Bacillus velezensis 20-1 and application thereof

Abstract

The invention relates to the field of microorganisms, and provides bacillus velezensis 20-1 and application thereof, the bacillus velezensis 20-1 is preserved in the China Center for Type Culture Collection on July 26, 2024, and the preservation number is CCTCC NO: M 20241694. The bacillus velezensis 20-1 can effectively inhibit the growth of common aquatic pathogenic fungi, has high thermal stability and protease tolerance, and can be used for preventing, relieving and / or treating diseases caused by aquatic pathogenic fungi infection.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a strain of Bacillus Velez 20-1 and an application thereof. Background Art

[0002] Aquaculture is an important aquaculture industry in my country, and the huge aquaculture output is facing serious threats of diseases. Saprolegnia is a highly pathogenic disease in aquaculture. Saprolegnia belongs to the Saprolegniales and Sprolegniaceae, mainly including the genera Saprolegnia and Achlya. Saprolegnia, also known as skin mold disease and white hair disease, is a common pathogenic bacteria in freshwater farmed animals. It is very popular both in time and region: Saprolegnia occurs in a wide temperature range, can occur all year round, and can occur in most aquaculture areas across the country. Saprolegnia spores exist in large quantities in the natural environment and have strong tolerance to the environment. At the same time, Saprolegnia has no strict selectivity for hosts, and injured hosts and eggs in the hatching process can be infected. As a common feature of fungal diseases, once parasitized by Saprolegnia, it is difficult to cure, so Saprolegnia has always been a major problem faced by aquaculture, causing huge economic losses to the aquaculture industry every year. Some species of the genus Achlysoma are aquatic pathogens of farmed fish and shrimp, common ones include Achlya americana and Achlya bisexualis.

[0003] The Megaloptera insects are holometabolous insects. The Protohermes xanthodes belongs to the Corydalidae family of the Megaloptera order. It is a rare edible and medicinal insect. Its commercial and scientific value has attracted the attention of entrepreneurs and researchers. In recent years, the number of Megaloptera insects in the wild has decreased sharply. The Protohermes xanthodes is a widely distributed species in China and a dominant species in artificial breeding. During its artificial breeding process, especially in the last stage, it is seriously infected by aquatic pathogenic fungi, which has a great impact on the development of the industry. At present, most aquatic pathogenic fungal diseases in the aquaculture industry are treated with antibiotics. It has been observed that the Protohermes xanthodes almost stop eating after being infected with bacteria, so it is difficult to achieve a preventive and therapeutic effect by feeding antibiotics, and the use of antibiotics has faced limited success and potential risks. Therefore, the development of alternatives to antibiotics in aquaculture is of great significance to effectively solve the problem of antibiotic resistance, and the use of intestinal probiotics to help the host prevent and control diseases is an effective means in the future. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0005] To this end, in a first aspect of the present invention, the present invention provides a Bacillus velezensis 20-1. According to an embodiment of the present invention, the Bacillus velezensis 20-1 was deposited in the China Center for Type Culture Collection on July 26, 2024, with a deposit number of CCTCC NO: M 20241694.

[0006] The Bacillus Velez 20-1 screened by the present invention can inhibit common aquatic pathogenic fungi and can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogenic fungi infection.

[0007] In a second aspect of the present invention, the present invention provides a fermentation broth. According to an embodiment of the present invention, the fermentation broth is fermented by the Bacillus Velez 20-1 described in the first aspect. The fermentation broth of the present invention can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogenic fungi infection.

[0008] In a third aspect of the present invention, the present invention provides a bacterial suspension. According to an embodiment of the present invention, the bacterial suspension comprises the Bacillus Velez 20-1 described in the first aspect. The bacterial suspension of the present invention can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogenic fungi infection.

[0009] In the fourth aspect of the present invention, the present invention proposes the use of the Bacillus Velez 20-1 described in the first aspect, the fermentation liquid described in the second aspect, or the bacterial suspension described in the third aspect in the preparation of food, beverage, health product, feed or additive that inhibits the activity of aquatic pathogenic fungi. The food, beverage, health product, feed or additive of the present invention can be used to prevent or inhibit diseases caused by aquatic pathogenic fungi infection.

[0010] In the fifth aspect of the present invention, the present invention provides the use of the Bacillus Velez 20-1 described in the first aspect, the fermentation liquid described in the second aspect, or the bacterial suspension described in the third aspect in the preparation of a drug for inhibiting the activity of aquatic pathogenic fungi. The drug of the present invention can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogenic fungi infection.

[0011] In a sixth aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises at least one of the Bacillus Velez 20-1 described in the first aspect, the fermentation broth described in the second aspect, and the bacterial suspension described in the third aspect. The composition of the present invention can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogenic fungal infections.

