Application of disodium fumarate as shrimp feed additive

Abstract

The invention discloses application of disodium fumarate as a shrimp feed additive, and belongs to the field of aquaculture. The invention provides application of disodium fumarate in inhibition of citrobacter freundii, and experimental results show that disodium fumarate has a good inhibition effect on aquatic pathogenic bacteria citrobacter freundii in vitro. Feeding experiment results show that the disodium fumarate can be used for improving the growth performance of shrimps, regulating immune-related gene expression and enhancing the anti-infection ability, and is beneficial to improving the diversity of intestinal flora and the stability of a tissue structure. The invention provides a safe and efficient feed additive suitable for antibiotic-free healthy culture of shrimps, and the feed additive has good popularization and application prospects.

Description

Technical Field

[0001] The present invention relates to the field of aquaculture, and in particular to the application of disodium fumarate as a shrimp feed additive. Background Art

[0002] Bacterial diseases are common in shrimp farming and spread rapidly, often causing large-scale deaths and bringing huge economic losses to the shrimp farming industry. Common pathogens include Vibrio parahaemolyticus, Citrobacter freundii, Aeromonas versii, and Citrobacter freundii. Procambarus clarkii is one of the important farmed shrimp species in my country. Citrobacter freundii is its main pathogen, often causing infection and disease. Currently, bacterial infections in shrimp mainly rely on chemical drugs such as antibiotics for prevention and treatment. However, Procambarus clarkii usually does not have obvious clinical symptoms in the early stages of infection, which often makes diagnosis difficult and medication is not administered in a timely manner.

[0003] The intestinal microbiome plays a vital role in the nutrition, immunity, and health of farmed shrimp. In a healthy state, the intestinal microbiome produces beneficial substances, maintains normal host metabolism and immune function, and helps the body resist invading pathogens. However, when disease occurs, the structure and function of the intestinal microbiome often become disrupted, leading to abnormal nutrient absorption and immune responses. In shrimp farming, probiotics are widely used to regulate water quality, enhance immunity, and regulate the intestinal microbiome.

[0004] Fumaric acid was first discovered in the tubers of the plant Corydalis yanhusuo and is currently synthesized in certain microorganisms through biosynthetic pathways. Fumaric acid is widely used in the pharmaceutical, food, and chemical industries, exhibiting anti-inflammatory, antioxidant, and immunomodulatory effects. Disodium fumarate is its sodium salt. Research has shown that disodium fumarate can improve rumen pH in ruminants, increase the activity of cellulolytic bacteria, and improve fermentation efficiency, thereby enhancing production performance and health.

[0005] At present, the use of antibiotics in the treatment of bacterial infections in shrimp can easily lead to the spread of resistance genes, increased susceptibility of pathogens, and imbalance of the host's intestinal microecology. Long-term use may also pose food safety risks. The application of probiotic preparations is still limited in its effectiveness in preventing and controlling bacterial infections in shrimp. In actual applications, probiotic strains often cannot successfully colonize in the shrimp intestine, thus affecting their effectiveness. In shrimp farming, there is still a lack of green feed additives or green alternatives to antibiotics for the prevention and control of bacterial infections. The wider role of disodium fumarate has not been developed and utilized.

[0006] Therefore, there is currently no fundamental solution to the problem of preventing and treating bacterial infections in shrimp and improving the structure of intestinal flora. Therefore, it is urgent to explore compounds with the function of preventing and treating diseases and improving growth status in shrimp farming. Summary of the Invention

[0007] The present invention aims to provide a use of disodium fumarate as a shrimp feed additive to solve the problems existing in the above-mentioned prior art. The present invention provides the antibacterial effect of disodium fumarate on Citrobacter freundii, and uses disodium fumarate as a feed additive to feed Procambarus clarkii, thereby improving its immunity, improving its intestinal flora structure, and enhancing its resistance to bacterial infection.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides an application of disodium fumarate in inhibiting Citrobacter freundii in vitro.

[0010] The present invention also provides an application of disodium fumarate in preparing a Citrobacter freundii antibacterial agent.

[0011] The present invention also provides a Citrobacter freundii antibacterial agent, which takes disodium fumarate as a main active ingredient.

[0012] Preferably, the bacteriostatic agent further comprises other bacteriostatic active substances.

[0013] The present invention also provides a use of disodium fumarate in preparing a medicament for preventing and / or treating Citrobacter freundii infection.

[0014] The present invention also provides a medicine for preventing and / or treating Citrobacter freundii infection, which contains disodium fumarate as a main active ingredient.

[0015] Preferably, a pharmaceutically acceptable excipient is also included.

[0016] The present invention also provides an application of disodium fumarate in the preparation of feed additives.

[0017] Preferably, the feed additive is a functional shrimp feed additive; the feed additive has at least one of the following functions:

[0018] Improve shrimp growth performance, improve the composition of shrimp intestinal flora, enhance shrimp disease resistance and maintain the integrity and stability of intestinal and hepatopancreatic tissue structure.

[0019] The present invention also provides a feed additive with disodium fumarate as a main active ingredient.

[0020] The present invention discloses the following technical effects:

[0021] The present invention provides the use of disodium fumarate in inhibiting Citrobacter freundii. Experimental results demonstrate that disodium fumarate has a significant inhibitory effect on the aquatic pathogen Citrobacter freundii in vitro. Feeding experiments also demonstrate that disodium fumarate can improve shrimp growth performance, regulate immune-related gene expression, enhance infection resistance, and contribute to improving intestinal flora diversity and tissue stability. This invention provides a safe and effective feed additive suitable for antibiotic-free, healthy shrimp farming, with promising prospects for widespread application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a statistical graph of the minimum inhibitory concentration of disodium fumarate against Citrobacter freundii; different lowercase letters indicate statistically significant differences;

[0024] Figure 2 This is the colony observation diagram of the minimum bactericidal concentration of disodium fumarate against Citrobacter freundii;

[0025] Figure 3 Time-kill curve of disodium fumarate against Freund's citric acid;

[0026] Figure 4 Freund's citric acid growth curve under the action of disodium fumarate; different lowercase letters indicate statistically significant differences;

[0027] Figure 5 Statistical graph showing the effect of disodium fumarate on the swarming and swimming ability of Citrobacter freundii; A represents the effect of disodium fumarate on swarming; B represents the diameter of swarming; C represents the effect of disodium fumarate on swimming; D represents the diameter of swimming; different lowercase letters indicate statistically significant differences.

