CN108220206A - A kind of bifidobacterium longum and its application - Google Patents

Abstract

The invention discloses a bifidobacterium longum and application thereof, belonging to the field of biotechnology. The bifidobacterium longum YS108R strain provided by the invention has the characteristic of producing viscous exopolysaccharide, and can obviously improve the colitis model of mice induced by DSS. The strain has a good ability to tolerate simulated gastrointestinal fluid, can significantly reduce the disease activity index of mice during DSS induction, and has an effective protective effect on colon tissue. Moreover, the Bifidobacterium longum YS108R of the present invention is isolated from the intestinal flora of healthy people, has no toxic and side effects on the human body, and has certain advantages over traditional drug treatment. The bacterial strain can be used to make probiotic bacteria powder, fermented milk, etc., and has broad market prospects.

Description

A kind of bifidobacterium longum and its application

technical field

The invention relates to a bifidobacterium longum and an application thereof, belonging to the field of biotechnology.

Background technique

Inflammatory bowel disease (IBD) affects the health of millions of people every year and seriously affects the quality of life of patients. IBD is a type of chronic intestinal inflammation with recurrent features, mainly including two phenotypes of ulcerative colitis (UC) and Crohn's disease (CD). At present, the pathogenesis of IBD is still unclear, but more and more studies have shown that the pathogenesis of IBD is related to factors such as susceptibility genes, environmental factors, immune response disorders, intestinal flora imbalance, and intestinal barrier damage. Gut flora is an important part of the human body, and the interaction between gut microbes and intestinal mucosal immunity affects the initiation and regulation of immune responses. Disturbance of intestinal flora may lead to excessive or dysregulated immune response, resulting in damage to the intestinal mucosa. With the popularization of high-pass sequencing technology and the development of bioinformatics analysis methods, more and more research results have shown that patients with inflammatory bowel disease generally have intestinal micro-ecosystem disorders and a decrease in the diversity of intestinal flora.

The human intestinal flora is one of the most complex micro-ecosystems. There are a huge number of microorganisms colonized in the human intestinal tract. These microorganisms interact and co-evolve with the host, and are closely related to human health. A large number of research results have shown that gut microbes affect the proliferation of host intestinal cells and the development of the immune system, and they are associated with many human diseases, such as obesity, inflammatory bowel disease (IBD), intestinal pathogen infection, liver cirrhosis, diabetes, etc. . As the dominant bacteria in the human intestinal flora, bifidobacteria have a symbiotic relationship with the host and play an important role in maintaining normal intestinal flora and human health. Therefore, some specific bifidobacteria strains can be used as probiotics for the intervention treatment of certain intestinal diseases. Microbial exopolysaccharide (EPS) is a kind of macromolecular polymer synthesized and secreted by some microorganisms, which can improve the tolerance of the bacteria to the gastrointestinal environment and protect the bacteria from the immune system of the host. influences. The extracellular polysaccharides produced by some bifidobacteria can regulate the host's immune response, reduce the host's inflammation level, and play a preventive role in colitis. The drugs currently used to treat inflammatory bowel disease mainly include anti-inflammatory drugs, immunosuppressants, and antibiotics. These drugs usually only relieve the symptoms of the disease, but cannot achieve the purpose of treatment, and long-term use may cause side effects, such as allergies and Liver Disease. Bifidobacteria, as symbiotic bacteria in the intestinal tract of healthy people, have a certain immune regulation effect on the host through the interaction with the intestinal immune system, and have no toxic side effects on the human body. Exopolysaccharide is an important mechanism for bifidobacteria to produce immune regulation on the host. The functions of exopolysaccharides produced by different microorganisms are also different due to the difference in monosaccharide composition and molecular structure. Studies have shown that viscous exopolysaccharides generally have a large molecular weight and can inhibit immune responses, while low-molecular-weight exopolysaccharides can usually promote immune responses. Therefore, screening a strain of bifidobacterium that can synthesize sticky exopolysaccharide has great significance and broad prospects for the development of drugs for the treatment of colitis or other intestinal inflammatory diseases.

