Culture medium for industrially producing bacillus velezensis TCS001 fermentation broth, culture process, lipopeptide-compound-containing mixture separated from fermentation broth, and use

Abstract

Optimization of the formula of a culture medium for industrially producing a Bacillus velezensis TCS001 fermentation broth and culture process conditions therefor. By using the culture medium and the culture process conditions, not only can the fermentation production cost be reduced, but also the effective viable bacterium count and the spore count in the Bacillus velezensis TCS001 fermentation broth can be improved, so that the fermentation broth can be used for industrial production and market promotion and application. Provided is a lipopeptide-compound-containing mixture separated from a Bacillus velezensis TCS001 fermentation broth. By further optimizing the formula of the culture medium for the Bacillus velezensis TCS001 fermentation broth and the culture fermentation process conditions, the output and yield of lipopeptide compounds in the fermentation broth are successfully improved, providing an efficient and cost-effective solution for the control of plant diseases.

Description

Culture medium and culture process for industrial production of Bacillus velez TCS001 fermentation broth, separation of mixture containing lipopeptide compounds from fermentation broth, and application thereof Technical Field

[0001] The present invention relates to the application fields of biological strain cultivation and separation of strain metabolites, and in particular to a culture medium for industrial production of Bacillus Velez TCS001 fermentation broth and separation of a mixture containing lipopeptide compounds from the special fermentation broth of Bacillus Velez TCS001. It also relates to optimization of cultivation and fermentation process conditions and application of the antibacterial activity of the mixture containing lipopeptide compounds. Background Art

[0002] Bacillus velezensis is a novel biocontrol bacterium. Spanish researchers Ruiz-García et al. first isolated two strains, CR-14b and CR-502T, in 2005. These strains are capable of producing large quantities of lipopeptides and exhibit strong antibacterial activity. Bacillus velezensis is widely distributed and can survive adverse conditions such as drought and salinity. However, few related products exist, suggesting promising development prospects.

[0003] Bacillus velezensis TCS001 was isolated from Bohai Sea silt. The original strain was Bacillus marineus CT2628. After mutagenesis and stabilization, it was identified and named Bacillus velezensis TCS001. It has been deposited with the General Microbiology Center of the China General Culture Collection (CGMCC) under the accession number CGMCC No. 8921 and was first published in Patent ZL201410168402.2. When grown on NA medium, TCS001 colonies are nearly round, light yellow, and opaque. Initially, the colonies are smooth with neat edges, but later develop wrinkles, slightly irregular edges, a central bulge, and a cloud-like appearance. Gram staining showed that the Bacillus Velezii TCS001 strain is Gram-positive and rod-shaped; it has a certain inhibitory effect on cucumber gray mold, cucumber vine blight, cucumber sclerotinia, cucumber brown spot, cotton wilt and banana leaf spot, among which the inhibition rate against cucumber gray mold is the highest, reaching 87.66%.

[0004] The inventors of this application have developed various types and formulations of Bacillus Velezii TCS001 culture media for laboratory culture dishes in previous studies:

[0005] NA medium: peptone 10.0 g / L, beef extract 3.0 g / L, glucose 10.0 g / L, yeast powder 1.0 g / L, agar powder 18.0 g / L, pH 7.0-7.2;

[0006] Seed culture medium: tryptone 7.0 g / L, yeast powder 2.0 g / L, glucose 2.0 g / L, NaCl 6.0 g / L, KCl 0.06 g / L, MgCl2·6H2O 0.5 g / L, pH 7.0-7.2;

[0007] Six basic fermentation media:

[0008] NB medium: peptone 10.0 g / L, beef extract 3.0 g / L, glucose 10.0 g / L, yeast powder 1.0 g / L, pH 7.0-7.2;

[0009] LB medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, pH 7.0-7.2;

[0010] BP medium: beef extract 5.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH 7.0-7.2;

[0011] SOB medium: peptone 20.0 g / L, yeast extract 5.0 g / L, NaCl 0.5 g / L, KCl 0.18 g / L, MgCl2·H2O 0.95 g / L, pH 7.0-7.2;

[0012] BPY medium: beef extract 5.0 g / L, peptone 10.0 g / L, yeast powder 5.0 g / L, glucose 5.0 g / L, pH 7.0-7.2;

[0013] YT medium: peptone 10.0 g / L, yeast powder 5.0 g / L, glucose 1.0 g / L, NaCl 5.0 g / L, pH 7.0-7.2;

[0014] Initial fermentation medium: peptone 10.0 g / L, yeast powder 1.0 g / L, beef extract 4.0 g / L, glucose 12.5 g / L, distilled water 1 L, pH 7.0;

[0015] The inventors of this application previously published a journal article (Optimization of fermentation conditions of Bacillus velezensis TCS001 using response surface methodology [J]. Journal of Pesticide Science, 2019, 21(4): 444-452.) disclosed that the optimal basal culture medium was screened from the above six basal fermentation media (NB, BPY, YT, BP, LB and SOB), and the optimal basal culture medium was finally obtained as NB medium. The fermentation conditions were: 10.0 g peptone, 12.5 g glucose, 4.0 g beef extract, 1.0 g yeast powder, 1000 mL distilled water, liquid volume (volume fraction) 40%, seed culture time 16 h, inoculation amount (volume fraction) 3%, rotation speed 164 r / min, temperature 25 ° C, pH 7.0, and fermentation culture time 36 h. The in vitro antibacterial activity test verified that the fermentation filtrate of the optimized culture had the strongest antibacterial activity, with an inhibition rate of 90.9%; the in vivo potted antibacterial test showed that the inhibition rate of the fermentation liquid of the optimized culture on cucumber gray mold lesions was 74.7%, an increase of 36.9% compared with before optimization.

[0016] However, the above culture medium and the optimal basal culture medium can only be used in laboratories and cannot meet the requirements of industrial production of Bacillus velez TCS001 fermentation broth.

[0017] Because microbial fermentation is the basis for obtaining a large amount of target products, the fermentation medium components and fermentation conditions (time, temperature, initial pH, inoculation size and shaker speed, etc.) vary depending on the desired target product. Optimizing these conditions can significantly affect the yield of the target product of the strain. In addition to considering the yield of the target product during the microbial fermentation process, a comprehensive analysis and evaluation of the fermentation cost should also be conducted to screen and obtain the optimal fermentation system suitable for the fermentation and application of the target strain. The purpose of optimizing the above-mentioned culture medium and conditions is to improve the antibacterial activity of the fermentation filtrate of the strain. The inhibitory effect of the fermentation filtrate on pathogenic fungi is mainly due to the secondary metabolites produced by the biocontrol strain during the stable growth period. In industrial production, the indicators considered for microbial preparations are mainly the number of viable cells or spores. Spores are a form formed by the strain in the later stage of growth and development and have characteristics such as resistance to stress. Therefore, obtaining the desired target product requires strict control of the strain growth period. The above-mentioned six culture media and fermentation process conditions cannot meet the needs of industrial large-scale spore production. The technical problem to be solved by the present invention is to optimize the culture medium components and fermentation conditions to obtain high spore yields.

[0018] In addition, Bacillus bacteria can secrete a variety of secondary metabolites with antibacterial activity. However, the metabolites in the fermentation broth of Bacillus velez TCS001 remain unclear. Another aspect of the present invention is to optimize the formulation of the Bacillus velez TCS001 culture medium and the culture and fermentation process to increase the yield of metabolites in the fermentation broth of Bacillus velez TCS001, extract and separate the metabolites, and perform bioactivity assays on the metabolites to elucidate the antibacterial mechanism of the fermentation broth of Bacillus velez TCS001.

[0019] Summary of the Invention

[0020] One of the objects of the present invention is to provide a culture medium formula and a culture process thereof that can be used for the industrial production of Bacillus velezensis TCS001 fermentation broth.

[0021] The culture medium for industrially producing Bacillus Velez TCS001 fermentation broth includes soluble peanut meal, one or more of glucose and soluble starch; the weight percentage of the soluble peanut meal is 1-5%, the weight percentage of glucose is 1-5%, and the weight percentage of the soluble starch is 1-5%; preferably, the weight percentage of the soluble peanut meal is 2%, the weight percentage of glucose is 3%, and the weight percentage of the soluble starch is 3%.

