CN102864091A - One-bacterium multiple-enzyme bacterial strain as well as screening method and application thereof - Google Patents
One-bacterium multiple-enzyme bacterial strain as well as screening method and application thereof Download PDFInfo
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Abstract
The invention relates to a one-bacterium multiple-enzyme bacterial strain as well as a screening method and an application thereof. The bacterial strain is bacillus subtilis (Bacillus subtilis 1.1111) and is collected in the China center for type culture collection with the collection number of CCTCC (China center for type culture collection) No: M2011286. The bacillus subtilis (Bacillus subtilis 1.1111) can be used for preparing the bacterial strains of nine enzymes, i.e. xylanase, protease, phytase, pectinase, lipase, sweet dew glucanase, glucoamylase and the like, and the yields of the protease, the sweet dew glucanase, amylase and the glucoamylase are very high. Meanwhile, the bacterial strain is proved to have strong endurance capacity on cholate, artificial gastric juice and artificial intestinal juice by simulating the internal cholate environment, the artificial gastric juice, artificial intestinal juice and the animal test, safety and growth simulation capability are shown to a tested animal, and a foundation is laid for effectively improving the enzyme production capability of the bacterial strain, simultaneously generating the xylanase, the protease, the phytase, the pectinase, the lipase, the sweet dew glucanase and the glucoamylase and realizing one-bacterium multiple-enzyme fermentation in the fermentation process. The mutual synergistic effect among various enzymes generated by the bacterial strain is strong, and the bacterial strain can be used as a feed additive to be applied to agricultural production for livestock, fowls, aquatic livestock and the like.
Description
Technical field
The present invention relates to an a kind of bacterium enzyme bacterial strain, also relate to simultaneously screening method and the application of this bacterial strain, belong to microbial technology field.
Background technology
Enzyme is a kind of biological catalyst; fodder enzyme preparation environment-friendly type " green " fodder additives efficient as a class, that have no side effect is widely used in animal produces; use digestibility and utilization ratio that fodder enzyme preparation can improve feed; improve the production performance of livestock and poultry and aquatic animal; can reduce the content of nitrogen and phosphorous in the movement again; protection water body and soil are avoided polluting, and very wide application prospect will be arranged.
Early stage zymin is directly extracted as raw material with animals and plants, but because the animal and plant growth cycle is long, is subjected to again the impact of the many factors such as geography, weather and season, is unsuitable for large-scale industrial production.At present people have turned to sight with the main source of microorganism as zymin, are used in suitability for industrialized production such as the microorganism preparation of amylase and proteolytic enzyme.Early stage compound enzymic preparation is to re-use after composite by single enzyme, and this kind mode easily causes production cost too high, and quality is unstable, uses inconvenient.
Summary of the invention
The object of the present invention is to provide an a kind of bacterium multienzyme bacterial strain.
Simultaneously, the present invention also aims to provide a kind of screening method of this bacterium multienzyme bacterial strain.
Further, the present invention also aims to provide a kind of application of this bacterium multienzyme bacterial strain.
To achieve these goals, technical scheme of the present invention has adopted an a kind of bacterium multienzyme bacterial strain, this bacterial strain is subtilis (Bacillus subtilis) 1.1111, is preserved in Chinese Typical Representative culture collection center, and its preserving number is CCTCC NO:M 2011286.
Use classical method for determining bacteria and molecular biology method 16S rDNA it is identified that it is subtilis (Bacillus subtilis) 1.1111, be deposited in the Chinese Typical Representative culture collection center of Hubei China province Wuhan City Wuhan University on August 11st, 2011, its preserving number is CCTCC NO:M 2011286.
This subtilis is to showing very strong tolerance to environment such as cholate, simulated gastric fluid, simulated intestinal fluids, the laboratory animal safety non-toxic is had no side effect, and can produce zytase, proteolytic enzyme, phytase, polygalacturonase, lipase, sweet dew dextranase, saccharifying enzyme plurality of enzymes, this is conducive to this bacterial strain as the further development and use of probiotic bacterium in fodder additives, for further character, the mutual relationship between each enzyme, the fermentation of realization one bacterium multienzyme of each enzyme of research are had laid a good foundation.
A described bacterium multienzyme bacterial strain can produce zytase, amylase, cellulase, proteolytic enzyme, phytase, polygalacturonase, lipase, sweet dew dextranase, saccharifying enzyme.
