CN114015607B - Bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid and application thereof - Google Patents
Bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid and application thereof, and relates to the technical field of microorganisms. The Bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid is named as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BOEN-305, which is preserved in Guangdong province collection center of microorganism strains at 11 months and 1 year 2021, and has the address: the microbial research institute of Guangdong province, no. 59 building, no. 5 building, of the university institute of Mieli Zhonglu, guangzhou, with the accession number GDMCC No.62024. The strain is derived from the soil of a vegetable planting base and belongs to natural strains, and the secreted 5-methyltetrahydrofolic acid is naturally synthesized, so that no side effect is generated, and the safety is high; and the bacillus amyloliquefaciens has good genetic stability and strong stress resistance, can resist damage in the production and transportation process, and has important industrial utilization value.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid and application thereof.
Background
Folic acid is used as a necessary vitamin in the growth process of a human body, the market application prospect is wide, folic acid in the market is mainly synthesized chemically, but the metabolic pathways of artificially synthesized folic acid and natural folic acid in the human body are different and have certain side effects, so that the reaction of the artificially synthesized folic acid product cannot be ignored, the folic acid generated by microbial fermentation is high in safety, is reduced and methylated before absorption, does not have side effects in a wide safe intake range, and has higher bioavailability, so that the synthesis of folic acid by using microorganisms is concerned in the research field, and more strains mainly including yeast, lactic acid bacteria and streptococcus thermophilus are researched.
5-methyltetrahydrofolic acid is the only active form of folic acid which plays a role in the body, folic acid is ubiquitous in human diet, the problem of folic acid deficiency still affects billions of people worldwide, folic acid has become a necessity as a food additive, adults recommend folic acid intake of 400 micrograms/day, and thus the market demand for folic acid is increasing. At present, the production of 5-MTHF is mainly synthesized industrially, but the problems of complicated resolution process, difficult purification and the like exist. And the precursor substance folic acid is also produced by industrial chemical synthesis, which causes pollution to the environment.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a beneficial bacterium widely used in animal breeding feed production, but no report on the application of the Bacillus amyloliquefaciens in the production of 5-methyltetrahydrofolic acid is found at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that: a bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid, which is named as: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BOEN-305, which was deposited in the Collection of microorganisms of Guangdong province on 11.1.2021, address: the microbial research institute of Guangzhou province, 5 th floor, of Mieli Zhonglu 100, guangzhou city, has a preservation number of GDMCC No.62024.
As a preferred embodiment of the Bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolate, the sequence of the Bacillus amyloliquefaciens BOEN-305 is shown as SEQ ID NO. 1.
As a preferred embodiment of the bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolate according to the invention, the bacillus amyloliquefaciens BOEN-305 is isolated from soil of a vegetable plantation site.
The invention also provides application of the bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid in preparation of products containing 5-methyltetrahydrofolic acid.
The invention also provides a bacillus amyloliquefaciens fermentation liquid which is prepared by fermenting the bacillus amyloliquefaciens with high yield of 5-methyltetrahydrofolic acid.
The invention also provides a preparation method of the bacillus amyloliquefaciens fermentation liquid, which comprises the following steps of:
(1) Preparing a seed solution: inoculating bacillus amyloliquefaciens BOEN-305 into a seed culture medium, and culturing for 14-20 h at 30-40 ℃ to obtain a seed solution;
(2) Preparing fermentation liquor: inoculating the seed solution obtained in the step (1) into a liquid fermentation culture medium, wherein the inoculation amount is 1% -7%, the culture temperature is 30-40 ℃, the air volume is 0.5-1.0 vvm, the stirring speed is 150-300 r/min, and the fermentation time is 24-48 h, so as to obtain the bacillus amyloliquefaciens fermentation liquid.
As a preferred embodiment of the method for preparing a bacillus amyloliquefaciens fermentation liquid of the present invention, the seed culture medium comprises: 3 to 10g/L of peptone, 0.2 to 1.5g/L of yeast extract, 8 to 25g/L of glucose, 0.05 to 0.5g/L of magnesium sulfate and 0.05 to 0.15g/L of ferric sulfate.
