WO2011154820A2 - Vitamin b12 producing probiotic bacterial strains - Google Patents

Vitamin b12 producing probiotic bacterial strains Download PDF

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Publication number
WO2011154820A2
WO2011154820A2 PCT/IB2011/001302 IB2011001302W WO2011154820A2 WO 2011154820 A2 WO2011154820 A2 WO 2011154820A2 IB 2011001302 W IB2011001302 W IB 2011001302W WO 2011154820 A2 WO2011154820 A2 WO 2011154820A2
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Prior art keywords
vitamin
dsmz
deposited
bacterial strain
number dsm
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PCT/IB2011/001302
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French (fr)
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WO2011154820A3 (en
Inventor
Giovanni Mogna
Gian Paolo Strozzi
Luca Mogna
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Probiotical S.P.A.
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Priority to EP11781589.4A priority Critical patent/EP2580316A2/en
Publication of WO2011154820A2 publication Critical patent/WO2011154820A2/en
Publication of WO2011154820A3 publication Critical patent/WO2011154820A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/42—Cobalamins, i.e. vitamin B12, LLD factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates to a composition comprising at least one probiotic bacterial strain belonging to the species Lactobacillus reuteri as a vitamin B12 producer.
  • the present invention relates to selected bacterial strains belonging to the species Lactobacillus reuteri as vitamin B12 producers.
  • the present invention relates to a food composition or supplement or pharmaceutical composition comprising a bacterial strain belonging to the species Lactobacillus reuteri.
  • Cobalamin (vitamin B12) plays an important role in the production of red blood cells and is essential for proper functioning of the nervous system .
  • Cobalamin vitamin B12
  • R chemical group which binds to the cobalt ion in its structural formula.
  • the group (R) can be: - hydrocyanic, -CN (cyanocobalamin)
  • the metabolically active forms are methyl- and 5 ' - deoxyadenosylcobalamin .
  • Hydroxocobalamin is the natural form in which vitamin B12 is usually taken in through the diet.
  • Cyanocobalamin does not exist in nature except in rare cases .
  • the cyanocobalamin produced synthetically is an artificial substance which is formed during industrial extraction processes, as use is made of papaya, a protease which is activated by the addition of CN- .
  • a transformation of hydroxocobalamin into cyanocobalamin is effected, because the latter form of vitamin B12 is more stable when exposed to air and more easily crystallisable , thus favouring production yields.
  • the majority of food products, food supplements and pharmaceutical products contain cyanocobalamin .
  • vitamin B12 naturally in the form of hydroxocobalamin as an alternative to vitamin B12 produced synthetically in the form of cyanocobalamin is viewed with much interest and responds to a strongly felt need of industry operators. Therefore, there remains a need to have a method for producing vitamin B12 naturally.
  • the subject matter of the present invention is a probiotic bacterial strain belonging to the species Lactobacillus reuteri, as set forth in the appended claim .
  • the subject matter of the present invention further relates to a selected probiotic bacterial strain belonging to the species Lactobacillus reuteri, as set forth in the appended claim.
  • the subject matter of the present invention further relates to a food composition or supplement or pharmaceutical composition as set forth in the appended claim .
  • the subject matter of the present invention further relates to a pharmaceutical composition for use in the preventive and/or curative treatment of subjects suffering from a pathology or a state of malaise due to vitamin B12 deficiency or deficit, as set forth in the appended claim.
  • the subject matter of the present invention further relates to a pharmaceutical composition for use in the preventive and/or curative treatment of subjects suffering from pernicious anaemia, as set forth in the appended claim.
  • the subject matter of the present invention further relates to a pharmaceutical composition for use in the treatment of subjects who take vitamin C, as set forth in the appended claim.
  • Table 1 shows the concentration values (mean value over a number of tests) of vitamin B12 (expressed in pg/litre) measured at OD 600 ⁇
  • the Applicant selected, among the probiotic bacterial strains selected from the group comprising the bacterial strains belonging to the species Lactobacillus reuteri , only those capable of producing vitamin B12.
  • the strain is selected from the group comprising:
  • the food composition or supplement or pharmaceutical composition comprises at least one bacterial strain, selected from among the above-mentioned ones.
  • the food composition or supplement or pharmaceutical composition can comprise two strains or three strains, selected from among the above-mentioned ones .
  • the food composition or supplement or pharmaceutical composition comprises the two strains Lactobacillus reuteri ATCC 55730 and Lactobacillus reuteri DSM 17938 or, alternatively, the three strains Lactobacillus reuteri ATCC 55730, Lactobacillus reuteri DSM 17938 and Lactobacillus reuteri DSM 16143.
  • the aforesaid bacterial species are present in an amount comprised from 0.1 to 75% by weight, preferably from 0.5 to 15% by weight; even more preferably from 1 to 10% by weight, relative to the total weight of the composition or supplement.
  • said percentage relative to the total weight of the composition depends on the product type of the composition or supplement.
  • the amount of said bacterial species is preferably greater than 40%.
  • the composition contains
  • the composition contains bacteria in a concentration comprised from lxlO 6 to lxlO 11 CFU/dose, preferably from lxlO 8 to lxlO 10 CFU/dose.
  • the dose may be comprised from 0.2 to 10 g, for example it is 0.25 g, 1 g, 3 g, 5 g or 7 g .
  • the probiotic bacteria used in the present invention can be in solid form, in particular in powder, dehydrated powder, spray or lyophilized form.
  • the food composition or supplement or pharmaceutical composition can further comprise prebiotic fibres and carbohydrates having a bifidogenic action, such as, for example, inulin, fructo-oligosaccharides (FOS) , galacto- and trans-galactooligosaccharides (GOS and TOS) , gluco- oligosaccharides (GOSx) , xylo-oligosaccharides (XOS) , chitosan oligosaccharides (COS) , soy oligosaccharides (SOS) , isomalto-oligosaccharides (IMOS) , resistant starch, pectin, psyllium, arabinogalactans , glucomannans , galactomannans , xylans, lactosucrose, lactulose, lactitol and various other types of gums, acacia, carob, oat or bamboo fibres and carbohydrates having a b
  • said fibre is selected from the group comprising FOS, inulin and citrus fibres, preferably in a ratio by weight of from 1:3 to 3:1.
  • the amount of the prebiotic fibres and/or carbohydrates having a bifidogenic action, if present, is comprised from 0.5 to 75% by weight, preferably from 1% to 40% by weight and even more preferably from 2 to 20% by weight relative to the total weight of the composition. In this case one has a supplement with symbiotic activity.
  • the food composition or supplement or pharmaceutical composition can further comprise one or more physiologically acceptable additives or excipients.
  • the food composition or supplement or pharmaceutical composition can further comprise other active ingredients and/or components, such as vitamins, minerals, bioactive peptides, substances having antioxidant, hypocholesterolemizing , hypoglycemizing, antiinflammatory or sweetening activity in an amount by weight generally comprised from 0.001% to 10% by weight, preferably from 0.5% to 5% by weight, depending in any case on the type of active component and the recommended daily dose thereof, if defined, relative to the total weight of the composition.
  • active ingredients and/or components such as vitamins, minerals, bioactive peptides, substances having antioxidant, hypocholesterolemizing , hypoglycemizing, antiinflammatory or sweetening activity in an amount by weight generally comprised from 0.001% to 10% by weight, preferably from 0.5% to 5% by weight, depending in any case on the type of active component and the recommended daily dose thereof, if defined, relative to the total weight of the composition.
  • the food composition or supplement or pharmaceutical composition of the present invention is prepared using already known techniques available to one of ordinary skill in the art who is capable of using known machinery and apparatus and the most suitable method.
