A fermentation method of high molecular weight gamma polyglutamic acid

Abstract

The present application belongs to the field of bioengineering fermentation technology, and particularly relates to a fermentation method of high molecular weight gamma polyglutamic acid. The method comprises the steps of slant seed culture, secondary seed culture and fermentation culture, and is characterized in that: the fermentation culture refers to inoculating the secondary seed liquid into the fermentation medium in the fermentation tank at a volume ratio of 10-15% for aerobic fermentation, the initial pH of the fermentation is set to 6.0-7.0, the pH 6.0-7.0 in the fermentation process is controlled by continuously adding 5-15% (WT) sodium hydroxide solution, the fermentation is carried out for 8-9 hours, and the pH in the fermentation process is not higher than 7.5 by intermittently adding the feed; the feed components are glutamic acid 30-70 g / L, potassium dihydrogen phosphate 2-8 g / L and ammonium sulfate 2-8 g / L. The present application can obtain high molecular weight gamma polyglutamic acid by maintaining the pH stability in the fermentation process, and on the other hand, the continuously added glutamic acid can be directly converted into polyglutamic acid, thereby improving the conversion rate of glutamic acid.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering fermentation, and specifically relates to a fermentation method of high-molecular-weight gamma-polyglutamic acid. BACKGROUND

[0002] Gamma-polyglutamic acid (γ-PGA) is a naturally occurring water-soluble biodegradable polymer composed of several D- and L-glutamic acid monomers connected by amide bonds between alpha-amino and gamma-carboxyl groups, with a relative molecular mass ranging from 50 KDa to 20,000 KDa. The main chain of gamma-polyglutamic acid contains a large number of free hydrophilic carboxyl groups, which form hydrogen bonds within and between molecules, thus having excellent water retention performance.

[0003] The final degradation product of gamma-polyglutamic acid is glutamic acid, which is friendly to the human body and the environment and has no toxic side effects. Since gamma-polyglutamic acid has good biocompatibility, it can be used as a fertilizer, a pesticide synergist, a water-retaining agent, a heavy metal ion adsorbent, a flocculant, a slow-release agent, and a drug carrier, and is widely used in the food, environmental, cosmetic, and pharmaceutical industries.

[0004] The production methods of gamma-polyglutamic acid include chemical synthesis, enzyme conversion, and biological fermentation. Compared with the chemical synthesis method and the enzyme conversion method, the biological fermentation method has the advantages of simple process, mild reaction conditions, and inexpensive raw materials, and is easy to realize industrial production, so it has shown a trend of vigorous development in recent years.

[0005] Su Guojin controls the pH of the fermentation liquid by adding sulfuric acid dropwise, and the fermentation cycle is 45-60h, and the content of polyglutamic acid in the fermentation liquid can reach 50-65g / L. The application does not mention the molecular weight of the obtained polyglutamic acid, and the invention patent publication number is CN111172212A; Yang Qi et al. invented a preparation process of cosmetic-grade gamma-polyglutamic acid, and the molecular weight of the obtained product is 800-1200kDa, and the invention patent publication number is CN110106212A. The biological fermentation method still has some defects, mainly the difficulty in stable control of the pH during the fermentation process, and the existence of gamma-polyglutamic acid decomposing enzyme in the fermentation system, which makes the polydispersity and molecular weight of the gamma-polyglutamic acid obtained by fermentation smaller, mostly below 1000kDa, so it is difficult to meet the requirements of high molecular weight and high yield at the same time in large-scale production. SUMMARY

[0006] In view of the above technical defects, the application provides a fermentation method of high-molecular-weight and high-yield gamma-polyglutamic acid.

[0007] To solve the above technical problems, the technical scheme provided by the present application is: a fermentation method of high molecular weight gamma polyglutamic acid, comprising the steps of slant seed culture, secondary seed culture and fermentation culture, characterized in that: the fermentation culture refers to inoculating the secondary seed liquid into the fermentation medium in the fermentation tank at a volume ratio of 10-15% for aerobic fermentation, setting the initial pH of fermentation to 6.0-7.0, controlling the pH 6.0-7.0 in the fermentation process by adding 5-15% (WT) sodium hydroxide solution, and when the pH naturally rises above the initial set pH value, inhibiting the continuous rise of pH by intermittent feeding, so that the pH in the fermentation process does not exceed 7.5; the feeding component is glutamic acid 30-70 g / L, potassium dihydrogen phosphate 2-8 g / L, and ammonium sulfate 2-8 g / L.

