CN104757275B - Bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, preparation method and its application as additive for farm animal feed - Google Patents
Bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, preparation method and its application as additive for farm animal feed Download PDFInfo
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- CN104757275B CN104757275B CN201510174676.7A CN201510174676A CN104757275B CN 104757275 B CN104757275 B CN 104757275B CN 201510174676 A CN201510174676 A CN 201510174676A CN 104757275 B CN104757275 B CN 104757275B
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- bacillus subtilis
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Abstract
The present invention relates to a kind of bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, bacillus subtilis seed liquor is seeded in radix glycyrrhizae fermentation medium by it, is cultivated and is obtained through shaking；The radix glycyrrhizae fermentative medium formula is：Licorice water extract 0.5-1.5mg/L, LB fluid nutrient medium 250mg/L, pH 7.4.The present invention utilizes bacillus subtilis liquid fermentation radix glycyrrhizae, production cost is low, while promotes the increase of bacillus subtilis viable bacteria amount and gemma rate to be formed, into new probiotics after fermentation, make an addition in livestock and fowl drinking water, improve livestock and poultry digestibility, production performance and immunity.
Description
Technical field
The invention belongs to probiotics technical field, and in particular to a kind of bacillus subtilis liquid fermentation radix glycyrrhizae Tiny ecosystem
Preparation, preparation method and applications.
Background technology
Bacillus subtilis is one kind of bacillus, can form gemma, gram-positive bacteria, is raised as a kind of livestock and poultry
Feed additives can produce a variety of digestive ferments, have the function that to improve animal digestion absorption, production performance and animal immune performance.Mesh
The preceding common bacillus subtilis probiotics of in the market is more with ordinary culture medium culture, is made an addition in the form of solid powder
It is ineffective in feed or drinking-water.
The content of the invention
A kind of present invention aims to overcome that prior art defect, there is provided bacillus subtilis liquid fermentation radix glycyrrhizae Tiny ecosystem system
Agent, the probiotics has the double effects of bacillus subtilis and radix glycyrrhizae after fermentation, and its effect is significantly greater than the two
Compounding.
Preparation method and its conduct present invention also offers above-mentioned bacillus subtilis liquid fermentation radix glycyrrhizae probiotics
The application of additive for farm animal feed.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, bacillus subtilis seed liquor is seeded to sweet by it
In careless fermentation medium, cultivate and obtain through shaking；The radix glycyrrhizae fermentative medium formula is：Licorice water extract 0.5-1.5mg/
L, LB fluid nutrient medium 250mg/L, pH 7.4.
The formula of LB fluid nutrient mediums：Tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH 7.4.
The radix glycyrrhizae fermentative medium formula is preferably：Licorice water extract 1.5mg/L, LB fluid nutrient medium 250mg/L,
pH 7.4。
Preferably, the licorice water extract is obtained through following methods：Extracting liquorice adds water to cook 3 times, for the first time decoct plus 4~
The water of 5 times of weight, second of the water for decocting the water for adding 3~4 times of weight, third time decoction plus 2~3 times of weight, decocts 30 every time
~60 minutes, merge decoction liquor three times, filter to obtain the final product.
The preparation method of above-mentioned bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, it includes the following steps：
1）Bacillus subtilis strain is inoculated in LB fluid nutrient mediums, 37 ± 3 DEG C of shaking 48 ± 12h of culture, obtain
Bacillus subtilis seed liquor；
2）Bacillus subtilis seed liquor is pressed 5%（v/v）Inoculum concentration be seeded in radix glycyrrhizae fermentation medium, 37 ± 3 DEG C
Shaking culture 24-72h to obtain the final product.
Application of the above-mentioned bacillus subtilis liquid fermentation radix glycyrrhizae probiotics as additive for farm animal feed, especially
Can be as the application of additive for feed for piglets.
