A composite antibacterial postbiotic preparation and its preparation method and application

Abstract

The present invention discloses a composite antibacterial postbiotic preparation and its preparation method and application. The composite antibacterial postbiotic preparation comprises 70% to 80% of Bacillus subtilis metabolites and 20% to 30% of inactivated Bacillus coagulans. The composite postbiotic preparation has a significant inhibitory effect on common pathogenic bacteria of Penaeus vannamei. The outdoor pond uses 100 to 200 g / mu / m, and the indoor factory cement pool uses 0.5 to 1 g / m 3 The average removal rate of Vibrio in water bodies is ≥71% in one day, ≥80% in two days, and ≥91% in three days; and it has a fast onset of effect, low oxygen consumption, and low cost in inhibiting pathogens in ponds; the storage process is not affected by high temperatures, and it can withstand high temperature, high pressure, and humid heat conditions in the processing of aquatic feed, and can meet the needs of aquatic feed addition. Its application in aquaculture can significantly improve the survival rate and economic benefits of aquaculture.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a composite antibacterial postbiotic preparation and a preparation method and application thereof. Background Art

[0002] Postbiotics refers to a preparation of inactive microorganisms and / or their components that are beneficial to the health of the host. It is considered to be the fourth generation of microecological products after probiotics, prebiotics and synbiotics. In the field of aquaculture, research on postbiotics has only just begun to appear and has gradually increased in the past two years.

[0003] In recent years, the occurrence of shrimp diseases such as bacterial hyalinosis (BVS) and acute hepatopancreatic necrosis syndrome (APHNS) of Litopenaeus vannamei fry is closely related to pathogenic bacteria such as Vibrio and Monospora of different species. These diseases are rapid in onset and have a high mortality rate. Therefore, the prevention and control of such pathogens is a key link in the success of aquaculture. As a biological control strategy for pathogenic Vibrio in aquatic products, microecological preparations such as lactic acid bacteria and Bacillus have gradually become an important part of antibiotic substitution and green aquaculture systems. Generally, probiotics can compete with pathogens for adhesion sites and nutritional resources, secrete antibacterial substances, and regulate the body's immunity and intestinal barrier function. However, when using traditional probiotic live bacteria preparations, they face challenges such as maintaining strain activity and stability and tolerating high temperatures in aquatic feed pelleting.

[0004] Postbiotics mainly contain inactive microorganisms and / or their components, which are little affected by ambient temperature. Studies have shown that they have great application potential in inhibiting pathogens, improving digestive health and immune function. In terms of improving the aquaculture environment and the intestinal tract of farmed animals, how to increase the content and stability of active ingredients through process optimization, and achieve a comprehensive prevention and control effect through the rational combination of different strains, is a difficult point in the development of innovative products. Summary of the invention

[0005] The present invention provides a composite antibacterial postbiotic preparation and a preparation method and application thereof. The present invention utilizes Bacillus subtilis ( Bacillus subtilis ) Under specific fermentation process conditions, the BSXE-1601 strain regulates metabolic pathways to produce highly active metabolites, which directly kills / inhibits pathogens and regulates the balance of host intestinal flora. Bacillus coagulans ) E21 inactivated bacteria to synergistically improve the host's immune resistance and intestinal barrier function and other mechanisms, effectively preventing and controlling the occurrence or outbreak of diseases caused by pathogenic bacteria such as Vibrio and Monocytogenes in aquatic animals.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0007] The present invention provides a composite antibacterial postbiotic preparation, which comprises, by volume percentage, 70% to 80% of Bacillus subtilis metabolites and 20% to 30% of inactivated Bacillus coagulans.

[0008] Furthermore, the Bacillus subtilis is selected from Bacillus subtilis BSXE-1601 with a deposit number of CGMCC NO.15949; the Bacillus subtilis metabolites include isocoumarins, soybean isoflavones, cyclolipopeptides, terpenes, fengyuansu, surfactin, iturin, bacillus lysin and subtilisin.

[0009] Furthermore, the Bacillus coagulans is selected from Bacillus coagulans E21 with a deposit number of CGMCC No.13044.