[0008] Further: in the fermentation method of high molecular weight gamma polyglutamic acid, the fermentation culture temperature is 30-38℃, the stirring speed is 200-650 rpm, the ventilation amount is 0.5-1.5vvm, and the tank pressure is 0.05-0.08MPa. When the fermentation culture is carried out for 20-24h, a glucose solution with a concentration of 500-750g / L is added to the fermentation liquid until the fermentation is completed.

[0009] The slant seed culture refers to taking out the gamma polyglutamic acid production glycerol seed from the -72℃ refrigerator, uniformly coating the tomato bottle slant, and culturing at a constant temperature of 30-38℃ for 18-24h, until the colonies on the surface of the culture medium are smooth, uniform, full and shiny, and the tomato bottle slant bacterial layer is prepared.

[0010] The secondary seed culture refers to washing, dispersing and shaking the tomato bottle slant bacterial layer with 100ml sterile water to prepare a bacterial suspension, taking 1ml of the bacterial suspension and placing it in a triangular flask containing 100ml sterile water to prepare a seed liquid, and then transferring the seed liquid to the secondary seed medium in the secondary seed tank to obtain a secondary seed liquid; the culture temperature is 30-38℃, and the culture time is 6-12h.721The OD600 is measured by spectrophotometer when it reaches 7-12.

[0011] The gamma polyglutamic acid production strain is Bacillus subtilis (B.subtilis).

[0012] The components of the secondary seed culture medium are: glucose 20-50g / L; magnesium sulfate 0.1-1.0g / L; yeast extract 5-12g / L; peptone 1-6g / L; sodium glutamate 20-70g / L; potassium dihydrogen phosphate 1-5g / L; and defoaming agent 0.2-1.0ml / L.

[0013] The fermentation medium components are: glucose 50-90 g / L; magnesium sulfate 0.1-1.0 g / L; yeast extract 5-12 g / L; peptone 2-10 g / L sodium glutamate 30-80 g / L; potassium dihydrogen phosphate 2-6 g / L; antifoam agent 0.2-1.0 ml / L.

[0014] The specific way of feeding is: intermittent feeding, feeding amount 150-400 g / h, with the principle of controlling pH not exceeding 7.5;

[0015] The specific way of feeding is: continuous feeding, feeding amount 150-300 g / h, controlling the concentration of reducing sugar in the fermentation broth 5-15 g / L.

[0016] Compared with the prior art, the fermentation culture refers to inoculating the secondary seed liquid into the fermentation medium in the fermentation tank at a volume ratio of 10-15% for aerobic fermentation, the initial pH of fermentation is set to 6.0-7.0, the pH 6.0-7.0 in the fermentation process is controlled by feeding 5-15% (WT) sodium hydroxide solution, and when the pH naturally rises above the initial set pH value after 8-9 h of fermentation, the pH continues to rise by intermittent feeding to inhibit the pH, so that the pH in the fermentation process does not exceed 7.5; the feeding components are glutamic acid 30-70 g / L, potassium dihydrogen phosphate 2-8 g / L, and ammonium sulfate 2-8 g / L. During the fermentation process, as the glutamic acid polymerizes, the free H+ in the system decreases, and in addition, the nitrogen source metabolizes to release amino acids, so that the pH of the fermentation broth gradually increases. When the pH rises above 7.5, under the action of the γ-polyglutamic acid decomposing enzyme in the fermentation system, the γ-amide bond between glutamic acid molecules breaks, the product is degraded into short peptides or even glutamic acid monomers, the regularity of the molecular structure is destroyed, and the γ-polyglutamic acid content is significantly reduced. By feeding the feeding liquid containing the substrate glutamic acid and the buffer potassium dihydrogen phosphate to the fermentation broth, on the one hand, the acidity of glutamic acid and potassium dihydrogen phosphate is used to neutralize the alkalinity of the fermentation system, so that the pH in the fermentation process is stable, the glutamic acid polymerization reaction can continue, the product in the fermentation system is not easy to degrade, the glutamic acid polymerization chain continues to lengthen, and high molecular weight γ-polyglutamic acid can be obtained; the greater the molecular weight, the stronger the water absorption and water retention of γ-polyglutamic acid, and the relatively broader application field. The γ-polyglutamic acid with a molecular weight of ≥2000 KDa has the functions of long-acting moisturizing and pollution isolation, and can effectively reduce the loss of water on the skin surface in a dry environment. On the other hand, the fermentation metabolic substrate glutamic acid is supplemented to the medium system, and the feedback inhibition caused by excessive substrate in the basic medium is weakened or eliminated, and the supplemented glutamic acid can be directly converted into polyglutamic acid, thereby improving the glutamic acid conversion rate. DETAILED DESCRIPTION

