CN110951798A - Biological fermentation method of gamma-polyglutamic acid and application thereof - Google Patents
Biological fermentation method of gamma-polyglutamic acid and application thereof Download PDFInfo
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Abstract
The invention provides a biological fermentation method of gamma-polyglutamic acid and application thereof, relating to the technical field of fermentation. The biological fermentation method of the gamma-polyglutamic acid comprises the steps of producing the gamma-polyglutamic acid by using bacillus subtilis and bacillus natto for fermentation; the ratio of viable bacteria of the bacillus subtilis to the bacillus natto is 1: (1-3). The biological fermentation method has the characteristics of short time consumption, high yield and the like, and the produced gamma-polyglutamic acid has good physiological and biochemical activities and has the effects of moisturizing, improving the texture of the skin, repairing the skin and the like.
Description
Technical Field
The invention relates to the technical field of fermentation, in particular to a biological fermentation method of gamma-polyglutamic acid and application thereof.
Background
Gamma-polyglutamic acid is a novel biological polymer material formed by connecting glutamic acid monomers through α -amino and gamma-carboxyl dehydration condensation, has good water absorption performance, and is found in bacillus anthracis at first and is a secondary metabolite of bacillus microorganisms.
The gamma-polyglutamic acid has good biocompatibility, ductility and plasticity. The final catabolite of the gamma-polyglutamic acid is glutamic acid, has no toxic or side effect on human bodies and environment, and cannot cause environmental pollution. The gamma-polyglutamic acid has the performances of gelling, film forming, moisture retention, adhesion, thickening, emulsification and the like, can be used as a thickening agent, a humectant, a biological adhesive, a drug carrier and the like, and is widely applied to the fields of cosmetics, medicines and foods.
The production method of gamma-polyglutamic acid mainly comprises a chemical synthesis method, an enzyme conversion method and a microbial fermentation method. The chemical synthesis method has the disadvantages of complex synthetic route, various intermediate products in the synthetic process, various easily oxidized groups, complex operation, harsh reaction conditions and high production cost. The enzymatic conversion method is to polymerize glutamic acid monomer into polyglutamic acid by utilizing enzymatic reaction, the reaction condition of the enzymatic conversion method is harsh, the polymerization degree and the molecular weight of the prepared gamma-polyglutamic acid are both low, and the physical, physiological and biochemical properties of the gamma-polyglutamic acid are influenced. Compared with a chemical synthesis method and an enzyme conversion method, the biological fermentation method has a simple process and is suitable for large-scale production. However, the biological fermentation method has the defects of long fermentation time and low production intensity, so that the improvement of the biological fermentation method and the improvement of the yield of the gamma-polyglutamic acid produced by biological fermentation are problems to be solved at present.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a biological fermentation method of gamma-polyglutamic acid, which can produce high yield of gamma-polyglutamic acid.
The second purpose of the invention is to provide the application of the biological fermentation method in the preparation of cosmetics.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the present invention, there is provided a method for biofermentation of gamma-polyglutamic acid, comprising producing gamma-polyglutamic acid using bacillus subtilis and bacillus natto for fermentation; the ratio of viable bacteria of the bacillus subtilis to the bacillus natto is 1: (1-3).
Preferably, the ratio of viable bacteria of the bacillus subtilis to the bacillus natto is 1: (1-2).
Preferably, the ratio of viable bacteria of the bacillus subtilis to the bacillus natto is 1:2.
preferably, the seed solution of the bacillus subtilis and the seed solution of the bacillus natto are respectively and independently prepared according to the following methods: inoculating a single colony in a strain activation culture medium, culturing for 15-16 h, and transferring to a seed liquid culture medium to culture to a logarithmic phase;
preferably, the seed liquid culture medium is cultured at the rotating speed of 140-160 rpm for 18-22 h to logarithmic phase;
preferably, the seed liquid culture medium comprises 18-22 g/L of glucose and 4.5-5.5 g/L, K of yeast extract2HPO41.5～2.5g/L、MgSO40.2-0.3 g/L and 9.5-10.5 g/L of sodium glutamate;
preferably, the pH of the seed liquid culture medium is 6.5-7.5.
