CN110373368B - Bifidobacterium longum strain ZJ1 and application thereof - Google Patents

Abstract

The invention discloses a bifidobacterium longum strain ZJ1 which is obtained by separating and screening excrements of elderly people with long life, is preserved in China center for culture collection of microorganisms in 2019, 6 and 26 months, and has the preservation number of CGMCC 18032. The survival rate of the strain is 78.47% after the strain is incubated in a pH 3.0 environment for 2h, 113.67% after the strain is incubated in a 0.1% cholate environment for 3h, 221% after the strain is incubated in a 0.1% trypsin environment for 3h, and 72.5% after the strain is treated in a 30mg/L hydrogen peroxide environment for 2 h; has good inhibition effect on escherichia coli, staphylococcus aureus, salmonella, pseudomonas aeruginosa, listeria monocytogenes and klebsiella pneumoniae.

Description

Bifidobacterium longum strain ZJ1 and application thereof

Technical Field

The invention relates to the technical field of probiotics, in particular to a bifidobacterium longum strain and application thereof.

Background

The probiotics is a kind of active microorganisms beneficial to the host, and is a general term for active beneficial microorganisms which are planted in the intestinal tract and the reproductive system of a human body to generate definite health efficacy, thereby improving the micro-ecological balance of the host and playing beneficial roles. The beneficial bacteria in human body and animal body mainly include lactobacillus, bifidobacterium and gram-positive coccus, wherein the number and species of bifidobacterium distributed in stomach and intestine are reduced with the increase of age stage, and the distribution is mostly in breast milk infant period.

Tissier, pasteur institute, france, 1899, first discovered and isolated an obligate anaerobe in the feces of breast-fed healthy infants, with a bifurcate end, named bifidobacterium, and set bifidobacterium in 1924. Currently, there are 32 species of bifidobacterium that have been identified, which are gram-positive bacteria, and have diverse forms, such as bent rod-like, L, V or Y-shaped forms.

The bifidobacteria are mainly inhabited in the gastrointestinal tract of a human body, are dominant bacteria in the intestinal tract of healthy people, have dynamic changes with age in type and quantity, and are closely related to a plurality of physiological and pathological changes in the organism. There is a natural tendency for the proportion of beneficial intestinal bacteria to the total number of bacteria to decrease year by year in a human lifetime. The infant time reaches 98%, the teenager time is kept about 40%, and the middle-aged time is reduced to 10%, so that people begin to show signs of body weakness and susceptibility to diseases; the number of bifidobacteria of the aged over 60 years is reduced to 1 percent, while the number of putrefactive bacteria such as clostridium aerogenes and escherichia coli is increased greatly, and finally, the intestinal tract is filled with the putrefactive bacteria, and the bifidobacteria almost disappear. Putrefying bacteria decompose food components in intestinal tract to generate toxic substances such as ammonia gas, amines, ammonium sulfide, skatole, indole, phenols and nitrosamine, and the toxic substances can accelerate aging and induce cancers to cause diseases such as arteriosclerosis and liver disorder after being absorbed by human bodies for a long time.

The Bifidobacterium can be used for treating chronic diarrhea, constipation, tumor, heart deficiency, and lactose digestion. Studies have shown that bifidobacteria have the following therapeutic effects: 1. maintaining normal intestinal flora balance, inhibiting pathogenic bacteria growth, preventing constipation, diarrhea and gastrointestinal disorder; 2. anti-tumor; 3. synthesizing vitamins and amino acids in intestinal tract and improving the absorption of calcium ions by organism; 4. reducing blood cholesterol level, and preventing and treating hypertension; 5. the lactose resistance of the dairy product is improved, and the digestibility is improved; 6. enhancing immunity, preventing antibiotic side effect, resisting aging, and prolonging life; 7. enhancing non-specific and specific immune response of the body; 8. control of endotoxemia; 9. the tolerance of the host to radiation is improved.

Disclosure of Invention

The invention protects bifidobacterium longum ZJ1 separated and screened from feces of the elderly with long life, which is preserved in China center for culture of microorganisms in 26.06.2019 with the preservation number of CGMCC 18032.

