Chinese herbal medicine preparation and application thereof in aquatic feed

Abstract

The invention relates to the technical field of feed additives, and particularly provides a Chinese herbal medicine preparation and application thereof in aquatic feed. The Chinese herbal medicine preparation is prepared by fermenting traditional Chinese medicine raw materials through clostridium butyricum, effective components in traditional Chinese medicine can be effectively released, and growth of aquatic animals is remarkably promoted and immunity of the aquatic animals is improved through the synergistic effect of the clostridium butyricum and the effective components of the traditional Chinese medicine, so that wide application of the Chinese herbal medicine preparation in aquatic feed is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of feed additives, and in particular to a Chinese herbal medicine preparation and application thereof in aquatic feed. Background Art

[0002] Chinese herbal medicines are extracted from plants, have rich and diverse chemical compositions, and contain a variety of nutrients and bioactive substances that can enhance the body's nonspecific immunity to fight viruses and bacteria.

[0003] In recent years, the application of Chinese herbal medicine as feed additives in aquaculture has become increasingly popular and extensive, and related research is very rich. Huang Jingjun et al.'s research shows that Chinese herbal medicine as feed additives can not only effectively improve the nonspecific immune performance of farmed fish and prevent the occurrence of diseases, but also improve fish growth performance and feed utilization; Ma Yanqun et al.'s research found that adding astragalus to feed for 48 days can significantly increase the weight gain rate and specific growth rate of channel catfish. Miao Yujia et al.'s research found that oridonin can inhibit the growth and reproduction of Aeromonas hydrophila by interfering with protein metabolism and increasing the permeability of cell membranes.

[0004] Herbal medicines have various ingredients. The compound Chinese herbal medicines used in combination with Chinese herbal medicines can promote the growth of aquatic animals by effectively stimulating the feeding of aquatic animals, promoting the growth and development of the digestive tract, accelerating nutrient absorption, improving the activity of digestive enzymes, and regulating the structure of intestinal flora under the synergistic effect of various active ingredients. Substances that play an immune role in Chinese herbal medicines include polysaccharides, alkaloids, organic acids, etc. Among them, the main immune active substances are polysaccharides, which can effectively promote cell phagocytosis and the body's immune regulation. Alkaloids have special biological activities and show anti-inflammatory and immunomodulatory effects. Organic acids can enhance the immune function of animal bodies. However, most Chinese herbal medicines are plant medicines, and most of their effective substances are surrounded by cell walls. How to make full use of their effective ingredients is the key issue we need to solve at present. Summary of the invention

[0005] The present invention solves the problems of the prior art and provides a Chinese herbal medicine preparation and its application in aquatic feed. The Chinese herbal medicine preparation is prepared by fermenting Chinese medicine raw materials with Clostridium butyricum, can effectively release the effective ingredients in the Chinese medicine, and can significantly promote the growth of aquatic animals and improve their immunity through the synergistic effect of Clostridium butyricum and the effective ingredients of the Chinese medicine, thereby facilitating its wide application in aquatic feed.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: In one aspect, the present invention provides a Chinese herbal medicine preparation comprising Clostridium butyricum.

[0007] The butyricum is Clostridium butyricum YYJ-1, which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on April 29, 2024, with the deposit number CCTCC NO: M2024807.

[0008] The Chinese herbal medicine preparation is prepared by fermenting Chinese medicinal raw materials with Clostridium butyricum.

[0009] The Chinese medicinal raw materials include dried tangerine peel, astragalus, wild hawthorn, calamus, artemisia annua, garlic and betel nut.

[0010] The components of the Chinese medicinal raw materials and their weight proportions are respectively 15 parts of dried tangerine peel, 25 parts of astragalus, 15 parts of wild hawthorn, 30 parts of calamus, 20 parts of artemisia annua, 5 parts of garlic and 8 parts of betel nut.

[0011] In one aspect, the present invention provides a method for preparing the Chinese herbal medicine preparation, comprising the following steps: (1) Prepare Chinese medicinal materials, the components and weight proportions of which are as follows: 15 parts of dried tangerine peel, 25 parts of astragalus, 15 parts of wild hawthorn, 30 parts of calamus, 20 parts of artemisia annua, 5 parts of garlic, and 8 parts of betel nut; (2) Grind the Chinese medicine raw materials and pass them through a 60-mesh sieve to obtain Chinese medicine powder; add 5-10 times the weight of water to the Chinese medicine powder, stir evenly, and soak for 24 hours to obtain a Chinese medicine suspension; (3) placing the Chinese medicine suspension and the fermentation medium in a fermentation container at a mass ratio of 1-2:1, and sterilizing them by high pressure wet heat at 121°C for 20 min to obtain the Chinese medicine fermentation matrix; (4) Inoculating the activated Clostridium butyricum YYJ-1 bacterial solution into the traditional Chinese medicine fermentation matrix at a volume ratio of 2-5%; filling the sealed fermentation container with nitrogen, exhausting the oxygen, and culturing at 37°C for 2-3 days; (5) Unseal the fermentation container, dry the fermentation product at 50-60°C, take it out, crush it, and pass it through a 60-mesh sieve to obtain the Chinese herbal medicine preparation.

[0012] The components and weight proportions of the fermentation medium in step (3) of the preparation method are as follows: 22 parts of peptone, 13 parts of glucose, 2.0 parts of soybean meal, 6 parts of sodium alginate, NH 4 NO 3 1 copy, K 2 HPO 4 1 part, MgSO 4 0.1 parts, MnSO 4 0.1 parts, CaCO 3 1 part, urea 5 parts, L-cysteine ​​0.5 parts, water 500 parts.

