Compound probiotic fermented traditional Chinese medicine preparation as well as preparation method and application thereof

Abstract

The invention discloses a compound probiotic fermented traditional Chinese medicine preparation as well as a preparation method and application thereof, and belongs to the technical field of microbial fermented traditional Chinese medicines. The compound probiotic fermented traditional Chinese medicine preparation is obtained by mixing lactobacillus plantarum and lactobacillus rhamnosus and fermenting pulsatilla chinensis. According to the compound probiotic fermented traditional Chinese medicine preparation, through the synergistic effect of multi-strain fermentation, the treatment effect of Chinese pulsatilla roots on calves suffering from escherichia coli diarrhea is improved. The prepared composite probiotic fermented traditional Chinese medicine preparation expands the application of the pulsatilla chinensis fermentation product in the field of healthy animal breeding, and has the effects of improving the growth performance, relieving the inflammatory response and improving the intestinal flora.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation of traditional Chinese medicine, in particular to a compound probiotic fermented traditional Chinese medicine preparation and a preparation method and application thereof. BACKGROUND

[0002] Diarrhea is a difficult problem that has plagued the animal husbandry industry, and calf diarrhea is one of the clinical diseases that seriously hinders the development of the global cattle industry. Due to high mortality, increased treatment costs, and production and processing losses, calf diarrhea causes an estimated damage of more than 1 billion US dollars to the cattle industry in North America each year. In China, Wang Xu et al. conducted a questionnaire survey on 19175 calves and 205414 adult cows in 57 dairy farms in 14 provinces and cities including Hebei Province, Qinghai Province, Beijing, Ningxia Hui Autonomous Region, and Heilongjiang Province. The results showed that the average annual incidence rate of calf diarrhea was 22.53% (2.50%-97.50%), and Escherichia coli (E. coli) is a very important bacterial pathogen that causes diarrhea in newborn calves. It can occur throughout the year, especially in winter and spring. Not only can it cause diarrhea in calves, but also septicemia, peritonitis, and other diseases in calves, and can cause acute death in severe cases. The incidence rate is as high as 90%, and the mortality rate is about 50%. The pathogenicity of pathogenic E. coli in the intestinal tract depends on two important virulence factors, the fimbriae on the surface of the bacteria (responsible for the ability of bacteria to adhere and colonize the small intestinal lumen surface) and the enterotoxin that affects the secretion of intestinal body fluid, and Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) are also the main causes of calf diarrhea.

[0003] At present, farms generally rely on large doses of antibiotics to treat calf diarrhea, which leads to the emergence of multiple drug-resistant strains, greatly increasing the difficulty of treatment. However, in recent years, people have gradually realized the various side effects brought about by the use of antibiotics, and the emergence of drug-resistant bacteria and the residue of antibiotics in meat are major concerns. More importantly, the misuse of antibiotics during the calf period and repeated diarrhea before weaning can lead to an immature intestinal flora, which can have a lasting negative impact on the digestion and absorption of the calf's growth diet. In order to effectively prevent and control E. coli-induced calf diarrhea, it is urgent to develop a new type of high-efficiency fermented traditional Chinese medicine preparation to improve the prevention and control effect of E. coli-induced calf diarrhea and promote the healthy and sustainable development of the cattle industry.

[0004] Pulsatillae radix is a plant of Ranunculaceae, also known as Yezhangren, Nahecao, and Baotougong, etc. The main medicinal part is the root, which is bitter and cold in nature and belongs to the stomach and large intestine channels. Pulsatillae radix is distributed in various parts of China and has high medicinal value due to its convenient collection and utilization. The effective components of Pulsatillae radix include triterpene saponins, triterpene acids, pulsatilla root, 2,3-hydroxy white birch acid, and daucosterol. From the perspective of modern pharmacology, Pulsatillae radix has the effects of clearing heat and resolving toxins, cooling blood and stopping diarrhea, treating yin itching, and clearing damp-heat of Yangming. It has biological activities such as antibacterial, anti-inflammatory, insecticidal, antitumor, sedative, analgesic, and others. With the increasing attention to natural medicines, Pulsatillae radix has attracted more and more attention due to its significant pharmacological effects in antibacterial, anti-inflammatory, and diarrhea prevention and control, especially in animal breeding. However, the traditional use of Pulsatillae radix is limited by its direct drying and crushing or simple decoction, which restricts its efficacy. Existing products are mainly single-component extracts or basic compound preparations, which are difficult to achieve stable and continuous efficacy in clinical practice. In addition, the use of Pulsatillae radix alone has problems such as bitter and cold components, poor palatability, and limited target points, which leads to a decrease in animal feeding willingness and poor treatment compliance, making it difficult to meet the current demand for efficient and safe preparations in animal health breeding. This not only limits the clinical application effect of Pulsatillae radix, but also affects its further development in the field of animal health breeding. SUMMARY

[0005] The purpose of the present application is to provide a compound probiotic fermented traditional Chinese medicine preparation and its preparation method and application, in order to solve the problems existing in the prior art.

