Method for effectively relieving soybean antigen protein Glycinin in fish feed and application

Abstract

The invention discloses a method for effectively relieving soybean antigen protein Glycinin in fish feed and application of the method. The invention discloses application of N-acetylcysteine as a functional feed additive capable of relieving carp enteritis induced by soybean antigen protein Glycinin. The feed disclosed by the invention comprises a soybean antigen protein Glycinin and N-acetylcysteine (NAC). According to the present invention, 1.5-6.0 g / kg of NAC is added to a feed containing 80 g / kg of Glycinin, such that the fish growth, the feed utilization rate, the digestion function, the protein metabolism function, the immune function, the oxidation resistance and the intestinal morphology can be significantly improved, and the soybean antigen protein Glycinin-induced growth inhibition and the intestinal injury can be effectively relieved; the functional fish feed additive provided by the invention is relatively low in cost and easy to popularize, and is beneficial to development of novel green antibiotic-replacing additives, improvement of the utilization rate of soybean protein sources, reduction of feed cost and promotion of healthy culture of fishes.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method and application of effectively alleviating soybean antigen protein Glycinin in fish feed, and application of N-acetylcysteine ​​as a functional feed additive capable of alleviating carp enteritis induced by soybean antigen protein Glycinin. Background Art

[0002] Soy protein sources have the characteristics of high digestibility and protein content, and have become one of the main plant protein sources for aquatic feed. However, excessive soybean meal significantly reduces the palatability and utilization of compound feed, and even causes serious intestinal diseases in fish. Current research shows that anti-nutritional factors are the main factor limiting the large-scale use of soy protein sources. Glycinin is one of the main protein components in soybeans. It has thermal stability, adhesion and strong immunogenicity, and is considered to be one of the main anti-nutritional factors. Soybean antigen protein Glycinin is one of the main anti-nutritional factors in soybeans, which partially limits the large-scale use of soy protein sources. Studies in aquatic animals have shown that high doses of Glycinin can cause intestinal damage in golden crucian carp (Cyprinus carpio×Carassius auratus), Jian carp (Cyprinus carpiovar.Jian), grass carp (Ctenopharyngodon idella), Luo's mullet (Rhynchocypris lagowskii Dybowski), pearl grouper (Epinephelusfuscoguttatus♀×E.lanceolatus♂) and Chinese mitten crab (Eriocheirsinensis), such as atrophy and damage of intestinal mucosal folds, and even enteritis. These negative effects ultimately lead to intestinal dysfunction and growth retardation. Studies have confirmed that the negative effects of Glycinin on the intestine of Luo's mullet are mainly concentrated in the hindgut, and similar results have been observed in grass carp and Jian carp. The antigen binding epitopes of fish intestines are mainly located in the hindgut, and the epithelial mucosal cells of the hindgut are most sensitive to antigen binding. The integrity of intestinal morphology and function is essential for maintaining the growth and health of fish. Therefore, in order to increase the use of soybean meal in fish feed, it is necessary to reduce or eliminate the growth inhibition and intestinal damage caused by dietary glycinin through dietary adjustment.

[0003] N-acetylcysteine ​​(NAC) is one of the important functional amino acids. Its biological activity mainly lies in the sulfhydryl group, and the acetyl group makes its activity more stable. It can be used as a sulfhydryl donor for cells and is considered to be an efficient free radical scavenger. In addition, NAC can be converted into biologically active substances such as cysteine, reduced glutathione, and taurine. It has physiological functions such as regulating fat metabolism, improving immune function, enhancing antioxidant capacity, improving intestinal health, regulating reproduction, and antibacterial. Studies on grass carp, Nile tilapia (Oreochromis niloticus), and Chinese mitten crab have confirmed that NAC can be used as a feed additive to improve the growth, health, and stress resistance of aquatic animals.

[0004] Nowadays, in the context of comprehensive "antibiotic ban", NAC has become a promising feed additive. Studies have confirmed that oxidative damage and inflammatory response are the main pathways of glycinin-induced intestinal damage. NAC may have a positive effect on reducing the negative effects of glycinin on fish. Summary of the invention

[0005] The purpose of the present invention is to provide a fish feed containing soybean antigen protein Glycinin and N-acetylcysteine ​​and a preparation method thereof.

[0006] Another object of the present invention is to provide the use of N-acetylcysteine ​​in the preparation of fish feed containing soybean antigen protein Glycinin. The use of N-acetylcysteine ​​as a functional feed additive that can alleviate carp enteritis induced by soybean antigen protein Glycinin can effectively alleviate growth inhibition and intestinal damage induced by soybean antigen protein Glycinin by improving fish growth and feed utilization, digestive function and protein metabolism function, immune function, antioxidant capacity and intestinal morphology.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The invention provides a method for effectively alleviating soybean antigen protein Glycinin in fish feed, comprising a feed additive, wherein the feed additive comprises soybean antigen protein Glycinin and N-acetylcysteine ​​(NAC).

[0009] The invention provides an application of a method for effectively alleviating soybean antigen protein Glycinin in fish feed, comprising application in fish feed, wherein the fish feed contains soybean antigen protein Glycinin and N-acetylcysteine.

[0010] The invention provides a fish feed containing soybean antigen protein Glycinin and N-acetylcysteine ​​(NAC).

[0011] Furthermore, when the added amount of soybean antigen protein Glycinin in the fish feed is 80 g / kg, 1.5-6.0 g / kg of N-acetylcysteine ​​is added.

[0012] Furthermore, when the added amount of soybean antigen protein Glycinin in the fish feed is 80 g / kg, 1.5, 3.0 or 6.0 g / kg of N-acetylcysteine ​​is added.

[0013] Furthermore, the fish feed comprises the following components in terms of mass percentage:

[0014] Fish meal 23%, corn gluten meal 7.85%, flour 25%, fish oil 3.05%, corn oil 3.05%, mineral and vitamin premix 0.5%, monocalcium phosphate 1%, choline chloride 0.5%, L-ascorbic acid-2-polyphosphate 0.08%, microcrystalline cellulose 4.29%, lysine 1.9%, methionine 0.31%, threonine 0.27%, aspartic acid 0-0.45%, casein 3.68%, gelatin 0.92%, dextrin 17.71%, N-acetylcysteine ​​0.15-0.6%, soybean globulin 8%.

[0015] Furthermore, the crude protein content of the fish feed is 36%, and the crude fat content is 9%.

[0016] The present invention provides a method for preparing the fish feed containing soybean antigen protein Glycinin and N-acetylcysteine ​​as described above, comprising the following steps:

[0017] According to the feed formula description, the feed raw materials are crushed and sieved, weighed according to the feed formula, mixed evenly, and made into granules. The feed granules are dried in an oven at 45°C for 24 hours, sealed in a sealed bag, and then stored in a -20°C refrigerator for later use.

