Traditional Chinese medicine fermentation preparation for preventing and treating microptera iridovirus disease and preparation method of traditional Chinese medicine fermentation preparation

Abstract

The invention belongs to the field of traditional Chinese medicines, and discloses a preparation method of a traditional Chinese medicine fermentation preparation for preventing and treating micropterus iridovirus, which is characterized in that a traditional Chinese medicine composition is fermented by compound bacteria to obtain the traditional Chinese medicine fermentation preparation, the compound bacteria are a combination of bacillus, clostridium butyricum, pediococcus pentosaceus and lactobacillus; the traditional Chinese medicine composition is a combination of codonopsis pilosula, astragalus membranaceus, liquorice, pithecellobium clypearia and isatis root. The preparation adopts compound bacteria to ferment the traditional Chinese medicine composition to obtain the traditional Chinese medicine fermentation preparation, and the preparation has the advantages of good palatability, easiness in digestion, convenience in disease control of secondary metabolites, quick effect taking and the like.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine, and in particular to a traditional Chinese medicine fermentation preparation for preventing and treating iris virus disease of the genus Rhacophorus and a preparation method thereof. Background Art

[0002] There are two types of California bass iridovirus reported so far, namely California bass frog iridovirus (LMBV) and California bass cytoma iridovirus (ISKNV). California bass frog iridovirus disease usually breaks out in the summer when the water temperature is 25℃~32℃. The symptoms vary greatly among different sizes, mainly manifested as redness and congestion at the base of the pelvic fins, redness, swelling and ulceration of the gill cover, a large number of small superficial ulcer spots on the body surface, or nearly circular, gray-white ulcer spots on the body surface, and white spots on the liver. Cytoma belongs to the iridovirus, and the suitable water temperature for the onset of the disease is 28℃~30℃. Under normal circumstances, the mortality rate will be higher than that of frog iridovirus disease. Its main symptoms are redness and congestion from the lower jaw to the abdomen of the diseased fish, severe liver lesions accompanied by bleeding and swelling, and gray or yellow color.

[0003] Prevention is the main approach to treating California sea bass iris virus disease. The incidence of California sea bass iris virus disease can be reduced through testing of fry, disinfection of fish ponds, water quality management, and feed management. However, ponds that have already been infected require treatment.

[0004] In the prior art, there are two main treatment methods for California perch iridovirus disease:

[0005] 1. Immunization, through vaccination;

[0006] 2. Treatment through medication, including probiotics, immune-boosting plant extracts, etc.

[0007] There are few existing technologies for the treatment of iris virus disease with Chinese herbal medicine. Chinese herbal medicine is generally in the form of plant extracts in the treatment of California perch iridovirus disease.

[0008] The main factors that hinder the use of Chinese herbal medicine in the treatment of California perch iridovirus disease include:

[0009] 1. Poor palatability, fish will not eat it, and the treatment effect will not be achieved;

[0010] 2. The content of active ingredients is low and the effect lasts too long;

[0011] 3. During the disease period, the intestinal digestion ability of California bass is poor and it is unable to absorb the active ingredients in Chinese herbal medicines.

[0012] Based on the above factors, there is no obviously effective Chinese herbal medicine preparation in the prior art to treat California sea bass iridovirus disease. Summary of the invention

[0013] The objective of the present invention is to provide a traditional Chinese medicine fermentation preparation for preventing and treating the iridovirus disease of Micropterus salmonides, and the preparation uses compound bacteria to ferment a traditional Chinese medicine composition to obtain the traditional Chinese medicine fermentation preparation, which has the advantages of good palatability, easy digestion, secondary metabolites being beneficial to disease control, fast onset time, etc.

[0014] The specific solution of the present invention is as follows:

[0015] A preparation method of a traditional Chinese medicine fermentation preparation for preventing and treating the iridovirus disease of Micropterus salmonides, which uses compound bacteria to ferment a traditional Chinese medicine composition to obtain the traditional Chinese medicine fermentation preparation;

[0016] The compound bacteria are a combination of Bacillus fermentation, Clostridium butyricum, Pediococcus pentosaceus, and Lactobacillus;

[0017] The traditional Chinese medicine composition is a combination of Codonopsis pilosula, Astragalus membranaceus, Glycyrrhiza uralensis, Archidendron clypearia, and Isatis indigotica.

