An antiviral sulfated mulberry leaf oligosaccharide, its preparation method, animal feed, and applications.

Abstract

This invention provides an antiviral sulfated mulberry leaf oligosaccharide, its preparation method, animal feed, and applications, belonging to the field of oligosaccharide technology. The invention involves microwave degradation and H2O2-Vc degradation of mulberry leaf polysaccharides, followed by sulfation modification of the degradation products to obtain sulfated mulberry leaf oligosaccharides. Experiments have shown that adding sulfated mulberry leaf oligosaccharides to feed for aquaculture can promote the growth of largemouth bass, improve feed utilization, antioxidant capacity, lipid metabolism, immune function, and enhance resistance to viral infections.

Description

Technical Field

[0001] This invention belongs to the field of oligosaccharide technology, specifically relating to an antiviral sulfated mulberry leaf oligosaccharide, its preparation method, animal feed, and applications. Background Technology

[0002] Largemouth bass (Micropterus salmoides) is one of China's most important freshwater economic fish, possessing significant aquaculture value. Farming largemouth bass not only provides my country with a large amount of high-quality animal protein but also brings substantial economic benefits. However, with the expansion of farming scale and increasing stocking density, the farming environment has gradually deteriorated, and disease problems in largemouth bass have become increasingly prominent, causing huge economic losses and severely restricting the sustainable development of the industry. To prevent, treat, and control diseases, antibiotics and chemicals are often used in aquaculture; however, excessive use of these agents leads to drug residues, adversely affecting the environment, human health, and animal safety. Currently, China has banned the addition of antibiotics to feed, and "antibiotic reduction and replacement" has become a major trend. Therefore, developing environmentally friendly and safe antibiotic alternatives has become a hot research topic in the industry. Among these, functional additives with preventative effects, such as prebiotics and plant extracts, have become a key research focus.

[0003] Prebiotics are organic substances that are not directly digested and absorbed by the host, but can selectively promote the growth or increase the activity of a small number of beneficial bacteria in the colon, thereby improving the host's health. With the deepening research on the functions and mechanisms of action of prebiotics, their application in the breeding of livestock and poultry such as chickens, pigs, cattle, and sheep is becoming increasingly widespread. Because prebiotics can improve the health of farmed aquatic animals, promote growth, and enhance immunity, they have been gradually used in aquaculture since the 1990s. Currently, prebiotics that can be used as feed additives are mainly oligosaccharides made from monosaccharide units such as fructose, galactose, glucose, or xylose, such as commercially available fructooligosaccharides, galactooligosaccharides, and isomaltooligosaccharides. Currently, commercially available oligosaccharides are generally chemically synthesized and have a single composition; research on functional oligosaccharides from plant sources is relatively limited.

[0004] Mulberry leaf polysaccharide (MLP) is a natural active ingredient extracted from mulberry leaves, possessing multiple functions including antioxidant, antibacterial, probiotic, hypoglycemic, and immune-stimulating effects. Studies have shown that dietary supplementation with MLP can improve the gut microbiota of weaned piglets, reduce diarrhea rates, and enhance the growth performance of early-weaned piglets. Furthermore, MLP can significantly promote the secretion of IL-2, IFN-γ, and sIgA in the jejunum and trachea of ​​chickens, promote IgA production in the cecum and tonsils, and enhance the immune function of the intestinal and tracheal mucosa. It is generally believed that the bioactivity of polysaccharides can be improved by degrading them into low-molecular-weight oligosaccharides through physical, chemical, and biological methods. MLP can be enzymatically hydrolyzed to obtain mulberry leaf oligosaccharides (MLO). Studies have shown that compared to mulberry leaf polysaccharide, mulberry leaf oligosaccharides have higher probiotic activity and antioxidant capacity. Furthermore, MLO can easily pass through the digestive system and reach the colon without being degraded. MLO also exhibits stronger activity in promoting the growth of probiotics and enhancing their ability to produce short-chain fatty acids. However, to date, there are no reports on the application of MLO as a feed additive in antiviral research in aquatic animals.

[0005] References

[0006] [1]LUSHCHAKV I.Environmentally induced oxidative stress in aquaticanimals[J].Aquatic Toxicology, 2011,101(1):13-30.

[0007] [2]RICO A,VAN DEN BRINK P J.Probabilistic risk assessment ofveterinary medicines applied to four major aquaculture species produced inAsia[J].Sci Total Environ,2014,468-469:630-41.

