Compound microbial inoculant for improving disease resistance of livestock and poultry and preparation method thereof

Abstract

The present application relates to the technical field of livestock and poultry breeding, and particularly relates to a compound microbial agent for improving the disease resistance of livestock and poultry and a preparation method thereof. The present application provides a microbial combination comprising Bacillus subtilis, Bacillus subtilis, Bacillus amyloliquefaciens, Yarrowia lipolytica and Trichoderma harzianum, and a compound microbial agent, which can effectively improve the disease resistance of livestock and poultry. In the specific embodiments of the present application, the survival rate of chicks infected with Escherichia coli can be significantly improved after treatment with the microbial combination provided by the present application, and the survival rate can reach more than 96%, and the body weight, chicken bursa index, spleen index and thymus index can be effectively recovered. Therefore, the microbial combination and the compound microbial agent provided by the present application can be used to improve the disease resistance of livestock and poultry, and provide a new idea for preparing products for improving the disease resistance of livestock and poultry.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of livestock and poultry breeding, in particular to a compound microbial agent for improving the disease resistance of livestock and poultry and a preparation method thereof. BACKGROUND

[0002] After the advent of antibiotics, they have gradually been applied in livestock and poultry production and have been widely accepted and used by breeding enterprises, and have made important contributions to the prevention and treatment of livestock and poultry diseases. The emergence of drug resistance and superbugs has damaged the immune function of animal bodies and has brought serious impacts on human food safety.

[0003] At present, more and more people in the industry realize the potential threat of conventional feed additives such as antibiotics, growth hormones, preservatives, antioxidants, disinfectants and the like to human health. And the state has ordered the prohibition of the addition of antibiotics in feed in 2020. Traditional feed additives have a variety of hazards: first, long-term addition of antibiotics in feed can induce the production of high-resistance microorganisms in poultry, and since these microorganisms have the ability to resist antibiotics, once they infect the human body, they will make patients have no medicine to cure, thereby posing a great threat to humans; second, the addition of artificially synthesized growth hormones in feed makes poultry grow abnormally, and the products lose their original nutritional value and flavor, the quality decreases, and the commodity value decreases; third, artificially synthesized chemical drugs such as antibiotics, growth hormones, preservatives, antioxidants, disinfectants often have toxic side effects, can cause normal tissue cells to be distorted, mutated, and even cause cancer, endangering human health, and these chemicals are enriched in poultry, accumulated in large quantities in the liver and muscle of poultry, and further exacerbate the harmfulness; in addition, these chemical drugs may also have other unknown toxic side effects, posing potential threats. Therefore, some developed countries have proposed initiatives to prohibit the use of antibiotics and growth hormones, and have successively formulated relevant laws and regulations, making this initiative legal.

[0004] Although some strains have been reported to be added to feed as feed additives to ultimately achieve the effect of improving the disease resistance of livestock and poultry, the effect is not significant. Therefore, it is necessary to find a compound microbial agent that can effectively improve the disease resistance of livestock and poultry. SUMMARY

[0005] The purpose of the present application is to provide a compound microbial agent for improving the disease resistance of livestock and poultry and a preparation method thereof to solve the problems existing in the prior art. The microbial combination and the compound microbial agent provided by the present application have the effect of improving the disease resistance of livestock and poultry.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0007] The application provides a microbial combination for improving the disease resistance of livestock and poultry, which comprises the following components in mass parts:

[0008] 1-3 parts of Bacillus siamensis, 3-7 parts of Bacillus subtilis, 2-5 parts of Bacillus amyloliquefaciens, 2-5 parts of Yarrowia lipolytica and 1-3 parts of Trichoderma harzianum.

[0009] Preferably, the ratio of the effective viable bacterial numbers of the Bacillus siamensis, the Bacillus subtilis, the Bacillus amyloliquefaciens, the Yarrowia lipolytica and the Trichoderma harzianum in the microbial combination is (2-6) x 10 9 :(15-25) x 10 9 :(8-20) x 10 9 :(2-10) x 10 8 :(2-6) x 10 7 .

[0010] The application provides application of the above microbial combination in preparation of a compound microbial agent with the effect of improving the disease resistance of livestock and poultry.

