CN103289915A - Stable and safe Bacillus subtilis preparation, its preparation method and application - Google Patents
Stable and safe Bacillus subtilis preparation, its preparation method and application Download PDFInfo
- Publication number
- CN103289915A CN103289915A CN2013100339444A CN201310033944A CN103289915A CN 103289915 A CN103289915 A CN 103289915A CN 2013100339444 A CN2013100339444 A CN 2013100339444A CN 201310033944 A CN201310033944 A CN 201310033944A CN 103289915 A CN103289915 A CN 103289915A
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- preparation
- solid
- solid fermentation
- culture
- 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
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention relates to a preparation method of a stable and safe Bacillus subtilis preparation and its application in breeding industry. The method includes: taking Bacillus subtilis as a fermentation strain, conducting culture at 37DEG C in an optimized solid medium, carrying out three-level amplification to perform large-scale fermentation production, conducting treatment by polyethylene glycol PEG4000 and sodium polyacrylate, thus obtaining the Bacillus subtilis preparation with stable activity. The Bacillus subtilis prepared by the method has stronger acid resistance and bile salt resistance, and can survive in severe environments with nutritional imbalance, has a higher survival rate in animal intestinal tracts and stronger colonization ability. Thus, adding of the Bacillus subtilis preparation as a feed additive into feed can promote absorption of nutrients in feed, improve the intestinal environment, and enhance the body immunity and disease resistance.
Description
Technical field
The present invention relates to the microbial fermentation engineering field, specifically a kind of solid medium prepares the method for probiotic feed additive and the application in aquaculture thereof through fermentation of bacillus subtilis.
Background technology
Probiotics becomes the comparatively desirable substitute of microbiotic.Genus bacillus is the internationally recognized microbial strains that can directly use in feed as the microorganism that probiotics uses.Subtilis is a kind of of bacillus, 0.7～0.8 * 2～3 microns of individual cells, and uniform coloring, no pod membrane, peritrichous can move.Gram-positive microorganism, 0.6～0.9 * 1.0～1.5 microns of gemma, oval to column, be positioned at thalline central authorities or inclined to one side slightly, gemma forms the back thalline and does not expand.The bacterium colony surface irregularity is opaque, dirty white or little yellow, and when growing in the liquid medium within, the normal wrinkle mould that forms.Aerophil, available protein, multiple sugar and starch decompose tryptophane and form indoles.
The subtilis mechanism of action: the subtilyne that produces in the subtilis thalli growth process, polymyxin, nystatin, linear gramicidins isoreactivity material, these active substances have the obvious suppression effect to the conditioned pathogen of pathogenic bacterium or autogenous infection; Subtilis consumes the free oxygen in the environment rapidly, causes the enteron aisle hypoxemia, promotes the growth of useful anerobe, and produces organic acid such as lactic acid, reduces the enteron aisle pH value, suppresses other pathogenic bacterium growth indirectly; Stimulate growing of animal immune organ, activate T, bone-marrow-derived lymphocyte, improve immunoglobulin (Ig) and antibody horizontal, strengthen cellular immunization and humoral immune function, improve herd immunity; Subtilis thalline self synthesizes enzymes such as α-Dian Fenmei, proteolytic enzyme, lipase, cellulase, in digestive tube with animal body in digestive enzymes together play a role; Multiple vitamin B group such as energy synthesise vitamins B1, B2, B6, nicotinic acid, the activity of raising animal body internal interference element and scavenger cell.
The formation of gemma is subjected to Effect of Environmental, and different strain forms the condition difference of gemma.The gemma of subtilis is easily preserved than other nourishing body cell, and the survival rate height be the desirable existence form of preparation bacillus subtilis formulation, and the gemma content in the preparation is the key factor that influences the product application effect.At present, the mode of production of bacillus preparation has two kinds: liquid state fermentation and solid state fermentation.Liquid state fermentation easily realizes pure culture and suitability for industrialized production, but to the equipment requirements height, complex manufacturing, and environmental pollution is serious; And the raw material that solid fermentation adopts generally is cheap agricultural byproducts (as wheat bran, husk etc.), and the equipment of employing is also simple than liquid fermenting, and production cost is significantly less than liquid fermenting.The present invention is research object with a bacillus subtilis, realizes large-scale industrial production by multistage amplification solid fermentation.