Contents of the invention

The first object of the present invention is to provide a kind of Bifidobacterium longum, said Bifidobacterium longum subsp.longum is Bifidobacterium longum subsp.longum YS108R, preserved on January 3, 2018 In Guangdong Microbial Culture Collection Center (GDMCC), the preservation address is Guangdong Institute of Microbiology, 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, and the preservation number is GDMCC No.60310.

In one embodiment of the present invention, the application of the bifidobacterium longum YS108R in the preparation of food and medicine with the effect of alleviating colitis.

The second object of the present invention is to provide a kind of bacterial powder and its preparation method, the main steps are as follows: After bifidobacterium longum YS108R is activated, inoculate into MRS culture medium according to the inoculation amount of 2-5%, 35-39 ℃ Anaerobic culture for 24h-36h, centrifuge at 8000-1200tmp for 20-30min, collect the sludge, wash it with normal saline for 2-3 times, add a freeze-drying protective agent according to 1-3 times the weight of the sludge, mix well and carry out vacuum freeze-drying , and finally obtain freeze-dried bacteria powder.

In one embodiment of the present invention, the MRS medium is MRS medium added with cysteine hydrochloride.

In one embodiment of the present invention, the components of the MRS medium are tryptone 8-10g, beef extract 8-10g, yeast powder 4-6g, glucose 18-22g, anhydrous sodium acetate 1.5-2.5g , Magnesium Sulfate Heptahydrate 0.4-0.6g, Manganese Sulfate Monohydrate 0.25-0.30g, Diammonium Hydrogen Citrate 1.5-2.5g, Dipotassium Hydrogen Phosphate Trihydrate 2.4-2.8g, Tween80 1-2mL, Cysteine Salt Acetate 0.4-0.6g, add distilled water to 1L, pH is 7.0-7.2.

In one embodiment of the present invention, the added amount of cystine hydrochloride is 0.02-0.08% by mass fraction.

In one embodiment of the present invention, the added amount of cystine hydrochloride is 0.05% by mass fraction.

In one embodiment of the present invention, the lyoprotectant consists of skim milk powder, trehalose, sucrose and water.

In one embodiment of the present invention, the components of the lyoprotectant are 100-120 g/L skim milk powder, 100-150 g/L trehalose, 150-200 g/L sucrose, and the rest water.

The third object of the present invention is to provide a kind of fermented milk and its preparation method, the preparation steps are as follows:

(1) Preparation of fermented milk raw materials: Mix milk powder, sucrose, and yeast powder with water at a ratio of 100-150g/L, 50-80g/L, and 1-2g/L, respectively, and then sterilize at 65-75°C 20-40min;

(2) Add 0.1-1‰ of a starter to the fermented milk raw material, ferment at 37-39°C for 4-8 hours to obtain a fermented milk product; Bacteria composition.

In one embodiment of the present invention, Bifidobacterium longum YS108R, Lactobacillus bulgaricus and Streptococcus thermophilus in the fermentation agent are 1-8:1:1.

The beneficial technical effects of the present invention are:

(1) The Bifidobacterium longum YS108R of the present invention is isolated from the intestinal flora of healthy people, and the bacterial strain has a better ability to tolerate simulated gastrointestinal fluid, and has no toxic and side effects on the human body. It has certain advantages over traditional drugs used to treat colitis. The bacterial strain can be used to make probiotic bacteria powder, fermented milk, etc., and has broad market prospects.

