Animal bifidobacterium and use thereof

Abstract

The animal bifidobacterium of the present invention relates to the field of calf breeding technology. Animal Bifidobacterium (Bifidobacterium animalis) has been deposited in the China Center for Type Culture Collection (CCTCC) on April 26, 2023, with a deposit number of CCTCC M 2023641. The animal bifidobacterium of the present invention has been subjected to safety evaluations such as drug resistance gene detection, amino acid decarboxylase activity and indigo matrix detection (toxic metabolite detection), pathogenicity test, hemolytic activity detection, and acid resistance test, bile salt resistance test, artificial gastrointestinal fluid tolerance test, in vitro antibacterial test, surface property determination, cell adhesion test and other related tests. The results all meet the requirements of microbial feed additives.

Description

Technical Field

[0001] The present invention relates to the technical field of calf breeding, in particular to an animal bifidobacterium and a use thereof. Background Art

[0002] Calf indigestion, also known as dyspepsia diarrhea, is a general term for gastrointestinal digestive disorders in lactating calves. Its main clinical features are significant digestive dysfunction and varying degrees of diarrhea. Calf indigestion can be categorized into simple indigestion and toxic indigestion based on clinical manifestations and course of disease. Simple indigestion occurs primarily in newborn calves within 15 days of age and manifests as acute digestive and nutritional disorders and mild systemic symptoms. Newborn calves have weak nervous system regulation and an immature digestive system. Factors such as improper feeding and management, untimely or excessive feeding of colostrum or milk, poor quality milk or milk replacer, malnutrition, poor housing hygiene, and stress can easily lead to gastrointestinal dysfunction and intestinal flora disturbances, resulting in simple indigestion. If simple indigestion is not effectively treated, it can progress to toxic indigestion, presenting with systemic symptoms such as severe digestive dysfunction, obvious autointoxication, and severe diarrhea. Indigestion seriously affects the absorption of nutrients by newborn calves, leading to delayed development and decreased resistance of calves. It makes them more susceptible to secondary infection by pathogens such as Escherichia coli, rotavirus, coronavirus, Cryptosporidium, and causes infectious diarrhea. Newborn calves with indigestion are also prone to secondary rumen bloat, gastritis, abomasal obstruction and torsion, respiratory tract infections, etc. In severe cases, they may even die, causing serious economic losses to the dairy and beef cattle industries every year.

[0003] Currently, large-scale ranches primarily prevent and treat indigestion and diarrhea in newborn calves by strengthening neonatal feeding and management, and using antibiotics and traditional Chinese medicine. However, the preventative use of antibiotics carries the potential for antibiotic overuse and bacterial resistance. Furthermore, long-term antibiotic use disrupts the development of the intestinal flora and digestive system in newborn calves, potentially having long-term negative impacts on their growth, development, and production performance. Recent studies have shown that the gastrointestinal microbiome plays a vital role in maintaining gastrointestinal health in humans and animals, nutrient digestion and metabolism, and regulating host immune function. New microbial agents developed based on the gut microbiome with precise therapeutic or preventive properties have become a new approach for the prevention and treatment of diseases in humans and animals, and hold broad market potential. In the early stages of life, the gut microbiome promotes the development and maturation of the digestive and immune systems in calves, with long-term impacts on their health and production performance in adulthood.

[0004] Other studies have shown that the composition of intestinal flora is closely related to the health and daily weight gain of calves. Calves with diarrhea, pneumonia or low daily weight gain also have lower intestinal flora diversity, and the number of probiotic bacteria such as Bifidobacterium is significantly reduced. Giovana S. Slanzon et al. analyzed the changes in the diversity of intestinal flora in calves with intestinal diseases and diarrhea. The results showed that the abundance of Bifidobacterium longum decreased and the abundance of Escherichia coli increased. The above studies show that probiotics such as Bifidobacterium play an important role in maintaining the intestinal health of calves. In recent years, probiotics of different sources and types have been gradually applied to the prevention of diarrhea and indigestion in calves, including Bifidobacterium longum, Enterococcus faecium, Pediococcus acidilactici, Bacillus subtilis, etc. Clinical application studies have shown that the above probiotics can reduce the incidence of diarrhea in calves to varying degrees and improve diarrhea symptoms.

[0005] Clinical manifestations of calf indigestion include loss of appetite, sometimes leading to anorexia; decreased or increased stool volume; and dry, sticky stool or diarrhea. Currently, the main treatments for calf indigestion are Western medicine and some traditional Chinese medicines. However, long-term use of Western medicines can damage organs such as the liver. While traditional Chinese medicines have fewer side effects, their preparation and processing are complex and tedious, and effective traditional Chinese medicines require authentic, expensive ingredients. Early production of feed additives was plagued by the overuse of antibiotics and hormones. Long-term use of antibiotics or sulfonamides to prevent livestock and poultry diseases or digestive disorders such as indigestion can lead to drug resistance in pathogens in the livestock intestine, disrupting gastrointestinal homeostasis. Although beneficial bacteria exist in the calf's intestine, isolated probiotics cannot be directly cultivated and used as feed additives. The large-scale use of probiotics containing heritable resistance genes for livestock farming could lead to the transfer of resistance genes to pathogens, causing drug resistance in diseased animals and reducing cure rates.

