CN102517235A - Bacillus subtilis - Google Patents
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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 has antagonistic action with collagen protein and to relevant pathogenic bacterium subtilis (Bacillus subtilis) CH-001.
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 a hydrophobic amino acid; Wherein the Gelucystine disulfide linkage is difficult to 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 water solution and biotechnology method at present.There are numerous defectives in conventional physical, chemical degradation method, such as the trophic component of product be prone to be destroyed, efficient is low, power consumption is high, pollution is big.And adopt the microbial degradation method effect remarkable, and both utilized protein resource, also help environment protection.The application of Keratin sulfate and M-Zyme simultaneously also is embodied in all trades and professions, holds out broad prospects and higher economic value and social value.Therefore utilize biotechnology approach exploitation feather keratin resource in recent years, become domestic and international research focus, the major technique approach comprises two kinds of microbiological deterioration and enzyme liberating.
At home and abroad to the screening of producing the M-Zyme bacterial strain, the research ten minutes fierceness of development and utilization.The mikrobe of the M-Zyme of having found at present secreted has the variety characteristics, and soil to anaerobism, comprises bacterium, fungi and actinomycetes etc. from aerobic to hot spring from the South Pole.
The many fungies of the occurring in nature Keratin sulfate of can both degrading is participated 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, like Trichophyton (Trichophyton) and sporidiole bacteria (Microsporum).Utilize the biotechnology keratic increasing demand of degrading obvious, found that successively some have the non-pathomycete of the product M-Zyme of development and application values, like 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 mikrobes 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 such as Streptomyces thermonitrificans (Mohamedin 1999), the treptomyces gulbarguensis (Syed et al.2009) etc. that also have more isolating thermophilic product M-Zymes from the soil of crater.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).
Screen and be separated to a lot of keratic bacteriums of degrading at present, concentrated on bacillus mostly, comprised 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 has nothing in common with each other from the different kind of bacillus, and this has fully showed the variety of mikrobe.Also possibility is provided 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); Oligotrophy Zymomonas mobilis (Stenotrophomonas sp Dl) (Yamamura S et al.2002 academic dissertation); Golden yellow bacillus (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.Thermophilus microorganism under 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, like extreme temperature, pH, salt and pressure (Kublanov et al.2009b).
In the application market field: utilize poultry feather as feed, must must pass through processing, and it is undesirable to handle physics, the chemical process method effect of feather keratin at present.Biofermentation technique is in the starting stage at present, 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 great 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; A large amount of high price imports external as Insect Biotech company in home market has added
health of Arazyme highly active protein enzyme and has raiseeed proteolytic enzyme feeds products such as precious feed, and to its demand still in increasing.
Keratin sulfate and collagen are good animal protein resource, but because both particular 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 with its hydrolysis, and this has limited to its development aspect feed.And microbe-derived protease activity is very strong, and scleroproein that can the multiple difficult degradation of hydrolysis is like 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 improve utilization ratio of raw materials greatly, lower production process, thereby lower production cost.
Summary of the invention
Technical problem to be solved by this invention is: the deficiency to above-mentioned prior art provides 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 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, like Fig. 1.In Electronic Speculum, observing thalline is: shaft-like, individual cells is big or small to be 0.6～0.7 * 2～3 microns, uniform coloring, and no 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 materials such as wheat bran, N.F,USP MANNITOL, the degraded of feather is had tangible promotion; And add glucose, an ammonium nitrate etc., it there is the obvious suppression effect; Add beans cake powder, bismuth subnitrate etc., faint restraining effect is arranged; Add Semen Maydis powder, fish meal, soybean cake powder etc., do not have obviously 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, under the sour environment less than pH5.0, does not grow the non-degradable feather; Under the alkaline environment of pH12, can grow, 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 DB, 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: shake bottle to OD
600Be 0.5, temperature 35-41 ℃, rotating speed 160r/min, fermentation time are 12-36h, pH 7.0-8.0; Preferable fermentation condition is: shake bottle 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 prodn 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 (last figure) is cultivated 12h with crude enzyme liquid (figure below); B: blank solution (last figure) is cultivated 24h with crude enzyme liquid (figure below); C: blank solution (last figure) is cultivated 36h with crude enzyme liquid (figure below).
The incubation time mensuration that Fig. 5 lives and reacts for the best enzyme of the crude enzyme liquid of bacterial strain of the present invention.
