Lactobacillus plantarum SCWLac-1 and application thereof in aquaculture

Abstract

The invention belongs to the technical field of microorganisms, and relates to lactobacillus plantarum SCWLac-1 and application thereof in aquaculture, in particular to application in the aspect of litopenaeus vannamei culture. The strain can be fermented on a large scale and can promote digestion and absorption of aquaculture animals so as to promote growth; in addition, the compound has a relatively strong inhibition effect on pathogenic bacteria, and the non-specific immune enzyme activity of the litopenaeus vannamei can also be improved; the microbial agent prepared from the strain and the aquaculture feed containing the strain are applied to aquaculture, diseases in aquaculture can be reduced, the use of antibiotics is indirectly reduced, the aquaculture environment is improved, and the economic benefits of farmers are further improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and relates to a strain of Lactobacillus plantarum SCWLac-1 and application thereof in aquaculture, in particular to application thereof in Litopenaeus vannamei aquaculture. Background Art

[0002] In recent years, with the development of breeding technology, the farming industry of Litopenaeus vannamei has flourished. However, various breeding problems have gradually become prominent: excessive breeding density leads to deterioration of water quality, frequent shrimp diseases, and long-term decline in production and quality of farmed shrimp; and artificial feed has low feeding efficiency, slow weight growth of shrimp, and low economic benefits. Lactic acid bacteria preparations can inhibit the growth of pathogens, improve water quality, and promote digestion of farmed animals. Although the application of probiotics can improve some problems, the phenomenon of irregular application of probiotics and uneven quality of strains is becoming more and more serious.

[0003] Aquaculture lactobacillus microecological preparations can promote digestion and absorption of farmed animals, improve the aquaculture environment, and inhibit the occurrence of diseases in aquaculture. However, with the development of the microecological preparation industry, there has been a phenomenon of uneven strains, and the screening of local probiotics in the aquaculture environment has become particularly important. Based on this, we carried out the screening of local probiotics in shrimp ponds, with the purpose of screening local lactic acid bacteria in shrimp ponds, exploring the antibacterial ability of the screened plant lactobacillus, and analyzing the effects on the relative growth rate, relative weight gain rate, nonspecific immunity, and intestinal flora structure of Penaeus vannamei, as well as the source of the antibacterial mechanism of the extracellular products of the screened strains. In order to develop new probiotic strain resources, improve the limitations of Penaeus vannamei aquaculture, and develop antibacterial active substances with good application prospects. Summary of the invention

[0004] The present invention provides a strain of Lactobacillus plantarum SCWLac-1, which is classified and named as Lactobacillus plantarum (Lactobacillus plantarum), has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, the deposit number is CGMCCNO.31693, and the deposit date is August 20, 2024.

[0005] The invention screens lactobacilli in water of a Litopenaeus vannamei culture pond and the intestinal tract of the shrimp, and obtains a plant lactobacillus strain SCWLac-1, which can be applied to aquaculture, especially to the culture of aquaculture animals such as Litopenaeus vannamei and turbot. The strain can be fermented on a large scale, and can promote the digestion and absorption of aquaculture animals, especially Litopenaeus vannamei, and thus promote the growth. The strain has a strong inhibitory effect on pathogenic bacteria such as Vibrio harveyi, Vibrio parahemolyticus and / or Vibrio alginolyticus, thereby reducing the occurrence of diseases in the aquaculture process, indirectly reducing the use of antibiotics, improving the aquaculture environment, and thus improving the economic benefits of farmers.

[0006] The present invention also provides a microbial agent, which is fermented and cultured using an MRS culture medium; the preparation method of the microbial agent is to ferment plant lactobacillus SCWLac-1 in an MRS culture medium at 36°C-39°C in a fermentation tank, centrifuge after fermentation, pour out the supernatant, resuspend with sterile physiological saline, and use a spectrophotometer to adjust the OD value of the resuspended bacterial solution to 600 equal to 1. The prepared microbial agent can be sprayed on feed, or directly sprayed into a breeding environment.

[0007] The plant lactobacillus SCWLac-1 of the present invention can also be used to improve the enzyme activity of non-specific immune enzymes of Penaeus vannamei, and the non-specific immune enzymes include superoxide dismutase (SOD), alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme and / or catalase.

[0008] The invention also provides an aquaculture feed, comprising the Lactobacillus plantarum SCWLac-1.

[0009] Beneficial technical effects produced by the present invention:

[0010] 1. Screening local probiotics from shrimp farming ponds. Experiments have verified that the extracellular products of the plant lactic acid bacteria SCWLac-1 have obvious antibacterial effects, and have a strong inhibitory effect on pathogenic bacteria Vibrio harveyi, Vibrio parahemolyticus and / or Vibrio alginolyticus; and the strain can improve the non-specific immune enzyme activity of Penaeus vannamei, including superoxide dismutase (SOD), alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme (Lysozyme) and catalase (Catalase). The experimental results show that the enzyme activity of the above enzymes can be significantly improved.

[0011] 2. The plant lactobacillus SCWLac-1 of the present invention not only improves the relative weight gain rate and relative growth rate of Penaeus vannamei, but also improves the relative weight gain rate of turbot, and the strain has a wide range of applications in aquatic animal breeding. Through practical application experiments, it is proved that the strain can significantly improve the relative weight growth rate and body length growth rate of aquatic animals.

