Composition for inhibiting saprolegniasis and application thereof

Abstract

The invention discloses a composition for inhibiting saprolegniasis. The composition comprises gallnut, tannic acid and lactic acid bacteria. Wherein gallnut and tannic acid have a broad-spectrum inhibition effect on various pathogenic microorganisms, so that the defect that the action range of lactic acid bacteria is limited is overcome. This combination can cope with more complex saprolegniasis and other potential pathogens. In addition, tannic acid has lasting water solubility and stability, active ingredients can be slowly released in water, and the lasting time of the inhibition effect is prolonged. Compared with gallnut and tannic acid, the lactic acid bacteria show slower pharmacodynamics in the aspect of water mildew inhibition. The lactic acid bacteria and the Chinese herbal medicines are combined to generate a synergistic effect, instant inhibition is realized through quick-acting components of the Chinese herbal medicines, and meanwhile, the action duration is prolonged by virtue of the slow-release characteristic of the lactic acid bacteria, so that a quick-acting-slow-release dual-action mechanism is constructed, and the continuous inhibition effect on the saprolegnia is remarkably improved.

Description

Technical Field

[0001] The present invention relates to aquaculture, in particular to an antibacterial agent for aquaculture, and specifically to a composition for inhibiting saprolegniasis. Background Art

[0002] Saprolegniasis is common in freshwater bodies where the water temperature is below 18°C for a long time in winter and spring; it is one of the common diseases of special economic fish. Since malachite green was banned at the beginning of the 21st century, many freshwater cultured fish worldwide have generally been suffering from long-term infection by pathogenic Saprolegnia, resulting in large-scale death of freshwater fish and their eggs and causing significant economic losses. The water temperature in Heyuan area is low in winter, and saprolegniasis is one of the important factors causing losses in aquaculture in Heyuan area. The outbreak of saprolegniasis results in an average loss of 10% of fish eggs and fry, and even some losses reach 50%.

[0003] Saprolegnia is a conditional pathogen; the occurrence of saprolegniasis usually requires low water temperature (especially in winter and spring), deteriorated water quality, imbalance of microbial flora, too high breeding density, physical damage on the fish body surface, etc. The infection of Saprolegnia generally occurs on the body surface of aquatic animals or the surface of their eggs. Aquatic animals living in freshwater environments are easily infected with saprolegniasis. The infected fish include mandarin fish, eel, carp, tilapia, bass, catfish, salmon and trout, etc. In freshwater aquaculture, the main pathogenic bacteria of saprolegniasis are Saprolegnia parasitica and Saprolegnia diclina. Saprolegnia parasitica mostly infects fish, while Saprolegnia diclina is mostly found in infected fish eggs. Saprolegnia belongs to oomycetes. Although oomycetes have many similarities with fungi, it is not a true fungus and belongs to Stramenopiles, Heterokonts. The symptoms of saprolegniasis usually show white or gray cottony mycelia attached to the fish body surface, fish fins or fish eggs ( Figure 1 ). The causes of death of fish and fish eggs infected with Saprolegnia are different. For fish, the growth of a large number of invasive Saprolegnia mycelia destroys the structure of the epidermis and thus disrupts the osmotic balance of the fish; for fish eggs, the Saprolegnia mycelia rupture the chorion and lead to osmotic imbalance. Saprolegnia can produce flagellated zoospores to spread in the water environment; Saprolegnia can also produce secondary zoospores and secondary cysts, and some secondary zoospores and secondary cysts contain boathook structures, which may help Saprolegnia attach to the fish skin or maintain buoyancy.

[0004] Before 2002, the effective prevention and control of Saprolegnia mainly relied on malachite green, a carcinogen that has been banned. Due to its problems such as being difficult to degrade, teratogenicity, and carcinogenicity, it has been prohibited. Therefore, it is urgent to develop new preventive drugs. Nowadays, the methods for controlling Saprolegnia in aquaculture mainly include using formalin, bronopol, seawater, etc., or manually removing individuals infected with Saprolegnia. However, the efficacy of these methods is not high. Experts studying Saprolegnia also predict that the current widespread use of formalin will be banned, which will exacerbate the harm of Saprolegnia. Currently, there is no vaccine available for the prevention and control of Saprolegnia. Therefore, the aquaculture industry urgently needs environmentally friendly, inexpensive, and highly active anti-Saprolegnia drugs to ensure the sustainable development of freshwater aquaculture and guarantee its economic benefits. Summary of the Invention

[0005] The object of the present invention is to provide a composition for inhibiting Saprolegnia.

[0006] The present invention provides the following technical solution: A composition for inhibiting Saprolegnia, the components of which include Chinese gallnut, tannic acid, and lactic acid bacteria.

[0007] In one embodiment, the ratio among Chinese gallnut, tannic acid, and lactic acid bacteria is: 5 - 30﹕20 - 150﹕50 - 1500.

[0008] Preferably, the ratio among Chinese gallnut, tannic acid, and lactic acid bacteria is: Chinese gallnut﹕tannic acid﹕lactic acid bacteria = 5 - 30﹕20 - 60﹕50 - 100.

[0009] Preferably, Chinese gallnut﹕tannic acid﹕lactic acid bacteria = 10﹕20﹕100; Chinese gallnut﹕tannic acid﹕lactic acid bacteria = 10﹕40﹕75; Chinese gallnut﹕tannic acid﹕lactic acid bacteria = 10﹕60﹕50.

[0010] The Chinese gallnut used is Chinese gallnut powder. The Chinese gallnut powder can be the powder of Chinese gallnut after being crushed without sieving, or the powder of Chinese gallnut after being crushed and sieved, and the powder with a mesh size of 60 - 240 is taken. Preferably, the Chinese gallnut powder is the powder with a mesh size of 120 - 240.

[0011] Preferably, the concentration of tannic acid is 75%.

[0012] Preferably, the lactic acid bacteria is Lactiplantibacillus plantarum L75a. The present invention also provides a preparation method of the above composition for inhibiting Saprolegnia.

[0013] The present invention provides the following technical solution: A preparation method of a composition for inhibiting Saprolegnia, which is obtained by crushing Chinese gallnut and then mixing it with tannic acid and lactic acid bacteria.

[0014] The present invention also provides the application of the above composition for inhibiting Saprolegnia.

[0015] Specifically, it relates to the application of the composition for inhibiting saprolegniasis as an aquaculture additive; and it relates to the application of the composition for inhibiting saprolegniasis as a component of aquaculture feed.

[0016] The present invention also provides an aquaculture feed.

[0017] Specifically, the aquaculture feed includes the composition for inhibiting saprolegniasis as its component.

[0018] Beneficial effects

[0019] 1. Significantly enhance the effect of inhibiting saprolegniasis: The concentration of gallnut for completely inhibiting the growth of saprolegnia is about 30 ppm, the concentration of tannic acid for completely inhibiting the growth of saprolegnia is about 100 ppm, and the concentration of Lactobacillus plantarum L75a for completely inhibiting the growth of saprolegnia is about 1500 ppm.

[0020] 2. Broad-spectrum antibacterial effect: Gallnut and tannic acid have a broad-spectrum inhibitory effect on a variety of pathogenic microorganisms (including bacteria, fungi and parasites), making up for the limitation of the action range of lactic acid bacteria. This combination can cope with more complex saprolegniasis infections and other potential pathogens.

[0021] 3. Synergistic effect and extended action time: Tannic acid has persistent water solubility and stability, and can slowly release active ingredients in water, extending the duration of the inhibitory effect. Lactobacillus plantarum L75a shows a slower pharmacokinetics in inhibiting saprolegnia compared with gallnut and tannic acid. The combination of lactic acid bacteria and Chinese herbal medicines produces a synergistic effect, achieving immediate inhibition through the quick-acting ingredients of Chinese herbal medicines, and at the same time extending the action duration by means of the slow-release characteristics of lactic acid bacteria, thus constructing a dual mechanism of quick-acting and slow-release, significantly enhancing the continuous inhibitory effect on saprolegnia.

