Bifidobacterium and application thereof

Abstract

The invention discloses a bifidobacterium strain and application thereof, and the bifidobacterium strain is separated from Bifidobacterium jiwang TF04 of mare milk yoghurt, the classification name of the bifidobacterium jiwang TF04 is Bifidobacterium jiwang TF04, the bifidobacterium strain is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No 32629. The bacterium has the characteristics of acid resistance, cholate resistance, broad-spectrum bacteriostasis, planting capacity and self-aggregation. (1) the method can be applied to preparation of a bacteriostatic agent; and (2) the beverage is also applied to preparation of the beverage and is beneficial to improvement of diarrhea, ozostomia, old bowel disease and the like after being taken, and the beverage can be obtained by fermentation of a JI liquid culture medium. And (3) the feed additive can also be applied to feed addition, and can improve the health level of animals, promote growth, reduce antibiotics and save cost. The preparation method is characterized by comprising the following steps: inoculating the TF04 into a JW solid-state fermentation culture medium, carrying out closed fermentation, drying, and adding the TF04 into a feed for application.

Description

Technical Field

[0001] The invention discloses a bifidobacterium and application thereof. Tianfu Bifidobacterium Jiwang TF04 is preserved in China General Microbiological Culture Collection Center with a preservation number of CGMCC No 32629. The bacterium has broad-spectrum antibacterial activity, nutritional function and intestinal regulation function, can be applied to the preparation of beverages, feeds and antibacterial agents, and belongs to the field of applied microbial technology. Technical Background

[0002] According to previous research results, the nutritional factors, bacteriocins, organic acids (such as lactic acid, acetic acid and butyric acid) and antibacterial substances such as hydrogen peroxide produced by probiotics during the fermentation process can not only nourish the human body, but also be used to inhibit pathogenic bacteria, inhibit common spoilage bacteria and pathogens in fruits, vegetables and cold fresh meat. [1-2] The physical and chemical methods can be used to select and breed Lactobacillus acidophilus NX2-6 to produce antibacterial agents. [3] Lactic acid bacteria form a competitive relationship with harmful microorganisms. In the process of colonizing mucosal epithelial cells, the number of harmful microorganisms is reduced through competition, reducing the occurrence of diseases. [4-5] Lactobacillus casei can produce extracellular polysaccharides to inhibit the adhesion of Staphylococcus aureus and thus achieve antibacterial effects [6] .

[0003] Probiotics and antibacterial agents can be applied to foods such as fruits and vegetables, fresh meat, aquatic products, dairy products, etc. In addition, probiotics and antibacterial agents are also used in medical and feed applications. Lactic acid bacteria can inhibit pathogenic bacteria in the intestines and reduce the risk of related diseases. At the same time, they can enhance gastrointestinal motility, promote food digestion, and enhance the body's immunity. [7-9] .

[0004] In the field of aquaculture technology, the misuse of antibiotics in animal feed has caused excessive antibiotic residues in animal food, which has put animals and humans at risk of developing antibiotic resistance. The EU has completely banned the consumption of antibiotic growth-promoting feed additives by food animals since 2006. In addition, the risks posed by antibiotics to animal and human health have also attracted the attention of my country's agricultural and rural departments. Strengthening the management of the rational use of antibiotics in the aquaculture process and excessive antibiotic residues in livestock and poultry food have become hot topics in the field of food safety.

[0005] Probiotics are normal flora in the intestines of animals, and have no toxic side effects on the animal body. They attach to the intestines of animals and produce some substances that inhibit the growth of pathogens. In addition, during the growth and metabolism process, they can secrete a variety of enzymes to promote the digestion and absorption of feed by animals, improve the feed-to-meat ratio, and promote the healthy growth of animals. Therefore, probiotic preparations have become a hot topic in the industry at home and abroad.

[0006] The Tianfu Bifidobacterium Jiwang TF04 isolated and disclosed by this technology is closely related to human life. It is a probiotic commonly found in cream, meat and many vegetable fermented products. It can pass through the stomach and colonize in the intestines to play a beneficial role. It has an important impact on intestinal microorganisms and has a wide range of applications in food fermentation, industrial lactic acid fermentation, feed additives and other fields.

[0007] References:

[0008] [1]WHO,FAO.Report of a joint FAO / WHO expert consultation onevaluation of health and nutritional properties of probiotics in foodincluding powder milk with live lactic acid bacteria[R].Amerian córdoba parkhotel córdoba argentina:WHO&FAO,2001.

[0009] [2] Gao Yuqi. Research on the breeding and application of high-yield bacteriocin strains of Lactobacillus acidophilus NX2-6[D]. Nanjing: Nanjing Agricultural University, 2015.

[0010] [3] Shan Chengjun, Xia Xiudong, Liu Xiaoli, et al. Isolation, purification and characterization of Lactobacillus panis C-M2 bacteriocin[J]. Food Science, 2017, 38(20): 20-26.

[0011] [4] Qu Yanfeng, Yue Yingxue, Wang Bingran, et al. Research progress on adhesion characteristics of lactic acid bacteria and their immunomodulatory effects[J]. Food Science and Technology, 2019, 44(12): 12-16.

[0012] [5]Bhat MI,Kapila S,Kapila R.Lactobacillus fermentum(MTCC-5898)supplementation rendersprophylactic action against Escherichia coli impairedintestinal barrier function through tightjunction modulation[J].LWT,2020,123(C):10911.

