METHODS OF PRODUCING UMAMI-FLAVORED FERMENTED PLANT-BASED PRODUCTS AND THEIR RELICS

Abstract

The invention relates to a method for producing an umami-flavored fermented plant-derived product. This method comprises a plant protein substrate supply step and a plant protein substrate fermentation step with a starter culture including Lactobacillus johnsonii. The invention relates to this fermented plant-derived product and the Lactobacillus johnsonii strains suitable for the invention. The invention also relates to peptides that confer and / or enhance umami flavor, a method for preparing such peptides, a composition containing such peptides, and the use of such peptides as flavoring agents.

Description

[0001] METHOD OF MAKING FERMENTED PLANT-BASED PRODUCTS HAVING UMAMI TASTE AND PRODUCTS THEREOF

[0002] TECHNICAL FIELD

[0003] The present invention relates generally to the field of fermentation of plant materials. In particular, the present invention relates to a method of making a fermented plant-based product having umami taste and fermented plant-based product thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Together with sweet, bitter, salty and sour, umami is one of the five basic tastes. Umami is a taste that spreads across and coats the tongue, lingers, and brings a mouthwatering sensation and a pleasant savory taste.

[0006] Umami imparting and umami enhancing substances, including peptides, are found in many animal foods like ham and cheese, as well as plant-based foods like soy sauce and sourdough bread.

[0007] Umami imparting and umami enhancing substances, in particular peptides, stimulate salivary secretion, appetite and increase food palatability. They are therefore valuable to improve the organoleptic quality of food.

[0008] Plant-based products are gaining increasing interest nowadays. Hence, it would be desirable to generate umami imparting and umami enhancing substances, in particular peptides, in plant-based products for offering an enhanced sensory experience in those products.

[0009] There are two ways to generate umami imparting and umami enhancing peptides.

[0010] On the one hand, these peptides may be produced by enzymatic hydrolysis. A variety of commercially available peptidase preparation were investigated for plant protein hydrolysis. For example, Flavourzyme (Novozyme) derived from Aspergillus niger, Alcalase from Bacillus subtilis, ProteAXK (AMANO) derived from Rhizopus oryzae, Umamyzyme from Aspergillus oryzae. However, the resulting hydrolysates generally exhibit unpleasant bitterness.

[0011] On the other hand, these peptides may be generated by fermentation. Such peptides are generally generated via process involving either more than one step or more than one strain. For example, Aspergillus, spp and lactic acid bacteria are needed to produce soy sauce, while fungal or malt protease and lactic acid baterial are needed for sourdough bread. It would therefore be desirable to provide a method for generating or enhancing umami taste in a plant-based product. It would desirable the method is natural and involves a limited number of steps and / or limited number of ingredients. It would also desirable that the method generates a plant-based product with limited bitterness.

[0012] It would also be desirable to provide molecules and compositions that can be used as flavouring agent, in particular to impart or enhance umami taste. It would be desirable that such molecules and compositions are derived from plant and are not artificial, i.e. not produced via chemical synthesis.

[0013] Any reference to prior art documents in this specification is not to be considered an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0014] SUMMARY OF THE INVENTION

[0015] The object of the present invention is to improve the state of the art, and in particular to provide methods, fermented plant-based products, Lactobacillus johnsonii strains, uses, composition and peptides that overcome the problems of the prior art and address the needs described above, or at least to provide a useful alternative.

[0016] The inventors were surprised to see that the object of the present invention could be achieved by the subject matter of the independent claims. The dependent claims further develop the idea of the present invention.

[0017] Accordingly, a first aspect of the invention of the invention proposes a method of making a fermented plant-based product, said method comprising providing a plant protein substrate, and fermenting the plant protein substrate with a starter culture comprising Lactobacillus johnsonii.

[0018] It has been discovered that the fermentation of plant proteins with Lactobacillus johnsonii effectively imparts and / or enhances umami taste in plant-based products while limiting generation of bitterness. The method of the invention is simple and involves a single step of fermentation.

[0019] A second aspect of the invention proposes a fermented plant-based product which comprises plant protein fermented with Lactobacillus johnsonii. The fermented plant-based product of the invention has a pleasant, significant, and enhanced umami taste while exhibiting limited bitterness.

[0020] A third aspect of the invention proposes Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-5910.

[0021] A fourth aspect of the invention proposes the use of Lactobacillus johnsonii in the production of a fermented plant-based product, wherein the Lactobacillus johnsonii is selected from the list consisting of Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-5910, Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-1225, Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-2474 or a combination thereof.

[0022] It has been discovered that the abovementioned Lactobacillus johnsonii strains effectively provide / enhance umami taste in plant-based products via fermentation of plant proteins.

[0023] A fifth aspect of the invention proposes the peptide which has 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.

[0024] A sixth aspect of the invention proposes a method for preparing one or more peptides comprising the step of: providing a plant protein substrate, fermenting the plant protein substrate with a starter culture comprising Lactobacillus johnsonii, isolating one or more peptides, wherein the one or more peptides are selected from the list consisting of peptide having the amino acid sequence of SEQ ID NO: 4, peptide having the amino acid sequence of SEQ ID NO: 5, peptide having the amino acid sequence of SEQ ID NO: 6, peptide having the amino acid sequence of SEQ ID NO: 7, peptide having the amino acid sequence of SEQ ID NO: 8, peptide having the amino acid sequence of SEQ ID NO: 9, peptide having the amino acid sequence of SEQ ID NO: 10, peptide having the amino acid sequence of SEQ ID NO: 11 or combination thereof.

[0025] A seventh aspect of the invention proposes a composition comprising one or more peptides, wherein the one or more peptides are selected from the list consisting of peptide having the amino acid sequence of SEQ ID NO: 4, peptide having the amino acid sequence of SEQ ID NO: 5, peptide having the amino acid sequence of SEQ ID NO: 6, peptide having the amino acid sequence of SEQ ID NO: 7, peptide having the amino acid sequence of SEQ ID NO: 8, peptide having the amino acid sequence of SEQ ID NO: 9, peptide having the amino acid sequence of SEQ ID NO: 10, peptide having the amino acid sequence of SEQ ID NO: 11 or a combination thereof.

[0026] An eighth aspect of the invention proposes the use of one or more peptides as flavouring agent, wherein the one or more peptides are selected from the list consisting of peptide having the amino acid sequence of SEQ ID NO: 4, peptide having the amino acid sequence of SEQ ID NO: 5, peptide having the amino acid sequence of SEQ ID NO: 6, peptide having the amino acid sequence of SEQ ID NO: 7, peptide having the amino acid sequence of SEQ ID NO: 8, peptide having the amino acid sequence of SEQ ID NO: 9, peptide having the amino acid sequence of SEQ ID NO: 10, peptide having the amino acid sequence of SEQ ID NO: 11 or a combination thereof.

[0027] A ninth aspect of the invention proposes the use of the composition of the seventh aspect of the invention as flavouring agent.

[0028] It has been discovered that the abovementioned peptides and composition comprising one or more of these peptides effectively impart and / or enhance umami taste in a product. These peptides or compositions may be used as umami flavour, umami taste enhancer and / or flavor masking agent.

[0029] These and other aspects, features and advantages of the invention will become more apparent to those skilled in the art from the detailed description of embodiments of the invention, in connection with the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 shows the sensory score for umami of fermented plant-based milk prepared with pea proteins and fermented with different starter cultures versus the sensory score for umami of the reference unfermented plant-based milk prepared with pea proteins (labelled unfermented pea protein isolate).

[0031] Figure 2 shows the umami peptide counts of the reference unfermented plant-based milk prepared with pea proteins (labelled unfermented pea protein isolate) and the umami peptide counts of fermented plant-based milks prepared with pea proteins and fermented with different Lactobacillus johnsonii strains.

