Method for producing gamma-polyglutamic acid by using fermentation culture medium

Abstract

The invention relates to a method for producing gamma-polyglutamic acid by using a fermentation culture medium, and belongs to the technical field of fermentation. The fermentation culture medium comprises the following components in parts by mass: 10 parts of brewer's grains, 0.15 part of monosodium glutamate and 10 parts of water; the water content of the brewer's grains is 62%. According to the bacillus subtilis disclosed by the invention, the brewer's grains can be used as a fermentation culture medium for fermentation production of the gamma-polyglutamic acid, and a new path is sought for comprehensive utilization of the brewer's grains; and the content of gamma-polyglutamic acid generated after fermentation is high. The culture medium for fermentation production of gamma-polyglutamic acid is simple in components, easy to obtain and low in cost, and a new way is provided for production of gamma-polyglutamic acid.

Description

[0001] This application is a divisional application. The application number of the original application is: 202210539026.8, the application date is 2022.05.18, and the name of the invention is: A fermentation medium for Bacillus subtilis and a method for producing γ-polyglutamic acid. Technical Field

[0002] The invention relates to the technical field of fermentation, and in particular to a method for producing gamma-polyglutamic acid by using a fermentation medium. Background Art

[0003] γ-Polyglutamic acid (γ-PGA) is a biodegradable, non-toxic anionic homopolymer composed of D- and L-glutamic acid monomers. It can be used as a water retaining agent, a storage and preservative for fruits, vegetables and flowers, a growth promoter, a fertilizer enhancer, an environmental stress resistance agent, and a soil conditioner. It is widely used in the agricultural field.

[0004] Brewer's grains are the most important by-products produced in the beer brewing process, accounting for 85% of all by-products of beer production. Brewer's grains are the undissolved residues of malt and auxiliary materials during the saccharification process. They are mainly composed of the seed coat, shell and a small amount of incompletely dissolved starch endosperm particles of malt. From the chemical composition point of view, they are mainly non-starch polysaccharides (cellulose, hemicellulose; 30% to 50%) and protein (19% to 30%). It is estimated that the annual average output of brewer's grains in the world is 39 million tons. Generally, 20kg of wet brewer's grains are produced for every 100L of beer produced; in my country, 1.1 to 1.3 tons of wet brewer's grains with a moisture content of 75% to 80% can be produced for every ton of malt. At present, the utilization of brewer's grains is mainly used as animal feed. With the annual growth of global beer production, the annual average output of brewer's grains produced has increased, while the market demand for feed is limited. Therefore, the excess brewer's grains are generally directly discharged as solid waste, which wastes resources and pollutes the environment. With the enhancement of human environmental awareness, the development and utilization of brewer's grains has become a research hotspot. A review of the prior art shows that no method for preparing γ-polyglutamic acid using brewer's grains has been found, nor has any precedent been found for which strain can use brewer's grains to ferment γ-polyglutamic acid. Summary of the invention

[0005] The object of the present invention is to provide a fermentation medium of Bacillus subtilis and a method for producing gamma-polyglutamic acid.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention also provides a fermentation medium for Bacillus subtilis, wherein the fermentation medium comprises the following components in parts by weight:

[0008] 5-10 parts of brewer's grains, 0.15-0.3 parts of monosodium glutamate, 10-30 parts of water;

[0009] The water content of the brewer's grains is 60-65%.

[0010] The present invention also provides a method for producing γ-polyglutamic acid using the fermentation medium, comprising the following steps:

[0011] (1) inoculating Bacillus subtilis into a liquid culture medium for activation culture to obtain a seed solution;

[0012] (2) inoculating the seed liquid into the fermentation medium for fermentation and culturing to obtain γ-polyglutamic acid;

[0013] The Bacillus subtilis is Bacillus subtilis YB18;

[0014] The Bacillus subtilis YB18 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number of CGMCC No. 17642, the classification name of Bacillus subtilis, and the deposit date of April 28, 2019;

[0015] The initial number of viable bacteria contained in the inoculated seed solution is 1.0 to 1.5×10 8 cfu / mL; the volume ratio of the seed liquid to the fermentation medium is 4-5:2500.

[0016] Preferably, the liquid culture medium in step (1) uses water as a solvent and comprises the following components in the following concentrations:

[0017] Peptone 8-12g / L, beef extract powder 2.5-3.5g / L, glucose 18-22g / L, sodium chloride 4-6g / L.

[0018] Preferably, the activation culture temperature in step (1) is 35-39°C;

[0019] The activation culture time is 16 to 20 hours;

[0020] The activation culture is shaking culture;

[0021] The oscillation speed is 140-160 rpm.

[0022] Preferably, the fermentation temperature in step (2) is 35-39°C;

[0023] The fermentation culture time is 45 to 72 hours;

[0024] The fermentation culture is a static culture.

