CN103060236A - Bacillus subtilis capable of producing high-yield protease and application of bacillus subtilis - Google Patents
Bacillus subtilis capable of producing high-yield protease and application of bacillus subtilis Download PDFInfo
- Publication number
- CN103060236A CN103060236A CN2012105942734A CN201210594273A CN103060236A CN 103060236 A CN103060236 A CN 103060236A CN 2012105942734 A CN2012105942734 A CN 2012105942734A CN 201210594273 A CN201210594273 A CN 201210594273A CN 103060236 A CN103060236 A CN 103060236A
- Authority
- CN
- China
- Prior art keywords
- hzd
- bacillus subtilis
- subtilis
- animals
- test
- 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.)
- Pending
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention relates to bacillus subtilis Hzd-bs-001 with the collection number of CGMCC No.6928. The bacillus subtilis Hzd-bs-001 can be applied to biological fermentation feed, microecological preparations and animal health-care products and is probiotics beneficial for growth of animals. The test indicates that the bacillus subtilis Hzd-bs-001 has high capacity of decomposing protein, can obviously increase the utilization ratio of animal protein, can effectively reduce the morbility of animal diseases, can balance intestinal bacterial colonies, particularly has extremely obvious effect of preventing and treating piglet diarrhea, improves the immunity of the animals, and promotes healthy and quick growth of the animals. The invention has the advantages that the bacillus subtilis Hzd-bs-001 is separated from soil for stacking beer vinasse for many years and has high degradation rate on biomass with high protein content such as beer vinasse and soy sauce dregs, so high-activity protease can be generated in the fermentation process; and the bacillus subtilis Hzd-bs-001 can effectively prevent and treat the piglet diarrhea.
Description
Technical field
The present invention relates to bacterial strain, particularly a kind of subtilis of high proteinase yield and the application in feed fermentation thereof
Background technology
Genus bacillus is widely distributed at occurring in nature, is generally aerobic or amphimicrobian Gram-positive bacillus, can produce the endogenous spore of circle, ellipse or column in the born of the same parents.In the gemma wall lamina corticalis is arranged, heat, ultraviolet ray and ionizing radiation and antibiotic are had very high resistance, can tolerate the advantages such as feed processing, storage, acidproof, high temperature, anti-extruding, therefore the research of pair genus bacillus in feed just arranged very early.
Subtilis has stronger product proteolytic enzyme, cellulase, amylase ability, and can secrete the secondary meta-bolitess such as multiple antibiotic; Can regulate the microbial population of animal intestinal tract balance by the enteron aisle occupy-place, improve intestine microenvironment, promote animal health; Growing of energy stimulating animal immune organ strengthens cellular immunization and humoral immune function, improves immunity of organisms.
In the 80's of 20th century, as probiotics, present many countries use bacillus feed additive to some scholar's research in a large number with genus bacillus, such as some country of Europe, the U.S., Japan etc., and have obtained great successes.China is relatively late to the research of microecological bacillus preparation, just enter industrialization development and exploitation recent years, and relatively single, functional poor, the easily microbiological contamination of the bacterial classification of these products, the practical application effect of little ecological product in livestock-raising is very little, and industrialized development is restricted.
Summary of the invention
For the defective that exists in the prior art, the object of the present invention is to provide a strain to be used for the subtilis of the high proteinase yield of fermented feed.This subtilis (Bacillus subtilis) Hzd-bs-001, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 3rd, 2012, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preserving number: CGMCC No.6928.It can be used for biological fermentation feed and animal health product, is a kind of probiotic bacterium with higher actual application value.The present invention shows by test: subtilis (Bacillus subtilis) Hzd-bs-001 has stronger protelytic ability, can significantly improve the livestock and poultry protein utilization rate, can effectively reduce the sickness rate of livestock and poultry, the balance intestinal colony, very significant action effect is especially arranged in the control baby pig diarrhoea, improve the immunizing power of livestock and poultry, promote the animal health Fast Growth.
Advantage of the present invention is: (1) subtilis (Bacillus subtilis) Hzd-bs-001 separates from the soil of old stacking brewer's grains, the higher biomass of protein content such as brewer's grains, soy sauce grain there is very high degradation rate, because producing highly active proteolytic enzyme during the fermentation.(2) subtilis (Bacillus subtilis) Hzd-bs-001 can prevent and treat baby pig diarrhoea very effectively.
Description of drawings:
Fig. 1 subtilis (Bacillus subtilis) Hzd-bs-001 colonial morphology photo;
Fig. 2 subtilis (Bacillus subtilis) Hzd-bs-001 microscopically form photo
Fig. 3 subtilis (Bacillus subtilis) Hzd-bs-001 16SrDNA electrophorogram;
The phylogenetic tree that the 16SrDNA of the bacterial strain that communicates of including among Fig. 4 subtilis (Bacillus subtilis) Hzd-bs-001 and the Genbank is constructed.