[0028] Figure 6 This is a statistical graph showing the effect of disodium fumarate on the cell activity of Citrobacter freundii; different lowercase letters indicate statistically significant differences;

[0029] Figure 7Figures 5 and 6 show the microscopic observation and statistical graphs of the effect of disodium fumarate on biofilm formation of Citrobacter freundii; AF are microscopic observations of biofilm morphology; A is the Control group; B is the 0.125MIC group; C is the 0.25MIC group; D is the 0.5MIC group; E is the 1MIC group; F is the 2MIC group; G is the quantitative detection result of biofilm; different lowercase letters indicate statistically significant differences;

[0030] Figure 8 This is a statistical graph showing the effect of disodium fumarate on the expression of intestinal immune-related genes; different lowercase letters indicate statistically significant differences;

[0031] Figure 9 This is a statistical graph showing the effect of disodium fumarate on the expression of hepatopancreatic immune-related genes; different lowercase letters indicate statistically significant differences;

[0032] Figure 10 This is a statistical graph showing the effect of disodium fumarate on the expression of immune-related genes in hemolymph supernatant; different lowercase letters indicate statistically significant differences;

[0033] Figure 11 Statistical graph of sample correlation analysis results; A is a heat map of correlation coefficients between samples, with colors representing the magnitude of the correlation coefficients; B is a principal component analysis graph, with blue representing the control group and red representing the experimental group;

[0034] Figure 12 Statistical graphs of differential gene analysis results; A is a histogram of differential gene changes; B is a volcano graph of differential gene changes;

[0035] Figure 13 This is a statistical graph of the GO function enrichment analysis results of differentially expressed genes;

[0036] Figure 14 Statistical graph of KEGG pathway enrichment analysis results of differentially expressed genes;

[0037] Figure 15 Statistical graph of qRT-PCR validation results;

[0038] Figure 16 This is a statistical graph showing the effect of 0.5% disodium fumarate on the number of OTUs in the intestinal flora of Procambarus clarkii;

[0039] Figure 17 This is a statistical graph showing the effect of 0.5% disodium fumarate on the α-diversity index of intestinal microorganisms in Procambarus clarkii; where A is the Chao1 index; B is the ACE index; C is the Shannon index; and D is the Simpson index.

[0040] Figure 18 The figure shows the analysis results of the effect of 0.5% disodium fumarate on the β-diversity of intestinal microorganisms of Procambarus clarkii; wherein A is the PCA column coordinate analysis diagram; B is the PcoA principal coordinate analysis diagram;

[0041] Figure 19 Statistical diagram of the effect of 0.5% disodium fumarate on the composition of the intestinal flora of Procambarus clarkii; A is the relative abundance analysis of dominant bacterial phyla; B is the relative abundance analysis of dominant bacterial genera; C is the significant difference analysis of the relative abundance of dominant bacterial phyla; D is the significant difference analysis of the relative abundance of dominant bacterial genera; * indicates P < 0.05;

[0042] Figure 20 This is the LEfSe analysis result diagram;

[0043] Figure 21 This is a statistical graph showing the effect of disodium fumarate on the ability of Procambarus clarkii to resist infection with Citrobacter freundii; different lowercase letters indicate statistically significant differences;

[0044] Figure 22 The microscopic observations show the effect of disodium fumarate on the pathological characteristics of hepatopancreas tissue after infection with Procambarus clarkii bacteria. A, B, and C represent the control group, 0.5% DSF group, 1.5% DSF group, and 4.5% DSF group, respectively. Arrows indicate: cellular vacuolization (HPV), hemosiderin deposition (HD), cell lysis (CL), and nuclear pyknosis (NP).

[0045] Figure 23 The microscopic observations show the effect of disodium fumarate on the pathological characteristics of the intestine of Procambarus clarkii after bacterial infection. A to D represent the control group, the 0.5% DSF group, the 1.5% DSF group, and the 4.5% DSF group, respectively. Arrows indicate separation of the mucosa from the lamina propria (SML), focal necrosis (FN), nuclear pyknosis (NP), and loosening of the mucus layer and connective tissue (N). DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] The purpose of the present invention is to utilize disodium fumarate, which has an antibacterial effect on the pathogenic bacteria of Procambarus clarkii (Citrobacter freundii), to explore its application as a feed additive. The present invention studies the in vitro antibacterial activity of disodium fumarate on the pathogenic bacteria of Procambarus clarkii (Citrobacter freundii), and uses it as a feed additive to evaluate its effects on the growth performance, immune indicators, and resistance to pathogen infection of Procambarus clarkii from indicators such as weight gain rate, specific growth rate, survival rate, and feed coefficient. By analyzing the hepatopancreas transcriptome, the effects on the body's immunity and metabolic capacity are evaluated. By high-throughput sequencing of 16S RNA, the effects on the composition and structure of intestinal flora are evaluated, thereby determining the effect of disodium fumarate as a feed additive on the immunity and disease resistance of Procambarus clarkii and on the regulation of the improvement of intestinal flora, providing a theoretical basis and technical support for the promotion and application of disodium fumarate as a green alternative to antibiotics in aquaculture.

