MXPA99001243A - Production and use of compositions comprising high concentrations of vitamin b12 activity - Google Patents

Abstract

The present invention provides a process for the preparation of a composition comprising natural vitamin B12, wherein said process comprises the steps of:a) obtaining microbial cells containing natural vitamin B12, b) causing opening of the microbial cells such that at least part of the soluble content of the cells comprising vitamin B12 is released in a liquid in which the cells are contained, c) separating the opened cells and the liquid comprising the vitamin B12, d) preparing a mixture of the vitamin B12 and at least a part of the opened cells, wherein the mixture has a vitamin B12 concentration on dry matter in excess of 0.1%(w/w).

Description

PRODUCTION AND USE OF COMPOSITIONS THAT COMPRISE HIGH ACTIVITY CONCENTRATIONS OF VITAMIN B12 Field of the Invention The present invention relates to the production of compositions comprising natural vitamin B12.

Background of the Invention Vitamin B12 is an important vitamin for humans and animals. It is used to treat pernicious anemia, and peripheral neuritis, and is used as a dietary supplement. Vitamin 12 is also a food supplement, important for animals as a growth enhancer. The term vitamin B12 is used to describe compounds of the cobalt corrinoid family, in particular those of the cobalamin group. The most used compound in this group is cyanocobalamin and as such the term vitamin B12 is sometimes used to refer to cyanocobalamin. In this specification, the term vitamin B12 should be attributed to its broad meaning REF: 029335 to include all the cobalt corrinoids of the cobalamin group, which includes in particular cyanocobalamin, hydroxocobalamin, methylcobalamin and 5'-deoxyadenosylcobalamin characterized by a cyano, hydroxy, methyl or 5'-deoxyadenosyl radical respectively. The compounds of methylcobalamin and 5'-deoxyadenosylcobalamin are known to be unstable to light in isolated form and are easily converted to hydroxocobalamin in aqueous solution. For this reason, almost all commercial vitamin B12 preparations consist of stable cyanocobalamin which as such is not the chemical form in which vitamin B12 can be found in nature. In this specification, the term natural vitamin B12 is defined to include all the chemical forms of vitamin B12 that occur naturally in nature, thus excluding cyanocobalamin. Vitamin B12 is produced industrially by microbial fermentation, using almost exclusively the species Pseudomonas s deni tri fi cans and Propi oniba ct eri um, then natural vitamin B12 is converted into the form of cyanocobalamin by chemical procedures that include cyanidation followed by the extraction and purification steps using organic solvents (as analyzed by Spalla and collaborators, 1989"Microbial production of vitamin B12", in: Biotechnology of vitamins, pigments and growth factors, EJ Vandamme ed., Elsevier, London, New York , pp. 257-284). The chemical conversion step and any subsequent purification step causes this production process to be costly, unsafe for operators and environmentally unfavorable. With ingestion, animals and humans convert cyanocobalamin to one of the natural forms of vitamin B12 such as methylcobalamin or 5'-deoxyadenosylcobalamin which are required to function as a coenzyme for several biochemical conversions (Ellenbogen, L., in: Handbook of vitamins, Nutritional, biochemical and clinical aspects, LJ Machlin, Marcel Dekker Inc., New York and Basel). In addition, the efficient growth of animals requires the presence of a sufficient amount of vitamin B12 activity in the animal's diet. Vitamin B12 preparations are often sold as food supplements as such or as part of a premix that contains additional vitamins and other food additives. Therefore, it is clear that a direct complement of 5'-deoxyadenosylcobalamin or methylcobalamin instead of cyanocobalamin would be of benefit. In view of the instability of these two compounds, these are not produced in isolation but are produced in dry formulation together with the biomass of the organism in which they are produced. Such formulations are well suited for use as a food supplement for animals. For the production of methylcobalamin and 'deoxyadenosilcobalamin, the bacteria of the genus Propi oniba cteri um are more preferred, because, unlike P. In addition, bacteria of this genus have obtained the GRAS status (Generally Recognized As Safe) from the United States Food and Drug Administration and are not known to produce endotoxins. Propi oniba ct eri um species are aerotolerant, non-motile, non-sporulating, gram-positive bacteria further characterized by the production of large amounts of propionic acid from carbohydrates, lactic acid and polyhydroxy alcohols. The genus Propi oniba cteri um fails in the high "GC" subdivision of gram-positive bacteria (TD Brock, MT Madigan, JM Martinko and J. Parker in: The Biology of Microorganisms 7th Edition, Prentice-Hall International Inc., 1994 ).

