High-density fermentation method of bacillus licheniformis liquid for producing polyglutamic acid

Abstract

The invention belongs to the technical field of biological fermentation, and particularly relates to a bacillus licheniformis liquid high-density fermentation method for producing polyglutamic acid. According to the bacillus licheniformis high-density fermentation method provided by the invention, by innovatively integrating staged application of coral mucus and symbiotic fermentation of chlorella FACHB-31, the problems that dissolved oxygen is difficult to regulate and control and the yield is low in the traditional production of producing polyglutamic acid by high-density fermentation of bacillus licheniformis are effectively solved; the fermentation efficiency and the product yield are obviously improved. The coral mucus is introduced as a carbon source and a growth promoter in the whole stage (activation, seed solution, fermentation and conversion), and the photosynthesis of chlorella FACHB-31 is combined, so that the yield of polyglutamic acid is greatly improved. The method is low in cost, high in efficiency, stable in yield and suitable for industrial fermentation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation, and particularly relates to a method for high-density liquid fermentation of Bacillus licheniformis for producing polyglutamic acid. Background Art

[0002] Poly-γ-glutamic acid is a polypeptide molecule formed by the binding of L-glutamic acid and D-glutamic acid through amide bonds, and has properties such as thickening, emulsifying, film-forming, moisturizing, flocculating, bonding, non-toxic, water-soluble, and biodegradable, and is applicable to fields such as agriculture, food, cosmetics, biomedicine, and environmental protection. Especially in the agricultural field, polyglutamic acid can be used as a slow-release carrier for agricultural chemicals. When using fertilizers, pesticides, herbicides, insect repellents, etc., adding an appropriate amount of polyglutamate can extend the residence time of these drugs on the surface of the target object and the action time of the active ingredients, improve the use effect, and reduce the usage amount of chemical fertilizers and pesticides. Polyglutamic acid itself can also be used as a new type of liquid fertilizer. There are literature reports that polyglutamic acid can degrade nitrogen and phosphorus in the soil, thereby enhancing the utilization rate of nitrogen fertilizer and phosphate fertilizer.

[0003] However, the existing production methods of polyglutamic acid mainly include chemical synthesis method, microbial fermentation method, and enzyme catalysis method. Among them, the microbial fermentation method has become the main production method at present due to its environmental protection, high efficiency, and scalable production characteristics. Especially the high-density liquid fermentation method of Bacillus licheniformis has received extensive attention due to its high yield and industrialization potential. However, the regulation of dissolved oxygen (DO) is a key limiting factor in the high-density liquid fermentation method of Bacillus licheniformis, and the ventilation volume and stirring speed need to be dynamically adjusted. Therefore, adopting a method of co-liquid fermentation of oxygen-producing algae and Bacillus licheniformis to increase the oxygen solubility and make up for the oxygen consumption becomes a feasible solution. There have been reports on the co-culture of algae (such as Chlorella) and nitrifying bacteria to treat wastewater, but there is no report on the high-density liquid fermentation of Bacillus licheniformis to produce polyglutamic acid. Then, how to break through the problems of algae-bacteria competition control and process integration in the process of high-density liquid fermentation of Bacillus licheniformis to produce polyglutamic acid, so as to achieve the balance of algae and bacteria and help the high-density liquid fermentation of Bacillus licheniformis to produce polyglutamic acid has become a new research direction. Summary of the Invention

[0004] In view of the deficiencies existing in the prior art, the present invention provides a method for high-density liquid fermentation of Bacillus licheniformis for producing polyglutamic acid.

[0005] A method for high-density liquid fermentation of Bacillus licheniformis for producing polyglutamic acid, comprising the following steps: (1)Activation of the strain: Inoculate Bacillus licheniformis on a nutrient agar slant and culture it at 35 - 37°C for 12 - 24 h to obtain the activated strain; (2)Preparation of the seed liquid: Inoculate the activated strain obtained in step (1) into a shake flask seed medium and perform shake flask culture to obtain the primary seed liquid; after the primary seed liquid is examined under a microscope and found to be free of contaminating bacteria, transfer it to a seed tank at an inoculation amount of 1 - 5% by volume. The liquid filling amount in the seed tank is 50 - 70% of the volume of the seed tank, and the medium is the same as the above shake flask seed medium to obtain the secondary seed liquid; (3)High - density live bacteria fermentation: After the secondary seed liquid obtained in step (2) is examined under a microscope and found to be free of contaminating bacteria, inoculate it into the fermentation medium in a fermenter at an inoculation amount of 1 - 5% by volume and culture for 12 - 48 h; (4)Polyglutamic acid conversion: After the culture in step (3) is completed, inoculate Chlorella FACHB - 31 with an initial OD680 of 0.500 - 0.520, and supplement it with light - dark cycles of 12 h / 12 h and light intensity of 9000 - 11000 Lux. Then, add sodium L - glutamate, coral mucus, and sucrose to the fermentation medium, and continue to ferment under the fermentation conditions of step (3) for 12 - 48 h. When more than 90% of the cell morphology in the fermentation broth is spores, stop fermentation, centrifuge to obtain the supernatant, add anhydrous ethanol with a volume 3 - 4 times that of the supernatant, stand at 1 - 4°C for 8 - 24 h and then centrifuge, and freeze - dry to obtain the polyglutamic acid.

[0006] The nutrient agar described in step (1) contains 100 - 400 g / L of coral mucus, 9 - 11 g / L of peptone, 4.5 - 5.5 g / L of NaCl, 0.6 - 1.2 g / L of KH2PO4, and the balance is water.