As a preferred embodiment of the method for preparing the Bacillus amyloliquefaciens fermentation liquid, the liquid fermentation medium comprises: 12 to 25g/L of molasses, 5 to 18g/L of yeast powder, 1.2 to 3.5g/L of calcium chloride, 0.1 to 1g/L of anhydrous magnesium sulfate, 0.01 to 0.5g/L of manganese sulfate, 2 to 5g/L of sodium chloride, 0.1 to 5g/L of disodium hydrogen phosphate, 0.01 to 0.5g/L of potassium dihydrogen phosphate and 0.05 to 0.2g/L of vitamin C.
As a preferred embodiment of the preparation method of the bacillus amyloliquefaciens fermentation liquid, the seed culture medium and the liquid fermentation culture medium are both prepared by distilled water; the pH value of the seed culture medium is 7.0-7.5; the pH value of the liquid fermentation medium is 7.2-7.5.
The invention also provides application of the bacillus amyloliquefaciens fermentation liquid or the bacillus amyloliquefaciens fermentation liquid prepared by the preparation method of the bacillus amyloliquefaciens fermentation liquid in preparation of products containing 5-methyltetrahydrofolic acid.
The invention has the beneficial effects that: the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BOEN-305 for high yield of 5-methyltetrahydrofolic acid can generate 5-methyltetrahydrofolic acid, and the strain is derived from soil of a vegetable planting base and belongs to natural strains, so that the secreted 5-methyltetrahydrofolic acid is naturally synthesized, no side effect is generated, and the safety is high; and the bacillus amyloliquefaciens has good genetic stability and strong stress resistance, can resist damage in the production and transportation process, and has important industrial utilization value. The invention also provides a bacillus amyloliquefaciens fermentation liquid produced by the fermentation of the bacillus amyloliquefaciens BOEN-305, and compared with other natural strains, the content of the 5-methyltetrahydrofolic acid in the bacillus amyloliquefaciens fermentation liquid is higher; and the fermentation operation of the bacillus amyloliquefaciens fermentation liquid is simple, and the industrial production is easy to realize.
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FIG. 1 shows the colony morphology of Bacillus amyloliquefaciens BOEN-305.
FIG. 2 is a gram stain of Bacillus amyloliquefaciens BOEN-305.
Detailed Description
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Example 1 deposit information of Bacillus amyloliquefaciens
The invention provides a Bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid, which is named as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BOEN-305 and is preserved in Guangdong province microorganism strain preservation center at 11/1 of 2021, with the address: the microbial research institute of Guangdong province, no. 59 building, no. 5 building, of the university institute of Mieli Zhonglu, guangzhou, with the accession number GDMCC No.62024.
Example 2 acquisition of Bacillus amyloliquefaciens BOEN-305 and identification of species
1. Obtaining the bacillus amyloliquefaciens BOEN-305: soil samples were collected from vegetable cultivation bases in Jiujiang city, jiangxi province. Weighing 5g of soil sample in a sterilized centrifuge tube, adding 5 times of sterile water, filtering to obtain filtrate, inoculating the filtrate in LB plate culture medium, and culturing at 37 ℃. Well-growing bacteria were picked and inoculated on an LB plate medium. And selecting colonies, carrying out streak culture, and repeatedly purifying for multiple times until a single colony is obtained.
2. Strain identification
(1) And (3) observing colony and thallus morphology: the strain BOEN-305 was streaked on LB plate medium and cultured at 37 ℃ for 48 hours, and then the growth of colonies on the plate was observed and recorded. The morphology of colonies of the strain of this example is shown in FIG. 1. The gram stain of the strain of this example was performed using the kit, and the results are shown in FIG. 2.
As can be seen from FIG. 1, the bacterial colony formed by the strain of this example is milky white, opaque, round, wrinkled, concave in the center, toothed in the edge, sticky and brittle, and easy to pick up. As can be seen from FIG. 2, the strains of this example are rod-shaped, single or short chain-shaped, spore-oval, subterminal, peritrichogenous, gram-positive.
