Method and medium for preserving lactic acid bacteria in a viable state

Field of the invention

The present invention relates to methods and media for the preservation of bacteria in a viable state. In particular, the present invention provides methods and media for the animal- free safe storage of preserved lactic acid bacteria.

Background

Lactic acid bacteria (LAB) are a group of gram-positive bacteria that produce lactic acid as a result of carbohydrate fermentation. They have been used for centuries in the fermentation of foods, not only for flavor and texture development, but also because of their ability to produce antimicrobial compounds, which prevent the growth of spoilage or pathogenic microorganisms.

LAB are best known for their role in the preparation of fermented food products, such as yogurt (Streptococcus spp. and Lactobacillus spp.), cheeses (Lactococcus spp.) and, sauerkraut (Leuconostoc spp.). In addition, LAB are also used for pickling of vegetables, baking, wine-making, curing fish, meats and sausages. Their growth lowers the pH due to lactic acid production. This acidification process inhibits the growth of most other microorganisms including the most common human pathogens, thus allowing these foods to obtain prolonged shelf life. The acidity also changes the texture of the foods due to precipitation of some proteins, and the biochemical conversions involved in growth enhance the flavor.

Over the years, microbiologists have developed various methods for the storage or preservation of lactic acid bacteria, including sub-culturing, drying, freezing-drying, and freezing. Other methods, such as storage under liquid paraffin, in distilled water, and liquid drying have also been used. Such methods aim to provide pure, viable and stable cultures of microorganisms on a continuous basis.

However, the methods used to preserve the strains of microorganisms in a collection are empirical and the bacteria are likely to evolve rapidly in response to changes in their environment, and preservation methods may have an influence on the stability of their properties. In the above-indicated methods, lactic acid bacteria are traditionally grown and maintained on media containing animal proteins such as milk, casein, meat peptone, horse serum, bovine

serum albumin, cheese whey, etc., and/or animal derived ingredients such as hemin chloride, etc. and/or ingredients obtained by using enzymes of animal origin such as trypsin, pepsin, pancreatin, etc. Moreover, it is well known in the art to cryo-preserve lactic acid bacteria by using animal components such as sweet whey solids, dried skim milk, dried whole milk, bovine serum albumin or horse serum.

For example, FR 2831395 discloses the use of a deposited Lactobacillus plantarum strain for the fermentation of soya juice and/or any other similar soya based product. The L. plantarum strain is isolated from vegetable sources and used for the fermentation of soymilk for the preparation of vegetable dairy analogues. This strain is preserved in liquid nitrogen on a medium containing animal-derived ingredients (MRS broth).

However, when using LAB that have been preserved and grown on media as indicated above for preparing dairy analogues, several problems may occur, including relatively long fermentation times, off-flavors, loss of specific metabolic activity of the LAB, less survival of the lactic acid bacteria in prepared dairy-free products and/or limited shelf life of the prepared dairy-free products.

In addition, the use of LAB that have been preserved and grown on media as indicated above in vegetarian or vegan foods is an ethical problem for specific consumer groups such as vegetarians and vegans and several religious groups such as in case of beef meat peptone for Jews and Hindi or in case of pork meat peptone for Muslims. Despite the number of methods available for preservation of lactic acid bacteria, it is clear that improved methods are needed. Improved methods should be economical, easy and safe to use, and provide for long-term viability of preserved cultures.

The present method therefore aims to provide an improved method and medium for preserving lactic acid bacteria in a viable state. It is a further object of the present invention to provide an improved bacteria culture of lactic acid bacteria for use in the preparation of food products, preferably fermented food products such as fermented dairy analogues. More in particular, it is an object of the present invention to provide lactic acid bacteria that can be used for preparing food products, e.g. fermented dairy analogues, in a more effective way and wherein preparation (fermentation) requires shorter fermentation times.

It is further an object of the invention to provide lactic acid bacteria that can be used for preparing food products, preferably fermented products, wherein said food products, e.g. dairy analogues, show improved quality, nutritional and/or organoleptic properties.

Summary The present invention provides an improved animal-free method for preserving bacteria, and in particular lactic acid bacteria, in a viable state. The invention provides animal-free pure, viable and stable cultures of lactic acid bacteria which are particularly suitable for preparing vegetable dairy analogues.

In a first aspect, the invention relates to a method for preserving lactic acid bacteria in a viable state. The method comprises the steps of: a) isolating the bacteria from their natural environment or from a culture collection on a suitable isolation medium; and b) adapting said isolated bacteria by growing said bacteria on a suitable adaptation medium. The method is in particular characterized in that the isolation and adaptation medium is an animal-free medium. In a preferred embodiment, the isolation medium and the adaptation medium are 100% vegetable media.

In a preferred embodiment, the method comprises the steps of: a) isolating said bacteria from their natural environment or from a culture collection on a suitable animal-free isolation medium; b) adapting said bacteria on a suitable animal-free adaptation medium; c) culturing the adapted bacteria at their optimal growth temperature in a suitable animal- free culture medium to increase cell mass; d) forming an animal-free cell concentrate from said grown bacteria; e) optionally forming an animal-free cell suspension by adding at least one cryopreservation agent to the animal-free cell concentrate obtained in step d), f) freezing the animal-free cell concentrate of step d) or the animal-free cell suspension of step e) to produce a frozen animal-free cell concentrate or cell suspension or freeze-drying the animal-free cell concentrate of step d) or the animal-free cell suspension of step e) in a vessel to produce a freeze-dried animal-free cell concentrate or cell suspension; g) storing the frozen animal-free cell concentrate or cell suspension or the freeze-dried animal-free cell concentrate or cell suspension at appropriate storing conditions until such time as it is needed;

characterized in that steps a) to e) are carried out under animal-free conditions. The method is characterized in that steps a) to e) are carried out under animal-free conditions.

In a further embodiment, the present method comprises the further step of re-animating said frozen animal-free cell concentrate or cell suspension or said freeze-dried animal-free cell concentrate or cell suspension by passing said cell concentrate or cell suspension before use on an animal-free or 100% vegetable medium.

In accordance with the methods of the present invention, the bacteria are preserved in an animal-free viable state in a depository institution according to the Budapest treaty. In another aspect, the invention also encompasses lactic acid bacteria that have been preserved according to the present method and a bacteria culture comprising one or more of such lactic acid bacteria.

In yet another aspect, the invention relates to the use of lactic acid bacteria or of a bacteria culture as defined herein in food preparation processes, in food fermentation processes, for the preparation of dietary supplements and preferably for preparing a fermented dairy analogue. The fermented dairy analogue preferably is a vegetable food product, a vegetable food ingredient, or a vegetable functional food. More preferably, the fermented dairy analogue is derived from Leguminosae (Fabaceae) such as soya, bean, pea, lupin, etc.

The present invention provides a method for preserving lactic acid bacteria wherein all steps in the preservation method are performed in animal-free conditions and/or media. Therefore, in accordance with the present method contamination of compounds of animal origin is avoided during the complete preservation and production process of the bacteria.

Moreover, the preserved bacteria according to the invention can be used in fermentation processes for preparing fermented dairy analogue. Provided that also all steps of such fermentation processes take place under animal-free conditions, the present invention thus provides a method for preparing fermented dairy analogues which is safer and more transparent, and which provides 100% traceability with regard to components of animal origin. In fact, contamination of compounds of animal origin is avoided during the complete fermentation process, including the initial stage of re-animating the lactic acid bacteria that have been preserved under animal-free conditions.

The applicants have further found that by using lactic acid bacteria that have been preserved and adapted in a 100% vegetable medium, reduced fermentation times are obtained for preparing fermented dairy analogues. Moreover, survival of the lactic acid bacteria during shelf life of prepared dairy analogues is also increased. Furthermore, using lactic acid bacteria that have been preserved and adapted in a 100% vegetable medium as a ferment in the present method permits to improve structural, nutritional and/or organoleptic properties of prepared dairy products, including improved digestibility,

Another advantage of the present bacteria culture is that it does not contain compounds of animal origin. The bacteria culture can advantageously be used to prepare dairy analogues that are suitable for specific consumer groups such as vegetarians, vegans, or religious groups such as Jews or Muslims. Furthermore, use of the present bacteria cultures removes the risk of transmitting animal derived contamination, such as bovine spongiform encephalopathy (BSE) or other infectious and harmful agents, into consumers of the prepared dairy analogues.