[0006] Currently, my country's Ministry of Agriculture has specified 34 beneficial microbial strains as acceptable feed additives. These beneficial bacteria are used in different ways for animal nutrition, depending on their distinct physiological and biochemical properties. In recent years, with the continuous advancement of microbiome and culturomics technologies, a growing number of new functional probiotics have been used in the prevention and treatment of human and animal diseases. According to the joint report of the Food and Agriculture Organization of the United Nations / World Health Organization (FAO / WHO) on the evaluation of probiotics in food, commercial probiotic strains must undergo in vitro and in vivo testing to assess their probiotic properties, including gastric acid tolerance, bile acid tolerance, adhesion and / or human epithelial cell adhesion, antagonism against opportunistic pathogens, ability to reduce pathogenic adhesion to the gastrointestinal mucosa, bile salt hydrolase activity, and spermicide resistance (for vaginal probiotics). Furthermore, candidate strains must undergo safety assessments, including hemolytic activity, toxic metabolites, drug resistance genes, drug susceptibility, pathogenicity, and cell and animal studies.

[0007] At present, the probiotics used as microbial preparations for preventing and treating calf indigestion are still very limited and cannot meet market demand. Summary of the Invention

[0008] In view of this, it is necessary to provide an animal Bifidobacterium that meets the requirements of food probiotics.

[0009] An animal Bifidobacterium (Bifidobacterium animalis) is deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number: CCTCC M 2023641 and the deposit date: April 26, 2023.

[0010] The animal bifidobacterium with the deposit number of CCTCC M 2023641 is used in a microbial preparation for calves.

[0011] The animal bifidobacterium with the deposit number of CCTCC M 2023641 is used in a microbial pharmaceutical preparation for preventing and treating calf indigestion.

[0012] The animal bifidobacterium with the deposit number of CCTCC M 2023641 is used as a microbial feed additive for calves.

[0013] Beneficial effects: The animal bifidobacterium of the present invention has been subjected to safety evaluations such as drug resistance gene detection, amino acid decarboxylase activity and indigo matrix detection (toxic metabolite detection), pathogenicity test, hemolytic activity detection, as well as acid resistance test, bile salt resistance test, artificial gastrointestinal fluid tolerance test, in vitro antibacterial test, surface property determination, cell adhesion test and other related tests, and the results all meet the requirements of microbial feed additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a phylogenetic evolutionary tree constructed based on the rRNA gene sequence of the test strain 64-N1 of the present invention. Figure 2 This is a comparison chart of the 64-N1 hemolytic test of the present invention, wherein A represents the positive control in which no hemolytic ring appears around the colony of the test strain, indicating no hemolysis. B represents the positive control in which a hemolytic ring appears around the colony of Enterococcus ATCC29212, indicating hemolysis.

[0015] Figure 3 These are the results of organ pathological sections of the strain of the present invention with acute oral toxicity; wherein, A, B, C, and D are respectively the results of pathological sections of the heart, liver, spleen, and kidney of mice gavaged with a high-concentration bacterial suspension of the strain; and E, F, G, and H are respectively the results of pathological sections of the heart, liver, spleen, and kidney of mice in the blank control group gavaged with sterile saline.

[0016] Figure 4 A picture of 64-N1 adhering to human colorectal cancer cells (Caco-2). DETAILED DESCRIPTION

[0017] The invention will be described in detail below with reference to specific examples. Unless otherwise specified, the test methods used in the examples are conventional methods.

[0018] The English and Chinese meanings of the various English abbreviations appearing in the present invention are shown in Table 1. For the convenience of statistics and description, the relevant words in the experiment will be replaced by English abbreviations.

[0019]

[0020]

[0021] The strains of the present invention are isolated from the milk of healthy Holstein cows and the fresh feces of healthy Holstein calves during the lactation period. In addition to animal Bifidobacterium 64-N1, other strains of probiotics isolated from the milk of healthy Holstein cows and the fresh feces of healthy Holstein calves during the lactation period are also present. However, these other strains did not meet the standards during the evaluation process for food probiotics and have been eliminated. Furthermore, since these eliminated strains have no practical value, it is not necessary to spend time and money on gene sequence testing. Furthermore, since these strains have differences in their gene sequences, they are likely not present in other cows. Comparing the strains of the present invention with these eliminated strains is meaningless. Subsequent experimental data are all experimental data for animal Bifidobacterium 64-N1 of the present invention. These experimental data are used to demonstrate that animal Bifidobacterium 64-N1 of the present invention meets the product requirements for food probiotics.

