CN104342390A - Sinorhizobium meliloti strain and composition and application of sinorhizobium meliloti strain - Google Patents

Sinorhizobium meliloti strain and composition and application of sinorhizobium meliloti strain Download PDF

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CN104342390A
CN104342390A CN201410529336.7A CN201410529336A CN104342390A CN 104342390 A CN104342390 A CN 104342390A CN 201410529336 A CN201410529336 A CN 201410529336A CN 104342390 A CN104342390 A CN 104342390A
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strain
sinorhizobium meliloti
vitamins
sinorhizobium
fermentation
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CN104342390B (en
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张大伟
李莎
夏苗苗
房欢
周文娟
郑平
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Tianjin Institute of Industrial Biotechnology of CAS
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Tianjin Institute of Industrial Biotechnology of CAS
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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
    • C12N1/205—Bacterial isolates
    • 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
    • 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

Abstract

The invention discloses a sinorhizobium meliloti strain capable of being applied to fermentation to generate vitamin B12, wherein the preservation number of the sinorhizobium meliloti strain is CGMCC No.9638. The invention also discloses a production and preparation method of vitamin 12 by the sinorhizobium meliloti strain in a fermentation manner. The method comprises a fermentation culture condition of the strain and an extraction method of the vitamin 12 in fermentation liquor; the method comprises the following steps: inoculating the strain on a solid culture medium into a triangular flask as a seed solution; inoculating the seed solution into a fermentation culture medium at 30 DEG C with 10% of inoculation amount; fermenting and cultivating at 200rpm for 200 hours, and then collecting the fermentation liquor; and adding a transforming agent to transform the fermentation liquor into cyano cobalt amine. The vitamin 12 is widely applied to the industries such as feeds, additives, medicines and cosmetics as a special factor in an adjusting organism growth process; and the requirement of the vitamin 12 rises year by year. The strain fermentation process is easy to control, so that the strain fermentation process is not affected by the factors such as seasons; the requirements on raw materials are simple; and fermentation production is developed into a main production mode.

Description

A kind of Sinorhizobium meliloti strain and composition thereof and application
Technical field
The invention belongs to Biological resources, fermentable and macromole natural product biosynthesis technology field.Relate to a strain and produce vitamins B 12bacterial strain, Sinorhizobium meliloti ( sinorhizobium medicae), and utilize this strain fermentation to prepare vitamins B 12method, be a kind of Sinorhizobium meliloti strain and composition thereof and application in particular.
Background technology
Vitamins B 12being again cobalami (Cobalamin), belonging to corrin compounds, is a unique vitamin compound containing metal ion at present, from vitamins B 12structure can find out, its basic structure comprises corrin ring and aglucon.Center corrin ring is made up of 4 pyrroles be connected and 1 cobalt atom, and cobalt is sequestered in 4 pyrroles centers.Kind of ligand above corrin ring is different, forms dissimilar cobalami, generally can form four types: (1) forms cyanocobalamin (Cyanocobalamin) when aglucon is cyano group; (2) hydroxycobalamine (Hydroxocobalamin) is formed when aglucon is hydroxyl; (3) deoxyadenosyl cobalamin (Adenosylcobalamin) is formed when aglucon is Desoxyadenosine; (4) methyl cobalamin (Methylcobalamin) is formed when aglucon is methyl.
In general medicine, the existence form of cobalami is cyanocobalamin, but the cobalami that can play a role in vivo is adenosylcobalamin, cyanocobalamin need to be converted into adenosylcobalamin after competence exertion therapeutic action.Therefore, for the patient that some are more serious, need adenosylcobalamin that direct injection keeps in Dark Place to reach the effect of fast treating.
Vitamins B 12synthetic method comprises synthetic method and microbe fermentation method, there is impassable problem in synthetic process, and therefore, scientists starts again to explore vitamins B 12building-up process in microbe, solves vitamins B 12production problem.Produce bacterial strain and mainly contain anaerobically fermenting bacterial strain propionibacterium freudenreichii, Xie Shi propionibacterium etc., aerobic fermentation bacterial strain Pseuomonas denitrifican, Rhodopseudomonas palustris, coarse Nocardia bacteria etc.Microbe synthesis vitamins B 12there are two approach: one is aerobic approach; Another kind is anaerobic pathway.First aerobic approach synthesizes corrin ring, is then chelated in ring by cobalt atom; And anaerobic pathway is chelated to after in porphyrin ring by cobalt atom, then form the corrin ring containing cobalt atom in conjunction with other atoms.In other building-up process, anaerobic pathway is identical with aerobic approach, and the enzyme of just often kind of approach catalysis is different.
