CN110373349B - Vitamin B production12Strain of (5) and use thereof - Google Patents

Abstract

The invention discloses a method for producing vitamin B₁₂The strain of (1) has a accession number of GDMCC No. 60674. The invention also discloses the vitamin B production₁₂The strain GDMCC No.60674 is used for producing the vitamin B₁₂The use of (1). The vitamin B is produced₁₂The strain GDMCC No.60674 can be used for producing vitamin B by fermentation through liquid stationary culture₁₂And the strain belongs to the genus Lysinibacillus, and no Lysinibacillus produces vitamin B at present₁₂Therefore, the strain GDMCC No.60674 has good industrial application value.

Description

Vitamin B production12Strain of (5) and use thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a vitamin B production method₁₂And its application.

Background

Vitamin B₁₂Also known as cobalamin (cobalamin), a generic name for a class of corrins containing cobalt, hydroxycobalamin (hydroxocobalamin), cyanocobalamin (cyanocobalamin), deoxyadenosylcobalamin (deoxyadenosylcobalamin) and methylcobalamin (methylcobalamin) are vitamins B₁₂Is mainly present. Vitamin B₁₂Is the most complex small molecular substance biosynthesized in nature, is involved in human cell metabolism, influences the synthesis and regulation of DNA, and is also involved in the synthesis of fatty acid and the generation of energy.

Since higher animals and plants cannot synthesize vitamin B by themselves₁₂Although eschen moser et al completed vitamin B in 1973₁₂(eschen moser a. organic natural stone synthesis of heutevitamine B)₁₂als Beispiel[J]Naturwissenschaften,1974,61(12):513-₁₂Almost exclusively by means of fermentation by microorganisms. At present, vitamin B is industrially produced by fermentation₁₂Mainly uses the anaerobic fermentation of P.freudenreichii and P.schermanii, and uses the aerobic fermentation of P.denitrificans to make vitamin B₁₂Industrial production of (SCOTT A. discovery nature's reverse pathways tovitamin B)₁₂:a 35-year odyssey[J]J Org Chem,2003,68(7): 2529) -2539). For example: aerobic synthesis of vitamin B for pseudomonas denitrificans₁₂The strain is transformed by means of genetic engineering, so that the final yield of the strain in a 50L fermentation tank reaches 239.7mg/L (Schhulin. Pseudomonas denitrificans genetic engineering bacteria construction and vitamin B)₁₂Optimization of fermentation Process [ D].2016.). For another example: separating the thallus cells of Propionibacterium freudenreichii by membrane separation technology, concentrating the fermentation liquid by 4 times, and making the conversion rate of intermediate Abo-cbi reach 78.8%, vitamin B₁₂The yield was 53.9mgL (Xuguxia, Du river bend, King's self-strength, et al, cell membrane separation Propionibacterium freudenreichii ex-situ conversion synthesis of vitamin B₁₂[J]Proceedings of process engineering, 2016(2).

The genus Lysinibacillus currently comprises 16 described species: bronitoleram, l.sphaericus, l.fusiformis, l.paravironic piens, l.xylaniliticus, l.masseliensis, l.odyse, l.sindbriensis, l.macroroides, l.mangiferihumi, l.meyeri, l.contineans, l.chunkkukjangi, l.manganicus, l.pakistaniensis, l.tabacifolii.

Cells of the genus Lysinibacillus are in the form of straight rods, often arranged in pairs or in chains, with rounded or square ends. Cell staining was mostly gram positive in young cultures with peritrichotic flagellar motility. Each cell produces a spore, and can adapt to a plurality of adverse environments. Aerobic or facultative anaerobic. Chemoheterotrophic bacteria of the fermentative or respiratory metabolic type. Species of the genus Lysinibacillus are isolated from various environments such as soil, human cerebrospinal fluid, spacecraft surfaces, tidal flat sediments, and the like. All strains of the genus have the common characteristic of being capable of producing spores and moving rods. The polar lipid component of the strain of Lysinibacillus genus is Diphosphatidylglycerol (DPG), Phosphatidylglycerol (PG), Phosphatidylethanolamine (PE) and Phospholipidic (PL). The research on the biodegradation of some strains of Lysinibacillus is reported in China, and the research shows that the strains have certain effect of degrading pollutants such as cresol, sodium sulfide and the like. However, in the prior art, there is no report on the production of vitamin B by Lysinibacillus₁₂The report of (1).