[0022] The above strains were isolated from the milk of healthy Holstein cows and fresh feces of healthy Holstein calves during lactation. Except for 64-N1, the other strains were used as control strains. 64-N1 is the strain to be protected by the present invention. The evolutionary tree of 64-N1 is as follows: Figure 1 As shown, the screened 64-N1 is Bifidobacterium animalis. 64-N1 has been deposited in the China Center for Type Culture Collection (CCTCC) on April 26, 2023, with the deposit number: CCTCC M2023641.

[0023] In addition, the bacterial strain ATCC15707 used in this article is Bifidobacterium longum, purchased from Beijing Beina Lianchuang Biotechnology Co., Ltd. Escherichia coli ATCC8739, Staphylococcus aureus ATCC33591, and Salmonella typhimurium ATCC14028 used in the experiments were purchased from the National Institute for the Authentication of Pharmaceutical and Biological Products. Human colorectal adenocarcinoma cells (Caco-2) were obtained from the College of Life Sciences, Ningxia University.

[0024] The main culture media required for the experiment are shown in Table 2.

[0025] Table 2: Culture media and their sources

[0026]

[0027]

[0028] The animal Bifidobacterium used in the experiment of the present invention was obtained by the following method: The screened animal Bifidobacterium was inoculated on a modified MRS agar plate and cultured anaerobically at 37°C for 48 hours, after which a single colony was picked and inoculated into fresh sterile MRS broth medium for anaerobically culture.

[0029] Modified MRS broth was prepared as follows: 10.0 g of peptone, 5.0 g of beef extract powder, 4.0 g of yeast extract powder, 20.0 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 2.0 g of triammonium citrate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 15.0 g of agar, and 1.0 g of Tween 80 were added to distilled water, the volume was adjusted to 1000 mL, the pH was adjusted to 6.2 ± 0.2, and the medium was sterilized by autoclaving at 121°C for 15–20 min. Filtered horse serum (50 mL / L), 0.5 g / L cysteine ​​hydrochloride solution, and mupirocin (50 mg / L) were then added to the MRS agar medium.

[0030] The following is the specific implementation process of the safety testing of Bifidobacterium animalis 64-N1.

[0031] Detection of transferable drug resistance genes in Bifidobacterium animalis 64-N1:

[0032] The drug-resistant genes of Bifidobacterium mainly include tetracycline resistance genes tet(W), tet(M), tet(S), tet(Q), tet(O), tet(W), tet(L), tet(P), ciprofloxacin resistance gene gyrA, archaemycin resistance gene vanX, kanamycin resistance gene aph(3“)-III., trimethoprim resistance gene dfrD, rifampicin resistance gene rpoB, clindamycin resistance gene lsa, aminoglycoside resistance genes aac(3), aph(3), aph(3′)-III, aadA, aadE, ant(6), ant(9), chloramphenicol resistance genes cat, cmlA and erythromycin resistance genes erm(B), mef, mef(E), mef(B), and β-lactam resistance genes blaCTX-M, blaSHV, blaTEM, blaZ, blaOXA-1, blaOXA-48.

[0033] Among them, the transferable drug-resistant genes that can be confirmed at present include tet(W), tet(M), aphA-1, aphA-2, aadA, addE, gyrA, aac6'-lb-cr, aac(6')-aph(2"), erm(B), str(A), str(B), sul2, and sul3 drug-resistant genes. After testing the animal Bifidobacterium 64-N1 of the present invention, no such transferable drug-resistant genes were found.

[0034] At the same time, tetracycline resistance genes will be widely spread through the sex pheromone-mediated coupling system. The present invention detected the sex pheromone virulence genes cpd, cob, and ccf of animal Bifidobacterium 64-N1, and the results showed that it did not contain sex pheromone virulence genes, that is, there was no transmission route for tetracycline resistance genes.

[0035] Minimum inhibitory concentration (MIC) testing

[0036] The drug sensitivity of Bifidobacterium animalis 64-N1, a quality control strain, was determined by the broth microdilution method. The MIC values ​​of Bifidobacterium animalis ATCC15707 against 13 antimicrobial drugs were all within the quality control range, and the test results are reliable. The 13 antimicrobial drugs are penicillin (100 mg), ampicillin (100 mg), ceftiofur (100 mg), gentamicin (100 mg), kanamycin (100 mg), neomycin (100 mg), streptomycin (150 mg), tetracycline hydrochloride (100 mg), erythromycin (200 mg), lincomycin (100 mg), florfenicol (100 mg), ciprofloxacin (100 mg), and sulfamethoxazole (100 mg). All of the above drugs were purchased from the China Veterinary Drug Inspection Institute.

[0037] The experimental results showed that animal Bifidobacterium 64-N1 was sensitive to all 13 drugs.

[0038] Hemolytic activity test of Bifidobacterium animalis 64-N1.

[0039] The activated suspension of Bifidobacterium animalis 64-N1 was streaked onto Columbia medium and incubated anaerobically at 37°C for 72 hours. The plate was then observed for α-hemolysis or β-hemolysis around the colonies. Enterococcus faecalis ATCC29212 was used as a positive control.

