Lactobacillus paracasei W1 and application thereof in inhibition of clostridium difficile infection

Abstract

The invention discloses a strain of lactobacillus paracasei W1 and application of the strain of lactobacillus paracasei W1 in inhibiting infection of clostridium difficile, and relates to the technical field of probiotic application, and the technical key point is that the preservation number of the lactobacillus paracasei W1 is CCTCC (China Center for Type Culture Collection) M 2024619, the preservation number of the lactobacillus paracasei W1 is CCTCC M 2024619, the preservation number of the lactobacillus paracasei W1 is CCTCC M 2024619, the preservation number of the lactobacillus paracasei W1 is CCTCC M 2024619, and the preservation number of the lactobacillus paracasei W1 is CCTCC M 2024619. The lactobacillus paracasei W1 has the capability of resisting low pH of gastrointestinal tracts and high-concentration bile salt, and the sterile fermentation supernatant of the lactobacillus paracasei W1 can inhibit the growth of clostridium difficile and the formation of a biological membrane. Animal experiments show that the lactobacillus paracasei W1 can inhibit colonization of clostridium difficile in vivo, improve injury of colonic mucosa, relieve shortening of colon and relieve inflammation caused by infection of the clostridium difficile. The bacterial strain has a wide application prospect in prevention and treatment of intestinal diseases caused by infection of C. difficile, as well as related dairy products, functional products and health-care products.

Description

Technical Field

[0001] The invention relates to the technical field of microbial engineering, and in particular to a strain of Lactobacillus paracasei (L. paracasei) W1 and application thereof in inhibiting Clostridium difficile (C. difficile) infection. Background Art

[0002] Clostridium difficile (C. difficile) is a Gram-positive anaerobic bacillus and a common pathogen of diarrhea in hospitalized patients. Clostridium difficile can form spores and has strong tolerance to physical and chemical factors such as heat, drying, and disinfectants, allowing it to survive for a long time in the environment. Therefore, spores are the main form of its spread among the population. Toxin A and toxin B are the main pathogenic factors of Clostridium difficile, both of which can act on the colon epithelium and immune cells, induce innate immunity and cause inflammatory responses. It is worth noting that toxin B may be a trigger for multiple organ dysfunction syndrome. At the same time, Clostridium difficile can acquire antibiotic resistance by forming biofilms and performing plasmid transfer, thereby affecting the therapeutic effect of antibiotics. Clostridium difficile infection (CDI) accounts for 30% of antibiotic-associated diarrhea (AAD). Clinical manifestations include mild to moderate diarrhea, pseudomembranous colitis, toxic megacolon, and even death. Currently, the treatment of Clostridium difficile infection (CDI) relies on antibiotics such as metronidazole, vancomycin, and fidaxomicin. However, with the increasing risk of bacterial resistance and recurrence of CDI, fecal microbiota transplantation (FMT) has gradually become a more advantageous alternative therapy for the treatment of recurrent CDI. FMT aims to treat CDI by restoring the diversity of intestinal microbiota, rebuilding the intestinal mucosal barrier, and restoring the metabolism of microbiota. Some studies have shown that FMT can not only treat colitis-related diseases, but also has the potential to be used in the treatment of irritable bowel syndrome, depression, and even cancer. Nevertheless, we should still consider the potential risks of FMT. FMT treatment may have some uncertain associations with certain diseases, including peripheral neuropathy, idiopathic thrombocytopenic purpura, Sjögren's syndrome, and rheumatoid arthritis. Therefore, new treatments with high safety are urgently needed in the clinic.

[0003] As the important role of probiotics in the treatment of colon inflammation becomes increasingly prominent, the antibacterial activity of specific probiotics against Clostridium difficile is still not fully understood. Studies have shown that a probiotic combination containing Lactobacillus acidophilus CL1285 and Lactobacillus casei LBC80R is well tolerated and can effectively reduce the risk of diarrhea associated with antibiotics. In addition, a meta-analysis showed that compared with other lactobacilli, Lactobacillus casei is more effective and safer in preventing antibiotic-associated diarrhea. However, there are currently no in vitro and in vivo studies on the inhibition of Clostridium difficile by Lactobacillus paracasei at home and abroad.

