阅读说明：本技术 农用低成本聚谷氨酸的生产方法 (Production method of agricultural low-cost polyglutamic acid ) 是由 程文焕 程贵强 刘路 杜鹏 位凤宇 高雷 王文强 于 2019-10-31 设计创作，主要内容包括：本发明属于氨基酸生产领域,公开了农用低成本聚谷氨酸的生产方法,其包括如下步骤：1)将枯草芽孢杆菌种子液按照5-10％的接种量接入含有发酵培养基的发酵罐中,发酵30小时,然后添加产酸培养基,继续培养15h,得到聚谷氨酸发酵液；2)聚谷氨酸发酵液于4000-6000rpm的条件下离心5-15min,取上清液,加入2-4倍体积的无水乙醇,4℃条件下沉淀10-20h,再以6000-8000rpm离心10-20min,收集沉淀,真空干燥得到农用聚谷氨酸。本发明生产的聚谷氨酸产量高,成本低,纯度为90%左右,完全能够满足农用需求。(The invention belongs to the field of amino acid production, and discloses a production method of agricultural low-cost polyglutamic acid, which comprises the following steps: 1) inoculating the bacillus subtilis seed solution into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 5-10%, fermenting for 30 hours, adding an acid-producing culture medium, and continuously culturing for 15 hours to obtain a polyglutamic acid fermentation broth; 2) centrifuging the polyglutamic acid fermentation liquor for 5-15min under the conditions of 4000-6000rpm, taking supernate, adding 2-4 times of volume of absolute ethyl alcohol, precipitating for 10-20h under the condition of 4 ℃, centrifuging for 10-20min at 6000-8000rpm, collecting precipitate, and drying in vacuum to obtain the agricultural polyglutamic acid. The polyglutamic acid produced by the method has high yield, low cost and purity of about 90 percent, and can completely meet the agricultural requirements.)

1. The production method of agricultural low-cost polyglutamic acid comprises the following steps:

1) inoculating the bacillus subtilis seed solution into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 5-10%, performing fermentation culture for 30 hours, adding an acid-producing culture medium, and continuing to perform fermentation culture for 15 hours to obtain a polyglutamic acid fermentation broth; the temperature in the whole fermentation culture process is controlled at 30-34 ℃, the pH is controlled at 6.9-7.0, the stirring speed is 200-;

2) centrifuging the polyglutamic acid fermentation liquor for 5-15min under the conditions of 4000-6000rpm, taking supernate, adding 2-4 times of volume of absolute ethyl alcohol, precipitating for 10-20h under the condition of 4 ℃, centrifuging for 10-20min at 6000-8000rpm, collecting precipitate, and drying in vacuum to obtain the agricultural polyglutamic acid.

2. The production method according to claim 1, characterized in that it comprises the steps of:

1) inoculating the bacillus subtilis seed solution into a 100L fermentation tank containing 60L of fermentation medium according to the inoculation amount of 5-10%, performing fermentation culture for 30 hours, adding 10L of acid-producing medium, and continuing to perform fermentation culture for 15 hours to obtain polyglutamic acid fermentation liquor; controlling the temperature at 30 ℃, the pH at 6.9-7.0, the stirring speed at 300rpm, the ventilation quantity at 1.2vvm, controlling the glucose concentration to be not lower than 10g/L by feeding 200g/L glucose solution, and feeding a defoaming agent for defoaming;

2) centrifuging the polyglutamic acid fermentation liquor for 10min at 5000rpm, taking supernate, adding 3 times of anhydrous ethanol, precipitating for 12h at 4 ℃, centrifuging for 15min at 8000rpm, collecting precipitate, and vacuum drying to obtain the agricultural polyglutamic acid.

3. The production method according to claim 1 or 2, characterized in that hydrogen peroxide is fed into the fermentation tank at a flow rate of 2-4ml/h per liter of fermentation liquor when the fermentation time is 20 hours, until the end of the fermentation.

