Yak-source composite microecological preparation and application thereof

Abstract

The invention provides a yak-source composite microecological preparation and application thereof, and belongs to the technical field of microbial composite inoculants. The yak-derived composite microecological preparation disclosed by the invention is prepared from the following components: bacillus coagulans, thiamine-solubilizing bacillus, bacillus subtilis and bacillus licheniformis according to a mass ratio of 1: (0.5 to 1.5): (0.5 to 1.5): (0.5 to 1.5). By adopting the yak-derived composite microecological preparation, the immune function and antioxidant capacity of yaks can be improved, the disease resistance of the yaks is further enhanced, in addition, the structure of intestinal flora of the yaks can be improved, the number and abundance of probiotics are increased, and the growth and reproduction of harmful bacteria are inhibited.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial complex agents, and particularly relates to a yak-derived complex microecological preparation and its application. Background Art

[0002] With the increasing demand for yak meat, the yak breeding industry has also witnessed rapid development, with the breeding scale and density continuously increasing. The livestock house environment is poor, which exacerbates the decline in the disease resistance of animals and further increases the risk of disease. The phenomenon of antibiotic abuse is becoming increasingly obvious, and at the same time, the problem of drug residues in livestock products occurs frequently, seriously threatening human health and causing damage to the ecological environment. With the gradual evolution of the trend of antibiotic-free breeding, it is urgent to find a healthy, green, non-toxic, harmless and pollution-free additive to replace antibiotics.

[0003] Microecological preparations can not only promote the growth of yaks, but also enhance their immunity, improve the intestinal ecological environment and reduce the occurrence of diseases. In addition, due to their non-toxic side effects and non-residual characteristics, they meet the requirements of modern breeding for safety and environmental protection. Therefore, in future breeding practices, strengthening the research and development and application of microecological preparations, as well as promoting scientific feeding management, will help improve the overall health and production performance of yaks. At present, the sources of complex microecological preparations sold on the market are complex, and there are few microecological preparations for yaks. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a yak-derived complex microecological preparation and its application. By mixing Bacillus coagulans, Bacillus thiaminolyticus, Bacillus subtilis and Bacillus licheniformis, the present invention prepares a complex microecological preparation, which can effectively enhance the disease resistance of yaks, improve the structure of the intestinal flora of yaks, increase the quantity and abundance of probiotics, and inhibit the growth and reproduction of harmful bacteria.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a yak-derived complex microecological preparation, and the yak-derived complex microecological preparation comprises components in the following mass ratio: Bacillus coagulans: Bacillus thiaminolyticus: Bacillus subtilis: Bacillus licheniformis = 1: 0.5-1.5: 0.5-1.5: 0.5-1.5.

[0007] Preferably, the yak-derived complex microecological preparation comprises components in the following mass ratio: Bacillus coagulans: Bacillus thiaminolyticus: Bacillus subtilis: Bacillus licheniformis = 1: 0.7-1.2: 0.7-1.2: 0.7-1.2.

[0008] Preferably, the yak-derived complex microecological preparation comprises components in the following mass ratio: Bacillus coagulans: Bacillus thiaminolyticus: Bacillus subtilis: Bacillus licheniformis = 1:1:1:1.

[0009] Preferably, the viable counts of Bacillus coagulans, Bacillus thiaminolyticus, Bacillus subtilis and Bacillus licheniformis in the yak-derived complex microecological preparation are all 1-5×10 8 CFU / g.

[0010] The present invention provides a method for using the yak-derived complex microecological preparation, which is to mix the yak-derived complex microecological preparation with the diet and directly feed it to cattle.

[0011] Preferably, the dosage of the yak-derived complex microecological preparation is 15-25 g / day.

[0012] The present invention provides an application of the yak-derived complex microecological preparation in the preparation of a product for improving the disease resistance of cattle.

