CN112322553A

CN112322553A - Clostridium difficile resistant lactococcus lactis and application thereof

Info

Publication number: CN112322553A
Application number: CN202011379482.8A
Authority: CN
Inventors: 郭刚; 刘开云; 孙敏; 李彦; 王宁; 舒月力
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2021-02-05
Anticipated expiration: 2040-11-30
Also published as: CN112322553B

Abstract

The invention discloses a lactic acid lactococcus resisting clostridium difficile and application thereof, wherein the lactic acid lactococcus is preserved in China general microbiological culture Collection center with the preservation number as follows: CGMCC No. 20064. The lactococcus lactis strain grows well in a high-acid and high-cholate concentration environment, has high acid resistance and cholate resistance, has an obvious growth inhibition effect on clostridium difficile, has an obvious inhibition effect on clostridium difficile adhesion to human intestinal epithelial cells, and can inhibit the colonization of clostridium difficile in vivo and relieve inflammation caused by pathogenic bacteria infection in animal experiments. The strain has wide application prospect in medicaments, functional products and health products for preventing or treating intestinal diseases caused by clostridium difficile infection.

Description

Clostridium difficile resistant lactococcus lactis and application thereof

Technical Field

The invention belongs to the fields of microbial technology and microbial pharmacy, relates to a newly screened microorganism, and particularly relates to a clostridium difficile resistant lactococcus lactis and application thereof.

Background

Lactic Acid Bacteria (LAB) are a general term for a group of bacteria that can utilize fermentable carbohydrates to produce large amounts of Lactic acid. Since the isolation of lactic acid bacteria in 1873, researchers have conducted intensive studies on such bacteria, and the most studies are currently conducted on Lactobacillus (Lactobacillus) and Bifidobacterium (Bifidobacterium), among others. The research of Wangchang and the like finds that the streptococcus cremoris has broad-spectrum antibacterial activity, not only has stronger inhibiting effect on gram-negative bacteria such as escherichia coli and the like, but also has stronger inhibiting effect on gram-positive strains such as bacillus and streptococcus and the like (congratulating on Wei, Wangchang and the like, giving full attention to the wave and the like, screening of novel lactobacillus producing strains and research on strain characteristics [ J Chang Wei, Wangchang and the like]Amino acids and biological resources 2002,24(1): 22-25.). Bernet et al found that Bifidobacterium could inhibit the adhesion of E.coli to intestinal epithelial cells (Bernet MF, brain D, Neeser JR, service AL. addition of human bipolar strains to cut human intestinal epithelial cells and inhibition of intestinal epithelial cells J]Applied Environmental microbiology.1993,59(12):4121-4128.) lactic acid bacteria as a probiotic, during fermentation, produce a variety of antibacterial substances including organic acids, H₂O₂And bacteriocin and the like, thereby stopping the invasion of pathogenic bacteria, further protecting and repairing intestinal flora barriers and maintaining the capability of intestinal microecological balance.

Lactococcus lactis (l.lactis) is an important model bacterium in lactic acid bacteria. Lactococcus lactis is a common facultative anaerobic gram-positive bacterium in the human and animal intestines, does not produce spores, is one of the important sources of probiotics, and is called a class of food-grade Safe (GRAS) microorganisms widely existing in nature. Lactococcus lactis is one of the commonly used fermentation formulations in the fermentation industry, especially in the fermented dairy industry. The research finds that the lactococcus lactis can be used as a live carrier vaccine for mucosal immunity to present bacterial and viral antigens; the expression of cytokines or other therapeutic molecules by the lactococcus lactis strain system for the treatment of respiratory diseases; lactococcus lactis can also be used as a cell factory to produce medicines (zilin, foot and mouth disease lactococcus lactis live vector vaccine construction and mucosal immune effect [ D ]. Chinese academy of agricultural sciences, 2016.) lactococcus lactis is one of lactic acid bacteria with the most extensive application, and has great potential for being developed into microecological preparations (clever, Lifenglin, research and development status of lactobacillus preparations [ J ]. Anhui agronomy report, 2007(16): 42-44)

