CN104789491A - Bacillus licheniformis strain and application thereof - Google Patents
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Abstract
The invention relates to a bacillus licheniformis strain and application thereof. The strain is a bacillus licheniformis strain BL14 (Bacillus licheniformis) which is preserved in CCMCC on 28th, February, 2014, wherein the preservation number is CCMCC NO. 8865. The application of the bacillus licheniformis strain comprises the following steps: collecting bean curd wastewater; adjusting the pH to 7.0; putting at 121 DEG C and autoclaving for 25 minutes; inoculating the bacillus licheniformis strain BL14 to the bean curd wastewater according to an inoculation quantity of 106cuf/mL; aerating; culturing for 3 days at 33 DEG C and fermenting the bean curd wastewater to a compound enzymatic liquid. The bacillus licheniformis can ferment and grow by taking the bean curd wastewater as a unique nutritional substance and produce an enzymatic liquid of endoglucanase and xylanase, so that a novel fermenting bacterial source is provided for producing endoglucanase and xylanase preparations.
Description
Technical field
The present invention is specifically related to a kind of lichem bacillus strain and application thereof.
Background technology
In order to make full use of resource, cost-saving, Mierocrystalline cellulose and high material such as rice bran, wheat time powder, cottonseed meal, straw powder, the herbage etc. of hemicellulose level are often used as the materials such as the high corn of raw material substitution price, soybean, wheat.Right a high proportion of robust fibre daily ration not only directly can not be absorbed by livestock and poultry, also easily causes the various problem such as livestock and poultry diarrhea, poor growth.For improving feed quality, feed factory and plant have been fully recognized that in feed and have added enzyme material, particularly the importance of non-digestive ferment that can not synthesize of the animal such as cellulase and hemicellulase self.
The main component of plant cell wall is made up of Mierocrystalline cellulose, hemicellulose and xylogen, Mierocrystalline cellulose and the mutual commissure of hemicellulose, after cellulase hydrolysis plant cellulose discharges free sugar, make Mierocrystalline cellulose and hemicellulose commissure body obtain loose, hemicellulose exposes and is resolved into the materials such as wood sugar rapidly by zytase.Because endoglucanase and zytase are one of main hydrolases of degraded cellulose and hemicellulose.Therefore by solubility small molecules class carbohydrate that the Mierocrystalline cellulose of high-content in feed, hemicellulose degradation Cheng Yi can be absorbed by animal by the dual hydrolytic action of endoglucanase and zytase, not only increase the palatability of feed, more improve the absorption rate of feed, greatly promote growing up healthy and sound of livestock and poultry.
For feed processing industry, add the demand that single zymin not only can not meet Mierocrystalline cellulose and hemicellulose in degrading plant cell walls simultaneously, and add the production cost that various zymin also considerably increases enterprise one by one, therefore be more and more subject to the favor of enterprise from all kinds of compound enzymic preparation of angle of effect and production cost, a large amount of experimentation on animalies also shows to add the utilising efficiency that compound enzymic preparation improves feed really greatly.The widely used excellent species selected at present mostly is the Mycophytas such as wood is mould, Penicillium, aspergillus niger, the production cycle used due to such bacterium production of cellulose enzyme is long, also a large amount of toxic substances is produced while producing enzyme, the thick enzyme that its metabolism produces can not directly add in feed, and therefore the removing such as such as membrane filtration toxic substance is requisite link in manufacture cellulase preparation with the technical program of purification cellulase substantially.Just not saying a large amount of losses appears in enzyme in this course, the use of this production sequence and the length of production cycle also greatly increase the production cost of enterprise.
Tofu wastewater is the bean curd draining that bean curd is formed in process of production, and its generation is 3-5 times of soybean dry weight.Bean curd water solid content accounts for 1%, primarily of compositions such as soluble protein, oligose, VITAMIN, lipid, trace elements.China is the big country of bean curd production and consumption, and it is produced a large amount of high concentrated organic wastewater of generation and enters the surface water, and fermentation causes the severe exacerbation of neighbouring water quality, serious threat environment for human survival in the environment.
Summary of the invention
One of the technical problem to be solved in the present invention, is to provide a kind of lichem bacillus strain.