[0013] The present invention also provides an application of the yak-derived complex microecological preparation in the preparation of a product for improving the intestinal flora of cattle.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a yak-derived complex microecological preparation and its application. The present invention prepares a complex microecological preparation by mixing Bacillus coagulans, Bacillus thiaminolyticus, Bacillus subtilis and Bacillus licheniformis. The complex microecological preparation in the present invention can be specifically targeted at the yak breed and has no biosafety problems. Using the yak-derived complex microecological preparation in the present invention can improve the immune function and antioxidant capacity of yaks, thereby enhancing the disease resistance of yaks. In addition, the complex microecological preparation can also improve the structure of the intestinal flora of yaks, increase the number and abundance of probiotics, and inhibit the growth and reproduction of harmful bacteria. Description of the Drawings

[0015] Figure 1 It is the cumulative box plot of sample species in the present invention;

[0016] Figure 2 It is the bar chart of species distribution at the phylum classification level of samples in the present invention;

[0017] Figure 3 It is the stacked impact plot of the relative abundance of species at the genus level in the present invention. Detailed Embodiments

[0018] The present invention provides a yak-derived compound probiotic preparation, and the yak-derived compound probiotic preparation comprises components in the following mass ratio: Bacillus coagulans: Bacillus thiaminolyticus: Bacillus subtilis: Bacillus licheniformis = 1: 0.5-1.5: 0.5-1.5: 0.5-1.5; preferably 1: 0.7-1.2: 0.7-1.2: 0.7-1.2; more preferably 1: 1: 1: 1.

[0019] In the present invention, the viable counts of Bacillus coagulans, Bacillus thiaminolyticus, Bacillus subtilis and Bacillus licheniformis in the yak-derived compound probiotic preparation are all 1-5×10 8 CFU / g.

[0020] The present invention provides a method for using the yak-derived compound probiotic preparation, which is to mix the yak-derived compound probiotic preparation with the daily diet and directly feed it to cattle.

[0021] In the present invention, the dosage of the yak-derived compound probiotic preparation is 15-25 g / day, preferably 18-22 g / day, and more preferably 20 g / day.

[0022] The present invention provides an application of the yak-derived compound probiotic preparation in the preparation of a product for improving the disease resistance of cattle.

[0023] The present invention also provides an application of the yak-derived compound probiotic preparation in the preparation of a product for improving the intestinal flora of cattle.

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] Example 1

[0026] Bacillus coagulans (purchased from Minsheng Zhongke Jiayi (Shandong) Bioengineering Co., Ltd.), Bacillus thiaminolyticus (purchased from Thermo Fisher Scientific Inc.), Bacillus subtilis and Bacillus licheniformis (both purchased from Beijing Beina Chuanglian Biotechnology Research Institute) were respectively placed in 100 mL of LB liquid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.), cultured in a shaker at 37 °C at 150 r / min for 24 h, the four strains were respectively subjected to scale-up culture, and then the four strains obtained by scale-up culture were respectively made into freeze-dried powders through a biological freeze-drying technology. According to the equal mass ratio, the freeze-dried powders of the four strains were mixed to obtain the yak-derived compound probiotic preparation.

[0027] Example 2 Strain Verification

[0028] Before freeze-drying the bacteria after enrichment culture in Example 1, the obtained strains were subjected to colony morphology, Gram staining, microscopic examination, determination of 16S rDNA gene sequence and bacterial identification, acid tolerance test, and bile salt tolerance test. The results are shown in Tables 1 to 3.

[0029] Colony morphology: Take 100 μL of the bacterial solution and dilute it to 10 -4 、10 -5 、10 -6 at three concentration gradients. Take 100 μL of each concentration of the bacterial solution and spread it on an LB solid medium with a sterile spreader. Incubate it upside down in an incubator at 37°C for 24 h. Take out the cultured plate and place it in a laminar flow hood to observe the morphology of single colonies.

[0030] Gram staining and microscopic examination: Dip an inoculation loop into the bacterial solution and smear it evenly on a glass slide. After fixation, perform Gram staining (Gram staining solution, purchased from Beijing Solarbio Science & Technology Co., Ltd.). Stain with crystal violet for 1 min, iodine solution for 1 min, 95% alcohol for 20 s, and safranin for 1 min. After drying, perform microscopic examination.

[0031] Determination of 16S rDNA gene sequence and bacterial identification: Use a pipette to aseptically aspirate 1 mL of the bacterial solution into a 1.5 mL sterilized centrifuge tube, seal it with a sealing film and make a mark, and send it to Tsingke Biotech Co., Ltd. for 16S rDNA gene sequence sequencing. The sequencing results are compared using the BLAST program in GenBank to identify the bacterial species.

