Bacillus subtilis MJ501 for producing gamma-polyglutamic acid from deep sea and application of bacillus subtilis MJ501

Abstract

The invention discloses bacillus subtilis MJ501 for producing gamma-polyglutamic acid from deep sea and application of the bacillus subtilis MJ501, and belongs to the technical field of microorganisms. The bacillus subtilis MJ501 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.31738. The bacillus subtilis MJ501 is a bacillus subtilis strain. Bacillus subtilis MJ501 is obtained through screening, and gamma-polyglutamic acid is obtained through fermentation production through the steps of strain activation, liquid fermentation and purification and extraction. The bacillus subtilis MJ501 provided by the invention can be used for converting glutamic acid and the like to generate polyglutamic acid, and also can be used for generating polyglutamic acid under the condition that a culture medium is not added with glutamic acid. Therefore, the bacillus subtilis MJ501 can enrich a strain resource library for producing the gamma-polyglutamic acid, can also improve the fermentation yield of the gamma-polyglutamic acid, has great research and development values, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a deep-sea Bacillus subtilis MJ501 strain producing gamma-polyglutamic acid and an application thereof. Background Art

[0002] γ-Polyglutamic acid (γ-PGA) is a homoanionic polypeptide composed of 500 to 5,000 glutamic acid monomers, consisting of D-glutamic acid and L-glutamic acid units linked by γ-amide bonds. Known as natto gum in the International Cosmetic Pharmacopoeia, γ-PGA is water-soluble, water-absorbent, metal-binding, and biodegradable. It is edible and non-toxic to humans and the environment. Therefore, γ-PGA has broad application prospects in medicine (drug sustained-release materials, tissue engineering materials, etc.), food (antifreeze materials, etc.), daily necessities (moisturizing, skin care, whitening, etc.), agriculture (biofertilizers, etc.), and the environment (water treatment, etc.).

[0003] The main methods for producing γ-polyglutamic acid include chemical synthesis, enzymatic conversion, and microbial fermentation. Compared with other methods, microbial fermentation offers mature production processes, easily regulated and optimized conditions, a shorter growth cycle, and easy access to the target product with high yields. However, currently, there are relatively few strains capable of efficiently producing polyglutamic acid, and further exploration and expansion of these production strains are needed. It is crucial to explore more bacterial strains, enrich the library of γ-polyglutamic acid-producing strains, and increase the fermentation yield of γ-polyglutamic acid.

[0004] Molecular weight is an important feature of the application of γ-polyglutamic acid. Different molecular weights are applicable to different fields. Different molecular weights can be obtained by using different fermentation strains and fermentation raw materials, or by changing the post-processing method. Polyglutamic acid produced by Bacillus generally has a higher molecular weight, with an average molecular weight of 10 5 and 8×10 6 Low- to medium-molecular-weight polyglutamic acid (PG) has better solubility and bioavailability, making it suitable for a variety of applications, such as food additives, drug carriers, and moisturizers. Most terrestrial γ-PG production strains exhibit high viscosity during fermentation, hindering further purification and extraction. Furthermore, the monomeric glutamic acid has a high degree of polymerization, with molecular weights ranging from several hundred to several thousand kDa. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention provides a deep-sea-derived Bacillus subtilis MJ501 strain that produces γ-polyglutamic acid and its applications. The Bacillus subtilis MJ501 can convert glutamate into polyglutamic acid and synthesize polyglutamic acid without exogenous glutamate addition, thereby enriching the resource base of γ-polyglutamic acid-producing strains and increasing the fermentation yield of γ-polyglutamic acid.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: The present invention provides a deep-sea Bacillus subtilis MJ501 that produces γ-polyglutamic acid. The classification of the Bacillus subtilis MJ501 is named Bacillus subtilis , and its deposit number is CGMCC No. 31738.

[0007] Furthermore, the colony of the Bacillus subtilis MJ501 is milky white, grows fast, is moist, has wrinkles, and can be drawn into threads; it can form a transparent circle in a positively charged neutral red plate, and droops like a stalactite due to the presence of mucus when inverted for culture.

[0008] Furthermore, the 16S rRNA sequence of the Bacillus subtilis MJ501 is shown as SEQ ID No. 1.

[0009] The present invention also provides the use of the Bacillus subtilis MJ501 in producing gamma-polyglutamic acid.

