An aquatic fermentation agent containing Lactobacillus plantarum, its preparation method and application

Abstract

The present invention relates to an aquatic fermentation agent containing Lactobacillus plantarum, its preparation method and application, including bacterial powder A, bacterial powder B and culture medium C; the bacterial powder A consists of Lactobacillus plantarum, Enterococcus faecalis, Pediococcus acidilactici, Bacillus subtilis, yeast and a carrier; the bacterial powder B consists of denitrifying bacteria and a carrier. The use method of the aquatic fermentation agent of the present invention is simple to operate, has a low fermentation cost and a high number of effective viable bacteria, effectively improves the problems of low viable bacteria number, poor storage stability and unobvious application effect caused by transportation and storage of traditional aquatic feed additives or biological water quality improvers, and can be widely applied to the aquaculture industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to an aquatic fermentation agent containing Lactobacillus plantarum, a preparation method thereof, and an application thereof. Background Art

[0002] In recent years, the aquaculture in China has developed rapidly, but many problems have also emerged, such as water pollution and imbalance of the intestinal flora of aquatic animals caused by some unreasonable measures during the high-density aquaculture process.

[0003] The live microbial ecological agent is a live microbial agent made of beneficial microorganisms or substances that promote the growth of microorganisms. The main active ingredients include live bacteria and their metabolites, which can improve the balance of the microecosystem and the aquaculture environment, and enhance the stress resistance and disease resistance of cultured aquatic animals.

[0004] Traditional microecological agents or water quality improvers for aquaculture are generally fermented by merchants themselves and packaged and sold in a liquid form. Although the quality can be strictly guaranteed during the production and packaging processes, due to the environmental changes during transportation and storage, the attenuation of the number of live bacteria, the growth of miscellaneous bacteria, the reduction of product quality, and the deterioration of effectiveness will occur, which will bring potential negative impacts to aquaculture. At the same time, the existing live microbial ecological products on the market have problems such as single function, poor stress resistance and activity stability of strains, and poor antibacterial effect, and cannot solve the four problems of water conditioning, intestinal tract conditioning, disease resistance, and growth promotion at the same time. Therefore, the use of microecological agents in the field of aquaculture has certain limitations. Summary of the Invention

[0005] In view of this, the main purpose of the present invention is to provide an aquatic fermentation agent containing Lactobacillus plantarum, a preparation method thereof, and an application thereof, so as to solve the problems of single product function, poor stress resistance of strains, poor storage stability, low effective viable bacteria count, and poor antibacterial effect in the existing microecological agents for aquaculture, and improve the growth performance of aquaculture animals by regulating water quality, balancing intestinal flora, and promoting feed digestion and absorption.

[0006] To achieve the above purpose, the present invention provides an aquatic fermentation agent containing Lactobacillus plantarum, which includes bacterial powder A, bacterial powder B, and culture medium C, and the weight ratio of the three is 0.05:(0.5 - 2):(20 - 40).

[0007] Preferably, the composition of the bacterial powder A by weight percentage is: Lactobacillus plantarum 0.2 - 1%; Enterococcus faecalis 8 - 10%; Pediococcus acidilactici 0.2 - 1%; Bacillus subtilis 4 - 10%; Saccharomyces cerevisiae 0.5 - 5%; glucose 10 - 20%; magnesium sulfate 1 - 5%; sodium chloride 5 - 20%; corn starch 30 - 35%.

[0008] Preferably, the total viable count of the bacterial powder A is 14.1-31 billion CFU / g, the viable count of Lactobacillus plantarum in the bacterial powder A is 1-5 billion CFU / g; the viable count of Enterococcus faecalis in the bacterial powder A is 8-10 billion CFU / g; the viable count of Pediococcus acidilactici in the bacterial powder A is 1-5 billion CFU / g; the viable count of Bacillus subtilis in the bacterial powder A is 4-10 billion CFU / g; the viable count of Saccharomyces cerevisiae in the bacterial powder A is 0.1-1 billion CFU / g.

[0009] Preferably, the composition of the bacterial powder B by weight percentage is: denitrifying bacteria 10-30%; wheat bran 20-30%; talcum powder 35-45%; the viable count of the denitrifying bacteria in the bacterial powder B is 1-3 billion / g.

[0010] Preferably, the composition of the culture medium C by mass-volume ratio is: brown sugar 10-50 g / L, corn peptone 10-40 g / L, yeast extract powder 5-10 g / L, sodium chloride 0.5-2 g / L, manganese sulfate 0.1-0.5 g / L, magnesium sulfate 0.25-1 g / L, light calcium carbonate 0.5-2 g / L.

[0011] To achieve the above object, the present invention also provides a preparation method of an aquatic fermentation agent containing Lactobacillus plantarum, comprising the following steps:

[0012] Preparation of bacterial powder A: Primarily premix the formula amounts of Lactobacillus plantarum, Enterococcus faecalis, Pediococcus acidilactici, Saccharomyces cerevisiae with one-half of the formula amount of corn starch to obtain a primary premix of bacterial powder A; then secondarily mix the primary premix of bacterial powder A with the formula amounts of Bacillus subtilis, glucose, magnesium sulfate, sodium chloride, and the remaining one-half of the formula amount of corn starch to obtain the bacterial powder A;

[0013] Preparation of bacterial powder B: Mix the formula amounts of denitrifying bacteria, wheat bran, and talcum powder evenly to obtain the bacterial powder B;

[0014] Preparation of culture medium C: Primarily premix the formula amounts of sodium chloride, manganese sulfate, magnesium sulfate, light calcium carbonate with one-third of the formula amount of brown sugar to obtain a primary premix of culture medium C. Then secondarily mix the primary premix of culture medium C with the formula amounts of yeast extract powder, corn peptone, and the remaining two-thirds of the formula amount of brown sugar to obtain the culture medium C.

[0015] Preferably, the rotation speed of the primary premixing of the bacterial powder A is 20-40 r / m, the mixing time is 200-300 seconds, the preferred rotation speed is 30 r / m, and the mixing time is 240 seconds. Further, the rotation speed of the secondary mixing of the bacterial powder A is 40-60 r / m, the mixing time is 120-180 seconds, the preferred rotation speed is 50 r / m, and the mixing time is 150 seconds.

[0016] Preferably, the mixing rotation speed of the bacterial powder B is 20-40 r / m, the mixing time is 200-300 seconds, the preferred rotation speed is 30 r / m, and the mixing time is 240 seconds.

