Goldfish feed for enhancing immunity and improving intestinal flora and preparation method thereof

Abstract

The application discloses goldfish feed for enhancing immunity and improving intestinal flora and a preparation method thereof. The goldfish feed is composed of a basic feed and added gambir acid in the basic feed, and the added amount of the gambir acid is 3%-10%. Through adding the gambir acid with a suitable concentration in the basic feed of the Tai Sih goldfish, the application realizes multi-dimensional and synergistic improvement of the growth performance of the fish body, serum biochemical immune indexes, liver antioxidant and damage protection functions, intestinal digestive enzyme activity and intestinal flora structure, and the optimal dosage is 5% of the added amount.

Description

Technical Field

[0001] This invention relates to the field of goldfish feed technology, and more specifically, to a goldfish feed that enhances immunity and improves intestinal flora, and a method for preparing the same. Background Technology

[0002] Currently, functional feeds commonly used in ornamental fish farming mainly enhance fish immunity, promote growth, and prevent diseases by adding vitamins, probiotics, chemically synthesized antioxidants, or antibiotics. For example, some commercial goldfish feeds add immune enhancers such as vitamin C, vitamin E, or β-glucan, or regulate the intestinal microecology through probiotics such as Bacillus and lactic acid bacteria. Some studies have also explored using extracts of traditional Chinese medicine such as astragalus polysaccharides and emodin as feed additives to leverage the antibacterial, anti-inflammatory, and growth-promoting effects of natural active substances.

[0003] However, the long-term use of chemically synthesized additives and antibiotics can easily lead to drug resistance in pathogens, drug residues, and water pollution, which is not in line with the development direction of green and ecological aquaculture. Secondly, the dosage of traditional Chinese herbal extracts often lacks precise experimental evidence, and the effects of different concentrations on growth promotion, anti-oxidation, and immune regulation vary greatly. Furthermore, excessive addition may burden the liver or produce toxic effects.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The technical objective of this application is to address the above-mentioned shortcomings by providing a goldfish feed that enhances immunity and improves gut microbiota, along with its preparation method. This application achieves multi-dimensional synergistic improvement in fish growth performance, serum biochemical immune indicators, liver antioxidant and damage protection functions, intestinal digestive enzyme activity, and gut microbiota structure by adding an appropriate concentration of chebulic acid to the basic feed for Thai Lion goldfish. Overall, the chebulic acid-added group outperformed the control group in terms of weight gain, specific growth rate, and feed utilization. It also significantly increased serum total protein, albumin, and globulin levels, enhanced the activity of key antioxidant enzymes in serum and liver, and effectively reduced the content of lipid peroxidation products, thereby alleviating oxidative stress damage. Furthermore, liver transaminase activity decreased significantly, while alkaline phosphatase and acid phosphatase activities increased significantly, indicating that liver function was protected and non-specific immune function was enhanced. Intestinal digestive enzyme activity was also significantly improved, and gut microbiota diversity and the proportion of dominant bacteria tended to be optimized. Considering the performance of various concentration gradients, a 5% addition level was the optimal dosage.

[0006] To achieve the above objectives, this application provides the following technical solution: According to one aspect of this application, a goldfish feed that enhances immunity and improves gut microbiota is provided, the goldfish feed consisting of a base feed and chebulic acid added to the base feed, the amount of chebulic acid added being 3% to 10%.

[0007] In some embodiments, the chebulic acid is coated on the surface of the base feed.

[0008] In some embodiments, the amount of chebulic acid added is 5%.

[0009] In some embodiments, the goldfish is a Thai lionfish.

[0010] In some embodiments, the basic feed has a crude protein content ≥35%, crude fiber content ≤8.5%, crude ash content ≤15%, crude fat content ≥3%, total phosphorus content ≥1%, lysine content ≥1.65%, and moisture content ≤12%.

[0011] In some embodiments, the purity of the chebulic acid is ≥98% by HPLC.

[0012] According to another aspect of this application, a method for preparing goldfish feed that enhances immunity and improves intestinal flora is provided, comprising: weighing an appropriate amount of chebulic acid powder, adding it to an appropriate amount of distilled water, heating and stirring until completely dissolved, then spraying it evenly on the surface of basic feed pellets, mixing thoroughly, and then drying.

