CN111803529A

CN111803529A - Composite microecological preparation for preventing calf diarrhea

Info

Publication number: CN111803529A
Application number: CN202010702682.6A
Authority: CN
Inventors: 张文举
Original assignee: Shihezi University
Current assignee: Shihezi University
Priority date: 2020-07-21
Filing date: 2020-07-21
Publication date: 2020-10-23

Abstract

The invention relates to a composite microecological preparation for preventing calf diarrhea. A composite microecological preparation for preventing calf diarrhea contains Lactobacillus acidophilus, Saccharomyces boulardii and Bacillus subtilis. According to the invention, through the comparison and analysis of the biological characteristics of acid resistance, cholate resistance and bacteriostasis of 3 probiotics including lactobacillus acidophilus, saccharomyces boulardii and bacillus subtilis, the pathogenic escherichia coli in the diarrhea calf feces is identified through separation, and the three probiotics have better tolerance and have better bacteriostasis effect on inhibiting escherichia coli k99 through the mixing of the three probiotics; the feed additive is applied to calves in lactation period, has the effects of increasing daily gain of the calves, increasing feed intake of the calves, reducing diarrhea rate, improving immunity and oxidation resistance of the calves, and can adjust intestinal flora of animals.

Description

Composite microecological preparation for preventing calf diarrhea

Technical Field

The invention belongs to the field of livestock, and particularly relates to a compound microecological preparation for preventing calf diarrhea.

Background

In the past decades, the use amount of antibiotics in preventing and treating livestock and poultry infection and resisting diseases is increased year by year, China is reported to be the country with the most antibiotic production and consumption in the world, and brings production benefits to the development of animal husbandry, and meanwhile, the ecological environment and health condition of human beings and livestock and poultry face huge risks, which also leads to the rise of the drug resistance of pathogens and common bacteria in the living environment of human beings, the livestock and poultry breeding environment, the livestock and poultry organism and the like. Therefore, in 2003, the use of antibiotics was gradually banned worldwide in sweden and in 1986, and in 2006 in the european union. With the increasing global trend towards banning the use of Antibiotic Growth Promoters (AGPS), the need for AGPS alternatives is becoming more stringent.

Calf is used as a reserve strength of the dairy cow breeding industry and has a crucial role in exerting the production potential of dairy cows, and Calf diarrhea (Calf diarrhea) is a main cause of death of calves. The calf diarrhea is a common disease, has great harm to the growth and development of calves, has complex etiology, is one of the frequently encountered diseases of the calves at present, and brings great economic loss to the cattle raising industry. According to statistics, the large-scale morbidity of the Xinjiang northern reclamation area is 40-60%, the mortality is 50-70%, and the microbial factors causing calf diarrhea mainly comprise bacterial (the average morbidity is 36.7%) and viral (the average morbidity is 7.5%) factors.

Researches find that pathogenic escherichia coli causing calf diarrhea in the near two years in Xinjiang area generate different degrees of drug resistance to antibiotics such as ampicillin, gentamicin, chloramphenicol, ciprofloxacin and the like. Therefore, there is an urgent need to find alternatives to antibiotics as preventive antibacterial agents. In order to improve animal welfare and protect the environment, the feeding microorganisms are concerned because the feeding microorganisms have the characteristics of no pollution, no residue and green and have the biological functions of improving the growth performance of livestock and poultry, optimizing intestinal flora, improving the immunity of animal organisms and the like, and a plurality of researches are just the feeding microecologics which are considered as potential substitutes.

In view of the above, the invention provides a novel compound microecological preparation for preventing calf diarrhea, which can replace antibiotics and prevent digestive tract diseases of young calves.

Disclosure of Invention

The invention aims to provide a compound microecological preparation for preventing calf diarrhea, which can replace antibiotics and prevent digestive tract diseases of young calves.

In order to realize the purpose, the adopted technical scheme is as follows:

a composite microecological preparation for preventing calf diarrhea contains Lactobacillus acidophilus, Saccharomyces boulardii and Bacillus subtilis.

Further, the ratio of the viable count of lactobacillus acidophilus, saccharomyces boulardii and bacillus subtilis is 1-3: 1-3: 1-3.

Still further, the ratio of the viable count of lactobacillus acidophilus, saccharomyces boulardii and bacillus subtilis is 1: 2: 1 or 3:3: 1.

still further, the ratio of viable count of lactobacillus acidophilus, saccharomyces boulardii and bacillus subtilis is 3:3: 1.

further, the composite microecological preparation is a liquid preparation.

Still further, the composite microecological preparation has viable count concentration of 1 × 10⁹cfu/mL lactobacillus acidophilus, Saccharomyces boulardii and Bacillus subtilis.

Further, the composite microecological preparation is used for preventing diarrhea caused by calf escherichia coli K99.

