CN109652342A - A method of Bt liquid fermentation medium is prepared by raw material of vegetable castoff - Google Patents
A method of Bt liquid fermentation medium is prepared by raw material of vegetable castoff Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 37
- 230000004151 fermentation Effects 0.000 title claims abstract description 37
- 235000013311 vegetables Nutrition 0.000 title claims abstract description 28
- 239000007788 liquid Substances 0.000 title claims abstract description 17
- 239000002994 raw material Substances 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title claims abstract description 13
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims abstract description 20
- 239000002699 waste material Substances 0.000 claims abstract description 14
- 239000000843 powder Substances 0.000 claims abstract description 12
- 229920002472 Starch Polymers 0.000 claims abstract description 9
- 235000019698 starch Nutrition 0.000 claims abstract description 9
- 239000008107 starch Substances 0.000 claims abstract description 9
- 240000007124 Brassica oleracea Species 0.000 claims abstract description 8
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 claims abstract description 8
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 claims abstract description 8
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 claims abstract description 8
- 230000001954 sterilising effect Effects 0.000 claims abstract description 8
- 244000013123 dwarf bean Species 0.000 claims abstract 5
- 238000002360 preparation method Methods 0.000 claims abstract 3
- 238000005273 aeration Methods 0.000 claims abstract 2
- 238000005119 centrifugation Methods 0.000 claims description 10
- 244000068988 Glycine max Species 0.000 claims description 9
- 235000010469 Glycine max Nutrition 0.000 claims description 9
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 238000005457 optimization Methods 0.000 claims description 2
- -1 Al 3+ 0.4 g/L Substances 0.000 claims 1
- 235000021331 green beans Nutrition 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 28
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 15
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 14
- 239000000654 additive Substances 0.000 abstract description 14
- 229910052799 carbon Inorganic materials 0.000 abstract description 14
- 229910021645 metal ion Inorganic materials 0.000 abstract description 8
- 230000000749 insecticidal effect Effects 0.000 abstract description 7
- 238000012360 testing method Methods 0.000 abstract description 7
- 241000193388 Bacillus thuringiensis Species 0.000 abstract description 3
- 229940097012 bacillus thuringiensis Drugs 0.000 abstract description 3
- 230000004071 biological effect Effects 0.000 abstract description 3
- 238000002156 mixing Methods 0.000 abstract description 2
- 235000019764 Soybean Meal Nutrition 0.000 abstract 1
- 238000003556 assay Methods 0.000 abstract 1
- 239000004455 soybean meal Substances 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 description 27
- 239000002609 medium Substances 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 244000046052 Phaseolus vulgaris Species 0.000 description 9
- 230000000996 additive effect Effects 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 7
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 7
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- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 239000000575 pesticide Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 229910001448 ferrous ion Inorganic materials 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 230000003519 ventilatory effect Effects 0.000 description 4
- 230000001018 virulence Effects 0.000 description 4
- 239000002351 wastewater Substances 0.000 description 4
- 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 description 3
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 3
- 240000003768 Solanum lycopersicum Species 0.000 description 3
- 235000009337 Spinacia oleracea Nutrition 0.000 description 3
- 244000300264 Spinacia oleracea Species 0.000 description 3
- REDXJYDRNCIFBQ-UHFFFAOYSA-N aluminium(3+) Chemical compound [Al+3] REDXJYDRNCIFBQ-UHFFFAOYSA-N 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000012137 tryptone Substances 0.000 description 3
- 229920002261 Corn starch Polymers 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000013405 beer Nutrition 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 229940099112 cornstarch Drugs 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000002917 insecticide Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 241000255967 Helicoverpa zea Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 235000019647 acidic taste Nutrition 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 229940088623 biologically active substance Drugs 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 235000013877 carbamide Nutrition 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
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- 238000007796 conventional method Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000004299 exfoliation Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000007269 microbial metabolism Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000015192 vegetable juice Nutrition 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
本发明涉及一种以蔬菜废弃物为原料制备Bt液体发酵培养基的方法，制备方法包括选择蔬菜废弃物、灭菌、配制发酵培养基、优化发酵条件。本发明以四季豆汁液和白菜汁液的重量比为2∶1混合后，作为苏云金芽胞杆菌发酵培养基的原料，通过添加氮源和碳源、单因素试验筛选合适的碳源、氮源添加物和最佳的金属离子，并且利用正交试验确定添加物的最佳浓度组合，确定可溶性淀粉（6 g/L）、黄豆饼粉（6 g/L）、Al3+（0.4 g/L）、Fe2+（0.4 g/L）作为最适外源添加物。优化发酵条件为：pH（7.0）、温度（35℃）、通气量（80 mL/250 mL装量），同时，生物活性测定结果表明其制备的Bt菌株的杀虫效果良好。
The invention relates to a method for preparing a Bt liquid fermentation medium by using vegetable waste as a raw material. The preparation method includes selecting vegetable waste, sterilizing, preparing a fermentation medium and optimizing fermentation conditions. In the present invention, after mixing the green bean juice and the cabbage juice in a weight ratio of 2:1, as the raw material of the Bacillus thuringiensis fermentation medium, the suitable carbon source and nitrogen source additives are screened by adding nitrogen source and carbon source, and single factor test. and the best metal ions, and use the orthogonal test to determine the best concentration combination of additives, determine soluble starch (6 g/L), soybean meal powder (6 g/L), Al 3+ (0.4 g/L) , Fe 2+ (0.4 g/L) as the most suitable exogenous additives. The optimized fermentation conditions were: pH (7.0), temperature (35 °C), and aeration (80 mL/250 mL volume). At the same time, the results of biological activity assay showed that the prepared Bt strain had good insecticidal effect.
