WO2025056442A1 - Method of making fermented plant-based products having kokumi characteristics and products thereof - Google Patents

Abstract

The method comprises a step of providing a plant protein substrate and a step of fermenting the plant protein substrate with a starter culture comprising Lactobacillus johnsonii. It also relates to such a fermented plant-based product and to Lactobacillus johnsonii strains suitable for the invention. It also relates to peptides that impart and/or enhance kokumi, method for preparing such peptides, composition comprising such peptides and use of such peptides as flavouring agent.

Description

METHOD OF MAKING FERMENTED PLANT-BASED PRODUCTS HAVING KOKUMI CHARACTERISTICS AND PRODUCTS THEREOF

TECHNICAL FIELD

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 kokumi, in particular kokumi sensation and fermented plant-based product thereof.

BACKGROUND OF THE INVENTION

Three key trends were identified driving consumer taste preference in the space of culinary and beverage space: a move toward taste complexity; increasing call for natural, clean label products; and reduced public acceptance of processed, industrialized food production.

To bring taste complexity in the culinary and beverage space, one opportunity relies on playing with the five different basic tastes which are umami, sweet, bitter, salty and sour. Another opportunity relies on bringing and/or modulating other taste dimensions. In particular, kokumi is a recently identified taste dimension. Kokumi is not considered as a sixth basic taste but is rather presented as a sensation or taste/flavour enhancer. In particular, kokumi enhances some basic taste such as umami, sweet or salty. In this view, the provision of kokumi peptides, i.e. peptides imparting or enhancing kokumi, appears as interesting to further bring/modulate taste complexity in the culinary and beverage space.

Generation of kokumi peptides increases the palatability of the ordinary-tasted food product. Kokumi peptides are promising substitutes for fat, MSG and flavor components such as caramel artificial flavorings and meat-like flavorings. Partial replacement of sodium chloride by kokumi peptides decreases the amount of salt added, but the saltiness of dishes may be at a similar level. It helps to prevent and control cardiovascular diseases like hypertension. Likewise, less sugary but sweet-tasted food with kokumi peptides may be formulated for diabetic patients.

Kokumi peptides are summarized and roughly classified into three groups according to their structural features. The first group consists of glutamyl peptides and poly glutamylated peptides with at least one glutamyl residue at the N-terminus; the second group contains short leucyl peptides with one leucyl residue at the N-terminus; while the peptides in the third group have few significant features in common. Kokumi imparting and kokumi enhancing substances, including peptides, may be prepared from or found in animal foods as well as plant-based foods.

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

Commercially available kokumi seasonings usually contain various combinations of broths of various foods, yeast extracts, maillard-reacted peptides, and protein hydrolysates, depending upon manufacturers. However, to use kokumi seasonings as additives is not preferred by consumers.

An alternative to the addition of kokumi seasonings is the in-situ/in-process generation of kokumi peptides during the manufacturing of food products. There are three approaches for generating kokumi peptides in-situ/in process: thermal treatment, enzymatic treatment, and fermentation.

Concerning the thermal treatment approach, it entails several challenges. The kokumi- inducing effect of thermal treatment (Millard Reaction) may have negative effect on food matrices. For example, it can generate certain level of browning or induce change of sensory/nutritional properties, which is not needed or desired in certain food products. Moreover, the requirement for a thermal treatment in this approach may also have negative effect on sensory, functional or nutritional properties of food products.

Concerning the enzymatic approach, it usually involves two reactions, protein hydrolysis by proteinase and y-glutamylation by y-Glutamyltranspeptidase (GGT) to generate kokumi peptides. This approach entails several challenges. First, this approach is not cost- effective. Second, this approach can be intricate to implement. For example, it may be complex to identify optimal reaction conditions suitable for the two groups of enzymes. Third, this approach leads to sensory defects. Indeed, the resulting hydrolysate exhibits unpleasant bitterness.