[0018] The invention provides application of N-acetylcysteine ​​in preparing fish feed containing soybean antigen protein Glycinin.

[0019] Furthermore, when the content of soybean antigen protein Glycinin in the fish feed is 80 g / kg, the added amount of N-acetylcysteine ​​is 1.5-6.0 g / kg.

[0020] Furthermore, when the content of soybean antigen protein Glycinin in the fish feed is 80 g / kg, the added amount of N-acetylcysteine ​​is 1.5, 3.0 or 6.0 g / kg.

[0021] By means of the above technical solution, the present invention has the following advantages and beneficial technical effects:

[0022] 1) The feed additive of the present invention can effectively alleviate the growth inhibition and intestinal damage induced by soybean antigen protein Glycinin by improving fish growth and feed utilization, digestive function and protein metabolism function, immune function, antioxidant capacity and intestinal morphology.

[0023] 2) The feed additive of the present invention comprises a carp feed additive of N-acetylcysteine, has simple components, readily available raw materials, is safe and harmless, can effectively alleviate carp growth inhibition and intestinal damage induced by soybean antigen protein Glycinin, and improves the utilization rate of soybean protein source.

[0024] 3) The method for preparing carp feed provided by the present invention has simple steps, low cost and is suitable for practical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 AK shows the effect of N-acetylcysteine ​​on the growth performance and feed utilization of carp fed with Glycinin;

[0026] Figure 2 AD shows the effect of N-acetylcysteine ​​on the nutritional composition of muscle of carp fed Glycinin;

[0027] Figure 3 AB shows the effect of N-acetylcysteine ​​on the activities of protein metabolic enzymes in carp fed Glycinin;

[0028] Figure 4 AC shows the effect of N-acetylcysteine ​​on the digestive enzyme activities of carp fed Glycinin;

[0029] Figure 5 AG shows the effect of N-acetylcysteine ​​on serum immune function of carp fed Glycinin;

[0030] Figure 6 AH shows the effect of N-acetylcysteine ​​on the expression of genes related to intestinal inflammation in carp fed Glycinin;

[0031] Figure 7 AH shows the effect of N-acetylcysteine ​​on the antioxidant capacity of the intestine of carp fed Glycinin;

[0032] Figure 8 AH shows the effect of N-acetylcysteine ​​on the expression of antioxidant-related genes in the intestine of carp fed Glycinin;

[0033] Fig. 9 AE shows the effect of N-acetylcysteine ​​on intestinal growth parameters of carp fed Glycinin;

[0034] Fig.10 AE shows the effect of N-acetylcysteine ​​on the morphology of intestinal tissues of carp fed Glycinin;

[0035] Fig.11 AD shows the effect of N-acetylcysteine ​​on the expression of genes related to tight junction proteins in the intestine of carp fed Glycinin;

[0036] Fig.12 AC shows the effect of N-acetylcysteine ​​on the intestinal mucosal permeability of carp fed with Glycinin. DETAILED DESCRIPTION

[0037] The purpose of the present invention is to evaluate the effects of adding different levels of NAC in feed on the growth performance, digestive function, protein metabolism function, immune function, antioxidant capacity and intestinal barrier function of carp consuming 80 g / kg Glycinin, in order to provide a theoretical reference for developing new green feed additives, improving the utilization rate of soybean meal in fish feed, and improving fish growth and health.

[0038] The invention discloses a functional feed additive and application capable of alleviating carp enteritis induced by soybean antigen protein Glycinin, and relates to the field of biotechnology. The additive feed disclosed by the invention comprises soybean antigen protein Glycinin and N-acetylcysteine ​​(NAC). In a feed containing 80g / kg Glycinin, adding 1.5-6.0g / kg NAC can significantly improve fish growth and feed utilization, digestive function and protein metabolism function, immune function, antioxidant capacity and intestinal morphology, and effectively alleviate growth inhibition and intestinal damage induced by soybean antigen protein Glycinin.

[0039] The functional fish feed additive provided by the present invention has low cost and is easy to promote, which is helpful for developing new green antibiotic substitute additives, improving the utilization rate of soybean protein sources, reducing feed costs, and promoting healthy fish breeding.

[0040] Examples 1-3

[0041] The Glycinin used in this experiment was extracted from defatted soybean powder using a simplified membrane intermediate test method. The total protein content of the extracted Glycinin was (94.30±0.11)% measured by the Kjeldahl method. Then, the Glycinin content was detected by electrophoresis (SDS-PAGE), and Glycinin accounted for 86.50% of the total protein content. With fish meal, casein and gelatin as the main protein sources, and fish oil and corn oil as fat sources, 5 kinds of nitrogen and fat compound feeds were prepared. Among them, the feed without Glycinin and NAC was used as the CK group, the feed containing only 80g / kg Glycinin was used as the Gly group, and the other three groups, on the basis of all containing 80g / kg Glycinin, were added with 1.50g / kg, 3.00g / kg and 6.00g / kg of NAC, respectively, and were named Gly-N1 group, Gly-N2 and Gly-N3 group respectively. The feed raw materials were crushed and passed through a 0.246 mm sieve, weighed according to the feed formula in Table 1, mixed evenly, and made into 1.5 mm diameter particles using a multifunctional screw extruder (YL90L4, Jilin Agricultural University, Changchun). The feed particles were dried in an oven at 45 °C for 24 h, sealed in a sealed bag, and then stored in a -20 °C refrigerator for later use.

[0042] Table 1 Feed formula and main nutritional composition (air-dried basis, g / kg)

[0043]

[0044]

[0045] Effect test example

[0046] Feeding experiment:

[0047] The carp required for the experiment was purchased from Changjia Fishery Aquatic Breeding Farm in Longtan District, Jilin City, and the feeding experiment was carried out in the aquaculture room of the Science and Technology Innovation Center of Jilin Agricultural University. Before the feeding experiment began, the fish were pre-fed for 15 days to adapt to the breeding environment and the control group feed. They were stopped for 24 hours before grouping, and 450 carp with strong physique and neat specifications (2.93±0.03g) were selected and randomly divided into 5 groups, with 3 replicates in each group, and 30 carp were released in each replicate (300L plastic tank) for an 8-week feeding experiment. During the breeding experiment, 1 / 4-1 / 3 of the water was replaced every day, and the water temperature was maintained at 24-28℃, dissolved oxygen>5mg / L, nitrite<0.05mg / L, ammonia nitrogen<0.30mg / L, and pH between 7.5-8.5. Artificial bait was spread every day, and the fish were fed 3 times (7:00, 12:00 and 17:00) when they were full, and the feeding rate was 3%-5% of their body weight.

[0048] Sample collection:

[0049] After the feeding experiment, the fish were deprived of food for 24 h, anesthetized with 100 mg / L MS-222 (ethyl 3-aminobenzoate, Sigma, USA), and the group mass of fish in each tank was weighed.

