CN114634894A - Bacillus subtilis for producing gamma-polyglutamic acid by salt-tolerant fermentation and application thereof - Google Patents
Bacillus subtilis for producing gamma-polyglutamic acid by salt-tolerant fermentation and application thereof Download PDFInfo
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Abstract
A Bacillus subtilis for realizing salt-tolerant fermentation production of gamma-polyglutamic acid and application thereof relate to the technical field of microbiology and bioengineering fermentation, and the Bacillus subtilis YZHY21.A05(Bacillus subtilis YZHY21.A05) is classified and stored in Guangdong province microorganism strain collection center, and the collection number is GDMCC NO: 62050, preservation date is 2021, 11 months and 9 days. The invention screens and obtains a bacillus subtilis for producing the gamma-polyglutamic acid by salt-tolerant fermentation from salted fish products, and the gamma-polyglutamic acid is fermented and extracted by three steps of strain activation, solid fermentation and purification extraction, so that the gamma-polyglutamic acid can be fermented, and the method can adapt to the high-salt and alkali environment in the fermentation process.
Description
Technical Field
The invention relates to the technical field of microbiology and bioengineering fermentation, in particular to a bacillus subtilis for producing gamma-polyglutamic acid by salt tolerant fermentation and application thereof.
Background
Gamma-polyglutamic acid (Gamma-PGA) is a Poly amino acid multifunctional environment-friendly biodegradable high polymer material, and the molecular weight is about 10-1000 kDa generally. The gamma-PGA has some unique physical, chemical and biological characteristics such as good water solubility, super strong adsorbability, complete natural degradation and biodegradation, no toxicity and harm to the environment, edible property and the like, can be used as raw materials of thickening agents, water retention agents, heavy metal adsorbents, flocculating agents, drug/fertilizer slow release agents, drug carriers and the like in industry, and is applied to the fields of food, cosmetics, agriculture, environmental protection, medicine, synthetic fibers, coating films and the like. With the enhancement of environmental protection consciousness, the research and application of γ -PGA have been receiving more and more attention from various academic circles around the world, and have become one of the research hotspots of biodegradable polymer materials.
At present, a microbial fermentation method is the best gamma-PGA preparation method generally adopted in industrial production, the gamma-PGA is prepared by strain screening, culture, separation and purification, the process is simple, the conditions are mild, and large-scale production can be carried out. Research shows that the increase of NaCl concentration is helpful to reduce the viscosity of the gamma-PGA, and may improve the oxygen and mass transfer rate of the fermentation system, thereby promoting the synthesis of the gamma-PGA, but the yield of the gamma-PGA is low. Since sodium ions have inhibitory effect on polyglutamic acid synthetase of strain, high concentration of sodium ions can replace calcium ions combined with membrane system of strain to cause Na+/Ca+The ratio is increased to thereby affect the γ -PGA yield.
Disclosure of Invention
The invention aims to provide a bacillus subtilis for producing gamma-polyglutamic acid by salt-tolerant fermentation and application thereof, and the bacillus subtilis is suitable for fermenting gamma-PGA in a high-salt environment while producing the gamma-PGA by high-yield fermentation.
The invention obtains a Bacillus subtilis strain for realizing salt-tolerant fermentation production of gamma-polyglutamic acid from salted fish products by screening, the Bacillus subtilis strain is classified and named as Bacillus subtilis YZHY21.A05, the Bacillus subtilis strain is preserved in Guangdong province microbial strain preservation center, and the preservation number is GDMCC NO: 62050, preservation date is 2021, 11 months and 9 days, and the address of the preservation unit is: the Michelson Dai No. 59 building 5, Ministry of sciences, Guangdong province, institute of microbiology, postal code 510070, Ministry of sciences, Michelson, Guangzhou, China.
The invention also provides application of the bacillus subtilis in producing the gamma-polyglutamic acid by salt-tolerant fermentation.
The invention also provides a technical scheme for preparing the gamma-polyglutamic acid by inoculating the bacillus subtilis YZHY21.A05 into a fermentation culture medium for fermentation.