  • the food composition or supplement or pharmaceutical composition can contain elements or substances with antioxidant activity as mentioned above, in an amount comprised from 0.0001% to 30% by weight relative to the weight of the final composition, depending on the concentration of substances with antioxidant activity and/or on the recommended daily amount (RDA) , where defined.
  • RDA recommended daily amount
  • Selenium can be present in the form of sodium selenate, L-selenomethionine , sodium selenite, sodium acid selenite and selenious acid, as well as in the form of selenium-enriched microorganisms, e.g. yeast, in an amount by weight comprised from 0.0005% to 0.005% relative to the weight of the final composition, in any case sufficient to contribute a quantity of selenium preferably comprised from 10 yg to 150 yg .
  • strains deposited by the company BIOMAN S.r.l., Via Alfieri 18, 10100 Turin, Italy have application; namely:
  • Said strains are capable of accumulating inside cells large quantities of selenium, especially in organic form, if grown in the presence of a suitable source of selenium in the culture medium.
  • the food composition or supplement or pharmaceutical composition can contain glutathione.
  • glutathione in antioxidant form relates to reduced glutathione (or GSH) .
  • the composition comprises glutathione in reduced form and selenium in an amount by weight comprised from 0.5% to 25%, relative to the weight of the final composition, in association with the vitamin B12 producing strains of the present invention.
  • the composition can comprise the sulphur amino acid cysteine and/or N- acetylcysteine and/or mixtures thereof.
  • the food composition or supplement or pharmaceutical composition can contain the above-mentioned probiotic bacteria of the present invention in microencapsulated form, i.e. coated with a composition containing at least one lipid (lipid composition) , preferably of vegetable origin.
  • lipid composition lipid composition
  • the food composition or supplement or pharmaceutical composition can comprise the above- mentioned probiotic bacteria of the present invention as microencapsulated bacteria and non-microencapsulated bacteria .
  • Said lipid composition comprises at least one lipid, and said at least one lipid is of vegetable origin.
  • said lipid of vegetable origin is selected from the group comprising saturated fats.
  • saturated fats are used having a melting point below 75°C, preferably comprised from 45 to 65°C.
  • said saturated fats are selected from the group comprising mono- and di- glycerides of saturated fatty acids, polyglycerols esterified with saturated fatty acids and free saturated fatty acids.
  • said saturated fats are selected from among polyglyceryl distearate, glyceryl palmitostearate or hydrogenated vegetable fats of non- lauric origin.
  • the above-mentioned probiotic bacteria of the present invention are mono-coated.
  • a single coating is produced with a same lipid.
  • the single coating is based on polyglyceryl distearate (commercial name Plurol Stearique WL 1009) .
  • the above-mentioned mono-coated probiotic bacteria of the present invention are placed in the food composition or supplement or pharmaceutical composition of the present invention.
  • the above-mentioned probiotic bacteria of the present invention are double-coated. In practice, a double coating is produced, in succession, with two lipids differing from each other.
  • a hydrogenated palm fat for example with Revel C
  • the glycerol dipalmi ostearate for example Precirol Ato 5
  • the pharmaceutical composition is indicated for use in the preventive and/or curative treatment of subjects suffering from a pathology or a state of malaise due to vitamin B12 deficiencies or deficits.
  • the pharmaceutical composition is indicated for use in the preventive and/or curative treatment of subjects suffering from pernicious anaemia.
  • Pernicious anaemia is a disease provoked by a deficiency or deficit of vitamin B12 , such as, for example, cobalamin. This disease is characterized by megaloblastic anaemia and nervous system disorders.
  • a pharmaceutical composition comprising at least one vitamin B12 producing strain of the present invention selected from the group comprising: Lactobacillus reuteri ATCC 53609, Lactobacillus reuteri DSM 20016, Lactobacillus reuteri ATCC 55730, Lactobacillus reuteri DSM 17938, Lactobacillus reuteri DSM 16143 and Lactobacillus reuteri NCIMB 701359, in association with at least one folic acid producing bacterial strain selected from the group comprising:
  • bacterial strain belonging to the species Bifidobacterium adolescentis, preferably Bifidobacterium adolescentis deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16595;
  • bacterial strain belonging to the species Bifidobacterium catenulatum/pseudocatenulatum, preferably Bifidobacterium catenulatum/pseudocatenulatum deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18350;
  • lactis preferably Bifidobacterium animalis subsp. lactis deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18352;
  • the pharmaceutical composition is indicated for use in the treatment of subjects who take vitamin C, since the intake of large amounts of vitamin C (greater than 1 g) can generate cobalamin deficiencies over time. This is due to the fact that vitamin C, in the presence of iron, can act as an oxidant and form free radicals which damage cobalamin and intrinsic factor.
  • a food composition or supplement or pharmaceutical composition of the present invention has valid application for those persons who take doses of vitamin C daily.
  • compositions or supplements of the present invention can be formulated in solid, lyophilized or dried form, for example in powder or granular form.
  • one pharmaceutical form of interest is tablets or hard or soft capsules.
  • tablets can comprise an inner part comprising the bacterial strains and an outer coating part.
  • the coating can comprise water-soluble polymers and/or polymers capable of resisting the pH variations in the stomach and enabling passage into the intestinal tract.
  • the probiotic bacterial strain is selected from the group comprising the bacterial strains belonging to the species Lactobacillus reuteri, as a vitamin B12 producer.
  • the vitamin B12 producing strain is selected from the group comprising: Lactobacillus reuteri ATCC 55730, Lactobacillus reuteri DSM 17938 and Lactobacillus reuteri DSM 16143.
  • a food composition or supplement or pharmaceutical composition comprises at least one bacterial vitamin B12 producing strain, and preferably at least one additional bacterial strain capable of accumulating selenium inside the bacterial cells is further present; preferably said bacterial strain is selected from the group comprising: Lactobacillus buchneri LB26BM, deposited with the DSMZ on 05/04/2004 and having the deposit number DSM 16341, Lactobacillus ferintoshensis LB6BM, deposited with the DSMZ on 17/01/2004 and having the deposit number DSM 16144, and Lactobacillus reuteri LB2BM, deposited with the DSMZ on 17/01/2004 and having the deposit number DSM 16143.
  • said composition further comprises glutathione in reduced form.
  • said composition further comprises an additional bacterial strain capable of producing folic acid; preferably said strain is selected from the group comprising: a bacterial strain belonging to the species Bifidobacterium adolescentis , preferably Bifidobacterium adolescentis deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16595; a bacterial strain belonging to the species Bifidobacterium catenulatum/pseudocatenulatum, preferably Bifidobacterium catenulatum/pseudocatenulatum deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18350; a bacterial strain belonging to the species Bifidobacterium animalis subsp .
  • lactis preferably Bifidobacterium animalis subsp. lactis deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18352; a bacterial strain belonging to the species Bifidobacterium breve, preferably Bifidobac erium breve deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16596; a bacterial strain belonging to the species Bifidobacterium pseudocatenulatum, preferably
  • said pharmaceutical composition is indicated for use in the preventive and/or curative treatment of subjects suffering from a pathology or a state of malaise due to vitamin B12 deficiencies or deficits .
  • said pharmaceutical composition is indicated for use in the preventive and/or curative treatment of subjects suffering from pernicious anaemia.
  • said pharmaceutical composition is indicated for use in the treatment of subjects who take vitamin C.
