Use of nicotinamide

Abstract

The present application relates to the field of biotechnology, and particularly relates to an application of nicotinamide. The present application increases the Vibrio parahaemolyticus resistance of crustaceans by using natural metabolites, avoids mass death of crustaceans caused by Vibrio parahaemolyticus disease, and has no toxicity and no side effects, and provides a green, efficient and environment-friendly feed additive for stimulating pathogenic stimulation of crustacean seedling feed.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of nicotinamide. Background Technology

[0002] Crustaceans are an important component of marine ecosystems. Shrimp and crabs are among the main aquaculture species consumed by humans. In the past decade, shrimp farming has become a pillar industry of my country's aquaculture sector, generating significant economic and social benefits. However, the accompanying disease problems severely restrict the healthy development of this industry. Bacterial diseases (such as acute hepatopancreatic necrosis, enteritis, red leg disease, eye rot, shell ulcers, black gill rot, etc.) are among the most common and severely impactful diseases in shrimp farming in recent years, causing billions of yuan in losses to the shrimp industry annually. The mainstream view is that Vibrio is the main pathogen causing bacterial diseases in shrimp and crabs, with Vibrio parahaemolyticus infection being the most prevalent. Currently, the main methods for controlling Vibrio diseases in shrimp and crab farming are the use of antibiotics, disinfectants, and antibacterial compounds. However, in the long run, this may lead to intestinal flora imbalance, the development of drug-resistant bacteria, and damage to the ecological environment. Recent studies have confirmed that certain metabolites can affect a host's susceptibility to pathogenic microorganisms in host-pathogen interactions, and that exogenous addition of metabolites can enhance a host's disease-fighting immunity. Therefore, screening and identifying metabolites with anti-Vibrio parahaemolyticus activity using metabolomics methods provides candidate targets and scientific basis for the development of green, efficient, and environmentally friendly aquatic seedling feeds.

[0003] Nicotinamide, a water-soluble amide derivative of vitamin B3, is widely found in various tissues of crustaceans, particularly in plasma, blood cells, hepatocellular carcinoma, heart, muscle, brain, and intestine. Nicotinamide participates in and functions in a variety of biological processes, including anti-inflammatory effects, energy provision, regulation of enzyme activity, regulation of apoptosis, and influencing the expression of antimicrobial peptides. Furthermore, nicotinamide is considered a safe therapeutic agent used to treat various human diseases.

[0004] In aquatic animal feed, nicotinamide is mainly used as a source of B vitamins. The addition amount is relatively small, and it can improve animal growth rate and weight to some extent, and reduce oxidative stress damage to the host; however, there are no research reports on its effects on disease resistance and immunity. Currently, there are no research reports on the use of nicotinamide in crustacean feed for combating Vibrio diseases. Summary of the Invention

[0005] The purpose of this invention is to solve the problem that crustaceans have weak resistance to Vibrio parahaemolyticus and are prone to large-scale mortality due to vibrio outbreaks, and to provide a green, efficient and environmentally friendly feed additive for crustacean seedlings to resist bacterial pathogens.

[0006] To solve the above technical problems, the following technical solution is adopted:

[0007] An application of nicotinamide: the use of nicotinamide in the preparation of formulations that enhance the ability of crustaceans to resist Vibrio parahaemolyticus.

[0008] Preferably, the nicotinamide is used in the preparation of formulations that promote the expression of antimicrobial peptide genes in hematopoietic cells; the antimicrobial peptide genes in hematopoietic cells include one or more of ALF1 and ALF4. Preferably, the nicotinamide is used in the preparation of formulations that promote the phagocytosis of Vibrio parahaemolyticus by hematopoietic cells.

[0009] Preferably, the amount of nicotinamide added is 4 to 100 μg / 6g relative to the body weight of the crustacean; the crustacean includes shrimp.

[0010] Preferably, the amount of nicotinamide added is 4 μg / 6g of the crustacean's body weight.

[0011] An application of nicotinamide as a feed additive for crustaceans.

[0012] A method for inhibiting bacterial growth in vitro with nicotinamide includes the following steps: mixing bacterial culture with nicotinamide solution at a volume ratio of 1:1, followed by incubation at a constant temperature.