Compared with the prior art, the invention has the beneficial effects that:

the invention takes separated pathogenic escherichia coli k99 and probiotics preserved in a laboratory as research objects, compares and analyzes biological characteristics among probiotic strains, and screens out a mixture ratio optimization combination of 3 strains of the compound microecological preparation for optimally inhibiting and preventing calf diarrhea. Provides a theoretical basis for developing a microecological preparation for preventing and treating calf diarrhea.

Meanwhile, the technical scheme of the invention has the effects of improving daily gain of calves, increasing feed intake of calves, reducing diarrhea rate, improving immunity and oxidation resistance of calves, and adjusting intestinal flora of animals.

Drawings

FIG. 1 is a microscopic image of Escherichia coli;

FIG. 2 is an amplification diagram of E.coli k 99;

FIG. 3 is a BLAST results from NCBI Genbank;

FIG. 4 shows the tolerance of Lactobacillus acidophilus to various pH values of artificial gastric juice;

FIG. 5 shows the tolerance of Bacillus to various pH values of artificial gastric juice;

FIG. 6 shows the tolerance of Saccharomyces boulardii to various pH values;

FIG. 7 shows the tolerance of 3 probiotic artificial intestinal fluids at different times;

FIG. 8 is a graph of the tolerability of 3 probiotic bovine bile salts.

Detailed Description

In order to further illustrate the composite microecological preparation for preventing calf diarrhea according to the present invention and achieve the intended objects, the following detailed description will be given to the specific embodiments, structures, characteristics and effects thereof of the composite microecological preparation for preventing calf diarrhea according to the present invention with reference to the preferred embodiments. In the following description, different "one embodiment" or "an embodiment" refers to not necessarily the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

The composite microecological preparation for preventing calf diarrhea according to the present invention will be described in further detail with reference to the following specific examples:

in the aspect, the pathogenic escherichia coli k99 in the feces of the diarrhea calf is separated and identified by adopting a molecular biological method. Meanwhile, the comparative analysis on the acid resistance, cholate resistance and bacteriostatic biological characteristics of 3 probiotics of lactobacillus acidophilus, saccharomyces boulardii and bacillus subtilis shows that the survival rate is 84.34% when the pH is 3. The survival rate of the bacillus is 142.24% when the pH value is 5; the survival rate in the artificial gastric juice is high at 9 hours and the OD at 0 hour₆₀₀Compared with the prior art, the survival rates of the saccharomyces boulardii and the lactobacillus acidophilus are respectively 108.7 percent and 129.73 percent, and the survival rate of the lactobacillus acidophilus reaches 96.37 percent. In the cattle bile salt culture medium containing different concentrations, the survival rate of the saccharomyces boulardii is 60.49 percent highest compared with the survival rate of the control group, the survival rate of the lactobacillus acidophilus is 85.1 percent highest compared with the survival rate of the control group, and the survival rate of the bacillus is 47.42 percent highest compared with the survival rate of the control group. The three probiotics have certain bacteriostatic characteristics on escherichia coli and salmonella. The three probiotics have better tolerance to artificial gastric juice, artificial intestinal juice and bovine bile salt and certain bacteriostatic property.

The inhibition of Escherichia coli k99 by different proportions of the three probiotics is found that after the three probiotics are cultured for 12 hours according to different proportions, the differences of the

groups

1, 2, 3, 4, 5, 6, 7, 8 and 9 in the test group are obvious (P is less than 0.05) compared with the control group, and the differences of the

groups

2, 4 and 8 are obvious (P is less than 0.05); after 24h of culture, the experimental group had significant differences (P <0.05) in the 2 nd, 3 rd, 4 th, 5 th, 6 th and 8 th groups compared with the control group, but no significant differences (P >0.05) between the 2 nd, 3 th, 4 th, 5 th, 6 th and 8 th groups. The 8 th treatment group has better bacteriostatic effect on escherichia coli K99 after being cultured for 12 hours, so that the optimal compounding ratio of lactobacillus acidophilus, bacillus subtilis and saccharomyces boulardii can be determined to be 3:3: 1. Provides theoretical basis for developing microecological preparation for replacing antibiotics and preventing digestive tract diseases of young calves.

The specific operation steps are as follows:

effect of a microbial preparation on digestive tract diseases in young calves

1 materials and methods

1.1 test materials

(1) Test strains

Probiotic bacterial strains: lactobacillus acidophilus (Lactobacillus acidophilus), Saccharomyces boulardii (Saccharomyces boulardii), and Bacillus subtilis (Bacillus subtilis) provided by biological feed laboratory of animal science institute of Stone river university.

Test germ, namely, autonomously separating escherichia coli (K99); salmonella and Staphylococcus aureus are purchased from Shanghai Lu micro-technology, Inc.

