FIELD OF INVENTION

The present invention relates to the field of Lactobacillus microbiology especially to vitaminology. The present invention in particular relates to production of vitamin B12 from Lactobacillus species.

BACKGROUND OF THE INVENTION

Lactic acid bacteria (LAB) are Gram-positive rods or cocci with a low chromosomal G+C content. They share the common feature of producing lactic acid as the major end product of carbohydrate metabolism. Lactic acid bacteria have been used to ferment or culture foods since the last 4000 years. They are of major economic significance and are of value in maintaining and promoting human health. LAB refers to a large group of beneficial bacteria that have similar properties and all produce lactic acid as an end product of the fermentation process. LAB is amongst the most important group of microorganisms used in the food industry. Their economic, industrial and nutritional value is clearly demonstrated by a wide variety of applications. Lactic acid and other metabolic products contribute to the organoleptic and textural profile of a food item. Historically, humans have domesticated LAB for thousands of years by incorporating them in our food fermentation processes. This long history of safe use contributed greatly to their prominent role in the food industry, being responsible for the manufacturing of several fermented foods and beverages. The industrial importance of the LAB is further evinced by their 'Generally recognized as safe' (GRAS) status, due to their ubiquitous appearance in food and their contribution to the healthy microflora of human mucosal surfaces. LAB as probiotic cultures supplement and help our normal gut bacteria to function more efficiently and helps in counteracting symptoms due to pathogenic bacteria. LAB optimizes the intestinal system and therefore also participates in the utilization of nutrients in food. Lactic acid bacteria (LAB) produce small amounts of vitamin B12. Since vitamin B12 biosynthesis is restricted only to microorganisms, potential ways of improving vitamin production are of great implication. The phenotypic traits of acidifying the environment and producing antimicrobial agents inhibit the growth of spoilage agents, leading to the establishment of a fortuitous symbiotic relationship with humans. LAB are the main components of dairy starter cultures, transforming milk into products such as yogurt or cheese, and extending its shelf-life by lowering the pH due to lactic acid production, they contribute to the wellbeing of human mucosal surfaces throughout most of the digestive tract, they may also produce bacteriocins (anti-microbial peptides or proteins) that improve the durability of fermented foods, they may contribute to the organoleptic profile of fermented foods, they can improve the texture and rheological properties of food by the synthesis of exopolysaccharides, and more recently, they have been studied to produce novel foods with improved nutritional value (nutraceuticals) [Vitamin B12 synthesis in Lactobacillus reuteri; Filipe Santos; Ph.D: thesis, Wageningen University, Wageningen, The Netherlands, 2008].

LAB are characterized by an increased tolerance to a lower pH range. This aspect partially enables LAB to outcompete other bacteria in a natural fermentation due to the ability of LAB to withstand the increased acidity from organic acid production (e.g., lactic acid). The lactic acid bacteria (LAB) comprise a clade of Gram-positive, low-GC, acid-tolerant, generally non-sporulating, non-respiring rod or cocci that are associated by their common metabolic and physiological characteristics. LAB also produces other bacteriostatic substances like bacteriocins that displace pathogenic bacteria from the intestinal mucosa. Lactic acid bacteria are also able to produce vitamin K and certain kinds of vitamin B. Some lactic acid bacteria produce amino acids that bind minerals in the intestines to make the absorption through the intestinal wall easier.
Vitamin B12, originally discovered as an anti-pernicious factor, is necessary for the formation of red blood cells, nerve sheaths, neurological function, various proteins, and DNA synthesis. Vitamin B12 is also essential for the prevention of nerve damage, neurological disturbances and certain forms of anaemia, has a coenzyme-function in the intermediary metabolism especially in cells of the nervous tissue, bone marrow and gastrointestinal tract and aids in proper digestion, fertility and growth. Vitamin B12 is also involved in fat and carbohydrate metabolism and is essential for growth.

Vitamin B12 is the largest and most complex of all the vitamins. The name, vitamin B12 is generic for a specific group of cobalt-containing corrinoids with biological activity in humans. Interestingly, it is the only known metabolite to contain cobalt, which gives this water-soluble vitamin its red colour. This group of corrinoids is also known as cobalamins. The main cobalamins in humans and animals are hydroxocobalamin, adenosylcobalamin and methylcobalamin, the last two being the active coenzyme forms. Cyanocobalamin is a form of vitamin B12 that has found wide clinical use due to its availability and stability. It is transformed into active factors in the body. In its methylcobalamin form, vitamin B12 is the direct cofactor for methionine synthase, the enzyme that recycles homocysteine back to methionine. Vitamin B12 is required in the synthesis of folate polyglutamates (active coenzymes required in the formation of nerve tissue) and in the regeneration of folic acid during red blood cell formation.
Adenosylcobalamin is also the coenzyme in ribonucleotide reduction, which provides building blocks for DNA synthesis.

