阅读说明：本技术 枯草芽孢杆菌YB18及其在发酵生产高分子量聚γ-谷氨酸中的应用 (Bacillus subtilis YB18 and application thereof in fermentation production of high molecular weight poly-gamma-glutamic acid ) 是由 王风青 罗惠波 毕长富 王川 于 2019-11-11 设计创作，主要内容包括：本发明公开了枯草芽孢杆菌YB18及其在发酵生产高分子量聚γ-谷氨酸中的应用,保藏编号为CGMCC No.17642,由中国微生物菌种保藏管理委员会普通微生物中心保藏,分类命名为枯草芽孢杆菌Bacillus subtilis,保藏日期为2019年4月28日。上述应用为将所述枯草芽孢杆菌YB18以白酒副产物黄水为基质经液态深层发酵合成高分子量的聚γ-谷氨酸。本发明筛选培育出的株枯草芽孢杆菌YB18可以利用白酒副产物黄水经液态深层发酵合成高产值的γ-PGA,可以使白酒产业的副产物资源化利用,得到一高产值新产品γ-PGA,有利于进一步提高白酒产业的效益。(The invention discloses Bacillus subtilis YB18 and application thereof in fermentation production of high molecular weight poly-gamma-glutamic acid, wherein the preservation number is CGMCC No.17642, the Bacillus subtilis is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation date is 2019, 4 and 28 days.)

1, kinds of Bacillus subtilis YB18, characterized in that the preservation number is CGMCC No.17642, the Bacillus subtilis is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, the classification name is Bacillus subtilis, and the preservation date is 2019, 4 months and 28 days.

2. The use of the bacillus subtilis YB18 of claim 1 in the production of high molecular weight poly-gamma-glutamic acid by using yellow water as a matrix and carrying out liquid submerged fermentation on microorganisms.

3. The application of the bacillus subtilis YB18 in the production of high molecular weight poly-gamma-glutamic acid by taking yellow water as a matrix through liquid submerged fermentation of microorganisms according to claim 2, wherein the bacillus subtilis YB18 is synthesized into the high molecular weight poly-gamma-glutamic acid by taking white spirit byproduct yellow water as a matrix through liquid submerged fermentation, and the pretreatment method of the yellow water is to adjust the pH value to 4-8 by using alkali liquor.

4. The application of the bacillus subtilis YB18 in the production of high molecular weight poly-gamma-glutamic acid by using yellow water as a matrix through liquid submerged fermentation of microorganisms according to claim 2 is characterized in that the bacillus subtilis YB18 on a slant culture medium is transferred to a liquid seed culture medium, and the liquid seed is obtained after shaking and constant temperature culture for 10-24 hours at 20-45 ℃ and 100-300 r/min.

5. The use of the bacillus subtilis YB18 in the liquid submerged fermentation of microorganisms using yellow water as a matrix to produce high molecular weight poly-gamma-glutamic acid according to claim 4, wherein the formula of the slant culture medium comprises: 5.0-15.0 g/L of peptone, 1.0-10.0 g/L of beef powder, 5.0-15.0 g/L of sodium chloride and 15.0-20.0 g/L of agar, and the pH value is 6.5-7.4.

6. The use of Bacillus subtilis YB18 in the production of high molecular weight poly-gamma-glutamic acid by the submerged fermentation of microorganisms using yellow water as a matrix according to claim 4, wherein the formula of the liquid seed culture medium comprises: 0.2-0.8L/L of yellow water, 1.0-10.0 g/L of peptone, 2.0-100.0 g/L of reducing sugar in corn saccharification liquid, 2-15.0 g/L of NaCl, and 4-8 of pH value.

7. The application of the bacillus subtilis YB18 in the production of the high molecular weight poly-gamma-glutamic acid by taking yellow water as a matrix through liquid submerged fermentation of microorganisms is characterized in that liquid seeds are inoculated into a fermentation culture medium according to the inoculation amount of 1-10% (v/v), and after shaking and constant temperature culture is carried out for 10-100 h at the temperature of 20-45 ℃ and the speed of 100-300r/min, the high molecular weight poly-gamma-glutamic acid is obtained through post-treatment.

