Application of biotin in preparation of feed or medicine for inhibiting bacteria and increasing apostichopus japonicus cell phagocytic activity

Abstract

The invention relates to application of biotin in preparation of feed or medicine for inhibiting bacteria and increasing apostichopus japonicus cell phagocytic activity, and belongs to the field of aquaculture, the application method is as follows: biotin is added into the medicine or feed to improve expression of superoxide dismutase and ferritin genes in apostichopus japonicus bodies, cells are protected from oxidative damage, and the apostichopus japonicus cell phagocytic activity is improved. Meanwhile, the expression of c3 and tlr3 genes is up-regulated, and the recognition capability of the stichopus japonicus on exogenous pathogens is promoted, so that the non-specific immunity of the stichopus japonicus can be remarkably enhanced, and the effect of preventing the skin ulceration syndrome of the stichopus japonicus is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of aquaculture, and particularly relates to the application of biotin in the preparation of feed or drugs for inhibiting bacteria and increasing the phagocytic activity of Apostichopus japonicus cells. Background Art

[0002] Apostichopus japonicus, also known as the sea cucumber, is the main aquaculture variety in China's sea cucumber aquaculture industry. In recent years, with the continuous expansion of the aquaculture scale and the improvement of the intensification level, the problem of diseases has become increasingly serious, posing challenges to the development of the industry. Apostichopus japonicus skin ulcer syndrome is the most common and most harmful disease in the process of Apostichopus japonicus aquaculture, causing economic losses of tens of billions of yuan every year and has become a key factor restricting the stable development of the industry. The disease was first discovered in Shandong Province in February 2003 and began to break out on a large scale along the northern coast of China in 2004. In recent years, affected by factors such as global climate change, germplasm degradation, and pathogen evolution, the occurrence of the disease has gradually become normal, posing a severe challenge to the Apostichopus japonicus aquaculture industry.

[0003] As a natural feed additive, biotin has received extensive attention for its potential in regulating metabolism. As a B vitamin, it is an essential coenzyme for five specific carboxylases, and biotin plays an important role in skin health, metabolic regulation, and immune response, and can effectively improve barrier function and antioxidant capacity. In humans and terrestrial animals, biotin deficiency may lead to skin lesions such as dermatitis and abnormal keratinization. Research shows that biotin is crucial for skin health and metabolic regulation. For Apostichopus japonicus, the function of biotin has not been studied. Summary of the Invention

[0004] The present invention provides the application of biotin in the preparation of feed or drugs for inhibiting bacteria and increasing the phagocytic activity of Apostichopus japonicus cells in view of the above technical problems. The biotin can significantly enhance the non-specific immunity of Apostichopus japonicus and has an inhibitory effect on pathogenic bacteria.

[0005] The present invention is realized through the following technical solutions:

[0006] The application of biotin in the preparation of feed or drugs for inhibiting bacteria and increasing the phagocytic activity of Apostichopus japonicus cells, and the application method is to add biotin to the drug or feed to increase the expression of superoxide dismutase and ferritin genes in Apostichopus japonicus, protect cells from oxidative damage, and at the same time up-regulate the expression of c3 and tlr3 genes.

[0007] Furthermore, the application is to add 1.5% by mass of biotin to the feed finally fed to Apostichopus japonicus.

[0008] The present invention also provides the application of the biotin in the preparation of feed for preventing Apostichopus japonicus from skin ulcer syndrome. The application is to add biotin with a mass ratio of 0.5% - 2% to the feed to increase the expression of superoxide dismutase and ferritin genes in Apostichopus japonicus, protect cells from oxidative damage, and at the same time up-regulate the expression of c3 and tlr3 genes, so as to enhance the recognition and clearance ability of Vibrio splendidus, and achieve the effect of preventing Apostichopus japonicus from skin ulcer syndrome.