[0010] The present invention also provides a method for preparing the composite antibacterial postbiotic preparation, which comprises the following steps:

[0011] (1) fermenting the activated Bacillus coagulans, inactivating the obtained Bacillus coagulans fermentation liquid at high temperature, and drying it to obtain inactivated Bacillus coagulans cells;

[0012] (2) activating and culturing Bacillus subtilis, fermenting the obtained Bacillus subtilis seed liquid, and then performing secondary fermentation on the obtained primary seed liquid to obtain a fermentation liquid;

[0013] (3) adding an adhesive carrier to the fermentation liquid and drying the fermentation liquid to obtain a spray-dried powder containing Bacillus subtilis metabolites;

[0014] (4) The inactivated Bacillus coagulans obtained in step (1) and the spray-dried powder containing the metabolites of Bacillus subtilis obtained in step (3) are mixed evenly to obtain a composite antibacterial postbiotic preparation.

[0015] Furthermore, the fermentation medium formula of the Bacillus coagulans is: 20-60 g / L bran, 10-30 g / L yeast powder, 10-30 g / L corn steep liquor powder, 2-5 g / L sodium chloride, and 0.5-1 g / L dipotassium hydrogen phosphate.

[0016] Preferably, the fermentation medium formula of Bacillus coagulans is: 40 g / L bran, 15 g / L yeast powder, 15 g / L corn steep liquor powder, 2 g / L sodium chloride, and 1 g / L dipotassium hydrogen phosphate.

[0017] Furthermore, the culture conditions of the Bacillus coagulans are: initial pH 7.0, temperature 40±1°C, fermenter speed 200 r / min, ventilation volume 1:0.85, and culture time 22 h.

[0018] The present invention also provides application of the composite antibacterial postbiotic preparation in the preparation of aquatic pathogen inhibitors.

[0019] Furthermore, the aquatic pathogens include Vibrio towerii, Photobacterium mermaidense, Vibrio new caledoniensis, and Vibrio erwensii.

[0020] The present invention also provides application of the composite antibacterial postbiotic preparation in shrimp farming.

[0021] Furthermore, the composite antibacterial postbiotic preparation can be mixed into shrimp diets as a feed additive, and can also be used by spraying into aquaculture water.

[0022] Furthermore, the amount of the composite antibacterial postbiotic preparation used as a feed additive is 0.25‰~5.00‰ of the weight of the shrimp diet; the amount of the composite antibacterial postbiotic preparation sprayed is 100 g / mu▪m~200 g / mu▪m.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] 1. The present invention utilizes Bacillus subtilis BSXE-1601 to be cultured and fermented under specific fermentation process conditions, and finally uses instantaneous spray drying post-treatment technology to eliminate potential factors such as enzymatic hydrolysis and hydrolysis reactions, thereby forming a powder preparation of Bacillus subtilis metabolites with high antibacterial activity, good stability and heat resistance. The Bacillus subtilis metabolites mainly contain three major types of antibacterial-related active ingredients, including: cyclolipopeptides, isocoumarins and soy isoflavones. Compared with traditional live bacterial preparations, the antibacterial metabolites of Bacillus subtilis have the following characteristics: (1) relatively clear functional components and stronger targeting; (2) high antibacterial activity and broad antibacterial spectrum; (3) no need for on-site activation / germination and faster effectiveness; (4) small bacterial count and lower oxygen consumption; (5) less interference from environmental and other limiting factors; (6) tolerance to wet and hot processing conditions of aquatic feed and more convenient application; and it does not involve animal pathogen resistance and aquatic product veterinary drug residue monitoring components, so it has the potential for application as a technology to reduce the amount of aquatic veterinary antibiotics.

[0025] 2. The present invention utilizes the inactivated bacteria of Bacillus coagulans E21 and the metabolites of Bacillus subtilis BSXE-1601 to further enhance the host immune disease resistance and intestinal barrier function, and effectively prevent and control the occurrence or outbreak of diseases caused by pathogenic bacteria such as Vibrio and Monocytogenes in aquatic animals.

