CN112813114A - Method for producing gamma-polyglutamic acid by solid-state fermentation of soybean curd residue - Google Patents
Method for producing gamma-polyglutamic acid by solid-state fermentation of soybean curd residue Download PDFInfo
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Abstract
The invention discloses a method for producing gamma-polyglutamic acid by solid-state fermentation of soybean curb residues. The method of the invention comprises the following steps: firstly, bacillus subtilis is revived and activated to obtain bacillus subtilis seed solution; preparing a solid fermentation substrate of the bean curd residue to prepare a solid fermentation culture medium, inoculating activated bacillus subtilis seed liquid, and fermenting to obtain a fermentation product; and separating the fermentation product and extracting to obtain the gamma-polyglutamic acid. The invention has short fermentation period, high utilization rate of the bean curd residue and high yield and production efficiency of the gamma-polyglutamic acid, and the produced gamma-polyglutamic acid can be widely applied to the fields of agriculture, food, medicine, chemical industry and the like.
Description
Technical Field
The invention relates to a method for producing gamma-polyglutamic acid by solid-state fermentation of soybean curb residues, belonging to the technical field of microbial engineering.
Background
The soybean curd residue is a main byproduct of soybean milk and bean curd production in soybean processing, and has high yield and low price. The bean curd refuse can be generally treated only as animal feed or garbage after industrial processing, and although the bean curd refuse still has high nutritive value, the bean curd refuse is not well utilized, so that resource waste and environmental pollution are caused. In order to improve the utilization rate of the soybean curb residue, the soybean curb residue is fermented by microorganisms, and the soybean curb residue fermentation method has the advantages of low cost, simplicity in operation and high feasibility.
Gamma-polyglutamic acid (gamma-PGA) is a homogeneous polypeptide polymerized by taking glutamic acid with left and right optical rotation as a unit body and an amido bond on the gamma-position. The gamma-polyglutamic acid has molecular weight distribution of 100kDa to 10000kDa, has the characteristics of excellent water solubility, super-strong adsorbability, good biodegradability, biocompatibility, lower cytotoxicity and the like, can be degraded into glutamic acid, and is an excellent biological safe and environment-friendly high polymer material. The existing research shows that the gamma-polyglutamic acid and the derivatives thereof can be applied to industries such as cosmetics, environmental protection, food, medicine, agriculture, desert control and the like due to good functions of thickening, moisturizing, film forming, adhesion and the like; the biological safety and degradability of the hydrogel and the microcapsule are more ideal materials for the hydrogel and the microcapsule, and the hydrogel and the microcapsule are widely applied to medical materials. However, because the yield of the gamma-polyglutamic acid is relatively low, the yield is still unsatisfactory even through the variant separation of strains, the separation and purification process is complex, time and labor are wasted, and the application research of the gamma-polyglutamic acid is mainly focused on scientific research experiments. The search for a production method with simple operation and low cost and a rapid and effective purification method is also a big problem to be solved by scientific research personnel.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to utilize the bean curd residue to produce the gamma-polyglutamic acid solves the utilization problem of the bean curd residue and the production problem of the gamma-polyglutamic acid.
In order to solve the technical problem, the invention provides a method for producing gamma-polyglutamic acid by solid state fermentation of soybean curd residue, which comprises the following steps:
step 1: recovering and activating the bacillus subtilis to obtain bacillus subtilis seed liquid;
step 2: preparing a bean curd residue fermentation medium, inoculating the bacillus subtilis seed liquid obtained in the step 1, and fermenting to obtain a bean curd residue fermentation product;
and step 3: separating the fermented product of the bean curd residue, and extracting the gamma-polyglutamic acid.
Preferably, the step 1 specifically comprises: inoculating the bacillus subtilis to a slant culture medium for recovery culture to obtain strains, and then selecting the strains to inoculate the strains to a shake flask filled with a liquid culture medium for activation culture to obtain bacillus subtilis fermentation seed liquid.
Preferably, the composition of the slant medium comprises: 40g of LB culture medium, 20g of agar, 1L of distilled water and natural pH; the composition of the liquid culture medium comprises: 30g of cane sugar, 10g of beef extract, 30g of sodium glutamate and MgSO4·7H2O 0.25g，K2HPO4 0.5g,CaCl21g, 1L of distilled water, natural pH.
Preferably, the resuscitation culture conditions are: the temperature is 37 ℃, and the time is 24 h; the conditions of the activation culture are as follows: 50mL of liquid culture medium, 220r/min of shaking bottle rotation speed, 37 ℃ and 18h of time.
Preferably, the prepared beancurd residue fermentation medium in the step 2 specifically comprises: and mixing the dry soybean curd residue with distilled water according to the mass ratio of 1: 2-4 to obtain the soybean curd residue fermentation medium.
