A novel composition comprising adenosyl cobalamin

Abstract

The invention relates to the field of microbial production of Vitamin B12.

Description

P62Q37Q27\NQ1A novel composition comprising adenosyl cobalaminField of the inventionThe invention relates to the field of microbial production of Vitamin B12.Background of the inventionVitamin B12 is chemically known as Coa-[a-5,6-dimethylbenzimidazolyl] - Co / 3-adenosyl cobamide or, in brief, adenosyl cobalamin. Herein, the terms Vitamin B12 and adenosyl cobalamin are used interchangeably herein.Vitamin B12 fermentations were traditionally carried out with a Propionibacterium, typically P freudenreichii which is a food grade bacterium that is present in dairy based foods (see e.g. WO199806868). Vitamin B12 as found in nature is adenosyl cobalamin. The adenosyl cobalamin in Propionibacterium is found in the cell and is not secreted into the fermentation medium. Downstream processing thus typically comprised a step of lysing the cells to release the adenosyl cobalamin which was subsequently purified. In US2006 / 0051323, vitamin B12 is produced in P freudenreichii. In the examples, see e.g.

[0075] , the biomass is pasteurized, as step which lyses the cells. In addition, the pasteurized biomass was spray dried at elevated temperature (see e.g.

[0080] , DE102013112046 relates to soy bean products such as tofu that is produced by cofermentation of vitamin B12-producing P freudenreichii. The resulting tofu comprises about 16 ng / g vitamin B12 (wet weight), see e.g.

