1. A production method of stable liquid gamma-polyglutamic acid is characterized in that: the method comprises the following steps:

step one, preparing a fermentation culture substance, uniformly mixing a plurality of nutrient substances to obtain a gamma-polyglutamic acid culture substance, and transferring the gamma-polyglutamic acid culture substance into a fermentation tank;

step two, adding fermentation strains into the fermentation tank with the culture substance of the gamma-polyglutamic acid in the step one, and fermenting the gamma-polyglutamic acid liquid under certain fermentation conditions after adding;

step three, after the fermentation in the step two is finished, the pH value of the liquid gamma-polyglutamic acid fermentation solution in the fermentation tank in the step two is 7.0, and then the fermentation liquid heating treatment is carried out on the gamma-polyglutamic acid fermentation solution which is normally fermented after the fermentation strain is added;

and step four, after the step three is finished, adding a stabilizer into the polyglutamic acid fermentation liquor of the fermentation tank after the heating is finished, and obtaining stable liquid gamma-polyglutamic acid.

2. The method of claim 1, wherein the stable liquid gamma-polyglutamic acid is prepared by the following steps: the liquid fermentation conditions of the gamma-polyglutamic acid in the second step are as follows: the fermentation temperature is 35-45 ℃, the ventilation volume of the fermentation tank is 1.0-2.0vvm during fermentation, the stirring speed of the fermentation liquid is 150-.

3. The method for producing a stabilized liquid gamma-polyglutamic acid according to claim 1 or 2, wherein: the fermentation strain in the second step is Bacillus velezensis PG1-2, the strain is preserved in China center for culture of microorganisms, the preservation number is CGMCC No.15718, the preservation address is No. 3 of No.1 Hospital No.1 of Xilu of Chaozhong in the Korean area of Beijing, and the preservation date is 5 and 2 days in 2018.

4. The method for producing a stabilized liquid gamma-polyglutamic acid according to claim 1 or 2, wherein: the fermentation culture substance (g/L) in the step is prepared from the following components in percentage by mass: 60-70 parts of cane sugar, 20-30 parts of yeast powder, 60-70 parts of sodium glutamate, 60-2 parts of KH2PO 41, 41-2 parts of K2HPO, 20.2-0.3 part of CaCl and 7.0-7.2 of initial pH.

5. The method of claim 1, wherein the stable liquid gamma-polyglutamic acid is prepared by the following steps: and when the heating treatment is carried out in the third step, the heating treatment temperature is 50-70 ℃, and the treatment time is 2-6 h.

6. The method of claim 1, wherein the stable liquid gamma-polyglutamic acid is prepared by the following steps: the stabilizer is sodium benzoate or potassium sorbate and calcium carbonate.

7. The method for producing a stabilized liquid gamma-polyglutamic acid according to claim 1 or 6, wherein: the dosage of the stabilizer is as follows: sodium benzoate or potassium sorbate: 0.5-1.5%, calcium carbonate: 0.5 to 2 percent.

Background

Gamma-polyglutamic acid is a neutral high molecular polymer formed by connecting glutamic acid monomers through peptide bonds, and the molecular weight is usually between 100KD and 1000 KD. The gamma-polyglutamic acid has good water solubility, super strong adsorption capacity and biodegradability, and the degradation product of the gamma-polyglutamic acid is non-toxic to human bodies and environment, and can be used as a thickening agent, a water-retaining agent, a slow-release agent and the like to be applied to the fields of industry, agriculture, food, medicine and the like.

The gamma-polyglutamic acid is mostly obtained by microbial fermentation synthesis, but the liquid gamma-polyglutamic acid has poor stability, is easily degraded by acid, alkali and microorganisms, is not easy to store in natural environment, and brings much inconvenience for subsequent industrial application.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a production method of stable liquid gamma-polyglutamic acid, which solves the problems that the liquid gamma-polyglutamic acid has poor stability, is easily degraded by acid, alkali and microorganisms and is not easy to store in natural environment.