(2) Culture medium:

LB nutrient agar medium, LB broth, Macconka agar medium, MRS liquid medium, MRS solid medium, YPD liquid medium, YPD solid medium, Bacillus medium were purchased from Qingdao Gaokoubo Biotech Ltd.

(3) Primary reagent

PBS buffer solution is used for diluting bacterial liquid. The bacterial DNA extraction kit is purchased from Tiangen Biochemical technology, Inc.; pepsin, trypsin, bovine bile salts were purchased from Beijing Biotopped Science & Technology co. The physiological and biochemical identification tube is purchased from Hangzhou Binhe microbial reagent GmbH. The K99 primer was synthesized by Shanghai Czeri Biotechnology, Inc., and the sequencing was performed by Huada Gene, Inc.

(4) Main equipment

A liquid transferring gun; electronic balance JM-500, Kunshan Keke electronics, Inc., electronic balance BS224S, Beijing Saedodes instruments, Inc.; acidimeters WTW PH3110, kaimen instruments; a vertical ultra-low temperature incubator GSP-9160MBE, a constant temperature incubator HPX-9162MBE, an ultra-clean workbench SW-CJ-2F, Shanghai Bocheng industries, Inc. medical equipment factory; ELIASA WD-2102B, BEIJING Beijing, six Biotech Co., Ltd; PCR instruments, Shanghai Naltz instruments, Inc. Centrifuge TG16-W, Hunan Xiang appearance laboratory Instrument development Co., Ltd

1.2 test methods

1.2.1 bacterial isolation and identification of calf feces Escherichia coli k99

The method comprises the steps of adopting a cotton swab to aseptically collect diarrhea feces samples of four calves which have diarrhea within 7 days of a certain scale cattle farm, diluting the diarrhea feces samples in sterile normal saline, culturing the diarrhea feces samples in LB broth culture medium at 37 ℃, further purifying the diarrhea feces samples in LB solid culture medium, separating and purifying the diarrhea feces samples, performing microscopic examination after gram staining, storing the purified bacterial colonies at-80 ℃, simultaneously adopting a kit to extract DNA of the purified bacteria, adopting Escherichia coli k99 specific primers (5'-TATTATCTGGTGGTATGG-3', 5'-GGTATCCTTTAGCAGCAGTATTTC-3') to perform PCR amplification, and adopting a 20uL reaction system, namely 2 x master PCR Mix lOuL, 3uL of a DNA template and 0.5uL of each of upstream and downstream primers (the concentration of the primers is 20umol/L), and adding sterilized ultrapure water to 20 uL. Pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 52.6 deg.C for 30 s; stretching at 72 ℃ for 30 s; the cycle was 35 times, and the extension was carried out at 72 ℃ for 10 min. And recovering and sequencing the amplification product, and performing physiological and biochemical identification on the Escherichia coli K99 strain.

1.2.2 activation of strains and determination of viable bacteria content

Strain activation: inoculating 3 strains in corresponding liquid culture media, performing shake culture at 37 ℃ for 18h, coating and streaking in MRS solid culture media, YPD solid culture media and bacillus culture media by using inoculating loops, selecting a single strain, inoculating the single strain into the MRS broth culture media, the YPD broth culture media and the bacillus broth culture media, wherein lactobacillus acidophilus is cultured and statically cultured at 37 ℃ for 24h, and saccharomyces boulardii and bacillus subtilis are cultured and statically cultured at 37 ℃ for 24h, and are activated for 2-3 generations for later use.

And (3) viable bacteria content determination: under aseptic condition, performing gradient dilution on activated 2 generation probiotic bacteria (Lactobacillus acidophilus, Bacillus subtilis, Saccharomyces boulardii, and Escherichia coli), and diluting the suspension 10 times to 10 times^-7、10^-8、10^-9Then, 100uL of each of the three dilutions of the bacterial solution was applied to a plate, three plates were applied to each dilution, and after 12-24 hours of inverted culture at 37 ℃, colonies were counted.

1.2.3 tolerance test for three probiotics at different pH values

Preparing artificial gastric juice: 16.4mL of hydrochloric acid with the mass fraction of 0.1kg/L is taken, and the pH value is adjusted to 2.0, 3.0, 4.0, 5.0 and 6.0. Then adding pepsin with the proportion of 0.01g/mL, and after the pepsin is dissolved uniformly, sterilizing the mixture by a microporous filter membrane for later use. Inoculating activated Lactobacillus acidophilus, Saccharomyces boulardii and Bacillus subtilis into pepsin solution with different pH values at an inoculation ratio of 1%, culturing in a constant temperature incubator at 37 deg.C for 3 hr, and measuring OD₆₀₀。

1.2.4 Artificial intestinal juice tolerance test

Preparation of artificial intestinal juice by accurately weighing KH₂P0₄3.4g, adding 250mL of distilled water for dissolving, and using the mass fractionAdjusting pH to 6.8 with 4g/L NaOH solution, adding pepsin to make its concentration be 0.01g/mL, after it is uniformly dissolved, sterilizing with microporous membrane, and placing in refrigerator at 4 deg.C for use. Inoculating the activated 3 probiotic strains into the artificial intestinal juice at an inoculum size of 10%, repeating the treatment for 3 times, culturing at 37 deg.C, sampling at 0, 3, 6, and 9 hr, and measuring OD of each strain with enzyme-labeling instrument₆₀₀The value is obtained.