4. The production method according to claim 1 or 2, wherein the fermentation medium component is: 30-40g/L of glucose, 5-10g/L of sodium citrate, 10-30g/L of corn steep liquor, 10-20g/L of ammonium chloride, 20-30g/L of sodium glutamate, 3-8g/L of calcium chloride, 0.1-0.2g/L of dipotassium hydrogen phosphate, 0.1-0.2g/L of potassium dihydrogen phosphate, 0.1-0.2g/L of magnesium sulfate heptahydrate and 0.1-0.2mg/L of ferrous sulfate heptahydrate.

5. The production method according to claim 1 or 2, wherein the acidogenic medium component is: 40-80mg/L of chitosan, 10-30g/L of sodium chloride and 3-7g/L of succinic acid.

6. The production method according to claim 4, wherein the fermentation medium comprises: 35g/L glucose, 10g/L sodium citrate, 20g/L corn steep liquor, 15g/L ammonium chloride, 25g/L sodium glutamate, 5g/L calcium chloride, 0.1g/L dipotassium hydrogen phosphate, 0.1g/L potassium dihydrogen phosphate, 0.1g/L magnesium sulfate heptahydrate and 0.1mg/L ferrous sulfate heptahydrate.

7. The production method according to claim 5, wherein the acid-producing medium comprises: 50mg/L of chitosan, 20g/L of sodium chloride and 5g/L of succinic acid.

8. An agricultural polyglutamic acid obtained by the production process according to claims 1 to 7.

Technical Field

The invention belongs to the field of amino acid production, and particularly relates to a production method of agricultural low-cost polyglutamic acid.

Background

Gamma-PGA full name gamma-Polyglutamic acid is a homogeneous polypeptide (Homo-polypeptide) polymerized by using glutamic acid with left and right optical rotation as a unit body and using amide bonds on gamma-positions, and the polymerization degree is about 1,000-15,000. γ - (D, L) -PGA, γ - (D) -PGA, γ - (L) -PGA, and the like are collectively referred to as γ -PGA. γ -PGA is named NATTO GUM (NATTO GUM) in International cosmetic pharmacopoeia, also known as plant collagen, collagene vegetale, phyto collage in European Union, Japan. It is called natto gum, polyglutamic acid or polyglutamic acid in China.

Polyglutamic acid (poly-gamma-glutamic acid, abbreviated as PGA) is a water-soluble polyamino acid produced by microbial fermentation in nature, and has a structure in which a glutamic acid unit forms a high-molecular polymer of a peptide bond through an alpha-amino group and a gamma-carboxyl group. The molecular weight distribution is between 100kDa and 10000 kDa. The poly-gamma-glutamic acid has excellent water solubility, super-strong adsorbability and biodegradability, the degradation product is pollution-free glutamic acid, the poly-gamma-glutamic acid is an excellent environment-friendly high polymer material, can be used as a water-retaining agent, a heavy metal ion adsorbent, a flocculating agent, a slow-release agent, a drug carrier and the like, and has great commercial value and social value in industries such as cosmetics, environmental protection, food, medicine, agriculture, desert control and the like.

Since the discovery of polyglutamic acid has been in history for several decades, the research on polyglutamic acid is mainly in the laboratory stage, and mainly includes the research on its properties, the improvement and gene research of producing bacteria, the research on fermentation process and extraction and purification process, and the research on the production and properties of derivatives. In recent years, due to the enhancement of people's environmental consciousness and the requirement of national sustainable development strategy, it is an industrial trend to develop environment-friendly materials and develop products for improving environmental problems, which also promotes the process of industrial research and exploration of polyglutamic acid. In this century, several international well-known companies began to study the production and application of polyglutamic acid, and some domestic universities and research institutes also actively developed related studies, and more domestic enterprises began to plan the large-scale production of polyglutamic acid. Due to the follow-up of these industrial studies, polyglutamic acid is one of the most interesting biological products at the present stage.