With the insight to better show the characteristics of the invention, some preferred embodiments and examples are described hereafter.

Description of the figures

FIG. 1 illustrates the pH course in a soya-milk that is being fermented with Lactococcus lactis S (LMG-23669).

FIG. 2 represents SDS PAGE profiles of L. casei T (LMG P-23506) on S medium (FIG. 2A), L. casei J (LMG P-23506) on MRS medium (FIG. 2B) and of L. casei 6904 on MRS medium (FIG. 2C).

FIG. 3 shows the gradual transition in typical IR spectrum when comparing L. casei V (LMG P-23504) on S medium (VS), adapted on S medium and re-adapted on MRS medium (VSM), adapted on MRS medium and re-adapted on S medium (VMS) and adapted to MRS medium (VM).

FIG. 4 represents a 2D-SDS PAGE profiles of L. casei V (LMG P-23504) on S medium (FIG. 4A), L casei V (LMG P-23504) on MRS medium (FIG. 4B). Arrows indicate the differences between both gels.

FIG. 5 illustrates the speed of fermentation of a soy-milk + 2% glucose when using Lactococcus lactis S (LMG P-23669) isolated, adapted and maintained on S or MRS medium.

FIG. 6 illustrates the speed of fermentation of a soy-milk + 2% glucose when using L. casei V (LMG P-23504) isolated, adapted and maintained on S or MRS medium.

FIG. 7 illustrates the speed of fermentation of a soy-milk + 2.5% raffinose when using B. infantis (LMG P-24096) isolated, adapted and maintained on S or MRS medium.

FIG. 8 illustrates fermentation at a constant pH of 5.9 of a soy-milk + 2.5% raffinose when using B. infantis (LMG P-24096) isolated, adapted and maintained on S or MRS medium.

Detailed description

The present invention provides an improved method for animal-free preservation of bacteria, and in particular lactic acid bacteria, in a viable state. The term "preservation" denotes the process of maintaining lactic acid bacteria under conditions in which their biological activity is reduced while they nonetheless remain viable and may resume essentially normal biological activity when taken out of the preservation state.

As used herein, the term "viability" or "a viable state" refers to the ability of the lactic acid bacteria to grow. For example, a "viable culture" is comprised of live lactic acid bacteria that are capable of metabolism and growth, while a "non-viable culture" is comprised of cells that are either dead or sufficiently damaged that they are not capable of good growth (i.e., no or abnormally small visible colonies are present), even under optimal conditions for their growth.

The present invention differs from prior art preservation methods in that all steps in the present method are performed under conditions and/or using media which are animal-free. The term "animal-free" in this context refers to conditions and/or media wherein components of animal origin are not added. Unintended traces of components of animal origin may be present but do not have any effect on the metabolism and/or enzyme expression pattern of the lactic acid bacteria. Preferably the lactic acid bacteria are preserved and grown according to the invention in an animal-free medium which is a substantially, and preferably a 100%, vegetable medium. The term "substantially" as used in this context refers to a vegetable medium wherein only 100 % vegetable ingredients are used but in which unintended traces of components of animal origin may be present, but wherein such traces do not have any effect

on the metabolism and/or enzyme expression pattern of the lactic acid bacteria. In a preferred embodiment, the animal-free or 100% vegetable medium comprise vegetable components and/or synthetic components derived from vegetable sources. Preferred examples of 100 % animal-free media include media wherein for instance the nitrogen source is a vegetable peptone such as soya peptone and the carbon source is a sugar moiety derived from plants such as fructose, or synthetic media with a defined amino acid composition and synthetic vitamins, which are only derived from non-animal sources.

Lactic acid bacteria

Preferably, the lactic acid bacteria which may be preserved in accordance with the present invention are selected from the genera of the Streptococcus, Lactobacillus, Lactococcus,

Leuconostoc. Pediococcus, Weisella, Oenococcus, Enterococcus, Propionibacterium,

Bifidobacterium, and preferably from the genera of Streptococcus, Lactobacillus, Lactococcus and Bifidobacterium. In an embodiment the lactic acid bacteria comprise Streptococcus salivarius or Streptococcus thermophilus. In another embodiment the lactic acid bacteria comprise Lactobacillus acidophilus, L. bulgaricus, L. casei, L. delbrueckii, L. fermentum, L. plantarum, or L. reuteri. In another embodiment the lactic acid bacteria comprise Lactococcus garvieae, L. lactis, L. cremoris, L. lactis subsp. diacetylactis, L. piscium, or L. raffinolactis. In yet another embodiment the lactic acid bacteria comprise Leuconostoc mesenteroides. In yet another embodiment the lactic acid bacteria comprise Propionibacterium freundchii or Propionibacterium shermani. In still another embodiment the lactic acid bacteria comprise

Bifidobacterium infantis, Bifidobacterium animalis (lactis), Bifidobacterium bifidus,

Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium adolescentis.

In a more preferred embodiment, the lactic acid bacteria comprise Lactobacillus casei V (LMG P-23504), Lactobacillus casei W (LMG P-23505), Lactobacillus casei T (LMG P- 23506), Lactococcus lactis S (LMG P-23669), Bifidobacterium infantis S (LMG P-24096) and/or Streptococcus salivarius ssp. thermophilus (LMG P-24095). The Lactobacillus casei V strain has been deposited to the BCCM/LMG Bacteria Collection, Ledeganckstraat 35 B-9000 Gent, Belgium on April 11 , 2006 (receipt of the deposit by the Depositary Authority on February 17, 2006). The deposited strain has the characteristics of the allocated accession number LMG P-23504. The Lactobacillus casei W strain has been deposited to the BCCM/LMG Bacteria Collection, Ledeganckstraat 35 B-9000 Gent, Belgium on April 11 , 2006 (receipt of the deposit by the Depositary Authority on February 17, 2006). The deposited

strain has the characteristics of the allocated accession number LMG P-23505. The Lactobacillus casei T strain has been deposited to the BCCM/LMG Bacteria Collection, Ledeganckstraat 35 B-9000 Gent, Belgium on April 11 , 2006 (receipt of the deposit by the Depositary Authority on February 17, 2006). The deposited strain has the characteristics of the allocated accession number LMG P-23506. In another embodiment said lactic acid bacteria is Lactococcus lactis S. This strain has been deposited to the BCCM/LMG Bacteria Collection, Ledeganckstraat 35 B-9000 Gent, Belgium on May 15, 2006. The deposited strain has the characteristics of the allocated accession number LMG P-23669. In another embodiment said lactic acid bacteria is Bifidobacterium infantis S. This strain has been deposited to the BCCM/LMG Bacteria Collection, Ledeganckstraat 35 B-9000 Gent, Belgium on April 12, 2007. The deposited strain has the characteristics of the allocated accession number LMG P-24096. In yet another embodiment said lactic acid bacteria is Streptococcus salivarius ssp. thermophilus. This strain has been deposited to the BCCM/LMG Bacteria Collection, Ledeganckstraat 35 B-9000 Gent, Belgium on April 12, 2007. The deposited strain has the characteristics of the allocated accession number LMG P-24095.

Animal-free Isolation and Adaptation medium

In accordance with the present invention, a method is provided wherein lactic acid bacteria are isolated and adapted by growing on a suitable animal-free isolation, and adaptation medium at their optimal growth temperature. The term "growing" as used herein refers to the growth of bacteria. Bacterial growth is herein defined as an increase in bacterial biomass.

Bacteria are isolated on an isolation medium, and preferably a 100% vegetable medium. The isolation step in the present method comprises the single step of bringing the bacteria on a suitable isolation medium, preferably a 100% vegetable isolation medium. The isolation step is the first step of bringing bacteria, e.g. from the environment or from a culture collection, onto a suitable isolation medium as defined herein. Thus, isolation in principle comprises substantially one handling step.

The subsequent adaptation step in the present method preferably comprises growing and frequently re-plating the bacteria on a suitable adaptation medium, preferably a 100% vegetable adaptation medium. The adaptation step implies at least one and preferably more than one re-plating of the bacteria onto a suitable adaptation medium as defined herein.

During this adaptation step, the bacteria are re-plated frequently, e.g. every 3 to 5 days, in order to keep the bacteria in a viable state. Preferably, during these re-plating operations, the largest bacterial colonies are selected. The bacteria are grown at a suitable temperature for a suitable time. For instance, for Lactococcus bacteria, the temperature is preferably around 3O ⁰ C; for Lactobacillus the temperature is preferably 3O ⁰ C or 37 ⁰ C dependent on the strain, for Streptococcus thermophilus the temperature is preferably 42 ⁰ C, while for Bifidobacerium the temperature is preferably around 37°.

