Two strains of lactic acid bacteria and applications thereof

Abstract

This invention discloses two strains of lactic acid bacteria and their applications. The two strains are *Enterococcus faecium* YY1-1 and *Enterococcus faecalis* YY9-1, deposited at the China Center for Type Culture Collection (CCTCC), with accession numbers CCTCC NO: M 2023685 and CCTCC NO: M 2023686, respectively. The two lactic acid bacteria isolated by this invention exhibit superior probiotic properties and strong safety. When mixed with feed, they can improve the growth performance, intestinal digestive enzyme activity, serum non-specific immune activity, and resistance to *Edwardsiella pneumoniae* infection in yellow catfish. Therefore, these two lactic acid bacteria have the potential to be used as feed additives for aquatic animals and also show broad prospects in the prevention and control of aquatic animal diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to two strains of lactic acid bacteria and their applications. Background Technology

[0002] The Vachelli yellow catfish (Pelteobagrus vachelli) belongs to the order Siluriformes, family Bagridae, and genus Pelteobagrus. It is also known as yellow thorn catfish or yellow wax catfish. Lacking intramuscular bones and possessing a delicious flavor, it is highly popular with consumers, resulting in high market demand and a high price. It has become a premium specialty fish widely promoted in my country in recent years. However, with the expansion of Vachelli's aquaculture scale and the continuous increase in stocking density, bacterial diseases in Vachelli have frequently broken out, causing increasingly prominent disease problems and significant economic losses. Edwardsiella tarda disease, caused by Edwardsiella tarda, is one of the most important diseases affecting the healthy growth of Vachelli, mainly causing "red head disease" or "ascites," with a mortality rate reaching over 90%, severely restricting the development of the Vachelli aquaculture industry. Currently, the use of antibiotics and other drugs remains the primary method for controlling aquatic animal diseases. However, long-term use or overuse of antibiotics can lead to drug-resistant strains, drug residues in water that harm human health, disrupt ecological balance, and pollute water quality, among other side effects. In recent years, antibiotic residues in aquatic products have frequently hindered exports, causing significant economic losses. Therefore, research into antibiotic alternatives is urgently needed.

[0003] Lactic acid bacteria (LAB) is a collective term for bacteria that can produce organic acids from fermentable sugars. Currently, *Lactobacillus* and *Bifidobacterium* are among the most widely used probiotics. Lactic acid bacteria lower the pH of the host's intestinal tract by producing organic acids, compete with pathogens for adhesion sites, and secrete antibacterial and bactericidal substances such as lactobacillusin to inhibit or kill pathogenic microorganisms. Simultaneously, lactic acid bacteria can strengthen the intestinal mucosal immune barrier function and enzyme activity, enhancing the host's immune function, and degrade ammonium and nitrite nitrogen in water bodies to purify water quality. Furthermore, the antibiotics produced by lactic acid bacteria have a broad antibacterial spectrum, are stable, do not harm humans, do not produce drug-resistant strains, and do not pollute water quality, giving them potential advantages for development as probiotics. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a strain of Edwardsiella tarda that prevents diseases in aquatic animals.

[0005] The technical solution of the present invention is: Enterococcus faecium YY1-1, which is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M 2023685.

[0006] The above-mentioned use of Enterococcus faecium YY1-1 in the preparation of drugs for treating aquatic animal diseases caused by Edwardsiella tarda.

[0007] Furthermore, the aquatic animal mentioned is the yellow catfish.

[0008] The above-mentioned application of Enterococcus faecium YY1-1 in promoting the growth of yellow catfish.

[0009] Enterococcus faecalis YY9-1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2023686.

[0010] The above-mentioned use of Enterococcus faecalis YY9-1 in the preparation of drugs for treating aquatic animal diseases caused by Edwardsiella tarda.

[0011] Furthermore, the aquatic animal mentioned is the yellow catfish.

[0012] The above-mentioned application of Enterococcus faecalis YY9-1 in promoting the growth of yellow catfish.

[0013] A compound microbial agent containing the aforementioned Enterococcus faecium YY1-1 and Enterococcus faecalis YY9-1.

[0014] The aforementioned compound microbial agent is used to promote the growth of yellow catfish or in the preparation of drugs for treating aquatic animal diseases caused by Edwardsiella tarda.

[0015] This invention isolated two lactic acid bacteria strains, Enterococcus faecalis and Enterococcus faecium, from the intestine of the yellow catfish (Pelteobagrus vachelli) that exhibit specific antagonistic activity against Edwardsiella tarda. After a series of probiotic characteristic analyses, it was preliminarily determined that both lactic acid bacteria possess the property of secreting proteases and can survive in the acidic, bile salt environment of the intestine. Because they were isolated from the intestine of the yellow catfish, they have the advantage of adapting to the intestinal environment of aquatic animals compared to lactic acid bacteria from other sources. Adding 0.1% concentration of whey protein microencapsulated lactic acid bacteria (Enterococcus faecalis, Enterococcus faecium, and a mixture of both) to the feed can promote the growth of *Pelteobagrus vachelli*, increase the activity of intestinal trypsin, pepsin, amylase, and lipase, and increase the total protein content in the intestine. It also promotes spleen growth in juvenile *Pelteobagrus vachelli*, increases the levels of lysozyme, catalase, glutathione peroxidase, and superoxide dismutase in the serum, while decreasing malondialdehyde levels. Furthermore, it enhances the resistance of juvenile *Pelteobagrus vachelli* to *Edwards tarda* infection. Therefore, these two lactic acid bacteria strains have the potential as feed additives for aquatic animals and also show broad prospects in the prevention and control of aquatic animal diseases.

