Compound Chinese herbal medicine fermentation preparation for preventing and treating white shrimp mc liver toxicity and preparation method thereof

Abstract

The application discloses a compound Chinese herbal medicine fermentation preparation for preventing and treating hepatotoxicity of Penaeus vannamei and a preparation method thereof. Spore bacillus is used as a fermentation strain to perform aerobic fermentation on four traditional Chinese herbal medicine stem and leaf wastes, i.e. Astragalus stem and leaf, Rhubarb stem and leaf, Codonopsis stem and leaf and Coptis stem and leaf, so as to obtain a compound Chinese herbal medicine fermentation product with higher active substances. The compound Chinese herbal medicine fermentation preparation of the application can be added into the feed of Penaeus vannamei, so as to effectively prevent and treat hepatotoxicity of Penaeus vannamei.

Description

Technical Field

[0001] This invention belongs to the field of feed additives for Litopenaeus vannamei, and in particular relates to a compound traditional Chinese medicine fermentation preparation and its preparation method for preventing and treating microcystin (hereinafter referred to as MCs) hepatotoxicity in Litopenaeus vannamei. Background Technology

[0002] Aquaculture is a crucial pillar of my country's fishery economy. In 2022, my country's aquaculture output reached 55.6546 million tons, accounting for 81% of the total aquatic output. With the prevalence of high-density, intensive farming methods, frequent outbreaks of aquatic animal diseases have severely hampered the healthy and sustainable development of the aquaculture industry. For decades, farmers have frequently used antibiotics to prevent and control diseases in aquatic animals. According to irregular statistics, my country's annual antibiotic production is approximately 210,000 tons, with antibiotic usage in aquaculture reaching 97,000 tons, accounting for about 46% of total production. In 2019, the Ministry of Agriculture and Rural Affairs issued Announcement No. 194, marking the official start of a comprehensive ban on antibiotics in my country. Compared with antibiotics, traditional Chinese medicine (TCM) has advantages such as no drug resistance, fewer toxic side effects, and no drug residues, and can improve animal growth performance and immune function. The cell wall structure of TCM is not conducive to the absorption and utilization of its active substances, but microbial fermentation can effectively decompose the cell wall, thereby allowing for the efficient release of its active ingredients.

[0003] The harvesting and processing of traditional Chinese medicinal herbs generates a large amount of waste. Statistics show that in recent years, my country's waste volume has reached 60-70 million tons, which is often directly discarded, wasting medicinal resources and causing environmental pollution. Research indicates that this waste is rich in active substances such as polysaccharides, saponins, and flavonoids. Using this waste as a raw material for herbal additives would effectively solve both the huge resource waste caused by direct disposal and the problem of high raw material prices in herbal additives. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a compound herbal fermentation preparation and its preparation method for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei, which can effectively prevent and treat MCs hepatotoxicity in Litopenaeus vannamei. The Bacillus used was deposited on August 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.28195 and classified as Bacillus sp.

[0005] To address the aforementioned technical problems, this invention provides a compound herbal fermentation preparation for preventing and treating macrophages (MCs) hepatotoxicity in Litopenaeus vannamei. The preparation comprises a compound herbal raw material for fermentation and a microbial agent for fermentation. The compound herbal raw material consists of the following components in parts by weight: 4-6 parts dried powder of Astragalus membranaceus stems and leaves, 3-5 parts dried powder of Rheum palmatum stems and leaves, 2-4 parts dried powder of Codonopsis pilosula stems and leaves, and 1-3 parts dried powder of Coptis chinensis stems and leaves. The microbial agent for fermentation is a diluted Bacillus spp. culture broth diluted to a viable cell density of 1.0 × 10⁻⁶. 9 CFU / mL -1.5×10 9 The fermentation microbial agent with a concentration of CFU / mL, the mass ratio of compound Chinese herbal medicine raw material to fermentation microbial agent is 1:(1-2), the Bacillus preservation number is CGMCC NO.28195, and the classification name is Bacillus sp.

