CN116287041A - Production method of gamma-polyglutamic acid fermentation broth with low chloride ion content - Google Patents
Production method of gamma-polyglutamic acid fermentation broth with low chloride ion content Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 71
- 230000004151 fermentation Effects 0.000 title claims abstract description 71
- 229920002643 polyglutamic acid Polymers 0.000 title claims abstract description 49
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 title claims abstract description 24
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000001963 growth medium Substances 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 18
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 18
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229940024606 amino acid Drugs 0.000 claims abstract description 16
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 15
- -1 compound amino acid Chemical class 0.000 claims abstract description 12
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- 229910052799 carbon Inorganic materials 0.000 claims abstract description 6
- 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 abstract description 6
- 235000013923 monosodium glutamate Nutrition 0.000 claims abstract description 6
- 229940073490 sodium glutamate Drugs 0.000 claims abstract description 6
- 230000003321 amplification Effects 0.000 claims abstract description 3
- 239000000463 material Substances 0.000 claims abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 3
- 230000001502 supplementing effect Effects 0.000 claims abstract description 3
- 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 14
- 235000001014 amino acid Nutrition 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 14
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 11
- 238000011218 seed culture Methods 0.000 claims description 11
- 230000001954 sterilising effect Effects 0.000 claims description 11
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 235000003704 aspartic acid Nutrition 0.000 claims description 6
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 238000005273 aeration Methods 0.000 claims description 5
- 239000004202 carbamide Substances 0.000 claims description 5
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 5
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 4
- 239000004254 Ammonium phosphate Substances 0.000 claims description 3
- 229910000148 ammonium phosphate Inorganic materials 0.000 claims description 3
- 235000019289 ammonium phosphates Nutrition 0.000 claims description 3
- 238000009629 microbiological culture Methods 0.000 claims description 3
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 2
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 2
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 2
- 235000013877 carbamide Nutrition 0.000 claims description 2
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 2
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 2
- 239000006012 monoammonium phosphate Substances 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 239000005696 Diammonium phosphate Substances 0.000 claims 1
- 150000001413 amino acids Chemical class 0.000 abstract description 5
- 239000003102 growth factor Substances 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 125000001477 organic nitrogen group Chemical group 0.000 abstract description 3
- 235000010633 broth Nutrition 0.000 description 23
- 239000003337 fertilizer Substances 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 9
- 239000002689 soil Substances 0.000 description 4
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 239000000460 chlorine Substances 0.000 description 3
- 229910052801 chlorine Inorganic materials 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 239000002994 raw material Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 1
- 244000241235 Citrullus lanatus Species 0.000 description 1
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 1
- 229930182847 D-glutamic acid Natural products 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-Glutamic acid Natural products OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 235000021536 Sugar beet Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
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- 230000035764 nutrition Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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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
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- 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
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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Abstract
The invention relates to the technical field of microbial fermentation, in particular to a production method of gamma-polyglutamic acid fermentation broth with low chloride ion content, which comprises the following steps: the bacillus subtilis is subjected to amplification culture to obtain seed liquid; a fermentation initial culture medium is prepared, and the components and the contents of the fermentation initial culture medium are as follows: 50-100 g/L sodium glutamate, 2-5 g/L, mgSO citric acid 4 0.1～0.5g/L、K 2 HPO 4 1-4 g/L, 0-5 g/L of compound amino acid, 1-10 g/L of inorganic nitrogen source and the balance of water; inoculating the seed liquid into a fermentation initial culture medium for culture, and adding a carbon source and an inorganic nitrogen source through material supplementing in the culture process to obtain the gamma-polyglutamic acid fermentation broth through culture. The invention greatly reduces the production by adding the inorganic nitrogen source with low cost and adding a small amount of amino acid growth factors to replace the organic nitrogen source with higher priceThe cost is low, and the chloride ion content in the prepared gamma-polyglutamic acid fermentation broth is low.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a production method of gamma-polyglutamic acid fermentation broth with low chloride ion content.