[0013] [6]Merghni A, Dallel I, Noumi E, et al. Antioxidant and antiproliferative potential of biosurfactants isolated from Lactobacillus casei and their anti-biofilm effect in oral Staphylococcus aureusstrains[J]. MicrobialPathogenesis, 2017, 104:84-89.

[0014] [7]Xu Bingzheng, Wang Ying, Liang Xiaoyue, et al. Research progress on application of lactic acid bacteria bacteriocins[J]. Journal of Heilongjiang Bayi Agricultural University, 2015(1):60-63.

[0015] [8] Thirumurugan A, Ramachandran S, Gobikrishnan S. Optimization of medium components for maximizing bacteriocin production by Lactobacillusplantarum ATM11 using statistical design [J]. International food researchjournal, 2015, 22(3): 1272-1279.

[0016] [9] Zhu Shuang, Zhang Aizhong, Jiang Ning, et al. Bacteriocins and their application in animal production [J]. Journal of Animal Nutrition, 2014, 26(2): 327-333. Summary of the invention

[0017] The applicant of the present invention "A strain of bifidobacterium and its application" deposited the Tianfu Bifidobacterium Jiwang TF04 strain in the China General Microbiological Culture Collection Center (CGMCC), which is established at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The collection center received the strain provided by the applicant on November 14, 2024. The collection number given to the culture by the collection center is CGMCC No 32629, and the classification name is: Tianfu Bifidobacterium Jiwang TF04 (Bifidobacterium jiwang TF04, referred to as: TF04). The deposited strain was identified as alive on November 18, 2024. Molecular biological strain identification and sequence comparison show that 57% of the TF04 bacteria are uncultivable microorganisms and unnamed bifidobacteria, indicating that Tianfu Bifidobacterium Jiwang TF04 has unique innovation.

[0018] Patent purpose: The purpose of "A strain of bifidobacterium and its application" is to provide Tianfu Bifidobacterium Jiwang TF04, which is classified and named Bifidobacterium jiwang TF04, and has been deposited in the China General Microbiological Culture Collection Center CGMCC, with the deposit number CGMCC No 32629.

[0019] The second purpose of "A strain of Bifidobacterium and its application" is to provide the application of the above-mentioned Tianfu Bifidobacterium Jiwang TF04 in the preparation of antibacterial agents.

[0020] The third purpose of "A strain of bifidobacterium and its application" is to provide the application of the above-mentioned Tianfu Bifidobacterium Jiwang TF04 in the preparation of beverages.

[0021] The fourth purpose of "A strain of bifidobacterium and its application" is to provide the application of the above-mentioned Tianfu Bifidobacterium Jiwang TF04 in the preparation of feed additives.

[0022] The technical scheme of the present invention is summarized as follows: 1. The traditional homemade mare's milk yogurt of herdsmen near Ordos City, Inner Mongolia Autonomous Region is used. Long-term consumers are healthy and energetic. The traditional homemade mare's milk yogurt is diluted with sterile water, and after hypoxic or anaerobic culture, colonies are picked. After four purifications, a high-quality strain, Tianfu Bifidobacterium Jiwang TF04, is isolated and obtained. The strain is preserved in the China General Microbiological Culture Collection Center, and the collection center gives the culture a collection number of CGMCCNo 32629.

[0023] 2. Application of Bifidobacteriumjiwang TF04 in antibacterial treatment. The preparation method thereof comprises the following steps:

[0024] (1) Tianfu Bifidobacterium Jiwang TF04 was activated on a conventional MRS agar plate, and a single colony was picked and inoculated into an MRS liquid culture medium tube. After culturing at 37 degrees Celsius for 24 hours, it was transferred to MRS liquid culture medium at a 2% inoculation rate and continued to be cultured for 24 hours as a seed culture liquid.

[0025] (2) MRS medium is mainly composed of peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, Tween 80 1.0 ml, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, distilled water 1000 ml, pH 6.2-6.6.

[0026] (3) The JI liquid fermentation medium for preparing the antibacterial agent is characterized by: 6g of puffed rice flour, 1.2g of soy milk powder, 10g of yeast extract powder, 12.6g of whole milk powder, 8g of glucose, 4g of sodium chloride, 1000mL of water, and sterilization. (4) Preferably, facultative anaerobic culture is performed with a liquid medium containing 50% MRS and 50% JI liquid fermentation medium, with an optimum pH of 6.0 and an optimum temperature of 37 degrees Celsius, and the culture is filtered, and the filtrate is a stable edible natural polypeptide broad-spectrum antibacterial agent.

[0027] 3. Application of Bifidobacterium jiwang TF04 in the preparation of a decoction, the preparation method comprising the following steps: inoculating the seeds of Bifidobacterium jiwang TF04 in a JI liquid fermentation medium at an inoculation rate of 1 to 10%, and performing closed fermentation at 30 to 37 degrees Celsius to obtain a decoction;

[0028] The formula (W / V) of the JI liquid fermentation medium is 0.2-0.85% of puffed rice flour, 0.4-0.88% of soy milk powder, 0.4-0.85% of yeast extract powder, 0.69-0.7% of whole milk powder, 0.2-1% of glucose and 0.2-0.3% of sodium chloride. During production, water is added to a fermentation volume of 10L-1800L and sterilized.

