A strain of fermented lactobacillus, its microecological preparation, and its preparation method and application

Abstract

A fermented lactobacillus, a microecological preparation thereof, a preparation method thereof and an application thereof. The fermented lactobacillus is deposited in the China Center for Type Culture Collection, and the strain deposit number is CCTCC NO: M 20241215; the classification name is: fermented lactobacillus PLF-6, the Latin name Lactobacillus fermentum PLF‑6. Its microecological preparation is produced by fermenting the fermented lactobacillus PLF‑6. The present invention also includes a method for preparing the fermented lactobacillus microecological preparation. The present invention also includes the application of the fermented lactobacillus and its microecological preparation in aquaculture. The fermented lactobacillus PLF‑6 and its microecological preparation of the present invention have good antibacterial effects on common fish pathogens such as Aeromonas vernix, Aeromonas sobria, Pseudomonas ayutans, Aeromonas hydrophila, etc.; feeding fish, especially crucian carp, with fermented lactobacillus PLF‑6 as a feed additive can improve the growth performance, immunity and intestinal flora of fish, especially crucian carp.

Description

Technical Field

[0001] The invention relates to the field of microecological preparations in aquaculture, and in particular to a strain of fermented lactobacillus, a microecological preparation thereof, and a preparation method and application thereof. Background Art

[0002] With the rapid development of aquaculture, aquaculture products play a vital role in food security, and their economic and social benefits are increasing. However, due to factors such as water quality deterioration and high-density farming, the incidence of fish in freshwater aquaculture has risen sharply, seriously affecting the health of fish, causing the cost of farming to continue to increase, thus endangering the sustainable development of my country's aquaculture industry. Aquaculture diseases are characterized by high incidence, diverse types, long onset time, and wide range of infection, and the number of fish infections during the farming process has shown an increasing trend year by year. To address this problem, people use antibiotics, vaccines, and Chinese herbal medicines as the main means of preventing and controlling fish diseases. Excessive use of antibiotics can lead to increased bacterial resistance, water pollution, and antibiotic residues in fish and aquatic products. Compared with antibiotics, vaccines do not leave harmful residues in aquatic products or aquaculture environments, but vaccine research and development require a lot of time, resources, and funds, making it difficult to respond to diseases caused by new pathogens in aquaculture in a timely manner. The effective active substances in traditional Chinese medicines are unstable, there are many types, extraction is difficult, and the cost is high. Summary of the invention

[0003] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a strain of Lactobacillus fermentum, its microecological preparation and its preparation method and application. Lactobacillus fermentum and its microecological preparation have antibacterial activity against a variety of fish pathogens, can improve the immunity and disease resistance of fish themselves, and can be applied to the prevention and control of fish diseases in aquaculture.

[0004] The technical solution adopted by the present invention to solve its technical problem is as follows: a strain of fermented Lactobacillus, which was deposited in the China Center for Type Culture Collection on June 13, 2024, with a strain collection number of CCTCC NO: M 20241215; it is classified and named: Lactobacillus fermentum PLF-6, with a Latin name of Lactobacillus fermentum PLF-6.

[0005] The 16S rRNA gene sequence of the Lactobacillus fermentum PLF-6 of the present invention is shown in the sequence listing SEQ ID NO: 1.

[0006] The fermented lactobacillus PLF-6 of the present invention was screened from the sludge of Taozi Lake Wetland in Changsha City, Hunan Province. The antibacterial active substances in the fermentation liquid of the strain have high tolerance to high temperature, acid-base and protease environment, and have strong stability in the actual use process; it has good antibacterial effect on various freshwater fish pathogens, can improve fish growth, enhance fish immunity, disease resistance and improve its intestinal flora.

[0007] The fermented lactobacillus of the present invention can be fermented to prepare microecological preparations for use in actual production.

[0008] Furthermore, fermented lactobacillus and its microecological preparations can be applied in aquaculture.

[0009] The fermented lactobacillus of the present invention is used in aquaculture as an antagonist against pathogenic bacteria of fish.