[0013] Compared with existing technologies, the advantages and positive effects of this application are as follows: By adding an appropriate concentration of chebulic acid to the basic feed of Thai goldfish, this application achieves multi-dimensional synergistic improvement in fish growth performance, serum biochemical immune indicators, liver antioxidant and damage protection functions, intestinal digestive enzyme activity, and intestinal flora structure. Overall, the chebulic acid-added group is superior to the control group without chebulic acid in terms of weight gain rate, specific growth rate, and feed utilization rate. It also significantly increases serum total protein, albumin, and globulin levels, enhances the activity of key antioxidant enzymes in serum and liver, and effectively reduces the content of lipid peroxidation products, thereby alleviating oxidative stress damage. Furthermore, liver transaminase activity significantly decreases, while alkaline phosphatase and acid phosphatase activities significantly increase, indicating that liver function is protected and non-specific immune function is enhanced. Intestinal digestive enzyme activity is also significantly improved, and intestinal flora diversity and the proportion of dominant bacteria tend to be optimized. Considering the performance of various concentration gradients, a 5% addition is the optimal dose.

[0014] Furthermore, chebulic acid can promote liver protein synthesis and metabolism, improve the nutritional status and humoral immune reserves of fish; effectively scavenge excess free radicals in the body, inhibit the lipid peroxidation chain reaction, and maintain cell membrane integrity; reduce the activity of hepatocyte damage marker enzymes while enhancing the activity of immune-related hydrolytic enzymes, thus having a dual effect of liver protection and immune enhancement; promote the synergistic secretion and activation of intestinal lipases, proteases, and amylases, improving the digestibility and absorption efficiency of feed nutrients; dynamically regulate the phylum-level structure of the dominant intestinal flora, enhancing the richness and diversity of the flora, thereby optimizing the intestinal microecological balance. Throughout the entire experimental period, the survival rate of each component remained at a high level, confirming that the concentration range of chebulic acid used was safe and harmless, providing a scientific formulation basis and systematic experimental support for the development of functional feeds specifically for Thai goldfish, and demonstrating clear prospects for industrial application. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This paper illustrates the effect of different concentrations of chebulic acid on the total serum protein of Thai goldfish in the embodiments of this application.

[0017] Figure 2 The effects of different concentrations of chebulic acid on serum albumin in Thai goldfish are shown in the embodiments of this application.

[0018] Figure 3 This paper illustrates the effect of different concentrations of chebulic acid on serum globulin in Thai goldfish in the embodiments of this application.

[0019] Figure 4 This paper illustrates the effect of different concentrations of chebulic acid on serum SOD in Thai goldfish in the embodiments of this application.

[0020] Figure 5 This paper illustrates the effect of different concentrations of chebulic acid on the serum CAT of Thai goldfish in the embodiments of this application.

[0021] Figure 6 This paper illustrates the effect of different concentrations of chebulic acid on serum MDA in goldfish from the embodiments of this application.

[0022] Figure 7 This paper illustrates the effect of different concentrations of chebulic acid on serum AKP in Thai goldfish in the embodiments of this application.

[0023] Figure 8 This paper illustrates the effect of different concentrations of chebulic acid on serum ACP in Thai goldfish in the embodiments of this application.

[0024] Figure 9 This paper illustrates the effect of different concentrations of chebulic acid on serum GPT in Thai goldfish in the embodiments of this application.

[0025] Figure 10 This paper illustrates the effect of different concentrations of chebulic acid on serum GOT in goldfish in the embodiments of this application.

[0026] Figure 11 This paper illustrates the effect of different concentrations of chebulic acid on the total protein in the liver of Thai goldfish in the embodiments of this application.

[0027] Figure 12 This paper illustrates the effect of different concentrations of chebulic acid on SOD in the liver of Thai goldfish in the embodiments of this application.

[0028] Figure 13 This paper illustrates the effect of different concentrations of chebulic acid on the CAT (caryotrophic lateral sclerosis) of the liver of the Thai goldfish in the embodiments of this application.

[0029] Figure 14 This paper illustrates the effect of different concentrations of chebulic acid on GSH-Px in the liver of Thai goldfish in the embodiments of this application.

[0030] Figure 15 This paper illustrates the effect of different concentrations of chebulic acid on GSH in the liver of Thai goldfish in the embodiments of this application.

[0031] Figure 16 This paper illustrates the effect of different concentrations of chebulic acid on T-AOC in the liver of Thai goldfish in the embodiments of this application.

