CN110229852B - Method for producing gamma-polyglutamic acid by fermenting soybean protein zymolyte - Google Patents
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Abstract
The invention belongs to the technical field of microbial engineering, and particularly relates to a method for producing gamma-polyglutamic acid by fermenting soybean protein zymolyte, which comprises the following steps: preparing soybean slurry by taking soybeans or soybean meal as a raw material; carrying out moderate enzymolysis on the soybean slurry by using protease to control the hydrolysis degree of the soybean protein to be 10-35%; adjusting the mass percentage concentration of solid matters in the soybean protein zymolyte to 1-6%, adding glucose or sucrose and sodium glutamate to be used as a culture medium for culturing the bacillus subtilis; inoculating the bacillus subtilis seed solution into a soybean protein zymolyte culture medium according to the inoculation amount of 1-8%, and stopping fermentation after carrying out shake culture or fermentation tank aeration culture for 36-54 h to obtain the gamma-polyglutamic acid. The method for producing the gamma-polyglutamic acid by fermenting the soybean protein zymolyte can obviously improve the yield of the gamma-polyglutamic acid, has cheap and easily obtained raw materials, and can effectively reduce the cost, thereby having good application prospect.
Description
Technical Field
The invention belongs to the technical field of microbial engineering, and particularly relates to a method for producing gamma-polyglutamic acid by fermenting soybean protein zymolyte.
Background
Gamma-polyglutamic acid (gamma-PGA for short), a high molecular amino acid polymer synthesized by microorganisms, has molecular mass of 5-200 ten thousand daltons generally. Gamma-polyglutamic acid is used as a novel natural polymer material, is matched with the characteristics of no toxicity and harmlessness, and is widely applied to a plurality of fields of cosmetics, environmental protection, food, medicine, agriculture, desert control and the like. The industrial production method of the gamma-polyglutamic acid generally adopts a microbial fermentation synthesis method. In the past decades, the industry and academia have conducted a great deal of research on the production process of gamma-polyglutamic acid, and although significant progress has been made, the price of gamma-polyglutamic acid products is still expensive, which greatly limits the wide application of gamma-polyglutamic acid. The reason for this is that gamma-polyglutamic acid has low production efficiency and high production cost. Therefore, improving the fermentation process is an important way to improve the production level of gamma-polyglutamic acid.
For fermentation processes, optimizing the culture medium is a necessary step to achieve high yields. The excellent strains must be combined with the culture medium and culture conditions suitable for the excellent strains to fully exert the production potential of the excellent strains. For example, Chinese patent application No. 02151746 discloses Bacillus subtilis NX-2 and a method for preparing gamma-polyglutamic acid by using the same; chinese patent application No. 200410010509 discloses Bacillus subtilis zju-7 and a method for preparing gamma-polyglutamic acid by using the same; chinese patent application No. 200610155278 discloses a method for preparing gamma-polyglutamic acid by mixing Bacillus subtilis and Corynebacterium glutamicum. The above prior art all relate to the optimization of the culture medium for the growth of the producer bacteria and the promotion of the synthesis of gamma-polyglutamic acid. In the metabolic process of synthesizing gamma-polyglutamic acid by fermenting bacillus subtilis, a culture medium has to have sufficient available carbon source and nitrogen source, and some glutamic acid dependent strains also need to provide sufficient glutamic acid or sodium glutamate as a precursor to synthesize the gamma-polyglutamic acid. The literature reports that various biochemicals such as peptone, yeast extract, beef extract and the like are commonly used as nitrogen source substances for increasing the yield of the gamma-polyglutamic acid, and the expensive biochemicals are one of the important reasons for high production cost of the gamma-polyglutamic acid. In addition, the nitrogen source of these biochemical reagents is not obvious to the Bacillus subtilis tested, and the yield increasing effect of gamma-polyglutamic acid is not obvious. Therefore, the development of a novel nitrogen source with low price and remarkable yield increasing effect has important significance for improving the production level of the gamma-polyglutamic acid and reducing the production cost.
