CN116445559A - Production method of glutamic acid independent gamma-polyglutamic acid - Google Patents
Production method of glutamic acid independent gamma-polyglutamic acid Download PDFInfo
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- 229920002643 polyglutamic acid Polymers 0.000 title claims abstract description 40
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 235000013922 glutamic acid Nutrition 0.000 title claims abstract description 32
- 239000004220 glutamic acid Substances 0.000 title claims abstract description 32
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 20
- 238000000855 fermentation Methods 0.000 claims abstract description 73
- 230000004151 fermentation Effects 0.000 claims abstract description 73
- 238000011218 seed culture Methods 0.000 claims abstract description 15
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 12
- 230000004913 activation Effects 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 239000002609 medium Substances 0.000 claims description 20
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 13
- 229940041514 candida albicans extract Drugs 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 239000012138 yeast extract Substances 0.000 claims description 12
- 238000009423 ventilation Methods 0.000 claims description 11
- 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 9
- 239000008103 glucose Substances 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 239000002054 inoculum Substances 0.000 claims description 6
- 230000003213 activating effect Effects 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 2
- 238000012136 culture method Methods 0.000 claims 1
- 238000001994 activation Methods 0.000 abstract description 6
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 27
- 229960002989 glutamic acid Drugs 0.000 description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 8
- 239000000843 powder Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000012827 research and development Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 description 2
- 229930182847 D-glutamic acid Natural products 0.000 description 2
- 108010020346 Polyglutamic Acid Proteins 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940049906 glutamate Drugs 0.000 description 2
- 229930195712 glutamate Natural products 0.000 description 2
- 229920001519 homopolymer Polymers 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000007888 film coating Substances 0.000 description 1
- 238000009501 film coating Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 229920005604 random copolymer Polymers 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
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- C12P13/00—Preparation of nitrogen-containing organic compounds
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Abstract
The invention provides a production method of glutamic acid independent gamma-polyglutamic acid, which uses bacillus licheniformis with the preservation number of CGMCC No.23967 to ferment and produce the glutamic acid independent gamma-polyglutamic acid, and comprises the steps of strain activation, seed culture, fermentation culture, gamma-polyglutamic acid determination and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a production method of glutamic acid independent gamma-polyglutamic acid.
Background
Gamma-polyglutamic acid (gamma-PGA) is a natural polymer, which is polymerized from D-glutamic acid and L-glutamic acid through gamma-amide bonds, and is significantly different from proteins formed by connecting different types of amino acids through amide bonds between alpha-amino groups and alpha-carboxyl groups.
Depending on the composition of the glutamic acid residues, γ -PGA can be divided into three classes: d-glutamic acid homopolymer (D-gamma-PGA), L-glutamic acid homopolymer (L-gamma-PGA) and random copolymer composed of D-and L-glutamic acid (D-L-gamma-PGA).
The gamma-PGA has good biocompatibility, ion adsorption and biodegradability, is harmless to human bodies and the environment, and is commonly used in the fields of cosmetics, food processing, agriculture, medicine, environmental protection and the like.
Bacillus producing γ -PGA can be divided into two classes: strains requiring external supply of glutamate produce γ -PGA, i.e., glutamate dependent strains; the other is a strain which can produce gamma-PGA without externally supplying glutamic acid and is independent of glutamic acid.
The present glutamic acid dependent strain has higher gamma-PGA yield, so the glutamic acid dependent strain is usually selected for fermentation production, for example, the invention patent CN114456980A in 2022 of the company discloses a bacillus with high gamma-polyglutamic acid yield, the strain is named as bacillus licheniformis (Bacillus licheniformis) BCSW-11052, and the strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO.23967 in 11 months 24 of 2021; wherein the produced high molecular polymer is identified as gamma-polyglutamic acid through analysis. Said invention uses molasses as carbon source, and under the condition of adding exogenous glutamic acid, the gamma-PGA content in unit volume fermentation liquor can be up to 86.3 g/L. However, the addition of exogenous glutamic acid theoretically increases the production cost. The glutamic acid independent strain has larger potential capability for producing the gamma-PGA, and the research on the production of the gamma-PGA by the glutamic acid independent strain is helpful for analyzing the biosynthesis and the regulation mechanism of the gamma-PGA.