Technical scheme of the present invention has also adopted a kind of screening method of a bacterium multienzyme bacterial strain, may further comprise the steps: to produce zytase, amylase, cellulase, proteolytic enzyme, phytase, polygalacturonase, lipase, the sweet dew dextranase, saccharifying enzyme is the screening target, use respectively each enzyme selectivity substratum, have or not transparent circle or hydrolysis circle according to periphery of bacterial colonies, and transparent circle or the diameter (D) of hydrolysis circle and the ratio of colony diameter (d), determine whether that tentatively bacterium producing multi enzyme preparation and high yield enzyme are alive, finally separate from the laboratory, screen the bacterial strain that a bacterium produces multienzyme in the microorganism resource storehouse that screening is preserved.
Technical scheme of the present invention also relates to the application of a kind of this bacterium multienzyme bacterial strain aspect the preparation probiotic agent.
Inclined-plane and seed culture medium are in g/L: peptone 10.0, and extractum carnis 5, NaCl 5, and the inclined-plane adds 15-20, pH7.2 again; Fermention medium is in g/L: Semen Maydis powder 36-44, soybean cake powder 36-44, Na
2HPO
48, (NH
4) 2SO
44, NH
4Cl 0.75, CaCl
21.0, pH7.0; Culture condition: 10L fermentation cylinder for fermentation volume is 7L, inoculum size is cumulative volume 10%, temperature is 35-40 ℃, air flow is 3L/L.min, stirring velocity is 200-300r/min, fermentation time 30-50h, a large amount of subtilis viable bacterias is arranged in the fermented liquid, take proteolytic enzyme, sweet dew dextranase, amylase and saccharifying enzyme as main, contain simultaneously the lytic enzymes such as zytase, amylase, cellulase, phytase, polygalacturonase, lipase, the concentrated 30%-60% starch drying that adds of fermented liquid is namely made probiotic bacterium and compound enzymic preparation.
Simultaneously, technical scheme of the present invention also relates to the application of an a kind of bacterium multienzyme bacterial strain aspect the preparation feed.
The addition of compound enzymic preparation in feed is that feed per ton adds 100-200g.
Described compound enzymic preparation consists of proteolytic enzyme 5000-10000U/g, sweet dew dextranase 1000-3000U/g, saccharifying enzyme 700-3000U/g, zytase 7500-15000U/g, amylase 500-3000U/g, cellulase 200-700U/g, phytase 50-80U/g, polygalacturonase 100-200U/g, lipase 200-500U/g.
Prozyme of the present invention has different mechanism of action, and the interpolation in feed can reduce its anti-oxidant action, improves efficiency of feed utilization, improves animal production efficiency, gives full play to the production performance of animal.The present invention proves that by cholate environment, simulated gastric fluid, simulated intestinal fluid and animal experiment in the analogue body this bacterial strain has very strong tolerance to cholate, simulated gastric fluid, simulated intestinal fluid, experimental animal is shown security and short growing ability, this is the enzymatic productivity of this bacterial strain of Effective Raise, can produce simultaneously zytase, proteolytic enzyme, phytase, polygalacturonase, lipase, sweet dew dextranase, saccharifying enzyme etc. during the fermentation, realize that bacterium multienzyme fermentation lays a good foundation.Synergistic action effect is strong mutually between the plurality of enzymes that this bacterial strain produces, and can be used as fodder additives and is applied to the agriculture productions such as poultry, fowl, aquatic animal.
The screening of bacterium producing multi enzyme preparation
1. produce the screening of zytase bacterial strain
The bacterial strain thorn that has activated is planted on the zytase screening culture medium, at 37 ℃ of constant temperature culture 24h, whether have transparent circle to produce according to periphery of bacterial colonies, judge whether it produces zytase.Measure respectively transparent circle diameter and colony diameter, determine the height of its secretion xylanase activity according to both ratios size, and sentence it as more than or equal to 2 o'clock with ratio and to produce the high bacterial strain of xylanase activity.
2. the screening of bacteria produced proteinase strain
The bacterial strain thorn that has activated is planted on the casein agar substratum, in 37 ℃ of incubators, cultivate 24h, whether have casein hydrolysis circle to produce according to periphery of bacterial colonies, judge whether it produces proteolytic enzyme.Measure respectively the diameter of hydrolysis circle and bacterium colony with ruler, determine the height of its secretory protein enzymic activity according to both ratios sizes, and with ratio more than or equal to sentencing it as the high bacterial strain of protease production at 2 o'clock.