[0018] Please comprehensively analyze the mechanism of the above five traditional Chinese medicines in the treatment of the iridovirus disease of Micropterus salmonides;

[0019] Codonopsis pilosula and Astragalus membranaceus have the effect of replenishing qi. Traditional Chinese medicine believes that when healthy qi exists inside, pathogenic factors cannot invade, and replenishing qi can improve immunity and fight against viruses. In addition, Astragalus membranaceus has a good effect in antiviral. Shang Lei et al. [1] Through the MTT colorimetric method, it was found that astragaloside IV has a direct inactivating effect on adenovirus (HAdV3), and the virus inhibition rate is positively correlated with the drug concentration. Wang Zhijie et al. [2] It was found that the total polysaccharides, total saponins, and total flavonoids of Astragalus membranaceus have significantly better effects on human HSV than the positive control group of acyclovir (ACV), and among them, the total saponins of Astragalus membranaceus have the best effect against HSV-1 (herpes simplex virus type 1).

[0020] Archidendron clypearia has the effects of clearing heat and detoxifying, cooling blood and detumescence. After iridovirus infection, organ enlargement and bleeding often occur, leading to organ failure and death. Archidendron clypearia clears heat and detoxifies, cools blood and detumescence to repair damaged tissues, which is beneficial to the recovery of diseased fish. In addition, modern pharmacological research shows that Archidendron clypearia also has the effect of antiviral. Zhang Zhongxian [3] Research on the anti-influenza virus effect of the water extract of Archidendron clypearia by Zhang Zhongxian et al. showed that the water extract of Archidendron clypearia can inhibit the cytopathic effect caused by influenza virus in vitro, suggesting that the water extract of Archidendron clypearia has an inhibitory effect on influenza virus in vitro.

[0021] Li Qiuyu et al. [4] In the construction of the anti-iridovirus efficacy model of Micropterus salmoides and the screening of antiviral traditional Chinese medicines, it was shown that Isatis indigotica has excellent performance in antiviral in vitro.

[0022] Citation source:

[0023] [1] Shang Lei, Qu Zhangyi, Ning Lili, et al. Study on the anti-adenovirus effect of astragaloside IV in vitro [J]. Chinese Pharmacological Bulletin, 2013, 29(06): 854.

[0024] [2] Wang Zhijie, Huang Tieniu, Liu Yanwen, Lu Suirong, Yang Zhanqiu. Preliminary experimental study on the anti-human herpesvirus effects of multiple components of Astragalus membranaceus [J]. Chinese Journal of Modern Applied Pharmacy, 2002(05): 356.

[0025] [3] Zhang Zhongxian, Huang Jianzhen. Study on the anti-influenza virus effect of the water extract of Pithecellobium clypearia [J]. China Tropical Medicine, 2008, 8(1): 30-34.

[0026] [4] Li Qiuyu, Huang Xiaohong. Construction of a pharmacodynamic model for Micropterus salmoides iridovirus and screening of antiviral traditional Chinese medicines [J]. Oceanologia et Limnologia Sinica, 2022, 53(7): 1513-1521.

[0027] Combined with compound bacteria fermentation, it increases the content of active substances, produces secondary metabolites, improves the drug efficacy, improves the palatability of Chinese herbal medicines, reduces toxicity, generally increases the curative effect, and reduces side effects. The traditional Chinese medicine fermentation preparation of the present invention is more scientific and perfect for the treatment of iridovirus disease of the genus Rana.

[0028] In the above preparation method of the traditional Chinese medicine fermentation preparation for preventing and treating iridovirus disease of the genus Rana of Micropterus salmoides, the traditional Chinese medicine composition contains 15-20 parts by weight of Codonopsis pilosula, 18-22 parts by weight of Astragalus membranaceus, 15-20 parts by weight of Glycyrrhiza uralensis, 15-20 parts by weight of Pithecellobium clypearia, and 15-20 parts by weight of Isatis indigotica.