[0001] , US7427397B2 relates inter alia to probiotic Propionibacterium strains in foods and supplements and their effect on mycobacteria such as Mycobacterium tuberculosis. Typically, about 21 ng / ml vitamin B12 is produced. US10857189B2 relates to Eubacterium hallii and Intestinimonas butyriciproducens comprising pseudovitamin B12; freeze dried bacteria are administered to a subject. EP2788511 B1 relates to a probiotic composition that may comprise Lactobacillus reuteri that may produce vitamin B12. A maximum of about 18ng / ml vitamin B12 is produced.Presently, commercially available vitamin B12 is largely cyano cobalamin, the cyanide salt of vitamin B12 which is not found in nature and is not biologically active in living organisms. The main reason to create cyano cobalamin as a 99% pure crystalline product after the fermentation is the removal of all (>99,9%) of the biomass. In this process the crystalline form and high affinity towards resins makes cyano cobalamin the preferred commercial form of vitamin B12. The crystalline form of cyano cobalamin to date is the form of choice. The crystalline cyano cobalamin is the starting point to create methyl cobalamin (for example US20040132687A1) and hydroxy cobalamin.The Propionibacterium based industrial production process was largely abandoned about 40 years ago, when new isolated and non-food grade bacteria like Pseudomonas denitrificans and later followed by Ensifer adhearans were discovered with a high yield of adenosyl cobalamin, which is partly kept inside the cell and partly excreted in the fermentation medium. With the introduction of a non-food grade bacterium and the adenosyl cobalamin divided over the cell and medium it became mandatory to produce a 99% pure cyano cobalamin to access the feed and food market with this form of vitamin B12. Cell fragments, toxins and DNA fragments of antibiotic resistanceP62Q37Q27\NQ2 need to be removed completely.Cyano cobalamin has several further drawbacks. It need to be shielded from light as it is not stable in the presence of light. It is a difficult to handle sticky crystal, which makes it difficult to handle in the application. Accordingly, in application it needs to be diluted with a solid phase powder to come to an evenly dispersed powder with a concentration of 0.1 % to 1 % w / w / cyano cobalamin for food and feed applications. The end concentrations in food and feed products are between about 30 and 10000 pg / kg product. The (heat and light) stability or survival during and after application in food and feed is important together with the biological availability in the digestive track of higher animals. This is particular important with elevated temperatures and moisture.Accordingly, there is need for an improved Vitamin B12 composition and a process to produce the improved composition.Description of the inventionThe inventors have established a novel solid composition comprising adenosyl cobalamin that solves the drawbacks listed herein above. The novel composition has unexpected high bioavailability, heat stability and light stability, and is uniformly distributed throughout the composition with typically 0.1 % w / w up to 1 % w / w adenosyl cobalamin.Accordingly, in a first aspect there is provided for a solid composition comprising: a. at least 0.1 % (w / w) adenosyl cobalamin, and b. adenosyl cobalamin producing microbial cells, wherein at least 20% of the microbial cells are intact microbial cells, wherein at least 20% (w / w) of the adenosyl cobalamin is present within the microbial cells, and wherein the microbial cells are food-grade, Generally Regarded As Safe (GRAS) and / or have a Qualified Assumption of Safety (QPS).In the embodiments herein, the term “solid composition” is defined a composition that predominantly contains solids and comprises at most 10% of a liquid, such as water. The solid composition may be in any form known to the person skilled in the art for solid compositions. The solid composition is herein referred to as “the solid composition according to the invention” or interchangeably “composition according to the invention”, “solid composition” or “composition”.In the embodiments herein, the microbial cells are cells that produce adenosyl cobalamin when cultured under conditions conducive to the production of adenosyl cobalamin. Preferably, at least 5 mg adenosyl cobalamin I litre fermentation broth can be produced. More preferably, at least 10mg / L, 15mg / L, 20mg / L, 50mg / L, 100mg / L, 200mg / L or more preferably at least 300mg / L fermentation broth cobalamin can be produced.In the embodiments herein, at least 20% (w / w), more preferably at least 25% (w / w), 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or most preferably at least 99% (w / w) of the microbial cells are intact microbial cells. Intact cells are defined herein as cells that have not been lysed during fermentation and / or downstream processing. The cells can be dead or alive (viable) with an intact cell wall that contains at least 90%, 95% or 99% ofP62Q37Q27\NQ3 the cell content, in other words, the cell membrane may be damaged if the cell content remains within the cell wall, it is regarded an intact cell.In the embodiments herein, at least 20% (w / w), more preferably at least 25% (w / w), 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95% or most preferably 99% w / w of the adenosyl cobalamin is present within the microbial cells and / or within the microbial cell wall.In the embodiments herein, the microbial cells are food-grade, Generally Regarded As