Preparation method and application of feed additive for improving growth performance and intestinal health of larimichthys crocea juveniles

Abstract

The invention discloses a preparation method of a feed additive for improving growth performance and intestinal health of larimichthys crocea juveniles, which comprises the following steps: taking dephenolized cottonseed protein with the protein content of 70% as a raw material, and sequentially carrying out step-by-step enzymolysis by using alkaline protease and flavourzyme to obtain the feed additive taking cottonseed peptide as a main component. The obtained feed additive is a functional feed additive capable of improving the digestive enzyme activity of the larimichthys crocea juveniles, enhancing the oxidation resistance and promoting intestinal development, and can effectively improve the growth performance and intestinal health of the larimichthys crocea juveniles.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of aquatic animal nutrition feed and feed additives, and particularly relates to a preparation method and application of a feed additive for improving the growth performance and intestinal health of large yellow croaker larvae. BACKGROUND

[0002] In aquaculture production, the larval stage is the most critical and vulnerable stage in the breeding cycle, and the growth and development status of this stage directly affects the survival rate, growth potential and subsequent economic benefits of the individual. Taking large yellow croaker, an important marine fish in China, as an example, although the breakthrough in artificial breeding technology has laid the foundation for large-scale breeding, the high mortality problem caused by the incomplete development of the digestive system of larvae still seriously restricts the sustainable development of the industry. On the other hand, traditional artificial micro-particle feed is difficult to meet the special nutritional needs of larvae. Complete protein molecules are difficult to be effectively broken down by the incomplete digestive system, and simply supplementing free amino acids has problems such as asynchronous absorption and low utilization efficiency. These problems directly lead to poor appetite, slow growth, and low survival rate of large yellow croaker larvae.

[0003] Therefore, in view of the physiological characteristics of large yellow croaker larvae, the development of functional feed additives that promote intestinal development and improve digestive enzyme activity is not only of important guiding significance for improving the success rate and quality of seed cultivation, but also is the key to promoting the healthy and sustainable development of large yellow croaker aquaculture. Studies have shown that cottonseed peptides, as high-quality plant protein hydrolysates, have multiple nutritional advantages: they are rich in low molecular weight oligopeptides, which can bypass the incomplete gastric digestion stage of larvae and be efficiently absorbed through intestinal peptide transporter proteins (PepT1); at the same time, they contain rich flavor amino acids such as glutamic acid and aspartic acid, which can significantly improve feed palatability and improve the feeding performance of larvae. Adding cottonseed peptides to feed can not only effectively solve the problem of digestive and absorptive disorders in larvae, but also reduce the dependence on expensive protein sources such as fish meal, providing an economic and efficient nutritional solution for industry development. SUMMARY

[0004] One of the purposes of the present application is to provide a preparation method of a feed additive for improving the growth performance and intestinal health of large yellow croaker larvae. The obtained feed additive is a functional feed additive with the functions of improving the digestive enzyme activity of large yellow croaker larvae, enhancing the antioxidant capacity and promoting the development of the intestinal tract, and can effectively improve the growth performance and intestinal health of large yellow croaker larvae.

[0005] The present application provides a preparation method of a feed additive for improving the growth performance and intestinal health of large yellow croaker larvae, which uses dephenolized cottonseed protein with a protein content of 70% as raw material, and sequentially uses alkaline protease and flavor protease for step-by-step enzymolysis to obtain a feed additive with cottonseed peptides as the main component.

[0006] The step-by-step enzymatic hydrolysis is: first, using alkaline protease to hydrolyze for 1-3 hours at pH 9.0-10.5 and temperature 50-60 DEG C; after inactivation of the enzyme, adjusting the pH to 6.5-7.5, adding flavor protease, and continuing the enzymatic hydrolysis for 1-2 hours at temperature 50-60 DEG C.

[0007] The addition amount of the alkaline protease is 6000-8000 U / g of protein substrate; and the addition amount of the flavor protease is 500-700 U / g of protein substrate.