Preferably, the fermentation comprises the steps of inoculating the seed liquid of the bacillus subtilis and the seed liquid of the bacillus natto into a fermentation culture medium according to the formula amount, and adding a fed-batch culture medium after fermenting for 12-16 h to maintain the final concentration of the glucose content in the fermentation culture medium at 0.15-0.25% m/v;
preferably, the total amount of inoculation of the seed solution of the bacillus subtilis and the seed solution of the bacillus natto is 8-10% v/v of the fermentation medium;
preferably, the fermentation conditions are: the fermentation medium accounts for 50-70% of the volume of the fermentation container, the rotating speed is 320-360 rpm, the pressure of the fermentation container is 0.02-0.04 MPa, and the total fermentation time is 75-85 h;
preferably, the fermentation medium comprises: 35-45 g/L glucose and 8-10 g/L, K yeast extract2HPO4·3H2O1.5～2.5g/L、MgSO40.2-0.3 g/L and 35-45 g/L of sodium glutamate; the pH value of the fermentation medium is preferably 7.0-8.0;
preferably, the feed medium comprises: 65-75 g/L glucose and 15-25 g/L, K yeast extract2HPO4·3H2O3.5～4.5g/L、MgSO40.4-0.6 g/L and 65-75 g/L of sodium glutamate.
Preferably, the biological fermentation process further comprises acidifying the fermentation broth after fermentation;
preferably, the acidification comprises adjusting the pH of the fermentation broth to 3-4, preferably to 3.5.
Preferably, after fermentation, separating thalli in the fermentation liquor, and reserving fermentation clear liquid; then concentrating the fermentation clear liquid to obtain a gamma-polyglutamic acid concentrated solution; purifying the gamma-polyglutamic acid concentrated solution to obtain gamma-polyglutamic acid;
preferably, the step of separating the thalli in the fermentation liquor comprises the steps of centrifuging the fermentation liquor, precipitating for 18-22 hours, and filtering by a filter membrane to obtain the fermentation clear liquid.
Preferably, concentrating the fermentation clear liquid to 25-35% of the original volume to obtain the gamma-polyglutamic acid concentrated solution; preferably concentrating the fermentation broth to 30% of its original volume;
preferably, the pH value of the gamma-polyglutamic acid concentrated solution is adjusted to 6-7, preferably 6;
preferably, the fermentation supernatant is concentrated using an ultrafiltration membrane, preferably using ultrafiltration membrane 6000.
Preferably, the purification treatment comprises using low carbon alcohol of C1-C4 to precipitate the gamma-polyglutamic acid in the gamma-polyglutamic acid concentrated solution;
preferably, the lower alcohol of C1-C4 preferably comprises ethanol;
preferably, the volume ratio of the gamma-polyglutamic acid concentrated solution to the ethanol is 1: (1-2); preferably 1: 1.5;
preferably, gamma-polyglutamic acid is precipitated at least twice by using low carbon alcohol of C1-C4.
According to another aspect of the invention, the invention also provides the application of the biological fermentation method of the gamma-polyglutamic acid in preparing cosmetics.
Compared with the prior art, the invention has the following beneficial effects:
the biological fermentation method of the gamma-polyglutamic acid provided by the invention has the characteristics of short time consumption and high yield. According to the biological fermentation method, the bacillus subtilis and the bacillus natto are cooperatively used for producing the gamma-polyglutamic acid through fermentation, the inoculation amount of the bacillus subtilis and the bacillus natto in a fermentation culture medium is optimized, the yield and the production efficiency of the gamma-polyglutamic acid are improved, and in some embodiments of the invention, the yield of the gamma-polyglutamic acid can reach 51.5g/L to the maximum.