Description
Technical field
The present invention relates to a kind of methods for preparing Bt liquid fermentation medium, and in particular to one kind is original with vegetable castoff
The method for expecting to prepare Bt liquid fermentation medium, belongs to biological pesticide technical field.
Background technique
Bacillus thuringiensis (Bacillus thuringiensis, Bt) as now efficiently, dosage it is big, safety
It is higher costly due to production cost to cause its price for biological insecticides.By the fermentation raw material of selection low cost, and
Its culture medium is optimized by the method for biostatistics, fermentability is improved and reaches or approach former expensive culture medium
Effect.
Since Bt culture medium has an apparent influence to Strain Virulence, thus for the research of Bt fermentation medium always into
Row, while the limitation for being widely used and promoting by cost of Bt bacterial strain, to based on guaranteeing bacterial strain normal growth and have preferable
The research of the low-cost culture medium of insecticidal effect is also taken seriously.
Yang Jianzhou is matched using in the research of gourmet powder waste water production Bt biological pesticide with the supernatant of gourmet powder waste water after pretreatment
Culture medium processed adds cornstarch 3%, and peptone 1%, dregs of beans 3%, initial p H is 8.0 before sterilizing, in 30 DEG C of temperature, 200rpm
Shaking table culture.Wu Liyun is that raw material carries out the research of Bt liquid state fermentation with 1/2 beer raw wastewater+1/2 using beer by-products
Acidified waste water is solution, adds glucose 0.2%, (NH4)2SO4 0.1%、ZnSO40.05%, Tween-80 0.15%, NaCl
0.6%, N substance 0.4%, yeast juice 30% (V/V), KH2PO40.12%.Tang Lingyun utilizes hair hydrolysis liquid, biogas slurry, municipal sludge
Raw material of industry cost has been significantly reduced etc. the Dipel for being respectively synthesized a variety of culture medium productions and ensure that its desinsection is imitated
Fruit.Wang Chenghui produces Bt using industrial wastewater using drunk fish culture Bt, Zhou Xueyong, Guo Ailian etc., and orchid, which is encouraged, etc. passes through corn
Fermentation alcohol culture Bt.These researchs have absolutely proved the feasibility that Bt culture medium is prepared by waste utilization, while can fill
Divide the cost for reducing industrial production Bt biological pesticide, substantially increase the generality of Bt insecticide, while also having handled indirectly certainly
The waste generated in right environment, life and industrial production operation, effectively reduces the pollution to environment.But maintaining Bt
The production cost that Bt biological pesticide is further decreased while original bioactivity is still the target of market economy pursuit.
Summary of the invention
The purpose of the present invention is to provide a kind of method for preparing Bt liquid fermentation medium as raw material using vegetable castoff,
Production cost in Bt fermentation process can be reduced, used raw materials for production are the vegetable castoff of the market of farm produce.
To achieve the purpose of the present invention, it adopts the following technical scheme that.
A kind of method that Bt liquid fermentation medium is prepared as raw material using vegetable castoff of the invention, it is characterised in that system
It is standby that steps are as follows:
(1) select vegetable castoff: the vegetable castoff for collecting the market of farm produce carries out classification sorting, selects vegetable castoff Chinese cabbage
And kidney bean, it squeezes by juice extractor after cleaning respectively and filters to take to obtain sticky juice, be centrifuged, take supernatant, obtain four seasons fermented bean drink
Liquid and Chinese cabbage juice；
(2) it sterilizes: by the kidney bean juice and Chinese cabbage juice of step (1), 121 DEG C of sterilizings；
(3) prepare fermentation medium: kidney bean juice and Chinese cabbage juice after taking step (2) to sterilize are in mass ratio 2: 1 mixing
Afterwards, 6 g/L of soluble starch, soybean cake powder 6 g/L, Al are added3+ 0.4 g/L、Fe2+ 0.4 g/L;
(4) optimization of fermentation conditions: pH 7.0,35 DEG C of temperature, 80 mL/250 mL loading amount of ventilatory capacity.
