Lactobacillus salivarius and berberine combined preparation for relieving diarrhea of yak calf as well as preparation method and application of lactobacillus salivarius and berberine combined preparation

Abstract

The invention discloses a lactobacillus salivarius and berberine combined preparation for relieving diarrhea of yak calves as well as a preparation method and application of the lactobacillus salivarius and berberine combined preparation, and belongs to the technical field of microbial compositions. The combined preparation consists of berberine and lactobacillus salivarius freeze-dried powder, the preparation method comprises the following steps: S1, adding coptis chinensis medicinal material coarse powder into an ethanol water solution, carrying out reflux extraction, and carrying out suction filtration to obtain a filtrate; adding hot water into the filtrate, heating to obtain thick slurry, adjusting the pH value, and adding sodium chloride to promote yellow crystals to separate out to obtain berberine; s2, inoculating lactobacillus salivarius into a liquid culture medium for anaerobic culture, separating by using a centrifugal machine, and retaining precipitated bacterial cells; adding a freeze-drying protective agent into the precipitated bacterial cells, and uniformly mixing to obtain a bacterial suspension; freeze-drying the obtained bacterial suspension to obtain lactobacillus salivarius freeze-dried powder; and mixing the berberine with the lactobacillus salivarius freeze-dried powder to obtain the combined preparation. The prepared combined preparation is efficient and safe, and drug resistance is not prone to being generated.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial compositions, and in particular relates to a combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks, and a preparation method and application thereof. Background Art

[0002] Yaks are the main breed of cattle unique to the Qinghai-Tibet Plateau and are vital to the lives and economy of local herders. They are highly adaptable and cold-resistant, can survive in high-altitude environments, and provide humans with products such as beef, milk and hides. China has the largest number of yaks in the world, with a total of about 15 million, accounting for more than 90% of the global total, mainly distributed in Qinghai, Tibet and Sichuan.

[0003] However, the yak industry faces serious challenges, especially the problem of yak diarrhea. Diarrhea is common in yaks and can be fatal in severe cases, causing huge economic losses to farmers. The causes of yak diarrhea are complex and can be divided into two types: infectious and non-infectious. Among them, infectious diarrhea is mainly caused by parasites, viruses or bacteria, which has a high morbidity and mortality rate. Due to their young age, low immunity and incomplete physical development, yaks are more susceptible to illness and have a higher mortality rate than adult yaks. In addition, the death of yaks is mainly caused by diarrhea, with the highest incidence rate in yaks under 2 weeks old. As an important foundation for the sustainable development of the yak industry, although various measures have been tried to reduce diarrhea in yaks caused by pathogens, the effect is not significant, which seriously restricts the sustainable development of the yak industry in the Qinghai-Tibet Plateau and its market competitiveness.

[0004] At present, the main means of treating yak diarrhea is the use of antibiotics, but the long-term and improper use of antibiotics has led to a series of problems, such as drug residues, increased drug resistance, and toxic side effects on the animal body. These problems not only affect the treatment effect, but may also lead to recurrence of the disease. As a traditional treatment method, Chinese herbal medicine has shown good anti-diarrhea, anti-inflammatory and immunomodulatory properties; Chinese herbal medicine does not contain chemical synthetic drugs, will not produce drug resistance, and can enhance the body's immune response, showing significant efficacy in preventing and treating yak calf diarrhea. In addition, probiotics are also considered to be an effective auxiliary treatment method; they inhibit the growth of intestinal pathogens, improve the intestinal environment, and maintain microbial balance by secreting antibacterial substances such as organic acids, bacteriocins and hydrogen peroxide; probiotics can also enhance the immunity of animals by producing metabolites such as short-chain fatty acids, which helps prevent and treat yak calf diarrhea.

