A crayfish intestinal microecological additive containing low-temperature protease and postbiotic preparations, and a preparation method and application thereof

Abstract

The present invention provides a kind of crayfish intestinal microecological additive containing low-temperature protease and postbiotic preparation and its preparation method and application. The present invention analyzes the alkaline protease gene derived from Bacillus subtilis and performs site-directed mutation to obtain a mutant aprEM, whose amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 2. The optimal reaction temperature of the mutant aprEM enzyme activity is reduced from 55 ° C to 45 ° C, and the enzyme activity is maintained at 20-40 ° C. More than 60%, more suitable for application under low temperature environment, especially suitable for the intestinal environment of aquaculture animals, thereby improving the digestion and utilization rate of feed raw materials. The microecological additive of the present invention also includes Bacillus subtilis metabolites and inactivated bacteria, which can purify water and inhibit some pathogens. The present invention proves through experiments that low-temperature protease combined with postbiotic preparations are applied in aquaculture, which can significantly improve its breeding survival rate and improve its economic benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic animal breeding, and in particular relates to a crayfish intestinal microecological additive containing low-temperature protease and a postbiotic preparation, and a preparation method and application thereof. Background Art

[0002] Protease preparations are widely used in the field of aquaculture. They can not only improve the digestion and absorption rate of protein feed raw materials and promote animal weight gain, but also reduce excrement and environmental pollution. However, the most commonly used proteases, especially alkaline proteases that have a good effect on protein degradation in feed raw materials, generally have an optimal reaction temperature of about 50-60℃, and the enzyme activity or catalytic activity under 20℃-40℃ is generally not high. However, animal breeding and intestinal environment are mostly 20℃-40℃, so optimizing and modifying protease genes to obtain enzyme preparations that are more suitable for maintaining high activity at low temperatures is an effective way to solve this problem.

[0003] Postbiotics refer to "preparations of inanimate microorganisms and / or their components that are beneficial to the health of the host", which includes inactivated probiotic cells and their metabolites. The specific components are extensive, mainly including: inactivated probiotic cell components: peptidoglycan (PG), teichoic acid (TA), lipoteichoic acid (LTA), cell membrane proteins, cell wall polysaccharides (CPSs), etc. Probiotic metabolites: enzymes, proteins, peptides, extracellular polysaccharides, organic acids, short-chain fatty acids, etc. Small molecule active substances released by enzyme-treated substrates of microbial metabolism: These substances have direct biological activity and can act on the host without the need for live bacteria. In farmed animals, the application of postbiotics has shown many positive effects: improving production performance, enhancing immunity, regulating intestinal microecology, improving stress response, promoting nutrient absorption, metabolic regulation, etc., further optimizing production performance. Summary of the invention

[0004] The purpose of the present invention is to provide a crayfish intestinal microecological additive containing low-temperature protease and postbiotic preparations, and a preparation method and application thereof. The present invention can not only improve the breeding efficiency and animal health level, but also improve the water body by applying a technical solution combining protease and postbiotic preparations in crayfish breeding, and has good market application prospects in the field of aquaculture animals.

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides a low-temperature protease mutant aprEM, whose amino acid sequence is shown in SEQ ID NO: 1. aprEM is obtained by changing the 38th amino acid of the protease with the amino acid sequence of SEQ ID NO: 3 from lysine to aspartic acid, the 102nd amino acid from isoleucine to valine, the 183rd amino acid from asparagine to threonine, and the 210th amino acid from tyrosine to methionine.

[0007] The present invention also provides a gene encoding a low-temperature protease mutant aprEM, and the nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0008] The present invention also provides a recombinant expression vector comprising the encoding gene, wherein the vector is pWB980 or pPIC9K.

[0009] The present invention provides a genetically engineered bacterium comprising the coding gene, wherein the genetically engineered bacterium is Pichia pastoris, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, or Bacillus licheniformis.

[0010] The invention provides a crayfish intestinal microecological additive containing low-temperature protease and postbiotic preparation. The microecological additive contains the low-temperature protease mutant aprEM enzyme preparation and the postbiotic preparation.

