CN105039230A - Biocontrol strain X1 fermentation medium and small-scale fermentation technology - Google Patents
Biocontrol strain X1 fermentation medium and small-scale fermentation technology Download PDFInfo
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- CN105039230A CN105039230A CN201510599801.9A CN201510599801A CN105039230A CN 105039230 A CN105039230 A CN 105039230A CN 201510599801 A CN201510599801 A CN 201510599801A CN 105039230 A CN105039230 A CN 105039230A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 53
- 230000004151 fermentation Effects 0.000 title claims abstract description 53
- 230000000443 biocontrol Effects 0.000 title claims abstract description 23
- 238000005516 engineering process Methods 0.000 title abstract description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims abstract description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 12
- 239000002609 medium Substances 0.000 claims abstract description 11
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 239000012138 yeast extract Substances 0.000 claims abstract description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 6
- 229920002472 Starch Polymers 0.000 claims abstract description 6
- 229910000019 calcium carbonate Inorganic materials 0.000 claims abstract description 6
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 6
- 229910000397 disodium phosphate Inorganic materials 0.000 claims abstract description 6
- 235000019800 disodium phosphate Nutrition 0.000 claims abstract description 6
- 239000008103 glucose Substances 0.000 claims abstract description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims abstract description 6
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 6
- 239000008107 starch Substances 0.000 claims abstract description 6
- 235000019698 starch Nutrition 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000013019 agitation Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 17
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 5
- 229940045641 monobasic sodium phosphate Drugs 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 3
- 238000011218 seed culture Methods 0.000 claims description 3
- 238000012546 transfer Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 abstract description 8
- 238000009776 industrial production Methods 0.000 abstract description 3
- 238000012258 culturing Methods 0.000 abstract description 2
- 230000003213 activating effect Effects 0.000 abstract 1
- 239000012153 distilled water Substances 0.000 abstract 1
- 229940099596 manganese sulfate Drugs 0.000 abstract 1
- 239000011702 manganese sulphate Substances 0.000 abstract 1
- 235000007079 manganese sulphate Nutrition 0.000 abstract 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 abstract 1
- 235000019799 monosodium phosphate Nutrition 0.000 abstract 1
- 238000004659 sterilization and disinfection Methods 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 13
- 238000011160 research Methods 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 7
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 6
- 238000011020 pilot scale process Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241001052560 Thallis Species 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 241000726221 Gemma Species 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 238000013461 design Methods 0.000 description 2
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- 238000005070 sampling Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
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- 239000000126 substance Substances 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical group [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
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- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
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- 239000002826 coolant Substances 0.000 description 1
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- 238000001228 spectrum Methods 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a biocontrol strain X1 fermentation medium and a small-scale fermentation technology. The fermentation medium comprises 1.0-1.5% of glucose, 0.5-0.8% of yeast extracts, 0.1-0.3% of starch, 0.2-0.4% of sodium hydrogen phosphate, 0.10-0.20% of sodium dihydrogen phosphate, 0.04-0.06% of magnesium sulfate, 0.05-0.15% of calcium carbonate, and 0.05-0.15% of manganese sulfate. The fermentation medium is obtained by adding 1000 mL distilled water, adjusting the pH value to be 7.5-8.0 and conducting sterilization for 25 min at the temperature of 115 DEG C. The small-scale fermentation technology comprises the steps of 1, activating biocontrol strains X1 stored in a refrigerator which is -70 DEG C with 4-6 mL NYD culture media, transferring the biocontrol strains X1 to a 500 mL triangular flask containing 80-120 mL seed media, and culturing the biocontrol strains X1 for 16-18 h at the temperature of 20-40 DEG C at the speed of 100-200 r/min; 2, inoculating seed fermentation liquor into a 100 L fermentation tank containing 60-80 L culture media for air agitation culture. The biocontrol strain X1 fermentation medium and the small-scale fermentation technology can provide experimental basis for industrial production of the biocontrol strains X1.
Description
Technical field
The invention belongs to microbial technology field, relate to the fermention medium of a kind of subtilis X1 and utilize this substratum to carry out the technique of fermenting.
Background technology
Antagonistic ability is strong and the excellent species of has a broad antifungal spectrum is the key that the biological control research of herbal medicine soil-borne disease and microbiobacterial agent are developed, and suitable production method and culture condition are then the guarantees playing excellent species performance.After screening obtains high-efficiency broad spectrum biocontrol strain, for finally reaching industry development object, needing to carry out liquid submerged fermentation research to object bacterial strain, comprising the determination of the Screening of Media of applicable suitability for industrialized production, the optimization of liquid fermentation condition and scale up test technique.