8. The use of bacillus subtilis YB18 in the production of high molecular weight poly-gamma-glutamic acid by using yellow water as a matrix microorganism through liquid submerged fermentation according to claim 7, wherein the formula of the fermentation medium comprises: 0.2-0.8L/L of yellow water, 2.0-100.0 g/L of reducing sugar in corn saccharification liquid, 5-100 g/L of monosodium glutamate and 2.0-15.0 g/L, K of sodium chloride₂HPO₄0.1-10.0 g/L, and pH value of 4-8.

9. The use of bacillus subtilis YB18 in the production of high molecular weight poly-gamma-glutamic acid by the submerged fermentation of microorganisms liquid with yellow water as a matrix according to claim 7, wherein the post-treatment comprises dilution, centrifugation, precipitation, dissolution and freeze-drying in sequence.

10. The high molecular weight poly-gamma-glutamic acid prepared by the application of the bacillus subtilis YB18 of in the liquid submerged fermentation of the microorganism taking yellow water as the matrix, as claimed in any one of claims 2 to 9, wherein the molecular weight of the high molecular weight poly-gamma-glutamic acid is 90-130 Da.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an application method of kinds of high molecular weight poly-gamma-glutamic acid produced by microbial fermentation with yellow water as a matrix and microorganisms thereof.

Background

Poly gamma-glutamic acid (hereinafter referred to as gamma-PGA) is biodegradable nontoxic anionic high molecular weight homopolymers formed by D-and L-glutamic acid monomers through α -amino and gamma-carboxyl, which can be synthesized by microbial fermentation⁺、Mg²⁺、K⁺、NH⁴⁺Or Ca²⁺) Has good biocompatibility and degradability, can be applied to various fields together with derivatives thereof, and has great commercial value.

1) Application of gamma-PGA in food industry: the gamma-PGA can be used as a novel food additive, an antifreeze, a mineral absorption promoter, an astringent, a stabilizer, etc. in foods.

2) The application of gamma-PGA in the field of medicine: Gamma-PGA with different molecular weights can be used as different drug sustained-release agents, nanoparticles (gamma-PGA-Phe NPs) synthesized by the gamma-PGA and the L-phenylalanine have the possibility of treating retinal diseases, chitosan/gamma-PGA PECs can effectively inhibit inflammatory cells, and the polymer material polymerized by the gamma-PGA can be used for packaging films of medicines, degradable operation lines and the like.

3) The application of gamma-PGA in the field of environmental protection: the gamma-PGA can interact with various harmful substances, and the polluted environment can be repaired by utilizing an adhesion and concentration mechanism between the gamma-PGA and the pollutants. If the water absorption of the gamma-PGA is combined with the soil, the moisture absorption capacity, the ventilation property and the fertilizer retention property of the soil can be improved, and the method can play an active role in the large-scale reconstruction of barren mountains and deserts. The gamma-PGA can absorb pesticide and fertilizer, prevent loss and release slowly, improve crop cultivation conditions and improve fertilizer utilization rate. The gamma-PGA is used as a biological flocculant to treat water pollution, can be biodegraded, and can cause secondary pollution to the environment after long-term use.

4) The application of gamma-PGA in the agricultural field: when the gamma-PGA is applied to the agricultural field as water-absorbent resin, pesticide slow-release , fertilizer embedding material and the like, the gamma-PGA can show better biocompatibility, ultrahigh water absorption and good biodegradability, can effectively absorb water and moisturize soil, inhibit the quick decomposition or loss of components of fertilizer or pesticide and play a good slow-release role.

5) Application of γ -PGA in the industrial field: the polymer material polymerized by gamma-PGA has similar properties to plastics, can be used for manufacturing leather, fiber, food packaging film and the like, and has proper strength, transparency and better elasticity.

In the traditional white spirit production process, a large amount of free water generated after microbial catabolism dissolves out acid, soluble starch, yeast dissolved-out substances, reducing sugar, tannin, alcohol and flavor precursor substances in fermented grains, then gradually settles with water which is not utilized by microorganisms in the fermented grains, and finally slowly deposits at the bottom of a cellar pool to form a brownish fluid liquid which is called yellow water as a by-product in the brewing process of the strong aromatic white spirit, wherein the yellow water is also called yellow serofluid and contains flavor and taste substances such as alcohol, acid, aldehyde, ester and the like, and also contains beneficial microorganisms domesticated for a long time, sugar substances, nitrogen-containing compounds, a small amount of tannin, pigments and other organic substances.