Microbial method, colorimetric method, Indirect Determination, high performance capillary electrophoresis, thin layer chromatography and high performance liquid chromatography detect B in fermented liquid 12content.But microbial method detects length consuming time, and sensitivity is low, be unfavorable for detecting in fermenting process; Colorimetry is only suitable for analyzing the simple samples of comparison of ingredients such as medicine, but there is interference effect for the sample of fermentation or complicated component; Indirect method calculates vitamins B by the change of cobalt contents 12content, although detection method is accurately quick, sample itself contains cobalt, thus can cause very big error; High performance capillary electrophoresis is low due to detectability, and food etc. can be caused to contain mcg vitamin B 12detection have limitation; Thin layer chromatography can not be unified, so have limitation in detection by quantitative due to applied sample amount; High performance liquid chromatography can detection level lower, vitamins B in the sample of complicated component 12content, there is easy, advantage fast and accurately.
Summary of the invention
The object of the invention is to the deficiency overcoming existing resource, provide separation in a strain soil to obtain Sinorhizobium meliloti TIB.SM.2013 preserving number CGMCC No. 9638 on the one hand.
Another aspect of the present invention is to provide a kind of vitamins B 12biosynthesis preparation method, by utilizing Sinorhizobium meliloti TIB.SM.2013 preserving number CGMCC No. 9638, liquid lengthy fermentation and prepare vitamins B 12.
The invention discloses following technology contents for achieving the above object:
A kind of Sinorhizobium meliloti strain ( sinorhizobium medicae) TIB.SM.2013, be separated from soil and obtain, by the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number CGMCC No. 9638, its detailed technology contents is as follows:
(1) the applicant plants separation in soil sample in June, 2013 Tianjin trifolium, screens the efficient bacterial strain TIB.SM.2013 of a strain.Through morphologic observation, physiological and biochemical test and 16SrDNA qualification, belong to Sinorhizobium meliloti strain ( sinorhizobium medicae).This bacterial strain is delivered the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms on September 4th, 2014 by applicant, preserving number CGMCC No. 9638.Preservation date: on September 4th, 2014; Unit address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
(2) Sinorhizobium meliloti strain ( sinorhizobium medicae) bacterial characteristics of TIB.SM.2013 and physiological and biochemical index:
1. this Sinorhizobium meliloti TIB.SM.2013 preserving number CGMCC No. 9638.Sucrose agar substratum grows comparatively slow, under 30 DEG C of aerobic conditions, grow 5 days, colony diameter is 3-5 mm, and in being sorrel, oval, bacterium colony is smooth; Colony morphology characteristic is see Fig. 1.This bacterial strain is rod-short, and centre has particle, and long 3-8 μm, without spore.
2. Sinorhizobium meliloti TIB.SM.2013 provided by the present invention, preserving number CGMCC No. 9638.The % content of G+C mol is 63.20%, and the suitable growth temperature of this bacterial strain is 28-30 DEG C, can biosynthesizing vitamins B during liquid lengthy fermentation 12.
The present invention further discloses Sinorhizobium meliloti strain ( sinorhizobium medicae) TIB.SM.2013, preserving number CGMCC No.9638, at preparation biosynthesizing vitamins B 12the application of aspect.It produces vitamins B in fermented liquid 12concentration be at least 50mg/L.
Sinorhizobium meliloti strain of the present invention ( sinorhizobium medicae) the % content of TIB.SM.2013, the G+C mol of its genomic dna is 63.20%; Wherein the mol ratio of G:C is 1:2.3.
The present invention further discloses a kind of composition, it comprise Sinorhizobium meliloti fermented liquid, Sinorhizobium meliloti strain ( sinorhizobium medicae) TIB.SM.2013, preserving number CGMCC No. 9638, composition comprises: vitamins B 12, corn steep liquor, sucrose, DMB, S-adenosylmethionine etc.
