CN116240151B - Fender-like bacillus and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a Fennella and application thereof. Experiments prove that the screened Afine-1 genome of the Afine-1 has no virulence genes, is not hemolytic in vitro and is sensitive to various antibiotics, and has good safety. The strain has obvious antibacterial capacity on clostridium difficile; can significantly improve diarrhea induced by chemotherapeutics 5-fluorouracil and irinotecan, and has anti-inflammatory effect. The preparation of the Fennella alternifolia provided by the invention can be used as a new generation of live bacteria preparation, and provides a new thought and application prospect for the prevention and/or treatment of chemotherapy-related diarrhea based on various mechanisms.

Description

Fender-like bacillus and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a Fennella and application thereof, in particular to application in treating and/or preventing diarrhea caused by chemotherapeutics.

Background

Chemotherapy-related diarrhea (Chemotherapy induced diarrhea, CID) is a common digestive tract toxic side effect caused by chemotherapy in tumor patients, and mild diarrhea can degrade the quality of life of patients, and frequent severe diarrhea can lead to dehydration, infection, shock, and even death of patients. Thus, patients with severe diarrhea need to reduce the dose of chemotherapy drugs or even interrupt chemotherapy, affecting tumor treatment.

Among the common chemotherapy regimens, the CapeIRI regimen (capecitabine + irinotecan) and the folfoxri regimen (fluorouracil + calcium folinate + oxaliplatin + irinotecan) most likely cause CID, with the incidence of grade 3-4 diarrhea as high as 20-47%. Among them, 5-fluorouracil (5-FU) and irinotecan are the drugs most likely to cause CID: most of 5-FU related diarrhea is watery or bloody, and is easy to cause sepsis, relatively heavy in degree and occasionally fatal; irinotecan can cause tardive diarrhea, typically occurring after 24 hours, for 6 to 14 days, regardless of dose and unpredictable.

The pathogenesis of CID is more complex than that of ordinary diarrhea. In one aspect, chemotherapy (e.g., 5-FU) can result in DNA damage and mitochondrial dysfunction, apoptosis of intestinal epithelial cells, excessive activation of transcription factor NF- κb, up-regulated expression of pro-inflammatory cytokines, and ultimately, damage to the epithelium, endothelium, and connective tissue. On the other hand, there are still other factors at risk of diarrhea in cancer patients treated with 5-FU or irinotecan, for example, due to damage to the intestinal barrier, disruption of mucosal integrity and intestinal flora, and proliferation of harmful bacteria such as clostridium difficile, a high risk of nosocomial infection with pathogenic bacteria.

According to 2018 ESMO (European Society for Medical Oncology, european society of oncology) guidelines for diarrhea in adult cancers, antidiarrheals, somatostatin analogues, glucocorticoids, antibiotics and the like are clinically used in many cases at present, and special medicines and preventive measures are lacking, so that side effects are large. For example, opioid (paradoxylamine) may pose a risk of paralytic ileus when used at large doses; somatostatin analogues (octreotide) can cause side effects such as cholelithiasis, hyperglycemia, abnormal glucose tolerance and the like; glucocorticoids may have a systemic effect, increase the risk of infection, exacerbate viral or bacterial infections, and the like. Epidemiological data show that the number of tumor patients worldwide increases substantially each year, about 300-700 tens of thousands of patients are predicted to suffer from chemotherapy-related side effects by 2040 years, but current treatment schemes lack special specific drugs and preventive measures, and are difficult to meet the increasing clinical demands.

The intestinal microecological system is the most huge and important microecological system of the organism, and intestinal microecological unbalance is closely related to pathogenesis of various diseases. A large number of researches show that intestinal probiotics can regulate organism tissues, organ functions and behaviors through intestinal nerves, immune systems, intestinal endocrine signals and other ways, thereby being beneficial to host health. However, for the curative effect of probiotics in preventing and treating the diarrhea related to radiotherapy and chemotherapy, a large-scale high-quality prospective random control test is lacking in recent years, and researches included in the current system analysis have obvious heterogeneity, strong deterministic evidence cannot be formed yet, and strict clinical researches are still needed to be carried out subsequently for definition. In addition, probiotic products for relieving diarrhea symptoms on the market are all traditional generation probiotics, and have a good treatment effect on CID complex pathogenesis.

At present, there is no study reporting that the bifidobacterium finesse can be used for treating and/or preventing the diarrhea related to chemotherapy. Patent document CN113999922a discloses an acute diarrhea marker microorganism and its application, which focuses on studying the change of microorganism abundance in stool samples of patients suffering from acute diarrhea. The pathogenesis of chemotherapy-related diarrhea is more complex than general diarrhea, and is manifested clinically as both acute and chronic diarrhea. Therefore, in order to meet the needs of patients with chemotherapy-related diarrhea for live bacteria preparations with targeted therapeutic effects, it is necessary to develop a novel probiotic preparation.

Disclosure of Invention

Aiming at diarrhea caused by a chemotherapeutic drug in the current clinical treatment of tumors, the invention provides a novel microbial preparation capable of remarkably improving diarrhea caused by the chemotherapeutic drug, and can remarkably reduce diarrhea scores and improve diarrhea symptoms. The microbial preparation can also inhibit clostridium difficile growth, and has antiinflammatory effect and antioxidant capacity.

The specific scheme of the invention is as follows:

the invention provides a kind of FennellaAlistipes finegoldii) The whole genome sequence and the preservation number are CCTCC No: the strain of M20221759 has an average nucleotide similarity of 99.3% or more.

