Preparation and application of two bifidobacterium longum subsp.infantis metabiotics for inhibiting clostridium perfringens

Abstract

The invention discloses preparation and application of two bifidobacterium longum subsp.infantis metabiotics for inhibiting clostridium perfringens, and belongs to the technical field of microorganisms. The two kinds of bifidobacterium longum subsp.infantis metabiotics provided by the invention have an excellent inhibition effect on the growth of clostridium perfringens, can destroy the integrity of cell walls and cell membranes and cause the release of intracellular substances, and are respectively fermentation supernatant of the bifidobacterium longum subsp.infantis CCFM1192 and CCFM1210. The two metabiotics provided by the invention can be used for remarkably relieving the symptom that a mouse is infected by clostridium perfringens, including effectively reducing the load of clostridium perfringens in excrement, relieving pathological changes of spleen, jejunum, ileum and colon tissues and improving the diversity of intestinal flora. Therefore, the bifidobacterium longum subsp. Infantis CCFM1192 and CCFM1210 metabiotics have a huge application prospect in the preparation of products for preventing and / or treating the clostridium perfringens related diseases.

Description

Technical Field

[0001] The invention relates to the preparation and application of two Bifidobacterium longum subspecies infantis postbiotics for inhibiting Clostridium perfringens, and belongs to the field of microbial technology. Background Art

[0002] Clostridium perfringens is an anaerobic, spore-forming Gram-positive bacillus. As an opportunistic pathogen, Clostridium perfringens exists in the intestinal microbiota of humans and animals. Clostridium perfringens can produce more than a dozen toxins, causing various serious gas gangrene and intestinal diseases, including food poisoning, non-foodborne diarrhea, and enterocolitis. More than 100 million cases of food poisoning are related to Clostridium perfringens infection each year, causing huge economic losses. At present, antibiotics are mainly used to treat or prevent Clostridium perfringens infection, but this approach will undoubtedly lead to the formation of drug-resistant strains and intestinal flora disorders, so there is an urgent need to find an effective and reliable alternative to antibiotics.

[0003] The prior art discloses the effect of fermentation broths from other probiotic sources on inhibiting the growth of Clostridium perfringens. For example, patent CN117448213B discloses a fermentation supernatant of Lactobacillus plantarum BLCC2-0072, but there is no in vivo verification of reducing the load of Clostridium perfringens. It is well known in the art that the results of in vitro experiments are difficult to serve as evidence of in vivo function. After oral administration, the substance will face the harsh environment of the gastrointestinal environment in the body, resulting in decreased stability, functional impairment, and the like. The document "Millifluidic-assisted ionic gelation technique for encapsulation of probiotics in double-layered polysaccharide structure" proposes that animal Bifidobacterium lactis subspecies and Lactobacillus plantarum need to be embedded to improve their survival rate in the harsh digestive conditions of the gastrointestinal tract, so it is impossible to prove its effectiveness in vivo. The document "Preparation of postbiotics from Lactobacillus paracasei TK1501 and its inhibitory effect on Helicobacter pylori" points out that postbiotics may be degraded by proteases and cannot tolerate acid-base environments in the body. Therefore, under the premise that the prior art has not yet disclosed postbiotics that can inhibit the growth of Clostridium perfringens in vivo, it is difficult to predict which postbiotics can have the function of inhibiting the growth of Clostridium perfringens in vivo.

[0004] Bacteriocins are highly effective, stable, and narrow-spectrum antimicrobial peptides produced by bacteria. Their excellent performance solves the problems caused by traditional antibiotics and circumvents the safety risks of using probiotics to transfer virulence factors / antibiotic resistance genes and the challenges of difficult probiotic colonization in adverse environments. Finding a postbiotic that contains a large amount of bacteriocins and has the function of inhibiting Clostridium perfringens is of great significance for the preparation of safe, side-effect-free antibiotic alternatives that can prevent / treat Clostridium perfringens infections. Summary of the invention

[0005] The invention provides preparation methods of two Bifidobacterium longum subsp. infantis postbiotics for inhibiting Clostridium perfringens and applications thereof in alleviating Clostridium perfringens infection symptoms.

[0006] The invention provides application of Bifidobacterium longum subsp. infantis CCFM1192 or Bifidobacterium longum subsp. infantis CCFM1210 in preparing medicines for inhibiting Clostridium perfringens.

[0007] In one embodiment of the present invention, the deposit number of the Bifidobacterium longum subsp. infantis CCFM1192 is GDMCC No: 61846, which has been disclosed in patent CN114276961A; the deposit number of the Bifidobacterium longum subsp. infantis CCFM1210 is GDMCC No: 62234, which has been disclosed in patent CN114574390A.

[0008] In one embodiment of the present invention, the medicine contains postbiotics prepared from Bifidobacterium longum subsp. infantis.

[0009] In one embodiment of the present invention, the postbiotic preparation method is (a) or (b):

[0010] (a) culturing the Bifidobacterium longum subspecies infantis, collecting the fermentation supernatant to obtain postbiotics;

[0011] (b) After culturing the Bifidobacterium longum subspecies infantis, collecting the fermentation supernatant, and extracting protein in the supernatant to obtain postbiotics.

[0012] In one embodiment of the present invention, the postbiotics may be used as a solution or after freeze-drying.

[0013] In one embodiment of the present invention, the postbiotic contains bacteriocin 1 and / or bacteriocin 2 and / or bacteriocin 3 and / or bacteriocin 4 and / or bacteriocin 5 and / or bacteriocin 6 and / or bacteriocin 7 and / or bacteriocin 8.