(2) Physiological and biochemical characteristics: the strain BOEN-305 provided by the embodiment can grow in an environment of 4-40 ℃ and is aerobic; can tolerate 10% (wt) of sodium chloride; can hydrolyze gelatin, starch, casein, and citrate; can utilize ammonium sulfate and ammonium nitrate; fructose, glucose, mannitol and xylose can be used as carbon sources; indole reaction, V.P. reaction, methyl red test, catalase and oxidase test are positive; can produce catalase without producing hydrogen sulfide.
(3) Molecular biological identification: the bacterial whole genome rapid extraction kit is adopted to extract the whole genome of the strain BOEN-305 of the embodiment, the extracted DNA is used for 16S rDNA sequence amplification, and the amplified sequence is sequenced. The amplified sequences were aligned by BLAST and the 16S rDNA database.
The experimental result shows that the nucleotide sequence of the 16S rDNA obtained by amplifying the strain BOEN-305 in the embodiment is shown in SEQ ID NO.1, and the strain is identified as bacillus amyloliquefaciens.
By combining the colony and thallus morphology, physiological and biochemical characteristics and molecular biological identification results of the strain BOEN-305 in the embodiment, the strain BOEN-305 can be determined to be Bacillus amyloliquefaciens and named as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BOEN-305.
Example 3
The preparation method of the bacillus amyloliquefaciens fermentation liquid comprises the following steps:
(1) Preparing a seed solution: inoculating the bacillus amyloliquefaciens BOEN-305 in the embodiment 2 into a seed culture medium, and culturing at 30 ℃ for 20 hours to obtain a seed solution;
(2) Preparing fermentation liquor: and (2) inoculating the seed solution obtained in the step (1) into a liquid fermentation culture medium, wherein the inoculation amount is 7%, the culture temperature is 30 ℃, the air volume is 0.5vvm, the stirring speed is 150r/min, and the fermentation time is 48h, so that the bacillus amyloliquefaciens fermentation liquid is obtained.
The seed medium of this example comprises: 3g/L of peptone, 1.5g/L of yeast extract, 8g/L of glucose, 0.05g/L of magnesium sulfate and 0.15g/L of ferric sulfate. The seed culture medium is prepared by distilled water, and the pH value of the seed culture medium is 7.0.
The liquid fermentation medium of this example comprises: 12g/L of molasses, 5g/L of yeast powder, 1.2g/L of calcium chloride, 0.1g/L of anhydrous magnesium sulfate, 0.1g/L of manganese sulfate, 3g/L of sodium chloride, 0.1g/L of disodium hydrogen phosphate, 0.01g/L of potassium dihydrogen phosphate and 0.1g/L of vitamin C. The liquid fermentation culture medium is prepared by distilled water, and the pH value of the liquid fermentation culture medium is 7.2.
Example 4
The preparation method of the bacillus amyloliquefaciens fermentation liquid comprises the following steps:
(1) Preparing a seed solution: inoculating the bacillus amyloliquefaciens BOEN-305 in example 2 into a seed culture medium, and culturing at 35 ℃ for 18h to obtain a seed solution;
(2) Preparing fermentation liquor: and (2) inoculating the seed solution obtained in the step (1) into a liquid fermentation culture medium, wherein the inoculation amount is 5%, the culture temperature is 35 ℃, the air volume is 0.8vvm, the stirring speed is 180r/min, and the fermentation time is 37h, so that the bacillus amyloliquefaciens fermentation liquid is obtained.
The seed medium of this example comprises: 5g/L of peptone, 1g/L of yeast extract, 16g/L of glucose, 0.1g/L of magnesium sulfate and 0.1g/L of ferric sulfate. The seed culture medium is prepared by distilled water, and the pH value of the seed culture medium is 7.2.