3. the screening of phytase-producing strain
The bacterial strain thorn that has activated is planted on phytase primary dcreening operation substratum, in 30 ℃ of incubators, cultivate 48h, whether produce transparent circle according to periphery of bacterial colonies and judge whether it produces phytase.Measure respectively diameter (D) and the colony diameter (d) of transparent circle with ruler, tentatively determine the height of its generation phytase activity according to both ratio sizes, and with ratio more than or equal to sentencing it as the high bacterial strain of phytase generating activity at 2 o'clock.
4. produce the screening of polygalacturonase bacterial strain
The bacterial strain thorn that has activated is planted on polygalacturonase primary dcreening operation substratum, in 37 ℃ of incubators, cultivate 36h, after the taking-up, after periphery of bacterial colonies drips several CTAB solution, leaves standstill 10min, observe transparent circle.Whether produce transparent circle according to periphery of bacterial colonies and judge whether it produces polygalacturonase.Measure respectively diameter (D) and the colony diameter (d) of transparent circle with ruler, tentatively determine the height of its generation pectinase activity according to both ratio sizes, and with ratio more than or equal to sentencing it as the high bacterial strain of product pectinase activity at 2 o'clock.
5. the screening of yielding lipase bacterial strain
The bacterial strain thorn that has activated is planted on lipase primary dcreening operation substratum, in 37 ℃ of incubators, cultivate 24h, whether produce the hydrolysis circle according to periphery of bacterial colonies and judge whether it produces lipase.Measure respectively diameter (D) and the colony diameter (d) of hydrolysis circle, tentatively determine the height of its generation lipase activity according to both ratio sizes, and with ratio more than or equal to sentencing it as the high bacterial strain of yielding lipase activity at 2 o'clock.
6. produce the screening of mannase bacterial strain
The bacterial strain thorn that has activated is planted on the mannase screening culture medium, behind 37 ℃ of cultivation 24h, whether produce transparent circle and size thereof with Congo red aqueous assay periphery of bacterial colonies, as the foundation of producing the mannase screening., and produce mannosans enzymic activity high bacterial strain with ratio more than or equal to sentencing it as at 2 o'clock.
7. produce the screening of saccharifying enzyme bacterial strain
On the thorn of the bacterial strain after the activation kind of Glycosylase screening culture medium, cultivate 36h for 37 ℃, adopt Gram's iodine solution dyeing, it produces saccharifying enzyme to have transparent circle to produce proof in periphery of bacterial colonies.Measure respectively transparent circle diameter and colony diameter, and produce diastatic activity high bacterial strain with ratio more than or equal to sentencing it as at 2 o'clock.
8. the screening of high yield amylase strain
On the thorn of the bacterial strain after the activation kind of Zulkovsky starch substratum plate, in 37 ℃ of incubators, cultivate 24h, adopt Gram's iodine solution dyeing, whether there is transparent circle to produce according to periphery of bacterial colonies and judges whether it produces amylase.Measure respectively transparent circle diameter and colony diameter, and with ratio more than or equal to sentencing it as the diastatic bacterial strain of high yield at 2 o'clock.9. the screening of High Cellulase Production bacterial strain
9. the screening of High Cellulase Production bacterial strain
On the thorn of the bacterial strain after the activation kind of cellulase screening culture medium, in 37 ℃ of incubators, cultivate 24h, adopt 1mg/ml congo red staining 1h, then use successively, the thorough flush away dye liquor of 1mol/LNaCl solution 1h, use again the 1mol/mlHCl fixation, whether form the dyeing circle according to periphery of bacterial colonies, judge whether genus bacillus produces cellulase.Measure respectively dyeing loop diameter and colony diameter, with ratio more than or equal to 2 sentence it as the higher cellulase bacterium producing multi enzyme preparation of specific activity.
Cost can be enhanced productivity, be saved to one bacterium multienzyme fermentation method greatly, and because each enzyme derives from same bacterial strain, its security and compound property seem and more have superiority.
