with a mixture of MeOH and H₂O (1+1, v+v = 1400 mL) at room temperature for 30 minutes, followed by filtration and centrifugation using a Buchner funnel (Rotilabo, 185 mm, Type 111A, Carl Roth GmbH + Co.KG, Karlsruhe, Germany). The filtrates were combined, the solvent was removed under vacuum at 40 °C, and the fractions were lyophilized to obtain the SPE fraction containing extractable metabolites. Regarding the fractionation procedure, an aliquot (1 g) of the active fraction was dissolved in water (50 mL) and applied to a Chromabond C18 ec polypropylene column (Macherey-Nagel, Düren, Germany) pre-conditioned with methanol (70 mL) followed by water (70 mL). Stepwise elution with water (75 mL) yielded a “polar” fraction, followed by stepwise elution with water (100%, v / v, 75 mL) yielded a “medium polar” fraction, and stepwise elution with methanol / water (50:50, v / v, 75 mL) yielded a nonpolar fraction, followed by stepwise elution with methanol / water (100%, v / v, 75 mL). The solvent was removed from the collected fractions by vacuum evaporation at 40 °C, the fractions were dissolved in water, lyophilized twice, and maintained at -20 °C until used for sensory analysis. The SPE fractionation procedure was taken from Hald et al., 2019.

[0172] Materials and Methods 5: MaxQuant Peptide Analysis (Computerized Peptide Identification)

[0173] Metabolites (including peptides) were separated and detected from the SPE fractions of materials and methods 4 obtained from fermented plant-based milk substitutes prepared with Lactobacillus johnsonii NCC533, NCC1584, NCC1657, NCC1680, and NCC2680, respectively, and from a reference unfermented plant-based milk substitute, using ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UPLC-ToF-MS). All measurements were obtained on a Sciex TripleTOF 6600 mass spectrometer (Sciex Darmstadt, Germany), which was connected to a Shimadzu Nexera X2 system (Shimadzu, Kyoto, Japan) operating in positive and negative electrospray ionization (ESI) modes. The ion spray voltage was set to 5500 eV; the source temperature was 550°C; the atomizing gas was 0.38 MPa; and the heating gas was 0.45 MPa. Metabolite (including peptide) separation was performed on two columns in separate and continuous runs: 1) a 150 × 2 mm, 1.7 µm Kinetex C18 column (Phenomenex, Aschaffenburg, Germany) with a gradient of 0.1% formic acid in water (A) and acetonitrile containing 0.1% formic acid (B) at a flow rate of 0.3 mL / min, with the following gradients: 0 min, 5% B; 2 min, 5% B; 18 min, 100% B; 21 min, 100% B; 22 min, 5% B; 25 min, 5% B; and 2) Acquity BEH amide (water) with a gradient of 5 mM NH4Ac in H2O, pH 3; B: 5 mM NH4Ac, 2% H2O in ACN, pH 3, with a gradient of 0 min, 95% B; 2 min, 95% B; 10 min, 50% B. B; 12 min, 0% B; 15 min, 0% B; 15.50 min, 95% B; 20 min, 95% B. The column oven was set to 40°C, and for C18 runs, TOF-MS scans were performed from m / z 50 to m / z 1500, while for HILIC chromatography, TOF-MS scans were performed from m / z 50 to m / z 1000. MS2 data were acquired using both data-dependent acquisition (IDA mode) and data-independent acquisition (SWATH mode).

[0174] For peptide identification, Maxquant software was used to process .wiff files (version 1.6.6.0) and genome-based sequences of pea storage proteins obtained from the UniProt database. Processing parameters were primarily set to default values. Due to the unpredictability of the wide range of fermenting agents and bacterial protease / peptidase production, non-specific searches were employed during this study. Among the parameters changed: minimum peptide length was set to 3, modifications included oxidation and acetyl groups (N-terminus of the protein), maximum peptide mass was set to 4600 Daltons, minimum peptide length for non-specific searches was set to 3, and maximum peptide length for non-specific searches was set to 25, and mass spectrometry was set to "Sciex qTOF". Protein FASTA files used for peptide searches were obtained from the UniProt database ("UniProt: A Universal Protein Knowledge Base for 2021", 2021). Protein / peptide searches were performed by searching the database for "pea (Pisum sativum)" and by downloading the FASTA file of the most abundant proteins from this organism found in the literature (Gueguen and Barbot, 1988, "Quantitative and qualitative variability of pea (Pisum sativum L.) protein composition").