Radix glycyrrhizae is a kind of conventional Chinese medicine, has and invigorates the spleen and benefits qi, clearing heat and detoxicating, expelling phlegm and arresting coughing, relieving spasm to stop pain, and coordinating the drug actions of a prescription is made
With.The present invention can make bacillus subtilis and radix glycyrrhizae interact using bacillus subtilis liquid fermentation radix glycyrrhizae, after fermentation
The radix glycyrrhizae activity substance content such as glycyrrhizic acid, licoflavone can be improved, reduces its toxic side effect；Bacillus subtilis can be made to live at the same time
Bacterial content substantially increases.
Compared to the prior art, beneficial effects of the present invention：
The present invention utilizes bacillus subtilis liquid fermentation radix glycyrrhizae, and production cost is low, while promotes bacillus subtilis to live
Bacterium amount increases and gemma rate is formed, and a kind of new probiotics is formed after fermentation, is made an addition in livestock and fowl drinking water, can significantly be carried
Digestion power, production performance and the immunity of high livestock and poultry.
Embodiment
The present invention is described further by the following examples, but protection scope of the present invention not limited to this.
Bacillus subtilis strain used is purchased from the management of Chinese industrial Microbiological Culture Collection in following each embodiments
The heart（CICC）, numbering 23759.The formula of LB fluid nutrient mediums：Tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/
L, pH 7.4.
The licorice water extract is obtained through following methods：Extracting liquorice adds water to cook 3 times, decocts add 5 times of weight for the first time
Water, second of the water for decocting the water for adding 4 times of weight, third time decoction plus 3 times of weight, every time decoction 50 minutes, merging are decocted three times
Boil liquid, is filtered to obtain the final product.
Comparative example 1
A kind of bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, bacillus subtilis seed liquor is seeded to sweet by it
In careless fermentation medium, cultivate and obtain through shaking；The radix glycyrrhizae fermentative medium formula is：Licorice water extract 0%, the training of LB liquid
Base 0.25% is supported, surplus is water, pH 7.4.
The preparation method of above-mentioned bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, it includes the following steps：
1）Bacillus subtilis strain is inoculated in LB fluid nutrient mediums, 37 DEG C of shaking culture 48h, obtain seed liquor；
2）Seed liquor is pressed 5%（v/v）Inoculum concentration be seeded in radix glycyrrhizae fermentation medium, 37 DEG C shaking culture 24-72h
To obtain the final product.
Embodiment 1
A kind of bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, bacillus subtilis seed liquor is seeded to sweet by it
In careless fermentation medium, cultivate and obtain through shaking；The radix glycyrrhizae fermentative medium formula is：Licorice water extract 0.5mg/L, LB liquid
Body culture medium 250mg/L, pH 7.4.
Licorice water extract 5%, LB fluid nutrient mediums 0.25%, surplus are water, pH 7.4.
The preparation method of above-mentioned bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, it includes the following steps：
1）Bacillus subtilis strain is inoculated in LB fluid nutrient mediums, 37 DEG C of shaking culture 48h, obtain withered grass gemma
Bacillus seed liquor；
2）Bacillus subtilis seed liquor is pressed 5%（v/v）Inoculum concentration be seeded in radix glycyrrhizae fermentation medium, 37 DEG C are shaken
Shake culture 24-72h to obtain the final product.
Embodiment 2
In the bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, radix glycyrrhizae fermentative medium formula is：Radix glycyrrhizae
Aqueous extracts 1.0mg/L, LB fluid nutrient medium 250mg/L, pH 7.4.Other and preparation method is the same as embodiment 1.
Embodiment 3
In the bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, radix glycyrrhizae fermentative medium formula is：Radix glycyrrhizae
Aqueous extracts 1.5mg/L, LB fluid nutrient medium 250mg/L, pH 7.4.Other and preparation method is the same as embodiment 1.
Embodiment 4
In the bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, radix glycyrrhizae fermentative medium formula is：Radix glycyrrhizae
Aqueous extracts 2.0mg/L, LB fluid nutrient medium 250mg/L, pH 7.4.Other and preparation method is the same as embodiment 1.
Bacillus subtilis biomass test in probiotics described in the various embodiments described above
Test sample：Comparative example 1 and the micro-ecology fermentation product prepared by embodiment 1-4.
