Use of allicin e for controlling white-spot disease in larimichthys crocea

Abstract

The use of allicin E for controlling white-spot disease in Larimichthys crocea, which use can effectively reduce the mortality of Larimichthys crocea, alleviate pathological changes in the spleen and liver of Larimichthys crocea caused by Pseudomonas plecoglossicida, and reduce the threats to food and environmental safety caused by the long-term use of antibiotics in Larimichthys crocea.

Description

Uses of garlic E in the prevention and treatment of white spot disease in large yellow croaker Technical Field

[0001] This invention relates to the field of prevention and treatment technology for white spot disease of large yellow croaker viscera, and proposes the use of garlic E to prevent and treat white spot disease of large yellow croaker. Background Technology

[0002] Large yellow croaker (Larimichthyscrocea) is currently the most widely farmed marine economic fish in my country, known as the "national fish of marine life." In 2022, the national production of large yellow croaker reached 227,600 tons, with approximately 2.585 billion fry raised, both ranking first among marine aquaculture fish in my country. However, with the expansion of large yellow croaker farming, losses caused by diseases are also increasing, reaching hundreds of millions of yuan annually, and posing a significant threat to food and environmental safety. The biggest threat to large yellow croaker currently is bacterial disease (deaths caused by Cryptocaryon irritans are mostly due to secondary bacterial infections, and the use of large net cages has expanded the swimming space of large yellow croakers and improved water exchange inside and outside the cages, greatly reducing the incidence of white spot disease on the body surface). The main bacterial disease of large yellow croaker is visceral white spot disease caused by Pseudomonas splecoglossicida. The disease has no obvious symptoms on the body surface. It is only discovered when dead or dying individuals float to the surface. At this time, most individuals in the net cage are already severely infected, and the diseased fish die one after another. The cumulative mortality rate can reach 50% to 90% or more.

[0003] Currently, the prevention and treatment of white spot disease in large yellow croaker mainly relies on antibiotics, such as enrofloxacin, doxycycline hydrochloride, and florfenicol. However, long-term use of antibiotics easily leads to drug resistance, and the amount of antibiotics used in large yellow croaker is increasing year by year, posing a significant threat to food and environmental safety. Reports of excessive antibiotic levels in fresh large yellow croaker are reported annually. Therefore, there is an urgent need in this field to develop novel methods for the prevention and treatment of white spot disease in large yellow croaker.

[0004] Summary of the Invention

[0005] Garlic E is a broad-spectrum biomimetic bactericide with more than 40 times the antibacterial activity of ordinary allicin. This project conducted research on the prevention and treatment of white spot disease in the viscera of large yellow croaker using garlic E, verifying the efficacy of garlic E in preventing and treating this disease.

[0006] A first aspect of the present invention provides a use of garlic E, characterized in that it is used to prepare a feed composition for reducing or improving *Pseudomonas proteus* infection in large yellow croaker; wherein the garlic E has a structure as shown in the following formula:

[0007] In another preferred embodiment, the feed composition is used to treat or reverse visceral white spot disease caused by Pseudomonas proteus infection.

[0008] In another preferred embodiment, the amount of garlic E in the feed composition is 100-800 mg / kg of feed.

[0009] In another preferred embodiment, the amount of garlic E in the feed composition is 300-500 mg / kg of feed.

[0010] In a second aspect of the invention, the feed composition is also used to downregulate the expression of pro-inflammatory factors in large yellow croaker.

[0011] In another preferred embodiment, the pro-inflammatory factor is selected from the group consisting of IL-1, IL-8, IFN-γ, and TNF-α.

[0012] In another preferred embodiment, the feed composition is also used to promote the healing of inflammation in large yellow croaker.

[0013] The present invention provides a feed composition, characterized in that the feed composition includes garlic E.

[0014] In another preferred embodiment, the content of allicin E in the feed composition is 100-800 mg / kg feed.

[0015] In another preferred embodiment, the amount of garlic E in the feed composition is 300-500 mg / kg of feed.

[0016] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0017] Figure 1 shows the protection rate of garlic E against Pseudomonas proteus infection in large yellow croaker. As shown in Figure 1, the mortality rate of large yellow croaker after being fed experimental feed containing 0 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg of garlic E was recorded.

[0018] Figure 2 shows the spleen bacterial content of large yellow croaker after feeding with garlic E. As shown in Figure 2, A represents the large yellow croaker group not fed with garlic E, and B represents the spleen bacterial content of the large yellow croaker group fed with 400 mg / kg of garlic E.

[0019] Figure 3 shows the pathological changes of garlic E in the spleen and liver of large yellow croaker. As shown in Figure 3, A and C are the spleen and liver of large yellow croaker in the group not fed garlic E, and B and D are the spleen and liver of large yellow croaker in the group fed garlic E 400 mg / kg feed. The red box represents the site of cell necrosis, the blue box represents the site of nodule formation, and the black arrow represents the edematous liver cells.

