Application of lactobacillus salivarius PC114 in preparation of product for removing intestinal drug-resistant bacteria
By preparing Lactobacillus salivarius PC114 bacterial agent, the problem of VRE resistance was solved, and the intestinal resistant bacteria were safely and effectively eliminated, the intestinal health was restored, and the structure and function of the flora were regulated.
Patent Information
- Application Number
- CN202510924059.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-04
- Publication Date
- 2025-10-14
AI Technical Summary
In existing technologies, the drug resistance problem of vancomycin-resistant enterococci (VRE) leads to limited treatment options, and traditional antibiotic treatment has safety and resistance risks. There is an urgent need to develop new safe and effective treatment strategies.
Lactobacillus salivarius PC114 bacterial agent is prepared through fermentation culture, precipitation and drying to eliminate intestinal drug-resistant bacteria, regulate intestinal flora, reduce the number of VRE, and repair intestinal barrier damage.
Lactobacillus salivarius PC114 significantly reduced the number of VRE, restored the diversity and uniformity of intestinal flora, regulated flora composition, and repaired intestinal damage, with better effects than other strains.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of microbial technology, in particular to the use of Lactobacillus salivarius PC114 in the preparation of products for clearing drug-resistant bacteria in the intestinal tract. BACKGROUND
[0002] Enterococcus is a bacterium widely colonized in the gastrointestinal tract of humans, which usually coexists with the host to maintain the balance of intestinal microecology. However, it also has potential harm and can cause various diseases as an opportunistic pathogen, especially for people with critical underlying diseases and low immune function, which is easy to cause serious infection and even threaten life safety.
[0003] The use of antibacterial drugs has continuously increased the global burden of enterococcal drug resistance. The discovery of the first vancomycin-resistant enterococcus (VRE) V583 suggests that vancomycin, the last effective antibacterial drug for enterococcal infection, has been conquered. Vancomycin was once considered the last effective drug against enterococcal infection, but now its effectiveness is no longer, resulting in extremely limited treatment options in the clinic, which not only brings great challenges to clinical prevention and treatment, but also becomes an important factor leading to global treatment difficulties and increased mortality. At present, the incidence of VRE is still high, therefore, it is urgent to develop a completely new safe and effective treatment strategy for VRE.
[0004] Lactic acid bacteria are a special group of microorganisms that exist in the human body, which usually have various probiotic functions. In recent years, lactic acid bacteria have attracted increasing attention for their role in improving intestinal diseases by reshaping the intestinal flora. Currently, lactic acid bacteria have become an auxiliary treatment method for some intestinal diseases in the clinic, and due to their non-toxicity, easy excretion from the human body, beneficial effects on indigenous intestinal flora, and non-resistance to drugs, etc. Therefore, finding an ideal lactic acid bacteria strain to clear vancomycin-resistant enterococci and repair the intestinal damage caused by them is an important breakthrough direction in this field, and also a promising treatment scheme to replace antibiotics in vivo. SUMMARY
[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide the use of Lactobacillus salivarius PC114 in the preparation of products for clearing drug-resistant bacteria in the intestinal tract, to solve the problems in the prior art.
[0006] To achieve the above-mentioned purposes and other related purposes, the present application first provides the use of Lactobacillus salivarius PC114 in the preparation of products for clearing drug-resistant bacteria in the intestinal tract.
[0007] The product is a product containing Lactobacillus salivarius PC114 bacterial agent.
[0008] In some embodiments of the present application, the preparation method of the bacterial agent comprises the following steps:
[0009] 1) inoculate the Lactobacillus salivarius PC114 seed liquid into the culture medium for fermentation culture to obtain a fermentation liquid;
[0010] 2) collect the precipitate in the fermentation liquid to obtain the Lactobacillus salivarius PC114 bacterial body;
[0011] 3) mix the obtained bacterial body with a protective agent, and dry to obtain the bacterial agent.
[0012] The application also provides a product for removing intestinal drug-resistant bacteria, which necessarily comprises the Lactobacillus salivarius PC114 and takes the Lactobacillus salivarius PC114 as an effective component for the aforementioned effects.
[0013] As described above, the use of the Lactobacillus salivarius PC114 in the preparation of the product for removing intestinal drug-resistant bacteria has the following beneficial effects:
[0014] The Lactobacillus salivarius PC114 can repair the intestinal barrier damage caused by vancomycin-resistant Enterococcus, regulate the intestinal flora composition and community diversity and evenness after vancomycin-resistant Enterococcus infection, and reduce the number of vancomycin-resistant Enterococcus in the body, indicating that the Lactobacillus salivarius PC114 has a good application prospect in the preparation of the product for removing vancomycin-resistant Enterococcus.