[0012] In a seventh aspect of the present invention, the present invention provides a method for inhibiting the growth of aquatic pathogenic fungi in vitro. According to an embodiment of the present invention, the method comprises: co-culturing at least one of the Bacillus Velez 20-1 described in the first aspect, the fermentation broth described in the second aspect, and the bacterial suspension described in the third aspect with aquatic pathogenic fungi. As mentioned above, the Bacillus Velez 20-1 of the present invention can effectively inhibit the growth of aquatic pathogenic fungi.

[0013] In the eighth aspect of the present invention, the present invention provides a culture device or production device for the Bacillus Velez 20-1 described in the first aspect, the fermentation liquid described in the second aspect, the bacterial suspension described in the third aspect, or the composition described in the sixth aspect. The culture device or production device according to the embodiment of the present invention can mass-produce the Bacillus Velez 20-1, fermentation liquid, bacterial suspension, or composition described in the present invention.

[0014] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 3. Figures 3 and 4 are morphological diagrams of healthy and diseased Acarina chinensis according to an embodiment of the present invention, wherein A shows the morphology of a healthy Acarina chinensis, B shows a diseased Acarina chinensis, C shows an enlarged view of the head of a diseased Acarina chinensis, D shows an enlarged view of the middle part of a diseased Acarina chinensis, and E shows an enlarged view of the tail of a diseased Acarina chinensis;

[0017] Figure 2 This is a colony morphology diagram of Bacillus velezensis 20-1 after being cultured on LB medium for 12 hours according to an embodiment of the present invention;

[0018] Figure 3 The figures are the result diagrams of the antagonism experiment of Bacillus velezensis 20-1 against Aspergillus hermaphroditus according to the embodiments of the present invention, wherein A shows the result diagram of the normally growing Aspergillus hermaphroditus, and B shows the result diagram of the detection of Bacillus velezensis 20-1 inhibiting the growth of Aspergillus hermaphroditus;

[0019] Figure 4 is a phylogenetic tree of Bacillus velezensis 20-1 according to an embodiment of the present invention;

[0020] Figure 5 The figures are test results of the effect of the fermentation liquid of Bacillus velezensis 20-1 on the germination of the spores of Amphoteric Aspergillus according to the embodiments of the present invention, wherein A shows the result of treating the spores of Amphoteric Aspergillus with the fermentation liquid of Bacillus velezensis 20-1 (1) and sterile water (2) for 12 hours respectively, B shows the morphology of the spores of Amphoteric Aspergillus under a 200x microscope after being treated with the fermentation liquid of Bacillus velezensis 20-1 for 12 hours; C shows the morphology of the spores of Amphoteric Aspergillus under a 200x microscope after being treated with sterile water for 12 hours;

[0021] Figure 6 Figures 1 and 2 are test results of the effect of the fermentation liquid of Bacillus velezensis 20-1 on the morphology of the hyphae of Amphoteric Aspergillus according to an embodiment of the present invention, wherein AC shows magnified images of different parts of the hyphae of Amphoteric Aspergillus growing normally under a 100-fold microscope; DF shows a method diagram of the hyphae of Amphoteric Aspergillus treated with the fermentation liquid of Bacillus velezensis 20-1 under a 100-fold microscope;

[0022] Figure 7 The results of the antibacterial effect of the fermentation liquid of Bacillus velezensis 20-1 on the amphoteric Aspergillus mold cake according to the embodiment of the present invention are shown in Figure A, wherein A shows the morphology of the normally grown amphoteric Aspergillus mold cake, and B shows the morphology of the amphoteric Aspergillus mold cake after being treated with the fermentation liquid of Bacillus velezensis 20-1;

[0023] Figure 8 The figure is a test result of the effect of temperature on the stability of Bacillus velezensis 20-1 fermentation broth against hermaphroditic fungi according to an embodiment of the present invention, wherein the data are expressed as the mean ± standard deviation of the inhibition rate, and different letters indicate significant differences at the P < 0.05 level tested by Duncan's new multiple range method;

[0024] Fig. 9 The figure is a result diagram of the effect of the protease according to the example of the present invention on the stability of the fermentation broth of Bacillus velezensis 20-1 against the amphoteric Aspergillus velezensis, wherein the data are expressed as the mean ± standard deviation of the diameter of the inhibition zone, and different letters indicate significant differences at the P < 0.05 level tested by Duncan's new multiple range method;

[0025] Fig.10The figure is a diagram of the antagonistic effect of the fermentation broth of Bacillus velezensis 20-1 on common aquatic pathogenic fungi according to an embodiment of the present invention, wherein the data are expressed as the mean ± standard deviation of the diameter of the inhibition zone, and the T test (Multiple ttests) is used to analyze the differences between the groups, and the "***" mark indicates that there is a very significant difference between the control group and the fermentation broth treatment group (P < 0.001, df = 4). DETAILED DESCRIPTION

[0026] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0027] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" and "multiple" is at least two or two, such as two, two, three, three, etc., unless otherwise clearly and specifically defined.

