Polysaccharide polymers, use of polysaccharide polymers and methods of making polysaccharide polymers

Abstract

The application discloses a polysaccharide polymer, application of the polysaccharide polymer and a preparation method of the polysaccharide polymer. The polysaccharide polymer QPS with significant selectivity in inhibiting harmful bacteria in aquaculture is obtained by being isolated and purified from water mould antagonistic actinomycete strain QHV2 for the first time. The polysaccharide polymer QPS can effectively inhibit mycelium growth and spore germination of saprolegnia / branchia mould and destroy mature biofilm. The polysaccharide polymer QPS is stable in activity, harmless to fish fry and good in biological safety, and can be used as a candidate material for research and development of a fish pathogenic fungus biological prevention and control agent. Meanwhile, the polysaccharide polymer QPS avoids pollution of harmful substances to water when the fermentation product of QHV2 is directly applied, and can selectively inhibit harmful bacteria such as staphylococcus aureus, saprolegnia and branchia mould in aquaculture, and has no inhibition or weak inhibition on beneficial bacteria such as pseudomonas pseudoalcaligenes and bacillus in aquaculture, and has better application effect.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic animal disease prevention and control, and in particular relates to a polysaccharide polymer and an application and a preparation method thereof. Background Art

[0002] Saprolegnia Saprolegnia spp.) and branchial molds ( Branchiomyces spp.) are two major pathogenic fungi of fish, spreading through water bodies in the form of zoospores. They possess epidemiological characteristics such as a wide host range, strong pathogenicity, and rapid spread. Saprolegnias are not only infectious to a variety of freshwater fish but can also infect fish eggs and induce secondary bacterial or viral infections, ultimately leading to large-scale mortality in the host population. Gill molds, on the other hand, primarily damage the respiratory physiology of fish by destroying the integrity of gill tissue structure. Because Saprolegnias and gill molds are invisible to the naked eye in the early stages of infection, they are often overlooked by aquaculture practitioners. Furthermore, Saprolegnias and gill molds can enhance their drug resistance by forming complex biofilm structures, making the prevention and control of Saprolegniasis and gill mold diseases extremely difficult. Since malachite green, a specific treatment for Saprolegniasis, was listed as a banned fishery drug due to its teratogenic, carcinogenic, and mutagenic effects, scholars at home and abroad have been dedicated to the search for alternatives to malachite green. However, existing anti-saprolegnia drugs, such as copper sulfate, preservatives, and clotrimazole, are prone to causing significant problems, including drug residues, ecological imbalance, and pathogenic resistance. Therefore, developing new, effective and safe anti-saprolegnia drugs to ensure the quality of aquatic products and protect the ecological safety of aquatic areas has become a top priority.

[0003] Aquatic animal disease control methods centered on biological antagonism have become a research hotspot in the aquaculture sector due to their environmental and environmentally friendly nature. Patent publication number CN106222104A discloses a Saprolegnia antagonistic actinomycete strain, QHV2, along with its isolation, identification, and applications. The Saprolegnia antagonistic actinomycete QHV2 described in the patent was isolated by the Hunan University of Arts and Sciences' Microbiology Technology Innovation Team from the sediment of a healthy pearl mussel aquaculture system in Changde, Hunan. The strain is currently deposited at the China Center for Type Culture Collection (strain name: Streptomyces montana). Streptomyces collinus , QHV2); deposited at Wuhan University, Wuhan, Hubei Province, China; strain accession number: CCTCC NO: M2016140. The Saprolegnia antagonistic actinomycete QHV2 provided in this patent plays an important role in Saprolegnia control. However, this patent directly utilizes the Saprolegnia antagonistic actinomycete QHV2 for Saprolegnia control. The fermentation product contains harmful substances that contaminate water bodies, limiting its application in Saprolegnia control. Furthermore, this fermentation product has an equally inhibitory effect on beneficial bacteria in aquaculture (such as Bacillus), potentially leading to an imbalance in the beneficial bacteria population. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a polysaccharide polymer for selectively inhibiting harmful bacteria in aquaculture, the application of the polysaccharide polymer and a method for preparing the polysaccharide polymer.

[0005] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0006] A polysaccharide polymer that selectively inhibits harmful bacteria in aquaculture, named QPS, has the following structural formula:

[0007] ;

[0008] Among them, x+y=9, and n is 50-70.

[0009] As a general technical concept, the present invention also provides a method for preparing the above-mentioned polysaccharide polymer for selectively inhibiting harmful bacteria in aquaculture, comprising the following steps:

[0010] (1) Take Saprolegnia antagonistic actinomycetes QHV2 ( Streptomyces collinus , QHV2) was inoculated into liquid culture medium for fermentation. The fermentation broth was precipitated with alcohol and protein was removed by trichloroacetic acid, and then the supernatant was collected by centrifugation.

[0011] (2) dialyzing the supernatant in a 14 KDa dialysis bag, collecting the dialysate and performing vacuum freeze drying to obtain a crude purified sample;

[0012] (3) purifying the crude purified sample by anion exchange column chromatography to obtain neutral polysaccharide;

[0013] (4) The neutral polysaccharide is further purified by gel column chromatography and vacuum freeze-dried to obtain the polysaccharide polymer.

