Enterococcus faecalis FS and application thereof

Abstract

The invention provides an enterococcus faecalis FS and application thereof. The enterococcus faecalis FS is preserved in the Guangdong Microbial Culture Collection Center on May 12, 2025, and the preservation number of the enterococcus faecalis FS is GDMCC (China General Microbiological Culture Collection Center) 66318. The novel enterococcus faecalis FS is separated from the intestinal tract of healthy adult largemouth bass, has good acid and alkali resistance, cholate resistance, acid production and antibacterial capacity, is good in biological safety and has no toxic or side effect on fishes. When the enterococcus faecalis FS provided by the invention is added into juvenile fish functional feed as a core component, not only can the growth performance and disease resistance of largemouth bass be improved, but also the use of antibiotics can be reduced, the environmental pollution can be reduced, the aquaculture industry can be promoted to develop towards a high-efficiency, environment-friendly and sustainable direction, and the enterococcus faecalis FS has important application value and market prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Enterococcus faecalis FS and its applications. Background Technology

[0002] As the global aquaculture industry rapidly shifts towards intensive and high-density models, frequent outbreaks of aquatic animal diseases have become a key bottleneck restricting the industry's sustainable development. Intensive aquaculture systems increase production by improving the biocapacity of water units, but the resulting ecological imbalances and pathogen accumulation problems are becoming increasingly prominent. High-density aquaculture leads to environmental stresses such as fluctuations in dissolved oxygen and ammonia nitrogen accumulation, causing a 2-3 fold increase in the proliferation rate of opportunistic pathogens such as *Aeromonas hydrophila* and *Vibrio parahaemolyticus*, triggering large-scale outbreaks of diseases such as enteritis and septicemia. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), aquaculture diseases cause more than US$6 billion in global economic losses annually, with diseases caused by *Vibrio* and *Aeromonas* pathogens accounting for more than 40% of these losses.

[0003] Largemouth bass (Micropterus salmoides) is a freshwater fish with extremely high economic value. Due to its rapid growth, strong adaptability, and delicious flesh, it has become one of the most important species in global aquaculture. In recent years, with the continuous expansion of aquaculture scale, disease problems in largemouth bass have become increasingly prominent, especially the frequent occurrence of bacterial and viral diseases, causing huge economic losses to the aquaculture industry. Traditional disease control methods mainly rely on antibiotics, but the overuse of antibiotics not only leads to increased drug resistance in pathogens but also causes serious harm to the aquatic environment, farmed animals, and human health. Therefore, the development of efficient and environmentally friendly alternative feed additives has become a research hotspot in the field of aquaculture.

[0004] Probiotics, as a novel and environmentally friendly feed additive, have attracted much attention in recent years. Endogenous lactic acid bacteria isolated from the intestines of aquatic animals have become a research focus due to their adaptability to the host and their probiotic potential. Enterococcus faecalis (… Enterococcus faecalis Enterococcus faecalis is a type of lactic acid bacteria, and some strains of Enterococcus faecalis have been used in the existing technology to regulate the intestinal flora of fish. However, the existing Enterococcus faecalis strains have the following significant defects in practical applications: (1) Most strains are isolated from non-host environments or laboratory screening systems, and have insufficient tolerance to the acidic environment (pH 3.0~4.0) and bile salts in the fish intestine, with a survival rate of less than 50%; (2) They have limited acid production capacity and cannot effectively inhibit the colonization of pathogens; (3) They have a narrow antibacterial spectrum and are not effective against Vibrio harveyi ( Vibrio harveyi Aeromonas versicolor ( Aeromonas veronii (4) The inhibitory activity against key pathogens such as ) is not significant; (5) The safety verification is insufficient, and there is a potential risk of hemolysis or toxin secretion.

[0005] Therefore, there is an urgent need to develop a novel Enterococcus faecalis strain that combines strong environmental adaptability, broad-spectrum antibacterial activity, and high biosafety, and to develop its application technology in aquatic pathogen inhibitors and functional feeds for juvenile fish, so as to break through existing technological bottlenecks and promote the transformation of aquaculture towards green and efficient directions. Summary of the Invention

[0006] To address the problems of increased antibiotic resistance in pathogens due to the use of antibiotics in existing aquatic disease control methods, insufficient acid resistance, bile salt resistance, and acid production capacity of existing Enterococcus faecalis strains, narrow antibacterial spectrum, and insufficient biosafety verification, this invention provides a strain of Enterococcus faecalis FS and its applications.

