Marine probiotics as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to marine probiotics as well as a preparation method and application thereof. According to the method, the marine probiotics are separated from the intestinal contents of the turbot, and the marine probiotics have high salt tolerance, so that the marine probiotics can well adapt to the marine environment. Growth characteristics and safety of the two are explored, a culture medium is optimized, and microcapsule powder is prepared through a spray drying method. In order to further explore the potential application of the probiotics, Sebastes schlegeli is selected as a research object, the microcapsule powder is activated and then directly splashed into aquaculture water, the water quality index, the fish growth performance and other indexes are measured, and reference is provided for the application of the marine probiotic preparation in mariculture.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to marine probiotics and a preparation method and application thereof. Background Art

[0002] In recent years, the marine fish farming industry has developed rapidly. The gradual expansion of the scale of farming and the continuous improvement of the degree of intensiveness have led to the outbreak of various bacterial diseases, causing significant economic losses, which has had an adverse impact on the green and sustainable development of marine fisheries. Antibiotics have been used in aquaculture for a long time, and there are potential safety hazards to the environment and aquatic products, especially the risk of the spread of drug-resistant genes is becoming increasingly serious. Antibiotics also pose a threat to human health. Therefore, it is particularly important to reduce the use of antibiotics, adopt biological control methods to reduce the infection rate of bacterial diseases in farmed fish, and improve the survival rate of farming. Microecological preparations can regulate the imbalance of the microecology, maintain the balance of the microecology, improve the health level of the body, improve the overall health level of the host organism, and achieve the effect of preventing and treating diseases. Specifically, lactic acid bacteria (LAB) are widely regarded as probiotic preparations because they can produce various beneficial compounds for the cultured species. In addition, they are recognized as safe microorganisms.

[0003] The fish microbiome can be diverse, including fungi, yeasts, viruses, as well as bacteria and archaea. The balance of bacteria in the intestine is essential for maintaining animal health because it affects nutrition and immunity. For example, the intestinal microbiome can protect the host by depriving invading pathogens and secreting various antimicrobial substances. Studies have shown that probiotics are developed from functional microorganisms in the intestine, which can effectively inhibit the growth of harmful bacteria, enhance enzyme activity in the body, regulate microecological balance, and improve water quality. Currently, LAB has been developed and applied in aquaculture. However, since some strains are not of fish origin, it is difficult to play an effective role in marine fish farming, which limits its application in this field.

[0004] Marine probiotics refer to strains isolated from marine organisms or marine environments. These strains have high salt tolerance and can therefore adapt well to the marine environment, play an effective role in marine fish farming, and have broad market prospects. Summary of the invention

[0005] The technical problem to be solved by the present invention is that lactic acid bacteria (LAB) are widely regarded as probiotic preparations because they can produce various beneficial compounds for cultured species. However, since some strains are not of fish origin, it is difficult for them to play an effective role in marine fish farming, which limits their application in this field.

[0006] To solve the above problems, the present invention isolates two strains of marine probiotics from the intestinal contents of turbot, which have high salt tolerance and can adapt well to the marine environment. Their growth characteristics and safety are explored, and their culture medium is optimized, and microcapsule powder is prepared by spray drying. In order to further explore the potential application of these probiotics, the present invention selects the xu's flathead as the research object. After activation, the microcapsule powder is directly sprinkled into the aquaculture water, and the water quality index, fish growth performance and other indicators are measured to provide a reference for the application of the marine probiotic preparation in seawater aquaculture.

[0007] To achieve the above-mentioned purpose, the present invention is achieved by the following technical means: marine probiotics, including two lactic acid bacteria, namely marine sake lactobacillus, named sake lactobacillus Lactobacillus sakei, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 11, 2024, with a preservation number of CGMCC No.32183; marine lactic acid Pediococcus, named Pediococcus acidilactici, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 11, 2024, with a preservation number of CGMCC No.32184.

[0008] A marine probiotic preparation comprises the above-mentioned marine probiotics, wherein preferably L. sakei and P. acidilactici each account for 50% by mass.