The present invention also disclose simultaneously Sinorhizobium meliloti strain ( sinorhizobium medicae) TIB.SM.2013, preserving number CGMCC No. 9638 produces vitamins B 12cultivation and extracting method, it is characterized in that being undertaken by following step:
(1) Sinorhizobium meliloti strain slant strains is inoculated into is equipped with in the triangular flask of substratum, under 28-32 DEG C, 150-220 rpm shaking table oscillating condition, cultivate 36 h, preparation seed liquor;
(2) seed liquor is seeded to fermention medium by the inoculum size (V/V) of 5-20%, then cultivates 6-9 days at 25-30 DEG C, obtain Sinorhizobium meliloti fermented liquid;
(3) 8% sodium nitrite solution and Glacial acetic acid 1:1(V/V is added in the fermented liquid obtained in step (2)), boiling water bath 20-30 min discharges the product in thalline and is converted into cyanocobalamin, and centrifugal 10 min of 10000-12000 rpm collect supernatant liquor separation detection product
Described substratum is composed of the following components by weight:
The carbon source of 6-40 part, the organic nitrogen source of 5-30 part,
The inorganic salt of 0.5-10 part, add water to 1000 parts, regulate pH=6.8-7.0.
Described carbon source is sucrose, maltose, lactose or trimethyl-glycine.Described organic nitrogen source is corn steep liquor, yeast extract, yeast leaching powder, yeast extract, yeast powder, yeast autolysis powder or corn starch.
Described inorganic salt are made up of the raw material of following portions by weight:
Magnesium sulfate heptahydrate MgSO 40.1-2 g/L, dipotassium hydrogen phosphate 0.5-3 g/L, zinc sulfate 0.2-1 g/L,
Manganous sulfate 0.1-3 g/L, cobalt chloride 0.3-5 g/L, 5,6-dimethylbenzimidazole 0.01-2 g/L.
The present invention is more detailed to be described below:
A kind of vitamins B 12preparation method, comprising:
Sinorhizobium meliloti TIB.SM.2013 is cultivated in (a) liquid medium within; (b) the Sinorhizobium meliloti TIB.SM.2013 of cultivation is seeded to fermention medium within its vegetative period, by Sinorhizobium meliloti TIB.SM.2013, fermention medium is fermented thus.Preferably, Sinorhizobium meliloti TIB.SM.2013 is available from Solid agar culture bacterial classification.Also preferably, the Sinorhizobium meliloti TIB.SM.2013 of cultivation is seeded to fermention medium by the inoculum size (V/V) of 5-15%.Further preferably, the cultivation of step (a) is carried out at least 5 days about 20-32 DEG C, preferably about 25-30 DEG C, such as about 5-10 days, preferably about 5-8 days, more preferably 5-6 days. the cultivation of step (b) is carried out at least 5 days about 20-32 DEG C, preferably about 25-30 DEG C, such as about 7-20 days, preferably about 8-15 days, more preferably from about 10-12 days.Closer, cultivate and carry out on shaking table.Still further, the rotating speed of described shaking table is set to about 150-220 rpm.By the method, vitamins B in fermented liquid 12concentration be at least 50 mg/L, be preferably at least 70 mg/L, be more preferably at least 80 mg/L, be also more preferably at least 100 mg/L, be preferably at least 120 mg/L further, 140 mg/L, 160 mg/L or 180 mg/L.
More specifically vitamins B 12preparation method comprise the steps:
(1) Sinorhizobium meliloti slant strains is inoculated into is equipped with in the triangular flask of substratum, under 20-32 DEG C, preferably 28-32 DEG C of shaking table 150-220 rpm oscillating condition, cultivate at least 1 day, such as about 1-10 days, preferably about 1-5 days, more preferably 1-2 days, preparation seed liquor;
(2) seed liquor is seeded to fermention medium by the inoculum size (V/V) of 5-20%, the liquid amount of fermention medium in 250 ml triangular flasks is 30-60 ml, then cultivate at least 5 days at 25-32 DEG C, preferably 28-30 DEG C of shaking table 150-220 rpm, such as about 6-15 days, preferably about 7-12 days, more preferably from about 9-12 days, obtains Sinorhizobium meliloti fermented liquid;
(3) after fermentation ends, 8% sodium nitrite solution and Glacial acetic acid 1:1(V/V is added) in fermented liquid, boiling water bath 20-30 min discharges the product in thalline and is converted into cyanocobalamin, and centrifugal 10 min of 10000-12000 rpm collect supernatant liquor separation detection vitamins B 12;
Consisting of of described liquid seed culture medium: trimethyl-glycine 0.1-10 g/L(preferred 1-6 g/L), the preferred 15-30 g/L of corn steep liquor 10-50 g/L(), K 2hPO 40.5-3 g/L(1-2 g/L), the preferred 50-80 g/L of sucrose 10-100 g/L(), MgSO 4the preferred 1-2 g/L of 1-4 g/L(), all the other are water, pH=6.8-7.2, and in 250 ml triangular flasks, liquid amount is 30-60 ml.
