A multi-factor-based fish feed formula screening method

Abstract

The application discloses a fish feed formula screening method based on multiple factors, which is based on a breeding environment, physiological indexes of fish bodies and characteristics of feed raw materials, optimizes multiple-factor experimental design, reduces experimental groups and simplifies an operation process; meanwhile, a mathematical model can be simulated and constructed, variation conditions of multiple combinations of different factors can be predicted, and optimal combinations of various variation factors are given; the breeding environment, the physiological indexes of the fish bodies and the feed raw materials are accurately combined, fish feed formula levels and quality are improved, and formula cost and feed cost are saved; application effects of new raw materials (new additives) in the formula can be quickly, simply and effectively evaluated. The application also provides a fish feed formula for Scophthalmus maximus breeding obtained by screening based on the above method and a fish antibacterial peptide with high thermal stability and sterilization activity. The method has the advantages of simplicity, effectiveness and rapidness.

Description

Technical Field

[0001] This invention relates to a method for screening fish feed, specifically a method for screening fish feed formulations based on multiple factors, belonging to the field of aquaculture feed technology. Background Technology

[0002] Current fish feed formulations primarily rely on single-factor or two-factor experiments to determine the optimal addition levels of one or two feed ingredients, which are then applied to the feed formulation using Excel spreadsheets or specialized formulation software. However, designs involving multiple factors (more than three feed ingredients) are rarely used due to their cumbersome operation and the significant resource investment required. Therefore, in actual fish feed formulation design, the actual addition levels for the synergistic effects of multiple ingredients are often judged based on experience, with few precise multi-factor experiments designed for verification. However, in situations requiring comprehensive consideration of complex factors such as the aquaculture environment, fish physiological indicators, and feed ingredient characteristics, multi-factor experiments can provide a reasonable combination of feed ingredient components, serving as an effective method to improve formulation quality. Furthermore, when introducing new ingredients, quickly determining the optimal addition level and the effective dosage for the synergistic effects of the new ingredient with the aquaculture environment, fish physiological indicators, and other feed ingredients is something that single-factor or two-factor experiments cannot simultaneously address. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a multi-factor-based fish feed formulation screening method, which has the advantages of being simple, effective, and rapid.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a method for screening fish feed formulations based on multiple factors, comprising the following steps: (1) Apply single-factor experiments to obtain suitable values ​​for each factor: Design a single-factor gradient experiment to obtain suitable values ​​for the factor (applicable to new raw materials or unknown factors); or obtain data by consulting literature (applicable to known factors); based on this data, use Design-Expert software to predict and apply the optimal combination of multiple factors. (2) Using Design-Expert software for multi-factor experimental design: The software will automatically generate multi-factor experimental groups. Usually, designing a 3×3 (i.e., 3 factors and 3 levels) multi-factor experiment requires 27 experimental groups, but through software optimization, it can be reduced to 17 experimental groups, reducing the use of experimental facilities and manpower. Then, according to the experimental design groups given by the software, the experiment is carried out, and the experimental results are added to the corresponding experimental group table. Finally, the software automatically generates function equations based on the experimental data and predicts the optimal combination of each variable factor according to the response surface analysis rules. (3) Conduct experiments on the optimal combination to verify the accuracy of the predicted combination data. Once the verification is successful, the data can be applied to the formulation design.

[0005] The second objective of this invention is to provide a fish feed formula for turbot farming (obtained after screening using the above-mentioned screening method), comprising, by weight percentage: fish meal 42.0%, fermented soybean meal 28.0%, fish antimicrobial peptides 0.025%~0.1%, wheat flour 15.40%~15.475%, phospholipids 2.0%, fish oil 3.0%, soybean oil 2.0%, choline 0.5%, calcium dihydrogen phosphate 1.0%, vitamin C 0.5%, vitamin mixture 2.5%, mineral mixture 2.5%, and sodium alginate 0.5%.

[0006] In the above technical solution, the fish feed formula preferably includes the following raw materials in the following weight percentages: fish meal 42.0%, fermented soybean meal 28.0%, fish antimicrobial peptides 0.025%, wheat flour 15.475%, phospholipids 2.0%, fish oil 3.0%, soybean oil 2.0%, choline 0.5%, calcium dihydrogen phosphate 1.0%, vitamin C 0.5%, vitamin mixture 2.5%, mineral mixture 2.5%, and sodium alginate 0.5%.

[0007] In the above technical solution, the fish feed formula preferably includes the following raw materials in the following weight percentages: fish meal 42.0%, fermented soybean meal 28.0%, fish antimicrobial peptides 0.05%, wheat flour 15.45%, phospholipids 2.0%, fish oil 3.0%, soybean oil 2.0%, choline 0.5%, calcium dihydrogen phosphate 1.0%, vitamin C 0.5%, vitamin mixture 2.5%, mineral mixture 2.5%, and sodium alginate 0.5%.