[0011] Furthermore, the preparation method of the microecological additive comprises the following steps:

[0012] (1) first fermenting the recombinant bacteria containing the protease mutant aprEM, and after the fermentation is completed, first centrifuging to obtain the supernatant and then concentrating and extracting to prepare a low-temperature protease preparation;

[0013] (2) The remaining fermentation metabolites and bacteria are inactivated and spray-dried to prepare postbiotic preparations;

[0014] (3) The low-temperature protease preparation and the postbiotic preparation are mixed in a mass ratio of 1:3-5 to prepare a microecological additive.

[0015] Furthermore, the fermentation medium formula of the protease mutant aprEM recombinant bacteria is: 2-5% soybean meal, 3-5% corn starch, 1000U / g neutral protease, 1000U / g high-temperature amylase, 3-5% glucose, 0.1-0.5% ammonium sulfate, 0.01-0.1% magnesium sulfate, 0.01-0.1% calcium chloride, 0.2-0.8% potassium dihydrogen phosphate, 0.02-0.05% Tween 80, and 3-10% corn steep liquor, calculated by mass ratio.

[0016] The present invention also provides the use of the low-temperature protease mutant aprEM or the intestinal microecological additive in feed additives for aquatic animal breeding.

[0017] Further: the aquatic animals include crayfish and prawns.

[0018] Further: the low-temperature protease mutant aprEM enzyme preparation or intestinal microecological additive is mixed into the crayfish diet as a feed additive and added. According to the mass ratio of the daily feeding amount to the crayfish body weight, the daily feeding ratio of the juvenile shrimp stage is 4-6%, and the daily feeding ratio of the mature crayfish is 3-5%. The intestinal microecological additive is added to the daily diet at an addition amount of 0.2%-0.5%; or it is used by spraying into the aquaculture water body, and the aquaculture water body is planted with Elodea and Verticillastera with a coverage rate of 40%-50%.

[0019] Furthermore: the intestinal microecological additive can inhibit Vibrio in aquaculture water.

[0020] Compared with the prior art, the advantages and technical effects of the present invention are: aprE The alkaline protease gene (GenBank: WP_029726299.1) was improved, multiple candidate sites were selected for site-directed mutagenesis by analysis, a mutant library and a high-throughput directional screening method were constructed, and a low-temperature protease mutant aprEM with improved enzyme activity under low-temperature conditions was obtained through a large number of screenings, and its mutation points were K38D / I102V / N183T / Y210M. The optimal reaction temperature of the mutant aprEM after the modification of the present invention was reduced from 55°C to 45°C, and the enzyme activity was maintained at more than 60% under the reaction conditions of 20-40°C. After fermentation in a 15L fermenter, the protease enzyme production level reached 6000U / mL.

[0021] The present invention applies low-temperature protease in combination with postbiotic preparations in crayfish farming, which can not only improve farming efficiency and animal health levels, but also improve water bodies. It shows good application potential in the field of aquatic animal farming and is of great significance to promoting green and healthy farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the electrophoresis result diagram of the low-temperature protease gene amplification and the genetic transformation diagram.

[0023] Figure 2 It is the optimal reaction temperature diagram of the low-temperature protease and its mutants.

[0024] Figure 3 The fermentation data of the low-temperature protease mutant in a 15 L fermenter.

[0025] Figure 4 This is a graph showing the effects of low-temperature proteases and postbiotics on Vibrio in crayfish farming water. DETAILED DESCRIPTION

[0026] In order to facilitate the understanding of the present invention, the present invention will be described in more comprehensive and detailed manner with reference to the accompanying drawings and embodiments below, but the protection scope of the present invention is not limited to the following specific embodiments.

[0027] The molecular biology experimental methods not specifically described in the following examples can be performed by referring to the specific methods listed in the book Molecular Cloning Laboratory Manual (3rd Edition) by J. Sambrook, or according to the kits and product instructions. The reagents and biological materials used in the specific examples can be obtained from commercial channels unless otherwise specified.