[0010] Furthermore, the microecological preparation is a liquid preparation or a solid preparation.

[0011] Furthermore, the aquaculture animals are freshwater fish.

[0012] Furthermore, the freshwater fish is crucian carp, grass carp or carp.

[0013] The preparation method of the microecological preparation of fermented lactobacillus of the present invention comprises the following steps:

[0014] (1) Inoculation and activation: inoculate Lactobacillus fermentum PLF-6 on a slant MRS solid culture medium, pick a single clone and culture it in an MRS liquid culture medium for fermentation, and collect the fermentation seed liquid, which is the activated seed liquid;

[0015] (2) First expansion culture: the seed liquid activated in step (1) is inoculated into a fermentation tank at an inoculation rate of 1-2 wt% for first expansion culture, to obtain the seed liquid after the first expansion culture; the first expansion culture fermentation medium formula is: peptone 8-12 g / L, beef powder 7-10 g / L, yeast powder 3-5 g / L, glucose 18-25 g / L, dipotassium hydrogen phosphate 1.5-3 g / L, diammonium hydrogen citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.3 g / L, manganese sulfate 0.03-0.05 g / L, Tween 80 0.5-2 g / L;

[0016] (3) Second expansion culture: inoculating the seed liquid after the first expansion culture in step (2) into a fermentation tank at an inoculation rate of 10-15wt% for second expansion culture, to obtain a fermentation product of the second expansion culture; the fermentation medium formula of the second expansion culture is the same as the fermentation medium formula of the first expansion culture in step (2), and the entire culture conditions of the first expansion culture and the second expansion culture are: anaerobic fermentation, temperature 35-37°C, and culture for 2 days;

[0017] During the fermentation process, 0.3% DF103 Dow fermentation defoamer can be added (conventional method).

[0018] (4) Concentration and collection: After tanking, collect the fermentation products of the second expanded culture and concentrate them to obtain a microecological liquid preparation; or spray dry them to obtain a microecological solid preparation.

[0019] Beneficial effects of the present invention:

[0020] (1) The fermented lactobacillus PLF-6 of the present invention has a good antibacterial effect on common fish pathogens such as Aeromonas hydrophila, Shewanella xiamenensis, Aeromonas anomala, Aeromonas salmonicida, Aeromonas sobria, Aeromonas vernix, and Pseudomonas ayutans;

[0021] (2) The antibacterial activity of Lactobacillus fermentum PLF-6 is relatively stable, and it can inhibit the growth and reproduction of Aeromonas veronii in fish, especially crucian carp, and improve the survival rate of fish, especially crucian carp, infected with Aeromonas veronii;

[0022] (3) Feeding fish, especially crucian carp, with fermented Lactobacillus PLF-6 as a feed additive can improve the growth, immunity and optimize the intestinal flora of fish, especially crucian carp.

[0023] (4) The method of preparing fermented lactobacillus into a microecological preparation is simple and has a low production cost. It can also effectively reduce the risk of dependence on antibiotics and drug resistance of pathogenic bacteria, and has good application prospects.

[0024] SEQ ID NO: 1:

[0025]

[0026]

[0027] Description of Microbiological Deposit

[0028] The fermented Lactobacillus of the present invention was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China) on June 13, 2024, with a strain accession number of CCTCC NO: M 20241215; the classification name is: fermented Lactobacillus PLF-6; Latin name: Lactobacillus fermentum PLF-6. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the antibacterial spectrum of Lactobacillus fermentum against 12 pathogens in the embodiment of the present invention;

[0030] Figure 2 is a morphological observation diagram of Lactobacillus fermentum in an embodiment of the present invention;

[0031] Figure 3 It is a phylogenetic tree diagram constructed from the 16S rRNA sequence of Lactobacillus fermentum in the embodiment of the present invention;

[0032] Figure 4 is a physiological and biochemical analysis diagram of fermented lactobacillus in an embodiment of the present invention;

[0033] Figure 5 1 is a colonization analysis of Lactobacillus fermentum in the intestine of crucian carp (a) and a heat map of species analysis at the species level (b) in an embodiment of the present invention;