[0025] The present invention provides a Bacillus subtilis and a fermentation medium thereof and a method for producing γ-polyglutamic acid. The strain and production method of the present invention have the following advantages:

[0026] (1) The Bacillus subtilis of the present invention can use brewer's grains as a fermentation medium to ferment and produce γ-polyglutamic acid, thus seeking a new path for the comprehensive utilization of brewer's grains; and the content of γ-polyglutamic acid produced after fermentation is high.

[0027] (2) The culture medium components for the fermentation production of γ-polyglutamic acid are simple, easy to obtain, and low in cost, providing a new approach for the production of γ-polyglutamic acid.

[0028] Collection Instructions

[0029] The Bacillus subtilis YB18 is deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC No.17642, a classification name of Bacillus subtilis, and a deposit date of April 28, 2019. DETAILED DESCRIPTION

[0030] The present invention provides a fermentation medium for Bacillus subtilis, wherein the fermentation medium comprises the following components in parts by weight:

[0031] 5 to 10 parts of brewer's grains, preferably 7.5 parts;

[0032] 0.15-0.3 parts of MSG, preferably 0.225 parts;

[0033] 10 to 30 parts of water, preferably 20 parts;

[0034] The water content of the brewer's grains is 60-65%, preferably 62.5%.

[0035] The present invention also provides a method for producing γ-polyglutamic acid using the fermentation medium, comprising the following steps:

[0036] (1) inoculating Bacillus subtilis into a liquid culture medium for activation culture to obtain a seed solution;

[0037] (2) inoculating the seed liquid into the fermentation medium for fermentation and culturing to obtain γ-polyglutamic acid;

[0038] The Bacillus subtilis is Bacillus subtilis YB18;

[0039] The Bacillus subtilis YB18 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number of CGMCC No. 17642, the classification name of Bacillus subtilis, and the deposit date of April 28, 2019;

[0040] The initial number of viable bacteria contained in the inoculated seed solution is 1.0 to 1.5×10 8 cfu / mL, preferably 1.25×10 8 cfu / mL; the volume ratio of the seed liquid to the fermentation medium is 4-5:2500, preferably 4.5:2500.

[0041] In the present invention, the liquid culture medium in step (1) uses water as a solvent and comprises the following components in the following concentrations:

[0042] Peptone 8-12 g / L, preferably 10 g / L;

[0043] Beef extract powder 2.5-3.5 g / L, preferably 3 g / L;

[0044] Glucose 18-22 g / L, preferably 20 g / L;

[0045] Sodium chloride 4-6 g / L, preferably 5 g / L.

[0046] In the present invention, the temperature of the activation culture in step (1) is 35-39°C, preferably 36-38°C, and more preferably 37°C; the time of the activation culture is 16-20h, preferably 17-19h, and more preferably 18h; the activation culture is shaking culture; the shaking speed is 140-160rpm, preferably 150rpm.

[0047] In the present invention, the temperature of the fermentation culture in step (2) is 35-39°C, preferably 36-38°C, and more preferably 37°C; the time of the fermentation culture is 45-72h, preferably 50-67h, more preferably 55-62h, and more preferably 58.5h; and the fermentation culture is static culture.

[0048] The solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0049] Example 1

[0050] The preparation method of each liter of liquid culture medium is as follows: take 800 mL of water, then add 8 g of peptone, 3.5 g of beef extract powder, 20 g of glucose, and 6 g of sodium chloride, then make up to 1 L with water, sterilize at 121° C. for 20 min, and obtain a liquid culture medium.

[0051] The preparation method of each liter of fermentation medium is as follows: 3000 mL of water is taken, then 800 g of brewer's grains with a water content of 60% and 30 g of monosodium glutamate are added, and the mixture is sterilized at 121° C. for 20 minutes to obtain the fermentation medium.

[0052] The Bacillus subtilis YB18 slant was selected and inoculated into 50 mL of liquid culture medium. The culture was shaken at 35°C and 160 rpm for 18 h to obtain a viable bacterial count of 1.20 × 10 8 cfu / mL of seed liquid. 4 mL of seed liquid was inoculated into 2500 mL of fermentation medium and statically cultured at 35°C for 60 h. The yield of γ-polyglutamic acid was detected by CTAB precipitation method, and 1000 g of brewer's grains could be fermented to obtain 30.75 g of γ-polyglutamic acid.

[0053] Example 2

[0054] The preparation method of each liter of liquid culture medium is as follows: take 800 mL of water, then add 12 g of peptone, 3 g of beef extract powder, 18 g of glucose, and 4 g of sodium chloride, then make up to 1 L with water, sterilize at 121° C. for 20 min, and obtain a liquid culture medium.

[0055] The preparation method of each liter of fermentation medium is as follows: 5000 mL of water is taken, and then 1250 g of brewer's grains with a water content of 65% and 50 g of monosodium glutamate are added, and the mixture is sterilized at 121° C. for 20 minutes to obtain the fermentation medium.

