CN102533579A - Culture medium for producing spores, and optimizing process and application thereof - Google Patents
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Abstract
The invention provides a culture medium for producing spores, and the optimizing process and application thereof, which are particularly applied to marine bacillus which hardly generates spores in a common liquid medium. The culture medium comprises the following components in percentage by weight: 0.5 to 3 percent of cornmeal, 0.1 to 1 percent of bran, 0.1 to 0.5 percent of soybean cake meal, 0.01 to 0.1 percent of potassium dihydrogen phosphate dodecahydrate, 0.05 to 0.3 percent of calcium carbonate anhydrous and the balance of water. The culture medium has the advantages of readily available raw materials, low cost and simple preparation, and can obviously improve the spore formation rate and total active spores of marine bacillus 9912 and marine bacillus subtilis 3728. By the culture medium and the application of the culture medium, essential conditions are provided for developing bacillus licheniformis 9912 active spore preparations and other related active spore preparations.
Description
Technical field
The invention belongs to the microbial fermentation field; More specifically; Relate to the substratum and the application thereof of the active gemma of bacillus marinus high yield of Response Surface Method optimization, be meant a kind of substratum and application thereof that sporiferous ocean Bacillus licheniformis produces the high reactivity gemma that be used to be difficult for especially.
Background technology
Fungal disease has brought tremendous loss for farming, forestry, and control of crop disease mainly relies on and uses the phosphoramidite chemical agricultural chemicals at present.Yet use chemical pesticide to cause the resistance of disease and pest constantly to increase in a large number, the agricultural and animal products pesticide residue are high, seriously polluted, and food and water source environment receive severe contamination, and human health and ecotope receive serious threat.Therefore; Gradually reduce the use of chemical pesticide; Accelerating exploitation has the microbial pesticide product innovation novel, high-efficiency low-toxicity of independent intellectual property right, very necessary and urgent, is the great strategic issue that is related to the national health of China, Economic development, protection environment and development green agriculture.
The biological pesticide of microbial source is the main source of higher effective and lower toxic pesticide, also is the main direction of studying of world's biological pesticide exploitation.Yet with the exploitation constantly of landing field mikrobe, it is fewer and feweri that discovery can produce the Microbial resources of new antibiotic, accelerates the common recognition that the abundant Living marine resources of exploitation not only become the World Science man, more becomes great powers in the world's competition focal point.China has also started marine biotechnology field 863 Program at Eleventh Five-Year Plan for this reason, seizes the strategic high ground of Living marine resources science and technology, accelerates to utilize Living marine resources and biotechnology to develop the innovative product with China's independent intellectual property right.
Especially in recent years, China's facilities vegetable is flourish on the one hand, and many fungal diseases frequently take place, and silborne fungal diseases increases the weight of year by year, lacks the biological pesticide that can effectively prevent and treat fungal disease on the other hand again.In addition, corresponding resistance pathogenic strain system has appearred in the life-time service of conventional pesticide, and the resistance problem of pathogenic bacterium has day by day seriously influenced the control effect of sterilant.Therefore press for the novel biological agent that effectively to prevent and treat fungal disease.
From 1998; People such as the Hu Jiangchun of Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences; The separation of the yellow Bohai Sea obtains ocean Bacillus licheniformis (Bacillus licheniformis) 9912 from Liaoning in succession; The separation of mangrove forest Bruguiera conjugata rhizosphere obtains bacillus marinus (Bacillus subtilis) 3728 and from the gorgonian of the South Sea, is separated to strain excellents such as sea bacillus subtilis (Bacillus subtilis) 3512A from the South Sea; Confirmed that also bacillus marinus 9912,3728,3512 can produce very strong antibacterial substance, the continuous cropping obstacle that booth vegetable soil-borne disease and fungal disease are caused has good biological control effect.Wherein the ocean Bacillus licheniformis 9912; Develop jointly goods by Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences and Ai Nuo ltd of North China pharmacy group; Field test shows crop pest biological controls such as graw mold of tomato, gray mold of cucumber, grape grey mould, ring rot of apple, pear scab, mites of grape and cotton verticillium wilts remarkably productive, is expected to be developed into novel biological bactericide.Yet ocean Bacillus licheniformis 9912 is difficult for producing gemma under conventional culture condition; This has seriously hindered it and has researched and developed into efficient, nontoxic, low-cost, as to be prone to storage and transport, shelf-lives length active cultivated spore preparation; Therefore this area presses for and filters out sporiferous cultivation limiting factor; Its fermentation condition of one-step optimization of going forward side by side improves that to produce the gemma rate total with gemma.