Safe (GRAS) and / or have a Qualified Assumption of Safety (QPS). These terms have the meaning as intended in their respective jurisdictions. Typically, the meaning of these terms is that the microbial cells are safe to be used in food and feed applications.In the embodiments herein, in the solid composition according to the invention, preferably at least 40% (w / w), more preferably 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% 85%, 90%, 95% or most preferably 99% w / w of the adenosyl cobalamin is present within the microbial cell wall. This means that the cell membrane may have been disrupted during fermentation or down stream processing such as drying, but that the cell wall is still intact such that the content of the cells, amongst which the adenosyl cobalamin, remains within the boundaries of the cell wall. When cells are lysed, the cell wall is disrupted and the contents of the cell is released to the outside of the cell.In the embodiments herein, at least 80%, preferably 90%, more preferably at least 95% of the Vitamin B12 in the composition will readily dissolve when contacted with water, illustrating the surprisingly high bioavailability of the Vitamin B12 in the solid composition according to the invention.In the embodiments herein, the solid composition surprisingly has very high stability. After prolonged storage at room temperature, almost all Vitamin B12 is still active. Preferably, after 3 months storage at room temperature of the solid composition according to the invention, at least 80%, more preferably at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or most preferably at least 99% of the Vitamin B12 in the solid composition is still active. Preferably, after 3 months storage at 20°C of the solid composition according to the invention, at least 80%, more preferably at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or most preferably at least 99% of the Vitamin B12 in the solid composition is still active. Preferably, after 3 months storage at 35°C of the solid composition according to the invention, at least 80%, more preferably at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or most preferably at least 99% of the Vitamin B12 in the solid composition is still active.In the embodiments herein, preferably at least 70%, more preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 89%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or most preferably at least 99% of the Vitamin B12 in the solid composition is still active after a pelleting process at 80°C, preferably a pelleting process as described in example 6 herein.In the embodiments herein, in the solid composition according to the invention, the adenosyl cobalamin is surprisingly light stable in view of adenosyl cobalamin as such which is very lightP62Q37Q27\NQ4 unstable. Preferably, the adenosyl cobalamin in the solid composition has at least 20%, 50%, 100%, 2-flod, 3-fold, 4-fold, 5 fold or more preferably at least 10-fold increased light stability compared to adenosyl cobalamin as such.In the embodiments herein, the solid composition preferably comprises at most 5% (w / w) cobyrinic acid, cobinamide, and / or aqua cobalamin, and / or wherein the composition comprises at most 0.05% (w / w) cyano cobalamin, methyl cobalamin and / or hydroxy cobalamin. More preferably, the levels of cobyrinic acid, cobinamide, aqua cobalamin are at most 4%, 3%, 2%, 1 %, 0.5%, or most preferably at most 0.1 % (w / w), More preferably, the levels of cyano cobalamin, methyl cobalamin and / or hydroxy cobalamin are at most 0.4%, 0.3%, 0.02%, 0.01 % (w / w) or most preferably these compounds are not detectable in the solid composition according to the invention.In the embodiments herein, the microbial cells may be any microbial cells that produce adenosyl cobalamin and are food-grade, Generally Regarded As Safe (GRAS) and / or have a Qualified Assumption of Safety (QPS). Preferably, the microbial cells comprise or consist of cells from the genus Propionibacterium. The cells of the genus Propionibacterium may be of any species of the genus Propionibacterium, such as but not limited to P.acidifaciens, P. australiense, P cyclohexanicum, P. freudenreichii, and P. westphaliense. A preferred species of Propionibacterium is Propionibacterium freudenreichii, more preferably P. freudenreichii ATCC6207 In the embodiments herein, the microbial cells may be recombinant cells or may be non-recombinant cells. In the embodiments herein, the adenosyl cobalamin is preferably distributed substantially homogeneously throughout the composition. Substantially homogeneous is herein construed as that there is less than 10% (w / w) difference in adenosyl cobalamin concentration within the solid composition.In the embodiments herein, the solid composition preferably is a food or feed product and may be formulated to accommodate being comprised in or as a food or feed product.In the embodiments herein, the solid composition may further comprise any further compound considered to be useful by the person skilled in the art, such as but not limited to: a carrier, a source of a carbohydrate, a fat, a protein and a vitamin. Such further compound may be inherently present in the microbial cells, such as folic acid (vitamin B11), protein and trehaloseIn the embodiments herein, the composition may be in the form of a tablet, a pellet, a powder, or is present in a capsule.In the embodiments herein the solid composition as defined herein above may conveniently be used to increase the amount of vitamin B12 in a subject. Accordingly, there