Traditional Chinese medicine composition, traditional Chinese medicine fermentation liquor and application of traditional Chinese medicine composition and traditional Chinese medicine fermentation liquor in aquaculture and prevention and treatment of fish blast

Abstract

The invention belongs to the technical field of fermented traditional Chinese medicines and aquaculture, and particularly relates to a traditional Chinese medicine composition, traditional Chinese medicine fermentation liquor and application of the traditional Chinese medicine composition and the traditional Chinese medicine fermentation liquor in aquaculture and fish blast prevention and treatment. According to the traditional Chinese medicine composition, Chinese herbal medicines and probiotics are compounded, and the variety and content of effective active ingredients in a fermented product of the traditional Chinese medicine composition are increased through the fermentation metabolism function of the probiotics. The Chinese herbal medicine generates effective components including malic acid and antibacterial peptide through decomposition and metabolism of probiotics, the content of the effective components is high, infection of common germs or viruses and the like of aquatic animals can be effectively resisted, the immunity of the aquatic animals can be improved, and therefore fish blast can be effectively prevented from occurring and spreading, and the survival rate of the aquatic animals is increased. The loss in the breeding process is reduced. Meanwhile, the Chinese herbal medicines are fermented, so that effective components in the Chinese herbal medicines are more convenient to absorb and utilize by aquatic animals, and the capability of resisting fish blast is enhanced. Furthermore, the traditional Chinese medicine fermentation liquor is natural and pollution-free, can be stored for a long time, and is convenient to transport and store.

Description

Technical Field

[0001] The invention belongs to the technical field of fermented Chinese medicine and aquaculture, and specifically relates to a Chinese medicine composition, a Chinese medicine fermentation liquid and applications thereof in aquaculture and prevention and treatment of fish plague. Background Art

[0002] Fish plague is a disease that affects fish, especially freshwater fish. Unlike commonly known fish diseases, "fish plague" does not refer to a single disease, but is a general term for a variety of fish diseases caused by one or more mixed infections of bacteria, viruses, fungi and parasites. Symptoms include gill and tail rot, ascites and bleeding. At the same time, there are often infections with parasites, water molds or aphanomyces, which can cause lesions similar to pox on the surface of the fish. Fish plague is distributed worldwide, and because of its rapid spread and relatively high mortality rate after infection, it poses a serious threat to both aquaculture and wild fish populations.

[0003] As a traditional medical resource, Chinese herbal medicine has a long history of application in the field of aquaculture. It has the characteristics of good biocompatibility and environmental protection and no pollution. Therefore, there are also many Chinese herbal medicine compositions for the prevention and treatment of fish plague. For example, some studies have used a Chinese herbal medicine composition composed of camptotheca bark, rhubarb, ground ivy, neem and fine lime powder to prevent and treat gill rot, enteritis, red skin and hemorrhagic disease of grass carp; in other studies, Chinese herbal medicines composed of wormwood, calamus, verbena, honeysuckle vine, lobelia, selfheal, wild chrysanthemum, plantain, houttuynia cordata, purslane and dandelion were used to prevent and treat fish plague.

[0004] Although the existing Chinese medicine compositions also have certain preventive and therapeutic effects on fish plague, most of the existing Chinese medicine compositions are simple combinations of several Chinese herbal medicines, and their active ingredients cannot be effectively stimulated and utilized, so their efficacy is low and the effect of preventing and treating fish plague is limited. Summary of the invention

[0005] The object of the present invention is to provide a Chinese medicine composition, a Chinese medicine fermentation liquid and their application in aquaculture and prevention and treatment of fish plague. The Chinese medicine composition and the Chinese medicine fermentation liquid can effectively prevent and treat fish plague with excellent effects, reduce the occurrence of farmed fish plague diseases, and improve the economic benefits of aquaculture.

[0006] The present invention provides a traditional Chinese medicine composition, comprising Chinese herbal medicine and probiotics;

[0007] The Chinese herbal medicine comprises the following raw materials in parts by weight: 30-50 parts of honeysuckle, 70-90 parts of forsythia, 30-50 parts of astragalus, 50-70 parts of isatis root and 30-50 parts of schisandra;

[0008] The probiotics include one or more of Saccharomyces cerevisiae, Pichia pastoris, Bacillus subtilis and probiotics EcN; the inoculation amount of the probiotics is 60% to 80% of the mass of the Chinese herbal medicine based on the probiotic seed liquid; the OD of the probiotic seed liquid is 600 It is 1.2 to 1.8.