[0007] According to a first aspect of the invention, a strain of Enterococcus faecalis is provided. Enterococcus faecalis FS was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 12, 2025, with accession number GDMCC 66318.

[0008] The inventors of this application isolated a novel strain FS from the intestine of a healthy adult largemouth bass. Its morphological characteristics were observed using colony morphology, cell morphology under an optical microscope, and scanning electron microscopy. The genomic DNA extracted from this strain was amplified and sequenced using universal primers for 16S rRNA gene alignment. BLAST alignment and 16S rRNA gene sequence analysis revealed that strain FS is related to Enterococcus faecalis (…). Enterococcus faecalis The homology with the strain was 99.8%, confirming it as *Enterococcus faecalis*. This *Enterococcus faecalis* FS strain was deposited on May 12, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at Guangdong Institute of Microbiology, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province. It was classified and named... Enterococcus faecalis FS, accession number GDMCC 66318.

[0009] Further systematic research on the biological characteristics, antibacterial ability, and growth-promoting effect of *Enterococcus faecalis* FS provided by this invention revealed the following: First, *Enterococcus faecalis* FS exhibits good acid and alkali resistance and bile salt tolerance, surviving in environments with a pH of 3.0–11.0 and growing well in bile salt concentrations of 0.1–0.4%. Second, *Enterococcus faecalis* FS demonstrates excellent acid-producing ability, producing acetic acid at concentrations as high as 9.1 mg / mL and propionic acid at concentrations as high as 4.0 mg / mL. Applying *Enterococcus faecalis* FS to the preparation of functional feed for juvenile largemouth bass can improve the intestinal health of juvenile largemouth bass. Third, *Enterococcus faecalis* FS exhibits good antibacterial activity, effectively inhibiting *Aeromonas hydrophila* (…). Aeromonas hydrophila ), Vibrio parahaemolyticus ( Vibrio parahaemolyticus ), Vibrio harveyi ( Vibrio Harvey Aeromonas versicolor ( Aeromonas veronii Fourth, Enterococcus faecalis FS has good biosafety and no toxic side effects on fish.

[0010] Adding Enterococcus faecalis FS provided by this invention as a core ingredient to functional feed for juvenile fish can not only improve the growth performance and disease resistance of largemouth bass, but also reduce antibiotic use, reduce environmental pollution, and promote the development of aquaculture towards high efficiency, environmental protection and sustainability. It has important application value and market prospects.

[0011] According to a second aspect of the present invention, the use of the above-mentioned Enterococcus faecalis FS in the preparation of aquatic pathogen inhibitors is provided.

[0012] Preferably, the aforementioned aquatic pathogens include at least one of Aeromonas hydrophila, Vibrio parahaemolyticus, Vibrio harveyi, and Aeromonas vesiculosus.

[0013] Preferably, the above-mentioned aquatic pathogen inhibitor includes at least one of the following: Enterococcus faecalis powder, Enterococcus faecalis fermentation broth, and Enterococcus faecalis fermentation extract.

[0014] According to a third aspect of the present invention, an aquatic pathogen inhibitor is provided, the aquatic pathogen inhibitor comprising the aforementioned Enterococcus faecalis FS.

[0015] Experiments have demonstrated that *Enterococcus faecalis* FS possesses good antibacterial activity. The *Enterococcus faecalis* FS provided by this invention was applied to the preparation of an aquatic pathogen inhibitor, which effectively inhibited *Aeromonas hydrophila* (…). Aeromonas hydrophila ), Vibrio parahaemolyticus ( Vibrio parahaemolyticus ), Vibrio harveyi ( Vibrio Harvey Aeromonas versicolor ( Aeromonas veronii The growth of aquatic pathogenic bacteria strains such as )

[0016] According to a fourth aspect of the present invention, the application of the above-mentioned Enterococcus faecalis FS in the preparation of functional feed for juvenile fish is provided.

[0017] According to a fifth aspect of the invention, a functional feed suitable for juvenile fish is provided, the functional feed comprising the aforementioned Enterococcus faecalis FS.

[0018] Preferably, the amount of Enterococcus faecalis FS added is 10. 8 CFU / g.