[0004] Through the above analysis, the problems and defects of the existing technology are as follows: At present, there is no probiotic product specifically used to inhibit Clostridium difficile on the domestic market. Therefore, the development of such lactic acid bacteria and using them as daily food to reduce the degree of infection has great application potential. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems and provide a strain of Lactobacillus paracasei W1 and an application thereof in inhibiting Clostridium difficile infection. The strain of Lactobacillus paracasei W1 has the ability to tolerate low pH and high concentration of bile salts in the gastrointestinal tract, and its sterile supernatant can inhibit the growth and biofilm formation of Clostridium difficile in vitro. In animal experiments, the bacteria can effectively reduce the mortality of mice infected with Clostridium difficile, and has a good antibacterial effect on Clostridium difficile both in vitro and in vivo.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] A strain of Lactobacillus paracasei (L. paracasei) W1 is characterized in that the Lactobacillus paracasei preservation number is CCTCC M 2024619.

[0008] Furthermore, the nucleotide sequence of 16S rRNA of Lactobacillus paracasei W1 is shown in SEQ ID No.1

[0009] Application of a strain of Lactobacillus paracasei (L. paracasei) W1 in inhibiting Clostridium difficile (C. difficile) infection.

[0010] Furthermore, the Lactobacillus paracasei (L. paracasei) W1 sterile supernatant inhibits the growth of Clostridium difficile and the formation of biofilm.

[0011] The invention discloses an application of a strain of Lactobacillus paracasei (L. paracasei) W1 in the preparation of dairy products, functional products and health products for preventing and treating intestinal diseases caused by Clostridium difficile (C. difficile) infection.

[0012] Furthermore, the content of Lactobacillus paracasei in the product is not less than 1.0×109 cfu / mL or 1.0×109 cfu / g.

[0013] Compared with the prior art, the beneficial effects of this solution are as follows: the Lactobacillus paracasei W1 of the present invention has good tolerance to gastrointestinal fluid, and its sterile supernatant can effectively inhibit the growth of Clostridium difficile in vitro, and has good therapeutic and preventive effects on infections caused by Clostridium difficile. It has application prospects in the preparation of dairy products, functional products and health products that can prevent or treat intestinal diseases caused by Clostridium difficile infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a colony morphology diagram of Lactobacillus paracasei W1 in an embodiment of the present invention;

[0015] Figure 2 is a Gram staining image of Lactobacillus paracasei W1 in an embodiment of the present invention;

[0016] Figure 3 Schematic diagram of the effect of the sterile supernatant of Lactobacillus paracasei W1 on the growth of Clostridium difficile in an embodiment of the present invention;

[0017] Figure 4 Schematic diagram of the effect of the sterile supernatant of Lactobacillus paracasei W1 on the biofilm formation of Clostridium difficile in an embodiment of the present invention;

[0018] Figure 5 Schematic diagram of the effect of Lactobacillus paracasei W1 on the colon length of CDI mice in an embodiment of the present invention;

[0019] Figure 6 Schematic diagram of the effect of Lactobacillus paracasei W1 on the survival rate of CDI mice in an embodiment of the present invention;

[0020] Figure 7 Schematic diagram of the effect of Lactobacillus paracasei W1 on CSS score of CDI mice in an embodiment of the present invention;

[0021] Figure 8 3 is a graph showing the effect of Lactobacillus paracasei W1 on intestinal tissue damage in CDI mice in an embodiment of the present invention. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0023] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0024] The Lactobacillus paracasei W1 provided by the present invention is taxonomically named Lacticaseibacillus paracaseiW1, was deposited in the China Center for Type Culture Collection (CCTCC) on April 3, 2024, with a deposit number of CCTCC M2024619, and the deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0025] The deposit number of the strain of Lactobacillus paracasei is CCTCC M 2024619.

[0026] The content of Lactobacillus paracasei provided by the present invention in the product is not less than 1.0×10 9 CFU / mL or 1.0×10 9 CFU / g.