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] As used in the specification, the words "comprise", "comprising" and the like are to be construed in an inclusive sense, that is to say, in the sense of "including, but not limited to", as opposed to an exclusive or exhaustive sense.

[0034] All numerical ranges should be understood to include each whole integer within the range.

[0035] As used in the specification, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0036] As used in the specification, the term "substantially free" or "substantially devoid of" means that no more than 10 weight percent, preferably no more than 5 weight percent, and more preferably no more than 1 weight percent of the excluded material is present. In a preferred embodiment, "substantially free" or "substantially devoid of" means that no more than 0.1 weight percent of the excluded material remains. "Entirely free" or "entirely devoid of" typically means that at most only trace amount of the excluded material is present, and preferably, no detectable amount is present.

[0037] Unless noted otherwise, all percentages in the specification refer to weight percent, where applicable.

[0038] Unless defined otherwise, all technical and scientific terms have and should be given the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0039] As used herein, the term "aqueous liquid" refers to a liquid comprising water.

[0040] Preferably, it refers to a liquid comprising at least 50%, more preferably at least 70wt.% water, more preferably at least 80wt% water, or even more preferably at least 90wt% water. Most preferably, it refers to a liquid consisting of water.

[0041] As used herein, the term "vegan" refers to an edible composition or product which is entirely devoid of animal products, or animal derived products.

[0042] As used herein, the term "vegetarian" refers to an edible composition or product which is devoid of meat, including fish.

[0043] As used herein, the term "plant flour" refers to a plant composition comprising a plant protein content from 5% to 49.9% proteins.

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

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

[0046] As used herein, the term "arginyl peptide" refers to a peptide with an amino acid sequence that comprises at least one arginine residue. In particular, the at least one arginine residue is accessible to umami taste receptors in the mouth, such as GluR4, mGluRl, taste receptor type 1 (TAS1R1 + TAS1R3) when the peptide is in its three-dimensional structure.

[0047] As used herein, the term "prolyl peptide" refers to a peptide with an amino acid sequence that comprises at least one proline residue. In particular, the at least one proline residue is accessible to umami taste receptors in the mouth, such as GluR4, mGluRl, taste receptor type 1 (TAS1R1 + TAS1R3) when the peptide is in its three-dimensional structure.

[0048] As used herein, the term "glutamyl peptide" refers to a peptide with an amino acid sequence that comprises at least one glutamine residue. In particular, the at least one glutamine residue is accessible to umami taste receptors in the mouth, such as GluR4, mGluRl, taste receptor type 1 (TAS1R1 + TAS1R3) when the peptide is in its three-dimensional structure.

[0049] For sake of clarity, in the context of the invention, "arginyl peptides", "prolyl peptides" and / or "glutamyl peptides" are not Maillard reaction products, in particular Maillard-reacted peptides. Likewise, for sake of clarity, in any of the relevant aspects of the invention (i.e. all aspects of the invention except the third aspect), any one of the peptides having amino sequence of SEQ ID NO:4, 5, 6, 7, 8, 9, 10 and 11 are not Maillard reaction products, in particular Maillard-reacted peptides. As used herein, the term "added reducing sugar" refers to a reducing sugar which is added and is not inherently present in the ingredients (e.g. plant protein substrate, plant protein) of the composition and the fermented-plant-based product of the invention.

[0050] As used herein, the term "high methoxyl pectin" is a pectin having a degree of esterification (DE) of at least 50%, preferably from 55% to 75%. The degree of esterification (DE) is defined as the number of methyl-esterified galacturonic acid units expressed as a percentage of the total galacturonic acid units in the pectin molecule.

[0051] In a first aspect, the invention relates to a method of making a fermented plant-based product. The fermented plant-based product may be fermented plant-based food or beverage. The fermented plant-based food or beverage may be selected from the list consisting of sauce, broth, dip, dressing, fermented dairy product analogue, meat analogue, pet food, confectionery, snack or soup. Preferably, the fermented plant-based food or beverage is vegetarian or vegan.

[0052] In a preferred embodiment, the fermented plant-based food or beverage is a fermented plant-based dairy product analogue or a meat analogue. In a more preferred, fermented plant-based food or beverage is a fermented plant-based dairy product analogue. The fermented plant-based dairy product analogue may be selected from the list consisting of cheese analogue, fresh cheese analogue, culinary cream analogue, cottage cheese analogue, yogurt analogue, kefir analogue, fermented milk analogue or a combination thereof. In a most preferred embodiment, the fermented plant-based food or beverage is a fermented plantbased beverage, in particular fermented plant-based dairy beverage analogue.

[0053] In some embodiment, the fermented plant-based product is free from pectin, in particular high methoxyl pectin.

[0054] The method comprises a 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 plant proteins into an aqueous liquid. In a preferred embodiment, the aqueous liquid is water.

[0055] In an embodiment, the plant protein substrate, preferably plant protein substrate comprises at least 0.5wt% plant protein, preferably 0.5 to 18wt%, more preferably 1.5 to 18wt% plant protein, more preferably 2 to 12wt%, even more preferably 5 to 12wt%. In an embodiment, the plant protein is different from soy protein and / or cereal protein.

[0056] Advantageously, the plant protein comprises, preferably consists of pulse protein. The pulse protein is selected from the list consisting of pea protein, adzuki bean protein, great northern bean protein, cannellini bean protein, fayot bean protein, navy bean protein, red bean protein, black bean protein, pinto bean protein, kidney bean protein, lima bean protein, mung bean protein, chickpea protein, faba bean protein, lentil protein or a combination thereof. More preferably, the plant protein comprises, preferably consists of pea protein.

[0057] The plant protein may be in the form of plant flour, plant protein concentrate, plant protein isolate or a combination thereof. Preferably, the plant protein is in the form of plant protein isolate, preferably pulse protein isolate, more preferably pea protein isolate.

[0058] The method further comprises a step of fermenting the plant protein substrate with a starter culture comprising Lactobacillus johnsonii.

[0059] It has been discovered that the fermentation of plant protein substrate with Lactobacillus johnsonii effectively imparts and / or enhances umami taste in plant-based products while limiting generation of bitterness. The method of the invention is simple and involves a single step of fermentation.

[0060] Advantageously, the starter culture consists only of Lactobacillus johnsonii. It has been observed that the umami taste generation / enhancement effect provided by the method of the invention can be achieved by fermenting the plant protein substrate with a single strain, in particular with Lactobacillus johnsonii only. In other words, the fermented plant-based product is free from bacteria other than Lactobacillus johnsonii, in particular other than Lactobacillus johnsonii as described below.

[0061] In a preferred embodiment, the Lactobacillus johnsonii expresses the proteinase PrtP and / orthe proteinase PrtM and / orthe oligopeptide permease OppA. The proteinase PrtP and the proteinase PrtM are cell-wall bound proteinases.

[0062] The proteinase PrtP has an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably 99% identity with SEQ ID NO:

[0063] 1. Most preferably, the proteinase PrtP has the amino acid sequence of SEQ ID NO: 1.

[0064] The proteinase PrtM has an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably 99% identity with SEQ ID NO:

[0065] 2. Most preferably, the proteinase PrtM has the amino acid sequence of SEQ ID NO: 2. The oligopeptide permease OppA has an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably 99% identity with SEQ ID NO: 3. Most preferably, the oligopeptide permease OppA has the amino acid sequence of SEQ ID NO: 3.

[0066] It has been discovered that Lactobacillus johnsonii strains which express proteinase PrtP and / or proteinase PrtM and / or oligopeptide permease OPPA effectively produce upon fermentation peptides that provide and / or enhance umami taste.

[0067] In particular, the Lactobacillus johnsonii may be selected from the list consisting of Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-5910, Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-1225, Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-2474 or a combination thereof.