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
Embodiment 1
The acquisition of subtilis (Bacillus subtilis) Hzd-bs-001 and culture condition thereof detect
1, screening and the evaluation of subtilis (Bacillus subtilis) Hzd-bs-001
1) screening of subtilis (Bacillus subtilis) Hzd-bs-001
(1) take by weighing 1g soil from the soil of brewer's grains is piled up by Qingdao Beer Brewery throughout the year place the 100ml sterilized water, after stirring with glass stick, at the vibrator 30min that vibrates, microorganism cells is disperseed again, leave standstill 20s～30s, its aqueous solution is 10
-2Diluent is drawn the 1ml diluent again in the test tube of 9ml sterilized water, and the vibration mixing is 10
-3Diluent is drawn 1ml 10 again
-3The diluent mixing that vibrates in the 9ml sterilized water is 10
-4Diluent makes 10
-2, 10
-3, 10
-4Serial dilutions.
(2) with culture medium flat plate numbering (each numbering is established 3 repetitions), dip each serial diluent one with the disinfection inoculation ring and encircle in the separation of having solidified and rule on culture medium flat plate.Line can be undertaken by following dual mode: a kind of is cross-streak method, one side be for the first time " Z " font line of doing at flat board.Rotate the about 70 ° of angles of culture dish, transfering loop after fire burnt and cooled off, by the part of ruling for the second time, is done for the second time " Z " font line.Carry out for the third time, rule for the 4th time with method.Another kind is continuous method of scoring, be from plate edge a bit. do continuously closely the wave line, until dull and stereotyped central authorities.Rotate 180 ° in culture dish, again from the same line of dull and stereotyped the other side (not burning transfering loop) to dull and stereotyped central authorities.
(3) with the flat board reversing, 37 ℃ of cultivations of constant temperature are observed two days later.
(4) repeatedly repeat (2) (3) 4～5 times, make and be entirely single culture in the substratum.
Separate and use substratum: adopt nutrient agar (NA) to cultivate, prescription: peptone 10g, extractum carnis 3g, sodium-chlor 5g, agar 15-20g, distilled water 1000ml, pH 7.0-7.2,121 ℃, 30min.
2) evaluation of bacterial classification
The single bacterial strain of picking separator well is in the 500ml triangular flask that 200ml liquid MNB substratum is housed from the NA substratum, and shaking culture 24h under 230r/min, the 35 ℃ of conditions carries out extraction, the evaluation of 16SrDNA again.
Described MNB culture medium prescription is as follows: glucose 5.0g, and peptone 5.0g, extractum carnis 3.0g, yeast extract paste 1.0g, magnesium sulfate heptahydrate 0.5g, manganous sulfate 0.005g, water 1000mL, pH 7.0-7.2, solid medium adds agar 15-20g, 115 ℃ of sterilization 30min.
Microbial characteristic: bacterium colony is irregular row, and surface drying grows into the later stage center and forms easily volcano sample projection, dirty white or faint yellow, as shown in Figure 1.Microscopically is observed visible thalline and is shaft-like single giving birth to or chain, the peritrichous gram-positive microorganism, and the tool gemma, often gemma is positioned at the thalline centre, ovalization, as shown in Figure 2.
The 16SrDNA sequence of subtilis (Bacillus subtilis) Hzd-bs-001 is shown in SEQ ID No.1.Institute is surveyed the 16SrDNA sequence contrast by BLAST, the constructing system evolutionary tree assert that it is subtilis as shown in Figure 4.
Biochemical Characteristics:
The Biochemical Characteristics of table 1 subtilis (Bacillus subtilis) Hzd-bs-001
Annotate: "+" expression growth or positive reaction; "-" expression is not grown or negative reaction.
Embodiment 2
The salt tolerance test of bacterial strain
Under the subtilis Hzd-bs-001 aseptic condition, by 5% inoculum size in different salinity fermention mediums, 230r/min, 35 ℃ of shaking culture are measured its OD600 value every 2h, and are as shown in table 2.Test-results shows that subtilis Hzd-bs-001 has stronger salt tolerance, is fit to the fodder production requirement.
The salt tolerance test of table 2 subtilis (Bacillus subtilis) Hzd-bs-001
Embodiment 3
The temperature tolerance test of bacterial strain
Under the subtilis Hzd-bs-001 aseptic condition, by 5% inoculum size in identical fermention medium, 230r/min, shaking culture under the differing temps is measured its OD600 value every 2h, and is as shown in table 2.Test-results surface subtilis Hzd-bs-001 grows the most vigorous in the time of 35 ℃, and preferably growth is also arranged in the time of 45 ℃, is fit to the fodder production requirement.