  • said composition or supplement can comprise said vitamin B12 producing bacterial strains and/or said bacterial strains capable of accumulating selenium and/or said folic acid producing bacterial strains coated with a composition containing at least one lipid, preferably of vegetable origin, said lipid being selected from the group comprising saturated fats having a melting point below 75°C, preferably comprised from 45 to 65°C.
  • the present microbiological method is used to determine the amount of vitamin B12 produced by the bacterial cells of the strains of the present invention.
  • the method is based on the use of the strain Lactobacillus delbrueckii subs, lactis (L. leichmannii) ATCC 7830-DSMZ 20355, which is auxotrophic for vitamin B12 and thus grows proportionally to the amount of vitamin B12 present in the culture medium. This growth is determined by spectrophotometric reading of the culture at a wavelength of 600 nm. The calibration line calculated from the growth of Lactobacillus delbrueckii subs, lactis (L. leichmannii) in media containing known scalar quantities of vitamin B12 is then used to determine the cobalamin produced by the bacterial strains of the present invention which were submitted to analysis .
  • the auxotrophic strain Lactobacillus delbrueckii subs, lactis (L. leichmannii) ATCC 7830-DSMZ 20355 is inoculated at a percentage of 1% in 15 ml of MRS broth (MRS Difco 55.00 g and 1000 ml of distilled water) and 1% cysteine hydrochloride is added to it (Merck code 1.02735.0100 - 50.00 g and 1000 ml of distilled water; 5% sol . ) .
  • the screw cap is screwed down taking care not to tighten it completely in order to enable the development of an anoxic environment.
  • the strain is incubated in a Gas-Pack provided with an Anaerocult A anaerobic system for 18-24 hours at 37 °C.
  • the pellet is resuspended in 10 ml of 0.85% NaCl saline solution.
  • the stock solution of cyanocobalamin is prepared as follows :
  • step (i) Add 1 ml of the solution obtained in step (i) to 99 ml of ultrapure water (final concentration 1 ⁇ g/ml) .
  • the tubes are autoclaved at 121°C for 15 minutes.
  • the tubes are subsequently vortexed in a vortex mixer .
  • the screw cap is screwed down taking care not to tighten it completely in order to enable the development of an anoxic environment.
  • the strain is incubated in a Gas-Pack provided with an Anaerocult A anaerobic system for 18-24 hours at 37°C.
  • the spectrophotometer is reset to 660 nm against 1 ml of vitamin B12 Assay medium (dilution with 0 ng/ml of cyanocobalamin) obtained as described above.
  • Vitamin B12 Assay Medium is dissolved in 100 ml of ultrapure water.
  • test tubes as described in step 1.4.3 are brought to a volume of 10 ml with the addition of ultrapure water.
  • the Bibby beakers as per step 1.4.5 are brought to a volume of 50 ml with the addition of ultrapure water.
  • the bacterial strains to be analyzed are inoculated in the test tubes belonging to the group described in step 1.4.3, at a percentage of 1%.
  • the pellet is resuspended in 1 ml of 0.1 M pH 4.5 sodium phosphate extraction buffer [Citric acid], 0.005% KCN.
  • the extraction buffer is prepared as follows:
  • the extract is boiled at 100°C for 5 minutes to permit protein denaturation and formation of cyanocobalamin .
  • the extract is diluted with ultrapure water according to the proportions shown below:
  • Each tube is brought to a volume of 10 ml with ultrapure water.
  • the tubes are subsequently vortexed in a vortex mixer .
  • the screw cap is screwed down taking care not to tighten it completely in order to enable the development of an anoxic environment.
  • the strain is incubated in a Gas-Pack provided with an Anaerocult A anaerobic system for 18-24 hours at 37°C.
  • test tubes are chilled at 4°C for 15-20 minutes to block bacterial growth.
  • the spectrophotometer is reset to 660 nm against 1 ml of vitamin B12 Assay medium (dilution with 0 ng/ml of cyanocobalamin) obtained as described above.
  • the absorbance values are read at the wavelength of 660 nm.
  • the present analytical method is used to determine the amount of vitamin B12 produced by the bacterial cells of the strains of the present invention.
  • the analytical determination of the vitamin B12 produced by the bacterial cultures of the strains of the present invention, which are the subject of the present analysis, is performed with the reverse phase HPLC method (column C18) using a UV/VIS detector.
  • 2.1.1.2 0.1 mg/ml solution take 1 ml of the solution as per step 2.2.1.1 and bring it to the required volume in a 10 ml flask with Milli-Q water.
  • step 2.2.1.4 The stock solution described in step 2.2.1.4 is used to prepare standard dilutions for creating the calibration line in the following manner:
  • Mobile phase Milli-Q water and Acetonitrile in elution gradient (as indicated in the table below) .
  • Elution gradient set the elution gradient from the keyboard of the HPLC Waters 625 LC System controller, as shown below:
  • Elution flow On the Waters 625 LC HPLC System controller, set a constant flow of 1.0 ml/min. - Column temperature: On the Waters 625 LC HPLC System controller set a temperature of 25°C for the temperature-controlled oven of the column.
  • Injection volume 20 ⁇ of standard and sample.
  • the wavelength is equal to 360 nm .
  • the software automatically integrates the peaks present in each chromatogram . However, it is fundamental to check that the automatic integration performed by the software is correct.
  • the retention time of cyanocobalamin is about 19 minutes.
  • the calibration line generated must have a value of R2 > 0.99.
  • the vitamin B12 present in the samples submitted to analysis is calculated by interpolation from the calibration line. 2.4 Preparation of samples for determining the vitamin B12 produced .
  • the samples are prepared following the same method as described in steps 1.4.1 to 1.4.20.
  • vitamin B12 vitamin B12 concentration concentration expressed in expressed in yg/litre] at OD 600 pg/litre] at OD 600
  • Lactobacillus reuteri 15.640 ⁇ 75% 18.030 ⁇ 12% ATCC 55730
  • Lactobacillus reuteri 10.950 ⁇ 68% 12.890 ⁇ 10%

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Abstract

The present invention relates to a composition comprising at least one probiotic bacterial strain belonging to the species Lactobacillus reuteri as a vitamin B12 producer. In particular, the present invention relates to selected bacterial strains belonging to the species Lactobacillus reuteri as vitamin B12 producers. Finally, the present invention relates to a food composition or supplement or pharmaceutical composition comprising a bacterial strain belonging to the species Lactobacillus reuteri.

Description

Title: "Vitamin B12 producing probiotic bacterial
strains"
DESCRIPTION
The present invention relates to a composition comprising at least one probiotic bacterial strain belonging to the species Lactobacillus reuteri as a vitamin B12 producer. In particular, the present invention relates to selected bacterial strains belonging to the species Lactobacillus reuteri as vitamin B12 producers. Finally, the present invention relates to a food composition or supplement or pharmaceutical composition comprising a bacterial strain belonging to the species Lactobacillus reuteri.
It is well known that Cobalamin (vitamin B12) plays an important role in the production of red blood cells and is essential for proper functioning of the nervous system .
Furthermore, it is well known that four chemical forms can make reference to Cobalamin (vitamin B12) depending on the type of chemical group (R) which binds to the cobalt ion in its structural formula. The group (R) can be: - hydrocyanic, -CN (cyanocobalamin)
- hydroxyl , -OH (hydroxocobalamin)
- methyl, -CH3 (methylcobalamin)
- 5 -deoxyadenosyl ( 5 ' -deoxyadenosylcobalamin) .
The metabolically active forms are methyl- and 5 ' - deoxyadenosylcobalamin .
Hydroxocobalamin is the natural form in which vitamin B12 is usually taken in through the diet.