[0005] Therefore, it is particularly important to develop a combined preparation that is efficient, safe, not prone to drug resistance, and can alleviate diarrhea in calves and yaks caused by Escherichia coli by combining the application of Chinese herbal medicine and probiotics. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention discloses a combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in yaks, and a preparation method and application thereof. The combined preparation prepared by mixing berberine with freeze-dried powder of Lactobacillus salivarius can enhance the physique of yaks, significantly improve the antioxidant capacity of yaks, regulate the composition and structure of intestinal flora, enhance intestinal barrier and immunity, etc., thereby alleviating diarrhea in yaks; and it will not cause drug resistance problems, and long-term use is safer and more reliable.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The invention discloses a combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks. The combined preparation is composed of berberine and freeze-dried powder of Lactobacillus salivarius; the Lactobacillus salivarius is classified and named as Lactobacillus salivarius (Lactobacillus salivarius GZLC1), which has been deposited in the China Center for Type Culture Collection on May 11, 2023, with a deposit number of CCTCC NO: M 2023735, and an address of the depository unit: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Postal Code: 430072.

[0009] The present invention also provides a method for preparing the combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks, comprising the following steps:

[0010] S1. Preparation of berberine: Crush the Chinese rhizome into coarse powder, add it to an ethanol aqueous solution, and reflux extract it. After the solution boils, continue to reflux extract for 50 minutes. After extraction, obtain a filtrate by suction filtration. Add an appropriate amount of hot water to the filtrate and heat it to a thick slurry. After cooling, adjust the pH to 2 with concentrated hydrochloric acid, and add an appropriate amount of sodium chloride to promote the precipitation of yellow crystals, thereby obtaining berberine.

[0011] S2. Preparation of lyophilized powder of Lactobacillus salivarius: taking an appropriate amount of Lactobacillus salivarius from a glycerol cryopreservation tube and inoculating it in MRS liquid culture medium, placing the inoculated MRS culture medium in a constant temperature incubator and anaerobic culture at 37°C, measuring the OD600 value using a spectrophotometer, stopping the culture when the OD600 value in the logarithmic growth phase is about 1.0, centrifuging using a high-speed refrigerated centrifuge, pouring off the supernatant, retaining the precipitated bacterial cells, and washing with sterile physiological saline; then adding a lyophilization protective agent to the precipitated bacterial cells, and mixing them evenly to obtain a bacterial suspension; the obtained bacterial suspension is divided into freeze-drying containers, and then placed in a refrigerator for pre-freezing at -20°C for 48 hours, and then placed in a -80°C freeze dryer for freeze drying for 48 hours to obtain lyophilized powder of Lactobacillus salivarius;

[0012] S3. Preparation of a combined preparation: The berberine prepared in step S1 and the freeze-dried powder of Lactobacillus salivarius prepared in step S2 are mixed evenly to obtain a combined preparation.

[0013] Preferably, in step S1, the ethanol aqueous solution is a 75 wt % ethanol aqueous solution.

[0014] Preferably, in step S1, the mass volume ratio of the coptis chinensis medicinal material coarse powder to the ethanol aqueous solution is 1 g:6 mL.

[0015] Preferably, in step S2, the rotation speed of the high-speed refrigerated centrifuge is 4000 rpm, the centrifugation time is 15 min, and the centrifugation temperature is 4°C.

[0016] Preferably, in step S2, the volume ratio of the precipitated bacterial cells to the lyophilization protectant is 1:1, and the lyophilization protectant is 10% skim milk or sucrose solution.

[0017] Preferably, in step S3, the mass ratio of berberine to Lactobacillus salivarius freeze-dried powder is 1:5.

[0018] Preferably, in step S3, the number of viable Lactobacillus salivarius per milligram of the combined preparation is 2.67×10 7 CFU.

[0019] The present invention also discloses the application of the combined preparation of Lactobacillus salivarius and berberine for alleviating calf yak diarrhea in the preparation of products for preventing, diagnosing, detecting or treating calf yak diarrhea.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] After the combined preparation of Lactobacillus salivarius and berberine provided by the present invention was applied to yak calves, it was found through experiments that it could not only improve their antioxidant capacity (such as Figure 8 ), and can also improve the intestinal performance of animals by enriching beneficial bacteria and reducing harmful bacteria, regulate the composition and structure of intestinal flora, and promote changes in the intestinal flora of yak calves, especially the increase in the relative abundance of beneficial bacteria and the decrease in the relative abundance of harmful bacteria (such as Fig.11 ); at the same time, it can restore the body function of yaks and enhance their immunity, providing a new reference for the breeding of yaks in plateau areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effect of the combined preparation prepared for the present invention on Escherichia coli;