[0010] Furthermore, the production steps of the γ-polyglutamic acid are: (1) culturing the activated marine Bacillus subtilis in liquid to obtain a seed solution; (2) fermenting the seed liquid to obtain a fermentation liquid; (3) The fermentation broth is centrifuged, the obtained precipitate is redissolved, ultrasonically disrupted, and then dialyzed for purification, and polyglutamic acid is obtained after drying.

[0011] Furthermore, in step (1), the temperature for activation and liquid culture is 25-30°C, the culture speed is 150 r / min, and the culture time is 4-8 h.

[0012] Furthermore, in step (2), the inoculation amount of the seed liquid is 3-5% v / v, the fermentation temperature is 28-37°C, the culture speed is 180 r / min, and the culture time is 48-72 h.

[0013] Furthermore, the composition of the liquid seed culture medium in the liquid culture is: 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

[0014] Furthermore, the composition of the fermentation medium in the fermentation culture is: 30 g / L glucose, 5 g / L yeast extract, 30 g / L sodium glutamate, 0.25 g / L magnesium sulfate, 2 g / L dipotassium hydrogen phosphate; or: 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

[0015] Furthermore, the molecular weight of the dialyzed solution in step (3) is 3500 Da, and the dialysis time is 1 week.

[0016] The present invention also provides the use of the marine Bacillus subtilis or the gamma-polyglutamic acid in plant preservation.

[0017] Furthermore, the application steps of the plant preservation are: first, dissolving the γ-polyglutamic acid, glycerol and casein in water to prepare a membrane liquid, and directly soaking the plant with the membrane liquid; or drying the membrane liquid into a film, and then using it to wrap the plant.

[0018] Furthermore, the mixing volume ratio of the γ-polyglutamic acid, glycerol and casein is 10-15:4:1.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: the present invention screens a strain of Bacillus subtilis that produces γ-polyglutamic acid from deep-sea sediments of the Indian Ocean. Bacillus subtilis MJ50 can convert glutamate into polyglutamate during fermentation and can also synthesize low-molecular-weight polyglutamate without exogenous glutamate addition. The extreme conditions of the deep-sea environment (such as high pressure and low temperature) make Bacillus subtilis MJ501 more adaptable to the environment.

[0020] The Bacillus subtilis MJ501 is capable of stable growth and fermentation within a wide range of temperature and pressure, reducing environmental control costs during the production process. Deep-sea strains generally have a lower risk of pathogenicity and are suitable for use in areas with high safety requirements, such as food and medicine. Bacillus subtilis MJ501 can not only efficiently utilize glutamate, but also achieve precise control of product molecular weight through fermentation condition optimization and enzymatic regulation. It also has good environmental adaptability and cost-effectiveness, simple process, mild conditions, and environmental friendliness, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a plate colony picture of the Bacillus subtilis MJ501.

[0022] Figure 2 It is a neutral red plate colony image of the Bacillus subtilis MJ501.

[0023] Figure 3 This is the SEM image (500 μm) of the Bacillus subtilis MJ501.

[0024] Figure 4 This is an amino acid detection diagram of polyglutamic acid produced by the Bacillus subtilis MJ501.

[0025] Figure 5 This is a diagram showing the FT-IR detection results of polyglutamic acid produced by the Bacillus subtilis MJ501.

[0026] Figure 6 The preservation effect of γ-polyglutamic acid film on fresh-cut apple slices, where (A) is the control; (B) is the soaking group; (C) is the film-wrapped group; (D) is the control after 9 days; (E) is the film-wrapped group after 9 days; (F) is the soaking group after 9 days. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0028] In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0029] In the present invention, the culture medium used is as follows: (1) The composition of 2216E agar medium is as follows: peptone 5.0 g / L, yeast extract powder 1.0 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.98 g / L, sodium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium carbonate 0.16 g / L, potassium bromide 0.08 g / L, strontium chloride 0.034 g / L, boric acid 0.022 g / L, sodium silicate 0.004 g / L, sodium fluoride 0.0024 g / L, ammonium nitrate 0.0016 g / L, sodium hydrogen phosphate 0.008 g / L, agar 15.0 g / L, pH 7.6±0.2.

[0030] (2) The composition of MRS agar medium is as follows: peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, pH 6.5±0.2.