[0007] The culture conditions for the primary seed liquid described in step (2) are 35 - 37°C, a shaking speed of 180 - 200 rpm, an aeration rate of 0.6 - 0.9 vvm, and culture for 8 - 24 h; the shake flask seed medium contains 200 - 400 g / L of coral mucus filtrate, 9 - 11 g / L of corn starch, 4 - 6 g / L of yeast extract, 0.8 - 1.2 g / L of KH2PO4, and the balance is water.

[0008] The culture conditions for the secondary seed liquid described in step (2) are 35 - 37°C, a stirring rate of 80 - 120 rpm, an aeration rate of 0.7 - 0.9 vvm, a tank pressure of 0.04 - 0.06 MPa, and culture for 8 - 24 h.

[0009] The fermentation medium described in step (3) contains 400 - 600 g / L of coral mucus, 4 - 6 g / L of glucose, 3 - 5 g / L of yeast extract, 0.1 - 0.3 g / L of MgSO4, 0.04 - 0.06 g / L of MnCl2, and the balance is water.

[0010] The high-density viable bacteria fermentation conditions described in step (3) are as follows: the fermentation temperature is 35 - 37°C; the air flow rate is adjusted as follows: controlled at 0.4 - 0.6 vvm from 0 h to 4 h, 0.9 - 1.1 vvm from 4 h to 12 h, and 1.4 - 1.6 vvm from 12 h to 24 h; the stirring rate is adjusted as follows: controlled at 80 - 90 rpm from 0 h to 4 h, 120 - 140 rpm from 4 h to 12 h, and 180 - 200 rpm from 12 h to 24 h.

[0011] The addition amount of sodium L-glutamate described in step (4) is 3 - 5 wt% of the mass of the fermentation medium, the addition amount of coral mucus is 9 - 11 wt% of the mass of the fermentation medium, and the addition amount of sucrose is 4 - 6 wt% of the mass of the fermentation medium.

[0012] The coral mucus is collected from an artificial aquaculture system of reef-building corals (Acropora pruinosa) under a simulated seawater environment. The salinity of this system is 33 - 35 wt‰, the temperature is 25 - 28°C, the light cycle is 10 h / 14 h, the light intensity is 1000 - 1500 Lux, and the continuous water flow rate is 0.1 - 0.3 m / s to simulate the natural reef area environment; using a sterile spatula or a silicone straw, gently scrape from the surface of the reef-building coral within 2 h after the coral feeds, that is, during the peak mucus secretion period; the collected mucus is sequentially pre-filtered through 100 - 150 μm, centrifuged at 6000 - 8000 rpm, and sterilized and filtered through 0.20 - 0.22 μm to obtain a filtrate, and the filtrate is the said coral mucus.

[0013] Advantages of the present invention: The method for high-density fermentation of Bacillus licheniformis provided by the present invention effectively solves the problems of difficult dissolved oxygen regulation and low productivity in the traditional high-density fermentation production of polyglutamic acid by Bacillus licheniformis through the innovative integration of the stage application of coral mucus and the symbiotic fermentation of Chlorella FACHB-31, and significantly improves the fermentation efficiency and product yield.

[0014] First of all, the present invention realizes a substantial increase in the polyglutamic acid productivity by introducing coral mucus as a carbon source and a growth promoter in the whole stage (activation, seed liquid, fermentation, conversion) and combining with the photosynthesis of Chlorella FACHB-31.

[0015] Secondly, the antibacterial properties of coral mucus (rich in polysaccharides and antibacterial peptides) effectively inhibit the growth of miscellaneous bacteria in the conversion stage, reduce the resource competition with Chlorella FACHB-31, thereby protecting the photosynthetic function of Chlorella FACHB-31 and providing a stable oxygen and metabolite supply. This method has low cost, high efficiency, and stable productivity, and is suitable for industrial fermentation. Specific embodiments

[0016] The coral mucus was collected from an artificial breeding system of reef-building corals (Acropora pruinosa) in a simulated seawater environment. The salinity of this system was 34 wt‰, the temperature was 26 °C, the light cycle was 10 h / 14 h, the light intensity was 1200 Lux, and the continuous water flow rate was 0.2 m / s to simulate the natural reef area environment. After 2 h of coral feeding, which was the peak mucus secretion period, it was gently scraped from the surface of the reef-building corals using a sterile spatula or a silica gel pipette. The collected mucus was successively pre-filtered through 100 μm, centrifuged at 6000 rpm, and sterilized by filtration through 0.22 μm to obtain a filtrate, and the filtrate was the coral mucus.

[0017] Bacillus licheniformis, with the preservation number: CGMCC 1.6510, was purchased from the China General Microbiological Culture Collection Center.

[0018] Chlorella sp. FACHB-31 was purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences.

