Application of mulberry leaf polysaccharide in preparation of product for improving adverse effects caused by feeding of largemouth bass with high-starch feed

Abstract

The invention provides application of mulberry leaf polysaccharide in preparation of a product for improving adverse effects caused by feeding largemouth bass with high-starch feed, and belongs to the technical field of feed. The invention provides application of mulberry leaf polysaccharide in preparation of a product for improving adverse effects caused by feeding of largemouth bass with high-starch feed. The adverse effects comprise growth performance reduction, oxidation resistance reduction, liver inflammation, liver glucose metabolism capability reduction, liver lipid metabolism capability reduction, intestinal flora disorder, intestinal muscle layer thickness reduction and / or intestinal short-chain fatty acid content reduction. According to the invention, the effect of the mulberry leaf polysaccharide on improving the adverse effects caused by feeding the micropterus salmoides with the high-starch feed is proposed for the first time, a brand new and effective solution is expected to be provided for green and healthy breeding of the micropterus salmoides, the blank in the field is filled, and the mulberry leaf polysaccharide has great significance on promoting sustainable development of aquaculture industry.

Description

Technical Field

[0001] The invention belongs to the technical field of feed, and particularly relates to the application of mulberry leaf polysaccharide in preparing a product for improving adverse effects caused by feeding largemouth bass with high-starch feed. Background Art

[0002] The largemouth bass (Micropterus salmoides), also known as the California bass, belongs to the order Perciformes, suborder Porcoidei, family Cehtrachidae, genus Micropterus, and is native to North America. Largemouth bass grows rapidly, has delicious meat, and is highly nutritious, making it an economically viable aquaculture fish. Largemouth bass are carnivorous, primarily consuming live bait fish, and are currently cultured on a fully formulated feed system. Starch, as the primary energy source in feed and an essential ingredient in the production of extruded feed, must be added to a certain amount of aquaculture feed. However, numerous studies on largemouth bass have shown that long-term feeding of diets containing more than 10% starch reduces digestibility, causes glycogen and lipid accumulation, and induces inflammation, oxidative stress, and immunosuppression, thereby inhibiting growth. Although chemically synthesized α-glucose inhibitors can achieve the effect of lowering blood sugar, they are highly toxic, costly, and have a single effect. There is an urgent need to find natural, healthy, and widely effective feed additives to protect the health of fish fed high-starch feed.

[0003] Mulberry leaf polysaccharides, a natural active ingredient extracted from mulberry leaves, possess multiple properties, including antioxidant, antibacterial, probiotic, and hypoglycemic properties. In livestock and poultry farming, they have been shown to improve intestinal microbiota and enhance immune function. However, their effectiveness in preventing and treating the adverse effects of high-starch feeds in largemouth bass farming has yet to be systematically studied and applied. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide the use of mulberry leaf polysaccharide in the preparation of a product for improving the adverse effects caused by feeding largemouth bass with high starch feed.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of mulberry leaf polysaccharide in the preparation of a product for improving the adverse effects caused by feeding largemouth bass with high-starch feed, wherein the adverse effects include decreased growth performance, decreased antioxidant capacity, liver inflammation, decreased liver glucose metabolism capacity, decreased liver lipid metabolism capacity, intestinal flora disorder, decreased intestinal muscle layer thickness and / or decreased intestinal short-chain fatty acid content.

[0007] Preferably, the decreased growth performance includes decreased fatness, increased organ-to-body ratio, increased liver-to-body ratio, increased feed conversion ratio and / or decreased specific growth rate.

[0008] Preferably, the decreased liver glucose metabolism ability includes an increase in liver glycogen content and / or an increase in serum glucose content.

[0009] Preferably, the decreased liver lipid metabolism ability includes an increased triglyceride content.

[0010] Preferably, the decreased antioxidant capacity includes an increase in malondialdehyde content, a decrease in total superoxide dismutase activity and / or a decrease in glutathione peroxidase activity.

[0011] Preferably, the intestinal short-chain fatty acids include propionic acid and / or butyric acid.

[0012] Preferably, the high-starch feed is a feed with a starch content of more than 10% by mass.

[0013] Preferably, the high-starch feed comprises 40% fish meal, 15% soy protein concentrate, 4% blood meal, 17% peeled soybean meal, 13% wheat starch, 1% monocalcium phosphate, 0.2% vitamin premix, 0.8% mineral element premix, 0.4% choline, 2% soybean lecithin oil, 3% soybean oil, 3% fish oil, and 0.6% microcrystalline cellulose.

