Application of composite probiotic agent in cultivation of cynoglossus semilaevis

Abstract

The invention provides application of a composite probiotic agent in cultivation of cynoglossus semilaevis, and belongs to the technical field of aquaculture. The composite probiotic agent provided by the invention is composed of Rhodospira sp., Rhodopseudomonas palustris and Rhodopseudomonas palustris. By mixing the feed for feeding, the intestinal function of the cynoglossus semilaevis can be enhanced, the absorption capacity of the cynoglossus semilaevis on nutrient substances is improved, the immunity of the cynoglossus semilaevis is enhanced, and the disease incidence is reduced; the proportion of harmful bacterium communities in the cynoglossus semilaevis benthic environment can be reduced by spraying the composite probiotic agent to the water bottom mud environment, and the morbidity of the cynoglossus semilaevis is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, and particularly to the application of a compound probiotic agent in the cultivation of Cynoglossus semilaevis. Background Art

[0002] Cynoglossus semilaevis is an important marine aquaculture fish. However, the current cultivation of Cynoglossus semilaevis faces many challenges. Under the traditional cultivation mode, the water quality is prone to deterioration, and harmful bacteria multiply in large numbers, which makes Cynoglossus semilaevis susceptible to diseases and affects its growth rate and quality. At the same time, the abuse of traditional prevention and control means such as antibiotics not only increases the cultivation cost, but also may lead to problems such as drug residues, endangering food safety and the ecological environment. Therefore, there is an urgent need for a green and efficient cultivation method to solve these problems, and the use of compound probiotics for cultivation has great potential.

[0003] Probiotics are a class of active microorganisms that can play a positive role in fish bodies or the cultivation environment. They can improve the cultivation environment by decomposing toxic substances (such as ammonia and nitrite) in the water body and bottom sediment. In addition, probiotics can promote the intestinal health of fish, enhance their immune systems, and make fish more resistant to diseases. Some probiotics can help fish grow better by improving the absorption of nutrients, such as Bacillus spp. and Lactobacillus spp. Moreover, by using probiotics, the dependence on chemical drugs and antibiotics in the cultivation environment can be reduced, which not only helps to maintain the ecological balance, but also can improve the cultivation efficiency and economic benefits, and produce safer and healthier aquatic products. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of a compound probiotic agent in the cultivation of Cynoglossus semilaevis, which can effectively improve the water quality, reduce the disease incidence rate of Cynoglossus semilaevis, and shorten the growth cycle by using the compound probiotic agent.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides the application of a compound probiotic agent in the cultivation of Cynoglossus semilaevis, which is characterized in that the compound probiotic agent is composed of Rhodospirillum, Rhodopseudomonas palustris and Chromatium.

[0007] Preferably, the bacterial concentration of Rhodospirillum in the compound probiotic agent is 3-5×10 7 CFU / mL, the bacterial concentration of Rhodopseudomonas palustris is 3-5×10 7 CFU / mL, and the bacterial concentration of Chromatium is 3-5×10 7 CFU / mL.

[0008] Preferably, the compound probiotic agent is used as follows: After feeding Cynoglossus semilaevis for 1 - 2 hours, the water in the culture pond is drained until there is 6 - 10 cm left, and then the compound probiotic agent is sprinkled.

[0009] Preferably, the feed for feeding contains 1% - 2% by mass of the compound probiotic agent.

[0010] Preferably, when culturing in the culture pond, the water level is maintained at 40 - 50 cm.

[0011] Preferably, the dosage of the compound probiotic agent is 250 - 350 mL sprinkled per cubic meter of water.