[0009] The beneficial effects of the present invention compared with the prior art: By adding biotin to the feed or medicine of Apostichopus japonicus, the present invention can up-regulate antioxidant-related genes (such as SOD and ferritin genes). Biotin enhances the antioxidant capacity of Apostichopus japonicus at the molecular level and reduces oxidative stress caused by infection. The up-regulation of SOD (superoxide dismutase) and ferritin genes can scavenge excessive reactive oxygen species (ROS) generated during the infection process and protect cells from oxidative damage. This gene regulation effect may relieve oxidative damage of Apostichopus japonicus under the pressure of pathogen infection more effectively than directly enhancing enzyme activity. On the other hand, the complement system and Toll-like receptor pathway show more targeted regulation effects in long-term defense. Adding biotin with a mass ratio of 1.5% to the feed of Apostichopus japonicus significantly up-regulates the expression of c3 and tlr3 genes. c3 is a key component in the complement system. The up-regulation of c3 gene expression in Apostichopus japonicus may mean that the complement system is activated, thus enhancing the recognition and clearance ability of Vibrio splendidus. Toll-like receptor 3 encoded by tlr3 gene is an important molecule for pathogen recognition, which can recognize the nucleic acid components of viruses and bacteria and activate the immune response. This regulation mechanism shows that biotin can not only relieve the pressure on the immune system, but also strengthen the immune defense ability when necessary, forming a more targeted protection effect. All these indicate that the addition amount of 1.5% biotin can significantly enhance the non-specific immunity of Apostichopus japonicus.

[0010] By adding 0.5% - 2% of biotin to the feed of Apostichopus japonicus, the present invention can effectively improve the non-specific immunity of Apostichopus japonicus, increase the survival rate of Apostichopus japonicus when facing the infection of Vibrio splendidus, and can also improve the water body flora structure of the Apostichopus japonicus breeding environment and reduce the proportion of the number of harmful bacteria. Description of the Drawings

[0011] Figure 1 It is the survival rate diagram of the cultured Apostichopus japonicus of the present invention;

[0012] Figure 2 It is the relative abundance diagram of the water body flora of the cultured Apostichopus japonicus before and after virus challenge of the present invention; a is before virus challenge, and b is after virus challenge;

[0013] Figure 3This is a comparison chart of the non-specific immunity of cultured sea cucumbers in the present invention; a shows the expression of ferritin gene in each experimental group, b shows the expression of tlr3 gene in each experimental group, c shows the expression of sod gene in each experimental group, d shows the expression of c3 gene in each experimental group, e shows the phagocytic activity of coelomocytes of sea cucumbers in each experimental group, and f shows the respiratory burst activity of coelomocytes of sea cucumbers in the experimental group. Detailed implementation mode

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. The following described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. The exemplified embodiments are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

[0015] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0016] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified. Example 1: Application of biotin in preparing feed for inhibiting bacteria and increasing the phagocytic activity of sea cucumber cells I. Feed preparation

[0017] (1) Mixing materials: First, stir 55 parts of sargassum powder, 15 parts of fish meal, 2 parts of guar gum, 2 parts of compound vitamins, 2 parts of compound mineral elements, and 23.5 parts of sea mud evenly according to the specified weight fractions of each component to make a basic feed; finally, add 0.5 part of biotin and stir evenly.

[0018] The basic feed is not limited to the above ratio, and any feed that can meet the healthy growth needs of sea cucumbers can be used as the basic feed; in this embodiment, the compound vitamins include vitamin A, D3, E, K3, B1, B2, B6, B12, C, H, nicotinic acid, folic acid, nicotinamide, etc., and the compound vitamins are purchased from Henan Huaxu Biotechnology Co., Ltd.

[0019] The compound minerals contain iron, copper, zinc, manganese, magnesium, iodine, selenium, cobalt, etc., and are purchased from Henan Huaxu Biotechnology Co., Ltd.

[0020] (2) Pelleting: Add 20% of pure water based on the weight of the basic raw materials, mix the raw materials into a dough-like shape, and use a feed pelletizer to press the uniformly mixed raw materials into pellet feed;

[0021] (3) Seal and store the pellets after drying.

[0022] II. Effect verification

[0023] Select healthy sea cucumbers weighing about 20 g. Set up 3 parallels for each experimental group. The initial feeding amount is 3% of the weight of the sea cucumbers. Feed the sea cucumbers once at 7:00 every morning. After feeding, conduct bottom suction and water change after 12 hours. Adjust the feeding amount every 10 days according to the growth of the sea cucumbers. At the same time, set the feeding of conventional feed products as the control group and feed in the same way for 60 days. At the same time, set the feeding of conventional feed products as the control group and feed in the same way for 60 days. The main components of the conventional feed products in this example are 55 parts of sargassum powder, 24 parts of sea mud, 15 parts of fish meal, 2 parts of guar gum, 2 parts of compound vitamins, and 2 parts of compound mineral elements.