[0022] The culture medium further comprises one or more of ferric phosphate, potassium chloride, sodium chloride, magnesium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, light calcium carbonate, and manganese sulfate. The mass percentage of the ferric phosphate is 0.002%, the mass percentage of the potassium chloride is 0.006%, the mass percentage of the sodium chloride is 0.6%, the mass percentage of the magnesium chloride is 0.05%, the mass percentage of the dipotassium hydrogen phosphate is 0.3%, the mass percentage of the potassium dihydrogen phosphate is 0.15%, the mass percentage of the light calcium carbonate is 0.05%, and the mass percentage of the manganese sulfate is 0.005%.

[0023] The present invention provides a process for producing Bacillus velez TCS001 fermentation broth based on the culture medium for industrially producing Bacillus velez TCS001 fermentation broth, wherein the fermentation culture time is 60-84 hours; preferably, the fermentation culture time is 68-76 hours; and particularly preferably, the fermentation culture time is 72 hours. The initial pH of the fermentation culture is 6-7.5; preferably, the initial pH of the fermentation culture is 7.0. The fermentation culture liquid volume is 16-32%, preferably, the fermentation culture liquid volume is 24%; the fermentation culture inoculum size is 0.5-2%, preferably, the fermentation culture inoculum size is 1%. The fermentation culture rotation speed is 160-200 r / min, preferably, the fermentation culture rotation speed is 180 r / min; and the fermentation culture temperature is 25-37° C., preferably, the fermentation culture temperature is 28° C.

[0024] The present invention also provides a method for improving the fermentation spore production of Bacillus velezensis TCS001, comprising the culture medium, soluble peanut powder having a weight percentage of 2-2.88%, an initial fermentation pH of 7.0, a fermentation inoculation amount of 1%, a fermentation liquid volume of 31-32%, a fermentation rotation speed of 173-180 r / min, and a fermentation temperature of 28°C.

[0025] The present invention also provides an application of glucose and soluble starch as a composite carbon source and / or soluble peanut cake powder as a nitrogen source in a culture medium for industrially producing Bacillus Velezii TCS001 fermentation broth.

[0026] The present invention also provides a Bacillus Velez subtilis TCS001 fermentation liquid obtained by fermentation using the culture medium.

[0027] Another object of the present invention is to extract, separate, purify and identify the lipopeptide antibacterial substances of the main metabolites of Bacillus Velez TCS001, and to clarify the types and contents of the lipopeptide antibacterial secondary metabolites of Bacillus Velez TCS001.

[0028] One of the purposes of the present invention is to optimize the formula of another Bacillus Velez TCS001 culture medium and the culture and fermentation process with the goal of improving the yield and productivity of metabolites in the fermentation broth of Bacillus Velez TCS001.

[0029] The present invention relates to a mixture containing lipopeptide compounds. The lipopeptide compounds are compounds having the following structure. The mixture contains at least one, two, three or more lipopeptide compounds.

[0030] Among them, the weight of the compound of formula (1), the compound of formula (2) and the compound of formula (3) respectively accounts for 19.5%, 48.8% and 31.7% of the total weight of the compound of formula (1), the compound of formula (2) and the compound of formula (3); preferably, the weight ratio of the compound of formula (1): the compound of formula (2): the compound of formula (3) is 19.5:48.8:31.7, and the weight ratio of the compound of formula (1): the compound of formula (2): the compound of formula (3) is approximately 2:5:3.

[0031] The weight of the compound of formula (IV) and the compound of formula (V) respectively account for 25% and 75% of the total weight of the compound of formula (IV) and the compound of formula (V). Preferably, the weight ratio of the compound of formula (IV) to the compound of formula (V) is about 25:75.

[0032] The mixture is isolated from the fermentation broth of Bacillus velez TCS001;

[0033] The separation method can be an acid precipitation method, a salting-out method, an organic solvent precipitation method, or an organic solvent extraction method. Preferably, the separation method is an acid precipitation method.

[0034] The Bacillus Velez TCS001 fermentation liquid is obtained by inoculating Bacillus Velez TCS001 seed liquid into a culture medium for fermentation;

[0035] The Bacillus Velez TCS001 seed liquid is obtained by fermenting the activated Bacillus Velez TCS001 strain with MLB culture medium;

[0036] The culture medium comprises 10.5 g / L of soluble starch, 18.5 g / L of peanut meal, and 3.0 g / L of NaCl.

[0037] The fermentation culture process conditions are as follows: liquid volume 32%, inoculation volume 3%, initial fermentation pH 6.0, fermentation temperature 31° C., inoculation age 16 hours, rotation speed 164 rpm, and fermentation culture time 48 hours.

[0038] Another object of the present invention is to determine the overall and partial biological activities of the lipopeptide antibacterial substances, which are the main metabolites in the fermentation broth of Bacillus velezensis TCS001.

[0039] The present invention relates to the use of lipopeptide compounds of formula (I) to formula (V) for inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi is gray mold, preferably Botrytis cinerea.

[0040] The present invention relates to the use of a mixture containing lipopeptide compounds in preventing and controlling pecan dry rot, tomato early blight, potato late blight, strawberry gray mold, rapeseed sclerotinia, apple leaf spot, cucumber target spot, cucumber black spot, wheat sheath blight, sorghum anthracnose, tea tree anthracnose, gloeosporium anthracnose, pepper phytophthora, dendrobium officinale anthracnose, pumpkin leaf spot, cucumber wilt, strawberry anthracnose, and apple brown spot.

[0041] Another object of the present invention is to improve the purity and concentration of active metabolites secreted by Bacillus through the separation and extraction of lipopeptide secondary metabolites, and because their physical and chemical properties are more stable than those of Bacillus Velez TCS001 itself, they have broad application value in field disease prevention and control.

[0042] The present invention relates to a method for increasing the content of lipopeptide compounds in a fermentation broth of Bacillus velezensis TCS001. The fermentation medium comprises 1% soluble starch, 2% peanut meal, and 0.3% NaCl. The fermentation process conditions are: a fermentation temperature of 31°C, a fermentation time of 48 hours, a pH of 6.0, an inoculum size of 3%, a liquid volume of 32%, and an acid precipitation pH of 2.0.

[0043] The invention relates to a method for increasing lipopeptide compounds in a fermentation broth of Bacillus velezensis TCS001. The fermentation medium comprises 10.5 g / L of soluble starch, 18.5 g / L of peanut meal and 3 g / L of NaCl.

[0044] Beneficial technical effects of the technical solution of the present invention

[0045] The present invention provides, for the first time, a culture medium formula capable of industrially producing a fermentation broth of Bacillus velez TCS001. The optimized culture process, validated through response surface optimization experiments and steepest-hill-climbing test models, reduces fermentation production costs while increasing the number of viable bacteria and spores in the fermentation broth. Furthermore, the formula can be used for the industrial production and widespread application of Bacillus velez TCS001. The fermentation broth of Bacillus velez TCS001 obtained through culture and fermentation meets the requirements for industrialization and widespread application.

[0046] The present invention is the first to isolate a mixture containing lipopeptide compounds from the fermentation broth of Bacillus velezensis TCS001 and to determine the biological activity of the mixture;

[0047] This invention clarifies for the first time the types of lipopeptide secondary metabolites produced by Bacillus Velez TCS001, and clarifies the chemical structure and biological activity of the main lipopeptide compounds. Moreover, because the physicochemical properties of lipopeptide active substances are more stable than those of Bacillus Velez TCS001 itself, they have broad application value in field disease prevention and control.

[0048] The invention improves the output and yield of metabolites in the fermentation liquid of Bacillus Velez TCS001 by optimizing the formula of the culture medium of Bacillus Velez TCS001 and the culture and fermentation process.

