CN117265050A - Bacillus bailii G-1-phendimeins for inhibiting alternaria alternata, and preparation method and application thereof - Google Patents
Bacillus bailii G-1-phendimeins for inhibiting alternaria alternata, and preparation method and application thereof Download PDFInfo
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- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- UCRLQOPRDMGYOA-DFTDUNEMSA-L zinc;(4r)-4-[[(2s)-2-[[(4r)-2-[(1s,2s)-1-amino-2-methylbutyl]-4,5-dihydro-1,3-thiazole-4-carbonyl]amino]-4-methylpentanoyl]amino]-5-[[(2s,3s)-1-[[(3s,6r,9s,12r,15s,18r,21s)-3-(2-amino-2-oxoethyl)-18-(3-aminopropyl)-12-benzyl-15-[(2s)-butan-2-yl]-6-(carbox Chemical compound [Zn+2].C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC([O-])=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2NC=NC=2)C(=O)N[C@H](CC([O-])=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 UCRLQOPRDMGYOA-DFTDUNEMSA-L 0.000 description 1
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Abstract
The invention provides bacillus belicus G-1 phendimeidin for inhibiting alternaria alternata, and a preparation method and application thereof, and belongs to the technical field of phendimeidin preparation. The invention starts from screening a low-cost culture medium formula by taking rapeseed meal as a fermentation raw material, comprehensively considers the culture medium composition, the component dosage and the external influence factors of lipopeptid produced by a fermentation process, optimizes the extraction and purification process, and forms a set of bacillus bailii G-1 phenjejunin high-efficiency preparation method with low cost, high yield and simple and convenient operation. The yield of the bacillus bailii G-1 phenedin in the fermentation liquid obtained by adopting the method disclosed by the invention is improved by 20.15% compared with that of a BPY culture medium, and the cost of raw materials is reduced by 13.14%. In addition, the invention proves that the strain G-1-fenaminocycline has good living body prevention effect on various fruit and vegetable alternaria alternate pathogens through a living body bacteriostasis test and a different temperature test, and can also play a role in bacteriostasis under the condition of low-temperature storage of fruits and vegetables.
Description
Technical Field
The invention belongs to the technical field of preparation of felines, and particularly relates to bacillus bailii G-1 felines for inhibiting alternaria alternata, and a preparation method and application thereof.
Background
As a main microorganism causing the putrefaction and deterioration of fruits, vegetables and refrigerated foods, the alternaria alternata not only has important influence on the storage and preservation of fruits, vegetables and refrigerated foods, but also is a potential toxin-producing bacterium, and threatens the health of human bodies and the safety of foods. In addition, the pathogens also have strong low temperature resistance, and the conidium of the pathogens still has strong pathogenicity under the ice temperature storage condition, and the risk of explosive morbidity and streptavidin exists, so that the disease control during the storage of fruits and vegetables has important significance for the storage and fresh keeping of fruits and vegetables and the safety of foods.
Fenstin (Fengycin) is taken as a cyclic lipopeptide substance which is synthesized by bacillus (Bacillus sp.) through a non-ribosomal peptide (NRPs) way and takes amino acid and beta-fatty acid as substrates, has the characteristics of low hemolytic activity, strong antioxidant capacity, nature, high efficiency, safety and green; the growth performance and the immunity performance of the broiler chickens can be improved; improving pharmacokinetic and pharmacodynamic properties; improving the flavor of white spirit, and has obvious application advantages in the fields of food preservation and fresh-keeping, medicine, washing products, cosmetic manufacture and the like. Therefore, the successful conversion application of the substance is not only a novel broad-spectrum and low-drug-resistance biological bactericide after chitosan, vancomycin, daptomycin, colistin, thiopeptidmycin, bacitracin zinc and the like, but also an ideal substitute product of chemical preservatives such as sodium nitrite, sodium benzoate, benzoic acid and the like, and has better application potential in the fields of agriculture, food, medicine and industry. However, the research of the antibacterial substances is mainly focused on the research of indoor and field antibacterial activity at present, and the problems of high production cost (mainly comprising expensive synthetic culture medium), low extraction and purification process efficiency, low yield and the like exist. Moreover, the related researches at present enable people to have deeper knowledge on the lipopeptide biosynthesis of the strain from external factors (culture conditions) and internal factors (gene regulation), but the current lipopeptide substance cheap carbon/nitrogen source fermentation technology, the efficient preparation method of the lipopeptide substance and the mutagenesis technology are not completely applicable to all strains, and the researches on the lipopeptide production technology taking agricultural wastes as raw materials and the related lipopeptide-producing strain excavation are weak. In addition, although the preparation methods related to lipopeptide substances at home and abroad are reported at present, a unified high-efficiency fermentation mode and high-yield engineering bacteria are not applied to production practice at present, and the problems of complex and various structures, high organic synthesis difficulty, high production cost, low preparation efficiency, low yield and the like found in the research process, which limit the later development and application of the lipopeptide substances, are still to be broken through.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for efficiently preparing a cyclic lipopeptide substance, namely, fenacet, with the effect of inhibiting alternaria alternata by using agricultural wastes as fermentation raw materials, which has the advantages of short time, low cost and high yield.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for efficiently preparing a fenamidone with the function of inhibiting alternaria alternata, which comprises the following steps: inoculating Bacillus bailii (Bacillus velezensis) G-1 into fermentation medium, fermenting, culturing, extracting, and purifying to obtain the final product; the bacillus bailii G-1 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.23750; the fermentation medium comprises rapeseed meal and glucose.
Preferably, the inoculation amount of the bacillus beijerinus G-1 is 1 to 2 percent of the volume of the fermentation medium.
Preferably, the fermentation time is 4-8 days, the fermentation temperature is 25-35 ℃, and the fermentation rotating speed is 120-200 rpm.
Preferably, the fermentation medium comprises the following raw materials by weight: 4-7% of rapeseed meal, 0.8-2% of glucose and K 2 HPO 4 ·3H 2 O0.03％～0.05％，MgSO 4 ·7H 2 O0.02％～0.05％，MnSO 4 ·7H 2 0.01 to 0.05 per mill of O and the balance of water; the pH value of the fermentation medium is 7-8.
Preferably, the extraction method is that methanol ultrasonic extraction is adopted after hydrochloric acid precipitation, and the pH value of the hydrochloric acid precipitation is 3; the dosage of methanol is 400-600mL, the ultrasonic power is 160w, the ultrasonic frequency is 40KHz, and the ultrasonic extraction time is 1h-1.5h based on 1L fermentation broth.
Preferably, the purified stationary phase is a dextran-sepharose tandem column and AQC18; the dextran-agarose gel series column consists of a DEAE-52 column, a phenyl-agarose gel column and a G-100 dextran gel column.
The invention also provides the finemetine prepared by the method.
The invention also provides the application of the method or the phendimedin in preventing and treating the infection of the alternaria alternata after fruit and vegetable harvest.
The invention also provides application of the bacillus beijerinus G-1 in preventing and treating the infection of the alternaria alternata after fruit and vegetable harvest.
Preferably, the fruit and vegetable comprises one or more of apples, pears, tomatoes and jujubes.
The invention has the beneficial effects that:
the invention provides a method for efficiently preparing the fenacet with the function of inhibiting the alternaria alternata by taking agricultural wastes as fermentation raw materials for the first time, and has the advantages of short time, low cost and high yield. The invention starts from screening a low-cost culture medium formula by taking agricultural wastes as fermentation raw materials, comprehensively considers the culture medium composition, the component amounts and the external factors of lipopeptid production of a fermentation process, optimizes the extraction and purification process, and forms a set of bacillus bailii G-1 phenmedine efficient preparation method with low cost, high yield and simple and convenient operation, thereby laying a theoretical foundation for large-scale production of the strain phenmedine.