is provided for the use of a solid composition as defined herein above to increase the physiological amount of adenosyl cobalamin (Vitamin B12) in a subject, wherein the subject may be an animal, preferably a mammal, a bird, a fish, shellfish or crustaceans. Preferably, the subject is a mammal.Further provided is the solid composition for use in the treatment or prevention of Vitamin B12 deficiency in a subject, wherein the subject is a subject as defined previously herein.Further provided is a method of treatment or prevention of Vitamin B12 deficiency in a subject, wherein the subject is a subject as defined previously herein.P62Q37Q27\NQ5In the embodiments herein, the Vitamin B12 deficiency in the subject may have any cause and may e.g. be associated with metformin treatment for type-2 diabetes, pernicious anaemia, atrophic gastritis, chronic inflammation of the pancreas, surgical procedure where part of the stomach and / or small intestine are removed, weight loss surgery, inflammatory bowel disease, such as Crohn's disease, ulcerative colitis, and the like, celiac disease, bacterial growth, heavy drinking, Graves' disease, lupus, long-term use of acid-reducing drugs, malnutrition, bulimia, and anorexia nervosa, HIV / AIDS, obesity, high body mass index, prediabetes state, insulin resistance, or a vegetarian or vegan diet.In a second aspect, there is provided for a process for the production of adenosyl cobalamin comprising: a. providing a adenosyl cobalamin producing microbial cell, , b. culturing the microbial cells under conditions conducive to the production of adenosyl cobalamin, and c. converting the culture broth into a solid composition, wherein the process does not comprise a step to lyse the microbial cells, and wherein the microbial cell is food-grade, Generally Regarded As Safe (GRAS) and / or has a Qualified Assumption of Safety (QPS).In this process according to the invention, preferably at most 10% (w / w), 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or most preferably at most 1 % (w / w) of the microbial cells in the final solid composition obtained is lysed.In this aspect, the features of the process are preferably the features of the first aspect herein.In the embodiments herein, step (c) of the process preferably comprises cell concentration and / or drying convert the broth into a solid composition.In the embodiments herein, the drying preferably is spray drying, freeze drying, or drying at low temperature and low pressure. Drying at low temperature and low pressure is typically performed at between 20 to 50 degrees Celsius and 1 to 10 kPa.In this aspect, the microbial cell is preferably a microbial cell as defined in the first aspect. A preferred microbial cell is a cell from the genus Propionibacterium, more preferably Propionibacterium freudenreichii, even more preferably P. freudenreichii ATCC6207.In this aspect, there is also provided for a solid composition comprising adenosyl cobalamin, obtainable by the process according to the invention, preferably the process according to this aspect.P62Q37Q27\NQ6DefinitionsIn this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of’ meaning that a product or a composition or a nucleic acid molecule or a peptide or polypeptide of a nucleic acid construct or vector or cell as defined herein may comprise additional component(s) than the ones specifically identified; said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 10% of the value.All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.Further embodimentsFurther embodiments of the invention are listed here below.1 . A solid composition comprising: a. at least 0.1 % (w / w) adenosyl cobalamin, and b. adenosyl cobalamin producing microbial cells, wherein at least 20% of the microbial cells are intact microbial cells, wherein at least 20% (w / w) of the adenosyl cobalamin is present within the microbial cells, and wherein the microbial cells are food-grade, Generally Regarded As Safe (GRAS) and / or have a Qualified Assumption of Safety (QPS).2. A solid composition according to embodiment 1 , wherein at least 40% (w / w) of the adenosyl cobalamin is present within the microbial cell wall.3. A solid composition according to claim 1 or 2, wherein at least 80% of the Vitamin B12 in the composition readily dissolves when contacted with water.4. A solid composition according to any one of embodiments 1 to 3, wherein after 3 months storage, at least 80% of the Vitamin B12 is still active.P62Q37Q27\NQ75. A solid composition according to any one of embodiments 1 to 4, wherein after 3 months storage at room temperature, preferably 20°C, at least 80% of the Vitamin B12 is still active.6. A solid composition according to any one of embodiments 1 to 5, wherein after 3 months storage at room temperature, preferably 35°C, at least 80% of the Vitamin B12 is still active.7. A solid composition according to any one of embodiments 1 to 6, wherein at least 70% of the Vitamin B12 in the solid composition is still active after a pelleting process at 80°C, preferably a pelleting process as described in example 6 herein.8. A solid composition according to any one of the preceding embodiments, wherein the adenosyl cobalamin in the solid composition has at least 20%, 50%, 100%, 2-fold, 3-fold, 4-fold, 5 fold or more preferably at least 10-fold increased light stability compared to adenosyl cobalamin as such.9. A solid composition according to any one of embodiments 1 to 8, wherein the composition comprises at most 5% (w / w) cobyrinic acid, cobinamide, and / or