The present invention is achieved in that a kind of lichem bacillus strain, described bacterial strain is lichem bacillus strain BL14 (Bacillus licheniformis), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on February 28th, 2014, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CCMCC NO.8865.
The technical problem to be solved in the present invention two, is the application providing a kind of described lichem bacillus strain.
The present invention is achieved in that a kind of application of described lichem bacillus strain, collect during bean curd makes the tofu wastewater discarded, adjust the pH to 7.0 of tofu wastewater, be placed in fermentor tank 121 DEG C of autoclaving 25min, by described lichem bacillus strain BL14 with 10
6the inoculum size of cfu/mL is inoculated in fermentor tank, aeration, and cultivate 3 days for 33 DEG C, namely tofu wastewater is fermented into the complex enzyme liquid containing endoglucanase and zytase.
Further, the measuring method of described endo-glucanase enzyme activity is as follows:
With the carboxymethylcellulose sodium solution 1.7ml that the phosphate buffered saline massfraction of 0.05mol/L pH 5.0 is l%, add complex enzyme liquid described in 0.5mL again, the phosphate buffered saline buffer of 0.3ml 0.05mol/L pH 5.5, after 50 DEG C of reaction 5min, add 1mL 3,5-edlefsen's reagent termination reaction, then boils 5min in boiling water bath, in 540nm colorimetric after dilution.
Further, the measuring method of described Xylanase activity is as follows: the xylan solution 1.0ml with the phosphate buffered saline massfraction of 0.05mol/L pH 5.0 being l%, add complex enzyme liquid described in 0.5mL again, the phosphate buffered saline buffer of 1.0ml 0.05mol/L pH 5.5, react 10min at 50 DEG C after, add 1mL 3,5-edlefsen's reagent termination reaction, then 5min is boiled in boiling water bath, in 540nm colorimetric after dilution.
The invention has the advantages that: lichem bacillus strain BL14 can be unique nutritive substance Fermentative growth with tofu wastewater, and produce the enzyme liquid of endoglucanase and zytase, thus provide new zymophyte source for the production of endoglucanase and xylanase preparation in the recycling of tofu wastewater and feed processing industry.
Accompanying drawing explanation
The present invention is further illustrated in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is the colonial morphology figure of lichem bacillus strain BL14 in the present invention.
Fig. 2 is the gramstaining figure of lichem bacillus strain BL14 in the present invention.
Fig. 3 is the JSM-6380LV scanning electron microscope thalli morphology observation figure of lichem bacillus strain BL14 in the present invention.
Fig. 4 is the pcr amplified fragment gel electrophoresis spectrum of lichem bacillus strain BL14 in the present invention.
Fig. 5 be in the present invention lichem bacillus strain BL14 based on the schematic diagram of the phylogenetic tree of 16S rRNA gene order.
Bacterial strain preservation
Bacterial strain in the present invention is lichem bacillus strain BL14 (Bacillus licheniformis), and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 28th, 2014, deposit number is CCMCC NO.8865.
Embodiment
A kind of lichem bacillus strain, described bacterial strain is lichem bacillus strain BL14 (Bacillus licheniformis), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 28th, 2014, deposit number is CCMCC NO.8865.
Described lichem bacillus strain can be applied in tofu wastewater and administer, also produce endoglucanase and zytase compound enzymic preparation simultaneously, concrete side is as follows: collect during bean curd makes the tofu wastewater discarded, adjust the pH to 7.0 of tofu wastewater, be placed in fermentor tank 121 DEG C of autoclaving 25min, by described lichem bacillus strain BL14 with 10
6the inoculum size of cfu/mL is inoculated in fermentor tank, aeration, and cultivate 3 days for 33 DEG C, namely tofu wastewater is fermented into the complex enzyme liquid containing endoglucanase and zytase.
The measuring method of described endo-glucanase enzyme activity is as follows:
With the carboxymethylcellulose sodium solution 1.7ml that the phosphate buffered saline massfraction of 0.05mol/L pH 5.0 is l%, add complex enzyme liquid described in 0.5mL again, the phosphate buffered saline buffer of 0.3ml 0.05mol/L pH 5.5, after 50 DEG C of reaction 5min, add 1mL 3,5-edlefsen's reagent termination reaction, then boils 5min in boiling water bath, in 540nm colorimetric after dilution.