[0009] Furthermore, the marine probiotic preparation adopts a spray drying process to prepare a microecological preparation, and whey protein powder or skimmed milk powder is used as a protective wall material. Spray drying is an effective method for preparing powdered products. The reason is that this technology has the advantages of simple equipment, low cost, fast speed, strong continuity, etc., and is suitable for large-scale production of probiotic powder.

[0010] A method for preparing a marine probiotic preparation comprises the following steps: after the strain is fermented in a culture medium, centrifuging at 4°C and 7000 rpm for 10 minutes, discarding the supernatant, washing the precipitate with sterile physiological saline, and finally retaining the bacterial body; preparing a spray-dried protective agent into a solution with a concentration of 20%, and fully mixing it with the bacterial body obtained by centrifugation; setting the inlet air temperature to 120°C and controlling the outlet air temperature to 65°C for spray drying.

[0011] Furthermore, the culture medium includes the following components: 20.0 g / L soybean meal, 15.0 g / L molasses, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1.0 g / L Tween-80.

[0012] Furthermore, the parameters of the culture medium fermentation are culturing the bacterial liquid at 37° C. and shaking at 200 r / min for 18 to 24 hours.

[0013] Application of the above marine probiotics in the preparation of water quality improvement preparations.

[0014] The application of the above-mentioned marine probiotics in aquaculture can significantly improve the growth performance and intestinal enzyme activity of fish.

[0015] Further, the culture medium is dissolved in tap water, boiled for 10 minutes, and then divided into three well-sealed plastic barrels with lids; after cooling, the bacterial powder is added, and the amount of bacterial powder added is 2g / L; it is fully stirred to dissolve, and sealed and fermented at room temperature for 3 days. The two marine probiotic powders are cultured separately, and then mixed in a mass ratio of 1:1, and the direct spraying method is adopted. Every 3L of bacterial liquid is sprinkled in the pool, and it is used once every 3 to 4 days. The culture medium preferably includes the following components: soybean meal 20.0g / L, molasses 15.0g / L, dipotassium hydrogen phosphate 2.0g / L, diammonium hydrogen citrate 2.0g / L, sodium acetate 5.0g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween-801.0g / L.

[0016] The above-mentioned marine probiotic preparation is used in the preparation of water quality improvement preparations or aquaculture. After the preparation is activated for 3 days, it is directly sprinkled on the aquaculture water surface, and the amount of water is increased every 2m 3 Add 1L of fermentation liquid to the breeding water.

[0017] The beneficial effects of the present invention are as follows: the present invention separates two strains of marine probiotics from the intestinal contents of turbot. They are identified as Bacillus sakei and Pediococcus acidilactici, and have obvious antibacterial effects, can inhibit some aquatic pathogens, have potential value for preventing and treating bacterial diseases, reduce the use of antibiotics, and are green, healthy and pollution-free. The MRS broth culture medium is optimized using soybean meal and molasses to achieve the purpose of cost saving. The marine probiotics are prepared into microcapsule powder for easy transportation and storage. The experimental results show that the use of the two strains of lactic acid bacteria can significantly improve the growth performance and intestinal enzyme activity of the mullet, while improving the water quality environment, providing a reference basis for the application of the marine probiotic preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shows the morphological characteristics and Gram staining of the strains; A is L. sakei and B is P. acidilactici.

[0019] Figure 2 Agarose gel electrophoresis diagram; A is L. sakei, B is P. acidilactici, and C is DNA Marker.

[0020] Figure 3It is the growth performance diagram of strains; A is L. sakei; B is P. acidilactici.

[0021] Figure 4 The figure shows the inhibitory effect of two strains of marine probiotics on four pathogenic bacteria; A is S. aureus, B is V. parahaemolyticus, C is E. coli, and D is S. typhimurium.

[0022] Figure 5 are hemolytic results; A is L. sakei, B is P. acidilactici, and C is S. aureus.

[0023] Figure 6 This is the growth curve of lactic acid bacteria in the optimized culture medium.

[0024] Figure 7 Screening of protective wall materials for spray drying.

