CN113957033A - Method for improving production capacity of polyglutamic acid-producing wild strain - Google Patents
Method for improving production capacity of polyglutamic acid-producing wild strain Download PDFInfo
- Publication number
- CN113957033A CN113957033A CN202111109424.8A CN202111109424A CN113957033A CN 113957033 A CN113957033 A CN 113957033A CN 202111109424 A CN202111109424 A CN 202111109424A CN 113957033 A CN113957033 A CN 113957033A
- Authority
- CN
- China
- Prior art keywords
- culture
- fermentation
- strain
- polyglutamic acid
- seed
- 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.)
- Pending
Links
- 229920002643 polyglutamic acid Polymers 0.000 title claims abstract description 47
- 108010020346 Polyglutamic Acid Proteins 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 17
- 238000000855 fermentation Methods 0.000 claims abstract description 76
- 230000004151 fermentation Effects 0.000 claims abstract description 76
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 54
- 238000011218 seed culture Methods 0.000 claims abstract description 28
- 230000008569 process Effects 0.000 claims abstract description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 42
- 239000001963 growth medium Substances 0.000 claims description 33
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 24
- 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 21
- 239000008103 glucose Substances 0.000 claims description 21
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 21
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 21
- 229940041514 candida albicans extract Drugs 0.000 claims description 17
- 239000012138 yeast extract Substances 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 14
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 14
- 239000004202 carbamide Substances 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 12
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 12
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 claims description 12
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 12
- 239000004223 monosodium glutamate Substances 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 239000004220 glutamic acid Substances 0.000 claims description 11
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 10
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 10
- 235000013922 glutamic acid Nutrition 0.000 claims description 10
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 9
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 9
- 230000003321 amplification Effects 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 8
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 8
- 238000011081 inoculation Methods 0.000 claims description 8
- 235000015097 nutrients Nutrition 0.000 claims description 8
- 230000001678 irradiating effect Effects 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 239000011790 ferrous sulphate Substances 0.000 claims description 4
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 4
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 4
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 4
- 238000009630 liquid culture Methods 0.000 claims description 4
- 229940099596 manganese sulfate Drugs 0.000 claims description 4
- 239000011702 manganese sulphate Substances 0.000 claims description 4
- 235000007079 manganese sulphate Nutrition 0.000 claims description 4
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 1
- 235000013305 food Nutrition 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 2
- 238000005457 optimization Methods 0.000 abstract description 2
- 238000010276 construction Methods 0.000 abstract 1
- 238000004904 shortening Methods 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 6
- 230000007613 environmental effect Effects 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007790 scraping Methods 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 241000193738 Bacillus anthracis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 229940065181 bacillus anthracis Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000008394 flocculating agent Substances 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of food microorganisms and fermentation, and particularly relates to a method for improving the production capacity of a polyglutamic acid production wild strain. The method comprises seed culture and expanded culture, wherein the seed culture comprises strain plate culture, strain slant culture and shake flask fermentation and the production of polyglutamic acid strain glycerol, and the expanded culture comprises the processes of primary seed culture, secondary seed culture and fermentation culture. According to the invention, through carrying out fermentation optimization model construction on the wild type strain, especially, ultraviolet lamp irradiation cannot be too long or too short, the distance of an ultraviolet lamp cannot be too high or too low, and then the aims of shortening the fermentation period and improving the fermentation yield are taken as targets, the invention further optimizes the fermentation regulation and control process, and as a result, the fermentation yield of the gamma-polyglutamic acid of the PGA-3 strain is improved from 0.3 percent to more than 3g/100ml in a 50L tank, the production cost is greatly reduced on the same ratio, and the strain becomes a strain with industrialization capability.
Description
Technical Field
The invention belongs to the technical field of food microorganisms and fermentation, and particularly relates to a method for improving the production capacity of a polyglutamic acid production wild strain.
Background
Polyglutamic acid (poly-gamma-glutamic acid, abbreviated as PGA) is a water-soluble polyamino acid produced by microbial fermentation in nature, and has a structure in which a glutamic acid unit forms a high-molecular polymer of a peptide bond through an alpha-amino group and a gamma-carboxyl group. The polyglutamic acid has excellent water solubility, super-strong adsorbability and biodegradability, the degradation product is pollution-free glutamic acid, the polyglutamic acid is an excellent environment-friendly high polymer material, can be used as a water-retaining agent, a heavy metal ion adsorbent, a flocculating agent, a slow-release agent, a drug carrier and the like, and has great commercial value and social value in industries such as cosmetics, environmental protection, food, medicine, agriculture, desert control and the like.