[0022] 4. Reduce the risk of drug resistance: Using gallnut, tannic acid, and lactic acid bacteria alone may increase the risk of saprolegnia developing resistance, while combined use can reduce this risk.

[0023] 5. Reduce ecological interference: Gallnut and tannic acid are natural plant extracts, with less interference to the water ecosystem. Combined use can reduce the dosage of lactic acid bacteria and avoid the negative impact of excessive use of lactic acid bacteria on the water microbial community.

[0024] 6. Reduce costs: The use of gallnut and tannic acid can enhance the overall inhibitory effect. While tannic acid is relatively inexpensive, reducing the usage amount of gallnut, thus reducing the overall cost. Moreover, gallnut, tannic acid, and Lactobacillus plantarum L75a have high efficiency in inhibiting saprolegnia, resulting in a low dosage concentration for achieving the minimum inhibition, low usage amount, and low cost.

[0025] 7. Improved adaptability: Galla chinensis and tannic acid have good stability in various water environments (such as high-salinity, low-pH, or severely polluted water bodies), compensating for the insufficient effects of lactic acid bacteria in certain environments. This combination is applicable to a wider range of water conditions.

[0026] 8. Higher safety: Galla chinensis and tannic acid are natural ingredients with low toxicity to aquatic organisms and the environment, and relatively high safety.

[0027] 9. Promote water body health: Galla chinensis and tannic acid have certain astringent and purifying effects, which can help improve water quality and reduce organic pollution. Lactic acid bacteria can promote the growth of beneficial microorganisms, and the combined use helps maintain the ecological balance of the water body.

[0028] 10. Anti-inflammatory and repair effects: Tannic acid has anti-inflammatory and antioxidant effects, which can reduce the tissue damage caused by Saprolegnia infection to aquatic organisms. This combination not only inhibits Saprolegnia but also promotes the recovery of aquatic organisms.

[0029] 11. Simple production process: The Chinese herbal medicine Galla chinensis only needs to be crushed, and lactic acid bacteria are directly expanded and cultured. Description of the drawings

[0030] Figure 1 Shows the symptoms of Saprolegnia disease on the body surface, fins or eggs of fish.

[0031] Figure 2 Shows the effect of Lactobacillus plantarum L75a on the mycelial growth of Saprolegnia F26;

[0032] In Figure (a), the vertical axis of the Saprolegnia inhibition rate is the quotient obtained by dividing the numerical difference between the mycelial radius of the Saprolegnia mold cake in the experimental group minus the mycelial radius of the mold cake in the blank control group by the mycelial radius of the mold cake in the blank control group, that is, (mycelial radius of the Saprolegnia mold cake in the experimental group - mycelial radius of the Saprolegnia mold cake in the blank group) / mycelial radius of the Saprolegnia mold cake in the blank group × 100%. The P value of each column represents the significant difference in the F26 colony diameter compared with the corresponding 24h or 48h blank control group.

[0033] Figure 3 Shows the effect of the fermentation broth, bacterial suspension and cell-free fermentation broth of Lactobacillus plantarum L75a on the mycelial growth of F26;

[0034] Note: The vertical axis of the colony diameter in Figure (a) is the numerical value after subtracting the diameter of the mold cake. * indicates a significant difference (P < 0.05) in the F26 colony diameter compared with the corresponding blank control group (C), and ** indicates a highly significant difference (P < 0.01).

[0035] Figure 4 Shows the 10 metabolites with the highest abundance detected in the fermentation broth of Lactobacillus plantarum L75a.

[0036] Figure 5Effect of four main metabolites of L75a on the mycelial growth of F26;

[0037] In Figures (a, b), the vertical axis of the colony diameter is the value after subtracting the diameter of the agar plug. Different letters indicate significant differences in the F26 colony diameter among different concentrations of the same metabolite (P<0.05). * indicates a significant difference in the F26 colony diameter compared with the corresponding blank control group (C) (P<0.05), and ** indicates a highly significant difference (P<0.01). Blue * indicates a growth-promoting effect compared with the blank control, and red * indicates a growth-inhibiting effect compared with the blank control.

[0038] Figure 6 Growth status of F26 fungal blocks without mycelial growth after being taken out and cultured for several days in the antibacterial experiment of L75a metabolites;

[0039] Culturing was stopped after the growth of Saprolegnia F26 colonies appeared.

[0040] Figure 7-1 Effect of different Chinese herbal medicines on the mycelial growth of Saprolegnia F26;

[0041] The vertical axis of the Saprolegnia inhibition rate is the quotient obtained by dividing the difference in the mycelial radius of the Saprolegnia fungal disc in the experimental group minus the mycelial radius of the fungal disc in the blank control group by the mycelial radius of the fungal disc in the blank control group, that is, (mycelial radius of the Saprolegnia fungal disc in the experimental group - mycelial radius of the Saprolegnia fungal disc in the blank group) / mycelial radius of the Saprolegnia fungal disc in the blank group × 100%. The P value of each column represents the significant difference in the F26 colony diameter compared with the corresponding 24h or 48h blank control group.

[0042] Figures 7-2 to 7-4 Effect of different Chinese herbal medicines on the mycelial growth of Saprolegnia F26.

[0043] Figure 8 Effect of Chinese gallnut on the mycelial growth of Saprolegnia F26

[0044] Note: In Figure (a), the vertical axis of the Saprolegnia inhibition rate is the quotient obtained by dividing the difference in the mycelial radius of the Saprolegnia fungal disc in the experimental group minus the mycelial radius of the fungal disc in the blank control group by the mycelial radius of the fungal disc in the blank control group, that is, (mycelial radius of the Saprolegnia fungal disc in the experimental group - mycelial radius of the Saprolegnia fungal disc in the blank group) / mycelial radius of the Saprolegnia fungal disc in the blank group × 100%. The P value of each column represents the significant difference in the F26 colony diameter compared with the corresponding 24h or 48h blank control group.

[0045] Figure 9 Inhibition of Saprolegnia by 10ppm Chinese gallnut with different mesh numbers.

[0046] Figure 10 Effect of tannic acid on the mycelial growth of Saprolegnia F26

[0047] Note: The vertical axis value of the inhibition rate of Saprolegnia in Figure (a) is the quotient obtained by dividing the numerical difference between the hyphal radius of the Saprolegnia cake in the experimental group minus the hyphal radius of the Saprolegnia cake in the blank control group by the hyphal radius of the Saprolegnia cake in the blank control group, that is, (hyphal radius of the Saprolegnia cake in the experimental group - hyphal radius of the Saprolegnia cake in the blank group) / hyphal radius of the Saprolegnia cake in the blank group × 100%. The P value of each column represents the significant difference in the colony diameter of F26 compared with the blank control group at the corresponding 24h or 48h.

[0048] Figure 11 For the hatching of colored carp fertilized eggs under different concentrations of Chinese gallnut (240 mesh)

[0049] Figure 12 For the α-diversity indices (Chao Index, Shannon Index, Ace Index, and Simpson Index) of each sample

[0050] Note: Chao Index and Ace Index are mainly used to represent the richness of bacterial species; Shannon Index and Simpson Index are used to represent the diversity of bacterial communities. * indicates a significant difference in the bacterial community indices corresponding to different concentrations of Chinese gallnut (P < 0.05).

[0051] Figure 13 For the bacterial abundance distribution of each sample at the phylum and class levels.

[0052] Figure 14 For the bacterial abundance distribution of each sample at the order and family levels.

[0053] Figure 15 For the bacterial abundance distribution of each sample at the genus and species levels.

[0054] Figure 16 For the fungal abundance distribution of each sample at the phylum and class levels.