[0015] The preservation information of the strain in the application is as follows:
[0016] Strain name: Lactobacillus salivarius;
[0017] Preservation number: CGMCC N.23518;
[0018] Preservation unit name: China General Microbiological Culture Collection Center;
[0019] Preservation unit address: No. 3, Beichen West Road, Chaoyang District, Beijing Preservation date: September 29, 2021. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 The figure shows the influence of the Lactobacillus salivarius strain PC114 intervention on the number of VRE in Example 3 of the application.
[0021] Figure 2Figure 1 shows the effect of Lactobacillus salivarius strain PC114 intervention on intestinal flora in Example 3 of the present application. Figure (a) shows the Chao 1 index and Shannon index of different groups. Figure (b) shows the distribution of intestinal flora structure of different groups in the principal coordinate analysis (PCoA) plot, and Figure (c) shows the relative abundance changes of different flora at the ASV (Amplicon Sequence Variant) level.
[0022] Figure 3 Figure 2 shows the effect of Lactobacillus salivarius strain PC114 intervention on ileum tissue in Example 3 of the present application. DETAILED DESCRIPTION
[0023] The present application first provides the use of Lactobacillus salivarius PC114 with the preservation number of CGMCC NO. 23518 in the preparation of a product for clearing drug-resistant bacteria in the intestine.
[0024] In the present application, the "clearing" refers to reducing or eliminating drug-resistant bacteria in the intestine.
[0025] In some embodiments of the present application, the drug-resistant bacteria are vancomycin-resistant Enterococcus.
[0026] In some embodiments of the present application, the product for clearing drug-resistant bacteria in the intestine has any one or more of the following effects:
[0027] 1) reducing the number of vancomycin-resistant Enterococcus;
[0028] 2) improving intestinal community diversity;
[0029] 3) improving the evenness of intestinal community;
[0030] 4) regulating the composition of intestinal flora;
[0031] 5) improving intestinal barrier damage.
[0032] In some embodiments of the present application, the composition of intestinal flora after VRE infection is effectively regulated by enriching ASVs including Bifidobacterium and Faecalibaculum.
[0033] Specifically, the reduction in the number of vancomycin-resistant Enterococcus refers to bringing the number of vancomycin-resistant Enterococcus of the subject close to or reaching the level of healthy individuals. For example, based on the number of vancomycin-resistant Enterococcus in healthy individuals, the product can restore the number of vancomycin-resistant Enterococcus of the subject to at least 10%, preferably at least 30%, more preferably at least 50%, even more preferably at least 70%, and most preferably at least 90% of that of healthy individuals.
[0034] Specifically, the improving intestinal flora diversity and the improving intestinal flora evenness refer to making the intestinal flora diversity and the intestinal flora evenness in the body of the subject close to or reach the level of healthy individuals. For example, taking the intestinal flora diversity and / or the intestinal flora evenness in healthy individuals as a benchmark, the product can restore the intestinal flora diversity and / or the intestinal flora evenness to at least 10%, preferably at least 30%, more preferably at least 50%, more preferably at least 70%, and most preferably at least 90% of that of healthy individuals.
[0035] In some embodiments of the present application, the flora diversity and evenness are characterized by Chao1 index and Shannon index.
[0036] Specifically, the adjusting the composition of intestinal flora refers to making the composition of intestinal flora in the body of the subject close to or reach the level of healthy individuals. For example, taking the composition of intestinal flora in healthy individuals as a benchmark, the product can restore the composition of intestinal flora to at least 10%, preferably at least 30%, more preferably at least 50%, more preferably at least 70%, and most preferably at least 90% of that of healthy individuals.
[0037] In some embodiments of the present application, the number of viable bacteria in the product is ≥1×10 9 CFU / g or the number of viable bacteria is ≥1×10 9 CFU / ml.
[0038] Further, the number of viable bacteria in the product is 1×10 9 CFU / g to 1×10 11 CFU / g or 1×10 9 CFU / ml to 1×10 11 CFU / ml.
[0039] In some embodiments of the present application, the product is a product containing Lactobacillus salivarius PC114 inoculant.