[0008] In the above technical solution, the fish feed formula preferably includes the following raw materials in the following weight percentages: fish meal 42.0%, fermented soybean meal 28.0%, fish antimicrobial peptides 0.075%, wheat flour 15.425%, phospholipids 2.0%, fish oil 3.0%, soybean oil 2.0%, choline 0.5%, calcium dihydrogen phosphate 1.0%, vitamin C 0.5%, vitamin mixture 2.5%, mineral mixture 2.5%, and sodium alginate 0.5%.

[0009] In the above technical solution, the fish feed formula preferably includes the following raw materials in the following weight percentages: fish meal 42.0%, fermented soybean meal 28.0%, fish antimicrobial peptides 0.1%, wheat flour 15.4%, phospholipids 2.0%, fish oil 3.0%, soybean oil 2.0%, choline 0.5%, calcium dihydrogen phosphate 1.0%, vitamin C 0.5%, vitamin mixture 2.5%, mineral mixture 2.5%, and sodium alginate 0.5%.

[0010] In the above technical solution, the formulas of the vitamin mixture and the mineral mixture are both general-purpose mixtures for marine fish, which are commercially available products commonly used in the field and were purchased from Qingdao Master Biotechnology Co., Ltd. (Qingdao, Shandong).

[0011] In the above technical solution, the fish antimicrobial peptide is a fish antimicrobial peptide with high thermal stability and bactericidal activity, and its amino acid sequence is: QSHISLCRLCCNCCKANKPCGFCCKF.

[0012] A third objective of this invention is to provide a method for preparing the aforementioned fish antimicrobial peptide, comprising the following steps: I. Antimicrobial peptide sequence modification: The amino acid sequence of the natural turbot antimicrobial peptide (mature peptide portion) is QSHISLCRWCCNCCKANKGCGFCCKF. The turbot antimicrobial peptide sequence was modified by changing tryptophan (W) at position 9 to leucine (L) and glycine (G) at position 19 to proline (P). The new sequence of the modified antimicrobial peptide is: QSHISLCRLCCNCCKANKPCGFCCKF. II. Artificially synthesized polypeptides: The swollen resin was placed in a reactor. The modified antimicrobial peptide from step I was placed in a DCM. Using the first C-terminal amino acid, phenylalanine F, as the artificial synthesis site, FMOC-Phe-OH, 1-hydroxybenzotriazole, and diisopropylcarbodiimide were added to the DCM. After stirring, the mixture was added to the reactor for reaction. Next, using the second C-terminal amino acid, lysine K, as the artificial synthesis site, Fmoc-Lys(Boc)-OH, HOBt, and DIC were added to the DCM. After stirring, the mixture was added to the reactor for reaction. The operation was repeated until the coupling reaction of the last amino acid residue, glutamine Q, was completed. A lysis buffer was added to remove the side chains of the synthesized peptide and cleave it from the resin to obtain the artificially synthesized peptide. III. Oxidative refolding of artificially synthesized peptides: A mixed solution of oxidized glutathione (GSSG) and reduced glutathione (GSH) was prepared. After adding buffer to the mixed solution, the artificially synthesized peptide in step II was refolded. The refolded product is a fish antimicrobial peptide with high thermal stability and bactericidal activity.

[0013] In the above technical solution, step II, the specific method for artificially synthesizing polypeptides includes the following steps: ① The FMOC-Wang-Resin resin swollen with dichloromethane DCM was placed into the reactor. Then, the first amino acid at the C-terminus of the modified antimicrobial peptide, phenylalanine F, was selected as the artificial synthesis site. The modified antimicrobial peptide was added to the DCM. Then, phenylalanine (FMOC-Phe-OH) protected by 9-fluorenylmethoxycarbonyl (FMOC) was dissolved in the DCM. The molar ratio of FMOC-Phe-OH to the modified antimicrobial peptide was (1.1):(1). Then, equimolar amounts of 1-hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) were added and stirred for 15 minutes. After 2 min, the resin was added to the reactor, along with a small amount of p-dimethylaminopyridine (DMAP) as a catalyst. The total amount of equimolar amounts of 1-hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) added was 35% of the mass of FMOC-Phe-OH, and the amount of p-dimethylaminopyridine (DMAP) added was 0.5% of the mass of FMOC-Phe-OH. After reacting with N2 for 2 h, the resin was washed with N,N-dimethylformamide (DMF) to remove unreacted amino acids and byproducts. Then, the prepared deprotection solution pyridine was added to remove the FMOC protecting groups on the resin. After reacting for 20 min, the resin was washed clean with DMF. The amount of pyridine used was 3 times the volume of the resin. ② Next, the second amino acid at the C-terminus, lysine K, was used as the artificial synthesis site. Fmoc-Lys(Boc)-OH, formed by protecting the amino group with FMOC and the side chain with tert-butyloxycarbonyl (Boc), was dissolved in DCM. The molar ratio of Fmoc-Lys(Boc)-OH to the modified antimicrobial peptide was (1.1): (1). Then, an equimolar amount of HOBt and DIC was added and stirred for 15 min before being added to the reactor. At the same time, a small amount of DMAP was added as a catalyst. The total amount of equimolar amounts of HOBt and DIC added was 35% of the mass of Fmoc-Lys(Boc)-OH, and the amount of DMAP added was 0.5% of the mass of Fmoc-Lys(Boc)-OH. After reacting with N2 for 2 h, it was washed with DMF. After reacting with pyridine as a deprotection solution for 20 min, it was washed clean with DMF. The amount of pyridine used was 3 times the volume of the resin. ③ Repeat the above operation until the glutamine Q coupling reaction of the last amino acid residue is completed; add lysis buffer to complete the side chain removal and cleavage of the synthesized peptide from the resin, and obtain the crude linear peptide. The crude linear peptide is separated and purified by high performance liquid chromatography (HPLC) to obtain a high-purity synthetic peptide. The lysis buffer is composed of the following components in the indicated mass percentages: 87.5% trifluoroacetic acid (TFA), 5% phenol, 2.5% mercaptoethanol, and 5% water.