[0012] 3. The present invention predicts the colonization change trend of Lactobacillus plantarum SCWLac-1 by predicting the results of 16S high-throughput sequencing; through in-depth mining of the strain genetic information, the pln JK gene cluster is obtained, which can synthesize bacteriocins with antibacterial effects, proving the antibacterial effect of Lactobacillus plantarum SCWLac-1 of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Electron microscope photo of Lactobacillus plantarum SCWLac-1;

[0014] Figure 2 Phylogenetic tree of Lactobacillus plantarum SCWLac-1;

[0015] Figure 3 Growth curve and pH change diagram of Lactobacillus plantarum SCWLac-1;

[0016] Figure 4 The inhibition zone of Lactobacillus plantarum SCWLac-1 against Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio alginolyticus;

[0017] Figure 5 The relative weight growth rate of Litopenaeus vannamei fed with Lactobacillus plantarum SCWLac-1 feed for 28 days in Hongqiang Aquaculture Farm, Laiyang, Yantai, Shandong Province;

[0018] Figure 6Effects of feeding Lactobacillus plantarum SCWLac-feed for 28 days on the relative body length growth rate of Litopenaeus vannamei in Hongqiang Aquaculture Farm, Yantai, Shandong

[0019] Figure 7 Effect of feeding Lactobacillus plantarum SCWLac-1 diet for 45 days on the relative weight growth rate of Penaeus vannamei at Linbo Aquaculture Farm in Qingdao, Shandong;

[0020] Figure 8 Effects of feeding Lactobacillus plantarum SCWLac-1 diet for 45 days on the relative body length growth rate of Penaeus vannamei at Linbo Aquaculture Farm in Jiaozhou, Qingdao, Shandong;

[0021] Fig. 9 Effects of feeding Lactobacillus plantarum SCWLac-1 diet for 40 days on the relative weight growth rate of Litopenaeus vannamei at Shandong Yantai Jialin Aquaculture Farm;

[0022] Fig.10 Effects of feeding Lactobacillus plantarum SCWLac-1 diet for 40 days on the relative body length growth rate of Penaeus vannamei at Yantai Jialin Aquaculture Farm in Shandong Province;

[0023] Fig.11 Effects of feeding Lactobacillus plantarum SCWLac-1 diet for 70 days on the relative weight growth rate of turbot in Yantai Jiajun Aquaculture Farm, Shandong Province;

[0024] Fig.12 Effects of Lactobacillus plantarum SCWLac-1 on serum acid phosphatase in Litopenaeus vannamei;

[0025] Fig.13 Effects of Lactobacillus plantarum SCWLac-1 on serum alkaline phosphatase in Litopenaeus vannamei;

[0026] Fig.14 Effects of Lactobacillus plantarum SCWLac-1 on serum lysozyme of Litopenaeus vannamei;

[0027] Fig.15 Effects of Lactobacillus plantarum SCWLac-1 on serum superoxide dismutase in Litopenaeus vannamei;

[0028] Fig.16 Effects of Lactobacillus plantarum SCWLac-1 on serum catalase in Litopenaeus vannamei;

[0029] Fig.17 Phylum distribution ratio in bacterial diversity OTUS;

[0030] Fig.18 Bacterial diversity: genus distribution ratio in OTUS;

[0031] Fig.19 Colonization trend chart of Lactobacillus plantarum SCWLac-1;

[0032] Fig. 20 Genome characteristics of Lactobacillus plantarum SCWLac-1;

[0033] Fig.21 The results of KEGG database comparison of Lactobacillus plantarum SCWLac-1 genome;

[0034] Fig. 22 Comparison results of the Lactobacillus plantarum SCWLac-1 genome with the CAZy database;

[0035] Fig.23 Structure of the class II bacteriocin Pln JK gene cluster in Lactobacillus plantarum SCWLac-1. DETAILED DESCRIPTION

[0036] The plant lactobacillus SCWLac-1 of the present invention is classified and named as Lactobacillus plantarum, and has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number being CGMCC NO.31693, and the deposit date being August 20, 2024.

[0037] The invention screens lactobacilli in water of a shrimp culture pond and shrimp intestines to obtain a plant lactobacillus strain SCWLac-1, which can be applied to aquaculture, especially to the culture of aquaculture animals such as shrimp and turbot. The strain can be fermented on a large scale, can promote digestion and absorption of aquaculture animals, especially shrimp (Litopenaeus vannamei), and further promote growth. The strain has a strong inhibitory effect on pathogenic bacteria Vibrio harveyi, Vibrio parahemolyticus and / or Vibrio alginolyticus.

[0038] The present invention also provides a microbial agent, which is fermented and cultured using an MRS medium; the preparation method of the microbial agent is to ferment plant lactobacillus SCWLac-1 using an MRS medium in a fermentation tank at 37°C, centrifuge after fermentation, pour out the supernatant, resuspend with sterile physiological saline, and use a spectrophotometer to adjust the OD value of the resuspended bacterial solution to 600 equal to 1. The prepared microbial agent can be sprayed on feed, or directly sprayed into a breeding environment.

[0039] The plant lactobacillus SCWLac-1 of the present invention can be used to improve the enzyme activity of non-specific immune enzymes of Penaeus vannamei, and the non-specific immune enzymes include superoxide dismutase (SOD), alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme and / or catalase.

