Application of loach G-type lysozyme rMa-LysG in preparation of loach breeding feed additive

Abstract

The invention discloses an application of loach G-type lysozyme rMa-LysG in preparation of a loach breeding feed additive. The loach G-type lysozyme rMa-LysG as the loach breeding feed additive can effectively improve the growth performance of loaches and improve the meat quality of the loaches. The G-type lysozyme rMa-LysG has remarkable bacteriostatic activity on various gram-negative bacteria and gram-positive bacteria, and by adding 30-60 mg / Kg of the loach G-type lysozyme rMa-LysG into loach breeding feed, the growth performance of loaches can be remarkably improved, the muscle texture of the loaches can be improved, the adhesiveness, chewiness and hardness can be improved, and the eating quality of the loaches can be effectively optimized. The loach G-type lysozyme rMa-LysG provides a basis for application of the loach G-type lysozyme rMa-LysG as a green feed additive in healthy loach breeding and meat quality improvement, and has important production practice value.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture feed additive technology, and relates to a new application of G-type lysozyme in the preparation of aquaculture feed additives, specifically the application of loach G-type lysozyme rMa-LysG in the preparation of loach aquaculture feed additives. Background Technology

[0002] With the large-scale and intensive development of aquaculture, loach, as a high-quality aquaculture species, has seen its farming scale continuously expand. However, under high-density farming models, loach is susceptible to environmental stress and pathogen infection, leading not only to a significant decline in growth performance (such as reduced weight gain and feed conversion ratio) but also to meat quality deterioration problems such as changes in muscle fiber structure and loss of flavor substances, severely restricting the improvement of quality and efficiency in the loach farming industry. Therefore, developing safe and efficient technologies to simultaneously improve the growth performance and meat quality of loach during the farming process has become a research hotspot and urgent need in the aquaculture and food industry.

[0003] Currently, healthy aquaculture technologies mainly include feed additive regulation and chemical preservative treatment. While feed additive regulation can improve meat quality to some extent, it suffers from drawbacks such as unstable effects and poor specificity. Lysozyme, as a natural alkaline protease, possesses various biological activities including antibacterial, anti-inflammatory, and antioxidant properties, and is non-toxic and highly safe, showing great application potential in medical and health, food industry, aquaculture, and agriculture.

[0004] Lysozyme type C and type G are research hotspots. Among them, type C is mainly derived from egg white and has the widest application. Gene recombination technology has achieved large-scale production, and there is strong demand in food preservation, medicine and other fields. Type G is mostly found in birds and fish and is a key immune factor for disease prevention and control in aquaculture. It has a broad antibacterial spectrum and participates in the innate immune response. It is an important part of the body's innate immune system. It can not only effectively inhibit the growth and reproduction of pathogens, but may also affect muscle quality formation by regulating the body's metabolism.

[0005] Patent document CN202111430200.7 discloses the application of a deep-sea g-type lysozyme, specifically its use in preparing bactericidal agents. The deep-sea g-type lysozyme is a recombinant protein expressed in *E. coli*, and the recombinant g-type lysozyme is used for bactericidal purposes. Patent document CN201110420479.0 discloses a mussel g-type lysozyme gene, its recombinant protein, and its applications. A gene fragment encoding the mature peptide of g-type lysozyme was amplified using PCR technology and cloned into the pET21(a) expression vector, achieving prokaryotic in vitro recombinant expression in *E. coli* BL21(DE3)plysS. The recombinant product showed inhibitory effects against various Gram-positive and Gram-negative pathogens, with the most significant effect against *Pseudomonas putida*, with a minimum inhibitory concentration (MIC) of 2.39–4.79 μmol / L. This provides a foundation for further research on the immune defense mechanisms of economically important shellfish and lays the groundwork for disease prevention and control, genetic selection, and feed additives in aquatic animals. However, none of the above patent documents involve the use of G-type lysozyme to improve the growth performance and meat quality of loach in loach farming.

[0006] Currently, there are no reports in existing technologies on the application of loach-derived G-type lysozyme (rMa-LysG) in improving loach meat quality. There is also a lack of technical solutions for simultaneously applying a single lysozyme resource to improve growth performance and regulate meat quality during the aquaculture process. This results in the underutilization of the application value of natural bioactive substances, failing to meet the urgent needs of the aquaculture industry for efficient and multifunctional natural aquaculture technologies. Therefore, exploring fish-derived natural bioactive substance resources and developing technologies for their dual application in improving growth performance and meat quality has significant theoretical and practical value.