(2) The Bifidobacterium longum YS108R described in the invention can significantly reduce the disease activity index of mice during DSS-induced colitis. Compared with the model group mice, myeloperoxidase ( The level of MPO) was significantly reduced, the expression levels of IL-1β, IL-6, TNF-α and other inflammatory factors were also significantly reduced, and the expression levels of tight junction proteins ZO-1, Claudin-1, Occludin and other genes related to intestinal barrier Significantly higher than the model group, the expression level of the mucin gene MUC-2 was also significantly higher. The observation results of colonic pathological sections also showed that the colonic tissue structure of mice in the YS108R intervention group was relatively complete without serious damage.

biological material deposit

A kind of Bifidobacterium longum subsp.longum YS108R was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on January 3, 2018. The preservation address is Building 59, Compound 59, No. 100 Xianlie Middle Road, Guangzhou City Lou Guangdong Institute of Microbiology, preservation number is GDMCC No.60310.

Description of drawings

Fig. 1 is the transmission electron microscope observation result of bacterial strain of the present invention.

Figure 2 shows the changes in DAI index during the DSS modeling of mice in each group.

Fig. 3 is the pathological section of the mouse colon tissue in each group.

Figure 4 shows the gene expression levels of inflammatory factors TNF-α, IL-1β and IL-6 in the colon tissue of mice in each group.

Figure 5 shows the expression levels of tight junction proteins ZO-1 and Claudin-1 genes in the colon tissue of mice in each group.

Detailed ways

The technical solution of the present invention will be further described below in conjunction with specific embodiments, but it is not limited to this. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered by the present invention. within the scope of protection. The experimental methods of not specifying specific conditions in the following examples are all according to routine methods and conditions in this area.

The composition of MRS medium: tryptone 10g, beef extract 10g, yeast powder 5g, glucose 20g, anhydrous sodium acetate 2g, magnesium sulfate heptahydrate 0.5g, manganese sulfate monohydrate 0.25g, diammonium hydrogen citrate 2g, trihydrate Dipotassium hydrogen phosphate 2.6g, Tween801mL, cysteine hydrochloride 0.5g, add distilled water to 1L, pH 7.0-7.2.

Embodiment 1: the separation method of Bifidobacterium longum YS108R

The method for isolating Bifidobacterium longum, the main steps are as follows:

(1) The preparation of medium: added the MRS medium of 0.05% cysteine hydrochloride and 100mg/L mupirocin;

(2) After gradient dilution of the feces samples collected from healthy people, select appropriate 3 gradient dilutions, spread on the solid plate of the above medium, and place the plate upside down in an anaerobic incubator at 37°C for 48 hours;

(3) pick colonies with good wire-drawing effect and carry out streaking purification;

(4) Pick the purified colonies and inoculate them in liquid medium for anaerobic culture for 24-48 hours, collect the fermentation broth for the determination of exopolysaccharides, and keep a small amount of fermentation broth, collect the bacteria by centrifugation, and use them for genome extraction and PCR and 16S rDNA sequencing;

(5) Determination of extracellular polysaccharides: Take 10 mL of fermentation broth and centrifuge at 10,000 g for 15 minutes at 4°C, collect the supernatant, remove protein components by trichloroacetic acid precipitation, mix with three times the volume of ice ethanol, and stand at 4°C After overnight, centrifuge at 12000g for 10min at 4°C, discard the supernatant, dialyze the precipitate with a dialysis bag with a molecular weight cut-off of 8000-14000Da for 48 hours, and freeze-dry to obtain crude polysaccharide. The content of exopolysaccharide was determined by phenol-sulfuric acid method, and a strain with high production of viscous exopolysaccharide was screened out.

(6) After physiological and biochemical identification and 16S rDNA sequencing analysis, the strain was identified as Bifidobacterium longum subsp.longum, and the strain number was YS108R. The physiological and biochemical test results are shown in Table 1, and the 16S rDNA sequencing results are shown in SEQ ID NO.1.

Table 1 Physiological and biochemical test results of strain YS108R

Note: where + indicates that it can grow and produce acid in the medium with the carbohydrate as the only carbon source, - indicates that it cannot grow and produce acid in the medium with the carbohydrate as the only carbon source.