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

[0007] In a first aspect, the present application provides a compound probiotic fermented traditional Chinese medicine preparation, which is obtained by inoculating Lactobacillus plantarum and Lactobacillus rhamnosus into Pulsatillae radix traditional Chinese medicine liquid and fermenting.

[0008] Preferably, the Lactobacillus plantarum and the Lactobacillus rhamnosus are mixed at a volume ratio of (1-2):(1-2).

[0009] Preferably, the fermentation time is 24-48 h.

[0010] In a second aspect, the present application also provides a preparation method of the compound probiotic fermented traditional Chinese medicine preparation, which comprises the steps of mixing Lactobacillus plantarum and Lactobacillus rhamnosus at a volume ratio of (1-2):(1-2), and inoculating into Pulsatillae radix liquid for fermentation.

[0011] Preferably, the inoculation amount of the mixed Lactobacillus plantarum and Lactobacillus rhamnosus is 2-6%.

[0012] Preferably, the mass concentration of the white head onion in the liquid medicine is 1%.

[0013] Preferably, the temperature of the fermentation is 37 DEG C.

[0014] In a third aspect, the application further provides application of the compound probiotic fermented traditional Chinese medicine preparation in preparing a product for preventing and treating E. coli diarrhea of calves.

[0015] In a fourth aspect, the application further provides a product for preventing and treating E. coli diarrhea of calves, wherein the product comprises the compound probiotic fermented traditional Chinese medicine preparation.

[0016] In a fifth aspect, the application further provides application of the compound probiotic fermented traditional Chinese medicine preparation in preparing a medicine for improving intestinal microecological environment of diarrhea calves.

[0017] The application discloses the following technical effects:

[0018] The application obtains a compound probiotic fermented traditional Chinese medicine preparation by screening probiotics and traditional Chinese medicine raw materials in the compound probiotic fermented traditional Chinese medicine preparation, and the compound probiotic fermented traditional Chinese medicine preparation improves the treatment effect of white head onion on calves suffering from E. coli diarrhea through the synergistic effect of multi-strain fermentation.

[0019] The compound probiotic fermented traditional Chinese medicine preparation prepared by the application expands the application of white head onion fermentation products in the field of animal health breeding, and has the effects of improving growth performance, reducing inflammatory reaction and improving intestinal flora. DETAILED DESCRIPTION

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0021] Figure 1 It is the separation and identification result of E. coli F2 in Example 1; wherein, A is the morphology of E. coli in LB culture medium; B is E. coli after Gram staining;

[0022] Figure 2 It is the identification result of virulence genes in Example 1; wherein, (A) is the identification result of ler virulence gene, (B) is the identification result of fimc virulence gene, (C) is the identification result of eae virulence gene, (D) is the identification result of bfpA virulence gene, and (E) is the identification result of irp2 virulence gene; M is DL1000 DNA Marker, PC is a positive control group, and C is a negative control group.

[0023] Figure 3 The influence of different treatment groups on the intestinal TNF-α content of diarrhea calves in Example 2; among them, A is the level of TNF-α of each group of calves on 0 d; B is the level of TNF-α of each group of calves on 7 d, C is the level of TNF-α of each group of calves on 14 d, the data is expressed as mean ± standard deviation, compared with C group, ** P<0.01, *P<0.05; compared with M group, ## P<0.01, # P<0.05;

[0024] Figure 4 The influence of different treatment groups on the intestinal IL-6 content of diarrhea calves in Example 2; among them, A is the level of IL-6 of each group of calves on 0 d; B is the level of IL-6 of each group of calves on 7 d, C is the level of IL-6 of each group of calves on 14 d, the data is expressed as mean ± standard deviation, compared with C group, ** P<0.01; compared with M group, ## P<0.01;

[0025] Figure 5 The influence of different treatment groups on the intestinal IL-1β content of diarrhea calves in Example 2; among them, A is the level of IL-1β of each group of calves on 0 d; B is the level of IL-1β of each group of calves on 7 d, C is the level of IL-1β of each group of calves on 14 d, the data is expressed as mean ± standard deviation, compared with C group, ** P<0.01, *P<0.05; compared with M group, ## P<0.01, # P<0.05;

[0026] Figure 6 The influence of different treatment groups on the intestinal IL-10 content of diarrhea calves in Example 2; among them, A is the level of IL-10 of each group of calves on 0 d; B is the level of IL-10 of each group of calves on 7 d, C is the level of IL-10 of each group of calves on 14 d, the data is expressed as mean ± standard deviation, compared with C group, ** P<0.01, *P<0.05; compared with M group, ## P<0.01, # P<0.05;