[0014] In one embodiment of the present invention, the amino acid sequence of bacteriocin 1 is: MNKIIDMQSIEEVNDDAVHDSGYSYSFCFGRPSSTSGFFC; the amino acid sequence of bacteriocin 2 is: MSVQTGKQSKLKMMAIALVTCASMTIIPVASASAAEADVIAPTTESGEYGIRPMWLNPEGKHTQYP AQGGIWEWGFWNVKIRSYYTHDTRTHGSSVSLNGDTVRSIDTVAKKQSIAEKYAVNYWGNNDAY YYRVS; the amino acid sequence of bacteriocin 3 is: MIAKKTTGGLLASMLALGASGTLIIPANANEVGTSVKVSCSVPTDRDAGISTVDVSYPYGGTWQYG TTLWGGCGDVYSNFLHNSRRHHSSVLNADNVYAYSGVKEAGIWAYARTYAVCGKVDQAFYGFD A; the amino acid sequence of bacteriocin 4 is: MSINEKSIVGESFEDLSAADMAMLTGRNDDGVAPASLSFAVSVLSVSFSACSVTVVTRLASC GNCK; the amino acid sequence of bacteriocin 5 is: MMNTAVEDEVVSEVWDADAAALDATPTDQKQIDKEFTKIVSPYMASSGWVCTLTQECGTFVCSCN; the amino acid sequence of bacteriocin 6 is: MGHTMSVQTGKQSKLKMMAVALVTCASMTIIPVASASAAEADVIAPTTESGEYGIRPMWL NPEGKHTQYPAQGGIWEWGFWNVKIRSYYTHDTRTHGSSVSLNGDTVRSIDTVAKKQSIAE KYAVNYWGNNDAYYYRVS; the amino acid sequence of bacteriocin 7 is: MIAKKITGGLLAATLALGASGTLIIPANANEVGTSVKVSCSVPTERDAGISTVDVSYPYGGT WQYGTTLWGGCGDVYSNFLHNSRRHHSSVLNADNVYAYSGVKEAGIWAYARTYAVCGKV DQAFYGFDA;The amino acid sequence of the bacteriocin 8 is: MRNNRLVKSTASLLLAVTVSFSSVMVASATETTSTNAAKTPQYAQSHYPSEAEIEAYLNDPQYKKYGRTREDVEAYLKANAEEDAATTTTMEAGLYSSWSTDLFSDGYWMNRSGVWSLSIMPRRALLWDTDRGWGQVYDRFHTSRHWTYYSAWADASMRKQFNCHAQYGMLKTPYNLEPSRSDVSPITCN. ;

[0015] In one embodiment of the present invention, the postbiotics can inhibit the growth of Clostridium perfringens and alleviate or treat the symptoms of diseases related thereto.

[0016] In one embodiment of the present invention, the drug has at least one of the following effects:

[0017] (1) Reduce the load of Clostridium perfringens in feces;

[0018] (2) Increase thymic index;

[0019] (3) Reduce spleen index;

[0020] (4) Alleviate spleen tissue lesions;

[0021] (5) Relieve lesions in the jejunum, ileum, and colon;

[0022] (6) Improve the diversity of intestinal flora and improve intestinal flora disorders.

[0023] In one embodiment of the present invention, the dosage form of the drug includes powder, granules, capsules, tablets, pills or oral liquid.

[0024] In one embodiment of the present invention, the medicine further contains a drug carrier and / or a pharmaceutical excipient.

[0025] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0026] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and / or an additive.

[0027] The present invention also provides the use of Bifidobacterium longum subspecies infantis CCFM1192 or Bifidobacterium longum subspecies infantis CCFM1210 in preparing Clostridium perfringens inhibitor, wherein the preservation number of Bifidobacterium longum subspecies infantis CCFM1192 is GDMCC No: 61846; the preservation number of Bifidobacterium longum subspecies infantis CCFM1210 is GDMCC No: 62234.

[0028] In one embodiment of the present invention, the inhibitor contains postbiotics prepared from Bifidobacterium longum subsp. infantis.

[0029] The present invention also provides a method for inhibiting the growth of Clostridium perfringens, which comprises adding postbiotics of Bifidobacterium longum infantis subspecies CCFM1192 or Bifidobacterium longum infantis subspecies CCFM1210 into a culture environment of Clostridium perfringens, wherein the preservation number of the Bifidobacterium longum infantis subspecies CCFM1192 is GDMCC No: 61846; the preservation number of the Bifidobacterium longum infantis subspecies CCFM1210 is GDMCC No: 62234.

[0030] Beneficial Effects

[0031] (1) The two postbiotics of the present invention are rich in bioactive substances, bacteriocins, and have excellent ability to inhibit the growth of Clostridium perfringens, but do not inhibit the growth of Lactobacillus plantarum, and have a narrow spectrum of antibacterial properties.

[0032] (2) The two postbiotics provided by the present invention have the effect of alleviating the symptoms of Clostridium perfringens infection, which is specifically reflected in:

[0033] Significantly reduced the load of Clostridium perfringens in the feces of mice infected with Clostridium perfringens;

[0034] Significantly increased the thymus index of mice infected with Clostridium perfringens;

[0035] Significantly reduced the spleen index of mice infected with Clostridium perfringens;

[0036] Alleviate spleen tissue lesions in mice infected with Clostridium perfringens;

[0037] Alleviate jejunal, ileal and colonic tissue lesions in mice infected with Clostridium perfringens;

[0038] Improve the diversity of intestinal flora in mice infected with Clostridium perfringens and improve intestinal flora disorders.