[0052] The present invention found that disodium fumarate has good in vitro antibacterial activity against Citrobacter freundii through the determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), growth curve, clustering and swimming ability and bacterial cell activity, as well as qualitative and quantitative analysis of biofilm. As a feed additive, disodium fumarate can significantly improve the growth performance, immune gene expression level and anti-pathogen infection ability of crayfish in the concentration range of 0.5% to 4.5%. Hepatopancreatic transcriptome analysis showed that the addition of 0.5% concentration of disodium fumarate can enhance the phagocytic and clearance ability of immune cells, improve protein processing efficiency and the activity of multiple metabolic pathways; further analysis of the intestinal flora structure found that it can increase the diversity of intestinal flora, increase the relative abundance of beneficial bacteria, and reduce the proportion of harmful bacteria. DSF (disodium fumarate) has the most suitable effect on the growth performance of crayfish at an addition of 1.5%, and the disease resistance is the strongest at additions of 1.5% and 4.5%. Overall, the addition of disodium fumarate can improve the immunity of crayfish, improve the structure of intestinal flora, and enhance the disease resistance against bacterial infections. It is a safe and efficient alternative to antibiotics. As a shrimp feed additive in aquaculture, it has excellent biosafety and broad application prospects.

[0053] The present invention's Citrobacter freundii strain was isolated by the inventors from the hepatopancreas tissue of diseased Procambarus clarkii crayfish at a breeding base in Tianjin. The strain was purified and cultured using the three-zone streak method in LB medium, and its pathogenicity was confirmed by recurrent infection experiments. The strain is currently maintained in the inventors' laboratory, and its 16S rRNA gene sequence has been published on the NCBI website (Accession Number: PV855883.1). The applicants commit to distributing the aforementioned biological material to the public within 20 years from the filing date of this application.

[0054] Example 1 Inhibitory ability of disodium fumarate on Citrobacter freundii

[0055] 1. Minimum inhibitory concentration (MIC) of disodium fumarate against Citrobacter freundii

[0056] 1.1 Strain culture and bacterial suspension preparation

[0057] Citrobacter freundii was inoculated into LB liquid medium and cultured in a 30°C constant temperature shaker (150 rpm) for 24 h until the logarithmic growth phase. The bacterial solution concentration was adjusted to 1×10 8 CFU / mL is reserved.

[0058] 1.2 Preparation of Disodium Fumarate (DSF) Solution and MIC Determination

[0059] Using LB liquid medium as the solvent, the DSF stock solution (640 mg / mL) was serially diluted to obtain a gradient concentration of DSF solutions with final concentrations of 640, 320, 160, 80, 40, 20, 10, 5, 2.5, 1.25, and 0.625 mg / mL. Subsequently, the MIC was determined in a 96-well cell plate using the broth microdilution method. 180 μL of bacterial suspension (1×10 8 CFU / mL) and 20 μL of DSF solution of different concentrations were added to make the final test concentrations 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.0625 mg / mL respectively. LB medium without DSF was set as a control, and 3 replicate wells were set for each concentration. The 96-well plate was placed in a 30°C constant temperature incubator and cultured for 24 hours. The absorbance of each well was measured at a wavelength of 600 nm using a microplate reader (OD 600 ) to determine the MIC of DSF against Citrobacter freundii.

[0060] 1.3 Results and Analysis

[0061] like Figure 1 As shown, compared with the control group, DSF concentrations of 2-64 mg / mL significantly inhibited the growth of Citrobacter freundii (P < 0.01). DSF concentrations of 0.125-1 mg / mL had no significant inhibitory effect on the growth of Citrobacter freundii. Therefore, the minimum inhibitory concentration of DSF against Citrobacter freundii is 2 mg / mL.

[0062] 2. Minimum bactericidal concentration (MBC) of disodium fumarate against Citrobacter freundii

[0063] The plate count method was used to determine the MBC of DSF against Citrobacter freundii. 10 μL of the reaction solution from the MIC experiment (containing different concentrations of DSF: 2, 4, 8, 16, 32, and 0 mg / mL) was evenly spread on the surface of LB solid culture medium and incubated at 30°C for 24 hours. The lowest drug concentration at which no colonies grew was determined as the MBC value.

[0064] The results are as follows Figure 2 As shown, no visible colonies were observed on the LB plates in the 8, 16, and 32 mg / mL DSF concentration groups, while bacterial growth was still visible in the 2 and 4 mg / mL concentration groups. Therefore, the MBC value of DSF against Citrobacter freundii was 8 mg / mL.

[0065] 3. Time-kill curve of the effect of disodium fumarate on Citrobacter freundii

[0066] 45 mL of bacterial suspension (3×10 6 CFU / mL) were mixed with 5 mL of DSF solution at different concentrations to achieve final DSF concentrations of 0 (control), 1, 2, 4, and 8 MIC, with three biological replicates for each concentration. Cultures were maintained at 30°C, and 1 mL samples were taken at 0, 1, 3, 5, 7, and 9 hours for determination of bacterial concentration using a McFarland turbidimeter.

[0067] The results are as follows Figure 3 As shown, the bactericidal effect of DSF was concentration-time dependent. DSF concentrations ranging from 1 to 8 MIC showed bactericidal activity against Citrobacter freundii. At 9 hours after incubation, no bacterial growth was observed at concentrations of 4 and 8 MIC.

[0068] 4. Determination of the Growth Curve of Citrobacter freundii under Different Concentrations of Disodium Fumarate

[0069] Take the culture of Citrobacter freundii in the logarithmic growth phase and adjust the bacterial solution concentration to 1×10 8 CFU / mL. In a 96-well plate, 20 μL of bacterial suspension (1×10 8 CFU / mL), and then 180 μL of LB liquid medium containing DSF was added to make the final concentrations 0.125, 0.25, 0.5 and 1 MIC, respectively. The control group was added with an equal volume of LB liquid medium without DSF, and 3 biological replicates were set for each group. The 96-well plate was placed in a 30°C constant temperature incubator and cultured for 48 hours. The OD was measured every 2 hours. 600 Record the data and draw a growth curve.

