A feed additive for repairing intestinal injury of aquatic animals, a feed and a preparation method thereof

Abstract

This invention proposes a feed additive, feed, and preparation method for repairing intestinal damage in aquatic animals, relating to the field of aquatic feed technology. This invention aims to address the problem of intestinal damage in aquatic animals caused by various factors in aquaculture. The feed additive of this invention comprises, by weight, 1-3 parts selenium nanoparticles, 5-10 parts astragalus polysaccharide, and 3-5 parts γ-aminobutyric acid (GABA). The compound aquatic feed additive and its formulated feed provided by this invention, through the optimized combination and synergistic effect of selenium nanoparticles with astragalus polysaccharide and GABA, improve the repair capacity of intestinal damage in aquatic animals through multiple pathways and levels, effectively alleviating the adverse effects of decreased survival rate caused by environmental stress and significantly improving the production performance of aquatic animals. This invention solves the problems of low bioavailability and narrow toxicity threshold of traditional selenium preparations, achieving safe and efficient intestinal protection.

Description

Technical Field

[0001] This invention relates to the field of aquatic feed technology, specifically to a feed additive, feed, and preparation method for repairing intestinal damage in aquatic animals. Background Technology

[0002] In aquaculture, gut health is a key factor affecting animal growth performance, immune function, and disease resistance. With the increasing intensive farming practices, aquatic animals face a variety of environmental stressors, including anti-nutritional factors in feed, water pollutants, pathogenic microorganisms, and endocrine disruptors. These factors can induce damage to intestinal structure, disruption of barrier function, and metabolic disorders, severely restricting farming efficiency and animal health.

[0003] Selenium (Se) is an essential trace element for aquatic animals and a key component of antioxidant enzymes such as glutathione peroxidase (GPX), playing a crucial role in maintaining redox homeostasis. However, traditional inorganic selenium (such as sodium selenite) and organic selenium (such as selenomethionine) suffer from low bioavailability and narrow toxicity thresholds, limiting their safe application in aquaculture. In recent years, selenium nanoparticles (SeNPs) have gradually become a hot topic in aquatic animal nutrition regulation and intestinal protection research due to their advantages such as small particle size, large specific surface area, high biocompatibility, and strong antioxidant activity. Existing studies have preliminarily confirmed that SeNPs have a positive effect on alleviating intestinal damage in fish induced by high-fat diets and heat stress, but whether they can effectively antagonize intestinal damage in aquatic animals caused by aquatic pollutants has not yet been reported. Currently, prevention and control measures for intestinal damage in aquatic animals mainly include the use of antibiotics, plant extracts, and probiotics. However, with the ban on antibiotics and the promotion of green fisheries, antibiotics have been banned in aquaculture, making the traditional antibiotic-dependent approach to controlling intestinal diseases unsustainable. While plant extracts possess certain antioxidant and anti-inflammatory activities, their active ingredients are complex, their targets are unclear, their stability is poor, and their dosage dependence is strong, making it difficult to achieve stable and repeatable intestinal protection effects in practical applications. Although probiotics help regulate the intestinal microecology, their colonization efficiency is greatly affected by host status, environmental conditions, and feed processing technology, and their ability to repair existing intestinal structural damage and metabolic disorders is limited when used alone. Especially when facing intestinal damage induced by strong environmental pollutants such as tetrabromobisphenol A, the above intervention methods are unable to target and regulate core pathological processes such as glucose metabolism reprogramming, mitochondrial dysfunction, and ferroptosis. In contrast, SeNPs exert their intestinal damage repair effect through a multi-pathway synergistic mechanism: On the one hand, SeNPs can regulate glucose metabolism reprogramming, shifting the abnormally enhanced glycolytic metabolic flux to the pentose phosphate pathway, promoting the production of NADPH and reduced glutathione, thereby enhancing the antioxidant defense capacity of intestinal epithelial cells; on the other hand, SeNPs restore the functional coupling between the tricarboxylic acid cycle and the electron transport chain by targeting glutamate dehydrogenase 1 (GLUD1), improving mitochondrial respiratory function and reducing the accumulation of mitochondrial reactive oxygen species; more importantly, SeNPs can simultaneously inhibit FUNDC1-mediated mitophagy and NCOA4-mediated ferritin autophagy, blocking the activation of the ferroptosis pathway, thus achieving systemic repair of intestinal damage at three levels: metabolic regulation, mitochondrial quality control, and cell death intervention. These mechanisms of action are not found in existing interventions such as antibiotics, plant extracts, and probiotics, revealing the unique advantages and broad application prospects of selenium nanoparticles as a novel feed additive in repairing intestinal damage in aquatic animals. Summary of the Invention