[0017] Preferably, the rotation speed of the primary premixing of the culture medium C is 20-40 r / m, the mixing time is 200-300 seconds, the preferred rotation speed is 30 r / m, and the mixing time is 240 seconds.

[0018] Preferably, the rotation speed of the secondary mixing of the culture medium C is 40-60 r / m, the mixing time is 120-180 seconds, the preferred rotation speed is 50 r / m, and the mixing time is 150 seconds.

[0019] In order to achieve the above object, the present invention also provides an application of an aquatic fermentation agent containing Lactobacillus plantarum in the preparation of aquatic fermentation feed and fermentation broth.

[0020] Preferably, the aquatic fermentation feed includes fish feed, shrimp feed, crab feed, frog feed, soft-shelled turtle feed or turtle feed, etc.

[0021] Preferably, for the application of the aquatic fermentation agent containing Lactobacillus plantarum in the preparation of aquatic fermentation feed, the application uses soybean meal, peanut meal, fish meal, flour, wheat bran, calcium dihydrogen phosphate, and sodium chloride as the fermentation substrate, and uses the aforementioned bacterial powder A with Lactobacillus plantarum, Enterococcus faecalis, Pediococcus acidilactici, Bacillus subtilis, and Saccharomyces cerevisiae as the main components as the fermentation inoculant, and the weight ratio of the fermentation substrate components to the fermentation inoculant components is (500-1000):1.

[0022] Preferably, the application of the aquatic fermentation agent containing Lactobacillus plantarum in the preparation of aquatic fermentation feed includes the following steps:

[0023] A) Activation of the fermentation inoculant: Activate 1-3 kg of the aforementioned bacterial powder A in 30-60 L of 10-50 g / L brown sugar water in warm water at 30-35 °C for 1-3 h to obtain the activated solution of the fermentation inoculant;

[0024] B) Preparation of the fermentation substrate mixture: First, crush soybean meal, rapeseed meal, and peanut meal and then sieve them through a 60 - 80 - mesh sieve to make soybean meal powder, rapeseed meal powder, and peanut meal powder. Then, thoroughly stir 20 - 35% soybean meal powder, 20 - 30% rapeseed meal powder, 10 - 20% peanut meal powder, 5 - 15% fish meal, 20 - 30% high - gluten flour, 4 - 8% wheat bran, 3 - 4% soybean oil, 1 - 2% calcium dihydrogen phosphate, 0.5 - 2% sodium chloride, and 0.25 - 0.5% magnesium sulfate by weight percentage to obtain the fermentation substrate mixture;

[0025] C) Fermentation: Evenly spray the activated liquid of the fermentation inoculant prepared in step A) above onto the fermentation substrate mixture prepared in step B). After packing it in a transparent sealed bag, place it in a fermentation barrel and ferment it at room temperature in a closed state for 3 - 5 days (2 - 3 days in summer and autumn, 3 - 5 days in winter and spring). After fermentation, the color of the material is uniform, the texture is soft, there is no mildew or caking, and the smell is mild (fruity, mellow, sour, or weak sour smell), then the fermented feed is made. Its moisture content is 30wt% - 40wt%, the pH value is less than 5.00, the crude protein content is 30 - 40wt%, and the acid - soluble protein content is 18 - 20wt%.

[0026] D) Evenly sprinkle the freshly prepared fermented feed in step C) above into the pond at 5 - 20% of the daily feeding amount of aquatic animals and feed once a day, which can improve the weight gain and growth rate of aquatic animals.

[0027] Preferably, the application of the aquatic fermentation agent containing Lactobacillus plantarum in the preparation of the fermentation broth includes the following steps:

[0028] 1) Expansion culture of bacterial powder A: Activate 0.25 - 0.5 kg of bacterial powder A with 5 - 10 L of 10 - 50 g / L brown sugar water at 30 - 35 °C for 0.5 - 3 h; dissolve culture medium C with boiling water at 100 °C, then cool it to 25 - 35 °C, adjust the pH value to 6.0 - 7.0, the liquid loading volume is 70 - 80% (v / v), and make up the volume to 90 - 95 L for standby; inoculate the activated bacterial powder A into the obtained culture medium C solution according to the mass - to - volume ratio of 1:400 - 1:200, stir evenly, and culture it in a closed state at 25 - 35 °C for 24 - 72 h to obtain the expanded culture solution of bacterial powder A;

[0029] 2) Activation of bacterial powder B: Activate 0.25 - 0.5 kg of bacterial powder B with 5.0 - 10 L of 10 - 50 g / L brown sugar warm water at 30 - 37 °C for 0.5 - 3 h;

[0030] 3) Thoroughly mix the expanded culture solution of bacterial powder A and the activated solution of bacterial powder B evenly according to the volume ratio of 20:1 - 5:1 to obtain the fermentation broth;

[0031] 4) Sprinkle the freshly prepared fermented bacterial liquid from step 3) into the water body of aquaculture ponds for fish, shrimp, crabs, frogs, soft-shelled turtles or turtles at a volume ratio of 1:2000 - 1:100, and increase or decrease the dosage according to the filtration feeding situation and growth; use it once every 7 - 10 days, and follow the principle of small amounts and multiple applications each time. The effect is better when used at noon on sunny days, and an aerator should be turned on on the night of the day of use.

[0032] The Lactobacillus plantarum in the above-mentioned bacterial powder A is Lactobacillus plantarum HEW-A490 screened and isolated from the intestinal contents of healthy animals. It was deposited on May 27, 2016 at the China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101), and the deposit number is CGMCC No. 12554, and the taxonomic name is Lactobacillus plantarum. This strain of Lactobacillus plantarum HEW-A490 has very strong acid production performance, extremely rich metabolites, and strong antibacterial performance; after 24h of fermentation culture, the pH value of the fermentation broth can be reduced to 3.37, and the acid production amount can reach 1.75 g / 100 mL (the determination of acid production amount refers to "GB 12456 National Food Safety Standard Determination of Total Acid in Foods", and the total acid is calculated as lactic acid). The metabolites produced are very rich, including lactic acid bacteria, lactic acid, short-chain fatty acids, β-glucosidase, etc. After analysis by liquid chromatography-mass spectrometry, this strain of Lactobacillus plantarum HEW-A490 has 170 metabolites, including 90 acids, 26 amino acids, and 10 oligosaccharides, and the content of phenyl lactic acid is very high.