[0013] Preferably, the method includes the following steps: mixing the bacterial culture and the nicotinamide solution at a volume ratio of 1:1 and incubating at 37°C for 2 hours; taking 30 μL of the incubated mixture and spreading it onto TSB or 2216E plates, with 3 biological replicates per group and 3 parallels for each biological replicate; incubating the spread plates at 37°C for 12 hours and recording the number of colonies on the plates.

[0014] The mixture was first incubated in solution for 2 hours without culture medium, allowing sufficient time and space for nicotinamide to come into contact with the bacteria. Afterward, it was spread and incubated for another 12 hours, during which bacteria grew on the culture medium, verifying the antibacterial effect of nicotinamide.

[0015] Preferably, the bacteria include one or more of Vibrio parahaemolyticus and Shewanella basalosa.

[0016] Preferably, the concentration of the nicotinamide solution is 30.53 μg / 100 μl.

[0017] Compared with the prior art, implementing the present invention has the following beneficial effects:

[0018] This invention provides a metabolite, nicotinamide, which, when exogenously derived, can improve the survival rate of shrimp infected with Vibrio parahaemolyticus. This metabolite was screened and identified from the plasma metabolome of Litopenaeus vannamei, which interferes with hemocyanin production under Vibrio parahaemolyticus infection. Recent studies have found that hemocyanin, in addition to its oxygen-carrying function, also regulates metabolic levels in crustaceans to maintain bacterial homeostasis. The inventors of this application used LC-MS / MS to determine the metabolic profile of shrimp plasma, screened and identified metabolites related to anti-Vibrio parahaemolyticus activity, and conducted multiple optimizations and experiments. Ultimately, the optimal dose of nicotinamide for in vivo anti-Vibrio parahaemolyticus activity in shrimp was determined to be 4 μg / 6 g, and the optimal dose for in vitro inhibition of Vibrio parahaemolyticus was 30.53 μg / 100 μL. The nicotinamide of this invention has the function of inhibiting the growth of Vibrio parahaemolyticus in shrimp, exhibits in vitro antibacterial activity against various aquatic pathogens, and can promote the phagocytosis of Vibrio parahaemolyticus by hemocytes, while also promoting the expression of hemocyte antimicrobial peptides ALF1 and ALF4.

[0019] This invention uses natural metabolites to increase the resistance of crustaceans to Vibrio parahaemolyticus, thus preventing large-scale mortality caused by vibrio infection in crustaceans. It is non-toxic and has no side effects, providing a green, efficient, and environmentally friendly feed additive for crustacean seedlings to resist pathogen stimulation. Attached Figure Description

[0020] Figure 1 This is a heatmap of cluster analysis of plasma samples before and after interference with hemocyanin in patients with Vibrio parahaemolyticus infection.

[0021] Figure 2 PLS-DA analysis of plasma samples before and after interference with hemocyanin in Vibrio parahaemolyticus infection.

[0022] Figure 3 This is a volcano analysis diagram of differential metabolites that interfere with hemocyanin before and after Vibrio parahaemolyticus infection.

[0023] Figure 4 This is a heatmap showing the differential metabolite analysis of hemocyanin before and after Vibrio parahaemolyticus infection.

[0024] Figure 5 (A) Comparative analysis of nicotinamide content based on ELISA, (B) Comparative analysis of nicotinamide content based on LC-MS / MS; ** indicates extremely significant difference (p<0.01).

[0025] Figure 6 This study analyzed the effects of different doses of nicotinamide on the abundance of Vibrio parahaemolyticus (A) and Warburgia (B) under physiological conditions and Vibrio parahaemolyticus infection conditions.

[0026] Figure 7The effect of different doses of nicotinamide on the phagocytic rate of Vibrio parahaemolyticus by hemocytes was analyzed; A is a flow cytometry analysis graph; B is a statistical graph of hemocyte phagocytic rate.

[0027] Figure 8 To analyze the effect of nicotinamide on the expression of antimicrobial peptides ALF1(A) and ALF4(B) in blood cells.

[0028] Figure 9 To analyze the antibacterial rate of different concentrations of nicotinamide against Vibrio parahaemolyticus.

[0029] Figure 10 Analysis of the inhibition rate of different concentrations of nicotinamide against Shewanella basalosa.

[0030] Figure 11 To determine the optimal inhibitory concentration of nicotinamide against Vibrio parahaemolyticus.

