CN117837697A - Application of nicotinamide - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to application of nicotinamide. The invention uses natural metabolite to increase the anti-vibrio parahaemolyticus ability of crustaceans, avoids the large-area death caused by vibriosis of crustaceans, has no toxicity or side effect, and provides a green, efficient and environment-friendly feed additive for the germchit feed of crustaceans against pathogenic stimulus.

Description

Application of nicotinamide

Technical Field

The present invention relates to the field of biotechnology, and in particular to an application of nicotinamide.

Background technique

Crustaceans are an important part of the marine ecosystem. Among them, shrimps and crabs are the main aquaculture species for human consumption. In the past decade, shrimp farming has become a pillar industry of aquaculture in my country, with significant economic and social benefits. However, the ensuing disease problems have seriously restricted the healthy development of the industry. Bacterial diseases (such as acute hepatopancreatic necrosis, enteritis, red leg disease, eye rot, shell ulcer, black gill rot, etc.) are one of the most common and most seriously affected diseases in shrimp farming in recent years, causing losses of tens of billions of yuan to the shrimp industry every year. The mainstream view is that Vibrio is the main pathogen of bacterial diseases of shrimps and crabs, among which Vibrio parahaemolyticus infection is the most common. At present, the methods for preventing and controlling Vibrio diseases in shrimp and crab farming are mainly the use of antibiotics, disinfectants and antibacterial compounds, but in the long run, it may lead to intestinal flora disorders, the production of drug-resistant bacteria and damage to the ecological environment. In recent years, studies have confirmed that in the interaction between host and pathogens, certain metabolites can affect the susceptibility of the host to pathogenic microorganisms, and exogenous addition of metabolites can enhance the host's anti-disease immunity. To this end, metabolites with anti-Vibrio parahaemolyticus activity were screened and identified through metabolomics methods, providing candidate targets and scientific basis for the development of green, efficient and environmentally friendly aquatic seed feed.

Nicotinamide, as an amide derivative of water-soluble vitamin B3, is widely present in various tissues of crustaceans, especially plasma, blood cells, hepatopancreas, heart, muscle, brain and intestine. Nicotinamide participates in various biological processes and plays functions including anti-inflammation, energy supply, regulation of enzyme activity, regulation of apoptosis, and influence on the expression of antimicrobial peptides. In addition, nicotinamide is also considered a safe therapeutic agent for the treatment of various human diseases.

In aquatic animal feed, nicotinamide is mainly used as a source of B vitamins. The added amount is small, which can increase the growth rate and weight of animals to a certain extent and reduce the damage of oxidative stress to the host; but there are no research reports on disease resistance and immunity. In the prior art, there are no research reports on adding nicotinamide to crustacean feed to resist Vibrio diseases.

Summary of the invention

The purpose of the present invention is to solve the problem that crustaceans have weak resistance to Vibrio parahaemolyticus and are prone to large-scale death due to vibriosis outbreaks, and to provide a green, efficient and environmentally friendly feed additive for crustacean seedling feed that can resist bacterial pathogens.

In order to solve the above technical problems, the following technical solutions are adopted:

An application of nicotinamide, and application of nicotinamide in the preparation of a preparation for increasing the ability of crustaceans to resist Vibrio parahaemolyticus.

Preferably, the nicotinamide is used in the preparation of a preparation for promoting the expression of blood cell antimicrobial peptide genes; the blood cell antimicrobial peptide genes include one or more of ALF1 and ALF4. Preferably, the nicotinamide is used in the preparation of a preparation for promoting blood cells to phagocytize Vibrio parahaemolyticus.

Preferably, the ratio of the added amount of nicotinamide to the body weight of the crustacean is 4-100 μg/6g; the crustacean includes shrimp.

Preferably, the ratio of the added amount of nicotinamide to the body weight of the crustacean is 4 μg/6g.

An application of nicotinamide, wherein nicotinamide is used as a feed additive for crustaceans.

A method for inhibiting bacterial growth in vitro with nicotinamide comprises the following steps: mixing bacterial liquid with nicotinamide solution in a volume ratio of 1:1, and then culturing at a constant temperature.

Preferably, the method comprises the following steps: mixing the bacterial liquid and the nicotinamide solution in a volume ratio of 1:1, and incubating in a 37°C incubator for 2 hours; taking 30 μL of the mixed solution after incubation to coat TSB or 2216E plates, with 3 biological replicates in each group and 3 parallels for each biological replicate; culturing the coated plates in a 37°C constant temperature incubator for 12 hours, and recording the number of colonies on the plates.