[0033] The Enterococcus faecalis HEW-A131 in the above-mentioned bacterial powder A was deposited on June 17, 2014 at the China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101), and the deposit number is CGMCC NO. 9353, and the taxonomic name is Enterococcus faecalis.

[0034] The Pediococcus acidilactici HEW-P27 in the above-mentioned bacterial powder A was deposited on March 7, 2018 at the China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101), and the deposit number is CGMCC NO. 15419, and the taxonomic name is Pediococcus acidilactici.

[0035] The present invention has the following beneficial effects:

[0036] The aquatic fermentation agent containing Lactobacillus plantarum involved in the present invention is composed of a variety of compound bacteria such as Lactobacillus plantarum, Enterococcus faecalis, Pediococcus acidilactici, Bacillus subtilis, Saccharomyces cerevisiae, and denitrifying bacteria. After amplification culture, the pH value of the fermentation broth is low and the viable bacteria count is high (the total viable bacteria count is greater than 20 billion CFU / mL). The viable bacteria can reduce nitrite in the water body to generate nitrogen gas, absorb ammonia nitrogen, degrade ammonia nitrogen, promote the establishment of the bacterial phase in the water body, inhibit the reproduction of harmful algae, maintain the balance of bacteria and algae, purify the water body, and stabilize the water quality; this method is simple to operate, the fermentation effect is stable, and effectively solves the problems of poor transportation, storage, and use effect of liquid aquatic viable bacteria microecological preparations;

[0037] The aquatic fermentation agent containing Lactobacillus plantarum involved in the present invention uses the activated bacterial powder A (Lactobacillus plantarum, Enterococcus faecalis, Pediococcus acidilactici, Bacillus subtilis, Saccharomyces cerevisiae) to prepare aquatic fermented feed. Since the Lactobacillus plantarum HEW-A490 in the bacterial powder A has strong acid-producing ability and rich metabolites, it can effectively inhibit pathogenic bacteria such as Aeromonas hydrophila, Vibrio parahaemolyticus, and Nocardia, reduce the incidence of diseases in aquaculture animals, and at the same time has extremely strong acid-producing, acid-resistant, and bile salt-resistant abilities, which can greatly improve the feed fermentation efficiency, making the prepared fermented feed rich in probiotics, organic acids such as acetic acid and lactic acid, biological enzymes such as β-glucosidase, active peptides, polysaccharides, growth promoting factors, immune stimulating factors, etc. After entering the intestines of aquatic animals, it can promote the proliferation of dominant intestinal flora, improve immunity. In addition, the fermented feed has a unique sour aroma, good palatability, high feed digestion and absorption rate, making the aquaculture animals have a high food intake, thick intestines, accelerated growth rate, a 10-30% increase in weight gain rate, a 10-15% reduction in feed coefficient, and a 15-20% reduction in the incidence of diseases. Specific embodiments

[0038] To further elaborate on the technical means and effects adopted by the present invention to achieve the intended invention purpose, the following is described in conjunction with preferred embodiments. Unless otherwise specified, the following various materials or reagents are all commercially available.

[0039] Example 1: Preparation of Lactobacillus plantarum HEW-A490 viable bacteria preparation and testing of its acid-producing performance, stress resistance performance, and antibacterial performance

[0040] 1.1 Preparation of Lactobacillus plantarum HEW-A490 fermentation broth

[0041] Streak the Lactobacillus plantarum HEW-A490 strain (deposit number: CGMCC No. 12554) on an MRS slant and culture it at 37°C for 20 h. Add 2 mL of sterilized normal saline to the slant to prepare a bacterial suspension, and transfer the bacterial suspension to 300 mL of seed medium for shake flask fermentation culture. Culture it at 37°C and 180 r / min with shaking for 15 h to prepare the Lactobacillus plantarum HEW-A490 seed liquid. After detection, the viable count of this seed liquid is 1.2×10 9 CFU / mL. After the shake flask fermentation culture is completed, inoculate the prepared Lactobacillus plantarum HEW-A490 seed liquid into a secondary fermentation culture tank for enlarged fermentation culture, and finally transfer it to a tertiary fermentation culture tank and culture it for 20 h before terminating the fermentation to prepare the Lactobacillus plantarum HEW-A490 fermentation liquid; after detection, the viable count of this fermentation liquid is 5.5×10 9 CFU / mL.

[0042] Among them, the seed medium is composed of the following components by weight percentage: glucose 0.5%, sucrose 1.2%, yeast extract 0.8%, tryptone 1.0%, dipotassium hydrogen phosphate 0.05%, magnesium sulfate 0.05%, sodium chloride 0.25%, calcium carbonate 0.17%, and the balance is water, and the pH value is 6.8±0.2. The conditions for shake flask fermentation are: the fermentation temperature is 37°C, the stirring speed is 180 r / m, and the fermentation time is 15 h.

[0043] Taking the culture in a 50 L fermentation tank as an example for secondary fermentation culture, the composition of the medium in the 50 L fermentation tank by weight percentage is: sucrose 1.5%, corn starch 2.0%, corn steep liquor dry powder 1.2%, yeast extract 1.4%, dipotassium hydrogen phosphate 0.08%, magnesium sulfate 0.05%, sodium chloride 0.2%, calcium carbonate 0.15%, manganese sulfate 0.03%, and the balance is water, and the pH value is 6.8±0.2. The fermentation conditions for the 50 L fermentation tank are: the liquid loading volume is 30 L of medium, the inoculation amount is 300 mL, the fermentation temperature is 37°C, the stirring speed is 120 r / m, and the fermentation time is 10 h.

[0044] Taking the culture in a 5 T fermentation tank as an example for tertiary fermentation culture, the medium in the 5 T fermentation tank is the same as the secondary fermentation medium. The fermentation conditions for the 5 T fermentation tank are: the liquid loading volume is 3.5 T of medium, the inoculation amount is 30 L, the fermentation temperature is 37°C, the stirring speed is 110 r / m, and the fermentation time is 20 h.