Disclosure of Invention

In the research and development process of long-term fermented food, the inventor separates microorganisms in the fermented food and screens a strain producing vitamin B from a separated strain₁₂The strain of (4) and deposited under the accession number GDMCCNo.60674. Meanwhile, the strain is determined to be a Lysinibacillus genus through molecular biological identification.

Accordingly, in a first aspect of the invention, there is provided a method of producing vitamin B₁₂The strain of (1) under accession number GDMCCNo.60674.

In a second aspect of the invention, said vitamin B is provided₁₂The strain GDMCC No.60674 is used for producing vitamin B by fermentation₁₂The use of (1).

The invention has the beneficial effects that: the invention provides a vitamin B-producing agent₁₂The strain GDMCC No.60674 has stable genetic characters. The strain GDMCC No.60674 is cultured by liquid standing, and vitamin B in the fermentation liquid₁₂The content of the (B) can reach 2.07 mu g/100 ml. The strain belongs to the genus Lysinibacillus, but no Lysinibacillus produces vitamin B at present₁₂Thus, the present inventors found that the genus Lysinibacillus has vitamin B-producing ability₁₂The method has potential industrial application value.

Drawings

FIG. 1 shows vitamin B₁₂And (5) qualitative experiment result chart.

FIG. 2 shows vitamin B₁₂The content of (3) is measured.

FIG. 3 shows the determination of vitamin B by enzyme-linked immunosorbent assay₁₂Experimental result chart of contents.

FIG. 4 is a phylogenetic tree of isolates and related strains.

Detailed Description

The present invention will be further described with reference to the following examples. It is to be understood that the experimental procedures in the following examples, which are not subject to specific conditions, are generally carried out according to conventional conditions, or conditions provided by the manufacturers.

The invention separates the microorganism in the stinky tofu milk and utilizes the established vitamin B₁₂Qualitative screening method for screening isolate having vitamin B production₁₂Competent isolates. Through molecular biological identification, isolates mainly accumulated in the genus Lysinibacillus. The enzyme-linked immunosorbent assay is utilized to verify that the isolate has vitamin B production₁₂The ability of the cell to perform. At the same time, the vitamin B is produced₁₂The strain C5-1 is subjected to passage experiments, and the strain is proved to have stable vitamin B production₁₂The strain C5-1 is deposited with the deposition number of GDMCC No. 60674.

1. Test apparatus, test reagents and strains used in the following examples

(1) Test apparatus

GI6-2 electrothermal constant temperature incubator, Seven Easy acidimeter, TECAN M200 full-wavelength multifunctional microplate analysis system, Merck Millipore ultrapure water machine, Thermo centrifuge, 1200 ultrahigh pressure liquid chromatograph, 6410B triple quadrupole mass spectrometer, C₁₈A chromatographic column, an ME215P electronic balance, a PHB-4pH meter and an HC-2517 high-speed centrifuge.

(2) Test reagent

Vitamin B₁₂(Vitamin B₁₂Or Cyanocobalamin) standard C₆₃H₈₈CoN₁₄O₁₄The purity of P is more than or equal to 99 percent; lactobacillus agar medium (Difco); lactobacillus broth (Difco); vitamin B₁₂Assay medium (Difco); MRS solid culture medium (land bridge), MRS liquid culture medium (land bridge),

FAST Vitamin B₁₂vitamin B₁₂Rapid test kit (Baifa).

(3) Bacterial strains

Lactobacillus gasseri leishmania: lactobacillus leichmannii ATCC 7830;

lactobacillus plantarum: lactobacillus plantarum ATCC 8014;

lactobacillus rhamnosus: lactobacillus casei spp. rhamnosus ATCC 7469;

lactobacillus reuteri: lactobacillus reuteri ATCC 23272.

EXAMPLE 1 Primary screening of isolates

(1) Obtaining of isolates

And (3) performing 10-time gradient series coefficient on the fermented bean curd sample, pouring 100 mu L of diluent into an MRS plate, coating the MRS plate, drying the plate, culturing for 3 days at 36 ℃, obtaining bacterial colonies with different morphological characteristics on the plate, selecting and transferring the isolates with different morphologies from the plates into an MRS liquid culture medium, marking, and culturing for 48 hours at 36 ℃.