Further, described Sinorhizobium meliloti TIB.SM.2013 seed culture medium is preferably 5 days.Consisting of of described fermention medium: carbon source 10-80 g/L (preferred 10-70 g/L), nitrogenous source 5-70g/L (preferred 5-30 g/L), inorganic salt 1.0-8 g/L (preferred 1.5-5 g/L), all the other are water.Further, the carbon source of described fermention medium can include but not limited to following one or more: sucrose, maltose, lactose, trimethyl-glycine, fructose, sorbyl alcohol; Be preferably: sucrose 10-80 g/L, trimethyl-glycine 0.1-10 g/L, maltose 10-30 g/L, lactose 20-40 g/L, fructose 10-80 g/L, sorbyl alcohol 2-40 g/L.
Described fermention medium nitrogenous source can include but not limited to following one or more: the leaching of corn steep liquor, yeast extract, yeast powder, yeast extract, yeast powder, yeast autolysis powder, corn starch; Corn steep liquor 10-80 g/L, corn starch 10-60 g/L, yeast autolysis powder 20-80 g/L.
The inorganic salt of described fermention medium comprise: MgSO 4, K 2hPO 4, ZnSO 4, MnSO 4, CoCl 2, DMB(5,6-dimethylbenzimidazole).Closer, described fermention medium is preferably:
Sucrose 20-80 g/L, trimethyl-glycine 0.1-10 g/L, corn steep liquor 10-50 g/L;
K 2HPO 40.5-3 g/L、MgSO 4 0.1-2 g/L、ZnSO 4 0.2-1 g/L；
MnSO 40.1-3 g/L, CoCl 20.3-5 g/L, DMB 0.01-2 g/L, all the other are water.
The inventive method is utilized to produce vitamins B 12, vitamins B in fermented liquid 12content be at least 30 mg/L, be preferably at least 70 mg/L, be more preferably at least 100 mg/L, be preferably at least 120 mg/L, 130 mg/L, 140 mg/L, 160 mg/L or 180 mg/L further.In order to reduce vitamins B 12loss in conversion process, can add sodium nitrite solution in fermented liquid increases transformation efficiency.In described fermented liquid, solid-liquid separating method can be filtration or centrifugal.
The invention still further relates to the composition comprising Sinorhizobium meliloti and fermented liquid, described fermented liquid comprises vitamins B 12, wherein vitamins B 12concentration in described fermented liquid is at least 30 mg/L, is preferably at least 70 mg/L, is more preferably at least 100 mg/L, is preferably at least 120 mg/L, 130 mg/L, 140 mg/L, 160 mg/L or 180 mg/L further.Described Sinorhizobium meliloti is Sinorhizobium meliloti TIB.SM.2013 preserving number CGMCC No. 9638 of the present invention.
The positively effect that the present invention is compared with prior art had is:
Compared with current microbial fermentation processes, the simple easy handling of this fermentative production mode; Bacterial strain is easy to cultivate, and raw material sources are extensive, and composition is simple, cheap and easy to get, does not have seasonal restriction; Product stability is good, is easy to shipping storage, and optimization culture based component and culture condition improve fermentation yield, make up current vitamins B 12output is too low, the deficiency that synthetic process cost is too high, can make vitamins B 12be widely used in the fields such as food, dyestuff, makeup.
accompanying drawing explanation
Fig. 1 is colonial morphology and the fermented liquid of Sinorhizobium meliloti TIB.SM.2013; Wherein a is Sinorhizobium meliloti form and size; B is fermented liquid;
Fig. 2 is Sinorhizobium meliloti growth tendency figure;
Fig. 3 is vitamins B 12change of production trend map.
embodiment
Following examples of the present invention are only used for explanation and realize the specific embodiment of the present invention, and these embodiments can not be interpreted as it is limitation of the present invention.Other any do not deviate from do under spirit of the present invention and principle change, modification, substitute, combine, simplify, be all considered as the substitute mode of equivalence, drop within protection scope of the present invention.
The experimental technique used in following embodiment, as without particular requirement, is ordinary method.