[0008] Preferably, the addition amount of the alkaline protease is 7000 U / g of protein substrate, and the addition amount of the flavor protease is 600 U / g of protein substrate.

[0009] In one preferred embodiment, the preparation method of the feed additive comprises the following steps: S1: preparing the dephenolated cottonseed protein into an aqueous solution, and adjusting the pH to 10.0 by using NaOH solution; S2: adding alkaline protease in an amount of 7000 U / g of protein, and stirring and reacting at constant temperature of 55 DEG C for 2 hours, during which the pH is kept constant by using pH-stat method; S3: inactivating the enzyme of the enzymatic hydrolysate of step S2 by heating at 90 DEG C for 10 min; S4: after the temperature of the enzymatic hydrolysate is reduced to 55 DEG C, adjusting the pH to 7.0 by using HCl solution; S5: adding flavor protease in an amount of 600 U / g of protein, and hydrolyzing at 55 DEG C for 1 hour; S6: inactivating the enzyme of the enzymatic hydrolysate of step S5 by heating at 95 DEG C for 10 min; S7: centrifuging (8000 r / min, 30 min, 4 DEG C) the inactivated enzymatic hydrolysate, and collecting the supernatant; S8: freeze-drying the supernatant to obtain the feed additive.

[0010] The second object of the present application is to provide a feed additive for improving the growth performance and intestinal health of large yellow croaker juveniles.

[0011] The feed additive for improving the growth performance and intestinal health of large yellow croaker juveniles is prepared by the above method.

[0012] The third object of the present application is to provide an aquatic feed for improving the growth performance and intestinal health of large yellow croaker juveniles.

[0013] Specifically, the aquatic feed for improving the growth performance and intestinal health of large yellow croaker juveniles comprises a basic feed and the feed additive prepared by the above method.

[0014] Preferably, the mass of the cottonseed peptides in the feed additive is 1-2 g / kg of the aquaculture feed addition level.

[0015] More preferably, the mass of the cottonseed peptides in the feed additive is 2 g / kg of the aquaculture feed addition level.

[0016] Beneficial effects The cottonseed peptides prepared by the present application are rich in various functional amino acids, and the content is significantly improved compared with the unenzymatic raw materials. Especially, the flavor amino acids (such as glutamic acid, aspartic acid, alanine) and the limiting amino acids (such as methionine, lysine, threonine) have a significant improvement range, especially the content of the methionine is improved by more than 80%.

[0017] The present application verifies through in vivo experiments that the addition of cottonseed peptides in feed can improve the digestive enzyme activity of large yellow croaker juveniles, enhance the antioxidant capacity and promote the development of the intestinal tract, improve the intestinal health, and thus improve the growth performance.

[0018] In addition, the research also found that the cottonseed peptides of the present application can also significantly optimize the intestinal flora structure of large yellow croaker juveniles, significantly improve the abundance of beneficial bacteria (such as Alphaproteobacteria, Ruegeria), and inhibit the abundance of conditional pathogenic bacteria (such as Gammaproteobacteria, Pseudomonas), thereby further promoting the intestinal health and growth performance through the "bacteria-intestine-health" axis. This effect is unexpected and embodies the innovation of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a response surface analysis graph of the mutual significant items of the hydrolysis parameters in Example 1 on the degree of hydrolysis of cottonseed protein.

[0020] Figure 2 It is a result graph of the influence of cottonseed peptides on the final body weight and specific growth rate of large yellow croaker juveniles.

[0021] Figure 3 It is a result graph of the influence of cottonseed peptides on the trypsin and amylase in the intestinal tract digestive and absorptive function of large yellow croaker juveniles.

[0022] Figure 4 It is a result graph of the influence of cottonseed peptides on the total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content of the intestinal tract of large yellow croaker juveniles.

