CN115109730A - Bacillus subtilis strain, bacillus powder and application thereof - Google Patents
Bacillus subtilis strain, bacillus powder and application thereof Download PDFInfo
- Publication number
- CN115109730A CN115109730A CN202210872869.XA CN202210872869A CN115109730A CN 115109730 A CN115109730 A CN 115109730A CN 202210872869 A CN202210872869 A CN 202210872869A CN 115109730 A CN115109730 A CN 115109730A
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- bacillus subtilis
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- fermentation
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Abstract
The invention provides a Bacillus subtilis strain which is Bacillus subtilis HY-001 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO.M2022622 and the preservation date of 2022 years, 05 months and 13 days. Obtained by ultraviolet mutagenesis, high temperature induction at 50 ℃ and screening of high-yield cellulase and amylase, wherein the number of the obtained product is GDMCC1.372 by the Guangdong province microorganism strain preservation center. The bacillus subtilis HY-001 can tolerate the high temperature of 50 ℃ and has obvious inhibition effect on various vibrios, so that the bacillus subtilis HY-001 has important significance in compost maturity and preventing and controlling vibrios in aquatic products.
Description
Technical Field
The invention relates to a high-temperature-resistant bacillus subtilis strain, and bacterial powder and application thereof.
Background
Bacillus subtilis is an aerobic gram-positive Bacillus, and active substances such as subtilin, polymyxin and the like generated in the growth process of the Bacillus subtilis can obviously inhibit pathogenic bacteria or pathogenic bacteria with endogenous infection. Bacillus subtilis can be metabolized to synthesize various enzymes such as alpha-amylase, protease, cellulase and the like, improve the digestion capability in the digestive tract, and decompose organic matters and toxic and harmful substances.
As can be seen from the current research reports, the growth temperature of the bacillus subtilis is generally 10-40 ℃, the high temperature resistance report is mainly the spore state formed by metabolic enzymes of the bacillus subtilis, and the report that the bacillus subtilis in the non-spore state can tolerate the temperature higher than 50 ℃ is basically absent. Therefore, the method has great significance in the application of compost maturity by screening the bacillus subtilis which can normally grow and metabolize at high temperature.
Since the use of bdellovibrio bacteriovorus microecological preparations is prohibited in 10 months of 2015, the agricultural department urgently needs safe and reliable substitutes for the control of pathogenic bacteria of the aquatic vibrios. The application of the bacillus subtilis in aquatic products is mainly reflected in the aspect of water quality regulation, and the application of the bacillus subtilis in bacteriostatic water body pathogenic bacteria is less, so that the screening of the bacillus subtilis capable of effectively inhibiting the aquatic product pathogenic bacteria has great significance.
Disclosure of Invention
The invention provides the bacillus subtilis HY-001 for overcoming the defects of the existing bacillus subtilis, and the bacillus subtilis HY-001 has the capabilities of tolerating high-temperature culture and inhibiting multiple strains of aquatic pathogenic bacteria and has wide application value in compost maturity and aquaculture.
The Bacillus subtilis HY-001 provided by the invention is Bacillus subtilis with a purchase number of GDMCC1.372 and is obtained by ultraviolet mutagenesis, high-temperature induction at 50 ℃ and a series of screening of high-yield cellulase and amylase, and is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO. M2022622 and a preservation date of 2022 years, 05 months and 13 days.
In a comparative test of the enzyme-producing capability of the bacillus subtilis HY-001, the enzyme activity of producing the cellulase and the amylase is higher than that of the bacillus subtilis GDMCC1.372, and the enzyme activity is obviously better than that of the bacillus subtilis GDMCC1.372 in temperature tolerance.
In the test for inhibiting aquatic pathogenic bacteria, the bacillus subtilis HY-001 has obvious inhibiting effect on aquatic pathogenic bacteria such as vibrio parahaemolyticus, vibrio alginolyticus, pseudomonas aeruginosa, aeromonas hydrophila and the like, and has higher inhibiting capability on aquatic pathogenic bacteria than bacillus subtilis GDMCC 1.372.
