CN111803529A - A compound microecological preparation for preventing calf diarrhea - Google Patents

Abstract

The invention is a composite microecological preparation for preventing calf diarrhea. A compound probiotic for preventing diarrhea in calves, containing Lactobacillus acidophilus, Saccharomyces bollardii and Bacillus subtilis. The present invention separates and identifies pathogenic Escherichia coli in the feces of calves with diarrhea, and has the biological characteristics of acid resistance, bile salt resistance and bacteriostasis to three probiotic bacteria of Lactobacillus acidophilus, Saccharomyces bodrum and Bacillus subtilis. The comparative analysis found that the three probiotics had good tolerance, and the mixture of the three probiotics had a good bacteriostatic effect on Escherichia coli k99; It can also increase the daily weight gain of calves, increase the feed intake of calves, reduce the rate of diarrhea, improve the immune performance and antioxidant performance of calves, and can regulate the intestinal flora of animals.

Description

A compound microecological preparation for preventing calf diarrhea

technical field

The invention belongs to the field of livestock, and in particular relates to a composite microecological preparation for preventing calf diarrhea.

Background technique

In the past few decades, the use of antibiotics in the prevention and control of livestock and poultry infections and disease resistance has increased year by year. It is reported that China is the country with the largest production and consumption of antibiotics in the world. While bringing production benefits to the development of animal husbandry, human and The ecological environment and health status of livestock and poultry also face huge risks, which has also led to the rise of drug resistance of pathogens and common bacteria in human living environment, livestock and poultry breeding environment, and livestock and poultry bodies. Therefore, in 2003, the world gradually banned the use of antibiotics in Sweden and 1986, and the European Union also banned the use of antibiotics in 2006. The growing global trend towards banning the use of antibiotic growth promoters (AGPS) has led to an increasingly urgent need for alternatives to AGPS.

As the backup force of dairy farming, calves play a vital role in the production potential of dairy cattle, and calf diarrhea is the main cause of calf death. Calf diarrhea is a common disease, which is harmful to the growth and development of calves, and its etiology is complex. According to statistics, the large-scale morbidity in the northern Xinjiang reclamation area accounts for 40-60%, and the mortality rate is 50-70%. The microbial factors leading to calf diarrhea are mainly bacterial (average morbidity rate 36.7%) and viral (average morbidity rate). 7.5%) factor.

The study found that the pathogenic Escherichia coli that caused diarrhea in calves in Xinjiang in the past two years had different degrees of resistance to antibiotics such as ampicillin, gentamicin, chloramphenicol and ciprofloxacin. Therefore, there is an urgent need to find alternatives to antibiotics as prophylactic antimicrobials. In order to improve animal welfare and protect the environment, feed microorganisms have attracted much attention because of their non-polluting, non-residue, green characteristics, and their biological functions such as improving the growth performance of livestock and poultry, optimizing intestinal flora, and improving animal immunity. , many studies are also that forage probiotics have been considered as potential substitutes.

In view of this, the present invention proposes a new compound microecological preparation for preventing calf diarrhea, which can replace antibiotics and prevent digestive tract diseases of young calves.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a composite microecological preparation for preventing calf diarrhea, which can replace antibiotics and prevent digestive tract diseases of young calves.

In order to achieve the above purpose, the adopted technical scheme is:

A composite microbial preparation for preventing diarrhea in calves, the composite microbial preparation contains Lactobacillus acidophilus, Saccharomyces bollardii and Bacillus subtilis.

Further, the ratio of the viable counts of Lactobacillus acidophilus, Saccharomyces bollardii and Bacillus subtilis is 1-3:1-3:1-3.

Still further, the ratio of the viable counts of Lactobacillus acidophilus, Saccharomyces bollardii and Bacillus subtilis is 1:2:1 or 3:3:1.

Still further, the ratio of the number of viable cells of the Lactobacillus acidophilus, Saccharomyces bollardii and Bacillus subtilis is 3:3:1.

Further, the composite microbial preparation is a liquid preparation.

Still further, the composite microecological preparation is formed by the proportion of Lactobacillus acidophilus, Saccharomyces bollardii and Bacillus subtilis, all of which have a viable bacterial count concentration of 1×10 ⁹ cfu/mL.

Further, the compound probiotic preparation is used to prevent diarrhea caused by Escherichia coli K99 in calves.

Compared with the prior art, the beneficial effects of the present invention are:

The invention takes the isolation of pathogenic Escherichia coli k99 and the probiotics preserved in the laboratory as the research objects, compares and analyzes the biological characteristics of the probiotic strains, and selects 3 strains of the compound microecological preparation that best inhibits and prevents calf diarrhea. Proportion optimization combination. It provides a theoretical basis for the development of microecological preparations for the prevention and treatment of calf diarrhea.