[0008] [3]CHU L,SU DQ,WANG HX,et al.Association between AntibioticExposure and Type 2Diabetes Mellitus in Middle-Aged and Older Adults[J].NUTRIENTS,2023,15(5).

[0009] [4]MEHDI Y,LETOURNEAU-MONTMINY M P,GAUCHER M L,et al.Use ofantibiotics in broiler production:Global impacts and alternatives[J].ANIMALNUTRITION,2018,4(2):170-8.

[0010] [5]PATEL S,GOYAL A.The current trends and future perspectives ofprebiotics research:a review[J].3BIOTECH,2012,2(2):115-25.

[0011] [6]YOUSEFIAN M,AMIRI M S.A review of the use of prebiotic inaquaculture for fish and shrimp[J].AFRICAN JOURNAL OF BIOTECHNOLOGY,2009,8(25):7313-8.

[0012] [7] E,OLSEN R E,GIFSTAD T et al.Prebiotics in aquaculture:areview[J].Aquaculture Nutrition,2010,16(2):117-36.

[0013] [8]GIBSON G R,PROBERT H M,VAN LOO J,et al.Dietary modulation ofthehuman colonic microbiota:updating the concept ofprebiotics[J].NUTRITIONRESEARCH REVIEWS,2004,17(2):259-75.

[0014] [9]AL-KHALAIFA H,AL-NASSERA,AL-SURAYEE T,et al.Effect of dietaryprobiotics and prebiotics on the performance of broiler chickens[J].POULTRYSCIENCE,2019,98(10):4465-79.

[0015]

[10] YAQOOB M U,ABD EL-HACK M E,HASSAN F,et al.The potentialmechanistic insights and future implications for the effect ofprebiotics onpoultry performance,gut microbiome,and intestinal morphology[J].POULTRYSCIENCE,2021,100(7).

[0016]

[11] CORINO C,DI GIANCAMILLO A,MODINA S C,et al.Prebiotic EffectsofSeaweed Polysaccharides in Pigs[J].ANIMALS,2021,11(6).

[0017]

[12] HOMOLKA M N,SMITH W N,HUSZ T C,et al.Evaluating performance ofbeef cattle consuming a supplemental probiotic-prebiotic during the first 21or 42 days after feedlot arrival[J].LIVESTOCK SCIENCE,2023,270.

[0018]

[13] CHASHNIDEL Y,BAHARI M,YANSARI A T,et al.The Effects of DietarySupplementation of Prebiotic and Peptide on Growth Performance and BloodParameters in Suckling Zell Lambs[J].SMALL RUMINANT RESEARCH,2020,188.

[0019]

[14] HANLEY F,BROWN,H.&CARBERY,J..First Observations on the Effects ofMannan Oligosaccharide Added to Hatchery Diets for Warmwater Hybrid RedTilapia.[J].Poster at the 11th Annual Symposium on Biotechnology in the FeedIndustry,Lexington,KY,USA.

[0020]

[15] GUERREIRO I,OLIVA-TELES A,ENES P.Prebiotics as functionalingredients:focus on Mediterranean fish aquaculture[J].Reviews inAquaculture,2017,10(4):800-32.

[0021]

[16] WU Y,LIU W B,LI H Y,et al.Effects ofdietary supplementation offructooligosaccharide on growth performance,body composition,intestinalenzymes activities and histology of blunt snout bream(Megalobramaamblycephala)fingerlings[J].Aquaculture Nutrition,2013,19(6):886-94.

[0022]

[17] GRISDALE-HELLAND B,HELLAND S J,GATLIN D M.The effects of dietarysupplementation with mannanoligosaccharide,fructooligosaccharide orgalactooligosaccharide on the growth and feed utilization of Atlantic salmon(Salmo salar)[J].AQUACULTURE,2008,283(1-4):163-7.

[0023]

[18] LI J Q,TAN B P,MAI K S.Dietary probiotic Bacillus OJ andisomaltooligosaccharides influence the intestine microbial populations,immuneresponses and resistance to white spot syndrome virus in shrimp(Litopenaeusvannamei)[J].AQUACULTURE,2009,291(1-2):35-40.