The preparation method of the bacillus subtilis powder comprises the following steps: activating a bacillus subtilis HY-001 shake flask, inoculating into a 10L seeding tank, filling 4L fermentation liquor, inoculating into a 200L fermentation tank according to 4% after fermentation is finished, and filling 100L fermentation liquor. Fermenting at 40 deg.C for 25h, stirring at 150r/min, and dissolving oxygen 80%. After fermentation, centrifugally collecting bacterial sludge by a disc centrifuge, and spraying by a centrifugal spray dryer to prepare bacterial powder, wherein the air inlet temperature is controlled to be 110 ℃ and the air outlet temperature is controlled to be 70 ℃.
Further, the fermentation liquor raw material comprises: 25g/L of glucose, 5g/L of yeast extract, 2g/L of compound amino acid powder, 2g/L of ammonium chloride, 0.1g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 3g/L of calcium carbonate and natural pH.
The bacillus subtilis HY-001 has the following advantages:
1. the bacillus subtilis has stable genetic property, can survive in the environment of 50-55 ℃ and carry out normal growth and metabolism, and is favorable for survival and playing the function in the high-temperature environment state, thereby expanding the application range of the bacillus subtilis.
2. Can effectively inhibit various aquatic pathogenic bacteria, so that the function is changed from general water regulating function to prevention and treatment of aquatic animal diseases, thereby partially replacing bdellovibrio bacteriovorus and antibiotic products.
3. Can produce a large amount of high-efficiency cellulase and amylase, and has obvious effect in compost maturity application.
Drawings
FIG. 1 is a growth curve of Bacillus subtilis HY-001 at different temperatures;
FIG. 2 is a high temperature resistance test coating plate for Bacillus subtilis HY-001 strain;
FIG. 3 is a high temperature resistance test coated plate of Bacillus subtilis GDMCC1.372 strain;
FIG. 4 is a photograph showing the inhibition zones of Bacillus subtilis HY-001 against E.coli and Vibrio parahaemolyticus;
FIG. 5 shows a plate coated with the strain before the test in the application of controlling vibrio in prawn culture with a dilution of 10 -1 ；
FIG. 6 is a plate coated with the strain at a dilution of 10 for 5 days in the application of the strain to control vibrio in prawn culture -1 。
Detailed Description
The following embodiments are provided for further description of the technical solution of the present invention, but the present invention is not limited to the scope of the embodiments, and all the modifications and equivalents thereof are within the scope of the present invention.
Example 1 UV mutagenesis and high temperature Induction culture
Taking the culture solution of the bacillus subtilis GDMCC1.372 cultured for 24h, and diluting the culture solution to 10 times by adopting the dilution method -3 Preparing into bacterial suspension. Taking a sterile plate 1 with a diameter of 9cm, adding the above 10 -3 2mL of the bacterial suspension was applied as a thin layer using a sterile coating rod. Irradiating for 3min under an ultraviolet lamp with a distance of 30cm and power of 15W, and preheating for 20min before irradiation. And (3) dipping the bacterial liquid by using a sterile coating rod, quickly coating the bacterial liquid on a beef extract peptone agar culture medium plate, and keeping dark light operation without turning on an incandescent lamp in the operation process. The evenly coated plate is wrapped by black cloth and cultured for 48h at 50 ℃. The bacillus subtilis colonies grown on the plate after the culture at 50 ℃ are all picked to 30mL of nutrient broth culture medium, bottled in a 100mL triangular flask, and cultured for 24h at 50 ℃ in a shaking way for later use.
The Bacillus subtilis HY-001 provided by the invention is Bacillus subtilis with a purchase number of GDMCC1.372 and is obtained by ultraviolet mutagenesis, high-temperature induction at 50 ℃ and a series of screening of high-yield cellulase and amylase, and is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO. M2022622 and a preservation date of 2022 years, 05 months and 13 days.
EXAMPLE 2 production of Amylase, screening of cellulase
1. Screening for producing amylase
Diluting the bacterial liquid after high-temperature induction culture at 50 ℃ by 10 times, and selecting 10 -4 、10 -5 、10 -6 0.1mL each was coated with an amylase medium, incubated at 50 ℃ for 48 hours, and the iodine solution was dropped onto the plate and observed to measure the size of the transparent circle. The strain producing the larger transparent circle is purified and separated for later use. Wherein the amylase screening culture medium comprises: 10g of soluble starch, 3g of beef extract, 10g of peptone, 5g of sodium chloride and 20g of agar.