Example 2: Determination of the properties of Bifidobacterium longum YS108R

Determination of biological characteristics of Bifidobacterium longum:

(1) Observation method of bacterial characteristics: pick a small amount of colony and smear it on a glass slide, carry out Gram staining, and observe under an optical microscope. Bacteria characteristics: Gram staining is positive, grows well in the environment of pH 3.0-8.0, does not form spores, the bacteria is about 0.5-1.5μm×2-8μm, rod-shaped, rod-shaped or bifurcated Y-shaped, transmission Electron microscope observation results showed that there were obvious exopolysaccharides on the surface of the bacteria (accompanying drawing 1).

(2) Observation method of colony characteristics: Streak the strain on the MRS solid medium, culture until a single colony grows, and observe the colony morphology.

Colony characteristics: Form round and raised colonies on MRS solid medium, milky white, opaque, smooth surface, colony diameter of 1-3mm, with a drawing effect.

(3) Liquid culture characteristics: During the culture process of MRS liquid medium, the bacteria were evenly suspended in the culture, and rarely deposited at the bottom.

(4) Determination of survival rate in simulated gastric juice:

Determination of survival rate in simulated intestinal fluid: Artificial simulated gastrointestinal fluid needs to be freshly prepared. Pepsin was dissolved in PBS with pH 3.0 to make the final concentration 3g/L, and filtered through a 0.22 μm filter membrane to prepare simulated gastric juice. Trypsin was dissolved in PBS with pH 8.0 to a final concentration of 1 g/L, and filtered through a 0.22 μm filter membrane to prepare simulated intestinal fluid. Centrifuge the cultured bifidobacterium at 6000rpm for 10min at 4°C, collect the sludge, resuspend it with 0.85% normal saline, and adjust the density of the bacteria in simulated gastric juice (pH 3.0) to 1×10 ⁹ CFU/ mL. After mixing, place it at 37°C and incubate for 2 hours, then count the number of live bacteria. Take 1 mL of simulated gastric juice-treated bacterial solution and add it to 9 mL of simulated intestinal juice (pH 8.0), mix well and incubate at 37°C, and detect the number of viable bacteria after 4 hours. The percentage of the ratio of the number of viable bacteria after treatment to the initial number of viable bacteria was the survival rate.

The survival rate in simulated gastric fluid was 86.33%, and the survival rate in simulated intestinal fluid was 79.38%

(5) Determination of extracellular polysaccharides: Take 10 mL of fermentation broth and centrifuge at 10,000 g for 15 minutes at 4°C, collect the supernatant, remove protein components by trichloroacetic acid precipitation, mix with three times the volume of ice ethanol, and store at 4°C After standing overnight, centrifuge at 12000g for 10min at 4°C, discard the supernatant, dialyze the precipitate for 48h with a dialysis bag with a molecular weight cut-off of 8000-14000Da, and freeze-dry to obtain crude polysaccharide. The content of exopolysaccharides was determined by the phenol-sulfuric acid method.

When cultured in MRS liquid medium, the exopolysaccharide production was 150mg/L.

Embodiment 3: the alleviating effect of Bifidobacterium longum YS108R on DSS-induced colitis mouse symptoms

Mouse pathology experiment, the main steps are as follows:

(1) Prepare 40 male C57 mice and randomly divide them into 5 groups: normal control group (Control), model group (Model), Bifidobacterium longum YS108R intervention group (YS108R+DSS), Bifidobacterium longum C11A10B intervention group (C11A10B+DSS) and the commercial strain Bifidobacterium lactis BB12 intervention group (BB12+DSS), 8 mice in each group, the experimental scheme and the treatment methods of mice in each group are shown in Table 2.