[0029] In the above preparation method of the traditional Chinese medicine fermentation preparation for preventing and treating iridovirus disease of the genus Rana of Micropterus salmoides, the compound bacteria are added to the traditional Chinese medicine composition in the form of an activated bacterial liquid; the preparation method of the activated bacterial liquid is:

[0030] The bacterial powder is added to the brown sugar solution for expansion culture so that the viable bacteria concentration in the activated bacterial liquid is not less than 1×10 9 CFU / g;

[0031] 0.01-0.02 g of Bacillus sp., 0.01-0.02 g of Clostridium butyricum, 0.03-0.05 g of Pediococcus pentosaceus, and 0.02-0.06 g of Lactobacillus are added to each liter of the brown sugar solution;

[0032] The concentration of the brown sugar solution is 50-100 g / L.

[0033] In the above preparation method of the traditional Chinese medicine fermentation preparation for preventing and treating iridovirus disease of the genus Rana of Micropterus salmoides, the expansion culture time is 24-36 h; the expansion culture temperature is 25-35 °C.

[0034] In the above preparation method of the traditional Chinese medicine fermentation preparation for preventing and treating the iridovirus disease of Micropterus salmonides, the fermentation is anaerobic fermentation; the fermentation time is 10 to 20 days; the fermentation temperature is 25 to 35 °C.

[0035] In the above preparation method of the traditional Chinese medicine fermentation preparation for preventing and treating the iridovirus disease of Micropterus salmonides, 200 to 300 g of activated bacterial liquid is added to each kilogram of the traditional Chinese medicine composition.

[0036] Meanwhile, the present invention also discloses a traditional Chinese medicine fermentation preparation prepared by using any one of the above methods.

[0037] The beneficial effects of the present application are as follows:

[0038] The traditional Chinese medicine composition is fermented by using a composite bacterium to obtain the traditional Chinese medicine fermentation preparation, which has the advantages of good palatability, easy digestion, secondary metabolites being beneficial to disease control, and fast onset time. Specific embodiments

[0039] Next, the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that for those embodiments not specifying specific conditions, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For the reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0040] The moisture content of Codonopsis pilosula, Astragalus membranaceus, Glycyrrhiza uralensis, Pithecellobium clypearia, and Isatis indigotica is 8 to 10 wt%.

[0041] Bacillus subtilis: purchased from Shandong Yihao Biotechnology Co., Ltd., product model: 400 billion water-soluble Bacillus subtilis;

[0042] Clostridium butyricum: purchased from Shandong Yihao Biotechnology Co., Ltd., product model: Clostridium butyricum for aquaculture;

[0043] Pediococcus pentosaceus: purchased from Jinan Qiyue Chemical Technology Co., Ltd., product model: 0631-68-1;

[0044] Lactobacillus: purchased from Jiangsu Caiwei Biotechnology Co., Ltd., product model: Lactobacillus plantarum.

[0045] Example 1

[0046] The preparation method of the traditional Chinese medicine fermentation preparation for preventing and treating the iridovirus disease of Micropterus salmonides includes the following steps:

[0047] Step 1, composite bacterium amplification culture: Add the bacterial powder to the brown sugar solution for amplification culture for 24 h, and the amplification culture temperature is 30 °C to make the viable bacteria concentration in the activated bacterial liquid not lower than 1×10 9CFU / g; 0.02 g of Bacillus sp., 0.02 g of Clostridium butyricum, 0.05 g of Pediococcus pentosaceus, and 0.05 g of Lactobacillus were added to each liter of brown sugar solution; the concentration of the brown sugar solution was 100 g / L;

[0048] Step 2: Fermentation: The activated bacterial liquid was added to the traditional Chinese medicine composition for fermentation. The fermentation conditions were: anaerobic fermentation; the fermentation time was 15 days; the fermentation temperature was 30 °C.

[0049] The composition of the traditional Chinese medicine composition was: 17 g of Codonopsis pilosula, 20 g of Astragalus membranaceus, 17 g of Glycyrrhiza uralensis, 17 g of Archidendron clypearia, and 17 g of Isatis indigotica.

[0050] The traditional Chinese medicines in the above traditional Chinese medicine composition were all pulverized into powders with a mesh number of 100.

[0051] 300 g of activated bacterial liquid was added to each kg of the traditional Chinese medicine composition.