Polyoxyethylene glycol PEG-4000 can be used for tablet, capsule, film-coat, dripping pill, the suppository in the medicine, because in the process of film-making, the ability that tablet discharges medicine, the PEG(PEG4000 of high molecular, PEG6000, PEG8000 can be improved in the plasticity-of PEG and it) be way of great use as the tackiness agent of making tablet.Food-grade polypropylene acid sodium is water-soluble high-molecular compound, has extremely strong thickening water retaining function, product purity height, performance are very stable, odorless, tasteless, stored for a long time not corrupt, can be widely used in wheaten food class, processed food and frozen product, be the foodstuff additive that U.S. FDA, Japanese health ministry and ministry of Health of China approval are used, harmless, be easy to preserve and storage.The subtilis solid fermentation culture that process PEG-4000 and sodium polyacrylate were handled, bacterial activity is more stable, loss of activity is littler in processing, transportation and storage, and can reach animal intestinal owing to its slow releasing function, improve the ability of subtilis field planting animal intestinal greatly, improved the bioactive stability of said preparation greatly.
Summary of the invention
The object of the present invention is to provide a kind of subtilis culture fermentative production and stabilization method.
Another object of the present invention is to provide the subtilis culture of this stabilization as the application of additive in aquaculture.
We improve its substratum selection, single factor experiment and the test of 4 factors, 3 horizontal quadratures that bacillus subtilis bacteria solid fermentation substratum carries out carbon source, nitrogenous source and buffering salt, obtain best solid culture based formulas, solid butt: be basestocks (ratio of quality is 3:1:1) with soybean cake powder, Semen Maydis powder and wheat-flour, glucose 5.0%, CaCO
31.5%, MnSO
4H
2O 0.04%.It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and the pH value is adjusted to 6.5, stirs.Solid medium through 121 ℃, 20min autoclave sterilization, namely be can be used as the solid fermentation substratum after cooling and uses.It is 35 ~ 37 ℃ that the fermentation of bacillus subtilis condition is optimized to obtain its optimum culturing temperature, initial pH value 6.5, inoculum size 10%.Behind the product process PEG4000 and sodium polyacrylate processing with solid fermentation, drying makes stable bacillus subtilis formulation.
Show that by experimentation on animals the solid fermentation culture of this bacterium is promoting appetite, putting on weight, prevents that aspect such as diarrhoea from having a significant effect.
Embodiment
The technical process of the bacillus subtilis formulation of embodiment 1 preparation stabilization
1. actication of culture:
The present invention adopts subtilis as fermented bacterium, purchases in microbial strains preservation center, Guangdong Province, preserving number, GIM 1.131, with the Bacillus subtilis strain of-80 ℃ of preservations at seed culture medium (peptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO
4Be added into 0.2%, agar powder 15g adds water to 1 liter, and autoclave sterilization is standby) rule on the solid plate, cultivated about 16 hours activation for 37 ℃.
2. preparation first order seed:
With the subtilis of activation, be inoculated in (peptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO in the 300 mL liquid seed culture mediums with transfering loop picking one ring
4Be added into 0.2%, add water to 1 liter, autoclave sterilization is standby), triangular flask is placed shaking table, 37 ℃, 250rpm, shaking culture made primary seed solution after 16 ~ 20 hours.
3. prepare the solid fermentation substratum:
The solid butt: be basestocks (ratio of quality is 3:1:1) with soybean cake powder, Semen Maydis powder and wheat-flour, glucose 5.0%, CaCO
31.5%, MnSO
4H
2O 0.04%.It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and the pH value is adjusted to 6.5, stirs.Solid medium through 121 ℃, 20min autoclave sterilization, namely be can be used as the solid fermentation substratum after cooling and uses.
