A composite shrimp feed additive and its application in shrimp farming

Abstract

The present invention discloses a compound penaeid shrimp feed additive and its application in penaeid shrimp farming. It is characterized by comprising Lactobacillus plantarum Ep-M17 and Tianxiangju powder. The preservation number of Lactobacillus plantarum Ep-M17 is CGMCC No. 24559, and the final addition concentration in the penaeid shrimp basal feed is 5×10<supgt;8< / supgt> CFU / g. The added mass percentage of Tianxiangju powder in the penaeid shrimp basal feed is 5%. The use of this compound penaeid shrimp feed additive in the preparation of drugs for inhibiting Enterocytozoon hepatopenaei in penaeid shrimp has the advantage of significantly improving the ability of penaeid shrimp to resist EHP disease and promoting the growth of penaeid shrimp.

Description

Technical Field

[0001] The invention relates to a composite shrimp feed additive, in particular to a composite shrimp feed additive and application thereof in shrimp farming. Background Art

[0002] Natural active ingredients such as polysaccharides, flavonoids, glycosides, etc. in plant feed additives can improve the immunity and disease resistance of aquatic animals and plants, thereby improving the ecological environment of aquaculture. Glehnia littoralis is a herb with wide adaptability, strong stress resistance, high yield, and rich nutritional value. It is rich in protein, amino acids, flavonoids, polyphenols, polysaccharides, vitamin C, vitamin E and other nutrients and bioactive functional substances. Feed additives based on Glehnia littoralis have low cost, good effects, and high nutrition and no antibiotics and disease prevention characteristics. They can be used as ideal antibiotic reduction and replacement products for aquaculture, which can promote the healthy and sustainable development of the aquaculture industry and increase economic benefits.

[0003] Probiotics are a type of beneficial microorganisms that exist in the intestines and can regulate the immune system, maintain the balance of the intestinal environment, and improve immunity. Since aquatic animals and plants are susceptible to infection by various pathogens, the addition of probiotics can help aquatic animals and plants establish healthy intestinal flora and enhance the intestinal defense against pathogens, thereby reducing the probability of disease. Studies have shown that probiotics have significant effects against a variety of aquaculture pathogens, such as Gram-positive bacteria, Gram-negative bacteria, and fungi. In addition, probiotics can also indirectly help prevent and treat parasitic diseases. For example, probiotics can promote food digestion and absorption, enhance the nutritional level and immune system of aquatic animals and plants, make them healthier, and resist the invasion of pathogens and parasites. At the same time, probiotics can also improve the decomposition of organic matter in water, maintain the stability and balance of water quality, and reduce the breeding and spread of parasitic diseases. Probiotics can be used in aquaculture through feeding, spraying, and input. Probiotics can also be made into compound preparations with other microorganisms, Chinese herbal medicines, and immune enhancers to enhance their disease prevention function. Therefore, the application of probiotics in aquaculture diseases can reduce the use of antibiotics, ensure the quality and safety of aquatic food, and improve aquaculture efficiency and sustainable development.

[0004] Enterocytozoon hepatopenaei (EHP) is one of the diseases that seriously affects the shrimp farming industry. Its main symptoms are yellow or brown spots on the shrimp body, and the mortality rate is as high as more than 30%. The diseased shrimp is susceptible to secondary infection by bacteria and fungi, which is the main cause of shrimp farming losses. The disease has various transmission pathways. At present, its transmission can be controlled by strengthening pond and equipment disinfection, shrimp feed management and drug treatment, but there is no effective prevention and control measure. The main components of existing cultured shrimp feed are protein, fat, vitamins and minerals, etc., which cannot effectively improve the disease resistance of cultured shrimp, nor can it effectively prevent and treat enterocytozoon hepatopenaei (EHP). Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method of an antibiotic-replacing feed additive based on chrysanthemum and lactic acid bacteria, which can significantly improve the anti-EHP disease ability of shrimp and promote the growth of shrimp, and its application in shrimp farming.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a composite shrimp feed additive, comprising plant lactobacillus Ep-M17 and chrysanthemum powder, wherein the preservation number of the plant lactobacillus Ep-M17 is CGMCC No. 24559, and the final concentration of the additive in the shrimp basic feed is 5×10 8 CFU / g, the mass percentage of the chrysanthemum powder added to the basic shrimp feed is 5%.

