Low-molecular-weight polyglutamic acid-producing strain and use thereof

Abstract

This invention discloses a strain capable of producing low molecular weight γ-polyglutamic acid and its applications. The invention provides *Bacillus subtilis*, strain number LM9, registered with the China General Microbiological Culture Collection Center (CGMCC) under number CGMCC No. 23791. This invention utilizes *Bacillus subtilis* LM9, a strain capable of synthesizing low molecular weight γ-polyglutamic acid, obtained from the black soil of Northeast China. It achieves the direct synthesis of low molecular weight γ-polyglutamic acid in microbial cells and further investigates its applications in plant protection, which has significant practical implications.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a low molecular weight polyglutamic acid producing strain and its application. Background Technology

[0002] γ-Polyglutamic acid (PGA) is a biopolymer composed of D-glutamic acid and L-glutamic acid monomers linked by amide bonds, with a molecular weight typically ranging from 100,000 to 1,000,000 Daltons. It is a water-soluble, biodegradable, and environmentally friendly polymer material, non-toxic to humans and the environment, and has a wide range of applications in food, cosmetics, pharmaceuticals, and water treatment. PGA is one of the few biopolymers that can be produced by microbial fermentation. Bacillus is the main producing strain of PGA. Through strain mutagenesis, optimization of culture medium composition, and optimization of fermentation methods, the fermentation level of PGA has reached 40–70 g / L (Chinese Invention Patent Application No.: 200910012392.2; Xu et al., Process Biochemistry, 2005, 40: 519-523).

[0003] Different molecular weights of γ-polyglutamic acid have different applications. High molecular weight γ-polyglutamic acid (molecular weight greater than 1 million Daltons) has stronger viscosity and higher water retention properties, and can be used as a flocculant in water treatment (Chinese Invention Patent Application No.: 200510040857.7). γ-polyglutamic acid with a molecular weight range of 700,000 to 1 million Daltons can be used as a moisturizer in cosmetics. Low molecular weight γ-polyglutamic acid (molecular weight less than 600,000 Daltons) can be used as a drug carrier in tumor treatment (Chinese Invention Patent Application No.: 202011047620.2). γ-polyglutamic acid with a molecular weight less than 50,000 Daltons can be used as a biofertilizer adjuvant to improve the soil environment, promote crop growth, and promote seed germination.

[0004] Microbial-derived γ-polyglutamic acid (γ-polyglutamic acid) has a molecular weight of over 600,000 Daltons, and most commercially available γ-polyglutamic acid is a medium- to high molecular weight polymer. Low molecular weight γ-polyglutamic acid is mainly obtained through physical, chemical, and enzymatic methods. A Chinese invention patent (application number: 201510191530.3) utilizes acid hydrolysis to prepare γ-polyglutamic acid with a molecular weight range of 10,000 to 500,000 Daltons. Another Chinese invention patent (application number: 202010455019.0) uses a dual-bacterial co-culture method to reduce the molecular weight of γ-polyglutamic acid using enzymes secreted by Bacillus cereus. The molecular weight of γ-polyglutamic acid significantly limits its applications, and the direct synthesis of low molecular weight γ-polyglutamic acid in microbial cells is becoming a research hotspot in the γ-polyglutamic acid industry both domestically and internationally. Currently reported methods for adjusting the molecular weight of γ-polyglutamic acid all suffer from cumbersome procedures and poor product stability. Summary of the Invention

[0005] The purpose of this invention is to provide a low molecular weight γ-polyglutamic acid producing strain and its application.

[0006] In a first aspect, the present invention claims protection for a strain of Bacillus subtilis.

[0007] The Bacillus subtilis strain claimed in this invention is LM9, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 23791.

[0008] Secondly, the present invention claims protection for cultures of Bacillus subtilis.

[0009] The culture of Bacillus subtilis claimed in this invention is the substance obtained by culturing Bacillus subtilis LM9 as described in the first aspect above on a bacterial culture medium (all substances in the culture container).

[0010] The substances in the above-mentioned cultures include Bacillus subtilis LM9 (the bacterial cell itself) and metabolites of Bacillus subtilis LM9.