This adaptation step is in particular characterized in that the LAB are grown on an animal- free, or 100% vegetable medium. The applicant has shown that by growing and frequently re- plating LAB on a 100% vegetable medium, LAB adapt to such medium. The term "adaptation", when used in the context refers to the phenomena that the LAB have undergone chemical and metabolic modifications, especially with regard to their enzyme metabolism, e.g. by adjusting their enzyme pattern, induction/down regulation of certain enzymes, enzyme activity, enzyme synthesis kinetics, etc., in function of the components present in the vegetable medium. As a result thereof, the LAB are more adapted to vegetable components, and obtain improved capabilities to subsequently ferment products of vegetable origin.

Any standard medium for the culture of bacteria may be used as the basis for preparing the present isolation and/or adaptation medium, insofar as the final composition of the medium is animal-free, or is substantially vegetable, as defined herein. The term "media" may be used in reference to solid plated media which support the growth of the bacteria. Also included within this definition are semi-solid and liquid microbial growth systems, as well as any type of media. Standard solid media can be prepared from any liquid media by the addition of a solidifying agent such as agar.

The isolation and/or adaptation media of the present invention preferably comprise a carbon source, a nitrogen source, a vitamin source, an essential mineral source and optionally a selecting agent, such as a reducing agent as L-cystein or an antibiotic, for selecting the microorganisms to be cultured.

In one embodiment the animal-free isolation medium and the animal-free adaptation medium are a same medium. In another embodiment, the animal-free isolation medium and the animal-free adaptation medium are different media. For example, the adaptation medium can be poorer than the isolation medium, or may comprise components (such as for instance specific peptides derived from a vegetable source, raffinose, stachyose, phytic acid, ...) which improve the adaptation process. In an example, the isolation medium comprises different

fermentable carbohydrates such as fructose, glucose and sucrose and a nitrogen source originating from various vegetable peptones (e.g. from soya and potato), while the adaptation medium is more selective and comprises for instance a specific soya peptone with a specific molecular weight distribution and one fermentable carbohydrate (e.g. fructose). As used herein, the term "carbon source" is used in reference to any compound which may be utilized as a source of carbon for bacterial growth and/or metabolism. Carbon sources may be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, and peptides.

As used herein, the term "nitrogen source" is used in reference to any compound which may be utilized as a source of nitrogen for bacterial growth and/or metabolism. As with carbon sources, nitrogen sources may be in various forms, such as free nitrogen, as well as compounds which contain nitrogen, including but not limited to amino acids, peptones, vitamins, ammonia and nitrogenous salts.

As used herein, the term "mineral source" may reference to the addition of one or more minerals. As used herein, the term "vitamin source" may reference to the addition of one or more vitamins.

As used herein, the term "selecting agent" is used in reference to any compound which inhibits the growth of, or kills microorganisms or stimulates the growth of the selected organism. It is intended that the term be used in its broadest sense, and includes, but is not limited to compounds such as L-cystein or antibiotics which are produced naturally or synthetically. It is also intended that the term includes compounds and elements that are useful for inhibiting the growth of, or for killing microorganisms or for stimulating selected organisms. Selecting agents may be incorporated into media used in the present invention. In this manner, organisms with undesirable characteristics (e.g., contaminants) may be inhibited or killed prior to or during preservation and/or revival of the organisms of interest. Alternatively, by adding stimulating agents, such agents stimulate growth of selected organisms so that the selected organisms have an advantage during their revival.

In a preferred embodiment, the medium according to the invention comprises a vegetable peptone; a yeast component (such as a yeast extract); a vegetable derived fermentable carbohydrate; and a buffer.

Vegetable peptones are complex mixtures of organic and inorganic compounds that are obtained by digestion of protein-containing tissues of plants such as e.g. soybean protein or

soybean meal. Peptones primarily contain peptides and single amino acids. Being crude digests of complex materials, they contain a great variety of other organic and inorganic materials. The peptones may be selected from the group comprising soya, cotton, wheat, malt, corn, potato, bean, lupin, sorghum and/or rice. Preferably the peptone is a soya peptone. Suitable examples of vegetable peptones include but are not limited to a commercial papaic digest of soybean protein (soybean meal), a commercial papaic digest of wheat flour, a commercial papaic digest of potato protein, etc...

Standard procedures well known to those skilled in the art can be used for the preparation of vegetable peptones. For instance, soy bean derived protein compositions can be prepared by enzymatic digestion of soy bean meal or soy isolate using standard vegetable enzymes such as papain. Soy bean derived protein compositions can also be obtained by acid hydrolysis of a soy isolate. To sum up, vegetable peptones are chemical or enzymatic digests of vegetable proteins and contain a mixture of amino acids, small peptides and polypeptides of different size. Preferably said vegetable peptone is present in the medium in an amount of between 0.1 and 10 % by weight, and preferably between 0.5 and 5 % by weight, and more preferably of about 1 % by weight.

In another embodiment, the isolation and/or adaptation medium comprises a yeast component. Such yeast component may be used as a source of amino acids, vitamins, coenzymes and purines and pyrimidines including many needed as growth factors by organisms. The concentration of yeast component in the present medium is preferably comprised between 0.1 and 10 % by weight, and preferably between 0.3 and 5 % by weight, and more preferably of about 0.5% by weight. Yeast components can be prepared by standard procedures well known to those skilled in the art. Furthermore, yeast components are commercially available. The yeast component may consist of an autolyzed yeast, yeast extract (i.e. extract of yeast cells) or an (ultra)filtrated yeast extract.

In a further embodiment, the present vegetable isolation and/or adaptation medium comprises a suitable buffer to maintain the optimum pH range of the organism in a solid medium or liquid medium in order to prevent marked changes in pH which otherwise would result from microbial production of organic acids or bases. Examples of suitable buffers include but are not limited to sodium and potassium phosphates and calcium carbonate. Crude organic preparations such as peptones (see below) also act as buffers. Chemical components such as tricine and MOPS may also be used as buffer components. The concentration of buffer in the present medium is preferably comprised between 0.1 and 10 % by weight; and preferably

between 0.3 and 5 % by weight, and preferably about 0.5% by weight. Preferably the vegetable medium is buffered to a pH of between 5 and 8, and preferably between 6 and 6.5. In an example the vegetable medium is buffered to a pH of 6.3 in the case of a medium for Lactococcus lactis or Lactobacillus casei. In yet a preferred embodiment, the present invention provides an isolation and/or adaptation medium which comprises a vegetable derived fermentable carbohydrate. Carbohydrates are chemical compounds that contain oxygen, hydrogen and carbon atoms. The term "vegetable derived" in this context refers to carbohydrates, which is a large group of sugars, starches, celluloses, and gums, that are substantially derived from a vegetable source such as corn, wheat, chicory, tapioca, rice, potato, beet or cane and that are substantially animal free. The fermentable carbohydrates may include glucose, fructose, sucrose, galactose, etc... This term excludes carbohydrates that are derived from milk or milk products, such as e.g. lactose. Preferably fructose or glucose is used as carbohydrate. The concentration of fermentable carbohydrate in the present medium is preferably comprised between 0.1 and 10 % by weight, and preferably between 0.5 and 5 % by weight, and preferably of 1 % by weight.

For preparing a solid medium, the vegetable medium further contains agar-agar, preferably in an amount of between 1 to 2% by weight, and for instance of 1.5% by weight.

In one embodiment, the invention comprises a method for preserving lactic acid bacteria in a viable state comprising the steps of a) isolating said bacteria from their natural environment or from a culture collection on a suitable animal-free isolation medium, and b) adapting said isolated bacteria by growing said bacteria on a suitable animal-free adaptation medium. Isolated and adapted bacteria that have been obtained according this method can be used in food preparation processes, preferably in food fermentation processes, and more preferably for preparing a fermented dairy analogue, as defined herein. The isolated and adapted bacteria can be added as such without further concentration or culturing to a dairy analogue as a starting material. For the preparation of such fermented dairy analogues the steps that are followed include: a) isolating one or more lactic acid bacteria as indicated above on an animal-free isolation medium; b) adapting said isolated bacteria by growing said bacteria on a suitable animal-free adaptation medium as indicated above; and c) preparing a dairy analogue by adding to said dairy analogue as a starting material a suitable amount of bacteria obtained in step b).