[0050] Eight carps were randomly selected from each tank, and their body weight, body length, visceral weight, hepatopancreas weight, intestinal weight and intestinal length were measured for the calculation of growth indicators.

[0051] Fifteen carps were randomly selected from each tank, and blood was collected from the tail vein using a syringe. The samples were allowed to stand at 4°C for 24 hours and then centrifuged at 3000g for 10 minutes at 4°C. The supernatant was collected and stored at -80°C for analysis of immune indicators and intestinal permeability. Muscles above the lateral line and below the dorsal fin were collected for the determination of muscle nutrient components. The liver, pancreas and intestines were collected, quick-frozen in liquid nitrogen, and stored at -80°C for the determination of digestive enzymes, protein metabolic enzyme activity, antioxidant capacity, and gene mRNA expression.

[0052] Four carps were randomly selected from each tank, and the hind intestines were collected. After being rinsed with saline, they were fixed in 4% neutral formaldehyde solution for intestinal growth index determination and tissue morphology analysis.

[0053] Effect Test Example 1 Determination of Growth Indicators and Muscle Nutritional Components

[0054] Growth performance and feed utilization indices were calculated according to the following formula:

[0055] Mass gain rate (WGR) = (Wt-W0) / W0×100%

[0056] Specific growth rate (SGR) = (ln Wt - ln W0) / t × 100%

[0057] Feed conversion ratio (FCR) = I t / (Wt-W0)

[0058] Feed efficiency (FER) = (Wt - W0) / I t ×100%

[0059] Protein efficiency (PER) = (Wt - W0) / (I t ×P)×100%

[0060] Liver to body ratio (HIS) = W H / Wt×100%

[0061] Visceral-to-body ratio (VSI) = W V / Wt×100%

[0062] Fullness (CF) = Wt / L 3 ×100

[0063] Survival rate (SR) = terminal number / initial number × 100%

[0064] Where: Wt, W0 are the final and initial fish weights (g), t is the test time (d); I t is the mass of dry feed ingested (g); P is the crude protein content of feed (%); L is the length of fish (cm); W H W is the mass of hepatopancreas (g); V is the visceral mass (g).

[0065] The nutritional composition of feed and muscle was determined using the standard method published by AOAC, namely, crude protein was determined by the Kjeldahl method; crude fat was determined by the Soxhlet extraction method; crude ash was determined by the 550°C muffle furnace burning method; and moisture was determined by the 105°C constant temperature drying weight loss method.

[0066] Effect Test Example 2 Enzyme Activity Determination

[0067] Protease activity was determined by Folin phenol reagent method. Amylase, lipase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), lysozyme (LZM), total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) and diamine oxidase activities, as well as complement 3 (C3), complement 4 (C4), immunoglobulin M (IgM), malondialdehyde (MDA), reduced glutathione (GSH), total antioxidant capacity (T-AOC), interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), D-lactic acid, and endothelin 1 content were all determined by kits.

[0068] Effect Test Example 3 Real-time Quantitative PCR Analysis

[0069] Total RNA was extracted from the intestine of carp using tissue lysis buffer (AC0101-B, Si Ke Jie), chloroform and isopropanol. Subsequently, the concentration and quality of total RNA were evaluated using a UV-visible spectrophotometer (NanoDrop2000, Thermo, USA) and gel electrophoresis, respectively. After ensuring that the total RNA met the specified standards, it was reverse transcribed into cDNA using a reverse transcription kit (AG0304-B, Si Ke Jie). Table 2 provides primer information, and β-actin was selected as the housekeeping gene. Fluorescence quantitative PCR used the TB Green dye method, and the reaction system was 20 μL, including 10 μL TransStart Green qPCR SuperMix (2×), 1 μL upstream primer, 1 μL downstream primer, 1 μL cDNA template, 7 μL nuclease-free water, and was performed in the ABI (7500) RT-PCR system. All samples were tested three times. After the qRT-PCR reaction was completed, 2 -ΔΔCT Methods The relative expression levels of all target gene mRNAs were calculated.

[0070] Table 2 Gene primer sequences

[0071]

[0072]

[0073] Effect Test Example 4 Intestinal Histological Analysis

[0074] The hindgut samples were processed according to the conventional procedures (trimming, washing, dehydration and transparentization) and embedded in paraffin blocks. Subsequently, the samples were cut into 5 μm thick slices, stained with hematoxylin-eosin, and then sealed. Finally, an Olympus optical microscope (IX71, Japan) was used for photography and measurement.

[0075] Statistical analysis:

[0076] The data were analyzed using SPSS (20.0) software. First, the CK group and the other groups were subjected to independent sample T test, "*" represents significant difference (P < 0.05), "**" represents extremely significant difference (P < 0.01). Then, the Gly group, Gly-N1 group, Gly-N2 group and Gly-N3 group were subjected to one-way analysis of variance. If the difference was significant (P < 0.05), Tukey's test was further used for multiple comparisons; orthogonal polynomial analysis was used to detect whether the data had significant linear and quadratic effects.

[0077] Results of effect test

[0078] 1. Effects of N-acetylcysteine ​​on growth performance and muscle nutrients of carp fed Glycinin

[0079] Figure 1 In the table, "*" and "**" indicate that there are significant differences (P<0.05) or extremely significant differences (P<0.01) between the treatment group and the CK group; different letters indicate significant differences (P<0.05). A: P value of one-way ANOVA; L: P value of linear effect; Q: P value of quadratic effect. Same below.

[0080] Depend on Figure 1 AK showed that compared with the CK group, the final body weight, weight gain rate, specific growth rate, feed efficiency and protein efficiency of the Gly group were significantly reduced (P<0.05), and the feed conversion rate was significantly increased (P<0.05). The NAC supplementation level was significantly linearly and quadratically correlated with the final body weight, weight gain rate, specific growth rate, feed conversion rate, feed efficiency and protein efficiency of the carp (P<0.05). Compared with the Gly group, the terminal body weight, weight gain rate and specific growth rate of the Gly-N1, Gly-N2 and Gly-N3 groups were significantly increased (P<0.05), and there was no significant difference between the Gly-N2 and Gly-N3 groups (P>0.05); the feed efficiency and protein efficiency of the Gly-N2 and Gly-N3 groups were significantly increased (P<0.05), and there was no significant difference between the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05); the feed conversion rate of the Gly-N2 and Gly-N3 groups was significantly decreased (P<0.05), and there was no significant difference between the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05).

[0081] Soybean antigen Glycinin is the main storage protein of soybean and is considered to be one of the main anti-nutritional factors. Excessive intake of Glycinin by aquatic animals can easily cause intestinal damage, leading to digestive dysfunction and growth inhibition. The results of this experiment showed that the addition of 80g / kg of Glycinin to the feed significantly reduced the final body weight, weight gain rate, specific growth rate, feed efficiency and protein efficiency of carp, indicating that Glycinin would inhibit carp growth performance and feed utilization. Previous studies in this laboratory also found that 40-80g / kg of Glycinin significantly reduced the weight gain rate, specific growth rate and feed efficiency of Rockfish. Similarly, similar results were found in studies of Chinese mitten crabs, Jian carp, pearl grouper, grass carp and golden crucian carp, revealing that excessive intake of Glycinin by aquatic animals generally has an adverse effect on growth.