At present, the comprehensive utilization of the yellow water has two approaches, wherein is used for improving the quality of the produced liquor, and the new product is developed for promoting the aging of pit mud, carrying out manual cellar culture and pit maintenance, and the secondary fermentation of the mixed fermented grains is a main application measure for improving the quality of the produced liquor by the yellow water.

Disclosure of Invention

The invention aims to overcome the problems in the prior art, and provides application methods for synthesizing high-molecular-weight polymer gamma-PGA by yellow water fermentation of a white spirit byproduct and microorganisms thereof, so that the byproducts in the white spirit industry can be recycled, and a high-yield new product gamma-PGA can be obtained.

The aspect of the invention provides bacillus subtilis YB18 with the preservation number of CGMCC No.17642, which is preserved by the common microorganism center of China Committee for culture Collection of microorganisms and is named as bacillus subtilis by classification, and the preservation date is 2019, 4 months and 28 days.

In another aspect of the invention, the application of the bacillus subtilis YB18 in the production of high molecular weight poly-gamma-glutamic acid by using yellow water as a matrix and carrying out liquid submerged fermentation on microorganisms is provided.

According to embodiments of the application of the bacillus subtilis YB18 in the production of high molecular weight poly-gamma-glutamic acid by using yellow water as a matrix through liquid submerged fermentation of microorganisms, the bacillus subtilis YB18 synthesizes high molecular weight poly-gamma-glutamic acid by using white spirit byproduct yellow water as a matrix through liquid submerged fermentation, wherein the pretreatment method of the yellow water is to adjust the pH value to 4-8 by using alkali liquor.

According to embodiments of the application of the bacillus subtilis YB18 in the production of high molecular weight poly-gamma-glutamic acid by using yellow water as a matrix through liquid submerged fermentation, the bacillus subtilis YB18 on a slant culture medium is transferred into a liquid seed culture medium, and the liquid seed is obtained after shaking constant-temperature culture for 10-24 hours at 20-45 ℃ and 100-300 r/min.

According to embodiments of the application of the bacillus subtilis YB18 in the production of high molecular weight poly-gamma-glutamic acid by using yellow water as a matrix and carrying out liquid submerged fermentation on microorganisms, the formula of the slant culture medium comprises 5.0-15.0 g/L of peptone, 1.0-10.0 g/L of beef powder, 5.0-15.0 g/L of sodium chloride and 15.0-20.0 g/L of agar, and the pH value is 6.5-7.4.

According to embodiments of the application of the bacillus subtilis YB18 in the production of high molecular weight poly-gamma-glutamic acid by using yellow water as a matrix through liquid submerged fermentation of microorganisms, the formula of the liquid seed culture medium comprises 0.2-0.8L/L of yellow water, 1.0-10.0 g/L of peptone, 2.0-100.0 g/L of reducing sugar in corn saccharification liquid and 2-15.0 g/L of NaCl, and the pH value is 4-8.

According to embodiments of the application of the bacillus subtilis YB18 in the production of the high molecular weight poly-gamma-glutamic acid by using yellow water as a matrix and carrying out liquid submerged fermentation on microorganisms, liquid seeds are inoculated into a fermentation culture medium according to the inoculation amount of 1-10% (v/v), and after shaking and constant-temperature culture is carried out at 20-45 ℃ and 100-300r/min for 10-100 h, the high molecular weight poly-gamma-glutamic acid is obtained through post-treatment.

According to embodiments of the application of the bacillus subtilis YB18 in the production of high molecular weight poly-gamma-glutamic acid by using yellow water as a matrix through liquid submerged fermentation of microorganisms, the formula of the fermentation medium comprises 0.2-0.8L/L of yellow water, 2.0-100.0 g/L of reducing sugar content of corn saccharification liquid, 5-100 g/L of monosodium glutamate and 2.0-15.0 g/L, K of sodium chloride₂HPO₄0.1-10.0 g/L, and pH value of 4-8.

According to embodiments of the application of the bacillus subtilis YB18 in the liquid submerged fermentation of microorganisms taking yellow water as a matrix to produce high molecular weight poly-gamma-glutamic acid, the post-treatment comprises dilution, centrifugation, precipitation, dissolution and freeze drying which are sequentially carried out.

In the aspect of , the invention provides high molecular weight poly-gamma-glutamic acid prepared by applying the bacillus subtilis YB18 in the liquid submerged fermentation production of the high molecular weight poly-gamma-glutamic acid by taking yellow water as a matrix microorganism, wherein the molecular weight of the high molecular weight poly-gamma-glutamic acid is 90-130 Da.

