Abalone viscera peptide capable of replacing fish meal as well as preparation method and application of abalone viscera peptide

Abstract

The invention belongs to the technical field of biology, and discloses abalone viscera peptide capable of replacing fish meal as well as a preparation method and application of the abalone viscera peptide. In the preparation method of the abalone viscera peptide provided by the invention, the alkaline protease and the neutral protease in a specific proportion are used as a compound enzyme system for realizing hydrolysis of the abalone viscera, and the compound enzyme system has high matching degree with the characteristics of proteins contained in the abalone viscera; according to the method, protein in the abalone viscera can be accurately hydrolyzed through synergistic cooperation of specific temperature, pH value and time conditions adopted in the enzymolysis treatment process, and the abalone viscera peptide with high purity and specific polypeptide composition is obtained while sufficient and non-excessive hydrolysis of the abalone viscera is achieved; in addition, the hydrolysis effect of the preparation method and the components of the obtained abalone viscera peptide are slightly influenced by operation and batch of abalone viscera raw materials, and the method has excellent process stability and has very excellent application prospects in industrial large-scale preparation of the abalone viscera peptide.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to an abalone viscera peptide that can replace fish meal, its preparation method, and its application. Background Technology

[0002] Fishmeal refers to a high-protein feed ingredient obtained from fish through processes such as degreasing, dehydration, and grinding. Fishmeal is high in protein and essential amino acids, and its amino acid composition is balanced, making it a primary protein source in aquatic feed. However, due to overfishing, environmental pollution, and the depletion of fishery resources, fishmeal production has been declining year by year. my country is the world's largest importer of fishmeal, and fishmeal shortages have become a key factor restricting the sustainable development of aquaculture. Therefore, developing alternative protein sources is urgently needed.

[0003] Abalone is an important aquaculture species in my country, with its farming and processing scale continuously expanding. Abalone viscera are one of the main byproducts generated during abalone processing, accounting for 15%-25% of the total weight of abalone, and are rich in nutrients such as protein, fat, and polysaccharides. Currently, abalone viscera are often discarded as processing waste. Therefore, the high-value utilization of abalone viscera, turning waste into treasure, is particularly urgent and necessary in this research field.

[0004] Abalone viscera contain approximately 50% protein; however, this type of protein has a low digestibility and absorption rate. Processing it into smaller molecular weight peptides can improve its digestibility and absorption. Enzymatic hydrolysis of abalone viscera using proteases offers advantages such as mild conditions, high specificity, high safety, and ease of control. While existing enzymatic hydrolysis methods have improved the utilization value of abalone viscera to some extent, the overall process is relatively complex, lacks precision, and fails to fully consider the growth requirements of aquaculture animals, thus limiting its widespread application in actual production and exhibiting certain limitations. Summary of the Invention

[0005] The primary objective of this invention is to address the problems of complex processes and insufficient precision in the preparation of abalone viscera peptides using protease hydrolysis, and to provide a method for preparing abalone viscera peptides.

[0006] The second objective of this invention is to provide an abalone viscera peptide.

[0007] A third objective of this invention is to provide the application of the above-mentioned abalone visceral peptides in feed production.

[0008] The fourth objective of this invention is to provide a feed.

[0009] Specifically, the preparation method of abalone visceral peptides provided by the present invention includes: enzymatically hydrolyzing abalone viscera with alkaline protease and neutral protease in a mass ratio of (1~2):(1~2) to obtain the abalone visceral peptides; wherein, the total amount of alkaline protease and neutral protease added is in the ratio of (5000~10000)U:5g to abalone visceral slurry; the enzymatic hydrolysis temperature is 40℃~60℃, the pH value is 6~10, and the time is not less than 1h.

[0010] Furthermore, the abalone viscera undergoes homogenization treatment at a speed of 10,000 rpm to 20,000 rpm for a time of 30 to 120 seconds.

[0011] Furthermore, in the enzymatic hydrolysis process, the concentration of abalone viscera added is 10wt%~80wt%.

[0012] Furthermore, the enzymatic hydrolysis treatment is carried out at a temperature of 45℃~50℃, a pH value of 7~9, and a time of 2h~4h.

[0013] Further, the preparation method includes: taking abalone visceral peptides obtained by the enzymatic hydrolysis and performing inactivation treatment and / or freeze-drying treatment; wherein, the inactivation treatment temperature is 95℃~105℃ and the time is 5min~20min; the freeze-drying treatment temperature is -20℃~-40℃ and the time is 48h~96h.

[0014] The abalone visceral peptides provided by this invention are prepared by the above-described method for preparing abalone visceral peptides.