In some embodiments, the whole genome sequence and accession number of the bacillus dysenteriae is cctccc No: the strain of M20221759 has an average nucleotide similarity of 99.4% or more.

In some embodiments, the whole genome sequence and accession number of the bacillus dysenteriae is cctccc No: the strain of M20221759 has an average nucleotide similarity of 99.5% or more.

In some embodiments, the whole genome sequence and accession number of the bacillus dysenteriae is cctccc No: the strain of M20221759 has an average nucleotide similarity of 99.6% or more.

In some embodiments, the whole genome sequence and accession number of the bacillus dysenteriae is cctccc No: the strain of M20221759 has an average nucleotide similarity of 99.7% or more.

In some embodiments, the whole genome sequence and accession number of the bacillus dysenteriae is cctccc No: the strain of M20221759 has an average nucleotide similarity of 99.8% or more.

In some embodiments, the whole genome sequence and accession number of the bacillus dysenteriae is cctccc No: the strain of M20221759 has an average nucleotide similarity of 99.9% or more.

In some embodiments, the whole genome sequence and accession number of the bacillus dysenteriae is cctccc No: the strain of M20221759 has 100% average nucleotide similarity.

The invention also provides a kind of FennellaAlistipes finegoldii) It is deposited in China center for type culture Collection with the accession name of Fenton's offeringsAlistipes finegoldiiAfine-1, with preservation number of CCTCC No: m20221759.

In some embodiments, the A.fensii isAlistipes finegoldii) For the preservation number of CCTCC No: cloning of strain M20221759.

The above-mentioned Fengshi's bacillusAlistipes finegoldii) Colony culture on bhi+mrs+modified GAM triple media is characterized by: smaller white opaque, convex circular, smooth-surfaced moist colonies; the strain morphology is identified by using a crystal violet staining method, and the Fennella is in a short rod shape under an optical microscope.

The above-mentioned A.fense is sensitive to at least one of the following 9 antibiotics: imipenem, vancomycin, tetracycline, erythromycin, clindamycin, levofloxacin, nitrofurantoin, high-concentration gentamicin, and rifampin.

The genome of the above-mentioned Fennella is free from virulence genes and is not hemolytic in vitro (i.e. gamma hemolysis).

The invention provides a pharmaceutical composition which contains the above-mentioned Fennella like bacillusAlistipes finegoldii) And any pharmaceutically acceptable carrier or adjuvant.

The invention provides the Fennella like bacillusAlistipes finegoldii) Or the use of a pharmaceutical composition for the preparation of a microbial preparation for the treatment and/or prevention of diarrhea caused by chemotherapeutic agents or/and for the inhibition of the proliferation of intestinal pathogenic bacteria.

Preferably, the microbial preparation has an inhibitory effect on clostridium difficile.

Preferably, the chemotherapeutic agents include, but are not limited to: fluorouracil, topoisomerase I inhibitors, tyrosine kinase inhibitors, taxanes, platinum chemotherapeutics and other chemotherapeutics.

Optionally, the fluorouracil comprises 5-fluorouracil, tegafur, capecitabine, and the like; the topoisomerase I inhibitor comprises irinotecan, topotecan and the like; the tyrosine kinase inhibitor comprises gefitinib, erlotinib, lapatinib, afatinib, dacatinib, vandetanib, lenatinib, oritinib, imatinib, dasatinib, nilatinib, bosutinib, panatinib and the like; the taxane chemotherapeutics comprise paclitaxel, docetaxel and the like; the platinum chemotherapeutic medicine comprises cisplatin, oxaliplatin and the like.

Preferably, the chemotherapeutic agent is 5-fluorouracil and/or irinotecan;

preferably, the microbial formulation is used to alleviate diarrhea symptoms caused by 5-fluorouracil and/or irinotecan;

preferably, the microbial preparation can remarkably reduce the expression of THP-1 cell pro-inflammatory factor TNF-alpha and has anti-inflammatory effect;

preferably, the microbial formulation has significant antioxidant capacity.

In a preferred embodiment of the present invention, the microbial preparation may comprise a strain of the bacterium African fenhnsonii and/or a strain of the bacterium African fenhnsonii fermentation supernatant and/or a strain of the bacterium African fenhnsonii lysate.

The invention has the following beneficial effects:

the Afine-1 of the invention is different from traditional probiotics and other existing on-ground products in the market, and belongs to a newer species. Experiments prove that the strain is sensitive to various antibiotics, has no hemolysis, and shows no virulence factor through whole genome analysis, thus indicating that the strain has good safety. In terms of effectiveness, the strain not only can remarkably improve diarrhea symptoms induced by chemotherapeutic drugs of 5-fluorouracil and irinotecan, but also has anti-inflammatory effect and antioxidant capacity, and can inhibit the growth of pathogen clostridium difficile. Therefore, the Afine-1 of the invention can be applied to a new generation of live bacteria preparation, and provides new ideas and strategies for the prevention and/or treatment of CID from various aspects.

Drawings

FIG. 1 is a graph showing the results of the antioxidant capacity of Afine-1 of B.fingii.

FIG. 2 is a graph showing the results of inhibiting the expression of the pro-inflammatory factor TNF- α in THP-1 cells by Afine-1 of A.fenhnsonii.