[0028] In this document, the terms “include”, “have” or “comprises” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0029] As used herein, the term "optionally" generally means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0030] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0031] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0032] As used herein, the terms "treatment" and "alleviation" both refer to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, especially humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a strain, fermentation broth, bacterial suspension or composition to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a strain, fermentation broth, bacterial suspension or composition described herein to an individual in need.

[0033] As used herein, the term "carrier" includes any solvent, drug stabilizer, or combination thereof, which are known to those skilled in the art. Except where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical compositions is contemplated.

[0034] Saprolegniasis is a common and extremely harmful fungal infectious disease in aquatic animals. As an aquatic animal disease caused by fungal parasites, Saprolegniasis has a wide range of temporal and geographical prevalence, and can occur in most aquaculture areas throughout the country throughout the year. At the same time, Saprolegnias have no strict selectivity for hosts. In recent years, the research on aquatic fungi has mainly focused on aquatic animals that suffer from Saprolegniasis, such as trout, carp, and silver carp. Saprolegniasis involves more than 40 species, including Saprolegnia, Corythuja, Microcystis, Cryptocystis, and Cystocystis. Among the more than ten species discovered in my country, Saprolegnia and Corythuja are the two genera that are most likely to cause the disease. Fish and amphibians are its main hosts. Under artificial breeding and reproduction conditions, especially in the stages of fish egg hatching and fish overwintering, Saprolegnia pathogens can cause large-scale deaths of fish, causing huge losses to the aquaculture industry. Saprolegnia is common in aquaculture waters, and its adaptability to temperature is very wide (5-26°C), and its optimal temperature for reproduction is 13-18°C. It has strong adaptability to the aquaculture water environment and strong resistance to adversity. It can form spores, male organs and egg-holding organs for sexual reproduction to resist adverse environments. Therefore, it is difficult to kill it with general drugs, which brings great difficulties to the prevention and treatment of Saprolegniasis. Chemical substances such as disinfectants and fungal drugs have broad-spectrum antibacterial properties and are widely used to treat fish diseases, but their residues lead to water quality deterioration, microbial resistance and the continuous increase in the amount of drugs used in aquaculture, which seriously threatens the quality and safety of aquatic products and public health safety.

[0035] strain

[0036] Therefore, in some embodiments, the present invention provides a Bacillus velezensis 20-1, which was deposited in the China Center for Type Culture Collection on July 26, 2024, with a deposit number of CCTCC NO: M 20241694.

[0037] According to some specific embodiments of the present invention, the above-mentioned Bacillus Velez 20-1 may further include at least one of the following additional technical features:

[0038] According to some specific embodiments of the present invention, the Bacillus Velezii 20-1 has a 16S rRNA sequence as shown in SEQ ID NO:1.

[0039]

[0040] The new strain of Bacillus Velez 20-1 of the present invention is screened from diseased Acanthoptera flavescens. Bacillus Velez 20-1 can inhibit common aquatic pathogenic fungi and can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogenic fungi infection, especially for aquatic animals, and can effectively treat saprolegniasis, with high application value.

[0041] As used herein, the terms "Bacillus velezensis 20-1", "Bacillus velezensis 20-1", "20-1 strain" and "20-1" are synonymous.

[0042] Fermentation Broth

[0043] In some embodiments, the present invention provides a fermentation broth, which is fermented by the aforementioned Bacillus Velez 20-1. The fermentation broth of the present invention can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogenic fungi infection.

[0044] It should be noted that the "fermentation broth" of the present invention refers to the solution obtained after culturing Bacillus Velez 20-1 for a period of time, which solution mainly contains Bacillus Velez 20-1 and its metabolites; or the supernatant after further treatment by centrifugation, filtration, etc., which supernatant mainly contains the metabolites of Bacillus Velez 20-1.

[0045] bacterial suspension

[0046] In some embodiments, the present invention provides a bacterial suspension, which includes the aforementioned Bacillus Velez 20-1. The bacterial suspension of the present invention can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogenic fungi infection.

[0047] It should be noted that the bacterial suspension can be obtained by treating the above fermentation broth by means of centrifugation, resuspension, etc.