[0006] The compound herbal raw materials consist of the following components in parts by weight: 5 parts dried powder of Astragalus membranaceus stems and leaves, 4 parts dried powder of Rheum palmatum stems and leaves, 3 parts dried powder of Codonopsis pilosula stems and leaves, and 2 parts dried powder of Coptis chinensis stems and leaves.

[0007] The fineness of the powdered stems and leaves of Astragalus membranaceus, Rheum palmatum, Codonopsis pilosula, and Coptis chinensis is ≥60 mesh.

[0008] The preparation method of the compound herbal fermentation preparation for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei, as described above, includes the following steps:

[0009] (1) Bacillus was cultured for 12-24 hours under the conditions of a carbon-to-nitrogen ratio of 8-12:1, an inoculum concentration of 3%-6%, a pH of 6.5-7.5, and a temperature of 31-37℃ to obtain a bacterial culture solution. The Bacillus culture solution in the late stage of exponential culture was diluted to a viable cell density of 1.0 × 10⁻⁶. 9 CFU / mL -1.5×10 9 CFU / mL fermentation microbial inoculants;

[0010] (2) The dried powders of Astragalus membranaceus stems and leaves, Rheum palmatum stems and leaves, Codonopsis pilosula stems and leaves, and Coptis chinensis stems and leaves were dried at 65°C, pulverized and passed through a 60-mesh sieve, and sterilized under high temperature and high pressure. The following proportions of each Chinese herbal medicine were accurately weighed: 4-6 parts of dried powder of Astragalus membranaceus stems and leaves, 3-5 parts of dried powder of Rheum palmatum stems and leaves, 2-4 parts of dried powder of Codonopsis pilosula stems and leaves, and 1-3 parts of dried powder of Coptis chinensis stems and leaves. They were mixed evenly to obtain the compound Chinese herbal medicine raw materials for later use.

[0011] (3) Under good breeding conditions, the compound Chinese herbal medicine raw materials and fermentation microbial inoculants were prepared by mass ratio of 1:(1-2), fermentation temperature of 31-37℃ and fermentation time of 48-96h to obtain the compound Chinese herbal medicine fermentation preparation for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei.

[0012] The beneficial effect of this invention is that adding the compound fermented Chinese herbal medicine preparation of this invention to the feed of Litopenaeus vannamei can effectively prevent and treat MCs hepatotoxicity in Litopenaeus vannamei. Attached Figure Description

[0013] Figure 1 Weight gain rate of Litopenaeus vannamei in different experimental groups using the compound herbal fermentation preparation of this invention;

[0014] Figure 2 Specific growth rates of Litopenaeus vannamei in different experimental groups using the compound herbal fermentation preparation of this invention;

[0015] Figure 3 Survival rates of Litopenaeus vannamei in different experimental groups using the compound herbal fermentation preparation of this invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0017] This invention discloses a compound herbal fermentation preparation for preventing and treating macrophages (MCs) hepatotoxicity in Litopenaeus vannamei, comprising a compound herbal raw material for fermentation and a microbial agent for fermentation. The compound herbal raw material consists of the following components in parts by weight: 4-6 parts dried powder of Astragalus membranaceus stems and leaves, 3-5 parts dried powder of Rheum palmatum stems and leaves, 2-4 parts dried powder of Codonopsis pilosula stems and leaves, and 1-3 parts dried powder of Coptis chinensis stems and leaves. The microbial agent for fermentation is a diluted solution of Bacillus spp. culture medium diluted to a viable cell density of 1.0 × 10⁻⁶. 9 CFU / mL -1.5×10 9 The fermentation microbial agent with a concentration of CFU / mL, the mass ratio of compound Chinese herbal raw materials to fermentation microbial agent is 1:(1-2), the Bacillus preservation number is CGMCC NO.28195, and the classification name is Bacillus sp.