[0023] Figure 5 It is a result graph of the influence of cottonseed peptides on the peptide transport carrier related to the intestinal tract digestive and absorptive function of large yellow croaker juveniles PepT1b and PepT2Figure of the results of the influence of the genes.

[0024] Figure 6 Figure of the results of the influence of cottonseed peptides on the relative abundance of the intestinal flora of juvenile large yellow croaker at the class and genus levels. ZO-1 and ZO-2 Influence of the genes Figure 7 Figure of the results of the influence of cottonseed peptides on the relative abundance of the intestinal flora of juvenile large yellow croaker at the class and genus levels. DETAILED DESCRIPTION

[0025] Example 1: Optimization of cottonseed protein enzymolysis conditions With de-phytenic cottonseed protein as raw material, the fixed hydrolysis time was 120 min, the alkaline protease addition amount (A), the hydrolysis temperature (B) and the pH value (C) were used as independent variables (condition variables), and the degree of hydrolysis (DH) was used as the response value. The Box-Behnken experimental design of three factors and three levels was used to optimize the alkaline protease hydrolysis process. The degree of hydrolysis was calculated by the pH-stat method. The factor and level design is shown in Table 1, and the response surface experimental scheme and results are shown in Table 2.

[0026] Table 1 Factors and levels of response surface experiment

[0027] Table 2 Response surface experiment scheme and results

[0028] The data in Table 2 were subjected to multiple regression fitting by using Design-Expert software, and a quadratic regression model of the degree of hydrolysis (Y) to the coded values of each factor was obtained: Y = 29.48 + 2.12A + 0.7738B - 0.1675C - 0.0275AB + 0.415AC + 0.035BC - 2.50A² - 2.65B² - 2.77C² Wherein, Y is the degree of hydrolysis (%), A, B and C are the coded variables of enzyme addition amount, hydrolysis temperature and pH, respectively.

[0029] Variance analysis was performed on the regression model to test its effectiveness, and the results are shown in Table 3. The analysis showed that the model was extremely significant (P < 0.0001), and the lack-of-fit term was not significant (P = 0.7857 > 0.05). The determination coefficient of the model R² = 0.9967, and the adjusted determination coefficient R²Adj = 0.9924, indicating that the model has good fitting degree and can be used for accurate prediction and analysis of the hydrolysis process.

[0030] Table 3 Variance analysis of the regression model

[0031] Note: * means significant difference (P<0.05); ** means extremely significant difference (P<0.01).

[0032] Response surface analysis of the effect of significant interaction of hydrolysis parameters on the degree of hydrolysis of cottonseed protein is shown in FIG. 1. Figure 1

[0033] According to the Box-Behnken experiment and regression model analysis, the alkaline protease hydrolysis process was optimized, and the optimal process parameters were determined as follows: the amount of alkaline protease added was 7000 U / g, the hydrolysis temperature was 55°C, and the pH was 10.0, and the maximum degree of hydrolysis was 29.87%.

[0034] This example is a single-enzyme method for preparation, and the subsequent double-enzyme method for preparation uses this optimized condition.

[0035] Example 2: Double-enzyme method for preparing cottonseed peptides and comparison with single-enzyme method S1: The dephenolized cottonseed protein was prepared into an aqueous solution, and the pH was adjusted to 10.0 with a NaOH solution; S2: The alkaline protease was added in an amount of 7000 U / g of protein, and the reaction was stirred at 55°C for 2 hours, during which the pH was maintained constant by the pH-stat method, to obtain an enzyme hydrolysate; S3: The enzyme hydrolysate of step S2 was heated at 90°C for 10 min to inactivate the enzyme; S4: After the temperature of the enzyme hydrolysate was reduced to 55°C, the pH was adjusted to 7.0 with an HC1 solution; S5: The flavor protease was added in an amount of 600 U / g of protein, and the enzyme hydrolysis was performed at 55°C for 1 hour; S6: The enzyme hydrolysate of step S5 was heated at 95°C for 10 min to inactivate the enzyme; S7: The enzyme hydrolysate after inactivation was centrifuged (8000 r / min, 30 min, 4°C) to collect the supernatant; S8: The supernatant was freeze-dried to obtain a feed additive with cottonseed peptides as the main component.