[0023] Figure 2 Effect of the combined preparation prepared by the present invention on the body weight (g) of mice;

[0024] Figure 3 The effects of the combined preparation prepared by the present invention on oxidative stress and inflammatory response in mice;

[0025] Figure 4 The effect of the combined preparation prepared by the present invention on the α diversity of the intestinal flora of mice;

[0026] Figure 5 Veen diagram of the OTU distribution of the combined preparation prepared by the present invention on the intestinal flora of mice;

[0027] Figure 6 Beta diversity analysis of mouse intestinal flora;

[0028] Figure 7 The effect of the combined preparation prepared by the present invention on the microbial community structure of mice at different taxonomic levels;

[0029] Figure 8 LEfSe analysis of mouse intestinal flora;

[0030] Fig. 9 Effects of the combined preparation prepared by the present invention on body weight (kg), growth rate (%) and diarrhea score of yak calves;

[0031] Fig.10 The effect of the combined preparation prepared by the present invention on oxidative stress and inflammatory response in yak calves;

[0032] Fig.11 The effect of the combined preparation prepared by the present invention on the α diversity of the intestinal flora of yak calves;

[0033] Fig.12 The effect of the combined preparation prepared by the present invention on the microbial community structure of calves and yaks at different taxonomic levels;

[0034] Fig.13 The beta diversity analysis of the intestinal flora of yak calves;

[0035] Fig.14 LEfSe analysis of intestinal flora in yak calves. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] The invention discloses a combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks. The combined preparation is composed of berberine and freeze-dried powder of Lactobacillus salivarius; the Lactobacillus salivarius is classified and named as Lactobacillus salivarius (Lactobacillus salivarius GZLC1), which has been deposited in the China Center for Type Culture Collection on May 11, 2023, with a deposit number of CCTCC NO: M 2023735, and an address of the depository unit: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Postal Code: 430072.

[0040] The present invention also provides a method for preparing the combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks, and the specific steps are as follows:

[0041] S1. Preparation of berberine: Crush the Chinese rhizome into coarse powder, add it to a 75wt% ethanol aqueous solution at a mass volume ratio of 1g:6mL, and reflux extract. After the solution boils, continue reflux extraction for 50min. After extraction, obtain a filtrate by suction filtration; add an appropriate amount of hot water to the filtrate and heat it to a thick slurry. After cooling, adjust the pH to 2 with concentrated hydrochloric acid, add an appropriate amount of sodium chloride to promote the precipitation of yellow crystals (stop adding when a small amount of yellow crystals precipitate), and wait until the yellow crystals are completely precipitated to obtain berberine.

[0042] S2. Preparation of lyophilized powder of Lactobacillus salivarius: Take an appropriate amount of Lactobacillus salivarius from a glycerol cryopreservation tube and inoculate it into MRS liquid culture medium, put the inoculated MRS culture medium into a constant temperature incubator and culture it anaerobically at 37°C, use a spectrophotometer to measure the OD600 value, stop the culture when the OD600 value in the logarithmic growth phase is about 1.0, use a high-speed refrigerated centrifuge to centrifuge at a speed of 4000 rpm, a centrifugation time of 15 min, and a centrifugation temperature of 4°C, pour out the supernatant, retain the precipitated bacterial cells, and wash them with sterile physiological saline; then add a lyophilization protective agent to the precipitated bacterial cells (wherein: the volume ratio of the precipitated bacterial cells to the lyophilization protective agent is 1:1), and mix them evenly to obtain a bacterial suspension; the obtained bacterial suspension is divided into lyophilization containers, and then placed in a refrigerator for pre-freezing at -20°C for 48 hours, and then placed in a -80°C freeze dryer for freeze drying for 48 hours to obtain lyophilized powder of Lactobacillus salivarius.

[0043] S3. Preparation of a combined preparation: The berberine prepared in step S1 and the freeze-dried powder of Lactobacillus salivarius prepared in step S2 are uniformly mixed in a mass ratio of 1:5, that is, a combined preparation is prepared.

[0044] Among them, in the above-prepared combined preparation, the number of viable Lactobacillus salivarius per mg of the combined preparation was 2.67×10 7 CFU.