[0034] Figure 6 1 is an analysis result diagram of the effect of the microecological preparation prepared by fermentation of Lactobacillus PLF-6 on the immune-related enzyme activities in crucian carp serum, a: acid phosphatase activity and alkaline phosphatase activity; b: peroxidase activity and glutathione peroxidase activity;

[0035] Figure 7 This is a diagram showing the effect of the probiotic preparation prepared by fermented Lactobacillus PLF-6 on the antioxidant-related genes of crucian carp in the embodiment of the present invention, a: kidney; b: spleen; c: intestine;

[0036] Figure 8 This is an analysis result of the effect of the microecological preparation prepared by fermented Lactobacillus PLF-6 on the immune factors of crucian carp in the embodiment of the present invention, a: liver; b: kidney; c: spleen; d: intestine;

[0037] Fig. 9 This is a diagram showing the protective effect of fermented lactobacillus PLF-6 on crucian carp infected with pathogenic bacteria Aeromonas veronii in an embodiment of the present invention;

[0038] Fig.10 This is an observation diagram of a tissue section of crucian carp after feeding with fermented Lactobacillus PLF-6 in an embodiment of the present invention;

[0039] Fig.11 : is a graph showing the effect of fermented lactobacillus PLF-6 on the length of intestinal villi and the thickness of the mucosal layer of crucian carp in an embodiment of the present invention;

[0040] Fig.12 Graph showing the effect of fermented Lactobacillus PLF-6 on species composition at the phylum level of crucian carp intestinal flora samples (a) and fungal species with significant differences (b) according to an embodiment of the present invention;

[0041] Fig.13 The effect of Lactobacillus fermentum PLF-6 in the present invention on the abundance of the top 20 species at the genus level in the intestinal flora of crucian carp (a) and the fungal species with significant differences (b);

[0042] Fig.14 This is a diagram of the protective effect of fermented lactobacillus on crucian carp visualized by using a small animal imaging system in an embodiment of the present invention. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with the embodiments and drawings.

[0044] Unless otherwise specified, the chemical reagents used in the embodiments of the present invention were obtained through conventional commercial channels.

[0045] The fermented Lactobacillus of this embodiment was deposited in the China Center for Type Culture Collection on June 13, 2024, with a strain accession number of CCTCC NO: M 20241215; the classification name is: fermented Lactobacillus PLF-6, and the Latin name is Lactobacillus fermentumPLF-6.

[0046] Example

[0047] (I) Screening of Lactobacillus fermentum PLF-6 and determination of its antibacterial spectrum

[0048] Different samples selected from the silt of Taozi Lake in Yuelu District, Changsha were diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5Different multiples were spread on MRS solid medium, marked, and cultured at 37°C for 2 days. Single clones with similar morphology to Lactobacillus were selected, streaked and purified on MRS solid medium, and cultured at 37°C for 2 days. Taking Erwinia spp., Aeromonas allosaccharophila, Citrobacter freundii, Edwardsiella tarda, Aeromonas caviae, Pseudomonas plecoglossicida, Plesiomonas Shigelloide, Aeromonas hydrophila, Aeromonas sobria, Aeromonas salmonicida, Aeromonas veronii, and Shewanella xiamenensis as indicator bacteria, it was found that the lactobacillus strain had an antibacterial effect on them (see Figure 1 ), named Lactobacillus fermentum PLF-6.

[0049] (II) Morphological characteristics of Lactobacillus fermentum PLF-6

[0050] When streaked on an MRS plate, the single colony of the strain was milky white, opaque, uneven, and rounded and smooth; under a phase contrast microscope, the strain was short rod-shaped; Gram staining was purple; under a cold-field scanning electron microscope, the overall morphology of the strain was short rod-shaped with elliptical ends (see Figure 2 ).