Separation and screening: taking 2g of a feces sample of the elderly with long life in an anaerobic workstation, carrying out gradient dilution by using sterile normal saline, and coating the feces sample on solid culture bases such as TPY, BL, BBL, BS, MRSL and the like for anaerobic culture at 37 ℃ for 24-48 h; selecting plates with moderate colony number, selecting milky white round colonies, and streaking for 2-3 times to obtain purified single colonies.

The invention also protects a microbial inoculum, and the active ingredients of the microbial inoculum contain the bifidobacterium longum strain.

The invention also provides a composition, which comprises the bifidobacterium longum strain; the invention also protects the application of the composition in food and health care products; the invention also protects the application of the composition in medicaments for inhibiting gastrointestinal pathogenic bacteria, medicaments for reducing cholesterol and anti-aging medicaments.

The survival rate of the bifidobacterium longum ZJ1 strain is 78.47% after being incubated in the environment with pH 3.0 for 2h, 113.67% after being incubated in the environment with 0.1% bile salt for 3h, 221% after being incubated in the environment with 0.1% trypsin for 3h, and 72.5% after being treated in the environment with 30mg/L hydrogen peroxide for 2 h; the degradation rate of cholesterol after 48h incubation with exogenously added 0.1mg/mL cholesterol was 24%.

The bifidobacterium longum ZJ1 strain has good inhibition effect on escherichia coli, staphylococcus aureus, salmonella, pseudomonas aeruginosa, listeria monocytogenes and klebsiella pneumoniae.

Bifidobacterium longum ZJ1 strain is sensitive to penicillin-G, vancomycin, rifampin, tetracycline and erythromycin, and resistant to streptomycin, kanamycin and norfloxacin.

Drawings

FIG. 1 is a schematic representation of the strain Bifidobacterium longum ZJ1 in morphology;

FIG. 2 is a schematic diagram of the colony of Bifidobacterium longum strain ZJ 1;

FIG. 3 is a phylogenetic tree of the 16S rDNA sequences of Bifidobacterium longum strain ZJ1 and related species.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention have been presented for purposes of illustration and description, and are not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.

Example 1

In an anaerobic workstation, 2g of a feces sample of Anhui Jinzhai Baisui old people in Changshouxiang is diluted with sterile normal saline in a gradient manner and coated on TPY, BL, BBL, BS, MRSL and other solid culture bases for anaerobic culture at 37 ℃ for 24-48 h; selecting plates with moderate colony number, selecting milky white round colonies, and streaking for 2-3 times to obtain purified single colonies.

According to the separation and screening conditions, liquid culture is respectively carried out on the separated strains, then the genome DNA of the strains is extracted, the 16S rDNA fragments are amplified by utilizing designed primers, the amplified 16S rDNA fragments are sequenced, the sequencing result is compared with NCBI database data, and the species classification of the strains is preliminarily judged.

According to the rules (trial implementation) of probiotic health food declaration and evaluation, the separated and screened strains are judged to belong to the strains of the probiotic health food, and the screened and purified strains are respectively subjected to solid culture and liquid culture.

Qualitative appraisal

According to Bergey's Manual of identification of bacteria and ' Manual of identification of common bacteria system ', the morphological characteristics and physiological and biochemical characteristics of the separated ZJ1 strain are combined with the prior observed strain, the primary identification of the separated ZJ1 strain can be carried out, the strain can be preliminarily judged to belong to the bifidobacteria, and the strain can not grow on a plate under the aerobic condition.

The strain is gram-positive bacteria, the cells of the strain are long and slightly bent, and the strain has various forms such as Y-shaped, V-shaped, long rod-shaped and bent arc-shaped, and is shown in figure 1. The colonies were smooth, convex, full-edged, creamy white, with a soft texture, as shown in fig. 2. The physiological and biochemical characteristics of the strain are shown in table 1.

Description of the symbols: "+", positive; "+^w", weak positive; "-", negative.

Table 1 identification of molecular biology

Extracting genome DNA by using a bacterial genome extraction kit, amplifying a 16S rDNA sequence by PCR, and comparing the 16S rDNA sequence in a database to obtain gene sequences of other strains with higher similarity, wherein the sequence sequences are detailed in a sequence table.