[0007] Furthermore, the preparation method of the chrysanthemum powder is to air-dry the chrysanthemum stems and leaves at room temperature or dry them at 40-60° C., and then grind them through a 100-mesh sieve.

[0008] The application of the composite shrimp feed additive in shrimp farming, the final concentration of the plant lactobacillus Ep-M17 added to the shrimp basic feed is 5×10 8 CFU / g, the mass percentage of the Tianxiangchrysanthemum powder added to the basic feed of shrimp is 5%, and after mixing, it is made into particles with a particle size of 1.5mm, or mixed with commercial feed and fed directly.

[0009] The composite shrimp feed additive is used in preparing shrimp hepatoenterozoan disease inhibitory drugs.

[0010] Compared with the prior art, the invention has the advantages that: the composite shrimp feed additive and the application thereof in shrimp farming of the invention, by adding appropriate amounts of chrysanthemum extract powder and plant lactobacillus Ep-M17 into shrimp feed, can obviously promote the growth of shrimp and improve the ability of shrimp to resist hepatoenterozoan disease through their synergistic effect, has good economic and social benefits, is conducive to the sustainable and healthy development of shrimp farming, and conforms to the development strategy trend of aquatic feed.

[0011] The above-mentioned Lactobacillus plantarum is the Ep-M17 strain, classified and named Lactobacillus plantarum (Lactobacillus plantarum), and was deposited in the General Microbiology Center of the China Culture Collection Administration on March 21, 2022, with the deposit number CGMCC No. 24559. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Effects of different feed additives on the growth performance and survival rate of shrimp. Effects of different feeds on the specific growth rate (A) and feed conversion rate (B), as well as the survival rate (C) of Penaeus vannamei. Ctr1: healthy shrimp fed with basic feed; Ctr2: EHP-infected shrimp fed with basic feed; Lp: EHP-infected shrimp fed with basic feed + Ep-M17; Cp: EHP-infected shrimp fed with basic feed + Glechoma longituba; LC: EHP-infected shrimp fed with basic feed + Ep-M17 and Glechoma longituba compound feed;

[0013] Figure 2 Effects of different feed additives on shrimp resistance to EHP infection. A: EHP load in the hepatopancreas of Penaeus vannamei; B: EHP load in the intestine of Penaeus vannamei;

[0014] Figure 3 The effects of different feed agents on shrimp resistance to Vibrio infection. A: Analysis of Vibrio load in the hepatopancreas of Penaeus vannamei (left: plate coating to detect the number of Vibrio; right: Max turbidimetry to detect the number of Vibrio); B: Analysis of Vibrio load in the intestine of Penaeus vannamei (left: plate coating to detect the number of Vibrio; right: Max turbidimetry to detect the number of Vibrio);

[0015] Figure 4 Effects of different feed additives on digestion and immunity-related enzyme activities of Penaeus vannamei. A: Digestion and immunity-related enzyme activities in the hepatopancreas of Penaeus vannamei, B: Digestion and immunity-related enzyme activities in the intestine of Penaeus vannamei. , where AKP: alkaline phosphatase (a); LZM: lysozyme (b); LPS: lipase (c); SOD: superoxide dismutase (d); α-AL: α-amylase (e); CAT: catalase (f). DETAILED DESCRIPTION

[0016] The present invention is further described in detail below with reference to the accompanying drawings.

