A soil conditioner for improving saline-alkali soil and its preparation method and application

Abstract

The present invention provides a soil conditioner for improving saline-alkali soil, and a preparation method and application thereof, and belongs to the technical field of biochemistry. The present invention provides a method for synthesizing glutamine, wherein Leuconostoc mesenteroides is subjected to anaerobically fermented culture in a fermentation medium containing its lysate and sodium glutamate to obtain a fermentation broth containing glutamine. The present invention provides a method for synthesizing γ-polyglutamic acid, wherein Bacillus coagulans is inoculated into a fermentation broth containing glutamine for aerobic fermentation to obtain a fermentation broth containing γ-polyglutamic acid. The present invention provides a method for preparing a soil conditioner, wherein a fermentation broth containing γ-polyglutamic acid is subjected to an esterification reaction with alcohols to obtain a soil conditioner. The obtained soil conditioner can significantly improve the physical and chemical properties of saline-alkali soil, improve the soil's ability to retain water and fertilizer, reduce soil salinity, promote plant growth, enhance soil microbial activity, and provide an effective solution for saline-alkali land improvement.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and in particular relates to a soil conditioner for improving saline-alkali soil, a preparation method and application thereof. Background Art

[0002] In recent years, soil salinization is one of the main problems of soil degradation. Saline-alkali soil (saline soil) is a general term for saline soil, alkaline soil, salinized and alkaline soil. Great progress has been made in improving saline-alkali land through physical measures, water conservancy engineering measures, biological measures and chemical measures. However, the salt content in saline-alkali soil is constantly changing with the influence of climate change, human activities, external pollution and other factors, and continuous improvement is still needed to improve the utilization rate of saline-alkali soil.

[0003] Glutamine is not only one of the basic amino acids for protein synthesis, but also an amino donor for many important compounds that have an ameliorative effect on saline-alkali soil, such as γ-polyglutamic acid. At present, the main production method of glutamine is microbial fermentation, which has mild reaction conditions and abundant product raw materials, and has gradually replaced the traditional enzymatic method and chemical synthesis method. People have done a lot of research on screening glutamine-producing bacteria from glutamate-producing bacteria. In fact, many of the current glutamine-producing bacteria are transformed from glutamate-producing bacteria Corynebacterium glutamicum, but their glutamine synthesis effect is still limited, and the yield of glutamine still needs to be further improved. Summary of the invention

[0004] In view of the defects in the prior art, the purpose of the present invention is to provide a method for synthesizing glutamine, which can significantly improve the synthesis effect of glutamine and increase the yield of glutamine; at the same time, the synthesized glutamine can be further used to prepare γ-polyglutamic acid, and then the γ-polyglutamic acid can be used to prepare a soil conditioner with a significant improvement effect on saline-alkali soil.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a method for synthesizing glutamine, comprising the following steps:

[0007] Leuconostoc mesenteroides ( Leuconostoc mesenteroides ) performing anaerobic fermentation in a fermentation medium containing Leuconostoc mesenteroides lysate, sodium glutamate and whey protein to obtain a fermentation liquid containing glutamine;

[0008] The content of sodium glutamate in the fermentation medium is 30~60g / L, and the content of whey protein is 3~6g / L;

[0009] The Leuconostoc mesenteroides lysate was prepared by adding 100 to 200 Leuconostoc mesenteroides to each liter of fermentation medium.

[0010] Preferably, the Leuconostoc mesenteroides includes Leuconostoc mesenteroides NTM048, and the preservation number of the Leuconostoc mesenteroides NTM048 is NITE BP-1519.

[0011] Preferably, the Leuconostoc mesenteroides lysate is obtained by high-pressure homogenization of Leuconostoc mesenteroides.

[0012] Preferably, the temperature of the anaerobic fermentation culture is 25-45° C.; the time of the anaerobic fermentation culture is 6-10 days; and the pH value of the anaerobic fermentation culture is 6.0-7.0.

[0013] The present invention provides a method for synthesizing γ-polyglutamic acid, comprising the following steps:

[0014] The fermentation liquid containing glutamine prepared by the method described in the above technical solution is used as the fermentation substrate;

[0015] After adding oxygen carriers to the fermentation medium, inoculation of Bacillus coagulans ( Bacillus coagulans ) for aerobic fermentation to obtain a fermentation solution containing γ-polyglutamic acid.

[0016] Preferably, the oxygen carrier comprises n-hexadecane; the amount of the oxygen carrier added is 0.3% to 1% of the volume of the fermentation substrate;

[0017] The temperature of the aerobic fermentation is 45-50° C.; the time of the aerobic fermentation is 10-15 days; and the pH value of the aerobic fermentation is 6.6-7.0.

[0018] The present invention provides a method for preparing a soil conditioner, comprising the following steps:

[0019] The fermentation bacteria liquid containing gamma-polyglutamic acid prepared by the synthesis method described in the above technical scheme is mixed with alcohols and a catalyst, and then subjected to an esterification reaction to obtain a soil conditioner.

[0020] Preferably, the alcohol includes any one or more of glycerol, ethanol and lactic acid; the catalyst includes any one or more of toluenesulfonic acid, sulfuric acid, carbonic acid and citric acid;

[0021] The temperature of the esterification reaction is 60-70° C.; the time of the esterification reaction is 24-48 hours.

[0022] The present invention provides a soil conditioner prepared by the preparation method described in the above technical solution.

[0023] The present invention provides the use of the soil conditioner described in the above technical solution in improving saline-alkali soil.

[0024] Beneficial effects of the present invention

[0025] The present invention provides a method for synthesizing glutamine, comprising the following steps: anaerobic fermentation and culturing of Leuconostoc mesenteroides in a fermentation medium containing Leuconostoc mesenteroides lysate, sodium glutamate and whey protein to obtain a fermentation liquid containing glutamine; the content of sodium glutamate in the fermentation medium is 30-60 g / L, and the content of whey protein is 3-6 g / L; Leuconostoc mesenteroides lysate prepared by adding 100-200 Leuconostoc mesenteroides per liter of fermentation medium. In the present invention, the Leuconostoc mesenteroides lysate contains glutamine synthetase. Glutamine synthetase is an enzyme that catalyzes the reaction of glutamate and ammonia to generate glutamine. In Leuconostoc mesenteroides, glutamine synthetase can utilize ammonia in the environment or ammonia generated by cell metabolism to react with glutamate to generate glutamine. The present invention adds its own lysate to the culture medium of Leuconostoc mesenteroides, adds sodium glutamate at the same time, and then performs anaerobic fermentation to control the fermentation process conditions, which is conducive to the growth of Leuconostoc mesenteroides and the synthesis of glutamine. At the same time, Leuconostoc mesenteroides can metabolize and synthesize more efficiently in an anaerobic environment, thereby further helping to increase the production of glutamine. After 6 to 10 days of anaerobic fermentation, a large amount of glutamine can be detected, and its content can be as high as 40 g / L or more.

[0026] Furthermore, the present invention provides a method for synthesizing γ-polyglutamic acid, which facilitates the further synthesis of γ-polyglutamic acid (γ-PGA) by adding Bacillus coagulans to the anaerobic fermentation culture solution for aerobic fermentation, thereby achieving high yield of γ-PGA.