[0045] The combined preparation prepared above is used in preparing products for preventing, diagnosing, detecting or treating diarrhea in calves and yaks.

[0046] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments:

[0047] Example 1

[0048] The preparation method of the combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks in this embodiment comprises the following specific steps:

[0049] S1. Preparation of berberine: Crush the Chinese rhizome into coarse powder, add it to a 75wt% ethanol aqueous solution at a mass volume ratio of 1g:6mL, and reflux extract. After the solution boils, continue reflux extraction for 50min. After extraction, obtain a filtrate by suction filtration; add an appropriate amount of hot water to the filtrate and heat it to a thick slurry. After cooling, adjust the pH to 2 with concentrated hydrochloric acid, and then add sodium chloride to promote the precipitation of yellow crystals (stop adding when a small amount of yellow crystals precipitate). When the yellow crystals are completely precipitated, berberine is obtained.

[0050] S2. Preparation of lyophilized powder of Lactobacillus salivarius: Take an appropriate amount of Lactobacillus salivarius from a glycerol cryopreservation tube and inoculate it into MRS liquid culture medium, put the inoculated MRS culture medium into a constant temperature incubator and culture it anaerobically at 37°C, use a spectrophotometer to measure the OD600 value, stop the culture when the OD600 value in the logarithmic growth phase is about 1.0, use a high-speed refrigerated centrifuge to centrifuge at a speed of 4000 rpm, a centrifugation time of 15 min, and a centrifugation temperature of 4°C, pour out the supernatant, retain the precipitated bacterial cells, and wash them with sterile physiological saline; then add a lyophilization protective agent to the precipitated bacterial cells (wherein: the volume ratio of the precipitated bacterial cells to the lyophilization protective agent is 1:1), and mix them evenly to obtain a bacterial suspension; the obtained bacterial suspension is divided into lyophilization containers, and then placed in a refrigerator for pre-freezing at -20°C for 48 hours, and then placed in a -80°C freeze dryer for freeze drying for 48 hours to obtain lyophilized powder of Lactobacillus salivarius.

[0051] S3. Preparation of a combined preparation: The berberine prepared in step S1 and the freeze-dried powder of Lactobacillus salivarius prepared in step S2 were mixed uniformly in a mass ratio of 1:5 to prepare a combined preparation, which is a combined preparation of Lactobacillus salivarius and berberine for relieving diarrhea in calves and yaks.

[0052] Example 2

[0053] This example is an in vitro antibacterial test of the combined preparation prepared in Example 1 against Escherichia coli.

[0054] After activating Escherichia coli and Lactobacillus salivarius on LB and MRS agar medium, a single colony of Escherichia coli was picked out and inoculated into LB broth medium, and a single colony of Lactobacillus salivarius was inoculated into a 10 mL centrifuge tube containing 3 mL of MRS broth medium, and cultured at 200 r / min and 37°C for 12 h; 25 mg of berberine was weighed and dissolved in 100 mL of LB broth medium to prepare an LB broth medium with a concentration of 0.25 mg / mL berberine. Escherichia coli group ( Figure 1 E) Take 100 μL MRS broth cultured in LB broth containing Escherichia coli, Lactobacillus salivarius group ( Figure 1 100 μL of overnight Lactobacillus salivarius supernatant was inoculated into LB broth containing Escherichia coli. Figure 1 B) 1% of Escherichia coli was inoculated into LB broth medium with berberine concentration of 0.25 mg / mL, and the combined preparation group ( Figure 1 In P+B), 100 μL of Lactobacillus salivarius supernatant and 1% of Escherichia coli inoculum were inoculated into LB broth medium with berberine concentration of 0.25 mg / mL; the above 4 groups were cultured at 37°C, and the OD value was detected at 600 nm with a spectrophotometer at regular intervals (shaken with a vortexer before each measurement), and the OD value within 0 to 12 h was measured. Three parallel tests were set for each group, and the data were recorded. The growth curves of Escherichia coli under different groups were drawn with OD value as the ordinate and time as the abscissa to explore the inhibitory effect of different groups on Escherichia coli. Figure 1 As shown (*p<0.05, **p<0.01, ***p<0.001).