[0031] Figure 12 Analysis of the optimal inhibitory concentration of nicotinamide against Shewanella basalosa.

[0032] Figure 13 The graph shows the effect of nicotinamide supplementation before and after interfering with hemocyanin on the survival rate of shrimp under physiological conditions.

[0033] Figure 14 The graph shows the effect of nicotinamide supplementation before and after interfering with hemocyanin in shrimp under Vibrio parahaemolyticus infection on the survival rate. Detailed Implementation

[0034] 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.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] Example 1

[0038] Shrimp plasma metabolomic profile analysis

[0039] First, LC-MS / MS was used to perform metabolomics analysis on shrimp plasma samples before and after interference with hemocyanin under Vibrio parahaemolyticus infection, namely "interference control + Vibrio parahaemolyticus infection group (dsGFP+Vp)" and "interference with hemocyanin + Vibrio parahaemolyticus infection group (dsLvHMC+Vp)," with 6 biological replicates in each group. The results showed that 142 metabolites were present in the plasma metabolomic profile ( Figure 1 ).

[0040] Subsequently, the stability and reproducibility of the LC-MS / MS method were evaluated by performing partial least squares discriminant analysis (PLS-DA) on all samples. The results showed that both groups exhibited good clustering with small intra-group differences and good stability; however, significant differences existed between the two groups, suggesting the possible existence of key differentially expressed metabolites affecting the shrimp's resistance to Vibrio parahaemolyticus. Figure 2 ).

[0041] Subsequently, the differences between the two groups of samples were compared by creating a volcano model, and 14 differentially expressed metabolites were obtained. Figure 3 Further comparison with the KEGG database and heatmap analysis ultimately identified seven differentially expressed metabolites with annotation information. Figure 4 ).

[0042] Finally, to analyze whether there were significant differences in the interference of metabolites screened by the above metabolomics methods with hemocyanin before and after Vibrio parahaemolyticus infection, ELISA technology was used for further verification. The results are as follows: Figure 5 A shows that the ELISA results of nicotinamide in plasma are consistent with the metabolomics results. Figure 5 B) Consistent, meaning that under Vibrio parahaemolyticus infection, the content of nicotinamide in shrimp plasma was significantly reduced after interfering with hemocyanin.

[0043] Example 2

[0044] Study on the reduction of Vibrio hemolymphoidis abundance in shrimp by nicotinamide

[0045] (1) Uniformly sized Litopenaeus vannamei (approximately 6g in weight) were randomly divided into four groups of 60 shrimp each. The first group served as the control group, with each shrimp receiving an intramuscular injection of 100μL of 0.65% saline solution. The second, third, and fourth experimental groups received intramuscular injections of 4μg, 20μg, and 100μg of nicotinamide, respectively. After 24 hours, each group was further divided into two subgroups. One subgroup received an injection of 100μL of 0.65% saline solution, while the other subgroup received an injection of 2×10⁻⁶ Vibrio parahaemolyticus. 5 CFU / Shrimp.

[0046] (2) Twenty-four hours after infection, hemolymph bacterial samples were collected from each group, with three biological replicates per group and at least three technical replicates per biological replicate. Hemolymph samples were collected from each shrimp, centrifuged at 800×g for 10 min to enrich hemolymph 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.

[0047] (3) Using hemolymph genomic DNA as a template, the abundance of Vibrio and Shewanella species in hemolymph was detected by qPCR primers for Vibrio and Shewanella-specific 16S rRNA genes, respectively. The obtained Ct values ​​were substituted into the bacterial absolute quantification standard curve to calculate the CFU value of bacteria per milliliter of hemolymph.

[0048] The results are as follows Figure 6 As shown, under Vibrio parahaemolyticus infection, the abundance of Vibrio hemolymphocytes in shrimp significantly decreased in the groups injected with nicotinamide at doses of 4 μg / 6g, 20 μg / 6g, and 100 μg / 6g, with the lowest abundance observed at a nicotinamide dose of 4 μg / 6g. Figure 6 A). Different doses of nicotinamide had no significant effect on the abundance of Shewanella. Figure 6 B). This demonstrates that adding nicotinamide can indeed reduce the number of Vibrio bacteria in the hemolymph of shrimp.

