CN106884031B - Method for optimizing shake flask fermentation process of bacillus subtilis T-500 for high yield of lipopeptide antibiotics - Google Patents

Method for optimizing shake flask fermentation process of bacillus subtilis T-500 for high yield of lipopeptide antibiotics Download PDF

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CN106884031B
CN106884031B CN201710298903.6A CN201710298903A CN106884031B CN 106884031 B CN106884031 B CN 106884031B CN 201710298903 A CN201710298903 A CN 201710298903A CN 106884031 B CN106884031 B CN 106884031B
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乔俊卿
刘永锋
刘邮洲
张荣胜
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Abstract

The invention particularly relates to a method for optimizing a shake flask fermentation process of bacillus subtilis T-500 for high-yield lipopeptide antibiotics, and belongs to the technical field of fermentation processes of bacillus subtilis T-500 lipopeptide antibiotics. The method takes rhizoctonia solani as an indicator bacterium, extracts lipopeptide antibiotics by an acid precipitation method based on 10 common fermentation culture media, analyzes the bacteriostatic effect of T-500 crude extract, and screens the components of the fermentation culture media which influence the bacteriostatic effect; the fermentation medium components and fermentation conditions of the T-500 high-yield lipopeptide antibiotics are optimized through Plackett-Burman experimental design, central combination experimental design (CCD) and a response surface method. The invention provides technical support and theoretical guidance for industrial production of the bacillus subtilis T-500 strain and acquisition of high-lipopeptide antibacterial substances.

Description

Method for optimizing shake flask fermentation process of bacillus subtilis T-500 for high yield of lipopeptide antibiotics
Technical Field
The invention belongs to the technical field of fermentation processes of bacillus subtilis T-500 lipopeptide antibiotics, and particularly relates to a method for optimizing a shake flask fermentation process of bacillus subtilis T-500 high-yield lipopeptide antibiotics.
Background
The bacillus is a biological control bacterium resource which is deeply researched at present, has a developed secretion system and stronger environmental adaptability, has antagonistic resistance to various plant pathogenic bacteria, is nontoxic, pollution-free and residue-free, is not easy to cause the pathogenic bacteria to generate drug resistance, and is widely applied to the creation and development of biological pesticides at present. The active strains popularized and applied at home and abroad comprise Bacillus amyloliquefaciens FZB42, Bacillus subtilis GB03, Bacillus pumilus GB34 and the like; the active component of the microbial bactericide, namely the Wenquning, developed by the Chenshiyibei and the like is bacillus subtilis Bs916, and the preparation has good prevention effect on rice sheath blight disease and false smut.
The existing biological control mechanism researches show that lipopeptide antibiotics generated by a non-ribosome synthetic pathway of bacillus, such as Surfactin, Iturin and Fengycin, have the effects of directly killing or inhibiting the growth of pathogens, competing with pathogenic bacteria for nutrition and space and inducing plants to generate disease resistance. At present, the main components of the common culture medium for bacillus fermentation are wheat flour, corn paste, soybean cake powder, fish meal, yeast extract (powder) and some trace elements (cations), however, the difference of the culture medium components has a great influence on the content of bacteria in the fermentation liquor and the synthesis of antibacterial substances aiming at different strains. Zhangingsheng and the like optimize the fermentation medium of the bacillus amyloliquefaciens Lx-11, so that the content of fermentation bacteria and the bacteriostatic effect are respectively improved by 180 percent and 30 percent. The research on the B.subtilis NJ-18 fermentation optimization finds that the bean pulp has the greatest influence on the bacteria content and the bacteriostatic activity.
Bacillus subtilis T-500 is a biocontrol bacterium which is obtained by early separation in the research laboratory and has good control effect on rice sheath blight and rice blast. The strain is preserved in China general microbiological culture Collection center, and an invention patent with the name of 'a strain of bacillus subtilis and application thereof' and the application number of 201210328716.5 is applied and authorized. In addition, the research room develops the dry suspension powder of the bacillus subtilis based on T-500 as an active ingredient, applies for an invention patent named as 'the dry suspension agent of the bacillus subtilis and a preparation method thereof' and with the application number of 201310301606.4, and obtains authorization. In order to further improve the content of lipopeptide antibiotics in dry suspension powder of bacillus subtilis T-500, the research screens key culture medium components and culture condition factors of lipopeptide antibiotics influencing T-500 by comparing the reported influences of 10 culture media and fermentation conditions commonly used for bacillus fermentation on lipopeptide antibiotics produced by T-500 strains; and the fermentation process is optimized by a response surface method so as to provide technical support and theoretical guidance for the industrial production and the acquisition of the high-lipopeptide antibacterial substance by the strain.
Disclosure of Invention
The invention aims to solve the defect that the content of lipopeptide antibiotics in the existing dry suspension powder of bacillus subtilis T-500 is low, and provides a method for optimizing a shake flask fermentation process for producing lipopeptide antibiotics with high yield by bacillus subtilis T-500.