[0056] The Bacillus subtilis YB18 slant was selected and inoculated into 50 mL of liquid culture medium. The culture was shaken at 39°C and 140 rpm for 16 h to obtain a viable cell count of 1.0 × 10 8 cfu / mL of seed liquid. 5 mL of seed liquid was inoculated into 2500 mL of fermentation medium and cultured statically at 39°C for 45 h. The yield of γ-polyglutamic acid was detected by CTAB precipitation method, and 31.25 g of γ-polyglutamic acid could be obtained from 1000 g of brewer's grains.

[0057] Example 3

[0058] The preparation method of each liter of liquid culture medium is as follows: take 800 mL of water, then add 10 g of peptone, 2.5 g of beef extract powder, 22 g of glucose, and 5 g of sodium chloride, then make up to 1 L with water, sterilize at 121° C. for 20 min, and obtain a liquid culture medium.

[0059] The preparation method of each liter of fermentation medium is as follows: 5000 mL of water is taken, then 5000 g of brewer's grains with a water content of 62% and 75 g of monosodium glutamate are added, and the mixture is sterilized at 121° C. for 20 minutes to obtain the fermentation medium.

[0060] The Bacillus subtilis YB18 slant was selected and inoculated into 50 mL of liquid culture medium. The culture was shaken at 37°C and 150 rpm for 20 h to obtain a viable count of 1.5 × 10 8 cfu / mL of seed liquid. 4 mL of seed liquid was inoculated into 2500 mL of fermentation medium and cultured statically at 38°C for 72 h. The yield of γ-polyglutamic acid was detected by CTAB precipitation method, and 33.45 g of γ-polyglutamic acid could be obtained from 1000 g of brewer's grains.

[0061] Comparative Example 1

[0062] The method of comparative example 1 of the present application was set up according to the method of Example 1. The difference from Example 1 was that the fermentation medium formula of comparative example 1 was: 0.5 L / L yellow water, 50 g / L reducing sugar content of corn saccharification liquid, 60 g / L monosodium glutamate, 9 g / L sodium chloride, 4 g / L K2HPO4, and pH value was 5. After culturing with the fermentation medium, the yield of γ-polyglutamic acid detected by CTAB precipitation method was 14.51 g / L.

[0063] Application Example 1

[0064] The cost of the fermentation medium of Examples 1 to 3 and Comparative Example 1 was calculated, and the cost per 100 g of γ-polyglutamic acid produced by fermentation was compared. The results are shown in Table 1.

[0065] Table 1100g γ-polyglutamic acid production raw material cost

[0066] Example 1 Example 2 Example 3 Comparative Example 1 Cost / Yuan 3.356084 3.3024 3.085201 9.264483

[0067] The brewer's grains are calculated at 0.0006 yuan / g; the monosodium glutamate is calculated at 0.01152 yuan / g; the yellow water is calculated at 1.001 yuan / mL; the corn saccharification liquid is calculated at 0.0018 yuan / mL; the sodium chloride is calculated at 0.0158 yuan / g; and the dipotassium hydrogen phosphate is calculated at 0.035 yuan / g.

[0068] As can be seen from the above embodiments, the present invention provides a fermentation medium of Bacillus subtilis and a method for producing γ-polyglutamic acid. The Bacillus subtilis of the present invention can use brewer's grains as a fermentation medium to ferment and produce γ-polyglutamic acid, which seeks a new path for the comprehensive utilization of brewer's grains; and the content of γ-polyglutamic acid produced after fermentation is high. The culture medium components for fermenting and producing γ-polyglutamic acid are simple, easy to obtain, and low in cost, providing a new way for the production of γ-polyglutamic acid.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for producing γ-polyglutamic acid using a fermentation medium, characterized in that: The steps include: (1) inoculating Bacillus subtilis into a liquid culture medium for activation culture to obtain a seed solution; (2) inoculating the seed liquid into the fermentation medium according to claim 1 for fermentation and culturing to obtain γ-polyglutamic acid; The Bacillus subtilis is Bacillus subtilis YB18; The Bacillus subtilis YB18 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number of CGMCC No. 17642, the classification name of Bacillus subtilis, and the deposit date of April 28, 2019; The initial number of viable bacteria contained in the inoculated seed solution was 1.5×10 8 cfu / mL; the volume ratio of the seed solution to the fermentation medium is 4:2500; The liquid culture medium in step (1) uses water as a solvent and comprises the following components in the following concentrations: Peptone 10g / L, beef extract powder 2.5g / L, glucose 22g / L, sodium chloride 5g / L; The activation culture temperature in step (1) is 37° C. The activation culture time is 20h; The activation culture is shaking culture; The oscillation speed is 150 rpm; The fermentation medium is composed of the following components in parts by weight: 10 parts of brewer's grains, 0.15 parts of monosodium glutamate, 10 parts of water; The water content of the brewer's grains is 62%; The fermentation temperature in step (2) is 38°C; The fermentation culture time is 72h; The fermentation culture is a static culture.