Disclosure of Invention
The invention aims to: aiming at the defects of high raw material cost and low production efficiency in the existing gamma-polyglutamic acid production technology, cheap and easily-obtained soybeans or soybean meal are used as raw materials for pulping, and after proper enzymolysis, soybean protein zymolyte is used as a nitrogen source substance for culturing bacillus subtilis to ferment and produce gamma-polyglutamic acid, so that the aims of increasing the yield of the gamma-polyglutamic acid and reducing the production cost are fulfilled.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for producing gamma-polyglutamic acid by fermenting soybean protein zymolyte, which comprises the following steps:
the method comprises the following steps: cleaning soybeans or soybean meal, adding deionized water with the mass of 8-15 times, soaking for 4-10 hours at 20-30 ℃, pulping by using a soybean milk machine, and filtering to remove residues to obtain soybean milk;
step two: adjusting the pH value of the soybean slurry obtained in the step one to 8-11, adding 20-200 mu/L of alkaline protease, carrying out heat preservation and enzymolysis for 1-8 h at 40-65 ℃, controlling the degree of protein hydrolysis to be 10-35%, then heating to inactivate enzyme, cooling, carrying out centrifugal separation for 20min at 5000r/min, and taking supernatant to obtain a soybean protein hydrolysate;
wherein the basic protease activity unit is defined as: at 40 ℃, under the measuring condition of pH 10, the enzyme quantity required for hydrolyzing casein to generate 1 microgram of tyrosine per minute is an enzyme activity unit which is expressed by u.
Step three: adjusting the concentration of the soybean protein zymolyte obtained in the step two, adding a carbon source and polyglutamic acid to synthesize a precursor substance, adjusting the pH to 6-8, sterilizing at 110-121 ℃ for 20-30 min, and cooling to room temperature for later use to obtain a soybean protein zymolyte culture medium;
wherein the soybean protein zymolyte culture medium comprises the following components: the mass percentage concentration of solid matters in the soybean protein zymolyte is 1-6%, 10-30 g/L of carbon source (glucose or sucrose) and 20-50 g/L of sodium glutamate (polyglutamic acid synthesis precursor) are added.
Step four: inoculating activated Bacillus subtilis slant strains to a seed liquid culture medium, wherein the liquid loading amount of the seed liquid culture medium in a 250mL conical flask is 50-70 mL, and performing shake culture or aeration culture at 37 ℃ for 16-24 h to obtain the Bacillus subtilis seed liquid.
The Bacillus subtilis is Bacillus subtilis DG02 (classified name: Bacillus subtilis; preservation date: 2019, 4-9 months; preservation number: GDMCC No. 60626; preservation unit full name: Guangdong province microorganism strain preservation center; preservation unit: GDMCC; preservation address: No. 59, 5 th of Juglans institute No. 100, Mr. Zhonglu, Guangzhou city). The bacillus subtilis seed liquid culture medium comprises the following components: 1000mL of bean sprout juice, 50g of cane sugar, natural pH and sterilization at 121 ℃ for 20 min. The preparation method of the bean sprout juice comprises the following steps: adding 1000mL of water into 500g of soybean sprout, boiling and concentrating to 500mL, and filtering to remove residues to obtain soybean sprout juice.
Step five: and (3) inoculating the bacillus subtilis seed solution obtained in the fourth step to the soybean protein zymolyte culture medium obtained in the third step according to the inoculation amount of 1-8%, and then, after shake cultivation or fermentation tank aeration cultivation for 36-54 h at 33-39 ℃, terminating fermentation to obtain fermentation liquor.
Step six: and D, centrifuging the fermentation liquor obtained in the fifth step to remove thalli, and measuring the content of the gamma-polyglutamic acid in the fermentation liquor by using a high performance liquid chromatography or a CTAB precipitation method.
The invention has the beneficial effects that: the soybean and the soybean meal have wide sources, high protein content and low price, and the soybean protein zymolyte is obtained by hydrolyzing the protein in the soybean milk by using protease after the soybean or the soybean meal is used as the raw material for pulping, and has the advantages of low price and easy obtainment of the raw material and stable quality compared with other protein zymolytes; the soybean milk contains rich soluble saccharides, mineral substances, phospholipid, vitamins and other nutrient components besides protein, and is more suitable for microbial growth than soybean protein isolate; compared with neutral protease, acid protease, papain and other proteases, the used alkaline protease is more suitable for hydrolyzing soybean protein and has the advantages of high hydrolysis rate and less enzyme dosage; compared with other biochemical reagents such as peptone, yeast extract (yeast powder), beef extract and the like as nitrogen sources, the soybean protein zymolyte is used as the nitrogen source of the culture medium, so that the raw material cost is obviously saved; because the soybean active peptide has biological activity of promoting the growth of various microorganisms and the synthesis of fermentation products, the yield of the gamma-polyglutamic acid can be improved by more than one time compared with the fermentation of a biochemical reagent culture medium by controlling the hydrolysis degree of soybean protein zymolyte and optimizing the composition and the culture condition of the culture medium.
