A water mold inhibitor, its preparation method and application

Abstract

This invention discloses a water mold inhibitor, its preparation method, and its application. The water mold inhibitor comprises Agaricus bisporus polysaccharide, *Epicoccumnigrum* Z01, *Lactobacillus paracasei* LY02, and ergothioneine. The preservation number of *Epicoccumnigrum* Z01 is CCTCC NO: M2024967. The concentration of Agaricus bisporus polysaccharide in the water mold inhibitor is 100-500 mg / L, and the spore concentration of *Epicoccumnigrum* Z01 is 1.0-8.0 × 10⁻⁶ mg / L. 8 / mL. The concentration of Lactobacillus paracasei LY02 in the water mold inhibitor is 1.0-5.0 × 10⁻⁶. 9 CFU / mL. The concentration of ergothioneine in the water mold inhibitor is 300-500 μmol / L. This invention can effectively inhibit water mold infection in aquatic animals.

Description

Technical Field

[0001] This invention relates to the technical field of microbial preparations, and more particularly to a saprolegnia inhibitor, its preparation method, and its application. Background Technology

[0002] In freshwater aquaculture, Saprolegnia, a widespread fungal disease, has long posed a serious threat to the health of aquatic animals, especially during cold seasons when its pathogenicity is particularly pronounced. Saprolegnia can infect aquatic animals at all life stages and has no strict host selectivity, greatly increasing the difficulty of its control. During infection, Saprolegnia, through its unique effector protein transport mechanism, can suppress the host's defense response, leading to epidermal cell damage and tissue decay, which in turn causes slow swimming, weakened vitality, and even death in aquatic animals, resulting in significant economic losses to the aquaculture industry.

[0003] Grass carp (Ctenopharyngodon idella), a traditional freshwater farmed fish in China, is farmed on a large scale domestically due to its wide adaptability and economic value. However, grass carp also face a serious challenge from saprolegniasis, especially under environmental stress and weakened immunity, making them highly susceptible to infection. Saprolegniasis not only affects the growth performance and quality of grass carp but can also lead to mass mortality at different growth stages, directly threatening the sustainable development of the aquaculture industry.

[0004] With increasing environmental awareness and stricter food safety standards, traditional chemical pesticides such as malachite green have been gradually banned or restricted due to their potential toxicity and environmental residues. This change has prompted researchers to turn to safer and more environmentally friendly biological control methods to explore alternative water mold control strategies. In recent years, a growing body of research has shown that various microorganisms in nature have antagonistic effects against water mold, such as Serratia marcescens, Aeromonas, Pseudomonas, Bacillus, and Actinomycetes. These microorganisms not only effectively inhibit the growth of water mold but also have advantages such as being residue-free and environmentally friendly, demonstrating enormous application potential. Summary of the Invention

[0005] The purpose of this invention is to provide a water mold inhibitor, its preparation method and application, for inhibiting water mold infection in aquatic animals.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a water mold inhibitor, comprising: button mushroom polysaccharide and Epicoccumnigrum Z01;

[0008] The preservation number of *Acetobacter nigricans* Z01 is CCTCC NO: M2024967.

[0009] Furthermore, the concentration of Agaricus bisporus polysaccharide in the water mold inhibitor is 100-500 mg / L, and the spore concentration of Acanthopanax niger Z01 is 1.0-8.0 × 10⁻⁶ mg / L. 8 per mL.

[0010] Furthermore, the water mold inhibitor also includes:

[0011] Lactobacillus paracasei LY02, wherein the concentration of Lactobacillus paracasei LY02 in the water mold inhibitor is 1.0-5.0 × 10⁻⁶. 9 CFU / mL.

[0012] Furthermore, the water mold inhibitor also includes:

[0013] Ergothioneine, wherein the concentration of ergothioneine in the water mold inhibitor is 300-500 μmol / L.

[0014] Secondly, the present invention provides a method for preparing a water mold inhibitor, comprising the following steps:

[0015] The *Plasmodium nigra* Z01 was inoculated into potato dextrose broth and cultured in a shaker at 175 rpm and 25°C for 5 days. The filtrate was then filtered through three layers of sterile gauze to obtain the *Plasmodium nigra* Z01 culture medium.

[0016] Add Agaricus bisporus polysaccharide to the culture medium of Acinetobacter niger Z01 and mix well to obtain a water mold inhibitor.

[0017] Furthermore, the preparation method of the water mold inhibitor also includes:

[0018] Lactobacillus paracasei LY02 was inoculated into Lactobacillus delbrueckii liquid culture medium and cultured in a shaker. The culture medium was then filtered through three layers of sterile gauze to obtain Lactobacillus paracasei LY02 culture medium.

[0019] Add Lactobacillus paracasei LY02 culture medium to Acinetobacter nigricans Z01 culture medium and mix well to obtain a water mold inhibitor.

[0020] Furthermore, the method for preparing the water mold inhibitor also includes: adding ergothionein to the culture medium of *Pseudomonas niger* Z01 and mixing it evenly to obtain the water mold inhibitor.

[0021] Furthermore, the OD600 of the culture medium of *Pseudomonas niger* Z01 is 1.2-1.5.

[0022] Furthermore, the OD600 of the Lactobacillus paracasei LY02 culture medium is 1.2-1.5.

[0023] Thirdly, the present invention also provides the application of a water mold inhibitor in the treatment or prevention of fungal infections, wherein the water mold inhibitor is the aforementioned water mold inhibitor.