[0016] When the two strains are combined into a compound bacterial agent, the growth-promoting effect and the ability to resist Edwardsiella tarda are significantly better than those of a single strain, showing a synergistic effect.

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

[0018] The two lactic acid bacteria isolated in this invention exhibit superior probiotic properties and strong safety. When mixed with feed, they can improve the growth performance, intestinal digestive enzyme activity, serum non-specific immune activity, and resistance to Edwardsiella tarda infection in yellow catfish. Therefore, these two lactic acid bacteria have the potential to serve as feed additives for aquatic animals and also show broad prospects in the prevention and control of aquatic animal diseases.

[0019] Preservation information:

[0020] Enterococcus faecium YY1-1 and Enterococcus faecalis YY9-1 were deposited on May 6, 2023, at the China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, with accession numbers CCTCC NO:M 2023685 and CCTCC NO:M 2023686, respectively. Attached Figure Description

[0021] Figure 1 Gram staining results of suspected lactic acid bacteria strains; a: Gram staining effect of strain YY9-1; b: Gram staining effect of strain YY1-1;

[0022] Figure 2Inhibitory effect of the initial screening strains on Edwardsiella tarda;

[0023] Figure 3 Results of protease activity analysis of the initial screening strains;

[0024] Figure 4 The relative survival rate of the initial screening strains after culturing in culture media with different pH values ​​for 1 hour;

[0025] Figure 5 The relative survival rates of each isolated strain after culturing in 10% bile for 5 hours;

[0026] Figure 6 Detection of acid-producing capacity of initial screening strains;

[0027] Figure 7 Results of PCR amplification and phylogenetic analysis of 16S rRNA gene of strains YY1-1 and YY9-1: a. PCR amplification results of 16S rRNA gene sequence of strain YY9-1: 1: marker; 2: DNA sequence of strain YY9-1; b. Phylogenetic tree of 16S rRNA gene sequence of YY9-1; c. PCR amplification results of 16S rRNA gene sequence of strain YY1-1: 1: marker; 2: DNA sequence of YY1-1; d. Phylogenetic tree of 16S rRNA gene sequence of YY1-1.

[0028] Figure 8 Differences in spleen specific gravity among different experimental groups;

[0029] Figure 9 The effect of different experimental group diets on the resistance of yellow catfish to Edwardsiella tarda infection. Detailed Implementation

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial channels.

[0031] 1. Materials and Methods

[0032] 1.1 Experimental Materials

[0033] 1.1.1 Materials Fish

[0034] The healthy yellow catfish were purchased from the Meishan Yellow Catfish Seedling Breeding Base. The average weight was 50g±5g. The body surface was intact during dissection, and there was no congestion or bleeding inside the body. The intestines were normal and there was no food residue.

[0035] 1.1.2 Experimental strains

[0036] The indicator bacterium was Edwardsiella tarda, provided by the Sichuan Provincial Key Laboratory of Fish Resources Conservation and Utilization in the Upper Yangtze River, and stored at -80℃.

[0037] 1.1.3 Main Reagents and Consumables

[0038] Brain-Heart Infusion (BHI) medium was purchased from Beijing Xinjingke Biotechnology Co., Ltd.; MRS medium and MRS broth were purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; PCR reaction system reagents and bacterial DNA extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd.; antimicrobial susceptibility test discs were purchased from Hangzhou Microbial Reagent Co., Ltd.; and Gram staining solution was purchased from Hangzhou Tianhe Biological Reagent Co., Ltd.

[0039] 1.1.4 Test Instruments and Materials

[0040] Pipettes, laminar flow hoods, vertical autoclaves, constant temperature water baths, PCR instruments, electrophoresis apparatus, gel imaging systems, constant temperature shakers, constant temperature biochemical incubators, Oxford cups, McFarland turbidimetric tubes, vernier calipers, low-temperature high-speed centrifuges, vortex mixers, etc.

[0041] 1.2 Experimental Methods

[0042] 1.2.1 Artificial Infection

[0043] Healthy yellow catfish were temporarily held for one week and then randomly divided into six groups (three replicates each for the experimental and control groups), with 20 fish in each group. They were fed 3% of their body weight in commercial feed daily and fasted for 24 hours before artificial infection. Aeration was maintained continuously for 24 hours during the temporary holding and experimental periods, and one-third of the water was changed daily. The concentration of Edwardsiella tarda was adjusted to LD50 (1.2 × 10⁻⁶). 7 The animals were injected intraperitoneally with CFU / mL, with 0.2 mL injected per animal. The control group was injected with the same amount of sterile saline.