[0014] Preferably, the preparation method of mulberry leaf polysaccharide includes the following steps: crushing the mulberry leaves and mixing them with water, performing low-temperature and high-pressure differential extraction, then taking the supernatant, mixing it with anhydrous ethanol, and taking the precipitate to obtain mulberry leaf polysaccharide; the temperature of the low-temperature and high-pressure differential extraction is 20-30°C, the pressure is 25-35MPa, and the extraction time is 30min each time.

[0015] The present invention also provides a high-starch feed for raising largemouth bass, comprising 40% fish meal, 15% soybean protein concentrate, 4% blood meal, 17% peeled soybean meal, 13% wheat starch, 1% calcium dihydrogen phosphate, 0.2% vitamin premix, 0.8% mineral element premix, 0.4% choline, 2% soybean lecithin oil, 3% soybean oil, 3% fish oil, and 0.6% mulberry leaf polysaccharide.

[0016] Beneficial effects of the present invention:

[0017] The present invention proposes for the first time that mulberry leaf polysaccharides have the function of improving the adverse effects caused by feeding largemouth sea bass with high-starch feed, and is expected to provide a new and effective solution for the green and healthy breeding of largemouth sea bass, filling the gap in this field, and having great significance for promoting the sustainable development of the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1This is the result of liver tissue section of largemouth bass (HE staining);

[0019] Figure 2 This is the result of intestinal tissue section of largemouth bass (HE staining);

[0020] Figure 3 Effects of adding mulberry leaf polysaccharide to high-starch feed on the expression of genes related to glucose metabolism and lipid metabolism in the liver of largemouth bass. A represents genes related to glucose metabolism; B represents genes related to lipid metabolism. Data columns with different lowercase letters indicate significant differences (P<0.05), and no letters or the same letters indicate no significant differences (P>0.05).

[0021] Figure 4 The effect of adding mulberry leaf polysaccharide to high starch diet on the expression of inflammation-related genes in the liver of largemouth bass. Data columns with different lowercase letters indicate significant differences (P<0.05), and no letters or the same letters indicate no significant differences (P>0.05).

[0022] Figure 5 Effects of adding mulberry leaf polysaccharide to high starch feed on the content of short-chain fatty acids in the intestine of largemouth bass. Data columns with different lowercase letters indicate significant differences (P<0.05), and no letters or the same letters indicate no significant differences (P>0.05).

[0023] Figure 6 The effect of adding mulberry leaf polysaccharide to high starch feed on the intestinal flora diversity of largemouth bass, where A is a Venn diagram; B is the β-diversity result, C is the α-diversity result, and from left to right and from top to bottom in C are the ACE index, Chao1 index, Shannon index and Simpson index results respectively;

[0024] Figure 7 The effect of adding mulberry leaf polysaccharide to high starch feed on the intestinal flora structure of largemouth bass. The left figure shows the results at the phylum level, and the right figure shows the results at the genus level.

[0025] Figure 8 These are the prediction results of intestinal microbial functions of largemouth bass, where A is the prediction result of microbial function at the L2 level, and B and C are the prediction results of microbial function at the L3 level. DETAILED DESCRIPTION

[0026] The present invention provides the use of mulberry leaf polysaccharide in the preparation of a product for improving the adverse effects caused by feeding largemouth bass with high-starch feed, wherein the adverse effects include decreased growth performance, decreased antioxidant capacity, liver inflammation, decreased liver glucose metabolism capacity, decreased liver lipid metabolism capacity, intestinal flora disorder, decreased intestinal muscle layer thickness and / or decreased intestinal short-chain fatty acid content.

[0027] In the present invention, the decreased growth performance preferably includes decreased fatness, increased organ-to-body ratio, increased liver-to-body ratio, increased feed conversion ratio and / or decreased specific growth rate; the decreased liver glucose metabolism ability preferably includes increased liver glycogen content and / or increased serum glucose content; the decreased liver lipid metabolism ability preferably includes increased triglyceride content; the decreased antioxidant capacity preferably includes increased malondialdehyde content, decreased total superoxide dismutase activity and / or decreased glutathione peroxidase activity; the intestinal short-chain fatty acids preferably include propionic acid and / or butyric acid.