[0039] (3) The postbiotic strains Bifidobacterium longum subsp. infantis CCFM1192 and Bifidobacterium longum subsp. infantis CCFM1210 of the present invention are isolated from the intestinal flora of healthy infants. The strains have no toxic side effects on the human body. Therefore, the postbiotic preparations prepared using Bifidobacterium longum subsp. infantis CCFM1192 and / or CCFM1210 provided by the present invention have broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 :Growth kinetics of Clostridium perfringens ATCC13124 in the presence of Bifidobacterium longum subsp. infantis postbiotics;

[0041] Figure 2 : Growth kinetics of Lactobacillus plantarum FJSWXM3IL1 in the presence of Bifidobacterium longum subsp. infantis postbiotics;

[0042] Figure 3 :Effects of Bifidobacterium longum subsp. infantis postbiotics on the cell membrane integrity of Clostridium perfringens ATCC13124;

[0043] Figure 4 :Effects of Bifidobacterium longum subsp. infantis postbiotics on the cell wall permeability of Clostridium perfringens ATCC13124;

[0044] Figure 5 : Scanning electron micrograph of Clostridium perfringens ATCC13124 treated with postbiotics from Bifidobacterium longum subsp. infantis;

[0045] Figure 6 : Transmission electron microscopy image of Clostridium perfringens ATCC13124 treated with postbiotics from Bifidobacterium longum subsp. infantis;

[0046] Figure 7 :Animal experiment flow chart;

[0047] Figure 8 :The fecal Clostridium perfringens load of mice in different groups during infection;

[0048] Fig. 9 : Thymus index of mice in different groups;

[0049] Fig.10 : Spleen index of mice in different groups;

[0050] Fig.11 :H&E staining of spleen tissues of mice in different groups;

[0051] Fig.12 : H&E staining of jejunum, colon and colon tissues of mice in different groups;

[0052] Fig.13 : Chao1 diversity index of intestinal flora of mice in different groups;

[0053] Fig.14 : Shannon diversity index of intestinal flora of mice in different groups;

[0054] Fig.15 : Simpson diversity index of intestinal flora of mice in different groups;

[0055] Fig.16 : Principal coordinate analysis of β-diversity index of intestinal flora in different groups of mice;

[0056] Figures 1 to 16 In the table, the values ​​after different letters in ac are significantly different, and the errors are presented in the form of Mean±SEM. DETAILED DESCRIPTION

[0057] The C57BL / 6 male mice involved in the following examples were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.

[0058] The culture medium involved in the following examples is as follows:

[0059] MRS liquid culture medium formula (L): peptone 10 g, beef extract 10 g, yeast powder 5 g, glucose 20 g, K2HPO4 2 g, diammonium citrate 2 g, sodium acetate 2 g, Tween 80 1 mL, MgSO4·7H2O 0.5 g, cysteine ​​hydrochloride 0.5 g, MnSO4·4H2O 0.25 g, pH 7.2-7.4.

[0060] BHI liquid culture medium formula (L): peptone 10g, dehydrated calf brain extract powder 12.5g, dehydrated buffalo heart extract powder 5g, sodium chloride 5g, glucose 2g, disodium hydrogen phosphate 2.5g, pH 7.2-7.6.

[0061] TSC agar medium formula (L): 15 g pancreatic acid, 5 g soytone, 5 g yeast extract, 1 g sodium metabisulfite, 1 g ammonium ferric citrate, 20 g agar, pH 7.5-7.7. After sterilization, add 20 ml of 0.5% D-cycloserine solution that has been sterilized by filtration.

[0062] The strains involved in the following embodiments: Bifidobacterium longum subsp. infantis CCFM1192 is deposited as GDMCC No: 61846 and has been disclosed in patent CN114276961A; Bifidobacterium longum subsp. infantis CCFM1210 is deposited as GDMCC No: 62234 and has been disclosed in patent CN114574390A; Bifidobacterium longum subsp. infantis CCFM1306 is deposited as GDMCC No: 63216 and has been disclosed in patent CN116590174A; Bifidobacterium longum subsp. infantis CCFM1306 is deposited as GDMCC No: 63216 and has been disclosed in patent CN116590174A; subsp. infantis) CCFM1270 has a preservation number of GDMCC No: 62840, which has been disclosed in patent CN115820488A; Lactobacillus plantarum (Lactiplantibacillus plantarum) FJSWXM3IL1 is preserved in the Food Biotechnology Strain Collection Center of the School of Food Science and Engineering of Jiangnan University.

[0063] Example 1: Preparation and component analysis of Bifidobacterium longum subsp. infantis postbiotics

[0064] The specific steps are as follows:

[0065] 1. Bacteria activation

[0066] Bifidobacterium longum subsp. infantis CCFM1192 and Bifidobacterium longum subsp. infantis CCFM1210 were respectively inoculated into MRS liquid culture medium and cultured at 37°C under anaerobic environment for 24 hours. The bacterial liquid was then transferred into fresh MRS liquid culture medium at an inoculation rate of greater than 10% (v / v) and activated to the third generation under 37°C under anaerobic environment.

[0067] 2. Preparation of postbiotics

[0068] The number of viable bacteria in the fermentation broth after the culture is completed is greater than 1×10 9CFU / mL, centrifuge and take the supernatant. Incubate the supernatant with 80% saturated ammonium sulfate at 4°C overnight to obtain a precipitate; redissolve the precipitate in pure water and pass it through an ultrafiltration membrane with a molecular weight cutoff between 1 and 30 kDa, and finally dry it to make a powder. When used, redissolve it according to the original fermentation broth volume concentration multiple of 10×, 20× and 40×.