[0015] Further, the abalone visceral peptides include one or more of the following protein peptides: (1) protein peptide 1 with the amino acid sequence GFAGDDAPR; (2) protein peptide 2 with the amino acid sequence GFAGDDAPRA; (3) protein peptide 3 with the amino acid sequence FAGDDAPRA; (4) protein peptide 4 with the amino acid sequence AGDDAPR; (5) protein peptide 5 with the amino acid sequence GINPEDVFHAD; (6) protein peptide 6 with the amino acid sequence PNIVFDHY; (7) protein peptide 7 with the amino acid sequence SPDILPQP; (8) (9) Protein peptide 8 with amino acid sequence SVDHGDYY; (10) Protein peptide 9 with amino acid sequence IANDPDSSF; (11) Protein peptide 10 with amino acid sequence LPIPNLP; (12) Protein peptide 11 with amino acid sequence YEPGVGHHEDHE; (13) Protein peptide 12 with amino acid sequence YSAYDPVFM; (14) Protein peptide 13 with amino acid sequence KLTTPTYGDL; (15) Protein peptide 14 with amino acid sequence NEDEFTRVN; (16) Protein peptide 15 with amino acid sequence WDWTRP.

[0016] This invention provides the application of the above-mentioned abalone visceral peptides in feed production.

[0017] The feed provided by this invention includes the aforementioned abalone visceral peptides.

[0018] Further, the feed comprises: abalone visceral peptides at a concentration of 1wt% to 20wt%, fish meal at a concentration of 10wt% to 40wt%, soybean meal at a concentration of 20wt% to 30wt%, chicken meal at a concentration of 10wt% to 20wt%, wheat flour at a concentration of 10wt% to 30wt%, fish oil at a concentration of 1wt% to 5wt%, soybean oil at a concentration of 1wt% to 5wt%, lecithin at a concentration of 1wt% to 5wt%, calcium dihydrogen carbonate at a concentration of 1wt% to 5wt%, vitamin C at a concentration of 0.05wt% to 0.2wt%, trace elements at a concentration of 0.5wt% to 2wt%, choline chloride at a concentration of 0.1wt% to 0.5wt%, and yttrium trioxide at a concentration of 0.05wt% to 0.1wt%.

[0019] Beneficial effects: The method for preparing abalone visceral peptides provided by this invention employs a specific ratio of alkaline protease and neutral protease as a composite enzyme system for hydrolyzing abalone viscera. This composite enzyme system has a high degree of compatibility with the protein characteristics contained in abalone viscera. Through its synergistic effect with the specific temperature, pH value, and time conditions used in the enzymatic hydrolysis process, it can precisely hydrolyze the proteins in abalone viscera, achieving sufficient but not excessive hydrolysis of abalone viscera and obtaining high-purity abalone visceral peptides with specific polypeptide compositions. Furthermore, the hydrolysis effect and the composition of the obtained abalone visceral peptides are minimally affected by the operation and the batch of abalone viscera raw materials, exhibiting excellent process stability. It has a very promising application prospect for the large-scale industrial preparation of abalone visceral peptides.

[0020] In some specific embodiments, a feed is provided, which includes abalone visceral peptides at a concentration of 1wt% to 20wt%. The abalone visceral peptides include a specific protein peptide composition. The feed made by partially replacing fishmeal with the abalone visceral peptides has no significant difference in utilization efficiency compared with the feed with only fishmeal added. It can well replace fishmeal in the feed and has a certain feeding attraction effect, and has good application prospects in feed production, especially aquatic feed production. Attached Figure Description

[0021] Figure 1 This is one of the experimental results of the abalone visceral peptide concentration test provided in Test Example 1 of the present invention; Figure 2The second figure shows the experimental results of the abalone visceral peptide concentration test provided in Test Example 1 of the present invention. Figure 3 Figure 3 shows the experimental results of the abalone visceral peptide concentration test provided in Test Example 1 of this invention; Figure 4 Figure 4 shows the experimental results of the abalone visceral peptide concentration test provided in Test Example 1 of this invention. Figure 5 Figure 5 shows the experimental results of the abalone visceral peptide concentration test provided in Test Example 1 of this invention; Figure 6 Figure 6 shows the experimental results of the abalone visceral peptide concentration test provided in Test Example 1 of the present invention; Figure 7 Figure 7 shows the experimental results of the abalone visceral peptide concentration test provided in Test Example 1 of the present invention. Figure 8 The experimental results of the sea bass weight gain rate test provided in Test Example 2 of the present invention are shown in the figure. Figure 9 The experimental results of the sea bass specific growth rate test provided in Test Example 2 of the present invention are shown in the figure. Figure 10 The figure shows the experimental results of the sea bass feed conversion ratio provided in Test Example 2 of the present invention. Detailed Implementation