[0011] The term "metabolite" refers to the primary and / or secondary metabolites produced during microbial metabolism. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the external environment and, through catabolism and anabolism, generate substances and energy to sustain life activities. The products of primary metabolism are called primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, and lipids. Secondary metabolism refers to the process by which microorganisms, at a certain growth stage, use primary metabolites as precursors to synthesize substances that have no clearly defined function for their life activities. The products of secondary metabolism are called secondary metabolites, and are mostly compounds with relatively complex molecular structures. Based on their functions, they can be classified into types such as antibiotics, hormones, alkaloids, and toxins.

[0012] In the above-mentioned cultures, the bacterial culture medium can be Bacillus subtilis culture medium, which can be a liquid culture medium or a solid culture medium.

[0013] The term "culture" refers to a liquid or solid product (all substances within the culture container) that has grown a microbial community after artificial inoculation and cultivation. It is a product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

[0014] In a specific embodiment of the present invention, the solvent of the bacterial culture medium is water, and the solutes and concentrations are as follows: glucose 10-30 g / L (e.g., 30 g / L), yeast extract 5-10 g / L (e.g., 8 g / L), sodium glutamate 20-50 g / L (e.g., 30 g / L), dipotassium hydrogen phosphate 1-4 g / L (e.g., 2 g / L), and magnesium sulfate 0.2-1 g / L (e.g., 0.25 g / L).

[0015] Thirdly, this invention claims protection for a microbial agent.

[0016] The microbial agent claimed in this invention contains Bacillus subtilis LM9 as described in the first aspect above, metabolites of Bacillus subtilis LM9, and / or the culture described in the second aspect above.

[0017] The microbial agent is a microbial agent for producing low molecular weight γ-polyglutamic acid, and / or a microbial agent for enhancing plant disease resistance, and / or a microbial agent for promoting plant growth.

[0018] In the above-mentioned bacterial agent, in addition to the active ingredient, the bacterial agent also contains a carrier. The carrier can be a biologically inert carrier. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, plant material, or polymer compound; the mineral material can be at least one selected from clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one selected from corn flour, soybean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0019] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0020] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the bacterial agent.

[0021] Fourthly, the present invention claims protection for the use of Bacillus subtilis LM9 as described in the first aspect above, or the culture described in the second aspect above, or the inoculant described in the third aspect above, in any of the following:

[0022] A1. Production of low molecular weight γ-polyglutamic acid;

[0023] A2. Prepare products for the production of low molecular weight γ-polyglutamic acid.

[0024] Fifthly, the present invention claims a method for preparing low molecular weight γ-polyglutamic acid.

[0025] The method for preparing low molecular weight γ-polyglutamic acid claimed in this invention may include the following steps: fermenting Bacillus subtilis LM9 as described in the first aspect above with a bacterial culture medium to obtain low molecular weight γ-polyglutamic acid from the fermentation broth.

[0026] Furthermore, the solvent of the bacterial culture medium is water, and the solutes and concentrations are as follows: glucose 10-30 g / L (e.g., 30 g / L), yeast extract 5-10 g / L (e.g., 8 g / L), sodium glutamate 20-50 g / L (e.g., 30 g / L), dipotassium hydrogen phosphate 1-4 g / L (e.g., 2 g / L), and magnesium sulfate 0.2-1 g / L (e.g., 0.25 g / L).

[0027] Furthermore, the fermentation culture can be carried out at a temperature of 37°C, a pH of 7.0, an aeration rate of 1 L / min, a rotation radius of 33 mm, and a dissolved oxygen level of 30%, with the dissolved oxygen level being related to the rotation speed.

[0028] Furthermore, the fermentation culture time can be 48 hours.

[0029] Furthermore, the fermentation culture is carried out by fed-batch fermentation, and the concentrations of glucose and L-glutamic acid in the fermentation broth are monitored. When the concentrations of glucose and L-glutamic acid are lower than 20 g / L, glucose and L-glutamic acid are added.

[0030] Further, the fermentation culture may include the following steps: centrifuging the fermentation broth to remove the bacterial cells, adding 3 times the volume of anhydrous ethanol to the supernatant, letting it stand at 4°C for more than 12 hours (overnight), centrifuging to collect the precipitate, adding an equal volume of distilled water to dissolve it, and obtaining a solution containing the low molecular weight γ-polyglutamic acid.