[0029] Preferably, a method for preparing Tianfu Bifidobacterium Jiwang TF04 fermented drink is characterized in that: the formula of the fermentation medium is 3.6 kg of puffed rice flour, 7.2 kg of soy milk powder, 15.3 kg of yeast extract powder, 12.6 kg of whole milk powder, 18 kg of glucose, 3.6 kg of sodium chloride, 1800 L of water is added, and sterilized. The TF04 strain is inoculated into the above-mentioned culture medium at an inoculum amount of 3%, and the drink is obtained by closed fermentation at 37 degrees Celsius.

[0030] 4. The solid fermentation product of Tianfu Bifidobacterium Jiwang TF04 is characterized in that: the Tianfu Bifidobacterium Jiwang TF04 strain is inoculated into a high-density JW solid fermentation medium at an inoculation amount of 1-10%, and the fermentation is carried out under closed conditions of 30-37 degrees Celsius, and the fermentation product is dried at 45-60 degrees Celsius to obtain the solid fermentation product of TF04. The formula of the high-density JW solid fermentation medium is (W / W mass percentage): 20.9-30% wheat bran, 5-24% soybean meal, 2% sucrose, 0.1% milk powder, 1-3% calcium carbonate, 5-7% beta-cyclodextrin and 45-55% water, and the fermentation is closed.

[0031] Preferably, a method for preparing a solid fermentation product of Tianfu Bifidobacterium Jiwang TF04 is characterized by (W / W mass percentage): 20.9% wheat bran, 24% soybean meal, 2% sucrose, 0.1% milk powder, 3% calcium carbonate, 5% β-cyclodextrin and 45% water, inoculated with 3% v / v seed bacterial liquid.

[0032] The invention has the advantages that: Bifidobacterium jiwang TF04 is provided, which is preserved in the China General Microorganism Culture Collection Management Center, and the preservation number is CGMCC No 32629. The bacterium can be applied to antibacterial agents, has a significant inhibitory effect on Escherichia coli, Staphylococcus aureus, Staphylococcus albus and Candida albicans, has good acid resistance, can survive under strong acid conditions of pH1-2, and has high bile salt tolerance. TF04 can also be used in the preparation of beverages, can regulate the microecological balance in the intestine, provide nutritional factors, promote the digestion and absorption of nutrients, improve old intestinal diseases, and enhance the role of non-specific immune function to prevent diseases. TF04 can also be applied in feed additives, which is suitable for the whole process of livestock and poultry and aquaculture, effectively improves the production performance of nursery pigs, can also significantly improve the health level of pigs, reduce the use of antibiotics and other drugs, provide nutritional factors, promote the digestion and absorption of nutrients, promote animal growth and improve feed conversion rate, and save costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The antibacterial test results of organic acids lactic acid and acetic acid were excluded.

[0034] Figure 2 Effect of proteases on antibacterial activity.

[0035] Figure 3 Effect of pH on the activity of antibacterial agents produced by TF04. DETAILED DESCRIPTION

[0036] The following is a further description of the Bifidobacterium bisporus TF04 of the present invention, referred to as TF04, with a preservation number of CGMCC No 32629, through specific examples.

[0037] In the following embodiments

[0038] The Escherichia coli (E. coli) (ATCC 25922) disclosed in the present invention (commercially available);

[0039] Staphylococcus albus (S. albus) (ATCC 8032) (commercially available);

[0040] Staphylococcus aureus (S. aureus) (ATCC 12600) (commercially available);

[0041] Candida albicans (C.albicans) (ATCC 76615 (commercially available)). The present invention discloses the above bacteria and fungi for a better understanding of the present invention, but does not limit the present invention.

[0042] Example 1

[0043] Isolation, Screening, Physiological and Biochemical Characteristics and Molecular Biological Identification of Bifidobacterium Jiwang TF04 from Tianfu

[0044] 1.1 Isolation of strains: Traditional homemade horse milk yogurt of herdsmen near Ordos City, Inner Mongolia Autonomous Region was used. Long-term consumption of this yogurt makes people healthy and energetic. The traditional homemade horse milk yogurt was diluted 10, 100, and 1000 times with sterile water, and 200 μL was spread on MRS solid culture medium, cultured at 37 degrees Celsius for 48 hours under reduced oxygen or anaerobic conditions, colonies were picked, streaked on MRS culture medium, and after 4 purifications, single colonies were picked and cultured in MRS liquid culture medium to the logarithmic phase, glycerol was added to a final concentration of 20%, and stored at -80 degrees Celsius for later use.

[0045] 1.2 Morphological characteristics of the strain, take a single colony of the purified strain, transfer it to MRS solid medium (agar), and culture it in a constant temperature incubator at 37 degrees Celsius for 24h, 36h and 48h, and observe the size, color, edge, protrusion, smoothness, viscosity, transparency and other characteristics of the colony. The results showed that the strain TF04 formed a colony with neat edges, smoothness, viscosity, glossy surface, and opaque on the MRS solid medium. Observed under a microscope, it was a bifidobacterium, named Tianda Tianfu Bifidobacterium 04, scientific name: Bifidobacterium jiwang TF04, abbreviated as: TF04.

[0046] 1.3 TF04 is a Gram-positive bacterium, and a fresh culture of strain TF04 cultured on MRS solid medium (agar) for 24 hours was selected for physiological and biochemical tests. The results showed that TF04 is a Gram-positive bacillus.