[0028] Feeding largemouth bass with a high-starch feed can induce oxidative stress and inflammatory responses in largemouth bass, causing disorders in sugar metabolism, lipid metabolism and intestinal flora, leading to a decrease in the growth performance of largemouth bass. Adding mulberry leaf polysaccharides to high-starch feed can improve the antioxidant capacity of largemouth bass, the diversity of intestinal flora and the relative abundance of beneficial bacteria, promote glycolysis and lipid oxidation and decomposition, and can also effectively alleviate the inflammatory response of the liver. In the present invention, adding mulberry leaf polysaccharides to high-starch feed can increase the fatness and specific growth rate of largemouth bass fed high-starch feed, and can reduce the organ-to-body ratio, liver-to-body ratio and feed coefficient of largemouth bass fed high-starch feed. Adding mulberry leaf polysaccharides to high-starch feed can reduce the levels of triglycerides and glucose in the serum of largemouth bass fed a high-starch diet, reduce the activity of alanine aminotransferase and aspartate aminotransferase in the serum, and reduce the levels of triglycerides, glycogen, and malondialdehyde in the liver. It can also increase the activity of total superoxide dismutase and glutathione peroxidase in the liver and the total antioxidant capacity. Adding mulberry leaf polysaccharides to high-starch feed can reduce liver inflammation and prevent liver necrosis in largemouth bass fed a high-starch diet, increase the thickness of the intestinal muscular layer, and increase the content of short-chain fatty acids in the intestine.

[0029] In the present invention, the high-starch feed is preferably a feed with a starch mass percentage of more than 10%, and more preferably a feed with a starch mass percentage of 12.68%. In the present invention, the high-starch feed preferably includes 40% fish meal, 15% soybean protein concentrate, 4% blood meal, 17% peeled soybean meal, 13% wheat starch, 1% monocalcium phosphate, 0.2% vitamin premix, 0.8% mineral element premix, 0.4% choline, 2% soybean lecithin oil, 3% soybean oil, 3% fish oil and 0.6% microcrystalline cellulose. In the application of the present invention, when mulberry leaf polysaccharide is added to the high-starch feed, the addition amount of the mulberry leaf polysaccharide is preferably 0.6%. When adding mulberry leaf polysaccharide, the microcrystalline cellulose in the high-starch feed can be removed. In the present invention, all "%" refer to mass percentages.

[0030] In the present invention, the preparation method of mulberry leaf polysaccharide preferably includes the following steps: crushing the mulberry leaves and mixing them with water, performing low-temperature and high-pressure differential extraction, then taking the supernatant, mixing it with anhydrous ethanol, and taking the precipitate to obtain mulberry leaf polysaccharide; the temperature of the low-temperature and high-pressure differential extraction is 20-30°C, the pressure is 25-35MPa, and the extraction time is 30min each time.

[0031] The present invention does not specifically limit the specific method of pulverization. In some embodiments, an ultrafine grinder is used for pulverization. After pulverization, the mulberry leaves are preferably passed through a 60-100 mesh screen, more preferably a 70-90 mesh screen. In the present invention, after obtaining mulberry leaf powder, the mass-to-volume ratio of mulberry leaf powder to water is preferably 1g:20-30mL, more preferably 1g:22-28mL. In the present invention, the temperature for low-temperature, high-pressure differential extraction is preferably 22-28°C, more preferably 24-26°C; the pressure for low-temperature, high-pressure differential extraction is preferably 26-33 MPa, more preferably 28-31 MPa. In the present invention, the number of low-temperature, high-pressure differential extractions is preferably 3-4 times. In the present invention, the volume ratio of supernatant to anhydrous ethanol is preferably 1:5, and anhydrous ethanol is added to the mixture for alcohol precipitation, preferably for 24 hours. In the present invention, the precipitate is preferably washed, and the washing solvent is preferably anhydrous ethanol. The invention adopts low temperature and high pressure difference to extract mulberry leaf polysaccharide from mulberry leaves, which has the advantage of high extraction yield, and the mulberry leaf polysaccharide extracted under low temperature conditions has high activity.

[0032] The present invention also provides a high-starch feed for raising largemouth bass, comprising 40% fish meal, 15% soybean protein concentrate, 4% blood meal, 17% peeled soybean meal, 13% wheat starch, 1% calcium dihydrogen phosphate, 0.2% vitamin premix, 0.8% mineral element premix, 0.4% choline, 2% soybean lecithin oil, 3% soybean oil, 3% fish oil, and 0.6% mulberry leaf polysaccharide.