[0018] The compound herbal raw materials consist of the following components in parts by weight: 5 parts dried powder of Astragalus membranaceus stems and leaves, 4 parts dried powder of Rheum palmatum stems and leaves, 3 parts dried powder of Codonopsis pilosula stems and leaves, and 2 parts dried powder of Coptis chinensis stems and leaves.

[0019] The fineness of the powdered stems and leaves of Astragalus membranaceus, Rheum palmatum, Codonopsis pilosula, and Coptis chinensis is ≥60 mesh.

[0020] The preparation method of the compound herbal fermentation preparation for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei, as described above, includes the following steps:

[0021] (1) Bacillus was cultured for 12-24 hours under the conditions of a carbon-to-nitrogen ratio of 8-12:1, an inoculum concentration of 3%-6%, a pH of 6.5-7.5, and a temperature of 31-37℃ to obtain a bacterial culture solution. The Bacillus culture solution in the late stage of exponential culture was diluted to a viable cell density of 1.0 × 10⁻⁶. 9 CFU / mL -1.5×10 9 CFU / mL fermentation microbial inoculants;

[0022] (2) The dried powders of Astragalus membranaceus stems and leaves, Rheum palmatum stems and leaves, Codonopsis pilosula stems and leaves, and Coptis chinensis stems and leaves were dried at 65°C, pulverized and passed through a 60-mesh sieve, and sterilized under high temperature and high pressure. The following proportions of each Chinese herbal medicine were accurately weighed: 4-6 parts of dried powder of Astragalus membranaceus stems and leaves, 3-5 parts of dried powder of Rheum palmatum stems and leaves, 2-4 parts of dried powder of Codonopsis pilosula stems and leaves, and 1-3 parts of dried powder of Coptis chinensis stems and leaves. They were mixed evenly to obtain the compound Chinese herbal medicine raw materials for later use.

[0023] (3) Under good breeding conditions, the compound Chinese herbal medicine raw materials and fermentation microbial inoculants were prepared by mass ratio of 1:(1-2), fermentation temperature of 31-37℃ and fermentation time of 48-96h to obtain the compound Chinese herbal medicine fermentation preparation for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei.

[0024] The Bacillus used was deposited on August 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.28195 and classified as Bacillus sp.

[0025] Example 1

[0026] A compound herbal fermentation preparation for preventing and treating hepatotoxicity of macrophages (MCs) in Litopenaeus vannamei is prepared according to the following steps:

[0027] (1) The optimal culture conditions for Bacillus were determined using orthogonal experiments: a carbon-to-nitrogen ratio of 8:1, an inoculum size of 3%, a pH of 6.5, and a temperature of 31℃. The culture was then incubated for 12 hours under these optimal conditions to obtain the bacterial culture solution. The Bacillus culture solution from the late stage of exponential growth was diluted to a viable cell density of 1.0 × 10⁻⁶. 9 CFU / mL fermentation microbial inoculants;

[0028] (2) Select dried powders of Astragalus membranaceus stems and leaves, dried powders of Rheum palmatum stems and leaves, dried powders of Codonopsis pilosula stems and leaves, and dried powders of Coptis chinensis stems and leaves. Dry them at 65°C, pulverize them and pass them through a 60-mesh sieve. Sterilize them under high temperature and high pressure. Accurately weigh each Chinese herbal medicine according to the following mass proportions: 4 parts of dried powder of Astragalus membranaceus stems and leaves, 3 parts of dried powder of Rheum palmatum stems and leaves, 2 parts of dried powder of Codonopsis pilosula stems and leaves, and 1 part of dried powder of Coptis chinensis stems and leaves. Mix them evenly to obtain the compound Chinese herbal medicine raw materials for later use.

[0029] (3) The optimal fermentation process of compound Chinese herbal medicine was determined by orthogonal experiment. Under good aquaculture conditions, the compound Chinese herbal medicine fermentation preparation for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei was obtained by using a mass ratio of compound Chinese herbal medicine raw materials to fermentation microbial inoculants of 1:1, fermentation temperature of 31℃, and fermentation time of 48h. The total polysaccharide content of the compound Chinese herbal medicine fermentation preparation after fermentation was 30mg / g.