[0024]

[19] MA Q Q,SANTHANAM R K,XUE Z H,et al.Effect of different dryingmethods on the physicochemical properties and antioxidant activities ofmulberry leaves polysaccharides[J].INTERNATIONAL JOURNAL OF BIOLOGICALMACROMOLECULES,2018,119:1137-43.

[0025]

[20] WANG F,LI J R,JIANG Y M.POLYSACCHARIDES FROM MULBERRY LEAF INRELATION TO THEIR ANTIOXIDANT ACTIVITY AND ANTIBACTERIAL ABILITY[J].JOURNALOF FOOD PROCESS ENGINEERING,2010,33(1):39-50.

[0026]

[21] WANG Y Q,SHAO S C,GUO C L,et al.The homogenous polysaccharideSY01-23 purified from leaf of Morus alba L.has bioactivity on human gutBacteroides ovatus and Bacteroides cellulosilyticus[J].INTERNATIONAL JOURNALOF BIOLOGICAL MACROMOLECULES,2020,158:698-707.

[0027]

[22] CHEN S K,XI M M,GAO F,et al.Evaluation of mulberry leaves'hypoglycemic properties and hypoglycemic mechanisms[J].FRONTIERS INPHARMACOLOGY,2023,14.

[0028]

[23] CHEN X L,YANG H F,JIA J P,et al.Mulberry leafpolysaccharidesupplementation contributes to enhancing the respiratory mucosal barrierimmune response in Newcastle disease virus-vaccinated chicks[J].POULTRYSCIENCE,2021,100(2):592-602.

[0029]

[24] ZHAO X J,LI L,LUO Q L,et al.Effects ofmulberry(Morus alba L.)leafpolysaccharides on growth performance,diarrhea,blood parameters,and gutmicrobiota of early-weanling pigs[J].LIVESTOCK SCIENCE,2015,177:88-94.

[0030]

[25] Purification,characterization and in vitro and in vivo immuneenhancement ofpolysaccharides from mulberry leaves[J].PLoS ONE,2019.

[0031]

[26] MARTINA M,BEATRIZ G,REMEDIOS Y,et al.Kinetic assessment on theautohydrolysis of pectin-rich by-products[J].Chemical Engineering Journal,2010,162(2).

[0032]

[27] BINGJIAN D,YI S,XIAOSONG H,et al.Oligosaccharides prepared byacid hydrolysis of polysaccharides from pumpkin(Cucurbita moschata)pulp andtheir prebiotic activities[J].International Journal of Food Science&Technology,2011,46(5).

[0033]

[28] A K S,N J,A P K,et al.Process for Enzymatic Production ofXylooligosaccharides from the Xylan of Corn Cobs[J].Journal of FoodProcessing and Preservation,2015,39(6).

[0034]

[29] HU TG, ZOU YX, LI EN, et al. Effects of enzymatic hydrolysis on the structural, rheological, and functional properties of mulberry leafpolysaccharide[J]. FOOD CHEMISTRY, 2021,355.

[0035]

[30] HU TG,WU H,YU YS,et al.Preparation,structural characterization and prebiotic potential of mulberry leaf oligosaccharides[J].Food&Function,2022,13(9):5287-98.

[0036]

[31] HU TG,WU H,YU YS,et al.Preparation,structural characterization and prebiotic potential of mulberry leaf oligosaccharides[J].FOOD&FUNCTION,2022,13(9):5287-98. Summary of the Invention

[0037] In view of this, the purpose of this invention is to provide a sulfated mulberry leaf oligosaccharide that has significant antiviral activity and improves the survival rate of largemouth bass after viral challenge.

[0038] This invention provides an antiviral sulfated mulberry leaf oligosaccharide, comprising the following types of monosaccharides: glucuronic acid, rhamnose, and mannose;

[0039] The molar ratio of glucuronic acid, rhamnose, and mannose is 2:(3-4):(4-7);

[0040] In the monosaccharide, the sulfonic acid group replaces the hydroxyl group in the monosaccharide, and the degree of substitution of the polysaccharide is 1.107 to 1.126;

[0041] The molecular weight of sulfated mulberry leaf oligosaccharides ranges from 2000 Da to 4000 Da.

[0042] Preferably, the molar ratio of glucuronic acid, rhamnose, and mannose is 2:3:4.

[0043] Preferably, the degree of substitution of the polysaccharide is 1.126.

