WO2022057741A1 - Bifidobacterium animalis, breeding method therefor and use thereof - Google Patents

Abstract

A bifidobacterium animalis, a breeding method therefor and a use thereof. The bifidobacterium animalis is bifidobacterium animalis IU-100 in Bifidobacterium, and the accession number thereof is CGMCC No. 12942. The bifidobacterium strain is obtained by continuously researching the intestinal floras of the long living elderly in Bama district in Guangxi of China for many years, selecting elder excrement samples having a high intestinal bifidobacteria quantity and carrying out separation and purification by a selective culture method. The bifidobacterium animalis can not only improve the intestinal health of populations, but also has a potential effect of improving immunity. Meanwhile, a simple and easy breeding method and use is provided, and the present invention thus has high efficiency and low cost.

Description

Bifidobacterium animalis and its breeding method and application technical field

The invention relates to an animal bifidobacteria, a breeding method and application thereof, and belongs to the technical field of Bifidobacterium lactis.

Background technique

Probiotics are a class of active microorganisms that can improve the host's micro-ecological balance and exert beneficial effects on the human body. Probiotics colonize the human body, such as the intestinal tract, and play an important physiological role in the digestion and absorption of nutrients, the growth and development of the body, immunity and antagonism. In a normal adult, the gut contains more than 1,000 species of microorganisms, which can be divided into three categories: origin flora, opportunistic pathogens and transit bacteria. The flora of origin is the dominant intestinal bacteria with immunomodulatory effects; opportunistic pathogens are harmless when the intestinal flora is balanced, but can be harmful to the human body under certain conditions; harmful bacteria, the number of such bacteria is small under normal circumstances, and the bacteria It can cause disease when the group is out of balance. People with constipation and diarrhea are often accompanied by an imbalance of intestinal flora.

With the development of society, people pay more and more attention to their own health, and physical maintenance through exercise and healthy diet has become the main way of health care. However, factors such as busy work, irregular daily routines, and physical changes during pregnancy have led to abnormal intestinal health in the current population, such as constipation and diarrhea, which have become the main sub-health manifestations of people of all ages. Intestinal abnormalities can be treated with medication, but they affect the gut microbiome and are not a healthy and sustainable way to convince most people.

Active probiotics with intestinal optimization function or yogurt products loaded with such active probiotics can also improve the human intestinal environment, relieve or eliminate constipation and diarrhea. It is a better choice for regulating and optimizing human intestinal health.

In addition, as a delicious and healthy food, low-temperature yogurt mainly functions from the microorganisms added and proliferated in the production of yogurt, especially functional probiotics; It is easier to keep active in medium and low temperature yogurt is a good carrier for the survival of probiotics.

The core of functional probiotics lies in the selection of strains, the verification and strengthening of their functions, and only strains that have been scientifically verified can play a safe and effective role in functional health.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a kind of animal bifidobacteria, which can not only improve the intestinal health of the population, but also has the potential to enhance immunity; meanwhile, the present invention provides a simple and easy breeding method and application, and the efficiency High and low cost.

The animal Bifidobacterium of the present invention, the strain is Bifidobacterium animalis IU-100 in the genus Bifidobacterium, and the animal Bifidobacterium has been preserved in China Microbial Species on September 5, 2016 The General Microbiology Center of the Preservation and Management Committee, Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, classified as Bifidobacterium animalis, and deposited as CGMCC No.12942.

Bifidobacterium animal strains are the Bifidobacterium strains obtained by selecting and purifying the fecal samples of the elderly with a high number of intestinal bifidobacteria through years of research on the intestinal flora of long-lived elderly people in Bama, Guangxi, China.

Using the animal bifidobacteria to improve the intestinal health of the population.

The breeding method of described animal bifidobacteria comprises the following steps:

(1) The fecal suspension of the longevity elderly immersed in the anaerobic diluent was vortexed to prepare a fecal suspension, serially diluted 10 times with the anaerobic diluent, poured into the plate with NPNL-X-Gal selective medium, and placed in an anaerobic bag. Incubate at 37°C for 40-60h in an anaerobic gas-producing bag to isolate potential bifidobacteria;

(2) Pick blue colonies from the plate, and after Gram stain microscopy, select Gram-positive colonies with irregular bacilli, and continue to separate and purify 2-3 times until potential bifids are isolated. Bacillus pure culture;

(3) carrying out a cell adhesion experiment with several pure cultures of bifidobacteria isolated above, and selecting a bacterial strain with excellent adhesion ability;

(4) Bifidobacterium animalis IU-100 is obtained by carrying out strain identification of the above-selected Bifidobacterium pure culture.