Concerning the fermentation approach, fermentation is a traditional way of food production with high public acceptance without any controlled additives and produced with environmentally friendly processes. However, in such an approach, peptides are generally generated via process involving either more than one step or more than one strain. It would therefore be desirable to provide a method for generating or enhancing kokumi 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.

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

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.

SUMMARY OF THE INVENTION

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.

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.

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.

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

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 pleasant, significant, and enhanced kokumi, in particular kokumi sensation while exhibiting limited bitterness.

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

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.

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

A fifth aspect of the invention proposes the peptide which has the amino acid sequence of SEQ ID NO: 1, or, SEQ ID NO: 2, or, SEQ ID NO: 3.

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: 1, peptide having the amino acid sequence of SEQ ID NO: 2, peptide having the amino acid sequence of SEQ ID NO: 3, or combination thereof.

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: 1, peptide having the amino acid sequence of SEQ ID NO: 2, peptide having the amino acid sequence of SEQ ID NO: 3, or a combination thereof.

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: 1, peptide having the amino acid sequence of SEQ ID NO: 2, peptide having the amino acid sequence of SEQ ID NO: 3, or a combination thereof.

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

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

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.

DETAILED DESCRIPTION OF THE INVENTION

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.

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

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

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.

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

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.

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

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

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

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

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

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

As used herein, the term "kokumi peptide" refers to a peptide that imparts and/or enhances kokumi, in particular kokumi sensation. In other words, it refers to a peptide that can enhance one or more of the following basic tastes: sweet taste, salty taste, sour taste, and umami, and can impart marginal tastes of the basic tastes, such as thickness, growth (mouthfulness), continuity, and harmony accompanying the basic taste. For sake of clarity, in the context of the invention, a kokumi peptide is not a Maillard reaction product, in particular Maillard-reacted peptide. 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:1, 2 and 3 are not Maillard reaction products, in particular Maillard-reacted peptides. As used herein, the terms "kokumi" and "kokumi sensation" are used interchangeably, "kokumi" refers to a sensation that cannot be expressed by the five basic tastes: sweet taste, salty taste, sour taste, bitter taste, and umami, and in which, not only one or more of the five basic tastes, marginal tastes of the basic tastes, such as thickness, growth (mouthfulness), continuity, and harmony are enhanced.

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.

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.The term "CaSR" used in this specification means the calcium sensing receptor which belongs to the class C of the 7- time transmembrane receptor and which is thus also referred to as "calcium receptor". The term "CaSR agonist" used in this specification means a substance which is linked with the foregoing CaSR to thus activate the receptor CaSR. In addition, the term "activate CaSR" used herein means that a ligand is linked with CaSR to thus activate a protein linked with guanine nucleotide and to transmit signals outputted from the same. Moreover, the ability of a substance to form a linkage with CaSR to thus activate the same is referred to as "CaSR agonist activity".

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.

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.

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

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.

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.

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

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.

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

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

Advantageously, the starter culture consists only of Lactobacillus johnsonii. It has been observed that the kokumi 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.

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.

These Lactobacillus johnsonii strains effectively provide/enhance kokumi 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 kokumi, in particular kokumi sensation.

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.

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.

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.

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

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 kokumi, in particular kokumi sensation 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.

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

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

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

The method of the invention allows the production of kokumi peptides, i.e. peptides that impart and/or enhance kokumi, in particular kokumi sensation. In particular, the fermented plant-based product comprises one or several kokumi peptides.

In some embodiment, the one or several kokumi peptides may be derived from plant, preferably from plant different from soy and/or cereal.

Advantageously, the one or several kokumi 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.

In some embodiment, the one or several kokumi peptides may comprise at least one leucyl residue and/or at least one glutamyl residue, preferably gamma-glutamyl residue. In some embodiment, the one or several kokumi peptides may comprise at least one leucyl residue at the N-terminus and/or C-terminus and/or at least one glutamyl residue at the N- terminus and/or C-terminus, preferably gamma-glutamyl residue at the N-terminus and/or C- terminus.

