Preparation and application of Lactobacillus plantarum strains with antagonistic properties against aquatic pathogens and preparations thereof

Abstract

The invention discloses a plant lactobacillus strain having antagonistic properties against aquatic pathogens and the preparation and application of the same, belonging to the field of microbial technology. Lactobacillus plantarum) FF34 has a wide range of carbon and nitrogen sources for fermentation, has amylase activity, and can utilize starchy carbon sources. It degrades nitrite quickly and has antagonistic effects on a variety of common aquatic pathogens. FF34 single bacterial agent is prepared, or mixed with a variety of Bacillus to prepare FF34 composite agent. FF34 single bacterial agent or composite agent is used to ferment fish, shrimp, crab feed and cottonseed meal and other feed raw materials, which significantly improves the nutritional content of feed. The resulting fermented feed is rich in probiotics, acid-soluble proteins and active small peptides, and the content of anti-nutritional factors such as tannins, free cottonphenol, and phytic acid is reduced; adding FF34 agent to feed promotes the growth of intestinal tissue of aquatic animals, improves liver function, improves antioxidant activity, improves the growth performance of farmed animals, and reduces feed coefficient. After the FF34 composite agent is immobilized and placed in the aquaculture water body, it can continuously improve water quality.

Description

Technical Field

[0001] The invention relates to a plant lactobacillus strain with antagonistic properties against aquatic pathogens and a single bacterial preparation thereof, a mixed fermentation culture method of the plant lactobacillus strain and bacillus, and a preparation method and application of a composite bacterial preparation thereof, belonging to the technical field of microorganisms. Background Art

[0002] Lactobacillus plantarum is a type of Gram-positive lactobacillus that is widely found in nature and is a probiotic flora in the human gastrointestinal tract. Studies have shown that Lactobacillus plantarum has a variety of beneficial effects on the human body, such as regulating immune function, regulating chronic metabolic diseases, antagonizing pathogenic bacteria infection, regulating intestinal function, regulating mental and neurological functions, etc. Due to its good physiological characteristics and probiotic functions, Lactobacillus plantarum is widely used in food industry production, medical care and other fields. In recent years, with the implementation of the policy of banning antibiotics in feed, the urgent need to reduce the amount of fish meal and soybean meal and develop grain substitutes, the application research of lactic acid bacteria in animal breeding has increased. As a national microbial feed additive catalog variety, Lactobacillus plantarum has expanded from the initial application of silage fermentation for ruminants such as cattle and sheep to beneficial application research in the field of aquaculture. Current research reports show that the beneficial effects of Lactobacillus plantarum in aquaculture are mainly manifested in the following ways: (1) regulating water quality by spraying Lactobacillus plantarum; and (2) improving the gastrointestinal environment of aquatic animals, inhibiting the growth of harmful intestinal bacteria, promoting nutrient absorption of feed, enhancing their resistance to pathogens, and promoting the growth of aquatic animals by feeding them mixed with bait or fermenting feed.

[0003] At present, lactic acid bacteria have not been widely used in aquaculture production practice. The application of Lactobacillus plantarum as a beneficial lactic acid bacteria in aquaculture is also very limited. The main reason is (1) high cost of use. Lactic acid bacteria, including Lactobacillus plantarum, are strict anaerobes or facultative anaerobes, without spores, poor environmental resistance, easy to inactivate, and have high nutritional requirements and large nitrogen source requirements when the bacteria proliferate and grow. The carbon source used for high-density bacterial culture is mainly sugar, and cheap starchy agricultural and sideline products such as wheat flour cannot be used. In addition, large-scale high-density pure strain cultivation of lactobacillus requires anaerobic fermentation equipment and anaerobic environmental conditions, and the cultivation cost is high. Compared with aerobic bacterial agents such as Bacillus subtilis, the aerobic fermentation of Bacillus uses a general aerobic fermentation tank, the fermentation system is mature, and the volume of the fermentation tank can reach more than 50 tons, while the fermentation of pure strain lactobacillus requires a dedicated anaerobic system. The scale of anaerobic fermentation tanks is small and cannot reach the volume scale of aerobic fermentation. Therefore, pure strain live lactobacillus preparations lack scale effect, high manufacturing costs, and high cost of use. (2) There are many types of aquaculture in my country, and the aquaculture species and regional environment are complex. There is a great lack of suitable excellent strains of Lactobacillus plantarum. Strains with antagonistic properties against aquatic pathogens are not only scarce in source, but also lack low-cost and large-scale cultivation methods, which cannot meet the demand for antibiotic substitutes. (3) There is a lack of multifunctional new Lactobacillus plantarum. At present, aquaculture is mainly based on high-density intensive static water aquaculture, with high aquaculture density and high-protein feed. Due to the low feed utilization rate of farmed animals, such as the utilization rate of feed protein is only about 30%, most of the nitrogen and phosphorus in the feed are discharged into the aquaculture water body with leftover bait and excrement, which deteriorates the ecological environment of the aquaculture water body, loses the self-purification and regulation ability of the pond, and the concentration of ammonia nitrogen and nitrite in the water body exceeds the standard, which endangers the health of farmed animals, reduces their own immune ability, causes frequent bacterial diseases, and brings great harm to aquaculture production. Therefore, it is urgent to develop new multifunctional Lactobacillus plantarum strains to play the purification and regulation function of reducing nitrite and other adverse water quality factors, antagonize pathogens, improve feed utilization, and promote the growth of farmed animals. (4) At present, there is an urgent need to reduce the amount of fish meal and soybean meal and develop feed substitutes. In terms of using the growth and metabolism of probiotics to improve and enhance the nutritional value of plant protein resources such as cottonseed meal, rapeseed meal, and mulberry leaves, Bacillus and lactic acid bacteria are both excellent candidate strains. However, the strains currently used in fermented feed are relatively single, and the compatibility and synergy between strains are weak, and the effect on intestinal function repair and inflammation regulation of aquatic animals is poor. (5) Although there are research reports that fermented feed with Lactobacillus plantarum or adding Lactobacillus plantarum to feed can improve the growth performance of aquatic animals, there is a lack of research on the mechanism of the effects of Lactobacillus plantarum on the nutrient utilization and metabolism of fish feed, which affects its promotion and application.(6) Lactobacillus plantarum is a facultative anaerobic bacterium. When applied by spraying, Lactobacillus plantarum generally has poor tolerance to oxygen and is easily inactivated in an oxygenated aquaculture water environment. Its effective time is short, and free Lactobacillus plantarum is easily lost with the water when the pond water is changed, requiring re-addition, which increases the aquaculture cost. Summary of the invention

[0004] The invention aims to provide a plant lactobacillus strain (Lactobacillus plantarum) FF34 with antagonistic properties against aquatic pathogens and a single-bacterium culture method thereof, a composite bacterial mixed culture method of FF34 and Bacillus, and a preparation method and application of a single-bacterium bacterial agent and a composite bacterial agent.

[0005] The plant lactobacillus FF34 of the present invention is facultatively anaerobic, has tolerance to oxygen, can ferment and utilize a wide range of carbon and nitrogen sources, has amylase activity, and can directly utilize starchy carbon sources. The plant lactobacillus FF34 rapidly degrades nitrite under anaerobic conditions, and has an antagonistic effect on common pathogenic bacteria in aquatic products such as Listonella anguillarum, Edwardsiella, Vibrio alginolyticus, Aeromonas hydrophila, and Escherichia coli, among which the inhibitory effect on Vibrio alginolyticus, Edwardsiella, Escherichia coli, and Aeromonas hydrophila is remarkable.

[0006] FF34 single bacteria aqueous solution or powder is obtained by strain activation, immobilized fermentation culture and preparation; Lactobacillus plantarum FF34 has good biocompatibility with a variety of beneficial Bacillus, such as Bacillus subtilis AB90008-15 or Bacillus amyloliquefaciens JSSW-LA or Bacillus coagulans JSSW-LA-07-1 or Clostridium butyricum JSIM-MCB20040312 or Zhangzhou Bacillus JSSW-BP44, which have antagonistic properties against aquatic pathogens, and can be synergistically fermented and co-cultured with one of these Bacillus to obtain FF34 composite bacterial agent.

[0007] Lactobacillus plantarum FF34 single bacteria agent or compound agent is used to ferment feed raw materials such as cottonseed meal, soybean meal, new leaf feed raw materials such as mulberry leaves, and fish, shrimp and crab feed, which can significantly improve the nutritional content and quality of feed raw materials. The resulting fermented feed is rich in probiotics, acid-soluble proteins and active small peptides, and the content of anti-nutritional factors such as tannins, free cottonphenol, and phytic acid is reduced; adding Lactobacillus plantarum FF34 single bacteria agent or compound agent to the basic diet or high-starch feed can promote the growth of intestinal tissue of aquatic animals, improve liver function, increase antioxidant activity, significantly improve the growth performance of aquatic animals, and reduce feed coefficient. After being adsorbed and fixed by pumice, activated carbon or ceramsite, Lactobacillus plantarum FF34 compound agent is placed in the aquaculture water body, which can degrade the ammonia nitrogen, nitrite, total phosphorus, and total nitrogen content of the water body, inhibit the growth of aquatic pathogens, and continuously improve the water quality of aquaculture ponds.

[0008] The technical solution of the present invention is a Lactobacillus plantarum FF34, which is classified and named as Lactobacillus plantarum (lactobacillus plantarum), and is preserved in China Center for Type Culture Collection CCTCC, address: Wuhan University, Wuhan, China, and preservation number CCTCC NO: M 20211221.

[0009] The plant lactobacillus strain FF34 of the present invention is a Gram-positive bacterium, which is rod-shaped under microscopic examination and does not produce spores. The strain belongs to a non-strict anaerobic bacterium and has a wide growth temperature range, and can grow at 15-45° C. After anaerobically culturing on an MRS plate for 48 hours, the diameter of the colony grown on the surface is 1.0-3.0 mm, and the colony is round, smooth, moist, opaque, milky white, and does not produce pigment.

[0010] Lactobacillus plantarum FF34 is a non-strict anaerobic bacterium, facultative anaerobic, oxygen-tolerant, homotypic fermentation of sugar substances to produce the beneficial substance DL-lactic acid, can adapt to the water and sediment environment of aquaculture ponds, can ferment and utilize a wide range of carbon and nitrogen sources, has amylase activity, and can directly utilize starch carbon sources. Lactobacillus plantarum FF34 can rapidly degrade nitrite under anaerobic conditions, and has antagonistic effects on common aquatic pathogens such as Listonella anguillarum, Edwardsiella, Vibrio alginolyticus, Aeromonas hydrophila, and Escherichia coli. Among them, the inhibitory effect on Vibrio alginolyticus, Edwardsiella, Escherichia coli, and Aeromonas hydrophila is significant.