[0030] Example 2

[0031] The difference between this embodiment and Embodiment 1 is that:

[0032] The Bacillus culture conditions were as follows: carbon-to-nitrogen ratio 10:1, inoculum size 5%, pH 7, temperature 34℃. Under these optimal conditions, the culture was incubated for 16 hours to obtain the bacterial culture broth. The Bacillus culture broth from the late exponential growth stage was then diluted to a viable cell density of 1.2 × 10⁻⁶. 9 CFU / mL fermentation microbial inoculants.

[0033] The traditional Chinese medicine composition comprises the following components in parts by weight: 5 parts of dried powder of Astragalus membranaceus stems and leaves (fineness ≥ 60 mesh), 4 parts of dried powder of Rheum palmatum stems and leaves (fineness ≥ 60 mesh), 3 parts of dried powder of Codonopsis pilosula stems and leaves (fineness ≥ 60 mesh), and 2 parts of dried powder of Coptis chinensis stems and leaves (fineness ≥ 60 mesh).

[0034] The optimal fermentation process for the traditional Chinese medicine is as follows: Under austerity conditions, the compound traditional Chinese medicine raw materials and fermentation microbial agents are fermented at a mass ratio of 1:1.5, a fermentation temperature of 34℃, and a fermentation time of 72h to obtain a compound traditional Chinese medicine fermentation preparation for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei. The total polysaccharide content of the compound traditional Chinese medicine fermentation preparation after fermentation is 35mg / g.

[0035] Example 3

[0036] The difference between this embodiment and Embodiment 1 is that:

[0037] The Bacillus culture conditions were as follows: carbon-to-nitrogen ratio 12:1, inoculum size 6%, pH 7.5, temperature 37℃. Under these optimal conditions, the culture was incubated for 24 hours to obtain the bacterial culture solution. The Bacillus culture solution from the late exponential growth stage was then diluted to a viable cell density of 1.5 × 10⁻⁶ cells / year. 9 CFU / mL fermentation microbial inoculants;

[0038] The traditional Chinese medicine composition comprises the following components in parts by weight: 5 parts of dried powder of Astragalus membranaceus stems and leaves (fineness ≥ 60 mesh), 5 parts of dried powder of Rheum palmatum stems and leaves (fineness ≥ 60 mesh), 4 parts of dried powder of Codonopsis pilosula stems and leaves (fineness ≥ 60 mesh), and 3 parts of dried powder of Coptis chinensis stems and leaves (fineness ≥ 60 mesh).

[0039] The herbal fermentation process is as follows: Under austerity conditions, the herbal raw materials and fermentation microbial agents are mixed at a mass ratio of 1:2, the fermentation temperature is 37℃, and the fermentation time is 96h to obtain a herbal fermentation preparation for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei. The total polysaccharide content of the herbal fermentation preparation after fermentation is 40mg / g.

[0040] To further illustrate the therapeutic effect of the compound fermented Chinese herbal medicine preparation of the present invention on MCs hepatotoxicity in Litopenaeus vannamei, animal experiments are provided below as an illustration; the fermented Chinese herbal medicine microecological preparations involved below were all prepared in accordance with Example 2.

[0041] 1. Materials and Methods

[0042] 1.1 Experimental Animals

[0043] 240 whiteleg shrimp weighing (3.08±0.03g); 240 whiteleg shrimp weighing (12.5±1.0g).

[0044] 1.2 Test Drugs

[0045] The compound herbal fermentation preparation obtained in Example 2 contains unfermented Chinese herbs (with the same proportions as the herbal components in the compound herbal fermentation preparation).