[0068] These Lactobacillus johnsonii strains effectively provide / enhance umami taste in plantbased products via fermentation of plant proteins. In particular, it is believed that, upon fermentation, they hydrolyse plant proteins into peptides that impart and / or enhance umami taste.

[0069] Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-5910 was deposited by Societe des Produits Nestle S.A. with the accession number CNCM 1-5910 on 05 October 2022, with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France, under the Budapest Treaty.

[0070] Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-1225 was deposited by Nestec S.A. with the accession number CNCM 1-1225 on 30 June 1992, with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France, under the Budapest Treaty.

[0071] Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-2474 was deposited by Societe des Produits Nestle S.A. with the accession number CNCM 1-2474 on 9 May 2000, with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France, under the Budapest Treaty.

[0072] The applicant Societe des Produits Nestle S.A. (SPN) is the successor in title of the depositor Nestec S.A..

[0073] In a preferred embodiment, the fermentation step of the plant protein substrate with the starter culture lasts at least 16 hours, preferably at least 18 hours, more preferably at least 24 hours, even more preferably at least 35 hours, most preferably at least 40 hours. Without wishing to be bound by theory, it is believed that this minimal fermentation time ensures enough time for proteolytic machinery of Lactobacillus johnsonii to set in and to produce during fermentation sufficient level of peptides that impart and / or enhance umami taste in the plant-based product. In an embodiment, the fermentation step of the plant protein substrate with the starter culture lasts at most 50 hours, more preferably at most 48 hours.

[0074] In an embodiment, the fermentation step is performed at a temperature of 35 to 45°C

[0075] In another embodiment, the fermentation step is performed under stirring, preferably under stirring of 200 to 1200rpm.

[0076] The fermentation step may be performed under anaerobic or aerobic conditions, preferably under aerobic conditions.

[0077] The method of the invention allows the production of arginyl peptides, prolyl peptides and / or glutamyl peptides that impart and / or enhance umami taste. In particular, the fermented plant-based product comprises arginyl peptides, prolyl peptides and / or glutamyl peptides.

[0078] In a preferred embodiment, the arginyl peptides, prolyl peptides and / or glutamyl peptides consists of 2 to 20 amino acids, preferably 2 to 15 amino acids. Arginyl peptides, prolyl peptides and / or glutamyl peptides with limited amino acid sequence length are more prone to impart and / or enhance umami.

[0079] The fermented dairy product comprises elevated levels of such peptides. In a preferred embodiment, the concentration of arginyl peptides, prolyl peptides and / orglutamyl peptides in the fermented plant-based product is of at least 0.2, preferably at least 0.5, more preferably at least 1.0, even more preferably at least 1.5, most preferably 2.0 pmol per litre of fermented plant-based product.

[0080] In a preferred embodiment, the concentration of arginyl peptides, prolyl peptides and / or glutamyl peptides in the fermented plant-based product is of at most 100, preferably at most 50, more preferably at most 30, even more preferably at most 20, most preferably at most 19 pmol per litre of fermented plant-based product.

[0081] In some embodiment, the arginyl peptides, prolyl peptides and / or glutamyl peptides may be derived from plant, preferably from plant different from soy and / or cereal.

[0082] Advantageously, the arginyl peptides, prolyl peptides and / or glutamyl peptides may be derived from pulse. The pulse is selected from the list consisting of pea, adzuki bean, great northern bean, cannellini bean, fayot bean, navy bean, red bean, black bean, pinto bean, kidney bean, lima bean, mung bean, chickpea, faba bean, lentil or a combination thereof. More preferably, the pulse is pea.

[0083] It has been discovered that the method of the invention allows the production of new peptides that effectively impart or enhance umami taste. The fermented plant-based product comprises peptides selected from the list consisting of peptide having amino acid sequence of SEQ ID NO: 4, peptide having amino acid sequence of SEQ ID NO: 5, peptide having amino acid sequence of SEQ ID NO: 6, peptide having amino acid sequence of SEQ ID NO: 7, peptides having amino acid sequence of SEQ ID NO: 8, peptide having amino acid sequence of SEQ ID NO: 9, peptide having amino acid sequence of SEQ ID NO: 10, peptide having amino acid sequence of SEQ ID NO: 11 or a combination thereof.

[0084] In some embodiment, the peptides listed above may be derived from plant, preferably from plant different from soy and / or cereal.

[0085] Advantageously, the peptides listed above may be derived from pulse. The pulse is selected from the list consisting of pea, adzuki bean, great northern bean, cannellini bean, fayot bean, navy bean, red bean, black bean, pinto bean, kidney bean, lima bean, mung bean, chickpea, faba bean, lentil or a combination thereof. More preferably, the pulse is pea.

[0086] In an embodiment, the fermented plant-based product may comprise at least 0.2, preferably at least 0.5, more preferably at least 1.0, even more preferably at least 1.5, most preferably 2.0 pmol of peptides per litre of fermented plant-based product, wherein the peptides are selected from the list consisting of peptide having amino acid sequence of SEQ ID NO: 4, peptide having amino acid sequence of SEQ ID NO: 5, peptide having amino acid sequence of SEQ ID NO: 6, peptide having amino acid sequence of SEQ ID NO: 7, peptide having amino acid sequence of SEQ ID NO: 8, peptide having amino acid sequence of SEQ ID NO: 9, peptide having amino acid sequence of SEQ ID NO: 10, peptide having amino acid sequence of SEQ ID NO: 11 or a combination thereof.

[0087] In an embodiment, the fermented plant-based product may comprise at most 100, preferably at most 50, more preferably at most 30, even more preferably at most 20, most preferably at most 19 pmol of peptides per litre of fermented plant-based product, wherein the peptides are selected from the list consisting of peptide having amino acid sequence of SEQ ID NO: 4, peptide having amino acid sequence of SEQ ID NO: 5, peptide having amino acid sequence of SEQ ID NO: 6, peptide having amino acid sequence of SEQ ID NO: 7, peptide having amino acid sequence of SEQ ID NO: 8, peptide having amino acid sequence of SEQ ID NO: 9, peptide having amino acid sequence of SEQ ID NO: 10, peptide having amino acid sequence of SEQ ID NO: 11 or a combination thereof.

[0088] The fermented plant-based product has umami taste. The umami taste may be assessed by a panel trained to evaluate the umami taste of food products by providing a score on a scale from 0 (not detectable) to 5 (strongly detectable). An example of method to assess umami taste is provided in the present examples.

[0089] The plant-based product is substantially free, preferably free from added sweetening ingredient. By "added sweetening ingredient", it refers to exogenous ingredients that impart sweet taste when added to a composition or a product, e.g., sucrose, honey, fruit. The term "exogenous" excludes sweetening ingredients which are inherently present in the plant protein substrate, preferably in the plant protein and the aqueous liquid of the plant protein suspension. In particular, the plant-based product is substantially free, preferably free from added sucrose and / or added fruit. This avoids predominant sweet taste that may cover the umami taste imparted and / or enhanced by the method of the invention.

[0090] In an embodiment, the fermented plant-based product has an acidic pH, in particular a pH of less than 5.5, preferably less than 5.0, more preferably less than 4.6, most preferably of 3.0 to 4.6.

[0091] In a further embodiment, the fermented plant-based product may further comprise, for example, an ingredient selected from the list consisting of vitamin, mineral, color, fiber, prebiotic, hydrocolloid, salt, buffering agent, fat, oil, emulsifier, plant-based milk alternative, plant-based cream alternative, solid food particles or a mixture thereof.

[0092] In some embodiment, the method does not comprise any step of Maillard reaction. Accordingly, the method does not comprise any step of Mail la rd reaction consisting of: a step of heat-treating the obtained fermented plant-based product at a temperature above 100°C for at least 30 minutes, preferably at least 1 hour, or a step of adding one or more added reducing sugar(s) into the obtained fermented plant-based product followed by heat treatment of said fermented plant-based product in which one or more reducing sugar(s) has been added at a temperature above 100°C for at least 30 minutes, preferably at least 1 hour.