[0032] Acid tolerance test: Use 1 mol / L HCl and 0.5 mol / L NaOH to adjust the pH of the PBS solution to 7.4, 4.0, 3.0, 2.0, sterilize it at 121°C for 30 min and set aside. Add the bacterial solution to the above-prepared PBS solution at a volume ratio of 1:10, incubate it in a shaker at 37°C for 2 h and then take it out. Dilute it 10-fold serially and take 100 μL to spread on a plate, invert it in an incubator at 37°C, culture for 24 h, count the viable bacteria on the plate, repeat each group 3 times, and calculate the survival rate by taking the average value.

[0033] Bile salt tolerance test: Use bovine bile salt to adjust the PBS solution to concentrations of negative, 0.15%, 0.3%, and 0.6%, sterilize it at 121°C for 30 min and set aside. Add the bacterial solution to the above-prepared PBS solution at a volume ratio of 1:10, incubate it in a shaker at 37°C for 2 h and then take it out. Dilute it 10-fold serially and take 100 μL to spread on a plate, invert it in an incubator at 37°C, culture for 24 h, count the viable bacteria on the plate, repeat each group 3 times, and calculate the survival rate by taking the average value.

[0034] Table 1 Colony morphology, Gram staining, and microscopic examination results of 4 strains of bacteria

[0035]

[0036] Table 2 Results of acid tolerance test of 4 strains of bacteria

[0037]

[0038]

[0039] Table 3 Results of bile salt tolerance test of 4 strains of bacteria

[0040]

[0041] Through the determination of 16S rDNA gene sequences and bacterial identification, it can be known that the strains after enlarged culture in Example 1 belong to Bacillus coagulans, Bacillus thiaminolyticus, Bacillus subtilis and Bacillus licheniformis respectively. And from the results of the acid tolerance test and bile salt tolerance test, it can be seen that the strains after enlarged culture can survive normally in the acidic environment and high bile salt environment of the intestine, indicating that the compound microecological preparation obtained from Bacillus coagulans, Bacillus thiaminolyticus, Bacillus subtilis and Bacillus licheniformis can be used to improve the intestinal flora of cattle.

[0042] Example 3

[0043] Twelve healthy yaks with similar body sizes were selected and randomly divided into 2 groups, namely the control group (CG group) and the experimental group (EG group). The groups were isolated from each other and from the cattle herd, and were fed in pens. The yak-derived compound microecological preparation obtained in Example 1 was fed once a day, and the feeding dose was 20 g per cow. The feeding was carried out at 8:00 in the morning every day. The yak-derived compound microecological preparation was evenly mixed into the daily diet (conventional yak diet can be used), and free drinking water was provided after each feeding. The CG group was fed a normal diet, and the compound microecological preparation was added to the diet of the EG group. The feeding method was to grind the compound microecological preparation into powder and evenly mix it into the diet, and then evenly distribute the mixed feed into the feed troughs of each group for feeding.

[0044] On the morning of the 0th day, 7th day and 14th day of the experiment, fasting jugular vein blood samples were collected from the cattle in each group, 6 in each group. The collected blood was allowed to stand for 2 h, centrifuged at 3000 r / min for 15 min, the blood collection tube was tilted slightly, and the serum was slowly sucked out with a pipette and aliquoted into 1.5 mL centrifuge tubes and stored at -20 °C for later measurement. The collected blood was tested for serum biochemical indexes, immune indexes, oxidation and antioxidant indexes, and the results are shown in Tables 4 to 6.

[0045] The detection method of serum biochemistry was as follows: ALT, AST, ALP, GGT, TP, ALB, GLB, AG, TBIL, UREA, Ca, and P in the collected blood were measured by the method described in the operation manual of the automatic blood biochemistry analyzer, and the results are shown in Table 4.

[0046] The detection method of immune indexes was as follows: IgA, IgM, and IgG were detected by the method described in the kit instruction manual. The results are shown in Table 5, and all the kits were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.

[0047] The detection method of oxidation and antioxidant indexes was as follows: T-AOC, SOD, GSH-Px, CAT, and MDA were measured by the method described in the kit instruction manual. The results are shown in Table 6, and all the kits were purchased from Shanghai Youxuan Biotechnology Co., Ltd.