At the same time, the technical solution of the present invention has the functions of increasing the daily weight gain of the calf, increasing the feed intake of the calf, reducing the diarrhea rate, improving the immune performance and the antioxidant performance of the calf, and can regulate the intestinal flora of animals.

Description of drawings

Fig. 1 is the microscope picture of Escherichia coli;

Fig. 2 is the amplification diagram of Escherichia coli k99;

Figure 3 is a graph of the results of BLAST performed by NCBI Genbank;

Fig. 4 is the tolerance of different pH values of Lactobacillus acidophilus artificial gastric juice;

Fig. 5 is the tolerance of different pH values of Bacillus artificial gastric juice;

Fig. 6 is the tolerance of different pH values of Saccharomyces boulardii artificial gastric juice;

Figure 7 shows the tolerance of three probiotic artificial intestinal fluids at different times;

Figure 8 is a graph showing the tolerance of 3 kinds of probiotic bovine bile salts.

Detailed ways

In order to further describe the composite microbial preparation for preventing calf diarrhea according to the present invention and achieve the intended purpose of the invention, the following specific implementation of the composite microbial preparation for preventing calf diarrhea proposed according to the present invention in conjunction with the preferred embodiments The method, structure, characteristics and efficacy thereof are described in detail as follows. In the following description, different "an embodiment" or "embodiments" do not necessarily refer to the same embodiment. Furthermore, the particular features, structures or characteristics in one or more embodiments may be combined in any suitable form.

Below in conjunction with specific embodiment, a kind of composite microecological preparation for preventing calf diarrhea of the present invention will be further introduced in detail:

In this aspect, pathogenic Escherichia coli k99 is isolated and identified in the feces of calves with diarrhea by using molecular biology methods. At the same time, the comparative analysis of the biological characteristics of acid resistance, bile salt resistance and bacteriostasis of Lactobacillus acidophilus, Saccharomyces bollardii and Bacillus subtilis showed that at pH 3, the survival rate was 84.34%. Bacillus at pH 5, the survival rate was 142.24%; in artificial gastric juice, the survival rate was high at 9 hours, compared with OD ₆₀₀ at 0 hours, the survival rate of Saccharomyces boulardii and Lactobacillus acidophilus were 108.7 %, 129.73%, Lactobacillus acidophilus reached 96.37%. In the medium containing bovine bile salts with different concentrations, the survival rate of Saccharomyces boulardii was the highest compared with the control group, which was 60.49%, the survival rate of Lactobacillus acidophilus was the highest compared with the control group, 85.1%, and the survival rate of Bacillus was higher than that of the control group. The highest survival rate of the group was 47.42%. The three strains of probiotics have certain antibacterial properties against Escherichia coli and Salmonella. The results indicated that the three probiotics had good tolerance to artificial gastric juice, artificial intestinal juice and bovine bile salts, and had certain bacteriostatic properties.

By inhibiting Escherichia coli k99 with different ratios of the three probiotics, it was found that the three probiotics were cultured for 12 hours according to different ratios, and the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, and 9th groups of the experimental group Compared with the control group, the difference was significant (P<0.05), and the difference was significant (P<0.05) between groups 2, 4, and 8; Compared with the control group, the difference was significant (P<0.05), but the difference between the 2nd, 3rd, 4th, 5th, 6th, and 8th was not significant (P>0.05). The eighth treatment group had better bacteriostatic effect on Escherichia coli K99 after culturing for 12 hours, so it could be determined that the optimal compounding ratio of Lactobacillus acidophilus, Bacillus subtilis, and Saccharomyces boulardii was 3:3:1. It provides a theoretical basis for the development of microecological preparations to replace antibiotics and prevent digestive tract diseases in young calves.

The specific operation steps are as follows:

The effect of microbiological preparations on digestive tract diseases of young calves

1 Materials and methods

1.1 Test material

(1) Test strains

Probiotic strains: Lactobacillus acidophilus, Saccharomyces boulardii, Bacillus subtilis, provided by Biofeed Laboratory, School of Animal Science and Technology, Shihezi University.

Test bacteria: Escherichia coli (K99) was independently isolated; Salmonella and Staphylococcus aureus were purchased from Shanghai Luwei Technology Co., Ltd.

(2) Culture medium:

LB nutrient agar medium, LB broth, MacConkey agar medium, MRS liquid medium, MRS solid medium, YPD liquid medium, YPD solid medium and Bacillus medium were purchased from Qingdao Hi-Tech Park Haibo Biotechnology Ltd.

