WO2018014832A1

WO2018014832A1 - Applications of bacteroides fragilis in preparing composition for preventing vibrio parahaemolyticus infection

Info

Publication number: WO2018014832A1
Application number: PCT/CN2017/093401
Authority: WO
Inventors: 王晔; 智发朝; 刘洋洋; 白杨; 李正超; 王从峰
Original assignee: 广州普维君健药业有限公司
Priority date: 2016-07-18
Filing date: 2017-07-18
Publication date: 2018-01-25
Also published as: CN107629977A

Abstract

Provided are applications of a Bacteroides fragilis, specifically related to applications of Bacteroides fragilis ZY-312 of deposit number CGMCC No. 10685 in preparing a medicament or food product for preventing Vibrio parahaemolyticus infection. The Bacteroides fragilis comprises viable Bacteroides fragilis, inactivated Bacteroides fragilis, Bacteroides fragilis lysate and/or Bacteroides fragilis culture supernatant.

Description

Application of Bacteroides fragilis in preparing composition for preventing and treating Vibrio parahaemolyticus infection

Technical field

The invention relates to the technical field of application of Bacteroides fragilis, in particular to the application of Bacteroides fragilis in the preparation of a composition for controlling Vibrio parahaemolyticus infection.

Background technique

Vibrio parahaemolyticus is a Gram-negative bacterium, widely distributed in seafood such as fish and shellfish, and is an important halophilic food-borne pathogen in marine aquaculture. Vibrio parahaemolyticus is widely found in marine products such as seawater, seabed sediments, and fish, shrimps and crabs. It can cause diseases in fish, shrimp, crab and shellfish, and not only cause serious economic losses to the aquatic industry. And eating seafood contaminated or improperly processed by the bacteria can cause typical gastroenteritis reactions such as diarrhea, intestinal cramps, nausea, vomiting, and cause serious harm to human health. Cases of food poisoning caused by the bacteria have been reported all over the world, and the most affected countries and regions mainly include Japan, South Korea, Hong Kong, Taiwan and mainland China. Marine products that have contaminated a large number of such bacteria and have not been processed well may cause acute gastroenteritis or food poisoning after being consumed by humans. The "stolen disease" of shrimp that broke out in 2010 was caused by Vibrio parahaemolyticus. China's foodborne disease surveillance network data show that food poisoning caused by Vibrio parahaemolyticus has become the first bacterial food poisoning in coastal areas in China. In addition, the latest data from the US Centers for Disease Control and Prevention website (http://www.cdc.gov/foodborneburden/trends-in-foodborne-illness.html) also shows that since 2005, food poisoning caused by Vibrio parahaemolyticus has been shown. There is a significant global upward trend.

The strain is widely distributed, mainly distributed in seawater and aquatic products. The detection rate of Vibrio parahaemolyticus in seawater along the coast of East China is 47.5%-66.5%, and the average prevalence rate of marine fish and shrimp is 45.6%-48.7%. Up to 90% in summer. In addition to seafood, livestock, poultry, salted eggs, freshwater fish, etc. have been found to have the presence of Vibrio parahaemolyticus. Seawater is the source of pollution of the bacteria. The main route of transmission is through food, such as seafood, sea salt, salt pickles, etc. The common ones are crabs, crayfish, oysters, squid, sea bream, fish, yellow mud snails, etc. Eggs, meat or vegetables. In addition, patients with a history of intestinal disease and coastal carriers are also one of the sources of infection.

If the tools for processing food (such as consoles, knives, containers, storage rooms, etc.) are not cooked, the Vibrio parahaemolyticus in the raw food can contaminate other foods, especially cooked foods, through the above tools. In China, the pollution of some oyster products can reach 24000MPN/100g, and the oyster-borne seafood can easily cause other foods to be contaminated by the bacteria through various ways such as consoles, cutting boards, tableware, containers and refrigerators. Food contaminated with Vibrio parahaemolyticus, stored at higher temperatures, does not heat before consumption, or does not heat thoroughly, can cause food poisoning. In addition, Vibrio parahaemolyticus in coastal seawater and seabed sediments can also contaminate aquatic foods, and may also cause pollution of rivers, ponds and well water near the sea, so that freshwater products in the region are also subject to para-hemolytic arcs. Bacterial contamination.