[0007] Therefore, the present invention aims to overcome the shortcomings of the prior art and provide a lysozyme-based feed additive and its application method to solve the problem of disease prevention and control in loach farming, while achieving a significant improvement in farming quality. Summary of the Invention

[0008] The purpose of this invention is to provide an application of loach G-type lysozyme rMa-LysG in the preparation of loach aquaculture feed additives. This loach G-type lysozyme rMa-LysG, as a loach aquaculture feed additive, can effectively improve the growth performance and meat quality of loach.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention is the application of loach G-type lysozyme rMa-LysG in the preparation of loach aquaculture feed additives, wherein the gene sequence of loach G-type lysozyme rMa-LysG is shown in the sequence listing SEQ ID NO.1.

[0010] Furthermore, the amino acid sequence of the loach G-type lysozyme rMa-LysG is shown in SEQ ID NO.2 of the sequence listing.

[0011] Furthermore, the amount of loach G-type lysozyme rMa-LysG added to loach farming feed as an additive is 30~60mg / Kg.

[0012] The second aspect of this invention relates to the application of loach G-type lysozyme rMa-LysG in the preparation of antibacterial feed additives for loach farming. This loach G-type lysozyme rMa-LysG exhibits inhibitory effects against both Gram-negative and Gram-positive bacteria. The minimum inhibitory concentration (MIC) of rMa-LysG against Gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus) and Gram-negative bacteria (Aeromonas hydrophila, Aeromonas vesiculosus, and Pseudomonas aeruginosa) was determined using a doubling method. The inhibitory effect against Aeromonas hydrophila was found to be the best, therefore Aeromonas hydrophila was used in subsequent experiments. The destructive effect of rMa-LysG on Aeromonas hydrophila was observed under a scanning electron microscope.

[0013] The third aspect of this invention relates to the application of loach G-type lysozyme rMa-LysG in the preparation of a loach aquaculture feed additive that inhibits Aeromonas hydrophila biofilm. Individual free-living bacteria have limited tolerance to the environment, while biofilm formation enhances bacterial resistance to external environmental stresses, helping the host escape the killing effects of antibacterial substances. Furthermore, biofilm contamination occurs in both pharmaceutical and food production processes; therefore, biofilm detection is a crucial indicator in the sterilization process. Using an enzyme-linked immunosorbent assay (ELISA) reader, optical microscope, fluorescence microscope, and laser confocal microscope, it was observed that rMa-LysG inhibited both the formation and maturation of Aeromonas hydrophila biofilms.

[0014] The fourth aspect of this invention relates to the application of loach G-type lysozyme rMa-LysG in the preparation of loach aquaculture feed additives to improve loach growth performance. A 60-day feeding trial was conducted by adding 30 mg / kg and 60 mg / kg of loach G-type lysozyme rMa-LysG to the diet. The condition factor (CF), weight gain (WGR), hepatic-to-body ratio (HIS), visceral-to-body ratio (VSI), and specific growth rate (SGR) of the loaches were measured. The results showed that all growth performance indicators were significantly improved after dietary supplementation, while there was no significant difference in weight gain and specific growth rate between the 30 mg / kg and 60 mg / kg groups.

[0015] The fifth aspect of this invention relates to the application of loach G-type lysozyme rMa-LysG in the preparation of a loach aquaculture feed additive that improves the nutritional composition of loach muscle. Muscle protein, water, and fat work synergistically to constitute the overall quality of muscle—protein determines the structure and nutritional basis of muscle, water ensures freshness and juiciness, and fat imparts a unique flavor and smooth texture. The balance of these three components is a core prerequisite for high-quality muscle. The protein and crude fat content of loach after 60 days of feeding were determined using the Kjeldahl method and Soxhlet extraction. The water content in the muscle was determined using an electric thermostatic drying oven, referring to GB5009.3-2016 and GB / T6435-2014. The results show that the addition of loach G-type lysozyme rMa-LysG to the feed significantly improved the protein, water, and fat content of loach muscle.