[0017] 1. Implementation

[0018] 1. Weigh the raw materials separately: based on the weight of the cultured shrimp feed, Lactobacillus plantarum Ep-M17 is 5×10 8 Weigh Lactobacillus plantarum Ep-M17 and Tianxiangju fine powder at a ratio of 5% CFU / g feed and Tianxiangju fine powder;

[0019] 2. Grind the above-mentioned chrysanthemum stems and leaves into fine powder and pass through a 100-mesh sieve;

[0020] 3. Fully mix the above raw material powders to obtain the composite feed additive for promoting the growth of shrimp and improving the disease resistance of shrimp;

[0021] 4. Add the above compound feed additives to the basic shrimp feed (Tongwei, 8838 sinker 1.0-1.2 mm) to make granular bait for feeding.

[0022] The above-mentioned Tianxiangju: is rich in a series of nutrients and biologically active functional substances such as protein (10-18%), amino acids, flavonoids, polyphenols, polysaccharides, vitamin C, vitamin E, carotene, superoxide dismutase (SOD), etc. Plant lactobacillus Ep-M17: can produce a variety of metabolic substrates, such as vitamins, short-chain fatty acids, organic acids and digestive enzymes, which promote the host's nutritional metabolism and development. At the same time, it induces an increase in the activity level of enzymes related to the hepatopancreas and intestinal immunity and nutritional metabolism of shrimp, thereby improving the immune and metabolic functions of the hepatopancreas and intestinal tract of shrimp.

[0023] 2. Comparison

[0024] Comparative Example 1: Basic shrimp feed.

[0025] Comparative Example 2: Basic shrimp feed with 5 wt% of Glehnia littoralis powder added.

[0026] Comparative Example 3: Basic shrimp feed supplemented with Lactobacillus plantarum Ep-M17, wherein the amount of Lactobacillus plantarum Ep-M17 added was 5×10 8 CFU / g feed.

[0027] Comparative Example 4: Adding Lactobacillus plantarum Ep-M17 and chrysanthemum powder to the shrimp basic feed, wherein the amount of chrysanthemum powder added is 5wt%, and the amount of Lactobacillus plantarum Ep-M17 added is 5×10 8 CFU / g feed.

[0028] 3. Breeding Experiment Analysis

[0029] The experiment was conducted at the pilot plant of Ningbo University. The experimental shrimp Penaeus vannamei was cultured in an ecological circulation culture system. Each bucket (1m in diameter and 0.8m in height) was used to culture 50 shrimps. Three parallels were set up for each feed group. Shrimp fry with an average body length of 3.0±0.20cm and an average weight of 0.65±0.04g were uniformly released. The culture density was 50 shrimps / m 3 , the experiment was officially started 7 days after seeding. Four experimental groups (example group, comparative example 1, comparative example 2, comparative example 3 and comparative example 4) of Penaeus vannamei were infected by feeding the shrimp tissue containing enterocytosis (EHP). Uninterrupted aeration, water temperature 27±2℃, pH7.8~7.9, salinity 5‰. The daily feed amount was about 4% of the shrimp weight, with no or a little surplus as the standard. Feed 3 times a day, at 7:00 in the morning, 12:00 noon and 6:00 in the afternoon, respectively. Residual bait and excrement were removed in time. The breeding cycle was 60 days.