[0047] 1.4TF04 has strong reproductive capacity and can grow at 28-45°, with the best culture temperature being 37°. With its strong reproductive capacity, it can survive and reproduce rapidly in the intestine, thus better regulating the balance of the flora.

[0048] 1.5TF04 has strong acid resistance. When ingested, TF04 can withstand the corrosion of gastric acid and oxygen and survive in sufficient quantities into the human intestine. Since the pH value of most human gastric juice is between 1.5 and 3.0, the strain must have a certain acid resistance to colonize in the stomach and intestine and exert its probiotic effect. The pH tolerance test of TF04 showed that the survival rate of TF04 strain was different at different pH values. After treatment with pH 1-5, the survival rate of the strain increased significantly with the increase of pH (P>0.05). In the acidic environment of pH 1, the survival rate of the strain was 25.65%. When the pH was 3, the survival rate of the strain reached more than 65%. When the pH was 5, the survival rate of the strain was 82.75%. In the acidic environment of pH 1, the strain still had a high survival rate, indicating that TF04 has good tolerance to the acidic environment (see Table 1).

[0049] Table 1 Survival rate of strains in acidic environment

[0050] pH %(average) 1 25.65 2 35.56 3 65.00 4 71.91 5 82.75

[0051] (P>0.05)

[0052] 1.6TF04 has colonization ability in the intestine. The experimental results show that the hydrophobicity of TF04 to toluene and xylene is above 30%, which shows high hydrophobicity. The higher the surface hydrophobicity of the strain, the stronger its interaction with epithelial cells, the greater its adhesion, and the stronger its colonization ability in the intestine. The research results show that good hydrophobicity is conducive to the growth and colonization of TF04 on the intestinal mucosa, and can better play its probiotic role.

[0053] 1.7 Self-aggregation of TF04 strain. The self-aggregation ability of the strain is related to its adhesion ability on the host cells. The strains with strong self-aggregation ability can better colonize the surface of cells such as intestinal cells. At the same time, the strong adhesion effect is not affected by fluid flushing, thereby improving the probiotic function. The self-aggregation rate of TF04 was low in the first 2 hours (see Table 2), with a self-aggregation rate of 7.37%. The self-aggregation rate of TF04 gradually increased from 2 to 8 hours. After 8 hours, TF04 showed a higher self-aggregation (40.10%).

[0054] Table 2 Self-aggregation of TF04

[0055] Hour %(average) 2 7.37 4 21.91 8 40.10

[0056] The 1.8TF04 strain has tolerance to bile salts. After being treated with different concentrations of ox bile salts, its survival rate gradually decreased with the increase of bile salt concentration, but the survival rate was still greater than 70% under 3% ox bile salt conditions, indicating that the strain grows well in an environment with high bile salt content.

[0057] 1.9 Molecular biological identification of strains

[0058] Tianfu Bifidobacterium jiwang TF04 (Bifidobacterium jiwang TF04), after strain identification and 16SrRNA gene sequencing by a third party Shenzhen BGI Gene Technology Service Co., Ltd., 100 similar strains were compared by blast sequence similarity on NCBI, and the sequence similarity reached about 93% (see Table 3), of which 31% were marked as microbial strains that exist in nature but cannot be cultured, indicating that the isolation and cultivation of this strain is difficult; in addition, undetermined species of Bifidobacterium accounted for 26%, which shows that Tianfu Bifidobacterium jiwang TF04 of the present invention has unique innovation.

[0059] In addition, the similarity with the sequences of Bifidobacterium fecal 10% and Bifidobacterium adolescentis strains is about 93%. Based on the physiological and biochemical indicators and molecular biological identification results of strain TF04, combined with its morphological characteristics, we named it: BifidobacteriumjiwangTF04, BifidobacteriumjiwangTF04, and it was deposited in the China General Microbiological Culture Collection Center on November 14, 2024, with the deposit number CGMCC No 32629.

[0060] Table 3 Identification of TF04 strains 16SrRNA gene sequencing Analysis of 100 similar strains

[0061]

[0062]

[0063] 57% of the TF04 bacteria are uncultivable microorganisms and unnamed bifidobacteria, indicating that Tianfu Bifidobacterium Jiwang TF04 has unique innovation.

[0064] Example 2. Application of TF04 in antibacterial agents and antibacterial effects

[0065] Application of Bifidobacterium jiwang TF04 in antibacterial agents. The preparation method thereof comprises the following steps:

[0066] 2.1 Activate the conventional MRS agar plate, pick a single colony and inoculate it into a MRS liquid culture tube, incubate it at 37 degrees Celsius for 24 hours, transfer it to MRS liquid culture medium with a 2% inoculation amount, and use it as a seed culture liquid after 24 hours of incubation. The main components of MRS culture medium are 10.0 grams of peptone, 10.0 grams of beef extract, 5.0 grams of yeast extract, 2.0 grams of diammonium hydrogen citrate, 20.0 grams of glucose, 1.0 milliliters of Tween 80, 5.0 grams of sodium acetate, 2.0 grams of dipotassium hydrogen phosphate, 0.58 grams of magnesium sulfate, 0.25 grams of manganese sulfate, 1000 milliliters of distilled water, pH 6.2-6.6. The optimum pH is 6.0, the optimum temperature is 37 degrees Celsius, and it is facultatively anaerobic.