[0009] The present invention also provides a traditional Chinese medicine fermentation broth, which is prepared from the traditional Chinese medicine composition described in the above technical solution.

[0010] Preferably, the effective ingredients of the traditional Chinese medicine fermentation broth include malic acid and / or antimicrobial peptides.

[0011] Preferably, the concentration of the malic acid in the traditional Chinese medicine fermentation broth is 2.157-5.896 g / L; the concentration of the antimicrobial peptide in the traditional Chinese medicine fermentation broth is 0.523-2.178 g / L.

[0012] The present invention also provides a method for preparing the Chinese medicine fermentation broth described in the above technical solution, comprising the following steps:

[0013] Mixing powders of honeysuckle, forsythia, astragalus, isatis root and schisandra chinensis to obtain a Chinese herbal medicine fermentation raw material;

[0014] The Chinese herbal medicine fermentation raw material is mixed with the probiotic seed liquid and fermented to obtain a Chinese herbal medicine fermentation crude liquid;

[0015] The crude Chinese medicine fermentation liquid is sterilized and filtered, and the filtrate is taken for solid-liquid separation to obtain the Chinese medicine fermentation liquid.

[0016] Preferably, the fermentation is anaerobic fermentation; the temperature of the anaerobic fermentation is 20-40°C, the rotation speed is 200 rpm, and the time is 24-72 hours.

[0017] Preferably, the sterilization temperature is 115-125° C. and the time is 10-20 min.

[0018] The present invention also provides a feed, which comprises the traditional Chinese medicine fermentation liquid described in the above technical solution or the traditional Chinese medicine fermentation liquid prepared by the preparation method described in the above technical solution.

[0019] Preferably, the mass percentage of the traditional Chinese medicine fermentation liquid in the feed is 1% to 3%.

[0020] The present invention also provides the use of the Chinese medicine composition described in the above technical solution, the Chinese medicine fermentation liquid described in the above technical solution, the Chinese medicine fermentation liquid prepared by the preparation method described in the above technical solution, or the feed described in the above technical solution in aquaculture and / or preparation of products for preventing and treating fish plague.

[0021] Preferably, the fish plague disease includes fish plague disease caused by bacterial and / or viral infection.

[0022] Preferably, the bacteria include one or more of Aeromonas hydrophila, Vibrio harveyi and Vibrio parahaemolyticus; and the virus includes iridovirus.

[0023] Preferably, the fish plague disease includes one or more of fish enteritis, red skin disease and gill rot.

[0024] Beneficial effects:

[0025] The invention provides a traditional Chinese medicine composition, comprising Chinese herbal medicine and probiotics; the Chinese herbal medicine comprises the following raw materials in parts by weight: 30-50 parts of honeysuckle, 70-90 parts of forsythia, 30-50 parts of astragalus, 50-70 parts of isatis root and 30-50 parts of schisandra chinensis; the probiotics comprise one or more of saccharomyces cerevisiae, pichia pastoris, bacillus subtilis and probiotics EcN; the inoculation amount of the probiotics is 60%-80% of the weight of the Chinese herbal medicine based on probiotic seed liquid, and the OD value of the probiotic seed liquid is 0.1%. 600 The present invention compounds Chinese herbal medicine with probiotics, and through the fermentation and metabolism function of the probiotics, improves the types and contents of effective active ingredients in the fermented product of the Chinese herbal medicine composition.