[0055] from Figure 1 It can be seen that: with the E. coli group ( Figure 1 Compared with the berberine group ( Figure 1 B) and Lactobacillus salivarius group ( Figure 1 The growth of Escherichia coli was inhibited to a certain extent (p<0.05, p<0.01). The combined preparation group ( Figure 1 The inhibitory effect of the combined preparation group ( Figure 1 P+B) relative to the berberine group ( Figure 1 B) and Lactobacillus salivarius group ( Figure 1 The inhibitory effect of medium P) on Escherichia coli was better (p<0.01).

[0056] Example 3

[0057] This example is an experiment on the effect of the combined preparation prepared in Example 1 on the body weight, serum indexes and intestinal flora diversity of mice.

[0058] Sixty ICR mice aged 4 weeks were selected, half male and half female, with a body weight of 18g±2g. The experimental mice were randomly divided into JC group (blank group), JM group (model group), and JT group (combination preparation group), and adaptive feeding was performed for 7 days. The experimental cycle was 22 days. On the 1st to 20th day of the experiment, the JT group (combination preparation group) was intragastrically administered with 7.5 mg (containing 2×10 8 CFU of Lactobacillus salivarius) were added to the JC group (blank group) and the JM group (model group) for gavage with an equal amount of saline. On the 21st day of the experiment, 1×10 7 CFU / kg of Escherichia coli suspension, the JC group (blank group) did not receive any treatment, and the mice were sampled after 24 hours. At the end of the experiment, blood was collected from all mice. The experimental mice were blooded by eyeball blood sampling. After the blood was left to stand at room temperature for 2-3 hours, it was centrifuged at 3500r / min for 15 minutes to separate serum, which was used to detect the antioxidant capacity and immune capacity of mice (total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α)), and endothelial relaxing factor (NO) indicators. Fresh rectal feces samples from each group of mice were collected for 16S rRNA high-throughput sequencing to analyze the effects on the intestinal flora of mice; the JC group was divided into C1-C3, the JM group was divided into M1-M3, and the JT group was divided into X1-X3; the specific detection and analysis are as follows.

[0059] Figure 2 This is a graph showing the effect of the combined preparation prepared in Example 1 of the present invention on the body weight (g) of mice. It can be seen from the figure that the body weight of the mice in the JM group (model group) was slightly lower than that of the JC group (blank group). After treatment with the combined preparation prepared by the present invention, the body weight of the mice in the JT group (combined preparation group) increased compared with the JM group (model group) and almost recovered to the level of the JC group (blank group).

[0060] Figure 3The effect of the combined preparation prepared in Example 1 of the present invention on oxidative stress and inflammatory response in mice (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). It can be seen from the figure that compared with the JC group (blank group), the levels of MDA (p<0.001) and NO (p<0.001) in the serum of the JM group (model group) increased significantly, while the levels of SOD (p<0.05) and T-AOC (p<0.001) decreased; after treatment with the combined preparation of the present invention, compared with the JC group (blank group), the levels of MDA (p<0.001) and NO (p<0.001) in the serum of the JT group (combined preparation group) mice returned to normal, and the JT The levels of T-AOC and SOD (p<0.01) in the JM group (model group) (combined preparation group) were slightly increased; compared with the JC group (blank group), the levels of IL-6 (p<0.001), IL-10 (p<0.01), and TNF-α (p<0.0001) in the serum of the JM group (model group) were extremely significantly increased, while the levels of IL-6 (p<0.05), IL-10 (p<0.05), and TNF-α (p<0.001) in the JT group (combined preparation group) were significantly decreased; this shows that the combined preparation of the present invention can not only balance the oxidative stress ability in mice, but also inhibit the increase in the level of pro-inflammatory cytokines and the decrease in the level of anti-inflammatory cytokines in the serum of diarrheal mice, thereby achieving the therapeutic effect of anti-inflammatory and alleviating diarrhea.

[0061] Figure 4 The effect of the combined preparation prepared by the present invention on the α diversity of the intestinal flora of mice can be seen from the figure: there was no significant difference in Chao 1 index, Faith_pd index, Goods_coverage index, Observed_cpecies index, Shannon index, Pielou_evenness index and Simpson index among the JC group, JM group and JT group (p>0.05), but after the combined treatment of Lactobacillus salivarius and berberine, the Goods_coverage index, Simpson index and Pielou_evenness index increased; this shows that the richness, uniformity and diversity of the intestinal flora of mice were slightly increased after treatment with the combined preparation of the present invention.