Since the discovery of polyglutamic acid has been in history for several decades, the research of polyglutamic acid is mainly in the laboratory stage. In recent years, due to the enhancement of people's environmental consciousness and the requirement of national sustainable development strategy, it is an industrial trend to develop environment-friendly materials and develop products for improving environmental problems, which also promotes the process of industrial research and exploration of polyglutamic acid.
Since Ivanovic et al discovered that the cell capsule of Bacillus anthracis contains gamma-polyglutamic acid in 1937, a plurality of scholars have successively studied gamma-polyglutamic acid, and a plurality of wild strains producing gamma-polyglutamic acid are discovered in the process, but the strains have the problems of low production capacity of gamma-polyglutamic acid and difficulty in realizing industrial production.
Disclosure of Invention
Aiming at the technical problems, the invention provides a method for gradually improving the productivity expression of low-productivity strains so as to realize transformation and upgrade of wild strains into industrial production strains.
In order to solve the technical problems, the technical scheme provided by the invention is as follows: a method for improving the production capacity of the wild-type polyglutamic acid-producing strain comprises seed culture and expanded culture, wherein the seed culture comprises strain plate culture, strain slant culture and shake flask fermentation and the production of polyglutamic acid strain glycerol,
the strain plate culture refers to that the wild strain for producing polyglutamic acid is taken as an initial strain, and 10 percent of the wild strain is prepared－2～10－8Coating the diluted bacteria liquid in a flat substrate, irradiating by an ultraviolet lamp, wrapping the flat with shading paper, and culturing in a constant temperature incubator at 37 +/-1 ℃ for 24 +/-2 h to obtain flat strains;
the slant culture means that starting strains are cultured on a flat plate for 24 +/-2 hours, the diameter of a single strain colony reaches 0.8-1.5 mm, a sticky single strain colony with a high protrusion is selected by an inoculating loop to be arranged in a slant culture medium, and then the single strain colony is cultured for 24 +/-2 hours at the temperature of 37 +/-1 ℃ in a constant temperature incubator to obtain an original slant strain of the single strain;
the shake flask fermentation is that a ring of bacterial lawn is inoculated from an original inclined plane of a single bacterial strain to a shake flask fermentation culture medium, and the single bacterial lawn is cultured in a reciprocating shaking table at a stroke of 75mm and a rotation speed of 110 times/minute at 37 +/-0.5 ℃ until the residual sugar content is less than or equal to 0.5% and put in a flask;
the preparation of the polyglutamic acid glycerol seeds refers to repeated shake flask fermentation culture, single strains with the polyglutamic acid content of more than 15g/l are selected after the first shake flask fermentation and the second shake flask fermentation, slant seeds are scraped into glycerol liquid with the volume ratio of 20 percent to prepare the glycerol seeds, and the glycerol seeds are preserved in a refrigerator at the temperature of minus 72 ℃.
Further: in the above method for improving the production capacity of the polyglutamic acid wild strain, the formula of the shake flask fermentation medium is as follows: 5-8% of glucose, 4-8% of glutamic acid, 0.5-1.5% of yeast extract, 0.1-0.3% of monopotassium phosphate, 0.05-0.3% of magnesium sulfate, 0.1-0.5% of ammonium sulfate and 4% of light calcium carbonate.
The pure plate culture medium and the slant culture medium have the following formula: 0.5-5% of glucose, 0.5-1.5% of yeast powder, 0.5-1.5% of peptone, 2-5% of monosodium glutamate, 0.05-0.3% of urea, 0.1-0.2% of dipotassium phosphate, 0.05-0.5% of magnesium sulfate, 2.0% of agar and pH 7.0.
The formula of the shake flask fermentation medium is as follows: 5-8% of glucose, 4-8% of glutamic acid, 0.5-1.5% of yeast extract, 0.1-0.3% of monopotassium phosphate, 0.05-0.3% of magnesium sulfate, 0.1-0.5% of ammonium sulfate and 4% of light calcium carbonate.
