CN114958646B - Bacillus amyloliquefaciens blue for producing polyglutamic acid - Google Patents
Bacillus amyloliquefaciens blue for producing polyglutamic acid Download PDFInfo
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- 108010020346 Polyglutamic Acid Proteins 0.000 title claims abstract description 50
- 229920002643 polyglutamic acid Polymers 0.000 title claims abstract description 50
- 241000193744 Bacillus amyloliquefaciens Species 0.000 title claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 37
- 230000004151 fermentation Effects 0.000 claims abstract description 37
- 244000068988 Glycine max Species 0.000 claims abstract description 32
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 32
- 230000001580 bacterial effect Effects 0.000 claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 13
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 13
- 239000004220 glutamic acid Substances 0.000 claims abstract description 13
- 244000005700 microbiome Species 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 239000002609 medium Substances 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000002244 precipitate Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 9
- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000008223 sterile water Substances 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 241001052560 Thallis Species 0.000 claims description 6
- 239000012043 crude product Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 4
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 229940073490 sodium glutamate Drugs 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 235000019270 ammonium chloride Nutrition 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000007790 scraping Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 abstract description 4
- 235000013336 milk Nutrition 0.000 abstract description 2
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- 238000012827 research and development Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 5
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- 235000014469 Bacillus subtilis Nutrition 0.000 description 4
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 240000007124 Brassica oleracea Species 0.000 description 1
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 1
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 1
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
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- 229920002307 Dextran Polymers 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
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- 239000002250 absorbent Substances 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229920000704 biodegradable plastic Polymers 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- -1 health care Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 229920006158 high molecular weight polymer Polymers 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
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- 238000000926 separation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 235000021404 traditional food Nutrition 0.000 description 1
Classifications
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- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The blue bacillus amyloliquefaciens for producing polyglutamic acid relates to the technical field of biological fermentation, the strain is in the form of a blue bacterial colony, the taxonomy of the strain is named as bacillus amyloliquefaciens, the Latin is named as Bacillus amyloliquefaciens, the autonomous number of the strain is YZHY21.A02, and the strain is preserved in the microorganism strain collection of Guangdong province, and the preservation number of the strain is GDMCC NO:61924. the bacillus amyloliquefaciens provided by the invention can convert glutamic acid and the like to generate polyglutamic acid, can utilize soybean milk to ferment to generate polyglutamic acid, and can also use soybean to ferment in solid state to generate polyglutamic acid. The yield of polyglutamic acid in the fermentation broth per unit volume of the glutamic acid culture medium can reach 10.8g/L, and the yield of polyglutamic acid by solid fermentation can reach 4.9g/100g of soybeans. Therefore, the strain is a strain with great research and development value.
Description
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a blue bacillus amyloliquefaciens for producing polyglutamic acid.
Background
Polyglutamic acid (PGA) is a water-soluble, anionic, biodegradable and edible biopolymer composed of 500 to 5000 glutamic acid monomers. It has various potential applications in the fields of food, medicine, health care, water treatment and the like. The application of polyglutamic acid in food is as main component of Japanese traditional food "natto", and natto is prepared by inoculating Bacillus subtilis or Bacillus natto into cooked soybean and fermenting, and has certain health promotion effect. The natto not only can effectively prevent and treat cardiovascular and cerebrovascular diseases, but also has the effects of protecting liver, resisting tumor, resisting oxidation, delaying aging and the like. The purified natto gel can also be used in cosmetics, and has effects of moistening skin. In the medical field, polyglutamic acid can be used for slow release of medicines and can also be applied to research on tissue structures. In terms of water treatment, polyglutamic acid can be used as a biopolymer flocculant and can be used as an absorbent for heavy metals and impurities. Meanwhile, the polyglutamic acid hydrogel subjected to crosslinking modification can be also used in the fields of agriculture, biodegradable plastics, health care and the like.