Humans have an auxotrophic requirement for vitamin B12 with a recommended nutrient intake (RNI) for healthy adults of 2.4 (|a.g/day) [Institute of Medicine. Food and Nutrition Board. Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, VitaminBn, Pantothenic Acid, Biotin, and Choline. Washington, DC: National Academy Press, 1998]. Interestingly, animals (including humans) and protists require cobalamin but apparently do not synthesize it, whereas plants and fungi are thought to neither synthesize nor use vitamin B12. Vitamin B12 is the only vitamin that cannot be obtained from eating a strict plant-based diet. Vitamin B12 is synthesized by a few representatives of bacteria and archaea and this is the only source of this vitamin for humans, which enters the human food chain mostly through incorporation into foods of animal origin. This essential vitamin is produced by bacteria that live in the gut of animals that eat grass and other kinds of vegetation. Humans too have bacteria in the
digestive systems that produce vitamin B12. However, the problem is that humans cannot absorb enough of it to meet the daily requirement. Animals are able to absorb the vitamin B12 made in their gut much better than humans and so they store the vitamin B12 in their tissues, milk, and eggs. That is why meat eaters and vegetarians who eat dairy products and eggs usually get enough vitamin B12, compared to those who follow a strict plant-based diet. Dietary sources with significant vitamin B12 content include meat (liver), poultry, milk, milk derivatives and eggs, amongst others.

More than half of the Indian population is vegetarian for religious or socio-economic reasons and therefore, much of the Indian population is at risk of having low vitamin B12 status throughout life. Indians in India as well as those who have migrated abroad are suggested to have high circulating homocysteine levels, as an indicator of vitamin B12 deficiency. The superimpositions of other conditions that perturb either vitamin B12 absorption like, partial gastrectomy or bypass, use of proton-pump inhibitors, and ileal disease or surgical resection, further aggravate the condition. Vegetarians and immune compromised individuals are most at risk of vitamin B12 deficiency. Vitamin B12 deficiency has been demonstrated to lead to pernicious anaemia, neurological dysfunction, megaloblastic anaemia, fatigue, weakness, constipation, loss of appetite, weight loss and neurological changes such as numbness and tingling in the hands and feet [Stabler, S. P., 1999; B12 and Nutrition, p. 343-365. In R. Banerjee (Ed.), Chemistry and Biochemistry of B12, John Wiley & Sons, Inc]. Deficiency of vitamin B12 results in a macrocytic anemia, elevated homocysteine, peripheral neuropathy, memory loss and other cognitive deficits. The autoimmune disease pernicious anaemia is another common cause. It can also cause symptoms of mania and psychosis. In rare extreme cases, paralysis can also result.

Supplementation of folic acid and vitamin B12 were found to be a cost-effective treatment for heart disease and was found to be useful in the reduction of associated deaths among the adult U.S. population. Homocysteine is suggested to be a possible risk factor for heart disease and B-vitamins (folic acid, B6 and B12) play an important role in maintaining healthy homocysteine levels [Tice et al., (2001) Cost-effectiveness of Vitamin Therapy to Lower Plasma Homocysteine Levels for the Prevention of Coronary Heart Disease. JAMA, 286 (8): 936-943]. Vitamin B12 is life saving and highly essential for patients suffering from chronic fatigue syndrome. Some medications are known to interfere with the absorption or metabolism of vitamin B12 like proton pump inhibitors (PPI), metformin, nitrous oxide anesthesia, colchicine and epileptic medications [Fiona O'Leary and Samir Samman (2010) Vitamin B12 in Health and Disease Nutrients, 2(3): 299-316; DOI:10.3390/nu2030299]. Therefore, people on such medication also require vitamin B12 supplementation to combat the loss of this vitamin. Another risk group are the elderly, most often not because of a diet poor in B12, but most often because of malabsorption phenomena [Morris et al. 2007; Folate and vitamin B-12 status in relation to anemia, macrocytosis, and cognitive impairment in older Americans in the age of folic acid fortification. Am. J. Clin. Nutr. 2007, 85, 193-200].

Strict vegetarian dietary regimes tend to be poor in vitamin B12, which has boosted the popularity of fortifying vegetarian foodstuffs with vitamin B12. In situ microbial vitamin B12 production is a convenient strategy to achieve natural enrichment of fermented foods, notably from vegetable sources. Vitamin B12-producing bacterial genera include Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium, Flavobacterium, Micromonospora, Mycobacterium, Norcardia, Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus and Xanthomonas. Production of the vitamin at an industrial scale is carried out by fermentation by action of selected microorganisms. Streptomyces griseus, a bacterium once thought to be yeast, has been the commercial source of vitamin B12 for many years. However, now-a-days, because of their naturally high vitamin B12 productivity and their rapid growth, strains of Propionibacterium shermanii and Pseudomonas denitrificans are often employed for industrial production of vitamin B12. The strains are frequently grown under special conditions to enhance yield and sometimes genetically engineered versions of one or both of these species are also employed. Today vitamin B12 production is exclusively limited to biosynthetic fermentation processes, using selected and genetically optimized micro-organisms as chemical synthesis was a highly complicated process which involved more than 70 synthesis steps. The industrial production of vitamin B12 could not rely on chemical synthesis, since it proved to be far too technically challenging and economically unviable. . In the light of the challenges faced in the chemical synthesis of the molecule, attention was turned to the natural synthesis of B12. Intriguingly, B12 biosynthesis is limited to a few representatives of bacteria and archaea while B12-dependent enzymes are widespread throughout all domains of life.

