CN104673727A - Bacillus subtilis Swl-19 and application thereof - Google Patents
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Abstract
The invention discloses bacillus subtilis Swl-19 and application thereof. The bacillus subtilis Swl-19 is separated from mesenteron of bombyx mori, the size of spore is (0.8-1.2)*(1.2-2.4) micron, the bacillus subtilis Swl-19 is elliptically columnar, the surface of the bacterial colony is dried, rough, non-transparent and milk white, the edge is neat, extracellular digestive enzymes such as alpha-amylase, protease, lipase, cellulase and the like can be generated, and the conversion ratio and health of domestic silkworm silk leaves can be further improved, so that the bacillus subtilis Swl-19 can be used as probiotics for silkworms and plays an important role on sericultural production.
Description
Technical field
The invention belongs to biological field, be specifically related to silkworm prebiotic subtilis (Bacillus subtilis) Swl-19 and application thereof.
Background technology
Probiotic bacterium (Probiotics) is the living microorganism that a class is useful to host, be colonizated in host intestine, reproductive system, energy produces definite health efficacy thus improves the active beneficial microorganism general name of host's microecological balance, performance beneficial effect, common probiotic bacterium genus bacillus, milk-acid bacteria, yeast, mould, photosynthetic bacterium etc.At present, probiotic bacterium has been widely used in each fields such as food, medicines and health protection and feed.Manage by U.S. food and safety of medicine management board (FDA) and U.S. feed the microorganism that can feed that meeting (AAFCO) approves and have 43 kinds, wherein genus bacillus has 5 kinds, is bacillus pumilus (Bacillus pumilus), slowly genus bacillus (Bacillus lentus), subtilis (Bacillus subtilis), Bacillus coagulans (Bacillus coagulans) and Bacillus licheniformis (Bacillus licheniformis) respectively.The feed microbe additive that the permission that the Ministry of Agriculture of China announces for 2006 uses has 16 kinds, and the genus bacillus of wherein adding has 2 kinds, is subtilis and Bacillus licheniformis respectively.Along with going deep into of research both at home and abroad, the prebiotic effect of more genus bacillus is proved, domestic and international report be used for genus bacillus in Production of Livestock and Poultry except above-mentioned several except, also have bacillus megaterium Bacillus megaterium, Bacillus circulans Bacillus circulans, Japan bacillus toyoi, bacillus firmus Bacillus firmus, Bacillus cereus Bacillus cereus, bacillus natto Bacillus natto.Silkworm is a kind of special economic animal, and raise and have certain singularity, its common probiotic bacterium is not also suitable for silkworm, and the bacillus therefore screened for silkworm rearing has potential prebiotic function, to promote sericultural production.
Summary of the invention
In view of this, an object of the present invention is to provide to be had promoter action to sericultural production and has probiotic bacterium, and this bacterial strain can as the bacterial strain producing proteolytic enzyme, amylase, lipase and cellulase, for sericultural production involved enzyme preparation band is helped; Two of object of the present invention is to provide subtilis Swl-19 in the application of preparation silkworm in probiotic bacterium; Three of object of the present invention is to provide subtilis Swl-19 improving the application in domestic silkworm silk leaf transformation efficiency.