[0036] In addition, the unhydrolyzed cottonseed protein was used as a control, and the test was performed under the same test method.

[0037] The crude protein content of the sample was determined by the Kjeldahl method according to the national standard GB / T6432 2018.

[0038] The amino acid composition of the sample was determined by drying in a freeze dryer for 48 h to a constant weight, digesting in a 6 mol / L HC1 solution at 110°C for 22 h, and finally automatically analyzing by a full-automatic amino acid analyzer (L-8900, HITACHI, Japan). ​

[0039] From Table 4, compared with the non-enzymatic cottonseed protein and the single-enzyme process, the cottonseed peptide prepared by the double-enzyme process has a significant increase in the contents of crude protein, small peptides, and key flavor amino acids (aspartic acid and glutamic acid) and limiting amino acids (methionine and lysine). This indicates that the double-enzyme process can more efficiently and comprehensively release the nutritional components of cottonseed protein, and the product quality is significantly better than that of the traditional single-enzyme process.

[0040] Table 4 Comparison of product ingredients of cottonseed peptides prepared by different processes (%)

[0041] Note: The data in the table is the mass percentage (%) of each amino acid in the total sample; the column of "increase ratio compared with the non-enzymatic group" is the calculation result of the data of the "double-enzyme method" compared with the data of the "non-enzymatic cottonseed protein"; "-" indicates no relevant data.

[0042] Example 3 Preparation of feed Based on the results of Example 2, the cottonseed peptide prepared by the double-enzyme method was selected for feed preparation.

[0043] The basic feed for large yellow croaker juveniles used contains the following components (in g / kg, see Table 5): white fish meal 450.0, krill meal 220.0, squid viscera meal 30.0, yeast extract 35.0, high-gluten flour 48.0, a-starch 30.0, fish oil 65.0, soy lecithin 50.0, sodium alginate 20.0, complex vitamin 15.0, complex mineral 10.0, ascorbic acid 2.0, calcium dihydrogen phosphate 20.0, mold inhibitor 0.5, antioxidant 0.5, and choline chloride 2.0.

[0044] To the above basic feed, 0 g / kg (CP0), 0.5 g / kg (CP0.5), 1 g / kg (CP1), and 2 g / kg (CP2) of cottonseed peptide were added respectively to prepare four experimental feeds with equal nitrogen and equal fat.

[0045] Table 5 Formulation of feeds in different groups (g / kg)

[0046] 1 Complex vitamin (mg / kg): retinyl acetate, 32; α tocopherol, 240; menadione, 10; vitamin B1, 25; pyridoxine hydrochloride, 20; vitamin B12, 10; riboflavin, 45; pantothenic acid, 60; vitamin D3, 5; folic acid, 20; nicotinic acid, 200; biotin, 60; inositol, 800; microcrystalline cellulose, 13473.

[0047] 2 Multiple minerals: MgSO4·7H2O, 1200; FeSO4·H2O, 80; ZnSO4·H2O, 50; CuSO4·5H2O, 10; MnSO4·H2O, 45; CoCl2·6H2O, 50; Na2SeO3, 20; H2CaIO4, 60; zeolite powder, 13485.

[0048] Example 4: Application effect of cottonseed peptide in feed for juvenile large yellow croaker For the experiment, 42,000 juvenile large yellow croakers with an initial weight of 3.83 ± 0.10 mg were selected and randomly divided into 4 groups, with three replicates per group and 3,500 fish per replicate. A 30-day feeding and growth experiment was conducted. The water temperature was maintained at 23–26 ℃. The control group was fed a basal diet without cottonseed peptides (CP0). The experimental groups were fed a basal diet supplemented with 0.5 g / kg, 1 g / kg, and 2 g / kg cottonseed peptides, respectively (hereinafter referred to as "cottonseed peptide diet"). Feeding was conducted 6 times daily, always at full capacity, at 06:00, 09:00, 12:00, 16:00, 20:00, and 23:00. After 30 days of rearing, the fish were fasted for 24 hours, and then samples were taken. The remaining quantity and weight in each container were recorded, and the intestines of the large yellow croakers from each container were collected for analysis.

