Method for preparing gamma-polyglutamic acid by microbial fermentation

Abstract

The present invention discloses a method for preparing γ-polyglutamic acid by microbial fermentation. In the method, enzymatic hydrolysate is used as the main carbon source of the fermentation medium, a magnetic oxide / epoxy resin porous composite material is added to the fermentation medium, and γ-polyglutamic acid is prepared by fermentation of Bacillus subtilis. Compared with the prior art, the yield of γ-polyglutamic acid prepared by the present invention is higher.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a method for preparing gamma-polyglutamic acid by microbial fermentation. Background Art

[0002] γ-Polyglutamic acid (γ-PGA) is an environmentally friendly multifunctional biodegradable polymer material of the polyamino acid class. As a polymer, γ-PGA has some unique physical, chemical and biological properties, such as good water solubility, super strong adsorption, can be completely biodegraded, non-toxic and harmless, edible, etc. It can be used as a raw material for water retaining agent, thickener, flocculant, heavy metal adsorbent, drug / fertilizer sustained release agent and drug carrier, etc., and has broad application prospects in agriculture, food, medicine, cosmetics, environmental protection, synthetic fiber and coating. The synthesis methods of γ-polyglutamic acid mainly include extraction method, chemical synthesis method, microbial fermentation method and enzyme conversion method. At present, the preparation of γ-polyglutamic acid is mainly carried out by microbial fermentation method, which has the advantages of relatively simple process and relatively easy separation and purification of products, but the yield of γ-polyglutamic acid in the fermentation process needs to be improved.

[0003] The invention patent with application number 202010536784.5 discloses a method for preparing γ-polyglutamic acid using resting cells of recombinant bacteria expressing polyglutamate synthetase and / or polyglutamate synthetase, which also provides polyglutamate synthetase, and then provides a coding gene for polyglutamate synthetase, and then provides a recombinant expression vector of the nucleic acid sequence of polyglutamate synthetase and a method for obtaining recombinant bacteria expressing polyglutamate synthetase. The method uses whole microbial cells as reaction catalysts, transforms exogenous substrates with structural modifications, and synthesizes resting free cells, which has mild reaction conditions, simple reaction system, short reaction time, high conversion rate, and no pollution, greatly accelerating the synthesis efficiency of the target product γ-polyglutamic acid, and the product molecular weight is relatively uniform, belonging to the low molecular weight range of γ-polyglutamic acid. However, this method has a large investment cost and is not economical.

[0004] The invention patent with application number 200610122640.5 discloses a γ-polyglutamic acid producing bacterium and a method for preparing γ-polyglutamic acid using the strain. The microbial strain provided by the invention is a γ-polyglutamic acid (γ-PGA) producing bacterium, Bacillus subtilis PGA-7 CCTCC M 206102, which has been selected. The method for producing γ-polyglutamic acid is to use Bacillus subtilis CCTCC M 206102 as a fermentation strain and to use sugar raw materials for shake flask fermentation. No L-glutamic acid is required to be added to the fermentation raw materials. This has a significant reduction in raw material cost compared to the conventional microbial fermentation that requires glutamic acid as a raw material. Under the optimal shake flask fermentation process conditions, the invention can produce up to 18 g / L of γ-PGA, and has good prospects for industrial application. However, the method has the problem that the yield of γ-polyglutamic acid is not high enough. Summary of the invention

[0005] In view of the problems of uneconomical production and low yield in the prior art, the technical problem to be solved by the present invention is to provide an economical microbial fermentation method for increasing the yield of γ-polyglutamic acid.

[0006] To achieve the above object, the present invention provides a method for preparing γ-polyglutamic acid by microbial fermentation, the steps of which are as follows:

[0007] Step 1, activating the strain: using an inoculation loop to pick 1 to 3 loops of Bacillus subtilis strains and inoculate them on a flat plate activation culture medium, and culturing them at a constant temperature of 35 to 38° C. for 11 to 13 hours to obtain activated strains;

[0008] Step 2, preparation of seed solution: pick 2 to 5 rings of activated bacteria prepared in step 1, inoculate them into seed culture medium, and culture them at a constant temperature of 35 to 38° C. with shaking for 14 to 16 hours at an shaking speed of 200 to 250 rpm to obtain seed solution; dilute the seed solution to prepare a bacterial suspension with a concentration of 130 million to 160 million per liter;

[0009] Step 3, preparation of fermentation liquid: inoculate the bacterial suspension prepared in step 2 into the fermentation medium, add the ultraviolet sterilized magnetic oxide / epoxy resin porous composite material, and culture at a constant temperature of 35-38° C. with shaking for 51-53 hours to obtain a fermentation liquid containing γ-polyglutamic acid;

[0010] Step 4, extraction of γ-polyglutamic acid: the fermentation liquid containing γ-polyglutamic acid obtained in step 3 is adjusted to pH 3-4, centrifuged at 4000-6000 rpm for 25-50 minutes, the supernatant is taken, anhydrous ethanol precooled at -1-3°C for 20-40 minutes is added, stirred at 300-500 rpm for 10-30 minutes at 3-4.5°C and allowed to stand for 14-16 hours, then centrifuged at 4000-6000 rpm for 12-25 minutes, the precipitate is collected, vacuum dried for 8-16 hours, then added to water, stirred at 300-500 rpm for 15-30 minutes, dialyzed in water for 23-25 ​​hours, and vacuum freeze-dried for 10-24 hours to obtain γ-polyglutamic acid.