It has been found that its virulence factors include heat-resistant hemolysin, heat-resistant hemolysin, adhesin, urease, lipopolysaccharide, type III secretion system, mucinase and iron-detecting system, but Vibrio parahaemolyticus causes food. The exact pathogenesis of poisoning has not been fully elucidated. Because Vibrio parahaemolyticus is one of the comorbidities of mermaid, it has attracted widespread attention from researchers in the prevention and treatment of gastroenteritis caused by Vibrio parahaemolyticus infection.

Bacteroides fragilis (B.f) is a model species of Bacteroides, which is commonly found in the lower digestive tract of mammals and is a commensal bacterium in the gut of humans and animals. It belongs to Gram-negative bacteria, rod-shaped, obtuse and densely stained at both ends, and there are obligate anaerobic bacteria with capsules, no spores and no power. In recent years, many studies have found that Bacteroides fragilis may have good development prospects as a potential probiotic strain. The infection of Vibrio parahaemolyticus in the treatment of Vibrio parahaemolyticus has not been reported yet.

Summary of the invention

Based on this, the present invention provides a novel application of Bacteroides fragilis.

The specific technical solutions are as follows:

Use of bacteroides fragilis in the preparation of drugs or foods for controlling Vibrio parahaemolyticus infection.

In some of these embodiments, the bacteroides fragilis is bacteroides fragilis ZY-312 with accession number CGMCC No. 10685.

In some of these embodiments, the Bacteroides fragilis comprises a Bacteroides fragilis live, a Bacteroides fragilis inactivated bacterium, a Bacteroides fragilis lysate, and/or a Bacteroides fragilis culture supernatant.

In some of these embodiments, the pharmaceutical dosage form is a pill, tablet, granule, capsule, oral solution, aerosol or tube feeding formulation. The medicament can be used in humans or animals.

In some of these embodiments, the food product is milk powder, ice cream, milk-based fermented food, or cereal fermented food. The food product may also be an animal food such as a feed or the like.

The invention also provides a medicament for preventing and treating Vibrio parahaemolyticus infection.

The specific technical solutions are as follows:

The invention relates to a medicament for preventing and treating Vibrio parahaemolyticus, wherein the active ingredient comprises bacteroides fragilis, and the bacteroides fragilis is bacteroides fragilis ZY with the preservation number CGMCC No. 10685. -312.

In some of these embodiments, the bacteroides fragilis comprises Bacteroides fragilis, Bacteroides fragilis inactivated, Bacteroides fragilis lysate, and/or Bacteroides fragilis culture supernatant.

The present invention also provides a food for preventing and treating Vibrio parahaemolyticus infection.

The specific technical solutions are as follows:

A food for controlling infection by Vibrio parahaemolyticus, comprising bacteroides fragilis, which is a bacteroides fragilis ZY-312 with the preservation number CGMCC No. 10685.

The bacteroides fragilis ZY-312 of the present invention has been deposited with the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on April 2, 2015, and its deposit number is CGMCC No. 10685, and the deposit address is No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing.

The inventors of the present invention have discovered a new application of Bacteroides fragilis through long-term experience accumulation and a large number of creative experimental studies, and have opened up a new application field of Bacteroides fragilis. The invention proves that the Bacteroides fragilis live bacteria, the fragile Bacteroides inactivated cells, the Bacteroides fragilis lysate solution and the Bacteroides fragilis culture supernatant have good control effects on the infection of Vibrio parahaemolyticus. This indicates that bacteroides fragilis have good edible and medicinal prospects. Bacteroides fragilis is an enteric bacterium, and its live bacteria, inactivated cells, lysate solution and culture supernatant can be used to prepare drugs or foods for controlling Vibrio parahaemolyticus infection.