The amplification culture comprises the processes of primary seed culture, secondary seed culture and fermentation culture, wherein the primary seed culture is to inoculate a glycerol seed into a primary seed culture medium, perform seed amplification culture by a reciprocating shaking table (stroke 75mm) and rotating speed of 100 times/min, and culture at 37 +/-0.5 ℃ until logarithmic phase; the first-level seed liquid culture medium comprises 0.5-3% of glucose, 1-3% of yeast extract, 2-5% of monosodium glutamate, 0.05-0.3% of urea, 0.1-0.2% of dipotassium phosphate, 0.05-0.5% of magnesium sulfate and pH7.0.
The stroke of the reciprocating shaking table is 75 mm.
The second-stage seed culture is to inoculate the first-stage seed solution to a fermentation tank of a second-stage seed culture medium according to the inoculation amount of 0.01-0.1%, and the rotation speed of the fermentation tank is 300rpm, and the seed is subjected to seed amplification culture at 37 +/-0.5 ℃ until the seed is cultured to a logarithmic phase;
the secondary seed liquid culture medium comprises 0.5-3% of glucose, 1-3% of yeast extract, 2-5% of monosodium glutamate, 0.05-0.3% of urea, 0.1-0.2% of dipotassium phosphate, 0.05-0.5% of magnesium sulfate and has a pH value of 7.0.
The fermentation culture is to inoculate the secondary seed liquid to a fermentation medium according to the inoculation amount of 5-10%, and perform fermentation culture to obtain polyglutamic acid fermentation liquid;
the formula of the fermentation medium is as follows: 5-8% of glucose, 4-8% of glutamic acid, 0.5-1.5% of yeast extract, 0.1-0.2% of monopotassium phosphate, 0.1-0.2% of magnesium sulfate and 0.1-0.5% of ammonium sulfate.
The fermentation culture conditions are as follows: the pH value is 6.5-7.0, the temperature is 37-39 ℃, the fermentation dissolved oxygen is controlled to be more than or equal to 30%, the residual sugar is maintained at 2-4% when the residual sugar is 3 +/-1%, the nutrient solution is supplemented when the viscosity is more than or equal to 1000CP, and the residual sugar is less than 0.5% and placed in a tank.
The nutrient solution comprises the following formula: 5-100 PPM ferrous sulfate and 5-100 PPM manganese sulfate mixed solution.
Compared with the prior art, the invention has the beneficial effects that: the invention relates to a method for improving the industrial production capacity of a polyglutamic acid-producing wild strain, which is characterized in that a fermentation optimization model is constructed on the wild strain, particularly, ultraviolet lamp irradiation cannot be too long or too short, the distance between ultraviolet lamps cannot be too high or too low, and the ultraviolet lamps are turned on and must be carried out in a dark room. On the basis of the optimal formula and culture conditions obtained by a shake flask test, the fermentation regulation and control process is further optimized by combining the advantages that the 5L tank and the 50L tank have controllable air volume, pH value, dissolved oxygen and feeding, and the fermentation yield of the PGA-3 strain is improved from 0.3 percent to more than 3g/100ml in the 50L tank, the production cost is greatly reduced on the same scale, and the strain becomes a strain with industrialization capability.
Detailed Description
The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention, but are merely illustrative, and the selection of the raw materials in the process can be made according to the circumstances without substantially affecting the result.
Example 1
Taking polyglutamic acid-producing wild strain as starting strain, streaking in slant culture medium, culturing at 37 + -1 deg.C for 24 hr, placing half test tube slant seed in 30ml 0.85% physiological saline, shaking for 5 min, and preparing into 10－2～10－8Coating the diluted bacterium liquid in a flat substrate, keeping the distance between the diluted bacterium liquid and an ultraviolet lamp at 20cm in a black room, opening a dish cover, opening the ultraviolet lamp, irradiating for 30 seconds, closing the ultraviolet lamp for 1 hour, then irradiating for 30 seconds, wrapping the flat plate by using shading paper, and culturing for 24 hours in a constant-temperature incubator at the temperature of 37 ℃ to obtain a flat strain;
selecting a single strain with a colony diameter of 0.8-1.5 mm from the flat strains, selecting the single strain in a slant culture medium by using an inoculating loop, streaking, and culturing at 37 ℃ for 24 hours to obtain a single strain slant seed;
inoculating a circular lawn from a single strain slant strain into a shake flask fermentation medium, culturing in a reciprocating shaking table at a stroke of 75mm and a rotation speed of 110 times/min at 37 +/-0.5 ℃ until the residual sugar content is less than or equal to 0.5%, and placing the bottle;
repeating the shake flask fermentation culture, selecting single strains with homo-polyglutamic acid content of more than 15g/l for the first shake flask fermentation and the second shake flask fermentation, scraping slant seeds into glycerol solution with volume ratio of 20%, making into glycerol seed (code number M36), and storing in refrigerator at-72 deg.C for use.