[0049] The lipopeptide secondary metabolites extracted and separated by the present invention are important antibacterial active substances secreted by Bacillus. The present invention increases the yield of lipopeptide secondary metabolites of Bacillus velezensis TCS001, thereby effectively improving its effect in preventing and controlling plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Effects of different fermentation times on the viable count, spore count, and spore rate of Bacillus velezensis TCS001;

[0051] Figure 2 Effects of different carbon sources (A) on the number of viable cells, number of spores, and spore rate in the fermentation broth of Bacillus velezensis TCS001;

[0052] Figure 3 Effects of glucose mass fraction (B) on viable cell count, spore count, and spore rate in fermentation broth of Bacillus velezensis TCS001;

[0053] Figure 4 Effects of soluble starch mass fraction (C) on viable cell count, spore count, and spore rate in fermentation broth of Bacillus velezensis TCS001;

[0054] Figure 5 Effects of organic nitrogen source (D) on viable cell count, spore count, and spore rate in fermentation broth of Bacillus velezensis TCS001;

[0055] Figure 6 Effects of the mass fraction of soluble peanut cake powder (E) on the number of viable cells, number of spores, and spore rate in the fermentation broth of Bacillus velezensis TCS001;

[0056] Figure 7 Effects of different initial pH (A) on the number of viable cells, number of spores, and spore rate in the fermentation broth of Bacillus velezensis TCS001;

[0057] Figure 8 Effects of different liquid volumes (C) on the number of viable cells, number of spores, and spore rate in the fermentation broth of Bacillus velezensis TCS001;

[0058] Figure 9 Effects of different inoculum sizes (B) on viable cell counts, spore counts, and spore rate in fermentation broth of Bacillus velezensis TCS001;

[0059] Figure 10 Effects of different rotation speeds (D) on the number of viable cells, number of spores, and spore rate in the fermentation broth of Bacillus velezensis TCS001;

[0060] Figure 11 Effects of different temperatures (E) on the number of viable cells, number of spores, and spore rate in the fermentation broth of Bacillus velezensis TCS001;

[0061] Figure 12. Corresponding contour and surface plots of the effects of AB factors (mass fraction of soluble peanut meal and rotation speed) on the spore count of Bacillus velez TCS001.

[0062] Figure 13. Corresponding contour and surface plots of the effects of AC (mass fraction of soluble peanut meal and liquid volume) on the spore count of Bacillus velez TCS001.

[0063] Figure 14. Corresponding contour and surface plots of the effects of BC (rotating speed and liquid volume) factors on the spore count of Bacillus velez TCS001.

[0064] FIG15 is a schematic diagram of extracting and separating a mixture containing lipopeptide compounds from the fermentation broth of Bacillus velez TCS001;

[0065] Figure 16: Lipopeptide production by Bacillus velezensis TCS001 under different fermentation conditions;

[0066] Figure 17: Lipopeptide production by Bacillus velezensis TCS001 under different fermentation medium components and contents;

[0067] Figure 18: The interactive effect of peanut meal content and soluble starch content on the production of lipopeptides by Bacillus Velez TCS001;

[0068] Figure 19: Effect of the interaction between soluble starch content and fermentation temperature on the production of lipopeptides by Bacillus velez TCS001;

[0069] Figure 20. Effect of the interaction between fermentation temperature and peanut meal content on the production of lipopeptides by Bacillus velez TCS001;

[0070] Figure 21 is a negative ion high-resolution mass spectrum of compound 1;

[0071] Figure 22 1H-NMR spectrum of compound 1;

[0072] Figure 23 13C-NMR of compound 1;

[0073] Figure 24 Structure of Compound 1;

[0074] Figure 25 is a negative ion high-resolution mass spectrum of compound 2;

[0075] Figure 26 1H-NMR spectrum of compound 2;

[0076] Figure 27 13C-NMR of compound 2;

[0077] Figure 28 Structure of Compound 2;

[0078] Figure 29 is a negative ion high-resolution mass spectrum of compound 3;

[0079] Figure 30 1H-NMR spectrum of compound 3;

[0080] Figure 31 13C-NMR spectrum of compound 3;

[0081] Figure 32 Structure of compound 3;

[0082] Figure 33 Bacillus velez TCS001 fermenter;

[0083] Figure 34 is a negative ion high-resolution mass spectrum of compound 4;

[0084] Figure 35 1H-NMR spectrum of compound 4;

[0085] Figure 36 shows the 13C-NMR spectrum of compound 4;

[0086] Figure 37 Structure of Compound 4;

[0087] Figure 38 is a negative ion high-resolution mass spectrum of compound 5;

[0088] Figure 39 1H-NMR spectrum of compound 5;

[0089] Figure 40 is the 13C-NMR spectrum of compound 5;

[0090] Figure 41 Structure of Compound 5;

[0091] Figure 42 Antibacterial activity assay results of compounds 1 to 3;

[0092] Figure 43 Antibacterial activity assay results of compounds 1 to 3;

[0093] Figure 44 Antibacterial activity assay results of compounds 4 to 5;

[0094] FIG45 shows the inhibitory effect of a mixture of lipopeptides extracted and isolated from the fermentation broth of Bacillus velezensis TCS001 on 18 plant pathogenic fungi;

[0095] Annotations in Figure 45: A: Colletotrichum gloeosporioides; B: Phytophthora capsici; C: Hickory bark rot; D: Gibberellic acid; E: Sclerotinia sclerotiorum; F: Apple brown spot; G: Colletotrichum truncatula; H: Botrytis cinerea; I: Potato late blight; J: Curvularia zeae leaf spot; K: Rhizoctonia solani; L: Apple ring rot; M: Sorghum anthracnose; N: Wheat root rot; O: Strawberry anthracnose; P: Cucumber brown spot; Q: Cucumber wilt; R: Torreya grandis root rot; AR is the blank control group; A'-R' is a 50 mg / L mixture containing lipopeptide compounds; A"-R" is 50 mg / L chlorothalonil; DETAILED DESCRIPTION

[0096] Example 1

[0097] A culture medium and culture process for industrial production of Bacillus Velez TCS001 fermentation broth are studied as follows:

[0098] Initial selection of test culture medium:

[0099] LB medium: 10.0 g of tryptone, 5.0 g of yeast powder, 3.0 g of beef extract, 10.0 g of NaCl, 18.0 g of agar, 1000 mL of distilled water, pH 7.0-7.2.

[0100] MLB medium: 7.0 g of tryptone, 2.0 g of yeast powder, 2.0 g of glucose, 6.0 g of NaCl, 0.06 g of KCl, 0.5 g of MgCl2·6H2O, 1000 mL of distilled water, pH 7.0-7.2.

[0101] Optimized initial culture medium: glucose 30 g, beef extract 20 g, FePO4 0.02 g, KCl 0.06 g, NaCl 6 g, MgCl2·6H2O 0.5 g, K2HPO4 3 g, KH2PO4 1.5 g, CaCO3 0.5 g, MnSO4 0.05 g, distilled water 1000 mL, pH 7.5.

[0102] Test methods

[0103] Strain activation

[0104] The Velezacacillus strain TCS001 was streaked onto an LB solid plate using the plate streak method and cultured in a bacterial incubator at 28° C. for 48 h to obtain a single colony.

[0105] Seed solution and fermentation liquid preparation

[0106] Use an inoculating loop to pick a single colony grown from a streak on the plate and transfer it to 150 / 250 mL of MLB liquid medium. Incubate in a constant temperature shaker at 27°C and 145 rpm for 16 hours to obtain a seed solution. Inoculate the seed solution at a 3% (volume fraction) inoculum into 100 / 250 mL of fermentation medium and incubate in a constant temperature shaker at 25°C and 164 rpm for 72 hours to obtain a fermentation broth.

[0107] Calculation of viable bacteria, spore count, and spore rate

[0108] The viable bacteria were counted using the dilution plate method, in which the fermentation broth was heated at 80°C for 10 min, serially diluted, and then spread on LB solid plates for counting to determine the number of spores.

[0109] Spore rate = number of spores / number of viable bacteria × 100%

[0110] Selection of optimal fermentation time for strains

[0111] The number of viable bacteria and spores in the fermentation broth of the strain was used as an indicator to determine the effects of different fermentation times on the number of viable bacteria and spores in the fermentation broth of the strain. The measurement was conducted every 24 hours until 96 hours, and the optimal fermentation time was selected based on the measurement results.

[0112] Single factor optimization experiment

[0113] Optimization of optimal culture medium components

[0114] Several carbon sources (glucose, sucrose, soluble starch, fructose, maltose) and organic nitrogen sources (beef extract, soybean powder, yeast powder, peptone, peanut meal) were selected and replaced in equal amounts with the corresponding components of the initial culture medium. In a 250mL Erlenmeyer flask filled with 40% (the same below), inoculated with 3% (volume fraction, the same below), cultured at 164 rpm and 25°C for 72 hours, the number of viable cells and spores per milliliter of the fermentation broth was measured, and the sporulation rate was calculated to determine the optimal carbon and organic nitrogen source types. The effects of different carbon and nitrogen source concentrations (mass fractions) on TCS001 sporulation were also screened to determine the optimal carbon and nitrogen source concentrations.