Background
The gamma-polyglutamic acid (gamma-PGA) is formed by combining D-glutamic acid or L-glutamic acid through an amide bond between alpha-amino and gamma-carboxyl, is an extracellular water-soluble high molecular amino acid polymer synthesized by certain Bacillus sp, and has excellent film forming property, cohesiveness and extremely strong water absorption. In the agricultural field, the gamma-polyglutamic acid serving as a fertilizer synergist not only can increase the yield of crops, but also can play a good slow-release role on fertilizer and moisture, and has remarkable effects of water retention, fertilizer retention, yield increase and fertilizer conservation. The gamma-polyglutamic acid fertilizer is high in the main agriculture developed countries in the world, wherein the gamma-polyglutamic acid fertilizer accounts for 55% of the fertilizer consumption in the United states, and more than 90% of crops in israel use the gamma-polyglutamic acid fertilizer.
At present, gamma-polyglutamic acid is mainly produced by microbial fermentation, and organic nitrogen sources such as peptone, yeast powder and the like are needed to be added into a fermentation medium, so that on one hand, the fermentation cost is increased, the price of the gamma-polyglutamic acid is high, and on the other hand, the chloride ion content in the produced gamma-polyglutamic acid fermentation liquor is high, and the fertilizer production, the fertilization effect and the soil environment are influenced. Firstly, the high-pressure system of the fertilizer production device has high requirement on chloride ion control, and when the gamma-polyglutamic acid stock solution is added into a urea evaporation system to produce novel urea, the chloride ion content in the raw materials is required to be controlled below 100 mg/kg. The chloride ions can promote the hydrolysis of carbohydrates, so that the sugar content of watermelons, sugar beets and glucose can be reduced; when chloride ions are more, the seedlings of sensitive crops are often damaged. Thirdly, a great amount of chloride ions remain in the soil due to the fact that the chlorine-containing fertilizer is applied in a great amount or for a long time, the conditions of hardening, salinization, alkalization and the like of the soil are easy to occur, the soil environment is deteriorated, and therefore the nutrient absorption capacity of crops is reduced.
Therefore, the search for a production method of the high-efficiency energy-saving gamma-polyglutamic acid fermentation broth with low chloride ion content is a problem to be solved by scientific researchers in the field.
Disclosure of Invention
Aiming at the technical problems of higher cost and higher chlorine content of the produced gamma-polyglutamic acid fermentation broth in the existing production method of the gamma-polyglutamic acid fermentation broth, the invention provides the production method of the gamma-polyglutamic acid fermentation broth with low chlorine ion content, which effectively improves the fermentation yield of the gamma-polyglutamic acid, reduces the chlorine ion content in the gamma-polyglutamic acid fermentation broth and simultaneously reduces the production cost.
The invention provides a production method of gamma-polyglutamic acid fermentation broth with low chloride ion content, which specifically comprises the following steps:
(1) The bacillus subtilis is subjected to amplification culture to obtain seed liquid;
(2) A fermentation initial culture medium is prepared, and the components and the contents of the fermentation initial culture medium are as follows: 50-100 g/L sodium glutamate, 2-5 g/L, mgSO citric acid 4 0.1～0.5g/L、K 2 HPO 4 1-4 g/L, 0-5 g/L of compound amino acid, 1-10 g/L of inorganic nitrogen source, and the balance of water, regulating pH to 7.2-7.5, and sterilizing at high temperature;
(3) Inoculating the seed liquid into a fermentation initial culture medium for culture, and adding a carbon source and an inorganic nitrogen source through material supplementing in the culture process to obtain the gamma-polyglutamic acid fermentation broth through culture.
Further, the bacillus subtilis is bacillus subtilis FRD518, and bacillus subtilis (Bacillus subtilis) FRD518 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 6772 in the year 11 and 02 of 2012.
Further, in the step (1), 1mL of frozen bacillus subtilis seed liquid is inoculated into a 500mL triangular flask filled with 100mL of seed culture medium, shake culture is carried out at 35-38 ℃ by a shaking table at 180-250 r/min until the OD600 of the seeds reaches 1.6-2.2, and first-stage seed liquid is obtained; inoculating the first-stage seed liquid into a seed tank, and culturing at 35-38 ℃ under stirring and aeration to obtain a second-stage seed liquid.