However, for food and pharmaceutical use of said vitamin, reliance must be made on the most stable form, represented by cyanocobalamin, which is chemically synthesized. Cyanocobalamin does not exist in nature except in rare cases .
Therefore, the cyanocobalamin produced synthetically is an artificial substance which is formed during industrial extraction processes, as use is made of papaya, a protease which is activated by the addition of CN- . In practice, during industrial synthetic processes, a transformation of hydroxocobalamin into cyanocobalamin is effected, because the latter form of vitamin B12 is more stable when exposed to air and more easily crystallisable , thus favouring production yields. For this reason, the majority of food products, food supplements and pharmaceutical products contain cyanocobalamin . Therefore, the possibility of producing vitamin B12 naturally in the form of hydroxocobalamin as an alternative to vitamin B12 produced synthetically in the form of cyanocobalamin is viewed with much interest and responds to a strongly felt need of industry operators. Therefore, there remains a need to have a method for producing vitamin B12 naturally.
In particular, there remains a need to have a method for producing hydroxocobalamin naturally.
Finally, it is well known that meat contains vitamin B12.
However, for people who do not consume meat -based products, such as vegetarians, it may be difficult to procure a daily amount of vitamin B12 sufficient to meet daily requirements.
Therefore, the availability of non-meat-based food products rich in vitamin B12 would enable vegetarians to take in more easily a daily dose of vitamin B12 sufficient to meet their daily requirements.
The subject matter of the present invention is a probiotic bacterial strain belonging to the species Lactobacillus reuteri, as set forth in the appended claim .
The subject matter of the present invention further relates to a selected probiotic bacterial strain belonging to the species Lactobacillus reuteri, as set forth in the appended claim.
The subject matter of the present invention further relates to a food composition or supplement or pharmaceutical composition as set forth in the appended claim .
The subject matter of the present invention further relates to a pharmaceutical composition for use in the preventive and/or curative treatment of subjects suffering from a pathology or a state of malaise due to vitamin B12 deficiency or deficit, as set forth in the appended claim.
The subject matter of the present invention further relates to a pharmaceutical composition for use in the preventive and/or curative treatment of subjects suffering from pernicious anaemia, as set forth in the appended claim.
The subject matter of the present invention further relates to a pharmaceutical composition for use in the treatment of subjects who take vitamin C, as set forth in the appended claim.
Further preferred embodiments of the present invention will be set forth and illustrated below without intending to limit the scope of the present invention in any way . Table 1 shows the concentration values (mean value over a number of tests) of vitamin B12 (expressed in pg/litre) measured at OD600 ·
After intense research activity the Applicant selected, among the probiotic bacterial strains selected from the group comprising the bacterial strains belonging to the species Lactobacillus reuteri , only those capable of producing vitamin B12.
In a preferred embodiment, the strain is selected from the group comprising:
- Lactobacillus reuteri ATCC 55730,
- Lactobacillus reuteri DSM 17938, and
- Lactobacillus reuteri DSM 16143.
In a preferred embodiment, the food composition or supplement or pharmaceutical composition comprises at least one bacterial strain, selected from among the above-mentioned ones.
Preferably, the food composition or supplement or pharmaceutical composition can comprise two strains or three strains, selected from among the above-mentioned ones .
Advantageously, the food composition or supplement or pharmaceutical composition comprises the two strains Lactobacillus reuteri ATCC 55730 and Lactobacillus reuteri DSM 17938 or, alternatively, the three strains Lactobacillus reuteri ATCC 55730, Lactobacillus reuteri DSM 17938 and Lactobacillus reuteri DSM 16143.
The aforesaid bacterial species are present in an amount comprised from 0.1 to 75% by weight, preferably from 0.5 to 15% by weight; even more preferably from 1 to 10% by weight, relative to the total weight of the composition or supplement. However, said percentage relative to the total weight of the composition depends on the product type of the composition or supplement. For example, in a capsule the amount of said bacterial species is preferably greater than 40%.
In a preferred embodiment, the composition contains
6 bacteria m a concentration comprised from 1x10 to
8 10 lxlO11 CFU/g of mixture, preferably from 1x10 to 1x10 CFU/g of mixture.
In a preferred embodiment, the composition contains bacteria in a concentration comprised from lxlO6 to lxlO11 CFU/dose, preferably from lxlO8 to lxlO10 CFU/dose. The dose may be comprised from 0.2 to 10 g, for example it is 0.25 g, 1 g, 3 g, 5 g or 7 g .
The probiotic bacteria used in the present invention can be in solid form, in particular in powder, dehydrated powder, spray or lyophilized form.
In a preferred embodiment of the invention, the food composition or supplement or pharmaceutical composition can further comprise prebiotic fibres and carbohydrates having a bifidogenic action, such as, for example, inulin, fructo-oligosaccharides (FOS) , galacto- and trans-galactooligosaccharides (GOS and TOS) , gluco- oligosaccharides (GOSx) , xylo-oligosaccharides (XOS) , chitosan oligosaccharides (COS) , soy oligosaccharides (SOS) , isomalto-oligosaccharides (IMOS) , resistant starch, pectin, psyllium, arabinogalactans , glucomannans , galactomannans , xylans, lactosucrose, lactulose, lactitol and various other types of gums, acacia, carob, oat or bamboo fibre, citrus fibres and, in general, fibres containing a soluble and an insoluble portion, in a variable ratio to each other.
Advantageously, said fibre is selected from the group comprising FOS, inulin and citrus fibres, preferably in a ratio by weight of from 1:3 to 3:1.
The amount of the prebiotic fibres and/or carbohydrates having a bifidogenic action, if present, is comprised from 0.5 to 75% by weight, preferably from 1% to 40% by weight and even more preferably from 2 to 20% by weight relative to the total weight of the composition. In this case one has a supplement with symbiotic activity.
In a preferred embodiment, the food composition or supplement or pharmaceutical composition can further comprise one or more physiologically acceptable additives or excipients.
In a preferred embodiment of the invention, the food composition or supplement or pharmaceutical composition can further comprise other active ingredients and/or components, such as vitamins, minerals, bioactive peptides, substances having antioxidant, hypocholesterolemizing , hypoglycemizing, antiinflammatory or sweetening activity in an amount by weight generally comprised from 0.001% to 10% by weight, preferably from 0.5% to 5% by weight, depending in any case on the type of active component and the recommended daily dose thereof, if defined, relative to the total weight of the composition.
The food composition or supplement or pharmaceutical composition of the present invention is prepared using already known techniques available to one of ordinary skill in the art who is capable of using known machinery and apparatus and the most suitable method.
In a preferred embodiment, the food composition or supplement or pharmaceutical composition can contain elements or substances with antioxidant activity as mentioned above, in an amount comprised from 0.0001% to 30% by weight relative to the weight of the final composition, depending on the concentration of substances with antioxidant activity and/or on the recommended daily amount (RDA) , where defined.
Selenium can be present in the form of sodium selenate, L-selenomethionine , sodium selenite, sodium acid selenite and selenious acid, as well as in the form of selenium-enriched microorganisms, e.g. yeast, in an amount by weight comprised from 0.0005% to 0.005% relative to the weight of the final composition, in any case sufficient to contribute a quantity of selenium preferably comprised from 10 yg to 150 yg .
Advantageously, the strains deposited by the company BIOMAN S.r.l., Via Alfieri 18, 10100 Turin, Italy, have application; namely:
- Lactobacillus buchneri LB26BM, deposited with the DSMZ on 05/04/2004 and having the deposit number DSM 16341, and/or
- Lactobacillus ferintoshensis LB6BM, deposited with the DSMZ on 17/01/2004 and having the deposit number DSM 16144, and/or
- Lactobacillus reuteri LB2BM, deposited with the DSMZ on 17/01/2004 and having the deposit number DSM 16143, and/or
in association with the vitamin B12 producing strains of the present invention.