[0082] Glycinin cannot be completely degraded in the intestines of young aquatic animals. The residual Glycinin enters the blood circulation in the form of large molecular antigens, leading to intestinal immune response and oxidative damage, and ultimately inhibiting growth and feed utilization. Supplementing appropriate concentrations of NAC in glycinin feed significantly improved carp growth performance and feed utilization. NAC is considered to be an effective free radical scavenger due to the presence of thiol groups. At the same time, NAC can be metabolized to produce a variety of bioactive substances such as cysteine, reduced glutathione, and taurine. It has physiological functions such as promoting amino acid absorption and transport, regulating lipid metabolism, improving immune function, enhancing antioxidant capacity, and improving intestinal health. It is considered to be a promising functional feed additive. In a study of Nile tilapia, it was pointed out that the addition of 2.00g / kg NAC to feed significantly improved the body's weight gain rate and specific growth rate, revealing that NAC can promote fish growth.

[0083] Similarly, the addition of 2.00g / kg NAC to the feed significantly improved the weight gain rate and specific growth rate of grass carp, revealing that NAC can promote fish growth. Methionine has long been considered a limiting amino acid in carp formula feed. Cysteine ​​is a metabolite of methionine in the body. Supplementing NAC in the feed can partially save feed methionine, which may be one of the reasons why NAC promotes fish growth.

[0084] In addition, the study of Chinese mitten crab pointed out that NAC can effectively alleviate the growth inhibition caused by T-2 toxin, glycinin or β-conglycinin by improving immune function and antioxidant capacity, inhibiting cell apoptosis and improving intestinal health. The results of this experiment revealed that supplementing the feed with appropriate levels of NAC can significantly improve the growth inhibition induced by glycinin in carp, which may be attributed to the multiple physiological regulatory effects of NAC.

[0085] Depend on Figure 2 AD showed that compared with the CK group, the crude protein content of carp muscle in the Gly group was significantly reduced (P<0.05). The NAC supplementation level was significantly linearly correlated with the crude protein content of carp muscle (P<0.05). Compared with the Gly group, the crude protein content of carp muscle in the Gly-N2 and Gly-N3 groups was significantly increased (P<0.05), and there was no significant difference between the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05).

[0086] Animal body composition can effectively reflect the nutritional level and physiological status of the body, and is easily affected by changes in feed ingredients. In this study, the addition of 80g / kg of Glycinin to the feed significantly reduced the crude protein content of carp muscle, but Glycinin had no significant effect on muscle moisture, crude fat and crude ash content. These results indicate that excessive Glycinin inhibits protein deposition in the body of Lophatherum, which may be related to Glycinin's reduced protein utilization. The digestive tract of young animals cannot completely degrade Glycinin, resulting in a reduction in the effective protein content in the feed. In addition, Glycinin can destroy the intestinal structure and affect the secretion of proteases, resulting in a decrease in feed protein utilization. The decrease in protein utilization may cause malnutrition and ultimately reduce the crude protein content of muscle. The study pointed out that the addition of 60g / kg, 90g / kg and 120g / kg of Glycinin to the feed significantly reduced the crude protein content of carp fry and carp muscle; the addition of 60g / kg, 90g / kg and 120g / kg of Glycinin to the feed significantly reduced the crude protein content of golden crucian carp muscle, but had no significant effect on the moisture, crude fat and crude ash content; the addition of 160g / kg of Glycinin to the feed significantly reduced the crude protein content of Chinese mitten crab, but had no significant effect on the moisture, crude fat and crude ash content. Supplementing 3.00-6.00g / kg of NAC to Glycinin feed significantly increased the crude protein content of carp muscle. Similarly, in the study of Chinese mitten crab, it was pointed out that β-conglycinin reduced the crude protein content of the body, while NAC supplementation tended to increase the crude protein content. At present, few studies have reported the effect of NAC on the nutritional composition of fish muscle. However, NAC has the effect of promoting amino acid absorption and transport and improving intestinal health, which may be a potential factor for NAC to increase the protein content of carp muscle fed with Glycinin.

[0087] 2. Effects of N-acetylcysteine ​​on the activities of digestive enzymes and protein metabolic enzymes in carp fed Glycinin

[0088] Depend on Figure 3 AB showed that compared with the CK group, the activities of AST and ALT in the hepatopancreas of carp in the Gly group were significantly decreased (P<0.05). The level of NAC addition was significantly linearly correlated with AST activity (P<0.05), and significantly linearly and quadratically correlated with ALT activity (P<0.05). Compared with the Gly group, the AST activity of the Gly-N2 and Gly-N3 groups was significantly increased (P<0.05), and there was no significant difference between the Gly-N2 and Gly-N3 groups (P>0.05); the ALT activity of the Gly-N1 and Gly-N2 groups was significantly increased (P<0.05), and there was no significant difference between the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05).

[0089] AST and ALT are key enzymes in the protein metabolism process of aquatic animals. They are mainly stored in liver cells, while the content in the blood is low. When liver cells are damaged, AST and ALT are released into the blood, thereby increasing the activity of AST and ALT in the blood. Therefore, AST and ALT activities are important indicators for monitoring fish liver / hepatopancreas damage and body protein utilization. In this study, 80g / kg Glycinin significantly reduced the AST and ALT activities in the hepatopancreas of carp, inhibiting the protein metabolism function of carp. Similarly, in the study of Luo's mullet, it was pointed out that 40-80g / kg Glycinin significantly reduced the ALT activity in the hepatopancreas, and 80g / kg Glycinin significantly reduced the AST activity in the hepatopancreas. However, 60-120g / kg Glycinin significantly reduced the ALT activity in the hepatopancreas of golden crucian carp, and 120g / kg Glycinin significantly reduced the AST activity in the hepatopancreas. These results may be related to the hepatopancreas damage caused by Glycinin and the lack of available amino acids. Excessive Glycinin reduces the utilization rate of feed protein, which may lead to the lack of available amino acids. Aminotransferases break down amino acids and transfer amino groups to α-keto acids (reversible catalysis). However, when available amino acids are insufficient, keto acids may decrease, thereby reducing AST and ALT activities. Supplementation of NAC in the diet increased ALT and AST activities in the hepatopancreas of carp, revealing that NAC can alleviate Glycinin-induced protein metabolism dysfunction. At the same time, this study observed that 3.00-6.00g / kg NAC improved feed efficiency and protein efficiency in carp fed Glycinin, which may be partly attributed to the enhanced protein metabolism function of the hepatopancreas. Similarly, previous studies in our laboratory have shown that NAC can effectively reduce lipid vacuoles in the hepatopancreas of carp and improve the morphological structure of the hepatopancreas. NAC can metabolize and produce bioactive compounds (such as cysteine, glutathione, and taurine, etc.), which have multiple prebiotic effects and detoxification properties, thereby reducing the burden and damage of the hepatopancreas.