[0011] Preferably, the plate activation culture medium comprises the following raw materials in weight percentage: 15-25 g / L agar powder, 8-12 g / L soy peptone, 3-8 g / L beef extract, 3-8 g / L sodium chloride, 1-3 g / L yeast powder, 0.5-1.5 g / L glucose, the solvent is water, and the pH is adjusted to 7.5.

[0012] Preferably, the seed culture medium comprises the following raw materials in weight percentage: 25-35 g / L sucrose, 25-35 g / L sodium glutamate, 5-15 g / L beef extract, 0.25-0.75 g / L dipotassium hydrogen phosphate trihydrate, 0.15-0.45 g / L magnesium sulfate heptahydrate, the solvent is water, and the pH is adjusted to 7.5.

[0013] Preferably, the inoculum volume in step 3 is 3-15% mL / mL.

[0014] Preferably, the fermentation medium comprises the following raw materials in weight percentage: 450-600 mL / L enzymolysate, 35-55 g / L sodium glutamate, 4-10 g / L beef extract, 0.5-1.5 g / L potassium hydrogen phosphate trihydrate, 0.15-0.35 g / L magnesium sulfate heptahydrate, 0.05-0.15 g / L sodium chloride, the solvent is water, and the pH is adjusted to 7.5.

[0015] Preferably, the fermentation medium filling volume is 120~180mL / 500mL.

[0016] Preferably, the pH of the culture medium is adjusted using a 1% to 1.5 wt % aqueous solution of dipotassium hydrogen phosphate and a 0.08% to 0.1 wt % aqueous solution of potassium dihydrogen phosphate.

[0017] Preferably, the above culture medium is sterilized by high temperature steam at 121°C for 18 to 25 minutes.

[0018] Preferably, the mass and volume ratio of the magnetic oxide / epoxy resin porous composite material to the fermentation medium is 0.3-1.6:9 g / mL.

[0019] Preferably, the pH in step 4 is adjusted using 0.01-0.1 mol / L hydrochloric acid.

[0020] Preferably, the ratio of the volume of water added in step 4 to the volume of the fermentation medium is 1-3:8-10.

[0021] Preferably, the dialysis cut-off molecular weight is 50 kD to 100 kD.

[0022] Further preferably, the enzymatic solution preparation method is:

[0023] 10 to 30 parts by weight of dry material are sequentially soaked in an acetic acid aqueous solution having a concentration of 4 to 8 wt%, an aqueous sodium hydroxide solution having a concentration of 8 to 12 wt%, and 10 to 30 parts by weight of water, and washed with water 1 to 3 times after each soaking. After the soaking treatment is completed, the dry material is sterilized at a high temperature of 119 to 125° C. for 1.5 to 2.5 hours, cooled to 20 to 30° C., washed 2 to 4 times in an ethanol aqueous solution having a concentration of 40 to 60 wt%, and then dried at 100 to 110° C. for 8 to 12 hours to obtain a pretreated dry material; 8 to 20 parts by weight of the pretreated dry material are mixed with a citric acid-sodium citrate buffer, and then cellulase and Aspergillus niger acid protease are added, and enzymolysis is carried out at a constant temperature of 35 to 38° C. for 8 to 12 hours, and then the mixture is water bathed at a temperature of 98 to 110° C. for 10 to 15 minutes, centrifuged at 4000 to 7000 rpm for 10 to 30 minutes, and the supernatant is collected to obtain an enzymolysis solution;

[0024] Preferably, the dry material is a mixture of soybean meal and sago palm pith sawdust in a mass ratio of 1:1 to 3.

[0025] Preferably, the mass volume ratio of the dry material to the acetic acid aqueous solution and the sodium hydroxide aqueous solution is independently 1:8-15 g / mL.

[0026] Preferably, the soaking time is independently 60 to 120 minutes.

[0027] Preferably, the mass volume ratio of the pretreated dry material to the citric acid-sodium citrate buffer is 80-200:1 g / L.

[0028] Preferably, the citric acid-sodium citrate buffer solution is obtained by mixing a citric acid aqueous solution and a sodium citrate aqueous solution, both of which have a concentration of 0.06-0.12 mol / L, in a volume ratio of 1.6-2.85:2.15-3.4.

[0029] Preferably, the amount of cellulase added is 9-14% of the mass of the sago palm pith sawdust.

[0030] Preferably, the added amount of the Aspergillus niger acid protease is 0.03-0.06% of the mass of the soybean meal.