The measuring method of described Xylanase activity is as follows: the xylan solution 1.0ml with the phosphate buffered saline massfraction of 0.05mol/L pH 5.0 being l%, add complex enzyme liquid described in 0.5mL again, the phosphate buffered saline buffer of 1.0ml 0.05mol/L pH 5.5, react 10min at 50 DEG C after, add 1mL 3,5-edlefsen's reagent termination reaction, then boils 5min in boiling water bath, in 540nm colorimetric after dilution.
Lichem bacillus strain BL14 of the present invention (Bacillus licheniformis) is separated containing the straw soil that rots and obtains from periphery farmland, awns Dangshan, Nanping City in Fujian province.
1, the acclimation and screening of lichem bacillus strain BL14 is separated:
The soil containing the straw that rots is gathered from periphery farmland, awns Dangshan, Nanping City in Fujian province, at room temperature seasoning 7 days, claim 5g soil in the triangular flask that 195ml acclimating nutrient solution is housed, 160r/min condition lower 30 DEG C of constant temperature culture domestication 5d, get 1g domestication bacterium liquid and dilute 10 respectively
-5, 10
-6, 10
-7after power, coat in Congo red solid medium, choose and can grow in Congo red solid medium and the bacterium colony of transparent circle can be produced, dull and stereotyped setting-out purifying is cultivated, the bacterium of purifying is inoculated in isolation medium after fermentation culture, draws bacterium liquid 0.1ml and cultivate in the hole of the Congo red solid medium with punch tool process, the formational situation of vision slit limit transparent circle, choose transparent circle and form obvious bacterial classification, preserve and called after lichem bacillus strain BL14.
Wherein the formula of each substratum is as follows:
(1) acclimating nutrient solution: (NH
4)
2sO
42g/L, KH
2pO
43g/L, MgSO
47H
203g/L, CaCl
23g/L, MnSO
4h
2o 0.16g/L, ZnSO
47H
2o 0.14g/L, CoCL
20.2/L, rice straw powder 150g/L, okara powder: 5g/L.
(2) isolation medium: (NH
4)
2s0
41.5g/L, KH
2pO
42.0g/L, MgSO
40.3g/L, CaC1
20.3g/L, FeSO
40.1g/L, ZnSO
40.1g/L, CMC-Na 10g/L, beech wood glycan 10g/L, pH is adjusted to 7.4.
(3) Congo red solid medium: (NH
4)
2sO
42g/L, MgSO
47H
203g/L, KH
2pO
41g/L, NaCl 0.5g/L, CMC-Na 10g/L, xylan 10g/L, Congo red 0.2g/L, pH 7.0, agar 20g/L.
2, the qualification of bacterial classification
The morphologic observation of 2.1 bacterium: colony morphological observation (see Fig. 1) is carried out to lichem bacillus strain BL14, the gramstaining form (see Fig. 2) of electron microscope observation thalline, JSM-6380LV scanning electron microscopic observation thalli morphology (see Fig. 3).Lichem bacillus strain BL14, bacterium colony decomposite leaf shape, opaque, wide: 0.6-0.8 μm, long: 1.5-3 μm, raw, oval in spore, Gram-positive, lines up chain.
The qualification of 2.2 physiological and biochemical properties:
Lichem bacillus strain BL14 bacterial strain physiological and biochemical property is in table 1, as seen from table: VP, Starch Hydrolysis and hydrogen peroxide experiment display is positive, illustrate that this bacterial strain can produce pyruvic acid by decomposition glucose, the further decarboxylation of pyruvic acid forms acetyl methyl carbinol, and can produce amylase by Starch Hydrolysis is glucide; Can not gelatin be utilized, produce ammonia experiment, MR experiment is negative; Can fermentation utilize D-Fructose to produce acid, aerogenesis, fermentation utilizes sucrose, glucose to produce acid, not aerogenesis.
Table 1 physiological and biochemical property
Note: physiologic character: ("+" represents positive; "-" represents negative); Carbohydrate fermentation experimental result recorder: ("+,+" represent and produce acid, aerogenesis; "-,-" represent and do not produce acid, not aerogenesis; " w – " represents that the weak positive produces acid, not aerogenesis.)
The molecular biology identification of 2.3 lichem bacillus strain BL14
(1) extraction of bacterial genomes DNA: adopt the bacterial genomes DNA extraction kit of TIANGEN company to extract.