Proof of deposit
And (3) classification and naming: bacillus subtilis
Latin literature name: bacillus subtilis DG02
The preservation date is as follows: 4 month and 9 days 2019
The preservation number is: GDMCC No: 60626
The preservation unit is called as follows: guangdong province microbial strain preservation center
The preservation unit is abbreviated as: GDMCC
And (4) storage address: guangzhou city first furious Zhonglu No. 100 large yard No. 59 building No. 5 building
Detailed Description
The present invention will be described in further detail with reference to specific embodiments, but the embodiments of the present invention are not limited thereto.
Example 1
(1) Preparation of seed liquid culture Medium
Adding 1000mL of water into 500g of soybean sprouts, boiling and concentrating to 500mL, and filtering the waste residues to obtain soybean sprout juice; 200mL of bean sprout juice and 10g of cane sugar are taken, the bean sprout juice is sterilized for 20min at the temperature of 121 ℃ under natural pH, and the culture medium of the bacillus subtilis seed liquid is obtained after cooling.
(2) Preparation of soy protein zymolyte culture medium
The method comprises the following steps: cleaning soybeans, adding 10 times of deionized water by mass, soaking for 8 hours at 25 ℃, pulping by using a soybean milk machine, filtering and removing slag to obtain soybean milk with the solid concentration of about 6 percent.
Step two: adding 200mL of soybean milk into a 500mL conical flask, adjusting the pH value to 10, adding 160 ten thousand u/L of alkaline protease, oscillating in a water bath at 60 ℃ and 120r/min for enzymolysis for 2h, heating at 100 ℃ for 10min to inactivate the enzyme, cooling, centrifuging at 5000r/min for 20min, filtering to remove precipitate and floating fat, and measuring the obtained light yellow clear liquid, namely the soybean protein zymolyte, wherein the hydrolysis degree is 18.34%.
Step three: diluting the soybean protein zymolyte to a solid mass percentage concentration of 3%, adding 20g/L of carbon source glucose and 50g/L of sodium glutamate (polyglutamic acid synthesis precursor), and adjusting the pH value to 7.0; the liquid loading of the culture medium in a 250mL conical flask is 60 mL; sterilizing at 110 deg.C for 20min, and cooling to room temperature to obtain soybean protein zymolyte culture medium.
(3) Fermentation synthesis of gamma-polyglutamic acid
Inoculating activated bacillus subtilis DG02 slant strains to a seed liquid culture medium, and performing shake culture at a constant temperature of 150r/min at 37 ℃ for 18h to obtain bacillus subtilis seed liquid; inoculating the seed solution into a soybean protein zymolyte culture medium according to the inoculation amount of 5%, performing shake culture at the constant temperature of 37 ℃ and 150r/min for 48h, and then stopping fermentation. The fermentation liquor is centrifuged (5000r/min, 20min) to remove thalli and solid particles, and the content of gamma-polyglutamic acid is measured to be 39.12g/L by a CTAB precipitation method.
Example 2
(1) Preparation of seed liquid
The same as in example 1.
(2) Preparation of soy protein zymolyte culture medium
The method comprises the following steps: the same as in example 1.
Step two: adding 2.0L of soybean milk into a 5L liquid fermentation tank, adjusting pH to 10, adding 160 ten thousand u/L of alkaline protease, carrying out heat preservation and enzymolysis at 60 ℃ and 120r/min for 2h, heating at 100 ℃ for 10min to inactivate enzyme activity, cooling, centrifuging at 5000r/min for 20min, filtering to remove precipitate and floating fat, and measuring the obtained light yellow clear liquid, namely the soybean protein zymolyte, wherein the hydrolysis degree is 17.53%.
Step three: diluting the soybean protein zymolyte until the mass percentage concentration of solid matters is 3%, adding 20g/L of carbon source sucrose and 50g/L of sodium glutamate (polyglutamic acid synthesis precursor), and adjusting the pH value to 7.0; the liquid loading of the culture medium in the fermentation tank is 3.0L; sterilizing at 121 deg.C for 30min, and cooling to room temperature to obtain soybean protein zymolyte culture medium.