[0051] (III) Identification of 16S rRNA gene and full gene of Lactobacillus fermentum PLF-6

[0052] The fermented Lactobacillus PLF-6 strain was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 1 day. The bacteria were collected and the genomic DNA of the strain PLF-6 was extracted using an Ezup column bacterial genomic DNA extraction kit (purchased from Shanghai Shenggong Biotechnology Co., Ltd.), and the 16S rRNA gene was amplified using primers.

[0053] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0054] 1492R:5'-CGGTTACCTTGTTACGACTT-3'

[0055] The 16S rRNA gene sequence was amplified using the above primers, and the expected length of the sequence was about 1500 bp.

[0056] PCR reaction system (20 μL): sterile double distilled water, 14 μL; 10× Buffer, 2 μL; dNTP, 1.6 μL; Bf-R (10 μM), 0.6 μL; Bf-F (10 μM), 0.6 μL; genomic template, 1 μL; PrimerSTAR DNA Polymerase (Takara), 0.2 μL;

[0057] PCR reaction program: pre-denaturation at 95°C for 5 min; 30 cycles: 95°C, 45 sec; 55°C, 45 sec; 72°C, 1.5 min; 72°C, 10 min.

[0058] After the 16S rRNA gene PCR products were detected by 1.0% agarose gel electrophoresis, the successfully amplified products were sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing.

[0059] The 16S rRNA gene sequence of the strain Lactobacillus fermentum PLF-6 was sequenced and showed to be 1482 bp in length. The amplified sequencing results were analyzed by BLAST comparison on NCBI, and the phylogenetic tree of the strain was constructed using the neighbor-joining method in MEGA 6.06 (see Figure 3 ). It is closest to Lactobacillus fermentum strain CIP 104927.1 and Lactobacillus fermentum strain NBRC 15885.

[0060] (IV) Analysis of the physicochemical properties of antibacterial active substances of Lactobacillus fermentum PLF-6

[0061] Activate the fermented Lactobacillus PLF-6 strain, centrifuge at 8000rpm for 10min, take out 100μL of the fermentation supernatant into an EP tube, and perform the following different treatments, which are repeated three times. After being treated in a constant temperature water bath at 40℃, 60℃, 80℃, and 100℃ for 1h, the antibacterial effect of the fermentation supernatant treated at different temperatures on Aeromonas hydrophila was detected by the punch method (see Figure 4 ); the fermentation supernatant was treated with trypsin and protease K at 37°C for 1 h, and the antibacterial effect of the fermentation supernatant treated with different proteases on Aeromonas hydrophila was detected by the punch method (see Figure 4 ); treated at room temperature for 1 h at pH 1, 3, 5, 7, 9, and 11, and then adjusted back to normal pH. The antibacterial effect of the fermentation supernatant treated with different pH buffers on Aeromonas hydrophila was detected by the punch method (see Figure 4 ).Depend on Figure 4 It can be seen that the antibacterial active substances in the fermentation broth of this strain have high tolerance to high temperature, acid, alkali and protease environment, and have strong stability in actual use.

[0062] (V) Preparation of Lactobacillus fermentum PLF-6 microecological preparation

[0063] (1) Inoculation and activation: inoculate Lactobacillus fermentum PLF-6 on a slant MRS solid culture medium, pick a single clone and culture it in an MRS liquid culture medium for fermentation, and collect the fermentation seed liquid, which is the activated seed liquid;

[0064] (2) First expansion culture: The seed liquid activated in step (1) is inoculated into a fermentation tank at an inoculum amount of 2 wt% for first expansion culture; the fermentation medium formula is: peptone 10 g / L, beef powder 8 g / L, yeast powder 4 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.03 g / L, Tween 80 1 g / L;

[0065] (3) Second expansion culture: The seed liquid activated in step (2) is inoculated into a fermentation tank at an inoculation rate of 15 wt% for second expansion culture. The fermentation medium formula is the same as the fermentation medium formula of the first expansion culture in step (2). The culture conditions are: the first expansion culture and the second expansion culture are all anaerobic fermentation conditions, the temperature is 35-37 ° C, and the culture is 2 days; 0.03% DF103 Dow fermentation defoamer (conventional means) is added during the fermentation process;

[0066] (4) Concentration and collection: After tanking, collect the fermentation products of the second expanded culture, concentrate them to obtain a microecological liquid preparation, or spray dry them to obtain a microecological solid preparation.