Preferably steps a) to c) are carried out under animal-free conditions.

Preservation method

In a preferred embodiment, the invention comprises a method for preserving lactic acid bacteria in a viable state comprising the steps of: a) isolating said bacteria from their natural environment or from a culture collection on a suitable animal-free isolation medium; b) adapting said bacteria on a suitable animal-free adaptation medium; c) culturing the adapted bacteria at their optimal growth temperature in a suitable animal- free culture medium to increase cell mass; and d) forming an animal-free cell concentrate from said grown bacteria;

The method is characterized in that steps a) to d) are carried out under animal-free conditions. Bacteria that have been obtained according to steps a) to d) of this method can be used in food preparation processes, preferably in food fermentation processes, and more preferably for preparing a fermented dairy analogue, as defined herein. The bacteria can be used directly following their preparation and have not been frozen or freeze-dried before use. The bacteria can be added as such as a starting material to a dairy analogue.

In accordance with the present method, isolated and adapted micro-organisms are cultured at their optimal growth temperature in a suitable animal-free culture medium to increase their cell mass.

Culturing involves the multiplication of adapted bacteria on a larger scale with the aim to obtain sufficient biomass which can be used in a next step for preparing dairy analogues.

In one embodiment the animal-free culture medium and the animal-free adaptation medium are a same medium. In another embodiment, the animal-free culture medium and the animal- free adaptation medium are different media. In a preferred embodiment, the culture medium is a 100% vegetable medium comprising at least a vegetable peptone and a vegetable derived carbohydrate. For example, the animal-free culture medium may consist of a nitrogen source such as soya-peptone, and a fermentable carbohydrate source such as fructose or complex carbohydrate sources such as corn steep liquor, molasses, and a yeast extract. Lactic acid bacteria can be isolated from their natural environment, e.g. from animal oral cavities and intestines, from milk, from plant leaves as well as decaying plant or animal matter

such as decaying vegetables, fecal matter, compost, naturally fermented vegetable products (pickles, sauerkraut, etc).

Lactic acid bacteria can also be isolated from a culture collection, such as culture collections that are present in depository institutions under the Budapest treaty. Isolated bacteria are subsequently grown on an isolation medium, and preferably a 100% vegetable medium.

The grown bacteria are harvested and an animal-free cell concentrate is prepared from the bacteria culture. The concentrate can be obtained, for example, by centrifuging the previously grown bacteria culture, and removing the aqueous supernatant. This step removes any metabolic by-products which may interfere with the bacteria's survival during a freezing or freeze-drying process and/or materials which may inhibit growth after subsequent re- animation. Other methods of preparing a cell concentrate such as microfiltration will be apparent to one skilled in this art.

Optionally, a cell suspension can be prepared from the cell concentrate. For this, the animal- free cell concentrate is suspended in fresh, sterile animal-free medium. The animal-free cell concentrate is preferably diluted with a predetermined volume of a known concentration of fresh animal-free (adaptation) medium to produce an animal-free cell suspension. Previous comments concerning the animal-free medium content used to grow the microorganism are equally applicable with respect to the fresh medium used to form the cell suspension. In particular, the fresh animal-free medium is a 100% vegetable medium.

In a further embodiment, the invention relates to a method for preserving lactic acid bacteria in a viable state comprising the steps a) to d) as defined above and further comprising the steps of: e) optionally forming an animal-free cell suspension by adding at least one cryopreservation agent to the animal-free cell concentrate obtained in step d), f) freezing the animal-free cell concentrate of step d) or the animal-free cell suspension of step e) to produce a frozen animal-free cell concentrate or cell suspension or freeze-drying the animal-free cell concentrate of step d) or the animal-free cell suspension of step e) in a vessel to produce a freeze-dried animal-free cell concentrate or cell suspension;

g) storing the frozen animal-free cell concentrate or cell suspension or the freeze-dried animal-free cell concentrate or cell suspension at appropriate storing conditions until such time as it is needed; characterized in that step e) is carried out under animal-free conditions. When the moisture content of a bacteria culture is removed during the freeze-drying process, molecules are virtually locked in position so that little or no opportunity exists for alteration of the physical or chemical properties of the product. Obviously, in preparing such products it is of prime importance that the viability of the bacteria culture be maintained. In order to protect the bacteria, the freeze-drying process generally is carried out in the presence of one or more cryoprotectant agents, designed to minimize cellular damage and increase survivability of the bacteria during the freeze-drying process.

In accordance with the present method, the animal-free (adaptation) medium may therefore further comprise at least one a cryopreservation agent. In a highly preferred embodiment, the cryopreservation agent is an animal-free agent. Suitable examples of cryopreservation agents are glycerol, mannitol, dimethylsulfoxide (DMSO), sugars such as fructose, sucrose, dextrose or trehalose, liquid carbon dioxide, dimethyl sulfoxide, ethylene glycol, poly ethylene glycol, propylene glycol, polyvinyl pyrrolidone (PVP), formamide, monosodium glutamate, hydroxy ethyl starch (HES), dextran, maltodextrins or any combinations thereof.

In a further step, the animal-free cell concentrate or the animal-free cell suspension is frozen to prepare an animal-free frozen cell concentrate or an animal-free frozen cell suspension, alternatively, the animal-free cell concentrate or the animal-free cell suspension are freeze- dried in a suitable vessel to prepare an animal-free freeze-dried cell concentrate or an animal- free freeze-dried cell suspension. Preferably, the cell concentrate or the cell suspension is frozen or freeze-dried as soon as possible after its preparation. Generally, a wide variety of glass and in some cases plastic ampoules and vials are typically used. Transparent vessels are particularly preferred as these permit visual inspection and optical analysis. The vessel typically has a volume below about one liter, and vessels with a volume below 100 ml will generally be used. The vessel is cleaned, sterilized and properly labeled before being used. A complete discussion of procedures used to freeze or freeze-dry the cell concentrate or the cell suspension is beyond the scope of the present invention and procedures readily available to the prior art have been found suitable.

In case of the freeze-drying of the cell concentrate or cell suspension, after the last desired amount of moisture has been removed, the vessel containing the freeze-dried preparation of

lactic acid bacteria and nutrients is properly sealed and is now ready for storage. The vessel containing the freeze-dried preparation can be stored under appropriate storing conditions, e.g. at room temperature, refrigerated, or frozen. One skilled in this art will recognize whether a particular bacterial strain requires additional special storage conditions, otherwise standard storage practices are proper.

The storage vessel contains a quantity of frozen or freeze-dried lactic acid bacteria and frozen or freeze-dried nutrients such that upon proper re-animation an active bacteria culture can be produced in situ, which culture undergoes substantial logarithmic growth from an initial cell population to an ending cell population. As used herein, the term "re-animation" refers to the process of reviving a preserved culture. While it is not intended that the present invention be limited to this method, re-animation is intended to encompass the thawing in case of a frozen preparation or the addition of liquid or fluid to the preserved culture in case of a freeze dried preparation. The suspension of organisms is then used to inoculate suitable microbiological media. The inoculated media are then incubated under conditions suitable for the growth of the organisms, and colonies of organisms present in the preserved culture observed.

In an example, the freeze-dried preparation is re-animated by mixing the freeze-dried preparation with suitable animal-free nutrient (adaptation) medium or a buffer solution, etc. Thereafter, propagation of the bacteria normally is accomplished by inoculating freshly prepared animal-free restoration (adaptation) media with all or part of the rehydrated preparation. Generally, the animal-free restoration medium employed for restoring the bacteria has the same animal-free nutrient components as the animal-free (adaptation) medium initially used to grow the culture.

The present invention provides animal-free bacteria cultures that have long shelf lives. The preserved bacteria are able to maintain their viability in animal-free conditions for long periods of time. The viability of the bacteria culture is measured following revival of the preserved organisms.

As mentioned above, the present invention provides a method for preserving lactic acid bacteria wherein all steps in the preservation method are performed in animal-free conditions and/or media. Therefore, contamination of compounds of animal origin is avoided during the complete preservation process of the bacteria.

Use of the preserved lactic acid bacteria

Lactic acid bacteria that have been preserved according to the present method and/or a bacteria culture comprising one or more of such lactic acid bacteria can advantageously be used in food preparation processes, in the preparation of dietary supplements and in food fermentation processes, for instance for preparing animal-free food preparations such as a fermented dairy analogue.