Test method：24h, 48h, 72h after fermentation above-described embodiment is sampled carry out bacillus subtilis,
Gemma rate and pH measure.Bacillus subtilis assay method：It is sterile equipped with 9mL physiological saline to draw the even liquid injection of 1mL samples
In test tube, mix, be made 1:The 10 even liquid of sample.According to aforesaid operations order, doubling dilution to 10 successively-10.Select 3 companies
Continuous acceptable diluent degree, each dilution factor take 0.1mL sample diluting liquids, are respectively placed on 2 plain agar tablets, and coating is equal
It is even.37 DEG C of culture 48h, count all bacterium colonies on tablet.According to formula：In every milliliter of sample on clump count=tablet
Bacterium solution additive amount in clump count × extension rate/tablet, to calculate total number of bacteria in fermented sample.Result of the test is shown in Table 1.
It can be seen that from above-mentioned table 1：It is withered with the increase of licorice water extract adding proportion in radix glycyrrhizae fermentation medium
Careless bacillus biomass also substantially increases；When licorice water extract additive amount is 1.5mg/L, the quantity of thalline is most, and ferment 72h
5.98 × 10 can be reached9；Subsequent thalline quantity is begun to decline again, illustrates that addition 1.5mg/L licorice water extracts are proper, together
When thalline gemma rate also reach highest.From the point of view of the result of the test：Appropriate addition licorice water extract is for bacillus subtilis bar
The propagation of bacterium and the generation of gemma have positive effect.
Relevant application test is carried out with the formula of embodiment 3：
Select weight, age in days（28 ages in days）Similar ternary（Du × length × big）Weanling pig 200, is randomly divided into 5 groups,
Every group of 4 repetitions, it is each to repeat 10（Male and female is fifty-fifty）, respectively test 1 group（0.2%）, test 2 groups（0.4%）, test 3 groups
（0.6%）, blank control group, bacillus subtilis control group（0.005%）.30 days experimental periods, whole process is using free choice feeding, drinking-water
Feeding method, carried out disinfection and be immunized by pig farm conventional program.Spirit, the appetite situation of pig are examined during experiment,
And carry out detailed record.Test pig is carried out one by one with the end of to weigh on an empty stomach respectively at on-test, is recorded in units of group
Feed intake, calculates average daily gain, average daily gain and feedstuff-meat ratio.
Average daily gain=（Total feed intake/pig's head number）/ experiment number of days
Average daily gain=（Last weight-initial weight）/ experiment number of days
Feedstuff-meat ratio=total feed intake/total augment weight
In the 2nd day morning after the test on an empty stomach to each test group piglet venous blood collection, -20 DEG C are stored in after preparing serum
In refrigerator, the content of IgG, IgA in each test group serum are detected respectively using kit and microplate reader.
As shown in Table 2, three average daily gain, average daily gain, feedstuff-meat ratio indexs, it is micro- to add present invention fermentation radix glycyrrhizae
The test group of ecological agent is better than blank control group and addition bacillus subtilis group effect, and significant difference.Addition 0.6%
3 groups of general effects of experiment it is preferable, average daily gain is respectively increased than blank control group, bacillus subtilis control group
13.64% and 9.08%；20.29% and is respectively increased than blank control group, bacillus subtilis control group in average daily gain
14.77%；7.69% and 6.25% is respectively increased than blank control group, bacillus subtilis control group in feedstuff-meat ratio.
As shown in Table 3, IgG, IgA content add the test group of present invention fermentation radix glycyrrhizae probiotics than sky in serum
White control group and addition bacillus subtilis group effect are good, and significant difference.3 groups of general effects of experiment of addition 0.6% are preferable,
12.60% and 5.69% is respectively increased than blank control group, bacillus subtilis control group in IgG content；IgA content groups are than blank pair
15.45% and 13.24% is respectively increased according to group, bacillus subtilis control group.
Therefore, selection addition 0.6% when present invention fermentation radix glycyrrhizae probiotics uses（Percentage by weight）Compare in feed
It is appropriate, animal immunizing power can be significantly improved.