The gamma-polyglutamic acid gamma-PGA can be used as a plant yield increasing nutrient and applied to the field of agriculture. Mainly comprises the following aspects: 1. the gamma-polyglutamic acid gamma-PGA Hydrogel and the gamma-PGA have super-strong hydrophilicity and water retention capacity, when the plants are flooded in soil, a layer of film is formed on the surface layer of plant root hairs, the function of protecting the root hairs is achieved, the optimal conveying platform for intimate contact of nutrients and water in the soil and the root hairs is provided, and dissolution, storage, conveying and absorption of fertilizers can be effectively improved. Prevent sulfate radicals, phosphate radicals and oxalate radicals from generating precipitation with metal elements, so that the crops can absorb phosphorus, calcium, magnesium and trace elements in soil more effectively. Promote the growth of crop root system and strengthen disease resistance. 2. The gamma-polyglutamic acid gamma-PGA Hydrogel and the gamma-PGA balance soil acid-base values, have excellent buffering capacity to acid and alkali, can effectively balance the soil acid-base values, and avoid acidic soil quality caused by long-term use of chemical fertilizers. 3. The gamma-polyglutamic acid can be combined with and precipitate toxic heavy metals, and has excellent chelating effect on toxic heavy metals such As Pb +2, Cu +2, Cd +2, Cr +3, Al +3, As +4 and the like. 4. The gamma-polyglutamic acid can enhance the disease resistance and stress resistance of plants, integrate plant nutrition and water active ingredients in soil, and enhance and resist symptoms caused by plant pathogens transmitted by the soil. 5. The yield is increased, the yield of agricultural products such as tea, melons, fruits, vegetables and the like can be increased rapidly, and the yield can be increased by 10-20%.

The previous patent technologies of the applicant have carried out a great deal of research on fermentation of polyglutamic acid, for example, a process for preparing, separating and purifying polyglutamic acid opens up a new strategy for improving a cheap nitrogen source in production of polyglutamic acid by hydrolyzing waste mycoprotein and utilizing crystallization mother liquor, and creates an innovative technology for producing polyamino acid by using carbon and nitrogen sources hydrolyzed by non-grain and waste biomass by combining 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. For example, the method for producing the polyglutamic acid by fermenting the waste thallus by utilizing the glutamic acid fermentation adopts two strains for mixed fermentation, and develops the waste thallus into protein hydrolysate by hydrolyzing the glutamic acid fermentation, so that the efficiency of producing the polyglutamic acid by fermentation is greatly improved, and the cost is also obviously reduced. However, the above techniques have the disadvantages that the components of the culture medium are complicated, unexpected risks are brought, and the controllability of mixed fermentation of the strains is poor. On the basis of the above results, the applicant has continued research on the fermentation process of polyglutamic acid.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a production method of agricultural low-cost polyglutamic acid.

The invention is realized by the following technical scheme.

The production method of agricultural low-cost polyglutamic acid comprises the following steps:

1) inoculating the bacillus subtilis seed solution into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 5-10%, fermenting for 30 hours, adding an acid-producing culture medium, and continuously culturing for 15 hours to obtain a polyglutamic acid fermentation broth; controlling the temperature at 30-34 ℃, the pH at 6.9-7.0, the stirring speed at 200-400rpm, the ventilation amount at 1.0-1.2vvm, controlling the glucose concentration to be not lower than 10g/L by feeding glucose solution, and feeding a defoaming agent for defoaming;

2) centrifuging the polyglutamic acid fermentation liquor for 5-15min under the conditions of 4000-6000rpm, taking supernate, adding 2-4 times of volume of absolute ethyl alcohol, precipitating for 10-20h under the condition of 4 ℃, centrifuging for 10-20min at 6000-8000rpm, collecting precipitate, and drying in vacuum to obtain the agricultural polyglutamic acid.

Further, the production method comprises the following steps:

1) inoculating the bacillus subtilis seed solution into a 100L fermentation tank containing 60L of fermentation medium according to the inoculation amount of 5-10%, fermenting for 30 hours, adding 10L of acid-producing medium, and continuously culturing for 15 hours to obtain polyglutamic acid fermentation liquor; controlling the temperature at 30 ℃, the pH at 6.9-7.0, the stirring speed at 300rpm, the ventilation quantity at 1.2vvm, controlling the glucose concentration to be not lower than 10g/L by feeding 200g/L glucose solution, and feeding a defoaming agent for defoaming;

2) centrifuging the polyglutamic acid fermentation liquor for 10min at 5000rpm, taking supernate, adding 3 times of anhydrous ethanol, precipitating for 12h at 4 ℃, centrifuging for 15min at 8000rpm, collecting precipitate, and vacuum drying to obtain the agricultural polyglutamic acid.