[0026] 3. The composite postbiotic preparation has a significant inhibitory effect on common pathogenic bacteria of Penaeus vannamei. The dosage for outdoor ponds is 100-200 g / mu / m, and for indoor factory cement pools is 0.5-1 g / m 3 The average removal rate of Vibrio in water bodies is ≥71% in one day, ≥80% in two days, and ≥91% in three days; and it has a fast onset of effect, low oxygen consumption, and low cost in inhibiting pathogens in ponds; the storage process is not affected by high temperatures, and it can withstand high temperature, high pressure, and humid heat conditions in the processing of aquatic feed, and can meet the needs of aquatic feed addition. Its application in aquaculture can significantly improve the survival rate and economic benefits of aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the key active ingredient of the metabolites of Bacillus subtilis BSXE-1601;

[0028] Figure 2 The stability tracking results of the metabolites of Bacillus subtilis BSXE-1601;

[0029] Figure 3 To evaluate the thermotolerance of metabolites of Bacillus subtilis BSXE-1601;

[0030] Figure 4 It is the plate inhibition zone of the composite antibacterial postbiotic preparation;

[0031] Figure 5 The changes in OD values ​​were determined for the MIC values ​​of the composite antibacterial postbiotic preparations;

[0032] Figure 6 For recirculating aquaculture experiments;

[0033] Figure 7 The mortality rate of Penaeus vannamei in the poison challenge test;

[0034] Figure 8 is the removal rate of Vibrio in cement pool after using different products;

[0035] Fig. 9 It is the removal rate of Vibrio in high-level pool after using composite antibacterial postbiotic preparation. Implementation

[0036] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0037] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0038] Bacillus coagulans ( Bacillus coagulans)E21 comes from the cecal digesta of healthy chickens. This strain is a Gram-positive bacterium, facultative anaerobic, and can decompose sugars to produce L-lactic acid. It is a homolactic fermentation bacterium; its optimal growth temperature is 45-50℃, and its optimal pH is 6.6-7.0. The depository of Bacillus coagulans E21 is the General Microbiology Center of China Microbiological Culture Collection (CGMCC); Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Deposit date: September 26, 2016; Bacillus coagulans Bacillus coagulans The deposit number is CGMCCNO.13044.

[0039] Bacillus subtilis ( Bacillus subtilis )BSXE-1601 was isolated from a shrimp farming pond. The strain is a Gram-positive bacterium with a rod-shaped body, spores, meso-spores, round or irregular colonies, irregular colony edges, opaque moss, off-white, moist surface, and easy to pick. The depository of Bacillus subtilis BSXE-1601 is: China General Microbiology Center (CGMCC); Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Deposit date: June 19, 2018; Bacillus subtilis Bacillus subtilis The deposit number is CGMCC NO.15949.

[0040] Example 1: Optimization of the process for inactivating Bacillus coagulans E21

[0041] (1) Screening of inorganic salts in fermentation medium

[0042] Take out the glycerol tube containing Bacillus coagulans E21 from the -20℃ refrigerator, streak it on the LB slant medium, and culture it in a constant temperature incubator at 37℃ for 3 days until spores form. The fermentation medium includes a basal medium and different inorganic salt components. The basal medium is 20g / L bran, 10g / L yeast powder, and pH 7.0. Different inorganic salts are added to the basal medium: sodium chloride (2g / L and 5g / L), magnesium sulfate (0.2g / L and 0.5g / L), manganese sulfate (0.5g / L and 1g / L), potassium dihydrogen phosphate (0.5g / L and 1g / L), and potassium dihydrogen phosphate (0.5g / L and 1g / L), and the volume is adjusted to 1L with water and sterilized at 116℃ for 30min. Take an appropriate amount of bacterial moss with an inoculating loop and add it to a small amount of sterile physiological saline, shake it evenly, and inoculate an equal amount into a 500mL baffled flask containing 100mL of different fermentation medium, and culture it in a constant temperature shaker at 40±1℃ and 200r / min for 22h. Determine the content of Bacillus coagulans in the fermentation medium, examine the spore production under a microscope, and determine the pH of the fermentation liquid.

[0043] The results are shown in Table 1. The type and amount of inorganic salts have a significant effect on the proliferation and sporulation of strain E21. Adding 2g / L sodium chloride and 1g / L potassium dihydrogen phosphate will reduce sporulation and have a higher bacterial count, while magnesium sulfate, manganese sulfate and potassium dihydrogen phosphate are easy to promote sporulation and cannot be used in the preparation of inactivated cells of Bacillus coagulans E21.