[0014] In the above technical solution, step III, the specific method for the oxidative refolding of the artificially synthesized peptide includes the following steps: A mixed solution is prepared by mixing 0.15 mmol / L oxidized glutathione (GSSG) and 1.5 mmol / L reduced glutathione (GSH) at a volume ratio of 1:10; a buffer solution is prepared containing 0.05 mol / L Tris-HCl and 0.05 mol / L NaCl, with a pH of 8.6; the buffer solution is added to the mixed solution, and then the artificially synthesized peptide is added to the system for refolding, with a volume ratio of 1:10 between the mixed solution and the buffer solution, and a mass ratio of 100:1 between the total mass of the mixed solution and the buffer solution and the artificially synthesized peptide; the refolding temperature is 4℃, the refolding time is 24 h, and after refolding, high-performance liquid chromatography (HPLC) is used for separation and purification. The collected elution peak is freeze-dried and stored at -80℃ for later use, thus obtaining the fish antimicrobial peptide.

[0015] Compared with existing technologies, it has the following characteristics: This invention provides a simple and effective method for screening fish feed formulations based on multiple factors. It optimizes the multi-factor experimental design and simplifies the operation process by reducing the number of experimental groups, based on the characteristics of the aquaculture environment, fish physiological indicators, and feed ingredients. Simultaneously, it can simulate and construct mathematical models to predict the changes in various combinations of different factors and provide the optimal combination for each changing factor. It precisely combines the aquaculture environment, fish physiological indicators, and feed ingredients to improve the level and quality of fish feed formulations while saving formulation and feed costs. It can also quickly, simply, and effectively evaluate the application effect of new raw materials (new additives) in the formulation. Attached Figure Description

[0016] Figure 1 The image shows the HPLC separation diagram of the fish antimicrobial peptide synthesized in Example 1. Figure 2 This is the HPLC separation chromatogram of the fish antimicrobial peptides after oxidative refolding in Example 1; Figure 3 This is a diagram illustrating the completion of the experimental design in Example 1; Figure 4 This is a diagram demonstrating how to fill in the dependent variable in Example 1; Figure 5 This is a demonstration diagram of the experimental design group in Example 1; Figure 6 Fill in the demonstration diagram for the experimental results in Example 1; Figure 7 This is a graph of the multivariate equations relating the dependent variable and factors in Example 1; Figure 8 This is a screenshot of the parameter input interface in Example 1; Figure 9 This is a diagram showing the optimal combination of parameters in Example 1. Detailed Implementation

[0017] The following describes in detail the specific embodiments of the technical solution of the present invention, but the present invention is not limited to the following description: Using turbot, a marine aquaculture species in northern my country, as the experimental fish, the experimental factors included culture temperature, culture density, fermented soybean meal content, and the content of newly developed fish antimicrobial peptides. The total antioxidant enzyme activity in the fish serum and the relative content of intestinal Vibrio were used as the evaluation indicators (i.e., dependent variables). The aim was to quickly and accurately evaluate the actual application effect of the newly developed fish antimicrobial peptides and their reasonable dosage in combination with the culture environment and other feed ingredients, and finally propose a precise and high-quality feed formula.

[0018] The technical solution of the present invention will be described below with reference to specific embodiments: Example 1: A multi-factor-based method for screening fish feed formulations The multi-factor-based fish feed formulation screening method in this embodiment includes the following steps: (1) Based on literature review, the suitable culture temperature for turbot is 18℃, the suitable culture density is 140 fish / m2, and the suitable replacement amount of fermented soybean meal in feed for fish meal is 40%.