aqua cobalamin, and / or wherein the composition comprises at most 0.05% (w / w) cyano cobalamin, methyl cobalamin and / or hydroxy cobalamin.10. A solid composition according to any one of embodiments 1 to 9, wherein the microbial cells comprise cells from the genus Propionibacterium.11. A solid composition according to embodiment 10, wherein the Propionibacterium is Propionibacterium freudenreichii, preferably Propionibacterium freudenreichii ATCC 6207.12. A solid composition according to any one of embodiment 1 to 11 , wherein the adenosyl cobalamin is distributed substantially homogeneously throughout the composition.13. A solid composition according to any one of embodiments 1 to 12, wherein the composition is a food or feed product.14. A solid composition according to any one of embodiments 1 to 13, wherein the composition further comprises a carrier, a source of a carbohydrate, a fat, a protein and / or a vitamin.15. A solid composition according to any one of embodiments 1 to 14, wherein the composition is in the form of a tablet, a pellet, a powder, or is present in a capsule.16. Use of a solid composition according to any one of embodiments 1 to 15 to increase the physiological amount of adenosyl cobalamin (Vitamin B12) in a subject17. A solid composition for use in the treatment or prevention of Vitamin B12 deficiency in a subject, comprising administration of the solid composition to the subject.P62Q37Q27\NQ818. A method of treatment or prevention of Vitamin B12 deficiency in a subject, comprising administration of the solid composition to the subject.19. A use according to embodiment 16, a composition for use according to embodiment 17, and a method of treatment or prevention according to embodiment 18, wherein the Vitamin B12 deficiency is associated with metformin treatment for type-2 diabetes, pernicious anaemia, atrophic gastritis, chronic inflammation of the pancreas, surgical procedure where part of the stomach and / or small intestine are removed, weight loss surgery, inflammatory bowel disease, such as Crohn's disease, ulcerative colitis, and the like, celiac disease, bacterial growth, heavy drinking, Graves' disease, lupus, long-term use of acid-reducing drugs, malnutrition, bulimia, and anorexia nervosa, HIV / AIDS, obesity, high body mass index, prediabetes state, insulin resistance, or a vegetarian or vegan diet.20. A process for the production of adenosyl cobalamin comprising: a. providing a adenosyl cobalamin producing microbial cell, , b. culturing the microbial cells under conditions conducive to the production of adenosyl cobalamin, and c. converting the culture broth into a solid composition, wherein the process does not comprise a step to lyse the microbial cells, and wherein the microbial cell is food-grade, Generally Regarded As Safe (GRAS) and / or has a Qualified Assumption of Safety (QPS)..21. A process according to embodiment 20, wherein step (c) comprises cell concentration and / or drying convert the broth into a solid composition.22. A process according to embodiment 21 , wherein the drying is freeze drying, or drying at low temperature and low pressure.23. A process according to any one of embodiments 20 to 22, wherein the microbial cell is from the genus Propionibacterium.24. A process according to embodiment 23, wherein the Propionibacterium is Propionibacterium freudenreichii, preferably Propionibacterium freudenreichii ATCC 6207.25. A solid composition comprising adenosyl cobalamin, obtainable by the process according to any one of claims 20 to 24.P62Q37Q27\NQ9ExamplesThe adenosyl cobalamin preparation according to the invention used in the examples herein is a solid composition, namely a 0.1 % up to 1 % adenosyl cobalamin containing spray dried biomass of Propionibacterium freudenreichii.Example 1 ; Production of a solid composition according to the invention IntroductionPropionibacterium freudenreichii ATCC 6207 was used to produce an adenosyl cobalamin containing composition according to the invention.A Propionibacterium freudenreichii fermentation is started with a 1 ml frozen vial in a shake flask comprising 20g / L Yeast extract, 20g / L dextrose, 30g / L CaCCh, 0.025g / L Ca pantothenate and 0.1 g / L inositol. Adjust pH to 7.1 with NH4OH. After inoculum the shake flask is incubated for 72 hours at 28°C. After 72 hours the content of the shake flask is divided over 4 times 2000ml shake flasks each containing 1500ml of the same medium. After 72 hours a fermenter with a working volume of 1 m3can be inoculated with the 6 liter inoculum.The 1 m3fermenter holds 600L sterile medium with yeast extract 15g / L, C0CI2.6H2O 5 mg / L, Mg SO4.7H2O 500mg / L, Ca-pantothenate 2mg / kg, ZnSO4.H20 5mg / L, MnSO4.H20 1 mg / L, FeSO4.7H20 20mg / L, Inositol 0,1 g / L and Methionine 1 g / L. The head space is flushed with nitrogen. There is a dextrose feed and the pH is controlled at 7.0 with NaOH and NH4OH. Mild stirring is sufficient to mix the fermentation broth. 