Due to restriction of vitamin B12 biosynthesis microorganisms and complications of chemical synthesis the use of microorganisms that naturally produce this complex vitamin can be exploited. Vitamin production processes by LAB would have huge advantages over the traditionally used processes as they could also be implemented for in situ production processes, such as food fermentations (Metabolic engineering of lactic acid bacteria; Hugenholtz and Kleerebezem; Current Opinion in Biotechnology 1999, 10:492-497). LAB also open up the safe, effective, tried and tested option of using food grade strains for the production of vitamin B12 when considered in the light of the other strains used in the industrial production of vitamin B12. The microorganism along with the vitamins may also be added in functional foods or used for the production of fortified food products.

US6492141 describes the process for production of vitamin B12 from Propionibacterium in a two-step culturing process. The patent describes the Propionibacterium-baseA fermentation process for an optimized yield of vitamin B12 keeping in view the inhibition of the organism's growth due to high production of propionic acid.

US2011/0159148 describes the method of production of a Lactobacillus fermentation medium for increased folate levels by Lactobacillus sp. The fermentation medium comprises extracts or juices or parts thereof of the melon fruit. The use of p-aminobenzoic acid is also mentioned for increasing the folate production and/or the folate: vitamin B12 ratio. In the prior art, although folate production was enhanced using co-culture with mutant strain, no significant effect on vitamin B12 levels was observed. A significant effect on production of only folate was achieved by using recombinant strain and by addition of folate precursor (PABA) in the media.
Industrial production of vitamin B12 is almost exclusively carried out by Pseudomonas denitirficans and Propionibacterium sp. However, the use of food-grade with GRAS labeled Lactobacillus offer more advantages in terms of use of the biomass along with the vitamin B12 produced by the microorganism. This process is also advantageous over the chemical synthesis as the process avoids the conversion of natural vitamin B12 into the cyanocobalamin form followed by extraction and purification process which render the production process expensive and probably unsafe to the operators and the environment.

SUMMARY OF THE INVENTION

An aspect of the present invention relates to a process for production of vitamin B12, wherein the process comprises co-culturing Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 under aerobic conditions at a temperature ranging from 35°C to 39°C for a period of 20 to 24 hours in a culture medium to obtain co-culture of the cells, and recovering vitamin B12 from the co-culture of the cells, wherein amount of vitamin B12 in the culture is in the range of 9 ng/L to 11 ng/L.
Another aspect of the present invention relates to a composition comprising Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 and a carrier.

OBJECTS OF THE PRESENT INVENTION

It is an object of the present invention to provide a process for the production of vitamin B12 from microbial co-cultures.

It is another object of the present invention to provide a novel co-culture for use in the production of vitamin B12.

It is another object of the present invention to provide a composition comprising vitamin B12 producing Lactobacillus strains to maintain and restore the natural flora in the gastrointestinal tract of a subject, preferably an animal, more preferably a human.

It is yet another object of the present invention to provide a composition comprising vitamin B12 producing Lactobacillus strains for use as a vitamin B12 probiotic supplement, for maintaining health and vitality of a subject.

It is yet another object of the present invention to provide a composition comprising vitamin B12 producing Lactobacillus strains, wherein the composition is used as a food, feed or nutritional supplement, food product or a pharmaceutical composition.

Another object of the present invention is to provide a process for the production of vitamin B12, wherein said process provides high yields in a shorter time frame than the conventional microbial processes.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

The following drawings form part of the present specification and are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

Figure 1 shows the Vitamin B12 production by monocultures of L. helveticus ATCC8018, monocultures of L. fermentum ATCC11976 and co-cultures of L. fermentum ATCC 11976 and L. helveticus ATCC8018. Data shows the results of three independent sets of experiments with three replicates each. (*) indicates values in different experimental groups are significantly different (p<0.05).

Figure 2 shows the vitamin B12 production by other bacterial monocultures and co-cultures of L. helveticus ATCC8018, L. fermentum ATCC 11976 and L. lactis. Synergistic effect in vitamin B12 production was observed only by the strains of L. fermentum ATCC 11976 and Z, helveticus ATCC8018.

Figure 3 shows the vitamin B12 production by monocultures and co-cultures of L. fermentum ATCC11976 and L. helveticus ATCC8018, Propionibacterium shermanii and P. freudenreichii.

DETAILED DESCRIPTION OF THE INVENTION

Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Definitions

For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only."
Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

The term "including" is used to mean "including but not limited to". The terms are used interchangeably.

The term "vitamin B12" is attributed to its broadest meaning so as to include all the cobalt corrinoids of the cobalamin group, which include in particular cyanocobalamin, hydroxocobalamin, methylcobalamin and 5'desoxyadenosylcobalamin, characterized by the cyano, hydroxyl, methyl or 5'desoxyadenosyl radical respectively. The term vitamin B12 further comprises any vitamin B12 precursor having vitamin B12 activity as detectable in the microbiological bioassay method based on the growth response of Lactobacillus delbrueckii var. lactis as described in AOAC (1995) [AOAC International, 1995; Official Methods of Analysis of AOAC International, 16 Ed. Method 960. 46. The Association, Arlington, VA].

The term "Colony forming unit (CFU)" used in the present invention refers to a measure of viable and/or bacterial cell present in the probiotic compositions as disclosed in the present invention.
For convenience the results are given as CFU/mL (colony-forming units per milliliter) for liquids and CFU/g (colony-forming units per gram) for solids.

The term "Lactic acid Bacteria" (LAB) refers to bacteria which produce lactic acid or another organic acid as an end product of fermentation such as, but not limited to bacteria of the genus Lactobacillus, Streptococcus, Lactococcus, Oenococcus, Leuconostoc, Pediococcus, Carnobacterium, Propionibacterium, Enterococcus and Bifidobacterium.

