The present invention relates generally to the field of plant-based food. In particular, the present invention relates to improving the amino acid profile of plant-based food products. One embodiment of the present invention relates to the use of a combination of Lacticaseibacillus paracasei and Bacillus amyloliquefaciens to enrich the L-lysine content in plant-based compositions through fermentation. The present invention further relates to the bacterial strain Bacillus amyloliquefaciens CNCM I- 5624. This strain can be used to ferment plant-based composition to increase their L- lysine content.

A growing number of consumers are choosing to enjoy food based on animal protein sources less often, be it as part of a flexitarian diet or as part of a vegetarian or vegan diet. A 2017 study found that 69% of Germans, for example, and 38% of Americans eat meatless meals once a week or more. Global plant-based foods sales are set to hit USD 5bn this year. Plant-based foods can have a positive impact on the environment as well, as they may use less water and CO ₂ in production.

One way to further increase the demand of plant-based foods is to further increase their nutritional profile. Oftentimes, today, for example plant-based milk alternatives do not yet achieve the nutritional profile of milk-based products, in particular if the plant-based milk alternative is based on a single-source plant-based material. Today the problem is usually overcome by combining several plant-based sources, or by supplementing the plant-based composition with specific micro- and/or macro nutrients.

Another way to further increase the demand of plant-based foods is to further increase their quality and taste profile to match the products of animal origin (e.g. milk or meat). Here the inventors propose to use fermentation to improve the taste, texture and nutritional profile of plant-based products.

In general, fermentation is known to be technology that can reduce off-flavours, liberate new flavours, optimize textural aspects, reduce anti-nutritional factors, and - also - to synthesize nutritional compounds such as vitamins and amino acids.

Advanced Materials Research (Volumes 343-344) Page 1042-1048 discloses that Peanut meal (PNM) is considered an inferior protein supplement and used as an inexpensive source of protein. In order to improve the nutritional qualities of PNM, it was fermented by Bacillus subtilis AS1.

L-lysine is the biologically active form of lysine. It plays a key role in proteinogenesis, connective tissues, calcium homeostasis, and fatty acid metabolism. Importantly, the human body cannot synthesize L-lysine. It is therefore considered an essential amino acid in humans and must be obtained from the diet. However, in vegetarian diets, the intake of lysine is sometimes insufficient due to lower quantities of lysine in cereal crops compared to meat and milk.

It would hence be desirable to have available a process that allows it to improve taste and texture of plant-based compositions, in particular, single-source plant-based compositions, while - at the same time - increasing their L-lysine content.

To achieve these benefits via fermentation, the right microbial strains have to be selected. Further improvements can be achieved by optimizing process conditions, especially to combine the optimization of flavour and nutritional aspects. It would therefore be desirable to have available a method to treat plant-based compositions, in particular, single-source plant-based compositions, for example, plant-based milk alternatives, that allows it to increase improve their amino-acid profile by increasing their L-lysine content while having a positive impact on taste and flavor.

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.

The objective of the present invention was it to provide a method to increase the L- lysine content in plant-based compositions, in particular single-source plant-based compositions, for example plant-based milk alternatives while improving their taste and/or texture profile, or to at least provide a useful alternative to existing solutions available in the art.

The inventors were surprised to see that the objective 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, the present invention provides a use of a composition comprising Lacticasei bacillus paracasei to enrich the L-lysine content in plant-based compositions through fermentation.

The present invention further provides a use of a composition comprising Bacillus amyloliquefaciens to enrich the L-lysine content in plant-based compositions through fermentation. The present invention further provides Bacillus amyloliquefaciens CNCM 1-5624. This strain can, for example, be used to ferment chickpea-based milk alternatives to increase the L-lysine content.

As used in this specification, the words "comprises", "comprising", and similar words, are not to be interpreted in an exclusive or exhaustive sense. In other words, they are intended to mean "including, but not limited to".

The present inventors have shown that the amino-acid profile of a plant-based composition can be improved through fermentation of the plant-based composition with Lacticaseibacillus paracasei. The amino acid profile is improved by increasing the L-lysine content of the plant-based composition through fermentation with Lacticaseibacillus paracasei. This could be achieved while - at the same time - removing and/or masking undesired off-flavours of the plant-based composition. Fermentation further allowed it to produce a pleasant texture in the plant-based composition.

The inventors could further show that Lacticaseibacillus paracasei and Bacillus amyloliquefaciens act synergistically to achieve the objective of the present invention. Finally, the inventors have identified certain specific Lacticaseibacillus paracasei and Bacillus amyloliquefaciens strains that allowed to produce particularly promising results.

Figure 1 shows lysine content in chickpea milk before and after fermentation by Lacticaseibacillus, and Bacillus.