[0027] Figure 7 The influence of different treatment groups on Alpha diversity in Example 2; ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05;

[0028] Figure 8For the impact of different treatment groups on Beta diversity in Example 2; ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05;

[0029] Figure 9 For the impact of different treatment groups on intestinal flora structure at the level of door in Experimental Example 2;

[0030] Figure 10 For the impact of different treatment groups on intestinal flora structure at the level of door in Experimental Example 2;

[0031] Figure 11 For the Lefse analysis results in Experimental Example 2;

[0032] Figure 12 For the Lefse analysis results in Experimental Example 2;

[0033] Figure 13 For the Lefse analysis results in Experimental Example 2; DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the application and not as limiting the application. It will be readily apparent to those skilled in the art that varying substitutions and modifications can be made to the application without departing from the scope and spirit of the application.

[0035] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for any numerical range recited herein, every narrower range that falls within the broader range is also contemplated as being within the scope of the application. These narrower ranges are also specifically recited herein. The upper and lower limits of these narrower ranges are independently combinable with one another as well as combinable with the upper and lower limits of the broader ranges.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0037] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all such modifications and variations warrant the patentable subject matter under the patent laws. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only and are not intended to limit the scope of the application.

[0038] As used herein, "comprises", "comprising", "includes", "including", "has", "having" or other similar words, are open- ended, that is, meaning "including but not limited to".

[0039] The traditional Chinese medicines used in the following examples were purchased from PPTH Traditional Chinese Medicine Herbal Pieces Co., Ltd. in Anguo City and stored at low temperature for future use; Lactobacillus plantarum (Lactobacillus plantarum 138404), Lactobacillus murinus (Lactobacillus murinus 185356), Bacillus subtilis (Bacillus subtilis 13894) and Bifidobacterium lactis (Bifidobacterium lactis 139258) were purchased from Shenzhen Yibaisun Technology Co., Ltd.

[0040] Traditional Chinese medicine preparation: Take 200 g of traditional Chinese medicine, crush the traditional Chinese medicine and pass it through a 60-mesh sieve, and then soak it in a 1000 mL beaker for 30 min. Add 800 mL of distilled water, pour it into a decoction kettle, and continue to heat it for 40 min after boiling. Filter it with 2 layers of gauze, pour the filtrate into another beaker, add 400 mL of distilled water to the decoction kettle, and heat it for 30 min. Filter it with 2 layers of gauze, pour the filtrate into the beaker containing the previous filtrate, and add 600 mL of distilled water to the decoction kettle. Heat it for 30 min, filter it with 2 layers of gauze, and pour the filtrate into the beaker containing the previous filtrate. Continue to heat and concentrate the collected filtrate to 200 mL, and its concentration is 1 g / mL. The required concentration in the experiment is prepared by diluting the above concentration. Use 100 mL of the traditional Chinese medicine liquid for traditional Chinese medicine test, and use 100 mL of the traditional Chinese medicine liquid for fermentation test.

[0041] Strain activation: Take a strain stored at -80°C, thaw it completely, and then pre-activate it in a 37°C incubator for 30 min for preliminary recovery. After recovery, use a pipette to take 1 mL of the bacterial solution in 100 mL of MRS broth medium in a clean bench, ensuring aseptic operation throughout the process. After inoculation, place it in a 37°C constant temperature incubator for stationary culture and activation for two generations (24 h). Take the activated second-generation bacterial solution for testing. The strains used in subsequent studies are controlled to have the same initial concentration of 10 9 CFU / mL by coating counting.

[0042] Example 1: Screening of strains and traditional Chinese medicine

[0043] 1. Escherichia coli isolation and identification

[0044] Collect the fecal samples of diarrhea cattle in a sterile container, take 5 g of the fecal sample, and dissolve it in a 50 mL sterile centrifuge tube with normal saline. Dilute it with normal saline by a factor of 4 to a concentration of 10-4 The 100 μL diluted sample was evenly coated on LB solid medium, and after the sample liquid was infiltrated in the medium, it was sealed with sealing film. It was placed in a 37°C incubator for 36-48 h. Single colonies suspected of E. coli were picked and purified. The purified strains were subjected to gram staining, and the morphology of the bacteria was observed under a microscope. Strains with E. coli morphological characteristics were selected for further identification. The bacterial DNA was extracted using a bacterial genomic DNA extraction kit according to the instructions. The genes were amplified using PCR with universal primers for bacteria: 27F: 5'-ACTGTGATGGGATACGCGTC-3' (SEQ ID NO. 17); 1492R: 5'-CTCCGTCAGCGTTTCAGCTA-3' (SEQ ID NO. 18); the PCR reaction system is shown in Table 1, and the reaction program is shown in Table 2. The PCR amplification product was subjected to gel electrophoresis, and the gel imaging results were observed. The PCR amplification product of the desired fragment was sent to Shanghai Sangon Company for sequencing. The sequencing results were compared and analyzed with the sequences in the GenBank database using BLAST.

