WO2022110282A1

WO2022110282A1 - Bifidobacterium longum subsp. longum i772, isolation and purification method therefor and use thereof

Info

Publication number: WO2022110282A1
Application number: PCT/CN2020/134226
Authority: WO
Inventors: 冯丽莉; 袁庆彬; 王世杰; 魏立华; 张栋; 薛玉玲; 荀一萍; 封肖颖; 刘尧尧
Original assignee: 君乐宝乳业集团有限公司
Priority date: 2020-11-27
Filing date: 2020-12-07
Publication date: 2022-06-02
Also published as: CN112458015B; CN112458015A

Abstract

A bifidobacterium longum subsp. longum i772, an isolation and purification method therefor and use thereof. The strain has been deposited in the China General Microbiological Culture Collection Center, with the deposit number of CGMCC NO. 19856. The bifidobacterium longum subsp. longum is obtained by enriching, screening and continuously purifying a target strain in the feces of breast-feeding infants or young children. Compared with the existing bifidobacterium longum subsp. longum, the screened bifidobacterium longum subsp. longum has the functions of regulating the intestinal flora balance and relieving the liver injury caused by alcohol. The strain is used for preparing beverages, foods, medicines or probiotic products having an intestinal regulation function.

Description

Bifidobacterium longum subsp. i772, its separation and purification method and application

technical field

The invention belongs to the technical field of bioengineering, and relates to a strain, a screening method and application thereof, in particular to a Bifidobacterium longum subsp. i772, a separation and purification method and application thereof.

Background technique

Drinking alcohol may lead to alcoholic liver disease (ALD). Ethanol can lead to the destruction of the intestinal barrier in ALD patients and the imbalance of flora. The levels of endotoxin in the plasma of patients without liver disease, alcoholic hepatitis and alcoholic liver cirrhosis are higher than those of normal people, which in turn triggers the body's inflammatory response.

The current measures to treat ALD mainly include alcohol abstinence and nutritional support as well as drug treatment. Drugs such as corticosteroids, pentoxifylline, anti-tumor necrosis factor alpha antibodies, and antioxidants have been used clinically, but the efficacy of these drugs is limited.

At present, probiotics such as lactic acid bacteria, bifidobacteria, and Lactobacillus acidophilus are widely used in the fields of bioengineering, food safety, and life and health. Therefore, the use of probiotics to regulate and intervene in the human body has become more and more extensive.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a kind of Bifidobacterium longum subsp. i772, so as to realize the purpose of regulating the balance of intestinal flora and relieving the liver damage caused by alcohol;

Another object of the present invention is to provide the preparation method of the above-mentioned Bifidobacterium longum subsp. longi i772, so as to achieve the purpose of rapid, effective and accurate screening of the bacterial strain;

The third object of the present invention is to provide the application of the above-mentioned Bifidobacterium longum subsp. i772.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A Bifidobacterium longum subspecies i772, the Bifidobacterium longum subsp. i772 is deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee, the preservation number is CGMCC NO.19856, and the Latin name is Bifidobacterium longum subsp.longum .

As a limitation of the present invention, the 16SrDNA gene sequence of the Bifidobacterium longum subsp. longi772 is as follows:

As another limitation of the present invention, the tuf gene sequence of described Bifidobacterium longum subsp. longi772 is as follows:

As a third limitation of the present invention, the Bifidobacterium longum subsp. i772 is isolated and screened from the feces of breast-fed infants or young children.