[0032] Figure 17 This paper illustrates the effect of different concentrations of chebulic acid on MDA in the liver of Thai goldfish in the embodiments of this application.

[0033] Figure 18 This paper illustrates the effect of different concentrations of chebulic acid on GPT in the liver of Thai goldfish in the embodiments of this application.

[0034] Figure 19 This paper illustrates the effect of different concentrations of chebulic acid on the liver GOT of the Thai goldfish in the embodiments of this application.

[0035] Figure 20 The effect of feed chebulic acid concentration on intestinal lipase in Thai lions is shown in the embodiments of this application (14d).

[0036] Figure 21 The effect of feed chebulic acid concentration on intestinal lipase in Thai lions is shown in the embodiments of this application (28 days).

[0037] Figure 22 The effect of feed chebulic acid concentration on intestinal trypsin in Thai lions is shown in the embodiments of this application (14d).

[0038] Figure 23 The effect of feed chebulic acid concentration on intestinal trypsin in Thai lions is shown in the embodiments of this application (28 days).

[0039] Figure 24 The effect of feed chebulic acid concentration on intestinal amylase in Thai lions is shown in the embodiments of this application (14d).

[0040] Figure 25 The effect of feed chebulic acid concentration on intestinal amylase in Thai lions is shown in the embodiments of this application (28 days).

[0041] Figure 26 The Venn diagrams of gut microbiota after 14 days and 28 days of feeding are shown in the embodiments of this application.

[0042] Figure 27 The PCoA diagram of gut microbiota is shown in the embodiments of this application.

[0043] Figure 28 The examples in this application show the relative abundance of chebulic acid concentration in the feed of Thai lions at the phylum level.

[0044] Figure 29 The examples in this application show the relative abundance of chebulic acid concentration in the feed of Thai lion gut microbiota at the genus level. Detailed Implementation

[0045] To better understand the above-mentioned objectives, features, and advantages of this application, the application will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

[0046] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0047] I. Experimental Materials and Methods: 1. Test materials: (1) Experimental fish, basic feed and chebulic acid.

[0048] The fish used in this experiment were transported from Tianjin Muchen Aquatic Technology Development Co., Ltd. to the laboratory of Tianjin Agricultural College. The Thai goldfish were temporarily kept in an aquarium for one week to allow them to acclimatize before their weight was measured. The average weight of the Thai goldfish was 50.96 ± 5.69 g. The basic feed was extruded compound feed for fish (153 Ornamental Fish Special) from Tongwei Co., Ltd., composed of soybean meal, fish meal, wheat flour, rapeseed meal, cottonseed meal, soybean oil, vitamins, and minerals. The product composition analysis showed crude protein ≥35%, crude fiber ≤8.5%, crude ash ≤15%, crude fat ≥3%, total phosphorus ≥1%, lysine ≥1.65%, and moisture ≤12%. Chebulinic acid (CAS: 18942-26-2) powder was obtained from Changqing Biotechnology Co., Ltd., grade: scientific research analysis reference standard, purity: HPLC ≥98%.

[0049] (2) Test instruments and equipment.

[0050] Electronic analytical balance (Sartorius, BSA224S, Beijing Sartorius Scientific Instruments Co., Ltd.); High-speed refrigerated centrifuge (JW-2017HR, Anhui Jiawen Instrument Equipment Co., Ltd.); High-speed tissue homogenizer (Tiss-24, Shanghai Cebo Biotechnology Development Center); Water bath (TW12, JULABO, Germany); Ultraviolet-visible spectrophotometer (UH5300, HITACHI Corporation, Japan); Fully automated biological microscope (BA600-4, Macody Industrial Group Co., Ltd.); Microplate reader (SpectraMax Mini, MOLECULAR DEVICES, USA). Pathological tissue embedding machine (HS-B7126, Shenyang Hengsong Technology Co., Ltd.); Paraffin slicer (RM-2255, Leica GmbH, Germany); Flat sheeter (HI-1220, Leica, Germany); Electric heating drying oven (BGZ-76, Shanghai Boxun Medical Bio-Instrument Co., Ltd.)

[0051] (3) Test reagents.