[0045] Table 1 Reaction system

[0046]

[0047] Table 2 Reaction conditions

[0048]

[0049] 2. Detection of E. coli virulence genes

[0050] According to the literature on E. coli virulence genes, eight primers for E. coli virulence genes ler, Stx1, Stx2, eae, ral, fimc, BfpA, and irp2 were designed and synthesized. The primers were synthesized by Shanghai Sangon Company, and the specific information of the primers is shown in Table 3. The products were subjected to gel electrophoresis, and E. coli containing the target gene was used as a positive control group (PC group), and deionized water was used as a negative control group (C group). The gel imaging results were observed.

[0051] Table 3 Primer sequences of different virulence genes

[0052]

[0053] 3. Screening of strains and traditional Chinese medicines

[0054] The probiotics selected B. salivarius, L. plantarum, L. rhamnosus and B. lactis were used to conduct the antibacterial test on the isolated E. coli, and the Chinese medicine selected Pulsatillae Decoction, Coptidis Decoction, Phellodendri Decoction and Cortex Fraxini Decoction was used to conduct the antibacterial test on the isolated E. coli. The Oxford cup quantitative diffusion method was adopted. The bacterial liquid with the viable count between 30-300 CFU / mL was adjusted in concentration, wherein the four probiotics and the Chinese medicine liquid were adjusted to the same concentration, and the E. coli concentration was adjusted. The antibacterial effect of the four probiotics and the Chinese medicine liquid on E. coli was evaluated by measuring the diameter of the inhibition zone.

[0055] 4. Experimental results

[0056] 4.1 Isolation and identification of E. coli

[0057] A total of 8 strains of E. coli were isolated from the fecal samples of diarrhea calves, which were named as F1-1, F1-2, F2, F4, F5, F6-1, F6-2, F8 and F10. The F2 strain was selected for morphological and staining analysis. On the LB solid culture medium, the E. coli showed uniform size, round, smooth surface, slightly convex, milky white colony morphology, and was accompanied by strong odor (Fig. 1A). Under the microscope observation of gram staining, the bacterial body was rod-shaped, red, single or paired arrangement of gram-negative bacteria (Fig. 1B). Figure 1 Figure 1 ​The PCR amplification products of the screened strains were sequenced and the results were subjected to BLAST comparison in GenBank. The results showed that F1-1, F1-2, F2, F4, F5, F6-1, F8 and F10 belonged to Escherichia coli. Among them, F1-1 had 100% similarity with the reference strain Escherichia coli strain SKLAN-202301; F2 had 100% similarity with the reference strain Escherichia coli strain SKLAN-202301; F1-2 had 99.80% similarity with the reference strain Escherichia coli strain EW2-81; F4 had 99.80% similarity with the reference strain Escherichia fergusonii strain E07; F5 had 99.80% similarity with the reference strain Escherichia coli strain 83-a-pink; F6-1 had 99.80% similarity with the reference strain Escherichia coli strain ECIUP_01; F6-2 had 99.81% similarity with the reference strain Bacillus licheniformis strain Y-1; F8 had 99.70% similarity with the reference strain Escherichia coli strain LZ-6; and F10 had 99.70% similarity with the reference strain Escherichia coli strain R24 (Table 4).

[0058] Table 4 Sequencing comparison results of strains

[0059]

[0060] 4.2 Virulence gene detection results

[0061] Five virulence genes, ler, eae, bfpA, Fimc and irp2, were detected in 8 strains of Escherichia coli. The PCR amplification products were identified by 1% agarose gel electrophoresis, and the target bands appeared at the same position as the positive control group Figure 2 ), among which the genes with higher carrying rate were ler (100%) and fimc (100%), while the detection rates of eae (37.5%), bfpA (37.5%) and irp2 (25%) were lower.

[0062] 4.3 Screening of bacteria and traditional Chinese medicines

[0063] The antibacterial effect of probiotics on E. coli isolated from the feces of diarrhea calves (Table 5) showed that L. rhamnosus and L. plantarum exhibited stronger antibacterial activity, especially in F2 and F4 samples, with inhibition zones of 15.66 ± 0.33 mm and 14.08 ± 1.24 mm, respectively. L. plantarum was particularly prominent in F4 and F8 samples, with inhibition zones of 15.35 ± 0.98 mm and 15.63 ± 1.45 mm, respectively. In contrast, other probiotics such as B. licheniformis and B. lactis had weaker antibacterial effects, and no inhibition zones were observed in some samples. Therefore, L. plantarum and L. rhamnosus were selected as the complex probiotics to study their inhibition of E. coli. The complex probiotics were prepared by mixing L. plantarum and L. rhamnosus (mixed culture) or by separately culturing L. plantarum and L. rhamnosus and then mixing them (pre-culture mixing). As shown in Table 5, the complex probiotics (mixed culture and pre-culture mixing) exhibited strong antibacterial effects in some samples, such as an inhibition zone of 18.56 ± 0.34 mm for the mixed culture in F2 sample.