4. solid fermentation:
To insert the first order seed bacterium according to 10% inoculum size through the solid fermentation substratum of sterilising treatment, and stir, keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37 ℃ cultivated 16 ~ 20 hours, made secondary seed.Secondary seed according to carrying out enlarged culturing in 10% the inoculum size adding sterilization solid fermentation substratum, is stirred, and keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37 ℃ cultivated 16 ~ 20 hours, made three grades of seeds.Can be according to concrete cultivation consumption, further with three grades of seeds according to the further enlarged culturing of 10% inoculum size.The subtilis culture is carried out enumeration, require the viable bacteria bulk concentration can reach 1.0 * 10
8More than the bright song of CFU/g, the gemma rate of formation is more than 90%.
5. stabilization treatment:
With the subtilis solid fermentation culture of oven dry, add mass ratio and be 0.25% sodium polyacrylate and 5% PEG4000(and be dissolved in water in advance), heat under the stirring and evenly mixing, airing or 65 ℃ and handle 60min and make finished product with inner drying.
The bacillus subtilis formulation of embodiment 2 stabilizations is to farrow in later stage of pregnancy to the lactation influence of performance and lactation power of sow
1 experimental design:
For verifying this bacillus subtilis formulation to the influence of growing-finishing pigs performance and immunizing power, select 64 antenatal sows of 30 days, the different varieties ratio is (white 10 of experiment group leader, 22 of Da Bai quite; White 10 of group leader of contrast, 22 of Da Bai) sow is divided into an experimental group and a control group at random, and experimental group and control group are set 4 repetitions respectively, and every group is respectively experimental group 5,7,10,10 according to the production practical situation; Control group 5,7,10,10.Experimental period is from sow extremely wean of warp in antenatal 30 days, about about 51 days of experimental period.Control group uses normal commercial feed, and experimental group is added 0.5% bacillus subtilis formulation on Normal Goods feed basis, experiment detailing such as table 1:
Table 1 daily ration structure
2 experiment statistics indexs:
The total litter size of experimental record sow, the strong young number of birth, piglet birth are heavy, piglet is transferred initial weight behind the hurdle, weaned piglet is heavy and piglet survival ratio.
3 experimental results:
3.1 bacillus subtilis formulation is to the farrowing sow Effect on Performance of farrowing
As table 2, shown in the table 3, experiment has been chosen 64 sows altogether as sample, divides 32 of experimental group, 32 of control groups.The farrowing situation is as shown in table 2, and experimental group is always farrowed 330,316 of strong young sums, and strong young rate 95.76%, it is 9.88 that every sow produces strong young number; 338 of the total farrowing of control group, 304 of strong young sums, strong young rate 89.84%, it is 9.50 that every sow produces strong young number; Strong young rate experimental group is higher 5.92 percentage points than control group, promotes 6.6%; The heavy experimental group of piglet birth promotes 8.1% than the heavy 0.12kg of control group.
Table 2 farrowing performance situation
The heavy situation of table 3 piglet birth
3.2 bacillus subtilis formulation is to the influence of milking sow lactation power
The index of estimating the sow milking capacity is piglet weightening finish during suckling.Because there is the hurdle situation of transferring in piglet birth back in the actual production, thus we add up transfer behind the hurdle every sow with the piglet number and the counterpoise of piglet as an important statistical indicator.From table 4, table 5 is transferred experimental group initial weight 1.82 kg behind the hurdle as can be seen, 21 age in days weaning weights, 5.49 kg, and 3.67 kg that increase weight, piglet is 93.60% in the surviving rate of nursery phase.Control group is transferred control group initial weight 1.98 kg behind the hurdle, 21 age in days weaning weights, 5.38 kg, and 3.40 kg that increase weight, piglet is 89.43 % in the surviving rate of nursery phase, 4.17 percentage points of surviving rate differences promote 4.7%.
Table 4 lactation power situation
Table 5 lactation surviving rate situation
4 interpretations
The strong young number of piglet birth, birth weight, weaning weight, the strong young number of wean all produce considerable influence to follow-up production achievement.The index that can directly estimate economic benefit is strong young number and the piglet weightening finish at nursery phase of weaning.Using bacillus subtilis formulation after 51 days, sow produces all that strong young number, birth are heavy, weightening finish between lactation, piglet survival ratio all are significantly improved.