5,6 dimethyl benzimidazole is added the last day of the fermentation in combination with mild aeration. Total fermentation takes approximately 7 days and reaches a biomass concentration of 30g / L and an adenosyl cobalamin concentration of 30 mg / L is reached.After further concentration of the fermenter content to 150gram biomass / L the biomass is spray dried and a 0.1 % (w / w) adenosyl cobalamin composition according to the invention is obtained.Example 2; Storage stability of the solid composition of the invention compared to a state of the art cyano cobalamin product.IntroductionStorage stability of the adenosyl cobalamin preparation according to the invention was tested at two relevant temperatures for twelve weeks against a commercial available cyano cobalamin product.Materials and Methods100 milligram of 0.1 % (w / w) adenosyl cobalamin preparation according to the invention and 100 milligram of a 0.1 % (w / w)commercial available cyano cobalamin preparation where both mixed with 99 gram of corncob powder. The mixtures are stored at 20°C and 35°C in an open tray for twelve weeks. The moisture content of the air was kept at 30% to mimic a realistic application environment.P62Q37Q27\NQ10Results and discussionAfter 12 weeks the vitamin B12 activity was measured with a commercial available assay.At 20°C the relative vitamin B12 activity of the adenosyl preparation was 90% of the initial value, while the relative vitamin B12 activity of the cyano cobalamin preparation was 76% of the initial value.Surprisingly, at 35°C the relative vitamin B12 activity of the adenosyl preparation was 95% of the initial value, while the relative vitamin B12 activity of the cyano cobalamin preparation was only 45% of the initial value.Example 3; Heat stability of the solid composition of the invention compared to a state of the art cyano cobalamin productIntroductionA 0.1 % (w / w) adenosyl cobalamin preparation according to the invention was tested at in a pelleting process as used in animal feed applications. This pelleting takes place at 80°C and the samples are heated and cooled within 20 minutes. The adenosyl cobalamin preparation was compared to a commercially available 0.1 % (w / w) cyano cobalamin preparation.Materials and Methods1000 milligram of 0.1 % (wZw)adenosyl cobalamin preparation and 1000 milligram of a 0.1 % (w / w) commercially available cyano cobalamin preparation where both mixed with 30kg of a commercial animal feed mix used for pig feed. The final feed mix used for the palletisation result in a 100 fold higher Vitamin B12 concentration compared to commonly used vitamin B12 concentration as used in pigs feed. This procedure was followed to be able to accurately measure the relative decrease in vitamin B12 with the commercial assay used.Results and discussionAfter cooling and resting the samples for 4 hours, the vitamin B12 activity was measured with a commercial available assay. The cyano cobalamin preparation lost 53 % of its initial activity, while, surprisingly, the adenosyl cobalamin preparation lost only 29% of its initial vitamin B12 activity.Example 4; Bioavailability of adenosyl cobalamin in a solid composition according to the invention.IntroductionBioavailability of adenosyl cobalamin in a solid composition according to the invention was compared to the bioavailability of cyano cobalamin in a state of the art composition, in broiler chickens. Given the carry-over of vitamin B12 via the egg yolk to new born chickens the test started with depleting one generation of broiler chickens on a vitamin B12 deficient diet. The 50 Ross 308P62Q37Q27\NQ11 broiler chicken hens received a feedstock without added vitamin B12. We collected 150 eggs and these eggs are incubated at an commercial hatchery. Out of this population of one-day chickens we random select 120 one day chickens, which are divided in 5 groups of 24. These groups are housed in a heated brooder unit for 7 days. After this the chickens are weighted and marked and housed in standard cages with each 6 chickens. A standard feedstock was used that lack vitamin B12 suppletion.The 5 variation in diets are as follows: A) standard feedstock without added vitamin 12, B) standard feedstock with 20mcg cyano cobalamin / kg, C) standard feedstock with 5mcg cyano cobalamin / kg, D) standard feedstock with 20mcg adenosyl cobalamin / kg, E) standard feedstock with 5mcg adenosyl cobalamin / kgMaterials and methodsAs of day 1 the standard feedstock was used, based on wheat / soy meal supplemented with linoleic acid, lime stone, phosphate, methionine, lysine, salt and a vitamin / minerals mix that did not contain vitamin B12. Overall composition contains: crude protein 22%, calcium 1 ,1 % and phosphorous 0,75%. Food and water are available ad libitum. Temperature kept at 23°C and the cage floors are cleaned daily.The variation in diets, which started at day 1 is as follows:Group A continue to receive the standard feedstock without added vitamin 12Group B receives the standard feedstock with 20mcg cyano cobalamin / kgGroup C receives the standard feedstock with 5mcg cyano cobalamin / kgGroup D receives the standard feedstock with 20mcg adenosyl cobalamine / kgGroup E receives the standard feedstock with 5mcg adenosyl cobalamin / kgGroup B represents the current practice for vitamin B12 supplementation in broiler chickens. The relative highest growth rate of these broiler chickens is between day 7 and 21 .We measured the full body weight at day 7 and day 21 of the chickens. After weighting at day 21 a blood sample was obtained and after beheading the chickens a liver sample was taken to determine the vitamin B12 concentrations.Vitamin B12 in blood was measured on a Roche Cobas e411 analyser using the sample preparation kit of Roche. The liver sample received a pre treatment with homogenization and centrifugation to obtain a liver serum sample.Results and discussionP62Q37Q27\NQ12Average weight at day 21 is not significantly different for group B, D, E (marked with an “a” in column 4). Group A show significant lower weight gain at day 21 compared to B, C, D, and E (marked with a “c” in column 4). There is a