[0012] The compound probiotic agent provided by the present invention can colonize in the intestine of Cynoglossus semilaevis by being mixed into the feed and forming beneficial flora, which can enhance the intestinal function of Cynoglossus semilaevis, improve its absorption ability of nutrients, enhance the immunity of the fish body at the same time, and reduce the incidence of diseases; by sprinkling the compound probiotic agent on the bottom mud environment, it can also reduce the proportion of harmful bacteria communities in the benthic environment of Cynoglossus semilaevis and reduce the incidence of Cynoglossus semilaevis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 For the accuracy analysis of metagenomic sequencing of the intestinal contents of Cynoglossus semilaevis in Test Example 1;

[0014] Figure 2 For the result chart of the number of genes in the intestinal contents of Cynoglossus semilaevis in Test Example 1;

[0015] Figure 3 For the proportion of bacterial species in the intestinal contents at the genus level of the control group and the experimental group in Test Example 1;

[0016] Figure 4 For the number of bacterial species in the bottom mud at the genus level of the control group and the experimental group in Test Example 2;

[0017] Figure 5 For the proportion of bacterial species in the bottom mud at the genus level of the control group and the experimental group in Test Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0018] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0019] Example 1

[0020] The healthy Cynoglossus semilaevis used in the breeding experiment were from Weizhuo Aquatic Products Co., Ltd., Caofeidian District, Hebei Province, with a specification of body weight 200±20 g and body length 29±2 cm. The area of the breeding pond was 5 m×7 m×1 m. 1000 Cynoglossus semilaevis of the above specifications were put into each breeding pond, with a total of 3 experimental groups and 3 control groups. The water level was maintained at 40 cm during breeding. Experimental group: Cynoglossus semilaevis were fed with Haitong No. 3 bait (containing 2% quality of compound probiotic agent) produced by Santong Biotechnology Co., Ltd. once every morning and evening, and 1 hour later, the water in the breeding pond was drained until there was 8 cm left. 500 L of the compound probiotic agent was sprinkled into the breeding pond, and the inlet valve was opened. The treatment method of the control group was the same as that of the experimental group, except that the feed did not contain the compound probiotic agent, and the corresponding amount of water was used to replace the compound probiotic agent for sprinkling.

[0021] Test Example 1

[0022] 1.1 After breeding in the manner of Example 1 for 1 month, 6 Cynoglossus semilaevis were randomly selected from the experimental group and the control group respectively. Under sterile conditions, intestinal contents were taken, and total intestinal microbial DNA was extracted using the Tiangen Bacterial Genomic DNA Extraction Kit (DP302), and metagenomic sequencing was carried out. The results are as Figure 1 and Figure 2 shown.

[0023] It can be seen from Figure 1 that 12 groups of raw data in the sequencing process were filtered to obtain the corresponding clean data. In the valid results, the sequencing error rate less than 1% (Q20) reached more than 95%, and the error rate less than 0.1% (Q30) reached more than 87%, indicating that the sequencing data has high reliability.

[0024] It can be seen from Figure 2 that the overall number of genes in the intestinal contents of the control group (C-II-1, C-II-2, C-II-3) was higher than that in the intestinal contents of the experimental group (E-II-1, E-II-2, E-II-3). The overall number of genes in the bottom mud of the control group (cdn-1, cdn-2, cdn-3) was slightly higher than that in the bottom mud of the experimental group (mdn-1, mdn-2, mdn-3). It shows that after using the compound probiotic agent, the dominant bacterial population has an advantage in the breeding water environment of Cynoglossus semilaevis.

[0025] 1.2 Analysis of the α-diversity index of the two kinds of intestinal contents

[0026] As can be seen from Table 1, the chao1 index and ace index of the intestinal content samples of Cynoglossus semilaevis in the control group (C-II-1, C-II-2, C-II-3) were both between 401 and 403, the shannon index was between 2.1832 and 2.1945, and the simpson index was between 0.5990 and 0.6007. The chao1 index and ace index of the intestinal content samples of Cynoglossus semilaevis in the experimental group (E-II-1, E-II-2, E-II-3) were both between 186 and 218, the shannon index was between 0.4742 and 0.5324, and the simpson index was between 0.0976 and 0.1097. Generally speaking, the chao1 and ace indexes of the control group were slightly higher than those of the experimental group, and the shannon index and simpson index were overall higher than those of the experimental group. This shows that the richness of intestinal content species is Control Group > Experimental Group, and the evenness of species is Control Group > Experimental Group. This indicates that after using the compound probiotic agent, the dominant bacterial population in the intestine of Cynoglossus semilaevis occupies an obvious advantage.

