CN114958646A - Blue bacillus amyloliquefaciens for producing polyglutamic acid - Google Patents
Blue bacillus amyloliquefaciens for producing polyglutamic acid Download PDFInfo
- Publication number
- CN114958646A CN114958646A CN202210340897.7A CN202210340897A CN114958646A CN 114958646 A CN114958646 A CN 114958646A CN 202210340897 A CN202210340897 A CN 202210340897A CN 114958646 A CN114958646 A CN 114958646A
- Authority
- CN
- China
- Prior art keywords
- polyglutamic acid
- bacillus amyloliquefaciens
- blue
- fermentation
- soybeans
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 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 30
- 238000000855 fermentation Methods 0.000 claims abstract description 37
- 230000004151 fermentation Effects 0.000 claims abstract description 36
- 244000068988 Glycine max Species 0.000 claims abstract description 35
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 35
- 239000001963 growth medium Substances 0.000 claims abstract description 19
- 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
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 244000005700 microbiome Species 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 29
- 230000001580 bacterial effect Effects 0.000 claims description 26
- 239000002609 medium Substances 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000002244 precipitate Substances 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 9
- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- 239000008223 sterile water Substances 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 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
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 239000012043 crude product Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000006916 nutrient agar Substances 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 238000011218 seed culture Methods 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000008272 agar Substances 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
- 229940073490 sodium glutamate Drugs 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 3
- 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
- 239000011324 bead Substances 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
- 238000007865 diluting 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
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 238000007790 scraping Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 claims description 2
- 238000012262 fermentative production Methods 0.000 claims 1
- 239000007787 solid Substances 0.000 abstract description 5
- 235000013336 milk Nutrition 0.000 abstract description 2
- 239000008267 milk Substances 0.000 abstract description 2
- 210000004080 milk Anatomy 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 244000063299 Bacillus subtilis Species 0.000 description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 235000013557 nattō Nutrition 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 229920001222 biopolymer Polymers 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000193755 Bacillus cereus Species 0.000 description 1
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 1
- 244000221633 Brassica rapa subsp chinensis Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 238000007792 addition Methods 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
- 239000002537 cosmetic Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 239000003337 fertilizer Substances 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
- 239000012535 impurity Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000013365 molecular weight analysis method Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 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
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000021404 traditional food Nutrition 0.000 description 1
Images
Classifications
-
- 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
Landscapes
- 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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The blue Bacillus amyloliquefaciens for producing polyglutamic acid relates to the technical field of biological fermentation, the strain form is blue colony, the taxonomic name is Bacillus amyloliquefaciens, the latin name is Bacillus amyloliquefaciens, the autonomous number is YZHY21.A02, the blue Bacillus amyloliquefaciens is preserved in Guangdong province microorganism strain preservation center, and the preservation number is GDMCC NO: 61924. the bacillus amyloliquefaciens provided by the invention can convert glutamic acid and the like into polyglutamic acid, can also be fermented by soybean milk to generate the polyglutamic acid, and can also be fermented by soybean solids to generate the polyglutamic acid. The yield of the polyglutamic acid in the fermentation liquor of the glutamic acid culture medium per unit volume can reach 10.8g/L, and the yield of the polyglutamic acid obtained by solid fermentation can reach 4.9g/100g of soybeans. Therefore, the strain is a strain with great research and development values.
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 consisting of 500-5000 glutamic acid monomers. It has many potential applications in the fields of food, medicine, health care, water treatment and the like. The application of the polyglutamic acid in food is used as a main component of Japanese traditional food natto, and the natto is a product prepared by inoculating bacillus subtilis or bacillus natto in cooked soybeans and fermenting, and has a certain health-care effect. The natto not only can effectively prevent and treat cardiovascular and cerebrovascular diseases, but also has various effects of protecting liver, resisting tumor, resisting oxidation, delaying senility and the like. The purified natto gum can also be used in cosmetics, and has effects of keeping moisture and caring skin. In the medical field, polyglutamic acid can be used for slow release of drugs and can also be applied to research on tissue structures. In the aspect of water treatment, polyglutamic acid can be used as a biopolymer flocculant and can be used as an absorbent of heavy metals and impurities. Meanwhile, the cross-linked and modified polyglutamic acid hydrogel can also be used in the fields of agriculture, biodegradable plastics, health care and the like.