Compared with the fermentation liquid of the BPY culture medium, the yield of the bacillus belicus G-1-phenmedine in the fermentation liquid obtained by adopting the method disclosed by the invention can be improved by about 20.15%, and the raw material cost is reduced by 13.14%. The invention also provides a method for preparing the bacillus subtilis G-1, which is characterized in that the bacillus subtilis G-1 has a good living body prevention effect on various fruit and vegetable alternaria alternate pathogens through a living body bacteriostasis test and a different temperature test, can play a role in bacteriostasis under the condition of low-temperature storage of fruits and vegetables, and evaluates the bacteriostasis effect and safety of the bacillus subtilis G-1 on the prevention and treatment of the fruit and vegetable post-harvest streptomyces diseases according to the relation between the temperature and the activity of the fenacet, the biological safety of the fenacet and 3 aspects of the living body test on the basis of the bacteriostasis effect and the safety of the fenacet on the fruit and vegetable post-harvest streptomyces disease lipopeptid biological preservative.
Preservation description
The bacillus beleiensis (Bacillus velezensis) G-1 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with the preservation time of 2021, 11 months and 08 days, and the preservation address of China general microbiological institute (CGMCC No. 23750) with the preservation number of North Chen West Lu No. 1, 3 in the Chart area of Beijing city.
Drawings
FIG. 1 shows the bacteriostatic activity of Alternaria alternata, which is a different agricultural waste lipopeptide extract, wherein A is the result of corncob meal, wheat bran, corn meal and wine lees, B is the result of bean pulp, rotted apples, vinegar residues and rotted pears, C is the result of germinated potatoes, walnut green husks and date pits, D is the result of rapeseed meal, rape straw and blank, and E is the result of wheat straw, corn straw, BPY culture solution and soybean meal;
FIG. 2 shows the oil drainage characteristics of 5 bacteriostatic agricultural waste fermentation broths, from top to bottom and from left to right, respectively showing the results of rapeseed meal, soybean meal, germinated potatoes, rape straw, soybean meal and BPY;
FIG. 3 shows the results of HPLC quantitative analysis of the G-1Fengycins content of the strain at different fermentation times, namely fermentation 1, 2, 3, 4, 5, 6, 7 and 8d from top to bottom and from left to right respectively;
FIG. 4 shows the bacteriostatic activity of strain G-1Fengycins against tomato early blight bacteria at different fermentation times, by: culturing at 26 ℃ for 2d, and obtaining a result;
FIG. 5 effect of fermentation time on the growth of strain G-1;
FIG. 6 effect of initial pH of fermentation broth on strain G-1 growth, bacteriostatic activity and Fengycins yield;
FIG. 7 effect of fermentation temperature on strain G-1 growth, bacteriostatic activity and Fengycins yield;
FIG. 8 effect of inoculum size on strain G-1 growth, bacteriostatic activity and Fengycins yield;
FIG. 9 effect of rotational speed on strain G-1 growth, bacteriostatic activity and Fengycins yield;
FIG. 10 shows the results of HPLC detection of the strain G-1Fengycins under different amounts of rapeseed meal, wherein A-M respectively represent 1G/100mL, 2G/100mL, 3G/100mL, 4G/100mL, 5G/100mL, 6G/100mL, 7G/100mL, 8G/100mL, 9G/100mL, 10G/100mL, 11G/100mL, 12G/100mL and 13G/100mL of the rapeseed meal;
FIG. 11 antibacterial activity of strain G-1Fengycins against tomato early blight bacteria at different meal dosages;
FIG. 12 biomass plate counts of strain G-1 at various meal levels;
FIG. 13 effects of different glucose amounts on strain G-1 growth (left panel) and on the inhibition zone (right panel);
FIG. 14 different K 2 HPO 4 ·3H 2 Influence of O usage on the growth of the strain G-1 and the synthesis of the phendimedin (left graph) and the inhibition zone (right graph); FIG. 15 different MgSO 4 ·7H 2 Influence of O usage on the growth of the strain G-1 and the synthesis of the phendimedin (left graph) and the inhibition zone (right graph); FIG. 16 different MnSO 4 ·7H 2 Influence of O usage on the growth of the strain G-1 and the synthesis of the phendimedin (left graph) and the inhibition zone (right graph);
fig. 17 shows a steep slope map (screen) test result in which the abscissa is: a factor; ordinate: characteristic root values;
FIG. 18 projection of principal components of strain G-1 growth and of the synthesis of the fenamidone under different fermentation conditions;
FIG. 19 results of PCA analysis of the effect of different fermentation conditions on strain G-1 growth and the synthesis of the finemetine;
FIG. 20 comprehensive scoring results of the extent of influence of different fermentation conditions on the growth of strain G-1 and the synthesis of the finemetine;
FIG. 21 interaction relationship between different fermentation conditions and strain G-1 growth and synthesis of fenaminon based on RDA analysis;
FIG. 22 effect of different extraction modes on the yield and bacteriostatic activity of strain G-1 fenamidone;
FIG. 23 effect of different precipitation pH values on strain G-1-fenaminocycline production and bacteriostatic activity;
FIG. 24 effect of methanol dose (left panel) and sonication time (right panel) on strain G-1-fenaminosulf production and bacteriostatic activity;
FIG. 25 influence of ultrasound power (left panel) and ultrasound frequency (right panel) on strain G-1-fenaminosulf production and bacteriostatic activity;
FIG. 26 shows the results of full wavelength scan of the maximum absorption wavelength of strain G-1-fenacet in different culture solutions, with the left graph showing the results of the BPY culture solution and the right graph showing the results of the culture solution of example 1 (rapeseed meal);
FIG. 27 shows a difference analysis of the detection effect of different detection wavelengths of the strain G-1 fenacet based on HPLC, wherein the upper graph shows the detection result at 215nm, and the lower graph shows the detection result at 244 nm;
FIG. 28 shows different stationary phase purification chromatograms and active peak oil extraction effects of crude lipopeptide extract of strain G-1;
FIG. 29 shows the results of electrophoresis detection of Tricine-SDS-PAGE of different stationary phase purification activity peak purities of crude lipopeptide extracts of the strain G-1, wherein A to E are D101 macroporous adsorption resin, sephadex LH-20, sephadex tandem column +AQC18, AQC18 and sephadex tandem column in sequence;
FIG. 30 effects of different flow rates on purification purity of strain G-1 lipopeptides;
FIG. 31 Activity of Strain G-1 Fenterleukin against Alternaria malis and Alternaria pyriformis pathogens;
FIG. 32 active control of strain G-1 Fenterleukin against Alternaria solani;
FIG. 33 active control of bacterial strain G-1 Fenjiedin against Alternaria alternata pathogens;
FIG. 34 effect of different temperatures on the bacteriostatic activity of the strain G-1 fenaminocycline on Alternaria alternata plates;
FIG. 35 effects of different temperatures on the bacteriostatic activity of the strain G-1 fenaminogin on the Alternaria alternata pathogens living body;
FIG. 36 effect of different concentrations of fenamidone on body weight change during 30d feeding of rats;
FIG. 37 bacterial strain G-1 meal and crude lipopeptide extract of BPY broth with antibacterial activity against Alternaria alternata (A. Sonali), control, BPY broth and meal, respectively, from left to right;
FIG. 38 LC detection profile of crude lipopeptide extract from strain G-1, and BPY broth;
FIG. 39 shows results of LC chromatographic peak mass spectrometry (ESI-MS) identification of strain G-1 rapeseed meal and BPY fermentation broth lipopeptide substances, wherein the results are BPY peak 1, rapeseed meal peak 1, BPY peak 2, rapeseed meal peak 2, BPY peak 3 and rapeseed meal peak 3 from top to bottom and from left to right respectively.