[0025] Figure 8 It is the moisture content and water activity of microcapsule powder. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] The Staphylococcus aureus, Vibrio parahaemolyticus, Escherichia coli and Salmonella typhimurium used in the present invention are all from the Marine Microbial Engineering Laboratory of Ocean University of China. MRS broth medium is used to culture LAB, and its composition includes 10.0g / L peptone, 8.0g / L beef extract, 4.0g / L yeast extract, 20.0g / L glucose, 2.0g / L dipotassium hydrogen phosphate, 2.0g / L diammonium hydrogen citrate, 5.0g / L sodium acetate, 0.2g / L magnesium sulfate, 0.04g / L manganese sulfate, and 1.0g / L Tween-80. 2% agar is added to the solid culture medium. Other main raw materials also include soybean meal, molasses, whey protein powder, skim milk powder, etc. All materials used in the embodiments of the present invention, unless otherwise specified, are purchased from the market.

[0028] Example 1: Screening and identification of strains

[0029] (1) Preliminary screening and purification of strains

[0030] First, anesthetize the turbot. Then disinfect the surface with 75% ethanol. Use sterile instruments to dissect the fish and remove the intestine. Cut the intestine longitudinally, take 1.0g of the intestinal contents, place it in 9.0mL of normal saline, vortex and mix for 3 minutes, and dilute it using the 10-fold dilution method. Select 3 diluents of appropriate concentrations, adsorb 100μL of each and evenly spread it on MRS solid culture medium. Incubate them upside down in a 37℃ incubator for 24 to 48h. Select a single colony and perform three streaking purifications.

[0031] (2) Strain morphology, physiological and biochemical identification

[0032] The purified strains were subjected to Gram staining (Beijing Solebow Biotechnology Co., Ltd.) to observe their morphological characteristics. Gram staining results were observed under a 100× oil microscope.

[0033] (3) 16S rDNA sequencing

[0034] DNA was extracted using a bacterial genomic DNA extraction kit (Tiangen Biotechnology Co., Ltd.), and the DNA concentration was determined using a Nanometer. Using genomic DNA as a template, universal primers were used: upstream primer (27F):

[0035] 5'-AGAGTTTGATCCTGGCTCAG-3', downstream primer (1492R): 5'-TACGGCTACCTTGTTACGACTT-3' for PCR amplification

[16] . The PCR amplification reaction system was: 2×Rapidtaq Mastermix 12.5μL, upstream and downstream primers 1.0μL each, DNA template 100-150ng, ddH2O complement 25μL. After verification by 1% agarose gel electrophoresis, the PCR product was sent to Bio-Tech (Shanghai) Co., Ltd. for sequencing.

[0036] (4) Growth characteristics measurement

[0037] A single colony was picked and inoculated into MRS medium, and then inoculated into a 100 mL Erlenmeyer flask containing 50 mL of liquid medium at a 5% inoculation volume and cultured in a shaker at 37°C. The growth characteristics of the strain were determined for 24 h using pH and OD600 as indicators.

[0038] (5) Antibacterial experiment

[0039] This experiment uses a double-layer agar plate method, with the lower layer being water agar medium. First, pour the lower layer of medium into a sterile plate and place it in a sterile Oxford cup after it solidifies. Activate Staphylococcus aureus, Vibrio parahaemolyticus, Escherichia coli, and Salmonella typhimurium to prepare 1.0×10 7CFU / mL bacterial suspension. Take 1mL of each of the four indicator bacteria suspensions, mix evenly with 20mL of semi-solid nutrient agar medium melted to 45°C, pour along the middle of the water agar plate, and remove all Oxford cups after solidification. Add 200μL of marine probiotic supernatant to each well, and use blank liquid culture medium as a control. After sealing, place in a 4°C refrigerator to diffuse for 4 to 5 hours, take out the culture dish and place it in a 37°C constant temperature incubator for 18 to 24 hours, and measure the diameter of the inhibition zone.

[0040] (6) Hemolytic activity test

[0041] The two lactic acid bacteria and Staphylococcus aureus (positive control) were streaked on Columbia blood agar plates and incubated at 37°C for 24 h to observe whether hemolysis occurred.