[0090] Depend on Figure 4 AC showed that compared with the CK group, the hepatopancreas and intestinal protease activities in the Gly group were significantly reduced (P<0.05). The NAC addition level was significantly linearly and quadratically correlated with the intestinal protease activity of carp (P<0.05). Compared with the Gly group, the intestinal protease activities of the Gly-N1, Gly-N2 and Gly-N3 groups were significantly increased (P<0.05), and there was no significant difference among the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05).

[0091] Digestive function directly affects the absorption and utilization of nutrients and is the main factor determining the normal growth and health of animals. Among them, protease, lipase and amylase are classic enzymes that directly reflect digestive function. In this study, 80g / kg Glycinin significantly reduced the intestinal protease activity of carp. The study revealed that when the amount of Glycinin added was 160g / kg, the intestinal trypsin activity of Chinese mitten crab decreased significantly. Similarly, in the study of Luo's mullet and golden crucian carp, it was revealed that excessive Glycinin added to the feed significantly reduced the intestinal and hepatopancreatic protease activity. It shows that high doses of Glycinin will reduce the digestive function of fish and the utilization of feed protein. As an antigen protein, excessive Glycinin intake by young animals will inhibit intestinal development and cause tissue damage, which may reduce the secretion of proteases. In addition, the effect of Glycinin on protease activity may also be related to its molecular structure. Glycinin contains -SH groups, which are easy to form stable food gels and are not easily hydrolyzed by proteases, thereby reducing the utilization of protein. However, supplementing 1.50-6.00g / kg NAC in the feed can increase the intestinal protease activity of carp fed with Glycinin. NAC significantly increased the intestinal and hepatopancreatic protease activity of Chinese mitten crabs fed with 7% β-conglycinin. Appropriate NAC supplementation can effectively alleviate the digestive dysfunction of carp caused by Glycinin. It is speculated that the anti-inflammatory and antioxidant properties of NAC avoid Glycinin-induced intestinal damage, thereby maintaining the normal physiological function of the intestine.

[0092] 3. Effects of N-acetylcysteine ​​on immune function and expression of inflammation-related genes in carp fed Glycinin

[0093] Depend on Figure 5AG showed that compared with the CK group, the serum LZM and AKP activities, IgM, C3 and C4 contents of carp in the Gly group were significantly reduced (P<0.05), and the IL-1β and TNF-α contents were significantly increased (P<0.05). The NAC supplementation level was significantly linearly correlated with the LZM and AKP activities, C3 and C4 contents (P<0.05), and was significantly quadratically correlated with the IgM content (P<0.05), and was significantly linearly and quadratically correlated with the IL-1β and TNF-α contents (P<0.05). Compared with the Gly group, the LZM activity in the Gly-N2 group was significantly increased (P<0.05), while there was no significant difference among the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05). The AKP activity and C4 content in the Gly-N2 and Gly-N3 groups were increased (P<0.05); the C4 content in the Gly-N1, Gly-N2 and Gly-N3 groups was increased (P<0.05). The IgM content in the ly-N2 group was significantly increased (P<0.05), while there was no significant difference among the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05); the IL-1β content and TNF-α content in the Gly-N1, Gly-N2 and Gly-N3 groups were significantly decreased (P<0.05), while there was no significant difference among the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05).

[0094] LZM is a key enzyme in the nonspecific immune system. It can destroy the cell wall by cleaving peptidoglycan, thereby resisting bacterial invasion. AKP is a marker regulatory enzyme of phagocytes and an important enzyme for maintaining physical health. Fish complement (mainly including C3 and C4, etc.) can not only participate in nonspecific immune responses, but also assist and supplement specific antibodies. It has the functions of conditioning, phagocytosis, mediating immune responses and inflammatory responses, and clearing pathogens. Proinflammatory cytokines (such as TNF-α, IL-1β, etc.) are an important class of immunoregulatory factors, and the inflammatory response mediated by them plays an important role in the process of animal immune regulation. In this study, 80g / kg Glycinin significantly reduced the activity of LZM and AKP in carp serum, as well as the content of C3, C4 and IgM. Similarly, in the study of Chinese mitten crab, it was shown that the addition of 80g / kg of Glycinin to the feed reduced the activity of intestinal lysozyme and increased the content of TNF-α. In addition, similar results were found in studies of grass carp, pearl grouper and golden crucian carp, revealing that excessive intake of glycinin by aquatic animals generally had a negative impact on immune function. It is worth noting that NAC supplementation reversed the negative impact of glycinin on the immune function of carp. Similarly, studies on golden pomfret (Trachinotus ovatus) showed that cysteine ​​had a positive effect on immune function by increasing the levels of serum C3, C4 and IgM and LZM activity. The immunomodulatory properties of NAC may be attributed to its metabolites (such as cysteine, glutathione, taurine, etc.), which have antibacterial and anti-inflammatory properties. Subsequently, the nuclear transcription factor-κB (NF-κB) signaling pathway, which is closely related to the inflammatory response, was studied in depth.

[0095] Depend on Figure 6AH showed that compared with the CK group, the mRNA expression of PI3K, AKT, NF-κB, TNF-α and IL-1β in the intestine of carp in the Gly group was significantly increased (P<0.05), and the mRNA expression of IκB, TGF-β and IL-10 was significantly decreased (P<0.05). The NAC addition level was significantly linearly correlated with the mRNA expression of PI3K, TGF-β and IL-10 (P<0.05), and was significantly linearly and quadratically correlated with the mRNA expression of AKT, NF-κB, IκB, TNF-α and IL-1β (P<0.05). Compared with the Gly group, the PI3K mRNA expression levels in the Gly-N2 and Gly-N3 groups were significantly decreased (P<0.05), while there was no significant difference in Gly-N1 (P>0.05); the AKT, NF-κB, TNF-α and IL-1β mRNA expression levels in the Gly-N1, Gly-N2 and Gly-N3 groups were significantly decreased (P<0.05); the TGF-β mRNA expression level in the Gly-N1, Gly-N2 and Gly-N3 groups was significantly increased (P<0.05), while there was no significant difference between the Gly-N2 and Gly-N3 groups (P>0.05); the IL-10 mRNA expression level in the Gly-N2 and Gly-N3 groups was significantly increased (P<0.05), while there was no significant difference between the Gly-N1 group and the Gly group (P>0.05).