[0040] The present invention further provides an aquaculture feed comprising the above-mentioned microbial agent.

[0041] The invention also provides an aquaculture feed, comprising the Lactobacillus plantarum SCWLac-1.

[0042] The MRS medium contains 10 g of casein digest, 10 g of beef extract powder, 10 g of yeast powder, 2 g of triammonium citrate, 5 g of ammonium acetate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 2 g of dipotassium hydrogen phosphate, 20 g of glucose, 1.08 g of Tween-80 and water per liter of medium, with a pH of 5.7±0.2 and is sterilized by high pressure at 121° C. for 15 minutes.

[0043] The aquaculture feed containing Lactobacillus plantarum SCWLac-1 prepared by the present invention is used for aquaculture:

[0044] After the experiment was completed at Hongqiang Aquaculture Farm in Laiyang, Yantai, Shandong, the survival rate of each group was 98%, the control group obtained a total of 69.20kg of Penaeus vannamei, and the experimental group obtained 78.30kg of Penaeus vannamei. The relative weight gain rate was also significantly improved, reaching 54.27%, significantly higher than the control group's 35.81% (p<0.05). The relative growth rate of body length was 15.58%, significantly higher than the control group's 8.91%.

[0045] After the experiment was completed at Linbo Aquaculture Farm in Jiaozhou, Qingdao, Shandong, the survival rate of each group was 97%, the control group obtained a total of 40.84kg of Penaeus vannamei, and the experimental group obtained 46.85kg of Penaeus vannamei. The relative weight gain rate was also significantly improved, reaching 490.83%, significantly higher than the control group's 415.12% (p<0.05). The relative growth rate of body length was 214.68%, significantly higher than the control group's 198.36%.

[0046] After the experiment was completed at Shandong Yantai Jialin Aquaculture Farm, the survival rate of each group was 97%, the control group obtained a total of 110.42kg of Vannamei shrimp, and the experimental group obtained 124.06kg of Vannamei shrimp. The relative weight gain rate was also significantly improved, reaching 379.88%, significantly higher than the control group's 327.16% (p<0.05). The relative growth rate of body length was 174.38%, significantly higher than the control group's 154.32%.

[0047] After the experiment at Shandong Yantai Jiajun Turbot Aquaculture Farm, the survival rate of the control group was 98.5%, and the survival rate of the experimental group was 99%. The control group obtained 484.03 kg of turbot, and the experimental group obtained 504.90 kg of turbot. The relative weight gain rate was also significantly improved, reaching 34.21%, which was significantly higher than the 28.95% of the control group (p<0.05).

[0048] The following is a further detailed introduction to the Lactobacillus plantarum SCWLac-1 of the present invention in conjunction with specific examples. The technical solutions of the present invention include but are not limited to the following examples.

[0049] Example 1: Screening and molecular biological identification of Lactobacillus plantarum SCWLac-1

[0050] This experiment was conducted at the Hongqiang Vannamei shrimp farm in Yantai, Shandong Province, with an experimental pond area of ​​2hm 2 , depth 1 ~ 1.5m, the sampling time was May 2018, the water temperature was 22 ℃, and the salinity was 30; the water of the target breeding pond and the healthy vannamei shrimp in the pond were collected respectively, and stored at 4 ℃. The collected samples were processed immediately after returning to the laboratory.

[0051] The collected aquaculture water samples were processed and diluted and applied with sterilized 0.85% saline (0.85% NaCl, w / v). Three dilution gradients were set: 10 -1 , 10 -2 and 10 -3 , take 200 μL of the original solution and samples of different dilution gradients and apply them on MRS lactic acid bacteria selective solid medium, set up three parallels for each group, and place them in a constant temperature incubator at 37°C for culture. Take the collected samples of Penaeus vannamei for processing, cut the shrimp along the back with sterile surgical scissors, peel off the entire intestine of the shrimp from the back with sterile tweezers, repeatedly rinse the intestine with sterile 0.85% saline (0.85% NaCl, w / v), grind the intestine with a grinder, and dilute and apply it with sterile 0.85% saline (0.85% NaCl, w / v). Set up three dilution gradients: 10 -1 , 10 -2 and 10 -3, 200 μL of the original solution and samples with different dilution gradients were spread on MRS lactic acid bacteria selective solid culture medium, three parallels were set up for each group, and cultured in a constant temperature incubator at 37°C.

[0052] During the culture period, observations were made several times. When visible colonies appeared on the plates, most lactic acid bacteria were white, smooth, and had a distinct sour taste. Therefore, plates with a distinct sour taste were selected for the separation and purification of lactic acid bacteria. According to the basic characteristics of the colonies, such as morphology, size, and color, milky white, round, smooth small colonies were selected and streaked three times on MRS solid medium to obtain single colonies.

[0053] The selected strains were observed under a JEM-1200EX electron microscope, and negatively stained electron microscope photos (×60000) were taken as shown in ( Figure 1 ), rod-shaped under electron microscope, without flagella, pili, or spores, and the length of the bacteria is about 2000nm.