The temperature tolerance test of table 3 subtilis (Bacillus subtilis) Hzd-bs-001
Embodiment 4
Acid resisting test:
Subtilis Hzd-bs-001 presses 5% inoculum size in identical fermention medium, 230r/min, and 35 ℃ of lower shaking culture are measured its OD every 2h
600Value, as shown in table 4.Test-results surface subtilis Hzd-bs-001 has preferably acid resistance, thus subtilis Hzd-bs-001 can with the acid-producing bacteria co-fermentation such as milk-acid bacteria, also can adapt to preferably the environment behind the fermentation and acid.
The acid resisting test of table 4 subtilis (Bacillus subtilis) Hzd-bs-001
Embodiment 5
Subtilis (Bacillus subtilis) Hzd-bs-001 is applied to fermented feed
Seed culture medium: adopt nutrient agar (NA) to cultivate, prescription: peptone 10g, extractum carnis 3g, sodium-chlor 5g, distilled water 1000ml, pH 7.0-7.2,121 ℃, 30min.
Fermention medium: glucose 20g, peptone 15g, sodium-chlor 5g, distilled water 1000ml, pH 7.0-7.2,121 ℃, 30min.
Picking list bacterium colony is in the 500ml triangular flask that the 200ml seed culture medium is housed from slant medium, behind 35 ℃ of shaking culture 24h, inoculum size access by 5% under the aseptic condition is equipped with in the 500ml triangular flask of 200ml fermention medium, 35 ℃ of shaking culture 24h, i.e. subtilis Hzd-bs-001 fermented liquid is finished in preparation.
Fermented feed proportioning raw materials (1kg): 0.351 part of peanut hull meal, 0.032 part in inferior powder, 0.02 part in wheat bran, husky 0.054 part of worm, 0.5 part of brewer's grains, 0.02 part of Preblend, 0.023 part of bacterium liquid.Composition and the proportioning of Preblend are as follows: 452 parts of stone flours; 330 parts of zeolite powders; 24 parts of compound vitamins; 50 parts of secondary calcium phosphates; 50 parts of whitewashing maize peels; 90 parts of salt; 2 parts of flavouring agents; 2 parts of sweeting agents.
The proportioning of described compound vitamin is as follows:
Vitamin A 20000IU/kg vitamins D
310000IU/kg
Vitamin-E 110mg/kg vitamin K
310mg/kg
Vitamins B
1The 8mg/kg vitamins B
212mg/kg
Vitamins B
6The 7mg/kg vitamins B
120.2mg/kg
Vitamin H 25mg/kg nicotinic acid 120mg/kg
Calcium pantothenate 60mg/kg folic acid 6mg/kg
Copper 3g/kg iron 3.5g/kg manganese 2g/kg
Zinc 3g/kg iodine 9mg/kg surplus is beer yeast powder;
Control group (CK) adds the 50ml sterilized water, and test group adds 50ml subtilis Hzd-bs-001 fermented liquid, puts into valve bag after mixing, and tightens, and puts into 35 ℃ of thermostatic protectiving incubators, detects the content of protein content and hydrolase behind the 5d.
Measurement result:
The variation of table 5 protein content
The variation of table 6 hydrolase
Test shows, in fermented feed, add subtilis Hzd-bs-001 separately, significantly improved the content of the water-soluble protein in the feed, bioactive peptide and total free aminoacids, various hydrolases have been significantly improved, especially protease content, have simultaneously very highly active cellulase, be conducive to the high material of degradation of fibers cellulose content.
Embodiment 6
The application test of subtilis hzd-001 fermented liquid in growing and fattening pigs
With the subtilis Hzd-bs-001 fermented liquid among the embodiment 5
Test period: on November 1,1 day to 2011 August in 2011, amount to 90 days.
The source of test pig and grouping: all from pig farm, villages and small towns, Shandong Province's Pingdu City south; Control group adopts the complete feed of buying on the market, and test group is that identical complete feed and subtilis hzd-bs-001 fermented liquid is combined with
Test site: pig farm, villages and small towns, Shandong Province's Pingdu City south
Test-results:
The application test of table 7 subtilis hzd-001 fermented liquid in growing and fattening pigs
Adopt automatic diet machine for food.Test group is used complete feed 1360kg altogether, and subtilis hzd-001 fermented liquid amounts to 8100ml.Control group is used the 1400kg complete feed altogether.
As seen from Table 7, by adding being used of subtilis hzd-bs-001 fermented liquid, the speed of growth of pig has been accelerated, and has reduced material meat, has optimized the effective rate of utilization of feed, has reduced cost, 0.58 yuan of 1 kilogram of pork Cost reduction of every growth.
Embodiment 7
Subtilis Hzd-001 fermented liquid is at the application test of prevention baby pig diarrhoea
With the subtilis Hzd-bs-001 fermented liquid among the embodiment 5
Test period: on August 31,1 day to 2011 August in 2011, amount to one month.