Detailed Description
The invention provides a method for efficiently preparing a fenamidone with the function of inhibiting alternaria alternata, which comprises the following steps: inoculating Bacillus bailii (Bacillus velezensis) G-1 into fermentation medium, fermenting, culturing, extracting, and purifying to obtain the final product; the bacillus bailii G-1 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.23750; the fermentation medium comprises rapeseed meal and glucose.
In the present invention, the inoculum size of Bacillus bailii G-1 is preferably 1% to 2% by volume based on the volume of the fermentation medium, the fermentation time is preferably 4 to 8 days, more preferably 7 days, the fermentation temperature is preferably 25 to 35 ℃, more preferably 30 ℃, and the fermentation speed is preferably 120rpm to 200rpm. In the present invention, the fermentation medium preferably comprises the following raw materials by weight: 4-7% of rapeseed meal, 0.8-2% of glucose and K 2 HPO 4 ·3H 2 O0.03％～0.05％，MgSO 4 ·7H 2 O0.02％～0.05％，MnSO 4 ·7H 2 0.01 to 0.05 per mill of O and the balance of water; more preferably comprises the following raw materials by weight: 5 to 6 percent of rapeseed meal, 0.9 to 1 percent of glucose and K 2 HPO 4 ·3H 2 O0.04％，MgSO 4 ·7H 2 O0.02％，MnSO 4 ·7H 2 0.01 to 0.05 per mill of O and the balance of water; the pH of the fermentation medium is preferably 7-8. The specific sources of the raw materials in the fermentation medium are not particularly limited, and the conventional commercial products in the field can be adopted.
In the invention, the extraction method is preferably that hydrochloric acid is precipitated and then methanol is adopted for ultrasonic extraction, and the pH value of the hydrochloric acid precipitation is preferably 3; the dosage of methanol is preferably 400-600mL, the ultrasonic power is preferably 160w, the ultrasonic frequency is preferably 40KHz, and the ultrasonic extraction time is preferably 1-1.5 h based on 1L fermentation broth. In the present invention, the purified stationary phase is preferably a sepharose tandem column and AQC18, wherein the sepharose tandem column is a primary purification and the AQC18 is a secondary purification. In the present invention, the sephadex tandem column is preferably composed of a DEAE-52 column, a phenyl-sephadex column and a G-100 sephadex column. The purification method of the stationary phase dextran-agarose gel tandem column +AQC18 of the invention is preferably as follows: the Tris buffer (pH 7.6-8.6) with 0.02mol/L mobile phase A contains 0.001mol/LEDTA; mobile phase B was 0.6mol/LNaCl; mobile phase C methanol; mobile phase D water. The elution procedure was: a is balanced for 10min, B10-70% is eluted linearly for 15min, C20-80% is eluted linearly for 15min, C100-10 min. Wherein A and B are primary purified mobile phases; c and D are secondary purified mobile phases with a detection wavelength of 244nm. In the invention, when the sephadex tandem column and the AQC18 stationary phase are adopted for purification, the preferred flow rate of the primary purification is 10mL/min, and the preferred flow rate of the secondary purification is 7mL/min.
The invention also provides the prepared phendimedin according to the method and the application of the phendimedin or the method in preventing and treating the infection of the fruit and vegetable post-harvest alternaria, and the invention also provides the prepared phendimedin according to the method and the application of the phendimedin or the method in preparing the product for preventing and treating the infection of the fruit and vegetable post-harvest alternaria. The invention also provides application of the bacillus beijerinus G-1 in preventing and treating the infection of the alternaria alternata after fruit and vegetable harvest or preparing related products. In the present invention, the fruits and vegetables preferably include one or more of apples, pears, tomatoes and dates.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the following examples, conventional methods are used unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
Inoculating Bacillus bailii (Bacillus velezensis) G-1 to fermentation medium (the weight percentage of each raw material in fermentation medium is 5% of rapeseed meal (i.e. rapeseed meal), 1% of glucose, and K based on the weight of fermentation medium 2 HPO 4 ·3H 2 O0.04％，MgSO 4 ·7H 2 O0.02％，MnSO 4 ·7H 2 O0.01 per mill, the balance being water, and the pH value of the fermentation medium is 7), fermenting and culturing for 7 days at 30 ℃ and 120rpm to obtain fermentation liquor.
Adjusting pH value of the supernatant fermentation liquor to 3 by using hydrochloric acid, precipitating for 12 hours at 4 ℃, centrifuging to collect precipitate, placing the precipitate in a 1000mL beaker, adding 600mL of methanol for ultrasonic extraction, performing ultrasonic extraction for 1 hour under the conditions that ultrasonic power is 160w and ultrasonic frequency is 40KHz, taking extract, purifying by using a stationary phase dextran-agarose gel tandem column and AQC18, wherein the dextran-agarose gel tandem column is primary purification, and the AQC18 is secondary purification. The purified mobile phase A contains 0.001mol/LEDTA in 0.02mol/L Tris buffer (pH 7.6-8.6); mobile phase B was 0.6mol/LNaCl; mobile phase C methanol; mobile phase D water. The elution procedure was: a is balanced for 10min, B10-70% is eluted linearly for 15min, C20-80% is eluted linearly for 15min, C100-10 min. Wherein A and B are primary purified mobile phases; c and D are secondary purified mobile phases with a detection wavelength of 244nm. The primary purification flow rate was 10mL/min, and the secondary purification flow rate was 7mL/min. And collecting the absorption peak with the retention time of the secondary purification at 11.5-13.0 min to obtain the high-purity Fencins (Fengycin) with the function of inhibiting the alternaria alternata.
Example 2
The preparation method comprises the steps of taking BPY culture solution (10G of peptone, 5G of beef extract, 5G of yeast extract, 5G of glucose, 5G of NaCl, and pH 7.0) as a basic formula, preparing different fermentation liquids by using different agricultural wastes (wheat bran, corn meal, corncob meal, soybean meal, rapeseed meal, wine lees, vinegar residue, rotten fruits and vegetables (16 types of apples, pears, date pits and walnut green husks), wheat straw, rape straw, germinated potatoes and corn straw) to replace nitrogen sources (namely, replacing peptone, beef extract and yeast extract) in the culture liquid, inoculating (inoculum size is the same as that of Bacillus beijeziensis G-1 (B.velezensisG-1) of example 1), fermenting at 28 ℃ for 72 hours at 160rpm, and collecting supernatant. Then, hydrochloric acid precipitation-methanol extraction method (6 mol/L hydrochloric acid is used for adjusting the pH value of the supernatant fermentation broth to be 2, precipitation is carried out at 4 ℃ for 12 hours, then the precipitate is collected by centrifugation, and is placed in a 1000mL beaker, 500mL of methanol is added for extraction, extraction is carried out for 3 times, each time 500mL of methanol is extracted for 4 hours, finally, 3 times of extraction liquid are combined and evaporated to dryness in a rotary manner, 10mL of sterile water is added for dissolving to obtain lipopeptide extract), each treatment of the lipotein is extracted, and the antibacterial activity of each treatment of the lipotein extract is measured by adopting an agar diffusion method with alternaria as a target.