[0017] The application will be further described below in combination with specific examples. In the following operations, those not described in detail can be performed according to the molecular biology experiment manual.

[0018] Example 1

[0019] The γ-polyglutamic acid glycerol was preserved and evenly coated on the bottle slant of eggplant, which was placed in a 36°C constant temperature incubator for 24h. 100ml sterile water was used to elute and disperse the slant bacterial lawn to prepare a bacterial suspension, which was inoculated into a 5L secondary seed tank (3L of culture medium was used for constant volume), the temperature was controlled at 35.0°C, the stirring speed was 600rpm, the ventilation volume was 1.3vvm, and the culture was performed for 10h. When the OD 600 When the OD reached 10, it was transferred to a 50L fermentation tank at a volume percentage of 10% for fermentation culture (the volume of the fermentation broth at 0h was 30L), the temperature was controlled at 37.0°C, the stirring speed was 650rpm, the ventilation volume was 1.4vvm, and the tank pressure was 0.07MPa. The initial pH was set to 6.1, and when the pH naturally rose to 6.5 after 8h, intermittent feeding of the culture medium was performed to stabilize the pH of the fermentation process at 6.5-6.8. When the fermentation was performed for 24h, a glucose solution with a concentration of 750g / L was stably fed to control the reducing sugar concentration of the fermentation system to 5-15g / L. The fermentation cycle was 58h, and the obtained fermentation broth was detected by high performance liquid chromatography (HPLC) at a wavelength of 220nm to detect the content of γ-polyglutamic acid, which was 40.2g / L, the molecular weight was 3450KDa, and the glutamic acid conversion rate was 89.92%.

[0020] The above-mentioned method for measuring the molecular weight is as follows: sodium sulfate solution is used as the mobile phase, different molecular weight dextran standard solutions are used as references, the peak time of γ-polyglutamic acid under the differential refractive index detector is detected, and the molecular weight of γ-polyglutamic acid is measured by high performance liquid chromatography. The measurement methods of the following examples are the same.

[0021] The above-mentioned glutamic acid conversion rate % = (G1+G2-G3) / (G1+G2)*100%

[0022] Wherein: G1 is the amount of glutamic acid in the raw material g; G2 is the amount of feeding glutamic acid g; G3 is the residual glutamic acid g;

[0023] G3 = residual glutamic acid content g / L * fermentation tank volume L; (the measurement methods of the following examples are the same)

[0024] The components of the above-mentioned secondary seed culture medium are as follows: glucose 20g / L; magnesium sulfate 0.4g / L; yeast extract 8g / L; peptone 5g / L; sodium glutamate 30g / L; potassium dihydrogen phosphate 1.5g / L; defoaming agent 0.5ml / L

[0025] The components of the fermentation culture medium are as follows: glucose 80g / L; magnesium sulfate 0.5g / L; yeast extract 10g / L; peptone 3g / L; sodium glutamate 40g / L; potassium dihydrogen phosphate 2g / L; defoaming agent 0.5ml / L

[0026] The components of the feed medium are: glutamic acid 50 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 5 g / L

