CN103283938A - Method for preparing bacillus subtilis active microbial agent - Google Patents
Method for preparing bacillus subtilis active microbial agent Download PDFInfo
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- CN103283938A CN103283938A CN2012104052222A CN201210405222A CN103283938A CN 103283938 A CN103283938 A CN 103283938A CN 2012104052222 A CN2012104052222 A CN 2012104052222A CN 201210405222 A CN201210405222 A CN 201210405222A CN 103283938 A CN103283938 A CN 103283938A
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- bacillus subtilis
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Abstract
The invention discloses a method for preparing a bacillus subtilis active microbial agent. The method comprises the following steps of: (1) performing solid fermentation on bacillus subtilis to obtain a bacillus subtilis bacteria solution; (2) uniformly mixing the bacillus subtilis bacteria solution and an additive according to a volume ratio of 85:15; (3) performing centrifugal bacteria collection on the mixed fermentation liquor, collecting the centrifugal bacteria, performing vacuum drying at the temperature of 60 DEG C, and obtaining the bacillus subtilis active microbial agent. The method has the beneficial effects that the invention provides an ideal method for preparing the bacillus subtilis active microbial agent, the preparation cost is low, and the prepared product is high in quality, can be preserved for a long time and can be preserved for over two years under sealing conditions; meanwhile, according to the prepared bacillus subtilis preparation, the growth of livestock and poultry is effectively promoted, the daily increasing weight of livestock and poultry is improved, and great economic benefits are brought to people.
Description
Technical field
The present invention relates to a kind of preparation method of probiotics, relate in particular to a kind of preparation method of bacillus subtilis active bacteria formulation.
Background technology
Probiotics is to adopt beneficial microbe, make the live bacteria agent that contains viable bacteria or comprise thalline and the preparation of metabolite through technologies such as cultivation, fermentation, drying, processing, have disease-resistant, cure the disease, function such as growth promotion, and have noresidue and pollute characteristics such as having no side effect and do not develop immunity to drugs.Bacillus subtilis is the desirable bacterial classification of probiotics, is included in the feed level microbe additive of the directly feeding animals that China Ministry of Agriculture announces.They are acidproof, and are alkaline-resisting, high temperature resistant, all can keep stable in pelletization and in the animal gastrointestinal tract; Bacillus subtilis energy extracellular proteinase, amylase, lipase and several amino acids, thus improve food conversion ratio, promote growth of animal, shorten breeding cycle; And can promote the growth of anaerobic bacterias such as Bifidobacterium, Bacillus acidi lactici and clostridium, effectively suppress the growth of aerobic bacteria such as enterobacteria and enterococcus in the enteron aisle, promote the growth of host's normal intestinal flora, keep the intestinal microecology balance.
In recent years, bacillus subtilis goods industry development is swift and violent both at home and abroad, becomes one of the fastest industry of global evolution, occupies more and more important position, and product category is various.Yet quality problems such as many bacillus subtilis goods ubiquity viable bacteria contents of present listing are low, the viable bacteria preservation term is short.
Therefore study the preparation method of bacillus subtilis active bacteria formulation highly significant, in the survival rate that keeps bacillus subtilis to greatest extent, make the bacillus subtilis active bacteria formulation be easy to long preservation and vigor does not reduce, be applicable to the demand of suitability for industrialized production more.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of bacillus subtilis active bacteria formulation is to overcome the problem that the active constituent content that production cost is high and production obtains is low and storage life is short that existing bacillus subtilis active bacteria formulation exists in preparation.
The technical scheme that realizes the object of the invention is as follows:
A kind of preparation method of bacillus subtilis active bacteria formulation is characterized in that, may further comprise the steps:
(1) bacillus subtilis is obtained bacillus subtilis bacterium liquid through solid fermentation;
(2) bacillus subtilis bacterium liquid and the additive volume ratio according to 85:15 is mixed;
(3) mixed zymotic fluid is carried out centrifugal collection thalline, collect centrifugal thalline, 60 ℃ of vacuum drying obtain the bacillus subtilis active bacteria formulation.
Further, consisting of of the additive in the described step (2): maize cob meal, potassium sorbate, vitamin E and vitamin C.
Preferably, maize cob meal, potassium sorbate, vitamin E and ascorbic ratio (W/V) are in the described additive: 98%:1%:0.4%:0.6%.
Beneficial effect of the present invention: (1) the invention provides a kind of preparation method of comparatively desirable bacillus subtilis active bacteria formulation, and not only preparation cost is low, and the good product quality of preparation; (2) the bacillus subtilis active bacteria formulation of the inventive method preparation can long preservation, can preserve more than 2 years under the condition of sealing; (3) the prepared bacillus subtilis formulation of the present invention, be mainly used in raiseeing, the feed addictive of fowl or aquaculture, for example: the feed addictive of chicken, duck, pig, ox, sheep, fish etc., effectively promote the growth of livestock and poultry, improve the weight that increases day by day of livestock and poultry, brought very big economic benefit to people.