The invention adopts the following technical scheme:
the method for optimizing the shake flask fermentation process of the bacillus subtilis T-500 for high yield of lipopeptide antibiotics comprises the following steps:
the method comprises the following steps: preparing a fermentation seed solution of bacillus subtilis T-500, and extracting lipopeptide antibiotics from a fermentation liquid obtained in the fermentation process, wherein the extraction of the lipopeptide antibiotics specifically comprises the following steps: centrifuging fermentation liquor obtained in the fermentation process of bacillus subtilis T-500, performing acid precipitation on supernate, adjusting pH, standing overnight, centrifuging, collecting precipitate, air drying, adding chromatographic grade methanol to dissolve precipitate, adjusting pH, centrifuging to obtain methanol solution, and filtering with a bacterial filter to obtain sterile methanol filtrate;
step two: inoculating the fermentation seed liquid to each culture medium for culture, respectively measuring the bacterium content and the bacteriostatic effect of lipopeptide antibiotics, comparing the influence of each culture medium on the bacterium content of T-500 strain fermentation liquid and the lipopeptide antibiotics, and screening better culture medium components;
step three: Plackett-Burman design of media composition: performing Plackett-Burman design on the basis of the composition of the better culture medium screened in the step two by combining the liquid loading amount and the rotating speed, and screening out key factors of the components of the culture medium; Plackett-Burman design of culture conditions: the Plackett-Burman design is adopted, and factors which have obvious influence on the antibacterial effect of the lipopeptide antibiotics of the strain T-500 are screened from the culture condition;
step four: (a) the method comprises the following steps Performing regression analysis on the Plackett-Burman experiment result of the culture medium components to obtain a main factor coding equation, performing variance analysis on the regression model, and further screening key factors of the culture medium components; (b) the method comprises the following steps Carrying out regression analysis on the Plackett-Burman design result of the culture condition to obtain a main factor coding equation, carrying out variance analysis on the regression model, and further screening key factors of the culture condition;
step five: (a) the method comprises the following steps According to the Plackett-Burman experiment result of the culture medium components, comprehensively selecting three key factors of the culture medium components which have obvious influence on the antibacterial effect of the lipopeptide antibiotics, determining the central point numerical value, further screening the optimal culture medium component combination by utilizing a three-factor five-horizontal-center combination experiment, and setting 20 groups of experiments in total, wherein each group is three in number; (b) selecting three factors which have significant influence on the antibacterial effect of the lipopeptide antibiotics according to the Plackett-Burman experiment result of the culture conditions, determining the numerical value of a central point, further screening the optimal culture medium condition combination by utilizing a three-factor five-horizontal-center combination experiment, and setting 20 groups of experiments in total, wherein each group is three in number;
step six: (a) the method comprises the following steps Selecting three key factors of the components of the culture medium obtained in the step four (a), performing three-factor five-level center combination design according to the step four (a), performing regression analysis on data of the width of the bacteriostatic zone obtained in the center combination experiment to obtain a main factor coding equation, and obtaining the optimal value of the three factors by the equation to obtain the optimal fermentation culture medium; (b) the method comprises the following steps Selecting three key factors of the culture conditions obtained in the step four (b), performing three-factor five-level center combination design according to the step four (b), performing regression analysis on data of the width of the bacteriostatic zone obtained in the center combination experiment to obtain a main factor coding equation, and obtaining the optimal value of the three factors by the equation to obtain the optimal culture conditions.
Further, the fermentation seed solution for preparing the bacillus subtilis T-500 in the step one is specifically: streaking a bacillus subtilis T-500 strain stored at low temperature into an LB solid culture medium plate, standing for culture, selecting a unit cell, inoculating the unit cell into YPG liquid, and performing shake culture to obtain a fermentation seed liquid; the pH at which the acid precipitated in step one was 2.0 and the temperature was 4 ℃ overnight.
Further, in the step one, the low temperature is-70 ℃, the static culture is static culture at 28 ℃ for 24 hours, and the shaking culture is shaking culture at 28 ℃ and 180rpm for 12 hours.
Further, the medium in step two is medium No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8, No. 9 or No. 10 as shown in Table 1.
Further, the step two specifically comprises the following steps: adopting a 10-time gradient multiple dilution plating method, diluting the fermentation seed liquid, plating the diluted fermentation seed liquid on a plate, recording the number of colonies per dish after culturing, and calculating the content of the fermentation liquid bacteria; the antibacterial activity determination specifically comprises the following steps: activating rhizoctonia solani on a PDA (personal digital assistant) plate by adopting a plate confronting method, making a bacterial cake after the plate is full of the rhizoctonia solani, inoculating the bacterial cake into the center of the PDA plate, punching a hole at a position away from the center in the cross direction by taking the bacterial cake as the center, dripping sterile methanol filtrate, drying in the air, culturing, and measuring the antibacterial bandwidth.
Further, in the second step, the fermentation liquid is diluted to 10%-8～10-7CFU/mL post-plated plates, incubated at 28 ℃ for 16hThen recording the number of colonies in each dish, and calculating the content of the fermentation broth; the antibacterial activity determination specifically comprises the following steps: activating rhizoctonia solani on a PDA (personal digital assistant) plate by adopting a plate confronting method, making a bacterial cake with the thickness of 5mm by using a puncher after the plate is fully grown, inoculating the bacterial cake into the center of the PDA plate, punching a hole (5mm) in a position 2.5cm away from the center in the cross direction by using the bacterial cake as the center, dripping 50 mu L of sterile methanol extraction filtrate, drying in the air, culturing at 28 ℃, and measuring the antibacterial bandwidth for 2-3 d.