[0040] like Figure 2 As shown, Bifidobacterium animalis 64-N1 is not hemolytic.

[0041] Detection of toxic metabolites of Bifidobacterium animalis 64-N1, namely, amino acid decarboxylase activity detection and indigo substrate detection:

[0042] a: Amino acid decarboxylase activity assay

[0043] Select a single colony cultured on modified MRS agar for 72 hours and inoculate it into test tubes of ornithine, arginine, and lysine aminodecarboxylase broth, as well as a control tube. Cover the surface of the culture medium with sterile liquid paraffin and incubate anaerobically at 37°C for 18–24 hours. If the control tube is yellow, the test tube is purple, indicating a positive result; if the test tube is yellow, it is negative.

[0044] b: Indigo matrix detection

[0045] Inoculate a single colony into a peptone (tryptophan broth) test tube and incubate at 37°C for 24-48 hours. First, add an appropriate amount of xylene to the culture medium and shake thoroughly to extract the indole produced by the bacterial growth metabolism. Then, add 2-3 drops of Kovacs indigo reagent and let it stand. If the upper layer appears red after the liquid is separated, it is positive; otherwise, it is negative.

[0046] As shown in Table 3, the results of the amino acid decarboxylase activity assay and the indole substrate assay indicated that Bifidobacterium animalis 64-N1 did not have ornithine, lysine, or arginine aminodecarboxylase activity and did not produce the toxic substance indole.

[0047] Table 3 Results of amino acid decarboxylase activity and indigo matrix assay in animal Bifidobacterium 64-N1

[0048]

[0049] Pathogenicity test

[0050] Healthy adult SPF-grade ICR mice (half male and half female, weighing 18.0-22.0 g) were administered Bifidobacterium animalis 64-N1 culture fluid via intraperitoneal injection and oral gavage to evaluate the pathogenicity of the test substance at different exposure routes. The methods and dosages were based on the "Technical Guidelines for Safety Testing and Evaluation of Bacteria Used in Health Food Ingredients (2020 Edition)."

[0051] The implementation process of intraperitoneal injection is as follows:

[0052] S1: Strain activation and bacterial suspension preparation

[0053] The purified bifidobacterium colony was thoroughly mixed with sterile saline, and the concentration of the bacterial suspension was adjusted with an appropriate amount of sterile saline to make the final bacterial concentration reach 5.0×10 7 CFU / mL was used for intraperitoneal injection in mice.

[0054] S2: intraperitoneal injection

[0055] Thirty-two mice, half male and half female, were randomly divided into four groups: two groups of female mice and two groups of male mice, with eight mice in each group. After one week of adaptive feeding, each mouse was injected with 0.2 mL of bacterial suspension or saline. The four groups of mice were: a female mouse sterile saline control group, a male mouse sterile saline control group, a 64-N1 female mouse bacterial suspension group, and a 64-N1 male mouse bacterial suspension group. The amount of bacteria injected into each mouse in the 64-N1 female mouse bacterial suspension group and the 64-N1 male mouse bacterial suspension group was no less than 1.0 × 10 7 CFU.

[0056] S3: Observation

[0057] After intraperitoneal injection, observe the animals once daily for 21 consecutive days. Observe and record any abnormalities in the mice's skin and fur, eyes and mucous membranes, respiratory status, limb movements, and behavior. Observe the mice for tremors, convulsions, diarrhea, lethargy, salivation, and coma. Weigh and record the weight of all mice before the experiment and after the experiment. For mice that die during the experiment, record the time of death as accurately as possible and weigh and record the weight.

[0058] The effects of intraperitoneal injection on the body weight of mice are shown in Tables 4 and 5.

[0059] Table 4 Effects of intraperitoneal injection of Bifidobacterium 64-N1 culture on body weight of female mice

[0060]

[0061] Table 5 Effects of intraperitoneal injection of Bifidobacterium 64-N1 culture on body weight in male mice

[0062]

[0063] During the experiment, there was no significant difference in the initial and final body weights of the mice between the bacterial suspension group and the normal saline control group (P>0.05), and no obvious activity or behavioral changes, illness or death were observed in the experimental mice.

[0064] S4: Collection and testing of mouse blood samples

[0065] After the experiment, two mice were randomly sampled from the experimental group and the blank group. The sample numbers were marked on PCR tubes containing EDTA-2K for later use. Blood was collected from the tail vein using a blood sampler into a PCR tube containing an anticoagulant (EDTA-2Na). The collected blood was placed in an ice box and stored. After returning to the laboratory, a blood test instrument was used to analyze the blood. The results are shown in Table 6.

[0066] Table 6 Blood routine of mice injected intraperitoneally

[0067]

[0068] There were no significant differences in the blood routine tests of mice in the bacterial suspension test group and the sterile saline control group (P>0.05).