[0014] In the above preparation method, preferably, step (3) includes the following steps: purifying the crude purified sample using a cellulose anion exchange column, with a sample concentration of 1 g / mL, eluting with pure water, controlling the flow rate to 1 mL / min, collecting the eluate at 10 mL / tube, collecting a total of 40 tubes, and combining 1 to 3 tubes to obtain neutral polysaccharides.

[0015] In the above preparation method, preferably, step (4) includes the following steps: further purifying the neutral polysaccharide using a dextran G-150 chromatography column, with a sample concentration of 1 g / mL, using pure water as the mobile phase, controlling the flow rate to 2 mL / min for elution, collecting the eluate at 30 mL / tube, collecting a total of 9 tubes, and combining 1 to 4 tubes to obtain the polysaccharide polymer.

[0016] In the above preparation method, preferably, the liquid culture medium is Gao's liquid culture medium No. 1, and during fermentation culture, the fermentation culture is carried out at 30° C. and 180 rpm for 5 to 7 days.

[0017] In the above preparation method, preferably, when collecting the supernatant by centrifugation, the supernatant is collected after centrifugation at 8500 rpm for 10 to 15 minutes.

[0018] As a general technical concept, the present invention also provides a use of the above-mentioned polysaccharide polymer in the preparation of a product for destroying Saprolegnia biofilm and / or Gill mold biofilm.

[0019] As a general technical concept, the present invention also provides an application of the above-mentioned polysaccharide polymer in the preparation of a product for selectively inhibiting harmful bacteria in aquaculture, wherein the harmful bacteria in aquaculture include Staphylococcus aureus ( Staphylococcus aureus ), water mold and gill mold, and the inhibition rate of the polysaccharide polymer on beneficial bacteria in aquaculture (the polysaccharide polymer has no inhibition or weak inhibition on beneficial bacteria in aquaculture) is lower than the inhibition rate on harmful bacteria in aquaculture, and the beneficial bacteria in aquaculture include Pseudomonas pseudoalcaligenes ( Pseudomonas pseudoalcaligenes ) and Bacillus ( Bacillus ). The present invention purifies a variety of polysaccharides, but not all polysaccharides exhibit the aforementioned selective antibacterial effect. This selective antibacterial effect is a unique property of the aforementioned polysaccharide polymers with specific structures.

[0020] Compared with the prior art, the advantages of the present invention are:

[0021] The present invention is the first to separate and purify a polysaccharide polymer QPS with significant selective inhibition of harmful bacteria in aquaculture from the antagonistic actinomycete strain QHV2 of Saprolegnia. The polysaccharide polymer QPS can effectively inhibit the mycelial growth and spore germination of Saprolegnia / Gill mold and destroy mature biofilms. It has stable activity, no toxic effects on fry, good biosafety, and can be used as a candidate material for the research and development of biological control agents for fish pathogenic fungi. At the same time, the polysaccharide polymer QPS has a clear structure, clear ingredients, high concentration of active ingredients, more precise dosage control, safety and controllability, and more significant antibacterial effect. In addition, the polysaccharide polymer QPS avoids the pollution of water bodies by harmful substances when the fermentation product of QHV2 is directly applied, and can selectively inhibit harmful fungi such as Staphylococcus aureus, Saprolegnia and Gill mold in aquaculture, while having no inhibition or weak inhibition on beneficial fungi such as Pseudomonas pseudoalcaligenes and Bacillus in aquaculture, and has better application effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is the tracking result of the anti-saprolegniatic activity of the sample after purification by Sephadex G-150 column chromatography.

[0024] Figure 2 This is the absorption peak diagram of the UV spectrum scanning of QPS.

[0025] Figure 3 This is the infrared spectrum of QPS.

[0026] Figure 4 is the 1D-NMR spectrum of QPS: where A is 1 H spectrum, B is 13 C spectrum.

[0027] Figure 5 2D-NMR spectrum of QPS: A is HSQC spectrum, B is COSY spectrum, C is HMBC spectrum, D is NOESY spectrum, and E is TOCSY spectrum.

[0028] Figure 6 The results of QPS inhibiting the growth of water mold mycelia, where A1 and A2 are QPS, B1 and B2 are malachite green, A1 and B1 are the effects of different concentrations of the solution on the growth of water mold mycelia, A2 and B2 are the comparisons of the activity of different concentrations of the solution on the growth of water mold mycelia, and different letters in the figure indicate significant differences between the groups ( P <0.05), groups marked with the same letter have no significant difference ( P >0.05).

[0029] Figure 7 The electron microscopic observation results of the effect of QPS on the morphological structure of Saprolegnia mycelium, where the upper row shows the morphological structure of the mycelium in the CK control group, and the lower row shows the morphological structure of the mycelium in the QPS bath group.

[0030] Figure 8 The results of QPS inhibiting Saprolegnia spore germination, where A represents QPS and B represents malachite green (the red arrows indicate the culture wells where no mycelium is visible to the naked eye).

[0031] Figure 9 The effects of different concentrations of QPS on mature Saprolegnia biofilm, where ae represents the difference in biofilm cell viability between different concentration treatment groups at the same time, and different letters indicate significant differences ( P<0.05, the same letters indicate no significant differences ( P >0.05).

[0032] Figure 10 is the inhibitory effect of QPS on the branchial mold pathogen GCM19, where A is the result of the confrontation growth test between QPS and branchial mold, and B is the comparison of the inhibitory activity of QPS on branchial mold. a and b represent the significant difference in the inhibition rate of the confrontation growth of QPS and branchial mold at different times. Different letters indicate significant differences ( P <0.05, the same letters indicate no significant differences ( P >0.05).