[0067] (VI) Application of Lactobacillus fermentum PLF-6 in fish farming

[0068] Example 1: Colonization analysis of Lactobacillus fermentum PLF-6 in crucian carp

[0069] Crucian carp weighing about 15 g were cultured in the frame for 14 days to adapt to the environment without feeding. After their growth state stabilized, they were starved for 48 hours and randomly divided into two groups. One group was fed with strain PLF-6 (feeding concentration was 1×10 9 CFU / mL), and the other group was fed with ordinary feed, with 20 fish in each group, and observed for 14 days. After the feeding, the contents of the front, middle and back intestines of the crucian carp in each group were taken out and smeared on the MRS plate containing erythromycin by dilution method. The results are as follows Figure 5As shown. The MRS plates of the PLF-6-fed group all had milky white, opaque strains, while the control group had no strains. The intestinal flora of crucian carp in each group of samples was determined by high-throughput sequencing technology, and fermented Lactobacillus was detected in the intestinal flora of crucian carp in the PLF-6-fed group, while fermented Lactobacillus was not found in the intestinal flora of the control group. The above data further showed that strain PLF-6 can colonize in the intestines of crucian carp, laying the foundation for the subsequent study of the effects of fermented Lactobacillus PLF-6 on the growth and immune defense ability of crucian carp.

[0070] Example 2: Effects of Lactobacillus fermentum PLF-6 on growth and disease resistance of crucian carp

[0071] The crucian carps in the experiment were divided into two groups, with 3 frames in each group and 15 crucian carps in each frame. They were fed with feed at 1% of their weight. 9 CFU / mL) of bacteria were mixed into the feed; the control group was fed with ordinary feed. The weight of the crucian carp in the experimental group and the control group was recorded before feeding, which was the initial weight (Wi). After the feeding was completed, the weight of the crucian carp in the two groups was recorded again after starvation acclimation for 12 hours, which was the final weight (Wf). After the feeding was completed, the expression levels of nonspecific immune indicators and specific immune-related factors in the serum of crucian carp were detected.

[0072] (1) Effects of fermented Lactobacillus PLF-6 on the growth performance of crucian carp

[0073] After continuous feeding of crucian carp for 30 days, the weight growth rate (WGR), specific growth rate (SGR) and survival rate (SR) were calculated using the following formula: WGR = ((W f -W i ) / W i )×100%;SGR=((lnW f -lnW i ) / feeding time)×100%; SR=(number of fish at the end of the experiment / number of fish at the beginning of the experiment)×100%. (See Table 1 for the results).

[0074] Table 1 Crucian carp growth performance parameters

[0075]

[0076] (2) Effects of fermented Lactobacillus PLF-6 on nonspecific immune parameters in crucian carp serum

[0077] After PLF-6 strain was mixed into the feed and fed to crucian carp for 30 days, the serum of crucian carp in the experimental group (PA) and the control group (DB) was collected and analyzed using enzyme activity detection kits (including ACP, AKP, GSH-PX and CAT test kits). The results are shown in Figure 6As shown. The strain PLF-6 significantly enhanced the activity of acid phosphatase (ACP) and alkaline phosphatase (AKP) in the serum of crucian carp (P < 0.001), which was 4.6 times and 6.6 times that of the control group, respectively; similarly, the activity of GSH-PX and CAT enzymes was also significantly increased (P < 0.001), which was 1.43 times and 2.18 times that of the control group, respectively. These results indicate that the strain PLF-6 can significantly increase the nonspecific immune response of crucian carp and improve its defense against external and internal pathogens.