The specific embodiment
Embodiment 1: the preparation of bacillus subtilis
Bacillus subtilis strain (bacillus subtilis that present embodiment adopts, available from Guangdong Province Institute of Micro-biology, deposit number is GIM1.131) activates bacterial classification inoculation on the beef extract-peptone solid medium.Put in the constant incubator then and cultivate, at 30 ℃, cultivate 20h;
The fermented and cultured of bacillus subtilis joins the bacterium colony wash-out of above-mentioned activation culture in the fermentation medium with aseptic manipulation, and 30 ℃, 200 commentaries on classics/min cultivate 20h.Consisting of of fermentation medium: sucrose 10g/L, peptone 5g/L, beef extract 5g/L, yeast soak powder 1.5g/L, manganese sulfate 0.0004g/L, pH 7.0.
Embodiment 2: add additive
According to following ratio additive preparation, consist of: maize cob meal, potassium sorbate, vitamin E and vitamin C, the ratio between them are 98:1:0.4:0.6
The above-mentioned additive for preparing is joined in the zymotic fluid among the embodiment 1, stir.Then mixed zymotic fluid is carried out centrifugal collection thalline, condition is: 4 ℃, 6000rpm, centrifugal 15min.Collect centrifugal thalline, 60 ℃ of vacuum drying get the bacillus subtilis dry bacterium powder, and the check viable count is 1.2 * 1011cfu/ml.
Embodiment 3: the Detection of Stability of bacillus subtilis active bacteria formulation
Get the bacillus subtilis active bacteria formulation sample that obtains among the embodiment 2, be divided into 2 parts, be positioned over room temperature and 37 ℃ of casees that keep sample, the sample that room temperature is placed sampling in per 3 months detects viable count and viable bacteria index, and sample sampling every month of 37 ℃ of placements detects a viable count.
Described viable count detection method may further comprise the steps:
(1) shake well fluid sample is drawn the 0.5ml fluid sample in test tube, adds the 4.5ml dilution, shake well, existing 1: 10 test liquid;
(2) dilution: draw the 0.5ml test liquid, add the 4.5ml dilution in another test tube, fully mixing makes into 1: 100 test liquid, repeats aforesaid operations, carries out 10 times of serial dilutions, until required dilution factor;
(3) drip sample: the bacterium liquid 0.2ml that draws the dilution factor (generally being 3 dilution factors reciprocal) of required mensuration drips in aseptic plate, pours the beef-protein medium that aseptic temperature is bathed into, leaves standstill behind the mixing and solidifies, and makees 6 plates altogether, and each dilution factor repeats 2 times;
(4) cultivate: treat to put upside down in incubator after plate solidifies, 30 ℃, 200 commentaries on classics/min cultivate 20h;
(5) colony counting: cultivate the growing state of each plate bacterium colony of back observation and remember clump count at plate point, overlap if any 2 or 2 above bacterium colonies, should all count, notice that the plate clump count should be between 20-300, otherwise should adjust dilution, redeterminate, behind the some meter, by formula calculate viable count, fill in record;
(6) computing formula: viable count (cfu/ml)=2 plate clump count sum/2 * 5 * dilution factor
The viable count testing result of the preparation that room temperature is placed is shown in table 1; The viable count testing result of the active bacteria formulation of 37 ℃ of placements is shown in table 2.
The active bacteria formulation room temperature preservation situation of table 1 the present invention preparation
Annotate: "-" expression viable count is lower than 1.0 * 109cfu/ml in the table.
37 ℃ of accelerated test preservations of active bacteria formulation situation of table 2 the present invention preparation
Annotate: "-" expression viable count is lower than 1.0 * 109cfu/ml in the table.
The result shows that bacillus subtilis active bacteria formulation of the present invention is preserved 15 months viable counts at normal temperatures and still remained on 2.5 * 109cfu/ml, preserves 9 months viable counts and still remain on 1.2 * 109cfu/ml under the higher environment of 37 ℃ of accelerated test temperature.
Embodiment 4: the effect of bacillus subtilis active bacteria formulation
(weight average is that 7.6 ± 0.15kg) principles by male and female half and half are divided into 4 groups at random with 28 ± 2 age in days weanling pigs of 72 health, every group of 3 repetitions, each repeats 6 pigs, group 1 is the blank group, the basal diet of feeding, the group 2 aureomycin additions of feeding are the basal diet of 75mg/kg, and group 3, group 4 are fed respectively and added the bacillus subtilis dry bacterium powder among 3 ‰ embodiment 1, the basal diet of the bacillus subtilis formulation among 3 ‰ embodiment 2.Free choice feeding and freely drinking water.Duration of test carries out feeding and management by the pig farm conventional method.Test to select the 28th day evening 8 and stop feeding, supply water as usual, 8 of mornings next day pig is only weighed on an empty stomach, calculate the average daily gain of test, average daily ingestion amount and feed efficiency.