[0013] The amount of live bacteria in the Clostridium butyricum liquid in step (4) of the preparation method is not less than 10 10 CFU / mL.

[0014] The invention also provides application of the Chinese herbal medicine preparation in aquatic feed production.

[0015] The present invention also provides a feed additive, comprising the Chinese herbal medicine preparation.

[0016] The present invention uses the screened Clostridium butyricum YYJ-1 to ferment the Chinese medicine raw materials to prepare the Chinese herbal medicine preparation, and the amount of live bacteria is not less than 10 9 CFU / g. Through the synergistic effect of Clostridium butyricum and the effective ingredients of traditional Chinese medicine, it can significantly improve the growth performance of aquatic animals, increase feed utilization, enhance the immunity and disease resistance of aquatic animals, and increase the survival rate, which is beneficial to improving the economic benefits of aquaculture.

[0017] Compared with the control group, the final weight, weight gain rate, specific growth rate, and survival rate of the pearl gentian grouper in the experimental group fed with the Chinese herbal preparation were significantly improved, and the feed coefficient was significantly reduced; among them, the pearl gentian grouper in group B fed with the Chinese herbal preparation described in Example 7 had the best growth performance, with the weight gain rate, specific growth rate, and survival rate increased by 69.09%, 46.59%, and 14.01%, respectively. The weight gain rate, survival rate, and specific growth rate of the vannamei shrimp in the experimental group fed with the Chinese herbal preparation were also significantly improved, and the feed coefficient was generally reduced.

[0018] After attacking with Vibrio harveyi, the mortality rate of pearl grouper in the control group was as high as 65.2%, while the mortality rate of pearl grouper in the experimental group fed with the Chinese herbal preparation of the present invention was significantly reduced, the resistance to Vibrio harveyi was significantly enhanced, and the relative immune protection rate was generally higher than 60%.

[0019] After the attack of Vibrio parahaemolyticus, the mortality rate of Penaeus vannamei in the control group was as high as 84.60%, while the mortality rate of Penaeus vannamei in the experimental group fed with the Chinese herbal preparation of the present invention was only 17.33%-27.54%; among them, the relative immune protection rate of Penaeus vannamei in group B fed with the Chinese herbal preparation of Example 7 was as high as 79.52%. In addition, the activities of phenoloxidase, superoxide dismutase and lysozyme in the serum of Penaeus vannamei in the experimental group were significantly improved; among them, the activities of phenoloxidase, superoxide dismutase and lysozyme in the serum of Penaeus vannamei in group B fed with the Chinese herbal preparation of Example 7 were the highest, which were increased by 77.2%, 13.1% and 34.8% respectively. It is shown that the Chinese herbal preparation provided by the present invention can effectively improve the immune indexes of Penaeus vannamei and enhance its disease resistance.

[0020] In summary, the Chinese herbal medicine preparation provided by the present invention can be widely used in aquaculture as a feed additive, and can also be used as an antibiotic substitute product to effectively reduce the amount of antibiotics used in the breeding process, which is beneficial to ensuring food safety and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the colony morphology of Clostridium butyricum YYJ-1. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The culture medium selected in the embodiment of the present invention has the following specific formula: Clostridium proliferation medium (RCM liquid medium): glucose 5.0 g / L, soluble starch 1.0 g / L, tryptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 3.0 g / L, sodium chloride 5.0 g / L, sodium acetate 3.0 g / L, L-cysteine ​​0.5 g / L; pH 7.0.

[0024] Clostridium proliferation medium (RCM solid medium): glucose 5.0 g / L, soluble starch 1.0 g / L, tryptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 3.0 g / L, sodium chloride 5.0 g / L, sodium acetate 3.0 g / L, L-cysteine ​​0.5 g / L, agar 0.5 g / L; pH 7.0.

[0025] Clostridium selective agar medium (TSN agar medium): 10 g yeast extract, 15 g tryptone, 0.5 g ferric citrate, 1 g sodium sulfite, 13.5 g agar, 1000 mL distilled water, adjust the pH to 7.0±0.1, sterilize with high pressure steam at 121℃ for 20 min, cool to 50-60℃, add 100 μg neomycin sulfate and 4 μg polymyxin B to every 100 mL of culture medium.

[0026] The technical solution disclosed in the present invention will be further described below in conjunction with specific embodiments.

[0027] Example 1 Screening, identification and performance analysis of Clostridium butyricum YYJ-1 The present invention screened a gas-producing anaerobic Clostridium from the fresh feces of pearl grouper. The strain was identified as Clostridium butyricum through molecular biology, physiological and biochemical and drug sensitivity tests and was named Clostridium butyricum YYJ-1 ( Clostridium butyricum YYJ-1), and was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on April 29, 2024, with the deposit number being CCTCC NO: M2024807.

[0028] The colony morphology of Clostridium butyricum YYJ-1 on RCM solid medium is as follows Figure 1 shown.

[0029] Clostridium butyricum YYJ-1 has good biosafety. It is insensitive to erythromycin, cefazolin, ciprofloxacin, and ofloxacin; it is moderately sensitive to amoxicillin; and it is highly sensitive to penicillin, ampicillin, norfloxacin, chloramphenicol, cefaclor, gentamicin, and amikacin.