1.2.5 bile salt tolerance test

Adding ox bile salt into MRS, YPD and Bacillus liquid culture medium respectively to obtain culture medium with ox bile salt concentration of 0.15g/L, 0.3g/L and 0.06g/L respectively. The 3 media were then sterilized at 118 ℃ for 15 minutes and cooled for use. Inoculating the activated strain into the corresponding ox bile salt culture medium, culturing at 37 deg.C for 8 hr with the medium without bile salt as reference, observing the growth of strain, and measuring OD₆₀₀And calculating the survival rate.

1.2.6 bacteriostatic test

Respectively culturing the separated escherichia coli k99, salmonella and staphylococcus aureus for 24h, diluting by 20 times, and uniformly coating 100uL of pathogenic bacteria liquid on a prepared sterile agar plate. Culturing at 37 deg.C for 12-24 hr by Oxford cup and punching method, measuring diameter of zone of inhibition, and comparing with sterile common liquid culture medium.

Proportioning optimization of 1.2.73 strain for inhibiting escherichia coli k99

Diluting the activated 3 probiotics to 10^-1To (3) is added. Then diluting to the same concentration (1X 10)⁹cfu/mL) were inoculated at different ratios in Table 1, and 11 groups of control groups were set, and added to a predetermined amount of LB liquid medium to give a final volume of 5mL, respectively, and shake-cultured at 37 ℃ and 165 rpm. The incubation times were set at 12h and 24 h. Then carrying out gradient dilution, and taking 10^-6、10^-7、10^-7100uL of the obtained product was spread on MacConkey agar medium, cultured in a 37 ℃ incubator for 24 hours, and then the number of Escherichia coli was counted, and a control group was set.

Table 1 inoculation amount units of different ratios of three probiotics: uL

1.2.8 detection index:

detection of OD₆₀₀Value and viable count of Escherichia coli K99.

1.2.9 data analysis:

the experimental data were analyzed by Spss21.0 statistical software for ONE-WAY analysis of variance (ONE-WAY ANONA) and Duncan's multiple analysis, and the results were expressed as "mean-squared standard deviation", with P <0.05 being significantly different.

2. Results and analysis

2.1 bacterial isolation and identification of feces Escherichia coli k99 from diarrhea calves

The Escherichia coli purified bacterial colony is gram-stained and microscopically checked to be G^-Short rod shape, results are shown in FIG. 1, E.coli k99 amplified in FIG. 2; BLAST was performed at NCBI Genbank and showed a similarity of 99.99%, as shown in FIG. 3. The biochemical identification is shown in table 2.

Meanwhile, the separated escherichia coli k99 is adopted to carry out virus challenge on the mice, 0.2mL of escherichia coli liquid cultured for 18 hours is injected into each mouse, and the result shows that 80% of mice die. The separated Escherichia coli k99 is proved to have stronger toxicity.

TABLE 2 physiological and biochemical identification table of Escherichia coli k99

2.2 tolerance test for different pH values of three probiotics

As shown in the results of the tests in figures 4-6, the three probiotics have better tolerance under the culture conditions with different pH values, wherein the survival rate of the lactobacillus acidophilus reaches 118.75% when the pH value is 5, and the survival rate reaches 84.34% when the pH value is 3.

The survival rate of the saccharomyces boulardii is over 100 percent for different pH values. The survival rate of the bacillus is 142.24% when the pH value is 5, the survival rate reaches more than 100% under different pH conditions, and the bacillus has better tolerance.

2.3 tolerance test for three probiotic artificial intestinal fluids

The results are shown in fig. 7, the three probiotics have better tolerance to the artificial intestinal juice under different culture time, the survival rate of the three probiotics in the artificial gastric juice is high at 9 hours, and the OD at 0 hour₆₀₀Compared with the prior art, the survival rates of the saccharomyces boulardii and the lactobacillus acidophilus are respectively 108.7 percent and 129.73 percent, and the survival rate of the lactobacillus acidophilus reaches 96.37 percent.

