Use of inosine

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to an application of inosine, the application of inosine in preparation of an immune response capacity of a crustacean, the application of inosine in enhancement of the ability of the crustacean to resist Vibrio parahaemolyticus, avoidance of infectious death of the crustacean caused by acute hepatopancreas necrosis disease (AHPND), and no toxicity and no side effects, and the inosine is a green, efficient and environment-friendly feed additive for seedling feed of the crustacean to resist pathogenic stimulation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to an application of inosine. BACKGROUND

[0002] In recent years, with the development of high-density intensive farming mode, frequent disease outbreaks have caused huge economic losses to the aquaculture industry (Dhar et al., 2014; Lafferty et al., 2015; Thamizhvanan et al., 2019), and seriously restricted the development of the industry. The main reasons are: large gap in shrimp breeding, serious degradation of germplasm and frequent disease outbreaks. As ectothermic animals, the dramatic changes in aquatic ecosystems increase the susceptibility of shrimp and other aquatic animals to bacteria (Prithvisagar et al., 2021; Wu et al., 2021; Zheng et al., 2021), viruses (Kibenge, 2019; Phuthaworn et al., 2016; Walker and Mohan, 2009) and parasites (Babu et al., 2021; Byers, 2021). In particular, acute hepatopancreatic necrosis disease (AHPND), which rapidly develops after infection and causes mass mortality in shrimp larvae, has become a major threat to global shrimp farming (Tran et al., 2013).

[0003] Inosine is a degradation product of purines (such as adenosine and ATP), and under cellular metabolic stress (such as inflammation), inosine can affect the host's immune process. Some scholars have reported that inosine can inhibit the production of pro-inflammatory cytokines in a mouse endotoxin model, thereby reducing the mortality rate of mice (Haskó et al., 2000). We previously found that inosine was significantly up-regulated in AHPND-resistant shrimp and significantly down-regulated in AHPND-sensitive shrimp after infection with acute hepatopancreatic necrosis disease (AHPND), indicating that inosine can be involved in the host's immune response to AHPND.

[0004] The large-scale and intensive shrimp farming mode has gradually exposed the problems of rough farming management. In particular, in a high-density farming environment, ecological environment disorders, such as vibrio infection, harmful algae outbreaks, parasitic or viral infections, etc. are all prone to induce the outbreak of AHPND in shrimp (Boonyawiwat et al., 2017).

[0005] In the prior art, the traditional method for relieving or treating AHPND of prawns is mainly to use antibiotics, disinfectants and bacteriostatic compounds. However, on the one hand, such drugs will destroy the intestinal flora homeostasis of prawns, and even cause other pathogenic bacteria to colonize in the host body (Xiong et al., 2016), on the other hand, long-term use of antibiotic drugs will produce the risk of drug-resistant bacteria, such as a strain of Vibrio harveyi causing acute hepatopancreas necrosis disease in Mexico carrying tetracycline resistance gene tetB (Han et al., 2015) and a strain of Vibrio campbellii in China carrying multiple drug resistance genes (Dong et al., 2017).

[0006] Therefore, in the prawn culture system, using traditional methods such as antibiotics to control AHPND is not a green and long-term effective prevention and control measure. SUMMARY

[0007] The purpose of the present application is to solve the problem that crustaceans have weak resistance to Vibrio parahaemolyticus and are prone to cause large-scale death due to AHPND.

[0008] In order to solve the above technical problems, the present application provides a nucleotide, inosine, which can improve the survival rate of prawns under the stimulation of AHPND pathogen Vibrio parahaemolyticus (with pVA1 plasmid). The additive amount is 20-100 μg / 6 g.

[0009] The following technical solutions are adopted:

[0010] The application of inosine in the preparation of a drug for increasing the resistance of crustaceans to Vibrio parahaemolyticus.

[0011] Preferably, the ratio of the amount of inosine added to the body weight of the crustacean is 20-100 μg / 6 g; and the crustacean is a prawn.

[0012] Preferably, the ratio of the amount of inosine added to the body weight of the crustacean is 100 μg / 6 g.

[0013] The application of inosine as a feed additive for crustaceans.

[0014] A method for inhibiting bacterial growth by inosine, comprising the following steps: mixing bacterial liquid with inosine solution at a volume ratio of 1:1, and then incubating at a constant temperature.