[0028] 1. Strains and vectors

[0029] Bacillus subtilis 168, plasmid pWB980, Escherichia coli BL21, and plasmid pET-21a(+) were purchased from Invitrogen.

[0030] 2. Reagents and Culture Media

[0031] Plasmid extraction kit, fragment purification and recovery kit, restriction endonuclease, protein marker: Blue PlusII Protein Marker (14-120 kDa), etc. were purchased from Nanjing Novozymes Co., Ltd.; kanamycin and ampicillin were purchased from Sangon Biotech (Shanghai) Co., Ltd.; and whole gene synthesis was performed by Shanghai Jierui Biotech Co., Ltd.

[0032] LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl.

[0033] Fermentation medium: soybean meal 2-5%, corn starch 3-5%, neutral protease 1000U / g, high temperature amylase 1000U / g, glucose 3-5%, ammonium sulfate 0.1-0.5%, magnesium sulfate 0.01-0.1%, calcium chloride 0.01-0.1%, potassium dihydrogen phosphate 0.2-0.8%, Tween 80 0.02-0.05%, corn steep liquor 3-10%. Calculated by mass ratio.

[0034] 3. Enzyme Activity Assay

[0035] Refer to the protease determination method in "GBT 23527-2009 Protease Preparations".

[0036] 4. Bacillus subtilis transformation method

[0037] The preparation and transformation of competent cells were performed using a modified Spizizen method, as detailed below:

[0038] Reagents:

[0039] SPI-A Salts Solution: (500 mL Solution)

[0040] 0.4% (NH4)2SO4 2 g

[0041] 2.8% K2HPO4×3H2O 14 g

[0042] 1.2% KH2PO4 6 g

[0043] 0.2% Trisodium Citrate Dihydrate 1 g

[0044] Sterilize at 121℃ for 20 min

[0045] SPI-B Salts Solution: (500 mL Solution)

[0046] 0.04% MgSO4×7H2O 0.2 g

[0047] Sterilize at 121℃ for 20 min

[0048] 100×CAYE Solution: (100 mL Solution)

[0049] 2% Casamino acid 2 g

[0050] 10% Yeast Extract 10 g

[0051] Sterilize at 121℃ for 20 min

[0052] SPI Medium: (20 mL)

[0053] 9.8 mL SPI-A Salts Solution

[0054] 9.8 mL SPI-B Salts Solution

[0055] 200 μL (1% V) Glucose (50% W, i.e. 125 g / 250 mL solution, sterilized at 115°C for 20 min)

[0056] 200 μL (1% V) 100×CAYE

[0057] SPII Medium: (6 mL)

[0058] 5.88 mL SPI Medium

[0059] 60 μL (1% V) 50 mM CaCl2 (100 mL: 5 mL 1 mol / L CaCl2×2H2O (MW:147.02) solution is added to make up to 100 mL)

[0060] 60 μL (1% V) 250 mM MgCl2 (100 mL: 5.1 g MgCl2×6H2O (MW: 203.30) solution is added to make up to 100 mL)

[0061] 100×EGTA Solution: 10 mmol / L EGTA solution (add a small amount of NaOH to pH 8.0 when dissolving), filter sterilize, and store at 4℃.

[0062] Conversion steps:

[0063] (1) The night before, pick the host bacteria and inoculate them into 3 mL of SPI liquid medium. Incubate at 37°C in a shaking incubator overnight.

[0064] (2) The next morning, take 100 μL of bacterial culture from the overnight culture and inoculate it into 5 mL of SPI Medium in a 50 mL centrifuge tube. Incubate at 37°C in a shaking incubator. After 3 h, measure the OD. 600 When the culture grows to the end of the logarithmic phase (about 4.5-5h), quickly take 200 μL and inoculate it into 2 mL SPII Medium, and culture it at 37℃ with a shaker at 100 rpm for 1.5 h.

[0065] (3) Add 20 μL of 100× EGTA solution and incubate at 37°C at 100 rpm for 10 min. Aliquot the solution into 1.5 mL centrifuge tubes, 500 μL per tube.

[0066] (4) Add an appropriate amount of plasmid or ligation product to the tube, mix gently, and incubate at 37°C at 100 rpm for 1.5 h.