[0011] The application provides a compound microbial agent with the effect of improving the disease resistance of livestock and poultry, and the preparation raw material of the compound microbial agent comprises the above microbial combination.

[0012] Preferably, the compound microbial agent further comprises an auxiliary material.

[0013] The application provides a preparation method of the above compound microbial agent, comprising the following steps:

[0014] After the strains in the above microbial combination are activated, fermentation culture is respectively performed to obtain Bacillus siamensis fermentation liquor, Bacillus subtilis fermentation liquor, Bacillus amyloliquefaciens fermentation liquor, Yarrowia lipolytica fermentation liquor and Trichoderma harzianum fermentation liquor.

[0015] The Bacillus siamensis fermentation liquor, the Bacillus subtilis fermentation liquor, the Bacillus amyloliquefaciens fermentation liquor, the Yarrowia lipolytica fermentation liquor and the Trichoderma harzianum fermentation liquor are uniformly mixed to obtain the compound microbial agent.

[0016] Preferably, the fermentation culture is performed at a temperature of 28-37 DEG C for 24-72 h, and the culture medium of the fermentation culture is LB culture medium.

[0017] And / or, the inoculation amount during the fermentation culture is 1-2 %.

[0018] The application provides application of the microbial combination or the compound microbial agent in preparation of a product for improving disease resistance of livestock and poultry.

[0019] The application provides a product for improving disease resistance of livestock and poultry, which comprises the microbial combination or the compound microbial agent.

[0020] The application provides a feeding method of livestock and poultry, which comprises the step of feeding the microbial combination or the compound microbial agent to the livestock and poultry.

[0021] The application discloses the following technical effects:

[0022] The application provides a microbial combination and a compound microbial agent comprising Bacillus subtilis, Bacillus subtilis, Bacillus amyloliquefaciens, Yarrowia lipolytica and Trichoderma harzianum, which can effectively improve the disease resistance of livestock and poultry. In specific embodiments of the application, the survival rate of chicks infected with Escherichia coli can be significantly improved after treatment with the microbial combination provided by the application, and the survival rate can reach more than 96%, and the body weight, chicken bursa index, spleen index and thymus index can be effectively recovered. Therefore, the microbial combination and the compound microbial agent provided by the application can be used to improve the disease resistance of livestock and poultry, and provide a new idea for preparing a product for improving the disease resistance of livestock and poultry. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings. The detailed description is not to be considered to limit the application in any way, but rather to explain certain aspects, features and embodiments of the application.

[0024] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or intermediate value in the stated range, is also encompassed within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In the case of conflict, the present specification will control.

[0026] Many modifications and variations of the described implementations of the present application can be made without departing from its spirit or scope, as will be apparent to those skilled in the art. Other implementations of the present application will be apparent to those skilled in the art from the foregoing description. The scope of the present application is to be limited only by the claims.

[0027] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having", and the like are open-ended terms that are intended to mean including, but not limited to.

[0028] Unless otherwise specified, the strains used in the present application are routinely purchased by those skilled in the art, and the methods used in the present application are well known to those skilled in the art.

[0029] Preparation Example:

[0030] 1. Preparation of strains

[0031] Bacillus siamensis: purchased from China Agricultural Microorganism Culture Collection Center, strain number ACCC 19948; Bacillus subtilis: purchased from China Agricultural Microorganism Culture Collection Center, strain number ACCC 19374; Bacillus amyloliquefaciens: purchased from China Agricultural Microorganism Culture Collection Center, strain number ACCC 10270; Yarrowia lipolytica: purchased from China Agricultural Microorganism Culture Collection Center, strain number ACCC 21176; Trichoderma harzianum: purchased from China Agricultural Microorganism Culture Collection Center, strain number ACCC 32533; Trichoderma reesei: purchased from China Agricultural Microorganism Culture Collection Center, strain number ACCC 32596; Bacillus megaterium: purchased from China Agricultural Microorganism Culture Collection Center, strain number ACCC 04288.

[0032] 2. Culture medium

[0033] The formula of LB medium is: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, and pH value is 7.4.