[0015] Preferably, the method comprises the following steps: mixing the bacterial solution and the inosine solution in a volume ratio of 1:1, incubating in a 37°C incubator for 2 hours; taking 30 μL of the above-mentioned mixed solution after incubation to coat TSB or LB plates, 3 biological replicates for each group, and 3 parallel replicates for each biological replicate; after incubating the coated plates in a 37°C constant temperature incubator for 12 hours, recording the number of bacteria on the plates.

[0016] The solution is mixed and incubated for 2 hours at first, at this time there is no culture medium, giving inosine and bacteria sufficient time and space to contact. Then coating and incubating for 12 hours, bacteria can grow on the culture medium, verifying the inhibitory effect of inosine on bacteria.

[0017] Preferably, the bacteria include one or more of Vibrio parahaemolyticus, Photobacterium damsel, Vibrio alginolyticus, Escherichia coli, (F) Streptococcus iniae, Staphylococcus aureus.

[0018] Preferably, the concentration of the inosine solution is 1 mg / ml.

[0019] Compared with the prior art, the implementation of the present application has the following beneficial effects:

[0020] AHPND is an important disease that threatens global shrimp farming. For shrimps lacking adaptive immune systems, green and safe acute hepatopancreas necrosis disease prevention and control measures are particularly important. Inosine, as a metabolic product of the host and microorganisms, is not only safe and green, but also can help the host resist the attack of pathogens and reshape the immune system of the host. The present application enhances the ability of crustaceans to resist Vibrio parahaemolyticus by using natural metabolite inosine, avoids infectious death of crustaceans caused by AHPND, and has no toxicity and side effects, providing green and efficient environmentally friendly feed additives for crustacean seedling feed to resist pathogenic stimulation. The present application lays a foundation for developing new acute hepatopancreas necrosis disease prevention and control measures, and promotes the green, healthy and sustainable development of shrimp farming. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 To increase the survival rate of shrimps under the stimulation of Vibrio parahaemolyticus (with pVA1 plasmid) with different doses of inosine.

[0022] Figure 2 To analyze the antibacterial rate of different concentrations of inosine on Vibrio parahaemolyticus (with pVA1 plasmid).

[0023] Figure 3 To analyze the antibacterial rate of different concentrations of inosine on Photobacterium damsel.

[0024] Figure 4 To analyze the antibacterial rate of different concentrations of inosine on Vibrio alginolyticus.

[0025] Figure 5To analyze the antibacterial rate of different concentrations of inosine against Escherichia coli.

[0026] Figure 6 To analyze the antibacterial rate of different concentrations of inosine against Staphylococcus aureus.

[0027] Figure 7 This is a graph showing the effect of the inosine functional package on the survival rate of shrimp in Example 3.

[0028] Figure 8 This is a graph showing the effect of the inosine functional package on the survival rate of shrimp in Example 4. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0030] Example 1

[0031] Study on the effect of inosine against AHPND in shrimp

[0032] To investigate the role of inosine in combating AHPND, 360 healthy shrimp (average weight approximately 6g) were randomly divided into 8 groups of 45 shrimp each. Referring to Table 1, each group received an intramuscular injection of inosine or saline one day before infection with Vibrio parahaemolyticus (carrying the pVA1 plasmid, the pathogen of AHPND). Then, starting on day 0, the shrimp were injected intramuscularly with either Vibrio parahaemolyticus (carrying the pVA1 plasmid) or saline. The number of surviving shrimp from day 0 to 1.5 after infection with Vibrio parahaemolyticus (carrying the pVA1 plasmid) was recorded, and the survival rate was calculated. This process was repeated three times.

[0033] Table 1. Grouping scheme for the effect of inosine on the survival rate of shrimp under Vibrio parahaemolyticus (with pVA1 plasmid) stress.

[0034]

[0035] Data from three repeated trials were analyzed by taking the average value. The results are as follows: Figure 1 As shown.

[0036] from Figure 1 It can be seen that in Vibrio parahaemolyticus (carrying pVA1 plasmid) (3×10 6Under stress of CFU / mL, the survival rate of shrimp injected with 0.2 mg / mL inosine was 27.27%, which was 23.27% higher than that of shrimp injected with saline (4%). The survival rate of shrimp injected with 1 mg / mL inosine was 13.04%, which was 9.04% higher than that of shrimp injected with saline (4%). However, the survival rate of shrimp injected with 5 mg / mL inosine was 0%, which was 4% lower than that of shrimp injected with saline (4%). Therefore, it is recommended to add 0.5-1 mg / mL inosine, which can significantly enhance the ability of shrimp to resist AHPND.