For achieving the above object, after deliberation, the invention provides following technical scheme:
1, subtilis (Bacillus subtilis) Swl-19, by the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is CGMCC No.10333.Subtilis is Swl-19 individual cells 1.0 ~ 1.4 × 3.4 ~ 4.4 μm, color even, without pod membrane, peritrichous, to move and aerobic; Gemma size is 0.7 ~ 0.9 × 1.2 ~ 2.4 μm, elliptical cylinder; Coarse opaque, the oyster white of bacterium colony surface drying, edge becomes neat; Available protein, multiple sugar and starch, can decompose tryptophane and form indoles.Subtilis is extensively distributed in the organism of soil and corruption, easily breeds therefore in withered grass leaching juice and gains the name; Some subtilises are the important production bacterium of α-amylase and neutral protease; Some subtilises have the enzyme system of strong degraded Nucleotide, therefore Chang Zuowei nucleosides is produced the parental plant of bacterium or produced the bacterial classification of 5'-NT.And subtilis has following effect: the subtilyne 1) produced in subtilis thalli growth process, polymyxin, nystatin, linear gramicidins isoreactivity material, the conditioned pathogen of these active substances to pathogenic bacterium or autogenous infection has obvious restraining effect; 2) subtilis consumes rapidly the free oxygen in environment, causes enteron aisle hypoxemia, promotes that useful anerobe grows, and produces the organic acids such as lactic acid, reduces gut pH, indirectly suppresses the growth of other pathogenic bacterium; 3) the growing of stimulating animal immune organ, activates T, bone-marrow-derived lymphocyte, improves immunoglobulin (Ig) and antibody horizontal, strengthens cellular immunization and humoral immune function, improves herd immunity; 4) subtilis thalline self synthesizes the enzymes such as α-amylase, proteolytic enzyme, lipase, cellulase, together plays a role in digestive tube with the digestive enzymes in animal body.5. the multiple vitamin B group such as energy synthesise vitamins B1, B2, B6, nicotinic acid, improves the activity of animal body internal interference element and scavenger cell.
Subtilis not only in feed Application comparison extensive, applying also quite extensive in sewage disposal and bio-fertilizer fermentation or fermentation bed make, is a kind of multi-functional microorganism.Range of application: 1. city has stable political situation Industrial Waste Water Treatments, industrial circulation water treatment, the process such as septic tank, septic tank, livestock-raising animal waste, stink process, excrement and urine treating system, the process such as rubbish, manure pit, manure pit.2. herding, poultry, special animals and pet cultivation, aquaculture.3. can with multiple bacterial classification mixture, in agriculture production, there is vital role.
2, described subtilis Swl-19 is as the application in silkworm probiotic bacterium.
Preferably, described probiotic bacterium is the probiotic bacterium producing extracellular digestion enzyme.
Preferably, described extracellular digestion enzyme is that proteolytic enzyme, amylase, lipase are or/and cellulase.
3, described subtilis Swl-19 is improving the application in domestic silkworm silk leaf transformation efficiency.
Preferably, described silk leaf transformation efficiency be the rate of being placed on small straw bundles to spin cocoons, Cocooning rate, cocoon weight or Cocoon layer ratio.
Beneficial effect of the present invention is: the invention discloses subtilis Swl-19, this bacterial strain is the dominant microflora in bombyx mori intestinal, the digestive enzymeses such as α-amylase, proteolytic enzyme, lipase and cellulase can be produced, promoter action is played to the health of silkworm and Sang Si transformation efficiency; Apply it to during silkworm industry is raised and can improve silkworm surviving rate, promote the amount of weaving silk of silkworm after being placed on small straw bundles to spin cocoons, have great help to sericultural production.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:
Fig. 1 is the scanning electron microscope result of subtilis Swl-19.
Fig. 2 is subtilis Swl-1916S rDNA sequence PCR electrophorogram (M:NMW012; Swl-19:16SrDNA sequence pcr amplification result).
Fig. 3 is N-J method Phylogenetic Analysis (the Bacillus circulans: bacillus circulans based on 16S rDNA sequence; Bacillus firmus: bacillus firmus; Bacillus azotoformans: Bacillus azotoformans; Bacillus lentus: Bacillus lentus; Bacillus fastidiosus: Bacillus fastidiosus; Bacillus megaterium: Bacillus megatherium; Virgibacillus pantothenticus: pantothenic acid broadshoot bacillus; Bacillus globisporus: silicate minerals decomposing bacteria; Bacillus pasteurii: Bacillus pasteurii; Bacillus alcalophilus: bacillus alcalophilus; Bacillus pumilus: bacillus pumilus; Bacillus licheniformis: Bacillus licheniformis; Paenibacillus popilliae: oyster white malignant bacteria; Paenibacillus lentimorbus: pantothenic acid broadshoot bacillus; Bacillus amyloliquefaciens: bacillus amyloliquefaciens; Bacillus tequilensis: Te Jila genus bacillus; Bacillus subtilis strain: bacillus subtilis strain; Bacillus coagulans: Bacillus coagulans; Geobacillus stearothermophilus: Geobacillus stearothermophilus; Bacillus mycoides: bacillus mycoides; Bacillus anthracis: anthrax bacillus; Bacillus cereus: bacillus cereus; Bacillus thuringiensis: Tribactur).