Multiple sequence alignment is carried out by using ClustalX 2.0 software, and then a phylogenetic tree is constructed by using MEGA5.0 software, and the taxonomic status of the strain is determined as shown in figure 3. The 16S rDNA sequence phylogenetic tree shows that the ZJ1 strain is located in Bifidobacterium longum.

Adopting MEGA5.0 software, adopting an ortho-ligation method to display that the bifidobacterium longum ZJ1 strain ZJ1 and a 16S rDNA sequence phylogenetic tree of related species are subjected to similarity repeated calculation for 1000 times, wherein the nodes of the phylogenetic tree only display Bootstrap values larger than 50% and the superscript T represents a model strain.

Example 2 animal toxicity test

80 SPF grade ICR mice (license number SCXK (Jing) 2016-. The animal is bred in an animal house (license number SYXK (Jing) 2017-.

Transferring the strain of Bifidobacterium longum ZJ1 after strain identification to MRS broth culture medium, placing at 36 deg.C, and anaerobically culturing for 2 days to obtain culture stock solution. And (4) taking part of the culture stock solution to concentrate by 5 times, and using the concentrated solution as a culture 5 times concentrated solution for later use.

80 ICR mice, half of the female and half of the male, were randomly selected into 4 groups of 10 mice each. The dose group is set as follows: a culture medium blank control group, a culture medium 5-time concentrated blank control group, a culture stock solution group and a culture 5-time concentrated group. The test substance is infused into the mouse with the BW dosage of 20.0mL/kg, the gastric perfusion is continuously carried out for 3 days, the observation is carried out for 7 days, and the weight change, the poisoning manifestation and the death condition of the mouse in the experimental process are recorded.

The results of viable cell count (0.1 mL of inoculum size) of Bifidobacterium longum ZJ1 strain in the stock culture are shown in Table 2, and the number of Bifidobacterium longum ZJ1 strains contained in the stock culture was 5.9X 107 CFU/mL.

The acute toxicity effect of the culture of the strain Bifidobacterium longum ZJ1 on mice is shown in Table 3, and the results show that the original culture solution and the 5-fold concentrated solution of the strain Bifidobacterium longum ZJ1 are continuously gavaged for 3 days at the dosage of 20.0mL/kg BW, and the mice are observed for 7 days, and no toxicity reaction or death of the tested mice is observed.

TABLE 2

TABLE 3

Example 3 tolerance test

After the probiotic product is taken in by oral cavity, probiotics enter the intestinal tract after staying for a short time (2-3h) in the stomach (pH is 2.0-3.0) along with food, and can suffer from stress factors in vivo such as gastric acid, pancreatic digestive enzyme, intestinal bile salt and the like. Bifidobacteria eventually colonize the large intestine downstream of the intestinal system, with about 0.03-0.3% of bile salts remaining under normal physiological conditions.

The experimental method comprises the following steps:

1. culturing the selected Bifidobacterium longum strain at 37 deg.C under anaerobic condition with the inoculum size of Bifidobacterium being 4%, respectively culturing 0h and 2h culture medium with different pH, diluting at appropriate ratio (observing turbidity condition of culture medium, adjusting), and plating for 24h for counting. The acid tolerance of bifidobacterium was evaluated, the results are shown in table 4, and it can be seen from table 4 that the survival rate of the ZJ1 strain after 2h incubation in an acidic environment which can tolerate pH 3.0 is 78.47%, indicating that it can normally survive at pH 3.0.

2. Culturing the selected Bifidobacterium longum strain at 37 deg.C under anaerobic condition with the inoculum size of Bifidobacterium being 4%, respectively culturing 0h and 3h bacterial solutions in different concentrations of bile salt culture medium (adjusting turbidity condition of culture medium), diluting at appropriate ratio, and plating for 24h for counting. The bile salt tolerance of bifidobacteria was evaluated and the results are shown in table 5. from table 5 it can be seen that the ZJ1 strain can divide and grow in an environment of 0.1% bile salt, 1% trypsin, which indicates that the bacteria can adapt to a simulated intestinal environment.

TABLE 4

TABLE 5

Example 4 bacteriostatic ability test

The experimental method comprises the following steps: the Oxford method determines the strong and weak inhibition effect of the bifidobacteria on escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, salmonella, klebsiella pneumoniae and listeria monocytogenes.