The material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1:
Root nodule bacterium synthesise vitamins B 12fermentation:
Liquid seed culture medium: trimethyl-glycine (Tianjin Science and Technology Ltd. of Silan) 2 g/L, corn steep liquor (Tianjin Guan Qian chemical industry sales department) 20 g/L, K 2hPO 4(Tianjin recovery chemical reagent) 1.65 g/L, sucrose (traditional Chinese medicines control interest Tianjin company limited) 60 g/L, MgSO 4(Tianjin recovery chemical reagent) 1.6 g/L, all the other are water, pH=6.8-7.2, and in 110 DEG C of sterilizing 15 min, in 250 ml triangular flasks, liquid amount is 30 ml.
Fermention medium: sucrose (traditional Chinese medicines control interest Tianjin company limited) 60 g/L, trimethyl-glycine (Tianjin Science and Technology Ltd. of Silan) 2 g/L, corn steep liquor (Tianjin Guan Qian chemical industry sales department) 20 g/L, K 2hPO 4(Tianjin recovery chemical reagent) 1.65 g/L, MgSO 4(Tianjin recovery chemical reagent) 1.6 g/L, ZnSO 4(Tianjin recovery chemical reagent) 0.8 g/L, MnSO 4(Tianjin recovery chemical reagent) 0.8 g/L, CoCl 2(Tianjin recovery chemical reagent) 0.6 g/L, DMB(traditional Chinese medicines are controlled interest Tianjin company limited) 0.05 g/L, all the other are water.In 110 DEG C of sterilizing 15 min, in 250 ml triangular flasks, liquid amount is 30 ml.
About 1 cm of picking from bacterial classification solid medium is inoculated in every bottle of liquid seed culture medium 2the Sinorhizobium meliloti TIB.SM.2013 lawn of size, cultivates 36 h at 30 DEG C of shaking table 200 rpm, obtains seed liquor.By seed liquor by volume 10% inoculum size access fermention medium, 30 DEG C of shaking table 200 rpm cultivate 8 days, obtain Sinorhizobium meliloti TIB.SM.2013 preserving number CGMCC No. 9638, thalline fermented liquid, vitamins B 12accumulate in thalline and supernatant liquor.
Embodiment 2:
Vitamins B 12extraction: add in thalline fermented liquid 10 ml 8% Sodium Nitrite and acetic acid each 2.5 ml mixing, boiling water bath 30 min, endobacillary vitamins B 12in born of the same parents, transfer to extracellular and be converted into cyanocobalamin, obtaining containing vitamins B 12mixed solution.
Embodiment 3:
High performance liquid chromatography detects vitamins B 12content, concrete grammar is as follows:
Vitamins B 12the pre-treatment of mixed solution: the vitamins B obtained will be processed by embodiment 2 12mixed solution is standing or centrifugal, and get supernatant liquor, via hole diameter is the membrane filtration of 0.22 μm, and permeate is vitamins B 12treat test sample.
The preparation of standard solution: accurately take vitamins B 12standard substance (Sigma, purity>=98%) 10 mg, are mixed with pure water the standard substance stock solution that concentration is 400 mg/L in 25 ml volumetric flasks.With pure water, stock solution is diluted to the standard solution that concentration is 40 mg/L, 80 mg/L, 120 mg/L, 160 mg/L, 200 mg/L respectively again.
Qualitative and quantitative detects: under identical chromatographic conditions, to vitamins B 12standard substance and testing sample carry out HPLC mensuration, by sample chromatogram figure and vitamins B 12standardized solution color atlas contrasts, according to vitamins B in retention time determination sample 12peak.With standard substance vitamins B 12concentration and corresponding peak area drawing standard curve, when standard substance are identical with sample feeding amount, with quantified by external standard method, calculate vitamins B in sample 12content, treat vitamins B in test sample 12concentration be vitamins B in fermented liquid 12concentration.
HPLC testing conditions: Agilent 1260 high performance liquid chromatograph, Agilent 518925-902 C18 chromatographic column (250 mm × 4.6 mm, particle diameter 5 μm); Moving phase is V(acetonitrile): V(pure water)=15:85, the pH to 3.0 of moving phase is regulated with acetic acid; Flow velocity 0.8 ml/min, column temperature 25 DEG C, sample size 15 μ L, ultraviolet detection wavelength 361 nm.