[0044] This invention provides a method for preparing the antiviral sulfated mulberry leaf oligosaccharide, comprising the following steps:

[0045] Mulberry leaf polysaccharides were subjected to microwave degradation and H2O2-Vc degradation to obtain degradation products;

[0046] The degradation products were modified by sulfation to obtain sulfated mulberry leaf oligosaccharides.

[0047] Preferably, during the microwave degradation, the microwave power is 500-750W, the microwave treatment time is 20-30 minutes, and the concentration of mulberry leaf polysaccharide is 10-100 mg / mL.

[0048] During the H2O2-Vc degradation, the final volume concentration of hydrogen peroxide is 0.5% to 3%; the final concentration of Vc is 5% to 10%; the degradation temperature of H2O2-Vc is 50 to 55°C, and the degradation time is 10 to 12 min.

[0049] The sulfation modification method involves reacting the degradation product with sulfur trioxide-pyridine in a water bath; the final concentration of sulfur trioxide-pyridine is 10 mg / ml; the temperature of the water bath reaction is 90–95 °C; and the reaction time is 1–1.5 h.

[0050] This invention provides the application of the antiviral sulfated mulberry leaf oligosaccharide or the sulfated mulberry leaf oligosaccharide obtained by the preparation method in aquaculture.

[0051] Preferably, the aquaculture includes the following applications:

[0052] 1) Enhance the antiviral resistance of aquatic animals;

[0053] 2) Enhance the antioxidant capacity of aquatic animals;

[0054] 3) Improve the immunity of aquatic animals;

[0055] 4) Improve feed utilization rate for aquatic animals;

[0056] 5) Improve the lipid metabolism capacity of aquatic animals.

[0057] Preferably, the virus includes largemouth bass iridovirus (LMBV);

[0058] The aquatic products include largemouth bass.

[0059] This invention provides an aquatic animal feed, comprising the antiviral sulfated mulberry leaf oligosaccharide or the sulfated mulberry leaf oligosaccharide obtained by the preparation method and a basic feed.

[0060] Preferably, the antiviral sulfated mulberry leaf oligosaccharide accounts for 0.5% to 1.2% of the mass of the basic feed.

[0061] This invention provides an antiviral sulfated mulberry leaf oligosaccharide, comprising the following monosaccharides: glucuronic acid, rhamnose, and mannose; the molar ratio of glucuronic acid, rhamnose, and mannose is 2:93-4):(4-7); in the monosaccharides, sulfonic acid groups replace the hydroxyl groups, and the degree of substitution is 1.107-1.126; the molecular weight of the sulfated mulberry leaf oligosaccharide is 2000 Da-4000 Da. The sulfated mulberry leaf oligosaccharide was added as a feed additive to basal feed for aquaculture. The effect of sulfated mulberry leaf oligosaccharide on the resistance of largemouth bass to LMBV virus was studied. The results showed that after feeding with feed containing sulfated mulberry leaf oligosaccharide, the survival rate of largemouth bass 14 days after challenge was over 70%, while the survival rate of the blank control group was only 25%. Therefore, sulfated mulberry leaf oligosaccharide can significantly improve the survival rate of largemouth bass infected with LMBV, indicating that sulfated mulberry leaf oligosaccharide has antiviral activity. Furthermore, this invention also investigated the effects of sulfated mulberry leaf oligosaccharides (MLOs) on the growth performance, antioxidant capacity, glucose and lipid metabolism, liver health, and gut microbiota of largemouth bass. The results showed that sulfated mulberry leaf oligosaccharides promote the growth of largemouth bass, improve feed utilization, and enhance the antioxidant capacity, lipid metabolism, and immune function of aquatic organisms. The sulfated mulberry leaf oligosaccharides provided by this invention offer a theoretical basis for the rational application of MLOs in formulated feeds for largemouth bass, enriching the biological functions of oligosaccharides. Attached Figure Description

[0062] Figure 1 The effect of MLO on the expression levels of inflammation-related genes; Note: Values ​​with different letters differed significantly (P<0.05 (mean±SEM, n=3)).

[0063] Figure 2 The effect of MLO on the expression levels of genes related to glucose and lipid metabolism; Note: Values ​​with different letters differed significantly (P<0.05 (mean±SEM, n=3)).

[0064] Figure 3 The effect of MLO on liver histology (H&E staining) of largemouth bass after 80 days.