Inoculating the glycerol seeds into a first-level seed culture medium, reciprocating a shaking table, performing seed amplification culture at 37 +/-0.5 ℃ at a stroke of 75mm and a rotating speed of 100-110 times/min until the culture reaches a logarithmic phase;
inoculating the primary seed liquid in the shake flask into a seed tank of a secondary seed culture medium according to the inoculation amount of 0.05%, carrying out seed amplification culture at the rotation speed of the seed tank of 150-300 rpm at 37 +/-0.5 ℃, and culturing to a logarithmic phase;
inoculating the secondary seed liquid into a fermentation tank for producing a fermentation culture medium according to the inoculation amount of 5 percent, wherein the fermentation culture conditions are as follows: and (3) controlling the pH to be 7.0 and the temperature to be 37 ℃, controlling the fermentation dissolved oxygen to be more than or equal to 30%, starting to supplement sugar when the residual sugar is 3% to maintain 0.5-4% of the residual sugar, supplementing the fermentation nutrient solution when the viscosity is more than or equal to 1000CP, and ending the fermentation when the residual sugar is less than 0.5% to obtain the polyglutamic acid fermentation broth. The content of the polyglutamic acid strain in a 50L tank by a liquid phase method is 3.2g/100 ml.
The pure plate culture medium and the slant culture medium have the following formula: 1% of glucose, 1.5% of yeast powder, 1% of peptone, 5% of monosodium glutamate, 0.1% of urea, 0.1% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate, 2.0% of agar and pH 7.0.
The formula of the shake flask fermentation medium is as follows: 5% of glucose, 4% of glutamic acid, 0.5% of yeast extract, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.1% of ammonium sulfate and 4% of light calcium carbonate.
The formula of the primary seed culture medium comprises 1.5% of glucose, 1% of yeast extract, 2% of monosodium glutamate, 0.05% of urea, 0.1% of dipotassium phosphate, 0.05% of magnesium sulfate and pH7.0.
The secondary seed culture medium comprises 3% of glucose, 1% of yeast extract, 2% of monosodium glutamate, 0.05% of urea, 0.1% of dipotassium hydrogen phosphate, 0.1% of magnesium sulfate and pH7.0.
The formula of the production fermentation medium is as follows: 10% of glucose, 4% of glutamic acid, 1.0% of yeast extract, 0.15% of monopotassium phosphate, 0.1% of magnesium sulfate and 0.3% of ammonium sulfate.
The formula of the fermentation nutrient solution is as follows: 5ppm of ferrous sulfate and 5ppm of manganese sulfate.
Example 2
Taking polyglutamic acid-producing wild strain as starting strain, streaking in slant culture medium, culturing at 37 + -1 deg.C for 24 hr, placing half test tube slant seed in 30ml 0.85% physiological saline, shaking for 5 min, and preparing into 10－2～10－8Coating the diluted bacterium liquid in a flat substrate, keeping the distance between the diluted bacterium liquid and an ultraviolet lamp at 20cm in a black room, opening a dish cover, opening the ultraviolet lamp for irradiating for 30 seconds, closing the ultraviolet lamp for 2 hours, then irradiating for 30 seconds, wrapping the flat plate by using shading paper, and culturing for 24 hours in a constant-temperature incubator at the temperature of 37 ℃ to obtain a flat strain;
selecting a single strain with a colony diameter of 0.8-1.5 mm from the flat strains, selecting the single strain in a slant culture medium by using an inoculating loop, streaking, and culturing at 37 ℃ for 24 hours to obtain a single strain slant seed;
inoculating a circular lawn from a single strain slant strain into a shake flask fermentation medium, culturing in a reciprocating shaking table at a stroke of 75mm and a rotation speed of 110 times/min at 37 +/-0.5 ℃ until the residual sugar content is less than or equal to 0.5%, and placing the bottle;
repeating the shake flask fermentation culture, selecting single strains with homo-polyglutamic acid content of more than 15g/l for the first shake flask fermentation and the second shake flask fermentation, scraping slant seeds into glycerol solution with volume ratio of 20%, making into glycerol strain (generation M60), and storing in refrigerator at-72 deg.C for use.
