CN114774488A - Production method of low-endotoxin gamma-polyglutamic acid - Google Patents
Production method of low-endotoxin gamma-polyglutamic acid Download PDFInfo
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Abstract
The invention relates to the technical field of microbial fermentation, in particular to a production method of low-endotoxin gamma-polyglutamic acid, which comprises the following steps: s1, preparing gamma-polyglutamic acid fermentation liquor; s2, carrying out acid treatment on the gamma-polyglutamic acid fermentation liquor to adjust the pH value to 3.0-5.0, carrying out high-temperature treatment, cooling, decoloring and filtering to obtain a filtrate; s3, adding acid into the filtrate to adjust the pH value to 1.0-3.0, standing to separate out a precipitate, and pumping away supernatant; s4, adding an organic solvent into the bottom sediment, stirring and precipitating, pumping the organic solvent on the upper layer, and drying the precipitate to obtain the organic compound. The method is not easy to be polluted by microorganisms in the process of treating the endotoxin, has simple process and is suitable for industrial production, and the content of the endotoxin in the production of the gamma-polyglutamic acid is effectively reduced.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a production method of low-endotoxin gamma-polyglutamic acid.
Background
The gamma-polyglutamic acid can be dissolved in water, can be biologically degraded, is edible, has no toxicity to human bodies and has no pollution to the environment. Nowadays, the biodegradable functional material is favored by people with the emphasis on environmental protection and sustainable development, and is gradually applied to medicine manufacturing, food processing, freezing prevention and fresh keeping of vegetables, fruits and marine products, development and application in many fields such as cosmetic industry, tobacco and leather manufacturing industry, plant seed protection and the like, and is a novel multifunctional biological product with great development value and wide prospect.
In order to use gamma-polyglutamic acid in the cosmetic industry and pharmaceutical manufacturing, it is necessary to ensure that gamma-polyglutamic acid has endotoxin at a sufficiently low concentration. Endotoxins are lipopolysaccharides that are present on the surface of the outer membrane of gram-negative bacteria. Endotoxins are highly toxic to mammals, especially humans, and are notoriously difficult to remove from materials. Endotoxin can become a pyrogen when it is released into the blood or other tissues where it is not normally found. Thus, endotoxins must be removed from pharmaceutically acceptable products.
The pyrogen removal technology for endotoxin-containing materials includes ion exchange chromatography, ultrafiltration, distillation, and various chemical methods aimed at destroying endotoxin, which have technical defects such as secondary microbial contamination, complicated process, and the like.
Disclosure of Invention
Aiming at the problems of secondary pollution, complex process and the like of the endotoxin removal method in the prior art, the invention provides the production method of the low-endotoxin-containing gamma-polyglutamic acid, the endotoxin treatment process is not easy to be polluted by microorganisms, the process is simple and suitable for industrial production, and the content of endotoxin in the production of the gamma-polyglutamic acid is effectively reduced.
The invention provides a method for producing gamma-polyglutamic acid containing low endotoxin, which comprises the following steps:
s1, preparing gamma-polyglutamic acid fermentation liquor;
s2, carrying out acid treatment on the gamma-polyglutamic acid fermentation liquor to adjust the pH value to 3.0-5.0, carrying out high-temperature treatment, cooling, decoloring and filtering to obtain a filtrate;
s3, adding acid into the filtrate to adjust the pH value to 1.0-3.0, standing to separate out a precipitate, and pumping out the supernatant;
s4, adding an organic solvent into the bottom sediment, stirring and precipitating, pumping the organic solvent on the upper layer, and drying the precipitate to obtain the organic compound.
Further, the gamma-polyglutamic acid fermentation liquor is prepared by fermenting Bacillus subtilis FRD518 serving as a production strain, wherein the Bacillus subtilis FRD518 is preserved in China general microbiological culture Collection center (CGMCC) No.6772 in 11/02/2012, and the strain is disclosed by the applicant's prior application patent CN 201210555304.5.
Further, the preparation method of the gamma-polyglutamic acid fermentation liquor comprises the following steps:
(1) inoculating bacillus subtilis FRD518 into a seed culture medium, wherein the inoculation amount is 1%, and performing shake culture at 35-38 ℃ and 180-220 r/min in a shaking table until OD600 reaches 1.6-2.2 to prepare a seed culture solution;
(2) inoculating the seed culture solution into a fermentation culture medium, wherein the inoculation amount is 2-5%, and culturing in a fermentation tank at 35-38 ℃ for 42-54 h under ventilation and stirring to obtain the gamma-polyglutamic acid fermentation liquor.