[0027] The present invention also provides a soil conditioner and a method for preparing the soil conditioner. The soil conditioner is prepared by esterification reaction of the γ-polyglutamic acid described in the above technical scheme with an alcohol substance. In the present invention, the soil conditioner includes esters containing γ-PGA, γ-polyglutamic acid, glutamine, alcohol substances, Leuconostoc mesenteroides and Bacillus coagulans. In the present invention, the γ-polyglutamic acid (γ-PGA) is a biopolymer with multiple application potentials. The esters containing γ-PGA prepared by esterification reaction can improve its hydrophobicity, thereby broadening its application range. After the esterification reaction, the generated product is an ester containing γ-PGA, which has improved thermal stability and processing performance. Esters containing γ-PGA have multiple benefits. They have better water retention, can enhance the water retention capacity of the soil, help the soil stay moist under drought conditions, promote the formation of a more stable agglomeration structure of soil particles, improve the porosity of the soil, and thus improve the ventilation and drainage capacity of the soil; γ-polyglutamic acid and / or esters containing γ-PGA, as biopolymers, can increase the organic matter content in the soil and help improve soil fertility; because γ-polyglutamic acid and / or esters containing γ-PGA are biodegradable, their decomposition in the soil will not have a negative impact on the environment, and they are an environmentally friendly soil conditioner. In the present invention, glutamine in the soil conditioner as an organic nitrogen source can promote soil microbial activity, help to form a good soil structure, improve soil ventilation and water retention capacity, and can also be used as a component of fertilizer to promote plant growth; γ-PGA in the soil conditioner helps to reduce the salt concentration in the soil solution, reduce the direct damage of salt to plant roots, and promote the growth of plants in saline-alkali soils; alcohol substances in the soil conditioner can combine with salt in the soil to form a complex, thereby reducing the salt concentration in the soil and reducing the impact of salt damage on plants. Saline-alkali soil has a compact structure, poor air permeability and water permeability, which is not conducive to the development of plant roots. Alcohol substances in soil conditioners can promote the coagulation of soil particles, improve soil structure, and increase porosity and air permeability. High salt and alkaline conditions in saline-alkali soils inhibit the activity of soil microorganisms, while alcohol substances, such as glycerol, lactic acid, etc., as a carbon source, can stimulate the growth of beneficial microorganisms and promote the activity of soil microorganisms, which is crucial for the recovery and improvement of saline-alkali soil fertility. The application of soil conditioners containing alcohol substances can alleviate the salinization process of saline-alkali soil, and by improving soil structure and promoting microbial activity, it helps to reduce soil salinity and alkalinity, and improve the crop growth environment. The Bacillus coagulans in the soil conditioner provided by the present invention has nitrogen fixation ability, and Leuconostoc mesenteroides may help release nutrients such as phosphorus and potassium, improve the effectiveness of nutrients in the soil, and provide them for plant absorption and utilization. It enhances the buffering capacity of the soil against acid-base changes and alleviates the soil acidification or alkalinization problems caused by agricultural activities such as fertilization.In summary, the soil conditioner provided by the present invention can change the distribution pattern of soil moisture, so that more moisture is accumulated in the soil layer area of ​​the crop root zone, which helps the crops to better absorb water and nutrients and promote the growth of crops. It can also improve the physical and chemical properties and soil structure of saline-alkali soil, reduce the pH value and sodium adsorption ratio (SAR) of the soil, thereby reducing the salt content of the soil and improving the permeability of the soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0029] Figure 1 Flow chart for preparing soil conditioner of the present invention. DETAILED DESCRIPTION

[0030] The present invention provides a method for synthesizing glutamine, comprising the following steps:

[0031] Leuconostoc mesenteroides ( Leuconostoc mesenteroides ) performing anaerobic fermentation in a fermentation medium containing Leuconostoc mesenteroides lysate, sodium glutamate and whey protein to obtain a fermentation liquid containing glutamine;

[0032] The content of sodium glutamate in the fermentation medium is 30~60g / L, and the content of whey protein is 3~6g / L;

[0033] The Leuconostoc mesenteroides lysate was prepared by adding 100 to 200 Leuconostoc mesenteroides to each liter of fermentation medium.

[0034] In the present invention, the Leuconostoc mesenteroides may include Leuconostoc mesenteroides NTM048; the accession number of the Leuconostoc mesenteroides NTM048 is NITE BP-1519; the Leuconostoc mesenteroides lysate may include a lysate of the Leuconostoc mesenteroides NTM048 strain. The present invention does not specifically limit the preparation method of the Leuconostoc mesenteroides lysate, and any conventional cell lysis method in the art may be used. In the present invention, the Leuconostoc mesenteroides lysate is obtained by high-pressure homogenization of Leuconostoc mesenteroides. The Leuconostoc mesenteroides lysed in the present invention is preferably obtained by streaking culture. The present invention does not specifically limit the method for streaking culture of the Leuconostoc mesenteroides, and any conventional culture method in the art may be used. In the present invention, the Leuconostoc mesenteroides streak culture is preferably carried out in a beef extract peptone medium. In the present invention, the temperature of the streaking culture may be 35-40°C. As an optional embodiment of the present invention, the temperature of the streaking culture may be 35°C, 36°C, 37°C, 38°C, 39°C or 40°C. In the present invention, the streaking culture time may be 24 to 48 hours. As an optional embodiment of the present invention, the streaking culture time may be 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h or 48h. After the streaking culture is completed, the present invention preferably takes the colony of Leuconostoc mesenteroides to separate and obtain 100 to 200 Leuconostoc mesenteroides for lysis. As an optional embodiment of the present invention, the amount of Leuconostoc mesenteroides used for lysis may be 100, 120, 140, 160, 180 or 200. After obtaining the Leuconostoc mesenteroides for lysis, the present invention preferably washes the Leuconostoc mesenteroides. The washing of the present invention is preferably carried out using phosphate buffer. The present invention does not specifically limit the number of washings, as long as the residual culture medium components can be removed. As an optional embodiment of the present invention, the number of washings may be 1 to 2 times. After washing, the present invention preferably uses a buffer solution containing EDTA to resuspend the intestinal membrane Leuconostoc cells to obtain a cell suspension. After obtaining the cell suspension, the present invention preferably performs high-pressure homogenization on the cell suspension; the high-pressure homogenization is preferably performed in a high-pressure homogenizer; the pressure of the high-pressure homogenization may be 10,000 to 20,000 psi. As an optional embodiment of the present invention, the pressure of the high-pressure homogenization may be 10,000, 12,000, 14,000, 16,000, 18,000 or 20,000 psi. In the present invention, the pressure of the high-pressure homogenization ensures effective cell lysis without damaging the activity of the enzyme. The high-pressure homogenization of the present invention may be performed multiple times until microscopic examination shows that most of the cells have been lysed to obtain an intestinal membrane Leuconostoc lysate. In the present invention, the preparation of the intestinal membrane Leuconostoc lysate must be carried out under sterile conditions.In the present invention, the Leuconostoc mesenteroides lysate contains glutamine synthetase; the glutamine synthetase is an enzyme that catalyzes the reaction of glutamate and ammonia to generate glutamine. In Leuconostoc mesenteroides, glutamine synthetase can utilize ammonia in the environment or ammonia produced by cell metabolism to react with glutamate to generate glutamine.