Clostridium difficile (c.difficile), also known as Clostridium difficile or bacillus pristinaespiralis, belongs to gram-positive anaerobic bacillus and is one of the main pathogenic bacteria of human intestinal infection. Difficile is normally present in the normal flora of the human gut, and other probiotics in the gut inhibit their excessive reproduction and degrade their produced toxins, thus not exhibiting pathogenicity. If the patient receives treatment of broad-spectrum antibacterial drugs, chemotherapeutic drugs or immunosuppressive agents for a long time, the intestinal flora can be disordered, Clostridium difficile overgrows and releases a large amount of toxins, Clostridium Difficile Infection (CDI) is caused, and clinical manifestations mainly include Clostridium difficile-associated diarrhea and pseudomembranous enteritis, and complications such as intestinal perforation, septic shock and the like are often accompanied, and even death is finally caused. Large-scale outbreaks of CDI have occurred for many times in developed countries such as Europe and North America, 30-40% of infected people die, and reports about CDI clinical cases have been reported in provinces such as Beijing, Shanghai and Henan of China, and experts predict that the CDI can become one of epidemic diseases threatening public health and safety in the future. Research suggests that the pathogenic mechanism of clostridium difficile is related to the toxin secreted by clostridium difficile, toxin a (TcdA) and toxin B (TcdB) are the main virulence factors of clostridium difficile, and toxin-specific antibodies can influence the infection and the titer of clostridium difficile in the body of clostridium difficile, and can effectively treat CDI and prevent the relapse of the clostridium difficile. However, currently, the formalin inactivated toxin vaccines of pasteur and feverfew which are the fastest in research progress are still in clinical trial stage, and no clostridium difficile vaccine is on the market.

According to statistics, China is one of the countries with serious abuse of antibiotics, and a series of problems are caused at present, such as appearance of super bacteria, diseases caused by dysbiosis of intestinal flora of human bodies, and the like. Therefore, finding alternatives to antibiotics is the focus of current research. Probiotics are widely proven to have the effects of inhibiting the growth of pathogenic bacteria and protecting intestinal mucosa, and researches show that lactic acid bacteria can adhere to host epithelial cells, compete and combine with adsorption sites of the pathogenic bacteria to form an effective biological barrier and inhibit the infection of the pathogenic bacteria (Caikaya, Huangzhangwang, Yedejun, Sun Chang Tao. probiotics regulate intestinal flora and immunoregulation action mechanism [ J ] Chinese feed 2011(18): 34-37.). Lactic acid bacteria products are used clinically for the treatment of diarrhea, peptic ulcer and constipation, without adverse effects, and are known as a safe and effective class of microbial agents (wu bin, smith. analysis of composition and rational use of common clinical microbial agents [ J ] journal of clinical drug therapy, 2010,8(05): 21-25.). Difficile is pathogenic only when the intestinal flora is disordered, so that the lactobacillus is used for inhibiting the colonization and growth of the difficile in the intestinal tract of a human body to become an attempted treatment method for CDI.

However, no probiotic product specially used for inhibiting clostridium difficile is available on the market, and considering that the incidence rate of clostridium difficile-associated diarrhea is continuously increased along with the wide use of broad-spectrum antibacterial drugs on the global scale, and outbreak prevalence appears in recent years, it is very necessary to develop the lactic acid bacteria and reduce the infection degree as daily food.

Disclosure of Invention

The invention aims to solve the problems and provides a lactococcus lactis strain with capacity of resisting clostridium difficile and application thereof, wherein the lactococcus lactis strain can tolerate pH 2.5 and 0.4% of cholate concentration, can inhibit growth of clostridium difficile in vitro and can be adhered to epithelial cells of human intestinal tracts, can effectively reduce death rate of mice infected by clostridium difficile in animal experiments, and has good bacteriostatic effect on clostridium difficile in vitro and in vivo.

In order to achieve the above purpose, the invention provides a clostridium difficile resistant lactococcus lactis which is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is as follows: CGMCC No. 20064.

In the invention, the Lactococcus lactis is separated from human gastric mucosal tissue, and the strain is identified by bacterial morphology, physiology and 16S rDNA sequencing, so that Lactococcus lactis (Lactococcus lactis) is named as HXLLC 20-1. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 6 months and 10 days in 2020, and the preservation unit is abbreviated as: CGMCC, storage unit address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a registration number: CGMCC No. 20064.