The source of test pig and grouping: all from pig farm, villages and small towns, Shandong Province's Pingdu City south, test pig is 10 age in days piglets, each 100 of control group and test group; Control group adopts the piglet perfect compound feed of buying on the market, and test group is that identical piglet perfect compound feed and subtilis Hzd-bs-001 fermented liquid is combined with
Test site: pig farm, villages and small towns, Shandong Province's Pingdu City south
Test-results:
Control group has 13 piglets the diarrhea situation to occur, wherein has 2 seriously to cause death, and the diarrhea rate is 15%; Test group only has 1 hair to give birth to the piglet of slight diarrhea situation, and the diarrhea rate is 1%.This shows that subtilis Hzd-001 fermented liquid can effectively prevent baby pig diarrhoea.
Sequence table
Claims (3)
1. a subtilis (Bacillus subtilis), what it is characterized in that this subtilis is subtilis (Bacillus subtilis) Hzd-bs-001, preserving number is: CGMCC No.6928.
2. the application of subtilis according to claim 1 (Bacillus subtilis) Hzd-bs-001 on decomposing protein.
3. the application of subtilis according to claim 1 (Bacillus subtilis) Hzd-bs-001 in fermented feed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012105942734A CN103060236A (en) | 2012-12-31 | 2012-12-31 | Bacillus subtilis capable of producing high-yield protease and application of bacillus subtilis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012105942734A CN103060236A (en) | 2012-12-31 | 2012-12-31 | Bacillus subtilis capable of producing high-yield protease and application of bacillus subtilis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103060236A true CN103060236A (en) | 2013-04-24 |
Family
ID=48103136
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012105942734A Pending CN103060236A (en) | 2012-12-31 | 2012-12-31 | Bacillus subtilis capable of producing high-yield protease and application of bacillus subtilis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103060236A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114164131A (en) * | 2020-12-22 | 2022-03-11 | 佛山市海天(高明)调味食品有限公司 | Salt-tolerant bacillus and application thereof |
-
2012
- 2012-12-31 CN CN2012105942734A patent/CN103060236A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114164131A (en) * | 2020-12-22 | 2022-03-11 | 佛山市海天(高明)调味食品有限公司 | Salt-tolerant bacillus and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106260540B (en) | Biological feed for creep feed and creep feed | |
| CN106479927A (en) | Method and its application using Bacillus licheniformis biosynthesiss nanometer selenium | |
| CN102517238B (en) | Acid-producing bacillus cereus and application thereof | |
| CN103098986B (en) | Beneficial microorganism antibiont-free feed and preparation method thereof | |
| CN109749957B (en) | Preparation and application of lactobacillus gasseri preparation with aquatic pathogenic bacteria antagonistic property | |
| CN103087964A (en) | Bacillus subtilis, microecological preparation and application of bacillus subtilis in animal feed | |
| CN103070285A (en) | Microbe feed additive and preparation method thereof | |
| CN110218688B (en) | Bacillus marinus LY-23, microbial inoculum and application thereof and product using same | |
| CN105385616A (en) | Bacillus subtilis for effectively degrading zearalenone and application thereof | |
| CN103184174B (en) | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium | |
| CN102229902B (en) | Bacillus pumilus, feed additive thereof, premix, and feed mildewproof agent | |
| CN103468594A (en) | Candidautilis strain and application thereof | |
| CN102517227B (en) | Enterococcus faecalis and applications and feed additive and leavening agent thereof | |
| CN106962594A (en) | A kind of Selenium-enriched fermentation dregs of beans, preparation method and application | |
| CN103013855A (en) | Compound microbial preparation and production process thereof | |
| CN111700157A (en) | Probiotic feed additive for improving immunity of aquatic animals | |
| CN103952336B (en) | Bacillus licheniformis, bacillus subtilis and lactobacillus casei preparation and preparation | |
| CN111685235B (en) | Preparation method of lactobacillus plantarum fermented liquid complete feed for piglets | |
| CN107821789A (en) | A kind of biologic ferment for improving fishes and shrimps intestinal health degree and its preparation method and application | |
| CN102321552A (en) | Clostridium butyricum used for feeding, and application thereof | |
| CN102524534B (en) | Composite microbial additive for feed and preparation method of composite microbial additive | |
| CN113215051A (en) | Method for preparing feed probiotics by using lactobacillus through rice flour wastewater and passion fruit peel | |
| CN102907585A (en) | Probiotics and vitamin premix and mixture for middle pig | |
| CN104642749A (en) | Composite micro-ecological preparation for promoting animal digestion and absorption | |
| CN105199978A (en) | Bacillus coagulans preparation for livestock breeding and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130424 |