There is very big-difference to microbial growth in shake flask fermentation and ferment tank, the experiment condition of shake flask fermentation is directly amplified to production tank, and the growth of bacterial strain is often not consistent.In order to industrial needs can be adapted to, eliminate the difference of these two kinds of scale fermentation results, the experimental result of shake flask fermentation is effectively applied in the middle of large-scale production, just have to pass through the research of laboratory room small-sized ferment tank condition.When understanding the metabolic rule of bacterial strain in fermentor tank, after rationally setting up the zymotechnique of small-sized fermentation tank, just the fermentation research of pilot scale or the fermentation research of plant size can be carried out.
Interim test is after lab scale completes, and conceptual design basis is carried out, and the research test between lab setup and full scale plant, being the basis of engineering research, is also an important step of process development.After pilot scale, full scale plant can be made to obtain relatively large safety coefficient, find the phenomenon that laboratory work cannot be observed early, correct the error that may occur in design.Pilot scale work must be undertaken by industrialized condition, and its object is as follows with effect: (1) sets up the Industrial Simulation device of certain scale, carries out comprehensive simulated investigation to New Process, specifies operational condition and control method.(2) technology that realizes at industrial scale of results of laboratory and economic feasibility is investigated.(3) investigate the impact of industrial fact on technological process and equipment, find and solve contingent various problem under actual production conditions.
In general the growth velocity of microorganism in the reactor of different volumes is different, and reason may be, the degree of depth of tank causes the solubleness of oxygen, air residence time different with distribution, and shearing force is different, during sterilizing caused by nutritive ingredient destructiveness difference.The scale change of fermentor tank, it is the change that absolute value or relative value all can cause much physics and biological parameter, thus be difficult to the external environment in large and small scale process residing for thalline is consistent, usually will occur that fermentative production horizontal instability or fermentation production rate are too low when amplifying and producing, finally cause achievement in research extended stationary periods in the conceptual phase in laboratory, cannot large-scale production and application be carried out.Optimization culture in laboratory study or fermentation results to be transferred in plant-scale fermentation production process and go, guarantee scale can obtain successful effect equally after amplifying in large-scale industrial production, then need to carry out the research of more larger fermentation condition or the interim test of plant size, with the zymotechnique of checking in the laboratory study stage, the fermentative production of plant size then just can be amplified to.
Summary of the invention
The object of this invention is to provide a kind of biocontrol strain X1 fermention medium and small-sized fermentation technique, for the suitability for industrialized production of carrying out this bacterial strain provides experiment basis and foundation.
The object of the invention is to be achieved through the following technical solutions:
A kind of biocontrol strain X1 fermention medium, comprise following composition: glucose 1.0 ~ 1.5%, yeast extract paste 0.5 ~ 0.8%, starch 0.1 ~ 0.3%, Sodium phosphate dibasic 0.2 ~ 0.4%, SODIUM PHOSPHATE, MONOBASIC 0.10 ~ 0.20%, magnesium sulfate 0.04 ~ 0.06%, calcium carbonate 0.05 ~ 0.15%, manganous sulfate 0.05 ~ 0.15%, adding distil water to 1000 milliliter, regulates pH7.5-8.0,115 DEG C of sterilizings 25 minutes.
Utilize above-mentioned substratum to carry out a technique for small-sized fermentation to biocontrol strain X1, comprise the steps:
(1) biocontrol strain X1 in-70 DEG C of refrigerator-freezers is stored in the activation of 4 ~ 6mLNYD substratum, then transfer and be equipped with in the 500mL triangular flask of 80 ~ 120mL seed culture medium, in 20 ~ 40 DEG C, cultivate 16 ~ 18h under 100 ~ 200r/min condition, make seed fermentation liquid.
(2) seed fermentation liquid is inoculated into aeration-agitation in the 100L fermentor tank that 60 ~ 80L substratum is housed to cultivate, keeps leavening temperature 28 ~ 30 DEG C; Stirring velocity 100 ~ 120r/min; Air flow 0.8 ~ 1.0 (V/Vmin); Inoculum size 1.5 ~ 2.0%; Fermentation time 36 ~ 40h.
Tool of the present invention has the following advantages:
1, substratum is simple and easy to get and cost is low, all the components solubilized, and process post-natal period of making a living facilitates feasible;
2, the present invention is fermented to the production that biocontrol strain X1 carries out small-sized fermentation tank, determines bacterial strain automatic control zymotechnique, for large-scale industrial production provides technical basis.
Accompanying drawing explanation
Fig. 1 is strain X 1 growth curve (100L);
Fig. 2 is the pH value (100L) of different incubation time bacterium liquid.