The evaluation of bacterium producing multi enzyme preparation
Use the plain agar substratum cultural characteristic of bacterium producing multi enzyme preparation is studied, utilize gram staining method that its dyeing property is studied; Utilize the biochemical tests such as sugar fermentating test, IMVIC test, Starch Hydrolysis, gelatine liquefication, hydrogen sulfide, lysine decarboxylase test, arginine dihydrolase test, ornithine decarboxylase test that 3 its physio-biochemical characteristics are analyzed, and utilize molecular biology method 16S rDNA that bacterium producing multi enzyme preparation is identified.
The tolerance of bacterium producing multi enzyme preparation and animal experiment
By cholate environment, simulated gastric fluid, simulated intestinal fluid environment in the analogue body, measure respectively the tolerance of bacterium producing multi enzyme preparation in the simulated intestinal fluid environment of the simulated gastric fluid of different cholate content, different pH values, different pH values.And to select healthy mice be laboratory animal, and bacterial strain is made bacteria suspension, and (the bacteria suspension viable count is 10
9CFU/mL), establish simultaneously blank, gavage is 5 days continuously, observes the healthy state of small white mouse.
Description of drawings
Fig. 1 is that bacterium producing multi enzyme preparation 16S rDNA pcr amplification is identified collection of illustrative plates; Swimming lane M is DL2000Marker; Swimming lane 1,2 is bacterium producing multi enzyme preparation 16S rDNA pcr amplification product; Swimming lane 3 negative controls.
The phylogenetic tree of Fig. 2 bacterium producing multi enzyme preparation; ▲ be the purpose bacterial strain.
Embodiment
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail
Bacterium producing multi enzyme preparation screening embodiment
1, produces the screening of zytase bacterial strain
The bacterium producing multi enzyme preparation thorn of activation is planted on the zytase screening culture medium, in 37 ℃ of incubators, cultivate 24h, whether there is transparent circle to produce according to periphery of bacterial colonies, through observing, discovery has this bacterial strain to produce transparent circle, measure transparent circle diameter (D), colony diameter (d) and the transparent circle diameter of bacterial strain and the ratio (D/d) of colony diameter, test-results sees Table 1.
Table 1 zytase decomposer enzyme the selection result alive
2. the screening of bacteria produced proteinase strain
The bacterium producing multi enzyme preparation thorn of activation is planted on the casein agar substratum, in 37 ℃ of incubators, cultivate 24h, observe and find to have this bacterial strain strain to produce the casein hydrolysis circle.Measure hydrolytic circle (D), colony diameter (d) and the hydrolytic circle of bacterial strain and the ratio (D/d) of colony diameter, test-results sees Table 2.
Table 2 proteases for decomposing bacterium enzyme primary dcreening operation alive
3. phytase-producing strain the selection result
The bacterium producing multi enzyme preparation thorn that activates is planted on phytase primary dcreening operation substratum plate, and behind the cultivation 48h, observation discovery bacterial strain produces transparent circle in 30 ℃ of incubators, and its transparent circle diameter (D), colony diameter (d) and the ratio of the two see Table 3.
Table 3 output value acid enzyme bacterial strain screening result
As shown in Table 3, the D/d value of this bacterial strain is 2.125, can determine tentatively that the activity of these bacterial strain phytase generatings is higher.
4. produce polygalacturonase bacterial strain screening result
The bacterium producing multi enzyme preparation thorn of activation is planted on polygalacturonase primary dcreening operation substratum plate, behind the cultivation 36h, observe and find that this bacterial strain produces transparent circle in 37 ℃ of incubators, its transparent circle diameter (D), colony diameter (d) and the ratio of the two see Table 4.
Table 4 produces polygalacturonase bacterial strain screening result
5. yielding lipase bacterial strain screening result
On bacterium producing multi enzyme preparation thorn kind of the lipase primary dcreening operation substratum with activation, behind the cultivation 24h, observe and find that this bacterial strain produces hydrolysis and encloses in 37 ℃ of incubators, its transparent circle diameter (D), colony diameter (d) and the ratio of the two see Table 5.
Table 5 yielding lipase bacterial strain screening result
6. produce the screening of mannase bacterial strain
The bacterial strain thorn that has activated is planted on the mannase screening culture medium, behind 37 ℃ of cultivation 24h, fall the Congo red aqueous solution of one deck at flat board and find that periphery of bacterial colonies produces transparent circle, its transparent circle diameter (D), colony diameter (d) and the ratio of the two see Table 6.