[0031] (3) The components of the polyglutamic acid screening medium are: glucose 10 g / L, yeast extract 5 g / L, sodium glutamate 5 g / L, KH2PO4 0.5 g / L, MgSO4 0.1 g / L, and agar 20 g / L.

[0032] (4) The composition of the liquid seed culture medium is: trypsin 10 g / L, yeast powder 5 g / L, and sodium chloride 10 g / L.

[0033] Example 1 1. Screening of Bacillus subtilis strains from marine sources Take 2 g of surface mud sample from the 2546 m deep sea of the Indian Ocean and put it into sterile water. Take 0.1 mL of the bacterial suspension and perform gradient dilution in a 10 mL sterile centrifuge tube and spread it on 2216E agar medium. Incubate it at 30°C in an inverted constant temperature. After a single colony grows, pick the colony and dilute it again and culture it at a constant temperature. Then, inoculate the single colony onto MRS agar medium for constant temperature screening and culture at 37°C. After the culture is completed, store the slant at 4°C and store it at -80°C with 25% glycerol.

[0034] like Figure 1 and Figure 2 Morphological identification of the strain is shown in Figure 2. The colonies are milky white, rapidly growing, moist, wrinkled, and capable of forming threads when lifted. They form transparent circles on positively charged neutral red plates and droop like stalactites when inverted due to the presence of mucus. This strain is suitable for growth at 20-40°C.

[0035] 2. Molecular biological identification of strains The DNA of the strain was extracted using a bacterial genomic DNA extraction kit (Tiangen®). 16S sequencing was commissioned to Bioengineering (Shanghai) Co., Ltd., and whole genome sequencing was commissioned to a sequencing company. The sequencing results showed that the length of the 16S rRNA gene of the strain was 1433 bp, and its nucleotide sequence is shown in SEQ ID No. 1. By comparing the sequencing results with the NCBI database, the known sequence with the highest homology to the 16S rDNA sequence of the bacteria was obtained. By comparing the phylogenetic tree, it can be seen that it is Bacillus subtilis Therefore, based on the above data, the isolated strain was identified as Bacillus subtilis ( Bacillus subtilis ), independently named Bacillus subtilis Bacillus subtilis MJ501.

[0036] The strain MJ501 was deposited in the China General Microbiology Center (CGMCC), China National Microbiology and Culture Collection Administration; Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: August 26, 2024; Bacillus subtilis MJ501 Bacillus subtilis The deposit number is CGMCC No. 31738.

[0037] 3. SEM identification of strains (1) Fixed material The Bacillus subtilis MJ501 bacterial liquid was used to prepare a sample. The cultured cells were centrifuged at 800-1500 rpm for 8-15 minutes to collect the precipitate. The precipitate was immersed in PBS (0.1M, NaCl-free), the cells or tissues were rinsed several times, and the supernatant was removed by centrifugation. 4°C pre-cooled 2.5-3% glutaraldehyde was added and fixed at 4°C for 4 hours or overnight (the time required depends on the size of the tissue, and this step can be extended).

[0038] (2) Dehydration Aspirate the fixative and rinse with PBS (0.1M, NaCl-free) 3-5 times, 15 minutes each time. Dehydrate with a series of graded alcohols (30%, 50%, 70%, 80%, 90%, 95%, 100%), dehydrating once with each concentration of alcohol for 10-20 minutes each time (usually 15 minutes). Dehydrate thoroughly with 100% alcohol 1-2 times, then replace with isoamyl acetate (banana aerator) twice, 20 minutes each time.

[0039] (3) Drying The critical point drying method is the most ideal, but a special instrument, a critical point dryer, is required. If the laboratory does not have such an instrument, natural drying can be used.

[0040] (4) Conductive treatment of samples Use vacuum spraying method. The spraying should be uniform and observed under a scanning electron microscope after completion.

[0041] Note: Except for fixation, 70% alcohol, 100% alcohol, and banana water, which can be kept for a long time or even overnight, all other conditions should follow the schedule.

[0042] like Figure 3 As shown, Bacillus subtilis MJ501 is short rod-shaped.