[0069] Body weight and blood test results indicate that intraperitoneal injection of Bifidobacterium 64-N1 is non-pathogenic. The oral gavage procedure is as follows:

[0070] S1: Strain activation and bacterial suspension preparation

[0071] The purified Bifidobacterium single colony was thoroughly mixed with sterile saline, and the concentration of the bacterial suspension was adjusted with an appropriate amount of sterile saline to make the final bacterial concentration reach 1×10 10 CFU / mL, 1×10 8 CFU / mL was used for oral gavage of mice.

[0072] S2: Oral gavage

[0073] Forty-eight mice, half of them female, half male, were randomly divided into six groups, three groups of female mice and three groups of male mice, with eight mice in each group. After one week of adaptive feeding, the six groups of mice were gavaged with physiological saline, high concentration bacterial suspension (1×10 10 CFU / mL), low concentration bacterial suspension (1×10 8 CFU / mL), with an oral gavage dose of 0.2 mL / mouse, once daily for 14 consecutive days. Six groups of mice were administered: a sterile saline control group for female mice, a sterile saline control group for male mice, a low-dose 64-N1 bacterial suspension group for female mice, a high-dose 64-N1 bacterial suspension group for female mice, a low-dose 64-N1 bacterial suspension group for male mice, and a high-dose 64-N1 bacterial suspension group for male mice. The mice were observed daily for activity, signs of poisoning, and weight changes.

[0074] S3: Observation

[0075] After oral gavage, the animals were observed once daily for 21 consecutive days. Abnormalities in the skin and fur, eyes and mucous membranes, respiratory status, limb movements, and behavior were observed and recorded. The mice were observed for tremors, convulsions, diarrhea, lethargy, salivation, and coma. All mice were weighed and recorded before the experiment and after the experiment. For mice that died during the experiment, the time of death was recorded as accurately as possible and the weight was also weighed and recorded.

[0076] The effects of oral administration on the body weight of mice are shown in Tables 7 and 8.

[0077] Table 7 Effects of oral administration on the body weight of female mice

[0078]

[0079] Table 8 Effects of oral administration of Bifidobacterium animalis 64-N1 culture on body weight of male mice

[0080]

[0081] During the experimental period, there was no significant difference in the initial body weight of female mice between the bacterial suspension group and the saline control group (P>0.05). However, a comparison of the final body weight between the bacterial suspension group and the saline control group showed a significant increase in the body weight of female 64-N1 mice in the high-dose bacterial suspension group (P<0.05). During the experimental period, there was no significant difference in the initial body weight of male mice between the bacterial suspension group and the saline control group (P>0.05). A comparison of the final body weight between the bacterial suspension group and the saline control group showed a highly significant increase in the body weight of male 64-N1 mice in the high-dose and low-dose bacterial suspension groups (P<0.01). No significant changes in activity or behavior, illness, or death were observed in the experimental mice.

[0082] S4: Blood routine index test

[0083] Blood was collected from the mice using the tail vein blood collection method into a PCR tube containing an anticoagulant, stored in an ice box and brought back to the laboratory for hematological parameters to be measured using an automatic blood cell analyzer. For organ sample collection, the heart, liver, spleen, and kidney of the mice in the bacterial suspension group and the normal saline control group were quickly removed using sterile tweezers and placed in a 50 mL sterile centrifuge tube. They were fixed with paraformaldehyde solution (4% PFA) and stored at room temperature. The collected organ samples were sent to a biopsy company for preparation of pathological sections, such as Figure 3 The test results are shown in Table 9.

[0084] Table 9 Blood routine of mice gavaged with Bifidobacterium animalis 64-N1 culture

[0085]

[0086] There were no significant differences in the blood routine tests of mice in the bacterial suspension test group and the sterile saline control group (P>0.05).

[0087] Body weight and blood test experiments showed that oral administration of Bifidobacterium 64-N1 was not pathogenic.

[0088] In summary, the intraperitoneal injection and gavage experiments indicate that the Bifidobacterium 64-N1 of the present invention is not pathogenic.

[0089] Evaluation of bacterial translocation ability

[0090] Analyze bacterial translocation in mouse blood and organ tissues (heart, liver, spleen, lung, and kidney). Spread 50 μL of mouse blood onto modified MRS plates and incubate anaerobically at 37°C for 72 hours. Count the number of colonies on the plates. Take tissue samples, homogenize them at 1 μg / mL in sterile saline, and spread 100 μL onto modified MRS plates. Incubate anaerobically at 37°C for 72 hours. Count the number of colonies on the plates. Any growth is considered positive.

[0091] After culturing the blood and organ tissues of mice in the control group and the experimental group, no colonies grew on the culture medium, indicating that animal Bifidobacterium 64-N1 did not transfer from the intestine to the blood and organ tissues, and no translocation occurred.

[0092] Furthermore, the present invention also conducted tolerance tests on animal Bifidobacterium 64-N1 through Test 1, Test 2, and Test 3.