[0031] Preferably, the magnetic oxide / epoxy resin porous composite material is prepared as follows:

[0032] S1. Add 30-60 parts by weight of magnesium nitrate hexahydrate and 80-120 parts by weight of ferric nitrate nonahydrate to 800-1500 parts by weight of water, stir at 300-500 rpm for 10-30 minutes to obtain a mixture A, then add 55-90 parts by weight of hexamethylenetetramine to the mixture A in 3-5 portions while stirring at 300-500 rpm, continue stirring at 300-500 rpm until the solution in the mixture A becomes transparent, then react at 118-205° C. for 9-11 hours, then cool naturally to 20-30° C., collect the solid, wash with water and anhydrous ethanol for 2-4 times respectively, dry at 55-80° C. for 8-12 hours, and then calcine at 650-750° C. for 3.5-4.5 hours to obtain a magnetic oxide;

[0033] S2, mix 60-80 parts by weight of the magnetic oxide prepared in step S1 and 45-60 parts by weight of water, stir at 300-500 rpm for 5-20 minutes to obtain a magnetic oxide dispersion; then mix 30-50 parts by weight of epoxy resin and 0.4-1.2 parts by weight of silicon carbide, stir at 300-500 rpm for 20-50 minutes, then ultrasonicate for 1.5-2.5 hours, add 4-25 parts by weight of curing agent, stir at 300-500 rpm for 1-4 minutes, then add the magnetic oxide dispersion, continue to stir at 300-500 rpm for 2-5 minutes, and cure at 15-30°C to obtain a magnetic oxide / epoxy resin porous composite material.

[0034] Preferably, the ultrasonic power is 200-300W.

[0035] Preferably, the curing agent is any one of diethylaminopropylamine, trimethylhexamethylenediamine, diethylamine and dipropylenetriamine.

[0036] Preferably, the formed particle size is 1-3 mm and can be any shape.

[0037] Sago palm tree pith sawdust: Sago palm is an evergreen tree of the genus Sago palm of the palm family. Its pith contains elements such as K, Ca, N, Mg, P and starch. The trunk is cut before flowering, the pith is taken out, and the sawdust is shaved to obtain Sago palm tree pith sawdust.

[0038] The present invention uses enzymatic hydrolysate as the main carbon source of the fermentation medium, adds a magnetic oxide / epoxy resin porous composite material to the fermentation medium, and prepares γ-polyglutamic acid through Bacillus subtilis fermentation. The prepared γ-polyglutamic acid has a high yield. The enzymatic hydrolysate uses soybean meal and sago palm pith sawdust as raw materials, and is sterilized at high temperature after being treated with acid, alkali and water, and then enzymatically hydrolyzed with cellulase and centrifuged to collect the supernatant after cleaning and drying. During the fermentation process, the strain can make full use of the enzymatic hydrolysate, and the amount of γ-polyglutamic acid produced increases; during the fermentation process, the viscosity of the fermentation liquid will affect the yield of γ-polyglutamic acid. When the viscosity of the fermentation liquid is relatively high, the dissolved oxygen content in the fermentation liquid will be insufficient. When the oxygen is insufficient, the metabolism of Bacillus subtilis will be affected, resulting in a decrease in the yield of γ-polyglutamic acid. Therefore, the dissolution of the fermentation liquid is increased during the fermentation process. Oxygen can increase the yield of γ-polyglutamic acid; for this purpose, the present invention prepares a magnetic oxide, and then uses a resin-water-filler multiphase suspension emulsion polymerization method to compound the magnetic oxide with epoxy resin to prepare a magnetic oxide / epoxy resin porous composite material. During the fermentation process, Bacillus subtilis is adsorbed on the surface of the magnetic oxide / epoxy resin porous composite material, increasing the contact area between Bacillus subtilis and the fermentation liquid, so that Bacillus subtilis can fully utilize nutrients. At the same time, the magnetic oxide / epoxy resin porous composite material increases the content of dissolved oxygen in the fermentation liquid during movement, which can increase the yield of γ-polyglutamic acid. At the same time, in the magnetic oxide / epoxy resin porous composite material, Fe 3+ and Mg 2+ , which can promote the production of γ-polyglutamic acid by Bacillus subtilis and increase the yield of γ-polyglutamic acid. The magnetic oxide / epoxy resin porous composite material has magnetic properties, which is conducive to the recovery of the magnetic oxide / epoxy resin porous composite material.

[0039] Due to the adoption of the above technical scheme, compared with the prior art, the present invention has the following advantages: 1) the enzymatic hydrolysate is used as the main carbon source of the fermentation medium, thereby increasing the yield of γ-polyglutamic acid; 2) during the fermentation process, the addition of the magnetic oxidant / epoxy resin porous composite material increases the content of dissolved oxygen in the fermentation process, thereby increasing the yield of γ-polyglutamic acid, and at the same time, Fe 3+ and Mg 2+ The precipitation of γ-polyglutamic acid was further promoted by Bacillus subtilis; 3) The magnetic oxide / epoxy resin porous composite material has magnetic properties, which is beneficial to the recovery of the magnetic oxide / epoxy resin porous composite material. DETAILED DESCRIPTION

[0040] Examples and Comparative Examples Partial raw materials:

[0041] Bacillus subtilis subsp.subtilis Bacillus subtilis subsp .subtilis Bacterial strain, CGMCC 1.3358.