[0027] Example 2

[0028] The γ-polyglutamic acid glycerol stock solution was uniformly coated on a potato bottle slant, which was placed in a 36°C constant temperature incubator for 24 hours. 100 ml of sterile water was used to elute and disperse the slant bacterial lawn to prepare a bacterial suspension, which was inoculated into a 5L secondary seed tank (culture medium was 3L), the temperature was controlled at 36°C, the stirring speed was 650 rpm, the ventilation volume was 1.5 vvm, and the culture was carried out for 8.5 hours. When the OD 600 When the OD reached 9.4, it was transferred to a 50L fermentation tank at a volume percentage of 10% for fermentation culture (fermentation liquid 0h volume 30L), the temperature was controlled at 37.0°C, the stirring speed was 650 rpm, the ventilation volume was 1.5 vvm, and the tank pressure was 0.08 MPa. The initial pH was set to 6.3, and when the pH naturally rose to 6.6 after 8h, intermittent flow feeding medium was added to keep the pH of the fermentation process stable at 6.6-7.0. When the fermentation was carried out to 23h, a glucose solution with a concentration of 600g / l was stably added, and the concentration of reducing sugar in the fermentation system was controlled at 5-15g / L. The fermentation period was 52h, and the content of γ-polyglutamic acid in the fermentation broth was 43.1g / L, the molecular weight was 3380KDa, and the glutamic acid conversion rate was 86.58% measured by HPLC method.

[0029] The components of the above secondary seed culture medium are: glucose 30 g / L; magnesium sulfate 0.5 g / L; yeast extract 12 g / L; peptone 3 g / L; sodium glutamate 40 g / L; potassium dihydrogen phosphate 2.5 g / L; defoaming agent 0.5 ml / L

[0030] The components of the fermentation medium are: glucose 90 g / L; magnesium sulfate 0.6 g / L; yeast extract 12 g / L; peptone 4 g / L; sodium glutamate 60 g / L; potassium dihydrogen phosphate 2.5 g / L; defoaming agent 0.7 ml / L

[0031] The components of the feed medium are: glutamic acid 40 g / L, potassium dihydrogen phosphate 4 g / L, ammonium sulfate 6 g / L

[0032] Example 3

[0033] The γ-polyglutamic acid glycerol stock solution was uniformly coated on a potato bottle slant, which was placed in a 36°C constant temperature incubator for 24 hours. 100 ml of sterile water was used to elute and disperse the slant bacterial lawn to prepare a bacterial suspension, which was inoculated into a 5L secondary seed tank (culture medium was 3L), the temperature was controlled at 36°C, the stirring speed was 650 rpm, the ventilation volume was 1.5 vvm, and the culture was carried out for 8.5 hours. When the OD 600When the pH reached 8.5, the fermentation culture was transferred to a 50L fermenter (fermentation broth 0h volume 30L) at a volume percentage of 10% for fermentation culture (fermentation broth 0h volume 30L), temperature control at 37.5°C, stirring speed 650rpm, ventilation volume 1.5vvm, tank pressure 0.08MPa. The initial pH was set at 6.5, and when the pH naturally rose to 6.7 at 8.5h, the feed medium was intermittently added to stabilize the pH at 6.7-7.1 during the fermentation process. When the fermentation reached 22h, a stable glucose solution with a concentration of 600g / l was added, and the reducing sugar concentration in the fermentation system was controlled at 5-15g / L. The fermentation period was 45h, and the HPLC method was used to measure the content of γ-polyglutamic acid in the fermentation broth, which was 40g / L, the molecular weight was 3480KDa, and the glutamic acid conversion rate was 94.02%.

[0034] The components of the above secondary seed medium are: glucose 40g / L; magnesium sulfate 0.6g / L; yeast extract 12g / L; peptone 3g / L; sodium glutamate 50g / L; potassium dihydrogen phosphate 2g / L; antifoam 0.5ml / L

[0035] The components of the fermentation medium are: glucose 70g / L; magnesium sulfate 0.4g / L; yeast extract 6g / L; peptone 8g / L; sodium glutamate 50g / L; potassium dihydrogen phosphate 3g / L; antifoam 0.6ml / L

[0036] The components of the feed medium are: glutamic acid 30g / L, potassium dihydrogen phosphate 3g / L, ammonium sulfate 4g / L