[0019] Example 1

[0020] A high-density fermentation method for producing a liquid of Bacillus licheniformis polyglutamic acid includes the following steps: (1) Strain activation: Inoculate Bacillus licheniformis onto a nutrient agar slant and culture it at 36 °C for 12 h to obtain an activated strain. (2) Seed liquid preparation: Inoculate the activated strain obtained in step (1) into a shake flask seed medium for shake flask culture to obtain a primary seed liquid. After the primary seed liquid is examined under a microscope and found to be free of contaminating bacteria, transfer it to a seed tank at an inoculation amount of 5% by volume. The liquid filling amount in the seed tank is 70% of the volume of the seed tank, and the medium is the same as the above shake flask seed medium to obtain a secondary seed liquid. (3) High-density viable bacteria fermentation: After the secondary seed liquid obtained in step (2) is examined under a microscope and found to be free of contaminating bacteria, inoculate it into the fermentation medium of a fermenter at an inoculation amount of 5% by volume and culture for 24 h. (4) Polyglutamic acid conversion: After the culture in step (3) is completed, inoculate Chlorella sp. FACHB-31 with an initial OD680 of 0.510, and supplement it with light and dark cycles of 12 h / 12 h and a light intensity of 10000 Lux. Then, add sodium L-glutamate, coral mucus, and sucrose to the fermentation medium, and continue to ferment for 24 h under the fermentation conditions of step (3). When more than 90% of the cell morphology in the fermentation broth is spores, stop fermentation, centrifuge to obtain the supernatant, add anhydrous ethanol with a volume three times that of the supernatant, let it stand at 4 °C for 12 h and then centrifuge, and freeze-dry to obtain the polyglutamic acid.

[0021] The nutrient agar described in step (1) contains 200 g / L of coral mucus, 10 g / L of peptone, 5 g / L of NaCl, 1 g / L of KH₂PO₄, and the balance is water.

[0022] The culture conditions for the primary seed liquid in step (2) are 37 °C, a shaking speed of 200 rpm, an aeration rate of 0.8 vvm, and a culture time of 12 h; the shake flask seed medium contains 300 g / L of coral mucus filtrate, 10 g / L of corn starch, 5 g / L of yeast extract, 1 g / L of KH₂PO₄, and the balance is water.

[0023] The culture conditions for the secondary seed liquid in step (2) are 37 °C, a stirring rate of 100 rpm, an aeration rate of 0.8 vvm, a tank pressure of 0.05 MPa, and a culture time of 12 h.

[0024] The fermentation medium described in step (3) contains 500 g / L of coral mucus, 5 g / L of glucose, 4 g / L of yeast extract, 0.2 g / L of MgSO₄, 0.05 g / L of MnCl₂, and the balance is water.

[0025] The high-density viable bacteria fermentation conditions in step (3) are: fermentation temperature 36 °C; air flow rate adjustment: controlled at 0.5 vvm from 0 h to 4 h, 1.0 vvm from 4 h to 12 h, and 1.5 vvm from 12 h to 24 h; stirring rate adjustment: controlled at 80 rpm from 0 h to 4 h, 120 rpm from 4 h to 12 h, and 180 rpm from 12 h to 24 h.

[0026] The addition amount of monosodium glutamate in step (4) is 4 wt% of the mass of the fermentation medium, the addition amount of coral mucus is 10 wt% of the mass of the fermentation medium, and the addition amount of sucrose is 5 wt% of the mass of the fermentation medium.

[0027] Example 2

[0028] The difference from Example 1 is that: the medium used for strain activation in step (1) does not contain coral mucus; A high-density fermentation method for producing polyglutamic acid by Bacillus licheniformis liquid, comprising the following steps: (1) Strain activation: Inoculate Bacillus licheniformis on a nutrient agar slant and culture at 36 °C for 12 h to obtain an activated strain; (2) Seed liquid preparation: Inoculate the activated strain obtained in step (1) into a shake flask seed medium for shake flask culture to obtain a primary seed liquid; after the primary seed liquid is examined by microscopy and found to be free of contaminating bacteria, transfer it to a seed tank at an inoculation amount of 5% by volume. The liquid filling amount in the seed tank is 70% of the volume of the seed tank, and the medium is the same as the above shake flask seed medium to obtain a secondary seed liquid; (3)High-density viable cell fermentation: After the secondary seed liquid obtained in step (2) is examined under a microscope and found to be free of contaminating bacteria, it is inoculated into the fermentation medium in a fermenter at an inoculation amount of 5% by volume and cultured for 24 h. (4)γ-PGA conversion: After the culture in step (3) is completed, Chlorella FACHB-31 with an initial OD680 of 0.510 is inoculated, and supplemented with light and dark cycles of 12 h / 12 h and light intensity of 10,000 Lux. Then, sodium L-glutamate, coral mucus, and sucrose are added to the fermentation medium, and fermentation is continued under the fermentation conditions of step (3) for 24 h. When more than 90% of the cell morphology in the fermentation broth is spores, fermentation is stopped, the supernatant is taken by centrifugation, anhydrous ethanol with a volume three times that of the supernatant is added, centrifuged after standing at 4 °C for 12 h, and freeze-dried to obtain the γ-PGA.

[0029] The nutrient agar described in step (1) contains 10 g / L of peptone, 5 g / L of NaCl, 1 g / L of KH2PO4, and the balance is water.

[0030] The culture conditions for the primary seed liquid described in step (2) are 37 °C, shaker speed of 200 rpm, aeration rate of 0.8 vvm, and culture for 12 h; the shake flask seed medium contains 300 g / L of coral mucus filtrate, 10 g / L of corn starch, 5 g / L of yeast extract, 1 g / L of KH2PO4, and the balance is water.

[0031] The culture conditions for the secondary seed liquid described in step (2) are 37 °C, stirring rate of 100 rpm, aeration rate of 0.8 vvm, tank pressure of 0.05 MPa, and culture for 12 h.