[0093] Test 1: Acid resistance test

[0094] The tolerance of bifidobacteria to acid was determined by plate colony counting method. Ordinary modified MRS culture medium was used as a control, and the pH of the modified MRS liquid culture medium was adjusted with 1 mol / L hydrochloric acid to 2.0, 3.0, 4.0, and 5.0, respectively. The bacterial solution was adjusted to OD600nm of 1±0.05 and then inoculated into modified MRS liquid culture medium of different pH values ​​at 2% inoculation amount, cultured at 37°C, and the number of viable bifidobacteria in each group of culture medium was determined at 0h, 2h, and 4h, respectively, with 3 parallel replicates set for each group. The test results are shown in Table 10, indicating that the strain still has growth activity at pH 4, indicating that the acid resistance of 64-N1 of the present invention is good.

[0095] Table 10: Acid resistance test results of strain 64-N1

[0096]

[0097] Test 2: Bile salt tolerance test

[0098] The tolerance of bifidobacteria to bile salts was determined by plate colony count. Using standard modified MRS medium as a control, modified MRS medium containing 0.1 g / L, 0.2 g / L, and 0.3 g / L ox bile salts was added. The bacterial suspension was adjusted to an OD600 nm of 1 ± 0.05 and inoculated at a 2% inoculum into 0.1%, 0.2%, and 0.3% bile salt-modified MRS medium, respectively. The cultures were incubated at 37°C. The viable bifidobacteria count in each medium was determined at 0, 2, and 4 hours, with three replicates per group. Survival rate = N1 / N0 × 100%. N1: viable cell count after 2 or 4 hours of bile salt treatment; N0: viable cell count at 0 hours. The results of the bile salt tolerance test of strain 64-N1 are shown in Table 11. The test results show that animal Bifidobacterium 64-N1 can grow and reproduce in culture media with bile salt concentrations of 0.1% and 0.2%, and its survival rate is low in culture media with 0.3% bile salt, that is, the animal Bifidobacterium of the present invention has a certain tolerance to bile salts.

[0099] Table 11: Bile salt tolerance test of strain 64-N1

[0100]

[0101] Experiment 3: Artificial gastrointestinal fluid tolerance test

[0102] Preparation of artificial gastric fluid: In 100 mL of simulated gastric fluid, add 0.32 g of pepsin and 0.2 g of NaCl, adjust the pH to 2.5 with HCl, and filter with a 0.22 μm sterile filter membrane.

[0103] Preparation of artificial intestinal fluid: In 100 mL of simulated intestinal fluid, add 1.0 g of trypsin, 0.68 g of potassium dihydrogen phosphate, and adjust the pH to 7.5 ± 0.1 with NaOH, and filter with a 0.22 μm sterile filter membrane.

[0104] Determination of tolerance to artificial gastric juice: The plate colony counting method was used to determine the tolerance of bifidobacteria to artificial gastric juice. The activated bacterial solution was centrifuged at 5000r / min and 4℃ for 5min, the supernatant was discarded, and the bifidobacteria were resuspended with sterile PBS and repeated three times. The bacterial solution was adjusted to OD600nm of 1±0.05 and inoculated into artificial gastric juice at a 2% inoculation volume and mixed. The culture was cultured at 37℃, and the number of live bifidobacteria in the culture medium of each group was determined at 0h, 2h and 4h, respectively. Three parallel replicates were set for each group. Survival rate = N1 / N0×100%. N1: The number of live bacteria after 2h or 4h of artificial gastric juice treatment; N0: The number of live bacteria at 0h;

[0105] Determination of tolerance to artificial intestinal fluid: The tolerance of bifidobacteria to artificial intestinal fluid was determined by plate colony counting method. The activated bacterial solution was centrifuged at 5000r / min and 4℃ for 5min, the supernatant was discarded, and the bifidobacteria were resuspended with sterile PBS and repeated three times. The bacterial solution was adjusted to OD600nm of 1±0.05 and inoculated into artificial gastric fluid at a 2% inoculation volume and mixed. The culture was cultured at 37℃, and the number of viable bifidobacteria in the culture medium of each group was determined at 0h, 2h and 4h, respectively. Three parallel replicates were set for each group. Survival rate = N1 / N0×100% N1: number of viable bacteria after 2h or 4h treatment with artificial intestinal fluid; N0: number of viable bacteria at 0h;

[0106] The gastrointestinal tolerance results of strain 64-N1 are shown in Table 12. After 2 hours of treatment with simulated gastric fluid, the survival rate of strain 64-N1 was 2.11±0.57%. After 4 hours of treatment with simulated intestinal fluid, the survival rate was 176.10±2.36%. It can be seen that the bacteria can tolerate a certain gastrointestinal environment.

[0107] Table 12: Gastrointestinal tolerance test of strain 64-N1

[0108]

[0109] The gastrointestinal tolerance test showed that the strain 64-N1 of the present invention was intolerant and met the requirements.