[0024] Example 2: Application of biotin in preparing feed for inhibiting bacteria and increasing the phagocytic activity of sea cucumber cells. The application method includes the following steps:

[0025] I. Feed preparation

[0026] (1) Mixing: First, stir 55 parts of sargassum powder, 15 parts of fish meal, 2 parts of guar gum, 2 parts of compound vitamins, 2 parts of compound mineral elements, and 23 parts of sea mud evenly according to the specified weight fractions of each component to make a basic feed; finally, add 1 part of biotin and stir evenly. The basic feed is not limited to the above ratio, and any feed that can meet the healthy growth needs of sea cucumbers can be used as the basic feed; in this example, the compound vitamins include vitamins A, D3, E, K3, B1, B2, B6, B12, C, H, nicotinic acid, folic acid, nicotinamide, etc., and the compound vitamins are purchased from Henan Huachu Biotechnology Co., Ltd.

[0027] The compound minerals include iron, copper, zinc, manganese, magnesium, iodine, selenium, cobalt, etc., and are purchased from Henan Huachu Biotechnology Co., Ltd.

[0028] (2) Pelleting: Add 20% of pure water based on the weight of the above basic raw materials, mix the raw materials into a dough-like shape, and use a feed pelletizer to press the uniformly mixed raw materials into pellet feed;

[0029] (3) Seal and store the pellets after drying.

[0030] II. Effect verification

[0031] Select healthy sea cucumbers weighing about 20 g. Set up 3 parallels for each experimental group. The initial feeding amount is 3% of the weight of the sea cucumbers. Feed the sea cucumbers once at 7:00 every morning. After feeding, carry out bottom suction and water change after 12 hours. Adjust the feeding amount every 10 days according to the growth of the sea cucumbers. At the same time, set the feeding of conventional feed products as the control group and feed in the same way for 60 days. At the same time, set the feeding of conventional feed products as the control group and feed in the same way for 60 days. The main components of the conventional feed product in this example are 55 parts of sargassum powder, 24 parts of sea mud, 15 parts of fish meal, 2 parts of guar gum, 2 parts of compound vitamins, and 2 parts of compound mineral elements.

[0032] Example 3: Application of biotin in the preparation of feed for inhibiting bacteria and increasing the phagocytic activity of sea cucumber cells. The application method includes the following steps:

[0033] I. Feed preparation

[0034] (1) Mixing: First, stir 55 parts of sargassum powder, 15 parts of fish meal, 2 parts of guar gum, 2 parts of compound vitamins, 2 parts of compound mineral elements, and 22.5 parts of sea mud evenly according to the specified weight fractions of each component to make a basic feed; finally, add 1.5 parts of biotin and stir evenly.

[0035] The basic feed is not limited to the above ratio, and any feed that can meet the healthy growth needs of sea cucumbers can be used as the basic feed; in this example, the compound vitamins include vitamin A, D3, E, K3, B1, B2, B6, B12, C, H, nicotinic acid, folic acid, nicotinamide, etc., and the compound vitamins are purchased from Henan Huachu Biotechnology Co., Ltd.

[0036] The compound minerals include iron, copper, zinc, manganese, magnesium, iodine, selenium, cobalt, etc., and are purchased from Henan Huachu Biotechnology Co., Ltd.

[0037] (2) Pelleting: Add 20% of pure water based on the weight of the basic raw materials, mix the raw materials into a dough-like shape, and use a feed pellet machine to press the uniformly mixed raw materials into pellet feed;

[0038] (3) The pellets are sealed and stored after drying.

[0039] II. Effect verification

[0040] Select healthy sea cucumbers weighing about 20 g. Set up 3 parallels for each experimental group. The initial feeding amount is 3% of the sea cucumber body weight. Feed the sea cucumbers once at 7:00 every morning. After feeding, perform bottom suction and water change after 12 hours. Adjust the feeding amount every 10 days according to the growth of the sea cucumbers. At the same time, set the feeding of the conventional feed product as the control group and feed it in the same way for 60 days. At the same time, set the feeding of the conventional feed product as the control group and feed it in the same way for 60 days. The main components of the conventional feed product in this example are 55 parts of sargassum powder, 24 parts of sea mud, 15 parts of fish meal, 2 parts of guar gum, 2 parts of compound vitamins, and 2 parts of compound mineral elements.

[0041] Example 4: Application of biotin in preparing feed for inhibiting bacteria and increasing the phagocytic activity of sea cucumber cells. The application method includes the following steps:

[0042] Mixing: First, stir 55 parts of sargassum powder, 15 parts of fish meal, 2 parts of guar gum, 2 parts of compound vitamins, 2 parts of compound mineral elements, and 22 parts of sea mud evenly according to the specified weight fractions of each component to make the basic feed; finally, add 2 parts of biotin and stir evenly.