The animal bifidobacteria is used for preparing yogurt products.

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

(1) The animal Bifidobacterium strain selected by the present invention has an excellent effect of improving the intestinal health of the population, and is specifically manifested in that by improving the intestinal microenvironment of the human body, the function of moistening the intestines and defecation of the human body is realized;

(2) The animal Bifidobacterium strain selected by the present invention also has the potential to enhance immunity.

detailed description

The present invention is further described below in conjunction with the examples, but does not limit the implementation of the present invention.

Example 1

Sample Collection:

In April 2007, he went to Bama County, Guangxi Zhuang Autonomous Region, to investigate the living conditions of the elderly with longevity and collect fecal samples of the elderly.

As shown in Table 1, all the people in group M are from Jiazhuan Township, where the long-lived elderly are concentrated in the suburbs of Bama. Group S is the direct or collateral descendants of Group M, who also live in Jiazhuan Township, and Group C is from Bama County Welfare Institute. And Baxu Urban Village.

Among them, group M is the longevity old people living in Jiazhuan Township, the suburb of Bama, group S is the generation of longevity old people living in the suburb of Bama, and group C is the old people living in the county town of Bama.

Table 1 Sampling population grouping

The 8 centenarians in group M came from villages such as Ping'an Village and Poyue Village, Jiazhuan Township. Among them, there were 6 longevity elders in Ping'an Village. The survey results showed that 7 centenarians could still perform housework and even farming activities, confirming that the aging symptoms of the long-lived elderly were delayed.

Before sampling, each elderly person was given a numbered, weighed and sterilized 50mL sampling tube and an ice box. For Group M elderly, an additional sampling tube containing 20mL of sterilized anaerobic diluent is issued for the isolation and screening of Bifidobacterium strains from long-lived elderly.

Each elderly person collects a fresh fecal sample and packs it in a collection tube (the elderly in group M divide the sample into two sampling tubes), put it in the ice box immediately, and notify the sample collector, and the sample will be transported back to the local experiment within 4 hours room.

The fecal samples were soaked in anaerobic diluent for bacterial isolation and culture immediately; another sample tube was stored in liquid nitrogen, then transferred to a dry ice incubator, airlifted back to Beijing, stored at -80°C, and processed within 7 days complete.

Example 2

Molecular biological identification of Bifidobacterium animalis:

1. Use the bacterial genome extraction kit to extract the genomic DNA of potential Bifidobacterium strains.

2. Use 27F and 1541R primers to amplify the full sequence of bacterial 16S rDNA by PCR. The product was recovered and purified by a DNA purification kit and sent to Invitrogen (Beijing) for sequencing.

3. Input the sequencing results into the NCBI database, and identify the strain species through BLASTn homology comparison.

The sequencing result is:

4. Comparison:

The above-mentioned 16s rDNA sequence was compared on NCBI, and it was found that the sequence homology with the 16s rDNA sequence of Bifidobacterium animalis subsp. lactis strain 2601 was 99%, and the E value was 0. Therefore, it can be determined that the isolated strain is Bifidobacterium animalis.

5. Preservation:

We then sent the strain to the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection and Management Committee for preservation, and the preservation number is CGMCC No.12942.

Example 3

Bacteria safety evaluation:

Safety evaluation is a prerequisite for the application of probiotics, and Bifidobacterium animalis IU-100 is a potential probiotic that has been extensively studied. The present invention conducts in vitro and in vivo safety evaluation of the strain, and provides a basic guarantee for the application of the strain.

Antibiotic susceptibility and biogenic amine production of IU-100 were evaluated in vitro; oral toxicity and translocation risk of IU-100 were evaluated in vivo.

The results showed that the strain IU-100 had no antibiotic-susceptibility peculiar to the strain, which ruled out the risk of lateral transfer of antibiotic resistance; no biogenic amine was found to be produced by the strain after culturing it in ^{decarboxylase} medium; ^9. After 10 ¹⁰ cfu/mouse was fed for 28 days, the mice did not have adverse reactions, and the strain was not found in blood, kidney and liver. The study found no unsafe factors for strain IU-100.