[0026] On this basis, the present invention also provides a Chinese medicine fermentation liquid, which is prepared by the above-mentioned Chinese medicine composition. The Chinese herbal medicine of the present invention is decomposed and metabolized by probiotics to produce effective ingredients including malic acid and antimicrobial peptides, and the content of the effective ingredients is relatively high, which can effectively resist the infection of common pathogens or viruses of aquatic animals, improve the immunity of aquatic animals, thereby effectively preventing the occurrence and spread of fish plague and reducing losses in the breeding process. At the same time, by fermenting the Chinese herbal medicine, the effective ingredients in the Chinese herbal medicine are more convenient for aquatic animals to absorb and utilize, and enhance the ability to resist fish plague. Furthermore, the Chinese medicine fermentation liquid is obtained by fermenting Chinese herbal medicine and probiotics, is natural and pollution-free, can be stored for a long time, and is convenient for transportation and storage; at the same time, the preparation process of the Chinese medicine fermentation liquid is simple and easy to mass produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0028] Figure 1 The graph shows the results of the antibacterial zone experiment of the Chinese medicine fermentation broth prepared in Example 8 and the blank control group, wherein the left graph is the blank control group and the right graph is the Chinese medicine fermentation broth group of Example 8. DETAILED DESCRIPTION

[0029] The present invention provides a traditional Chinese medicine composition, comprising Chinese herbal medicine and probiotics; the Chinese herbal medicine comprises the following raw materials in parts by weight: 30-50 parts of honeysuckle, 70-90 parts of forsythia, 30-50 parts of astragalus, 50-70 parts of isatis root and 30-50 parts of schisandra chinensis;

[0030] The probiotics include one or more of Saccharomyces cerevisiae, Pichia pastoris, Bacillus subtilis and probiotics EcN; the inoculation amount of the probiotics is 60% to 80% of the mass of the Chinese herbal medicine based on the probiotic seed liquid; the OD of the probiotic seed liquid is 600 It is 1.2 to 1.8.

[0031] As an embodiment, in the Chinese medicine composition of the present invention, the mass fraction of the honeysuckle is 40-50 parts; the mass fraction of the forsythia is 80-90 parts; the mass fraction of the astragalus is 40-50 parts; the mass fraction of the isatis root is 60-70 parts; the mass fraction of the schisandra chinensis is 40-50 parts. As an embodiment, the probiotics are Saccharomyces cerevisiae, Pichia pastoris, Bacillus subtilis or probiotics EcN; as another embodiment, the probiotics are probiotics EcN. The present invention does not specifically limit the source of the probiotics, and probiotics from conventional commercial sources in the field can be used. As an embodiment, the inoculation amount of the probiotics is 65% to 75% of the mass of the Chinese herbal medicine. As an embodiment, the OD of the probiotic seed solution is 600 is 1.4 to 1.6; as another embodiment, the OD of the probiotic seed solution 600 is 1.5.

[0032] The probiotic EcN in the present invention is a genetically modified Escherichia coli strain, which can improve the growth performance and immunity of animals, reduce the occurrence of diseases and the use of antibiotics; and can improve the growth performance, immunity and intestinal health of aquatic animals when used in combination with the Chinese herbal medicine of the present invention. The brewer's yeast of the present invention can promote the intestinal health of aquatic animals, balance the intestinal flora, reduce the growth of harmful bacteria, and thus reduce the incidence of diseases. The Bacillus subtilis of the present invention can promote the growth of aquatic animals, improve feed utilization, improve the intestinal health of aquatic animals, increase the number of beneficial intestinal bacteria, and thus improve the absorption and utilization of nutrients. The Pichia pastoris of the present invention has a fast fermentation speed and rich nutrients, thereby improving the antiviral ability of aquatic animals, improving the survival rate after virus infection, and further promoting the weight growth of aquatic animals.

[0033] As an embodiment, the preparation method of the probiotic seed liquid comprises the following steps: inoculating a probiotic monoclone into a liquid culture medium for shaking culture to obtain the probiotic culture liquid. As an embodiment, the temperature of the shaking culture is 20-40°C; as another embodiment, the temperature of the shaking culture is 25-37°C. As an embodiment, the rotation speed of the shaking culture is 200rpm. As an embodiment, the shaking culture time is 8-12h; as another embodiment, the shaking culture time is 9-10h. As an embodiment, when the probiotic is Saccharomyces cerevisiae or Pichia pastoris, the liquid culture medium is YPD culture medium; when the probiotic is Bacillus subtilis or probiotic EcN, the liquid culture medium is LB culture medium. As an embodiment, the pH value during the shaking culture process is 5-8; as another embodiment, when the probiotic is Saccharomyces cerevisiae or Pichia pastoris, the pH value is 5; when the probiotic is Bacillus subtilis or probiotic EcN, the pH value is 7.