[0027] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0028] Isolation and purification of Lactobacillus paracasei W1:

[0029] Take 1 mL of normal human oral saliva sample and add it to 9 mL of sterile water. After fully shaking, dilute it. Take 100 μL of the sample suspension with a dilution of 10-7 times and spread it on MRS solid culture medium. Incubate it upside down in an anaerobic incubator at 37°C for 48 hours. After a single colony grows on the plate, pick a single colony for purification. Repeat this process more than three times until a pure colony is cultured.

[0030] Embodiment 1: Lactobacillus paracasei W1 has the following microbiological characteristics:

[0031] (1) Colony characteristics: The bacterium forms milky white colonies on MRS solid medium. The colony surface is smooth, moist, and has protrusions. The edges are neat, opaque, and do not produce pigment. See Appendix. Figure 1 .

[0032] (2) Staining characteristics: Gram-positive, blue, rod-shaped or bar-shaped under light microscopy, see Appendix Figure 2 .

[0033] (3) Growth characteristics: The cells reached the late logarithmic phase after being cultured in MRS medium at 37°C under aerobic or anaerobic conditions for about 16 hours.

[0034] (4) Carbohydrate fermentation test of Lactobacillus paracasei W1. Lactic acid bacteria biochemical identification tube (purchased from Hangzhou Microbiological Reagent Co., Ltd.) was used to identify the reaction of Lactobacillus paracasei W1 to 11 kinds of carbohydrates.

[0035] The results are shown in Table 1. It can be seen from Table 1 that Lactobacillus paracasei W1 can utilize, decompose and metabolize 11 carbohydrates including esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, inulin, lactose and 1% sodium hippurate in the 11 carbohydrate fermentation tests.

[0036] Table 1 Biochemical identification results of Lactobacillus paracasei W1

[0037]

[0038] Example 2: Lactobacillus paracasei W1 has good tolerance to simulated gastrointestinal fluid

[0039] The lactobacillus paracasei W1 of cryopreservation is inoculated in the MRS solid medium, anaerobic culture is carried out at 37 ℃ for 48h, after MRS solid medium passage culture 3 times, get single colony inoculation in the liquid MRS medium and cultivate 24h, get the culture fluid of 1mL lactobacillus paracasei W1 respectively afterwards and be inoculated in the MRS liquid medium of 9.0mL pH=2 and pH=3 and set control group. Take out after anaerobic culture 3h at 37 ℃ then, be coated with 100 μ L on the MRS solid medium after the doubling dilution, take out after anaerobic culture 48h. Finally, count viable count on the plate and calculate its survival rate (see Table 2).

[0040] Survival rate = viable bacterial count on the plate after MRS treatment at pH = 2 or pH = 3 / viable bacterial count on the MRS plate × 100%.

[0041] Table 2 Determination of acid resistance of Lactobacillus paracasei W1

[0042]

[0043]

[0044] 1 mL of the culture fluid of Lactobacillus paracasei W1 was inoculated into 9.0 mL of MRS liquid medium containing 0.3% ox bile salt and MRS liquid medium without ox bile salt, respectively, and taken out after anaerobic culture at 37°C for 3 h. After multiple dilution, 100 μL was spread on MRS solid medium, and taken out after anaerobic culture for 48 h. The plate colony count was performed, the number of viable bacteria was determined, and the survival rate was calculated (see Table 3).

[0045] Survival rate = live bacteria count on the plate after MRS treatment with 0.3% ox bile salt / live bacteria count on the MRS plate × 100%,

[0046] Table 3 Determination of bile salt resistance of Lactobacillus paracasei W1

[0047]

[0048] The experimental results are shown in Table 2 and Table 3. The results show that Lactobacillus paracasei W1 has good acid resistance and bile salt resistance.