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 the antagonistic action figure of bacterial strain of the present invention to 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 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, is inoculated in the inorganic salt feather nutrient solution, cultivates 48h for 37 ℃; Observe the feather palliating degradation degree, just sift out effective strain, dip in that to get 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 the pure preferably bacterial strain of degradation effect, inoculates single bacterium colony at last in gelatine culture; Cultivate 12h, promptly 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, like Fig. 1.In Electronic Speculum, observing thalline is: shaft-like, individual cells is big or small to be 0.6～0.7 * 2～3 microns, uniform coloring.No pod membrane is like 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 preparatory sex change 4min; 95 ℃ of sex change 30s, 55 ℃ of annealing 45s, 72 ℃ of extension 1.5min circulate 30 times, 72 ℃ of last eventually 15min that extend, and 4 ℃ to ∞.After PCR accomplishes; Through electrophoresis detection brighter purpose band must be arranged in about 1500p place; Cutting glue with QlAquick Gel Extraction Kit and reclaim purifying, is that carrier is connected with the purpose fragment of purifying and carries out A-T and clone with PUM-T, utilizes the CaCl2 conversion method again; Recon is changed in the XL1-blue intestinal bacteria, add 1.0ml LB nutrient solution and (do not add Amp
+) recover to cultivate 2h after, 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 cultures, preserve each bacteria suspension after, extract plasmid PCR checking, will contain the segmental corresponding bacterium liquid of preserving of the purpose of the 1500bp that has an appointment and deliver 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 DB, do sequence homology relatively, carry out molecular genetics and detect, confirm 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 that bacterial strain of the present invention is the novel species of subtilis (Bacillus subtilis), called after subtilis (Bacillus subtilis) CH-001.Grow tree with clustal software comparison homologous sequence and constructing system, 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 ℃.Single bacterium colony of promptly inoculating bacterial strain of the present invention is after 30ml LB liquid nutrient medium is cultivated 12h, and 60% glycerine of getting 500ul culture bacteria suspension and 300ul high-temperature sterilization just can place-20 ℃ of preservations in 1.5ml centrifuge tube mixing.
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 basically, has less spherical feather particle shape to become, but also remaining pinna rachis; After cultivating 45h, pinna rachis is degraded and the weak point that attenuates by part.
Embodiment 5: different substrate cultivation (C source, N source) characteristic
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, gets its supernatant and obtains 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 the degraded of feather significantly; Add glucose, an ammonium nitrate etc. it is had restraining effect significantly; Add beans cake powder, bismuth subnitrate etc. faint restraining effect is arranged; Add Semen Maydis powder, fish meal, soybean cake powder etc. and do not have obviously effect.Add the alpha keratin class respectively: pig's feet etc. and β Keratin sulfate class: chicken feather, hair etc. and collagen protein class: Cowhells, gelatin etc. all has Degradation.
Through measuring the influence of different pH values and metals ion to 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, under the sour environment less than pH 5.0, does not grow, 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 the degradation effect of M-Zyme to 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.After cultivating the identical time with the direct shaking table of inorganic salt feather nutrient solution of not inoculating bacterial strain of the present invention, through 4 ℃, the centrifugal 5min of 10000rpm, the filtrating 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, can know the increase along with incubation time, and the nutrient solution mesoptile is by degraded gradually; And the content of total free aminoacids increases in the liquid; But Degradation is not obvious after cultivating 24h, explains 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 total free aminoacids in table 1 incubation time and the nutrient solution
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 1.0ml 10%TCA immediately; Leave standstill 30min in 4 ℃; The centrifugal 3min of 10000rpm gets the absorption value that its supernatant 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 the calibration zeroing at 335nm wave band place of its supernatant.
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, like Fig. 6.
, the test tube of 1.5ml10%Azocasein adds 1.0ml in being housed with the crude enzyme liquid of the different pH of 5 times of inorganic salt solution dilutions (in crude enzyme liquid, adding a certain amount of phosphoric acid buffer makes reaction pH be respectively acquisitions such as 4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0); Add 1.0ml 10%TCA at once behind 37 ℃ of reaction 2h; 4 ℃ leave standstill 30min; The centrifugal 3min of 10000rpm gets the absorption value that its supernatant is measured 335nm wave band place.Observe the variation of enzyme work with pH, the pH that draws the reaction alive of best enzyme is 8.0, like 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 falls dull and stereotyped.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 can observe the inhibition zone size, detect its antagonistic action; Like Fig. 8, the result shows that bacterial strain of the present invention has obvious inhibition zone to streptococcus aureus.