(2) pcr amplification of 16S rDNA sequence and order-checking: amplification 16SrDNA gene V6-V8 variable region, the primer is F968:5 '-AAC GCG AAG CTT AC-3 ' (SEQ ID NO:2); L1401:5 '-CGG TGT GTA CAA GAC CC-3 ' (SEQ ID NO:3).
PCR reaction system: 2*Taq PCR MasterMix:12.5ul, primer and DNA each 1.0ul, ddH
20:9.5ul.
Pcr amplification program: 94 DEG C of denaturation 5min, then 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C extend 1min, totally 35 circulations, and last 72 DEG C fully extend 10min, 4 DEG C of preservations.
(3) PCR primer detects and sequencing analysis: the PCR primer of getting 5ul, add EB 1.0% agarose in gel electrophoresis be separated inspection, amplification obtains object fragment and is about about 400bp (see Fig. 4), entrust Shanghai biotechnology company limited to complete order-checking the product containing target fragment, the actual nucleotide sequence obtained is 397bp (sequence is shown in SEQ ID NO:1).
(4) Phylogenetic Analysis: in NCBI data, Blast compare of analysis is carried out to each 16S rRNA sequence, the sequence homology obtaining sequence and Bacillus licheniformis is greater than 99%, the creation analysis that Maximum Parsimony in employing MEGA 4.1 carries out growing tree is same, the results are shown in Figure 5.
(5) acquisition of the bacterium number of logining: gene order submitted in ncbi database, carries out the application of the bacterium number of logining, and applies for that the accession number obtained is KJ627224.
In conjunction with morphologic observation and the bio-chemical characteristics of bacterial strain, and the homology analysis in phylogenetic tree, bacterial strain is defined as lichem bacillus strain BL14 (Bacillus licheniformis) bacterial classification.
3, the characteristic research of lichem bacillus strain BL14
Prepare bacterial classification mother liquor, this lichem bacillus strain of transfering loop picking 1 ring BL14 is in 100mL basic medium, and 150r/min, cultivates 24h, obtain bacterial classification mother liquor for 33 DEG C;
Get the above-mentioned bacterial classification mother liquor of 0.1mL respectively and be inoculated in several bottles containing in the triangular flask of 100mL tofu wastewater substratum, in 150r/min, cultivate 1d to 20d for 33 DEG C, take basic medium as control group, get this lichem bacillus strain BL14 fermented liquid at 1d, the 2d to 20d of fermentation respectively and measure the activity of endoglucanase and zytase respectively, the content situation of glucose and xylose in fermented liquid described in Simultaneously test.
Described basic medium (g/L) is: peptone 10, extractum carnis 5, sodium-chlor 5, PH:7.6.
Tofu wastewater substratum: soya bean and quality, than being 1:10, after making bean curd, being collected the tofu wastewater flowed out in last pressure bean curd program, adjusted pH 7.0, be placed in 121 DEG C of autoclaving 25min, be tofu wastewater substratum by traditional bean curd manufacture craft legal system.
(1) complex enzyme liquid preparation: get described fermented liquid in 4 DEG C at the different incubation time of cultivation 1d to 20d respectively, the centrifugal 15min of 5000r/min, gets supernatant liquor and be crude enzyme liquid.To boil the complex enzyme liquid of 15min for contrast.
(2) mensuration of endo-glucanase enzyme activity (CMCase): Xylo-Mucine (CMC-Na) the solution 1.7ml with the phosphate buffered saline massfraction of 0.05mol/L pH 5.0 being l%, add complex enzyme liquid described in 0.5mL again, the phosphate buffered saline buffer of 0.3ml 0.05mol/L pH 5.5, after 50 DEG C of reaction 5min, add 1mL DNS reagent termination reaction, then 5min is boiled in boiling water bath, in 540nm colorimetric after suitable proportion dilution.
(3) mensuration of Xylanase activity: the xylan solution 1.0ml with the phosphate buffered saline massfraction of 0.05mol/L pH 5.0 being l%, add complex enzyme liquid described in 0.5mL again, the phosphate buffered saline buffer of 1.0ml 0.05mol/L pH 5.5, react 10min at 50 DEG C after, add 1mL 3,5-edlefsen's reagent termination reaction, then boils 5min in boiling water bath, in 540nm colorimetric after suitably diluting.