[0044] 1.2.2 Isolation and Identification of Intestinal Lactic Acid Bacteria

[0045] (1) Isolation of lactic acid bacteria

[0046] Fourteen days after artificial infection, five surviving *Pelteobagrus vachelli* from each group were selected. After anesthetizing with phenoxyethanol, their intestines were harvested under aseptic conditions in a laminar flow hood. The intestines were dissected using sterile scissors and washed three times in sterile physiological saline to remove any intestinal residue. 0.5 g of the intestinal segment was accurately weighed into a sterile homogenizer, and 4.5 mL of sterile physiological saline was added to prepare a stock solution. After serial dilution, 0.1 mL of the stock solution was spread onto MRS agar medium containing 1% CaCO3. After incubation at 28°C for 24–48 h, single colonies with a clear calcium dissolution ring were picked and re-inoculated onto fresh MRS agar medium for purification. After purification, Gram staining was performed. Bacteria that stained positive were tested using oxidase test strips, and all strains showing positive results in both tests were retained for future use.

[0047] (2) Molecular biological identification

[0048] Bacterial DNA was extracted and isolated using the Tiangen Bacterial Genomic DNA Extraction Kit (DP302), and PCR amplification was performed using universal 16S rRNA primers. The upstream primer 5′-AGAGTTTGATCCTGGCTCAG-3′ and the downstream primer 5′-CGTCCTGGCAACAAAGGACAG-3′ were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The total reaction volume was 50 μL, including 25 μL of Taq PCR Master mix, 2 μL of each primer, 19 μL of DNase and RNase-free water, and 2 μL of DNA template. PCR amplification conditions were: pre-denaturation for 5 min (94℃); denaturation for 30 s (94℃), annealing for 30 s (55℃), extension for 1 min 30 s (72℃), 30 cycles; extension for 5 min (72℃); and final storage at 12℃. The amplified products were detected by electrophoresis using a 1% agarose gel. Successfully amplified PCR products were sent to Chengdu Qingke Zixi Biotechnology Co., Ltd. for sequencing. The sequencing results were compared using BLAST in NCBI.

[0049] 1.2.3 Screening of antagonistic bacteria against pathogens

[0050] Following the method of Hjelm et al., the Oxford cup method was used to determine the antagonistic ability of lactic acid bacteria against Edwardsiella tarda. The isolated and purified lactic acid bacteria and Edwardsiella tarda were revived and cultured for 24 h, and the concentration was adjusted to 1 × 10⁻⁶ using McFarland turbidimetric tubes. 6CFU / mL. Spread 100 μL of Edwardsiella tarda suspension onto a BHI agar plate. After air-drying, use sterile forceps to place a sterile Oxford cup (7.8 mm outer diameter, 6 mm inner diameter) on the plate and gently press to ensure the bottom of the cup is firmly attached to the culture medium. Add 100 μL of lactic acid bacteria suspension to each Oxford cup. Then incubate the plates at 28°C for 24 hours. Measure the diameter of the inhibition zone (including the Oxford cup diameter) using calipers. Each strain was repeated three times, with one negative control group (where the lactic acid bacteria were replaced with sterile double-distilled water).

[0051] 1.2.4 Analysis of the Probiotic Characteristics of Isolated Lactic Acid Bacteria

[0052] 1.2.4.1 Enzyme Activity Analysis

[0053] Using MRS as the basal medium, and following the method described in the literature, 8% skim milk powder, 1% Tween-80, and 1% starch were added to prepare protease, lipase, and amylase test media, respectively. Following the method of Liu Guanbin et al., sterile perforators were used to punch holes, and 100 μL of medium cooled to 60°C was injected into each hole, sealing the bottom. After the medium in the holes had completely solidified, the concentration of probiotics cultured overnight was adjusted to 1 × 10⁻⁶ using McFarland turbidimetric tubes. 6 CFU / mL, add 50 μL of lactic acid bacteria solution to the wells of the prepared enzyme activity detection medium, and incubate at 28℃ for 24 h. Check for the formation of a clear ring around the wells. Perform three replicates for each strain, and one negative control. For the negative control, replace the lactic acid bacteria with sterile double-distilled water.

[0054] 1.2.4.2 Drug sensitivity test

[0055] Antimicrobial susceptibility testing was performed using the disk diffusion method. The initially screened strains were cultured overnight at 28°C on MRS agar. Single colonies were then inoculated into MRS broth and cultured at 120 rpm for 24 hours at 28°C. The bacterial concentration was then adjusted to 1 × 10⁻⁶ using 0.75% physiological saline and a McFarland turbidimetric tube. 8 CFU / mL, take 200μL and spread it evenly on MRS agar medium using a sterile spreader. After air drying, use sterile forceps to flatly place the drug sensitivity paper onto the medium. Incubate upside down at 28℃ for 24h. Measure the diameter of the inhibition zone using a vernier caliper and determine the drug sensitivity range of the lactic acid bacteria according to the instructions.

[0056] 1.2.4.3 Acid resistance test

[0057] Following the method of Jiang Qihuan et al., lactic acid bacteria were inoculated into 150 mL of MRS broth culture medium and cultured on a shaker at 28°C for 24 h. The concentration was then adjusted to 1 × 10⁻⁶. 6CFU / mL. Lactic acid bacteria suspensions were inoculated at a ratio of 1:20 into MRS broth at pH 3.0, 4.0, and 5.0, respectively. After incubation at 28°C and 120 rpm for 1 h on a shaker, 100 μL of each suspension was plate-spread onto MRS agar plates for plate counting. The relative survival rate was calculated (relative survival rate (%) = CFU / mL). α ÷CFU MRS )×100%. CFU α : Viable bacterial count (CFU) after 1 hour of treatment with culture media at different pH values MRS The number of viable bacteria after 1 hour of incubation in MRS liquid medium. The experiment was independently repeated three times.

