CN102517235B - Bacillus subtilis - Google Patents
Bacillus subtilis Download PDFInfo
- Publication number
- CN102517235B CN102517235B CN 201110443948 CN201110443948A CN102517235B CN 102517235 B CN102517235 B CN 102517235B CN 201110443948 CN201110443948 CN 201110443948 CN 201110443948 A CN201110443948 A CN 201110443948A CN 102517235 B CN102517235 B CN 102517235B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- feather
- bacillus subtilis
- bacterial strain
- present
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
The invention relates to bacillus subtilis CH-001, which was collected in China Center for Type Culture Collection (CCTCC) on April 8th, 2011. The collection number is CCTCC NO: M 2011111. The bacillus subtilis has the characteristic of degrading keratin in a broad spectrum manner and has mild degradation and fermentation conditions, high enzyme production activity and high stability. The bacillus subtilis can generate high-activity keratinase, can degrade collagen, has antagonism on pathogenic bacteria such as staphylococcus aureus and the like, has the characteristics of probiotics, and is applicable to engineering bacteria development for fermentation production of animal protein feed additives and keratinase. The bacillus subtilis avoids the conflict between high-activity keratinase generated by most microorganisms and pathogenicity of the microorganisms on human or poultry and the broad spectrum characteristic on the keratin, and the antagonism on the related pathogenic bacteria is researched. The bacillus subtilis is a low-carbon, economic, high-efficiency, safe and green biological resource and has huge potential value for creation of economic and social benefits.
Description
Technical field
The invention belongs to the development and utilization field of microorganism germ plasma resource, but relate in particular to a kind of efficient degradation Keratin sulfate and collagen protein and relevant pathogenic bacterium are had subtilis (Bacillus subtilis) CH-001 of antagonistic action.
Background technology
Keratin sulfate is at the occurring in nature aboundresources, and China produces tens of many ten thousand tons per year, and it mainly is present in various animal feathers, hair, hoof etc.Wherein contain 75%～85% hard Keratin sulfate in the poultry feather, amino acid major part in the feather keratin is hydrophobic amino acid, wherein the Gelucystine disulfide linkage is difficult to be opened by proteolytic enzyme, so feather keratin character is extremely stable, facile hydrolysis not in the diluted acid diluted alkaline is if only depend on the digestive ferment in the animal autodigestion road can't be with its absorption.
The treatment process of feather keratin mainly contains traditional hydrolysis method and biotechnology method at present.There are numerous defectives in conventional physical, chemical degradation method, such as the trophic component of product easily destroyed, efficient is low, the power consumption is high, it is large to pollute.And adopt the microbial degradation method effect remarkable, and both utilized protein resource, also be conducive to environment protection.Keratin sulfate and Keratin sulfate application of enzymes also are embodied in all trades and professions simultaneously, hold out broad prospects and higher economic worth and social value.Therefore utilize in recent years biotechnology approach exploitation feather keratin resource, become domestic and international study hotspot, Major Technology comprises two kinds of microbiological deterioration and enzyme liberating.
Very fierce to the research of the screening of producing the M-Zyme bacterial strain, development and utilization at home and abroad.The microorganism of the M-Zyme secreted of having found at present has the diversity characteristics, from South Pole soil to hot spring, from aerobic to anaerobism, comprises bacterium, fungi and actinomycetes etc.
The many fungies of the occurring in nature Keratin sulfate of can both degrading participates in the circulation of keratic carbon, nitrogen and sulphur.Because the application demand of medical science and animal doctor aspect, the fungi of the product M-Zyme of early stage research is mainly skin class fungi, such as Trichophyton (Trichophyton) and sporidiole bacteria (Microsporum).Utilize the biotechnology keratic demand of degrading day by day obvious, found that successively some have the non-pathomycete of the product M-Zyme of development and application values, such as aspergillus (Aspergillus) (Santos et al.1996; Farag and Hassan2004), paecilomyces (Paecilomyces) (Gradisar et al.2005), Curvularia (Curvularia), wood mould (Trichoderma) (Cao et al.2008), Myrothecium (Myrothecium) (Moreira-Gasparin et al.2009) etc.