[0033] The present invention has no special restrictions on the specific sources of the raw materials except mulberry leaf polysaccharide. Any commercially available product in the art can be used. The fish meal is preferably Peruvian fish meal. In the present invention, the composition of the vitamin premix is ​​preferably as follows: per kilogram of vitamin premix contains: vitamin A 66666666.7 IU, vitamin D 400000000 IU, vitamin E 1g, vitamin K2 g, vitamin B15 g, vitamin B2 5g, vitamin B6 5g, vitamin B 121 g, calcium pantothenate 20 g, folic acid 10 g, biotin 1 g, niacin 20 g, choline chloride 200 g, defatted rice bran 700 g. The composition of the mineral element premix is ​​preferably as follows: per kilogram of the mineral element premix contains: CuCO3 4 g, FeC6H5O 715 g, MgO 26 g, MnSO4 5 g, KCl 250 g, ZnSO4 50 g, NaCl 50 g, zeolite powder 600 g.

[0034] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] In the following examples, unless otherwise specified, all methods are conventional.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] The data in the following examples were processed using SPSS 22.0 software for one-way analysis of variance (ANOVA). Significant differences were detected using Duncan's method for multiple comparisons between groups. Results are presented as mean ± standard error (SE). Significant differences were considered at P < 0.05.

[0038] Example 1

[0039] Preparation of mulberry leaf polysaccharide:

[0040] The freshly picked mulberry leaves were washed and dried, and then pulverized with an ultrafine grinder and passed through an 80-mesh sieve to obtain ultrafine mulberry leaf powder. The ultrafine mulberry leaf powder was weighed, and 25 mL of distilled water was added per 1 g of ultrafine mulberry leaf powder. The distilled water was added to the ultrafine mulberry leaf powder and mixed. The mixture was then added to a differential pressure extractor and subjected to low-temperature and high-pressure differential extraction at a temperature of 25° C. and a pressure of 30 MPa. Each extraction time was 30 minutes, and the extraction was repeated 3 times. The mixture was centrifuged at 10,000 r / min for 20 minutes. The supernatant was taken, and anhydrous ethanol 5 times the volume of the supernatant was added, and the mixture was precipitated in a refrigerator at 4° C. for 24 hours. After centrifugation, the supernatant was removed, and the precipitate was washed 4 times with anhydrous ethanol. The mixture was centrifuged and freeze-dried to obtain mulberry leaf polysaccharide.

[0041] Example 2

[0042] Preparation of mulberry leaf polysaccharide:

[0043] The freshly picked mulberry leaves were washed and dried, and then pulverized with an ultrafine grinder and passed through a 60-mesh sieve to obtain ultrafine mulberry leaf powder. The ultrafine mulberry leaf powder was weighed, and 20 mL of distilled water was added per 1 g of ultrafine mulberry leaf powder. The distilled water was added to the ultrafine mulberry leaf powder and mixed. The mixture was then added to a differential pressure extractor and subjected to low-temperature and high-pressure differential extraction at a temperature of 20° C. and a pressure of 25 MPa. Each extraction time was 30 minutes, and the extraction was repeated 3 times. The mixture was centrifuged at 10,000 r / min for 20 minutes. The supernatant was taken, and anhydrous ethanol 5 times the volume of the supernatant was added, and the mixture was precipitated in a refrigerator at 4° C. for 24 hours. After centrifugation, the supernatant was removed, and the precipitate was washed 4 times with anhydrous ethanol. The mixture was centrifuged and freeze-dried to obtain mulberry leaf polysaccharide.

[0044] Example 3

[0045] Preparation of mulberry leaf polysaccharide:

[0046] The freshly picked mulberry leaves were washed and dried, and then pulverized with an ultrafine grinder and passed through a 100-mesh sieve to obtain ultrafine mulberry leaf powder. The ultrafine mulberry leaf powder was weighed, and 30 mL of distilled water was added per 1 g of ultrafine mulberry leaf powder. The distilled water was added to the ultrafine mulberry leaf powder and mixed. The mixture was then added to a differential pressure extractor and subjected to low-temperature and high-pressure differential extraction at a temperature of 30° C. and a pressure of 35 MPa. Each extraction time was 30 min, and the extraction was repeated 3 times. The mixture was centrifuged at 10,000 r / min for 20 min, the supernatant was taken, and anhydrous ethanol 5 times the volume of the supernatant was added, and the mixture was precipitated in a refrigerator at 4° C. for 24 h. After centrifugation, the supernatant was removed, and the precipitate was washed 4 times with anhydrous ethanol, centrifuged, and the precipitate was freeze-dried to obtain mulberry leaf polysaccharide.