The Applicant has shown that lactic acid bacteria that have been preserved according to the present method obtain particular characteristics which are not observed in lactic acid bacteria that have been preserved according to a method different from that disclosed herein. It was shown that lactic acid bacteria that have been preserved according to the present method show an increase in activity of one or more enzymes compared to lactic acid bacteria that have not been preserved according to the present method. More in particular, such lactic acid bacteria show an increase in activity of one or more enzymes selected from the group comprising acid phosphatase, alkaline phosphatase, α-chymotrypsin, α-galactosidase and β- glucosidase.

In a preferred embodiment, the invention provides lactic acid bacteria as disclosed herein showing an increase in activity with at least 5%, preferably at least 10%, preferably with at least 15%, preferably with at least 20% and more preferably with at least 30% of one or more of the above given enzymes. Enzyme activity may for instance be increased with 15, 18, 20, 22, 25, 27, 30, 35, 40, 45 or even 50%.

The term "dietary supplement" as used herein is intended to refer to products comprising nutrients such as vitamins, minerals, fatty acids or amino acids, that are missing or not consumed in sufficient quantity in a person's diet. Dietary supplements may have medical utility. An example hereof is for instance a cell concentrate of probiotic bacteria such as Bifidobacterium, which is most often freeze-dried and packed in individual capsules. These capsules can be taken for therapeutic reasons, to prevent certain disease conditions or to improve gut health.

The term "dairy analogue" as used herein is meant to refer to a product of vegetable origin and may include a vegetable food product, a vegetable food ingredient, or a vegetable functional food. The term "of vegetable origin" indicates that the product only contains compounds derived from plants.

The term "food" or "food product" is used herein in a broad sense, and covers food for humans as well as food for animals (i.e. a feed). In a preferred aspect, the food is for human consumption. The food may be in the form of a solution or as a solid, depending on the use and/or the mode of application and/or the mode of administration. Non limitative examples of "vegetable food products" which may be obtained using a bacteria culture according to the present invention include for instance, yoghurts and drinking yoghurts; cheese, cheese sauce, (sour) cream, whipping cream, whipped topping, ice cream, water ices and desserts, confectionery, biscuits cakes and cake mixes, snack foods, fruit fillings, cake glaze, chocolate bakery filling, cake fillings, cake and doughnut icing, instant bakery filling creams, filing for cookies, ready-to-use bakery filling, reduced calorie filling, nutritional beverage, acidified soy/juice beverage, beverage powders, calcium fortified soymilk, aerated frozen desserts. In a preferred embodiment, preferably the present invention may be used in connection with soya based yoghurt and cheese production, such as fermented soya yoghurt drink, soya yoghurt, soya drinking yoghurt, soy cheese, fermented soy cream, soya based desserts and others.

The term "vegetable food ingredient as used herein includes a formulation which is or can be added in the preparation of other foodstuffs. The food ingredient may be in the form of a solution or as a solid-depending on the use and/or the mode of application and/or the mode of administration. As an example a fermented soy cream can be used as ingredient for the preparation of ready-meals; a fermented soy yoghurt can be used as an ingredient for soya milk shake or for soya ice cream which is based on soya yoghurt.

As used herein, the term "vegetable functional food' means vegetable food which is capable of providing not only a nutritional effect and/or a taste satisfaction, but is also capable of delivering a further beneficial effect to consumer. Accordingly, functional foods are ordinary foods that have components or ingredients (such as those described herein) incorporated into them that impart to the food a specific functional, e.g. medical or physiological benefit, other than a purely nutritional effect. Some functional foods are nutraceuticals. Here, the term "nutraceutical" means a food which is capable of providing not only a nutritional effect and/or a taste satisfaction, but is also capable of delivering a therapeutic (or other beneficial) effect to the consumer. Nutraceuticals cross the traditional dividing lines between foods and medicine. As an example an animal-free Bifidobacterium preparation can be cited, which is added to soy yoghurt or fruit preparations for the preparation of a vegetable functional food.

Such a vegetable functional food could be a nutraceutical in that it contributes to a better gut health, lowering cholesterol, stimulate immunity, etc.

For the preparation of fermented dairy analogues the steps that are followed include: a) isolating one or more lactic acid bacteria as indicated above on an animal-free isolation medium; b) adapting said isolated bacteria by growing said bacteria on a suitable animal-free adaptation medium as indicated above; c) culturing said adapted bacteria in a suitable animal-free culture medium, and d) preparing a dairy analogue by adding to said dairy analogue as a starting material a suitable amount of bacteria obtained in step c).

Preferably steps a) to d) are carried out under animal-free conditions.

Step a) and b) can be carried out as explained above. Step c) comprises the multiplication or culturing of adapted or re-animated bacteria in a suitable animal-free culture medium. Such animal-free culture medium may comprise vegetable peptones as defined herein, e.g. from soya, wheat, rice, potato; yeast components as defined herein; vegetable derived fermentable carbohydrates; or complex carbohydrate sources such as corn steep liquor, molasses, etc...

It will be clear that temperature and time required for the culturing step will depend on the type of lactic acid bacteria. Also, the culturing is carried out at the optimal temperature of the respective adapted bacteria, e.g. for Lactococcus lactis S at 3O ⁰ C whereas for Lactobacillus casei V at 30° or 37 ⁰ C, depending on the strain. pH is followed and kept constant by addition of alkaline like sodium hydroxide, ammonium hydroxide, calcium hydroxide, ... Optimal pH depends on the culture and is preferably comprised between 5 and 8 The pH is for instance about 6.3 in the case of Lactococcus lactis S or Lactobacillus caseiV .

In a following step d), a dairy analogue is prepared by adding to said dairy analogue as a starting material a suitable amount of bacteria that have been cultured in step c). A suitable amount may comprise between 10 ⁵ and 10 ¹⁰ bacteria per ml, and preferably comprise 10 ⁷ bacteria/ml. The preparation step d) may or may not include a fermentation step: the bacteria from step c) can be added as an ingredient to the dairy analogue without fermentation or can be added as a bacteria culture which is subsequently fermented. Temperature and time required for step d) will depend on the type of dairy analogue to be prepared or fermented. In a preferred example the dairy analogue is soya milk (prepared from soybeans as described as Tonyu or prepared from Soy Isolates). Also, the fermentation is carried out at the optimal

temperature of the respective adapted bacteria, e.g. for Lactococcus lactis S at 3O ⁰ C whereas for Lactobacillus caseiV at 37 ⁰ C. During fermentation, pH decreases and proteins coagulate.

Optionally, the product to be fermented may be pasteurized or sterilized prior to the fermentation thereof. In a preferred embodiment, the present invention may comprise the further step(s) of mixing of the prepared product in order to obtain a soft and smooth product, and/or admixing one or more additional ingredients such as for example pieces of fruit(s) or mashed fruit(s), vegetables, herbs, spices, sugar and sweeteners, flavors, colorants, stabilizers and thickeners, salt, preservatives,... Further in accordance with the present invention, a method is provided for preparing a dairy analogue, wherein fermentation time during the preparation of said dairy analogue is significantly reduced. Example 3 for instance illustrates how the use of a culture of lactic acid bacteria that have been isolated, adapted, cultured, preserved, and re-animated according to the invention permits to improve the preparation of a dairy analogue, and in particular to accelerate its fermentation and thus to reduce fermentation time during the preparation of such dairy analogue. In a preferred embodiment, a method is provided wherein fermentation time during the preparation of said dairy analogue is reduced with at least 10%, preferably with at least 15%, more preferably with at least 20% and even more preferably with at least 30%. The fermentation time may for instance be reduced with 15, 18, 20, 22, 25, 27, 30, 35, 40, 45 or even 50%.

In another aspect the invention further provides a dairy analogue obtainable by one of the above described method. The present dairy analogue can be characterized as follows.

In one embodiment a dairy analogue is provided having a reduced amount of phytic acid. A dairy analogue is provided wherein more phytic acid has been degraded during fermentation than in a dairy analogue not obtained by a method according to the invention. The invention thus provides a dairy analogue wherein degradation of phytic acid is higher, and preferably at least 10%, more preferably at least 25% and even more preferably at least 50% higher, than in a dairy analogue not obtained by a method according to the invention. In a preferred embodiment, a dairy analogue is provided wherein at least 10%, preferably at least 15%, more preferably at least 20% and even more preferably at least 25% more phytic acid is degraded than in a dairy analogue not obtained by a method according to the invention. In an example a dairy analogue is provided wherein degradation of phytic acid is at least 1.5 times,

and preferably at least 2 times, or even at least 3 times higher than the degradation of phytic acid in a dairy analogue not obtained by a method according to the invention.