[0033] Figure 11 Figure 3 shows the effect of different QPS immersion times and immersion times on the resistance of Xiangyun crucian carp eggs to Saprolegnia infection. A represents a single immersion of Xiangyun crucian carp eggs at the 24th hour of hatching, and B represents a double immersion of Xiangyun crucian carp eggs at the 24th and 48th hours of hatching. The capital letters (A, B) on the broken line in the figure represent the significant differences in the emergence rate of Xiangyun crucian carp among different QPS immersion treatment times. The lowercase letters (a, b) on the columns in the figure represent the significant differences in the Saprolegnia infection rate of Xiangyun crucian carp eggs among different QPS immersion treatment times. The same letters indicate no significant differences. P >0.05, different letters indicate significant differences, P <0.05.

[0034] Figure 12 These are the results of the toxicity test of different concentrations of QPS on Xiangyun crucian carp fry. DETAILED DESCRIPTION

[0035] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0036] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0037] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0038] α-D-Glcp (α-D-glucose) is a stereoisomer of glucose. It serves as the basic structural unit in polysaccharide polymers and is connected to other monosaccharide molecules through glycosidic bonds to form a complex structure.

[0039] Example:

[0040] A polysaccharide polymer that selectively inhibits harmful bacteria in aquaculture, with the following structural formula:

[0041] ;

[0042] Among them, x+y=9, and n is 50-70.

[0043] The polysaccharide polymer of this embodiment can be prepared by the following method:

[0044] (1) Determine the test strain and target strain

[0045] Test strain: Saprolegnia antagonistic actinomycetes QHV2 was isolated by the Microbial Technology Innovation Team of Hunan University of Arts and Sciences from the bottom mud of the healthy pearl oyster aquaculture water body in Changde, Hunan.

[0046] Target strain: Saprolegnia pathogen JM19 ( Saprolegnia JM19), branchial mold pathogen GCM19 ( Branchiomyces GCM19) were isolated from the body surface of crucian carp infected with saprolegniasis and the gill filaments of grass carp infected with gill mold disease, and preserved in the Hunan Key Laboratory of Molecular Immunology of Important Aquatic Animal Diseases of Hunan University of Arts and Science.

[0047] (2) Preparation of culture medium

[0048] Gao's medium No. 1 (g / L): soluble starch 20, KNO31, NaCl 0.5, K2HPO40.5, MgSO4·7H2O0.5, FeSO4·7H2O 0.01, agar powder 20 (added when making solid culture medium); potato dextrose agar synthetic medium (PDA, g / L): potato extract powder 12, glucose 20, agar 14; Sabouraud dextrose liquid synthetic medium (SDB, g / L): a mixture of equal parts of animal tissue pepsin hydrolysate and trypsin casein 10, glucose 20; potato dextrose hydrate synthetic medium (PDW, g / L): potato extract powder 6, glucose 20.

[0049] (3) Fermentation production and purification of active substances from QHV2 strain

[0050] A single QHV2 colony was inoculated into Gao's No. 1 liquid medium and cultured at 30°C and 180 rpm for 7 days. The culture was then purified in the following steps:

[0051] a) Ethanol precipitation: add anhydrous ethanol to the fermentation broth to make the alcohol content reach 80% (v / v), incubate at 4°C for 24 h to precipitate polysaccharides, centrifuge at 8000 rpm for 20 min, and collect the precipitate;

[0052] b) Trichloroacetic acid deproteinization: Dissolve the precipitate completely in ultrapure water, add trichloroacetic acid to a final concentration of 10% (v / v), shake at 180 rpm for 4 hours, and centrifuge at 8000 rpm for 30 minutes to remove the protein precipitate. Retain the supernatant and adjust the pH to approximately neutral with 4 M NaOH solution.

[0053] c) Dialysis: The supernatant was dialyzed through a 14 kDa cut-off dialysis bag at room temperature for 48 h to remove small molecules and oligosaccharides. The retained dialysate was collected and freeze-dried under vacuum to obtain crude polysaccharides.

[0054] d) Anion Exchange Column Chromatography: The crude polysaccharide sample was purified using a cellulose anion exchange column (5 cm × 50 cm, 10 cm height). A 1 g / mL solution was prepared from 4 g of crude polysaccharide sample and loaded onto the column. The solution was eluted with pure water at a controlled flow rate of 1 mL / min, and the eluate was collected at a rate of 10 mL per tube, for a total of 40 tubes. Using Saprolegnia sp. JM19 as the target strain, the anti-Saprolegnia activity of the eluate from each tube was tracked using the agar-pipette stand-off growth method. Neutral polysaccharides with anti-Saprolegnia activity were obtained (the eluates from tubes 1-3).

[0055] e) Gel column chromatography: The neutral polysaccharide sample obtained in step d) was further purified using a Sephadex G-150 column (2.4 cm × 100 cm, 50 cm height). The sample concentration was 1 g / mL, and elution was performed using pure water as the mobile phase at a controlled flow rate of 2 mL / min. The eluate was collected in 9 tubes at a volume of 30 mL per tube. Using Saprolegnia japonica JM19 as the target strain, the agar-pilot standoff growth method was used to track the anti-Saprolegnia active components (e.g., Figure 1 As shown, the eluates from tubes 1 to 4 showed anti-saprolegnia activity, and the eluates from tubes 1 to 4 were combined and freeze-dried in vacuo to obtain a purified polysaccharide polymer sample, which was named QPS.