[0115] Optimization of optimal culture conditions

[0116] The optimized culture medium components were used, with an inoculum size of 3%, an initial pH of 7.5, a liquid volume of 40%, a temperature of 25°C, and a rotation speed of 164 r / min as the initial culture conditions. Single-factor experiments were conducted on five factors, including fermentation temperature (25°C, 28°C, 32°C, 35°C, 37°C), initial pH (6.0, 6.5, 7.0, 7.5, 8.0), inoculum size (0.5%, 1%, 2%, 3%, 4%, 5%), liquid volume (16%, 24%, 28%, 32%, 36%, 40%), and rotation speed (140r / min, 160r / min, 180r / min, 200r / min, 220r / min). The number of spores in the TCS001 fermentation broth was used as the experimental indicator to determine the effect of each factor on spore production.

[0117] Response surface optimization experiment

[0118] Plackett-Burman (PB) test

[0119] Based on the results of the single-factor optimization experiments, we conducted further optimization through a PB test. Using Design Expert 10.0, we designed the experimental plan, setting two levels for each factor: a high level of "1" and a low level of "-1" within the range. Each experiment was replicated three times. Using the spore count in the fermentation broth of strain TCS001 as the response value, we screened for factors that significantly affected spore count.

[0120] Steepest climb test

[0121] Based on the results of the PB test, three relatively significant factors affecting spore production were screened out. The climbing direction was determined by the positive or negative effect value, and the climbing step size was determined according to the size of the factor effect value. The maximum response surface area was quickly approached and the center point of the box-behnken design (BBD) was determined.

[0122] BBD test

[0123] Using the BBD experimental design principle, taking spore yield as the response value and combining the center point obtained from the steepest climbing test, Design expert 10.0 software was used to design 17 groups of experiments with 3 factors and 3 levels, and the results were analyzed and processed.

[0124] Response surface optimization results verification test

[0125] After the response surface optimization was completed, repeated experiments were carried out under the optimal fermentation conditions. The fermentation broth was plated and counted, and the initial culture medium was used as the control to detect the increase rate of the spore number after optimization.

[0126] Test results

[0127] As shown in Figure 1, the number of live bacteria and spores of Bacillus velezensis TCS001 cultured in the initial culture medium increased with time, reaching a peak at 72 hours, with the number of live bacteria being 6.6×10 9 CFU / mL, spore count was 4.7×10 9 CFU / mL, and then the number of live bacteria and spores decreased significantly after 96 hours. Therefore, 72 hours was selected as the optimal time for the fermentation broth culture of this strain.

[0128] The analysis showed (Figure 2) that the number of spores of Bacillus velez TCS001 in different carbon sources was glucose > soluble starch > fructose > sucrose > maltose. When glucose was used as the carbon source, the number of viable bacteria and spores was the highest, at 5.13×10 9 CFU / mL and 3.50×10 9CFU / mL, and the spore rate was 68.22%. When soluble starch was used as the carbon source, although the number of viable bacteria and spores decreased, the spore rate increased to 83.6%. Therefore, glucose and soluble starch can be selected as the composite carbon source for spore production in the fermentation broth of Bacillus Velezii TCS001.

[0129] As shown in Figure 3, the number of spores and viable bacteria in the fermentation broth varies at different glucose concentrations. The number of spores and the spore rate both reach their highest values ​​when the glucose concentration is 3%, with the number of viable bacteria and spores reaching 7.66×10 9 CFU / mL and 5.2×10 9 CFU / mL, and the spore rate was 67.88%.

[0130] As shown in Figure 4, the number of spores and the number of viable bacteria in the fermentation broth were different at different soluble starch mass fractions. The number of spores and the spore rate in the fermentation broth reached the highest value when the soluble starch mass fraction was 3%, and the number of spores was 4.1×10 9 CFU / mL, and the spore rate was 68.33%. Therefore, 3% mass fraction of soluble starch and 3% concentration of glucose were selected as the fermentation medium for the subsequent spore production of Bacillus velez TCS001.

[0131] The results of the analysis showed that (Figure 5) the number of spores produced in the fermentation broth of Bacillus velez TCS001 under different organic nitrogen sources was as follows: soluble peanut cake powder > beef extract > yeast powder > peptone > soybean powder. When soluble peanut cake powder was used as the nitrogen source, the number of viable bacteria and spores was the highest, which were 6.23×10 9 CFU / mL and 3.76×10 9 CFU / mL, and the spore rate was 60.4%. Therefore, soluble peanut cake powder was selected as the optimal nitrogen source for the fermentation medium for spore production of Bacillus Velezii TCS001.

[0132] As shown in Figure 6, the number of spores in the fermentation broth is the highest when the mass fraction of soluble peanut powder is 2%, reaching 6.2×10 9 CFU / mL, the spore rate was 82.19%. Therefore, 2% by mass of soluble starch was selected as the subsequent nitrogen source concentration for the spore formation fermentation culture of Bacillus Velezii TCS001.

[0133] As shown in Figure 7, when the initial pH was 7.0, the number of viable bacteria and spores in the fermentation broth reached the highest, which were 9.46×10 9 CFU / mL and 6.46×10 9CFU / mL, and the spore rate was 68.28%. The number of viable bacteria and spores were significantly better than those of other treatments. Both higher and lower pH values ​​were not suitable for the production of viable bacteria and spores. Therefore, the pH value of 7.0 was selected for the subsequent spore production in the fermentation broth of Bacillus velez TCS001.

[0134] As shown in Figure 8, when the liquid volume was 24%, the number of viable bacteria and spores reached the highest, which were 7.9×10 9 CFU / mL and 6.5×10 9 CFU / mL, the spore rate was 82.27%. Therefore, the subsequent culture volume of TCS001 spore fermentation was selected to be 24%.

[0135] As shown in Figure 9, when the inoculum amount was 1%, the number of viable bacteria and spores was the highest, which were 8.6×10 9 CFU / mL and 6.53×10 9 CFU / mL, the spore rate was 75.93%. When the inoculation amount exceeded 1%, the spore number decreased. Therefore, the inoculation amount of the subsequent culture of spore-forming fermentation in the fermentation broth of Bacillus Velezii TCS001 was selected to be 1%.

[0136] As shown in Figure 10, when the speed is 180 r / min, the number of viable bacteria and spores is the highest, which are 8.34×10 9 CFU / mL and 6.3×10 9 CFU / mL, and the spore rate was 75.53%. Therefore, the subsequent culture rotation speed of 180r / min was selected for the spore fermentation in the fermentation broth of Bacillus Velezii TCS001.

[0137] As shown in Figure 11, Bacillus velezensis TCS001 can grow and reproduce in the temperature range of 25-37°C. The number of viable bacteria, spore counts, and spore rate vary significantly at different temperatures. When the culture temperature is 28°C, the number of viable bacteria and spore counts reach the highest values, which are 8.63×10 9 CFU / mL and 6.56×10 9 CFU / mL, and the spore rate was 76.01%. Therefore, the subsequent culture temperature for spore production fermentation in the fermentation broth of Bacillus Velezii TCS001 was selected as 28°C.

[0138] By analyzing the results of the single-factor experiments, we determined the factors for the PB experiment. Following the PB experimental design, we set each factor at two levels: -1 and 1. Using the number of spores in the fermentation broth of Bacillus velezensis TCS001 as the response, we determined the impact of each factor on spore production. We then selected factors with significant effects for the next step, the ramp-up experiment. The PB experiment was designed using Design Expert 10.0 software (Table 1), with 12 treatment groups, each with three replicates.

[0139] Table 1 PB experimental design and results

[0140] Table 2 Analysis of variance of each factor in PB test

[0141] From Table 1 and Table 2, we can see that the inoculation amount, liquid volume, temperature, glucose, and rotation speed are positive effects, while the initial pH, soluble peanut powder, and soluble starch are negative effects. 2 =98.49%, R 2 adj=94.48%, P value=0.0118<0.05, indicating that the model is relatively reliable. Rotation speed, peanut cake powder, liquid volume and inoculation amount have significant effects on TCS001 fermentation spore production (P<0.05), while other factors have no significant effect on TCS001 fermentation spore production (P>0.05).