Microbial fermentation is a simple, cost effective process compared to other methods of producing polyglutamic acid. Polyglutamic acid can be produced by various microorganisms, wherein the most extensive microorganism source is bacillus subtilis, bacillus amyloliquefaciens is quite similar to the bacillus subtilis and is a gram-positive rod-shaped bacterium, at present, a lot of researches on producing the polyglutamic acid by using the bacillus amyloliquefaciens exist, and Pingxiang Guiying and other people use the bacillus amyloliquefaciens and use sodium citrate and sodium glutamate as raw materials to ferment to obtain the polyglutamic acid, and the yield can reach 15-20 g/L (patent application number: 201010150439.4). Wang forest et al use Bacillus amyloliquefaciens to ferment polyglutamic acid and prepare fertilizer synergist, have better effect after applying to crops such as pakchoi, tomato, etc. (patent application number: 201810865558.4). The Honglizhi adopts a method of co-fermentation of bacillus amyloliquefaciens and bacillus cereus to produce and prepare the low-molecular-weight polyglutamic acid, and the yield reaches 15-20 g/L (patent application number: 202010455019.0).
Molecular weight is an important characteristic of microbial polyglutamic acid because molecular size has an effect on polymer properties. Different use purposes require polymers with different molecular weights, and different fermentation strains and fermentation raw materials are used, or different post-treatment methods are used for obtaining products with different molecular weights. The polyglutamic acid produced by bacillus generally has higher molecular weight, and the average molecular weight is 10 5 And 8X 10 6 In between. This high molecular weight polymer is a useful tackifier.
The Bacillus amyloliquefaciens YZHY21.A02 strain is characterized in that blue colonies can be formed, and a glutamic acid culture medium and a soybean culture medium can be utilized to produce high-molecular-weight polyglutamic acid.
Disclosure of Invention
The invention discloses a blue Bacillus amyloliquefaciens for producing polyglutamic acid, wherein the strain is blue colony, the taxonomic name is Bacillus amyloliquefaciens, the latin name is Bacillus amyloliquefaciens, the independent number is YZHY21.A02, the blue Bacillus amyloliquefaciens is preserved in Guangdong province microorganism strain preservation center, and the preservation number is GDMCC NO: 61924, the preservation date is 2021, 9 months and 9 days, the preservation unit address is No. 59 building 5 of Dazhou No. 100 Jic of Jie Cedrury, Guangdong province institute of microbiology, and postal code 510070.
The strain is isolated from fermented soybean food in Yangjiang area of Guangdong province. The following method is adopted for separation: placing 2g of fermented soya beans into a 300mL triangular flask filled with 50mL of sterile water and glass beads, shaking for 1h, and carrying out water bath at 80 ℃ for 20 min; taking 0.1mL of the bacterial suspension into a 10mL sterilizing centrifuge tube, carrying out gradient dilution and coating on a nutrient agar culture medium, carrying out inversion constant temperature culture at 35 ℃, picking blue-green bacterial colonies after single bacterial colonies grow out, carrying out dilution coating again, carrying out constant temperature culture, then inoculating the single bacterial colonies to a nutrient agar inclined plane for 35 ℃ constant temperature culture, and storing in a 4 ℃ refrigerator after the culture is finished.
Preferably, the agar medium used consists of: 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride, 18g/L of agar and 7.4 of pH value.
The invention discloses a specific application of blue bacillus amyloliquefaciens, which is to ferment and produce polyglutamic acid in a glutamic acid fermentation culture medium or a soybean fermentation culture medium.
The method for producing the polyglutamic acid by fermenting the glutamic acid fermentation medium comprises the following steps: inoculating slant strains to a liquid seed culture medium, culturing at 35 ℃ and 150r/min for 12-24 h, inoculating to a shake flask fermentation culture medium, and carrying out shake flask fermentation at 35-40 ℃ and 150r/min for 48-72 h; adding water into the fermentation liquor for diluting by 2-3 times, centrifuging at 8000r/min for 20-25 min to remove thalli and other insoluble substances, adding 2-3 times of 95% ethanol into the supernatant for precipitating 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 comprises the following components: 10g/L of glucose, 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride and 7.4 of pH value; the 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 phosphate, 0.3g/L of magnesium sulfate, 0.4g/L of calcium chloride and pH value of 7.2-7.5.