[0030] Clostridium butyricum YYJ-1 grew well in RCM liquid culture medium and entered the logarithmic growth phase after 3 to 4 hours. The bacterial count increased rapidly, and the organic acids produced by metabolism during the growth process caused the pH value of the culture medium to drop sharply. The logarithmic growth phase of Clostridium butyricum YYJ-1 strain was maintained for about 8 hours, and then entered the stable growth phase. The bacterial count no longer increased, the OD 600 nm value of the culture solution remained at about 1.7, and the pH value was stable at 4.5 to 4.7.

[0031] Clostridium butyricum YYJ-1 has strong stress resistance and can tolerate strong acid, high temperature or aerobic environment. After being cultured in a strong acidic environment of pH 1.0 for 3 hours, the survival rate of the strain was still higher than 95%; after being treated at 70℃ for 20 minutes and 80℃ for 10 minutes, the survival rates were as high as 80.37% and 53.29% respectively, and after being treated at 90℃ for 5 minutes, the survival rate could still reach 12.34%; after being exposed to an aerobic environment for 10 minutes and 30 minutes, the survival rate could still reach 68.29% and 55.21%, achieving unexpected technical results.

[0032] Example 2 Analysis of the acid production ability of Clostridium butyricum YYJ-1 A single colony of Clostridium butyricum YYJ-1 was picked and inoculated into RCM liquid medium, and cultured anaerobically at 37℃ and 220 r / min for 14-16 h. An appropriate amount of fermentation broth was transferred to 100 mL of sterile RCM liquid medium at a ratio of 1%, and cultured anaerobically at 37℃ and 220 r / min for 24 h. The fermentation broth of Clostridium butyricum YYJ-1 was centrifuged at 10000 r / min for 5 min, and the supernatant was collected and sterilized by ultrafiltration. At the same time, a commercial Clostridium butyricum sample was used as a control. The organic acids in the fermentation supernatants of the above two Clostridium butyricum were quantitatively analyzed by high-performance liquid chromatography. The detector was an ultraviolet detector, and the determination conditions were: wavelength 210 nm; mobile phase: 0.1% phosphoric acid aqueous solution. The blank RCM liquid medium was filtered through a 0.22μm filter before loading as a blank control. The results are shown in Table 1.

[0033] Table 1 Analysis of acid production by fermentation of Clostridium butyricum YYJ-1 Grouping lactic acid Acetic acid Propionic acid Butyric acid Hexanoic acid Blank control 0.117 g / L 0.037 g / L 0 0 0 Clostridium butyricum YYJ-1 4.375g / L 6.782 g / L 3.170 g / L 10.326 g / L 0 Commercially available Clostridium butyricum control 7.332 g / L 3.791 g / L 0.019 g / L 8.219 g / L 0 It can be seen from the data in Table 1 that the butyric Clostridium butyricum YYJ-1 provided by the present invention has a strong acid production ability, and the organic acids produced by its fermentation mainly include lactic acid, acetic acid, propionic acid and butyric acid, among which acetic acid and butyric acid have the highest contents, reaching 6.782 g / L and 10.326 g / L, respectively. The total content of the four organic acids is 27.3% higher than that of commercially available Clostridium butyricum, achieving unexpected technical effects.

[0034] Example 3 Scavenging effect of Clostridium butyricum YYJ-1 on DPPH free radicals Take 1 mL of the fermentation supernatant of Clostridium butyricum YYJ-1 and mix it evenly with 1 mL of 1.50 mmol / L DPPH anhydrous ethanol solution, react at room temperature in the dark for 30 min, and measure the absorbance at 517 nm. The blank group was replaced with an equal volume of ethanol instead of DPPH solution, and the control group was replaced with an equal volume of sterile water instead of the fermentation supernatant of Clostridium butyricum YYJ-1, and 0.125 g / L vitamin C was used as a positive control.

[0035] The calculation formula of clearance rate is: Clearance rate = [1-(A1-A) / A0]×100%.

[0036] Where A is the absorbance value of the blank group, A0 is the absorbance value of the control group, and A1 is the absorbance value of the sample group.

[0037] Table 2 The scavenging effect of Clostridium butyricum YYJ-1 on DPPH free radicals sample DPPH clearance Fermentation supernatant of Clostridium butyricum YYJ-1 95.81% 0.125 g / L Vitamin C 94.75% As shown in Table 2, the fermentation supernatant of Clostridium butyricum YYJ-1 has a strong antioxidant capacity, and the scavenging rate of DPPH free radicals is as high as 95.81%, which is equivalent to the effect of 0.125 g / L vitamin C.

[0038] Example 4 Analysis of the antibacterial ability of Clostridium butyricum YYJ-1

[0039] Use sterilized medical cotton swabs to dip into the bacterial liquid of four common aquatic pathogens, Staphylococcus aureus, Vibrio harveyi, Vibrio splendens and Vibrio parahaemolyticus, and evenly spread them on RCM solid culture medium. Use a 1mL pipette tip (outer diameter: 9mm) to punch holes in nutrient agar and RCM solid culture medium, and seal the bottom with flame. Pipette 200μL of Clostridium butyricum YYJ-1 fermentation liquid and add it to the corresponding well. Put the culture dish with the sample in a 4℃ refrigerator for 2h, move it to a 37℃ incubator, and culture it anaerobically for 12h. Measure and record the diameter of the inhibition zone in the plate to determine the antibacterial effect of Clostridium butyricum YYJ-1. The results showed that the Clostridium butyricum YYJ-1 provided by the present invention had a significant inhibitory effect on four pathogens: Staphylococcus aureus, Vibrio harveyi, Vibrio splendens and Vibrio parahaemolyticus, with the maximum inhibition zone diameter reaching 23.9 mm.