[0049] The experimental results show that: The final body weight of juvenile large yellow croaker fed with a diet supplemented with 1-2 g / kg cottonseed peptide was significantly higher than that of the control group and the group supplemented with 0.5 g / kg. P <0.05)( Figure 2 A). Furthermore, the specific growth rate of juvenile large yellow croaker fed with a diet supplemented with 1-2 g / kg cottonseed peptides was significantly higher than that of the control group (A). P <0.05)( Figure 2 b).

[0050] The intestinal trypsin activity of juvenile large yellow croaker fed with a diet supplemented with 2 g / kg cottonseed peptide was significantly higher than that of other groups. Furthermore, the intestinal lipase activity of juvenile large yellow croaker fed with a diet supplemented with 1-2 g / kg cottonseed peptide was significantly higher than that of the control group and the 0.5 g / kg group. P <0.05)( Figure 3 This indicates that cottonseed peptides can effectively improve the digestive capacity of juvenile fish for nutrients, thereby promoting intestinal health in juvenile large yellow croaker.

[0051] Large yellow croaker fry fed with 1-2 g / kg cottonseed peptides showed significantly higher total antioxidant capacity and superoxide dismutase activity in their intestines compared to the control group. P <0.05)( Figure 4A and B). Furthermore, the malondialdehyde (MDA) content in the intestines of juvenile large yellow croaker fed a diet supplemented with 1-2 g / kg cottonseed peptides was significantly lower than that in the control group (A and B). P <0.05) Figure 4 C) This indicates that cottonseed peptides can effectively increase the activity of intestinal antioxidant enzymes, alleviate damage caused by oxidative stress, and thus promote intestinal health in juvenile large yellow croaker.

[0052] Large yellow croaker fry fed with 2 g / kg cottonseed peptide feed contain intestinal peptide transporter protein. PepT1b The relative expression level of the gene was significantly higher than that of other groups. P <0.05)( Figure 5 A). In addition, juvenile large yellow croaker fed with a diet supplemented with 1-2 g / kg cottonseed peptides contain intestinal peptide transporters. PepT1b The relative expression level of the gene was significantly higher than that of the control group. P <0.05)( Figure 5 B) This indicates that cottonseed peptides can be directly and rapidly absorbed by the intestines.

[0053] Large yellow croaker fry fed with 1-2 g / kg cottonseed peptide feed have intestinal tight junction protein. ZO-1 and ZO-2 The relative gene expression level was significantly higher than that of the control group and the group with an added amount of 0.5 g / kg. P <0.05) Figure 6 This indicates that cottonseed peptides can effectively promote intestinal epithelial development, thereby promoting intestinal health in juvenile large yellow croaker.

[0054] Example 5: Effects of cottonseed peptides on the intestinal flora of juvenile large yellow croaker As described in Example 4, after the feeding experiment was completed, intestinal samples of juvenile large yellow croaker from the CP0 group (fed with basic feed) and the CP2 group (fed with feed supplemented with 2g / kg cottonseed peptide) were taken for 16S rRNA gene high-throughput sequencing analysis.

[0055] Depend on Figure 7 As shown in Tables A and 6, at the class level, after ingestion of cottonseed peptides, the relative abundance of α-Proteobacteria, which have potential beneficial functions, in the intestine of large yellow croaker significantly increased, while the relative abundance of γ-Proteobacteria, which contains various pathogens, significantly decreased. Figure 7 As shown in B and Table 7, at the genus level, the abundance of beneficial bacterial genera (such as Rugellellae) increased significantly, while the abundance of opportunistic pathogenic bacterial genera (such as Pseudomonas) was significantly suppressed. This change is consistent with the results at the class level, which further demonstrates the significant effect of the product of this invention in inhibiting pathogenic bacteria and promoting beneficial bacteria.