The results are shown in FIG. 1: the lipopeptide extracts of the treatment groups of only soybean meal, rapeseed meal, germinated potatoes, rape straw and soybean meal in 16 different agricultural wastes show good antibacterial activity on Alternaria alternata, the diameters of the antibacterial circle are respectively 26.3mm, 35.4mm, 27.1mm, 13.6mm and 14.3mm, and compared with BPY (16.8 mm), the antibacterial circle of the crude extracts of soybean meal, rapeseed meal and germinated potato lipopeptide is obviously higher than that of a control. The bacillus velezensisg-1 can be used for producing the phendimedin by using 3 agricultural wastes, and the 3 agricultural wastes have obvious promotion effect on the synthesis of the phendimedin.
To further determine whether the bacterial velezensisg-1 can synthesize the fenaminon by using 5 agricultural wastes, the oil extraction characteristics of the lipopeptide extract thereof were analyzed, and the results show that: the bacillus velezensisg-15 agricultural waste fermentation broths all show good oil discharge activity, and the diameter of the oil discharge ring is as follows: 84.3mm (rapeseed meal), 63.1mm (germinated potato), 28.6mm (soybean meal), 26.6mm (soybean meal) and 19.3mm (rape straw), and compared with control BPY (26.4 mm), the oil extraction activity of the broth of the rapeseed meal, germinated potato and soybean meal is obviously higher than that of the control (figure 2), further showing that the Bacillus velezensisg-1 can synthesize the phenmedine by using 5 agricultural wastes, and the yield of the phenmedine in the rapeseed meal is highest.
Example 3
Effect of different fermentation conditions on biosynthesis of B.velezensisG-1 with agricultural waste as Nitrogen Source Shi Fenjie
B.velezensisG-1 was inoculated into a fermentation medium (80 g/L of rapeseed meal, 20g/L of glucose, pH 7.0) for fermentation. And (3) adopting a single factor test, and comparing the influence of fermentation conditions such as fermentation time, initial pH value, temperature, inoculation amount, rotating speed and the like on the production of the fenamic acid by using the growth amount of the strain, the content of the fenamic acid (Fengycins) and the size of a bacteriostasis zone as evaluation indexes.
(1) Effect of fermentation time on growth of Strain G-1 and Fengycins Synthesis
When different fermentation times are studied, the rest fermentation conditions are as follows: 1% inoculum size (1% inoculum size indicates 1mL inoculum size based on 100mL broth volume, for example), 28℃at 160rpm. The results are shown in FIGS. 3-5. Under different fermentation time, the yield and the antibacterial activity of the strain G-1Fengycins are increased firstly and then decrease along with the fermentation time, but the yield and the antibacterial activity of the fermentation 3d and 4dFengycins are not obviously increased, and the yield and the antibacterial activity are highest at the fermentation 7d (figures 3 and 4); the growth of the strain G-1 gradually increases along with the extension of the fermentation time, and the strain G-1 tends to be stable at the 4 th day, but the content of the strain G-1 phenmedine begins to rapidly increase after the 4 th day, which means that the rapid synthesis time of the strain G-1 phenmedine is 4d after fermentation when the rapeseed meal is a nitrogen source, and the fermentation cost and the phenmedine yield are comprehensively considered, and then 7d is selected as the optimal fermentation time (figures 3 and 5).
(2) Effect of initial pH of fermentation broth on growth of Strain G-1 and Fengycins Synthesis
When different initial pH values are studied, the rest of fermentation conditions are: the inoculum size was 1%, 28 ℃, 7d, 160rpm. Inoculating strain G-1 into fermentation culture medium with different initial pH values, fermenting for 7d, and determining its Fengycins content and OD 600 And zone size, the results are shown in figure 6: the different initial pH values of fermentation have a significant effect on the growth of the strain G-1 and the yield of Fengycins; the growth rate of the strain G-1 at the pH value of 6-8 is obviously higher than other pH values, but the antibacterial activity and the Fengycins yield only show good effects at the pH values of 7 and 8, which shows that at the pH value of 6, the strain G-1 is not obviously influenced by the propagation, but the Fengycins synthesis is inhibited. In addition, the Fengycins yield and the antibacterial zone diameter of the strain G-1 under alkaline conditions are higher than those of the strain G-1 under acidic conditions, which indicates that the strain G-1Fengycins yield can be improved by properly increasing the pH value.
(3) Effect of fermentation temperature on growth of Strain G-1 and Fengycins Synthesis
When different fermentation temperatures are studied, the rest fermentation conditions are as follows: 1% inoculum size, 7d, 160rpm, pH7. The fermentation temperature of the strain G-1 was optimized with OD600 value, antibacterial activity and Fengycins content as evaluation indexes, and the results are shown in FIG. 7: along with the increase of the fermentation temperature, the OD600 value, the antibacterial activity and the Fengycins peak area of the strain G-1 show a trend of increasing firstly and then decreasing secondly, wherein the growth and propagation rate, the antibacterial activity and the Fengycins peak area of the strain G-1 are obviously higher than those of other fermentation temperatures within the range of 25-35 ℃, and the OD600 value, the antibacterial activity and the Fengycins peak area of the strain G-1 at low temperature (15-20 ℃) are higher than 40 ℃, so that the growth and the Fengycins synthesis of the strain G-1 can be obviously inhibited after the temperature is higher than 35 ℃. The recommended temperature is 25-35 ℃ which is the optimal fermentation temperature of the strain G-1 high-yield Fengycins.
(4) Effect of inoculum size on growth of Strain G-1 and Fengycins Synthesis
When different inoculum sizes are studied, the rest of fermentation conditions are: the results of fermentation at 30℃and pH7 for 7d at 160rpm are shown in FIG. 8: the inoculation amount has no obvious influence on the propagation rate of the strain G-1, but the antibacterial activity of the strain G-1 fermentation broth and the Fengycins peak area have certain difference between different inoculation amounts, and the strain G-1 fermentation broth and the Fengycins peak area all show a trend of rising and then falling along with the increase of the inoculation amount; the bacterial inhibition activity and the Fengycins peak area of the strain G-1 fermentation broth with the inoculation amount of 1 to 2 percent in the range of 0.5 to 4 percent show that the inoculation amount of the strain G-1 high-yield Fengycins needs to be controlled in the range of 1 to 2 percent.
(5) Influence of the rotational speed on the growth of the Strain G-1 and on the Fengycins synthesis
When different rotation speeds are studied, the rest fermentation conditions are as follows: 1% inoculum size, 7d fermentation, pH7.0, 30℃temperature, and results are shown in FIG. 9: the propagation rate, the Fengycins peak area and the yield of the strain G-1 all show a trend of rising first and then tending to be stable along with the increase of the rotating speed, and the difference between the rotating speeds of 120-200 rpm is not obvious, but is obviously higher than 80rpm, which indicates that the rotating speed of the strain G-1 needs to be ensured to be not lower than 120rpm.
Example 4
Effect of the amount of Medium Components on B.velezensisG-1 growth and Fenimepirin biosynthesis
(1) Influence of different meal amounts on the growth of the strain G-1 and the synthesis of the finemetine
When different quantities of rapeseed meal are studied, the quantities of the rest components in the fermentation medium are respectively 2% of glucose, and the fermentation conditions are as follows: 1% inoculum size, 7d fermentation, pH7.0, 30℃and 120rpm rotation speed. After analysis of bacterial inhibition activity, colony count and production of the finemens of the strain G-1 fermentation broth at different meal levels (1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% and 13%) by plate-counter method, coating count method and HPLC, it was found that: the strain G-1 has a tendency that the yield of the phendimedin increases and decreases with the increase of the amount of the vegetable meal, wherein the yield of the phendimedin is highest when the amount of the vegetable meal is 5 percent (figure 10); the bacteriostatic activity of the strain G-1 fermentation liquor also shows a trend of rising and then falling along with the increase of the amount of the vegetable pulp, wherein the bacteriostatic activity in the range of 4% -7% is better, and the bacteriostatic zone is obviously higher than the amount of the rest vegetable pulp (figure 11); the biomass of strain G-1 then showed a gradual decrease with increasing meal, with growth inhibition of strain G-1 being most pronounced with >5% meal (FIG. 12). The optimal amount of the rapeseed meal for high-yield of the bacterial strain G-1 is 5%.