Claims (3)
1. a kind of bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, it is characterised in that by bacillus subtilis seed liquor
It is seeded in radix glycyrrhizae fermentation medium, cultivates and obtain through shaking；The radix glycyrrhizae fermentative medium formula is：Licorice water extract
0.5-1.5mg/L, LB fluid nutrient medium 250mg/L, pH 7.4；
The licorice water extract is obtained through following methods：Extracting liquorice adds water to cook 3 times, decocts add 4~5 times of weight for the first time
Water, second of the water for decocting the water for adding 3~4 times of weight, third time decoction plus 2~3 times of weight, decocts 30~60 minutes every time,
Merge decoction liquor three times, filter to obtain the final product；
The preparation method of the bacillus subtilis liquid fermentation radix glycyrrhizae probiotics, includes the following steps：
1）Bacillus subtilis strain is inoculated in LB fluid nutrient mediums, 37 ± 3 DEG C of shaking 48 ± 12h of culture, obtain withered grass
Bacillus seed liquor；
2）Bacillus subtilis seed liquor is seeded in radix glycyrrhizae fermentation medium by 5% inoculum concentration, 37 ± 3 DEG C of shaking cultures
24-72h to obtain the final product.
2. the preparation method of bacillus subtilis liquid fermentation radix glycyrrhizae probiotics described in claim 1, it is characterised in that bag
Include following steps：
1）Bacillus subtilis strain is inoculated in LB fluid nutrient mediums, 37 ± 3 DEG C of shaking 48 ± 12h of culture, obtain withered grass
Bacillus seed liquor；
2）Bacillus subtilis seed liquor is seeded in radix glycyrrhizae fermentation medium by 5% inoculum concentration, 37 ± 3 DEG C of shaking cultures
24-72h to obtain the final product.
3. bacillus subtilis liquid fermentation radix glycyrrhizae probiotics described in claim 1 should as additive for farm animal feed
With.
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| CN109364114B (en) * | 2018-11-30 | 2021-08-10 | 河北征宇制药有限公司 | Fermented traditional Chinese medicine for improving growth and production performance of cattle and sheep and preparation method thereof |
| CN111000031A (en) * | 2019-12-26 | 2020-04-14 | 湖北三华生态农业科技发展有限公司 | Sweet potato leaf fermentation resource utilization method |
| CN111388527A (en) * | 2020-04-10 | 2020-07-10 | 聊城创新畜禽养殖有限公司 | Preparation for preventing and treating mycoplasma of laying hens and preparation method thereof |
| CN112011483B (en) * | 2020-08-13 | 2022-07-19 | 山东天润和生物工程有限公司 | Culture method suitable for enterococcus faecalis enrichment |
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|---|---|---|---|---|
| CN101116473A (en) * | 2006-08-01 | 2008-02-06 | 济南亿民动物药业有限公司 | Method for preparing glycolysis Chinese herbal medicine preparations for feed |
| CN102232998A (en) * | 2011-04-15 | 2011-11-09 | 北京康华远景科技有限公司 | Micro-ecological traditional Chinese medicine preparation for enhancing immunity of livestock and poultry, and preparation method thereof |
| CN104351570A (en) * | 2014-11-29 | 2015-02-18 | 郑州后羿制药有限公司 | Compound Chinese herbal medicine probiotics for piglets and preparation method of compound Chinese herbal medicine probiotics |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101116473A (en) * | 2006-08-01 | 2008-02-06 | 济南亿民动物药业有限公司 | Method for preparing glycolysis Chinese herbal medicine preparations for feed |
| CN102232998A (en) * | 2011-04-15 | 2011-11-09 | 北京康华远景科技有限公司 | Micro-ecological traditional Chinese medicine preparation for enhancing immunity of livestock and poultry, and preparation method thereof |
| CN104351570A (en) * | 2014-11-29 | 2015-02-18 | 郑州后羿制药有限公司 | Compound Chinese herbal medicine probiotics for piglets and preparation method of compound Chinese herbal medicine probiotics |
Non-Patent Citations (1)
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| "黄芪提取液发酵枯草芽孢杆菌工艺优化研究";乔宏兴等;《家畜生态学报》;20141231;第35卷(第7期);第58-62页 * |
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