[0040] In some embodiments of the present application, the preparation method of the inoculant comprises the following steps:
[0041] 1) inoculating Lactobacillus salivarius PC114 seed liquid into culture medium for fermentation culture to obtain fermentation broth;
[0042] 2) collecting the precipitate in the fermentation broth to obtain Lactobacillus salivarius PC114 bacterial bodies;
[0043] 3) mixing the obtained bacterial bodies with a protective agent, and drying to obtain the inoculant.
[0044] In some embodiments of the present application, in step 1), the inoculation amount is 1.25×10 7~1x10 8 CFU / mL.
[0045] In some embodiments of the present application, in step 1), the temperature of the fermentation culture is 25-45℃.
[0046] In some embodiments of the present application, in step 1), the time of the fermentation culture is 12-36h.
[0047] In some embodiments of the present application, in step 2), the precipitate in the fermentation broth is collected by centrifugation.
[0048] Further, the speed of the centrifugation is 4,000-12,000g.
[0049] Further, the time of the centrifugation is 8-12min.
[0050] In some embodiments of the present application, in step 3), the protective agent is skimmed milk.
[0051] Further, the skimmed milk is obtained by mixing skimmed milk powder with water.
[0052] Further, the mass fraction of the skimmed milk powder is 6-12% based on the mass of the skimmed milk.
[0053] In some embodiments of the present application, the viable cell count of Lactobacillus salivarius PC114 in the bacterial agent is ≥1x10 9 CFU / g or the viable cell count is ≥1x10 9 CFU / ml.
[0054] Further, the viable cell count of Lactobacillus salivarius PC114 in the bacterial agent is 1x10 9 CFU / g-1x10 11 CFU / g or 1x10 9 CFU / ml-1x10 11 CFU / ml.
[0055] The present application also provides a product for eliminating drug-resistant bacteria in the intestinal tract, which necessarily comprises the Lactobacillus salivarius PC114 and takes the Lactobacillus salivarius PC114 as the effective component for the aforementioned efficacy.
[0056] In the product, the effective component for the efficacy can only be the Lactobacillus salivarius PC114 or can comprise other substances.
[0057] That is, the Lactobacillus salivarius PC114 is the only effective component or one of the effective components of the product.
[0058] The product can be a single-component substance or a multi-component substance.
[0059] The dosage form of the product is not particularly limited, and can be various such as solid, liquid, gel, semi-liquid, aerosol, powder, etc.
[0060] The product is mainly aimed at mammals. The mammals are preferably rodents, even-toed ungulates, odd-toed ungulates, lagomorphs, primates, etc. The rodents are preferably mice. The primates are preferably monkeys, apes or humans.
[0061] The product includes but is not limited to drugs, health products, food, etc.
[0062] In some embodiments of the present application, the product is a drug, and the drug further comprises a pharmaceutically acceptable carrier or excipient.
[0063] "Pharmaceutically acceptable" means that when the drug is properly administered to animals or humans, they do not produce adverse, allergic or other adverse reactions.
[0064] The "pharmaceutically acceptable carrier or excipient" should be compatible with the active ingredient, i.e. it can be blended with the active ingredient without substantially reducing the effect of the drug under normal circumstances. Some specific examples of substances that can be used as pharmaceutically acceptable carriers or excipients are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help the stability of the formulation or to help improve the activity or its bioavailability or to produce an acceptable taste or smell in the case of oral administration.
[0065] The product in the present application can also be used in combination with other methods of eliminating intestinal drug-resistant bacteria. The combination can be simultaneous use or sequential use. Other methods of eliminating intestinal drug-resistant bacteria include but are not limited to other drug therapy or non-drug therapy, such as dietary adjustment, weight control, regular exercise, smoking cessation and alcohol restriction, stress management, adequate sleep.
[0066] Following along with the specific examples, other advantages and benefits of the present application will become apparent to those skilled in the art from the following description and accompanying drawings. The present application may be implemented or performed in other different ways than described in the present embodiments without departing from the spirit of the present application. The description of the present embodiments is intended for purposes of illustration and is not intended to limit the scope of the present application.
[0067] Before further description of the present application, it is understood that the present application is not limited to the particular examples described herein which are presented for purposes of illustration only, and as such, each of the particular examples presented herein is not intended to be limiting, and that the scope of the present application will be limited only by the appended claims. In the description of the present application, unless otherwise indicated, the singular forms "a", "an", and "the" include plural referents.
[0068] When numerical ranges are given, it should be understood that every numerical range encompasses any number falling within the range, including the endpoints, and any intervening range. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Except in the Examples, or where otherwise explicitly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, exemplary methods and materials are described herein.