[0056] Of course, the polysaccharide polymer QPS in this embodiment can also be prepared by other conventional chemical synthesis methods.

[0057] The structure of the polysaccharide polymer QPS was characterized by UV spectroscopy, infrared spectroscopy, and nuclear magnetic resonance analysis, as follows:

[0058] 1. UV spectral scanning analysis: Weigh 1.0 mg of QPS sample to prepare a 1 mg / mL QPS sample solution. Use dH2O as a blank control. Detect the absorbance of the sample solution at wavelengths of 185-400 nm. Determine the type of QPS compound based on the absorption peak at a specific wavelength.

[0059] 2. Infrared spectroscopy analysis: 2.0 mg of QPS sample and 200 mg of potassium bromide were accurately weighed and dried in an oven to constant weight. The samples were then crushed with potassium bromide powder in a mortar. Fourier transform infrared spectrometer (Nicolet is10, ThermoFisher Scientific, MA, USA) was used to analyze the samples at 4000–500 cm -1 The FTIR spectrum of QPS was obtained by scanning within the range.

[0060] 3. Monosaccharide Composition Determination: The monosaccharide composition of QPS was analyzed using high-performance liquid chromatography (HPLC) derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP). Sample preparation: Accurately weigh 5.0 mg of QPS sample and add 1 mL of 2 mol / L trifluoroacetic acid solution. Hydrolyze at 110°C for 2 h, cool to room temperature, and dry at 60°C for 2 h. Dissolve the sample before analysis. Analyze on an Agilent 1200 liquid chromatograph using a Waters C18 column (4.6 mm × 250 mm, 5 μm). Injection volume: 10 μL, flow rate: 1.0 mL / min, column temperature: 25°C, mobile phase A: 0.1 mol / L KH2PO4 (pH 6.8), mobile phase B: acetonitrile, detection wavelength: 245 nm.

[0061] 4. Methylation Analysis: After methylation, acid hydrolysis, and acetylation, QPS was analyzed by GC-MS to determine the glycosidic linkage type. The GC-MS analysis was performed using an Agilent Technologies Inc. (CA, UAS) 7890A-5977B gas chromatograph (GC-MS). The GC system utilized an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA). The carrier gas was high-purity helium (≥99.999%) at a flow rate of 1.0 mL / min and an inlet temperature of 260°C. The injection volume was 1 μL, and a split injection method with a split ratio of 10:1 and a solvent delay of 2.2 min was used. The mass spectrometer used was an Agilent 5977B quadrupole mass spectrometer (Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. The EI source was operated at an inlet temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The scan mode was full scan (SCAN), with a mass scan range (m / z) of 30 to 600.

[0062] 5. NMR: The lyophilized QPS sample was dissolved in 0.5 mL of D2O and measured using a 400 MHz Bruker NMR spectrometer for one-dimensional (1H)-NMR and (13)-C-NMR, as well as two-dimensional (2D) NMR COSY, HSQC, HMBC, NOESY, and TOCSY. Calibration: HDO hydrogen δH = δ 4.70 ppm, TMS carbon δC = δ 0.00 ppm. Deuterated water (D2O), spectral grade, Shanghai Aladdin Biochemical Technology Co., Ltd.; tetramethylsilane (TMS), ≥99.5%, Sigma. Instrument: 400 MHz Bruker NMR spectrometer, Bruker, Germany.

[0063] QPS UV spectrum scanning results are shown in Figure 2 , QPS has an obvious absorption peak at 190 nm, but no absorption peak at wavelengths of 260 nm and 280 nm, indicating that QPS is a polysaccharide compound that does not contain macromolecular substances such as proteins and nucleic acids.

[0064] QPS infrared spectrum scanning results are shown in Figure 3 The infrared spectrum shows the typical characteristics of polysaccharides. It can be seen from the figure that at 3404 cm -1 The broad and strong absorption peak at 2928 cm is the stretching vibration of OH in sugars. -1The smaller absorption peak at 1400~1200 cm is the stretching vibration of methyl or methylene CH. -1 The peaks between 1648 cm and 1648 cm should be the CH vibration of sugars. From the above, it can be determined that the sample is a polysaccharide. -1 1153 cm -OH bending vibration absorption peak, which is the characteristic absorption peak of polysaccharide molecules combined with water. -1 、1080 cm -1 、1027 cm -1 The three absorption peaks indicate that the polysaccharide sample contains pyranose rings. The above infrared test results can confirm that the polysaccharide sample contains neutral pyranose.

[0065] The results of the QPS monosaccharide composition are shown in Table 1. QPS is composed of three monosaccharide components: mannose, glucosamine, and glucose. Glucose has the highest content, accounting for 93.37%, followed by mannose and glucosamine, accounting for 4.75% and 1.87%, respectively (the two are interfering impurities).

[0066] Table 1: QPS monosaccharide composition determination results

[0067]

[0068] The results of QPS methylation analysis are shown in Table 2. After QPS was methylated and acetylated, the names of its derivatives showed that the glycosidic bond types of QPS included terminal glucose not involved in the connection, 1,4-glycosidic bond, 1,6-glycosidic bond, and 1,4- and 1,6-glycosidic bonds. The relative molar percentages showed that QPS was mainly connected by 1,4-glycosidic bond, with 1,6-glycosidic bond being less common, and branched structures connected by 1,4- and 1,6-glycosidic bonds existed.