The liquid fermentation medium of this example comprises: 15g/L of molasses, 8g/L of yeast powder, 2.5g/L of calcium chloride, 0.5g/L of anhydrous magnesium sulfate, 0.01g/L of manganese sulfate, 2g/L of sodium chloride, 2g/L of disodium hydrogen phosphate, 0.04g/L of potassium dihydrogen phosphate and 0.1g/L of vitamin C. The liquid fermentation culture medium is prepared by distilled water, and the pH value of the liquid fermentation culture medium is 7.3.
Example 5
The preparation method of the bacillus amyloliquefaciens fermentation liquid comprises the following steps:
(1) Preparing a seed solution: inoculating the bacillus amyloliquefaciens BOEN-305 in the embodiment 2 into a seed culture medium, and culturing for 14 hours at 40 ℃ to obtain a seed solution;
(2) Preparing fermentation liquor: and (2) inoculating the seed solution obtained in the step (1) into a liquid fermentation culture medium, wherein the inoculation amount is 1%, the culture temperature is 40 ℃, the air volume is 1.0vvm, the stirring speed is 300r/min, and the fermentation time is 24 hours, so that the bacillus amyloliquefaciens fermentation liquid is obtained.
The seed medium of this example comprises: 10g/L of peptone, 0.2g/L of yeast extract, 25g/L of glucose, 0.5g/L of magnesium sulfate and 0.05g/L of ferric sulfate. The seed culture medium is prepared by distilled water, and the pH value of the seed culture medium is 7.5.
The liquid fermentation medium of this example comprises: 25g/L of molasses, 18g/L of yeast powder, 3.5g/L of calcium chloride, 1g/L of anhydrous magnesium sulfate, 0.5g/L of manganese sulfate, 5g/L of sodium chloride, 5g/L of disodium hydrogen phosphate, 0.5g/L of monopotassium phosphate and 0.2g/L of vitamin C. The liquid fermentation medium is prepared by distilled water, and the pH value of the liquid fermentation medium is 7.5.
Example 6
In this example, the content of 5-methyltetrahydrofolate in the bacillus amyloliquefaciens fermentation liquid prepared by the preparation method of the bacillus amyloliquefaciens fermentation liquid of example 3, example 4 and example 5 was detected by high performance liquid chromatography-tandem mass spectrometry. The results are shown in Table 1.
TABLE 1
Example 7
This example demonstrates the genetic stability of Bacillus amyloliquefaciens BOEN-305 of example 2.
The experimental method comprises the following steps: the bacillus amyloliquefaciens BOEN-305 strain in the example 2 is cultured for 7 times of continuous passage, the capability of the bacillus amyloliquefaciens BOEN-305 for producing 5-methyltetrahydrofolate is respectively tested after each passage, and the content of the 5-methyltetrahydrofolate produced by the bacillus amyloliquefaciens BOEN-305 after passage is used as an index of the genetic stability of the strain. The results are shown in Table 2.
TABLE 2
As can be seen from Table 2, the content of the generated 5-methyltetrahydrofolate of the Bacillus amyloliquefaciens BOEN-305 strain still reaches more than 1.2ug/mL after being cultured for 7 times of continuous passage, which indicates that the strain has better genetic stability.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Yaen group stocks Co., ltd, foshan Yaen Biotechnology Co., ltd
<120> bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid and application thereof
<130> 2021.11.19
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1139
<212> DNA
<213> Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BOEN-305
<400> 1
actgaagacc acggtctcag actccgtacc ggaggcagca gtagggatct cgcaatgacg 60
gaaagtctga cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt 120
tgttagggaa gaacaagtgc cgttcaaata gggcggcacc ttgacggtac ctaaccagaa 180
agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg 240
aattattggg cgtaaagggc tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc 300
tcaaccgggg agggtcattg gaaactgggg aacttgagtg cagaagagga gagtggaatt 360
ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct 420
ctggtctgta actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct 480
ggtagtccac gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct 540
gcagctaacg cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg 600
aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga 660
accttaccag gtcttgacat cctctgacaa tcctagagat aggacgtccc cttcgggggc 720
agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 780
cgcaacgagc gcaacccttg atcttagttg ccagcattca gttgggcact ctaaggtgac 840
tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc 900
tgggctacac acgtgctaca atggacagaa caaagggcag cgaaaccgcg aggttaagcc 960
aatcccacaa atctgttctc agttcggatc gcagtctgca actcgactgc gtgaagctgg 1020
aatcgctagt aatcgcggat cagcatgccg cggtgaatac gcaccctgga tctagtgcca 1080
gcattcactg gcactcgaag gtgactgcgg tggacaaccg aggaagtgtg aggacggtc 1139
Claims (10)
1. The bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid is characterized in that the bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid is named as bacillus amyloliquefaciens（Bacillus amyloliquefaciens）BOEN-305, deposited at the Guangdong province Collection of microorganisms at 11.1.2021, address: the microbiological research institute of Guangzhou city, jifura Zhongluo No. 100, dazhou No. 59, building, guangdong province,the preservation number is GDMCC No.62024.