[0022] The method for preparing abalone visceral peptides provided by this invention includes: enzymatically hydrolyzing abalone viscera with alkaline protease and neutral protease to obtain the abalone visceral peptides. Specifically, the mass ratio of the alkaline protease to the neutral protease is (1~2):(1~2), such as 1:1, 1:1.3, 1:1.5, 1:1.8, 1:2, 1.5:1, 1.8:1, 2:1, or any value between them. The conditions for the enzymatic hydrolysis include a temperature of 40℃~60℃, such as 40℃, 42℃, 44℃, 46℃, 47.6℃, 48℃, 49℃, 50℃, 55℃, 60℃, or any value between them; a pH value of 6~10, such as 6, 6.3, 6.5, 6.8, 7, 7.2, 8, 9, 10, or any value between them; and a time of not less than 1 hour.

[0023] In this invention, the abalone viscera are preferably homogenized. By breaking down the abalone viscera, the contact between the abalone viscera tissue and alkaline and neutral proteases is increased, thereby achieving a better enzymatic hydrolysis effect.

[0024] In some specific embodiments, the homogenization conditions specifically include a rotation speed preferably between 10,000 rpm and 20,000 rpm, such as 10,000 rpm, 12,000 rpm, 14,000 rpm, 16,000 rpm, 18,000 rpm, 20,000 rpm or any value between them; and a time preferably between 30 s and 120 s, such as 30 s, 32 s, 35 s, 38 s, 40 s, 45 s, 60 s, 80 s, 100 s, 120 s or any value between them.

[0025] In some specific embodiments, the concentration of the abalone viscera added is preferably 10wt% to 80wt%, such as 10wt%, 12.5wt%, 15wt%, 18wt%, 20wt%, 35wt%, 40wt%, 50wt%, 60wt%, 70wt%, 80wt%, or any value between them.

[0026] In some specific embodiments, the temperature of the enzymatic hydrolysis treatment is preferably 45℃~50℃, such as 45℃, 47℃, 49℃, 50℃ or any value between them; the pH value is preferably 7~9, such as 7, 7.2, 7.5, 7.8, 8, 8.3, 8.5, 8.9, 9 or any value between them; the time is preferably 2h~4h, such as 2h, 2.3h, 2.5h, 2.8h, 3h, 3.3h, 3.7h, 4h or any value between them.

[0027] In this invention, the preferred preparation method includes: taking abalone visceral peptides obtained by the enzymatic hydrolysis treatment and performing inactivation treatment and / or freeze-drying treatment, so as to effectively inactivate enzyme molecules such as alkaline protease and neutral protease, and the resulting abalone visceral peptides are more conducive to preservation.

[0028] In some specific embodiments, the inactivation treatment conditions specifically include a temperature preferably between 95°C and 105°C, such as 95°C, 98°C, 99°C, 100°C, 101°C, 103°C, 105°C or any value between them; and a time preferably between 5 min and 20 min, such as 5 min, 8 min, 9 min, 10 min, 12 min, 15 min, 18 min, 20 min or any value between them.

[0029] In some specific embodiments, the freeze-drying conditions specifically include a temperature preferably between -20°C and -40°C, such as -20°C, -25°C, -30°C, -35°C, -40°C or any value between them; and a time between 48 min and 96 min, such as 48 min, 60 min, 72 min, 84 min, 96 min or any value between them.

[0030] The abalone visceral peptides provided by this invention are prepared by the above-described method for preparing abalone visceral peptides.

[0031] In this invention, the abalone visceral peptide specifically includes one or more of the following protein peptides: (1) protein peptide 1 with the amino acid sequence GFAGDDAPR; (2) protein peptide 2 with the amino acid sequence GFAGDDAPRA; (3) protein peptide 3 with the amino acid sequence FAGDDAPRA; (4) protein peptide 4 with the amino acid sequence AGDDAPR; (5) protein peptide 5 with the amino acid sequence GINPEDVFHAD; (6) protein peptide 6 with the amino acid sequence PNIVFDHY; (7) protein peptide 7 with the amino acid sequence SPDILPQP; (8) Protein peptide 8 with amino acid sequence SVDHGDYY; (9) Protein peptide 9 with amino acid sequence IANDPDSSF; (10) Protein peptide 10 with amino acid sequence LPIPNLP; (11) Protein peptide 11 with amino acid sequence YEPGVGHHEDHE; (12) Protein peptide 12 with amino acid sequence YSAYDPVFM; (13) Protein peptide 13 with amino acid sequence KLTTPTYGDL; (14) Protein peptide 14 with amino acid sequence NEDEFTRVN; (15) Protein peptide 15 with amino acid sequence WDWTRP.

[0032] This invention provides the application of the above-mentioned abalone visceral peptides in feed production.