[0042] (7) Optimization of MRS broth culture medium

[0043] The MRS broth medium used to culture lactic acid bacteria is expensive and not suitable for large-scale culture of strains. Therefore, some of the ingredients need to be replaced with other low-cost substances. The nitrogen sources such as peptone, beef powder, and yeast extract powder in MRS broth are replaced by soybean meal, and glucose is replaced by molasses. First, weigh a certain amount of soybean meal, add 2% neutral protease and alkaline protease, add distilled water and enzymolyze at 50°C for 3h. Then add molasses, sodium acetate and other ingredients, and sterilize at 115°C for 30min.

[0044] After activation, the lactic acid bacteria were inoculated into liquid culture medium at a 5% inoculation rate, cultured at 22±0.5°C for 2-3 days, and plated to determine the number of live bacteria.

[0045] Experimental results:

[0046] (1) Preliminary screening of strains

[0047] Probiotics have the characteristics of rich product variety, low cost, no toxic side effects, safety and no pollution. They have gradually replaced antibiotics and are widely used in aquaculture. Intestinal flora has a great influence on fish health by stimulating the immune system and intestinal epithelial development. Therefore, the present invention separates probiotics from the intestinal contents of marine fish.

[0048] Colony characteristics such as Figure 1 As shown above, the colonies cultured on the MRS plate for 48 hours were small, milky white, convex, shiny on the surface, and had a distinct sour taste. Gram staining, physiological and biochemical identification ( Figure 1 , Table 1), Figure 1 The following is the result of Gram staining, which is purple, proving that they are all Gram-positive bacteria. From the results in Table 1, it can be seen that the experimental strains are consistent with the physiological and biochemical characteristics of lactic acid bacteria.

[0049] Table 1 Physiological and biochemical identification

[0050]

[0051] Note: ﹢ indicates positive, ﹣ indicates negative

[0052] (2) 16S rDNA identification

[0053] The PCR amplification products of the two strains were initially screened and subjected to agarose gel electrophoresis, and the band was approximately 1500 bp ( Figure 2 ). The sequencing and splicing results of the PCR products were compared on the NCBI website. After identification, the strains were finally determined to be Lactobacillus sakei and Pediococcus acidilactici.

[0054] (3) Growth curve

[0055] The bacterial culture method used in this experiment was static culture. The growth rate of the two lactic acid bacteria was relatively slow in 0-4 hours, faster in 6-12 hours, and stable after 14 hours ( Figure 3 A). The acid production is relatively low during 0-4 hours, but increases during 6-10 hours, and the pH changes significantly ( Figure 3 B).

[0056] (4) Antibacterial experiment

[0057] The results of the double agar plate method showed that L. sakei had an inhibitory effect on Staphylococcus aureus, Vibrio parahaemolyticus, Escherichia coli, and Salmonella. P. acidilactici had an inhibitory effect on Staphylococcus aureus and Vibrio parahaemolyticus ( Figure 4 , Table 2). By measuring the diameter of the inhibition zone, it can be seen that the strains have different degrees of inhibition on different pathogens (Table 3).

[0058] Table 2 Inhibitory effect of isolated strains on pathogenic bacteria

[0059]

[0060] Note: ﹢ indicates antibacterial effect, ﹣ indicates no antibacterial effect

[0061] Table 3 Inhibition zone diameter

[0062]

[0063] (4) Hemolytic test

[0064] Staphylococcus aureus (positive control) had a significant hemolytic effect. However, no hemolytic effect was observed for L. sakei and P. acidilactici ( Figure 5). The hemolysis test showed that the two lactic acid bacteria had no hemolysis effect on Columbia blood agar plates, which provided a certain basis for proving its safety.

[0065] (5) Optimization of MRS broth culture medium

[0066] MRS broth medium is expensive and not suitable for large-scale microecological production. Therefore, soybean meal and molasses are used as nitrogen and carbon sources, and the viable bacteria can reach 10 under static culture conditions at room temperature. 8 CFU / mL.