FIG. 3 is a graph showing the effect of Afine-1 of E.fenhnsonii on improvement of diarrhea induced by 5-fluorouracil in mice (daily diarrhea score for each group of mice during the A experiment; day 9 diarrhea score for each group of mice; total diarrhea score for each group of mice during the C experiment).

FIG. 4 is a graph showing the effect of Afine-1 of Embectomium Fender on improvement of diarrhea induced in mice (daily diarrhea score for each group of mice during the A-test; total diarrhea score for each group of mice during the B-test).

Description of the embodiments

The technical solutions in the embodiments of the present invention will be clearly and completely described below: it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments, and that all other embodiments obtained by a person skilled in the art without making creative efforts based on the embodiments in the present invention shall fall within the protection scope of the present invention.

The preparation method of the culture medium used in the following examples is as follows:

preparation of YCFA liquid culture medium: weighing 10.0. 10.0 g g casein peptone, 2.5 g yeast extract, 10 mg/mL magnesium sulfate heptahydrate solution 0.45 mL, 0.45 mL (10% mother liquor), TE141 10 mL, dipotassium hydrogen phosphate 0.45 g, potassium dihydrogen phosphate 0.45 g, sodium chloride 0.90 g, VFA-mix 3.2 mL, dissolving in 1L distilled water, N ₂ Removing oxygen, packaging, sterilizing at 121deg.C under moist heat for 30 min, and storing in shade and dry place.

Preparation of TE 141: 1.50g of nitrilotriacetic acid (Nitrilotriacetic acid, NTA) is weighed and added into 200 mL pure water, a proper amount of NaOH is added until the solution becomes clear, then 800 mL of water is added, 50% HCl is used for adjusting the pH value to 5.5, and then MgSO is weighed in sequence ₄ ·7H ₂ O 3.00 g，MnSO ₄ ·H ₂ O 0.50 g，NaCl 1.00 g，FeSO ₄ ·7H ₂ O 0.10 g，CoSO ₄ ·7H ₂ O 0.18 g，CaCl ₂ ·2H ₂ O 0.10 g，ZnSO ₄ ·7H ₂ O 0.18 g，CuSO ₄ ·5H ₂ O 0.006 g，KAl(SO ₄ ) ₂ ·12H ₂ O 0.02 g，H ₃ BO ₃ 0.01 g，Na ₂ MoO ₄ ·2H ₂ O 0.01 g，NiCl ₂ ·6H ₂ O0.03 g,10 mg/mL Na ₂ SeO ₃ ·5H ₂ O solution 0.03 mL,10 mg/mL Na ₂ WO ₄ ·2H ₂ And adding the O solution 0.03 and mL into the test solution, and continuously stirring in the adding process to keep the solution clear for later use.

Preparation of VFA-mix: taking 90 mL parts of acetic acid, 30 mL parts of propionic acid, 10 mL parts of n-valeric acid, 10 mL parts of isobutyric acid and 10 mL parts of butyric acid, mixing for later use, and adjusting the mixture to be neutral by using 5M parts of NaOH before use.

Preparation of triple mixed media (bhi+mrs+modified GAM): weighing 19.25 g of BHI broth powder (Qingdao sea Bo Biotechnology Co., ltd., HB 8297-5), 13.5 g of MRS broth powder (Guangdong CycloKai Biotechnology Co., ltd., 027312), 15 g of modified GAM broth powder (Qingdao sea Bo Biotechnology Co., ltd., HB 8518-3) (agar powder 12 g was added when preparing a triple-mixed solid culture medium), dissolving in distilled water of 1L, N ₂ Removing oxygen, packaging, sterilizing at 121deg.C under moist heat for 30 min, and storing in shade and dry place.

Preparation of BF839 culture medium: weighing potato extract powder (Beijing Soy Bao technology Co., ltd., FA 0270), 6.0G, multivalent peptone (Beijing Soy Bao technology Co., ltd., P8950-250), 10.0G, lunar peptone (Qingdao Hi-Tech, haibo Biotechnology Co., ltd., HB 8277), 5.0G, sodium thioglycolate (Shanghai Altin Biotechnology Co., ltd., S105664-25G) 0.3G, yeast extract powder (British OXOID Co., LP 0021B) 5.0G, glucose (Cheng chemical Co., ltd., 50-99-7), 1.5G, disodium hydrogen phosphate (Cheng chemical Co., ltd., 7558-79-4), 4.0G, dissolved in distilled water of 1L, N ₂ Removing oxygen, packaging, sterilizing at 121deg.C under moist heat for 30 min, and storing in shade and dry place.

The preparation of GAM solid culture medium (Qingdao sea Bo Biotechnology Co., ltd., HB 8462), TSB (tryptone Soy Broth, qingdao sea Bo Biotechnology Co., ltd., HB 4114) and TSA (tryptone Soy agar, qingdao sea Bo Biotechnology Co., ltd., HB 4138) were all weighed and dissolved according to the instructions, and sterilized by high-temperature moist heat at 121℃for 30 min, and stored in a cool and dry place.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

EXAMPLE 1 isolation, purification and colony identification of Afine-1 from Afine-1

Fresh fecal samples of healthy volunteers are collected, resuspended in an appropriate amount of oxygen-free PBS, and shaken, preferably with complete resuspension of the samples. At N ₂ Filtering with single-layer gauze under protection, packaging the filtrate into 50 mL centrifuge tube, and thenAfter filtration through double gauze, the supernatant was discarded after centrifugation at 10000 rpm for 20 min. Adding proper amount of anaerobic PBS to resuspend thallus, adding equal volume of 50% anaerobic glycerol, mixing, and packaging into 2 mL screw cap tubes with 0.5 mL each. And bagging, vacuumizing, and storing in a refrigerator at-80 ℃ for standby.