The animal experiments and bacterial displacement experiments are described in detail below.

1. Experimental materials

Strain: Bifidobacterium animalis IU-100, isolated from the long-lived old man in Bama; Staphylococcus aureus ATCC25923 was purchased from China General Microorganism Collection.

2. Experimental method

(1) Animal experimental design:

SPF grade Balb/C mice, 6 to 8 weeks old, female. Raised in SPF animal room: temperature 22±2°C, humidity 55%±2%, 12h light/dark cycle, free water and food intake. Mice were randomly divided into 10 groups (10 mice in each group), and the groups were as follows:

In the intragastric administration group (3 groups), each mouse was intragastrically administered 2×10 ⁸ , 2×10 ⁹ and 2×10 ¹⁰ cfu/mouse for 28 days (0.2 mL per mouse per day);

Control group, gavage with normal saline; intraperitoneal injection group (3 groups): inject bacterial suspension with a concentration of 2×10 ⁹ cfu/mouse, 0.2 mL per mouse;

Intraperitoneal injection control group (3 groups): inject normal saline, 0.2 mL per mouse.

The three groups of intraperitoneal injection group and control group were sacrificed at 2, 5, and 10 days after injection, respectively, to detect the translocation of strains. During the experiment, the general physical signs, behavior, poisoning performance and death of the mice were observed every day, and the weight and food intake of the mice were recorded every week.

(2) Evaluation of bacterial translocation ability:

Bacterial translocation in blood, liver, spleen and kidney of mice was analyzed. 50uL blood was spread on MRS plate and BHI plate, MRS and BHI were cultured anaerobic and aerobic respectively at 37℃ for 48h, and the number of colonies on the plate was counted. Tissue samples were suspended in sterile physiological saline at 1 g/mL, and after homogenization, 100 uL were spread on MRS plates and BHI plates respectively, and cultured in the same way as blood samples. Colony growth was recorded as positive.

(3) Statistical analysis Test results Data are expressed as mean ± standard deviation (± SD). One-way analysis of variance was used to test the significance of differences between the means of different treatments, and the chi-square test was used to compare the shift rates between different treatments. All statistical analyses were performed using the statistical software SPSS version 17.0, and the significance level was P<0.05.

3. Analysis of results

(1) Oral IU-100

Blood routine indexes reflect the side effects of experimental strains in blood, and blood biochemical indexes reflect nutritional deficiencies and metabolic imbalances. Changes in these indicators usually precede other clinical symptoms.

In this experiment, the blood routine and blood biochemical indexes of the experimental group and the control group did not have any significant changes, indicating that the experimental strains had no adverse effect on the experimental animals.

Table 2 Blood routine indexes of oral gavage group (n=10)

Table 3 Translocation rates of test bacteria to liver, spleen, kidney and blood in mice in oral gavage group

4 Conclusion

Strain IU-100 has no antibiotic resistance other than species-specific resistance, and there is no risk of antibiotic transfer; the amine production experiment did not find the production of biogenic amines. Oral administration of 10 ⁸ , 10 ⁹ , and 10 ¹⁰ cfu/day in mice did not develop translocation in blood, liver, spleen and kidney for 28 days; it showed high safety of translocation. The safety evaluation of Bifidobacterium animalis IU-100 found no unsafe factors of the strain.

Example 4

Bacterial tolerance evaluation:

The tolerance of IU-100 was evaluated by comparing the laboratory strain IU-100 with the commercial strain BB12.

Using acid resistance test, bile salt resistance test and simulated artificial gastrointestinal fluid test on IU-100 and BB12, the tolerance of IU-100 was evaluated by plate count and growth curve.

The following conclusions can be drawn from the experimental results:

Within 0-3h, the survival numbers of IU-100 at pH 6.5, 3.0 and 2.5 all maintained the original order of magnitude;

Survival numbers decreased by about 2.5 orders of magnitude at pH 2.0.

After 14h treatment under different conditions, the OD _600nm value of IU-100 increased by about 2.0 and 0.5 when the bile salt concentration was 0% and 0.1%, respectively; when the bile salt concentration was 0.2% and 0.3%, there was basically no increase.

After IU-100 was cultured in artificial gastric juice and artificial intestinal juice for 2h and 6h, the survival rate increased by about 1 order of magnitude.