Disclosure of Invention
The invention aims to develop a production method for producing glutamic acid independent gamma-PGA on the basis of producing glutamic acid independent gamma-PGA by using bacillus licheniformis (CGMCC No. 23967) with the preservation number of 40.60 g/L.
The invention provides a production method of glutamic acid independent gamma-polyglutamic acid, which uses bacillus licheniformis for fermentation to produce the glutamic acid independent gamma-polyglutamic acid, and comprises the following steps:
(1) Activating strains: inoculating the bacillus licheniformis strain into a solid slant LB culture medium for activation;
(2) Seed culture: transferring the strain activated in the step (1) into a seed culture medium, and culturing seeds to obtain a seed culture solution;
(3) Fermentation culture: inoculating the seed culture solution prepared in the step (2) into a fermentation culture medium for fermentation culture;
(4) Gamma-polyglutamic acid assay: centrifuging the fermentation liquor obtained in the step (3), taking supernatant, diluting the supernatant 100 times by using a mobile phase, and detecting the supernatant by an HPLC method.
Preferably, in the step (1), the solid slant culture medium comprises the following components: 10.00g/L tryptone, 5.00g/L yeast extract powder, 10.00g/L sodium chloride, 20.00g/L agar powder and pH 7.2.
Preferably, in the step (1), the conditions for activating the strain are: culturing in a constant temperature incubator at 37 ℃ for 14-16h.
Preferably, the LB medium in the step (1) is sterilized for 20min at 121 ℃.
Preferably, in step (2), the seed medium comprises the following components: glucose 20.00g/L, yeast extract 25.00g/L, K 2 HPO 4 ·3H 2 O1.00 g/L、MgSO 4 ·7H 2 O0.50 g/L. The solvent is deionized water, and the seed culture medium is sterilized by a high-pressure steam sterilizing pot at 121 ℃ for 20min.
Preferably, in step (2), the seed culture conditions are: shake culturing at 34-40deg.C and pH7.0-7.2 at 180-240rpm for 10-12 hr; more preferably, in step (2), the seed culture conditions are: the culture was carried out at a shaking speed of 220rpm for 10 hours at 37℃and pH 7.0.
Preferably, in step (3), the fermentation medium comprises the following components: grape10-70g/L sugar, 60-100g/L citric acid, 30-60g/L yeast extract powder, 10-30g/L, mgSO peptone 4 ·7H 2 O1.00-1.6g/L、K 2 HPO 4 ·3H 2 O 0.1-0.3 g/L、MnSO 4 ·H 2 O0.1-0.3g/L、ZnSO 4 ·7H 2 O0.5-1.0 g/L. The pH value is 7.2, the solvent is deionized water, and the fermentation medium is sterilized for 20min at 121 ℃ by a high-pressure steam sterilizing pot.
Preferably, the seed solution in step (3) is inoculated into the fermentation medium in an inoculum size of 7% -12% (V/V). More preferably, the seed liquid is inoculated in an amount of 8% to 10% (V/V).
Preferably, in the step (3), the fermentation culture in the step (3) is shake flask fermentation culture or fermenter culture;
when shaking the bottle and fermenting and culturing, fermenting and culturing conditions are as follows: culturing for 72-96 h at 34-40 ℃ and pH7.0-7.2 and rotating speed 200-220 rpm;
when the fermentation tank is used for culturing, the fermentation culture conditions are as follows: culturing for 60-96 h at a rotating speed of 300-450 rpm under the conditions that the temperature is 34-40 ℃, the tank pressure is 0.02-0.05MPa, the ventilation ratio is 1:1-1:1.5 and the pH is 6.3-7.8; the ventilation ratio in the application refers to the ratio of ventilation per minute to the volume of fermentation broth, such as 3L fermentation broth, the ventilation ratio is 1:1.25, and the ventilation is 3.75L/min.
More preferably, in the case of fermentation in a fermenter, the conditions for fermentation are as follows: the temperature is 37 ℃ and the pH value is 7.2, the pH value can be obviously reduced in the process of 20 hours before fermentation, when the pH value is lower than 6.0, the pH value is regulated to 6.3 by using 6M NaoH solution, namely the pH value can not be lower than 6.3, the pH value in a fermentation tank is not interfered any more after 20 hours, the pH value can be slowly increased until the pH value is basically stable around 7.5, the rotating speed is 400rpm, and the ventilation ratio is 1:1.25 for 72 hours.