Further, the seed culture medium comprises the following components in concentration: 5-10 g/L glucose, 3-5 g/L yeast powder and 2-10 g/L, MgSO ammonium sulfate40.1-1.0 g/L, the pH of the culture medium is 7.2-7.5, and the culture medium is sterilized at 115 ℃ for 30 min.
Further, the fermentation medium is composed of the following components in concentration: 5-30 g/L peptone, 5-30 g/L yeast extract, 5-20 g/L ammonium sulfate, 30-100 g/L sodium glutamate and 2-5 g/L, MgSO citric acid40.1～0.5g/L、K2HPO41-4 g/L glucose 100-200 g/L, pH of the culture medium 7.2 ∞7.5, sterilizing at 115 ℃ for 30 min.
Further, in step S2, the acid is concentrated hydrochloric acid; the high-temperature treatment process conditions are that the treatment is carried out for 0.5 to 3 hours at the temperature of 121 ℃ and under the pressure of 0.1 MPa; the temperature of the fermentation liquor after cooling is 60 ℃.
Further, the decolorizing agent adopted for decolorizing is activated carbon, the adding amount of the activated carbon is 1% (w/v), the filtering adopts plate-and-frame filtering, and the filter aid added during the filtering is diatomite.
Further, in step S4, the organic solvent is absolute ethanol.
The invention has the beneficial effects that:
(1) in the process of treating the fermentation liquor, the invention adopts the high-temperature and acid degradation process to inactivate endotoxin and simultaneously reduce the molecular weight of polyglutamic acid so as to realize the production of polyglutamic acid with different molecular weights;
(2) the invention keeps the acid environment by adjusting the pH value in the treatment process, can prevent the pollution of microorganisms, avoids the secondary pollution of endotoxin in the treatment process and is suitable for industrial production;
(3) the invention utilizes the characteristic that only the gamma-polyglutamic acid is precipitated and separated out at a lower pH value to achieve the aim of separating and purifying the high-purity gamma-polyglutamic acid, and simultaneously, the recovery rate of the product is improved because the gamma-polyglutamic acid is completely separated out at the lower pH value;
(4) the gamma-polyglutamic acid prepared by the invention has good stability, is not easy to be polluted by microorganisms, and effectively reduces the generation of endotoxin.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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.
Example 1
The production method of the low endotoxin-containing gamma-polyglutamic acid comprises the following steps:
s1, preparation of Gamma-polyglutamic acid fermentation liquor
(1) Seed culture: inoculating bacillus subtilis FRD518 into a triangular flask filled with a seed culture medium, wherein the seed culture medium comprises the following components in concentration: 5g/L glucose, 3g/L yeast powder and 2g/L, MgSO ammonium sulfate40.1g/L, the pH value of the culture medium is 7.2, the culture medium is obtained by sterilizing for 30min at the temperature of 115 ℃, the inoculation amount is 1 percent, and the culture medium is shake-cultured in a shaking table at the temperature of 35 ℃ and 180r/min until the OD600 reaches 1.6;
(2) fermentation culture: inoculating the seed solution prepared in the step (1) into a fermentation medium, wherein the fermentation medium comprises the following components in concentration: 5g/L peptone, 5g/L yeast extract, 5g/L ammonium sulfate, 50g/L sodium glutamate, and 2g/L, MgSO citric acid40.1g/L、K2HPO41g/L, 100g/L glucose and 7.2 pH of culture medium, sterilizing at 115 deg.C for 30min, inoculating 2%, and culturing in a fermentation tank at 35 deg.C for 42h under aeration and stirring to obtain gamma-polyglutamic acid fermentation broth;
s2, processing fermentation liquor
Adjusting pH of the gamma-polyglutamic acid fermentation liquor to 5.0 by using concentrated hydrochloric acid, treating the gamma-polyglutamic acid fermentation liquor at the temperature of 121 ℃ and the pressure of 0.1MPa for 0.5h at high temperature, cooling circulating water to 60 ℃, adding 1% (w/v) of activated carbon, stirring and decoloring the mixture for 1h, then adding diatomite and starting plate-frame filtration to obtain filtrate;
s3, precipitating
Adjusting pH of the filtrate to 1.0 with concentrated hydrochloric acid, controlling hydrochloric acid addition speed to prevent rapid particle size increase, standing for 12 hr to precipitate, and removing supernatant;
s4, dehydrating and drying
Adding absolute ethyl alcohol into the bottom precipitate, stirring for 1 hour, standing until the materials are completely precipitated, pumping away the ethyl alcohol, vacuum drying the materials, discharging, packaging, and temporarily storing to obtain the finished product. The molecular weight of the gamma-polyglutamic acid obtained in the embodiment is 600-800 KDa through gel method, and the content of endotoxin is 55EU/g through gel method.