[0045] 1.2 Preparation of the viable bacteria preparation of Lactobacillus plantarum HEW-A490

[0046] The Lactobacillus plantarum HEW-A490 fermentation liquid obtained in 1.1 was centrifuged at 10000r / m for 30min to obtain active bacterial mud of Lactobacillus plantarum HEW-A490. The active bacterial mud and the protective agent were mixed uniformly at a weight ratio of 1:0.2 to form a semi-fluid bacterial suspension. The bacterial suspension was placed on a sterilized clean tray and then put into a freeze dryer. After pre-freezing, sublimation drying and vacuum drying, a loose block of Lactobacillus plantarum was obtained. The whole process included cooling to -40°C for 4h, heating to -10°C for 14h, heating to 0°C for 8h, and heating to 25°C for 10h. The block of Lactobacillus plantarum was collected and put into an extrusion granulator for crushing. After sieving through a 30-mesh standard sieve, a powdered active bacterial preparation of Lactobacillus plantarum HEW-A490 was obtained, and the effective number of active bacteria could reach 5.0×10 11 CFU / g.

[0047] The protective agent is composed of 11.5% soft sugar, 8.4% whey powder, 4.5% glycerol, 3.2% skimmed milk powder, 1.5% mannitol, 2.2% vitamin E, 0.2% vitamin C, and the balance is water.

[0048] 1.3 Test of acid production performance of Lactobacillus plantarum HEW-A490 live bacteria preparation

[0049] Table 1 pH changes and acid production of Lactobacillus plantarum HEW-A490 fermentation broth at different culture times

[0050]

[0051] Note: The determination of acid production refers to GB / T12456, and the total acid is calculated based on lactic acid bacteria.

[0052] It can be seen from the data in Table 1 that after 24 hours of fermentation and cultivation of the Lactobacillus plantarum HEW-A490 strain provided by the present invention, the pH value of the fermentation liquid decreased from 6.80 to 3.37, and the acid production reached 1.75 g / 100 mL, which is stronger than the control strain (the number of live bacteria of Lactobacillus plantarum is 100 billion CFU / g) and faster acid production speed. In particular, the acid production is 23% higher than that of the control strain (the number of live bacteria of Lactobacillus plantarum is 100 billion CFU / g), achieving unexpected results. 1.4 Test of stress resistance of Lactobacillus plantarum HEW-A490 live bacterial preparation

[0053] Table 2 Acid and bile resistance test of plant lactobacillus HEW-A490 live bacteria preparation of Example 1

[0054]

[0055]

[0056] Note: (1) Acid resistance test process: Immerse the sample to be tested in a phosphate buffered saline solution with a pH of 3.0 for 2 hours, and then detect the viable count. The viable count of the sample after treatment is divided by the viable count of the untreated sample to obtain the acid resistance survival rate of the sample in this treatment solution. Among them, the preparation steps of the phosphate buffered saline solution with a pH of 3.0 are as follows: First step, weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 3.63 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, and 1000 mL of distilled water; Second step, dissolve the above components in distilled water, adjust the pH value to 3.0 with 6 mol / L hydrochloric acid, sterilize at 121 °C for 30 min, and cool to room temperature for standby.

[0057] (2) Bile salt resistance test process: Immerse the sample to be tested in a phosphate buffered saline solution containing 0.15 wt% bile salt for 2 hours, and then detect the viable count. The viable count of the sample after treatment is divided by the viable count of the untreated sample to obtain the bile salt resistance survival rate of the sample in this treatment solution. Among them, the preparation of the 0.15 wt% bile salt solution is divided into three steps: First step, prepare a phosphate buffered saline solution with a pH of 7.4. Dissolve 8 g of sodium chloride, 0.2 g of potassium chloride, 3.63 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate in 1000 mL of distilled water in sequence, adjust the pH value to 7.4 with 6 mol / L hydrochloric acid, sterilize at 121 °C for 30 min, and cool for standby. Second step, accurately weigh 5.00 g of bile salt (biological reagent, purchased from Beijing Boao Xing Biotechnology Co., Ltd.), dissolve it in 100 mL of pure water to prepare a 5 wt% bile salt solution, sterilize at 121 °C for 30 min, and cool for standby. Third step, add 9 mL of the 5 wt% bile salt solution to 300 mL of the phosphate buffered saline solution with a pH of 7.4, and vortex and mix well to prepare a phosphate buffered saline solution containing 0.15 wt% bile salt.

[0058] It can be seen from the data in Table 2 that the viable bacteria preparation of Lactobacillus plantarum HEW-A490 provided in Example 1 of the present invention has a tolerance 11 percentage points higher and 2 percentage points higher than that of Lactobacillus plantarum on the market under the low-acid environment of the digestive tract and high bile salt concentration, indicating that more viable bacteria of Lactobacillus plantarum in Example 1 can reach the intestine for colonization and reproduce into dominant flora after passing through the digestive tract of animals, protecting the health of the animal intestine.

[0059] 1.5 Antibacterial performance test of Lactobacillus plantarum HEW-A490 fermentation broth

[0060] Using Aeromonas hydrophila, Vibrio parahaemolyticus, and Nocardia as pathogenic bacteria, the differences in the antibacterial effects between the fermented liquid of Lactobacillus plantarum HEW-A490 obtained in Example 1 (the active ingredient is nisin) and antibiotics were compared. The antibiotics used were enrofloxacin susceptibility test discs (the active ingredient is enrofloxacin) and florfenicol susceptibility test discs (the active ingredient is florfenicol). Holes were punched on the plates coated with pathogenic bacteria, 200 μL of the fermented liquid of Lactobacillus plantarum HEW-A490 was spotted into the holes, and at the same time, enrofloxacin susceptibility test discs and florfenicol susceptibility test discs were placed on the plates coated with pathogenic bacteria. Incubate at a constant temperature of 30 °C. After 24 h of incubation, observe whether clear and transparent antibacterial zones appear around the spotted area and the area where the susceptibility test paper is placed. The specific results are shown in Table 3.

[0061] The medium formula for the antibacterial test of Aeromonas hydrophila is calculated by weight-volume ratio as follows: glucose 10 g / L, peptone 5 g / L, beef extract 5 g / L, NaCl 5 g / L, agar powder 16 g / L. The medium formula for the antibacterial test of Vibrio parahaemolyticus is calculated by weight-volume ratio as follows: peptone 10 g / L, beef extract 5 g / L, NaCl 35 g / L, agar powder 16 g / L. The medium for the antibacterial test of Nocardia is BHI brain heart infusion broth.