Table 6 produces mannase bacterial strain screening result
7. produce the screening of saccharifying enzyme bacterial strain
On the thorn of the bacterial strain after the activation kind of Glycosylase screening culture medium, cultivate 36h for 37 ℃, adopt Gram's iodine solution dyeing, in periphery of bacterial colonies transparent circle is arranged, its transparent circle diameter (D), colony diameter (d) and the ratio of the two see Table 7.
Table 7 produces saccharifying enzyme bacterial strain screening result
8. produce amylase genus bacillus the selection result
On the thorn of the bacterial strain after the activation kind of Glycosylase screening culture medium, cultivate 36h for 37 ℃, adopt Gram's iodine solution dyeing, in periphery of bacterial colonies transparent circle is arranged, its transparent circle diameter (D), colony diameter (d) and the ratio of the two see Table 8.
Table 8 produces the amylase strain the selection result
9. High Cellulase Production genus bacillus the selection result
Bacterial strain thorn after the activation kind on cellulase screening screening culture medium, is cultivated 24h for 37 ℃, and dyed, decolouring and fixation have transparent circle in periphery of bacterial colonies, and its transparent circle diameter (D), colony diameter (d) and the ratio of the two see Table 9.
Table 9 cellulase decomposer enzyme is lived and is screened
The evaluation embodiment of bacterium producing multi enzyme preparation
1. the observation of bacterium producing multi enzyme preparation cultural characters
Bacterium producing multi enzyme preparation is inoculated on the plain agar substratum, cultivate 18～24h at 37 ℃, to its bacterium colony size, edge shape, colony colour, protuberance degree and whether the cultural characters such as dry observe, and utilize gram staining method that its dyeing property etc. is studied.Test-results sees Table 10.
Table 10 bacterium producing multi enzyme preparation cultural characters and dyeing property
2. the research of bacterium producing multi enzyme preparation physio-biochemical characteristics
With bacterium producing multi enzyme preparation carry out respectively V.P test, MR test, carbohydrate fermentation test,, catalase test, Starch Hydrolysis test, arginine dihydrolase test, lysine decarboxylase test, ornithine decarboxylase test, Citrate trianion utilization test, hydrogen sulfide production test, indole test, gelatin test etc.Its test-results sees Table 11.
Table 11 bacterium producing multi enzyme preparation physiological and biochemical test result
Annotate :+expression positive reaction ,-expression negative reaction.
3. the preliminary evaluation of bacterium producing multi enzyme preparation
Cultural characters, form, dyeing proterties and physiological and biochemical test result according to bacterium producing multi enzyme preparation, with reference to " uncle's Jie Shi Bacteria Identification handbook (the 8th edition) and " common bacteria system identification handbook belongs to kind of an evaluation to it, but this bacterium producing multi enzyme preparation of preliminary judgement is subtilis.
4. the molecular biology identification of bacterium producing multi enzyme preparation
Extract in the bacterium producing multi enzyme preparation with bacterial genomes DNA rapid extraction test kit and to carry out the 16SrDNA pcr amplification behind total DNA, electrophoresis result as shown in Figure 1, the PCR product is given birth to the worker by Shanghai and is carried out sequencing.By sequencer address as can be known, the size of this sequence is 1.501Kb.Sequence among sequence and the GenBank is compared, and set up phylogenetic analysis, the result as shown in Figure 2.
5. the final evaluation of bacterium producing multi enzyme preparation
According to classical method for determining bacteria, with reference to " the outstanding Bacteria Identification handbook of uncle is with " common bacteria system identification handbook in conjunction with the 16SrDNA identification systems relatively, can determine that this bacterium producing multi enzyme preparation is subtilis.
The tolerance of bacterium producing multi enzyme preparation and animal experiment embodiment
1. the bile tolerance of bacterium producing multi enzyme preparation test
With bacterium producing multi enzyme preparation be seeded in respectively contain 0.1%, 0.2%, 0.3% cholate LB solid medium cultivate cultivate 4～5 days after, observe the colony growth situation.The result shows that this bacterial strain still can grow.
2. the anti-simulated gastric fluid test of bacterium producing multi enzyme preparation
Bacterium producing multi enzyme preparation is made bacteria suspension, and the inoculum size by 1% is linked into respectively in the simulated gastric fluid of different pH2, pH3, pH4, measures its viable count at different time, and it the results are shown in Table 12.