[0043] Example 2 1. Liquid fermentation production of polyglutamic acid (glutamate dependent) The Bacillus subtilis MJ501 slant was inoculated into 5 mL of liquid seed medium for activation and cultured at 25-30°C, 150 rpm, for 4-8 hours. The slant was then inoculated into 30 mL of liquid seed medium for secondary activation and cultured at 25-30°C, 150 rpm, for 4-8 hours. The inoculum was then shaken in a flask at 28-37°C, 180 rpm, for 48-72 hours. After fermentation, the precipitate was precipitated overnight with 3-5 volumes of ethanol and centrifuged to remove impurities. The supernatant was discarded, and the resulting precipitate was re-dissolved in water and ultrasonically disrupted for 30 minutes using a 2-second sonication cycle followed by a 2-second on / off cycle. The precipitate was dialyzed (3500 Da) for approximately one week, depending on the specific conditions. The volume after dialysis was calculated and freeze-vacuum dried to obtain the crude PGA product.

[0044] The fermentation medium composition was as follows: glucose 30 g / L, yeast extract 5 g / L, sodium glutamate 30 g / L, magnesium sulfate 0.25 g / L, potassium dihydrogen phosphate 2 g / L, and natural pH.

[0045] 2. Liquid fermentation production of polyglutamic acid (non-glutamic acid dependent) A slant of Bacillus subtilis MJ501 was inoculated into 5 mL of liquid seed medium for activation and cultured at 25-30°C and 150 rpm for 4-8 hours. The culture was then inoculated into 30 mL of liquid seed medium for secondary activation and cultured at 25-30°C and 150 rpm for 4-8 hours. The inoculum was then shaken in a flask at 3-5% (v / v) of the seed medium and fermented at 28-37°C and 180 rpm for 48-72 hours. After fermentation, the mixture was precipitated overnight with 3-5 volumes of ethanol and centrifuged to remove impurities. The supernatant was discarded, and the resulting precipitate was re-dissolved in water and sonicated for 30 minutes using a 2-second sonication cycle followed by a 2-second on / off cycle. The solution was dialyzed (3500 Da) for approximately one week, depending on the specific conditions. The volume after dialysis was calculated and freeze-vacuum-dried to obtain the crude PGA. The fermentation medium at this point was the same ratio as the seed medium. The fermentation medium consists of 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

[0046] 3. Polyglutamic acid detection method The strain producing the largest amount of γ-polyglutamic acid with the highest purity was determined by testing. The testing method was as follows: after obtaining a culture rich in γ-polyglutamic acid through the above fermentation (the above two groups of products), the culture was centrifuged to remove the bacteria, the supernatant was taken, 3 to 5 times the volume of anhydrous ethanol was added to precipitate overnight, the mixture was centrifuged at 4°C, the supernatant was removed, and the precipitate was dissolved in ultrapure water to obtain a test sample, which was tested using an amino acid analyzer.

[0047] The extracted product was treated by hydrolysis: 2 g of the sample was placed in an ampoule, and 2 mL of 12 M concentrated hydrochloric acid and 8 mL of 6 M hydrochloric acid were added. The amount and concentration of hydrochloric acid were closely related to the sample. The seal was melted with an alcohol burner and hydrolysis was performed in a high-temperature, forced-air drying oven (110°C, 24 h). Filtering was performed as needed to remove impurities, and the ampoule was washed three times with ultrapure water, approximately 5 mL each time. A ceramic evaporating dish was evaporated to dryness in a boiling water bath. 0.02 M dilute hydrochloric acid was added to the volume. The dilution was determined by the volume of the volume, which was adjusted according to the experimental process to ensure that the highest amino acid content did not exceed the instrument threshold (upper detection limit). In this experiment, a dilution of 5.000 (i.e., 5 mL of the volume of solution) was used. The evaporating dish was ground with a glass rod and homogenized by ultrasound. 0.5–1 mL of the solution was aspirated using a sterile disposable syringe through a polyethersulfone (PES) filter membrane and injected into a liquid phase vial. Amino acid content was determined using an amino acid analyzer. The injection volume was 50 μL, and the program used the default sodium salt amino acid detection system.

[0048] like Figure 4 As shown, the glutamate content of Bacillus subtilis MJ501 reached 94.977%.

[0049] 4. Analysis of crude polyglutamic acid Since amino acid testing indicated that the product from the glutamate-independent culture medium was of higher purity, 1 g of the resulting polyglutamic acid was weighed, dissolved, and filtered through a 0.45 μm membrane. Molecular weight analysis was performed using gel permeation chromatography (GPC). The instrument was an Agilent 1260 GPC / SEC MDS, with a PL aquagel-OH 30 column and a PL aquagel-OH mixed-H column connected in series. The mobile phase consisted of 0.3 M Na₂SO₄ solution at a flow rate of 1 mL / min. The column and detector temperatures were both 40°C, the injection volume was 100 μL, and the run time was 30 min.