Taking out 1 frozen sample tube during separation, weighing the thawed sample according to the ratio of 1:10, re-suspending in anaerobic PBS, vortex oscillating for 5 min, introducing into an anaerobic glove box, taking 0.5 mL bacterial suspension, oscillating and mixing in 4.5 mL anaerobic PBS, and gradient diluting to 10 ^-6 Mixing the bacterial liquid with YCFA culture medium, and packaging into 384-well plate for culturing for one week. Selecting the bacterial liquid of the hole site which grows, transferring, culturing 48 and h, detecting one part by using MALDI-TOF-MS, and primarily classifying the separated bacterial strains; the other plate is transferred into a 96-well plate again according to the mass spectrum result, after 48-h culture, one plate is subjected to 16S rDNA gene amplification and sent to Beijing qing biological science and technology Co., ltd for sequencing, and 50% glycerol is added into the other plate according to the ratio of 1:1 for mixing and temporary storage, and the PCR result is confirmed and then used.

Analysis of the 16S rDNA gene sequencing results, comparison of the sequences with NCBI nucleic acid database, and the results show that the sequence is identical to that of a strain of Eimeria fenhnsoniiAlistipes finegoldiiSequence similarity of the highest degree>99.9%) to thereby preliminarily identify it asAlistipes finegoldiiThe strain is named as Afine-1 of the Emamectin Fender, and is a small white opaque, convex circular colony with smooth and moist surface on a three-mixed solid culture medium (BHI+MRS+modified GAM); the strain morphology is identified by using a crystal violet staining method, and the Afine-1 strain of the Emotion fens is in a short rod shape under an optical microscope. The strain is preserved in China center for type culture collection (CCTCC, university of Wuhan collection) for 11 months and 10 days in 2022, and the preservation number of the strain is CCTCC No: m20221759.

EXAMPLE 2 Whole genome analysis of Afine-1

The Afine-1 of the Afine-like bacillus is inoculated into a5 mL anaerobic triple mixed culture medium according to the inoculation amount of 2 percent, the culture is carried out until the late logarithmic growth phase, the whole genome DNA of the strain is extracted, and the NovaSeq 6000 of an Illumina high-throughput sequencing platform is utilized for carrying out whole genome sequencing. After assembly and annotation, the protein sequences were entered into virulence gene bank Virulence Factor Databases for virulence factor analysis. The results show that the bacteria do not have virulence factors.

The novel analysis of the strain was performed using the average nucleotide similarity (Average Nucleotide Identity, ANI). By searching in Genbank, 37 published results were foundAlistipes finegoldiiThe whole genome, by comparison with fastANI (v 1.33), shows that the two strains closest to the whole genome of the A.facilis Afine-1 are GCA_014845615.1 (ANI=99.27%) and GCA_928722145.1 (ANI=99.25%) respectively, which are lower than 99.9%, so that the A.facilis Afine-1 can be considered as a new strain.

EXAMPLE 3 haemolysis assay of Afine-1 of A.fense

The deposited Afine-1 strain of the Emamectin Fender was inoculated into 5 mL anaerobic triple mixed medium (BHI+MRS+modified GAM) in an inoculum size of 2%, enterococcus faecalis (beta hemolysis, CICC23658, china industry microbiological culture Collection center) was used as a positive control, and a blank medium was used as a negative control. All strains were anaerobically cultured in an anaerobic triple medium at 37℃for 12 h to give activated strains. 2.5. Mu.L of each of the activated bacterial solutions was inoculated onto a Columbia plate (Ama Biotechnology Co., shanghai) and 3 strains were placed in parallel. After anaerobic culture at 37 ℃ for 48 h, a completely transparent hemolytic ring with obvious limit is formed around the enterococcus faecalis colony as a positive control, and the colony is subjected to beta hemolysis; the culture medium surrounding the Afine-1 colony of the A.fenicola was unchanged, and was gamma hemolyzed, i.e., not hemolyzed.

Example 4 antibiotic susceptibility test of Afine-1 of A.fensonii

According to the requirement of antibiotic sensitivity test in the third section of "microecological live bacteria preparation general theory" of Chinese pharmacopoeia (2020 edition), the sensitivity of the strain to the antibiotic is measured by adopting an agar diffusion paper sheet method, and the sensitivity level of the strain to the antibiotic is judged according to the size of the inhibition zone.

Strain activation and streaking: activating the Afine-1 strain in a triple mixed culture medium (BHI+MRS+modified GAM), performing anaerobic culture at 37 ℃ for 24 h, selecting a one-ring bacteria liquid, streaking on a Buchner solid culture medium, and placing a plate in the anaerobic culture at 37 ℃; staphylococcus aureus (CMCC (B) 26003, chinese food and drug verification institute) is activated in TSB liquid culture medium, aerobically cultured at 37 ℃ for 24 h, and a cyclic fungus liquid is selected and streaked on TSA solid culture medium, and the plate is placed in aerobically cultured at 37 ℃.

Preparing a bacterial suspension: several single colonies are selected from the cultured solid plate and inoculated into physiological saline to prepare bacterial suspension, and the turbidity of the suspension is regulated to 0.5 McO concentration.