(2) After the above strains were cultured for 24 hours, the bacteria were collected by centrifugation, and the bacteria were resuspended with normal saline, and the number of viable bacteria was adjusted to 5×10 ⁹ CFU/mL to make a bacterial suspension for intragastric administration of mice , orally administered dose of 0.2mL. On the 14th day, dextran sodium sulfate (DSS) was made into a solution with a concentration of 2.5% with physiological saline, and the mice were given to drink instead of drinking water for modeling, once every two days. During the DSS modeling period (15-21 days), the body weight, fecal status, and fecal occult blood of the mice were recorded every day to calculate the disease activity index (DAI). The scoring criteria are shown in Table 3.

Table 2 Mouse treatment scheme

Table 3DAI Scoring Criteria

The disease activity index of the mice in each group during the DSS modeling process is shown in Figure 2. From the second day after modeling, the DAI index of the mice in the other groups except the normal group began to increase, and the DAI index of the model group increased the fastest. At the end of modeling, the DAI index of the YS108R intervention group was significantly lower than that of the model group, while the DAI indices of the C11A10B intervention group and the BB12 intervention group were between the YS108R intervention group and the model group, but there was no significant difference with the model group, indicating that the present invention The strain YS108R has a certain improvement effect on the DSS-induced colitis mouse model.

Example 4: The protective effect of Bifidobacterium longum YS108R on the colon tissue of colitis mice

After DSS induction, the colonic tissues of mice showed obvious damage, including inflammatory cell infiltration, destruction of crypts and glandular structures, etc. The pathological sections of the colonic tissues of the treated mice in each group in Example 2 are shown in Figure 3. It can be seen that the colonic tissue structure of the YS108R intervention group is similar to that of the normal group (Control), with complete glands and crypts. cells were not significantly reduced. In the model group, the mucosal structure of the colon was almost completely destroyed, the inflammatory infiltration was severe, the crypt and gland structure disappeared, and the goblet cells almost completely disappeared. The colon tissue of the C11A10B intervention group was severely damaged, which was close to that of the model group. Compared with the model group, the colonic tissue of the BB12 intervention group was improved to some extent, but there was still obvious inflammatory infiltration, and the crypt structure was also severely damaged. It shows that YS108R has a good protective effect on mouse colon tissue.

Example 5: Effect of Bifidobacterium longum YS108R on the expression level of inflammatory factors in the colon tissue of colitis mice

The expression levels of inflammatory factors TNF-α, IL-1β and IL-6 genes in the colon tissue of mice in each group are shown in Figure 4. The expression levels of TNF-α, IL-1β and IL-6 genes in the YS108R intervention group were significantly lower than those in the model group, while the expression levels of TNF-α, IL-1β and IL-6 genes in the C11A10B intervention group were not the same as those in the model group. Significant difference, indicating that YS108R strain can effectively alleviate the inflammation level of DSS-induced colitis model.

Example 6: Effect of Bifidobacterium longum YS108R on the expression level of tight junction protein in the colon tissue of colitis mice

Tight junction proteins are an important part of the intestinal barrier and play an important role in maintaining the integrity of the intestinal epithelial barrier function. The expression levels of tight junction proteins ZO-1 and Claudin-1 genes in colon tissue of mice in each group are shown in Fig. 5 . The expression levels of ZO-1 and Claudin-1 genes in the YS108R intervention group were significantly higher than those in the model group, while the expression levels of ZO-1 and Claudin-1 genes in the BB12 intervention group were even lower than those in the model group, indicating that Bifidobacterium longum YS108R It has a good effect on maintaining the colonic barrier structure.

Combined with the results in Examples 2-6, it can be shown that Bifidobacterium longum YS108R can improve DSS-induced colitis by inhibiting the expression of inflammatory factors and maintaining the colonic barrier structure.