[0040] Example 5 Application of Clostridium butyricum YYJ-1 in the cultivation of Penaeus vannamei 1. Preparation of bacterial powder The Clostridium butyricum YYJ-1 strain was activated, expanded, and fermented in liquid form, centrifuged, concentrated, and freeze-dried to produce a culture medium with a live bacterial content of about 10 10 CFU / g of bacterial powder.

[0041] 2. Experimental Design 600 whiteleg shrimps with an average body length of 2.5 cm were selected and randomly divided into 2 groups, with 3 parallels in each group and 100 shrimps in each parallel. The shrimps in group A were fed with basic feed, and the shrimps in group B were fed with basic feed and 5‰ of the above-mentioned Clostridium butyricum YYJ-1 powder was added. The aquaculture water used natural seawater filtered by sand. The test water temperature was 26~28℃, pH was 7.5~7.8, and salinity was 26‰~28‰. During the test, oxygen was continuously supplied, the dissolved oxygen was not less than 5mg / L, and the ammonia nitrogen was not higher than 0.5mg / L. Feeding was done twice a day. The feeding cycle was 6 weeks. After the feeding, the hepatopancreas tissue of the shrimp was taken and stored at -80℃ for subsequent experiments.

[0042] 3. Growth performance Before the experiment, the number and weight of shrimp in each culture box were counted. On the last day of the experiment, the shrimp were deprived of feed for 24 hours, and the shrimp in each culture box were counted and weighed to calculate the survival rate (SR), weight gain rate (WGR), specific growth rate (SGR) and feed coefficient (FCR).

[0043] Survival rate (%) = number of shrimps at the end of the experiment / number of shrimps at the beginning of the experiment × 100%; Weight gain rate (%) = (final weight - initial weight) / initial weight × 100%; Specific growth rate (%) = (ln final weight - ln initial weight) / number of test days × 100%; Feed coefficient = (dry weight of fed feed - dry weight of remaining feed) / (final weight - initial weight).

[0044] The results showed that compared with the control group, the survival rate, weight gain rate and specific growth rate of the experimental group fed with Clostridium butyricum YYJ-1 powder increased by 25.8%, 98.0% and 59.0% respectively, and the feed coefficient decreased by 14.6%, and the effect was very significant (P<0.05). This shows that the Clostridium butyricum YYJ-1 provided by the present invention can effectively improve the growth performance of southern shrimp, which is beneficial to increase the breeding benefits.

[0045] 4. Hepatopancreas antioxidant index

[0046] The hepatopancreas tissue of Penaeus vannamei obtained in the above experiment was taken and ground evenly, and the superoxide dismutase (SOD), catalase (CAT), polyphenol oxidase (PPO) activities and malondialdehyde (MDA) content in the hepatopancreas of Penaeus vannamei were detected using a kit (Nanjing Jiancheng Bioengineering Co., Ltd.). The results showed that compared with the control, the malondialdehyde (MDA) content in the hepatopancreas of the experimental group of shrimp fed with Clostridium butyricum YYJ-1 powder decreased by 50.0% (P<0.05), and the activities of superoxide dismutase (SOD), catalase (CAT), and polyphenol oxidase (PPO) increased by 53.2%, 37.2%, and 65.5%, respectively, and the effect was very significant (P<0.05). This shows that the Clostridium butyricum YYJ-1 provided by the present invention can significantly enhance the nonspecific antioxidant capacity of shrimp itself, thereby improving its body immunity and resistance, and reducing the occurrence of aquaculture diseases.

[0047] In summary, the Clostridium butyricum YYJ-1 provided by the present invention has strong acid production and antioxidant capacity, strong tolerance to strong acid, high temperature and aerobic environment, and can significantly inhibit the growth of pathogens such as Vibrio splendens and Vibrio parahaemolyticus. The strain can be added alone, or it can be compounded with a variety of probiotics such as Bacillus licheniformis, Bacillus subtilis, Saccharomyces cerevisiae, Bifidobacterium, Enterococcus faecium, Lactobacillus acidophilus, Enterococcus faecalis, Pediococcus acidilactici and added to aquatic feed, which can effectively improve the growth performance of aquatic animals, enhance their body immunity, help reduce the occurrence of diseases, increase breeding benefits, and has broad application prospects.

[0048] Example 6 A Chinese herbal medicine preparation and its preparation method 1. Prepare Chinese medicinal raw materials, whose components and weight proportions are: 15 parts of dried tangerine peel, 25 parts of astragalus, 15 parts of wild hawthorn, 30 parts of calamus, 20 parts of artemisia annua, 5 parts of garlic, and 8 parts of betel nut; 2. Grind the Chinese medicine raw materials and pass them through a 60-mesh sieve to obtain Chinese medicine powder; add 5 times the weight of water to the Chinese medicine powder, stir evenly, and soak for 24 hours to obtain a Chinese medicine suspension; 3. Prepare fermentation medium: its components and weight proportions are: 22 parts of peptone, 13 parts of glucose, 2.0 parts of soybean meal, 6 parts of sodium alginate, NH 4 NO 3 1 copy, K 2 HPO 4 1 part, MgSO 4 0.1 parts, MnSO 4 0.1 parts, CaCO 3 1 part, urea 5 parts, L-cysteine ​​0.5 parts, water 500 parts; 4. Place the Chinese medicine suspension and the fermentation medium in a fermentation container at a mass ratio of 1:1, and sterilize them by high pressure wet heat at 121°C for 20 minutes to obtain the Chinese medicine fermentation matrix; 5. Inoculate the activated Clostridium butyricum YYJ-1 bacterial solution into the traditional Chinese medicine fermentation matrix at a volume ratio of 2%, and the amount of live bacteria in the bacterial solution should not be less than 10 10 CFU / mL; nitrogen was filled into the sealed fermentation container, oxygen was exhausted, and cultured at 37°C for 3 days; 6. Unseal the fermentation container, dry the fermentation product at 50-60°C, take it out, crush it, and pass it through a 60-mesh sieve to obtain the Chinese herbal medicine preparation.