[0069] 3. Analysis of the components of postbiotics

[0070] To further confirm the expression of bacteriocins in postbiotics CCFM1192 and CCFM1210, LC-MS / MS was used to confirm the bacteriocin sequences in the extracts. After reduction, alkylation, desalting and freeze-drying, the postbiotics were analyzed and identified by Nano-LC-ESI-LTQ-Obitrap MS / MS. Liquid chromatography conditions: chromatographic column: Reprosil-Pur 120C18-AQ (100 μm×180 mm, 3 μm); mobile phase: A water / formic acid (99.9:0.1, v / v), B acetonitrile / water / formic acid (80:19.9:0.1, v / v); gradient elution: 0-2 minutes (96%-92% A, 4%-8% B); 2-35 minutes (92%-72% A, 8%-28% B); 35-55 minutes (72%-60% A, 28%-40% B); 55-56 minutes (60%-5% A, 40%-95% B), 56-66 minutes (5% A, 95% B). Flow rate: 600 nL / min. Mass spectrometry conditions: The primary mass spectrometry was scanned in the Orbitrap, with a scanning range of 100-1500 m·z-1, an AGCtarget of 400000, and a resolution of 120,000; the secondary mass spectrometry used dynamic data-dependent scanning, an AGCtarget of 500000, and a resolution of 15,000; the collision energy was 30 eV. The information obtained was used for De Novo sequencing using PEAKSStudio 10.6, and the measured sequence was matched with the search target protein database.

[0071] As shown in Table 1 , polypeptide fragments of bacteriocin 2 and bacteriocin 3 were detected in the postbiotic CCFM1192, and polypeptide fragments of bacteriocin 4, bacteriocin 5, bacteriocin 6, bacteriocin 7, and bacteriocin 8 were detected in the postbiotic CCFM1210, proving that these bacteriocins exist in postbiotics.

[0072] Table 1 LC-MS / MS identification results

[0073]

[0074]

[0075]

[0076] Example 2: Antibacterial activity of Bifidobacterium longum subsp. infantis postbiotics

[0077] 1. Bacteria activation

[0078] Clostridium perfringens ATCC13124 was inoculated into BHI liquid culture medium and cultured at 37°C in an anaerobic environment for 18 hours, then the bacterial solution was transferred into fresh BHI liquid culture medium at an inoculum volume of 1% (v / v) and activated to the third generation at 37°C in an anaerobic environment.

[0079] Lactobacillus plantarum FJSWXM3IL1 was inoculated into MRS liquid culture medium and cultured at 37°C for 24 h, then the bacterial solution was transferred into fresh MRS liquid culture medium at a 1% (v / v) inoculation volume and activated at 37°C to the third generation.

[0080] 2. Effects of Bifidobacterium longum subsp. infantis postbiotics on the growth kinetics of Clostridium perfringens ATCC13124 and Lactobacillus plantarum FJSWXM3IL1

[0081] Bifidobacterium longum subsp. infantis CCFM1192, CCFM1210, CCFM1306 and CCFM1270 were prepared as postbiotics according to the postbiotic preparation method in Example 1.

[0082] Clostridium perfringens ATCC13124 was inoculated into BHI liquid medium at a concentration of 1×10 6 CFU / mL, Bifidobacterium longum subsp. infantis postbiotics were added to make the final concentration reach 20×. The growth was measured at 600nm using a multifunctional microplate reader under anaerobic conditions at 37°C.

[0083] Lactobacillus plantarum FJSWXM3IL1 was inoculated into MRS liquid medium at a concentration of 1×10 6CFU / mL, two Bifidobacterium longum subsp. infantis postbiotics CCFM1192 and CCFM1210 were added to make the final concentration reach 20×. The growth of Lactobacillus plantarum FJSWXM3IL1 was measured at 600nm using a multifunctional microplate reader at 37℃.

[0084] like Figure 1 As shown in the figure, compared with the control group, after adding postbiotics CCFM1192 and CCFM1210, the OD600 value of Clostridium perfringens ATCC13124 hardly increased, and basically remained at the initial value; after adding postbiotics CCFM1306 and CCFM1270, the growth of Clostridium perfringens ATCC13124 was inhibited, but the inhibitory effect was not as good as that of postbiotics CCFM1192 and CCFM1210. This shows that postbiotics CCFM1192 and CCFM1210 have excellent inhibitory effects on the growth of Clostridium perfringens ATCC13124.

[0085] like Figure 2 As shown in the figure, after adding CCFM1192 postbiotics and CCFM1210 postbiotics, the growth curve of Lactobacillus plantarum FJSWXM3IL1 was no different from that of the control group, showing typical growth curve characteristics, indicating that the two postbiotics had no effect on the growth of Lactobacillus plantarum FJSWXM3IL1. It further shows that the postbiotics of Bifidobacterium longum subsp. infantis CCFM1192 and CCFM1210 have narrow spectrum.