(2) Effect of different glucose usage on growth of Strain G-1 and Synthesis of Fenimepirin
When different glucose amounts (0.5%, 0.8%, 1%, 2%, 3% and 4%) were studied, the amounts of the remaining components in the fermentation medium were respectively: 5% of rapeseed meal, and the fermentation conditions are the same as (1). The results are shown in FIG. 13, in which strain G-1OD was increased with the increase in glucose consumption 600 The values, the antibacterial zone and the area of the peak of the phendimedin all show the trend of rising and then falling; strain G-1OD when glucose is used in 0.8% -2% 600 The value and the yield (peak area) of the phendimedin are both obviously higher than other dosage, and the difference is not obvious in the range of 0.8-2%, but the effect of the fermentation liquor antibacterial zone with the 1% glucose addition amount is best, and the value is obviously higher than the dosage of other glucose, so that the preferred dosage of the glucose is recommended to be 1%.
(3)K 2 HPO 4 ·3H 2 Effect of O usage on growth of Strain G-1 and Synthesis of Fenimepirin
Study of different K 2 HPO 4 ·3H 2 When the amount of O is 0, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, the amounts of the other components in the fermentation medium are respectively as follows: 5% of rapeseed meal and 1% of glucose, and the fermentation conditions are the same as (1). The results are shown in FIG. 14, where K in the medium 2 HPO 4 ·3H 2 The O dosage has a certain influence on the growth of the strain G-1 and the synthesis of the phenmedine, and along with K 2 HPO 4 ·3H 2 The increasing amount of O shows the trend of increasing before decreasing, but the increasing amount of O shows the trend of decreasing obviously after the increasing amount of G-1OD600 and the increasing amount of the O exceeds 0.06%, the antibacterial zone starts to drop significantly after exceeding 0.05%, and it is presumed that the reason why the antibacterial zone is inconsistent with the area change rule of the lipopeptide peak is probably that the composition of the fenaminogen is changed, so that the antibacterial activity is changed. After comprehensive consideration, 0.03% -0.05% of the additive is recommended to be a better additive amount.
(4)MgSO 4 ·7H 2 Effect of O usage on growth of Strain G-1 and Synthesis of Fenimepirin
Study of different MgSO' s 4 ·7H 2 When O is used (0, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%), fermentation culture is performedThe dosages of the rest components in the nutrient medium are respectively as follows: 5% of rapeseed meal, 1% of glucose and K 2 HPO 4 ·3H 2 O0.03%, and the fermentation conditions are the same as (1). The results are shown in FIG. 15, along with MgSO 4 ·7H 2 The increase of the dosage of O, the increase of the strain G-1OD600 value, the area of the peak of the phendimin and the antibacterial zone are all in a trend of rising and then descending, and the differences are not obvious in the range of 0.02% -0.05%, but are obviously higher than other dosages, so that the cost, the production of the phendimin and the antibacterial activity are comprehensively considered, and 0.02% is selected as the preferable addition.
(5)MnSO 4 ·7H 2 Effect of O usage on growth of Strain G-1 and Synthesis of Fenimepirin
Investigation of different MnSOs 4 ·7H 2 When the dosage of O is 0, 0.01 per mill, 0.02 per mill, 0.03 per mill, 0.04 per mill, 0.05 per mill, 0.06 per mill, 0.07 per mill and 0.08 per mill, the dosages of the rest components in the fermentation medium are respectively as follows: 5% of rapeseed meal, 1% of glucose and K 2 HPO 4 ·3H 2 O0.03％、MgSO 4 ·7H 2 O0.02%, and the fermentation conditions are the same as (1). The results are shown in FIG. 16, where the MnSO is different in the medium 4 ·7H 2 The addition of O has a significant effect on the growth of the strain G-1 and the synthesis of the phenmedine; along with MnSO 4 ·7H 2 The O addition amount is increased, the strain G-1OD600 value, the area of the Fenace element peak and the antibacterial zone all show a tendency of descending after the Mr. is high, but the change rule of the antibacterial zone has a certain difference with the Fenace element peak area and the OD600 value; the bacteria inhibition zone of the strain G-1 is not obviously different in the range of 0.01 to 0.04 per mill, the peak area and OD600 value of the felinine are not obviously different in the range of 0.03 to 0.05 per mill, but are obviously higher than those of other additive amount treatment, so that the total consideration of cost, the felinine yield and the bacteria inhibition activity is recommended to be 0.01 per mill as MnSO 4 ·7H 2 O is preferably added in an amount.
Example 5
Comprehensive optimization of component amounts and fermentation conditions of velezensisg-1 finemedin-producing medium
On the basis of single factor optimization of the component consumption of the culture medium and the fermentation condition, the influence of the component consumption of the culture medium and the fermentation condition on the growth, the production and the antibacterial activity of the strain G-1 is comprehensively evaluated by a method of combining SPSS principal component analysis and RDA redundancy analysis and related significance test, and the aim of finding out key factors influencing the production and the antibacterial activity of the strain G-1 is fulfilled.
(1) Principal component analysis results
To comprehensively analyze the relation between fermentation conditions and medium components and the growth of the strain G-1 and the synthesis of the phendimedin, PCA visualization was introduced and the contribution of the fermentation conditions and the medium components to the growth of the strain G-1 and the synthesis of the phendimedin was quantified. Kmo= 0.743 (greater than 0.60), bartlett's test P < 0.001 indicated that PCA was suitable for this experiment (table 1). According to the interpretation degree of each principal component on the data variation, a steep slope map (screen) is drawn, and according to the position of 'steep slope is gradually gentle', the extracted principal components 1 and 2 can be judged to interpret the data-related variation (figure 17). As can be seen from the total variance interpretation results, the extracted 2 main components can respectively interpret 82.37% (PC 1) and 11.54% (PC 2) of the variation, and the cumulative contribution rate accounts for 93.91% of the total variance, which shows that 2 PCs can interpret 93.91% of the total information, wherein PC1 accounts for 82.37%, and can better reflect the growth of the strain G-1 and the synthesis condition of the phendimedin (Table 2). As can be seen from FIG. 18, all fermentation conditions had a positive correlation with the growth of strain G-1 and the synthesis of fenacet, but the degree of influence of the different fermentation conditions was somewhat different. In addition, the PCA not only can visualize and quantify the contribution of the fermentation conditions to the growth of the strain G-1 and the synthesis of the phenmedine, but also can reflect the stability of the growth of the strain G-1 and the synthesis of the phenmedine under different fermentation conditions. In the PCA score plot, the closer the data fall points, the more closely it affects each condition. As shown in FIG. 19, the rotation speed, fermentation time, glucose, rapeseed meal, dipotassium hydrogen phosphate and magnesium sulfate fall points are concentrated (only span 2 quadrants), which shows that the fermentation conditions have a more approximate effect on the growth of the strain G-1 and the synthesis of the phenmedine and are main factors mainly influencing the growth of the strain G-1 and the synthesis stability of the lipopeptid.