Figure 2 shows lysine content in sunflower seed milk before and after fermentation by single and combined Lacticaseibacillus and Bacillus and Propionibacteria (2M: mix fermentation by a P. freudenreichii strain and L. paracasei NCC 2511; 3M: a P. freudenreichii strain + L. paracasei NCC 2511 + B. natto NCC 156).

Figure 3 shows sensory improvement in chickpea milk after fermentation by Lacticaseibacillus and Bacillus.

Figure 4 shows how the addition of small amounts of citrate into the fermentation medium significantly increased the l-lysine biosynthesis, for example when fermenting with NCC156.

Consequently, the present invention relates in part to the use of of a composition comprising Lacticaseibacillus paracasei to enrich the L-lysine content in plant-based compositions through fermentation.

The present invention relates in part to the use of of a composition comprising Lacticaseibacillus amyloliquefaciens to enrich the L-lysine content in plant-based compositions through fermentation.

The plant-based compositions may be a single-source plant-based composition. A plant-based composition may be considered a single source plant-based composition, if at least at least 75 weight-%, at least 90 weight-%, at least 95 weight-%, at least 99 weight-% or 100 weight-% of its protein content is derived from one plant origin, for example from chickpea.

Lacticaseibacillus paracasei (L. paracasei) is a well-studied gram-positive, facultatively heterofermentative species of lactic acid bacteria. They are frequently used in food technology, for example in dairy product fermentation and as probiotics. For the purpose of the present invention, a composition shall be considered as plantbased, if at least 75 weight-%, at least 90 weight-%, at least 95 weight-%, at least 99 weight-% or 100 weight-% of its protein content is not from animal origin. For example, a composition may be considered as plant-based, if at least 75 weight-%, at least 90 weight-%, at least 95 weight-%, at least 99 weight-% or 100 weight-% of its protein content is from plant origin.

The plant-based composition may be a plant-based food composition. For the purpose of the present invention, the term "food" shall mean in accordance with Codex Alimentarius any substance, whether processed, semi-processed or raw, which is intended for human consumption, and includes drink, chewing gum and any substance which has been used in the manufacture, preparation or treatment of "food" but does not include cosmetics or tobacco or substances used only as drugs.

The inventors were able to demonstrate, that by fermentation with Lacticaseibacillus paracasei the L-lysine content in plant-based compositions could be increased significantly, for example, by at least 4 %, by at least 5 %, by at least 7 %, or by at least 10 %.

The inventors have carried out further studies to even further improve the L-lysine generation and were surprised that the addition of Bacillus amyloliquefaciens allowed to even further increase the L-lysine generation. The inventors were surprised to see that Lacticaseibacillus paracasei and Bacillus amyloliquefaciens acted synergistically in improving the L-lysine content in a plant-based composition, for example in a chickpeabased milk alternative.

Consequently, in the use of the present invention the composition may further comprise Bacillus amyloliquefaciens. Natto is a traditional food obtained from fermented boiled soybeans. Bacillus subtilis natto (Bacillus amyloliquefaciens) is a potential probiotic bacterium cultured from natto, and is a gram-positive spore-forming bacterium, for example described in Journal of Dairy Science, Volume 93, Issue 12, December 2010, Pages 5851-5855.

The inventors have obtained particular promising results when the Lacticaseibacillus paracasei was Lacticaseibacillus paracasei NCC 2511.

All strains were deposited under the Budapest treaty.

Lacticaseibacillus paracasei NCC 2511 equals ATCC 25302 (American Type Culture Collection) / DSM 5622 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. This strain is commercially available from many sources, for example from JCM RIKEN, Japan.

The inventors have obtained particular promising results when the Bacillus amyloliquefaciens was Bacillus amyloliquefaciens NCC156.

Bacillus amyloliquefaciens NCC156 is identical to Bacillus amyloliquefaciens NCC199 and was deposited under the Budapest treaty with the Institut Pasteur (28 rue du Docteur Roux 75024 Paris Cedex 15) and was designated CNCM 1-5624.

Hence, the subject matter of the present invention comprises Bacillus amyloliquefaciens NCC156.

The inventors have tested different combinations of Bacillus amyloliquefaciens and Lacticaseibacillus paracasei. While they all achieved the objective of the present invention and were surprisingly more effective than Lacticaseibacillus paracasei alone a combination of Bacillus amyloliquefaciens NCC156 and Lacticaseibacillus paracasei NCC 2511 stood out in terms of L-lysine generation. Hence, in one embodiment of the use of the present invention the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens NCC156 and the Lacticaseibacillus paracasei is Lacticaseibacillus paracasei NCC 2511.