[0056] This corroborates the results observed in Example 4 regarding improved growth performance (weight gain rate, feed conversion ratio) and improved intestinal morphology, together elucidating the mechanism by which cottonseed peptides exert their effects through the "microbe-gut-health" axis: cottonseed peptides improve intestinal health by optimizing the intestinal flora, reducing competition and consumption by pathogens, and promoting nutrient absorption, thereby ultimately achieving the technical effect of promoting growth.

[0057] Table 6. Effects of cottonseed peptide supplementation in feed on the relative abundance of gut microbiota in juvenile large yellow croaker (%)

[0058] Table 7. Effects of cottonseed peptide supplementation in feed on the relative abundance of gut microbiota in juvenile large yellow croaker (%)

[0059] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a feed additive to improve the growth performance and intestinal health of juvenile large yellow croaker, characterized in that, Using phenol-free cottonseed protein with a protein content of 70% as raw material, the feed additive with cottonseed peptides as the main component was obtained by stepwise enzymatic hydrolysis using alkaline protease and flavor protease.

2. The method for preparing the feed additive for improving the growth performance and intestinal health of juvenile large yellow croaker according to claim 1, characterized in that, The stepwise enzymatic hydrolysis is as follows: first, alkaline protease is used for enzymatic hydrolysis for 1-3 hours at pH 9.0-10.5 and temperature 50-60℃; after enzyme inactivation, the pH is adjusted to 6.5-7.5, flavor protease is added, and enzymatic hydrolysis is continued for 1-2 hours at temperature 50-60℃.

3. The method for preparing the feed additive for improving the growth performance and intestinal health of juvenile large yellow croaker according to claim 1 or 2, characterized in that, The amount of alkaline protease added is 6000-8000 U / g protein substrate.

4. The method for preparing the feed additive for improving the growth performance and intestinal health of juvenile large yellow croaker according to claim 1, characterized in that, The alkaline protease was added at a rate of 7000 U / g protein substrate, and the flavor protease was added at a rate of 600 U / g protein substrate.

5. The method for preparing the feed additive for improving the growth performance and intestinal health of juvenile large yellow croaker according to claim 1, characterized in that, Includes the following steps: S1: Prepare an aqueous solution of dephenolized cottonseed protein and adjust the pH to 10.0 with NaOH solution; S2: Add alkaline protease at a protein concentration of 7000 U / g and stir at 55°C for 2 hours, maintaining a constant pH using the pH-stat method during the process; S3: Inactivate the enzyme by heating the enzymatic hydrolysate from step S2 at 90 °C for 10 min; S4: After the temperature of the enzymatic hydrolysate drops to 55 ℃, adjust the pH to 7.0; S5: Add flavor protease at a rate of 600 U / g protein and hydrolyze at 55 °C for 1 hour; S6: Inactivate the enzyme by heating the enzymatic hydrolysate from step S5 at 95 °C for 10 min; S7: Centrifuge the enzyme hydrolysate after enzyme inactivation and collect the supernatant; S8: Freeze-dry the supernatant to obtain the final product.

6. A feed additive that improves the growth performance and intestinal health of juvenile large yellow croaker, characterized by: It is prepared by the preparation method according to any one of claims 1-5.

7. An aquatic feed that improves the growth performance and intestinal health of juvenile large yellow croaker, characterized by: Includes basic feed and the feed additives as described in claim 6.

8. The aquatic feed according to claim 7, characterized in that, The mass of cottonseed peptide in the feed additive is 1-2 g / kg at the aquatic feed addition level.

9. The aquatic feed according to claim 7, characterized in that, The mass of cottonseed peptide in the feed additive is 2 g / kg at the aquatic feed addition level.

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