The mixture was first incubated for 2 hours without culture medium, giving nicotinamide and bacteria enough time and space to contact. Then the mixture was incubated for 12 hours, and bacteria were allowed to grow on the culture medium, verifying the antibacterial effect of nicotinamide.

Preferably, the bacteria include one or more of Vibrio parahaemolyticus and Shewanella basaltica.

Preferably, the concentration of the nicotinamide solution is 30.53 μg/100 μl.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides a metabolite, nicotinamide, which can improve the survival rate of shrimp under Vibrio parahaemolyticus infection. The metabolite is screened and identified from the plasma metabolome of Penaeus vannamei that interferes with hemocyanin under Vibrio parahaemolyticus infection. In recent years, studies have found that hemocyanin has the function of regulating the metabolic level in crustaceans to maintain the homeostasis of the flora in addition to the oxygen-carrying function. The inventors of the present application determined the plasma metabolic spectrum of shrimp by LC-MS/MS, screened and identified metabolites related to anti-Vibrio parahaemolyticus, and conducted multiple optimizations and experiments, and finally determined that the optimal dose of nicotinamide against Vibrio parahaemolyticus in shrimp is 4μg/6g, and the optimal dose of in vitro inhibition of Vibrio parahaemolyticus is 30.53μg/100μL. The nicotinamide of the present invention has the function of inhibiting the growth of Vibrio parahaemolyticus in shrimp hemolymph, has in vitro antibacterial activity against a variety of aquatic pathogens, and can promote the phagocytosis of Vibrio parahaemolyticus by blood cells, while promoting the expression of blood cell antimicrobial peptides ALF1 and ALF4.

The invention increases the ability of crustaceans to resist Vibrio parahaemolyticus with natural metabolites, avoids large-scale death caused by Vibrio disease of crustaceans, is non-toxic and has no side effects, and provides a green, efficient and environmentally friendly feed additive for crustacean seedling feed that can resist pathogen stimulation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a heat map of cluster analysis of plasma samples before and after interference with hemocyanin under Vibrio parahaemolyticus infection.

Figure 2 is a PLS-DA analysis of plasma samples before and after hemocyanin interference under Vibrio parahaemolyticus infection.

FIG. 3 is a volcano analysis diagram of differential metabolites before and after interference with hemocyanin under Vibrio parahaemolyticus infection.

FIG4 is a heat map of differential metabolite analysis before and after interference with hemocyanin under Vibrio parahaemolyticus infection.

FIG5 (A) is a comparative analysis of nicotinamide content based on ELISA analysis, and (B) is a comparative analysis of nicotinamide content based on LC-MS/MS analysis; ** indicates a very significant difference (p<0.01).

FIG6 is an analysis of the effects of different doses of nicotinamide on the abundance of Vibrio (A) and Vascella (B) in hemolymph under physiological conditions and Vibrio parahaemolyticus infection conditions.

Figure 7 is an analysis of the effect of different doses of nicotinamide on the phagocytic rate of Vibrio parahaemolyticus by blood cells; A is a flow cytometer analysis chart; B is a statistical chart of the blood cell phagocytic rate.

FIG8 is an analysis of the effect of nicotinamide on the expression of blood cell antimicrobial peptides ALF1 (A) and ALF4 (B).

FIG. 9 is an analysis of the inhibition rate of different concentrations of nicotinamide against Vibrio parahaemolyticus.

FIG. 10 is an analysis of the inhibition rate of nicotinamide at different concentrations against Shewanella basaltica.

FIG. 11 is an analysis of the optimal inhibitory concentration of nicotinamide against Vibrio parahaemolyticus.

FIG. 12 is an analysis of the optimal inhibitory concentration of nicotinamide against Shewanella basaltica.

FIG. 13 is an analysis of the effect of supplementing nicotinamide on the survival rate of shrimp before and after interfering with hemocyanin under physiological conditions.