[0004] This invention addresses the problem of intestinal damage in aquatic animals caused by various factors during aquaculture, and proposes a feed additive, feed, and its preparation method for repairing intestinal damage in aquatic animals.

[0005] The technical solution adopted by the present invention to solve the above problems is as follows:

[0006] Firstly, this invention proposes a feed additive for repairing intestinal damage in aquatic animals. The additive uses selenium nanoparticles as the main active ingredient, comprising 1-3 parts by weight; the particles are monodisperse spherical with a diameter of 180 nm or less. In addition, the additive also contains the following compound components in parts by weight: 5-10 parts of Astragalus polysaccharide and 3-5 parts of γ-aminobutyric acid (GABA). Astragalus polysaccharide, as an immunomodulator, can synergistically enhance the intestinal mucosal barrier function with selenium nanoparticles; GABA, as a neurotransmitter regulator, can alleviate intestinal peristalsis abnormalities caused by stress. The above compound components and selenium nanoparticles form a synergistic effect, jointly achieving multi-target repair of intestinal damage.

[0007] Furthermore, the selenium nanoparticles are monodisperse spherical with a particle size of 180 nm or less.

[0008] Secondly, the present invention provides a compound feed for aquatic animals, wherein the feed, by weight, comprises 5-10 parts of the feed additive as described in claim 1 or 2 and 500-1000 parts of the basic feed.

[0009] Furthermore, the basic feed consists of the following components: 14% fish meal, 25% soybean meal, 25% cottonseed meal, 6% soybean oil, 25% chicken meal, 0.2% choline chloride, 2% calcium dihydrogen phosphate, 2.3% bentonite, 0.1% vitamin premix, 0.05% mineral premix, 0.12% lysine hydrochloride, 0.12% arginine hydrochloride, 0.08% DL-methionine, and 0.03% threonine.

[0010] Thirdly, the present invention provides a method for preparing aquatic compound feed, comprising the following steps: Step 1: Mix selenium nanoparticles, astragalus polysaccharide and γ-aminobutyric acid evenly according to the weight ratio to obtain a premix; Step 2: Add the carrier to the premix obtained in Step 1 and mix using a stepwise expansion mixing method, successively expanding the mixing ratio to 1:10, stirring for 30 minutes each time in a three-dimensional mixer at 20 rpm. After the final mixture is homogeneous, pass it through an 80-mesh sieve, vacuum package it, and obtain the feed additive.

[0011] Preferably, the carrier is bentonite.

[0012] Step 3: Grind the raw materials for the basic feed according to the weight proportions and sieve them through a 60-mesh sieve; mix them thoroughly after sieving to obtain the basic feed; Step 4: Take the basic feed obtained in Step 3 and the aquatic feed additive obtained in Step 2 according to the weight proportions, add deionized water, mix thoroughly, and obtain a feed mixture. Preferably, the amount of deionized water added is 100-200 mL / kg of the feed mixture. Step 5: Use a twin-screw extruder to process the feed mixture obtained in step 4 into pellets with a diameter of 2-3 mm. After sieving, pack the pellets into self-sealing bags and store them at -20℃.

[0013] Fourthly, the present invention also proposes the application of the above-mentioned aquatic compound feed in the preparation of products for the repair of intestinal damage in aquatic animals. The application achieves the repair of intestinal damage by improving the intestinal antioxidant capacity of aquatic animals and maintaining the integrity of the intestinal barrier.