[0032] The fermentation medium described in step (3) contains 500 g / L of coral mucus, 5 g / L of glucose, 4 g / L of yeast extract, 0.2 g / L of MgSO4, 0.05 g / L of MnCl2, and the balance is water.

[0033] The high-density viable cell fermentation conditions described in step (3) are: fermentation temperature of 36 °C; air flow rate adjustment: controlled at 0.5 vvm from 0 h to 4 h, 1.0 vvm from 4 h to 12 h, and 1.5 vvm from 12 h to 24 h; stirring rate adjustment: controlled at 80 rpm from 0 h to 4 h, 120 rpm from 4 h to 12 h, and 180 rpm from 12 h to 24 h.

[0034] The addition amount of sodium L-glutamate described in step (4) is 4 wt% of the mass of the fermentation medium, the addition amount of coral mucus is 10 wt% of the mass of the fermentation medium, and the addition amount of sucrose is 5 wt% of the mass of the fermentation medium.

[0035] Example 3

[0036] It is different from Example 2 in that the shake flask seed medium used in step (2) does not contain coral mucus. A method for high-density fermentation of liquid Bacillus licheniformis for producing polyglutamic acid, comprising the following steps: (1) Strain activation: Inoculate Bacillus licheniformis on a nutrient agar slant and culture at 36°C for 12 h to obtain an activated strain. (2) Seed liquid preparation: Inoculate the activated strain obtained in step (1) into a shake flask seed medium and perform shake flask culture to obtain a primary seed liquid; after the primary seed liquid is examined under a microscope and found to be free of contaminating bacteria, transfer it to a seed tank at an inoculation volume ratio of 5%, and the liquid filling volume in the seed tank is 70% of the volume of the seed tank. The medium is the same as the above shake flask seed medium to obtain a secondary seed liquid. (3) High-density viable bacteria fermentation: After the secondary seed liquid obtained in step (2) is examined under a microscope and found to be free of contaminating bacteria, inoculate it into the fermentation medium in a fermenter at an inoculation volume ratio of 5% and culture for 24 h. (4) Polyglutamic acid conversion: After the culture in step (3) is completed, inoculate Chlorella FACHB-31 with an initial OD680 of 0.510, and supplement with light and dark cycles of 12 h / 12 h and light intensity of 10,000 Lux. Then, add sodium L-glutamate, coral mucus, and sucrose to the fermentation medium, and continue to ferment for 24 h under the fermentation conditions of step (3). When more than 90% of the cell morphology in the fermentation broth is spores, stop fermentation, centrifuge to obtain the supernatant, add anhydrous ethanol with a volume three times that of the supernatant, stand at 4°C for 12 h, then centrifuge, and freeze-dry to obtain the polyglutamic acid.

[0037] The nutrient agar described in step (1) contains 10 g / L of peptone, 5 g / L of NaCl, 1 g / L of KH2PO4, and the balance is water.

[0038] The culture conditions for the primary seed liquid described in step (2) are 37°C, a shaking speed of 200 rpm, an aeration rate of 0.8 vvm, and culture for 12 h; the shake flask seed medium contains 10 g / L of corn starch, 5 g / L of yeast extract, 1 g / L of KH2PO4, and the balance is water.

[0039] The culture conditions for the secondary seed liquid described in step (2) are 37°C, a stirring rate of 100 rpm, an aeration rate of 0.8 vvm, a tank pressure of 0.05 MPa, and culture for 12 h.

[0040] The fermentation medium described in step (3) contains 500 g / L of coral mucus, 5 g / L of glucose, 4 g / L of yeast extract, 0.2 g / L of MgSO4, 0.05 g / L of MnCl2, and the balance is water.

[0041] The high-density live bacteria fermentation conditions described in step (3) are as follows: the fermentation temperature is 36°C; the air flow rate is adjusted as follows: controlled at 0.5 vvm from 0 h to 4 h, 1.0 vvm from 4 h to 12 h, and 1.5 vvm from 12 h to 24 h; the stirring rate is adjusted as follows: controlled at 80 rpm from 0 h to 4 h, 120 rpm from 4 h to 12 h, and 180 rpm from 12 h to 24 h.

[0042] The added amount of monosodium glutamate in step (4) is 4 wt% of the mass of the fermentation medium, the added amount of coral mucus is 10 wt% of the mass of the fermentation medium, and the added amount of sucrose is 5 wt% of the mass of the fermentation medium.

[0043] Example 4

[0044] The difference from Example 3 is that the fermentation medium used in step (3) does not contain coral mucus; A high-density fermentation method for producing polyglutamic acid with Bacillus licheniformis liquid, comprising the following steps: (1) Strain activation: Inoculate Bacillus licheniformis onto a nutrient agar slant and culture at 36°C for 12 h to obtain an activated strain; (2) Seed liquid preparation: Inoculate the activated strain obtained in step (1) into a shake flask seed medium for shake flask culture to obtain a primary seed liquid; after the primary seed liquid is examined under a microscope and found to be free of contaminating bacteria, transfer it to a seed tank at an inoculation volume ratio of 5%, and the liquid filling amount in the seed tank is 70% of the volume of the seed tank. The medium is the same as the above shake flask seed medium to obtain a secondary seed liquid; (3) High-density live bacteria fermentation: After the secondary seed liquid obtained in step (2) is examined under a microscope and found to be free of contaminating bacteria, inoculate it into the fermentation medium in a fermenter at an inoculation volume ratio of 5% and culture for 24 h; (4) Polyglutamic acid conversion: After the culture in step (3) is completed, inoculate Chlorella FACHB-31 with an initial OD680 of 0.510, and supplement with light and dark cycles of 12 h / 12 h and light intensity of 10,000 Lux. Then, add monosodium glutamate, coral mucus, and sucrose to the fermentation medium, and continue to ferment for 24 h under the fermentation conditions of step (3). When more than 90% of the cell morphology in the fermentation broth is spores, stop fermentation, centrifuge to obtain the supernatant, add anhydrous ethanol with a volume three times that of the supernatant, stand at 4°C for 12 h and then centrifuge, and freeze-dry to obtain the polyglutamic acid.