[0035] After obtaining the Leuconostoc mesenteroides lysate, the present invention adds the Leuconostoc mesenteroides lysate and sodium glutamate to the fermentation medium. The present invention preferably adds the Leuconostoc mesenteroides lysate and sodium glutamate to the sterilized fermentation medium. In the present invention, the Leuconostoc mesenteroides lysate is preferably prepared under aseptic conditions; the sodium glutamate is preferably sterile sodium glutamate. In the present invention, the Leuconostoc mesenteroides lysate prepared by adding 100 to 200 Leuconostoc mesenteroides per liter of fermentation medium. In the present invention, the amount of sodium glutamate added to the fermentation medium can be 30 to 60 g / L. As an optional embodiment of the present invention, the amount of sodium glutamate added to the fermentation medium can be 30 g / L, 40 g / L, 50 g / L or 60 g / L. In the present invention, the fermentation medium may contain whey protein, i.e. whey, which contains lactose and other nutrients, is suitable as a nutrient for Leuconostoc mesenteroides, promotes the anaerobic fermentation process, and is beneficial to increase the yield of glutamine. As an optional embodiment of the present invention, the content of whey protein in the fermentation medium can be 3-6 g / L, or 3 g / L, 4 g / L, 5 g / L or 6 g / L. In the present invention, ammonium nitrate is preferably also added to the fermentation medium; the amount of ammonium nitrate added can be 10-20 g / L. As an optional embodiment of the present invention, the amount of ammonium nitrate added can be 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L. The present invention adds ammonium nitrate to the fermentation medium mainly to provide a nitrogen source for the anaerobic fermentation of Leuconostoc mesenteroides. In the present invention, the pH of the fermentation medium can be 6-7, or 6.5.

[0036] In the present invention, the composition of the basic culture medium of the fermentation medium can be 16-24g of glucose, 55-65g of milk powder, 3-6g of whey protein, 3-6g of yeast extract powder, 1.5-2.5g of dipotassium hydrogen phosphate, 1.5-2.5g of anhydrous sodium acetate, 0.54-0.62g of magnesium sulfate, 0.45-0.55g of L-cysteine ​​hydrochloride and 0.8-1.2g of Tween per 1L of culture medium. In the present invention, 16-24g of glucose is contained in each 1L of culture medium. As an optional embodiment of the present invention, 16g, 17g, 18g, 19g, 20g, 21g, 22g, 23g or 24g of glucose can be contained in each 1L of culture medium. In the present invention, 55-65g of milk powder is contained in each 1L of culture medium. As an optional embodiment of the present invention, 55g, 56g, 57g, 58g, 59g, 60g, 61g, 62g, 63g, 64g or 65g of milk powder may be contained in each 1L of culture medium. In the present invention, 3-6g of whey protein may be contained in each 1L of culture medium. As an optional embodiment of the present invention, 3g, 4g, 5g or 6g of whey protein may be contained in each 1L of culture medium. In the present invention, 3-6g of yeast extract powder may be contained in each 1L of culture medium. As an optional embodiment of the present invention, 3g, 4g, 5g or 6g of yeast extract powder may be contained in each 1L of culture medium. In the present invention, 1.5-2.5g of dipotassium hydrogen phosphate may be contained in each 1L of culture medium. As an optional embodiment of the present invention, 1.5g, 1.7g, 2.0g, 2.2g, 2.3g or 2.5g of dipotassium hydrogen phosphate may be contained in each 1L of culture medium. In the present invention, 1.5 to 2.5 g of anhydrous sodium acetate may be contained in each 1 L of culture medium. As an optional embodiment of the present invention, 1.5 g, 1.7 g, 2.0 g, 2.2 g, 2.3 g or 2.5 g of anhydrous sodium acetate may be contained in each 1 L of culture medium. In the present invention, 0.54 to 0.62 g of magnesium sulfate may be contained in each 1 L of culture medium. As an optional embodiment of the present invention, 0.54 g, 0.56 g, 0.58 g, 0.60 g or 0.62 g of magnesium sulfate may be contained in each 1 L of culture medium. In the present invention, 0.45 to 0.55 g of L-cysteine ​​hydrochloride may be contained in each 1 L of culture medium. As an optional embodiment of the present invention, 0.45 g, 0.47 g, 0.50 g, 0.53 g or 0.55 g of L-cysteine ​​hydrochloride may be contained in each 1 L of culture medium. In the present invention, 0.8 to 1.2 g of Tween may be contained in each 1 L of culture medium. As an optional embodiment of the present invention, each 1L of culture medium may contain 0.8g, 0.9g, 1.0g, 1.1g or 1.2g of Tween. In the present invention, the fermentation medium preferably uses water as a solvent, and the water may be distilled water.

[0037] After obtaining a fermentation medium containing Leuconostoc mesenteroides lysate, sodium glutamate and whey protein, the present invention preferably inoculates Leuconostoc mesenteroides into the fermentation medium for anaerobic fermentation culture. The present invention preferably performs activation culture and expansion culture on the Leuconostoc mesenteroides before anaerobic fermentation culture. In the present invention, the activation culture can be carried out in MRS culture medium; the temperature of the activation culture can be 35-40°C, or 35°C, 36°C, 37°C, 38°C, 39°C or 40°C; the time of the activation culture can be 24-48h, or 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h or 48h; the activation culture is preferably anaerobic culture. In the present invention, after the activation culture is completed, an activated bacterial solution of Leuconostoc mesenteroides is obtained. After obtaining the activated bacterial solution of Leuconostoc mesenteroides, the present invention preferably takes the activated bacterial solution of Leuconostoc mesenteroides for expansion culture to obtain Leuconostoc mesenteroides seed solution. In the present invention, the temperature of the expansion culture can be 35~40°C; the time of the expansion culture can be 24~48h, or 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h or 48h; the expansion culture can be aerobic culture. As an optional embodiment, the composition of the culture medium used for the expansion culture can be: 10g beef extract, 10g peptone, 20g glucose, 5g yeast extract powder, 2g dipotassium hydrogen phosphate, 2g ammonium citrate, 0.1g anhydrous sodium sulfite, 0.2g magnesium sulfate, 5g anhydrous sodium acetate, 0.05g manganese sulfate, Tween-80 1mL and 1L of distilled water; as another optional embodiment, the composition of the culture medium used for the expanded culture can also be: 10g glucose, 1g corn steep liquor hydrolyzate, 3g ammonium chloride, 0.02g potassium dihydrogen phosphate, 0.02g potassium hydrogen phosphate, 20μg vitamin B1, 4mg zinc sulfate, 4mg ferrous sulfate and 4μg biotin and 1L of distilled water; as another optional embodiment, the composition of the culture medium used for the expanded culture can also be: 3g chitosan with a molecular weight of 8000-10000Da, 7g yeast extract, 10g glucose, 0.01g inositol, 0.03mg biotin, 7g thiamine hydrochloride, 5g riboflavin and vitamin B 12 3.0 mg and 1 L of distilled water. In the present invention, the pH value of the culture medium used for the expanded culture may be 5.5.