IU-100 is similar to the commercial strain BB12 in acid resistance, bile salt resistance and artificial gastrointestinal fluid resistance, and has the potential to develop into a commercial strain.

Example 5

Animal experiment (mouse laxative mouse experiment):

Animal experiments prove that Bifidobacterium animalis IU100 strain has a laxative effect on constipated mice.

The experimental animals of the present invention are: Balb/c female mice, 6-8 weeks old, weighing 18-22 g, SPF grade, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

Detection methods include:

(1) Determination of defecation time, number of fecal particles and weight of feces: 7 days after the test samples were administered, mice in each group were fasted for 16 hours. The model control group, the positive control group and the three dose groups were given compound diphenoxylate (10 mg/kgBW) by gavage, and the blank control group was given distilled water. After 0.5 hours of compound diphenoxylate, the mice in the blank control group and the model control group were gavaged with ink. They were kept in single cages and fed with normal drinking water. From the time of filling the ink, the time of the first black stool, the number and weight of the black stool and the water content of the feces of each animal were recorded within 5 hours.

(2) Small intestine propulsion test: 15 days after the test samples were given, the mice in each group were fasted for 16 hours. The model control group and the treatment group were given compound diphenoxylate (5mg/kgBW) by gavage, and the blank control group was given distilled water. After 0.5 hours after administration of compound diphenoxylate, the treatment group was respectively given ink containing the test sample (containing 5% activated carbon powder, 10% gum arabic), and the control group was given the ink by gavage. Immediately after 25 minutes, the animals were sacrificed by dislocating the cervical vertebrae. The abdominal cavity was opened to separate the mesentery. The upper end of the intestinal tube from the pylorus and the lower end to the ileocecal area was cut, placed on a tray, and the small intestine was gently pulled into a straight line. , from the pylorus to the front of the ink is the "ink advance length".

Calculate the ink advance rate as follows:

Ink propelling rate % = ink propelling length (cm)/total length of small intestine (cm) × 100%

results and analysis:

1. Defecation time, number of stool particles and stool weight

IU-100 bacterial strains were orally gavaged in experimental mice, and after 7 days, the influence of defecation time, particle, weight and fecal water content on constipation model mice was measured, and the results are shown in the following table:

Table 4 Effects of IU-100 on the first defecation time, particle size, weight and fecal water content in constipated mice

^▲ Compared with blank group, ^▲▲ p<0.01; *Compared with model group, *p<0.05;**p<0.01; ^■ Compared with positive control ^■ p<0.05

Compared with the blank control group, there were significant differences in the time of first black stool, stool particles and stool weight in the model group, indicating that the constipation mouse model was established. Different doses of IU100 bacterial solution shortened the first defecation time of constipated mice, and increased the weight of defecation particles and feces in mice, especially in the middle dose group, the first defecation time was significantly different from the model group, and the effect was better than the positive control strain.

2. Results of the Small Intestine Propulsion Test Study

The experimental animals were given IU100 viable bacteria for 15 days, and the small intestine propulsion test was performed.

Table 5 The effect of IU100 on small intestinal motility in mice

^▲ Compared with blank group, ^▲▲ p<0.01; *Compared with model group, *p<0.05;**p<0.01.

The results showed that the ink propulsion rate of the model group was significantly lower than that of the blank group, and the mouse constipation model was established. After IU100 administration for 15 days, the ink propulsion rate of mice in each dose group was significantly higher than that in the model group, and the low and high dose groups reached extremely significant levels, which were close to the blank control. There was no difference between the mice in the IU100 group and the positive control group. The research results showed that the IU100 strain could significantly increase the peristalsis of the small intestine in constipated mice, promote the advancement of intestinal contents, and have a laxative effect on the constipated mice.

The final results showed that the administration of IU100 for 7 days could significantly improve the time of first melena excretion, increase the number and weight of melena grains in constipated mice, and for 15 days, it could significantly improve the ink propulsion rate in the small intestine of constipated mice. It shows that Bifidobacterium animalis IU100 strain has a laxative effect on constipation mice.

Example 6

Gut-improving effects of Bifidobacterium animalis-containing yogurt in healthy adults:

1. Experimental procedure

This experiment is divided into the following three stages:

(1) Preliminary screening of volunteers:

Select volunteers who meet the experimental requirements.