[0049] Example 3: Effect of Lactobacillus paracasei W1 sterile supernatant on the growth of Clostridium difficile

[0050] After Lactobacillus paracasei W1 was revived and subcultured 2-3 times, it was inoculated in MRS liquid medium, and after anaerobic culture at 37°C for 48h, the fermentation supernatant of Lactobacillus paracasei W1 was collected, centrifuged at 12000g for 10min, filtered with a 0.22μm filter membrane to obtain a sterile supernatant (Cell-free supernatant, CFS), and then fresh BHI broth was used to prepare (1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32) sterile supernatants of different dilution ratios, and placed in a 4°C refrigerator for standby use. After the experimental strain Clostridium difficile ATCC43255 was taken out of a -80°C refrigerator and melted, 100μL was drawn and coated on a CCFA plate, activated and subcultured 3 times, and a single colony was picked and inoculated in BHI broth and cultured to an OD600 of about 0.5. 400 μL of the bacterial solution in the exponential growth phase was taken out and inoculated into test tubes containing 4 mL of sterile supernatant at different dilution ratios (1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32), and a sterile supernatant blank control group and a positive control group containing only Clostridium difficile (CD) were set up. This time was recorded as time 0, and then anaerobically cultured at 37°C, with culture turbidity (OD 600nm) as the growth index of CD. 200 μL of samples were taken at different time points, and the absorbance value was measured at a wavelength of 600nm, and the growth curve was drawn (see Appendix). Figure 3 ). The results showed that CFS at 1 / 2, 1 / 4, and 1 / 8 dilutions significantly inhibited the growth of Clostridium difficile.

[0051] Example 4: Effect of Lactobacillus paracasei W1 sterile supernatant on Clostridium difficile biofilm formation

[0052] The lowest dilution ratio without turbidity change in the process of Example 3 was taken as the inhibitory concentration, and the dilution ratio with turbidity change was taken for the experiment. 400 μL was taken from the bacterial solution in the exponential growth phase and inoculated into a test tube containing 4 mL of sterile supernatant of different dilutions (1 / 16, 1 / 32), and three parallels were made for each dilution. 1 mL was taken from each well and inoculated into a 24-well polystyrene plate and cultured for 72 hours under anaerobic conditions at 37°C. After the culture was completed, the 24-well plate cover was opened, the supernatant was sucked off, and the biofilm was gently washed twice with sterile distilled water to remove the residual bacterial solution and dried at 37°C for 30 minutes. The biofilm was gently washed twice with sterile distilled water again and dried at 37°C for 30 minutes. Then, 0.2% crystal violet staining solution was added for staining for 30 minutes, the excess stain was poured off and gently washed 6 times with distilled water to remove the residual stain, and dried at 37°C. Finally, 200 μL of 95% ethanol was added to dissolve the dye solution bound to the biofilm (30 min), 200 μL was added to a 96-well plate, and the absorbance of each well was measured at a wavelength of 570 nm (see Appendix). Figure 4 The results showed that CFS at a dilution of 1 / 16 significantly inhibited the biofilm formation of Clostridium difficile.

[0053] Example 5: Effect of Lactobacillus paracasei W1 on colon shortening in CDI mice

[0054] Thirty-one C57 female mice (6 weeks, 14-16g) were randomly divided into 4 groups, with 7 mice in the control group and 8 mice in each of the other groups, namely: control group (Control), model group (CDI), Lactobacillus paracasei W1 prevention group (LP W1 pre) and Lactobacillus paracasei W1 treatment group (LP W1 post). For the three experimental groups, an antibiotic mixture was prepared as the drinking water for mice. The antibiotic mixture consisted of kanamycin (0.4mg / mL), gentamicin (0.035mg / mL), colistin (850U / mL), metronidazole (0.215mg / mL), and vancomycin (0.045mg / mL). The treatment lasted for 5 consecutive days, and then the mice were fed with sterile drinking water for 1 day. The Lactobacillus paracasei W1 prevention group (W1 pre) was gavaged with 200μL of Lactobacillus paracasei W1 suspension (1×10 9 CFU / mL), and mice in the control group drank sterile water all the time.