[0078] (3) Effect of Lactobacillus fermentum PLF-6 on the antioxidant capacity of crucian carp

[0079] After feeding crucian carp for 30 days, the effect of strain PLF-6 on crucian carp kidney ( Figure 7 a) in the spleen ( Figure 7 b) in the intestines Figure 7 The results are shown in Figure 3. Figure 7 As shown. The expression levels of Nrf-2 (nuclear transcription factor) and Keap-1 (antioxidant protein) genes in the experimental group (PA) were significantly higher than those in the control group (DB). The above experimental results show that strain PLF-6 can increase the expression level of antioxidant-related genes in crucian carp and improve its antioxidant capacity.

[0080] (4) Effects of Lactobacillus fermentum PLF-6 on the expression of immune-related genes in crucian carp tissues

[0081] After continuous feeding of crucian carp for 30 days, the effect of strain PLF-6 on the expression levels of IgM (immunoglobulin), C3 (complement), LSZ (lysozyme), and IL-1β (interleukin 1β) in crucian carp tissues was determined. Figure 8 As shown, the liver ( Figure 8 a) in the kidneys ( Figure 8 b) Spleen ( Figure 8 c) in the intestines ( Figure 8 d), compared with the control group (DB), the IgM of the experimental group (PA) crucian carp increased significantly in the liver and kidney; the C3 expression level was significantly upregulated in the spleen; the LSZ expression level increased in the liver, kidney, spleen, and intestine; and IL-1β had no significant changes in the four immune tissues. This shows that strain PLF-6 can improve the immunity of crucian carp without causing inflammatory response.

[0082] (5) Experiment on the protective effect of Lactobacillus fermentum PLF-6 on crucian carp

[0083] After the strain PLF-6 was mixed into the feed and fed to crucian carp for 30 days, the protective effect of the strain on crucian carp infected with Aeromonas vermiformis was determined. Fig. 9The survival rate of crucian carp fed with strain PLF-6 after being infected with Aeromonas veroni was significantly higher than that of the control group, which proved that strain PLF-6 can increase the resistance of crucian carp to pathogens and improve its survival rate.

[0084] Example 3: Effect of Lactobacillus fermentum PLF-6 on intestinal flora of crucian carp

[0085] The crucian carp in the experiment were divided into two groups, with 3 frames in each group and 15 crucian carp in each frame. The experimental group (feed with strain PLF-6 bacteria, PA) and the control group (feed without strain PLF-6 bacteria, DB). 9 CFU / mL); 1% of the bacteria was mixed into the feed for 30 consecutive days. After 30 days, 6 crucian carps were randomly selected from each group, dissected, and their intestines were taken out and placed in 1.5mL EP tubes, marked, stored at -80℃, and then sent to Beijing Aoweisen Gene Technology Co., Ltd. for high-throughput sequencing analysis; 2 crucian carps were randomly selected from each group for dissection, and the dissected liver, kidney, spleen, and intestine were sent to Wuhan Saiweier Biotechnology Co., Ltd. for HE staining and determination of intestinal structure.

[0086] (1) Effects of strain PLF-6 on the tissue structure of crucian carp

[0087] In this experiment, the liver, kidney, spleen and intestinal tissues of crucian carp in the common feed feeding group and the strain PLF-6 feed feeding group were sliced ​​and observed. Fig.10 As shown. It was found that the tissue cells of crucian carp fed with strain PLF-6 had the same morphology as those of the control group, and were in good growth condition, without showing pathological characteristics, indicating that the strain did not damage the crucian carp tissue. The effect of strain PLF-6 on the length of villi and the thickness of the mucosal layer in the intestine of crucian carp was then determined, as shown in Fig.11 As shown, it was found that compared with the control group, the villus length and mucosal layer thickness in the intestine of crucian carp in the experimental group were significantly increased (p<0.05).

[0088] (2) Analysis of intestinal microbiome abundance characteristics

[0089] Through OTU cluster analysis, 18 phyla were identified from 12 samples, among which the abundance of Firmicutes, Bacteroidota, Actinobacteria, Verrucomicrobiota, Fusobacteriota, Patescibacteria, Proteobacteria, and Chloroflex exceeded 0.1%. Fig.12As shown in the figure, the Verrucomicrobia in the experimental group (PA) was significantly higher than that in the control group (DB); while the Bacteroidetes was significantly lower than that in the control group (P<0.01). Fig.12 shown.