Example 2
The production method of the low endotoxin-containing gamma-polyglutamic acid comprises the following steps:
s1, preparation of Gamma-polyglutamic acid fermentation liquor
(1) Seed culture: inoculating bacillus subtilis FRD518 into a triangular flask filled with a seed culture medium, wherein the seed culture medium comprises the following components in concentration: 7.5g/L glucose, 4g/L yeast powder and 6g/L, MgSO ammonium sulfate40.5g/L, the pH value of the culture medium is 7.3, the culture medium is obtained by sterilizing for 30min at the temperature of 115 ℃, the inoculation amount is 1 percent, and the culture medium is shake-cultured by a shaking table at the temperature of 36 ℃ and 200r/min until the OD600 reaches 2.0;
(2) fermentation culture: inoculating the seed liquid prepared in the step (1) into a fermentation medium, wherein the fermentation medium comprises the following components in concentration: 15g/L of peptone, 15g/L of yeast extract, 15g/L of ammonium sulfate, 80g/L of sodium glutamate and 3g/L, MgSO of citric acid40.3g/L、K2HPO42.0g/L, 150g/L glucose, pH 7.3, sterilizing at 115 deg.C for 30min to obtain fermentation broth, inoculating 4%, and culturing in a fermentation tank at 36 deg.C under aeration and stirring for 43 hr to obtain gamma-polyglutamic acid fermentation broth;
s2, processing fermentation liquor
Adjusting pH of the gamma-polyglutamic acid fermentation liquor to 4.0 with concentrated hydrochloric acid, treating at 121 deg.C and 0.1MPa for 1.5h, cooling with circulating water to 60 deg.C, adding 1% (w/v) active carbon, stirring for decolorizing for 1h, adding diatomaceous earth, and filtering with plate frame to obtain filtrate;
s3, precipitating
Adjusting pH of the filtrate to 2.0 with concentrated hydrochloric acid, controlling hydrochloric acid addition speed to prevent rapid particle size increase, standing for 18 hr to precipitate, and removing supernatant;
s4, dehydrating and drying
Adding absolute ethyl alcohol into the bottom precipitate, stirring for 1 hour, standing until the materials are completely precipitated, pumping away the ethyl alcohol, vacuum drying the materials, discharging, packaging, and temporarily storing to obtain the finished product. The molecular weight of the gamma-polyglutamic acid obtained in the example was determined by gel method to be 100kDa-300kDa, and the content of endotoxin was determined by gel method to be 45 EU/g.
Example 3
The production method of the gamma-polyglutamic acid containing low endotoxin comprises the following steps:
s1, preparation of Gamma-polyglutamic acid fermentation liquor
(1) Seed culture: inoculating bacillus subtilis FRD518 into a triangular flask filled with a seed culture medium, wherein the seed culture medium comprises the following components in concentration: 10g/L glucose, 5g/L yeast powder and 10g/L, MgSO ammonium sulfate41.0g/L, the pH of the culture medium is 7.5, the culture medium is obtained by sterilizing for 30min at the temperature of 115 ℃, the inoculum size is 1 percent, and shaking culture is carried out on a shaking table at 220r/min at the temperature of 38 ℃ until the OD600 reaches 2.2;
(2) fermentation culture: inoculating the seed liquid prepared in the step (1) into a fermentation medium, wherein the fermentation medium comprises the following components in concentration: 30g/L of peptone, 30g/L of yeast extract, 20g/L of ammonium sulfate, 100g/L of sodium glutamate and 5g/L, MgSO of citric acid40.5g/L、K2HPO44.0g/L, 200g/L glucose, pH 7.5 of culture medium, sterilizing at 115 ℃ for 30min, inoculating 5%, and culturing in a fermentation tank at 38 ℃ for 54h under aeration and stirring to obtain gamma-polyglutamic acid fermentation broth;
s2, processing fermentation liquor
Adjusting pH of the gamma-polyglutamic acid fermentation liquor to 5.0 by using concentrated hydrochloric acid, performing high-temperature treatment for 3h at the temperature of 121 ℃ and the pressure of 0.1MPa, cooling by circulating water, adding 1% (w/v) of activated carbon when the temperature is reduced to 60 ℃, stirring and decoloring for 1h, then adding diatomite, and starting plate-and-frame filtration to obtain filtrate;
s3, precipitating
Adjusting pH of the filtrate to 3.0 with concentrated hydrochloric acid, controlling hydrochloric acid addition speed to prevent rapid particle size increase, standing for 24 hr to precipitate, and removing supernatant;
s4, dehydrating and drying
Adding absolute ethyl alcohol into the bottom precipitate, stirring for 1 hour, standing until the materials are completely precipitated, pumping away the ethyl alcohol, vacuum drying the materials, discharging, packaging, and temporarily storing to obtain the finished product. The molecular weight of the gamma-polyglutamic acid obtained in the example was 5kDa to 30kDa as determined by gel method, and the content of endotoxin was 39EU/g as determined by gel method.