[0054] 16S rDNA was selected as the target gene for amplification and sequencing, and the screened strains were subjected to strain biological identification. The selected primers were 27F: 5'-AGAGTTTGATCCTGGCTCA-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'. After the amplification, the PCR product sequence was determined. PCR reagents, primer sequences, and sequence determination were from Shanghai Qingke Biotechnology Co., Ltd. The 16S rDNA sequencing results of the strain are shown in Sequence 1 of the nucleotide sequence table; the sequences obtained by the determination were compared in NCBI, and it was determined that the screened strain was Lactobacillus plantarum, belonging to the Lactobacillaceae family (Lactobacillus) genus (Lactobacillus), and the phylogenetic tree relationship of this strain with other strains (see Figure 2 ). It was identified as Lactobacillus plantarum SCWLac-1 by the General Microbiology Center of China Microbiological Culture Collection Administration and was preserved with the preservation number CGMCC NO.31693.

[0055] The above PCR reaction system:

[0056]

[0057] The above PCR reaction conditions:

[0058]

[0059] The isolated and purified Lactobacillus plantarum strain was inoculated on a 2216E solid slope, preserved with a sterile 15% glycerol preservation solution (15% glycerol: 85% saline, v / v) and placed in a -80°C refrigerator for low-temperature preservation. Three tubes of each strain were placed in different preservation boxes to ensure the safety of the strain.

[0060] Example 2. Analysis of growth characteristics of strains

[0061] The selected plant lactobacillus SCWLac-1 was inoculated into sterilized MRS liquid culture medium (three parallels) and cultured at a constant temperature of 180r / min according to its optimal culture temperature. During the culture process, the bacterial suspension was diluted and spread with sterile physiological saline every 4 hours, and cultured and counted. According to the national standard, 30 to 300 cells / plate were selected for counting. Its growth curve and pH value were determined. The results can be found in ( Figure 3 ), from the growth curve of plant lactobacillus SCWLac-1, it showed exponential growth in 6h-18h, reached its peak in 24h, some bacteria aged and died after 24h, and showed a slow downward trend in 24h-54h. The pH value decreased fastest in the logarithmic growth phase of bacteria, from 5.81 to 3.84, and it was speculated that a large amount of acidic substances were produced in this stage.

[0062] Example 3: Screening of the antagonistic ability of extracellular products of Lactobacillus plantarum SCWLac-1 against pathogens

[0063] Current studies have shown that bacterial extracellular products contain a large number of antibiotics, bacteriocins and other antibacterial substances. Therefore, this experiment used the extracellular product concentrate of the screening strain SCWLac-1 cultured for 36 hours to conduct an antibacterial experiment on the main pathogens of farmed shrimp, Vibrio harveyi, Vibrio Parahemolyticus and Vibrio alginolyticus as indicator bacteria.

[0064] First, use an inoculation loop to scrape the isolated strain Lactobacillus plantarum SCWLac-1 activated for 36 hours, transfer to liquid MRS medium, shake at 37 ° C, culture at 180rpm for 36 hours, centrifuge at 6000rpm, discard the bacterial sediment, collect the bacterial supernatant in a sterile 50mL centrifuge tube, and use a sterilized 0.22um filter membrane to filter to completely remove the bacteria, avoid the influence of bacterial growth on the experiment, and store at 4 ° C for use. Calculate the culture time difference between probiotics and indicator bacteria before the experiment, try to complete the collection of Lactobacillus plantarum SCWLac-1 supernatant when the indicator bacteria are activated and coated, and use the freshest supernatant possible for antibacterial experiments. The indicator bacteria were inoculated on a 2216E plate, cultured at 28 ° C for 12 hours, and a small amount of indicator bacteria was scraped with an inoculation loop, and diluted with sterilized 0.85% saline and evenly coated on the 2216E plate punched with an Oxford cup. 3 parallels were added to each plate, and the culture supernatant of Lactobacillus plantarum SCWLac-1 strain was added. Pipette 100 μL of supernatant into the well, and incubate at 28°C for 24 hours to observe whether there is an obvious antibacterial zone around the well. The antibacterial results are shown in ( Figure 4 ), the results showed that the supernatant of the screened Lactobacillus plantarum SCWLac-1 strain had a good antibacterial effect on three indicator bacteria, Vibrio harveyi, Vibrio parahaemolyticus and Vibrio alginolyticus.

[0065] Example 4: Experiment on feeding Lactobacillus plantarum SCWLac-1 feed to shrimps from Hongqiang Aquaculture Farm in Yantai, Shandong