[0042] Citric acid-sodium citrate buffer: Mix 2.3 L of 0.1 mol / L citric acid aqueous solution with 2.7 L of 0.1 mol / L sodium citrate aqueous solution.

[0043] Cellulase: enzyme activity: 100000 u / g.

[0044] Aspergillus niger acid protease: enzyme activity: 100000 u / g.

[0045] Epoxy resin: Model: E44, epoxy equivalent G / EQ: 210~244.

[0046] Silicon carbide: particle size: 325 mesh.

[0047] Example 1

[0048] A method for preparing γ-polyglutamic acid by microbial fermentation, comprising the following steps:

[0049] Step 1, activating the bacteria: adding 20g agar powder, 10g soy peptone, 5g beef extract, 5g sodium chloride, 2g yeast powder, and 1g glucose to 1L water, stirring at 500 rpm for 45 minutes, adjusting the pH to 7.5 with a 1.2wt% dipotassium hydrogen phosphate aqueous solution and a 0.09wt% potassium dihydrogen phosphate aqueous solution, respectively, to obtain a plate activation culture medium mixture, and then sterilizing it with high-temperature steam at 121°C for 20 minutes, taking it out, pouring it into a sterilized plate culture container, and cooling it to obtain a plate activation culture medium; picking 2 rings of Bacillus subtilis subsp. subtilis with an inoculation loop Bacillus subtilis subsp .subtilis The bacteria were inoculated on a plate activation culture medium and cultured at a constant temperature of 37°C for 12 hours to obtain activated bacteria.

[0050] Step 2, preparation of seed solution: add 30g sucrose, 30g sodium glutamate, 10g beef extract, 0.5g potassium dihydrogen phosphate trihydrate, and 0.25g magnesium sulfate heptahydrate into 1L water, stir at 500 rpm for 45 minutes, adjust the pH to 7.5 with a 1.2wt% potassium dihydrogen phosphate aqueous solution and a 0.09wt% potassium dihydrogen phosphate aqueous solution, respectively, to obtain a seed culture medium mixture, and then sterilize it with high-temperature steam at 121°C for 20 minutes, take it out, pour it into a sterilized eggplant slant culture container, and obtain a seed culture medium after cooling; pick 3 rings of activated strains from step 1 with an inoculation loop, inoculate them into the seed culture medium, and culture them at 37°C for 15 hours with a constant temperature shaking speed of 220 rpm to obtain a seed solution; dilute the seed solution to prepare a strain suspension with a concentration of 150 million / L;

[0051] Step 3, preparation of fermentation broth: 550mL of enzymatic hydrolyzate, 40g of sodium glutamate, 8g of beef extract, 1g of potassium dihydrogen phosphate trihydrate, 0.25g of magnesium sulfate heptahydrate, and 0.1g of sodium chloride are added to 450mL of water, and the pH is adjusted to 7.5 with a 1.2wt% aqueous solution of potassium dihydrogen phosphate and a 0.09wt% aqueous solution of potassium dihydrogen phosphate, respectively, to obtain a fermentation culture mixture, and then sterilized at 121°C for 20 minutes with high-temperature steam, taken out, 900mL of the fermentation culture mixture is poured into a 3L sterilized fermentation tank, and cooled to obtain a fermentation medium; the strain suspension prepared in step 2 is inoculated into the fermentation medium, the inoculation amount is 10%mL / mL, 120g of a UV-sterilized magnetic oxide / epoxy resin porous composite material is added, and the mixture is cultured at 37°C for 52 hours with constant temperature shaking to obtain a fermentation broth containing γ-polyglutamic acid;

[0052] Step 4, extraction of γ-polyglutamic acid: the fermentation liquid containing γ-polyglutamic acid obtained in step 3 is adjusted to pH 3.5 with 0.05 mol / L hydrochloric acid, centrifuged at 5000 rpm for 30 minutes, the supernatant is taken, anhydrous ethanol precooled at 2°C for 30 minutes is added, stirred at 400 rpm for 20 minutes at 4°C and allowed to stand for 15 hours, then centrifuged at 5000 rpm for 15 minutes, the precipitate is collected, vacuum dried for 12 hours, then added to 200 mL of water, stirred at 400 rpm for 20 minutes, dialyzed in water with a dialysis bag with a molecular cutoff of 50 kD for 24 hours, and vacuum freeze-dried for 20 hours to obtain γ-polyglutamic acid.

[0053] The enzymatic solution preparation method is as follows:

[0054] 100 g of soybean meal was mixed with 100 g of sago palm pith sawdust to obtain dry material, and the dry material was soaked in 2000 mL of 5 wt% acetic acid aqueous solution, 2000 mL of 10 wt% sodium hydroxide aqueous solution and 2000 mL of water in sequence, the soaking time was 90 minutes for each soaking, and the dry material was washed with water twice after each soaking. After the soaking treatment, it was sterilized at 121° C. for 2 hours, cooled to 25° C., washed 3 times in 50 wt% ethanol aqueous solution, and then dried at 105° C. for 10 hours to obtain pretreated dry material; 150 g of the pretreated dry material was mixed with 1 L of citric acid-sodium citrate buffer, and then 0.04 mg of cellulase and 10 g of Aspergillus niger acid protease were added, and the dry material was enzymolyzed at a constant temperature of 37° C. for 10 hours, and then water bathed at 100° C. for 12 minutes, and then centrifuged at 5000 rpm for 15 minutes, and the supernatant was collected to obtain an enzymolysis solution.

