The present invention relates generally to the field of plant-based food. In particular, the present invention relates to improving the vitamin B12 content of plant-based food products. One embodiment of the present invention relates to the use of a combination of Bacillus amyloliquefaciens and Propionibacterium freudenreichii to enrich the vitamin B12 content in plant-based compositions through fermentation. The present invention further relates to a bacterial strain selected from the group consisting of Bacillus amyloliquefaciens CNCM I-5624, Propionibacterium freudenreichii CNCM I-5623, and Propionibacterium freudenreichii CNCM I-5639.

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.

For example, EP1625794 discloses a fermented milk product containing an increased content of in situ produced vitamin B12, wherein at least bacteria capable of producing vitamin B12 are present.

Vitamin B12 is also known as cobalamin. Vitamin B12 is one of the B-vitamins and is soluble in water. It plays a key role in human metabolism. For example, it is involved in the maturation of developing red blood cells in the bone marrow, in fatty acid metabolism, in amino acid metabolism, in DNA synthesis, and in the myelin synthesis and - consequently - in the normal functioning of the nervous system.

Vitamin B12 is usually obtained in the daily diet from consuming animal-sourced foods, including meat, fish, fowl, milk and eggs. Very few non-animal-based food sources are rich in vitamin B12. As a consequence, many grain-based products are fortified with vitamin B12. Also, consumers electing not to consume animal-based food are frequently advised to consume a dietary supplement comprising vitamin B12 or vitamin B12 fortified foods.

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 vitamin B12 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 their vitamin B12 content while also having a positive impact on taste and flavor. As different bacterial strains deliver different taste and textures in fermentation, it would be desirable to have available an alternative method to what is described in EP1625794. It would also be desirable to have available a fermentation method that delivers particularly high vitamin B12 contents.

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 particular effective method to increase the vitamin B12 content in plant-based compositions, in particular singlesource 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 combination of Bacillus amyloliquefaciens and Propionibacterium freudenreichii to enrich the vitamin B12 content in plant-based compositions through fermentation.

The present invention further provides Bacillus amyloliquefaciens CNCM 1-5624, Propionibacterium freudenreichii CNCM 1-5623, and Propionibacterium freudenreichii CNCM 1-5639. These strains can be used, for example, to ferment sunflower-based plant-based ingredients to increase their vitamin B12 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 vitamin B12 content of a plant-based composition can be improved through fermentation of the plant-based composition with a combination of Bacillus amyloliquefaciens and Propionibacterium freudenreichii. The micronutrient profile of the plant-based composition is improved by increasing the vitamin B12 content through fermentation. 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 Bacillus amyloliquefaciens and Propionibacterium freudenreichii act synergistically to achieve the objective of the present invention. Finally, the inventors have identified certain specific Bacillus amyloliquefaciens and Propionibacterium freudenreichii strains that allowed to produced particularly promising results. The inventors could further show that the use of a combination of Bacillus amyloliquefaciens, Propionibacterium freudenreichii and Lacticaseibacillus paracasei is particularly effective in achieving the objective of the present invention, for example in enriching the vitamin B12 content in plant-based compositions through fermentation.

Figure 1 shows Vitamin B12 content in three kinds of plant milk before and after fermentation. Single means NCC1177, double means NCC1177+NCC2511, triple means NCC1177+NCC2511+NCC156.

Figure 2 shows that mixed-culture fermentation yielded a more balanced volatile compounds composition.

Consequently, the present invention relates in part to the use of a combination of Bacillus amyloliquefaciens and Propionibacterium freudenreichii to enrich the vitamin B12 content in plant-based compositions through fermentation.

The inventors were surprised to see that Bacillus amyloliquefaciens and Propionibacterium freudenreichii acted synergistically in improving the vitamin B12 content in a plant-based composition, for example in a sunflower-based plant-based composition.

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. Bacillus amyloliquefaciens is a well-studied species of bacterium in the genus Bacillus, frequently used in agriculture to strengthen the resistance of roots to pathogens or high salt conditions.

Propionibacterium freudenreichii is another well-studied gram-positive bacterium frequently used in food manufacturing. It has a particularly long usage history in cheese making, in particular in the making of the famous Emmental cheese.

