CN112655827A - Clostridium butyricum preparation special for ruminants and preparation method and application thereof - Google Patents
Clostridium butyricum preparation special for ruminants and preparation method and application thereof Download PDFInfo
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- CN112655827A CN112655827A CN202011514842.0A CN202011514842A CN112655827A CN 112655827 A CN112655827 A CN 112655827A CN 202011514842 A CN202011514842 A CN 202011514842A CN 112655827 A CN112655827 A CN 112655827A
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Abstract
The invention relates to the field of animal feed additives, in particular to a clostridium butyricum preparation special for ruminants and a preparation method and application thereof. The clostridium butyricum preparation special for the ruminant can effectively improve the digestibility of the feed, the production performance of the animal and the health of the organism, reduce the breeding cost of the ruminant and increase the economic benefit, and the product is a green, safe, pollution-free and residue-free feed additive product.
Description
Technical Field
The invention relates to the field of animal feed additives, in particular to a clostridium butyricum preparation special for ruminants and a preparation method and application thereof.
Background
Ruminants rely on the powerful rumen to convert fibrous plant resources that are not digestible by humans into high quality meat and milk. Growth-promoting antibiotics are prohibited in the current feed. In the face of the development dilemma of the ruminant breeding industry and the problem of the breeding production safety, the selection of the green, safe, pollution-free and residue-free feed additive becomes important. The enzyme preparation and the probiotic product are considered as safe, green and reliable feed additives, and a large amount of research shows that the enzyme preparation and the probiotic product have remarkable effects on improving the feed digestibility and the breeding production efficiency.
Clostridium butyricum is a butyric acid-producing gram-positive anaerobic bacterium of the genus clostridium of the family bacillaceae, is a normal intestinal bacterium of animals, and widely exists in animal wastes and soil. The clostridium butyricum growth metabolites comprise amino acid, short-chain fatty acid and various digestive enzymes, and can promote the growth of beneficial bacteria such as lactobacillus and bifidobacterium, regulate the intestinal microecological balance and promote the absorption of nutrient substances. The clostridium butyricum forms spores in the later growth stage, can tolerate adverse environments such as high temperature, high pressure, mechanical friction, acid and alkali and the like in the feed processing process, and is less influenced by gastrointestinal fluids and bile salts.
Clostridium butyricum performs its function mainly through four aspects: firstly, the microecological balance of animal intestinal tracts is maintained, the proliferation of beneficial bacteria (bifidobacteria and lactobacillus) is promoted, and the growth of harmful bacteria (salmonella and escherichia coli) is inhibited; secondly, various enzymes, vitamins and the like which are useful for animals are generated, and the digestion and absorption capacity of the animals is improved; thirdly, butyric acid is produced, regeneration and repair of animal small intestine epithelial cell tissues are promoted, and intestinal health is maintained; enhancing non-specific immunity of animals, resisting infection and reducing the occurrence of diseases; fourthly, the diarrhea rate of animals is reduced, and the growth performance is improved.
At present, the application research of clostridium butyricum is mainly focused on monogastric animals, and the clostridium butyricum has obvious improvement effect on the production performance of animals, but the research on ruminants is less, and the effect is not clear; meanwhile, a large amount of bacteria and fungi exist in the rumen of the ruminant, the internal environment is complex, and common probiotics are difficult to grow and metabolize in the rumen and cannot play a role, so that the screening of clostridium butyricum strains capable of adapting to the gastrointestinal tract of the ruminant is very important.
Disclosure of Invention
The invention provides a method for screening clostridium butyricum strains from rumen of ruminant and a green and safe additive product for overcoming the defects of the prior art; meanwhile, animal tests of multiple batches and multiple doses prove that the clostridium butyricum preparation product provided by the invention effectively improves calf diarrhea and rumen health level, and improves the dietary digestibility and production performance of ruminants.
The invention aims to provide a special clostridium butyricum preparation for ruminants.