The vitamins B of every gram of dry cell weight 12the calculation formula of content:
Embodiment 4:
Different fermentations carbon source is to vitamins B 12the impact of productive rate
The carbon source of described fermention medium is replaced with glucose, maltose, sucrose, fructose, sorbyl alcohol, lactose, Semen Maydis powder respectively, carries out fermentation culture according to the method for embodiment 1, after fermentation ends, measure vitamins B in fermented liquid 12content.When sucrose is as carbon source, vitamins B 12output the highest, can be used as Sinorhizobium meliloti TIB.SM.2013 strain liquid fermentative production vitamins B 12preferred carbon source, be secondly maltose.
Embodiment 5:
Different fermentations nitrogenous source is to vitamins B 12the impact of productive rate
The nitrogenous source of described fermention medium is replaced with respectively corn steep liquor, yeast extract, yeast leaching powder, yeast extract, yeast powder, yeast autolysis powder, corn starch, extractum carnis, peptone, casein freeze, Tryptones, ammonium sulfate, volatile salt, fermentation culture is carried out according to the method for embodiment 1, when after fermentation ends, corn steep liquor is as nitrogenous source, vitamins B 12output increase substantially, can be used as Sinorhizobium meliloti TIB.SM.2013 strain liquid fermentative production vitamins B 12preferred nitrogenous source, be secondly yeast powder.
Embodiment 6:
In fermention medium, the content of carbon source and nitrogenous source is to vitamins B 12the impact of productive rate
Embodiment 7:
The optimization fermentative medium formula of Sinorhizobium meliloti TIB.SM.2013
Sinorhizobium meliloti TIB.SM.2013 is through optimizing fermention medium carbon source, the kind of nitrogenous source and the content of carbon source and nitrogenous source, and be optimized culture medium prescription, fills a prescription as follows:
Fermention medium: sucrose 60 g/L, trimethyl-glycine 2 g/L, corn steep liquor 20 g/L, K 2hPO 41.65 g/L, MgSO 41.6 g/L, ZnSO 40.8 g/L, MnSO 40.8 g/L, CoCl 20.6 g/L, DMB0.05 g/L, all the other are water.In 110 DEG C of sterilizing 15 min, in 250 ml triangular flasks, liquid amount is 30 ml.
Embodiment 8:
The mensuration of the G+C mol% content of Sinorhizobium meliloti TIB.SM.2013 genomic dna
According to prokaryotic gene group feature, genome sequencing is carried out to Sinorhizobium meliloti, pattern recognition (Base calling) is carried out to sequencing result, preliminary quality is analyzed, use two generation sequencing data statistic of attribute software NGSQCToolkit(v2.3) remove inferior quality and joint sequence etc., filtration parameter-cutOffQualScore30.
Treating processes is divided into following several step:
(1) primer and joint sequence is removed;
(2) only retain mass value higher than 30 site account for the encoding sequence of encoder block overall length more than 70%;
(3) reads that unidentified base sum accounts for coding total length more than 5% is removed;
gene sequencing data statistics
Embodiment 9
The separation of bacterial strain, screening and identification
(1) strains separation:
Sample collecting: gather peanut, red bean, mung bean, soya bean, soybean, trifolium, alfalfa growing soil sample separation root nodule bacterium.Every kind of plant collected specimens 10 parts, gets root system 200 g and soil 500 g respectively.Gathered plant root nodule bacterium surface sterilization is crushed.Pick juice, YMA Viola crystallina plate culture medium is rule, cultivate under 30 DEG C of conditions.After growing bacterial plaque, picking bacterial plaque is rule in the Congo red substratum of YMA, then from YMA Congo red substratum picking do not inhale single bacterium colony of look streak culture, in culturing process, observe the growing state of root nodule bacterium bacterium colony.Repeat aforesaid operations, until be separated to purer single bacterium colony, be stored in glycerine for subsequent use.
(2) screening of strain excellent
By cultural method in porous plate the fermentation culture of the bacterial classification after purifying according to embodiment (1), extract vitamins B according to embodiment (2) method 12, according to embodiment (3) detection method, filter out a strain vitamins B 12produce bacterial strain.
(3) qualification of bacterial strain:
BTB reaction qualification rhizobial growth speed: if YMA medium turns yellow, be then acid-producing bacteria strain, belong to fast raw root nodule bacterium; If substratum becomes blue, then bacterial strain produces alkali, belongs to Slow_growing rhizobia.