[0037] Example 4

[0038] The γ-polyglutamic acid glycerol preservation seed liquid was uniformly coated on the potato bottle slant, and was incubated in a 36°C constant temperature incubator for 24h. 100ml sterile water was used to elute and disperse the slant bacterial lawn to prepare a bacterial suspension, which was inoculated into a 5L secondary seed tank (medium volume 3L), and the temperature was controlled at 37.5°C, the stirring speed was 650rpm, and the ventilation volume was 1.5vvm. The culture was incubated for 7.5h, and the OD 600 When the pH reached 8.5, the fermentation culture was transferred to a 50L fermenter (fermentation broth 0h volume 30L) at a volume percentage of 10% for fermentation culture (fermentation broth 0h volume 30L), temperature control at 37.5°C, stirring speed 650rpm, ventilation volume 1.5vvm, tank pressure 0.08MPa. The initial pH was set at 6.5, and when the pH naturally rose to 6.7 at 8.5h, the feed medium was intermittently added to stabilize the pH at 6.7-7.1 during the fermentation process. When the fermentation reached 22h, a stable glucose solution with a concentration of 600g / l was added, and the reducing sugar concentration in the fermentation system was controlled at 5-15g / L. The fermentation period was 45h, and the HPLC method was used to measure the content of γ-polyglutamic acid in the fermentation broth, which was 40g / L, the molecular weight was 3480KDa, and the glutamic acid conversion rate was 94.02%.

[0039] The components of the secondary seed culture medium are: glucose 30 g / L; magnesium sulfate 0.5 g / L; yeast extract 10 g / L; peptone 4 g / L; sodium glutamate 30 g / L; potassium dihydrogen phosphate 1.5 g / L; antifoam agent 0.5 ml / L

[0040] The components of the fermentation medium are: glucose 80 g / L; magnesium sulfate 0.7 g / L; yeast extract 8 g / L; peptone 6 g / L; sodium glutamate 45 g / L; potassium dihydrogen phosphate 3.5 g / L; antifoam agent 0.6 ml / L

[0041] The components of the feed medium are: glutamic acid 40 g / L, potassium dihydrogen phosphate 4 g / L, ammonium sulfate 5 g / L

[0042] Example 5

[0043] The γ-polyglutamic acid glycerol stock solution was uniformly coated on a potato bottle slant, which was placed in a 36°C constant temperature incubator for 24 h. 100 ml of sterile water was used to elute and disperse the slant bacterial lawn to prepare a bacterial suspension, which was inoculated into a 5L secondary seed tank (3L of medium was used to make up the volume), the temperature was controlled at 37°C, the stirring speed was 650 rpm, the ventilation volume was 1.5 vvm, and the culture was carried out for 8 h. When the OD 600 When the OD reached 8.8, the culture was transferred into a 50L fermentation tank at a volume percentage of 10% for fermentation culture (the volume of the fermentation broth at 0 h was 30L), the temperature was controlled at 37.5°C, the stirring speed was 650 rpm, the ventilation volume was 1.5 vvm, and the tank pressure was 0.08 MPa. The initial pH was set to 6.7, and when the pH naturally rose to 7.0 at 8.5 h, the feed medium was intermittently added to stabilize the pH at 7.0-7.3 during the fermentation process. When the fermentation was carried out for 24 h, a glucose solution with a concentration of 600 g / L was stably added to control the concentration of reducing sugar in the fermentation system to 5-15 g / L. The fermentation period was 51 h, and the content of γ-polyglutamic acid in the fermentation broth was 40.7 g / L, the molecular weight was 3320 KDa, and the glutamic acid conversion rate was 80.63% as determined by HPLC.