[0052] The following kits were purchased from Nanjing Jiancheng Bioengineering Institute: Superoxide Dismutase (SOD) Assay Kit, Malondialdehyde (MDA) Assay Kit, Glutathione Peroxidase (GSH-Px) Assay Kit, Reduced Glutathione (GSH) Assay Kit, Total Antioxidant Capacity (T-AOC) Assay Kit, Catalase (CAT) Assay Kit, Alanine Aminotransferase (ALT / GPT) Assay Kit, Aspartate Aminotransferase (AST / GOT) Assay Kit, Alkaline Phosphatase (ALP / AKP) Assay Kit, Acid Phosphatase (ACP) Assay Kit, Total Protein (TP) Assay Kit, and Albumin (ALB) Assay Kit.

[0053] Formaldehyde (Tianjin Jinke Fine Chemical Research Institute, batch number: 20250923); Sodium dihydrogen phosphate (Tianjin Guangfu Technology Development Co., Ltd., batch number: 20200402). Disodium hydrogen phosphate (Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.); Anhydrous ethanol (Tianjin Jindong Tianzheng Fine Chemical Reagent Factory); tert-Butanol (Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.); Xylene (Tianjin Fengchuan Chemical Reagent Technology Co., Ltd., batch number: 202209102); Paraffin wax (Shanghai Huayong Paraffin Wax Co., Ltd.); Optical resin (Shanghai Specimen Model Factory).

[0054] 2. Experimental methods.

[0055] (1) Preparation of experimental feed.

[0056] This experiment consisted of four groups: a control group (basic feed only), experimental group 1 (basic feed supplemented with 3% chebulic acid), experimental group 2 (basic feed supplemented with 5% chebulic acid), and experimental group 3 (basic feed supplemented with 10% chebulic acid). When preparing the feed, an appropriate amount of chebulic acid powder was weighed according to the corresponding proportion and added to an appropriate amount of distilled water. The mixture was heated and stirred until completely dissolved. Then, it was evenly sprayed onto the surface of the basic feed pellets, thoroughly mixed, and then dried for later use.

[0057] (2) Experimental fish farming and sample collection.

[0058] After temporary rearing and acclimatization, 360 healthy and disease-free Thai goldfish were randomly selected and grouped into three parallel groups of 30 fish each. During the experiment, the water temperature was maintained at 24±1℃, one-third of the water was changed daily, and the fish were fed twice a day (10:00 and 17:00). Uneaten food and feces in the aquarium were cleaned.

[0059] On day 14 of rearing and day 28 after rearing, samples were collected after a 24-hour fast. Before blood collection, the centrifuge tubes and syringes were moistened with heparin, and blood was drawn from the ventral side of the goldfish's tail stalk. The blood was centrifuged at 3000 r / min for 10 min at 4°C to separate the serum, which was then stored at -80°C for later use. After blood collection, the goldfish were dissected. A portion of the liver tissue was fixed in 10% neutral formalin solution for histopathological section preparation; the remaining liver tissue was placed in a sealed bag and stored at -80°C for liver function assays.

[0060] (3) Sample determination.

[0061] Blood test indicators: TP, ALB, GLB, SOD, CAT, MDA, AKP, ACP, GPT, GOT.

[0062] Liver function indicators: TP, SOD, CAT, MDA, GSH, GSH-PX, GPT, GOT, T-AOC.

[0063] Before measuring liver markers, homogenize the sample using a homogenizer. Centrifuge the supernatant of the homogenate at 3000 r / min for 10 min at 4℃, and then measure each marker. Blood and liver markers were measured according to the instructions of the kits sold by Nanjing Jiancheng Biotechnology Institute.

[0064] Enzyme activity assay: Follow the instructions of the Nanjing Jiancheng assay kit.

[0065] Liver tissue sections: Liver tissue underwent dehydration with tert-butanol, clearing with xylene, and paraffin embedding. The sections were then cut into continuous sections 5-7 μm thick using a microtome. After hematoxylin-eosin (HE) staining, the sections were mounted and observed and photographed using a slide scanner.

[0066] Intestinal flora determination: The collected intestinal contents were entrusted to Novogene Corporation for intestinal flora determination using 16S rRNA amplicon sequencing technology. The determination included sequencing analysis and bacterial species identification.

[0067] (4) Data processing and analysis.

[0068] Experimental data are expressed as mean ± standard error (Mean ± SE). One-way ANOVA was performed using SPSS 26.0 software. P When the value is less than 0.05, the difference is considered significant, and a chart is created using Excel software.