The actinomycetes that produce M-Zyme mainly are streptomyces (Streptomyces), and these microorganisms are from various soil.The actinomycetes Streptomyces flavis 2BG (mesophilic) of two kinds of tool angle of elevation protease activities that from the soil of the South Pole, are separated to and Microbispora aerata IMBAS-11A (thermophilic), (Gushterova et al.2005); The actinomycetes of some thermophilic product M-Zymes that from the soil of crater, separate in addition such as Streptomyces thermonitrificans (Mohamedin 1999), treptomyces gulbarguensis (Syed et al.2009) etc.The actinomycetes kind of mesophilic product M-Zyme also has Streptomyces albidoflavus K1-02 (Bressollier et al.1999) and Streptomyces graminofaciens (Szabo et al.2000).
A lot of keratic bacteriums of degrading have been screened at present and have been separated to, mostly concentrate on bacillus, comprise Bacillus licheniformis (Bacillus lincheniformis) and subtilis (Bacillus subtilus) (Lin et al.1999; Suh and Lee 2001; Manczinger et al.2003; Balaji et al.2008; Cai et al.2008; Zhang et al.2009), bacillus pumilus (Bacillus pumilus), bacillus cereus (Baeillus cereus) (Kim et al.2001; Werlang and Brandelli 2005; Kumar et al.2008; Ghosh et al.2008), thermophilic, Alkaliphilic bacillus (Bacillus halodurans) (Takami et al.1992,1999), Bacillis pseudofirmus AL-8 (Gessesse et al.2003), Bacillis pseudofirmusFA30-01 (Kojima et al.2006) etc.The more mesophilic genus bacillus that are separated to from the Amazon basin.The M-Zyme of separation and purification is different from the different kind of bacillus, and this has fully showed heterotrophic bacteria diversity.Also provide possibility for different biotechnology applications.Except bacillus, vibrios (Vivrio sp Kr2) (Sangali et al.2000), Xanthomonas campestris (Xanthomonas maltoPhilia) (De Toni et al.2002; Wang Jing etc. 2007), Stenotrophomonas (Stenotrophomonas sp Dl) (Yamamura S et al.2002 academic dissertation), Chryseobacterium sp (Chryseobacterium sp.Kr6) (Riffel et al.2007 academic dissertation), microbacterium (Microbacterium sp.) (Gupta et al.2006; The Ji Jindian academic dissertation) etc. also has the keratic ability of degraded.The thermophilic microorganism of some extreme environment as, Fervidobacterium pennavorans (Friedrich and Antranikian 1996), Fervidobacterium islandicum (Nam etal.2002), Meiothermus ruber H328 (Matsui et al.2009), Clostridium sporogenes (Ionata et al.2008) etc. have report to have the keratic ability of degraded.
In addition, also reported the archeobacteria of the product M-Zyme that some are separated from extreme environment, their existence is at general biological nonviable extreme environment, such as extreme temperature, pH, salt and pressure (Kublanov et al.2009b).
In the Application Areas of market: utilize poultry feather as feed, must be through processing, and it is undesirable to process at present physics, the chemical process method effect of feather keratin.Biofermentation technique now is in the starting stage, and is also less in production application.At the beginning of 21 century, Korea S Insect Biotech company successfully isolates a kind of product highly active protein enzyme Arazyme; This proteolytic enzyme has stronger proteolysis ability, and high temperature resistant, wide pH value; Can be good at the degradation of feather by using Keratin sulfate.Successful Application has represented huge application prospect to fields such as fodder additives, clinical medicine, Industrial Wastewater Treatment, food-processing, makeup.And China's research in this respect and application ground zero, domestic market a large amount of high price imports have added Arazyme highly active protein enzyme such as Insect Biotech company abroad
Health is raiseeed the proteolytic enzyme feeds products such as precious feed, and its demand is still being strengthened.
Keratin sulfate and collagen are good animal protein resource, but because both special chemical structures, character is more stable, and animal is difficult to a large amount of directly absorptions, and many proteolytic enzyme also can't be hydrolyzed it, and this has limited to its development aspect feed.And microbe-derived protease activity is very strong, can be hydrolyzed the scleroproein of multiple difficult degradation, such as Keratin sulfate and collagen etc.If can obtain the stronger bacterial strain of comprehensive degradation capability that single bacterium has degraded Keratin sulfate and collagen protein simultaneously, and after applying in the feed, will greatly improve the utilization ratio of raw material, lower production process, thereby lower production cost.
Summary of the invention
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, provide a kind of probiotic bacterium---subtilis (Bacillus subtilis) CH-001.Bacterial strain of the present invention can not only the efficient degradation Keratin sulfate, and the degradable collagen protein to human body, animal toxicological harmless effect, has antagonistic action to relevant pathogenic bacterium simultaneously.Solved most of microbe degraded Keratin sulfate or collagen protein and it has pathogenic contradiction to people or poultry, and the list of only degrade Keratin sulfate or collagen protein is functional.