(II) technical scheme

In order to achieve the purpose, the invention is realized by the following technical scheme: a production method of stable liquid gamma-polyglutamic acid comprises the following steps:

step one, preparing a fermentation culture substance, uniformly mixing a plurality of nutrient substances to obtain a gamma-polyglutamic acid culture substance, and transferring the gamma-polyglutamic acid culture substance into a fermentation tank;

step two, adding fermentation strains into the fermentation tank with the culture substance of the gamma-polyglutamic acid in the step one, and fermenting the gamma-polyglutamic acid liquid under certain fermentation conditions after adding;

step three, after the fermentation in the step two is finished, the pH value of the liquid gamma-polyglutamic acid fermentation solution in the fermentation tank in the step two is 7.0, and then the fermentation liquid heating treatment is carried out on the gamma-polyglutamic acid fermentation solution which is normally fermented after the fermentation strain is added;

and step four, after the step three is finished, adding a stabilizer into the polyglutamic acid fermentation liquor of the fermentation tank after the heating is finished, and obtaining stable liquid gamma-polyglutamic acid.

The liquid fermentation conditions of the gamma-polyglutamic acid in the second step are as follows: the fermentation temperature is 35-45 ℃, the ventilation volume of the fermentation tank is 1.0-2.0vvm during fermentation, the stirring speed of the fermentation liquid is 150-.

The fermentation strain in the second step is Bacillus velezensis PG1-2, the strain is preserved in China center for culture of microorganisms, the preservation number is CGMCC No.15718, the preservation address is No. 3 of No.1 Hospital No.1 of Xilu of Chaozhong in the Korean area of Beijing, and the preservation date is 5 and 2 days in 2018.

The fermentation culture substance (g/L) in the step is prepared from the following components in percentage by mass: 60-70 parts of cane sugar, 20-30 parts of yeast powder, 60-70 parts of sodium glutamate, 60-2 parts of KH2PO 41, 41-2 parts of K2HPO, 20.2-0.3 part of CaCl and 7.0-7.2 of initial pH.

And when the heating treatment is carried out in the third step, the heating treatment temperature is 50-70 ℃, and the treatment time is 2-6 h.

The stabilizer is sodium benzoate or potassium sorbate and calcium carbonate.

The dosage of the stabilizer is as follows: sodium benzoate or potassium sorbate: 0.5-1.5%, calcium carbonate: 0.5 to 2 percent.

(III) advantageous effects

The invention provides a production method of stable liquid gamma-polyglutamic acid. The method has the following beneficial effects:

the invention carries out fermentation liquor heating treatment on the normally fermented gamma-polyglutamic acid fermentation solution after adding the fermentation strain in the fermentation tank, and treats the liquid gamma-polyglutamic acid by using stabilizing agents such as sodium benzoate or potassium sorbate and calcium carbonate, the treated gamma-polyglutamic acid can be kept stable at 37 ℃, the content of the gamma-polyglutamic acid can be kept at 88-95% after being placed for 12 months, the highest content of the gamma-polyglutamic acid can be kept at 78-88% after being placed for 24 months, and the treated liquid gamma-polyglutamic acid has good stability, is not easily degraded by acid, alkali and microorganisms, is easy to store in natural environment, brings convenience for subsequent industrial application, and is worthy of vigorous popularization.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The first embodiment is as follows:

the embodiment of the invention provides a production method of stable liquid gamma-polyglutamic acid, which comprises the following steps:

step one, preparing a fermentation culture substance, uniformly mixing a plurality of nutrient substances to obtain a gamma-polyglutamic acid culture substance, and transferring the gamma-polyglutamic acid culture substance into a fermentation tank;

step two, adding fermentation strains into the fermentation tank with the culture substance of the gamma-polyglutamic acid in the step one, and fermenting the gamma-polyglutamic acid liquid under certain fermentation conditions after adding;

step three, after the fermentation in the step two is finished, the pH value of the liquid gamma-polyglutamic acid fermentation solution in the fermentation tank in the step two is 7.0, and then the fermentation liquid heating treatment is carried out on the gamma-polyglutamic acid fermentation solution which is normally fermented after the fermentation strain is added;

and step four, after the step three is finished, adding a stabilizer into the polyglutamic acid fermentation liquor of the fermentation tank after the heating is finished, and obtaining stable liquid gamma-polyglutamic acid.