[0019] Preferably, the juvenile fish mentioned above include juvenile largemouth bass.

[0020] Frequent disease outbreaks in largemouth bass farming, coupled with antibiotic overuse leading to increasing drug resistance and environmental pollution, highlight the challenges posed by this invention. Enterococcus faecalis FS possesses excellent probiotic properties. Applying this FS to the preparation of functional feed for juvenile fish, and feeding the resulting functional feed to juvenile fish, effectively inhibits Aeromonas hydrophila (…). Aeromonas hydrophila ), Vibrio parahaemolyticus ( Vibrio parahaemolyticus This invention inhibits the growth of pathogens such as [specific pathogens not listed here], significantly improving the growth performance, immunity, and intestinal health of juvenile fish, thus addressing the economic losses and environmental pollution caused by diseases and antibiotic overuse in largemouth bass farming. Furthermore, Enterococcus faecalis FS has no toxic side effects on largemouth bass and exhibits good biosafety, making it a potential candidate strain for probiotic preparations. The functional feed for juvenile fish provided by this invention can replace antibiotics, reduce disease incidence, promote green and environmentally friendly aquaculture, and offer a new solution for the sustainable development of largemouth bass farming. It also provides a green and environmentally friendly feed additive for aquaculture, possessing significant application value.

[0021] Preferably, the above-mentioned functional feed is prepared by the following steps: mixing the Enterococcus faecalis FS with feed raw materials, and then granulating and drying the mixture to obtain the functional feed. Attached Figure Description

[0022] Figure 1 The morphological observation results of Enterococcus faecalis FS provided in Example 1.

[0023] Figure 2 The results of 16S rRNA gene sequence analysis and BLAST alignment of Enterococcus faecalis FS provided in Example 1.

[0024] Figure 3 The reaction results of Enterococcus faecalis FS provided in Example 2 on the Biolog GEN Ⅲ identification plate.

[0025] Figure 4 The growth curve of Enterococcus faecalis FS provided in Example 3 under MRS medium.

[0026] Figure 5 The growth of Enterococcus faecalis FS provided in Example 3 at different pH values.

[0027] Figure 6 The growth of Enterococcus faecalis FS provided in Example 3 under different bile salt concentrations.

[0028] Figure 7 The image shows the growth of Enterococcus faecalis FS provided in Example 5 after 24 hours of culture on Columbia agar blood plates.

[0029] Figure 8The graph shows the protective effect (survival rate curve) of Enterococcus faecalis FS against pathogen infection provided in Example 7. Detailed Implementation

[0030] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1: Isolation, purification and identification of Enterococcus faecalis FS In this embodiment, a novel *Enterococcus faecalis* strain was isolated from the intestine of a healthy adult largemouth bass and named *Enterococcus faecalis* FS. This strain was deposited on May 12, 2025, at the Guangdong Provincial Institute of Microbiology, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province. Enterococcus faecalis FS, with accession number GDMCC No: 66318.

[0032] The main instruments, reagents, experimental methods, and results used in the isolation and identification of Enterococcus faecalis (FS) are as follows: 1. Main instruments and reagents Optical microscope, MRS agar medium, Gram staining kit, universal primers for 16S rRNA gene (27F and 1492R).

[0033] 2. Experimental Methods (1) Separation and purification Healthy adult largemouth bass were taken, and their bodies were cleaned with 75% alcohol. The abdominal cavity was opened with sterile scissors, and the intestines were cut into 5 mL centrifuge tubes and chopped. Sterile deionized water was added, and the mixture was thoroughly homogenized by shaking. The mixture was then centrifuged at 1000 r / min at room temperature for 30 s. The supernatant was collected to obtain a bacterial suspension. The bacterial suspension was diluted using a serial dilution method and spread onto plates containing MRS agar. The plates were then incubated at 37°C for 48 h. Single colonies were then picked for purification and culture to obtain the dominant strain FS (i.e., Enterococcus faecalis FS of this invention).

[0034] (2) Identification The morphological characteristics of the dominant strain FS were observed by Gram staining and optical microscopy. Genomic DNA was extracted, and PCR amplification and sequencing were performed using universal primers for the 16S rRNA gene. The strain classification of the dominant strain FS was determined by BLAST comparison.