[0069] Preparation method of the seed (fermentation strain) of Example 1
[0070] The Lactobacillus salivarius PC114 strain was picked up with an inoculation loop to a single colony and put into 10 mL of MRS liquid medium (purchased from Merck Co., Germany), and the colony was uniformly dispersed in the liquid medium by using a vortex oscillator. After 48 h of anaerobic culture at 37°C, the culture was removed, inoculated into MRS liquid medium at a 2% (v / v) inoculation amount, and cultured anaerobically at 37°C for 24 h. The culture was collected, centrifuged at 15,000 rpm for 10 min, and the supernatant was discarded. The bacterial cells were washed twice with sterile distilled water and suspended with sterile distilled water in an original culture volume to obtain a seed for fermentation. The bacterial concentration of the seed liquid was 2.5 x 10 9 CFU / mL.
[0071] The preparation method of the seed liquid of Lactobacillus salivarius BD8459 (accession number: CGMCC NO. 34253) was the same as that of Lactobacillus salivarius PC114 strain.
[0072] Preparation method of the bacterial agent of Example 2
[0073] 2.1 Preparation of skim milk
[0074] Skim milk powder and distilled water were mixed at a ratio of 10% (w / w), and after being fully dissolved, sterilized at 125℃ for 5 min, and cooled to room temperature to obtain the required concentration of skim milk.
[0075] 2.2 Preparation of the bacterial agent
[0076] 1) The fermentation seed liquid of Lactobacillus salivarius PC114 was aseptically inoculated into MRS liquid medium at an inoculation amount of 2% (v / v, the same below), and incubated at 37℃ for 24 h to obtain a fermentation liquid;
[0077] 2) The fermentation liquid obtained in step 1) was centrifuged at 10,000 g for 10 min, and the supernatant was discarded. The precipitate was washed twice with sterile distilled water, and the obtained bacterial cells were resuspended in an original volume of skim milk (10% of the mass percentage of skim milk powder).
[0078] 3) The bacterial cells obtained in step 2) were resuspended in an original volume of skim milk (10% of the mass percentage of skim milk powder).
[0079] The preparation method of the bacterial agent of Lactobacillus salivarius BD8459 was the same as that of Lactobacillus salivarius PC114.
[0080] Example 3 Effect of Lactobacillus salivarius PC114 on removing vancomycin-resistant Enterococcus in the intestinal tract
[0081] 3.1 Experimental design and grouping
[0082] Animal experiment: 32 SPF male C57BL / 6 mice aged 6 weeks were purchased (Jiangsu Jicai Yekang Biotechnology Co., Ltd.), and randomly divided into 4 groups with 8 mice in each group. After adaptive feeding for 1 week, 0.25 g / L vancomycin was added to the drinking water for 10 days. On the 7th day, 1×10 8 CFU / mL of VRE was administered by gavage to 3 groups, and the remaining 1 group served as a negative control. After 10 days, vancomycin was no longer added to the drinking water, and the remaining 3 groups were administered by gavage with PC114 (live bacteria number of 1×10 9 CFU / mL), PBS 0.2 mL, and Lactobacillus salivarius BD8459 (live bacteria number of 1×10 9 CFU / mL), respectively, once a day for three weeks. The information table of experimental animals and the gavage method are shown in Table 1.
[0083] Table 1 Information table of experimental animals and gavage method
[0084]
[0085] 3.2 Effect of Lactobacillus salivarius PC114 on the number of VRE
[0086] Feces collected at different time points from each group of mice were diluted with PBS, then plated on plates containing vancomycin 32 mg / L, and the counting results are shown in Figure 1 Compared with the control group, both PC114 and BD8459 can significantly reduce the number of VRE after 21 days of intervention, but the results of PC114 are relatively better. The experimental results show that Lactobacillus salivarius PC114 can significantly reduce the content of VRE, and the effect is better than that of Lactobacillus salivarius BD8459.
[0087] 3.3 Effect of Lactobacillus salivarius PC114 on intestinal flora structure
[0088] The intestinal contents of the ileum of each group of mice were analyzed for flora composition and structure, and the results are shown in Figure 2 The Chao 1 index shows that both Lactobacillus salivarius PC114 and BD8459 can significantly improve the diversity of the mouse ileum community after 21 days of intervention, and the Shannon index shows that PC114 can also significantly improve the evenness of the mouse ileum community, but BD8459 has a relatively poor effect on improving the evenness of the ileum community Figure 2 a).