Furthermore, the culture conditions in the third step are temperature, rotating speed, liquid loading amount, inoculation amount and fermentation time, the preferable culture medium components in the third step are wheat flour (5-10 g/L), soybean cake powder (10-20 g/L), corn paste (5-10 g/L), peptone (2.5-10 g/L), yeast powder (1.25-5 g/L), sodium chloride (1-5 g/L) and cations, in the Plackett-Burman design in the third step, each factor is 2 levels, and each group of experiments is repeated for 3 times.
Furthermore, the encoding equation of the main factor in the step four (a) is 8.54-0.017A-1.07B-0.21C-0.76D-0.32E-0.12F +0.30G-0.85H +0.21I, wherein gamma is the predicted value of the width of the inhibition zone; and (C) in the step four (B), the main factor coding equation is gamma which is 8.83-0.057A-0.17B +0.33C-0.44D-0.66E, and gamma is a predicted value of the width of the inhibition zone in the equation.
Further, in step six (a), the prime factor coding equation is: gamma 9.90-0.60A +0.58B-0.033C +0.21AB-0.29AC-0.29 BC-0.94A2-0.61B2-0.41C2，R2R is corrected 0.961020.9276, wherein gamma is the predicted value of the width of the inhibition zone; the encoding equation of the main factors in the step six (b) is as follows: gamma 10.95+0.65A-0.13B-0.51C-0.45AB +0.80AC +0.55BC-0.89A2-0.53B2-0.53C2，R2R is corrected to 0.963520.9306, wherein gamma is the predicted value of the width of the inhibition zone.
Further, in the sixth step (a), the optimal fermentation medium is 7.00g/L of soybean cake powder, 4.92g/L of peptone, 1.90g/L of yeast powder, 5.00g/L of wheat flour, 5.00g/L of corn paste, 1.00g/L of NaCl, and MgSO40.20g/L，MnSO45.0mg/L，FeSO40.5mgL; the optimal culture conditions in the step six (b) are as follows: liquid loading amount: 105mL/500mL triangular flask, inoculum size: 0.87%, fermentation time: 41.35h, temperature 28 ℃, rotation speed: 180 rpm.
The invention has the beneficial effects that: according to the invention, the composition of the culture medium which has a large influence on the lipopeptide substances produced by the T-500 strain is screened out by comparing the antibacterial difference of the lipopeptide substances produced by the strain T-500 in the shake flask fermentation in different culture media, the optimal culture medium component of the T-500 shake flask fermentation is optimized through Plackett-Burman design and a response surface experiment, and then the fermentation culture condition is optimized by utilizing a response surface method. Compared with the No. 3 culture medium with the best performance when the components of the T-500 optimal culture medium are initially screened, the lipopeptide antibiotics generated by the T-500 fermentation after the fermentation process is optimized have the bacteriostasis bandwidth of rice sheath blight increased by 20.4%, and the content of zymophyte is also increased by 62.9%. The invention provides technical support and theoretical guidance for industrial production of the bacillus subtilis T-500 strain and acquisition of high-lipopeptide antibacterial substances.
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FIG. 1 is a graph showing the effect of different media on the bacterial content of fermentation broth of T-500 strain;
FIG. 2 is a graph showing the effect of different culture media on the bacteriostatic ability of lipopeptide antibiotics produced by T-500 strain;
FIG. 3 is a three-dimensional graph of response surfaces of the effects of soybean cake powder and peptone (a), soybean cake powder and yeast powder (b), peptone and yeast powder (c) on the bacteriostatic effect of lipopeptide antibiotics produced by shake flask fermentation of T-500 strain;
FIG. 4 is a three-dimensional graph of response curves of the effects of liquid loading and inoculation amount (a), liquid loading and fermentation time (b), and inoculation amount and fermentation time (c) on the bacteriostatic effect of lipopeptide antibiotics produced by shake flask fermentation of T-500 strain.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention.
Examples
Bacillus subtilis T-500 and Rhizoctonia solani RH-2 are preserved in the plant protection research institute of agricultural academy of sciences of Jiangsu province in the research laboratory of rice disease and biological control.
The conventional activation of Bacillus subtilis T-500 adopts LB culture medium (tryptone 10.0g, yeast powder 5.0g, NaCl 10.0g), and the fermentation seed liquid is prepared by common culture medium YPG (tryptone 5.0g/L, yeast extract 5.0g/L, glucose 5.0 g/L). The rhizoctonia solani RH-2 is activated and cultured by PDA culture medium (potato 200.0g/L, glucose 20.0g/L, agar 15.0 g/L). The details of the 10 common basal fermentation media used in this study are shown in Table 1.
TABLE 1 type and composition of culture Medium
Figure GSB0000188519370000061
Figure GSB0000188519370000062
Preparing a fermented seed solution: selecting T-500 strain stored in a refrigerator at-70 ℃, streaking the strain into an LB solid plate, standing and culturing at 28 ℃ for 24h, selecting unit cell, inoculating the unit cell into a 100mL triangular flask containing 20mL YPG, and performing shake culture at 28 ℃ and 180rpm for 12h to obtain fermentation seed liquid.
And (3) measuring the bacteria content and the bacteriostatic activity of the yeast liquid: the content of the fermentation broth bacteria is as follows: diluting the fermentation liquid to 10 times by adopting a 10-time gradient multiple dilution plating method-8～10-7And CFU/mL, then uniformly coating 0.1mL of LB plate, culturing at 28 ℃ for 16h, recording the colony number of each dish, and calculating the bacterium content of the fermentation liquid.