In some embodiment, the one or several kokumi peptide consists of 2 to 20 amino acids, preferably 2 to 15, more preferably 2 to 10 amino acids, more preferably 4 to 8 amino acids

The fermented dairy product comprises elevated levels of such peptides. In a preferred embodiment, the concentration of kokumi peptides in the fermented plant-based product is of at least 0.2, preferably at least 0.3, more preferably 0.5, more preferably at least 1.0, even more preferably at least 1.5, most preferably 2.0 pmol per litre of fermented plantbased product.

In a preferred embodiment, the concentration of kokumi 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 15 pmol per litre of fermented plant-based product.

It has been discovered that the method of the invention allows the production of new peptides that effectively impart or enhance kokumi. The fermented plant-based product comprises peptides selected from the list consisting of peptide having amino acid sequence of SEQ ID NO: 1, peptide having amino acid sequence of SEQ ID NO: 2, peptide having amino acid sequence of SEQ ID NO: 3, or a combination thereof. In an embodiment, the fermented plant-based product may comprise at least 0.2, preferably at least 0.3, more preferably 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, wherein the peptides are selected from the list consisting of peptide having amino acid sequence of SEQ ID NO: 1, peptide having amino acid sequence of SEQ ID NO: 2, peptide having amino acid sequence of SEQ ID NO: 3or a combination thereof.

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 15 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: 1, peptide having amino acid sequence of SEQ ID NO: 2, peptide having amino acid sequence of SEQ ID NO: 3, or a combination thereof.

The fermented plant-based product has kokumi, in particular kokumi sensation. The kokumi may be assessed by a panel trained to evaluate the kokumi of food products by providing a score on a scale from 0 (not detectable) to 5 (strongly detectable). An example of method to assess kokumi is provided in the present examples. Another example of sensory evaluation is provided in Example 15 of EP1946110 Bl. Other methods to assess the effect of molecules/compounds on kokumi are known in the art. For example, this can be assessed by measuring the activity of calcium sensing receptor (CaSR): molecules/compounds that impart or enhance kokumi generally show an agonist effect on the CaSR activity. An example of such a method is also provided in EP1946110 Bl.

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.

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.

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.

In some embodiment, the method does not comprise any step of Maillard reaction. Accordingly, the method does not comprise any step of Mai Ila rd reaction consisting of: a step of heat-treating the obtained fermented plant-based product with 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.

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.

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 Ila rd reaction brings further complexity to the process and suggests higher energy consumption.

In a second aspect, the invention relates to a fermented plant-based product which comprises plant protein fermented with Lactobacillus johnsonii. The plant protein and Lactobacillus johnsonii may be as described in the first aspect of the invention.

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.

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

The fermented plant-based product of the invention has pleasant, significant, and enhanced kokumi sensation while exhibiting limited bitterness.

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.

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.

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.

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

This Lactobacillus johnsonii strain effectively generates/enhances kokumi, in particular kokumi sensation 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 kokumi.

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.

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

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

In a fifth aspect, the invention relates to a peptide which has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

These peptides effectively impart and/or enhance kokumi in a product.

In some preferred embodiment, the peptide which has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 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.

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 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 Maillard reaction brings further complexity and suggests higher energy consumption.

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: 1, peptide having the amino acid sequence of SEQ ID NO: 2, peptide having the amino acid sequence of SEQ ID NO: 3, or combination thereof.

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.

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.

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 present 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...

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: 1, peptide having the amino acid sequence of SEQ ID NO: 2, peptide having the amino acid sequence of SEQ ID NO: 3, or a combination thereof.

The composition has pleasant, significant, and enhanced kokumi sensation while exhibiting limited bitterness. The composition may also be added to a product, in particular a food product, to impart and/or enhance kokumi.

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.