[0044] Table 1 Proliferation, spore production and pH changes of strain E21 in different culture media

[0045]

[0046] (2) Orthogonal test of carbon and nitrogen sources in fermentation medium

[0047] Take out the glycerol tube containing Bacillus coagulans E21 from the -20℃ refrigerator, streak it on the LB slant medium, and culture it in a constant temperature incubator at 37℃ for 3 days until spores are formed. The culture medium selected was bran (20~60 g / L), yeast powder (10~30 g / L), corn steep liquor powder (10~30 g / L), sodium chloride (2g / L) and potassium dihydrogen phosphate (1g / L). The orthogonal experimental scheme with different carbon and nitrogen raw material addition ratios was designed by using the orthogonal design assistant software, and the corresponding culture medium was prepared and sterilized at 116℃ for 30min. Use an inoculation loop to take an appropriate amount of bacterial moss from the LB slant and shake it with a small amount of sterile physiological saline. Inoculate an equal amount into a 500mL baffled triangular flask containing 100mL of different fermentation medium, and culture it on a constant temperature shaker at 40℃±1℃ and 200r / min for 22h. Determine the content of Bacillus coagulans in the fermentation medium, examine the spore production under a microscope, and determine the pH of the fermentation liquid.

[0048] The experimental results are shown in Table 2. Among the three carbon and nitrogen sources, the factor with the greatest impact on the bacterial quantity is bran, followed by corn syrup powder, and finally yeast powder. In order to obtain a higher bacterial quantity and the lowest sporulation rate, the optimized carbon and nitrogen source scheme was analyzed to be 40g / L bran, 10g / L yeast powder, and 10g / L corn syrup powder.

[0049] Table 2 Orthogonal experimental design groups and results

[0050]

[0051] (3) Pilot fermentation and powder spraying production

[0052] The medium composition optimized by baffled shake flask was 40g / L bran, 10g / L yeast powder, 10g / L corn steep liquor powder, 2g / L sodium chloride and 1g / L potassium dihydrogen phosphate for 7.5 ton pilot fermentation tank production. The initial pH of the medium was 7.0, and the pH range was between 6.2 and 7.2 during the fermentation process. The temperature was 40±1℃, the fermentation tank speed was 200 r / min, the ventilation rate was 1:0.85, and the fermentation broth of Bacillus coagulans was obtained after 22h of culture. The cell number of Bacillus coagulans E21 in the fermentation broth was 8.00×10 9 CFU / mL ~1.00×10 10 CFU / mL, and the sporulation rate is ≤10%. The fermented Bacillus coagulans fermentation liquid is heated to 90~95℃ and maintained for 30min, and then cooled to room temperature within 30min using circulating water. Add 10% anhydrous glucose carrier and use a spray drying tower for spray drying. The sample flow rate is 20~30 mL / min, the atomization frequency is 40 Hz, the inlet temperature is 160~170℃, and the outlet temperature is 70~80℃. The obtained dry powder is the inactivated bacteria of Bacillus coagulans. The pilot yield of 3 batches is 9.5%~11.5%, that is, one ton of Bacillus coagulans fermentation liquid produces 95~115kg of inactivated bacteria of Bacillus coagulans.

[0053] Example 2: Optimization of pilot fermentation process for metabolites of Bacillus subtilis BSXE-1601 strain

[0054] 1. Seed liquid culture: Take Bacillus subtilis BSXE-1601 strain storage liquid (about 1×10 6 -1×10 7 cfu / ml) were streaked on nutrient agar medium and cultured at 30°C for 24 h. A single colony was picked and inoculated into liquid nutrient medium and cultured at 30°C and 100 r / min for 24 h to obtain activated Bacillus subtilis BSXE-1601 seed liquid.