[0055] The preparation of the magnetic oxide / epoxy resin porous composite material is as follows:

[0056] S1. Add 46.1 g of magnesium nitrate hexahydrate and 100.8 g of ferric nitrate nonahydrate to 900 mL of water, stir at 400 rpm for 20 minutes to obtain a mixture A, then add 75.69 g of hexamethylenetetramine to the mixture A in 4 portions while stirring at 400 rpm, continue stirring at 400 rpm until the solution in the mixture A becomes transparent, then react at 200° C. for 10 hours, then cool naturally to 25° C., collect the solid, wash with water and anhydrous ethanol 3 times respectively, dry at 65° C. for 10 hours, and then calcine at 700° C. for 4 hours to obtain a magnetic oxide;

[0057] S2. Mix 76.8 g of the magnetic oxide prepared in step S1 and 51.2 g of water, and stir at 400 rpm for 15 minutes to obtain a magnetic oxide dispersion; then mix 40 g of epoxy resin and 1 g of silicon carbide, and stir at 400 rpm for 35 minutes, then ultrasonicate for 2 hours with an ultrasonic power of 250 W, add 20 g of diethylaminopropylamine, and stir at 400 rpm for 3 minutes, then add the magnetic oxide dispersion, continue to stir at 400 rpm for 4 minutes, and cure at 25°C to form spheres with a particle size of 2 mm to obtain a magnetic oxide / epoxy resin porous composite material.

[0058] Comparative Example 1

[0059] A method for preparing γ-polyglutamic acid by microbial fermentation is basically the same as that in Example 1, the only difference being the method for preparing the enzymatic hydrolysate.

[0060] The enzymatic solution preparation method is as follows:

[0061] 200g of sago palm pith sawdust was soaked in 2000mL of 5vt% acetic acid aqueous solution, 2000mL of 10wt% sodium hydroxide aqueous solution and 2000mL of water in sequence, the soaking time was 90 minutes respectively, and the mixture was washed with water twice after each soaking. After the soaking treatment, the mixture was sterilized at 121°C for 2 hours, cooled to 25°C, washed 3 times in 50vt% ethanol aqueous solution, and then dried at 105°C for 10 hours to obtain a pretreated dry material; 150g of the pretreated dry material was mixed with 1L of citric acid-sodium citrate buffer, and then 0.04mg of cellulase and 10g of Aspergillus niger acid protease were added, and the mixture was enzymolyzed at a constant temperature of 37°C for 10 hours, and then water bathed at 100°C for 12 minutes, and then centrifuged at 5000 rpm for 15 minutes, and the supernatant was collected to obtain an enzymolysis solution.

[0062] The preparation of the magnetic oxide / epoxy resin porous composite material is the same as that in Example 1.

[0063] Comparative Example 2

[0064] A method for preparing γ-polyglutamic acid by microbial fermentation is basically the same as that in Example 1, the only difference being that the method for preparing the enzymatic hydrolysate is different.

[0065] The enzymatic solution preparation method is as follows:

[0066] 200 g of soybean meal was soaked in 2000 mL of 5vt% acetic acid aqueous solution, 2000 mL of 10wt% sodium hydroxide aqueous solution and 2000 mL of water in sequence, with the soaking time being 90 minutes respectively. After each soaking, it was washed with water twice. After the soaking treatment, it was sterilized at 121°C for 2 hours, cooled to 25°C, washed 3 times in 50vt% ethanol aqueous solution, and then dried at 105°C for 10 hours to obtain pretreated dry material; 150 g of the pretreated dry material was mixed with 1L of citric acid-sodium citrate buffer, and then 0.04 mg of cellulase and 10 g of Aspergillus niger acid protease were added, and the mixture was enzymolyzed at a constant temperature of 37°C for 10 hours, and then bathed in water at 100°C for 12 minutes, and then centrifuged at 5000 rpm for 15 minutes, and the supernatant was collected to obtain an enzymolysis solution.

[0067] The preparation of the magnetic oxide / epoxy resin porous composite material is the same as that in Example 1.

[0068] Comparative Example 3

[0069] A method for preparing γ-polyglutamic acid by microbial fermentation is basically the same as that in Example 1, the only difference being that the magnetic oxide / epoxy resin porous composite material is replaced by an epoxy resin composite material.

[0070] The method for preparing the enzymatic hydrolysate is the same as that in Example 1.

[0071] The epoxy resin composite material is prepared as follows:

[0072] 40 g of epoxy resin and 1 g of silicon carbide were mixed, stirred at 400 rpm for 35 minutes, and then ultrasonicated for 2 hours with an ultrasonic power of 250 W. 20 g of diethylaminopropylamine was added, stirred at 400 rpm for 3 minutes, and cured at 25° C. to form spheres with a particle size of 2 mm to obtain an epoxy resin composite material.