[0031] In the above text, the low molecular weight γ-polyglutamic acid can be γ-polyglutamic acid with a molecular weight of less than 50,000 Daltons. For example, γ-polyglutamic acid with a molecular weight between 29,000 and 41,000 Daltons.

[0032] In a specific embodiment of the present invention, the low molecular weight γ-polyglutamic acid has an average molecular weight of 34,000 Daltons.

[0033] The molecular weight dispersion coefficient of the low molecular weight γ-polyglutamic acid is 4.8-6.4.

[0034] In a specific embodiment of the present invention, the average molecular weight dispersion coefficient of the low molecular weight γ-polyglutamic acid is 5.4.

[0035] Sixthly, the present invention claims protection for the use of Bacillus subtilis LM9 as described in the first aspect above, or the culture as described in the second aspect above, or the inoculum as described in the third aspect above, or the low molecular weight γ-polyglutamic acid or a solution containing the low molecular weight γ-polyglutamic acid or a dilution thereof prepared by the method described in the fifth aspect above, in any of the following:

[0036] B1. Enhance plant disease resistance;

[0037] B2. Prepare products to enhance plant disease resistance;

[0038] B3. Promotes plant growth;

[0039] B4. Prepare products for promoting plant growth.

[0040] Seventhly, the present invention claims protection for any of the following methods:

[0041] Method I: A method for enhancing plant disease resistance, comprising the following steps: applying to the plant the low molecular weight γ-polyglutamic acid or a solution containing the low molecular weight γ-polyglutamic acid or a dilution thereof prepared by the method described in the fifth aspect above.

[0042] Method II: A method for promoting plant growth, comprising the steps of applying to the plant the low molecular weight γ-polyglutamic acid or a solution containing the low molecular weight γ-polyglutamic acid or a dilution thereof prepared by the method described in the fifth aspect above.

[0043] Specifically, applying the low molecular weight γ-polyglutamic acid or a solution containing the low molecular weight γ-polyglutamic acid or a dilution thereof to the plant can be done by adding the low molecular weight γ-polyglutamic acid or a solution containing the low molecular weight γ-polyglutamic acid to the plant's culture medium (such as a culture medium or soil), for example, by watering it into the plant's rhizosphere soil.

[0044] In a specific embodiment of the present invention, when the low molecular weight γ-polyglutamic acid or a solution containing the low molecular weight γ-polyglutamic acid or a dilution thereof is applied to the plant, the working concentration of the low molecular weight γ-polyglutamic acid is 1-5 g / L, preferably 1 g / L.

[0045] In the above text, the disease resistance mentioned can refer to resistance to diseases caused by pathogenic fungi that infect plants.

[0046] Furthermore, the pathogenic fungus may be Verticillium dahliae (such as Verticillium dahliae V592), and the corresponding disease may be Verticillium wilt.

[0047] In the above text, promoting plant growth can be manifested as promoting root elongation and / or promoting plant height increase.

[0048] The plant can be a dicotyledonous plant or a monocotyledonous plant. The dicotyledonous plant can be a Brassicaceae plant. The Brassicaceae plant can be Arabidopsis thaliana.

[0049] This invention relates to a strain of Bacillus subtilis LM9, obtained from the black soil of Northeast China, capable of synthesizing low molecular weight γ-polyglutamic acid. This achievement realizes the direct synthesis of low molecular weight γ-polyglutamic acid in microbial cells, and further investigates its applications in plant protection, which has significant practical implications.

[0050] Preservation Instructions

[0051] Classification and nomenclature: Bacillus subtilis;

[0052] Biomaterials used for ginseng: LM9;

[0053] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0054] The abbreviation for the depository institution is CGMCC.

[0055] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0056] Date of deposit: November 12, 2021;

[0057] Registered with the China National Collection Center (CGMCC) No. 23791. Attached Figure Description

[0058] Figure 1 The results of the 16S rDNA sequence alignment for strain LM9 are shown.

[0059] Figure 2 The effect of low molecular weight γ-polyglutamic acid on plant disease resistance.

[0060] Figure 3 Disease index of Arabidopsis thaliana plants after inoculation with Verticillium dahliae (V592) and different treatments.