The present invention also provides a method for separating and purifying the above-mentioned Bifidobacterium longum species i772, comprising the following steps performed in turn:

S1. Collection of samples

Take the feces of breastfed infants or young children, add them into normal saline and mix thoroughly to obtain sample A;

S2. Sample enrichment

Take sample A, add it to the modified MRS liquid medium, and culture it anaerobically for 62-82 hours at 35-40°C to obtain culture solution B; wherein:

①The improved MRS liquid medium is an MRS liquid medium supplemented with 0.5‰ by mass cysteine;

② The volume ratio of sample A to the modified MRS liquid medium is 1:10-100;

S3. Strain isolation and screening

Take the culture solution B, dilute it with a 0.9% sterile physiological saline with a 10-fold gradient, and successively dilute it by 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ times, correspondingly to obtain bacterial suspensions C1 to C5 ;

Take the improved MRS solid medium, melt it, and pour it into the first and the fifth petri dishes respectively. After cooling and complete solidification, medium D1 to D5 are obtained; wherein, the improved MRS solid medium is per 1000 mL of the improved MRS. The solid medium obtained by adding 15% mass fraction of agar to the liquid medium;

Draw 0.1 mL of each of the bacterial suspensions C1 to C5, and apply them to the medium D1 to D5 one by one, then invert the plate and place it in an anaerobic culture at 35 to 40 °C for 62 to 82 hours to observe the growth of the colonies. ;

After typical colonies appear on the plate, select a typical single colony E;

S4. Strain purification

Pick the selected single colony E, streak the single colony E culture onto the modified MRS solid medium, and cultivate it in an anaerobic environment at 35 to 40 °C for 62 to 82 hours; cultivate three times continuously; obtain a pure culture F, which is The Bifidobacterium longum subsp. i772.

As the limitation of the above separation and purification method, the preservation method of the Bifidobacterium longum subsp. longi i772 strain is as follows: the pure culture F is mixed with sterile glycerol with a mass fraction of 50% in a ratio of 1:1, Store in a sterile environment at -80～-70°C, and inoculate it into the slope of a modified MRS solid medium test tube for temporary storage.

As another limitation of the above separation and purification method, the improved MRS liquid medium is composed of 10 parts by weight of casein peptone, 10 parts by weight of beef extract, 5 parts by weight of yeast extract, 20 parts by weight of glucose, 5 parts by weight of sodium acetate, 2 parts by weight of lemon Acid diamine, ₁ part by weight Tween _- 80, ₂ parts by weight _K2HPO4 , 0.2 part by weight MgSO4.7H2O, 0.05 part by weight _MnSO4.7H2O , _0.5 part by weight cysteine and 1000 volumes parts of distilled water; wherein, the corresponding ratio between parts by weight and parts by volume is g:mL.

The bacteriological properties of the Bifidobacterium longum subsp. i772 of the present invention are as follows:

(1) Strain Characteristic Experiment

The isolated and purified strains were cultured anaerobically at 35-40°C for 62-82 hours, and the characteristics of the strains were observed and tested respectively. The results are as follows:

实验项目experimental project	结果result	实验项目experimental project	结果result
革兰氏染色a	阳性positive	细胞形态Cell morphology	多形态杆状polymorphic rod

oxidase

-

contact enzyme

-

Note: "-" means no relevant basic features;

(2) Molecular biological identification

The above-mentioned strains were identified by molecular biology, extracted by DNA, amplified by PCR, and sequenced by 16Sr RNA and tuf gene sequences and the sequenced results were as follows:

The 16Sr gene sequence of Bifidobacterium longum subsp. longi i772 is:

The thf gene sequence of Bifidobacterium longum subsp. longi i772 is:

The gene sequence alignment was finally determined as Bifidobacterium longum subsp. longi i772.

The invention also provides the application of the above-mentioned Bifidobacterium longum subsp. i772, which is used for regulating the balance of intestinal flora and relieving the damage caused by alcohol to the liver.

As a limitation of the application of the above-mentioned Bifidobacterium longum subsp. longi i772, the Bifidobacterium longum subsp. longum i772 strain can be used to prepare beverages, foods, medicines or probiotic products with intestinal regulating function.

As a further limitation of the application of the above-mentioned Bifidobacterium longum subsp. longi i772, the probiotic product prepared by the Bifidobacterium longum subsp. longum i772 strain is a probiotic bacteria containing only the Bifidobacterium longum subsp. longum i772; or The compound probiotic is prepared by mixing the Bifidobacterium longum subsp. i772 with Lactobacillus paracasei N1115, Lactobacillus rhamnosus X253 and animal Bifidobacterium subsp. i797.