2. Screening of cellulase production
The strains purified in the amylase screening test are activated and cultured for 18h, diluted by 10 times,choose 10 -4 、10 -5 、10 -6 Each 0.1mL of the cellulase screening medium was coated, incubated at 50 ℃ for 48 hours, and the size of the transparent circle was observed and measured. The strain producing the larger transparent circle is purified and separated for later use. Wherein the cellulase screening culture medium: 10g of sodium carboxymethylcellulose, 1g of yeast extract, 0.2g of Congo red, 4g of ammonium sulfate, 2g of monopotassium phosphate, 0.5g of magnesium sulfate, 0.5g of manganese sulfate, 20g of agar and pH 5.5.
Example 3 rescreening of selected strains
Streaking and purifying the strains obtained after a series of screening to obtain pure Bacillus subtilis HY-001, streaking the strains with nutrient agar, culturing at 50 deg.C and 55 deg.C for 24 hr, and observing the growth of aseptic colony. Adopting a fermentation culture medium to culture liquid for 24h, carrying out cellulase biopsy detection by using NY/T2321-2013, carrying out protease activity detection by using NY/T1847-2010, and comparing with the Bacillus subtilis GDMCC 1.372. Wherein the fermentation medium comprises 25g/L of glucose, 5g/L of yeast extract, 2g/L of compound amino acid powder, 2g/L of ammonium chloride, 0.1g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 3g/L of calcium carbonate and natural pH.
TABLE 12 comparative test results for Bacillus subtilis
As can be seen from the above table 1, Bacillus subtilis HY-001 can grow normally at 50 ℃ and 55 ℃, while Bacillus subtilis GDMCC1.372 cannot grow, and Bacillus subtilis HY-001 is better than Bacillus subtilis GDMCC1.372 in temperature resistance. It is also far better than Bacillus subtilis GDMCC1.372 in enzyme production ability.
EXAMPLE 4 Strain growth temperature detection
Inoculating the screened Bacillus subtilis HY-001 into 30mL fermentation medium, bottling in 100mL triangular bottle, culturing at 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, and 60 deg.C for 30h, sampling every 6h to detect OD600nm (light absorption value OD600nm is proportional to the concentration of light absorption substance in solution), and comparing with Bacillus subtilis GDMCC 1.372. Wherein the fermentation medium comprises 25g/L of glucose, 5g/L of yeast extract, 2g/L of compound amino acid powder, 2g/L of ammonium chloride, 0.1g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 3g/L of calcium carbonate and natural pH.
The optimum growth temperature of the bacillus subtilis HY-001 is 40 ℃, the bacillus subtilis HY-001 can slowly and normally grow at 50 ℃ and 55 ℃, and cannot grow at 60 ℃ and is in an apoptosis state. While the optimum growth temperature of the bacillus subtilis GDMCC1.372 is 35 ℃, the bacillus subtilis GDMCC cannot normally grow at the temperature higher than 50 ℃, and the activities of strains at other temperatures are shown in the table 2, the table 3 and the figure 1. Therefore, the bacillus subtilis HY-001 is better than the bacillus subtilis GDMCC1.372 in high-temperature environment resistance.
TABLE 2 growth of Bacillus subtilis HY-001 at different temperatures
TABLE 3 growth of Bacillus subtilis GDMCC1.372 at different temperatures
Example 5 genetic stability test
Taking bacillus subtilis HY-001 obtained by initial screening as the initial generation 1, performing streak nutrient agar plate subculture test every day, after the streak growth on the day 1 and day 2, streaking and inoculating the next plate again for 1 generation, continuously calculating the working time, testing for half a year, totaling about 150 times, and re-testing whether the strain can tolerate 50 ℃ culture growth and the cellulase and amylase activities are weakened or not in the last time, wherein the detection method is the same as that in example 3.
TABLE 4 genetic stability test results for Bacillus subtilis HY-001
| The temperature tolerance is 50 DEG C | The temperature tolerance is 55 DEG C | Cellulase U/mL | Amylase U/mL |
| + | + | 164±3.65 | 858±1.64 |
As can be seen from the above table 4, after the streaking subculture for 150 times, the strain can still normally grow at 50 ℃ and 55 ℃ after the retest, and the difference between the cellulase activity and the amylase activity is not great from the data of the strain rescreening example 3, which indicates that the strain characteristics are not changed after the streaking subculture for 150 times, and the inheritance of the strain is relatively stable.
