Application and method of aspergillus cristatus in preparation of medicine for treating and / or preventing bacterial infection

Abstract

The invention relates to the field of microorganisms, and discloses application and a method of aspergillus cristatus in preparation of a medicine for treating and / or preventing bacterial infection, and the preservation number of the aspergillus cristatus is CGMCC No.41727. The aspergillus cristatus provided by the invention has an inhibition effect on streptococcus mutans, escherichia coli, staphylococcus aureus and salmonella typhimurium, can secrete various active enzymes, and can relieve the symptoms of bacterial infection, including inhibition of expression of proinflammatory factors, promotion of expression of anti-inflammatory factors and reduction of the number of neutrophils; the aspergillus cristatus provided by the invention can also relieve the symptoms of colitis. The aspergillus cristatus provided by the invention has an application prospect in preparation of drugs for treating and / or preventing bacterial infection.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more specifically to the use and methods of Aspergillus cristatus in the preparation of medicaments for the treatment and / or prevention of bacterial infections. Background Technology

[0002] Bacterial infections pose a significant challenge to human health and safety. However, the problem of bacterial resistance is becoming increasingly serious, with the emergence of multidrug-resistant bacteria, pan-drug-resistant bacteria, and even pan-drug-resistant bacteria frequently rendering traditional antibiotic treatments ineffective.

[0003] Antibiotic development faces challenges such as long development cycles, high costs, and low success rates, and often lags behind the evolution of bacterial resistance. Against this backdrop, extracting substances with antibacterial activity from natural biological resources has become an important approach to finding new anti-infective drugs. Fungi, as a diverse group of organisms in nature rich in metabolic products, have played a landmark role in the history of antibiotic development with their secondary metabolites (such as penicillin and cephalosporins), providing key weapons for humanity to combat bacterial infections.

[0004] Aspergillus cristatus ( Aspergillus cristatus As a filamentous fungus widely distributed in nature, *Aspergillus cristatus* has gradually come into the research field in recent years. It plays an important role in the production of traditional fermented foods (such as Fu brick tea), and studies have found that its metabolites possess rich biological activities, including antioxidant, antitumor, and immunomodulatory effects. More importantly, increasing evidence suggests that *Aspergillus cristatus* can produce a variety of compounds with antibacterial or bactericidal activities. These substances may inhibit bacterial growth through unique mechanisms of action and even show significant activity against drug-resistant bacteria. However, the number of *Aspergillus cristatus* strains currently proven to have antibacterial properties is insufficient to meet the antibacterial demand.

[0005] Therefore, there is an urgent need to provide Aspergillus cristatus that can inhibit bacterial growth and to use it in the preparation of drugs for the treatment or prevention of bacterial infections. Summary of the Invention

[0006] The purpose of this invention is to overcome the aforementioned problems in the prior art and to provide the application and method of Aspergillus cristatus in the preparation of medicaments for the treatment and / or prevention of bacterial infections.

[0007] The first aspect of the present invention provides Aspergillus cristatus ( Aspergillus cristatus The use of Aspergillus cristatus in the preparation of medicaments for the treatment and / or prevention of bacterial infections, wherein the accession number of Aspergillus cristatus is CGMCC No. 41727.

[0008] A second aspect of the present invention provides a method for inhibiting bacteria, the method comprising: contacting Aspergillus cristatus with bacteria, the specimen having accession number CGMCC No. 41727.

[0009] Through the above technical solution, the Aspergillus cristatus provided by the present invention can inhibit a variety of bacteria, alleviate the increase of neutrophils caused by inflammatory infection, downregulate TNF-α and IL-6, upregulate IL-10, and relieve colitis symptoms, and has the prospect of preparing drugs for the treatment and / or prevention of bacterial infections.

[0010] Biological Preservation The strain provided by this invention is classified and named *Aspergillus cristatus*. Aspergillus cristatus It was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41727 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0011] Figure 1 These are the fluorescence microscopy results of the zebrafish bacterial inflammation model in Example 2; Figure 2 This is a graph showing the colon length of mice in Example 3; Figure 3 This is a diagram showing the colonic pathological staining results of mice in Example 3. Detailed Implementation

[0012] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0013] The inventors of this invention unexpectedly obtained a strain of Aspergillus cristatus that can colonize the intestine and can be used to prepare fermented dairy products. Aspergillus cristatus The strain, named CCNH008, can enter the intestines and colonize through fermented dairy products, thereby exerting a probiotic effect on the organism. Due to these characteristics, CCNH008 is more suitable for use in drugs for the prevention and / or treatment of intestinal-related bacterial infections than conventional Aspergillus cristatus.