[0175] During processing, the SPE fractions of materials and methods 4 were filtered based on sequences from milk substitute samples fermented with Lactobacillus johnsonii NCC533, NCC1584, NCC1657, NCC1680 and NCC2680, respectively, and these sequences were not present in the SPE fractions of materials and methods 4 obtained from reference unfermented milk substitute samples.

[0176] Materials and Methods 6: Umami Prediction Tool: iUmami-SCM

[0177] Umami peptides were identified and characterized using a sequence-based predictor called iUmami-SCM. This tool is publicly available at http: / / camt.pythonanywhere.com / iUmami-SCM. The Scorecard method (SCM) is the methodology behind the propensity scoring tool used to evaluate peptides. The range of peptides identified in a sample was from 20-30 peptides (for low-protein hydrolysis starter cultures) to up to 200-500 peptides (in high-protein hydrolysis starter cultures), such as NCC533 in pea protein isolate solution fermented for 48 hours. The strongest peptides present in the evidence files of these strains were collected and submitted to the iUmamiSCM software to obtain a possible prediction of whether these molecules are likely to have human taste activity, particularly potential umami activity. Peptides with an iUmami SCM score higher than 590 were considered to have significant umami activity.

[0178] Materials and Methods 7: Peptide Quantification via SWATH

[0179] The quantification of peptides using the SWATH method in different fermented plant-based milk substitute samples (refer to Example 5) is based on the work published by Sepalde and colleagues, “Mapping Taste-Relevant Food Peptidomesby Means of Sequential Window Acquisition of All Theoretical Fragment Ion-Mass Spectrometry”, J. Agric. Food Chem. 2020, 68 (38), 10287–10298.

[0180] UHPLC-ToF-MS measurements were obtained on a TripleTOF 6600 mass spectrometer (Sciex GmbH, Darmstadt, Germany), connected to a Shimadzu Nexera X2 system (Shimadzu Corporation, Kyoto, Japan) operating in positive ESI mode. The ion spray voltage was set to 5500 eV; the source temperature to 550 °C; the nebulizing gas to 55 psi; the heating gas to 65 psi; and nitrogen to 35 psi as the curtain gas to effectively desolvate the ions. Aliquots (10 µL) of the prepared sample were injected into a UHPLC-ToF-MS system connected to a 150 × 2 mm, 1.7 µm Kinetex C18 column (Phenomena GmbH, Aschaffenburg, Germany) equipped with the same type of guard column. Eluent A consisted of 1% formic acid in water, and eluent B consisted of 1% formic acid in acetonitrile. Chromatography was performed at a flow rate of 0.3 mL / min, starting with the following intervals: 0–0.5 min, 5% B; 14 min, 40% B; 15 min, 100% B; 17 min, 5% B; and 20 min, 5% B. In SWATH mode, a full-spectrum ToF-MS scan was acquired (m / z 100–1000, 150 ms), followed by 20 product ion scans with a variable Q1 isolation window from m / z 200 to 1000, overlapping at 1 Da: m / z. 199.5−256.7, 255.7−296.6, 295.6−326.7, 325.7−353.0, 352.0−378.2, 377.2−402.5, 401.5−429.8, 428.8−455.0, 454.0−483.4, 482.4−514.4, 513.4−543.7, 542.7−578.3, 577.3−615.3, 614.3−659.0, 658.5−703.9, 702.9−756.2, 755.2−812.8, 811.8−870.8, 869.8−939.1 and 938.1−1000.5. Product ion spectra were accumulated in high-resolution mode for 50 ms using DP at 80 V, CE at 35 V, and CES at 15 V. Quantification was performed using external standard calibration, with standard solutions starting at 0.5 mmol / L for each peptide and diluted 16-fold 1:1 in a 50:50 MeOH: Millipore solution. System control and data collection were performed using AnalystTF 1.7.1 (Sciex GmbH, Darmstadt, Germany). Data analysis was performed using MultiQuant 3.0.2 (Sciex GmbH, Darmstadt, Germany).

[0181] Materials and Methods 8: Genomic-peptidomic analysis of Lactobacillus species

[0182] The list of GenInfo identifiers (GI) for proteolysis-related genes was obtained from supplemental materials in the following literature: Liu et al., “The proteolytic system of lactic acid bacteria revisited: a genomic comparison”, BMC Genomics 2010, 11:36. Since GI numbers are not consecutive, they were searched in UniParc (https: / / www.uniprot.org / uniparc / ), then in UniProt (https: / / www.uniprot.org / ), and the protein sequences in FASTA format were downloaded. GI numbers are often associated with more than one UniProt accession number, which may generate redundant mappings via BLAST search.