Preferably, in the step (4), the method for detecting the production of gamma-polyglutamic acid using the HPLC method comprises: wavelength 210nm, mobile phase: 0.05mol/L sodium sulfate solution, collecting the fermentation liquor in the step (3), centrifuging the fermentation liquor for 20min at 15000rpm, taking 1mL of supernatant, diluting to 50mL by using a mobile phase, and performing film coating to detect the content of gamma-PGA in the fermentation liquor in unit volume.
The beneficial effects of the invention are as follows:
according to the invention, bacillus licheniformis (Bacillus licheniformis) with the preservation number of CGMCC No.23967 is taken as an initial strain, a culture medium suitable for synthesizing glutamic acid independent polyglutamic acid is prepared by regulating and controlling a fermentation culture medium, the temperature is changed, so that the activity of polyglutamic acid synthase is influenced, the rotation speed and ventilation are controlled, so that dissolved oxygen is determined, the fermentation pH is controlled, and the gamma-polyglutamic acid yield is finally improved. Compared with other glutamic acid independent strains, the invention has higher gamma-polyglutamic acid yield and simple process, and the strain bacillus licheniformis CGMCC No.23967 can produce both glutamic acid dependent gamma-polyglutamic acid and glutamic acid independent gamma-polyglutamic acid.
Detailed Description
The following examples are provided to clearly and fully describe the technical aspects of the present invention, and it is apparent that the described examples are some, but not all, examples of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention is further illustrated with reference to specific embodiments.
Example 1
A method for producing glutamic acid independent gamma-polyglutamic acid, comprising the following steps:
(1) Activating strains: inoculating an original strain of bacillus licheniformis with the preservation number of CGMCC No.23967 on a solid slant LB culture medium, and culturing at 37 ℃ and pH7.2 for 14 hours;
the inclined plane culture medium comprises the following components: 10.00g/L tryptone, 5.00g/L yeast extract powder, 10.00g/L sodium chloride and 20.00g/L agar powder.
(2) Preparing seed liquid: inoculating the activated strain into a 500ml triangular flask filled with 100ml of liquid seed culture medium, and culturing at 37 ℃ and pH7.0 at 220rpm for 10 hours;
the liquid seed culture medium comprises the following components: glucose 20.00g/L, yeast extract 25.00g/L, K 2 HPO 4 ·3H 2 O 1.00g/L、MgSO 4 ·7H 2 O0.50 g/L, and deionized water as solvent.
(3) Shaking and fermenting: inoculating the seed solution in the step (2) into 500mL shaking bottles with baffles according to the inoculum size of 10% v/v, wherein the liquid loading amount of a fermentation medium is 50mL, the temperature is 34 ℃, the pH is 7.8, the rotating speed is 220rpm, and the fermentation time is 72 hours;
the fermentation medium comprises the following components in percentage by weight: glucose 50g/L, yeast extract 45 g/L, peptone 20g/L, mgSO 4 ·7H 2 O 1.4g/L、K 2 HPO 4 ·3H 2 O0.2 g/L、MnSO 4 ·H 2 O 0.25g/L、ZnSO 4 ·7H 2 O 0.75 g/L。
And centrifuging to remove thalli after fermentation is finished, taking supernatant, and detecting the content of gamma-polyglutamic acid in fermentation broth. The results showed that the yield of gamma-polyglutamic acid in this example was 13.53g/L.
Example 2
Strain activation and seed liquid preparation the same as in example 1
(3) Shaking and fermenting: inoculating the seed liquid in the step (2) into 500mL shaking bottles with baffles according to the inoculum size of 10% v/v, wherein the liquid loading amount of a fermentation medium is 50mL, the temperature is 37 ℃, the rotating speed is 220rpm, the pH is 7.2, and the fermentation time is 72 hours;
the fermentation medium comprises the following components in percentage by weight: glucose 47 g/L, citric acid 60g/L, yeast extract 45 g/L, peptone 25g/L, mgSO 4 ·7H 2 O1.4 g/L、K 2 HPO 4 ·3H 2 O 0.2g/L、MnSO 4 ·H 2 O 0.22 g/L、ZnSO 4 ·7H 2 O 0.75 g/L。
In this example, the yield of gamma-polyglutamic acid was 30.01. 30.01 g/L.