(3) Main reagents

PBS buffer for bacterial dilution. Bacterial DNA extraction kits were purchased from Tiangen Biochemical Technology Co., Ltd.; pepsin, trypsin, and ox bile salts were purchased from Beijing Biotopped Science&Technology Co., Ltd. Physiological and biochemical identification tubes were purchased from Hangzhou Binhe Microbial Reagent Co., Ltd. The K99 primer was synthesized by Shanghai Jierui Biotechnology Co., Ltd., and the sequencing was completed by Huada Gene Co., Ltd.

(4) Main equipment

Pipette gun; electronic balance JM-500, Kunshan Jinkehua Electronics Co., Ltd., electronic balance BS224S, Beijing Sartorius Instrument Co., Ltd.; acidity meter WTW PH3110, Kaiming state instrument; vertical ultra-low temperature incubator GSP-9160MBE, constant temperature incubator HPX-9162MBE, ultra-clean workbench SW-CJ-2F, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; microplate reader WD-2102B, BEIJING Beijing Liuyi Biotechnology Co., Ltd.; PCR instrument, Shanghai Naz Instrument Co., Ltd. Centrifuge TG16-W, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.

1.2 Test method

1.2.1 Bacterial isolation and identification of Escherichia coli k99 from feces of calves with diarrhea

Using cotton swabs to aseptically collect diarrhea fecal samples from four calves with diarrhea within 7 days of age in a large-scale cattle farm, dilute in sterile saline and culture in LB broth medium at 37 °C, and further in Purified in LB solid medium, and separated and purified. After Gram staining, microscopic examination was performed, and the purified colonies were stored at -80 °C. At the same time, the DNA of bacteria was extracted and purified by a kit, and the specific primer (5'-TATTATCTGGTGGTATGG) of Escherichia coli k99 was used. -3', 5'-GGTATCCTTTAGCAGCAGTATTTC-3') for PCR amplification, the PCR reaction system and reaction conditions are: 20uL reaction system: 2×master PCR Mix 10uL, DNA template 3uL, upstream and downstream primers each 0.5uL (primer concentration 20umol/L), add sterile ultrapure water to 20uL. Pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s; annealing at 52.6°C for 30s; extension at 72°C for 30s; cycle 35 times, extension at 72°C for 10min. The amplified product was recovered and sequenced, and the physiological and biochemical identification of Escherichia coli K99 strain was carried out.

1.2.2 Activation of strains and determination of viable bacterial content

Strain activation: Inoculate the 3 strains in the corresponding liquid medium at 37°C for 18h in a shaker, use the inoculation loop to coat and streak in MRS solid medium, YPD solid medium, and Bacillus medium, pick a single strain Inoculated into MRS broth, YPD broth, and Bacillus broth, where Lactobacillus acidophilus was cultured at 37°C for 24 hours, and Saccharomyces cerevisiae and Bacillus subtilis were cultured at 37°C. Cultivated for 24h, activated for 2-3 generations, and set aside.

Determination of viable bacteria content: Under sterile conditions, the activated probiotics of the second generation (Lactobacillus acidophilus, Bacillus subtilis, Saccharomyces boulardii, Escherichia coli) were serially diluted, and the suspension was diluted 10 times to 10 ^-7 , 10 ^-8 , 10 ^-9 , then take 100uL of each of the three dilutions of bacterial liquid for plate coating, spread three plates for each dilution, and invert at 37 °C for 12-24h After culturing for 12-24 hours, count the colonies .

1.2.3 Tolerance test of three probiotics at different pH values

Preparation of artificial gastric juice: take 16.4 mL of hydrochloric acid with a mass fraction of 0.1 kg/L, and adjust its pH to 2.0, 3.0, 4.0, 5.0, and 6.0. Then, pepsin was added at a ratio of 0.01 g/mL, and after it was evenly dissolved, the microporous membrane was sterilized and used for later use. The activated Lactobacillus acidophilus, Lactobacillus acidophilus, Saccharomyces boulardii, and Bacillus subtilis were inoculated into the above pepsin solutions with different pH values according to the inoculation ratio of 1%, and cultivated in a constant temperature incubator at 37 °C for 3 hours. _OD600 .

1.2.4 Tolerance test of artificial intestinal fluid

Preparation of artificial intestinal juice: accurately weigh 3.4 g of KH ₂ P0 ₄ , add 250 mL of distilled water to dissolve, adjust the pH to 6.8 with a mass fraction of 4 g/L NaOH solution, add pepsin to make its concentration 0.01 g/mL, and wait for it to dissolve evenly. , sterilized by a microporous filter, and placed in a 4°C refrigerator for later use. The three activated probiotic strains were added to the above artificial intestinal fluid with 10% inoculum respectively, and each treatment was repeated 3 times, cultured at 37°C, and sampled at 0, 3, 6, and 9 hours, respectively, with a microplate reader. The OD ₆₀₀ value of each strain was determined.