The invention also aims to provide a preparation method of the special clostridium butyricum preparation for the ruminant.
The clostridium butyricum preparation special for the ruminants comprises clostridium butyricum VTSJ-2, wherein rumen contents are screened, and the clostridium butyricum is separated and purified by adopting Hungat anaerobic rolling tube technology, and the preservation number is GDMCC NO. 61311. The clostridium butyricum VTSJ-2 has the biological characteristics of acid production, hydrogen production and spore structure, can be well adapted to the gastrointestinal environment of animals, and can play the physiological function through growth and metabolism; meanwhile, a large number of in vitro tests and animal tests prove that the clostridium butyricum can obviously improve the diarrhea and rumen health level of calves and improve the digestibility of feed and the production performance of animals.
The clostridium butyricum preparation special for the ruminant is prepared by the following method:
fermenting clostridium butyricum liquid; treating, drying and granulating.
According to the specific embodiment of the invention, the special clostridium butyricum preparation for ruminants is prepared by the following method: inoculating the activated seed liquid into a production tank for liquid fermentation for 42h, adding a carrier and other components into the fermentation liquid, and then carrying out boiling drying, including the processes of uniformly mixing, extruding, rounding, granulating and the like.
Preferably, the clostridium butyricum preparation for ruminant according to the present invention, wherein the carrier is selected from corn starch, maltodextrin or silica; the binder is selected from microcrystalline cellulose or ethyl cellulose.
The product of the invention is suitable for ruminants, comprising: cows, beef cattle and sheep.
The feed additive can be directly added into animal ration to be mixed uniformly and fed, and recommended dosage is 10-20 g/d of cow head, 5-10 g/d of beef cattle head and 2-4 g/d of sheep head.
The invention has the advantages that: compared with the prior art and products, the clostridium butyricum preparation special for ruminants is prepared by mixing and granulating clostridium butyricum with a carrier, an adhesive and other components, and the clostridium butyricum strain is screened from rumen contents of the ruminants and can be well adapted to the gastrointestinal environment of the ruminants. Application research shows that the clostridium butyricum preparation can not only promote the growth of ruminants, adjust the micro-ecological balance of animal intestinal tracts, protect intestinal epithelial tissues, generate a plurality of beneficial substances, but also reduce the use of antibiotic products in feed.
The clostridium butyricum preparation special for the ruminant can effectively improve the digestibility of the feed, the production performance of the animal and the health of the organism, reduce the breeding cost of the ruminant and increase the economic benefit, and the product is a green, safe, pollution-free and residue-free feed additive product.
Drawings
Figure 1 shows the effect of clostridium butyricum on rumen pH;
FIG. 2 shows the growth curve of Clostridium butyricum in rumen fluid;
FIG. 3 shows the effect of a product according to the invention on daily weight gain in calves of 1-2 months of age;
FIG. 4 shows the effect of a product according to the invention on daily weight gain in calves of 2-3 months of age;
figure 5 shows the effect of the product of the invention on daily weight gain of calves of 5-7 months old;
FIG. 6 shows the effect of the product of the invention on daily weight gain of 5-7 month old cow calves;
FIG. 7 shows the effect of the product of the invention on the daily gain of a fattening cattle;
FIG. 8 shows the effect of the product of the invention on the number of viable Clostridium butyricum bacteria in cow dung.