[0055] 2. Secondary fermentation process: Inoculate the activated Bacillus subtilis BSXE-1601 seed liquid into the first seed tank containing sterilized fermentation medium at an inoculation rate of 4-6% (bacterial volume is about 1×10 6 -1×10 7cfu / ml), and the fermentation parameters were adjusted as follows: temperature 30°C, stirring speed 100 r / min, tank pressure 0.05 MPa, natural pH, and culture for 16-20 h. Then, the first-level seed liquid was inoculated into a fermentation tank containing sterilized fermentation medium at a volume ratio of 1:10, and cultured for 20-24 h at 30°C, 100 r / min speed, tank pressure 0.05 MPa, and natural pH. After stopping the tank, 10% of the adherent carrier was added, and the airflow high-temperature spray drying technology was used to obtain a low viable bacterial content (10~30 ×10 8 CFU / g) of Bacillus subtilis metabolite spray-dried powder.

[0056] The fermentation medium formula is as follows: corn starch 10 g / L, peptone 10 g / L, glucose 10 g / L, sodium chloride 5 g / L, KH 2 PO 4 5 g / L, pH adjusted to 7.0, sterilized the prepared culture medium at 121℃, 101KPa for 30min, cooled to room temperature and inoculated with seed solution.

[0057] Example 3: Analysis of metabolites of Bacillus subtilis BSXE-1601

[0058] Experiment 1: Analysis of active ingredients of metabolites

[0059] Liquid chromatography-mass spectrometry (LC-MS) was used to quantitatively analyze the key active small molecule components (relative molecular weight less than about 1000) in the metabolites of Bacillus subtilis BSXE-1601 in Example 2. The differential metabolites related to antibacterial activity mainly include: Amicoumacin (Ami A / B / C), soybean isoflavones, cyclolipopeptides (Cyclic lipopeptides), terpenes (Epothilone A), Fengycin, Surfactin, Iturin, Bacilysin and other small peptides such as subtilisin ( Figure 1 ).

[0060] Experiment 2: Stability Tracking

[0061] In order to evaluate the stability and shelf life of the metabolites of Bacillus subtilis BSXE-1601, the spray-dried powder was placed in a 4°C refrigerator and a 30°C incubator, and the antibacterial activity was detected by the plate Oxford cup antibacterial method, targeting Vibrio alginolyticus and benchmarking the potency of chlortetracycline hydrochloride (ppm). Figure 2) showed that under room temperature (20-30°C) storage conditions, the antibacterial activity retention rate of Bacillus subtilis BSXE-1601 metabolites was above 90% for 6 months and above 70% for 12 months, indicating that the metabolites of Bacillus subtilis BSXE-1601 do not require low-temperature refrigeration and have a shelf life of more than 12 months.

[0062] Test 3: Heat resistance evaluation

[0063] In order to simulate the high temperature and wet heat pelleting conditions of aquatic feed, the pilot sample of Bacillus subtilis BSXE-1601 metabolites was added to the conventional aquatic feed diet formula (Table 3), and the feed pellets were prepared with a laboratory small pelletizer. The antibacterial activity of BSXE-1601 metabolites before and after pelleting under wet heat conditions was detected. The raw materials were weighed and mixed according to the formula. The formula is shown in Table 3, and the pellets were made using a laboratory small feed pelletizer. After pelleting, 10 g of the sample was weighed and placed in a 250 mL conical flask containing 100 mL sterile water and small glass beads, and treated in an oven at 100°C for 10 min and 30 min, and 110°C for 5 min and 10 min, respectively. Results ( Figure 3 ) showed that the antibacterial activity retention rate of Bacillus subtilis BSXE-1601 metabolites after 100℃ wet heat treatment for 30 min was 75%, and the antibacterial activity retention rate after 110℃ wet heat treatment for 10 min was 83%. This shows that the metabolites of Bacillus subtilis BSXE-1601 have the potential for the development of feed additive products.

[0064] Table 3 Simulated formula of aquatic feed diet

[0065]

[0066] Example 4: Preparation of composite antibacterial postbiotic preparation and evaluation of in vitro antibacterial effect

[0067] The inactivated bacteria of Bacillus coagulans E21 obtained in Example 1 and the metabolites of Bacillus subtilis BSXE-1601 obtained in Example 2 were mixed and compounded in a ratio of 3:7 to obtain a composite antibacterial postbiotic preparation.