[0142] The gradient direction of the test values ​​of the important influencing factors, rotation speed, liquid volume and peanut cake powder, was used as the climbing direction. The change step size was determined according to the response value of each factor. The culture was carried out at 28°C, initial pH value of 7.0 and inoculation size of 1%. The results of the steepest climbing test are shown in Table 3.

[0143] Table 3 Steepest climbing test

[0144] As can be seen from Table 3, with the increase of the concentration of important influencing factors, the trend of the change of the number of spores in the fermentation broth is first increased and then decreased. The number of spores in the fermentation broth of Experiment 4 was the highest, reaching 6.75×10 9 CFU / mL. Therefore, a rotation speed of 170 r / min, a liquid volume of 32%, and 3% peanut meal were used for subsequent optimization experiments. Other conditions included 3.0% glucose, 3.0% soluble starch, 0.002% FePO4, 0.006% KCl, 0.6% NaCl, 0.05% MgCl2·6H2O, 0.3% K2HPO4, 0.15% KH2PO4, 0.05% CaCO3, 0.005% MnSO4, a culture temperature of 28°C, an inoculum size of 1.0%, and a pH of 7.0.

[0145] The peanut cake powder, rotation speed and liquid volume in the steepest climbing test are marked as A, B and C respectively. The BBD test results and analysis are shown in Table 4, and the regression model variance analysis results are shown in Table 5.

[0146] Table 4 Box-Behnken central composite experimental design and results

[0147] Table 5 BBD variance analysis results Note: R 2 =94.50%, R 2 adj =87.73%, *p<0.05, **p<0.01.

[0148] Model R 2 =94.50%, R 2 adj = 87.73%, P = 0.0012 < 0.01, lack of fit P = 0.1410 > 0.05, indicating that the model is reliable and can be used to analyze and predict response values. AB, A 2 、B 2 、C 2 The results showed that the effect of B on spore production in the fermentation broth of Bacillus velez TCS001 was extremely significant (P < 0.01), and B had a significant effect on spore production in the fermentation broth of Bacillus velez TCS001 (P < 0.05). A, C, A×C, and B×C had some effects but were not significant (P > 0.05). The experimental results were analyzed using Design expert 10.0 software, and the quadratic regression equation for the response value and the experimental factors was fitted as follows: Y = 6.98-0.037A + 0.60B-0.057C-0.13AB + 0.013AC-0.0025BC-0.15A 2 -0.17B 2 -0.21C 2 .

[0149] Contour maps and 3D surface plots were drawn based on the regression equation, and the results are shown in Figures 12, 13, and 14. The results show that the interaction effect between peanut meal and rotational speed is characterized by an elliptical contour map and a steep 3D surface plot, indicating the strongest interaction. The interaction effect between rotational speed and liquid volume is characterized by a circular contour map and a smooth 3D surface plot, indicating a relatively weak interaction.

[0150] The optimal fermentation parameters for Bacillus velez TCS001 obtained from the model and software analysis were 2.88% peanut meal, 31% liquid volume, 173 r / min rotation speed, 3.0% glucose, 3.0% soluble starch, 0.002% FePO4, 0.006% KCl, 0.6% NaCl, 0.05% MgCl2·6H2O, 0.3% K2HPO4, 0.15% KH2PO4, 0.05% CaCO3, 0.005% MnSO4, culture temperature 28℃, inoculum size 1.0%, and pH 7.0. The predicted spore number under these conditions was 6.995×10 9 CFU / mL.

[0151] In order to verify the accuracy of the above optimized conditions, three repeated experiments were carried out under the optimized conditions, and the actual spore production number was 6.70×10 9 The measured values ​​of CFU / mL were close to those predicted by the regression equation, indicating that the mathematical model established by the research institute was reliable.

[0152] The above specific implementation methods further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made on the basis of the technical solutions of the present invention should be included in the scope of protection of the present invention.

[0153] Example 2

[0154] The research on separation of mixtures containing lipopeptides from fermentation broth and their application is as follows:

[0155] A mixture containing lipopeptides was isolated from the fermentation broth of Bacillus velezensis TCS001, and the structures of the lipopeptides in the mixture were identified.

[0156] Test materials

[0157] Test strains

[0158] Biocontrol bacteria: Bacillus velezensis TCS001 was isolated from Bohai silt and has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) with the deposit number CGMCC No.8921.

[0159] culture medium

[0160] PDA solid medium: anhydrous glucose 20.0 g / L, potato 200.0 g / L, agar powder 18.0 g / L, distilled water 1 L, natural pH.

[0161] LB solid medium: agar powder 18.0 g / L, tryptone 10.0 g / L, yeast powder 5.0 g / L, NaCl 10.0 g / L, distilled water 1 L, pH 7.0.

[0162] MLB seed culture medium: 2.0 g / L anhydrous glucose, 7.0 g / L tryptone, 2.0 g / L yeast extract, 6.0 g / L NaCl, 0.06 g / L KCl, 0.5 g / L MgCl2·6H2O, 1 L distilled water, pH 7.0.

[0163] Fermentation medium: soluble starch 10.5 g / L, peanut powder 18.5 g / L, NaCl 3.0 g / L, distilled water 1 L, pH 6.0.

[0164] Main instruments

[0165] MGC-250BP artificial climate chamber (Shanghai Yiheng Scientific Instrument Co., Ltd.);

[0166] BSA233S electronic precision balance (Sartorius Scientific Instruments Co., Ltd.);

[0167] HH-4 digital display constant temperature water bath (Changzhou Guohua Electric Co., Ltd.);

[0168] FE28 pH meter (Mettler-Toledo Instruments Co., Ltd.);

[0169] CT15RE desktop refrigerated centrifuge (Hitachi, Japan);

[0170] SW-CJ-2FD clean workbench (Suzhou Antai Air Technology Co., Ltd.);

[0171] ZQZY-CF oscillating incubator (Shanghai Zhichu Instrument Co., Ltd.);

[0172] YB102 electronic balance (Hangzhou Weimipai Technology Co., Ltd.);

[0173] Thin layer chromatography silica gel plates (Hangzhou Kaiying Instrument Business Department);

[0174] Shaker (New Brunswick Scientific);

[0175] Silica gel (Qingdao Ocean Chemical Plant);

[0176] Diaion HP-20 resin (Mitsubishi Chemical Corporation);

[0177] Ultrasonic oscillator (Hangzhou Farante Ultrasonic Technology Co., Ltd.);

[0178] Hot air constant temperature drying oven (Shanghai Shibei Instrument Equipment Factory);

[0179] Rotary evaporator (Digital water bath SB-1000) (EYELA, Japan);

[0180] Fermentation tank (30 L) (Hangzhou Huihe Machinery Equipment Co., Ltd.);

[0181] High performance liquid chromatography (Shimadzu LC-8A, Shimadzu-C18, 5 μm, 250 × 20 mm id) (Shimadzu Corporation, Japan);

[0182] superconducting nuclear magnetic resonance instrument (Bruber AVANCE-400) (Bruker, Rheinstetten, Germany);

[0183] UV spectrometer (Varian Cary 300 Bio spectrophotometer) (Varian Technologies China Co., Ltd., USA);

[0184] (VARIAN) infrared spectrometer (Nicolet Magna FT-IR 750 spectrometer) (Varian, Palo Alto, CA, USA);

[0185] Test methods and results

[0186] Activation culture of test strains

[0187] Activation of plant pathogenic fungi: Inoculate the plant pathogens on a PDA plate, culture it upside down in a 25°C incubator, and select the strains with strong vitality on the periphery of the culture dish for use.

[0188] Activation of TCS001 strain: Take 10 μL of Bacillus velezensis TCS001 bacterial solution from the thawed glycerol tube and spread it on LB plate. After incubation at 27°C in the dark for 24 hours, streak culture on LB plate again and incubate at 27°C in the dark for 48 hours before use.

[0189] Extraction of lipopeptides produced by fermentation of Bacillus velez TCS001

[0190] As shown in Figure 15, a loopful of TCS001 strain, activated after 48 hours of culture, was inoculated into a 250 mL Erlenmeyer flask containing 150 mL of MLB broth. The broth was then incubated at 145 rpm and 27°C for 16 hours to prepare a seed solution. The seed solution was then inoculated into a 250 mL Erlenmeyer flask containing 80 mL of fermentation broth at a 3% inoculum level and incubated at 31°C and 164 rpm for 48 hours. Following the incubation period, lipopeptides were extracted using acid precipitation. The fermentation broth was centrifuged at 8000 rpm for 10 minutes at 4°C to obtain a supernatant. The pH of the supernatant was adjusted to 2.0 with 6 mol / L HCl and the precipitate was allowed to stand at 4°C overnight. The acid-precipitated solution was then centrifuged at 8000 rpm for 10 minutes, the supernatant discarded, and the precipitate was dried and weighed.