(3) Fermentation synthesis of gamma-polyglutamic acid
Inoculating activated bacillus subtilis DG02 slant strain to a seed liquid culture medium, filling 120mL of 500mL conical bottles with liquid, and performing shake culture at 37 ℃ and 150r/min for 18h to obtain bacillus subtilis seed liquid; inoculating the seed solution into a soybean protein zymolyte culture medium according to the inoculation amount of 5%, and culturing at 37 ℃; in the culture stage, the aeration ratio is gradually increased and maintained at 0.3-0.6 (v/v) within 1-6 h, and the stirring speed is 150-200 r/min; in the culture stage, the aeration ratio is maintained at 0.6-0.8 (v/v) for 7-18 h, and the stirring speed is 200-300 r/min; in the culture stage, the aeration ratio is gradually reduced and maintained at 0.5-0.3 (v/v) within 19-42 h, and the stirring speed is 150-200 r/min; and (4) monitoring and adjusting the pH value to 6.5-7.5 in the culture process, adding a proper amount of defoaming agent to eliminate foam, and stopping fermentation after the total culture time is 42 hours. The fermentation liquor is centrifuged (5000r/min, 20min) to remove thalli and solid particles, and the content of gamma-polyglutamic acid is measured to be 38.86g/L by a CTAB precipitation method. Compared with the shake flask experiment, the fermentation period of the 5L fermentation tank is shortened by about 6h, and the yield of the gamma-polyglutamic acid is close to that of the shake flask fermentation result.
Comparative example 1
(1) Preparation of seed liquid culture Medium
The same as in example 1.
(2) Preparation of Soy protein Medium
The method comprises the following steps: the soybean pulping process was the same as in example 1.
Step two: directly diluting the soybean milk until the mass percentage concentration of solid matters is 3%, adding 20g/L of carbon source glucose and 50g/L of sodium glutamate (polyglutamic acid synthesis precursor), and adjusting the pH value to 7.0; the liquid loading of the culture medium in a 250mL conical flask is 60 mL; sterilizing at 110 deg.C for 20min, and cooling to room temperature to obtain soybean protein culture medium.
(3) Fermentation synthesis of gamma-polyglutamic acid
The procedure was as in example 1. The content of gamma-polyglutamic acid measured by CTAB precipitation method was 11.43 g/L.
Comparative example 2
(1) Preparation of seed liquid culture Medium
The same as in example 1.
(2) Preparation of Biochemical reagent Medium
The biochemical reagent culture medium with yeast extract as nitrogen source consists of: 35g/L glucose, 10g/L yeast extract, 40g/L sodium glutamate and MgSO4 1.0g/L，K2HPO4 2.0g/L，MnSO40.5g/L, pH7.0, and 50mL of 250mL conical flask liquid; sterilizing at 110 deg.C for 20min, and cooling to room temperature.
(3) Fermentation synthesis of gamma-polyglutamic acid
Inoculating activated bacillus subtilis DG02 slant strains to a seed liquid culture medium, and culturing at 37 ℃ and a shaking table rotating speed of 150r/min for 24 h; inoculating the seed solution into a soybean protein zymolyte culture medium according to the inoculation amount of 3 percent, culturing at 37 ℃ and 150r/min for 48h, and then terminating fermentation. The fermentation liquor is centrifuged (5000r/min, 20min) to remove thalli and solid particles, and the content of gamma-polyglutamic acid is measured to be 17.83g/L by a CTAB precipitation method.
As can be seen from comparison of examples 1 and 2 with comparative example 1, the soybean protein hydrolysate culture medium using soybean protein as a nitrogen source after moderate enzymolysis can significantly improve the yield of gamma-polyglutamic acid, which is increased by more than 200% compared with the soybean protein culture medium directly using soybean milk as a nitrogen source; the reason is that various bioactive peptides generated by moderate hydrolysis of soybean protein by alkaline protease have promotion effect on synthesis of gamma-polyglutamic acid by bacillus subtilis, and the soybean protein does not have the bioactivity.
As can be seen from the comparison of examples 1 and 2 with comparative example 2, the soybean protein zymolyte culture medium can promote the bacillus subtilis to synthesize gamma-polyglutamic acid better than the biochemical reagent culture medium taking yeast extract as nitrogen source, and the yield of the gamma-polyglutamic acid is improved by more than 100%; the yeast extract is the best nitrogen source selected in the previous optimization experiment of the common nitrogen source, and comparison shows that the soybean protein zymolyte has more advantages than the common nitrogen source in the fermentation industry such as the yeast extract.