[0034] 3. Preparation of fermentation product

[0035] After activation of Bacillus siamensis, Bacillus subtilis, Bacillus amyloliquefaciens, Yarrowia lipolytica, Trichoderma harzianum, Trichoderma reesei and Bacillus megaterium, fermentation culture was carried out on the activated strains respectively, the culture medium used for fermentation culture was LB culture medium, the temperature was controlled between 28-42℃, the fermentation time was 48h, and the inoculation amount was 1%, to obtain Bacillus siamensis fermentation broth, Bacillus subtilis fermentation broth, Bacillus amyloliquefaciens fermentation broth, Yarrowia lipolytica fermentation broth, Trichoderma harzianum fermentation broth, Trichoderma reesei fermentation broth and Bacillus megaterium fermentation broth;

[0036] The obtained Bacillus siamensis fermentation broth, Bacillus subtilis fermentation broth, Bacillus amyloliquefaciens fermentation broth, Yarrowia lipolytica fermentation broth, Trichoderma harzianum fermentation broth, Trichoderma reesei fermentation broth and Bacillus megaterium fermentation broth were diluted, so that the viable cell count of Bacillus siamensis fermentation broth reached 2×10 9 CFU / mL, the viable cell count of Bacillus subtilis fermentation broth reached 5×10 9 CFU / mL, the viable cell count of Bacillus amyloliquefaciens fermentation broth reached 4×10 9 CFU / mL, the viable cell count of Yarrowia lipolytica fermentation broth reached 2×10 8 CFU / mL, the viable cell count of Trichoderma harzianum fermentation broth reached 2×10 7 CFU / mL, the viable cell count of Trichoderma reesei fermentation broth reached 2×10 7 CFU / mL, and the viable cell count of Bacillus megaterium fermentation broth reached 2×10 9 CFU / mL.

[0037] Preparation of compound microbial agent with different combinations in Example 1

[0038] The fermentation broth of different strains was mixed according to the mass parts in Table 1 to obtain different compound microbial agents.

[0039] Table 1 Different compound microbial agents

[0040]

[0041] Note: In this example, 1mL of strain fermentation broth is recorded as 1g of strain fermentation broth.

[0042] Example 2 Effect verification experiment

[0043] 1. Test strain

[0044] Enterotoxigenic Escherichia coli K99.

[0045] 2. Test animals and case model establishment

[0046] 2.1. Test animals

[0047] 9-day-old healthy chicks 270.

[0048] 2.2, case model establishment

[0049] Randomly selected 240 healthy chicks, body weight in 250 g or so, using the prepared enterotoxigenic E. coli K99 liquid to each chick intraperitoneal injection, injection dose was 0.2 mL / one, enterotoxigenic E. coli K99 in enterotoxigenic E. coli K99 liquid effective viable count was 2x10 9 CFU / mL; 30 healthy chicks as control group. Subsequently observed the chicks mental state, food intake, body weight and feces, etc.; chicken spirit depressed, two wings drooping, stand out of the crowd, lethargy, yellow and white loose stool, or liver surface moist, with red and yellow interlaced strip or heart, liver surface with jelly-like or fibrinous exudate, judged as case model establishment success.

[0050] 2.3, feeding management

[0051] The test was carried out in Jiangxi Agricultural University, and the test chicken was in cage. The test used 4 layers of cage, free feeding, free drinking water. The indoor temperature, light according to the conventional feeding management requirements. Every day at 9:00 and 18:00, add water and feed, take the remaining drinking water, add to the fixed volume, weigh the remaining feed, add to the fixed weight. The diet used in each group was the same, which was produced by a well-known feed company in China.

[0052] 3, test method

[0053] 3.1, test animal grouping and treatment

[0054] 30 healthy chicks as blank control group, 0.5 mL / one of physiological saline was given by gavage, the rest of the sickling (step "2.2" to establish the case model) were randomly divided into 8 groups, 30 in each group, respectively, model group, complex bacteria 1 group-complex bacteria 7 group. Among them, the model group was given 0.5 mL / one of physiological saline by gavage; complex bacteria 1 group-complex bacteria 7 group was given complex bacteria 1-7 prepared by gavage, the dose was 1 mL / one. Once a day, for 3 days, during the experiment (complex bacteria treatment for 3 days), the mental state, appetite, feces and other clinical symptoms of each group were observed and recorded, and the cure rate, effective rate and mortality rate were calculated.