[0061] Figure 4 The effects of γ-polyglutamic acid on plant growth (A) and on plant root length (B) are shown. **: P < 0.01. Detailed Implementation

[0062] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0064] Example 1: Isolation, screening, and identification of Bacillus subtilis LM9

[0065] I. Strain Screening

[0066] 1. Initial screening

[0067] Weigh 1g of a soil sample from the black soil region of Northeast China and dissolve it in 100mL of sterile physiological saline. Shake for 30 minutes to obtain a sample suspension. Under aseptic conditions, serially dilute the sample to different factors. Spread 0.1mL of each dilution of the bacterial suspension onto LB agar plates containing 20g / L L-glutamate (formulation: 10g / L peptone, 5g / L yeast extract, 10g / L NaCl, 20g / L agar). Incubate at 37℃ for 24h. Observe the colony morphology, transparency, viscosity, and other characteristics. Select colonies with viscous edges for streak purification, number, and store.

[0068] 2. Secondary screening

[0069] The pure colonies obtained by streaking were inoculated into LB liquid medium and cultured at 37°C and 220 rpm for 17 h. Then, 1% (v / v) inoculum was added to seed culture medium (formula: glucose 20 g / L, yeast extract 5 g / L, monosodium glutamate 10 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.25 g / L, balance water), and cultured in shake flasks at 37°C and 220 rpm for 17 h. Finally, 1% (v / v) inoculum was added to fermentation medium (formula: glucose 30 g / L, yeast extract 8 g / L, monosodium glutamate 30 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.25 g / L, balance water), and cultured in shake flasks at 37°C and 220 rpm for 48 h. The consumption of glucose and L-glutamate, as well as the formation of γ-polyglutamate, were measured (see Example 2 for specific methods).

[0070] A total of 260 strains with γ-polyglutamic acid synthesis ability were screened, and the strain with the highest yield was named LM9.

[0071] II. Identification of strain LM9

[0072] Genomic DNA was extracted from the bacterial strain using a bacterial genomic DNA extraction kit, and the 16S rDNA of the strain was amplified by PCR using universal primers 27-F (5'-AGAGTT TGA TCC TGG CTC AG-3') and 1492-R (5'-GGT TAC CTT GTT ACG ACT T-3').

[0073] The amplification conditions were: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 10 s, 52℃ annealing for 20 s, 72℃ extension for 1 min, for 30 cycles; and 72℃ final extension for 10 min.

[0074] PCR products were sent to Beijing Ruiboxing Technology Co., Ltd. for sequencing. The sequencing assembly results were analyzed by BLAST alignment on the NCBI website (http: / / www.ncbi.nlm.nih.gov / blast / blast.cgi). The 16S rDNA sequence of strain LM9 is shown in SEQ ID No. 1. Figure 1 ).

[0075] Morphological characteristics: Individual cells are (0.7–0.8) μm × (2–3) μm, Gram-positive; spores are (0.6–0.9) μm × (1.0–1.5) μm, located in the center or slightly off-center of the bacterial cell. The colony surface is viscous and translucent, with moist edges.

[0076] Physiological and biochemical characteristic identification:

[0077] Strain LM9 can utilize maltose, glucose, sucrose, and fructose. It cannot utilize lactose, xylose, or arabinose, and the citrate test is negative.

[0078] The results of the comparison of physiological and biochemical characteristics of strain LM9 are shown in Table 1. In Table 1, + indicates a positive result or growth, and - indicates a negative result or no growth.

[0079] Table 1. Physiological and biochemical characteristics of strain LM9

[0080]

[0081]

[0082] Based on the above morphological, physiological and biochemical characteristics and 16S rDNA sequencing, strain LM9 was identified as Bacillus subtilis, and was deposited on November 12, 2021, at the China General Microbiological Culture Collection Center (CGMCC; address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; postcode: 100101), with accession number CGMCC No. 23791.