[0047] Example 4

[0048] Three experimental feeds were prepared using Peruvian fish meal, soy protein concentrate, and dehulled soybean meal as the primary protein sources, and fish oil, soybean oil, and soybean lecithin oil as the primary fat sources: a low-starch group (CK group, starch content: 7.58%), a high-starch group (HD group, starch content: 12.68%), and a high-starch group supplemented with mulberry leaf polysaccharides obtained in Example 1 (MLP group, starch content: 12.68%, MLP added at 0.6%). All raw materials were ground and passed through a 60-mesh sieve, weighed and mixed according to the ratios in Table 1, conditioned with 102°C steam for 8 minutes, then extruded into 3mm pellets, dried at 55°C, and stored at -20°C for later use. The experimental feed composition and nutritional levels are shown in Table 1.

[0049] Table 1 Composition of experimental feed and its nutritional level (dry matter basis)

[0050]

[0051]

[0052] Each kilogram of vitamin premix in Table 1 contains: Vitamin A 666666666.7 IU, Vitamin D 400000000 IU, Vitamin E 1 g, Vitamin K2 g, Vitamin B15 g, Vitamin B25 g, Vitamin B65 g, Vitamin B 12 1 g, calcium pantothenate 20 g, folic acid 10 g, biotin 1 g, niacin 20 g, choline chloride 200 g, defatted rice bran 700 g. Each kilogram of mineral element premix contains: CuCO3 4 g, FeC6H5O 715 g, MgO 26 g, MnSO4 5 g, KCl 250 g, ZnSO4 50 g, NaCl 50 g, zeolite powder 600 g. The nutrient levels in Table 1 are measured values. The crude protein, crude fat, crude ash, and starch contents of the feed were determined according to GB / T 6432-2018, GB / T 6433-2006, GB / T 6438-2007, and GB / T 42491-2023, respectively.

[0053] Example 5

[0054] The three experimental feeds obtained in Example 4 were respectively used to feed largemouth bass, specifically:

[0055] Largemouth bass (Black Bass) were provided by Guangdong Liangshi Aquatic Seed Co., Ltd. (Foshan) and maintained in an indoor recirculating aquaculture system at the Institute of Sericulture and Agricultural Products Processing, Guangdong Academy of Agricultural Sciences, for one week. They were fed twice daily with the CK diet. Following a 24-hour fast, 270 juveniles with an average initial weight of 35.34 ± 0.52 g were selected and randomly assigned to nine 75-cm-diameter culture tanks (350 L of aquaculture water volume), with 30 fish per tank. The nine tanks were then randomly divided into three groups, each with three replicates. The fish were fed twice daily (at 9:00 AM and 4:30 PM) to indicate apparent satiation. Any uneaten feed was removed 0.5 h after feeding, dried, and weighed. Daily feed intake and mortality were accurately recorded. The experimental period lasted 60 days. During the experiment, the water was changed three times a week, with each water change amounting to one-third of the water volume in each culture tank. The water temperature was maintained between 25.4 and 30.2°C, the dissolved oxygen content was ≥6.5 mg / L, the nitrite content was ≤0.05 mg / L, the ammonia nitrogen content was ≤0.2 mg / L, the pH was 6.5 to 8.0, and natural light was used. The animal experiments were approved by the Animal Welfare and Ethics Committee of the Guangdong Academy of Agricultural Sciences (Approval No. GDAAS2022015).

[0056] After the feeding experiment, the fish were fasted for 24 hours, and the number, total weight and total food intake of each replicate test fish were counted, and the terminal body weight, weight gain rate, specific growth rate and feed coefficient were calculated; 9 test fish were randomly selected from each replicate to measure body length and weight, calculate fatness, and then blood was collected from the tail vein, kept at 4℃ for 2 hours, centrifuged at 1324×g for 10 minutes, and the supernatant was taken to measure biochemical indicators; the internal organs and liver were separated and weighed, and the organ-to-body ratio and liver-to-body ratio were calculated; the foregut and liver of 6 test fish were randomly selected from each replicate and stored in a refrigerator at -80℃ for the determination of intestinal flora, short-chain fatty acids, liver biochemical indicators, sugar metabolism and lipid metabolism, and inflammation-related genes; the midgut and liver of 3 test fish were randomly selected from each replicate, and fixed with 4% paraformaldehyde for tissue section analysis.