[0038] After obtaining the Leuconostoc mesenteroides seed solution, the present invention preferably inoculates the Leuconostoc mesenteroides seed solution into a fermentation medium for anaerobic fermentation. The present invention can adjust the bacterial activity of the Leuconostoc mesenteroides seed solution to 6×10 6 ~9×10 6CFU / mL can also be adjusted to 7.6×10 6 CFU / mL. After adjusting the bacterial activity of the intestinal membrane seed liquid, the obtained intestinal membrane seed liquid is inoculated into the fermentation medium for anaerobic fermentation culture. The present invention can inoculate the intestinal membrane seed liquid into the fermentation medium at a volume fraction of 5% of the medium. In the present invention, the temperature of the anaerobic fermentation culture can be 25~45°C. As an optional embodiment of the present invention, the temperature of the anaerobic fermentation culture can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or 45°C. In the present invention, the time of the anaerobic fermentation culture can be 6~10d. As an optional embodiment of the present invention, the time of the anaerobic fermentation culture can be 6d, 7d, 8d, 9d or 10d. In the present invention, the pH value of the anaerobic fermentation culture can be 6~7. As an optional embodiment of the present invention, the pH value of the anaerobic fermentation culture can be 6.5. After the anaerobic fermentation culture is completed, the present invention obtains a fermentation broth of Leuconostoc mesenteroides. In the present invention, the fermentation broth of Leuconostoc mesenteroides includes glutamine. In the process of anaerobic fermentation culture of Leuconostoc mesenteroides, the present invention uses whey as a fermentation substrate, and simultaneously adds Leuconostoc mesenteroides lysate and sodium glutamate as a fermentation culture method for transforming Leuconostoc mesenteroides, which is beneficial to increasing the yield of glutamine.

[0039] The method for synthesizing glutamine provided by the present invention is to add its own lysate to the culture medium of Leuconostoc mesenteroides, add sodium glutamate at the same time, then perform anaerobic fermentation, control the fermentation process conditions, and be beneficial to the growth of Leuconostoc mesenteroides and the synthesis of glutamine. At the same time, Leuconostoc mesenteroides can be more efficiently metabolized and synthesized in an anaerobic environment, thereby further contributing to increasing the yield of glutamine. After 6 to 10 days of anaerobic fermentation, a large amount of glutamine can be detected, and its content can be as high as 40 g / L or more.

[0040] The present invention provides a method for synthesizing γ-polyglutamic acid, comprising the following steps:

[0041] The fermentation liquid containing glutamine prepared by the method described in the above technical solution is used as the fermentation substrate;

[0042] After adding oxygen carriers to the fermentation substrate, Bacillus coagulans is inoculated to carry out aerobic fermentation to obtain a fermentation bacterial liquid containing gamma-polyglutamic acid.

[0043] In the present invention, the oxygen carrier includes n-hexadecane; the amount of the oxygen carrier added may be 0.3% to 1% of the volume of the fermentation substrate. As an optional embodiment of the present invention, the amount of the oxygen carrier added may be 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1% of the volume of the fermentation substrate. The present invention does not specifically limit the inoculation method of Bacillus coagulans, and any conventional inoculation method in the art may be used. In the present invention, the Bacillus coagulans may be the Bacillus coagulans YJY21-05 strain preserved with accession number CGMCC No. 22760. In the present invention, the temperature of the aerobic fermentation may be 45 to 50°C. As an optional embodiment of the present invention, the temperature of the aerobic fermentation may be 45°C, 46°C, 47°C, 48°C, 49°C or 50°C. In the present invention, the time of the aerobic fermentation may be 10 to 15 days. As an optional embodiment of the present invention, the aerobic fermentation time can be 10d, 11d, 12d, 13d, 14d or 15d. In the present invention, the pH value of the aerobic fermentation can be 6.6-7.0. As an optional embodiment of the present invention, the pH value of the aerobic fermentation can be 6.6, 6.7, 6.8, 6.9 or 7.0. After the aerobic fermentation is completed, the present invention obtains a fermentation broth of Bacillus coagulans. In the present invention, the fermentation broth of Bacillus coagulans contains γ-polyglutamic acid.

[0044] In the synthesis method of γ-polyglutamic acid of the present invention, Bacillus coagulans is added to the fermentation liquid containing glutamine for aerobic fermentation, so as to facilitate the further synthesis of γ-polyglutamic acid (γ-PGA). In the aerobic fermentation process, the oxygen carrier n-hexadecane is added to increase the dissolved oxygen content in the fermentation liquid, thereby increasing the yield of γ-PGA. In the aerobic fermentation process, 0.5% volume ratio of n-hexadecane is added, and the fermentation for 48 hours can increase the yield of γ-PGA by more than 30%. The γ-polyglutamic acid (γ-PGA) prepared by the present invention is a biopolymer with multiple application potentials. In the process of preparing γ-polyglutamic acid of the present invention, anaerobic fermentation is first performed, and then aerobic fermentation is performed by adding Bacillus coagulans. This anaerobic-aerobic combined fermentation strategy can make full use of the metabolic characteristics of different microorganisms and improve the synthesis efficiency of the target product. The present invention achieves high yield of γ-PGA by means of genetic engineering, anaerobic fermentation followed by aerobic fermentation, and the like.

[0045] The present invention provides a method for preparing a soil conditioner, comprising the following steps:

[0046] After the fermentation liquid containing γ-polyglutamic acid prepared by the above technical solution is mixed with alcohols and a catalyst, an esterification reaction is carried out to obtain a soil conditioner. In the present invention, the alcohol includes any one or more of glycerol, ethanol and lactic acid; the catalyst includes any one or more of toluenesulfonic acid, sulfuric acid, carbonic acid and citric acid. In the present invention, the addition amount of the alcohol can be 10~30g / L; the addition amount of the catalyst can be 0.15~5g / L. In the present invention, the temperature of the esterification reaction is 60~70℃. As an optional embodiment of the present invention, the temperature of the esterification reaction can be 60℃, 62℃, 64℃, 65℃, 66℃, 68℃ or 70℃. In the present invention, the time of the esterification reaction can be 24~48h. As an optional embodiment of the present invention, the esterification reaction time can be 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h or 48h. After the esterification reaction is completed, a soil conditioner is obtained.

[0047] In the present invention, the soil conditioner contains γ-polyglutamic acid esters, namely modified γ-polyglutamic acid; the soil conditioner also contains γ-polyglutamic acid, glutamine, alcohol substances, Leuconostoc mesenteroides and Bacillus coagulans.