Example 3
Strain activation and seed liquid preparation the same as in example 1
(3) Fermenting in a fermentation tank: inoculating the seed solution in the step (2) into a 5L fermentation tank with an inoculum size of 10%v/v, wherein the liquid loading amount of a fermentation medium is 3L, the fermentation temperature is 37 ℃, the tank pressure is 0.02Mpa, the ventilation ratio is 1:1.25, the rotating speed is 350 rpm, the pH value in the fermentation process is reduced, and 6M NaOH is added to adjust each time the pH value is reduced, so that the pH value is kept at about 7.2, and the culture is carried out for 72 hours.
The fermentation medium comprises the following components in percentage by weight: glucose 47 g/L, citric acid 60g/L, yeast extract 45 g/L, peptone 25g/L, mgSO 4 ·7H 2 O1.4 g/L、K 2 HPO 4 ·3H 2 O0.2 g/L、MnSO 4 ·H 2 O 0.22 g/L、ZnSO 4 ·7H 2 O 0.75 g/L。
The yield of gamma-polyglutamic acid in this example was 19.77 g/L.
Example 4
Strain activation and seed liquid preparation the same as in example 1
(3) Fermenting in a fermentation tank: inoculating the seed liquid in the step (2) into a 5L fermentation tank with 10% of inoculation amount, wherein the liquid loading amount of a fermentation medium is 3L, the fermentation temperature is 37 ℃, the tank pressure is 0.02Mpa, the ventilation ratio is 1:1.25, the rotating speed is 400rpm, the initial pH value is 7.2, the pH is obviously reduced in the process of 20 hours before fermentation, when the pH value is lower than 6.0, the pH is regulated to 6.3 by using 6M NaOH, the pH is 7.2-7.3 after 20 hours, then the fermentation medium slowly rises to be about 7.5, the fermentation is basically stable, and the total culture time is 72 hours.
The fermentation medium comprises the following components in percentage by weight: glucose 47 g/L, citric acid 60g/L, yeast extract 45 g/L, peptone 25g/L, mgSO 4 ·7H 2 O1.4 g/L、K 2 HPO 4 ·3H 2 O0.2 g/L、MnSO 4 ·H 2 O 0.22 g/L、ZnSO 4 ·7H 2 O 0.75 g/L。
The gamma-polyglutamic acid yield in this example was 36.08 g/L.
In the research and development process, the pH value can be obviously reduced in the first 20 hours in the fermentation culture process, after several failed experimental training, research and development personnel continuously search and adjust the proper pH value condition, the effect of adjusting the pH value to about 6.3 in the first 20 hours is better, after 20 hours, the pH value can be slowly increased without interference to the pH value in the tank body, and finally, the pH value can be basically stabilized at about 7.5.
Example 5
Strain activation and seed liquid preparation the same as in example 1
(3) Fermenting in a fermentation tank: inoculating the seed solution in the step (2) into a 30L fermentation tank with an inoculum size of 10%, wherein the liquid loading amount of a fermentation medium is 15L, the fermentation temperature is 37 ℃, the tank pressure is 0.03Mpa, the ventilation ratio is 1:1.25, the rotating speed is 400rpm, the initial pH is 7.2, when the pH value is lower than 6.0 in the process of 20 hours before fermentation, the pH value is regulated to 6.3 by using 6M NaoH solution, the pH value can slowly rise after 20 hours, the rise is basically stable to about 7.5, and the total culture is carried out for 72 hours.
The fermentation medium comprises the following components in percentage by weight: glucose 47 g/L, citric acid 60g/L, yeast extract 45 g/L, peptone 25g/L, mgSO 4 ·7H 2 O1.4 g/L、K 2 HPO 4 ·3H 2 O0.2 g/L、MnSO 4 ·H 2 O 0.22 g/L、ZnSO 4 ·7H 2 O 0.75 g/L。
The gamma-polyglutamic acid yield in this example was 40.60. 40.60 g/L.