[0033] The feed provided by this invention includes the aforementioned abalone visceral peptides. More specifically, the feed specifically includes: abalone visceral peptides at a concentration preferably of 1wt% to 20wt%, such as 1wt%, 2wt%, 3.81wt%, 5wt%, 7.61wt%, 10wt%, 11.42wt%, 15.23wt%, 18wt%, 20wt%, or any value between them; fishmeal at a concentration preferably of 10wt% to 40wt%, such as 10wt%, 12.5wt%, 15wt%, 18wt%, 20wt%, 21wt%, 24wt%, 27wt%, 30wt%, or any value between them; and soybean meal at a concentration preferably of 20wt% to 30wt%, such as 20wt%, 23wt%, 25wt%, 26wt%. The concentration is preferably 10wt% to 20wt% of chicken meal, such as 10wt%, 12wt%, 13wt%, 15wt%, 18wt%, 20wt%, or any value between them; the concentration is preferably 10wt% to 30wt% of flour, such as 10wt%, 12wt%, 15wt%, 18.27wt%, 19.08wt%, 19.89wt%, 20.69wt%, 21.5wt%, 25wt%, 30wt%, or any value between them; the concentration is preferably 1wt% to 5wt% of fish oil, such as 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%. The preferred concentrations are: 1 wt%, 4 wt%, 5 wt%, or any value between them; soybean oil, such as 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 4 wt%, 5 wt%, or any value between them; lecithin, such as 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 4 wt%, 5 wt%, or any value between them; calcium dihydrogen carbonate, such as 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 4 wt%, 5 wt%, or any value between them; and calcium dihydrogen carbonate, such as 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 4 wt%, 5 wt%, or any value between them; and calcium dihydrogen carbonate, such as 0.05 wt% to 0.2 wt%. Vitamin C at a concentration of t%, such as 0.05wt%, 0.08wt%, 0.1wt%, 0.15wt%, 0.2wt%, or any value thereto; trace elements at a concentration preferably of 0.5wt% to 2wt%, such as 0.5wt%, 0.8wt%, 1wt%, 1.5wt%, 2wt%, or any value thereto; choline chloride at a concentration preferably of 0.1wt% to 0.5wt%, such as 0.1wt%, 0.2wt%, 0.3wt%, 0.5wt%, or any value thereto; and yttrium trioxide at a concentration preferably of 0.05wt% to 0.1wt%, such as 0.05wt%, 0.08wt%, 0.1wt%, or any value thereto.

[0034] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0035] The abalone viscera used in the following examples and comparative examples are from the same manufacturer and the same batch, that is, the abalone viscera used in each example and comparative example are the same, and are provided by Xiamen Qicheng Marine Technology Co., Ltd. Alkaline protease (Yuanye Biotechnology Co., Ltd., product number S10154); Neutral protease (Yuanye Biotechnology Co., Ltd., product number S10013); Papain (Yuanye Biotechnology Co., Ltd., product number S10011); Flavor protease (Yuanye Biotechnology Co., Ltd., product number S10153); Trypsin (Yuanye Biotechnology Co., Ltd., product number S10034).

[0036] Example 1 This embodiment illustrates a method for preparing abalone visceral peptides, specifically including: S1. Take the cleaned abalone viscera and remove the contents, add it to a high-speed tissue homogenizer, and homogenize it at 18000 rpm for 1 min to obtain abalone viscera paste.

[0037] S2. Mix abalone viscera slurry with distilled water at a mass ratio of 1:1. Add mixed protease agent-1 (including alkaline protease and neutral protease at a mass ratio of 1:1) at a ratio of 7882U:5g to 5g. Perform enzymatic hydrolysis at 47.6℃ and pH=8 for 3.3h to obtain the hydrolysate.

[0038] S3. Take the enzymatic hydrolysate and inactivate it in a boiling water bath for 10 min, cool it to room temperature, and freeze-dry it at -35℃ for 72 h to obtain abalone visceral peptides.

[0039] Comparative Example 1 This comparative example uses the method provided in Example 1 to prepare abalone visceral peptides, except that in S2, an equal amount of papain is added instead of mixed protease agent-1, while other conditions remain the same, to obtain abalone visceral peptides.

[0040] Comparative Example 2 This comparative example uses the method provided in Example 1 to prepare abalone visceral peptides, except that in S2, an equal amount of flavor protease is added instead of mixed protease agent-1, while other conditions remain the same, to obtain abalone visceral peptides.

[0041] Comparative Example 3 This comparative example uses the method provided in Example 1 to prepare abalone visceral peptides, except that in S2, an equal amount of neutral protease is added instead of mixed protease agent-1, while other conditions remain the same, to obtain abalone visceral peptides.