[0055] Figure 17 For the fungal abundance distribution of each sample at the order and family levels.

[0056] Figure 18 For the fungal abundance distribution of each sample at the genus and species levels.

[0057] Figures 19-1 to 19-3 For the effects of compound combinations and their individual components on the mycelial growth of Saprolegnia F26. Detailed implementation methods

[0058] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the following will fully describe the technical solutions in the embodiments of this application in combination with the accompanying drawings in the embodiments of this application to fully understand the objectives, effects, and application prospects of the present invention. The following embodiments are only used to clarify the present invention and are not used to limit the scope of application of the present invention.

[0059] Unless otherwise specified, the test methods used in the following embodiments are carried out according to conventional methods or the conditions recommended by the manufacturer; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0060] 1. Preparation of Chinese gallnut (240 mesh): After the Chinese gallnut (purchased from Kangmei Pharmaceutical Co., Ltd.) was pulverized by a pulverizer, there was an uneven particle size. It was speculated that there were differences in the inhibitory effects of Chinese gallnuts with different mesh numbers on Saprolegnia at the same concentration, and experiments were conducted on this. Chinese gallnuts with different mesh numbers were obtained by screening through different mesh sieves. Among them, the Chinese gallnut that passed through 60 mesh but could not pass through 80 mesh was defined as 60-mesh Chinese gallnut; the Chinese gallnut that passed through 120 mesh but could not pass through 140 mesh was defined as 120-mesh Chinese gallnut; the Chinese gallnut that passed through 180 mesh but could not pass through 200 mesh was defined as 180-mesh Chinese gallnut; the Chinese gallnut that passed through 240 mesh was defined as 240-mesh Chinese gallnut.

[0061] 2. 75% tannic acid: Directly purchased from Guangdong Huihua Biotechnology Co., Ltd.

[0062] 3. Preparation of the fermentation broth of Lactiplantibacillus plantarum L75a:

[0063] The lactic acid bacterium is Lactiplantibacillus plantarum L75a (Accession number: NZ_CP074423), abbreviated as L75a, which is preserved in the Guangdong Provincial Microbial Culture Collection Center, with the preservation registration number GDMCC No: 63183, and the preservation date is February 22, 2023, and the taxonomic name is: Lactiplantibacillus plantarum.

[0064] Adjust the initial concentration of the lactic acid bacterium solution to 10 9 cells / mL, and transfer it into MRS liquid medium at an inoculation amount of 1‰ (5 mL / 50 mL, V / V), and shake-culture it in a shaker at 37 °C and a rotation speed of 200 r / min for 24 h to obtain the fermentation broth of Lactiplantibacillus plantarum L75a.

[0065] 4. Isolation source and preservation of Saprolegnia

[0066] The prepared PDA medium was autoclaved and cooled to 50 °C, penicillin and streptomycin were added, and the final concentration of both antibiotics was 100 μg / mL. After mixing, pour the plate to obtain the antibiotic PDA plate.

[0067] Use sterile scissors to cut off the mycelia of Saprolegnia from the fins and body surfaces of farmed Micropterus salmonides infected with Saprolegnia. After rinsing with sterile water, place them on the above-mentioned antibiotic PDA plates and culture at 20 °C for several days. After the Saprolegnia grows, transfer the tip of the mycelia to a new antibiotic PDA plate for purification. Repeat the operation until there is no bacterial contamination on the plate.

[0068] Transfer the purified Saprolegnia to a PDA plate. After it covers the entire plate, cut it into pieces and store them in sterile mineral oil, and store them in a 4 °C refrigerator. At the same time, amplify and sequence the ITS rRNA gene of the isolated Saprolegnia strain to obtain the Saprolegnia parasitica strain F26 (Accession number: MW819628), which is called Saprolegnia F26, or simply F26.

[0069] I. In vitro anti-Saprolegnia concentration range of the fermentation metabolites of Lactobacillus plantarum L75a

[0070] 1. In vitro anti-Saprolegnia concentration range of the fermentation liquid of Lactobacillus plantarum L75a

[0071] After adding 0.5 mL of 1 / 5 PDB to each well of a 24-well plate, add 0.5 mL of the diluted lactic acid bacteria fermentation broth to adjust the concentration of the fermentation broth of Lactobacillus plantarum L75a in the 6-well plate to 50, 100, 250, 500, 1000, and 1500 ppm, with sterile ddH2O as the blank control. Use a sterile punch with a diameter of 3 mm to punch circular bacterial cakes on the 1 / 5 PDA plate covered with Saprolegnia F26, place the mycelia facing up in the center of the 24-well plate, set 3 parallels for each treatment group, and statically culture in a 20 °C incubator for 48 h. Take pictures and record at 24 h and 48 h respectively, and measure the longest side diameter of the Saprolegnia as the colony diameter. The lowest drug concentration that completely inhibits mycelial growth is determined as the minimum inhibitory concentration (MIC). Transfer the bacterial cakes in the wells showing no visible mycelial growth to a new 1 / 5 PDA plate. The drug concentration at which no visible mycelial growth is still observed on the plate after culturing at 20 °C for 7 days is regarded as the minimum bactericidal concentration (MFC). The effects of different concentrations of the fermentation broth of Lactobacillus plantarum L75a on the colony growth diameter of Saprolegnia F26 are as follows Figure 2As shown. At the 24-hour culture time, compared with the blank control, L75a at a concentration of 50 ppm and above had a highly significant inhibitory effect on the growth of F26 colonies (P<0.0001), and the inhibition rates corresponding to the concentrations of 50 ppm, 100 ppm, 250 ppm, 500 ppm, 1000 ppm, and 1500 ppm were 10.2%, 24.0%, 31.4%, 55.2%, 91.0%, and 94.7% respectively. After 48-hour culture, compared with the blank control, L75a at a concentration of 50 ppm and above had a highly significant inhibitory effect on the growth of F26 colonies (P<0.0001), and the inhibition rates corresponding to the concentrations of 50 ppm, 100 ppm, 250 ppm, 500 ppm, 1000 ppm, and 1500 ppm were 28.6%, 48.5%, 54.3%, 61.7%, 93.7%, and 91.6% respectively. Lactobacillus plantarum L75a inhibited Saprolegnia sp. F26. Compared with 24 hours, at the same concentration, the inhibition rate at 48 hours was higher than that at 24 hours, that is, the inhibitory effect on Saprolegnia sp. at 48 hours was better.

[0072] 2. Inhibitory effects of the fermentation broth, cell suspension and cell-free supernatant of Lactobacillus plantarum L75a on the growth of parasitic Saprolegnia

[0073] After adding 2.7 mL of 1 / 5 PDB to each well of a 6-well plate, 300 μL of the diluted lactic acid bacteria fermentation broth or cell suspension or cell-free supernatant was added to adjust its concentration in the 6-well plate to 1000 ppm and 100 ppm, with sterile ddH2O as the blank control. A sterile puncher with a diameter of 3 mm was used to take circular bacterial cakes on the 1 / 5 PDA plate covered with Saprolegnia sp. F26, and the mycelial side was placed face up in the center of the 6-well plate. Three parallels were set for each treatment group and incubated statically in an incubator at 20°C for 48 hours. Photos were taken and the longest diameter of the Saprolegnia sp. was measured as the colony diameter at 24 hours and 48 hours respectively. The growth inhibitory abilities of the three on Saprolegnia sp. F26 were compared.