The biological fermentation method of the gamma-polyglutamic acid provided by the invention has high yield, and can reduce the production cost of cosmetics when being used in the cosmetics. The gamma-polyglutamic acid produced by the biological fermentation method has good physiological and biochemical activities, and has the effects of moisturizing, improving the texture of the skin, repairing the skin and the like. The gamma-polyglutamic acid prepared by the preparation method can be used in cosmetics to improve the content of the horny layer of the skin, improve the elasticity of the skin and reduce the loss of moisture in the skin, and has the effects of locking water, preserving moisture and keeping the skin bright and elastic.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. 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.
According to one aspect of the present invention, there is provided a biofermentation method of gamma-polyglutamic acid, the biofermentation method comprising producing gamma-polyglutamic acid by fermenting using bacillus subtilis and bacillus natto, the ratio of viable bacteria of the bacillus subtilis to the bacillus natto being 1: (1-3).
Bacillus subtilis belongs to the genus Bacillus, is non-capsular, has peritrichogenous flagella, is a gram-positive bacterium, and can form endophytic spores. The bacillus subtilis can be fermented to produce gamma-polyglutamic acid, and is a glutamic acid dependent gamma-polyglutamic acid production strain. Bacillus natto (Bacillus natto) is a subspecies of Bacillus subtilis, the Bacillus natto cells are long rod-shaped, gram-positive bacteria, can form spores under adverse environment, the spores are columnar or elliptical, the cysts are expanded, and the Bacillus natto has flagella, is acid-resistant and heat-resistant. According to the invention, experiments show that the bacillus subtilis and the bacillus natto are fermented together, and the fermentation conditions are shown in the following ratio of 1: (1-3) the bacillus subtilis and the bacillus natto are inoculated in a fermentation medium, so that the yield of the gamma-polyglutamic acid is high. In some embodiments of the invention, the yield of gamma-polyglutamic acid is up to 51.5 g/L. The ratio of viable bacteria of the bacillus subtilis to the bacillus natto is 1: (1-3). For example, but not limited to, 1:1, 1:1.5, 1:2, 1:2.5, or 1: 3. The yield of gamma-polyglutamic acid can be further improved by optimizing the using amount of the bacillus subtilis and the bacillus natto during fermentation, and the ratio of the viable bacteria amount of the bacillus subtilis to the viable bacteria amount of the bacillus natto is preferably 1: (1-2), the yield can reach about 50g/L, more preferably 1:2, and the yield can reach 51.5 g/L.
In some preferred embodiments, the seed solution of Bacillus subtilis and the seed solution of Bacillus natto are prepared independently as follows: inoculating the single colony in a strain activation culture medium, culturing for 15-16 h, transferring to a seed liquid culture medium, and culturing to logarithmic phase to obtain a seed liquid. The condition for culturing the strain in the seed liquid culture medium is preferably that the culture speed is 140-160 rpm, and the strain is cultured for 18-22 h to logarithmic phase. The preferred formulation of seed liquid medium is as follows: 18-22 g/L glucose, 4.5-5.5 g/L, K yeast extract2HPO41.5～2.5g/L、MgSO40.2-0.3 g/L and 9.5-10.5 g/L of sodium glutamate.
In some preferred embodiments, the fermentation is carried out in the following manner: inoculating a seed solution of bacillus subtilis and a seed solution of bacillus natto into a fermentation culture medium according to a formula amount, fermenting for 12-16 h, and then adding a fed-batch culture medium to maintain the final concentration of the glucose content in the fermentation culture medium at 0.15-0.25% m/v; the final concentration of glucose content is preferably maintained at 0.2% m/v. Since various nutrients in the fermentation medium are consumed rapidly as the cells grow, it is necessary to supplement nutrients to maintain the growth of the cells and to synthesize gamma-polyglutamic acid. Since high glucose concentration produces polysaccharide by-products, which are not favorable for the production of gamma-polyglutamic acid, the glucose concentration in the culture medium needs to be optimized.