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

In some embodiment, the composition is not fermented. In some embodiment, the composition is free from pectin, in particular high methoxyl pectin.

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 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 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...

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: 1, peptide having the amino acid sequence of SEQ ID NO: 2, peptide having the amino acid sequence of SEQ ID NO: 3, or a combination thereof.

In particular, these one or more peptides may be used as kokumi-imparting flavour, kokumi enhancer, taste enhancer and/or flavor masking agent.

In some embodiment, the taste enhancer is a basic taste enhancer. In some embodiment, the taste enhancer is sweet taste enhancer and/or salty taste enhancer and/or umami taste enhancer and/or sourness enhancer.

In some embodiment, the taste enhancer is marginal taste enhancer. For example, the taste enhancer is thickness enhancer and/or growth (mouthfulness) enhancer and/or continuity enhancer, and/or harmony enhancer.

In some preferred embodiment, the 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, the peptides of the composition have not undergone Maillard reaction. Maillard reaction is undesirable in the context of the present 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...

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 kokumi-imparting flavour, kokumi enhancer, taste enhancer and/or flavor masking agent. In some embodiment, the taste enhancer is sweet taste enhancer and/or salty taste enhancer and/or umami taste enhancer.

Peptide sequences

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.

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.

EXAMPLES

Example 1: Material and Method

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

Fermented plant-based milk alternatives were prepared. To do so, 200 ml emulsions at 10% pea protein were first prepared by mixing pea protein isolate (85% pea protein) in water.

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.

Table 1

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

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.

Material and Method 3: Sensory analysis

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

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.

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 (cf. example 2). Alternatively, one unfermented sample of Material and Method 2 (reference, no peptide spiking) and two unfermented samples of Material and Method 2 that were spiked with an identified peptide, i.e. a total of three samples, were evaluated at each sensory session (cf. example 3).

The unfermented (non-spiked or spiked with peptide) and the fermented plant-based milk alternatives were stirred the whole time during the test to avoid sedimentation.

For the sensory evaluation, the panel was asked to describe the sensory attributes of the different samples and/or to evaluate specifically the kokumi or bitterness perception in mouth by providing a score (=sensory score) on a scale from 0 to 5 for each sample.

Material and Method 4: Solid-phase extraction fractionation of the plant-based milk alternatives metabolome to obtain SPE fractions with metabolites, including kokumi- imparting and/or enhancing peptides

The fermented plant-based milk alternatives prepared with respectively L. johnsonii NCC533, NCC1657 and NCC2680 according to Material and Method 1 and the reference unfermented plant-based milk alternative milk according to Material and Method 2 were 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 (incl. peptides).

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 "medium-polar", 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.

Material and Method 5: Peptides separation and identification Metabolites (incl. peptides) separation and detection from SPE fractions of Material and Method 4 obtained from the fermented plant-based milk alternatives prepared with respectively L. johnsonii NCC533, NCC1657 and NCC2680 and obtained 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).

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").

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, NCC1657 and NCC2680 and which were not present in the SPE fraction of Material and Method 4 obtained from the reference unfermented milk alternative sample.

Material and Method 6: Peptide quantification via SWATH

Quantification of peptides using SWATH methodology in the different fermented plantbased milk alternative samples (cf. example 4) 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.

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,

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 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).

Material and Method 7: Sensory analysis of SPE fractions

The SPE fractions obtained in Material and Method 4 were reconstituted in water and then tasted by a panel.

The panel was asked to describe the sensory attributes of the different samples and/or to evaluate specifically the kokumi perception in mouth by providing a score (=sensory score) on a scale from 0 to 5 for each SPE fraction sample. If the sensory score was of 0.5 or above, it was considered as kokumi active SPE fraction.

Example 2: Sensory assessment of Kokumi and bitterness of fermented plant-based milk alternatives prepared with L. johnsonii.

A fermented plant-based milk alternative sample was prepared with NCC533 according to the Material and Method 1 of example 1.