[0049] After testing, the amount of live bacteria in the Chinese herbal medicine preparation is not less than 10 9 CFU / g.

[0050] Example 7 A Chinese herbal medicine preparation and its preparation method 1. Prepare Chinese medicinal raw materials, whose components and weight proportions are: 15 parts of dried tangerine peel, 25 parts of astragalus, 15 parts of wild hawthorn, 30 parts of calamus, 20 parts of artemisia annua, 5 parts of garlic, and 8 parts of betel nut; 2. Grind the Chinese medicine raw materials and pass them through a 60-mesh sieve to obtain Chinese medicine powder; add 5 times the weight of water to the Chinese medicine powder, stir evenly, and soak for 24 hours to obtain a Chinese medicine suspension; 3. Prepare fermentation medium: its components and weight proportions are: 22 parts of peptone, 13 parts of glucose, 2.0 parts of soybean meal, 6 parts of sodium alginate, NH 4 NO 3 1 copy, K 2 HPO 4 1 part, MgSO 4 0.1 parts, MnSO4 0.1 parts, CaCO 3 1 part, urea 5 parts, L-cysteine ​​0.5 parts, water 500 parts; 4. Place the Chinese medicine suspension and the fermentation medium in a fermentation container at a mass ratio of 1:1, and sterilize them by high pressure wet heat at 121°C for 20 minutes to obtain the Chinese medicine fermentation matrix; 5. Inoculate the activated Clostridium butyricum YYJ-1 bacterial solution into the traditional Chinese medicine fermentation matrix at a volume ratio of 4%, and the amount of live bacteria in the bacterial solution should not be less than 10 10 CFU / mL; nitrogen was filled into the sealed fermentation container, oxygen was exhausted, and cultured at 37°C for 2 days; 6. Unseal the fermentation container, dry the fermentation product at 50-60°C, take it out, crush it, and pass it through a 60-mesh sieve to obtain the Chinese herbal medicine preparation.

[0051] After testing, the amount of live bacteria in the Chinese herbal medicine preparation is not less than 10 9 CFU / g.

[0052] Example 8 A Chinese herbal medicine preparation and its preparation method 1. Prepare Chinese medicinal raw materials, whose components and weight proportions are: 15 parts of dried tangerine peel, 25 parts of astragalus, 15 parts of wild hawthorn, 30 parts of calamus, 20 parts of artemisia annua, 5 parts of garlic, and 8 parts of betel nut; 2. Grind the Chinese medicine raw materials and pass them through a 60-mesh sieve to obtain Chinese medicine powder; add 10 times the weight of water to the Chinese medicine powder, stir evenly, and soak for 24 hours to obtain a Chinese medicine suspension; 3. Prepare fermentation medium: its components and weight proportions are: 22 parts of peptone, 13 parts of glucose, 2.0 parts of soybean meal, 6 parts of sodium alginate, NH 4 NO 3 1 copy, K 2 HPO 4 1 part, MgSO 4 0.1 parts, MnSO 4 0.1 parts, CaCO 3 1 part, urea 5 parts, L-cysteine ​​0.5 parts, water 500 parts; 4. Place the Chinese medicine suspension and the fermentation medium in a fermentation container at a mass ratio of 2:1, and sterilize them by high pressure wet heat at 121°C for 20 minutes to obtain the Chinese medicine fermentation matrix; 5. Inoculate the activated Clostridium butyricum YYJ-1 bacterial solution into the traditional Chinese medicine fermentation matrix at a volume ratio of 5%, and the amount of live bacteria in the bacterial solution should not be less than 10 10 CFU / mL; nitrogen was filled into the sealed fermentation container, oxygen was exhausted, and cultured at 37°C for 2 days; 6. Unseal the fermentation container, dry the fermentation product at 50-60°C, take it out, crush it, and pass it through a 60-mesh sieve to obtain the Chinese herbal medicine preparation.

[0053] After testing, the amount of live bacteria in the Chinese herbal medicine preparation is not less than 10 9 CFU / g.

[0054] Example 9 Application of Chinese herbal medicine preparation in pearl grouper breeding In this example, the Chinese herbal medicine preparation prepared above was used to feed pearl grouper, and the growth performance and immune ability of the pearl grouper were tested.

[0055] Experimental Group: Group A: The Chinese herbal medicine preparation prepared in Example 6 was added to the basic bait at a mass ratio of 3%; Group B: The Chinese herbal medicine preparation prepared in Example 7 was added to the basic bait at a mass ratio of 3%; Group C: The Chinese herbal medicine preparation prepared in Example 8 was added to the basic bait at a mass ratio of 3%; Control group: basic bait.