[0024] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0025] The water mold inhibitor of this invention can effectively inhibit the activity and reproduction of fungi, especially preventing water mold disease caused by pathogenic water mold. Therefore, this invention not only enriches the types of biocontrol bacteria but also broadens their application in the control of water mold diseases in aquaculture. Furthermore, this invention not only provides a new solution for the prevention and control of water mold diseases in freshwater farmed animals but also effectively avoids the problem of drug residues. Attached Figure Description

[0026] Figure 1 : is a graph showing the water mold infection rate of each group of grass carp in the embodiments of the present invention.

[0027] Figure 2 : These are comparative images of liver tissue sections from different groups of grass carp in embodiments of the present invention.

[0028] Figure 3 : These are comparative images of intestinal tissue slices from different groups of grass carp in embodiments of the present invention.

[0029] Figure 4 : These are comparative images of muscle tissue slices from various groups of grass carp in embodiments of the present invention. Detailed Implementation

[0030] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, they are provided to make the invention more comprehensive and complete, and to fully convey the concept of the exemplary embodiments to those skilled in the art. The same reference numerals in the drawings denote the same or similar structures, and therefore repeated descriptions of them will be omitted.

[0031] The terms used to express position and direction in this invention are illustrated with reference to the accompanying drawings, but changes can be made as needed, and all such changes are included within the scope of protection of this invention.

[0032] This application includes a description of the strains, drugs, and instruments involved:

[0033] Potato glucose agar medium was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.

[0034] Potato glucose broth culture medium was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.

[0035] Lactobacillus delbrueckii solid culture medium, Qingdao Haibo Biotechnology Co., Ltd.

[0036] Liquid culture medium for Lactobacillus delbrueckii, Qingdao Haibo Biotechnology Co., Ltd.

[0037] Bacterial genomic DNA extraction kit (DP302) was purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0038] Agaricus bisporus polysaccharide, purchased from Xi'an Huilin Biotechnology Co., Ltd.

[0039] Ergothioneine, purchased from Guangdong Mingcheng Biotechnology Co., Ltd.

[0040] Giemsa staining solution was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0041] The sterile gauze is gauze that has been sterilized at high temperature. The gauze was purchased from Caoxian Hualu Sanitary Materials Co., Ltd., with production batch number 23082501 and specifications of 21s x 32s.

[0042] The Ezup column-type fungal genomic DNA extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0043] Epicoccumnigrum Z01 was collected from the laboratory of the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, and deposited at the China Center for Type Culture Collection, Luojia Mountain, Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCCNO: M2024967 and deposit date of May 16, 2024.

[0044] This invention uses Giemsa staining to observe blood cell morphology, including:

[0045] Boil the slide with soap or laundry detergent for 20 minutes, then rinse repeatedly with hot water and tap water. Finally, rinse 3 to 5 times with distilled water, or soak in 95% alcohol for 1 hour and then wipe or dry. Draw blood from the tail of a grass carp and drop it onto one end of a prepared slide about 4-5 mm from the tip. Use a spreader to evenly spread the blood onto the slide, forming a thin, uniform blood film. After the blood film dries, fix it with anhydrous methanol for 3 minutes, then stain with Giemsa staining solution at room temperature for 15 minutes. Finally, slowly rinse the slide with distilled water from one end, air dry, and examine under a microscope.

[0046] The method for calculating the number of white blood cells in this invention includes (generally, counting in parallel 5 times and taking the average):

[0047] White blood cell count = A / N × M1 × 10 × 106

[0048] In the formula, N is the number of squares observed under the microscope, and N is 4 in this invention; M1 is the magnification of the blood smear, and M1 is 20 in this invention; A is the total number of white blood cells in the N squares observed under the microscope.

[0049] The preparation of paraffin sections according to the present invention includes:

[0050] After collecting liver, intestine, and muscle tissue from grass carp, the tissues were immediately fixed in Born's solution. After 20 hours of fixation, the tissues were transferred to an alcohol solution for dehydration, followed by paraffin embedding, sectioning, hematoxylin-eosin staining, and neutral resin mounting. The tissues were then observed and photographed under a microscope (Olympus BX51).

[0051] (I) Separation and Identification

[0052] 1.1 Isolation of strains:

[0053] 1.1.1 Isolation of Saprolegnia pathogen and antagonistic fungal strains:

[0054] Grass carp with wounds on their bodies were collected from aquaculture ponds in Jingzhou, Hubei Province, and labeled as Grass Carp A. For application, refer to... Figure 1 Grass carp A had congestion around its eyes and at the base of its pelvic fins, and some scales were missing from its body surface. Grass carp A was then transported to the laboratory of the Yangtze River Fisheries Research Institute of the Chinese Academy of Fishery Sciences.

[0055] Spray the body surface of grass carp A with alcohol, remove the eyes of grass carp A, puncture the lens with sterile tweezers, and place the punctured lens on potato dextrose agar medium. Incubate at 25°C for 3 days, and mycelia will grow on the potato dextrose agar medium.

[0056] Using a sterile scalpel, cut a 5mm x 5mm rectangular piece covered with mycelium. Invert the 5mm x 5mm rectangular piece covered with mycelium onto a fresh potato dextrose agar medium (with the mycelium facing the agar medium) and incubate at 25°C for 3 days. Repeat this step until a single strain grows on the potato dextrose agar medium. The white strain with mycelium is the pathogen of Saprolegnia.