[0066] On July 18, 2020, two square cement pools with a length, width and height of 6m×6m×2m were selected at Hongqiang Aquaculture Farm in Yantai, Shandong. Before the experiment, the breeding ponds were disinfected with potassium permanganate, and 3,000 vannamei shrimp with an average weight of 17g were placed in each pond. The control group was fed with normal feed, and the experimental group was fed with feed sprayed with Lactobacillus plantarum SCWLac-1; they were fed continuously for 28 days. During the experiment, the water temperature was 28°C and the salinity was 30. The daily feed was fed according to 3.5% of the weight of the shrimp. The aerator maintained sufficient oxygen, the bottom of the pool was funnel-shaped, and the residual bait and feces were discharged while changing the water. The water was changed once a day in the morning and evening, and the water change volume was 20% each time. At the beginning and end of the experiment, the weight of the shrimp in each group was measured, and the number and health status of the shrimp were recorded. Survival rate (SR, %) = (final number of shrimp / initial number of shrimp) × 100. Calculate the average weight W0 and W of the shrimp at the beginning and end of the experiment t , calculate the relative weight gain rate. The calculation formula is: relative weight gain rate (%) = (W t =W0) / W0×100. At the beginning and end of the experiment, the body length of the shrimp in each group was measured. The average body length L0 and L0 of the shrimp at the beginning and end of the experiment were calculated. t , calculate the relative growth rate. The calculation formula is: relative body length growth rate (%) = (L t-L0) / L0×100. After the experiment, the survival rate of each group was 98%, the control group obtained 69.20kg of Penaeus vannamei, and the experimental group obtained 78.30kg of Penaeus vannamei. The relative weight gain rate was also significantly improved, reaching 54.27%, which was significantly higher than the control group's 35.81% (p<0.05) ( Figure 5 The relative growth rate of body length was 15.58%, significantly higher than that of the control group (8.91%). Figure 6 ).

[0067] The preparation method of the Lactobacillus plantarum SCWLac-1 feed described in this embodiment is as follows: fermentation is carried out in a fermentation tank at 37°C using an MRS medium. After fermentation, the feed is centrifuged at 8000 rpm for 5 minutes, the supernatant is discarded, and the feed is resuspended with sterile saline. This step is repeated 3 times. The resuspended bacterial solution is adjusted to OD600=1 using a spectrophotometer, and the feed is sprayed evenly with a sprayer at a ratio of 300 ml of bacterial solution to 3 kg of feed, and the feed is naturally dried for 30 minutes. The effective concentration of the feed is 1×10 8 CFU / g. The prepared feed was placed in a -20 degree refrigerator and must be used up before 7 days. Feed was orally fed 3 times a day, at 8:00 in the morning, 12:00 noon, and 17:00 in the evening, and the daily feeding amount was 3.5% of the shrimp body weight.

[0068] Example 5: Experiment on feeding Lactobacillus plantarum SCWLac-1 feed to shrimps at Linbo Aquaculture Farm in Qingdao, Shandong

[0069] On April 10, 2023, two rectangular cement pools with a length, width and height of 6m×5m×2m were selected at Linbo Farm in Qingdao, Shandong. Before the experiment, the breeding ponds were disinfected with potassium permanganate, and 2,500 Vannamei shrimp with an average weight of 3.27g were placed in each pond. The control group was fed normal feed, and the experimental group was fed feed sprayed with SCWLac-1 for 45 consecutive days. During the experiment, the water temperature was 28°C and the salinity was 30. The daily feed was fed according to 3.5% of the weight of the shrimp. The aerator maintains sufficient oxygen, the bottom of the pool is funnel-shaped, and the residual bait and feces are discharged while changing the water. The water is changed once a day in the morning and evening, and the amount of water changed each time is 20%. At the beginning and end of the experiment, the weight of the shrimp in each group was measured, and the number and health status of the shrimp were recorded. Survival rate (SR, %) = (final number of shrimp / initial number of shrimp) × 100. Calculate the average weight W0 and W of the shrimp at the beginning and end of the experiment t , calculate the relative weight gain rate. The calculation formula is: relative weight gain rate (%) = (W t =W0) / W0×100. At the beginning and end of the experiment, the body length of the shrimp in each group was measured. The average body length L0 and L0 of the shrimp at the beginning and end of the experiment were calculated. t, calculate the relative growth rate. The calculation formula is: relative body length growth rate (%) = (L t -L0) / L0×100. After the experiment, the survival rate of each group was 97%, the control group obtained 40.84kg of Penaeus vannamei, and the experimental group obtained 46.85kg of Penaeus vannamei. The relative weight gain rate was also significantly improved, reaching 490.83%, which was significantly higher than the control group's 415.12% (p<0.05) ( Figure 7 The relative growth rate of body length was 214.68%, significantly higher than that of the control group (198.36%). Figure 8 ).

[0070] The preparation method of the plant lactobacillus SCWLac-1 feed described in this embodiment is as follows: fermentation is carried out in a fermentation tank at about 37°C using an MRS medium. After fermentation, the feed is centrifuged at 8000 rpm for 5 minutes, the supernatant is discarded, and the feed is resuspended with sterile saline. This step is repeated, and the resuspended bacterial solution is adjusted to OD600=1 using a spectrophotometer. The feed is sprayed evenly with a sprayer at a ratio of 300 ml of bacterial solution to 3 kg of feed, and the feed is dried naturally for 30 minutes. The effective concentration of the feed is 1×10 8 CFU / g. The prepared feed was placed in a -20 degree refrigerator and must be used up before 7 days. Feed was orally fed 3 times a day, at 8:00 in the morning, 12:00 noon, and 17:00 in the evening, and the daily feeding amount was 3.5% of the shrimp body weight.