[0014] The first aspect of the present invention provides Aspergillus cristatus ( Aspergillus cristatus The use of Aspergillus cristatus in the preparation of medicaments for the treatment and / or prevention of bacterial infections, wherein the accession number of Aspergillus cristatus is CGMCC No. 41727.

[0015] Preferably, the bacteria include streptococci ( Streptococcus Escherichia coli ( ), Escherichia coli Escherichia ),staphylococcus( Staphylococcus) and Salmonella ( Salmonella At least one of the following.

[0016] More preferably, the bacteria include Streptococcus mutans (Streptococcus mutans) Streptococcus mutans ), Escherichia coli ( Escherichia coli Staphylococcus aureus ( Staphylococcus aureus subsp. golden ) and Salmonella typhimurium ( Salmonella enterica subsp. enterica serovar Typhimurium At least one of the following.

[0017] A second aspect of the present invention provides a method for inhibiting bacteria, the method comprising: contacting Aspergillus cristatus with bacteria, the specimen having accession number CGMCC No. 41727.

[0018] The present invention will be described in detail below through embodiments.

[0019] Streptococcus mutans ( Streptococcus mutans (CGMCC 1.2499), purchased from China General Microbiological Culture Collection Center; Staphylococcus aureus ( Staphylococcus aureus subsp. golden The item, numbered CMCC(B)26001, was purchased from the China Medical Bacteriological Culture Collection Center. Salmonella typhimurium ( Salmonella enterica subsp. enterica serovar Typhimurium (ATCC14028), purchased from the American Center for Type Culture Collection; Escherichia coli (Escherichia coli) Escherichia coli (CICC 10421), purchased from the China Industrial Microbial Culture Collection Center; Absorbance was measured using a visible light spectrophotometer.

[0020] PDA culture medium: purchased from Qingdao Haibo Biotechnology, catalog number HB0233-5.

[0021] Example 1 The inhibitory effects of Aspergillus cristatus fermentation broth on Streptococcus mutans, Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli were evaluated using the MIC method.

[0022] Streptococcus mutans, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium were inoculated into LB liquid medium, cultured at 37°C for 18 h (Streptococcus mutans was cultured anaerobically, the others aerobically), and then transferred to their respective fresh media and cultured until the viable count reached 10^6. 8 CFU / mL, centrifuged to separate bacterial cells, and resuspended in LB liquid medium to 10⁻⁶. 5 CFU / mL was used as the indicator bacterial solution.

[0023] Aspergillus cristatus was activated to the second generation on PDA medium, then transferred to malt extract sporulation medium and cultured for 10-15 days. The plates were washed with physiological saline and the mycelium was filtered off to prepare 10... 8 CFU / ml spore suspension (suspension of the test strain).

[0024] Mix the bacterial suspension of the test strain with the indicator bacterial suspension according to the volumes shown in Table 1 below; seal the ELISA plate, cap it, and incubate it at 37℃ and 200rpm for 18h, then measure the absorbance at 600nm. Replace the bacterial suspension of the test strain with an equal volume of culture medium as a negative control, and replace the bacterial suspension of the test strain with an equal volume of kanamycin (100ug / mL) as a positive control.

[0025] Table 1

[0026] Inhibition rate = (A0 - A) / A0 × 100%; Wherein, A0 is the OD of the negative control water sample after the experiment. 600 Value, A is the OD of the experimental group sample after the experiment. 600 The inhibition rate results are shown in Table 2.

[0027] Table 2

[0028] The results showed that Aspergillus cristatus CCNH008 had significantly higher inhibition rates against Streptococcus mutans, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium than the reference strain.

[0029] Example 2 3-dpf transgenic neutrophil green fluorescent zebrafish were randomly selected and placed in 6-well plates with a volume of 3 mL per well and 30 zebrafish per well.

[0030] Aspergillus cristatus CCNH008 group was given water-soluble samples of the corresponding strain (final concentration 2×10). 5 (CFU / mL) The positive control group was given indomethacin in water (final concentration of 100 μM, also known as the indomethacin group). Set up a normal control group and a model control group that did not receive the strain or indomethacin.