[0074] The effects of the fermentation broth, cell suspension and cell-free supernatant of Lactobacillus plantarum L75a on the colony growth diameter of Saprolegnia sp. F26 are as Figure 3As shown in the figure. Under the conditions of culturing for 24 h and 48 h, compared with the blank control, the fermentation broth and cell suspension of L75a at 100 ppm and 1000 ppm both had extremely significant inhibitory effects on the growth of the mycelium of Saprolegnia F26 (P<0.01). The cell-free supernatant had a weaker inhibitory ability on the growth of F26 and a shorter effective action time. Only at a concentration of 1000 ppm and a culturing time of 24 h did it have a significant inhibitory effect on the growth of F26 mycelium. At the same time, under the conditions of the same concentration and culturing time, through comparison among the three, it can be obtained that the inhibitory effect of the cell-free supernatant was the worst, and the colony diameter of F26 was significantly higher than that of the fermentation broth and cell suspension; at a high concentration (1000 ppm), the inhibitory effect of the fermentation broth was the best, and the colony diameter of F26 was significantly lower than that of the cell suspension, but its significant effect decreased after the concentration was reduced (100 ppm) (the P value increased). At a culturing time of 24 h, there was no significant difference between the fermentation broth and the cell suspension at a concentration of 100 ppm (P = 0.06). At a culturing time of 48 h, the colony diameter of F26 in the fermentation broth group at a concentration of 100 ppm was significantly lower than that in the cell suspension group (P = 0.043), but the significant effect was lower than that at 1000 ppm (P = 0.021).

[0075] 3. Determination of the main metabolites of Lactobacillus plantarum L75a

[0076] Lactobacillus plantarum L75a stored at -80 °C was inoculated into a 50 mL centrifuge tube containing 5 mL of liquid MRS medium. After culturing for 24 h in a shaker at 28 °C and a rotation speed of 200 r / min, the OD 600 value was measured. The bacterial liquid was transferred to a centrifuge tube containing fresh 5 mL of MRS medium to make the initial concentration of the bacterial liquid 10 5 cells / mL. Then the centrifuge tube was placed in a shaker at 28 °C and a rotation speed of 200 r / min and cultured for 24 h. The obtained L75a fermentation broth was centrifuged at a rotation speed of 5000 xg for 5 min to separate the supernatant and bacterial cells. The supernatant was filtered and sterilized through a 0.22 μM needle filter to obtain the cell-free supernatant of L75a. The MRS medium without inoculating L75a was used as a control, and liquid chromatography-mass spectrometry (LC-MS, N = 5) and gas chromatography-mass spectrometry (GC-MS, N = 6) metabolite analyses (Suzhou Panomic Biotechnology Co., Ltd.) were carried out together with the cell-free supernatant of L75a.

[0077] The Fold Chang was calculated by comparing the peak intensity of each metabolite in the cell-free supernatant of L75a with the average peak intensity of the corresponding compound in the control group. The abundance of metabolites was determined by the Fold Chang of the peak intensity (Fold Chang>100). The results of the top 10 metabolites with the highest abundance detected in the cell-free supernatant extract of L75a are as Figure 4As shown in the figure. According to GC-MS analysis, a total of 23 metabolites were identified, among which the Fold Chang (L75a / Control) was greater than 100 for pyruvic acid (PA); according to LC-MS analysis, a total of 96 metabolites were identified, among which the Fold Chang (L75a / Control) was greater than 100 for gluconic acid (GA), ethyl (4S,5S)-4,5-dihydroxy-2,6-dioxohexanoate, (S)-beta-tyrosine, 1D-chiro-inositol, cellobiono-1,5-lactone, 3-aminopentanedioate, urocanic acid, pyrrolidonecarboxylic acid, 4-hydroxybutanoic acid.

[0078] 3. Ability test of the main metabolites of Lactobacillus plantarum L75a to inhibit Saprolegnia F26

[0079] According to the above analysis results of the metabolites of Lactobacillus plantarum L75a, the metabolites with higher concentration and antibacterial potential were selected, and the ability test of inhibiting Saprolegnia F26 was carried out based on their chemical standards. The relevant chemical standards were prepared into a concentration of 1000 μmol / L. After measuring the pH value with a pH meter (Dinghai Technology) and adjusting the pH to neutral (6.5 - 7.5) using NaOH, the relevant chemical standard sterile mother liquor was obtained by filtering and sterilizing with a 0.22 μM needle filter. Subsequently, the mother liquor was diluted with sterile ddH2O according to the required concentration for use in the experiment.

[0080] 1 / 5PDA was prepared, cooled to 55 °C after sterilization, and the adjusted-concentration chemical standards were fully mixed with 1 / 5PDA at a ratio of 1:4, so that the final concentration of the chemical standards in the medium was 10, 20, 50, 100, 200 μmol / L. At the same time, ddH2O and 35‰ NaCl were used as blank control and positive control respectively. Pour 6 mL of each into a petri dish (60 mm), let it stand and solidify. Then, use a sterile hole punch with a diameter of 3 mm to take circular bacterial cakes on the 1 / 5PDA plate covered with Saprolegnia F26, and place the mycelium side down in the center of the medium. Set 3 parallels for each treatment group and incubate statically in an incubator at 20 °C for 48 h. Take pictures and record at 24 h and 48 h respectively, and measure the longest diameter of the Saprolegnia as the colony diameter.

[0081] According to the above experimental results and based on their main functions, four metabolites, namely the parent butyric acid (BA) of gluconic acid, pyruvic acid, and 4-hydroxybutyric acid, and its derivative 3-hydroxybutyric acid (3-HA), were selected to test the growth inhibitory ability of Saprolegnia F26. The experimental results are as Figure 5 shown.

[0082] Under the condition of culturing for 24 h, GA at concentrations of 20 μmol / L and 50 μmol / L significantly promoted the growth of F26 (P < 0.05), and GA at concentrations of 100 μmol / L and 200 μmol / L extremely significantly promoted the growth of F26 (P < 0.01); PA at a concentration of 20 μmol / L significantly promoted the growth of F26 (P < 0.05), PA at concentrations of 50 μmol / L and 100 μmol / L extremely significantly promoted the growth of F26 (P < 0.01), and PA at concentrations of 10 μmol / L and 200 μmol / L had no significant effect on the growth of F26 (P > 0.05); all tested concentrations of BA extremely significantly inhibited the growth of F26 (P < 0.01); 3-HA at a concentration of 10 μmol / L had no significant effect on the growth of F26 (P > 0.05), 3-HA at concentrations of 20 μmol / L and 50 μmol / L extremely significantly promoted the growth of F26 (P < 0.01), and 3-HA at concentrations of 100 μmol / L and 200 μmol / L extremely significantly inhibited the growth of F26 (P < 0.01). GA reached the strongest promoting effect at 200 μmol / L, PA and 3-HA reached the strongest promoting effect at 50 μmol / L, the minimum inhibitory concentration (MIC) of BA was 10 μmol / L, and the minimum inhibitory concentration (MIC) of 3-HA was 200 μmol / L.

[0083] Under the condition of culturing for 48 h, the colony diameters of F26 in the experimental groups supplemented with GA and PA were all extremely significantly larger than those in the blank control group, showing an extremely significant promoting effect on the growth of F26 (P < 0.01); the colony diameters of F26 in the experimental groups supplemented with BA were all extremely significantly smaller than those in the blank control group, showing an extremely significant inhibitory effect on the growth of F26 (P < 0.01), the minimum inhibitory concentration (MIC) was 20 μmol / L, and the minimum bactericidal concentration (MFC) was 200 μmol / L ( Figure 6) In the experimental group supplemented with 3-HA, there was no significant difference in the colony diameter of F26 at 10 μmol / L and 100 μmol / L compared with the blank control group (P>0.05). The colony diameters of F26 at 10 μmol / L and 100 μmol / L were extremely significantly higher than those of the blank group, showing an extremely significant promoting effect on the growth of F26 (P<0.01). The colony diameter of F26 at 200 μmol / L was extremely significantly lower than that of the blank control group, showing an extremely significant inhibitory effect on the growth of F26 (P<0.01). The experimental test concentrations did not reach the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MFC). The promoting effect of each metabolite on F26 was still significant at 48 h, and the significant inhibitory effect of 3-HA on F26 weakened at 48 h.