[0019] (2) Fish antimicrobial peptides: The fish antimicrobial peptides were developed by our research team. Experiments determined that the appropriate addition level in turbot feed is 500 mg / kg. Details are as follows: A. Development of Fish Antimicrobial Peptides: Natural antimicrobial peptides generally exhibit good thermal stability below 60℃. Extruded aquatic feed is currently the main type of aquatic feed, accounting for over 60% of the total feed volume, and will continue to maintain a steady growth trend. The processing temperature in extruded feed is typically between 70-120℃, which greatly limits the application of antimicrobial peptides in extruded feed. To improve the thermal stability of turbot antimicrobial peptides, enabling them to withstand the high-temperature processes of extruded feed production while maintaining their bactericidal efficacy, it is proposed to modify existing natural sequences to develop new antimicrobial peptides with higher thermal stability and bactericidal activity, including the following steps: I. According to literature review, the amino acid sequence of the natural turbot antimicrobial peptide (mature peptide portion) is QSHISLCRWCCNCCKANKGCGFCCKF. Based on the characteristics of proline (Pro) and leucine (Leu), (Pro is an amino acid that can significantly improve the thermal stability of proteins. The mechanism is that proline can reduce the skeletal entropy of protein unfolding and increase protein rigidity. Based on this, Pro can be introduced into the α-helix or β-sheet structure to increase the rigidity of the peptide chain; the hydrophobic amino acid Leu can increase the interaction between the antimicrobial peptide and the bacterial cell membrane, thereby improving antimicrobial activity, and helps to form the α-helix or β-sheet structure, which is crucial for the stability and function of the antimicrobial peptide. Some non-critical amino acids in the sequence can be replaced with Leu.) The turbot antimicrobial peptide sequence was modified to the following new sequence: QSHISLCRLCCNCCKANKPCGFCCKF, with tryptophan (W) at position 9 replaced by leucine (L) and glycine (G) at position 19 replaced by proline (P).

[0020] II. Artificial synthesis of modified peptides using solid-phase chemical synthesis: Synthesis was performed using a Tetras peptide synthesizer (ThuraMed, USA). The basic reaction process is as follows: ① The FMOC-Wang-Resin resin swollen with dichloromethane DCM was placed into the reactor. Then, the first amino acid at the C-terminus of the modified antimicrobial peptide, phenylalanine F, was selected as the artificial synthesis site. The modified antimicrobial peptide was added to the DCM. Then, phenylalanine (FMOC-Phe-OH) protected by 9-fluorenylmethoxycarbonyl (FMOC) was dissolved in the DCM. The molar ratio of FMOC-Phe-OH to the modified antimicrobial peptide was (1.1):(1). Then, equimolar amounts of 1-hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) were added and stirred for 15 minutes. After 2 min, the resin was added to the reactor, along with a small amount of p-dimethylaminopyridine (DMAP) as a catalyst. The total amount of equimolar amounts of 1-hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) added was 35% of the mass of FMOC-Phe-OH, and the amount of p-dimethylaminopyridine (DMAP) added was 0.5% of the mass of FMOC-Phe-OH. After reacting with N2 for 2 h, the resin was washed with N,N-dimethylformamide (DMF) to remove unreacted amino acids and byproducts. Then, the prepared deprotection solution pyridine was added to remove the FMOC protecting groups on the resin. After reacting for 20 min, the resin was washed clean with DMF. The amount of pyridine used was 3 times the volume of the resin. ② Next, the second amino acid at the C-terminus, lysine K, was used as the artificial synthesis site. Fmoc-Lys(Boc)-OH, formed by protecting the amino group with FMOC and the side chain with tert-butyloxycarbonyl (Boc), was dissolved in DCM. The molar ratio of Fmoc-Lys(Boc)-OH to the modified antimicrobial peptide was (1.1): (1). Then, an equimolar amount of HOBt and DIC was added and stirred for 15 min before being added to the reactor. At the same time, a small amount of DMAP was added as a catalyst. The total amount of equimolar amounts of HOBt and DIC added was 35% of the mass of Fmoc-Lys(Boc)-OH, and the amount of DMAP added was 0.5% of the mass of Fmoc-Lys(Boc)-OH. After reacting with N2 for 2 h, it was washed with DMF. After reacting with pyridine as a deprotection solution for 20 min, it was washed clean with DMF. The amount of pyridine used was 3 times the volume of the resin. ③ Repeat the above operation until the glutamine Q coupling reaction of the last amino acid residue is completed; add lysis buffer to complete the side chain removal and cleavage of the synthesized polypeptide from the resin to obtain the crude linear polypeptide. The lysis buffer is composed of the following components in mass percentage: 87.5% trifluoroacetic acid (TFA), 5% phenol, 2.5% mercaptoethanol, and 5% water.

[0021] The synthesized linear peptide crude product was separated and purified by high-performance liquid chromatography (HPLC) using an Agilent C8 reversed-phase column (5 μm, 300A). Eluent A was an aqueous solution containing 0.1% TFA, and eluent B was an acetonitrile solution containing 0.1% TFA. The elution gradient was such that the proportion of eluent B increased from 10% to 40% over 35 minutes at a flow rate of 1.0 ml / min. Detection was performed using ultraviolet light at a wavelength of 220 nm. The HPLC results showed a single elution peak, indicating high purity (e.g., ...). Figure 1 (As shown). The eluted target peak was collected and analyzed by mass spectrometry, and the results showed that its molecular weight was 2910 Da.