[0070] The results are as follows Figure 4 As shown, the control group (0 mg / mL) entered the logarithmic growth phase after 2 hours of culture and the stationary phase after 24 hours. DSF at a concentration of 0.125 MIC delayed the entry of Citrobacter freundii into the logarithmic growth phase and the stationary phase and had a certain inhibitory effect on growth. At a DSF concentration of 0.25-1 MIC, the bacteria entered the logarithmic growth phase after 8 hours of culture and gradually entered the stationary phase at 46 hours. Furthermore, after 48 hours of culture, the bacterial liquid concentration in the 0.25-1 MIC concentration group was significantly lower than that in the DSF control group (P < 0.05).

[0071] 5. Effects of disodium fumarate on the colonization and swimming ability of Citrobacter freundii

[0072] 5.1 Swarming and swimming experiments

[0073] The experiment selected bacterial solution in logarithmic growth phase (1×10 8CFU / mL) were spotted on the surface of cluster solid culture medium (tryptone 1g / L, sodium chloride 0.5g / L, agar 0.5g / L, glucose 0.5g / L) and swimming solid culture medium (tryptone 1g / L, sodium chloride 3g / L, agar 0.3g / L) containing different concentrations of DSF (0.125, 0.25, 0.5, 1 and 2MIC), with 3 parallels per group. After the inoculated plates were incubated at 30°C for 24 hours, the colony spread diameter (mm) was accurately measured using a vernier caliper, and the average of three measurements was taken as the final result. All experiments were set up with a culture medium without DSF as a negative control.

[0074] 5.2 Cluster Capabilities

[0075] like Figure 5 As shown in Figures A and B, compared with the control group, the diameter of bacterial clusters was significantly reduced at a concentration of 0.5 MIC, while there was no significant difference at other concentrations (P < 0.05).

[0076] 5.3 Swimming ability

[0077] like Figure 5 As shown in C and D, at MIC concentrations of 0.125-0.5, the swimming diameter of Citrobacter freundii was significantly reduced compared with the control group (P<0.05), indicating that the bacterial swimming ability was significantly inhibited.

[0078] 6. Effect of disodium fumarate on the activity of Citrobacter freundii cells

[0079] Take 90 μL of bacterial solution in logarithmic growth phase (1×10 8 CFU / mL) were added to a 96-well microtiter plate, and 10 μL of DSF solution of different concentrations was added to make the final concentrations of 0 (blank control), 0.125, 0.25, 0.5 and 1 MIC, respectively. Three parallels were set up for each group. The culture plate was placed in a 30°C constant temperature incubator for 24 hours, and then 10 μL of CCK-8 solution was added to each well. After incubation in the dark for 1 hour, the OD was measured. 430 value.

[0080] The results are as follows Figure 6 As shown in the figure, compared with the control group, the cell activity of the 1MIC concentration group was significantly lower than that of the control group (P<0.05); there was no significant difference in the cell activity of the other concentration groups compared with the control group.

[0081] 7. Effect of disodium fumarate on biofilm formation of Citrobacter freundii

[0082] 7.1 Microscopic observation of biofilm morphology

[0083] In a 24-well culture plate, a sterile cell slide (14 mm) was pre-placed. Subsequently, 900 μL of bacterial suspension and 100 μL of DSF solution of different concentrations were added to make the final DSF concentration reach 0 (control), 0.125, 0.25, 0.5, 1, and 2 MIC, respectively, with 3 replicates per group. After static incubation at 30°C for 24 hours, the coverslips were removed and washed 3 times with PBS, fixed with anhydrous methanol for 15 minutes, and stained with 0.2% crystal violet in the dark for 20 minutes. The crystal violet dye was discarded, and the cells were washed with PBS and dried. The bacterial biofilm morphology was observed under an optical microscope.

[0084] 7.2 Quantitative detection of biofilm

[0085] In a 96-well microplate, 20 μL of DSF solution was added in sequence and mixed with 180 μL of Citrobacter freundii suspension to make the final DSF concentration reach 0.125, 0.25, 0.5, 1 and 2 MIC, respectively, with 3 replicates for each group. LB liquid culture medium without DSF was used as a negative control. After static culture at 30°C for 24 hours, the culture medium was discarded. Excess culture medium and floating bacteria were washed away with sterile PBS, 200 μL of anhydrous methanol was added for fixation for 15 minutes, and the fixative was discarded. 200 μL of 0.2% crystal violet solution was added and stained in the dark for 20 minutes. After discarding the staining solution, the free dye was washed three times with PBS. After washing with PBS, 300 μL of 33% glacial acetic acid was added for desorption for 10 minutes, and the OD was measured. 630 The biofilm formation amount was quantitatively evaluated.

[0086] 7.3 Morphological observation and quantitative analysis results

[0087] like Figure 7 As shown in Figure A, in the control group, the biofilm formed by Citrobacter freundii showed a dense network structure with close adhesion between cells; at a concentration of 0.125 MIC, the density of the bacterial biofilm decreased slightly, and the degree of bacterial aggregation weakened, but the overall community structure remained relatively intact ( Figure 7 At 0.25 MIC concentration, DSF caused the bacterial biofilm to become more dispersed and the bacterial density to decrease, but local aggregation was still observed ( Figure 7 In the 0.5MIC and 1MIC concentration groups, it was observed that the bacterial biofilm was more sparse, with increased bacterial spacing and significantly dispersed community structure ( Figure 7 D and E); bacteria in the 2MIC concentration group hardly formed biofilm ( Figure 7 Middle F).

[0088] The quantitative analysis results showed that compared with the control group, DSF at a concentration of 0.125-2MIC had a significant inhibitory effect on the biofilm formation of Citrobacter freundii ( Figure 7 (G, P < 0.05).