Microbial fermentation is a simple, cost-effective process compared to other processes for producing polyglutamic acid. Polyglutamic acid can be produced by various microorganisms, wherein the most extensive microbial source is bacillus subtilis, bacillus amyloliquefaciens is very similar to bacillus subtilis, is a gram-positive and rod-shaped bacterium, and at present, many researches for producing polyglutamic acid by using bacillus amyloliquefaciens exist, zhao Xiangying et al use bacillus amyloliquefaciens, and ferment sodium citrate and sodium glutamate as raw materials to obtain polyglutamic acid, and the yield can reach 15-20 g/L (patent application number: 201010150439.4). Wang Senlin et al used Bacillus amyloliquefaciens to ferment polyglutamic acid and prepare fertilizer potentiators, which had good effects on crops such as cabbage, tomato, etc. (patent application No. 201810865558.4). Hong Lizhi the low molecular weight polyglutamic acid is produced and prepared by adopting a method of co-fermenting bacillus amyloliquefaciens and bacillus cereus, and the yield reaches 15-20 g/L (patent application number: 202010455019.0).
Molecular weight is an important feature of microbial polyglutamic acid because molecular size has an effect on polymer properties. Different molecular weight polymers are needed for different purposes, and products with different molecular weights can be obtained by different fermentation strains and fermentation raw materials or by different post-treatment methods. The polyglutamic acid produced by bacillus generally has higher molecular weight and average molecular weight of 10 5 And 8X 10 6 Between them. Such high molecular weight polymers are useful tackifiers.
The bacillus amyloliquefaciens Bacillus amyloliquefaciens YZHY21.A02 strain is characterized by being capable of forming blue colonies and producing high-molecular-weight polyglutamic acid by using a glutamic acid culture medium and a soybean culture medium.
Disclosure of Invention
The invention discloses a blue bacillus amyloliquefaciens for producing polyglutamic acid, which is in a form of a blue bacterial colony, and has the taxonomy of being named as bacillus amyloliquefaciens, the Latin name is Bacillus amyloliquefaciens, the autonomous number is YZHY21.A02, and the strain is preserved in the microorganism strain preservation center of Guangdong province, and the preservation number is GDMCC NO:61924 the date of deposit is 2021, 9 and 9, and the address of the deposit is 5 th floor of No. 59 of No. 100 institute of Mitsui, guangdong university, and postal code 510070.
The strain is isolated from fermented soybean food in Yangjiang area of Guangdong. The separation method comprises the following steps: putting 2g of fermented soya beans into a 300mL triangular flask containing 50mL of sterile water and provided with glass beads, oscillating for 1h, and carrying out water bath at 80 ℃ for 20min; and (3) taking 0.1mL of the bacterial suspension in a 10mL sterilization centrifuge tube, carrying out gradient dilution and coating on a nutrient agar culture medium, carrying out inverted constant temperature culture at 35 ℃, picking up a bluish-green colony after a single colony grows out, diluting and coating again, carrying out constant temperature culture, inoculating the single colony to a nutrient agar inclined plane, carrying out constant temperature culture at 35 ℃, and storing in a refrigerator at 4 ℃ after the culture is completed.
Preferably, the agar medium used has the following composition: 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride, 18g/L agar and pH value of 7.4.
The invention discloses a specific application of the bacillus amyloliquefaciens blue, which is to ferment and produce polyglutamic acid in a glutamic acid fermentation medium or a soybean fermentation medium.
Wherein, the steps of producing polyglutamic acid by adopting a glutamic acid fermentation culture medium for fermentation are as follows: inoculating the slant strain to a liquid seed culture medium, culturing for 12-24 h at 35 ℃ at 150r/min, and then inoculating to a shake flask fermentation culture medium for shake flask fermentation for 48-72 h at 35-40 ℃ at 150 r/min; diluting the fermentation liquid with water for 2-3 times, centrifuging at 8000r/min for 20-25 min to remove thalli and other insoluble matters, adding 2-3 times of 95% ethanol into supernatant to precipitate for 12-24 h, centrifuging again to collect precipitate, adding deionized water to dissolve the precipitate, and freeze-drying to obtain a polyglutamic acid crude product.
The liquid seed culture medium comprises the following components: glucose 10g/L, peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, and pH 7.4; the fermentation medium consists of: 40g/L of glucose, 50g/L of sodium glutamate, 3g/L of yeast powder, 2g/L of ammonium chloride, 3g/L of dipotassium hydrogen phosphate, 0.3g/L of magnesium sulfate, 0.4g/L of calcium chloride and pH value of 7.2-7.5.