[0042] Comparative Example 4 This comparative example uses the method provided in Example 1 to prepare abalone visceral peptides, except that in S2, an equal amount of alkaline protease is added instead of mixed protease agent-1, while other conditions remain the same, to obtain abalone visceral peptides.

[0043] Comparative Example 5 This comparative example uses the method provided in Example 1 to prepare abalone visceral peptides, except that in S2, an equal amount of trypsin is added instead of mixed protease agent-1, while other conditions remain the same, to obtain abalone visceral peptides.

[0044] Comparative Example 6 This comparative example uses the method provided in Example 1 to prepare abalone visceral peptides, except that in S2, mixed protease agent-2 (including alkaline protease and flavor protease in a mass ratio of 1:1) is added in equal amounts instead of mixed protease agent-1, and other conditions are the same, to obtain abalone visceral peptides.

[0045] Comparative Example 7 This comparative example uses the method provided in Example 1 to prepare abalone visceral peptides, except that in S2, mixed protease agent-3 (including alkaline protease and trypsin in a 1:1 mass ratio) is added in equal amounts instead of mixed protease agent-1, and other conditions are the same, to obtain abalone visceral peptides.

[0046] Comparative Example 8 This comparative example uses the method provided in Example 1 to prepare abalone visceral peptides. The difference is that in S2, mixed protease agent-4 (including alkaline protease and papain in a 1:1 mass ratio) is added in equal amounts instead of mixed protease agent-1, and other conditions are the same, to obtain abalone visceral peptides.

[0047] Comparative Example 9 This comparative example uses the method provided in Example 1 to prepare abalone visceral peptides, except that in S2, mixed protease agent-5 (including neutral protease and flavor protease in a mass ratio of 1:1) is added in equal amounts instead of mixed protease agent-1, and other conditions are the same, to obtain abalone visceral peptides.

[0048] Comparative Example 10 This comparative example uses the method provided in Example 1 to prepare abalone visceral peptides, except that in S2, mixed protease agent-1 is replaced with an equal amount of mixed protease agent-6 (including neutral protease and trypsin in a 1:1 mass ratio), and other conditions are the same, to obtain abalone visceral peptides.

[0049] Comparative Example 11 This comparative example uses the method provided in Example 1 to prepare abalone visceral peptides. The difference is that in S2, mixed protease agent-7 (including neutral protease and papain in a 1:1 mass ratio) is added in equal amounts instead of mixed protease agent-1, and other conditions are the same, to obtain abalone visceral peptides.

[0050] Examples 2 and 3 Examples 2 and 3 use the method provided in Example 1 to prepare abalone visceral peptides. The difference is that in S2, the mass ratio of alkaline protease and neutral protease in mixed protease agent-1 is different, as shown in Table 1. Other conditions are the same, and abalone visceral peptides are obtained.

[0051] Comparative Examples 12 and 13 Comparative Examples 12 and 13 were prepared using the method provided in Example 1 to obtain abalone visceral peptides. The difference was that in S2, the mass ratio of alkaline protease and neutral protease in mixed protease agent-1 was different, as shown in Table 1. Other conditions were the same, and abalone visceral peptides were obtained.

[0052] Examples 4-6 Examples 4-6 use the method provided in Example 1 to prepare abalone visceral peptides. The difference is that the amount of mixed protease agent-1 added in S2 is different, as shown in Table 1. Other conditions are the same, and abalone visceral peptides are obtained.

[0053] Comparative Examples 14 and 15 Comparative Examples 14 and 15 were prepared using the method provided in Example 1 to obtain abalone visceral peptides. The difference was that the amount of mixed protease agent-1 added in S2 was different, as shown in Table 1. Other conditions were the same, and abalone visceral peptides were obtained.

[0054] Examples 7-9 Examples 7-9 use the method provided in Example 1 to prepare abalone visceral peptides. The difference is that the temperature used in the enzymatic hydrolysis in S2 is different, as shown in Table 1. Other conditions are the same, and abalone visceral peptides are obtained.

[0055] Comparative Examples 16-18 Comparative Examples 16-18 were prepared using the method provided in Example 1 to obtain abalone visceral peptides. The difference was that the temperature used in the enzymatic hydrolysis in S2 was different, as shown in Table 1. Other conditions were the same, and abalone visceral peptides were obtained.

[0056] Examples 10-13 Examples 10-13 use the method provided in Example 1 to prepare abalone visceral peptides. The difference is that the enzymatic hydrolysis time in S2 is different, as shown in Table 1. Other conditions are the same, and abalone visceral peptides are obtained.