[0096] NF-κB protein usually binds to the inhibitory protein IκB-α in the cytoplasm to form a trimer complex. After being phosphorylated and entering the nucleus, it can promote the expression of downstream inflammatory cytokines (such as TNF-α, IL-1β, etc.), and can also regulate the maturation of B lymphocytes and T lymphocytes. In this study, Glycinin downregulated the expression of IκB, TGF-β and IL-10 mRNA in the hind intestine of carp, and upregulated the expression of NF-κB, IL-1β and TNF-α mRNA, revealing that Glycinin induces inflammation by activating the NF-κB signaling pathway. Similar results were also found in the study of Jian carp, grass carp, golden crucian carp and grouper. It is worth noting that NAC can effectively intervene in this process. NAC supplementation upregulated the expression of IκB, TGF-β and IL-10 mRNA in the hind intestine of carp, and downregulated the expression of NF-κB, IL-1β and TNF-α mRNA, revealing that NAC effectively inhibited the activation of the NF-κB signaling pathway, thereby inhibiting the inflammatory response induced by Glycinin. Similarly, the study on tilapia showed that the addition of NAC to feed significantly downregulated the expression of IL-1β in the head kidney tissue after challenge with Streptococcus iniae. In addition, NAC downregulated the expression of intestinal proinflammatory cytokines (p38MAPK, ALF3, EsRelish, LITAF and ILF2) in Chinese mitten crabs that ingested β-conglycinin. NAC can regulate the NF-κB pathway through multiple pathways. The PI3K-AKT signaling pathway is involved in the regulation of cell growth, immunity, metabolism and apoptosis, and is also an important signaling pathway upstream of the NF-κB pathway. Overexpression of AKT can activate the IKK complex, promote the phosphorylation and degradation of IκB, thereby releasing NF-κB into the cell nucleus and inducing inflammation. In this study, the addition of NAC downregulated the mRNA expression of PI3K and AKT, revealing that NAC can inhibit the transcription of downstream NF-κB by inhibiting the PI3K-AKT signaling pathway.

[0097] At the same time, NAC can scavenge ROS and inhibit the nuclear translocation of IκB kinase β and NF-κB protein to inhibit NF-κB activation, indicating that NAC can upregulate IκB mRNA expression to exert anti-inflammatory effects. In addition, TNF-α can act as an activator of the NF-κB signaling pathway to aggravate the inflammatory response. NAC can regulate the interaction between TNF-α and TNF receptor 1 and TNF receptor 2, reduce the binding of TNF-α to its receptors, and thus inhibit the activation of the NF-κB signaling pathway. In this study, the expression of TNF-α mRNA was downregulated, further revealing that the anti-inflammatory effect of NAC was partly attributed to blocking the activation of the NF-κB signaling pathway induced by TNF-α. The effects of NAC on the NF-κB signaling pathway in the intestine of carp involve multiple pathways, and NAC can inhibit the inflammatory response induced by Glycinin.

[0098] 4. Effects of N-acetylcysteine ​​on antioxidant capacity and related gene expression in carp fed Glycinin

[0099] Depend on Figure 7 AH showed that compared with the CK group, the MDA content in the hind intestine of the Gly group was significantly increased (P<0.05), and the T-AOC and T-SOD, GPx, GR and GST activities were significantly decreased (P<0.05). The NAC addition level was significantly linearly correlated with the T-AOC, GSH content, GPx, GR and GST activities (P<0.05), and was significantly linearly and quadratically correlated with the MDA content and T-SOD activity (P<0.05). Compared with the Gly group, the MDA content in the Gly-N1, Gly-N2 and Gly-N3 groups was significantly decreased (P<0.05), while there was no significant difference between the Gly-N2 and Gly-N3 groups (P>0.05); the T-AOC, GPx and GST activities in the Gly-N2 and Gly-N3 groups were significantly increased (P<0.05), while there was no significant difference between the Gly-N2 and Gly-N3 groups (P>0.05); the T-SOD and GR activities in the Gly-N1, Gly-N2 and Gly-N3 groups were significantly increased (P<0.05), while there was no significant difference between the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05); the GSH content in the Gly-N2 group was significantly increased (P<0.05), while there was no significant difference between the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05).

[0100] The antioxidant defense system plays a vital role in protecting the body from oxidative stress. MDA is the final decomposition product of lipid peroxidation caused by ROS and is a biomarker reflecting the degree of oxidative stress. GPx, GR, GST, CAT and T-SOD are important enzymes in the enzymatic antioxidant defense system and play an important role in scavenging ROS and maintaining redox homeostasis in the body. GSH is a tripeptide antioxidant that can neutralize ROS and assist in restoring the activity of other antioxidant enzymes. T-AOC is a comprehensive indicator for evaluating the antioxidant status of the body. In this study, Glycinin increased the content of MDA in the hind intestine of carp and decreased T-AOC, CAT, T-SOD, GPx, GR and GST activities and GSH content. These results were supported by studies on Chinese mitten crab, golden crucian carp, Jian carp and grass carp, revealing that oxidative damage is one of the main ways in which Glycinin induces intestinal damage. Previous studies have shown that soybean globulin increased ROS generation associated with the NOX signaling pathway and reduced ROS scavenging capacity associated with Nrf2 translocation, leading to increased ROS content in the intestine of grass carp. Inflammation can aggravate oxidative stress by producing ROS. At the same time, ROS can increase the production of inflammatory mediators through the MAPK / NF-κB signaling pathway and produce ROS under the action of the NADPH oxidase complex, thereby amplifying the oxidative stress response and further stimulating the activation of inflammatory cells. Therefore, there is a vicious cycle between oxidative stress and inflammation, which may be a potential mechanism for soybean globulin-induced intestinal damage in fish. NAC supplementation reduced the MDA content in the hindgut of carp fed Glycinin and increased T-AOC, CAT, T-SOD, GPx, GR and GST activities and GSH content. Similarly, NAC also effectively enhanced the antioxidant capacity of grass carp, Nile tilapia, rainbow trout, European eel (Anguilla anguilla L.) and Chinese mitten crab. NAC is a potent antioxidant with multiple antioxidant mechanisms. Its thiol groups can directly provide electrons to neutralize ROS; as a precursor, NAC can enhance the synthesis of intracellular GSH; in addition, NAC can chelate certain heavy metal ions, thereby reducing the accumulation of peroxides and alleviating oxidative stress. In this study, NAC supplementation significantly increased hindgut GSH content and GPx, GR, and GST activities, indicating that GSH metabolism plays a key role in the antioxidant properties of NAC.