[0022] III. Oxidative refolding of the synthetic peptide: A mixed solution of oxidized glutathione (GSSG) (0.15 mmol / L) and reduced glutathione (GSH) (1.5 mmol / L) was prepared at a volume ratio of 1:10. A buffer solution containing 0.05 mol / L Tris-HCl and 0.05 mol / L NaCl with a pH of 8.6 was prepared. The buffer solution was added to the mixed solution, and then the synthetic peptide was added to the system for refolding. The volume ratio of the mixed solution to the buffer solution was 1:10, and the total mass ratio of the mixed solution and the buffer solution to the synthetic peptide was 100:1. The refolding temperature was 4℃, and the refolding time was 24 h.

[0023] HPLC was used to detect the refolded product, and mass spectrometry was used to analyze the molecular weight of the refolded product. The HPLC results showed a single elution peak, indicating high purity of the refolded product (e.g., ...). Figure 2 (As shown). The target elution peak was collected and analyzed by mass spectrometry. The results showed a molecular weight of 2902 Da. The reduction of 8 Da was due to the formation of 4 disulfide bonds by 8 cysteine ​​residues in the sequence, resulting in a reduction of 8 H atoms. The collected elution peak was freeze-dried and stored at -80℃ for subsequent experiments.

[0024] B. Antimicrobial activity assay of fish antimicrobial peptides: The bacteria used for testing were Vibrio anguillarum, a common pathogen of turbot. The bacterial culture, prepared in LB broth, was added to each well of a sterilized 96-well plate at a concentration of 90 μL. The synthesized and refolded fish antimicrobial peptides were dissolved in sterile purified water to a concentration of 500 μmol / L, and then serially diluted to a minimum concentration of 0.49 μmol / L. Each concentration of fish antimicrobial peptide was added to the bacterial culture at a concentration of 10 μL / well of the 96-well plate. Purified water was used as a negative control. The 96-well plate was gently shaken on a shaker to promote uniform mixing of the bacterial culture and the fish antimicrobial peptides. The plate was then incubated at 37°C for 10 h. The OD630 value of each well was then measured using a microplate reader, and compared with the absorbance value before incubation to assess the inhibitory effect of the fish antimicrobial peptides on bacterial growth. The results are shown in Table 1. When the concentration of fish antimicrobial peptide was 31.25 μmol / L, Vibrio anguillarum continued to grow, while when the concentration of fish antimicrobial peptide was greater than or equal to 62.5 μmol / L, the growth of Vibrio anguillarum was inhibited.

[0025] Table 1. Detection of antibacterial activity of synthetic fish antimicrobial peptides 90 μL bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution 10 μL peptide concentration (µmol / L) 500 250 125 62.5 31.25 15.63 7.81 3.91 1.95 0.98 0.49 pure water OD630 value before culture 1.27 1.25 1.26 1.21 1.21 1.19 1.19 1.20 1.20 1.21 1.20 1.22 OD630 value after cultivation 1.22 1.21 1.21 1.20 1.31 1.35 1.40 1.42 1.45 1.50 1.50 1.55 C. Combined detection of the high-temperature resistance and antibacterial activity of fish antimicrobial peptides: In the above experimental detection system, different concentrations of fish antimicrobial peptides were incubated in constant temperature incubators at 60℃, 90℃, and 120℃ for 30 min, respectively, and then added to 96-well plates containing Vibrio anguillarum bacterial solution. The OD630 value of each well was measured. The results are shown in Table 2. After treatment at different temperatures of 60℃, 90℃, and 120℃, the antibacterial effect of the modified fish antimicrobial peptides against Vibrio anguillarum did not decrease, indicating that the modified fish antimicrobial peptides can withstand temperatures of 60-120℃ and exert antibacterial function, and the modification was successful.