Summary of the invention
The present invention is exactly to the problems referred to above, and a kind of substratum and optimization method and application of bacillus marinus gemma of Response Surface Method optimization are provided.
In order to realize above-mentioned purpose of the present invention, the present invention adopts following technical scheme:
The substratum of the bacillus marinus gemma that Response Surface Method is optimized, its composition and weight proportion are Semen Maydis powder 0.5～3%; Wheat bran 0.1～1%, soybean cake powder 0.1～0.5%, ten dihydrogen phosphate dihydrate potassium 0.01～0.1%; Carbon Dioxide calcium 0.05～0.3%, surplus are water.
Can add glucose and/or molasses in the described substratum, its whole weight is 0.5～1.5% of substratum gross weight.
Its composition and weight proportion are glucose 0.5～1.0%, Semen Maydis powder 1.5～2%; Wheat bran 0.3～0.5%, soybean cake powder 0.1～0.2%, ten dihydrogen phosphate dihydrate potassium 0.03～0.07%; Carbon Dioxide calcium 0.06～0.09%, surplus are water, the pH=6.5 of system.
Optimizing process: at first from the initial pH of state, inoculum size, culture temperature, dissolved oxygen amount, fermented liquid of inoculation thalline, add and to utilize the single-factor multilevel test to screen decision the factors such as divalent-metal ion, incubation time to produce the state that the gemma key factor is the inoculation thalline, and confirmed optimum inoculum size and incubation time.Secondly according to 2 times Plackett-Burman Design, filter out three principal elements that influence gemma rate and gemma sum in the medium component: Semen Maydis powder, wheat bran, CaCO
3(Response surface methodology RSM) optimizes Semen Maydis powder, wheat bran, CaCO after the steepest hill climbing test confirms to adopt Response Surface Method after the response surface method central horizontal
3The righttest cultivation concentration.
The concrete steps of utilizing the substratum of described bacillus marinus that bacillus marinus is cultivated do,
(1) the bacillus marinus bacterial strain of activation preservation at first; Single colony inoculation after the picking activation carries out slant culture on test tube or Kolle flask; Cultivate 48h～96h down for 28 ℃; Sterile sampling, its culture amount of cure of microscopic examination select central gemma to form homogeneous then, and the slant culture of two ends color tinted clear is as ferment-seeded;
(2) gemma of step (1) being selected forms homogeneous, and the culture aseptic inoculation of two ends color tinted clear is cultivated 12～18h down for 36 ℃ and reached logarithmic phase and thalline quantity is not less than 10 to thalline in the described substratum of claim 1
7CFU/mL can obtain fermentation seed liquid;
(3) fermentation seed liquid that step (2) is obtained is inoculated in the described substratum of claim 1 according to 8～10% volume ratio once more; In producing jar, carry out fermentation culture; Culture condition is tank pressure 0.05～0.06Mpa, air quantity 100: 10～100: 150vvm; Rotating speed 220～250rpm, culture temperature is 35 ℃～37 ℃; When fermented liquid gemma rate reaches 80～99%, effectively the gemma number reaches 10
9CFU/mL～10
10During CFU/mL, can stop fermentation, what obtain is the bacillus marinus after cultivating.
The inoculative proportion of seeded process described in the step (2) is that the culture that hooks up a transfering loop is inoculated in 250mL～500mL nutrient solution; 1 18mm*180mm test tube slant culture can be inoculated in 5L～10L fermentor tank; And can be amplified to 70L in this ratio.
Be suitable for the amplification step by step of the scale of fermenting.Both be suitable for of the amplification of 500mL shake-flask seed liquid to the fermentation of 10L jar; Be suitable for of the amplification of 10L jar seed fermentation liquid to the fermentation of 70L jar; Be suitable for of the amplification of 70L jar seed fermentation liquid, also be suitable for of the amplification of 500L jar seed fermentation liquid to the fermentation of 5000L jar to the fermentation of 500L jar.Beneficial effect of the present invention:
After utilizing substratum of the present invention to cultivate; Be difficult for its gemma rate of sporiferous bacillus marinus in the conventional liq substratum by original 1～20%; No matter be at shake-flask culture; Still 10L, 70L, its gemma rate of 500L fermentor cultivation have all surpassed 80～99%, and the gemma sum reaches 10
9More than CFU/mL reached, total active gemma number improves tens to tens of times than other substratum fermentations, and was significant for the development of novel sea genus bacillus sterilant.