Further, the seed culture medium used for the primary seed and the secondary seed comprises the following components in percentage by weight: glucose 5-10 g/L, yeast powder 3-5 g/L, ammonium sulfate 2-10 g/L, mgSO 4 0.1-1.0 g/L, water as the rest, pH 7.2-7.5, and sterilizing at 115 deg.C for 30min.
Further, in the step (2), the high-temperature sterilization is specifically performed at 115 ℃ for 30min.
Further, in the step (3), the inoculation amount is 3% -5%, and the fermentation tank is aerated and stirred for culture at 35-38 ℃.
Further, the carbon source is glucose, the feeding mode is fed-batch feeding, and the concentration of the glucose is controlled to be 5-15 g/L.
Further, the inorganic nitrogen source is one or more of ammonium sulfate, ammonium nitrate, ammonium phosphate, diammonium hydrogen phosphate, monoammonium phosphate and urea, and the feeding mode is fed-batch feeding, and the feeding rate is 0.1-1 g/(L.h).
Further, the compound amino acid is two or more of tyrosine, phenylalanine and aspartic acid.
The invention has the beneficial effects that:
1. according to the production method of the gamma-polyglutamic acid fermentation broth with low chloride ion content, provided by the invention, the low inorganic nitrogen source is added in a flowing manner, and a small amount of amino acid growth factors are added to replace the organic nitrogen source with higher price to ferment and produce the gamma-polyglutamic acid, so that the production cost is greatly reduced, the fermentation medium does not contain chloride ions, and the chloride ion content in the prepared gamma-polyglutamic acid fermentation broth is lower.
2. According to the invention, the fermentation medium and the fermentation process are optimized, the carbon and nitrogen levels in the fermentation medium are controlled, so that bacillus subtilis is in a starvation state, the bacterial growth is balanced with the synthesis of gamma-polyglutamic acid, and the fermentation raw material utilization rate is improved. Meanwhile, the culture medium used by the invention has simple components, is favorable for the subsequent treatment of fermentation liquor, and has higher purity for preparing the cosmetic-grade gamma-polyglutamic acid.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
The bacillus subtilis (Bacillus subtilis) FRD518 used in the following examples was deposited in China general microbiological culture Collection center, with the accession number CGMCC No.6772, on the 11 th month 02 of 2012, which strain was disclosed in the applicant's prior application CN 201210555304.5.
Example 1
(1) Preparing a seed culture medium, wherein the seed culture medium comprises the following components in percentage by weight: 10g/L glucose, 5g/L yeast powder, 8g/L ammonium sulfate and MgSO 4 0.8g/L, water as the rest, pH 7.2, sterilizing at 115 deg.C for 30min.
(2) The primary seed and the secondary seed are inoculated into 1mL of frozen bacillus subtilis FRD518 seed liquid in a 500mL triangular flask filled with 100mL of seed culture medium, shake culture is carried out at 35 ℃ and 180r/min shaking table, and the seed is cultivated until the seed OD 600 Reaching 1.6 to obtain first-level seed liquid; inoculating the first-stage seed liquid into a seed tank, and culturing at 36 ℃ under stirring and aeration to obtain a second-stage seed liquid.
(3) A fermentation initial culture medium is prepared, and the components and the contents of the fermentation initial culture medium are as follows: 80g/L sodium glutamate, 4g/L, mgSO citric acid 4 0.5g/L、K 2 HPO 4 3g/L, 3g/L of compound amino acid, 8g/L of ammonium sulfate and the balance of water, adjusting pH to 7.2-7.5, and sterilizing for 30min at 115 ℃; wherein the compound amino acid comprises tyrosine, phenylalanine and aspartic acid, and the content ratio of the compound amino acid to the aspartic acid is 1:1:1.