The liquid fermentation conditions of the gamma-polyglutamic acid in the second step are as follows: the fermentation temperature is 35 ℃, the ventilation volume of the fermentation tank during fermentation is 1.0vvm, the stirring speed of the fermentation liquid during fermentation is 150rpm, the fermentation pH is 6.5, and the fermentation time is 48 hours, so that liquid gamma-polyglutamic acid is obtained, wherein the content of the gamma-polyglutamic acid is 21.0 g/L.

The fermentation strain in the second step is Bacillus velezensis PG1-2, the strain is preserved in China center for culture of microorganisms, the preservation number is CGMCC No.15718, the preservation address is No. 3 of No.1 Hospital No.1 of Xilu of Beijing, Chaoyang, and the preservation date is 5 months and 2 days in 2018.

The fermentation culture substance (g/L) comprises the following components in percentage by mass: 60 parts of cane sugar, 20 parts of yeast powder, 60 parts of sodium glutamate, 41 parts of KH2PO, 41 parts of K2HPO, 20.2 parts of CaCl and 7.0-7.2 of initial pH.

When the heating treatment is carried out in the third step, the heating treatment temperature is 50 ℃, after the water bath treatment lasts for 6 hours, the content of the gamma-polyglutamic acid is detected to be 20.5g/L, compared with the fermentation product liquid, the content is reduced by 2.4%, the treated liquid gamma-polyglutamic acid has good stability, is not easily degraded by acid, alkali and microorganisms, is easy to store in the natural environment, and brings convenience for subsequent industrial application.

The stabilizer is sodium benzoate or potassium sorbate and calcium carbonate.

The dosage of the stabilizer is as follows: sodium benzoate 0.5%, calcium carbonate 0.5%;

under the condition of keeping the conditions unchanged, after the gamma-polyglutamic acid is stored for 12 months at 37 ℃, the content of the gamma-polyglutamic acid is detected to be 18.5g/L, and is reduced by 12 percent compared with the fermented product liquid; after being stored for 24 months at 37 ℃, the content of the gamma-polyglutamic acid is detected to be 16.4g/L, and compared with the fermentation product liquid, the content is reduced by 22 percent.

Example two:

the embodiment of the invention provides a production method of stable liquid gamma-polyglutamic acid, which comprises the following steps:

step one, preparing a fermentation culture substance, uniformly mixing a plurality of nutrient substances to obtain a gamma-polyglutamic acid culture substance, and transferring the gamma-polyglutamic acid culture substance into a fermentation tank;

step two, adding fermentation strains into the fermentation tank with the culture substance of the gamma-polyglutamic acid in the step one, and fermenting the gamma-polyglutamic acid liquid under certain fermentation conditions after adding;

step three, after the fermentation in the step two is finished, the pH value of the liquid gamma-polyglutamic acid fermentation solution in the fermentation tank in the step two is 7.0, and then the fermentation liquid heating treatment is carried out on the gamma-polyglutamic acid fermentation solution which is normally fermented after the fermentation strain is added;

and step four, after the step three is finished, adding a stabilizer into the polyglutamic acid fermentation liquor of the fermentation tank after the heating is finished, and obtaining stable liquid gamma-polyglutamic acid.

The liquid fermentation conditions of the gamma-polyglutamic acid in the second step are as follows: the fermentation temperature is 37 ℃, the ventilation volume of the fermentation tank during fermentation is 1.5vvm, the stirring speed of the fermentation liquid during fermentation is 200rpm, the fermentation pH is 7.0, the fermentation time is 48 hours, liquid gamma-polyglutamic acid is obtained, the content of the gamma-polyglutamic acid is 23.2g/L, the stability of the treated liquid gamma-polyglutamic acid is good, the gamma-polyglutamic acid is not easily degraded by acid, alkali and microorganisms, the gamma-polyglutamic acid is easy to store in a natural environment, and convenience is brought to subsequent industrial application.