The culture medium prescription of mentioning in the literary composition is following:
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 (Bacillus subtilis) CH-001, is preserved in Chinese typical culture collection center on April 8th, 2011, and deposit number is: CCTCC NO.M 2011111.
2. the effect of bacterial strain as claimed in claim 1 in the antagonism streptococcus aureus.
3. the method for a fermentative prepn fermentation of bacillus subtilis liquid is characterized in that: the fermentation bacterial strain uses therefor is the described bacterial strain of claim 1, and the used substratum that ferments is an inorganic salt feather nutrient solution, and fermentation condition is: shake bottle 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 is: NaCl 0.5g/L, KH
2PO
40.4g/L, K
2HPO
40.3g/L, feather meal 10g/L, pH8.0.
4. the method for fermentative prepn fermentation of bacillus subtilis liquid as claimed in claim 3 is characterized in that: fermentation condition is: shake bottle 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 like claim 3 or 4 described methods.
6. the effect of fermented liquid as claimed in claim 5 in the degraded Keratin sulfate.
7. the effect of fermented liquid as claimed in claim 5 in degrade collagen albumen.
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| CN103642735A (en) * | 2013-12-12 | 2014-03-19 | 江南大学 | Bacillus pumilus capable of producing keratinase and application method thereof |
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| CN104694440A (en) * | 2015-03-24 | 2015-06-10 | 东华大学 | Feather degrading bacterium and application thereof |
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| CN107118984A (en) * | 2017-05-09 | 2017-09-01 | 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 |
| CN109517750A (en) * | 2017-09-20 | 2019-03-26 | 财团法人农业科技研究院 | Bacillus subtilis strain for preparing fermented feather powder and application thereof |
| CN115399403A (en) * | 2022-09-14 | 2022-11-29 | 江苏三仪生物工程有限公司 | Production method and application of feather oligopeptide chelating type mineral elements |
| CN115606704A (en) * | 2022-11-02 | 2023-01-17 | 上海福贝宠物用品股份有限公司 | Hair bulb remover for improving gastrointestinal tract health of pets and preparation method thereof |
| CN116650380A (en) * | 2023-06-21 | 2023-08-29 | 广州中妆美业化妆品有限公司 | Anti-aging tea fermentation product for improving skin microcirculation and preparation method and application thereof |
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| CN103642735A (en) * | 2013-12-12 | 2014-03-19 | 江南大学 | Bacillus pumilus capable of producing keratinase and application method thereof |
| CN103667155B (en) * | 2013-12-23 | 2015-07-29 | 华南农业大学 | One bacillus subtilis Bacillus subtilis 3-2 and application thereof |
| CN103667155A (en) * | 2013-12-23 | 2014-03-26 | 华南农业大学 | 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 |
| CN105255771A (en) * | 2015-11-05 | 2016-01-20 | 鲁东大学 | Staphylococcus succinus for generating collagenase and application thereof |
| CN107118984A (en) * | 2017-05-09 | 2017-09-01 | 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 |
| CN109517750A (en) * | 2017-09-20 | 2019-03-26 | 财团法人农业科技研究院 | Bacillus subtilis strain for preparing fermented feather powder and application thereof |
| CN115399403A (en) * | 2022-09-14 | 2022-11-29 | 江苏三仪生物工程有限公司 | Production method and application of feather oligopeptide chelating type mineral elements |
| 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 |
| CN115606704A (en) * | 2022-11-02 | 2023-01-17 | 上海福贝宠物用品股份有限公司 | Hair bulb remover for improving gastrointestinal tract health of pets and preparation method thereof |
| 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 |
| CN116650380A (en) * | 2023-06-21 | 2023-08-29 | 广州中妆美业化妆品有限公司 | Anti-aging tea fermentation product for improving skin microcirculation and preparation method and application thereof |
| CN116650380B (en) * | 2023-06-21 | 2024-05-17 | 广州中妆美业化妆品有限公司 | Anti-aging tea fermentation product for improving skin microcirculation and preparation method and application thereof |
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