[0071] Example 6: Experiment on feeding of Penaeus vannamei at Jialin Aquaculture Farm in Yantai, Shandong

[0072] On May 8, 2024, two square cement pools with a length, width and height of 10m×10m×2m were selected at the Jialin Farm in Yantai, Shandong. Before the experiment, the breeding ponds were disinfected with bleaching powder, and 5,000 Vannamei shrimps with an average weight of 5.33g were placed in each pool. The control group was fed with normal feed, and the experimental group was fed with feed sprayed with Lactobacillus plantarum SCWLac-1 for 40 consecutive days. During the experiment, the water temperature was 28°C and the salinity was 30. The daily feed was fed according to 3.5% of the weight of the shrimp. The aerator maintained sufficient oxygen, the bottom of the pool was funnel-shaped, and the residual bait and feces were discharged while changing the water. The water was changed once in the morning and evening every day, and the amount of water changed each time was 20%. At the beginning and end of the experiment, the weight of the shrimp in each group was measured, and the number and health status of the shrimp were recorded. Survival rate (SR, %) = (final number of shrimp / initial number of shrimp) × 100. Calculate the average body weight W0 and Wt of the shrimp at the beginning and end of the experiment, and calculate the relative weight gain rate. The calculation formula is: Relative weight gain rate (%) = (Wt-W0) / W0×100. Measure the body length of shrimp in each group at the beginning and end of the experiment. Calculate the average body length L0 and Lt of shrimp at the beginning and end of the experiment. t, calculate the relative growth rate. The calculation formula is: relative body length growth rate (%) = (L t -L0) / L0×100. After the experiment, the survival rate of each group was 97%, the control group obtained a total of 110.42kg of Penaeus vannamei, and the experimental group obtained 124.06kg of Penaeus vannamei. The relative weight gain rate was also significantly improved, reaching 379.88%, which was significantly higher than the control group's 327.16% (p<0.05) ( Fig. 9 The relative growth rate of body length was 174.38%, significantly higher than that of the control group (154.32%). Fig.10 ).

[0073] The preparation method of the Lactobacillus plantarum SCWLac-1 feed described in this embodiment is as follows: fermentation is carried out in a fermentation tank at 37°C using an MRS medium. After fermentation, the feed is centrifuged at 7000 rpm for 5 minutes, the supernatant is discarded, and the feed is resuspended with sterile saline. This step is repeated 4 times. The resuspended bacterial solution is adjusted to OD600=1 using a spectrophotometer, and the feed is sprayed evenly with a sprayer at a ratio of 300 ml of bacterial solution to 3 kg of feed, and the feed is naturally dried for 30 minutes. The effective concentration of the feed is 1×10 8 CFU / g. The prepared feed was placed in a -20 degree refrigerator and must be used up before 7 days. Feed was orally fed 3 times a day, at 8:00 in the morning, 12:00 noon, and 17:00 in the evening, and the daily feeding amount was 3.5% of the shrimp body weight.

[0074] Example 7: Experiment on feeding turbot with Lactobacillus plantarum SCWLac-1 feed at Jiajun Aquaculture Farm in Yantai, Shandong

[0075] On July 8, 2023, two cement pools with a length, width and depth of 15m×10m×1m were selected from Jiajun Aquaculture Farm in Yantai, Shandong. Before the experiment, the culture ponds were disinfected by spraying bleaching powder. 2000 turbots with an initial weight of 190g were placed in each pool. The control group was fed with normal feed, and the experimental group was fed with feed sprayed with Lactobacillus plantarum SCWLac-1. During the 70-day feeding trial, the photoperiod was 2h light / 22h dark cycle, and 30% of the water was replaced every two days to maintain water quality. The water temperature, salinity and pH value varied between 13 and 15℃, 28 and 30, and 7.8 and 8.2, respectively. The fish were fed manually twice a day, and visual satiety was achieved at 08:00 and 17:00. At the beginning and end of the experiment, the weight of turbot in each group was measured, and the number and health status of turbot were recorded. Survival rate (SR, %) = (final number of turbot / initial number of turbot) × 100; calculation formula: relative weight gain rate (%) = (Wt-W0) / W0 × 100. After the experiment, the survival rate of the control group was 98.5%, and the survival rate of the experimental group was 99%. The control group obtained a total of 484.03 kg turbot, and the experimental group obtained 504.90 kg turbot. The relative weight gain rate was also significantly improved, reaching 34.21%, which was significantly higher than the 28.95% of the control group (p < 0.05) ( Fig.11 ).

[0076] The preparation method of the Lactobacillus plantarum SCWLac-1 feed described in this embodiment is as follows: fermentation is carried out in a fermentation tank at 37°C using an MRS medium. After fermentation, the feed is centrifuged at 9000 rpm for 5 minutes, the supernatant is discarded, and the feed is resuspended with sterile saline. This step is repeated 5 times. The resuspended bacterial solution is adjusted to OD600=1 using a spectrophotometer, and the feed is sprayed evenly with a sprayer at a ratio of 300 ml of bacterial solution to 3 kg of feed, and the feed is dried naturally for 30 minutes. The effective concentration of the feed is 1×10 8 CFU / g. The prepared feed was placed in a -20 degree refrigerator and must be used up before 7 days. Feed was orally fed 3 times a day, at 8:00 in the morning, 12:00 noon, and 17:00 in the evening, and the daily feeding amount was 3.5% of the shrimp body weight.