Preferably, the conditions of the fermentation culture in the step 2 are as follows: inoculating 2% of the strain, keeping the temperature at 37 ℃ for 24-28 h, and standing for fermentation.
Preferably, the separation of step 3 is specifically: soaking the fermented product of bean curd residue in 0.9 wt% physiological saline water for 10min, centrifuging, and removing thallus and part of bean flour precipitate to obtain supernatant; the extraction specifically comprises the following steps: adjusting the pH value of the separated supernatant to 3, then adding 4 times of isopropanol, putting the mixture into a refrigerator for precipitation for 12 hours, centrifuging the mixture, and centrifuging the precipitate to obtain the gamma-polyglutamic acid.
Preferably, the Bacillus subtilis is Bacillus subtilis GIM 1.286.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention adopts the bean curd residue which is an industrial waste with high yield and low price as a solid fermentation substrate, effectively improves the industrial value and provides an effective utilization mode for the bean curd residue;
2. the invention has simple production mode, lower required cost, no need of large-scale production facility investment and stronger feasibility and operability.
3. The method has the advantages of short fermentation period, high utilization rate of the soybean curb residue and high yield and production efficiency of the gamma-polyglutamic acid.
Drawings
FIG. 1 is a process flow diagram of the production of gamma-polyglutamic acid by solid state fermentation of soybean curd residue according to the present invention.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.
Example 1
A method for producing gamma-polyglutamic acid by solid state fermentation of soybean curd residue is shown in figure 1, and comprises the following steps:
(1) preparation of a culture medium: firstly, preparing a culture medium of bacillus subtilis, wherein the slant culture medium comprises the following steps: 40g of LB culture medium, 20g of agar, 1L of distilled water, natural pH and 121 ℃ for 20 min; liquid culture medium: 30g of cane sugar, 10g of beef extract, 30g of sodium glutamate and MgSO4·7H2O 0.25g，K2HPO4 0.5g,CaCl21g, natural pH, 1L of distilled water; preparing a bean curd residue fermentation culture medium, adding distilled water into the dried bean curd residue, mixing uniformly, adjusting the water content of the bean curd residue to be 60%, and sterilizing at 121 ℃ for 15 min.
(2) Activating and culturing the bacillus subtilis: inoculating the bacillus subtilis preserved at 4 ℃ to a slant culture medium under the aseptic condition, culturing at 37 ℃ for 24h to recover the strains, selecting seeds, inoculating the seeds to a shake flask (250mL) filled with 50mL of liquid culture medium, and culturing at 37 ℃ for 18h at 220r/min to obtain the bacillus subtilis fermented seed liquid.
(3) Fermenting and culturing the bean curd residue, namely inoculating the activated seed liquid into a bean curd residue fermentation culture medium according to the inoculation amount of 2 percent, and performing fermentation culture at 37 ℃ for 24 hours; the Bacillus subtilis is Bacillus subtilis GIM 1.286.
(4) Soaking fermented bean curd residue in 0.9% normal saline for 10min, centrifuging, removing thallus and part of bean flour precipitate, collecting supernatant, and adjusting pH to 3.
(5) Adding 4 times volume of isopropanol into the extractive solution, and precipitating in refrigerator for 12 h.
(6) Centrifuging the alcohol precipitation solution to obtain a precipitate, namely a gamma-polyglutamic acid crude extract; the yield of the prepared gamma-polyglutamic acid crude extract is 19.96 percent.
For comparison, soybean was fermented under the same process conditions as the above preparation method, and the yield of the crude extract of gamma-polyglutamic acid was 10.12%.
Example 2
A method for producing gamma-polyglutamic acid by solid state fermentation of soybean curd residue is shown in figure 1, and comprises the following steps:
(1) preparation of a culture medium: firstly, preparing a culture medium of bacillus subtilis, wherein the slant culture medium comprises the following steps: 40g of LB culture medium, 20g of agar, 1L of distilled water, natural pH and 121 ℃ for 20 min; liquid culture medium: 30g of cane sugar, 10g of beef extract, 30g of sodium glutamate and MgSO4·7H2O 0.25g，K2HPO4 0.5g,CaCl21g, natural pH, 1L of distilled water; preparing a bean curd residue fermentation culture medium, adding distilled water into the dried bean curd residue, mixing uniformly, adjusting the water content of the bean curd residue to 80%, and sterilizing at 121 ℃ for 15 min.
(2) Activating and culturing the bacillus subtilis: inoculating the bacillus subtilis preserved at 4 ℃ to a slant culture medium under the aseptic condition, culturing at 37 ℃ for 24h to recover the strains, selecting seeds, inoculating the seeds to a shake flask (250mL) filled with 50mL of liquid culture medium, and culturing at 37 ℃ for 18h at 220r/min to obtain the bacillus subtilis fermented seed liquid.