[0044] The components of the secondary seed culture medium are: glucose 40 g / L; magnesium sulfate 0.6 g / L; yeast extract 10 g / L; peptone 5 g / L; sodium glutamate 40 g / L; potassium dihydrogen phosphate 2.0 g / L; antifoam agent 0.5 ml / L

[0045] The components of the fermentation medium are: glucose 90 g / L; magnesium sulfate 0.8 g / L; yeast extract 10 g / L; peptone 4 g / L; sodium glutamate 50 g / L; potassium dihydrogen phosphate 3 g / L; antifoam agent 0.6 ml / L

[0046] The components of the feed medium are: glutamic acid 40 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 6 g / L

[0047] Comparative Example 1

[0048] The γ-polyglutamic acid glycerol preservation seed solution was uniformly coated on the potato bottle slant, and was placed in a 36°C constant temperature incubator for 24h. 100ml sterile water was used to elute and disperse the slant bacterial lawn to prepare a bacterial suspension, which was inoculated into a 5L secondary seed tank (culture medium was 3L), the temperature was controlled at 36.0°C, the stirring speed was 650rpm, the ventilation volume was 1.5vvm, and the culture was performed for 8h. The OD 600 When the OD reached 9, it was transferred to a 50L fermentation tank at a volume percentage of 10% for fermentation culture (the volume of the fermentation broth was 30L at 0h), the temperature was controlled at 37.0°C, the stirring speed was 650rpm, the ventilation volume was 1.5vvm, and the tank pressure was 0.08MPa. The initial pH was set to 6.3, and no feeding was added during the fermentation process. The pH naturally rose to 6.65 at 8.5h, and the pH naturally rose to 7.86 at 36h, while the viscosity of the fermentation broth decreased from 1248cps to 552cps. When the fermentation was performed for 23h, a glucose solution with a concentration of 600g / L was stably fed to control the reducing sugar concentration of the fermentation system to be 5-15g / L. The fermentation period was 38h, and the γ-polyglutamic acid content in the fermentation broth was 25g / L, the molecular weight was 2280KDa, and the glutamic acid conversion rate was 70.46% measured by HPLC.

[0049] The components of the above secondary seed culture medium are: glucose 30g / L; magnesium sulfate 0.5g / L; yeast extract 12g / L; peptone 3g / L; sodium glutamate 40g / L; potassium dihydrogen phosphate 2.5g / L; defoaming agent 0.5ml / L

[0050] The components of the fermentation culture medium are: glucose 80g / L; magnesium sulfate 0.5g / L; yeast extract 10g / L; peptone 3g / L; sodium glutamate 40g / L; potassium dihydrogen phosphate 2g / L; defoaming agent 0.5ml / L;

[0051] Comparative Example 2

[0052] The γ-polyglutamic acid glycerol preservation seed solution was uniformly coated on the potato bottle slant, and was placed in a 36°C constant temperature incubator for 24h. 100ml sterile water was used to elute and disperse the slant bacterial lawn to prepare a bacterial suspension, which was inoculated into a 5L secondary seed tank (culture medium was 3L), the temperature was controlled at 36.0°C, the stirring speed was 650rpm, the ventilation volume was 1.5vvm, and the culture was performed for 8h. The OD 600When the pH reaches 9.5, the fermentation culture is switched to a 50L fermenter (fermentation broth 0h volume 30L) at a volume percentage of 10%, the temperature is controlled at 37.5℃, the stirring speed is 650rpm, the ventilation volume is 1.5vvm, and the tank pressure is 0.08MPa. The initial pH is set at 6.5, the pH naturally rises to 6.9, and the feed medium without glutamic acid is intermittently added to maintain the pH at 6.9-7.2. It can be observed that the pH can only be maintained at about 7.5 after 24h and cannot continue to decrease, and the viscosity of the fermentation broth decreases from 1428cps to 320cps during this process. When the fermentation is carried out for 24h, a glucose solution with a concentration of 700g / L is stably added to control the reducing sugar concentration of the fermentation system to 5-15g / L. The fermentation period is 37h, and the content of γ-polyglutamic acid in the fermentation broth is 22g / L, the molecular weight is 2130KDa, and the glutamic acid conversion rate is 62.44% measured by HPLC.