[0030] After the feeding experiment, the weight of the shrimp was measured, and the specific growth rate of the shrimp was calculated using the formula: WGR = (final weight - initial weight) / initial weight; the feed conversion rate of the shrimp was calculated using the formula: FCR = feed amount / (final weight - initial weight); the survival rate of the shrimp infected with EHP was calculated; and the number of enterocytosis and Vibrio in the hepatopancreas and intestines of the shrimp was detected. The specific growth rate results are shown in Figure 2. Figure 1 As shown in A, the specific growth rate of shrimp in the Ctr1 group was (304.92±29.77), the specific growth rate of shrimp in the Ctr2 group infected with EHP was (241.17±92.70), the specific growth rate of shrimp in the Lp group fed with Ep-M17 was (285.67±85.11), the specific growth rate of shrimp in the Cp group fed with Gastrodia elata was (265.00±97.26), and the specific growth rate of shrimp in the LC group fed with Ep-M17 and Gastrodia elata compound feed was (305.83±56.69). The feed conversion rate results are shown in Figure 1 As shown in B, the feed conversion rate of shrimp in the Ctr1 group was (70.25±6.04), the feed conversion rate of shrimp in the Ctr2 group was (66.64±22.83), the feed conversion rate of shrimp in the Lp group was (68.75±23.53), and the feed conversion rate of shrimp in the LC group was (71.54±12.02). Figure 1The results showed that after five weeks of infection with EHP, the survival rate of shrimp in the Ctr1 group was 91%, the survival rate of shrimp in the Ctr2 group dropped to 36%, the survival rates of shrimp in the Lp group and the Cp group were 51% and 53%, respectively, which were 15% and 17% higher than those in the Ctr2 group, respectively, while the survival rate of shrimp in the LC group was 72%, which was 36% higher than that in the Ctr2 group. In summary, the above study found that adding a mixture of chrysanthemum powder and Lactobacillus plantarum to the basic feed for feeding shrimp can significantly increase the specific growth rate of shrimp and the survival rate of infected shrimp, and its effect is higher than that of the basic feed and the addition of chrysanthemum or Ep-M17 alone, indicating that the chrysanthemum and Ep-M17 composite additive has good palatability, can improve the ability of Penaeus vannamei to resist EHP infection, significantly promote the growth of shrimp, and has a synergistic effect.

[0031] After the feeding experiment, qPCR was used to detect the EHP load in the hepatopancreas and intestine of shrimp. Five shrimps were randomly selected from each experimental group to obtain the hepatopancreas and intestine. After extracting bacterial DNA, specific primers were used to quantitatively detect the amount of EHP. The numbers with different letters indicate significant differences (p < 0.05). Figure 2 A shows that the EHP load of the Ctr2 group has been maintained at a high level. At the fifth week, the EHP load of the Ctr2 group was close to 10 8 The EHP load in the Lp, Cp and LC groups fed with Ep-M17 and Tianxiangju decreased with the extension of feeding time. In the fifth week, the EHP load in the hepatopancreas of the three groups was less than 10 3 copies / μL. The results of intestinal tissue qPCR are as follows Figure 2 B shows that the content of EHP in the intestine of shrimp is much lower than that in the hepatopancreas. In the fifth week, the EHP load of the Ctr2 group was about 1000 copies / μL, the EHP load of the Lp group was less than 10 copies / μL, the EHP load of the Cp group was about 100 copies / μL, and the EHP load of the LC group was less than 5 copies / μL. The EHP load in the hepatopancreas and intestine of shrimp fed with chrysanthemum and Ep-M17 additives was significantly lower than that of the group fed with ordinary feed. This shows that the addition of chrysanthemum and Ep-M17 can effectively inhibit the proliferation of EHP in shrimp.

[0032] After the feeding experiment, three shrimps were randomly selected from each group to obtain the hepatopancreas and intestines. The shrimp tissue homogenate was resuspended and diluted with TSB, and then the same volume of diluted sample solution was spread on the plate for comparison. At the same time, the absorbance of the sample solution was detected at a wavelength of 600nm. The comparison results of the number of Vibrio in the hepatopancreas of shrimp are shown in Figure 2. Figure 3As shown in A, the number of Vibrio in the hepatopancreas of the Ctr1 group was less, while the number of Vibrio in the hepatopancreas of the Ctr2 group was more. After feeding Ep-M17 and Tianxiangju, the number of Vibrio in the Lp group, Cp group and LC group decreased. Similarly, the absorbance results of the hepatopancreatic Vibrio sample showed that the OD of the Ctr1 group was (0.54±0.05), and the OD of the Ctr2 group was (1.04±0.14). The Lp group and Cp group were (0.45±0.09) and (0.84±0.15), respectively. The LC group fed with the combined feed was (0.34±0.08). The comparison results of the number of Vibrio in the shrimp intestine are shown in Figure 3 As shown in B, the results of the coating test showed that the number of Vibrio in the intestines of different groups of shrimps was similar to the change trend of Vibrio in the hepatopancreas, but the number was higher. Similarly, the absorbance results of the intestinal Vibrio sample showed that the OD value of the Ctr1 group was (0.77±0.09) and the OD value of the Ctr2 group was (1.08±0.14). The Lp group and the Cp group were (0.84±0.15) and (0.91±0.17), respectively. The LC group was (0.55±0.14). The Vibrio load in the hepatopancreas and intestines of shrimps fed with the composite additive of Tianxiangju and Ep-M17 was significantly lower than that of shrimps fed with the basic feed, indicating that the addition of the composite additive of Tianxiangju and Ep-M17 can inhibit the proliferation and translocation of Vibrio in the intestines of shrimps and improve the resistance of shrimps to Vibrio.