[0064] Table 5 Antibacterial effect of probiotics on E. coli

[0065]

[0066] The antibacterial effect of traditional Chinese medicine on E. coli isolated from the feces of diarrhea calves (Table 6) showed that the traditional Chinese medicine liquid from Pulsatilla chinensis exhibited stronger antibacterial activity, with an average inhibition zone diameter of 15.60 mm in 8 samples, which was higher than that of the other three traditional Chinese medicine liquids, indicating that it had strong inhibitory ability against E. coli. The traditional Chinese medicine liquid from Cortex Fraxini had the weakest antibacterial effect, with an average inhibition zone of only 10.92 mm, and small inhibition zones were formed in some samples, suggesting that it had weak antibacterial activity. Therefore, the traditional Chinese medicine liquid from Pulsatilla chinensis was selected for further experiments.

[0067] Table 6 Antibacterial effect of traditional Chinese medicine on E. coli

[0068]

[0069] Example 2 Preparation of complex probiotic fermented traditional Chinese medicine preparation

[0070] Based on the screening results of the antibacterial activity of traditional Chinese medicine and probiotics against E. coli in Example 1, the traditional Chinese medicine liquid from Pulsatilla chinensis was used as the nutrient fermentation substrate, and L. plantarum and L. rhamnosus were used as the fermentation strains. L. plantarum and L. rhamnosus were mixed at a volume ratio of 1:1, and then inoculated into the traditional Chinese medicine liquid from Pulsatilla chinensis (concentration of 1%, w / v) at an inoculation amount of 4%. The mixture was fermented at 37°C for 36 h to obtain a complex probiotic fermented traditional Chinese medicine preparation.

[0071] Example 3 Preparation of compound probiotic fermented traditional Chinese medicine preparation

[0072] Lactobacillus plantarum and Lactobacillus rhamnosus were mixed at a volume ratio of 1:2, inoculated into the traditional Chinese medicine liquid of Bai Tou Weng (the concentration of the traditional Chinese medicine liquid was 1%, w / v) at an inoculation amount of 4%, and fermented at 37°C for 36 h to obtain the compound probiotic fermented traditional Chinese medicine preparation.

[0073] Example 4 Preparation of compound probiotic fermented traditional Chinese medicine preparation

[0074] Lactobacillus plantarum and Lactobacillus rhamnosus were mixed at a volume ratio of 2:1, inoculated into the traditional Chinese medicine liquid of Bai Tou Weng (the concentration of the traditional Chinese medicine liquid was 1%, w / v) at an inoculation amount of 4%, and fermented at 37°C for 36 h to obtain the compound probiotic fermented traditional Chinese medicine preparation.

[0075] Example 5 Preparation of compound probiotic fermented traditional Chinese medicine preparation

[0076] Lactobacillus plantarum and Lactobacillus rhamnosus were mixed at a volume ratio of 1:1, inoculated into the traditional Chinese medicine liquid of Bai Tou Weng (the concentration of the traditional Chinese medicine liquid was 1%, w / v) at an inoculation amount of 2%, and fermented at 37°C for 36 h to obtain the compound probiotic fermented traditional Chinese medicine preparation.

[0077] Example 6 Preparation of compound probiotic fermented traditional Chinese medicine preparation

[0078] Lactobacillus plantarum and Lactobacillus rhamnosus were mixed at a volume ratio of 1:1, inoculated into the traditional Chinese medicine liquid of Bai Tou Weng (the concentration of the traditional Chinese medicine liquid was 1%, w / v) at an inoculation amount of 6%, and fermented at 37°C for 36 h to obtain the compound probiotic fermented traditional Chinese medicine preparation.

[0079] Experimental Example 1 Analysis of traditional Chinese medicine ingredients

[0080] The compound probiotic fermented traditional Chinese medicine preparation prepared in Example 1 was used for subsequent experiments.

[0081] Non-target metabolome detection was performed on Bai Tou Weng and the compound probiotic fermented traditional Chinese medicine preparation, and the chromatographic column was Xselect HSS T3, 2.5 μm, 2.1 x 150 mm, the mobile phase A was 0.1% formic acid-water, B was 0.1% formic acid-acetonitrile, the column temperature was 50°C, and the flow rate was 0.4 mL / min.

[0082] The results are shown in Table 7. Compared with the Bai Tou Weng group, L-lactic acid, isofraxidin, baicalein, ginsenoside F2, adenocaulonoside, wuyanxiansaoside D, and methyl gallate were significantly up-regulated in the compound probiotic fermented traditional Chinese medicine preparation group.