[0110] Furthermore, the present invention also conducted other tests to meet the requirements of microbial feed additives.

[0111] In vitro antibacterial test

[0112] Preparation of fermentation supernatant: Pick a single colony and inoculate it into modified MRS liquid medium and culture it for 72 hours. Centrifuge the bacterial liquid at 12000 r / min for 1 minute, take the supernatant, filter it with a 0.22 μm sterile filter membrane and set aside.

[0113] Antibacterial test: Pour 20 mL of autoclaved TSA medium into a 10 cm diameter plate, dilute overnight cultured Escherichia coli ATCC8739, Staphylococcus aureus ATCC33591, and Salmonella enterica serovar Typhimurium ATCC14028 to 10 6 CFU / mL, use a cotton swab to spread the bacterial solution on the agar surface, and after it is completely absorbed, place 3 Oxford cups on each dish, add 200 μL of the supernatant of the bifidobacterium to be tested into the Oxford cups, place them in a 4°C refrigerator for 2 hours, and then transfer them to a 37°C incubator for incubation for 24 hours, and then measure the diameter of the inhibition zone with a vernier caliper.

[0114] Table: 13 Results of in vitro antibacterial test of strain 64-N1

[0115]

[0116] Surface activity assay

[0117] Hydrophobicity determination: Overnight bacterial cultures were centrifuged, washed twice with PBS, and the OD600nm value was adjusted to 1±0.02, recorded as A0. 2 mL of chloroform was added to equal volumes of the bacterial suspension and vortexed for 1 min. The suspensions were incubated at 37°C for 2 h. After separation of the two phases, the aqueous phase was collected and its absorbance at OD600nm was measured and recorded as A2. The experiment was repeated three times, and the hydrophobicity of each strain was calculated. The mean ± standard deviation of the hydrophobicity values ​​of the three experiments was calculated. The hydrophobicity (%) of the strains was expressed as: (A0-A2) / A0×100%. The results are shown in Table 14.

[0118] Determination of autoaggregation rate: Centrifuge the overnight culture, wash twice with PBS, and adjust the OD600nm to 1±0.02, recording it as A0. A 4 mL suspension was incubated at 37°C for 24 hours. The absorbance of the culture supernatant was measured at OD600nm and recorded as A1. Three replicates were performed, and the autoaggregation rate of each strain was calculated. The mean ± standard deviation of the autoaggregation rates of the three experiments was calculated. The autoaggregation rate (%) of each strain was expressed as: (A0-A1) / A0×100%. The results are shown in Table 14.

[0119] Table 14: Surface property determination results of Bifidobacterium animalis 64-N1

[0120] strain number Hydrophobic chloroform (%) Autopolymerization rate (%) 64-N1 66.86±1.85％ 3.93±1.14％

[0121] Cell adhesion assay

[0122] Cell culture: After Caco-2 cells have recovered, resuspend them in 10% fetal bovine serum medium and inoculate them into cell culture flasks. Place the cells in a 37°C, 5% CO2 cell culture incubator. Once the cells have grown to the bottom of the flask, perform a 1:3 passage. After the cells have been passaged for at least five generations, perform the following experiments.

[0123] Cell adhesion and observation test: Pour off the cell culture medium, co-culture the adherent cells with the bacterial suspension for 4 hours, then pour off the bacterial suspension, wash off the non-adherent bifidobacteria with PBS buffer, remove the cell slide, fix it with cell fixative for 15 minutes, rinse it with PBS 2-3 times, and then perform Gram staining on the cells. Observe and take pictures with an oil immersion lens. The results are as follows: Figure 4 As shown, 20 fields of view were randomly selected under the microscope, and the number of bifidobacteria adhering to each cell was counted and recorded. The results were expressed as mean ± standard deviation. The adhesion rate of animal Bifidobacterium 64-N1 is shown in Table 15.

[0124] Table: 15 Adhesion rate of animal Bifidobacterium 64-N1

[0125] strain number Adhesion rate (%) 64-N1 147.4±59.90％

[0126] The above test results show that the animal Bifidobacterium 64-N1 of the present invention can be used as a microbial feed additive for calves.