[0093] For example, the added reducing sugar may be selected from xylose, arabinose, ribose, glucose, galactose and mixture thereof. In some embodiment, the fermented plant-based product may be free from one or more added reducing sugar. For example, the one or more added reducing sugar may be selected from xylose, arabinose, ribose, glucose, galactose and mixture thereof.

[0094] Maillard reaction is undesirable in the context of the present invention as it requires addition of added reducing sugars but also the performance of a harsh heat-treatment at high temperature for a long time that may generate undesirable flavour compounds in the fermented plant-based product. In addition, this harsh heat-treatment may result in other undesirable consequences on the fermented plant-based products: loss of compounds of interest (including macro- and micro-nutrients), protein aggregation, protein denaturation etc... In addition, the implementation of a Mai I la rd reaction brings further complexity to the process and suggests higher energy consumption.

[0095] In a second aspect, the invention relates to a fermented plant-based product which comprises plant protein fermented with Lactobacillus johnsonii.

[0096] The plant protein and Lactobacillus johnsonii may be as described in the first aspect of the invention.

[0097] The features of the fermented plant-based product disclosed in the first aspect invention apply to the fermented plant-based product of the second aspect of the invention, and vice versa.

[0098] In an embodiment, the fermented plant-based product may be obtained or obtainable by the method of the first aspect of the invention.

[0099] The fermented plant-based product of the invention has a pleasant, significant, and enhanced umami taste while exhibiting limited bitterness.

[0100] In some embodiment, the fermented plant-based product has not undergone Mai Ila rd reaction, in particular Maillard reaction as described in the first aspect of the invention.

[0101] In some embodiment, the fermented plant-based product may be free from one or more added reducing sugars. For example, the one or more added reducing sugars may be selected from xylose, arabinose, ribose, glucose, galactose and mixture thereof.

[0102] Maillard reaction is undesirable in the context of the present invention as it requires addition of added reducing sugars but also the performance of a harsh heat-treatment at high temperature for a long time that may generate undesirable flavour compounds in the fermented plant-based product. In addition, this harsh heat-treatment may result in other undesirable consequences on the fermented plant-based products: loss of compounds of interest (including macro- and micro-nutrients), protein aggregation, protein denaturation etc... In addition, the implementation of a maillard reaction brings further complexity and suggests higher energy consumption.

[0103] In a third aspect, the invention relates to Lactobacillus johnsonii which deposited at the CNCM under reference number CNCM 1-5910.

[0104] This Lactobacillus johnsonii strain effectively generates / enhances umami taste in plant-based products via fermentation of plant proteins while generating limited bitterness. It is believed that, upon fermentation, this strain hydrolyzes plant proteins into peptides that impart and / or enhance umami taste.

[0105] In a fourth aspect, the invention relates to the use of Lactobacillus johnsonii in the production of a fermented plant-based product, wherein the Lactobacillus johnsonii is selected from the list consisting of Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-5910, Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-1225, Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-2474 or a combination thereof.

[0106] The fermented plant-based product may be a fermented plant-based product as described in the first and the second aspect of the invention.

[0107] The benefit of such strains is described in the first and third aspects of the invention.

[0108] In a fifth aspect, the invention relates to a peptide which has 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.

[0109] These peptides effectively impart and / or enhance umami taste in a product.

[0110] In some preferred embodiment, the peptide which has 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 embodiment, said peptide is not combined with one or more reducing sugars, in particular one or more added reducing sugars. For example, the one or more reducing sugars, in particular one or more added reducing sugars may be selected from xylose, arabinose, ribose, glucose, galactose and mixture thereof. In some embodiment, said peptide has not undergone Maillard reaction, in particular Maillard reaction as described in the first aspect of the invention.

[0111] Maillard reaction is undesirable in the context of the invention as it requires addition of added reducing sugars but also the performance of a harsh heat-treatment at high temperature for a long time that may generate undesirable flavour compounds. In addition, this harsh heat-treatment may result in other undesirable consequences: loss of compounds of interest, protein aggregation, protein denaturation etc... In addition, the implementation of a Mai Ila rd reaction brings further complexity and suggests higher energy consumption.

[0112] In a sixth aspect, the invention relates to a method for preparing one or more peptides comprising the step of: providing a plant protein substrate, fermenting the plant protein substrate with a starter culture comprising Lactobacillus johnsonii, isolating one or more peptides, optionally, purifying the one or more peptides, wherein the one or more peptides are selected from the list consisting of peptide having the amino acid sequence of SEQ ID NO: 4, peptide having the amino acid sequence of SEQ ID NO: 5, peptide having the amino acid sequence of SEQ ID NO: 6, peptide having the amino acid sequence of SEQ ID NO: 7, peptide having the amino acid sequence of SEQ ID NO: 8, peptide having the amino acid sequence of SEQ ID NO: 9, peptide having the amino acid sequence of SEQ ID NO: 10, peptides having the amino acid sequence of SEQ ID NO: 11 or combination thereof.

[0113] The plant protein substrate, the plant protein, the starter culture, the Lactobacillus johnsonii and the fermentation step are as provided in the first aspect of the invention.

[0114] In some embodiment, the peptides listed above may be derived from plant, preferably from plant different from soy and / or cereal.

[0115] Advantageously, the peptides listed above may be derived from pulse. The pulse is selected from the list consisting of pea, adzuki bean, great northern bean, cannellini bean, fayot bean, navy bean, red bean, black bean, pinto bean, kidney bean, lima bean, mung bean, chickpea, faba bean, lentil or a combination thereof. More preferably, the pulse is pea. The isolation and the purification of the peptides may be done by methods well known to the person skilled in the art to isolate and purify peptides.

[0116] In some preferred embodiment, after isolation, said one or more peptides are not combined with one or more reducing sugars, in particular one or more added reducing sugars. For example, the one or more reducing sugars, in particular one or more added reducing sugars may be selected from xylose, arabinose, ribose, glucose, galactose and mixture thereof. In some embodiment, said one or more peptides have not undergone Maillard reaction. Maillard reaction is undesirable in the context of the invention as it requires addition of reducing sugars but also the performance of a harsh heat-treatment at high temperature for a long time that may generate undesirable flavour compounds as reaction products. In addition, this harsh heat-treatment may result in other undesirable consequences: loss of compounds of interest, protein aggregation, protein denaturation etc...

[0117] In a seventh aspect, the invention relates to a composition comprising one or more peptides, wherein the one or more peptides are selected from the list consisting of peptide having the amino acid sequence of SEQ ID NO: 4, peptide having the amino acid sequence of SEQ ID NO: 5, peptide having the amino acid sequence of SEQ ID NO: 6, peptide having the amino acid sequence of SEQ ID NO: 7, peptide having the amino acid sequence of SEQ ID NO: 8, peptide having the amino acid sequence of SEQ ID NO: 9, peptide having the amino acid sequence of SEQ ID NO: 10, peptide having the amino acid sequence of SEQ ID NO: 11 or a combination thereof.

[0118] The composition has a pleasant, significant, and enhanced umami taste while exhibiting limited bitterness. The composition may also be added to a product, in particular a food product, to impart and / or enhance umami taste.

[0119] In some embodiment, the peptides listed above may be derived from plant, preferably from plant different from soy and / or cereal.

[0120] Advantageously, the peptides listed above may be derived from pulse. The pulse is selected from the list consisting of pea, adzuki bean, great northern bean, cannellini bean, fayot bean, navy bean, red bean, black bean, pinto bean, kidney bean, lima bean, mung bean, chickpea, faba bean, lentil or a combination thereof. More preferably, the pulse is pea.