The bacillus subtilis YB18 screened and cultured by the invention can utilize yellow water of a white spirit byproduct to synthesize high-yield gamma-PGA through liquid submerged fermentation, thereby not only recycling the byproduct of the white spirit industry, but also obtaining a high-yield new product gamma-PGA, and being beneficial to further and improving the benefit of the white spirit industry.

Drawings

Fig. 1 shows a uv scanning analysis pattern of the γ -PGA sample obtained in the example.

FIG. 2 shows an infrared scanning analysis spectrum of the γ -PGA sample obtained in the example.

Detailed Description

All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.

Any of the features disclosed in this specification may be replaced by alternative features serving an equivalent or similar purpose unless expressly stated otherwise, i.e. each feature is simply examples of the series of equivalent or similar features unless expressly stated otherwise.

According to an exemplary embodiment of the invention, strains of Bacillus subtilis YB18(Bacillus subtilis YB18) are obtained by screening firstly, are preserved in the China general microbiological culture Collection center (CGMCC, zip code: 100101, address: Kogyang district Tunglu, China academy of sciences) of China Committee of culture Collection of microorganisms, have the preservation number of CGMCC No.17642, are classified and named as Bacillus subtilis, and have the preservation time of 2019, 4 months and 28 days.

First, a method for screening a strain will be specifically described.

1. Sample processing

On an ultra-clean workbench, scooping spoons of natto food or sucking a proper amount of yellow sauce water in a sterilized 250mL triangular flask filled with glass beads and 50mL of sterile water by using a small spoon, sealing the triangular flask by using a sealing film, placing the triangular flask in a constant temperature oscillator at 30-37 ℃ for l 0-30 min, taking 1mL of the treated sample, inoculating the sample into the 250mL triangular flask filled with 50mL of basal medium, and performing constant temperature shaking enrichment culture at 30-37 ℃ for 10-24 h to obtain an enrichment culture solution.

2. Separating and purifying strains

(1) Separation: taking 1mL of the enrichment culture solution, coating the enrichment culture solution on a flat plate filled with a selective culture medium by adopting a gradient dilution method, culturing at a constant temperature of 30-39 ℃ for 20-36 h, observing and recording the colony condition, selecting a single colony with a large diameter and viscosity, and carrying out streaking separation for multiple times until a pure colony is obtained and used as a primary screening strain.

(2) Re-screening: inoculating the primary screened strains into a fresh liquid seed culture medium, carrying out shake culture at a constant temperature of 30-39 ℃ for 12-24 h, then respectively inoculating the primary screened strains into 250mL triangular flasks filled with 50mL of sterilized basic fermentation culture medium according to the inoculation amount of 1-10% (V/V), and carrying out shake culture at a constant temperature of 100-250 r/min at 30-39 ℃ for 24-100 h. And (4) performing a fermentation experiment for three times, detecting the concentration of the gamma-PGA in the fermentation liquor, and preserving the gamma-PGA with high yield on an inclined plane for later use.

3. Mutagenesis and breeding of strains

Re-screening the strain preserved on the inclined plane, inoculating rings into a 250mL triangular flask filled with 25mL seed culture medium, carrying out constant temperature shaking culture at 30-40 ℃ and 100-300r/min for 12-24 h to enable the strain to be in logarithmic phase, then taking 50mL culture solution, centrifuging at 4000r/min for 10min to collect the strain, washing the strain 3-4 times with normal saline, adjusting the concentration of the strain with normal saline to prepare the strain with the cell concentration of 10^6-8Taking 5mL of bacterial liquid in a culture dish (with a magnetic rod) with the diameter of 90mm, placing the culture dish on a magnetic stirrer in ultra-clean work, adjusting the distance between the culture dish and an ultraviolet lamp to be 10-30 cm, starting the magnetic stirrer, opening a dish cover, carrying out mutagenesis treatment for 5-15 min under the ultraviolet lamp with the power of 15-30W in the dark, taking the treatment liquid, coating and inoculating the treatment liquid in a selective culture medium, carrying out constant-temperature culture at the temperature of 30-39 ℃ for 24-48 h, and picking a single bacterial colony to transfer to a slant culture medium for constant-temperature culture at the temperature of 30-39 ℃ for 24-48 h for later use.