[0026] Table 2. Combined detection of the high-temperature resistance and antibacterial activity of synthetic fish antimicrobial peptides 90 μL bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution bacterial solution 10 μL peptide concentration (µmol / L) 500 250 125 62.5 31.25 15.63 7.81 3.91 1.95 0.98 0.49 pure water OD630 value before incubation (after treatment at 60℃) 1.26 1.25 1.26 1.22 1.21 1.19 1.20 1.20 1.19 1.21 1.21 1.22 OD630 value after incubation (after treatment at 60℃) 1.20 1.19 1.21 1.21 1.30 1.35 1.40 1.40 1.44 1.48 1.52 1.55 OD630 value before incubation (after treatment at 90℃) 1.25 1.26 1.27 1.21 1.20 1.19 1.19 1.19 1.20 1.20 1.20 1.22 OD630 value after incubation (after treatment at 90℃) 1.19 1.21 1.21 1.19 1.32 1.36 1.40 1.41 1.45 1.49 1.50 1.56 OD630 value before culture (after treatment at 120℃) 1.25 1.27 1.26 1.20 1.19 1.21 1.19 1.20 1.19 1.21 1.19 1.22 OD630 value after incubation (after treatment at 120℃) 1.22 1.20 1.21 1.20 1.31 1.36 1.41 1.40 1.47 1.50 1.51 1.54 D. Appropriate addition amount of fish antimicrobial peptides: A basic feed formulation was designed so that the content of the synthetic and well-refolded freeze-dried fish antimicrobial peptide powder added to the feed was 0, 250, 500, 750, and 1000 mg / kg, respectively. The corresponding proportions in the formulation were 0 (control group), 0.025, 0.05, 0.075, and 0.1%, respectively. Detailed formulations are shown in Table 3. All solid raw materials were sieved through a 60-mesh sieve. After thorough mixing, five groups of feeds were prepared for use. The experiment consisted of 5 groups, with 3 replicates per group. Each tank contained 20 juvenile turbot (8.31±0.12 g), all hatched from the same batch that year. The experimental fish were fed the control group feed for 3 days before the experiment officially began. The experiment was conducted in a small-scale recirculating aquaculture system within the farm. The tanks were 50L fiberglass containers with a water flow rate of 75L / h, a water temperature of 18±1℃, and dissolved oxygen not less than 7.0 mg / L. Fish were fed twice daily, at 08:00 and 17:00, for a period of 30 days. Feeding and mortality data were recorded during the experiment. At the end of the experiment, after a 24-hour fast, the fish body weight was measured. Blood was collected from the tail vein of 5 fish and incubated overnight at 4℃. Serum was obtained by centrifugation and tested using a kit purchased from Nanjing Jiancheng. Intestinal samples were collected from the other 5 fish and sent to Novogene Biotechnology Co., Ltd. for microbial testing. The results (Table 4) showed that when the amount of fish antimicrobial peptides added to the feed reached 500 mg / kg, compared with the control group, the weight gain rate of juvenile turbot was significantly increased (P<0.05), the total superoxide dismutase activity in serum was significantly increased (P<0.05), and the relative content of intestinal Vibrio was significantly decreased (P<0.05). Considering the cost, it is recommended that the minimum amount of fish antimicrobial peptides added to turbot feed be 500 mg / kg.

[0027] Table 3. Experimental feed formulations

[0028] *The vitamin and mineral blend is a general formula for marine fish and is a commonly available commercial product in this field. It was purchased from Qingdao Master Biotechnology Co., Ltd. (Qingdao, Shandong).

[0029] Table 4. Effects of adding synthetic fish antimicrobial peptides to feed on growth, immunity, and intestinal Vibrio in juvenile turbot.

[0030] (3) Design and implement multi-factor experiments using Design-Expert software. A. First, run Design-Expert 10.0.7 software on your computer. Select "New Design," and in the new window that appears, select "Response Surface" on the left, then click "Box-Behnken." In the experimental design table that appears, select the number 4 for "Numeric Factors." Next, based on the appropriate values ​​from the single-factor experimental results, fill in the factor name, its corresponding unit, and the low and high ranges of factor variation. Leave the other options as default and click "Continue." Figure 3 (As shown).

[0031] B. Next, on the newly appeared page, select number 2 for "Responses (Dependent Variable)," fill in the dependent variable name and its corresponding unit, and click "Finish" (e.g., ...). Figure 4 (As shown). At this point, the software will automatically generate experimental design groups with different levels of each factor (such as...). Figure 5 (As shown). Typically, a 4*3 (i.e., 4 factors and 3 levels) multifactor experiment requires 81 experimental groups, but through software optimization, it can be reduced to 29 experimental groups, greatly reducing the use of experimental facilities and manpower.

[0032] C. Conduct experiments according to the experimental design groups provided by the software, and supplement the experimental results into the corresponding experimental group tables. Specifically, first, conduct the experiments according to the experimental design group tables generated by the software. The experimental conditions and operations are the same as described in 2D (using three identical aquaculture systems, with temperatures of 15, 18, and 21℃ respectively, and a temperature control error within ±1℃). The radius of the bottom of the aquaculture tank is 0.4 m (stocking densities of 9, 18, and 27 fish respectively). The experimental feed formula is shown in Table 5. At the end of the experiment, collect the corresponding data according to the settings of "Responses (dependent variable)". Finally, fill in the corresponding experimental results in the "Responses (dependent variable)" column of the experimental design group table and perform subsequent analysis. See Table 5 for the experimental results. Figure 6 .

[0033] Table 5. Multifactorial experimental feed formulations

[0034] *The vitamin and mineral blend is a general formula for marine fish, as above.