Further, the fermentation medium is a soybean medium.
As the preferred technical scheme of the invention, the method for producing the gamma-polyglutamic acid by utilizing the bacillus subtilis to realize salt-tolerant fermentation comprises the following steps:
(1) and (3) activation of thalli: taking out the bacillus subtilis inclined plane from a refrigerated cabinet at 4 ℃, and placing the bacillus subtilis inclined plane in an incubator at 28 ℃ for activation for 2-6 hours;
(2) solid fermentation: taking the bacterial slant in the step (1), cleaning the slant lawn with 5mL of 12%, 16% and 24% of sterile saline water on each slant under the aseptic condition, inoculating the slant lawn with 1-4% of inoculum size into a soybean culture medium, and fermenting at constant temperature of 28-35 ℃ for 24-48 h;
(3) extracting gamma-polyglutamic acid: adding sterile water into a soybean culture medium after fermentation for 24-48 h, adjusting the pH to 7.0-8.0, shaking for 15min, centrifuging the filtrate, removing thalli and a small amount of soybean residues to obtain a supernatant, adding a proper amount of ethanol into the supernatant for precipitation, finally collecting the precipitate, and drying the precipitate to constant weight to obtain gamma-polyglutamic acid;
(4) preparation of gamma-polyglutamic acid Gel Permeation Chromatography (GPC) samples: and (4) collecting the sample filtrate in the step (3), centrifuging, extracting by using 95% absolute ethyl alcohol, drying at the low temperature of 60 ℃ to obtain pure gamma-polyglutamic acid (gamma-PGA), and dissolving in 3mol/L sodium sulfate solution.
Further preferably, the volume ratio of the ethanol and the supernatant liquid adopted in the step (3) is 2-4: 1, and the mass fraction of the ethanol is 95%.
Compared with the prior art, the invention has the beneficial effects that:
the invention obtains the bacillus subtilis for producing the gamma-polyglutamic acid by high-salt fermentation from salted fish products through screening, and the gamma-polyglutamic acid is fermented and extracted through three steps of strain activation, solid fermentation and purification extraction, so that the gamma-polyglutamic acid can be fermented, and the method is suitable for high-salt and alkali environment in the fermentation process.
Drawings
FIG. 1 is a phylogenetic tree constructed by the 16SrDNA sequence of the salt-tolerant Bacillus subtilis strain of the invention.
FIG. 2 is a histogram comparing the contents of fermented polyglutamic acid of salt-tolerant Bacillus subtilis and commercially available daily-produced high-yield natto essence.
FIG. 3 is a salt tolerant (12%) Bacillus subtilis Gel Permeation Chromatography (GPC) chromatogram of the present invention.
Detailed Description
Example 1
Separating and screening strains:
the separation and screening comprises the following steps:
(1) and (3) thallus enrichment: adding 1g of commercially available salted fish product into nutrient broth culture medium (liquid content 20mL/250mL triangular flask) with NaCl concentration of 12%, heating in water bath at 80 deg.C for 20min, and shake culturing for 24h (30 deg.C, 150r/min) with shaking table.
(2) Primary screening: adding 1mL of the enrichment solution obtained in the step (1) into 9mL of sterile water to obtain 10-1Sample diluent, and different gradient diluents are obtained in sequence in the method. Each of the dilutions was applied in an amount of 0.1mL to a nutrient agar plate and incubated at a constant temperature of 35 ℃ for 24 hours.
(3) Separation and purification: and selecting the strains with high growth speed, large bacterial colonies and sticky surfaces to corresponding plates, and further separating and purifying.
(4) Re-screening: the separated and purified strain was inoculated into a soybean solid medium (soybean content: 50g soybean/300 mL Erlenmeyer flask), and left to ferment at a constant temperature of 35 ℃ for 48 hours. And inoculating the screened strain into soybean, observing and analyzing the condition of producing the gamma-PGA, and obtaining the strain with better salt tolerance and excellent performance of producing the gamma-PGA for enlarged culture and preservation.
