FIELD OF INVENTION

The present invention relates to the field of yeast microbiology especially to vitaminology. The present invention in particular is related to production of vitamin B12 from the yeast, Kluyveromyces.

BACKGROUND OF THE INVENTION

In 1934, three researchers won the Nobel Prize in medicine for discovering the lifesaving properties of vitamin B12. They found that eating large amounts of raw liver, which contains high amounts of vitamin B12, could save the life of previously incurable patients with pernicious anaemia. This finding saves 10,000 lives a year in the US alone. Vitamin B12 was isolated from liver extract in 1948 and its structure was elucidated 7 years later.

Vitamin B12 is the largest and most complex of all the vitamins. Vitamin B12, an anti-pernicious factor, is required for the healthy functioning of the body, for memory and concentration, to maintain normal energy levels, for normal growth and development, to help the immune system function properly, as an essential growth factor, in the regeneration of folic acid, to help prevent cancer and to metabolize macronutrients properly. Vitamin B12 is also helpful during pregnancy and lactation. Vitamin B12 is necessary for the formation of red blood cells, nerve sheaths, neurological function, various proteins, and DNA synthesis; 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.

The name, vitamin B12 is generic for a specific group of cobalt-containing corrinoids with biological activity in humans. 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 is widely used clinically 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.

In humans, vitamin B12 functions primarily as a coenzyme in intermediary metabolism. Two metabolic reactions are dependent on vitamin B12 where it functions as a cofactor in (a) the methionine synthase reaction with methylcobalamin and (b) the methylmalonyl CoA mutase reaction with adenosylcobalamin. Methionine synthase catalyzes the conversion of homocysteine to methionine. Methionine is required for the formation of S-adenosylmethionine, a universal methyl donor for various substrates, including DNA, RNA, hormones, proteins, and lipids. Methylmalonyl CoA mutase converts 1-methylmalonyl CoA to succinyl CoA (an important reaction in lipid and carbohydrate metabolism). L-methylmalonyl-CoA mutase converts L-mefhylmalonyl-CoA to succinyl-CoA in the degradation of propionate, an important step in fat and protein metabolism. Succinyl-CoA is also required for haemoglobin synthesis.

Vitamin B12 is the only vitamin that cannot be obtained from eating a strict plant-based diet. Though there is some vitamin B12 in plants, it is not nearly enough to meet the requirements of the human body. Humans have an auxotrophic requirement for vitamin B12 with a recommended nutrient intake (RNI) for healthy adults of 2.4 (g/day) [Institute of Medicine, Food and Nutrition Board, Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington, DC: National Academy Press, 1998]. Animals, including humans, and protists require cobalamin but apparently do not synthesize it, whereas plants and fungi are thought to neither synthesize nor use it. Vitamin B12 synthesized by a few representatives of bacteria and archaea 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 made 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 make vitamin B12. However, the problem is that humans cannot absorb enough of it to meet the daily requirement. Human faeces contain appreciable quantities of vitamin B12 or vitamin B12-like material which is suggested to be produced by bacteria in the colon. However, vitamin B12 produced by colonic bacterial fermentations is most likely not reachable to the host. It has been suggested that vitamin B12 produced by a microorganism capable of colonizing proximal intestine would potentially be host accessible [Albert et al. 1980, Vitamin B12 synthesis by human small intestinal bacteria. Nature; Feb 21; 283(5749):781-2]. 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. Vegetarians and immune compromised individuals are the most at risk of vitamin B12 deficiency.

Vitamin B12 bound to protein in food is released by the activity of hydrochloric acid and gastric protease in the stomach. Fortified foods or synthetic vitamin B12 does not require this separation step. However, the free vitamin B12 (extrinsic factor) has to combine with intrinsic factor, a glycoprotein secreted by the stomach's parietal cells, and the resulting complex undergoes absorption within the distal ileum by receptor-mediated endocytosis. This absorption is observed to decrease drastically when the capacity of intrinsic factor is exceeded, at 1-2 meg of vitamin B12 [Carmel, 2008, How I treat cobalamin (vitamin B12) deficiency. Blood, 112: 2214-21].
Vitamin B12 deficiency leads 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. It is most likely to occur among elderly people, as absorption through the gut declines with age. The autoimmune disease pernicious anemia is another common cause. It can also cause symptoms of mania and psychosis. In rare extreme cases, paralysis can result. Supplementation of folic acid and vitamin B12 were found to be cost-effective treatment for heart disease and in the reduction of associated deaths among the adult U.S. population as 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 also 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 orrsuch medication also require vitamin B12 supplementation to combat the loss of this vitamin. In elderly with low vitamin B12 status, restoration of vitamin B12 concentration was found to be protective against cognitive impairment. [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].