[0057] 5.1 Growth performance

[0058] Weight gain rate (WGR, %) = (W f -W i ) / W i ×100; specific growth rate (SGR, % / d) = (lnW f -lnW i ) / T×100; Food intake (FI, g / fish) = F t (g) / [(M i +M f ) / 2]; Feed coefficient (FC) = Ft (g) / (W ft -W it ); Fullness (CF, g / cm 3 )=W f / L f 3 ×100; Visceral-to-body ratio (VSI, %) = W v / W f ×100; liver-to-body ratio (HSI, %) = W h / W f ×100; where: W i is the initial body weight, W f is the final body weight, T is the culture time, F t is the total food intake, M i is the initial mantissa, M f is the final digit, W ft is the final total weight, W it is the initial total weight, L f is the terminal body length, W v is the wet weight of visceral mass, W h It is caused by dampness in the liver.

[0059] The results are shown in Table 2. Compared with the HD group, the final body weight, WGR, and CF were significantly increased in the CK and MLP groups (P < 0.05), and the VSI and HSI were significantly decreased (P < 0.05). Compared with the HD group, the SGR was significantly increased in the MLP group (P < 0.05), and the FC was significantly decreased (P < 0.05). There was no significant difference in FI among the groups (P > 0.05).

[0060] Table 2 Effects of adding mulberry leaf polysaccharide to high starch feed on the growth performance of largemouth bass

[0061]

[0062] Note: Data in the same row with different lowercase letters indicate significant differences (P<0.05), while the same lowercase letters or no letters indicate no significant differences (P>0.05). The meanings of the letters in the same row in the following tables are the same as in this table and will not be repeated here.

[0063] 5.2 Determination of serum and liver biochemical and antioxidant indicators

[0064] According to the manufacturer's instructions, commercial kits from Nanjing Jiancheng Bioengineering Institute were used to measure serum triglyceride (TG), glucose (GLU), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities, as well as liver TG and glycogen (GLY) contents, total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) activities, total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content.

[0065] The results of serum biochemical analysis are shown in Table 3. Compared with the HD group, the TG and CLU contents, ALT and AST activities in the CK and MLP groups were significantly decreased (P<0.05); compared with the CK group, the CLU content in the MLP group was significantly decreased (P<0.05).

[0066] Table 3 Effects of adding mulberry leaf polysaccharide to high starch feed on serum biochemistry of largemouth bass

[0067]

[0068] The results of liver biochemical and antioxidant index analysis are shown in Table 4. Compared with the HD group, the CK and MLP groups had significantly decreased liver TG, GLY, and MDA contents (P<0.05), and significantly increased T-SOD, GSH-Px, and T-AOC activities (P<0.05). Compared with the CK group, the MLP group had significantly increased T-AOC activity (P<0.05).

[0069] Table 4 Effects of adding mulberry leaf polysaccharide to high starch feed on biochemical indices and antioxidant capacity of largemouth bass liver

[0070]

[0071] 5.3 Intestinal and liver tissue section analysis

[0072] The midgut and liver were carefully removed, the fat was carefully removed, and the tissue sections were rinsed with 4°C saline and fixed in 4% paraformaldehyde. The preparation of intestinal and liver tissue sections and HE staining were based on the methods in the article (Fu Bing, Peng Kai, Chen Bing, et al. Effects of broad bean supplementation on growth performance, intestinal digestive enzyme activity, intestinal morphology, and intestinal flora of tilapia [J]. Journal of Animal Nutrition, 2022, 35(1): 494-504.).

[0073] Liver pathological section results Figure 1 As shown, the hepatocytes of largemouth bass in the CK and MLP groups were tightly arranged, with normal cell morphology and no obvious necrosis or inflammatory reaction. However, the hepatocytes of largemouth bass in the HD group showed a large number of vacuolation (black triangles) and occasional inflammatory cell infiltration (red arrows).

[0074] The results of intestinal tissue morphology are shown in Table 5 and Figure 2 As shown, the intestinal structure of each group was intact. Compared with the HD group, the thickness of the muscular layer in the CK and MLP groups was significantly increased (P<0.05). There was no significant difference in the height and width of the intestinal villi among the groups (P>0.05).

[0075] Table 5 Effects of adding mulberry leaf polysaccharide to high starch feed on intestinal tissue morphology of largemouth bass

[0076]

[0077]

[0078] 5.4 Expression of genes related to hepatic glucose metabolism, lipid metabolism, and inflammation

[0079] Liver RNA extraction, reverse transcription, RT-qPCR, and calculation of relative gene expression were performed according to the description of the article (ZHOU DL, ZHONG WH, FU B, et al. Dietary supplementation of mulberry leaf oligosaccharides improves the growth, glucose and lipid metabolism, immunity, and virus resistance in largemouth bass (Micropterus salmoides) [J]. Frontiers in Immunology, 2025, 16: 1525992.). Primers for interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB), transforming growth factor-β (TGF-β), glucokinase (GK), phosphofructokinase (PFK), acetyl-CoA carboxylase (ACC), glycerol acyltransferase (DGAT), fatty acid synthase (FAS), peroxisome proliferator-activated receptor-α (PPAR-α), carnitine acyltransferase-1 (CPT-1), and β-actin were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer information is shown in Table 6.