Patent application RU 2001953 (Antibiotics Enzymes Research Technical Institute) refers to a method of spray dehydration for PropioniJacteriujn shermanii which has been used to produce the biomass containing 5'-deoxyadenosylcobalamin. Methylcobalamin in the biomass of methanogenic bacteria is commercially available from Gedeon Richter company. However, the content of vitamin B12 in these preparations is limited by the level at which vitamin B12 occurs during fermentation and, as a consequence, the vitamin B12 content in these preparations does not exceed 0.1% (w / w) . The concentration of vitamin B12 in a composition is commonly measured as the dry weight of vitamin B12 as a percentage of the dry weight of the composition. Concentrated products (ie> 0.1% w / w) of cyanocobalamin are commercially available. However, food mills and premix manufacturers can not use this material because as a consequence of their electrostatic properties the cyanocobalamin is segregated from its carrier in the processing into food mills and premixture manufacturers when formulated in concentrations > 0.1% p / p. Segregation is a particular problem during sieving, sifting or allowing the composition to rest for a prolonged period of time. The present invention describes a process by which it becomes possible to produce a composition having a relatively high concentration (> 0.1% w / w) of the natural vitamin B12. These new concentrated products can be used for example in animal feed, human food or as an ingredient in cosmetics. These new concentrated products can be more convenient for use and for transportation, thus allowing a reduction in costs. These can be advantageously used by food mills and premix manufacturers such as these can show a reduced segregation of the carrier B12 vitamin during processing. The process can be carried out easily on an industrial scale and is relatively environmentally friendly as there is no need to use organic solvents or cyanidation.