Vegetarianism has been widely practiced for several millennia in India. 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. Lower serum vitamin B12 levels in Indian lacto-vegetarians were confirmed by studies from different geographic regions in India. 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. Thus, in the Indian scenario it is vital, especially for vegetarians to routinely take cobalamin or vitamin B12 supplements.

Strict vegetarian dietary regimes poor in vitamin B12, 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. The production of vitamin B12 by bacterial cultures is well documented. 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. However, because of their already naturally high vitamin B12 productivity and their rapid growth, mainly Propionibacterium shermanii and Pseudomonas denitrificans strains are employed for industrial production of vitamin B12.
Since vitamin B12 biosynthesis is restricted only to microorganisms and chemical synthesis is a highly complicated process involving more than 70 synthesis steps, the use of yeast cells that naturally produce this complex vitamin can be exploited. In comparison to bacteria, yeast is eukaryotic, larger in size, has a thicker cell wall, passes through the GI tract and does not have to be refrigerated for viability.

Yeasts have been used by humans for making bread and beverage products since 2500BC. They have a history of safe use and are now popularly used as single cell proteins. Yeasts are an excellent source of protein containing essential amino acids, rich in vitamins, dietary fibres, minerals, and naturally low in fat and salt. Yeast is easy to use and blends well with liquids. It can be used in almost any recipe and in small amounts are known to enhance the flavour of food and food products. The most popular yeast strain in the industry is Saccharomyces cerevisiae. However, the potential of other yeast strains as producers of important metabolites and fine chemicals is yet to be explored completely.

The yeast, Kluyveromyces lactis (Kluyveromyces yeast), has derived its name from its ability to assimilate lactose and convert it into lactic acid. Although it is ascomycetous budding yeast closely related to Saccharomyces cerevisiae, K. lactis has become an attractive alternative model owing to distinct metabolic and physiological properties. K. lactis is of great industrial interest since it is able to grow to a high cell density and to secrete heterologous proteins efficiently. Lactase from K. lactis has got a GRAS status and K. lactis itself has a history of human consumption, as it is a component of kefir, a fermented milk drink that originated with shepherds of the North Caucasus. Kefir contains a mixture of organisms including Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. diacetylactis, Leuconostoc mesenteroides subsp. cremoris, Lactobacillus kejyr, Saccharomyces unisporus and Kluyveromyces lactis; however the potential of yeast for production of vitamin B12 is unexplored so far.

Yeast are known to produce B-complex vitamins (Giri K. V. and Krishnaswamy, P. R.; 1954, Studies on the synthesis of riboflavin by a mutant yeast, Saccharomyces cerevisiae. J. Bacteriol., 67, 309-313). However, they are usually considered as non producers of vitamin B12 [Martens J.H., Barg H., Warren M., Jahn D. (2002) Microbial production of vitamin B12. Applied Microbiology and Biotechnology. 58 (3): 275-285, DOI: 10.1007/s00253-001-0902-7]. However, the yeast Kluyveromyces waltii is known to possess genes for producing cobalamin. Saccharomyces cerevisiae, commonly referred to as Brewer's yeast and a highly exploited yeast in industrial biotechnology, is grown specifically to make nutritional supplements which is a rich source of minerals, particularly chromium, selenium; protein; and the B-complex vitamins. The B-complex vitamins in brewer's yeast include Bl (thiamine), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), B6 (pyridoxine), B9 (folic acid), and H or B7 (biotin) but does not contain vitamin B12, an essential vitamin found in meat and dairy products. Vegetarians sometimes take Brewer's yeast mistakenly believing that it provides B12, which can be lacking in their diet. Although US patent 4,310,553 (Odintsova, 1982) describes a process for producing food vitamin concentrate from the wine yeast, Saccharomyces vini (ellipsoideus), it is specifically mentioned that the food vitamin concentrate may be added with vitamin B12 in the required levels since vitamin B12 is absent in the yeast and the grapes. As mentioned in the prior art, beer yeast and pressed baker's yeast produce only minor amounts of vitamins which are insufficient as a stand-alone source required for human metabolism. The resources for the beer yeast as food vitamin producers is also scarce since the amount of yeast accumulated is used up for the next cycle of fermentation.