2. The bacillus amyloliquefaciens capable of highly producing 5-methyltetrahydrofolate according to claim 1, wherein a 16S rDNA sequence of the bacillus amyloliquefaciens BOEN-305 is shown as SEQ ID No. 1.
3. The bacillus amyloliquefaciens highly yielding 5-methyltetrahydrofolate according to claim 1, wherein the bacillus amyloliquefaciens BOEN-305 is isolated from vegetable plantation soil.
4. Use of the bacillus amyloliquefaciens producing 5-methyltetrahydrofolate according to any one of claims 1~3 in high yield in the manufacture of a product comprising 5-methyltetrahydrofolate.
5. A Bacillus amyloliquefaciens fermentation broth prepared from the Bacillus amyloliquefaciens producing 5-methyltetrahydrofolate in high yield according to any one of claims 1~3 by liquid fermentation.
6. The method of preparing a bacillus amyloliquefaciens fermentation broth of claim 5, comprising the steps of:
(1) Preparing a seed solution: inoculating bacillus amyloliquefaciens BOEN-305 into a seed culture medium, and culturing at 30-40 ℃ for 14-20h to obtain a seed solution;
(2) Preparing fermentation liquor: and (2) inoculating the seed solution obtained in the step (1) into a liquid fermentation culture medium, wherein the inoculation amount is 1% -7%, the culture temperature is 30-40 ℃, the air volume is 0.5-1.0 vvm, the stirring speed is 150-300r/min, and the fermentation time is 24-48h, so as to obtain the bacillus amyloliquefaciens fermentation liquid.
7. The method of claim 6, wherein the seed medium comprises: 3 to 10g/L of peptone, 0.2 to 1.5g/L of yeast extract, 8 to 25g/L of glucose, 0.05 to 0.5g/L of magnesium sulfate and 0.05 to 0.15g/L of ferric sulfate.
8. The method of claim 6, wherein the liquid fermentation medium comprises: 12 to 25g/L of molasses, 5 to 18g/L of yeast powder, 1.2 to 3.5g/L of calcium chloride, 0.1 to 1g/L of anhydrous magnesium sulfate, 0.01 to 0.5g/L of manganese sulfate, 2 to 5g/L of sodium chloride, 0.1 to 5g/L of disodium hydrogen phosphate, 0.01 to 0.5g/L of potassium dihydrogen phosphate and 0.05 to 0.2g/L of vitamin C.
9. The method for preparing Bacillus amyloliquefaciens fermentation liquid according to claim 6, wherein the seed culture medium and the liquid fermentation culture medium are both prepared by distilled water; the pH value of the seed culture medium is 7.0 to 7.5; the pH value of the liquid fermentation medium is 7.2 to 7.5.
10. Use of a bacillus amyloliquefaciens fermentation broth according to claim 5 for preparing a product comprising 5-methyltetrahydrofolate.
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| WO2021036348A1 (en) * | 2019-08-23 | 2021-03-04 | Chifeng Pharmaceutical Co., Ltd. | Folate producing strain and the preparation and application thereof |
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