[0011] Lactobacillus plantarum FF34 is obtained by activation of preserved strains, high-density immobilized fermentation culture in a fermentation tank and preparation of preparations to obtain its single-bacteria aqueous solution or powder; Lactobacillus plantarum FF34 can also be mixed with Bacillus subtilis AB90008-15, Bacillus amyloliquefaciens JSSW-LA, Bacillus coagulans JSSW-LA-07-1, Bacillus Zhangzhou JSSW-BP44 or Clostridium butyricum JSIM-MCB20040312, which have antagonistic properties against aquatic pathogens, for co-fermentation and co-culture to obtain a functional composite bacterial agent. Lactobacillus plantarum FF34 single bacteria agent or compound agent is used to ferment feed raw materials such as cottonseed meal, soybean meal, new leaf feed raw materials such as mulberry leaves, and fish, shrimp and crab feed, which can significantly improve the nutritional content of feed raw materials. The resulting fermented feed is rich in probiotics, acid-soluble proteins and active small peptides, and the content of anti-nutritional factors such as tannins, free cottonphenol, and phytic acid is reduced; adding Lactobacillus plantarum FF34 single bacteria agent or compound agent to the basic diet or high-starch feed can promote the growth of intestinal tissue of aquatic animals, improve liver function, increase antioxidant activity, significantly improve the growth performance of aquatic animals, and reduce feed coefficient. After being adsorbed and fixed by pumice, activated carbon or ceramsite, Lactobacillus plantarum FF34 compound agent can be put into the aquaculture water to degrade the ammonia nitrogen, nitrite, total phosphorus, and total nitrogen content of the water, inhibit the growth of aquatic pathogens, and continuously improve the water quality of aquaculture ponds.

[0012] In summary, Lactobacillus plantarum FF34 is a multifunctional microbial strain that can improve the nutritional value of feed, enhance the growth performance of aquaculture animals, and improve the water quality of aquaculture ponds.

[0013] The method for preparing a single bacterial agent having antagonistic properties against aquatic pathogens by using Lactobacillus plantarum FF34 comprises the following steps:

[0014] (1) Bacteria activation:

[0015] Aseptically open the freeze-dried preserved strain of Lactobacillus plantarum FF34, inoculate it into a test tube containing MRS broth, and culture it at 30-35℃ for 24-48h, then transfer it to a MRS broth triangular bottle, and culture and activate it at 30-35℃ for 20-48h; repeat the activation 2-3 times, examine it under a microscope, and count it. When the bacterial concentration reaches 10 9 CFU / mL as seed solution;

[0016] MRS broth medium composition (g / L): casein digest 10, beef extract powder 10, yeast extract powder 4, triammonium citrate 2, sodium acetate 5, magnesium sulfate 0.2, manganese sulfate 0.05, dipotassium hydrogen phosphate 2, glucose 20, Tween-80 1.08, made up to volume with distilled water, pH 5.7±0.2;

[0017] (2) Immobilized fermentation culture:

[0018] Preparation of seed culture solution in triangular flask: The seed solution obtained in step (1) is inoculated with 1% to 5% by volume into a 3L triangular flask filled with immobilized fermentation medium, and the volume is 90% by volume. The solution is cultured at 30 to 35°C for 24 to 40 hours. When the bacterial cell concentration reaches 10 9 When the concentration of CFU / mL is 0.04, it is used as the seed culture medium of the triangular flask; it is connected to the common aerobic fermenter for primary and secondary fixed fermentation and gradually expanded, that is, 3L triangular flask, then 500L fermenter, then 10T fermenter, and the inoculation is completed by sterile air pressure delivery between fermenters;

[0019] Primary immobilized culture: the immobilized culture seed liquid is inoculated into a 500L fermenter at a volume ratio of 1% to 5%, the volume ratio of the fermenter is 80% to 90%, the tank pressure is kept at 0.01 to 0.05Mpa with sterile air, intermittent stirring, a rotation speed of 80 to 100 r / min, and cultured at 30 to 35°C for 20 to 40 hours. When the pH is 4.5 to 5.5, the culture is terminated to obtain a primary immobilized culture liquid, in which the plant lactobacillus FF34 bacteria are adsorbed and fixed in the gel particles formed by calcium alginate + silicon dioxide, and the concentration of live bacteria is ≥5×10 9 CFU / mL;

[0020] Secondary immobilized culture: The primary immobilized culture solution is sent by sterile air pressure, and inoculated into a 10T fermenter at a volume ratio of 1% to 5%. The volume ratio of the fermenter is 70% to 90%. The sterile air is used to keep the tank pressure at 0.01 to 0.05Mpa, and the fermentation solution is stirred intermittently at a speed of 80 to 100 r / min and cultured at 30 to 38°C for 20 to 40 hours. When the pH of the fermentation solution drops to 4.0 to 5.5, the culture is terminated to obtain a secondary immobilized culture solution, in which the plant lactobacillus FF34 cells are adsorbed and fixed in the gel particles formed by calcium alginate + silicon dioxide, and the cell concentration is ≥5×10 9 CFU / mL;

[0021] The composition of the fixed fermentation medium is calculated in g / L: 10-40 carbon source, 3-15 nitrogen source, 0.2-5 potassium dihydrogen phosphate, 0-0.2 magnesium sulfate, 0-0.05 manganese sulfate, 5-40 calcium carbonate, 3-10 sodium alginate, 1-10 silicon dioxide, prepared with tap water, pH 6.0-6.5, sterilized at 121°C for 30 minutes; wherein the carbon source includes one or a combination of carbon sources such as wheat flour, corn starch, dextrin, molasses, glucose, fructose syrup, etc. The nitrogen source includes one or a combination of nitrogen sources such as fish meal peptone, tryptone, soy peptone, soy protein isolate, yeast extract, yeast powder, corn steep liquor, cottonseed meal, rapeseed meal, soybean meal, peanut meal and blood meal, etc.;

[0022] (3) Preparation of a single bacterial agent of Lactobacillus plantarum FF34 with antagonistic properties against aquatic pathogens:

[0023] (A) Preparation of Lactobacillus plantarum FF34 single bacterial solution: The secondary immobilized culture solution obtained in step (2) is filled to obtain a Lactobacillus plantarum FF34 single bacterial solution, wherein the concentration of live FF34 bacteria in the bacterial solution is ≥5.0×10 9 CFU / mL;

[0024] (B) Preparation of plant lactobacillus FF34 single bacterial powder: the secondary immobilized culture solution obtained in step (2) is centrifuged to collect wet bacterial cells, and the mixture is mixed in a mass ratio of wet bacterial cells: calcium carbonate: dextrin: silicon dioxide = 1:0.1-2:0.5-2:0.01, and spray-dried or vacuum-dried to obtain plant lactobacillus FF34 powder, wherein the concentration of live FF34 bacteria in the powder is not less than 1×10 10 CFU / g;

[0025] The method for preparing a composite bacterial agent by co-fermentation and co-culturing of Lactobacillus plantarum FF34 and Bacillus subtilis, comprises the following steps:

[0026] (1) The Bacillus species mixed with strain FF34 for fermentation are: aerobic Bacillus species, facultative aerobic Bacillus species and anaerobic Bacillus species, i.e., species with antagonistic properties against aquatic pathogens:

[0027] Bacillus subtilis AB90008-15, its antagonistic properties refer to patent number ZL 201911117444.2; deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, deposit number: CGMCC NO.18684, deposit date October 15, 2019.

[0028] Or Bacillus amyloliquefaciens JSSW-LA, its antagonistic properties refer to patent number ZL 201510733305.8; deposited in China Center for Type Culture Collection, deposit number: CCTCC NO:M 2015602, deposit date is October 12, 2015.

[0029] Or Bacillus coagulans JSSW-LA-07-1, its antagonistic properties refer to patent ZL 201510057403.4, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, deposit number: CGMCC NO.10182, deposit date December 15, 2014.

[0030] Or Clostridium butyricum JSIM-MCB20040312, see patent ZL201110083328.0, its antibacterial performance see the paper "Chen Qiuhong et al. Study on the biological characteristics of probiotic Clostridium butyricum CB-7 [J]. Anhui Agricultural Science, 2011, 39(10): 5922-5925", deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, deposit number CGMCC NO.1647, deposit date March 10, 2006.

[0031] Or Zhangzhou Bacillus JSSW-BP44, see patent application number 202010066397.X, deposited in China Center for Type Culture Collection, deposit number: CCTCC NO: 2019989, deposit date is December 2, 2019.

[0032] (2) The mixed fermentation of FF34 and the above-mentioned Bacillus was carried out in two steps: the first fermentation step was to activate the above-mentioned Bacillus by slant or triangular flask and then inoculate it into the mixed fermentation medium, the inoculation amount was 1% to 10% by volume, the culture temperature was 30 to 37°C, the culture was carried out for 24 to 48 hours, and the spore formation in the bacteria was observed under a microscope, indicating that the first fermentation was completed; the second fermentation step was to first adjust the pH of the fermentation liquid obtained in the first fermentation to 5.0 to 5.4 with hydrochloric acid, and then inoculate the activated Lactobacillus plantarum FF34, the inoculation amount was 1% to 10% by volume, the culture temperature was 30 to 35°C, intermittent stirring was performed, and the concentration of 20% (w / v) glucose or 30% calcium carbonate or ammonia water was fed to control the pH of the fermentation liquid to 4.5 to 5.0, and the culture was carried out for about 24 hours to obtain a mixed fermentation liquid of FF34 and Bacillus. Microscopic examination showed that lactobacillus and spores coexisted, and the concentration of live FF34 in the mixed fermentation liquid was ≥1.0×10 9 CFU / mL, Bacillus live bacteria concentration ≥5.0×10 8 CFU / mL, spore rate ≥90%;

[0033] The composition of the mixed fermentation medium is in g / L: 0-50 wheat flour, 0.1-5 peptone, 0-5 yeast extract, 0-3 sodium acetate, 0-10 corn starch, 0-2 ammonium sulfate, 0-5 sodium chloride, 0-5 calcium carbonate, 0-0.5 potassium dihydrogen phosphate, 0-0.5 magnesium sulfate, 0-0.5 manganese sulfate, 0-0.3 sodium alginate, tap water to volume, pH 7.0, sterilized at 121°C for 30 min;

[0034] (3) Preparation of plant lactobacillus FF34 composite bacterial agent:

[0035] (A) The mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus obtained in step (2) above is directly bottled to obtain a Lactobacillus plantarum FF34 composite bacterial liquid, wherein the live bacteria concentration of FF34 in the composite bacterial liquid is ≥1.0×10 9 CFU / mL, Bacillus live bacteria concentration ≥5.0×10 8 CFU / mL, spore rate ≥90%;

[0036] (B) The mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus obtained in the above step (2) is centrifuged to collect wet cells, mixed in a mass ratio of wet cells: calcium carbonate: silicon dioxide = 1:0.1 to 3:0.01, and spray-dried or vacuum-dried to obtain a Lactobacillus plantarum FF34 composite bacterial agent powder, wherein the live bacteria concentration of FF34 in the composite bacterial agent powder is ≥5×10 9 CFU / g, Bacillus live bacteria concentration ≥5.0×10 9 CFU / g.