[0069] III. Results Analysis: 1. Effect of feed chebulic acid concentration on the growth performance of Thai lions: The effects of adding different concentrations of chebulic acid to the feed on the growth performance of Thai goldfish are shown in Table 1-2. The survival rate of all groups reached 100%, indicating that the addition of chebulic acid to the feed did not negatively affect the survival of the Thai goldfish. In terms of weight gain rate, specific growth rate, and liver-to-body ratio, experimental groups B, C, and D were all higher than control group A, and the feed conversion ratio of the experimental groups was lower than that of the control group. As shown in Table 1, by day 14 of feeding, there were no significant differences in any of the indicators among the different groups. P >0.05). Group B achieved the highest values ​​in weight gain rate, specific growth rate, and offal-to-body ratio, while its feed conversion ratio was the lowest among all experimental groups. Group C had the highest liver-to-body ratio. Table 2 shows that after 28 days of feeding, Group B maintained the highest levels of weight gain rate, specific growth rate, and offal-to-body ratio among all groups, with Group B's offal-to-body ratio significantly higher than Group A ( P >0.05), which is 1.17 times that of group A. The liver-to-body ratio also reached its maximum in group B, and there were no significant differences in other indicators except for the organ-to-body ratio. P >0.05).

[0070] Table 1. Effect of feed chebulic acid concentration on the growth performance of Thai lions (14 days)

[0071] Table 2. Effect of feed chebulic acid concentration on the growth performance of Thai lions (28 days)

[0072] 2. Effects of adding different concentrations of chebulic acid to feed on serum-related indicators of Thai goldfish.

[0073] (1) Effects on serum protein metabolism indicators.

[0074] The effects of adding different concentrations of chebulic acid to feed on the serum total protein (TP), albumin (ALB), and globulin (GLB) content of Thai goldfish are as follows: Figure 1 , Figure 2 , Figure 3 As shown. By Figure 1-3 It can be seen that on day 14 of the experiment, there was no significant difference between the 3% chebulic acid addition group and the control group. P >0.05), the 5% chebulic acid addition group was significantly higher than the control group ( P <0.05), TP and ALB levels in the 10% chebulic acid-added group were significantly higher than those in the control group ( P <0.05), GLB showed no significant difference ( P >0.05). By Figure 1-3 It can be seen that on day 28 of the experiment, the levels of chebulic acid in all three groups were significantly higher than those in the control group. P<0.05, among which the 5% chebulic acid addition group showed the most significant improvement, while there was no significant difference between the 3% chebulic acid addition group and the 10% chebulic acid addition group. P >0.05). Comparing the two sampling time points, the serum TP, ALB, and GLB levels in each experimental group increased with the extension of the experimental period. The levels on day 28 were all higher than those on day 14, with the 5% chebulic acid addition group showing the most significant increase.

[0075] (2) Effects on serum antioxidant indicators.

[0076] The effects of adding different concentrations of chebulic acid to feed on the activity of SOD, CAT and MDA in the serum of Thai goldfish are as follows: Figure 4 , Figure 5 , Figure 6 As shown. By Figure 4-6 It can be seen that on day 14 of the experiment, there was no significant difference between the 3% concentration chebulic acid group and the control group. P >0.05); the 5% concentration chebulic acid addition group was significantly better than the control group ( P <0.05%, the 10% concentration chebulic acid addition group was significantly better than the control group ( P <0.05), but inferior to the 5% concentration chebulic acid supplement group. (By...) Figure 4-6 As shown, on day 28 of the experiment, all three groups were significantly better than the control group. P <0.05), the 5% concentration of chebulic acid added group showed the best effect. As the experimental period was extended, the activities of SOD and CAT in each experimental group gradually increased, while the content of MDA gradually decreased. The antioxidant effect on day 28 was better than that on day 14.

[0077] (3) Effects on serum immunity and liver damage-related indicators.

[0078] The effects of adding different concentrations of chebulic acid to feed on the serum AKP, ACP, GPT, and GOT activities of Thai goldfish are as follows: Figure 7 , Figure 8 , Figure 9 , Figure 10 As shown. By Figure 7-10 It can be seen that on day 14 of the experiment, there was no significant difference between the 3% concentration chebulic acid addition group and the control group. P >0.05); the 5% concentration chebulic acid addition group was significantly better than the control group ( P <0.05, the AKP and GPT levels in the 10% concentration chebulic acid-added group were significantly higher / lower than those in the control group ( P <0.05), there was no significant difference in ACP and GOT ( P >0.05), and all indicators were inferior to the 5% concentration chebulic acid supplement group. (From...) Figure 7-10 As shown, on day 28 of the experiment, all three groups were significantly better than the control group. P<0.05, the 5% concentration of chebulic acid added group showed the most significant increase / decrease, while there was no significant difference between the 3% concentration of chebulic acid added group and the 10% concentration of chebulic acid added group. P >0.05). The activities of AKP and ACP in each experimental group increased with the extension of the experimental period, while the activities of GPT and GOT decreased with the extension of the experimental period.