[0057] Comparative Example 19 Comparative Example 19 prepared abalone visceral peptides using the method provided in Example 1, except that the enzymatic hydrolysis time in S2 was different, as shown in Table 1. Other conditions were the same, and abalone visceral peptides were obtained.

[0058] Examples 14-17 Examples 14-17 use the method provided in Example 1 to prepare abalone visceral peptides. The difference is that the pH value of the enzymatic hydrolysis treatment in S2 is different, as shown in Table 1. Other conditions are the same, and abalone visceral peptides are obtained.

[0059] Comparative Example 20 Comparative Example 20 prepared abalone visceral peptides using the method provided in Example 1, except that the pH value of the enzymatic hydrolysis treatment in S2 was different, as shown in Table 1. Other conditions were the same, and abalone visceral peptides were obtained.

[0060] Table 1.

[0061] Test Example 1 This test example illustrates the preparation effect of the abalone visceral peptide preparation methods provided in the above embodiments and comparative examples. The test specifically includes: 1. Polypeptide concentration of abalone viscera peptides In both the examples and comparative examples, the enzymatic hydrolysate obtained in step S3 was inactivated and cooled to room temperature. Then, at a 1:1 volume ratio, the hydrolysate and 10% TCA solution were mixed thoroughly and allowed to stand for 10 min. The mixture was then centrifuged at 4000 rpm for 15 min. The supernatant was collected and diluted 10-fold with 5% TCA solution. The diluted supernatant was then mixed with biuret reagent at a 3:2 volume ratio and reacted for 10 min. The mixture was centrifuged at 2000 rpm for 10 min. The absorbance of the supernatant was measured at 540 nm. The peptide concentration was calculated using a standard curve (prepared with GGYR tetrapeptide as the standard reagent). The specific details are as follows: Figures 1-7 As shown.

[0062] Depend on Figures 1-7The results shown indicate that, as can be seen from the comparison of the results in Comparative Examples 1 to 5, alkaline protease and neutral protease have excellent enzymatic hydrolysis effects, indicating that abalone viscera are rich in peptide bonds that are sensitive to alkaline protease or neutral protease, which can effectively achieve the hydrolysis of abalone viscera.

[0063] As can be seen from the comparison between Examples 1-3 and Comparative Examples 12 and 13, when the mass ratio of alkaline protease to neutral protease is (1-2):(1-2), the alkaline protease and neutral protease in the enzymatic hydrolysis system can better cooperate to act on different peptide bond sites, resulting in a better hydrolysis effect.

[0064] As can be seen from the comparison between Examples 1 and 4-6 and Comparative Examples 14 and 15, the hydrolysis effect is excellent in the range of 5000U / 5g to 10000U / 5g, and the peptide concentration in the obtained abalone viscera peptide is high; however, when the amount of enzyme added is 12500U / 5g, the amount of enzyme added is too high, which causes excessive hydrolysis of protein and produces free amino acids.

[0065] As can be seen from the comparison between Examples 1 and 7-9 and Comparative Examples 16-18, the preferred temperature range for enzymatic hydrolysis of abalone viscera using a complex enzyme system including alkaline protease and neutral protease is 40℃~60℃.

[0066] As can be seen from the comparison of Examples 1 and 10-13 with Comparative Example 19, when the enzymatic hydrolysis time is no more than 4 hours, the peptide concentration increases with time. This is because in the early stage of the reaction, there is sufficient protein in the abalone viscera, and the protease has more opportunities to bind with the abalone viscera protein, thus increasing the reaction rate. When the enzymatic hydrolysis time is longer than 4 hours, most of the abalone viscera protein is hydrolyzed, the enzymatic hydrolysis reaction reaches a steady state, and extending the reaction time has no significant effect on the peptide concentration.

[0067] As can be seen from the comparison between Examples 1 and 14-17 and Comparative Example 20, the preferred pH range for enzymatic hydrolysis of abalone viscera using a complex enzyme system including alkaline protease and neutral protease is 6-10.

[0068] 2. Polypeptide composition of abalone viscera peptides (1) The preparation method provided in Example 1 was used to prepare abalone viscera in parallel to obtain abalone viscera peptides. The abalone viscera peptides were dissolved in distilled water to obtain a test solution with a protein concentration of 1 mg / mL.

[0069] (2) The test solution was analyzed by LC-MS / MS to obtain the polypeptide composition of each abalone visceral peptide, as shown in Table 2. The LC-MS / MS analysis conditions included: reversed-phase column; aqueous phase was 0.1% formic acid aqueous solution, and organic phase was a mixture of formic acid, acetonitrile and water with a volume ratio of 0.1:80:19.9; flow rate was 300 mL / min; and analysis time was 60 min.

[0070] The mass spectrometry intensity value is calculated using the maxquant software based on the data analyzed by LC-MS / MS. The higher the value, the higher the abundance of protein peptides in the sample.