[0191] Single-factor optimization of fermentation conditions for lipopeptides derived from strain TCS001

[0192] The single-factor experimental method was used to study the effects of different fermentation temperatures (25℃, 28℃, 32℃, 37℃), fermentation time (24h, 36h, 48h, 60h, 72h), initial fermentation liquid pH (5.0, 6.0, 7.0, 8.0 and 9.0), liquid volume (60 / 250ml, 80 / 250ml, 100 / 250mL, 120 / 250mL and 150 / 250mL), and inoculation size (1%, 2%, 3%, 5% and 8%) on the production of lipopeptides, and the optimal fermentation conditions were obtained. Based on this, single-factor optimization experiments were conducted on fermentation medium components, optimizing the types and contents of carbon sources (maltose, sucrose, mannitol, soluble starch, fructose, and glucose), nitrogen sources (peanut meal, peptone, yeast extract, and beef extract), and inorganic salt ions (MgSO4·7H2O, FeSO4·7H2O, NaCl, CaCl2, MnSO4, K2HPO4, and KH2PO4). Based on the initial fermentation conditions, each variable was varied individually, with five replicates for each experiment.

[0193] The TCS001 lipopeptide precipitates obtained under different fermentation conditions were collected, dried in an oven at 60°C for 18 h, and weighed to obtain the dry weight of the TCS001 lipopeptide.

[0194] The dry weight of lipopeptides under different fermentation conditions was compared to obtain the optimal fermentation conditions.

[0195] Test results

[0196] Effects of fermentation conditions on the yield of lipopeptides

[0197] The initial fermentation conditions were as follows: fermentation temperature of 25°C, fermentation time of 36h, liquid volume of 40% (100 / 250mL), inoculum size of 3%, pH 7.0, acid precipitation pH 2, and shaker speed of 164rpm. With fermentation temperature as the only variable, four experimental groups were set at 25°C, 28°C, 32°C, and 37°C, with other fermentation parameters remaining unchanged. As can be seen from Figure 16, when the fermentation temperature was 28°C, the fermentation time was 48h, the initial fermentation pH was 6.0, the liquid volume was 80 / 250mL, and the inoculum size was 3%, the yields of lipopeptides reached their maximum, which were 0.464g / L, 0.540g / L, 0.498g / L, 0.595g / L, and 0.524g / L, respectively.

[0198] The optimized fermentation conditions were as follows: fermentation temperature of 28°C, fermentation time of 48 h, initial fermentation pH of 6.0, liquid volume of 32% (80 / 250 mL), and inoculation size of 3%.

[0199] An optimization experiment of fermentation medium components was carried out on basic fermentation medium.

[0200] The results of the optimization test of the fermentation medium components showed that when the carbon source was soluble starch, the production of lipopeptides reached the maximum, which was 0.615 g / L. Compared with the initial carbon source peptone, the production increased by 10.8%. Therefore, the optimal carbon source was soluble starch (Figure 17).

[0201] The soluble starch content was optimized, and six concentration gradients of 0.75%, 1.0%, 1.25%, 1.5%, 2.0% and 2.5% were set respectively. The results are shown in Figure 17. When the soluble starch content was 1%, the production of lipopeptides reached the maximum, which was 0.633 g / L. Therefore, the optimal content of soluble starch was 1%.

[0202] When the nitrogen source was peanut cake powder, the yield of lipopeptides reached the maximum, which was 0.645 g / L. Compared with the initial carbon source, the yield increased by 16.2%. Therefore, the optimal nitrogen source was peanut cake powder (Figure 17).

[0203] The peanut cake powder content was optimized, and six concentration gradients were set at 0.75%, 1.0%, 1.5%, 2.0%, 2.5% and 3%. The results showed that when the peanut cake powder content was 2%, the production of lipopeptides reached the maximum, which was 0.655 g / L. Therefore, the optimal content of peanut cake powder was 2% (Figure 17).

[0204] As shown in Figure 17 , taking into account various factors such as cost, NaCl was finally selected as the inorganic salt in the fermentation medium, with a yield of 0.663 g / L, which was 5.23% higher than that without adding inorganic salts. When the NaCl content was 0.3%, the yield reached a maximum of 0.68 g / L ( Figure 17 ).

[0205] Optimization of the process for isolating crude lipopeptides from fermentation of Bacillus velez TCS001

[0206] Test methods

[0207] Based on the results of the single-factor optimization test, the effects of fermentation conditions and culture medium components were comprehensively considered, and the optimal fermentation conditions for the production of lipopeptides by TCS001 were preliminarily determined. Six factors that significantly affected the production of lipopeptides by TCS001 were selected. The six selected factors were then screened using the Placket and Burman Design method. Each condition was set at two levels, high (+1) and low (-1), and three key factors affecting the production of lipopeptides were screened for further study.

[0208] The importance of fermentation conditions and six components of the fermentation medium were analyzed: fermentation temperature, fermentation time, liquid volume, peanut meal, soluble starch, and NaCl. Each component was tested with five replicates, using lipopeptide production as the response. The experimental factors and levels are shown in Table 6.

[0209] Table 6 Placket-Burman test factors and levels

[0210] Test results

[0211] The results of the PB screening test showed that fermentation temperature, peanut meal content, and soluble starch content had significant effects on lipopeptide production. A three-factor, three-level Box-Benhnken central composite experimental design was conducted using Design Expert software for fermentation temperature (A), soluble starch (B), and peanut meal (C). The factors and levels are shown in Table 7. The response surface design and experimental results are shown in Table 8.

[0212] Table 7 Box-Behnken design variables and levels

[0213] Table 8 Box-Behnken central composite experimental design and results

[0214] The response surface analysis was performed using the software Design Expert. With the lipopeptide yield Y as the response value, and the fermentation temperature A, soluble starch B, and peanut powder C as the center points, the multivariate quadratic polynomial regression model equation was obtained as follows: Y = 0.68 + 0.00125A - 0.03B + 0.0065C - 0.0015AB + 0.003AC + 0.011BC - 0.052A 2 -0.023B 2 -0.044C 2 .

[0215] The model was subjected to variance analysis and difference significance test. It can be seen from Table 4 that the model regression is significant (P < 0.001). Observing the regression equation, it can be seen that the order of the factors affecting the lipopeptide fermentation conditions is peanut cake powder > soluble starch > fermentation temperature. In order to illustrate the degree of influence of the direct effect of each factor and its interaction on the impact value, the test results were subjected to variance analysis. The F value of the model is 6.74, and the P value is 0.0099, which reached the significant level. The lack of fit term is 0.6855 and is less than 0.05, indicating that the quadratic model used in this experiment is significant. The P value of soluble starch A is less than 0.01, indicating that the soluble starch content has a highly significant effect on the fermentation titer, and the fermentation temperature A and peanut cake powder content C are not significant. The coefficient of determination R of the model 2 The corrected determination coefficient R 2 (Adj) is 0.8637, indicating that the model has a high degree of fit with the experimental data. Therefore, the model can be used to predict the effect of fermentation conditions on the production of lipopeptides of Bacillus velezinis TCS001.

[0216] Table 9 Box-Behnken experimental design regression analysis results Note: R 2 =0.9866, R 2 (Adj)=0.8637,*p<0.05,**p<0.01

[0217] Based on the multivariate quadratic polynomial regression model equation and combined with Design Expert software, response surface curves and contour lines were drawn. By fixing any one of the factors, fermentation temperature, soluble starch content, and peanut meal content, at zero, a 3D interaction model of the other two factors was generated, allowing for visual analysis. Figure 18 shows the interactive relationship between peanut meal content and soluble starch content on TCS001 lipopeptide production, Figure 19 shows the interactive relationship between soluble starch content and fermentation temperature on TCS001 lipopeptide production, and Figure 20 shows the interactive relationship between peanut meal content and fermentation temperature on TCS001 lipopeptide production.

[0218] The results were further analyzed by software, and the predicted fermentation conditions were fermentation temperature of 31°C, soluble starch content of 10.5g / L, peanut cake powder content of 18.5g / L, and predicted lipopeptide production of 0.693g / L.