[0067] (5) Collect the cells by centrifugation at 4000 rpm, discard part of the supernatant, and resuspend the cells in about 200 μL by gently pipetting. Spread the cells on a LB solid medium plate containing 10 μg / mL kanamycin and culture at 37°C overnight.

[0068] Example 1: Construction of recombinant bacteria expressing low-temperature protease gene

[0069] The amino acid sequence of the protease from Bacillus subtilis (GenBank: WP_029726299.1) is shown in SEQ ID NO: 3, and the codons are optimized to synthesize the corresponding nucleotide sequence, as shown in SEQ ID NO: 4. Primers were designed, with a BamH I restriction enzyme site at the 5' end and a Pst I restriction enzyme site at the 3' end.

[0070] Perform PCR amplification of the target band: Use the following primers:

[0071] AF: CGC GGATCC ATGAGAAGCAAAAAATTGTGG (SEQ ID NO: 5);

[0072] AR: AAAA CTGCAG TTATTGTGCAGCAGCTTGTAC (SEQ ID NO: 6)

[0073]

[0074] The reaction conditions were as follows: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 10 sec, annealing at 58°C for 30 sec, extension at 72°C for 1 min, 30 cycles, post-extension at 72°C for 10 min, and storage at 15°C.

[0075] The PCR product was subjected to electrophoresis. Figure 1 As shown, the single PCR product of the band was purified, double-digested, connected with the pWB980 vector (according to the steps in the kit instructions), and transformed into Bacillus subtilis 168, and coated on a plate containing kanamycin (final concentration 20μg / mL) antibiotics to screen the recombinant bacteria. Figure 1 As shown, the successfully transformed recombinant bacteria have obvious hydrolysis circles. The recombinant bacteria are purified and preserved, and shake flask fermentation is carried out.

[0076] SEQ ID No: 3

[0077] MRSKKLWISLLFALTLIFTMAFSNMSAQAAGKSSTEKKYIVGFKQTMSAMSSAKKKDVISEKGGKVQKQFKYVNAAAATLDEKAVKELKKDPSVAYVEEDHIAHEYAQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVAPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSSSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMAPGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ

[0078] SEQ ID No.: 4

[0079]

[0080] Example 2: Construction of the low-temperature protease mutant aprEM

[0081] The mutants were constructed by site-directed mutagenesis, specifically by synthesizing the corresponding primers, performing several rounds of PCR, mutating the selected sites one by one, and then verifying them by connecting to the T vector and sequencing. The gene of SEQ ID NO: 4 was used as a template, and the primer sequences used were as follows:

[0082]

[0083] For example: The steps to mutate K38D are as follows

[0084] (1) Use primers AF and 38R to amplify the upstream gene fragment;

[0085] (2) Primers 38F and AR were used to amplify the downstream gene fragment; the amplification program was 95°C pre-denaturation for 3 min, 94°C denaturation for 10 sec, 58°C annealing for 30 sec, 72°C extension at 1 Kb / min, 30 cycles, 72°C, post-extension for 10 min, and storage at 15°C;

[0086] (3) Overlap PCR was performed on the amplified upstream and downstream gene fragments: equal amounts of diluted upstream and downstream fragments were added to the PCR system as templates; the amplification program was 95°C pre-denaturation for 3 min, 94°C denaturation for 10 sec, 68°C annealing for 1 min 30 sec, 30 cycles (primers AF and AR were added after 5 cycles), 72°C, post-extension for 10 min, and storage at 15°C;

[0087] (4) The amplified gene fragments were subjected to electrophoresis detection and connected to the T vector. After transformation to DH5a, the transformants were selected for sequencing verification;

[0088] (5) After sequence analysis, the gene sequence with the correct mutation was obtained, namely the K38D mutant.

[0089] The correctly mutated gene sequence was then used as a template for site-directed mutagenesis of the next site until all selected sites were mutated, thereby obtaining the mutant aprEM (K38D / I102V / N183T / Y210M), whose amino acid sequence is shown in SEQ ID NO: 1 and the corresponding nucleotide sequence of the coding gene is shown in SEQ ID NO: 2.