[0055] One day before Clostridium difficile infection, all experimental groups of mice were intraperitoneally injected with clindamycin (10 mg / kg). They were fasted and deprived of water from 17 h before infection to 2 h after infection. On the first day after infection, the Lactobacillus paracasei W1 treatment group (LP W1post) was treated with a bacterial suspension (1×10 9 CFU / mL) by intragastric administration, once a day for 6 consecutive days, and observe whether the mice have diarrhea or death, and weigh and record the situation.

[0056] In addition, the fecal characteristics, behavioral status and weight changes of the mice were observed daily, and the clinical sickness score (CSS) of the mice was calculated. The scores of the specific scoring items are shown in Table 4. On the 6th day, the mice were dissected, their colons were taken and the colon length was measured ( Figure 5 ) and calculated the survival rate of mice ( Figure 6 ) and CSS score ( Figure 7 ).

[0057] Table 4 Clinical sickness score (CSS)

[0058]

[0059] The experimental results are as follows Figure 5 As shown. Compared with the control group, the colon length of mice in the CDI group was significantly shortened, while the colon length of the LP W1post and LP W1 pre groups was significantly restored. This indicates that Lactobacillus paracasei W1 has an improving effect on colon shortening in Clostridium difficile colitis. The survival rate of mice in the CDI group was significantly reduced, while the survival rates of the LP W1 post and LP W1 pre groups were improved, especially the LP W1 pre group. The CSS score of the CDI group was significantly increased, while the LP W1 post and LP W1 pre groups were both decreased compared with the CDI group, among which the LP W1 pre group had the most significant decrease. This indicates that Lactobacillus paracasei W1 has a relieving and repairing effect on Clostridium difficile colitis. As shown Figure 6 , Figure 7 .

[0060] Example 6: Effect of Lactobacillus paracasei W1 on intestinal tissue damage in CDI mice

[0061] The cecum and colon tissues of mice were obtained, and approximately 1 cm of the cecum and colon tissues were cut and immersed in 4% paraformaldehyde fixative for 24 hours. After sectioning, HE staining was performed to observe tissue changes.

[0062] HE staining of colon and cecum tissues showed that ( Figure 8 ), the epithelial structure of the colon and cecum in the CDI group was severely damaged, goblet cells and crypts were missing, and the colon and cecum showed obvious inflammation, with a large number of inflammatory cells infiltrating the submucosal layer. In contrast, the colon mucosal epithelial cells of the mice in the Control, LP W1 treatment and LP W1 prevention groups were intact, the glands of the lamina propria were normal in morphology, the crypts were normal, and only a small amount of inflammatory cells infiltrated the submucosal layer. This indicates that Lactobacillus paracasei W1 can prevent and treat the symptoms of colitis caused by Clostridium difficile.

[0063] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A strain of Lactobacillus paracasei (L. paracasei) W1, characterized in that: The Lactobacillus paracasei preservation number is CCTCC M 2024619.

2. a strain of Lactobacillus paracasei (L.paracasei) W1 as claimed in claim 1, is characterized in that: The nucleotide sequence of 16S rRNA of the Lactobacillus paracasei (L. paracasei) W1 is shown in SEQ ID No.

1.

3. Application of a strain of Lactobacillus paracasei (L. paracasei) W1 in inhibiting Clostridium difficile (C. difficile) infection.

4. The use of a strain of Lactobacillus paracasei W1 as claimed in claim 3 in inhibiting Clostridium difficile infection, characterized in that: The Lactobacillus paracasei (L. paracasei) W1 sterile supernatant inhibits the growth of Clostridium difficile and the formation of biofilm.

5. Application of a strain of Lactobacillus paracasei (L. paracasei) W1 in the preparation of dairy products, functional products and health products for preventing and treating intestinal diseases caused by Clostridium difficile (C. difficile) infection.

6. The use of a strain of Lactobacillus paracasei W1 as claimed in claim 5 in the preparation of dairy products, functional products and health products for preventing and treating intestinal diseases caused by Clostridium difficile infection, characterized in that: The content of Lactobacillus paracasei in the product is not less than 1.0×10 9 CFU / mL or 1.0×10 9 CFU / g.

Images