[0034] The present invention also provides a Chinese medicine fermentation liquid, which is prepared from the Chinese medicine composition described in the above technical solution. As an embodiment, the effective ingredients of the Chinese medicine fermentation liquid of the present invention may include malic acid and / or antimicrobial peptides; as another embodiment, the effective ingredients of the Chinese medicine fermentation liquid may be malic acid and antimicrobial peptides. As an embodiment, the concentration of malic acid in the Chinese medicine fermentation liquid is 2.157-5.896 g / L; the concentration of antimicrobial peptides in the Chinese medicine fermentation liquid is 0.523-2.178 g / L. The malic acid in the Chinese medicine fermentation liquid of the present invention has anti-stress activation and can better prevent and treat fish plague, especially fish enteritis, red skin disease or gill rot. The antimicrobial peptides of the present invention have the effect of stimulating or enhancing the body's immune response through a variety of immunomodulatory effects.

[0035] The present invention also provides a method for preparing the Chinese medicine fermentation broth described in the above technical solution, comprising the following steps:

[0036] Mixing powders of honeysuckle, forsythia, astragalus, isatis root and schisandra chinensis to obtain a Chinese herbal medicine fermentation raw material;

[0037] The Chinese herbal medicine fermentation raw material is mixed with the probiotic seed liquid and fermented to obtain a Chinese herbal medicine fermentation crude liquid;

[0038] The crude Chinese medicine fermentation liquid is sterilized and filtered, and the filtrate is taken for solid-liquid separation to obtain the Chinese medicine fermentation liquid.

[0039] The present invention mixes powders of honeysuckle, forsythia, astragalus, isatis root and schisandra chinensis to obtain Chinese herbal medicine fermentation raw materials. As an embodiment, the particle sizes of the powders of honeysuckle, forsythia, astragalus, isatis root and schisandra chinensis of the present invention are 80 to 200 meshes respectively; as another embodiment, the particle sizes of the powders of honeysuckle, forsythia, astragalus, isatis root and schisandra chinensis are 120 to 160 meshes respectively.

[0040] After obtaining the Chinese medicine fermentation raw material, the present invention mixes the Chinese herbal medicine fermentation raw material with the probiotic seed liquid and ferments to obtain a Chinese medicine fermentation crude liquid. As an embodiment, the fermentation of the present invention is anaerobic fermentation; the temperature of the anaerobic fermentation is 20-40°C, the rotation speed is 200rpm, and the time is 24-72h; as another embodiment, the temperature of the anaerobic fermentation is 25-37°C; the time is 36-48h.

[0041] 4) After obtaining the crude Chinese medicine fermentation liquid, the present invention sterilizes and filters the crude Chinese medicine fermentation liquid, takes the filtrate for solid-liquid separation, and obtains the Chinese medicine fermentation liquid. As an embodiment, the sterilization temperature of the present invention is 115-125°C, and the time is 10-20min; as another embodiment, the sterilization temperature is 121°C, and the time is 15min. As an embodiment, the filtering condition can be filtering with a conventional 20μm pore size filter paper, and the filtration can remove the blocky Chinese medicine powder in the crude Chinese medicine fermentation liquid. As an embodiment, the solid-liquid separation method can be centrifugation; the centrifugal speed is 2000-4000rpm; the time is 10-20min; as another embodiment, the centrifugal speed is 2000-3000rpm; the time is 15-20min.

[0042] The present invention also provides a feed, which includes the Chinese medicine fermentation liquid described in the above technical solution or the Chinese medicine fermentation liquid prepared by the preparation method described in the above technical solution. As an embodiment, the mass percentage of the Chinese medicine fermentation liquid in the feed is 1% to 3%; as another embodiment, the mass percentage of the Chinese medicine fermentation liquid in the feed is 2%.

[0043] The present invention also provides the use of the Chinese medicine composition described in the above technical solution, the Chinese medicine fermentation liquid described in the above technical solution, the Chinese medicine fermentation liquid prepared by the preparation method described in the above technical solution, or the feed described in the above technical solution in aquaculture and / or the preparation of products for preventing and treating fish plague. As an embodiment, the aquatic product is fish, shrimp, turtle or tongue sole; as another embodiment, the aquatic product is fish. As an embodiment, the fish plague includes fish plague caused by bacterial and / or viral infection; as another embodiment, the bacteria are one or more of Aeromonas hydrophila, Vibrio harveyi and Vibrio parahaemolyticus; the virus is iridovirus.