Table 3 bacillus subtilis active bacteria formulation is to the influence of ablactation piglet growth performance
| ? | Group 1(blank) | Group 2(antibiotic) | Group 3(dry bacterium powder) | Group 4(active bacteria formulation) |
| Daily gain | 179.31±4.18 | 224.41±2.73 | 220.19±3.17 | 231.28±2.53 |
| The material anharmonic ratio | 1.85±0.27 | 1.65±0.07 | 1.66±0.31 | 1.53±0.14 |
The result shows: bacillus subtilis formulation can effectively improve the growth performance of wean sucking pig.The whole test phase, the daily gain of adding the bacillus subtilis formulation group is significantly higher than blank group and dry bacterium powder group (P＜0.05), and the material anharmonic ratio significantly is lower than blank group and dry bacterium powder group (P＜0.05) (table 2).Evidence, the bacillus subtilis active bacteria formulation that adds amount of the present invention in the daily ration of piglet can significantly promote piglet growth, promotes efficiency of feed utilization, improves growth in piglets speed and daily gain, pig production performance only is greatly improved, also alternative part antibiotic.
The present invention is not limited to above-mentioned preferred forms; anyone can draw other various forms of products under enlightenment of the present invention; no matter but do any variation in its shape or structure; every have identical with a application or akin technical scheme, all drops within protection scope of the present invention.
Claims (3)
1. the preparation method of a bacillus subtilis active bacteria formulation is characterized in that, may further comprise the steps:
(1) bacillus subtilis is obtained bacillus subtilis bacterium liquid through solid fermentation;
(2) bacillus subtilis bacterium liquid and the additive volume ratio according to 85:15 is mixed;
(3) mixed zymotic fluid is carried out centrifugal collection thalline, collect centrifugal thalline, 60 ℃ of vacuum drying obtain the bacillus subtilis active bacteria formulation.
2. the preparation method of bacillus subtilis active bacteria formulation according to claim 1 is characterized in that: the consisting of of the additive in the described step (2): maize cob meal, potassium sorbate, vitamin E and vitamin C.
3. the preparation method of bacillus subtilis active bacteria formulation according to claim 1 and 2, it is characterized in that: maize cob meal, potassium sorbate, vitamin E and ascorbic ratio (W/V) are in the described additive: 98%:1%:0.4%:0.6%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232526A (en) * | 2014-08-29 | 2014-12-24 | 湖北省生物农药工程研究中心 | Process for preparing viable bacillus subtilis preparation |
| CN104605139A (en) * | 2015-01-08 | 2015-05-13 | 河北农业大学 | Bacillus subtilis-acanthopanax senticosus polysaccharide synbiotic, as well as preparation method and application thereof as feed additive |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101109008A (en) * | 2006-07-18 | 2008-01-23 | 上海四季生物科技有限公司 | Aquifer amendment containing multiple active microorganisms and method of preparing the same |
| CN101965902A (en) * | 2010-11-05 | 2011-02-09 | 辽宁邦成曙光生物科技有限公司 | Method for preparing viable bacillus subtilis preparation for feed |
| CN102000343A (en) * | 2009-09-01 | 2011-04-06 | 天津瑞普生物技术股份有限公司 | Protective agent of live bacillus subtilis preparation |
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- 2012-10-23 CN CN2012104052222A patent/CN103283938A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101109008A (en) * | 2006-07-18 | 2008-01-23 | 上海四季生物科技有限公司 | Aquifer amendment containing multiple active microorganisms and method of preparing the same |
| CN102000343A (en) * | 2009-09-01 | 2011-04-06 | 天津瑞普生物技术股份有限公司 | Protective agent of live bacillus subtilis preparation |
| CN101965902A (en) * | 2010-11-05 | 2011-02-09 | 辽宁邦成曙光生物科技有限公司 | Method for preparing viable bacillus subtilis preparation for feed |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232526A (en) * | 2014-08-29 | 2014-12-24 | 湖北省生物农药工程研究中心 | Process for preparing viable bacillus subtilis preparation |
| CN104605139A (en) * | 2015-01-08 | 2015-05-13 | 河北农业大学 | Bacillus subtilis-acanthopanax senticosus polysaccharide synbiotic, as well as preparation method and application thereof as feed additive |
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Application publication date: 20130911 |