[0020] Figure 4 shows the effect of allicin E on the expression levels of inflammatory factors in large yellow croaker. As shown in Figure 4, A, B, C, and D represent the mRNA expression levels of IL-1, IL-8, IFN-γ, and TNF-α in large yellow croaker as detected by real-time quantitative PCR, respectively; A: IL-1, B: IL-8, C: IFN-γ, D: TNF-α; # indicates significant difference (n=12, P<0.05).

[0021] Figure 5 shows the MIC experiment results of allicin inhibiting Pseudomonas proteus.

[0022] Figure 6 shows the results of the MBC experiment on the inhibition of Pseudomonas proteus by allicin E. Detailed Implementation

[0023] Through long-term and in-depth research, the inventors discovered that adding garlic E to feed can effectively improve the infection of large yellow croaker with *Pseudomonas proteus*, alleviate edema and inflammation caused by *Pseudomonas proteus* infection, and promote the healing of inflammation caused by *Pseudomonas proteus*. Based on the above findings, the inventors completed this invention.

[0024] Garlic E

[0025] ALE (Allicin E) is a single compound synthesized biomimetically by modifying the structure outside the antibacterial active group of ordinary allicin. Its structure is shown in the following formula:

[0026] Garlic E has broad-spectrum bactericidal and green safety characteristics, and can effectively replace traditional antibiotics, supporting environmental disinfection, animal and plant protection, preservation, and upgrading of the biopharmaceutical industry.

[0027] Feed composition with added garlic E

[0028] This invention provides a feed composition; the feed composition is fortified with garlic E, the garlic E having the structure shown in the following formula:

[0029] Because allicin E has good bactericidal properties, the feed composition described above is used to treat or reverse visceral white spot disease caused by Pseudomonas proteus infection. In a preferred embodiment, the amount of allicin E in the feed composition is 100-800 mg / kg feed, more preferably 300-500 mg / kg feed.

[0030] The feed composition described above can also improve inflammatory responses, for example, by downregulating the expression of pro-inflammatory factors in large yellow croaker, such as those selected from the group consisting of IL-1, IL-8, IFN-γ, and TNF-α. After reversing the expression of inflammatory factors, the feed composition is also used to promote the healing of inflammation in large yellow croaker.

[0031] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0032] Example 1: Effect of Garlic E on the Resistance of Large Yellow Croaker to Pseudomonas Proteus Infection

[0033] The experiment was conducted at the College of Fisheries, Jimei University. 360 healthy large yellow croakers (approximately 5g each) were randomly divided into three gradient treatment groups, with three parallel groups (30 large yellow croakers per group) in each gradient. The groups were fed experimental feed supplemented with 0 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg of allicin, respectively. Three days after feeding the medicated feed, the fish were artificially infected with *Pseudomonas proteus*, and the medicated feed was continued for another 10 days. Mortality rates were recorded. Statistical analysis, as shown in Figure 1, revealed that the mortality rate was lowest in the 400 mg / kg medicated feed group (25.56%), while the mortality rate in the control group (without allicin) was 71.11%, significantly higher than the experimental groups.

[0034] Example 2: Effect of Garlic E on Bacterial Content in the Spleen of Large Yellow Croaker

[0035] The spleens of surviving large yellow croakers from Example 1 were collected, cut into pieces, and rinsed with 1 mL of physiological saline. 100 mL of the rinsing solution was spread on LB agar plates. It was found that the bacterial content in the spleens of surviving large yellow croakers in the control group was still very high, while no bacteria were detected in the experimental group (added amount 400 mg / kg) (Figure 2). This suggests that bacterial growth in the spleens of large yellow croakers was completely inhibited after being added to the feed.

[0036] Example 3: Effects of Garlic E on the Morphology of Spleen and Liver of Large Yellow Croaker

[0037] Spleen and liver of large yellow croaker were collected in Example 1 and pathological sections were prepared, as shown in Figure 3. In the control group, the spleen of the large yellow croaker showed obvious nodules, cysts, and necrotic lesions; while in the experimental group (added at 400 mg / kg), the spleen cells showed normal morphology and distribution. Furthermore, the liver of the control group showed significant edema, while the liver morphology of the experimental group was relatively normal, suggesting that adding garlic E to the feed can effectively reduce necrosis of the liver and spleen tissues in infected large yellow croaker.

[0038] Example 4: Effect of Garlic E on the Expression Levels of Pro-inflammatory Factors in Large Yellow Croaker

[0039] Liver, spleen, and gills of large yellow croaker were collected in Example 1. Real-time quantitative PCR was used to detect the mRNA expression levels of IL-1, IL-8, IFN-γ, and TNF-α in the large yellow croaker, as shown in Figure 4. Compared to the control group without garlic E, the expression levels of IL-1 and IL-8 in the liver, spleen, and gills of the experimental group fed with 400 mg / kg medicated feed were significantly reduced (P<0.05, Figures 4A and 4B); while the expression level of IFN-γ was significantly reduced in the liver and spleen (P<0.05), but remained unchanged in the gills (Figure 4C). The expression level of TNF-α transcript remained essentially unchanged in all organs (Figure 4D). In conclusion, garlic E can significantly reduce the expression levels of pro-inflammatory factors in large yellow croaker and promote the healing of inflammation caused by *Pseudomonas proteus*.