Inoculating the glycerol seeds into a first-level seed culture medium, reciprocating a shaking table, performing seed amplification culture at 37 +/-0.5 ℃ at a stroke of 75mm and a rotating speed of 100-110 times/min until the culture reaches a logarithmic phase;
inoculating the primary seed liquid in the shake flask into a seed tank of a secondary seed culture medium according to the inoculation amount of 0.05%, carrying out seed amplification culture at the rotation speed of the seed tank of 150-300 rpm at 37 +/-0.5 ℃, and culturing to a logarithmic phase;
inoculating the second-stage seed liquid into a fermentation tank for producing a fermentation culture medium according to the inoculation amount of 10%, wherein the fermentation culture conditions are as follows: and (3) controlling the pH to be 6.5-7.0 and the temperature to be 37-39 ℃, controlling the fermentation dissolved oxygen to be more than or equal to 30%, starting to supplement sugar when the residual sugar is 3 +/-1%, maintaining 0.5-4% of the residual sugar, supplementing the fermentation nutrient solution when the viscosity is more than or equal to 1000CP, and ending the fermentation when the residual sugar is less than 0.5% to obtain the polyglutamic acid fermentation broth. The content of the polyglutamic acid strain in a 50L tank by a liquid phase method is 3.1g/100 ml.
The pure plate culture medium and the slant culture medium have the following formula: 1% of glucose, 1% of yeast powder, 1% of peptone, 5% of monosodium glutamate, 0.1% of urea, 0.1% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate, 2.0% of agar and pH7.0.
The formula of the shake flask fermentation medium is as follows: 5% of glucose, 4% of glutamic acid, 0.5% of yeast extract, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.1% of ammonium sulfate and 4% of light calcium carbonate.
The formula of the primary seed culture medium comprises 1.5% of glucose, 1% of yeast extract, 2% of monosodium glutamate, 0.05% of urea, 0.1% of dipotassium phosphate, 0.05% of magnesium sulfate and pH7.0.
The secondary seed culture medium comprises 3% of glucose, 1% of yeast extract, 2% of monosodium glutamate, 0.05% of urea, 0.1% of dipotassium hydrogen phosphate, 0.1% of magnesium sulfate and pH7.0.
The formula of the production fermentation medium is as follows: glucose 8%, glutamic acid 4%, yeast extract 1.0%, potassium dihydrogen phosphate 0.15%, magnesium sulfate 0.1%, and ammonium sulfate 0.3%.
The formula of the fermentation nutrient solution is as follows: 50ppm ferrous sulfate and 50ppm manganese sulfate.
The foregoing is only a preferred embodiment of this invention and any obvious combination of alternatives, modifications and variations thereof are within the scope of the invention without departing from the spirit of the invention. It should be understood that the examples are merely for illustrative purposes and are not intended to limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A method for improving the production capacity of a wild-type strain for producing polyglutamic acid comprises seed culture and expanded culture, wherein the seed culture comprises strain plate culture, strain slant culture, shake flask fermentation and glycerol strain production of polyglutamic acid;
the strain plate culture is to prepare 10 of wild strain for producing polyglutamic acid as an initial strain－2～10－8Coating the diluted bacteria liquid in a flat substrate, irradiating by an ultraviolet lamp, wrapping the flat with shading paper, and culturing in a constant temperature incubator at 37 +/-1 ℃ for 24 +/-2 h to obtain flat strains;
the slant culture is to select a plate strain with a colony diameter of 0.8-1.5 mm, select the plate strain in a slant culture medium by using an inoculating loop, streak the plate strain, and culture the plate strain at 37 +/-1 ℃ for 24 +/-2 hours to obtain a single strain slant seed;
the shake flask fermentation is that a ring bacterial lawn is inoculated from an original inclined plane of a single bacterial strain to a shake flask fermentation medium, the mixture is cultured in a reciprocating shaking table at the rotating speed of 110 times/minute at 37 +/-0.5 ℃ until the residual sugar content is less than or equal to 0.5% and put in a flask;
the method for preparing the polyglutamic acid glycerol seeds refers to repeated shake flask fermentation culture, single strains with the polyglutamic acid content of more than 15g/l are selected after the first shake flask fermentation and the second shake flask fermentation, and slant seeds are scraped into glycerol liquid with the volume ratio of 20% to prepare the glycerol seeds.