[0069] Table 2: QPS sugar bond structure analysis

[0070]

[0071] The results of polysaccharide methylation analysis showed that the molar ratio of sugar residues 1,4-Glcp (65.566%) and 1,4,6-Glcp (4.488%) was approximately 11:1, indicating that the polysaccharide sample was a polysaccharide with →4)-α-D-Glcp-(1→ as the main chain, a side chain attached to the O-6 position of →4)-α-D-Glcp-(1→, and a molar ratio of 1,4,6-Glcp (4.488%) and 1,6-Glcp (2.946%) of approximately 1:1.

[0072] The monosaccharide composition of the polysaccharide samples, the polysaccharide methylation analysis results and the one-dimensional ( Figure 4 ) and 2D NMR ( Figure 5) information analysis, the inferred structure of the polysaccharide sample is a →4)-α-D-Glc p -(1→main chain, in part α-Glc p of O -6 position is connected to the side chain α-D-Glc p -(1→6)-α-D-Glc p -(1→α-D-glucan.

[0073] The polysaccharide polymer QPS of this embodiment has a wide range of applications in inhibiting the growth of Saprolegnia mycelia and spore germination, inhibiting the growth of Gill mold mycelia and spore germination, destroying Saprolegnia biofilm and Gill mold biofilm, and selectively inhibiting harmful bacteria in aquaculture. The specific applications are as follows:

[0074] 1. QPS inhibition of Saprolegnia mycelium growth test

[0075] Using the serial dilution method, QPS was prepared at concentrations of 4.0, 2.0, 1.0, 0.5, 0.25, 0.125, 0.0625, and 0.0313 mg / mL, respectively. 250 μL of each concentration of QPS was evenly applied to the surface of a PDA culture plate. The plate was then placed upright to air-dry. An 8 mm diameter Saprolegnia spp. was then inoculated onto the center of the PDA plate using the agar transfer method. After incubation at 25°C for 36 h, mycelial diameters were measured and recorded using the cross-hatch method, and the inhibition rate was calculated. Three replicates were set up for each concentration. A PDA plate coated with 250 μL of sterile water served as a blank control (CK), and malachite green (drug concentrations of 0.64, 0.32, 0.16, 0.08, 0.04, 0.02, 0.01, and 0.005 mg / mL, respectively) was used as a positive control. Inhibition rate (%) = [(diameter of Saprolegnia mycelia in the blank control group - diameter of Saprolegnia mycelia in the experimental group) / diameter of Saprolegnia mycelia in the blank control group] × 100%. Figure 6 shown.

[0076] Depend on Figure 6 A1 and A2 show that QPS has a significant inhibitory effect on the growth of Saprolegnia mycelium in the range of 0.25-4 mg / mL ( P <0.05), and the inhibitory effect was positively correlated with its concentration. When the concentration was 0.25 mg / mL, the inhibition rate on the growth of Saprolegnia mycelium reached 60.24%, which was significantly lower than that of the positive control malachite green ( Figure 6 Compared with B1 and B2 in the samples, its antibacterial effect was equivalent to that of malachite green at a concentration of 0.020 mg / mL (inhibition rate was 52.78%). When the concentration of QPS was 2 mg / mL, it could completely inhibit the growth of Saprolegnia mycelium ( Figure 6 A1 and A2 in the middle), and the antibacterial effect was equivalent to that of the positive control malachite green 0.080 mg / mL ( Figure 6B1 and B2 in the middle). This shows that QPS has a good effect in inhibiting the growth of Saprolegnia mycelium.

[0077] To further observe the effect of QPS on the mycelial structure of Saprolegnia, an in vitro culture method was used in 24-well plates with SDB culture medium: under sterile conditions, an appropriate amount of Saprolegnia JM19 mycelia was transferred to culture wells containing 0.2 mg / L QPS. Mycelia in culture wells without QPS were used as controls. After culturing at 25°C for 7 days, scanning electron microscopy was used to observe the changes in the mycelial structure of Saprolegnia after the action of QPS. The experimental results are shown in Figure 2. Figure 7 shown.

[0078] Depend on Figure 7 The results showed that after 7 days of continuous QPS bathing, the mycelial morphology of Saprolegnia spp. differed significantly from that of the control group. The mycelial structure of the bathing group was dry, bent, shrunken, and uneven, while that of the CK control group was plump, flat, smooth, and uniform in thickness. This suggests that QPS inhibits mycelial growth by disrupting mycelial structure.