2.4 three probiotic bile salt tolerance test

The results are shown in FIG. 8: with the increase of the concentration of the bovine bile salts, the survival rates of the saccharomyces boulardii and the lactobacillus acidophilus are respectively 50.64%, 60.49% and 46.37% compared with the survival rates of the control group, 81.65%, 85.1% and 81.19% compared with the survival rate of the lactobacillus acidophilus, and the survival rates of the bacillus are respectively 45.1%, 45.11% and 47.42% compared with the survival rate of the control group. The saccharomyces boulardii and lactobacillus acidophilus have stronger tolerance to bovine bile salts, while the bacillus is weaker.

2.5 bacteriostatic test for three probiotics

The results are shown in table 3, and the saccharomyces boulardii, the lactobacillus acidophilus, the bacillus subtilis have certain bacteriostatic characteristics on escherichia coli and salmonella, the lactobacillus acidophilus has stronger bacteriostatic activity on staphylococcus aureus, and the saccharomyces boulardii and the lactobacillus acidophilus have poorer bacteriostatic activity on staphylococcus aureus.

TABLE 3 zone of inhibition diameters of three probiotics

2.6 ratio optimization of three strains for inhibiting Escherichia coli k99

The results of in vitro bacteriostatic tests on bovine Escherichia coli K99 after culturing for 12h and 24h by combining lactobacillus acidophilus, Saccharomyces boulardii and Bacillus in different proportions are shown in Table 4.

Table 4 in vitro bacteriostatic test (n-3) logcfu/mL of escherichia coli k99 with three probiotic preparations in different ratios

The same row without letters or data shoulder with the same letter indicates no significant difference (P >0.05), and different lower case letters indicate significant difference (P < 0.05). The following table is the same.

As can be seen from table 4, after the three probiotics are cultured for 12 hours according to different ratios, the differences of the

groups

1, 2, 3, 4, 5, 6, 7, 8 and 9 in the test group are significant (P is less than 0.05) compared with the control group, the differences of the group 10 and the control group are insignificant (P is greater than 0.05), the differences of the

groups

1, 3, 5, 6, 7, 9 and 10 are insignificant (P is greater than 0.05), and the differences of the

groups

2, 4 and 8 are significant (P is less than 0.05); after 24h of culture, the differences of the 2 nd, 3 rd, 4 th, 5 th, 6 th and 8 th groups are significant (P <0.05) compared with the control group, but the differences of the 2 nd, 3 rd, 4 th, 5 th, 6 th and 8 th groups are not significant (P >0.05), and the differences of the 1 st, 2 nd, 7 th, 9 th and 10 th groups are not significant (P >0.05) compared with the control group.

In conclusion, in combination with practical application, the composite microbial preparation is discharged after the cattle intestinal tract wriggles for 12 hours, and the 8 th treatment group has a good bacteriostatic effect on escherichia coli K99 after being cultured for 12 hours, so that the optimal compounding ratio of lactobacillus acidophilus, bacillus subtilis and saccharomyces boulardii can be determined to be 3:3: 1.

Analysis of probiotic properties of probiotics:

as the research is directed at the research of the compound probiotics of calf Escherichia coli K99, according to the imperfect development characteristics of the digestive system of the newborn calf, nutrients in daily ration are digested and absorbed mainly by virtue of the abominable stomach and small intestine, and the calf within two weeks does not have rumination behavior, the digestive function characteristics of the calf are basically similar to that of a monogastric animal, the fluctuation of the pH value of the digestive tract acidity of the calf is large at the moment, and the calf has great influence on the digestive physiology, the research shows that the pH value of the abomasum is greatly changed for 24h before and after the calf is fed, the pH value of the abomasum is between 1.5 and 2.0 before the calf is fed, the pH value of the abomasum can be increased from 1.5 to 6.0 after the calf is fed, and then can be reduced to between 1.5 and 2.0, and the pH value of the abomasum of the calf can be greatly fluctuated before and after the calf is fed, but can also be in a. Trypsin is also different in different intestine sections of calves, wherein the activity is lowest in duodenum, highest in jejunum and second in ileum. The concentration of bile juice in intestinal tract of calf is constantly changing, so it is difficult to predict its concentration in a certain period of time, but generally speaking, the content of bile salt in small intestine is usually 0.03-0.3%.

In vitro identification of probiotics has been widely used to evaluate the probiotic potential of candidate bacteria. Including tolerance to gastric juice and bile salt content, antibacterial action, etc. An important feature of the ability of probiotics to survive in the animal body is their ability to withstand the acidic conditions of the stomach and bile salts in the small intestine.

The experimental result shows that the three probiotics, namely the lactobacillus acidophilus, the bacillus and the saccharomyces boulardii have higher tolerance to acid conditions, bovine bile salts and trypsin.