The fermentation strain in the second step is Bacillus velezensis PG1-2, the strain is preserved in China center for culture of microorganisms, the preservation number is CGMCC No.15718, the preservation address is No. 3 of No.1 Hospital No.1 of Xilu of Beijing, Chaoyang, and the preservation date is 5 months and 2 days in 2018.

The fermentation culture substance (g/L) comprises the following components in percentage by mass: 60 parts of cane sugar, 20 parts of yeast powder, 60 parts of sodium glutamate, 41 parts of KH2PO, 41 parts of K2HPO, 20.2 parts of CaCl and 7.0-7.2 of initial pH.

And when the heating treatment is carried out in the third step, the heating treatment temperature is 60 ℃, and the water bath treatment time is 4 hours.

The stabilizer is sodium benzoate or potassium sorbate and calcium carbonate.

The dosage of the stabilizer is as follows: potassium sorbate: 0.5%, calcium carbonate: 1 percent;

under the condition of unchanging the above conditions, after the liquid is stored for 12 months at 37 ℃, the content of the gamma-polyglutamic acid is detected to be 21.6g/L, and compared with the fermented product liquid, the content is reduced by 7 percent; after being stored for 24 months at 37 ℃, the content of the gamma-polyglutamic acid is detected to be 19.5g/L, and compared with the fermentation product liquid, the content is reduced by 16 percent.

Example three:

the embodiment of the invention provides a production method of stable liquid gamma-polyglutamic acid, which comprises the following steps:

step one, preparing a fermentation culture substance, uniformly mixing a plurality of nutrient substances to obtain a gamma-polyglutamic acid culture substance, and transferring the gamma-polyglutamic acid culture substance into a fermentation tank;

step two, adding fermentation strains into the fermentation tank with the culture substance of the gamma-polyglutamic acid in the step one, and fermenting the gamma-polyglutamic acid liquid under certain fermentation conditions after adding;

step three, after the fermentation in the step two is finished, the pH value of the liquid gamma-polyglutamic acid fermentation solution in the fermentation tank in the step two is 7.0, and then the fermentation liquid heating treatment is carried out on the gamma-polyglutamic acid fermentation solution which is normally fermented after the fermentation strain is added;

and step four, after the step three is finished, adding a stabilizer into the polyglutamic acid fermentation liquor of the fermentation tank after the heating is finished, and obtaining stable liquid gamma-polyglutamic acid.

The liquid fermentation conditions of the gamma-polyglutamic acid in the second step are as follows: the fermentation temperature is 42 ℃, the ventilation volume of the fermentation tank during fermentation is 2.0vvm, the stirring speed of the fermentation liquid during fermentation is 250rpm, the fermentation pH is 7.0, and the fermentation time is 60 hours, so that liquid gamma-polyglutamic acid is obtained, wherein the content of the gamma-polyglutamic acid is 25.6 g/L.

The fermentation strain in the second step is Bacillus velezensis PG1-2, which is preserved in China center for culture Collection of microorganisms with the preservation number of CGMCC No. 15718.

The fermentation culture substance (g/L) comprises the following components in percentage by mass: 60 parts of cane sugar, 20 parts of yeast powder, 60 parts of sodium glutamate, KH2PO 41 parts, 41 parts of K2HPO, 20.2 parts of CaCl and initial pH of 7.0-7.2, and the treated liquid gamma-polyglutamic acid has good stability, is not easily degraded by acid, alkali and microorganisms, is easy to store in natural environment and brings convenience for subsequent industrial application.

When the heating treatment is carried out in the third step, the heating treatment temperature is 70 ℃, and the treatment time is 2 hours.

The stabilizer is sodium benzoate or potassium sorbate and calcium carbonate.

The dosage of the stabilizer is as follows: sodium benzoate: 1.5%, calcium carbonate: 2 percent;

under the condition of unchanging the above conditions, after the liquid is stored for 12 months at 37 ℃, the content of the gamma-polyglutamic acid is detected to be 24.3g/L, and compared with the fermented product liquid, the content is reduced by 5 percent; after being stored for 24 months at 37 ℃, the content of the gamma-polyglutamic acid is detected to be 22.5g/L, and compared with the fermentation product liquid, the content is reduced by 12 percent.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.