The term "food grade" refers to being regarded as safe for human and/or animal consumption by the relevant regulatory authorities. The term also refers to components which can be safely ingested by humans or animals.

The term "food" or "food product" refers to liquid, semi-solid and/or solid food products such as nutritional compositions suitable for human and/or animal consumption.

The term "food product ingredient" or "food supplement ingredient" refers to a product which is suitable for being added to a final food product, or a food supplement, or during the production process of a food product, or a food supplement.

The term "probiotic", "probiotics" or "probiotic strains" refers to microorganisms such as Lactobacillus, which have a beneficial effect on the host when ingested by the subject and which are generally regarded as safe (GRAS) to humans. The term also means a live microorganism that survives passage through the gastrointestinal tract and has a beneficial effect on the subject.
The terms "probiotic composition(s)," "composition(s) comprising blend of microorganism", "composition(s) comprising mixture of microorganisms" "composition(s) comprising blend of Lactobacillus strains", "composition(s) comprising Lactobacillus strains" and "composition(s)" are used interchangeably.

The term "probiotic lactic acid bacteria" are those LAB strains which also have probiotic properties, i.e. which are probiotic strains.

The term "fermentation" or "fermentation culture" refers to growth cultures used for growth of bacteria which convert carbohydrates into alcohol and/or acids, usually but not necessarily, under anaerobic conditions.

The term "growth phase" refers to commonly known growth phases of microorganism, for ex., in batch fermentation cultures, such as the lag-phase, exponential growth phase and the subsequent stationary phase, followed by the death phase.

The term "subject" means a living organism, including a plant, a microbe, a human, an animal such as domestic, agricultural, or exotic etc.

The term "treating" is art recognized and includes preventing a disease, disorder or condition from occurring in a patient which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the invention, as described herein.
The present invention provides a process for the production of vitamin B12 by microbial fermentation by employing a two-component co-culture which maintains a relatively constant ratio of species population over multiple passages. Both the components of the co-culture belong to the Lactobacillus genus. The first co-culture component is L. helveticus ATCC 8018 and the second co-culture component is L. fermentum ATCC 11976. In the endeavor to investigate the production of vitamin B12 in microorganisms which have GRAS status and are also successful vitamin B12 producers, surprising and unexpected results were obtained when the novel co-culture combination of the Lactobacillus strains, L. helveticus ATCC 8018 and L. fermentum ATCC 11976 showed synergistic effect in the production of vitamin B12 compared to other co-cultures and monocultures (Figure 1 and 2). These two vitamin B12 producing Lactobacillus strains also displayed better adhesion and colonization attributes to plastic surface in co-cultured conditions compared to monoculture conditions. Since these strains are natural strains, there is no added expenditure involved in the form of recombinant technology or use of supplements for the production of vitamin B12. Natural synergism of the two strains, L. helveticus ATCC 8018 and L. fermentum ATCC 11976 for the production of vitamin B12 is shown in the present invention. No added supplements are required for the present invention.

The present invention provides a process for production of vitamin B12, said process comprising co-culturing Lactobacillus helveticus ATCC 8018 and Lactobacillus fermentum ATCC 11976 cells in a culture medium, growing the cell cultures for a required time period, recovering the vitamin B12 from the culture medium and optionally purifying the recovered vitamin B12 obtained by the process. The process using co-cultured Lactobacillus helveticus ATCC 8018 and Lactobacillus fermentum ATCC 11976 cells have the advantage that they allow to formulate natural vitamin B12 together with the biomass in which it is produced. The process provides for an economical, efficient and industrially useful method of producing vitamin B12 which comprises co-culturing Lactobacillus helveticus ATCC 8018 and Lactobacillus fermentum ATCC 11976 cells which is capable of producing vitamin B12 and recovering the produced vitamin B12.

The present invention provides a synergistic potential of lactic acid bacteria for production of vitamin B12. The process for production of vitamin B12, wherein the process comprises co-culturing Lactobacillus helveticus ATCC 8018 and Lactobacillus fermentum ATCC 11976 cells in a culture medium, growing the L. helveticus and L. fermentum cells for a period in the range of 18 hours to 24 hours and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in the range from 9 -11 ng/L.

The process for production of vitamin B12 comprises optionally purifying the recovered vitamin B12 obtained by this process. The two Lactobacillus strains, L. helveticus ATCC 8018 and L. fermentum ATCC 11976 were co-cultured in a ratio of 1:1 under aerobic conditions, temperature in the range of 35-39°C and pH 5.0 to 6.0 for a fermenter residence period of 18-24 hours in a micro-inoculum broth. The micro-inoculum broth comprises proteose peptone, yeast extract, D (+) glucose, potassium di-hydrogen phosphate and Tween-80. The two strains, L. helveticus ATCC 8018 and L. fermentum ATCC 11976, were first cultured separately in Man, Rogosa and Sharpe (MRS) media for 24 hr to the cell number approximately 108 CFU/ml for preparation of feedstock. The feedstock of both cultures thus prepared was mixed in 1:1 ratio and was added at a concentration of 0.1% level (50ul in 50 ml) in micro-inoculum broth and incubated for 18-24 hours. The two bacterial culture feedstocks were mixed in 1:1 (v/v) ratio before each co-culture experiment. Suitable feedstocks well known in the art can be used for the growth of the bacterial strains.