The fermentation may be carried out in an aerobic state or in an anaerobic state. In a particular preferred embodiment of the present invention, the fermentation is carried out in two steps, wherein one step is carried out in an aerobic state and the other step is carried out in an anaerobic state. The inventors have obtained best results when the first step of the fermentation was carried out in an aerobic state and the second step of the fermentation was carried out in an anaerobic state.

In the use of the present invention the fermentation may be carried out using the steps of preparing a suspension of the plant protein with water, preparation of a preculture of the microorganisms, and the fermentation of the plant protein suspension with the preculture.

The inventors have obtained very good results when the fermentation of the plant protein suspension comprised an aerobic and an anaerobic fermentation step. For example, the fermentation of the plant protein suspension may be carried out at room temperature or at temperature in the range of about 25°C to 35°C, for example 28°C to 32°C. The inventors have obtained good results when the fermentation of the plant protein suspension was carried out for about 12 - 72 hours, for example 24 - 48 hours.

In one embodiment, the fermentation of the plant protein suspension comprises a first aerobic fermentation step for 12-48 hours, for example 20-28 hours and a second anaerobic fermentation step for 24-60 hours, for example 44-52 hours. In another embodiment, the fermentation of the plant protein suspension comprises a first anaerobic fermentation step for 24-60 hours, for example 44-52 hours and a second anaerobic fermentation step for 12-48 hours, for example 20-28 hours.

For example, the fermentation may be carried out as described in the Examples.

Surprisingly, the inventors have also found that the biosynthesis of L-lysine could be significantly enhanced, when small amounts of citrate were added to the fermentation medium. The inventors have obtained very good results, when the fermentation medium contained about 5 - 50 mM citrate, for example, about 5 - 30 mM citrate, further for example about 5-20 mM citrate. In one embodiment the fermentation medium contained about 8 - 12 mM citrate.

Hence in one embodiment of the use of the present invention, the fermentation is carried out in the presence of about 5 - 50 mM citrate, for example, about 5 - 30 mM citrate, further for example about 5-20 mM citrate.

In a particular preferred embodiment of the present invention the composition used in the present invention comprises Bacillus amyloliquefaciens, for example Bacillus amyloliquefaciens NCC156 and the fermentation is carried out in the presence of about 5 - 50 mM citrate, for example, about 5 - 30 mM citrate, further for example about 5- 20 mM citrate.

After fermentation the plant-based compositions with an enriched L-lysine content may be used as a natural source of L-lysine for vegan products such as meat alternatives, dairy alternatives, cheese alternatives, milk alternatives, bacon alternatives, or seafood alternatives, for example, while improving taste due by flavor generation and/or removal of off-flavor. The fermented plant-based compositions may be used as a final fermented product (e.g., as cheese alternative, or as a plant-based milk alternative) or as an ingredient to be added to other products (e.g., as ingredient for a meat alternative products).

Hence, in the use of the present invention the plant-based composition may be selected from the group consisting of ingredients for plant-based products and plantbased milk product alternatives, for example, plant-based milk alternatives or plantbased cheese alternatives.

Depending on the intended purpose of the fermented composition and its desired properties, the plant-based composition may be based on a single plant protein source or on a combination of plant protein sources.

For example, in the use of the present invention the plant-based composition may comprise plant proteins derived from sources selected from the group consisting of the Fabacea family, the Leguminosa family, nuts, seeds, sasha inshi, sunflower, chia or combinations thereof.

In one embodiment of the present invention, the plant proteins may be derived from pulses. For example, the pulses may be selected from the group consisting of peas, chickpeas, lentils, beans, or combinations thereof.

Typically, the peas may be selected from the group consisting of garden peas, cow peas, blackeye peas, field peas or combinations thereof. The beans may be selected from the group consisting of dry beans, kidney deans, haricot beans, butter beans, adzuki beans, mungo beans, green gram, black gram, scarlet runner beans, broad beans, field beans, horse beans, faba beans, or combinations thereof. For example, the plant-based composition is based on chick-pea. A composition shall be considered as based on chick-pea, if at least 75 weight-%, at least 90 weight-%, at least 95 weight-%, at least 99 weight-% or 100 weight-% of its protein content is from chick-pea origin.

In the use of the present invention the plant protein in the plant-based composition may contribute at least 75 weight-% of the total protein content of the composition, at least 85 weight-% of the total protein content of the composition, or at least 95 weight-% of the total protein content of the composition. In one embodiment of the present invention, the plant protein in the plant-based composition may contribute 100 weight-% of the total protein content of the composition.