The method for producing the polyglutamic acid by fermenting the soybean fermentation medium comprises the following steps: cleaning soybeans, soaking the soybeans in water for 12-18 h, and putting 50g of the soybeans in a 300mL triangular flask for sterilization at 121 ℃ for 25 min; pouring a proper amount of sterile water into the inclined test tube, scraping the bacterial colony into the sterile water, pouring the bacterial colony into a triangular flask filled with soybeans, shaking the bacterial colony evenly, and placing the bacterial colony in an incubator at the temperature of 35-40 ℃ for culturing for 48-72 hours; adding deionized water 6-10 times of the amount of the soybeans to wash the soybeans, centrifuging at 8000r/min for 20-25 min to remove thalli and other insoluble substances, taking supernate, adding 95% ethanol 2-3 times of the supernate 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 blue bacterial colony, can convert glutamic acid and the like to generate polyglutamic acid, can also be fermented by soybean milk to generate the polyglutamic acid, and can also be fermented by soybean solids to generate the polyglutamic acid. The yield of the polyglutamic acid in the fermentation liquor of the glutamic acid culture medium per unit volume can reach 10.8g/L, and the yield of the polyglutamic acid obtained by solid fermentation can reach 4.9g/100g of soybeans. Therefore, the strain is a strain with great research and development values.
Drawings
FIG. 1 is a plate colony map.
FIG. 2 is a test tube colony image.
FIG. 3 shows the phylogenetic tree constructed from the strain 16SrDNA sequence.
Detailed Description
Example 1 screening of Bacillus amyloliquefaciens blue strains
2g of fermented soya beans food in Guangdong Yangjiang area was put into a 300mL triangular flask with glass beads and 50mL of sterile water, shaken for 1h, and water-bathed at 80 ℃ for 20 min. Taking 0.1mL of the bacterial suspension into a 10mL sterilizing centrifuge tube, carrying out gradient dilution and coating on a nutrient agar culture medium, carrying out inversion constant temperature culture at 35 ℃, picking a blue bacterial colony for secondary dilution and coating after a single bacterial colony grows out, carrying out constant temperature culture, then inoculating the single bacterial colony to a nutrient agar inclined plane for 35 ℃ constant temperature culture, and storing in a 4 ℃ refrigerator after the culture is finished.
Referring to FIGS. 1 and 2, the morphological identification of the strain is: the colonies were blue in color.
Molecular biological identification of the strains: extracting the DNA of the obtained strain by using a bacterial genome DNA extraction kit, amplifying 16S rRNA, recovering and purifying a product gel, 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 1389bp (the splicing 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. By comparing phylogenetic trees, the similarity between the phylogenetic trees and Bacillus amyloliquefaciens is the highest, and the matching degree reaches 100 percent (shown in figure 3). Therefore, according to the above data, the isolated strain was identified as Bacillus amyloliquefaciens, which was autonomously named Bacillus amyloliquefaciens yyyyyyyyyyy21.a02 (Bacillus amyloliquefaciens yyyyyyyyy21.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 slant strains to a liquid seed culture medium, culturing at 35 ℃ and 150r/min for 12-24 h, inoculating to a shake flask fermentation culture medium, and carrying out shake flask fermentation at 35-40 ℃ and 150r/min for 72 h. Adding water into the fermentation liquor for diluting by 2-3 times, centrifuging at 8000r/min for 20-25 min to remove thalli and other insoluble substances, adding 2-3 times of 95% ethanol into the supernatant for precipitating 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.
Wherein, the seed culture medium comprises the following components: 10g/L of glucose, 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride and 7.4 of pH value. The liquid fermentation culture medium is glutamic acid fermentation culture 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 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 soybeans, soaking the soybeans in water for 12-18 h, and placing 50g of the soybeans in a 300mL triangular flask for sterilization at 121 ℃ for 25 min. Pouring a proper amount of sterile water into the inclined test tube, scraping the bacterial colony into the sterile water, pouring the bacterial colony into a triangular flask filled with soybeans, shaking the bacterial colony evenly, and placing the bacterial colony in an incubator at 35-40 ℃ for culturing for 72 hours. Adding deionized water 6-10 times of the amount of the soybeans to wash the soybeans, centrifuging at 8000r/min for 20-25 min to remove thalli and other insoluble substances, taking supernate, adding 95% ethanol 2-3 times of the supernate 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 analysis of crude polyglutamic acid
1g of polyglutamic acid prepared as described above was weighed, dissolved, filtered with a 0.45 μm water film, and subjected to molecular weight analysis using Gel Permeation Chromatography (GPC). Wherein the mobile phase adopts 0.3M Na 2 SO 4 The solution was dextran at a flow rate of 0.4mL/min and a temperature of 30 ℃.