[0048] use

[0049] In some embodiments, the present invention provides the use of the aforementioned Bacillus Velez 20-1, fermentation broth or bacterial suspension in the preparation of food, beverage, health product, feed or additive that inhibits the activity of aquatic pathogenic fungi. The food, beverage, health product, feed or additive of the present invention can be used to inhibit or prevent related diseases caused by aquatic pathogenic fungi, and assist in regulating metabolic diseases caused by aquatic pathogenic fungi.

[0050] In some embodiments, the present invention provides the use of the aforementioned Bacillus Velez 20-1, fermentation broth or bacterial suspension in the preparation of a drug for inhibiting the activity of aquatic pathogenic fungi. The drug of the present invention can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogenic fungi infection.

[0051] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0052] According to an embodiment of the present invention, the aquatic pathogenic fungi include at least one of Amphiporium hermosus, Amphiporium americanum, Mucor solani, Mucor circinelloides, Pythium sutchuenensis, Glehnia crassa, Aeromonas punctata, Aeromonas sobria, Aeromonas hydrophila, Pseudomonas fluorescens, Pseudomonas albicans, Pseudomonas aquaticus, Flexibacter columnaris, Edwardsiella, Nocardia asteroides and Streptococcus agalactiae.

[0053] According to an embodiment of the present invention, the aquatic pathogenic fungi include at least one of Aspergillus niger, Aspergillus americanus, Mucor solani, Mucor circinelloides, Pythium incognita and Corynebacterium compactum.

[0054] Food, beverage, medicine, health product, feed or additive

[0055] The present invention provides a food, beverage, medicine, health product, feed or additive, wherein the food, beverage, medicine, health product, feed or additive comprises at least one of the aforementioned Bacillus Velez 20-1, fermentation broth or bacterial suspension. The food, beverage, health product, feed or additive of the present invention can be used to inhibit or prevent related diseases caused by aquatic pathogenic fungi, assist in regulating metabolic disorders caused by aquatic pathogenic fungi, and the medicine of the present invention can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogenic fungi infection.

[0056] According to an embodiment of the present invention, the above-mentioned food, beverage, medicine, health product, feed or additive may further include at least one of the following additional technical features:

[0057] According to an embodiment of the present invention, it further comprises pharmaceutically acceptable excipients or carriers, excipients or carriers acceptable in food or health products, or excipients or carriers acceptable in animal feed.

[0058] As used herein, "acceptable in food" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the food requirements of different countries.

[0059] As used herein, "acceptable in health products" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the health product requirements of different countries.

[0060] In this article, "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammals treated therewith. Preferably, "pharmaceutically acceptable" of the present invention refers to those approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals or humans.

[0061] In this context, "adjuvant or carrier acceptable in animal feed" refers to a substance or composition that can be consumed by animals, which may be adjusted according to the animal feed requirements of different countries.

[0062] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, drug stabilizer, or combination thereof, which are known to those skilled in the art. Except where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical compositions is covered.

[0063] In this article, the term "pharmaceutically acceptable excipient" may include any solvent suitable for a particular target dosage form. Except for any conventional excipients that are incompatible with the Velezella 20-1 disclosed in the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated by the present disclosure.

[0064] According to an embodiment of the present invention, at least one of the aforementioned Bacillus Velez 20-1, fermentation broth and bacterial suspension is added or inoculated into food, beverages, health products, feed or additives, or added to medicines, thereby further obtaining food, beverages, health products, feed or additives having the activity of inhibiting aquatic pathogenic fungi, or obtaining medicines that can prevent, alleviate and / or treat aquatic pathogenic fungal infections or diseases caused by aquatic pathogenic fungal infections.

[0065] Exemplarily, the aforementioned foods include but are not limited to: probiotic tablets, fermented dairy products (such as probiotic yogurt), probiotic solid beverages, probiotic milk powder, probiotic cheese, probiotic soy products, probiotic candies, probiotic fermented vegetables, etc.

[0066] For example, the aforementioned medicines include but are not limited to: medicines for human use and medicines for veterinary use. The aforementioned medicines for veterinary use may be for pets, livestock, wild animals, and aquatic animals.

[0067] Exemplarily, the aforementioned health products include, but are not limited to: health products for humans, health products for animals (especially health products for aquatic animals).

[0068] It should be noted that the characteristics and advantages described above for Bacillus Velez 20-1 are also applicable to the food, beverage, medicine, health product, feed or additive, and will not be repeated here.

[0069] Composition

[0070] In some embodiments, the present invention provides a composition comprising at least one of the aforementioned Bacillus Velez 20-1, fermentation broth and bacterial suspension. The composition of the present invention can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogenic fungi infection.

[0071] According to an embodiment of the present invention, the above composition may further include at least one of the following additional technical features:

[0072] According to an embodiment of the present invention, the composition further comprises an ingestible excipient and / or carrier.