[0071] Table 2.

[0072] As shown in Table 2, the types of protein peptides obtained by enzymatic hydrolysis of different batches of abalone viscera are basically the same, and the mass spectrometry intensity values ​​of the 15 protein peptides are similar, indicating that the process has excellent process stability and has good application prospects in realizing the large-scale industrial production of abalone viscera peptides.

[0073] Examples 18-21 Examples 18-21 illustrate the preparation of a sea bass feed, specifically including: taking the abalone viscera peptides, fish meal, soybean meal, chicken meal, flour, fish oil, soybean oil, lecithin, calcium dihydrogen carbonate, vitamin C, trace element premix (Guangdong Haiyinte Biotechnology Group Co., Ltd., product number A601), choline chloride and yttrium trioxide provided in Example 1 according to the addition amounts shown in Table 3, and mixing them to obtain the sea bass feed.

[0074] Comparative Example 21 Comparative Example 21 prepared sea bass feed using the method provided in Example 18, except that an equal amount of fishmeal was added instead of the abalone visceral peptides provided in Example 1, while other conditions remained the same, and sea bass feed was obtained.

[0075] Table 3.

[0076] Test Example 2 This test example illustrates the relevant performance of the sea bass feed provided in the above embodiments and comparative examples. The tests specifically include: 1. Nutritional level of sea bass feed The nutritional levels in sea bass feed were tested according to the methods provided in GB / T 6432-2018 Determination of Crude Protein in Feed - Kjeldahl Method and GB / T 6433-2025 Determination of Crude Fat in Feed. The results are shown in Table 4.

[0077] Table 4.

[0078] 2. Feeding effect of sea bass feed (1) 225 healthy and uniformly sized juvenile sea bass (141.22±0.38g) were randomly assigned to 15 culture tanks with a room temperature control system, with 15 fish in each tank. The 15 tanks were randomly divided into 5 treatments (3 replicates per treatment) and the following treatments were performed: i. Feed the sea bass feed provided in Example 18 twice a day (6:30 and 17:30), feeding each time until the fish no longer have a strong appetite and there is no uneaten food. Continue to raise the fish for 8 weeks. The specific raising conditions include: 12 hours of light / 12 hours of darkness, water temperature of 27.6℃~28.4℃, and pH of 8.0. ii. Feed the sea bass food provided in Example 19 twice a day (6:30 and 17:30), each time until the fish no longer have a strong appetite and there is no uneaten food. Continue to raise the fish for 8 weeks. The specific conditions for raising the fish include: 12 hours of light / 12 hours of darkness, water temperature of 27.6℃~28.4℃, and pH of 8.0. iii. Feed the sea bass food provided in Example 20 twice a day (6:30 and 17:30), each time until the fish no longer have a strong appetite and there is no uneaten food. Continue to raise the fish for 8 weeks. The specific conditions for raising the fish include: 12 hours of light / 12 hours of darkness, water temperature of 27.6℃~28.4℃, and pH of 8.0. iv. Feed the sea bass food provided in Example 21 twice a day (6:30 and 17:30), each time until the fish no longer have a strong appetite and there is no uneaten food. Continue to raise the fish for 8 weeks. The specific conditions for raising the fish include: 12 hours of light / 12 hours of darkness, water temperature of 27.6℃~28.4℃, and pH of 8.0. v. Feed the sea bass food provided in Comparison 21 twice a day (6:30 and 17:30), feeding until the fish no longer have a strong appetite and there is no uneaten food. Continue this feeding for 8 weeks. The specific feeding conditions include: 12 hours of light / 12 hours of darkness, water temperature of 27.6℃~28.4℃, and pH of 8.0.

[0079] (2) Record the amount of sea bass feed given each day and calculate the total amount of sea bass feed given during the 8-week continuous feeding period, as shown in Table 5.

[0080] Table 5

[0081] As shown in Table 5, compared with Comparative Example 21, the amount of food consumed (reflected by the amount of food fed during satiated feeding) was significantly increased when the sea bass feed provided in Example 20 was used. This indicates that the sea bass feed with a fishmeal replacement rate of 30% can improve the appetite of juvenile sea bass and has a certain feeding-enhancing effect.

[0082] (3) After the feeding period, all experimental fish were starved for 24 hours to empty their gastrointestinal contents. Fifteen fish were randomly selected from each bucket, anesthetized with eugenol, weighed, and their body length was measured. The weight gain rate (in %), specific growth rate (in % / day), and feed conversion ratio were calculated according to the following formulas. The results are as follows: Figures 8-10 As shown.