The total amount of inoculation of the seed solution of the bacillus subtilis and the seed solution of the bacillus natto is preferably 8-10% v/v of the fermentation medium, and the too small inoculation amount can prolong the culture time and reduce the production efficiency; too large an amount of inoculation will cause insufficient dissolved oxygen, affecting the product synthesis. The fermentation conditions are preferably: the fermentation medium accounts for 50-70% of the volume of the fermentation container, the rotating speed is 320-360 rpm, the pressure of the fermentation container is 0.02-0.04 MPa, and the total fermentation time is 75-85 h. The liquid loading amount, the rotating speed and other parameters in the fermentation container can influence the dissolved oxygen amount of the culture medium, and the liquid loading amount, the rotating speed and other optimized technological parameters are selected to be beneficial to the production of the gamma-polyglutamic acid.
The preferred formulations of the fermentation medium and the feed medium are as follows: fermentation medium: 35-45 g/L glucose and 8-10 g/L, K yeast extract2HPO4·3H2O 1.5～2.5g/L、MgSO40.2-0.3 g/L and 35-45 g/L of sodium glutamate; the pH of the fermentation medium is preferably 7.0-8.0. Feeding a culture medium: 65-75 g/L glucose and 15-25 g/L, K yeast extract2HPO4·3H2O3.5～4.5g/L、MgSO40.4-0.6 g/L and 65-75 g/L of sodium glutamate.
In some preferred embodiments, the biological fermentation method further comprises acidifying the fermentation broth after fermentation, and then separating the gamma-polyglutamic acid in the fermentation broth, wherein the acidification preferably adjusts the pH of the fermentation broth to 3-4, preferably 3.5.
In some preferred embodiments, isolating the gamma-polyglutamic acid from the fermentation broth comprises isolating the biomass from the fermentation broth after fermentation and retaining the fermentation broth; then concentrating the fermentation clear liquid to obtain a gamma-polyglutamic acid concentrated solution; and purifying the gamma-polyglutamic acid concentrated solution to obtain the gamma-polyglutamic acid.
The method for separating the thalli in the fermentation liquor preferably comprises the steps of centrifuging the fermentation liquor, precipitating for 18-22 hours, and centrifuging for 15-25 min at 3000-5000 rpm preferably; and filtering the precipitate by a filter membrane to obtain the fermentation clear liquid, wherein the filter membrane is preferably a filter membrane of 0.4-0.5 mu m.
In some preferred embodiments, the fermentation clear liquid is concentrated to 25-35% of the original volume to obtain the gamma-polyglutamic acid concentrated solution; preferably the fermentation broth is concentrated to 30% of its original volume. The concentration mode is preferably ultrafiltration membrane concentration, and the ultrafiltration membrane is preferably ultrafiltration membrane 6000. Before separating the gamma-polyglutamic acid in the gamma-polyglutamic acid concentrated solution, preferably, the pH of the gamma-polyglutamic acid concentrated solution is adjusted to 6-7, preferably 6.
In some preferred embodiments, the gamma-polyglutamic acid is separated, and the gamma-polyglutamic acid in the gamma-polyglutamic acid concentrated solution is preferably precipitated by using low carbon alcohol of C1-C4. The C1-C4 lower alcohol preferably includes ethanol with good precipitation effect and less side effect. When ethanol is used for precipitating the gamma-polyglutamic acid, the volume ratio of the gamma-polyglutamic acid concentrated solution to the ethanol is 1: (1-2); preferably, the ratio of 1:1.5 volume ratio. In order to sufficiently precipitate the gamma-polyglutamic acid in the gamma-polyglutamic acid concentrated solution and improve the yield of the gamma-polyglutamic acid, the gamma-polyglutamic acid is preferably precipitated twice by using low-carbon alcohol of C1-C4. The mode of precipitating the gamma-polyglutamic acid for multiple times is to re-dissolve the gamma-polyglutamic acid precipitated for the previous time and then precipitate the gamma-polyglutamic acid by using low carbon alcohol of C1-C4 so as to improve the purity of the gamma-polyglutamic acid in the precipitated product.