[0037] Example 2

[0038] In vitro antibacterial activity of inosine

[0039] To investigate whether inosine possesses antibacterial properties in vitro, 60 μL of Vibrio parahaemolyticus (with pVA1 plasmid) (2 × 10⁻⁶) was collected. 4 CFU / mL)( Figure 2 ), Mermaid luminescent bacteria (5×10) 3 CFU / mL)( Figure 3 ), Vibrio alginolyticus (5×10) 3 CFU / mL)( Figure 4 ), Escherichia coli (3×10) 4 CFU / mL)( Figure 5 (F) Dolphin Streptococcus (5×10) 3 CFU / mL), Staphylococcus aureus (5×10⁻⁶ CFU / mL), Staphyl 3 CFU / mL)( Figure 6 A suspension of inosine was mixed with equal volumes of different concentrations (0.2 mg / ml, 1 mg / ml, 5 mg / ml) and incubated at 37°C for 2 hours. 30 μL of the incubated mixture was then spread onto TSB or LB plates, with three biological replicates per group and three parallel replicates per biological replicate. After incubation at 37°C for 12 hours, the colony counts on the plates were photographed and recorded. Bacterial inhibition rate = (number of colonies in blank control - number of colonies in experimental group) / number of colonies in blank control × 100%. Results are as follows: Figures 2 to 6 As shown, inosine at concentrations of 0.2–1 mg / mL can inhibit the proliferation of Vibrio parahaemolyticus (with pVA1 plasmid). Inosine at concentrations of 0.20 mg / mL can inhibit the growth and reproduction of *Bacillus melanogaster*. However, inosine at concentrations of 0.2–1 mg / mL is ineffective against *Vibrio alginolyticus* (5 × 10⁻⁶). 3 CFU / mL), Escherichia coli (3×10) 4 CFU / mL), Staphylococcus aureus (5×10⁻⁶ CFU / mL), Staphyl 3 Inosine (CFU / mL) has no antibacterial activity. Therefore, inosine at concentrations of 0.2–1 mg / mL can specifically inhibit the proliferation of AHPND-pathogenic Vibrio parahaemolyticus in vitro.

[0040] Example 3

[0041] In order to prevent and control the harm of AHPND to prawn culture and improve the antibacterial function of prawn under intensive large-scale culture, a functional package containing inosine (inosine: 50-80%, vitamin A (500,000 IU / kg): 0.3-0.5%, sodium selenite (1%): 0.3-0.8%, dextrin or corn flour or wheat flour or rice husk powder: 7%-50%) is designed.

[0042] Taking the functional package with the minimum dose of inosine (inosine: 50%, vitamin A (500,000 IU / kg): 0.3%, sodium selenite (1%): 0.3%, dextrin: 49.4%) as an example, the effect of the functional package on the resistance of Litopenaeus vannamei to AHPND was detected. First, healthy Litopenaeus vannamei (average weight about 6 g) were randomly divided into five groups, the first group was the control group (fed with feed without functional package), and the second group, the third group, the fourth group and the fifth group were the test groups, and a 2-fold gradient test was performed, that is, each ton of full price material contained 2.5 kg, 5 kg, 10 kg and 20 kg of functional package respectively, and each group contained 50 shrimps. Then after feeding the shrimps with the feed for 72 hours, the shrimps were stimulated with Vibrio parahaemolyticus (4x10 5 shrimp) with pVA1 plasmid for 48 hours, and the survival number of the shrimps was recorded. The results are shown in Table 1. Figure 7 As shown in Table 1, after feeding the shrimps with the feed for 72 hours, the survival rates of the test groups 1, 2, 3 and 4 were increased to 84.00%, 90.38%, 88.24% and 89.80% respectively compared with the control group (the survival rate was 76.47%). At the same time, after adding 2.5-20 kg of functional package per ton of full price material, the survival rates of the test groups 1-4 were significantly increased compared with the control group (the survival rate was 5.88%) after the shrimps were stimulated with Vibrio parahaemolyticus with pVA1 plasmid for 96 hours, and the survival rates were 30.00% (p=0.003), 38.46% (p<0.001), 43.14% (p<0.001) and 32.65% (p<0.001) respectively. As can be seen from the survival rate results, the test group 3, that is, the addition amount of 10 kg, is the best choice, but the results of the test group 3, the test group 1, the test group 2 and the test group 4 are not significantly different, therefore, from the perspective of cost performance, the formula of the test group 1 with less addition amount and the same effect can be selected. The above results show that adding 2.5-20 kg of functional package can significantly improve the survival rate of the shrimps after being infected with Vibrio parahaemolyticus (with pVA1 plasmid).