Table 12 bacterium producing multi enzyme preparation is deposited viable count (mL after the gastric juice effect
-1)
Annotate: the data in the table are the logarithmic value of viable count.
3. the anti-simulated intestinal fluid test of bacterium producing multi enzyme preparation
It is 7.6 simulated intestinal fluid that bacterium producing multi enzyme preparation is linked into respectively the pH value by 1% inoculum size, measures viable count at different time, and it the results are shown in Table 13.
Table 13 bacterium producing multi enzyme preparation is deposited viable count (mL after the intestinal juice effect
-1)
Annotate: the data in the table are logarithmic value.
4. the animal experiment of bacterium producing multi enzyme preparation
Bacterium producing multi enzyme preparation is made bacteria suspension, and continuous irrigation was fed 5 days, result such as the table 14 observed afterwards at the 10th day.
Table 14 bacterium producing multi enzyme preparation animal test results
Claims (8)
1. a bacterium multienzyme bacterial strain is characterized in that, this bacterial strain is subtilis (Bacillus subtilis) 1.1111, is preserved in Chinese Typical Representative culture collection center, and its preserving number is CCTCC NO:M 2011286.
2. a bacterium multienzyme bacterial strain according to claim 1, it is characterized in that: can produce zytase, amylase, cellulase, proteolytic enzyme, phytase, polygalacturonase, lipase, sweet dew dextranase, saccharifying enzyme, wherein proteolytic enzyme, sweet dew dextranase, amylase and saccharifying enzyme are that the high yield enzyme is lived.
3. the screening method of a bacterium multienzyme bacterial strain, it is characterized in that: may further comprise the steps: to produce zytase, amylase, cellulase, proteolytic enzyme, phytase, polygalacturonase, lipase, the sweet dew dextranase, saccharifying enzyme is the screening target, use respectively each enzyme selectivity substratum, have or not transparent circle or hydrolysis circle according to periphery of bacterial colonies, and transparent circle or the diameter (D) of hydrolysis circle and the ratio of colony diameter (d), determine whether that tentatively bacterium producing multi enzyme preparation and high yield enzyme are alive, finally separate from the laboratory, screen the bacterial strain that a bacterium produces multienzyme in the microorganism resource storehouse that screening is preserved.
4. the application of a bacterium multienzyme bacterial strain aspect the preparation probiotic agent.
5. application according to claim 4 is characterized in that: inclined-plane and seed culture medium are in g/L: peptone 10.0, and extractum carnis 5, NaCl 5, and the inclined-plane adds 15-20, pH7.2 again; Fermention medium is in g/L: Semen Maydis powder 36-44, soybean cake powder 36-44, Na
2HPO
48, (NH
4) 2SO
44, NH
4Cl 0.75, CaCl
21.0, pH7.0; Culture condition: 10L fermentation cylinder for fermentation volume is 7L, inoculum size is cumulative volume 10%, temperature is 35-40 ℃, air flow is 3L/L.min, stirring velocity is 200-300r/min, fermentation time 30-50h, a large amount of subtilis viable bacterias is arranged in the fermented liquid, take proteolytic enzyme, sweet dew dextranase, amylase and saccharifying enzyme as main, contain simultaneously the lytic enzymes such as zytase, amylase, cellulase, phytase, polygalacturonase, lipase, the concentrated 30%-60% starch drying that adds of fermented liquid is namely made probiotic bacterium and compound enzymic preparation.
6. the application of a bacterium multienzyme bacterial strain aspect the preparation feed.
7. application according to claim 6 is characterized in that: the addition of compound enzymic preparation in feed is that feed per ton adds 100-200g.
8. application according to claim 6, it is characterized in that: described compound enzymic preparation consists of proteolytic enzyme 5000-10000U/g, sweet dew dextranase 1000-3000U/g, saccharifying enzyme 700-3000U/g, zytase 7500-15000U/g, amylase 500-3000U/g, cellulase 200-700U/g, phytase 50-80U/g, polygalacturonase 100-200U/g, lipase 200-500U/g.
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| CN104328097A (en) * | 2014-07-17 | 2015-02-04 | 河南科技大学 | Method for producing cellulase, mannase and glucoamylase from bacillus subtilis |
| CN104762229A (en) * | 2015-03-24 | 2015-07-08 | 福建省农业科学院农业工程技术研究所 | A bacillus subtilis strain and applications thereof |
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