[0056] Farming management The experimental fish was pearl grouper. Before the experiment, the purchased grouper was placed in a temporary holding pond (5.0mx3.0mx2.5m) for 1 week and fed with basic bait during the period. After it was stably acclimated, it was fasted for 24h. Groupers with a size of 22.0±1.00g and no abnormalities were selected and randomly assigned to 0.3m 3 The fish were placed in breeding barrels, with 30 fish in each barrel and 3 replicates in each experimental group, and cultured for 4 weeks. According to the weekly weighing, the fish were fed at 3%-5% of their body weight, and fed twice a day (8:30 and 17:30) at apparent satiation. The water temperature was 28-30°C, the salinity was 28~29, and the dissolved oxygen was 7mg / L. During the culture period, 50% of the water was changed every day. After 28 days of culture, 6 fish were randomly selected from each experimental group, their intestines were taken out, and after weighing, they were quickly divided into the cryopreservation tubes of the RNA-fr control group and quickly frozen with liquid nitrogen, and stored at -80°C. Determination indicators and methods 1. Growth performance indicators Weight gain rate (WG, g) = (final weight - initial weight) / initial weight × 100%; Specific growth rate (SGR, % / d) = (ln final average weight - ln initial average weight) / number of days of feeding × 100%; Feed coefficient ratio (FCR) = dry weight of feed consumed / (final body weight - initial body weight); Survival rate (SR, %) = final number of fish / initial number of fish × 100%.

[0057] See Table 3 for specific results.

[0058] Table 3 Effects of Chinese herbal medicine preparations on growth performance of pearl grouper Group Initial body weight IW (g) Final body weight FW (g) Weight gain rate WG (%) Survival rate SR (%) Feed Coefficient FCR Specific growth rate SGR (%) A 23.06±0.05 <![CDATA[48.57±2.87 b ]]> <![CDATA[110.62±5.87 b ]]> <![CDATA[90.59±3.44 b ]]> <![CDATA[0.73±0.15 b ]]> <![CDATA[2.66±0.22 c ]]> B 23.34±0.02 <![CDATA[52.34±0.33 c ]]> <![CDATA[124.25±3.01 c ]]> <![CDATA[94.87±2.42 b ]]> <![CDATA[0.64±0.04 c ]]> <![CDATA[2.88±0.10 a ]]> C 23.31±0.08 <![CDATA[48.78±0.42 bc ]]> <![CDATA[109.78±1.92 bc ]]> <![CDATA[90.33±0.05 b ]]> <![CDATA[0.73±0.04 bc ]]> <![CDATA[2.65±0.02 b ]]> Comparison 23.08±0.06 <![CDATA[40.04±0.31 a ]]> <![CDATA[73.48±1.34 a ]]> <![CDATA[83.21±0.00 a ]]> <![CDATA[1.10±0.02 a ]]> <![CDATA[1.97±0.04 d ]]> As shown in Table 3, compared with the control group, the final body weight, weight gain rate, specific growth rate, and survival rate of the pearl gentian grouper in the experimental group fed with the Chinese herbal medicine preparation were significantly improved, and the feed coefficient was significantly reduced; among them, the pearl gentian grouper in Group B fed with the Chinese herbal medicine preparation described in Example 7 had the best growth performance, and the weight gain rate, specific growth rate, and survival rate increased by 69.09%, 46.59%, and 14.01%, respectively. It is thus shown that the Chinese herbal medicine preparation prepared by fermenting Chinese medicinal raw materials with Clostridium butyricum YYJ-1 in the present invention can significantly improve the growth performance of aquatic animals, improve feed utilization, and achieve unexpected technical effects.

[0059] 2. Attack the virus After continuously feeding the feed containing the Chinese herbal medicine preparation of the present invention for 28 days, a challenge experiment was conducted. The challenge concentration was 5.54×10 8 cfu / mL, 20 pearl groupers were selected from each group and injected intraperitoneally with 200 µL of Vibrio harveyi solution. After the injection, they were kept in normal feeding and the number of deaths in each group was recorded every day. The observation lasted for 14 days. The mortality rate was counted and the relative immune protection rate was calculated. The specific results are shown in Table 4.

[0060] The relative immune protection rate (RPS) calculation formula is as follows: RPS=(1-mortality rate of experimental group / mortality rate of control group)×100%.

[0061] Table 4 Effects of Chinese herbal medicine preparations on the immune performance of pearl grouper Group Mortality of pearl grouper Relative immune protection rate A 26.0% 60.1% B 23.1% 64.6% C 25.9% 60.3% Control group 65.2% 0 As shown in Table 4, after the attack of Vibrio harveyi, the mortality rate of the pearl grouper in the control group was as high as 65.2%, while the mortality rate of the pearl grouper in the experimental group fed with the Chinese herbal medicine preparation of the present invention was significantly reduced, and the resistance to Vibrio harveyi was significantly enhanced, and the relative immune protection rate was generally higher than 60%. This shows that the Chinese herbal medicine preparation provided by the present invention can effectively improve the immune performance and disease resistance of aquatic animals, and the effect is remarkable.

[0062] Example 10 Application of Chinese herbal medicine preparations in the cultivation of Penaeus vannamei In this example, the Chinese herbal medicine preparation prepared above was used to feed Penaeus vannamei, and the growth performance and immune ability of Penaeus vannamei were tested.