[0067] 2.2 The preparation of the antibacterial agent is characterized by: mixing 50% MRS liquid culture medium and 50% JI liquid fermentation culture medium, culturing together, filtering the culture, and the filtrate is a stable edible natural polypeptide broad-spectrum antibacterial agent.

[0068] The JI liquid fermentation medium is characterized by: the prepared medium is 6g of puffed rice flour, 1.2g of soy milk powder, 10g of yeast extract powder, 12.6g of whole milk powder, 8g of glucose, 4g of sodium chloride, 1000mL of water is added, and sterilized. The TF04 strain is inoculated at a 10% inoculation amount into a mixed medium containing 50% of MRS liquid medium and 50% of JI liquid fermentation medium, and the fermentation is carried out in a closed manner at 37 degrees Celsius, and the supernatant is filtered to obtain the antibacterial agent.

[0069] 2.3 Antibacterial effect of the antibacterial agent prepared by using TF04 on various pathogenic bacteria: The comprehensive antibacterial effect of TF04 on four pathogenic bacteria, namely, Escherichia coli, Staphylococcus aureus, Staphylococcus albicans and Candida albicans, was analyzed. The pH values ​​of different fermentation supernatants of TF04 were measured to be 3.2 and 2.8, respectively. The pH of distilled water was adjusted to the same pH as the antibacterial agent (pH 3.2 and 2.8) using lactic acid and acetic acid with concentrations of 15% and 20%, respectively. The antibacterial activity was determined using Staphylococcus aureus and Escherichia coli as indicator bacteria using the punching method. The results are shown in Tables 4 and Figure 1 , Figure 1 No inhibition zone was produced in the areas treated with acetic acid or lactic acid (see Figure 1 ), indicating that the antibacterial active substance of the antibacterial agent is not the organic acid produced during the metabolism of the strain, but the metabolite of the TF04 strain, which has an inhibitory effect on four pathogenic bacteria: Escherichia coli, Staphylococcus aureus, Staphylococcus albus and Candida albicans.

[0070] Table 4 Antibacterial results of the antibacterial agents produced by TF04 strain excluding organic acids

[0071]

[0072] Note: The diameter of the inhibition zone is the mean ± square error (n = 3); "—" means no obvious inhibition zone is produced

[0073] 2.4 Properties of antibacterial substances produced by strain TF04

[0074] The properties of the antibacterial substances produced by the TF04 strain were studied, and it was found that the antibacterial substances were protein antibacterial substances secreted by the bacteria. The verification method was to use 1.0 mg / mL pepsin, proteinase K, papain and trypsin, respectively, added to the antibacterial agent, and placed in a 45℃ water bath for 2 hours, and then placed in a 95℃ constant temperature water bath for 10 minutes to terminate the enzyme activity. Then, Escherichia coli, Staphylococcus aureus and Staphylococcus albus were used as indicator bacteria, and the supernatant culture fluid without protease treatment was used as a control, and the antibacterial activity was tested by the punching method. The antibacterial agent whose antibacterial activity was reduced or lost after protease hydrolysis was selected, that is, the protein antibacterial agent.

[0075] The experimental results show that pepsin and proteinase K make the antibacterial activity of the antibacterial agent against Escherichia coli, Staphylococcus aureus and Staphylococcus albus "zero". The antibacterial activity of the antibacterial agent treated with papain and trypsin against Escherichia coli, Staphylococcus aureus and Staphylococcus albus is reduced to about 50%, respectively. Therefore, the antibacterial substance produced by TF04 is a natural protein antibacterial agent (see Figure 2 ).

[0076] 2.5 Acid-base stability of the antibacterial agent produced by strain TF04

[0077] Take 10 ml of the TF04 antibacterial agent prepared in 2.4, adjust the pH value to 2, 4, 6, 7, 8, 9, 10 with 1 mol / L HCl and 2 mol / L NaOH respectively, treat for 24 hours, take the untreated antibacterial agent (pH = 3.2) as the control group, use Escherichia coli, Staphylococcus aureus and Staphylococcus aureus as indicator bacteria, and use the punch method to determine the activity of the antibacterial agent. The independent test was repeated three times. The results are as follows Figure 3 shown.

[0078] The antibacterial agent produced by TF04 has antibacterial activity in the pH range of 2-4, and the antibacterial performance decreases rapidly or even disappears completely as the pH value increases. TF04 has the widest range of antibacterial activity against Escherichia coli and can maintain good antibacterial performance in a wide range of acidic and weakly alkaline environments. As for Staphylococcus aureus and Staphylococcus albus, it has antibacterial activity under acidic conditions, but loses its activity when pH>7. This shows that TF04 has a good effect on pathogenic bacteria in the intestine under gastric acid conditions.

[0079] Example 3: Application of Tianfu Bifidobacterium Jiwang TF04 in a drink

[0080] The method for preparing a liquid beverage fermentation product by using Tianfu Bifidobacterium Jiwang TF04 is characterized by comprising the following steps:

[0081] After activation on a conventional MRS agar plate, pick a single colony and inoculate it into a MRS liquid culture medium tube. After culturing at 37 degrees Celsius for 24 hours, transfer it to MRS liquid culture medium at a 2% inoculation rate and use it as a seed culture solution after culturing for 24 hours.