Extracting lipopeptide antibiotics: centrifuging the fermentation liquor obtained in the T-500 fermentation process at 6000rpm for 15min, precipitating the supernatant by HCl, adjusting the pH to 2.0, standing overnight at 4 ℃, centrifuging at 10000rpm for 5min, collecting the precipitate, air drying, adding chromatographic grade methanol according to 1/10 volume of the fermentation liquor to dissolve the precipitate, adjusting the pH to 7.0, dissolving at room temperature for 24h, centrifuging to obtain a methanol solution, and filtering by a bacterial filter (phi ═ 0.22 mu m) to obtain a sterile filtrate for measuring the antibacterial activity.
And (3) determination of antibacterial activity: activating rhizoctonia solani on a PDA (personal digital assistant) plate by adopting a plate confronting method, making a bacterial cake with the thickness of 5mm by using a puncher after the plate is fully grown, inoculating the bacterial cake into the center of the PDA plate, punching a hole (5mm) in a position 2.5cm away from the center in the cross direction by using the bacterial cake as the center, then dripping 50 mu L of sterile methanol extraction filtrate into the hole, drying the filtrate in the air, culturing the filtrate at 28 ℃, and measuring the antibacterial bandwidth for 2-3 d.
Optimizing a fermentation process:
screening of culture medium: inoculating 1% of seed solution into a 500mL triangular flask containing 100mL of culture medium, culturing at 28 ℃ and 180r/min, respectively measuring the bacterial content and the bacteriostatic effect of lipopeptide antibiotics after 48 hours, comparing the influence of each culture medium on the bacterial content of T-500 strain fermentation liquor and the lipopeptide antibiotics, and screening the optimal culture medium components.
Screening key factors of culture medium components: according to the experimental result, the T-500 bacteria content of the fermentation of the culture mediums No. 2, No. 3 and No. 5 is higher; the fermentation liquid of the culture medium No. 3 and the culture medium No. 5 has the best bacteriostatic effect. For this purpose, the design of the Plackett-Burman shake flask fermentation experiments was carried out using design-expert software, based on medium compositions No. 2, No. 3 and No. 5, from A: wheat flour (5-10 g/L), B: soybean cake powder (10-20 g/L), C: corn paste (5-10 g/L), D: peptone (2.5-10 g/L), E: yeast powder (1.25-5 g/L), F: sodium chloride (1-5G/L), G: cation (51.37-205.5 mg/L, MgSO)4，MnSO4And FeSO4Scaled as a whole), H: liquid loading amount (80-120 mL/500mL triangular flask) and I: and (4) screening key factors from 9 factors of the rotating speed (150-180 rpm). The level of each factor in the test was 2: i.e., high level "1" (maximum range interval) and low level "-1" (minimum range interval), with 3 replicates per set of experiments.
Screening key factors of culture conditions: using Plackett-Burman design from a: temperature (25-30 ℃), B: rotating speed (120-180 r.min < -1 >), C: liquid loading amount (80-120 mL/500mL triangular flask), D: inoculation amount (1% -3%), E: and (3) screening factors which have obvious influence on the bacteriostatic ability of the lipopeptide antibiotics of the strain T-500 from 5 factors such as fermentation time (48-72 h). The level of each factor in the test was 2: i.e., high level "1" and low level "-1", each set of experiments was repeated 3 times.
Center combination experiment (CCD) response surface method test design:
culture medium key factor center combination experiment: according to the Plackett-Burman experiment result of the culture medium components, three key factors (soybean cake powder, peptone and yeast powder) of the culture medium components which have obvious influence on the content of fermentation bacteria and the bacteriostatic effect of lipopeptide antibiotics are comprehensively selected, and the central point value is determined. Then, a three-factor experiment is carried out by using a central combination experiment, according to the design principle, the obtained axial point a value is 1.68179, the angular point value is 1, five levels of "-1.68179, -1, 0, 1, 1.68179" are obtained, and then experiments are designed according to different levels, and 20 groups of experiments are set, wherein each group is repeated three times (table 2).
TABLE 2 key media ingredient center combination Experimental design Each factor level
Figure GSB0000188519370000081
Figure GSB0000188519370000083
Culture condition key factor center combination experiment: according to the Plackett-Burman experiment result of the culture condition, three factors (liquid loading amount, inoculation amount and fermentation time) which have significant influence on the antibacterial effect of the lipopeptide antibiotics are selected, and the central point value is determined. Then, a three-factor experiment is carried out by using a central combination experiment, according to the design principle, the obtained axial point a value is 1.68179, the angular point value is 1, five levels of "-1.68179, -1, 0, 1, 1.68179" are obtained, and then experiments are designed according to different levels, and 20 groups of experiments are set, wherein each group is repeated three times (table 3).
TABLE 3 Key culture Condition center Combined Experimental design Each factor level
Figure GSB0000188519370000082
The Plackett-Burman test, the center combination test, and the response surface analysis in this study were designed and analyzed using the design expert.v.8.0.6.1 software.
