Process for the production of a low-chloride-content gamma-polyglutamic acid fermentation broth

Abstract

The present application relates to the technical field of microbial fermentation, and particularly relates to a production method of a low-chloride-ion-content gamma-polyglutamic acid fermentation liquor, and specifically comprises the following steps: after the bacillus subtilis is expanded and cultured, a seed liquid is obtained; a fermentation initial culture medium is prepared, and the components and contents of the fermentation initial culture medium are as follows: sodium glutamate 50-100 g / L, citric acid 2-5 g / L, MgSO4 0.1-0.5 g / L, K2HPO4 1-4 g / L, compound amino acid 0-5 g / L, inorganic nitrogen source 1-10 g / L, and the rest is water; the seed liquid is connected to the fermentation initial culture medium for culture, and in the culture process, the carbon source and the inorganic nitrogen source are added through feeding, and the gamma-polyglutamic acid fermentation liquor is obtained through culture. In the present application, the low-cost inorganic nitrogen source is added through flow addition, and a small amount of amino acid growth factor is added to replace the high-price organic nitrogen source, so that the production cost is greatly reduced, and the prepared gamma-polyglutamic acid fermentation liquor has a low chloride ion content.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a method for producing γ-polyglutamic acid fermentation broth with low chloride ion content. Background Technology

[0002] γ-Polyglutamic acid (γ-PGA) is an extracellular, water-soluble high-molecular-weight amino acid polymer synthesized by certain Bacillus sp., composed of D-glutamic acid or L-glutamic acid linked by amide bonds between α-amino and γ-carboxyl groups. It possesses excellent film-forming properties, adhesiveness, and strong water absorption. In agriculture, γ-polyglutamic acid, as a fertilizer synergist, not only increases crop yield but also provides excellent slow-release of fertilizer and water, exhibiting significant water and fertilizer retention, yield-increasing, and fertilizer-saving effects. In major agriculturally developed countries worldwide, γ-polyglutamic acid fertilizers account for a high proportion of fertilizer use; in the United States, γ-polyglutamic acid fertilizers account for 55% of fertilizer usage, and in Israel, over 90% of crops use γ-polyglutamic acid fertilizers.

[0003] Currently, γ-polyglutamic acid (γ-polyglutamic acid) is mainly produced through microbial fermentation. This requires the addition of organic nitrogen sources such as peptone and yeast powder to the fermentation medium, increasing fermentation costs and keeping γ-polyglutamic acid prices high. Furthermore, the resulting γ-polyglutamic acid fermentation broth has a high chloride ion content, negatively impacting fertilizer production, fertilization effectiveness, and the soil environment. Firstly, the high-pressure systems in fertilizer production facilities have stringent requirements for chloride ion control. When adding γ-polyglutamic acid stock solution to the urea evaporation system to produce new types of urea, the chloride ion content in the raw materials must be controlled below 100 mg / kg. Secondly, chloride ions promote the hydrolysis of carbohydrates, reducing the sugar content of watermelons, sugar beets, and grapes; high chloride ion levels often harm seedlings of sensitive crops. Thirdly, the application of large amounts or prolonged exposure to chloride-containing fertilizers leads to high levels of residual chloride ions in the soil, easily causing soil compaction, salinization, and alkalization, deteriorating the soil environment, and thus reducing the crop's ability to absorb nutrients.

[0004] Therefore, finding a highly efficient and energy-saving method for producing γ-polyglutamic acid fermentation broth with low chloride ion content is an urgent problem for researchers in this field. Summary of the Invention

[0005] To address the technical problems of high cost and high chloride content in existing γ-polyglutamic acid fermentation broth production methods, this invention provides a method for producing γ-polyglutamic acid fermentation broth with low chloride ion content, which effectively increases the fermentation yield of γ-polyglutamic acid, reduces the chloride ion content in the γ-polyglutamic acid fermentation broth, and lowers production costs.