Wherein, the steps of fermenting and producing polyglutamic acid by adopting a soybean fermentation medium are as follows: cleaning soybeans, soaking the soybeans in water for 12-18 hours, and placing 50g of the soybeans in a 300mL triangular flask for sterilization at 121 ℃ for 25 minutes; pouring a proper amount of sterile water into the inclined plane test tube, scraping a bacterial colony into the sterile water, pouring the bacterial colony into a triangular flask filled with soybeans, shaking uniformly, and placing the triangular flask in an incubator at 35-40 ℃ for culturing for 48-72 h; adding deionized water with the quantity of 6-10 times of that of the soybeans to wash the soybeans, centrifuging for 20-25 min at 8000r/min to remove thalli and other insoluble matters, taking supernatant, adding 2-3 times of 95% ethanol to precipitate for 12-24 h, centrifuging to collect precipitate, adding deionized water to dissolve the precipitate, and freeze-drying to obtain a polyglutamic acid crude product.
Compared with the prior art, the invention has the beneficial effects that:
the bacillus amyloliquefaciens provided by the invention has a bacterial strain form of blue bacterial colony, can convert glutamic acid and the like to generate polyglutamic acid, can utilize soybean milk to ferment to generate polyglutamic acid, and can also use soybean to ferment to generate polyglutamic acid. The yield of polyglutamic acid in the fermentation broth per unit volume of the glutamic acid culture medium can reach 10.8g/L, and the yield of polyglutamic acid by solid fermentation can reach 4.9g/100g of soybeans. Therefore, the strain is a strain with great research and development value.
Drawings
FIG. 1 is a plate colony map.
FIG. 2 is a colony chart of test tubes.
FIG. 3 is a phylogenetic tree constructed by the sequence of the strain 16 SrDNA.
Detailed Description
EXAMPLE 1 screening of Bacillus blue Amylase Strain
2g of fermented soybean food in Yangjiang area of Guangdong was placed in a 300mL triangular flask with 50mL of sterile water and glass beads, shaken for 1h, and water-bath was performed at 80℃for 20min. And (3) taking 0.1mL of the bacterial suspension in a 10mL sterilization centrifuge tube, carrying out gradient dilution and coating on a nutrient agar culture medium, carrying out inverted constant temperature culture at 35 ℃, picking up a blue bacterial colony after a single bacterial colony grows out, diluting and coating again, carrying out constant temperature culture, inoculating the single bacterial colony to a nutrient agar inclined plane, carrying out constant temperature culture at 35 ℃, and storing in a refrigerator at 4 ℃ after the culture is completed.
Referring to fig. 1 and 2, the morphological identification results of the strain are: colonies were blue.
Molecular biological identification of strains: extracting the DNA of the obtained strain by using a bacterial genome DNA extraction kit, amplifying 16S rRNA, recovering and purifying the product gel, and sending the product gel to Guangzhou Ai Ji biotechnology limited company for gene sequencing. Sequencing results show that the length of the 16S rRNA gene of the strain is 1389bp (the splicing sequence is shown as SEQ ID No. 1). And comparing the sequencing result with NCBI database to obtain the known sequence with highest homology with the 16S rDNA sequence of the bacterium. As can be seen by comparing the phylogenetic tree, the similarity with Bacillus amyloliquefaciens is highest, and the matching degree reaches 100% (shown in figure 3). Thus, according to the above data, the isolated strain was identified as bacillus amyloliquefaciens Bacillus amyloliquefaciens, autonomously designated bacillus amyloliquefaciens yzhy21.a02 (Bacillus amyloliquefaciens yzhy 21.a02). The strain can resist high temperature of 70-90 ℃ for 10-20 minutes and is suitable for growth at 30-40 ℃.
EXAMPLE 2 liquid fermentation production of polyglutamic acid
Inoculating the slant strain to a liquid seed culture medium, culturing at 35 ℃ and 150r/min for 12-24 h, and then inoculating to a shake flask fermentation culture medium, and shake flask fermentation at 35-40 ℃ and 150r/min for 72h. Diluting the fermentation liquid with water for 2-3 times, centrifuging at 8000r/min for 20-25 min to remove thalli and other insoluble matters, adding 2-3 times of 95% ethanol into supernatant to precipitate for 12-24 h, centrifuging again to collect precipitate, adding deionized water to dissolve the precipitate, and freeze-drying to obtain a polyglutamic acid crude product.