[0048] In the present invention, the modified γ-polyglutamic acid in the soil conditioner has better water retention, can enhance the water retention capacity of the soil, help the soil to remain moist under drought conditions, promote the formation of a more stable agglomeration structure of soil particles, improve the porosity of the soil, and thus improve the ventilation and drainage capacity of the soil. As a biopolymer, the γ-polyglutamic acid and the modified γ-polyglutamic acid can increase the organic matter content in the soil, which is helpful for improving the fertility of the soil, and the γ-polyglutamic acid and the modified γ-polyglutamic acid are biodegradable, and their decomposition in the soil will not have a negative impact on the environment, and is an environmentally friendly soil conditioner. Glutamine in the soil conditioner, as an organic nitrogen source, can promote soil microbial activity, help form a good soil structure, improve the ventilation and water retention capacity of the soil, and can also be used as a component of fertilizer to promote plant growth. The γ-polyglutamic acid (γ-PGA) in the soil conditioner also helps to reduce the salt concentration in the soil solution, reduce the direct damage of salt to the plant root system, and promote the growth of plants in saline-alkali soils. The alcohol substances in the soil conditioner, such as glycerol, can combine with the salt in the soil to form a complex, thereby reducing the salt concentration in the soil and alleviating the impact of salt damage on plants. The saline-alkali soil has a compact structure, poor air permeability and water permeability, which is not conducive to the development of plant roots. Alcohol substances such as glycerol can promote the coagulation of soil particles, improve soil structure, and increase porosity and air permeability. The high salt and alkaline conditions in saline-alkali soil inhibit the activity of soil microorganisms, and alcohol substances such as glycerol, as a carbon source, can stimulate the growth of beneficial microorganisms and promote the activity of soil microorganisms, which is crucial for the recovery and improvement of saline-alkali soil fertility. The application of alcohol substances such as glycerol can alleviate the salinization process of saline-alkali soil, and by improving soil structure and promoting microbial activity, it helps to reduce the salt and alkalinity of the soil and improve the crop growth environment. In addition, the Bacillus coagulans in the soil conditioner has the ability to fix nitrogen, and the intestinal membrane leuconostoc may help release nutrients such as phosphorus and potassium, improve the effectiveness of nutrients in the soil, and provide plants with absorption and utilization. Enhance the soil's buffering capacity against acid-base changes and reduce soil acidification or alkalinization problems caused by agricultural activities such as fertilization.

[0049] The present invention also provides a soil conditioner prepared by the preparation method described in the above technical solution. In the present invention, the soil conditioner includes esters containing γ-PGA, γ-polyglutamic acid, glutamine, alcohol substances, Leuconostoc mesenteroides and Bacillus coagulans.

[0050] The soil conditioner provided by the present invention can change the distribution pattern of soil moisture, so that more moisture is accumulated in the soil layer area of ​​the crop root zone, which helps the crops to better absorb water and nutrients and promote the growth of crops. It can also improve the physical and chemical properties and soil structure of saline-alkali soil, reduce the pH value and sodium adsorption ratio (SAR) of the soil, thereby reducing the salt content of the soil and improving the permeability of the soil.

[0051] The present invention also provides the use of the soil conditioner described in the above technical solution in improving saline-alkali soil. The results of the examples show that the soil conditioner provided by the present invention can promote the growth of crops in saline-alkali soil, increase the yield and plant height of crops; the soil conditioner provided by the present invention can improve the physical and chemical properties and soil structure of saline-alkali soil, reduce the pH value and sodium adsorption ratio of the soil, and increase the content of available potassium, available phosphorus, organic matter, etc. in saline-alkali soil.

[0052] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] In the following technical scheme, the Leuconostoc mesenteroides strain NTM048 with a preservation number of NITE BP-1519 is taken as an example, and the Bacillus coagulans strain YJY21-05 with a preservation number of CGMCC No. 22760 is taken as an example to specifically illustrate the technical scheme of the present invention. Among them, the Leuconostoc mesenteroides strain NTM048 with a preservation number of NITE BP-1519 is specifically referred to as the Leuconostoc mesenteroides strain with a preservation number of NITE BP-1519 in CN105229142A. The Bacillus coagulans strain YJY21-05 with a preservation number of CGMCC No. 22760 is specifically referred to as the Bacillus coagulans strain with a preservation number of GMCC No. 22760 in CN114437975A.

[0054] The process flow of the present invention for synthesizing glutamine, then synthesizing γ-polyglutamic acid from glutamine, and then preparing a soil conditioner from γ-polyglutamic acid is as follows: Figure 1 shown.

[0055] The method for obtaining Leuconostoc mesenteroides used in the Leuconostoc mesenteroides lysate in the following examples is as follows:

[0056] First, thoroughly burn the inoculation loop over the flame of an alcohol lamp to sterilize it to ensure aseptic operation. Gently touch the sterilized inoculation loop to the edge of the culture medium and let it cool to near room temperature. Gently touch the selected single colony with the cooled inoculation loop. Slowly lift the inoculation loop and transfer it to the new culture medium surface together with the colony. Repeat the above steps as needed until enough single colony samples are obtained.

[0057] The EDTA-containing buffer solution used in the following examples was obtained by adding 1.86 g of EDTA to 100 mL of deionized water.

[0058] Example 1

[0059] A method for synthesizing glutamine, comprising the following steps:

[0060] 1. Preparation of Leuconostoc mesenteroides Lysate

[0061] The lysate of Leuconostoc mesenteroides was prepared by high pressure homogenization method, and the specific steps are as follows:

[0062] The Enterobacter mesenteroides was streaked on beef extract peptone medium at 37°C for 48 hours. After the culture was completed, about 160 Enterobacter mesenteroides were taken for lysate preparation. About 160 Enterobacter mesenteroides were resuspended in PBS. Then centrifuged at 5500 rpm for 10 minutes at 4°C. The supernatant was removed and the precipitated cells were retained. The cell pellet was resuspended in 75mL of phosphate buffered saline (PBS) and centrifuged and washed again twice to remove the residual culture medium components. The washed cell pellet was resuspended in a buffer solution containing EDTA and the cell suspension was transferred to a high-pressure homogenizer. A pressure of 18,000 psi was set to ensure effective cell lysis without damaging the activity of the enzyme. The high-pressure treatment was performed three times until microscopic examination showed that most of the cells had been lysed. The Enterobacter mesenteroides lysate was obtained.

[0063] 2. Cultivation of Leuconostoc mesenteroides

[0064] The Leuconostoc mesenteroides was activated and cultured in MRS medium at 35°C for 40 hours. The activation culture was anaerobic. After the activation culture was completed, an activated bacterial solution of Leuconostoc mesenteroides was obtained.

[0065] The activated bacterial liquid of Leuconostoc mesenteroides was cultured in the culture medium (1) at a temperature of 35°C for 36 hours and aerobic culture was performed to obtain the seed liquid of Leuconostoc mesenteroides. The composition of the culture medium (1) is: 10 g beef extract, 10 g peptone, 20 g glucose, 5 g yeast extract powder, 2 g dipotassium hydrogen phosphate, 2 g ammonium citrate, 0.1 g anhydrous sodium sulfite, 0.2 g magnesium sulfate, 5 g anhydrous sodium acetate, 0.05 g manganese sulfate, 1 mL Tween-80 and 1 L distilled water. The pH value of the culture medium (1) is 5.5 and it is sterilized at 108°C for 20 minutes.