Fig. 4 is the hemolytic test-results of bacterial strain Swl-19.
Fig. 5 is the hydrolysis circle (A: the hydrolysis circle that bacteria produced proteinase strain is formed in screening culture medium that bacterial strain Swl-19 is formed in screening culture medium; B: the hydrolysis circle that yielding lipase bacterial strain is formed in screening culture medium; C: the hydrolysis circle that cellulase producing strain is formed in screening culture medium; D produces the hydrolysis circle that amylase strain is formed in screening culture medium).
Fig. 6 is that bacterial strain Swl-19 produces extracellular digestion enzyme ability.
Fig. 7 is the DGGE collection of illustrative plates adding bombyx mori intestinal flora after food bacterial strain Swl-19.
Culture presevation
In the present invention, subtilis (Bacillus subtilis) Swl-19 is separated from Midgut of Silkworm, Bombyx Mori to obtain, send China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, deposit number is CGMCC No.10333, address is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Chinese science institute of microbiology, preservation date is on January 9th, 2015, and Classification And Nomenclature is subtilis Bacillus subtilis.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (third edition, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.
The culture medium prescription that the present invention uses is as follows:
NA solid medium: extractum carnis 5.0g; Peptone 10.0g; Sodium-chlor 5.0g; Agar 15.0g; Sterilized water 1000mL; The neutral 7.0-7.2 of pH.
NB solid medium: extractum carnis 5.0g; Peptone 10.0g; Sodium-chlor 5.0g; Sterilized water 1000mL; The neutral 7.0-7.2 of pH.
Embodiment 1, the silkworm selection systems of prebiotic subtilis (Bacillus subtilis) Swl-19
1, the silkworm screening of prebiotic subtilis (B.subtilis) Swl-19
Screen according to the bacterial strain screening scope that can be applicable to probiotic bacterium that National agricultural portion announces, finally genus bacillus is decided to be in conjunction with silkworm particular case, the concrete grammar of screening is: raise to 3 days 5 ages after being hastened the hatching of silkworms according to a conventional method by silkworm egg, get after hungry 24h 50 boss silkworms with 75% alcohol body surface sterilize, and calcination gently under spirit lamp.Dissect silkworm under aseptic condition, get 90 DEG C of heating 15min in the triangular flask that middle intestines and content (about 5g) join containing 45mL sterilized water.The solution drawn after heating makes gradient dilution, and extent of dilution is 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6with 10
-7, get 0.1mL respectively and be uniformly coated on NA culture medium flat plate, cultivate 48h for 37 DEG C.Select the discrepant bacterium colony of form to rule further separation and purification, until microscopy is for subsequent use in-20 DEG C of Refrigerator stores after single culture.Then the bacterial strain of separation is carried out scanning electron microscope, result as shown in Figure 1.Result shows, and the size of isolated strains is 1.0 ~ 1.4 × 3.4 ~ 4.4 μm, and gemma size is 0.8 ~ 1.2 × 1.2 ~ 2.4 μm, ovalize column, coarse opaque, the oyster white of bacterium colony surface drying, and edge becomes neat.
2, the silkworm qualification of prebiotic subtilis (B.subtilis) Swl-19
Morphology is carried out to being separated the bacterial strain obtained, Physiology and biochemistry qualification (concrete grammar is with reference to the book such as " common bacteria system identification handbook " and " Microbiology Experiment ") and molecular biology identification, the i.e. kind of this bacterial strain of 16S rDNA Sequence Identification, concrete grammar is as follows: by bacterial universal primers 27F and 1492R amplification 16S rrna sequence, the sequence of primer 2 7F is 5 '-agagtttgatcctggctcag-3 ', the sequence of primer 1492R is 5 '-ggctaccttgttacgactt-3 ', amplification condition is: (TaKaRa Taq is set with R001B to 50 μ L amplification systems, precious biotechnology (Dalian) company limited): 4 μ L DNA profilings, forward and each 1 μ L of reverse primer (10mol/L), 10 × PCR buffer (Mg
2+plus) 5 μ L, dNTP Mixture (each 2.5mM) 4 μ L, Taq DNA polymerase (5U/ μ L) 0.3 μ L, sterilizing ultrapure water complements to 50 μ L, amplification program is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 40s, 55 DEG C of annealing 40s, and 72 DEG C extend 1min, 35 circulations, and 72 DEG C extend 10min, 4 DEG C of preservations, amplified production is carried out agarose gel electrophoresis, and result as shown in Figure 2.