Bacterial strain of the present invention is subtilis (Bacillus subtilis) CH-001, be preserved in Chinese Typical Representative culture collection center on April 8th, 2011, deposit number is: CCTCC NO.M 2011111, depositary institution address: China, Wuhan, Wuhan University.
Bacterial strain CH-001 of the present invention is in 37 ℃ of streak culture growths in the LB substratum, bacterium colony circle, surface wettability, coarse opaque, and the edge is irregular, white or little yellow.Picking single bacterium colony wherein carries out gramstaining, and being purple, rod-short explanation bacterial strain of the present invention behind the gramstaining is gram-positive microorganism, such as Fig. 1.Observing thalline in Electronic Speculum is: shaft-like, individual cells is big or small to be 0.6～0.7 * 2～3 microns, and uniform coloring without pod membrane, is seen Fig. 2.
The cultural characteristic of bacterial strain of the present invention when different substrate: in inoculation has the inorganic salt feather nutrient solution of bacterial strain of the present invention, add the materials such as wheat bran, N.F,USP MANNITOL, the degraded of feather is had obvious promotion; And add glucose, ammonium nitrate etc., it there is obvious restraining effect; Add beans cake powder, bismuth subnitrate etc., faint restraining effect is arranged; Add Semen Maydis powder, fish meal, soybean cake powder etc., without obvious effect.Metal Ca
2+, Mg
2+Its degradation of feather by using there is promoter action, Zn
2+Its degradation of feather by using there is restraining effect.The optimum pH of strains for degrading feather of the present invention is about 8.0, does not grow the non-degradable feather under the sour environment less than pH5.0; Can grow under the alkaline environment of pH12, degradation of feather by using is good.
The 16SrDNA sequence of the bacterial strain of the present invention that obtains is carried out the blast comparison in the genebank database, the result shows that the sequence homology of the 16SrDNA of bacterial strain of the present invention and subtilis Bacillus subtilis subsp.subtilis strain DSM 10 is 99%.Identify to show that bacterial strain of the present invention is the novel species of subtilis (Bacillus sub tilis), called after subtilis (Bacillus subtilis) CH-001, the phylogenetic tree of bacterial strain of the present invention is seen Fig. 3.
Bacterial strain of the present invention has antagonistic action to streptococcus aureus, and inoculation 0.1-0.5% bacterial strain of the present invention fermentation culture in inorganic salt feather nutrient solution, its fermented liquid degradable Keratin sulfate and collagen protein, and fermentation condition is: shaking flask is to OD
600Be 0.5, temperature 35-41 ℃, rotating speed 160r/min, fermentation time are 12-36h, pH 7.0-8.0; Better fermentation condition is: shaking flask is to OD
600Be 0.5, by 0.1% inoculation fermentation, 37 ℃ of temperature, rotating speed 160r/min, fermentation time are 24h, and pH 8.0.
So, bacterial strain of the present invention has the wide spectrum keratic characteristic of degrading, degradable fermented mild condition, and inulinase-producing activity is high, good stability.Can not only produce the high reactivity M-Zyme, the degradable collagen protein has antagonistic action to pathogenic bacterium such as streptococcus aureuses simultaneously, has probiotic properties, is applicable to the engineering bacteria exploitation of animal protein feed additive, M-Zyme fermentative production etc.Having solved most of microbe product high reactivity M-Zyme has pathogenic conflicting to reach keratic wide spectrum degradation characteristic with its tool to people or poultry, has studied its antagonistic action to relevant pathogenic bacterium.Be the green bio resource of a kind of low-carbon economy, highly effective and safe, have the huge potential value of creating economical, societal benefits.
Description of drawings
Fig. 1 is bacterial strain gramstaining figure of the present invention.
Fig. 2 is the Electronic Speculum figure of bacterial strain of the present invention.
Fig. 3 is the phylogenetic tree of bacterial strain of the present invention.
Fig. 4 is bacterial strain of the present invention degradation effect to feather after different time is cultivated.
Wherein: A: blank solution (upper figure) is cultivated 12h with crude enzyme liquid (figure below); B: blank solution (upper figure) is cultivated 24h with crude enzyme liquid (figure below); C: blank solution (upper figure) is cultivated 36h with crude enzyme liquid (figure below).