[0121] In particular, the one or more peptides are added peptides, i.e. that they were added in the composition and not inherently present in the composition. In some embodiment, the one or more peptides, in particular one or more added peptides are isolated and / or purified, i.e. that they were added isolated and / or purified form.

[0122] In some embodiment, the composition is an oral composition, preferably food composition or beverage composition or nutritional composition.

[0123] In some embodiment, the composition is not fermented.

[0124] In some embodiment, the composition is free from pectin, in particular high methoxyl pectin.

[0125] In some preferred embodiment, the composition does not comprise one or more added reducing sugars. For example, the one or more added reducing sugars may be selected from xylose, arabinose, ribose, glucose, galactose and mixture thereof. In some embodiment, the one or more peptides of the composition have not undergone Maillard reaction. Maillard reaction is undesirable in the context of the invention as it requires addition of added reducing sugars but also the performance of a harsh heat-treatment at high temperature for a long time that may generate undesirable flavour compounds as reaction products. In addition, this harsh heat-treatment may result in other undesirable consequences: loss of compounds of interest, protein aggregation, protein denaturation etc...

[0126] In an eighth aspect, the invention relates to the use of one or more peptides as flavouring agent, wherein the one or more peptides are selected from the list consisting of peptide having the amino acid sequence of SEQ ID NO: 4, peptide having the amino acid sequence of SEQ ID NO: 5, peptide having the amino acid sequence of SEQ ID NO: 6, peptide having the amino acid sequence of SEQ ID NO: 7, peptide having the amino acid sequence of SEQ ID NO: 8, peptide having the amino acid sequence of SEQ ID NO: 9, peptide having the amino acid sequence of SEQ ID NO: 10, peptide having the amino acid sequence of SEQ ID NO: 11 or a combination thereof.

[0127] In some embodiment, the peptides listed above may be derived from plant, preferably from plant different from soy and / or cereal.

[0128] Advantageously, the peptides listed above may be derived from pulse. The pulse is selected from the list consisting of pea, adzuki bean, great northern bean, cannellini bean, fayot bean, navy bean, red bean, black bean, pinto bean, kidney bean, lima bean, mung bean, chickpea, faba bean, lentil or a combination thereof. More preferably, the pulse is pea.

[0129] In particular, these peptides may be used as umami flavour, umami taste enhancer and / or flavor masking agent. In some preferred embodiment, said one or more peptides are not combined with one or more reducing sugars, in particular one or more added reducing sugars. For example, the one or more reducing sugars, in particular one or more added reducing sugars may be selected from xylose, arabinose, ribose, glucose, galactose and mixture thereof. In some embodiment, said one or more peptides of the composition have not undergone Maillard reaction. Maillard reaction is undesirable in the context of the invention as it requires addition of added reducing sugar but also the performance of a harsh heat-treatment at high temperature for a long time that may generate undesirable flavour compounds as reaction products. In addition, this harsh heat-treatment may result in other undesirable consequences: loss of compounds of interest, protein aggregation, protein denaturation etc...

[0130] In a ninth aspect, the invention relates to the use of the composition of the seventh aspect of the invention as flavouring agent. In particular, it may be used as umami flavour, umami taste enhancer and / or flavor masking agent.

[0131] Protein sequences

[0132] • SEQ ID NO: 1 (length: 298)

[0133] MKRIIKVGALAASSFSLLFLAGCSSNNKEVAQYGDKKISQEQFYQELNSSPTSKTILANMLIYGALKEAYGSK LNKDQVEADYNSYKKRYGSQFDSFLKENSYTKKSFKQMIELNHLSKIALKKQMKPTTSQLKEEWKDYQPKI TVQHISTTSRDTANTVIEKLNNGESFASLASKYSVDSLTSSNGGKLPAFDMQNRRYDSTFKKGAYKLKNDE YTKEPIKVTNGYEVIKMINHPKKGNFADKQKELTEAIYNKWASNSRIMNNVISQVLKEQHVSIKDKDLKSA LDMYKGSNKANF

[0134] • SEQ ID NO: 2 (length: 2209)

[0135] MKKKREKYTWLLLSTALITSGQILGGGEQIIKASVESQTNSVKKSKTAVEHSTTALSRQAVEAQLAAQGVN FERLTPEEQQEVYVDVIVQLEALPASENGSIDSQTASRAEIEQASNKVIAAQSGIKDEVQKITNQAIDKSYG YVVNGFATKAKVGDIKKLREIKGVKSVTLAKVYFAADTSANNMANVSTVWSNYQYKGEGTVVSIIDTGID PNHKDLRLSDESKVKLTAKDIDGFTENSGYGRYFTSKVPFGHNYSDNNDIITDDDPKEQHGMHVAGIVAA NGTGKNSASSVVGVAPEAQLLAMKAFSNSDSSSTTDSTSVIGAVDDSAKLGADVLNMSLGSVSGEQTED DPEIAAVEKAVKHGTAAVISAGNSGTSTSNQEGVNKDFYGNPDMETIGSPGTSRSATTVASAENTKVTTD GMTVSTADGKKIFGPSVTQLSPNTSHDAFDSKKFYIVKDASGKLGMGTPSQYTADVKGKVAVVSRGEITF TDKQKYAQAAGAAGLIIVNNAGGNTPLTSVLYNEGFPTAGLSTDDGNKLVAYVEAHPDELLRVNVEVQP LNNVIREEDLMSSFTSYGPVSDLSFKPDITAPGGNIWSLQNNNGYINMSGTSMASPFIAGSQALLVQAM NDKTGKFYETYQKMSGSERAALIKNIQMNTANIEVDVDHGSVIESPRRQGAGLVNVEAAINAILHNPSTV