[0058] 1.2.4.4 Anticholic Ability Test

[0059] The bile resistance of the initial screening strains was tested according to the method of Estefanía Muoz-Atienza et al. Bile from *Pelteobagrus vannamei* was collected under aseptic conditions. Lactic acid bacteria suspensions were prepared according to method 1.2.4.3. Inoculation was performed at a rate of 10% (V / V) in MRS broth with bile contents of 0%, 3%, 5%, and 10% (V / V), respectively. After incubation at 28°C for 0 h, 1 h, 3 h, and 5 h, the viable bacteria count in each treatment group was determined using the plate count method, and the relative survival rate was calculated. Relative survival rate (%) = (CFU) / (Volume of bacteria in each treatment group). α ÷CFU 0h ×100%. That is, the number of viable bacteria in the corresponding bile concentration treatment group after the corresponding treatment time divided by the number of viable bacteria in the bile concentration group after 0 h of treatment. Each treatment group was set up with three independent replicates to observe its tolerance to bile salts.

[0060] 1.2.4.5 Acid production capacity test

[0061] Prepare a lactic acid bacteria suspension according to method 1.2.4.3. Inoculate 1 mL into 100 mL of MRS broth culture medium and incubate at 28°C and 120 rpm on a shaker. Measure the pH of each lactic acid bacteria culture medium using a pH meter at 0 h, 2 h, 4 h, 6 h, 12 h, 24 h, and 48 h to determine the acid-producing capacity of the isolated lactic acid bacteria.

[0062] 1.2.5 Security Testing

[0063] 1.2.5.1 Hemolytic Analysis

[0064] Prepare rabbit or fish blood MRS agar medium. After sterilizing the MRS medium, cool it to 50–60°C, add 8% fresh rabbit blood (or blood from healthy yellow catfish), cool, punch holes, and seal the bottom. Incubate the probiotics overnight, then adjust the concentration to 1 × 10⁻⁶ using McFarland turbidimetric tubes. 6CFU / mL, add 50μL of probiotics to each well of a blood agar plate and observe for any hemolytic reaction.

[0065] 1.2.5.2 Artificial Infection Experiment

[0066] Lactic acid bacteria were cultured statically on MRS agar medium at 28°C for 24 hours. After washing the bacteria with physiological saline, the concentration was adjusted to 1×10⁻⁶. 9 CFU / mL. The experiment was set up with four groups: one positive control, two experimental groups, and one negative control. Each group consisted of 10 yellow catfish (10g ± 2g). Each experimental group received an intraperitoneal injection of 0.1mL of bacterial culture, while the positive control received an intraperitoneal injection of 0.1mL of *Vibrio mimicus* derived from yellow catfish preserved in our laboratory (concentration 1×10⁻⁶). 9 (CFU / mL), with the negative control group receiving an intraperitoneal injection of 0.1 mL of 0.9% physiological saline. The water temperature was maintained at 25℃ throughout the experiment, with ample oxygen supply. The health status of the yellow catfish in each experimental group was observed daily for 7 consecutive days. On the 7th day, the surviving fish were dissected to observe for any lesions in their internal organs.

[0067] 1.2.6 Effects of two strains of lactic acid bacteria on intestinal digestive function and non-specific immunity in yellow catfish (Pelteobagrus vachelli).

[0068] 1.2.6.1 Feed Preparation

[0069] The basic feed was compound feed (expanded feed) for yellow catfish from Sichuan Meishan Pingchun Feed Co., Ltd., with the following nutrient composition by mass fraction: crude protein 43%, crude fat 12%, crude fiber 3%, crude ash 10%, moisture 8%, calcium 2.0%, and total phosphorus 1.6%.

[0070] Following the method of Heidebach et al., whey protein microcapsules were prepared. A 15% concentration of whey protein was prepared, magnetically stirred for 2 hours, and then the pH was adjusted to 7.0 with 5 mol / L NaOH and stirred overnight at 4°C. 2 mL of concentrated bacterial culture (1×10⁻⁶) was then added... 10After mixing 28 mL of the above whey protein solution with CFU / mL, TGase (10 U / g whey protein) was quickly added and vortexed to disperse it evenly. 150 mL of soybean oil was added to a sterile 250 mL Erlenmeyer flask and preheated to 40 °C. The bacterial protein mixture was then added to the flask, and the mixture was stirred at 900 rpm at 50 °C for 180 min. After the droplets were converted into gel particles by the enzyme, the mixture was centrifuged at 500 × g for 1 min, and the microcapsule particles were collected and washed twice with 100 mL of sterile saline. The mixture was then centrifuged at 1200 × g for 5 min, and the colloidal particles were collected again and stored at 4 °C for later use. The prepared microcapsule bacterial particles were placed in a gelatinized starch slurry (5 g starch dissolved in 70 mL of pure water) and stirred at 1200 rpm for 5 min. The starch slurry containing the bacterial microcapsule particles was then thoroughly mixed with the basic feed (the final bacterial concentration in the feed was 1 × 10⁻⁶). 10 (CFU / Kg), air-dried and packaged, stored at 4℃ for later use.