[0035] (4) The software automatically generates function equations based on experimental data and predicts the optimal combination of each variable factor according to the response surface analysis rules. Specifically, first select the "R1, R2..." option under "Analysis" on the left side of the page. After clicking the "ANOVA" option above the dialog box that appears, a new small dialog box will appear on the left. After clicking "Equations", the generated multivariate equations about the dependent variable and factors will appear under "Final Equation in Terms of Actual Factors" (see...). Figure 7 The multivariate equations for this experiment are: Serum total antioxidant enzyme activity = -290.28 + 32.62 * temperature + 0.08 * stocking density + 0.20 * fermented soybean meal replacement amount + 0.03 * fish antimicrobial peptide content - 2.01 * temperature * stocking density + 0.01 * temperature * fermented soybean meal replacement amount - 2.81 * temperature * fish antimicrobial peptide content - 4.76 * stocking density * fermented soybean meal replacement amount + 2.85 * stocking density * fish antimicrobial peptide content - 3.5 * fermented soybean meal replacement amount * fish antimicrobial peptide content - 0.91 * temperature 2 - 2.20 * stocking density 2 - 4.41 * fermented soybean meal replacement amount 2 - 2.41 * fish antimicrobial peptide content 2 Then click the "Numerical" option under "Optimization" on the left side of the page. In the new dialog box, click "Criteria" at the top, and set the "Goal" for the corresponding "Responses". Choose either "maximize" or "minimize" based on the actual situation, and enter the highest possible value and the lowest possible value accordingly (e.g., ...). Figure 8 As shown in the image, in this experiment, the serum total antioxidant enzyme activity was selected as "maximum," and the relative content of Vibrio ingeri was selected as "minimum." Finally, after clicking the "Solutions" option above, the resulting combination represents the maximum or minimum "Responses" value that can be achieved after taking the optimal values ​​for each factor. The values ​​of each factor are the best combination predicted by the software (e.g., ...). Figure 9 As shown in the figure, the optimal combination of factors in this experiment is as follows: temperature 17.792℃, stocking density 143.686 fish / m³. 2 The amount of fishmeal replaced by fermented soybean meal was 41.284%, and the content of fish antimicrobial peptides was 614.245 mg / kg. Under these conditions, the highest serum total antioxidant enzyme activity (21.916 U / mg Protein) and the lowest relative content of Vibrio intestinal bacteria (2.035%) could be obtained.

[0036] (5) Conduct a practical application experiment on the optimal combination to verify the accuracy of the predicted combination data and apply it in practice. Specifically, the optimal combination data of each factor predicted by the software (rounded to the nearest integer according to the actual situation) is used to repeat the experiment under the previous experimental conditions. In this experiment, the values ​​of each factor are as follows: temperature is 18℃, and stocking density is 144 fish / m³. 2 The fermented soybean meal replaced 41% of the fishmeal, and the fish antimicrobial peptide content was 614 mg / kg. The experimental results are as follows: serum total antioxidant enzyme activity was 22.01 U / mg Protein, and the relative content of intestinal Vibrio was 2.04%. The experimental results (22.01 U / mg Protein, 2.04%) showed little difference from the software-predicted results (21.916 U / mg Protein, 2.035%), with errors of 0.43% and 0.25% respectively. This indicates that the software-predicted data is accurate and reliable and can be practically applied to feed formulations.

[0037] (6) The factors obtained in this experiment are as follows: temperature was 18℃, and stocking density was 144 fish / m³. 2 Fermented soybean meal replaced 41% of fishmeal, and the fish antimicrobial peptide content was 614 mg / kg. Under these conditions, the maximum serum total antioxidant enzyme activity could be obtained, maintaining the body's optimal immune function, and the minimum relative content of intestinal Vibrio was obtained at 2.04%, reducing the occurrence of Vibrio infection.

[0038] Example 2: A multi-factor-based fish feed formulation

[0039] The fish feed formulation comprises the following raw materials in weight percentages: fish meal 42.0%, fermented soybean meal 28.0%, fish antimicrobial peptides 0.025%, wheat flour 15.475%, phospholipids 2.0%, fish oil 3.0%, soybean oil 2.0%, choline 0.5%, calcium dihydrogen phosphate 1.0%, vitamin C 0.5%, vitamin mixture 2.5%, mineral mixture 2.5%, and sodium alginate 0.5%. The vitamin mixture and mineral mixture are the same as in Example 1.

[0040] Example 3: A multi-factor-based fish feed formulation The fish feed formulation comprises the following raw materials in the indicated weight percentages: fish meal 42.0%, fermented soybean meal 28.0%, fish antimicrobial peptides 0.05%, wheat flour 15.45%, phospholipids 2.0%, fish oil 3.0%, soybean oil 2.0%, choline 0.5%, calcium dihydrogen phosphate 1.0%, vitamin C 0.5%, vitamin mixture 2.5%, mineral mixture 2.5%, and sodium alginate 0.5%. The vitamin mixture and mineral mixture are the same as in Example 1.

[0041] Example 4: A multi-factor-based fish feed formulation The fish feed formulation comprises the following raw materials in weight percentages: fish meal 42.0%, fermented soybean meal 28.0%, fish antimicrobial peptides 0.075%, wheat flour 15.425%, phospholipids 2.0%, fish oil 3.0%, soybean oil 2.0%, choline 0.5%, calcium dihydrogen phosphate 1.0%, vitamin C 0.5%, vitamin mixture 2.5%, mineral mixture 2.5%, and sodium alginate 0.5%. The vitamin mixture and mineral mixture are the same as in Example 1.

[0042] Example 5: A multi-factor-based fish feed formulation The fish feed formulation comprises the following raw materials in weight percentages: fish meal 42.0%, fermented soybean meal 28.0%, fish antimicrobial peptides 0.1%, wheat flour 15.4%, phospholipids 2.0%, fish oil 3.0%, soybean oil 2.0%, choline 0.5%, calcium dihydrogen phosphate 1.0%, vitamin C 0.5%, vitamin mixture 2.5%, mineral mixture 2.5%, and sodium alginate 0.5%. The vitamin mixture and mineral mixture are the same as in Example 1.