[0027] Table 2 Results of α-diversity index of intestinal contents of Cynoglossus semilaevis in the control group and experimental group

[0028]

[0029]

[0030] 1.3 Bacterial community structure of Cynoglossus semilaevis intestine at the genus level

[0031] As Figure 3 can be seen, from the perspective of genera, among the intestinal bacteria of Cynoglossus semilaevis in the control group (C-II), bacteria of the genus Vibrio accounted for the highest proportion, about 86.38%, followed by the genus Photobacterium, accounting for about 7.66%. Among the intestinal bacteria of Cynoglossus semilaevis in the experimental group (E-II), bacteria of the genus Photobacterium accounted for about 83.77% and were the dominant bacterial population, followed by the genus Vibrio, accounting for about 5.90%. The proportion of bacteria of the remaining genera was relatively low. The genus Vibrio is widely distributed in marine and freshwater environments. This genus contains various species, including some important pathogens such as Vibrio cholerae (causing cholera) and Vibrio parahaemolyticus (causing seafood-related food poisoning). The genus Photobacterium is a genus in which most bacteria are beneficial. It can be seen that after using the compound probiotic agent, it is beneficial to the maintenance and stability of beneficial intestinal flora microorganisms of Cynoglossus semilaevis.

[0032] Experimental Example 2

[0033] After culturing in the manner of Example 1 for 1 month, 3 bottom sediment samples were taken from three sides at a position 40 cm above the water surface in the culture ponds of Cynoglossus semilaevis in the control group and the experimental group respectively, and metagenomic sequencing was carried out.

[0034] 2.1 Analysis of α-diversity indices of the two bottom sediments

[0035] The α-diversity index can reflect the evenness and richness of microorganisms. Generally speaking, the larger the values of the three indices of chao1, ace, and shannon, the higher the species richness; the closer the simpson index is to 1, the higher the species evenness. As can be seen from Table 2, the chao1 index of the bottom sediment samples (cdn-1, cdn-2, cdn-3) in the control group was between 1453.4615 and 1495, the ace index was between 1455.0812 and 1493, the shannon index was between 6.2223 and 6.3339, and the simpson index was between 0.9631 and 0.9673. The chao1 index of the bottom sediment samples (mdn-1, mdn-2, mdn-3) in the experimental group was between 1324 and 1458.4737, the ace index was between 1324 and 1458.5263, the shannon index was between 4.7967 and 5.9531, and the simpson index was between 0.8741 and 0.9499. Generally speaking, the chao1 and ace indices of the bottom sediment in the control group were slightly higher than those in the experimental group, and the shannon index and simpson index were overall higher than those in the experimental group. It shows that the richness of the bottom sediment bacteria species is control group bottom sediment > experimental group bottom sediment, and the evenness of the bottom sediment species is control group bottom sediment > experimental group bottom sediment. This also shows that after using the compound probiotic agent, the number of dominant bacteria in the aquaculture water environment of Cynoglossus semilaevis occupies an advantage.