[0006] This invention provides a method for producing γ-polyglutamic acid fermentation broth with low chloride ion content, specifically including the following steps:

[0007] (1) Seed culture of Bacillus subtilis was obtained after large-scale culture;

[0008] (2) Prepare the initial fermentation culture medium with the following components and contents: sodium glutamate 50-100 g / L, citric acid 2-5 g / L, MgSO4 0.1-0.5 g / L, K2HPO4 1-4 g / L, complex amino acids 0-5 g / L, inorganic nitrogen source 1-10 g / L, the remainder being water, adjust the pH to 7.2-7.5, and sterilize at high temperature;

[0009] (3) The seed liquid was inoculated into the initial fermentation medium and cultured. During the culture process, carbon source and inorganic nitrogen source were added by feeding to obtain γ-polyglutamic acid fermentation broth.

[0010] Furthermore, the Bacillus subtilis used was Bacillus subtilis FRD518, which was deposited on November 2, 2012 at the China General Microbiological Culture Collection Center with accession number CGMCC No. 6772.

[0011] Further, in step (1), 1 mL of frozen Bacillus subtilis seed solution is inoculated into a 500 mL Erlenmeyer flask containing 100 mL of seed culture medium and cultured on a shaker at 35–38 °C and 180–250 r / min until the seed OD600 reaches 1.6–2.2 to obtain primary seed solution; the primary seed solution is inoculated into a seed tank and cultured at 35–38 °C with stirring and aeration to obtain secondary seed solution.

[0012] Furthermore, the composition and content of the seed culture medium used for primary and secondary seeds are as follows: glucose 5-10 g / L, yeast powder 3-5 g / L, ammonium sulfate 2-10 g / L, MgSO4 0.1-1.0 g / L, and the remainder is water, adjusted to pH 7.2-7.5, and sterilized at 115℃ for 30 min.

[0013] Furthermore, in step (2), the high-temperature sterilization specifically refers to sterilization at 115°C for 30 minutes.

[0014] Furthermore, in step (3), the inoculum amount is 3%-5%, and the fermentation tank is aerated and stirred at 35-38℃.

[0015] Furthermore, the carbon source is glucose, the feeding method is fed in a feed-by-feed manner, and the glucose concentration is controlled at 5–15 g / L.

[0016] Furthermore, the inorganic nitrogen source is one or more of ammonium sulfate, ammonium nitrate, ammonium phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, and urea. The feeding method is feed-feed, and the feeding rate is 0.1 to 1 g / (L·h).

[0017] Furthermore, the complex amino acid consists of two or more of tyrosine, phenylalanine, and aspartic acid.

[0018] The beneficial effects of this invention are as follows:

[0019] 1. The method for producing γ-polyglutamic acid fermentation broth with low chloride ion content provided by the present invention uses a low-cost inorganic nitrogen source and adds a small amount of amino acid growth factor to replace the more expensive organic nitrogen source for fermentation to produce γ-polyglutamic acid, which greatly reduces the production cost. In addition, the fermentation medium used does not contain chloride ions, and the prepared γ-polyglutamic acid fermentation broth has a low chloride ion content.

[0020] 2. This invention optimizes the fermentation medium and fermentation process, controlling the carbon and nitrogen levels in the fermentation medium to put Bacillus subtilis in a starved state, thus balancing cell growth and γ-polyglutamic acid synthesis and improving the utilization rate of fermentation raw materials. Furthermore, the simple composition of the culture medium used in this invention facilitates subsequent processing of the fermentation broth, resulting in higher purity cosmetic-grade γ-polyglutamic acid. Detailed Implementation

[0021] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0022] The Bacillus subtilis FRD518 used in the following examples was deposited at the China General Microbiological Culture Collection Center on November 2, 2012, with accession number CGMCC No. 6772. This strain has been published in the applicant's prior patent application CN201210555304.5.

[0023] Example 1

[0024] (1) Prepare seed culture medium. The components and contents of the seed culture medium are as follows: glucose 10g / L, yeast powder 5g / L, ammonium sulfate 8g / L, MgSO4 0.8g / L, and the rest is water. Adjust the pH to 7.2 and sterilize at 115℃ for 30min.

[0025] (2) For primary and secondary seeds, use the above-mentioned seed culture medium. Inoculate 1 mL of frozen Bacillus subtilis FRD518 seed solution into a 500 mL Erlenmeyer flask containing 100 mL of seed culture medium. Incubate at 35°C with shaking on a shaker at 180 r / min until the seed OD reaches the specified level. 600 When the concentration reaches 1.6, a primary seed culture is obtained. The primary seed culture is then inoculated into a seed tank and cultured at 36°C with stirring and aeration to obtain a secondary seed culture.