Table 1 statistics test (KMO) and Bartlett Li Qiuti test (Bartlett's test)
Table 2 TotalVarianceExplained Total variance interpretation
Finally, the linear combination coefficient (factor absolute value/square root of the characteristic root of the corresponding principal component) corresponding to the 2 principal components obtained by principal component analysis, the comprehensive score model coefficient ([ first principal component variance ] the first linear combination coefficient + the second principal component variance ] the second linear combination coefficient ]/the sum of the two component variances) are obtained, the principal component contribution rate weight (sum of each comprehensive score model coefficient/comprehensive score model coefficient) is calculated, and the influence degree sequencing of each factor on the growth of the strain G-1 and the synthesis influence of the finemens is obtained according to the weight in descending order. As shown in FIG. 20, the fermentation conditions of 10 strains G-1 and the synthesis of the fenacet are greatly influenced by the rotation speed, glucose, vegetable meal, fermentation time, dipotassium hydrogen phosphate and fermentation temperature, the weights are all more than 0.1, and the 6 fermentation conditions are the key factors for influencing the growth of the strains G-1 and the synthesis of the fenacet.
(2) RDA analysis + correlation significance analysis results
As shown in FIG. 21, the RDA analysis shows that the included angle between the rotation speed, fermentation time, inoculum size, glucose, dipotassium hydrogen phosphate and OD600 is acute, which indicates that the related fermentation conditions have a positive correlation with the growth of the strain G-1, but the rapeseed meal, pH, fermentation temperature, magnesium sulfate and OD 600 The included angle is an obtuse angle and shows a negative correlation; the included angle between the area of the Fenjieisu peak and pH, fermentation temperature and rapeseed meal is an acute angle and shows a positive correlation, while the included angle between the rest fermentation conditions is an obtuse angle and shows a negative correlation; the included angles between the antibacterial zone and the rotating speed, the inoculation amount, the dipotassium hydrogen phosphate, the magnesium sulfate, the manganese sulfate, the rapeseed meal and the pH are in positive correlation, while the other fermentation conditions are in negative correlation (the included angle is an obtuse angle); from the aspect of line length, the rotation speed, the vegetable meal and the dipotassium hydrogen phosphate are obviously larger than other conditions, which shows that 3 are very closely related to the growth of the strain G-1 and the synthesis of the phenmedine.
In addition, the results of the correlation significance analysis showed that strain G-1 grew (OD 600 ) And the antibacterial activity of the phendimedin are obviously and positively related to the fermentation time and the rotation speed (P<0.05 And the vegetable meal can obviously influence the antibacterial activity (P)<0.05 It was demonstrated that the three are the core factors affecting the growth of strain G-1 and the synthesis and bacteriostatic activity of the finemens (Table 3).
TABLE 3 Spearman-related significance analysis between fermentation conditions and growth of strain G-1 and synthesis of fenaminon
(3) Key limiting factor screening affecting bacterial strain G-1 growth and fenaminon synthesis based on principal component analysis, RDA analysis and related significance
After comprehensively evaluating the main component analysis result and the RDA+ related significance analysis result, the 2 analysis modes can obtain the rotation speed, fermentation time and rapeseed meal which are key influence factors for influencing the growth of the strain G-1 and the synthesis of the fenacet; although it cannot be clearly obtained by RDA+ related significance analysis that glucose, dipotassium hydrogen phosphate and fermentation temperature can significantly influence the growth of the strain G-1 and the synthesis of the fenaminopeptide, the included angle between the fermentation temperature and the lipopeptide peak area is obviously smaller than other conditions, and the growth (OD) of the glucose and the strain G-1 600 ) The included angle between the main component analysis results and the RDA+ related significance analysis results are comprehensively considered, and then the rotating speed, the glucose, the rapeseed meal, the fermentation time, the dipotassium hydrogen phosphate and the fermentation temperature are judged as key fermentation conditions affecting the growth of the strain G-1 and the synthesis of the phendimedin, so that a clear target is provided for further targeted optimization of the later fermentation process.
Example 6
Optimization of extraction Process of velezensisG-1 Fenjie-Su
After fermentation was completed according to the method of example 1, the extraction amounts of the felbinac under the conventional extraction mode (hydrochloric acid precipitation and methanol extraction process), methanol ultrasonic extraction process and supercritical fluid extraction process were respectively compared and analyzed, and the extraction time, cost, yield and antibacterial activity were used as evaluation indexes.
The conventional extraction method comprises the following steps: the pH of the supernatant broth was adjusted to 2 using 6mol/L hydrochloric acid, and the precipitate was collected by centrifugation after 12h precipitation at 4℃and placed in a 1000mL beaker, and extracted with 500mL methanol. The extraction was performed 3 times with 500mL methanol for 4h each. Finally, the extracts are combined for 3 times and distilled to dryness by rotating, and 10mL of sterile water is added for dissolution to obtain the lipopeptide extract.
Methanol ultrasonic extraction process: the pH value of the supernatant fermentation broth is regulated to be 3 by using 6mol/L hydrochloric acid, the supernatant is precipitated at 4 ℃ for 12 hours, the precipitate is collected by centrifugation, and is placed in a 1000mL beaker, 500mL of methanol is added for ultrasonic extraction, the ultrasonic power is 160w, the ultrasonic frequency is 40KHz, and the ultrasonic extraction time is 1 hour.
Supercritical fluid extraction process: the pH value of the supernatant fermentation broth is regulated to be 3 by using 6mol/L hydrochloric acid, and the supernatant is centrifugally collected after being precipitated for 12 hours at the temperature of 4 ℃, and the precipitate is placed in an extraction kettle, 100mL of methanol is added into a carrier adding middle port, and supercritical fluid extraction is carried out. Extraction conditions: the temperature is 50 ℃ and the extraction pressure is 28Mpa; separation conditions: separation conditions: the separation pressure is 4Mpa, the temperature of the separation 1 port is 40 ℃, and the temperature of the separation 2 port is 30 ℃. The extraction time was 2h.
The results are shown in FIG. 22: compared with the conventional extraction mode, the peak area and the antibacterial zone of the phendimedin extracted for 1h by ultrasonic extraction are obviously increased, but supercritical CO 2 The lipopeptide peak area and the antibacterial zone of the extraction technology are lower than those of the control, which shows that the ultrasonic extraction can not only effectively shorten the extraction time of the lipopeptide, but also improve the yield to a certain extent, so that the ultrasonic is selected as a better extraction mode of the strain G-1 lipopeptide.
Example 7
Influence of different pH values on lipopeptide precipitation effect in strain G-1 fermentation liquor by adopting acidification precipitation process
After comparative analysis of the yield of the strain G-1-fenacet at different precipitation pH values, the other steps are the same as in example 1, the yield of the strain G-1-fenacet shows a tendency of rising and then falling along with the increase of the pH value, the difference is not obvious in the range of pH values from 1 to 3, but the yield is obviously reduced when the pH value is more than 3, which indicates that the optimal precipitation maximum pH value of the strain G-1-fenacet is 3 (figure 23).