Lactococcus lactis HXL LLC20-1 has the following microbiological properties:

(1) morphological characteristics

The bacterium is a gram-positive strain, and is oval or spherical under a light mirror; an opaque milky colony is formed on an MRS solid culture medium, the surface is smooth and has bulges, and the edge is neat.

(2) Physiological properties

The lactococcus lactis can grow well under the conditions of pH 2.5 and 0.4% of cholate concentration, can inhibit the growth of clostridium difficile in-vitro culture experiments, has the diameter of an inhibition ring of 15.7 +/-0.3 mm, has the inhibition rate of 84.43% on the adhesion of clostridium difficile to human intestinal epithelial cells, and has obvious inhibition effect on the in-vivo colonization of clostridium difficile in animal experiments.

The method for culturing the lactococcus lactis resisting clostridium difficile comprises the steps of inoculating the lactococcus lactis HXL LLC20-1 into a culture medium, and culturing for 24-48 h at 28-30 ℃. The culture medium can be a culture medium conventional in the art, and can grow the lactococcus lactis HXL LLC20-1, preferably a MRS culture medium. The MRS culture medium is one of the conventional culture media in the field, and is prepared from 10.0g of beef extract, 5.0g of peptone, 5.0g of yeast extract, 20.0g of glucose, 2.0g of diammonium hydrogen citrate, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 801 mL of Tween, 0.2g of magnesium sulfate and 0.05g of manganese sulfate by adding water to dissolve the beef extract, and then fixing the volume to 1L, wherein the pH value is 6.4. The culture temperature is preferably 30 ℃ and the culture time is preferably 28 h.

The invention also provides application of the lactobacillus lactis resisting clostridium difficile in medicaments, functional products and health care products for preventing or treating intestinal diseases caused by clostridium difficile infection.

The medicament comprises various medicines, microbial preparations and other medicines, and the functional products comprise functional fermented fruit juice, functional yoghourt and other products. In addition, the foreign gene fragment is inserted into lactobacillus expression vector plasmids (such as pLA, pPG612, pMG36c, pMG36e and the like) to obtain recombinant plasmids; the lactococcus lactis which is used for expressing the recombinant protein coded by the foreign gene is obtained by transforming and introducing the recombinant plasmid into the lactococcus lactis, and the lactococcus lactis also belongs to the protection scope of the invention.

Compared with the prior art, the technical scheme provided by the invention has the beneficial effects that: the invention provides lactococcus lactis with clostridium difficile resistance and application thereof in medicaments, functional products and health-care products for preventing or treating intestinal diseases caused by clostridium difficile infection. The strain grows well in the environment with high acid and high cholate concentration, has higher acid resistance and cholate resistance, and is suitable for the environment of the digestive tract; the strain has obvious growth inhibition effect on clostridium difficile, has obvious inhibition effect on clostridium difficile adhered to intestinal epithelial cells of human, and also has obvious inhibition effect on clostridium difficile in vivo in animal experiments, thereby playing a role in regulating the microbial flora of the digestive tract, and having prevention and treatment effect on clostridium difficile infection.

Drawings

FIG. 1 is a colony morphology of lactococcus lactis HXL LLC20-1 on MRS medium in example 1;

FIG. 2 is an agarose gel electrophoresis of the PCR product of the pure strain isolated from example 1, wherein M is DNA marker III, lane 1 is about 1500bp of the amplified band of 16s rDNA of the strain, and lane 2 is a negative control;

FIG. 3 is a graph comparing the growth of lactococcus lactis HXL LLC20-1 in example 2 under different acid conditions;

FIG. 4 is a graph comparing the growth of lactococcus lactis HXL LLC20-1 in example 3 at different concentrations of bile salts;

fig. 5 shows the inhibition of lactococcus lactis HXLLC20-1 against clostridium difficile adhering to human intestinal epithelial cells in example 5, wherein experiment group I is a mixture of lactococcus lactis and clostridium difficile, experiment group II is a mixture of lactococcus lactis and clostridium difficile, and experiment group III is a mixture of clostridium difficile and lactobacillus lactis;

FIG. 6 shows the inhibition of lactococcus lactis HXL LLC20-1 on Clostridium difficile in the model of mice infected with Clostridium difficile in example 6, wherein A is the survival rate of mice with different dosages of Clostridium difficile, and B is the inhibition rate of lactococcus lactis HXL LLC20-1 on Clostridium difficile.