Inoculation plate: dipping a sterile cotton swab in the bacterial liquid, and rotationally squeezing out the redundant bacterial liquid on the inner wall of the tube; the surface of the Brucella plate (the Fennell-like bacillus group) or the TSA plate (the staphylococcus aureus group) is uniformly coated for 3 times, the plate is rotated for 60 degrees each time, and finally the agar edge in the plate is coated for one circle (the completion is achieved within 15 minutes).

Sticking an antibacterial drug paper sheet: the drug sensitive paper sheets are distributed and placed on the flat plates by using a drug sensitive paper sheet distributor, 3 drug sensitive paper sheets are placed on each flat plate, and 3 drug sensitive paper sheets are arranged in parallel. Specific information of the 14 drug sensitive paper sheets (Kangtai Biotechnology Co., ltd.) is shown in Table 1.

TABLE 1 information on 14 drug sensitive paper sheets

Chinese name of paper

Goods number

Content of

Quality control bacteria quality control drug sensitivity range

Drug resistance

Intermediary(s)

Sensitivity to

Reference bacteria

Penicillin

Z51031

10 units

26-37

≤14

-

≥15

Enterococcus genus

Ampicillin (Amoxicillin)

Z21011

10 μg

27-35

≤16

-

≥17

Enterococcus genus

Imipenem

Z21049

10 μg

-

-

-

≥16

Haemophilus genus

Vancomycin

Z21026

30 μg

17-21

≤14

15-16

≥17

Enterococcus genus

Ceftriaxone

Z21002

30 μg

22-28

≤13

14-20

≥21

Enterobacteriaceae (enterobacteriaceae)

Tetracycline

Z21036

30 μg

24-30

≤14

15-18

≥19

Enterococcus genus

Erythromycin

Z21030

15 μg

22-30

≤13

14-22

≥23

Enterococcus genus

Clindamycin

Z21029

2 μg

26-32

≤15

16-18

≥19

Streptococcus pneumoniae

Levofloxacin

Z21014

5 μg

25-30

≤13

14-16

≥17

Enterococcus genus

Trimethoprim

Z21045

25 μg

/

≤10

-

≥16

Staphylococcus genus

Nitrofurantoin

Z21047

300 μg

18-22

≤14

15-16

≥17

Enterococcus genus

High concentration gentamicin

Z22021

120 μg

/

6

7-9

≥10

Enterococcus genus

Streptomycin

Z21035

300 μg

/

6

7-9

≥10

Enterococcus genus

Rifampicin

Z21037

15 μg

26-34

≤16

17-19

≥20

Staphylococcus genus

The experimental results are as follows: staphylococcus aureus is used as a quality control strain, and the results all accord with the drug sensitivity range. Afine-1 of Earthworum is sensitive to 9 antibiotics of imipenem, vancomycin, tetracycline, erythromycin, clindamycin, levofloxacin, nitrofurantoin, high-concentration gentamicin, rifampin; 2 antibiotics resistance to penicillin and ampicillin; 3 antibiotics, ceftriaxone, trimethoprim, streptomycin.

Example 5 antioxidant assay of Afine-1 of A.fensis

After the deposited Afine-1 strain was activated, it was inoculated into 5 mL anaerobic BF839 medium at an inoculum size of 2%, and LGG (Lactobacillus rhamnosus GG, CICC6141, china industry microbiological culture Collection center) was used as a positive control, and all strains were anaerobically cultured in the anaerobic BF839 medium at 37℃for 24 h.

Sample processing: taking 0.5 mL culture bacteria liquid, centrifuging at 12000 rpm for 20 min, discarding supernatant, and re-suspending with 0.5 mL precooled extracting solution; transferring to sterilized screw cap tube containing beads (Sigma Co., U.S.A., G4649-1 KG), shaking to break wall once (parameter set: 4.5 m/s,30 s), centrifuging at 12000 rpm at 4deg.C for 10 min, and collecting supernatant, and placing on ice for testing.

BCA protein concentration assay (PC 0020, beijing solibao technologies limited):

1) Preparing a solution: according to the number of standard substances and samples, adding 1 volume of Cu reagent (50:1) into 50 volumes of BCA reagent to prepare BCA working solution, and fully and uniformly mixing.

2) Drawing a standard curve: 10. Mu.L BSA standard was diluted to 100. Mu.L with PBS to a final concentration of 0.5. 0.5 mg/mL; standards were added to protein standard wells of 96-well plates at 0, 2, 4, 6, 8, 12, 16, 20 μl, respectively, with PBS added to make up to 20 μl. 200 mu L of BCA working solution is added to each hole site, the mixture is placed at 37 ℃ for 15-30 min, and the absorbance at A562 nm is measured.

3) Sample detection: 200 mu L of BCA working solution is added to each sample to be tested, the sample is placed for 15-30 min at 37 ℃, and the absorbance at A562 nm is measured.

Detection of Strain Total antioxidant Capacity (Beijing Soy Bao technology Co., ltd., BC 1315):

1) Preparing a solution: the extract is placed in a refrigerator or on ice for precooling at 2-8deg.C before use; 10 mg FeSO is added immediately before use ₄ ·7H ₂ Adding 0.9 mL distilled water and 20 mu L concentrated sulfuric acid into the O standard substance to prepare 40 mu mol/mL FeSO ₄ Standby standard solution; the first reagent, the second reagent and the third reagent in the kit are mixed according to the proportion of 7:1:1 to prepare working solution, and the working solution is prepared for use and is placed in a constant temperature incubator at 37 ℃ for preheating for 10 min before use.