[0086] Example 3: Inhibitory Mechanism of Bifidobacterium longum subsp. infantis Postbiotics on Clostridium perfringens ATCC13124

[0087] 1. Effects of Bifidobacterium longum subsp. infantis postbiotics on the cell membrane integrity of Clostridium perfringens ATCC13124

[0088] The propidium iodide (PI) fluorescent probe method was used to detect the integrity of the cell membrane of Clostridium perfringens ATCC13124. Clostridium perfringens ATCC13124 cultured to the exponential phase was washed three times in 5 mM HEPES buffer (pH = 7.0) containing 5 mM glucose, and the cell concentration was adjusted to 10 8 CFU / mL. Subsequently, 100 μL of bacterial suspension was anaerobically incubated with 100 μL CCFM1192 postbiotics and CCFM1210 postbiotics at 10×, 20× and 40× final concentrations at 37°C for 1 hour. After adding 20 μM PI at a final concentration and incubating at 37°C in the dark for 30 minutes, the fluorescence intensity was measured at an excitation wavelength of 535 nm and an emission wavelength of 615 nm. The data were corrected by subtracting the fluorescence signal of untreated cells containing PI.

[0089] like Figure 3 As shown, under the action of different concentrations of postbiotics, PI of Clostridium perfringens ATCC13124 showed different fluorescence intensities. Under the action of 10×, 20× and 40× CCFM1192 postbiotics, the fluorescence intensities of PI released by the strain were 21193.00±383.33RFU, 28883.33±236.12RFU and 38954.33±429.24RFU, respectively. Under the action of 10×, 20× and 40× CCFM1210 postbiotics, the fluorescence intensities of PI released by the strain were 31542.00±457.09RFU, 34018.66±748.73RFU and 50976.66±979.74RFU, respectively. When the same postbiotic was exposed to concentrations of 10×, 20×, and 40×, the measured fluorescence intensity increased successively. That is, the higher the concentration of the postbiotic, the higher the released fluorescence intensity, indicating that the ability to destroy the integrity of the bacterial cell membrane is stronger, that is, it is dose-dependent.

[0090] 2. Effect of Bifidobacterium longum subsp. infantis postbiotics on the cell wall permeability of Clostridium perfringens ATCC13124

[0091] The permeability of the cell wall of Clostridium perfringens ATCC13124 was evaluated by measuring the concentration of alkaline phosphatase (AKP) in the periplasmic space. Clostridium perfringens ATCC13124 cultured to the exponential phase was washed three times in 10 mM PBS (pH = 7.0) and the cell concentration was adjusted to 10 8 CFU / mL. Subsequently, 100 μL of bacterial suspension was anaerobically incubated with 100 μL of crude bacteriocin at 10×, 20×, and 40× final concentrations at 37°C for 1 h. The leakage of alkaline phosphatase was detected using an alkaline phosphatase assay kit (Nanjing Jiancheng, China).

[0092] like Figure 4 As shown in the figure, the concentration of AKP leakage of Clostridium perfringens ATCC13124 was different under the action of different concentrations of postbiotics. The concentrations of AKP leakage of the strain under the action of 10×, 20× and 40× CCFM1192 postbiotics were 5.83±0.10U / L, 12.54±0.34U / L and 22.61±0.30U / L, respectively. The concentrations of AKP leakage of the strain under the action of 10×, 20× and 40× CCFM1210 postbiotics were 8.89±0.40U / L, 14.22±0.10U / L and 28.24±0.34U / L, respectively. Similar to the results of the PI test, the AKP concentration of the same postbiotic increased in a step-like manner as the concentration increased, indicating that the ability to destroy the bacterial cell wall was stronger. The above results indicate that Bifidobacterium longum subsp. infantis postbiotics CCFM1192 and CCFM1210 destroyed the integrity of the cell wall and cell membrane of Clostridium perfringens ATCC13124 in a dose-dependent manner.

[0093] 3. Scanning electron microscopy image of Bifidobacterium longum subsp. infantis postbiotic treatment (Clostridium perfringens ATCC13124)

[0094] Scanning electron microscopy was used to observe the changes in the surface microstructure of Clostridium perfringens ATCC13124 treated with postbiotics from Bifidobacterium longum subsp. infantis. Clostridium perfringens ATCC13124 cultured to the exponential phase was washed three times in 5 mM HEPES buffer (pH = 7.0) containing 5 mM glucose, and the bacterial concentration was adjusted to 10 8 CFU / mL. Add 200 μL of bacterial suspension to 20× final concentration of CCFM1192 postbiotics and CCFM1210 postbiotics and incubate anaerobically at 37°C for 1 hour. Untreated cells were used as controls. Harvest the bacterial sludge by centrifugation at 3000 rpm for 5 minutes, and then resuspend the bacteria in 2.5% glutaraldehyde and fix overnight at 4°C. Wash with PBS buffer 3-4 times to remove the fixative, and then use 30%-100% ethanol for gradient dehydration, dehydrating for 20 minutes at each concentration. The dehydrated bacteria were dried and sprayed with gold, and finally the morphology of the bacteria was observed using a SU8100 scanning electron microscope.

[0095] 4. Transmission electron microscopy image of Clostridium perfringens ATCC13124 after postbiotic treatment of Bifidobacterium longum subsp. infantis

[0096] Transmission electron microscopy was used to observe the effects of postbiotic treatment of Bifidobacterium longum subsp. infantis on the intracellular organization and structure of Clostridium perfringens ATCC13124. The pretreatment and scanning electron microscopy conditions of the samples were consistent. The bacteria were fixed with 2.5% glutaraldehyde at 4°C overnight, and then fixed with 1% osmium acid for 2h. The bacterial cells were washed with PBS buffer to remove the residual osmium acid, and then dehydrated with 30%-100% ethanol in a gradient manner, and dehydrated for 20min at each concentration. The dehydrated bacterial cells were infiltrated according to the following steps: acetone: 812 embedding agent = 1:1 for 2-4h, acetone: 812 embedding agent = 1:2 for overnight penetration, and 812 embedding agent for 5-8h. Put it in an oven at 37°C overnight to complete the embedding of the bacteria. After the wax block was trimmed, the sample was cut into 70 nm ultrathin sections using a Leica UC7 ultrathin microtome and fixed on a copper grid. The sections were then stained with a 2% uranyl acetate saturated alcohol solution for 8 min and a 2.6% lead citrate solution in the absence of carbon dioxide for 8 min. After drying, the sections were observed and photographed using an H-7650 transmission microscope.