[0033] Three shrimps were randomly selected from the five experimental groups. Their hepatopancreas and intestines were disinfected with 75% alcohol and then homogenized with 1:10 (g / mL) 4°C saline. The hepatopancreas and intestinal homogenates were centrifuged at 3000r / min for 10min, and the supernatant was aspirated to detect enzyme activity. Enzyme-linked immunosorbent assay (ELISA) was used to detect alkaline phosphatase (AKP), lysozyme (LZM), superoxide dismutase (SOD), catalase (CAT), α-amylase (α-AL) and lipase (LPS). The results are as follows Figure 4 As shown in the figure, after feeding Ep-M17 and Tianxiangchrysanthemum, the digestive enzyme activities and immune-related enzyme activities in the hepatopancreas and intestine of shrimp were higher than those in the Ctr2 group. Figure 4 As shown in A, after feeding Ep-M17 and Tianxiangchrysanthemum, the activities of the six enzymes tested were significantly increased, among which the superoxide dismutase activity and lysozyme activity of the LC group were (175108.75±10508.01) and (173.55±4.90), respectively, while the SOD activity and LZM activity of the Ctr2 group were only (21952.07±392.46) and (35.27±1.97). Figure 4As shown in B, the activities of enzymes in the intestines of shrimps in the Ctr2 group were significantly lower than those in the intestines of healthy shrimps in the Ctr1 group. After feeding Ep-M17 and Glehnia littoralis, the activities of these six enzymes in the intestines of diseased shrimps increased significantly. Among them, the activities of superoxide dismutase, α-amylase, and catalase in the Ctr2 group were (416644.53±13215.50), (161.35±6.93), and (211.58±14.49), respectively. After feeding the combined feed, the activities of superoxide dismutase, α-amylase, and catalase in the LC group increased to (1225863.89±2938.91), (426.85±10.96), and (544.70±20.65), respectively. Feeding Glehnia littoralis powder and Lactobacillus plantarum to shrimps can significantly increase the activities of digestive and immune-related enzymes in the hepatopancreas and intestines of diseased shrimps. This indicates that adding the composite additive of chrysanthemum and Ep-M17 can enhance the digestion and immunity of shrimp.

[0034] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A composite shrimp feed additive, Features: The invention comprises plant lactobacillus Ep-M17 and chrysanthemum powder, wherein the preservation number of the plant lactobacillus Ep-M17 is CGMCC No.24559, and the final concentration of the plant lactobacillus Ep-M17 added to the basic feed of shrimp is 5×10 8 CFU / g, the mass percentage of the chrysanthemum powder added to the basic shrimp feed is 5%.

2. A composite shrimp feed additive according to claim 1, Features: The preparation method of the chrysanthemum powder is as follows: air-dry the chrysanthemum stems and leaves at room temperature or dry at 40-60 DEG C, and grind them to pass through a 100-mesh sieve.

3. Use of the composite shrimp feed additive according to claim 1 in shrimp farming, Features: The final concentration of Lactobacillus plantarum Ep-M17 added to the basic shrimp feed is 5×10 8 CFU / g, the mass percentage of the Tianxiangchrysanthemum powder added to the basic feed of shrimp is 5%, and after mixing, it is made into particles with a particle size of 1.5mm, or mixed with commercial feed and fed directly.

4. Use of the composite shrimp feed additive according to claim 1 in preparing a drug for inhibiting hepatoenterocytosis of shrimp.

Images