[0183] The strain genome was used as the target for the downloaded protein query, with a sequence having an e-value of 0.001 (best hit). BLAST results were filtered to hits with at least 80% query coverage. A heatmap was constructed using query ID and identity percentage. Grouping of genomes (vertical axis) and proteins (horizontal axis) was performed using hierarchical clustering. The clustering of genomes reflects gene-by-gene similarity, rather than overall genomic differences.

[0184] A BLAST search was performed on proteins from the *Lactobacillus johnsonii* proteolytic system using a nonredundant protein database (https: / / www.ncbi.nlm.nih.gov / refseq / about / nonredundantproteins / ), and the results were filtered to extract accession numbers for "multi-species" Lactobacillus, corresponding to potential homologs across the entire genus. Protein sequences from the retrieved accession numbers were downloaded from the batch Entrez database (https: / / www.ncbi.nlm.nih.gov / sites / batchentrez), and the genomes of *Lactobacillus johnsonii*, *Lactobacillus fermentum*, and *Lactobacillus rhamnosus* were queried. *Lactococcus lactis*, *Lactococcus faecium* subsp. *lactococcus*, and *Lactococcus lactis* subsp. *lactococcus* were compared with *Lactobacillus johnsonii*, as reported in the following literature review regarding the search for proteolytic systems of *Lactococcus lactis*: Liu et al., BMCGenomics 2010, 11:36.

[0185] NCBI accession numbers for proteolysis-related genes were extracted from the following literature: Broadbent et al., J., “Genetic diversity in proteolytic enzymes and amino acid metabolism among Lactobacillus helveticus strains”, Dairy Sci. 2011.94:4313–4328 and downloaded from the Entrez batch. Strains of the Lactobacillus helveticus genome were used as objects for proteins downloaded via BLAST query. The results were filtered as described above, and non-clustered heatmaps were generated. Peptidome data were used for correlation with the BLAST data.

[0186] Example 2: Sensory evaluation of fermented plant-based milk substitutes prepared with strains of different genera and species, including umami flavor.

[0187] Different fermented plant-based milk substitute samples were prepared using NCC533, NCC2705, NCC660, NCC2378, NCC4007 or NCC3059, respectively, according to the method provided in Materials and Methods 1 of Example 1.

[0188] A reference unfermented plant-based milk substitute sample (hereinafter referred to as the reference sample) was also prepared according to the materials and methods of Example 1.

[0189] Based on the materials and methods of Example 1, samples of different fermented plant-based milk substitutes and reference samples were evaluated by sensory analysis.

[0190] Results are provided Figure 1 In the middle. Reference sample in Figure 1 The protein isolate is labeled as unfermented pea protein.

[0191] exist Figure 1 It can be observed that Lactobacillus johnsonii, especially Lactobacillus johnsonii NCC533, obtained the highest sensory score for umami, and therefore the highest umami flavor. The sensory score for umami, and thus the umami flavor, was significantly lower for other starter cultures.

[0192] In particular, Lactobacillus johnsonii, especially Lactobacillus johnsonii NCC533, allows for the highest enhancement of umami flavor compared to the reference sample (reference). Figure 2 ).

[0193] These data indicate that Lactobacillus johnsonii significantly enhances the umami flavor of fermented plant-based products through fermentation.

[0194] Example 3: Counting of umami peptides in fermented plant-based milk substitutes prepared with different Lactobacillus johnsonii.

[0195] After identifying the ability of Lactobacillus johnsonii NCC 533 to generate umami flavor in Example 2, the ability of four other Lactobacillus johnsonii strains to generate umami peptides was tested.

[0196] Fermented plant-based milk substitute samples were prepared using NCC533, NCC1584, NCC1657, NCC1680, and NCC2680, according to the method provided in Materials and Methods 1 of Example 1.

[0197] A reference unfermented plant-based milk substitute sample (hereinafter referred to as the reference sample) was also prepared according to the materials and methods of Example 1.