[0073] Comparative Example 4

[0074] A method for preparing γ-polyglutamic acid by microbial fermentation, the preparation is as follows:

[0075] Step 1, activating the bacteria: adding 20g agar powder, 10g soy peptone, 5g beef extract, 5g sodium chloride, 2g yeast powder, and 1g glucose to 1L water, stirring at 500 rpm for 45 minutes, adjusting the pH to 7.5 with a 1.2wt% dipotassium hydrogen phosphate aqueous solution and a 0.09wt% potassium dihydrogen phosphate aqueous solution, respectively, to obtain a plate activation culture medium mixture, and then sterilizing it with high-temperature steam at 121°C for 20 minutes, taking it out, pouring it into a sterilized plate culture container, and cooling it to obtain a plate activation culture medium; picking 2 rings of Bacillus subtilis subsp. subtilis with an inoculation loop Bacillus subtilis subsp .subtilis The bacteria were inoculated on a plate activation culture medium and cultured at a constant temperature of 37°C for 12 hours to obtain activated bacteria.

[0076] Step 2, preparation of seed solution: add 30g sucrose, 30g sodium glutamate, 10g beef extract, 0.5g potassium dihydrogen phosphate trihydrate, and 0.25g magnesium sulfate heptahydrate into 1L water, stir at 500 rpm for 45 minutes, adjust the pH to 7.5 with a 1.2wt% potassium dihydrogen phosphate aqueous solution and a 0.09wt% potassium dihydrogen phosphate aqueous solution, respectively, to obtain a seed culture medium mixture, and then sterilize it with high-temperature steam at 121°C for 20 minutes, take it out, pour it into a sterilized eggplant slant culture container, and obtain a seed culture medium after cooling; pick 3 rings of activated strains from step 1 with an inoculation loop, inoculate them into the seed culture medium, and culture them at 37°C for 15 hours with a constant temperature shaking speed of 220 rpm to obtain a seed solution; dilute the seed solution to prepare a strain suspension with a concentration of 150 million / L;

[0077] Step 3, preparation of fermentation broth: 550mL of enzymatic hydrolyzate, 40g of sodium glutamate, 8g of beef extract, 1g of dipotassium hydrogen phosphate trihydrate, 0.25g of magnesium sulfate heptahydrate, and 0.1g of sodium chloride are added to 450mL of water, and the pH is adjusted to 7.5 with a 1.2wt% dipotassium hydrogen phosphate aqueous solution and a 0.09wt% potassium dihydrogen phosphate aqueous solution, respectively, to obtain a fermentation culture mixture, and then sterilize it with high-temperature steam at 121°C for 20 minutes, take it out, pour 900mL of the fermentation culture mixture into a 3L sterilized fermentation tank, and obtain a fermentation medium after cooling; the strain suspension prepared in step 2 is inoculated into the fermentation medium, the inoculation amount is 10%mL / mL, and the fermentation broth containing γ-polyglutamic acid is obtained at 37°C for 52 hours with constant temperature shaking;

[0078] Step 4, extraction of γ-polyglutamic acid: the fermentation liquid containing γ-polyglutamic acid obtained in step 3 is adjusted to pH 3.5 with 0.05 mol / L hydrochloric acid, centrifuged at 5000 rpm for 30 minutes, the supernatant is taken, anhydrous ethanol precooled at 2°C for 30 minutes is added, stirred at 400 rpm for 20 minutes at 4°C and allowed to stand for 15 hours, then centrifuged at 5000 rpm for 15 minutes, the precipitate is collected, vacuum dried for 12 hours, then added to 200 mL of water, stirred at 400 rpm for 20 minutes, dialyzed in water with a dialysis bag with a molecular cutoff of 50 kD for 24 hours, and vacuum freeze-dried for 20 hours to obtain γ-polyglutamic acid.

[0079] The preparation method of the enzymatic hydrolysate is the same as that in Example 1.

[0080] Test Example 1

[0081] γ-Polyglutamic acid production determination:

[0082] The yield of γ- prepared by the microbial fermentation method of the present invention is determined, and the determination steps are as follows: take 100 mL of the fermentation broth in step 3, adjust the pH to 3.5 with 0.05 mol / L hydrochloric acid, centrifuge at 5000 rpm for 30 minutes, take the supernatant, add anhydrous ethanol precooled at 2°C for 30 minutes, stir at 400 rpm at 4°C for 20 minutes and stand for 15 hours, then continue to centrifuge at 5000 rpm for 15 minutes, collect the precipitate, vacuum dry for 12 hours, and then add The mixture was stirred at 400 rpm for 20 minutes in 200 mL of water, dialyzed in water with a dialysis bag having a molecular cutoff of 50 kD for 24 hours, and vacuum-frozen for 20 hours to obtain γ-polyglutamic acid. The prepared γ-polyglutamic acid was then vacuum-dried at 60°C to constant weight, weighed, and the mass was recorded as m / mg. The concentration C (mg / mL) of γ-polyglutamic acid in the fermentation broth was calculated according to Formula 1. The concentration of γ-polyglutamic acid in the fermentation broth was used to represent the yield of γ-polyglutamic acid in the fermentation. The test results are shown in Table 1:

[0083]

[0084] Where: C is the concentration of γ-polyglutamic acid in the fermentation broth; m is the mass of γ-glutamic acid in 100 mL of fermentation broth (mg).