[0036] Table 2 Results of α-diversity indices of the bottom sediments in the control group and the experimental group

[0037]

[0038]

[0039] 2.2 Bacterial community structure of the bottom sediment at the genus level

[0040] From the perspective of genera, in the species stacking diagram ( Figure 4) Except for unidentified and other bacteria, the dominant bacteria in the sediment of the control group (cdn) were not obvious. Among them, the relative abundances of Leucothrix (5.1242%), Roseovarius (2.7389%), Aliiroseovarius (4.8071%), Polaribacter (3.2835%), Ardenticatena (3.8461%), Hanstruepera (2.7333%), and Tenacibaculum (2.8052%) accounted for approximately 25.34% of the total bacteria in the sediment of the control group. In the sediment of the experimental group (mdn), Leucothrix (18.1382%), Roseovarius (8.3857%), Aliiroseovarius (5.9824%), Photobacterium (8.9183%), and Vibrio (4.5522%) were the dominant bacteria, and the sum of their relative abundances accounted for approximately 45.98% of the total bacteria in the sediment of the experimental group ( Figure 5 ).

[0041] Through comparison, it was found that after using the compound probiotic agent, bacteria genera such as Leucothrix, Roseovarius, Aliiroseovarius, and Photobacterium, which can degrade various organic substances, dominated in the aquaculture pond, leaving no living space for harmful bacteria genera, thus greatly improving the aquaculture environment of Cynoglossus semilaevis.

[0042] Test Example 3

[0043] After culturing in the manner of Example 1 for 1 month, the body weight (randomly selecting 30 fish to calculate the average body weight) and the number of dead fish of Cynoglossus semilaevis were measured. The results are shown in Table 3. The average body weight of the control group was 271.5 g, and the average body weight of the experimental group was 283.7 g. After using the compound probiotic agent, the body weight of Cynoglossus semilaevis increased by 6.1% compared with the control group, and the mortality rate decreased by 1.3%, from 4.1% to 2.8%.

[0044] Table 3 Body weight and number of dead fish of Cynoglossus semilaevis

[0045] Average body weight (g) Number of deaths (tails) Experimental group 283.7 28 Control group 271.5 41

[0046] Test Example 4

[0047] After using the compound probiotic agent for 1 month, the pH, ammonia nitrogen, nitrite, and dissolved oxygen in the aquaculture environment were detected. As shown in Table 4, the statistical results indicated that the aquaculture environment of Cynoglossus semilaevis was greatly improved, and the compound probiotic agent had the effects of stabilizing pH, reducing ammonia nitrogen and nitrite, and increasing dissolved oxygen.

[0048] Table 4 Effects of compound probiotic on the aquaculture environment of Cynoglossus semilaevis

[0049] pH value Ammonia nitrogen (mg / L) Nitrite (mg / L) Dissolved oxygen (mg / L) Experimental group 7.9 0.02 0.08 6.0 Control group 7.0 0.08 0.13 5.5

[0050] In summary, the present method can effectively inhibit the damage of harmful bacteria to the cultivation of Cynoglossus semilaevis, ensure that beneficial bacteria predominate in the cultivation environment and intestinal microenvironment of Cynoglossus semilaevis, thereby improving the survival rate of Cynoglossus semilaevis cultivation.

[0051] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Application of a compound probiotic agent in culturing Cynoglossus semilaevis, characterized in that, The composite probiotic agent is composed of Rhodospirillum, Rhodopseudomonas palustris and Thiospirillum.

2. The application according to claim 1, characterized in that The bacterial concentration of Rhodospirillum in the compound probiotic agent is 3-5×10 7 CFU / mL, the bacterial concentration of Rhodopseudomonas palustris is 3-5×10 7 CFU / mL, and the bacterial concentration of Chromatium is 3-5×10 7 CFU / mL.

3. The application according to claim 1, characterized in that, The usage method of the composite probiotic agent is as follows: After feeding Cynoglossus semilaevis for 1 - 2 hours, drain the water in the culture pond until there is 6 - 10 cm left, and then sprinkle the composite probiotic agent.

4. The application according to claim 3, characterized in that The feed for feeding contains 1% - 2% by mass of the composite probiotic agent.

5. The application according to claim 3, characterized in that, When culturing in the culture pond, the water level is maintained at 40 - 50 cm.

6. The application according to claim 3, wherein The dosage of the composite probiotic agent is 250 - 350 mL sprinkled per cubic meter of water.

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