[0136] SGSNGYPGVELKDFQDRQHQFTIKFTNRTNKDIEYGLNENGKFSDVYTSETDPKTGVLFEKKIDGASLTPS

[0137] EKIVVPANSTKEVTINLSLPDNFKENQYVEGFMAFTGSDNSHLKIPYMGFFGDWAAPAIFDGLNGLAFNP

[0138] GNNNLGTIVTAGNKNGAVGYAGLNQDEDGNYRVDPDAIALSTADGASVSWVRPQYFLFRNANDVKAEI

[0139] LNQDGEVINTLVSLAHVTKSYWAASSQRYAKFNYAPAWDGTYFNQQTNKTEKVPDGTYTYRVTGTVDG

[0140] TKKQQHYDIKVKVDSVKPEVKNLKLGSHKDQTGKVSYVLKAEAKDNFSGLNGQANTYVNGELNRSVAYD

[0141] IVGSSSDGYQKIEVPLSDEQVKTLRAGKNDLAIAVFDNATNAGTNSGTSNKPGEINFGLIIDNNLPQKITTV

[0142] SDGYDMTDDSYTISGTYPEKVYGTYTDKDGKEHDLNISYDEASERFVTKLPLSVSDYDTNVKFYADEEHET

[0143] LITQKRINVSLVPPKLESLKVDDQETYTGNEEAKLSQTSEDTVEVSGKVSDDTDKVAVKVAGKTYSAKPTKE

[0144] HTFKVKVPVSYGENTMNIVLTDKDGNSSSVKQIVKSSDRGKTVVSAKDVTFDNGIKFGTTSVNTETENYD

[0145] PKTGKLTLTGKVNRPTTTVRIGDHTVKVKADGTFKLVLDLGKHGAKVFPVLIGDTTVNDTVQERLTFYVDS

[0146] NNPELTLNQEKDQSGYVPVYTNKEEFKLQGTISDDYPYYSLLINDNNVDANWDDVDYNGNKNLKKSFSH

[0147] SVKLKEGKNTFNVVVVDNNDNRSEVQTLVVYYKKAQKLASPQITATTASDKKSVTVTGKAKDGNVLYSTD

[0148] NGNKYNVLPEDGVTVKNNGKLLFKTVDKYGNESEVVEYDVKTIGKEESTVDKSVAQARKDLRKKLDQAR

[0149] ALGNTGKYTHESAKKLAQARQEASKALKDKNATLQELKQASEQLEEAIKNLVEKPVDQNKDKDKVEDKD

[0150] SQVNALKEKLEETVKAGEKFDKDKYTDDSVEKVTKALDEAKVVLANKDANSTDVQDAIDSIVNATKSLKEK

[0151] QVSPEKTTQQEDTPKENKDTEEVLAAKNALKEKADKLSHLDTTKYTSESAENLSNALKKVNQVLTNKDAN

[0152] KAQVQEALDSLSQAEKDLVEKTESNKDVETAKEQLREELNKHKDEDKSQYTDDSAKVKDNAEKISEGVLD

[0153] SKDAQADEVNKAKDSLVEAEKGLVKKEENKPAESDTQEVDKAREALEQEVNKNANVNLDGYTPESQDKF

[0154] KEILNGVRDVLNDKNASASSLEKAEKVLETATGVLTQVEHQVELPKVEQPVVTPEKKQ.TEQEEAKKSESSS

[0155] VSTSKDEVKEPEEKKQDAHSVSDKGTGTSVEKKGQTPADALSQVEHQAELSKNEQEEAKKSESHSASTSK

[0156] DEVKEPEEKKQDAESVSDKESSTLVGKKHQVARSAQESSKKNADNKVPTHKNSNKAQNTTNSSTRSTKSS

[0157] NNKELPKTGERETFFGLLVTGITALFASLGTVLRIKNKK

[0158] • SEQ ID NO: 3 (length : 584)

[0159] MKMSKSKLLYVTLASSLTIFATACGAKKTDSSNQTIKFSQNVPKKAIKKGGTLTYALENESPFTGIFLSEIADT

[0160] SPDVEAAAPGDESLFSINDQYQITDKGAATLKLNHHDNTAKIKVKDKVRWSDGKPVVAKDLEYSYEILAN

[0161] PKVQTTQYTSSLENIKGMAEYHQGKAKEISGIEMPDGPNGKTLILHFKELKPAMLNAASGFYWEHAAPY

[0162] HYLKKVPFEKLVSSDQVRKHPLYFGPYKMDKTVQGQSTSWSRNPYYWRGKPNFAHIYMSNITNSNVSQ

[0163] AIKSHKFDVASVLDSQWLQVKNTKGVNFVGKKTLSYNYLAFKVGKWDQKLGKNVENPHAKMNNPALR

[0164] KAMAYAMNVDVINNRFYHGLKFRVNSLIPSQFTPYYDKNIPTYSYNLNKANELLDKAGYKKAPGALYRRQ

[0165] PNGKPLVINIAVRGSGENSEAIWRNYIQQWKKAGLNVKFLGGRPMEFNRWVAAVKSSDPKIDVLEGSW GAAGDPSPSVFYGEKMPYNFARFVSPTNTKLLDEIDSAKSFDKNYRVQKFHEWQRWMYNKAYVVPTSG

[0166] AYSVTAVNSKVTGWSLKPSANVWYEAGFSK

[0167] Those skilled in the art will understand that they can freely combine all features of the present invention disclosed herein. In particular, features described for the products of the present invention may be combined with the uses or the methods of the present invention and vice versa. Further, features described for different embodiments of the present invention may be combined.

[0168] Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification. Further advantages and features of the present invention are apparent from the figures and non-limiting examples.

[0169] EXAMPLES

[0170] Example 1: Material and Method

[0171] Material and Method 1: Preparation of fermented plant-based milk.

[0172] Fermented plant-based milk alternatives were prepared.

[0173] To do so, 200 ml emulsions at 10% pea protein were first prepared by mixing pea protein isolate (85% pea protein) in water.

[0174] The different emulsions were inoculated by a starter culture comprising one of the strains listed in table 1 and fermented with said starter culture under the condition provided in table 1 for 48 hours and under a stirring speed of 120 rpm to obtain fermented plant-based milk alternatives. All fermentation were performed in 500 ml sterile Erlenmeyer flask using a laminar flow and sterile pipettes. For the genus Bifidobacterium, an anaerobic incubator was employed for the fermentation step and the oxygen levels were set at 0.5%. Table 1

[0175] Material and Method 2: Preparation of reference unfermented plant-based milk alternative

[0176] A reference unfermented plant-based milk alternative was prepared as follows. Pea protein isolate (85% pea protein) was mixed with water to obtain 200 ml of an emulsion at 10% pea protein. This emulsion corresponds to the reference unfermented plant-based milk alternative.

[0177] Material and Method 3: Sensory analysis

[0178] The sensory of the different samples was assessed by a panel trained to assess the taste of plant-based milk.

[0179] The sensory assessment was performed through different sensory sessions in different days to avoid sensory fatigue. In addition, bottled water was provided during the sensory analysis to be used as mouth washer at the beginning of the sensory evaluation and inbetween the tested samples.

[0180] In particular, an aliquot of 5 ml of the reference unfermented plant-based milk alternative of Material and Method 2 was presented to the panel together with a same aliquot of the fermented plant-based milk alternatives of Material and Method 1. One unfermented sample (reference) and two fermented samples, for a total of three samples were evaluated at each sensory session.

[0181] The unfermented and the fermented plant-based milk alternatives were stirred the whole time during the test to avoid sedimentation.

[0182] For the sensory evaluation, the panel was asked to evaluate the umami or bitterness taste perception by providing a score (=sensory score) on a scale from 0 to 5 for each sample.

[0183] Material and Method 4: Solid-phase extraction fractionation of the plant-based milk alternatives to obtain SPE fractions with metabolites, including umami-imparting and / or

[0184] Fermented plant-based milk alternative prepared with different strains of Material and Method 1 and the reference unfermented plant-based milk alternative of Material and Method 2 were both subjected to solvent extraction. The extraction protocol consisted of 340 g of the dried proteins extracted three times with a mixture of MeOH and H2O (1+1, v +v; 1400 mL) by stirring for 30 min at room temperature, followed by filtration using a Buchner funnel (Rotilabo, 185 mm, type 111A, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and centrifugation. The filtrates were combined, freed from solvent in a vacuum at 40 °C, and freeze-dried to give SPE fraction with extractable metabolites. Regarding the fractionation procedure, an aliquot (1 g) of active fraction was taken up in water (50 mL) and applied on a Chromabond C18 ec polypropylene cartridge (Macherey-Nagel, Duren, Germany) preconditioned with methanol (70 mL), followed by water (70 mL). After stepwise elution with water (75 mL) to give fraction "polar", water (100%, v / v, 75 mL) to give fraction "mediumpolar", methanol / water (50:50, v / v, 75 mL) to give fraction non-polar, methanol / water (100%, v / v, 75 mL) . The collected fractions were freed from the solvent by vacuum evaporation at 40 °C, taken up in the water, lyophilized twice, and kept at -20 °C until used for sensory analysis. The SPE fractionation procedure was taken from Hald et al., 2019.