Interestingly, two strains of different Lactobacillus species namely L. helveticus ATCC 8018 and L. fermentum ATCC 11976 were found to synergize each other, thereby leading to enhanced vitamin B12 production as compared to their monocultures. A significant enhancement of 1.25 folds was observed in vitamin B12 production in co-culture of L. helveticus ATCC 8018 and L. fermentum ATCC 11976 compared to their monocultures. L. helveticus ATCC 8018 monoculture gave a yield of 8.2 pg/ml, L. fermentum ATCC 11976 monoculture gave a yield of 8.4 pg/ml while the co-culture of these strains gave a yield of 10.5pg/ml of vitamin B12. Figure 1 shows the level of vitamin B12 production by Lactobacillus strains under monoculture and co-culture conditions. Figure 2 shows the amount of vitamin B12 produced by the co-cultures of Lactobacillus strains and the microbial strains, Propionibacterium shermanii and P. freudenreichii used in industrial production of vitamin B12. The use of Lactobacillus strain co-cultures can give add-on functional benefits to the host.

The present invention provides a process for production of vitamin B12, wherein the process comprises co-culturing Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 under aerobic conditions at a temperature ranging from 35°C to 39°C in a culture medium to obtain co-culture of Lactobacillus helveticus ATCC 8018 and Lactobacillus fermentum ATCC 11976, growing the co-culture of Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 for a period of 20 to 24 hours, and recovering vitamin B12 from the co-culture; wherein amount of vitamin B12 in the culture is in the range of 9 ng/L to 11 ng/L.

Further embodiment of the present invention provides a process for production of vitamin B12, wherein the process comprises co-culturing Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 under aerobic conditions at a temperature ranging from 35°C to 39°C for a period of 20 to 24 hours in a culture medium to obtain co-culture of the cells and recovering vitamin B12 from the co-culture of the cells, wherein amount of vitamin B12 in the culture is in the range of 9 ng/L to 11 ng/L.

Another embodiment of the present invention provides a process for production of vitamin B12, wherein the process comprises co-culturing Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 under aerobic conditions at a temperature ranging from 35°C to 39°C in a culture medium to obtain co-culture of Lactobacillus helveticus ATCC 8018 and Lactobacillus fermentum ATCC 11976, growing the co-culture of Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 for a period of 20 to 24 hours, and recovering vitamin B12 from the co-culture; wherein the amount of vitamin B12 in the culture is in the range of 10 ng/L to 11 ng/L.

In an embodiment of the present invention, there is provided a process for the production of vitamin B12, said process comprising co-culturing Lactobacillus helveticus and Lactobacillus fermentum cells in a culture medium, growing the L. helveticus and L. fermentum cells for a period in the range of 18 hours to 24 hours and recovering vitamin B12 from the culture medium of the previous step, wherein vitamin B12 is produced in an amount of 10.5 ng/L.

In an embodiment of the present invention, there is provided a process for the production of vitamin B12, said process comprising co-culturing Lactobacillus helveticus ATCC 8018 and Lactobacillus fermentum ATCC 11976 cells in a culture medium, growing the L. helveticus and L. fermentum cells for a period in the range of 18 hours to 24 hours and recovering vitamin B12 from the culture medium of the previous step, wherein vitamin B12 is produced in an amount in the range of 9 - 11 ng/L.

In yet another embodiment of the present invention, there is provided a process for production of vitamin B12, wherein the process comprises co-culturing Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 under aerobic conditions at a temperature ranging from 35°C to 39°C in a culture medium to obtain co-culture of Lactobacillus helveticus ATCC 8018 and Lactobacillus fermentum ATCC 11976, growing the co-culture of Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 for a period of 20 to 24 hours, and recovering vitamin B12 from the co-culture; wherein amount of vitamin B12 in the culture is in the range of 9 ng/L to 11 ng/L, wherein the process further comprises purifying vitamin B12.

In still another embodiment of the present invention, there is provided a process for the production of vitamin B12, said process comprising co-culturing Lactobacillus helveticus and Lactobacillus fermentum cells in a culture medium, growing the L. helveticus and L. fermentum cells for a period in the range of 18 hours to 24 hours and recovering vitamin B12 from the culture medium of the previous step, wherein vitamin B12 is produced in an amount of 10.5 ng/L, wherein the culture medium comprises a carbon source, a nitrogen source, a phosphorus source, trace elements, growth factors, surfactants and emulsifiers.

In an embodiment of the present invention, there is provided a process for production of vitamin B12, wherein the process comprises co-culturing Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 under aerobic conditions at a temperature ranging from 35°C to 39°C in a culture medium to obtain co-culture of Lactobacillus helveticus ATCC 8018 and Lactobacillus fermentum ATCC 11976, growing the co-culture of Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 for a period of 20 to 24 hours, and recovering vitamin B12 from the co-culture; wherein amount of vitamin B12 in the culture is in the range of 9 ng/L to 11 ng/L, wherein the culture medium comprises proteose peptone, yeast extract, D(+) glucose, potassium dihydrogen phosphate and Tween-80.