[0079] 3. Effects of adding different concentrations of chebulic acid to feed on liver-related indicators of Thai goldfish.

[0080] (1) Effects on liver protein metabolism and antioxidant indicators.

[0081] The effects of adding different concentrations of chebulic acid to feed on the total protein (TP), SOD, CAT, GPX activity, and GSH, T-AOC, and MDA content in the liver of Thai goldfish are as follows: Figure 11-17 As shown. By Figure 11-17 It can be seen that on day 14 of the experiment, there were no significant differences in various indicators between the 3% concentration chebulic acid addition group and the control group. P >0.05); the 5% concentration chebulic acid added group had significantly higher levels of TP, SOD, CAT, GPX, GSH, and T-AOC than the control group ( P <0.05), MDA was significantly lower than that of the control group ( P <0.05); the 10% concentration chebulic acid addition group showed significantly better performance in all indicators than the control group ( P <0.05), but inferior to the 5% concentration chebulic acid supplement group. (By...) Figure 11-17 As shown, on day 28 of the experiment, the 5% concentration chebulic acid addition group reached the highest levels of all indicators, with highly significant differences compared to the control group. P <0.01); All indicators in the 3% and 10% chebulic acid addition groups were lower than those in the 5% chebulic acid addition group, and there was no significant difference. P >0.05).

[0082] (2) Effects on liver injury-related indicators.

[0083] The effects of adding different concentrations of chebulic acid to feed on the activities of alanine aminotransferase (GPT) and aspartate aminotransferase (GOT) in the liver of Thai goldfish, as shown in the following figures. Figure 18 , Figure 19 As shown. By Figure 18 , 19 It can be seen that on day 14 of the experiment, there was no significant difference between the 3% concentration chebulic acid addition group and the control group. P >0.05); the 5% concentration of chebulic acid added group was significantly lower than the control group ( P <0.05, the GPT in the 10% concentration chebulic acid addition group was significantly lower than that in the control group ( P <0.05), GOT showed no significant difference (P >0.05), and the activities of both enzymes were higher than those in the 5% chebulic acid-added group. (From...) Figure 18 , 19 As shown, on day 28 of the experiment, the difference between the 5% chebulic acid addition group and the control group was extremely significant. P <0.01), all three groups were significantly lower than the control group ( P <0.05, there was no significant difference between the 3% concentration chebulic acid addition group and the 10% concentration chebulic acid addition group ( P >0.05). The activities of GPT and GOT in each experimental group gradually decreased with the extension of the experimental period.

[0084] 4. Effect of feed chebulic acid concentration on digestive enzymes in the intestines of Thai lions.

[0085] (1) Effect of feed chebulic acid concentration on intestinal lipase in Thai lions.

[0086] The effects of adding different concentrations of chebulic acid to feed on intestinal lipase in Thai goldfish, such as Figure 20-21 As shown. The lipase activities in both the foregut and hindgut of the experimental group were higher than those in the control group. At 14 days of feeding, the foregut lipase activity reached its maximum in group B, significantly higher than in the other groups ( P <0.05, which is 1.97 times that of group A. Midgut lipase activity was highest in group A, significantly exceeding the other three experimental groups ( P <0.05. Hindgut lipase activity reached its maximum value in group B, significantly higher than in groups A and D. P <0.05, which is 1.96 times that of group A. At 28 days of feeding, the foregut lipase activity reached its maximum in group B, significantly higher than in other groups ( P <0.05). The maximum midgut lipase activity was observed in group B, significantly higher than in other groups (P<0.05). The hindgut lipase activity in group B was significantly higher than in other groups (P<0.05).

[0087] (2) Effect of feed chebulic acid concentration on intestinal trypsin in Thai lions.