[0049] Example 3

[0050] Research on nicotinamide promoting phagocytosis of Vibrio parahaemolyticus by hemocytes

[0051] (1) Activate and expand the culture of Vibrio parahaemolyticus in advance. Centrifuge at 5000×g for 5 min, remove the supernatant, and resuspend the obtained precipitate of bacteria in 0.01M PBS at a volume ratio of 5:1. Inactivate the bacteria at 72℃ for 20 min, and centrifuge again to enrich the bacteria. Wash the bacteria three times with 0.1M NaHCO3 solution (pH 9.0), and then resuspend the bacteria in an equal volume of 0.01M PBS. Measure the OD using an ELISA reader. 600 The bacterial concentration was adjusted to 3×10⁻⁶. 8 CFU / mL.

[0052] (2) Add fluorescein isothiocyanate (FITC) (100 mg / mL) to the bacterial culture at a volume ratio of 1000:1, and incubate at 37°C for 2 h. Centrifuge for 5 min to enrich the labeled bacteria. Wash the bacteria three times with 0.01 M PBS in the dark, and finally resuspend them with an equal volume of 0.01 M PBS. Store the bacteria at -20°C.

[0053] (3) Nicotinamide was administered intramuscularly according to Example 2. Shrimp were randomly divided into four groups of 15 shrimp each. The first group was the control group, with each shrimp receiving an intramuscular injection of 100 μL of physiological saline (0.65%). The second, third, and fourth experimental groups received intramuscular injections of 4 μg, 20 μg, and 100 μg of nicotinamide per shrimp, respectively. Twenty-four hours later, each group of shrimp was injected intramuscularly with FITC-labeled Vibrio parahaemolyticus (3 × 10⁻⁶). 8 (CFU / mL), protected from light for 1 hour, a blood lymph sample was taken from each shrimp and mixed with an equal volume of anticoagulant, 3 biological replicates per group, and 5 shrimp per biological replicate.

[0054] (4) Centrifuge the collected blood cells at 800×g for 10 min, resuspend the blood cells in 300 μL of 0.01M PBS, and perform flow cytometry. TM C6 Plus (BD Biosciences) statistical analysis of phagocytic cell ratio (FITC channel, excitation 485nm, emission 529nm).

[0055] The results are as follows Figure 7 As shown, the proportion of green fluorescent positive cells phagocytosing Vibrio parahaemolyticus in shrimp hemolymphocytes increased in the groups injected with nicotinamide at 4 μg / 6g, 20 μg / 6g, and 100 μg / 6g. Figure 7 A); Statistical analysis of phagocytosis rate revealed that, compared to the control group, the phagocytosis rate of blood cells was highest at a nicotinamide dose of 4 μg / 6 g. Figure 7 B). This indicates that the addition of nicotinamide can enhance the phagocytic activity of shrimp hemocellular cells against Vibrio parahaemolyticus.

[0056] Example 4

[0057] Nicotinamide promotes the expression of the antimicrobial peptide ALF in blood cells.

[0058] (1) As in Example 2, each shrimp in the control group was injected intramuscularly with 100 μL of physiological saline (0.65%), while each shrimp in the experimental group was injected intramuscularly with 4 μg of nicotinamide. After 24 hours, the shrimp in both the control and experimental groups were further divided into two groups: one group was injected with 100 μL of physiological saline (0.65%) per shrimp, and the other group was injected with 2 × 10⁻⁶ Vibrio parahaemolyticus bacteria. 5 CFU / Shrimp.

[0059] (2) Twenty-four hours after infection, RNA was extracted from shrimp hemocyte tissue using the Flytek TRNAfast 200 RNA extraction kit, and then analyzed according to the full-length gold standard. The One-Step gDNA Removal and cDNA Synthesis Kit instructions state that the total RNA described above is reverse transcribed to synthesize first-strand cDNA, and then... 480 Real-Time PCR Instrument, according to Genstar Green TM I. Detect the expression of the antimicrobial peptide ALF gene using qPCR according to the kit instructions. Figure 8 ).

[0060] The results showed that, under both physiological and Vibrio parahaemolyticus infection conditions, nicotinamide supplementation significantly increased the levels of the antimicrobial peptide ALF1 in blood cells. Figure 8 A) and ALF4 expression ( Figure 8 B).