2) Drawing a standard curve: distilled water is used for 40 mu mol/mL standard solutionDiluting into standard solutions of 0.15, 0.1, 0.05, 0.025, 0.0125, 0.00625 and 0.003125 mu mol/mL for use, wherein Fe ²⁺ Final concentrations were 0.075, 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.00156. Mu. Mol/mL, respectively. 100. Mu.L of standard solution (distilled water as blank) was sucked up, added to 100. Mu.L of reagent II in the kit, thoroughly mixed, reacted at room temperature for 10 min, and the absorbance at 593 nm was measured.

3) Sample detection: 180 mu L of working solution, 6 mu L of sample to be detected and 18 mu L of distilled water are respectively added into a 96-well plate, fully and uniformly mixed, reacted for 10 min at room temperature, and the absorbance at 593 nm is measured.

The calculation formula is as follows:

1) The protein concentration of the sample was calculated from the protein standard curve.

2) Calculating sample Fe according to an antioxidant standard curve ²⁺ Concentration.

3) Total antioxidant capacity calculation:

total antioxidant capacity (μmol/mg prot) =x×v inverse total/(V-like×cpr) =34×x/Cpr

x: fe in sample ²⁺ Final concentration, μmol/mL; v inverse total: total reaction volume, 0.204mL;

v sample: sample volume in reaction, 0.006mL; cpr: sample protein concentration, mg/mL.

Experimental results: as can be seen from FIG. 1, the total antioxidant capacity of the positive control strain LGG reaches 0.25 mu mol/mg prot, and the total antioxidant capacity of Afine-1 reaches 0.36 mu mol/mg prot, which is significantly stronger than that of LGG #p<0.05 Shows that Afine-1 has stronger antioxidant capacity.

EXAMPLE 6 evaluation of antibacterial Activity of Afine-1 of Equisqualis for Clostridium difficile

After the Afine-1 of the Emotion fens is activated, the Afine-1 is inoculated into an anaerobic three-mixed culture medium (BHI+MRS+modified GAM) according to an inoculum size of 2 percent, and is cultured for 48 h at 37 ℃ to obtain a fermentation broth. Meanwhile, clostridium difficile (CICC 22951, china center for type culture collection of Industrial microorganisms) is subjected to activation and transfer, and then diluted 50 times in a sterile anaerobic triple-mixed liquid culture medium to serve as pathogenic bacteria liquid; 0.2. 0.2 mL pathogenic bacteria liquid was spread on a sterile anaerobic GAM solid medium (+5% horse serum). 3 sterilized oxford cups are placed on the coated flat plate, and 0.2 mL Fender-like bacillus fermentation liquor is added into the oxford cups. Placing the bacteria inhibition zone into an anaerobic culture box, adding an anaerobic gas producing bag, and measuring the size of the bacteria inhibition zone by using a vernier caliper after the plates are vertically cultured for 24 h. The result shows that the diameter of the inhibition zone of the Afine-1 of the alike bacterium fenhnsonii on clostridium difficile is 15.00 mm, which shows that the Afine-1 of the alike bacterium fensonii has the antibacterial capability on clostridium difficile.

Example 7 in vitro cell inflammation inhibition assay of Afine-1 of A.fensonii

THP-1 cell polarization: THP-1 was suspended cells (BNCC No. 358410, north Nata-Aoshima Biotech Co., ltd.) and polarized to mature macrophages using PMA (phorbol 12-tetradecanoate 13-acetate, phorbol 12-myristate1 3-acetate, sigma-Aldrich Company, P1585). Upon differentiation of THP-1 into mature macrophages, further use was made of lipopolysaccharides (LPS, sigma-Aldrich Company, L3024) in combination with interferon-gamma (IFN-gamma, pai Tex Biotechnology, inc., AF-30-02-20) to induce polarization of mature macrophages into inflammatory macrophages. RPMI-1640 medium (Celaster technologies (China) C11875500 BT) containing 10% FBS (Celaster technologies (China) SH 30084.03) and a final concentration of 100ng/mL PMA was used at 1×10 ⁵ Seeding Density of wells THP-1 cells were seeded in 96 well plates and placed in 5% CO ₂ After 24-h culture at 37℃in an incubator, the medium RPMI-1640 containing 10% FBS, 100ng/mL LPS and 20 ng/mL IFN-. Gamma.was replaced and placed in 5% CO ₂ In an incubator, 24. 24 h was cultured at 37 ℃.

Strain culture: from the bacterial preservation, 200 mu L of Afine-1 bacterial liquid of the Fender-like bacillus was taken to 5 mL anaerobic three-mixed culture medium (BHI+MRS+modified GAM), and the anaerobic culture was performed in an electrothermal incubator at 37 ℃ for 24 h. After one transfer, anaerobic culture 8 h. Taking 1 mL bacterial liquid, and centrifuging at 5000 rpm/min for 15 min. The strain was diluted to 3.67×10 with RPMI-1640 medium containing 10% FBS ⁶ CFU/mL was ready for use.