(2) Qualitative preliminary screening of isolates

Taking out of each labeled test tubeMixing the strain-divided fermentation liquid with distilled water at a ratio of 1:9 (i.e. adding 9mL of distilled water into 1mL of strain fermentation liquid, mixing well), and adding 4mL of distilled water and 5mL of vitamin B into 1mL of diluent₁₂Test broth, sterilized at 121 ℃ for 5 min. Sterilizing to crack thallus, and adding vitamin B₁₂After lysis of the bacteria, vitamin B₁₂The cells are dissolved out. Inoculating to vitamin B₁₂Sensitive strain L.leichmannii ATCC 7830, cultured at 36 ℃ for 24 h. At the same time can accumulate vitamin B₁₂As a positive control, with no vitamin B production, as well as L.reuteri ATCC 23272₁₂The strain (such as Lactobacillus plantarum ATCC 8014) was used as a negative control, and a non-inoculated culture medium was used as a blank control. If L.leichmanii ATCC 7830 growth indicates vitamin B₁₂The qualitative result is positive, which indicates that the strain possibly produces vitamin B₁₂And used as the analytical strain for the next test. Vitamin B₁₂The qualitative test can be used as the preliminary judgment of whether the isolate produces vitamin B₁₂A simple and convenient method of (1). This method is feasible for use in primary screening of sample isolates. The results are shown in FIG. 1. Wherein, test tube number 1: blank control; 2: reuteri ATCC 23272 positive control; 3: plantarumatcc 8014 negative control; 4-19 are isolates.

As can be seen from FIG. 1, the positive control tube 2 is turbid, the negative control tube 3 is clear, and the turbidity of the test tubes (4-19) of the isolate refers to the turbidity of the positive and negative tubes, which indicates that the isolate may produce vitamin B₁₂And the strain is marked as positive, and the strain can be identified and other follow-up researches.

Isolate was initially screened by visual inspection of the turbidity of the test tube, where the turbidity was higher in the test tube: strains C5-1, C5-2, C5-8 and C7-1, etc., which are presumed to have vitamin B producing ability₁₂Capability.

Wherein, the strain numbers of C5-1, C5-2, C5-8, C7-1 and the like correspond to the sample numbers of the strains, such as the strain C5-1, and are derived from the first colony separated from the sample C5; c5-2, derived from a second colony isolated in sample C5.

Example 2 isolated plant-produced vitaminsElement B₁₂Verification and content determination of

Due to vitamin B₁₂The qualitative screening and the content determination are both established in the determination of vitamin B by a microbiological method₁₂Based on the above, for the stability and reliability of results, the invention adopts multiple methods to participate in the production of vitamin B by the strain together₁₂And (5) verifying the capability.

(1) Microbiological method verification

Selecting vitamin B₁₂Qualitative result in the test tube of higher turbidity isolates C5-1, C5-2, C5-8, C7-1 and negative strains C8-2, C8-2 and L.plantarum ATCC 8014, the determination of vitamin B in fermentation liquor by microbiological method₁₂And (4) content. The method comprises the following specific steps:

isolates were inoculated into 10mL MRS broth and cultured for 5 days at 36 ℃. Adding 10mL vitamin B into 5mL fermentation liquid₁₂The pretreatment solutions (1.3 g of anhydrous disodium hydrogen phosphate, 1.0g of anhydrous sodium metabisulfite, 1.2g of citric acid, dissolved in 100mL of water) were mixed, then 150mL of water was added, hydrolysis was carried out at 121 ℃ for 10min, and after cooling, the pH was adjusted to 4.5. + -. 0.2. Then the volume is adjusted to 250mL by water and the mixture is filtered. L.leichmannii ATCC 7830 strain fermentation liquor is subjected to vitamin B₁₂And (4) measuring the content. Meanwhile, lactobacillus plantarum ATCC 8014 is used as a negative control, and lactobacillus reuteri ATCC 23272 is used as a positive control. The results are shown in FIG. 2.

The results show that: the isolated strain numbers C5-1, C5-2, C5-8 and C7-1 fermentation liquor produce vitamin B₁₂The content of (B) is 2.07. mu.g/100 ml, 1.90. mu.g/100 ml, 1.98. mu.g/100 ml and 1.79. mu.g/100 ml respectively, and the content of vitamin B is more than that of the positive strain L.reuteriATCC 23272₁₂The content is slightly higher. Meanwhile, the strains C8-2 and C8-2 act as vitamin B₁₂The negative strain of the qualitative experiment can not detect the vitamin B₁₂Vitamin B was not detected in the negative strain Lactobacillus plantarum L.plantarum ATCC 8014 either₁₂。

And (4) conclusion: vitamin B₁₂The results of the qualitative screening experiment are consistent with the results of the content determination, and the isolate can be metabolized to generate stable vitamin B after the result culture₁₂And yields were close to that of the positive strain l.reuteri ATCC 23272.