A flavor analysis and a sensory evaluation has shown the fermentation of the use of the present invention allows it to improve the taste and/or the smell of the plant-based composition. Hence, the framework of the present invention comprises the use of the present invention to improve the smell of plant-based compositions.

In particular, it was possible to impart a freshness and a sweetness to the smell of the plant-based composition. Hence, in the use of the present invention the fermentation with Lacticaseibacillus paracasei and/or Bacillus amyloliquefaciens improves the freshness and sweetness of the smell of the plant-based compositions.

The present inventors have also tasted the plant-based composition fermented in accordance with the present invention and have found that the fermentation leads to an improved taste and an improved mouthfeel of the tested composition. Hence, the framework of the present invention further comprises the use of the present invention to improve the taste and/or the texture of plant-based compositions. Those skilled in the art will understand that they can freely combine all features of the present invention disclosed herein. Features described for different embodiments of the present invention may be combined. 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.

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:

Plant milk fermentation

1. Plant milk media preparation

A chickpea suspension (in the following named chickpea milk due to its milk-like appearance) was prepared by mixing 10 % (w/w) chickpea flour with deionized water. A two-step heat treatment was applied for sterilization. First, the chickpea suspension was stirred (250 rpm) for 2 h at 75 °C, followed by autoclaving (121 °C, 15 min). Pea milk was prepared by dissolving 10 % (w/w) pea flour in deionized water, and was autoclaved (121 °C, 15 min).

Sunflower seed milk was prepared by mixing 7 % (w/w) of 55 % sunflower protein with deionized water, then pasteurized under 95 °C for 6 h. Prior to microbial fermentation, the suspension was manually homogenized.

2. Preculture preparation o Bacillus amyloliquefaciens NCC 156 (identical to Bacillus amyloliquefaciens NCC 199):

Preculture condition (Pl, from glycerol stock):

Media: Modified tryptic soy broth, shaker (30 degree, 80 % humidity, 230 rpm), overnight

Preculture condition (P2, 2 % from Pl):

Media: Modified tryptic soy broth, shaker (30 degree, 80 % humidity, 230 rpm), around 8 h

Modified tryptic soy broth containing 17.0 g of tryptone (Becton Dickinson), 5.0 g of NaCI, 3.0 g of soytone (Becton Dickinson), 2.5 g of K ₂ HPO ₄ , and 1.0 mL of 30% silicone antifoam (Sigma-Aldrich) per litre. o Lacticaseibacillus paracasei N CC 2511 :

Preculture condition (Pl, from glycerol stock):

MRS (De Man, Rogosa and Sharpe broth), incubator, aerotolerant cultivation (30 degree, no humidity control, no shaking) overnight Preculture condition (P2, 2 % from pl):

MRS, incubator (30 degree, no humidity control, no shaking) overnight o A Propionibacterium freudenreichii strain:

Preculture condition (Pl, from glycerol stock):

M91, anaerobic jar in incubator (30 degree, no humidity control, no shaking), 48 h

Preculture condition (P2, 2 % from pl):

M91, anaerobic jar in incubator (30 degree, no humidity control, no shaking), overnight

3. Main culture fermentation related to Figure 2

Lacticaseibacillus paracasei NCC 2511+ a Propionibacterium freudenreichii strain:

Anaerobic jar in incubator (30 degree, no humidity control, no shaking), 48 h

Lacticaseibacillus paracasei NCC 2511+ a Propionibacterium freudenreichii strain + Bacillus amyloliquefaciens NCC 156; a Propionibacterium freudenreichii strain + Bacillus amyloliquefaciens NCC 156 :

1) Anaerobic jar in incubator (30 degree, no humidity control, no shaking), 48 h; then shaker (30 degree, 80 % humidity, 130 rpm), 24 h

2) Shaker (30 degree, 80 % humidity, 130 rpm), 24 h; then anaerobic jar in incubator (30 degree, no humidity control, no shaking), 48 h.

During the main fermentation, 130 rpm instead of 230 rpm, to avoid evaporation and to keep the balance between anaerobic and aerobic strains.

4. Addition of citrate

The inventors have carried out experiments where citrate (10 mM) was spiked into the fermentation medium. For B. amyloliquefaciens NCC 156, this resulted in 27% increased L-lysine production to 0.257mg/g fermented chickpea milk after 24 h.

Analysis of lysine

• Hydrolysis of the test portion in 6 mol/L hydrochloric acid under nitrogen.

Evaporation of an aliquot and dilution in appropriate buffer. • After separation by ion-exchange chromatography, individual amino acids are detected at 570nm (except for Proline detected at 440nm) after a post-column derivatization with ninhydrin reagent. Quantification is performed by comparison of the signal obtained for amino acids in sample hydrolysates with standard solutions (external standard).

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