[0065] Figure 4 The results of the MLO survival curve analysis against LMBV in largemouth bass. Detailed Implementation

[0066] This invention provides an antiviral sulfated mulberry leaf oligosaccharide, comprising the following types of monosaccharides: glucuronic acid, rhamnose, and mannose;

[0067] The molar ratio of glucuronic acid, rhamnose, and mannose is 2:3 to 4:4 to 7.

[0068] In the monosaccharide, the sulfonic acid group replaces the hydroxyl group in the monosaccharide, and the degree of substitution of the polysaccharide is 1.107 to 1.126;

[0069] The molecular weight of sulfated mulberry leaf oligosaccharides ranges from 2000 Da to 4000 Da.

[0070] In this invention, the molar ratio of glucuronic acid, rhamnose, and mannose is preferably 2:3:4. The degree of substitution of the polysaccharide is preferably 1.126.

[0071] This invention provides a method for preparing the antiviral sulfated mulberry leaf oligosaccharide, comprising the following steps:

[0072] Mulberry leaf polysaccharides were subjected to microwave degradation and H2O2-Vc degradation to obtain degradation products;

[0073] The degradation products were modified by sulfation to obtain sulfated mulberry leaf oligosaccharides.

[0074] This invention involves microwave degradation and H2O2-Vc degradation of mulberry leaf polysaccharides to obtain degradation products.

[0075] This invention does not impose any particular restriction on the source of the mulberry leaf polysaccharide; it can be prepared using extraction methods well-known in the art or purchased commercially. The extraction method for the mulberry leaf polysaccharide is preferably water extraction followed by alcohol precipitation. After obtaining the mulberry leaf polysaccharide, its concentration is preferably determined. The preferred method for concentration determination is the phenol-sulfuric acid method.

[0076] In this invention, during microwave degradation, the microwave power is preferably 500-750W, the microwave treatment time is preferably 20-30 minutes, and the concentration of mulberry leaf polysaccharides is preferably 10-100 mg / mL, more preferably 20-80 mg / mL, further preferably 30-70 mg / mL, and most preferably 50 mg / mL. This microwave degradation facilitates the breakdown of mulberry leaf polysaccharides into mulberry leaf oligosaccharides under microwave action, while simultaneously reducing the viscosity of the reaction system.

[0077] In this invention, during the H2O2-Vc degradation, the final volume concentration of hydrogen peroxide is preferably 0.5% to 3%, more preferably 1%; the final concentration of Vc is preferably 5% to 10%, more preferably 6%. The H2O2-Vc degradation temperature is preferably 50 to 55°C, and the degradation time is preferably 10 to 12 minutes. The H2O2-Vc degradation facilitates the further degradation of mulberry leaf oligosaccharides prepared by microwave degradation under H2O2-Vc conditions.

[0078] In this invention, the sulfation modification method preferably involves reacting the degradation product with sulfur trioxide-pyridine in a water bath; the final concentration of the sulfur trioxide-pyridine is preferably 10 mg / ml; the water bath reaction temperature is preferably 90–92°C, and the water bath reaction time is preferably 1–1.5 h. After the water bath reaction, the pH of the reaction product is preferably adjusted to neutral, desalted, and the solid phase is separated. The desalting method is preferably dialysis. The molecular weight cutoff of the dialysis bag is preferably 2000 Da. The dialysis time is preferably 48 h. The method for separating the solid phase is preferably centrifugation. The centrifugation speed is preferably 8000 × g to 13000 × g, and most preferably 10000 × g. After obtaining the solid phase, it is preferably freeze-dried.

[0079] Based on the above-mentioned activity of sulfated mulberry leaf oligosaccharides, the present invention provides an aquatic animal feed, comprising the antiviral sulfated mulberry leaf oligosaccharides or sulfated mulberry leaf oligosaccharides obtained by the above preparation method and a basic feed.

[0080] In this invention, the antiviral sulfated mulberry leaf oligosaccharide is preferably present in the basal feed at a mass percentage of 0.5% to 1.2%.

[0081] This invention provides the application of the antiviral sulfated mulberry leaf oligosaccharide or the sulfated mulberry leaf oligosaccharide obtained by the preparation method in aquaculture.