Wherein, the seed culture medium comprises the following components: glucose 10g/L, peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, and pH 7.4. The liquid fermentation medium is glutamic acid fermentation medium. The glutamic acid fermentation medium comprises the following components: 40g/L of glucose, 50g/L of sodium glutamate, 3g/L of yeast powder, 2g/L of ammonium chloride, 3g/L of dipotassium hydrogen phosphate, 0.3g/L of magnesium sulfate, 0.4g/L of calcium chloride and pH value of 7.2-7.5.
EXAMPLE 3 solid fermentation production of polyglutamic acid
Cleaning soybean, soaking in water for 12-18 h, placing 50g into a 300mL triangular flask, and sterilizing at 121 ℃ for 25min. Pouring a proper amount of sterile water into the inclined plane test tube, scraping the bacterial colony into the sterile water, pouring the bacterial colony into a triangular flask filled with soybeans, shaking uniformly, and placing the triangular flask in an incubator at 35-40 ℃ for culturing for 72 hours. Adding deionized water with the quantity of 6-10 times of that of the soybeans to wash the soybeans, centrifuging for 20-25 min at 8000r/min to remove thalli and other insoluble matters, taking supernatant, adding 2-3 times of 95% ethanol to precipitate for 12-24 h, centrifuging to collect precipitate, adding deionized water to dissolve the precipitate, and freeze-drying to obtain a polyglutamic acid crude product.
EXAMPLE 4 crude polyglutamic acid analysis
1g of polyglutamic acid prepared as described above was weighed, dissolved, filtered through a 0.45 μm water film, and analyzed for molecular weight by Gel Permeation Chromatography (GPC). Wherein the mobile phase adopts 0.3M Na 2 SO 4 The flow rate of the solution is 0.4mL/min, the temperature is 30 ℃, and the standard sample is dextran.
The average molecular weight of polyglutamic acid obtained by fermenting glutamic acid liquid is 3.5X10 by GPC 5 The average molecular weight of polyglutamic acid obtained by fermenting soybean is 2.13×10 6 。
The foregoing is merely illustrative and explanatory of the principles of the invention, as various modifications and additions may be made to the specific embodiments described, or similar thereto, by those skilled in the art, without departing from the principles of the invention or beyond the scope of the appended claims.
Sequence listing
<110> Anhui Guangdong Intelligence badge source biotechnology Co., ltd
<120> Bacillus amyloliquefaciens blue for producing polyglutamic acid
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1389
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
aaaggttacc tcaccgactt cgggtgttac aaactctcgt ggtgtgacgg gcggtgtgta 60
caaggcccgg gaacgtattc accgcggcat gctgatccgc gattactagc gattccagct 120
tcacgcagtc gagttgcaga ctgcgatccg aactgagaac agatttgtgg gattggctta 180
acctcgcggt ttcgctgccc tttgttctgc ccattgtagc acgtgtgtag cccaggtcat 240
aaggggcatg atgatttgac gtcatcccca ccttcctccg gtttgtcacc ggcagtcacc 300
ttagagtgcc caactgaatg ctggcaacta agatcaaggg ttgcgctcgt tgcgggactt 360
aacccaacat ctcacgacac gagctgacga caaccatgca ccacctgtca ctctgccccc 420
gaaggggacg tcctatctct aggattgtca gaggatgtca agacctggta aggttcttcg 480
cgttgcttcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc aattcctttg 540
agtttcagtc ttgcgaccgt actccccagg cggagtgctt aatgcgttag ctgcagcact 600
aaggggcgga aaccccctaa cacttagcac tcatcgttta cggcgtggac taccagggta 660
tctaatcctg ttcgctcccc acgctttcgc tcctcagcgt cagttacaga ccagagagtc 720
gccttcgcca ctggtgttcc tccacatctc tacgcatttc accgctacac gtggaattcc 780
actctcctct tctgcactca agttccccag tttccaatga ccctccccgg ttgagccggg 840
ggctttcaca tcagacttaa gaaaccgcct gcgagccctt tacgcccaat aattccggac 900
aacgcttgcc acctacgtat taccgcggct gctggcacgt agttagccgt ggctttctgg 960
ttaggtaccg tcaaggtgcc gccctatttg aacggcactt gttcttccct aacaacagag 1020
ctttacgatc cgaaaacctt catcactcac gcggcgttgc tccgtcagac tttcgtccat 1080