significant lower weight gain at day 7 and day 21 in group C (marked with a “b” in column 4) compared to group E.In the liver tissue values of vitamin B12 in group D and E are significantly higher than the vitamin B12 liver values in group B and C.It is surprising to observe that adenosyl cobalamin uptake seems better in group D and E, despite lower blood plasma levels of vitamin B12 in group D and E. These observations support the improved bioavailability adenosyl cobalamin in a composition according to the invention over cyano cobalamin.Example 5; Storage stability of the solid composition of the invention compared to a composition where the wet biomass at the end of fermentation is pasteurized before the drying process. The pasteurization step disrupts the cells and cell wall. As a result, the adenosyl cobalamin is released into the wet biomass and fermentation broth. The pasteurized preparation was subjected to the same drying process as described in example 1 .IntroductionStorage stability of the adenosyl cobalamin preparation according to the invention and the separate preparation that included a pasteurization step before the drying step were analysed at two relevant temperatures for twelve weeks. The pasteurization caused cell disruption and release of more than 60% of the adenosyl cobalamin into the medium before further downstream processing.Materials and Methods The portion of the adenosyl cobalamin preparation that was pasteurized, had a lower concentration of adenosyl cobalamin per gram dry mass. To compensate and reach the same adenosyl cobalamin concentration at the start of the experiment in both preparations the initial amount of the pasteurized preparation was adjusted.100 milligram of 0.1 % (w / w) adenosyl cobalamin preparation according to the invention and 220 milligram of a 0.045% (w / w) pasteurized adenosyl cobalamin preparation where both mixed with 99 gram of corncob powder. The initial adenosyl cobalamin concentration was thus equal for both test mixtures. The mixtures were stored at 20°C and 35°C in an open tray for twelve weeks. The moisture content of the air was kept at 30% to mimic a realistic application environment.Results and discussionAfter 12 weeks the vitamin B12 activity was measured with a commercially available assay.P62Q37Q27\NQ13At 20°C, the relative vitamin B12 activity of the adenosyl preparation according to the invention was 87% of the initial value, while the relative vitamin B12 activity of the pasteurized adenosyl cobalamin preparation was 81 % of the initial value.At 35°C, the relative vitamin B12 activity of the adenosyl preparation according to the invention was 91 % of the initial value, while the relative vitamin B12 activity of the pasteurized adenosyl cobalamin preparation was only 62% of the initial value.This result demonstrates the stabilizing effect of intact cells in the composition according to the invention on adenosyl cobalamin over the exposure of free adenosyl cobalamin that is not within the boundaries of the cell wall, despite the presence of the dried biomass.Example 6; Heat stability of the solid composition of the invention compared to a composition where the wet biomass end of fermentation is pasteurized before the drying process. The pasteurization step disrupts the cells and cell wall. As a result, the adenosyl cobalamin is released into the wet biomass and present fermentation broth. The pasteurized preparation followed the same drying process as described in example 1 .IntroductionA 0.1 % (w / w) adenosyl cobalamin preparation according to the invention and a separate preparation that included a pasteurization step before the drying step were compared in a pelleting process as used in animal feed applications. The pasteurization resulted in cell disruption while releasing more than 60% of the adenosyl cobalamin in the medium before further downstream processing.Materials and MethodsThe portion of the adenosyl cobalamin preparation that was pasteurized, had a lower amount of adenosyl cobalamin per gram dry mass. To compensate and reach the same adenosyl cobalamin concentration at the start of the experiment in both preparations the initial amount for the pasteurized preparation was adjusted. The initial adenosyl cobalamin concentration was thus equal for both test mixtures.The pelleting process took place at 80°C and the samples were heated and cooled within 20 minutes. 1000 milligram of 0.1 % (w / w) adenosyl cobalamin preparation according to the invention and 2200 milligram of a 0.045% (w / w) pasteurized cobalamin preparation where both mixed with 30kg of a commercial animal feed mix used for pig feed. The final feed mix used for the palletisation resulted in a 100-fold higher Vitamin B12 concentration compared to the commonly used vitamin B12 concentration in pigs feed. This procedure was followed intentionally to be able to accurately measure the relative decrease in vitamin B12 with the commercial assay used.Results and discussionAfter cooling and resting the samples for 4 hours, the vitamin B12 activity was measured with a commercial available assay. The pasteurized adenosyl cobalamin preparation lost 43 % of its initial vitamin B12 activity, while, surprisingly, the adenosyl cobalamin preparation according to the invention lost only 26% of its initial vitamin B12 activity.This result demonstrates the stabilizing effect of the intact cells on adenosyl cobalamin over the exposure of free adenosyl cobalamine that is not within the boundaries of the cell wall, despite the present of the dried biomass.