Screening components of an optimal T-500 fermentation medium: FIG. 1 shows the effect of different basal fermentation media on T-500 fermentation broth content and on the inhibition of Rhizoctonia solani by lipopeptide antibiotics. Of the 10 culture mediums, the T-500 strain grows better in the culture mediums No. 3, 5 and 2, particularly the culture medium No. 3, and the strain content reaches 4.55 multiplied by 109CFU/mL. Different culture mediums also have obvious influence on the antibacterial effect of the lipopeptide antibiotics generated by the T-500, wherein the antibacterial bandwidths of the No. 3 culture medium and the No. 5 culture medium are respectively 9.33 mm and 9.13 mm. Based on the effect of the above medium, the primary screening is considered as follows: 2. components in the culture media No. 3 and No. 5 are main components influencing the content and the bacteriostatic effect of T-500 zymophyte, and the next step is to screen key factors of Plackett-Burman of the culture medium components.
Plackett-Burman screens media key factors: the design of the Plackett-Burman experiment at the 9-factor 2 level and the results are shown in table 4. Regression analysis is carried out on the data in the table 4, and the obtained main factor coding equation is as follows: γ ═ 8.54-0.017A-1.07B-0.21C-0.76D-0.32E-0.12F +0.30G-0.85H +0.21I, corrected for R20.9798, wherein gamma is the predicted value of the width of the inhibition zone. 97.98% of the changes in gamma, as seen by the correction coefficients, can be explained by the regression equation. Analysis of variance with Design-Expert on the regression model showed (table 5), first that the regression model was significant (P ═ 0.0164); in addition, the soybean cake powder (B), peptone (D), yeast powder (E) and liquid loading amount (H) have significant influence (P is less than 0.05) on the effect of inhibiting the rhizoctonia solani of the lipopeptide antibiotics produced by the T-500 strain, and the wheat flour (A), the corn paste (C), NaCl (F), the cation (G) and the rotating speed (I) have no significant influence on the bacteriostatic effect of the T-500 strain. In the next step of central combined experimental design, the culture medium components (soybean cake powder, peptone and yeast powder) are mainly used as research objects for optimization, and culture condition factors are further screened and optimized in the following steps.
TABLE 4 culture Medium composition partial factor Plackett-Burman test design and test results
Figure GSB0000188519370000101
TABLE 5 analysis of variance analysis of the Medium composition part factor Plackett-Burman test design
Figure GSB0000188519370000102
Figure GSB0000188519370000103
Culture medium key factor center combined design experiment and response surface analysis
According to the experimental results, three key factors (soybean cake powder, peptone and yeast powder) of the components of the culture medium are obtained, then the central combination design (table 2) of three factors and five levels is carried out, and the optimal combination of the components of the culture medium which influences the T-500 shake flask fermentation lipopeptide antibiotics to inhibit the rhizoctonia solani is further optimized. Performing regression analysis on data of the width of the bacteriostatic band obtained by the central combined experiment to obtain a main factor coding equation: gamma 9.90-0.60A +0.58B-0.033C +0.21AB-0.29AC-0.29 BC-0.94A2-0.61B2-0.41C2，R2R is corrected 0.961020.9276, wherein gamma is the predicted value of the width of the inhibition zone. Analysis of variance of the regression equation shows (Table 6) that the P value of the model is less than 0.0001, which indicates that the model is correct, and the actual measured value of the width of the bacteriostatic zone generated by T-500 has significant correlation with the predicted value of the equation. The coefficient of variation CV of the model is 3.95 percent and is less than 10 percent, and the model belongs to weak variation, which further indicates the reliability of experimental data. The value of the missing fitting term (representing the part of the regression equation which can not be fitted) is 2.05(P is more than 0.05), which indicates that the missing fitting of the regression equation is not significant and the fitted degree of the established regression equation is considered to be high. The above results indicate that the model can be used to describe the effect of fermentation medium composition on the bacteriostatic effect of lipopeptide antibiotics produced by T-500 strains.
TABLE 6 analysis of variance of regression equation of Central combination of key factors in culture Medium (CCD)
Figure GSB0000188519370000111
Figure GSB0000188519370000112
FIGS. 3a to 3C are three-dimensional graphs of response surfaces of the effects of three factors, namely A (soybean cake powder), B (peptone) and C (yeast powder), on the bacteriostatic effect of lipopeptide antibiotics produced by shake flask fermentation of T-500 strains. 3a, 3b and 3c all have a convex peak, the optimum value range of the three variables can be determined. According to the obtained second-order multivariate equation, the optimal values of three factors are obtained, namely 7.00g/L of soybean cake powder, 4.92g/L of peptone and 1.90g/L of yeast powder, and under the condition, the inhibition bandwidth of the lipopeptide antibiotics for inhibiting rhizoctonia solani reaches 10.11 mm. Based on this, we obtained the best fermentation medium: 7.00g/L of soybean cake powder, 4.92g/L of peptone, 1.90g/L of yeast powder, 5.00g/L of wheat flour, 5.00g/L of corn paste, 1.00g/L of NaCl and MgSO40.20g/L，MnSO45.0mg/L，FeSO40.5mg/L。
Plackett-Burman screens culture condition key factors: the experimental design and results of the PlackettBurman experiment at 5 factor 2 levels of culture conditions are shown in table 7. Regression analysis was performed on the data of table 7, and the resulting encoding equation for the main factors was: and gamma is 8.83-0.057A-0.17B +0.33C-0.44D-0.66E, and gamma is a predicted value of the width of the inhibition zone in the formula. The 87.25% of the changes in gamma known from the correction coefficients can be explained by the regression equation. Analysis of variance of the regression equation showed that the P value for the regression model was < 0.05, indicating that the model is correct. In addition, the liquid loading amount (C), the inoculation amount (D) and the fermentation time (E) have significant influence on the effect of inhibiting the rhizoctonia solani of the lipopeptide antibiotics produced by the T-500 strain (P is less than 0.05), and the temperature (A) and the rotating speed (B) have no significant influence on the bacteriostatic effect of the T-500 strain. We continue to use the central combinatorial experimental design (CCD) to further optimize the optimal combination of liquid loading, inoculum size and fermentation time.