[0043] The basic feed is not limited to the above ratio, and any feed that can meet the healthy growth needs of sea cucumbers can be used as the basic feed; in this example, the compound vitamins include vitamin A, D3, E, K3, B1, B2, B6, B12, C, H, nicotinic acid, folic acid, nicotinamide, etc., and the compound vitamins are purchased from Henan Huaxu Biotechnology Co., Ltd.

[0044] The compound minerals include iron, copper, zinc, manganese, magnesium, iodine, selenium, cobalt, etc., and are purchased from Henan Huaxu Biotechnology Co., Ltd.

[0045] (2) Pelleting: Add 20% of pure water based on the weight of the basic raw materials, mix the raw materials into a dough-like shape, and use a feed pelletizer to press the uniformly mixed raw materials into pellet feed;

[0046] (3) After drying the pellets, store them sealed.

[0047] II. Effect verification

[0048] Select healthy sea cucumbers weighing about 20 g. Set up 3 parallels for each experimental group. The initial feeding amount is 3% of the sea cucumber weight. Feed the sea cucumbers once at 7:00 every morning. After feeding, perform bottom suction and water change after 12 hours. Adjust the feeding amount every 10 days according to the growth of the sea cucumbers. At the same time, set the feeding of conventional feed products as the control group and feed in the same way for 60 days. At the same time, set the feeding of conventional feed products as the control group and feed in the same way for 60 days. The main components of the conventional feed product in this example are 55 parts of sargassum powder, 24 parts of sea mud, 15 parts of fish meal, 2 parts of guar gum, 2 parts of compound vitamins, and 2 parts of compound mineral elements.

[0049] As Figure 1 This is the survival rate chart of the sea cucumbers cultured by the present invention. Compared with the control group, the survival rate of the experimental group increased significantly. Especially in the experimental group with 1.5% biotin added, the survival rate reached 90% (P < 0.05), indicating that the feed of the present invention has a significant positive effect on the disease resistance of sea cucumbers.

[0050] Take some of the cultured sea cucumbers in Examples 1-4 for the challenge experiment. The challenge method is to add a suspension of Vibrio splendidus with a concentration of 1×10 7 CFU / mL into each water tank so that the concentration of Vibrio splendidus in the seawater is 1×10 5 CFU / ml. Soak continuously for 14 days, change half of the water every day, and re-add the suspension of Vibrio splendidus after water change to maintain the vibrio concentration. Observe the situation of sea cucumbers every day during this period. As Figure 2 shown, this is the relative abundance chart of the water body bacterial community at the phylum level of the challenged cultured sea cucumbers of the present invention. In the analysis result of the relative abundance of the water body bacterial community, the abundance of Proteobacteria was relatively high before the challenge (87.9516%), but decreased to 65.5885% after the challenge, showing an obvious decrease compared with the control group (90.504% after the challenge). Some conditional pathogenic bacteria are often contained in Proteobacteria, indicating that biotin may play a disease prevention role by inhibiting the relative dominance of potential pathogenic bacteria. This dynamic optimization of the water body bacterial community provides a stable external barrier for sea cucumbers, reducing both the infection risk of Vibrio splendidus and providing a benign environment for the immune system of sea cucumbers. The abundance of Bacteroidetes was relatively low in the 1.5% group before the challenge (4.3214%), but increased to 21.237% after the challenge. Bacteroidetes has the function of decomposing organic matter in the ecosystem. The increase in its abundance may reflect changes in nutrient utilization and competitive pressure, which helps to inhibit the growth of pathogenic bacteria or inhibit their activity through products.