Said strains, in fact, are capable of accumulating inside cells large quantities of selenium, especially in organic form, if grown in the presence of a suitable source of selenium in the culture medium.
In another preferred embodiment, the food composition or supplement or pharmaceutical composition can contain glutathione. In particular, glutathione in antioxidant form relates to reduced glutathione (or GSH) . In a preferred embodiment, the composition comprises glutathione in reduced form and selenium in an amount by weight comprised from 0.5% to 25%, relative to the weight of the final composition, in association with the vitamin B12 producing strains of the present invention. Advantageously, since glutathione can be partially inactivated if taken orally, the composition can comprise the sulphur amino acid cysteine and/or N- acetylcysteine and/or mixtures thereof.
In one embodiment of the present invention, the food composition or supplement or pharmaceutical composition can contain the above-mentioned probiotic bacteria of the present invention in microencapsulated form, i.e. coated with a composition containing at least one lipid (lipid composition) , preferably of vegetable origin.
Alternatively, the food composition or supplement or pharmaceutical composition can comprise the above- mentioned probiotic bacteria of the present invention as microencapsulated bacteria and non-microencapsulated bacteria .
Said lipid composition comprises at least one lipid, and said at least one lipid is of vegetable origin.
Advantageously, said lipid of vegetable origin is selected from the group comprising saturated fats.
Advantageously, saturated fats are used having a melting point below 75°C, preferably comprised from 45 to 65°C. In a preferred embodiment, said saturated fats are selected from the group comprising mono- and di- glycerides of saturated fatty acids, polyglycerols esterified with saturated fatty acids and free saturated fatty acids. Preferably, said saturated fats are selected from among polyglyceryl distearate, glyceryl palmitostearate or hydrogenated vegetable fats of non- lauric origin.
In a first embodiment, the above-mentioned probiotic bacteria of the present invention are mono-coated.
In practice, a single coating is produced with a same lipid. Advantageously, the single coating is based on polyglyceryl distearate (commercial name Plurol Stearique WL 1009) .
The above-mentioned mono-coated probiotic bacteria of the present invention are placed in the food composition or supplement or pharmaceutical composition of the present invention. In a second embodiment, the above-mentioned probiotic bacteria of the present invention are double-coated. In practice, a double coating is produced, in succession, with two lipids differing from each other.
Advantageously, the two lipids are selected from among a hydrogenated palm fat (Tm=60°C) and a glycerol dipalmitostearate (Tm=57-60°C) , which are sprayed onto the lyophilizate in succession, i.e. a double covering is applied on the lyophilizate: the first with the hydrogenated palm fat (for example with Revel C) and the second with the glycerol dipalmi ostearate (for example Precirol Ato 5) in a ratio of 3:1 to each other, advantageously 2:1, for example 2/3 by weight of the former and 1/3 by weight of the latter.
In a preferred embodiment, the pharmaceutical composition is indicated for use in the preventive and/or curative treatment of subjects suffering from a pathology or a state of malaise due to vitamin B12 deficiencies or deficits.
In another preferred embodiment, the pharmaceutical composition is indicated for use in the preventive and/or curative treatment of subjects suffering from pernicious anaemia.
Pernicious anaemia is a disease provoked by a deficiency or deficit of vitamin B12 , such as, for example, cobalamin. This disease is characterized by megaloblastic anaemia and nervous system disorders.
In these cases it is also important to evaluate the concentration of vitamin B12 and folic acid, since a deficiency of the latter similarly provokes a condition of megaloblastic anaemia, without, however, affecting the nervous system.
Therefore, in the case of pernicious anaemia it is very important to improve the anaemic condition by administering a pharmaceutical composition comprising at least one vitamin B12 producing strain of the present invention selected from the group comprising: Lactobacillus reuteri ATCC 53609, Lactobacillus reuteri DSM 20016, Lactobacillus reuteri ATCC 55730, Lactobacillus reuteri DSM 17938, Lactobacillus reuteri DSM 16143 and Lactobacillus reuteri NCIMB 701359, in association with at least one folic acid producing bacterial strain selected from the group comprising:
- a bacterial strain belonging to the species Bifidobacterium adolescentis, preferably Bifidobacterium adolescentis deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16595;
- a bacterial strain belonging to the species Bifidobacterium catenulatum/pseudocatenulatum, preferably Bifidobacterium catenulatum/pseudocatenulatum deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18350;
- a bacterial strain belonging to the species Bifidobacterium animalis subsp . lactis, preferably Bifidobacterium animalis subsp. lactis deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18352;
- a bacterial strain belonging to the species Bifidobacterium breve, preferably Bifidobacterium breve deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16596;
- a bacterial strain belonging to the species Bifidobacterium pseudocatenulatum, preferably Bifidobacterium pseudocatenulatum deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16597, Bifidobacterium pseudocatenulatum deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16598 and Bifidobacterium catenulatum/pseudocatenulatum deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18353.
The above-mentioned strains were deposited by the company Anidral Sri (now Probiotical SpA, Via Mattei 3, Novara 28100 Italy) with the DSMZ in Germany on 21.07.2004 and 15.06.2006.
In another preferred embodiment, the pharmaceutical composition is indicated for use in the treatment of subjects who take vitamin C, since the intake of large amounts of vitamin C (greater than 1 g) can generate cobalamin deficiencies over time. This is due to the fact that vitamin C, in the presence of iron, can act as an oxidant and form free radicals which damage cobalamin and intrinsic factor.
Therefore, a food composition or supplement or pharmaceutical composition of the present invention has valid application for those persons who take doses of vitamin C daily.
Hence the use of probiotic bacterial strains which are producers of vitamin B12 (in the form of hydroxocobalamin, which is the natural form taken in most through the diet) , is an advantageous alternative compared to the use of foods or supplements or drugs containing vitamin B12 in the form of cyanocobalamin . All of the above-mentioned compositions or supplements of the present invention can be formulated in solid, lyophilized or dried form, for example in powder or granular form.
Moreover, one pharmaceutical form of interest is tablets or hard or soft capsules.
Insofar as tablets are concerned, they can comprise an inner part comprising the bacterial strains and an outer coating part. The coating can comprise water-soluble polymers and/or polymers capable of resisting the pH variations in the stomach and enabling passage into the intestinal tract.
In a preferred embodiment, the probiotic bacterial strain is selected from the group comprising the bacterial strains belonging to the species Lactobacillus reuteri, as a vitamin B12 producer.
In another preferred embodiment, the vitamin B12 producing strain is selected from the group comprising: Lactobacillus reuteri ATCC 55730, Lactobacillus reuteri DSM 17938 and Lactobacillus reuteri DSM 16143.
In another preferred embodiment, a food composition or supplement or pharmaceutical composition comprises at least one bacterial vitamin B12 producing strain, and preferably at least one additional bacterial strain capable of accumulating selenium inside the bacterial cells is further present; preferably said bacterial strain is selected from the group comprising: Lactobacillus buchneri LB26BM, deposited with the DSMZ on 05/04/2004 and having the deposit number DSM 16341, Lactobacillus ferintoshensis LB6BM, deposited with the DSMZ on 17/01/2004 and having the deposit number DSM 16144, and Lactobacillus reuteri LB2BM, deposited with the DSMZ on 17/01/2004 and having the deposit number DSM 16143.
In another preferred embodiment, said composition further comprises glutathione in reduced form.