[0077] Example 8: Determination of immune-related enzyme activities

[0078] Sampling was performed 0 days before feeding the experimental diet and 7 days, 14 days, and 28 days after feeding the diet. Hemolymph was slowly extracted from the abdominal blood sinus of Penaeus vannamei with a 1 ml sterile syringe and placed in a 1.5 ml sterile centrifuge tube with an equal amount of anticoagulant. Centrifuge at 4°C 2000 rpm for 10 min, and the supernatant was taken and measured using a kit (Nanjing Jiancheng Bioengineering Institute, China) according to the instruction manual. Serum acid phosphatase, alkaline phosphatase, lysozyme, superoxide dismutase, and catalase (see Figure 12-16 ) Feeding Lactobacillus SCWLac-1 can improve the activities of shrimp intestinal immune-related enzymes. After feeding for 14 days, the levels of acid phosphatase, alkaline phosphatase, superoxide dismutase, and catalase were significantly higher than those in the control group. After feeding for 28 days, the levels of alkaline phosphatase, lysozyme, and superoxide dismutase were significantly higher than those in the control group.

[0079] The preparation method of the experimental feed described in this embodiment is to use MRS medium to ferment in a fermenter at 39°C. After fermentation, centrifuge at 10000rpm for 3 minutes, pour out the supernatant, resuspend with sterile saline, repeat this step 4 times, adjust the resuspended bacterial solution OD600=1 with a spectrophotometer, spray evenly with a sprayer according to the ratio of 100ml bacterial solution to 1kg feed, and dry naturally for 30 minutes; the effective concentration of the feed is 1×10 9 CFU / g. The prepared feed was placed in a -20 degree refrigerator and must be used up before 7 days. Feed was orally fed 3 times a day, at 8:00 in the morning, 12:00 noon, and 17:00 in the evening, and the daily feeding amount was 3.5% of the shrimp body weight.

[0080] Example 9: Detection of intestinal microbial diversity in Penaeus vannamei fed with Lactobacillus SCWLac-1

[0081] Intestinal samples of Penaeus vannamei continuously fed with Lactobacillus SCWLac-1 feed for different days were taken, and DNA was extracted for in vitro amplification of 16S rRNA gene sequence. The PCR primers used were the universal primers B8F (5'-AGAGTTTGATCCTGGCTCAG-3') and B1510R (5'-GGTTACCTTGTTACGACTT-3') of bacterial 16S rRNA gene sequence. The PCR results were detected by 1% agarose gel electrophoresis, and the samples had a bright band at 1500bp, proving that the sample PCR was successful. The PCR reaction system and reaction conditions are as follows.

[0082] PCR reaction system:

[0083]

[0084] PCR reaction conditions:

[0085]

[0086] Enzyme digestion reaction system:

[0087]

[0088] The PCR product was digested with the rapid restriction endonuclease HaeⅢ, and the digestion reaction system was as follows. The above reaction system was placed in a 37℃ metal bath and incubated for 10 minutes. The digestion results were detected using 2% agarose gel electrophoresis, and different types of PCR products were selected by bands at different positions and sent to BGI Genomics Co., Ltd. for sequencing. The results showed that Proteobacteria accounted for the highest proportion, followed by Bacteroidota, Firmicutes, and Actinobacteriota. Firmicutes accounted for 42.5% after feeding Lactobacillus SCWLac-1 for 7 days, while the control group accounted for only 5.8%, an increase of more than 6 times. There was a clear downward trend from 14d to 28d, from 20.5% to 3.6% from 14d to 21d. It dropped to 2.8% on 28d, which was lower than the control group of 5.8% ( Fig.17 ). At the genus level, the top 10 of the 30 genera are Rhodobacteraceae, Ralstonia, Vibrio, Lactobacillus, Maribacter, Xanthomarina, Acinetobacter, Ruegeria, Photobacterium, and Sulfitobacter. It is worth mentioning that the Vibrio control group was 1.4%, which rose to 17.5% after 7 days of feeding with Lactobacillus, and then dropped rapidly to 0.6% on 14 days. It remained basically stable on 21 days and 28 days, which were 12.8% and 11.9% respectively, which were 9.1 times and 8.5 times that of the control group ( Fig.18 ). In order to more intuitively show the change trend of Lactobacillus plantarum SCWLac-1, see ( Fig.19 ) The percentage of Lactobacillus in the control group was 0.8%, which became 30.48% and 12.2% after 7 and 14 days of feeding respectively, and then dropped to 0.4% and 0.3% after 21 and 28 days.

[0089] Example 10: In-depth mining of genetic information of Lactobacillus plantarum SCWLac-1

[0090] The bacterial solution was picked from the -80℃ refrigerator on MRS medium and activated twice. Then, cultured at 37℃ for 48h. The bacteria were washed with saline, centrifuged and collected. The genomic DNA was extracted using the classic phenol / chloroform / isoamyl alcohol extraction method (Marmur, 1961). The concentration and quality of the extracted genomic DNA were measured using a Qubit fluorometer and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Qualified DNA samples were sent to Wuhan BGI Co., Ltd. for library construction and sequencing analysis. The genome sequencing used the DNBSEQ platform and the PacBio joint assembly strategy to obtain a complete genome map. The target data collection volume for each sample was 100X clean data for the BGISEQ platform and 100X for the PacBio platform. The library construction types were 800bp conventional library and 20Kb.