TABLE 7 culture Condition part factor Plackett-Burman test design and test results
Figure GSB0000188519370000121
TABLE 8 analysis of variance of the partial factor Plackett-Burman test design for culture conditions
Figure GSB0000188519370000131
The method comprises the following steps of (1) culture condition key factor center combined design experiment and response surface analysis: according to the experimental results, three key factors (liquid loading amount, inoculation amount and fermentation time) of the culture conditions are obtained, then three-factor five-level central combination design is carried out (table 3), and the optimal combination of the culture conditions which influence the T-500 shake flask fermentation of the lipopeptide antibiotics to inhibit the rhizoctonia solani is further optimized. Performing regression analysis on data of the width of the bacteriostatic band obtained by the central combined experiment to obtain a main factor coding equation: gamma 10.95+0.65A-0.13B-0.51C-0.45AB +0.80AC +0.55BC-0.89A2-0.53B2-0.53C2，R2R is corrected to 0.963520.9306, wherein gamma is the predicted value of the width of the inhibition zone. Analysis of variance of the regression equation showed (table 9) that the P value for the model was less than 0.0001, indicating that the model is correct. The coefficient of variation CV of the model is 3.80 percent and less than 10 percent, which belongs to weak variation and further indicates the reliability of experimental data. The missing fitting term value (representing the part of the regression equation which can not be fitted) is 3.33(P is more than 0.05), which indicates that the fitted degree of the established regression equation is high. The above analysis shows that the regression model of the fermentation culture conditions can be used for describing the influence of the fermentation culture conditions on the bacteriostatic effect of the lipopeptide antibiotics generated by the T-500 strain.
TABLE 9 analysis of variance of regression equation of key factor center combination experiment under culture conditions
Figure GSB0000188519370000141
FIGS. 4a to 4C are response surface three-dimensional graphs of the influence of three factors A (liquid loading amount), B (inoculation amount) and C (fermentation time) on the bacteriostatic effect of lipopeptide antibiotics produced by shake flask fermentation of T-500 strain. Fig. 4a, 4b and 4c show that three factors interact two by two with a convex peak, so that the optimal value range of three variables can be determined. According to the second-order multivariate equation obtained by 2.5, the optimal values of three factors are obtained, wherein the liquid loading amount is 105mL/500mL of triangular flask, the inoculation amount is 0.87%, the fermentation time is 41.35h, and under the condition, the inhibition bandwidth of the lipopeptide antibiotics for inhibiting the rhizoctonia solani reaches 11.20 mm. Based on this, we obtained the optimal fermentation culture conditions as: liquid loading amount: 105mL/500mL triangular flask, inoculum size: 0.87%, fermentation time: 41.35h, temperature 28 ℃, rotation speed: 180 rpm.
Experimental verification of the fermentation optimization process: based on the optimal fermentation medium formula (7.00 g/L of soybean cake powder, 4.92g/L of protein, 1.90g/L of yeast powder, 5.00g/L of wheat flour, 5.00g/L of corn paste, 1.00g/L of NaCl, MgSO 540.20g/L，MnSO45.0mg/L，FeSO40.5mg/L) and under the optimal fermentation culture conditions (liquid loading volume of 105mL/500mL triangular flask, inoculation amount of 0.87%, fermentation time of 41.35h, temperature of 28 ℃ and rotation speed of 180rpm), the shaking flask fermentation culture experiment is carried out, the band width of the lipopeptide antibiotics generated by the fermentation of the strain T-500 for inhibiting the rice sheath blight germ is 11.23 +/-0.15 mm, the band width is not significantly different from the predicted value of 11.20mm, the content of the fermentation germs is 7.41 +/-1.18 multiplied by 109CFU/mL. Compared with the No. 3 culture medium with the best performance when the components of the T-500 optimal culture medium are initially screened, the lipopeptide antibiotics generated by the T-500 fermentation after the fermentation process is optimized have the bacteriostasis bandwidth of rice sheath blight increased by 20.4%, and the content of zymophyte is also increased by 62.9%.
The secretion of antibacterial substances is one of important biocontrol mechanisms of biocontrol bacillus, lipopeptide antibiotics are main antibacterial active ingredients of bacillus, have an antagonistic effect, can influence the formation of a biological membrane so as to influence the colonization capacity of biocontrol bacteria, and can also be used as an exciton to cause the induced disease resistance of plants, so that the improvement of the yield of the lipopeptide substances has important significance for enhancing the control effect of the biocontrol bacillus. Fermentation is an important link in the industrialization process of biocontrol microorganisms, and a PlackettBurman (PB) test design method and a response surface analysis (RSM) are widely applied to screening and optimization of a microbial fermentation process. Liu Jingland and the like optimize and know the Iturin A synthesis condition of the bacillus amyloliquefaciens CC09 strain by using a PB and a central combination test method, and after optimization, the yield of the Iturin A synthesized by the CC09 reaches 501mg/L and is improved by 4.2 times compared with 138mg/L before optimization. Sen et al respectively carried out optimization research on key culture medium components, fermentation conditions, inoculation liquid bacterial age and inoculation amount which influence surfactin yield by using a response surface method (response surface method). The method includes the steps of screening 13 factors affecting B.amyloliquefaciens ES-2-4 lipopeptide antibacterial substances by using PB test design, optimizing fermentation medium components, and increasing the yield of a crude extract of the lipopeptide antibacterial substances to 6.76g/L and 39.7% compared with the yield of the crude extract before optimization.