[0084] II. In vitro anti-Saprolegnia concentration range of Chinese herbal medicines

[0085] 1. Screening of Chinese herbal medicines

[0086] Test the in vitro anti-Saprolegnia effects of different Chinese herbal medicines at different concentrations, and screen out the most effective Chinese herbal medicines against Saprolegnia. Grind Chinese gallnut, Ligusticum chuanxiong, Kochia scoparia, Dictamnus dasycarpus, clove, Morinda officinalis and Angelica sinensis into powder and put them into bottles ( Figures 7-1 to 7-4 ). Weigh 0.04 g of the above different Chinese herbal medicine powders into 50 ml centrifuge tubes, sterilize them with ultraviolet rays for 20 min in a laminar flow hood. After ultraviolet sterilization, dilute them with sterile water to form Chinese herbal medicine solutions. In a 24-well plate, add 0.5 mL of 1 / 5 PDB to each well, and then add 0.5 ml of the diluted Chinese herbal medicine solution to adjust the concentration of the Chinese herbal medicine solution in the 24-well plate to 1, 10, 100, 1000 ppm, with sterile ddH2O as the blank control. Use a sterile punch with a diameter of 2 mm to take circular bacterial cakes on the PDA plate covered with Saprolegnia F26, place the mycelium facing up and closely attached to the lower edge of the 24-well plate. Set 3 parallels for each treatment group, and incubate them statically in a 20 °C incubator for 48 h. Take pictures and record at 24 h and 48 h respectively, and measure the longest side radius of the Saprolegnia as the colony radius. Determine the minimum inhibitory concentration (MIC) as the lowest drug concentration that completely inhibits mycelial growth.

[0087] The results are as follows Figure 7-2As shown, at 24 hours, when the concentration of Chinese gallnut was 10 ppm and above, it had an extremely significant inhibitory effect on Saprolegnia F26 (p < 0.0001), and the inhibition rate reached 24.6% at 10 ppm, and 100% at 100 ppm and 1000 ppm; Ligusticum wallichii had no significant inhibitory effect when the concentration was 100 ppm and below, and had a significant inhibitory effect on Saprolegnia F26 at a concentration of 1000 ppm (p < 0.0001), with an inhibition rate of 18.9%; Kochia scoparia had a significant inhibitory effect on Saprolegnia F26 when the concentration was 1 ppm and above (1 ppm and 10 ppm: p < 0.05; 100 ppm and 1000 ppm: p < 0.0001), the inhibition rate was 8.3% at 1 ppm and 10 ppm, and 21.7% and 45% at 100 ppm and 1000 ppm respectively; Dictamnus dasycarpus Turcz. had no significant inhibitory effect on Saprolegnia F26 when the concentration was 10 ppm and below, and had an extremely significant inhibitory effect when the concentration was 100 ppm and above (p < 0.0001), and the inhibition rates at 100 ppm and 1000 ppm were 27.8% and 100% respectively; Syzygium aromaticum had a significant inhibitory effect on Saprolegnia F26 when the concentration was 10 ppm and above (10 ppm: p < 0.05, 100 ppm and 1000 ppm: p < 0.0001), and the inhibition rates at 10 ppm, 100 ppm and 1000 ppm were 8.3%, 32.8% and 81.7% respectively; Morinda officinalis How had no significant inhibitory effect on Saprolegnia F26 when the concentration was 100 ppm and below, and had a significant inhibitory effect at 1000 ppm (p < 0.0001), and the inhibition rate at 1000 ppm was 18.3%; Angelica sinensis had no significant inhibitory effect on F26 when the concentration was 1000 ppm and below.At 48 h, when the concentration of Chinese gallnut was 10 ppm and above, it had an extremely significant inhibitory effect on Saprolegnia F26 (p < 0.0001), and the inhibition rates at 1 ppm and 10 ppm were 9.4% and 30.9% respectively, and the inhibition rates at 100 ppm and 1000 ppm reached 100%; Chuanxiong had no significant inhibitory effect when the concentration was 100 ppm and below, and had a significant inhibitory effect on Saprolegnia F26 at a concentration of 1000 ppm (p < 0.05), with an inhibition rate of 10.9%; Kochia scoparia had a significant inhibitory effect on Saprolegnia F26 when the concentration was 1 ppm and above (1 ppm and 10 ppm: p < 0.05; 100 ppm and 1000 ppm: p < 0.0001), and the inhibition rates at 1 ppm and 10 ppm were 9.4% and 10.2% respectively, and the inhibition rates at 100 ppm and 1000 ppm were 43.9% and 62.8% respectively; Dictamnus dasycarpus had no significant inhibitory effect on Saprolegnia F26 when the concentration was 10 ppm and below, and had an extremely significant inhibitory effect when the concentration was 100 ppm and above (p < 0.0001), and the inhibition rates at 100 ppm and 1000 ppm were 27.9% and 88.6% respectively; Clove had a significant inhibitory effect on Saprolegnia F26 when the concentration was 1 ppm and above (1 ppm: p < 0.05, 10 ppm, 100 ppm and 1000 ppm: p < 0.0001), and the inhibition rates at 1 ppm, 10 ppm, 100 ppm and 1000 ppm were 10.5%, 20.8%, 27.9% and 88.6% respectively; Morinda officinalis had no significant inhibitory effect on Saprolegnia F26 when the concentration was 100 ppm and below, and had a significant inhibitory effect at 1000 ppm (p < 0.0001), and the inhibition rate at 1000 ppm was 16.6%; Angelica sinensis had a significant inhibitory effect on F26 at a concentration of 10 ppm (p < 0.05), and the inhibition rate was 10%. Finally, Chinese gallnut was selected as the Chinese herbal medicine for inhibiting Saprolegnia.

[0088] 2. Concentration range of Chinese gallnut inhibiting Saprolegnia

[0089] Weigh 0.04 g of Chinese gallnut powder into a 50-ml centrifuge tube and sterilize it with ultraviolet light for 20 min in a laminar flow hood. After ultraviolet sterilization, dilute it with sterile water to form a Chinese gallnut medicinal solution. In a 24-well plate, add 0.5 mL of 1 / 5 PDB to each well, and then add 0.5 ml of the diluted Chinese gallnut medicinal solution to adjust the concentration of the Chinese gallnut medicinal solution in the 24-well plate to 5, 10, 15, 20, 25, and 30 ppm, with sterile ddH2O as the blank control. Use a sterile punch with a diameter of 2 mm to punch circular fungal discs on a PDA plate covered with Saprolegnia F26, place the hyphae facing up and close to the lower edge of the 24-well plate. Set up 3 parallels for each treatment group and incubate them statically in an incubator at 20 °C for 48 h. Take pictures and record at 24 h and 48 h respectively, and measure the longest side radius of the Saprolegnia as the colony radius. The minimum inhibitory concentration (MIC) is determined as the lowest drug concentration that completely inhibits hyphal growth.

[0090] The results are as follows Figure 8 As shown, at 24 h, when the concentration of Chinese gallnut was 5 ppm - 30 ppm, it had a significant inhibitory effect on Saprolegnia F26 (p < 0.0001), and the inhibition rates at concentrations of 5, 10, 15, 20, 25, and 30 ppm were 20.3%, 38.8%, 49.9%, 73.0%, 85.3%, and 94.4% respectively; at 48 h, when the concentration of Chinese gallnut was 5 ppm - 30 ppm, it had a significant inhibitory effect on Saprolegnia F26 (p < 0.0001), and the inhibition rates at concentrations of 5, 10, 15, 20, 25, and 30 ppm were 24.3%, 36.3%, 54.5%, 64.1%, 71.0%, and 88.3% respectively.