Claims (7)
1. the preparation method of the bacillus subtilis formulation of a stability and safety may further comprise the steps:
(1) actication of culture:
The present invention adopts subtilis as fermented bacterium, purchases in microbial strains preservation center, Guangdong Province, preserving number, GIM 1.131, with the Bacillus subtilis strain of-80 ℃ of preservations at seed culture medium (peptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO
4Be added into 0.2%, agar powder 15g adds water to 1 liter, and autoclave sterilization is standby) solid rules on the inclined-plane, cultivates about 16 hours activation for 37 ℃;
(2) preparation first order seed:
With the subtilis of activation, be inoculated in (peptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO in the 300 mL liquid seed culture mediums with transfering loop picking one ring
4Be added into 0.2%, add water to 1 liter, autoclave sterilization is standby), triangular flask is placed shaking table, 37 ℃, 250rpm, shaking culture made primary seed solution after 16 ~ 20 hours;
(3) preparation solid fermentation substratum:
The solid butt: be basestocks (ratio of quality is 3:1:1) with soybean cake powder, Semen Maydis powder and wheat-flour, glucose 5.0%, CaCO
31.5%, MnSO
4H
2O 0.04%.It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and the pH value is adjusted to 6.5, stirs.Solid medium through 121 ℃, 20min autoclave sterilization, namely be can be used as the solid fermentation substratum after cooling and uses;
(4) solid fermentation:
To insert the first order seed bacterium according to 10% inoculum size through the solid fermentation substratum of sterilising treatment, and stir, keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37 ℃ cultivated 16 ~ 20 hours, made secondary seed.Secondary seed according to carrying out enlarged culturing in 10% the inoculum size adding sterilization solid fermentation substratum, is stirred, and keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37 ℃ cultivated 16 ~ 20 hours, made three grades of seeds.Can be according to concrete cultivation
Consumption, further with three grades of seeds according to the further enlarged culturing of 10% inoculum size.The subtilis culture is carried out enumeration, require the viable bacteria bulk concentration can reach 1.0 * 10
8More than the bright song of CFU/g, the gemma rate of formation is more than 90%;
(5) stabilization treatment:
With the subtilis solid fermentation culture of oven dry, add mass ratio and be 0.25% sodium polyacrylate and 5% PEG4000(and be dissolved in water in advance), heat under the stirring and evenly mixing, airing or 65 ℃ and handle 60min and make finished product with inner drying.
2. the bacillus subtilis formulation of stability and safety according to claim 1 is characterized in that described solid butt: be basestocks (ratio of quality is 3:1:1) with soybean cake powder, Semen Maydis powder and wheat-flour, and glucose 5.0%, CaCO
31.5%, MnSO
4H
2O 0.04%.
3. bacillus subtilis bacteria solid fermentation according to claim 1, it is characterized in that: culture temperature is that temperature is 35 ~ 37 ℃, and humidity is 50 ~ 70%.
4. bacillus subtilis bacteria solid fermentation according to claim 1 is characterized in that: at different levels kinds of daughter bacteria inoculum sizes are 10% inoculum size.
5. bacillus subtilis bacteria solid fermentation according to claim 1, it is characterized in that: used bacterial classification is the subtilis of bacillus.
6. bacillus subtilis bacteria solid fermentation according to claim 1, it is characterized in that: the addition as fodder additives is 0.5 ~ 2%.