[0062] Table 3 Antibacterial diameter of the fermented liquid of Lactobacillus plantarum HEW-A490 in Example 1 (unit: cm)

[0063]

[0064] From the data in Table 3, it can be seen that the Lactobacillus plantarum HEW-A490 provided in Example 1 of the present invention has significant inhibitory effects on Aeromonas hydrophila, Vibrio parahaemolyticus, and Nocardia. Moreover, its antibacterial effects on Aeromonas hydrophila and Nocardia are better than those of enrofloxacin and florfenicol, and its antibacterial effect on Vibrio parahaemolyticus is better than that of enrofloxacin and slightly weaker than that of florfenicol. Thus, it shows that the Lactobacillus plantarum HEW-A490 strain in Example 1 has extremely strong antibacterial and antimicrobial abilities and can be widely used in aquaculture to prevent and reduce enteritis in aquatic animals, improve the absorption and utilization rate of feed, and enhance the efficiency of aquaculture.

[0065] Example 2: Preparation method of an aquatic fermentation agent containing Lactobacillus plantarum

[0066] The aquatic fermentation agent containing Lactobacillus plantarum includes bacterial powder A, bacterial powder B, and medium C, which are prepared respectively according to the following methods:

[0067] 2.1 Preparation method of bacterial powder A

[0068] The raw materials of Bacterial Powder A, by weight percentage, contain the following components: Lactobacillus plantarum HEW-A490 0.5%; Enterococcus faecalis 10%; Pediococcus acidilactici 0.5%; Bacillus subtilis 10%; Saccharomyces cerevisiae 0.5%; glucose 20%; magnesium sulfate 5%; sodium chloride 20%; corn starch 33.5%; the viable count of Lactobacillus plantarum HEW-A490 is 5.0×10 11 CFU / g, the viable count of Enterococcus faecalis is 1.0×10 11 CFU / g, the viable count of Pediococcus acidilactici is 5.0×10 11 CFU / g, the viable count of Bacillus subtilis is 1.0×10 11 CFU / g, the viable count of Saccharomyces cerevisiae is 2.0×10 10 CFU / g; The specific preparation method of the viable bacteria preparation of Lactobacillus plantarum HEW-A490 is shown in Example 1. The difference in the preparation methods of the viable bacteria preparations of Enterococcus faecalis and Pediococcus acidilactici from that of Example 1 lies in the strain types (the corresponding strains are Enterococcus faecalis and Pediococcus acidilactici) and the medium formulations.

[0069] The seed medium of Enterococcus faecalis HEW-A131 (preservation number: CGMCC No.9353) is the same as the seed medium of Lactobacillus plantarum HEW-A490 in Example 1, and the viable count of the obtained Enterococcus faecalis seed liquid is 1.0×10 9 CFU / mL; Taking the cultivation in a 50L fermenter as an example for its secondary fermentation culture, the composition of the medium in the 50L fermenter, by weight percentage, is: white sugar 2.8%, corn steep liquor dry powder 0.9%, yeast extract 0.6%, magnesium chloride 0.09%, dipotassium hydrogen phosphate 0.08%, sodium chloride 0.1%, calcium carbonate 0.16%, manganese sulfate 0.04%, Tween-80 0.08%, and the balance is water, and the pH value is 6.8±0.2. The fermentation conditions in the 50L fermenter are: the liquid loading volume is 35L of the medium, the inoculation amount is 300mL, the fermentation temperature is 37°C, the stirring speed is 120r / m, and the fermentation time is 8h.

[0070] Taking the cultivation in a 5T fermenter as an example for its tertiary fermentation culture, the medium in the 5T fermenter is the same as the secondary fermentation medium. The fermentation conditions in the 5T fermenter are: the liquid loading volume is 3.5T of the medium, the inoculation amount is 30L, the fermentation temperature is 37°C, the stirring speed is 80r / m, and the fermentation time is 16h; the viable count of the obtained Enterococcus faecalis fermentation broth is 4.5×10 9 CFU / mL.

[0071] The seed medium of Pediococcus acidilactici HEW-P27 (deposit number: CGMCC No. 15419) consists of the following components by weight percentage: glucose 2.80%, tryptone 0.45%, yeast extract powder 0.55%, magnesium chloride 0.09%, manganese sulfate 0.035%, sodium chloride 0.12%, calcium carbonate 0.15%, Tween-80 0.08%, with the balance being water, and the pH value being 6.8 ± 0.2; the viable cell count of the prepared Pediococcus acidilactici seed liquid is 2.0×10 9 CFU / mL.

[0072] Taking the 50L fermenter culture as an example for its secondary fermentation culture, the composition of the medium in the 50L fermenter by weight percentage is: sucrose 2.5%, corn steep liquor dry powder 1.2%, yeast extract 0.5%, magnesium chloride 0.09%, sodium chloride 0.1%, calcium carbonate 0.16%, manganese sulfate 0.04%, Tween-80 0.08%, with the balance being water, and the pH value being 6.8 ± 0.2. The fermentation conditions in the 50L fermenter are: the liquid loading volume is 35L of the medium, the inoculation amount is 300mL, the fermentation temperature is 37°C, the stirring speed is 120r / m, and the fermentation time is 8h.

[0073] Taking the 5T fermenter culture as an example for its tertiary fermentation culture, the medium in the 5T fermenter is the same as the secondary fermentation medium. The fermentation conditions in the 5T fermenter are: the liquid loading volume is 4.0T of the medium, the inoculation amount is 30L, the fermentation temperature is 37°C, the stirring speed is 80r / m, and the fermentation time is 16h; the viable cell count of the prepared Pediococcus acidilactici fermentation broth is 5.6×10 9 CFU / mL.

[0074] Bacillus subtilis and Saccharomyces cerevisiae are both commercially available products.

[0075] The preparation steps of powder A are as follows: respectively, the formula amounts of Lactobacillus plantarum, Enterococcus faecalis, Pediococcus acidilactici, and Saccharomyces cerevisiae are premixed with half of the formula amount of corn starch to obtain the primary premix of powder A; then the primary premix of powder A is mixed with the formula amounts of Bacillus subtilis, glucose, magnesium sulfate, sodium chloride, and the remaining half of the formula amount of corn starch for the second time to obtain the powder A, where the effective viable cell count of Lactobacillus plantarum is 2.5×10 9 CFU / g, the effective viable cell count of Enterococcus faecalis is 1.0×10 10 CFU / g, the effective viable cell count of Pediococcus acidilactici is 2.5×10 9 CFU / g, the effective viable cell count of Bacillus subtilis is 1.0×10 10 CFU / g, and the effective viable cell count of Saccharomyces cerevisiae is 1.0×10 8CFU / g; the rotation speed of the primary premix of the bacterial powder A is 30r / m and the mixing time is 240 seconds, and the rotation speed of the secondary mixing of the bacterial powder A is 50r / m and the mixing time is 150 seconds.