Embodiment
Below in conjunction with accompanying drawing, technical scheme of the present invention is further described, but is not limited thereto,
Everyly technical solution of the present invention modified or equivalent to replace, and not departing from the spirit and scope of technical solution of the present invention, all should be encompassed in protection scope of the present invention.
The invention provides the small-sized fermentation technique of a kind of biocontrol strain X1, comprise following content:
1 materials and methods
1.1 bacterial strain
Biocontrol strain X1:CGMCCNO.4037.
1.2 substratum
Glucose 1.5%, yeast extract paste 0.5%, starch 0.1%, Sodium phosphate dibasic 0.3%, SODIUM PHOSPHATE, MONOBASIC 0.15%, magnesium sulfate 0.05%, calcium carbonate 0.01%, manganous sulfate 0.01%.Regulate PH7.5-8.0,115 DEG C of sterilizings 25 minutes.
The 100L automatic control tank liquid submerged fermentation research of 1.3 biocontrol strain X1
Adopt fermentor tank (Zhenjiang, Jiangsu MCGS-100L) to carry out fermentation culture, its operation is as follows:
(1) ready work wanted by the fermentor tank before cultivating, and guarantees power supply supply, checks water coolant water source, checks air source of the gas; Substratum is injected, the necessary submergence agitating vane of substratum in fermentor tank; Open the vent valve at discharge filter top;
(2) on temperature regulator, leavening temperature and sterilising temp is set;
(3) select " sterilizing " item in master mode, sterilizing starts;
(4) after sterilizing terminates, be cooled to after leavening temperature until temperature, above inoculation mouth, light the iron ring twining alcohol swab, open inoculation mouth, access cultured seed liquor; Select " fermentation " item in master mode, fermentation starts;
(5) fermented liquid is got in timing, detects bacteria concentration and sporulation situation, determines best incubation time.
Be stored in Bacillus subtilis strain in-70 DEG C of refrigerator-freezers with the activation of 5mLNYD substratum, then transfer and be equipped with in the 500mL triangular flask of 100mL seed culture medium, in 30 DEG C, under 150r/min condition, cultivate 18h, make seed fermentation liquid.By seed fermentation liquid by 1.5% inoculum size be inoculated into and be equipped with in (MCGS-100L) fermentor tank of 75L substratum, under the condition of 30 DEG C, keep oxygen-supply quantity 50% level, aeration-agitation is cultivated.During the fermentation, every the OD of 6h sampling and measuring fermented liquid
600value, draws strain growth curve.In addition every the pH value of 8h sampling and measuring fermented liquid.
2 results and analysis
In the pilot scale fermentation of biocontrol strain X1, in order to reduce production cost, improving spore forming rate, with the extractum carnis in yeast extract paste substitutive medium, adding MgSO simultaneously
4, CaCO
3with, MnSO
43 kinds of inorganic salts ingredients, with the culture condition optimized (culture temperature 30 DEG C, initial pH value 7.5, inoculation 16 ~ 18h in age) be primary condition, at 75L liquid amount, with rotating speed 180r/min under 1.5% inoculum size, air flow 1:1VVM carries out the automatic control fermentation of X1 bacterial strain 100L tank.
The mensuration of 2.1 strain growth curves
In liquid fermenting process, thalli morphology and bacterial concentration directly reflect the growing state of bacterial strain.Thalli morphology is by microscopic examination; The mensuration of bacterial concentration weighs the change of producing bacterium biomass in whole culturing process.General earlier fermentation bacteria concentration increases very fast, mid-term bacteria concentration substantially constant.Thalli growth lag phase is very short in the present invention, enters logarithmic phase afterwards, terminates through 24h logarithmic phase, the growth of thalline proceeds to the stage of stable development (see Fig. 1), thalline starts to form gemma at about 30h, and 40h reaches maximum value, and spore forming rate reaches more than 95%.
The mensuration of 2.2 fermented liquid pH value
Fermented liquid pH value is the concentrated expression of microbial metabolism, affects again the carrying out of metabolism, so be very important parameter.During the fermentation, along with the utilization of thalline to nutritive substance and the accumulation of meta-bolites, the pH of fermented liquid will inevitably change, by observing the whether normal of the passable hydrolysis and fermentation of pH value Changing Pattern.
The change of pH value is the concentrated expression of microbial metabolism situation---matrix metabolism, Product formation, cell state, nutritional status, oxygen supply situation.In X1 bacterial strain 100L automatic control tank fermenting process, nutrient solution initial pH value is adjusted to 7.5, and earlier fermentation is along with the Growth and reproduction of thalline, and the pH value of fermented liquid declines gradually, illustrates that thalline utilizes sugar to create acidic substance in a large number.When fermenting to 20h, the pH value of fermented liquid drops to 6.5, then starts progressively to go up, now gemma starts to be formed gradually, and when fermenting to 30h, the pH value of fermented liquid rises to 7.8, when continuing to lower tank, the pH value of fermented liquid remains on about 8.0 (see Fig. 2).