[0082] The Tianfu Bifidobacterium Jiwang TF04 strain is inoculated into a JI liquid fermentation medium at an inoculation rate of 1-10%, and the medium is fermented in a closed manner at 30-37 degrees Celsius to obtain a drink; the formula (W / V) of the JI liquid fermentation medium is 0.2-0.85% puffed rice flour, 0.4-0.88% soy milk powder, 0.4-0.85% yeast extract powder, 0.69-0.7% whole milk powder, 0.2-1% glucose, and 0.2-0.3% sodium chloride. During production, water is added to a fermentation volume of 10L-1800L, and the medium is sterilized.

[0083] 3.1 Preparation of medium for 10L of potion: The medium is 0.06kg of puffed rice flour, 0.12kg of soy milk powder, 0.10kg of yeast extract powder, 0.126kg of whole milk powder, 0.08kg of glucose, 0.04kg of sodium chloride, add 10L of water, and sterilize. Inoculate TF04 strain into the above medium at a 10% inoculation rate, and ferment in a closed environment at 30 degrees Celsius to obtain a potion that can be drunk directly.

[0084] 3.2 Preparation of medium for producing 180L of potion: The prepared medium is 1.53kg of puffed rice flour, 1.59kg of soy milk powder, 0.72kg of yeast extract powder, 1.26kg of whole milk powder, 0.36kg of glucose, 0.54kg of sodium chloride, add water to 180L, and sterilize. Inoculate TF04 strain into the above medium at a 4% inoculum, and ferment in a closed environment at 36 degrees Celsius to obtain a potion that can be drunk directly.

[0085] 3.3 Preparation of medium for the production of 1800L of beverage: The medium is 3.6 kg of puffed rice flour, 7.2 kg of soy milk powder, 15.3 kg of yeast extract powder, 12.6 kg of whole milk powder, 18 kg of glucose, and sodium chloride.

[0086] 3.6 kg, add 1800 L of water, sterilize. Inoculate TF04 strain into the above culture medium at a rate of 1%, and ferment in a closed environment at 37 degrees Celsius to obtain a drink that can be drunk directly.

[0087] The effects of the drinks prepared in Examples 3.1, 3.2 and 3.3 are consistent.

[0088] Example 4. Application effect of the decoction prepared by TF04

[0089] 4.1 Beneficial to the recovery from diarrhea. Clinical observation studies on volunteers with chronic diarrhea who drank the decoction prepared by TF04 showed that after two weeks of medication, the frequency and shape of abnormal stools in patients disappeared, and the clinical symptoms disappeared. The total effective rate was 91.3%, and the recurrence rate was low.

[0090] Diarrhea recovery test: Take the drink produced in Example 3.1, select 32 volunteers, and divide them into two groups, a drinking group and a control group, with 17 people in each group. Take 50 ml of the drink prepared in Example 3.1 to drink. During the drinking period and two weeks after drinking, the frequency and shape of stools of the patients in the drinking group were abnormal, and the clinical symptoms disappeared. The total effective rate was 91.3% (Note: the experiment was repeated 3 times, and the results are the average of three times), and the recurrence rate was low.

[0091] The effects of the drinks prepared in Examples 3.2 and 3.3 are consistent.

[0092] 4.2 Drinking TF04 prepared drinks can improve bad breath. Bad breath is mostly caused by the imbalance of gastrointestinal flora in the human body. Diseases in the stomach and intestines will produce a large amount of toxins, which will cause strong bad breath. Drinking TF04 prepared drinks will make the intestines acidic, which can effectively inhibit the abnormal fermentation caused by harmful bacteria, thereby eliminating the occurrence of bad breath. Drinking TF04 prepared drinks or spraying them can 100% improve bad breath.

[0093] Improvement of halitosis test: Take the drink prepared in Example 3.1, select 32 volunteers, divide them into two groups, a drinking group and a control group, with 17 people in each group. Take 50 ml of the drink prepared in Example 3.1 to drink. During the drinking period and two weeks after drinking, the halitosis symptoms of the drinking group disappeared, and the total effective rate was 100% (Note: the experiment was repeated 3 times, and the results are the average of the three times).

[0094] The effects of the drinks prepared in Examples 3.2 and 3.3 are consistent.

[0095] 4.3 Improve old intestinal diseases

[0096] Chronic intestinal diseases refer to chronic intestinal diseases such as enteritis, intestinal ulcers, and irritable bowel syndrome. TF04 secretes acetic acid and lactic acid to create an acidic, anaerobic environment, which kills a large number of harmful bacteria and re-forms a bifidobacterium biofilm on the intestinal wall, making it impossible for foreign pathogens and transient bacteria to colonize on the intestinal surface, thus promoting the healing of intestinal ulcers and lesions.

[0097] Test for improving enteritis: Take the drink prepared in Example 3.1, select 32 volunteers, divide them into two groups, a drinking group and a control group, with 17 people in each group. Drink 50 ml of the drink prepared in Example 3.1. After drinking for two weeks, the symptoms of the drinking group disappeared, and the total effective rate was 100% (Note: the experiment was repeated 3 times, and the results are the average of the three times).

[0098] The effects of the drinks prepared in Examples 3.2 and 3.3 are consistent.