[0080] Table 6 RT-qPCR primers

[0081]

[0082] The results of liver glucose metabolism and lipid metabolism related gene detection are as follows Figure 3 As shown in the data, compared with the HD group, the expression levels of pfk, gk, ppar-α and cpt-1 in the CK and MLP groups were significantly increased (P<0.05), and the expression levels of dgat, fas and acc were significantly decreased (P<0.05); compared with the CK group, the expression levels of pfk, gk, ppar-α and cpt-1 in the MLP group were significantly increased (P<0.05), and the expression levels of dgat and fas were significantly decreased (P<0.05).

[0083] Liver inflammation-related gene test results Figure 4 As shown in the data, compared with the HD group, the expression levels of IL-10 and TGF-β in the CK and MLP groups were significantly increased (P<0.05), and the expression levels of IL-8, NF-κB, and TNF-α were significantly decreased (P<0.05); compared with the CK group, the expression levels of IL-10 and TGF-β in the MLP group were significantly increased (P<0.05), and the expression levels of IL-8 and TNF-α were significantly decreased (P<0.05).

[0084] 5.5 Determination of short-chain fatty acids

[0085] Intestinal short-chain fatty acid content was determined using a gas chromatograph (GC-2010PLUS, Shimadzu, Japan). 0.2 g of the chopped sample was placed in a 2 mL centrifuge tube, and 1 mL of 10 mmol / L sodium hydroxide solution (containing 5 mmol / L crotonic acid solution) was added. The tube was shaken for 30 seconds and centrifuged at 13201 × g for 2 minutes. The supernatant was filtered through a 0.22 μm membrane and stored at -20°C overnight.

[0086] The chromatographic conditions were carried out according to the article (HU TG, YU YS, WU JJ, et al. Structural elucidation of mulberry leaf oligosaccharide and its selective promotion of gut microbiota to alleviate type 2 diabetes mellitus[J]. Food Science and Human Wellness, 2024, 13(4): 2161-2173.), with the following differences: chromatographic column: DB-FFAP column (30m×0.25μm ID, 0.25μm), nitrogen and hydrogen as carrier gas, split ratio of 1:10, flow rate set to 30.0mL / min, and injector and detector temperatures were both set to 240℃.

[0087] Short-chain fatty acid determination results Figure 5 As shown in the results, compared with the HD group, the intestinal propionic acid content in the CK and MLP groups was significantly increased (P<0.05), and the butyric acid content in the MLP group was significantly increased (P<0.05). There was no significant difference in the acetic acid content among the groups (P>0.05).

[0088] 5.6 Intestinal flora analysis

[0089] After extracting intestinal DNA using a kit, primers were designed based on sequences from the 16S DNA "V3+V4" hypervariable regions for PCR amplification of total intestinal bacterial DNA. The primers used were 338F (5′-ACTCCTACGGGAGGCAGCAG-3′ (SEQ ID NO. 27)) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′ (SEQ ID NO. 28)). PE reads generated by MiSeq high-throughput sequencing were first spliced ​​based on overlap relationships, and sequence quality control and filtering were performed. After sample differentiation, operational taxonomic unit (OTU) cluster analysis and species taxonomy analysis were performed. Community composition, alpha diversity, and beta diversity were statistically analyzed based on OTUs. PICRUSt 2 software was used to predict the function of the intestinal microbiome in largemouth bass.

[0090] The results of intestinal flora diversity are as follows Figure 6 As shown, a total of 1440 OTUs were detected in the three groups, of which 138 were shared among the three groups. The numbers of unique OTUs in the CK, HD, and MLP groups were 352, 241, and 525, respectively. Principal component analysis (PCoA) results showed that the CK group clustered clearly with the HD and MLP groups, with some overlap between the HD and MLP groups, suggesting that MLP can improve the intestinal microbial composition of largemouth bass fed a high-starch diet. There were no significant differences in the ACE index, Chao1 index, Shannon index, and Simpson index among the groups (P>0.05).