[0083] Table 7 Screening results of up-regulated differential metabolites

[0084]

[0085] Experimental Example 2 Animal Experiment

[0086] The subsequent experiment was carried out using the composite probiotic fermented traditional Chinese medicine preparation prepared in Example 1.

[0087] The control group was obtained by adding 100 mL of normal saline to the milk replacer;

[0088] The positive treatment group was obtained by dissolving the antibiotic in the milk replacer at a dose of 5 mg / kg based on the body weight of the calf;

[0089] The composite probiotic group was obtained by mixing Lactobacillus plantarum and Lactobacillus rhamnosus at a volume ratio of 1:1 and culturing for 36 h, and then adding 100 mL (viable bacterial concentration > 10 9 CFU / mL) to the milk replacer;

[0090] The traditional Chinese medicine group was obtained by adding 100 mL of Pulsatillae Decoction (traditional Chinese medicine liquid concentration 1%, w / v) to the milk replacer;

[0091] The composite probiotic fermented traditional Chinese medicine preparation group was obtained by adding 100 mL of the composite probiotic fermented traditional Chinese medicine preparation to the milk replacer;

[0092] Fifty 5-day-old calves with similar body weights and suffering from E. coli diarrhea were selected as test objects and randomly divided into 5 treatments with 10 replicates in each treatment. In addition, 10 healthy 5-day-old calves with similar body weights were selected as a control group. The control group (group C): normally fed with milk replacer; the positive treatment group (group A): adding antibiotics to the milk replacer; the diarrhea group (group M): normally fed with milk replacer; the composite probiotic group (group CP): adding composite probiotics to the milk replacer; the traditional Chinese medicine group (group P): adding Pulsatillae Decoction to the milk replacer; the composite probiotic fermented traditional Chinese medicine preparation group (group FP): adding the composite probiotic fermented traditional Chinese medicine preparation to the milk replacer. Feeding was carried out once a day in the morning.

[0093] 1. Growth performance determination

[0094] The average daily gain (ADG) and body weight of the calves were determined every 7 days;

[0095] Average daily gain (ADG): The body weight of the calf was weighed before morning feeding on an empty stomach, and the difference between the body weight at the end of the test and the body weight at the beginning of the test divided by the test time was the average daily gain of the calf.

[0096] Body length: the distance from the front edge of the scapula to the base of the tail.

[0097] Body length: the distance from the highest point of the hip joint to the base of the tail.

[0098] Chest circumference: the vertical circumference of the body at the angle of the scapula, which is tight enough to insert the index finger and middle finger up and down.

[0099] 2. Inflammatory index determination

[0100] The ELISA kit was selected, and the kit used a double antibody one-step sandwich enzyme-linked immunoassay (ELISA). Into the coated micro-wells coated with calf IL-6 (interleukin-6), IL-1β (interleukin-1β), IL-10 (interleukin-10) and TNF-α (tumor necrosis factor α) capture antibody, standard sample, HRP labeled detection antibody was added in turn, incubated and washed thoroughly. Color development with substrate TMB, TMB is converted to blue under the catalysis of peroxidase, and is converted to the final yellow under the action of acid. The color depth is positively correlated with the amount of calf IL-6 (interleukin-6), IL-1β (interleukin-1β), IL-10 (interleukin-10) and TNF-α (tumor necrosis factor α) in the sample. The absorbance (OD value) was measured at 450 nm wavelength by enzyme-labeled instrument, and the sample concentration was calculated.

[0101] 3. Intestinal fecal flora detection

[0102] Using 16S rRNA amplicon sequencing technology, first, total microbial genomic DNA was extracted from each fecal sample using a DNA extraction kit. The V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR using bacterial universal primers. Then, the PCR amplification product was purified. Finally, sequencing was performed using the constructed sequencing library. After sequencing, the obtained sequences were quality controlled and filtered, and clustered into OTU representative sequences to generate an OTU table. After distinguishing the samples, OTU clustering analysis and species taxonomic analysis were performed. Based on the results of OTU clustering analysis, α diversity analysis, β diversity analysis, and species composition and correlation analysis were performed. The α diversity analysis calculated Good's coverage, Chao1, Observed species, shannon and simpson index for diversity index difference significance analysis. The β diversity analysis calculated principal component analysis (PCA) for the difference of bacterial community composition between different groups. The influence of the relative abundance of calf intestinal flora was drawn on the community column chart at the taxonomic door level, and the bacteria with an abundance of less than 1% in all samples were classified as others. The community column chart was drawn at the taxonomic genus level, and the bacteria with less than 1% in all samples were classified as others. The linear discriminant analysis (LDA) threshold was set to 3, and the LEfSe multi-level species difference discriminant analysis was performed from the door level to the genus level.