[0127] In order to verify the effect of animal Bifidobacterium 64-N1 in preventing and treating calf indigestion and diarrhea, the present invention uses animal Bifidobacterium 64-N1 freeze-dried powder for the prevention of calf indigestion and diarrhea. Animal Bifidobacterium 64-N1 is deposited in the China Center for Type Culture Collection (CCTCC), with the deposit number: CCTCC M2023641, and the deposit date is April 26, 2023. The experimental group (T group) consists of 15 newborn calves, each of which is fed with a bacterial content of 1×10 93-5 g of animal Bifidobacterium 64-N1 freeze-dried powder (CFU / g) was fed daily to each calf between 16 and 35 days of age at a concentration of 1 × 109 CFU / g. A control group (Group C) consisted of 15 newborn calves not fed with animal Bifidobacterium 64-N1 freeze-dried powder. The incidence of dyspepsia and diarrhea, as well as fecal scores, was recorded. Blood samples were collected and analyzed for immune-related factors and oxidative stress markers. The effects of animal Bifidobacterium 64-N1 on calf health and blood biochemical parameters were compared. Results showed that only two calves in Group T developed dyspepsia between 1 and 15 days of age, which resolved after 3-5 days of continuous feeding. The remaining calves in Group T, fed with Animal Bifidobacterium 64-N, did not develop diarrhea or other illnesses, nor did any calves die, within 35 days of age. In Group C, seven calves developed indigestion between days 1 and 15, and ten developed diarrhea between days 16 and 35, three of whom died. As shown in Table 16, the pro-inflammatory cytokines IL-1β and TNF-α in the blood of calves in Group T were significantly lower than those in Group C from days 7 to 27 and from days 7 to 14, respectively (P < 0.05), while the anti-inflammatory cytokine IL-10 was significantly higher than that in Group C (P < 0.05). The antioxidant stress markers GSH-Px and SOD in the blood of calves in Group T were significantly higher than those in Group C from days 7 to 27 (P < 0.05). The levels of antibodies IgA and IgG in the blood of calves in Group T were significantly higher than those in Group C from days 7 to 27 and from days 7 to 14, respectively (P < 0.05). The above research results show that feeding Bifidobacterium animalis 64-N1 can significantly reduce the incidence of indigestion and diarrhea in calves, and significantly increase the levels of anti-inflammatory cytokines, anti-oxidative stress, and antibodies in the blood of calves during the early lactation stage. Therefore, Bifidobacterium animalis 64-N1 of the present invention can be used as a probiotic to prevent and treat indigestion in calves.

[0128] Table 16 Effects of Bifidobacterium 64-N1 on calf serum immune factors and oxidative stress indicators

[0129]

[0130] In order to verify the effect of animal Bifidobacterium 64-N1 in preventing and treating calf indigestion, the present invention combines animal Bifidobacterium 64-N1 with freeze-dried powder of laboratory-isolated Bifidobacterium P4 in a mass ratio of 1:1 to form a composite probiotic preparation, which is applied to the prevention and treatment of calf indigestion. Animal Bifidobacterium 64-N1 is deposited with the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC M 2023641 and a deposit date of April 26, 2023. Bifidobacterium P4 was deposited with the China Center for Type Culture Collection (CCTCC) on April 26, 2023, with a deposit number of CCTCC M 2023640. The results, shown in Table 17, were obtained from a large-scale dairy farm in Ningxia. Fifteen healthy calves aged 12 to 28 days and 15 calves with indigestion were fed a combination of 1.75 to 2.25 grams of the compound probiotics with each meal for seven consecutive days. The calves were observed for 21 consecutive days. Abnormalities in the calves' skin and fur, eyes and mucous membranes, respiratory status, limb movements, and behavior were observed and recorded. Particular attention was paid to observing for tremors, convulsions, diarrhea, lethargy, salivation, and coma. On the second day of feeding, indigestion symptoms (loss of appetite, thick yellow or transparent stools, and diarrhea) were significantly alleviated in the indigestible calves. After three to five days of continuous feeding, the calves were essentially cured. No animals died during the feeding period. The animals showed increased appetite, normal stools, no diarrhea, and no food-grabbing behavior. The cure rate reached 99%, with one calf dying from severe dehydration. Healthy calves do not have indigestion phenomena, and there are no abnormalities in the calf's skin and hair, eyes and mucous membranes, respiratory conditions, limb movements, behavioral patterns, etc., and there are no phenomena such as tremor, convulsion, diarrhea, lethargy, salivation and coma. In the above-mentioned feeding test, the probiotic preparation is effective in curing calf indigestion, and there are no phenomena such as relapse of the disease in recovered animals. In addition, the above-mentioned composite probiotic preparation is initially applied to calves with diarrhea, and all have the effect of alleviating and curing diarrhea. Therefore, animal Bifidobacterium 64-N1 of the present invention can be used as a composite probiotic preparation composite bacterial strain to be applied to the prevention and treatment of calf indigestion disease.

[0131] Table 17 Effect of Bifidobacterium 64-N1 in preventing and treating indigestion in calves.

[0132]

[0133] The above disclosure is only a preferred embodiment of the present invention, and it is certainly not intended to limit the scope of the present invention. A person skilled in the art can understand that all or part of the processes of the above embodiment and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A strain of Bifidobacterium animalis, deposited with the China Center for Type Culture Collection (CCTCC), with accession number: CCTCC M 2023641, and a deposit date of April 26, 2023.

2. Use of the animal bifidobacterium according to claim 1 in the preparation of a microbial preparation for calves.

3. Use of the animal bifidobacterium according to claim 1 in the preparation of a microbial pharmaceutical preparation for preventing and treating calf indigestion.

4. Use of the animal bifidobacterium according to claim 1 in the preparation of a microbial feed additive for calves.

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