[0016] The sixth aspect of this invention relates to the application of loach G-type lysozyme rMa-LysG in the preparation of a loach farming feed additive that improves the texture of loach meat. Muscle texture is a key quality indicator for loach and significantly influences consumer acceptance. After a 60-day feeding trial, the muscle characteristics of the loach were analyzed using a texture analyzer. The results showed that the addition of loach G-type lysozyme rMa-LysG to the feed significantly improved the adhesiveness, chewiness, and firmness of the loach meat.

[0017] Compared with existing technologies, this invention has the following advantages and beneficial effects: The loach G-type lysozyme rMa-LysG in this invention has a dual application in improving loach growth performance and meat quality. Verification has shown that this G-type lysozyme rMa-LysG has significant antibacterial activity against various Gram-negative and Gram-positive bacteria. Furthermore, adding 30-60 mg / kg of loach G-type lysozyme rMa-LysG to loach farming feed can significantly improve loach growth performance, improve loach muscle texture, enhance adhesiveness, chewiness, and firmness, effectively optimizing the edible quality of loach. This invention provides a basis for the application of loach G-type lysozyme rMa-LysG as a green feed additive in healthy loach farming and meat quality improvement, and has significant practical production value. Attached Figure Description

[0018] Figure 1 This is a protein expression diagram of loach G-type lysozyme rMa-LysG in Example 1.

[0019] Figure 2 The image shows the antibacterial effect of loach G-type lysozyme rMa-LysG in Example 2. A is a representative plate image from the antibacterial experiment; B is the growth curve of liquid antibacterial activity; and C is the state of Aeromonas hydrophila under SEM.

[0020] Figure 3Example 3 illustrates the inhibitory effect of loach G-type lysozyme rMa-LysG on Aeromonas hydrophila biofilm, where A represents the inhibitory effect on the formation process of Aeromonas hydrophila biofilm, and B represents the inhibitory effect on the maturation of Aeromonas hydrophila biofilm.

[0021] Figure 4 The results of changes in loach growth performance after adding 30 mg / kg and 60 mg / kg of loach G-type lysozyme rMa-LysG for 60 days in Example 4 are shown.

[0022] Figure 5 The results show the changes in the nutritional composition of loach muscle after adding 60 mg / kg loach G-type lysozyme rMa-LysG for 60 days in Example 5.

[0023] Figure 6 The graph shows the results of improving the muscle texture of loach by adding 30 mg / kg and 60 mg / kg loach G-type lysozyme rMa-LysG in Example 6. In the graph, A represents hardness, B represents adhesiveness, C represents chewiness, D represents adhesion, E represents elasticity, and F represents muscle texture parameters presented using radar images. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified. Example 1

[0025] cDNA cloning of loach G-type lysozyme rMa-LysG and construction of rMa-LysG recombinant strain: (1) RNA extraction and cDNA synthesis: Total RNA was extracted from loach (liver, intestine, stomach, gills, and blood) tissues using Trizol reagent, and RNA concentration was assessed using NanoDrop 2000. Subsequently, first-strand cDNA was synthesized using the PrimeScript first-strand cDNA synthesis kit.

[0026] (2) cDNA cloning: This invention designed a pair of specific primers to amplify the Ma-LysG gene: Ma-LysG-F (5'-TACTCACTCGAGGACATCATGAACATAGAC-3') and Ma-LysG-R (5'-TACTCAGAATTCAAAAGCAAAGGTTACTGA-3').

[0027] (3) Perform the following PCR amplification: 94℃ for 5 min; 94℃ for 30 s, 48℃ for 30 s, 72℃ for 30 s, 34 cycles; 72℃ for 10 min.

[0028] (4) After PCR amplification, the product was recovered by 1wt% agarose gel electrophoresis at 120V for 20 min. The gel showing the correct band was cut off and the agarose gel was recovered using a gel small-dose recovery kit according to the manufacturer's instructions to obtain the purified Ma-LysG fragment.

[0029] (5) Expression vector construction: The expression vector in (3) was constructed by double digestion of the Ma-LysG fragment and the pET30a plasmid with EcoRI and XhoI respectively.