EXAMPLE 6 Strain temperature resistance test
And (3) placing the culture solution after the strain activation in a water bath at the temperature of 60 ℃, 80 ℃ and 100 ℃ for heat preservation for 2min, 5min, 10min, 20min, 30min, 1h and 2h, then performing viable bacteria detection, sucking 0.1mL of a coated nutrient agar plate, taking the viable bacteria number after heat preservation for 30min at the temperature of 60 ℃ as the initial bacteria concentration, and comparing with the Bacillus subtilis GDMCC 1.372.
The test result data show that the bacillus subtilis GDMCC1.372 is completely inactivated after being treated at 80 ℃ for 1h and 100 ℃ for more than 5min, while the bacillus subtilis HY-001 has activity under the same treatment conditions, and the test results are shown in Table 5 and figures 2 and 3 in detail, which shows that the bacillus subtilis HY-001 has good high temperature resistance.
TABLE 5 comparison of the results of the high temperature resistance tests of the strains
Example 7 bacterial Strain inhibition of the aquatic pathogen indicator test
Adding 1mL of aquatic pathogen indicator bacterium suspension into nutrient agar culture medium prepared from 100mL of seawater at 50 deg.C, and shaking to even (final concentration of bacterium is 10) 5 CFU/mL), pouring the plate, placing the Oxford cup in the plate after cooling and solidification, respectively adding 0.1mL of fermentation supernatant obtained after fermentation and centrifugation of Bacillus subtilis HY-001 and Bacillus subtilis GDMCC1.372 into the Oxford cup, placing the mixture at 30 ℃ for culturing for 24h, and comparing with Bacillus subtilis GDMCC 1.372. And observing whether the bacteriostatic zone is generated or not, and measuring the diameter of the bacteriostatic zone by using a vernier caliper.
In the inhibition test of the strain fermentation liquid on the aquatic product pathogenic indicator bacteria, the result shows that the sensitivity of the fermentation liquid of the bacillus subtilis HY-001 to the tested pathogenic indicator bacteria is more than medium sensitivity, most of the sensitivity of the bacillus subtilis GDMCC1.372 is low sensitivity, and the specific inhibition results of related indicator bacteria are shown in Table 6 and figure 4, which indicates that the quality of related antibacterial substances generated by the fermentation of the bacillus subtilis HY-001 is higher than that of the bacillus subtilis GDMCC1.372, and the inhibition effect of the related antibacterial substances is better than that of the bacillus subtilis GDMCC 1.372.
TABLE 6 test results of inhibition of strains on pathogenic bacteria of aquatic products
Note: diameter of zone of inhibition (mm): insensitivity: 0, low sensitivity: <10, medium sensitivity: 10-14, high sensitivity: 15-20, extremely sensitive: >20
Example 8 use of the Strain in the control of Vibrio
In a certain prawn farm, 6 prawn culture ponds are selected, wherein ponds with consistent water depth, area, stocking amount and management mode are used as tests. Divided into 3 groups of 2 culture ponds. The bacterial powder of bacillus subtilis HY-001 and bacillus subtilis GDMCC1.372 in 2 groups of test groups respectively has a bacterial powder specification of 200 hundred million CFU/g, the usage amount is 10 g/mu, and no treatment is performed on a control group. The test time was 5 days, during which 6 pond management modes were consistent. And 5 days later, detecting and analyzing the total vibrio indexes in the 6 pond water qualities, and detecting the total vibrio by adopting TCBS culture medium flat plate dilution coating.
In the control group without using Bacillus subtilis, the total number of vibrios before and after the control group is not changed greatly, the total number of vibrios using Bacillus subtilis HY-001 is reduced by 98.8% on average, the total number of vibrios using Bacillus subtilis GDMCC1.372 is reduced by 59.3% on average, and the specific test results are shown in Table 7 and FIGS. 5 and 6. The bacillus subtilis has certain effect on preventing and treating vibrio, and the bacillus subtilis HY-001 has better effect than bacillus subtilis GDMCC 1.372.