Description of the invention The present invention provides a process for the preparation of a composition comprising natural vitamin B12 which has a concentration of vitamin B12 in the dry matter in excess of 0.1% (w / w). The method comprising the steps of a) obtaining microbial cells containing natural vitamin B12, b) causing the opening of the microbial cells such that at least part of the soluble content of the cells comprising vitamin B12 is released in a liquid in which the cells are contained, c) separating the open cells and the liquid comprising vitamin B12, and optionally d) preparing a mixture of vitamin B12 and at least a portion of the open cells. Microbial cells containing vitamin B12 are preferably obtained in an industrial fermentation process using known microorganisms to produce vitamin B12. These include bacteria belonging to the genera of Acetobacter ium, Aerobacter, Agrobacterium, Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Clostridium, Corynebacterium, Escherichia, Eubacterium, Flavobacterium, Methanobacillus, Methanosarcina, Mycobacterium, Propionibacterium, Proteus, Pseudomonas, Rhizobium, Rhodopseudomonas, Salmonella, Serratia, Streptococcus, Streptomyces and Santomonas. Preferably, the bacteria to be used in the method of the invention is safe for human and / or animal consumption and does not produce endo- or exotoxins. More preferably, the bacterium has obtained the GRAS (Generally Recognized As Safe) condition from the Food and Drug Administration of the United States. The most preferred bacterium for use in the method of the invention is the genus Propi oniba ct eri um, the preferred species of which are P. freudenrei chi i or P. shermani i. It is well known to those skilled in the art that industrial production of vitamin B12, similar to that of many microbial metabolites, is carried out using strains resulting from programs designed to improve the desired qualities of any particular strain. These programs essentially consist of the treatment of the production strain with a mutagenic agent and the selection of mutants that exhibit improved productivity or other advantages. Several techniques have been described for the rational selection of mutant microorganisms of overproduction of vitamin B12 (Spalla, C, Grein, A., Garofo, L and Ferni, G. Microbial production of vitamin B12 In: Biotechnology of vitamins, pigments and growth factors pp. 257-284 (EJ Vandamme, ed.) Elsevier, London / NY, 1989). Such mutant strains are used in the preferred methods of the invention and are capable of further increasing the concentrations of vitamin B12 in the compositions obtained. In the invention, the term "natural vitamin B12" is defined to comprise all the chemical forms of vitamin B12 that occur naturally in nature, the cyanocobalamin form of vitamin B12 being excluded. In the preferred methods and compositions of the invention, naturally occurring vitamin B12 comprises 5'-deoxyadenosylcobalamin and / or methylcobalamin. In the method of the invention, microbial cells containing vitamin B12 are treated to cause lysis or other disruption of the cell membrane. Lysis describes the opening of the microbial cells such that (at least part of) the soluble content of the cells comprising vitamin B12 is released in a liquid in which the cells are contained. Preferred treatments to open the cells are heat treatments that include pasteurization or heating in an autoclave; treatment with bacteriolytic enzymes such as lysozyme; Mechanical disruption of the cells that includes crushing or the use of shearing forces; the treatment with chemicals that cause the lysis of cells such as detergents or organic solvents; as well as combinations of these treatments. Lysis or other disruption of the membrane produces a lysis product which can be separated into solid and liquid phases. In a preferred process of the invention, at least part of the solid phase (open cells) is added to the liquid phase containing the natural vitamin B12 so that a composition is obtained which is a mixture comprising biomass, i.e. preferably open microbial cells and natural vitamin B12. The content of vitamin B12 or the concentration of these compositions is expressed as percentage by weight of vitamin B12 based on the content of the dry matter of the composition. The process of the invention produces compositions having a concentration of vitamin B12 in excess of 0.1% (w / w) in the dry matter. In the preferred methods, the composition has a concentration of natural vitamin B12 in excess of 0.2% (p / p), more preferably in excess of 0.4% (w / w), even more preferably in excess of 0.6%, even more preferably in excess of 0.8% (w / w), and much more preferred in excess of 1.0% (w / w). It is preferable that the vitamin B12 concentrations of the compositions of the invention do not exceed 10% (w / w), more preferably the concentrations of vitamin B12 do not exceed 5%. In the process of the invention, the solid phase of the lysis product comprising the waste of the cells resulting from the opening of the cells is separated from the liquid containing the released vitamin B12. A number of different solid-liquid separation techniques are available to the skilled person to perform this separation, including centrifugation and filtration techniques. A preferred method for separating solid waste from cells from the liquid containing vitamin B12 is ultrafiltration. In a preferred process of the invention, the open microbial cells are washed and the washed products are combined with the vitamin B12 obtained after the separation of the cells and the liquid containing the vitamin B12. Preferably, washing is effected by diafiltration with ion-free water which is preferably used for washing the open cells. The diafiltered product containing vitamin B12 is then combined with the liquid phase containing vitamin B12. In a further preferred method of the invention, the liquid phase containing the natural vitamin B12 is subjected to a drying treatment. Any suitable means of drying such as, for example, spray drying, fluid bed drying, freeze drying, or vacuum drying can be used. In a preferred process of the invention, the microbial cells containing vitamin B12 are washed before they are lysed, to further increase the concentration of vitamin B12 in the dry matter by removing components from the medium. Preferably, the washing is carried out using diafiltration, with which ion-free water is preferably used. In a further aspect of the invention there are provided compositions comprising vitamin B12, which can be obtained in a process of the invention. Preferably, most of the activity of vitamin B12 in these compositions is in the form of natural vitamin B12. The compositions of the invention are characterized in that vitamin B12 is present in a concentration based on dry matter in excess of 0.1% (w / w) • In a preferred composition of the invention the concentration of vitamin B12 is in excess 0.2% (p / p), preferably in excess of 0.4% (p / p), more preferably in excess of 0.6%, even more preferably in excess of 0.8% (p / p), and much more preferably in excess of 1.0% (w / w). However, preferably, the concentrations of vitamin B12 of the compositions of the invention do not exceed 10% (w / w), more preferably concentrations of vitamin B12 do not exceed 5%. The compositions of the invention comprise vitamin B12 and a carrier which preferentially comprises biomass, for example, whole cells and / or cell debris. The biomass comprised in these compositions is derived preferentially from a microorganism capable of producing vitamin B12, such as the bacteria mentioned above, of which Propi onibacterium species are most preferred. The compositions of the invention are preferably dry compositions, wherein dry is defined as having a water content of less than 15% by weight, more preferably less than 10%, much more preferably less than 5% . In the preferred dry compositions of the invention, the activity of vitamin B12 is distributed substantially homogeneously throughout the powder or granulated powder containing the carrier and vitamin B12. As a result, the activity of vitamin B12 in these compositions advantageously does not segregate to a large degree from the other substituents in the composition, especially when exposed to gravitational forces, sieving, sifting or electrostatic forces during processing of the compositions, even when they are used in concentrations in excess of 0.1% (w / w). The processing is understood herein to comprise the processing of the compositions of the invention in food or food premixes. In a further aspect of the invention, the composition of the invention is used as, or in the production of a food supplement to promote growth for animals. To this end, compositions containing vitamin B12 are added to the other food components, either directly or in the form of a premix which also contains other vitamins, minerals and / or bioactive ingredients. As shown in the specific example, feeding an animal with a diet comprising a composition of the invention promotes its growth.