[0073] According to an embodiment of the present invention, the excipient includes at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent and a sustained-release agent.

[0074] According to an embodiment of the present invention, the carrier includes at least one selected from sugars, cellulose and its derivatives, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols and cereal hydrolyzed solids.

[0075] According to an embodiment of the present invention, the dosage form of the composition includes at least one selected from oral liquid, powder, granule, capsule, tablet, pill, aqueous emulsion, microemulsion, suspoemulsion and spray.

[0076] method

[0077] In some embodiments, the present invention provides a method for inhibiting the growth of aquatic pathogenic fungi in vitro, the method comprising: co-culturing the aquatic pathogenic fungi with at least one of the aforementioned Bacillus Velez 20-1, fermentation broth and bacterial suspension. As mentioned above, the Bacillus Velez 20-1 of the present invention can effectively inhibit the growth of aquatic pathogenic fungi.

[0078] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0079] According to an embodiment of the present invention, the aquatic pathogenic fungi include at least one of Amphiporium hermosus, Amphiporium americanum, Mucor solani, Mucor circinelloides, Pythium sutchuenensis, Glehnia crassa, Aeromonas punctata, Aeromonas sobria, Aeromonas hydrophila, Pseudomonas fluorescens, Pseudomonas albicans, Pseudomonas aquaticus, Flexibacter columnaris, Edwardsiella, Nocardia asteroides and Streptococcus agalactiae.

[0080] According to an embodiment of the present invention, the aquatic pathogenic fungi include at least one of Aspergillus niger, Aspergillus americanus, Mucor solani, Mucor circinelloides, Pythium incognita and Corynebacterium compactum.

[0081] Device

[0082] In some embodiments, the present invention provides a culture device or production device for the aforementioned Bacillus Velez 20-1, fermentation broth, bacterial suspension or composition. The culture device or production device according to the embodiment of the present invention can realize mass production of the Bacillus Velez 20-1, fermentation broth, bacterial suspension or composition described in the present invention.

[0083] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0084] Example 1: Isolation and screening of bacteria antagonistic to Amphiprion from the intestinal tract of Acanthus chinensis

[0085] 1.1 Isolation and screening of strains

[0086] 1.1.1 Isolation of Aspergillus hermaphroditicus: Isolate and culture aquatic pathogenic fungi on the diseased insect bodies, and identify the species of aquatic pathogenic fungi in combination with ITS. Rinse the diseased insect bodies three times in sterile pure water and place them in a sterilized culture dish (d=9 cm). Keep them moisturized and culture them in a constant temperature incubator at 25°C. After the conidia of Aspergillus hermaphroditicus grow noticeably on the surface of the insect bodies, use an inoculation loop to gently pick a small amount of spores or hyphae from the surface of the diseased insect bodies in a disinfected and sterilized clean bench and inoculate them on PDA culture medium. After culturing for 2 days, pick a small amount of spores or hyphae and purify them in a new culture medium until the morphological characteristics of the colonies grown on the plate are consistent. Place the purified strain in 25% glycerol and store it in a -80°C refrigerator. See the morphology of the diseased insect bodies for details. Figure 1 .

[0087] 1.1.2 Isolation and screening of insect-derived bacteria: Fifteen last-instar larvae of Acanthidae with good growth conditions were randomly selected and starved for 24 h, and randomly divided into 3 groups. They were washed three times with sterile water and disinfected with 75% alcohol for 200 s on a clean bench sterilized with ultraviolet light for 30 min. After rinsing with distilled water, the intestines of the last-instar larvae of Acanthidae were dissected under sterile conditions. Sterile scissors were used to cut open the abdomen along the dorsal midline, the entire digestive tract was taken out, the midgut tissue was cut, and the dissected midgut was placed in a 2 mL centrifuge tube containing 1 ml of sterile water. 1-2 sterile metal balls with a diameter of 5 mm were added, and the intestinal tissue was broken up on an oscillator at 20 Hz / min for 3 min to obtain an intestinal homogenate.

[0088] The intestinal contents extracted above were diluted in 5 gradients according to 10-1, 10-2, 10-3, 10-4, and 10-5, and the dilutions of the intestinal homogenate were spread on four culture media, namely MRS culture medium (lactic acid bacteria culture medium, M8330, purchased from Beijing Solebow Technology Co., Ltd.); EC culture medium (enterococcus culture medium, HB0133-3, purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.); NA culture medium (general bacterial culture medium, HB0109, purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.); TSA culture medium (Coxiella bacteria, LA0170, purchased from Beijing Solebow Technology Co., Ltd.); LB culture medium (general bacterial culture medium): 10g peptone, 5g yeast extract, 10g sodium chloride, 15g agar, dissolved in 1L sterile water, adjusted to pH 7.0; and then cultured at 37°C. The plates were observed every 12 hours to obtain the original bacterial strains. The isolates were classified according to differences in colony size, color and morphology. Colonies of different morphologies were purified on LB plates for more than five generations to obtain monoclonal strains. The purified strains were stored in a 50% glycerol solution in a -80°C refrigerator for future use.