(4) Inoculating the secondary seed liquid into the fermentation initial culture medium, wherein the inoculum size is 5%, ventilating and stirring the fermentation tank at 37 ℃ for culture, feeding glucose and ammonium sulfate in the culture process, controlling the glucose concentration to be 5-15 g/L, controlling the feeding rate of ammonium sulfate to be 0.2 g/(L.h), and culturing for 44h to obtain the gamma-polyglutamic acid fermentation broth.
Example 2
(1) Preparing a seed culture medium, wherein the seed culture medium comprises the following components in percentage by weight: glucose 5g/L, yeast powder 3g/L, ammonium sulfate 3g/L, mgSO 4 0.2g/L, water as the rest, pH 7.5, sterilizing at 115 deg.C for 30min.
(2) The primary seed and the secondary seed are inoculated in a 500mL triangular flask filled with 100mL of seed culture medium by 1mL of frozen bacillus subtilis FRD518 seed liquid by using the seed culture medium, and shake culture is carried out at 37 ℃ and 220r/min by a shaking table until the seed OD is obtained 600 Reaching 2.0 to obtain first-level seed liquid; inoculating the first-stage seed liquid into a seed tank, and culturing at 37 ℃ under stirring and aeration to obtain a second-stage seed liquid.
(3) A fermentation initial culture medium is prepared, and the components and the contents of the fermentation initial culture medium are as follows: 60g/L sodium glutamate, 2g/L, mgSO citric acid 4 0.1g/L、K 2 HPO 4 1.0g/L, 1g/L of compound amino acid, 8g/L of ammonium phosphate and the balance of water, adjusting the pH value to 7.5, and sterilizing for 30min at 115 ℃; wherein the compound amino acid comprises tyrosine, phenylalanine and aspartic acid, and the content ratio of the compound amino acid to the aspartic acid is 3:4:4.
(4) Inoculating the secondary seed liquid into the fermentation initial culture medium, wherein the inoculum size is 3%, ventilating and stirring the fermentation tank at 35 ℃ for culture, feeding glucose and inorganic nitrogen source (the mixture of ammonium sulfate and urea with the mass ratio of 1:2) in the culture process, controlling the glucose concentration to be 5-15 g/L, and the feeding rate of the inorganic nitrogen source to be 0.3 g/(L.h), and culturing for 48 hours to obtain the gamma-polyglutamic acid fermentation broth.
Comparative example
(1) The seed liquid was prepared in the same manner as in example 2.
(2) According to the components of the fermentation medium described in patent application CN202210521932.5, a fermentation medium is prepared, and the components and contents thereof are as follows: 15g/L peptone, 15g/L yeast extract, 15g/L ammonium sulfate, 60g/L sodium glutamate, 2g/L citric acid, mgSO 4 0.1g/L，K 2 HPO 4 1.0g/L, dextran150g/L glucose, adjusting pH to 7.5, and sterilizing at 115deg.C for 30min.
(3) Inoculating the seed liquid into the fermentation culture medium, wherein the inoculum size is 3%, and culturing in a 35 ℃ fermentation tank under aeration and stirring for 48h to obtain the gamma-polyglutamic acid fermentation broth.
Test case
The gamma-polyglutamic acid content and the chloride ion content in the gamma-polyglutamic acid fermentation broths prepared in example 2 and comparative example were examined, and the results obtained are shown in table 1.
TABLE 1 quality comparison of fermentation broths of gamma-polyglutamic acid produced by fermentation using two methods
| Sample of | Gamma-polyglutamic acid content/% | Chloride ion content/(mg/L) in fermentation broth |
| Example 2 | 7.65 | 43 |
| Comparative example | 5.51 | 2897 |
As can be seen from Table 1, the content of chloride ions in the fermentation broth of gamma-polyglutamic acid prepared in example 2 was less than 100mg/L, whereas the content of chloride ions in the fermentation broth of gamma-polyglutamic acid prepared in comparative example was higher, reaching 2897mg/L, mainly because peptone and yeast powder contained chlorine in the fermentation medium used in comparative example, whereas the fermentation medium of example 2 used inorganic nitrogen source and added small amounts of amino acid growth factors, and contained no or very little chloride ions. In addition, the fermentation yield of gamma-polyglutamic acid of example 2 was higher than that of the comparative example, and thus it was found that the inorganic nitrogen source and amino acid used in the fermentation medium of example 2 maintained the normal metabolism of the cells to produce a large amount of gamma-polyglutamic acid, whereas the fermentation medium of the comparative example was rich in nutrition, and the growth of the cells was large, and the production of gamma-polyglutamic acid was relatively small.