Fig. 5 is the incubation time mensuration that the best enzyme of the crude enzyme liquid of bacterial strain of the present invention is lived and reacted.
Fig. 6 is that the reaction Best Times of crude enzyme liquid and azo-casein is measured.
Fig. 7 is that the best pH of the reaction of crude enzyme liquid and azo-casein measures.
Fig. 8 is that bacterial strain of the present invention is to the antagonistic action figure of streptococcus aureus.
Embodiment
Embodiment 1:
Get the soil that Agricultural University Of Hunan stacks feather in the school for a long time, put into the sterile distilled water mixing, leave standstill, get supernatant liquor after the clarification and coat in the solid inorganic salt feather substratum, cultivated 2-3 days for 37 ℃, obtain bacterium colony.The bacterium colony that coating is obtained separates with the toothpick picking, be inoculated in the inorganic salt feather nutrient solution, cultivate 48h for 37 ℃, observe the feather palliating degradation degree, just sift out effective strain, dip its nutrient solution repeatedly streak culture, and picking list bacterium colony is cultivated in inorganic salt feather nutrient solution and is carried out repeated screening again, obtains preferably pure bacterial strain of degradation effect, inoculates at last single bacterium colony in gelatine culture, cultivate 12h, namely get bacterial strain of the present invention for 37 ℃.
Embodiment 2: the thalline dyeing of bacterial strain of the present invention, electron microscopic observation, colony morphology characteristic observation experiment
Bacterial strain of the present invention is in 37 ℃ of streak culture growths in the LB liquid nutrient medium, bacterium colony circle, surface wettability, coarse opaque, and the edge is irregular, white or little yellow.Picking single bacterium colony wherein carries out gramstaining, and being purple, rod-short explanation bacterial strain of the present invention behind the gramstaining is gram-positive microorganism, such as Fig. 1.Observing thalline in Electronic Speculum is: shaft-like, individual cells is big or small to be 0.6～0.7 * 2～3 microns, uniform coloring.Without pod membrane, such as Fig. 2.
Embodiment 3: the 16SrDNA evaluation of bacterial strain of the present invention and the structure of phylogenetic tree
Adopt the EDTA method to extract strain gene group DNA of the present invention among the present invention, utilize 16S rDNA primers F: agtggccattacggccagagtttgatcmtggctcag (shown in SEQ ID No:1); R:agaggccgaggcggcctacgggytaccttgttacgactt (shown in SEQ ID No:2) obtains dominant strain high-fidelity sequence, and the PCR system is: ddH
2O:40.0ul, 10 * Buffer (Mg
2+Plus): 5ul, d NTPs:1ul, F:1.0ul, R:1.0ul, Taq enzyme: 1ul, dna profiling: 1ul, total volume50ul, 95 ℃ of denaturation 4min, 95 ℃ of sex change 30s, 55 ℃ of annealing 45s, 72 ℃ of extension 1.5min circulate 30 times, 15min is extended at 72 ℃ of whole ends, and 4 ℃ to ∞.After PCR finishes, must brighter purpose band be arranged in about 1500p place through electrophoresis detection, cut glue with QlAquick Gel Extraction Kit and reclaim purifying, be connected with the purpose fragment of purifying take PUM-T as carrier and carry out the A-T clone, recycling CaCl2 conversion method, recon is changed in the XL1-blue intestinal bacteria, add 1.0ml LB nutrient solution and (do not add Amp
+) behind the renewal cultivation 2h, coated plate is in adding Amp
+LB solid medium (Amp
+Ultimate density 100mg/ml) cultivate 12h for 37 ℃ in, picking list bacterium colony adds Amp in 30ml
+The LB liquid nutrient medium in (Amp
+Ultimate density 100mg/ml) 37 ℃ of shaking table overnight incubation, preserve each bacteria suspension after, extract the plasmid PCR checking, the correspondence that will contain the purpose fragment of the 1500bp that has an appointment is preserved bacterium liquid and is delivered to gene sequencing company and check order.The 16SrDNA sequence of bacterial strain of the present invention is shown in SEQ ID No:3.
In the Genbank database, do sequence homology relatively, carry out Molecular genetic test, determine that the sequence homology of the 16SrDNA of bacterial strain of the present invention and subtilis Bacillus subtilis subsp.subtilis strain DSM 10 is 99%.The result shows, bacterial strain of the present invention is the novel species of subtilis (Bacillus subtilis), called after subtilis (Bacillus subtilis) CH-001.With clustal software comparison homologous sequence and phylogenetic tree construction, as shown in Figure 3.