[0089] In terms of intestinal flora composition analysis, it is found that PC114 and BD8459 can effectively change the structure of part of the mouse ileum flora after intervention, and can be significantly distinguished from the control group. Specifically, in the control group, after vancomycin treatment and intragastric administration of VRE, the amplicon sequence variant (ASV) of Enterococcus belonging to unclassified is significantly enriched in the mouse ileum, which further proves the success of the modeling.
[0090] Compared with the control group, the relative abundance of Lactobacillus in the PC114 and BD8459 groups has increased, but the intestinal flora change trend of the two groups of mice is different. The PC114 group significantly enriched ASVs including Bifidobacterium and Faecalibaculum, while the BD8459 group significantly enriched ASVs of Dubosiella. The above results show that although PC114 and BD8459 can regulate the composition of intestinal flora after VRE infection, the types of regulating flora are different, especially, the effect of PC114 on improving community evenness is better than that of BD8459.
[0091] 3.4 Effect of Lactobacillus salivarius PC114 on ileum tissue morphology
[0092] The ileum tissue of each group of mice at the end of the experiment was selected for staining and microscopic examination, and the results are shown in Figure 3As shown, in the model group G3, the ileum tissue of mice showed local degeneration and necrosis (black arrow), shedding of intestinal villi (yellow arrow), slight pathological changes of a small amount of inflammatory cell infiltration (green arrow) and fibrous tissue hyperplasia (blue arrow). In contrast, no obvious pathological changes were observed in the PC114 and BD8459 intervention groups. This indicates that PC114 and BD8459 have good effects in repairing the pathological damage caused by vancomycin enterococci.
[0093] The above examples are intended to illustrate the embodiments disclosed in the present application and should not be understood as limiting the present application. In addition, various modifications listed herein and changes in the method of the invention are obvious to those skilled in the art without departing from the scope and spirit of the present application. Although the present application has been specifically described in conjunction with various specific preferred embodiments thereof, it should be understood that the present application should not be limited to these specific embodiments. In fact, various modifications as described above to obtain the invention that are obvious to those skilled in the art should be included within the scope of the present application.
Claims
1. Use of Lactobacillus salivarius PC114 with a preservation number of CGMCC NO. 23518 in the preparation of a product for eliminating drug-resistant intestinal bacteria.
2. The use according to claim 1, characterized in that The drug-resistant bacteria are vancomycin-resistant enterococci.
3. The use according to claim 1, characterized in that The product for eliminating drug-resistant intestinal bacteria has any one or more of the following effects: 1) Reduce the number of vancomycin-resistant enterococci; 2) Improve intestinal community diversity; 3) Improve the uniformity of intestinal flora; 4) Regulate the composition of intestinal flora; 5) Improve intestinal barrier damage.
4. The use according to claim 1, characterized in that The number of viable bacteria in the product is ≥1×10 9 CFU / g or viable bacteria count ≥1×10 9 CFU / ml.
5. The use according to claim 1, characterized in that The product is selected from medicines, health products, and foods; preferably, the medicine further includes a pharmaceutically acceptable carrier or excipient.
6. The method according to claim 1, wherein: The product contains Lactobacillus salivarius PC114 bacterial agent.
7. The use according to claim 6, characterized in that The preparation method of the Lactobacillus salivarius PC114 bacterial agent comprises the following steps: 1) inoculating a Lactobacillus salivarius PC114 seed solution into a culture medium for fermentation to obtain a fermentation solution; 2) collecting the precipitate in the fermentation broth to obtain Lactobacillus salivarius PC114 bacteria; 3) mixing the obtained bacterial cells with a protective agent and drying the mixture to obtain the bacterial agent.
8. The use according to claim 7, characterized in that In step 1), any one or more of the following features are included: 11) The inoculum size is 1.25×10 7 ~1×10 8 CFU / mL; 12) The fermentation culture temperature is 25°C to 45°C; 13) The fermentation culture time is 12 to 36 hours.
9. The use according to claim 7, characterized in that In step 2), the precipitate in the fermentation broth is collected by centrifugation; Preferably, the centrifugal speed is 4,000 to 12,000 g; preferably, the centrifugal time is 8 to 12 min.
10. The use according to claim 7, characterized in that In step 3), the protective agent is skim milk; preferably, the skim milk is obtained by mixing skim milk powder with water; more preferably, the mass fraction of the skim milk powder is 6 to 12% based on the mass of the skim milk.