[0045] The nutrient agar described in step (1) contains 10 g / L of peptone, 5 g / L of NaCl, 1 g / L of KH2PO4, and the balance is water.

[0046] The culture conditions of the primary seed liquor described in step (2) are 37°C, shaking speed of 200 rpm, aeration rate of 0.8 vvm, and culture for 12 h; the shake flask seed medium contains 10 g / L of corn starch, 5 g / L of yeast extract, 1 g / L of KH2PO4, and the balance is water.

[0047] The culture conditions of the secondary seed liquor described in step (3) are 37°C, stirring rate of 100 rpm, aeration rate of 0.8 vvm, tank pressure of 0.05 MPa, and culture for 12 h.

[0048] The fermentation medium described in step (3) contains 5 g / L of glucose, 4 g / L of yeast extract, 0.2 g / L of MgSO4, 0.05 g / L of MnCl2, and the balance is water.

[0049] The high-density viable bacteria fermentation conditions described in step (3) are: fermentation temperature of 36°C; air flow rate adjustment: controlled at 0.5 vvm from 0 h to 4 h, 1.0 vvm from 4 h to 12 h, and 1.5 vvm from 12 h to 24 h; stirring rate adjustment: controlled at 80 rpm from 0 h to 4 h, 120 rpm from 4 h to 12 h, and 180 rpm from 12 h to 24 h.

[0050] The addition amount of monosodium glutamate in step (4) is 4 wt% of the mass of the fermentation medium, the addition amount of coral mucus is 10 wt% of the mass of the fermentation medium, and the addition amount of sucrose is 5 wt% of the mass of the fermentation medium.

[0051] Example 5

[0052] The difference from Example 4 is that no coral mucus is added to the fermentation medium used in step (4); A method for high-density fermentation of liquid Bacillus licheniformis for producing polyglutamic acid, comprising the following steps: (1) Strain activation: Inoculate Bacillus licheniformis onto a nutrient agar slant and culture at 36°C for 12 h to obtain an activated strain; (2) Seed liquor preparation: Inoculate the activated strain obtained in step (1) into a shake flask seed medium for shake flask culture to obtain a primary seed liquor; after the primary seed liquor is examined under a microscope and found to be free of contaminating bacteria, transfer it to a seed tank at an inoculation amount of 5% by volume. The liquid filling amount in the seed tank is 70% of the volume of the seed tank, and the medium is the same as the above shake flask seed medium to obtain a secondary seed liquor; (3) High-density viable bacteria fermentation: After the secondary seed liquor obtained in step (2) is examined under a microscope and found to be free of contaminating bacteria, inoculate it into the fermentation medium in a fermenter at an inoculation amount of 5% by volume and culture for 24 h; (4)Polyglutamic acid conversion: After the end of the cultivation in step (3), Chlorella FACHB-31 with an initial OD680 of 0.510 was inoculated, and supplemented with light and dark cycles of 12 h / 12 h and light intensity of 10,000 Lux. Then, sodium L-glutamate and sucrose were added to the fermentation medium, and fermentation was continued under the fermentation conditions of step (3) for 24 h. When more than 90% of the cell morphology in the fermentation broth was spores, fermentation was stopped, the supernatant was centrifuged, anhydrous ethanol with a volume three times that of the supernatant was added, centrifuged after standing at 4 °C for 12 h, and freeze-dried to obtain the polyglutamic acid.

[0053] The nutrient agar described in step (1) contains 10 g / L of peptone, 5 g / L of NaCl, 1 g / L of KH2PO4, and the balance is water.

[0054] The cultivation conditions for the primary seed liquid in step (2) are 37 °C, shaking speed of 200 rpm, aeration rate of 0.8 vvm, and cultivation for 12 h; the shake flask seed medium contains 10 g / L of corn starch, 5 g / L of yeast extract, 1 g / L of KH2PO4, and the balance is water.

[0055] The cultivation conditions for the secondary seed liquid in step (2) are 37 °C, stirring rate of 100 rpm, aeration rate of 0.8 vvm, tank pressure of 0.05 MPa, and cultivation for 12 h.

[0056] The fermentation medium described in step (3) contains 5 g / L of glucose, 4 g / L of yeast extract, 0.2 g / L of MgSO4, 0.05 g / L of MnCl2, and the balance is water.

[0057] The high-density viable cell fermentation conditions in step (3) are: fermentation temperature 36 °C; air flow adjustment: controlled at 0.5 vvm from 0 h to 4 h, 1.0 vvm from 4 h to 12 h, and 1.5 vvm from 12 h to 24 h; stirring rate adjustment: controlled at 80 rpm from 0 h to 4 h, 120 rpm from 4 h to 12 h, and 180 rpm from 12 h to 24 h.