In another embodiment said composition further comprises an additional bacterial strain capable of producing folic acid; preferably said strain is selected from the group comprising: a bacterial strain belonging to the species Bifidobacterium adolescentis , preferably Bifidobacterium adolescentis deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16595; a bacterial strain belonging to the species Bifidobacterium catenulatum/pseudocatenulatum, preferably Bifidobacterium catenulatum/pseudocatenulatum deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18350; a bacterial strain belonging to the species Bifidobacterium animalis subsp . lactis, preferably Bifidobacterium animalis subsp. lactis deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18352; a bacterial strain belonging to the species Bifidobacterium breve, preferably Bifidobac erium breve deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16596; a bacterial strain belonging to the species Bifidobacterium pseudocatenulatum, preferably
Bifidobacterium pseudocatenulatum deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16597, Bifidobacterium pseudocatenulatum deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16598 and Bifidobacterium catenulatum/pseudocatenulatum deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18353. In another preferred embodiment, said pharmaceutical composition is indicated for use in the preventive and/or curative treatment of subjects suffering from a pathology or a state of malaise due to vitamin B12 deficiencies or deficits .
In another preferred embodiment, said pharmaceutical composition is indicated for use in the preventive and/or curative treatment of subjects suffering from pernicious anaemia.
In another preferred embodiment, said pharmaceutical composition is indicated for use in the treatment of subjects who take vitamin C.
In another preferred embodiment, said composition or supplement can comprise said vitamin B12 producing bacterial strains and/or said bacterial strains capable of accumulating selenium and/or said folic acid producing bacterial strains coated with a composition containing at least one lipid, preferably of vegetable origin, said lipid being selected from the group comprising saturated fats having a melting point below 75°C, preferably comprised from 45 to 65°C.
Experimental part - Microbiological method
The present microbiological method is used to determine the amount of vitamin B12 produced by the bacterial cells of the strains of the present invention.
The method is based on the use of the strain Lactobacillus delbrueckii subs, lactis (L. leichmannii) ATCC 7830-DSMZ 20355, which is auxotrophic for vitamin B12 and thus grows proportionally to the amount of vitamin B12 present in the culture medium. This growth is determined by spectrophotometric reading of the culture at a wavelength of 600 nm. The calibration line calculated from the growth of Lactobacillus delbrueckii subs, lactis (L. leichmannii) in media containing known scalar quantities of vitamin B12 is then used to determine the cobalamin produced by the bacterial strains of the present invention which were submitted to analysis .
Microbiological method
1.1 Preparation of the auxotrophic strain for calculating the calibration line and concentrations of vitamin B12 produced.
1.1.1 The auxotrophic strain Lactobacillus delbrueckii subs, lactis (L. leichmannii) ATCC 7830-DSMZ 20355 is inoculated at a percentage of 1% in 15 ml of MRS broth (MRS Difco 55.00 g and 1000 ml of distilled water) and 1% cysteine hydrochloride is added to it (Merck code 1.02735.0100 - 50.00 g and 1000 ml of distilled water; 5% sol . ) .
1.1.2 It is subsequently vortexed in a vortex mixer.
1.1.3 The screw cap is screwed down taking care not to tighten it completely in order to enable the development of an anoxic environment. The strain is incubated in a Gas-Pack provided with an Anaerocult A anaerobic system for 18-24 hours at 37 °C.
1.1.4 Subsequently, the culture obtained is transplanted two consecutive times using the same medium and the same percentage of inoculum.
1.1.5 At the end of the incubation period, centrifuge the culture of Lactobacillus delbrueckii subs, lactis (L. leichmannii) ATCC 7830-DSMZ 20355 at 6000 rpm for 15 minutes .
1.1.6 At the end of centrifugation , the supernatant is discarded and the pellet is washed three consecutive times with 10 ml of 0.85% NaCl saline solution.
1.1.7 The pellet is resuspended in 10 ml of 0.85% NaCl saline solution.
1.1.8 Then a 1:100 dilution is made with 0.85% NaCl saline solution so as to obtain the inoculum necessary for setting up tubes at known concentrations of vitamin 3312, necessary for calculating the calibration line and setting up tubes from which the amount of vitamin B12 produced will be determined.
1.2 Preparation of the tubes necessary for calculating the calibration line .
1.2.1 7.6 g of vitamin B12 Assay Medium (Difco code 60179) is dissolved in 100 ml of ultrapure water.
1.2.2 It is heated and stirred simultaneously and allowed to boil for about 2-3 minutes to completely dissolve all the powder.
1.2.3 5 ml of vitamin B12 Assay Medium is distributed into seven glass test tubes previously washed with ultrapure water and placed in an oven at 100°C overnight .
1.2.4 The stock solution of cyanocobalamin (prepared as described below) is added to the test tubes, which are brought to the final volume of 10 ml with ultrapure water, as described below:
- ml of stock solution per tube:
0, 0.5, 1, 2, 3, 4, 5
- ml of ultrapure water per tube: 5, 4.5, 4, 3, 2, 1, 0
- Concentration of vitamin B12 per tube (ng/ml) :
0.0 0.025 0.05 0.10 0.15 0.20 0.25
The stock solution of cyanocobalamin is prepared as follows :
i) Dissolve 10 mg of cyanocobalamin standard (Sigma code C3607) in a 100 ml flask, bringing it to the required volume with a 25% ethanol solution in ultrapure water (final concentration 0.1 mg/ml) .
ii) Add 1 ml of the solution obtained in step (i) to 99 ml of ultrapure water (final concentration 1 μg/ml) .
iii) Add 1 ml of the solution obtained in step (ii) to 99 ml of ultrapure water (final concentration 10 ng/ml) iv) Add 1 ml of the solution obtained in step (iii) to 199 ml of ultrapure water (final concentration 0.05 ng/ml) .
1.2.5 The tubes are autoclaved at 121°C for 15 minutes.
1.2.6 At the end of the heat treatment, add 100 μΐ of the suspension of Lactobacillus delbrueckii subsp. lactis (L. leichmannii) ATCC 7830-DSMZ 20355, obtained as described above in 1.1.8 , to each test tube.
1.2.7 The tubes are subsequently vortexed in a vortex mixer .
1.2.8 The screw cap is screwed down taking care not to tighten it completely in order to enable the development of an anoxic environment. The strain is incubated in a Gas-Pack provided with an Anaerocult A anaerobic system for 18-24 hours at 37°C.
1.2.9 At the end of the incubation period, chill the test tubes at 4°C for 15-20 minutes to block bacterial growth .
1.3 Calculation of the calibration line .
1.3.1 The spectrophotometer is reset to 660 nm against 1 ml of vitamin B12 Assay medium (dilution with 0 ng/ml of cyanocobalamin) obtained as described above.
1.3.2 Add 1 ml of the culture obtained as per step 1.2.9 in 1.5 ml plastic cuvettes according to the ascending order of the concentration of cyanocobalamin present .
1.3.3 The absorbance values are read at the wavelength of 660 nm.
1.3.4 The absorbance values obtained were used to determine the calibration line, whose equation will be used to extrapolate data related to the unknown quantities of vitamin B12 present in the cultures obtained as per step 1.4.18.
1.4 Preparation of samples for determining the vitamin B12 produced .
1.4.1 7.6 g of Vitamin B12 Assay Medium is dissolved in 100 ml of ultrapure water.
1.4.2 It is heated and stirred simultaneously and allowed to boil for about 2-3 minutes to completely dissolve all the powder.