[0037] Application of the plant lactobacillus FF34 single bacterial agent and its composite bacterial agent prepared in the above steps:

[0038] (1) Plant Lactobacillus FF34 single bacteria agent or composite bacteria agent is used to ferment fish, shrimp and crab feed or feed raw materials such as cottonseed meal, soybean meal, mulberry leaves, etc. The plant Lactobacillus FF34 single bacteria agent or composite bacteria agent is mixed with the feed or feed raw materials at a mass percentage of 0.1% to 5%, and water is added to adjust the moisture content of the material to 25% to 40%, and the material is sealed and fermented at 28 to 35° C. for 3 to 5 days. After the above feed or feed raw materials are fermented by plant Lactobacillus FF34 single bacteria agent or composite bacteria agent, there is no mildew, and the nutritional components of fish, shrimp and crab feed or feed raw materials are significantly improved. The obtained fermented feed is rich in probiotics, acid-soluble proteins and active small peptides, and the content of anti-nutritional factors such as tannins, free cottony phenol, and phytic acid is reduced.

[0039] (2) Adding 0.05% to 1% by weight of Lactobacillus plantarum FF34 single bacteria agent or compound bacteria agent to the basic diet or high-starch feed can promote the growth of intestinal tissue of aquatic animals, improve liver function, enhance antioxidant activity, significantly improve the growth performance of aquatic animals, and reduce the feed coefficient.

[0040] (3) After the plant lactobacillus FF34 composite bacterial agent aqueous solution is mixed with 2% to 4% sodium alginate by mass volume ratio, pumice, activated carbon or ceramsite is added for infiltration and adsorption for 1 to 4 hours, and then taken out, and then immersed in 2% to 3% calcium chloride solution, and cured and cross-linked at 4 to 8°C for 1 to 10 hours to obtain the plant lactobacillus FF34 composite bacterial immobilized bacterial pellets (blocks), and the bacterial cell concentration in the bacterial pellets (blocks) is not less than 5×10 8 CFU / g. The immobilized bacterial pellets (blocks) of Lactobacillus plantarum FF34 composite bacteria are placed in the aquaculture water body and used continuously for more than 20 days. They can continuously degrade the content of ammonia nitrogen, nitrite, total phosphorus and total nitrogen in the water body, inhibit the growth of aquatic pathogens such as Aeromonas, and continuously improve the water quality of aquaculture ponds.

[0041] Determination of the antibacterial activity of Lactobacillus plantarum FF34: The pathogenic bacteria and Lactobacillus plantarum FF34 culture liquid were inoculated into LB broth to make the initial concentration of FF34 in the culture medium 10 5 ~10 6 CFU / mL, the initial concentration of pathogenic bacteria is 10 5 ~10 7 CFU / mL, incubate at 30℃ for 24h, take samples and count at 0h and 24h, and observe the antagonistic effect of FF34 on pathogenic bacteria. The results showed that it had antagonistic effects on common aquatic pathogens such as Listonella anguillarum, Edwardsiella, Vibrio alginolyticus, Aeromonas hydrophila and Escherichia coli, among which the inhibitory effect on Vibrio alginolyticus, Edwardsiella, Escherichia coli and Aeromonas hydrophila was significant.

[0042] Determination of the ability of Lactobacillus plantarum FF34 to degrade nitrite: MRS broth medium was divided into 50 mL / test tube, and sterilized with 2 mol / L sodium nitrite solution at 121°C for 15 min. Then, 0.1 mL of 2 mol / L sodium nitrite solution was added to each test tube containing MRS broth medium, and then 5 mL of activated Lactobacillus plantarum FF34 suspension was inoculated. The control group was inoculated with 5 mL of sterile distilled water. Each group had 3 parallels and was cultured at 35°C for 20 h. Samples were taken at 0 h, 7 h, and 20 h, and centrifuged at 4000 r / min for 10 min. The supernatant was taken and the nitrite nitrogen concentration was detected using the American Hach reagent. Data were statistically analyzed using SPSS18.0, and all results were expressed as mean ± standard error. Indicates that the independent sample T test was used to compare the differences between the groups. Under anaerobic conditions, nitrite nitrogen can be significantly degraded within 20 hours, and the degradation rate reaches 96%.

[0043] Beneficial effects of the present invention:

[0044] (1) General lactobacillus (including plant lactobacillus) has complex nutritional requirements and lacks amylase activity. It not only needs monosaccharides or disaccharides such as glucose, fructose or maltose as carbon sources, but also needs amino acids, peptides, nucleic acid derivatives, salts, fatty acids or fatty acid lipids, and each species has special nutritional requirements. This characteristic limits the source of raw materials for large-scale cultivation of beneficial lactobacillus, resulting in increased culture medium cost. Plant lactobacillus FF34 of the present invention has amylase activity, can directly utilize starchy carbohydrates, and has a wide range of available carbon and nitrogen sources. This characteristic is different from general lactobacillus (including general plant lactobacillus), so that the carbon source that plant lactobacillus FF34 can utilize is expanded from monosaccharides or disaccharides such as glucose, fructose or maltose to starchy carbohydrates, thereby making cheap agricultural and sideline products such as wheat flour, cottonseed meal, rapeseed meal, soybean meal, etc. can be directly used as carbon sources and nitrogen sources for the proliferation and cultivation of FF34, greatly reducing the culture medium cost.

[0045] (2) Lactobacillus plantarum FF34 is a facultative lactobacillus with good tolerance to dissolved oxygen. It can grow without strict anaerobic conditions. This characteristic makes it possible to use ordinary aerobic fermentation tanks to cultivate Lactobacillus plantarum FF34 on a large scale. However, the tank pressure maintenance of the aerobic fermentation tank and the use of sterile air pressure to transfer the seeds between the seed tank and the fermentation tank still result in the risk of high dissolved oxygen during the fermentation and cultivation process, thereby inhibiting the growth of FF34. The present invention adopts a high-density and low-cost immobilized fermentation and expansion method. In an ordinary aerobic fermentation tank, a culture method in which the bacterial body is immobilized and wrapped with sodium alginate while fermenting is used. The growth of Lactobacillus plantarum FF34 utilizes the acid-producing characteristics of starch or sugar to dissolve the calcium carbonate in the culture medium to release free calcium ions, and form calcium alginate gel with sodium alginate in the culture medium. The bacterial body is wrapped in small particles of calcium alginate gel to isolate the dissolved oxygen transmission and protect the growth activity of the bacterial body, thereby obtaining a high concentration of bacterial body. The concentration of Lactobacillus plantarum FF34 in the fermentation broth exceeds 5×10 9 CFU / mL; then spray drying was used to obtain FF34 bacterial concentrations exceeding 1×10 10 CFU / g, thereby further reducing the cost of using Lactobacillus plantarum FF34 aqueous solution and powder preparation, which is conducive to the promotion and application of Lactobacillus plantarum FF34 in ecological breeding of livestock, poultry and fishery.

[0046] (3) Lactobacillus plantarum FF34 has good compatibility with a variety of beneficial Bacillus species and can be co-cultured with multiple strains for mixed fermentation. Lactobacillus plantarum FF34 has low nutritional requirements for growth, can utilize a wide range of carbon and nitrogen sources, and can directly utilize starchy carbohydrates. Since starchy carbon sources such as wheat bran are suitable and cheap carbon sources for Bacillus, the ability of Lactobacillus plantarum FF34 to directly utilize starchy carbon sources enables FF34 to be mixed and fermented with a variety of Bacillus. The types of Bacillus covered are wide, involving aerobic Bacillus, facultative aerobic Bacillus and anaerobic Bacillus, such as Bacillus subtilis AB90008-15 (aerobic bacteria) or Bacillus amyloliquefaciens JSSW-LA (aerobic bacteria) or Bacillus coagulans JSSW-LA-07-1 (facultative aerobic bacteria) or Clostridium butyricum JSIM-MCB20040312 (anaerobic bacteria) or Bacillus Zhangzhouensis JSSW-BP44 (facultative aerobic bacteria) with antagonistic properties to aquatic pathogens. When FF34 and Bacillus are co-cultured in a mixed fermentation medium, after these Bacillus form spores, the pH is adjusted to put the spores into a dormant state, and then FF34 is continuously inoculated to fully utilize the remaining nutrients in the fermentation medium for FF34 proliferation culture, and finally a composite bacterial agent containing a high concentration of Bacillus (spore rate ≥ 90%) + Lactobacillus plantarum FF34 is obtained, which reduces the fermentation cost of the composite bacterial agent and reduces the discharge of fermentation waste liquid.

[0047] (4) Lactobacillus plantarum FF34 has antagonistic effects on common aquatic pathogens such as Listonella anguillarum, Edwardsiella, Vibrio alginolyticus, Aeromonas hydrophila and Escherichia coli, among which the inhibitory effects on Vibrio alginolyticus, Edwardsiella, Escherichia coli and Aeromonas hydrophila are significant. In addition, Lactobacillus plantarum FF34 can be mixed and fermented with a variety of Bacillus with antagonistic properties against aquatic pathogens, such as Bacillus subtilis AB90008-15, Bacillus amyloliquefaciens JSSW-LA, Bacillus coagulans JSSW-LA-07-1 or Clostridium butyricum JSIM-MCB20040312. The obtained composite bacterial agent further enriches the types of aquatic pathogens that the bacterial agent can antagonize, enhances the effectiveness of aquatic animal disease prevention and control, and promotes the healthy growth of aquatic animals.