[0076] 2.2 Preparation method of bacterial powder B

[0077] The raw materials of bacterial powder B, in terms of weight percentage, contain the following components: 30% denitrifying bacteria, 25% bran, and 45% talcum powder, wherein the denitrifying bacteria are commercially available products, and the effective viable bacteria count is 1.0×10 10 CFU / g;

[0078] Preparation steps of bacterial powder B: respectively mix the denitrifying bacteria, bran and talcum powder in the formula amount to obtain the bacterial powder B. The effective viable count of the denitrifying bacteria in the bacterial powder B is 3.0×10 9 CFU / g; the mixing speed of the bacterial powder B is 30r / m and the mixing time is 240 seconds.

[0079] 2.3 Preparation of Medium C

[0080] The raw material composition of culture medium C is calculated by mass volume ratio, and contains the following components: 50 g / L brown sugar, 36.25 g / L corn peptone, 10 g / L yeast extract powder, 1.5 g / L sodium chloride, 0.25 g / L manganese sulfate, 0.5 g / L magnesium sulfate, and 1.5 g / L light calcium carbonate, that is, 50 g brown sugar, 36.25 g corn peptone, 10 g yeast extract powder, 1.5 g sodium chloride, 0.25 g manganese sulfate, 0.5 g magnesium sulfate, and 1.5 g light calcium carbonate, a total of 100 g, after being fully mixed and evenly, quantitatively packaged to obtain culture medium C. When used, each 100 g culture medium C is dissolved in 1000 mL water.

[0081] The preparation steps of culture medium C are as follows: first, each raw material is weighed separately according to the formula amount, and then the formula amount of sodium chloride, manganese sulfate, magnesium sulfate, light calcium carbonate and one-third of the formula amount of brown sugar are pre-mixed to obtain the culture medium C primary premix; then, the culture medium C primary premix is secondarily mixed with the formula amount of yeast extract powder, corn peptone, and the remaining two-thirds of the formula amount of brown sugar to obtain the culture medium C; the rotation speed of the primary premix of the culture medium C is 30r / m and the mixing time is 240 seconds, and the rotation speed of the secondary mixing of the culture medium C is 50r / m and the mixing time is 150 seconds.

[0082] Example 3: A method for preparing fermented bacterial liquid using aquatic fermentation agent containing Lactobacillus plantarum

[0083] The steps of preparing fermented bacterial liquid from an aquatic fermentation agent containing plant lactobacillus include activating and expanding bacterial powder A, activating bacterial powder B, and uniformly mixing the expanded bacterial powder A liquid and the activated bacterial powder B liquid to obtain the fermented bacterial liquid.

[0084] 3.1 Activation and Amplification Cultivation of Bacterial Powder A

[0085] Take 0.05 kg of bacterial powder A prepared in Example 2, activate it with 1 L of brown sugar water with a mass - volume ratio of 50 g / L at 32 °C warm water for 3 h; dissolve 20 kg of culture medium C prepared in Example 2 with boiling water at 100 °C, then cool it to 35 °C, adjust the pH value to 7.0, the liquid - filling amount is 70% (v / v), and make up the volume to 200 L for standby; inoculate the activated bacterial powder A activation solution into the previously prepared culture medium C solution according to a volume ratio of 1:200, stir evenly, and cultivate it in a sealed fermentation barrel at 35 °C for 48 h to obtain the amplified culture solution of bacterial powder A, and the pH value of this amplified culture solution is lower than 3.5.

[0086] 3.2 Activation of Bacterial Powder B

[0087] Take 1.0 kg of bacterial powder B prepared in Example 2, activate it with 20 L of brown sugar warm water with a mass - volume ratio of 50 g / L at 35 °C for 2 h to obtain the activation solution of bacterial powder B.

[0088] 3.3 Preparation of Fermented Bacterial Liquid

[0089] Fully mix the amplified culture solution of bacterial powder A prepared in Step 3.1 and the activation solution of bacterial powder B prepared in Step 3.2 according to a volume ratio of 10:1 to prepare the aquatic - product fermented bacterial liquid containing Lactobacillus plantarum.

[0090] Example 4: Method for Preparing Aquatic - Product Fermented Feed with an Aquatic - Product Fermenting Agent Containing Lactobacillus plantarum

[0091] The steps for an aquatic - product fermenting agent containing Lactobacillus plantarum to prepare aquatic - product fermented feed include three steps: activation of the fermenting agent, preparation of the fermentation substrate mixture, and sealed fermentation.

[0092] 4.1 Activation of the Fermenting Agent:

[0093] Take 1 kg of bacterial powder A prepared in Example 2, activate it with 30 L of brown sugar water with a mass - volume ratio of 50 g / L in warm water at 32 °C for 3 h to obtain the activation solution of the fermenting agent.

[0094] 4.2 Preparation of the Fermentation Substrate Mixture

[0095] First crush soybean meal, rapeseed meal, and peanut meal and then pass them through a 60 - mesh sieve to make soybean meal powder, rapeseed meal powder, and peanut meal powder. Then, fully stir 20% soybean meal powder, 25% rapeseed meal powder, 10% peanut meal powder, 8% fish meal, 25% high - gluten flour, 6.75% wheat bran, 3% soybean oil, 1.5% calcium dihydrogen phosphate, 0.5% sodium chloride, and 0.25% magnesium sulfate by mass fraction to obtain the fermentation substrate mixture.

[0096] 4.3 Sealed Fermentation

[0097] The activated liquid of the fermentation inoculant prepared in step 4.1 above was evenly sprayed (using an agricultural manual reciprocating sprayer) on the fermentation substrate mixture prepared in step 4.2, then packed in a transparent sealed bag and placed in a fermentation barrel for sealed fermentation at room temperature for 3 days to obtain the aquatic fermentation feed; after testing, the moisture content of the fermentation feed was 32 wt%, the pH value was 4.85, the crude protein content was 36.71 wt%, and the acid-soluble protein content was 18.5 wt%.