[0030] The enzyme digestion system is as follows: DNA fragment digestion (20 μL system) DNA 2μL <![CDATA[dd H2O]]> 14μL 10× Qustone buffer (colorless) 2μL Enzymes (1 μL each) 2μL Enzyme digestion of plasmid (20 μL system) plasmid 2μL <![CDATA[dd H2O]]> 16μL 10× Qustone buffer (colored) 2μL Enzymes (1 μL each) 2μL Samples were added on ice, and the enzyme digestion was performed entirely on ice. Three tubes were used for each gene / plasmid digestion. PCR was performed at 37°C for 45 minutes.

[0031] (6) After enzyme digestion, the digested pET30a vector was detected and recovered using a gel microparticle kit, and the Ma-LysG fragment was recovered as a PCR product. The recovered Ma-LysG fragment was ligated to the pET30a vector to construct the expression vector pET30a-Ma-LysG.

[0032] (7) The ligation product of the above steps was transformed into competent Escherichia coli DH5α, plated on LB solid medium containing X-gal and ampicillin sodium, and positive strains were screened and inoculated into LB medium containing ampicillin sodium. The strains were cultured at 37°C and 180 rpm for 10 h, and then the expression plasmid was extracted using a kit.

[0033] (8) Construction of Ma-LysG recombinant strain: The above expression plasmid was transformed into Escherichia coli BL21 competent cells, inoculated into LB solid medium containing kanamycin sulfate, and cultured at 37°C until colonies grew and positive strains were screened.

[0034] (9) Highly expressed protein: The rMa-LysG recombinant strain was grown in LB medium at 37°C and 180 rpm. 3 mL of the bacterial culture was transferred to 300 mL of LB medium at a volume ratio of 1:100 and cultured with shaking at 37°C and 180 rpm for 3 h. Then, IPTG inducer with a final concentration of 0.5 mM was added and cultured for another 4 h. After that, the culture was centrifuged at 4°C and 6000 rpm for 10 min. The supernatant was discarded and the precipitate was collected. The precipitate was suspended with 20 mL of denaturing lysis buffer and sonicated in an ice bath at 35% power for 3 s on and 3 s off for 45 min. Then, the culture was centrifuged at 4°C and 12000 rpm for 10 min and the supernatant was collected. After purification, dialyze (dialysis buffer is 50 times the volume of supernatant) at 4℃ for more than 16 hours. The dialyzing buffer is changed the next day and dialyze for another day. Finally, centrifuge at 4℃ and 10,000 rpm for 10 minutes. The supernatant containing the target protein is retained and stored at -80℃ until use.

[0035] Figure 1 SDS-PAGE was used to detect the induced expression of loach G-type lysozyme rMa-LysG; lane M: standard protein molecular marker; lane 1: uninduced protein; lane 2: recombinant protein induced by 0.5 mM IPTG; lane 3: purified Ma-LysG. Example 2

[0036] Antibacterial activity assay of loach G-type lysozyme rMa-LysG: (1) Antibacterial activity test: Six commonly used fish bacteria were selected, namely Gram-negative bacteria: Aeromonas hydrophila ( Aeromonas hydrophila Aeromonas versicolor ( Aeromonas veronii ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Aeromonas salmonidae ( ) Aeromonas salmonicida Gram-positive bacteria: Staphylococcus aureus ( Staphylococcus aureus Micrococcus luteus ( Micrococcus luteus Six bacterial strains were inoculated onto LB medium and cultured on a shaker at 37 / 28℃ and 180 rpm for 12 h. The six bacterial suspensions were then centrifuged at 6000 rpm for 5 min, the supernatant was discarded, and the precipitates were resuspended in sterile PB medium and diluted. 10 μL of the bacterial suspension (1 × 10⁻⁶) was taken. 5 50 μL of recombinant protein (50 μg / mL) was mixed with 140 μL of sterile PB medium and then added to a sterile 96-well plate. The control group was prepared by mixing sterile PBS with bacterial suspension and PB medium. Finally, the 96-well plate was incubated at 37 / 28℃ and 110 rpm with slow shaking. The bacterial density was detected by measuring the absorbance at 600 nm using a microplate reader at multiple time points from 0 to 40 h. The experimental data were repeated three times.