[0079] 2. QPS inhibits the germination of Saprolegnia spores

[0080] Mature Saprolegnia mycelia were obtained by PDA plate culture. After confirming the release of zoospores by microscopic observation, the Saprolegnia block was punctured with a sterile yellow pipette tip and transferred to a 2 mL centrifuge tube containing 1 mL of PDW medium. The centrifuge tube was gently shaken and inverted for 10 min to fully release the Saprolegnia spores. The supernatant was collected and combined to obtain a Saprolegnia spore suspension. The number of spores per milliliter of the suspension was counted using a phytoplankton counting plate, and the concentration was adjusted to 2 × 10 6 ~ 2×10 7 CFU / mL, set aside. Using the 48-well plate microdilution method, QPS was prepared at concentrations of 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0156, and 0.0078 mg / mL, and malachite green was used as a positive control to prepare QPS at concentrations of 1.25×10 -3 , 0.625×10 -3 , 0.32×10 -3 , 0.16×10 -3 , 0.08×10 -3 , 0.04×10 -3 , 0.02×10 -3 , 0.01×10 -3mg / mL. 300 μL of QPS at different concentrations and an equal volume of Saprolegnia spore suspension were added to the wells of the well plate, and the mixture was gently blown and mixed with a pipette. 300 μL of sterile water and an equal volume of Saprolegnia spore suspension were added as the blank control group. Three parallels were set up for each group, and cultured at 25°C for 7 days. The spore germination was observed every day. The concentration corresponding to the culture well with no visible mycelial growth was the minimum inhibitory concentration of the drug for inhibiting the germination of Saprolegnia spores. The test results are shown in Figure 2. Figure 8 and as shown in Table 3 below.

[0081] Depend on Figure 8 As shown in Table 3, when the mass concentration of QPS is 0.125 mg / mL, it can effectively inhibit the germination of Saprolegnia spores. Below this concentration, hyphae formation can be seen with the naked eye. 0.125 mg / mL is the minimum inhibitory concentration (MIC) of QPS for inhibiting spore germination. Compared with the positive control malachite green ( Figure 7 B), which is equivalent to a concentration of 0.32×10 -3 The results showed that QPS has a good inhibitory effect on the germination of Saprolegnia spores.

[0082] Table 3: Minimum inhibitory concentration test results of QPS treatment on Saprolegnia spore germination

[0083]

[0084] 3. QPS destroys mature biofilm of Saprolegnia

[0085] Saprolegnia biofilms were constructed, and mature Saprolegnia biofilms cultured for 72 h were used as test materials. Taking the MIC of QPS for inhibition of Saprolegnia spore germination = 0.125 mg / mL as a reference, QPS was prepared at concentrations of 0.250, 0.125, 0.063, 0.031, and 0.016 mg / mL (i.e., 2 MIC, 1 MIC, 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC), respectively. Under sterile conditions, excess culture medium in a 24-well plate was discarded, and the biofilms were slowly rinsed three times with sterile 1× PBS buffer to remove free hyphae. Treatments were treated with 1 mL of QPS at different concentrations, while the control group was treated with 1 mL of sterile water. Three replicates were set up for each concentration. After incubation at 25°C for 12, 24, 36, and 48 h, the drug solution in the wells was discarded and the biofilms were slowly rinsed three times with sterile 1× PBS buffer to fully remove the drug solution. The CCK-8 assay was used to detect the viability (%) of the biofilm cells. The destructive effects of different concentrations of QPS on the mature Saprolegnia biofilm were compared and calculated as follows:

[0086] ;

[0087] Where: A(0): absorbance of blank culture medium reaction solution; A(1): absorbance of mature biofilm reaction solution treated with QPS; A(2): absorbance of mature biofilm reaction solution not treated with QPS.

[0088] Effects of QPS on mature biofilms of Saprolegnia Figure 9 After the biofilm was treated with QPS at concentrations of 0.031, 0.063, 0.125, and 0.25 mg / mL for 36 h, the cell viability of the Saprolegnia biofilm decreased significantly compared with the control group ( P <0.05), their cell viability decreased by 70.6%, 76.3%, 88.5%, and 83.7%, respectively. After 48 h of QPS treatment of Saprolegnia biofilms, biofilm cell viability further decreased across all treatment groups, reaching an 80% decrease in the 0.031 mg / mL treatment group and approximately 90% decreases in the 0.063, 0.125, and 0.25 mg / mL treatment groups. This suggests that QPS treatment at concentrations above 0.031 mg / mL significantly disrupts mature Saprolegnia biofilms.

[0089] 4. Inhibition of QPS on the growth of gill mold hyphae

[0090] The branchial mold pathogen GCM19 was used as the target bacteria. The agar transfer method was used to inoculate the branchial mold block to the center of the PDA plate. Four sampling points were set at equal intervals about 1 cm away from the center, and 20 μL of QPS (1 mg / mL) was spotted on each point. 20 μL of sterile water was used as the blank control. The plates were placed upright and allowed to air-dry before being inverted and cultured at 25°C. The diameters of the branchial mold hyphae after 24 h and 48 h of confrontation growth were observed and measured, and the inhibition rate was calculated. The experiment was set up in triplicate.

[0091] Inhibition rate (%) = [(diameter of gill mold hyphae in the control group - diameter of gill mold hyphae in the experimental group) / diameter of gill mold hyphae in the control group] × 100%.

[0092] The test results are as follows Figure 10 As shown. Figure 10 It can be seen that QPS has a significant inhibitory effect on the branchial mold pathogen GCM19 ( Figure 10 Middle A). When QPS and Gill mold were grown against each other for 24 h and 48 h, the inhibition rates of Gill mold mycelium growth were 50.56% and 70.58%, respectively, with significant differences between the two ( P <0.05, Figure 10 (B) This indicates that QPS has a stable inhibitory effect on branchial mold pathogens.