Example 8
Single-factor optimization of methanol ultrasonic extraction process parameters
The results of single-factor optimization of the methanol dosage, ultrasonic time, ultrasonic power and ultrasonic frequency in the ultrasonic extraction process of the bacterial strain G-1 lipopeptide (all other conditions are the same as in example 6 when single-factor optimization is carried out) show that the peak area and the antibacterial zone of the bacterial strain G-1 are both in a trend of increasing firstly and then tending to be stable with the increase of the methanol dosage, and the increasing effect is not obvious when the methanol dosage is 600mL, which indicates that the preferred methanol dosage is 600mL (figure 24); the peak area and the zone of inhibition of the strain G-1-fenamidone both show a trend of rising and then falling with the increase of ultrasonic time and power, and reach the maximum value respectively at ultrasonic time of 1.5h and 160w (figures 24-25); the high frequency (40 KHz) of the fenamidone peak area and the zone of inhibition were significantly higher than the low frequency (20 KHz) (fig. 25), so that the methanol usage amounts of 600mL, 1.5h, 160w and 40KHz were chosen as the preferred parameters for ultrasonic extraction of the strain G-1 fenamidone.
Example 9
Detection wavelength optimization
As a result of scanning the extract (crude lipopeptide extract) obtained in example 1 at a full wavelength in the range of 200-700nm by means of an UP-2500 ultraviolet spectrophotometer, it was found that the crude lipopeptide extract of strain G-1 forms a stable absorption peak at 244nm, and the difference was insignificant compared with the maximum absorption wavelength (243 nm) of the fermentation broth obtained by fermentation culture with a BPY fermentation medium (FIG. 26).
In addition, by using the ultraviolet absorption wavelength 215nm adopted in most of the prior detection of the phendimediate as a control, and respectively carrying out dual-wavelength detection at 215nm and 244nm by preparing liquid phase, the result shows that the absorption peak height and the area of the strain G-1 phendimediate at 244nm are higher than 215nm (figure 27), so 244nm is selected as the detection wavelength.
Example 10
Optimizing a stationary phase:
a series column of 244nm detection wavelength versus dextran-agarose gel as determined in example 9 was used: DEAE-52 (negatively charged in lipopeptide neutral environment) +phenyl-sepharose (having a strong hydrophobicity) +g-100 sephadex (molecular weight); sephadex LH-20 (SephadexLH 20); AQC18 (20-35 um,40g, agelatechnogies); and (3) comparing and analyzing the efficiency of the purification process of 5 stationary phases, namely macroporous adsorption resin (D101 macroporous adsorption resin) and sephadex tandem column plus AQC18, detecting the purity by Tricine-SDS-PAGE electrophoresis, and finally optimizing the purification process parameters. The stationary phase optimization was performed in the same manner as in example 1, except for the purification step.
(1) Dextran-sepharose tandem column: the mobile phase A of DEAE-52, phenyl-sepharose (with stronger hydrophobicity) and G-100 sephadex (molecular weight) contains 0.001 mol/LETTA in Tris buffer (pH 7.6-8.6) with 0.02mol/L, the mobile phase B is 0.6mol/LNaCl solution for linear elution, the elution program is A100% equilibrium 10min, B (NaCl) 10% -90% linear elution 30min, B100%10min.
(2) The mobile phases of the dextran gel LH-20 (SephadexLH 20), the AQC18 and the macroporous adsorption resin are methanol (B) and water (A); the elution procedure was: 20% B is balanced for 10min,20% -80% B is eluted linearly for 30min, and 100% B is eluted for 10min.
(3) Dextran-agarose gel tandem column +AQC18, mobile phase A0.02 mol/L Tris buffer (pH 7.6-8.6) contains 0.001mol/LEDTA; mobile phase B was 0.6mol/LNaCl; mobile phase C methanol; mobile phase D water. The elution procedure was: a is balanced for 10min, B10-70% is eluted linearly for 15min, C20-80% is eluted linearly for 15min, C100-10 min.
After comparative analysis of the yield and purity of the strain G-1, which was purified by different stationary phases, it was found that in terms of the mass yield and the oil extraction activity: the single stationary phase purification aspect uses AQC18 column material with highest yield and oil extraction activity, and then uses sephadex column, and uses macroporous adsorption resin with worst purification efficiency (figure 28); the purification of each stationary phase alone did not work well in terms of purity, although a single peak was shown in the HPLC profile, multiple bands were found to be still present after electrophoresis detection, indicating that the peak was of lower purity (fig. 29).
After the combined application of the 2 purification modes with higher material yield and oil drainage activity, the material yield is reduced by 51.7% compared with the highest AQC18, but the purity is obviously improved, which shows that the combined purification mode based on 2 stationary phases can greatly improve the purity of the fenamic acid, but the material yield loss rate is as high as more than 50%, so that the later stage needs to be further explored how to improve the material yield (FIGS. 28-29). In addition, although the HPLC profile of the Sephadex LH-20 column was similar to that of the tandem column, the material yield was low and the peak time was late (FIG. 28), so that Sephadex tandem column +AQC18 was selected as strain G-1 high purity lipopeptide to prepare a superior stationary phase.
Example 11
Mobile phase flow rate optimization of dextran-sepharose tandem column + AQC18 stationary phase
The flow rates of the mobile phases (0.02 mol/L Tris buffer (pH 7.6-8.6) for the primary purification (dextran-agarose gel series column) and the secondary purification AQC18 (mobile phase A) were optimized for 0.001 mol/LETTA, 0.6mol/LNaCl for mobile phase B, methanol for mobile phase C, and water for mobile phase D were evaluated for 10min for A equilibrium, 15min for B10-70% for linear elution, 15min for C20-80% for linear elution, and 10min for C100), wherein A and B are the primary purification mobile phases, and C and D are the secondary purification mobile phases for detection at 244 nm), and the results are shown in Table 4 and FIG. 30: the retention time of the strain G-1-fenacet is gradually shortened along with the increase of the flow rate, but the peak area is in a descending trend, and the separation degree is gradually reduced; the retention time and peak area of the primary purification in the flow rate range of 5-10 mL are not significantly different, but the purity is higher by 10mL, so 10mL/min is selected as the optimal flow rate of the primary purification; the difference between the retention time and the peak area of the lipopeptides in the second stage is not obvious in 7-10 min, but the separation degree is 7mL/min, so that 7mL/min is selected as the optimal flow rate for the second-stage purification.
TABLE 4 influence of different flow rates on the retention time and peak area of strain G-1Fengycin
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Example 12
Analysis of bacteriostatic Effect of velezensisG-1 Fenimedium on Alternaria alternata after fruit and vegetable harvest
(1) In-vivo antibacterial effect of fenamidone on alternaria alternata after fruit and vegetable harvest
The antibacterial activity of B.velezensisG-1 finemein on Alternaria is determined on living fruits and vegetables by adopting a surface disinfection (firstly, 75% ethanol is used for soaking for 1min, sterile water is used for washing for 3 times, secondly, sodium hypochlorite is used for soaking for 3min, sterile water is used for washing for 3 times, finally, a super clean bench is used for airing) and puncture inoculation (a 5mm puncher is used for manufacturing wounds with the diameter of 5mm and the depth of 3 mm. Then 100 mu.L of lipopeptide extract with the concentration of 600 mu.g/mL is added to the wounds (prepared in example 1), after the wounds are dried, alternaria alternata cake with the diameter of 5mm is inoculated to the wounds, different treatment morbidity degrees are observed after the culture is carried out for 10 days at room temperature (25 ℃), apples, pears, tomatoes and jujubes are used as study objects). The lipopeptide extract obtained in example 1 was used as a control.
The results are shown in figures 31-33, and the results show that the strain G-1 substance has good antibacterial effect after the in-vivo antibacterial effect of the strain G-1 is measured on apples, pears, tomatoes and jujubes, so that the substance has good application potential in the aspect of preventing and treating the fruit and vegetable alternaria alternate.