Example 2
Identification of the strains:
and extracting and rescreening the DNA of the obtained strain by using a bacterial genome DNA extraction kit, and carrying out PCR amplification on 16S rRNA by using an upstream primer 27F and a downstream primer 1492R. And recovering and purifying the product gel after amplification, and sending the product gel to Guangzhou Egyptian biotechnology limited company for gene sequencing.
Sequencing results show that the length of the 16S rRNA gene of the strain is 1400bp (the sequence is shown as SEQ ID No. 1). The sequencing result is compared with NCBI database, and the known sequence with the highest homology with the 16S rDNA sequence of the bacterium can be obtained. As can be seen from the comparison, Bacillus subtilis has the highest similarity as shown in FIG. 1. Therefore, the Bacillus subtilis separated and screened by the invention is considered to be Bacillus subtilis, and is specifically named as Bacillus subtilis YZHY21.A05(Bacillus subtilis YZHY 21.A05).
Example 3
Method for producing gamma-PGA (poly-glycolic acid) by utilizing bacillus subtilis to realize salt-tolerant fermentation
(1) And (3) activation of thalli: the bacillus subtilis inclined plane is taken out of a refrigerated cabinet at 4 ℃, and is placed in an incubator at 28 ℃ for activation for 2 hours.
(2) Solid fermentation: taking the strain slant in the step (1), cleaning the slant lawn with 5mL of 12%, 16% and 24% sterile saline water on each slant under the aseptic condition, inoculating the slant lawn with 4% of inoculum size into a soybean culture medium (bean filling amount: 50g of wet soybeans/300 mL triangular flask), and fermenting at constant temperature for 48h, wherein the fermentation temperature is 30 ℃.
(3) Extracting gamma-polyglutamic acid: adding sterile water into a soybean culture medium fermented for 48 hours, adjusting the pH to 7.0-8.0, shaking for 15min, and filtering. The filtrate was centrifuged (8000r/min, 5min, 25 ℃), and the cells and a small amount of soybean residue were removed to obtain a supernatant. Adding 95% ethanol (1: 3 ═ V: V) into the supernatant for precipitation, finally collecting the precipitate, and drying the precipitate to constant weight to obtain the gamma-PGA.
Comparative examples
Comparing the yield of the bacillus subtilis YZHY21.A05 and the yield of the gamma-polyglutamic acid produced by commercial Gaoqiana bean powder by contrast screening at different NaCl concentrations, and fermenting for 48 hours under the environment that the NaCl concentration is 12 percent, wherein the yields of the gamma-polyglutamic acid produced by the bacillus subtilis YZHY21.A05 and the commercial Gaoqiana bean powder are almost the same; when the NaCl concentration is 16% and 24%, the high-bridge fermentation condition is slightly better than that of the bacillus subtilis YZHY21.A05, and meanwhile, the bacillus subtilis YZHY21.A05 strain is excellent in performance and has the potential of fermentation optimization in a high-saline-alkali environment. Please refer to fig. 2.
Example 4
The molecular weight of gamma-PGA produced by the bacillus subtilis YZHY21.A05 is determined:
respectively preparing spore suspension of screened bacillus subtilis by adopting saline water with NaCl concentration of 12%, 16% and 24%, inoculating the spore suspension into a soybean solid culture medium (the soybean charging amount is 50g of soybeans per 300mL of triangular flask), fermenting for 48 hours, dissolving a fermentation product into sterile water, centrifuging, adding 95% absolute ethyl alcohol with three times of volume, and centrifuging at high speed for 5 min; after the operations, the pure gamma-PGA is obtained by low-temperature drying, the number average molecular weight of the gamma-PGA is measured by Gel Permeation Chromatography (GPC), and the standard curve of the Gel Permeation Chromatography (GPC) is Y-1.57468X +18.7684 (correlation factor: 0.99954616).