FIG. 14 is an analysis of the effect of supplementing nicotinamide on the survival rate of shrimp before and after interfering with hemocyanin under Vibrio parahaemolyticus infection.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

Example 1

Analysis of metabolic profiles in shrimp plasma

First, LC-MS/MS technology was used to perform metabolomics analysis on shrimp plasma samples before and after hemocyanin interference under Vibrio parahaemolyticus infection, namely "interference control + Vibrio parahaemolyticus infection group (dsGFP+Vp)" and "interference hemocyanin + Vibrio parahaemolyticus infection group (dsLvHMC+Vp)", with 6 biological replicates in each group. The results showed that there were 142 metabolites in the plasma metabolome profile (Figure 1).

Then, the stability and reproducibility of the LC-MS/MS method were evaluated by partial least squares discriminant analysis (PLS-DA) of all samples. The results showed that the two groups could be well clustered, with small differences within the groups and good stability; at the same time, the differences between the two groups were significant, suggesting that there may be key differential metabolites that affect the ability of shrimp to resist Vibrio parahaemolyticus (Figure 2).

Subsequently, the differences between the two groups of samples were compared by creating a volcano model, and 14 differential metabolites were obtained (Figure 3). After further comparison with the KEGG database, 7 differential metabolites with annotation information were finally obtained through heat map analysis (Figure 4).

Finally, in order to analyze whether there are significant differences in the metabolites screened by the above metabolomics method before and after the interference of hemocyanin under Vibrio parahaemolyticus infection, ELISA technology was further used to verify it. The results are shown in Figure 5A. The ELISA test results of nicotinamide in plasma are consistent with the metabolomics results (Figure 5B), that is, under Vibrio parahaemolyticus infection, the content of nicotinamide in shrimp plasma is significantly reduced after the interference of hemocyanin.

Example 2

Study on the effect of nicotinamide on reducing the abundance of Vibrio in shrimp hemolymph

(1) Uniform-sized Litopenaeus vannamei (average weight of about 6 g) were randomly divided into four groups, each with 60 shrimp. The first group was the control group, in which each shrimp was injected intramuscularly with 100 μL of saline (0.65%). The second, third and fourth experimental groups were injected intramuscularly with 4 μg, 20 μg and 100 μg of nicotinamide, respectively. After 24 hours, the shrimps in groups one, two, three and four were divided into two groups, one of which was injected with 100 μL of saline (0.65%) and the other with 2×10 ⁵ CFU/shrimp of Vibrio parahaemolyticus.

(2) 24 hours after infection, hemolymph bacterial samples were collected from each group, with 3 biological replicates in each group and no less than 3 technical replicates for each biological replicate. Hemolymph samples were collected from each shrimp and centrifuged at 800×g for 10 min to enrich blood cells. The supernatant was centrifuged at 5000×g for 10 min to enrich bacteria, and hemolymph genomic DNA was extracted using a marine animal genomic DNA extraction kit.

(3) Using hemolymph genomic DNA as a template, qPCR primers specific to the 16S rRNA gene of Vibrio and Shewanella were used to detect the abundance of Vibrio and Shewanella in hemolymph, respectively. The obtained Ct value was inserted into the bacterial absolute quantitative standard curve to calculate the CFU value of bacteria per milliliter of hemolymph.

The results are shown in Figure 6. Under the infection of Vibrio parahaemolyticus, the abundance of Vibrio in the hemolymph of shrimps injected with nicotinamide at 4μg/6g, 20μg/6g, and 100μg/6g was significantly reduced, among which the abundance of Vibrio was the lowest when the nicotinamide dose was 4μg/6g (Figure 6A). Different doses of nicotinamide had no significant effect on the abundance of Shewanella (Figure 6B). This shows that the addition of nicotinamide can indeed reduce the number of Vibrio in the hemolymph of shrimps.

Example 3

Study on the effect of nicotinamide on the phagocytosis of Vibrio parahaemolyticus by blood cells

(1) Activate and expand the culture of Vibrio parahaemolyticus in advance, centrifuge at 5000×g for 5 min, remove the supernatant, resuspend the precipitated bacteria with 0.01M PBS at a volume ratio of 5:1, inactivate at 72℃ for 20 min, and centrifuge again to enrich the bacteria. Wash the bacteria three times with 0.1M NaHCO ₃ solution (pH 9.0), then resuspend the bacteria with an equal volume of 0.01M PBS, measure the OD ₆₀₀ value with an enzyme marker, and adjust the bacterial concentration to 3×10 ⁸ CFU/mL.