[0091] The relative abundance of intestinal flora of largemouth bass at the phylum and genus levels are as follows: Figure 7 As shown. At the phylum level, the dominant bacterial groups were Fusobacteriota, Proteobacteria, Firmicutes, Actinobacteriota, and Bacteriodota, respectively. These five phyla accounted for over 90% of the intestinal microbiota in each group. Compared with the HD group, the relative abundances of Bacteroidetes, Actinobacteria, and Firmicutes increased in the CK and MLP groups (P>0.05), while the relative abundance of Fusobacteria decreased (P>0.05). At the genus level, compared with the HD group, the relative abundance of Cetobacterium decreased in the CK and MLP groups (P>0.05), while the relative abundance of Lactobacillus increased (P>0.05). The relative abundance of Aeromonas increased in the MLP group (P>0.05).

[0092] The prediction results of intestinal microbial function of largemouth bass are as follows Figure 8As shown. At the L2 level, compared with the HD group, the MLP group showed significant increases in amino acid metabolism, lipid metabolism, xenobiotic biodegradation and metabolism, transport, and catabolism (P < 0.05). At the L3 level, compared with the CK group, the HD group showed significant decreases in histidine metabolism, limonene and pinene degradation, lipid metabolism, D-arginine and D-ornithine metabolism, and amebiasis (P < 0.05). Compared with the HD group, the MLP group showed significant increases in glycine, serine, and threonine metabolism, histidine metabolism, tricarboxylic acid cycle, glutathione metabolism, fatty acid degradation, arginine and proline metabolism, valine, leucine, and isoleucine degradation, glyoxylate and dicarboxylic acid metabolism, cyanoamino acid metabolism, β-alanine metabolism, geraniol degradation, unsaturated fatty acid biosynthesis, oxidative phosphorylation, tryptophan metabolism, tyrosine metabolism, phenylalanine metabolism, inositol phosphate metabolism, ascorbate and oxyde metabolism, peroxisome, and novobiocin biosynthesis (P < 0.05).

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of mulberry leaf polysaccharide in the preparation of a product for improving the adverse effects of feeding largemouth bass with high starch feed, characterized in that: The adverse effects include decreased growth performance, decreased antioxidant capacity, liver inflammation, decreased liver glucose metabolism capacity, decreased liver lipid metabolism capacity, intestinal flora disturbance, decreased intestinal muscle layer thickness and / or decreased intestinal short-chain fatty acid content.

2. The use according to claim 1, characterized in that The decreased growth performance includes decreased fatness, increased organ-to-body ratio, increased liver-to-body ratio, increased feed conversion rate and / or decreased specific growth rate.

3. The use according to claim 1, characterized in that The decreased liver glucose metabolism ability includes an increase in liver glycogen content and / or an increase in serum glucose content.

4. The use according to claim 1, characterized in that The decreased liver lipid metabolism ability includes an increased triglyceride content.

5. The use according to claim 1, characterized in that The decreased antioxidant capacity includes increased malondialdehyde content, decreased total superoxide dismutase activity and / or decreased glutathione peroxidase activity.

6. The use according to claim 1, characterized in that The intestinal short-chain fatty acids include propionic acid and / or butyric acid.

7. The use according to claim 1, characterized in that The high-starch feed is a feed with a starch content of more than 10% by mass.

8. The use according to claim 1, characterized in that The high-starch feed comprises 40% fish meal, 15% soybean protein concentrate, 4% blood meal, 17% peeled soybean meal, 13% wheat starch, 1% monocalcium phosphate, 0.2% vitamin premix, 0.8% mineral element premix, 0.4% choline, 2% soybean lecithin oil, 3% soybean oil, 3% fish oil, and 0.6% microcrystalline cellulose.

9. The use according to claim 1, characterized in that The preparation method of mulberry leaf polysaccharide comprises the following steps: crushing mulberry leaves and mixing them with water, performing low-temperature and high-pressure differential extraction, then taking the supernatant, mixing it with anhydrous ethanol, and taking the precipitate to obtain mulberry leaf polysaccharide; the temperature of the low-temperature and high-pressure differential extraction is 20-30°C, the pressure is 25-35MPa, and the extraction time is 30 minutes each time.

10. A high starch feed for raising largemouth bass, characterized in that: Including fish meal 40%, soy protein concentrate 15%, blood meal 4%, peeled soybean meal 17%, wheat starch 13%, calcium dihydrogen phosphate 1%, vitamin premix 0.2%, mineral element premix 0.8%, choline 0.4%, soybean lecithin oil 2%, soybean oil 3%, fish oil 3%, mulberry leaf polysaccharide 0.6%.

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