[0048] At 0d, 7d, and 14d in the morning, sterile feces were collected from each group of cows. After quenching with liquid nitrogen, they were stored at -80°C and sent to a biotechnology company for intestinal flora sequencing. The results of the analysis of the changes in the flora structure are shown in Table 7 and Figures 1 to 3 .

[0049] Table 4 Effects of the compound probiotic preparation on serum biochemical indexes

[0050]

[0051]

[0052]

[0053] Note: In the same row, if the superscript letters are the same or not marked, it means that the difference is not significant (P>0.05); if the superscript is different lowercase letters, it indicates that the difference is significant (P<0.05); if the superscript is different capital letters, it means that the difference is extremely significant (P<0.01).

[0054] As can be seen from Table 4, at 0d, 7d, and 14d, compared with the CG group, there were no significant differences in ALT, TP, ALP, GGT, ALB, GLB, AG, TBIL, CREA, Ca, and P in the EG group. However, the levels of TP, ALB, CREA, Ca, and P all showed an upward trend. At 14d, the contents of AST and TBIL in the EG group were significantly lower than those in the CG group (P<0.05); at 7d, the UREA level in the EG group was also significantly lower than that in the CG group (P<0.05). At 0d, 7d, and 14d, no significant differences were observed in the other biochemical indexes between the CG group and the EG group.

[0055] Table 5 Effects of the compound probiotic preparation on serum immune indexes

[0056]

[0057] Note: In the same row, if the superscript letters are the same or not marked, it means the difference is not significant (P > 0.05); if the superscript is different lowercase letters, it indicates a significant difference (P < 0.05); if the superscript is different uppercase letters, it means a highly significant difference (P < 0.01).

[0058] As can be seen from Table 5, at 7d and 14d, compared with the CG group, the IgG content in the EG group showed a highly significant increase (P < 0.01). At 14d, the IgM content in the EG group was extremely significantly higher than that in the CG group (P < 0.01). Compared with the CG group at 7d, although the IgM content in the EG group tended to increase, the difference was not significant. At 14d, compared with the CG group, the IgA content in the EG group was extremely significantly increased (P < 0.01). From the experimental results of the invention, feeding microecological preparations to animals can effectively increase the content of IgG and IgM in the body. During the experiment, no disease infection occurred in yaks, which may be related to the stimulating effect of microecological preparations on intestinal epithelial cells.

[0059] Table 6 Effects of compound microecological preparations on antioxidant indexes

[0060]

[0061] Note: In the same row, if the superscript letters are the same or not marked, it means the difference is not significant (P > 0.05); if the superscript is different lowercase letters, it indicates a significant difference (P < 0.05); if the superscript is different uppercase letters, it means a highly significant difference (P < 0.01).

[0062] As can be seen from Table 6, compared with the CG group, the T-AOC, GSH-Px, and SOD in the CG and EG groups at 7d and 14d were all extremely significantly increased (P < 0.01). At 7d, compared with the CG group, the CAT content in the EG group was significantly increased (P < 0.05). At 14d, compared with the CG group, the CAT content in the EG group was extremely significantly increased (P < 0.01). At 7d and 14d, compared with the CG group, the MDA content in the EG group was extremely significantly decreased (P < 0.01). This indicates that the compound microecological preparation can enhance the antioxidant ability of animals.

[0063] Table 7 Alpha diversity analysis of samples

[0064]

[0065] Alpha diversity analysis is the diversity analysis of species in a single sample. Among them, the Observed species index and the Shannon index can reflect the number and evenness of species in the sample community. The larger the index, the higher the richness of the sample; the Chao1 index and the ACE index can reflect the number of OTUs in the sample. The larger the index, the higher the richness of the sample; an index for estimating the number of OTUs contained in the sample; the Simpson index: the probability that two randomly selected OTUs in the sample are different species. The smaller the index, the higher the richness of the sample; the Good-coverage index: represents the coverage rate of each sample library. The closer it is to 1, the lower the probability of not being detected. It can be seen from Table 7 that the Observed species index of the EG group at 7d and 14d is slightly higher than that at 0d, and the Shannon index at 14d is slightly higher than that at 0d and 7d; the Simpson index, Pielou_J index, ACE index, and Pd_faith index show basically the same performance at 0d, 7d, and 14d; the Chao1 index at 14d is significantly higher than that at 0d and 7d.