[0083] Example 2: Identification of the ability of Bacillus subtilis LM9 to produce low molecular weight γ-polyglutamic acid

[0084] I. Fermentation Culture

[0085] Bacillus subtilis LM9 was inoculated into a 100 mL Erlenmeyer flask containing 20 mL of seed culture medium (formulation as described in Example 1) and cultured at 37°C with shaking at 220 rpm for 12 hours. 4 mL of this culture was then inoculated into a 1 L Erlenmeyer flask containing 400 mL of seed culture medium and cultured at 37°C with shaking at 220 rpm for 12 hours to obtain the seed culture (OD). 600nm =10-14). The seed culture was inoculated at a rate of 10% (v / v) into a 5L fermenter (Shanghai Baoxing BIOTECH 5L fermenter) containing 2L of fermentation medium (formulation as shown in Example 1). The fermentation temperature was 37℃, pH was 7.0, aeration rate was 1L / min, rotation radius was 33mm, and dissolved oxygen was 30%, with dissolved oxygen correlated with rotation speed. The fermentation method was fed-batch fermentation. The concentrations of glucose and L-glutamic acid in the fermentation broth were monitored, and glucose and L-glutamic acid were added when the concentrations were below 20g / L.

[0086] II. Measurement Method

[0087] 1. Determination methods for glucose and L-glutamate

[0088] An SBA-40C analyzer was used. After centrifugation of the fermentation broth, the supernatant was collected, appropriately diluted, and 25 μL was drawn up by the injection needle and injected into the reaction chamber. The substrates (glucose and L-glutamate) permeated through the enzyme membrane ring, contacted the immobilized enzyme layer, and reacted, generating a current signal. This current signal was linearly proportional to the concentration of the substrate. The signal was controlled by a microcomputer, and the results could be directly displayed and printed.

[0089] 2. Method for determining γ-polyglutamic acid content

[0090] The CTAB method was used to determine the content of γ-polyglutamic acid. The specific steps were as follows: The fermentation broth was centrifuged (12000 rpm, 20 min) to remove bacterial cells. The supernatant was collected, and three volumes of anhydrous ethanol were added. The mixture was incubated overnight at 4°C. The precipitate was collected by centrifugation (12000 rpm, 20 min), dissolved in an equal volume of distilled water, and diluted 100 times before analysis. 0.1 mL of CTAB solution (5 g / L) and 0.1 mL of the sample were added to a 96-well plate. After thorough mixing for 3 min, the absorbance was measured at 250 nm using a microplate reader. A standard curve for γ-polyglutamic acid was plotted, and the concentration of γ-polyglutamic acid in the sample was calculated.

[0091] 3. Method for determining the molecular weight of γ-polyglutamic acid

[0092] The molecular weight of γ-polyglutamic acid was determined using aqueous gel permeation chromatography (GPC) with three columns in series: Waters Ultrahydrogel™ 2000 (7.8 × 300 mm), Waters Ultrahydrogel™ 250 (7.8 × 300 mm), and Waters Ultrahydrogel™ 120 (7.8 × 300 mm). Detection was performed using deionized water as the mobile phase at a flow rate of 0.6 mL / min and a column temperature of 65 °C, with a differential refractive index (RI) detector. A standard equation relating molecular weight and retention time was established using dextran of different molecular weights as standards to calculate the molecular weight of the γ-polyglutamic acid in the samples.

[0093] Molecular weight dispersion factor (D) = weight-average molecular weight (Daltons) / number-average molecular weight (Daltons)

[0094] III. Results and Analysis

[0095] At the end of fermentation, the fermentation broth was collected, centrifuged at 12,000 rpm for 20 min to remove the bacterial cells, and the supernatant was collected to determine the content and molecular weight of γ-polyglutamic acid in the fermentation broth. The experiment was repeated three times, and the average value of the results was taken.

[0096] Results: In a 5L fermenter, after 48 hours of fermentation, the yield of γ-polyglutamic acid was 26 g / L, the molecular weight of the product was 34,000 Daltons, and the molecular weight dispersion index was 5.4 (Table 2).

[0097] Table 2. Results of three repeated experiments in the fermenter

[0098]

[0099] Example 3: Application of low molecular weight γ-polyglutamic acid from Bacillus subtilis LM9 in plant protection.

[0100] Test γ-polyglutamic acid: The γ-polyglutamic acid prepared in Example 2 was concentrated to 50 g / L and set aside for later use.