[0044] As an embodiment, the fish plague disease includes one or more of fish enteritis, red skin disease and gill rot disease.

[0045] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] In the following examples, if no specific conditions are specified, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0047] In the following examples, unless otherwise specified, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market;

[0048] In the following examples, the Saccharomyces cerevisiae (BNCC142548) used was purchased from Beina Biotechnology, Pichia pastoris (BNCC141852) was purchased from Beina Biotechnology, Bacillus subtilis (CGMCC 1.12939) was provided by China General Microbiological Culture Collection Center, and probiotics EcN was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.;

[0049] In the following examples, Aeromonas hydrophila (CICC 10868) was provided by China Industrial Microbiological Culture Collection Administration Center, Vibrio harveyi (BB170) was purchased from Wuhan Warner Biotechnology Co., Ltd., iridovirus was isolated from largemouth bass infected in a sea bass farm in Guangdong, and Vibrio parahaemolyticus (CICC 21617) was provided by China Industrial Microbiological Culture Collection Administration Center.

[0050] Examples 1 to 12

[0051] 1. A Chinese medicine composition for preventing and treating fish plague, the formula of which is shown in Table 1:

[0052] Table 1 Prescriptions of the Chinese medicine compositions of Examples 1 to 12

[0053]

[0054]

[0055] 2. A method for preparing a Chinese medicine fermentation broth using the Chinese medicine composition of step 1, comprising the following steps:

[0056] 1) Grind honeysuckle, forsythia, astragalus, isatis root and schisandra chinensis into 120 meshes, mix well and obtain a mixed material.

[0057] 2) Add water at a material-liquid ratio of 1 g:5 mL and mix well to obtain a fermentation substrate.

[0058] 3) Inoculate the fermentation substrate with a seed solution of Saccharomyces cerevisiae or a seed solution of Pichia pastoris according to the corresponding inoculation amount in Table 1, and ferment at 37° C. and 200 rpm for 48 h. During the fermentation process, real-time detection of the fermentation pH value is maintained between 4 and 6 to obtain a crude Chinese medicine fermentation solution.

[0059] Alternatively, the fermentation substrate is inoculated with Bacillus subtilis seed solution or probiotic EcN seed solution according to the corresponding inoculation amount in Table 1, and fermented at 37° C. and 200 rpm for 48 h. During the fermentation process, the fermentation pH is detected in real time and maintained between 7 and 8 to obtain a crude Chinese medicine fermentation liquid.

[0060] The method for preparing the brewer's yeast seed solution is as follows: a single clone of the selected brewer's yeast is inoculated into 50 mL of YPD medium, and cultured at 37°C and 200 rpm for 10 h to obtain the brewer's yeast seed solution. 600 The value is 1.5;

[0061] Method for preparing Pichia pastoris seed solution: inoculate a single clone of Pichia pastoris picked into 50 mL of YPD medium and culture at 37°C and 200 rpm for 10 h to obtain Pichia pastoris seed solution. 600 The value is 1.5;

[0062] Preparation method of probiotic EcN seed solution: inoculate a single clone of probiotic EcN strain into 50 mL of LB medium and culture at 37°C and 200 rpm for 10 h to obtain probiotic EcN seed solution. 600 The value is 1.5;

[0063] Preparation method of Bacillus subtilis seed solution: inoculate a single clone of Bacillus subtilis strain into 50 mL of LB medium and culture at 37°C and 200 rpm for 10 h to obtain Bacillus subtilis seed solution. 600 The value is 1.5.

[0064] 5) The crude Chinese medicine fermentation liquid in step 3) is sterilized at a high temperature of 121° C. for 15 min, the sterilized crude Chinese medicine fermentation liquid is first filtered with a 25 μm pore size filter paper to remove larger pieces of Chinese medicine powder, and then centrifuged at 4000 rpm for 20 min to obtain the Chinese medicine fermentation liquid.