Wherein, step (1) centrifugation, centrifugal intensity are 10000 turns/min, centrifugation time 10min.
Step (1) described centrifugation is repeated centrifugation 5 times.
Sterilization time described in step (2) is 20min.
Advantages of the present invention and usefulness:
1, the present invention selects suitable vegetable castoff alternative materials for the purpose of reducing Bt biological pesticide fermentation raw material cost,
Part nutriment required for its normal physiological is metabolized is provided.
2, in view of different carbon source nitrogen source and mineral matter element are to the weight of strain growth and synthesis of biologically active substance
The property wanted has carried out comprehensive analysis for the selection of additive.
3, the fermentation level of Bt and virulence are improved again by optimizing to culture medium, before guaranteeing its insecticidal effect
It puts, provides technical support for a large amount of cheap cultures of Bt bacterial strain.
Detailed description of the invention
Fig. 1 is schematic diagram of the different vegetable waste content to the Bt influence grown；
Fig. 2 is different carbon source and the influence schematic diagram that nitrogen source grows Bt；
Fig. 3 is the influence schematic diagram that different metal ions grow Bt；
Fig. 4 is that orthogonal test intuitively analyzes schematic diagram；
Fig. 5 is the influence schematic diagram that pH value grows Bt；
Fig. 6 is the influence schematic diagram that temperature and culture medium loading amount grow Bt.
Specific embodiment
In order to which the present invention is furture elucidated rather than the limitation present invention, it is illustrated with reference to embodiments.Following implementations
Experimental method described in example is unless otherwise specified conventional method.
Embodiment 1: ferment effect of the different vegetable waste to Bt
Different types of vegetable castoff (tomato, Chinese cabbage, kidney bean, cabbage and spinach) is collected in Foochow market of farm produce, is carried out
Classification sorting, squeezes by juice extractor after cleaning respectively and filters to take to obtain sticky juice, and is carried out 5 centrifugations, and centrifugation is strong
Degree is 10000 turns/min, centrifugation time 10min, obtains supernatant, and sample is carried out 121 DEG C of sterilizing 20min.Take standard bacteria
Strain Bt 8010, is inoculated in above-mentioned juice and LB liquid medium, in 30 DEG C, 200 rpm incubators by 2% volume ratio
Shaken cultivation, interval time point samples microscopy, until the content of bacillus exfoliation amount and parasporal crystal is 50%, obtains Bt hair
Zymotic fluid.A) show that kidney bean and Chinese cabbage can more preferably promote Bt strain growth relative to tomato, spinach and cabbage in Fig. 1, and
This it appears that the effect of cabbage and spinach is substantially suitable, Binding experiment phenomenon finds also while tomato less effective
Cause to generate more sediment, therefore subsequent experimental selection Chinese cabbage and kidney bean.By in Fig. 1 b) it is found that when kidney bean and white
When vegetable juice liquid proportional is 2: 1, Bt strain growth is preferable, and in order to enable waste source is wider, therefore this ratio is selected to prepare
Culture medium.
The screening of embodiment 2, different carbon source, nitrogen source, metal ion additive
4 kinds of carbon sources (glucose, sucrose, soluble starch, sweet dews are separately added into pretreated vegetable castoff culture medium
Alcohol) and 4 kinds of nitrogen sources (tryptone, corn flour, urea, soybean cake powder), concentration is 6 g/L, by its respectively with LB culture medium
Group is compareed.Fig. 2 the result shows that, for carbon source additive, mannitol can not be obviously promoted strain growth, excess-three species diversity
It is relatively small, while soluble starch is best for the facilitation effect of strain growth, select soluble starch i.e. cornstarch for
Carbon source additive；For nitrogen source additive, the facilitation of urea is smaller, may be inorganic nitrogen-sourced related, tryptone with it
Effect it is best, secondly be soybean cake powder, the two gap is smaller, plays the role of good promotion strain growth, it is contemplated that
The factor of culture medium cost selects soybean cake powder for nitrogen source additive.In addition, passing through the control group with utilization LB culture medium fermentation
It was found that, individually add carbon source or nitrogen source and be difficult to play quite or preferably facilitation effect, therefore, it is necessary to consider by
Carbon source and nitrogen source additive cooperate simultaneously, expand its effect collectively promoted to strain growth.