(2) Add fluorescein isothiocyanate (FITC) (100 mg/mL) in a volume ratio of 1000:1 to the bacterial solution in the dark to label Vibrio parahaemolyticus, incubate at 37°C for 2 h, centrifuge for 5 min, and enrich the labeled bacteria. Wash the bacteria three times with 0.01 M PBS in the dark, and finally resuspend with an equal volume of 0.01 M PBS and store the bacteria at -20°C.

(3) Nicotinamide was injected intramuscularly according to Example 2, and the shrimps were randomly divided into four groups, each with 15 shrimps. The first group was the control group, and each shrimp was injected intramuscularly with 100 μL of physiological saline (0.65%). The second, third, and fourth experimental groups were injected intramuscularly with 4 μg, 20 μg, and 100 μg of nicotinamide, respectively. After 24 hours, each group of shrimps was injected intramuscularly with FITC-labeled Vibrio parahaemolyticus (3×10 ⁸ CFU/mL) and protected from light for 1 hour. Hemolymph samples were taken from each shrimp and mixed with an equal volume of anticoagulant. Each group had 3 biological replicates, and every 5 shrimps were one biological replicate.

(4) The collected blood cells were centrifuged at 800×g for 10 min, resuspended in 300 μL 0.01 M PBS, and the proportion of phagocytic cells was counted using a flow cytometer BD Accuri ^TM C6 Plus (BD Biosciences) (FITC channel, excitation 485 nm, emission 529 nm).

The results are shown in Figure 7. The proportion of green fluorescence positive cells of shrimp hemocytes phagocytosing Vibrio parahaemolyticus increased in the groups injected with nicotinamide 4μg/6g, 20μg/6g, and 100μg/6g (Figure 7A). The phagocytosis rate was statistically found to be the highest when the nicotinamide dose was 4μg/6g compared with the control group (Figure 7B). This shows that the addition of nicotinamide can improve the phagocytosis of shrimp hemocytes against Vibrio parahaemolyticus.

Example 4

Nicotinamide promotes the expression of antimicrobial peptide ALF in blood cells

(1) According to Example 2, each shrimp in the control group was injected intramuscularly with 100 μL of saline (0.65%), and each shrimp in the experimental group was injected intramuscularly with 4 μg of nicotinamide. After 24 hours, the shrimps in the control group and the experimental group were divided into two groups, one of which was injected with 100 μL of saline (0.65%) per shrimp, and the other was injected with 2×10 ⁵ CFU/shrimp of Vibrio parahaemolyticus.

(2) 24 hours after infection, RNA from shrimp hemocytes was extracted using the Filitech TRNAfast 200 RNA extraction kit and then analyzed according to the full-scale gold One-Step gDNA Removal and cDNA Synthesis Kit Instructions: The total RNA was reverse transcribed to synthesize the first-strand cDNA, and then the cDNA was synthesized by using/> 480 fluorescence quantitative PCR instrument, according to Genstar/> The expression of antimicrobial peptide ALFs gene was detected by qPCR according to the instructions of Green ^TM I kit (Figure 8).

The results showed that under physiological conditions and Vibrio parahaemolyticus infection conditions, the addition of nicotinamide could significantly increase the expression of antimicrobial peptides ALF1 ( FIG. 8A ) and ALF4 ( FIG. 8B ) in blood cells.

Example 5

Study on the antibacterial effect of nicotinamide in vitro

60 μL of Vibrio parahaemolyticus (2×10 ⁵ CFU/mL) and Shewanella basaltica (1×10 ⁵ CFU/mL) were mixed with an equal volume of nicotinamide of different concentrations (15.3μg/100μL, 30.5μg/100μl, 61μg/100μl, 122μg/100μL, 244μg/100μL), and an equal volume of saline (0.65%) was mixed with an equal amount of bacteria as a negative control, and incubated in a 37°C incubator for 2 hours. 30 μL of the above incubated mixture was coated on TSB or 2216E plates, with 3 biological replicates in each group, and 3 parallels for each biological replicate. After 12 hours of incubation in a 37°C incubator, the number of colonies on the plate was scanned and recorded. Bacterial inhibition rate = (negative control colony number - experimental group colony number) / blank control colony number × 100%.