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions should be within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present disclosure and the scope of the present invention.
Claims (8)
1. The production method of the low endotoxin-containing gamma-polyglutamic acid is characterized by comprising the following steps of:
s1, preparing gamma-polyglutamic acid fermentation liquor;
s2, carrying out acid treatment on the gamma-polyglutamic acid fermentation liquor to adjust the pH value to 3.0-5.0, carrying out high-temperature treatment, cooling, decoloring and filtering to obtain a filtrate;
s3, adding acid into the filtrate to adjust the pH value to 1.0-3.0, standing to separate out a precipitate, and pumping away supernatant;
and S4, adding an organic solvent into the bottom precipitate, stirring and precipitating, pumping the upper-layer organic solvent, and drying the precipitate to obtain the organic silicon fertilizer.
2. The method for producing gamma-polyglutamic acid with low endotoxin as claimed in claim 1, wherein the gamma-polyglutamic acid fermentation broth is prepared by fermenting Bacillus subtilis FRD518 as a production strain, the Bacillus subtilis FRD518 is preserved in China general microbiological culture Collection center at 11.02.2012, the preservation address is Beijing city rising area north Chen Xilu No. 1 Hospital No. 3, and the preservation number is CGMCC No. 6772.
3. The method for producing gamma-polyglutamic acid with low endotoxin of claim 2, wherein the preparation method of the gamma-polyglutamic acid fermentation broth comprises:
(1) inoculating bacillus subtilis FRD518 into a seed culture medium, wherein the inoculation amount is 1%, and performing shake culture at 35-38 ℃ and 180-220 r/min in a shaking table until OD600 reaches 1.6-2.2 to prepare a seed culture solution;
(2) inoculating the seed culture solution into a fermentation culture medium, wherein the inoculation amount is 2-5%, and culturing in a fermentation tank at 35-38 ℃ for 42-54 h under ventilation and stirring to obtain the gamma-polyglutamic acid fermentation liquor.
4. The method for producing low endotoxin-containing gamma-polyglutamic acid as claimed in claim 3, wherein, in the step (1), the seed culture medium is composed of the following components in the following concentrations: 5-10 g/L glucose, 3-5 g/L yeast powder and 2-10 g/L, MgSO ammonium sulfate40.1-1.0 g/L, the pH of the culture medium is 7.2-7.5, and the culture medium is sterilized at 115 ℃ for 30 min.
5. The process for producing gamma-polyglutamic acid with low endotoxin as claimed in claim 3, wherein, in the step (2), the fermentation medium is composed of the following components in the following concentrations: 5-30 g/L peptone, 5-30 g/L yeast extract, 5-20 g/L ammonium sulfate, 30-100 g/L sodium glutamate and 2-5 g/L, MgSO citric acid40.1～0.5g/L、K2HPO41-4 g/L glucose, 100-200 g/L glucose, and 7.2-7.5 pH of culture medium, and sterilizing at 115 deg.C for 30 min.
6. The method for producing γ -polyglutamic acid with low endotoxin of claim 1, wherein in step S2, the acid is concentrated hydrochloric acid; the high-temperature treatment process conditions are that the treatment is carried out for 0.5 to 3 hours at the temperature of 121 ℃ and under the pressure of 0.1 MPa; the temperature of the fermentation liquor after cooling is 60 ℃.
7. The method for producing gamma-polyglutamic acid with low endotoxin as claimed in claim 1, wherein the decolorizing agent used for decolorizing is activated carbon, the adding amount of the activated carbon is 1% (w/v), the filtration is performed by plate-and-frame filtration, and the filter aid added during the filtration is diatomite.
8. The method of claim 1, wherein the organic solvent is absolute ethanol in step S4.
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