[0066] The bacterial activity of the Leuconostoc mesenteroides seed solution was adjusted to 7.6×10 6CFU / mL. The seed liquid of Enterobacter mesenteroides was inoculated into the fermentation medium at a volume fraction of 5% for anaerobic fermentation. During the anaerobic fermentation, the culture temperature was maintained at 35°C, the pH value of the reaction system was maintained at 6.5, and a 15% hydrochloric acid solution was used as a pH regulator to maintain the pH value. The anaerobic fermentation time was 10 days. After the anaerobic fermentation was completed, the fermentation broth was obtained, and the metabolites in the fermentation broth were detected by high performance liquid chromatography (HPLC), and the content of glutamine was 42.9 g / L.

[0067] The basic culture medium composition of the fermentation medium is as follows: 21g of glucose, 62g of milk powder, 6g of whey protein, 6g of yeast extract powder, 2.5g of dipotassium hydrogen phosphate, 2.5g of anhydrous sodium acetate, 0.62g of magnesium sulfate, 0.45g of L-cysteine ​​hydrochloride, and 0.8g of Tween are added to each liter of culture medium. The lysate of Leuconostoc mesenteroides prepared in step 1, 60g / L of sodium glutamate, and 20g / L of ammonium nitrate are also added to each liter of fermentation medium.

[0068] Example 2

[0069] A method for synthesizing glutamine, comprising the following steps:

[0070] The preparation of the lysate of Leuconostoc mesenteroides is the same as step 1 in Example 1, and the culture of Leuconostoc mesenteroides is the same as step 2 in Example 1. The only difference from step 2 is that the culture medium used for the expanded culture of Leuconostoc mesenteroides is different, and the expanded culture is carried out using culture medium (2).

[0071] The composition of the culture medium (2) is as follows: 10 g of glucose, 1 g of corn syrup hydrolyzate, 3 g of ammonium chloride, 0.02 g of potassium dihydrogen phosphate, 0.02 g of potassium hydrogen phosphate, 20 μg of vitamin B1, 4 mg of zinc sulfate, 4 mg of ferrous sulfate, 4 μg of biotin and 1 L of distilled water.

[0072] The glutamine content in the fermentation liquid obtained by anaerobic fermentation culture was 37.6 g / L.

[0073] Example 3

[0074] A method for synthesizing glutamine, comprising the following steps:

[0075] The preparation of the lysate of Leuconostoc mesenteroides is the same as step 1 in Example 1, and the culture of Leuconostoc mesenteroides is the same as step 2 in Example 1. The only difference from step 2 is that the culture medium used for the expanded culture of Leuconostoc mesenteroides is different, and the expanded culture is carried out using culture medium (3).

[0076] The composition of the culture medium (3) is: 3 g chitosan with a molecular weight of 8000-10000 Da, 7 g yeast extract powder, 10 g glucose, 0.01 g inositol, 0.03 mg biotin, 7 g thiamine hydrochloride, 5 g riboflavin, vitamin B 12 3.0mg and 1L distilled water.

[0077] The glutamine content in the fermentation liquid obtained by anaerobic fermentation culture was 36.8 g / L.

[0078] Example 4

[0079] A method for synthesizing glutamine, comprising the following steps:

[0080] The preparation of the Leuconostoc mesenteroides lysate is the same as step 1 in Example 1, except that 200 Leuconostoc mesenteroides are used to prepare the lysate to obtain the Leuconostoc mesenteroides lysate.

[0081] The Leuconostoc mesenteroides lysate prepared in this example was also added to each liter of fermentation medium; the amount of sodium glutamate added to the fermentation medium was 60 g / L; and the amount of ammonium nitrate added was 20 g / L.

[0082] The activation culture and expansion culture of Leuconostoc mesenteroides were the same as in Example 1. After obtaining the Leuconostoc mesenteroides seed solution, the bacterial activity of the Leuconostoc mesenteroides seed solution was adjusted to 7.6×10 6 CFU / mL. The seed liquid of Enterobacter mesenteroides was inoculated into the fermentation medium of this embodiment at a volume fraction of 5% of the fermentation medium for anaerobic fermentation culture. During the anaerobic fermentation culture, the culture temperature was maintained at 30°C, the pH value of the reaction system was maintained at 6.5 during the culture process, and a 15% hydrochloric acid solution was used as a pH regulator to maintain the pH value. The anaerobic fermentation culture time was 8d. After the anaerobic fermentation culture was completed, a fermentation broth was obtained, and the metabolites in the fermentation broth were detected by high performance liquid chromatography (HPLC), and the content of glutamine was found to be 35.7g / L.

[0083] Example 5

[0084] A method for synthesizing glutamine, comprising the following steps:

[0085] The preparation of the Leuconostoc mesenteroides lysate is the same as step 1 in Example 1, except that 150 Leuconostoc mesenteroides are used to prepare the lysate to obtain the Leuconostoc mesenteroides lysate.

[0086] The Leuconostoc mesenteroides lysate prepared in this example was also added to each liter of fermentation medium; the amount of sodium glutamate added to the fermentation medium was 30 g / L; and the amount of ammonium nitrate added to the fermentation medium was 10 g / L.

[0087] The activation culture and expansion culture of Leuconostoc mesenteroides were the same as in Example 1. After obtaining the Leuconostoc mesenteroides seed solution, the bacterial activity of the Leuconostoc mesenteroides seed solution was adjusted to 7.6×10 6 CFU / mL. The seed liquid of Enterobacter mesenteroides was inoculated into the fermentation medium of this embodiment at a volume fraction of 5% of the fermentation medium for anaerobic fermentation culture. During the anaerobic fermentation culture, the culture temperature was maintained at 25°C, the pH value of the reaction system was maintained at 7.0 during the culture process, and a 15% hydrochloric acid solution was used as a pH regulator to maintain the pH value. The anaerobic fermentation culture time was 10d. After the anaerobic fermentation culture was completed, a fermentation broth was obtained, and the metabolites in the fermentation broth were detected by high performance liquid chromatography (HPLC), and the content of glutamine was found to be 32.8g / L.

[0088] Example 6

[0089] A method for synthesizing glutamine, comprising the following steps:

[0090] The preparation of the Leuconostoc mesenteroides lysate is the same as step 1 in Example 1, except that 100 Leuconostoc mesenteroides are used to prepare the lysate to obtain the Leuconostoc mesenteroides lysate.

[0091] The Leuconostoc mesenteroides lysate prepared in this example was also added to each liter of fermentation medium; the amount of sodium glutamate added to the fermentation medium was 50 g / L; and the amount of ammonium nitrate added was 15 g / L.