TABLE 7 comparison of viable count of Vibrio bacteria before and after
Example 9 application of strains to compost composting of Chicken manure
The chicken manure main material and the auxiliary material rice bran are mixed according to the proportion of 3:1, the water content is controlled to be 50-60%, and the materials are held by hands to form a ball without water drops. Respectively mixing bacterial powder of bacillus subtilis HY-001 and bacillus subtilis GDMCC1.372 according to the use ratio of 500 g/ton material, wherein the specification of the bacterial powder is 200 hundred million CFU/g. Controlling 5 tons of piled materials per pile and 1 meter in height, starting to overturn once a day when the pile is heated to 60 ℃, decomposing for 30 days, sampling and detecting every 10 days, and recording related test data, wherein the fecal coliform number is detected according to GB/T19524.1, the ascarid egg death rate is detected according to GB/T19524.2, and simultaneously, no bacillus subtilis is added as a control group.
The test results show that the high-temperature-resistant bacillus subtilis HY-001 can rapidly increase the temperature to more than 50 ℃ in 4 days and last for 25 days, so that the faecal coliform and ascaris eggs are rapidly killed by the continuous high-temperature action, so that the odor disappears, while the control group and the bacillus subtilis GDMCC1.372 cannot increase the rotten temperature of chicken manure to more than 50 ℃, so that the rotten effect is poor or cannot achieve the rotten effect, and the specific relevant test item data are shown in Table 8. Test results show that the decomposing effect of the bacillus subtilis HY-001 is far better than that of the bacillus subtilis GDMCC 1.372.
TABLE 8 data of the test results of the strains in the maturation of chicken manure
While some embodiments of the present invention have been described above, the present invention is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
Claims (10)
1. The Bacillus subtilis strain is characterized by being Bacillus subtilis HY-001 and preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO. M2022622 and the preservation date of 2022 years, 05 months and 13 days.
2. The bacillus subtilis strain of claim 1, wherein the bacillus subtilis HY-001 is obtained by ultraviolet mutagenesis, high-temperature induction at 50 ℃ and screening of high-yield cellulase and amylase from Guangdong province culture Collection with the number GDMCC 1.372.
3. The bacillus subtilis strain of claim 2, wherein the uv mutagenesis: taking the culture solution of the bacillus subtilis GDMCC1.372 cultured for 24h, and diluting the culture solution to 10 times by adopting the dilution method -3 Preparing a bacterial suspension; taking a sterile plate 1 with a diameter of 9cm, adding the above 10 -3 2mL of the bacterial suspension is coated into a thin layer by using a sterile coating rod; irradiating for 3min under an ultraviolet lamp with a distance of 30cm and power of 15W, and preheating for 20min by turning on an ultraviolet lamp switch before irradiation; and (3) dipping the bacterial liquid by using a sterile coating rod, quickly coating the bacterial liquid on a beef extract peptone agar culture medium plate, and keeping dark light operation without turning on an incandescent lamp in the operation process.
4. A bacillus subtilis strain according to claim 2 wherein the induction at a high temperature of 50 ℃ is: wrapping the uniformly coated flat plate with black cloth, and culturing at 50 ℃ for 48 h; the bacillus subtilis colonies grown on the plate after the culture at 50 ℃ are all picked to 30mL of nutrient broth culture medium, bottled in a 100mL triangular flask, and cultured for 24h at 50 ℃ in a shaking way for later use.
5. The bacillus subtilis strain of claim 2, wherein the amyloidogenic enzyme is selected from the group consisting of: diluting the bacterial liquid after high-temperature induction culture at 50 ℃ by 10 times, and selecting 10 -4 、10 -5 、10 -6 Coating 0.1mL of amylase culture medium on each of the cells, culturing at 50 ℃ for 48h, dropwise adding iodine solution onto a flat plate, and observing and measuring the size of a transparent ring; purifying and separating the strains which generate larger transparent circles for later use; wherein the amylase screening culture medium comprises: 10g of soluble starch, 3g of beef extract, 10g of peptone, 5g of sodium chloride and 20g of agar.
6. The bacillus subtilis strain of claim 2, wherein the cellulase production screening comprises: activating and culturing the strain purified in the amylase screening test for 18h, diluting by 10 times, and selecting 10 -4 、10 -5 、10 -6 Coating 0.1mL of each cellulase screening culture medium, culturing at 50 ℃ for 48h, and observing and measuring the size of a transparent ring; purifying and separating the strains which generate larger transparent circles for later use; wherein the cellulase screening culture medium: 10g of sodium carboxymethylcellulose, 1g of yeast extract, 0.2g of Congo red, 4g of ammonium sulfate, 2g of monopotassium phosphate, 0.5g of magnesium sulfate and manganese sulfate0.5g agar 20g, pH 5.5.