tgcggaagat tccctactgc tgcctcccgt aggagtctgg gccgtgtctc agtcccagtg 1140
tggccgatca ccctctcagg tcggctacgc atcgtcgcct tggtgagccg ttacctcacc 1200
aactagctaa tgcgccgcgg gtccatctgt aagtggtagc cgaagccacc ttttatgttt 1260
gaaccatgcg gttcaaacaa gcatccggta ttagccccgg tttcccggag ttatcccagt 1320
cttacaggca ggttacccac gtgttactca cccgtccgcc gctaacatca gggagcaagc 1380
tcccatctg 1389
Claims (2)
1. Bacillus amyloliquefaciens blue for producing polyglutamic acid, which is in the form of blue bacterial colony, and is named as Bacillus amyloliquefaciens, and Latin is named as LatinBacillus amyloliquefaciensAutonomous number yzhy21.a02, deposited with the collection of microorganism strains, cantonese province, accession No. GDMCC NO:61924 the date of deposit is 2021, 9 and 9, and the address of the deposit is 5 th floor of No. 59 of No. 100 institute of Mitsui, guangdong university, and postal code 510070.
2. The use of the bacillus amyloliquefaciens blue according to claim 1 for fermentation production of polyglutamic acid, wherein the fermentation medium is a glutamic acid fermentation medium or a soybean fermentation medium;
the steps of producing polyglutamic acid by adopting a glutamic acid fermentation medium for fermentation are as follows: inoculating the slant strain to a liquid seed culture medium, culturing for 12-24 h at 35 ℃ at 150r/min, and then inoculating to a shake flask fermentation culture medium, and shake flask fermentation for 48-72 h at 35-40 ℃ at 150 r/min; diluting the fermentation liquor with water for 2-3 times, centrifuging for 20-25 min at 8000r/min to remove thalli and other insoluble matters, adding 2-3 times of 95% ethanol into supernatant to precipitate for 12-24 h, centrifuging to collect precipitate, adding deionized water to dissolve the precipitate, and freeze-drying to obtain a polyglutamic acid crude product; the liquid seed culture medium consists of: glucose 10g/L, peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, and pH 7.4; the fermentation medium consists of: 40g/L of glucose, 50g/L of sodium glutamate, 3g/L of yeast powder, 2g/L of ammonium chloride, 3g/L of dipotassium hydrogen phosphate, 0.3g/L of magnesium sulfate, 0.4g/L of calcium chloride and pH value of 7.2-7.5;
the steps of fermenting and producing polyglutamic acid by adopting a soybean fermentation medium are as follows: cleaning soybeans, soaking the soybeans in water for 12-18 hours, and placing 50g of the soybeans in a 300mL triangular flask for sterilization at 121 ℃ for 25 minutes; pouring a proper amount of sterile water into the inclined plane test tube, scraping a bacterial colony into the sterile water, pouring the bacterial colony into a triangular flask filled with soybeans, shaking uniformly, and placing the triangular flask in a 35-40 ℃ incubator for culturing for 48-72 h; adding deionized water with the amount of 6-10 times of the amount of the soybeans to wash the soybeans, centrifuging for 20-25 min at 8000r/min to remove thalli and other insoluble matters, adding 2-3 times of 95% ethanol into supernatant to precipitate for 12-24 h, centrifuging to collect precipitate, adding deionized water to dissolve the precipitate, and freeze-drying to obtain a polyglutamic acid crude product.
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| CN111465685A (en) * | 2017-08-31 | 2020-07-28 | Cj第一制糖株式会社 | Novel bacillus amyloliquefaciens strain and method for preparing fermented soybean product by using same |
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| CN106591190A (en) * | 2016-12-16 | 2017-04-26 | 大连理工大学 | Bacillus and application in preparing Gama-polyglutamic acid |
| CN111465685A (en) * | 2017-08-31 | 2020-07-28 | Cj第一制糖株式会社 | Novel bacillus amyloliquefaciens strain and method for preparing fermented soybean product by using same |
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