[0089] The obtained monoclonal strain was grown in 500 μL liquid LB medium at 37°C for 2-3 hours. The 16S rRNA sequence was amplified using the bacterial culture as a template, and the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTAC GACTT-3') were used to clone the 16S rRNA of the bacteria. The PCR reaction system was 25 μL: ddH2O 8.5 μL, Mix 12.5 μL, DNA template 2 μL, 27F (10 mmol / L) and 1492R (10 mmol / L) each 1 μL. PCR conditions: 95°C 5 min, (94°C 30 s, 56°C 30 s, 72°C 1 min) × 30 cycles, 72°C 10 min. The amplified product was detected by 1.0% agarose gel electrophoresis, and the amplified product (about 1.5 kb) was sent to Shanghai Bioengineering Co., Ltd. for sequencing.

[0090] The plate confrontation method was used to screen the antagonistic effect of isolated strains on amphoteric Aspergillus. After the isolated monoclonal strain was activated, it was inoculated on the PDA plate in a parallel streak manner. The distance between the two bacterial lines was 5 cm. It was cultured at 30℃ for 1 day. After the colony grew out, an agar block with pathogenic fungi (8 mm in diameter) was inoculated in the middle of the bacterial line. It was cultured at 25℃ for 3 days. The shortest distance between the pathogenic fungal colony and the double bacterial lines was determined. The average of the shortest distances of three repetitions was taken as the final inhibition distance. The plates with antagonistic effects were cultured for 15 days to observe the stability and persistence of the antagonistic effect. Each group was set up with 3 repetitions. In the middle of the PDA plate of the control group, a pathogenic fungus agar block (8 mm in diameter) was inoculated. It was cultured at 25℃ for 3 days. The pathogenic fungi had covered the entire plate. The number of the strain with antagonistic effects and the final inhibition distance were recorded.

[0091] A total of 26 bacterial strains were isolated from the midgut of the last instar larvae of Acanthoptera yanhuangensis. After fermentation, 5 bacterial strains were found to have antagonistic effects on Amphotericin B. Among them, the antagonistic effect of the bacteria labeled 20-1 was the best, with an antagonistic distance of 4.25±0.20mm. The colony morphology of bacteria 20-1 after 12 hours of culture on LB medium is shown in Figure 2 The results of the preliminary screening experiment of antagonism of bacteria 20-1 to amphoteric fungi are shown in Figure 3 .

[0092] 1.2 Homologous comparison of isolated strains and construction of phylogenetic tree

[0093] The 16S rRNA sequence obtained by sequencing was blasted against the NCBI GenBank database, and the closely related sequences of each strain were selected and sequenced using clustalX1.83. The phylogenetic tree was constructed using MEGA6.0 software and the Neighbor-Joining algorithm, and the reliability of the phylogenetic tree was tested using bootstrap. Strain 20-1 was identified as Bacillus velezensis, with a total sequence length of 1452 bp.

[0094] The 16S sequencing results of the isolated 20-1 strain are as follows:

[0095]

[0096] The phylogenetic tree of strain 20-1 is shown in Figure 4 .

[0097] Example 2: Determination of Physiological and Biochemical Indexes of Bacillus velezensis 20-1

[0098] The Bacillus velezensis 20-1 bacteria isolated in Example 1 were identified for their carbohydrate metabolism ability and sensitivity to other compounds. The pure culture of Bacillus velezensis 20-1 was inoculated into BiologBUG+B medium and cultured at 37°C. The purified single colony was picked and added to the Inoculating Fluid inoculation solution. The bacterial concentration was adjusted to 90%-98% using a turbidity meter. The bacterial suspension was added to the BIOLOG GENIII microplate, and 100 μL was added to each well. The microplate was placed at 37°C and cultured for 48 hours. The metabolism of Bacillus velezensis 20-1 bacteria to different carbon sources and the sensitivity to different antibiotics, different pH values ​​and different concentrations of NaCl were evaluated according to the OD595 value, and the changes after incubation of the negative wells and the positive wells were used as controls for verification. The experimental results are recorded in Table 1.