[0083] Weight gain rate = (W1 - W0) / W0 × 100%; Specific growth rate = 100 × (lnW1 - lnW0) / t; Feed conversion ratio = W D / (W1-W0) In the above formula, W1 is the final weight of the fish, in grams; W0 is the initial weight of the fish, in grams; W D This refers to the amount of feed given, expressed in grams.

[0084] Depend on Figures 8-10 The results show that when sea bass were fed with the sea bass feed provided in Examples 18-21 of this invention, the weight gain rate and specific growth rate of sea bass were not significantly different from those in Comparative Example 21, indicating that the abalone visceral peptides provided by this invention can be used to replace fish meal in feed, and the fish meal replacement rate can reach more than 40%.

[0085] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A method for preparing abalone visceral peptide, characterized in that, The preparation method comprises: subjecting abalone viscera to enzymatic hydrolysis treatment by using alkaline protease and neutral protease in a mass ratio of (1-2):(1-2), so as to obtain the abalone viscera peptide. In the enzymatic hydrolysis treatment, the total enzyme addition amount of the alkaline protease and the neutral protease is (5000-10000) U:5 g with respect to the addition amount of the abalone viscera slurry; the temperature is 40-60 DEG C, the pH value is 6-10, and the time is not less than 1 h.

2. The method of claim 1, wherein the abalone visceral peptide is prepared by the steps of: (a) extracting abalone visceral peptide from abalone visceral tissues; (b) purifying the abalone visceral peptide; and (c) drying the abalone visceral peptide. The abalone viscera is subjected to homogenization treatment, and the homogenization treatment is performed at a speed of 10 000-20 000 rpm for 30-120 s.

3. The method for preparing abalone visceral peptides according to claim 1, characterized in that, In the enzymatic hydrolysis treatment, the abalone viscera is added at a concentration of 10-80 wt%.

4. The method for preparing abalone visceral peptides according to claim 1, characterized in that, The enzymatic hydrolysis treatment is performed at a temperature of 45-50 DEG C, a pH value of 7-9, and a time of 2-4 h.

5. The method for preparing abalone visceral peptides according to claim 1, characterized in that, The preparation method comprises: subjecting the abalone viscera peptide obtained through the enzymatic hydrolysis treatment to inactivation treatment and / or freeze-drying treatment. In the inactivation treatment, the temperature is 95-105 DEG C, and the time is 5-20 min; in the freeze-drying treatment, the temperature is -20 DEG C to -40 DEG C, and the time is 48-96 h.

6. A haliotis viscera peptide, characterized by, The abalone viscera peptide is prepared by the preparation method of the abalone viscera peptide according to any one of claims 1-5.

7. The abalone visceral peptide according to claim 6, characterized in that, The abalone viscera peptide comprises one or more of the following protein peptides: (1) a protein peptide 1 with an amino acid sequence GFAGDDAPR; (2) a protein peptide 2 with an amino acid sequence GFAGDDAPRA; (3) a protein peptide 3 with an amino acid sequence FAGDDAPRA; (4) a protein peptide 4 with an amino acid sequence AGDDAPR; (5) a protein peptide 5 with an amino acid sequence GINPEDVFHAD; (6) a protein peptide 6 with an amino acid sequence PNIVFDHY; (7) a protein peptide 7 with an amino acid sequence SPDILPQP; (8) a protein peptide 8 with an amino acid sequence SVDHGDYY; (9) a protein peptide 9 with an amino acid sequence IANDPDSSF; (10) a protein peptide 10 with an amino acid sequence LPIPNLP; (11) a protein peptide 11 with an amino acid sequence YEPGVGHHEDHE; (12) a protein peptide 12 with an amino acid sequence YSAYDPVFM; (13) a protein peptide 13 with an amino acid sequence KLTTPTYGDL; (14) a protein peptide 14 with an amino acid sequence NEDEFTRVN; and (15) a protein peptide 15 with an amino acid sequence WDWTRP.

8. The abalone viscera peptide according to claim 6 or 7 is used in the production of feed.

9. A feed, characterized in that, The feed comprises the abalone viscera peptide according to claim 6 or 7.

10. The feed according to claim 9, characterized in that, The feed comprises: 1wt%-20wt% of abalone viscera peptide, 10wt%-40wt% of fish meal, 20wt%-30wt% of soybean meal, 10wt%-20wt% of chicken meal, 10wt%-30wt% of flour, 1wt%-5wt% of fish oil, 1wt%-5wt% of soybean oil, 1wt%-5wt% of lecithin, 1wt%-5wt% of calcium bicarbonate, 0.05wt%-0.2wt% of vitamin C, 0.5wt%-2wt% of trace elements, 0.1wt%-0.5wt% of choline chloride and 0.05wt%-0.1wt% of yttrium trioxide.

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