In a still further aspect of the invention, compositions comprising vitamin B12 are used as, or in the production of a food supplement for humans and / or incorporated into cosmetic preparations, such as shampoos or body lotions.

Examples Experimental Vitamin B12 Assay The activity of vitamin B12 was assayed using a turbidimetric bioassay based on the growth response of Lactoba cill us lei chmani i ATCC 7830 as described in detail in: The United Stated Pharmacopoeia, The National Formulary , 1995, pp. 1719-1721, United Stated Pharmacopoeial Convention, Inc., Rockville MD. 20 Concentrations of B12 in Plasma B12 in plasma was measured using commercially available radioassay equipment (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK). The sample of ? ^ plasma is combined with vitamin Bi2 (57Co) in a solution containing dithiothreitol and cyanide. The mixture is boiled for 30 minutes to inactivate the endogenous binding proteins and to convert the various forms of vitamin B 2 to cynocobalamin. The mixture is cooled and then combined with porcine intrinsic factor, purified affinity, immobilized (attached to polymer beads). This adjusts and damps the pH of the reaction mixture to 9.35. The reaction mixture is then incubated for 60 minutes at room temperature. During incubation, endogenous and labeled vitamins compete for the limited number of binding sites based on their relative concentrations. The reaction mixture is then centrifuged at 1500 g for 10 minutes. The labeled and unlabeled vitamins that bind immobilized binding proteins are concentrated in the bottom of the tube in the form of a pellet and the unbound vitamins remain in the supernatant. The supernatant is aspirated and the radioactivity associated with the pellet is counted. Normal curves are prepared using the standards of vitamin Bi2 in a human serum albumin base. The concentration of vitamin Bi2 in the plasma sample is determined from the normal curves. The normal curves were plotted and the unknown curves were determined, using the AssayZap universal trial calculator, version 2.32 (Biosoft, Cambridge, UK.). Plasma samples were required to be diluted 10-50 times to bring them within the most sensitive range of the assay.

Comparative Example 1 Fermentation was carried out with Propi on iba cteri um freudenrei chi i using a procedure known to those skilled in the art (see for example Spalla et al., 1989"Microbial production of vitamin B12", in: Biotechnology of vitamins, pigments and growth Factors, EJ Vandamme ed., Elsevier, London, New York, pp. 257-284). A strain of P. Freudenrei chi i suitable is available from the American Type Culture Collection under accession number ATCC 6207. A broth was obtained at the end of the fermentation which had a potency of 5'-deoxyadenosilcobalamin of 10 mg / l and a content of matter dry of 7.5%. The dehydration by sprinkling this broth resulted in a product with a vitamin B12 concentration of 0.01%. In order to obtain a higher concentration of vitamin B12, a solid-liquid separation of the broth was attempted. Vitamin B12 is intracellular in P. freudenrei chii, which is why the removal of the components of the medium (extracellular) and the dehydration by subsequent sprinkling of the biomass should result in a dehydrated product by sprinkling with a concentration of vitamin B12 more high. In the laboratory scale, a centrifugation step was performed at a maximum g force of 5000-6000 * g. This force g is comparable to the centrifugal forces of centrifugal machines used in the industrial scale. The aliquots of 1000 ml of broth were centrifuged in a Beckmann JM / 6E centrifugal machine for 10 minutes at 5000 rpm. However, no separation of the biomass and the broth was obtained under these conditions. Because the higher centrifugal forces are less attractive on an industrial scale for economic reasons, it was analyzed if it is feasible to separate the liquid and the solids in the broth using ultrafiltration. 2000 ml of broth was ultrafiltrated, with an initial concentration of 4.5 mg of vitamin B12 / 1 (vitamin B12 in the dry matter 002%), by means of a coiled winding module 30 kD AMICON (0.09 m2) with a feed pressure of 1 bar. Under these conditions it was possible to concentrate the sample of vitamin B12 5 times. The average permeate product flow was 30 l / m2h. The concentration of vitamin B12 in the concentrated product was 20 mg / l, while in the permeate less than 0.2 mg / l of vitamin B12 was detected. The concentration of vitamin B12 in the concentrated product, when expressed in terms of the dry matter content, was 0.05%, compared to 0.02% before ultrafiltration. To further increase the titre of vitamin B12 in the dry matter, then the diafiltration of the biomass was analyzed. The concentrated product of the ultrafiltration as obtained in the experiment described above was washed six times with ion-free water, which resulted in a potency of vitamin B12 in the concentrated product of 20 mg / l. No vitamin B12 activity was detected in the diafiltrate. Subsequent spray drying of the concentrated product resulted in a spray dried product with a vitamin B12 concentration of 0.06%. It is concluded from the previous experiments that it is not possible to obtain a product with a concentration of vitamin B12 of more than 0.06% based on the dry matter starting from a broth with a titre of vitamin B12 of 5-10 mg / l.