Owing to adopting the above-mentioned technical scheme, the technical progress achieved by the present invention compared with the prior art is:

Bifidobacterium longum subsp. i772 can effectively alleviate alcohol-induced liver damage. Lactine aminotransferase (also known as alanine aminotransferase, ALT) is mainly distributed in the hepatic cytoplasm, and aspartate aminotransferase (also known as aspartate aminotransferase, AST) is mainly distributed in the liver cytoplasm and hepatocyte mitochondria, γ- Glutamyl transpeptidase (GGT) is mainly distributed in capillary bile ducts and hepatocyte microsomes. When pathogenic factors (such as alcohol) cause hepatocyte degeneration and increased cell membrane permeability, ALT, AST and GGT are released , resulting in a significant increase in the levels of the three in serum. After using this strain, it can be detected that the content of ALT, AST and GGT in the patient's liver decreases, which proves that this strain has the function of alleviating the degree of liver damage caused by alcohol;

The bacteria also has a good role in regulating the intestinal tract;

The Bifidobacterium longum subsp. i772 provided by the invention has a wide application range, and can be used not only for preparing probiotic bacteria products, but also for preparing beverages, foods or medicines.

The present invention will be further described in detail below through specific embodiments, and it should be understood that the described embodiments are only used to explain the present invention, and do not limit the present invention.

Embodiment 1 A kind of Bifidobacterium longum subsp. longi i772 and its separation and purification method

1. Bacteria information

[Corrected 05.01.2021 according to Rule 26]
Bifidobacterium longum subsp. 1772 was isolated and screened from the feces of breastfed infants or young children. The bacteria have been deposited in the General Microbiology Center of the China Microorganism Culture Collection on May 20, 2020. The address of the depository : China Microorganism Culture Collection, Mailbox 2714, Beijing, China, Postal Code: 100080, Beijing (CN), deposit number CGMCCNO.19856, Latin name is Bifidobacterium longum subsp.longum.

2. Isolation and purification method of Bifidobacterium longum subsp. i772

The separation and purification method is carried out in sequence according to the following steps:

S1. Collection of samples

Using a sterile sampling spoon, take 20 g of feces from breast-fed infants or young children, add it to 100 mL of normal saline and mix thoroughly to obtain sample A;

S2. Sample enrichment

Take 2 mL of sample A, add it to 100 mL of modified MRS liquid medium, and culture it anaerobically for 62 to 82 hours at 35 to 40 °C to obtain culture solution B;

The improved MRS liquid medium is an MRS liquid medium supplemented with 0.5‰ by mass of cysteine;

The volume ratio of sample A to the modified MRS liquid medium is 1:10-100;

S3. Strain isolation and screening

Take 1 mL of culture solution B, and dilute it with 0.9% sterile physiological saline in a 10-fold gradient, successively by gradient dilution ^10-1 , ^10-2 , ^10-3 , ^10-4 , ^10-5 times, correspondingly to obtain Bacterial suspension C1～C5;

Take the modified MRS solid medium, melt it, and pour it into the first to fifth petri dishes respectively. After cooling and complete solidification, obtain medium D1 to D5, and absorb 0.1 mL of each bacterial suspension C1 to C5 and add them one by one. Correspondingly coated on the medium D1 ~ D5, then inverted the plate, placed in the environment of 35 ~ 40 ℃ anaerobic culture for 62 ~ 82h, observe the colony growth;

The modified MRS homogenous medium is a solid medium obtained by adding 15% by mass of agar to every 1000 mL of the modified MRS liquid medium;

After typical colonies appear on the plate, select a typical single colony E;

S4. Strain purification

Pick the selected single colony E, streak the single colony E culture onto the modified MRS solid medium, and cultivate it in an anaerobic environment at 35 to 40 °C for 62 to 82 hours; culture it continuously for three times; obtain a pure culture F, namely For the Bifidobacterium longum subsp. i772.