Embodiment 7: the making of Bifidobacterium longum YS108R freeze-dried bacteria powder

The preparation method of Bifidobacterium longum YS108R freeze-dried bacterial powder, concrete steps are as follows:

(1) Preparation of medium: MRS medium added with 0.05% cysteine hydrochloride;

(2) Preparation of protective agent: 120g/L skimmed milk powder, 100g/L trehalose, 150g/L sucrose and the rest of the water;

(3) After continuously activating the bacterial strain for 3 generations, inoculate it into the above-mentioned culture medium with an inoculum size of 2%, culture it anaerobically at 37°C for 24 hours, centrifuge at 8000g for 30 minutes, collect the sludge, wash it twice with normal saline, and press the bacteria Three times the weight of the mud is added with a freeze-drying protective agent, mixed evenly, vacuum freeze-dried, and finally the freeze-dried bacteria powder is vacuum-packed. The prepared bacteria powder has a live bacteria concentration of up to 1×1010 CFU/g, and can be used as a starter for the production of fermented milk.

Example 8: Production of Bifidobacterium longum YS108R and commercially available yogurt starter compound fermented milk

(1) Preparation of fermented milk raw materials: Mix milk powder, sucrose, and yeast powder with water at a ratio of 120g/L, 50g/L, and 1g/L, respectively, and then sterilize at 70°C for 30 minutes.

Add the bacteria powder described in Example 5 to the above-mentioned fermented milk raw material at 0.5‰, add commercially available yogurt starter (Lactobacillus bulgaricus and Streptococcus thermophilus) at the same time, and ferment at 37°C for 6-8 hours to obtain a fermented milk product . Gained fermented milk has good sensory and physical and chemical properties (as shown in Table 4), and mouthfeel is fine and smooth, slimy, has no bad taste, and local flavor has no obvious difference with traditional fermentation.

Table 4 Physicochemical indicators and sensory evaluation results of mixed fermented milk

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

sequence listing

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Claims (10)

1. Bifidobacterium longum, characterized in that, said Bifidobacterium longum (Bifidobacterium longum) is Bifidobacterium longum (Bifidobacterium longum) YS108R, preserved in Guangdong Microbial Culture Collection Center on January 3, 2018 (GDMCC), the preservation address is Guangdong Institute of Microbiology, 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, and the preservation number is GDMCC No.60310. 2. a preparation method of bacterial powder, it is characterized in that, the main steps of described method are as follows: after Bifidobacterium longum YS108R described in claim 1 is activated, inoculate in MRS culture medium by 2～5% inoculum size , anaerobic culture at 35-39°C for 24h-36h, centrifuge at 8000-1200tmp for 20-30min, collect the sludge, wash it with normal saline for 2-3 times, add freeze-dried protective agent according to 1-3 times the weight of the sludge, and mix well Carry out vacuum freeze-drying afterward, finally obtain freeze-dried bacteria powder. 3. The method according to claim 2, characterized in that, the MRS medium is the MRS medium added with cysteine hydrochloride. 4. The method according to claim 2 or 3, characterized in that the added amount of cystine hydrochloride is 0.02-0.08% by mass fraction. 5. The method according to any one of claims 2-4, characterized in that, the lyoprotectant consists of skimmed milk powder, trehalose, sucrose and water. 6. The method according to claim 5, characterized in that, the freeze-drying protective agent components are 100-120g/L skim milk powder, 100-150g/L trehalose, 150-200g/L sucrose, and the balance water. 7. the bacterial powder that application claim 2-6 arbitrary described method prepares. 8. the application of the bacterium powder described in claim 7 in preparing the food and medicine of the effect of alleviating colitis. 9. a fermented milk, characterized in that, the production steps are as follows: (1) Preparation of fermented milk raw materials: Mix milk powder, sucrose, and yeast powder with water at a ratio of 100-150g/L, 50-80g/L, and 1-2g/L, respectively, and then sterilize at 65-75°C 20-40min; (2) Add 0.1-1‰ of starter to the fermented milk raw material, ferment at 37-39°C for 4-8 hours to obtain fermented milk products; Bacteria composition. 10. fermented milk according to claim 9, is characterized in that, Bifidobacterium longum YS108R, Lactobacillus bulgaricus and Streptococcus thermophilus in the starter are 1-8:1:1 by volume.