[0098] Example 5: Application of Fermentation Bacterial Liquid in Grass Carp Ponds

[0099] Grass carp with consistent specifications (average tail weight 4.21 ± 0.03 g) were selected. After the test fish were raised in a cement pond for 2 weeks, they were randomly assigned to two groups, with 3 parallel replicates in each group in 6 fish ponds of 2 mu each. 400 fish were stocked in each fish pond. The fish ponds were kept aerated continuously (aerated, and the aerator was turned on according to the weather conditions every day to increase the dissolved oxygen in the water body, and the rotation speed of the aerator was 140 revolutions per minute). Feeding was carried out once at 9:00 am and 16:00 pm every day until full satiety; the aquaculture water body of the control group was not treated. The test group was splashed with the aquatic fermentation bacterial liquid containing Lactobacillus plantarum prepared in Example 3. When used, 0.01 L of the fermentation bacterial liquid was evenly splashed (using a water ladle or a manual sprayer) on each square meter of the water surface, that is, 13.34 L of the fermentation bacterial liquid was splashed into each 2-mu fish pond, and it was splashed once every 7 days. During the feeding process, the water temperature was maintained at 25 ± 0.5 °C, and the feeding process lasted for 6 weeks; the content of nitrite and ammonia nitrogen in the water was measured once a week using a portable water quality analyzer, and the experimental results are shown in Table 4.

[0100] Table 4. Changes in Nitrite and Ammonia Nitrogen in Water

[0101]

[0102]

[0103] It can be seen from the data in Table 4 that with the addition of the aquatic fermentation bacterial liquid containing Lactobacillus plantarum, the content of nitrite and ammonia nitrogen in the water body gradually decreased; in the 6th week of the test, the content of nitrite and ammonia nitrogen in the test group decreased by 72.2% and 75.4% respectively compared with the control group, indicating that adding an aquatic fermentation bacterial liquid containing Lactobacillus plantarum in the pond improved the utilization rate of nitrogen and phosphorus in the feed, and at the same time could continue to decompose pollution sources such as organic matter in residual baits and feces in the water body, purified the water quality, and reduced the content of toxic and harmful substances in the water body.

[0104] Example 6: Application of Aquatic Fermentation Feed in Grass Carp

[0105] Select grass carps with consistent specifications (average tail weight is 82 g), divide the experimental fish into two treatments, with 3 replicates for each treatment and 60 fish for each replicate. Feed them 3 times a day to satiety for 12 weeks. The control group is fed with a conventional aquatic compound feed (its formula composition by weight percentage is: soybean meal 21%, rapeseed meal 24%, peanut meal 10%, fish meal 8%, flour 25%, wheat bran 3.15%, soybean oil 3%, bentonite 2%, calcium dihydrogen phosphate 1.2%, compound multivitamins 1%, compound multiminerals 1%, choline chloride 0.25%, crystalline methionine 0.1%, vitamin C 0.3%). The experimental group is additionally fed with the aquatic fermented feed prepared in Example 4 at 10 wt% of the daily feeding amount. After the feeding is completed, the weight gain and feed coefficient of the experimental fish in each group are measured respectively. The experimental results are shown in Table 5. The daily feeding amount refers to feeding 3% of the fish body weight as the feed amount.

[0106] Table 5 Growth of fish body

[0107] Control group Experimental group Initial body weight of fish (g / fish) 82.15±1.04 82.08±1.07 Final body weight of fish (g / fish) 229.15±3.12 271.28±3.85 Weight gain rate (%) 178.94±11.63 230.51±10.25 Feed conversion ratio 1.52±0.04 1.35±0.08

[0108] Note: Weight gain rate = 100% × (final weight - initial weight) / initial weight, feed coefficient = feed intake / (final weight - initial weight)

[0109] It can be seen from the data in Table 5 that the weight gain rate of the experimental group is 28.8% higher than that of the control group, and the feed coefficient is 11.18% lower than that of the control group, indicating that the aquatic fermented feed prepared in Example 4 of the present invention can significantly promote the growth of grass carps, improve the feed digestibility and reduce the feed coefficient.

[0110] The above is only the preferred embodiment of the present invention, and it does not impose any form of limitation on the present invention. Any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An aquatic fermentation agent containing Lactobacillus plantarum, characterized in that, It includes bacterial powder A, bacterial powder B and culture medium C, and the weight ratio of the three is 0.05: (0.5 - 2): (20 - 40); the composition of the bacterial powder A by weight percentage is: Lactobacillus plantarum 0.2 - 1%; Enterococcus faecalis 8 - 10%; Pediococcus acidilactici 0.2 - 1%; Bacillus subtilis 4 - 10%; Saccharomyces cerevisiae 0.5 - 5%; glucose 10 - 20%; magnesium sulfate 1 - 5%; sodium chloride 5 - 20%; corn starch 30 - 35%; the composition of the bacterial powder B by weight percentage is: denitrifying bacteria 10 - 30%; wheat bran 20 - 30%; talcum powder 35 - 45%; the viable count of the denitrifying bacteria in the bacterial powder B is 1 - 3 billion / g.

2. The aquatic fermenting agent according to claim 1, characterized in that, The total viable count of the bacterial powder A is 14.1 - 31 billion CFU / g, and the viable count of Lactobacillus plantarum in the bacterial powder A is 1 - 5 billion CFU / g; the viable count of Enterococcus faecalis in the bacterial powder A is 8 - 10 billion CFU / g; the viable count of Pediococcus acidilactici in the bacterial powder A is 1 - 5 billion CFU / g; the viable count of Bacillus subtilis in the bacterial powder A is 4 - 10 billion CFU / g; the viable count of Saccharomyces cerevisiae in the bacterial powder A is 0.1 - 1 billion CFU / g.

3. The aquatic fermentation agent according to claim 1, characterized in that, The viable count of the denitrifying bacteria in the bacterial powder B is 1 - 3 billion / g; the composition of the culture medium C by mass - volume ratio is: brown sugar 10 - 50 g / L, corn peptone 10 - 40 g / L, yeast extract powder 5 - 10 g / L, sodium chloride 0.5 - 2 g / L, manganese sulfate 0.1 - 0.5 g / L, magnesium sulfate 0.25 - 1 g / L, light calcium carbonate 0.5 - 2 g / L.