The antibacterial effect of lactobacillus acidophilus, bacillus subtilis and saccharomyces boulardii on escherichia coli (K99) in different proportions is as follows:

the feed microbial additive is an active preparation, has green and pollution-free properties, has unprecedented development in the aspects of promoting animal growth, intestinal health and the like, plays a great role in controlling the health track (including and exceeding immune components) of a host, and has biological functions of promoting nutrient absorption, regulating the structure of intestinal flora, improving the immunity of livestock organisms and the like.

Lactobacillus acidophilus is a typical probiotic bacterium, and has physiological effects of inhibiting intestinal pathogens, promoting immune cell homeostasis and intestinal health, inhibiting enterotoxigenic escherichia coli K99, and the like. Research shows that the Lactobacillus acidophilus has higher survival rate of the strain in pH3 and stronger viability in a culture medium containing bile salt, and the critical concentration is 0.3 percent, which shows that the strain has the capability of resisting the antibacterial action when passing through the small intestine.

Saccharomyces buchneri is one of the best studied probiotics in Acute Gastroenteritis (AGE) and has been shown to be safe. The saccharomyces boulardii has antitoxin, and phosphatase secreted by the saccharomyces boulardii can obviously destroy endotoxin secreted by escherichia coli. Has growth inhibiting effect on Salmonella typhimurium, Escherichia coli, Candida albicans, etc., and can improve activity of short chain fatty acid in intestinal tract, regulate metabolism function of intestinal tract, promote nutrition effect of intestinal mucosa, enhance immunity, and increase SCFA content in organism. Because it is aerobic bacteria and has high adhesiveness, oxygen in intestinal tract can be consumed and hydrogen peroxide can be decomposed at the same time, so that the proliferation of anaerobic bacteria is promoted, and the flora imbalance caused by improper use of antibiotics can be regulated, thereby promoting the intestinal flora balance. The application research on poultry, pigs and aquatic products has been reported, but the research on ruminants, particularly calves, is less. The application of the composition on calves has the effects of increasing daily gain of calves, reducing diarrhea rate, improving immunity and the like.

The content of Bacillus subtilis in animal digestive tract is low. Because the oxygen consumption bacteria are actually consumed, the oxygen in the digestive tract of the animal can be quickly consumed, so that the animal can generate an anaerobic environment and inhibit the growth of harmful pathogenic bacteria. Meanwhile, the bacillus subtilis has a bactericidal effect, and bacteriocin in the metabolite generated by the bacillus subtilis can effectively kill pathogenic floras such as escherichia coli, staphylococcus aureus, salmonella and the like, so that the health of an animal body is maintained.

In the test, the separation and identification of pathogenic escherichia coli in the feces of the diarrhea calf are carried out, the comparison and analysis of the acid resistance, cholate resistance and bacteriostatic biological characteristics of 3 probiotics including lactobacillus acidophilus, saccharomyces boulardii and bacillus subtilis are found, the three probiotics have better tolerance, and the inhibition of escherichia coli k99 by different proportions of the three probiotics is found, so that the optimal compounding ratio of the lactobacillus acidophilus, the bacillus subtilis and the saccharomyces boulardii is determined to be 3:3:1, the better bacteriostatic effect is achieved, and the method can be used for later-stage development of alternative antibiotics and prevention of digestive tract diseases of young calves and provides a theoretical basis.

The influence of the two microecologics on calf growth performance, diarrhea rate, blood immunity index and oxidation resistance

40 calves of 5 days old are selected for the test, and are randomly divided into 4 groups of 10 calves, and 1 calf is repeated for each calf. The group I is a control group, the same amount of normal saline is fed, the group II is fed with 0.5g of microbial preparation, the group III is fed with 1g of microbial preparation, the group IV is fed with 2g of microbial preparation, the test period is 8 weeks, and test data are collected respectively with the 0 th week, the 2 th week, the 4 th week, the 6 th week and the 8 th week of the test. Growth performance, diarrhea rate, blood immunity index and oxidation resistance are used as evaluation indexes.

The microbial preparation was the 8 th microbial preparation in table 4.

Test method and data acquisition

1. Growth performance, feed intake, stool score

(1) Food intake: the calves formula, starter diet and alfalfa feed intake were recorded daily.

(2) And (3) grading the feces: the feces status of the test calves was evaluated according to scoring criteria (table). 1 minute, normal shape; 2 min, soft and loose; 3, loosening to form a water sample; 4, dividing into a water sample, mucus and a small amount of blood silk; 5 points, water sample, mucus, and abundant blood streak. When feces were rated 3 points and above, diarrhea was noted.

The diarrhea rate (%) (+) Σ (number of diarrhea heads × number of diarrhea days)/(total number of heads × number of test days) × 100.