[0185] Material and Method 5: MaxQuant Peptide analysis (in-silico peptide identification)

[0186] Metabolites (incl. peptides) separation and detection from SPE fractions of Material and Method 4 obtained from the fermented plant-based milk alternatives prepared respectively with L. johnsonii NCC533, NCC1584, NCC1657, NCC1680 and NCC2680 and from the reference unfermented plant-based milk alternative were done by using Ultraperformance Liquid Chromatography Time-of-Flight Mass spectrometry (UPLC-ToF-MS). All measurements were acquired on Sciex TripleTOF 6600 mass spectrometers (Sciex Darmstadt, Germany) connected to a Shimadzu Nexera X2 system (Shimadzu, Kyoto, Japan) operating in positive and negative electrospray ionization mode (ESI mode). The ion spray voltage was set at 5500 eV; the source temperature was 550 °C, nebulizing gas (0.38 MPa), and heating gas (0.45 Mpa). Metabolites (incl. peptides) separation was performed on two chromatographic columns in distinctive and consecutive runs: 1) 150 x 2 mm, 1.7 pm 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 gradient: 0 min, 5 % B; 2 min, 5 % B; 18 min, 100 %B; 21 min, 100 % B; 22 min, 5 % B; 25min, 5 % B and 2) Acquity BEH amide (water) with a gradient of 5mM NH4Ac in H20 at pH3; B: 5mM NH4Ac, 2% H2O In ACN at pH3 with a gradient of 0 min, 95% B; 2 min, 95% B; 10 min, 50% B; 12 min, 0% B; 15 min, 0% B; 15.50 min, 95% B; 20 min, 95% B. Column oven was set at 40 °C, and TOF-MS scan was performed from m / z 50 to m / z 1500 acquiring for C18 run while from m / z 50 to m / z 1000 for HILIC chromatography. MS2 data were acquired in data-dependent acquisition (IDA mode) and data-independent acquisition (SWATH mode).

[0187] For peptide identification, Maxquant software was operated to process the .wiff files (Versionl.6.6.0) and the genome-based sequences of the pea storage protein obtained from the UniProt database. Processing parameters were taken mostly as default. Unspecific search was employed during this study due to the unpredictability of such a broad fermentation agent and proteinase / peptidase production by the bacteria. Among the parameters that changed: Minimum peptide length was set as 3, modification included Oxidation and Acetyl (Protein N-term), Max peptides mass set to 4600 Dalton, minimum peptide length for unspecific search 3 and maximum peptides length for unspecific search set to 25, Mass spec setting was set as "Sciex qTOF". Protein fasta files used for peptide search were obtained from uniprot database ("UniProt: the universal protein knowledgebase in 2021", 2021). The protein / peptide search was performed by searching "Pisum Sativum" in the database and by downloading the fasta files of the most abundant protein from this organism found in literature (Gueguen & Barbot, 1988, "Quantitative and qualitative variability of pea (Pisum sativum L.) protein composition").

[0188] During processing, the .wiff file was filtered based on the sequences in the SPE fraction of Material and Method 4 obtained from the milk alternative sample fermented respectively with L. johnsoniii NCC533, NCC1584, NCC1657, NCC1680 and NCC2680 and which were not present in the SPE fraction of Material and Method 4 obtained from the reference unfermented milk alternative sample.

[0189] Material and Method 6: Tool for prediction of umaminess: iUmami-SCM

[0190] A sequence-based predictor named iUmami-SCM was used for the identification and characterization of umami peptides. The tool is publicly available at http: / / camt.pythonanywhere.com / iUmami-SCM. Scoring card method (SCM) is the methodology behind the tool for the estimation of the propensity score of peptides. Peptides identified in the samples range from 20-30 peptides for low-proteolytic starter cultures up to 200-500 in the highly proteolytic starter cultures such as NCC533 at 48h fermentation time in pea protein isolate solution. The most intense peptides present in the evidence file of these strains were collected and submitted to the iUmami SCM software to gain a possible prediction of whether these molecules might possess a human taste activity and, in particular, a possible umami activity. A peptide which has iUmami SCM score above 590 is considered to have a significant umami activity.

[0191] Material and Method 7: Peptide quantification via SWATH

[0192] Quantification of peptides using SWATH methodology in the in the different fermented plant-based milk alternative samples (cf. example 5) was based on the work published by Sebald and colleagues, "Mapping Taste-Relevant Food Peptidomes by Means of Sequential Window Acquisition of All Theoretical Fragment Ion-Mass Spectrometry", J. Agric. Food Chem. 2020, 68 (38), 10287-10298.

[0193] UHPLC-ToF-MS measurements were acquired on a TripleTOF 6600 mass spectrometer (Sciex, Darmstadt, Germany) connected to a Shimadzu Nexera X2 system (Shimadzu, Kyoto, Japan) operating in the positive ESI mode. Ion spray voltage was set at 5500 eV; the source temperature was 550 °C; the nebulizing gas was at 55 psi; the heating gas was at 65 psi, and nitrogen served as the curtain gas at 35 psi to effectively desolvate ions. An aliquot (10 pL) of the prepared sample was injected into the UHPLC-ToF-MS system connected to a 150 x 2 mm, 1.7 pm, Kinetex C18 column (Phenomenex, Aschaffenburg, Germany) equipped with a guard column of the same type. Eluent A consisted of 1% formic acid in water, and eluent B was 1% formic acid in acetonitrile. Using a flow rate of 0.3 mL / min, chromatography was performed starting with 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, ToF- MS survey scans were acquired (m / z 100-1000, 150 ms), followed by 20 product ion scans with variable QI isolation windows from m / z 200 to 1000 with an overlap of 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,

[0194] 513.4-543.7, 542.7-578.3, 577.3-615.3, 614.3-659.0, 658.5-703.9, 702.9-756.2,

[0195] 755.2-812.8, 811.8-870.8, 869.8-939.1, and 938.1-1000.5 Product ion spectra were accumulated in the high-resolution mode for 50 ms using DP of 80 V, CE of 35 V, and CES of 15 V. Quantitation was performed using external standard calibration with standard solutions starting from 0.5 mmol / L for each peptide and diluting 1:1 for 16 times in 50:50 MeOH: Millipore. System control and data acquisition were 54 performed using AnalystTF 1.7.1 (Sciex, Darmstadt, Germany). Data analysis was performed using MultiQuant 3.0.2 (Sciex, Darmstadt, Germany).

[0196] Material and Method 8: Genome-peptidome analysis for L. johnsonii species A list of Geninfo Identifier (Gl) numbers of proteolysis-related genes was obtained from the supplementary material of Liu et al., "The proteolytic system of lactic acid bacteria revisited: a genomic comparison", BMC Genomics 2010, 11:36. As Gl numbers are discontinued, they were searched for in UniParc (https: / / www.uniprot.org / uniparc / ), the UniParc identifiers were then searched for in UniProt (https: / / www.uniprot.org / ), and the protein sequences in fasta format were downloaded. A Gl number is often linked to more than one UniProt accession, which may generate redundant mapping via the BLAST search.

[0197] The strains genomes were used as subjects querying the downloaded proteins targeting one sequence (best hit) with an e-value of 0.001. BLAST results were filtered to hits having at least 80% of coverage of the query. Query ID and identity percentage were used to build heatmaps. Grouping of genomes (vertical axis) and proteins (horizontal axis) was done by hierarchical clustering. The clustering of genomes reflects the gene-by-gene similarity and not the overall genomic differences.

[0198] L. johnsonii proteolysis system proteins were BLAST searched against the non- redundant database of proteins

[0199] (https: / / www.ncbi.nlm.nih.gov / refseq / about / nonredundantproteins / ), and results were filtered to extract accession numbers of "MULTISPECIES" hits of Lactobacillus, corresponding to potential homologous genes across the genus. The protein sequences from the retrieved accessions were downloaded from batch Entrez

[0200] (https: / / www.ncbi.nlm.nih.gov / sites / batchentrez) and queried against genomes of L. johnsonii, L. fermentum, and L. rhamnosus. Lactococcus lactis, L. lactis subspp cremoris and L. lactis subspp lactis were compared to L. johnsonii, querying the proteolytic systems of L. lactis as reported by Liu et al. BMC Genomics 2010, 11:36.

[0201] NCBI accessions of proteolysis-related genes were extracted from 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 batch Entrez. Strains of L. helveticus genomes were used as subjects querying the downloaded proteins via BLAST. Results were filtered as described above, and unclustered heatmaps were produced. The peptidome data that were used as a connection with the BLAST data.