[0063] Experimental Group: Group A: The Chinese herbal medicine preparation prepared in Example 6 was added to the basic bait at a mass ratio of 2%; Group B: The Chinese herbal medicine preparation prepared in Example 7 was added to the basic bait at a mass ratio of 2%; Group C: The Chinese herbal medicine preparation prepared in Example 8 was added to the basic bait at a mass ratio of 2%; Control group: basic bait.

[0064] 2. Experimental Design Litopenaeus vannamei was obtained from Chengyang Aquatic Products Market, with a body weight of 3.39±0.3 g.

[0065] The purchased Penaeus vannamei were first temporarily reared in a 2m×2m×1m temporary rearing pond for 1 week, during which time they were fed with basic bait. After the temporary rearing, shrimps of uniform size and good health were selected and randomly divided into 5 groups, with 300 shrimps in each group, 3 replicates in each group, and 100 shrimps in each replicate. The shrimps were fed according to the above sample groups. The feeding experiment lasted for 28 days. During the experiment, the water temperature was controlled at (28±2)℃, the pH was 7.5~7.8, and the salinity was 26‰~28‰. During the experiment, oxygenation was continuously added, the dissolved oxygen was not less than 5mg / L, and the ammonia nitrogen was not higher than 0.5mg / L. Feed was added 4 times a day, once every 6h.

[0066] 3. Growth performance Before the experiment, the number and weight of shrimp in each culture box were counted. On the last day of the experiment, the shrimp were fed for 24 hours, and the shrimp in each culture box were counted and weighed to calculate the survival rate (SR), weight gain rate (WGR), specific growth rate (SGR) and feed coefficient (FCR). The specific results are shown in Table 5.

[0067] Survival rate (%) = number of shrimps at the end of the experiment / number of shrimps at the beginning of the experiment × 100%; Weight gain rate (%) = (final weight - initial weight) / initial weight × 100%; Specific growth rate (%) = (ln final weight - ln initial weight) / number of days of feeding × 100%; Feed coefficient = dry weight of feed consumed / (final weight - initial weight).

[0068] Table 5 Effects of Chinese herbal medicine preparations on the growth performance of Penaeus vannamei Group Initial weight g Final body weight (g) Weight gain% Survival rate% Feed Coefficient Specific growth rate% A 3.24±0.03 <![CDATA[9.08±0.09 b ]]> <![CDATA[180.25±2.32 b ]]> <![CDATA[81±1.35 ab ]]> <![CDATA[1.36±0.18 b ]]> <![CDATA[3.68±0.24 b ]]> B 3.04±0.07 <![CDATA[10.33±0.15 a ]]> <![CDATA[239.80±1.85 a ]]> <![CDATA[92±1.06 a ]]> <![CDATA[1.27±0.12 b ]]> <![CDATA[4.37±0.13 a <!-- 8 -->]]> C 3.21±0.04 <![CDATA[8.79±0.26 b ]]> <![CDATA[173.83±2.35 ab ]]> <![CDATA[84.43±0.27 a ]]> <![CDATA[1.43±0.11 b ]]> <![CDATA[3.60±0.15 b ]]> Comparison 3.18±0.09 <![CDATA[7.24±0.23 ab ]]> <![CDATA[127.67±1.34 ab ]]> <![CDATA[72.37±0.02 b ]]> <![CDATA[1.68±0.18 a ]]> <![CDATA[2.94±0.22 ab ]]> From the results in Table 5, it can be seen that compared with the control group, the weight gain rate, survival rate and specific growth rate of the experimental group fed with Chinese herbal preparations were significantly improved, and the feed coefficient was generally reduced.

[0069] 4. Attack the virus After continuously feeding the feed containing the Chinese herbal medicine preparation of the present invention for 28 days, a challenge experiment was conducted. Vibrio parahaemolyticus (1×10 7CFU / mL) for virus attack, 0.02 mL / animal, and after the injection, the animals were kept in normal feeding and the number of deaths in each group was recorded every day. The observation was continued for 6 days, the mortality rate was counted, and the relative immune protection rate was calculated. The specific results are shown in Table 6.

[0070] The relative immune protection rate (RPS) calculation formula is as follows: RPS=(1-mortality rate of experimental group / mortality rate of control group)×100%.

[0071] Table 6 Effects of Chinese herbal medicine preparations on the immune performance of Penaeus vannamei Group Vannamei shrimp mortality rate (%) Relative immune protection rate (%) A 27.54% 67.45% B 17.33% 79.52% C 23.69% 72.00% Control group 84.60% 0 As shown in Table 6, after the challenge with Vibrio parahaemolyticus, the mortality rate of Penaeus vannamei in the control group was as high as 84.60%, while the mortality rate of Penaeus vannamei in the experimental group fed with the Chinese herbal medicine preparation of the present invention was only 17.33%-27.54%; among them, the relative immune protection rate of Penaeus vannamei in Group B fed with the Chinese herbal medicine preparation of Example 7 was as high as 79.52%. This shows that the Chinese herbal medicine preparation provided by the present invention can effectively improve the resistance of aquatic animals to Vibrio parahaemolyticus, and the immune effect is significant.

[0072] 3. Determination of serum biochemical indicators Five shrimps were randomly selected from each test pool, and blood was drawn from the shrimp's venous sinus with a syringe. After standing for 2 hours, the blood was centrifuged at 4°C for 10 minutes at a speed of 4000 r / min. The supernatant was retained and the activities of serum phenoloxidase, superoxide dismutase and lysozyme were measured using the kit of Nanjing Jiancheng Bioengineering Research Institute. The specific results are shown in Table 7.