In another embodiment a dairy analogue is provided having a reduced amount of raffinose. A dairy analogue is provided wherein more raffinose has been degraded during fermentation than in a dairy analogue not obtained by a method according to the invention. The invention thus provides a dairy analogue wherein degradation of raffinose is higher, and preferably at least 10%, more preferably at least 25% and even more preferably at least 50% higher, than in a dairy analogue not obtained by a method according to the invention. In a preferred embodiment, a dairy analogue is provided wherein at least 25%, preferably at least 50%, more preferably at least 75% and even more preferably at least 100% more raffinose is degraded than in a dairy analogue not obtained by a method according to the invention. In an example a dairy analogue is provided wherein degradation of raffinose is at least 3 times, and preferably at least 5 times, or even at least 7 times higher than the degradation of raffinose in a dairy analogue not obtained by a method according to the invention. In yet another embodiment, a dairy analogue is provided having an increased amount of aglucon forms of isoflavones. A dairy analogue is provided wherein more glucoside form(s) of isoflavones have been transformed during fermentation into aglucon form(s) of isoflavones than in a dairy analogue not obtained by a method according to the invention. The invention thus provides a dairy analogue wherein transformation of glucoside form(s) of isoflavones into aglucon form(s) of isoflavones is higher, and preferably at least 10%, more preferably at least 25% and even more preferably at least 50% higher, than in a dairy analogue not obtained by a method according to the invention. In a preferred embodiment, a dairy analogue is provided wherein at least 5%, preferably at least 10%, more preferably at least 20% and even more preferably at least 25% more transformation of glucoside form(s) of isoflavones into aglucon form(s) of isoflavones than in a dairy analogue not obtained by a method according to the invention. In an example, a dairy analogue is provided wherein transformation of a glucoside form into an aglucon form of isoflavones is at least 1.1 times, and preferably at least 1.2 times, or even at least 1.3 times higher than in a dairy analogue not obtained by a method according to the invention. In another aspect, the invention provides lactic acid bacteria that have been isolated, adapted, and optionally cultured as disclosed herein. In another aspect, the invention provides lactic acid bacteria that have been isolated, adapted, cultured, and optionally preserved and re-animated as disclosed herein.

In one preferred embodiment, the invention provides lactic acid bacteria as disclosed herein, which are capable of increasing degradation of phytic acid in a dairy analogue during fermentation thereof. The invention thus provides lactic acid bacteria showing an increase in phosphatase activity, e.g. of acid phosphatase and/or alkaline phosphatase activity, with at least 5%, preferably with at least 10%, preferably with at least 20% and more preferably with at least 30%. For instance, lactic acid bacteria are provided that are capable of degrading at least 1.5 times, and preferably at least 2 times, or even at least 3 times more phytic acid than lactic acid bacteria that have not been grown on a 100% vegetable isolation and adaptation medium in accordance with the present invention. In another preferred embodiment, the invention provides lactic acid bacteria as disclosed herein, which are capable of increasing degradation of raffinose in a dairy analogue during fermentation thereof. The invention thus provides lactic acid bacteria showing an increase in α-galactosidase activity with at least 5%, preferably with at least 10%, preferably with at least 20% and more preferably with at least 30%. For instance lactic acid bacteria are provided that are capable of degrading at least 3 times, and preferably at least 5 times, or even at least 7 times more raffinose than lactic acid bacteria that have not been grown on a 100% vegetable isolation and adaptation medium in accordance with the present invention.

In yet another embodiment, the invention provides lactic acid bacteria as disclosed herein, which are capable of increasing transformation of (inactive) glucoside form(s) into (active) aglucon form(s) of isoflavones in a dairy analogue during fermentation thereof. The invention thus provides lactic acid bacteria showing an increase in β-glucosidase activity, with at least 5%, preferably with at least 10%, preferably with at least 20% and more preferably with at least 30%. For instance lactic acid bacteria are provided that are capable of transforming at least 1.1 times, and preferably at least 1.2 times, or even at least 1.3 times more glucoside form(s) into aglucon form(s) of isoflavones than lactic acid bacteria that have not been grown on a 100% vegetable isolation and adaptation medium in accordance with the present invention.

A bacteria culture or lactic acid bacteria as defined herein are particularly suitable for being used for improving the preparation of dairy analogue. The preparation can be improved in the sense that dairy analogues can be obtained having improved quality, nutritional and organoleptic properties, such as for instance, enhanced taste and/or flavor stability, enhanced digestibility, improved nutritional value, enhanced availability of minerals such as Ca, Fe, Mg, etc... The invention for instance relates to the use of a bacteria culture or lactic acid bacteria

as defined herein for preparing a dairy analogue having a reduced amount of phytic acid and/or raffinose, and/or for preparing a dairy analogue having an increased amount of aglucon form(s) of isoflavones.

In addition the preparation process of the dairy analogue as such can also be improved. In particular, the invention also relates to the use of a bacteria culture or lactic acid bacteria as defined herein for reducing fermentation time during the preparation of a dairy analogue.

Preferably a bacteria culture or lactic acid bacteria according to the invention are used for reducing fermentation time during the preparation of a dairy analogue with at least 10%, preferably with at least 15%, preferably with at least 20% and more preferably with at least 30%. The fermentation time may for instance be reduced with 15, 18, 20, 22, 25, 27, 30, 35,

40, 45 or even 50%.

The invention further relates to the use of lactic acid bacteria or a culture thereof as disclosed herein for increasing degradation of phytic acid in a dairy analogue during fermentation thereof, and/or for increasing degradation of raffinose in a dairy analogue during fermentation thereof and/or for increasing transformation of glucoside form(s) into aglucon form(s) of isoflavones in a dairy analogue during fermentation thereof.

The following examples are intended to illustrate particular embodiments of the invention, and do not limit the scope of the invention.

Examples Example 1 : 100% vegetable adaptation medium according to the present invention

The present example illustrates a 100% vegetable medium according to the present invention. The medium consists of: 10 g Peptone S (= commercial papaic digest of soybean meal from Acumedia, 5 g Yeast Extract, ultrafiltrated ( Ultrafiltrated autolysate of bakers' yeast) from Acumedia, 10g Fructose, 5 g of a Phosphate buffer pH=7,2 ( from Acumedia) 15 g Agar-agar ( bacteriological), and demineralised water up to 1 liter.

For preparing broth, the above-mentioned medium wherein agar-agar is omitted may be used.

Example 2: Characterization of bacteria cultures according to the present invention

Lactic acid bacteria that were used in the following examples as starting material were either isolated from natural sources (e.g. food products) or obtained from a culture collection (BCCM - Gent Belgium).

2.1 Enzyme induction pattern

^* Lactobacillus casei V (LMG P-23504)

In a first experiment Lactobacillus casei V (LMG P-23504) was grown and maintained in the following ways: 1 ) grown and maintained on a S medium (VS); 2) grown and maintained on a MRS medium (VM); 3) grown and maintained on a S medium and once re-plated on a MRS medium (VSM); and 4) and grown and maintained on a MRS medium and once re-plated on a S medium (VMS).

The S medium is a substantially vegetable medium according to the invention comprising soya peptone, yeast extract, a phosphate buffer and a sugar source. The MRS medium corresponds to medium comprising a meat peptone. The LAB were re-plated twice a week on the MRS or the S media whereby a selection was made of the largest bacterial colonies (=breeding).

The enzyme induction patterns of the bacteria grown on the above-stated media were analyzed in a semi-quantitative way. Using a commercial kit (Biomerieux) enzyme activity was determined by means of a color code (0: no enzyme activity to 5: strongest enzyme activity). Enzyme activities were measured after incubation at 37° during 4 hours.

Results of this experiment indicated that by adapting Lactobacillus casei V (LMG P-23504) to a substantially vegetable medium (S medium) according to the invention, certain enzymes were significantly induced or increased, e.g. alkaline phosphatase, acid phosphatase, β- glucosidase and α-fucosidase. These enzymes were practically not induced when adapting the bacteria to a MRS medium. However, when the bacteria, adapted to a substantially vegetable medium were re-adapted to a MRS medium, there was a transition in the enzyme pattern: the enzyme induction pattern was reversed.