In still another embodiment of the present invention, there is provided a process for the production of vitamin B12, said process comprising co-culturing Lactobacillus helveticus and Lactobacillus fermentum cells in a culture medium, growing the L. helveticus and L. fermentum cells for a period in the range of 18 hours to 24 hours and recovering vitamin B12 from the culture medium of the previous step, wherein vitamin B12 is produced in an amount of 10.5 ng/L, wherein Lactobacillus helveticus and Lactobacillus fermentum cells are grown at a temperature in the range of 35°C to 39°C.

In still another embodiment of the present invention, there is provided a process for the production of vitamin B12, said process comprising co-culturing Lactobacillus helveticus and Lactobacillus fermentum cells in a culture medium, growing the L. helveticus and L. fermentum cells for a period in the range of 18 hours to 24 hours and recovering vitamin B12 from the culture medium of the previous step, wherein vitamin B12 is produced in an amount of 10.5 ng/L, wherein Lactobacillus helveticus and Lactobacillus fermentum cells are grown at a temperature of 35°C.

An embodiment of the present invention provides a process for production of vitamin B12, wherein the process comprises co-culturing Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 under aerobic conditions in a culture medium to obtain co-culture of Lactobacillus helveticus ATCC 8018 and Lactobacillus fermentum ATCC 11976, growing the co-culture of Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 for a period of 20 to 24 hours, and recovering vitamin B12 from the co-culture; wherein amount of vitamin B12 in the culture is in the range of 9 ng/L to 11 ng/L, wherein Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 cells are grown at a temperature of 37°C.

In another embodiment of the present invention, there is provided a process for production of vitamin B12, wherein the process comprises co-culturing Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 under aerobic conditions at a temperature ranging from 35°C to 39°C in a culture medium to obtain co-culture of Lactobacillus helveticus ATCC 8018 and Lactobacillus fermentum ATCC 11976, growing the co-culture of Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 for a period of 20 to 24 hours, and recovering vitamin B12 from the co-culture; wherein amount of vitamin B12 in the culture is in the range of 9 ng/L to 11 ng/L, wherein the pH of the culture medium is maintained in the range of 5.0 to 6.0.

In still another embodiment of the present invention, there is provided a process for the production of vitamin B12, said process comprising co-culturing Lactobacillus helveticus and Lactobacillus fermentum cells in a culture medium, growing the L. helveticus and L. fermentum cells for a period in the range of 18 hours to 24 hours and recovering vitamin B12 from the culture medium of the previous step, wherein pH of the culture medium is in the range of 5.4-5.8.

In still another embodiment of the present invention, there is provided a process for the production of vitamin B12, said process comprising co-culturing Lactobacillus helveticus and Lactobacillus fermentum cells in a culture medium, growing the L. helveticus and L. fermentum cells for a period in the range of 18 hours to 24 hours and recovering vitamin B12 from the culture medium of the previous step, wherein pH of the culture medium is 5.6.

A major advantage of the present invention is to enhance the bioavailability of vitamin B12 using Lactobacillus strains that can be added as food supplements and have potential colonizing ability since they already have GRAS status and are well-known probiotics with various health benefits. Vitamin B12 supplements in form of LAB in foods can benefit people suffering from deficiency symptoms, chronic illnesses and those who are under medication that interfere with vitamin B12 absorption. The present invention can find extensive application in the area of functional foods, nutraceuticals, pharmaceutical industries and other industries. Lactobacillus culture is employed in the food industry and already has GRAS (Generally Regarded as Safe) status. Lactobacillus cultures can be used for large scale biosynthesis of vitamin B12 and can be added to foods as a source of vitamin B12. The present invention also relates to a composition comprising the Lactobacillus strains for the production of vitamin B12 and could be used in the production of food products, food additives, food supplements and as fortifying components, nutritional supplements and probiotics. The co-cultures can also be explored for the production of other metabolites besides vitamin during its growth like folate, conjugated linoleic acids, bacteriocins and beneficial fatty acids like eicosapentaenoic acid and docosahexaenoic acid.

An embodiment of the present invention provides a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum with or without carrier.

In an embodiment of the present invention, there is provided a composition comprising Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 and a carrier.

In yet another embodiment of the present invention there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum without a carrier.

In yet another embodiment of the present invention there is provided a composition comprising Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976, wherein the composition comprises a carrier selected from the group consisting of dietary fibers, inulins, carbohydrates, proteins, lipids, phytochemicals, glycosylated proteins, a pharmaceutically acceptable carrier and combinations thereof.

In yet another embodiment of the present invention there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum wherein the composition comprises a pharmaceutically acceptable carrier.

In one embodiment of the present invention, there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum with or without carrier, wherein the Lactobacillus helveticus is Lactobacillus helveticus ATCC 8018 and Lactobacillus fermentum Lactobacillus fermentum ATCC 11976.

In another embodiment of the present invention, there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum with or without a carrier, wherein the composition is used to maintain and restore the natural flora in the gastrointestinal tract.

In still another embodiment of the present invention, there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum with or without a carrier, wherein the composition is used as a vitamin B12 probiotic supplement.

In still another embodiment of the present invention, there is provided a composition comprising vitamin B12 producing Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 with or without a carrier, wherein the composition is a probiotic supplement, food, feed or nutritional supplement, food products or pharmaceutical composition.

In still another embodiment of the present invention, there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum with or without a carrier, wherein the composition is a food supplement.

In still another embodiment of the present invention, there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum with or without a carrier, wherein the composition is a feed supplement.

In still another embodiment of the present invention, there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum with or without a carrier, wherein the composition is a nutritional supplement.