Effect of the Afine-1 bacterium of the type Fenton on the expression of TNF- α by THP-1 cells: mature giant phagocytosisCytostatic medium, 100. Mu.L of RPMI-1640 medium containing 10% FBS was added as a normal control group, pre-polarized inflammatory macrophages, and 100. Mu.L of RPMI-1640 medium containing 10% FBS was added as a model group; as a positive control group, 100. Mu.L of RPMI-1640 medium containing 10% FBS and dexamethasone (Sigma-Aldrich Company, D4902-25) at a final concentration of 25. Mu.g/mL was added to the pre-polarized inflammatory macrophages, and 100. Mu.L of the pre-prepared Afine-1 strain solution of Afine-like Fennella was added to the experimental group. Placed in 5% CO ₂ Incubator, incubation 24 h at 37 ℃. mu.L of the cell culture broth was aspirated, centrifuged at 4℃at 5000 rpm/min for 15 min, and the supernatant was collected and assayed for TNF-alpha content (E-EL-H0109 c, wuhan Iretto Biotechnology Co., ltd.) using the Human TNF-alpha (Tumor Necrosis Factor Alpha) ELISA kit.

Experimental results: as shown in FIG. 2, compared with the model group, the Afine-1 strain of the Emotion-like bacillus can obviously inhibit the expression of the pro-inflammatory factor TNF-alpha in the inflammatory macrophage THP-1, which shows that the strain has very obvious anti-inflammatory effect.

Example 8 therapeutic Effect of Afine-1 of Eimeria fenhnsonii on mice with 5-fluorouracil diarrhea

And (3) preparing a freeze-drying protective agent: trehalose 10 g, fructo-oligosaccharide 5g, skim milk powder 10 g are weighed, dissolved in 70 g purified water and sterilized at 115 ℃ for 20 min. Sodium ascorbate 5g is added before use.

Preparing bacterial powder: inoculating the preserved Afine-1 of the Emotion fens into an anaerobic triple mixed culture medium (BHI+MRS+modified GAM) according to an inoculation amount of 10%, and performing anaerobic culture at 37 ℃ and 150 rpm for 18-24 hours until reaching OD ₆₀₀ The value is more than or equal to 0.8, and the first-stage seed liquid is obtained. Transferring the culture medium to anaerobic three-mixed culture medium again according to 1.5% of inoculation amount, and performing anaerobic culture at 37 ℃ and 150 rpm for 18-24 hours until reaching OD ₆₀₀ The value is more than or equal to 1.0, and the secondary seed liquid is obtained. The fermentation broth was pumped into a fermenter with the modified GAM broth at 1% inoculum size by peristaltic pump, and fermentation parameters (37℃at pH 6.5, 150 rpm, 0.06 MPa pressure control) were set to initiate fermentation. Fermentation broth OD ₆₀₀ The value is not less than 1.4 or OD ₆₀₀ Stopping fermentation when the value is increased to be less than or equal to 0.1, setting the fermentation temperature to be 20 ℃, and collecting the bacterial liquid. Fungus liquid split chargingInto a centrifuge bowl, the supernatant was discarded by centrifugation. According to bacterial mud: the weight ratio of the freeze-drying protective agent is 1:2, and the freeze-drying protective agent is added and mixed uniformly to emulsify the bacterial mud. And (3) putting the emulsified bacterial suspension on a plate layer of a freeze dryer cooled to the temperature of minus 40 ℃ for freeze-drying, taking out bacterial cakes after the freeze-drying procedure is finished, and crushing to obtain bacterial powder. Animals were dosed with 1X 10 saline using 0.2 mL saline ⁹ The CFU bacterial powder is prepared into bacterial suspension.

Experimental animals: in the study, 20 SPF-grade male Balb/c mice are selected for experiment, the weight of the mice is 18-22 g, the mice are purchased from Beijing Vetong Lihua laboratory animal technology Co., ltd, and the mice are bred in SPF-grade animal houses. The mice were randomly divided into 4 groups of 5 mice based on initial body weight.

Experiment design: the 5-FU (Tianjin JinYao pharmaceutical Co., ltd., specification 10 mL/count, 0.25 g/10 mL) solution was used to induce the mice chemotherapy-related diarrhea model, and the 4 groups were a normal control group, a model control group, a positive control loperamide group and an Afine-1 group, respectively. Except normal control group, the other groups are subjected to 5-FU single intraperitoneal injection molding treatment, and the molding amount is 350 mg/kg. All groups were administered by gavage, normal and model control with lyoprotectant, positive control with loperamide (Siampelopsis pharmaceutical Co., ltd.) at a dose of 20 mg/kg, afine-1 with a dose of 1X 10 ⁹ The CFU/dose alone was administered with Afine-1 of Afine-like bacteria. The overall experimental period was 9 days, D1-D9. D3, performing single molding treatment on the 5-FU. The normal control group, the model control group and the Afine-1 group were continuously administered by gastric lavage for 5 days, and the loperamide group was continuously administered for 9 days once daily. The specific experimental groupings and dosing regimens are shown in table 2.

Diarrhea detection: mice were placed in 1 mouse cage with clean filter paper placed in each cage. Hard feces, normally considered 0 minutes; slightly wet or soft stool was regarded as 1 minute; moderately wet feces, fecal and anal Zhou Bujie are considered as 2 minutes; severe, thin stool and severe anus Zhou Bujie were considered 3 minutes. During the experimental period, the mouse feces were observed and scored daily. Total diarrhea score was the sum of daily diarrhea scores.

The specific experimental groupings and dosing regimens are shown in table 2.