Then be connected to by amplified production on pMD19-T Vector, transform DH5 α competent cell, then picking positive colony checking order, result is as shown in SEQ ID NO.1.16S rDNA sequence order-checking obtained submits to NCBI (http://www.ncbi.nlm.nih.gov) to carry out BLAST comparison and to its homologous sequence MEGA 5.05 software N-J method phylogenetic tree construction.The 16S rDNA of BLAST comparison result display acquisition bacterial strain and Bacillus subtilis strain YQH 60 has the homology of 99%; As shown in Figure 3, it is a class that result shows that acquisition bacterial strain and subtilis Bacillus subtilis gather to phylogenetic tree, and this illustrates that the genetic distance of this bacterial strain and subtilis is nearer.In conjunction with morphological specificity, the physio-biochemical characteristics of bacterial strain, and with reference to " common bacteria system identification handbook " by its called after subtilis (B.subtilis) Swl-19, referred to as Swl-19.
Select 14 kinds of common antibiotics to adopt K-B method to carry out antibiotic drug sensitive detection to subtilis Swl-19, carry out Physiology and biochemistry detection, to determine its drug susceptibility and physio-biochemical characteristics (table 1) simultaneously.Antibiotic drug sensitive detects concrete grammar: the subtilis Swl-19 after activation 16h is got 100 μ L and is uniformly coated on NA media surface, evenly place drug sensitive test paper (drug susceptability test paper is purchased from Hangzhou microorganism reagent company limited) in media surface, check that zymogenic bacteria is to antibiotic resistance.
The Physiology and biochemistry of table 1, bacterial strain Swl-19 and microbiotic detect
+: positive;-: negative+: Positive reaction;-: Negative reaction; S: responsive; M: medium; R: resistance S:sensitive; M:middle; R:resistant
Utilize the haemolysis of Columbia Blood Agar plate culture medium detection Swl-19 pathogenic, concrete implementation step: by the bacterial strain Swl-19 dibbling of activation culture (blood agar is purchased from Jiangmen city Khellinum trade Co., Ltd) on blood agar, observe after 48h and whether have haemolysis circle, result as shown in Figure 4.Result shows, and bacterial strain Swl-19, without hemolytic (producing without haemolysis circle), illustrates that it does not have pathogenic to silkworm.
Produce digestive ferment ability outward with the born of the same parents producing proteolytic enzyme screening culture medium, amylase screening culture medium, lipase screening culture medium and cellulase screening culture medium detection SW-19, result as shown in Figure 5 and Figure 6.Result shows, and Swl-19 can produce the extracellular digestion enzymes such as extracellular protease, amylase, lipase and cellulase, and it is the strongest to produce born of the same parents' exoamylases and cellulase ability, and it is relatively weak to produce extracellular lipase ability.In above-mentioned detection, the screening culture medium of bacteria produced proteinase strain is that in NA substratum, add casein to mass percent be 1.0%, pH9.2 ~ 9.8; To produce amylase screening culture medium be that in NA substratum, add Zulkovsky starch to massfraction be 1%, pH is 9.2 ~ 9.8; Yielding lipase screening culture medium is that in NA substratum, add sweet oil emulsion to concentration be 120mL/L and final concentration is 0.005 (wt) % toluylene red, pH7.2, the compound method of sweet oil emulsion is: get sweet oil and mix with the ratio of volume ratio 1:3 with the polyvinyl alcohol (PVA) of 0.02g/mL, 10000r/min stirring and emulsifying 5min, obtains sweet oil emulsion; Cellulase-producing screening culture medium is that in NA substratum, add carboxymethyl cellulose (CMC) to massfraction be 1%, pH is 9.2 ~ 9.8.