[0052] Example 2

[0053] Generally the same as Example 1, the difference was that: the composition of the traditional Chinese medicine composition was: 15 g of Codonopsis pilosula, 22 g of Astragalus membranaceus, 20 g of Glycyrrhiza uralensis, 15 g of Archidendron clypearia, and 20 g of Isatis indigotica; 300 g of activated bacterial liquid was added to each kg of the traditional Chinese medicine composition.

[0054] Example 3

[0055] Generally the same as Example 1, the difference was that: the composition of the traditional Chinese medicine composition was: 20 g of Codonopsis pilosula, 18 g of Astragalus membranaceus, 15 g of Glycyrrhiza uralensis, 20 g of Archidendron clypearia, and 15 g of Isatis indigotica; 300 g of activated bacterial liquid was added to each kg of the traditional Chinese medicine composition.

[0056] Comparative Example 1

[0057] Generally the same as Example 1, the difference was that the bacteria used in the activated bacterial liquid were Bacillus sp. and Clostridium butyricum.

[0058] Comparative Example 2

[0059] Generally the same as Example 1, the difference was that the bacteria used in the activated bacterial liquid were Pediococcus pentosaceus and Lactobacillus.

[0060] Comparative Example 3

[0061] Generally the same as Example 1, the difference was that the traditional Chinese medicine composition was a combination of Codonopsis pilosula, Astragalus membranaceus, Glycyrrhiza uralensis, and Isatis indigotica; the composition of the traditional Chinese medicine composition was: 17 g of Codonopsis pilosula, 20 g of Astragalus membranaceus, 17 g of Glycyrrhiza uralensis, and 17 g of Isatis indigotica.

[0062] Performance Test

[0063] 1. Breeding Experiment 1

[0064] Ranavirus was found in a pond in Jiangmen, Guangdong in August 2024;

[0065] The diseased fish were divided into 7 ponds (volume: 6 cubic meters) for breeding, with 100 fish in each pond;

[0066] Among them, the above-mentioned traditional Chinese medicine fermentation preparations of the examples and comparative examples were sprinkled into 6 ponds respectively, and the remaining one pond was used as a blank group and sprinkled with distilled water.

[0067] Observe the death and body rot conditions of each pond, and take samples to detect the CT value of the virus;

[0068] The entire breeding cycle was 10 days, and the temperature was controlled at 25°C; the water body control standards were: the control standards for aquaculture water bodies also included ammonia nitrogen not exceeding 0.2, nitrite between 0.02 - 0.05, pH value between 7.6 - 8.6, dissolved oxygen between 5 - 8, and total alkalinity between 80 - 150.

[0069] The addition amount of the above fermentation preparation was 1 g / m³ of water body.

[0070] The test results are shown in Table 1 below;

[0071] Table 1 Test Results

[0072] Death situation of Micropterus salmoides Rotting body situation of Micropterus salmoides Virus detection situation Example 1 8 9 CT value 31 Example 2 15 10 CT value 30 Example 3 13 12 CT value 29 Comparative example 1 18 11 CT value 30 Comparative example 2 28 14 CT value 28 Comparative example 3 25 10 CT value 26 Blank group 39 15 CT value 25

[0073] Result Analysis

[0074] From the number of dead Micropterus salmonides, according to the mean difference, relative index, and t and P test values, it can be seen that Example 1 has a good therapeutic effect. From the perspective of controlling death, the order is Example 1, Example 3, Example 2, Comparative Example 1, Comparative Example 3, Comparative Example 2, and the control group. The P value < 0.05 indicates significant difference. The incidence of body rot, according to the mean difference and relative index, with P < 0.05 showing significant difference, shows that using this formula can significantly reduce the incidence of body rot.

[0075] From the above experimental cases, it can be seen that Bacillus subtilis and Clostridium butyricum are crucial for the performance of traditional Chinese medicine components, and their importance is greater than that of Pediococcus pentosaceus and Lactobacillus; Comparative Example 3 proves that Abrus cantoniensis is very important in the whole traditional Chinese medicine formula. In the prior art, only the conclusion that the water extract of Abrus cantoniensis has anti-influenza virus effect is disclosed. Since influenza virus is a virus of the Orthomyxoviridae family, and iridovirus is a cytoplasmic DNA virus, the former has Single-stranded negative-sense RNA as the genetic material, while the latter has linear double-stranded DNA as the genetic material. The present application newly discovers that the traditional Chinese medicine component containing Abrus cantoniensis can significantly inhibit the proliferation of iridovirus after fermentation.