The mensuration of 2.3 fermented liquid living spores numbers
After the every a collection of 100L automatic control tank fermentation ends of X1 bacterial strain, get the living spores number in bacterium liquid mensuration fermented liquid.The living spores number of 5 Batch fermentation bacterium liquid is 14.1 ~ 16.3 hundred million/mL as a result, average out to 14.9 hundred million/mL (see table 1).
Table 1X1 bacterial strain 100L automatic control tank fermentation results
Claims (4)
1. a biocontrol strain X1 fermention medium, it is characterized in that described culture medium prescription is as follows: glucose 1.0 ~ 1.5%, yeast extract paste 0.5 ~ 0.8%, starch 0.1 ~ 0.3%, Sodium phosphate dibasic 0.2 ~ 0.4%, SODIUM PHOSPHATE, MONOBASIC 0.10 ~ 0.20%, magnesium sulfate 0.04 ~ 0.06%, calcium carbonate 0.05 ~ 0.15%, manganous sulfate 0.05 ~ 0.15%, adding distil water to 1000 milliliter.
2. biocontrol strain X1 fermention medium according to claim 1, is characterized in that described culture medium prescription is as follows: glucose 1.5%, yeast extract paste 0.5%, starch 0.1%, Sodium phosphate dibasic 0.3%, SODIUM PHOSPHATE, MONOBASIC 0.15%, magnesium sulfate 0.05%, calcium carbonate 0.01%, manganous sulfate 0.01%.
3. a biocontrol strain X1 small-sized fermentation technique, is characterized in that described processing step is as follows:
(1) biocontrol strain X1 in-70 DEG C of refrigerator-freezers is stored in the activation of 4 ~ 6mLNYD substratum, then transfer and be equipped with in the 500mL triangular flask of 80 ~ 120mL seed culture medium, in 20 ~ 40 DEG C, cultivate 16 ~ 18h under 100 ~ 200r/min condition, make seed fermentation liquid.
(2) seed fermentation liquid is inoculated into aeration-agitation in the 100L fermentor tank that 60 ~ 80L substratum is housed to cultivate, culture medium prescription is as follows: glucose 1.0 ~ 1.5%, yeast extract paste 0.5 ~ 0.8%, starch 0.1 ~ 0.3%, Sodium phosphate dibasic 0.2 ~ 0.4%, SODIUM PHOSPHATE, MONOBASIC 0.10 ~ 0.20%, magnesium sulfate 0.04 ~ 0.06%, calcium carbonate 0.05 ~ 0.15%, manganous sulfate 0.05 ~ 0.15%, adding distil water to 1000 milliliter, keeps leavening temperature 28 ~ 30 DEG C; Stirring velocity 100 ~ 120r/min; Air flow 0.8 ~ 1.0V/Vmin; Inoculum size 1.5 ~ 2.0%; Fermentation time 36 ~ 40h.
4. biocontrol strain X1 small-sized fermentation technique according to claim 3, is characterized in that described culture temperature 30 DEG C, liquid amount 75L, inoculum size 1.5%, rotating speed 180r/min, air flow 1.0V/Vmin.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834122A (en) * | 2015-12-03 | 2017-06-13 | 曾贵华 | Biocontrol bacterial strain X1 fermentation mediums |
| CN108239602A (en) * | 2017-12-04 | 2018-07-03 | 连云港巨森农业科技发展有限公司 | A kind of production technology of complex microbial inoculum |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974462A (en) * | 2010-10-22 | 2011-02-16 | 黑龙江省科学院微生物研究所 | Bacillus subtilis for preventing and controlling Chinese herbal medicine soil-borne diseases and preparation method of bactericide thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974462A (en) * | 2010-10-22 | 2011-02-16 | 黑龙江省科学院微生物研究所 | Bacillus subtilis for preventing and controlling Chinese herbal medicine soil-borne diseases and preparation method of bactericide thereof |
Non-Patent Citations (3)
| Title |
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| 李晶: ""黄瓜枯萎病高效拮抗枯草芽孢杆菌的筛选及生防机制研究"", 《中国博士学位论文全文数据库（电子期刊）农业科技辑》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834122A (en) * | 2015-12-03 | 2017-06-13 | 曾贵华 | Biocontrol bacterial strain X1 fermentation mediums |
| CN108239602A (en) * | 2017-12-04 | 2018-07-03 | 连云港巨森农业科技发展有限公司 | A kind of production technology of complex microbial inoculum |
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