Litmus milk reaction is identical with BTB reactivity: if substratum reddens, be then acid-producing bacteria strain, belongs to fast raw root nodule bacterium; If substratum becomes blue, then bacterial strain produces alkali, belongs to Slow_growing rhizobia.
3-ketone group lactose reaction qualification edaphic bacillus and root nodule bacterium: root nodule bacterium can not utilize 3-ketone group lactose, citric acid and starch.
Extract bacterial strain DNA, according to conserved regions sequence in database, amplification 16SrDNA fragment, through identify this bacterial strain be Sinorhizobium meliloti ( sinorhizobium medicae) TIB.SM.2013.
Embodiment 10
One comprise Sinorhizobium meliloti fermented liquid, Sinorhizobium meliloti strain ( sinorhizobium medicae) composition of TIB.SM.2013, preserving number CGMCC No. 9638, wherein vitamins B 1250 mg/L, corn steep liquor 3 g/L; Sucrose 5 g/L, DMB 0.5 mg/L; S-adenosylmethionine 2 g/L.
Embodiment 11
Sinorhizobium meliloti strain ( sinorhizobium medicae) safety evaluation of TIB.SM.2013
Select healthy Kun ming white mouse 50, male and female half and half.Respectively be divided into 5 groups according to sex, often organize 5.By the vitamins B extracted 12powders in mice is disposable feeds, and dosage is 1 mg, 2 mg, 3 mg, 4 mg, and clear water is control group.Continuous Observation 14 days after feeding, observes mouse with or without intoxication conditions performance and dead generation.Through observing each Ten Female and male mice does not all produce toxic manifestations and death condition, show the vitamins B of bacterial strain TIB.SM.2013 fermented extracted 12can use safely.

Claims (10)

  1. A 1. Sinorhizobium meliloti strain ( sinorhizobium medicae) TIB.SM.2013, be separated from soil and obtain, by the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number CGMCC No. 9638.
  2. 2. Sinorhizobium meliloti strain described in claim 1 ( sinorhizobium medicae) TIB.SM.2013, preserving number CGMCC No. 9638 is at preparation biosynthesizing vitamins B 12the application of aspect.
  3. 3. application according to claim 2, it produces vitamins B in fermented liquid 12concentration be at least 50 mg/L.
  4. 4. a composition, it comprise Sinorhizobium meliloti fermented liquid, Sinorhizobium meliloti strain ( sinorhizobium medicae) TIB.SM.2013, preserving number CGMCC No. 9638.
  5. 5. Sinorhizobium meliloti strain described in an employing claim 1 ( sinorhizobium medicae) TIB.SM.2013, preserving number CGMCC No. 9638 produces vitamins B 12cultivation and extracting method, it is characterized in that being undertaken by following step:
    (1) Sinorhizobium meliloti strain slant strains is inoculated into is equipped with in the triangular flask of substratum, under 28-32 DEG C, 150-220 rpm shaking table oscillating condition, cultivate 36 h, preparation seed liquor;
    (2) seed liquor is seeded to fermention medium by the inoculum size (V/V) of 5-20%, then cultivates 6-9 days at 25-30 DEG C, obtain Sinorhizobium meliloti fermented liquid;
    (3) 8% sodium nitrite solution and Glacial acetic acid 1:1(V/V is added in the fermented liquid obtained in step (2)), boiling water bath 20-30 min discharges the product in thalline and is converted into cyanocobalamin, and centrifugal 10 min of 10000-12000 rpm collect supernatant liquor separation detection product;
    Described substratum is composed of the following components by weight:
    6-40 part carbon source, 5-30 part organic nitrogen source,
    0.5-10 part inorganic salt, add water to 1000 parts, regulate pH=6.8-7.0.
  6. 6. adopt Sinorhizobium meliloti strain to produce vitamins B according to claim 5 12cultivation and extracting method, it is characterized in that described cultivation and extracting method are for all vitamins B 12produce bacterial strain, comprise Bacillaceae, Rhodopseudomonas, rhizobium, salmonella, streptomyces, serratia, suis, X xanthomonas; Preferred bacterial strain is rhizobium, more preferably Sinorhizobium meliloti.
  7. 7. described in claim 5, adopt Sinorhizobium meliloti strain to produce vitamins B 12cultivation and extracting method, it is characterized in that described carbon source is sucrose, maltose, lactose or trimethyl-glycine.