(2) Enzyme linked immunosorbent assay verification

Enzyme linked immunosorbent assay for vitamin B₁₂Qualitative results in the fermentation of the isolates C5-1, C5-8, C7-1 and L.reuteri ATCC 23272 with higher turbidity in the test tube were subjected to vitamin B₁₂The results of the content measurement are shown in FIG. 3 and Table 1.

TABLE 1 enzyme-linked immunosorbent assay for vitamin B₁₂Content (wt.)

Note: l15 is the strain number of l.reuteri ATCC 23272.

By using

FAST Vitamin B₁₂Vitamin B₁₂Rapid detection kit

The principle of the kit is as follows: vitamin B is coated in the microporous plate₁₂The specific antibody of (1). Adding vitamin B₁₂Standard substance and enzyme-labeled vitamin B₁₂(enzyme linker). Free and enzyme-labeled vitamin B₁₂Competitive binding of vitamin B₁₂Antibody binding sites (competitive enzyme linked immunosorbent assay). Unbound enzyme-labeled vitamin B₁₂Are removed in a subsequent washing step. A substrate/chromogen is added to the wells and the bound enzyme conjugate converts the chromogen to a blue color. The color changed from blue to yellow upon addition of the reaction stop solution. Absorbance value measured at 450nm with vitamin B in the sample₁₂Is inversely proportional.

Vitamin B according to the operation flow of the kit₁₂The standard substance concentration is in the range of 0.5-30.0. mu.g/l, the linearity is good, and R is²0.9978. Vitamin B in fermentation broths of strains C5-1, C5-8, C7-1 and L.reuteri ATCC 23272₁₂The results of the content detection were 7.54. mu.g/L, 6.39. mu.g/L, 7.37. mu.g/L and 11.72. mu.g/L, respectively. The conversion to each 100ml score is respectively: 0.754. mu.g/100 ml, 0.639. mu.g/100 ml, 0.737. mu.g/100 ml and 1.172. mu.g/100 ml. (Note: fermentation of the strains by enzyme-Linked immunosorbent assayLiquid vitamin B₁₂The result is slightly lower than that measured microbiologically, probably because it is the vitamin B that can be utilized by the microorganism that is measured microbiologically₁₂The enzyme linked immunosorbent assay is used for quantitatively determining vitamin B in food by competition₁₂So that it is possible that the presence of metabolic products of the strain in the fermentation broth inhibits the binding of antigen and antibody in the enzyme-linked immunosorbent assay to some extent resulting in vitamin B₁₂The final result is slightly lower).

And (4) conclusion: vitamin B is present in the products metabolically synthesized by strains C5-1, C5-8 and C7-1₁₂。

Example 3 extraction and amplification of DNA

The genomic DNA of isolates C5-1, C5-8 and C7-1 verified in example 2 was subjected to PCR amplification using bacterial universal primers 27f and 1492r, the primer sequences of which are shown in Table 2, to obtain a fragment of 1.5kb in size. The method comprises the following specific steps:

1. extraction of DNA

1) Centrifuging 2-5mL of bacterial liquid at 12000rpm for 5min, and removing the supernatant;

2) adding lysozyme (10mg/mL) into the precipitate, 400 mu L, and carrying out water bath at 37 ℃ for 1.5 h;

3) adding 400 mu L CTAB and 20 mu L proteinase K, and carrying out water bath at 56 ℃ for 2 h;

4) after the water bath, Tris-phenol: isoamyl alcohol: mixing chloroform 25:24:1, 800 μ L, centrifuging at 12000rpm for 10 min;

5) the supernatant (about 400. mu.L) was removed and an equal volume of isoamyl alcohol: mixing chloroform at a ratio of 24:1, and centrifuging at 12000rpm for 10 min;

6) collecting supernatant (about 600 μ L), adding 2 times volume of anhydrous ice ethanol, and standing in refrigerator at-20 deg.C for 20-30 min;

7) centrifuging at 12000rpm for 5min, and discarding the supernatant;

8) washing the precipitate with 75% ethanol for 1-2 times, and drying in a fume hood;

9) the dried pellet was added 50. mu.L of 0.1 XTE (10mM Tris-HCl, 1mM EDTA; pH 8.0) water, and storing at-20 ℃.