[0093] 5. QPS test on sticky fish eggs against water mold infection

[0094] Fertilized eggs of Xiangyun crucian carp (Carassius auratus) that had been evenly adhered to mesh and artificially incubated for 24 hours were used as the test material (produced at the Changde base of Xiangyun Biotechnology Co., Ltd.). According to the experimental design and the dimensions of the suspended incubation box (φ = 6.5 cm, h = 10 cm), 21 small mesh pieces (10 cm × 6 cm) were cut and numbered T0-1 to T0-3, T1-1 to T1-6, T2-1 to T2-6, and T3-1 to T3-6. The total number of eggs and the number of fertilized eggs on the mesh pieces were counted. Groups T1, T2, and T3 were designated as QPS-treated groups. Eggs were treated with a single immersion at 24 hours of incubation and two immersions at 24 and 48 hours of incubation, with immersion times of 20, 40, and 60 minutes, respectively. Group T0 served as the control group (CK) without QPS treatment. Three replicates were performed for each treatment. At the 24th hour of incubation, all the eggs in groups T1, T2, and T3 were immersed in 0.3 mg / mL QPS for the first time. After the immersion, the small meshes were inserted into the incubation box and placed in the incubation tank used in actual production (water temperature 21°C) for further incubation (the experimental site was provided by the Changde base of Xiangyun Biotechnology Co., Ltd.). At the 48th hour of incubation, the small meshes of eggs (T1-4 to T1-6 in group T1, T2-4 to T2-6 in group T2, and T3-4 to T3-6 in group T3) were removed from the incubation tank and placed in 0.3 mg / mL QPS for the second immersion. After the immersion, the small meshes were transferred to the incubation tank for further incubation. After 60 hours of incubation, wrap the hatching box with a permeable cloth bag with an internal spring. Continue incubation for 1-2 days until all the fry have hatched. On the fifth day of incubation, count the number of eggs infected with Saprolegniae and the number of fry in each hatching box. Calculate the Saprolegniae infection rate and fry emergence rate using the following formula:

[0095] Fish egg Saprolegnia infection rate (%) = number of fish eggs infected with Saprolegnia on the small mesh / total number of fish eggs on the mesh × 100%;

[0096] Fry emergence rate (%) = number of fry hatched in the hatching box / number of fertilized eggs on the small mesh in the box × 100%.

[0097] The sticky eggs of Xiangyun crucian carp were used as the test materials, and the infection rate of Saprolegnia and fry emergence rate were used as the investigation indicators. The effects of different bathing times and bathing times of QPS at a concentration of 0.3 mg / mL on the resistance of sticky eggs of Xiangyun crucian carp to Saprolegnia infection were shown. Figure 11 .

[0098] like Figure 11As shown in the results, different soaking times and soaking times of QPS significantly affected the resistance of Xiangyun crucian carp sticky fish eggs to Saprolegnia infection. Compared with the control group (CK), the infection rate of Saprolegnia in the experimental groups was significantly reduced, whether the eggs were soaked once only 24 hours after hatching or twice 24 and 48 hours after hatching ( P <0.05, but there was no significant difference in the infection rate of Saprolegnia among the treatment groups ( P >0.05). In a single immersion treatment ( Figure 11 Middle A), with the extension of soaking time (20, 40, 60 min), the fry emergence rate showed a slight upward trend, but the difference between the groups was not significant ( P >0.05). In the two immersion treatments ( Figure 11 The germination rates of the 20 min and 40 min treatment groups were significantly higher than those of the control group ( P <0.05), the germination rate of the 20 min treatment group was the highest, reaching an average of 87.57%, which was 30.15% higher than that of the control group (57.42%). The germination rate of the 60 min treatment group (50.53%) was not significantly different from that of the control group ( P The above results showed that two QPS immersion treatments (immersion time 20 min) at 24 h and 48 h after hatching of Xiangyun crucian carp sticky fish eggs could effectively reduce the infection rate of Saprolegnia and significantly increase the fry emergence rate.

[0099] 6. Biosafety testing of QPS

[0100] Xiangyun crucian carp fry (swim bladder inflated) 3 days after artificial hatching were used as test subjects. Fifteen sterile plastic Petri dishes (φ = 60 mm, h = 10 mm) were divided into five groups. Pure water and QPS were added to a 9 mL immersion system at concentrations of 1.0, 0.5, 0.25, and 0.03 mg / mL, respectively. A Petri dish without QPS served as a blank control (CK). Three replicates were set up for each group. Ten actively swimming fry were randomly placed in each Petri dish and observed for 7 consecutive days without feeding or water changes. The activity and survival of the fry were observed and recorded daily. The final survival rate was calculated using the following formula:

[0101] Survival rate (%) = (number of surviving fry / 10) × 100%.

[0102] The results of the challenge test on Xiangyun crucian carp fry with different concentrations of QPS were shown in the table. Figure 12 .like Figure 12As shown in the QPS-induced immersion test, fry in the control group (CK) exhibited sluggish swimming and mortality starting on day 5. Mortality rates increased dramatically between days 5 and 7, ultimately leading to complete death. This is likely due to depletion of the yolk sac and starvation in the absence of food. Fry survival in the 0.03 and 0.25 mg / mL QPS-treated groups generally mirrored the control group, but the survival rate on day 5 in both groups was 85%, higher than the 65% survival rate of the CK group. Notably, the survival rate of fry in the 1 mg / mL QPS-treated group remained high at 90% on day 7, and surviving individuals displayed normal swimming activity. This is likely due to the high concentration of QPS active polysaccharides providing energy to the fry. In summary, QPS is nontoxic to fry in the 0.03–1 mg / mL concentration range, demonstrating good biosafety.