[0071] 1.2.6.2 Experimental Fish Rearing and Management

[0072] The experiment was conducted in an outdoor cement tank (2m×1.5m×1.5m) with 24-hour continuous aeration in the Sichuan Provincial Key Laboratory for the Protection and Utilization of Fish Resources in the Upper Yangtze River. Aerated groundwater was used for the experiment. The experimental fish were purchased from the local Washi Yellow Catfish fry base in Neijiang. Healthy, disease-free juvenile Yellow Catfish of uniform size with an initial body weight of (5.33±0.05) g were fed a basal diet and three experimental diets, designated as the control group, the Enterococcus faecalis group (YY1-1), the Enterococcus faecalis group (YY9-1), and the mixed bacteria group (YY9-1 + YY1-1 mixed at a 1:1 ratio, with the final concentration in the feed consistent with the single-bacterial groups, abbreviated as HH). Feeding was conducted daily at 8:00 AM and 6:00 PM, with a feed amount of 3% of body weight, for 14 days. During the experiment, the water was exposed to natural light, with a temperature of 25–28℃, dissolved oxygen >5 mg / L, pH 7.8–8.2, ammonia nitrogen ≤0.2 mg / L, and nitrite ≤0.02 mg / L.

[0073] 1.2.6.3 Sample Collection and Measurement

[0074] 1.2.6.3.1 Sample Collection

[0075] After the aquaculture experiment, fish were withheld from feed for 24 hours. Ten fish from each of the control group and the lactic acid bacteria-added group were randomly collected and anesthetized with ethylene glycol phenyl ether. After anesthesia, body length, weight, and spleen weight were measured. Blood was drawn from the tail vein using a 1ml syringe (sterile), and then combined and placed in a sterile EP tube. After storage at 4°C for 12 hours, the supernatant was obtained by centrifugation at 3000r / min for 10 minutes. The supernatant (serum) was aliquoted into new, numbered sterile EP tubes and stored at -80°C for later use in the detection of serum immunoenzyme activity.

[0076] 1.2.6.3.2 Measurement of growth indicators

[0077] Growth indicators were measured for specific growth rates, weight gain rates, and spleen-to-body ratios, using the following formulas:

[0078] Specific growth rate (SGR) = (lnWt - lnW0) / t × 100%,

[0079] Weight gain rate (WGR) (%) = 100 × (Wt – W0) / W0

[0080] In the formula: W0 and Wt are the average initial body weight and average final body weight (g); t is the experimental time (d).

[0081] Spleen-to-body weight ratio / % = Spleen weight (g) / Body weight (g) × 100.

[0082] 1.2.6.3.3 Determination of intestinal digestive enzyme levels

[0083] After the aquaculture experiment, fish were starved for 24 hours. Five yellow catfish were randomly selected from each experimental group, euthanized, and their foreguts were quickly dissected on an ice tray. Intestinal contents and fat masses were removed, and the fish were washed with ice-cold saline. After being blotted dry with filter paper, the fish were weighed (g) and shredded. Physiological saline was added at a ratio of 1:9, and the mixture was homogenized in an ice-water bath to prepare a 10% tissue homogenate. The homogenate was centrifuged at 2500 r / min for 10 min at 4°C, and the supernatant (crude enzyme solution) was carefully aliquoted and stored at 4°C for testing. The activities of pepsin, trypsin, lipase, amylase, and total protein content were measured using kits from Nanjing Jiancheng Bioengineering Institute: pepsin assay kit, trypsin assay kit (UV colorimetric method), lipase (LPS) assay kit, α-amylase (AMS) test kit, and total protein (TP) assay kit. Enzyme activity was measured in U·mgpro. -1 or U·gpro -1 .

[0084] 1.2.6.3.4 Serum Immune Index Measurement

[0085] The activity indicators of serum glutathione peroxidase, lysozyme, catalase, malondialdehyde, and superoxide dismutase were determined using kits from Nanjing Jiancheng Bioengineering Institute.

[0086] 1.2.6.3.5 Disease Resistance Test

[0087] Edwardsiella tarda used in the experiment was obtained from the Sichuan Provincial Key Laboratory of Fish Resource Conservation and Utilization in the Upper Yangtze River. The bacterial strain was cultured on BHI agar medium in a bacterial incubator at 28℃ for 24 h, followed by two rejuvenation cycles. The bacteria were then washed with sterile physiological saline, and the cells were collected. The cells were centrifuged at 4000 rpm for 15 min, and the precipitated bacterial cells were collected and serially diluted to a concentration of 50-fold LD50 (5.7 × 10⁻⁶). 8 After (CFU / mL), the samples were stored at 4℃ for later use. After the culture experiment, 20 fish from each group were challenged with the virus. Each fish was injected intraperitoneally with 0.2 ml of Edwardsiella tarda solution. After the injection, each group continued to be fed the previous experimental feed. After 7 days of experiment, the cumulative number of dead fish in each experimental group was counted and the survival rate was calculated.

[0088] Mortality (%) = 100 × (N0 – N) t ) / N0

[0089] In the formula: N0, N t These represent the initial number of fish and the final number of fish, respectively.

[0090] 1.2.7 Experimental Data Processing

[0091] Experimental data were analyzed using Microsoft Excel 2019 and IBM SPSS Statistics 19, and the results are expressed as mean ± standard deviation.