[0067] L. sakei and P. acidilactici grew well with soybean meal as nitrogen source and molasses as carbon source. The results of the orthogonal experiment showed that the number of viable bacteria of the two lactic acid bacteria in the fifth group of culture medium was the highest (P<0.05) (Table 4). The growth curve showed that when the culture medium was cultured at room temperature for 48 hours, the number of viable bacteria reached the highest ( Figure 6 ).

[0068] Table 4 Optimization of MRS broth orthogonal experiment

[0069]

[0070] Example 2: Spray Drying

[0071] (1) Screening of protective agents

[0072] Spray drying is an effective method for preparing powdered products. The reason is that this technology has the advantages of simple equipment, low cost, fast speed, and strong continuity, and is suitable for large-scale production of probiotic powder. After the strain is fermented in the optimized culture medium, it is centrifuged at 4°C and 7000rpm for 10 minutes, the supernatant is discarded, and the precipitate is washed 3 times with sterile saline, and finally the bacteria are retained. The protective agent for spray drying is selected from maltodextrin, skim milk powder, whey protein powder, soluble starch, and trehalose, and a solution with a concentration of 20% is prepared and mixed thoroughly with the bacteria obtained by centrifugation. The dilution and coating method is used to determine the number of viable bacteria in the sample before and after spray drying. The optimal protective agent is determined based on the survival rate of the number of viable bacteria.

[0073] (2) Determination of moisture content and water activity

[0074] Determination of moisture content: Accurately weigh 1.00g of bacterial powder, put it into a weighing bottle with a lid, put it into a constant temperature air drying oven, dry it at 105℃ for 4-6 hours, cover it, take it out, cool it in a desiccator for 0.5 hours, and then weigh it. Repeat the operation until the difference between the previous and the next weighing is less than 2mg.

[0075] The formula is as follows: Water content = (m1-m2) / m1×100% (1). In formula (1), m1 is the mass before drying (g), and m2 is the dry mass (g). The water activity was measured using an ST-3A water activity meter (Xiamen Yishite Instrument Co., Ltd.).

[0076] Experimental results:

[0077] (1) Selection of protective agent

[0078] Microcapsule powders of two bacterial strains were prepared by spray drying. Five commonly used spray drying protective wall materials were selected. When soluble starch was used as a protective agent, the survival rate of the strains was the lowest ( Figure 7 ). When whey protein powder was used as the wall material, the survival rate of L. sakei and P. acidilactici before and after spray drying was the highest, about 60%, and the protection effect was the best (P<0.05). The price of skim milk powder used in this experiment was twice that of whey protein powder. Considering the protection effect and cost comprehensively, whey protein powder was finally selected as the optimal spray drying protection wall material for the strain.

[0079] (2) Determination of water activity and moisture content

[0080] With whey protein powder as the protective agent, the water content of the two microcapsule powders after spray drying was about 4.2%. The water activity of P.acidilactici is 0.13, and the water activity of L.sakei is 0.15, which is conducive to storage ( Figure 8 ).

[0081] Example 3: Marine fish farming experiment

[0082] The experiment was conducted at the aquaculture base of Qingdao Beibao Marine Technology Co., Ltd. The experimental subject was the Xu's flathead. 200 juvenile fish weighing (50.12±2.65)g were taken and evenly divided into a control group (marked as A) and an experimental group (marked as B). The breeding environment was a (2×2×1)m pond. During the entire experimental period, the water was changed every 3 days, and half of the water was changed each time. Feeding was done once a day (15:00). The water temperature was 20-25℃, and feeding was 45 days.

[0083] Dissolve the optimized culture medium in tap water, boil for 10 minutes, and then divide into three well-sealed plastic buckets with lids. After cooling, add bacterial powder at an amount of 2g / L. Stir thoroughly to dissolve, seal and ferment at room temperature for 3 days. Cultivate the two marine probiotic powders separately. Mix them in a 1:1 ratio (marked as MIXMP), use the direct spraying method, sprinkle 3L of bacterial liquid in the pool, and use it once every 3 to 4 days (twice a week).