[0090] The mutant aprEM was extracted from the T vector by double enzyme digestion and gel recovery to obtain the mutant target gene, and then connected with the vector pWB980 that had been digested with the same double enzymes (BamH I and Pst I) and transformed into Bacillus subtilis 168. Positive clones were screened on kanamycin-resistant LB plates to obtain mutant recombinant bacteria.

[0091] SEQ ID NO: 1

[0092] MRSKKLWISLLFALTLIFTMAFSNMSAQAAGKSSTEKDYIVGFKQTMSAMSSAKKKDVISEKGGKVQKQFKYVNAAAATLDEKAVKELKKDPSVAYVEEDHVAHEYAQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNTSIGVLGV APSASLYAVKVLDSTGSGQMSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSSSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMAPGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ

[0093] SEQ ID NO: 2

[0094]

[0095] Example 3: Shake flask fermentation expression verification of low-temperature protease mutant recombinant bacteria

[0096] The recombinant bacteria of the above-mentioned proteases and their mutants were activated, that is, streaked on LB plates containing 1% casein and kanamycin resistance, and cultured at 37 degrees overnight; the next day, single colonies with larger hydrolysis circles were selected and inoculated into LB (final concentration of kanamycin 20µg / mL) liquid culture medium, and cultured at 37 degrees for 12 hours; according to the inoculation amount of 5%, they were inoculated into the fermentation medium: 2-5% soybean meal, 3-5% corn starch, 3-5% glucose, 0.1-0.5% ammonium sulfate, 0.01-0.1% magnesium sulfate, 0.01-0.1% calcium chloride, 0.2-0.8% potassium dihydrogen phosphate, 3-10% corn steep liquor, calculated by mass ratio. After 78 hours of shake flask fermentation, the culture medium was centrifuged to obtain the supernatant, and the average enzyme activity and the most suitable reaction temperature of the fermentation supernatant of each mutant were determined according to the national standard alkaline egg method. The results are as follows Figure 2 .

[0097] After enzyme activity determination, the enzyme activities of the protease and its mutant were comparable, but the optimal reaction temperature of the mutant gene dropped from 55°C to 45°C, and the enzyme activity remained above 60% at 20-40°C. Compared with the starting gene, the mutant aprEM could better exert its enzymatic catalytic function under low temperature conditions.

[0098] Example 4: Fermentation of low-temperature protease mutants in a 30 L fermenter and preparation of postbiotic products

[0099] The genetically engineered bacteria expressing the protease mutant aprEM in the above example were streaked onto LB plates containing kanamycin resistance (final concentration of 20 μg / mL), cultured at 37° C. until single colonies grew, and single colonies with good growth were selected for fermentation.

[0100] Seed solution: LB liquid medium, containing kanamycin resistance (final concentration 20 μg / mL);

[0101] Fermentation medium (without resistance): soybean meal 2-5%, corn starch 3-5%, neutral protease 1000U / g, high temperature amylase 1000U / g, glucose 3-5%, ammonium sulfate 0.1-0.5%, magnesium sulfate 0.01-0.1%, calcium chloride 0.01-0.1%, potassium dihydrogen phosphate 0.2-0.8%, Tween 80 0.02-0.05%, corn steep liquor 3-10%, calculated by mass ratio.

[0102] Fermentation steps: The recombinant bacteria were inoculated into LB liquid medium, and cultured overnight with shaking at 37°C and 200 rpm; the overnight cultured seed liquid was inoculated into a 15L fermenter with a liquid volume of 8L;

[0103] Control conditions: 37℃, 300-600rpm; dissolved oxygen 20%-60%; tank pressure 0.05Mpa; ventilation volume 0-8h 0.6 m 3 / h; 8h to tank stop 0.8-0.9 m 3 / h. Ferment until the spore formation rate under microscopic examination is above 90%. The pH value of the fermentation process is natural. The enzyme activity is measured after 24h of fermentation and after the fermentation is completed (generally 48h).