[0092] 2 Experimental Results

[0093] 2.1 Isolation of lactic acid bacteria strains

[0094] Fourteen days after intraperitoneal injection of Edwardsiella tarda into yellow catfish, intestinal tissue homogenate was collected and cultured on MRS agar medium containing 1% calcium carbonate. Gram-positive bacteria with a distinct calcium-dissolving ring were isolated from the intestines of yellow catfish. Figure 1 22 suspected lactic acid bacteria strains that were catalase negative.

[0095] 2.2 Inhibitory effect of isolated strains on indicator bacteria

[0096] The inhibitory spectrum of 22 suspected lactic acid bacteria strains against Edwardsiella tarda was determined using the Oxford cup method, and the results are shown in Table 1. Four strains with significant inhibitory effects on Edwardsiella tarda were screened out, namely YY1-1, YY9-1, YY10-1, and 56-1. Figure 2 In the figure, 'a' represents the inhibitory effect of strains YY1-1, YY9-1, 56-1, 4-11, and YY10-1 on the indicator bacteria. It can be seen that the antibacterial effects of YY9-1, YY10-1, and 56-1 are very significant. Figure 2 In the figure, b represents the inhibition range of YY1-1, YY9-1, YY10-1, and 56-1 against Edwardsiella tarda. Among them, YY9-1, YY10-1, and 56-1 showed the best antibacterial effects, reaching 18.55±1.57 mm, 18.81±0.75 mm, and 18.87±0.40 mm, respectively; YY1-1 had an inhibition diameter of 18.04±1.11 mm, indicating a strong inhibitory effect.

[0097] Table 1. Inhibition diameters of 22 suspected lactic acid bacteria strains against Edwardsiella tarda.

[0098]

[0099] 2.3 Analysis of the beneficial characteristics of the isolated strains

[0100] YY10-1, YY9-1, YY1-1, and 56-1, which have strong antibacterial activity, were selected for probiotic characteristic analysis.

[0101] 2.3.1 Initial screening of lactic acid bacteria enzyme activity analysis

[0102] The isolated strains were inoculated into protease, amylase, and lipase test media and cultured at 28°C for 24 hours. The enzyme activity results of the initial screening strains are shown in Table 2. It can be seen that strains YY10-1, YY9-1, YY1-1, and 56-1 were positive for protease, but negative for lipase and amylase. Based on this, it can be preliminarily determined that strains YY10-1, YY9-1, YY1-1, and 56-1 possess the property of secreting proteases.

[0103] Table 2 Enzyme activities of initially screened strains

[0104]

[0105]

[0106] Note: "—" indicates the absence of a transparent ring.

[0107] 2.3.2 Drug sensitivity test

[0108] Drug susceptibility testing was conducted on the four initially screened strains using the paper disc method. After incubation at 28℃ for 24 hours, the diameter of the inhibition zone for each drug was measured to determine the susceptibility. The results are shown in Table 3. It can be seen that among the four isolates, YY1-1 and YY9-1 were sensitive to commonly used antibiotics such as enrofloxacin, doxycycline, and florfenicol; while strain YY10-1 showed resistance to all commonly used antibiotics except doxycycline; and strain 56-1 showed intermediate resistance to doxycycline and florfenicol, but was sensitive to enrofloxacin.

[0109] Table 3 Drug susceptibility of initially screened strains

[0110]

[0111]

[0112] Note: R stands for "resistance", I for "intermediate", and S for "sensitive".

[0113] 2.3.3 Acid resistance test

[0114] The four bacterial strains were inoculated at a ratio of 1:20 into MRS broth medium with pH values ​​of 3.0, 4.0, and 5.0, respectively, and cultured at 28°C for 1 hour. The counting results are shown in Table 4. Figure 4 It can be seen that strains 56-1 and YY10-1 cannot grow or their numbers are significantly reduced under pH=3 conditions. The relative survival rates of strains YY9-1 and YY1-1 after 1 hour of cultivation at pH 3 reached 60% and 43.6%, respectively. YY9-1 and YY1-1 exhibit good acid resistance.

[0115] Table 4. Tolerance of the initially screened strains to acidic environments.

[0116]

[0117] Note: "—" indicates no bacterial growth.

[0118] 2.3.4 Anti-cholesterol ability test

[0119] Four initially screened bacterial strains were cultured in MRS broth containing 0%, 3%, 5%, and 10% *Pelteobagrus vachelli* bile at 28°C for 0 h, 1 h, 3 h, and 5 h, respectively. 100 μL of each culture was then plated onto MRS agar medium for counting. The results are shown in Table 5. Figure 5 As shown in the table, all four initially screened strains were able to survive in the presence of yellow catfish bile. Strains YY1-1 and YY9-1 still maintained a bacterial count of 2.06 × 10⁻⁶ after culturing in MRS broth with a bile concentration of 10% for 5 hours. 3 CFU / mL, 1.02×10 3The viable count of CFU / mL was 100%, and the survival rate continued to increase with the extension of culture time, indicating strong resistance to bile. Although the viable count of strains YY10-1 and 56-1 also increased with the extension of time under the same conditions, the viable count was only 6.14 × 10⁻⁶. 2 CFU / mL, 7.16×10 2 The relative survival rates were 84.34% and 80.81% for CFU / mL, respectively, indicating that the anti-bile ability was weaker than the former two.