1.2.5 Bile salt tolerance test

Bovine bile salts were added to MRS, YPD and Bacillus liquid medium respectively to prepare the medium with bovine bile salt concentrations of 0.15g/L, 0.3g/L and 0.06g/L respectively. The three media were then sterilized at 118°C for 15 minutes, and then used after cooling. The above-mentioned activated strains were inoculated into the corresponding bovine bile salts medium prepared above, and the medium without bile salts was used as a reference, and cultivated for 8h at a constant temperature of 37 ° C to observe the growth of the strains, and measure the OD ₆₀₀ , to calculate the survival rate.

1.2.6 Bacteriostatic test

The isolated Escherichia coli k99, Salmonella, and Staphylococcus aureus were cultured for 24h respectively, diluted 20 times, and 100uL of the pathogenic bacteria solution was evenly spread on the sterile agar plate prepared in advance. Using Oxford cup and punching method, cultured at 37°C for 12-24h, the diameter of inhibition zone was measured, and sterile common liquid medium was used as control.

1.2.73 strains to inhibit Escherichia coli k99 ratio optimization

Dilute the activated 3 strains of probiotics into a 10 ^-1 dilution suspension. Then it was diluted to the same concentration (1×10 ⁹ cfu/mL), inoculated according to the different proportions of the species in Table 1, and a control group was set up, a total of 11 groups were added to a certain amount of LB liquid medium, respectively. The final volume was 5 mL, and the culture was shaken at 37°C and 165 rpm. The incubation time was set to 12h and 24h. Then carry out gradient dilution, take 10 ^-6 , 10 ^-7 , 10 ^-7 100uL to spread on MacConkey agar medium, culture in a constant temperature incubator at 37°C for 24 hours, and count E. coli, and set a control group at the same time.

Table 1 The inoculum unit of different proportions of three kinds of probiotics: uL

1.2.8 Detection indicators:

The OD ₆₀₀ value and the viable count of Escherichia coli K99 were detected.

1.2.9 Data Analysis:

The test data were analyzed by one-way analysis of variance (ONE-WAY ANONA) and Duncan's multiple analysis using Spss21.0 statistical software.

2. Results and Analysis

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

The purified E. coli colonies were Gram-stained, and the microscopic examination was G ^- , short rod-shaped, the results are shown in Figure 1, and E. coli k99 was amplified in Figure 2; BLAST was performed on NCBI Genbank, and the results showed that the similarity was 99.99%, as shown in Figure 3. The biochemical identification is shown in Table 2.

At the same time, the isolated Escherichia coli k99 was used to challenge the mice, and each mouse was injected with 0.2 mL of Escherichia coli bacteria cultured for 18 hours. The results showed that 80% of the mice died. It is proved that the isolated E. coli k99 has strong virulence.

Table 2 Physiological and biochemical identification of Escherichia coli k99

2.2 Tolerance test of three probiotics at different pH values

The test results are shown in Figure 4-6. The three probiotics have good tolerance under different pH values. Among them, Lactobacillus acidophilus has a survival rate of 118.75% at pH 5. When it was 3, the survival rate was 84.34%.

Saccharomyces boulardii showed a survival rate of more than 100% at different pH values. When the pH of Bacillus is 5, the survival rate is 142.24%, and under different pH conditions, the survival rate reaches more than 100%, and has good tolerance.

2.3 Tolerance test of three kinds of probiotics in artificial intestinal fluid

The results are shown in Figure 7. The three probiotics have good tolerance to artificial intestinal juice at different incubation times, and the three probiotics have a high survival rate in artificial gastric juice at 9 hours, which is comparable to the OD ₆₀₀ of 0 hours. In contrast, the survival rates of Saccharomyces boulardii and Lactobacillus acidophilus were 108.7% and 129.73%, respectively, and Lactobacillus acidophilus reached 96.37%.

2.4 Bile salt tolerance test of three probiotics

The results are shown in Figure 8: with the increase of bovine bile salt concentration, the survival rate of Saccharomyces boulardii was 50.64%, 60.49%, and 46.37% compared with the control group, and the survival rate of Lactobacillus acidophilus was higher than that of the control group, respectively. were 81.65%, 85.1%, and 81.19%, and the survival rate of Bacillus was 45.1%, 45.11%, and 47.42%, respectively, compared with the control group. Saccharomyces boulardii and Lactobacillus acidophilus were more resistant to bovine bile salts, while Bacillus was weaker.