1.4.3 5 ml of vitamin B12 Assay Medium is distributed into "n" glass test tubes (n = number of samples to be activated x number of activation subcultures) previously washed with ultrapure water and placed in an oven at 100°C.
1.4.4 5 ml of vitamin B12 Assay Medium is distributed into "n" glass test tubes (n = number of samples to be analyzed x 5) previously washed with ultrapure water and placed in an oven at 100°C overnight.
1.4.5 25 ml of vitamin B12 Assay medium is distributed into "n" 100 ml Bibby beakers (Pyrex glass bottles, generally 100 or 250 ml, suitable for autoclave sterilization of culture media) (n = number of samples to be analyzed) previously washed with ultrapure water and placed in an oven at 100°C.
1.4.6 The test tubes as described in step 1.4.3 are brought to a volume of 10 ml with the addition of ultrapure water.
1.4.7 The Bibby beakers as per step 1.4.5 are brought to a volume of 50 ml with the addition of ultrapure water.
1.4.8 They are autoclaved at 121 °C for 15 minutes.
1.4.9 The bacterial strains to be analyzed are inoculated in the test tubes belonging to the group described in step 1.4.3, at a percentage of 1%.
1.4.10 The tubes are subsequently vortexed in a vortex mixer .
1.4.11 The screw cap is firmly screwed down and the tubes are incubated in a temperature-controlled bath for 18-24 hours at 37°C.
1.4.12 The culture obtained is transplanted three consecutive times using the same medium and the same percentage of inoculum.
1.4.13 The bacterial strains in the Bibby beakers belonging to the group described in step 1.4.5 are inoculated, in a percentage of 1%.
1.4.14 The beakers are subsequently vortexed in a vortex mixer .
1.4.15 The screw cap is firmly screwed down and the cultures are incubated in a temperature-controlled bath for 18-24 hours at 37°C.
1.4.16 At the end of the incubation period, centrifuge at 6000 rpm for 15 minutes.
1.4.17 At the end of centrifugation, the supernatant is removed and the pellet is washed three consecutive times with 10 ml of 0.1 M pH 7 sodium phosphate buffer.
1.4.18 The pellet is resuspended in 1 ml of 0.1 M pH 4.5 sodium phosphate extraction buffer [Citric acid], 0.005% KCN. The extraction buffer is prepared as follows:
al) Bring 100 ml of 0.1 M pH 4.5 sodium phosphate buffer solution to a pH of 4.5 by adding citric acid.
a2) Add 0.005% KCN to the solution obtained in step al) . a3) Submit to stirring for 10 minutes.
1.4.19 The solution is submitted to stirring at 4°C with 1 g of glass beads for three 5 -minute cycles.
1.4.20 At the end of the stirring period, the extract is boiled at 100°C for 5 minutes to permit protein denaturation and formation of cyanocobalamin .
1.4.21 At the end of the heat treatment, the extract is diluted with ultrapure water according to the proportions shown below:
- dilution: 1:2 1:5 1:10 1:100 1:1000
- μΐ extracted: 500 200 100 10 1
- μΐ ddH20: 500 800 900 990 999
1.4.22 Each dilution is added to the tubes as per step 1.4.4.
1.4.23 Each tube is brought to a volume of 10 ml with ultrapure water.
1.4.24 100 μΐ of the suspension of Lactobacillus delbrueckii subs, lactis (L. leichmannii) ATCC 7830-DSMZ 20355 as per step 1.4.8 is added.
1.4.25 The tubes are subsequently vortexed in a vortex mixer . 1.4.26 The screw cap is screwed down taking care not to tighten it completely in order to enable the development of an anoxic environment. The strain is incubated in a Gas-Pack provided with an Anaerocult A anaerobic system for 18-24 hours at 37°C.
1.4.27 At the end of the incubation period, the test tubes are chilled at 4°C for 15-20 minutes to block bacterial growth.
1.5 Determination of the vitamin B12 produced.
1.5.1 The spectrophotometer is reset to 660 nm against 1 ml of vitamin B12 Assay medium (dilution with 0 ng/ml of cyanocobalamin) obtained as described above.
1.5.2 1 ml of the culture obtained as described in step 1.2.9 is added to 1.5 ml plastic cuvettes according to the ascending order of the dilutions of the extract as described in step 1.4.21.
1.5.3 The absorbance values are read at the wavelength of 660 nm.
1.5.4 The absorbance values obtained were used in the equation of the line calculated above in step 1.3.4 to calculate the concentration of vitamin B12 produced by the strains considered. The results are expressed in ng/1.
Experimental part - Analytical method (HPLC)
The present analytical method is used to determine the amount of vitamin B12 produced by the bacterial cells of the strains of the present invention.
The analytical determination of the vitamin B12 produced by the bacterial cultures of the strains of the present invention, which are the subject of the present analysis, is performed with the reverse phase HPLC method (column C18) using a UV/VIS detector.
Analytical method (HPLC)
2.1 Preparation of standard solutions for calculating the calibration line .
2.1.1 10 mg of Cyanocobalamin Standard (Sigma code
C3607) is weighed and the following serial dilutions are made :
2.1.1.1 1 mg/ml solution: weigh 10 mg of Cyanocobalamin Standard (Sigma code C3607) in a 10 ml flask and bring to the required volume with Milli-Q water and 25% ethanol (ethanol serves to increase the solubility of the cyanocobalamin) .
2.1.1.2 0.1 mg/ml solution: take 1 ml of the solution as per step 2.2.1.1 and bring it to the required volume in a 10 ml flask with Milli-Q water.
2.1.1.3 0.01 mg/ml solution: take 1 ml of the solution as per step 2.2.1.2 and bring it to the required volume in a 10 ml flask with Milli-Q water.
2.1.1.4 Stock solution (1000 ng/ml) : take 5 ml of the solution as per step 2.2.1.3 and bring it to the required volume in a 50 ml flask with Milli-Q water.
2.1.2 The stock solution described in step 2.2.1.4 is used to prepare standard dilutions for creating the calibration line in the following manner:
2.1.2.1 1000 ng/ml solution or stock solution as such.
2.1.2.2 800 ng/ml solution: take 4 ml of stock solution and bring to the required volume in a 5 ml flask with Milli-Q water.
2.1.2.3 500 ng/ml solution: take 5 ml of stock solution and bring to the required volume in a 10 ml flask with Milli-Q water.
2.1.2.4 200 ng/ml solution: take 1 ml of stock solution and bring to the required volume in a 5 ml flask with Milli-Q water.
2.1.2.5 100 ng/ml solution: take 1 ml of stock solution and bring to the required volume in a 10 ml flask with Milli-Q water.
2.1.2.6 50 ng/ml solution: take 1 ml of the 500 ng/ml solution (see step 2.2.23) and bring to the required volume in a 10 ml flask with Milli-Q water.
2.1.2.7 25 ng/ml solution: take 5 ml of the 50 ng/ml solution (see step 7.2.2.6) and bring to the required volume in a 10 ml flask with Milli-Q water.
2.2 Main parameter settings for the HPLC method . In order to use the HPLC Waters 625 LC System, TotalChrom (Perkin Elmer) data acquisition software and the UV/VIS 785 A detector (Perkin Elmer) correctly, the directions provided in the usage procedures must be followed carefully. The fundamental test parameters for determining the amount of Vitamin B12 produced by the bacterial strains submitted to analysis are indicated below :
2.2.1 HPLC parameters:
Type of chromatography column :
Spherisorb ODS 2 column (Waters) C18 250x4.6 mm, diameter 5μπι.
Mobile phase: Milli-Q water and Acetonitrile in elution gradient (as indicated in the table below) .