[0035] 3. Experimental Results (1) Morphological observation results Enterococcus faecalis (FS) colonies were observed using colony morphology, cell morphology under an optical microscope, and scanning electron microscopy, respectively. The results are as follows: Figure 1 As shown, where, Figure 1 A represents the colony morphology of Enterococcus faecalis FS on agar plates containing MRS. Figure 1 B indicates the Gram staining result of Enterococcus faecalis FS. Figure 1 C represents the observation results of Enterococcus faecalis FS under a scanning electron microscope.

[0036] Depend on Figure 1 The colony morphology observation results of A showed that Enterococcus faecalis FS formed multiple relatively uniform colonies on the culture medium plate. These colonies were round, with neat and smooth edges, raised surfaces, white color, and relatively uniform texture. Moreover, the colonies were relatively dispersed and did not merge with each other, indicating that the strain grew well on MRS agar medium and had a certain degree of independence.

[0037] Depend on Figure 1 The Gram staining results of B indicate that Enterococcus faecalis FS is a Gram-positive coccus. Specifically, the Gram staining result of Enterococcus faecalis FS is purple. Under an optical microscope, the cell morphology of Enterococcus faecalis FS can be observed to be spherical or nearly spherical. The cells are usually arranged in pairs or short chains, which is consistent with the general morphological characteristics of enterococci. After staining, the color contrast of the cells is obvious and the morphology is clearly distinguishable, which is helpful for the morphological identification of this strain.

[0038] Depend on Figure 1 As shown in the scanning electron microscope image of C, the surface details of Enterococcus faecalis FS cells are clearly visible. The cells are spherical or ellipsoidal with relatively smooth surfaces. Some cell surfaces may have some fine protrusions or structures. The cells are close to each other, and some cells have just completed division, showing obvious traces of intercellular connections or septa. In addition, the cell size is in the micrometer range. The above scanning electron microscope images provide intuitive information for the study of the ultrastructure of Enterococcus faecalis FS.

[0039] (2) Results of 16S rRNA gene sequence analysis and BLAST alignment Genomic DNA extracted from the dominant strain FS was amplified by PCR and sequenced using universal primers for the 16S rRNA gene, followed by BLAST alignment. The results are as follows: Figure 2 As shown.

[0040] Depend on Figure 2The 16S rRNA gene sequence analysis showed that the dominant strain FS clustered with Enterococcus faecalis MZ520858, and the homology between the dominant strain FS and Enterococcus faecalis MZ520858 was 99.8%. Based on the 16S rRNA sequence analysis results, the dominant strain FS was identified as Enterococcus faecalis.

[0041] Example 2: Detection of physiological and biochemical characteristics of Enterococcus faecalis (FS) This embodiment aims to detect the physiological and biochemical characteristics of Enterococcus faecalis FS isolated and purified in Example 1. The main instruments and reagents, experimental methods, and experimental results used are as follows: 1. Main instruments and reagents Biolog GEN Ⅲ identification plate, high performance liquid chromatograph (HPLC).

[0042] 2. Experimental Methods The carbon source utilization capacity of Enterococcus faecalis FS was detected using the Biolog GEN III identification plate, and the content of short-chain fatty acids (SCFA) in its metabolites was determined by HPLC.

[0043] 3. Experimental Results The reaction results of Enterococcus faecalis FS on the Biolog GEN Ⅲ identification plate are as follows: Figure 3 As shown. By Figure 3 It is known that Enterococcus faecalis FS can utilize 15 kinds of carbon sources such as dextrin, propionic acid, and acetic acid, and grows well under conditions such as 1-8% NaCl, lincomycin, and vancomycin.

[0044] The content of short-chain fatty acids (SCFAs) in the metabolites of Enterococcus faecalis FS was determined by HPLC, and the results are shown in Table 1. As can be seen from the HPLC results in Table 1, the concentration of acetic acid produced by Enterococcus faecalis FS was 9.1 mg / mL, and the concentration of propionic acid was 4.0 mg / mL.

[0045] The above results indicate that Enterococcus faecalis FS has a good ability to produce short-chain fatty acids. Applying Enterococcus faecalis FS to the preparation of functional feed for juvenile largemouth bass can improve the intestinal health of juvenile largemouth bass.