[0103] 7. Comparison of the antibacterial effects of actinomycete QHV2 fermentation broth and QPS

[0104] In order to verify the selective antibacterial effect of QPS, the following tests were conducted on common harmful bacteria in aquaculture (Staphylococcus aureus ATCC29213) and common beneficial bacteria in aquaculture (Bacillus HUAS2407 and Pseudomonas pseudoalcaligenes HUAS2406):

[0105] (1) Preparation of bacterial suspension

[0106] Take four 12 mL bacterial culture tubes and add 3 mL of LB liquid medium to each. Pick a single colony from the solid culture medium of Staphylococcus aureus, Bacillus, and Pseudomonas pseudoalcaligenes and add it to the liquid medium. Use one tube as a blank control. Incubate the tubes in a constant temperature shaker at 37°C, 200 rpm, and shake overnight for 15 hours.

[0107] Appropriate amounts of freeze-dried samples of the fermentation supernatant of actinomycetes QHV2 and QPS samples were weighed and placed in 12 mL culture tubes to a working concentration of 50 mg / mL. Sterilize under ultraviolet light for 30 min and set aside.

[0108] (2) Inhibition zone test

[0109] Use sterile PBS solution to dilute the three strains to 10 6 CFU / mL, then evenly spread 100 μL onto LB solid medium. Use a 10 mm borer to punch holes, add 100 μL of the sterilized sample to each well, and incubate the culture dish in a constant temperature incubator at 37°C for 24 hours. After incubation, remove the dish, photograph it with a standard camera, and measure and record the size of the inhibition zone.

[0110] (3) Experimental results

[0111] As shown in Table 4, the actinomycete QHV2 fermentation broth exhibited significant antibacterial activity against Staphylococcus aureus, Bacillus, and Pseudomonas pseudoalcaligenes. QPS also exhibited more pronounced antibacterial activity against Staphylococcus aureus. However, QPS had no inhibitory effect on Pseudomonas pseudoalcaligenes, a dominant bacterial species in the water, and only weakly inhibited the beneficial Bacillus. Compared to the actinomycete QHV2 fermentation broth, QPS exhibited significant selective antibacterial activity, enabling selective inhibition of harmful bacteria in aquaculture without the risk of water pollution associated with direct use of the QHV2 fermentation broth. These results demonstrate that, compared to QHV2 fermentation broth, the use of QPS to control water molds avoids the inhibition of beneficial and dominant bacteria in the water, making it safer for aquaculture ecosystems.

[0112] Table 4: Determination of inhibition zone size of QHV2 fermentation broth and QPS

[0113]

Claims

1. A method for preparing a polysaccharide polymer for selectively inhibiting harmful bacteria in aquaculture, characterized in that: The following steps are involved: (1) Take Saprolegnia antagonistic actinomycetes QHV2 ( Streptomyces collinus , QHV2) were inoculated into liquid culture medium for fermentation. The fermentation broth was precipitated with alcohol and protein was removed by trichloroacetic acid, and then the supernatant was collected by centrifugation. (2) dialyzing the supernatant in a 14 KDa dialysis bag, collecting the dialysate and performing vacuum freeze drying to obtain a crude purified sample; (3) purifying the crude purified sample by anion exchange column chromatography to obtain neutral polysaccharide; (4) further purifying the neutral polysaccharide by gel column chromatography, and vacuum freeze-drying to obtain the polysaccharide polymer; Step (3) comprises the following steps: purifying the crude purified sample using a cellulose anion exchange column, loading the sample at a concentration of 1 g / mL, eluting with pure water, controlling the flow rate to be 1 mL / min, collecting the eluate at 10 mL / tube, collecting a total of 40 tubes, and combining 1 to 3 tubes to obtain a neutral polysaccharide; Step (4) comprises the following steps: further purifying the neutral polysaccharide using a dextran G-150 chromatography column, with a loading concentration of 1 g / mL, using pure water as the mobile phase, controlling the flow rate to 2 mL / min for elution, collecting the eluate at 30 mL / tube, collecting a total of 9 tubes, and combining tubes 1 to 4 to obtain the polysaccharide polymer.

2. The preparation method according to claim 1, characterized in that The liquid culture medium is Gao's No. 1 liquid culture medium. During fermentation, the culture is carried out at 30° C. and 180 rpm for 5 to 7 days.

3. The preparation method according to claim 1, characterized in that When collecting the supernatant by centrifugation, centrifuge at 8500 rpm for 10 to 15 minutes and collect the supernatant.

4. Use of a polysaccharide polymer prepared by the method according to any one of claims 1 to 3 in preparing a product for destroying Saprolegnia biofilm.

5. Use of a polysaccharide polymer prepared by the method according to any one of claims 1 to 3 in preparing a product for selectively inhibiting harmful bacteria in aquaculture, characterized in that: The harmful bacteria in aquaculture include Staphylococcus aureus, Saprolegnia and Gill mold, and the inhibition rate of the polysaccharide polymer on beneficial bacteria in aquaculture is lower than the inhibition rate on harmful bacteria in aquaculture. The beneficial bacteria in aquaculture include at least one of Pseudomonas pseudoalcaligenes and Bacillus.

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