[0082] In this invention, the aquaculture preferably includes the following applications:

[0083] 1) Enhance the antiviral resistance of aquatic animals;

[0084] 2) Enhance the antioxidant capacity of aquatic animals

[0085] 3) Improve the immunity of aquatic animals;

[0086] 4) Improve feed utilization rate for aquatic animals;

[0087] 5) Improve the lipid metabolism capacity of aquatic animals.

[0088] In this invention, the virus preferably includes largemouth bass iridovirus (LMBV). The aquatic organism preferably includes largemouth bass, more preferably largemouth bass. In embodiments of this invention, sulfated mulberry leaf oligosaccharides were used to prepare aquatic feeds (MLOL and MLOH) at two different dosages to study the resistance of sulfated mulberry leaf oligosaccharides to viruses in aquatic animals. The results showed that the blank control group (largemouth bass fed only with a basic diet supplemented with sulfated mulberry leaf oligosaccharides) began to die 3 days after challenge, the low-dose group (MLOL) died on the 4th day, and the high-dose group (MLOH) died on the 6th day; at the same time, the number of deaths did not change after 14 days of challenge, the survival rate of the control group was 25%, the survival rate of the MLOL group was 65%, and the survival rate of the MLOH group was 70%. Compared with the control group, the relative protection rate of the MLOL treatment group was 42.86%, and the relative protection rate of the MLOH treatment group was 50%. It can be seen that sulfated mulberry leaf oligosaccharides are beneficial in reducing the mortality rate of largemouth bass after challenge, indicating that sulfated mulberry leaf oligosaccharides have antiviral activity.

[0089] In this embodiment of the invention, the effect of sulfated mulberry leaf oligosaccharide on the growth performance of largemouth bass was also studied. The results showed that sulfated mulberry leaf oligosaccharide could reduce the feed conversion ratio and visceral-to-body ratio of largemouth bass, and the high-dose group (MLOH) also significantly reduced the hepato-to-body ratio.

[0090] In this embodiment of the invention, the effects of sulfated mulberry leaf oligosaccharides on blood biochemical parameters of largemouth bass were also studied. The results showed that sulfated mulberry leaf oligosaccharides were beneficial in reducing blood triglyceride and blood glucose levels, and decreasing the enzyme activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The effects of sulfated mulberry leaf oligosaccharides on the liver tissue of largemouth bass were also investigated. Sulfated mulberry leaf oligosaccharides were beneficial in improving cell vacuolation and nuclear displacement in liver tissue, resulting in intact liver cell structure and clear outlines. This indicates that sulfated mulberry leaf oligosaccharides can significantly improve liver health in largemouth bass.

[0091] In this embodiment of the invention, the effect of sulfated mulberry leaf oligosaccharides on the antioxidant capacity of largemouth bass was also studied. The results showed that sulfated mulberry leaf oligosaccharides could effectively increase the enzyme activity of total superoxide dismutase, increase GSH content, and decrease malondialdehyde content, indicating that sulfated mulberry leaf oligosaccharides can effectively improve the antioxidant capacity of the blood and liver of largemouth bass.

[0092] In this embodiment of the invention, the effect of sulfated mulberry leaf oligosaccharide on the inflammatory response of largemouth bass was also studied. The results showed that sulfated mulberry leaf oligosaccharide could effectively increase the expression of anti-inflammatory factors and decrease the expression of pro-inflammatory factors. It can be seen that sulfated mulberry leaf oligosaccharide has an inhibitory effect on the inflammatory response of largemouth bass.

[0093] In this embodiment of the invention, the effects of sulfated mulberry leaf oligosaccharides on the hepatic glycolipid metabolism of largemouth bass were investigated. The results showed that sulfated mulberry leaf oligosaccharides increased the expression levels of glycolysis genes and lipid metabolism-related genes, indicating that sulfated mulberry leaf oligosaccharides can significantly improve the hepatic glycolipid metabolism function of largemouth bass.

[0094] The following detailed description, in conjunction with embodiments, illustrates an antiviral sulfated mulberry leaf oligosaccharide, its preparation method, its application in animal feed, and other aspects provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0095] Example 1

[0096] Preparation method of sulfated mulberry leaf oligosaccharides

[0097] 1. Mulberry leaf polysaccharide: After crushing mulberry leaves, extract with water at 70℃ for 5 hours. After the liquid is concentrated to 1 / 3, precipitate with 3 times the amount of 95% ethanol for 48 hours. Collect the precipitate to obtain mulberry leaf polysaccharide.