2. Serum sample collection and determination

Collecting 5mL of blood by jugular vein of heparin sodium-containing vacuum blood collection tube before feeding on the 2 nd, 4 th and 6 th week-old day, standing at room temperature for 30min, centrifuging at 3000r/min for 15min, collecting serum, placing into 1.5mL freezing tube, labeling group and cattle number, sampling date, and preserving with liquid nitrogen. Serum concentrations of Ig A, Ig G, Ig M, IL-2, IL-4, IL-1 β and TNF- α were measured, and serum total protein ((TP), globulin (Glb), albumin (Alb), glucose (Glu), urea nitrogen (BUN), alkaline phosphatase (ALP), creatinine, aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) were measured.

3. Collecting a feces sample:

(1) freezing and storing the excrement sample by liquid nitrogen: collecting dung samples before feeding to calves of 0, 2, 4, 6 and 8 weeks old in the morning, placing the dung samples in 50ml centrifuge tubes, randomly selecting 6 cattle in each group for rectum dung taking, and respectively packaging the dung samples collected by each cattle into 6 small tubes. And (5) freezing and storing by using liquid nitrogen.

4. Blood immunity index and antioxidation performance

And (5) detecting by using a kit.

(II) results:

1. effect of composite probiotics on growth performance of calves

As shown in Table 5, at week 2, the average daily gain was higher in trial group III than in control group I, with significant differences (P <0.05) but no significant differences between groups (P > 0.05). The average daily intake of the groups II, III and IV is higher than that of the group I (P <0.05), but the difference among the groups is not significant (P > 0.05). At 4wk, there was no significant difference in average daily gain and average daily food intake between groups (P > 0.05). At 6wk, the daily gain of the groups II and III is obviously higher than that of the groups I and IV (P <0.05), and the differences of the groups II and IV and the group I are obvious (P < 0.05). At 8wk, there was no significant difference in ADG between groups (P >0.05), but ADG was higher in groups II and IV than in group I. Compared with the groups I, II and III, the admi of the group IV has significant difference (P <0.05), and the group II and the group III have no significant difference (P > 0.05).

TABLE 5 influence of Complex Microecological Agents on Calf growth Performance

In the same row, different lower case letters are marked to indicate that the difference is significant (P <0.05), and the same or no letter is marked with no significant difference (P >0.05), and the following are the same. ADG (average daily gain), ADMI (average daily food intake).

2. Effect of composite probiotics on calf feces score and diarrhea rate

TABLE 6 Effect of Complex Microecological Agents on Calf fecal Scoring and diarrhea Rate

As can be seen from Table 6, when the stool scores are 2WK and 4WK, the stool scores of the groups I, II and III have no significant difference (P >0.05), the stool scores of the groups II and IV have no significant difference (P >0.05), and the stool scores of the groups I and III have significant difference (P <0.05) compared with the stool scores of the groups IV; at 6WK and 8WK, the differences between groups were not significant (P > 0.05).

The diarrhea rate of the test group is reduced compared with the control group at 2WK and 4WK, the diarrhea rate of the IV group is reduced by 51.65% compared with the I group at 2WK, and the diarrhea rate of the IV group is 0 at 4 WK.

3. Effect of composite probiotics on immune function of lactating calves

As shown in Table 7, the serum IgA content varies among calves of different ages. At 2 weeks, the difference between II and IV is obvious (P <0.05), and III and IV are obviously higher than I (P < 0.05); in the 4 th week, the difference between III and IV is extremely obvious (P <0.001), the difference between II and III is obvious (P <0.05), the difference between I and II is not obvious (P >0.05) and 0.05), the difference between III and IV is obvious (P <0.001), the difference between II and III is obvious (P <0.05), the difference between I and II is not obvious (P >0.05), and the difference between III and IV is not obvious (P > 0.05); at 6wk, the difference between II and IV groups is very obvious (P <0.05) (P <0.001), the difference between III and I and II is obvious (P <0.05), and the difference between IV and I and II is obvious (P < 0.001); at 8wk, the difference between IV and I is significant (P <0.05), and the difference between I, II and III is not significant (P > 0.05).

The serum IgM levels of calves of different weeks are different. At 2 weeks, the difference between II and IV is obvious (P <0.05), and III and IV are obviously higher than I (P < 0.05); in the 4 th week, the difference between III and IV is extremely obvious (P <0.001), the difference between II and III is obvious (P <0.05), the difference between I and II is not obvious (P >0.05) and 0.05), the difference between III and IV is obvious (P <0.001), the difference between II and III is obvious (P <0.05), the difference between I and II is not obvious (P >0.05), and the difference between III and IV is not obvious (P > 0.05); at 6wk, the difference between II and IV groups is very obvious (P <0.05) (P <0.001), the difference between III and I and II is obvious (P <0.05), and the difference between IV and I and II is obvious (P < 0.001); at 8wk, the difference between IV and I is significant (P <0.05), and the difference between I, II and III is not significant (P > 0.05).