[0073] Table 7 Effects of Chinese herbal medicine preparations on serum immune indexes of Penaeus vannamei project Control group A B C <![CDATA[Phenol oxidase (U·min －1 ·mL －1 )]]> <![CDATA[2.85±0.35 b ]]> <![CDATA[4.68±0.76 ab ]]> <![CDATA[5.05±0.81 a ]]> <![CDATA[4.71±0.62 ab ]]> <![CDATA[Superoxide dismutase (U·mL －1 )]]> <![CDATA[130.72±2.37 b ]]> <![CDATA[142.97±2.58 ab ]]> <![CDATA[147.81±2.17 a ]]> <![CDATA[142.28±2.61 ab ]]> <![CDATA[Lysozyme (μg·mL －1 )]]> <![CDATA[28.45±2.16 b ]]> <![CDATA[35.49±2.48 ab ]]> <![CDATA[38.36±2.29 a ]]> <![CDATA[34.89±2.12 ab ]]> From the results in Table 7, it can be seen that compared with the control group, the activities of phenoloxidase, superoxide dismutase and lysozyme in the serum of Penaeus vannamei in the experimental group fed with the Chinese herbal medicine preparation were significantly increased; among them, the activities of phenoloxidase, superoxide dismutase and lysozyme in the serum of Penaeus vannamei in Group B fed with the Chinese herbal medicine preparation described in Example 7 were the highest, increasing by 77.2%, 13.1% and 34.8% respectively. It can be seen that the Chinese herbal medicine preparation provided by the present invention can effectively improve the immune indexes of Penaeus vannamei and enhance its disease resistance.

[0074] In summary, the Chinese herbal medicine preparation prepared by fermenting Chinese medicinal raw materials with Clostridium butyricum YYJ-1 can be widely used in aquatic feed, can significantly improve the growth performance of aquatic animals, increase feed utilization, enhance the immune performance and disease resistance of aquatic animals, and reduce the occurrence of diseases, with very significant effects.

Claims

1. A Chinese herbal medicine preparation, characterized in that: The Chinese herbal medicine preparation contains Clostridium butyricum ( Clostridium butyricum ).

2. The Chinese herbal medicine preparation according to claim 1, characterized in that: The deposit number of the Clostridium butyricum is CCTCCNO: M2024807.

3. The Chinese herbal medicine preparation according to claim 2, characterized in that: The Chinese herbal medicine preparation is prepared by fermenting Chinese medicinal raw materials with Clostridium butyricum.

4. The Chinese herbal medicine preparation according to claim 3, characterized in that: The Chinese medicinal raw materials include dried tangerine peel, astragalus, wild hawthorn, calamus, artemisia annua, garlic and betel nut.

5. The Chinese herbal medicine preparation according to claim 4, characterized in that: The components of the Chinese medicinal raw materials and their weight proportions are respectively 15 parts of dried tangerine peel, 25 parts of astragalus, 15 parts of wild hawthorn, 30 parts of calamus, 20 parts of artemisia annua, 5 parts of garlic and 8 parts of betel nut.

6. The Chinese herbal medicine preparation according to claim 5, characterized in that: The preparation method of the Chinese herbal medicine preparation comprises the following steps: (1) Prepare Chinese medicinal materials, the components and weight proportions of which are as follows: 15 parts of dried tangerine peel, 25 parts of astragalus, 15 parts of wild hawthorn, 30 parts of calamus, 20 parts of artemisia annua, 5 parts of garlic, and 8 parts of betel nut; (2) Grind the Chinese medicine raw materials and pass them through a 60-mesh sieve to obtain Chinese medicine powder; add 5-10 times the weight of water to the Chinese medicine powder, stir evenly, and soak for 24 hours to obtain a Chinese medicine suspension; (3) placing the Chinese medicine suspension and the fermentation medium in a fermentation container at a mass ratio of 1-2:1, and sterilizing them by high pressure wet heat at 121°C for 20 min to obtain the Chinese medicine fermentation matrix; (4) inoculating the activated Clostridium butyricum liquid into the traditional Chinese medicine fermentation matrix at a volume ratio of 2-5%; filling the sealed fermentation container with nitrogen, exhausting the oxygen, and culturing at 37°C for 2-3 days; (5) Unseal the fermentation container, dry the fermentation product at 50-60°C, take it out, crush it, and pass it through a 60-mesh sieve to obtain the Chinese herbal medicine preparation.

7. The Chinese herbal medicine preparation according to claim 6, characterized in that: The components and weight proportions of the fermentation medium in step (3) of the preparation method are respectively: 22 parts of peptone, 13 parts of glucose, 2.0 parts of soybean meal, 6 parts of sodium alginate, 1 part of NH4NO3, 1 part of K2HPO4, 0.1 part of MgSO4, 0.1 part of MnSO4, 1 part of CaCO3, 5 parts of urea, 0.5 part of L-cysteine, and 500 parts of water.

8. The Chinese herbal medicine preparation according to claim 7, characterized in that: In the preparation method, the amount of live bacteria in the Clostridium butyricum liquid in step (4) is not less than 10 10 CFU / mL.

9. Use of the Chinese herbal medicine preparation according to any one of claims 1 to 8 in the production of aquatic feed.

10. A feed additive, characterized in that: The feed additive comprises the Chinese herbal medicine preparation according to any one of claims 1-8.

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