In conclusion, the soybean protein zymolyte is obtained by using the protease to hydrolyze the protein in the soybean milk after the soybean or the soybean meal is used as the raw material for pulping, and has the advantages of low price and easy obtainment of the raw material compared with other protein sources; the soybean milk contains rich soluble saccharides, mineral substances, phospholipid, vitamins and other nutrient components besides protein, and is more suitable for microbial growth than soybean protein isolate; compared with neutral protease, acidic protease and papain, the alkaline protease is more suitable for hydrolyzing soybean protein and has the advantages of high hydrolysis rate and less enzyme consumption; compared with various biochemical reagents such as peptone, yeast extract (yeast powder), beef extract and the like, the soybean protein zymolyte is used as the nitrogen source of the culture medium, so that the raw material cost is obviously saved; the yield of the gamma-polyglutamic acid can be improved by more than one time compared with the fermentation yield of a biochemical reagent culture medium by controlling the hydrolysis degree of the soybean protein zymolyte and optimizing the composition and the culture conditions of the culture medium.
Variations and modifications to the above-described embodiments will be apparent to those skilled in the art in light of the above teachings and teachings. Therefore, the present invention is not limited to the above-mentioned embodiments, and any obvious improvement, replacement or modification made by those skilled in the art based on the present invention is within the protection scope of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
Claims (7)
1. A method for producing gamma-polyglutamic acid by fermenting soybean protein zymolyte is characterized by comprising the following steps:
the method comprises the following steps: cleaning soybeans or soybean meal, adding deionized water with the mass of 8-15 times, soaking for 4-10 hours at 20-30 ℃, pulping by using a soybean milk machine, and filtering to remove residues to obtain soybean milk;
step two: adjusting the pH value of the soybean slurry obtained in the step one to 8-11, adding 20-200 mu/L of alkaline protease, carrying out heat preservation and enzymolysis for 1-8 h at 40-65 ℃, controlling the degree of protein hydrolysis to be 10-35%, then heating to inactivate enzyme, cooling, carrying out centrifugal separation for 20min at 5000r/min, and taking supernatant to obtain a soybean protein hydrolysate;
step three: adjusting the concentration of the soybean protein zymolyte obtained in the step two, adding a carbon source and polyglutamic acid to synthesize a precursor substance, adjusting the pH to 6-8, sterilizing at 110-121 ℃ for 20-30 min, and cooling to room temperature for later use to obtain a soybean protein zymolyte culture medium;
step four: inoculating Bacillus subtilis to a seed liquid culture medium, and performing shake culture or aerobic culture at 37 ℃ for 16-24 hours to obtain a Bacillus subtilis seed liquid;
step five: inoculating the bacillus subtilis seed solution obtained in the fourth step to the soybean protein zymolyte culture medium obtained in the third step according to the inoculation amount of 1-8%, carrying out shake culture at the constant temperature of 33-39 ℃ or carrying out aeration culture for 36-54 h, and stopping fermentation to obtain a fermentation liquid;
step six: and D, performing centrifugal separation and thallus removal on the fermentation liquor obtained in the step five, and determining the content of the gamma-polyglutamic acid in the fermentation liquor.
2. The method for producing gamma-polyglutamic acid by using soybean protein zymolyte fermentation according to claim 1, wherein: in the third step, the mass percentage concentration of the solid matters in the soybean protein zymolyte is 1-6%.
3. The method for producing gamma-polyglutamic acid by using soybean protein zymolyte fermentation according to claim 1, wherein: in the third step, the carbon source is 10-30 g/L, and the precursor substance for synthesizing the polyglutamic acid is 20-50 g/L.
4. The method for producing gamma-polyglutamic acid by using soybean protein zymolyte fermentation according to claim 1, wherein: in the third step, the carbon source is glucose or sucrose, and the precursor for synthesizing polyglutamic acid is sodium glutamate.
5. The method for producing gamma-polyglutamic acid by using soybean protein zymolyte fermentation according to claim 1, wherein: in step four, the Bacillus subtilis (Bacillus subtilis) is Bacillus subtilis DG02 with the deposit number: GDMCC No: 60626, the depository is called: guangdong province microbial strain preservation center.
6. The method for producing gamma-polyglutamic acid by using soybean protein zymolyte fermentation according to claim 1, wherein: in the fourth step, the preparation method of the seed liquid culture medium comprises the steps of adding 50g of cane sugar into 1000mL of bean sprout juice, naturally adjusting the pH value, and sterilizing at 121 ℃ for 20 min.
7. The method for producing gamma-polyglutamic acid by using soybean protein zymolyte fermentation according to claim 6, wherein: the bean sprout juice is prepared by adding 1000mL of water into 500g of soybean sprout, boiling and concentrating to 500mL, and filtering to remove residue.
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