In still another embodiment of the present invention, there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum with or without a carrier, wherein the composition is a food product.

In still another embodiment of the present invention, there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum with or without a carrier, wherein the composition is a pharmaceutical composition.

In still another embodiment of the present invention, there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum with or without a carrier, wherein the composition is a nutraceutical composition.

In another embodiment of the present invention, there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum with or without a carrier, wherein the composition is encapsulated and/or coated.

In another embodiment of the present invention, there is provided a composition comprising co-cultures of vitamin B12 producing Lactobacillus helveticus and Lactobacillus fermentum with or without a carrier, wherein the composition is in the form of tablets, sucking tablets, sweets, chewing gums, capsules, cream, gel, ointment, lotion, melting strips and sprays.

Another embodiment of the present invention provides a process for the production of vitamin B12 and precursors thereof having detectable vitamin B12 activity, said process comprising: co-culturing Lactobacillus helveticus and Lactobacillus fermentum cells in a culture medium, harvesting the cultures upon growing for 18 hours to 24 hours, recovering of vitamin B12 from the culture medium and purifying the recovered vitamin B12, wherein the yield of the recovered vitamin B12 is 10.5 ng/L, wherein Lactobacillus helveticus and Lactobacillus fermentum strains are present in the initial concentration of 108 CFU/ml.

Another embodiment of the present invention provides a process for the production of vitamin B12 and precursors thereof having detectable vitamin B12 activity, said process comprising: co-culturing Lactobacillus helveticus and Lactobacillus fermentum cells in a culture medium, harvesting the cultures upon growing for 18 hours to 24 hours, recovering of vitamin B12 from the culture medium and purifying the recovered vitamin B12, wherein the yield of the recovered vitamin B12 is about 10.5 ng/L, wherein Lactobacillus helveticus and Lactobacillus fermentum cells are present in the initial concentration ratio range of 3:1 to 1:3.

Another embodiment of the present invention provides a process for the production of vitamin B12 and precursors thereof having detectable vitamin B12 activity, said process comprising: co-culturing Lactobacillus helveticus and Lactobacillus fermentum cells in a culture medium, harvesting the cultures upon growing for 18 hours to 24 hours, recovering of vitamin B12 from the culture medium and purifying the recovered vitamin B12, wherein the yield of the recovered vitamin B12 is about 10.5 ng/L, wherein Lactobacillus helveticus and Lactobacillus fermentum cells are present in the initial concentration ratio range of 2:1 to 1:2.

Another embodiment of the present invention provides a process for the production of vitamin B12 and precursors thereof having detectable vitamin B12 activity, said process comprising: co-culturing Lactobacillus helveticus and Lactobacillus fermentum cells in a culture medium, harvesting the cultures upon growing for 18 hours to 24 hours, recovering of vitamin B12 from the culture medium and purifying the recovered vitamin B12, wherein the yield of the recovered vitamin B12 is about 10.5 ng/L, wherein Lactobacillus helveticus and Lactobacillus fermentum cells are present in the initial concentration ratio of 1:1.

Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible. As such, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiment contained therein.

EXAMPLES

The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure.

Example 1

Preparation of culture medium for L. helveticus ATCC 8018 and L. fermentum ATCC 11976 and formation of co-cultures

Culture media for monocultures and feedstock preparation

The monocultures of L. helveticus ATCC 8018 and L. fermentum ATCC 11976 were grown in De Man, Rogosa and Sharpe broth (MRS broth; Sigma). The broth was prepared by dissolving 10 g peptone, 8 g meat extract, 4 g yeast extract, 20 g D(+)-glucose, 2 g di-potassium hydrogen phosphate, 5 g sodium acetate trihydrate, 2 g triammonium citrate, 0.2 g magnesium sulfate heptahydrate and 0.05 g manganous sulfate tetrahydrate in One liter distilled water. The media was then sterilized by autoclaving at 121°C for 15 min at 15 psi. The monocultures were grown at 37°C for 20-24 hrs under aerobic conditions. These cultures were further used for the preparation of the feedstock cultures. The cells were grown to a desired cell density of 10 CFU/ml.

Culture media for co-cultures

The micro-inoculum broth for co-culturing of the Lactobacillus strains was prepared by dissolving 5 g proteose peptone, 20 g yeast extract, 10 g D(+) glucose, 2 g potassium dihydrogen phosphate and 0.1 g Tween-80 in one liter of double distilled water. The media was then sterilized by autoclaving at 121°C for 15 min at 15 psi.

Example 2

Inoculation and growth of L. helveticus ATCC 8018 and L. fermentum ATCC 11976 and maintenance of co-cultures

The co-culture of the strains, L. helveticus ATCC 8018 and L. fermentum ATCC 11976 was carried out by obtaining aliquots of 50 ul each of L. fermentum ATCC 11976 and L. helveticus ATCC 8018 cultures from the feedstock when the cell density of 108 CFU/ml was reached as measured spectro-photometrically at an optical density of 600 nm. These aliquots were mixed and cultured together in 50 ml micro-inoculum broth (Fluka) at 37°C for 18-24 hrs under aerobic conditions. The co-cultures were grown to the cell density of approximately 108 CFU/ml.