[0061] Example 5

[0062] In vitro antibacterial activity of nicotinamide

[0063] Take 60 μL of Vibrio parahaemolyticus (2 × 10⁶) each time. 5 CFU / mL), Shewanella basalis (1×10⁻⁶) 5 The bacterial culture (CFU / mL) was mixed with equal volumes of nicotinamide at different concentrations (15.3 μg / 100 μL, 30.5 μg / 100 μL, 61 μg / 100 μL, 122 μg / 100 μL, 244 μg / 100 μL), and an equal volume of physiological saline (0.65%) was mixed with an equal volume of bacteria as a negative control. The mixture was incubated at 37°C for 2 hours. 30 μL of the incubated mixture was spread onto TSB or 2216E plates, with three biological replicates per group and three parallel replicates per biological replicate. After incubation at 37°C for 12 hours, the colony count was scanned and recorded. Bacterial inhibition rate = (Negative control colony count - Experimental group colony count) / Blank control colony count × 100%.

[0064] The results are as follows Figures 9-12 As shown, a certain concentration of nicotinamide can inhibit bacterial growth. When the concentration is 30.5 μg / 100 μl, nicotinamide inhibits the growth of Vibrio parahaemolyticus (Vibrio parahaemolyticus). Figure 9 ) and Shewanella basaltii ( Figure 10 The inhibitory effect on colony growth was most significant, with an inhibition rate of 99.71%. Figure 11 ) and 59.77% Figure 12 Subsequently, as the concentration of nicotinamide increased, its inhibition rate against both bacteria decreased. This indicates that the optimal dose of nicotinamide for inhibiting the growth of pathogenic bacteria in vitro is 30.53 μg / 100 μL.

[0065] Example 6

[0066] Analysis of shrimp survival rate after nicotinamide addition

[0067] To analyze whether nicotinamide, regulated by hemocyanin, affects the shrimp's resistance to Vibrio parahaemolyticus infection, RNAi, metabolite reinjection, and pathogen stimulation techniques were used to compare and analyze the shrimp survival rates before and after hemocyanin interference and nicotinamide reinjection under physiological and Vibrio parahaemolyticus infection conditions. First, under physiological conditions, shrimp were intramuscularly injected with dsRNA (10 μg / 6g), the control group received dsGFP, and the hemocyanin-interfered group received dsLvHMC. Forty-eight hours later, the shrimp in the control and interference groups were randomly divided into two groups: the experimental group received an intramuscular injection of 4 μg nicotinamide per shrimp, while the control group received an equal volume of physiological saline (0.65%). Shrimp survival rates were monitored within 96 hours after nicotinamide injection. Figure 13Furthermore, 24 hours after nicotinamide injection, each group of shrimp was injected with 2×10⁻⁶ Vibrio parahaemolyticus bacteria. 5 CFU / shrimp, and monitor shrimp survival rate within 48 hours of Vibrio parahaemolyticus infection. Figure 14 The results showed that under physiological conditions, the survival rate of shrimp in the non-nicotinamide hemocyanin-interfering group was 40% after 96 hours, while the survival rate of shrimp in the nicotinamide-injected group was 74.29%. Under Vibrio parahaemolyticus infection conditions, the survival rate of shrimp in the non-nicotinamide hemocyanin-interfering group was 0% after 48 hours, while the survival rate of shrimp in the nicotinamide-injected group was 22.73%. This indicates that adding nicotinamide can improve the survival rate of shrimp under Vibrio parahaemolyticus infection.

[0068] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, any equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. An application of nicotinamide, characterized in that, The application of nicotinamide in the preparation of injectable formulations that enhance the resistance of crustaceans to Vibrio parahaemolyticus; wherein the amount of nicotinamide added is 4~100 μg / 6g of the body weight of the crustacean; wherein the crustacean is shrimp.

2. The application of nicotinamide according to claim 1, characterized in that, The application of nicotinamide in the preparation of injectable formulations that promote the expression of antimicrobial peptide genes in blood cells; wherein the antimicrobial peptide gene in blood cells is one or more of ALF1 and ALF4.

3. The application of nicotinamide according to claim 1, characterized in that, The application of nicotinamide in the preparation of injectable formulations that promote the phagocytosis of Vibrio parahaemolyticus by hemocytes.

4. The application of nicotinamide according to claim 1, characterized in that, The amount of nicotinamide added is 4 μg / 6g relative to the body weight of the crustacean.

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