The glycerol-preserved pathogenic indicator bacteria were inoculated into TSB liquid medium and cultured for 8h, and diluted to OD600 ═ 1.0, followed by addition of saline 1: 103, diluting, coating the TSB solid culture medium with 200uL of the diluted bacterial suspension, and carrying out a bacteriostatic experiment after air drying. 3 Oxford cups are uniformly placed on each plate, wherein two Oxford cups are added with cultured bifidobacteria (OD is 1.0), the other Oxford cup is added with non-inoculated culture medium as a control, the cells are cultured for 24h at 37 ℃, the size of a bacteriostatic circle is measured by a caliper, the bacteriostatic ability of the bifidobacteria is evaluated, the cells are shown in table 6, the Bifidobacterium longum JZ1 has inhibitory action on escherichia coli, salmonella, pseudomonas aeruginosa, klebsiella pneumoniae, staphylococcus aureus and listeria monocytogenes, wherein the inhibitory action on the escherichia coli is strongest, and the inhibitory action on the klebsiella pneumoniae is weakest.

Indication of bacterial species	Bacteriostatic diameter (mm)
		Escherichia coli	18.9±0.4
Listeria monocytogenes	18.6±0.4
		Pseudomonas aeruginosa	17.2±0.2
Salmonella	12.0±0.5
		Klebsiella pneumoniae	11.8±0.7
Staphylococcus aureus	11.8±0.3

TABLE 6

Example 5 antibiotic susceptibility test

The antibiotics exert antibacterial or bactericidal effects by interfering cell wall synthesis (penicillin, vancomycin), damaging cell membranes (aminoglycosides: streptomycin, kanamycin), inhibiting bacterial protein synthesis (tetracycline: tetracycline; macrolide: erythromycin) and inhibiting nucleic acid synthesis (rifampin; quinolones: norfloxacin), and the like, and the sensitivity of bifidobacteria to 8 antibiotics is detected by a MIC method.

The experimental method comprises the following steps:

preparing a tested bacterium solution: the tested strain (bifidobacterium) is inoculated and cultured for 18h according to 1 percent, the OD600nm value of the tested strain is measured, the tested strain is firstly diluted to OD600 nm-0.4 by using MRS culture medium, and then the tested strain is diluted by 200 times by using MRS culture medium until the concentration of the strain liquid is 5.0 multiplied by 104 to 1.0 multiplied by 105 cfu/mL.

Preparing antibiotics: the maximum concentration for each antibiotic experiment (4096 ug/mL kanamycin and streptomycin, 256ug/mL erythromycin and rifampin, 128ug/mL penicillin and tetracycline, 1024ug/mL vancomycin and norfloxacin) was diluted with distilled water or absolute ethanol, and further diluted in 8-fold steps (each step required 0.5 mL).

Adding 75uL of antibiotics with different concentrations and 75uL of prepared bacteria liquid into each hole of a 96-hole plate, uniformly mixing, performing 3 parallel antibiotics, performing anaerobic culture at 37 ℃ for 24h, and observing. The minimum concentration of the bacterial suspension that did not grow was recorded as the MIC of the antibiotic and the results are shown in Table 7.

The results show that bifidobacterium longum ZJ1 is sensitive to cell wall targeted antibiotics including penicillin-G and vancomycin; resistant to antibiotics, including streptomycin and kanamycin, which are aminoglycosides, by damaging the cell membrane and inhibiting the synthesis of bacterial proteins; but are sensitive to erythromycin, including tetracyclines and macrolides, by inhibiting bacterial protein synthesis; sensitive to rifampin, an antibiotic that inhibits nucleic acid synthesis, and resistant to norfloxacin, quinolones.

Note: EFSA, the European food safety agency, ≧ the standard value is judged as "tolerated"; the < standard is judged as "intolerant".