[0057] For a single pale yellow bacterial strain (with an inhibition zone) on potato dextrose agar medium, use a sterile scalpel to cut a 5mm x 5mm rectangular piece. Invert this 5mm x 5mm rectangular piece onto a fresh potato dextrose agar medium (with the pale yellow strain facing the agar medium) and incubate at 25°C for 3 days. Repeat this process until a single bacterial strain grows on the potato dextrose agar medium.

[0058] 1.1.2 Isolation of Lactobacillus:

[0059] Dilute Da Vinci Pure Yogurt (Original Flavor) from New Hope Dairy Co., Ltd. by 10 times, take 100 μL and spread it evenly on Lactobacillus delbrueckii culture medium. Seal the culture medium plate to ensure that it is airtight and incubate it in a constant temperature incubator at 37°C for 48 hours.

[0060] Select single colonies that are white or pale yellow, viscous in texture, round, slightly raised, with neat edges, and conform to the typical morphology of Lactobacillus. Streak them on Lactobacillus delbrueckii agar and incubate them in a 37°C incubator for 48 hours.

[0061] 1.2 Purification of Saprolegnia pathogen and antagonistic superbug strains

[0062] 1.2.1 Purification of Saprolegnia pathogen and antagonistic superbug strains

[0063] After typical colonies appear on potato dextrose agar, select single white colonies exhibiting the colony characteristics of standard Saprolegnia, standard Epicoccus nigrum colonies, and single yellow colonies producing inhibition zones (inhibiting the Saprolegnia pathogen), respectively, for reference. Figure 2 .

[0064] The two selected single colonies were cultured three times consecutively. The colony morphology in the culture medium was consistent, and the cell morphology of the strain was uniform. This yielded a pure strain of the Saprolegnia pathogen and a pure strain of the antagonistic true strain.

[0065] 1.2.2 Purification of Lactobacillus

[0066] After typical colonies appear on the Lactobacillus delbrueckii culture medium, single colonies with the colony characteristics of standard Lactobacillus are picked and cultured three times consecutively. If the colony morphology in the culture medium is consistent and the cell morphology of the strain is uniform, then a pure strain of Lactobacillus is obtained.

[0067] 1.3 Biological identification of the strain:

[0068] 1.3.1 Biological identification of pathogenic bacteria and antagonistic fungal strains:

[0069] The specific method refers to the instructions of the Fungal Extraction Kit (Ezup Column-Based Fungal Genomic DNA Extraction Kit) for extracting DNA from Saprolegnia pathogens and antagonistic fungal strains.

[0070] ITSrDNA analysis

[0071] The universal primers ITS1 and ITS4 for the fungal ITS region were used to identify pathogenic bacteria and antagonistic fungal strains: primer ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'; primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3').

[0072] PCR reaction system: Using the extracted genome as a template, the total system volume is 50 μl: 25 μl ExTaq enzyme, 2 μl primer ITS1, 2 μl primer ITS4, 2 μl template, and double-distilled water to bring the volume to 50 μl.

[0073] PCR amplification conditions: Pre-denaturation at 95℃ for 3 min. 35 reaction cycles: denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min. Final extension at 72℃ for 10 min. Terminate the reaction at 4℃.

[0074] After amplification, 5 μl of the PCR product was subjected to 1% agarose gel electrophoresis, and the amplified product was sent to Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing. The sequencing results were aligned using GenBank blast, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA 5.0 software.

[0075] Figure 3 In the diagram, M represents the standard template; 1, 2, 3, and 4 are pathogenic bacteria; and 5 and 6 are antagonistic fungal strains. (Reference) Figure 3 It can be seen that the 16S rDNA sequence length of the pathogen is 708 bp, while the 16S rDNA sequence length of the antagonistic euphemism is 527 bp. (Reference) Figure 4 The pathogen clustered with *Saprolegnia ferax* (accession number: GQ119935.1), and based on morphological and molecular biological characteristics, the pathogen was identified as *Saprolegnia ferax* and named *Saprolegnia ferax* CY01. (Reference) Figure 4 The antagonistic euphratica strain showed 100% homology with Epicoccumnigrum and clustered with Epicoccumnigrum (accession number: OQ555113.1). Based on morphological and molecular biological characteristics, the antagonistic euphratica strain was identified as Epicoccumnigrum and named Epicoccumnigrum Z01.

[0076] The nucleotide sequence of *Streptococcus niger* Z01 is as follows:

[0077] GGGTGACCTGCGGAAGGATCATTACCTAGAGTTTGTAGACTTCGGTCTGCTACCTCTT

[0078] ACCCATGTCTTTTGAGTACCTTCGTTTCCTCGGCGGGTCCGCCCGCCGATTGGACAAC

[0079] ATTCAAACCCTTTGCAGTTGCAATCAGCGTCTGAAAAAACATAATAGTTACAACTTTC

[0080] AACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTA

[0081] GTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTG

[0082] GTATTCCATGGGGCATGCCTGTTCGAGCGTCATTTGTACCTTCAAGCTCTGCTTGGTGT

[0083] TGGGTGTTTTGTCTCGCCTCTGCGTGTAGACTCGCCTTAAAACAATTGGCAGCCGGCG

[0084] TATTGATTTCGGAGCGCAGTACATCTCGCGCTTTGCACTCATAACGGCGACGTCCAAA

[0085] AGTACATTTTTACACTCTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCAT

[0086] ATCT

[0087] The nucleotide sequence of Saprolegnia multisporea CY01 is as follows:

[0088] GAAGGATCATTACCACACCAAAAAACACCCCACGTGAATGTACTCTTTATGAGGCTTG

[0089] CGCTGCCCTTGTGGCGGCTAGCCGAAGGTTTCGCAAGAAGCCGATGTCAATTTGAATC

[0090] CTTTTTAAACTACGACTGATCAAAACTGCAGATAGAAATGTCTGCATGCAATTGAAATA

[0091] CAACTTTCAACAGTGGATGTCTAGGCTCGCACACCGATGAAGAACGCTGCGAACTGC

[0092] GATACGTAATGCGAATTGCAGAATTCAGTGAGTCATCAAAATTTTGAACGCATATTGCA

[0093] CTTCCGGGTTAGTCCTGGGAGTATGTTTGTATCAGTGTCCGTGAACACAACCTTGTTTC

[0094] ATTTCTTGATTGAGATGGAGCAGAATGTGAAGGTCTTGTAATTACAAGTCCTTTTAAAC

[0095] GACGGTACCTATGCGTCCTCGTGAGATGTATTATTTAAAGGTATGCCTGCGCTTCTTTC

[0096] GAGAGTTTTGTGTGGCGGCACACAGCATTCAAAGAGAGAGCAAATCGCGGTAGTTTT

[0097] GCTTGTATTTCGGTACGAGTGGACACATATTGCTTTTTGTGATTTCTGCGAGTCTGTTG

[0098] TTTAAGCACAGGACACGTAAGGAGAGTGAGTATGCTGGTGCATTTCTTGGCGCATGGA

[0099] GGCAAATTGGGAATTCAATCCAATTTGGACCTGATATCAAACAAGACTACCCGCTGAA

[0100] CTTAAGCA

[0101] 1.3.2 Biological identification of lactobacilli:

[0102] Lactobacillus DNA was extracted according to the instructions of the bacterial DNA extraction kit (bacterial genomic DNA extraction kit (DP302)).

[0103] Specifically, purified single Lactobacillus colonies are used as templates for amplification using universal 16S rRNA sequencing primers, such as the commonly used primer pair 1492r / F27. The sequence of the forward primer 27F is 5'-TACGGYTACCTTGTTACGACTT-3'; the sequence of the reverse primer 1492R is 5'-AGAGTTTGATCMTGGCTCAG-3'.

[0104] PCR reaction system: Using the extracted genome as a template, the total PCR reaction system is 50 μl: 25 μl Taq DNA polymerase, 1 μl forward primer, 1 μl reverse primer, 2 μl template, and double-distilled water to bring the volume to 50 μl.

[0105] PCR conditions: Pre-denaturation at 95℃ for 5 min. Then, 35 cycles were performed: denaturation at 95℃ for 30 s; annealing at 55℃ for 45 s; extension at 72℃ for 1 min 20 s; extension at 72℃ for 15 min; and termination of the reaction at 4℃.

[0106] After amplification, 5 μl of the amplification product was verified by 1% agarose gel electrophoresis, sequenced by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd., and compared with BLAST sequence in NCBI. The isolated lactobacillus was identified as Lacticaseibacillus paracasei and named Lacticaseibacillus paracasei LY02.

[0107] The nucleotide sequence of Lactobacillus paracasei LY02 is as follows:

[0108] TGCAAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAA

[0109] CGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACA

[0110] TTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAA

[0111] GATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGT

[0112] AATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTG

[0113] GGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAAT

[0114] GGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAA

[0115] AACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTAT

[0116] CCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGC

[0117] AAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGAT

[0118] GTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGC

[0119] AGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAA

[0120] CACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATG

[0121] GGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGT

[0122] GTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGG

[0123] AGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTG

[0124] GAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTT

[0125] GATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGT

[0126] TGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTA

[0127] TGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGA

[0128] GGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGC

[0129] TACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCC

[0130] ATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAA

[0131] TCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCA

[0132] CACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGC

[0133] C

[0134] (II) Preparation of water mold inhibitor

[0135] 2.1 Water mold inhibitor A

[0136] Water mold inhibitor A: comprises Agaricus bisporus polysaccharide and *Gnaphalium affine* Z01 culture medium. The OD600 of the *Gnaphalium affine* Z01 culture medium is 1.2-1.5, and the concentration of Agaricus bisporus polysaccharide is 100-500 mg / L. In a specific embodiment of this invention, the concentration of Agaricus bisporus polysaccharide is 300 mg / L.

[0137] In application, the primary *Pseudomonas nigricans* Z01 purified in step 1.2 was inoculated into 200 mL of potato dextrose broth and cultured in a shaker at 175 rpm and 25°C for 5 days. After filtration through three layers of sterile gauze, the *Pseudomonas nigricans* Z01 culture broth was obtained, with an OD600 of 1.2-1.5. In a specific embodiment of this invention, the OD600 of the *Pseudomonas nigricans* Z01 culture broth was 1.3.

[0138] In practical applications, 1-5 mg of Agaricus bisporus polysaccharide is mixed with 10 mL of *Pseudomonas niger* Z01 culture medium and stirred evenly to obtain water mold inhibitor A. In a specific embodiment of the present invention, 3.0 mg of Agaricus bisporus polysaccharide is mixed with 10 mL of *Pseudomonas niger* Z01 culture medium and stirred evenly to obtain water mold inhibitor A.

[0139] 2.2 Water mold inhibitor B

[0140] Water mold inhibitor B comprises Agaricus bisporus polysaccharide, Agaricus niger Z01 culture medium, and Lactobacillus paracasei LY02 culture medium. The OD600 of the Lactobacillus paracasei LY02 culture medium is 1.2-1.5. In a specific embodiment of this invention, the OD600 of the Lactobacillus paracasei LY02 culture medium is 1.4.