[0101] Depend on Figure 8AH showed that compared with the CK group, the expression levels of Nrf2, HO1, SOD, GPx and GSTP1 mRNA in the hind intestine of carp in the Gly group were significantly decreased (P<0.05), and the expression level of Keap1 mRNA was significantly increased (P<0.05). The NAC supplementation level was significantly linearly correlated with the expression levels of Nrf2, HO1, GPx and GSTP1 mRNA (P<0.05), and was significantly linearly and quadratically correlated with the expression levels of Keap1 and GS mRNA (P<0.05). Compared with the Gly group, the mRNA expression levels of Nrf2, GPx and GSTP1 in the Gly-N2 and Gly-N3 groups were significantly increased (P<0.05), while there was no significant difference between the Gly-N2 and Gly-N3 groups (P>0.05); the mRNA expression level of Keap1 in the Gly-N1, Gly-N2 and Gly-N3 groups was significantly decreased (P<0.05), while there was no significant difference between the Gly-N2 and Gly-N3 groups (P>0.05); the mRNA expression level of HO1 in the Gly-N3 group was significantly increased (P<0.05), while there was no significant difference between the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05); the mRNA expression level of GR in the Gly-N1, Gly-N2 and Gly-N3 groups was significantly increased (P<0.05). The mRNA expression level was significantly increased (P<0.05), while there was no significant difference among the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05).

[0102] The Nrf2-Keap1 signaling pathway is an important endogenous antioxidant signaling pathway in animals. During oxidative stress, a large amount of ROS is produced, which stimulates the dissociation of Nrf2 from Keap1. Subsequently, Nrf2 is transported to the nucleus and binds to Maf protein, and then binds to antioxidant response elements, thereby mediating the expression of downstream antioxidants (including HO-1, CAT, GPx, GR, GST, etc.). However, in this study, Glycinin significantly reduced the expression of Nrf2, HO-1, SOD, GPx, GS and GSTP1 mRNA in the hind intestine of carp, and increased the expression of Keap1 mRNA, revealing that Glycinin reduces the antioxidant capacity of carp intestine by inhibiting the activation of the Nrf2-Keap1 signaling pathway. These inconsistent results may be related to the breeding time. During the 8-week breeding trial, Glycinin continuously stimulated the production of intestinal ROS, which may have exceeded the threshold of carp intestinal antioxidant defense, destroyed the structure and function of the intestine, and finally inhibited the expression of antioxidant enzyme-related genes. Similarly, studies on grass carp and Lophatherum robur found that glycinin reduced the expression of antioxidant enzyme-related genes by inhibiting the Nrf2-Keap1 signaling pathway. This study showed that the antioxidant effect of NAC involved the Nrf2 pathway. NAC upregulated the expression of Nrf2, HO-1, SOD, GPx, GS, and GSTP1 mRNA in the hind intestine of carp by activating the Nrf2-Keap1 signaling pathway. Although NAC exhibits strong antioxidant properties in aquatic animals, there are very limited studies on its effects on the Nrf2 signaling pathway. We speculate that the ability of NAC to regulate the Nrf2-Keap1 signaling pathway may be related to its metabolites. Cysteine ​​is a metabolite of NAC, and studies have shown that L-cysteine ​​upregulated the expression of Nrf2 mRNA in the intestine of golden pomfret, thereby increasing the mRNA expression of intestinal CAT, SOD, and GPx. Similarly, L-cysteine ​​promoted the nuclear translocation of Nrf2 in the intestine of LPS-challenged piglets, thereby alleviating oxidative stress. In addition, the further product GSH can promote enzymatic reactions and participate in Nrf2-Keap1 signal transduction, indicating that NAC can participate in intercellular signal transduction, thereby regulating the antioxidant capacity of carp intestine.

[0103] 5. Effects of N-acetylcysteine ​​on intestinal mucosal barrier function in carp fed Glycinin

[0104] Depend on Fig. 9AE showed that compared with the CK group, the number of mucosal folds, height of mucosal folds and thickness of muscle layer in the hind intestine of carp in the Gly group were significantly reduced (P<0.05). The NAC supplementation level was significantly linearly correlated with the number of mucosal folds and height of mucosal folds (P<0.05), and was significantly linearly and quadratically correlated with the thickness of muscle layer (P<0.05). Compared with the Gly group, the number of mucosal folds in the Gly-N2 and Gly-N3 groups was significantly increased (P<0.05), while there was no significant difference between the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05); the height of mucosal folds and thickness of muscle layer in the Gly-N1, Gly-N2 and Gly-N3 groups were significantly increased (P<0.05), while there was no significant difference between the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05).

[0105] Fig.10 Middle, MF, mucosal folds; MH, mucosal fold height; MW, mucosal fold width; ML, muscle layer thickness; LP, lamina propria; white frame, epithelial mucosa separated from lamina propria; yellow frame, epithelial mucosa rupture.

[0106] Depend on Fig.10 AE showed that the morphological structure of the intestinal mucosal folds in the CK group was intact, the nuclei of the intestinal mucosal epithelial cells were arranged tightly and neatly, the muscle layer was thick, the mucosal folds were well developed, and there was no sign of morphological damage. However, in the Gly group, it was observed that the nuclei of the intestinal mucosal epithelial cells were arranged in a disordered manner, part of the epithelial mucosa was separated from the lamina propria, and the mucosal folds were damaged and atrophied. A slight improvement was observed in the Gly-N1 group, but there were still signs of mucosal damage. The morphological structure of the intestinal mucosa in the Gly-N2 and Gly-N3 groups was similar to that of the control group, and no obvious damage was observed.

[0107] Depend on Fig.11 AD showed that compared with the CK group, the expression levels of ZO-1, Occludin1 and Claudin3 mRNA in the hind intestine of carp in the Gly group were significantly reduced (P<0.05). The NAC supplementation level was significantly linearly correlated with the expression levels of Occludin1 and Claudin7 mRNA (P<0.05), and was significantly linearly and quadratically correlated with the expression levels of ZO-1 and Claudin3 mRNA (P<0.05). Compared with the Gly group, the mRNA expression levels of ZO-1 and Claudin3 in the Gly-N1, Gly-N2 and Gly-N3 groups were significantly increased (P<0.05), while there was no significant difference between the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05); the mRNA expression levels of Occludin1 and Claudin7 in the Gly-N2 and Gly-N3 groups were significantly increased (P<0.05), while there was no significant difference between the Gly-N2 and Gly-N3 groups (P>0.05).

[0108] Depend on Fig.12 AC showed that compared with the CK group, the levels of serum diamine oxidase, D-lactic acid and endothelin-1 in the Gly group were significantly increased (P>0.05). The NAC supplementation level was significantly linearly and quadratically correlated with the diamine oxidase content (P<0.05), and was significantly quadratically correlated with the D-lactic acid content (P<0.05). Compared with the Gly group, the diamine oxidase content in the Gly-N1, Gly-N2 and Gly-N3 groups was significantly reduced (P<0.05), and the Gly-N2 group was significantly lower than that in the Gly-N1 and Gly-N3 groups (P<0.05); the D-lactic acid content in the Gly-N1 and Gly-N2 groups was significantly reduced (P<0.05), while there was no significant difference between the Gly-N1, Gly-N2 and Gly-N3 groups (P>0.05).