[0037] (2) Antibacterial test: 50 μL of overnight bacterial culture (1×10⁻⁶) was used. 5 The mixture (CFU / mL) was incubated with 50 μL of protein (0.5 mg / mL) at 37°C for 4 h. The mixture was then diluted 10-fold, and 100 μL of the diluted solution was plated on LB agar plates. After incubation for 12 h, colonies were counted and the average value was calculated.

[0038] Test results as follows Figure 2 As shown, loach G-type lysozyme rMa-LysG exhibits significant antibacterial activity against the tested bacteria: Gram-negative bacteria: Aeromonas hydrophila, Aeromonas vesiculosus, Pseudomonas aeruginosa, Aeromonas salmonidae; and Gram-positive bacteria: Staphylococcus aureus, Micrococcus luteus, with the strongest antibacterial activity against Aeromonas hydrophila. Example 3

[0039] The ability of loach G-type lysozyme rMa-LysG to inhibit biofilm: (1) Since loach G-type lysozyme rMa-LysG has the strongest antibacterial ability against Aeromonas hydrophila, Aeromonas hydrophila was selected as the infecting strain in subsequent experiments. The activated test strain Aeromonas hydrophila was inoculated into 24-well plates at a volume fraction of 1%, and loach G-type lysozyme rMa-LysG was added. The plates were co-cultured with Aeromonas hydrophila for 72 h, and the OD was detected by an enzyme-linked immunosorbent assay (ELISA) reader at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. 595 , to represent the biomass in the biofilm during the biofilm formation process.

[0040] (2) The activated Aeromonas hydrophila strain was inoculated into 24-well plates at a volume fraction of 1% and cultured at 30°C for 48 h. Half of the culture medium was replaced every 12 h to form a mature biofilm. The biofilm was treated with rMa-LysG at concentrations of 1 / 8 MIC, 1 / 4 MIC, and 1 / 2 MIC. The dissipation effect of rMa-LysG on the biofilm was observed by optical microscopy using crystal violet staining. Live and dead cells were stained with FITC and PI, and the survival status of cells in the biofilm after rMa-LysG treatment was observed by fluorescence microscopy. The thickness and density of the mature Aeromonas hydrophila biofilm were observed by laser confocal microscopy using FITC-labeled bacteria.

[0041] Test results as follows Figure 3 As shown, Aeromonas hydrophila is inhibited by loach G-type lysozyme rMa-LysG during both the formation and maturation of biofilms, resulting in an increase in dead cells, a decrease in biofilm density and thickness, and biofilm dissipation. Example 4

[0042] The effect of loach G-type lysozyme rMa-LysG as a feed additive on loach growth performance: (1) Two experimental feeds were prepared by adding 30 mg / Kg and 60 mg / Kg of loach G-type lysozyme rMa-LysG to commercial feed, respectively. The control feed was a standard commercial feed (without loach G-type lysozyme rMa-LysG). The commercial loach feed was thoroughly ground into powder using an electric grinder, and then different proportions of loach G-type lysozyme rMa-LysG were added, along with distilled water to make the mixture completely homogenized. The feed was then granulated using a pellet mill, and subsequently sealed and dried at 25°C for 12 hours to complete the feed preparation. It was then stored at 4°C until use.

[0043] (2) Loaches (approximately 3-5 cm each) were randomly divided into three groups. The control group was fed a diet without loach G-type lysozyme rMa-LysG, while the experimental groups were fed diets containing 30 mg / kg and 60 mg / kg of loach G-type lysozyme rMa-LysG, respectively. The feeding was repeated for 60 days. The loaches were fed twice daily (8:00 AM and 5:00 PM) at a rate of 2 wt% of their body weight. After the feeding experiment, their growth performance indicators were measured.

[0044] Test results as follows Figure 4 As shown, adding loach G-type lysozyme rMa-LysG to feed can effectively improve the growth performance of loach. Example 5

[0045] The effect of loach G-type lysozyme rMa-LysG as a feed additive on the nutritional composition of loach muscle: After 60 days of feeding with a specific diet, the protein, fat, and water content in the muscle were measured. The results are as follows: Figure 5 As shown, loach G-type lysozyme rMa-LysG, when added to feed, can effectively increase the protein, moisture, and fat content of loach muscle. Example 6

[0046] The effect of loach G-type lysozyme rMa-LysG as a feed additive on loach meat quality: After 60 days of feeding, muscle texture was analyzed using a texture analyzer (XT Plus, Stable Micro Systems, UK). Muscle samples (12 per group) were processed into standardized test blocks (1cm × 1cm × 0.5cm) and compressed using an 8mm diameter cylindrical probe (25N range). The TPA was set as follows: descent speed 30mm / min, compression level 50%, and interval 2s. Parameters such as adhesiveness, chewiness, hardness, adhesion, and elasticity were calculated based on the TPA force-time curve.