(3) Fermenting and culturing the bean curd residue, namely inoculating the activated seed liquid into a bean curd residue fermentation culture medium according to the inoculation amount of 2 percent, and performing fermentation culture at 37 ℃ for 24 hours; the Bacillus subtilis is Bacillus subtilis GIM 1.286.
(4) Soaking fermented bean curd residue in 0.9% normal saline for 10min, centrifuging, removing thallus and part of bean flour precipitate, collecting supernatant, and adjusting pH to 3.
(5) Adding 4 times volume of isopropanol into the extractive solution, and precipitating in refrigerator for 12 h.
(6) Centrifuging the alcohol precipitation solution to obtain a precipitate, namely a gamma-polyglutamic acid crude extract; the yield of the prepared gamma-polyglutamic acid crude extract is 19.34 percent.
For comparison, soybean was fermented under the same process conditions as the above preparation method, and the yield of the crude extract of gamma-polyglutamic acid was 10.12%.
Example 3
A method for producing gamma-polyglutamic acid by solid state fermentation of soybean curd residue is shown in figure 1, and comprises the following steps:
(1) preparation of a culture medium: firstly, preparing a culture medium of bacillus subtilis, wherein the slant culture medium comprises the following steps: 40g of LB culture medium, 20g of agar, 1L of distilled water, natural pH and 121 ℃ for 20 min; liquid culture medium: 30g of cane sugar, 10g of beef extract, 30g of sodium glutamate and MgSO4·7H2O 0.25g，K2HPO4 0.5g,CaCl21g, natural pH, 1L of distilled water; preparing a bean curd residue fermentation culture medium, adding distilled water into the dried bean curd residue, mixing uniformly, adjusting the water content of the bean curd residue to 70%, and sterilizing at 121 ℃ for 15 min.
(2) Activating and culturing the bacillus subtilis: inoculating the bacillus subtilis preserved at 4 ℃ to a slant culture medium under the aseptic condition, culturing at 37 ℃ for 24h to recover the strains, selecting seeds, inoculating the seeds to a shake flask (250mL) filled with 50mL of liquid culture medium, and culturing at 37 ℃ for 18h at 220r/min to obtain the bacillus subtilis fermented seed liquid.
(3) Fermenting and culturing bean curd residue, namely inoculating the activated seed liquid into a bean curd residue fermentation culture medium according to the inoculation amount of 2 percent, and performing fermentation culture at 37 ℃ for 28 hours; the Bacillus subtilis is Bacillus subtilis GIM 1.286.
(4) Soaking fermented bean curd residue in 0.9% normal saline for 10min, centrifuging, removing thallus and part of bean flour precipitate, collecting supernatant, and adjusting pH to 3.
(5) Adding 4 times volume of isopropanol into the extractive solution, and precipitating in refrigerator for 12 h.
(6) Centrifuging the alcohol precipitation solution to obtain a precipitate, namely a gamma-polyglutamic acid crude extract; the yield of the prepared gamma-polyglutamic acid crude extract is 24.9%.
For comparison, soybeans were fermented under the same process conditions as the above preparation method, and the yield of the crude extract of gamma-polyglutamic acid was 15.424%.
Example 4
A method for producing gamma-polyglutamic acid by solid state fermentation of soybean curd residue is shown in figure 1, and comprises the following steps:
(1) preparation of a culture medium: firstly, preparing a culture medium of bacillus subtilis, wherein the slant culture medium comprises the following steps: 40g of LB culture medium, 20g of agar, 1L of distilled water, natural pH and 121 ℃ for 20 min; liquid culture medium: 30g of cane sugar, 10g of beef extract, 30g of sodium glutamate and MgSO4·7H2O 0.25g，K2HPO4 0.5g,CaCl21g, natural pH, 1L of distilled water; preparing a bean curd residue fermentation culture medium, adding distilled water into the dried bean curd residue, mixing uniformly, adjusting the water content of the bean curd residue to 80%, and sterilizing at 121 ℃ for 15 min.
(2) Activating and culturing the bacillus subtilis: inoculating the bacillus subtilis preserved at 4 ℃ to a slant culture medium under the aseptic condition, culturing at 37 ℃ for 24h to recover the strains, selecting seeds, inoculating the seeds to a shake flask (250mL) filled with 50mL of liquid culture medium, and culturing at 37 ℃ for 18h at 220r/min to obtain the bacillus subtilis fermented seed liquid.
(3) Fermenting and culturing bean curd residue, namely inoculating the activated seed liquid into a bean curd residue fermentation culture medium according to the inoculation amount of 2 percent, and performing fermentation culture at 37 ℃ for 28 hours; the Bacillus subtilis is Bacillus subtilis GIM 1.286.
(4) Soaking fermented bean curd residue in 0.9% normal saline for 10min, centrifuging, removing thallus and part of bean flour precipitate, collecting supernatant, and adjusting pH to 3.