Detailed Description

The technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, belong to the present invention.

In the following examples, n repetitions are set for each treatment involved, i.e. each treatment is repeated n times with exactly the same operation, in order to increase the accuracy of the test; the n parallel experiments are set, namely n identical samples are taken from the same batch number and are tested under completely identical conditions, so that the single experiment is prevented from being in error and no control exists.

EXAMPLE 1 screening and identification of lactococcus lactis HXL LLC20-1

1. Screening of lactococcus lactis HXL LLC20-1

1.1 sample sources

The strain used in the present invention was collected from a healthy human gastric mucosal tissue as a sample.

1.2 preparation of the culture Medium

The culture medium used for sample separation and strain screening is MRS solid culture medium, and the components of the MRS solid culture medium are shown in Table 1; the agar in Table 1 was removed, and MRS liquid medium was obtained under the sterilization condition of 115 ℃ for 15 min. The reagents involved in the culture medium were purchased from Biotechnology (Shanghai) Inc.

TABLE 1 MRS solid Medium formulation

1.3 isolation and purification of the Strain

Human stomach tissue was grasped with forceps, and one side of the gastric mucosa was spread on an MRS agar plate (MRS solid medium) while streaking with an inoculating loop. The growth was observed after 48h incubation at 30 ℃. And (3) selecting a single colony on the plate according to different colony characteristics, further carrying out zonal streaking culture on an MRS plate, and repeating the steps twice to obtain a pure cultured strain.

2. Identification of lactococcus lactis HXL LLC20-1

2.1 morphological characteristics

Recording the morphological characteristics of the bacterial colony of the strain on an MRS agar plate, wherein the bacterial colony is a milky opaque bacterial colony as shown in figure 1, the surface of the bacterial colony is smooth and has bulges, and the edges of the bacterial colony are regular; and fixing a target strain smear, and observing cell characteristics by gram-stain microscopy, wherein the strain is gram-positive and is oval or spherical under a light microscope.

2.2 physiological and Biochemical identification

The physiological and biochemical characteristics of lactococcus lactis HXL LLC20-1 were identified with reference to the Manual of identification of common bacteria systems and the Manual of identification of Bergey bacteria, and a reference lactococcus lactis standard strain (BNCC195305) purchased from North Nay Bio Inc. was used as a control, and the results are shown in Table 2.

TABLE 2 physiological and biochemical identification results

Note: + positive and-negative

2.316S rDNA identification

Extracting single colony DNA of the screened strain as a template to amplify the strain 16S rDNA, adopting bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') to carry out a PCR experiment of the 16S rDNA, taking a PCR product to carry out 1.5% agarose gel detection and photographing after the PCR reaction amplification is finished, wherein the agarose gel electrophoresis detection result is shown in figure 2, and the size of a target band is about 1500 bp. Meanwhile, the PCR product is purified and then sent to Chengdu microgram-Tiantai company for sequencing, and the homology of the measured gene sequence result of the strain and the gene sequence in the National Center for Biotechnology Information (NCBI) of America is compared, and the result shows that the homology of the sequence and the 16S rDNA sequence of the lactococcus lactis is over 99 percent.

And determining the strain to be a Lactococcus lactis strain according to the sequence alignment result of the Lactococcus lactis HXL LLC20-1 and the combination of the observed colony characteristics, microscopic cell characteristics and physiological and biochemical identification results of the isolated strain.

Example 2 detection of acid resistance of lactococcus lactis HXL LLC20-1

The overall pH condition in the human gastric environment is strong acid, so the acid resistance of the strain is an important index for evaluating whether the strain can survive and colonize in the gastric acid environment. In order to simulate gastric acid environment, the isolated lactococcus lactis HXL LLC20-1 (hereinafter referred to as strain HXL 20-1) is cultured and analyzed in different acidic pH environments.