According to another aspect of the invention, the invention also provides the application of the biological fermentation method of the gamma-polyglutamic acid in preparing cosmetics. The biological fermentation method of the gamma-polyglutamic acid provided by the invention has high yield, and can reduce the production cost of cosmetics when being used in the cosmetics. The gamma-polyglutamic acid prepared by the biological fermentation method has good physiological and biochemical activities, and can improve the content of the horny layer of the skin, improve the elasticity of the skin and reduce the water loss through skin when being used in cosmetics.
The technical solution and the advantages of the present invention will be further explained with reference to the preferred embodiments.
The formulations of seed broth, fermentation medium and feed medium used in the following examples were as follows:
seed liquid culture medium: glucose 20g/L, yeast extract 5g/L, K g2HPO43g/L、MgSO40.25g/L and 10g/L of sodium glutamate, and the pH is adjusted to 7.0.
Fermentation medium: glucose 40g/L and yeast extract 8g/L, K g2HPO4·3H2O 2g/L、MgSO40.25g/L and 40g/L of sodium glutamate; adjusting the pH value to 7.0-8.0.
Feeding a culture medium: glucose 70g/L, yeast extract 20g/L, K g2HPO4·3H2O 4g/L、MgSO40.5g/L and 70g/L of sodium glutamate.
Example 1
Example 1 provides a method for biofermentation of gamma-polyglutamic acid, comprising the steps of:
(1) preparing a seed solution of bacillus subtilis and a seed solution of bacillus natto: transferring the test tube slant strain on the TSA culture medium stored at 4 ℃ to room temperature for activation for 10h, inoculating 3 single colonies into the sterile TSA culture medium by using a sterile inoculating loop on a sterile operating platform, culturing for 16h, transferring the single colonies into a triangular flask containing the sterile seed culture medium, culturing at 37 ℃, rotating at 150rpm, and culturing for 20h to logarithmic phase.
(2) Fermentation: inoculating the seed solution of the bacillus subtilis and the seed solution of the bacillus natto into a fermentation tank according to the volume ratio of 1:2, wherein the total amount of the seed solution of the bacillus subtilis and the seed solution of the bacillus natto in the inoculation is 8% v/v of a fermentation culture medium. The liquid loading of the fermentation medium in a 500L fermentation tank is 300L, the fermentation temperature is 37 ℃, the tank pressure is 0.03Mpa, the rotating speed is 350rpm, after the fermentation is carried out for 15h, the feeding of the medium is started, the feeding speed of the medium is regulated through the feedback of glucose, the final concentration of the glucose is controlled to be 0.2% m/v, and the total fermentation time is 80 h.
(3) Acidifying the fermentation liquor: 3000mL of the fermented broth was adjusted to pH3.5 with concentrated sulfuric acid.
(4) Degerming and concentrating gamma-polyglutamic acid: acidifying the fermentation liquor, centrifuging at 4000r/min for 20 minutes, precipitating for 20 hours, and treating with a 0.45-micrometer microfiltration membrane to obtain a clear liquid; and (3) concentrating the sterilized fermentation liquor by using an ultrafiltration membrane 6000, diluting the sterilized liquid by using distilled water by 10 times in the concentration process, finally concentrating the fermentation liquor to 30% of the original volume, and adjusting the pH value of the concentrated solution to 6.5.
(5) Purification of gamma-polyglutamic acid: adding ethanol into the gamma-polyglutamic acid concentrated solution according to the proportion of 1:1.5, precipitating, removing supernatant, adding 1000mL of water into the precipitate, adding ethanol again according to the proportion of 1:1.5, precipitating, and freeze-drying to obtain a pure gamma-polyglutamic acid product.