[0043] The above examples are merely illustrative of the technical concept and features of the present invention and should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the essence of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a fish antimicrobial peptide, characterized in that, Includes the following steps: I. Antimicrobial peptide sequence modification: The amino acid sequence of the mature peptide of the natural turbot antimicrobial peptide is QSHISLCRWCCNC CKA NKGCGFCCKF. The turbot antimicrobial peptide sequence was modified by changing tryptophan (W) at position 9 to leucine (L) and glycine (G) at position 19 to proline (P). The new sequence of the modified antimicrobial peptide is: QSHISLCRLCCNCCKANKPCGFCCKF. II. Artificially synthesized polypeptides: ① The FMOC-Wang-Resin resin swollen in dichloromethane DCM was placed in the reactor. Then, the first amino acid at the C-terminus of the modified antimicrobial peptide, phenylalanine F, was selected as the artificial synthesis site. The modified antimicrobial peptide was added to the DCM, and then 9-fluorenylmethoxycarbonyl (FMOC)-protected phenylalanine (FMOC-Phe-OH) was dissolved in the DCM. The molar ratio of FMOC-Phe-OH to the modified antimicrobial peptide was 1.1:

1. Then, equimolar amounts of 1-hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) were added and stirred for 15 minutes. After 2 min, the resin was added to the reactor, along with a small amount of p-dimethylaminopyridine (DMAP) as a catalyst. The total amount of equimolar amounts of 1-hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) added was 35% of the mass of FMOC-Phe-OH, and the amount of p-dimethylaminopyridine (DMAP) added was 0.5% of the mass of FMOC-Phe-OH. After reacting with N2 for 2 h, the resin was washed with N,N-dimethylformamide (DMF) to remove unreacted amino acids and byproducts. Then, the prepared deprotection solution pyridine was added to remove the FMOC protecting groups on the resin. After reacting for 20 min, the resin was washed clean with DMF. The amount of pyridine used was 3 times the volume of the resin. ② Next, the second amino acid at the C-terminus, lysine K, was used as the artificial synthesis site. Fmoc-Lys(Boc)-OH, formed by protecting the amino group with FMOC and the side chain with tert-butyloxycarbonyl (Boc), was dissolved in DCM. The molar ratio of Fmoc-Lys(Boc)-OH to the modified antimicrobial peptide was 1.1:

1. Then, equimolar amounts of HOBt and DIC were added and stirred for 15 min before being added to the reactor. At the same time, a small amount of DMAP was added as a catalyst. The total amount of equimolar amounts of HOBt and DIC added was 35% of the mass of Fmoc-Lys(Boc)-OH, and the amount of DMAP added was 0.5% of the mass of Fmoc-Lys(Boc)-OH. After reacting with N2 for 2 h, the mixture was washed with DMF. After adding the deprotecting solution pyridine and reacting for 20 min, the mixture was washed clean with DMF. The amount of pyridine used was 3 times the volume of the resin. ③ Repeat the above operation until the glutamine Q coupling reaction of the last amino acid residue is completed; add lysis buffer to complete the side chain removal and cleavage of the synthesized peptide from the resin, and obtain the crude linear peptide. The crude linear peptide is separated and purified by high performance liquid chromatography (HPLC) to obtain a high-purity synthetic peptide; the lysis buffer is composed of the following components in the indicated mass percentages: 87.5% trifluoroacetic acid (TFA), 5% phenol, 2.5% mercaptoethanol, and 5% water; III. Oxidative refolding of artificially synthesized peptides: A mixed solution of oxidized glutathione (GSSG) and reduced glutathione (GSH) was prepared. After adding buffer to the mixed solution, the artificially synthesized peptide in step II was refolded. The refolded product is a fish antimicrobial peptide with high thermal stability and bactericidal activity.

2. The method for preparing fish antimicrobial peptides according to claim 1, characterized in that, In step III, the specific method for the oxidative refolding of the artificially synthesized peptide includes the following steps: A mixed solution is prepared by mixing 0.15 mmol / L oxidized glutathione (GSSG) and 1.5 mmol / L reduced glutathione (GSH) at a volume ratio of 1:10; a buffer solution is prepared containing 0.05 mol / L Tris-HCl and 0.05 mol / L NaCl, with a pH of 8.6; the buffer solution is added to the mixed solution, and then the artificially synthesized peptide is added to the system for refolding. The volume ratio of the mixed solution to the buffer solution is 1:10, and the total mass ratio of the mixed solution and the buffer solution to the artificially synthesized peptide is 100:1; the refolding temperature is 4℃, and the refolding time is 24 h. After refolding, high-performance liquid chromatography (HPLC) is used for separation and purification. The collected elution peak is freeze-dried and stored at -80℃ for later use, thus obtaining the fish antimicrobial peptide.

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