[0092] The activation culture and expansion culture of Leuconostoc mesenteroides were the same as in Example 1. After obtaining the Leuconostoc mesenteroides seed solution, the bacterial activity of the Leuconostoc mesenteroides seed solution was adjusted to 7.6×10 6 CFU / mL. The seed liquid of Enterobacter mesenteroides was inoculated into the fermentation medium of this embodiment at a volume fraction of 5% of the fermentation medium for anaerobic fermentation culture. During the anaerobic fermentation culture, the culture temperature was maintained at 45°C, the pH value of the reaction system was maintained at 6.0 during the culture process, and a 15% hydrochloric acid solution was used as a pH regulator to maintain the pH value. The anaerobic fermentation culture time was 6d. After the anaerobic fermentation culture was completed, a fermentation broth was obtained, and the metabolites in the fermentation broth were detected by high performance liquid chromatography (HPLC), and the content of glutamine was found to be 28.2g / L.

[0093] Comparative Example 1

[0094] Prepare a strain of Leuconostoc mesenteroides, activate and culture the Leuconostoc mesenteroides in an MRS culture medium at 37° C. for 40 hours, and obtain an activated bacterial solution of Leuconostoc mesenteroides after the activation culture is completed.

[0095] The bacterial activity of the Leuconostoc mesenteroides seed solution was adjusted to 7.6×10 6CFU / mL. The seed liquid of Enterobacter mesenteroides was inoculated into the fermentation medium at a volume fraction of 5% for anaerobic fermentation. The fermenter was sealed and nitrogen was introduced for anaerobic fermentation. The culture temperature was maintained at 37°C during the anaerobic fermentation. The pH value of the reaction system was maintained at 6.5 during the culture. A 15% hydrochloric acid solution was used as a pH regulator to maintain the pH value. The anaerobic fermentation time was 10 days. After the anaerobic fermentation was completed, the fermentation broth was obtained. The metabolites in the fermentation broth were detected by high performance liquid chromatography (HPLC), and the content of glutamine was found to be 26.8 g / L.

[0096] The basic medium of the anaerobic fermentation medium is beef extract peptone medium, and the specific composition is: beef extract 10g, peptone 10g, glucose 20g, yeast extract powder 5g, dipotassium hydrogen phosphate 2g, ammonium citrate 2g, anhydrous sodium sulfite 0.1g, magnesium sulfate 0.2g, anhydrous sodium acetate 5g, manganese sulfate 0.05g, Tween-80 1mL and distilled water 1L. The anaerobic fermentation medium is based on the beef extract peptone medium, and sodium glutamate 60g / L is added. The pH value of the anaerobic fermentation medium is 5.5, and it is sterilized at 108℃ for 20min.

[0097] Comparative Example 2

[0098] A method for synthesizing glutamine, the steps are the same as those in Example 1, the only difference being:

[0099] After obtaining the Leuconostoc mesenteroides lysate, the Leuconostoc mesenteroides lysate is added to 1 L of sodium glutamate solution, wherein the mass concentration of sodium glutamate in the sodium glutamate solution is 60 g / L.

[0100] The lysate of Leuconostoc mesenteroides loses its activity during the glutamine synthesis process, and the effect of synthesizing glutamine is very poor, with the amount of glutamine synthesized being only 11.7 g / L.

[0101] Comparative Example 3

[0102] A method for synthesizing glutamine, the steps are the same as those in Example 1, the only difference being:

[0103] No preparation of Leuconostoc mesenteroides lysate was performed, and no Leuconostoc mesenteroides lysate was added to the Leuconostoc mesenteroides fermentation medium.

[0104] The amount of glutamine synthesized is only 27.5g / L.

[0105] Example 7

[0106] A method for synthesizing γ-polyglutamic acid, comprising the following steps:

[0107] Take the fermentation liquid prepared in Example 1, add n-hexadecane to the fermentation liquid at a volume ratio of 0.5%, and then add Bacillus coagulans for aerobic fermentation. Aerobic fermentation is carried out at a temperature of 45° C. and a pH of 7.0, and the aerobic fermentation time is 15 days. After the aerobic fermentation is completed, a fermentation liquid of Bacillus coagulans is obtained. The content of γ-polyglutamic acid in the fermentation liquid of Bacillus coagulans is 43.1 g / L.

[0108] Example 8

[0109] A method for synthesizing γ-polyglutamic acid, comprising the following steps:

[0110] Take the fermentation liquid prepared in Example 1, add n-hexadecane to the fermentation liquid at a volume ratio of 0.3%, and then add Bacillus coagulans for aerobic fermentation. Aerobic fermentation is carried out at a temperature of 50° C. and a pH of 6.6, and the aerobic fermentation time is 15 days. After the aerobic fermentation is completed, a fermentation liquid of Bacillus coagulans is obtained. The content of γ-polyglutamic acid in the fermentation liquid of Bacillus coagulans is 35.9 g / L.

[0111] Example 9

[0112] A method for synthesizing γ-polyglutamic acid, comprising the following steps:

[0113] Take the fermentation liquid prepared in Example 1, add n-hexadecane to the fermentation liquid at a volume ratio of 1.0%, and then add Bacillus coagulans for aerobic fermentation. Aerobic fermentation is carried out at a temperature of 45° C. and a pH of 7.0, and the aerobic fermentation time is 10 days. After the aerobic fermentation is completed, a fermentation liquid of Bacillus coagulans is obtained. The content of γ-polyglutamic acid in the fermentation liquid of Bacillus coagulans is 34.2 g / L.

[0114] Comparative Example 4

[0115] A method for synthesizing γ-polyglutamic acid, comprising the following steps:

[0116] Bacillus coagulans was cultured on a medium containing sodium L-glutamate, the molar concentration of sodium L-glutamate in the medium was 400 mM / L, n-hexadecane was added to the medium at a volume ratio of 0.5%, and the culture method and culture time were the same as those in Example 8, thereby preparing γ-polyglutamic acid.

[0117] The composition of the culture medium is: 52 mg / L leucine, 2.5 g / L glucose, 4 g / L K2SO4 solution, and 0.11 mg / L vitamin B1.

[0118] The production amount of γ-polyglutamic acid was 32.8 g / L.

[0119] Example 10

[0120] A soil conditioner, the preparation method steps are as follows:

[0121] Glycerol and toluenesulfonic acid were added to the Bacillus coagulans fermentation broth prepared in Example 7, with the glycerol addition amount being 30 g / L and the toluenesulfonic acid addition amount being 5 g / L. Glycerol and γ-polyglutamic acid (γ-PGA) in the Bacillus coagulans fermentation broth underwent esterification reaction at 65° C. under the action of toluenesulfonic acid, and a soil conditioner was obtained after the reaction for 48 hours.

[0122] Embodiment 11

[0123] A soil conditioner, the preparation method steps are as follows:

[0124] Ethanol and toluenesulfonic acid were added to the fermentation broth of Bacillus coagulans prepared in Example 7, the ethanol addition amount was 30 g / L, and the toluenesulfonic acid addition amount was 5 g / L. Ethanol and γ-polyglutamic acid (γ-PGA) in the fermentation broth of Bacillus coagulans underwent esterification reaction at 65° C. under the action of toluenesulfonic acid, and a soil conditioner was obtained after the reaction for 48 hours.