[0099] Example 5 Solid fermentation product of TF04 and its application in feed additives

[0100] The preparation method of the solid fermentation product of Tianfu Bifidobacterium Jiwang TF04 is characterized in that: after activation on a conventional MRS agar plate, a single colony is picked and inoculated in an MRS liquid culture medium test tube, and after culturing at 37 degrees Celsius for 24 hours, it is transferred to an MRS liquid culture medium with a 2% inoculation amount, and then cultured for 24 hours as a seed bacterial liquid. The formula (W / W mass percentage) of the high-density JW solid fermentation medium is: 20.9-30% wheat bran, 5-24% soybean meal, 2% sucrose, 0.1% milk powder, 1-3% calcium carbonate, 5-7% beta-cyclodextrin and 45-55% water. The Tianfu Bifidobacterium Jiwang TF04 strain is inoculated into the JW solid fermentation medium at an inoculation amount of 1-10%, and fermented in a closed manner at 30-37 degrees Celsius for 48 hours. Then the fermentation product is dried at 45-60 degrees Celsius for 8 hours to obtain the solid fermentation product of strain TF04.

[0101] 5.1 The prepared seed culture liquid was inoculated into the configured high-density JW solid fermentation medium (W / W mass percentage): 20.9% wheat bran, 24% soybean meal, 2% sucrose, 0.1% milk powder, 3% calcium carbonate, 5% β-cyclodextrin and 45% water, and the Tianfu Bifidobacterium Jiwang TF04 strain was inoculated into the medium at an inoculation rate of 10%, and the culture medium was sealed and fermented at 37 degrees Celsius for 48 hours, and then the fermentation product was dried at 50 degrees Celsius for 8 hours to obtain the solid fermentation product of strain TF04.

[0102] 5.4 Prepare the formula of medium-density JW solid-state fermentation medium (W / W mass percentage): 22.9% wheat bran, 14% soybean meal, 2% sucrose, 0.1% milk powder, 1% calcium carbonate, 7% β-cyclodextrin and 53% water. Inoculate the Tianfu Bifidobacterium Jiwang TF04 strain into the medium at an inoculum rate of 3%, ferment in a closed chamber at 36 degrees Celsius for 48 hours, and then dry the fermentation product at 60 degrees Celsius for 8 hours to obtain the solid fermentation product of strain TF04.

[0103] 5.5 Prepare the formula of low-density JW solid-state fermentation medium (W / W mass percentage): 30% wheat bran, 5% soybean meal, 2% sucrose, 0.1% milk powder, 2% calcium carbonate, 5.9% β-cyclodextrin and 55% water. Inoculate the Tianfu Bifidobacterium Jiwang TF04 strain into the medium at an inoculum size of 1%, ferment in a closed chamber at 30 degrees Celsius for 48 hours, and then dry the fermentation product at 45 degrees Celsius for 8 hours to obtain the solid fermentation product of strain TF04.

[0104] Example 6 Effect of solid fermentation products of strain TF04 on the production performance of nursery pigs

[0105] 6.1 Experimental animals and grouping: 216 32-day-old weaned piglets were selected as experimental subjects, including 108 in the experimental group and 108 in the control group, and fed to 77 days of age. The experiment started on March 4, 2024 and ended on April 18, 2024, with a duration of 45 days.

[0106] 6.2 Experimental design

[0107] Control group 1: 0.1% of a similar product from the market was added to the basic diet;

[0108] Control group 2: The basal diet was not supplemented with the solid fermentation product of strain TF04;

[0109] Experimental group: The solid fermentation product of TF04 prepared in Example 5.1 was used for the experiment. 0.1% (mass percentage) of the solid fermentation product of strain TF04 was added to the basic diet. The detailed design of the experiment is shown in Table 5.

[0110] Table 5. Diet structure

[0111] Group Diet structure Experimental Group Basal diet containing 0.1% TF04 Control group 1 Basic diet containing 0.1% of a similar product on the market Control group 2 Basic diet

[0112] The experimental design of the TF04 solid fermentation product prepared in Examples 5.2 and 5.3 is the same.

[0113] 6.3 Effect of fermentation strains on weight gain in nursery pigs

[0114] The TF04 solid fermentation product prepared in Example 5.1 was used for the experiment. The weight gain of nursery pigs is shown in Table 6. The initial weight of the experimental group was 8.89 kg / head, the final weight was 25.42 kg / head, and the daily weight gain was 367.33 g; the initial weight of the control group was 9.09 kg / head, the final weight was 25.21 kg / head, and the daily weight gain was 358.22 g. The daily weight gain of the experimental group was 9.11 g more than that of the control group 1, and the net weight gain per head was 0.37 kg more during the experiment.

[0115] Table 6. Effect of fermentation strains on weight gain in nursery pigs

[0116]

[0117] The TF04 solid fermentation products prepared in Examples 5.2 and 5.3 have the same effects.

[0118] 6.4 Effect of strain TF04 on the survival rate of nursery pigs

[0119] The TF04 solid fermentation product prepared in Example 5.1 was tested. The nursery pig breeding rate is shown in Table 7. 108 piglets were selected in the experimental group. During the experiment, 4 piglets were eliminated and 1 piglet died. At the end of the experiment, 103 piglets were kept, and the breeding rate was 95.37%. 108 weaned piglets were selected in the control group. During the experiment, 4 piglets were eliminated and 5 piglets died. At the end of the experiment, 99 piglets were kept, and the breeding rate was 91.67%. The breeding rate of the experimental group was significantly higher than that of the control group.

[0120] Table 7 Effect of fermentation strains on the growth rate of nursery pigs

[0121] Group Initial number of experiments Number of heads at the end of the experiment Survival rate (%) Experimental Group 108 103 95.37 Control group 1 108 100 92.59 Control group 2 108 99 91.67

[0122] The TF04 solid fermentation products prepared in Examples 5.2 and 5.3 have the same effects.