[0014] The beneficial effects of this invention are: 1. This invention solves the problems of low bioavailability and narrow toxicity threshold of traditional selenium preparations, and achieves safe and efficient intestinal protection.

[0015] 2. This invention uses monodisperse spherical selenium nanoparticles with a particle size of 180 nm or less as the core active ingredient. Compared with traditional inorganic selenium (sodium selenite) and organic selenium (selenomethionine), it has higher intestinal absorption efficiency and bioavailability.

[0016] 3. The compound aquatic feed additive and its compound feed provided by the present invention, through the optimized combination and synergistic effect of SeNPs with Astragalus polysaccharide and γ-aminobutyric acid, improve the repair ability of intestinal damage in aquatic animals through multiple pathways and levels, effectively alleviate the adverse effects of environmental stress such as decreased survival rate, and significantly improve the production performance of aquatic animals.

[0017] 4. The compound feed provided by this invention is used to improve intestinal damage in aquatic animals. According to experimental results, it can significantly improve intestinal antioxidant capacity, maintain intestinal barrier integrity, and realize the intestinal damage repair capacity of aquatic animals. Attached Figure Description

[0018] Figure 1 This is a comparison chart of the survival rates of carp in different treatment groups in this embodiment of the invention; Figure 2 This is a graph showing the detection results of inflammatory factors IL-1β and TNF-α in the serum and intestinal tissue of carp in different treatment groups in this embodiment of the invention; Figure 3This is a graph showing the detection results of antioxidant enzyme activity and malondialdehyde content in the intestinal tissue of carp in different treatment groups in this embodiment of the invention; Figure 4 These are ultrastructural scans of the intestinal tissue surface of carp in different treatment groups in this embodiment of the invention; Figure 5 This is a graph showing the relative mRNA expression levels of tight junction proteins closure protein, tight junction protein 1, and tight junction protein 1 in the intestinal tissues of carp in various groups of embodiments of the present invention. Detailed Implementation

[0019] This embodiment proposes a feed additive for repairing intestinal damage in aquatic animals, aiming to address intestinal damage in aquatic animals caused by various factors during aquaculture. The feed additive for repairing intestinal damage in aquatic animals proposed in this invention will be described below in specific embodiments: Example: I. Material Preparation: Control group feed, low-dose additive (experimental group feed 1), and high-dose additive (experimental group feed 2) were prepared separately. The control group feed was the basal feed. Experimental group feed 1 and experimental group feed 2 were both aquatic compound feeds of this invention, differing only in the ratio of aquatic feed additives. The formulas of the three feeds are shown in Table 1. SeNPs were purchased from Xianfeng Nanotechnology Co., Ltd., and can be prepared by mixing sodium selenite with lactic acid bacteria, culturing, and filtering.

[0020] Table 1 Experimental feed formulation and its nutritional components

[0021] The control group, low-dose additive, and high-dose additive feeds were dried at 105℃ until their quality became constant. Changes in feed moisture content were analyzed, and the Kjeldahl nitrogen determination method was used to determine crude protein content. Crude fat was extracted using petroleum ether extraction. Simultaneously, feed powder samples were thoroughly carbonized in a carbonization furnace and then calcined in a crucible furnace at 550℃ to determine ash content. The moisture, crude protein, crude fat, and ash content results for the three feeds are shown in Table 2.

[0022] Table 2 Results of feed moisture and dry-basis composition (%)

[0023] Therefore, it can be seen that there was no significant change in the crude protein and crude fat content of the three feeds.