The collection number of clostridium butyricum (clostridium butyricum) strain VTSJ-2 is GDMCC NO: 61311, deposited on 25.11.2020 and collected in Guangdong province, where the culture Collection was Anhui, building No. 59, building No. 5 of Middledo 100, Guangzhou, institute of microbiology, Guangdong province, post code 510070.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1 preparation of a novel Clostridium butyricum preparation product for ruminants
The specific screening process of clostridium butyricum is as follows:
1. fully mixing the collected rumen contents, and taking 1mL of the mixed rumen contents by using a sterile syringe for inoculation;
2. injecting the sample into an anaerobic bottle containing 90mL of deoxidized sterile water, placing in 80 ℃ water bath for 10min, killing the nutriment and activating spore germination, transferring into a liquid clostridium multiplication culture medium for amplification culture, and standing and culturing at 37 ℃ for 48 h;
3. the culture solution was placed in a 80 ℃ water bath for 10min with a sterile anaerobic syringe (filled with nitrogen), transferred to Clostridium liquid selective medium, and subjected to static culture at 37 ℃ for 48 h. 4. Quickly injecting the three-parallel 9-gradient diluted culture solution into a solid clostridium selective culture medium, rolling the medium on ice until the medium is solidified and is uniformly distributed on the tube wall, vertically placing the tube wall at 37 ℃, and statically culturing for 24 hours until an obvious single bacterial colony is formed;
5. selecting a rolling tube with moderate single colony number, good diversity and uniform distribution for single colony picking, determining a target single colony to be picked through colony morphological characteristic observation, quickly picking the target single colony to a liquid storage culture medium, and performing static culture at 37 ℃ for 48 hours;
6. and (4) rolling the preliminarily screened strains for multiple times, and selecting the strains to finally obtain pure bacteria.
The special clostridium butyricum preparation product for the ruminant is produced mainly by a clostridium butyricum liquid fermentation technology and a post-treatment drying granulation process, and the specific production process comprises the following steps: the clostridium butyricum is prepared by adding carrier adhesives such as microcrystalline cellulose, maltodextrin and corn starch and other components into fermentation liquor, mixing and granulating, wherein the corn starch, the microcrystalline cellulose and the maltodextrin are combined according to the proportion of 40:5:8 and are uniformly mixed with the fermentation liquor, and then extruding, rolling and drying, wherein the particle size of the clostridium butyricum product is 2-5 mm.
The clostridium butyricum liquid fermentation medium comprises 2% (W/V) of soybean meal, 1% (W/V) of yeast powder, 1.6% (W/V) of corn steep liquor, 0.32% (W/V) of trisodium citrate, 0.04% (W/V) of potassium dihydrogen phosphate, 0.03% (W/V) of sodium chloride, 0.06% (W/V) of manganese sulfate, 0.06% (W/V) of calcium chloride and 0.04% (W/V) of magnesium sulfate.
The method for measuring clostridium butyricum in fermentation liquor and products adopts a plate counting method, and mainly comprises the following steps: preparation of sample dilutions, inoculation, culture and enumeration. The specific implementation process comprises the following steps:
(1) weighing 25g (or mL) of sample by aseptic technique, placing in a triangular flask containing 225mL of sterile physiological saline (the flask is internally provided with a proper amount of sterile glass beads), placing on a vortex oscillator, oscillating for 30min, and mixing well to obtain a sample homogeneous solution at a ratio of 1: 10. Sucking 0.5mL of the 1:10 sample homogenizing solution by using a sterile pipette, adding the sample homogenizing solution into a sterile test tube containing 4.5mL of sterile physiological saline, and uniformly mixing to prepare a mixture of 1:102The sample of (1) was homogenized, and the dilution was performed in this order to obtain 1:10, respectively3～1:109Equal concentration (sterile pipette must be replaced for each dilution) of sample.
(2) And (3) respectively sucking 1mL of bacterial suspensions with different dilutions by using a 1mL sterile pipette, adding the bacterial suspensions into a sterilized culture dish, respectively pouring the melted Clostridium butyricum culture medium (cooled to 50 ℃) into the culture dish, rotating and uniformly mixing 15-20 mL of each culture medium, and standing and solidifying. 2-3 continuous suitable dilutions are taken from each sample, 2-3 plates are repeatedly inoculated to each dilution, and sterile distilled water is added to make blank control of 2-3 plates.
(3) And (4) inverting the inoculated culture dish, and culturing for 18-24 hours under the constant-temperature anaerobic condition at the temperature of 37 +/-1 ℃.
(4) After culturing, counting is carried out, and if the colony number of all the dilutions put on the plate is not between 30CFU and 300CFU, and when a part of the colonies is less than 30CFU or more than 300CFU, the colony number is calculated by multiplying the average colony number closest to 30CFU or 300CFU by the dilution factor. When the colony number is more than or equal to 100CFU, after the 3 rd digit is reduced by adopting the principle of 'rounding off', the first 2 digits are taken, and the digits are replaced by 0; it can also be expressed in the form of an index of 10, reduced by the "rounding" principle, followed by two significant digits.
The measured viable count of the clostridium butyricum in the fermentation liquor is 2.1 multiplied by 109cfu/mL, the viable count after drying and granulation is 2108～200×108cfu/g。
EXAMPLE 2 in vitro test Effect of the product
1. Materials and methods
Collecting rumen contents more than 2 heads respectively by using an oral cavity sampler, mixing in equal volume, filtering with four layers of gauze, and rapidly adding CO2A mixed culture solution (the ratio of rumen fluid to buffer solution is 1:1) is prepared in a glass bottle of saturated and preheated (39 ℃) buffer solution. 70mL of mixed solution and 2g of bacterial powder (actually measured viable count is 4.3 hundred million CFU/g) are added into a glass tube of a test group, and the initial bacterial content is 12.3x10 theoretically6CFU/mL。
2. Test results
The results of the time course of the effect of clostridium butyricum products on rumen pH are shown in fig. 1. As can be seen from fig. 1, clostridium butyricum decreased the rumen pH at each sampling time point and finally stabilized at a pH of about 6.0, compared to the control group. The growth of Clostridium butyricum in rumen fluid is shown in FIG. 2. As can be seen from FIG. 2, compared with the control group, the concentration of the bacteria at each sampling time point of the Clostridium butyricum group is higher than that of the control group, and shows a trend of increasing and then stabilizing, which indicates that the Clostridium butyricum in the product can recover and proliferate in rumen environment, and can reduce the pH value of rumen fluid and reduce the proliferation of harmful bacteria
The effect of clostridium butyricum products on feed digestibility is listed in table 1. As can be seen from table 1, compared with the control group, the clostridium butyricum product significantly improves the Dry Matter (DM) digestibility and the neutral detergent fiber (NDFF and Acid Detergent Fiber (ADF) digestibility by 1.7%, 1.54% and 0.48%, respectively, which indicates that the clostridium butyricum product can effectively improve the degradation rate of the feed in rumen.
TABLE 1 influence of Clostridium butyricum products on feed digestibility
Example 3 application effect of the product on beef cattle of 1-3 months old
1. Materials and methods
45 rouges and cattle with the weight of about 54kg are selected for the test, and are randomly and evenly divided into three treatment groups: feeding basic ration to a control group, adding 4 g/d.head of probiotic product on the basic ration in a 4g test group, adding 8 g/d.head of probiotic product on the basic ration in an 8g test group, wherein the viable count of the product is 5x 108CFU/g. The test was carried out in a pasture in the lotus of Shandong for a total of 64 days.
2. Test results
The effect of the product on daily calf weight gain is shown in figures 3 and 4. As can be seen from FIG. 3, compared with the control group, the 4g additive group (C4 test group) of the product of the invention can increase the daily gain of calves at 1-2 months by 90g, and the 8g additive group (C8 test group) can increase the daily gain of calves at 1-2 months by 120 g. As can be seen from FIG. 4, compared with the control group, the 4g additive group (C4 test group) of the product of the invention can increase the daily gain of calves at 2-3 months of age more than 40g of the control group, and the 8g additive group (C8 test group) can increase the daily gain of calves at 2-3 months of age more than 60g of the control group.
Example 4 application effect of the product on beef cattle of 5-7 months old
1. Materials and methods
56 cattle with the weight of about 200kg, 28 bulls of male calves and 28 cows of female calves are selected and divided into four groups. Feeding the control group of male calf and female calf with basic ration, adding Clostridium butyricum preparation 10 g/d.head into the basic ration of male calf and female calf test group, and increasing the viable count of the product to 5x 108CFU/g. The test was carried out in a pasture in Shandong for a total of 67 days.