The use of Kluyveromyces yeast is highly advantageous since they allow formulating natural vitamin B12 together with the biomass in which it is produced. Although Propionibacterium sp are the prime microorganisms used in the fermentation industry for the microbial synthesis of vitamin B12, there are several limiting factors in their exploitation. Propionic acid produced by these bacteria act as a limiting factor for biomass growth and thereby for high yield of vitamin B12. A circumvention of this problem of propionic acid toxicity and hampering of vitamin B12 yield may require the deployment of more than one type of fermentation techniques, use of immobilization techniques etc. leading to an escalation of costs in the manufacture of the vitamin B12.

The present invention aims to exploit a hitherto unexploited source of microbial synthesis of vitamin B12. The present invention does not involve any tedious and laborious procedure of autolysis of the prior art and is probably the first report of production of vitamin B12 from yeast since no yeast strain is yet reported to produce vitamin B12.

SUMMARY OF THE INVENTION

An aspect of the present invention relates to a process for production of vitamin B12, wherein said process comprises culturing Kluyveromyces lactis ATCC 8585 or Kluyveromyces waltii ATCC 56500 cells under aerobic condition at 25°C to 27°C in a culture medium comprising proteose peptone, yeast extract, glucose, potassium di-hydrogen phosphate and Tween-80 to obtain a culture of Kluyveromyces lactis ATCC 8585 or Kluyveromyces waltii ATCC 56500; growing the Kluyveromyces culture for a period of 18 to 25 hours; and recovering vitamin B12 from the Kluyveromyces culture, wherein amount of vitamin B12 in the culture is in the range of 16 ng/L to 24 ng/L.

Another aspect of the present invention relates to a composition comprising vitamin B12 producing Kluyveromyces lactis having accession number ATCC 8585, Kluyveromyces waltii ATCC 56500 or combination thereof 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 Kluyveromyces yeast.

It is another object of the present invention to provide a composition comprising vitamin B12 producing Kluyveromyces yeast.

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

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

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 Lactobacillus sp, Bacillus sp and Kluyveromyces lactis ATCC 8585.

Figure 2 shows the Vitamin B12 production by industrial strains of Propionibacterium shermanii, Propionibacterium freudenrechii and Kluyveromyces lactis ATCC 8585. Figure 3: Vitamin B12 production by two Kluyveromyces lactis ATCC 8585 and Kluyveromyces waltii ATCC 56500

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." "Including" and "including but not limited to" are used interchangeably.

The term "vitamin B12" should be 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 [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 "probiotic" 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 Kluyveromyces strains", "composition(s) comprising Kluyveromyces strains" and "composition(s)" are used interchangeably.

The term "subject" means a living organism, including a plant, a microbe, a human, an animal (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 production of vitamin B12, said process comprising culturing Kluyveromyces yeast 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 Kluyveromyces yeast 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 cultivating Kluyveromyces yeast cells which is capable of producing vitamin B12 and recovering the produced vitamin B12.

During a screening process of microorganisms for vitamin B12 production potential, the inventors surprisingly found two yeast strains namely Kluveromyces lactis ATCC 8585 and Kluveromyces waltii ATCC 56500 producing an unexpected significantly high amount of vitamin B12 compared to Lactobacillus sp and Bacillus sp, as shown in Figure 1. This might be a first report on the production of the vitamin by a yeast culture which is apathogenic, produces no toxins and already has GRAS status. Kluyveromyces lactis ATCC 8585 has a history of safe usage in the food industry and is being actively used for production of lactase that is added in food. Thus, Kluyveromyces lactis ATCC 8585 and Kluveromyces waltii ATCC 56500 can be further used for industrial production of vitamin B12 or as vitamin B12 source in food and nutritional supplements.
The present invention provides a process for production of vitamin B12, wherein the process comprises culturing Kluyveromyces cells in a culture medium, growing the Kluyveromyces cells for a period in the range of 18 hours to 25 hours and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in an amount in the range of 18 ng/L to 22 ng/L, more preferably in the range of 19 ng/L to 21 ng/L, most preferably in the range of 19.5 ng/L to 20.5 ng/L. Interestingly, the inventors obtained an unexpected and surprising result when the level of vitamin B12 production by Kluyveromyces lactis ATCC 8585 was found to be 19.9 pg/ml and Kluveromyces waltii ATCC 56500 was found to be 19.8 ng/L which was almost double to that produced by Lactobacillus and Bacillus sp in the range of 0.5 to 7.5 pg/ml, at the same
Ft inoculum level i.e. 10 CFU/ml. Kluyveromyces lactis is as good a vitamin B12 producer as other industrial strains, including Propionibacterium shermanii and Propionibacterium freudenreichii, as shown in Figure 2.