Preferably, when the fermentation time is 20 hours, adding hydrogen peroxide into the fermentation tank in a feeding flow manner at a flow rate of 2-4ml/h in each liter of fermentation liquid until the fermentation is finished.

Preferably, the fermentation medium components are: 30-40g/L of glucose, 5-10g/L of sodium citrate, 10-30g/L of corn steep liquor, 10-20g/L of ammonium chloride, 20-30g/L of sodium glutamate, 3-8g/L of calcium chloride, 0.1-0.2g/L of dipotassium hydrogen phosphate, 0.1-0.2g/L of potassium dihydrogen phosphate, 0.1-0.2g/L of magnesium sulfate heptahydrate and 0.1-0.2mg/L of ferrous sulfate heptahydrate.

Preferably, the acidogenic medium component is: 40-80mg/L of chitosan, 10-30g/L of sodium chloride and 3-7g/L of succinic acid.

More preferably, the fermentation medium components are: 35g/L glucose, 10g/L sodium citrate, 20g/L corn steep liquor, 15g/L ammonium chloride, 25g/L sodium glutamate, 5g/L calcium chloride, 0.1g/L dipotassium hydrogen phosphate, 0.1g/L potassium dihydrogen phosphate, 0.1g/L magnesium sulfate heptahydrate and 0.1mg/L ferrous sulfate heptahydrate.

More preferably, the acidogenic medium component is: 50mg/L of chitosan, 20g/L of sodium chloride and 5g/L of succinic acid.

Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:

the culture medium of the invention is composed of two parts, the fermentation culture medium is more important than the proliferation of the bacterial strain, and the acid-producing culture medium is beneficial to the synthesis and the secretion of the polyglutamic acid;

according to the invention, the fermentation process is optimized in multiple aspects by optimizing culture conditions such as carbon and nitrogen source types and combining metabolic flow modification and secretion mechanism, so that the cost is reduced, and the fermentation efficiency of polyglutamic acid is improved;

in the middle and later period of fermentation, the proliferation speed of the strain is slowed down, acid production is taken as the main part, amino on chitosan is combined with teichoic acid or lipopolysaccharide with negative charges in the bacterial cell wall, and metal cations are chelated, so that the permeability of the cell wall is changed, and the secretion of polyglutamic acid to the outside of cells is promoted. The sodium chloride can generate certain osmotic pressure, the strain generates stress response to a hypertonic environment, and the accumulation of the polyglutamic acid is promoted through a self-feedback mechanism.

The glyoxylate circularly consumes a large amount of ATP and causes waste of carbon sources, and the fed-in succinic acid has an inhibiting effect on isocitrate lyase, so that the flux entering the glyoxylate circular metabolism is reduced; ATP activates glutamic acid, then AMP formed by removing ppi from ATP binds with glutamic acid, and elongation of polyglutamic acid fragment is continuously formed through the action of a series of enzymes; the succinic acid is added in the middle and later stages of fermentation, so that the succinic acid has an inhibiting effect on a glyoxylate cycle pathway, and intermediate metabolites are more flowed to a tricarboxylic acid cycle pathway, so that the increase of the yield of the glutamic acid is promoted, the consumption of ATP can be reduced, and the yield of the polyglutamic acid is improved.

The invention adopts the combination of hydrogen peroxide feeding and oxygen supply by aeration in the middle and later stages of fermentation, improves the mass transfer rate of oxygen, and can improve the cell density and metabolic efficiency of a fermentation system by certain addition concentration and addition mode.

The polyglutamic acid produced by the method has high yield, low cost and purity of about 90 percent, and can completely meet the agricultural requirements.

Drawings

FIG. 1: the influence of chitosan on the yield of polyglutamic acid;

FIG. 2: the effect of sodium chloride on polyglutamic acid production;

FIG. 3: effect of succinic acid on polyglutamic acid production.

Detailed Description

Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.