[0046] 1.3 Test Methods

[0047] Growth performance index determination: 240 whiteleg shrimp treated with the drug were randomly divided into 4 groups of 20 shrimp each: negative control group, positive control group, experimental group 1, and experimental group 2, with 3 replicates per group. The positive control group, experimental group 1, and experimental group 2 were all treated with the same dose of the drug. The negative control group and positive control group were fed with whiteleg shrimp feed, while experimental group 1 was fed with 1% compound fermented Chinese herbal medicine preparation in addition to the feed for the control group, and experimental group 2 was fed with 1% unfermented Chinese herbal medicine in addition to the feed for the control group. All whiteleg shrimp were raised under the same conditions and fed 3 times a day at a daily feed amount of 2% of the shrimp's body weight for 30 days.

[0048] Determination of hepatopancreatic toxin content in Litopenaeus vannamei: 240 Litopenaeus vannamei shrimp weighing (12.5±1.0g) were randomly divided into 4 groups of 20 shrimp each: negative control group, positive control group, experimental group 1, and experimental group 2, with 3 replicates per group. The positive control group, experimental group 1, and experimental group 2 were injected with 20μL of MC-LR solution at a concentration of 230.0μg / kg, while the negative control group was injected with an equal volume of PBS solution. The negative control group and positive control group were fed Litopenaeus vannamei shrimp feed, while experimental group 1 was fed with 1% compound fermented Chinese herbal medicine preparation, and experimental group 2 was fed with 1% unfermented Chinese herbal medicine preparation. All Litopenaeus vannamei shrimp were raised under the same conditions, fed 3 times a day at a daily feed amount of 2% of the shrimp's body weight, for a period of 48 hours. 1.4 Efficacy assessment

[0049] Growth performance index determination: The initial weight of the Litopenaeus vannamei was recorded before the start of the experiment, and the final weight was recorded 24 hours after feeding was stopped. The following indicators were calculated after the experiment:

[0050] Weight gain rate (%) = [final weight (g) - initial weight (g)] / initial weight (g) × 100%;

[0051] Specific growth rate (%) / d = 100% × [ln final body weight (g) / ln initial body weight (g)] / number of feeding days;

[0052] Survival rate (%) = (Number of terminal tails / Initial tails) × 100%;

[0053] Determination of hepatopancreatic toxin content: 48 hours after injection, 5 shrimp from each group were taken, their hepatopancreas were dissected, weighed, and freeze-dried for 72 hours. The MC-LR content in the hepatopancreas was determined by high performance liquid chromatography.

[0054] 2. Analysis of the effects of fermented traditional Chinese medicine microecological preparations on the growth performance of Litopenaeus vannamei.

[0055] Table 1 shows the treatment results of each group of Litopenaeus vannamei during the experiment. As shown in Table 1, the survival rate of Litopenaeus vannamei was 96.67% when only fed with Litopenaeus vannamei feed, but only 38.33% after treatment with the toxin, indicating that Microcystin toxins seriously harm the growth of Litopenaeus vannamei. Compared with the positive control group, the survival rate of the unfermented herbal medicine group (Group 1 and Group 2) was 58.33% after treatment, showing a certain therapeutic effect on MCs hepatotoxicity in Litopenaeus vannamei. The compound fermented herbal medicine preparation of this invention has a significant therapeutic effect on MCs hepatotoxicity in Litopenaeus vannamei, with a survival rate as high as 91.67%. The above experimental results demonstrate that the compound fermented herbal medicine preparation of this invention can effectively prevent and treat MCs hepatotoxicity in Litopenaeus vannamei.

[0056] Table 1

[0057]

[0058] 3. Analysis of the efficacy of fermented traditional Chinese medicine preparations in preventing and treating MCs hepatotoxicity in Litopenaeus vannamei.

[0059] The test results showed that the hepatopancreatic toxin content in the negative control group of Litopenaeus vannamei was (0.09±0.01) μg / kg, while the hepatopancreatic toxin content in the positive control group was as high as (2.80±0.02) μg / kg. The hepatopancreatic toxin content in experimental group 1 was (0.50±0.02) μg / kg, and the hepatopancreatic toxin content in experimental group 2 was (1.81±0.02) μg / kg. These test results indicate that the compound herbal fermentation preparation of this invention can effectively reduce the accumulation of MCs in the hepatopancreas of Litopenaeus vannamei and can effectively prevent and treat MCs hepatotoxicity in Litopenaeus vannamei.