[0219] Using the software Design Expert to analyze the data and solve the regression equation, it was determined that this model has a maximum response value. The actual values ​​of the three factors were converted: fermentation temperature of 31°C, soluble starch of 10.5g / L, and peanut meal of 18.5g / L. The predicted maximum response value, Y, was 0.693g / L. To verify the accuracy of the model's predictions, the experiment was repeated three times using the optimized fermentation conditions. The results showed that the average lipopeptide production of TCS001 bacteria was 0.653g / L, reaching 94.23% of the model's predicted value, and there was a good fit between the predicted value and the actual value. Therefore, this model can well reflect the influence of various factors of TCS001 fermentation conditions on the production of lipopeptides.

[0220] Extraction, separation and identification of lipopeptides

[0221] The yellow solid extracted after acid precipitation was mixed with silica gel, placed in a fume hood and allowed to stand. After the silica gel powder was dried, it was loaded onto the column. After the solvent was completely evaporated, the column was loaded with a volume ratio of sample silica gel to column silica gel of 1:4. Dichloromethane / methanol was used as the elution phase for gradient elution (CH2Cl2 / CH3OH=95:5 / 90:10 / 85:15 / 80:20 / 70:30 / 50:50) with an elution volume of 800 mL. The fractions were collected in a 250 mL conical flask with 150 mL in each bottle. The components were analyzed by TLC thin-layer chromatography. According to UV and color development, the fractions were combined and concentrated to obtain four parts I, II, III, and IV. Compound III was eluted using a 1L gel chromatography column with a CH2Cl2 / CH3OH (1:1, v / v) system. The elution volume was 1L, and the fractions were collected in 10mL test tubes. After TLC analysis, the following fractions were obtained: I-1, I-2, and I-3. I-2 was subjected to semi-preparative HPLC to yield two compounds, while I-3 was subjected to semi-preparative HPLC to yield one compound. Semi-preparative HPLC was performed at a flow rate of 1.5mL / min and a detection wavelength of λ = 220nm using a nitrile:0.1% TFA ratio of 40:60. The structures of the compounds were primarily identified using NMR nuclear magnetic resonance (NMR) and ESI-MS mass spectrometry, combined with high-resolution mass spectrometry and NMR spectroscopy.

[0222] Compound 1 (TCS001-I-1) is a white powder identified as iturin with a molecular formula of C49H76N12O14. The negative ion high-resolution mass spectrum, 1H-NMR spectrum, 13C-NMR spectrum, and structure of Compound 1 are shown in Figures 21-24.

[0223] Compound 2 (TCS001-I-2) is a white powder identified as iturin with a molecular formula of C49H76N12O14. The negative ion high-resolution mass spectrum, 1H-NMR spectrum, 13C-NMR spectrum, and structure of compound 2 are shown in Figures 25-28.

[0224] Compound 3 (TCS001-I-3) is a white powder identified as iturin with a molecular formula of C48H74N12O14. The negative ion high-resolution mass spectrum, 1H-NMR spectrum, 13C-NMR spectrum, and structure of compound 3 are shown in Figures 29-32.

[0225] Large-scale fermentation of lipopeptides by Bacillus velezensis TCS001

[0226] As shown in Figure 19, a 30L fermenter was used, with a 30L fermentation volume. The fermentation recipe consisted of 10.5g / L soluble starch, 18.5g / L peanut meal, 3.0g / L NaCl, and 1L distilled water. The pH was adjusted to 6.0, and the distilled water was used. The seed solution (3%) was added to the fermenter, and the fermentation temperature was 31°C, the rotation speed was 164 rpm, and the fermentation time was 48 hours.

[0227] Isolation, extraction and identification of large amounts of fermentation compounds

[0228] After tanking, the fermentation broth is centrifuged at 4000 rpm to separate the cells from the supernatant. The supernatant is then adsorbed twice on a pre-prepared HP-20 macroporous resin. After complete adsorption, the column is rinsed with deionized water for approximately three column volumes. Elution is then performed with 95% industrial ethanol, and the eluate is collected. The cells are extracted with methanol two to three times. The previously collected supernatant is combined with the cell extract and concentrated under reduced pressure. The fractions are analyzed by TLC thin-layer chromatography. Based on UV and color development, the fractions are combined and concentrated to yield five fractions: I, II, III, IV, and V. Fraction III is subjected to silica gel chromatography, and TLC analysis yields the following fractions: I-1, I-2, and I-3.

[0229] Two compounds were isolated from I-2 using semi-preparative HPLC. Semi-preparative HPLC: flow rate v = 1.5 mL / min, detection wavelength λ = 220 nm, methanol:pure water 40:60 ratio. The structures of the compounds were primarily identified using techniques such as NMR nuclear magnetic resonance and ESI-MS, combined with high-resolution mass spectrometry and NMR spectroscopy.

[0230] Compound 4 (TCS001-I-4), a white powder, was identified as the macrolide macrolactin. The negative ion high-resolution mass spectrum, 1H-NMR spectrum, 13C-NMR spectrum, and structure of compound 4 are shown in Figures 34-37.

[0231] Compound 5 (TCS001-I-5) is a white powder identified as the macrolide macrolactin. The negative ion high-resolution mass spectrum, 1H-NMR spectrum, 13C-NMR spectrum, and structure of compound 5 are shown in Figures 38-41.

[0232] Example 3

[0233] Determination of antibacterial activity of lipopeptides

[0234] Antibacterial activity assay of compounds 1 to 3

[0235] The antibacterial activity of the isolated compounds was determined, and the growth rate method was used to determine the inhibitory effects of the compounds on plant pathogenic fungi.

[0236] First, the compound was prepared into a 1% stock solution, which was then serially diluted to 50, 25, and 12.5 mg / L. Boscalid at the same concentration was used as a control. The drug solutions were quantitatively pipetted from low to high concentrations and added to sterile PDA to create the corresponding drug-containing culture media. PDA without the drug served as a blank control.

[0237] Inoculate a vigorously growing pathogenic bacteria cake with a diameter of 8 mm in the center of the plate with the mycelium facing downwards and incubate at 25°C for 24-96 hours. When the diameter of the pathogenic bacteria in the control group is 3 / 4 of the culture dish, measure the diameter of the pathogenic bacteria colony using the cross-cross method and calculate the inhibition rate. The inhibition rate is calculated according to formula (2): Inhibition rate (%) = (control group colony diameter - treatment group colony diameter) / (control group colony diameter - 0.8 cm) × 100 (2)

[0238] The antibacterial activity was determined using Botrytis cinerea as the target. As shown in Figures 42 and 43, the results showed that the activities of the three compounds were all above 75%, among which TCS001-I-2 had the best activity, which also indicated that the main active substance of the strain was iturin.

[0239] Determination of antibacterial activity of compounds 4 to 5

[0240] The antibacterial activity of the isolated compounds was determined, and the growth rate method was used to determine the inhibitory effects of the compounds on plant pathogenic fungi.

[0241] First, the compound was prepared into a 1% stock solution, which was then serially diluted to 10, 5, and 2.5 mg / L. Boscalid at the same concentration was used as a control. The drug solutions were quantitatively pipetted from low to high concentrations and added to sterile PDA to create the corresponding drug-containing culture media. PDA without the drug served as a blank control.

[0242] Inoculate a vigorously growing pathogenic bacteria cake with a diameter of 8 mm in the center of the plate with the mycelium facing downwards and incubate at 25°C for 24-96 hours. When the diameter of the pathogenic bacteria in the control group is 3 / 4 of the culture dish, measure the diameter of the pathogenic bacteria colony using the cross-cross method and calculate the inhibition rate. The inhibition rate is calculated according to formula (2): Inhibition rate (%) = (control group colony diameter - treatment group colony diameter) / (control group colony diameter - 0.8 cm) × 100 (2)

[0243] The antibacterial activity was determined using cucumber gray mold as the target. As shown in Figure 44, the results showed that the two compounds had an inhibitory effect on the growth of gray mold, and the inhibitory effect increased with increasing concentration, with the inhibition rate reaching more than 80% at 10 mg / L.