[0091] 3. Inhibition of Saprolegnia by Chinese gallnut with different mesh numbers

[0092] After the Chinese gallnuts were ground into powder by a pulverizer, the particle sizes were uneven. It was speculated that there were differences in the inhibitory effects of Chinese gallnuts with different mesh numbers on Saprolegnia at the same concentration, and an experiment was conducted on this. Chinese gallnuts with different mesh numbers were obtained by screening through different mesh sieves. Among them, Chinese gallnuts that passed through 60 mesh but not 80 mesh were defined as 60-mesh Chinese gallnuts; Chinese gallnuts that passed through 120 mesh but not 140 mesh were defined as 120-mesh Chinese gallnuts; Chinese gallnuts that passed through 180 mesh but not 200 mesh were defined as 180-mesh Chinese gallnuts; Chinese gallnuts that passed through 240 mesh were defined as 240-mesh Chinese gallnuts. 0.04 g of Chinese gallnut powder of no sieve mesh number (abbreviated as no sieve), 60 mesh, 120 mesh, 180 mesh, and 240 mesh were respectively weighed into 50 ml centrifuge tubes and sterilized with ultraviolet rays for 20 min in a laminar flow hood. After the ultraviolet sterilization was completed, they were diluted with sterile water to form Chinese gallnut liquid medicines of no sieve, 60 mesh, 120 mesh, 180 mesh, and 240 mesh respectively. In a 24-well plate, after adding 0.5 mL of 1 / 5 PDB to each well, 0.5 ml of the diluted Chinese gallnut liquid medicine of each mesh number was added to adjust the concentration of the Chinese gallnut liquid medicine of each mesh number in the 24-well plate to 10 and 20 ppm, with sterile ddH2O as the blank control. A sterile puncher with a diameter of 2 mm was used to take a circular bacterial cake on a PDA plate covered with Saprolegnia F26, and the mycelium was placed facing up close to the lower edge of the 24-well plate. Three parallels were set for each treatment group and incubated statically in an incubator at 20 °C for 48 h. Photos were taken and recorded at 24 h and 48 h respectively, and the longest side radius of the Saprolegnia was measured as the colony radius.

[0093] The results are shown in Table 3. When the Chinese gallnuts at 10 ppm inhibited Saprolegnia at 24 h, the inhibition rates of the Chinese gallnuts of no sieve, 60 mesh, 120 mesh, 180 mesh, and 240 mesh on Saprolegnia were 29%, 25.1%, 37.7%, 38.4%, and 48.3% ( Figure 9 ), respectively. There were significant differences between the Chinese gallnuts of no sieve and 60 mesh and those of 120 mesh, 180 mesh, and 240 mesh; there were significant differences between the Chinese gallnuts of 120 mesh and 180 mesh and those of no sieve, 60 mesh, and 240 mesh; there were significant differences between the Chinese gallnuts of 240 mesh and those of no sieve, 60 mesh, 120 mesh, and 180 mesh. When the Chinese gallnuts at 10 ppm inhibited Saprolegnia at 48 h, the inhibition rates of the Chinese gallnuts of no sieve, 60 mesh, 120 mesh, 180 mesh, and 240 mesh on Saprolegnia were 25.6%, 20.1%, 37.3%, 42.9%, and 45.7%, respectively. There were significant differences between the Chinese gallnuts of no sieve and those of 60 mesh, 120 mesh, 180 mesh, and 240 mesh; there were significant differences between the Chinese gallnuts of 60 mesh and those of no sieve, 180 mesh, and 240 mesh; there were significant differences between the Chinese gallnuts of 120 mesh and those of no sieve, 60 mesh, 120 mesh, 180 mesh, and 240 mesh; there were significant differences between the Chinese gallnuts of 180 mesh and 240 mesh and those of no sieve, 60 mesh, and 120 mesh.

[0094] Table 3 Differences in the inhibition rates of Saprolegnia by Chinese gallnuts with different mesh numbers

[0095]

[0096] Note: Mean ± standard deviation (n = 3); Superscript letters represent the results of pairwise comparisons within the same column. The same superscript letter indicates no significant difference (P > 0.05), while different superscript letters indicate a significant difference (P < 0.05).

[0097] 4. Concentration range of tannic acid inhibiting Saprolegnia

[0098] Tannic acid is the main component of Chinese gallnut. It was hypothesized that tannic acid might also have good inhibitory effects on Saprolegnia, and an experiment was conducted. 0.04 g of tannic acid powder (with a content of 75%, from Guangdong Huihua Biotechnology Co., Ltd.) was weighed into a 50 ml centrifuge tube and sterilized under ultraviolet light for 20 min in a laminar flow hood. After ultraviolet sterilization, it was diluted with sterile water to form a tannic acid solution. In a 24-well plate, 0.5 mL of 1 / 5 PDB was added to each well, and then 0.5 ml of the diluted tannic acid solution was added to adjust the concentration of the tannic acid solution in the 24-well plate to 25, 50, 75, 100, and 150 ppm, with sterile ddH2O as the blank control. A sterile puncher with a diameter of 2 mm was used to punch circular fungal discs from a PDA plate covered with Saprolegnia F26. The mycelium side was placed facing up and closely attached to the lower edge of the 24-well plate. Three replicates were set for each treatment group and incubated statically in an incubator at 20 °C for 48 h. Photos were taken and the longest side radius of Saprolegnia was measured as the colony radius at 24 h and 48 h respectively. The minimum inhibitory concentration (MIC) was determined as the lowest drug concentration that completely inhibited mycelial growth.

[0099] The results are as follows Figure 10 As shown, at 24 h, tannic acid at concentrations of 25 ppm - 150 ppm had significant inhibitory effects on Saprolegnia F26 (p < 0.0001), and the inhibition rates at 25, 50, 75, 100, and 150 ppm were 52.5%, 71.0%, 92.9%, 100%, and 100% respectively; at 48 h, tannic acid at concentrations of 25 ppm - 150 ppm had significant inhibitory effects on Saprolegnia F26 (p < 0.0001), and the inhibition rates at 25, 50, 75, 100, and 150 ppm were 52.6%, 71.1%, 86.9%, 93.4%, and 100% respectively.

[0100] III. In vivo anti-Saprolegnia activity of Chinese gallnut

[0101] 1. Effects of different concentrations of Chinese gallnut (240 mesh) on the hatching rate of colored carp fertilized eggs

[0102] Galla chinensis showed good inhibitory effects in the in vitro experiment of inhibiting Saprolegnia, but its effect in in vivo experiments is unknown. It is speculated that Galla chinensis will affect the microbial community structure of fish and their eggs. Fish hatching is a core link in aquaculture, and the hatching rate of fish eggs is closely related to the balance of the microbial community on the egg membrane surface. During the hatching process, pathogenic microorganisms (such as bacteria, fungi, parasites) on the egg membrane surface are likely to cause infections, leading to embryo death or deformities. Traditional chemical drugs, such as formaldehyde and antibiotics, although having certain effects, have problems such as drug resistance and residual pollution. As a Chinese herbal medicine with strong ability to inhibit Saprolegnia in vitro, Galla chinensis may be effective in preventing and controlling the occurrence of Saprolegnia, which usually occurs on fish eggs and fish bodies. It is also necessary to evaluate whether Galla chinensis will cause other effects on fish eggs and fish bodies while preventing and controlling Saprolegnia. In the actual prevention and control of Saprolegnia, the hatching rate of fish eggs can be increased by reducing the risk of fish eggs being infected with Saprolegnia. Therefore, it is necessary to explore whether Galla chinensis will affect the hatching rate of fish and fish eggs, and it is speculated that Galla chinensis will affect the microbial community structure on fish eggs.