The following example illustrates the increased phosphatase activity of the L. casei V (LMG P-23504) when grown and maintained on S medium, compared to L. casei V (LMG P-23504)

grown and maintained on MRS medium. Soy milk + 2% added glucose was inoculated with 1X10 ⁷ / ml_ of L. casei V that was maintained on S or that was maintained on MRS medium and the inoculated soy milk was fermented at 30° until pH of 4.5.

The content of phytic acid was measured in the soy milk before and after fermentation. Results are presented in Table 1.

Table 1

Table 1 demonstrates that the L. casei V (LMG P-23504) that was maintained on S medium induces a higher breakdown of the anti-nutritional factor phytic acid. This is a nutritional advantage for the consumer. The amount of phytic acid in the dairy analogue was reduced with more than 67% when using L. casei V that had been maintained on S medium compared with a reduction of 40 % when using L. casei V that had been maintained on MRS medium. This example further indicates that there is more than 27% more phytic acid degraded in the dairy analogue prepared with L. casei V that was maintained on S medium than in a dairy analogue prepared with L. casei V that was maintained on MRS medium. L. casei V that was maintained on S medium is capable of increasing the degradation of phytic acid in a dairy analogue with a factor of at least 1.675.

* Lactococcus lactis LMG 8522 In another experiment a Lactococcus lactis LMG 8522 was adapted for several months either on a S medium or on a MRS medium. After several months of culture, enzyme activity was determined using the kit as defined above.

Results hereof indicated that by adapting Lactococcus lactis on a substantially vegetable medium according to the invention, certain enzymes were induced, e.g. alkaline phosphatase and α-chymotrypsine. These enzymes were not induced when adapting the lactic acid bacterium on a MRS medium.

^* Lactococcus lactis S (LMG P-23669)

In yet another experiment a Lactococcus lactis S (LMG P-23669) was adapted for several months either on S medium or on MRS medium. After several months of culture, enzyme activity was determined using the kit as defined above.

Results hereof indicated that by adapting Lactococcus lactis S (LMG P-23669) on a substantially vegetable medium according to the invention, certain enzymes were induced or increased, e.g. alkaline phosphatase and α-chymotrypsine. These enzymes were not induced when adapting the lactic acid bacterium on a MRS medium.

The following example illustrates the increased phosphatase activity of Lactococcus lactis S (LMG P-23669) when maintained on S medium, compared to Lactococcus lactis S (LMG P- 23669) maintained on a MRS medium. Soy milk + 2% added glucose was inoculated with 1X10 ⁷ / mL of Lactococcus lactis S that was maintained on S or that was maintained on MRS medium and the inoculated soy milk was fermented at 30° until pH of 4.5.

The content of phytic acid was measured in the soy milk before and after fermentation. Results are presented in Table 2. Table 2

Table 2 demonstrates that the L. lactis S (LMG P-23669) that was maintained on S medium induces a higher breakdown of the anti-nutritional factor phytic acid. The amount of phytic acid in the dairy analogue was reduced with 27% when using L. lactis S that had been maintained on S medium compared with a reduction of 7% when using L. lactis S that had been maintained on MRS medium. This example further indicates that there is about 20% more phytic acid degraded in the dairy analogue prepared with L. lactis S that was maintained on S medium than in a dairy analogue prepared with L. lactis S that was maintained on MRS medium. L. lactis S that was maintained on S medium is capable of increasing the degradation of phytic acid in a dairy analogue with a factor of at least 3.86.

^* Bifidobacterium infantis (LMG P-24096)

In yet another experiment Bifidobacterium infantis LMG P-24096 was adapted for several months either on a S medium or on a MRS medium in anaerobic conditions. After several months of culture, enzyme activity was determined using the kit as defined above.

Results hereof indicated that by adapting Bifidobacterium infantis (LMG P-24096) on a substantially vegetable medium according to the invention, certain enzymes were induced or increased, e.g. α-galactosidase and β-glucosidase. These enzymes were not induced when adapting the lactic acid bacterium on MRS medium.

The following example illustrates for instance the increased α-galactosidase and β- glucosidase activity of the Bifidobacterium infantis (LMG P-24096) when maintained on S medium, compared to Bifidobacterium infantis (LMG P-24096) maintained on a MRS medium.

Soy milk + 2.5 % raffinose was inoculated with 1X10 ⁷ / mL of B. infantis that was maintained on S or that was maintained on MRS medium and the inoculated soy milk was fermented at 37° under anaerobic conditions.

During fermentation the content of raffinose was measured before and after 6 hours of fermentation by B. infantis that was maintained on S or that was maintained on MRS medium. The B. infantis culture adapted to the S medium showed a higher breakdown of raffinose due to the increased α-galactosidase activity: 6 hours after the start of the fermentation, B. infantis that was maintained on S medium had broken down 6 % of the raffinose compared with the B. infantis that was maintained on MRS medium and that had broken down 0.8 % of the raffinose. This example indicates that there is about 5.2% more raffinose degraded in the dairy analogue prepared with B. infantis that was maintained on S medium than in a dairy analogue prepared with B. infantis that was maintained on MRS medium. This example further indicates that B. infantis that was maintained on S medium is capable of increasing the degradation of raffinose in a dairy analogue with a factor of at least 7.5. During fermentation the content of isoflavones was also measured before and after 6 hours of fermentation by B. infantis that was maintained on S or that was maintained on MRS medium. The B. infantis culture adapted to the S medium showed higher transformation of the biological inactive glucoside-form of the isoflavones to the biologically active aglucon-form due to increased β-glucosidase activity: 6 hours after the start of the fermentation, B. infantis that was maintained on S medium had transformed 98 % of the glycosides daidzin and genistin to the aglucons daidzein and geistein while B. infantis that was maintained on MRS medium had transformed only 89%. This example indicates that there is about 9% more

glucoside-form of the isoflavones that are transformed in the dairy analogue prepared with B. infantis that was maintained on S medium than in a dairy analogue prepared with B. infantis that was maintained on MRS medium. This example further indicates that B. infantis that was maintained on S medium is capable of increasing transformation of (inactive) glucoside form(s) into (active) aglucon form(s) of isoflavones with a factor of at least 1.1.

Activities of the induced enzymes can be correlated to improvements with regard to quality and organoleptic properties of dairy analogues produced with the LAB.

For instance, the enzymes alkaline phosphatase and acid phosphatase may play a role in the degradation of phytic acid, which is an anti-nutritional factor present in e.g. soya. Phytic acid binds minerals such as Ca, Fe and Mg and thus reduces their bio-availability. Degradation of phytic acid therefore increases availability of such minerals.

Another example is that of β-glucosidase. This enzyme transforms glucosides to a glucose- unit and an aglucon. In a soya-matrix the β-glucosidase will transform biological inactive glucoside-form of isoflavones to biological active aglucon-moiety. An increased β-glucosidase activity increases the bio-availability of isoflavones that are present in soya in the glucoside- form.

Yet another example is that of α-galactosidase. This enzyme is capable of breaking down raffinose and stacchyose. These are undigestable sugars that are typically present in soy milk and that cannot be digested by humans. This can cause unwanted gas formation in the gastro-intestinal tract. An increased breakdown of these flatulence-sugars in soy milk increases the digestability for the consumers.

Still another example is α-chymotrypsin. This proteolytic enzyme has a function in pre- digestion, formation of bio-active peptides, aroma formation, structure formation.

^* Conclusion

Summarized, these results indicate that lactic acid bacteria that are adapted to a substantially vegetable medium undergo significant metabolic changes. These metabolic changes of the strain are however reversed when the strain is re-adapted to a medium containing compounds of animal origin. The induced enzymes play a role in the quality, nutritional and organoleptic improvements of dairy analogues produced with the present LAB.

2.2 DNA fingerprinting using RAPD analysis

A RAPD analysis was performed using DNA from L. casei T (LMG P-23506) adapted to a S medium and from L. casei T adapted to a MRS medium. For a definition of the S and MRS media, see 2.1 above.

Results of this analysis revealed that the bacterial cultures did not show differences in their DNA finger print for the selected primers. Results further indicated that adapting the bacteria to a S medium compared to a MRS medium did not induce any differences on a DNA level (mutations) for the selected primers, but as further shown under point 2.1 above, resulted in differences in enzyme activity and induction patterns and thus in adaptation.