Description of drawings
The gemma state graph that Fig. 1 generates after for bacillus marinus fermentation 18h;
Fig. 2 is the active gemma quantity of the every milliliter of fermented liquid in fermentation back.
Bacterial strain is preserved in Chinese typical culture collection center; The preservation address is a China. Wuhan. and Wuhan University; Postcode is 430072, and the preservation time is on December 9th, 2010, and deposit number is CCTCC NO:M2010343; Strain name is a Bacillus licheniformis 9912, and latin name is Bacillus licheniformus 9912.
Embodiment:
Embodiment 1
1, the activation of bacterial classification
With ocean Bacillus licheniformis 9912 (deposit number: CCTCC M2010343); Bacillus marinus 3728 (deposit number: CCTCC M207052, Hu Jiangchun, Xu Hongli, Liu Li etc., a kind of sea bacillus subtilis and screening method thereof; Number of patent application: 200710011179.0); Sea bacillus subtilis 3512A (deposit number: CCTCC M20705, Hu Jiangchun, Li Wei, Liu Li, Xue Delin, Wang Nan, Pan Huaqi, Wang Shujin, control cucumber fusarium axysporum sea bacillus subtilis preparation; Number of patent application: 200810011342.8) streak inoculation is cultivated 48h for 28 ± 1 ℃ in the modified starch nutrient agar respectively; Picking is rule the single bacterium colony of typical case once more and to be inoculated the inclined-plane behind the purifying after microscopic examination, and 28 ± 1 ℃ to cultivate 48h subsequent use.Wherein the colonies typical form of ocean Bacillus licheniformis on the modified starch nutrient agar is the edge irregularity, and surface drying purses up slightly, and bacterium colony is thicker; Mirror is observed form down: the shaft-like or column of cell, and size 0.6～0.8 * 1.5～3.0 μ m give birth to column or ellipse in the gemma, do not expand, and 1 flagellum, end are given birth to or side is given birth to.
The modified starch substratum consists of: Zulkovsky starch 2%, Carnis Bovis seu Bubali cream 0.5%, yeast extract paste 0.5%, NaCl0.5%, agar 1.8%.PH nature 115 ℃ of steam sterilizing 30min (or 121 ℃ of steam sterilizing 20min).
2, the preparation of seed liquor
Asepticly hook up one and encircle that the activatory gemma forms good bacillus marinus, the inoculation liquid fermentation medium is cultivated 12～18h for 36 ℃ and is not less than 10 to thalline logarithmic phase and thalline quantity
7CFU/mL is as fermentation seed liquid.Its seed culture medium can be inoculated in eggplant bottle modified starch substratum with activated spawn during pilot scale, then with sterilized water wash spore inoculating in 30～70L fermentor tank as seed liquor.Also can be through shaking the amplification step by step of bottle, at last with the seed fermentation liquid of canister as pilot scale.
The seeding tank liquid fermentation medium can increase glucose content relatively and reduce calcium carbonate content greatly with respect to the fermention medium that amplifies jar.
3, fermentor tank amplifies fermentation
Fermentation seed liquid is inoculated into an amplification jar liquid fermentation medium according to 10%.Wherein producing a jar fermentation culture conditions is: tank pressure 0.05Mpa, and air quantity 100: 150vvm, rotating speed 250rpm, culture temperature is 36 ℃.At last, when fermentation time is 18h when above, the growth conditions of sterile sampling microscopic cell, the formation of gemma, and living contaminants situation.When fermented liquid gemma rate reaches 80～99%, estimate effective gemma number and reach 10
9During CFU/mL, can stop fermentation.Gemma when Fig. 1 is ocean Bacillus licheniformis 10L jar fermentation 18h forms situation.As can be seen from the figure form the central gemma of homogeneous, thalline two ends uniform coloring, the brood cell leads and reaches 95%.
Liquid fermentation medium: Semen Maydis powder: 1.8%, wheat bran: 0.35%, soybean cake powder: 0.1%, ten dihydrogen phosphate dihydrate potassium: 0.05%, Carbon Dioxide calcium: 0.07%, all the other are water, pH 6.5.
