CN105936924B - Novel process for preparing polyglutamic acid by using crop straws - Google Patents
Novel process for preparing polyglutamic acid by using crop straws Download PDFInfo
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Abstract
The invention belongs to the field of amino acid production, and discloses a novel process for preparing polyglutamic acid by using crop straws, which comprises the following steps: preparing crop straw hydrolysate, preparing rice bran extract in step 2), preparing waste thallus hydrolysate in step 3), concentrating mother liquor in step 4), taking fermentation medium raw materials in step 5), preparing a fermentation medium in step 6), and preparing polyglutamic acid in step 7) by fermentation. The invention carries out biochemical treatment on crop straws, combines waste thalli, improves fermentation efficiency and reduces raw material cost.
Description
Technical Field
The invention belongs to the field of amino acid production, and particularly relates to a novel process for preparing polyglutamic acid by using crop straws.
Background
Polyglutamic acid (abbreviated as PGA) is a homogeneous polypeptide formed by polymerization of glutamic acid monomers through an amide bond at the γ -position. It has water solubility, biodegradability and no toxicity, can be widely applied to the fields of food industry, cosmetics, health care, water treatment, wastewater treatment, sanitary products, medical treatment and the like, and can be used as a thickening agent, a cryoprotectant, a slow release agent, a drug carrier, a biological adhesive, a humectant, biodegradable fiber, super absorbent resin, a biological flocculant, a heavy metal ion absorbent and the like.
The prior art researches on the fermentation process of the polyglutamic acid mainly focus on strain improvement, and the improvement of a fermentation culture medium is rare. The development of the culture medium with low cost and high acid production quantity is a research direction for reducing the enterprise cost to the maximum extent. Based on the above technical problems, new hydrolysis technologies of advanced enzymatic and chemical methods are developed, and it is very likely to make breakthrough progress in preparing high-yield carbon sources and utilizable nitrogen sources.
Corn is a main grain crop planted in northern areas, and corn straws serving as agricultural wastes are generally subjected to simple crushing treatment or combustion treatment, can not be fully utilized, and are easy to cause environmental pollution. Research shows that the corn stalks contain more than 30 percent of carbohydrate, 2 to 4 percent of protein and 05 to 1 percent of fat, and can be ensiled and also can be directly fed. For herbivores, the net energy gain of 2kg of corn straws is equivalent to that of 1kg of corn grains, and particularly, after the corn straws are subjected to ensiling, yellow storage, ammoniation, saccharification and the like, the utilization rate can be improved, and the benefits can be more considerable. According to research and analysis, the digestion energy contained in the corn straws is 2235.8kJ/kg, the nutrition is rich, and the total energy is equivalent to that of pasture. The fine processing treatment of the corn straws is needed, so that the corn straws have good ecological benefit and economic benefit.
Disclosure of Invention
The invention aims to solve the defect of high cost of a fermentation culture medium in the prior art, and provides a novel process for preparing polyglutamic acid by using crop straws.
The invention is realized by the following technical scheme:
a new process for preparing polyglutamic acid by using crop straws comprises the following steps: preparing crop straw hydrolysate, preparing rice bran extract in step 2), preparing waste thallus hydrolysate in step 3), concentrating mother liquor in step 4), taking fermentation medium raw materials in step 5), preparing a fermentation medium in step 6), and preparing polyglutamic acid in step 7) by fermentation.