[0103] 4. Experimental results

[0104] 4.1 The effect of compound probiotics fermented traditional Chinese medicine preparation on growth performance of diarrhea calf

[0105] As shown in Table 8, compared with the control group (C group), the body weight and average daily gain of diarrhea group (M group) were reduced by 3.3 kg and 176.96 g (P<0.01), respectively, and the body length was reduced by 5.6 cm (P<0.05) at 14 d; compared with the diarrhea group (M group), the body weight and average daily gain of the positive treatment group (A group) were increased by 2.64 kg and 180.36 g (P<0.05), respectively, the body weight and average daily gain of the traditional Chinese medicine group (P group) were increased by 2.64 kg and 206.61 g (P<0.05), respectively, the body weight and average daily gain of the compound probiotics fermented traditional Chinese medicine preparation group (FP group) were increased by 2.87 kg and 183.33 g (P<0.05), respectively, the body length was increased by 6.82 cm (P<0.01), the body weight and average daily gain of the compound probiotics group (CP group) were increased by 3.44 kg and 205.71 g (P<0.05), respectively, the body length was increased by 7.5 cm (P<0.01), and the chest circumference was increased by 5.15 cm (P<0.05).

[0106] Table 8 The effect of probiotics fermented traditional Chinese medicine on growth performance of calf

[0107]

[0108] Note: The data is expressed as mean ± standard deviation. Compared with the control group (C group), **P<0.01, *P<0.05; compared with the diarrhea group (M group), ## P<0.01, # P<0.05.

[0109] 4.2 The effect of compound probiotics fermented traditional Chinese medicine preparation on intestinal inflammation of diarrhea calf

[0110] 4.2.1 The effect on intestinal TNF-α content

[0111] As shown in the detection results, compared with the C group, the TNF-α content of the M group was extremely significantly increased (P<0.01) at 0 d (A); Figure 3 compared with the C group, the TNF-α content of the M group was extremely significantly increased (P<0.01) at 7 d, and the TNF-α content of the FP and CP groups was significantly decreased (P<0.05), and the TNF-α content of the A group was extremely significantly decreased (P<0.05) (B); Figure 3 compared with the C group, the TNF-α content of the M group was extremely significantly increased (P<0.01) at 14 d, and the TNF-α content of the P, FP and CP groups was significantly decreased (P<0.05), and the TNF-α content of the A group was extremely significantly decreased (P<0.05) (C).Figure 3 M group was significantly higher than that of C group (P<0.01) (Fig. 4.2.2-1).

[0112] 4.2.2 Effect on IL-6 content in the intestine

[0113] The results showed that at 0 d, the IL-6 content of M group was significantly higher than that of C group (P<0.01) (Fig. 4.2.2-1). Figure 4 A group, P group, FP group and CP group were significantly lower than that of M group (P<0.01) (Fig. 4.2.2-2). Figure 4 B group) (Fig. 4.2.2-3). Figure 4 C).

[0114] 4.2.3 Effect on IL-1β content in the intestine

[0115] The results showed that at 0 d, the IL-1β content of M group was significantly higher than that of C group (P<0.01) (Fig. 4.2.3-1). Figure 5 A group was significantly lower than that of M group (P<0.01) (Fig. 4.2.3-2). Figure 5 B group) (Fig. 4.2.3-3). Figure 5 C).

[0116] 4.2.4 Effect on IL-10 content in the intestine

[0117] The results showed that at 0 d, the IL-10 content of M group was significantly lower than that of C group (P<0.01) (Fig. 4.2.4-1). Figure 6 A group was significantly higher than that of M group (P<0.01) (Fig. 4.2.4-2). Figure 6 B group) (Fig. 4.2.4-3). Figure 6 C).

[0118] 4.3 Effects of compound probiotic fermented traditional Chinese medicine preparations on the intestinal flora of diarrheal calves

[0119] 4.3.1 Impact on Alpha and Beta Diversity

[0120] Coverage was represented using the Good's coverage index, richness using the Chao1 and Observed species indices, and diversity using the Simpson and Shannon indices. The analysis results show that ( Figure 7 In the Good's coverage index, the sample coverage of each group reached 97%-99%, meeting the needs of sequencing. In the Chao1 index, Observed species index, Simpson index, and Shannon index, the diversity and abundance of gut microbiota in the diarrheal calf M group decreased. After treatment intervention, all indices in the A, P, and CP groups increased significantly (P<0.01), and all indices in the FP group also rebounded, corresponding to an increase in the abundance and diversity of gut microbiota. This indicates that the intervention of compound probiotic fermented traditional Chinese medicine preparation can significantly improve the abundance and diversity of gut microbiota in diarrheal calves. Principal Coordinates Analysis (PCoA) was performed on the gut microbiota of calves using the bray-curtis distance. The analysis results show that ( Figure 8 Diarrhea affects the beta diversity of gut microbiota. The gut microbiota community of group M is significantly different from that of other groups. At the same time, group M has a larger area within the 95% confidence interval and relatively discrete PCoA feature vectors within the group, suggesting that there is a large heterogeneity and variability in the gut microbiota of diarrheal calves.