[0084] (1) Growth performance measurement

[0085] Survival rate = (total number of fish at the end of the test / total number of fish at the beginning of the test) × 100%;

[0086] Weight gain rate = (final weight - initial weight) / number of breeding days × 100%;

[0087] Specific growth rate = (1n final weight - 1n initial weight) / number of culture days × 100%;

[0088] Fatness = (final weight / final body length 3 )×100;

[0089] Liver-to-body ratio = liver body weight / body weight at the end of each tail × 100%.

[0090] (2) Determination of intestinal digestive enzyme activity

[0091] The intestine was weighed and placed in a 1.5 mL centrifuge tube. Sterile saline was added at a ratio of 1:9 of intestinal weight (g): saline volume (mL). The centrifuge tube was placed on ice for homogenization and centrifuged at 4°C, 2500 r / min for 10 min in a low-temperature centrifuge. The supernatant was taken and the activities of protease, lipase and amylase were determined using the kit from Nanjing Jiancheng Bioengineering Institute.

[0092] (3) Water quality measurement

[0093] During the experiment, water samples were taken every 9 days to measure pH, nitrite, chemical oxygen demand (COD) and dissolved oxygen (DO) content. pH was measured using a portable pH meter (PHSJ-4A, Shanghai INESA Scientific Instrument Co., Ltd.). COD was determined using the alkaline potassium permanganate method (GB 17378.4-2007). Nitrite was determined using the diazo coupling spectrophotometry method (GB / T5750.5-2023). Dissolved oxygen was determined using a portable dissolved oxygen analyzer GBP-607 (HJ 506-2009).

[0094] Experimental results:

[0095] (1) Effect of MIXMP on the growth performance of P. schrenkiana

[0096] In addition to the above basic theory, the present invention also carried out an application test of marine fish farming. The effect of MIXMP on the growth of H. schrenkiana was explored through a comparative test (Table 5). As can be seen from the table, after 45 days of the experiment, the survival rate, weight gain rate and fatness of H. schrenkiana in group B were significantly higher than those in group A (P<0.05), and the liver-body ratio of group B was significantly lower than that of group A (P<0.05). The survival rate of group B was 5% higher than that of group A, and the weight gain rate was 6% higher. The specific growth rate of group B was higher than that of group A, but the difference was not significant (P>0.05).

[0097] Table 5 Differences in growth performance between two groups of P. schrenkiana

[0098]

[0099] The values ​​are mean ± standard deviation. Different letters in the same index indicate significant differences (P<0.05).

[0100] (2) Effects of MIXMP on intestinal enzyme activity

[0101] Digestive enzymes include proteases, lipases and amylases. After the test, the intestinal enzyme activities of animals in groups A and B were measured (Table 6). The results showed that the protease and lipase activities of group B were higher than those of group A (P<0.05), but there was no significant difference in amylase activity between group B and group A (P>0.05). In the present invention, the intestinal activities of the three digestive enzymes in the test group were all improved compared with those in the control group, indicating that adding two strains of marine probiotics to water can significantly improve the intestinal digestive enzyme activities of P. xu'sii.

[0102] Table 6 Differences in intestinal enzyme activities between the two groups of P. xu

[0103]

[0104] The values ​​are mean ± standard deviation. Different letters in the same index indicate significant differences (P<0.05).

[0105] (3) Impact of MIXMP on water quality

[0106] The test results (Table 7) show that in the selected 5 test stages, the pH, COD and nitrite of group B were significantly lower than those of group A, and the DO content was higher than that of group A (P<0.05). In the process of aquaculture, microecological preparations can not only promote animal growth, but also reduce harmful substances in water and improve water quality. In the present invention, the pH value of the experimental group decreased, mainly due to the growth and reproduction of lactic acid bacteria in water. L.saki and P.acidilactici produce organic acids, causing pH changes. The COD and nitrite contents of the experimental group were significantly lower than those of the control group. The degradation of organic matter and nitrite nitrogen in water by MIXMP increased DO, indicating that MIXMP improved water quality conditions.

[0107] Table 7 Differences in water quality between the two experimental groups

[0108]

[0109] The values ​​are mean ± standard deviation. Different letters in the same index indicate significant differences (P<0.05).