Specifically, the process comprises the following steps:
step 1) preparing crop straw hydrolysate: putting crop straws into a pulverizer to be pulverized, sieving the pulverized crop straws with a 100-mesh sieve, adding hydrochloric acid with the concentration of 5M and the weight of two times of the hydrochloric acid, stirring the mixture at 200rpm to hydrolyze the mixture for 6 hours, and finally adding ammonia water to adjust the pH value of the solution to 6.9-7.1 to obtain the fertilizer;
step 2) preparing a rice bran extract:
spreading rice bran into a flat layer with a thickness of 1cm, and irradiating with ultraviolet rays with an intensity of 1000uw/cm for 8min2Is then put intoAdding water with twice weight into a container, soaking for 1 hour, then adding alpha-amylase accounting for 1 percent of the weight of the rice bran, heating to 70 ℃, maintaining the temperature at 70 ℃ for hydrolysis for 1 hour, then inactivating the enzyme at 100 ℃, and finally concentrating the enzymatic hydrolysate into paste to obtain the rice bran-containing enzymatic hydrolysate;
step 3) preparing waste thallus hydrolysate: preparing glutamic acid fermentation liquor by utilizing microbial fermentation, filtering and collecting waste thalli, wherein the filtrate is used for extracting glutamic acid, and mother liquor after the glutamic acid is extracted is reserved; drying the waste thalli, crushing the waste thalli into powder by a crusher, then placing the powder into a reaction tank, adding 5mol/L hydrochloric acid, stirring and hydrolyzing for 24 hours at the temperature of 60 ℃, wherein the stirring speed is 300 r/min, neutralizing residual hydrochloric acid by ammonia water after the reaction is finished, and controlling the pH value of the solution to be 7.0 to obtain the microbial inoculum;
step 4) concentrating the mother liquor: concentrating the mother liquor obtained in the step 3) to obtain a concentrated solution with the glutamic acid content of 15 g/L;
step 5), taking fermentation medium raw materials: taking the raw materials according to the weight percentage for standby, wherein: 15% of crop straw hydrolysate, 13% of waste thallus hydrolysate, 5% of glucose, 1% of rice bran extract, 0.01% of shell powder, 0.01% of magnesium sulfate, 0.01% of potassium dihydrogen phosphate and the balance of concentrated solution obtained in the step 4);
step 6) preparing a fermentation medium: sequentially adding crop straw hydrolysate, waste thallus hydrolysate, glucose, rice bran extract, shell powder, magnesium sulfate and potassium dihydrogen phosphate into the concentrated solution, uniformly stirring, then carrying out sterilization treatment at the temperature of 108-;
step 7), fermentation preparation of polyglutamic acid: culturing bacillus subtilis to obtain a seed solution, inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 9%, and continuously fermenting for 42 hours to obtain polyglutamic acid fermentation liquor.
Preferably, the crop plant is corn.
Preferably, the particle size of the shell powder is 100 meshes.
The beneficial effects obtained by the invention mainly comprise:
the invention directly hydrolyzes the waste thalli to be used as fermentation raw materials, provides rich ammonium chloride and amino acid nitrogen sources, and can be used as microbial fermentation nutrients.
The corn stalk waste is crushed and hydrolyzed, so that nitrogen, phosphorus, potassium, calcium, magnesium, cellulose polysaccharide and the like are effectively utilized; the rice bran belongs to agricultural wastes, contains a large amount of protein, fat, sugar, vitamins and the like, but has low utilization rate of bacterial strains, and improves the leaching rate of various nutrients and the utilization rate of the bacterial strains greatly after biochemical treatment;
the novel process for producing polyglutamic acid without adding glutamic acid is established by utilizing the glutamic acid production crystallization mother liquor (containing a small amount of glutamic acid and a large amount of ammonium salt), so that the production cost is reduced, and the overall production efficiency of glutamic acid fermentation and polyglutamic acid co-production can be improved;
by hydrolyzing the waste mycoprotein and utilizing the crystallization mother liquor, a new strategy for improving the cheap nitrogen source in the production of the polyglutamic acid is developed, and an innovative technology for producing the polyamino acid by utilizing the carbon-nitrogen source hydrolyzed by non-grain and waste biomass is developed by combining the combined application of agricultural wastes such as corn straw hydrolysate and the like, so that the cost is greatly reduced, and the enterprise profit is improved.
Detailed Description
The invention will be further explained with reference to specific examples, which should not be construed as limiting the innovative teachings of the invention.