Preparing MRS liquid culture medium with pH of 2.5, 3.0, 3.5, 4.0, 4.5, culturing strain HXL LLC20-1 in 30 deg.C incubator for 18 hr, centrifuging at 4000rpm for 10min to collect thallus, washing thallus with Phosphate Buffer Solution (PBS) for three times, suspending thallus in PBS to obtain 1 × 10⁸CFU/mL bacterial suspension. The strain is sequentially inoculated in the five MRS liquid culture media with different pH values according to the inoculation amount of 2 percent, each MRS liquid culture medium with different pH values comprises 3 parts, then the MRS liquid culture media with the same pH values are respectively cultured for 2h, 4h and 6h at 30 ℃ and 220rpm, 100 mu L of bacterial liquid is respectively and uniformly coated on an MRS agar plate (MRS solid culture medium), the plate bacterial colony counting is carried out after the culture for 48h at 30 ℃, and the MRS liquid culture media with the pH value of 7.0 are used as a control, thereby determining the acid resistance of the strain. Each treatment was set to 3 replicates.

The growth of strain HXL LLC20-1 in MRS medium with different pH values is shown in FIG. 3, and it can be seen from the figure that the growth pattern of the strain is similar under the condition of pH 2.5-4.5, the number of bacteria at 2h is the lowest, but the growth activity is still maintained at 4h, and the number of bacteria at 6h is still slowly increased. It can be seen that the strain HXL LLC20-1 has a high degree of low acid tolerance.

EXAMPLE 3 determination of the bile salt resistance of lactococcus lactis HXL LLC20-1

After gastric digestion, the thalli can come to the small intestine, the small intestine has high concentration of bile salts, and the cell membrane function of the thalli can be damaged by the presence of the bile salts. Therefore, the tolerance degree of the tested thalli bile salt is also an important index for evaluating the survival efficiency of the probiotic agent, and in order to simulate the inhibition effect of the bile salt in the small intestine, the separated strain HXLLC20-1 is cultured and analyzed under the environment of different bile salt concentrations.

Preparing MRS liquid culture medium with bile salt concentration of 0.1%, 0.2%, 0.3%, 0.4%, culturing strain HXL LLC20-1 at 30 deg.C in incubatorCulturing for 18h, centrifuging at 4000rpm for 10min, collecting thallus, washing with PBS buffer solution for three times, and suspending thallus in PBS to obtain 1 × 10⁸CFU/mL bacterial suspension. Sequentially inoculating the cells in MRS liquid culture media with different cholate concentrations according to the inoculation amount of 2%, wherein each MRS liquid culture medium with each cholate concentration is 3 parts, then respectively culturing the MRS liquid culture media with the same cholate concentrations for 2h, 4h and 6h at 30 ℃ and 220rpm, and taking the MRS liquid culture media without adding cholate as a control. And 100 mul of the total bacterial strains are uniformly coated on an MRS agar plate, and the plate bacterial colonies are counted after being cultured for 48 hours at 30 ℃, so that the bile salt resistance of the bacterial strains is determined. Each treatment was set to 3 replicates. The survival rate formula is shown as formula (1):

survival rate (%) (live count after bile salt action/initial live count x 100% (1)

The change of viable count of lactococcus lactis HXL LLC20-1 after acting in MRS culture media with different concentrations of bile salts is shown in FIG. 4. It can be seen from the figure that bile salts have a certain inhibitory effect on the growth of the strain HXL LLC20-1, and the effect is enhanced with increasing concentration of bile salts. Furthermore, it can be seen that the number of viable bacteria in the experimental group after 2h slowly increased with time, indicating that the strain can tolerate this concentration of bile salts and maintain stable growth. The survival rates of the strains are respectively 93.67 percent and 79.24 percent when the concentration of the bile salt is 0.3 percent and 0.4 percent, and the viable count is 10⁶Above CFU/mL, the lactococcus lactis HXL LLC20-1 has better bile salt tolerance and meets the concentration of the bacteria of the lactic acid bacteria which play a probiotic role.