[0097] To further verify the bactericidal action mode of bacteriocins against Clostridium perfringens ATCC13124, SEM was used to observe the changes in the bacterial surface morphology and TEM was used to observe the changes in the internal microstructure of the bacteria. Figure 5 SEM results show that the untreated control group of Clostridium perfringens has a complete cell structure and a smooth and plump surface. The bacterial surfaces treated with different postbiotics showed varying degrees of roughness, wrinkles, and shortened lengths, and debris appeared around the bacteria. Figure 6 TEM results show that the cytoplasm of untreated Clostridium perfringens cells is densely and evenly distributed, full of ribosomes and fibrous nucleoids, and the cell wall is tightly attached to the cell membrane. When the cells were exposed to Bifidobacterium longum subsp. infantis CCFM1192 postbiotics and CCFM1210 postbiotics, the cell contours were severely deformed, the cell structure was destroyed, the intracellular substances were unevenly distributed, gaps appeared between the cell membrane and the cell wall, and plasmolysis occurred. The cells were obviously ruptured and the cell contents leaked. These results confirm that Bifidobacterium longum subsp. infantis CCFM1192 postbiotics and CCFM1210 postbiotics cause the release of intracellular substances by destroying the integrity of the cell wall and cell membrane, leading to the death of Clostridium perfringens ATCC13124.

[0098] Example 4: Bifidobacterium longum subsp. infantis postbiotics effectively reduce the load of Clostridium perfringens in the feces of mice infected with Clostridium perfringens

[0099] The specific steps are as follows:

[0100] This experiment was approved by the Experimental Animal Care and Use Committee of Jiangsu Institute of Parasitic Diseases (IACUC-JlPD-20240157). Forty male C57BL / 6 mice were randomly divided into five groups, namely the control group, model group, CCFM1192 group, CCFM1210 group and positive drug vancomycin group, with 8 mice in each group. The first week was the adaptation period, the second week was the modeling period, and the third week was the intervention period. The specific process of animal experiments can be found in Figure 7 shown.

[0101] After the adaptation period, mixed antibiotics (0.4 mg / mL kanamycin, 0.035 mg / mL gentamicin, 850 U / mL colistin, 0.215 mg / mL metronidazole, 0.045 mg / mL vancomycin) were added to the sterile drinking water of mice in the model group, CCFM1192 group, CCFM1210 group, and vancomycin group, and they were allowed to drink freely for 3 days. Then they were replaced with normal sterile drinking water, and 50 mg / kg of clindamycin was orally administered 2 days later. This stage will disrupt the mouse microbiota, which is closer to the clinical situation of intestinal dysfunction. The next day, 1×10 9 CFU / mL Clostridium perfringens ATCC13124100 μL, and all mice were closely monitored for signs of disease symptoms.

[0102] During the intervention period, CCFM1192 group and CCFM1210 group were intragastrically administered with 100 μL and rectal administration with 50 μL of the corresponding 40× postbiotics, and the vancomycin group was replaced with an equal volume of 5 mg / mL vancomycin; the model group and the control group were replaced with sterile saline, and the intervention was continued for 5 days.

[0103] Feces of mice were collected on days 14-19 during the intervention period, homogenized and serially diluted. The mixture was spread on a fresh TSC agar plate containing 0.4% D-cycloserine, and another layer of TSC agar was spread on the plate surface after it dried to prevent H2S dispersion. After anaerobic culture for 18 hours, the black colonies on the plate were counted.

[0104] like Figure 8 As shown in Figure 2, infection caused a continuous increase in the load of Clostridium perfringens in the feces of mice until day 16, reaching 7.52×10 9 The peak of CFU / g was 2.34, and then it began to decline. The fecal load of Clostridium perfringens in mice treated with CCFM1192 postbiotics and CCFM1210 postbiotics began to decline after the 15th day. On the 16th day, the fecal load of Clostridium perfringens in the CCFM1192 group and the CCFM1210 group was 6.68×10 8 CFU / g and 7.23×10 8 CFU / g. On the 17th day, the fecal loads of Clostridium perfringens in the CCFM1192 group and the CCFM1210 group were 1.56×10 7 CFU / g and 7.47×10 6 CFU / g, which was lower than that of the model group on day 19 (1.71×10 7CFU / g). The load of Clostridium perfringens in the vancomycin group decreased rapidly in the early stage; however, after the fourth day, its inhibitory effect entered a plateau phase, and the load of Clostridium perfringens in feces on the 17th, 18th, and 19th days was 6.59×10 6 CFU / g, 1.83×10 7 CFU / g and 1.18×10 7 CFU / g. On the 19th day, the fecal loads of Clostridium perfringens in the CCFM1192 group and the CCFM1210 group were 1.61×10 6 CFU / g and 1.83×10 6 The above results show that Bifidobacterium longum subsp. infantis postbiotics can effectively reduce the load of Clostridium perfringens in the intestine of mice, and the long-term intervention effect is better than vancomycin.