According to the invention, the composition of the culture medium which has a large influence on the lipopeptide substances produced by the T-500 strain is screened out by comparing the antibacterial difference of the lipopeptide substances produced by the strain T-500 in the shake flask fermentation in different culture media, the optimal culture medium component of the T-500 shake flask fermentation is optimized through Plackett-Burman design and a response surface experiment, and then the fermentation culture condition is optimized by utilizing a response surface method. Compared with the No. 3 culture medium with the best performance when the components of the T-500 optimal culture medium are initially screened, the lipopeptide antibiotics generated by the T-500 fermentation after the fermentation process is optimized have the bacteriostasis bandwidth of rice sheath blight increased by 20.4%, and the content of zymophyte is also increased by 62.9%.
The invention only screens and optimizes the fermentation medium components and the culture conditions of the bacillus subtilis T-500 in a shake flask, provides an optimization method, and needs further small test and pilot test to perfect the fermentation process so as to provide reliable theoretical parameters for industrial fermentation production.
It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (5)

1. The method for optimizing the shake flask fermentation process of the bacillus subtilis T-500 for high yield of lipopeptide antibiotics is characterized by comprising the following steps:
the method comprises the following steps: preparing a fermentation seed solution of bacillus subtilis T-500, and extracting lipopeptide antibiotics from a fermentation liquid obtained in the fermentation process, wherein the extraction of the lipopeptide antibiotics specifically comprises the following steps: centrifuging fermentation liquor obtained in the fermentation process of bacillus subtilis T-500, performing acid precipitation on supernatant with the pH of 2.0, standing overnight at 4 ℃, centrifuging, collecting precipitate, air drying, adding chromatographic grade methanol to dissolve the precipitate, adjusting the pH, centrifuging after dissolution to obtain a methanol solution, and filtering by using a bacterial filter to obtain sterile methanol filtrate;
step two: inoculating the fermentation seed liquid to each culture medium for culture, respectively measuring the bacterium content and the bacteriostatic effect of lipopeptide antibiotics, comparing the influence of each culture medium on the bacterium content of T-500 strain fermentation liquid and the lipopeptide antibiotics, and screening better culture medium components;
step three: Plackett-Burman design of media composition: performing Plackett-Burman design on the basis of the composition of the better culture medium screened in the step two by combining the liquid loading amount and the rotating speed, and screening out key factors of the components of the culture medium; Plackett-Burman design of culture conditions: adopting Plackett-Burman design to screen factors which have obvious influence on the lipopeptide antibiotics produced by the strain T-500 from culture conditions;
step four: (a) the method comprises the following steps Performing regression analysis on the Plackett-Burman experiment result of the culture medium components to obtain a main factor coding equation, performing variance analysis on the regression model, and further screening key factors of the culture medium components; (b) the method comprises the following steps Carrying out regression analysis on the Plackett-Burman design result of the culture condition to obtain a main factor coding equation, carrying out variance analysis on the regression model, and further screening key factors of the culture condition;
step five: (a) the method comprises the following steps According to the Plackett-Burman experiment result of the culture medium components, comprehensively selecting three key factors of the culture medium components which have obvious influence on the antibacterial effect of the lipopeptide antibiotics, determining the central point numerical value, further screening the optimal culture medium component combination by utilizing a three-factor five-horizontal-center combination experiment, and setting 20 groups of experiments in total, wherein each group is three in number; (b) selecting three factors which have significant influence on the antibacterial effect of the lipopeptide antibiotics according to the Plackett-Burman experiment result of the culture conditions, determining the numerical value of a central point, further screening the optimal culture medium condition combination by utilizing a three-factor five-horizontal-center combination experiment, and setting 20 groups of experiments in total, wherein each group is three in number;
step six: (a) the method comprises the following steps Selecting three key factors of the components of the culture medium obtained in the step four (a), performing three-factor five-level center combination design according to the step four (a), performing regression analysis on data of the width of the bacteriostatic zone obtained in the center combination experiment to obtain a main factor coding equation, and obtaining the optimal value of the three factors by the equation to obtain the optimal fermentation culture medium; (b) the method comprises the following steps Selecting three key factors of the culture condition obtained in the step four (b), performing three-factor five-level center combination design according to the step four (b), performing regression analysis on data of the width of the bacteriostatic band obtained in the center combination experiment to obtain a main factor coding equation, and obtaining the optimal value of the three factors by the equation to obtain the optimal culture condition;