When IgM is at 2wk, the differences of the group III and the group IV are extremely obvious (P <0.001), the difference of the group III is obvious (P <0.05), the difference of the group III and the group IV is not obvious (P >0.05), and the difference of the group I and the group II is not obvious (P > 0.05);

at 4wk, the differences among the groups II, III and IV are obvious (P <0.05) compared with the group I, and the differences among the groups II, III and IV are not obvious (P > 0.05);

at 6wk, the IV has obvious difference (P >0.05) compared with I, and the difference between I, II and III is not obvious (P > 0.05);

at 8wk, IV is more obvious than I and II (P is more than 0.05), II is more obvious than I (P is less than 0.05), III is more obvious than I (P is less than 0.001), and III and IV are not obvious (P is more than 0.05);

TABLE 7 Effect of Complex Microecological preparations on serum immunoglobulin content (ng/L) of lactating calves

4. Influence of composite probiotics on serum cytokine content of lactating calves

As shown in Table 8, the IL-1. beta. levels were different in the serum of calves of different weeks of age. At week 2, group 2 had significant differences compared to group 2 (P <0.05), whereas the differences among

groups

1, 3, 4 were not significant (P > 0.05); at 4 weeks, the difference between groups I, II, III and IV is not significant (P >0.05), while the difference between groups III and IV is significant (P < 0.05); at 6wk, the differences among the groups II, III and IV are significant (P <0.05), the differences are not significant (P >0.05), but the differences between the groups III and IV are significant (P < 0.05); no significance was observed at 8wk (P <0.05) between groups;

differences in IL-2 between groups were not significant at weeks 2 and 4 (P > 0.05); at week 6, the IL-2 difference between group 4 and group 4 was significant (P < 0.05); the difference in IL-2 between

groups

3 and 3 was not significant (P > 0.05); at week 8, the difference between the two groups was not significant (P >0.05) the group IV had significant differences compared to the groups II, III, IV (P <0.05), while the differences between the groups II, III, IV were not significant (P > 0.05);

no significant difference in IL-2 levels between groups at weeks 2 and 6 (P > 0.05); at week 4, there was no significant difference between groups II, III, and IV (P >0.05), but significant difference between groups III and IV (P < 0.05). At 8wk, the difference between the group III and the group IV is significant (P <0.05), the difference between the group III, the group IV, the group II and the group IV is not significant (P >0.05), and the difference between the group I and the group II is not significant (P > 0.05).

At 2 weeks, the serum TNF-alpha of II, III and IV groups has no significant difference (P is more than 0.05), and the difference between II and IV groups is significant (P is less than 0.05); at 4 weeks, no significant difference (P >0.05) exists among I, II and III groups, no significant difference exists between II and IV groups, and significant difference (P <0.05) exists between III and IV groups, and the IV group and I group are compared; at 6wk, there was no significant difference between groups (P < 0.05); when the total weight is 8wk, the difference among I, II and III groups is not significant (P is more than 0.05), and the difference among IV groups is significant (P is less than 0.05);

TABLE 8 influence of the Complex Microecological preparation on the antioxidant index of serum of lactating calves (ng/L)

As can be seen from tables 5-6, the microbial preparation of the present application not only can reduce the frequency of diarrhea, but also stimulate the appetite of calves and increase the feed intake. The feed can supplement nutrition in time while stopping diarrhea, and can rapidly eliminate adverse effects of calf diarrhea from two aspects. Tables 7-8 also demonstrate that cellular and humoral immunity of calves is rapidly enhanced after using the microbial preparation of the present invention.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.

Claims

1. A composite microecological preparation for preventing calf diarrhea is characterized by comprising lactobacillus acidophilus, saccharomyces boulardii and bacillus subtilis.

2. The complex microecological formulation according to claim 1, wherein,

the ratio of the viable count of the lactobacillus acidophilus, the saccharomyces boulardii and the bacillus subtilis is 1-3: 1-3: 1-3.

3. The composite microecological formulation according to claim 2,

the ratio of the viable count of the lactobacillus acidophilus, the saccharomyces boulardii and the bacillus subtilis is 1: 2: 1 or 3:3: 1.

4. the composite microecological formulation according to claim 3,

the ratio of the viable count of the lactobacillus acidophilus, the saccharomyces boulardii and the bacillus subtilis is 3:3: 1.

5. the complex microecological formulation according to claim 1, wherein,

the compound microecological preparation is a liquid preparation.

6. The composite microecological formulation according to claim 5,

the composite microecological preparation has viable bacteria concentration of 1 × 10⁹cfu/mL lactobacillus acidophilus, Saccharomyces boulardii and Bacillus subtilis.

7. The complex microecological formulation according to claim 1, wherein,

the compound microecological preparation is used for preventing diarrhea caused by calf Escherichia coli K99.