[0068] The composite antibacterial postbiotic preparation was weighed and dissolved in sterile distilled water at a mass volume ratio of 10%. The mixture was shaken at 30°C and 200 r / min for 1 h, centrifuged at 12000 r / min for 10 min, and the supernatant was taken for in vitro Oxford cup antibacterial test. Different Vibrio were selected as target pathogens, and the logarithmic value (log) of chlortetracycline hydrochloride concentration (400, 800, 1600, 3200 ppm) was used as the abscissa and the diameter of the inhibition zone (in millimeters) was used as the ordinate for linear analysis. The regression equation and correlation coefficient r (R 2>0.9 can be considered valid). The diameter of the inhibition zone of the composite antibacterial postbiotic preparation of the present invention is converted into the potency of chlortetracycline hydrochloride according to the regression equation.

[0069] As shown in Table 4 and Figure 4 As shown in Figure 2, the direct inhibitory / killing effect of the composite antibacterial postbiotic preparation on Vibrio towerii is equivalent to 8.1×10 4 The antibacterial activity exhibited by ppm chloramphenicol hydrochloride can also effectively inhibit Photorhabditis elegans, and also show good inhibitory effects on important aquatic pathogens such as Vibrio New Caledoniensis and Vibrio Owensii.

[0070] Table 4 Inhibitory effect of compound antibacterial postbiotic preparation on aquatic pathogens

[0071]

[0072] Note: “﹢﹢﹢”: high sensitivity, inhibition zone ≥20 mm; “﹢﹢”: medium sensitivity, inhibition zone 15 ~20 mm; “﹢”: low sensitivity, inhibition zone 10 ~15 mm.

[0073] Example 5: Minimum inhibitory concentration (MIC) detection of composite antibacterial postbiotic preparation

[0074] In order to quantitatively determine the in vitro antibacterial activity of the composite antibacterial postbiotic preparation, the minimum inhibitory concentration (MIC) was determined by the 96-well plate microdilution method. Weigh 5 g of the composite antibacterial postbiotic preparations of different pilot batches, No. S6-2109 and S6-2112 powder, add 95 mL of sterile water, shake on a shaker at 30℃ and 200 r / min for 4 h, centrifuge at 12000 r / min for 20 min, take the supernatant, and filter through a 0.22 μM microporous filter membrane to obtain a 5% concentration extract. Vibrio towerii, Photobacterium mermaidii, Vibrio erwinii and Vibrio caledonia were used as the targeted pathogens for inhibition. The pathogens were inoculated into 2216E liquid culture medium at a 1% addition amount, placed in a shaker at 30℃ and 200 r / min for 17 h, and the cultured indicator bacteria suspension was diluted to 10 6CFU / mL for later use. In a 96-well sterile plate, dilute the sample using the 2-fold dilution method. Add 100 μL of sterile 2216E liquid culture medium to wells A1-A11, add 100 μL of 5% S6-2109 batch extract to well A1, pipette and mix, then pipette 100 μL to well A2, pipette and mix, then pipette 100 μL to well A3 in the same row, and so on, until well 11, mix and discard 100 μL, rows BCD are the same as row A. Add 100 μL of sterile 2216E liquid culture medium to wells A1-A11, add 100 μL of Vibrio dilution to wells B1-B11, and rows CD are parallel to row B. Add 100 μL of 5% S6-2109 batch extract to rows EFGH, and other operations are the same as rows ABCD. A11-H11 was used as a positive control, i.e., 100 μL of sterile 2216E liquid culture medium and 100 μL of Vibrio dilution were added. The prepared 96-well plate was placed on a shaker at 30°C and 100 r / min and incubated for 16 h. The OD was measured using an enzyme reader. 600 Absorbance value.

[0075] As shown in Table 5 and Figure 5 As shown in the figure, the MIC values ​​of different batches of composite antibacterial postbiotic preparations S6-2109 and S6-2112 against Vibrio towerii, Photobacterium mermaidii, Vibrio caledoniensis and Vibrio erwinii were 1.56‰, 1.25%, 1.25% and 2.5%, respectively. The results showed that there was no significant difference in the antibacterial activity between different batches of composite antibacterial postbiotic preparations.