[0053] The components of the above secondary seed culture medium are as follows: glucose 20g / L; magnesium sulfate 0.4g / L; yeast extract 8g / L; peptone 5g / L; sodium glutamate 30g / L; potassium dihydrogen phosphate 1.5g / L; defoaming agent 0.5ml / L

[0054] The components of the fermentation culture medium are as follows: glucose 70g / L; magnesium sulfate 0.4g / L; yeast extract 6g / L; peptone 8g / L; sodium glutamate 50g / L; potassium dihydrogen phosphate 2g / L; defoaming agent 0.6ml / L

[0055] The components of the feed medium are as follows: potassium dihydrogen phosphate 3g / L, ammonium sulfate 4g / L

[0056] As can be seen from the above examples 1-5 and comparative examples 1-2, the feed liquid containing the substrate glutamic acid and the buffer potassium dihydrogen phosphate is added to the fermentation broth by feeding, the acidity of glutamic acid and potassium dihydrogen phosphate can be used to neutralize the alkalinity of the fermentation system, and the fermentation metabolic substrate is supplemented to the system, so that the pH is stable during the fermentation process, the glutamic acid polymerization reaction can continue, the product in the fermentation system is not easily degraded, the glutamic acid polymerization chain is continuously extended, and high molecular weight γ-polyglutamic acid can be obtained.

[0057] Obviously, the examples of the present application are only examples for clearly illustrating the present application, and are not a limitation on the embodiments of the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A fermentation method for high molecular weight γ-polyglutamic acid, comprising the steps of slant seed culture, secondary seed culture, and fermentation culture, characterized in that: The fermentation culture refers to inoculating the secondary seed culture into the fermentation medium of the fermenter at a volume ratio of 10% to 15% for aerobic fermentation. The initial pH of the fermentation is set to 6.0 to 7.0, and the pH of the fermentation process is controlled at 6.0 to 7.0 by adding 5% to 15% (wt) sodium hydroxide solution. After fermentation for 8-9 hours, the pH of the fermentation process is kept below 7.5 by intermittent feeding. The feed components are glutamic acid 30-70 g / L, potassium dihydrogen phosphate 2-8 g / L, and ammonium sulfate 2-8 g / L. The slant seed culture refers to taking out the γ-polyglutamic acid producing strain from a -72℃ freezer, coating it on the slant of an eggplant bottle, and incubating it at a constant temperature of 30-38℃ for 18-24 hours to prepare an eggplant bottle slant mycelium. The secondary seed culture refers to washing, breaking up, and shaking the bacterial growth on the slant of an eggplant flask with 100ml of sterile water to prepare a bacterial suspension. 1ml of this suspension is then placed in an Erlenmeyer flask containing 100ml of sterile water to prepare a seed culture, which is then transferred to the secondary seed culture medium in the secondary seed tank to obtain the secondary seed culture. The culture temperature is 30-38℃, and the culture time is 6-12 hours. The γ-polyglutamic acid producing strain is Bacillus subtilis. The secondary seed culture medium consists of: glucose 20–50 g / L; magnesium sulfate 0.1–1.0 g / L; yeast extract 5–12 g / L; peptone 1–6 g / L; sodium glutamate 20–70 g / L; potassium dihydrogen phosphate 1–5 g / L; and antifoaming agent 0.2–1.0 ml / L.

2. The fermentation method for high molecular weight γ-polyglutamic acid according to claim 1, characterized in that: The fermentation culture temperature is 30-38℃, the stirring speed is 200-650rpm, the ventilation rate is 0.5-1.5vvm, and the tank pressure is 0.05~0.08MPa.

3. The fermentation method for high molecular weight γ-polyglutamic acid according to claim 2, characterized in that: During the 20-24 h fermentation period, a glucose solution with a concentration of 500-750 g / L is added to the fermentation broth until fermentation is complete.

4. The fermentation method for high molecular weight γ-polyglutamic acid according to claim 1, characterized in that: The fermentation medium consists of: glucose 50-90 g / L; magnesium sulfate 0.1-1.0 g / L; yeast extract 5-12 g / L; peptone 2-10 g / L; sodium glutamate 30-80 g / L; potassium dihydrogen phosphate 2-6 g / L; and defoamer 0.2-1.0 ml / L.

5. The fermentation method for high molecular weight γ-polyglutamic acid according to claim 4, characterized in that: The specific feeding method is: intermittent feeding, with a feeding rate of 150-400 g / h.

6. The fermentation method for high molecular weight γ-polyglutamic acid according to claim 3, characterized in that: The specific method for adding glucose solution is as follows: continuous addition at a rate of 150-300 g / h, controlling the reducing sugar concentration in the fermentation broth to be 5-15 g / L.