[0058] The added amount of sodium L-glutamate in step (4) is 4 wt% of the mass of the fermentation medium, and the added amount of sucrose is 5 wt% of the mass of the fermentation medium.

[0059] Example 6

[0060] The difference from Example 4 is that in step (4), Chlorella FACHB-31 is not inoculated; A high-density fermentation method for producing polyglutamic acid by Bacillus licheniformis liquid, comprising the following steps: (1)Strain activation: Inoculate Bacillus licheniformis on a nutrient agar slant and culture at 36 °C for 12 h to obtain an activated strain; (2)Seed liquid preparation: The activated strain obtained in step (1) is inoculated into a shake flask seed culture medium for shake flask culture to obtain a primary seed liquid; after the primary seed liquid is examined under a microscope and found to be free of contaminating bacteria, it is transferred to a seed tank at an inoculation amount of 5% by volume. The liquid filling amount in the seed tank is 70% of the volume of the seed tank, and the culture medium is the same as the above shake flask seed culture medium to obtain a secondary seed liquid; (3)High-density viable bacteria fermentation: After the secondary seed liquid obtained in step (2) is examined under a microscope and found to be free of contaminating bacteria, it is inoculated into the fermentation culture medium of a fermenter at an inoculation amount of 5% by volume and cultured for 24 h; (4)γ-PGA conversion: After the culture in step (3) is completed, Chlorella FACHB-31 with an initial OD680 of 0.510 is inoculated, and supplemented with a light-dark cycle of 12 h / 12 h and a light intensity of 10,000 Lux. Then, sodium L-glutamate and sucrose are added to the fermentation culture medium, and fermentation is continued under the fermentation conditions of step (3) for 24 h. When more than 90% of the cell morphology in the fermentation broth is spores, fermentation is stopped, the supernatant is taken by centrifugation, anhydrous ethanol with a volume three times that of the supernatant is added, centrifuged after standing at 4 °C for 12 h, and freeze-dried to obtain the γ-PGA.

[0061] The nutrient agar in step (1) contains 10 g / L of peptone, 5 g / L of NaCl, 1 g / L of KH2PO4, and the balance is water.

[0062] The culture conditions for the primary seed liquid in step (2) are 37 °C, a shaking speed of 200 rpm, an aeration rate of 0.8 vvm, and a culture time of 12 h; the shake flask seed culture medium contains 10 g / L of corn starch, 5 g / L of yeast extract, 1 g / L of KH2PO4, and the balance is water.

[0063] The culture conditions for the secondary seed liquid in step (2) are 37 °C, a stirring rate of 100 rpm, an aeration rate of 0.8 vvm, a tank pressure of 0.05 MPa, and a culture time of 12 h.

[0064] The fermentation culture medium in step (3) contains 5 g / L of glucose, 4 g / L of yeast extract, 0.2 g / L of MgSO4, 0.05 g / L of MnCl2, and the balance is water.

[0065] The high-density viable bacteria fermentation conditions in step (3) are: the fermentation temperature is 36 °C; air flow adjustment: controlled at 0.5 vvm from 0 h to 4 h, 1.0 vvm from 4 h to 12 h, and 1.5 vvm from 12 h to 24 h; stirring rate adjustment: controlled at 80 rpm from 0 h to 4 h, 120 rpm from 4 h to 12 h, and 180 rpm from 12 h to 24 h.

[0066] The addition amount of sodium L-glutamate described in step (4) is 4 wt% of the mass of the fermentation medium, the addition amount of coral mucus is 10 wt% of the mass of the fermentation medium, and the addition amount of sucrose is 5 wt% of the mass of the fermentation medium.

[0067] Comparative Example 1

[0068] The difference from Example 5 is that in step (4), Chlorella FACHB-31 is not inoculated. A method for high-density fermentation of Bacillus licheniformis liquid for producing polyglutamic acid, comprising the following steps: (1) Strain activation: Inoculate Bacillus licheniformis onto a nutrient agar slant and culture at 36 °C for 12 h to obtain an activated strain. (2) Seed liquid preparation: Inoculate the activated strain obtained in step (1) into a shake flask seed medium for shake flask culture to obtain a primary seed liquid; after the primary seed liquid is examined under a microscope and found to be free of contamination by miscellaneous bacteria, transfer it to a seed tank at an inoculation amount of 5% by volume. The liquid filling amount in the seed tank is 70% of the volume of the seed tank, and the medium is the same as the above shake flask seed medium to obtain a secondary seed liquid. (3) High-density viable bacteria fermentation: After the secondary seed liquid obtained in step (2) is examined under a microscope and found to be free of contamination by miscellaneous bacteria, inoculate it into the fermentation medium of the fermentation tank at an inoculation amount of 5% by volume and culture for 24 h. (4) Polyglutamic acid conversion: After the culture in step (3) is completed, supplement with a light-dark cycle of 12 h / 12 h and a light intensity of 10000 Lux. Then, supplement sodium L-glutamate and sucrose in the fermentation medium and continue to ferment under the fermentation conditions of step (3) for 24 h. When more than 90% of the cell morphology in the fermentation broth is spores, stop fermentation, centrifuge to obtain the supernatant, add anhydrous ethanol with a volume three times that of the supernatant, stand at 4 °C for 12 h and then centrifuge, and freeze-dry to obtain the polyglutamic acid.

[0069] The nutrient agar described in step (1) contains 10 g / L of peptone, 5 g / L of NaCl, 1 g / L of KH2PO4, and the balance is water.