[0103] The experimental object used in the fish activity experiment was fish eggs. The fertilized eggs of colored carp and the breeding water used to cultivate the fertilized eggs of colored carp in the experiment were all from Guangdong Junyi Koi Breeding Co., Ltd., Dongguan City, Guangdong Province. The Galla chinensis powder that could pass through a 240-mesh sieve was marked as 240-mesh Galla chinensis. The 240-mesh Galla chinensis powder was evenly spread on a disposable culture dish, placed in a ultra-clean workbench and sterilized with ultraviolet light for 20 minutes, and then put into a tube for standby. Weighed 0.18 g of 240-mesh Galla chinensis powder into a 50-ml centrifuge tube and diluted it with breeding water to form a 240-mesh Galla chinensis medicinal liquid. In a disposable culture dish, added the 240-mesh Galla chinensis medicinal liquid to adjust the concentration of the 240-mesh Galla chinensis medicinal liquid in the culture dish to 20 ppm and 10 ppm, with breeding water as the blank control. Put 20 fertilized eggs of colored carp in each culture dish. After the fertilized fish eggs were completely hatched, record and statistically analyze the data and take pictures. Set three parallels for each treatment group. The data obtained from the experiment were analyzed using the software SPSS 22.0. In the significant difference analysis, the independent samples T-test was used for pairwise comparison, and one-way analysis of variance (One-way ANOVA) was used for comparisons of three groups or more, and the Tukey multiple comparison method was used for post hoc tests. The data were expressed as mean ± standard error (SE).

[0104] As shown in Table 4, compared with the blank control group at 0 ppm, when using 10 ppm and 20 ppm of Galla chinensis for soaking and hatching, the hatching rate of fertilized eggs of colored carp increased from 83.3% to 96.7%, showing a significant difference. For details, see Figure 11 .

[0105] Table 4 Effects of different concentrations of Galla chinensis (240-mesh) on the hatching rate of fertilized eggs of colored carp

[0106]

[0107] Note: Mean ± standard deviation (n = 3); superscript letters indicate the results of pairwise comparisons within the same column. Values with the same superscript letter indicate no significant difference (P > 0.05), while different superscript letters indicate significant differences (P < 0.05). Hatching rate of fish eggs 1 (%) is the ratio of the number of hatched colored carp fertilized eggs to the total number of colored carp fertilized eggs.

[0108] 2. Effects of different concentrations of gallnut (240 mesh) on the microbial flora on the surface of unfertilized eggs of colored carp

[0109] After the experiment on the effects of different concentrations of gallnut (240 mesh) on the growth of Saprolegnia in unfertilized eggs of colored carp, the fish eggs were taken with sterilized forceps and placed in cryotubes. All samples were immediately stored in liquid nitrogen after collection and then stored in a -80 °C refrigerator after sampling. Then, the samples were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) for DNA extraction, PCR amplification of 16S rRNA and ITS rRNA genes, Illumina MiSeq sequencing, and sequencing data processing to obtain the composition of bacterial and fungal flora. The UPARSE algorithm was used to classify all sequences into OUTs, and bioinformatics statistical analysis of OUTs was performed at a 97% similarity level.

[0110] The sequencing results were subjected to cluster analysis. After rarefaction, the number of bacterial and fungal OTUs obtained were 4049 and 1203, respectively. The α-diversity (Chao Index, Shannon Index, Ace Index, and Simpson Index) indices of each group of samples are as Figure 12 shown. The species richness of fungi on the egg membranes of colored carp eggs treated with three different concentrations of gallnut was lower than that of bacteria. According to the comparison of the species richness and diversity of the bacterial flora among groups, different concentrations of gallnut treatment did not cause significant changes in the species richness and diversity of the flora; according to the comparison of the species richness and diversity of the fungal flora among groups, an increase in the gallnut concentration led to a decrease in the species richness and diversity of the fungal flora, and there were significant differences in the fungal flora diversity between the blank group treated with 0 ppm gallnut and the group treated with 20 ppm gallnut.

[0111] At the phylum and class levels, the compositional abundances of each bacterial species are as Figure 13As shown in the figure. At the phylum level, the dominant bacterial flora mainly includes Proteobacteria and Bacteroidota. Specifically, the relative abundances of Proteobacteria in the gallnut 0 ppm group, 10 ppm group, and 20 ppm group are 51.97%, 72.62%, and 77.4% respectively, while the relative abundances of Bacteroidota in these three groups are 35.4%, 19.09%, and 12.71% respectively. In addition, the relative abundances of Proteobacteria, Bacteroidota, and Deinococcus in the gallnut 10 ppm group and 20 ppm group are significantly higher than those in the gallnut 0 ppm group (P < 0.01). At the class level, the dominant bacterial flora is mainly Gammaproteobacteria and Bacteroidia. Among them, the relative abundances of Gammaproteobacteria in the gallnut 0 ppm group, 10 ppm group, and 20 ppm group are 36.9%, 55.6%, and 62.9% respectively, while the relative abundances of Bacteroidia are 35.37%, 19.05%, and 12.61% respectively. Compared with the gallnut 0 ppm group, the relative abundance of Gammaproteobacteria in the 10 ppm group and 20 ppm group increased significantly (P < 0.01), while the relative abundances of Bacteroidia and Deinococcus decreased significantly (P < 0.01 and P < 0.001).

[0112] At the order and family levels, the compositional abundances of each bacterial species are as Figure 14 shown. At the order level, the dominant bacterial flora is mainly Burkholderiales, and its relative abundances in the gallnut 0 ppm group, 10 ppm group, and 20 ppm group are 25.41%, 26.07%, and 18.62% respectively. Compared with the gallnut 0 ppm group, the relative abundance of Flavobacteriales in the 10 ppm group and 20 ppm group decreased significantly (P < 0.01), while the relative abundance of Enterobacterales in the 20 ppm group increased significantly (P < 0.05). At the family level, the dominant bacterial flora is Comamonadaceae, and its relative abundances in the gallnut 0 ppm group, 10 ppm group, and 20 ppm group are 15.96%, 23.5%, and 16.61% respectively. In addition, the relative abundance of Oxalobacteraceae in the gallnut 10 ppm group and 20 ppm group decreased significantly (P < 0.001), while the relative abundance of Enterobacteriaceae in the 20 ppm group increased significantly (P < 0.05).

[0113] At the order and family levels, the compositional abundances of each bacterial species are as Figure 15As shown in the figure. At the species level, the dominant bacterial flora was mainly Chryseobacterium indologenes, and its relative abundances in the 0 ppm, 10 ppm, and 20 ppm groups of Chinese gallnut were 25.41%, 11.06%, and 4.54%, respectively. Compared with the 0 ppm group of Chinese gallnut, the relative abundances of Chryseobacterium indologenes and Deinococcus soli in the 10 ppm and 20 ppm groups were significantly reduced (P<0.01), while the relative abundance of Pseudomonas mosselii in the 20 ppm group was significantly increased (P<0.05). At the genus level, the dominant bacterial flora was Chryseobacterium, and its relative abundances in the 0 ppm, 10 ppm, and 20 ppm groups of Chinese gallnut were 31.69%, 13.4%, and 8.29%, respectively. In addition, the 10 ppm and 20 ppm groups of Chinese gallnut significantly reduced the relative abundances of Chryseobacterium and Acidovorax (P<0.001 and P<0.01), while the 10 ppm and 20 ppm groups significantly increased the relative abundance of Comamonas (P<0.05).