Embodiment 4: the preservation of bacterial classification of the present invention, cultivation, fermentative degradation feather
Bacterial strain of the present invention can be about 20% glycerine LB substratum with ultimate density and be stored in-20 ℃.Namely inoculate single bacterium colony of bacterial strain of the present invention after 30ml LB liquid nutrient medium is cultivated 12h, get 60% glycerine of 500ul cultivation bacteria suspension and 300ul high-temperature sterilization in 1.5ml centrifuge tube mixing, just can place-20 ℃ of preservations.
Get 30ml inorganic salt feather nutrient solution in the triangular flask of 100ml, after the sterilization, single bacterium colony 0.1% of picking bacterial strain of the present invention is inoculated in this nutrient solution, 37 ℃, the 160rpm/min shaking table is cultivated, can be observed: after cultivating 8h, nutrient solution becomes muddy and a large amount of fracture feather sheets is arranged, but does not separate with pinna rachis; After cultivating 12h, media surface more superfine feather powder occurs and is adsorbed on the glass wall, and fracture feather sheet shortens, and base portion does not separate with pinna rachis; After cultivating 16h, fracture feather sheet is degraded fully substantially, has less spherical feather particle shape to become, but also is left pinna rachis; After cultivating 45h, pinna rachis is attenuated short by Partial digestion.
Embodiment 5: different substrate cultivation (C source, N source) feature
Have in inoculation and to add 0.1% different carbon sources or nitrogenous source in the inorganic salt feather nutrient solution of 0.1% bacterial strain of the present invention, in 37 ℃, the 160rpm/min shaking table is cultivated 24h, get its supernatant liquor and obtain crude enzyme liquid, and measure the activity of each crude enzyme liquid, can obtain adding wheat bran, N.F,USP MANNITOL etc. has higher enzymic activity, has promoted significantly the degraded of feather; Add glucose, ammonium nitrate etc. it is had significantly restraining effect; Add beans cake powder, bismuth subnitrate etc. faint restraining effect is arranged; Add Semen Maydis powder, fish meal, soybean cake powder etc. without obvious effect.Add respectively the alpha keratin class: pig's feet etc. and β cyokeratin: chicken feather, hair etc. and collagen protein class: Cowhells, gelatin etc. has Degradation.
By measuring different pH values and metal ion to the impact of strains for degrading feather speed of the present invention, and initial optimization growth conditions.The optimum pH of strains for degrading feather of the present invention is about 8.0, does not grow under the sour environment less than pH 5.0, and the non-degradable feather can be grown under the alkaline environment of pH12, and degradation of feather by using is good, metal Ca
2+, Mg
2+Its degradation of feather by using there is promoter action, Zn
2+Its degradation of feather by using there is restraining effect.
Embodiment 6: detect M-Zyme to the degradation effect of feather
Adopt ninhydrin method to measure the crude enzyme liquid of different time (12h, 24h, 36h) taking-up (after the inorganic salt feather nutrient solution shaking table cultivation of inoculation bacterial strain of the present invention, through 4 ℃, the centrifugal 5min of 10000rpm, the 0.22um filtration sterilization obtains) in total free a-amino acid content.Behind the direct shaking table cultivation of the inorganic salt feather nutrient solution of not inoculating bacterial strain of the present invention same time, through 4 ℃, the centrifugal 5min of 10000rpm, the filtrate that the 0.22um filtration sterilization obtains is blank solution.Obtain the relation of total free a-amino acid content in the feather degraded situation solution corresponding with it, the result sees table 1 and Fig. 4, as can be known along with the increase of incubation time, the nutrient solution mesoptile is degraded gradually, and the content of liquid Free Amino Acids increases, but Degradation is not obvious after cultivating 24h, illustrates in the fermented liquid of bacterial strain of the present invention to have produced the M-Zyme that feather is had Degradation, and the degradation effect of this M-Zyme to feather also has been described simultaneously.
The relation with contents of table 1 incubation time and nutrient solution Free Amino Acids
Embodiment 7: the crude enzyme liquid determination of activity
Experimental group: the crude enzyme liquid in the test tube that 1.5ml 10%Azocasein is housed behind adding 1.0ml difference fermentation culture 8h, 14h, 24h, 30h, the 36h (is inoculated 0.1% bacterial strain of the present invention after 150ml inorganic salt feather nutrient solution shaking table is cultivated, through 4 ℃, the centrifugal 5min of 10000rpm, the 0.22um filtration sterilization obtains.) 5 times of diluents.Behind 37 ℃ of reaction different times (0min, 30min, 60min, 90min, 120min, 150min, 180min), add immediately 1.0ml 10%TCA, leave standstill 30min in 4 ℃, the centrifugal 3min of 10000rpm gets the absorption value that its supernatant liquor is measured 335nm wave band place, result such as Fig. 5.