[0090] To further analyze the effect of strain PLF-6 on the intestinal flora of crucian carp at the genus classification level, the genera with an abundance of more than 0.1% were clustered after annotation analysis. Then, after integrating the duplicates in the two groups, the top 20 genera with the highest abundance were as follows: Fig.13 As shown. The abundance of Lactobacillus in the experimental group (PA) was significantly higher than that in the control group (DB) (P<0.05); while the abundance of Klebsiella was significantly lower than that in the control group (P<0.05). This indicates that strain PLF-6 can reduce the abundance of potential pathogenic bacteria in the intestinal flora of crucian carp, increase the abundance of probiotics, and regulate the intestinal flora, thereby improving the resistance of crucian carp to pathogens.

[0091] Example 4: Antagonistic effect of the probiotic preparation prepared by the novel fermented Lactobacillus PLF-6 on Aeromonas veronii in crucian carp

[0092] Crucian carp weighing about 15 g were cultured in the frame for 14 days to adapt to the environment without feeding. After their growth state stabilized, they were divided into 3 groups, with 20 fish in each group. The concentrations of AvX005 (Aeromonas vervetii) and strain Lactobacillus fermentum PLF-6 were adjusted to 1×10 9 CFU / mL. Control group 1 (+): 200 μL AvX005 (1×10 9 CFU / mL) of fermentation supernatant was injected into crucian carp. Control group 2 (-): 200 μL PBS was injected into crucian carp. Experimental group: 200 μL fermentation supernatant of Lactobacillus fermentum PLF-6 (1×10 9 CFU / mL) were injected into crucian carp, and the health status of the fish was observed.

[0093] Experimental group 1 ( Fig.14 Middle control group): injection of 200 μL AvX005 mCherry (1×10 9 CFU / mL) fermentation supernatant into crucian carp, and then fed with ordinary feed; experimental group 2 ( Fig.14 PLF-6 (experimental group): 200 μL AvX005 mCherry (1×10 9 CFU / mL) fermentation supernatant into crucian carp, and the bacteria of strain PLF-6 (1×10 9 CFU / mL) were fed to crucian carp. AvX005 was observed for 7 consecutive days. mCherryThe distribution of red fluorescence in the crucian carp and its interaction with strain PLF-6 were photographed every 1 day.

[0094] The results are shown in Table 2. Compared with the control group 1 (AvX005), the mortality rate of the test group (PLF-6) was significantly reduced, and it was the same as the mortality rate of the control group 2 (PBS). In addition, the crucian carp in the test group did not show obvious damage, while the crucian carp in the control group 1 (AvX005) showed obvious pathological characteristics such as abdominal swelling and fin congestion. The above experimental results further indicate that the strain PLF-6 is safe and has no toxic effects on crucian carp.

[0095] The red fluorescence distribution is as follows Fig.14 As shown. On the first day, it was observed that the gills, fins, and abdomen of the crucian carp in the experimental group and the control group showed red fluorescence signals; on the third day, it was observed that the red fluorescence signals in the crucian carp in the control group shifted to the area where the internal organs were located, while the red fluorescence in the crucian carp in the experimental group did not shift significantly compared with the red fluorescence in the control group, and was significantly reduced; on the fifth day, it was observed that the red fluorescence of the crucian carp in the control group was all over the body. The crucian carp in the experimental group only had a small amount of fluorescence in the gills and fins, indicating that strain PLF-6 can inhibit AvX005 mCherry Proliferation in crucian carp (see Fig.14 ).

[0096] Table 2 Animal live safety test

[0097]

Claims

1. A strain of Lactobacillus fermentum, characterized in that: Classification name: Lactobacillus fermentum ( Lactobacillus fermentum )PLF-6; deposited in China Center for Type Culture Collection on June 13, 2024, the strain collection number is CCTCC NO: M 20241215.

2. The microecological preparation of Lactobacillus fermentum as claimed in claim 1, characterized in that The preparation is completed by fermentation of the fermented lactobacillus PLF-6.