Example 1
A new process for preparing polyglutamic acid by using crop straws comprises the following steps:
step 1) preparing crop straw hydrolysate: putting crop straws into a pulverizer to be pulverized, sieving the pulverized crop straws with a 100-mesh sieve, adding hydrochloric acid with the concentration of 5M and the weight of two times of the hydrochloric acid, stirring the mixture at 200rpm to hydrolyze the mixture for 6 hours, and finally adding ammonia water to adjust the pH value of the solution to 6.9-7.1 to obtain the fertilizer; the crops are corns;
step 2) preparing a rice bran extract:
spreading rice bran into a flat layer with a thickness of 1cm, and irradiating with ultraviolet rays with an intensity of 1000uw/cm for 8min2Then put into a container, added with water with twice weight to soak for 1 hourThen, adding alpha-amylase (36U/mg, Sigma company) accounting for 1% of the weight of the rice bran, heating to 70 ℃, keeping the temperature at 70 ℃ for hydrolysis for 1 hour, then inactivating the enzyme at 100 ℃, and finally concentrating the enzymatic hydrolysate into paste to obtain the rice bran-containing enzymatic hydrolysate;
step 3) preparing waste thallus hydrolysate: preparing glutamic acid fermentation liquor by utilizing microbial fermentation, filtering and collecting waste thalli, wherein the filtrate is used for extracting glutamic acid, and mother liquor after the glutamic acid is extracted is reserved; drying the waste thalli, crushing the waste thalli into powder by a crusher, then placing the powder into a reaction tank, adding 5mol/L hydrochloric acid, stirring and hydrolyzing the mixture at the temperature of 60 ℃ for 24 hours with the stirring speed of 300 r/min based on the waste thalli, neutralizing the residual hydrochloric acid by ammonia water after the reaction is finished, and controlling the pH value of the solution to be 7.0 to obtain the microbial inoculum;
step 4) concentrating the mother liquor: concentrating the mother liquor obtained in the step 3) to obtain a concentrated solution with the glutamic acid content of 15 g/L;
step 5), taking fermentation medium raw materials: taking the raw materials according to the weight percentage for standby, wherein: 15% of crop straw hydrolysate, 13% of waste thallus hydrolysate, 5% of glucose, 1% of rice bran extract, 0.01% of shell powder, 0.01% of magnesium sulfate, 0.01% of potassium dihydrogen phosphate and the balance of concentrated solution obtained in the step 4); the particle size of the shell powder is 100 meshes;
step 6) preparing a fermentation medium: sequentially adding crop straw hydrolysate, waste thallus hydrolysate, glucose, rice bran extract, shell powder, magnesium sulfate and potassium dihydrogen phosphate into the concentrated solution, uniformly stirring, then carrying out sterilization treatment at the temperature of 108-;
step 7), fermentation preparation of polyglutamic acid: culturing Bacillus subtilis (CGMCC No. 2108) to obtain a seed solution, inoculating the seed solution into a fermentation medium according to the inoculation amount of 9 percent (volume ratio), and continuously fermenting for 42 hours to obtain polyglutamic acid fermentation liquor; the temperature in the fermentation process is controlled at 30 ℃, the pH is controlled at 6.9-7.0, and the concentration of glucose is controlled to be not less than 20 g/L.
Example 2
Example 2 the fermentation medium used was: 40g/L glucose, 25g/L corn steep liquor, 15g/L yeast extract, 20g/L sodium glutamate, 10g/L ammonium sulfate, 0.2g/L potassium dihydrogen phosphate, 0.1g/L magnesium sulfate heptahydrate and 0.1mg/L ferrous sulfate; the other processes were the same as in example 1.