[0024] II. Experimental Design and Sample Collection Carp (Cyprinus carpio L.) were purchased and acclimatized in a laboratory for two weeks. During this period, the water temperature was maintained at 26.0±0.2℃, and the dissolved oxygen level was maintained at 5.8±0.2 mg / L. After the acclimatization period, 180 fish (weighing 432.87±0.5 g) were divided into six groups and placed in corresponding breeding tanks: control group 1, control group 2, experimental group 1, experimental group 2, experimental group 3, and experimental group 4, with 30 fish in each group. During the experiment, control group 1 and control group 2 were fed control feed, experimental group 1 and experimental group 3 were fed low-dose additive feed, and experimental group 2 and experimental group 4 were fed high-dose additive feed. On day 30 of the experiment, all carp were fasted, and control group 2, experimental group 3, and experimental group 4 were intraperitoneally injected with 500 μL / fish of 3% sodium dextran sulfate (DSS) to induce an enteritis model. After continuing to be raised for 5 days, the fish were immediately sacrificed, and serum and intestinal tissue samples were collected and preserved at -80℃.

[0025] III. Biochemical Indicator Analysis Serum was diluted with physiological saline at a ratio of 1:9, and inflammatory factors (such as IL-1β and TNFα) were detected using an enzyme-linked immunosorbent assay (ELISA) kit. Simultaneously, intestinal tissue from each group was collected and homogenized at -4°C at a tissue mass (9) to physiological saline volume (mL) ratio of 1:9. The homogenate was then centrifuged at 1500g for 15 minutes, and the supernatant was collected and used to detect pro-inflammatory factors (IL-1β and TNFα) using an ELISA kit. Furthermore, the activities of antioxidant enzymes (T-AOC, SOD, and MDA) in serum and intestinal tissue were also measured. IV. Histopathological Examination Carp midgut tissue was collected, rinsed with PBS to remove contents, fixed in 2.5% glutaraldehyde at 4°C for 24 hours, rinsed three times with PBS, dehydrated with a gradient of ethanol (50%, 70%, 80%, 90%, 100%, 15 minutes each), replaced with isoamyl acetate for 30 minutes, critical point dried, sputtered with gold, and the intestinal mucosal surface structure was observed and photographed under a scanning electron microscope.

[0026] V. Gene Expression Analysis Total RNA was extracted from preserved intestinal tissue samples using TRIzo1 reagent. RNA was reverse transcribed into cDNA using a reverse transcription kit. The reaction volume was 20 μL, including 10 μL 2×SYBR Green PCR Master Mix, 0.8 μL forward and reverse primers (10 μM), 1 μL cDNA template, and 8.2 μL nuclease-free water. The expression of intestinal barrier function-related genes (Occludin, ZO-1, and Claudin-1) was detected by qRTPCR using a real-time quantitative PCR system, with β-actin as an internal control gene. The relative mRNA expression levels among groups were compared. All data are expressed as mean ± standard error (SEM), and statistical analysis was performed using SPSS 20.0 software. One-way ANOVA was used. If one-way ANOVA showed significant differences, Duncan's multiple comparison method was used to assess the significance between groups. Furthermore, t-tests were used to compare differences between control group 1 and control group 2, experimental group 1, experimental group 2, experimental group 3, and experimental group 4. A p-value < 0.05 is considered statistically significant. Values ​​with different superscripts on the same row indicate significant differences.

[0027] 6. Results Analysis 1. Effects of formulated aquatic feed on the survival rate of carp with DSS-induced enteritis Figure 1 It can be concluded that, compared with control group 1, the survival rate of carp with DSS-induced enteritis in control group 2 was significantly decreased (p<0.01). When the aquatic compound feed of the present invention was used, the survival rate of carp with DSS-induced enteritis in experimental groups 2 and 4 was significantly increased (p<0.05). The survival rate of carp with DSS-induced enteritis fed with high doses of the supplementary feed was significantly higher than that of carp with DSS-induced enteritis in other groups (p<0.05). This indicates that SeNPs, Astragalus polysaccharides, and γ-aminobutyric acid in the compound feed of the present invention can effectively alleviate the occurrence of carp enteritis.

[0028] 2. Effects of feed additives on serum and intestinal tissue levels of inflammatory factors in a DSS-induced carp enteritis model To evaluate the alleviating effect of the formulated aquatic feed of this invention on intestinal inflammation, the levels of inflammatory factors IL-1β and TNF-α in the serum and intestinal tissue of carp in each group were detected by ELISA. The results are as follows: Figure 2 As shown.