[0089] The experimental results of this example show that disodium fumarate can significantly inhibit the growth, motility, biofilm formation and cell activity of Citrobacter freundii in vitro, and its minimum inhibitory concentration is 2 mg / mL.

[0090] Example 2 Effects of different concentrations of DSF on growth performance and immune-related gene expression in Procambarus clarkii

[0091] 1. Experimental Animal Husbandry and Sample Collection

[0092] 1.1 Feed preparation

[0093] Basal diets were purchased from Tongwei Co., Ltd. The basal feed dry powder was thoroughly mixed with DSF and pelleted using a pellet mill to prepare pellets (approximately 3 mm in diameter) containing 0%, 0.5%, 1.5%, and 4.5% DSF. The DSF-containing feeds were dried at room temperature and stored sealed at 4°C. The nutritional composition of the basal feed is shown in Table 1.

[0094] Table 1 Nutritional distribution of basic feed

[0095]

[0096] 1.2 Animal husbandry and grouping

[0097] The healthy Procambarus clarkii (average weight 4±2g) used in this experiment were purchased from a farm in Jinghai District, Tianjin, and transported to a recirculating aquaculture laboratory for temporary rearing at a water temperature of 23±2℃ and continuous feeding. After 14 days of temporary rearing, the Procambarus clarkii were randomly divided into four groups, with 3 parallels in each group. The four groups of Procambarus clarkii were fed with feed containing 0%, 0.5%, 1.5%, and 4.5% DSF, respectively. The breeding experiment lasted for 28 days, and the crayfish were fed once a day at 8:30 am and 5:30 pm, with a feeding amount of 2% of their body weight. After 2 hours of feeding, the feed residues at the bottom of the tank were collected, dried in an oven, and weighed to ensure accurate recording of feed consumption.

[0098] 1.3 Sample collection

[0099] On the 28th day after feeding DSF, hepatopancreas, intestine and hemolymph samples of each group were collected for qRT-PCR experiments. The specific operation is as follows: when collecting blood, use a 1mL sterile syringe to draw 0.5mL of pre-cooled sodium heparin anticoagulant, and then draw 0.5mL of hemolymph sample, mix well and centrifuge at 4℃ (12000rpm, 15min), and collect the supernatant for use. Subsequently, the surface of the crayfish was wiped with 75% ethanol, and the dissection was performed under sterile conditions to separate the intestinal and hepatopancreatic tissues. Three tails were randomly selected for each parallel, and the tissues were mixed separately as a biological replicate sample, with three parallel samples in each group (n=3). The samples were quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for real-time fluorescence quantitative PCR experiments.

[0100] 2. Effects of disodium fumarate on the growth performance of Procambarus clarkii

[0101] On the 28th day of the culture experiment, the crayfish were counted and weighed (accurate to 0.01g), and their survival rate, weight gain rate, specific growth rate and feed conversion rate were statistically analyzed.

[0102] The experimental results (Table 2) showed that the weight gain rate and specific growth rate of crayfish in the DSF-fed group were significantly higher than those in the control group. Specifically, the weight gain rate and specific growth rate of the low-concentration (0.5%) and high-concentration (4.5%) groups were significantly lower than those in the medium-concentration (1.5%) group (P < 0.05). In addition, the feed conversion rate of the DSF-fed group was significantly lower than that of the control group (P < 0.05), indicating that feed utilization was significantly improved. However, the survival rate did not show significant differences among the groups (P > 0.05). In summary, within the concentration range of 0.5% to 4.5%, DSF addition at a level of 1.5% was most effective in affecting the growth performance of crayfish.

[0103] Table 2 Effects of disodium fumarate on growth indicators of Procambarus clarkii

[0104]

[0105] Note: Different letters indicate significant differences (P<0.05), n=3, the calculation formula of relevant growth performance indicators refers to: Ning Keyuan. Effects of adding taurine and marine red yeast in feed on growth, immunity and intestinal health of bigeye fish [D]. Dalian Ocean University, 2024.

[0106] 3. Effects of DSF feeding on immune gene expression in the intestine, hepatopancreas, and hemolymph supernatant of Procambarus clarkii

[0107] 3.1 RNA extraction and cDNA synthesis

[0108] Total RNA was extracted from the hemolymph supernatant, intestine, and hepatopancreas of Procambarus clarkii using RNAiso Plus reagent (Takara) according to the manufacturer's instructions. RNA quality was tested for integrity by 1% agarose gel electrophoresis, and purity and concentration were determined using an ultramicro spectrophotometer (NanoDrop 2000, Thermo). Subsequently, PrimeScript TM RT reagent Kit with gDNA Eraser (Takara) was used to reverse transcribe RNA samples into cDNA.

[0109] 3.2 Real-time quantitative PCR (qRT-PCR)

[0110] qRT-PCR was used to measure the expression levels of MYD88, Bax, Bcl2, Caspase 3, CuZnSOD, and Astacidin using 18S rRNA as an internal reference gene. Primer sequences for each gene were designed using NCBI Primer-BLAST. See Table 3 for the specific sequences.

[0111] The total volume of the PCR reaction system was 20 μL, including BeyoFast TM SYBR Green qPCR Mix (2×, Beyotime), 2 μL cDNA template, 1.6 μL primers (0.8 μL each of forward and reverse primers), and 6.4 μL RNase-free water (ddH2O). The qRT-PCR amplification program was set as follows: 95°C pre-denaturation for 3 minutes, followed by 40 cycles, each cycle consisting of 95°C denaturation for 15 seconds, annealing for 1 minute, and extension at 72°C for 2 minutes. The relative expression levels of genes were calculated using 2 -△△Ct The results were calculated and normalized using the control group as the benchmark.