An embodiment of the present invention provides a process for production of vitamin B12, wherein the process comprising culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces cells for a period in the range of 18 hours to 25 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 18 ng/L to 22 ng/L.
In an embodiment of the present invention, there is provided a process for production of vitamin B12, wherein the process comprising culturing Kluyveromyces lactis ATCC

8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces cells for a period in the range of 18 hours to 25 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 18 ng/L to 22 ng/L, more preferably in the range of 19 ng/L to 21 ng/L, most preferably in the range of 19.5 ng/L to 20.5 ng/L.

In yet another embodiment of the present invention, there is provided a process for production of vitamin B12, wherein the process comprising culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces cells for a period in the range of 18 hours to 25 hours and recovering vitamin B12 from the culture medium of the previous step, wherein vitamin B12 is produced in an amount of 19.5 ng/L to 20.5 ng/L.

In another embodiment of the present invention, there is provided a process for production of vitamin B12, the process comprising culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces cells for a period in the range of 18 hours to 25 hours; and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in an amount in the range of 18 ng/L to 22 ng/L, wherein the process optionally comprises purifying the vitamin B12 after recovery from the culture medium.

Another embodiment of the present invention provides a process for production of vitamin B12 and precursors thereof having detectable vitamin B12 activity, said process comprising: culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium, harvesting the cultures upon growing for 20 hrs, recovering of vitamin B12 from the culture medium and purifying the recovered vitamin B12, wherein the yield of the recovered vitamin B12 is in the range of 18 ng/L to 22 ng/L, wherein the initial inoculum of the Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells is 108 CFU/ml.
Another embodiment of the present invention provides a process for production of vitamin B12 and precursors thereof having detectable vitamin B12 activity, said process comprising: culturing Kluyveromyces yeast cells in a culture medium, harvesting the cultures upon growing for 20 hrs, recovering of vitamin B12 from the culture medium and purifying the recovered vitamin B12, wherein the yield of the recovered vitamin B12 is 19.9 ng/L, wherein the initial inoculum of the Kluyveromyces lactis ATCC 8585 cells or Kluveromyces waltii ATCC 56500 is 108 CFU/ml.

In another embodiment of the present invention, there is provided a process for production of vitamin B12, the process comprising culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces cells for a period in the range of 18 hours to 25 hours; and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in an amount in the range of 18 ng/L to 22 ng/L, wherein the culture medium comprises a carbon source, a nitrogen source, a phosphorus source, trace elements, growth factors, surfactants and emulsifiers.

In still another embodiment of the present invention, there is provided a process for production of vitamin B12, the process comprising culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces cells for a period in the range of 18 hours to 25 hours; and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in an amount in the range of 18 ng/L to 22 ng/L, wherein the culture medium comprises Proteose peptone, yeast extract, glucose, potassium di-hydrogen phosphate and Tween-80.

In another embodiment of the present invention, there is provided a process for production of vitamin B12, the process comprises culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces cells for a period in the range of 18 hours to 25 hours; and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in an amount in the range of 18 ng/L to 22 ng/L, wherein the Kluyveromyces cells are grown at a temperature in the range of 25°C to 27°C.

In yet another embodiment of the present invention, there is provided a process for production of vitamin B12, the process comprising culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces cells for a period in the range of 18 hours to 25 hours; and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in an amount of 18 ng/L to 22 ng/L, wherein the Kluyveromyces cells are grown at a temperature of 25 °C.

In still another embodiment of the present invention, there is provided a process for production of vitamin B12, the process comprising culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces lactis cells for a period in the range of 18 hours to 25 hours; and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in an amount of 18 ng/L to 22 ng/L, wherein the Kluyveromyces cells are grown at a temperature of 26°C.