S5. Preservation of strains

Mix the pure culture F with sterile 50% glycerol in a ratio of 1:1, store it in a sterile environment at -80 to -70 °C, and at the same time inoculate it into the slope of a modified MRS solid medium test tube for use. Save temporarily.

Example 2 Bacteriological characteristics of the bacterial species of Bifidobacterium longum subsp. i772

1. Strain Characteristic Experiment

The isolated and purified strains were cultured anaerobic at 35 to 40 °C for 62 to 82 hours, and the characteristics of the strains were observed and tested respectively. The results are shown in Table 1:

Table 1. Basic characteristics of Bifidobacterium longum subsp. i772

experimental project

result

experimental project

result

革兰氏染色a	阳性positive	细胞形态Cell morphology	多形态杆状polymorphic rod
氧化酶oxidase	--	接触酶contact enzyme	--

Note: "-" means no relevant basic features.

2. Molecular biological identification

The above strains were identified by molecular biology, DNA extraction, PCR amplification, and 16Sr RNA sequencing of 16Sr RNA and tuf gene sequence of Bifidobacterium longum subsp. longi i772 were performed. in:

The 16S rDNA gene sequence of Bifidobacterium longum subsp. i772 is as follows:

The thf gene sequence of Bifidobacterium longum subsp. longi i772 is:

Example 3 Experiment of using Bifidobacterium longum subsp. longi i772 to alleviate alcoholic liver injury

①Experimental animals and materials

Experimental animals: 80 clean-grade healthy male SD rats, all weighing 130±10g

Experimental materials: basic feed stored at room temperature (Experimental Animal Center of Hebei Medical University), experimental bedding and rat cage

②Experimental strain: Bifidobacterium longum subsp. i772

③ Animal model establishment and grouping

All rats were housed in separate cages in the same room, 5 rats in each cage, with free access to food and water, and the circadian light cycle was 12h light and 12h dark. The ambient temperature is controlled at 23±2℃, and the humidity is controlled at 55%-65%. After the experimental animals were purchased, the experiment was started after 1 week of adaptive feeding in the animal breeding room.

Forty male SD rats were randomly divided into normal control group (n=8) and alcohol intervention group (n=32). The alcohol intervention group was divided into group A (blank group), group B (low-dose treatment group), group C (medium-dose treatment group), and group D (high-dose treatment group), with 8 rats in each group. The control group had a normal diet; the drinking water in the alcohol intervention group was replaced with 20% food-grade ethanol by volume.

In addition, group B was fed an aqueous solution of Bifidobacterium longum subsp. longi i772 probiotic powder with a viable count of 3×10 ¹⁰ CFU/mL at 10 mg/Kg every day according to the body weight of mice.

Group C was fed an aqueous solution of Bifidobacterium longum subsp. longi i772 probiotic powder with a viable count of 3×10 ¹⁰ CFU/mL at 50 mg/Kg every day according to the body weight of mice.

Group D was fed an aqueous solution of Bifidobacterium longum subsp. longum i772 probiotic powder with a viable count of 100 mg/Kg of 3×10 ¹⁰ CFU/mL every day according to the body weight of mice.

After 6 weeks of feeding, the eyeballs were removed after fasting for 12 hours, and blood was collected. After standing at room temperature for coagulation, centrifugation at 3000 r for 15 min, serum was collected, and stored at -80 °C for testing. After blood collection, the liver was quickly discarded, the wet weight of the liver was weighed, and the liver coefficient was calculated (liver coefficient=liver weight/body weight×100%).

The right lobe of mouse liver was taken and placed in 10% neutral formalin for fixation, routinely harvested, dehydrated, embedded in paraffin, sliced (4 μm thick), stained with HE, and examined by pathologists under a light microscope. .

Use detection kits to detect alanine aminotransferase (also known as alanine aminotransferase, ALT), aspartate aminotransferase (also known as aspartate aminotransferase, AST), γ-glutamyl transpeptidase (GGT).