Average molecular weight of polyglutamic acid obtained by liquid fermentation of glutamic acid by GPC detectionThe amount is 3.5X 10 5 The average molecular weight of polyglutamic acid obtained by fermenting soybean is 2.13 × 10 6 。
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> a blue bacillus amyloliquefaciens 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 (7)
1. The blue Bacillus amyloliquefaciens for producing polyglutamic acid has a strain form of blue colony, is named as Bacillus amyloliquefaciens by taxonomy, is named as Bacillus amyloliquefaciens by latin, has an autonomous number of YZHY21.A02, is preserved in Guangdong province microorganism strain preservation center, and has a preservation number of GDMCC NO: 61924, the preservation date is 2021, 9 months and 9 days, the preservation unit address is No. 59 building 5 of Dazhou No. 100 Jic of Jie Cedrury, Guangdong province institute of microbiology, and postal code 510070.
2. The bacillus coelentus blue according to claim 1, wherein the strain is isolated by: putting 2g of fermented soya beans in Yangjiang area of Guangdong into a 300mL triangular flask filled with 50mL of sterile water and glass beads, shaking for 1h, and carrying out water bath at 80 ℃ for 20 min; taking 0.1mL of the bacterial suspension in a 10mL sterile centrifuge tube, carrying out gradient dilution and coating on a nutrient agar culture medium, carrying out inversion constant-temperature culture at 35 ℃, picking out blue-green bacterial colonies after single bacterial colonies grow out, carrying out dilution coating again, carrying out constant-temperature culture, then inoculating the single bacterial colonies to a nutrient agar inclined plane, carrying out constant-temperature culture at 35 ℃, and storing in a 4 ℃ refrigerator after the culture is finished.
3. The bacillus coelentus blue according to claim 2, wherein the agar medium used consists of: 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride, 18g/L of agar and 7.4 of pH value.
4. The use of Bacillus amyloliquefaciens blue for the fermentative production of polyglutamic acid according to claim 1, wherein the fermentation medium is glutamic acid fermentation medium or soybean fermentation medium.
5. The use according to claim 4, wherein the step of producing polyglutamic acid by fermentation using glutamic acid fermentation medium comprises: inoculating slant strains to a liquid seed culture medium, culturing at 35 ℃ and 150r/min for 12-24 h, inoculating to a shake flask fermentation culture medium, and carrying out shake flask fermentation at 35-40 ℃ and 150r/min for 48-72 h; adding water into the fermentation liquor for diluting by 2-3 times, centrifuging at 8000r/min for 20-25 min to remove thalli and other insoluble substances, adding 2-3 times of 95% ethanol into the supernatant for precipitating 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.
6. The use of claim 5, wherein the liquid seed medium consists of: 10g/L of glucose, 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride and 7.4 of pH value; the 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 phosphate, 0.3g/L of magnesium sulfate, 0.4g/L of calcium chloride and pH value of 7.2-7.5.
7. The use of claim 4, wherein the step of producing polyglutamic acid by fermenting soybean fermentation medium comprises: cleaning soybeans, soaking the soybeans in water for 12-18 h, and putting 50g of the soybeans in a 300mL triangular flask for sterilization at 121 ℃ for 25 min; pouring a proper amount of sterile water into the inclined test tube, scraping the bacterial colony into the sterile water, pouring the bacterial colony into a triangular flask filled with soybeans, shaking the bacterial colony evenly, and placing the bacterial colony in an incubator at the temperature of 35-40 ℃ for culturing for 48-72 hours; adding deionized water 6-10 times of the amount of the soybeans to wash the soybeans, centrifuging at 8000r/min for 20-25 min to remove thalli and other insoluble substances, taking supernate, 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210340897.7A CN114958646B (en) | 2022-04-02 | 2022-04-02 | Bacillus amyloliquefaciens blue for producing polyglutamic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210340897.7A CN114958646B (en) | 2022-04-02 | 2022-04-02 | Bacillus amyloliquefaciens blue for producing polyglutamic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114958646A true CN114958646A (en) | 2022-08-30 |