Example 3
The specific results of the polyglutamic acid yield in the fermentation broth of examples 1-2 of the present invention are shown in Table 1:
TABLE 1
| Group of | Polyglutamic acid yield (g/L) |
| Example 1 | 34.2 |
| Example 2 | 32.3 |
And (4) conclusion: the acid yield difference between the group 1 of the embodiment of the invention and the group 2 of the embodiment of the invention is not large, and the group 1 of the embodiment of the invention is slightly higher; the cost of the fermentation culture medium in the embodiment 1 of the invention only accounts for about 40% of the cost of the culture medium in the embodiment 2 through cost verification, and the waste is changed into valuable, so that the investment of enterprises is saved, and the net income of the enterprises is improved.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (2)
1. A process for preparing polyglutamic acid by using crop straws comprises the following steps:
step 1) preparing crop straw hydrolysate: putting crop straws into a pulverizer to be pulverized, sieving the pulverized crop straws with a 100-mesh sieve, adding hydrochloric acid with the concentration of 5M and the weight of two times of the hydrochloric acid, stirring the mixture at 200rpm to hydrolyze the mixture for 6 hours, and finally adding ammonia water to adjust the pH value of the solution to 6.9-7.1 to obtain the fertilizer;
step 2) preparing a rice bran extract:
spreading rice bran into a flat layer with a thickness of 1cm, and irradiating with ultraviolet rays with an intensity of 1000uw/cm for 8min2Putting the mixture into a container, adding water with twice weight, soaking for 1 hour, then adding alpha-amylase accounting for 1 percent of the weight of the rice bran, heating to 70 ℃, maintaining the temperature at 70 ℃ for hydrolysis for 1 hour, then inactivating the enzyme at 100 ℃, and finally concentrating the enzymatic hydrolysate into paste to obtain the rice bran-containing enzymatic hydrolysate;
step 3) preparing waste thallus hydrolysate: preparing glutamic acid fermentation liquor by utilizing microbial fermentation, filtering and collecting waste thalli, wherein the filtrate is used for extracting glutamic acid, and mother liquor after the glutamic acid is extracted is reserved; drying the waste thalli, crushing the waste thalli into powder by a crusher, then placing the powder into a reaction tank, adding 5mol/L hydrochloric acid, stirring and hydrolyzing for 24 hours at the temperature of 60 ℃, wherein the stirring speed is 300 r/min, neutralizing residual hydrochloric acid by ammonia water after the reaction is finished, and controlling the pH value of the solution to be 7.0 to obtain the microbial inoculum;
step 4) concentrating the mother liquor: concentrating the mother liquor obtained in the step 3) to obtain a concentrated solution with the glutamic acid content of 15 g/L;
step 5), taking fermentation medium raw materials: taking the raw materials according to the weight percentage for standby, wherein: 15% of crop straw hydrolysate, 13% of waste thallus hydrolysate, 5% of glucose, 1% of rice bran extract, 0.01% of shell powder, 0.01% of magnesium sulfate, 0.01% of potassium dihydrogen phosphate and the balance of concentrated solution obtained in the step 4);
step 6) preparing a fermentation medium: sequentially adding crop straw hydrolysate, waste thallus hydrolysate, glucose, rice bran extract, shell powder, magnesium sulfate and potassium dihydrogen phosphate into the concentrated solution, uniformly stirring, then carrying out sterilization treatment at the temperature of 108-;
step 7), fermentation preparation of polyglutamic acid: bacillus subtilis CGMCC No.2108 to obtain seed liquid, inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 9%, and continuously fermenting for 42 hours to obtain polyglutamic acid fermentation liquid;
the crop is corn.
2. The process according to claim 1, wherein the shell powder has a particle size of 100 mesh.
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| CN100999745A (en) * | 2006-12-18 | 2007-07-18 | 浙江大学 | Process of preparing gamma-poly glutaminic acid |
| CN101109010A (en) * | 2007-07-17 | 2008-01-23 | 秦皇岛领先科技发展有限公司 | Mycopremna generating gamma- polyglutamic acid and culturing method thereof |
| CN101979627A (en) * | 2010-10-08 | 2011-02-23 | 天津科技大学 | Method for preparing glutamic acid fermentation organic nitrogen additive from glutamic acid fermentation waste thalli |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100999745A (en) * | 2006-12-18 | 2007-07-18 | 浙江大学 | Process of preparing gamma-poly glutaminic acid |
| CN101109010A (en) * | 2007-07-17 | 2008-01-23 | 秦皇岛领先科技发展有限公司 | Mycopremna generating gamma- polyglutamic acid and culturing method thereof |
| CN101979627A (en) * | 2010-10-08 | 2011-02-23 | 天津科技大学 | Method for preparing glutamic acid fermentation organic nitrogen additive from glutamic acid fermentation waste thalli |
Non-Patent Citations (2)
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