EXAMPLE 4 determination of inhibition level of Clostridium difficile by lactococcus lactis HXL LLC20-1

1. Culturing of bacterial strains

Clostridium difficile (BNCC186115) was purchased from North Nam Bio, inoculated in liquid Carex Medium (purchased from CYCLOKAY) and incubated at 37 ℃ in an anaerobic workstation for 48 h. The strain HXLLC20-1 is inoculated in MRS liquid culture medium, cultured for 24h at 30 ℃ and 220rpm, and centrifuged at 12000rpm for 10min to collect supernatant.

2. Bacteriostasis test

Performing Clostridium difficile on the separated strain HXL LLC20-1 by an Oxford cup bacteriostasis circle methodAnd (3) in vitro bacteriostasis test. Prepare a water agar (2%) plate as a lower culture medium, and uniformly place 4 sterilized oxford cups at a distance of about 2.5cm from the center of the plate after solidification. Adjusting the concentration of clostridium difficile bacterial liquid to 1 × 10⁸CFU/mL, and add it to the chilled 50 ℃ meat medium, shake it up and pour it slowly onto water agar that has been placed in an Oxford cup. 200 mu L of culture solution supernatant of lactococcus lactis is added into each Oxford cup, MRS liquid culture medium is added as negative control, the mixture is placed in an anaerobic workstation at 37 ℃ for culturing for 48 hours, the existence of the inhibition zone is observed, and the diameter of the inhibition zone is measured. 3 replicates were set up.

The diameter of the bacterial strain HXL LLC20-1 on the inhibition ring for the growth of clostridium difficile is shown in Table 1, and the result shows that the bacterial strain HXL LLC20-1 has a certain inhibition effect on the growth of clostridium difficile, the diameter of the inhibition ring can reach 15.7 +/-0.3 mm, and the bacterial strain has strong bacteriostasis.

TABLE 1 lactococcus lactis supernatant inhibiting the diameter of the zone of inhibition of Clostridium difficile

Example 5 lactococcus lactis HXL LLC20-1 inhibits Clostridium difficile adhesion to human intestinal epithelial cells

The bacterial strain with strong adhesiveness can stay in the digestive tract for a relatively long time, which is beneficial to the colonization of the bacterial strain in vivo, and simultaneously competes the adhesive site of pathogenic bacteria to weaken the pathogenicity of pathogenic bacteria. The inhibition effect of lactococcus lactis HXL LLC20-1 on the adhesive capacity of clostridium difficile is studied in vitro by taking human intestinal epithelial cell (HT29) cells as a model. HT29 cells (BNCC255089) were purchased from north nah bio and used to simulate in vivo adhesion of intestinal epithelium using a monolayer of HT29 cells.

Resuscitated activated HT29 cells were plated at 10⁵Perwell plated in 24-well cell culture plates at 37 ℃ with 5% CO₂The cells were cultured until 80% cells were adherent, and the cells were washed three times with PBS buffer. The inhibitory capacity against c.difficile was analyzed in different addition modes of strain HXLLC 20-1:

group I: to HT29 cells 200. mu.L of strain HXL LLC20-1 was addedBacterial suspension (1X 10)⁸CFU/mL), 5% CO at 37 ℃₂After co-culturing for 1 hour in the incubator, the cells were washed three times with PBS, and 200. mu.L of Clostridium difficile suspension (1X 10) was added thereto⁸CFU/mL), and continuously culturing for 1h under the same conditions;

group II: 200. mu.L of a suspension of strain HXL LLC20-1 (1X 10) was added to HT29 cells⁸CFU/mL) and Clostridium difficile (1X 10)⁸CFU/mL), 5% CO at 37 ℃₂Co-culturing for 2h in an incubator;

group III: to HT29 cells, 200. mu.L of a Clostridium difficile suspension (1X 10) was added⁸CFU/mL), 5% CO at 37 ℃₂After co-cultivation for 1h in the incubator, the cells were washed three times with PBS, and 200. mu.L of a suspension of strain HXL LLC20-1 (1X 10) was added thereto⁸CFU/mL), and continuously culturing for 1h under the same conditions;