[0085] Table 1 Test results

[0086] Sample Yield (mg / mL) Example 1 43.55 Comparative Example 1 39.53 Comparative Example 2 28.26 Comparative Example 3 41.26 Comparative Example 4 39.75

[0087] By comparing Example 1 with Comparative Examples 1 to 4, it can be seen that the yield of γ-polyglutamic acid in Example 1 is greater than that in Comparative Examples 1 to 4. This may be because the enzymolyzate in Example 1 is obtained by enzymolyzing soybean meal and sago palm pith sawdust with cellulase and protease. The enzymolyzate may contain not only glucose and small peptides as carbon sources and nitrogen sources, but also xylose and other carbon sources. During the fermentation process, the bacteria fully utilize the enzymolyzate, which increases the γ-polyglutamic acid produced by the bacteria fermentation and improves the yield of γ-polyglutamic acid.

[0088] Test Example 2

[0089] Flocculation activity assay:

[0090] Reference journal articles ( Bacillus subtilis ZJUTZY Research on the molecular weight distribution and flocculation characteristics of γ-polyglutamic acid produced by the present invention, author: Zhang Yi et al., Journal of Zhejiang University of Technology, December 2008) The flocculation activity of the γ-polyglutamic acid prepared by the present invention was determined, and the steps are as follows: 0.08g of purchased γ-polyglutamic acid (molecular weight greater than 700,000) and 0.08g of the γ-polyglutamic acid prepared by the present invention were added to 1L of water, stirred at 500 rpm until dissolved, to obtain a γ-polyglutamic acid aqueous solution; then 80mL of water and 5mL of 10g / L calcium chloride aqueous solution were added at 40°C. The supernatant was stirred at 500 rpm for 5 minutes, and then the mixture was allowed to stand for 10 minutes. The liquid layer 1 cm below the surface of the supernatant was aspirated with a pipette, and the absorbance A at a wavelength of 570 nm was measured with an ultraviolet spectrophotometer. The kaolin solution without the addition of the γ-polyglutamic acid solution was used as a control, and the absorbance A0 at a wavelength of 550 nm was measured with an ultraviolet spectrophotometer. The flocculation rate was calculated according to Formula 1, and the flocculation activity was expressed by the flocculation rate. The results are shown in Table 2:

[0091]

[0092] Table 2 Test results

[0093] Sample Flocculation rate / % Example 1 47.62 Comparative Example 1 31.86 Comparative Example 2 21.55 Comparative Example 3 36.31 Comparative Example 4 32.25

[0094] By comparing Example 1 with Comparative Examples 1 to 4, it can be seen that the flocculation effect of Example 1 is better than that of Comparative Examples 1 to 4, which may be due to the high conversion rate of γ-polyglutamic acid in Example 1, which improves the flocculation activity. In the prior art, some researchers used microbial fermentation technology to produce γ-polyglutamic acid and tested its flocculation activity, and its flocculation rate could reach 46.99%. The flocculation effect of the present invention is better.

Claims

1. A method for preparing γ-polyglutamic acid by microbial fermentation, characterized in that: The steps include: Step 1, activating the strain: using an inoculation loop to pick 1-3 rings of Bacillus subtilis strains and inoculate them on a flat plate activation culture medium, and culturing them at a constant temperature of 35-38° C. for 11-13 hours to obtain activated strains; Step 2, preparation of seed solution: pick 2-5 rings of activated bacteria prepared in step 1, inoculate them into seed culture medium, and culture them at 35-38°C constant temperature with shaking for 14-16 hours at an shaking speed of 200-250 rpm to obtain seed solution; dilute the seed solution to prepare a bacterial suspension with a concentration of 130-160 million / L; Step 3, preparation of fermentation liquid: inoculate the bacterial suspension prepared in step 2 into the fermentation medium, add the ultraviolet sterilized magnetic oxide / epoxy resin porous composite material, and culture at 35-38° C. with constant temperature shaking for 51-53 hours to obtain a fermentation liquid containing γ-polyglutamic acid; Step 4, extraction of γ-polyglutamic acid: the fermentation liquid containing γ-polyglutamic acid obtained in step 3 is adjusted to pH 3-4, centrifuged at 4000-6000 rpm for 25-50 minutes, the supernatant is taken, anhydrous ethanol precooled at -1-3°C for 20-40 minutes is added, stirred at 300-500 rpm for 10-30 minutes at 3-4.5°C and allowed to stand for 14-16 hours, then centrifuged at 4000-6000 rpm for 12-25 minutes, the precipitate is collected, vacuum dried for 8-16 hours, then added to water, stirred at 300-500 rpm for 15-30 minutes, dialyzed in water for 23-25 ​​hours, and vacuum freeze-dried for 10-24 hours to obtain γ-polyglutamic acid; The fermentation medium uses enzymatic hydrolysate as the main carbon source; The enzymatic hydrolysate is obtained by washing soybean meal and sago palm pith sawdust with acid, alkali and water, and then enzymolyzing with cellulase and Aspergillus niger acid protease; The preparation method of the magnetic oxide / epoxy resin porous composite material is as follows: S1. Add magnesium nitrate hexahydrate and ferric nitrate nonahydrate into water and stir evenly to obtain a mixture A. Then, under stirring, add hexamethylenetetramine into the mixture A in 3-5 portions, continue stirring until the solution in the mixture A becomes transparent, then react at 118-205° C. for 9-11 hours, then naturally cool to 20-30° C., collect the solid, wash with water and anhydrous ethanol respectively, dry, and then roast at 650-750° C. for 3.5-4.5 hours to obtain a magnetic oxide; The mass ratio of magnesium nitrate hexahydrate, ferric nitrate nonahydrate, water and hexamethylenetetramine in step S1 is 30-60:80-120:800-1500:55-90; S2, mixing the magnetic oxide prepared in step S1 with water, stirring evenly to obtain a magnetic oxide dispersion; then mixing epoxy resin and silicon carbide, stirring, and then ultrasonicating, adding a curing agent, stirring for 1-4 minutes, then adding the magnetic oxide dispersion, stirring for 2-5 minutes, and curing to obtain a magnetic oxide / epoxy resin porous composite material; The mass ratio of the magnetic oxide, water, epoxy resin, silicon carbide and curing agent in step S2 is 60-80:45-60:30-50:0.4-1.2:4-25.