7. A bacillus subtilis HY-001 shake flask as defined in claim 1 is activated, inoculated into a 10L seed tank, filled with 4L fermentation liquor, inoculated into a 200L fermentation tank at 4% after fermentation is finished, and filled with 100L fermentation liquor; fermenting at 40 deg.C for 25h under stirring at 150r/min with dissolved oxygen of 80%; after fermentation, centrifugally collecting bacterial sludge by a disc centrifuge, and spraying by a centrifugal spray dryer to prepare bacterial powder, wherein the air inlet temperature is controlled to be 110 ℃ and the air outlet temperature is controlled to be 70 ℃.
8. The bacillus subtilis powder of claim 7, wherein the fermentation broth raw material comprises: 25g/L of glucose, 5g/L of yeast extract, 2g/L of compound amino acid powder, 2g/L of ammonium chloride, 0.1g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 3g/L of calcium carbonate and natural pH.
9. The bacillus subtilis powder of claim 7, which is used for inhibiting vibrio parahaemolyticus, vibrio alginolyticus, pseudomonas aeruginosa and aeromonas hydrophila.
10. The bacillus subtilis strain of claim 1, which is used for inhibiting vibrio parahaemolyticus, vibrio alginolyticus, pseudomonas aeruginosa and aeromonas hydrophila.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834596A (en) * | 2014-03-10 | 2014-06-04 | 上海海洋大学 | Bacillus subtilis shou003, anti-vibrio protein and preparation method and applications of bacillus subtilis shou003 and anti-vibrio protein |
| CN104328066A (en) * | 2014-03-28 | 2015-02-04 | 杭州法莫西生物医药科技有限公司 | Bacillus subtilis H4, decomposed inoculant prepared therefrom and application of the decomposed inoculant |
| CN108865953A (en) * | 2018-07-25 | 2018-11-23 | 中国海洋大学 | One plant of wide spectrum inhibits bacillus and its composite bacteria preparation of aquatic products vibrio pathogen |
| CN108949616A (en) * | 2018-06-25 | 2018-12-07 | 青岛农业大学 | Yielding lipase and bacillus subtilis and the application method for inhibiting Vibrio splindidus |
-
2022
- 2022-07-21 CN CN202210872869.XA patent/CN115109730B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834596A (en) * | 2014-03-10 | 2014-06-04 | 上海海洋大学 | Bacillus subtilis shou003, anti-vibrio protein and preparation method and applications of bacillus subtilis shou003 and anti-vibrio protein |
| CN104328066A (en) * | 2014-03-28 | 2015-02-04 | 杭州法莫西生物医药科技有限公司 | Bacillus subtilis H4, decomposed inoculant prepared therefrom and application of the decomposed inoculant |
| CN108949616A (en) * | 2018-06-25 | 2018-12-07 | 青岛农业大学 | Yielding lipase and bacillus subtilis and the application method for inhibiting Vibrio splindidus |
| CN108865953A (en) * | 2018-07-25 | 2018-11-23 | 中国海洋大学 | One plant of wide spectrum inhibits bacillus and its composite bacteria preparation of aquatic products vibrio pathogen |
Non-Patent Citations (3)
| Title |
|---|
| GUO Z, PAN S, LIU T, ZHAO Q, WANG Y, GUO N, CHANG X, LIU T, DONG Y, YIN Y.: "Bacillus subtilis Inhibits Vibrio natriegens-Induced Corrosion via Biomineralization in Seawater.", 《FRONT MICROBIOL.》, pages 1111 * |
| 林松泉,庄若飞,刘兵 等: "1株凝结芽孢杆菌ZC-1生物学特性研究", 《饲料研究》, pages 70 - 74 * |
| 谢凤行;赵玉洁;周可;张峰峰;李亚玲;: "产胞外淀粉酶枯草芽孢杆菌的分离筛选及其紫外诱变育种", 华北农学报, no. 03, pages 78 - 82 * |
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