4, the mensuration of fermentation activity gemma
With 80 ℃ of insulations of fermented liquid 10min, kill nourishing body, adopt continuous gradient dilution spread plate method counting again.The gemma sum that 500L fermentor tank different time is measured is as shown in Figure 2.Can find that from Fig. 2 active brood cell's number has reached 10 when fermenting 12h
8More than the CFU/mL, and in 15h, increase sharply, after this tend towards stability basically, reach 10 during 24h
9More than the CFU/mL
Embodiment 2 above-mentioned bacillus marinus high yield gemma Optimum of culture medium
1. single-factor multilevel test design
With industrial cheap substrate (Semen Maydis powder 1.0%, soybean cake powder 0.1%, wheat bran 0.3%, potassium primary phosphate 0.05%, pH nature) is basic medium; The initial pH of state, inoculum size, culture temperature, dissolved oxygen amount, fermented liquid through changing the inoculation thalline, add factors such as divalent-metal ion, incubation time, investigated various factors ocean Bacillus licheniformis 9912 gemma are formed and the total influence of gemma.Screening with the inoculation of gemma state is the key factor that the restriction gemma generates, gemma production rate less than 20% when inoculating with non-gemma state, and the gemma production rate surpasses more than 80% after inoculating with the gemma state.Preliminary definite 36 ℃ of 180rpm shake-flask culture 20h～36h can satisfy the needs of high yield gemma; Compare and optimize preceding 28 ℃ of 180rpm shake-flask culture shortening fermentation time, 10～15h; Optimum inoculation amount after optimizing during amplification culture is 8～10%; The dissolved oxygen increase can postpone the generation of gemma, and the initial pH that ferments is that gemma formation in 6～7 o'clock and gemma sum are higher.
2.Plackett-Burman?Design
Through the first time Plackett-Burman Design from Semen Maydis powder, soybean cake powder, wheat bran, KH
2PO
4, CaCO
3, MgSO
4, MnSO
4, FeSO
4, ZnSO
4, filter out three principal elements that influence gemma rate and gemma sum in the medium component in 11 factors such as the initial pH of fermented liquid, culture temperature: Semen Maydis powder, wheat bran, CaCO
3Influence gemma rate and two total principal elements of gemma with fermentation condition, confirm that in conjunction with aforesaid single-factor multilevel test design result optimum fermentation condition does, leavening temperature is 6.5 with the initial pH of fermented liquid for 36 ℃.And can find out from the effect value that gemma generates, in substratum, add Mn
2+And Zn
2+Ion is unfavorable for that the gemma of ocean Bacillus licheniformis generates, and adds Fe
2+And Mg
2+It is little that its gemma is formed influence.
What confirm fermention medium on this basis must be Semen Maydis powder, soybean cake powder, wheat bran, potassium primary phosphate, lime carbonate with necessary component; And they are carried out the Plackett-Burman Design second time, thereby calculate their generate influence to gemma effect value (as shown in table 1) more accurately.Can find out that the Semen Maydis powder and the wheat bran of carbon source can significantly improve the gemma sum the most, and lime carbonate influences also obviously to the generation of gemma.
Each factorial effect and the estimation of table 1Plackett-Burman Design
3, curved surface response experimental design
Response Surface Method (Response surface methodology; RSM) be a kind of statistics test design of optimizing bioprocess; This method is estimated the factor and the interaction thereof that influence bioprocess to set up the continuous variable surface model, and then definite optimum level scope.The inventor at first uses the steepest hill climbing to confirm its response surface method central horizontal before Response Surface Method, only in this way approaches as far as possible and could set up effective response surface fit equation after optimum produces the gemma zone.Interpretation of result is as shown in table 2, and losing the plan value is 0.456, and much larger than 0.05, R-Sq is 90.45%, explains that the predictor confidence level of corresponding surface is very high.In addition, three principal elements have stronger interaction.Wheat bran wherein as shown in table 3 and lime carbonate have significant interactions (p=0.035).Set up fit equation according to respective face method test-results
Y=135.7X
1+ 463.6X
2+ 1777.6X
3-36.7X
1 2-980.5X
2 2-19209.8X
3 2-11.7X
1X
2-58.3X
1X
3+ 3200X
2X
3+ 227.0 (X
1: Semen Maydis powder; X
2: wheat bran; X
3: lime carbonate) respectively to X
1, X
2, X
3Ask local derviation, get maximum value.Utilize the substratum after optimizing, through shaking bottle, 10L, 70L, the checking of 500L fermentor tank, its gemma rate surpasses 90%, and the gemma sum reaches 10
9CFU/mL (fitting error is 6.4%).