(5) Adding 4 times volume of isopropanol into the extractive solution, and precipitating in refrigerator for 12 h.
(6) Centrifuging the alcohol precipitation solution to obtain a precipitate, namely a gamma-polyglutamic acid crude extract; the yield of the prepared gamma-polyglutamic acid crude extract is 20.9%.
For comparison, soybeans were fermented under the same process conditions as the above preparation method, and the yield of the crude extract of gamma-polyglutamic acid was 15.424%.
In examples 1 to 4, the yield of gamma-polyglutamic acid was compared between soybean curd refuse and soybean fermented under the same process conditions, and the results are shown in table 1, wherein the increase rate of gamma-polyglutamic acid was the increase rate of soybean curd refuse relative to the yield of gamma-polyglutamic acid obtained by soybean fermentation.
TABLE 1 comparison of the yield of gamma-polyglutamic acid
As can be seen from Table 1, the yield of gamma-polyglutamic acid can be improved by more than 90% compared with soybean with the same quality in a shorter 24-hour fermentation period and can be improved by more than 30% in a 28-hour fermentation period after the soybean curd residue is fermented. And the bean curd residue is used as a waste byproduct, so that the utilization value of the bean curd residue is improved, and the residual nutritional value of the bean curd residue is fully utilized.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, which should also be construed as a protection scope of the present invention.
Claims (8)
1. A method for producing gamma-polyglutamic acid by solid state fermentation of soybean curb residue is characterized by comprising the following steps:
step 1: recovering and activating the bacillus subtilis to obtain bacillus subtilis seed liquid;
step 2: preparing a bean curd residue fermentation culture medium, inoculating the bacillus subtilis seed solution obtained in the step 1, and performing fermentation culture to obtain a fermentation product;
and step 3: and separating the fermentation product and extracting to obtain the gamma-polyglutamic acid.
2. The method for producing gamma-polyglutamic acid by solid state fermentation of soybean curd refuse according to claim 1, wherein the step 1 is specifically: inoculating the bacillus subtilis to a slant culture medium for recovery culture to obtain strains, and then selecting the strains to inoculate the strains to a shake flask filled with a liquid culture medium for activation culture to obtain bacillus subtilis fermentation seed liquid.
3. The method for producing gamma-polyglutamic acid by solid state fermentation of soybean curd refuse according to claim 2, wherein the composition of the slant medium comprises: 40g of LB culture medium, 20g of agar, 1L of distilled water and natural pH; the composition of the liquid culture medium comprises: 30g of cane sugar, 10g of beef extract, 30g of sodium glutamate and MgSO4·7H2O 0.25g，K2HPO4 0.5g,CaCl21g, 1L of distilled water, natural pH.
4. The method for producing gamma-polyglutamic acid by solid state fermentation of soybean curd refuse according to claim 2, wherein the recovery culture conditions are: the temperature is 37 ℃, and the time is 24 h; the conditions of the activation culture are as follows: 50mL of liquid culture medium, 220r/min of shaking bottle rotation speed, 37 ℃ and 18h of time.
5. The method for producing gamma-polyglutamic acid by solid state fermentation of soybean curd residue according to claim 1, wherein the prepared soybean curd residue fermentation medium in step 2 is specifically: and mixing the dry soybean curd residue with distilled water according to the mass ratio of 1: 2-4 to obtain the soybean curd residue fermentation medium.
6. The method for producing gamma-polyglutamic acid by solid state fermentation of soybean curd refuse according to claim 1, wherein the conditions of the fermentation culture in the step 2 are: inoculating 2% of the strain, keeping the temperature at 37 ℃ for 24-28 h, and standing for fermentation.
7. The method for producing gamma-polyglutamic acid by solid state fermentation of soybean curd refuse according to claim 1, wherein the separation of step 3 is specifically: soaking the fermentation product in 0.9 wt% physiological saline water for 10min, centrifuging, and removing thallus and part of semen glycines powder precipitate to obtain supernatant; the extraction specifically comprises the following steps: adjusting the pH value of the separated supernatant to 3, then adding 4 times of isopropanol, putting the mixture into a refrigerator for precipitation for 12 hours, centrifuging the mixture, and centrifuging the precipitate to obtain the gamma-polyglutamic acid.
8. The method for producing gamma-polyglutamic acid by solid state fermentation of soybean curd residue according to any one of claims 1 to 7, wherein the Bacillus subtilis is Bacillus subtilis GIM 1.286.
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| CN114195557A (en) * | 2021-12-18 | 2022-03-18 | 蒋中领 | Method for preparing amino acid chelated organic calcium fertilizer |
| CN114854635A (en) * | 2022-05-18 | 2022-08-05 | 四川生力源生物工程有限公司 | Fermentation medium of bacillus subtilis and method for producing gamma-polyglutamic acid |
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