Contrast liquid: the blank solution in the test tube that 1.5ml 10%Azocasein is housed behind adding 1.0ml difference fermentation culture 8h, 14h, 24h, 30h, the 36h is not (after inoculating the direct shaking table cultivation of 150ml inorganic salt feather nutrient solution of bacterial strain of the present invention, through 4 ℃, the centrifugal 5min of 10000rpm, the 0.22um filtration sterilization obtains.) 5 times of diluents, 37 ℃ of reactions do not add 1.0ml 10%TCA after (0min, 30min, 60min, 90min, 120min, 150min, 180min) not simultaneously immediately, 4 ℃ leave standstill 30min, and the centrifugal 3min of 10000rpm gets its supernatant liquor in the calibration zeroing of 335nm wave band place.
The work of observation enzyme is with the variation in reaction times, and the best enzyme of the crude enzyme liquid fermented incubation time alive that records bacterial strain of the present invention is 24h, sees Fig. 5, and the reaction Best Times of crude enzyme liquid and azo-casein is about 2h, such as Fig. 6.
In being housed, the test tube of 1.5ml10%Azocasein adds 1.0ml with the crude enzyme liquid of the different pH of 5 times of inorganic salt solution dilutions (adding a certain amount of phosphoric acid buffer in crude enzyme liquid makes reaction pH be respectively the acquisitions such as 4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0), add at once 1.0ml 10%TCA behind 37 ℃ of reaction 2h, 4 ℃ leave standstill 30min, the centrifugal 3min of 10000rpm gets the absorption value that its supernatant liquor is measured 335nm wave band place.Observe enzyme work with the variation of pH, the pH that draws the reaction alive of best enzyme is 8.0, such as Fig. 7.
Definition enzyme unit alive is: under optimum condition, wavelength is in the mensuration of 335nm, and it is the note 1U of enzyme unit alive that the absorbancy of reaction 1h increases by 0.1.Comprehensive above-mentioned each enzyme reaction conditions alive records the highest enzyme work and is: 10.19U, comparing bacterial strain of the present invention with the activity of other these type of bacterium has higher enzymic activity.
Embodiment 8: bacterial strain of the present invention is to the antagonistic action of streptococcus aureus
The beef extract-peptone solid medium is behind high-temperature sterilization, and is to be cooled during to non-scald on hand and ot-yet-hardened, draws streptococcus aureus solution that 200ul broken up with aseptic deionized water to this substratum with the imbibition rifle, shakes up fast and be down flat plate.After the culture medium solidifying in the flat board, single bacterium colony of the more excellent bacterial strain that filters out among the picking embodiment 1 is inoculated in dull and stereotyped central authorities, behind 37 ℃ of cultivation 12h, just observable inhibition zone size detects its antagonistic action, such as Fig. 8, result's demonstration, bacterial strain of the present invention has obvious inhibition zone to streptococcus aureus.
The culture medium prescription of mentioning in the literary composition is as follows:
Inorganic salt feather nutrient solution is: NaCl 0.5g/L, KH
2PO
40.4g/L, K
2HPO
40.3g/L, (or feather 60-70 root/L), pH 8.0 for feather meal 10g/L; Solid-solid inorganic salt feather substratum then adds agar 15g/L.
Gelatine culture is: peptone 1.5g, beef extract powder 0.3g, Sodium phosphate dibasic 0.2g, agar powder 1.5g, water 100ml, gelatin 1.0g, Ph8.0.
LB liquid nutrient medium (LB nutrient solution): yeast extract 5g/L, Tryptones 10g/L, sodium-chlor 10g/L, pH 7.2.The LB solid medium then adds agar 15g/L.
The feather meal substratum is: NaCl 0.5g/L, KH
2PO
40.4g/L, K
2HPO
40.3g/L, feather meal 10g/L (or feather 60-70 root/L), pH 8.0, and solid medium then adds agar 15g/L.
Beef-protein medium: beef extract 3g/L, peptone 10g/L, NaCl 5g/L, pH7.2-7.4, solid medium then add agar 15g/L.