In the research and development process, a large-capacity fermentation tank is used, the yield is increased even though the concentration of each component of the fermentation medium is unchanged, and the analysis proves that the large-capacity fermentation tank is probably due to the fact that the dissolved oxygen in the tank is increased, and the efficient conversion of fermentation culture is facilitated.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (9)
1. A method for producing glutamic acid independent gamma-polyglutamic acid is characterized in that bacillus licheniformis with the preservation number of CGMCC No.23967 is used for fermenting and producing the glutamic acid independent gamma-polyglutamic acid, and the method comprises the following steps:
(1) Activating strains: inoculating bacillus licheniformis strain into LB solid slant culture medium for activation;
(2) Seed culture: transferring the strain activated in the step (1) into a seed culture medium, and culturing seeds to obtain a seed culture solution;
(3) Fermentation culture: inoculating the seed culture solution prepared in the step (2) into a fermentation culture medium for fermentation culture; the fermentation medium is as follows: glucose 10-70g/L, citric acid 60-100g/L, yeast extract 30-60g/L, peptone 10-30g/L, mgSO 4 ·7H 2 O 1.00-1.6g/L、K 2 HPO 4 ·3H 2 O 0.1-0.3g/L、MnSO 4 ·H 2 O 0.1-0.3g/L、ZnSO 4 ·7H 2 O0.5-1.0g/L；
(4) Gamma-polyglutamic acid assay: centrifuging the fermentation liquor obtained in the step (3), taking supernatant, diluting the supernatant by 100 times by using a mobile phase, and detecting the content of gamma-polyglutamic acid in the fermentation liquor in unit volume by using an HPLC method.
2. The method for producing glutamic acid-independent gamma-polyglutamic acid according to claim 1, wherein the activation condition in the step (1) is cultivation in a constant temperature incubator at 37℃for 14 to 16 hours.
3. The method for producing glutamic acid-independent gamma-polyglutamic acid according to claim 1, wherein the seed medium in the step (2) is: glucose 20g/L, yeast extract 25g/L, K 2 HPO 4 ·3H 2 O1g/L、MgSO 4 ·7H 2 O0.5 g/L, and deionized water as solvent.
4. The method for producing glutamic acid-independent gamma-polyglutamic acid according to claim 1, wherein the seed culture conditions in the step (2) are as follows: shaking culture at 34-40deg.C and pH7.0-7.2 at 180-240rpm for 10-12 hr.
5. The method for producing glutamic acid-independent gamma-polyglutamic acid according to claim 1, wherein the seed liquid in the step (3) is inoculated into the fermentation medium in an inoculum size of 7% -12% (V/V).
6. The method for producing glutamic acid-independent gamma-polyglutamic acid according to claim 1, wherein the fermentation culture method in the step (3) is a shake flask fermentation culture or a fermenter culture.
7. The method for producing glutamic acid-independent gamma-polyglutamic acid according to claim 6, wherein the fermentation culture conditions in shake flask fermentation culture are as follows: culturing at 34-40 deg.c, pH7.0-7.2 and rotation speed 200-220rpm for 72-96 hr.
8. The method for producing glutamic acid-independent gamma-polyglutamic acid according to claim 6, wherein the fermentation culture conditions are as follows: culturing at 34-40 deg.c, tank pressure of 0.02-0.05MPa, ventilation ratio of 1:1-1:1.5 and pH of 6.3-7.8 at 300-450 rpm for 60-96 hr.
9. The method for producing glutamic acid-independent gamma-polyglutamic acid according to claim 8, wherein the conditions of the fermenter culture are: the fermentation temperature was 37℃and the initial pH was 7.2, and during 20 hours before fermentation, when the pH was below 6.0, the pH was adjusted to 6.3 at a rotation speed of 400rpm at a draft ratio of 1:1.25 for 72 hours.
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| CN107760732A (en) * | 2017-11-16 | 2018-03-06 | 天津北洋百川生物技术有限公司 | A kind of production method of agriculture level γ polyglutamic acids |
| CN114456980A (en) * | 2022-02-28 | 2022-05-10 | 天津科技大学 | Gamma-polyglutamic acid high-yield strain and application thereof |
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| CN107760732A (en) * | 2017-11-16 | 2018-03-06 | 天津北洋百川生物技术有限公司 | A kind of production method of agriculture level γ polyglutamic acids |
| CN114456980A (en) * | 2022-02-28 | 2022-05-10 | 天津科技大学 | Gamma-polyglutamic acid high-yield strain and application thereof |
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