Elution gradient: set the elution gradient from the keyboard of the HPLC Waters 625 LC System controller, as shown below:
Milli-Q Water Acetonitrile
95 5
85 15
70 30
95
95
Elution flow: On the Waters 625 LC HPLC System controller, set a constant flow of 1.0 ml/min. - Column temperature: On the Waters 625 LC HPLC System controller set a temperature of 25°C for the temperature-controlled oven of the column.
Injection volume: 20 μΐ of standard and sample.
2.2.2 Parameters for the UV/VIS detector. The wavelength is equal to 360 nm .
2.3 Calibration line .
2.3.1 Inject all the standard solutions prepared as described in step 2.2, one after the other.
2.3.2 Acquire the associated chromatograms via the TotalChrom software.
2.3.3 The software automatically integrates the peaks present in each chromatogram . However, it is fundamental to check that the automatic integration performed by the software is correct. The retention time of cyanocobalamin is about 19 minutes.
2.3.4 The areas of each single peak associated with vitamin B12 are recorded. Based on the value of the areas of the peaks in relation to the starting concentrations, a scatter plot is created.
2.3.5 The calibration line generated must have a value of R2 > 0.99.
2.3.6 The vitamin B12 present in the samples submitted to analysis is calculated by interpolation from the calibration line. 2.4 Preparation of samples for determining the vitamin B12 produced .
The samples are prepared following the same method as described in steps 1.4.1 to 1.4.20.
2.5 Determination of the vitamin B12 produced .
2.5.1 20 μΐ of the extract prepared in step 1.4.20 is injected in HPLC .
2.5.2 The value of the area of the peak associated with the sample analyzed is interpolated from the calibration line prepared as per step 1.4.
2.5.3 Calculate the final vitamin B12 value of each individual sample based on the quantity of the injected sample (20 μΐ) . The results are expressed in ng/1.
Table 1
VITAMIN B12 producing Microbiological HPLC
strains assay assay
(mean of a number (mean of a number of tests) of tests)
[vitamin B12 [vitamin B12 concentration concentration expressed in expressed in yg/litre] at OD600 pg/litre] at OD600
Lactobacillus reuteri 15.640 ± 75% 18.030 ± 12% ATCC 55730
Lactobacillus reuteri 10.950 ± 68% 12.890 ± 10%
DSM 17938
Lactobacillus reuteri 2.790 ± 76% 8.000 ± 13%
DSM 16143

Claims

1. A probiotic bacterial strain selected from the group comprising the bacterial strains belonging to the species Lactobacillus reuteri, as a vitamin B12 producer.
2. The strain according to claim 1, wherein said strain is selected from the group comprising: Lactobacillus reuteri ATCC 55730, Lactobacillus reuteri DSM 17938 and Lactobacillus reuteri DSM 16143.
3. A food composition or supplement or pharmaceutical composition comprising at least one bacterial strain according to claim 1 or 2.
4. The food composition or supplement or pharmaceutical composition according to claim 3, wherein at least one further bacterial strain capable of accumulating selenium inside the bacterial cells is additionally present; said bacterial strain is preferably selected from the group comprising: Lactobacillus buchneri LB26B , deposited with the DSMZ on 05/04/2004 and having the deposit number DSM 16341, Lactobacillus ferintoshensis LB6BM, deposited with the DSMZ on 17/01/2004 and having the deposit number DSM 16144, and Lactobacillus reuteri LB2BM, deposited with the DSMZ on 17/01/2004 and having the deposit number DSM 16143.
5. The food composition or supplement or pharmaceutical composition according to claim 3 or 4, wherein glutathione in reduced form is additionally present.
The food composition or supplement or pharmaceutical composition according to any one of claims 3-5, wherein a further bacterial strain capable of producing folic acid is additionally present, preferably said strain is selected from the group comprising: a bacterial strain belonging to the species Bifidobacterium adolescentis, preferably Bifidobacterium adolescentis deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16595; a bacterial strain belonging to the species Bifidobacterium catenulatum/pseudocatenulatum, preferably Bifidobacterium catenulatum/pseudocatenulatum deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18350; a bacterial strain belonging to the species Bifidobacterium animalis subsp. lactis, preferably Bifidobacterium animalis subsp. lactis deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18352; a bacterial strain belonging to the species Bifidobacterium breve, preferably Bifidobacterium breve deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16596; a bacterial strain belonging to the species Bifidobacterium pseudocatenulatum, preferably Bifidobacterium pseudocatenulatum deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16597, Bifidobacterium pseudocatenulatum deposited with the DSMZ on 21.07.2004 and having the accession number DSM 16598 and Bifidobacterium catenulatum/pseudocatenulatum deposited with the DSMZ on 15.06.2006 and having the accession number DSM 18353.
7. The pharmaceutical composition according to any one of claims 3-6, for use in the preventive and/or curative treatment of subjects suffering from a pathology or a state of malaise due to vitamin B12 deficiencies or deficits
8. The pharmaceutical composition according to any one of claims 3-6, for use in the preventive and/or curative treatment of subjects suffering from pernicious anaemia.
9. The pharmaceutical composition according to any one of claims 3-6, for use in the treatment of subjects who take vitamin C.
10. The composition according to any one of claims 3 to 9, wherein said vitamin B12 producing bacterial strains and/or said bacterial strains capable of accumulating selenium and/or said folic acid producing bacterial strains are coated with a composition containing at least one lipid, preferably of vegetable origin, said lipid being selected from the group comprising saturated fats having a melting point below 75°C, preferably comprised from 45 to 65°C.
PCT/IB2011/001302 2010-06-11 2011-06-10 Vitamin b12 producing probiotic bacterial strains WO2011154820A2 (en)

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EP2609814A1 (en) * 2011-12-30 2013-07-03 Nestec S.A. Lactobacillus reuteri DSM 17938 for the development of cognitive function
ES2604355A1 (en) * 2016-12-13 2017-03-06 Europastry,S.A. New strains of the lactobacillus reuteri species for the preparation of panarian masses (Machine-translation by Google Translate, not legally binding)
EP3655520B1 (en) 2017-07-21 2023-09-06 Frettlöh, Martin Sequential co-culturing method for producing a vitamin- and protein-rich food product

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Publication number Priority date Publication date Assignee Title
EP2609813A1 (en) * 2011-12-30 2013-07-03 Nestec S.A. Lactobacillus reuteri DSM 17938 for the development of the enteric nervous system
EP2609814A1 (en) * 2011-12-30 2013-07-03 Nestec S.A. Lactobacillus reuteri DSM 17938 for the development of cognitive function
WO2013098033A1 (en) * 2011-12-30 2013-07-04 Nestec S.A. Lactobacillus reuteri dsm 17938 for the development of cognitive function
WO2013098032A1 (en) * 2011-12-30 2013-07-04 Nestec S.A. Lactobacillus reuteri dsm 17938 for the development of the enteric nervous system
CN104185427A (en) * 2011-12-30 2014-12-03 生命大地女神有限公司 Lactobacillus reuteri DSM 17938 for the development of cognitive function
CN104244736A (en) * 2011-12-30 2014-12-24 生命大地女神有限公司 Lactobacillus reuteri dsm 17938 for the development of the enteric nervous system
ES2604355A1 (en) * 2016-12-13 2017-03-06 Europastry,S.A. New strains of the lactobacillus reuteri species for the preparation of panarian masses (Machine-translation by Google Translate, not legally binding)
EP3655520B1 (en) 2017-07-21 2023-09-06 Frettlöh, Martin Sequential co-culturing method for producing a vitamin- and protein-rich food product

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