Claims

1. P62Q37Q27\NQ15Claims1 . A solid composition comprising: a. at least 0.1 % (w / w) adenosyl cobalamin, and b. adenosyl cobalamin producing microbial cells, wherein at least 20% of the microbial cells are intact microbial cells, wherein at least 20% (w / w) of the adenosyl cobalamin is present within the microbial cells, and wherein the microbial cells are food-grade, Generally Regarded As Safe (GRAS) and / or have a Qualified Assumption of Safety (QPS).

2. A solid composition according to claim 1 , wherein at least 40% (w / w) of the adenosyl cobalamin is present within the microbial cell wall.

3. A solid composition according to claim 1 or 2, wherein at least 80% of the Vitamin B12 in the composition readily dissolves when contacted with water.

4. A solid composition according to any one of claims 1 to 3, wherein after 3 months storage, at least 80% of the Vitamin B12 is still active.

5. A solid composition according to any one of claims 1 to 4, wherein after 3 months storage at room temperature, preferably 20°C, at least 80% of the Vitamin B12 is still active.

6. A solid composition according to any one of claims 1 to 5, wherein the adenosyl cobalamin in the solid composition has at least 20%, 50%, 100%, 2-fold, 3-fold, 4-fold, 5 fold or more preferably at least 10-fold increased light stability compared to adenosyl cobalamin as such.

7. A solid composition according to any one of claims 1 to 6, wherein the composition comprises at most 5% (w / w) cobyrinic acid, cobinamide, and / or aqua cobalamin, and / or wherein the composition comprises at most 0.05% cyano cobalamin, methyl cobalamin and / or hydroxy cobalamin.

8. A solid composition according to any one of claims 1 to 7, wherein the microbial cells comprise cells from the genus Propionibacterium.

9. A solid composition according to claim 8, wherein the Propionibacterium is Propionibacterium freudenreichii.

10. A solid composition according to any one of claim 1 to 9, wherein the adenosyl cobalamin is distributed substantially homogeneously throughout the composition.

11. A solid composition according to any one of claims 1 to 10, wherein the composition is a food or feed product.

12. A solid composition according to any one of claims 1 to 11 , wherein the composition further comprises a carrier, a source of a carbohydrate, a fat, a protein and / or a vitamin.P62Q37Q27\NQ1613. A solid composition according to any one of claims 1 to 12, wherein the composition is in the form of a tablet, a pellet, a powder, or is present in a capsule.

14. Use of a solid composition according to any one of claims 1 to 13 to increase the physiological amount of adenosyl cobalamin (Vitamin B12) in a subject.

15. A solid composition for use in the treatment or prevention of Vitamin B12 deficiency in a subject, comprising administration of the solid composition to the subject.

16. A method of treatment or prevention of Vitamin B12 deficiency in a subject, comprising administration of the solid composition to the subject.

17. A use according to claim 14, a composition for use according to claim 15, and a method of treatment or prevention according to claim 16, wherein the Vitamin B12 deficiency is associated with metformin treatment for type-2 diabetes, pernicious anaemia, atrophic gastritis, chronic inflammation of the pancreas, surgical procedure where part of the stomach and / or small intestine are removed, weight loss surgery, inflammatory bowel disease, such as Crohn's disease, ulcerative colitis, and the like, celiac disease, bacterial growth, heavy drinking, Graves' disease, lupus, long-term use of acid-reducing drugs, malnutrition, bulimia, and anorexia nervosa, HIV / AIDS, obesity, high body mass index, prediabetes state, insulin resistance, or a vegetarian or vegan diet.

18. A process for the production of adenosyl cobalamin comprising: a. providing a adenosyl cobalamin producing microbial cell, , b. culturing the microbial cells under conditions conducive to the production of adenosyl cobalamin, and c. converting the culture broth into a solid composition, wherein the process does not comprise a step to lyse the microbial cells, and wherein the microbial cell is food-grade, Generally Regarded As Safe (GRAS) and / or has a Qualified Assumption of Safety (QPS)..

19. A process according to claim 16, wherein step (c) comprises cell concentration and / or drying convert the broth into a solid composition.

20. A process according to claim 19, wherein the drying is freeze drying, or drying at low temperature and low pressure.21 . A process according to any one of claims 18 to 20, wherein the microbial cell is from the genus Propionibacterium.

22. A process according to embodiment 21 , wherein the Propionibacterium is Propionibacterium freudenreichii.

23. A solid composition comprising adenosyl cobalamin, obtainable by the process according to any one of claims 18 to 22.

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