[0091] The obtained PacBio library data was assembled using Canu version 1.7.1. After using REAPR software (Hunt et al., 2013) to detect incorrectly assembled sequences, the assembled genome was manually corrected using Pilon software, and then the assembly data was checked using CheckM to evaluate the integrity of the assembly data. The open reading frames (ORFs) in the genome were predicted and annotated using the prokaryotic genome annotation system RAST (RAST, https: / / rast.nmpdr.org / ). Protein annotation was performed using GhostKOALA (https: / / www.kegg.jp / ghostkoala / ) using the Kyoto encyclopedia of genes and genomes (KEGG). In addition, BLASTp and HMMER 3.0 software were used for carbohydrate CAZy database comparison. Based on the genome RAST annotation results, the bacteriocin-related genes and proteins in Lactobacillus plantarum SCWLac-1 were mined.

[0092] The genome size of Lactobacillus plantarum SCWLac-1 was 3243795 bp, the GC content was 44.59%, and the predicted protein coding sequence (CDS) was 3019. The genomic features were predicted in the genome sequence of Lactobacillus plantarum SCWLac-1, including 16 rRNAs, 65 tRNAs, 5 16S rRNAs, 38 sRNAs, 31 House-keeping Genes ( Fig. 20 , Table 1).

[0093] Table 1:

[0094]

[0095] The protein sequences obtained by analyzing the RAST annotation results of Lactobacillus plantarum SCWLac-1 were subjected to KEGG metabolic analysis, and the results are as follows: Fig.21 ). Through KEGG annotation, a total of 3099 genes (98.69%) were annotated in SCWLac-1, and the coding genes were mainly located in carbohydrate metabolism, aminoacid metabolism, membrane transport, nucleic acid metabolism, translation, and metabolism of cofactors and vitamins and other related processes.

[0096] The CAZy database is a professional database related to carbohydrate enzymes, including enzyme families that can catalyze carbohydrate degradation, modification, and biosynthesis. The database contains four main categories: Glycoside Hydrolases (GH), Glycosyl Transferases (GT), Polysaccharide Lyases (PL) and Carbohydrate Esterases (CE). In addition, it also contains carbohydrate-related modules (Carbohydrate-Binding Modules, CBM) and auxiliary module enzymes (Auxiliary Activities, AA). By using the CAZy database for analysis, we can gain an in-depth understanding of the carbohydrate utilization of strain SCWLac-1, as shown in ( Fig. 22 ). In the genome of Lactobacillus plantarum SCWLac-1, 175 genes were annotated to the glycosyltransferase module, 146 genes were annotated to the glycoside hydrolase module, followed by 55 and 37 genes annotated to the carbohydrate-related module and the carbohydrate esterase module, respectively, and 3 and 2 genes were annotated to the auxiliary module enzymes and polysaccharide lyases, respectively. Therefore, it is shown that Lactobacillus plantarum SCWLac-1 has a strong polysaccharide utilization activity and can hydrolyze large molecular sugars into small molecules for its own use.

[0097] At present, more than 20 bacteriocin proteins have been found in Lactobacillus plantarum, and studies have shown that Lactobacillus plantarum usually contains a gene cluster that can encode class II bacteriocins, and the related genes are continuously distributed on the genome. After analysis, the bacteriocin genes in the genome of Lactobacillus plantarum SCWLac-1 mainly include genes encoding class I bacteriocin Nisin and dipeptide bacteriocin pln JK gene cluster, and do not contain dipeptide plnEF. Among them, the gene structure diagram of the pln JK gene cluster is shown in ( Fig.23 ) is shown. Pln KJ is the key gene for synthesizing bacteriocin in SCWLac-1, while plnL is an immune protein that protects Lactobacillus plantarum SCWLac-1 from the damage of its own bacteriocin. The remaining genes are responsible for transporting the synthesized bacteriocin to the outside of the cell and exerting specific antibacterial effects.

Claims

1. A strain of Lactobacillus plantarum SCWLac-1, classified and named Lactobacillus plantarum (Lactobacillus plantarum), has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number CGMCC NO.31693 and the deposit date August 20, 2024.

2. Application of Lactobacillus plantarum SCWLac-1 in aquaculture as claimed in claim 1.

3. Application of plant lactobacillus SCWLac-1 in cultivation of Litopenaeus vannamei as claimed in claim 2.

4. Application of Lactobacillus plantarum SCWLac-1 in turbot farming as claimed in claim 2.

5. Use of Lactobacillus plantarum SCWLac-1 as claimed in claim 1 in inhibiting pathogenic bacteria Vibrio harveyi, Vibrio parahemolyticus and / or Vibrio alginolyticus.

6. The use of Lactobacillus plantarum SCWLac-1 as claimed in claim 1 in improving the enzymatic activity of non-specific immune enzymes of Penaeus vannamei; the non-specific immune enzymes include superoxide dismutase (SOD), alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme and / or catalase.

7. A microbial agent comprising the Lactobacillus plantarum SCWLac-1 according to claim 1.

8. The microbial agent as claimed in claim 7 is fermented using MRS culture medium.

9. The method for preparing the microbial agent as claimed in claim 8 is as follows: fermenting Lactobacillus plantarum SCWLac-1 in a fermentation tank at 36°C-39°C using MRS medium, centrifuging after fermentation, discarding the supernatant, resuspending with sterile saline, and adjusting the OD value of the resuspended bacterial solution to 1 at 600 using a spectrophotometer. 10 . An aquaculture feed comprising the Lactobacillus plantarum SCWLac-1 according to claim 1 .

Images