[0141] In application, *Lactobacillus paracasei* LY02 was inoculated into 200 ml of *Lactobacillus delbrueckii* liquid culture medium and cultured in a shaker at 175 rpm and 25°C for 5 days. Then, the culture was filtered through three layers of sterile gauze to obtain the *Lactobacillus paracasei* LY02 culture medium. The OD of the *Lactobacillus paracasei* LY02 culture medium was 1.2-1.5. In a specific embodiment of the present invention, the OD600 of the *Lactobacillus paracasei* LY02 culture medium was 1.4.

[0142] In practical applications, 1-5 mg of Agaricus bisporus polysaccharide, 10 mL of *Pseudomonas niger* Z01 culture medium, and 10 mL of *Lactobacillus paracasei* LY02 culture medium are mixed and stirred evenly to obtain water mold inhibitor B. In a specific embodiment of the present invention, 3.0 mg of Agaricus bisporus polysaccharide, 10 mL of *Pseudomonas niger* Z01 culture medium, and 10 mL of *Lactobacillus paracasei* LY02 culture medium are mixed and stirred evenly to obtain water mold inhibitor B.

[0143] 3.3 Water mold inhibitor C

[0144] Water mold inhibitor C comprises Agaricus bisporus polysaccharide, Agaricus niger Z01 culture medium, Lactobacillus paracasei LY02 culture medium, and ergothioneine. The concentration of ergothioneine is 300-500 μmol / L. In a specific embodiment of this invention, the concentration of ergothioneine is 400 μmol / L.

[0145] In application, 1-5 mg of Agaricus bisporus polysaccharide, 3-5 μmol ergothioneine, 10 ml of *Pseudomonas niger* Z01 culture medium, and 10 ml of *Lactobacillus paracasei* LY02 culture medium are mixed and stirred evenly to obtain water mold inhibitor C. In a specific embodiment of the present invention, 3.0 mg of Agaricus bisporus polysaccharide, 4 μmol ergothioneine, 10 ml of *Pseudomonas niger* Z01 culture medium, and 10 ml of *Lactobacillus paracasei* LY02 culture medium are mixed and stirred evenly to obtain water mold inhibitor C.

[0146] (III) Application of Saprolegnia inhibitors

[0147] Grass carp weighing 70-100g were temporarily raised for 14 days and then divided into a blank control group, an infection group, a button mushroom polysaccharide group, an ergothionein group, a lactobacillus group, a Z01 group, a water mold inhibitor group A, a water mold inhibitor group B, and a water mold inhibitor group C.

[0148] Blank control group: Aerated culture water was added to the culture water body and the water temperature was maintained at 18℃.

[0149] Infection group: Aerated aquaculture water was added to the aquaculture water body, and the water temperature was maintained at 18℃.

[0150] Agaricus bisporus polysaccharide group: Agaricus bisporus polysaccharide was added to the aquaculture water to a final concentration of 300 mg / L, and the water temperature was maintained at 18℃. Soaking was performed for 1 hour each time, for 3 consecutive days. Simultaneously, Agaricus bisporus polysaccharide was mixed into the fish feed to a concentration of 300 mg / kg. Feeding was performed once daily for 3 consecutive days.

[0151] Ergothioneine group: Ergothioneine was added to the aquaculture water to a final concentration of 400 μmol / L, and the water temperature was maintained at 18℃. Each soaking lasted 1 hour, for 3 consecutive days. Simultaneously, ergothioneine was mixed into the fish feed to a concentration of 400 μmol / kg. This was done once daily for 3 consecutive days.

[0152] Lactobacillus group: Lactobacillus paracasei LY02 culture medium was added to the aquaculture water, with a final concentration of Lactobacillus paracasei LY02 of 1.0-5.0 × 10⁻⁶. 9 CFU / mL (In a specific embodiment of the present invention, the final concentration of Lactobacillus paracasei LY02 is 3.0 × 10⁻⁶ CFU / mL) 9 (CFU / mL) and maintain the water temperature at 18℃. Soak for 1 hour each time, for 3 consecutive days. Simultaneously, add *Lactobacillus paracasei* LY02 culture medium to the fish bait; the concentration of *Lactobacillus paracasei* LY02 in the fish bait should be 1.0-5.0 × 10⁻⁶ CFU / mL. 9 In one specific embodiment of the present invention, the concentration of *Lactobacillus paracasei* LYO2 in the fish bait is 3.0 × 10⁻⁶ CFU / g. 9(CFU / g). Administer once daily for 3 consecutive days.

[0153] Group Z01: *Pseudomonas nigricans* Z01 culture medium was added to the aquaculture water, with a final concentration of *Pseudomonas nigricans* Z01 spores of 2.0 × 10⁻⁶. 8 Z01 spores / ml, and the water temperature was maintained at 18℃. Soaking was performed for 1 hour each time, for 3 consecutive days. Simultaneously, *Pseudomonas nigricans* Z01 culture medium was stirred into the fish bait, with a Z01 spore concentration of 2.0 × 10⁻⁶ spores in the bait. 8 Feed once a day for 3 consecutive days.