(2) Analysis of correlation between temperature and antibacterial activity of Alternaria tenuifolia
(1) Determination of the bacteriostatic Activity of the finemens (obtained in example 1) on Alternaria alternata at different temperatures (0 ℃, 4 ℃, 10 ℃ and room temperature) on plates using the growth rate method: pathogenic bacteria cake with diameter of 5mm is obtained on a PDA plate of Alternaria alternata which is activated and cultured for 5d at 28 ℃ by using a puncher with diameter of 5mm, inoculated on a PDA plate with concentration of 600ug/mL of the finemein by using an inoculating needle, and respectively cultured for 7d, 10d, 20d and 40d at room temperature, 10 ℃ and 4 ℃ and 0 ℃ and then the finemein antibacterial activity at different temperatures is observed.
(2) Verifying the influence of temperature on the bacteriostatic activity of Alternaria tenuifolia on living fruits and vegetables: temperature based on Yulu pear living body test affects experimental design on bacteriostasis activity of the phenmedine: firstly, selecting Yulu pear with approximate size and maturity, and manufacturing a wound with a diameter of 5mm and a depth of 3mm by using a puncher with a diameter of 5 mm; secondly, adding 100uL of the felbinac with the concentration of 600ug/mL, and drying for 1h in an ultra-clean bench; thirdly, using a puncher with the diameter of 5mm to punch and inoculate Streptomyces bacterial cakes with the diameter of 5mm to a wound; finally, after culturing for 7d, 10d, 20d and 40d at room temperature, 10 ℃, 4 ℃ and 0 ℃ respectively, the antibacterial activity of the fenaminosulin at different temperatures is observed.
As shown in fig. 34 and 35, with the decrease of the storage temperature, the growth rate of the control group (the control group is not added with the phendimetate and the equivalent phendimetate solvent is used for replacing the phendimetate) and the treated group of the alternaria alternate gradually decreases, and the colony diameter of the alternaria alternate after the phendimetate treatment is obviously smaller than that of the control group, which indicates that the strain G-1 phendimetate can also play a better antibacterial effect at a low temperature, and has a better application potential in the field of low-temperature storage and preservation of fruits and vegetables.
(3) Biological safety evaluation of Strain G-1 and lipopeptides thereof
The biosafety of b.velezensisg-1 phendimedin (obtained in example 1) was designed and tested with reference to the national standard "methods of pesticide registration toxicology test". Acute toxicity: strain G-1 test doses 0, 2, 4, 6, 8, 10G/kg; the test doses of the phendimedin are 0, 500mg/kg, 1000mg/kg, 2000mg/kg, 4000mg/kg and 5000mg/kg. Subacute toxicity: the test dose of the phendimedin is 0, 500mg/kg, 1000mg/kg and 2000mg/kg.
Acute oral toxicity results indicate that: no death cases appear during feeding of rats treated differently, the LD50 of the strain G-1 and the phendimedin are respectively more than 10G/kg and 5000mg/kg, and the daily increase of the body weight of rats treated differently by the strain G-1 and the lipopeptides thereof is not significantly different from that of the control (Table 5-6), which indicates that the acute oral toxicity of the strain G-1 and the phendimedin is lower and the biological safety is better.
TABLE 5 results of acute oral toxicity test of Strain G-1
TABLE 6 results of acute oral toxicity test of lipopeptides
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Subacute oral toxicity results: after oral feeding is carried out on the subacute toxicity of rats, the rats in the treated group are found to have weight increase within 30 days after feeding, and the changes of various indexes of blood routine and biochemistry are not obviously different from those of a control, so that the subacute toxicity of the strain G-1 phendimedin is lower (figure 36; tables 7-8), and the strain G-1 phendimedin has higher biological safety and can be used in the field of fruit and vegetable storage and preservation.
TABLE 7 influence of different concentrations of fenamidone on blood normative index during the 30d feeding period of rats
TABLE 8 Effect of different concentrations of fenaminocycline on Biochemical indicators during 30d feeding of rats
Example 13
The difference from example 1 is that the fermentation medium is BPY broth (peptone 10g, beef extract 5g, yeast extract 5g, glucose 5g, naCl5g, pH 7.0) and the other fermentation conditions are the same as in example 1. The effect of the meal fermentation broth of the strain G-1 (example 1) and the BPY fermentation broth on the antibacterial activity of Alternaria alternata (A. Sonali) is compared by adopting a growth rate method, the comparison group is a group without any fermentation broth added, and the result is shown in a figure 37, the antibacterial activity of the meal fermentation broth lipopeptide crude extract of the strain G-1 is obviously higher than that of the same BPY fermentation broth, and the result shows that the meal fermentation broth of the strain G-1 can produce high-yield lipopeptide antibacterial substances after fermentation.
After qualitative and relative quantitative analysis of the lipopeptides (Surfactin, fengycin and Bacillus) in the 2 fermentation broths of example 1 and this example, LC-MS showed that the relative peak area, peak height and signal intensity of Fengycin in the meal fermentation broth were significantly higher than those of the BPY fermentation broth, but the relative peak area, peak height and signal intensity of Surfacin and Bacillus D were lower than those of the BPY fermentation broth (FIGS. 38-39), indicating that the meal fermentation broth was favorable for the synthesis of Fengycin by strain G-1, while the synthesis of Surfacin and Bacillus D was inhibited.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for efficiently preparing a fenamidone with the function of inhibiting alternaria alternata, which is characterized by comprising the following steps: inoculating bacillus bailii (Bacillus velezensis) G-1 into a fermentation medium, fermenting and culturing, extracting and purifying to obtain the final product; the bacillus bailii G-1 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.23750; the fermentation medium comprises rapeseed meal and glucose.
2. The method according to claim 1, wherein the inoculum size of bacillus beijerinus G-1 is 1% to 2% by volume of the inoculation based on the volume of the fermentation medium.
3. The method according to claim 1, wherein the fermentation time is 4-8 days, the fermentation temperature is 25-35 ℃, and the fermentation speed is 120-200 rpm.
4. The method according to claim 1, characterized in that the fermentation medium comprises the following raw materials by weight: 4-7% of rapeseed meal, 0.8-2% of glucose and K 2 HPO 4 ·3H 2 O 0.03％～0.05％，MgSO 4 ·7H 2 O 0.02％～0.05％，MnSO 4 ·7H 2 0.01 to 0.05 per mill of O and the balance of water; the pH value of the fermentation medium is 7-8.
5. The method according to claim 1, wherein the extraction method is that hydrochloric acid is precipitated and then methanol is adopted for ultrasonic extraction, and the pH value of the hydrochloric acid precipitate is 3; the dosage of methanol is 400-600mL, the ultrasonic power is 160w, the ultrasonic frequency is 40KHz, and the ultrasonic extraction time is 1h-1.5h based on 1L fermentation broth.
6. The method of claim 1, wherein the purified stationary phase is a dextran-sepharose tandem column and AQC18; the sephadex tandem column consists of a DEAE-52 column, a phenyl-sepharose column and a G-100 sephadex column.
7. The felines prepared by the process according to any one of claims 1 to 6.
8. Use of the method according to any one of claims 1 to 6 or the phenmedine according to claim 7 for controlling alternaria alternata infection after fruit and vegetable harvest.
9. An application of bacillus bailii G-1 in preventing and controlling the infection of alternaria alternata after fruit and vegetable harvest.
10. The use according to claim 8 or 9, wherein the fruit and vegetable comprises one or more of apples, pears, tomatoes and jujubes.
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| CN117660266B (en) * | 2024-01-29 | 2024-04-09 | 南京农业大学三亚研究院 | Bacillus bailii, antibacterial lipopeptide, biological control microbial agent and application thereof |
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