[0198] The formation of umami peptides was assessed by measuring the umami peptide count. The umami peptide count corresponds to the number of different peptides with umami activity identified in the sample. In the SPE fractions of fermented and unfermented samples obtained according to the materials and methods of Example 1, peptides were identified using the materials and methods of Example 1, and their tendency to impart / improve umami was assessed using the materials and methods of Example 1, and their tendency to impart / improve umami was evaluated using the materials and methods of Example 1, and their tendency to impart / improve umami.

[0199] Results are provided Figure 2 In the middle. Reference sample in Figure 2 The protein isolate is labeled as unfermented pea protein.

[0200] It can be seen that different strains of Lactobacillus johnsonii exhibit different abilities to produce umami peptides and thus umami flavors.

[0201] In particular, only Lactobacillus johnsonii NCC533, NCC1657, and NCC2680 showed increased umami peptide production during fermentation compared to the reference. NCC533 obtained the highest peptide count.

[0202] The samples were tasted by a panel. Compared to the reference in the oral cavity, the samples fermented with Lactobacillus johnsonii NCC533, NCC1657, and NCC2680 showed an increased umami flavor. Furthermore, those samples exhibited limited bitterness. No increase in bitterness was perceived compared to the reference.

[0203] Example 4: Identification of peptides that impart and / or enhance umami flavor

[0204] As disclosed above, Lactobacillus johnsonii NCC533 has been shown to impart the highest umami flavor. To further investigate umami flavor generation, the potential of peptides generated by Lactobacillus johnsonii NCC533 to impart and / or enhance umami flavor was evaluated.

[0205] To conduct this evaluation, peptides generated by *Lactobacillus johnsonii* NCC533 were first identified in an SPE fraction prepared according to the materials and methods of Example 1, method 5, using materials and methods 4 of Example 1. This SPE fraction was prepared from a sample of Example 3 fermented with *Lactobacillus johnsonii* NCC533. The potential of the identified peptides to impart and / or enhance umami flavor was then evaluated according to the materials and methods of Example 1, method 6.

[0206] The predictive tool identified eight novel peptides (listed in Table 2) with high iUmami SCM scores in samples fermented with Lactobacillus johnsonii NCC533. This suggests that these peptides contribute to and / or enhance the umami flavor in the fermented samples.

[0207]

[0208] Table 2

[0209] It is believed that Lactobacillus johnsonii, particularly Lactobacillus johnsonii NCC533, has proteolytic activity during plant protein fermentation that tends to generate peptides that significantly impart and / or enhance umami flavor, including those listed in Table 2.

[0210] Example 5: Development of peptides that impart and / or enhance umami flavor over fermentation time

[0211] As fermentation time progresses, the production of some peptides in Table 2 that impart and / or enhance umami flavor is assessed.

[0212] Different plant-based milk substitute samples were prepared using NCC533 according to the methods provided in Material and Method 1 of Example 1, but with different fermentation times: 0 hours, 24 hours and 48 hours.

[0213] According to the materials and methods of Example 1, the peptides listed in Table 3 were quantified in different milk substitute samples. The results are provided in Table 3.

[0214]

[0215] Table 3

[0216] It can be observed that the amount of peptides that impart and / or enhance umami increases with fermentation time.

[0217] Example 6: Genes encoding proteolytic proteins in Lactobacillus strains that impart and / or enhance umami flavor

[0218] The proteolytic genes of *Lactobacillus johnsonii* NCC533, NCC1657, and NCC2680 were compared with the proteolytic genes of *Lactobacillus johnsonii* (i.e., *Lactobacillus johnsonii* NCC1584 and NCC1585), which impart a limited umami flavor. The genes were compared according to the materials and methods of Example 1.

[0219] In this study, genes encoding the proteolytic proteins listed in Table 4 were considered.

[0220]

[0221] Table 4

[0222] Compared to Lactobacillus johnsonii NCC533, NCC1657, and NCC2680, Lactobacillus johnsonii NCC1584 and NCC1585 lack the cell wall-binding protease PrtP. Furthermore, compared to NCC1657 and NCC2680, Lactobacillus johnsonii NCC1584 and NCC1585 exhibit highly dissimilar cell wall-binding protease PrtM and oligopeptide permease OppA (less than 50% identity).

[0223] No major differences were observed for the other proteolytic proteins listed in Table 4.

[0224] Therefore, based on the above, it is believed that PrtM, PrtP, and OppA contribute to the proteolysis of plant proteins to produce peptides that impart / enhance umami flavor.

[0225] Although the invention has been described by way of example, it should be understood that variations and modifications may be made without departing from the scope of the invention as defined in the claims.