[0043] Example 4

[0044] In order to prevent and control the harm of AHPND to the prawn culture and improve the antibacterial function of prawn under the large-scale intensive culture, a functional package containing inosine (inosine: 50-80%, vitamin A (500,000 IU / kg): 0.3-0.5%, sodium selenite (1%): 0.3-0.8%, dextrin or corn flour or wheat flour or rice hull powder: 7%-50%) is designed.

[0045] Taking the functional package with the highest dose of inosine (inosine: 80%, vitamin A (500,000 IU / kg): 0.5%, sodium selenite (1%): 0.8%, dextrin: 7%) as an example, the influence of the functional package on the survival rate of the brine shrimp is detected. First, healthy brine shrimp (about 6g in average weight) are randomly divided into five groups, the first group is a control group (fed with feed without the functional package), and the second to fifth groups are test groups, and a 2-fold gradient test is performed, that is, 2.5 kg, 5 kg, 10 kg and 20 kg of the functional package are respectively added to each ton of full-price feed, and 50 shrimps are in each group. Then, after feeding the shrimps with the feed for 72 hours, the shrimps are stimulated by Vibrio parahaemolyticus with pVA1 plasmid (4x10 5 / shrimp) for 48 hours, and the survival number of the shrimps is recorded, and the results are shown in Figure 8 As shown in the results, after feeding the shrimps with the feed for 72 hours and before injecting the pathogenic bacteria, the survival rates of the test group 1, the test group 2, the test group 3 and the test group 4 are respectively increased to 90.38%, 88.24%, 89.80% and 86.00% compared with the control group (the survival rate is 84.31%). At the same time, after adding 2.5-20 kg of the functional package to each ton of full-price feed, the survival rates of the test group 1, the test group 2, the test group 3 and the test group 4 are significantly increased compared with the control group (the survival rate is 3.92%) after the brine shrimp is stimulated by Vibrio parahaemolyticus with pVA1 plasmid for 48 hours, and the survival rates are respectively 40.38% (p<0.001), 45.10% (p<0.001), 36.73% (p<0.001) and 34.00% (p=0.003). As can be seen from the survival rate, the test group 1 and the test group 2, that is, the addition amount of 2.5 kg and 5 kg, are the optimal choices, but from the perspective of cost performance, the test group 1 with a smaller addition amount and the same effectiveness can be selected.

[0046] As can be seen from the results of the example 3 and the example 4, the functional package containing inosine (inosine: 50-80%, vitamin A (500,000 IU / kg): 0.3-0.5%, sodium selenite (1%): 0.3-0.8%, dextrin or corn flour or wheat flour or rice hull powder: 7%-50%) designed in the present application has the functions of improving the survival rate of the brine shrimp and especially improving the survival rate of the brine shrimp after being infected by Vibrio parahaemolyticus with pVA1 plasmid under the same feed addition ratio, which proves that the functional package designed in the present application is safe and effective.

[0047] The above merely provides the preferred embodiment of the present application, and cannot allude the protection scope of the present application, therefore, any equivalent changes made according to the claims of the present application shall be within the scope of the present application.

Claims

1. Use of inosine, characterized in that, Use of inosine in the preparation of an additive functional package for enhancing the ability of a crustacean to resist Vibrio parahaemolyticus, the additive functional package comprising the following components by mass fraction: 50-80% inosine, 0.3-0.5% vitamin A, 3×10 -3 ~8×10 -3 % sodium selenite, 7%-50% filler.

2. Use according to claim 1, characterized in that, The additive functional package is added in the base feed in an amount of 5-20 kg / ton.

3. A method of inhibiting bacterial growth in vitro by inosine, characterized by, The method comprises the following steps: mixing bacterial liquid and inosine solution in a volume ratio of 1:1, and then incubating at a constant temperature; the bacteria are one or more of Vibrio parahaemolyticus and Photinus pyralis.

4. The method of claim 3, wherein the inosine inhibits bacterial growth in vitro. The method comprises the following steps: mixing the bacterial liquid and the inosine solution in a volume ratio of 1:1, incubating in a 37 DEG C incubator for 2 hours; taking 30 muL of the mixed solution after incubation to coat TSB or LB plates, 3 biological repeats in each group, and 3 parallel in each biological repeat; after the coated plates are cultured in a 37 DEG C constant temperature incubator for 12 hours, the number of bacteria on the plates is recorded.

5. The method of claim 3, wherein the inosine inhibits bacterial growth in vitro. The concentration of the inosine solution is 1 mg / ml.

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