[0031] After incubating at 25℃ for 3 hours under the same conditions, except for the normal control group, all experimental groups had lipopolysaccharide (LPS) injected into the yolk sac of zebrafish (100 ng / fish) to establish a zebrafish bacterial inflammation model. After treatment at 28℃ for 2 hours, 10 zebrafish from each group were randomly selected and photographed under a fluorescence microscope (results are shown below). Figure 1As shown in Table 3, images were analyzed and data were collected using the advanced image processing software NIS-Elements D3.20 to analyze the number of neutrophils in the zebrafish yolk sac. The relative expression levels (folds) of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10 and IL-6) RNA were detected by real-time quantitative PCR. The test results for different groups are shown in Table 3.

[0032] Table 3

[0033] In Table 3, for different groups of data with the same parameters, those with the same letter indicate no significant difference between groups. P >0.05), different letters indicate significant differences between groups ( P <0.05).

[0034] Table 3 shows that Aspergillus cristatus CCNH008 can significantly reduce the number of neutrophils in zebrafish, downregulate the expression of pro-inflammatory factors TNF-α and IL-6, and upregulate the expression of anti-inflammatory factor IL-10 to alleviate bacterial inflammation.

[0035] Example 3 Thirty mice of strain C57BL / 6, aged 7-9 weeks, were used to establish a mouse model of colitis by inducing the disease in drinking water with 1.5% sodium dextran sulfate (DSS) for 7 days. The presence of weight loss, loose stools, diarrhea, bloody stools or fecal occult blood, and ulcers in the mice was considered a successful DSS model.

[0036] After successful modeling, the mice were randomly divided into three groups: 1. Normal group: Sterile water for drinking, and PBS buffer was administered by gavage once a day; 2. Model group: DSS solution was administered orally, and PBS buffer was administered by gavage once a day; 3. CCNH008 group: Administered DSS solution orally and once daily by gavage CCNH008 *Aspergillus cristatus* dissolved in PBS buffer, with a final concentration of 10... 7 CFU / kg BW; The experiment lasted four weeks, with timely replenishment of feed and drinking water to ensure the mice had sufficient food and water. The mice's weight, fecal condition, and any instances of blood in their stool were recorded. At the end of the experiment, blood, feces, and colon tissue and its contents were collected from the mice for subsequent testing.

[0037] (1) Mouse body weight, food intake, and water intake, once a day; at the beginning of the experiment, the initial body weight of the mice was weighed and recorded, as well as the initial weight of the feed and water. The weight of the mice, the weight of the remaining feed, and the weight of the remaining water were recorded daily. The body weight data are shown in Table 4 (in g). The food intake or water intake was calculated according to the following formula, and the results are shown in Table 5.

[0038] Food intake or water intake = (initial feed or water weight - remaining feed or water weight) / (day × number of mice).

[0039] Table 4

[0040] Table 5

[0041] (2) The Disease Activity Index (DAI) of mice was assessed based on body weight, fecal condition, and blood in stool. The percentage of body weight loss was scored as 0, 1-5%, 5-10%, 10-20%, and >20%, respectively. The fecal viscosity was scored as 0, 1, 2, and 3, respectively, for normal, soft, mucous, and loose stools. The fecal occult blood test was scored as 0, 1, 2, 3, and 4, respectively, for negative, light blue, blue, dark blue, and visible blood in stool. The results are shown in Table 6.

[0042] Table 6

[0043] As shown in Tables 4-6, compared with the normal control group, the model group had a lower body weight, indicating that the model was successfully established. Compared with the model group, gavage with CCNH008 did not affect the mice's food and water intake, but could inhibit the weight loss caused by enteritis, improve the enteritis condition, and reduce the DAI score.

[0044] (3) After 28 days of intervention, the mice were euthanized, the abdominal cavity was opened, and the entire colon was separated and cut off, neatly placed on filter paper, and the colon length was measured and recorded. A section of about 0.5-1.0 cm from the middle of the colon was carefully cut off, quickly rinsed with physiological saline, and fixed overnight in a general-purpose tissue fixative. After complete tissue fixation, routine dehydration, embedding, sectioning, and H&E staining were performed. The degree of colonic inflammation, crypt damage, depth of inflammation, area of ​​inflammatory infiltration, and depth of lesions were observed and scored under a microscope.

[0045] Colon length results as follows Figure 2 As shown (from left to right: normal group, model group, and CCNH008 group). The colon lengths of the normal group, model group, and CCNH008 group were 8.88±0.46, 8.00±0.75, and 8.21±0.48, respectively (significant differences were observed between the groups).