[0046] Table 1. Content of various short-chain fatty acids produced by Enterococcus faecalis FS

[0047] Example 3: Growth of Enterococcus faecalis (FS) at different pH and bile salt concentrations This embodiment aims to study the growth of Enterococcus faecalis FS isolated and purified in Example 1 under different pH and bile salt concentrations. The main instruments and reagents used, experimental methods, and experimental results are as follows: 1. Main instruments and reagents pH meter, spectrophotometer, constant temperature incubator, bile salts (ox bile salts), MRS culture medium.

[0048] 2. Experimental Methods (1) Growth curve determination: Enterococcus faecalis FS was inoculated into MRS medium and cultured at 37℃. The absorbance OD of the culture medium at a wavelength of 600 nm was measured every 2 hours. 600 Values ​​are used to plot growth curves.

[0049] (2) Acid and alkali resistance test: The pH of the MRS liquid medium was adjusted to 1.0, 3.0, 5.0, 7.0, 9.0, 11.0 and 13.0 using 5 mol / L HCl solution and 5 mol / L NaOH solution, and the pH was measured again after sterilization. Enterococcus faecalis FS culture in the exponential growth phase was collected and centrifuged at 5000 g for 10 min. The precipitate was collected, washed twice with sterile PBS buffer, and resuspended in sterile PBS buffer to obtain a bacterial suspension. The bacterial suspension was inoculated into the pH-adjusted MRS liquid medium at an inoculation rate of 10% (v / v). After culturing on a shaker at 37℃ and 220 r / min for 4 h, samples were taken, the bacterial suspension was diluted to a suitable concentration and spread on plates, and cultured at 37℃ for 36 hours. After h, colony counts were performed and survival rates were calculated. Each experimental group was set up with 3 replicates and Enterococcus faecalis FS cultured in MRS liquid medium at pH=7.0 as the control group.

[0050] (3) Bile salt tolerance test: MRS liquid medium containing 0.0%, 0.1%, 0.2%, 0.3%, and 0.4% (w / v) bovine bile salts was prepared. Enterococcus faecalis FS in the exponential growth phase was collected and centrifuged at 5000 g for 10 min. The precipitate was collected and washed twice with sterile PBS buffer and then resuspended in sterile PBS buffer to obtain a bacterial suspension. The bacterial suspension was inoculated into MRS liquid medium containing different bile salt concentrations at an inoculation amount of 10% (v / v). After culturing on a shaker at 37℃ and 220 r / min for 4 h, samples were taken, the bacterial suspension was diluted to an appropriate concentration and spread on a plate. After culturing at 37℃ for 36 h, colony counts were performed and the survival rate was calculated. Each experimental group was set up with 3 replicates and Enterococcus faecalis FS cultured in MRS liquid medium with a bovine bile salt concentration of 0.0% was used as the control group.

[0051] 3. Experimental Results The growth curve of Enterococcus faecalis FS cultured on MRS medium is as follows: Figure 4 As shown. By Figure 4 It can be seen that Enterococcus faecalis FS enters the logarithmic growth phase after 2 h of culture and enters the stationary phase after 22 h, with the maximum OD value reaching 100%. 600 The value is 1.2.

[0052] The acid and alkali resistance test results of Enterococcus faecalis FS are as follows: Figure 5 As shown. By Figure 5 It can be seen that after culturing for 4 h under different pH conditions, Enterococcus faecalis FS can survive in MRS liquid medium with pH values ​​ranging from 3.0 to 11.0. In the pH range of 3.0 to 9.0, the survival rate of Enterococcus faecalis FS increases with increasing pH value, reaching as high as 186.5% at pH 9.0 (P<0.05). The above results indicate that Enterococcus faecalis FS has a wide pH range of adaptability. However, when the pH value is between 11.0 and 13.0, the survival rate of Enterococcus faecalis FS decreases with increasing pH value.

[0053] The results of the bile salt tolerance test for Enterococcus faecalis FS are as follows: Figure 6 As shown. By Figure 6 It can be seen that Enterococcus faecalis FS strain can survive after treatment under different bile salt concentrations for 4 h. When the bile salt concentration is 0.1%, the survival rate of Enterococcus faecalis FS strain is significantly improved (P<0.05). When the bile salt concentration is 0.2% and 0.3%, the survival rate of FS is still as high as 90.7% and 67.1%, respectively. The above results indicate that Enterococcus faecalis FS strain has a strong bile salt tolerance.