2. The method for improving the productivity of a polyglutamic acid-producing wild type strain according to claim 1, wherein: the ultraviolet lamp irradiation means opening the dish cover, opening the ultraviolet lamp for 30 +/-2 seconds, closing the ultraviolet lamp for 1.5 +/-0.5 hours, and then irradiating the ultraviolet lamp for 30 +/-2 seconds.
3. The method for improving the productivity of a polyglutamic acid-producing wild type strain according to claim 1, wherein:
the pure plate culture medium and the slant culture medium have the following formula: 0.5-5% of glucose, 0.5-1.5% of yeast powder, 0.5-1.5% of peptone, 2-5% of monosodium glutamate, 0.05-0.3% of urea, 0.1-0.2% of dipotassium phosphate, 0.05-0.5% of magnesium sulfate, 2.0% of agar and pH 7.0.
4. The method for improving the productivity of a polyglutamic acid-producing wild type strain according to claim 1, wherein: the formula of the shake flask fermentation medium is as follows: 5-8% of glucose, 4-8% of glutamic acid, 0.5-1.5% of yeast extract, 0.1-0.3% of monopotassium phosphate, 0.05-0.3% of magnesium sulfate, 0.1-0.5% of ammonium sulfate and 4% of light calcium carbonate.
5. The method for improving the productivity of a polyglutamic acid-producing wild type strain according to claim 1, wherein: the enlarged culture comprises the processes of primary seed culture, secondary seed culture and fermentation culture.
6. The method for improving the productivity of a polyglutamic acid-producing wild type strain according to claim 5, wherein: the first-stage seed culture is to inoculate the glycerol seeds into a first-stage seed culture medium, perform seed amplification culture by reciprocating a shaking table at a rotating speed of 100 times/min, and culture at 37 +/-0.5 ℃ until the logarithmic phase; the first-level seed liquid culture medium comprises 0.5-3% of glucose, 1-3% of yeast extract, 2-5% of monosodium glutamate, 0.05-0.3% of urea, 0.1-0.2% of dipotassium phosphate, 0.05-0.5% of magnesium sulfate and 7.0% of PH.
7. The method for improving the productivity of a polyglutamic acid-producing wild type strain according to claim 1 or 6, wherein: the stroke of the reciprocating shaking table is 75 mm.
8. The method for improving the productivity of a polyglutamic acid-producing wild type strain according to claim 5, wherein: the second-stage seed culture is to inoculate the first-stage seed solution to a fermentation tank of a second-stage seed culture medium according to the inoculation amount of 0.01-0.1%, and the rotation speed of the fermentation tank is 300rpm, and the seed is subjected to seed amplification culture at 37 +/-0.5 ℃ until the seed is cultured to a logarithmic phase;
the secondary seed liquid culture medium comprises 0.5-3% of glucose, 1-3% of yeast extract, 2-5% of monosodium glutamate, 0.05-0.3% of urea, 0.1-0.2% of dipotassium phosphate, 0.05-0.5% of magnesium sulfate and has a pH value of 7.0.
9. The method for improving the productivity of a polyglutamic acid-producing wild type strain according to claim 5, wherein: the fermentation culture is to inoculate the secondary seed liquid to a fermentation medium according to the inoculation amount of 5-10%, and perform fermentation culture to obtain polyglutamic acid fermentation liquid;
the formula of the fermentation medium is as follows: 5-8% of glucose, 4-8% of glutamic acid, 0.5-1.5% of yeast extract, 0.1-0.2% of monopotassium phosphate, 0.1-0.2% of magnesium sulfate and 0.1-0.5% of ammonium sulfate;
the fermentation culture conditions are as follows: the pH value is 6.5-7.0, the temperature is 37-39 ℃, the fermentation dissolved oxygen is controlled to be more than or equal to 30%, the residual sugar is maintained at 2-4% when the residual sugar is 3 +/-1%, the nutrient solution is supplemented when the viscosity is more than or equal to 1000CP, and the residual sugar is less than 0.5% and placed in a tank.