In an embodiment of the present invention, there is provided a process for production of vitamin B12, the process comprising culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces lactis cells for a period in the range of 18 hours to 25 hours; and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in an amount of 19.9 ng/L, wherein the Kluyveromyces cells are grown at a temperature of 27°C.

In another embodiment of the present invention, there is provided a process for production of vitamin B12, the process comprising culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces cells for a period in the range of 18 hours to 25 hours; and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in an amount in the range of 18 ng/L to 22 ng/L, wherein the pH of culture medium is in the range of 6.0 to 6.8.

In yet another embodiment of the present invention, there is provided a process for production of vitamin B12, the process comprising culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces cells for a period in the range of 18 hours to 25 hours; and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in an amount in the range of 18 ng/L to 22 ng/L, wherein the pH of culture medium is in the range of 6.3 to 6.8.

In yet another embodiment of the present invention, there is provided a process for production of vitamin B12, the process comprising culturing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 cells in a culture medium; growing the Kluyveromyces cells for a period in the range of 18 hours to 25 hours; and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in an amount in the range of 18 ng/L to 22 ng/L, wherein the pH of culture medium is in the range of 6.5 to 6.8.

In still another embodiment of the present invention, there is provided a process for production of vitamin B12, the process comprising culturing Kluyveromyces lactis ATCC 8585 cells or Kluveromyces waltii ATCC 56500 in a culture medium; growing the Kluyveromyces cells for a period in the range of 18 hours to 25 hours; and recovering vitamin B12 from the culture medium, wherein vitamin B12 is produced in an amount of 19.9 ng/L, wherein the pH of culture medium is in the range of 6.5 to 6.8.

Kluyveromyces lactis is employed in the food industry and already has GRAS (Generally Regarded as Safe) status. K lactis can be used for large scale biosynthesis of vitamin B12, can be added to foods as a source of vitamin and may be used for the production of nutritional yeast. The microorganism can also be explored for the production of other metabolites besides vitamin during its growth. The present invention also relates to a composition comprising the strain Kluyveromyces lactis ATCC 8585 wherein the strain is a vitamin B12 producing strain and could be used in the production of food products, food additives, food supplements, as fortifying components, nutritional supplements and probiotics. The present invention opens up a method for using Kluyveromyces lactis as probiotic supplements for vitamin B12, which can easily make way in vegetarian diets and can also, protect against and treat vitamin B12 deficiency.
An embodiment of the present invention provides a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585 or Kluveromyces waltii ATCC 56500 with or without a carrier.

In still another embodiment of the present invention, there is provided a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585 and Kluveromyces waltii ATCC 56500 with or without a carrier.

In yet another embodiment of the present invention there is provided a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585, Kluveromyces waltii ATCC 56500 or combination thereof, wherein the composition comprises a carrier selected from the group of dietary fibers, inulins, carbohydrates, proteins, lipids, phytochemicals, glycosylated proteins and combinations thereof.

In yet another embodiment of the present invention there is provided a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585 wherein the composition comprises a pharmaceutically acceptable carrier.

In another embodiment of the present invention, there is provided a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585 with or without a carrier, wherein the composition maintains and restores the natural flora in the gastrointestinal tract.

In still another embodiment of the present invention, there is provided a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585 with or without a carrier, wherein the composition is a vitamin B12 probiotic supplement.

In still another embodiment of the present invention, there is provided a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585 with or without a carrier, wherein the composition is used in the treatment of vitamin B12 deficiency.

In still another embodiment of the present invention, there is provided a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585 with or without a carrier, wherein the composition is a food, feed or nutritional supplement, food product or pharmaceutical composition.

In still another embodiment of the present invention, there is provided a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585 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 vitamin B12 producing Kluyveromyces lactis ATCC 8585 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 vitamin B12 producing Kluyveromyces lactis ATCC 8585 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 vitamin B12 producing Kluyveromyces lactis ATCC 8585 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 vitamin B12 producing Kluyveromyces lactis ATCC 8585 with or without a carrier, wherein the composition is a pharmaceutical composition.

In another embodiment of the present invention there is provided a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585 with or without a carrier, wherein the composition is encapsulated or coated.

In still another embodiment of the present invention there is provided a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585 with or without a carrier, wherein the composition is encapsulated.

In another embodiment of the present invention there is provided a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585 with or without a carrier, wherein the composition is coated.

In another embodiment of the present invention there is provided a composition comprising vitamin B12 producing Kluyveromyces lactis ATCC 8585 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.