Example 3

Detection and Estimation of Vitamin B12

The growth of Lactobacillus delbrueckii var. lactis is limited by the concentration of vitamin B12 in defined medium as described above. This principle was used to measure the amount of vitamin B12 production by bacteria cultures as per AOAC International, 1995. The assay involved spectrophotometric examination of the growth of Lactobacillus delbrueckii.

The medium comprises D(+)-Glucose anhydrous (40.0 g/L), casein hydrolysate "Vitamin-free" (15.0 g/L), L-asparagine (0.2 g/L), L-cystinium chloride (0.2 g/L), L-Cystine (0.4 g/L), DL-tryptophan (0.4 g/L), adenine (0.02 g/L), guanine (0.02 g/L), uracil (0.02 g/L), xanthine (0.02 g/L), 4-aminobenzoic acid (0.002 g/L), L(+)-ascorbic acid (4.0 g/L), D(+)-biotin (Vitamin H) (0.00001 g/L), calcium D(+)-pantothenate (0.001 g/L), folic acid (0.0002 g/L), nicotinic acid (0.002 g/L), pyridoxal hydrochloride (0.004 g/L), pyridoxamine hydrochloride (0.0008 g/L), riboflavin (0.001 g/L), thiaminium dichloride (0.001 g/L), potassium phosphate dibasic (1.0 g/L), iron(II)sulfate (0.02 g/L), potassium phosphate monobasic (1.0 g/L), magnesium sulfate (0.4 g/L), manganese(II) sulfate (0.02 g/L), sodium acetate anhydrous (20.0 g/L) and sodium chloride (0.02 g/L). Preparation of standard solution 100 mg of vitamin B12 (Fluka 95190) was dissolved in 1 litre of distilled water (lOOug/ml) to prepare vitamin B12 stock. Standard curve was then prepared using concentrations ranging from 0 to 50 pg/ml.

Preparation of preparatory culture

Lactobacillus delbrueckii subsp. lactis (ATCC 7830) was inoculated in the Micro-Inoculum-Broth (Fluka), and incubated for 20 hours at 37°C under aerobic conditions. The culture was then centrifuged at 3500 rpm for 10 min at 4°C. The culture pellet was washed three times with physiological saline and suspended in 5ml of saline. Microbial counts were adjusted to 108 bacteria/ml using optical density (OD) measurements at 600nm.

Preparation of assay solution

The test cultures of microorganisms, L. fermentum (ATCC 11976) and L. helveticus (ATCC 8018) and the co-cultures of L. fermentum and L. helveticus, were grown in 50ml Micro-inoculum broth at 37°C for 18 hrs (OD at 600nm, 108 cells/ml). The cultures were centrifuged at 3500 rpm for 10 min at 4°C. The pellets were discarded and the supernatants were used as the assay solution for test. Different volumes (1ml, 1.5ml, 2ml, 2.5ml, 3ml, and 4ml) of the assay solution were taken in test tubes and the final volume was made upto 5 ml using distilled water. Dehydrated vitamin B12 assay medium (83g) was dissolved with 2ml Tween-80 in 1 litre distilled water and sterilized.

Photometric estimation of vitamin B12

The standard solution as described above, the assay solution and controls (excluding the sterile controls) were incubated with 0.1 mi of preparatory culture and incubated in dark for 24 hours at 37°C. The calibration standards and samples were measured photometrically at 546 nm against the culture control. A calibration curve was recorded with the OD values at 546 nm on the linear ordinate against the vitamin B12 concentration on the logarithmic abscissa. Standard curve was prepared using known concentrations of vitamin B12 standard ranging from 0-50 pg/ml. Vitamin content was calculated in pg/mL or ng/L for all the different volumes of the assay solution. The amount of vitamin B12 produced by the co-cultures of L.helveticus ATCC 8018 and L. fermentum ATCC 11976 is 10.5 ng/L.

In further continuation, the recovery and extraction of vitamin B12 produced by the co-cultures of L. helveticus and L. fermentum was carried out. The purification and concentration of recovered vitamin B12 and evaluation of the purified vitamin B12 was done.

1/ We Claim:

1. A process for production of vitamin B12, wherein said process comprises

a. co-culturing Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 under aerobic conditions at a temperature ranging from 35°C to 39°C for a period of 20 to 24 hours in a culture medium to obtain co-culture of the cells, and

b. recovering vitamin B12 from said co-culture of the cells wherein amount of vitamin B12 in the culture is in the range of 9 ng/L to 11 ng/L.

2. The process as claimed in claim 1, wherein the temperature is 37°C

3. The process as claimed in claim 1, wherein the culture medium comprises proteose peptone, yeast extract, glucose, potassium dihydrogen phosphate and Tween-80.

4. The process as claimed in claim 1, wherein the process further comprises purifying vitamin B12.

5. The process as claimed in claim 1, wherein pH of the culture medium is in the range of 5.0 to 6.0.

6. A composition comprising Lactobacillus helveticus having accession number ATCC 8018 and Lactobacillus fermentum having accession number ATCC 11976 and a carrier.

7. The composition as claimed in claim 6, wherein said carrier is selected from a group consisting of dietary fibers, inulins, carbohydrates, proteins, lipids, phytochemicals, glycosylated proteins, a pharmaceutically acceptable carrier and combinations thereof.

8. The composition as claimed in claim 6, wherein said composition is probiotic supplement, food, feed or nutritional supplement, vitamin supplement, food products or pharmaceutical composition.