[0048] (5) After the feed is fermented by Lactobacillus plantarum FF34 single bacteria or composite bacteria, there is no mildew, the fermented feed is rich in probiotics, the nutritional components of the feed are significantly improved, the soluble protein is increased by 42.38% to 116.31%, the acid-soluble protein is increased by 118.54% to 218.53%, the active small peptides are increased by 42.14% to 168.41%, and the content of anti-nutritional factors such as phytic acid, tannins, and free cottony phenol is significantly reduced. The preliminary study of the mechanism of the influence of Lactobacillus plantarum FF34 on the nutrient utilization and metabolism of fish feed by the present invention shows that adding Lactobacillus plantarum FF34 single bacteria or composite bacteria at a mass percentage of 0.05% to 1% in the basic diet or high-starch feed can promote the growth of intestinal tissue of aquatic animals, improve liver function, increase antioxidant activity, significantly improve the growth performance of aquatic animals, and reduce the feed coefficient.

[0049] (6) The plant lactobacillus FF34 compound bacterial agent is immobilized by adsorption on pumice, activated carbon or expanded clay and then placed in the aquaculture water. Compared with the direct spraying of unimmobilized bacterial agents, the immobilized bacteria are not easily inactivated or lost in the oxygen-containing aquaculture water environment. The effective time is more than 20 days. It can sustainably degrade the ammonia nitrogen, nitrite, total phosphorus and total nitrogen content in the water, inhibit the growth of aquatic pathogens, and improve the water quality of aquaculture ponds.

[0050] Biomaterial sample storage

[0051] (1) Lactobacillus plantarum FF34, classified as Lactobacillus plantarum, is deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: M20211221, deposit date: October 11, 2021. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1Effects of Lactobacillus plantarum and high starch supplementation on Oil Red O-stained sections of liver of young Megalobrama amblycephala. Figure A shows Oil Red O-stained sections of the 15WM group (×200 times), Figure B shows Oil Red O-stained sections of the 35WM+LAB group (×200 times), and Figure C shows Oil Red O-stained sections of the 35WM group (×200 times).

[0053] Figure 2 Neighbor-Joining phylogenetic tree of strain FF-34 constructed based on 16S rRNA gene sequence alignment with Enterococcus faecalis (Y18293.1) as the outbranch. DETAILED DESCRIPTION

[0054] Example 1: Screening of bacterial strains

[0055] The muddy water was collected from the crab breeding pond in Mashan Town, Binhu District, Wuxi City, and physiological saline was added to grind to make a homogenate. 0.1 mL was taken by a pipette and spread on MRS solid medium for anaerobic culture at 25-35°C. The colonies with good growth were selected according to the colony size and inoculated and screened repeatedly until a uniform single colony was obtained, which was named FF34.

[0056] Example 2: Bacterial species identification

[0057] (1) Morphological characteristics: Lactobacillus plantarum strain FF34 was Gram-positive. After being spread on an MRS plate and cultured anaerobically at 35°C for 48 h, the colonies grown were raised, smooth, dense, white, opaque, and 1 to 3 mm in diameter. The deep colonies in MRS pour-over culture were lens-shaped or diamond-shaped, and the colonies at the bottom of the plate were flat and round. Under a microscope (16×100), the bacteria were short rod-shaped with rounded and slightly square ends, arranged in pairs or short chains, (0.9 to 1.2) μm × (2 to 4) μm, without flagella, and able to move.

[0058] (2) Biochemical characteristics:

[0059] Table 1 Physiological and biochemical characteristics of strain FF-34 – enzyme activity, carbon source oxidation

[0060]

[0061]

[0062] +: positive reaction; -: negative reaction; W: weak positive reaction

[0063] Table 2 Physiological and biochemical characteristics of strain FF-34 – Acid production using carbon sources

[0064]

[0065]

[0066] +: positive reaction; -: negative reaction; W: weak positive reaction

[0067] (3) 16S rRNA sequence analysis and phylogenetic tree construction:

[0068] The 16S rRNA gene sequence of Lactobacillus plantarum strain FF34 is shown in SEQ ID NO.1.

[0069] Sequencing results of the gene sequence of Lactobacillus plantarum strain FF34: The 16S rRNA gene sequence amplified by the strain was searched for homology in NCBI by Blast, and the 16S rRNA gene sequence of the genus Lactobacillus was retrieved. The strain phylogenetic tree was constructed by the neighbor-joining method. The isolated strain belongs to the same branch as Lactobacillus plantarum subsp. plantarum ATCC 14917 (accession number: ACGZ01000098) on the phylogenetic tree. The isolated strain was identified as Lactobacillus plantarum based on morphological and physiological and biochemical characteristics (see Figure 2 ).

[0070] Example 3: Determination of the antibacterial ability of Lactobacillus plantarum FF34 against aquatic pathogens

[0071] The pathogenic bacteria and Lactobacillus plantarum FF34 bacterial solution were inoculated into LB medium and cultured at 30°C for 24 hours. Samples were taken and counted at 0 hours and 24 hours to observe the antagonistic effect of Lactobacillus plantarum FF34 on the pathogenic bacteria. The results are shown in Table 3:

[0072] Table 3 Co-culture determination results of Lactobacillus plantarum FF34 and pathogenic bacteria

[0073]

[0074]

[0075] Under co-culture conditions, Lactobacillus plantarum FF34 had the strongest inhibitory ability against Vibrio alginolyticus and Edwardsiella, followed by Escherichia coli and Aeromonas hydrophila, and had a weaker inhibitory ability against Listonella anguillarum. Lactobacillus plantarum FF34 completely inhibited the growth of Vibrio alginolyticus within 24 hours, reducing the proliferation of Edwardsiella by about 2 orders of magnitude, and the proliferation of Escherichia coli and Aeromonas hydrophila by one order of magnitude respectively.

[0076] Example 4: Determination of the ability of Lactobacillus plantarum FF34 to degrade nitrite

[0077] MRS broth medium was divided into 50mL / test tubes, sterilized with 2mol / L sodium nitrite solution at 121℃ for 15min, and then 0.1mL of 2mol / L sodium nitrite solution was added to each test tube containing MRS broth medium, and then 5mL of activated plant lactobacillus FF34 suspension was inoculated. The control group was connected with 5mL of sterile distilled water. Each group had 3 parallels and was cultured at 35℃ for 20h. Samples were taken at 0h, 7h and 20h, centrifuged at 4000r / min for 10min, and the supernatant was taken. The nitrite nitrogen concentration was detected with the American Hach reagent. The data were statistically analyzed using SPSS18.0, and all results were expressed as mean ± standard error. Indicates that the independent sample T test was used to compare the differences among the groups.

[0078] The results in Table 4 show that Lactobacillus plantarum FF34 has high nitrite reductase activity and can rapidly degrade nitrite nitrogen under anaerobic conditions. The degradation is extremely significant within 20 hours, with a degradation rate of 82.30±0.50% in 7 hours and 96.00±0.01% in 20 hours.

[0079] Table 4 Nitrite degradation ability of Lactobacillus plantarum FF34

[0080] Grouping Control group FF34 test group time Nitrite nitrogen mg / L Nitrite nitrogen mg / L 0h <![CDATA[135.22±2.34 Aa ]]> <![CDATA[135.89±2.34 Aa ]]> 7h <![CDATA[135.69±0.55 Aa ]]> <![CDATA[24.05±0.99 Bb ]]> 20h <![CDATA[134.31±0.20 Aa ]]> <![CDATA[5.22±1.24 Cb ]]> 20h nitrite nitrogen degradation rate <![CDATA[1.12±1.37% b ]]> <![CDATA[96.00±0.01% a ]]>

[0081] Note: Different lowercase letters in the same row indicate significant differences (P<0.05), and different capital letters in the same column indicate significant differences (P<0.05).

[0082] Example 5 Preparation of a single bacterial preparation of Lactobacillus plantarum FF34 having antagonistic properties against aquatic pathogens

[0083] 1) Bacteria activation:

[0084] Aseptically open the freeze-dried preserved strain of Lactobacillus plantarum FF34, inoculate it into a test tube containing MRS broth, and culture it at 30-35℃ for 24-48h, then transfer it to a MRS broth triangular bottle, and culture and activate it at 30-35℃ for 20-48h; repeat the activation 2-3 times, examine it under a microscope, and count it. When the bacterial concentration reaches 10 9 CFU / mL as seed solution;

[0085] MRS broth medium composition (g / L): casein digest 10, beef extract powder 10, yeast extract powder 4, triammonium citrate 2, sodium acetate 5, magnesium sulfate 0.2, manganese sulfate 0.05, dipotassium hydrogen phosphate 2, glucose 20, Tween-80 1.08, made up to volume with distilled water, pH 5.7±0.2;

[0086] (2) Immobilized fermentation culture:

[0087] Preparation of seed culture solution in triangular flask: The seed solution obtained in step (1) is inoculated with 1% to 5% by volume into a 3L triangular flask filled with immobilized fermentation medium, and the volume is 90% by volume. The solution is cultured at 30 to 35°C for 24 to 40 hours. When the bacterial cell concentration reaches 10 9 When the CFU / mL is reached, it is used as the seed culture medium of the triangular flask and introduced into a common aerobic fermenter for primary and secondary fixed fermentation and step-by-step expansion, i.e., 3L triangular flask, then 500L fermenter, then 10T fermenter, and the inoculation is completed by sterile air pressure delivery between fermenters;

[0088] Primary immobilized culture: the immobilized culture seed liquid is inoculated into a 500L fermenter at a volume ratio of 1% to 5%, the volume ratio of the fermenter is 80% to 90%, the tank pressure is kept at 0.01 to 0.05Mpa with sterile air, intermittent stirring, a rotation speed of 80 to 100 r / min, and cultured at 30 to 35°C for 20 to 40 hours. When the pH is 4.5 to 5.5, the culture is terminated to obtain a primary immobilized culture liquid, in which the plant lactobacillus FF34 bacteria are adsorbed and fixed in the gel particles formed by calcium alginate + silicon dioxide, and the concentration of live bacteria is ≥5×10 9 CFU / mL;

[0089] Secondary immobilized culture: The primary immobilized culture solution is sent by sterile air pressure, and inoculated into a 10T fermenter at a volume ratio of 1% to 5%. The volume ratio of the fermenter is 70% to 90%. The sterile air is used to keep the tank pressure at 0.01 to 0.05Mpa, and the fermentation solution is stirred intermittently at a speed of 80 to 100 r / min and cultured at 30 to 38°C for 20 to 40 hours. When the pH of the fermentation solution drops to 4.0 to 5.5, the culture is terminated to obtain a secondary immobilized culture solution, in which the plant lactobacillus FF34 cells are adsorbed and fixed in the gel particles formed by calcium alginate + silicon dioxide, and the cell concentration is ≥5×10 9 CFU / mL;

[0090] The composition of the fixed fermentation medium is calculated in g / L: carbon source 10-40, nitrogen source 3-15, dipotassium hydrogen phosphate 0.2-5, magnesium sulfate 0-0.2, manganese sulfate 0-0.05, calcium carbonate 5-40, sodium alginate 3-10, silicon dioxide 1-10, prepared with tap water, pH 6.0-6.5, sterilized at 121°C for 30 minutes; wherein the carbon source includes one or a combination of carbon sources such as wheat flour, corn starch, dextrin, molasses, glucose, fructose syrup, etc. The nitrogen source includes one or a combination of nitrogen sources such as fish meal peptone, tryptone, soy peptone, soy protein isolate, yeast extract, yeast powder, corn steep liquor, cottonseed meal, rapeseed meal, soybean meal, peanut meal and blood meal, etc.