4. A preparation method of the aquatic fermentation agent containing Lactobacillus plantarum according to any one of claims 1-3, characterized in that, It includes the following steps: Preparation of bacterial powder A: Primarily premix the formula - amount of Lactobacillus plantarum, Enterococcus faecalis, Pediococcus acidilactici, Saccharomyces cerevisiae with one - half of the formula - amount of corn starch to obtain the primary premix of bacterial powder A; then secondarily mix the primary premix of bacterial powder A with the formula - amount of Bacillus subtilis, glucose, magnesium sulfate, sodium chloride, and the remaining one - half of the formula - amount of corn starch to obtain the bacterial powder A. Preparation of bacterial powder B: Mix the formula - amount of denitrifying bacteria, wheat bran and talcum powder evenly to obtain the bacterial powder B. Preparation of culture medium C: Primarily premix the formula - amount of sodium chloride, manganese sulfate, magnesium sulfate, light calcium carbonate with one - third of the formula - amount of brown sugar to obtain the primary premix of culture medium C. Then secondarily mix the primary premix of culture medium C with the formula - amount of yeast extract powder, corn peptone, and the remaining two - thirds of the formula - amount of brown sugar to obtain the culture medium C.

5. The preparation method of the aquatic fermentation agent containing Lactobacillus plantarum according to claim 4, characterized in that, The rotation speed of the primary premixing of the bacterial powder A is 20-40 r / m, and the mixing time is 200-300 seconds; the rotation speed of the secondary mixing of the bacterial powder A is 40-60 r / m, and the mixing time is 120-180 seconds; the mixing rotation speed of the bacterial powder B is 20-40 r / m, and the mixing time is 200-300 seconds; the rotation speed of the primary premixing of the culture medium C is 20-40 r / m, and the mixing time is 200-300 seconds; the rotation speed of the secondary mixing of the culture medium C is 40-60 r / m, and the mixing time is 120-180 seconds.

6. Use of the lactic acid bacteria-fermented aquatic product agent containing Lactobacillus plantarum according to any one of claims 1-3 in the preparation of a fermented bacterial liquid and the preparation of a lactic acid bacteria-fermented aquatic feed.

7. Use of the lactic acid bacteria-fermented aquatic product agent containing Lactobacillus plantarum according to claim 6 in the preparation of a fermented bacterial liquid and the preparation of a lactic acid bacteria-fermented aquatic feed, characterized in that the lactic acid bacteria-fermented aquatic feed includes fish feed, shrimp feed, crab feed, frog feed, soft-shelled turtle feed or turtle feed; in the said application, soybean meal, peanut meal, fish meal, flour, wheat bran, calcium dihydrogen phosphate, and sodium chloride are used as the fermentation substrate, and the bacterial powder A mainly composed of Lactobacillus plantarum, Enterococcus faecalis, Pediococcus acidilactici, Bacillus subtilis, and Saccharomyces cerevisiae is used as the fermentation agent, and the weight ratio of the fermentation substrate components to the fermentation agent components is (500-1000):

1.

8. Use of the lactic acid bacteria plantarum-containing aquaculture starter in preparing fermented bacterial liquid and preparing aquaculture fermented feed, characterized in that, The use of the lactic acid bacteria-fermented aquatic product agent containing Lactobacillus plantarum in the preparation of a lactic acid bacteria-fermented aquatic feed includes the following steps: A) Activation of the fermentation agent: Activate 1-3 kg of the bacterial powder A in 30-60 L of 10-50 g / L brown sugar water in warm water at 30-35 °C for 1-3 h to obtain the activated liquid of the fermentation agent; B) Preparation of the fermentation substrate mixture: First, crush soybean meal, rapeseed meal, and peanut meal and then pass through a 60-80 mesh sieve to make soybean meal powder, rapeseed meal powder, and peanut meal powder. Then, fully stir 20-35% soybean meal powder, 20-30% rapeseed meal powder, 10-20% peanut meal powder, 5-15% fish meal, 20-30% high-gluten flour, 4-8% wheat bran, 3-4% soybean oil, 1-2% calcium dihydrogen phosphate, 0.5-2% sodium chloride, and 0.25-0.5% magnesium sulfate by mass fraction to obtain the fermentation substrate mixture; C) Fermentation: Evenly spray the activated liquid of the fermentation agent prepared in step A) above into the fermentation substrate mixture prepared in step B), pack it in a transparent sealed bag and place it in a fermentation barrel, and ferment it at room temperature and in a closed state for 3-5 days. After fermentation, the color and luster of the material are uniform, the texture is soft, there is no mildew or caking, and the smell is gentle, that is, the fermented feed is made, and its moisture content is 30 wt%-40 wt%, the pH value is lower than 5.00, the crude protein content is 30-40 wt%, and the acid-soluble protein content is 18-20 wt%; D) Sprinkle the freshly prepared fermented feed in step C) above into the pond at 5-20% wt of the daily feeding amount of aquatic animals, and feed once a day.

9. Use of the lactic acid bacteria plantarum-containing aquatic fermenting agent according to claim 6 in the preparation of a fermented bacterial liquid and the preparation of an aquatic fermented feed, characterized in that, The use of the lactic acid bacteria-fermented aquatic product agent containing Lactobacillus plantarum in the preparation of a fermented bacterial liquid includes the following steps: 1) Expansion of bacterial powder A: 0.25-0.5 kg of bacterial powder A is activated in 30-35°C warm water with 5-10 L of 10-50 g / L brown sugar water for 0.5-3 h; after dissolving culture medium C in 100°C boiling water, the temperature is lowered to 25-35°C, the pH value is adjusted to 6.0-7.0, the liquid volume is 70-80% (v / v), and the volume is fixed to 90-95 L for standby; the activated bacterial powder A is inoculated into the obtained culture medium C solution at a mass volume ratio of 1:400-1:200, stirred evenly, and cultured in a closed manner at 25-35°C for 24-72 h to obtain bacterial powder A expansion solution; 2) Activation of bacterial powder B: Activate 0.25-0.5 kg of bacterial powder B with 5.0-10 L of 10-50 g / L, 30-37°C brown sugar warm water for 0.5-3 h; 3) Mix the culture solution of bacterial powder A and the activation solution of bacterial powder B in a volume ratio of 20:1-5:1 to obtain a fermentation solution; Sprinkle the freshly prepared fermentation liquid in step 3) at a volume ratio of 1:2000-1:100 into the water of aquaculture ponds for fish, shrimp, crab, frog, turtle or tortoise once every 7-10 days.

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