On the basis of adding carbon source and nitrogen source, 4 kinds are added into pretreated vegetable castoff culture medium respectively and commonly uses
Metal ion (Mg2+、Fe2+、Al3+、Na+), additive amount is 1.2 g/L, it is compareed with LB culture medium group respectively.
Fig. 3 the result shows that: the strain growth effect for adding the culture medium of ferrous ion is best, and fermentation level and LB culture medium are close, together
Shi Mingxian than remaining metal ion facilitation effect more preferably, secondly for aluminium ion therefore, select ferrous ion and aluminium addition be
Metal ion.
Embodiment 3, carbon source, nitrogen source, metal ion additive optimal combination (orthogonal test)
On the basis of initial medium (including additives), in order to improve fermentation level again, need to culture medium into
Row suboptimization again, using the horizontal orthogonal test of four factor three to carbon source (soluble starch), nitrogen source (soybean cake powder), metal from
Sub (ferrous ion, aluminium ion) is added in pretreated vegetable castoff culture medium according to different proportion, filters out its best group
It closes.
Table 1 is carbon source, nitrogen source, the concentration level design of metal ion, and it is orthogonal to design its using statistics software SPSS17
Test, such as table 2.The result shows that: variance analysis shows that the conspicuousness of soluble starch and soybean cake powder (value < 0.05 sig) is obvious,
Illustrate that the influence of soluble starch and soybean cake powder is more obvious.It is examined using Deng Kenshi, and intuitively analyzes (Fig. 4), as a result
Show that the optimal combination that four Factor Selections go out be A3B3C2D3(6 g/L of glucose, 6 g/L of soybean cake powder, ferrous ion are
0.4 g/L, aluminium ion are 0.4 g/L).Meanwhile the fermentation level of the optimal medium and LB culture medium filtered out is suitable, and
Compared with other 3 culture mediums selected at random, obviously play the role of promoting strain growth.
1 orthogonal test factor of table and level design
2 medium additives Orthogonal Experiment and Design result of table
The influence that embodiment 4, different fermentations condition grow Bt
It is right in microorganism itself by adjusting the initial pH value of culture medium since pH has a significant effect to microorganism growth metabolism
On the basis of environment a certain range pH is adjusted, the indirect influence that medium pH grows it is controlled.The culture medium of experiment is carried out
The setting of different initial pH determines its optimal initial pH by its upgrowth situation in order to more preferably promote the fermentation of microorganism raw
It is long.Fig. 5 shows that optimal initial pH range grows it for the living environment of 7.0 ~ 7.5 slant acidities and meta-alkalescence and plays inhibition
Effect, especially initial pH is lower than 6.5 and under the fermentation condition higher than 8.0.
Meanwhile the speed that grows to microbial metabolism of a certain range fermentation temperature and substance synthesis and energy production play
Decisive role, therefore to the control of fermentation temperature not only its adjustable growth metabolism speed, while its virulence can be produced
It is raw to influence.Too low or too high temperature does not only result in the low of fermentation level, while microorganism can be caused impaired or dead
It dies.When Fig. 6 shows 35 DEG C, rate of bacterial growth is most fast, and 25-35 DEG C of other opposite temperature have more apparent facilitation.
In addition, Bt bacterium is aerobic bacteria, the ventilatory capacity of culture medium can guarantee that its normal growth is metabolized, pass through control 250
The loading amount of bacterium solution in mL conical flask studies influence of the ventilatory capacity to strain growth.Fig. 6 shows that too low ventilatory capacity is raw to bacterial strain
With influence is inhibited, after loading amount is 80 mL/250 mL, due to there is sufficient oxygen, because strain growth is preferable.
Embodiment 5, the result of biological activity determination
Through biological activity determination (table 3), 8010 bacterial strain of Bt of vegetable castoff culture medium fermentation has a good insecticidal effect, and 48
The result shows that the bacterium solution killing rate that stoste and extension rate are 5 reaches 100% after hour, trained by the standard of comparative experiments room
Support base (10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of sodium chloride；PH=7.0) fermentation Bt Strain Virulence, two
Person's insecticidal effect is suitable, and the bacterium solution of vegetable castoff culture medium, which is diluted to after 20 times, still to be had preferable insecticidal effect (80%), and when dilute
It releases death rate when multiple reaches 50 times and reaches 56.7%.The μ L/mL of bacterium solution LC50=16.783 of standard LB culture medium, vegetables are discarded
The μ L/mL of bacterium solution LC50=17.420 of object shows that the insecticidal effect of the two is close.
Bioassay results of the table 3 to bollworm newly hatched larvae
Claims (4)
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| CN110923187A (en) * | 2019-10-31 | 2020-03-27 | 长江大学 | Agricultural waste product culture medium suitable for growth of piriformospora indica |
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