(2) Empty period:

The emptying period lasted a total of 10 days. During the period, volunteers could not drink any fermented products (including yogurt, cheese, active lactic acid bacteria drinks, etc.), but could drink milk. Day 10±1 was the time of the first stool sample collection.

(3) Yogurt drinking period:

The drinking period lasts for a total of 20 days. During the period, the volunteers were not allowed to drink any fermented products (including yogurt, cheese, active lactic acid bacteria drinks, etc.) except for drinking a bottle of IU-100 yogurt within one hour after lunch every day. In addition, it is necessary to fill in the meal statistics form on time every day. Day 10±1 was the time of the second stool sample collection, and day 20±1 was the time of the third stool sample collection, and the body weight was recorded.

The preparation method of the fermented milk rich in Bifidobacterium lactis IU-100 in the present invention comprises the following steps:

(1) The raw milk is heated up to 60°C, and uniformly mixed white granulated sugar, agar, whey protein concentrate, and starch material are added, the homogenization and sterilization are completed, and the temperature is lowered to 43°C;

(2) Inoculate Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, IU-100 Bifidobacterium lactis to the raw milk treated in step (1), stir and mix well, and at a temperature of 43° C., stand for fermentation until titration The acidity is greater than 70°T to obtain fermented milk;

(3) the fermented milk obtained in step (2) is cooled to 4° C. after demulsification, and then transferred to an environment with a temperature of 4° C. for refrigerating and ripening for 8 hours to obtain Bifidobacterium lactis IU- 100 of fermented milk.

2. Experimental sample and population grouping

After screening, a total of 110 people were enrolled in the group. During the period, 20 people withdrew due to factors such as influenza and medication. The grouping of the group is shown in the following table:

Table 6

group	male	Female	Total number of people	average age	The number of valid people at the end of the experiment
Ordinary yogurt control group-A group	12	40	52	23.2±1.4	42
Bifidobacterium-containing yogurt group-B group	twenty three	35	58	25.3±5.0	48

Experimental sample:

(1) Ordinary yogurt: solidified yogurt, 200g/bottle, fermented strains: Streptococcus thermophilus and Lactobacillus bulgaricus.

(2) Bifidobacterium-containing yogurt group: coagulated yogurt, 200g/bottle, fermented strains: Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium animalis (maintain more than 1×10 ⁸ CFU/ml during the experiment).

3. Defecation Condition Scoring Criteria

Table 7

4. Experimental results

Table 8 Statistics of the number of bowel movements

The number of bowel movements per day decreased by 0.41 times/day in the group taking regular yogurt, and the number of bowel movements improved in 9.5% of the people. The frequency of defecation increased by 0.4 times/day in the group of people who took yogurt containing bifidobacteria, and 33.3% of the population improved the frequency of defecation. Compared with the control group A, the difference was significant, indicating that taking yogurt containing bifidobacteria, the frequency of defecation was significantly improved. .

Table 9 Bristol stool score (4 is the most ideal score, the closer to 4 the more ideal)

The Bristol stool score is an important indicator for evaluating the health status of stool, of which 4 is the most ideal score, and the closer to 4, the higher the health. The table above shows the changes in Bristol score before and after 20 days of consumption of yogurt. There is no significant difference between groups A and B. Among them, the improvement rate of fecal morphogenesis in group A was 21.4%, while that in group B was 46.5. There was a significant difference between the two. The results show that yogurt containing bifidobacteria can significantly improve the fecal morphology of the population compared with ordinary yogurt.

Table 10 Proportion of people with ideal feces (4 points) shape

grouping	0 days	20 days	Change
Group A	35.7% (15/42)	38.1% (16/42)	2.4% (1/42)
Group B	37.5% (18/48)	52.0% (26/48)	16.6% (8/48)
Statistical Analysis	P>0.05	P>0.05	P<0.05

Table 10 counts the proportion of people with ideal feces (4 servings) before and after taking yogurt for 20 days. The proportion of people with ideal feces (4 servings) in group A only increased by 2.4%, and the proportion of people in group B with ideal feces (4 servings) increased by only 2.4%. Compared with ordinary yogurt, the yogurt containing bifidobacteria can significantly improve the fecal morphology of the crowd.