[0051] Take a part of the cultured sea cucumbers from Examples 1-4 for real-time fluorescence quantitative qPCR detection. Take a certain amount of the inner body wall of each sea cucumber, add 1 ml of TRIzol reagent, and grind evenly. Let it stand at room temperature for 5 minutes to ensure cell lysis and release of RNA. Then add 0.2 ml of chloroform, mix well by shaking, and let it stand at room temperature for 5 minutes. Subsequently, centrifuge at 12,000 r / min for 10 minutes, which is divided into three layers. Carefully transfer the supernatant to a new tube, add an equal volume of isopropanol and mix well, and let it stand at room temperature for 10 minutes to promote RNA precipitation. Centrifuge again at 12,000 r / min for 10 minutes to remove the supernatant. Add 75% ethanol to wash the RNA precipitate, and centrifuge at 12,000 r / min for 5 minutes. After removing the ethanol, air-dry the RNA precipitate naturally and dissolve it in RNase-free water. Detect its concentration and quality by spectrophotometer and gel electrophoresis. For cDNA synthesis, use the FastKing One-Step Genomic DNA Removal and cDNA First Strand Synthesis Premix Kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. to reverse transcribe RNA into cDNA. The qPCR reaction uses the FastReal Fast Fluorescent Quantitative PCR Premix Kit (SYBR Green) from Tiangen Biochemical Technology (Beijing) Co., Ltd. for real-time fluorescence quantitative PCR. To ensure the reliability of the data, each experimental group is performed with 3 technical replicates. That is, each sample is subjected to 3 technical replicates for the qPCR reaction to ensure the accuracy and consistency of the results. The gene expression level is analyzed and compared using the 2 -△△CT -ΔΔCt method, which can effectively eliminate operation errors and technical biases, thereby improving the reliability of experimental data. Figure 3This is a comparison chart of the non-specific immunity of Apostichopus japonicus cultured in this invention. The coelomocytes of Apostichopus japonicus have phagocytic function and can recognize and phagocytize invading Vibrio splendidus. During the process of phagocytosis of Vibrio splendidus by phagocytes, a respiratory burst phenomenon will occur, generating a large amount of reactive oxygen species (such as superoxide anion, hydrogen peroxide, etc.). These reactive oxygen species have strong oxidation ability and can effectively kill Vibrio splendidus. In this study, there was no significant difference in the phagocytic activity of coelomocytes and the respiratory burst activity of coelomocytes of Apostichopus japonicus in all experimental groups compared with the control group, but the 1.5% group was relatively high among the experimental groups. The antioxidant function is an important part of the immune system. Although long-term feeding of biotin did not significantly increase the activity of antioxidant enzymes (such as SOD), by upregulating antioxidant-related genes (such as SOD and ferritin genes), biotin enhanced the antioxidant ability of Apostichopus japonicus at the molecular level and reduced the oxidative stress caused by infection. The upregulation of SOD (superoxide dismutase) and ferritin genes can scavenge the excessive reactive oxygen species (ROS) generated during the infection process and protect cells from oxidative damage. This gene regulation effect may more effectively relieve the oxidative damage of Apostichopus japonicus under the pressure of pathogen infection than directly increasing the enzyme activity. On the other hand, the complement system and Toll-like receptor pathway show more targeted regulatory effects in long-term defense. The moderate concentration of biotin (1.5% group) significantly upregulated the expression of c3 and tlr3 genes. C3 is a key component in the complement system. The upregulation of c3 gene expression in Apostichopus japonicus may mean that the complement system is activated, thus enhancing the ability to recognize and clear Vibrio splendidus. Toll-like receptor 3 encoded by the tlr3 gene is an important molecule for pathogen recognition, which can recognize the nucleic acid components of viruses and bacteria and activate the immune response. This regulatory mechanism indicates that biotin can not only relieve the pressure on the immune system but also strengthen the immune defense ability when necessary, forming a more targeted protective effect. All these indicate that biotin at an addition amount of 1.5% can significantly enhance the non-specific immunity of Apostichopus japonicus.

Claims

1. Application of biotin in preparing feed or medicine for inhibiting bacteria and increasing phagocytic activity of sea cucumber cells, characterized in that, The application method described is to add biotin to drugs or feeds to increase the expression of superoxide dismutase and ferritin genes in sea cucumbers, protect cells from oxidative damage, and at the same time up-regulate the expression of c3 and tlr3 genes.

2. The application according to claim 1, wherein The application described is to add biotin with a mass ratio of 1.5% to the feed for finally feeding sea cucumbers.

3. Application of biotin in preparing feed for preventing Apostichopus japonicus Selenka from suffering from skin ulcer syndrome, characterized in that, The application described is to add biotin with a mass ratio of 0.5% - 2% to the feed to increase the expression of superoxide dismutase and ferritin genes in sea cucumbers, protect cells from oxidative damage, and at the same time up-regulate the expression of c3 and tlr3 genes, so as to enhance the recognition and clearance ability of Vibrio splendidus, and achieve the effect of preventing the skin ulcer syndrome of sea cucumbers.

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