[0065] 3. Determine the content of antimicrobial peptides and malic acid in the traditional Chinese medicine fermentation broth obtained in step 2

[0066] 3.1 Determination of antimicrobial peptide content, the steps are as follows:

[0067] 1) The supernatant obtained by centrifugation (i.e., the Chinese medicine fermentation liquid) was evaporated at 45° C. using a rotary evaporator until the mass ratio of the concentrate to the original supernatant was 1:4.

[0068] 2) Add 80 mL of distilled water to the obtained sample to make up the volume, and then stir with a magnetic stirrer at 25-37° C. and 100-500 r / min for 10-15 min.

[0069] 3) Take an appropriate amount of the supernatant in step 2), add an equal volume of 10 g / 100 mL trichloroacetic acid solution, centrifuge at 6000 rpm for 10 min to obtain the supernatant, then filter it through a 0.45 μm microporous membrane to remove the residual bacteria and macromolecular proteins in the fermentation, and then freeze-dry it in a vacuum, and store it in a refrigerator at 4°C for later use.

[0070] 4) The sample obtained in step 3) was dissolved in ultrapure water to a concentration of 10 mg / mL, filtered through a 0.22 μm water filter membrane, and loaded into an affinity filler Ni-Resin HP chromatography column. The sample was eluted with ultrapure water, the absorbance was measured at a wavelength of 280 nm, and the antimicrobial peptides were collected every 2 minutes, and the content of the antimicrobial peptides was calculated.

[0071] The content of antimicrobial peptides in the Chinese medicine fermentation broth obtained in Examples 1 to 12 in step 2 was measured according to the method in 3.1. The results are shown in Table 2.

[0072] 3.2 Determination method and data analysis of malic acid content. The steps of content determination are as follows:

[0073] 1) First, prepare 0.2% phosphoric acid, and then place it on ice for 15 minutes. Take 5 ml of the obtained fermentation liquid, add 20 mL of pre-cooled phosphoric acid, vortex to mix, and then place it in a pre-cooled centrifuge and centrifuge at 6000 rpm / min for 30 minutes until it is clear.

[0074] 2) The supernatant obtained in step 1) was filtered through a 0.22 μm microporous filter membrane using a 1 mL syringe to obtain a test solution.

[0075] 3) The malic acid content was determined using Shimadzu LC-20A liquid chromatography.

[0076] 4) The chromatographic conditions are as follows: the chromatographic column is a reverse phase C18 column, the mobile phase is 0.2% phosphoric acid and methanol, 0.2% phosphoric acid accounts for 96%, methanol accounts for 4%, the flow rate is 1.2 mL / min, the injection volume is 10 μL, the column temperature is 30°C, the detection wavelength is 210 nm, and the results of measuring the content of malic acid in the traditional Chinese medicine fermentation broth are shown in Table 2.

[0077] Table 2 Determination results of antimicrobial peptides and malic acid in the traditional Chinese medicine fermentation broth of Examples 1 to 12

[0078]

[0079]

[0080] It can be concluded from Table 2 that the traditional Chinese medicine fermentation broth of the present invention contains antimicrobial peptides and malic acid, and the concentrations of the antimicrobial peptides and malic acid in the traditional Chinese medicine fermentation broth are 0.523-2.178 g / L and 2.157-5.896 g / L, respectively.

[0081] Embodiment 13

[0082] The antibacterial effect of the Chinese medicinal fermentation broth prepared in Examples 1 to 12 on Aeromonas hydrophila, Vibrio harveyi, iridovirus, and Vibrio parahaemolyticus was verified by the following steps:

[0083] Freshly prepared Aeromonas hydrophila, Vibrio harveyi, iridovirus or Vibrio parahaemolyticus seed stock was diluted to 1 × 10 6 cfu / mL, draw 150μL and evenly spread it on the LB solid plate; clamp three sterile filter paper pieces and place them on the surface of the LB medium coated with bacterial solution in a triangular symmetry, add 20μL of the Chinese medicine fermentation liquid to the sterile filter paper pieces respectively, and use the blank control group without adding Chinese medicine fermentation liquid; let it stand for 15 minutes, and after the liquid is fully diffused, transfer it to a 37℃ constant temperature incubator and culture it for 12 hours; after the incubation, observe and measure the size of the inhibition zone with a ruler;

[0084] The preparation steps of the seed solution of Aeromonas hydrophila, Vibrio harveyi, iridovirus or Vibrio parahaemolyticus are as follows: single clones are picked from the four pathogen plates respectively, and then the bacteria are shaken in LB medium. 600 When the concentration of 1:1 is 0.6, the seed solutions of the four pathogens mentioned above are obtained respectively.