Example 3
By using the method for separating and purifying gamma-polyglutamic acid from fermentation broth described in example 3 of patent application CN201510193843.2, cosmetic-grade high-purity gamma-polyglutamic acid was separated and purified from the gamma-polyglutamic acid fermentation broth prepared in example 2 and comparative example, and the purity of gamma-polyglutamic acid finally obtained from the gamma-polyglutamic acid fermentation broth prepared in example 2 was 97.84%, and the purity of gamma-polyglutamic acid obtained from the gamma-polyglutamic acid fermentation broth prepared in comparative example was 96.43%.
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (9)
1. The production method of the gamma-polyglutamic acid fermentation broth with low chloride ion content is characterized by comprising the following steps of:
(1) The bacillus subtilis is subjected to amplification culture to obtain seed liquid;
(2) A fermentation initial culture medium is prepared, and the components and the contents of the fermentation initial culture medium are as follows: 50-100 g/L sodium glutamate, 2-5 g/L, mgSO citric acid 4 0.1～0.5g/L、K 2 HPO 4 1-4 g/L, 0-5 g/L of compound amino acid, 1-10 g/L of inorganic nitrogen source, and the balance of water, regulating pH to 7.2-7.5, and sterilizing at high temperature;
(3) Inoculating the seed liquid into a fermentation initial culture medium for culture, and adding a carbon source and an inorganic nitrogen source through material supplementing in the culture process to obtain the gamma-polyglutamic acid fermentation broth through culture.
2. The method of claim 1, wherein the bacillus subtilis is bacillus subtilis FRD518, and bacillus subtilis (Bacillus subtilis) FRD518 is deposited in China general microbiological culture collection center (CGMCC) No.6772, 11/02/2012.
3. The production method according to claim 1, wherein in the step (1), 1mL of the frozen bacillus subtilis seed liquid is inoculated into a 500mL triangular flask filled with 100mL of seed culture medium, shake culture is carried out at 35-38 ℃ at 180-250 r/min, and the culture is carried out until the OD of the seeds is reached 600 Reaching 1.6 to 2.2 to obtain first-stage seed liquid; inoculating the first-stage seed liquid into a seed tank, and culturing at 35-38 ℃ under stirring and aeration to obtain a second-stage seed liquid.
4. A method according to claim 3, wherein the primary seed and the secondary seed are used in a seed culture medium comprising the following components and contents: glucose 5-10 g/L, yeast powder 3-5 g/L, ammonium sulfate 2-10 g/L, mgSO 4 0.1-1.0 g/L, water as the rest, pH 7.2-7.5, and sterilizing at 115 deg.C for 30min.
5. The method according to claim 1, wherein in step (2), the high temperature sterilization is specifically carried out at 115℃for 30min.
6. The process according to claim 1, wherein in the step (3), the inoculation amount is 3% -5%, and the fermentation tank is aerated and stirred at 35-38 ℃.
7. The production method according to claim 1, wherein the carbon source is glucose, the feeding mode is fed-batch feeding, and the glucose concentration is controlled to be 5-15 g/L.
8. The production method according to claim 1, wherein the inorganic nitrogen source is one or more of ammonium sulfate, ammonium nitrate, ammonium phosphate, diammonium phosphate, monoammonium phosphate and urea, and the feeding mode is fed-batch feeding, and the feeding rate is 0.1-1 g/(L.h).
9. The method according to claim 1, wherein the compound amino acid is two or more of tyrosine, phenylalanine and aspartic acid.
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