[0091] (3) Preparation of plant lactobacillus FF34 single bacterial preparation:

[0092] (A) Preparation of Lactobacillus plantarum FF34 single bacterial solution: The secondary immobilized culture solution obtained in step (2) is filled to obtain a Lactobacillus plantarum FF34 solution, wherein the concentration of live FF34 bacteria in the bacterial solution is ≥5.0×10 9 CFU / mL;

[0093] (B) Preparation of plant lactobacillus FF34 single bacterial powder: the secondary immobilized culture solution obtained in step (2) is centrifuged to collect wet bacterial cells, and the mixture is mixed in a mass ratio of wet bacterial cells: calcium carbonate: dextrin: silicon dioxide = 1:0.1-2:0.5-2:0.01, and spray-dried or vacuum-dried to obtain plant lactobacillus FF34 powder, wherein the concentration of live FF34 bacteria in the powder is not less than 1×10 10 CFU / g.

[0094] Example 6: Mixed fermentation and co-cultivation of Lactobacillus plantarum FF34 and Clostridium butyricum JSIM-MCB20040312 (Accession No. CGMCC No. 1647) and preparation of composite bacterial agent

[0095] (1) Activation of bacterial strains and cultivation of triangular flask seed solution

[0096] (A) Activation of Clostridium butyricum JSIM-MCB 20040312 and culture of seed solution in triangular flask:

[0097] Aseptically opening the freeze-dried preserved strain of Clostridium butyricum JSIM-MCB 20040312, inoculating it with a test tube filled with an activation culture medium, and performing static anaerobic culture at 30-37° C. for 18-24 hours, then transferring it to a fresh activation culture medium at an inoculation amount of 1%-10% by volume, and culturing it at 30-37° C. for 20-24 hours. After microscopic examination, when more than 90% of the bacteria form spores, it is mature, and the activation is repeated 2-3 times. The seed bacterial suspension is inoculated with an inoculation amount of 1%-10% by volume into several 3L triangular flasks filled with seed culture medium, and the volume of the triangular flask is 80%-90% by volume. It is statically cultured in a constant temperature incubator at 30-37° C. for 20-24 hours, and after microscopic examination, when more than 90% of the bacteria form spores, it is used as triangular flask seed liquid;

[0098] Clostridium butyricum JSIM-MCB 20040312 activation and seed culture medium composition (in g / L):

[0099] Yeast extract 3-5, beef extract 5-20, peptone 5-20, glucose 5-10, soluble starch 1-2, sodium chloride 5, sodium acetate 3-5, cysteine ​​hydrochloride 0.5-1, calcium carbonate 1-7, prepared with deionized water, pH 6.0-7.5;

[0100] (B) Activation of Lactobacillus plantarum FF34 and culture of seed solution in triangular flask

[0101] Aseptically open the freeze-dried preserved strain of Lactobacillus plantarum FF34, inoculate it into a test tube containing MRS broth, and culture it at 30-35℃ for 24-48h, then transfer it to a small triangular bottle of MRS broth, and culture and activate it at 30-35℃ for 20-48h; repeat the activation 2-3 times, examine it under a microscope, and count it. When the bacterial concentration is 10 9 CFU / mL was used as seed solution, and the inoculation volume was 1% to 5% in several 3L triangular flasks filled with MRS fermentation medium, and the volume was 90% by volume. The culture was kept at 30 to 35°C for 24 to 40 hours. When the bacterial concentration reached 10 9 CFU / mL, used as seed solution for triangular flask;

[0102] MRS broth medium composition (g / L): casein digest 10, beef extract powder 10, yeast extract powder 4, triammonium citrate 2, sodium acetate 5, magnesium sulfate 0.2, manganese sulfate 0.05, dipotassium hydrogen phosphate 2, glucose 20, Tween-80 1.08, made up to volume with distilled water, pH 5.7±0.2;

[0103] (2) Mixed fermentation and co-culture

[0104] The mixed fermentation of FF34 and Clostridium butyricum JSIM-MCB 20040312 was carried out in two steps: the first fermentation step: the Clostridium butyricum JSIM-MCB obtained in step (A) above was The 20040312 fermentation tank seed liquid is inoculated into a 500L fermentation tank filled with a mixed fermentation medium at a volume ratio of 1% to 10%, and the liquid volume is 70% to 80% (V / V). The mixture is cultured at 30 to 37° C. for 24 to 48 hours. The spore formation in the bacterial body is observed under a microscope, and the first fermentation is completed. The second fermentation is firstly to adjust the pH of the fermentation liquid obtained by the first fermentation to 5.0 to 5.4 with hydrochloric acid, and then the plant lactobacillus FF34 triangular flask seed liquid obtained in the above step (B) is inoculated with an inoculation volume of 1% to 10%, the culture temperature is 30 to 35° C., and the mixture is stirred intermittently. The concentration of glucose or calcium carbonate or ammonia water is 20% (w / v). The pH of the fermentation liquid is controlled to 4.5 to 5.0. The mixture is cultured for about 24 hours to obtain a mixed fermentation liquid of FF34 and bacillus. The coexistence of lactobacillus and spores can be seen under a microscope, and the concentration of live FF34 bacteria in the mixed fermentation liquid is ≥1.0×10 9 CFU / mL, Bacillus live bacteria concentration ≥5.0×10 8 CFU / mL, spore rate ≥90%;

[0105] The composition of the mixed fermentation medium is in g / L: 0-50 wheat flour, 0.1-5 peptone, 0-5 yeast extract, 0-3 sodium acetate, 0-10 corn starch, 0-2 ammonium sulfate, 0-5 sodium chloride, 0-5 calcium carbonate, 0-0.5 potassium dihydrogen phosphate, 0-0.5 magnesium sulfate, 0-0.5 manganese sulfate, 0-0.3 sodium alginate, tap water to volume, pH 7.0, sterilized at 121°C for 30 min;

[0106] (3) Preparation of plant lactobacillus FF34 composite bacterial agent:

[0107] (A) The mixed fermentation liquid of Lactobacillus plantarum FF34 and Clostridium butyricum JSIM-MCB2004031 obtained in the above step (2) is directly bottled to obtain a Lactobacillus plantarum FF34 composite bacterial liquid, wherein the live bacteria concentration of FF34 in the composite bacterial liquid is ≥1.0×10 9 CFU / mL, Bacillus live bacteria concentration ≥5.0×10 8 CFU / mL, spore rate ≥90%;

[0108] (B) The mixed fermentation liquid of Lactobacillus plantarum FF34 and Clostridium butyricum JSIM-MCB2004031 obtained in the above step (2) is centrifuged to collect wet cells, mixed in a mass ratio of wet cells: calcium carbonate: silicon dioxide = 1:0.1 to 3:0.01, and spray-dried or vacuum-dried to obtain Lactobacillus plantarum FF34 composite bacterial agent powder, wherein the live bacteria concentration of FF34 in the powder is ≥5×10 9 CFU / g, Bacillus live bacteria concentration ≥5.0×10 9 CFU / g.

[0109] Example 7 Mixed fermentation and co-cultivation of Lactobacillus plantarum FF34 and Bacillus subtilis AB90008-15 (Accession No. CGMCC NO.18684) and preparation of composite bacterial agent

[0110] (1) Activation of bacterial strains and cultivation of triangular flask seed solution

[0111] (A) Activation and seed preparation of Bacillus subtilis AB90008-15:

[0112] Aseptically opening the Bacillus subtilis AB90008-15 freeze-dried tube preserved bacteria, inoculating it on bran nutrient agar, culturing it at 30-37° C. for 24-48 hours, then transferring it to the bran nutrient agar slant, repeatedly activating it 2-3 times, and inspecting it under a microscope. When more than 90% of the Bacillus subtilis bacteria form spores, it is mature, scraping the bacterial lawn, and preparing a bacterial suspension with sterile water as a Bacillus subtilis seed liquid;

[0113] The composition of the bran nutrient agar slant medium is in g / L: peptone 10, beef extract 3, NaCl 5, bran 10, agar 15-20, prepared with distilled water to a fixed volume, pH 7.0-7.2;

[0114] (B) Activation of Lactobacillus plantarum FF34 and culture of seed solution in triangular flask

[0115] Aseptically open the freeze-dried preserved strain of Lactobacillus plantarum FF34, inoculate it into a test tube containing MRS broth, and culture it at 30-35℃ for 24-48h, then transfer it to a small triangular bottle of MRS broth, and culture and activate it at 30-35℃ for 20-48h; repeat the activation 2-3 times, examine it under a microscope, and count it. When the bacterial concentration is 10 9 CFU / mL was used as seed solution, and the inoculation volume was 1% to 5% in several 3L triangular flasks filled with fixed fermentation medium, and the volume was 90%. The culture was kept at 30 to 35 °C for 24 to 40 hours. When the bacterial concentration reached 10 9 CFU / mL, used as seed solution for triangular flask;

[0116] MRS broth medium composition (g / L): casein digest 10, beef extract powder 10, yeast extract powder 4, triammonium citrate 2, sodium acetate 5, magnesium sulfate 0.2, manganese sulfate 0.05, dipotassium hydrogen phosphate 2, glucose 20, Tween-80 1.08, made up to volume with distilled water, pH 5.7±0.2;

[0117] The composition of the fixed fermentation medium is calculated in g / L: carbon source 10-40, nitrogen source 3-15, dipotassium hydrogen phosphate 0.2-5, magnesium sulfate 0-0.2, manganese sulfate 0-0.05, calcium carbonate 5-40, sodium alginate 3-10, silicon dioxide 1-10, prepared with tap water, pH 6.0-6.5, sterilized at 121°C for 30 minutes; wherein the carbon source includes one or a combination of carbon sources such as wheat flour, corn starch, dextrin, molasses, glucose, fructose syrup, etc. The nitrogen source includes one or a combination of nitrogen sources such as fish meal peptone, tryptone, soy peptone, soy protein isolate, yeast extract, yeast powder, corn steep liquor, cottonseed meal, rapeseed meal, soybean meal, peanut meal and blood meal, etc.