DRAWINGS

1 is a colony morphology of anaerobic culture of Bacteroides fragilis according to the present invention;

Figure 2 is a microscopic examination of the Bacteroides fragilis of the present invention (1000 ×);

Fig. 3 is a graph showing the results of inhibition of B. parahaemolyticus-infected cells by Bacteroides fragilis of Example 2.

detailed description

The invention is further described in detail below with reference to the accompanying examples, which are intended to illustrate the invention and not to limit the scope of the invention.

The Bacteroides fragilis described in the following examples is bacteroides fragilis ZY-312. It was deposited on April 2, 2015 at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee, and its deposit number is CGMCC No. 10685, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

Example 1 Isolation of Bacteroides fragilis and preparation of samples

(1) Enrichment culture and sample preparation of Bacteroides fragilis

The strain was streaked into a blood plate, and cultured at 37 ° C for 48 hours under anaerobic conditions.

Characteristics of colony: Bacteroides fragilis ZY-312 showed round convexity, translucency, white, smooth surface and no hemolysis after culture for 48 hours on blood plate. The colony diameter was 1-3 mm, see Figure 1.

Microscopic morphology: Bacteroides fragilis ZY-312 was subjected to Gram staining microscopy. It was a Gram-negative bacterium with a typical rod shape. Both ends were obtuse and densely stained. The non-colored part of the cells was shaped like a vacuole. figure 2.

(2) Preparation of samples

1) Enrichment: A single colony was inoculated into TSB (tryptone soy broth containing 5% fetal bovine serum) for enrichment culture, and the obtained bacterial solution was stored for use.

2) Bacteroides fragilis live liquid: The bacterial liquid prepared in step 1) is determined by the number of bacteria in the M. turbidity tube, diluted with physiological saline to 10 ⁶ CFU/ml, 10 ⁸ CFU/ml, 10 ¹⁰ CFU/ Ml, the samples are labeled as sample A, sample B and sample C, respectively, and stored for later use.

3) Bacteroides fragilis inactivated bacteria solution: The Bacteroides fragilis inactivated bacteria solution is prepared by high temperature or ultraviolet irradiation. Specific steps: separately take 10 ⁶ CFU/mL, 10 ⁸ CFU/mL, 10 ¹⁰ CFU/mL of the prepared Bacteroides fragilis live liquid 5 mL in a beaker, and place the beaker containing the bacterial liquid in the step 2) The Bacteroides fragilis inactivated bacteria solution can be prepared by using a constant temperature water bath at 100 ° C for 20-30 min or in an ultraviolet environment for 30-60 min, and the samples are labeled as sample D, sample E and sample F, respectively.

4) Bacteroides fragilis lysate: The Bacteroides fragilis lysate was prepared by ultrasonication. Specific steps: using 10 ⁶ CFU/mL, 10 ⁸ CFU/mL, 10 ¹⁰ CFU/mL prepared by step 2), 5 mL of Bacteroides fragilis live bacteria, lysed by ultrasound for 30 minutes, On 10 seconds, Off 10 seconds The lysate efficiency of the system is >99%, and after centrifugation, it is centrifuged at 6000 rpm, 10 min, 4 ° C, filtered by a 0.22 μm filter, and used as sample G, sample H and sample I, respectively.

5) Bacteroides fragilis culture supernatant: The bacterial liquid prepared in step 1) is determined by the number of bacteria in the M. turbidity tube, diluted with physiological saline to 10 ⁶ CFU/ml, 10 ⁸ CFU/ml, 10 ¹⁰ CFU/ The above bacterial solution was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected, and the supernatant was filtered through a 0.22 μm microporous membrane, and the filtered supernatant was stored at 4 ° C until use. The samples were labeled as sample J, sample K, and sample L, respectively, and stored for later use.