Table 11 Defecation related symptoms

Table 11 counts the changes in the defecation somatosensory state of the crowd before and after taking yogurt for 20 days. It is subdivided into six aspects, including defecation pain, defecation incompleteness, defecation effort, defecation time, unsuccessful defecation times, and defecation discomfort. It shows that before and after taking yogurt, there is no significant change in defecation body sensation, and there is no significant change in yogurt in groups A and B, which may be related to the fact that the experimental population itself has no defecation disorder.

In summary, the experiment selected 110 healthy adults for the population experiment, and at the end of the experiment there were 90 valid populations. After a 20-day double-blind oral experiment, the results showed that compared with ordinary yogurt, taking yogurt containing Bifidobacterium IU-100 did not significantly affect the state of defecation, but the frequency of defecation and the score of stool morphology were significantly improved.

Example 7

Probiotic yogurt with Bifidobacterium IU-100 to make:

1. Materials and instruments

Raw milk: Shandong Deyi Dairy Co., Ltd. Gaoqing Ranch raw milk, white sugar, agar, starch, whey protein concentrate: protein content 34.5%, pectin, strains (commercial freeze-dried strains, Streptococcus thermophilus , Lactobacillus delbrueckii subsp. bulgaricus), IU-100 Bifidobacterium lactis (Shandong Deyi Dairy Co., Ltd. own strain).

DHP-9272 type electric heating constant temperature incubator, constant temperature water bath, JHG-Q54-P70 high pressure homogenizer, ZHJH-1109B sterile operating table, LDZX-40CI type vertical automatic electric heating pressure steam sterilizer, Haier refrigerator, IKA RW20digital Digital mixer.

2. Sample preparation

(1) Preparation of 0.1mol/L NaOH standard solution: Prepare according to the preparation of GB/T 601-2016 standard titration solution of chemical reagents.

3. Process flow

The process flow of product production is as follows:

4. Determination of physicochemical and microbial indicators

Acidity: Determined according to GB5413.34-2010 Determination of acidity in milk and other dairy products.

Fat: determined according to the Soxhlet extraction method in GB5009.6-2016.

Protein: Determined according to the Kjeldahl method in GB2009.5-2016.

Microorganisms: test according to GB4789.18-2010.

5. Test results

IU100 probiotic yogurt stops acidity 75°T;

Fat content 3.7g/100g;

Protein content 3.0g/100g;

Mold: not detected;

Yeast: not detected;

Pathogenic bacteria: 5, 0, 0, -;

Escherichia coli: 5, 2, 0, 0;

All indicators meet the requirements of GB19302-2010.

The number of viable Bifidobacterium lactis in IU100 probiotic yogurt during the shelf life:

The number of viable bacteria in 1 day was 9.2×10 ⁷ CFU/g;

The number of viable bacteria in 7 days was 8.8×10 ⁷ CFU/g;

The number of viable cells in 21 days was 5.4×10 ⁷ CFU/g.

Claims (4)

1. A Bifidobacterium animalis, characterized in that: the strain is Bifidobacterium animalis IU-100 in the genus Bifidobacterium, and the preservation number of the strain is CGMCC No.12942.
2. The animal bifidobacteria according to claim 1, characterized in that: using the animal bifidobacteria to improve the intestinal health of the crowd.
3. A method for selection and breeding of animal bifidobacteria described in claim 1, is characterized in that: comprise the following steps:

   (1) The fecal suspension of the longevity elderly immersed in the anaerobic diluent was vortexed to prepare a fecal suspension, serially diluted 10 times with the anaerobic diluent, poured into the plate with NPNL-X-Gal selective medium, and placed in an anaerobic bag. Incubate at 37°C for 40-60h with an anaerobic gas-producing bag to isolate potential bifidobacteria;

   (2) Pick blue colonies from the plate, and after Gram stain microscopy, select Gram-positive colonies with irregular bacilli, and continue to separate and purify 2-3 times until the potential bifidum is isolated. Bacillus pure culture;

   (3) carrying out a cell adhesion experiment with several pure cultures of bifidobacteria isolated above, and selecting a strain with excellent adhesion ability;

   (4) Bifidobacterium animalis IU-100 is obtained by carrying out strain identification of the above-selected Bifidobacterium pure culture.
4. An application of the animal bifidobacteria of claim 1, characterized in that: applied to the preparation of yogurt products.