3. The microecological preparation of fermented lactobacillus as claimed in claim 2, characterized in that, Prepared into liquid preparation or solid preparation.

4. The method for preparing the microecological preparation of the fermented lactobacillus as claimed in claim 2 or 3, characterized in that, The following steps are involved: (1) Inoculation and activation: inoculate the fermented Lactobacillus PLF-6 on a slant MRS solid culture medium, pick a single clone and culture it in an MRS liquid culture medium for fermentation, and collect the fermented seed liquid, which is the activated seed liquid; (2) First expansion culture: The seed liquid activated in step (1) is inoculated into a fermentation tank at an inoculation rate of 1-2 wt% for first expansion culture, thereby obtaining the seed liquid after the first expansion culture; the first expansion culture fermentation medium formula is: peptone 8-12 g / L, beef powder 7-10 g / L, yeast powder 3-5 g / L, glucose 18-25 g / L, dipotassium hydrogen phosphate 1.5-3 g / L, diammonium hydrogen citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.3 g / L, manganese sulfate 0.03-0.05 g / L, Tween 80 0.5-2 g / L; (3) Second expansion culture: the seed liquid after the first expansion culture in step (2) is inoculated into a fermentation tank at an inoculation rate of 10-15wt% for second expansion culture to obtain a fermentation product of the second expansion culture; the fermentation medium formula of the second expansion culture is the same as the fermentation medium formula of the first expansion culture in step (2), and the entire culture conditions of the first expansion culture and the second expansion culture are: anaerobic fermentation, temperature 35-37°C, and culture for 2 days; (4) Concentration and collection: After tanking, collect the fermentation products of the second expanded culture and concentrate them to obtain a microecological liquid preparation; or spray dry them to obtain a microecological solid preparation.

5. The use of Lactobacillus fermentum according to claim 1 or 2, characterized in that, It is used in aquaculture as an antagonist against fish pathogenic bacteria, the fish pathogenic bacteria being Erwinia ( Erwinia spp. ), Aeromonas anomala Aeromonas allosaccharophila ) and Citrobacter freundii ( Citrobacter freundii ), Edwardsiella tarda ( Edwardsiella tarda )、Aeromonas caviae( Aeromonas caviae )、Pseudomonas ayuensis( Pseudomonas plecoglossicida ), Pleistomonas shigelloides ( Plesiomonas Shigelloide )、Aeromonas hydrophila( Aeromonas hydrophila )、Aeromonas sobriae( Aeromonas sobria ), Aeromonas salmonicida ( Aeromonas salmonicida )、Aeromonas vervetii( Aeromonas veronii ) or Shewanella xiamenensis ( Shewanella xiamenensis ).

6. The application of the probiotic preparation of Lactobacillus fermentum as claimed in claim 2 or 3, characterized in that, It is used in aquaculture to inhibit fish pathogenic bacteria, the fish pathogenic bacteria is Erwinia ( Erwinia spp. ), Aeromonas anomala Aeromonas allosaccharophila ) and Citrobacter freundii ( Citrobacter freundii ), Edwardsiella tarda ( Edwardsiella tarda )、Aeromonas caviae( Aeromonas caviae )、Pseudomonas ayuensis( Pseudomonas plecoglossicida ), Pleistomonas shigelloides ( Plesiomonas Shigelloide )、Aeromonas hydrophila( Aeromonas hydrophila )、Aeromonas sobriae( Aeromonas sobria ), Aeromonas salmonicida ( Aeromonas salmonicida )、Aeromonas vervetii( Aeromonas veronii ) or Shewanella xiamenensis ( Shewanella xiamenensis ).

7. The use of the probiotic preparation of Lactobacillus fermentum as claimed in claim 6, characterized in that, Applications in aquaculture The animals used in aquaculture are freshwater fish.

8. The application of the probiotic preparation of Lactobacillus fermentum as claimed in claim 7, characterized in that, The freshwater fish is crucian carp, grass carp or carp.

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