[0101] I. Impact on plant disease resistance

[0102] When the Arabidopsis thaliana (Col-0) plants in the greenhouse have grown for about two weeks, they are removed from the pots, their roots are rinsed with tap water to remove vermiculite, and then they are soaked in a solution of 10%... 6The *Verticillium dahliae* spore suspension was incubated for 30 minutes. Arabidopsis thaliana was then replanted in soil and treated twice weekly with different concentrations of γ-polyglutamic acid (10-fold diluted, final concentration 5 g / L), γ-polyglutamic acid (50-fold diluted, final concentration 1 g / L), and commercially available γ-polyglutamic acid (final concentration 1 g / L, molecular weight approximately 700,000). Phenotypic characteristics were observed, and the disease index of Arabidopsis thaliana plants inoculated with *Verticillium dahliae* (V592) after different treatments was calculated.

[0103] Disease index = [Σ(Disease level × Number of plants at that level)] / (Total number of plants × 4) × 100%.

[0104] Arabidopsis disease is classified into 5 levels. Level 0 indicates no pathogen infection; Level 1 indicates that less than 25% (including the leaf tips) of the leaves are yellow and diseased; Level 2 indicates that 25%-50% (excluding the left leaf tip but including the right leaf tip) of the leaves are yellow and diseased; Level 3 indicates that 50%-75% (excluding the left leaf tip but including the right leaf tip) of the leaves are yellow and withered; Level 4 indicates that more than 75% (excluding the leaf tips) of the leaves are diseased.

[0105] Each group treated 10 plants, with 3 replicates, and the results were averaged.

[0106] II. Impact on plant growth

[0107] Arabidopsis seeds were placed in solid plates containing water, 10-fold diluted γ-polyglutamic acid (final concentration of γ-polyglutamic acid was 5 g / L), and 50-fold diluted γ-polyglutamic acid (final concentration of γ-polyglutamic acid was 1 g / L). After about two weeks, seedlings sprouted, were taken out, photographed, and root length was measured.

[0108] Each group treated 10 plants, with 3 replicates.

[0109] III. Results and Analysis

[0110] 1. Impact on plant disease resistance

[0111] from Figure 2 It can be seen that Arabidopsis thaliana without root infection by Verticillium dahliae V592 showed good growth, but growth slowed after root infection by Verticillium dahliae V592. The plants with added low molecular weight γ-polyglutamic acid showed better growth than those without. Furthermore, the low molecular weight γ-polyglutamic acid synthesized by Bacillus subtilis LM9 in this invention is superior to commercially available γ-polyglutamic acid (molecular weight around 700,000) in enhancing plant disease resistance.

[0112] The disease index of Arabidopsis thaliana plants infected with Verticillium dahliae (V592) was 70%. After treatment with a 50-fold dilution of γ-polyglutamic acid (final concentration of γ-polyglutamic acid: 1 g / L), the disease index of Arabidopsis thaliana plants infected with Verticillium dahliae (V592) decreased to 53%. After treatment with commercially available γ-polyglutamic acid (final concentration of γ-polyglutamic acid: 1 g / L, molecular weight approximately 700,000), the disease index of Arabidopsis thaliana plants infected with Verticillium dahliae (V592) was 68%. Figure 3 ).

[0113] The above results indicate that the low molecular weight γ-polyglutamic acid synthesized by Bacillus subtilis LM9 in this invention can enhance the disease resistance of plants.

[0114] 2. Impact on plant growth

[0115] from Figure 4 As shown in Figure A, the plant growth in the solid culture medium containing low molecular weight γ-polyglutamic acid was better than that in the control group without γ-polyglutamic acid. Compared with the water-treated control group, the plants in the low molecular weight γ-polyglutamic acid treatment group had longer roots and taller plants. Root length measurements showed that the root length of Arabidopsis thaliana treated with 50-fold dilution of γ-polyglutamic acid (final concentration of γ-polyglutamic acid was 1 g / L) was 1.7 ± 0.2 cm, the root length of Arabidopsis thaliana treated with 10-fold dilution of γ-polyglutamic acid (final concentration of γ-polyglutamic acid was 5 g / L) was 1.1 ± 0.2 cm, while the root length of Arabidopsis thaliana without γ-polyglutamic acid was only 0.1 ± 0.1 cm. Figure 4 (B) The above results demonstrate that the low molecular weight γ-polyglutamic acid obtained by this technique can promote the growth of plant roots and stems even at relatively low concentrations.