Example 2
Example 2 provides a method for biofermentation of gamma-polyglutamic acid, which is different from example 1 in that the seed solution of bacillus subtilis and the seed solution of bacillus natto are inoculated into a fermentation tank in a volume ratio of 1:1 in step (2).
Example 3
Example 3 provides a method for biofermentation of gamma-polyglutamic acid, which is different from example 1 in that the seed solution of bacillus subtilis and the seed solution of bacillus natto are inoculated into a fermentation tank in a volume ratio of 1:1.5 in step (2).
Example 4
Example 4 provides a method for biofermentation of gamma-polyglutamic acid, which is different from example 1 in that the seed solution of bacillus subtilis and the seed solution of bacillus natto are inoculated into a fermentation tank in a volume ratio of 1:2.5 in step (2).
Example 5
Example 5 provides a method for biofermentation of gamma-polyglutamic acid, which is different from example 1 in that the seed solution of bacillus subtilis and the seed solution of bacillus natto are inoculated into a fermenter in the step (2) in a volume ratio of 1: 3.
Example 6
Example 6 provides a method for biofermentation of gamma-polyglutamic acid, comprising the steps of:
(1) preparing a seed solution of bacillus subtilis and a seed solution of bacillus natto: transferring the test tube slant strain on the TSA culture medium stored at 4 ℃ to room temperature for activation for 10h, inoculating 3 single colonies into the sterile TSA culture medium by using a sterile inoculating loop on a sterile operating platform, culturing for 16h, transferring the single colonies into a triangular flask containing the sterile seed culture medium, culturing at 37 ℃, rotating at 150rpm, and culturing for 20h to logarithmic phase.
(2) Fermentation: inoculating the seed solution of the bacillus subtilis and the seed solution of the bacillus natto into a fermentation tank according to the volume ratio of 1:2, wherein the total amount of the seed solution of the bacillus subtilis and the seed solution of the bacillus natto in the inoculation is 10% v/v of a fermentation culture medium. The liquid loading of the fermentation medium in a 500L fermentation tank is 350L, the fermentation temperature is 37 ℃, the tank pressure is 0.02Mpa, the rotating speed is 350rpm, after the fermentation is carried out for 15h, the feeding of the medium is started, the feeding speed of the medium is regulated through the feedback of glucose, the final concentration of the glucose is controlled to be 0.25% m/v, and the total fermentation time is 75 h.
(3) Acidifying the fermentation liquor: 3000mL of the fermented broth was adjusted to pH3.5 with concentrated sulfuric acid.
(4) Degerming and concentrating gamma-polyglutamic acid: acidifying the fermentation liquor, centrifuging at 4000r/min for 20 minutes, precipitating for 20 hours, and treating with a 0.45-micrometer microfiltration membrane to obtain a clear liquid; and (3) concentrating the sterilized fermentation liquor by using an ultrafiltration membrane 6000, diluting the sterilized liquid by using distilled water by 10 times in the concentration process, finally concentrating the fermentation liquor to 30% of the original volume, and adjusting the pH value of the concentrated solution to 6.5.
(5) Purification of gamma-polyglutamic acid: adding ethanol into the gamma-polyglutamic acid concentrated solution according to the proportion of 1:2, precipitating, removing supernatant, adding 1000mL of water into the precipitate, adding ethanol again according to the proportion of 1:1, precipitating, and freeze-drying to obtain a pure gamma-polyglutamic acid product.
Example 7
Example 7 provides a method for biofermentation of gamma-polyglutamic acid, comprising the steps of:
(1) preparing a seed solution of bacillus subtilis and a seed solution of bacillus natto: transferring the test tube slant strain on the TSA culture medium stored at 4 ℃ to room temperature for activation for 10h, inoculating 3 single colonies into the sterile TSA culture medium by using a sterile inoculating loop on a sterile operating platform, culturing for 16h, transferring the single colonies into a triangular flask containing the sterile seed culture medium, culturing at 37 ℃, rotating at 150rpm, and culturing for 20h to logarithmic phase.