[0109] The intestine is an important digestive organ of fish and the most direct part of the body stimulated by pathogens or antigens in food. The integrity of intestinal morphology and function is essential for maintaining the growth, development and health of the body. In this study, 80g / kg Glycinin significantly reduced the height, width and muscle layer thickness of the hind intestinal mucosal folds of carp and caused mucosal damage. Similarly, Glycinin also caused nuclear translocation and goblet cell congestion in the intestinal mucosa of grass carp, and caused atrophy and damage to the intestinal mucosal folds of Chinese mitten crab and Luo's mullet. Intestinal damage and dysplasia reduce the functional surface area of ​​the intestinal mucosa, thereby weakening the digestive function. Many studies have confirmed that Glycinin-induced intestinal damage in aquatic animals is related to inflammatory response and oxidative stress. NAC has anti-inflammatory, antioxidant and intestinal health-improving effects and is a promising intestinal repair agent. In this study, 3.00-6.00g / kg NAC supplementation improved the intestinal mucosal development of carp fed Glycinin and inhibited intestinal damage. In the study of another antigen protein β-conglycinin, it was confirmed that supplementing 0.50-1.00g / kg NAC in the feed inhibited the damage caused by β-conglycinin to the peritrophic membrane and intestinal epithelial cells of Chinese mitten crab. Adding 0.50g / kg NAC to the feed can reduce the ulcerative mucosal damage of piglets caused by acetic acid, increase the number and density of goblet cells, and reduce the number and density of intraepithelial lymphocytes.

[57] When piglets were challenged with LPS, adding 0.50g / kg NAC to the feed reduced the depth of small intestinal crypts and increased the surface area of ​​intestinal villi. NAC can promote the development of intestinal mucosa and alleviate the atrophy and damage of intestinal mucosa in animals under stress.

[0110] Intestinal damage also reduces the intestinal mucosal barrier function and increases the possibility of antigen invasion. D-lactic acid is a unique metabolite of intestinal bacteria, and diamine oxidase is a unique enzyme present in intestinal epithelial cells. When the intestinal mucosa is damaged, D-lactic acid and diamine oxidase are released into the blood circulation system and participate in inducing the synthesis and release of endothelin-1 by vascular endothelial cells. Normally, serum D-lactic acid, diamine oxidase and endothelin-1 levels are low. In this study, 80g / kg Glycinin significantly increased the concentrations of D-lactic acid, diamine oxidase and endothelin-1 in carp serum. The results of the study were also supported by Jian carp and grass carp, indicating that Glycinin destroyed the tight junctions between intestinal epithelial cells and induced cell apoptosis through oxidative damage and inflammation, thereby increasing intestinal permeability. At the same time, this study found that 80g / kg Glycinin significantly downregulated the expression of ZO-1, Occludin1 and Claudin3 mRNA in the hind intestine of carp, revealing that the increase in intestinal mucosal permeability induced by Glycinin may be related to the decrease in the expression of tight junction proteins. NAC also plays an important role in maintaining the integrity of the intestinal mucosal barrier. The results of this study showed that the addition of appropriate concentrations of NAC upregulated the expression of ZO-1, Occludin1, Claudin3 and Claudin7 mRNA in the hind intestine of carp, and reduced the serum D-lactate, diamine oxidase and endothelin-1 concentrations. At present, no studies have reported on the effects of NAC on intestinal tight junction proteins in aquatic animals. In terrestrial animals, 0.50g / kg NAC increased the expression of claudin-1 and occludin proteins in the small intestinal mucosa of piglets under LPS challenge, and reduced the activity of plasma diamine oxidase. NAC can inhibit Glycinin-induced intestinal barrier function damage by promoting the expression of tight junction proteins in the carp intestine.

[0111] In summary, 80g / kg Glycinin significantly reduced the intestinal digestive function, protein metabolism function, immune function and antioxidant capacity of carp, and ultimately led to growth inhibition and intestinal damage. Supplementing 3.00-6.00g / kg NAC in the feed significantly improved the growth performance and digestive function of carp fed with soybean antigen protein Glycinin. It also activated the Nrf2 signaling pathway by enhancing glutathione metabolism, improved the body's antioxidant capacity, inhibited the NF-κB signaling pathway, improved the body's anti-inflammatory and immune regulation, and promoted the development of intestinal mucosa.

[0112] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Therefore, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for effectively alleviating soybean antigen protein Glycinin in fish feed, comprising a feed additive, wherein the feed additive comprises soybean antigen protein Glycinin and N-acetylcysteine ​​(NAC).

2. An application of a method for effectively alleviating soybean antigen protein Glycinin in fish feed, comprising application in fish feed, wherein the fish feed contains soybean antigen protein Glycinin and N-acetylcysteine.

3. A functional fish feed capable of alleviating carp enteritis induced by soybean antigen protein Glycinin, the fish feed comprising soybean antigen protein Glycinin and N-acetylcysteine.

4. The fish feed according to claim 3, characterized in that: When the added amount of soybean antigen protein Glycinin in the fish feed is 80 g / kg, the added amount of N-acetylcysteine ​​is 1.5-6.0 g / kg.

5. The fish feed according to claim 3, characterized in that: When the added amount of soybean antigen protein Glycinin in the fish feed is 80 g / kg, the added amount of N-acetylcysteine ​​is 1.5, 3.0 or 6.0 g / kg.

6. The fish feed according to claim 3, characterized in that: The fish feed comprises the following components in terms of mass percentage: Fish meal 23%, corn gluten meal 7.85%, flour 25%, fish oil 3.05%, corn oil 3.05%, mineral and vitamin premix 0.5%, monocalcium phosphate 1%, choline chloride 0.5%, L-ascorbic acid-2-polyphosphate 0.08%, microcrystalline cellulose 4.29%, lysine 1.9%, methionine 0.31%, threonine 0.27%, aspartic acid 0-0.45%, casein 3.68%, gelatin 0.92%, dextrin 17.71%, N-acetylcysteine ​​0.15-0.6%, soybean globulin 8%.

7. The fish feed according to claim 3, characterized in that: According to the mass percentage, The crude protein content of the fish feed is 36%, and the crude fat content is 9%.

8. The method for preparing fish feed containing soybean antigen protein Glycinin and N-acetylcysteine ​​according to any one of claims 3 to 7, characterized in that: The following steps are involved: According to the feed formula description, the feed raw materials are crushed and sieved, weighed according to the feed formula, mixed evenly, and then made into granules; the feed granules are dried in an oven at 45°C for 24 hours, sealed in a sealed bag, and then stored in a -20°C refrigerator for later use.

9. Application of N-acetylcysteine ​​in the preparation of fish feed containing soybean antigen protein Glycinin.

10. The use according to claim 7, characterized in that: When the added amount of soybean antigen protein Glycinin in the fish feed is 80 g / kg, the added amount of N-acetylcysteine ​​is 1.5-6.0 g / kg.

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