Test results:

Note: * represents a significant difference compared with group A (p<0.05); ** represents a very significant difference compared with group A (p<0.01)

④Results and Analysis

ALT is mainly distributed in liver cytoplasm, and AST is mainly distributed in liver cytoplasm and liver cell mitochondria (the "machine" in liver cells that is specially responsible for providing cellular power). When the pathogenic factors lead to the degeneration of hepatocytes and the increase of cell membrane permeability, ALT is mainly released from the cells; when the hepatocytes are severely damaged and necrotic, the AST in the mitochondria is released, resulting in a significant increase in serum AST. GGT in the liver is mainly confined to capillary bile ducts and hepatocyte microsomes. It can reflect the necrosis and damage of hepatocytes, and can be used to diagnose and observe liver endocrine disorders, extrahepatic obstruction, and the process of alcoholic liver damage in cirrhosis.

After testing, high-dose use of Bifidobacterium longum subsp. i772 can significantly reduce the liver coefficient of mice; improve the characteristics of liver cases; and reduce the effects of ALT, AST, and GGT three liver disease indicators.

Example 4 Application of Bifidobacterium longum subsp. i772

Bifidobacterium longum subsp. longi i772 can be used to prepare beverages, foods, medicines or probiotic products with intestinal regulating function, and the preparation methods are all commonly used techniques in the art.

Claims

A Bifidobacterium longum subspecies i772 is characterized in that: the Bifidobacterium longum subspecies i772 is preserved in the General Microorganism Center of the China Microorganism Culture Collection Management Committee, and the preservation number is CGMCC NO.19856.
根据权利要求1所述的长双歧杆菌长亚种i772，其特征在于：其16SrDNA基因序列如下：AGTCGAACGGGATCCATCAGGCTTTGCTTGCTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCAGTTGATCGCATGGTCTTCTGGGAAAGCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGACGGCGGGGTAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGAGCAAGCGAGAGTGAGTTTACCCGTTGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGGTCGTAGAGATACGGCTTCCCTTCGGGGC GGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTATGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACGCGGCGACGCGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACCCCTTGTGGGATGGAGCC。
Bifidobacterium longum longum subspecies i772 according to claim 1, is characterized in that: its tuf gene sequence is as follows:

CCGCGGTATCACCATCAACATCGCCCACATCGAGTACCAGACCGAGAAGCGTCACTACGCTCACGTCGACTGCCCGGGCCACGCCGACTTCGTGAAGAACATGATTACCGGTGCTGCC CAGATGGATGGCGCTATCCTCGTTGTGGCCGCCACCGACGGCCCGATGGCCCAGACTCGCGAGCACGTGCTGCTCGCCCGTCAGGTTGGCGTTCCGAAGATCCTCGTCGCCCTGAACAAGTGCGACATGGTCGACGATGAAGAGCTCATCGAGCTCGTCGAAGAAGAGGTCCGCGACCTCCTCGACGAGAACGGCTTCGACCGTGACTGCCCGGTCATCCACACCTCCGCTTACGGTGCTCTGCACGACGACGCTCCGGACCACGAGAAGTGGGTCCAGTCCGTTAAGGACCTCATGGACGCTGTCGACGACTACATCCCGACCCCGGTTCACGACCTGGACAAGCCGTTCCTGATGCCGATCGAGGACGTCTTCACCATCTCCGGCCGTGGTACCGTTGTCACCGGTCGTGTCGAGCGTGGCCAGCTGGCCGTCAACACCCCGGTCGAGATCGTTGGTATCCGTCCGACCCAGCAGACCACCGTCACCTCCATCGAGACCTTCCACAAGACCATGGACGCCTGCGAGGCTGGCGACAACACCGGTCTGCTTCTGCGTGGTCTCGGCCGTGACGATGTCGAGCGTGGCCAGGTTGTGGCCAAGCCGGGCTCCGTCACCCCGCACACCAAGTTCGAGGGCGAAGTCTACGTGCTGACCAAGGACGAAGGCGGCCGTCACTCGCCGTTCTTCTCCAACTACCGTCCGCAGTTCTACTTCCGCACCACCGACGTCACCGGCGTCATCGAGCTGCCGG。
The Bifidobacterium longum subsp. i772 according to any one of claims 1-3, characterized in that it is isolated and screened from the feces of breast-fed infants or young children.
The method for separating and purifying Bifidobacterium longum subsp. i772 according to any one of claims 1-4, wherein the method for separating and purifying comprises the following steps that are carried out in turn:

S1 collects samples

Take the feces of breastfed infants or young children, add them into normal saline and mix thoroughly to obtain sample A;

S2. Sample enrichment

Take sample A, add it to the modified MRS liquid medium, and culture it anaerobically for 62-82 hours at 35-40°C to obtain culture solution B; wherein:

①The improved MRS liquid medium is an MRS liquid medium supplemented with 0.5‰ by mass cysteine;

② The volume ratio of sample A to the modified MRS liquid medium is 1:10-100;

S3. Strain isolation and screening

Take the culture solution B, dilute it with a 10-fold gradient with sterile normal saline with a concentration of 0.9%, and sequentially dilute 10-1 , 10-2 , 10-3 , 10-4 , and 10-5 times, corresponding to the obtained bacteria. Suspension C1～C5;

Take the modified MRS solid medium, melt it, and pour it into the first to fifth petri dishes respectively, and after cooling and complete solidification, obtain the medium D1-D5; wherein, the modified MRS solid medium is per 1000 mL of modified MRS The solid medium obtained by adding 15% agar by mass to the liquid medium;

Draw 0.1 mL of each of the bacterial suspensions C1 to C5, and apply them to the medium D1 to D5 one by one, then invert the plate and place it in an anaerobic culture at 35 to 40 °C for 62 to 82 hours, and observe the growth of the colonies. ;

After typical colonies appear on the plate, select a typical single colony E;

Purification of S4 strain

Pick the selected single colony E, streak the single colony E culture onto the modified MRS solid medium, and cultivate it in an anaerobic environment at 35 to 40 °C for 62 to 82 hours; culture it continuously for three times; obtain a pure culture F, which is The Bifidobacterium longum subsp. i772.
The method for separating and purifying Bifidobacterium longum subsp. i772 according to claim 5, characterized in that: the step

The following steps are also included after S4:

S5. Mix the pure culture F with sterile 50% glycerol in a ratio of 1:1, store it in a sterile environment of -80 to -70°C, and inoculate it on the slope of the modified MRS solid medium test tube at the same time. for temporary storage.
The method for separating and purifying Bifidobacterium longum subsp. longi i772 according to claim 5, wherein in the step S2, the improved MRS liquid medium is composed of 10 parts by weight of casein peptone, 10 parts by weight of beef extract, 5 parts by weight of yeast paste, 20 parts by weight of glucose, 5 parts by weight of sodium acetate, 2 parts by weight of diamine citrate, 1 part by weight of Tween-80, 2 parts by weight of K 2 HPO 4 , 0.2 parts by weight of MgSO 4 ·7H 2 O , 0.05 parts by weight of MnSO 4 ·7H 2 O, 0.5 parts by weight of cysteine and 1000 parts by volume of distilled water; wherein, the corresponding ratio between parts by weight and parts by volume is g:mL.
The application of Bifidobacterium longum subsp. longi i772 according to any one of claims 1-4, characterized in that: the bacterial strain is used for regulating the balance of intestinal flora and relieving the damage caused by alcohol to the liver.
According to the application of Bifidobacterium longum subsp. longi i772 described in claim 8, it is characterized in that: this strain is used for preparing beverage, food, medicine or probiotic product with intestinal regulating function.
The application of Bifidobacterium longum subsp. i772 according to claim 9, characterized in that: the probiotic product is a probiotic containing only the Bifidobacterium longum subsp. i772; Bifidobacterium longum subsp. i772 is mixed with Lactobacillus paracasei N1115, Lactobacillus rhamnosus X253 and animal bifidobacteria subsp. i797 to prepare compound probiotics.