Example 2 In order to prevent the presence of viable production organisms, ie live cells, in the dried product, a pasteurization step of 65 ° C was carried out for 30 minutes in a broth which was obtained as described in Example 1. The treated broth was ultrafiltered as described above. A permeated pink product was observed with a concentration of vitamin B12 (dry matter) of 0.2 - 0.4%. It is thought that pasteurization causes the lysis of (at least part of) the cells P. freudenrei chi i and the release of intracellular vitamin B12 in the medium. This may allow one to obtain dry products that comprise vitamin B12 in concentrations ranging from 0.06% to 0.3% (based on dry matter), by combining the permeate product and the pasteurized broth before the dehydrated by spraying. In more detail, 350 ml of the heat-treated broth was ultrafiltered under the conditions described above in Example 1. The concentrated product was diafiltered with 2500 ml of ion-free water. The light pink permeate and the diafiltered product were combined and mixed with 750 ml of concentrated ultrafiltration product, pasteurized. The mixture was dehydrated by spraying in a Büichi lab spray dryer whereby the inlet temperature was adjusted to 180 ° C and the outlet was adjusted to a temperature of 104 ° C. The concentration of vitamin B12 of the dehydrated product by spraying in this way obtained was 0.13%.

Example 3 1200 l of broth with a concentration of vitamin B12 of 8 mg / l in a pilot plant fermentation of P. freudenreichii were obtained and ultrafiltered in an MDS DDS ultra filtration module using 20 kD membranes and an initial water flow of 38 l / m2h. The temperature during ultrafiltration was maintained at room temperature. The circulation flow was approximately 15 m3 / h. The feed pressure was adjusted to approximately 7 bar. The broth was concentrated 11.5 times (average flow of 23 l / m2h). The concentrated product was washed with 500 1 of ion-free water at room temperature until a conductivity of about 3 mS / cm was reached.

The resulting concentrated product was treated at 90 ° C for 6 hours. Part of the concentrated, heat-treated product was diafiltered, which resulted in approximately 300 1 of permeate product containing vitamin B12. The retained product was stored at 4 ° C. The permeate was concentrated on a pilot scale in a glass evaporator at 40 ° C under vacuum, resulting in approximately 6 1 of concentrated product. By combining and mixing an appropriate amount of the retained product with the permeate, concentrate, a mixture was compounded which was dehydrated by sprinkling in a NIRO spray drier. The air temperature at the inlet was adjusted to 160 ° C and the outlet temperature was adjusted to approximately 90 95 ° C. In an operation time of 5 hours, approximately 45 1 of the mixture (about 9 kg / hour) was spray dried. This resulted in 1307 g of spray-dried material with an activity of vitamin B12 of 1100 mg / kg, ie 0.11%.