| CN114958646B CN114958646B (en) | 2023-12-05 |
Family
ID=82978186
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210340897.7A Active CN114958646B (en) | 2022-04-02 | 2022-04-02 | Bacillus amyloliquefaciens blue for producing polyglutamic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114958646B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875910A (en) * | 2010-04-20 | 2010-11-03 | 山东省食品发酵工业研究设计院 | Bacillus amyloliquefaciens for producing gamma-polyglutamic acid |
| CN106591190A (en) * | 2016-12-16 | 2017-04-26 | 大连理工大学 | Bacillus and application in preparing Gama-polyglutamic acid |
| CN108048499A (en) * | 2018-02-09 | 2018-05-18 | 烟台市佳益有机肥料有限公司 | A kind of method of solid fermentation production gamma-polyglutamic acid |
| WO2020036400A1 (en) * | 2018-08-13 | 2020-02-20 | 씨제이제일제당(주) | BACILLUS AMYLOLIQUEFACIENS CJBA1 AND METHOD FOR PRODUCING γ-POLYGLUTAMIC ACID USING SAME |
| 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 |
-
2022
- 2022-04-02 CN CN202210340897.7A patent/CN114958646B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875910A (en) * | 2010-04-20 | 2010-11-03 | 山东省食品发酵工业研究设计院 | Bacillus amyloliquefaciens for producing gamma-polyglutamic acid |
| 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 |
| CN108048499A (en) * | 2018-02-09 | 2018-05-18 | 烟台市佳益有机肥料有限公司 | A kind of method of solid fermentation production gamma-polyglutamic acid |
| WO2020036400A1 (en) * | 2018-08-13 | 2020-02-20 | 씨제이제일제당(주) | BACILLUS AMYLOLIQUEFACIENS CJBA1 AND METHOD FOR PRODUCING γ-POLYGLUTAMIC ACID USING SAME |
Non-Patent Citations (1)
| Title |
|---|
| 张浩;杜万根;舒旭晨;赵世光;: "解淀粉芽孢杆菌PI142产γ-聚谷氨酸发酵条件优化", 淮南师范学院学报, no. 02 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114958646B (en) | 2023-12-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110016446B (en) | Bacillus belgii and method for producing polyglutamic acid by using same | |
| CN110577915B (en) | Bacillus licheniformis CZ186 strain and application thereof | |
| BR112013033322B1 (en) | PROCESS TO PRODUCE CHITIN DERIVATIVES | |
| CN109609408B (en) | Gamma-polyglutamic acid high-yield strain and method for preparing gamma-polyglutamic acid by using strain for liquid fermentation | |
| Pyar et al. | Potentials of pineapple waste as growth medium for Lactobacillus species | |
| CN110923144B (en) | High selenate tolerant bacteria and screening method and application thereof | |
| CN110200018B (en) | Optimal DSE inoculation amount for promoting plant rooting | |
| CN104845896B (en) | Produce the bacterial strain and method of Weilan gum | |
| CN101363014B (en) | Method for producing gamma-glutamyl transpeptidase using bacillus subtilis | |
| CN114540252B (en) | Microbacterium P6 for converting livestock and poultry breeding waste and application | |
| CN113789280B (en) | Lysine bacillus fusiformis preparation for degrading uric acid and preparation method and application thereof | |
| CN102994430A (en) | Bacterial cellulose production strain and application thereof | |
| RU2639557C1 (en) | Xanthomonas campestris bacteria strain - xanthan producer | |
| CN111621435B (en) | Myxobacteria and application thereof | |
| CN108841748A (en) | Sinorhizobium nitrogen-fixing bacteria strain H6 and its application | |
| CN108546663A (en) | One boar source book song lactobacillus and its application | |
| CN115418337B (en) | Lignin degrading bacterium and application thereof in rice straw micro-storage | |
| CN114958646B (en) | Bacillus amyloliquefaciens blue for producing polyglutamic acid | |
| CN116536195A (en) | Bacillus clausii and application thereof in preparation of organic microbial fertilizer | |
| CN110305819A (en) | One plant of feather efficient degrading bacterial strain and its application | |
| CN113755377B (en) | Paramycosis bacillus preparation for degrading uric acid and preparation method and application thereof | |
| CN113046249B (en) | Verticillium lecanii LL-01 and biocontrol application thereof | |
| KR100842450B1 (en) | -2 Kluyvera sp. CL-2 and Method of solubilizing insoluble-phosphate immobilized in soils by using the same | |
| CN109234328B (en) | Method for producing gamma-polyglutamic acid | |
| CN101392226A (en) | Grass green streptomycete |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |