CN110974851A - Tibetan pig manure bacterium capsule for relieving diarrhea of weaned piglets and preparation method thereof - Google Patents
Tibetan pig manure bacterium capsule for relieving diarrhea of weaned piglets and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/4841—Filling excipients; Inactive ingredients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
Abstract
The invention discloses a Tibetan pig manure bacterium capsule for relieving diarrhea of weaned piglets and a preparation method thereof. Collecting fresh Tibetan pig rectal feces, adding a phosphate buffer solution, and preparing Tibetan pig fecal bacteria suspension in an anaerobic operating platform; centrifuging and purifying the Tibetan pig manure bacterial suspension, extracting supernatant, and uniformly mixing the supernatant, a sodium alginate solution, whey protein isolate, sucrose, mannitol and glycerol in proportion; and dropping the mixed solution into a calcium chloride solution at a constant speed to form pig manure preservation bacterium gel particles, filtering the gel particles out of an anaerobic box, standing for 0.5-1h, and then packaging the gel particles into a methyl cellulose capsule shell to obtain the pig manure preservation bacterium capsule. The preparation method is simple in preparation process, high in efficiency and low in cost, the diversity and the activity of bacteria in the Tibetan pig manure are completely preserved in the prepared Tibetan pig manure capsule, and meanwhile, the activity of the bacteria in the Tibetan pig manure capsule can be improved by adding the glycerol, the whey protein isolate, the sucrose and the mannitol, the application effect of the intestinal flora preparation is improved, and the death rate of diarrhea of weaned piglets is remarkably reduced.
Description
Technical Field
The invention belongs to the technical field of veterinary medicines, and particularly relates to a preparation method of Tibetan pig manure bacteria capsules for relieving diarrhea of weaned pigs and Tibetan pig manure bacteria capsules for relieving diarrhea of weaned pigs prepared by the method.
Background
Weaning is a critical period in the life cycle of pigs, and weaned piglets often suffer from serious intestinal disease infection, resulting in reduced productivity and economic loss in the live pig industry. The intestinal flora imbalance phenomenon caused by the sudden change of the daily ration of the piglets is one of the main causes of the diarrhea of the piglets after weaning. During the weaning period of the piglets, the nutritional sources are changed from breast milk to solid feed, and the levels and the sources of protein and fiber of the piglets influence the intestinal flora structure and diversity of the piglets. The development of disturbed intestinal flora and loss of diversity in pigs early in life may increase the risk of contracting intestinal diseases. For example, the frequent occurrence of severe diarrhea in weaned piglets is closely related to intestinal flora disturbance.
In order to solve the problem of diarrhea caused by intestinal flora disorder of weaned piglets, the addition of probiotics into the feed is the existing main technology. The probiotics can be used for relieving the diarrhea of the weaned pigs to a certain extent, but the recent research shows that the treatment effect of the probiotics on the diarrhea of the weaned pigs has obvious individual difference and has potential risk. The prior art (such as patent application numbers 201811617527.3, 201811636148.9) adds probiotics into the diet, but the probiotics disclosed by the technology have complex components and have no obvious effect on relieving the weaning diarrhea of the piglets.
Scientific research shows that after flora in excrement of healthy pigs is separated by fecal strain transplantation and transplanted to gastrointestinal tracts of weaned pigs, diarrhea of the weaned pigs can be prevented by establishing intestinal flora with normal functions. The published technology shows (for example, patent application No. 201811024719.3) that the research on coprophila transplantation treatment is in the beginning stage, and the screening standard of related donors, the quality and safety of bacterial liquid preparation, the standard operation of transplantation process and the like need to be further researched and determined.
The characteristics of the fecal flora donor are the key of the technology, and fecal microorganisms of local pig species with strong disease resistance have great development potential. The fecal strain selected by the invention is derived from healthy Tibetan pigs, and the Tibetan pigs have the characteristics of strong stress resistance, strong diarrhea resistance, coarse feeding resistance and the like. Although a good fecal strain donor exists, the transplanting mode is also a key step related to whether the later popularization is smooth or not. The fecal strain transplantation technology at the present stage is mainly an instrument intervention method, such as a gastroscope, a stomach tube, an enteroscope, a jejunum tube and the like. Fecal microorganisms need to pass through the stomach and the duodenum through a catheter and be injected into the far end of the duodenum, so that the process is complex, the application of fecal bacteria intervention for long-term use is correspondingly limited, and the popularization and the utilization of the Tibetan pig fecal bacteria are not facilitated. For example, CN201510442182.2 discloses a method for improving growth performance and reducing diarrhea rate of weaned pigs by using fecal strain transplantation, the fecal strain donor adopted in the patent is only a conventional piglet, and the transplantation mode is oral drenching, and long-term drenching can cause the treatment mismatch of piglets, which is not beneficial to long-term intervention treatment. The problem also exists in the method for preventing and treating piglet diarrhea by using the coprophilous fungus transplantation technology disclosed in CN 201510148458.6.
In view of the above disadvantages, a Tibetan pig feces bacterium capsule which has the advantages of simple operation process and low preparation cost, completely preserves the bacterial diversity and activity in Tibetan pig feces, effectively controls piglet diarrhea, maintains intestinal flora of piglets, is convenient to use, and has no individual difference after treatment is urgently needed by the industry.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides an enteric Tibetan pig manure bacterium capsule. The Tibetan pig fecal strain is used as a content to prepare an enteric capsule for oral administration, the capsule keeps the diversity and activity of microorganisms in the fecal strain not to be reduced, and the operation is simple.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a preparation method of Tibetan pig manure bacterium capsules for relieving diarrhea of weaned piglets comprises the following steps:
(1) preparing a filtrate: collecting fresh Tibetan pig rectum feces, quickly transferring to an anaerobic workbench (Thermo Scientific 1029), adding phosphate buffer solution, pulverizing, coarsely filtering the pulverized solution twice with medical gauze, and sequentially sieving with 80 mesh and 100 mesh sieves to obtain filtrate;
(2) extracting a supernatant: the filtrate is subpackaged in a 50ml centrifuge tube for centrifugal purification, and supernatant is extracted for standby;
(3) preparing a pig manure bacterium mixed solution: mixing the supernatant, sodium alginate solution, whey protein isolate, sucrose, mannitol and glycerol to obtain mixed solution of pig manure bacteria;
(4) preparing Tibetan pig manure bacterium capsules: and (3) dropping a calcium chloride solution with the mass fraction of 1-3% into the pig manure bacterium mixed solution at a constant speed to form pig manure bacterium storage gel particles, filtering the gel particles out of an anaerobic box, standing, and then packaging into a methyl cellulose capsule shell to obtain the pig manure bacterium storage capsule.
Further, the day age of the Tibetan pigs in the step (1) is 100-150 days, and the corn-soybean meal type feed meeting the nutritional requirements of the Tibetan pigs is fed one month before excrement is collected; selecting healthy Tibetan pigs with no abnormal body surface and negative rectal fecal virus detection results one week before collecting feces.
Further, adding phosphate buffer solution into the Tibetan pig rectal excrement in the step (1) according to the ratio of 1:3-5 (m/v).
Further, the centrifugal force in the step (2) is 5000-7000g, and the centrifugal time is 10-15 min.
Further, adding 2-5g of sodium alginate, 1-4g of whey protein isolate, 0.5-3g of sucrose, 1-3.5ml of mannitol and 8-16ml of glycerol into every 100ml of supernatant in the step (3), and mixing for 40-60min by using a magnetic stirrer to obtain a fully and uniformly mixed Tibetan pig manure bacterial suspension.
Further, the gel particles in the step (4) are placed in an anaerobic workbench and kept still for 30-60 min.
Further, the mass of the gel particles in each capsule subpackaged in the step (4) is 0.8-1.2 g.
The invention also discloses a Tibetan pig manure bacterium capsule prepared by any one of the preparation methods.
The invention also discloses application of the Tibetan pig manure bacterium capsule in preparing a veterinary drug for treating diarrhea.
The invention also discloses a use method of the Tibetan pig manure bacterium capsule in relieving diarrhea of weaned pigs, and the weaned pigs of 21-28 days old take 2-4 Tibetan pig manure bacterium capsules every day.
The invention has the beneficial effects that:
1. the fecal bacteria capsule prepared by the method completely keeps the diversity and activity of fecal microorganisms of a fecal donor, has good application effect, and effectively reduces the diarrhea condition of weaned piglets.
2. The application of the materials such as glycerol, whey protein isolate, sucrose, mannitol and the like is adopted, so that the activity of bacteria in the Tibetan pig manure bacteria capsule can be effectively improved, and the application effect of the intestinal flora preparation is improved.
3. The fecal bacteria capsule prepared by the invention is not required to be drenched, is convenient to use, is easier to popularize and apply, and is more beneficial to diarrhea piglets needing long-term intervention treatment.
Drawings
FIG. 1 shows the number of piglets in each group that suffered from diarrhea.
Detailed Description
The technical solution of the present invention is further described below, but the present invention is not limited to the following embodiments.
Example 1
Fresh rectal faeces of Tibetan pigs were quickly transferred to an anaerobic bench (Thermo Scientific 1029) and crushed after addition of phosphate buffer at a mass/volume ratio of 1: 4. Coarse-filtering the pulverized liquid twice with medical gauze, and filtering with 80-mesh sieve; the filtrate is subpackaged in a 50ml centrifuge tube, 5000g is centrifuged for 15min, and the supernatant is extracted. Weighing 100ml of supernatant, adding 4g of sodium alginate, 1g of whey protein isolate, 0.5g of sucrose, 3ml of mannitol and 14.5ml of glycerol, and mixing for 40min by using a magnetic stirrer to obtain a fully and uniformly mixed Tibetan pig manure bacterial suspension. And uniformly dropping the fully and uniformly mixed Tibetan pig manure bacterial suspension into a calcium chloride solution with the mass fraction of 3% to form gel particles. And standing the gel particles in an anaerobic workbench for 50 min. And the gel particles after standing are distributed in the shells of the methylcellulose capsules, so that the mass of the gel particles in each capsule is ensured to be 0.8 g.
Example 2
Fresh rectal faeces of Tibetan pigs were quickly transferred to an anaerobic bench (Thermo Scientific 1029) and crushed after addition of phosphate buffer at a mass/volume ratio of 1: 5. Coarse-filtering the pulverized liquid twice with medical gauze, and filtering with 80-mesh sieve; the filtrate was dispensed into 50ml centrifuge tubes, 7000g, centrifuged for 10min and the supernatant was extracted. Weighing 100ml of supernatant, adding 1g of sodium alginate, 4g of whey protein isolate, 1g of sucrose, 3ml of mannitol and 10.5ml of glycerol, and mixing for 60min by using a magnetic stirrer to obtain a fully and uniformly mixed Tibetan pig manure bacterial suspension. And uniformly dropping the fully and uniformly mixed Tibetan pig manure bacterial suspension into a calcium chloride solution with the mass fraction of 2% to form gel particles. And standing the gel particles in an anaerobic workbench for 30 min. And the gel particles after standing are distributed in the shells of the methylcellulose capsules, so that the mass of the gel particles in each capsule is ensured to be 1.2 g.
Test example 1
The Tibetan pig manure bacterium capsule prepared by the invention has the treatment effect on the diarrhea condition of piglets
The test selects 48 healthy 21-day-old weaned piglets, and the piglets are divided into three groups (a control group, a probiotic group and a fecal bacteria transplanting group) according to sex and weight. The control group, the probiotic group and the fecal strain transplanting group are fed with the same basic ration, the fecal strain transplanting group takes 3 fecal strain capsules described in the embodiment 1 eight times a day in the morning, the control group also takes the same number of empty capsules at the same time, and the probiotic group additionally adds 500mg/kg of bacillus subtilis in the feed. All pigs were fed and drunk freely, and the diarrhea of piglets in each group was counted daily during the test period, and the specific results are shown in table 1 and fig. 1.
Effect of the fecal bacteria capsules described in Table 1 on diarrhea rate and diarrhea index of weaned piglets
Note: compared with the fecal bacteria transplanting group,b P<0.01，a P<0.05。
as can be seen from the results in table 1, the diarrhea rates of the probiotic group and the fecal bacteria transplant group are both significantly reduced compared to the blank intervention of the control group, but the degree of the reduction of the fecal bacteria transplant group is more significant, and the reduction range of the performance of the fecal bacteria transplant group in the diarrhea index is more significant, and is the lowest of the three groups (lower than the probiotic group and significantly lower than the control group). The fecal bacteria capsule prepared by the invention has the most obvious and best effect on relieving the diarrhea of the weaned piglets.
Test example 2
The initial weights of 3 groups of piglets were counted, as well as the productivity and mortality of each group of piglets at 45 days after the corresponding treatment. And recording the daily weight gain grams and daily defecation conditions of the piglets within 45 days, and calculating an average value. The criteria for daily bowel movement scores described in the table were: 1 point represents that the excrement is in a strip shape or a granular shape; score 2 indicates that the feces can be shaped, but are softer; 3 points represent that the feces are unformed and thinner; 4, separating the liquid manure and keeping the color normal; and 5 points represent the separation of liquid dung and abnormal color. The results are shown in Table 2, and the mortality of each group of piglets is shown in Table 2.
Influence of the fecal bacteria capsules described in Table 2 on productivity and mortality of weaned piglets
Note: compared with the fecal bacteria transplanting group,b P<0.01，a P<0.05。
according to the results in table 2, the initial weights of 3 groups of piglets are not obviously different, but after 1 month of treatment of each component, the daily gain gram of the fecal strain transplantation group is obviously better than that of the probiotic group and the control group, and although the daily gain gram of the probiotic group is also improved compared with that of the control group, the improvement range is not as great as that of the fecal strain transplantation group. On the daily defecation score statistics, the relative shaping of the pigling defecation of the fecal strain transplanting group is carried out, the score median is 1.5 points, the probiotic group is 2.8 points, the feces are soft, and the shaping degree is lower than that of the fecal strain transplanting group; however, the feces of the piglets in the control group are thin, and even the feces and water are separated. On the data monitoring that the piglets die, the mortality rate of the weaned piglets is reduced by the coprophilous fungus capsules compared with the control group and the probiotic group, and the results of figure 1 clearly show that the diarrhea head number of the coprophilous fungus transplanted group is always at a lower level and is obviously lower than that of the probiotic group and the control group, wherein the diarrhea head number level of the control group is the highest. The fecal strain capsule can promote the absorption of piglets on feed, reduce the diarrhea and death probability of piglets, maintain the molding degree of piglet feces, promote the growth of piglets and improve the daily gain of piglets.
Test example 3
The Tibetan pig manure bacterium capsule prepared by the invention is used for detecting the diversity and activity of microorganisms
And (3) detecting microbial diversity: the glycolysis culture DNA collected at 0h and 48h was extracted using Fast DNA SPIN kit (MP Biomedicals, USA) and then analyzed for microbial composition in intestinal chyme at Sichuan university institute of animal nutrition.
The results of the microbial diversity measurements are shown in Table 3
TABLE 3 relative abundance (log (copy/g)) of beneficial and opportunistic bacteria in the intestine after 48h for each group of piglets
Note: compared with the fecal bacteria transplanting group,b P<0.01，a P<0.05。
table 3 the results show that: the coprophilous fungus capsule can obviously promote the proliferation of the Lactobacillus in the intestinal tract of the piglet (the effect is close to that of the positive control group 1 of the comparative example 1 and is obviously superior to that of the positive control group 2 of the comparative example 2); significantly promoting the proliferation of Bifidobacterium (less effective than the positive control group 1 of comparative example 1 but slightly more effective than the positive control group 2 of comparative example 2); the growth of Escherichia coli-Shigella Escherichia coli is obviously inhibited (the effect is equivalent to that of the positive control group 1 of the comparative example 1 and is obviously better than that of the positive control group 2 of the comparative example 2).
In conclusion, the Tibetan pig manure bacteria capsule prepared by the method completely keeps the diversity and activity of manure microorganisms of a manure donor, has a good application effect, improves the application effect of an intestinal flora preparation, and effectively reduces the diarrhea condition of weaned piglets. The fecal bacteria capsule is not required to be drenched, is convenient to use, facilitates the popularization and application of the medicine, and is more beneficial to diarrhea piglets needing long-term intervention treatment.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (10)
1. A preparation method of Tibetan pig manure bacterium capsules for relieving diarrhea of weaned piglets is characterized by comprising the following steps:
(1) preparing a filtrate: collecting fresh rectal excrement of the Tibetan pigs, quickly transferring the excrement to an anaerobic workbench, adding phosphate buffer solution, then carrying out crushing treatment, carrying out coarse filtration on the crushed solution twice by using medical gauze, and then sequentially screening by using 80-mesh and 100-mesh screens to obtain filtrate for later use;
(2) extracting a supernatant: the filtrate is subpackaged in a 50ml centrifuge tube for centrifugal purification, and supernatant is extracted for standby;
(3) preparing a pig manure bacterium mixed solution: mixing the supernatant, sodium alginate solution, whey protein isolate, sucrose, mannitol and glycerol to obtain pig manure bacteria mixed solution;
(4) preparing Tibetan pig manure bacterium capsules: and (3) dropping a calcium chloride solution with the mass fraction of 1-3% into the pig manure bacterium mixed solution at a constant speed to form pig manure bacterium storage gel particles, filtering the gel particles out of an anaerobic box, standing, and then packaging into a methyl cellulose capsule shell to obtain the pig manure bacterium storage capsule.
2. The method of claim 1, wherein: the age of the Tibetan pigs in the step (1) is 100-150 days, and the Tibetan pigs are healthy and have no intestinal diseases.
3. The method of claim 1, wherein: adding phosphate buffer solution into the Tibetan pig rectum excrement in the step (1) according to the ratio of 1:3-5 (m/v).
4. The method of claim 1, wherein: the centrifugal force in the step (2) is 5000-7000g, and the centrifugal time is 10-15 min.
5. The method of claim 1, wherein: and (3) adding 2-5g of sodium alginate, 1-4g of whey protein isolate, 0.5-3g of sucrose, 1-3.5ml of mannitol and 8-16ml of glycerol into each 100ml of supernatant, and mixing for 40-60min by using a magnetic stirrer to obtain a fully and uniformly mixed Tibetan pig manure bacterial suspension.
6. The method of claim 1, wherein: and (4) placing the gel particles in the step (4) in an anaerobic workbench for standing for 30-60 min.
7. The method of claim 1, wherein: and (4) the mass of the gel particles in each capsule subpackaged in the step (4) is 0.8-1.2 g.
8. A Tibetan pig manure bacterium capsule prepared by the preparation method according to any one of claims 1 to 7.
9. Use of the Tibetan pig manure bacterium capsule according to claim 8 in the preparation of a veterinary medicament for the treatment of diarrhea.
10. A method of using the Tibetan pig manure capsule of claim 8 in alleviating diarrhea in weaned piglets, characterized by: the 21-28-day-old weaned piglets take 2-4 Tibetan pig manure bacteria capsules every day.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010005352.1A CN110974851A (en) | 2020-01-03 | 2020-01-03 | Tibetan pig manure bacterium capsule for relieving diarrhea of weaned piglets and preparation method thereof |
| PCT/CN2020/000336 WO2021134740A1 (en) | 2020-01-03 | 2020-12-31 | Tibetan pig fecal microbiota capsule for relieving diarrhea in weaned piglet, and preparation method therefor |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010005352.1A CN110974851A (en) | 2020-01-03 | 2020-01-03 | Tibetan pig manure bacterium capsule for relieving diarrhea of weaned piglets and preparation method thereof |
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| WO2021134740A1 (en) * | 2020-01-03 | 2021-07-08 | 四川农业大学 | Tibetan pig fecal microbiota capsule for relieving diarrhea in weaned piglet, and preparation method therefor |
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| WO2021134740A1 (en) * | 2020-01-03 | 2021-07-08 | 四川农业大学 | Tibetan pig fecal microbiota capsule for relieving diarrhea in weaned piglet, and preparation method therefor |
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Application publication date: 20200410 |