Claims

1. A method for preparing a fermented plant-based product, the method comprising providing a plant protein substrate and fermenting the plant protein substrate with a starter culture containing Lactobacillus johnsonii.

2. The method according to claim 1, wherein the plant protein substrate is a legume protein, preferably pea protein.

3. The method according to claim 1 or 2, wherein the plant protein substrate is a plant protein suspension.

4. The method according to any one of the preceding claims, wherein the Lactobacillus johnsonii expresses protease PrtP and / or protease PrtM and / or oligopeptide permease OppA.

5. The method according to any one of the preceding claims, wherein the *Lactobacillus johnsonii* is selected from the list consisting of: *Lactobacillus johnsonii* deposited in CNCM with accession number CNCM I-5910, *Lactobacillus johnsonii* deposited in CNCM with accession number CNCM I-1225, *Lactobacillus johnsonii* deposited in CNCM with accession number CNCM I-2474, or a combination thereof.

6. The method according to any one of the preceding claims, wherein the fermentation step of the plant protein substrate with the starter culture lasts for at least 16 hours, preferably at least 18 hours.

7. The method according to any one of the preceding claims, wherein the fermented plant-based product is a fermented plant-based food or beverage, preferably a fermented plant-based dairy analog or a plant-based meat analog.

8. A fermented plant-based product comprising plant proteins fermented with Lactobacillus johnsonii.

9. The fermented plant-based product according to claim 8, wherein the plant protein is a legume protein, preferably pea protein.

10. The fermented plant-based product according to claim 8 or 9, wherein the fermented plant-based product comprises argininoyl peptide, prolyl peptide, and / or glutamyl peptide.

11. Lactobacillus johnsonii, which is deposited in CNCM with accession number CNCM I-5910.

12. Use of *Lactobacillus johnsonii* in the production of fermented plant-based products, wherein said *Lactobacillus johnsonii* is selected from the list consisting of: *Lactobacillus johnsonii* deposited in CNCM with accession number CNCM I-5910, *Lactobacillus johnsonii* deposited in CNCM with accession number CNCM I-1225, *Lactobacillus johnsonii* deposited in CNCM with accession number CNCM I-2474, or a combination thereof.

13. Peptides having the following amino acid sequence: SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11。 14. A method for preparing one or more peptides, the method comprising the following steps: Provides plant protein substrates, The plant protein substrate was fermented using a starter culture containing Lactobacillus johnsonii. Separate one or more peptides, The one or more peptides described herein are selected from the list consisting of: peptides having the amino acid sequence of SEQ ID NO: 4, peptides having the amino acid sequence of SEQ ID NO: 5, peptides having the amino acid sequence of SEQ ID NO: 6, peptides having the amino acid sequence of SEQ ID NO: 7, peptides having the amino acid sequence of SEQ ID NO: 8, peptides having the amino acid sequence of SEQ ID NO: 9, peptides having the amino acid sequence of SEQ ID NO: 10, peptides having the amino acid sequence of SEQ ID NO: 11, or combinations thereof.

15. A composition comprising one or more peptides, wherein the one or more peptides are selected from the list consisting of: a peptide having the amino acid sequence of SEQ ID NO: 4, a peptide having the amino acid sequence of SEQ ID NO: 5, a peptide having the amino acid sequence of SEQ ID NO: 6, a peptide having the amino acid sequence of SEQ ID NO: 7, a peptide having the amino acid sequence of SEQ ID NO: 8, a peptide having the amino acid sequence of SEQ ID NO: 9, a peptide having the amino acid sequence of SEQ ID NO: 10, a peptide having the amino acid sequence of SEQ ID NO: 11, or combinations thereof.

16. Use of one or more peptides as a flavoring agent, wherein said one or more peptides are selected from the list consisting of: a peptide having the amino acid sequence of SEQ ID NO: 4, a peptide having the amino acid sequence of SEQ ID NO: 5, a peptide having the amino acid sequence of SEQ ID NO: 6, a peptide having the amino acid sequence of SEQ ID NO: 7, a peptide having the amino acid sequence of SEQ ID NO: 8, a peptide having the amino acid sequence of SEQ ID NO: 9, a peptide having the amino acid sequence of SEQ ID NO: 10, a peptide having the amino acid sequence of SEQ ID NO: 11, or combinations thereof.

17. Use of the composition according to claim 15 as a flavoring agent.

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