[0054] The above results indicate that Enterococcus faecalis FS has good resistance to acids, alkalis and bile salts.

[0055] Example 4: In vitro antibacterial experiment of Enterococcus faecalis FS This embodiment aims to study the in vitro antibacterial effect of Enterococcus faecalis FS isolated and purified in Example 1. The main instruments and reagents used, experimental methods, and experimental results are as follows: 1. Main instruments and reagents Aeromonas hydrophila ( Aeromonas hydrophila ), Vibrio parahaemolyticus ( Vibrio parahaemolyticus ), Vibrio harveyi ( Vibrio Harvey Aeromonas versicolor ( Aeromonas veronii Four common aquatic pathogen strains, LB medium, and a punch.

[0056] 2. Experimental Methods The above four pathogens were inoculated into LB liquid medium and cultured at 37°C until the logarithmic growth phase. Then, the antibacterial activity of Enterococcus faecalis FS against the above four common aquatic pathogens was determined by the well-drilling method. The culture medium of Enterococcus faecalis FS was centrifuged and the supernatant was added to the wells of agar plates inoculated with aquatic pathogens. After culturing at 37°C for 24 h, the diameter of the inhibition zone was measured.

[0057] 3. Experimental Results The results of the antibacterial activity of Enterococcus faecalis FS against four common aquatic pathogens are shown in Table 2. As can be seen from Table 2, Enterococcus faecalis FS has significant antibacterial activity against Aeromonas hydrophila, Vibrio parahaemolyticus, Vibrio harveyi, and Aeromonas vesiculosus, with inhibition zone diameters of 8 mm, 10 mm, 10 mm, and 9 mm, respectively.

[0058] Table 2. Results of the antibacterial activity of Enterococcus faecalis FS against four common aquatic pathogens.

[0059] Example 5 Biosafety assessment of Enterococcus faecalis FS This embodiment aims to evaluate the biosafety of Enterococcus faecalis FS isolated and purified in Example 1. The main instruments and reagents, experimental methods, and experimental results used are as follows: 1. Main instruments and reagents Juvenile largemouth bass, sterile saline solution.

[0060] 2. Experimental Methods (1) Take a culture of Enterococcus faecalis FS in the logarithmic growth phase and serially dilute it with sterile physiological saline to a concentration of approximately 1.5 × 10⁻⁶. 8 Using a pipette, take 10-100 μL of the adjusted bacterial suspension at CFU / mL and inoculate it onto the surface of a Columbia agar (CNA) plate by spotting or streaking. Then, invert the inoculated Columbia agar plate and incubate it in a 37°C incubator for 24 hours.

[0061] (2) Juvenile largemouth bass were randomly divided into a control group and an experimental group, with 10 fish in each group. The experiment was conducted in triplicate. The juvenile fish in the experimental group were injected intraperitoneally with Enterococcus faecalis FS solution (1×10⁻⁶). 8 The experimental group (CFU / mL) and the control group (equal volume of sterile saline) were observed for 14 consecutive days. The survival rate, behavior and pathological changes of the juvenile fish in the experimental group and the control group were recorded.

[0062] 3. Experimental Results The growth of Enterococcus faecalis FS on Columbia agar blood plates after 24 hours is shown in the following results. Figure 7 As shown. By Figure 7It can be seen that numerous round, white colonies grow on Columbia agar blood plates. The colony surfaces are smooth and the edges are neat. At the same time, the hemolysis phenomenon around the colonies is carefully observed to judge the hemolysis situation. According to the classification criteria of hemolysis type, it was found that no obvious hemolysis ring appeared around these colonies.

[0063] Within 14 days of intraperitoneal injection of Enterococcus faecalis FS solution into juvenile largemouth bass, no fish in the experimental group showed mortality, abnormal behavior, or pathological changes, indicating that Enterococcus faecalis FS has no toxic side effects on largemouth bass and has good biosafety.