[0114] The abundance of each fungal species composition at the phylum and class levels is as follows Figure 16 As shown in the figure. At the phylum level, the dominant bacterial flora was Ascomycota and Basidiomycota. The relative abundances of Ascomycota in the 0 ppm, 10 ppm, and 20 ppm groups of Chinese gallnut were 46.29%, 81.19%, and 72.81%, respectively, while the relative abundances of Bacteroidetes in these three groups were 24.64%, 10.92%, and 14.36%, respectively. Compared with the 0 ppm group of Chinese gallnut, the relative abundances of Ascomycota in the 10 ppm and 20 ppm groups were significantly increased (P<0.05). At the class level, the dominant bacterial flora was mainly Dothideomycetes, and its relative abundances in the 0 ppm, 10 ppm, and 20 ppm groups of Chinese gallnut were 28.75%, 59.71%, and 71.61%, respectively. Compared with the 0 ppm group of Chinese gallnut, the relative abundance of Dothideomycetes in the 20 ppm group was significantly increased (P<0.05).

[0115] The abundance of each fungal species composition at the order and family levels is as follows Figure 17As shown in the figure. At the order level, the dominant bacterial flora is Capnodiales, and the relative abundances of Capnodiales in the 0 ppm, 10 ppm, and 20 ppm groups of Chinese gallnut are 22.27%, 47.01%, and 67.98% respectively. Compared with the 0 ppm group of Chinese gallnut, the relative abundance of Capnodiales in the 20 ppm group is significantly increased (P < 0.05). At the family level, the dominant bacterial flora is mainly Cladosporiaceae, and its relative abundances in the 0 ppm, 10 ppm, and 20 ppm groups of Chinese gallnut are 22.26%, 46.58%, and 67.89% respectively. Compared with the 0 ppm group of Chinese gallnut, the relative abundance of Cladosporiaceae in the 20 ppm group is significantly increased (P < 0.05).

[0116] The abundance of each fungal species composition at the genus and species levels is as follows Figure 18 As shown in the figure. At the order level, the dominant bacterial flora is Cladosporium, and the relative abundances of Cladosporium in the 0 ppm, 10 ppm, and 20 ppm groups of Chinese gallnut are 22.26%, 45.9%, and 67.24% respectively. Compared with the 0 ppm group of Chinese gallnut, the relative abundance of Cladosporium in the 20 ppm group is significantly increased (P < 0.05). At the species level, the dominant bacterial flora is mainly Cladosporium halotolerans, and its relative abundances in the 0 ppm, 10 ppm, and 20 ppm groups of Chinese gallnut are 20.15%, 42.71%, and 58.33% respectively. Compared with the 0 ppm and 10 ppm groups of Chinese gallnut, the relative abundance of Cladosporium delicatulum in the 20 ppm group is significantly increased (P < 0.05).

[0117] IV. Exploration of the anti-Saprolegnia concentration range and the best compounding scheme of the metabolites of Lactobacillus plantarum L75a fermentation broth and Chinese herbal medicines

[0118] The anti-Saprolegnia effects obtained through different compounding schemes were compared, and the best compounding scheme was finally obtained. The compounding scheme was adjusted according to the minimum bactericidal concentration of Lactobacillus plantarum L75a fermentation broth and Chinese herbal medicines, and there were the following 3 compounding schemes (Table 3): (1) Chinese gallnut 10 ppm + tannic acid 20 ppm + L75a 100 ppm; (2) Chinese gallnut 10 ppm + tannic acid 40 ppm + L75a 75 ppm; (3) Chinese gallnut 10 ppm + tannic acid 60 ppm + L75a 50 ppm. The final best compounding scheme was obtained based on the comprehensive evaluation of the prices and Saprolegnia inhibition effects of different compounding schemes.

[0119] The results are as follows Figures 19-1 to 19-3As shown, at 24 h, the first compounding scheme combination (10 ppm gallnut, 20 ppm tannic acid, and 100 ppm Lactobacillus plantarum L75a) had a highly significant inhibitory effect compared with the blank group (p < 0.0001), and the inhibition rate of Saprolegnia was 68.7%; the second compounding scheme combination (10 ppm gallnut, 40 ppm tannic acid, and 75 ppm Lactobacillus plantarum L75a) had a highly significant inhibitory effect compared with the blank group (p < 0.0001), and the inhibition rate of Saprolegnia was 91.8%; the third compounding scheme combination (10 ppm gallnut, 60 ppm tannic acid, and 50 ppm Lactobacillus plantarum L75a) had a highly significant inhibitory effect compared with the blank group (p < 0.0001), and the inhibition rate of Saprolegnia was 100%. At 48 h, the first compounding scheme combination (10 ppm gallnut, 20 ppm tannic acid, and 100 ppm Lactobacillus plantarum L75a) had a highly significant inhibitory effect compared with the blank group (p < 0.0001), and the inhibition rate of Saprolegnia was 72.5%; the second compounding scheme combination (10 ppm gallnut, 40 ppm tannic acid, and 75 ppm Lactobacillus plantarum L75a) had a highly significant inhibitory effect compared with the blank group (p < 0.0001), and the inhibition rate of Saprolegnia was 87.6%; the third compounding scheme combination (10 ppm gallnut, 60 ppm tannic acid, and 50 ppm Lactobacillus plantarum L75a) had a highly significant inhibitory effect compared with the blank group (p < 0.0001), and the inhibition rate of Saprolegnia was 98%. All three compounding combination schemes had a significant inhibitory effect on Saprolegnia. The price of gallnut is relatively expensive, about 240 yuan / kg in the market. These three schemes can all reduce the demand for gallnut, thereby reducing costs. Among them, the third combination scheme reached an inhibition rate of about 100%. The usage amount of gallnut with the same Saprolegnia inhibition rate was about 35 ppm, that is, the third combination reduced the usage amount of gallnut by 25 ppm.

[0120] Table 5 Compounding Scheme

[0121]

[0122] The above-described embodiments only represent one implementation manner of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

Claims

1. A composition for inhibiting saprolegniasis, characterized in that, The components include Chinese gallnut, tannic acid and lactic acid bacteria.

2. The composition for inhibiting saprolegniasis according to claim 1, wherein The ratio among the Chinese gallnut, tannic acid and lactic acid bacteria is: Chinese gallnut﹕tannic acid﹕lactic acid bacteria = 5 - 30﹕20 - 150﹕50 - 1500.

3. The composition for inhibiting saprolegniasis according to claim 1, characterized in that, The ratio among the Chinese gallnut, tannic acid and lactic acid bacteria is: Chinese gallnut﹕tannic acid﹕lactic acid bacteria = 5 - 30﹕20 - 60﹕50 - 100.

4. The composition for inhibiting saprolegniasis according to claim 1, wherein, The ratio among the Chinese gallnut, tannic acid and lactic acid bacteria is: Chinese gallnut﹕tannic acid﹕lactic acid bacteria = 10﹕20﹕100; or Chinese gallnut﹕tannic acid﹕lactic acid bacteria = 10﹕40﹕75; or Chinese gallnut﹕tannic acid﹕lactic acid bacteria = 10﹕60﹕50.

5. The composition for inhibiting saprolegniasis according to claim 1, characterized in that, The Chinese gallnut is in the form of Chinese gallnut powder, and the lactic acid bacteria is in the form of lactic acid bacteria fermentation broth; the concentration of tannic acid is 75%; the lactic acid bacteria is Lactiplantibacillus plantarum L75a.

6. The composition for inhibiting saprolegniasis according to claim 5, characterized in that, The Chinese gallnut powder is the powder of Chinese gallnut without sieving after pulverization, or the powder of Chinese gallnut after pulverization and sieving, taking the powder with a mesh size of 60 - 240 meshes.

7. A method for preparing a composition for inhibiting Saprolegniasis, characterized in that, Pulverize the Chinese gallnut, and then mix it with tannic acid and lactic acid bacteria to obtain the product.

8. Use of the composition for inhibiting saprolegniasis according to any one of claims 1 - 6 as an aquaculture additive.

9. Use of the composition for inhibiting saprolegniasis according to any one of claims 1 - 6 as an aquaculture feed ingredient.

10. Aquaculture feed, characterized in that, Its components include the composition for inhibiting saprolegniasis according to any one of claims 1 - 6.

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