[0123] 6.5 Effect Discussion and Explanation

[0124] This experiment shows that the average weight of each nursery pig in the experimental group increased by 0.37 kg, the average daily weight gain increased by 9.11 grams, and the death rate decreased by 2.78% compared with the basic feed control group. The experiment proves that strain TF04 can effectively improve the production performance of nursery pigs and is significantly better than similar products on the market. In addition, the use of strain TF04 can significantly improve the health level of the pig herd, reduce the use of antibiotics and other drugs, and also save costs.

[0125] In summary, "A strain of bifidobacterium and its application" Tianfu Bifidobacterium Jiwang TF04 can be used to regulate the balance of microecology in the intestine of animals, has the effect of enhancing nonspecific immune function to prevent diseases, and can also provide nutritional factors, promote digestion and absorption of nutrients, promote animal growth and improve feed conversion rate.

[0126] The above embodiments are preferred implementation modes of "A strain of bifidobacterium and its application", but the implementation modes of "A strain of bifidobacterium and its application" are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of "A strain of bifidobacterium and its application" shall be equivalent replacement modes and shall be included in the protection scope of "A strain of bifidobacterium and its application".

Claims

1. Tianfu Bifidobacterium Jiwang TF04, which is classified and named Bifidobacterium jiwang TF04 , has been deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit number CGMCC No 32629.

2. The Tianfu Bifidobacterium Jiwang TF04 according to claim 1 Bifidobacterium jiwang Application of TF04 in antibacterial agent.

3. The Tianfu Bifidobacterium Jiwang TF04 according to claim 2 Bifidobacterium jiwang TF04 is used as an antibacterial agent, which is characterized by being activated on a conventional MRS agar plate, picking a single TF04 colony and inoculating it in a common MRS liquid culture medium test tube, culturing it at 37 degrees Celsius for 24 hours, transferring it to an MRS liquid culture medium at a 2% inoculation amount, and culturing it for another 24 hours as a seed bacterial liquid. Further, it is mixed with 50% MRS liquid culture medium and 50% JI liquid fermentation medium, and cultured together. The JI liquid fermentation medium is characterized by: 6g puffed rice flour, 1.2g soy milk powder, 10g yeast extract powder, 12.6g whole milk powder, 8g glucose, 4g sodium chloride, adding 1000mL of water, and sterilizing. TF04 strains are inoculated at an inoculation amount of 1-10% into a mixed culture medium containing 50% MRS liquid culture medium and 50% JI liquid fermentation medium, and fermented in a closed manner at 37 degrees Celsius. The supernatant is filtered to obtain the antibacterial agent.

4. The Tianfu Bifidobacterium Jiwang TF04 according to claim 1 Bifidobacterium jiwang Application of TF04 in preparation of beverages.

5. A method for preparing a liquid drink fermentation product of Bifidobacterium jiwang TF04, characterized in that: The Tianfu Bifidobacterium Jiwang TF04 strain described in claim 1 is inoculated into the JI liquid fermentation medium at an inoculation rate of 1-10%, and the mixture is fermented in a closed manner at 30-37 degrees Celsius to obtain a drink; the formula (W / V) of the JI liquid fermentation medium is 0.2-0.85% puffed rice flour, 0.4-0.88% soy milk powder, 0.4-0.85% yeast extract powder, 0.69-0.7% whole milk powder, 0.2-1% glucose, and 0.2-0.3% sodium chloride. During production, water is added to a fermentation volume of 10L-1800L and sterilized.

6. A method for preparing Tianfu Bifidobacterium Jiwang TF04 fermented beverage according to claim 4, characterized in that: The formula of the fermentation medium is 3.6 kg of puffed rice flour, 7.2 kg of soy milk powder, 15.3 kg of yeast extract powder, 12.6 kg of whole milk powder, 18 kg of glucose, 3.6 kg of sodium chloride, 1800 L of water, and sterilization. TF04 strain is inoculated into the above medium at an inoculum rate of 3%, and fermented in a closed environment at 37 degrees Celsius to obtain a drink.

7. The solid fermentation product of Bifidobacterium tianfuensis TF04 according to claim 1, characterized in that: Inoculate the Tianfu Bifidobacterium Jiwang TF04 strain into the JW solid fermentation medium at a rate of 1-10%, perform closed fermentation at 30-37 degrees Celsius, and then dry the fermentation product at 45-60 degrees Celsius; The formula of the JW solid-state fermentation medium (W / W mass percentage) is: 20.9-30% wheat bran, 5-24% soybean meal, 2% sucrose, 0.1% milk powder, 1-3% calcium carbonate, 5-7% beta-cyclodextrin and 45-55% water, and the fermentation is closed.

8. A method for preparing a solid fermentation product of Bifidobacterium bisporus TF04 according to claim 7, characterized in that (W / W mass percentage): 20.9% wheat bran, 24% soybean meal, 2% sucrose, 0.1% milk powder, 3% calcium carbonate, 5% β-cyclodextrin and 45% water, inoculated with 3% v / v seed bacterial liquid, closed fermentation, and dried at 45-60 degrees Celsius to obtain a solid fermentation product.

9. A solid fermentation product of Bifidobacterium jiwang TF04, characterized in that: The preparation method is the method described in claim 7, and the formed solid fermentation product is used in the preparation of feed additives.

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