[0099] Table 1: Physiological and biochemical indexes of Bacillus velezensis 20-1

[0100]

[0101]

[0102] Note: "+" indicates positive, "-" indicates negative, and "w" indicates weak positive Example 3: Study on the antibacterial effect of Bacillus velezensis20-1

[0103] 1. In vitro inhibitory effect of Bacillus velezensis 20-1 on Amphotericin B.

[0104] 1.1 Effect of bacterial fermentation broth on the growth of Amphotericin B spores

[0105] The bacteria were inoculated in LB liquid culture medium at a rate of 5% and cultured at 37° C., 200 r / min for 48 h, centrifuged at 15000 rpm / min for 15 min, and filtered through a 0.22 nm bacterial filter membrane.

[0106] The amphoteric fungus stored in -80℃ glycerol was activated in PDA medium in a 25℃ incubator for 3 days. The surface of the medium was rinsed with sterile water several times, filtered with sterilized multi-layer gauze several times, and the spore concentration was adjusted to 2x10 using a hemocytometer. 4 Pieces / ml.

[0107] In 6 sterilized 1.5 mL centrifuge tubes, add 500 μL PDB (potato broth) and 200 μL of Amphotericinus spores (spore concentration of 2 × 10 4 The cells were randomly divided into two groups with three replicates each. 500 μL of bacterial fermentation broth was added to the treatment group, and 500 μL of sterile water was added to the control group. The cells were cultured at 25°C and the germination of the spores of Amphotericinus was observed under a microscope.

[0108] 1.2 Effects of bacterial fermentation broth on the growth of Amphotericin B.

[0109] The Amphotericum spp. stored in glycerol at -80°C was activated in PDA medium in a 25°C incubator for 3 days for use.

[0110] Mix the fermentation liquid of the strain with the PDA medium at a volume ratio of 1:10 to prepare a medium containing the fermentation liquid of Bacillus velezensis20-1. Use sterile water as a blank control and pipette an appropriate amount onto a clean glass slide to make the medium evenly attached to the slide. Use sterilized tweezers to pick up a small amount of hyphae and place it on the slide. Place the slide in a 90mm medium, seal it, and culture it in a 25℃ incubator overnight. Observe the growth of hyphae under a microscope.

[0111] The fermentation liquid of the strain was mixed with the PDA medium in a volume ratio of 1:10 to prepare a medium containing the fermentation liquid of Bacillus velezensis 20-1, and sterile water was used as a blank control. An 8 mm amphoteric mold cake was inoculated in the center of a 90 mm PDA plate, and three replicates were made for each treatment, and cultured at 25°C for 24 hours. When the colonies of the control group filled the culture dish, the growth diameters of the colonies of the control group and the treatment group were measured by the cross method, and the antibacterial rate of the fermentation liquid of the 20-1 strain was calculated. Antibacterial rate = (growth diameter of the colonies of the control group - growth diameter of the colonies of the treatment group) / (growth diameter of the colonies of the control group - diameter of the cake) × 100%.

[0112] The results are shown in Table 2 and Figures 5 to 7 As shown, the bacterial fermentation broth can effectively inhibit the spore germination and hyphae growth of Amphoteric Mildew, and the inhibition rate reaches 80.17±0.85%.

[0113] Table 2: Inhibition rate of 20-1 bacterial fermentation broth on the growth of Amphoteric Mildew

[0114]

[0115] Example 4: Stability of Bacillus velezensis 20-1 against Amphotericin Bacillus

[0116] 1.1 Determination of thermal stability of fermentation broth

[0117] The fermentation broth of the strain was treated at 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 30 minutes respectively. After the temperature cooled down, the fermentation broth that had not been treated with temperature was used as the control, and the sterile water that had not been treated with temperature was used as the blank control. The culture was carried out at 25℃ for 24 hours. Each treatment was repeated 3 times. The relative inhibition rate of the fermentation broth of the 20-1 strain under different temperature treatments was determined. Relative inhibition rate = (blank control colony growth diameter - treated colony growth diameter) / (blank control colony growth diameter - untreated colony growth diameter) × 100%. The results are as follows Figure 8 As shown, the bacterial fermentation broth has good temperature stability, and its relative antibacterial rate is above 80% at 40-90°C.

[0118] 1.2 Fermentation broth protease stability

[0119] The fermentation broth of the strain was treated with proteinase K (PK) and trypsin (Trypsin) at a final concentration of 30U / mg at 37℃ for 1h, and then inactivated in a metal bath at 80℃. After the temperature cooled, the fermentation broth without protease treatment was used as the control, and sterile water without temperature treatment was used as the blank control. The culture was carried out at 25℃ for 24h. Each treatment was repeated 3 times. The diameter of the inhibition zone (mm) was used as an indicator of the effect of inhibiting aquatic pathogenic fungi. The experimental results are shown in Figure 2. Fig. 9 As shown, the bacterial fermentation broth has good protease stability, and the growth diameter of the inhibition zone of the fermentation broth treated with enzyme protein is greater than 20 mm.