2. Test results
The effect of the product on the daily gain of 5-7 month old beef cattle is shown in fig. 5 and 6. As can be seen from fig. 5 and fig. 6, compared with the control group, the daily gain of the bull calf of 5-7 months old can be increased by adding 10g of the product per day to be higher than that of the control group 170g, and the daily gain of the cow calf can be increased to be higher than that of the control group 140 g.
Example 5 application Effect of the product in fattening cattle
1. Materials and methods
12 Simmental hybrid cattle with the weight of about 520kg are selected for the test, and are randomly and averagely divided into two treatment groups: the control group is fed with basic ration, and the test group is added with 20 g/d.head of the product of the invention on the basic ration. The test is carried out in a rumination test base of the research of the excessive labor bearing schools for 36d, and the cattle are weighed respectively on the first day and the last day of the test.
2. Test results
The effect of the product on the daily gain of fattening cattle and the number of live clostridium butyricum in the feces is shown in fig. 7 and 8. As can be seen from FIG. 7, compared with the control group, the daily gain of beef cattle increased by adding 20g of the product per day compared with 170g of the control group. As can be seen from FIG. 8, the viable count of Clostridium butyricum in beef cattle feces of the test group is 7.2 times that of the control group, and reaches 15.9x104 CFU/g。
The foregoing is a detailed description of the product of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific embodiments.
Claims (10)
1. The clostridium butyricum preparation special for the ruminant is characterized by comprising clostridium butyricum with the preservation number of GDMCC NO: 61311.
2. the clostridium butyricum preparation for ruminants according to claim 1, which comprises a carrier and a binder.
3. The clostridium butyricum formulation for ruminants according to claim 2, wherein the carrier is selected from corn starch, maltodextrin or silica.
4. The clostridium butyricum formulation for ruminants according to claim 2, wherein the binder is selected from microcrystalline cellulose or ethyl cellulose.
5. The Clostridium butyricum preparation for ruminants according to claim 1, wherein the viable count of Clostridium butyricum contained in the Clostridium butyricum preparation for ruminants is 2 x108～200×108cfu/g。
6. A method of preparing a clostridium butyricum preparation specific for ruminants, comprising the steps of: fermenting clostridium butyricum liquid; treating, drying and granulating.
7. The method of preparing a clostridium butyricum preparation for ruminants according to claim 6, wherein the method comprises the steps of:
inoculating the activated clostridium butyricum seed liquid into a production tank for liquid fermentation for 42h, adding a carrier and other components into the fermentation liquid, and then drying and granulating.
8. The method for preparing Clostridium butyricum preparation for ruminants according to claim 6, wherein the carrier is selected from corn starch, maltodextrin or silica.
9. The method for preparing Clostridium butyricum formulation for ruminants as claimed in claim 6, wherein the binder used in the granulation process is selected from microcrystalline cellulose or ethyl cellulose.
10. A method for improving the productivity of a ruminant, characterized in that the Clostridium butyricum preparation for ruminant specified in claim 1 is fed to a ruminant.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611273A (en) * | 2015-02-11 | 2015-05-13 | 成都与康科技有限公司 | Novel strain Clostridium butyricum UCN-12 and composition and application thereof |
| CN110951640A (en) * | 2019-12-06 | 2020-04-03 | 山东蔚蓝生物科技有限公司 | Probiotic clostridium butyricum and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611273A (en) * | 2015-02-11 | 2015-05-13 | 成都与康科技有限公司 | Novel strain Clostridium butyricum UCN-12 and composition and application thereof |
| CN110951640A (en) * | 2019-12-06 | 2020-04-03 | 山东蔚蓝生物科技有限公司 | Probiotic clostridium butyricum and application thereof |
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