Above enzyme activity all deducts the sugared content of fermented liquid and substrate, makes standardized solution with glucose/wood sugar, generates 1ug glucose/wood sugar as a Ge Meihuo unit (U) using every milliliter of enzyme liquid per minute.
(4) mensuration of glucose/Xylose Content in fermented liquid: get complex enzyme liquid 0.5ml (with the sterilized water of 0.5ml for contrast), add the phosphate buffered saline buffer of the 0.05mol/L pH 5.5 of 2ml, add in 1mL DNS reagent boiling water bath and boil 5min, in 540nm colorimetric after suitably diluting.
The change of glucose in 3.1 endoglucanase and fermented liquid
With base culture base lichem bacillus strain BL14, the maximum inscribe glucanase vigor of generation appears at the 3rd day of fermentation, and vigor is 64.73 ± 7.45 (U/ml), then declines rapidly, ferments to during 6d and can't detect enzyme activity.When taking tofu wastewater as substratum, maximum enzyme is lived and is appeared at the 3d of fermentation, vigor is 213.80 ± 9.74, improve 2.30 times compared to basic medium, fermentation to enzyme work during 4d be 112.63 ± 24.03 (U/ml), then start rapid decline (see table 2), illustrate that lichem bacillus strain BL14 can be not only that direct nutritive substance grows with tofu wastewater, and compared with basic medium, the inducing action of tofu wastewater Middle nutrition material makes it produce endoglucanase ability to reach obvious raising, the hold-time of endo-glucanase enzyme activity also extends to 10d from the 4d of basic medium, namely the endo-glucanase adopting tofu wastewater to cultivate the generation of this bacterium is more easily preserved.
Detect glucose content changing conditions in substratum simultaneously, as known from Table 2: in basic medium, in general trend, drop to 192.84 ± 7.68 (μ g/ml) from 263.836 ± 7.31 (μ g/ml) before fermentation, occur the situation that small size consumption declines.In tofu wastewater substratum, compared with before fermentation, rise to 3204.68 ± 39.61 at fermentation 1d glucose content by 1283.06 ± 31.54 (μ g/ml) before fermenting, illustrate that the macromolecular carbohydrate in tofu wastewater is resolved into glucose by a large amount of endoglucanase that this lichem bacillus strain BL14 is induced to secrete.Along with longer fermentation times, in fermented liquid, the content of glucose starts to decline gradually, when fermentation is to 20d, drops to 303.00 ± 9.15 (μ g/ml), illustrate that this lichem bacillus strain BL14 can utilize glucose to grow as the energy and nutritive substance.
The change of glucose in table 2 endo-glucanase enzyme activity and fermented liquid
Note: ND is not for detect
The change of wood sugar in 3.2 zytases and fermented liquid
As shown in Table 3, with basic medium and tofu wastewater culture medium culturing lichem bacillus strain BL14, produce the 3rd day that the highest xylanase activity force value all appears at fermentation, vigor is respectively 424.48 ± 9.24,487.13 ± 11.06 (U/ml), thereafter decline gradually, fermentation also all can detect enzyme activity to during 20d, is respectively 93.31 ± 10.76,199.540 ± 25.32 (U/ml).As shown in Table 3, the vigor of zytase that produces in tofu wastewater of lichem bacillus strain BL14 is all apparently higher than basic medium generally, and the highest enzyme is lived and improve 15%.
Detect Xylose Content changing conditions in substratum: in basic medium, the content of wood sugar presents straight line downward trend substantially simultaneously, drop to 1074.64 ± 94.70 (the μ g/ml) of fermentation 20d from 5519.64 ± 223.96 (μ g/ml) before fermentation gradually; And in tofu wastewater substratum, wood sugar slightly rises when fermenting 1d, 9330.02 ± 276.72 (μ g/ml) are risen to by 8169.84 ± 194.02 (μ g/ml) before fermenting, illustrate that the zytase that this lichem bacillus strain BL14 produces is degraded to the macromole hemicellulose class material in tofu wastewater, decline gradually afterwards, fermentation dropped to 2215.73 ± 105.95 (μ g/ml) by the 20th day, illustrated that this lichem bacillus strain BL14 has carried out metabolism to wood sugar.