[0062] Figure 5 This is a Veen diagram of the OTU distribution of the combined preparation of the present invention on the intestinal flora of mice. It can be seen from the figure that the JC group, the JM group and the JT group have a total of 187 common ASVs, among which the JC group, the JM group and the JT group have 625, 922 and 769 unique ASVs, respectively.

[0063] Figure 6This is a β-diversity analysis diagram of the intestinal flora of mice. It can be seen from the figure that the points among the JC group, the JM group and the JT group all showed different degrees of separation, indicating that after the treatment of the combined preparation of the present invention, the intestinal flora structure of the mice changed; and the stress value (<0.2) of the NMDS analysis also provides reliable results for further research and analysis.

[0064] Figure 7 The figure is an effect diagram of the combined preparation of the present invention on the structure of the microbial community of mice at different classification levels. It can be seen from the figure that there are differences in the intestinal flora of mice at both the phylum and genus levels. From the phylum level, after infection with Escherichia coli, the relative abundance of Proteobacteria and Verrucomicrobiota in the intestine of mice increased significantly, and the relative abundance of Campylobacterota and Firmicutes decreased significantly. After treatment with the combined preparation of the present invention, the relative abundance of Proteobacteria and Verrucomicrobiota in the intestine of mice decreased significantly, and the relative abundance of Firmicutes and Bacteroidota increased significantly ( Figure 7 a); From the phylum level, after infection with Escherichia coli, the relative abundance of Escherichia (Escherichia_710834) and Akkermansia in the intestine of mice increased significantly, and the relative abundance of Helicobacter (Helicobacter_C_479931), Lactobacillus (Lactobacillus), and Ralstonia (Ralstonia) decreased significantly. After treatment with the combined preparation of the present invention, the relative abundance of Escherichia (Escherichia_710834) in the intestine of mice decreased significantly, and the relative abundance of Lactobacillus (Lactobacillus) increased significantly ( Figure 7 b).

[0065] Figure 8 This is the LEfSe (Linear Discriminant Analysis Effect Size) analysis of the mouse intestinal flora. It can be seen from the figure that according to the LEfSe analysis, o_Mycoplasmatales, g_Malacoplasma_A_271108 and f_Mycoplasmoidaceae were detected in the JC group; the biomarker of the JM group was g_Eybacterium_R.

[0066] Example 4

[0067] This example is a test of the effect of the combined preparation prepared in Example 1 on the body weight, serum indexes and intestinal flora diversity of yak calves. During the test, all animals were given normal water and feed.

[0068] Eighteen healthy male yak calves from the Qinghai-Tibet Plateau, aged 4 months and weighing about 25 kg, were selected and randomly divided into the JC group (control group), the JM group (model group) and the JT group (combined preparation group). The experimental period was 12 days, with the first 3 days being adaptive feeding. On the 4th to 10th day of the experiment, the JT group (combined preparation group) was given 120 mg of the combined preparation (containing 3.2×10 9 CFU of Lactobacillus salivarius), the JC group (control group) and the JM group (model group) were given an equal amount of distilled water; on the 11th day of the experiment, the JM group (model group) and the JT group (combination preparation group) were induced with multidrug-resistant Escherichia coli (PP859186), and the JC group was induced with an equal volume of LB medium (Solarbio, Beijing-China); on the 12th day, samples were taken from the above groups of yaks respectively. At the end of the experiment, blood was collected from all the yaks in the above groups. The blood was collected from the jugular vein. After the blood was allowed to stand at room temperature for 2-3 hours, it was centrifuged at 3500r / min for 15 minutes to separate the serum, which was used to detect the antioxidant capacity and immune capacity of the yaks (total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β)). Fresh rectal feces samples were collected from each group of yaks for 16S rRNA high-throughput sequencing to analyze the effects on the intestinal flora of yaks. The specific test results are analyzed as follows.