2.5 Bacteriostatic test of three probiotics

The results are shown in Table 3. Saccharomyces boulardii, Lactobacillus acidophilus, Bacillus, Escherichia coli and Salmonella have certain antibacterial properties, and Lactobacillus acidophilus has strong antibacterial properties against Staphylococcus aureus. Saccharomyces boulardii and Lactobacillus acidophilus have poor bacteriostatic activity against Staphylococcus aureus.

Table 3 Diameter of inhibition zone of three probiotics

2.6 Optimization of the ratio of three strains to inhibit Escherichia coli k99

Table 4 shows the results of the in vitro antibacterial test of Lactobacillus acidophilus, Saccharomyces bovis, and Bacillus in different proportions to bovine Escherichia coli K99 after culturing for 12h and 24h.

Table 4 In vitro antibacterial test of three kinds of probiotic preparations according to different ratios to Escherichia coli k99 (n=3) logcfu/mL

If there is no letter in the same line or the data with the same letter, it means that the difference is not significant (P>0.05), and different lowercase letters means the difference is significant (P<0.05). The table below is the same.

It can be seen from Table 4 that after culturing the three probiotics in different proportions for 12 hours, the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, and 9th groups of the experimental group were significantly different from those of the control group (P <0.05), there was no significant difference between the 10th group and the control group (P>0.05), and there was no significant difference between the 1st, 3rd, 5th, 6th, 7th, 9th and 10th groups (P>0.05). , 4 and 8 groups were significantly different (P<0.05); after 24 hours of culture, the 2, 3, 4, 5, 6 and 8 of the experimental group were significantly different from those of the control group (P<0.05), but the second, , 3, 4, 5, 6, and 8 were not significantly different (P>0.05), and the first, 2, 7, 9, and 10 groups in the experimental group had no significant differences compared with the control group (P>0.05).

In summary, combined with the practical application, the composite microbial preparation was discharged after 12 hours of peristalsis in the bovine intestinal tract, and the eighth treatment group had better bacteriostatic effect on E. The optimal compounding ratio of Bacillus and Saccharomyces bodrum is 3:3:1.

Analysis of the probiotic properties of probiotics:

Since this study is aimed at calf Escherichia coli K99 to study compound probiotics, according to the characteristics of the immature digestive system of newborn calves, the abomasum and small intestine mainly rely on the abomasum and small intestine to digest and absorb nutrients in the diet, and within two weeks of age The calf does not appear ruminant behavior, and the characteristics of its digestive function are basically similar to those of monogastric animals. At this time, the acidity and pH value of the calf's digestive tract fluctuates greatly, which has a greater impact on its digestive physiology. Studies have shown that calves The pH value of the abomasum changes greatly before and after eating for 24 hours. The pH value of the abomasum of calves before feeding is between 1.5 and 2.0. Between 2.0, it can be seen that the pH of the calf's abomasum fluctuates greatly before and after eating, but it also shows a stable state with the time of eating. The levels of trypsin in different intestinal breaks of calves are also different, the activity of which is the lowest in the duodenum, the highest in the jejunum, and the second in the ileum. The intestinal bile concentration of calves is constantly changing, making it difficult to predict its concentration over a period of time, but in general, bile salts in the small intestine are typically 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 effect, etc. An important feature of probiotics' survival in animals is their ability to tolerate the acidic conditions of the stomach and bile salts in the small intestine.

The results of this experiment showed that the three probiotic strains Lactobacillus acidophilus, Bacillus and Saccharomyces bradii had high tolerance to acidic conditions, bovine bile salts and trypsin.

The antibacterial effects of different ratios of Lactobacillus acidophilus, Bacillus subtilis, and Saccharomyces bollardii on Escherichia coli (K99):

Feed microbial additive is an active formulation that has achieved unprecedented development in applications in promoting animal growth, intestinal health, etc. due to its green and non-polluting properties, playing a role in manipulating the host's health trajectory (including and beyond immune components) It plays a great role, and has biological functions such as promoting the absorption of nutrients, regulating the structure of intestinal flora and improving the immunity of livestock and poultry.

Lactobacillus acidophilus is a typical probiotic with physiological functions such as inhibiting intestinal pathogens, promoting immune cell homeostasis and intestinal health, and inhibiting enterotoxin-producing Escherichia coli K99. Studies have shown that Lactobacillus acidophilus has a high survival rate in pH 3 and medium containing bile salts, and the critical concentration is 0.3%, indicating that the strain has resistance to its passage through the small intestine. Antibacterial ability.