[0088] The effects of adding different concentrations of chebulic acid to feed on intestinal trypsin in Thai goldfish, such as Figure 22-23 As shown. There was no significant difference in foregut trypsin activity among the groups (P<0.05). After 14 days of feeding, the foregut trypsin activity in group B was higher than that in the other groups, and the midgut and hindgut trypsin activities in group B were significantly higher than those in the other groups. P<0.05), midgut activity was 1.52 times that of group A, and hindgut activity was 1.64 times that of group A. At 28 days of feeding, the foregut trypsin activity in group B was higher than that in the other groups, the midgut trypsin activity was highest in group D, and the hindgut trypsin activity was highest in group B, significantly higher than that in other groups (P<0.05), and was 1.46 times that of group A.

[0089] (3) Effect of feed chebulic acid concentration on intestinal amylase in Thai lions.

[0090] The effects of adding different concentrations of chebulic acid to feed on intestinal amylase in Thai goldfish, such as Figure 24-25 As shown in the figure, the intestinal amylase activity of the experimental fish showed a trend of increasing, decreasing, and then increasing again with the increase of chebulic acid concentration in the feed, with the lowest activity in group A and the highest activity in group B. As can be seen from the figure, at 14 days of feeding, the maximum amylase activity in the foregut, midgut, and hindgut was found in group B, and group B was significantly higher than the other groups (P<0.05), being 1.29 times, 1.47 times, and 1.46 times that of group A, respectively. At 28 days of feeding, group B still possessed the highest amylase activity in all intestinal segments. The foregut amylase activity in group B was significantly higher than that in group A (…). P <0.05), which is 1.17 times that of group A. The midgut and hindgut amylase activities in group B were significantly higher than those in the other treatment groups (P<0.05).

[0091] 5. The effect of feed chebulic acid concentration on the intestinal flora of Thai lions.

[0092] (1) Venn diagram analysis.

[0093] The Venn diagram of the gut microbiota in this experiment is shown below. Figure 26 .in, Figure 26 A is 14d. Figure 26 B is 28d. For example... Figure 26 As shown, after 14 days of feeding, groups A, B, C and D had a total of 74 OTUs, with the number of unique OTUs being 141 (A), 355 (B), 280 (C) and 194 (D), respectively; after 28 days of feeding, groups A, B, C and D had a total of 142 OTUs, with the number of unique OTUs being 488 (A), 274 (B), 431 (C) and 249 (D), respectively.

[0094] (2) Alpha diversity analysis.

[0095] The degree of species isolation within a community can be evaluated using alpha diversity, which is primarily assessed from two perspectives: species richness (i.e., species composition) and species evenness (i.e., distribution characteristics). This study used the total number of species in the sample (Chao1), the number of observed species (Sob), the coverage of each sample library (Good's coverage), evenness (Pielou), Shannon index, and Simpson index to analyze the differences in alpha diversity among the sample groups. Among these, Chao1 and Sob were positively correlated with bacterial community richness, while the Shannon index was positively correlated with bacterial community diversity.

[0096] The effects of adding different concentrations of chebulic acid to the feed on the gut microbiota of Thai goldfish are shown in Table 3-4. The coverage rate of samples in each experimental group reached 1, indicating that the sequencing depth met the requirements and was sufficient to accurately represent the actual composition of the gut microbiota of Thai goldfish. Although there were some differences among the groups in the Chao1 index, Sob index, Pielou index, Shannon index, and Simpson index, most of these differences did not reach statistical significance. P <0.05). After 14 days of feeding, with increasing feed chebulic acid concentration, the Alpha diversity index of the experimental fish, except for Coverage, showed a trend of first increasing and then decreasing, reaching its maximum in group B. The Pielou index of group B was significantly higher than that of groups C and D. P <0.05, the Shannon index and Simpson index of group B were significantly higher than those of group D. P <0.05). Group A had the lowest Chao1 and Sob indices. At 28 days of feeding, Group C had the highest Chao1 and Sob indices, but Group C's Pielou and Shannon indices were lower than Group A's. Group A had the highest Pielou, Shannon, and Simpson indices, while Group B had the lowest Simpson index.

[0097] Table 3. Effect of feed chebulic acid concentration on the Alpha diversity index of intestinal flora in Thai lions (14 days)

[0098] Table 4. Effect of feed chebulic acid concentration on the Alpha diversity index of intestinal flora in Thai lions (28 days)

[0099] (3) Beta diversity analysis.