[0154] Group A (Saprolegnia inhibitor): Water mold inhibitor A was added to the aquaculture water, resulting in a final concentration of 300 mg / L for Agaricus bisporus polysaccharide and 2.0 × 10⁻⁶ spores for Acinetobacter nigricans Z01. 8 Z01 spores / ml, and maintain the water temperature at 18℃. Soak for 1 hour each time, for 3 consecutive days. Simultaneously, add water mold inhibitor A to the fish bait. The concentration of Agaricus bisporus polysaccharide in the fish bait is 300 mg / kg, and the concentration of Agaricus bisporus Z01 spores is 2.0 × 10⁻⁶. 8 Feed once a day for 3 consecutive days.

[0155] Group B of water mold inhibitors: Water mold inhibitor B was added to the aquaculture water, with a final concentration of 300 mg / L for Agaricus bisporus polysaccharide and 3.0 × 10⁻⁶ mg / L for Lactobacillus paracasei LY02. 9 The final concentration of *Pseudomonas niger* Z01 spores was 2.0 × 10⁻⁶ CFU / mL. 8 Z01 spores / ml, and maintain the water temperature at 18℃. Soak for 1 hour each time, for 3 consecutive days. Simultaneously, add water mold inhibitor B to the fish bait. The concentration of Agaricus bisporus polysaccharide in the fish bait is 300 mg / kg, and the concentration of Agaricus bisporus Z01 spores is 2.0 × 10⁻⁶. 8 The final concentration of Lactobacillus paracasei LY02 was 3.0 × 10⁻⁶. 9 CFU / g. Administer once daily for 3 consecutive days.

[0156] Group C (Saprolegnia inhibitor): Saprolegnia inhibitor C was added to the aquaculture water, resulting in a final concentration of 300 mg / L for Agaricus bisporus polysaccharide and 3.0 × 10⁻⁶ mg / L for Lactobacillus paracasei LY02. 9 The final concentration of *Pseudomonas niger* Z01 spores was 2.0 × 10⁻⁶ CFU / mL. 8 The concentration of Z01 spores / ml was adjusted, with a final ergothioneine concentration of 400 μmol / L, and the water temperature was maintained at 18℃. Simultaneously, a water mold inhibitor B was added to the fish bait, resulting in a Agaricus bisporus polysaccharide concentration of 300 mg / kg and an Agaricus niger Z01 spore concentration of 2.0 × 10⁻⁶. 8 The final concentration of Lactobacillus paracasei LY02 was 3.0 × 10⁻⁶. 9CFU / g, with a final ergothioneine concentration of 400 μmol / kg. Administer once daily for 3 consecutive days.

[0157] When used, the fish bait is an extruded compound feed that does not contain any drug additives. The extruded compound feed is produced by Jiangmen Xinjian Feed Co., Ltd., and the product composition is as follows: crude protein ≥32%, crude fiber ≤15%, crude fat ≥3.5%, crude ash ≤15%, total phosphorus ≥0.5%, moisture ≤12%, and lysine ≥1.4%.

[0158] After soaking and feeding grass carp with a saprolegnia inhibitor, wounds of equal size were artificially created on the same part of each grass carp in the infection group, button polysaccharide group, ergothionein group, lactobacillus group, Z01 group, saprolegnia inhibitor A group, saprolegnia inhibitor B group, and saprolegnia inhibitor C group. The fish were then infected with Saprolegnia CY01 (for details, refer to the method of infection with Saprolegnia described in Xu Jialu's 2012 paper "Construction and Application of Fish Saprolegnia Disease Model" published in the Journal of Shanghai Ocean University). The fish were then observed for 7 consecutive days, and the infection rate, changes in red blood cell count, white blood cell count, hemoglobin, blood cell count, and tissue changes were recorded.

[0159] refer to Figure 1 It can be seen that when the fish has no wounds, grass carp are unlikely to be infected with saprolegniasis even if the aquaculture water contains saprolegnia pathogens; however, when the fish has wounds and the water contains saprolegnia pathogens, grass carp are easily infected with saprolegniasis. Specifically, the infection rate in the blank control group was 0%, while the infection rate in the infected group was 100%, indicating that the experimental environment met the conditions for saprolegnia infection, and the experimental design was successful. Furthermore, the infection rate in the button mushroom polysaccharide group was 90%, and the infection rate in the ergothioneine group was 80%, which is likely due to the increased nutrients in the aquaculture water, which improved the resistance of the grass carp. The infection rates in the lactobacillus group and the Z01 group decreased significantly, indicating that both *Lactobacillus paracasei* LY02 and *Pseudomonas niger* Z01 can inhibit the activity of *Saprolegnia multifiliis* CY01, and the inhibitory effect of *Pseudomonas niger* Z01 is significantly better than that of *Lactobacillus paracasei* LY02. After combination, the infection rate in the saprolegnia inhibitor C group was only 10%, indicating that the saprolegnia inhibitor C group can effectively inhibit the infection of injured fish by *Saprolegnia multifiliis* CY01.

[0160] As shown in Table 1, compared with the blank control group, the changes in white blood cell, red blood cell, and hemoglobin levels in grass carp from the infection group, Agaricus bisporus polysaccharide group, ergothioneine group, Lactobacillus group, Z01 group, Saprolegnia inhibitor A group, Saprolegnia inhibitor B group, and Saprolegnia inhibitor C group did not reach statistical significance. This indicates that early Saprolegnia infection generally does not prevent bacterial infection. Furthermore, the changes in white blood cell, red blood cell, and hemoglobin levels in each group showed that the drug components of the Saprolegnia inhibitors did not cause significant changes in white blood cell, red blood cell, and hemoglobin levels, indicating that they are safe for grass carp.

[0161] Table 1. Changes in red blood cells, white blood cells, and hemoglobin in grass carp.