Then, inoculating the strain cultured on the inclined plane into a liquid basic fermentation culture medium, measuring the concentration of the gamma-PGA after finishing the culture, repeating the fermentation experiment for 3 times, and selecting the corresponding strain with high content of the gamma-PGA as a good strain to be bred to obtain the strain YB 18. And continuously passaging the strain YB18 on a slant culture medium for 10 times, performing a genetic stability experiment on the strain YB18, performing a fermentation experiment after each passage, and detecting the content of gamma-PGA.

4. Strain identification

The strain YB18 is characterized by cell morphology observation and physiological and biochemical characteristic experiments, wherein the strain has a rod shape, is gram-positive, forms spores, has no cyst expansion, is catalase-positive and oxidase-positive, and can utilize α -D-glucose, gelatin, D-maltose, D-fructose-6-phosphate, D-trehalose, D-galacturonic acid, galactonolactone, glucuronamide, β -methyl-D-glucoside, N-acetyl-D-glucosamine, sucrose, gentiobiose, glycerol, inositol, D-mannitol, D-sorbitol, pinodipond, D-mannose, D-fructose, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-pyroglutamic acid, L-serine, pectin, D-gluconic acid, mucic acid, glycouronic acid, L-lactic acid, citric acid, L-malic acid, dextrin and formic acid for growth, D-glucose-6-phosphate, D-cellobiose, D-glucurone, L-proline, L-hydroxypivalic acid, L-glutamic acid, D-hydroxypivalic acid, D-D.

16S rDNA sequencing of the above YB18 strain resulted in the following:

TCGGCGGCTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTGCA

the identification result is that kinds of Bacillus subtilis YB18(Bacillus subtilis YB18) are obtained by screening and cultivating.

The bacillus subtilis YB18 is applied to the production of high molecular weight poly-gamma-glutamic acid by using yellow water as a matrix and carrying out liquid submerged fermentation on microorganisms. Specifically, bacillus subtilis YB18 is subjected to liquid submerged fermentation by taking yellow water which is a white spirit byproduct as a matrix to synthesize poly-gamma-glutamic acid with high molecular weight, wherein the pretreatment method of the yellow water is to adjust the pH value to 4-8 by using alkali liquor.

During specific fermentation production, the bacillus subtilis YB18 on the slant culture medium is transferred to a liquid seed culture medium, and the liquid seed is obtained after shaking constant-temperature culture for 10-24 h at 20-45 ℃ and 100-300 r/min.

Wherein, the formula of the slant culture medium preferably comprises: 5.0-15.0 g/L of peptone, 1.0-10.0 g/L of beef powder, 5.0-15.0 g/L of sodium chloride and 15.0-20.0 g/L of agar, and the pH value is 6.5-7.4; the formulation of the liquid seed culture medium preferably comprises: 0.2-0.8L/L of yellow water, 1.0-10.0 g/L of peptone, 2.0-100.0 g/L of reducing sugar in corn saccharification liquid, 2-15.0 g/L of NaCl, and 4-8 of pH value.

Then inoculating the liquid seeds into a fermentation medium according to the inoculation amount of 1-10% (v/v), carrying out shake constant-temperature culture at 20-45 ℃ and 100-300r/min for 10-100 h, and carrying out post-treatment to obtain the high molecular weight poly-gamma-glutamic acid, wherein the formula of the fermentation medium preferably comprises: 0.2-0.8L/L of yellow water, 2.0-100.0 g/L of reducing sugar in corn saccharification liquid, 5-100 g/L of monosodium glutamate and 2.0-15.0 g/L, K of sodium chloride₂HPO₄0.1-10.0 g/L, and pH value of 4-8.

The post-treatment can comprise dilution, centrifugation, precipitation, dissolution and freeze drying which are sequentially carried out, for example, after the fermentation is finished, distilled water with 2-3 times of volume is added for dilution, the centrifugation is carried out for 10-20 min at 8000-10000 r/min, supernatant is poured out, absolute ethyl alcohol with 2-4 times of volume is added for precipitation, the supernatant is discarded, the distilled water is added for full dissolution, and after pre-freezing is carried out for 5-6 h at-80 ℃, vacuum freeze drying is carried out at-60 ℃ to obtain the poly-gamma-glutamic acid.

Through detection, the poly-gamma-glutamic acid prepared by the invention has high molecular weight of 90-130 Da.

In order to make the technical means, inventive features, objectives and effects achieved by the present invention easily understandable, the present invention is further described in by examples.