[0026] (3) Prepare the initial fermentation culture medium with the following components and contents: 80 g / L sodium glutamate, 4 g / L citric acid, 0.5 g / L MgSO4, 3 g / L K2HPO4, 3 g / L complex amino acids, 8 g / L ammonium sulfate, and the remainder is water. Adjust the pH to 7.2-7.5 and sterilize at 115℃ for 30 min. The complex amino acids include tyrosine, phenylalanine and aspartic acid, with a ratio of 1:1:1.

[0027] (4) The secondary seed liquid was inoculated into the above-mentioned initial fermentation medium at an inoculation amount of 5%. The fermentation was carried out in a fermenter at 37°C with aeration and stirring. During the culture, glucose and ammonium sulfate were added, and the glucose concentration was controlled at 5-15 g / L. The ammonium sulfate feeding rate was 0.2 g / (L·h). The γ-polyglutamic acid fermentation broth was obtained after 44 h of culture.

[0028] Example 2

[0029] (1) Prepare seed culture medium. The components and contents of the seed culture medium are as follows: glucose 5g / L, yeast powder 3g / L, ammonium sulfate 3g / L, MgSO4 0.2g / L, and the remaining components are water. Adjust the pH to 7.5 and sterilize at 115℃ for 30min.

[0030] (2) For primary and secondary seeds, use the above-mentioned seed culture medium. Inoculate 1 mL of frozen Bacillus subtilis FRD518 seed solution into a 500 mL Erlenmeyer flask containing 100 mL of seed culture medium. Incubate at 37°C and 220 r / min on a shaker until the seed OD reaches the specified level. 600 Once the viscosity reaches 2.0, a primary seed culture is obtained. The primary seed culture is then inoculated into a seed tank and cultured at 37°C with stirring and aeration to obtain a secondary seed culture.

[0031] (3) Prepare the initial fermentation medium with the following components and contents: 60 g / L sodium glutamate, 2 g / L citric acid, 0.1 g / L MgSO4, 1.0 g / L K2HPO4, 1 g / L complex amino acids, 8 g / L ammonium phosphate, and the remainder is water. Adjust the pH to 7.5 and sterilize at 115℃ for 30 min. The complex amino acids include tyrosine, phenylalanine and aspartic acid, with a content ratio of 3:4:4.

[0032] (4) The secondary seed liquid was inoculated into the above-mentioned initial fermentation medium at an inoculation amount of 3%. The fermentation was carried out in a fermenter at 35°C with aeration and stirring. During the culture, glucose and inorganic nitrogen source (a mixture of ammonium sulfate and urea in a mass ratio of 1:2) were added. The glucose concentration was controlled at 5-15 g / L and the inorganic nitrogen source was fed at a rate of 0.3 g / (L·h). After 48 h of culture, γ-polyglutamic acid fermentation broth was obtained.

[0033] Comparative Example

[0034] (1) The preparation method of seed liquid is the same as in Example 2.

[0035] (2) According to the composition of the fermentation medium described in patent application CN202210521932.5, the fermentation medium was prepared with the following composition and content: peptone 15g / L, yeast extract 15g / L, ammonium sulfate 15g / L, sodium glutamate 60g / L, citric acid 2g / L, MgSO4 0.1g / L, K2HPO4 1.0g / L, glucose 150g / L, pH adjusted to 7.5, and sterilized at 115℃ for 30min.

[0036] (3) Inoculate the seed liquid into the above fermentation medium at an inoculation amount of 3%, and culture it in a fermenter at 35°C with aeration and stirring for 48 hours to obtain γ-polyglutamic acid fermentation broth.

[0037] Test case

[0038] The γ-polyglutamic acid content and chloride ion content in the γ-polyglutamic acid fermentation broth prepared in Example 2 and the comparative example were detected, and the results are shown in Table 1.

[0039] Table 1 Comparison of product quality of fermentation broth for producing γ-polyglutamic acid using two methods.