[0125] Example 12

[0126] A soil conditioner, the preparation method steps are as follows:

[0127] Lactic acid and toluenesulfonic acid were added to the Bacillus coagulans fermentation broth prepared in Example 7, with the lactic acid added in an amount of 30 g / L and the toluenesulfonic acid added in an amount of 5 g / L. Lactic acid and γ-polyglutamic acid (γ-PGA) in the Bacillus coagulans fermentation broth underwent esterification reaction at 65° C. under the action of toluenesulfonic acid, and a soil conditioner was obtained after the reaction for 48 hours.

[0128] Application Example 1

[0129] The effect of the soil conditioner prepared in Examples 10 to 12 on improving saline-alkali soil was determined by using a rice greenhouse test

[0130] The saline-alkali soil in Dingxiang County, Shanxi Province was used as the test soil. The volume of the plastic pot was 1L, and 10 rice grains were planted in each pot. The treatment step was to irrigate with 50mL of distilled water every day. The air temperature in the growth room was maintained at 22±2℃ during the day and 18±2℃ at night. The photoperiod was 12h and the light intensity was 950μmol / m 2 ·s. Relative humidity is 45±5% during the day and 70±5% at night.

[0131] The following test was conducted in a plastic pot with a volume of 1 L. Before planting rice, four treatments were performed on the soil for planting rice. The soil in treatment 1 was saline-alkali soil, and no soil conditioner was applied, which was used as a control group; in treatment 2, the soil conditioner prepared in Example 10 was applied in the saline-alkali soil, and the application amount was 100 g / pot, which was recorded as the glycerol soil conditioner group; in treatment 3, the soil conditioner prepared in Example 11 was applied in the saline-alkali soil, and the application amount was 100 g / pot, which was recorded as the ethanol soil conditioner group; in treatment 4, the soil conditioner prepared in Example 12 was applied in the saline-alkali soil, and the application amount was 100 g / pot, which was recorded as the lactic acid soil conditioner group. Three parallel tests were performed for each treatment.

[0132] The rice plants were harvested after 18 weeks of the experiment, and the physical and chemical properties of the soil in each treatment group were tested.

[0133] At the same time, the height and yield of rice plants were counted by randomly using a 5-point sampling method.

[0134] The statistical results of rice plant height and yield in each treatment group after 18 weeks of the experiment are shown in Table 1. The results of the physical and chemical properties of the soil in each treatment group after 18 weeks of the experiment are shown in Table 2.

[0135] Table 1 Statistical results of rice plant height and yield in each treatment group

[0136]

[0137] Table 2 Physical and chemical properties of soil in each treatment group

[0138]

[0139] From Tables 1 and 2, (1) the effect of glycerol soil conditioner was the most significant. Compared with the control group, the soil available potassium, available phosphorus, and organic matter contents increased by 45.7%, 36.3%, and 29.5%, respectively. The pH value decreased to 6.7, the exchangeable sodium decreased by 16.9%, the plant height increased by an average of 2.35 cm, and the yield increased by 37.4%. 3- 、SO4 2- 、Na + The contents of Cl increased by 11.9%, 16.4% and 18.5% respectively; - , Ca 2+ Mg 2+ The content decreased by 12.7%, 11.4% and 15.1%.

[0140] (2) In the ethanol soil conditioner group, compared with the control group, the soil available potassium, available phosphorus, and organic matter contents increased by 32.5%, 28.8%, and 30.6%, respectively, the pH decreased to 7.4, the exchangeable sodium decreased by 13.9%, the plant height increased by an average of 1.98 cm, and the yield increased by 20.7%. 3- 、SO4 2- 、Na + The contents of Cl increased by 10.8%, 14.4% and 16.5% respectively; - , Ca 2+ Mg 2+ The content decreased by 16.3%, 10.3% and 12.9%.

[0141] (3) In the lactic acid soil conditioner group, compared with the control group, the soil available potassium, available phosphorus, and organic matter contents increased by 40.6%, 32.9%, and 29.7%, respectively, the pH decreased to 6.2, the exchangeable sodium decreased by 18.5%, the plant height increased by an average of 2.09 cm, and the yield increased by 31.9%. 3- 、SO4 2- 、Na + The contents of Cl increased by 15.8%, 10.4% and 15.8% respectively; - , Ca 2+ Mg 2+ The content decreased by 13.6%, 17.3% and 19.9%.

[0142] The addition of glycerin soil conditioner, ethanol soil conditioner and lactic acid soil conditioner to the soil increased the soil moisture content by 18%, 9% and 11% respectively.

[0143] In summary, the soil conditioner provided by the present invention can significantly improve the physical and chemical properties of saline-alkali soil, increase the soil's ability to retain water and fertilizer, reduce soil salinity, promote plant growth, and enhance soil microbial activity, providing an effective solution for saline-alkali land improvement.

[0144] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a soil conditioner, characterized in that: The following steps are involved: (1) Leuconostoc mesenteroides ( Leuconostoc mesenteroides ) performing anaerobic fermentation in a fermentation medium containing a lysate of Leuconostoc mesenteroides, sodium glutamate and whey protein to obtain a fermentation liquid containing glutamine; the Leuconostoc mesenteroides includes Leuconostoc mesenteroides NTM048, and the preservation number of the Leuconostoc mesenteroides NTM048 is NITE BP-1519; The content of sodium glutamate in the fermentation medium is 30~60g / L, and the content of whey protein is 3~6g / L; Leuconostoc mesenteroides lysate prepared by adding 100 to 200 Leuconostoc mesenteroides to each liter of fermentation medium; (2) Using fermentation broth containing glutamine as the fermentation substrate; After adding oxygen carriers to the fermentation medium, inoculation of Bacillus coagulans ( Bacillus coagulans ) to perform aerobic fermentation to obtain a fermentation solution containing γ-polyglutamic acid; the Bacillus coagulans is Bacillus coagulans YJY21-05 with a preservation number of CGMCC No. 22760; (3) The fermentation liquid containing γ-polyglutamic acid is mixed with an alcohol and a catalyst, and then subjected to an esterification reaction to obtain a soil conditioner.

2. The preparation method according to claim 1, characterized in that: The Leuconostoc mesenteroides lysate is obtained by high-pressure homogenization of Leuconostoc mesenteroides.

3. The preparation method according to claim 1, characterized in that: The temperature of the anaerobic fermentation culture is 25-45° C.; the time of the anaerobic fermentation culture is 6-10 days; and the pH value of the anaerobic fermentation culture is 6.0-7.

0.

4. The preparation method according to claim 1, characterized in that: The oxygen carrier includes n-hexadecane; the added amount of the oxygen carrier is 0.3% to 1% of the volume of the fermentation substrate; The temperature of the aerobic fermentation is 45-50° C.; the time of the aerobic fermentation is 10-15 days; and the pH value of the aerobic fermentation is 6.6-7.

0.

5. The preparation method according to claim 1, characterized in that: The alcohol includes any one or more of glycerol, ethanol and lactic acid; the catalyst includes any one or more of toluenesulfonic acid, sulfuric acid, carbonic acid and citric acid; The temperature of the esterification reaction is 60-70° C.; the time of the esterification reaction is 24-48 hours.

6. A soil conditioner prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the soil conditioner according to claim 6 in improving saline-alkali soil.

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