TABLE 7

Example 6 Cholesterol degradation experiment

The majority of cholesterol in the human body comes from the diet. When food with high fat and high cholesterol is excessively taken, the content of blood cholesterol exceeds the standard, and various cardiovascular and cerebrovascular diseases are caused. Research shows that the serum cholesterol content of normal adults is 2.9-6.00mmol/L (1.1-2.3mg/mL), the high serum cholesterol is in positive correlation with the burst rate of coronary heart disease, and when the normal cholesterol level is higher than 1mmol, the burst rate of coronary heart disease is about 35% higher, and the death rate of coronary heart disease is about 45% higher; serum cholesterol is reduced by 1%, and the risk of coronary heart disease is reduced by 2-3%.

The experimental method comprises the following steps: inoculating the cultured Bifidobacterium for 18h into MRSL-CHOL culture medium at an inoculation amount of 1%, anaerobically culturing at 37 deg.C for 24h with an equal volume of uninoculated MRSL-CHOL culture medium as blank control, and determining the degradation rate of Bifidobacterium on cholesterol.

Centrifuging 3ml of bacterial liquid 5000rmp for 10min, putting 2ml of supernate into a 50ml centrifuge tube, adding absolute ethyl alcohol to 10ml, fully and uniformly mixing, centrifuging 4500 for 10min, putting 1ml of supernate into a 12ml centrifuge tube, slowly adding 2ml of ferric ammonium sulfate color developing agent (if the color developing agent is directly added into the supernate of the bacterial liquid to show dark color), immediately shaking up, standing at room temperature for 30min (for cooling to room temperature), measuring the OD 560nm value of each tube, and calculating the degradation rate of the bifidobacteria on cholesterol, wherein the result is shown in Table 8.

Formula for calculating degradation rate of cholesterol

Wherein A is the content of cholesterol in MRSL-CHOL medium before inoculation (namely blank control), B is the content of cholesterol after fermentation of bifidobacterium in MRSL-CHOL medium, and W is the cholesterol reduction rate.

The result shows that the degradation rate of cholesterol after the exogenous addition of 0.1mg/mL cholesterol is incubated for 48 hours is 24%, and the bifidobacterium longum ZJ1 can be used for developing the blood fat reducing product.

TABLE 8

Example 7 Hydrogen peroxide tolerance test

The strain to be tested is inoculated into a liquid MRSL culture medium in an inoculation amount of 1 percent, and is anaerobically cultured to the middle logarithmic phase (about 16h) under the condition of 37 ℃. Respectively diluting 30% hydrogen peroxide into solutions with different concentrations of 0-300mg/L by using MRS culture medium, inoculating 18h cultured Bifidobacterium longum with MRS culture medium without hydrogen peroxide as a control according to 4%, culturing for 2h under anaerobic condition at 37 ℃, diluting the solution in proper proportion, plating the solution, culturing for 24h for counting, and calculating and comparing the growth conditions of the Bifidobacterium longum under different hydrogen peroxide concentrations, wherein the results are shown in Table 9.

The survival rate was calculated as Sr% (a/a0) × 100, where a0 and a represent H, respectively₂O₂Viable count before and after the action, Sr% represents survival rate.

The results showed 0.3mg/L of H₂O₂Almost does not inhibit cell division of ZJ1 strain, and the cell can still proliferate; and 30mg/L of H₂O₂After the action, the cell division and the apoptosis of the strain reach the balance, and the total number of the cells is kept unchanged.

TABLE 9

It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by one of ordinary skill in the art and related arts based on the embodiments of the present invention without any creative effort, shall fall within the protection scope of the present invention.

Sequence listing

<110> Zhuo Yuan health science and technology Co., Ltd

<120> Bifidobacterium longum strain ZJ1 and application thereof

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Claims (6)

1. A Bifidobacterium longum strain is prepared from the excrement of longevity old people through separating and screening, named Bifidobacterium longum (Bifidobacterium longum)Bifidobacterium longum) ZJ1, and has been preserved in China center for culture Collection of microorganisms in 26.6.2019 with the preservation number CGMCC 18032.

2. A microbial preparation comprising the Bifidobacterium longum strain as claimed in claim 1 as an active ingredient.

3. A composition comprising the Bifidobacterium longum strain of claim 1 as an ingredient.

4. Use of the composition of claim 3 in food and health care products.

5. Use of a composition according to claim 3 for the manufacture of a medicament for the inhibition of gastrointestinal pathogenic bacteria.

6. Use of a composition according to claim 3 for the preparation of a cholesterol-lowering medicament.

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