[0112] Table 3 qRT-PCR primer information

[0113]

[0114] 3.3 Analysis of intestinal immune gene expression

[0115] like Figure 8As shown, after 28 days of DSF feeding, the expression levels of Astacidin and Bax genes were significantly higher in the 0.5% DSF group than in the other groups (P<0.05), while no significant differences were found among the other groups. CuZnSOD gene expression was significantly higher in the 0.5% and 1.5% DSF groups than in the other groups (P<0.05), with no statistical difference between the 4.5% group and the control group (P>0.05). MyD88 gene expression was significantly upregulated in the 0.5% group compared with the other groups, while expression levels in the 1.5% and 4.5% groups were significantly lower than in the control group (P<0.05). Furthermore, Caspase 3 gene expression was significantly higher in the control group than in the 4.5% group (P<0.05). Bcl2 gene expression was also significantly higher in the control group than in the 0.5%-4.5% DSF treatment groups (P<0.05).

[0116] 3.4 Analysis of hepatopancreatic immune gene expression

[0117] like Figure 9 As shown, in the 0.5% DSF group, the expression levels of MyD88, Bcl2, Caspase 3, CuZnSOD, and Astacidin genes were significantly higher than those in the control, 1.5% DSF, and 4.5% DSF groups (P<0.05); whereas the expression levels of these genes in the 1.5% and 4.5% DSF groups were significantly lower than those in the control group (P<0.05). Compared with the control group, the expression of the Bax gene was significantly upregulated in the 0.5% group, while it was significantly downregulated in the 4.5% group (P<0.05).

[0118] 3.5 Analysis of immune gene expression in hemolymph supernatant

[0119] like Figure 10 As shown, the Astacidin gene expression level in the 0.5% DSF group was significantly higher than that in the control and 4.5% groups, but significantly lower than that in the 1.5% group (P < 0.05). The CuZnSOD gene expression level in the 4.5% group was significantly higher than that in the other groups (P < 0.05). The MyD88, Bax, and Bcl2 gene expression levels in the 1.5% and 4.5% groups were significantly higher than that in the control group, but significantly lower than those in the 0.5% group (P < 0.05). Furthermore, the MyD88 expression level in the 1.5% group was significantly higher than that in the 4.5% group (P < 0.05). Furthermore, the Caspase3 gene expression level in the hemolymph supernatant of the 0.5% DSF group was the highest among all groups and significantly different from that in the other treatment groups (P < 0.05).

[0120] The experimental results of this example show that adding 0.5%, 1.5%, and 4.5% disodium fumarate to the feed can significantly increase the weight gain rate, specific growth rate, and feed utilization rate of Procambarus clarkii after feeding for 28 days; at the same time, it can significantly increase the expression levels of immune genes in the intestine, hepatopancreas, and plasma.

[0121] Example 3 Effect of 0.5% DSF in feed on hepatopancreas transcriptome analysis

[0122] 1. Collection of Hepatopancreas Samples from Procambarus clarkii

[0123] The crayfish from Example 2 were cultured for 28 days. Hepatopancreas tissue samples were collected from two groups of crayfish fed a diet supplemented with 0% and 0.5% DSF, designated the CG and TG groups, respectively. Three crayfish were randomly selected from each replicate and aseptically dissected. The hepatopancreas was collected and pooled into a single sample. Three samples were collected from each group and stored at -80°C for hepatopancreatic tissue transcriptomic sequencing.

[0124] 2. RNA-seq Sequencing and Data Analysis

[0125] The sample was placed in 1 mL of Trizol lysis buffer and mixed thoroughly. Total RNA from the hepatopancreas of Procambarus clarkii was extracted according to standard procedures. RNA purity was determined using an ultramicrospectrophotometer. Libraries were constructed using the VAHTS Universal V5 RNA-seq LibraryPrep Kit and sent to Shanghai Ouyi Biotechnology Co., Ltd. for sequencing. Sequencing data were aligned to the reference genome using HISAT2, and gene quantification analysis was performed. Differentially expressed genes were screened using DESeq2 software, and genes with significant differences (q value < 0.05 and fold difference ≥ 2 or ≤ 0.5) were further analyzed for GO and KEGG pathway significance.

[0126] 3. Sequencing basic data

[0127] After sequencing, a total of 6 samples were obtained, generating an average of 46.95M effective sequences (Clean reads). The results of Person correlation analysis showed that the correlation coefficients of samples within the two groups were greater than 0.93, indicating the reliability of the experimental procedure and sample selection ( Figure 11 The results of principal component analysis showed that the samples of the CG group and the TG group were significantly separated and clustered, further verifying the differences of the samples ( Figure 11 934 differentially expressed genes were identified between the two groups, of which 537 were upregulated and 397 were downregulated ( Figure 12 ).

[0128] 4.GO functional enrichment analysis

[0129] The differentially expressed genes were significantly enriched in the three major functional categories of GO (cellular components, molecular functions and biological processes) ( Figure 13In biological processes, differentially expressed genes were mainly involved in negative regulation of endopeptidase activity, retinol metabolism, negative regulation of coagulation, sodium ion transmembrane transport, and cellular calcium ion homeostasis. In terms of cellular components, they were mainly enriched in the extracellular region, endoplasmic reticulum membrane, and organelle membranes. In terms of molecular function, they were mainly manifested in oxygen carrier activity, oxidoreductase activity, iron ion binding, heme binding, and monooxygenase activity.