[0060] like Figure 1-3 As shown, the compound fermented Chinese herbal medicine preparation of the present invention for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei can effectively prevent and treat MCs hepatotoxicity in Litopenaeus vannamei.

[0061] This invention uses Bacillus as the fermentation strain to aerobic ferment the stems and leaves of four traditional Chinese medicinal herbs—Astragalus membranaceus stems and leaves, Rheum palmatum stems and leaves, Codonopsis pilosula stems and leaves, and Coptis chinensis stems and leaves—to obtain a compound Chinese medicinal herb fermentation product with higher levels of active substances.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound fermented traditional Chinese medicine preparation for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei, characterized in that, The product includes compound herbal raw materials for fermentation and microbial agents for fermentation. The compound herbal raw materials consist of the following components in parts by weight: 4-6 parts dried powder of Astragalus membranaceus stems and leaves, 3-5 parts dried powder of Rheum palmatum stems and leaves, 2-4 parts dried powder of Codonopsis pilosula stems and leaves, and 1-3 parts dried powder of Coptis chinensis stems and leaves. The microbial agent for fermentation is a diluted solution of Bacillus spp. culture medium, diluted to a viable cell density of 1.0 × 10⁻⁶. 9 CFU / mL -1.5×10 9 The fermentation microbial agent with CFU / mL, the mass ratio of compound Chinese herbal raw materials to fermentation microbial agent is 1:(1-2), the Bacillus preservation number is CGMCC NO.28195, and the classification name is Bacillus. Bacillus sp. .

2. The compound herbal fermentation preparation for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei according to claim 1, characterized in that, The compound herbal raw materials consist of the following components in parts by weight: 5 parts dried powder of Astragalus membranaceus stems and leaves, 4 parts dried powder of Rheum palmatum stems and leaves, 3 parts dried powder of Codonopsis pilosula stems and leaves, and 2 parts dried powder of Coptis chinensis stems and leaves.

3. The compound herbal fermentation preparation for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei according to claim 1 or 2, characterized in that, The fineness of the powdered stems and leaves of Astragalus membranaceus, Rheum palmatum, Codonopsis pilosula, and Coptis chinensis is ≥60 mesh.

4. The preparation method of the compound herbal fermentation preparation for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei as described in claim 1, characterized in that, Includes the following steps: (1) Bacillus was cultured for 12-24 hours under the conditions of carbon-nitrogen ratio of 8-12:1, inoculation amount of 3%-6%, pH of 6.5-7.5, and temperature of 31-37℃ to obtain a bacterial culture medium. The Bacillus culture medium in the late stage of exponential culture was diluted to a viable cell density of 1.0×10⁻⁶. 9 CFU / mL -1.5×10 9 CFU / mL fermentation microbial inoculants; (2) The dried powders of Astragalus membranaceus stems and leaves, Rheum palmatum stems and leaves, Codonopsis pilosula stems and leaves, and Coptis chinensis stems and leaves were dried at 65°C, pulverized and passed through a 60-mesh sieve, and sterilized under high temperature and high pressure. The following proportions of each Chinese herbal medicine were accurately weighed: 4-6 parts of dried powder of Astragalus membranaceus stems and leaves, 3-5 parts of dried powder of Rheum palmatum stems and leaves, 2-4 parts of dried powder of Codonopsis pilosula stems and leaves, and 1-3 parts of dried powder of Coptis chinensis stems and leaves. They were mixed evenly to obtain the compound Chinese herbal medicine raw materials for later use. (3) Under aerobic conditions, the compound Chinese herbal medicine raw materials and fermentation microbial inoculants were prepared in a mass ratio of 1: (1-2), fermentation temperature of 31-37℃, and fermentation time of 48-96 h to obtain the compound Chinese herbal medicine fermentation preparation for preventing and treating MCs hepatotoxicity in Litopenaeus vannamei.

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