Table 1 sample preparation method

Example 2 Effect of Bacteroides fragilis on cells infected by Vibrio parahaemolyticus

In this embodiment, the Bacteroides fragilis viable liquid, the inactivated bacterial solution, the lysate solution, and the culture supernatant prepared in Example 1 were used for the experiment. In this embodiment, Real Time Cell Analysis (RTCA) is used to observe the growth of adherent cells and the response state of adherent cells to external stimuli in real time. In this example, five sets of experiments were set up, taking LoVo cells and RAW264.7 cells as examples. The specific experimental steps are as follows:

1. Cell pretreatment

The cultured human colon cancer-derived LoVo cells (or RAW264.7 cells) were discarded, and the cells were digested with 2.5 mL trypsin solution at 37 ° C for 1 to 3 minutes. The cells were slightly rounded and loosened under the microscope. The pancreatin solution was discarded, and 5 mL of cell culture medium (DMEM/F12 (1:1) + 10% fetal bovine serum, the same below) was aspirated by a sterile pipette to blow down the digested cells to prepare a cell suspension. The suspension was centrifuged at 1000 rpm for 5 min at room temperature, and the supernatant was discarded. Resuspend the cells in a sterile pipette in 5 mL of medium, mix well, and count the blood cell count plate. After the counting, the cell suspension concentration was adjusted to 2 × 10 ⁵ /mL.

2. RTCA detector detects cell viability

1) Baseline assay: 100 μL of cell culture medium was added to each well of the RTCA cell culture plate, and the RTCA cell culture plate was placed in an RTCA detector to measure the baseline;

2) 300 μL of the above-mentioned LoVo cell (or RAW264.7 cell) suspension having a concentration of 2 × 10 ⁵ /mL was further added to each well of the above plate. The RTCA cell culture plate was placed in an RTCA detector, and the RTCA detector was set to be tested every 3 minutes, and placed in a 37 ° C, 5% CO ₂ incubator for 14 hours.

3) The RTCA detector was paused, the RTCA cell culture plate was taken out, 300 μl of the cell culture solution was discarded per well, and 300 μl of the LoVo cell (or RAW264.7 cell) suspension prepared in the step 1 was added to the test groups 1 to 5, respectively. The RTCA plates were re-inserted into the RTCA detector and placed in a 37 ° C, 5% CO ₂ incubator for 3 hours.

4) Wild Vibrio parahaemolyticus strain J5421 was inoculated into 5 ml of autoclaved 3.5% sodium chloride in LB liquid medium, and placed in a 37 ° C incubator for 8 hours. After transferring the passage 2 times at 1:50, the logarithmic bacterial solution was centrifuged at 4000 rpm for 10 minutes at room temperature, the supernatant was discarded, and the PBS was washed twice. The cells were resuspended in PBS and the bacterial solution OD600=1.0 was adjusted. The concentration of the bacteria was about 1×10 ⁹ CFU/mL. The wild Vibrio parahaemolyticus strain J5421 was diluted 10-fold with a cell culture medium at a concentration of about 1 × 10 ⁸ CFU/mL.

5) The RTCA detector was suspended, and the RTCA cell culture plate was taken out, and 1×10 ⁸ CFU/mL of the Vibrio hemolyticus wild strain J5421 25 μl prepared in the above step 4) was added to each of the 5 groups of RTCA cell culture plates. . Then, 25 μl of the sample B prepared in Example 1 (bacteria of Bacteroides fragilis) was added to each well of the RTCA cell culture plate of the experimental group 1; 25 μl of the preparation of Example 1 was added to each well of the RTCA cell culture plate of the experimental group 2 Sample E (Bacteroides fragilis inactivated bacterial solution); 25 μl of sample H (Bacteroides fragilis lysate solution) prepared in Example 1 was added to each well of RTCA cell culture plate of Experimental Group 3; RTCA cell culture in Experimental Group 4 25 μl of the sample K prepared in Example 1 (Bacteroides fragilis culture supernatant) was added to each well of the plate; 25 μl of PBS was added to each well of the RTCA cell culture plate of the experimental group 5 (control group). The RTCA plate was placed in the RTCA detector, placed in a 37 ° C, 5% CO ₂ incubator, and the adherent growth of LoVo cells (or RAW264.7 cells) and the response of adherent cells to external stimuli. Perform real-time dynamic observations.