[0069] Fig. 9 The combined preparation of the present invention has an effect on the weight (kg), growth rate (%) and diarrhea score of yak calves. It can be seen from the figure that there is no significant difference in the initial (4th day) and final (12th day) weights of the JC group, the JM group and the JT group ( Fig. 9 a); The weight growth rate of yaks in the JM group was significantly lower than that in the JC group (p<0.01), while the weight growth rate of yaks in the JT group was significantly higher than that in the JM group (p<0.05) ( Fig. 9 b), indicating that the combined preparation can effectively improve the growth rate of yak calves, making it close to or reach the normal level; the diarrhea score of the JM group was significantly higher than that of the JC group (p<0.0001) and the JT group (p<0.01) ( Fig. 9 c), indicating that the combined preparation of the present invention has an anti-diarrhea effect.

[0070] Fig.10The effect of the combined preparation prepared by the present invention on oxidative stress and inflammatory response in yak calves can be seen from the figure: the levels of T-AOC (p<0.001), SOD (p<0.001), GSH-Px (p<0.001), and IL-10 (p<0.01) in the JM group were significantly lower than those in the JC group, and the levels of MDA (p<0.0001), TNF-α (p<0.01), IL-1β (p<0.0001), and IL-6 (p<0.01) in the JM group were significantly lower than those in the JC group. 0.0001) levels in the JT group were significantly higher than those in the JC group. Compared with the JM group, the levels of T-AOC (p<0.01), SOD (p<0.0001), GSH-Px (p<0.0001) and IL-10 (p<0.01) in the JT group were significantly increased, and the levels of MDA (p<0.01), TNF-α (p<0.05), IL-1β (p<0.001) and IL-6 (p<0.0001) in the JT group were significantly decreased compared with those in the JM group.

[0071] Fig.11 The figure shows the effect of the combined preparation prepared by the present invention on the α diversity of the intestinal flora of yak calves. It can be seen from the figure that there are 434 common ASVs in the yak calves in the JC group, JM group and JT group, among which there are 2278, 2124 and 2052 unique ASVs in the JC group, JM group and JT group, respectively. Fig.11 a); The rarefaction curves of all yak calf fecal samples quickly reached a peak and remained stable, indicating that the yak calf intestinal microbiome had a high richness, and the grade abundance of yak calf fecal samples in each group was smooth, indicating a high degree of uniformity ( Fig.11 b, c); In addition, there were no significant differences in Chao1 index, Faith_pd index, Goods_coverage index, Observed_cpecies index, Shannon index, Pielou_evenness index and Simpson index among the JC group, JM group and JT group (p>0.05)( Fig.11 d).

[0072] Fig.12 The figure is the effect of the combined preparation of the present invention on the microbial community structure of yak calves at different classification levels. It can be seen from the figure that the intestinal flora of yak calves is different at the phylum, class, order, family and genus levels. From the phylum level, Firmicutes, Bacteroidetes and Actinobacteriota are dominant in the JC group (53.01%, 29.00% and 13.29%), JM group (54.13%, 22.77% and 17.86%) and JT group (44.54%, 25.18% and 26.81%). Fig.12a); At the genus level, the relative abundance of g_UCG-005, g_Bifidobacterium, and g_Bacteroidota in the JM group increased significantly, and the relative abundance of g_Rikenellaceae_RC9_gut_group decreased significantly; after treatment with the combined preparation of Lactobacillus salivarius and berberine, the relative abundance of g_UCG-005 and g_Bacteroidota in the intestine of yak calves decreased significantly, and the relative abundance of g_Bifidobacterium and g_Rikenellaceae_RC9_gut_group increased significantly ( Fig.12 e).

[0073] Fig.13 The β diversity analysis diagram of the intestinal flora of yak calves shows that according to the β diversity analysis, the points between the JC group, the JM group and the JT group all show different degrees of separation. The stress value of the NMDS analysis (<0.2) also provides reliable results for further research and analysis, indicating that there are significant differences ( Fig.13 ac); and Kruskal-Wallis analysis confirmed this, showing significant differences among the three groups (p<0.05) ( Fig.13 d).