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 a combination of Bacillus amyloliquefaciens and Propionibacterium freudenreichii the vitamin B12 content in plant-based compositions could be increased by at least 200% compared to unfermented plant milk. The inventors have carried out further studies to even further improve the vitamin B12 generation and were surprised that the addition of Lacticaseibacillus paracasei allowed to even further increase the vitamin B12 generation.

The inventors were surprised to see that Bacillus amyloliquefaciens, Propionibacterium freudenreichii and Lacticaseibacillus paracasei acted synergistically in improving the vitamin B12 content in a plant-based composition, for example in a sunflower-based plant-based composition.

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.

Consequently, in the use of the present invention the composition may further comprise Lacticaseibacillus paracasei.

The inventors have obtained particular promising results when the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens CNCM 1-5624; the Propionibacterium freudenreichii is selected from the group consisting of CNCM 1-5639, CNCM 1-5623, or combinations thereof; and Lacticaseibacillus paracasei is ATCC 25302.

For example, the Bacillus amyloliquefaciens may be Bacillus amyloliquefaciens CNCM 1-5624; Propionibacterium freudenreichii may be selected from the group consisting of CNCM 1-5639, CNCM 1-5623, or combinations thereof; and Lacticaseibacillus paracasei is ATCC 25302.

All strains were deposited under the Budapest treaty. Bacillus amyloliquefaciens NCC 156 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.

Propionibacterium freudenreichii NCC 1177 was deposited under the Budapest treaty with the Institut Pasteur (28 rue du Docteur Roux 75024 Paris Cedex 15) and was designated CNCM 1-5623.

Propionibacterium freudenreichii NCC 1186 was deposited under the Budapest treaty with the Institut Pasteur (28 rue du Docteur Roux 75024 Paris Cedex 15) and was designated CNCM 1-5639.

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

Lacticaseibacillus rhamnosus NCC 4007 is described, for example in W02010091992, was deposited under the Budapest Treaty as Lacticaseibacillus rhamnosus CGMCC 1 .3724 with the China General Microbiological Culture Collection Center (CGMCC).

Hence, the subject matter of the present invention comprises Bacillus amyloliquefaciens CNCM 1-5624 and Propionibacterium freudenreichii CNCM 1-5623.

The inventors have tested different combinations of Bacillus amyloliquefaciens and Propionibacterium freudenreichii. While they all achieved the objective of the present invention and were surprisingly more effective than one of the strains alone, a combination of Bacillus amyloliquefaciens is NCC156 and Propionibacterium freudenreichii is NCC 1177 stood out in terms of vitamin B12 generation. Hence, in one embodiment of the use of the present invention the Bacillus amyloliquefaciens is CNCM 1-5624 and the Propionibacterium freudenreichii is CNCM I- 5623.

The inventors have also tested different combinations of Bacillus amyloliquefaciens, Propionibacterium freudenreichii and Lacticaseibacillus paracasei. While they all achieved the objective of the present invention and were surprisingly more effective than one or two of the strains alone, a combination of Bacillus amyloliquefaciens is NCC156, the Propionibacterium freudenreichii is NCC1177 and the Lacticaseibacillus paracasei is NCC2511 stood out in terms of vitamin B12 generation.

Hence, in one embodiment of the use of the present invention Bacillus amyloliquefaciens is CNCM 1-5624, the Propionibacterium freudenreichii is CNCM I- 5623 and the Lacticaseibacillus paracasei is ATCC 25302.

The fermentation may be carried out in an anaerobic state or in an aerobic 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 anaerobic state and the other step is carried out in an aerobic state. The inventors have obtained best results when the first step of the fermentation was carried out in an anaerobic state and the second step of the fermentation was carried out in an aerobic state.

In the use of the present invention the fermentation may be carried out usingthe 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 anaerobic and an aerobic 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 anaerobic fermentation step for 12-48 hours, for example 20-28 hours and a second aerobic 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 aerobic fermentation step for 12-48 hours, for example 20-28 hours.

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

After fermentation the plant-based compositions with an enriched vitamin B12 content may be used as a natural source of vitamin B12 for vegan products such as meat alternatives, dairy alternatives, cheese alternatives, milk alternatives, bacon alternatives, or seafood alternatives, for example, while improving taste by flavor generation and/or the removal or masking of undesired off-flavors.