[0029] In control group 2, the serum and intestinal tissue levels of IL-1β and TNF-α after DSS-induced enteritis were significantly higher than those in control group 1 (p<0.01), indicating that the enteritis model was successfully established. However, after using the aquatic compound feed of this invention, compared with control group 2, the serum IL-1β level in experimental group 2 decreased by 50.7% (p<0.01), the TNF-α level decreased by 53.7% (p<0.01), the intestinal IL-1β level decreased by 55.2% (p<0.01), and the TNF-α level decreased by 67.1% (p<0.01). In experimental group 4, the serum IL-1β level decreased by 64.4% (p<0.01), the TNF-α level decreased by 59.9% (p<0.01), the intestinal IL-1β level decreased by 43.9% (p<0.01), and the TNF-α level decreased by 52.8% (p<0.01). This demonstrates that the synergistic effect of multiple functional components in the aquatic compound feed of the present invention can effectively alleviate the effects of DSS-induced enteritis in carp at the molecular level.

[0030] 3. Effects of feed additives on the activity of antioxidant enzymes in serum and intestinal tissue of a DSS-induced carp enteritis model To evaluate the antioxidant effect of the aquatic compound feed of this invention on the intestines, the content of antioxidant enzymes in the intestinal tissues of each group was measured. The results are as follows: Figure 3 As shown.

[0031] Compared with control group 1, the activity of antioxidant enzymes in the intestinal tissue of carp in control group 2 was significantly reduced, specifically, the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and catalase (CAT) were decreased (p<0.01), while the content of malondialdehyde (MDA) was increased (p<0.05). However, after adding the aquatic feed additive of this invention to the basal feed, the activities of T-AOC, SOD, and CAT in the intestinal tissue of carp in experimental groups 2 and 3 were significantly increased (p<0.01), while the activity of MDA was significantly decreased (p<0.05). This indicates that the aquatic feed additive of this invention, through the combination of SeNPs with astragalus polysaccharide and γ-aminobutyric acid, effectively enhances the antioxidant capacity of carp under intestinal damage conditions and reduces the damage to the body caused by oxidative stress through the synergistic effect of multiple antioxidant components.

[0032] 4. Effects of feed additives on intestinal tissue structure in a DSS-induced carp enteritis model To evaluate the effect of the aquatic compound feed of the present invention on intestinal damage, the ultrastructure of the intestinal tissue surface was analyzed by scanning electron microscopy, and the results are as follows: Figure 4 As shown.

[0033] Compared to control group 1, the midgut surface integrity of carp in control group 2 was significantly impaired, with severe loss of microvilli, complete exposure of columnar cells, and obvious gaps. Furthermore, the midgut surface of experimental groups 1 and 3 was intact and smooth, with tightly packed and uniformly thick microvilli, consistent with the results of control group 1, indicating that the formulated aquatic feed of this invention has no effect on the carp intestine. However, after adding the aquatic feed additive of this invention, the midgut surface integrity of experimental groups 2 and 4 was significantly improved compared to control group 2, with a significant increase in the number of microvilli and partial exposure of columnar cells. This indicates that the aquatic feed additive of this invention can protect the intestinal tissue structure through the combination of SeNPs, Astragalus polysaccharide, and γ-aminobutyric acid, allowing the carp intestinal tissue to maintain its structural integrity even under DSS-induced enteritis conditions.

[0034] 5. Effects of feed additives on the expression of genes related to intestinal barrier function in a DSS-induced carp enteritis model To evaluate the effect of the aquatic compound feed of the present invention on intestinal barrier function, the relative mRNA expression levels of the tight junction proteins Occludin, ZO-1, and Claudin-1 in the intestinal tissue of carp in each group were detected by qRT-PCR. The results are shown in Table 5.

[0035] Compared with control group 1, the intestinal barrier function of carp in control group 2 was significantly reduced, specifically manifested by a significant decrease in the expression of Occludin, ZO-1, and Claudin-1 (p<0.05). However, after adding the aquatic feed additive of this invention to the basal diet, the expression of Occludin, ZO-1, and Claudin-1 in the intestinal tissue of carp in experimental groups 2 and 3 was significantly increased (p<0.05). This indicates that the aquatic feed additive of this invention achieves multiple protections for the intestine through various active ingredients, effectively reducing intestinal damage caused by DSS-induced enteritis and improving the intestinal physiological state of carp under intestinal damage.