[0118] (2) Mixed fermentation and co-culture

[0119] The mixed fermentation of FF34 and Bacillus subtilis AB90008-15 was carried out in two steps: the first fermentation step: the Bacillus subtilis seed suspension obtained in the above step (A) was inoculated at a volume ratio of 1% to 10% into a 500L fermentation tank filled with fermentation medium for primary seed tank culture, the liquid volume was 50% to 60% by volume, and the ventilation volume was 15m 3 / h, tank pressure 0.05~0.1Mpa, rotation speed 100~150r / min, 30~37℃, ventilation culture 24~48h, microscopic examination of spore formation in the bacteria, that is, the first fermentation is completed; the second fermentation is to adjust the pH of the fermentation liquid to 5.0~5.4 with hydrochloric acid, then access the plant lactobacillus FF34 triangular flask seed liquid obtained in the above step (B), the inoculation amount is 1%~10% by volume, the culture temperature is 30~35℃, intermittent stirring, the flow addition concentration is 20% (w / v) glucose or 30% calcium carbonate or ammonia water, the pH of the fermentation liquid is controlled to 4.5~5.0, and the culture is carried out for about 24h to obtain a mixed fermentation liquid of FF34 and bacillus, and microscopic examination shows that lactobacillus and spores coexist, and the concentration of FF34 live bacteria in the mixed fermentation liquid is ≥1.0×10 9 CFU / mL, Bacillus live bacteria concentration ≥5.0×10 8 CFU / mL, spore rate ≥90%;

[0120] The composition of the mixed fermentation medium is in g / L: 0-50 wheat flour, 0.1-5 peptone, 0-5 yeast extract, 0-3 sodium acetate, 0-10 corn starch, 0-2 ammonium sulfate, 0-5 sodium chloride, 0-5 calcium carbonate, 0-0.5 potassium dihydrogen phosphate, 0-0.5 magnesium sulfate, 0-0.5 manganese sulfate, 0-0.3 sodium alginate, tap water to volume, pH 7.0, sterilized at 121°C for 30 min;

[0121] (3) Preparation of plant lactobacillus FF34 composite bacterial agent:

[0122] (A) The mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus subtilis AB90008-15 obtained in the above step (2) is directly bottled to obtain a Lactobacillus plantarum FF34 composite bacterial liquid, wherein the live bacteria concentration of FF34 in the composite bacterial liquid is ≥1.0×10 9 CFU / mL, Bacillus live bacteria concentration ≥5.0×10 8 CFU / mL, spore rate ≥90%;

[0123] (B) The mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus subtilis AB9008-15 obtained in the above step (2) is centrifuged to collect wet cells, and the wet cells: calcium carbonate: silicon dioxide = 1:0.1 to 3:0.01 are mixed, and spray drying or vacuum drying is performed to obtain Lactobacillus plantarum FF34 composite bacterial agent powder, and the concentration of live FF34 in the powder is ≥5×10 9 CFU / g, Bacillus live bacteria concentration ≥5.0×10 9 CFU / g.

[0124] Example 8: Results of fermentation of feed with Lactobacillus plantarum FF34 composite bacterial agent

[0125] The Lactobacillus plantarum FF34 composite bacterial agent powder prepared in Example 7 was mixed with shrimp feed, crab feed, lobster feed and cottonseed meal in an addition amount of 0.1-5% by mass, and water was added to adjust the moisture content of the materials to 25%-40%, and the materials were respectively packed into fermentation bags with a volume capacity of 5L (commercially available) at 1kg / bag. Shrimp feed, crab feed, lobster feed and cottonseed meal fermented without adding bacterial powder were used as controls. Three parallels were added in each group and fermented in a closed fermentation bag at 28-35°C for 3-5 days. The fermentation results are shown in Table 5 below. The data were statistically analyzed using SPSS18.0. All results are expressed as mean±standard error Indicates that the independent sample T test was used to compare the differences among the groups.

[0126] Table 5 Results of Lactobacillus plantarum FF34 composite bacterial agent shrimp and crab feed

[0127]

[0128] Note: * indicates significant difference (p<0.05).

[0129] The results in Table 5 show that after the shrimp feed, crab feed and crayfish feed were fermented with the plantarum Lactobacillus FF34 composite bacterial agent, the fermented feed was rich in beneficial lactic acid bacteria and Bacillus, and the nutritional value of the feed was significantly improved. The soluble protein content increased by 116.31%, 92.57% and 42.38% respectively, the acid-soluble protein content increased by 218.53%, 155.21% and 118.41% respectively, and the small peptide content increased by 45.20%, 76.13% and 168.41% respectively.

[0130] Table 6 Results of cottonseed meal fermentation with Lactobacillus plantarum FF34 composite bacterial agent

[0131]

[0132]

[0133] Note: * indicates significant difference (p<0.05).

[0134] The results in Table 6 show that after the cottonseed meal was fermented with the compound bacterial agent of Lactobacillus plantarum FF34, it was rich in probiotics such as lactic acid bacteria, among which lactic acid bacteria proliferated in large quantities, with a concentration as high as 10 9 CFU / g, the Bacillus in cottonseed meal fermented with FF34 compound bacterial agent can reach 10 7CFU / g. In the process of cottonseed meal fermentation, plant lactobacillus FF34 composite bacterial agent inhibited the growth of mold and prevented the mildew caused by cottonseed meal after adding water. After cottonseed meal fermented by plant lactobacillus FF34 composite bacteria, the nutritional components of cottonseed meal were improved, and the crude protein, soluble protein, acid-soluble protein and active small peptide contents of cottonseed meal were significantly increased (p < 0.05), soluble protein increased by 85.13%, acid-soluble protein increased by 155.42%, and active small peptides increased by 42.14%. Although the crude protein of fermented cottonseed meal increased by 9.02%, the difference was not significant (p > 0.05). In addition, after cottonseed meal fermented by plant lactobacillus FF34 composite bacterial agent, the content of anti-nutritional factors such as phytic acid, tannin and free cottonpol in cottonseed meal was significantly reduced (p < 0.05), phytic acid decreased by 63.74%, tannin decreased by 50.61%, and free cottonpol decreased by 54.17%.

[0135] Example 9: Effects of adding Lactobacillus plantarum FF34 to feed on growth and feed utilization of young Megalobrama amblycephala

[0136] In this embodiment, fish meal, soybean meal, rapeseed meal and cottonseed meal were used as protein sources, and soybean oil was used as fat source to prepare two groups of test feeds, namely a conventional control feed group (CN) and a Lactobacillus plantarum FF34 added group LAB. The LAB group was prepared by adding the Lactobacillus plantarum FF34 single bacterial preparation prepared in Example 5 to the conventional control feed (CN) at a mass percentage of 0.01% to 1%, so that the FF34 bacterial concentration was 10 6 cfu / g feed. The crude protein content of each group of feed was 32.5%, crude fat was 6.2%, and gross energy was 17.4 kJ / Kg. The composition of the conventional control feed was (mass percentage): fish meal 6.4%, soybean meal 25.4%, rapeseed meal 16.2%, cottonseed meal 15.3%, wheat starch 16.6%, rice bran 5.7%, bran 5.8%, soybean oil 3.1%, monocalcium phosphate 1.0%, premix 1.0%, vitamin C 0.5%, choline chloride 0.4%, microcrystalline cellulose 2.1%, bentonite 0.5%.

[0137] 120 healthy and uniform juveniles of Megalobrama amblycephala (initial weight 13.5±0.5g) were selected and kept for one week, and then randomly divided into 6 outdoor cages (1m×1m×1m), 20 in each cage, divided into 2 groups, 3 replicates in each group, and fed the experimental feed 3 times a day (8:00, 12:00 and 17:00) for 8 weeks. During the culture period, the water temperature was maintained at 28-31℃, dissolved oxygen ≥7mg / L, ammonia nitrogen ≤0.1mg / L, pH 7.3-7.8, and a natural light cycle (12L:12D) was used. The water quality of the culture was measured every week. After the 8-week culture experiment, the fish were fasted for 24h, and the growth index, organ index and blood index were measured.

[0138] The growth performance index is calculated as follows:

[0139] Weight gain rate (WGR) (%) = 100 × (Wt-W0) / W0;

[0140] Specific growth rate (SGR) (% / d) = 100 × (ln Wt-ln W0) / t;

[0141] Feed coefficient (FCR) = F / (Wt-W0);

[0142] Where: W0 is the initial weight (g); Wt is the final weight (g); t is the number of feeding days (d); F is the total amount of feed consumed (air-dried basis) (g).

[0143] Serum blood glucose (GLU), cholesterol (TC), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels were measured using Shenzhen Mindray automatic biochemical analyzer (BS-400Q2080, Shenzhen, China), and the kits used were purchased from Shenzhen Mindray Co., Ltd. The data of the CN control group and LAB (Lactobacillus plantarum test group) were statistically analyzed using SPSS18.0, and all results are expressed as mean ± standard error. Indicates that the independent sample T test was used to compare the differences between the groups, and * indicates that the difference between the two groups was significant, with the test level being P < 0.05. The results are shown in Table 7.

[0144] Table 7 Effects of adding Lactobacillus plantarum FF34 to feed on growth and blood physiology of young bream

[0145] index CN (control group) LAB (Lactobacillus plantarum test group) Growth performance indicators WGR, % 126.87±3.85 155.48±2.69* SGR, % / d 1.64±0.04 11.88±0.02* FCR 2.18±0.06 11.89±0.05* Serum biochemical indexes GLU, mmol / L 9.93±0.56 7.14±0.70* LDL, mmol / L 0.61±0.06 0.66±0.03 HDL, mmol / L 1.04±0.09 0.64±0.02* TG, mmol / L 1.43±0.11 11.87±0.07* TC, mmol / L 5.83±0.20 7.03±0.17* ALT, mmol / L 4.74±0.37 2.78±0.27* AST, mmol / L 185.01±13.34 142.32±6.81*

[0146] Note: * indicates significant difference (p<0.05).

[0147] The results in Table 7 show that: adding 10 6cfu / g of Lactobacillus plantarum FF34 significantly increased the weight gain rate (P<0.05) and specific growth rate (P<0.05), significantly reduced the feed coefficient (P<0.05), and improved the feed utilization and growth performance of young bream. Alanine aminotransferase and aspartate aminotransferase are indicators of liver metabolic function. High-density lipoprotein can export cholesterol and promote cholesterol metabolism. Total cholesterol is an important raw material for synthesizing physiologically active substances such as adrenaline, bile acid and vitamin D. The results of this application example show that the addition of Lactobacillus plantarum can significantly reduce the content of blood glucose, alanine transferase and aspartate aminotransferase in the blood (P<0.05), indicating that it has the function of improving liver metabolism and interpretation, and improving the blood physiological health of bream. At the same time, the increase in lipid substances such as total cholesterol also suggests that the addition of Lactobacillus plantarum FF34 can promote the metabolism of lipid substances in fish and provide precursor substances for lipid metabolism in the liver and the body.