control group: the control experiments in groups I, II and III were set up to add 200. mu.L of C.difficile suspension (1X 10) to HT29 cells⁸CFU/mL), 5% CO at 37 ℃₂Co-culturing for 2h in an incubator;

finally, the cells were washed three times with PBS in each of the three experiments to prepare cell suspensions, 100. mu.L of each of the experimental group and the control group was applied to a cycloserine-cefoxitin-fructose-yolk agar (CCFA) plate as an identification medium for Clostridium difficile, and the cells were cultured at 37 ℃ in an anaerobic workstation for 2-3 days, followed by comparison with the experimental group (N) on the plate₁) And control group (N)₂) C.difficile count. The formula of the inhibition rate is shown as formula (2):

inhibition ratio (%) - (1-N)₁/N₂)×100％ (2)

The results are shown in fig. 5, and it can be seen from the graph that the group I adhesion inhibition rate is 84.43%, the group II adhesion inhibition rate is 71.33%, and the group III adhesion inhibition rate is 43.67%, and it can be seen that the group I lactococcus lactis HXLLC20-1 has a significant inhibitory effect on clostridium difficile.

EXAMPLE 6 inhibition of Clostridium difficile by Strain HXL LLC20-1 in a model of mouse infection with Clostridium difficile

(1) Determination of Clostridium difficile bacterial liquid dose in mouse infection model

25 SPF-grade BALB/C female mice, 6 weeks old, after being adapted to feed for one week, are randomly divided into 5 groups (N is 5), wherein 4 experimental groups and one control group are respectively marked as an A group, a B group, a C group and a D group; the control group was designated as group E. For 4 experimental groups, the drinking water is added with antibiotic mixed liquor, and the antibiotic mixed liquor comprises the following components: kanamycin (0.4mg/mL), gentamicin (0.035mg/mL), colistin (850U/mL), metronidazole (0.215mg/mL), vancomycin (0.045mg/mL), treatment was continued for 3 days, followed by 2 days of sterile drinking water. The control mice had been drinking sterile water.

In the first 1 day of Clostridium difficile infection, all mice (experimental and control) were injected intraperitoneally with clindamycin (10 mg/Kg). Fasting and water deprivation are carried out from 17h before infection to 2h after infection. Feeding 0.3mL of gastric acid neutralizing solution to each mouse half an hour before infection, and feeding 0.4mL of bacterial solution with different concentration gradients to each mouse half an hour after infection, wherein the A group: 1X 10⁴CFU/mL, group B: 1X 10⁵CFU/mL, group C: 1X 10⁶CFU/mL, group D: 1X 10⁷CFU/mL, control mice (group E) gavage 0.4mL of PBS buffer. The mice were observed for diarrhea, arch back, death, etc. and were weighed for recording. The results are shown in FIG. 6A (in which the data of the control group is compared with 1X 10)⁴Coincidence of CFU/mL data), it can be seen from the figure that the infected bacterial fluid is 10⁶The mortality rate of CFU/mL mice on day six was 60%, and all mice in this group showed diarrhea within 1-2 days after infection, so 10 was selected⁶CFU/mL is the experimental infection dose.

(2) Inhibition rate of strain HXLLC20-1 on clostridium difficile

Re-taking 20 SPF-grade BALB/c female mice of 6 weeks old, randomly dividing into experimental group and control group (N ═ 10), and dividing into 10 groups⁶The bacterial liquid concentration of CFU/mL is the infection concentration, and the bacterial liquid concentration is used for carrying out clostridium difficile infection on the experimental group in the same treatment mode as that in the step (1). On day 2 post-infection, mice surviving the experimental group were randomly divided into two groups, group a with a suspension of strain HXL LLC20-1 (1X 10)⁸CFU/mL), once a day, for 6 consecutive days; group b did not do anything. Mice were observed for diarrhea, death and weighed for recording. The results are as followsAs shown in FIG. 6B, it can be seen that the mortality rate of the mice in the group is obviously reduced compared with that of untreated experimental group mice when the mice are perfused with lactococcus lactis liquid HXL LLC20-1 the next day after infection, which indicates that the strain plays an inhibiting effect on Clostridium difficile in the intestinal tracts of the mice and prevents Clostridium difficile from further attacking the intestinal tracts. From the aspect of diarrhea, the diarrhea symptoms of the mice in the group a are obviously relieved, and the change value of the body weight of the mice in the group a is increased by 37.16 percent compared with that in the group b, which shows that lactococcus lactis HXL LLC20-1 has an obvious inhibiting effect on clostridium difficile in vivo and can help to relieve the symptoms of clostridium difficile infection.