[0076] Table 5 MIC values ​​of different batches of composite antibacterial postbiotic preparations

[0077]

[0078] Example 6: Application of composite antibacterial postbiotic preparation in cultivation of Penaeus vannamei

[0079] The experiment used Litopenaeus vannamei as the breeding object, and carried out breeding experiments and virus attack experiments in the aquaculture laboratory of Qingdao Genyuan Biological Group ( Figure 6 ).

[0080] Experiment 1: Farming experiment

[0081] A total of 288 healthy Penaeus vannamei (average weight of a single shrimp: 1.06±0.08 g) were selected and randomly divided into 4 groups. The blank control group was fed with normal compound feed, and the experimental groups were mixed with compound feed and added with 0.5 g / kg, 1.0 g / kg, and 5.0 g / kg of the compound antibacterial postbiotic preparation, respectively. Each group was set up with 6 replicates, and 12 shrimps were stocked in each aquarium ( Figure 6). The shrimps were fed three times a day, with a feeding time of 90±10 min. The air was continuously aerated with an air pump, and the flow rate of the circulating water pump was 12000 L / h. The water was changed by 3% every day. The water quality conditions were always maintained as follows: water temperature 27±1℃, pH 8.2±0.2, ammonia nitrogen <0.1mg / L, nitrite ≤0.05 mg / L. After 30 days of feeding under the above conditions, the survival rate, weight gain rate, feed conversion rate, and changes in the number of Vibrio in the water and shrimp bodies were observed and counted.

[0082] The results (Table 6) showed that adding 0.25‰, 1.00‰, and 5.00‰ of the compound antibacterial postbiotic preparation to the shrimp diet could significantly improve the survival rate of Penaeus vannamei ( p <0.05), had no significant effect on weight gain rate (%) and feed conversion rate.

[0083] Table 6 Growth performance of Penaeus vannamei fed with compound antibacterial postbiotic preparations

[0084]

[0085] Experiment 2: Poison attack test

[0086] After the sampling of the aquaculture experiment was completed, the remaining shrimp in each treatment group were divided into three replicates and cultured for 3 days. Each shrimp was injected intramuscularly with 1.0×10 5 The toxicity test of CFU Vibrio tower was carried out, the mortality of shrimp was observed and counted, and the activities of serum immune enzymes: superoxide dismutase (SOD), phenoloxidase (PO), alkaline phosphatase (ALP) and lysozyme (LYS) were detected.

[0087] Results (Table 7 and Figure 7 ) showed that under the conditions of Vibrio tower virus test, feeding 0.25‰, 1.00‰, 5.00‰ composite antibacterial postbiotic preparations can significantly reduce the mortality of Penaeus vannamei ( p <0.05). In terms of shrimp serum immunity and antioxidant indicators, the three test doses can significantly increase the activity of superoxide dismutase (SOD), 0.25‰ and 1.00‰ significantly increase the activity of alkaline phosphatase (ALP), and 1.00‰ and 5.00‰ significantly increase the activity of lysozyme (LYS).

[0088] Table 7 Serum immune enzyme activity levels of Penaeus vannamei

[0089]

[0090] Example 7: Composite antibacterial postbiotic preparation pond application test

[0091] Experiment 1: Application in cement ponds for factory shrimp farming

[0092] The experiment was carried out at the Vannamei shrimp factory breeding base in Rizhao City, Shandong Province. The shrimp size was about 8 cm (80 days after stocking), the breeding pond size was 5 m × 6 m × 1.0 m, the water temperature was 28℃, nitrite = 0.3 mg / L, ammonia nitrogen = 3 mg / L, pH = 8.0, and the daily water exchange volume was 1 / 3. In the case of Vibrio outbreak (Vibrio number ≥ 10000 CFU / mL) and shrimp death, potassium persulfate group, Bdellovibrio group and composite antibacterial postbiotic preparation group were set up respectively to compare the effect of Vibrio prevention and control. The experimental grouping and antibacterial preparation use scheme are shown in Table 8.