[0130] 5. KEGG pathway enrichment analysis

[0131] The analysis results showed that the differentially expressed genes were mainly involved in important biological functions such as cellular processes, genetic information processing and metabolism. Among the top 20 significantly enriched KEGG pathways ( Figure 14 ), phagosome pathway, endoplasmic reticulum protein processing pathway, lysosome pathway, and tyrosine metabolism pathway showed significant enrichment (P<0.05). Specifically, the comparison between the CG group and the TG group found that: in terms of cellular processes, differentially expressed genes were significantly enriched in the phagosome, lysosome, and peroxisome pathways; in terms of genetic information processing, they were mainly enriched in the endoplasmic reticulum protein processing pathway; in terms of metabolism, they were involved in pathways such as tyrosine metabolism, fatty acid biosynthesis, unsaturated fatty acid biosynthesis, riboflavin metabolism, pantothenic acid and coenzyme A biosynthesis, linoleic acid metabolism, retinol metabolism, niacin and nicotinamide metabolism, starch and sucrose metabolism, butyrate metabolism, alanine metabolism, glycolysis / gluconeogenesis, lipid metabolism, glyceride metabolism, and pyruvate metabolism.

[0132] 6. qRT-PCR Validation

[0133] Among the significantly enriched KEGG pathways, six differentially expressed genes were screened for qRT-PCR validation, including cathepsin, calreticulin, cholesterol transporter (NPC2), N-acetylsulfatase (Galns), N-acetylgalactosaminidase (NAGA), and cysteine ​​protease (LCP1). The experimental results showed that the expression patterns of the above six genes were consistent with the results of RNA-Seq analysis, verifying the accuracy of transcriptome sequencing ( Figure 15 ).

[0134] The experimental results of this example show that adding 0.5% DSF to the feed can significantly enhance the phagocytic and clearance capabilities of cells in the hepatopancreas tissue of Procambarus clarkii, promote protein processing and folding, and activate multiple metabolic pathways, thereby improving overall immune function and metabolic efficiency. It has good growth-promoting and stress-resistant potential and can be used as a functional feed additive in Procambarus clarkii farming.

[0135] Example 4 Effect of adding 0.5% DSF to feed on the composition of the intestinal flora of Procambarus clarkii

[0136] 1. Collection of intestinal contents samples of Procambarus clarkii

[0137] The crayfish from Example 2 were cultured for 28 days. Intestinal contents were collected from two groups of crayfish fed a diet supplemented with 0% and 0.5% DSF, designated the CK and TL groups, respectively. Seven crayfish were randomly selected from each replicate and aseptically dissected. The intestinal contents were collected and pooled to form a single sample. Three samples were obtained from each group and stored at -80°C for subsequent analysis of the intestinal flora composition.

[0138] 2.16S rDNA high-throughput sequencing and data analysis

[0139] DNA extraction, gene amplification, and sequencing were performed by Guangzhou Kidio Biotechnology Co., Ltd. Total DNA from intestinal contents was extracted using HiPure Stool DNA Kits, and the V3-V4 region was amplified using barcoded specific primers. After purification, libraries were constructed and high-throughput sequencing was performed on the Illumina PE250 platform. Raw data were quality-cut using FASTP software, and after removing chimeras, paired-end reads were spliced ​​into tags, which were then screened for clean tags. Clean tags were clustered into operational taxonomic units (OTUs) with 97% consistency, and OTU sequences were annotated to species using the RDP database. Alpha diversity indices were calculated using QIIME software, and principal coordinate analysis (PCoA) was performed using the Vegan package in R based on unweighted Unifrac distances. Species that differed between groups were identified using LEfSe analysis, and heat maps were drawn to compare species differences.

[0140] 2.1 Sequencing basic data

[0141] A total of 785,963 high-quality data were obtained from the two groups of 6 samples, which were assigned to 1922 OTUs ( Figure 16 Among them, there were 591 OTUs in common between the two groups, and 746 and 585 unique OTUs in the CK and TL groups, respectively.

[0142] 2.2 Analysis of intestinal flora diversity

[0143] The results of α diversity analysis showed that the Simpson index of the TL group was significantly higher than that of the CK group (P<0.05), and there was no significant difference in Chao1, ACE, and Shannon index between the two groups ( Figure 17 ). PCA analysis showed that the sample distribution of CK group and TL group was significantly separated, and the sample distribution of CK group was more dispersed. The results of PCoA analysis were consistent with PCA ( Figure 18 ).

[0144] 2.3 Analysis of the composition and structure of the intestinal microbiome of Procambarus clarkii

[0145] At the phylum level, the dominant phyla in both groups were Proteobacteria, Firmicutes, and Bacteroidetes, and there was no significant difference in relative abundance ( Figure 19 At the genus level, Candidatus_Bacilloplasma, Tyzzerella, Vibrio, Anaerorhabdus_furcosa_group, Flavobacterium, Pseudomonas, Cloacibacterium, Lysobacter and Bacteroides were the main dominant genera in both groups ( Figure 19 Among them, the relative abundance of Candidatus_Bacilloplasma in the treatment group (TL) was significantly higher than that in the control group (P<0.05) ( Figure 19 C and D).

[0146] 2.4LEfSe Analysis

[0147] LEfSe analysis results show that ( Figure 20 , LDA>4, P<0.05). In the CK group, the abundance of Enterobacterales, Flavobacterium, Lachnospiraceae, Proteobacteria, Clostridia, Tyzzerella, Alphaproteobacteria, Rhodobacterales, and Rhizobiales was higher. In the TL group, the abundance of Bacilli, Mycoplasmataceae, Candidatus_Bacilloplasma, Mycoplasmatales, Cloacibacterium, Weeksellaceae, Bacteroidales, RsaHf231, and Dysgonomonadaceae was higher.

[0148] The addition of 0.5% DSF to the feed significantly altered the composition of the gut microbiota of Procambarus clarkii. 16S rDNA high-throughput sequencing revealed that the gut microbiota diversity of the TL group was enhanced compared to the control group, with a significant increase in the relative abundance of the Candidatus_Bacilloplasma genus. LEfSe analysis further revealed significant differences in the abundance of specific bacterial genera between the TL and CK groups, indicating that 0.5% DSF significantly modulated the gut microbial community.