[0120] Table 5. Growth of the initially screened strains under different bile concentrations (CFU / mL)

[0121]

[0122] 2.3.5 Determination of acid production capacity

[0123] Strains YY1-1, YY9-1, YY10-1, and 56-1 were inoculated into MRS broth medium, and the pH of the culture medium was measured at 0h, 1h, 3h, 6h, 9h, 12h, 24h, 48h, and 72h, respectively. The experimental results are shown below. Figure 6 Of the four isolated strains, strain YY9-1 exhibited the strongest acid-producing capacity, with its pH dropping to 5.3 within 24 hours; the pH of strain YY1-1's culture medium also decreased to 5.3 within 48 hours. In contrast, strains 56-1 and YY10-1 showed a slower pH decrease, eventually remaining at 5.4–5.5.

[0124] 2.4 Safety Testing

[0125] 2.4.1 Hemolytic Analysis

[0126] After inoculating the isolated strains onto rabbit blood BHI agar and yellow catfish blood BHI agar, and incubating them at 28℃ for 24 h, the solubility of the strains on rabbit blood and yellow catfish blood was observed. The experimental results are shown in Table 6. It can be seen that the four initially screened strains showed no solubility in yellow catfish blood, exhibiting γ-hemolysis (non-hemolysis); while on rabbit blood agar plates, they produced slight grass-green hemolysis, exhibiting α-hemolysis (incomplete hemolysis).

[0127] Table 6. Solubility of the initial screening strains in rabbit and fish blood.

[0128]

[0129] Note: α stands for "incomplete hemolysis", γ stands for "non-hemolysis".

[0130] 2.4.2 Artificial Infection Experiment

[0131] Based on the above probiotic characteristics analysis, YY1-1 and YY9-1 were selected for safety analysis. The bacterial cultures were cultured at a rate of 1×10⁻⁶. 9 After intraperitoneal injection of 0.1 mL CFU / mL into *Pelteobagrus vachelli*, the survival rates of each experimental group were observed for 7 days, as shown in Table 7. It can be seen that the survival rates of experimental groups YY1-1 and YY9-1 reached 100%, the survival rate of the positive control was 0%, and the survival rate of the negative control was 100%. This indicates that the mortality rates of experimental groups YY1-1 and YY9-1 were within the normal range. Necropsy of the deceased *Pelteobagrus vachelli* in the experimental groups revealed no congestion, hemorrhage, or ulceration on the body surface; the gills were bright red and normal; and the internal organs were normal.

[0132] Table 7. Safety test results of the screened strains against yellow catfish (Pelteobagrus vachelli).

[0133]

[0134] 2.5 PCR amplification and phylogenetic analysis of 16S rRNA genes in strains YY1-1 and YY9-1

[0135] After extracting genomic DNA from YY1-1 and YY9-1, which exhibited the best probiotic properties, PCR amplification of the 16S rRNA gene yielded a target fragment of approximately 1500 bp for each strain. The PCR amplification products were sent to Chengdu Qingke Zixi Biotechnology Co., Ltd. for sequencing. The sequence lengths obtained were 1455 bp (SEQ ID No. 1) and 1448 bp (SEQ ID No. 2), respectively. Blast alignment of the 16S rRNA gene sequences of the two strains was performed using NCBI. The results showed that YY1-1 had the highest homology with Enterococcus faecium, and YY9-1 had the highest homology with Enterococcus faecalis. A phylogenetic tree constructed from the 16S rRNA gene sequences of the strain with the highest homology in the Blast alignment and other Enterococcus species showed that YY1-1 clustered with Enterococcus faecium, and YY9-1 clustered with Enterococcus faecalis. Figure 7 Based on the morphological, physiological and biochemical characteristics, and 16S rRNA gene sequence homology analysis of the strains, YY1-1 was identified as Enterococcus faecium, and YY9-1 as Enterococcus faecalis.

[0136] Enterococcus faecium YY1-1 and Enterococcus faecalis YY9-1 were deposited on May 6, 2023, at the China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, with accession numbers CCTCC NO:M 2023685 and CCTCC NO:M 2023686, respectively.

[0137] 2.6 Effects of adding probiotics YY1-1 and YY9-1 to feed on the growth of yellow catfish (Pelteobagrus vachelli)

[0138] There were no significant differences in the initial average body length and weight of the yellow catfish among the groups (P > 0.05). After a two-week breeding experiment, as shown in Table 8, the addition of compound probiotics to the feed had varying degrees of effect on the body length, weight, specific growth rate, and weight gain rate of the yellow catfish. The main effect was that in the short term, the mixed treatment group significantly increased the growth of body length, weight, specific growth rate, and weight gain rate of the yellow catfish compared with the other three groups (P < 0.05). There were no significant differences between the YY1-1 treatment group and the YY9-1 treatment group (P > 0.05), but both were significantly higher than the control group (P < 0.05).

[0139] Table 8 Growth Indicators of Yellow Catfish

[0140]

[0141] Note: Different lowercase superscript letters in the same row indicate significant differences (P < 0.05).