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. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

Example 1

Vitamin B12 production using Kluyveromyces lactis ATCC 8585 and Kluyveromyces Waltii ATCC 56500

Micro-inoculum broth culture medium was prepared by dissolving 5 g Proteose peptone, 20 g Yeast extract, 10 g D (+)-glucose, 2 g potassium di-hydrogen phosphate and 0.1 g Tween-80 in one liter double distilled water. The media was sterilized by autoclaving at 121 °C for 15 min at 15 psi. lOul of inoculum of Kluyveromyces lactis having accession number ATCC 8585 was taken in 100 ml sterile micro-inoculum broth and incubated at 25±2 °C for 20 hrs. The cell count was adjusted to 108 CFU/ml for assay of vitamin B12 production.

The growth of Lactobacillus delbrueckii subspecies lactis having accession ATCC 7830 is limited by the concentration of vitamin B12 in defined medium. This principle was used to measure the amount of vitamin B12 production by bacteria cultures as per AOAC International, 1995.

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 vitamin B12 concentrations ranging from 0 to 50 pg/ml. This standard curve was then used for the estimation of vitamin B12 production.

Preparation of preparatory culture

Lactobacillus delbrueckii subsp. lactis having accession number 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 Kluyveromyces lactis having accession number ATCC 8585 and Kluyveromyces waltii having accession number ATCC 56500 were grown in 50ml Micro-inoculum broth culture medium as described in above at 37°C for 18±2 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 (lml, 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 one litre distilled water and was 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 ml of preparatory culture of Lactobacillus delbrueckii subsp. lactis having accession number ATCC 7830 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 on the linear ordinate against the vitamin B12 concentration on the logarithmic abscissa.

Vitamin content was calculated in pg/mL for all the different volumes of the assay solution. The amount of vitamin B12 produced by K. lactis ATCC 8585 was found to be 19.9 ng/L (Figure 1 and 2). The vitamin B12 production by K. waltii ATCC 56500 was estimated to be 19.8 pg/ml (19.8ng/L) (Figure 3).

In further continuation, the recovery and extraction of vitamin B12 produced by Kluyveromyces lactis ATCC 8585 and K. waltii ATCC 56500 was carried out. The purification and concentration of recovered vitamin B12 and evaluation of the purified vitamin B12 was done. Vitamin B12 was extracted and purified from the sample as per AOAC procedure (Kirchner U, Degenhardt K, Raffler G, Nelson M. (2012) Determination of vitamin B12 in infant formula and adult nutritionals using HPLC after purification on an immunoaffinity column: first action 2011.09. J AOAC Int.; 95(4):933-6). Vitamin B12 was extracted from sample using sodium acetate buffer in the presence of sodium cyanide. Briefly, cobalamin present in the fermentation media (pH adjusted to 5.5) was converted to cynocobalamin using potassium cyanide (0.001%). Cynocobalamin was then purified using affinity column (EASY-EXTRACT VITAMIN B12), eluted into methanol and then concentrated using rotary evaporator.

I/We Claim:

1. A process for production of vitamin B12, wherein said process comprises
a. culturing Kluyveromyces lactis ATCC 8585 or Kluyveromyces waltii ATCC 56500 cells under aerobic condition at 25 °C to 27°C in a culture medium comprising proteose peptone, yeast extract, glucose, potassium di-hydrogen phosphate and Tween-80 to obtain a culture of Kluyveromyces lactis ATCC 8585 or Kluyveromyces waltii ATCC 56500;
b. growing the Kluyveromyces culture for a period of 18 to 25 hours; and
c. recovering vitamin B12 from the culture of step (b); wherein amount of vitamin B12 in the culture is in the range of 16 ng/L to 24 ng/L.

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

3. The process as claimed in claim 1, wherein pH of the culture medium is in the range of6.0to6.8.

4. The process as claimed in claim 1, wherein amount of vitamin B12 in the culture is in the range of 18 ng/L to 22 ng/L

5. A composition comprising vitamin B12 producing Kluyveromyces lactis having accession number ATCC 8585, Kluyveromyces waltii ATCC 56500 or combination thereof and a carrier.

6. The composition as claimed in claim 5, 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.

7. The composition as claimed in claim 5, wherein said composition is probiotic supplement, food, feed or nutritional supplement, food product or pharmaceutical composition.