[0076] 2. Breeding Experiment 2

[0077] California bass are raised in a fish pond in Zhuhai. The fish pond is about 8 mu in size and 1.8 meters deep. There are 5,000 fry per mu, and they are now about one tael in size. On April 15, 2024, dead fish appeared, about thirty per day. After April 18, 100 - 200 fish died, and on April 20, it increased to about 400. Upon examination of the fish, there were ulcers on the body surface, bleeding on the gill covers, and the gill filaments were also bleeding and turning white. Upon dissection, it was found that the liver was white with bleeding points, the gallbladder was enlarged, and there was no food in the intestines.

[0078] PCR detection was carried out on the fish, and frog iridovirus was found. No bacteria were found in the bacterial culture. First, feeding was stopped. On April 20, compound iodine was splashed at 150 mL / mu * 1 meter water depth on the first day. Life No. 1 (i.e., the traditional Chinese medicine fermentation preparation of Example 1 of the present invention) was splashed at 1 kg / mu * 1 meter water depth. From April 21 to April 24, Life No. 1 was splashed at 1 kilogram / mu * 1 meter water depth.

[0079] The number of dead California bass gradually increased from more than 400 to more than 500 and then gradually decreased to about 100. Five days later, it was used for another three days and gradually decreased to 30 - 40.

[0080] From the case, it can be seen that Example 1 has a certain effect in treating frog iridovirus disease in California bass.

Claims

1. A preparation method of a traditional Chinese medicine fermentation preparation for preventing and treating the iridovirus disease of Micropterus salmonides, characterized in that, A traditional Chinese medicine fermentation preparation is obtained by fermenting a traditional Chinese medicine composition with a compound bacterium; The compound bacterium is a combination of Bacillus, Clostridium butyricum, Pediococcus pentosaceus, and Lactobacillus; The traditional Chinese medicine composition is a combination of Codonopsis pilosula, Astragalus membranaceus, Glycyrrhiza uralensis, Archidendron clypearia, and Isatis indigotica.

2. The preparation method of the traditional Chinese medicine fermentation preparation for preventing and treating Micropterus salmonides ranavirus disease according to claim 1, wherein, The traditional Chinese medicine composition contains 15-20 parts by weight of Codonopsis pilosula, 18-22 parts by weight of Astragalus membranaceus, 15-20 parts by weight of Glycyrrhiza uralensis, 15-20 parts by weight of Archidendron clypearia, and 15-20 parts by weight of Isatis indigotica.

3. The preparation method of the traditional Chinese medicine fermentation preparation for preventing and treating Micropterus salmonides ranavirus disease according to claim 1, characterized in that The compound bacterium is added to the traditional Chinese medicine composition in the form of an activated bacterial solution; the preparation method of the activated bacterial solution is: Add the bacterial powder to the brown sugar solution for expansion culture so that the viable bacteria concentration in the activated bacterial liquid is not less than 1×10 9 CFU / g; Add 0.01-0.02 g of Bacillus, 0.01-0.02 g of Clostridium butyricum, 0.03-0.05 g of Pediococcus pentosaceus, and 0.02-0.06 g of Lactobacillus to each liter of brown sugar solution; The concentration of the brown sugar solution is 50-100 g / L.

4. The preparation method of the traditional Chinese medicine fermentation preparation for preventing and treating Micropterus salmonides ranavirus disease according to claim 3, characterized in that, The expansion culture time is 24-36 h; the expansion culture temperature is 25-35 °C.

5. The preparation method of the traditional Chinese medicine fermentation preparation for preventing and treating Micropterus salmonides ranavirus disease according to claim 1, wherein, The fermentation is anaerobic fermentation; the fermentation time is 10-20 days; the fermentation temperature is 25-35 °C.

6. The preparation method of the traditional Chinese medicine fermentation preparation for preventing and treating the iridovirus disease of Micropterus salmonides, as claimed in claim 3, is characterized in that, Add 200-300 g of the activated bacterial solution to each kilogram of the traditional Chinese medicine composition.

7. A traditional Chinese medicine fermentation preparation, characterized in that, Prepared by the method according to any one of claims 1 to 6.