[0050] GPC analysis showed that the weight-average molecular weight of polyglutamic acid produced by the fermentation of the bacteria was 392 kDa.

[0051] 5. Polyglutamic acid structure characterization Sample preparation: KBr tableting method (applicable to solid samples): Mix 1-2 mg of dry γ-polyglutamic acid sample with 100-200 mg of spectroscopic grade KBr powder and grind until uniform. Press into transparent thin tablets using a tablet press (pressure 10-15 MPa for 1-2 minutes).

[0052] Fourier transform infrared spectrometer (FT-IR), scanning range: 4000-400 cm⁻¹. Resolution: 4 cm⁻¹. Number of scans: 32 times (optimized for signal-to-noise ratio). Background correction: blank KBr plate or air was used as background before each measurement. The results are shown in Figure 2. Figure 5 As shown in Table 1, the characteristic peaks are consistent with the specific functional groups of γ-PGA given in the literature.

[0053] Table 1 FT-IR results of polyglutamic acid produced by Bacillus subtilis MJ501

[0054] Example 3: Effect of polyglutamic acid on the preservation of fresh-cut apples The purified γ-polyglutamic acid was dissolved in deionized water in the following proportions: 5% polyglutamic acid + 2% glycerol + 0.5% casein to prepare a film-forming solution, which can be appropriately concentrated in an oven. The experiment was divided into two groups: a film-wrapping group and an infiltration group. The film-wrapping group first stirred the film-forming solution evenly, poured it into a glass dish, and dried it at 40°C to form a homogeneous milky white film, which was then wrapped onto fresh-cut apple slices. The infiltration group directly immersed the fresh-cut apple slices in the film-forming solution and dried it at 40°C to form a film. The state of the apple slices was observed after 9 days, and the results were as follows: Figure 6 As shown, the preservation effect of fresh-cut apple slices at room temperature was as follows: soaked group > film-wrapped group > control.

[0055] The above content is merely an example and explanation of the concept of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present invention.

Claims

1. A deep-sea Bacillus subtilis MJ501 strain producing γ-polyglutamic acid, characterized by: The classification of the Bacillus subtilis MJ501 is named Bacillus subtilis , and its deposit number is CGMCC No. 31738.

2. The Bacillus subtilis MJ501 according to claim 1, characterized in that The colony of the Bacillus subtilis MJ501 is milky white, grows fast, is moist, has wrinkles, and can be drawn into threads; it can form a transparent circle in a positively charged neutral red plate, and droops in a stalactite shape due to the presence of mucus when cultured in an inverted manner.

3. Use of the Bacillus subtilis MJ501 according to claim 1 in the production of γ-polyglutamic acid.

4. The use according to claim 3, characterized in that The production steps of the γ-polyglutamic acid are: (1) culturing the activated Bacillus subtilis MJ501 in liquid to obtain a seed solution; (2) fermenting the seed liquid to obtain a fermentation liquid; (3) The fermentation broth is centrifuged, the obtained precipitate is redissolved, ultrasonically disrupted, and then dialyzed for purification, and γ-polyglutamic acid is obtained after drying.

5. The use according to claim 4, characterized in that The temperature of activation and liquid culture in step (1) is 25-30° C., and the culture time is 4-8 h.

6. The use according to claim 4, characterized in that In step (2), the inoculation amount of the seed liquid is 3-5% v / v, the fermentation temperature is 28-37° C., and the culture time is 48-72 h.

7. The use according to claim 4, characterized in that The molecular weight of the dialyzed protein in step (3) is 3500 Da, and the dialysis time is 3-8 days.

8. Use of the Bacillus subtilis MJ501 according to claim 1 or the γ-polyglutamic acid produced according to claim 3 in plant preservation.

9. The use according to claim 8, characterized in that The application steps of the plant preservation method are as follows: first, dissolving gamma-polyglutamic acid, glycerol and casein in water to prepare a membrane liquid, and directly soaking the plant with the membrane liquid; or drying the membrane liquid into a film, and then using it to wrap the plant.

10. The application according to claim 8, characterized in that: The volume ratio of the mixture of γ-polyglutamic acid, glycerol and casein is 10-15:4:1.

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