The estimation regression coefficient of table 2 response curved design
The variance analysis of table 3 response curved design
Comparative Examples 1
Use initial fermention medium, fermentation ocean Bacillus licheniformis 9912 bacillus marinuses such as grade.
The fermentation culture based component, according to the weight meter: Semen Maydis powder: 1.0%, wheat bran: 0.3%, soybean cake powder: 0.1%, ten dihydrogen phosphate dihydrate potassium: 0.05%, all the other are water, the pH nature.
Fermentation process: asepticly hook up one and encircle activatory bacillus marinus, the inoculation liquid fermentation medium, 28 ℃ cultivate 18h to the thalline logarithmic phase as fermentation seed liquid.Amplify fermentation according to 10% inoculum size.Wherein producing a jar fermentation culture conditions is: tank pressure 0.05Mpa, and air quantity 100: 150vvm, rotating speed 220rpm, culture temperature is 28 ℃.Fermentation 48h, sterile sampling detects, and the gemma rate is 20%, and active gemma test result is 3.0 * 10
7CFU/mL.
Comparative Examples 2
Use the fermention medium after Response Surface Method is optimized, fermentation ocean Bacillus licheniformis 9912 bacillus marinuses such as grade.
The fermentation culture based component, according to the weight meter: Semen Maydis powder: 1.8%, wheat bran: 0.35%, soybean cake powder: 0.1%, ten dihydrogen phosphate dihydrate potassium: 0.05%, Carbon Dioxide calcium: 0.07%, all the other are water, pH 6.5.
Fermentation process: asepticly hook up one and encircle that the activatory gemma forms good bacillus marinus, the inoculation liquid fermentation medium is cultivated 18h for 36 ℃ and is not less than 10 to thalline logarithmic phase and thalline quantity
7CFU/mL is as fermentation seed liquid.Fermentation seed liquid is inoculated into an amplification jar liquid fermentation medium according to 10%.Wherein producing a jar fermentation culture conditions is: tank pressure 0.05Mpa, and air quantity 100: 150vvm, rotating speed 250rpm, culture temperature is 36 ℃.Fermentation 24h, sterile sampling detects, and the gemma rate is 90～99%, and active gemma test result is 1.0 * 10
9CFU/mL.
Comparative Examples 3
Use and add glucose or molasses, seed liquor and fermentation different culture medium, fermentation ocean Bacillus licheniformis 9912 bacillus marinuses such as grade.
The seed liquor medium component, according to the weight meter: glucose 1.0%, Semen Maydis powder: 1.8%, wheat bran: 0.35%, soybean cake powder: 0.1%, ten dihydrogen phosphate dihydrate potassium: 0.05%, all the other are water, pH 6.5.
The fermentation culture based component, according to the weight meter: glucose 0.5%, Semen Maydis powder: 1.8%, wheat bran: 0.35%, soybean cake powder: 0.1%, ten dihydrogen phosphate dihydrate potassium: 0.05%, Carbon Dioxide calcium: 0.08%, all the other are water, pH 6.5.
Fermentation process: asepticly hook up one and encircle that the activatory gemma forms good bacillus marinus, the inoculation liquid fermentation medium is cultivated 16h for 36 ℃ and is not less than 5.0 * 10 to thalline logarithmic phase and thalline quantity
7CFU/mL is as fermentation seed liquid.Fermentation seed liquid is inoculated into an amplification jar liquid fermentation medium according to 10%.Wherein producing a jar fermentation culture conditions is: tank pressure 0.05Mpa, and air quantity 100: 150vvm, rotating speed 250rpm, culture temperature is 36 ℃.Fermentation 24h, sterile sampling detects, and the gemma rate is 80～99%, and active gemma test result is 4.2 * 10
9CFU/mL.
Claims (6)
1. one kind is produced the gemma substratum, it is characterized in that, its composition and weight proportion do, Semen Maydis powder 0.5～3%, and wheat bran 0.1～1%, soybean cake powder 0.1～0.5%, ten dihydrogen phosphate dihydrate potassium 0.01～0.1%, Carbon Dioxide calcium 0.05～0.3%, surplus is a water.
2. substratum according to claim 1 is characterized in that, can add glucose and/or molasses in the described substratum, and its whole weight is 0.5～1.5% of substratum gross weight.
3. substratum according to claim 2 is characterized in that its composition and weight proportion do; Glucose 0.5～1.0%, Semen Maydis powder 1.5～2%, wheat bran 0.3～0.5%; Soybean cake powder 0.1～0.2%, ten dihydrogen phosphate dihydrate potassium 0.03～0.07%, Carbon Dioxide calcium 0.06～0.09%; Surplus is a water, the pH=6.5 of system.