[0120] Example 5: Broad spectrum of antagonism against Bacillus velezensis 20-1

[0121] Referring to the preparation method of bacterial fermentation broth of Bacillus velezensis 20-1 and the in vitro inhibition experimental steps of Bacillus velezensis 20-1 on Amphisexualus in Example 3, bacterial fermentation broth was prepared, and the in vitro antibacterial ability of Bacillus velezensis 20-1 on four aquatic pathogenic fungi was evaluated. The four aquatic pathogenic fungi were Mucor hiemalis, Mucor circinelloides, Saprolegnia australis, and Phanerochaete concrescens. The pathogenic fungi were inoculated into PDA medium and activated for 3 days for use.

[0122] The diameter of the inhibition zone (mm) was used as an indicator of the effect of inhibiting aquatic pathogenic fungi. The diameters of the inhibition zones of the control group and the fermentation liquid treatment group were compared and the differences were statistically analyzed. Fig.10 The relative inhibitory effect of bacterial fermentation broth on permafrost Mucor and southern Pythium was above 80%, and it also had a good antagonistic effect on compact light-stalked Corynebacterium, with an inhibitory effect of more than 70%, but the antagonistic effect on Mucor circinelloides was poor.

[0123] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "embodiment" or "specific embodiment" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment are included in at least one embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments and features of the different embodiments described in this specification without contradiction.

[0124] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A Bacillus velezensis 20-1, characterized in that: The Bacillus Velez 20-1 was deposited in the China Center for Type Culture Collection on July 26, 2024, with the deposit number being CCTCC NO:M20241694.

2. A fermentation broth, characterized in that: The fermentation broth is obtained by fermenting the Bacillus Velez 20-1 described in claim 1.

3. A bacterial suspension, characterized in that: The bacterial suspension includes the Bacillus Velez 20-1 described in claim 1.

4. Use of the Bacillus Velez 20-1 according to claim 1, the fermentation liquid according to claim 2 or the bacterial suspension according to claim 3 in the preparation of food, beverage, health product, feed or additive that inhibits the activity of aquatic pathogenic fungi.

5. Use of the Bacillus Velez 20-1 according to claim 1, the fermentation liquid according to claim 2 or the bacterial suspension according to claim 3 in the preparation of a medicine for inhibiting the activity of aquatic pathogenic fungi.

6. The use according to claim 4 or 5, characterized in that The aquatic pathogenic fungi include at least one of Amphiporium amphotericum, Amphiporium americanum, Mucor solani, Mucor circinelloides, Pythium incense, Coneculus compactus, Aeromonas punctata, Aeromonas sobria, Aeromonas hydrophila, Pseudomonas fluorescens, Pseudomonas albicans, Pseudomonas aquaticus, Flexibacter columnaris, Edwardsiella, Nocardia asteroides and Streptococcus agalactiae.

7. A composition, characterized in that The composition comprises at least one of the Bacillus Velezii 20-1 according to claim 1, the fermentation broth according to claim 2, and the bacterial suspension according to claim 3.

8. The composition according to claim 7, characterized in that The dosage form of the composition includes at least one selected from oral liquid, powder, granule, capsule, tablet, pill, aqueous emulsion, microemulsion, suspoemulsion and spray.

9. The composition according to claim 7, characterized in that The composition further comprises an excipient and / or a carrier.

10. The composition according to claim 9, characterized in that The excipient comprises at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent and a sustained-release agent.

11. The composition according to claim 10, characterized in that The carrier comprises at least one selected from sugars, cellulose and its derivatives, calcium phosphates, stearic acid alkaline earth metal salts, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols and cereal hydrolyzed solids.

12. A method for inhibiting the growth of aquatic pathogenic fungi in vitro, characterized in that: include: At least one of the Bacillus Velez 20-1 according to claim 1, the fermentation liquid according to claim 2, and the bacterial suspension according to claim 3 is co-cultured with aquatic pathogenic fungi.

13. The method according to claim 12, characterized in that The aquatic pathogenic fungi include at least one of Amphiporium amphotericum, Amphiporium americanum, Mucor solani, Mucor circinelloides, Pythium incense, Coneculus compactus, Aeromonas punctata, Aeromonas sobria, Aeromonas hydrophila, Pseudomonas fluorescens, Pseudomonas albicans, Pseudomonas aquaticus, Flexibacter columnaris, Edwardsiella, Nocardia asteroides and Streptococcus agalactiae.

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