[0149] The experimental results of this example show that adding 0.5% DSF to the feed can increase the diversity of intestinal flora, improve the composition and structure of the intestinal flora of Procambarus clarkii, and regulate intestinal health.

[0150] Example 5 Effect of disodium fumarate on the ability of Procambarus clarkii to resist infection with Citrobacter freundii

[0151] 1. Preparation of Bacterial Suspension

[0152] The Citrobacter freundii isolated and preserved in this laboratory was inoculated into LB liquid medium and cultured under suitable conditions until the logarithmic growth phase. Subsequently, the bacterial suspension was centrifuged to collect the bacterial precipitate and resuspended in sterile saline to adjust the bacterial suspension concentration to 2.73×10 7 CFU / mL, used for infection experiments.

[0153] 2. Infection Experiment and Sample Collection

[0154] As in the culture experiment described in Example 2, crayfish were fed diets supplemented with 0%, 0.5%, 1.5%, and 4.5% DSF. After 28 days of culture, 30 crayfish were randomly selected from each group (three replicates per group, 10 crayfish per replicate) for intraperitoneal infection. The experimental group received 100 μL of the bacterial suspension, while the control group received an equal volume of sterile saline. During the infection period, symptoms and mortality were observed and recorded daily. During the experiment, crayfish exhibiting pathological symptoms or near-death status were promptly sampled, and hepatopancreas and intestinal tissues were collected and fixed in Bonn's solution for histopathological analysis.

[0155] 3. Mortality Statistical Analysis

[0156] like Figure 21As shown, 7 days after infection with Citrobacter freundii, the mortality rates of the control group and the 0.5%, 1.5%, and 4.5% DSF groups were 75%, 50%, 25%, and 25%, respectively. Compared with the control group, the mortality rate of all DSF-fed groups was significantly reduced (P<0.05). Among them, the mortality rate of the 0.5% group was significantly higher than that of the 1.5% and 4.5% groups (P<0.05), but there was no significant difference between the 1.5% and 4.5% groups (P>0.05). These results show that DSF feeding can effectively improve the resistance of Procambarus clarkii to Citrobacter freundii infection.

[0157] 4. Histopathological characteristics after infection

[0158] After the culture period, one Procambarus clarkii crayfish was randomly selected from each replicate, and the intestinal and hepatopancreatic tissues were isolated and fixed in Bonn's solution for 24 hours. Following fixation, the tissues were dehydrated by immersion in different concentrations of tert-butyl alcohol, with each dehydration period lasting 2 hours. The dehydrated tissues were then immersed in first, second, and third grade paraffin wax, each for 2 hours, and then embedded using an embedding machine. The embedded tissue blocks were cut into 5 μm-thick sections on a paraffin microtome, spread, and stained with hematoxylin and eosin (H&E).

[0159] After infection with Citrobacter freundii, the intestinal and hepatopancreatic tissues of Procambarus clarkii showed concentration-dependent pathological changes with increasing DSF feeding concentration. The hepatopancreatic lesions in the control group were most obvious, with highly dilated hepatic ducts, disappearance of the star-shaped lumen structure, severe cavitation in the lumen, blurred boundaries between hepatic ducts, disordered cell arrangement, and obvious pathological changes such as hemosiderin deposition, cell lysis, and nuclear condensation. Figure 22 In the DSF treatment group, as the feeding concentration increased, the degree of tissue damage gradually decreased, especially in the high-concentration group (4.5% DSF), where the hepatic tubule structure was basically intact, the cells were arranged more regularly, and the tissue morphology was close to normal ( Figure 22 Chinese BD).

[0160] The control group showed that the intestinal villi were arranged in disorder, with blunting, fusion or shedding of the villus tips; the submucosal structure was loose or dissolved and separated from the lamina propria; the epithelial cells were arranged in disorder and the boundaries were unclear ( Figure 23 As the DSF concentration increased, the pathological damage to the intestinal mucosa gradually decreased. In the 0.5% DSF group, although the structure of the lamina propria was loose and the tips of the intestinal villi were blunted, the overall structure remained intact and the epithelial cells were neatly arranged. In the 1.5% and 4.5% DSF concentration groups, the intestinal villi were generally neatly arranged, with significant wrinkles and clear cell boundaries, but the mucosal layer was slightly separated from the lamina propria (see Figure 23 Chinese BD).

[0161] The experimental results of this example show that the addition of 0.5%, 1.5%, and 4.5% disodium fumarate to feed significantly enhanced the resistance of Procambarus clarkii to infection with Citrobacter freundii after 28 days of feeding. The 1.5% and 4.5% additions significantly enhanced disease resistance compared to the 0.5% addition. The addition of disodium fumarate also improved tissue tolerance to infection-induced damage, suggesting that DSF has a potential preventive protective effect.

[0162] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An application of disodium fumarate in inhibiting Citrobacter freundii in vitro.

2. Application of disodium fumarate in the preparation of a Citrobacter freundii antibacterial agent.

3. A Citrobacter freundii antibacterial agent, characterized in that The main active ingredient is disodium fumarate.

4. The antibacterial agent according to claim 3, wherein The bacteriostatic agent also includes other bacteriostatic active substances.

5. Use of disodium fumarate in the preparation of a medicament for preventing and / or treating Citrobacter freundii infection.

6. A drug for preventing and / or treating Citrobacter freundii infection, characterized in that: The main active ingredient is disodium fumarate.

7. The drug according to claim 6, characterized in that Pharmaceutically acceptable excipients are also included.

8. Use of disodium fumarate in the preparation of feed additives.

9. The use according to claim 8, characterized in that The feed additive is a functional shrimp feed additive; The feed additive has at least one of the following functions: Improve shrimp growth performance, improve the composition of shrimp intestinal flora, enhance shrimp disease resistance and maintain the integrity and stability of intestinal and hepatopancreatic tissue structure.

10. A feed additive, characterized in that The main active ingredient is disodium fumarate.

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