4. said Optimum of culture medium method of claim 1 is characterized in that:
At first from the initial pH of state, inoculum size, culture temperature, dissolved oxygen amount, fermented liquid of inoculation thalline, add and to utilize the single-factor multilevel test to screen decision the factors such as divalent-metal ion, incubation time to produce the state that the gemma key factor is the inoculation thalline, and confirmed optimum inoculum size and incubation time; Secondly according to 2 times Plackett-Burman Design, filter out three principal elements that influence gemma rate and gemma sum in the medium component: Semen Maydis powder, wheat bran, CaCO
3(Response surface methodology RSM) optimizes Semen Maydis powder, wheat bran, CaCO after the steepest hill climbing test confirms to adopt Response Surface Method after the response surface method central horizontal
3The righttest cultivation concentration.
5. the application of the described substratum of claim 1 is characterized in that: utilize the substratum of the described bacillus marinus of claim 1 that bacillus marinus is cultivated, concrete steps do,
(1) the bacillus marinus bacterial strain of activation preservation at first; Single colony inoculation after the picking activation carries out slant culture on test tube or Kolle flask; Cultivate 48h～96h down for 28 ℃; Sterile sampling, its culture amount of cure of microscopic examination then select slant culture that central gemma forms homogeneous, two ends color tinted clear as ferment-seeded;
(2) gemma of step (1) being selected forms homogeneous, and the slant culture aseptic inoculation of two ends color tinted clear is cultivated 12～18h down for 36 ℃ and reached logarithmic phase and thalline quantity is not less than 10 to thalline in the described substratum of claim 1
7CFU/mL can obtain fermentation seed liquid;
(3) fermentation seed liquid that step (2) is obtained is inoculated in the described substratum of claim 1 according to 8～10% volume ratio once more; In producing jar, carry out fermentation culture; Culture condition is tank pressure 0.05～0.06Mpa, air quantity 100:10～100:150vvm; Rotating speed 220～250rpm, culture temperature is 35 ℃～37 ℃; When fermented liquid gemma rate reaches 80～99%, effectively the gemma number reaches 10
9CFU/mL～10
10During CFU/mL, can stop fermentation, what obtain is the bacillus marinus after cultivating.
6. according to the application of the described substratum of claim 5, its characteristic also is:
The inoculative proportion of seeded process described in the step (2) is that the culture that hooks up a transfering loop is inoculated in 250mL～500mL nutrient solution; 1 18mm*180mm test tube slant culture can be inoculated in 5L～10L fermentor tank; And can be amplified to 70L in this ratio.
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| CN103131653A (en) * | 2013-02-21 | 2013-06-05 | 海南大学 | Bacillus culture media with cane molasses as main raw material |
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| CN104732084A (en) * | 2015-03-20 | 2015-06-24 | 上海交通大学 | Model construction method for reducing rapidly digestible starch content on basis of response surface method |
| CN104732084B (en) * | 2015-03-20 | 2017-07-11 | 上海交通大学 | The model building method of rapid digestion content of starch is reduced based on response phase method |
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| CN107760641A (en) * | 2017-12-11 | 2018-03-06 | 天津市农业生物技术研究中心 | A kind of bacillus subtilis microbial agent and its application |
| CN108641996B (en) * | 2018-06-19 | 2020-12-01 | 广东容大生物股份有限公司 | Fermentation medium of bacillus licheniformis and production method thereof |
| CN108641996A (en) * | 2018-06-19 | 2018-10-12 | 广东容大生物股份有限公司 | A kind of fermentation medium and its production method of bacillus licheniformis |
| CN110628676A (en) * | 2019-09-29 | 2019-12-31 | 郑燕鹏 | Strain monitoring method for preparing live bacteria preparation by fermentation expansion |
| CN113308382A (en) * | 2021-05-14 | 2021-08-27 | 海南大学 | Biocontrol bacterium preparation for enhancing stress resistance of crops |
| CN116536368A (en) * | 2023-06-26 | 2023-08-04 | 广东迪美新材料科技有限公司 | Bacillus marinus fermentation product, preparation method and application thereof |
| CN116536368B (en) * | 2023-06-26 | 2023-10-13 | 广东迪美新材料科技有限公司 | Bacillus marinus fermentation product, preparation method and application thereof |
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