[0142] 2.7 Effects of adding probiotics YY1-1 and YY9-1 to feed on intestinal digestive enzymes in yellow catfish (Pelteobagrus vachelli)

[0143] The effects of adding compound probiotics to the feed on intestinal digestive enzymes in *Pelteobagrus vachelli* are shown in Table 9. The intestinal trypsin activity of the mixed-treatment group was significantly higher than that of the control group, YY1-1, and YY9-1 (P < 0.05). There was no significant difference in trypsin activity between the control group and the YY1-1 and YY9-1 treatment groups (P > 0.05). The pepsin activity of the mixed-treatment group and the YY1-1 treatment group was significantly higher than that of the control group (P < 0.05); the pepsin activity of the YY9-1 treatment group was higher than that of the control group, but lower than that of the mixed-treatment group and the YY1-1 treatment group, but the difference was not significant (P > 0.05). The total intestinal protein content of the mixed-treatment group was significantly higher than that of the control group, YY1-1, and YY9-1 (P < 0.05). There was no significant difference in total intestinal protein content between the control group and the YY1-1 and YY9-1 treatment groups (P > 0.05). The intestinal lipase activity of the mixed-treatment group of *Pelteobagrus vachelli* was significantly higher than that of the control group, the YY1-1 treatment group, and the YY9-1 treatment group (P < 0.05). There was no significant difference in intestinal lipase activity between the YY1-1 and YY9-1 treatment groups (P > 0.05), but both were significantly higher than the control group (P < 0.05) and significantly lower than the mixed-treatment group (P < 0.05). The intestinal amylase activity of the mixed-treatment group of *Pelteobagrus vachelli* was significantly higher than that of the control group, the YY1-1 treatment group, and the YY9-1 treatment group (P < 0.05). There was no significant difference in intestinal amylase activity between the YY1-1 and YY9-1 treatment groups (P > 0.05), but both were significantly higher than the control group (P < 0.05) and significantly lower than the mixed-treatment group (P < 0.05).

[0144] Table 9. Effects of adding compound probiotics to the feed on intestinal digestive enzymes in yellow catfish.

[0145]

[0146] Note: Different lowercase superscript letters in the same row indicate significant differences (P < 0.05).

[0147] 2.8 Effects of adding probiotics YY1-1 and YY9-1 to feed on non-specific immune indicators of yellow catfish (Pelteobagrus vachelli)

[0148] 2.8.1 Results of spleen-to-body ratio measurement

[0149] After 14 days of feeding, the body weight and spleen weight of the juvenile fish were dissected and measured. Calculations showed that the spleen-to-body ratio of the lactic acid bacteria-added group was significantly higher than that of the control group (P < 0.05). Figure 8 The HH group had the highest value, showing a significant difference from the control group.

[0150] 2.8.2 Effects of different experimental group diets on serum immunity and antioxidant indicators of yellow catfish (Pelteobagrus vachelli)

[0151] Compared with the control group, serum lysozyme activity increased to varying degrees in all other groups (Table 10). The HH group showed a 58.08% increase compared to the control group (P < 0.05), while the YY1-1 and YY9-1 groups showed a 51.16% increase compared to the control group, with significant differences (P < 0.05). Serum catalase activity was highest in the YY9-1 group, followed by the HH group, both significantly higher than the control group (P < 0.05). Glutathione peroxidase activity was significantly higher in the lactic acid bacteria-added groups than in their control group (P < 0.05). Serum superoxide dismutase activity was significantly lower in the control group than in the lactic acid bacteria-added groups (P < 0.05), while there were no significant differences among the other lactic acid bacteria-added groups (P > 0.05). Except for the control group, serum malondialdehyde (MDA) levels were significantly decreased in all added groups, with the YY9-1 group showing the largest decrease (P < 0.05).

[0152] Table 10 Effects of different experimental group diets on serum immunity and antioxidant indicators of yellow catfish.

[0153]

[0154] Note: Different lowercase superscript letters in the same row indicate significant differences (P < 0.05).

[0155] 2.8.3 Effects of different experimental diets on the resistance of yellow catfish to Edwardsiella tarda infection.

[0156] Seven days after injection of Edwardsiella tarda, sporadic deaths of yellow catfish began to occur in the initial experimental group. As the infection time increased, the number of surviving yellow catfish decreased significantly, and the mortality rate began to rise rapidly. The mortality rate of the control group increased the most, followed by the YY1-1 group, then the YY9-1 group, and the HH group had the most survivors.

[0157] In the later stages, except for the control group, the mortality rate in the other lactic acid bacteria-added groups decreased and tended to stabilize. Seven days after infection, the survival rate of yellow catfish in all groups was higher than that in the control group, with the HH group showing the highest survival rate, significantly higher than the other groups. At this point, the survival rate of the control group dropped to 100%, and all the experimental fish died. Figure 9 ).

Claims

1. A compound microbial agent containing Enterococcus faecalis ( Enterococcus faecium YY1-1 and Enterococcus faecalis ( Enterococcus faecalis YY9-1, Enterococcus faecalis ( Enterococcus faecium YY1-1 and Enterococcus faecalis ( Enterococcus faecalis The ratio of YY9-1 is 1:1; the Enterococcus faecalis ( Enterococcus faecium YY1-1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2023685. The Enterococcus faecalis (…) Enterococcus faecalis YY9-1 is deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M 2023686.

2. The use of the compound microbial agent according to claim 1 in promoting the growth of yellow catfish or in the preparation of drugs for treating aquatic animal diseases caused by Edwardsiella tarda.

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