Claims (7)
1. subtilis, it is characterized in that: this bacterial strain is subtilis
2. the application of bacterial strain as claimed in claim 1 in the antagonism streptococcus aureus.
3. one kind ferments and prepares the method for fermentation of bacillus subtilis liquid, it is characterized in that: the fermentation bacterial strain uses therefor is bacterial strain claimed in claim 1, and the fermentation used medium is inorganic salt feather nutrient solution, and fermentation condition is: shaking flask is to OD
600Be 0.5, press the 0.1-0.5% inoculation fermentation, temperature 35-41 ℃, rotating speed 160r/min, fermentation time are 12-36h, pH 7.0-8.0; This inorganic salt feather nutrient solution prescription is: NaCl 0.5g/L, KH
2PO
40.4g/L, K
2HPO
40.3g/L, feather meal 10g/L, pH8.0.
4. fermentation as claimed in claim 3 prepares the method for fermentation of bacillus subtilis liquid, it is characterized in that: fermentation condition is: shaking flask is to OD
600Be 0.5, by 0.1% inoculation fermentation, 37 ℃ of temperature, rotating speed 160r/min, fermentation time are 24h, and pH 8.0.
5. the fermented liquid that obtains such as claim 3 or 4 described methods.
6. the application of fermented liquid as claimed in claim 5 in the degraded Keratin sulfate.
7. the application of fermented liquid as claimed in claim 5 in degrade collagen albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110443948 CN102517235B (en) | 2011-12-27 | 2011-12-27 | Bacillus subtilis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110443948 CN102517235B (en) | 2011-12-27 | 2011-12-27 | Bacillus subtilis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102517235A CN102517235A (en) | 2012-06-27 |
| CN102517235B true CN102517235B (en) | 2013-01-16 |
Family
ID=46288306
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110443948 Expired - Fee Related CN102517235B (en) | 2011-12-27 | 2011-12-27 | Bacillus subtilis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102517235B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015028701A1 (en) * | 2013-08-27 | 2015-03-05 | Teknologian Tutkimuskeskus Vtt | Microbiological keratin processing |
| CN105255771B (en) * | 2015-11-05 | 2018-07-13 | 鲁东大学 | It is a kind of production Collagenase amber staphylococcus and its application |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232963B (en) * | 2013-05-27 | 2014-10-15 | 天津益丽康生物科技有限公司 | Collagenase producing strain |
| CN103589674B (en) * | 2013-12-02 | 2015-07-01 | 国家海洋局第三海洋研究所 | Bacillus subtilis Pc3 and use of bacillus subtilis Pc3 in preparation of fermentation supernatant for preventing and controlling plant pathogenic fungi |
| CN103589673B (en) * | 2013-12-02 | 2015-05-27 | 国家海洋局第三海洋研究所 | Bacillus subtilis A053 and use of bacillus subtilis A053 in preparation of fermentation liquor for preventing and controlling plant pathogenic fungi |
| CN103642735B (en) * | 2013-12-12 | 2016-01-27 | 江南大学 | Bacillus pumilus and the application method thereof of M-Zyme are produced in one strain |
| CN103667155B (en) * | 2013-12-23 | 2015-07-29 | 华南农业大学 | One bacillus subtilis Bacillus subtilis 3-2 and application thereof |
| CN104694440A (en) * | 2015-03-24 | 2015-06-10 | 东华大学 | Feather degrading bacterium and application thereof |
| CN107118984B (en) * | 2017-05-09 | 2018-06-08 | 山东省农业科学院生物技术研究中心 | A kind of ferment tank technique of keratin degrading bacteria and its application in live pig loses hair or feathers |
| TW201915165A (en) * | 2017-09-20 | 2019-04-16 | 行政院農業委員會農業藥物毒物試驗所 | Bacillus subtilis strain for preparing fermented feather meal and use thereof for producing feather powder and proteolytic enzyme and keratinolytic enzyme strains |
| CN115399403B (en) * | 2022-09-14 | 2023-12-22 | 江苏三仪生物工程有限公司 | Production method and application of feather oligopeptide chelated mineral element |
| CN115606704B (en) * | 2022-11-02 | 2023-11-07 | 上海福贝宠物用品股份有限公司 | Hair bulb remover for improving gastrointestinal health of pets and preparation method thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100740973B1 (en) * | 2005-02-18 | 2007-07-20 | 씨.에프. 주식회사 | -1 Bacillus subtilis PL-1 having keratinolytic activity and the protease therefrom |