The results show that the number average molecular weight of the fermented gamma-PGA with NaCl concentration of 12%, 16% and 24% is 480000-530000. The fermentation yielded γ -PGA with a NaCl concentration of 12% having a number average molecular weight of 480901 (as shown in FIG. 3). The number average molecular weights of the gamma-PGA obtained by fermentation at NaCl concentrations of 16% and 24% are 491061 and 521320, respectively.
The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.
Sequence listing
<110> Anhui Yuanzhihui Biotechnology Limited
<120> bacillus subtilis for producing gamma-polyglutamic acid by salt-tolerant fermentation and application thereof
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<170> SIPOSequenceListing 1.0
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agatgggagc ttgctccctg atgttagcgg cggacgggtg agtaacacgt gggtaacctg 60
cctgtaagac tgggataact ccgggaaacc ggggctaata ccggatggtt gtttgaaccg 120
catggttcaa acataaaagg tggcttcggc taccacttac agatggaccc gcggcgcatt 180
agctagttgg tgaggtaacg gctcaccaag gcaacgatgc gtagccgacc tgagagggtg 240
atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc agtagggaat 300
cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa ggttttcgga 360
tcgtaaagct ctgttgttag ggaagaacaa gtaccgttcg aatagggcgg taccttgacg 420
gtacctaacc agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg 480
caagcgttgt ccggaattat tgggcgtaaa gggctcgcag gcggtttctt aagtctgatg 540
tgaaagcccc cggctcaacc ggggagggtc attggaaact ggggaacttg agtgcagaag 600
aggagagtgg aattccacgt gtagcggtga aatgcgtaga gatgtggagg aacaccagtg 660
gcgaaggcga ctctctggtc tgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca 720
ggattagata ccctggtagt ccacgccgta aacgatgagt gctaagtgtt agggggtttc 780
cgccccttag tgctgcagct aacgcattaa gcactccgcc tggggagtac ggtcgcaaga 840
ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 900
aagcaacgcg aagaacctta ccaggtcttg acatcctctg acaatcctag agataggacg 960
tccccttcgg gggcagagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat 1020
gttgggttaa gtcccgcaac gagcgcaacc cttgatctta gttgccagca ttcagttggg 1080
cactctaagg tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca 1140
tgccccttat gacctgggct acacacgtgc tacaatggac agaacaaagg gcagcgaaac 1200
cgcgaggtta agccaatccc acaaatctgt tctcagttcg gatcgcagtc tgcaactcga 1260
ctgcgtgaag ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc 1320
gggccttgta cacaccgccc gtcacaccac gagagtttgt aacacccgaa gtcggtgagg 1380
taacctttta ggagccagcc 1400
Claims (3)
1. The Bacillus subtilis is classified and named as Bacillus subtilis YZHY21.A05(Bacillus subtilis YZHY21.A05) and is preserved in Guangdong province microorganism strain preservation center, and the preservation number is GDMCC NO: 62050, preservation date is 2021, 11 months and 9 days, and preservation unit addresses are: xieli Zhonglu 100 Dazhou 59 th 5 th, Guangzhou province institute of microbiology, Guangdong province institute of sciences, postal code 510070.
2. The use of the bacillus subtilis of claim 1 for the production of gamma-polyglutamic acid by salt tolerant fermentation.
3. The use of claim 2, wherein the bacillus subtilis is capable of producing gamma-polyglutamic acid by fermentation in an environment with a NaCl concentration of 12% to 24%.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1416461A (en) * | 2001-01-11 | 2003-05-07 | 生物领袖公司 | Bacillus subtilis Var. chungkookjang producing high molecular weight poly-gumma-glutamic acid |
| US20090126045A1 (en) * | 2005-03-04 | 2009-05-14 | Eiichiro Ono | Transgenic Plant for Producing Polyglutamic Acid |
| KR20160017797A (en) * | 2014-08-05 | 2016-02-17 | 엠제이바이오 주식회사 | Polyglutamic Acid production method use Bacillus subtillis MJ80 strain |
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