[0036] In summary, existing technologies have limited research on the application of multi-component compound additives in aquatic animals, particularly those with intestinal damage. This invention employs a unique feed additive formulation that leverages the combined effects of multiple active substances and synergistic interactions between different components to achieve a multi-pathway, multi-target synergistic effect. This effectively mitigates the adverse effects of intestinal damage on carp from multiple perspectives, including growth performance, endocrine regulation, antioxidant function, and intestinal health. Furthermore, the feed additive components used in this invention are all natural, safe, and non-toxic raw materials, making it more environmentally friendly than conventional intestinal damage mitigation methods such as antibiotics. This has significant implications for guiding production practices and promoting industry development.

[0037] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent substitutions, and improvements made to the above embodiments without departing from the scope of the present invention, based on the technical essence of the present invention and within the spirit and principles of the present invention, shall still fall within the protection scope of the present invention.

Claims

1. A feed additive for repairing intestinal damage in aquatic animals, characterized in that, By weight, it includes: 1-3 parts selenium nanoparticles, 5-10 parts astragalus polysaccharide and 3-5 parts γ-aminobutyric acid.

2. The feed additive for repairing intestinal damage in aquatic animals according to claim 1, characterized in that, The selenium nanoparticles are monodisperse spherical with a particle size of 180 nm or less.

3. A compound feed for aquatic life, characterized in that, The feed, by weight, includes 5-10 parts of the feed additive as described in claim 1 or 2 and 500-1000 parts of the base feed.

4. The aquatic compound feed according to claim 3, characterized in that, The basic feed consists of the following components: 14% fish meal, 25% soybean meal, 25% cottonseed meal, 6% soybean oil, 25% chicken meal, 0.2% choline chloride, 2% calcium dihydrogen phosphate, 2.3% bentonite, 0.1% vitamin premix, 0.05% mineral premix, 0.12% lysine hydrochloride, 0.12% arginine hydrochloride, 0.08% DL-methionine, and 0.03% threonine.

5. A method for preparing aquatic compound feed, characterized in that, Includes the following steps: Step 1: Mix selenium nanoparticles, astragalus polysaccharide and γ-aminobutyric acid evenly according to the weight ratio to obtain a premix; Step 2: Add the carrier to the premix obtained in Step 1 and mix. After mixing evenly, pass the mixture through an 80-mesh sieve and vacuum package it to obtain the feed additive. Step 3: Grind the raw materials for the basic feed according to the weight proportions and sieve them through a 60-mesh sieve; mix them thoroughly after sieving to obtain the basic feed; Step 4: Take the basic feed obtained in Step 3 and the aquatic feed additive obtained in Step 2 according to the weight proportions, add deionized water, mix thoroughly to obtain a feed mixture; the amount of deionized water added is 100-200 mL / kg of the feed mixture mass. Step 5: Use a twin-screw extruder to process the feed mixture obtained in step 4 into pellets with a diameter of 2-3 mm. After sieving, pack the pellets into self-sealing bags and store them at -20℃.

6. The method for preparing aquatic compound feed according to claim 5, characterized in that, The carrier mentioned in step 2 is bentonite.

7. The method for preparing aquatic compound feed according to claim 5, characterized in that, In step 2, a stepwise expansion mixing method is adopted, and the mixture is expanded in stages at a ratio of 1:10 and 1:

10. Each time, the mixture is stirred in a three-dimensional mixer for 30 minutes at a speed of 20 rpm.

8. The application of the aquatic compound feed according to claim 3 or 4 in the preparation of products for the repair of intestinal damage in aquatic animals, characterized in that, The application achieves intestinal repair by enhancing the intestinal antioxidant capacity and maintaining the integrity of the intestinal barrier in aquatic animals.

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