the fermentation seed liquid for preparing the bacillus subtilis T-500 in the step one is specifically as follows: streaking a bacillus subtilis T-500 strain stored at-70 ℃ into an LB solid medium flat plate, carrying out static culture at 28 ℃ for 24 hours, selecting a single cell, inoculating the single cell into YPG liquid, and carrying out shake culture at 28 ℃ and 180rpm for 12 hours to obtain a fermentation seed liquid;
the culture medium in the second step is No. 1 culture medium: peptone 5.0g, yeast powder 5.0g, glucose 5.0g, K2HPO40.2g，MgSO4·7H20.2g of O and 1000mL of distilled water; medium No. 2: 10.0g of wheat flour, 20.0g of soybean cake powder and MgSO40.2g，MnSO45.0mg，FeSO40.5mg, 1000mL of distilled water; medium No. 3: 10.0g of corn paste, 20.0g of soybean cake powder and MgSO40.2g，MnSO45.0mg，FeSO40.5mg, 1000mL of distilled water; number 4Culture medium: rice flour 10.0g, soybean cake powder 20.0g, MgSO40.2g，MnSO45.0mg，FeSO40.5mg, 1000mL of distilled water; medium No. 5: 10.0g of peptone, 5.0g of yeast powder, 5.0g of NaCl and 1000mL of distilled water; medium No. 6: peptone 5.0g, yeast powder 5.0g, glucose 5.0g, MgSO 540.5g，KCl 0.5g，KH2PO41.0g，FeSO40.15mg，MnSO45.0mg，CuSO40.16mg, 1000mL of distilled water; medium No. 7: 10.0g of soybean cake powder, 20.0g of corn paste and NH4SO42.0g，Na2HPO42.0g, 1000mL of distilled water; medium No. 8: 1.0g of soluble starch, 15.0g of glucose, 30.0g of soybean cake powder, 0.2g of yeast powder and KH2PO41.5g，K2HPO43.0g，MgSO4·7H2O 0.5g，CaCO30.1g，FeSO40.1g, 1000mL of distilled water; medium No. 9: glucose 20.0g, L-glutamic acid 5.0g, MgSO40.5g，KCl0.5g，KH2PO41.0g，FeSO40.15mg，MnSO45.0mg，CuSO40.16mg, 1000mL of distilled water; medium No. 10: 5.0g of peptone, 5.0g of yeast powder, 5.0g of glucose and 1000mL of distilled water.
2. The method for optimizing the shake flask fermentation process of bacillus subtilis T-500 for high yield of lipopeptide antibiotics according to claim 1, wherein the step two comprises the specific steps of: adopting a 10-time gradient multiple dilution plating method, diluting the fermentation seed liquid, plating the diluted fermentation seed liquid on a plate, recording the number of colonies per dish after culturing, and calculating the content of the fermentation liquid bacteria; the antibacterial activity determination specifically comprises the following steps: activating rhizoctonia solani on a PDA (personal digital assistant) plate by adopting a plate confronting method, making a bacterial cake after the plate is full of the rhizoctonia solani, inoculating the bacterial cake into the center of the PDA plate, punching a hole at a position 2.5cm away from the center in the cross direction by taking the bacterial cake as the center, dripping sterile methanol filtrate, drying in the air, culturing, and measuring the antibacterial bandwidth.
3. The shake flask fermentation process for producing lipopeptide antibiotics with high yield of bacillus subtilis T-500 according to claim 2The method is characterized in that the fermentation liquor is diluted to 10 in the step two-8～10-7Coating a flat plate after CFU/mL, culturing at 28 ℃ for 16h, recording the colony number of each dish, and calculating the bacterium content of the fermentation liquid; the antibacterial activity determination specifically comprises the following steps: activating rhizoctonia solani on a PDA (personal digital assistant) plate by adopting a plate confronting method, making a bacterial cake with the thickness of 5mm by using a puncher after the plate is full of the rhizoctonia solani, inoculating the bacterial cake into the center of the PDA plate, punching 5mm by using the puncher at the position 2.5cm away from the center in the cross direction by taking the bacterial cake as the center, dripping 50 mu L of sterile methanol extraction filtrate into the filtrate, drying the filtrate in the air, culturing the filtrate at 28 ℃, and measuring the antibacterial bandwidth for 2-3 d.
4. The method for optimizing shake flask fermentation process of Bacillus subtilis T-500 for high yield of lipopeptide antibiotic according to claim 1, wherein the major factor encoding equation of step six (a) is: y ═ 9.90-0.60A +0.58B-0.033C +0.21AB-0.29AC-0.29 BC-0.94A2-0.61B2-0.41C2，R2R is corrected 0.961020.9276, wherein Y is the predicted value of the width of the inhibition zone; the encoding equation of the main factors in the step six (b) is as follows: y is 10.95+0.65A-0.13B-0.51C-0.45AB +0.80AC +0.55BC-0.89A2-0.53B2-0.53C2，R2R is corrected to 0.963520.9306, wherein Y is the predicted value of the width of the inhibition zone;
in the coding equation of the main factors in the step six (a), A is soybean cake powder, B is peptone and C is yeast powder; in the coding equation of the main factors in the step six (B), A is the liquid loading amount, B is the inoculation amount, and C is the fermentation time.
5. The method for optimizing shake flask fermentation process for high lipopeptide antibiotic production by bacillus subtilis T-500 according to claim 1, wherein the optimal fermentation medium in the sixth step (a) is soybean meal 7.00g/L, peptone 4.92g/L, yeast powder 1.90g/L, wheat flour 5.00g/L, corn paste 5.00g/L, NaCl 1.00g/L, MgSO 1.00g/L40.20g/L，MnSO45.0mg/L，FeSO40.5 mg/L; the optimal culture conditions in the step six (b) are as follows: liquid loading amount: 105mL/500mL triangular flask, inoculum size:0.87%, fermentation time: 41.35h, temperature 28 ℃, rotation speed: 180 rpm.
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