[0105] Example 5: Effect of Bifidobacterium longum subsp. infantis postbiotics on the reduction of thymic index in mice infected with Clostridium perfringens

[0106] Specific implementation method Refer to Example 4. On the 19th day, the mice were killed, the complete thymus of the mice was collected, and after the excess blood was drawn, it was weighed with a micro electronic scale to calculate the thymus index of the mice.

[0107] Thymus index (%) = thymus mass (g) / mouse body weight (g) × 100%.

[0108] like Fig. 9 As shown in the figure, the thymus index of the control group was 0.24±0.01%, while that of the model group was 0.18±0.01%, which was significantly lower than that of the control group (P<0.05). The thymus index recovered to 0.21±0.01% under the intervention of CCFM1192 postbiotics, and recovered to 0.20±0.01% under the intervention of CCFM1210 postbiotics. Therefore, CCFM1192 postbiotics and CCFM1210 postbiotics can increase the thymus index of mice and alleviate infection symptoms.

[0109] Example 6: Effect of Bifidobacterium longum subsp. infantis postbiotics on the increased spleen index in mice infected with Clostridium perfringens

[0110] Specific implementation method Refer to Example 4. After the mice were killed on the 19th day, the complete spleen of the mice was collected, and after the excess blood was absorbed, it was weighed with a micro electronic scale to calculate the spleen index of the mice.

[0111] Spleen index (%) = spleen mass (g) / mouse body weight (g) × 100%.

[0112] like Fig.10 As shown, the spleen index of the control group was 0.26±0.01%, while the spleen index of the model group was 0.30±0.01%, which was significantly higher than that of the control group (P<0.05). The spleen index was significantly reduced to 0.26±0.01% (P<0.05) under the intervention of CCFM1192 postbiotics, which was 13.62% lower than that of the model group. The spleen index recovered to 0.28±0.02% under the intervention of CCFM1210 postbiotics, which was 7.01% lower than that of the model group. Therefore, CCFM1192 postbiotics and CCFM1210 postbiotics can reduce the spleen index of mice and relieve infection symptoms.

[0113] Example 7: Effect of Bifidobacterium longum subsp. infantis postbiotics on the alleviation of spleen tissue lesions in mice infected with Clostridium perfringens

[0114] Specific implementation method Refer to Example 4. The weighed mouse spleen was immersed in 4% paraformaldehyde overnight for making hematoxylin & eosin (H&E) tissue sections. The sections were scanned using a Pannoramic MIDI digital section scanner and photographed.

[0115] like Fig.11 As shown in the figure, the red and white pulp structures of the spleen in the control group were intact and clearly visible. The spleen structure in the model group was severely abnormal, with severe disorder in arrangement, unclear boundaries between the red and white pulps, severely irregular shapes of splenic bodies in the white pulp, reduced volume of white pulp, significantly reduced number, or even absent; fewer lymphocytes and scattered multinucleated giant cells in the red pulp; severe extramedullary hematopoiesis in the red pulp. The spleen under the intervention of CCFM1192 postbiotics, CCFM1210 postbiotics, and vancomycin basically restored its normal morphological structure.

[0116] Example 8: Effect of Bifidobacterium longum subsp. infantis postbiotics on the severity of lesions in the jejunum, ileum and colon of mice infected with Clostridium perfringens

[0117] Specific implementation method Refer to Example 4. After the mice were killed on the 19th day, the jejunum, ileum and colon of the mice were collected and immersed in 4% paraformaldehyde overnight for making hematoxylin & eosin (H&E) tissue sections. The sections were scanned using a Pannoramic MIDI digital slice scanner and photographed.

[0118] like Fig.12 As shown in the figure, the villi of the jejunum in the control group were long finger-shaped, and the free edge of the absorptive cells showed clear striated edges. In the model group, the glandular structure of the jejunum was moderately disordered, the glands in the surface mucosa layer were moderately eroded, missing, and exuded in large quantities, the goblet cells and the proper glands were significantly reduced, the villus structure was disordered, fused, and fell off; the submucosal layer was moderately edematous and sparse; the blood vessels in the interstitium were slightly dilated, congested, and hemorrhagic; and there were more lymphocytes infiltrating in the lamina propria. The above symptoms of the jejunum in the CCFM1192 group, CCFM1210 group, and vancomycin group were greatly improved.

[0119] The villi in the ileum of the control group were short and conical, with more goblet cells and abundant lymphoid tissue in the lamina propria. In the model group, the glandular structure of the ileum was slightly disordered, with focal erosion and loss of glands in the surface mucosal layer, fewer goblet cells and glands in the lamina propria, disordered arrangement and fusion of the villi, moderate edema and looseness of the submucosal layer, moderate dilation, congestion and bleeding of blood vessels in the interstitium, and more lymphocyte infiltration in the lamina propria. The above symptoms of the ileum in the CCFM1192 group, CCFM1210 group and vancomycin group were greatly improved.

[0120] The control group had normal colonic tissue structure, flat mucosal surface, and no villi; the colon glands were dense and there were many goblet cells. In the model group, the colon gland structure was moderately disordered, the surface mucosal glands were moderately eroded and missing, a small amount of mucus secretion, goblet cells were reduced, the villus structure was disordered and fused; the thickness of the submucosal layer and the muscular layer was focally thinned, highly edematous and loose; the blood vessels in the interstitium were highly dilated, congested, and hemorrhagic; a large number of lymphocytes infiltrated and lymph follicles aggregated in the lamina propria. The above symptoms of the colon in the CCFM1192 group, CCFM1210 group, and vancomycin group were greatly improved.