[0244] Example 4

[0245] The lipopeptides produced by fermentation of Bacillus velez TCS001 were extracted as described in Example 1;

[0246] Determination of the antibacterial activity of a mixture containing lipopeptides produced by fermentation of Bacillus velez TCS001

[0247] The growth rate method was used to determine the inhibitory effect of lipopeptides produced by Bacillus velezensis TCS001 on plant pathogenic fungi. The lipopeptides from the TCS001 strain were first prepared into a 1% stock solution and then serially diluted to 50 mg / L. Chlorothalonil at the same concentration was used as a control. The drug solutions were then quantitatively pipetted from low to high concentrations and added to sterile polydimethylsiloxane (PDA) to prepare drug-containing culture media of corresponding concentrations. PDA without the drug served as a blank control.

[0248] Inoculate a vigorously growing pathogen cake with a diameter of 8 mm in the center of the plate with the mycelium side facing down and culture it upside down at 25°C for 24-96 hours. When the diameter of the pathogen in the control group is 3 / 4 of the culture dish, use the cross method to measure the diameter of the pathogen colony and calculate the inhibition rate.

[0249] The inhibition rate was calculated according to the following formula: Inhibition rate (%) = (colony diameter of control group - colony diameter of treated group) / (colony diameter of control group - 0.8 cm) × 100

[0250] Test results

[0251] A 50 mg / L mixture containing lipopeptides exhibited significant inhibitory effects against 18 tested plant pathogens, including pecan stem rot, apple ring rot, rapeseed sclerotinia, wheat root rot, and tomato gray mold, with inhibition rates ranging from 56.45% to 97.15% (Table 10, Figure 45). The inhibitory effects against pecan stem rot, apple ring rot, wheat root rot, sorghum anthracnose, corn leaf spot, sorghum anthracnose, and torreya root rot were superior to those of 50 mg / L chlorothalonil. These test results demonstrate that the mixture containing lipopeptides exhibits broad-spectrum antifungal activity.

[0252] Table 10 Inhibitory effect of mixtures containing lipopeptides produced by fermentation of Bacillus velezensis TCS001 on plant pathogenic fungi Note: Data in the table are mean ± SD. Different lowercase letters after the data in the same column indicate significant differences at the P < 0.05 level.

[0253] The above specific implementation methods further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made on the basis of the technical solutions of the present invention should be included in the scope of protection of the present invention.

Claims

1. A culture medium for industrial production of Bacillus Velez TCS001 fermentation broth, characterized in that: The culture medium comprises one or more of soluble peanut powder, glucose and soluble starch.

2. The culture medium for industrial production of Bacillus Velez TCS001 fermentation broth according to claim 1, characterized in that: The weight percentage of the soluble peanut powder is 1-5%, the weight percentage of the glucose is 1-5%, and the weight percentage of the soluble starch is 1-5%; preferably, the weight percentage of the soluble peanut powder is 2%, the weight percentage of the glucose is 3%, and the weight percentage of the soluble starch is 3%.

3. The culture medium for industrial production of Bacillus Velez TCS001 fermentation broth according to claim 2, characterized in that: The culture medium further comprises one or more of iron phosphate, potassium chloride, sodium chloride, magnesium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, light calcium carbonate, and manganese sulfate.

4. The culture medium for industrial production of Bacillus Velez TCS001 fermentation broth according to claim 3, characterized in that: The mass percentage of the iron phosphate is 0.002%, the mass percentage of the potassium chloride is 0.006%, the mass percentage of the sodium chloride is 0.6%, the mass percentage of the magnesium chloride is 0.05%, the mass percentage of the dipotassium hydrogen phosphate is 0.3%, the mass percentage of the potassium dihydrogen phosphate is 0.15%, the mass percentage of the light calcium carbonate is 0.05%, and the mass percentage of the manganese sulfate is 0.005%.

5. A process for producing Bacillus Velez TCS001 fermentation broth using the culture medium according to any one of claims 1 to 4, characterized in that: The fermentation culture time is 60-84 hours; preferably, the fermentation culture time is 68-76 hours; particularly preferably, the fermentation culture time is 72 hours.

6. The process according to claim 5, characterized in that The initial pH of the fermentation culture is 6-7.5; preferably, the initial pH of the fermentation culture is 7.0; the fermentation culture liquid volume is 16-32%, preferably, the fermentation culture liquid volume is 24%; the fermentation culture inoculation amount is 0.5-2%, preferably, the fermentation culture inoculation amount is 1%.

7. The process according to claim 5, characterized in that The fermentation culture speed is 160-200r / min, preferably, the fermentation culture speed is 180r / min; the fermentation culture temperature is 25-37°C, preferably, the fermentation culture temperature is 28°C.

8. A method for improving spore production by fermentation of Bacillus Velez TCS001, characterized in that: The method comprises the culture medium according to any one of claims 1 to 4, wherein the weight percentage of soluble peanut cake powder is 2-2.88%, the initial pH of the fermentation culture is 7.0, the inoculation amount of the fermentation culture is 1%, the liquid volume of the fermentation culture is 31-32%, the fermentation culture rotation speed is 173-180 r / min, and the fermentation culture temperature is 28°C.

9. Application of glucose and soluble starch as composite carbon source and / or soluble peanut meal as nitrogen source in culture medium for industrial production of Bacillus Velez TCS001 fermentation broth.

10. A fermentation liquid of Bacillus Velez TCS001 obtained by fermentation according to the culture medium according to any one of claims 1 to 4.

11. A mixture containing lipopeptide compounds and use thereof for inhibiting plant pathogenic fungi, characterized in that: The lipopeptide compound is a compound having the following structure, wherein the mixture contains at least one, two, three or more lipopeptide compounds, 12. The mixture comprising lipopeptide compounds according to claim 11, characterized in that: The mixture was isolated from the fermentation broth of Bacillus velez TCS001.

13. The mixture comprising lipopeptide compounds according to claim 12, characterized in that: The method for separating lipopeptide compounds is an acid precipitation method.

14. The mixture comprising lipopeptide compounds according to claim 12, characterized in that: The Bacillus Velez TCS001 fermentation liquid is obtained by inoculating Bacillus Velez TCS001 seed liquid into a fermentation medium for fermentation culture; preferably, the fermentation medium comprises a carbon source of soluble starch, the mass / volume of which is 1%; a nitrogen source of peanut cake powder, the mass / volume of which is 2%; and an inorganic salt of NaCl, the mass / volume of which is 0.3%; particularly preferably, the fermentation medium comprises 10.5 g / L of soluble starch, 18.5 g / L of peanut cake powder, and 3 g / L of NaCl.

15. The mixture comprising lipopeptide compounds according to claim 14, characterized in that: The fermentation culture process conditions include one or more of the following: fermentation temperature of 31° C., fermentation culture time of 48 h, initial fermentation pH of 6.0, and liquid volume of 32%; the fermentation culture process conditions also include inoculation volume of 3%, seed age of 16 h, and rotation speed of 164 rpm.

16. The mixture comprising lipopeptide compounds according to claim 14, characterized in that: The Bacillus Velez TCS001 seed liquid is obtained by fermenting the activated Bacillus Velez TCS001 strain through MLB culture liquid.

17. Use of the lipopeptide compound according to claim 11 for inhibiting plant pathogenic fungi; preferably, the plant pathogenic fungi is Botrytis cinerea, and particularly preferably, the plant pathogenic fungi is Botrytis cinerea.

18. Use of the mixture comprising lipopeptide compounds according to claim 11 in preventing and controlling pecan dry rot, tomato early blight, potato late blight, strawberry gray mold, rapeseed sclerotinia, apple leaf spot, cucumber target spot, cucumber black spot, wheat sheath blight, sorghum anthracnose, tea tree anthracnose, gloeosporium anthracnose, pepper phytophthora, Dendrobium officinale anthracnose, pumpkin leaf spot, cucumber wilt, strawberry anthracnose, and apple brown spot.

19. A method for increasing the lipopeptide compounds in the fermentation broth of Bacillus Velez TCS001, characterized in that: The fermentation medium comprises 1% soluble starch by mass / volume, 2% peanut powder by mass / volume, and 0.3% NaCl by mass / volume; preferably, the fermentation medium comprises 10.5 g / L soluble starch, 18.5 g / L peanut powder, and 3 g / L NaCl.

20. The method for increasing the lipopeptide compounds in the fermentation broth of Bacillus Velezii TCS001 according to claim 19, characterized in that: The fermentation process conditions include one or more of the following: fermentation temperature of 31° C., fermentation time of 48 h, pH of 6.0, and liquid volume of 32%.

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