Brucella is one of the best-studied probiotics in acute gastroenteritis (AGE) and has been shown to be safe. Saccharomyces boulardii is antitoxin, and the phosphatase secreted by Saccharomyces boulardii can significantly destroy the endotoxin secreted by Escherichia coli. It has the effect of inhibiting the growth of Salmonella typhimurium, Escherichia coli, Candida albicans, etc. It can improve the activity of short-chain fatty acids in the intestinal tract of the body, regulate the metabolic function of the intestinal tract, promote the nutritional effect of the intestinal mucosa, and enhance the body's The role of immune function, prevention and treatment of pathogenic infection increases the content of SCFA in the body. Because it is aerobic and has high adhesion, it can consume oxygen in the intestines and decompose hydrogen peroxide, promote the growth of anaerobic bacteria, and regulate the imbalance of flora caused by improper use of antibiotics, thereby Promotes intestinal flora balance. There have been relevant reports on applied research on poultry, pigs, and aquaculture, but less research has been done on ruminants, especially calves. Application on calves can increase the daily weight gain of calves, reduce the rate of diarrhea, and improve immunity.

The content of Bacillus subtilis in the digestive tract of animals is less. In fact, aerobic bacteria can quickly consume oxygen in the digestive tract of animals, creating an anaerobic environment and inhibiting the growth of harmful pathogenic bacteria. At the same time, Bacillus has bactericidal effect, and the bacteriocin in its metabolites can effectively kill pathogenic flora such as Escherichia coli, Staphylococcus aureus and Salmonella, thereby maintaining the health of the animal body.

In this experiment, through the isolation and identification of pathogenic Escherichia coli in the feces of calves with diarrhea, the three probiotic bacteria strains of Lactobacillus acidophilus, Saccharomyces bollardii, and Bacillus subtilis have the acid resistance, bile salt resistance and bacteriostatic activity. The comparative analysis of biological characteristics found that the three probiotics had good tolerance, and by inhibiting Escherichia coli k99 with different ratios of the three probiotics, Lactobacillus acidophilus, Bacillus subtilis, Brady When the optimal compounding ratio of yeast is 3:3:1, it has a good bacteriostatic effect, which can be used for the later development of alternative antibiotics and to provide a theoretical basis for the prevention of digestive tract diseases in young calves.

Effects of two microecological preparations on growth performance, diarrhea rate, blood immune indexes and antioxidant properties of calves

40 5-day-old calves were selected for the experiment and randomly divided into 4 groups with 10 calves in each group and 1 calf in each replicate. Group I was the control group, fed the same amount of normal saline, group II was fed 0.5 g of microbial preparations, group III was fed 1 g of microbial preparations, and group IV was fed 2 g of microbial preparations. The experimental data were collected at 0, 2, 4, 6, and 8 weeks. Growth performance, diarrhea rate, blood immune index and antioxidant performance were used as evaluation indicators.

The microbial preparation was the eighth microbial preparation in Table 4.

(1) Test method and data collection

1. Growth performance, feed intake, stool score

(1) Feed intake: The feed intake of calf milk replacer, starter and alfalfa was recorded every day.

(2) Feces score: Evaluate the feces of the test calves according to the scoring standard (table). 1 point, normal shape; 2 points, soft and loose; 3 points, loose and watery; 4 points, watery, mucus, a little blood; 5 points, watery, mucus, a lot of blood. Diarrhea was recorded when stool was rated 3 and above.

Diarrhea rate (%) = Σ (diarrhea head count×diarrhea days)/(total head count×test days)×100.

2. Serum sample collection and determination

5 mL of blood was collected from the jugular vein with a vacuum blood collection tube containing heparin sodium before the morning feeding on the 2nd, 4th, 6th, and 6th weeks of feeding, and left standing at room temperature for 30 minutes, then centrifuged at 3000 r/min for 15 minutes, and the serum was collected into 1.5 mL of cryopreservation. In the tube, mark the group and cattle number, sampling date, and store in liquid nitrogen. Measure the concentrations of Ig A, IgG, Ig M, IL-2, IL-4, IL-1β and TNF-α in serum, and measure serum total protein ((TP), globulin (Glb), albumin (Alb) , glucose (Glu), urea nitrogen (BUN), alkaline phosphatase (ALP), creatinine, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations.

3. Fecal sample collection:

(1) Liquid nitrogen cryopreservation of fecal samples: fecal samples were collected before morning feeding of calves at 0, 2, 4, 6, and 8 weeks of age and placed in 50ml centrifuge tubes. Six cows were randomly selected from each group to collect feces from the rectum. The fecal samples collected from the head cows were divided into 6 small tubes. Freeze in liquid nitrogen.

4. Blood immune indexes and antioxidant properties

Tested with a kit.