[0121] Example 9: Effect of Bifidobacterium longum subsp. infantis postbiotics on the restoration of intestinal flora structure in mice infected with Clostridium perfringens

[0122] Specific implementation method Refer to Example 4. Collect mouse feces on the 19th day, extract total genomic DNA from the feces, amplify, purify and sequence the V3-V4 region of 16S rDNA, and use QIIME2 to analyze the collected data. The analysis results of the α diversity and β diversity of the microbiota are shown in Figures 13-16 .

[0123] Antibiotic modeling can cause disturbances in the intestinal flora of mice, so the effect of Bifidobacterium longum subsp. infantis postbiotic intervention on the intestinal flora structure of mice infected with Clostridium perfringens was further evaluated. Figure 13-15 As shown, the Chao1 index, Shannon index and Simpson index of the control group were 334.69±19.61, 6.17±0.19 and 0.96±0.01, respectively. Compared with the control group, the three α diversity indices of the model group, including Chao1 index, Shannon index and Simpson index, were significantly reduced (P<0.05), which were 41.88±2.24, 3.05±0.11 and 0.80±0.02, respectively. This indicates that antibiotic treatment and Clostridium perfringens infection reduce the species richness and uniformity of the intestinal microbiota of mice. However, the treatment of postbiotics CCFM1192 and CCFM1210 significantly increased the three α diversity indices of the intestinal microbiota of mice (P<0.05). The chao1 index, Shannon index and Simpson index of the CCFM1192 group were 135.88±14.87, 3.77±0.16 and 0.87±0.01, respectively, and the chao1 index, Shannon index and Simpson index of the CCFM1210 group were 115.88±10.99, 3.69±0.15 and 0.86±0.01, respectively, making the species composition of the intestinal flora richer and more uniform. There was no significant difference between the vancomycin group and the model group (P>0.05). The chao1 index, Shannon index and Simpson index of the vancomycin group were 41.38±2.20, 2.82±0.13 and 0.76±0.03, respectively.

[0124] like Fig.16 As shown in the figure, principal coordinate analysis (PCoA) based on unweighted Unifrac distance showed that the intestinal microbiota belonging to the same group tended to cluster together. The intestinal flora of mice in the vancomycin group was very similar to that of the modeling group, but after the intervention of postbiotics CCFM1192 and postbiotics CCFM1210, the intestinal flora of mice moved towards the control group.

[0125] The above results indicate that Bifidobacterium longum subsp. infantis postbiotics can effectively increase the diversity of intestinal flora in mice infected with Clostridium perfringens and improve intestinal flora disorders caused by antibiotic treatment and Clostridium perfringens infection.

[0126] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of Bifidobacterium longum subsp. infantis CCFM1192 or Bifidobacterium longum subsp. infantis CCFM1210 in the preparation of a medicament for inhibiting Clostridium perfringens, characterized in that: The deposit number of the Bifidobacterium longum subsp. infantis CCFM1192 is GDMCC No: 61846; the deposit number of the Bifidobacterium longum subsp. infantis CCFM1210 is GDMCC No: 62234.

2. The use according to claim 1, characterized in that: The medicine contains postbiotics prepared from the Bifidobacterium longum subspecies infantis.

3. The use according to claim 2, characterized in that: The preparation method of the postbiotics is (a) or (b): (a) culturing the Bifidobacterium longum subspecies infantis, collecting the fermentation supernatant to obtain postbiotics; (b) After culturing the Bifidobacterium longum subspecies infantis, collecting the fermentation supernatant, and extracting protein in the supernatant to obtain postbiotics.

4. The use according to claim 3, characterized in that: The drug has at least one of the following effects: (1) Reduce the load of Clostridium perfringens in feces; (2) Increase thymic index; (3) Reduce spleen index; (4) Alleviate spleen tissue lesions; (5) Relieve lesions in the jejunum, ileum, and colon; (6) Improve the diversity of intestinal flora and improve intestinal flora disorders.

5. The use according to claim 4, characterized in that: The dosage form of the medicine includes powder, granule, capsule, tablet, pill or oral solution.

6. The use according to claim 5, characterized in that The medicine also contains a drug carrier and / or a pharmaceutical excipient.

7. The use according to claim 6, characterized in that The drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes; the pharmaceutical excipients comprise excipients and / or additives.

8. Use of Bifidobacterium longum subspecies infantis CCFM1192 or Bifidobacterium longum subspecies infantis CCFM1210 in the preparation of Clostridium perfringens inhibitors, characterized in that: The deposit number of the Bifidobacterium longum subsp. infantis CCFM1192 is GDMCC No: 61846; the deposit number of the Bifidobacterium longum subsp. infantis CCFM1210 is GDMCC No: 62234.

9. The use according to claim 8, characterized in that The inhibitor contains postbiotics prepared from the Bifidobacterium longum subspecies infantis.

10. A method for inhibiting the growth of Clostridium perfringens, characterized in that: Postbiotics of Bifidobacterium longum subspecies infantis CCFM1192 or Bifidobacterium longum subspecies infantis CCFM1210 are added to the culture environment of Clostridium perfringens, wherein the preservation number of Bifidobacterium longum subspecies infantis CCFM1192 is GDMCC No: 61846; the preservation number of Bifidobacterium longum subspecies infantis CCFM1210 is GDMCC No: 62234.

Images