[0040] sample γ-polyglutamic acid content in fermentation broth / % Chloride ion content in fermentation broth (mg / L) Example 2 7.65 43 Comparative Example 5.51 2897

[0041] As shown in Table 1, the chloride ion content in the γ-polyglutamic acid fermentation broth prepared in Example 2 was less than 100 mg / L, while the chloride ion content in the γ-polyglutamic acid fermentation broth prepared in the comparative example was higher, reaching 2897 mg / L. This is mainly because the peptone and yeast extract in the fermentation medium used in the comparative example contained chloride, while the fermentation medium in Example 2 used an inorganic nitrogen source and added a small amount of amino acid growth factors, containing little or no chloride ions. Furthermore, the γ-polyglutamic acid fermentation yield in Example 2 was higher than that in the comparative example. This indicates that the inorganic nitrogen source and amino acids used in the fermentation medium of Example 2 could maintain the normal metabolism of the cells and produce a large amount of γ-polyglutamic acid, while the nutrient-rich fermentation medium in the comparative example may have led to excessive cell growth, resulting in relatively less γ-polyglutamic acid production.

[0042] Example 3

[0043] Using the method for separating and purifying γ-polyglutamic acid from fermentation broth described in Example 3 of patent application CN201510193843.2, cosmetic-grade high-purity γ-polyglutamic acid was separated and purified from the γ-polyglutamic acid fermentation broth prepared in Example 2 and the comparative example. The final purity of γ-polyglutamic acid obtained from the γ-polyglutamic acid fermentation broth prepared in Example 2 was 97.84%, and the purity of γ-polyglutamic acid obtained from the γ-polyglutamic acid fermentation broth prepared in the comparative example was 96.43%.

[0044] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A method for producing γ-polyglutamic acid fermentation broth with low chloride ion content, characterized in that, Specifically, the following steps are included: (1) Seed culture of Bacillus subtilis was obtained after large-scale culture; (2) Prepare the initial fermentation culture medium with the following components and contents: sodium glutamate 50~100g / L, citric acid 2~5g / L, MgSO4 0.1~0.5g / L, K2HPO4 1~4g / L, complex amino acids 0~5g / L, inorganic nitrogen source 1~10g / L, the remainder being water, adjust the pH to 7.2~7.5, and sterilize at high temperature; (3) The seed culture was inoculated into the initial fermentation medium and cultured. During the culture process, carbon source and inorganic nitrogen source were added by feeding to obtain γ-polyglutamic acid fermentation broth. The carbon source was glucose, and the feeding method was fed in a fed-batch manner, with the glucose concentration controlled at 5~15g / L. The inorganic nitrogen source was a mixture of ammonium sulfate and urea in a mass ratio of 1:2, and the feeding method was fed in a fed-batch manner, with a feeding rate of 0.1~1g / (L·h).

2. The production method as described in claim 1, characterized in that, The Bacillus subtilis used was Bacillus subtilis FRD518, Bacillus subtilis ( Bacillus subtilis FRD518 was deposited on November 2, 2012, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 6772.

3. The production method as described in claim 1, characterized in that, In step (1), 1 mL of frozen Bacillus subtilis seed culture was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of seed culture medium and cultured at 35-38°C with shaking at 180-250 r / min until the seed OD reached the specified level. 600 When the pH reaches 1.6~2.2, a primary seed culture is obtained. The primary seed culture is inoculated into a seed tank and cultured at a temperature of 35~38℃ with stirring and aeration to obtain a secondary seed culture.

4. The production method as described in claim 3, characterized in that, The components and contents of the seed culture medium used for primary and secondary seeds are as follows: glucose 5~10g / L, yeast powder 3~5g / L, ammonium sulfate 2~10g / L, MgSO4 0.1~1.0g / L, and the remainder is water. The pH is adjusted to 7.2~7.5 and sterilized at 115℃ for 30min.

5. The production method as described in claim 1, characterized in that, In step (2), high-temperature sterilization specifically means sterilization at 115℃ for 30 minutes.

6. The production method as described in claim 1, characterized in that, In step (3), the inoculum amount is 3%-5%, and the fermentation tank is ventilated and stirred at 35-38℃.

7. The production method as described in claim 1, characterized in that, The complex amino acid is two or more of tyrosine, phenylalanine, and aspartic acid.