Example 4 In an experiment similar to that described in Example 3, fermentation was performed using the P. freudenrei chi i strain improved CBS 929.97. The concentration of vitamin B12, resulting in the fermentation broth was 40 mg / l. Ultrafiltration and diafiltration were performed essentially as described in Example 3. Using ultrafiltration, they were concentrated 100 1 of the broth from 40 mg / l to about 40 1. Using the diafiltration, the concentrated product was washed with 200 1 of ion-free water. The resulting concentrated, washed product had an activity of vitamin B12 of 96.5 mg / kg and 6.72% of dry matter (the dehydration by sprinkling of this concentrated product, washing would have given an activity of vitamin B12 of 0.14%). The concentrated, washed product was subsequently lysed at 90 ° C for 5 minutes, followed by a separation of the waste from the cell and the liquid by ultrafiltration. The concentration of vitamin B12, potential of the permeate product. reached 0.87% dry matter (calculated from a vitamin B12 activity of 52.1 mg / kg and a dry matter content of 0.6%). After preconcentration of the permeate product in vacuum at 65 ° C, different amounts of permeate and concentrated product were combined and dried. The potencies of vitamin B12 of the dry products, obtained were given below: concentrated product activity of vitamin B12 per product permeate dry matter 1 0.5% 7 0.65% 8 0.72% 2 0.29% P. freudenreichii CBS 292.97 was deposited on July 10, 1997 in Centraalbureau voor Schimmelcutures, Baarn, The Netherlands.

Example 16 Application of natural vitamin B12 in animal feed.

A test was conducted with young chickens to compare the efficacy of natural vitamin B12 with cyanocobalamin. Young chicks, one-day-old males were randomly assigned to cages. They kept eight animals per box. The cages were placed in a house for young chickens heated, ventilated and artificially illuminated. The animals were vaccinated against New Castle disease at the ages of one and fourteen days. The animals received the experimental diets ad libi tum from day 1, the first 10 days in the form of powder material, then in pellet form. The water was freely available. The experiment lasted 28 days. On day 28 the animals were weighed, the food consumption was determined and blood samples were taken. Blood samples were taken from 4 randomly selected young chickens per cage. The blood was collected in heparinized tubes. The tubes were centrifuged and the plasma was frozen (-18 ° C) until the analysis of vitamin B12 was carried out. This analysis was performed as described above under Esperimental (concentrations of B12 in Plasma). Three treatments were included in this experiment: I. Basic diet without addition of vitamin B12 II. Basic diet with addition of cyanocobalamin (30 ppb of active substance) III. Basic diet with addition of natural vitamin B12 (30 ppb of active substance) Each treatment was repeated 5 times. The composition of the basic diet is present in table 1, the results in table 2.

Table 1. Composition of the basic diet (in%). Maize 55 Tapioca 1 Soybean meal 30 Beans, heat treated 5 Feather meal 1 Soybean oil 2 Animal fat 3 Vitamins *, minerals, amino acids 3 Contents calculated: Metabolic Energy 13.3 MJ / kg Raw protein 22.0% Lysine (total) 1.27% Methionine + Cysteine (total) 0.95% *: Without vitamin B12.

Table 2. Effect of cyanocobalamin and natural vitamin B12 on growth, food intake and feed conversion ratio, average between 1 and 28 days of age and on vitamin B12 contents in blood plasma on day 28.

Example 6 Segregation of a carrier's vitamin B12 activity The segregation of the active compound of the carrier can be caused by the shape of the particles, density and density in volume, fluidity, adhesive and electrostatic properties, moisture content and hygroscopicity. These segregation procedures may occur during the preparation of premixes and compound feed and during transportation and subsequent storage. This segregation is particularly critical in the case of premixes comprising micro-components such as vitamins and elements of small particles, macro-components such as minerals and carriers. The separation properties of the commercial, existing cyanocobalamin preparations (obtained from Rhóne Poulenc under the trade name Microvit® B12) have been compared with the preparation of the natural vitamin B12 prepared as described in the example. Limestone is used as a carrier for existing commercial cyanocobalamin preparations. The test substances were expelled from a storage container by means of a vibration channel to form a stack as shown in Figure 1. Three samples were taken from the center and mixed. The procedure was repeated with the samples of the base of the pile. Samples were analyzed for the activity of vitamin B12 as described above and compared with the homogenous, original mixture. The deviations in the samples of the homogeneous, original mixture were calculated as follows: Deviation in the base (%) =. { (concentration as base - original mix concentration.} * 100 Center deviation (%) =. {(concentration in the center - original mix concentration) / original mix concentration.}. * 100 The comparison of the deviations at the base of the stack and the center of the stack of the natural cyanocobalamin and vitamin B12 compositions of the invention are shown in Table 3.