Embodiment 2, the silkworm application of prebiotic subtilis (Bacillus subtilis) Swl-19
Food silkworm is added with after prebiotic subtilis (Bacillus subtilis) Swl-19 to silkworm, detect the colonisation of this strain bacterium in Silkworm, Bombyx mori by DGGE molecular engineering method and on the multifarious impact of dominant microflora in enteron aisle, and silk leaf transformation efficiency and the health of silkworm after food Swl-19 are added in investigation.Its method adopts artificial spray to freshen food method, and freshen food to 5 length of times outside Silkworm, Bombyx mori, to detect the probiotic effects of this bacterium to silkworm, concrete steps are as follows:
After silkworm is normally hastened the hatching of silkworms, collecting ants, raise to 4 length of times with new fresh mulberry leaf, then by 4 the length of time silkworm first day be divided into 2 groups, every component 3 district.(bacterial concentration is 10 to the mulberry leaf that test group silkworm feeding sprays containing Swl-19 bacterial strain
7cfu/mL), the silkworm feeding clear water sprinkling mulberry leaf respectively of control group, adopt even spray pattern, to determine to test accuracy.Feed 2 groups of silkworms to the 3rd day 5 ages, get silkworm blood and middle intestines and content thereof, Bing Jiangmei district determines silkworm head number and continues to feed to being placed on small straw bundles to spin cocoons.Finally detect the colonisation of bacterial strain Swl-19 in bombyx mori intestinal and the influence to bombyx mori intestinal microenvironment with denaturing gradient gel electrophoresis technique, result as shown in Figure 7.DGGE band and ncbi database are contrasted, result is as shown in table 2.
Table 2, DGGE band and ncbi database comparing result
From Fig. 7 and table 2, the dominant microflora after adding food bacterial strain Swl-19 in bombyx mori intestinal there occurs change, adds bacillus in food rear intestinal and Enterobacter bacteria obviously increases.These dominant microfloras increased can play promoter action to the growth of silkworm and to digesting and assimilating of nutritive substance.
Silkworm is raised according to above-mentioned identical method, and adopt weighting method and the counting process investigation rate of being placed on small straw bundles to spin cocoons of silkworm, Cocooning rate, health and cocoon weight and Cocoon layer ratio, the professional statistical analysis software SPSS 20 of data acquisition analyzes, and otherness is done to each index significantly analyze, result is as shown in table 3.
Table 3, Item Survey List
As shown in Table 3, bacterial strain Swl-19 can improve a leaf transformation efficiency in sericultural production, improves silkworm resistance against diseases and Cocooning rate etc.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (6)
1. subtilis (Bacillus subtilis) Swl-19, by the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is CGMCC No.10333.
2. subtilis Swl-19 described in claim 1 is as the application in silkworm probiotic bacterium.
3. application according to claim 2, is characterized in that: described probiotic bacterium is the probiotic bacterium producing extracellular digestion enzyme.
4. application according to claim 3, is characterized in that: described extracellular digestion enzyme is that proteolytic enzyme, amylase, lipase are or/and cellulase.
5. subtilis Swl-19 described in claim 1 is improving the application in domestic silkworm silk leaf transformation efficiency.
6. application according to claim 5, is characterized in that: described silk leaf transformation efficiency is the rate of being placed on small straw bundles to spin cocoons, Cocooning rate, cocoon weight or Cocoon layer ratio.
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| CN112301055A (en) * | 2020-11-04 | 2021-02-02 | 西南大学 | Application and method of silkworm amylase gene BmAmy1 |
| CN117530371A (en) * | 2023-12-15 | 2024-02-09 | 山东农业大学 | Silkworm artificial feed additive containing alkaline protease and preparation method and application thereof |
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| CN110184332A (en) * | 2019-06-27 | 2019-08-30 | 福建省致青生态环保有限公司 | It is a kind of handle high microsteping material compost bacterium preparation method and its application |
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| CN112301055A (en) * | 2020-11-04 | 2021-02-02 | 西南大学 | Application and method of silkworm amylase gene BmAmy1 |
| CN117530371A (en) * | 2023-12-15 | 2024-02-09 | 山东农业大学 | Silkworm artificial feed additive containing alkaline protease and preparation method and application thereof |
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