The results are shown in Figures 9 to 12. A certain concentration of nicotinamide can inhibit the growth of bacteria. When the concentration is 30.5 μg/100 μl, nicotinamide has the most significant inhibitory effect on the growth of Vibrio parahaemolyticus (Figure 9) and Shewanella basaltica (Figure 10), with inhibition rates of 99.71% (Figure 11) and 59.77% (Figure 12), respectively. After that, as the concentration of nicotinamide increases, the inhibition rates of the two bacteria decrease. This shows that the optimal dose of nicotinamide to inhibit the growth of pathogenic bacteria in vitro is 30.53 μg/100 μL.

Example 6

Analysis on the survival rate of shrimp supplemented with nicotinamide

In order to analyze whether nicotinamide affects the ability of shrimp to resist Vibrio parahaemolyticus infection under the regulation of hemocyanin, RNAi, metabolite complementation and pathogen stimulation techniques were used to compare the survival rate of shrimp supplemented with nicotinamide before and after hemocyanin interference under physiological conditions and Vibrio parahaemolyticus infection. First, shrimps were injected with dsRNA (10μg/6g) intramuscularly under physiological conditions, the control group was dsGFP, and the hemocyanin interference group was dsLvHMC; 48 hours later, the control group and the interference group shrimps were randomly divided into two groups, among which each shrimp in the experimental group was injected with 4μg nicotinamide intramuscularly, and the control group was injected with an equal volume of saline (0.65%), and the survival rate of shrimps was monitored within 96 hours after nicotinamide injection (Figure 13). Furthermore, 24 hours after the injection of nicotinamide, each group of shrimps were injected with 2×10 ⁵ CFU/shrimp of Vibrio parahaemolyticus, and the survival rate of shrimps was monitored within 48 hours after infection with Vibrio parahaemolyticus (Figure 14). The results showed that under physiological conditions, the survival rate of shrimp in the hemocyanin interference group without nicotinamide injection was 40% at 96 hours, while the survival rate of shrimp in the hemocyanin interference group injected with nicotinamide was 74.29%. Under Vibrio parahaemolyticus infection, the survival rate of shrimp in the hemocyanin interference group without nicotinamide injection was 0% at 48 hours, while the survival rate of shrimp in the hemocyanin interference group injected with nicotinamide was 22.73%. This shows that the addition of nicotinamide can improve the survival rate of shrimp under Vibrio parahaemolyticus infection.

The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims (10)

1. An application of nicotinamide, characterized in that nicotinamide is used in the preparation of a preparation for increasing the ability of crustaceans to resist Vibrio parahaemolyticus. 2. The use of nicotinamide according to claim 1, characterized in that the nicotinamide is used in the preparation of a preparation for promoting the expression of blood cell antimicrobial peptide genes; the blood cell antimicrobial peptide genes include one or more of ALF1 and ALF4. 3. The use of nicotinamide according to claim 1, characterized in that the nicotinamide is used in the preparation of a preparation for promoting blood cells to phagocytose Vibrio parahaemolyticus. 4. The use of nicotinamide according to claim 1, characterized in that the ratio of the added amount of nicotinamide to the body weight of the crustacean is 4 to 100 μg/6g; and the crustacean includes shrimp. 5. The use of nicotinamide according to claim 4, characterized in that the ratio of the added amount of nicotinamide to the body weight of the crustacean is 4 μg/6g. 6. An application of nicotinamide, characterized in that nicotinamide is used as a feed additive for crustaceans. 7. A method for inhibiting bacterial growth in vitro with nicotinamide, characterized in that it comprises the following steps: mixing bacterial culture and nicotinamide solution in a volume ratio of 1:1, and then incubating at a constant temperature. 8. The method for inhibiting bacterial growth in vitro with nicotinamide according to claim 7, characterized in that it comprises the following steps: mixing the bacterial liquid and the nicotinamide solution in a volume ratio of 1:1, and incubating in a 37°C incubator for 2 hours; taking 30 μL of the incubated mixture to coat TSB or 2216E plates, with 3 biological replicates in each group and 3 parallels for each biological replicate; after incubating the coated plates in a 37°C constant temperature incubator for 12 hours, the number of colonies on the plates was recorded. 9. The method for inhibiting bacterial growth in vitro with nicotinamide according to claim 7, characterized in that the bacteria include one or more of Vibrio parahaemolyticus and Shewanella basaltica. 10 . The method for inhibiting bacterial growth in vitro using nicotinamide according to claim 7 , wherein the concentration of the nicotinamide solution is 30.53 μg/100 μl.