2. The method for preparing γ-polyglutamic acid by microbial fermentation as claimed in claim 1, characterized in that: The plate activation culture medium comprises the following raw materials in weight percentage: 15-25 g / L agar powder, 8-12 g / L soy peptone, 3-8 g / L beef extract, 3-8 g / L sodium chloride, 1-3 g / L yeast powder, 0.5-1.5 g / L glucose, the solvent is water, and the pH is adjusted to 7.5; the seed culture medium comprises the following raw materials in weight percentage: 25-35 g / L sucrose, 25-35 g / L sodium glutamate, 5-15 g / L beef extract, 0.25-0.75 g / L potassium hydrogen phosphate trihydrate, 0.15-0.45 g / L magnesium sulfate heptahydrate, the solvent is water, and the pH is adjusted to 7.

5.

3. The method for preparing γ-polyglutamic acid by microbial fermentation as claimed in claim 1, characterized in that: The inoculation amount in step 3 is 3%-15% mL / mL; the fermentation medium comprises the following raw materials in weight percentage: 450-600 mL / L enzymatic hydrolysate, 35-55 g / L sodium glutamate, 4-10 g / L beef extract, 0.5-1.5 g / L dipotassium hydrogen phosphate trihydrate, 0.15-0.35 g / L magnesium sulfate heptahydrate, 0.05-0.15 g / L sodium chloride, the solvent is water, and the pH is adjusted to 7.5; the fermentation medium liquid volume is 120-180 mL / 500 mL.

4. The method for preparing γ-polyglutamic acid by microbial fermentation as claimed in claim 1, characterized in that: The mass and volume ratio of the magnetic oxide / epoxy resin porous composite material to the fermentation medium is 0.3-1.6:9 g / mL; the volume ratio of the added water to the volume of the fermentation medium is 1-3:8-10.

5. The method for preparing γ-polyglutamic acid by microbial fermentation as claimed in claim 1, characterized in that: The preparation method of the enzymatic hydrolysate is as follows: 10-30 parts by weight of dry material are soaked in 4vt%-8vt% acetic acid aqueous solution, 8wt%-12wt% sodium hydroxide aqueous solution and 10-30 parts by weight of water in sequence, washed with water after each soaking, sterilized at 119-125°C for 1.5-2.5 hours, cooled, washed in ethanol aqueous solution and then dried to obtain pretreated dry material; 8-20 parts by weight of pretreated dry material are mixed with citric acid-sodium citrate buffer, cellulase and Aspergillus niger acid protease are added, and enzymolysis is carried out at a constant temperature of 35-38°C for 8-12 hours, and then water bathed at 98-110°C for 10-15 minutes, centrifuged, and the supernatant is collected to obtain an enzymolysis solution.

6. The method for preparing γ-polyglutamic acid by microbial fermentation as claimed in claim 5, characterized in that: The dry material is a mixture of soybean meal and sago palm pith sawdust in a mass ratio of 1:1-3; the mass volume ratio of the dry material to the acetic acid aqueous solution and the sodium hydroxide aqueous solution is independently 1:8-15 g / mL; the mass volume ratio of the pretreated dry material to the citric acid-sodium citrate buffer solution is 80-200:1 g / L; the added amount of the cellulase is 9%-14% of the mass of the sago palm pith sawdust; and the added amount of the Aspergillus niger acid protease is 0.03%-0.06% of the mass of the soybean meal.

7. The method for preparing γ-polyglutamic acid by microbial fermentation as claimed in claim 1, characterized in that: The molded particles have a diameter of 1-3 mm and are of any shape.

8. A γ-polyglutamic acid, characterized in that: Prepared by the preparation method described in any one of claims 1 to 7.