[0162] Group <![CDATA[Red blood cell count × 10 12 / L]]> <![CDATA[White blood cell count × 10 10 / L]]> Hemoglobin × g / L Blank control group 2.29±0.19a 4.43±0.47a 75.13±4.37a Infection group 2.38±0.24a 4.26±0.53a 74.77±3.34a button mushroom polysaccharide group 2.51±0.22a 4.25±0.42a 73.74±3.62a Ergothionein group 2.55±0.24a 4.28±0.41a 72.44±2.91a Lactobacillus group 2.19±0.19a 4.93±0.47a 72.03±4.37a Group Z01 2.88±0.24a 4.26±0.53a 71.77±3.34a Water mold inhibitor group A 2.21±0.22a 4.15±0.42a 73.74±3.62a Water mold inhibitor group B 2.35±0.24a 4.28±0.41a 72.44±2.91a Water mold inhibitor group C 2.25±0.25a 4.78±0.42a 77.44±2.92a

[0163] Figures 2-4 In the table, a represents the blank control group; b represents the infection group; c represents the button mushroom polysaccharide group; d represents the ergothioneine group; e represents the lactobacillus group; f represents the Z01 group; g represents the water mold inhibitor A group; h represents the water mold inhibitor B group; and i represents the water mold inhibitor C group.

[0164] refer to Figure 2 It can be seen that: no obvious abnormalities were found in the liver tissue sections of grass carp in each group; the liver cells were arranged in double rows in a plate-like shape; the liver plates were distributed in a radiating pattern with the central vein as the center; and the liver cells were irregular polygons.

[0165] Figure 2 In case a: Hepatocytes in the liver tissue are arranged in a plate-like pattern, with the hepatic plates radiating outwards from the central vein. The hepatocytes are irregular polygons with large, round nuclei. The adjacent hepatocyte cords are irregularly connected to form a network of hepatic sinusoids, which are not significantly dilated. No obvious abnormalities were observed in the tissue. Figure 2 In b: The liver tissue shows hepatocytes and sinusoids arranged in a roughly radial pattern around the central vein. Diffuse hydropic degeneration of hepatocytes is visible, with loose and pale cytoplasm. The liver plates are arranged regularly and neatly, and there is no obvious dilation or compression of the sinusoids. There are no obvious abnormalities in the portal areas between adjacent liver lobules. No obvious inflammatory changes are seen. Figure 2 In c: Hepatocytes in the liver tissue are arranged in double rows in a plate-like pattern. The hepatic plates are distributed in a radiating pattern with the central vein as the center. The hepatocytes are irregular polygons with little cytoplasm and are vacuolated. The adjacent hepatocyte cords are irregular and interconnected to form a network of hepatic sinusoids. No obvious dilation of the hepatic sinusoids was observed. No obvious inflammatory cell infiltration was observed. Figure 2 In d: hepatocytes in liver tissue were arranged in double rows in a plate-like pattern, with the hepatic plates radiating outwards from the central vein. The hepatocytes were irregular polygons. Compared with the blank control, the cytoplasm content of hepatocytes was slightly increased, and no other significant differences were observed. Figure 2In the e-control: hepatocytes in the liver tissue are arranged in double rows in a plate-like pattern, with the hepatic plates radiating outwards from the central vein. The hepatocytes are irregular polygons. Compared with the blank control, the cytoplasm content of hepatocytes is slightly increased, and no other significant differences are observed. Figure 2 In the f-sample: hepatocytes in the liver tissue were arranged in double rows in a plate-like pattern, with the hepatic plates radiating outward from the central vein. The hepatocytes were irregular polygons with little cytoplasm and were vacuolated. Compared with the blank control, a small number of lymphocytes were infiltrated around local blood vessels, but no other significant differences were observed. Figure 2 In the g-cell analysis: numerous hepatocytes with vacuolated cytoplasm were observed in the liver tissue; venous congestion was rare; and no obvious inflammatory cell infiltration was observed. Figure 2 In the h-strain: numerous hepatocytes with vacuolated cytoplasm were observed in the liver tissue; no obvious inflammatory cell infiltration was observed. Figure 2 In the i-th cell: hepatocytes are arranged in double rows in a plate-like pattern, with the hepatic plates radiating outwards from the central vein. The hepatocytes are irregular polygons with large, round nuclei. The adjacent hepatocyte cords are irregularly connected to form a network of hepatic sinusoids, which are not significantly dilated. No obvious abnormalities were observed in the tissue.

[0166] refer to Figure 3 It can be seen that: no obvious abnormalities were found in the intestinal sections of grass carp in each group; the structure of each layer of intestinal tissue was clear; the number of intestinal villi in the mucosal layer was abundant; the mucosal epithelium was intact; the cells were arranged regularly; the surface was a single layer of columnar epithelium with normal morphology and structure and neat arrangement; goblet cells were distributed between the epithelial cells; and the muscle layer and serosa layer composed of smooth muscle cells were visible under the intestinal wall.