[0040] Example 5: Experiment on the antibacterial and bactericidal activity of allicin against Pseudomonas proteus.

[0041] Garlic E-in was provided by Shanghai Laishen Biotechnology Co., Ltd. *Pseudomonas proteus* (sample number ZDL) was isolated from the liver tissue of a diseased large yellow croaker and identified as *Pseudomonas proteus*. MH medium and agar powder were purchased from Guangdong Huankai Microbial Technology Co., Ltd. 96-well plates were from Corning.

[0042] Remove *Pseudomonas proteus* from the refrigerator and inoculate it onto an MHA plate for activation. Pick a single colony and inoculate it onto MH liquid medium. Culture it to the logarithmic growth phase and determine its bacterial concentration D60 and the corresponding colony number. Calculate the colony number when OD600 = 0.5.

[0043] MIC determination: Collect *Pseudomonas proteus* cultured to the logarithmic growth phase and adjust the bacterial concentration to 1 x 10⁻⁶. 6CFU / mL. Allicin E was diluted with sterile water to 4096 mg / L for later use. Using a 96-well plate method, allicin E was serially diluted 2-fold to final concentrations of 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 mg / L. No drug was added to the control wells; instead, an equal volume of sterile physiological saline was added. Then, *Pseudomonas proteus* was added to each well to a final concentration of 5 x 10⁻⁶. 5 CFU / mL. After incubating at 28°C for 24 hours, observe the bacterial growth and determine the MIC.

[0044] The results of the antibacterial test of garlic E showed that the culture medium with a drug concentration of 16 mg / L was clear, indicating that no bacteria grew in this concentration group. This concentration is the minimum inhibitory concentration, i.e., MIC = 16 mg / L; as shown in Figure 5.

[0045] MBC assay: Take the bacterial culture from the corresponding wells in the drug group where no bacteria grow, spread it on MHA medium, incubate at 28 degrees Celsius for 24 hours, and observe the bacterial growth. The concentration of the drug where no bacteria grow is the MBC.

[0046] MBC: The culture medium was spread onto MHA plates and observed for bacterial growth. The results showed that no bacteria grew in the culture medium with a drug concentration of 16 mg / L, indicating that this concentration was the minimum bactericidal concentration, i.e., MBC = 16 mg / L, as shown in Figure 6.

[0047] In summary, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MCC) of allicin against Pseudomonas proteus are both 16 mg / L.

[0048] *Pseudomonas proteus* is a major pathogen causing white spot disease in large yellow croaker, and its high mortality rate causes serious economic losses to the large yellow croaker aquaculture industry. Previous studies have shown that total saponins of Panax notoginseng (mainly containing ginsenosides Rb1, Rg1, and notoginsenoside R1, among other monomeric saponins) have an inhibitory effect on *Pseudomonas proteus*, but a significant inhibitory effect is only observed when the in vitro antibacterial concentration reaches 25 mg / L or even 50 mg / L. The allicin E of this invention exhibits significantly superior activity against *Pseudomonas proteus* compared to other non-antibiotic active ingredients.

[0049] discuss

[0050] The experimental results showed that the optimal dosage of garlic E in preventing and treating white spot disease of large yellow croaker caused by Pseudomonas proteus was 400 mg / kg of feed. This dosage of garlic E could significantly reduce the mortality rate of large yellow croaker, reduce the bacterial load in the spleen, and alleviate the pathological changes in the spleen and liver of large yellow croaker caused by Pseudomonas proteus. In addition, garlic E could also reduce the expression levels of pro-inflammatory factors IL-1, IL-8 and IFN-γ, thereby reducing the occurrence of inflammation.

[0051] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A use of garlic E, characterized in that, A feed composition for preparing feeds that reduce or improve *Pseudomonas proteus* infection in large yellow croaker; wherein the allicin E has the structure shown in the following formula:

2. The use as described in claim 1, characterized in that, The feed composition is used to treat or reverse visceral white spot disease caused by Pseudomonas proteus infection.

3. The use as described in claim 1, characterized in that, In the feed composition, the amount of garlic E is 100-800 mg / kg of feed.

4. The use as described in claim 1, characterized in that, In the feed composition, the amount of garlic E is 300-500 mg / kg of feed.

5. The use as described in claim 1, characterized in that, The feed composition is also used to downregulate the expression of pro-inflammatory factors in large yellow croaker.

6. The use as described in claim 5, characterized in that, The pro-inflammatory factors mentioned are selected from the following group: IL-1, IL-8, IFN-γ and TNF-α.

7. The use as described in claim 1, characterized in that, The feed composition is also used to promote the healing of inflammation in large yellow croaker.

8. A feed composition, characterized in that, The feed composition includes garlic E.

9. The composition according to claim 7, characterized in that, The feed composition contains 100-800 mg / kg of allicin.

10. The composition according to claim 7, characterized in that, In the feed composition, the amount of garlic E is 300-500 mg / kg of feed.

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