[0104] Protease preparation and detection:

[0105] After the fermentation liquid is centrifuged at 5000 rpm for 5 min, the supernatant enzyme liquid is obtained, or the fermentation liquid is processed by a plate and frame filter to obtain a crude enzyme liquid and then spray-dried into a powder preparation. It is used for application testing. The fermentation process curve is shown in Figure 3 As shown: samples were taken every 4 hours to measure the enzyme production level, and the antibacterial activity reached the highest point after 48 hours of fermentation.

[0106] Preparation of postbiotic preparations:

[0107] After 50 hours of fermentation, the tank is stopped, the fermentation liquid is first centrifuged to obtain the supernatant, and then the protease enzyme liquid is concentrated and extracted, and the remaining fermentation metabolites and bacteria are inactivated and spray-dried to prepare a postbiotic preparation, which contains 70-80% of Bacillus subtilis metabolites and 20-30% of inactivated bacteria. The present invention makes both the protease liquid and the postbiotic preparation into products, basically without wasting the fermentation process products, and they are used in combination in applications, which significantly improves the production efficiency.

[0108] Example 5: Experimental application of low-temperature protease and its postbiotics in crayfish farming

[0109] The on-site breeding base was randomly selected from 6 adjacent crayfish breeding ponds (100m 2 / , water depth 1-1.5 m) as the experimental group, and the other 3 as the control group, 4-5 g / 3kg of crayfish were put into each pond. The experimental group and the control group were fed according to the following table. The experimental period was 90 days, the culture temperature was 20-30°C, and they were fed twice a day, with a daily feeding amount of 3-6% of their body weight (according to the mass ratio of daily feeding amount to the body weight of crayfish, the daily feeding ratio of juvenile shrimp stage is 4-6%, and the daily feeding ratio of mature crayfish is 3-5%).

[0110] The main raw materials of feed are fish meal, high-gluten flour, soybean meal, rapeseed meal, shrimp shell meal, meat meal, complex vitamins, trace elements, etc. The nutritional level is crude protein 37-40%, moisture 10-12%, crude fiber 8-11%, crude ash 9-13%, calcium 2-5%, and total phosphorus 0.2-0.5%.

[0111] Additives containing low-temperature protease and postbiotic preparations: low-temperature protease preparations and postbiotic preparations are mixed in a mass ratio of 1:5 to prepare a microecological additive.

[0112] Table 1 Experimental design

[0113]

[0114] At the beginning and end of the experiment, the initial and final weights of crayfish were counted, and the weight gain rate and feed coefficient were calculated. The experimental results are shown in Table 2.

[0115] Weight gain rate = (W 末 -W 初 ) / W 初 ×100% ;

[0116] Feed coefficient = feed consumption / weight gain = W 饲料 / (W 末 -W 初 );

[0117] Table 2 Effects of low-temperature protease and postbiotic preparations on the growth performance of crayfish

[0118]

[0119] It can be seen from Table 2 that the addition of low-temperature proteases and postbiotic preparations can increase the weight gain rate of crayfish to a certain extent, while reducing the feed coefficient, thereby improving the utilization rate of feed and improving economic benefits.

[0120] Example 6: Effects of low-temperature proteases and their postbiotics on Vibrio in crayfish aquaculture water

[0121] Environmental microorganisms affect the composition of intestinal microorganisms and the establishment of microecology in aquatic animals. The aquaculture water environment is closely related to the intestinal flora and health of crayfish. According to Example 5, in crayfish farming, low-temperature protease and postbiotic preparations (70-80% Bacillus subtilis metabolites and 20-30% inactivated bacteria) are used daily, mixed with feed at an addition rate of 0.2% and fed twice. Water samples are taken 12h and 24h after feeding to detect the number of Vibrio, and sampling is repeated 3 times.