[0066] The species accumulation boxplot is mainly used to describe the impact of increasing sample size on species diversity. From Figure 1 it can be seen that the abscissa represents the sample size, and the ordinate is the number of operational taxonomic units (OTUs) obtained after sampling. When the sample size is in the range of 1 to 2, the number of OTUs increases rapidly, indicating that there are many species in the submitted samples. As the sample size continues to increase, the number of OTUs gradually levels off, meaning that the number of species does not increase sharply with the increase in sample size.

[0067] The species distribution bar chart can intuitively reflect the proportion of each phylum-level species in the sample. From Figure 2 it can be more intuitively seen the proportion of each phylum-level species in the sample. It can be found that the top 10 most abundant phyla are listed in all 9 samples. Among them, the abundances of Firmicutes, Bacteroidetes, and Proteobacteria are relatively high. From Figure 2 it can be known that in the phylum Firmicutes, the abundances at 7d and 14d in the EG group are higher than that at 0d; in the phylum Bacteroidetes, the abundances at 7d and 14d in the EG group are higher than that at 0d; in the phylum Proteobacteria, the abundances at 7d and 14d in the EG group are lower than that at 0d.

[0068] Figure 3showed the top 10 genera with the highest relative abundances at the genus level, namely Arthrobacter, Anaerobacter, Romboutsia, Micromonospora, Sarcina, Klebsiella, Paeniclostridium, Methanobrevibacter, Bacteroides, and candidatus saccharimonas. From Figure 3 it can be seen that at 7 d, the relative abundances of Arthrobacter and Micromonospora increased, and at 14 d, the relative abundance of Anaerobacter increased.

[0069] In summary, the strains used in the preparation of the composite probiotic preparation of the present invention can tolerate the acidic environment and bile salt environment in the animal body. Through clinical trials, it is confirmed that the composite probiotic preparation prepared by the present invention can improve the immune function and antioxidant capacity of yaks, thereby enhancing the disease resistance of yaks. In addition, the composite probiotic preparation can also improve the intestinal flora structure of yaks, increase the number and abundance of probiotics, and inhibit the growth and reproduction of harmful bacteria.

[0070] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A yak-derived compound microecological preparation, characterized in that, The yak-derived compound microecological preparation comprises components in the following mass ratio: Bacillus coagulans: Bacillus thiaminolyticus: Bacillus subtilis: Bacillus licheniformis = 1: 0.5-1.5: 0.5-1.5: 0.5-1.

5.

2. The yak-derived compound microecological preparation according to claim 1, wherein The yak-derived compound microecological preparation comprises components in the following mass ratio: Bacillus coagulans: Bacillus thiaminolyticus: Bacillus subtilis: Bacillus licheniformis = 1: 0.7-1.2: 0.7-1.2: 0.7-1.

2.

3. The yak-derived compound microecological preparation according to claim 2, wherein The yak-derived compound microecological preparation comprises components in the following mass ratio: Bacillus coagulans: Bacillus thiaminolyticus: Bacillus subtilis: Bacillus licheniformis = 1: 1: 1:

1.

4. The yak-derived compound microecological preparation according to claim 3, wherein The viable counts of Bacillus coagulans, Bacillus thiaminolyticus, Bacillus subtilis and Bacillus licheniformis in the yak-derived compound probiotic preparation are all 1-5×10 8 CFU / g.

5. The method for using the yak-derived compound microecological preparation according to any one of claims 1 to 4, characterized in that Mix the yak-derived compound microecological preparation with the daily ration and directly feed it to cattle.

6. The usage method according to claim 5, characterized in that, The dosage of the yak-derived compound microecological preparation is 15-25 g / day.

7. Use of the yak-derived compound microecological preparation according to any one of claims 1 to 4 in the preparation of a product for improving the disease resistance of cattle.

8. Use of the yak-derived compound microecological preparation according to any one of claims 1 to 4 in the preparation of a product for improving the intestinal flora of cattle.

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