(2) Results:

1. The effect of compound probiotics on the growth performance of calves

As shown in Table 5, in the second week, the average daily weight gain of the experimental group III was higher than that of the control group I, and the difference was significant (P<0.05), but there was no significant difference between the groups (P>0.05). The average daily feed intake of group Ⅱ, Ⅲ and Ⅳ was higher than that of group Ⅰ (P<0.05), but there was no significant difference between groups (P>0.05). At 4wk, there was no significant difference in the average daily gain and average daily feed intake among the groups (P>0.05). At 6wk, the daily weight gain of Ⅱ and Ⅲ groups was significantly higher than that of Ⅰ and Ⅳ groups (P<0.05). At 8wk, there was no significant difference in ADG among the groups (P>0.05), but the ADG in groups Ⅱ and Ⅳ was higher than that in group Ⅰ. Compared with group Ⅰ, Ⅱ, Ⅲ, the admi of group Ⅳ had significant difference (P<0.05), but there was no significant difference between group Ⅱ and group Ⅲ (P>0.05).

Table 5 Effects of compound probiotics on growth performance of calves

In the same row, different lowercase letters indicate significant differences (P < 0.05), and there is no significant difference with the same or no superscript letters (P > 0.05), the same below. ADG (average daily gain), ADMI (average daily feed intake).

2. The effect of compound probiotics on calf fecal score and diarrhea rate

Table 6 Effects of compound probiotics on calf manure score and diarrhea rate

It can be seen from Table 6 that there was no significant difference in stool score between groups I, II and III at 2WK and 4WK (P>0.05), and there was no significant difference between groups II and IV (P>0.05). 0.05), there were significant differences between groups Ⅰ and Ⅲ compared with group Ⅳ stool score (P<0.05); at 6WK and 8WK, there was no significant difference between groups (P>0.05).

At 2WK and 4WK, the diarrhea rate in the experimental group was lower than that in the control group. At 2WK, the rate of diarrhea in group IV decreased by 51.65% compared with group I. At 4WK, the diarrhea rate in group IV was 0.

3. The effect of compound probiotics on the immune function of lactating calves

As shown in Table 7, the content of IgA in the serum of calves of different ages was different. At the second week, the difference between II and IV was significant (P<0.05), and III and IV were significantly higher than I (P<0.05); at the fourth week, the difference between III and IV was extremely significant (P<0.001), and the difference between II and III was significant. (P<0.05), there was no significant difference between I and II (P>0.05) 0.05), there was a significant difference between III and IV (P<0.001), and the difference between II and III was significant (P<0.05), and the difference between I and II was not significant (P<0.05). P>0.05), there was no significant difference between Ⅲ and Ⅳ (P>0.05); at 6wk, the difference between Ⅱ and Ⅳ groups was extremely significant (P<0.05) (P<0.001), and the difference between Ⅲ and Ⅰ, Ⅱ was significant (P<0.001). <0.05), there was significant difference between IV and I, II (P<0.001); at 8wk, IV and I were significantly different (P<0.05), and I, II, III had no significant difference (P>0.05).

The serum IgM levels of calves of different weeks were different. At the second week, the difference between II and IV was significant (P<0.05), and III and IV were significantly higher than I (P<0.05); at the fourth week, the difference between III and IV was extremely significant (P<0.001), and the difference between II and III was significant. (P<0.05), there was no significant difference between I and II (P>0.05) 0.05), there was a significant difference between III and IV (P<0.001), and the difference between II and III was significant (P<0.05), and the difference between I and II was not significant (P<0.05). P>0.05), there was no significant difference between Ⅲ and Ⅳ (P>0.05); at 6wk, the difference between Ⅱ and Ⅳ groups was extremely significant (P<0.05) (P<0.001), and the difference between Ⅲ and Ⅰ, Ⅱ was significant (P<0.001). <0.05), there was significant difference between IV and I, II (P<0.001); at 8wk, IV and I were significantly different (P<0.05), and I, II, III had no significant difference (P>0.05).