2.3 Protein patterns using SDS-page

A one-dimensional SDS-PAGE analysis was performed on Lactobacillus casei LMG 6904, and L. casei J (LMG P-23506) adapted to a S medium and on L. casei J adapted to a MRS medium.

The protein pattern of the L. casei T (LMG P-23506) adapted to the S medium was different from the protein pattern of the other two bacterial cultures, pointing at a difference in enzyme activity between the studied bacterial cultures due to adaptation.

FIG. 2 illustrates the differences in protein expression. The arrows point to differences in specific protein expressions. As is shown by comparison of FIG. 2A showing the SDS PAGE profile of L. casei J on S medium and FIG. 2B showing the SDS PAGE profile of L. casei J on

MRS medium, differences in specific protein expressions occur by adaptation to the respective adaptation medium. As is shown by comparison of FIG. 2A showing the SDS

PAGE profile of L. casei T on S medium and FIG. 2C showing the SDS PAGE profile of L. casei 6904, the adapted strain L. casei J has become different from the parent strain L. casei

6904 which was obtained from the culture collection BCCM.

In another example, a two-dimensional SDS page analysis was performed in which proteins were separated according to their size and iso-electric point (iso-electric focussing). Figure 4 illustrates the results of such analysis. The results of this 2D analysis for L. casei V (LMG- 23504) grown on S medium (FIG. 4A) or MRS medium (FIG. 4B) demonstrate the difference

in protein expression between the two samples. On FIG. 4 the arrows indicate the differences between both gels.

2.4 Total cell content analysis (FTIR-UATR) (Perkin Elmer) Using Infra Red Spectroscopy, the total cell content of four bacterial cultures was studied. The cell content is considered as a characteristic for a certain strain under certain growth conditions. Using databases, it is possible to identify bacteria using IR spectra.

In this experiment Lactobacillus casei V (LMG P-23504), L. casei T (LMG P-23506), Lactococcus lactis S (LMG P-23669) and Lactococcus lactis LMG 9452 were grown and maintained in several ways including: 1 ) adapted to S medium; 2) adapted to MRS medium; 3) adapted to S medium and re-adapted to MRS medium; and 4) adapted to MRS medium and re-adapted to S medium.

Results of this experiment showed significant differences between bacteria that were adapted to S medium and bacteria that were adapted to MRS medium. Results are expressed in % similarity (with a standard deviation of ≤1 %). The difference between Lactobacillus casei V (LMG-23504) adapted to S medium and adapted to MRS was 3.5 %. When the L. casei V adapted to S (VS) was re-adapted to MRS (VSM) the difference was 1.4 %. When the L. casei V adapted to MRS (VM) was re-adapted to S medium (VMS), the difference was 3%. FIG. 3 shows a gradual transition in typical IR spectrum when comparing VS, VSM, VMS and VM.

The difference between L. casei T (LMG P-23506) adapted to S medium and adapted to MRS was 3.3 %.

The difference between Lactococcus lactis S (LMG P-23669) adapted to S medium and adapted to MRS was 4.3 %. The difference between Lactococcus lactis LMG 9452 adapted to S medium and adapted to MRS was 3.1 %.

Example 3: Fermentation studies

The following examples illustrate that the use of a culture of lactic acid bacteria that have been isolated, adapted, cultured, preserved and re-animated according to the present invention permits to improve the preparation of dairy analogue, and in particular to improve

(accelerate) its fermentation and thus to reduce fermentation time during the preparation of a dairy analogue.

* Lactococcus lactis S (LMG P-23669) Bacteria cultures of Lactococcus lactis S (LMG P-23669) were prepared according to the present invention by culturing said strain on an S medium or on an MRS medium, as defined in example 2. Similar inoculation amounts, comparable to McFarland scale n ⁰ 5, of these bacteria cultures were then used for the fermentation of a soy based composition consisting of 85 % by weight of soy milk, 2% by weight of fructose and 13% by weight of water. The time necessary to obtain a pH value of 4.6 was determined. Such pH value is considered as a "safe" value for suppressing the development of pathogens. As illustrated on FIG. 1, the time required for reaching this pH value using a bacteria culture of Lactococcus lactis S cultured to S medium was approximately 60 minutes shorter than when using a bacteria culture of Lactococcus lactis S cultured on an MRS medium. Results of this experiment thus indicated that the use of LAB that were grown on a substantially vegetable medium according to the invention permits to reduce times required for the preparation (fermentation) of dairy analogues. In this example the time required for reaching a pH value which is considered as "safe" for suppressing the development of pathogens is reached about 1.2 times faster when using a Lactococcus strain that has been isolated, adapted and cultured on a 100% vegetable medium. This example demonstrates the technological advantage of adaptation of the bacteria strain to the S medium. Fermentation time during the preparation of said dairy analogue can be reduced with about 20%.

^* Lactococcus lactis S (LMG P-23669) Bacteria cultures of Lactococcus lactis S (LMG P-23669) were prepared according to the present invention by culturing said strain on an S medium or on an MRS medium, as defined in example 2.

Soy milk with 2% fructose was inoculated with 1X10 ⁷ / mL of Lactococcus lactis adapted to S or MRS medium and fermented at 30° until the critical pH of 4.6 was reached. Figure 5 shows that L. lactis S adapted to S medium reached the pH of 4.6 one hour earlier than the traditionally grown MRS bacteria. In this example the time required for reaching a pH value which is considered as "safe" for suppressing the development of pathogens is reached about

1.2 times faster when using a Lactococcus strain that has been isolated, adapted and cultured on a 100% vegetable medium. Fermentation time during the preparation of said dairy analogue can be reduced with about 20%. This example demonstrates the technological advantage of adaptation of the bacteria strain to the S medium.

* Lactobacillus casei V (LMG P-23504^

Bacteria cultures of Lactobacillus casei V (LMG P-23504) were prepared according to the present invention by culturing said strain on an S medium or on an MRS medium, as defined in example 2. Soy milk with 2% added glucose was inoculated with 5X10 ⁷ / mL L. casei V grown on S or MRS medium. The fermentation was performed at 37° until the safe pH limit of 4.6 was reached. Figure 6 shows that the L. casei V that was adapted to the S medium already reached the desired pH of 4.6 after 2.5 hours of fermentation, while the MRS grown L. casei V reached this pH after 6.5 hours of fermentation. The time required for reaching this pH value using a bacteria culture of Lactobacillus casei V cultured on S medium was approximately 4 hours shorter than when using a bacteria culture of Lactobacillus casei V cultured on an MRS medium. Thus, the time required for reaching a pH value which is considered as "safe" for suppressing the development of pathogens is reached more than 2 times faster, and in this example 2.6 times faster when using a Lactobacillus strain that has been isolated, adapted and cultured on a 100% vegetable medium. Fermentation time during the preparation of said dairy analogue can be reduced with more than 100%. This example demonstrates the technological advantage of adaptation of the bacteria strain to the S medium.

^* Bifidobacterium infantis (LMG P-24096)

Bacteria cultures of Bifidobacterium infantis (LMG P-24096) were prepared according to the present invention by culturing said strain on an S medium or on an MRS medium, as defined in example 2.

Soy milk with 2.5 % raffinose was inoculated with 1X10 ⁷ / mL of B. infantis adapted to S or MRS medium and fermented at 37° under anaerobic conditions. Figure 7 shows that B. infantis adapted to the S medium grows faster than the MRS culture. After 6 hours the fermentation was stopped because the B. infantis inhibits itself due to a decreasing pH.

During fermentation B. infantis inhibits itself due to a decreasing pH and the formation of lactic and acetic acid. To examine the acid forming capacity without an inhiting effect of the decreasing pH, the fermentation test described above was repeated but the pH was kept constant at 5.9 by addition of NaOH 1 M. Figure 8 demonstrates that the B. infantis that was adapted to S medium reached a pH of 5.9 approximately 3.5 hours earlier than B. infantis that was adapted to MRS medium and that more NaOH was consumed during fermentation. This demonstrates the higher speed of growth and the higher acid forming capacity of B. infantis, when adapted to S medium. Thus, the time required for reaching a pH value of 5.9 is reached more than 2 times faster, and in this example 2.2 times faster when using a B. infantis strain that has been isolated, adapted and cultured on a 100% vegetable medium. This example demonstrates the technological advantage of adaptation of the bacteria strain to the S medium.