[0085] The Chinese medicine fermentation broth prepared in Examples 1 to 12 were all subjected to antibacterial verification using the above steps, and the results are shown in Table 3; the antibacterial zone experimental results of the Chinese medicine fermentation broth in Example 8 and the blank control group are shown in Figure 3. Figure 1 As shown, Figure 1 The three inhibition zones in the middle represent three biological replicates.

[0086] Table 3 Antibacterial test results of 12 kinds of Chinese medicine fermentation broths prepared in Examples 1 to 12

[0087]

[0088]

[0089] It can be concluded from Table 3 that the sizes of the inhibition zones of the 12 kinds of Chinese medicine fermentation broths are all different. First, the component content analysis is performed. The inhibition zones of the four kinds of Chinese medicine fermentation broth strains in Examples 1 to 4 are the smallest overall, and the antibacterial ability is the weakest. The inhibition zones of the four kinds of Chinese medicine fermentation broth strains in Examples 5 to 8 are the largest overall, and the antibacterial ability is the strongest. The inhibition zones of the four kinds of fermentation broth strains in Examples 9 to 12 are in the middle as a whole, and the antibacterial ability is average. Analysis of the antibacterial ability of different strains shows that the antibacterial ability of the 12 kinds of fermentation broths is respectively anti-Vibrio parahaemolyticus > anti-Vibrio harveyi > anti-Aeromonas hydrophila > anti-iridovirus.

[0090] From the overall analysis results, it can be seen that the Chinese medicine fermentation broths in Examples 1 to 12 all have a certain antibacterial effect, and the Chinese medicine fermentation broths in Examples 4, 8 and 12 are all obtained by probiotic EcN fermentation, and their antibacterial effects are relatively better, which means that the probiotic EcN fermentation effect is the best.

[0091] Embodiment 14

[0092] The aquaculture test of the Chinese medicinal fermentation liquid of Examples 4, 8 and 12 is as follows:

[0093] A total of 400 carps with fish enteritis, red skin disease or gill rot caused by bacterial infection were taken and divided into 4 groups, each with 100 carps. The experimental groups were fed with the Chinese medicine fermentation liquid prepared in Example 4, Example 8 and Example 12 respectively; the blank control group was fed with bait without any drug; the dosage of the Chinese medicine fermentation liquid was 10g of Chinese medicine fermentation liquid / 1kg carp per day, and the drug was used for 7 consecutive days and then observed for 3 days. The disappearance of disease symptoms and the return of spirit and eating to normal were considered cured, and the absence of improvement or death was considered ineffective. The results are shown in Table 4.

[0094] Table 4 Effect of the Chinese medicinal fermentation broth prepared in Examples 4, 8 and 12 on the treatment of fish enteritis

[0095]

[0096] It can be concluded from Table 4 that the Chinese medicinal fermentation broth has a significant therapeutic effect on the treatment of fish enteritis. The Chinese medicinal fermentation broth prepared in Example 8 has a better effect on the treatment of enteritis, with a cure rate of 91%, followed by the Chinese medicinal fermentation broth prepared in Example 4 with a cure rate of 81%, and finally the Chinese medicinal fermentation broth prepared in Example 12 with a cure rate of 76%.

[0097] Embodiment 15

[0098] The aquaculture test of the Chinese medicinal fermentation liquid of Examples 5, 6, 7 and 8 is as follows:

[0099] 500 carps with fish enteritis, red skin disease or gill rot caused by bacterial infection were divided into 5 groups, each with 100 carps. The experimental groups were fed with the Chinese medicine fermentation liquid prepared in Examples 5, 6, 7 and 8 respectively; the blank control group was fed with bait without any medicine; the dosage of the Chinese medicine fermentation liquid was 10g medicine / 1kg carp per day, and the drug was used for 7 consecutive days and then observed for 3 days. The disappearance of the disease symptoms and the return of spirit and eating to normal were considered as cure, and the absence of improvement or death were considered as ineffective. The results are shown in Table 4.