10. The method for improving the productivity of a polyglutamic acid-producing wild type strain according to claim 9, wherein: the nutrient solution comprises the following formula: 5-100 PPM ferrous sulfate and 5-100 PPM manganese sulfate mixed solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111109424.8A CN113957033A (en) | 2021-09-22 | 2021-09-22 | Method for improving production capacity of polyglutamic acid-producing wild strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111109424.8A CN113957033A (en) | 2021-09-22 | 2021-09-22 | Method for improving production capacity of polyglutamic acid-producing wild strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113957033A true CN113957033A (en) | 2022-01-21 |
Family
ID=79462143
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111109424.8A Pending CN113957033A (en) | 2021-09-22 | 2021-09-22 | Method for improving production capacity of polyglutamic acid-producing wild strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113957033A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385718A (en) * | 2014-05-21 | 2016-03-09 | 武汉慧宝康源医学研究有限责任公司 | Fermentation preparation method of gamma-polyglutamic acid and strain |
| CN110129216A (en) * | 2019-04-16 | 2019-08-16 | 东莞理工学院 | A kind of bacillus subtilis mutagenic strain and its cultural method suitable for solid fermentation production gamma-polyglutamic acid |
| CN110129225A (en) * | 2019-05-14 | 2019-08-16 | 山东汉泰生物科技有限公司 | γ~polyglutamic acid producing strains and breeding prepare γ~polyglutamic acid method |
-
2021
- 2021-09-22 CN CN202111109424.8A patent/CN113957033A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385718A (en) * | 2014-05-21 | 2016-03-09 | 武汉慧宝康源医学研究有限责任公司 | Fermentation preparation method of gamma-polyglutamic acid and strain |
| CN110129216A (en) * | 2019-04-16 | 2019-08-16 | 东莞理工学院 | A kind of bacillus subtilis mutagenic strain and its cultural method suitable for solid fermentation production gamma-polyglutamic acid |
| CN110129225A (en) * | 2019-05-14 | 2019-08-16 | 山东汉泰生物科技有限公司 | γ~polyglutamic acid producing strains and breeding prepare γ~polyglutamic acid method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102168115A (en) | Industrialized production method of coenzyme Q10 | |
| CN109082448B (en) | Escherichia coli and application thereof in fermentation production of 1, 5-pentanediamine | |
| CN107267422B (en) | Comamonas testosteroni HHALA-001 and method for producing L-alanine by using same | |
| CN111363707A (en) | Method for improving acid production rate and conversion rate of butyric acid fermentation | |
| CN103451244B (en) | A kind of faecium is preparing the application in Pfansteihl | |
| CN101565723B (en) | Fermentation production technique of L-tryptophan | |
| CN112831437B (en) | Bacillus subtilis and fermentation method for high yield of indoleacetic acid and high spore formation rate | |
| CN101153294B (en) | Immobilized cell single-tank high-strength continuous fermentation process for succinic acid | |
| CN110964760A (en) | Mutagenesis breeding method of high-yield gamma-aminobutyric acid strain | |
| CN110656065B (en) | Streptomyces for producing epsilon-polylysine and application thereof | |
| CN116333948B (en) | Clostridium aerophilum enrichment medium and preparation method thereof | |
| CN111019995B (en) | Method for producing vanillin by fermentation with eugenol as substrate | |
| CN102719502A (en) | Method for producing L-alanine by mutating lactate-production bacteria | |
| CN112852891A (en) | Artificial dual-bacterium system for producing mcl-PHA and application thereof | |
| CN108641961B (en) | Method for high-density culture of guava leaf endophytes | |
| RU2439143C1 (en) | Cupriavidus eutrophus VKPM B-10646 BACTERIA STRAIN - PRODUCER OF POLYHYDROXY ALKANOATES AND PRODUCTION METHOD THEREOF | |
| CN113957033A (en) | Method for improving production capacity of polyglutamic acid-producing wild strain | |
| CN1952164A (en) | Combined fermentation process for producing D-lactic acid | |
| CN103667107B (en) | A kind of manure enterococcin strain producing Pfansteihl | |
| CN104561139A (en) | Method for increasing final cell density of microorganisms and shortening culture time | |
| CN110452862B (en) | Pseudomonas fluorescens strain and application thereof | |
| CN111172212A (en) | Fermentation method of high-content polyglutamic acid | |
| CN114058514A (en) | Method for accumulating starch by using marine green algae, Qingdao and Pantoea galbana | |
| CN113604390A (en) | Corynebacterium glutamicum and application thereof in fermentation production of L-ornithine | |
| CN115637233A (en) | Bacterial strain for high yield of dodecanedioic acid and fermentation method |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220121 |
|
| RJ01 | Rejection of invention patent application after publication |