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

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.

The Results are provided in table 2.

Table 2

It can be observed in table 2 that the sensory score for kokumi is increased after fermentation with L. johnsonii, in particular !, johnsonii NCC533. In addition, the sensory score for bitterness is decreased after fermentation with L. johnsonii, in particular L. johnsonii NCC533. These data suggest that Lactobacillus johnsonii significantly enhanced the kokumi sensation while reducing the bitterness perception in fermented plant-based product via fermentation.

Example 3: Identification of peptides imparting and/or enhancing kokumi

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

To undertake this assessment, the peptides generated by L. johnsonii NCC533 were identified via Material and Method 5 of example 1 in the SPE fractions of the milk alternative sample fermented with L. johnsonii NCC533 of Material and Method 4 of example 1. The identification of peptides via Material and Method 5 of example 1 was performed only on SPE fractions that were identified as kokumi active SPE fraction after sensory analysis according to Material and Method 7.

The potential of the identified peptides to impart and/or enhance kokumi was assessed by sensory assessment according to Material and Method 3 of example 1. In particular, the identified peptides were produced and provided by GenScript (Leiden, the Netherlands). Then, the unfermented samples according to the Material and Method 2 of example 1 were spiked respectively with identified peptides. The peptides were spiked in the unfermented samples at the same level as they were present in the fermented sample after 48hours of fermentation (cf. example 4).

11 peptides were identified in the kokumi active SPE fraction of the fermented plantbased milk alternative obtained with NCC553.

Among the 11 peptides, 3 sequences were identified as imparting and/or enhancing kokumi during sensory assessment. Indeed, according to the panellists, the samples spiked respectively with the 3 following peptides: GQIEEL, GSAQEVD and GSSHEVD were described as more intense, smoother, more mouthcoating, and had a more long-lasting mouthfeel. In other words, these 3 peptides impart and/or enhance kokumi in the samples.

The peptides generated by L. johnsonii NCC1657 and NCC2680 were also identified via

Material and Method 5 of example 1 in the different SPE fractions of the milk alternative samples fermented respectively with L. johnsonii NCC1657 and NCC2680 of Material and Method 4 of example 1. The 3 peptides GQIEEL, GSAQEVD and GSSHEVD were also identified in samples fermented with L. johnsonii NCC1657 and NCC2680. This suggests that kokumi sensation is likely present in these milk alternative samples fermented with L. johnsonii NCC1657 and NCC2680.

Example 4: Production over fermentation time of peptides imparting and/or enhancing kokumi

The production of three peptides imparting and/or enhancing kokumi identified in example 3 was assessed over time of fermentation.

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.

The peptides listed in table 3 were quantified in the different milk alternative samples according to Material and Method 6. The results are provided in Table 3.

Table 3

It can be observed that the amount of peptides imparting and/or enhancing kokumi increase with the fermentation time.

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

1. 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 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.

5. 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.

6. 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.

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

8. The fermented plant-based product according to claim 7, wherein said plant protein is pulse protein, preferably pea protein.

9. The fermented plant-based product according to claim 7 or 8, which comprises kokumi peptides.

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

11. 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.

12. A peptide which has the amino acid sequence of:

- SEQ ID NO: 1, or,

- SEQ ID NO: 2, or,

- SEQ ID NO: 3.

13. 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: 1, peptide having the amino acid sequence of SEQ ID NO: 2, peptide having the amino acid sequence of SEQ ID NO: 3, or combination thereof.

14. 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: 1, peptide having the amino acid sequence of SEQ ID NO: 2, peptide having the amino acid sequence of SEQ ID NO: 3, or a combination thereof.

15. 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: 1, peptide having the amino acid sequence of SEQ ID NO: 2, peptide having the amino acid sequence of SEQ ID NO: 3, or a combination thereof.

16. Use of the composition of claim 14 as flavouring agent.