In conclusion, the invention screens out 1 strain of lactococcus lactis by isolated culture of human gastric mucosal tissue, and the strain is named as HXLLC 20-1. Experiments of in vitro acid resistance, cholate resistance and inhibition of growth and adhesion cells of clostridium difficile are carried out on the human gastric lactococcus lactis, the lactococcus lactis is proved to be strong in gastric environment resistance and have the effects of inhibiting growth and adhesion of clostridium difficile to gastric tissue cells, and then the lactococcus lactis is found to have an obvious inhibition effect on clostridium difficile in vivo through an SPF-grade BALB/c mouse model. Therefore, the strain is suitable for the digestive tract environment, has excellent capacity of resisting clostridium difficile, and has wide application prospect in resisting the pathogenic bacteria clostridium difficile in the intestinal condition.

It will be appreciated by those of ordinary skill in the art that the embodiments described herein are intended to assist the reader in understanding the principles of the invention and are to be construed as being without limitation to such specifically recited embodiments and examples. Those skilled in the art can make various other specific changes and combinations based on the teachings of the present invention without departing from the spirit of the invention, and these changes and combinations are within the scope of the invention.

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caaagatgat cccgcgttgt attagctagt tggtgaggta aaggctcacc aaggcgatga 240

tacatagccg acctgagagg gtgatcggcc acattgggac tgagacacgg cccaaactcc 300

tacgggaggc agcagtaggg aatcttcggc aatggacgaa agtctgaccg agcaacgccg 360

cgtgagtgaa gaaggttttc ggatcgtaaa actctgttgg tagagaagaa cgttggtgag 420

agtggaaagc tcatcaagtg acggtaacta cccagaaagg gacggctaac tacgtgccag 480

cagccgcggt aatacgtagg tcccgagcgt tgtccggatt tattgggcgt aaagcgagcg 540

caggtggttt attaagtctg gtgtaaaagg cagtggctca accattgtat gcattggaaa 600

ctggtagact tgagtgcagg agaggagagt ggaattccat gtgtagcggt gaaatgcgta 660

gatatatgga ggaacaccgg tggcgaaagc ggctctctgg cctgtaactg acactgaggc 720

tcgaaagcgt ggggagcaaa caggattaga taccctggta gtccacgccg taaacgatga 780

gtgctagatg tagggagcta taagttctct gtatcgcagc taacgcaata agcactccgc 840

ctggggagta cgaccgcaag gttgaaactc aaaggaattg acgggggccc gcacaagcgg 900

tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatactcg 960

tgctattcct agagatagga agttccttcg ggacacggga tacaggtggt gcatggttgt 1020

cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccctattgtt 1080

agttgccatc attaagttgg gcactctaac gagactgccg gtgataaacc ggaggaaggt 1140

ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga 1200

tggtacaacg agtcgcgaga cagtgatgtt tagctaatct cttaaaacca ttctcagttc 1260

ggattgtagg ctgcaactcg cctacatgaa gtcggaatcg ctagtaatcg cggatcagca 1320

cgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgggagttgg 1380

gagtacccga agtaggttgc ctaaccgcaa ggagggcgct tcctaaggta agaccgatga 1440

ctggggtgaa gtcgtaacaa 1460

Claims

1. The lactococcus lactis resisting clostridium difficile is preserved in the China general microbiological culture Collection center with the preservation number as follows: CGMCC No. 20064.

2. Use of lactococcus lactis against clostridium difficile according to claim 1, characterized in that: the application of the compound in medicaments, functional products and health care products for preventing or treating intestinal diseases caused by clostridium difficile infection.

3. Use of lactococcus lactis against clostridium difficile according to claim 2, characterized in that: the functional product comprises functional fermented fruit juice and functional yoghourt.