| CN100419072C (en) * | 2005-12-27 | 2008-09-17 | 云南师范大学 | Keratinase-proudicng bacterium and its preparation method |
| CN101497910A (en) * | 2008-01-31 | 2009-08-05 | 海城市宏大牧业有限公司 | Method for preparing enzymolysis oligopeptide protein |
| CN101240302B (en) * | 2008-03-14 | 2010-11-10 | 山东省农业科学院高新技术研究中心 | Method for producing L-phenylalanine by using bacillus subtilis to ferment feather |
| BRPI0900748A2 (en) * | 2009-03-09 | 2010-11-09 | Serraria Uniao De Bariri Ltda | Process for the manufacture of enzymatic preparations obtained from poultry feather, enzymatic preparations so manufactured, their use, animal feed and hair transformation agents |
| CN101869184B (en) * | 2010-06-13 | 2012-06-27 | 山东省农业科学院高新技术研究中心 | Microbial feed additive and preparation method thereof |
-
2011
- 2011-12-27 CN CN 201110443948 patent/CN102517235B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015028701A1 (en) * | 2013-08-27 | 2015-03-05 | Teknologian Tutkimuskeskus Vtt | Microbiological keratin processing |
| EP3041950A4 (en) * | 2013-08-27 | 2017-03-22 | Teknologian tutkimuskeskus VTT Oy | Microbiological keratin processing |
| CN105255771B (en) * | 2015-11-05 | 2018-07-13 | 鲁东大学 | It is a kind of production Collagenase amber staphylococcus and its application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102517235A (en) | 2012-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102517235B (en) | Bacillus subtilis | |
| Vaseekaran et al. | Isolation and identification of a bacterial strain producing thermostable α-amylase. | |
| CN103667155B (en) | One bacillus subtilis Bacillus subtilis 3-2 and application thereof | |
| CN103695354B (en) | One strain bacillus cereus Bacillus cereus J2 and application thereof | |
| CN105543149B (en) | One plant of new bacillus megaterium and its application | |
| KR102494770B1 (en) | Novel Paenibacillus sp. Daeji-L2 strain | |
| CN102399726B (en) | Sporosarcina and application thereof | |
| CN101245331B (en) | South pole marine microorganism bacterial strain n2a of high-production low-temperature catalase | |
| CN105802892B (en) | It is a kind of produce keratinase germ oligotrophy unit cell and its application | |
| CN104894024A (en) | Pseudoalteromonas mutant strain and application thereof | |
| CN109321500B (en) | Bacillus amyloliquefaciens strain and application thereof in prevention and treatment of camellia oleifera anthracnose disease | |
| KR20210013995A (en) | Bacillus sp, a microorganism product for treating food waste and a treating method of food waste using it | |
| Tiwari et al. | Diversity and molecular characterization of dominant Bacillus amyloliquefaciens (JNU-001) endophytic bacterial strains isolated from native Neem varieties of Sanganer region of Rajasthan | |
| CN104560817B (en) | Thermophilic bacillus licheniformis UTM102 for producing phytase and application of thermophilic bacillus licheniformis UTM102 | |
| CN105950497A (en) | Keratinase producing bacillus cereus and application thereof | |
| CN103695355B (en) | One pseudomonas Pseudomonas otitidis H3 and application thereof | |
| CN105861373B (en) | It is a kind of produce keratinase pseudomonas aeruginosa and its application | |
| Parhamfar et al. | Purification and Characterization of an Extracellular Phosphatase Enzyme From Bacillus spp. | |
| CN101245325A (en) | Stenotrophomonas maltophilia of strain producing novel alkali proteinase | |
| CN104560818A (en) | Bacillus licheniformis UTM118 for producing high-temperature-resistant acid alpha-amylase and application of bacillus licheniformis UTM118 | |
| CN103667156B (en) | One strain Chryseobacterium sp Chryseobacterium ureilyticum R1 and application thereof | |
| KR101477886B1 (en) | Bacillus spp., identified from lugworm and microbial cleaning agent. | |
| CN108441437A (en) | A kind of composite bacteria agent and its application | |
| CN114874953B (en) | Peanut rhizosphere biocontrol bacterium-Bacillus beleisi SW-1 and application thereof | |
| De et al. | An insight into the celluloytic potential of three strains of Bacillus spp. isolated from benthic soil of aquaculture farms in East Kolkata Wetlands, India. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130116 Termination date: 20141227 |
|
| EXPY | Termination of patent right or utility model |