(2) Fermentation: inoculating the seed solution of the bacillus subtilis and the seed solution of the bacillus natto into a fermentation tank according to the volume ratio of 1:2, wherein the total amount of the seed solution of the bacillus subtilis and the seed solution of the bacillus natto in the inoculation is 5% v/v of a fermentation culture medium. The liquid loading of the fermentation medium in a 500L fermentation tank is 300L, the fermentation temperature is 37 ℃, the tank pressure is 0.04Mpa, the rotating speed is 300rpm, when the fermentation is carried out for 12h, the feeding of the medium is started, the feeding speed of the medium is regulated through the feedback of glucose, the final concentration of the glucose is controlled to be 0.15% m/v, and the total fermentation time is 85 h.
(3) Acidifying the fermentation liquor: 3000mL of the fermented broth was adjusted to pH3.5 with concentrated sulfuric acid.
(4) Degerming and concentrating gamma-polyglutamic acid: acidifying the fermentation liquor, centrifuging at 4000r/min for 20 minutes, precipitating for 20 hours, and treating with a 0.45-micrometer microfiltration membrane to obtain a clear liquid; and (3) concentrating the sterilized fermentation liquor by using an ultrafiltration membrane 6000, diluting the sterilized liquid by using distilled water by 10 times in the concentration process, finally concentrating the fermentation liquor to 30% of the original volume, and adjusting the pH value of the concentrated solution to 6.5.
(5) Purification of gamma-polyglutamic acid: adding ethanol into the gamma-polyglutamic acid concentrated solution according to the proportion of 1:1, precipitating, removing supernatant, adding 1000mL of water into the precipitate, adding ethanol again according to the proportion of 1:1, precipitating, and freeze-drying to obtain a pure gamma-polyglutamic acid product.
Comparative example 1
Comparative example 1 provides a biological fermentation method of gamma-polyglutamic acid, which is different from example 1 in that only seed solution of bacillus subtilis is inoculated into a fermenter in step (2).
Comparative example 2
Comparative example 2 provides a biological fermentation method of gamma-polyglutamic acid, which is different from example 1 in that only the seed solution of bacillus natto is inoculated into the fermentation tank in step (2).
Comparative example 3
Comparative example 3 provides a biological fermentation method of gamma-polyglutamic acid, which is different from example 1 in that the seed solution of bacillus subtilis and the seed solution of bacillus natto are inoculated into a fermentation tank in a volume ratio of 1:5 in step (2).
Effect example 1
Comparing the production efficiency of the gamma-polyglutamic acid biological fermentation methods provided in examples 1 to 7 and comparative examples 1 to 3, specifically, the method comprises the steps of after fermentation is completed, obtaining the quality and purity of a pure gamma-polyglutamic acid product by the content of gamma-polyglutamic acid in fermentation liquor and freeze drying, wherein the content of gamma-polyglutamic acid in the fermentation liquor is detected by a glutamic acid analyzer, the purity is detected by an HPLC method, and the results are shown in Table 1:
TABLE 1
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A biological fermentation method of gamma-polyglutamic acid is characterized by comprising the steps of producing the gamma-polyglutamic acid by using bacillus subtilis and bacillus natto for fermentation;
the ratio of viable bacteria of the bacillus subtilis to the bacillus natto is 1: (1-3).
2. The biological fermentation method according to claim 1, wherein the ratio of viable bacteria of bacillus subtilis to bacillus natto is 1: (1-2).
3. The biological fermentation method according to claim 2, wherein the ratio of viable bacteria of bacillus subtilis to bacillus natto is 1:2.