[0064] Example 6: Application of Enterococcus faecalis FS in functional feed for juvenile fish This embodiment aims to apply the Enterococcus faecalis FS isolated and purified in Example 1 to the preparation of functional feed for juvenile fish. Specifically, Enterococcus faecalis FS was added to ordinary feed and mixed, then granulated and dried to obtain functional feed for juvenile fish. This functional feed was used to feed juvenile largemouth bass to study the effect of Enterococcus faecalis FS on the growth performance of juvenile largemouth bass. The main instruments and reagents used, experimental methods, and experimental results are as follows: 1. Main instruments and reagents Largemouth bass juveniles, regular feed, functional feed (add 1×10 to regular feed) 8 CFU / g Enterococcus FS), of which the ordinary feed was purchased from Guangdong Haid Group Co., Ltd., and its main components include 700 g / kg fish meal, 200 g / kg corn starch, 30 g / kg fish oil, 20 g / kg mineral premix, 20 g / kg vitamin premix, and 30 g / kg sodium carboxymethyl cellulose.

[0065] 2. Experimental Methods Largemouth bass juveniles were randomly divided into a control group and an experimental group, with 50 fish in each group. The control group was fed with ordinary feed, while the experimental group was fed with functional feed. After 8 weeks of continuous feeding, the weight and length of the largemouth bass juveniles were measured, and the weight gain rate (WGR), visceral index (VSI), hepatosome index (HIS), condition factor (K), and feed conversion ratio (FE) of the largemouth bass juveniles were calculated.

[0066] 3. Experimental Results Table 3. Effects of Enterococcus faecalis (FS) on the growth performance of juvenile largemouth bass.

[0067] Note: If the same letter appears in the data for the same indicator in the control group and the experimental group, it indicates that there is no significant difference between the two groups.

[0068] The effects of Enterococcus faecalis (FS) on the growth performance of juvenile largemouth bass are shown in Table 3.

[0069] Analysis of the data in Table 3: (1) The initial weight of the juvenile fish in the experimental group and the control group was 8.04±0.05 g, indicating that there was no significant difference in the weight of the two groups of largemouth bass at the beginning of the experiment. The final weight of the juvenile fish in the experimental group was 23.33±2.29 g, which was significantly higher than the final weight of the juvenile fish in the control group of 18.44±3.09 g (P<0.05), indicating that adding Enterococcus faecalis FS (functional feed) to ordinary feed can significantly promote the weight gain of juvenile largemouth bass. (2) The weight gain rate of the experimental group juvenile fish (191.63±28.59%) was significantly higher than that of the control group juvenile fish (130.53±38.59%) (P<0.05), further proving that Enterococcus faecalis FS has a promoting effect on the growth of largemouth bass juveniles. (3) The visceral body ratio (i.e., visceral index, VSI) of the experimental group juvenile fish was 6.53±0.71%, and the visceral body ratio of the control group juvenile fish was 6.35±0.63% (P>0.05). There was no significant difference in the visceral body ratio between the two groups, indicating that Enterococcus faecalis FS had little effect on the ratio of visceral organs to body weight of largemouth bass juveniles. (4) The liver-to-body ratio (i.e., liver-to-body index, HSI) of the juvenile fish in the control group was 1.58±0.31%, and the liver-to-body ratio of the juvenile fish in the experimental group was 1.6±0.26%. There was no significant difference in the liver-to-body ratio between the two groups, indicating that Enterococcus faecalis FS had no significant effect on the ratio of liver to body weight of largemouth bass. (5) The condition rating (K) of the juvenile fish in the control group and the experimental group was 2.08±0.2 g / g and 2.18±0.13 g / g, respectively, with no significant difference (P>0.05). This may be because the experimental period was short or the basic feed was nutritious enough, so the effect of Enterococcus faecalis on condition rating had not yet been shown.

[0070] (6) The feed efficiency (FE) of the experimental group of juvenile fish was 47.19±7.04%, which was significantly higher than that of the control group of juvenile fish (37.02±10.95%) (P<0.05). This indicates that the addition of Enterococcus faecalis FS improved the feed utilization efficiency of juvenile largemouth bass and enabled them to convert more feed into their own body weight.

[0071] Example 7: Protective effect of Enterococcus faecalis FS against pathogen infection This embodiment aims to investigate whether the Enterococcus faecalis FS isolated and purified in Example 1 has a protective effect against pathogen infection. The main instruments and reagents used, experimental methods, and experimental results are as follows: 1. Main instruments and reagents Largemouth bass juveniles, Aeromonas hydrophila strain, regular feed, functional feed (add 1×10) 8The feed (CFU / g Enterococcus faecalis FS) was purchased from Guangdong Haid Group Co., Ltd., and its main components include 700 g / kg fish meal, 200 g / kg corn starch, 30 g / kg fish oil, 20 g / kg mineral premix, 20 g / kg vitamin premix, and 30 g / kg sodium carboxymethyl cellulose.