[0110] In summary, the present invention uses selective culture medium to separate Lactobacillus sakei and Pediococcus acidilactici from the healthy intestine of turbot, and neither of them shows hemolytic activity. The results show that Lactobacillus sakei can effectively inhibit the growth of Staphylococcus aureus, Vibrio parahaemolyticus, Escherichia coli and Salmonella typhimurium. The diameters of the inhibition zones are 16.75 ± 0.14 mm, 16.68 ± 0.22 mm, 11.90 ± 0.10 mm and 14.12 ± 0.08 mm, respectively. The results show that Pediococcus acidilactici has an inhibitory effect on Staphylococcus aureus (21.04 ± 0.28 mm) and Vibrio parahaemolyticus (15.13 ± 0.13 mm). Soybean meal and molasses were used to replace the nitrogen and carbon sources in the culture medium. Spray drying was used to prepare microecological preparations, and whey protein powder was selected as the protective wall material. The survival rate of Lactobacillus sakei and Pediococcus acidilactici was about 60%. The activated marine probiotics were added to the culture water of Hsu's flathead. The results showed that the survival rate, weight gain rate, fatness, protease and lipase levels of the experimental group were significantly higher than those of the control group (P<0.05). The pH value, chemical oxygen demand and nitrite concentration of the experimental group were significantly lower than those of the control group (P<0.05). The dissolved oxygen content was significantly higher than that of the control group (P<0.05).

[0111] Finally, it should be noted that although the above embodiments describe the specific implementation of the present invention, they are not intended to limit the present invention; those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. All modifications or equivalent replacements should be included in the protection scope of the present invention.

Claims

1. Marine probiotics, characterized by It includes two lactic acid bacteria: marine sake lactobacillus, named L. sakei, which was deposited in the General Microbiology Center of China Culture Collection Administration on October 11, 2024, with the deposit number CGMCC No. 32183; marine lactic acid Pediococcus, named P. acidilactici, which was deposited in the General Microbiology Center of China Culture Collection Administration on October 11, 2024, with the deposit number CGMCC No. 32184.

2. A marine probiotic preparation comprising the marine probiotic according to claim 1.

3. The marine probiotic preparation according to claim 2, characterized in that: The marine probiotic preparation adopts a spray drying process to prepare a microecological preparation, and whey protein powder or skimmed milk powder is used as a protective wall material.

4. The method for preparing the marine probiotic preparation according to claim 2, characterized in that The following steps are involved: After the strain is fermented in the culture medium, it is centrifuged at 4°C and 7000rpm for 10 minutes, the supernatant is discarded, the precipitate is washed with sterile saline, and finally the bacteria are retained; the spray-drying protective agent is prepared into a solution with a concentration of 20%, and is fully mixed with the bacteria obtained by centrifugation; the inlet air temperature is set at 120°C, and the outlet air temperature is controlled at 65°C for spray drying.

5. The preparation method according to claim 4, characterized in that The culture medium includes the following components: 20.0 g / L soybean meal, 15.0 g / L molasses, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1.0 g / L Tween-80.

6. The preparation method according to claim 4, characterized in that: The parameters of the culture medium fermentation are culturing the bacterial liquid at 37°C and shaking at 200r / min for 18 to 24 hours.

7. Use of the marine probiotics according to claim 1 in the preparation of water quality improvement preparations.

8. Use of the marine probiotics according to claim 1 in aquaculture.

9. The use according to claim 7 or 8, characterized in that: Dissolve the culture medium in tap water, boil for 10 minutes, and then divide it into three well-sealed plastic buckets with lids; add bacterial powder after cooling, with the amount of bacterial powder added being 2g / L; stir thoroughly to dissolve it, and ferment it at room temperature in a sealed state for 3 days; culture the two marine probiotic powders separately, and then mix them in a mass ratio of 1:1, and use the direct spraying method to sprinkle 3L of bacterial liquid in the pool, and use it once every 3 to 4 days.

10. Use of the marine probiotic preparation according to claim 2 in the preparation of water quality improvement preparations or aquaculture.

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