3. Experimental results and analysis

The results of LoVo cells are shown in Fig. 3. As can be seen from Fig. 3, samples B (experiment group 1), sample E (test group 2), sample H (test group 3), and samples prepared in Example 1 of the present invention were added. After K (test group 4), although the normalized cell index (NCI) curve showed a downward trend, the decrease was much smaller than that of the experimental group 5 (control group). The experimental results of the experimental group 5 showed that the standardized cell index curve began to decrease gradually after adding Vibrio parahaemolyticus and PBS, indicating that LoVo cells could not properly adhere to the electrode at the bottom of the RTCA cell culture plate, suggesting that the LoVo cells were subjected to hemolysis. Vibrio virulence damage begins to loosen and fall off. The above results indicate that Vibrio parahaemolyticus has a strong toxic effect on normal growth of LoVo cells, and the addition of Bacteroides fragilis live bacteria, Bacteroides fragilis inactivated bacteria, Bacteroides fragilis lysate Both the solution and the culture supernatant have protective effects on LoVo cells, which can inhibit the infection of cells by Vibrio parahaemolyticus and reduce the damage of Vibrio parahaemolyticus to LoVo cells.

The results of RAW264.7 cells were similar to those of LoVo cells, and Bacteroides fragilis could also alleviate the infection damage of RAW264.7 cells by Vibrio parahaemolyticus.

The other samples provided in Table 1 of Example 1 of the present invention can also inhibit the cells infected by Vibrio parahaemolyticus, and have similar protective effects on LOVO cells and RAW264.7 cells, and the specific results are omitted.

The technical features of the above-described embodiments may be arbitrarily combined. For the sake of brevity of description, all possible combinations of the technical features in the above embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be considered as the scope of this manual.

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims

Use of bacteroides fragilis in the preparation of drugs or foods for controlling Vibrio parahaemolyticus infection.
The use according to claim 1, characterized in that the bacteroides fragilis is bacteroides fragilis ZY-312 with the accession number CGMCC No. 10685.
The use according to claim 1, characterized in that the bacteroids fragilis comprise live Bacteroides fragilis, Bacteroides fragilis inactivated bacteria, Bacteroides fragilis lysate and/or Bacteroides fragilis culture supernatant liquid.
The use according to any one of claims 1 to 3, characterized in that the pharmaceutical dosage form is a pill, a tablet, a granule, a capsule, an oral solution, an aerosol or a tube feeding preparation.
The use according to any one of claims 1 to 3, characterized in that the food product is milk powder, ice cream, milk-based fermented food or cereal fermented food.
A medicament for controlling infection by Vibrio parahaemolyticus, characterized in that the active ingredient comprises bacteroides fragilis, and the bacteroides fragilis is Bacteroides fragilis deposited under the accession number CGMCC No. 10685 ( Bacteroides fragilis) ZY-312.
The medicament according to claim 6, wherein the bacteroids fragilis comprises live Bacteroides fragilis, Bacteroides fragilis inactivated bacteria, Bacteroides fragilis lysate and/or Bacteroides fragilis culture supernatant liquid.
A food for preventing and treating Vibrio parahaemolyticus, characterized in that it comprises bacteroides fragilis, and the bacteroides fragilis is bacteroides fragilis with the preservation number CGMCC No. 10685. ZY-312.
The food according to claim 8, wherein the bacteroids fragilis comprises live Bacteroides fragilis, Bacteroides fragilis inactivated bacteria, Bacteroides fragilis lysate and/or Bacteroides fragilis culture supernatant liquid.