The change of wood sugar in table 3 xylosidase vigor and fermented liquid
In sum, described lichem bacillus strain BL14 is after screening domestication, can take tofu wastewater as sole nutrition material, high yield endoglucanase and zytase simultaneously, the vigor of the two kinds of enzymes produced significantly improves compared to basic medium, illustrating that lichem bacillus strain BL14 is more suitable for growth in tofu wastewater is cultivated, is that the suitableeest fermentation time of substratum production endoglucanase and zytase is the 3d that ferments with tofu wastewater.
Claims (4)
1. a lichem bacillus strain, it is characterized in that: described bacterial strain is lichem bacillus strain BL14 (Bacillus licheniformis), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 28th, 2014, deposit number is CCMCC NO.8865.
2. the application of a lichem bacillus strain as claimed in claim 1, it is characterized in that: collect during bean curd makes the tofu wastewater discarded, adjust the pH to 7.0 of tofu wastewater, be placed in fermentor tank 121 DEG C of autoclaving 25min, by described lichem bacillus strain BL14 with 10
6the inoculum size of cfu/mL is inoculated in fermentor tank, aeration, and cultivate 3 days for 33 DEG C, namely tofu wastewater is fermented into the complex enzyme liquid containing endoglucanase and zytase.
3. the application of lichem bacillus strain as claimed in claim 2, is characterized in that: the measuring method of described endo-glucanase enzyme activity is as follows:
With the carboxymethylcellulose sodium solution 1.7ml that the phosphate buffered saline massfraction of 0.05mol/L pH 5.0 is l%, add complex enzyme liquid described in 0.5mL again, the phosphate buffered saline buffer of 0.3ml 0.05mol/L pH 5.5, after 50 DEG C of reaction 5min, add 1mL 3,5-edlefsen's reagent termination reaction, then boils 5min in boiling water bath, in 540nm colorimetric after dilution.
4. the application of lichem bacillus strain as claimed in claim 2, it is characterized in that: the measuring method of described Xylanase activity is as follows: the xylan solution 1.0ml with the phosphate buffered saline massfraction of 0.05mol/L pH 5.0 being l%, add complex enzyme liquid described in 0.5mL again, the phosphate buffered saline buffer of 1.0ml 0.05mol/LpH 5.5, react 10min at 50 DEG C after, add 1mL 3,5-edlefsen's reagent termination reaction, then 5min is boiled in boiling water bath, in 540nm colorimetric after dilution.
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Cited By (4)
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| CN106591264A (en) * | 2017-03-02 | 2017-04-26 | 福建省农业科学院农业工程技术研究所 | Endoglucanase promotion factor |
| CN107475233A (en) * | 2017-09-30 | 2017-12-15 | 南京工业大学 | Method for producing nattokinase by using bean curd yellow serofluid |
| CN108271785A (en) * | 2018-01-30 | 2018-07-13 | 南京工业大学 | Biopesticide produced by fermenting bean curd yellow water and preparation method thereof |
| CN114480180A (en) * | 2022-01-04 | 2022-05-13 | 山东蔚蓝生物科技有限公司 | Bacillus licheniformis for straw degradation and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591264A (en) * | 2017-03-02 | 2017-04-26 | 福建省农业科学院农业工程技术研究所 | Endoglucanase promotion factor |
| CN107475233A (en) * | 2017-09-30 | 2017-12-15 | 南京工业大学 | Method for producing nattokinase by using bean curd yellow serofluid |
| CN107475233B (en) * | 2017-09-30 | 2021-01-12 | 南京工业大学 | Method for producing nattokinase by using bean curd yellow serofluid |
| CN108271785A (en) * | 2018-01-30 | 2018-07-13 | 南京工业大学 | Biopesticide produced by fermenting bean curd yellow water and preparation method thereof |
| CN114480180A (en) * | 2022-01-04 | 2022-05-13 | 山东蔚蓝生物科技有限公司 | Bacillus licheniformis for straw degradation and application thereof |
| CN114480180B (en) * | 2022-01-04 | 2023-08-04 | 山东蔚蓝生物科技有限公司 | Bacillus licheniformis for straw degradation and application thereof |
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| CN104789491B (en) | 2018-03-23 |
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