TABLE 2 modeling and dosing regimen for the therapeutic Effect of Equipped Fenton Afine-1 treatment of 5-FU diarrhea mice

Group of

Quantity of

Molding agent

Amount of modeling agent

Test article

Administration volume

Dosage for administration

Days of administration

Normal control group

5

Physiological saline

/

Freeze-drying protective agent

0.2 mL/only

/

5 d

Model control group

5

5-FU

350mg/kg

Freeze-drying protective agent

0.2 mL/only

/

5 d

Loperamide

5

5-FU

350mg/kg

Loperamide

10 mL/kg

20 mg/kg

9 d

Afine-1

5

5-FU

350mg/kg

Afine-1

0.2 mL/only

1×10 9 CFU/only

5 d

Note that: 5-FU 5-fluorouracil; CFU colony forming unit colony forming units; d, tiantian

Experimental results: as shown in fig. 3, both loperamide and the like bacillus fenhnsonii Afine-1 significantly reduced the diarrhea score on day 9 (P < 0.01) compared to the model control group; loperamide significantly reduced the total diarrhea score (P < 0.05), afine-1 significantly reduced the total diarrhea score (P < 0.01) in 5-FU-induced mice (FIGS. 3A, B and C).

The above results demonstrate that the Afine-1 strain of the present invention significantly improves diarrhea symptoms in mice caused by 5-fluorouracil.

EXAMPLE 9 therapeutic Effect of Afine-1 of Embectonia cerealis on diarrhea mice

Experimental animals: in the study, 24 SPF-grade male Balb/c mice are selected for experiment, the weight of the mice is 18-22 g, the mice are purchased from Beijing Vetong Lihua experiment animal technology Co., ltd, and the mice are bred in SPF-grade animal houses. The mice were randomly divided into 4 groups based on initial body weight.

Experiment design: mice were induced with an i Li Tikang (CPT-11, shanghai-derived leaf biotechnology limited, specification 5 g) solution for chemotherapy-related diarrhea models, with 4 groups being normal control (n=5), model control (n=7), positive control prednisone (n=6) and a. Fene-1 (n=6), respectively. The other groups except the normal control group were subjected to CPT-11 continuous 4-day intraperitoneal injection molding treatment with a molding amount of 60 mg/kg. All the groups are administrated by stomach irrigation, normal control group and model control group, and positive control group is administrated with prednisone (Tianjin Yijin medicine Co., ltd.) at a dosage of 100 mg/kg, and Afine-1 group is administrated with Eheater-like bacillus fens 1×10 ⁹ CFU/CFU. The overall experimental period was 9 days, D1-D9. D3, D4, D5 and D6 were subjected to CPT-11 molding treatment once a day. The normal control group, the model control group and the Afine-1 group were continuously administered by gastric lavage for 9 days, and prednisone was continuously administered from D3 to D9 for 7 days once daily. The specific experimental groupings and dosing regimens are shown in table 3.

Diarrhea detection: mice were placed in 1 mouse cage with clean filter paper placed in each cage. Hard feces, normally considered 0 minutes; slightly wet or soft stool was regarded as 1 minute; moderately wet feces, fecal and anal Zhou Bujie are considered as 2 minutes; severe, thin stool and severe anus Zhou Bujie were considered 3 minutes. During the experimental period, the mouse feces were observed and scored daily.

TABLE 3 modeling and dosing regimen for the therapeutic Effect of Afine-1 of A.fense on CPT-11 diarrhea mice

Group of

Quantity of

Molding agent

Amount of modeling agent

Test article

Administration volume

Dosage for administration

Frequency of administration

Normal control group

5

Physiological saline

60 mg/kg

Freeze-drying protective agent

0.2 mL/only

/

9 d

Model control group

7

CPT-11

60 mg/kg

Freeze-drying protective agent

0.2 mL/only

/

9 d

Prednisone positive control group

6

CPT-11

60 mg/kg

Prednisone

10 mL/kg

100 mg/kg/dose

7 d

Afine-1 group

6

CPT-11

60 mg/kg

Afine-1

0.2 mL/only

1×10 9 CFU/only

9 d

Note that: CPT-11 irinotecan; CFU colony forming unit colony forming units; d, tiantian

Experimental results: as shown in FIG. 4, both prednisone and Afine-1 bacteria were able to start decreasing diarrhea scores the next day after molding (A); prednisone can significantly reduce the total diarrhea score (P < 0.01), and the Afine-1 strain of the Equipped can significantly reduce the total diarrhea score (P < 0.05) (B).

The above description is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto. Any person skilled in the art, within the scope of the present disclosure, may apply to the present invention, and any equivalent or modified embodiments thereof are included in the scope of the present invention.

Claims (4)

1. Fender's bacillusAlistipes finegoldii) It is deposited in China center for type culture Collection with the accession name of Fenton's offeringsAlistipes finegoldiiAfine-1, with preservation number of CCTCC NO: m20221759.

2. A pharmaceutical composition comprising the bacterium of the species Emotion of the formula of claim 1Alistipes finegoldii) And a pharmaceutically acceptable carrier or adjuvant.

3. The method of claim 1Alistipes finegoldii) Or the use of the pharmaceutical composition according to claim 2 in the preparation of a microbial preparation for the treatment and/or prophylaxis of diarrhea caused by chemotherapeutic agents or/and inhibition of clostridium difficileClostridium difficile) Is a breeding of (a) a strain.

4. The use according to claim 3, wherein the chemotherapeutic agent comprises at least one of the following agents: fluorouracil, topoisomerase I inhibitor, tyrosine kinase inhibitor, taxane, and platinum chemotherapeutics.