[0070] The culture conditions for the primary seed liquid described in step (2) are 37 °C, a shaking speed of 200 rpm, an aeration rate of 0.8 vvm, and culture for 12 h; the shake flask seed medium contains 10 g / L of corn starch, 5 g / L of yeast extract, 1 g / L of KH2PO4, and the balance is water.

[0071] The culture conditions for the secondary seed liquid described in step (2) are 37 °C, a stirring rate of 100 rpm, an aeration rate of 0.8 vvm, a tank pressure of 0.05 MPa, and culture for 12 h.

[0072] The fermentation medium described in step (3) contains 5 g / L of glucose, 4 g / L of yeast extract, 0.2 g / L of MgSO4, 0.05 g / L of MnCl2, and the balance is water.

[0073] The high-density live bacteria fermentation conditions described in step (3) are as follows: fermentation temperature is 36 °C; air flow adjustment: controlled at 0.5 vvm from 0 h to 4 h, 1.0 vvm from 4 h to 12 h, and 1.5 vvm from 12 h to 24 h; stirring rate adjustment: controlled at 80 rpm from 0 h to 4 h, 120 rpm from 4 h to 12 h, and 180 rpm from 12 h to 24 h.

[0074] The addition amount of monosodium L-glutamate described in step (4) is 4 wt% of the mass of the fermentation medium, and the addition amount of sucrose is 5 wt% of the mass of the fermentation medium.

[0075] Test Example 1

[0076] Weigh the polyglutamic acid produced by the high-density fermentation method of the liquid of Bacillus licheniformis for producing polyglutamic acid in Examples 1-5 and Comparative Example 1. The volume of the fermentation medium used in the high-density fermentation stage is 1 L, and the polyglutamic acid yield is shown in Table 1; Table 1: Polyglutamic acid yield

[0077] It can be seen from Table 1 that in Example 1 (the whole stage includes coral mucus), the polyglutamic acid yield is the highest because coral mucus provides nutrients from the activation stage, shortens the lag phase, and improves the spore formation rate and polyglutamic acid synthesis efficiency; coral mucus is rich in polysaccharides (such as arabinose and xylose) and proteins, and can be used as a high-quality carbon source and microbial growth promoter. Its presence significantly increases the cell density and metabolic activity.

[0078] In Example 2, coral mucus is missing in the activation stage: the yield drops to 11.2 g because the incomplete activation of the strain affects subsequent growth.

[0079] In Example 3, coral mucus is missing in the activation and seed liquid fermentation stages, and the yield drops to 9.0 g. The decrease in the quality of the seed liquid results in insufficient strain quantity at the initial stage of fermentation.

[0080] In Example 4, coral mucus is added only in the polyglutamic acid conversion stage, and the yield is 7.3 g. Because of the lack of nutrients in the early stage, the proliferation of bacteria is restricted, and the addition in the conversion stage only partially compensates.

[0081] In Example 5, there is no mucus at all, and the yield is the lowest. Only relying on the basic carbon source (glucose), the synthesis of polyglutamic acid is restricted.

[0082] In Example 6, coral mucus was supplemented only during the conversion of polyglutamic acid, and Chlorella FACHB-31 was not inoculated. The yield of polyglutamic acid also decreased significantly compared to Example 4. The present invention believes that without Chlorella FACHB-31, the mucus directly promotes bacterial growth but cannot provide additional oxygen, resulting in a decrease in yield.

[0083] Chlorella FACHB-31 undergoes photosynthesis under a light-dark cycle, providing dissolved oxygen and metabolites, promoting the microaerobic fermentation and polyglutamic acid conversion of Bacillus licheniformis. In Comparative Example 1, Chlorella FACHB-31 was not inoculated, and the yield was only 3.8 g, which further decreased compared to Example 5 (containing Chlorella FACHB-31), confirming that Chlorella FACHB-31 improves the conversion efficiency through oxygen supply and symbiotic metabolism.

[0084] It can be seen from the test example data that, firstly, the present invention realizes a significant increase in the yield of polyglutamic acid by introducing coral mucus as a carbon source and growth promoter in the whole stage (activation, seed liquid, fermentation, conversion) and combining it with the photosynthesis of Chlorella FACHB-31. As shown in Example 1, the yield reached 12.5 g, which was 229% higher than that of Comparative Example 1 (3.8 g) without coral mucus and without Chlorella FACHB-31, reflecting the optimization of resource utilization.

[0085] Secondly, the antibacterial properties of coral mucus (rich in polysaccharides and antibacterial peptides) effectively inhibit the growth of miscellaneous bacteria during the conversion stage, reducing the resource competition with Chlorella FACHB-31, thereby protecting the photosynthetic function of Chlorella FACHB-31 and providing a stable supply of oxygen and metabolites. The comparison between Example 4 (coral mucus was supplemented during the conversion stage and Chlorella FACHB-31 was inoculated, with a yield of 7.3 g) and Example 5 (without mucus but with Chlorella FACHB-31 inoculated, with a yield of 5.6 g) shows that coral mucus increased the yield by 30.4%, proving its core role in competitive inhibition; Example 6 (with coral mucus but without Chlorella FACHB-31, with a yield of 5.5 g) further confirmed that coral mucus alone inhibits miscellaneous bacteria, and the yield was 44.7% higher than that of Comparative Example 1.