Table 3 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following claims is claimed as property.

Claims (22)

1. A method for preparing natural vitamin B12, the method is characterized in that it comprises: (a) cultivating microbial cells under conditions such that they produce, intracellularly, the natural vitamin B12; and (b) otherwise lyse or disrupt the outer membrane of the microbial cells that have produced natural vitamin B12 to release vitamin B12.

2. A process according to claim 1, characterized in that the lysis produces a lysed product and the solid and liquid phases of the lysed product are separated, the liquid phase containing the natural vitamin B123.

A method according to claim 2, characterized in that it further comprises washing the solid phase of the lysed product and combining the washed products with the liquid phase of the lysed product.

4. A process according to claim 3, characterized in that the washing of the solid phase of the lysed product is carried out by diafiltration.

5. A method according to any of claims 2 to 4, characterized in that it further comprises adding at least part of the solid phase of the lysed product to the liquid phase containing the natural vitamin B12.

6. A method according to any of the preceding claims, characterized in that it further comprises washing the microbial cells before lysis.

7. A method according to claim 6, characterized in that the washing of the microbial cells before lysis is carried out by diafiltration.

8. A method according to any of claims 2 to 7, characterized in that it also comprises drying the liquid phase containing the natural vitamin B12.

9. A method according to any of the preceding claims, characterized in that the microbial cells are cells of one or more of the bacterial genera Acetobacterium, Acetobacter, Agrobacterium, Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Clostridium, Corynebacterium, Escherichia, Eubacterium, Flavobacterium, Methanobacillus, Metranosarcina, Mycobacterium, Propionibacterium, Proteus, Pseudomonas, Rhizobium, Rhodopseudomonas, Salmonella, Serratia, Streptococcus, Streptomyces and Xanthomas.

10. A method according to claim 9, characterized in that the microbial cells are cells of the bacterial genus Propionijacteriu / n.

11. A method according to any of the preceding claims, characterized in that the lysis is caused by heat treatment, pasteurization, treatment with bacterial enzymes, mechanical disruption or chemical treatment, either individually or in combination with each other.

12. A process according to any of claims 2 to 11, characterized in that the separation of the solid and liquid phases of the lysed product is effected by ultrafiltration or microfiltration.

13. A process according to any of the preceding claims, characterized in that the natural vitamin B12 is 5'-deoxyadenosilcobalamin or methylcobalamin.

14. A composition characterized in that it comprises natural vitamin B12 in an amount greater than 0.1% by weight based on the dry matter content of the composition.

15. A composition according to claim 13, characterized in that it can be obtained by the process defined in any of claims 1 to 13.

16. A composition according to claim 14 or 15 characterized in that it is a dry composition, wherein the natural vitamin B12 is substantially homogeneously distributed throughout the composition and where vitamin B12 does not segregate from the other components when exposed to sieving, sifting, gravitational forces or electrostatic forces.

17. A composition according to any of claims 14 to 16, characterized in that it comprises biomass.

18. A composition according to claim 16, characterized in that the biomass is derived from bacteria of the genera, Acetobacterium, Acetobacter, Agrobacterium, Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Clostridium, Corynebacterium, Escherichia, Eubacterium, Flavobacterium, Methanobacillus, Methanosarcina, Mycobacterium, Propionibacterium, Proteus, Pseudomonas, Rhizobium, Rhodopseudomonas, Salmonella, Serratia, Streptococcus, Streptomyces and Xanthomonas either individually or in combination of two or more.

19. A composition according to claim 18, characterized in that the genus is Propionibacterium.

20. A composition according to any of claims 14 to 19 wherein the natural vitamin B12 is 5 'deoxyadenosylcobalamin or methylcobalamin.

21. The use of a composition according to any of claims 14 to 20 in the production of a food supplement.

22. The use of a composition according to any of claims 14 to 20 in the production of a cosmetic preparation.