[0148] Example 10: Effects of adding Lactobacillus plantarum FF34 to high-starch feed on high-starch feed utilization and growth performance of young bream

[0149] In this embodiment, fish meal, soybean meal, rapeseed meal and cottonseed meal were used as protein sources, and soybean oil was used as a fat source to prepare two groups of test feeds, namely a high starch control feed (HWM) group and a high starch + Lactobacillus plantarum FF34 (HWM + LAB) group. The Lactobacillus plantarum FF34 single bacterial preparation prepared in Example 5 was added to the high starch control feed (HWM) at a mass percentage of 0.01% to 1%, that is, the (HWM + LAB) group, wherein the FF34 bacterial concentration was 10 6 cfu / g feed. The crude protein content of each group of feed was 32.5%, crude fat was 6.2%, carbohydrate content was 43%, and gross energy was 17.4 kJ / Kg. The composition of the conventional control feed was (mass percentage): fish meal 6.4%, soybean meal 21.6%, rapeseed meal 16.2%, cottonseed meal 15.3%, wheat starch 33.2%, soybean oil 3.9%, calcium dihydrogen phosphate 1.0%, premix 1.0%, vitamin C 0.5%, choline chloride 0.4%, bentonite 0.5%.

[0150] 120 healthy and uniform juveniles of Megalobrama amblycephala (initial weight 13.5±0.5g) were selected and kept for one week, and then randomly divided into 6 outdoor cages (1m×1m×1m), 20 in each cage, divided into 2 groups, 3 replicates in each group, and fed the experimental feed 3 times a day (8:00, 12:00 and 17:00) for 8 weeks. During the culture period, the water temperature was maintained at 28-31℃, dissolved oxygen ≥7mg / L, ammonia nitrogen ≤0.1mg / L, pH 7.3-7.8, and a natural light cycle (12L:12D) was used. The water quality of the culture was measured every week. After the 8-week culture experiment, the fish were fasted for 24h, and the growth index and organ index were measured.

[0151] The growth performance index is calculated as follows:

[0152] Weight gain rate (WGR) (%) = 100 × (Wt-W0) / W0;

[0153] Specific growth rate (SGR) (% / d) = 100 × (ln Wt-ln W0) / t;

[0154] Feed coefficient (FCR) = F / (Wt-W0);

[0155] Intestine to body ratio (VI) = 100 × W i / W b

[0156] Where: W0 is the initial weight (g); Wt is the final weight (g); t is the number of feeding days (d); F is the total amount of feed ingested (air-dried basis) (g); Wb is the final individual weight of each fish (g); Wi is the intestinal weight of each fish (g).

[0157] In this example, the data of HWM (high starch control feed group) and HWM+LAB (high starch feed+FF34 test group) were statistically analyzed using SPSS18.0, and all results were expressed as mean ± standard error. Indicates that the independent sample T test was used to compare the differences between the groups. * indicates that the difference between the two groups was significant, and the test level was P < 0.05. The results are shown in Table 8.

[0158] Table 8 Effects of adding Lactobacillus plantarum FF34 to high-starch feed on growth and high-starch feed utilization of young bream

[0159] index HWM (high starch control diet group) HWM+LAB (high starch feed+FF34 test group) Final weight, g 38.26±0.61 42.60±1.56* WGR, % 139.59±7.50 189.48±5.03* SGR, % / d 1.74±0.06 2.12±0.03* FCR 2.13±0.12 1.45±0.03* Intestine to body ratio 3.02±0.22 3.75±0.18*

[0160] Note: * indicates significant difference (p<0.05)

[0161] The bighead carp is one of the major freshwater fish in my country. Its tolerance and utilization of carbohydrates are relatively weak. Excessive carbohydrates in feed can cause sugar intolerance in fish, leading to growth retardation. The results in Table 8 show that the addition of Lactobacillus plantarum FF34 to high-starch feed significantly improved the growth performance and feed utilization efficiency of young bighead carp. Its average individual final weight increased significantly by 11.3% (P < 0.05), the weight gain rate increased significantly by 35.7% (P < 0.05), the specific growth rate increased significantly by 21.8% (P < 0.05), and the feed coefficient decreased significantly by 31.9% (P < 0.05). Moreover, the addition of Lactobacillus plantarum FF34 to high-starch feed can increase the intestinal body ratio index and promote intestinal growth and development. The ratio of the intestinal tract to the individual increased significantly by 24.2% (P < 0.05).

[0162] Example 11: Effect of adding Lactobacillus plantarum FF34 to high-starch feed on the antioxidant effect of the liver of young bream

[0163] In this embodiment, fish meal, soybean meal, rapeseed meal and cottonseed meal were used as protein sources, and soybean oil was used as a fat source to prepare two groups of test feeds, namely, high starch feed (HCH) and high starch feed + Lactobacillus plantarum FF34 (HCH + LAB). The high starch feed (HCH) was used as the control group, and the Lactobacillus plantarum FF34 single bacterial preparation prepared in Example 5 was added to the high starch feed (HCH) at a mass percentage of 0.01% to 1%, namely the (HCH + LAB) test group, wherein the FF34 bacterial concentration was 10 6 cfu / g feed. The crude protein content of each test group was 33.4%, the crude fat content was 6.5%, and the carbohydrate content was 47%. The conventional control feed composition was: fish meal 7%, soybean meal 23%, rapeseed meal 15%, cottonseed meal 12%, wheat starch 36%, soybean oil 4%, calcium dihydrogen phosphate 1.0%, premix 1.0%, choline chloride 0.5%, bentonite 0.5%.

[0164] 120 healthy and uniform young bream (initial weight 13.1±0.4g) were selected and kept for one week, and then randomly divided into 6 outdoor cages (1m×1m×1m), 20 in each cage, divided into 2 groups, 3 replicates in each group, and fed with the test feed 3 times a day (8:00, 12:00 and 17:00) for 8 weeks. During the culture period, the water temperature was maintained at 26-30℃, dissolved oxygen ≥7mg / L, ammonia nitrogen ≤0.1mg / L, pH 6..9-7.5, and a natural light cycle (12L:12D) was used. The water quality of the culture was measured every week. After the 8-week culture experiment, the fish were fasted for 24h and the antioxidant index of the fish liver was measured. The total antioxidant capacity (TAOC), catalase (CAT), total superoxide dismutase (SOD), reduced glutathione (GSH) and malondialdehyde (MDA) contents of the liver were determined using kits from Nanjing Jiancheng Bioengineering Institute.

[0165] In this example, the data of HCH (high starch feed control group) and HCH+LAB (high starch feed+Lactobacillus plantarum FF34 test group) were statistically analyzed using SPSS18.0, and all results were expressed as mean ± standard error. Indicates that the independent sample T test was used to compare the differences between the groups. * indicates that the difference between the two groups was significant, and the test level was P < 0.05. The results are shown in Table 9.

[0166] Table 9 Effects of adding Lactobacillus plantarum FF34 to high starch feed on liver antioxidant capacity of young bream

[0167]

[0168] Fish are naturally intolerant to high levels of sugar in feed. Excessive carbohydrates in feed will reduce fish growth performance and immunity, cause sugar metabolism disorders, show fish sugar intolerance, and cause the liver's antioxidant capacity to weaken. This leads to impaired liver antioxidant function. The results in Table 9 show that adding Lactobacillus plantarum FF34 to high-starch feed significantly increased the levels of GSH, CAT, and SOD in the liver (P < 0.05). Therefore, it is speculated that Lactobacillus plantarum FF34 can use the rich starch in the feed to increase cholesterol levels to promote bile acid circulation metabolism, activate liver antioxidant capacity, and maintain growth and health.

[0169] Example 12: Effect of adding Lactobacillus plantarum FF34 to high-starch feed on fat deposition in muscle and liver of young bream

[0170] In this embodiment, fish meal, soybean meal, rapeseed meal and cottonseed meal were used as protein sources, and soybean oil was used as fat source to prepare three groups of test feeds, namely control feed (15WM), high starch feed (35WM) and high starch feed + Lactobacillus plantarum FF34 (35WM + LAB). The Lactobacillus plantarum FF34 single bacterial preparation prepared in Example 5 was added to the high starch feed (35WM) at a mass percentage of 0.01% to 1%, that is, the 35WM + LAB test group, wherein the FF34 bacterial concentration was 10 6 cfu / g feed, feed formula is shown in Table 10.

[0171] Table 10 Feed formula composition of this embodiment

[0172]

[0173] 180 healthy and uniform young bream (initial weight 13.6±0.7g) were selected and kept for one week, and then randomly divided into 9 outdoor cages (1m×1m×1m), 20 in each cage, and divided into 3 groups, with 3 replicates in each group. The experimental feed was fed 3 times a day (8:00, 12:00 and 17:00) for 8 weeks. During the culture period, the water temperature was maintained at 25-30℃, dissolved oxygen ≥7mg / L, ammonia nitrogen ≤0.1mg / L, pH 6.9-7.5, and a natural light cycle (12L:12D) was used. The water quality of the culture was measured every week. After the 8-week culture experiment, the fish were fasted for 24h, the muscle fat content of the fish was measured, and the lipid droplet deposition in the liver was analyzed. The analysis of muscle nutrient components was determined according to the national standard method. The dry matter content was calculated by drying in an oven at 105°C under normal pressure until constant weight; the crude protein content was determined by the Kjeldahl method (GB / T6432-1994); the crude fat content was determined by the Soxhlet extraction method (GB / T6433-1994); and the crude ash content was determined by the 560°C burning method (GB / T6438-1992). The liver tissue blocks fixed in 4% paraformaldehyde were taken out, frozen liver sections were made, and stained with Oil Red O, and the Oil Red O lipid droplet area was analyzed using Image J software. In this embodiment, the data comparison between 15WM (control feed), 35WM (high starch feed) and 35WM+LAB (high starch feed+Lactobacillus plantarum FF34) was performed using a one-way ANOVA turkey's test, with a test level of P<0.05, and the results are shown in Table 11.