[0072] 2. Experimental Methods Juvenile largemouth bass were randomly divided into a control group and an experimental group, with 10 fish in each group. The experiment was conducted in triplicate. The experimental group was fed with 1×10 8 The control group was fed a normal diet (functional feed) with CFU / g Enterococcus faecalis FS, while the control group was fed a normal diet. After 4 weeks of continuous feeding, the control group was injected intraperitoneally with Aeromonas hydrophila (1×10⁻⁶). 7 (CFU / mL), recording the survival rate and pathological changes of juvenile fish.

[0073] 3. Experimental Results The survival rate curves of juvenile largemouth bass in the control and experimental groups are as follows: Figure 8 As shown, the negative control group represents the group that did not undergo challenge treatment (i.e., did not receive intraperitoneal injection of Aeromonas hydrophila strain) and was not fed probiotics (i.e., did not receive Enterococcus faecalis FS) as a blank reference. The positive control group represents the group that underwent challenge treatment (i.e., intraperitoneal injection of Aeromonas hydrophila strain) but was not fed probiotics. The FS group represents the group that underwent challenge treatment and was fed probiotics (i.e., was fed Enterococcus faecalis FS).

[0074] Depend on Figure 8 It can be seen that the positive control group of largemouth bass juveniles began to die in large numbers from day 3, and all of them died by day 4, verifying the high lethality of the pathogen. The survival rate of the FS group of largemouth bass juveniles was the same as that of the negative control group of largemouth bass juveniles in the early stage of the experiment (days 1-2), both being 100%. Mortality began to occur from day 3, but the mortality rate was significantly slower than that of the positive control group of largemouth bass juveniles. By day 7, the survival rate of the FS group of largemouth bass juveniles was as high as 30%, which was significantly higher than the survival rate of 0% of the positive control group of largemouth bass juveniles.

[0075] The results above show that the survival rate of juvenile largemouth bass in the experimental group was significantly higher than that in the control group (P<0.05), and there were no obvious pathological changes. This indicates that Enterococcus faecalis FS can effectively improve the disease resistance of juvenile largemouth bass. This is mainly because Enterococcus faecalis FS is a probiotic, which can significantly delay the mortality process of fish and reduce the lethal effect of pathogens on fish, indicating that it has the potential to enhance the resistance of fish and protect the health of fish.

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A strain of Enterococcus faecalis ( Enterococcus faecalis FS was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 12, 2025, with accession number GDMCC 66318.

2. The application of Enterococcus faecalis FS as described in claim 1 in the preparation of aquatic pathogen inhibitors.

3. The application of Enterococcus faecalis FS as described in claim 2 in the preparation of aquatic pathogen inhibitors, characterized in that: The aquatic pathogens include at least one of Aeromonas hydrophila, Vibrio parahaemolyticus, Vibrio harveyi, and Aeromonas viride.

4. The application of Enterococcus faecalis FS as described in claim 2 in the preparation of aquatic pathogen inhibitors, characterized in that: The aquatic pathogen inhibitor includes at least one of the following: Enterococcus faecalis powder, Enterococcus faecalis fermentation broth, and Enterococcus faecalis fermentation extract.

5. An inhibitor of aquatic pathogens, characterized in that: The aquatic pathogen inhibitor includes Enterococcus faecalis FS as described in claim 1.

6. The application of Enterococcus faecalis FS as described in claim 1 in the preparation of functional feed for juvenile fish.

7. A functional feed suitable for juvenile fish, characterized in that: The functional feed includes Enterococcus faecalis FS as described in claim 1.

8. The functional feed for juvenile fish as described in claim 7, characterized in that: The amount of Enterococcus faecalis FS added was 10. 8 CFU / g.

9. The functional feed for juvenile fish as described in claim 7, characterized in that: The juvenile fish include juvenile largemouth bass.

10. The functional feed for juvenile fish as described in claim 7, characterized in that, The functional feed is prepared by the following steps: the Enterococcus faecalis FS is mixed with feed ingredients, and then granulated and dried to obtain the functional feed.

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