[0055] 3.2, curative effect determination standard

[0056] Cure: spirit, diet, excretion returned to normal, symptoms completely disappeared;

[0057] Effective: spirit, diet, excretion improved, symptoms significantly reduced;

[0058] Invalid: no improvement of symptoms;

[0059] Death: death, and different degrees of pericarditis, perihelitis and other lesions were found in autopsy.

[0060] 3.3, weight measurement

[0061] On the second day of stopping treatment, the weight of all chickens was measured.

[0062] 3.4, immune organ measurement

[0063] Before the test, 10 nine-day-old chickens were randomly selected from each group, and on the second day of stopping treatment, the bursa of each group of chickens, spleen and thymus were collected and weighed.

[0064] 4, results

[0065] 4.1, statistical results of curative effect

[0066] The statistical results of curative effect are shown in Table 2.

[0067] Table 2 statistical results of curative effect (n=30)

[0068] Treatment Cured (only) Effective (only) Ineffective (only) Death (only) Blank control group 30 - - - Model group - - - 30 Compound inoculant 1 group 28 1 0 1 Compound inoculant 2 group 25 3 1 1 Compound inoculant 3 group 24 4 1 1 Compound inoculant 4 group 18 5 5 2 Compound inoculant 5 group 14 6 7 3 Compound inoculant 6 group 11 6 8 5 Compound inoculant 7 group 8 10 10 2

[0069] As can be seen from the results in Table 2, the mortality rate of chicken E. coli disease is as high as 100%. After treatment in different ways, the chicks in each group are improved to different degrees, but the number of deaths of chicks treated with complex bacterial agent 1-complex bacterial agent 3 is 1. The application also sets complex bacterial agents 4-7, and the results show that: although complex bacterial agents 4-5 are also composed of 5 strains, the number of dead chicks reaches 2-3; the number of deaths of complex bacterial agents 6-7 is also 2-5, and the number of invalid chicks is also significantly increased. As can be seen from the above, when one of the strains in the application is missing or replaced, the treatment effect is affected, and the survival rate of chicks treated with complex bacterial agents 1-3 provided by the application can reach 96.7%.

[0070] 4.2, weight measurement results

[0071] The weight measurement results are shown in Table 3.

[0072] Table 3 weight measurement results (n=30)

[0073] Treatment Body weight (g) Blank control group 450.12 Model group 0 Compound inoculant 1 group 420.37 Compound inoculant 2 group 400.98 Compound inoculant 3 group 385.52 Compound inoculant 4 group 327.67 Compound inoculant 5 group 314.14 Compound inoculant 6 group 306.50 Compound inoculant 7 group 300.63

[0074] Note: the data in the table are average values, and the following table is the same; because the model group is dead, the weight is recorded as 0, and the initial weight is about 250g.

[0075] As shown in Table 4, the body weight of each group of chicks is increased after different treatments, but the increase of the chicks treated by the complex microbial agent 1 to 3 is the most.

[0076] 4.3, immune organ measurement results

[0077] The immune organ measurement results are shown in Table 4.

[0078] Table 4 immune organ detection results

[0079] Treatment Bursa of fabricius index Spleen index Thymus index Blank control group 0.34 0.11 0.39 Model group 0.24 0.03 0.31 Compound inoculant 1 group 0.33 0.1 0.45 Compound inoculant 2 group 0.31 0.08 0.41 Compound inoculant 3 group 0.32 0.07 0.43 Compound inoculant 4 group 0.28 0.07 0.36 Compound inoculant 5 group 0.26 0.07 0.34 Compound inoculant 6 group 0.25 0.05 0.31 Compound inoculant 7 group 0.23 0.05 0.33

[0080] Note: the calculation formula of the bursa of fabricius index is: bursa of fabricius index = bursa of fabricius weight / body weight*100; the calculation formula of the spleen index is: spleen index = spleen weight / body weight*100; the calculation formula of the thymus index is: thymus index = thymus weight / body weight*100.

[0081] As shown in Table 4, the complex microbial agent 1 to 3 provided by the application can promote the recovery of the bursa of fabricius index, the spleen index and the thymus index of the chicks after treating the chicks. Therefore, the complex microbial agent 1 to 3 provided by the application can improve the structure and function of the immune system of animals.