[0116] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. <110> Institute of Microbiology, Chinese Academy of Sciences <120> A low molecular weight polyglutamic acid producing strain and its application <130> GNCLN213360 <160> 1 <170> Patent In version 3.5 <210> 1 <211> 1428 <212> DNA <213> Bacillus subtilis <400> 1 tatacatgca gtcgagcgga cagatgggag cttgctccct gatgttagcg gcggacgggt 60 gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac cggggctaat 120 accggatggt tgtttgaacc gcatggttca aacataaaag gtggcttcgg ctaccactta 180 cagatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg 240 cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc cagactccta 300 cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg 360 tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtaccgttc 420 gaatagggcg gtaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca 480 gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggctcgca 540 ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac 600 tgggaactt gagtgcagaa gaggagtg gattccacg tgtagcggtg aaatgcgtag 660 agatgtggag gaacaccagt ggcgaggcg actctctggt ctgtactga cgctgaggag 720 cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag 780 tgctaagtgt taggggtt ccgcccctta gtgctgcagc taacgcatta agcactccgc 840 ctggggagta cggtcgcaag actgaactc aaaggaattg acggggggccc gcacaagcgg 900 tggagcatgt ggtttaattc gaagcaacgc gagaacctt accaggctt gataccct 960 ɣaatccta gagataggac gtccccttcg ggggcagagtzgaggtggt gcatggttgt 1020 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt 1080 agttgccagc attcagttgg gcactctaag gtgactgccg gtgacaacc ggaggaggt 1140 ggggatgacg tcaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga 1200 cagaacaag ggcagcgaaa ccgcgaggtt aagccaatcc cacaaatctg ttctcagttc 1260 ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg cggatcagca 1320 tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg 1380 taacacccga agtcggtgag gtaaccttta ggagccagcc gccgaagg 1428

Claims

1. Bacillus subtilis, strain number LM9, is registered at the China General Microbiological Culture Collection Center (CGMCC) with the number CGMCC No. 23791.

2. A culture of Bacillus subtilis, which is a substance obtained by culturing Bacillus subtilis as described in claim 1 on a bacterial culture medium; The substance includes the Bacillus subtilis as described in claim 1 and its metabolites.

3. A microbial agent, characterized in that: The bacterial agent contains Bacillus subtilis as described in claim 1 or the culture as described in claim 2.

4. The use of Bacillus subtilis as described in claim 1, the culture as described in claim 2, or the inoculum as described in claim 3 in any of the following: A1. Production of low molecular weight γ-polyglutamic acid; A2. Prepare products for the production of low molecular weight γ-polyglutamic acid.

5. A method for preparing low molecular weight γ-polyglutamic acid, comprising the following steps: fermenting and culturing Bacillus subtilis as described in claim 1 with a bacterial culture medium to obtain low molecular weight γ-polyglutamic acid from the fermentation broth.

6. The method according to claim 5, characterized in that: The bacterial culture medium is water as the solvent, and the solutes and concentrations are as follows: glucose 10-30 g / L, yeast extract 5-10 g / L, monosodium glutamate 20-50 g / L, dipotassium hydrogen phosphate 1-4 g / L, magnesium sulfate 0.2-1 g / L; and / or The fermentation culture was carried out at a temperature of 37°C, a pH of 7.0, an aeration rate of 1 L / min, and a dissolved oxygen content of 30%; and / or The fermentation culture time is 48 hours; and / or The fermentation process further includes the following steps: centrifuging the fermentation broth to remove the bacterial cells, adding anhydrous ethanol to the supernatant, allowing it to stand at 4°C for more than 12 hours, centrifuging to collect the precipitate, adding distilled water to dissolve it, and obtaining a solution containing the low molecular weight γ-polyglutamic acid.

7. The application according to claim 4 or the method according to claim 5 or 6, characterized in that: The low molecular weight γ-polyglutamic acid is γ-polyglutamic acid with a molecular weight of less than 50,000 Daltons.

8. The use of Bacillus subtilis as described in claim 1, the culture as described in claim 2, or the inoculum as described in claim 3 in any of the following: B1. Enhance plant disease resistance; B2. Prepare products to enhance plant disease resistance; B3. Promotes plant growth; B4. Prepare products for promoting plant growth; The disease resistance refers to resistance to diseases caused by pathogenic fungi infecting plants; the pathogenic fungus is Verticillium dahliae; The promotion of plant growth is manifested in promoting the elongation of plant roots and / or promoting the increase of plant height.

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