[0122] The experimental results are as follows Figure 4As shown, before use, the number of Vibrio in the water of crayfish breeding ponds was about 5000-10000 CFU / mL. After using the postbiotic compound enzyme preparation, compared with the control group, the removal rates of Vibrio in the water were 61.64% and 70.51% at 12h and 24h, respectively, and the number of Vibrio was controlled within the safety threshold. Because the protease postbiotic compound preparation (70-80% of Bacillus subtilis metabolites and 20-30% of inactivated bacteria) contains Bacillus subtilis' own antibacterial metabolites, it can inhibit certain pathogenic bacteria and play a role in purifying the water environment. The present invention uses low-temperature protease and its postbiotic compound, which not only improves the weight gain rate of crayfish, but also is more conducive to the healthy breeding of crayfish.

[0123] The present invention further experiments on the culture effect of planting aquatic plants in the water for breeding crayfish. The coverage rate of aquatic plants in the water for breeding is 40%-50%. For example, planting Elodea and Hydrilla verticillata, and combining the use of the intestinal microecological additives provided by the present invention in the water for breeding will have a better culture effect.

[0124] 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 low-temperature protease mutant aprEM, characterized in that The amino acid sequence of the low-temperature protease mutant aprEM is shown in SEQ ID NO:

1. aprEM is obtained by changing the 38th amino acid of the protease with the amino acid sequence of SEQ ID NO: 3 from lysine to aspartic acid, the 102nd amino acid from isoleucine to valine, the 183rd amino acid from asparagine to threonine, and the 210th amino acid from tyrosine to methionine.

2. The gene encoding the low-temperature protease mutant aprEM according to claim 1, characterized in that The nucleotide sequence thereof is shown in SEQ ID NO:

2.

3. A recombinant expression vector comprising the coding gene according to claim 2, characterized in that: The expression vector is pWB980 or pPIC9K.

4. A genetically engineered bacterium comprising the coding gene according to claim 2, characterized in that: The genetically engineered bacteria are Pichia pastoris, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Bacillus licheniformis.

5. A crayfish intestinal microecological additive containing low-temperature protease and postbiotic preparation, characterized in that: The microecological additive comprises the low-temperature protease mutant aprEM enzyme preparation and the postbiotic preparation according to claim 1, and the preparation method of the microecological additive comprises the following steps: (1) first fermenting Bacillus subtilis containing the protease mutant aprEM, and after the fermentation is completed, first centrifuging to obtain the supernatant and then concentrating and extracting to prepare the protease mutant aprEM; (2) Bacillus subtilis expressing the protease mutant aprEM is inactivated and spray-dried to prepare a postbiotic preparation by removing the remaining fermentation metabolites and bacteria of the protease mutant aprEM; (3) The protease mutant aprEM and the postbiotic preparation are mixed in a mass ratio of 1:3-5 to prepare a microecological additive.

6. The crayfish intestinal microecological additive according to claim 5, characterized in that: The fermentation medium formula of the Bacillus subtilis expressing the protease mutant aprEM is: 2-5% soybean meal, 3-5% corn starch, 1000U / g neutral protease, 1000U / g high-temperature amylase, 3-5% glucose, 0.1-0.5% ammonium sulfate, 0.01-0.1% magnesium sulfate, 0.01-0.1% calcium chloride, 0.2-0.8% potassium dihydrogen phosphate, 0.02-0.05% Tween 80, and 3-10% corn steep liquor, calculated by mass ratio.

7. Use of the low-temperature protease mutant aprEM according to claim 1 or the intestinal microecological additive according to claim 5 in the preparation of an additive for crayfish farming, characterized in that: The postbiotic preparation of the intestinal microecological additive comprises 70-80% of Bacillus subtilis metabolites and 20-30% of inactivated bacteria.

8. The use according to claim 7, characterized in that: A low-temperature protease mutant aprEM enzyme preparation or an intestinal microecological additive is mixed into the daily diet of crayfish as a feed additive and added. The daily feeding ratio of the juvenile stage is 4-6%, and the daily feeding ratio of the mature crayfish is 3-5%. The intestinal microecological additive is added to the daily diet at an addition amount of 0.2%-0.5%; or it can be used by spraying into the aquaculture water body, and the aquaculture water body is planted with Elodea and Verticillastera with a coverage rate of 40%-50%.

9. The use according to claim 7, characterized in that: The intestinal microecological additive can inhibit Vibrio in aquaculture water.

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