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 plant- based milk product alternatives, for example, plant-based milk alternatives or plantbased cheese alternatives.

For example, the plant-based composition may be sunflower seed milk. Sunflower seed milk can be prepared, for example, by mixing 7 % (w/w) of sunflower protein Heliaflor 55 (All Organic Treasures GmbH) with deionized water, followed by pasteurization at about 95 °C for about 6 h. Prior to the microbial fermentation, the suspension can then be homogenized by shaking.

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 may be 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.

Further for example, the plant-based composition may be based on sunflower. A composition shall be considered as based on sunflower, 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 Bacillus amyloliquefaciens, Propionibacterium freudenreichii and/or

Lacticaseibacillus paracasei 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:

Example 1

1. Plant milk media preparation o 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). o Pea milk was prepared by dissolving 10 % (w/w) pea flour in deionized water, and was autoclaved (121 °C, 15 min). o 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:

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 (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 Propionibacterium freudenreichii NCC 1177:

Preculture condition (Pl, from glycerol stock):

Medium 91, anaerobic jar in incubator (30 degree, no humidity control, no shaking), 48 h

Preculture condition (P2, 2 % from pl):

Medium 91, anaerobic jar in incubator (30 degree, no humidity control, no shaking), overnight

Composition of Medium 91

Casein peptone, tryptic digest 10.0 g

Yeast extract 5.0 g

Na-lactate 10.0 g

Distilled water 1000.0 ml

Adjust pH to 7.0 - 7.2.

3. Main culture fermentation

Fermentation stage 1) Anaerobic jar in incubator (30 degree, no humidity control, no shaking), 48 h;

Fermentation stage 2) shaker (30 degree, 80 % humidity, 130 rpm), 24 h

4. Analysis of Vitamin B12

Vitamin B12 was analysed following AOAC 2014.02 with small modifications. Briefly, 6 mL of sample were mixed with an extraction buffer (2.5 mL) and cyanide solution (0.1 mL). The solutions were heated at 107 ⁵ C for 20 min. After making up to 10 mL with water, the extracts were centrifuged, and 8.5 mL of supernatant was subjected to automated immunoaffinity clean-up on a Gilson GX-271 ASPEC system. The final eluate was evaporated to dryness and reconstituted in 0.2 mL of water. Analysis was performed by UHPLC-UV, the chromatographic conditions were modified to allow separation of cyanocobalamin and tentatively identified pseudocobalamin. Separation took place on a Waters Atlantis T3 column, 3.0 pm, 4.6 x 150 mm on isocratic mode. Mobile phase was methanol-water (25:75), flow rate 0.3 mL/min.

Example 2

Fermentation condition optimization

1. Preculture preparation of strains are the same as example 1. Precultures were grown in the respective medium under anaerobic or aerobic conditions in flasks.

2. Main fermentation

The main fermentation was conducted in the 1.8L DASGIP Bioreactor system (DASGIP AG, Germany). The plant milk was transferred into glass bioreactor, incubated on the bench top control system for a minimum of 6 hours with the vessel temperature at 30°C. During this time the DO probe was calibrated between 0% and 100%. After the calibration of the DO probe, precultures of Propionibacterium freudenreichii NCC 1177 and Bacillus amyloliquefaciens NCC 156 are inoculated into plant milk to start the fermentation. The pH was monitored using an on-line pH sensor. The culture temperature was controlled at 30°C. For the control of dissolved oxygen (DO) level, pO2 sensor (02 Sen lnPro6800G) was utilized the DO control.

All sensor signals were amplified and then transmitted to a personal computer interfaced with an A/D converter. For anaerobic fermentation (stage 1), filter-sterilized nitrogen gas was sparged through the fermentation broth to maintain an anaerobic environment, whereas air was used to maintain an aerobic condition (stage 2). The DO concentration (or ORP) was controlled by adjusting the volume of air pumped in and the rotation speed of the stirrer for the aerobic fermentation.

3. Sample analysis of vitamin B12

During and after fermentation, samples are taken for vitamin B12 analysis. The analysis method is the sample as in example 1. (Original in Electronic Form)

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