  8. 8. described in claim 5, adopt Sinorhizobium meliloti strain to produce vitamins B 12cultivation and extracting method, it is characterized in that described organic nitrogen source be corn steep liquor, yeast extract, yeast leaching powder, yeast extract, yeast powder, yeast autolysis powder or corn starch.
  9. 9. adopt Sinorhizobium meliloti strain to produce vitamins B according to claim 5 12cultivation and extracting method, it is characterized in that described inorganic salt are made up of following raw material:
    Magnesium sulfate heptahydrate 0.1-2 g/L, dipotassium hydrogen phosphate 0.5-3 g/L, zinc sulfate 0.2-1 g/L, manganous sulfate 0.1-3 g/L, cobalt chloride 0.3-5 g/L, 5,6-dimethylbenzimidazole 0.01-2 g/L.
  10. 10. Sinorhizobium meliloti strain described in claim 1 ( sinorhizobium medicae) TIB.SM.2013, preserving number CGMCC No. 9638 is at preparation biosynthesizing vitamins B 12the application of aspect.
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CN106754565A (en) * 2017-02-15 2017-05-31 黑龙江省科学院生物肥料研究中心 The method for improving the alfalfa Phylloxera microbial inoculum term of validity
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CN113621803A (en) * 2021-06-28 2021-11-09 中山大学 Method for separating lanthanum and neodymium from ionic rare earth tailings by bioleaching
CN113905999A (en) * 2019-06-05 2022-01-07 雅拉英国有限公司 Chemical composition for seed treatment

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CN105638219A (en) * 2016-01-28 2016-06-08 山西省农业科学院高寒区作物研究所 Cultivation method for increasing yield of alfalfa by means of biological inoculation agent
CN105950520A (en) * 2016-07-18 2016-09-21 武汉市农业科学技术研究院作物科学研究所 Rhizobium capable of efficiently solubilizing phosphorus and application of rhizobium
CN105950520B (en) * 2016-07-18 2019-05-10 武汉市农业科学技术研究院作物科学研究所 A kind of rhizobium of efficient phosphate-solubilizing and its application
CN106636258A (en) * 2016-12-20 2017-05-10 安徽省新康饲料有限公司 Rice straw fermentation culture medium for generating vitamin B12 by rumen fluid, and method and application thereof
CN106636258B (en) * 2016-12-20 2018-06-01 安徽省新康饲料有限公司 Vitamin B is produced using rumen fluid12Rice straw fermented culture medium and its methods and applications
CN106754565A (en) * 2017-02-15 2017-05-31 黑龙江省科学院生物肥料研究中心 The method for improving the alfalfa Phylloxera microbial inoculum term of validity
CN113905999A (en) * 2019-06-05 2022-01-07 雅拉英国有限公司 Chemical composition for seed treatment
CN110950938A (en) * 2019-12-23 2020-04-03 中国科学院天津工业生物技术研究所 Bi-component regulation and control system and application thereof in preparing vitamin B12In (1)
CN111041020A (en) * 2019-12-23 2020-04-21 中国科学院天津工业生物技术研究所 Isocitrate lyase mutant, mutant gene and application thereof in preparation of vitamin B12In (1)
CN111057133A (en) * 2019-12-23 2020-04-24 中国科学院天津工业生物技术研究所 Response regulator and its mutant and its application in preparing vitamin B12In (1)
CN111041020B (en) * 2019-12-23 2021-09-07 中国科学院天津工业生物技术研究所 Isocitrate lyase mutant, mutant gene and application thereof in preparation of vitamin B12In (1)
CN110950938B (en) * 2019-12-23 2021-09-28 中国科学院天津工业生物技术研究所 Bi-component regulation and control system and application thereof in preparing vitamin B12In (1)
CN111057133B (en) * 2019-12-23 2021-09-28 中国科学院天津工业生物技术研究所 Response regulator and its mutant and its application in preparing vitamin B12In (1)
CN110819605A (en) * 2020-01-07 2020-02-21 中国科学院天津工业生物技术研究所 Methionine synthetase mutant, mutant gene and application thereof in preparation of vitamin B12In (1)
CN110804598B (en) * 2020-01-07 2020-04-07 中国科学院天津工业生物技术研究所 Precorrin-2C (20) -methyltransferase mutant, mutant gene and application thereof in preparing vitamin B12In (1)
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CN113621803A (en) * 2021-06-28 2021-11-09 中山大学 Method for separating lanthanum and neodymium from ionic rare earth tailings by bioleaching

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