[0047] Test results as follows Figure 6As shown, adding 30 mg / Kg and 60 mg / Kg of loach G-type lysozyme rMa-LysG to the feed can significantly optimize the adhesiveness, chewiness and hardness of loach meat, thereby effectively improving the edible quality of loach.

[0048] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

[0049] SEQUENCE LISTING <110> Henan Normal University <120> Application of loach G-type lysozyme rMa-LysG in the preparation of loach aquaculture feed additives <130> 2026 <160> 4 <170> Patentin version 3.3 <210> 1 <211> 591 <212> DNA <213> Artificial sequence <400> 1 atgaggtttccggtgatattttttatattgtaccagcatgcatttatggacatcatgaacacacaactggagcatcagaaaacagccaaacagacaaattaactgtaaagggtgttgcagcttctaaaaactggct gagactgatttgacccgaatggggaaatacaagagtaaaatcattaaagttggcaaagcaagcaaatggacccagctgtgatcgctgccatccagagagtctagctggagctgcactgaagaatgggtggggtgatcgtg gaaatggttcggcctcatgcaggttgacaaacgctatcaatcctttgcttgggacagtgaagagcatattaaaaggaactgatatactcgtcaactcttaaagatattaaagcaaaatttcccaaatggacagaga tcaatgtttaaaaggaggaatatcggcttacaatgcaggtgtgaggaatgtgcgctcatatcgcatggacatcggcaccacaggaaacgactacgctaatgatgttgccagagcccagtggtataaaagcaaaggttactga 591 <210> 2 <211> 196 <212> RNA <213> artificial sequence <400> 2 MRFPVIFFILLPACIYGDIMNIDTTGASEKTAKQDKLTVKGVAASKKLAETDLTRMGKYKSKIIKVGKAKQMDPAVIAAIISRAGAALKNGWGDR GNGFGLMQVDKRYHNPLGAWDSEEHIKQGTDILVNSIKDIKFPKWTEDQCLKGGISAYNAGVRNVRSYDRMDIGTTGNDYANDVVARAQWYKSKGY 196 <210> 3 <211> 30 <212> DNA <213> Artificial sequence <400> 3 tactcactcgaggacatcatgaacatagac 30 <210> 4 <211> 30 <212> DNA <213> Artificial sequence <400> 4 tactcagaattcaaaagcaaaggtactga 30

Claims

1. Application of loach G-type lysozyme rMa-LysG in the preparation of loach aquaculture feed additive, wherein the gene sequence of loach G-type lysozyme rMa-LysG is shown in the sequence listing SEQ ID NO.

1.

2. The application according to claim 1, characterized in that: The amino acid sequence of the loach G-type lysozyme rMa-LysG is shown in the sequence listing SEQ ID NO.

2.

3. The application according to claim 1, characterized in that: The amount of loach G-type lysozyme rMa-LysG added to loach farming feed as an additive is 30~60mg / Kg.

4. Application of loach G-type lysozyme rMa-LysG in the preparation of antibacterial feed additives for loach farming. This loach G-type lysozyme rMa-LysG has inhibitory effects on both Gram-negative and Gram-positive bacteria.

5. Application of loach G-type lysozyme rMa-LysG in the preparation of loach aquaculture feed additives that inhibit Aeromonas hydrophila biofilm.

6. Application of loach G-type lysozyme rMa-LysG in the preparation of loach aquaculture feed additives to improve loach growth performance.

7. Application of loach G-type lysozyme rMa-LysG in the preparation of loach aquaculture feed additives that improve the nutritional composition of loach muscle.

8. Application of loach G-type lysozyme rMa-LysG in the preparation of loach aquaculture feed additives to improve loach meat texture.

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