At 2wk, the difference in IgM between groups III and IV was extremely significant (P<0.001), the difference between group III and II was significant (P<0.05), and the difference between groups III and IV was not significant (P>0.05). The difference is not significant (P>0.05);

At 4wk, there were significant differences between groups II, III, and IV compared with group I (P<0.05), but no significant differences between groups II, III, and IV (P>0.05);

At 6wk, there was a significant difference between IV and I (P>0.05), and there was no significant difference between groups I, II, and III (P>0.05);

At 8wk, Ⅳ was significantly different from Ⅰ and Ⅱ (P>0.05), Ⅱ group was significantly different from Ⅰ group (P<0.05), Ⅲ group was significantly different from Ⅰ group (P<0.001), and the difference between Ⅲ and Ⅳ group was not significant. Significant (P>0.05);

Table 7 Effects of compound probiotics on serum immunoglobulin content of lactating calves (ng/L)

4. Effects of compound probiotics on serum cytokine levels in lactating calves

As shown in Table 8, the serum IL-1β levels of calves of different ages were different. At the second week, there was a significant difference between the second group and the second group (P<0.05), but there was no significant difference between the first, third, and fourth groups (P>0.05); at the fourth week, I, II There was no significant difference among groups Ⅲ and Ⅳ (P>0.05), while the difference between groups Ⅲ and Ⅳ was significant (P<0.05); at 6wk, there was significant difference among groups Ⅱ, Ⅲ and Ⅳ (P<0.05) , there was no significant difference (P>0.05), but there was a significant difference between group III and group IV (P<0.05); at 8wk, there was no significant difference between groups (P<0.05);

In the 2nd and 4th week, there was no significant difference in IL-2 among the groups (P>0.05); in the 6th week, there was a significant difference in IL-2 between the 4th and 4th groups (P<0.05). ); there was no significant difference in IL-2 between groups 3 and 3 (P>0.05); at week 8, there was no significant difference between the two groups (P>0.05). There was significant difference between groups Ⅲ and Ⅳ (P<0.05), but there was no significant difference among groups Ⅱ, Ⅲ and Ⅳ (P>0.05).

At the 2nd and 6th week, there was no significant difference in IL-2 levels among the groups (P>0.05); However, there were significant differences between groups Ⅲ and Ⅳ (P<0.05). At 8wk, there was a significant difference between group III and group IV (P<0.05), but there was no significant difference between groups III, IV, II, and IV (P>0.05), and there was no significant difference between groups I and II (P<0.05). >0.05).

At 2 weeks, there was no significant difference in serum TNF-α between groups Ⅱ, Ⅲ and Ⅳ (P>0.05), while the difference between groups Ⅱ and Ⅳ was significant (P<0.05); at 4 weeks, there was no significant difference between groups Ⅰ, Ⅱ, Ⅲ. There was a significant difference (P>0.05), but there was no significant difference between groups II and IV. There was a significant difference between groups III and IV (P>0.05), and group IV was significantly different from group I (P<0.05). There was no significant difference between groups (P<0.05); at 8wk, there was no significant difference between groups I, II and III (P>0.05), and there was significant difference between groups IV (P<0.05);

Table 8 Effects of compound probiotics on serum antioxidant indexes of lactating calves (ng/L)

It can be seen from Tables 5-6 that the microbial preparation of the present application can not only reduce the number of diarrhea, but also stimulate the appetite of calves and increase feed intake. At the same time of stopping diarrhea, timely supplement nutrition can quickly eliminate the adverse effects of calf diarrhea from two aspects. Tables 7-8 also illustrate the rapid enhancement of cellular and humoral immunity of calves after using the microbial preparation of the present invention.

The above descriptions are only preferred embodiments of the embodiments of the present invention, and are not intended to limit the embodiments of the present invention in any form. Any simple modifications, equivalent changes and Modifications still fall within the scope of the technical solutions of the embodiments of the present invention.

Claims (7)

1. a composite microecological preparation for preventing calf diarrhea, is characterized in that, described composite microecological preparation contains Lactobacillus acidophilus, Saccharomyces boulardii and Bacillus subtilis. 2. composite probiotic preparation according to claim 1, is characterized in that, The ratio of the viable bacterial counts of the Lactobacillus acidophilus, Saccharomyces bollardii and Bacillus subtilis is 1-3:1-3:1-3. 3. composite probiotic preparation according to claim 2, is characterized in that, The ratio of the number of viable bacteria of Lactobacillus acidophilus, Saccharomyces bollardii and Bacillus subtilis is 1:2:1 or 3:3:1. 4. composite probiotic preparation according to claim 3, is characterized in that, The ratio of the number of viable bacteria of the Lactobacillus acidophilus, Saccharomyces bodii and Bacillus subtilis is 3:3:1. 5. composite probiotic preparation according to claim 1, is characterized in that, The composite microecological preparation is a liquid preparation. 6. composite probiotic preparation according to claim 5, is characterized in that, The composite microecological preparation is formed by the proportion of Lactobacillus acidophilus, Saccharomyces bodii and Bacillus subtilis, all of which have a viable bacteria concentration of 1×10 ⁹ cfu/mL. 7. composite probiotic preparation according to claim 1, is characterized in that, The compound probiotic preparation is used for preventing diarrhea caused by Escherichia coli K99 in calves.

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