[0100] The results of the PCoA analysis of gut microbiota in this experiment are shown below. Figure 27The distance between samples was inversely correlated with the similarity of their species composition; the smaller the distance, the higher the similarity. Therefore, samples with highly similar community structures tended to cluster together, while samples with significantly different community compositions tended to be more dispersed. Overall, the samples in each group showed varying degrees of dispersion, but no significant dispersion was observed between the groups themselves. These results indicate that no significant differences were detected in Beta diversity among the experimental groups.

[0101] (4) Analysis of gut microbiota composition.

[0102] The top 10 species with the highest relative abundance of gut microbiota at the phylum level in this experiment are listed below. Figure 28 .Depend on Figure 28 It was found that at the phylum level, the dominant bacterial groups were mainly Fusobacteriota, Bacillota, Verrucomicrobiota, Pseudomonadota, Bacteroidota, Chlamydiota, Actinomycetota, Dependentiae, Chloroflexota, and Planctomycetota, but the content of each group was not the same. After 14 days of feeding, as the concentration of chebulic acid in the feed increased, the relative abundance of Fusobacteriota and Verrucomicrobiota showed a trend of first decreasing and then increasing. Fusobacteriota was the phylum with the highest proportion in groups A, C, and D, accounting for 31.64%, 43.97%, and 63.32% respectively, while the highest proportion in group B was Firmicutes, accounting for 38.25%. At 28 days of feeding, Proteobacteria was the most abundant phylum in groups A and C, accounting for 37.03% and 35.01% respectively; Fusobacteria was the most abundant phylum in groups B and C, accounting for 56.01% and 43.33% respectively. The abundance of phyla in each group fluctuated with increasing chebulic acid concentration in the feed. These results indicate that differences in chebulic acid concentration in the feed affect the phylum-level composition of the intestinal microbiota.

[0103] Figure 29 This describes the species composition of each group of gut microbiota at the genus level. The dominant genera are primarily *Cetobacter* (*Cetacea*). Cetobacterium ), Microbacteria ( Exiguobacterium ), Akkermania ( Akkermansia Bacteroides ( Bacteroides ), Chlamydia genus ( Neochlamydia Aeromonas spp. Aeromonas ), Cumulus ( ) Culicoidibacter ), Methylerythrophytes ( Methylorubrum ).in accordance with Figure 10 Data shows that the dominant bacterial genus in groups A, C, and D was *Cetacea*, with proportions of 31.64%, 43.97%, and 63.32% respectively after 14 days of feeding, and 31.05%, 15.33%, and 43.33% respectively after 28 days of feeding. In group B, *Microbacterium* had the highest relative abundance at 14 days (24.86%), followed by *Cetacea* at 9.10%; *Cetacea* had the highest relative abundance at 28 days (56.01%).

[0104] Unless otherwise specified in the examples, the procedures shall be performed in accordance with conventional conditions in the field or product instructions; reagents and instruments whose manufacturers are not specified shall be conventional products that can be purchased through legitimate channels.

[0105] Through the above specific embodiments, those skilled in the art can easily implement this application. However, it should be understood that this application is not limited to the specific embodiments described above. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. A goldfish feed that enhances immunity and improves gut microbiota, characterized in that, The goldfish feed consists of a base feed and chebulic acid added to the base feed, wherein the amount of chebulic acid added is 3% to 10%.

2. The goldfish feed for enhancing immunity and improving intestinal flora according to claim 1, characterized in that, The chebulic acid is coated on the surface of the base feed.

3. The goldfish feed for enhancing immunity and improving intestinal flora according to claim 1, characterized in that, The amount of chebulic acid added is 5%.

4. The goldfish feed for enhancing immunity and improving intestinal flora according to claim 1, characterized in that, The goldfish in question is a Thai lion goldfish.

5. The goldfish feed for enhancing immunity and improving intestinal flora according to claim 1, characterized in that, The basic feed contains ≥35% crude protein, ≤8.5% crude fiber, ≤15% crude ash, ≥3% crude fat, ≥1% total phosphorus, ≥1.65% lysine, and ≤12% moisture.

6. The goldfish feed for enhancing immunity and improving intestinal flora according to claim 1, characterized in that, The purity of the chebulic acid is ≥98% by HPLC.

7. A method for preparing goldfish feed that enhances immunity and improves intestinal flora, characterized in that, include: Weigh an appropriate amount of chebulic acid powder and add it to an appropriate amount of distilled water. Heat and stir until completely dissolved. Then spray it evenly on the surface of the basic feed pellets, mix thoroughly, and then air dry.

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