7. fermentation of bacillus subtilis according to claim 1, it is characterized in that: the Application Areas as fodder additives is additive for farm animal feed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100339444A CN103289915A (en) | 2013-01-29 | 2013-01-29 | Stable and safe Bacillus subtilis preparation, its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100339444A CN103289915A (en) | 2013-01-29 | 2013-01-29 | Stable and safe Bacillus subtilis preparation, its preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103289915A true CN103289915A (en) | 2013-09-11 |
Family
ID=49091453
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013100339444A Pending CN103289915A (en) | 2013-01-29 | 2013-01-29 | Stable and safe Bacillus subtilis preparation, its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103289915A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103535544A (en) * | 2013-09-29 | 2014-01-29 | 菏泽和美华饲料有限公司 | Feed for improving farrowing weight of farrowing sow |
| CN103981118A (en) * | 2013-12-24 | 2014-08-13 | 北京伟嘉人生物技术有限公司 | Bacillus subtilis feed additive and preparation method and application thereof |
| CN110637925A (en) * | 2018-06-27 | 2020-01-03 | 无锡三智生物科技有限公司 | Preparation process of fermentation microbial inoculum for fermenting feed |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101298593A (en) * | 2008-05-23 | 2008-11-05 | 厦门天工生化有限公司 | Straw microorganism composite transformation agent, preparation and use in protein feed |
| CN102352334A (en) * | 2011-10-21 | 2012-02-15 | 山东省农业科学院高新技术研究中心 | Method for producing viable bacillus subtilis by way of solid fermentation |
-
2013
- 2013-01-29 CN CN2013100339444A patent/CN103289915A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101298593A (en) * | 2008-05-23 | 2008-11-05 | 厦门天工生化有限公司 | Straw microorganism composite transformation agent, preparation and use in protein feed |
| CN102352334A (en) * | 2011-10-21 | 2012-02-15 | 山东省农业科学院高新技术研究中心 | Method for producing viable bacillus subtilis by way of solid fermentation |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103535544A (en) * | 2013-09-29 | 2014-01-29 | 菏泽和美华饲料有限公司 | Feed for improving farrowing weight of farrowing sow |
| CN103535544B (en) * | 2013-09-29 | 2015-09-09 | 菏泽和美华饲料有限公司 | A kind of feed improving farrowing sow farrowing and weigh |
| CN103981118A (en) * | 2013-12-24 | 2014-08-13 | 北京伟嘉人生物技术有限公司 | Bacillus subtilis feed additive and preparation method and application thereof |
| CN110637925A (en) * | 2018-06-27 | 2020-01-03 | 无锡三智生物科技有限公司 | Preparation process of fermentation microbial inoculum for fermenting feed |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102199590B (en) | Method for preparing butyric acid bacteria powder by microencapsulated propagation culture and application | |
| CN101580799B (en) | Micro-ecological preparation and application thereof | |
| CN101690545B (en) | Method for producing complex micro-ecological preparation with microbial agents and enzyme | |
| CN101449744B (en) | Multifunctional biology aquatic feedstuff additive | |
| CN103275907B (en) | Bacillus amyloliquefacien and preparation method and application thereof | |
| CN103039699B (en) | Mixed fermentation feed and preparation method and application method of mixed fermentation feed | |
| CN102067941B (en) | Method for producing compound microbial preparation serving as fodder | |
| CN102696860B (en) | Highly efficient and low-cost microbiological feed proteins based on vinegar residue and miscellaneous meal | |
| CN103289910A (en) | Solid fermentation production method of bacillus coagulans | |
| CN101971920B (en) | Porcine lactobacillus reuteri lyophilized preparation and preparation method thereof | |
| CN105002116A (en) | Microorganism straw feed microbial agent and preparation method thereof | |
| CN102813091A (en) | Compound premix for pigs and mixed batch of compound premix | |
| CN102517227B (en) | Enterococcus faecalis and applications and feed additive and leavening agent thereof | |
| CN101912056A (en) | Method for preparing drug residue free feed for feeding live pigs | |
| CN101589771A (en) | The preparation method of bio-additive premixed feed special for pigs and bioactive substance carrier thereof | |
| CN105199978A (en) | Bacillus coagulans preparation for livestock breeding and preparation method thereof | |
| JP2010029117A (en) | Animal feed composition containing lactobacillusplantarum, animal mixed feed containing the same, and method for retaining or proliferating the lactobacillusplantarum in animal intestinal canal | |
| CN103289915A (en) | Stable and safe Bacillus subtilis preparation, its preparation method and application | |
| CN105211632A (en) | A kind of multiple-effect microbial feed addictive and preparation method thereof | |
| CN103960479A (en) | High-protein orange pomace fermented feed and preparation technique thereof | |
| CN103283939A (en) | Solid fermentation preparation method for yeast protein feed | |
| CN103305435A (en) | Method for preparing lactobacillus plantarum culture | |
| CN114190486A (en) | Microbial fermented feed for preventing and treating constipation of sows and sow breeding method | |
| CN111690573B (en) | Lactobacillus johnsonii for animal intestinal probiotics and ferment prepared by fermenting same and application thereof | |
| CN103289909A (en) | Solid fermentation production method of bacillus licheniformis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130911 |