[0093] Table 8 Grouping of the industrialized Penaeus vannamei farming experiment

[0094]

[0095] result( Figure 8 ) showed that under the condition of Vibrio outbreak, the Vibrio removal rate of the composite antibacterial postbiotic preparation was 71.47% after one day of use, showing a gradually decreasing trend. On the fourth day, the Vibrio removal rate reached 93.70%; the Vibrio removal rate of the potassium persulfate group and the Bdellovibrio group decreased significantly after the third day, and Vibrio rebound occurred. Therefore, the Vibrio prevention and control efficiency (timeliness and sustainability) of the composite antibacterial postbiotic preparation is significantly better than the conventional potassium persulfate and Bdellovibrio treatment technology.

[0096] Experiment 2: Application in high-level shrimp farming ponds

[0097] The experiment was carried out at the Vannamei high-level pond breeding base in Zhangpu City, Fujian Province. The shrimp size was about 50 heads / jin, the pond area was 1.5 mu, the water depth was 1.8 m, the breeding water temperature was 28℃, the nitrite was 0.3 mg / L, the ammonia nitrogen was 1.2 mg / L, the salinity was 28‰, the pH was 7.1, there was no water change, and 3 to 4 meals were fed daily. Three days before the pond thinning, a composite antibacterial postbiotic preparation was used, and 150 g / mu▪m was sprinkled on the water. In the evening, 1 g / kg was added to the feed and fed once. The number of Vibrio hepatopancreas in the water and shrimp was tested 24 h and 48 h after use.

[0098] Before use, the number of Vibrio in the water of shrimp culture ponds was about 5000-10000 CFU / mL. After using the composite antibacterial postbiotic preparation, the number of Vibrio in the water decreased by 71.66% and 80.52% at 24 h and 48 h, respectively, and the number of Vibrio in the hepatopancreas of shrimp decreased by 44.65% and 90.28%, respectively ( Fig. 9 ), the number of Vibrio is controlled within the safety threshold.

[0099] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A composite antibacterial postbiotic preparation, characterized in that: Measured by volume percentage, the composite antibacterial postbiotic preparation includes 70% to 80% of Bacillus subtilis metabolites and 20% to 30% of inactivated Bacillus coagulans. The Bacillus subtilis is selected from Bacillus subtilis BSXE-1601 with a deposit number of CGMCC NO.15949; the Bacillus subtilis metabolites include isocoumarins, soybean isoflavones, cyclolipopeptides, terpenes, fengyuansu, surfactin, iturin, bacillus lysin and subtilisin; The Bacillus coagulans is selected from Bacillus coagulans E21 with a deposit number of CGMCC No. 13044; The preparation method of the composite antibacterial postbiotic preparation comprises the following steps: (1) fermenting the activated Bacillus coagulans, inactivating and drying the obtained Bacillus coagulans fermentation liquid, and obtaining inactivated Bacillus coagulans cells; (2) activating and culturing Bacillus subtilis, fermenting the obtained Bacillus subtilis seed liquid, and then performing secondary fermentation on the obtained primary seed liquid to obtain a fermentation liquid; (3) adding an adhesive carrier to the fermentation liquid and drying the fermentation liquid to obtain a spray-dried powder containing Bacillus subtilis metabolites; (4) mixing the inactivated Bacillus coagulans obtained in step (1) and the spray-dried powder containing the metabolites of Bacillus subtilis obtained in step (3) to obtain a composite antibacterial postbiotic preparation; The fermentation medium of the bacillus coagulans is composed of: 20-60 g / L bran, 10-30 g / L yeast powder, 10-30 g / L corn steep liquor powder, 2-5 g / L sodium chloride, and 0.5-1 g / L dipotassium hydrogen phosphate.

2. The use of the composite antibacterial postbiotic preparation according to claim 1 in the preparation of aquatic pathogen inhibitors, characterized in that: The aquatic pathogens include Vibrio towerii, Photobacterium mermaidii, Vibrio new caledoniensis, and Vibrio erwensii.

3. The use of the composite antibacterial postbiotic preparation according to claim 1 in shrimp farming, characterized in that: The composite antibacterial postbiotic preparation can be mixed into the shrimp diet as a feed additive, and can also be used by spraying into the aquaculture water; the dosage of the composite antibacterial postbiotic preparation as a feed additive is 1‰~5.00‰ of the weight of the shrimp diet; the dosage of the composite antibacterial postbiotic preparation for spraying is 100 g / mu•m~200 g / mu•m.

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