[0082] Example 3

[0083] In June 2024, 220 30-day-old piglets with an average weight of 8.5 kg raised by a certain county breeder, 30 piglets died, some piglets had persistent diarrhea, discharged paste-like loose stools, reduced appetite, and emaciated body, and spirit. When the dead piglets were dissected, it was found that the intestinal wall of the dead piglets was thin, the intestinal mucosa was congested and easy to peel off, there was yellow liquid and gas in the intestine, and there were different degrees of bleeding points in the heart, liver and kidney. The intestinal contents of the dead piglets were collected aseptically, cultured and identified in the laboratory, and the pathogenic bacteria of the dead piglets were diagnosed as Escherichia coli. The piglets were treated by gavage with the complex microbial agent 1 prepared in Example 1, the dose was 5 mL / head / day, and the treatment was continued for 3 days. The diarrhea symptoms of the piglets disappeared, the feed intake gradually stabilized, and the mental state gradually recovered. Among the 190 treated piglets, 3 died, 1 still had diarrhea, reduced appetite or mental apathy, 186 were cured, the cure rate was 97.9%, and follow-up was conducted at half a month and 1 month, respectively. The breeder feedback: the piglets with diarrhea, reduced appetite or mental apathy recovered correctly, and all piglets did not relapse, the feed intake, spirit, feces, etc. were normal, the hair color was smooth, the growth performance was normal, and there was no piglet.

[0084] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. The use of a microbial combination in the preparation of a compound microbial agent with the effect of improving the disease resistance of livestock and poultry, characterized in that, The microbial combination is composed of the following components by mass parts: 1-3 parts Bacillus siamensis (Bacillus siamensis) Bacillus siamensis , 3-7 parts Bacillus subtilis (Bacillus subtilis) Bacillus subtilis , 2-5 parts Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Bacillus amyloliquefaciens , 2-5 parts Yarrowia lipolytica (Yarrowia lipolytica) Yarrowia lipolytica , and 1-3 parts Trichoderma harzianum (Trichoderma harzianum) Trichoderma harzianum ; The strain number of the Bacillus siamensis is ACCC 19948; the strain number of the Bacillus subtilis is ACCC 19374; the strain number of the Bacillus amyloliquefaciens is ACCC 10270; the strain number of the Yarrowia lipolytica is ACCC 21176; and the strain number of the Trichoderma harzianum is ACCC 32533.

2. Use according to claim 1, characterized in that, The ratio of the effective viable cell number of the Bacillus siamensis, the Bacillus subtilis, the Bacillus amyloliquefaciens, the Yarrowia lipolytica, and the Trichoderma harzianum in the microbial combination is (2-6)×10 9 : (15-25)×10 9 : (8-20)×10 9 : (2-10)×10 8 : (2-6)×10 7 .

3. A complex microbial agent having the effect of improving the disease resistance of livestock and poultry, characterized by comprising the following bacteria: The preparation raw material of the complex microbial agent comprises the microbial combination in claim 1 or 2. ​ 4. The complex bacterial agent according to claim 3, characterized by, The complex microbial agent further comprises auxiliary materials.

5. The preparation method of the complex bacterial agent of claim 3, characterized in that, The method comprises the following steps: After the strains in the microbial combination in claim 1 are activated, the strains are respectively subjected to fermentation culture to obtain Bacillus siamensis fermentation liquor, Bacillus subtilis fermentation liquor, Bacillus amyloliquefaciens fermentation liquor, Yarrowia lipolytica fermentation liquor and Trichoderma harzianum fermentation liquor; The Bacillus siamensis fermentation liquor, the Bacillus subtilis fermentation liquor, the Bacillus amyloliquefaciens fermentation liquor, the Yarrowia lipolytica fermentation liquor and the Trichoderma harzianum fermentation liquor are mixed uniformly to obtain the complex microbial agent.

6. The production method according to claim 5, wherein The fermentation culture is carried out at a temperature of 28-37℃ for 24-72h; and the culture medium for the fermentation culture is LB culture medium. And / or, the inoculation amount during the fermentation culture is 1-2%.

7. Use of the complex microbial agent in claim 3 or 4 in the preparation of a product for improving the disease resistance of livestock and poultry.

8. A product for improving the disease resistance of livestock and poultry, characterized by, The product comprises the complex microbial agent in claim 3 or 4.

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