[0086] Finally, the introduction of coral mucus in the activation stage (Example 1) is more crucial than in the later stage (Example 4). The yield gradient (Example 1: 12.5 g > Example 2: 11.2 g > Example 3: 9.0 g) shows that early application shortens the lag phase and increases the spore formation rate to over 90%, solving the low-efficiency problem of stage disconnection in the prior art.

[0087] This method has low cost, high efficiency, stable yield, and is suitable for industrial fermentation.

Claims

1. A high-density fermentation method for a liquid of Bacillus licheniformis for producing polyglutamic acid, characterized in that, It includes the following steps: (1) Strain activation: Inoculate Bacillus licheniformis on a nutrient agar slant for cultivation to obtain an activated strain; (2) Seed liquid preparation: Inoculate the activated strain obtained in step (1) into a shake flask seed culture medium for shake flask cultivation to obtain a primary seed liquid; after the primary seed liquid is examined under a microscope and found to be free of contaminating bacteria, transfer it to a seed tank at an inoculation amount of 1-5% by volume. The liquid filling amount in the seed tank is 50-70% of the volume of the seed tank, and the culture medium is the same as the above shake flask seed culture medium to obtain a secondary seed liquid; (3) High-density viable cell fermentation: After the secondary seed liquid obtained in step (2) is examined under a microscope and found to be free of contaminating bacteria, inoculate it into the fermentation culture medium in a fermenter at an inoculation amount of 1-5% by volume and culture for 12-48 h; (4) Polyglutamic acid conversion: After the cultivation in step (3) is completed, inoculate Chlorella FACHB-31 and supplement with light. Then, add sodium L-glutamate, coral mucus and sucrose to the fermentation culture medium, and continue to ferment under the fermentation conditions of step (3). When more than 90% of the cell morphology in the fermentation broth is spores, stop fermentation, centrifuge to obtain the supernatant, add anhydrous ethanol with a volume 3-4 times that of the supernatant, let it stand at 1-4 °C for 8-24 h and then centrifuge, and freeze-dry to obtain the polyglutamic acid.

2. The high-density fermentation method of Bacillus licheniformis liquid for producing polyglutamic acid according to claim 1, characterized in that, The nutrient agar in step (1) contains 100-400 g / L of coral mucus, 9-11 g / L of peptone, 4.5-5.5 g / L of NaCl, 0.6-1.2 g / L of KH2PO4, and the balance is water.

3. The high-density fermentation method of the lichen bacillus liquid for producing polyglutamic acid according to claim 1, characterized in that, The cultivation conditions for the primary seed liquid in step (2) are 35-37 °C, a shaker speed of 180-200 rpm, an aeration rate of 0.6-0.9 vvm, and cultivation for 8-24 h; the shake flask seed culture medium contains 200-400 g / L of coral mucus filtrate, 9-11 g / L of corn starch, 4-6 g / L of yeast extract, 0.8-1.2 g / L of KH2PO4, and the balance is water.

4. The high-density fermentation method of the lichen bacillus liquid for producing polyglutamic acid according to claim 1, characterized in that, The cultivation conditions for the secondary seed liquid in step (2) are 35-37 °C, a stirring rate of 80-120 rpm, an aeration rate of 0.7-0.9 vvm, a tank pressure of 0.04-0.06 MPa, and cultivation for 8-24 h.

5. The high-density fermentation method of the liquid of Bacillus licheniformis for producing polyglutamic acid according to claim 1, characterized in that, The fermentation culture medium in step (3) contains 400-600 g / L of coral mucus, 4-6 g / L of glucose, 3-5 g / L of yeast extract, 0.1-0.3 g / L of MgSO4, 0.04-0.06 g / L of MnCl2, and the balance is water.

6. The high-density fermentation method of the Bacillus licheniformis liquid for producing polyglutamic acid according to claim 1, characterized in that, The high-density viable cell fermentation conditions in step (3) are: fermentation temperature 35-37 °C; air flow rate adjustment: controlled at 0.4-0.6 vvm from 0 h to 4 h, 0.9-1.1 vvm from 4 h to 12 h, and 1.4-1.6 vvm from 12 h to 24 h; stirring rate adjustment: controlled at 80-90 rpm from 0 h to 4 h, 120-140 rpm from 4 h to 12 h, and 180-200 rpm from 12 h to 24 h.

7. The high-density fermentation method of the Bacillus licheniformis liquid for producing polyglutamic acid according to claim 1, characterized in that, The additional amount of sodium L-glutamate described in step (4) is 3-5 wt% of the mass of the fermentation medium, the additional amount of coral mucus is 9-11 wt% of the mass of the fermentation medium, and the additional amount of sucrose is 4-6 wt% of the mass of the fermentation medium.

8. The high-density fermentation method of the liquid of Bacillus licheniformis for producing polyglutamic acid according to claim 1, characterized in that, The coral mucus is collected from an artificial aquaculture system of reef-building corals (Acropora pruinosa) in a simulated seawater environment. The salinity of the system is 33-35 wt‰, the temperature is 25-28 °C, the light cycle is 10 h / 14 h, the light intensity is 1000-1500 Lux, and the continuous water flow rate is 0.1-0.3 m / s to simulate the natural reef area environment; using a sterile spatula or silicone straw, gently scrape from the surface of the reef-building coral within 2 h after the coral feeds, that is, during the peak mucus secretion period; the collected mucus is successively pre-filtered through 100-150 μm, centrifuged at 6000-8000 rpm, and sterilized and filtered through 0.20-0.22 μm to obtain a filtrate, and the filtrate is the coral mucus.

Images