4. the biological fermentation method according to any one of claims 1 to 3, wherein the seed solution of Bacillus subtilis and the seed solution of Bacillus natto are prepared separately according to the following method: inoculating a single colony in a strain activation culture medium, culturing for 15-16 h, and transferring to a seed liquid culture medium to culture to a logarithmic phase;
preferably, the seed liquid culture medium is cultured at the rotating speed of 140-160 rpm for 18-22 h to logarithmic phase;
preferably, the seed liquid culture medium comprises 18-22 g/L of glucose and 4.5-5.5 g/L, K of yeast extract2HPO41.5～2.5g/L、MgSO40.2-0.3 g/L and 9.5-10.5 g/L of sodium glutamate;
preferably, the pH of the seed liquid culture medium is 6.5-7.5.
5. The biological fermentation method according to any one of claims 1 to 3, wherein the fermentation comprises inoculating a seed solution of Bacillus subtilis and a seed solution of Bacillus natto in a fermentation medium according to a formula amount, and adding a fed-batch medium after fermenting for 12 to 16 hours so that the final concentration of the glucose content in the fermentation medium is maintained at 0.15 to 0.25% m/v;
preferably, the total amount of inoculation of the seed solution of the bacillus subtilis and the seed solution of the bacillus natto is 8-10% v/v of the fermentation medium;
preferably, the fermentation conditions are: the fermentation medium accounts for 50-70% of the volume of the fermentation container, the rotating speed is 320-360 rpm, the pressure of the fermentation container is 0.02-0.04 MPa, and the total fermentation time is 75-85 h;
preferably, the fermentation medium comprises: 35-45 g/L glucose and 8-10 g/L, K yeast extract2HPO4·3H2O 1.5～2.5g/L、MgSO40.2-0.3 g/L and 35-45 g/L of sodium glutamate; the pH value of the fermentation medium is preferably 7.0-8.0;
preferably, the feed medium comprises: glucose 65-75 gYeast extract 15-25 g/L, K2HPO4·3H2O 3.5～4.5g/L、MgSO40.4-0.6 g/L and 65-75 g/L of sodium glutamate.
6. The biofermentation process of any one of claims 1-3, further comprising acidifying the fermentation broth after fermentation;
preferably, the acidification comprises adjusting the pH of the fermentation broth to 3-4, preferably to 3.5.
7. A biofermentation method according to any one of claims 1 to 3, characterized in that the biomass in the fermentation broth is separated after fermentation and the fermentation supernatant is retained; then concentrating the fermentation clear liquid to obtain a gamma-polyglutamic acid concentrated solution; purifying the gamma-polyglutamic acid concentrated solution to obtain gamma-polyglutamic acid;
preferably, the step of separating the thalli in the fermentation liquor comprises the steps of centrifuging the fermentation liquor, precipitating for 18-22 hours, and filtering by a filter membrane to obtain the fermentation clear liquid.
8. The biological fermentation method according to claim 7, wherein the fermentation clear liquid is concentrated to 25 to 35% of the original volume to obtain the gamma-polyglutamic acid concentrated solution; preferably concentrating the fermentation broth to 30% of its original volume;
preferably, the pH value of the gamma-polyglutamic acid concentrated solution is adjusted to 6-7, preferably 6;
preferably, the fermentation supernatant is concentrated using an ultrafiltration membrane, preferably using ultrafiltration membrane 6000.
9. The biological fermentation method according to claim 7, wherein the purification treatment comprises using low carbon alcohol of C1-C4 to precipitate the gamma-polyglutamic acid in the gamma-polyglutamic acid concentrated solution;
preferably, the lower alcohol of C1-C4 preferably comprises ethanol;
preferably, the volume ratio of the gamma-polyglutamic acid concentrated solution to the ethanol is 1: (1-2); preferably 1: 1.5;
preferably, gamma-polyglutamic acid is precipitated at least twice by using low carbon alcohol of C1-C4.
10. Use of a process for the biological fermentation of gamma-polyglutamic acid according to any one of claims 1 to 9 for the preparation of cosmetics.
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