[0167] Figure 3 The intestinal tissue showed clear structure in each layer, with abundant villi in the mucosal layer, intact mucosal epithelium, and regular cell arrangement. Occasionally, the epithelium at the tip of the villi was separated from the lamina propria. The myocytes in the muscular layer were arranged regularly and had normal morphology. No obvious inflammatory changes were observed. Figure 3 The structure of each layer of the intestinal tissue was clear, the mucosa layer had abundant villi and intact mucosal epithelium; compared with the blank control, the lamina propria showed connective tissue hyperplasia with edema, loose connective tissue arrangement, and a small amount of lymphocyte infiltration, but no other significant differences were observed. Figure 3 The intestinal tissue of the control group showed clear structure in each layer, with abundant villi in the mucosa. Compared with the blank control, the mucosal epithelium was locally absent, and the villi epithelium was rarely separated from the lamina propria. The lamina propria showed connective tissue hyperplasia with edema, loose connective tissue arrangement, and a small amount of lymphocyte infiltration. No other significant differences were observed. Figure 3 The intestinal tissue of the control group showed clear structure in each layer, with abundant villi in the mucosal layer and intact mucosal epithelium. Compared with the blank control, the villi epithelium was often separated from the lamina propria, but no other significant differences were observed. Figure 3A small number of epithelial cells were shed from the mucosa of the intestinal tissue; no obvious abnormalities were observed in the lamina propria; the muscular layer was stained evenly; the muscle fibers had normal morphology and structure and were arranged regularly; no obvious inflammatory cell infiltration was observed. Figure 3 The intestinal mucosa layer was rich in villi and goblet cells. Necrosis and shedding of epithelial cells at the villi tips were rare, and the nuclei were condensed, deeply stained, and fragmented. No obvious hyperplasia or obvious inflammatory cell infiltration was observed. Figure 3 The surface of the small intestine is covered with villi, which are abundant and covered with a single layer of columnar epithelium. The villi are normal in morphology and structure and are arranged in an orderly manner. Goblet cells are distributed between the epithelial cells. The muscular layer and serosa layer composed of smooth muscle cells are visible under the intestinal wall, and no obvious abnormalities are observed. Figure 3 The intestinal tissue showed clear layers. The intestinal villi epithelium was a single layer of columnar epithelium with densely packed cells. Goblet cells were numerous but slightly reduced in size, mostly arranged in 2-3 rows on the mucosal surface, and locally clustered together. The intestinal villi were abundant, mostly finger-shaped, cone-shaped, and dentate, arranged regularly, and without intestinal glands. The muscular layer was relatively thick, and the morphology and structure of the muscle fibers were normal. No obvious inflammatory cell infiltration was observed.

[0168] refer to Figure 4 It can be seen that there are no obvious differences in the muscle tissue sections of grass carp in each group. The muscle cells in the muscle tissue are arranged regularly, uniformly stained, and have clear boundaries. The cell nuclei are located at the edge. Several muscle cells are clustered into bundles, and each muscle bundle is wrapped by a perimysium composed of loose connective tissue. No obvious abnormalities were found in the tissue.

[0169] Figure 4 The muscle cells in muscle tissues a, b, c, d, e, and f were arranged regularly, uniformly stained, and clearly demarcated, with the nuclei located at the edges. Several muscle cells were clustered into bundles, and each bundle was wrapped by a perimysium composed of loose connective tissue. No obvious abnormalities were observed in the tissues. No significant differences were observed compared with the blank control. Figure 4 The muscle tissue of g, h, and i was stained evenly, the muscle fibers had normal morphology and structure, clear boundaries and regular arrangement, no obvious abnormalities were seen in the interstitium, and no obvious inflammatory cell infiltration was seen.

[0170] In summary, the water mold inhibitor of this invention can effectively inhibit the activity and reproduction of fungi, especially preventing water mold disease caused by pathogenic water mold. Therefore, this invention not only enriches the types of biocontrol bacteria but also broadens their application in the prevention and control of water mold diseases in aquaculture. This invention not only provides a new solution for the prevention and control of water mold disease in freshwater farmed animals but also effectively avoids the problem of drug residues.

[0171] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the invention without departing from the principles and spirit of the invention, and all such changes should fall within the protection scope of the claims of the present invention.

Claims

1. A water mold inhibitor, characterized in that, The water mold is a polypody water mold; the water mold inhibitor includes: Agaricus bisporus polysaccharide and Acetobacter nigricans (… Epicoccumnigrum Z01; The preservation number of *Acetobacter nigricans* Z01 is CCTCC NO: M2024967; The concentration of Agaricus bisporus polysaccharide in the water mold inhibitor is 100-500 mg / L, and the spore concentration of Acanthopanax niger Z01 is 1.0-8.0 × 10⁻⁶ mg / L. 8 per mL.

2. The water mold inhibitor according to claim 1, characterized in that, Also includes: Ergothioneine, wherein the concentration of ergothioneine in the water mold inhibitor is 300-500 μmol / L.

3. A method for preparing the saprolegnia inhibitor as described in claim 1, characterized in that, Includes the following steps: The *Plasmodium nigra* Z01 was inoculated into potato dextrose broth and cultured in a shaker. After filtration through three layers of sterile gauze, the culture medium of *Plasmodium nigra* Z01 was obtained. Add Agaricus bisporus polysaccharide to the culture medium of Acinetobacter niger Z01 and mix well to obtain a water mold inhibitor.

4. The method for preparing the water mold inhibitor according to claim 3, characterized in that, Also includes: Ergothioneine was added to the culture medium of *Pseudomonas niger* Z01 and mixed thoroughly to obtain a water mold inhibitor.

5. The method for preparing the water mold inhibitor according to claim 3, characterized in that, The OD600 of the culture medium of *Acetobacter nigricans* Z01 is 1.2-1.

5.

6. The use of a water mold inhibitor in the preparation of an agent for treating or preventing polymyxa infection, wherein the water mold inhibitor is the water mold inhibitor according to any one of claims 1-2.

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