[0074] Fig.14 This is the LEfSe analysis of the intestinal flora of yaks. It can be seen from the figure that according to the LEfSe analysis, the three groups have significantly different phyla and genera. c_Saccharimonadia, o_Saccharimonadales, f_Saccharimonadaceae, g_Saccharofermentans, g_Lactobacillus, f_Lactobacillaceae, etc. were detected in the JC group; in the JM group, g_Enterorhabdus, g_UBA1819, g_Solobacterium, etc. were highly enriched; and in the JT group, Actinomycetes (c_Actinobacteria) and f_Bifidobacteriaceae, etc. were significantly enriched.

[0075] The above is a detailed introduction to the combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks disclosed in the present invention, as well as its preparation method and application. Specific examples are used herein to illustrate the principles and implementation methods of the present invention, and the description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks, characterized in that: The combined preparation is composed of berberine and Lactobacillus salivarius freeze-dried powder; the Lactobacillus salivarius is classified and named Lactobacillus salivarius GZLC1, which was deposited in the China Center for Type Culture Collection on May 11, 2023, with a deposit number of CCTCC NO: M 2023735, and the address of the deposit unit is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Postal Code: 430072.

2. The method for preparing the combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks according to claim 1, characterized in that: The following steps are involved: S1. Preparation of berberine: Crush the Chinese rhizome into coarse powder and add it to an ethanol aqueous solution for reflux extraction. After the solution boils, continue to reflux extraction for 50 minutes. After extraction, obtain a filtrate by suction filtration. Add an appropriate amount of hot water to the filtrate and heat it to a thick slurry. After cooling, adjust the pH to 2 with concentrated hydrochloric acid, and then add sodium chloride to promote the precipitation of yellow crystals to obtain berberine. S2. Preparation of lyophilized powder of Lactobacillus salivarius: taking an appropriate amount of Lactobacillus salivarius from a glycerol cryopreservation tube and inoculating it in MRS liquid culture medium, placing the inoculated MRS culture medium in a constant temperature incubator and anaerobic culture at 37°C, measuring the OD600 value using a spectrophotometer, stopping the culture when the OD600 value in the logarithmic growth phase is about 1.0, centrifuging using a high-speed refrigerated centrifuge, pouring off the supernatant, retaining the precipitated bacterial cells, and washing with sterile physiological saline; then adding a lyophilization protective agent to the precipitated bacterial cells, and mixing them evenly to obtain a bacterial suspension; the obtained bacterial suspension is divided into freeze-drying containers, and then placed in a refrigerator for pre-freezing at -20°C for 48 hours, and then placed in a -80°C freeze dryer for freeze drying for 48 hours to obtain lyophilized powder of Lactobacillus salivarius; S3. Preparation of a combined preparation: The berberine prepared in step S1 and the freeze-dried powder of Lactobacillus salivarius prepared in step S2 are mixed evenly to obtain a combined preparation.

3. The method for preparing the combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks according to claim 2, characterized in that: In step S1, the ethanol aqueous solution is a 75 wt % ethanol aqueous solution.

4. The method for preparing the combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks according to claim 3, characterized in that: In step S1, the mass volume ratio of the crude powder of Coptis chinensis medicinal material to the ethanol aqueous solution is 1 g:6 mL.

5. The method for preparing the combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks according to claim 2, characterized in that: In step S2, the rotation speed of the high-speed refrigerated centrifuge is 4000 rpm, the centrifugation time is 15 min, and the centrifugation temperature is 4°C.

6. The method for preparing the combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks according to claim 2, characterized in that: In step S2, the volume ratio of the precipitated bacterial cells to the lyophilization protectant is 1:1, and the lyophilization protectant is 10% skim milk or sucrose solution.

7. The method for preparing the combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks according to claim 2, characterized in that: In step S3, the mass ratio of berberine to Lactobacillus salivarius freeze-dried powder is 1:

5.

8. The method for preparing the combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks according to claim 2, characterized in that: In step S3, the number of viable Lactobacillus salivarius per milligram of the combined preparation is 2.67×10 7 CFU.

9. Use of the combined preparation of Lactobacillus salivarius and berberine for alleviating diarrhea in calves and yaks as claimed in claim 1 in the preparation of products for preventing, diagnosing, detecting or treating diarrhea in calves and yaks.

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