[0046] Colon pathological staining results as follows Figure 3As shown (from left to right: normal group, model group, and CCNH008 group; from top to bottom: results at 4x, 10x, and 40x magnification), the colon pathology scores for the normal group, model group, and CCNH008 group were 1.5±0.7, 6.2±1.6, and 3.2±0.7, respectively (significant differences between the groups).

[0047] (4) Inflammatory factor markers: GAPDH was selected as an internal control, and the relative expression levels of inflammatory factors TNF-α, IL-1β and IL-6 were detected using a kit. The results are shown in Table 7.

[0048] Table 7

[0049] The results in Table 7 show that CCNH008 can inhibit the increase of inflammatory factors caused by enteritis.

[0050] Example 4 CCNH008 and CICC2650 were activated to the second generation on PDA medium, then transferred to malt extract sporulation medium and cultured for 10-15 days. The plates were washed with physiological saline and the mycelium was filtered off to prepare 10... 8 CFU / ml spore suspension. The spore suspension was inoculated onto PDB medium and cultured for 4 days. The supernatant was collected to obtain the crude enzyme solution. The precipitated mycelium was dried and weighed to calculate the specific enzyme activity. Enzyme activity is defined as follows: Ferulic acid esterase activity: One enzyme activity unit is defined as the amount of p-nitrophenol produced per minute by hydrolyzing ferulic acid p-nitrophenyl ester per gram of bacteria at 40℃ and pH 6.0. Ferulic acid esterase catalyzes the decomposition of the substrate ferulic acid p-nitrophenyl ester to p-nitrophenol, which has a maximum absorption peak at 405 nm.

[0051] Polyphenol oxidase activity: One unit of enzyme activity is defined as a change of 0.01 in absorbance at 525 nm per minute per g of bacteria per mL of reaction system at 25℃ and pH 6.0.

[0052] Tanninase activity: One unit of enzyme activity is defined as the amount of enzyme required per gram of bacteria to hydrolyze and reduce 0.01 µmol of propyl gallate per minute under conditions of 40°C and pH 5.0. Using the antioxidant propyl gallate as the substrate for the tanninase enzymatic reaction, the optical density of the substrate before and after the reaction was measured at 270 nm.

[0053] Cellulase activity: The amount of enzyme required per gram of bacteria to catalyze the production of 1 μg of reducing sugar from sodium carboxymethyl cellulose per minute at 37℃ and pH 5.5 is defined as one unit of enzyme activity. The content of reducing sugar produced by cellulase-catalyzed degradation of sodium carboxymethyl cellulose was determined by the anthrone colorimetric method, and the absorbance at 620 nm was measured.

[0054] Glutaminase activity: One unit of enzyme activity is defined as the rate at which each gram of bacteria catalyzes the production of 1 nmol of ammonia from glutamine per minute under conditions of 37°C and pH 7.0. Glutaminase catalyzes the hydrolysis of glutamine into L-glutamic acid and ammonia. The rate of ammonia increase is detected using Nessler's reagent, and the absorbance at 420 nm is measured.

[0055] Aminopeptidase activity: The level of aminopeptidase in the sample was determined using a double-antibody sandwich method. Purified aminopeptidase antibody was coated onto a microplate to prepare a solid-phase antibody. Crude enzyme solution (fermentation supernatant of the bacterial strain) was added sequentially to the microplate coated with monoclonal antibody, followed by binding with HRP-labeled aminopeptidase antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB was converted to blue under the catalysis of HRP enzyme, and then to yellow under acidic conditions. The color intensity was positively correlated with the aminopeptidase level in the sample. Absorbance was measured at 450 nm.

[0056] A standard curve was established to measure the enzyme activity of the tested strains by comparing the concentration of the degradation products (or the concentration after degradation) of the above-mentioned enzyme activity-related substrates with absorbance values. Enzyme activity is measured in nmol / (min g mycelial dry weight).

[0057] Table 8

[0058] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. Aspergillus cristatus ( Aspergillus cristatus Its use in the preparation of medicaments for treating and / or preventing bacterial infections, characterized in that, The preservation number of the *Aspergillus cristatus* is CGMCC No. 41727.

2. The application according to claim 1, wherein, The bacteria include at least one of streptococci, Escherichia coli, staphylococci, and Salmonella.

3. The application according to claim 2, wherein, The bacteria include at least one of Streptococcus mutans, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium.

4. A method for inhibiting bacteria, characterized in that, The method involves contacting Aspergillus cristatus with bacteria, which has the accession number CGMCC No.41727.

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