[0100] Table 5 Effect of the Chinese medicinal fermentation broth prepared in Examples 5, 6, 7 and 8 on the treatment of fish enteritis

[0101]

[0102] It can be concluded from the data in Table 5 that the Chinese medicinal fermentation broth has a significant therapeutic effect on the treatment of fish enteritis. The Chinese medicinal fermentation broth prepared in Examples 5, 6, 7 and 8 is fermented by Saccharomyces cerevisiae, Pichia pastoris, Bacillus subtilis and probiotics EcN, respectively. The experimental results show that the Chinese medicinal fermentation broth fermented by probiotics EcN has the best ability to treat enteritis, with a cure rate of 91%.

[0103] It can be concluded from the above examples that the Chinese medicine composition and Chinese medicine fermentation liquid of the present invention can effectively prevent and treat fish plague with excellent effects, reduce the occurrence of farmed fish plague diseases, and improve the economic benefits of aquaculture.

[0104] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Chinese medicine composition, characterized in that: Including Chinese herbs and probiotics; The Chinese herbal medicine comprises the following raw materials in parts by weight: 30-50 parts of honeysuckle, 70-90 parts of forsythia, 30-50 parts of astragalus, 50-70 parts of isatis root and 30-50 parts of schisandra; The probiotics include one or more of Saccharomyces cerevisiae, Pichia pastoris, Bacillus subtilis and probiotic EcN; The inoculation amount of the probiotics is 60% to 80% of the mass of the Chinese herbal medicine based on the probiotics seed solution. 600 It is 1.2 to 1.

8.

2. A Chinese medicine fermentation liquid, characterized in that: The traditional Chinese medicine fermentation broth is prepared from the traditional Chinese medicine composition according to claim 1.

3. The Chinese medicine fermentation liquid according to claim 2, characterized in that: The effective ingredients of the traditional Chinese medicine fermentation liquid include malic acid and / or antimicrobial peptides.

4. The Chinese medicine fermentation liquid according to claim 3, characterized in that: The concentration of the malic acid in the traditional Chinese medicine fermentation broth is 2.157-5.896 g / L; the concentration of the antimicrobial peptide in the traditional Chinese medicine fermentation broth is 0.523-2.178 g / L.

5. The method for preparing the Chinese medicine fermentation broth according to any one of claims 2 to 4, characterized in that: The steps include: Mixing powders of honeysuckle, forsythia, astragalus, isatis root and schisandra chinensis to obtain a Chinese herbal medicine fermentation raw material; The Chinese herbal medicine fermentation raw material is mixed with the probiotic seed liquid and then fermented to obtain a Chinese herbal medicine fermentation crude liquid; The crude Chinese medicine fermentation liquid is sterilized and filtered, and the filtrate is taken for solid-liquid separation to obtain the Chinese medicine fermentation liquid.

6. The preparation method according to claim 5, characterized in that: The fermentation is anaerobic fermentation; the temperature of the anaerobic fermentation is 20-40°C, the rotation speed is 200rpm, and the time is 24-72h.

7. The preparation method according to claim 5, characterized in that: The sterilization temperature is 115-125° C. and the sterilization time is 10-20 minutes.

8. A feed, characterized in that: The feed comprises the Chinese medicinal fermentation liquid as described in any one of claims 2 to 4 or the Chinese medicinal fermentation liquid prepared by the preparation method as described in any one of claims 5 to 7.

9. The feed according to claim 8, characterized in that: The mass percentage of the traditional Chinese medicine fermentation liquid in the feed is 1% to 3%.

10. Use of the Chinese medicine composition according to claim 1, the Chinese medicine fermentation liquid according to any one of claims 2 to 4, the Chinese medicine fermentation liquid prepared by the preparation method according to any one of claims 5 to 7, or the feed according to claim 8 or 9 in aquaculture and / or preparation of products for preventing and treating fish plague.

11. The use according to claim 10, characterized in that: The fish plague disease includes fish plague disease caused by bacteria and / or virus infection.

12. The use according to claim 11, characterized in that: The bacteria include one or more of Aeromonas hydrophila, Vibrio harveyi and Vibrio parahaemolyticus; the virus includes iridovirus.

13. The use according to any one of claims 10 to 12, characterized in that: The fish plague diseases include one or more of fish enteritis, red skin disease and gill rot.

Images