[0121] 4.3.2 Effects on the gut microbiota structure of diarrheal calves

[0122] At the level of the door ( Figure 9), compared with group C, the relative abundance of Bacteroidetes in group M showed a downward trend, and the relative abundance of Actinobacteria, Proteobacteria and Fusobacteria showed an upward trend; compared with group M, the relative abundance of Bacteroidetes in group A showed an upward trend, and the relative abundance of Actinobacteria, Proteobacteria and Fusobacteria showed a downward trend, the relative abundance of Bacteroidetes in group P showed an upward trend, and the relative abundance of Actinobacteria, Proteobacteria and Fusobacteria showed a downward trend, the relative abundance of Bacteroidetes and Actinobacteria in group FP showed an upward trend, and the relative abundance of Proteobacteria and Fusobacteria showed a downward trend, the relative abundance of Bacteroidetes in group CP showed an upward trend, and the relative abundance of Actinobacteria, Proteobacteria and Fusobacteria showed a downward trend.

[0123] At the genus level ( Figure 10), compared with group C, the relative abundance of Blautia, Bacteroides, Prevotella, Bifidobacterium, Faecalibacterium decreased, and the relative abundance of Collinsella, Dorea increased in group M; after treatment with traditional Chinese medicine and probiotics, compared with group M, the relative abundance of Blautia, Lactobacillus, Prevotella, Faecalibacterium increased, and the relative abundance of Collinsella, Dorea decreased in group A, the relative abundance of Blautia, Bifidobacterium, Faecalibacterium increased, and the relative abundance of Collinsella, Dorea decreased in group P, the relative abundance of Prevotella, Bifidobacterium, Faecalibacterium increased, and the relative abundance of Collinsella, Dorea decreased in group FP, the relative abundance of Blautia, Lactobacillus, Bifidobacterium, Faecalibacterium increased, and the relative abundance of Collinsella, Dorea decreased in group FP, the relative abundance of Blautia, Lactobacillus, Bifidobacterium, Faecalibacterium increased, and the relative abundance of Collinsella, Dorea decreased in group CP.

[0124] 4.3.3 Effect on intestinal microbial composition of diarrhea calf

[0125] From Figures 11-13It can be seen that the difference analysis identified significant differences in Prevotella, Oscillospira, Parabacteroides, Collinsella, Dorea, Bifidobacterium in each group. Similarly, Clostridium, Dorea, Prevotella, Bifidobacterium were found in the top twenty random forest important genera, which played an important role in the intervention of complex probiotic fermented Chinese medicine preparations on diarrhea.

[0126] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A composite probiotic fermented traditional Chinese medicine preparation, characterized in that, The compound probiotic fermented traditional Chinese medicine preparation is obtained by inoculating Lactobacillus plantarum and Lactobacillus rhamnosus into a traditional Chinese medicine liquid of Bletilla striata to perform fermentation.

2. The composite probiotic fermented traditional Chinese medicine preparation according to claim 1, characterized in that, The Lactobacillus plantarum and the Lactobacillus rhamnosus are mixed at a volume ratio of (1-2):(1-2).

3. The composite probiotic fermented traditional Chinese medicine preparation according to claim 1, characterized in that, The fermentation time is 24-48 h.

4. A method for preparing the composite probiotic fermented traditional Chinese medicine preparation according to any one of claims 1-3, characterized in that, The method comprises the step of inoculating Lactobacillus plantarum and Lactobacillus rhamnosus mixed at a volume ratio of (1-2):(1-2) into a traditional Chinese medicine liquid of Bletilla striata to perform fermentation.

5. The production method according to claim 4, characterized by, The inoculation amount of the Lactobacillus plantarum and the Lactobacillus rhamnosus after mixing is 2-6%.

6. The preparation method according to claim 4, characterized in that, The mass concentration of the Bletilla striata in the traditional Chinese medicine liquid is 1%.

7. The preparation method according to claim 4, characterized in that, The fermentation temperature is 37℃.

8. Use of the compound probiotic fermented traditional Chinese medicine preparation according to any one of claims 1-3 in the preparation of a product for preventing and treating colibacillosis diarrhea of a calf.

9. A product for the control of colibacillosis in calves, characterized in that, The product comprises the compound probiotic fermented traditional Chinese medicine preparation according to any one of claims 1-3.

10. Use of the compound probiotic fermented traditional Chinese medicine preparation according to any one of claims 1-3 in the preparation of a medicine for improving the intestinal microecological environment of a diarrhea calf.

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