[0174] Since fish are naturally intolerant to high levels of sugar in their feed, excess carbohydrates in the feed can cause disorders in sugar and lipid metabolism, and excessive deposition of lipids in the fish's body can lead to oxidative stress damage and decreased disease resistance. Figure 1 In the results, the Oil Red O staining images of lipid droplets in liver tissue sections showed that the lipid droplet deposition in the liver tissue of the amblycephala group fed with the 35WM group feed increased significantly, while the addition of Lactobacillus plantarum (35WM+LAB) to the high-starch feed reduced the area of ​​lipid droplets in the liver. Further analysis of muscle fat and liver lipid droplet area of ​​amblycephala group (Table 11) showed that compared with the basic control group (15WM), the high-starch group (35WM) significantly increased the amount of crude fat in the muscle and the deposition of lipid droplets in the liver (P<0.05). The addition of Lactobacillus plantarum FF34 (35WM+LAB) to the high-starch feed could significantly reduce the crude fat content in the muscle of amblycephala group (P<0.05), which was not significantly different from the control group; while the liver lipid droplet deposition in the 35WM+LAB group was higher than that in the control group, but significantly lower than that in the high-starch group (P<0.05).

[0175] Figure 1Effects of Lactobacillus plantarum and high starch addition on Oil Red O-stained sections of liver of young bream. Figure A shows Oil Red O-stained sections of the 15WM group (×200 times), Figure B shows Oil Red O-stained sections of the 35WM+LAB group (×200 times), and Figure C shows Oil Red O-stained sections of the 35WM group (×200 times). Red in the figure indicates lipid droplets, and blue indicates cell nuclei.

[0176] Table 11 Effects of high starch supplementation with Lactobacillus plantarum FF34 on muscle fat content and liver lipid droplet deposition in juvenile Megalobrama amblycephala

[0177] Grouping 15WM 35WM 35WM+LAB Muscle fat content, % <![CDATA[4.88±0.61 a ]]> <![CDATA[6.59±0.35 b ]]> <![CDATA[4.75±0.48 a ]]> Hepatic lipid droplet deposition, % <![CDATA[2.50±0.64 a ]]> <![CDATA[28.48±3.01 c ]]> <![CDATA[20.48±0.92 b ]]>

[0178] Note: The same lowercase letters in the same row indicate no significant difference (p>0.05), and different lowercase letters in the same row indicate significant difference (p<0.05).

Claims

1. A Lactobacillus plantarum FF34, classified and named Lactobacillus plantarum ( lactobacillus plantarum ), deposited in China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, with deposit number CCTCC NO: M20211221.

2. A method for preparing a single bacterial agent of Lactobacillus plantarum FF34 having antagonistic properties against aquatic pathogens using the strain described in claim 1, characterized in that Here are the steps: (1) Bacteria activation: Aseptically open the freeze-dried preserved strain of Lactobacillus plantarum FF34, inoculate it into a test tube containing MRS broth, and culture it at 30-35℃ for 24-48h, then transfer it to a MRS broth triangular bottle, and culture and activate it at 30-35℃ for 20-48h; repeat the activation 2-3 times, examine it under a microscope, and count it. When the bacterial concentration reaches 10 9 CFU / mL as seed solution; MRS broth medium composition (g / L): casein digest 10, beef extract powder 10, yeast extract powder 4, triammonium citrate 2, sodium acetate 5, magnesium sulfate 0.2, manganese sulfate 0.05, dipotassium hydrogen phosphate 2, glucose 20, Tween-80 1.08, made up to volume with distilled water, pH 5.7±0.2; (2) Immobilized fermentation culture: Preparation of seed culture solution in triangular flask: The seed solution obtained in step (1) is inoculated at a volume ratio of 1% to 5% into a 3L triangular flask filled with immobilized fermentation medium, with the volume ratio of 90%, and cultured at 30 to 35°C for 24 to 40 hours. When the bacterial cell concentration reaches 10 9 CFU / mL, used as seed culture medium in triangular flasks; Scale-up and expansion: The seed culture solution in the triangular flask is connected to the common aerobic fermentation tank for primary and secondary fixed fermentation and scaled up step by step, i.e.: 3L triangular flask, then 500L fermentation tank, then 10T fermentation tank, and the inoculation is completed by sterile air pressure delivery between fermentation tanks; Primary immobilized culture: The triangular flask seed culture solution is inoculated into a 500L fermenter at a volume ratio of 1% to 5%, and the volume of the fermenter is 80% to 90%. Sterile air is used to maintain the tank pressure at 0.01 to 0.05 MPa. Intermittent stirring is performed at a speed of 80 to 100 r / min. The culture is cultured at 30 to 35°C for 20 to 40 hours. When the pH is 4.5 to 5.5, the culture is terminated to obtain a primary immobilized culture solution, in which the Lactobacillus plantarum FF34 cells are adsorbed and fixed in the small gel particles formed by calcium alginate and silica, and the concentration of live cells is ≥5×10 9 CFU / mL; Secondary immobilized culture: The primary immobilized culture solution is sent by sterile air pressure and inoculated into a 10T fermenter at a volume ratio of 1% to 5%. The volume of the fermenter is 70% to 90%. The sterile air is used to keep the tank pressure at 0.01 to 0.05 MPa. Stir intermittently, rotate at 80 to 100 r / min, and culture at 30 to 38 ° C for 20 to 40 hours. When the pH of the fermentation solution drops to 4.0 to 5.5, the culture is terminated to obtain the secondary immobilized culture solution, in which the Lactobacillus plantarum FF34 cells are adsorbed and fixed in the small gel particles formed by calcium alginate and silica, and the cell concentration is ≥5×10 9 CFU / mL; The composition of the fixed fermentation medium is calculated in g / L: 10-40 g of carbon source, 3-15 g of nitrogen source, 0.2-5 g of dipotassium hydrogen phosphate, 0-0.2 g of magnesium sulfate, 0-0.05 g of manganese sulfate, 5-40 g of calcium carbonate, 3-10 g of sodium alginate, and 1-10 g of silicon dioxide, which is prepared with tap water to a fixed volume, pH 6.0-6.5, and sterilized at 121°C for 30 min; wherein the carbon source comprises one or a combination of wheat flour, corn starch, dextrin, molasses, glucose, and fructose syrup; the nitrogen source comprises one or a combination of fish meal peptone, tryptone, soy peptone, soy protein isolate, yeast extract, yeast powder, corn steep liquor, cottonseed meal, rapeseed meal, soybean meal, peanut meal, and blood meal; (3) Preparation of Lactobacillus plantarum FF34 single bacterial agent with antagonistic properties against aquatic pathogens: (A) Preparation of Lactobacillus plantarum FF34 single-strain aqueous solution: The secondary immobilized culture solution obtained in step (2) is filled to obtain a Lactobacillus plantarum FF34 single-strain aqueous solution, wherein the live bacteria concentration of FF34 in the single-strain aqueous solution is ≥ 5.0 × 10 9 CFU / mL; (B) Preparation of Lactobacillus plantarum FF34 single bacterial powder: The secondary immobilized culture solution obtained in step (2) is centrifuged to collect wet bacterial cells, and the mixture is mixed in a mass ratio of wet bacterial cells: calcium carbonate: dextrin: silicon dioxide = 1:0.1 to 2:0.5 to 2:0.01, and spray-dried or vacuum-dried to obtain Lactobacillus plantarum FF34 powder, wherein the concentration of live Lactobacillus plantarum FF34 in the powder is not less than 1×10 10 CFU / g.

3. A method for preparing a composite bacterial agent by mixed fermentation culture of Lactobacillus plantarum FF34 and Bacillus according to claim 1, characterized in that Here are the steps: (1) The Bacillus spores mixed with Lactobacillus plantarum FF34 for fermentation are: Bacillus subtilis AB90008-15 with antagonistic properties against aquatic pathogens with a preservation number of CGMCC NO.18684, or Clostridium butyricum JSIM-MCB20040312 with a preservation number of CGMCC NO.1647; (2) Mixed fermentation of Lactobacillus plantarum FF34 and the Bacillus subtilis described in step (1): It is carried out in two steps. The first fermentation step is to activate the Bacillus subtilis described in step (1) by slant or triangular flask and then inoculate it into the mixed fermentation medium. The inoculation amount is 1% to 10% by volume. The culture temperature is 30 to 37°C. The culture is carried out for 24 to 48 hours. The formation of spores in the bacteria is observed under a microscope, indicating that the first fermentation step is completed. The second fermentation step is to first adjust the pH of the fermentation liquid obtained in the first fermentation step to 5.0 to 5.4 with hydrochloric acid, and then inoculate the activated Lactobacillus plantarum FF34. The inoculation amount is 1% to 10% by volume. The culture temperature is 30 to 35°C, and intermittent stirring is performed. During this period, 20% w / v glucose or 30% calcium carbonate or ammonia water is added to control the pH of the fermentation liquid. 4.5~5.0, culture for 24h, obtain the mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus, microscopic examination shows that Lactobacillus plantarum and Bacillus coexist, and the concentration of live Lactobacillus plantarum FF34 in the mixed fermentation liquid is ≥1.0×10 9 CFU / mL, Bacillus live bacteria concentration ≥5.0×10 8 CFU / mL, spore rate ≥90%; The composition of the mixed fermentation medium is in g / L: 0-50 wheat flour, 0.1-5 peptone, 0-5 yeast extract, 0-3 sodium acetate, 0-10 corn starch, 0-2 ammonium sulfate, 0-5 sodium chloride, 0-5 calcium carbonate, 0-0.5 potassium dihydrogen phosphate, 0-0.5 magnesium sulfate, 0-0.5 manganese sulfate, 0-0.3 sodium alginate, tap water to volume, pH 7.0, sterilized at 121°C for 30 min; Preparation of composite bacterial agent of Lactobacillus plantarum FF34 and Bacillus subtilis: (A) The mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus obtained in the above step (2) is directly bottled to obtain a composite bacterial solution of Lactobacillus plantarum FF34 and Bacillus plantarum, wherein the live bacterial concentration of Lactobacillus plantarum FF34 in the composite bacterial solution is ≥1.0×10 9 CFU / mL, Bacillus live bacteria concentration ≥5.0×10 8 CFU / mL, spore rate ≥90%; (B) The mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus obtained in the above step (2) is centrifuged to collect wet bacteria, mixed in a mass ratio of wet bacteria: calcium carbonate: silicon dioxide = 1:0.1 to 3:0.01, and spray-dried or vacuum-dried to obtain a composite bacterial powder of Lactobacillus plantarum FF34 and Bacillus, wherein the live bacterial concentration of Lactobacillus plantarum FF34 in the composite bacterial powder is ≥ 5×10 9 CFU / g, Bacillus live bacteria concentration ≥5.0×10 9 CFU / g.

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