[0202] Example 2: Sensory assessment, including Umami taste, of fermented plant-based milk alternatives prepared with strains having different genus and species. Different fermented plant-based milk alternative samples were prepared respectively with NCC533, NCC2705, NCC660, NCC2378, NCC4007 or NCC3059 according to the method provided in Material and Method 1 of example 1.

[0203] A reference unfermented plant-based milk alternative sample (hereinafter, reference sample) was also prepared according to the Material and Method 2 of example 1.

[0204] The different fermented plant-based milk alternative samples and the reference sample were assessed via sensory analysis according to Material and Method 3 of example 1.

[0205] The Results are provided in Figure 1. The reference sample is labelled unfermented pea protein isolate in figure 1.

[0206] It can be observed in figure 1 that the highest sensory score for umami, and so the highest umami taste is obtained with Lactobacillus johnsonii, in particular Lactobacillus johnsonii NCC533. The sensory score for umami, and so the umami taste is significantly lower for the other starter cultures.

[0207] In particular, Lactobacillus johnsonii, in particular Lactobacillus johnsonii NCC533 allows the highest enhancement of the umami taste compared to the reference sample (cf. figure 2).

[0208] These data suggest that Lactobacillus johnsonii significantly enhanced the umami taste of fermented plant-based product via fermentation.

[0209] Example 3: Umami peptide count of fermented plant-based milk alternatives prepared with different Lactobacillus johnsonii.

[0210] After L. johnsonii NCC 533 was identified for capability for generating umami taste in example 2, four other strains of L. johnsonii were tested for the capability for generating umami peptides.

[0211] Fermented plant-based milk alternative samples were prepared with NCC533, NCC1584, NCC1657, NCC1680 and NCC2680 according to the method provided in Material and Method 1 of example 1.

[0212] A reference unfermented plant-based milk alternative sample (hereinafter, reference sample) was also prepared according to the Material and Method 2 of example 1.

[0213] The generation of umami peptides was assessed by measuring the umami peptide counts. The umami peptide counts correspond to the number of different peptides identified in the sample and which has umami activity. The peptides were identified with Material and Method 5 of example 1 in the SPE fractions of fermented and unfermented samples obtained according to Material and Method 4 of example 1 and their propensity to i m pa rt / im prove umami taste was assessed with Material and Method 6 of example 1.

[0214] The results are provided in figure 2. The reference sample is labelled unfermented pea protein isolate in figure 2.

[0215] It can be seen that the different L.johnsonii strains show different capacity to generate umami peptide, and so umami taste.

[0216] In particular, only L. johnsonii NCC533, NCC1657 and NCC2680 exhibit increase umami peptide generation upon fermentation compared to the reference. The highest peptide count was obtained for NCC533.

[0217] The samples were tasted by the panel. The samples fermented with L. johnsonii NCC533, NCC1657 and NCC2680 exhibit increased umami taste compared to the reference in mouth. In addition, those samples exhibit limited bitterness. No bitterness increase is perceived in comparison with the reference.

[0218] Example 4: Identification of peptides imparting and / or enhancing umami taste

[0219] As disclosed above, L.johnsonii NCC533 was shown to impart the highest umami taste. To further investigate about umami taste generation, the peptides generated by L. johnsonii NCC533 were assessed for their potential to impart and / or enhance umami taste.

[0220] To undertake this assessment, the peptides generated by L. johnsonii NCC533 were first identified via Material and Method 5 of example 1 in the SPE fraction prepared according to Material and Method 4 of example 1, said SPE fraction being prepared from the samples of example 3 fermented with L. johnsonii NCC533. The potential to impart and / or enhance umami taste of the identified peptides was assessed according to Material and Method 6 of example 1.

[0221] The prediction tool identified 8 new peptides (listed in Table 2) in the sample fermented with L. johnsonii NCC533 that have high iUmami SCM score. This suggested that these peptides contribute to impart and / or enhance of the umami taste in the fermented sample. Table 2

[0222] It is believed that the proteolytic activity of L. johnsonii, in particular L. johnsonii NCC533 during fermentation of plant protein tends to generate peptides that significantly impart and / or increase umami taste, including the ones listed in table 2.

[0223] Example 5: Production over fermentation time of peptides imparting and / or enhancing umami taste

[0224] The production of some peptides imparting and / or enhancing umami taste of table 2 was assessed over time of fermentation.

[0225] Different fermented plant-based milk alternative samples were prepared with NCC533 according to the method provided in Material and Method 1 of example 1 but with different fermentation time: 0, 24 and 48 hours.

[0226] The peptides listed in table 3 were quantified in the different milk alternative samples according to Material and Method 7 of example 1. The results are provided in table 3.

[0227] Table 3

[0228] It can be observed that the amount of peptides imparting and / or enhancing umami increase with the fermentation time. Example 6: Gene coding proteolytic proteins in L.johnsonii strains imparting and / or enhancing umami taste

[0229] The proteolytic genes of L. johnsonii NCC533, NCC1657 and NCC2680 were compared with the proteolytic genes of L. johnsonii that impart limited umami taste, i.e. L. johnsonii NCC1584 and NCC1585. The genes were compared according to the Material and Method 8 of example 1.

[0230] The gene encoding the proteolytic proteins of table 4 were considered in this study. Table 4

[0231] L. johnsonii NCC1584 and NCC1585 lack the cell-wall bound proteinase PrtP compared to L. johnsonii NCC533, NCC1657 and NCC2680. In addition, L. johsonii NCC1584 and NCC1585 showed highly dissimilar cell-wall bound proteinase PrtM and oligopetipe permease OppA (less tham 50% identity) compared to NCC1657 and NCC2680.

[0232] No major differences were observed for the other proteolytic proteins listed in table 4.

[0233] Hence, based on the above, it is believed that PrtM, PrtP and OppA contribute to the proteolysis of plant proteins to produce peptides imparting / enhancing umami taste.

[0234] Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims.

Claims

CLAIMS1. A method of making a fermented plant-based product, said method comprising providing a plant protein substrate, and fermenting the plant protein substrate with a starter culture comprising Lactobacillus johnsonii.

2. The method according to claim 1, wherein the plant protein of the plant protein substrate is pulse 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 the proteinase PrtP and / or the proteinase PrtM and / or the 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 at the CNCM under reference number CNCM 1-5910, Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-1225, Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-2474 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 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 fermented plant-based dairy analogue or plant-based meat analogue.

8. A fermented plant-based product which comprises plant protein fermented with Lactobacillus johnsonii.

9. The fermented plant-based product according to claim 8, wherein said plant protein is pulse protein, preferably pea protein.

10. The fermented plant-based product according to claim 8 or 9, which comprises arginyl peptides, prolyl peptides and / or glutamyl peptides.

11. Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-5910.

12. Use of Lactobacillus johnsonii in the production of a fermented plant-based product, wherein the Lactobacillus johnsonii is selected from the list consisting of Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-5910, Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-1225, Lactobacillus johnsonii deposited at the CNCM under reference number CNCM 1-2474 or a combination thereof.

13. A peptide which has 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.

14. A method for preparing one or more peptides comprising the step of: providing a plant protein substrate, fermenting the plant protein substrate with a starter culture comprising Lactobacillus johnsonii, isolating one or more peptides, wherein the one or more peptides are selected from the list consisting of peptide having the amino acid sequence of SEQ ID NO: 4, peptide having the amino acid sequence of SEQ ID NO: 5, peptide having the amino acid sequence of SEQ ID NO: 6, peptide having the amino acidsequence of SEQ ID NO: 7, peptide having the amino acid sequence of SEQ ID NO: 8, peptide having the amino acid sequence of SEQ ID NO: 9, peptide having the amino acid sequence of SEQ ID NO: 10, peptide having the amino acid sequence of SEQ ID NO: 11 or combination thereof.

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

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

17. Use of the composition of claim 15 as flavouring agent.