2. Determination of DNA content

For extractedDNA content and purity were determined as A₂₆₀/A₂₈₀This indicates that the extracted DNA satisfies the following experimental requirements (generally, A is considered to be₂₆₀/A₂₈₀1.8-2.0, the extracted DNA template was of better quality). The concentration of the DNA template in each sample was also diluted so that the final concentration was 50 ng/. mu.L.

3. The primer of the present invention was synthesized by Shanghai Junjun Biotechnology services Co., Ltd, and the sequence of the primer is shown in Table 2.

TABLE 2 primer sequences

4. PCR amplification system and conditions

25 μ L PCR System: 2.5. mu.L of 10 XBuffer, 0.15. mu.L of Taq enzyme (2.5U/. mu.L), 1.5. mu.L of MgCl₂(25mmol/L), 1. mu.L dNTP (20mmol/L), 0.5. mu.L forward primer (10. mu. mol/L), 0.5. mu.L reverse primer (10. mu. mol/L), 17.85. mu.L re-distilled water, 1. mu.L template DNA. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, and 34 cycles; extension at 72 ℃ for 10 min. Negative control was prepared using 1. mu.L of sterile distilled water as template. The PCR product was obtained from Shanghai Yingjun Biotech Co., Ltd.

Example 4 construction of phylogenetic Tree of isolates

The PCR amplification products were manually corrected for DNA sequence using BioEdit software, the corrected sequences were subjected to sequence determination, and the sequences were compared for homology (BLAST) on NCBI website (http:// www.ncbi.nlm.nih.gov).

The sequence of each strain is input into NCBI gene library, and the similarity comparison analysis is carried out on the isolate and the 16SrRNA sequence in the database by using Blast program, so as to construct phylogenetic tree, as shown in figure 3. The construction process of the phylogenetic tree comprises the following steps: and selecting the species with closer relationship as the inner group, and selecting the species with farther relationship as the outer group. Then, the sequences of the test strain and the reference model strain were subjected to matching analysis (Alignment) using MEGA 6.0, the phylogenetic tree was obtained by using neighbor-joining method and the evolutionary distance thereof was calculated according to Kimural's two-parameter model, and the computational analysis of Bootstrap value was performed according to 1000 random sampling.

From a phylogenetic tree, the isolates C5-1, C5-2, C5-8 and C7-1 have higher similarity with the genus Lysinibacillus, but cannot be distinguished from Lysinibacillus fusiformis (Lysinibacillus cereus) and Lysinibacillus sphaericus (Lysinibacillus sphaericus) of the genus by 16S rRNA, and probably the two strains have high homology and need to be classified and distinguished by other conserved regions, and which strain belongs to the genus cannot be specifically distinguished.

And (4) conclusion: strains C5-1, C5-2, C5-8 and C7-1 are of the genus Lysinibacillus.

Example 5 passage stability test

In order to study the genetic stability of the strain, the selected strain C5-1 was inoculated once at regular intervals as the next generation strain, and vitamin B production by the strains of different generations was examined₁₂The ability of the cell to perform. Inoculating the strain C5-1 into 5mL of MRS culture medium from a slant, culturing at 36 deg.C for 48h, and determining vitamin B in the fermentation broth by using microbiological method₁₂The above steps were repeated until the 5 th generation was determined. The results are shown in Table 3.

TABLE 3 passage stability test

The results show that the strain passes 5 times of passages and vitamin B₁₂The average value of the content of (B) is 1.56 ug/100mL, the maximum value is 1.60ug/100mL, the minimum value is 1.49ug/100mL, and vitamin B is produced₁₂The ability of (c) did not change significantly with increasing number of passages.

And (4) conclusion: the strain C5-1 has good passage stability.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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Claims (2)

1. Vitamin B production₁₂The strain (Lysinibacillus sp.) of (1), wherein said vitamin B is produced₁₂The strain of (2) is deposited under the accession number GDMCC No. 60674.

2. Vitamin B-producing formulation as claimed in claim 1₁₂The strain GDMCC No.60674 for producing vitamin B₁₂The use of (1).

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