[0098] 2. Concentration determination and dilution: The polysaccharide concentration was determined using the phenol-sulfuric acid method, and appropriately diluted to 70 mg / mL.

[0099] 3. Microwave degradation: Take 100 mL of polysaccharide liquid and microwave at 500 W for 20 min.

[0100] 4. H2O2-Vc degradation: Add 1 mL of 30% H2O2, stir at 50℃ for 10 min; then add 1 mL of 4 mol / L Vc, stir at 50℃ for 10 min.

[0101] 5. Sulfation modification: Add 1g of sulfur trioxide-pyridine, incubate in a water bath at 90℃ for 1h, then cool to room temperature with ice water. Add 5mol / L NaOH to adjust the pH to 7, desalinate through a 2000Da dialysis bag for 48h, centrifuge, and collect the precipitate.

[0102] 6. Freeze-drying: The sulfated mulberry leaf oligosaccharides were freeze-dried to obtain sulfated mulberry leaf oligosaccharides.

[0103] The degree of substitution of polysaccharides with sulfonic acid groups was determined by the barium chloride gelatin method, and the types and proportions of monosaccharides in sulfated mulberry leaf oligosaccharides were determined by GC-MS.

[0104] The results showed that the degree of polysaccharide substitution was 1.126, and the monosaccharide composition consisted of glucuronic acid, rhamnose, and mannose in a molar ratio of 2:3:4.

[0105] Example 2

[0106] A method for preparing animal feed

[0107] Commercial California bass feed (Foshan Nanhai District Jieda Feed Co., Ltd., Foshan, China) was used as the base feed. The sulfated mulberry leaf oligosaccharide prepared in Example 1 was weighed and added to the base feed (formula and nutrient levels are shown in the table) to prepare feeds containing 0.5% MLO (MLOL) and 1% MLO (MLOH) by mass concentration, respectively.

[0108] Table 1. Basal Diet Formulas and Nutritional Levels

[0109]

[0110]

[0111] 1 CK, basal diet; MLOL, supplemented with 0.5% MLO; MLOH, supplemented with 1.0% MLO.

[0112] 2) Each kilogram of vitamin premix contains: Vitamin A 66,666,666.7 IU, Vitamin D 400,000,000 IU, Vitamin E 1 g, Vitamin K 2 g, Vitamin B1 5 g, Vitamin B2 5 g, Vitamin B6 5 g, Vitamin B12 1 g, Calcium pantothenate 20 g, Folic acid 10 g, Biotin 1 g, Niacin 20 g, Choline chloride 200 g, and defatted rice bran 700 g.

[0113] 3) Each kilogram of mineral premix contains: 4 g CuCO3, 15 g FeC6H5O7, 26 g MgO, 5 g MnSO4, 250 g KCl, 50 g ZnSO4, 50 g NaCl, and 600 g zeolite powder.

[0114] Example 3

[0115] Application of sulfated mulberry leaf oligosaccharides in aquaculture

[0116] I. Experimental Methods

[0117] 1. Feeding and Management

[0118] Largemouth bass used in the experiment were purchased from Guangdong Liangshi Aquatic Seed Industry Co., Ltd. After being transported back to Guangzhou, the experimental fish were temporarily held for two weeks in the indoor recirculating aquaculture system of the Sericulture and Agricultural Products Processing Research Institute of the Guangdong Academy of Agricultural Sciences, fed daily with a basal feed. Before the formal experiment, feeding was stopped for 24 hours. 450 juvenile fish with an average initial weight of 26.89±1.16g were selected and randomly divided into nine 75cm diameter rearing tanks (350L volume), with 50 fish per tank. The nine tanks were randomly divided into three groups, with three replicates per group. The two experimental groups were fed basal feeds containing 0.5% MLO (MLOL) and 1% MLO (MLOH), respectively, while the control group (CK) was fed the basal feed. During the formal experiment, feeding was done twice daily (09:00 and 16:30), with a feed amount of 2-4% of the fish's body weight. The experimental period was 80 days. The indoor recirculating aquaculture system consists of a microfiltration system, a biochemical reaction system, a protein separator, an ultraviolet disinfection device, and a culture tank. The culture water source is disinfected and aerated tap water. During the experiment, the water was changed 3 times a week, with 1 / 4 of the water being replaced. The water temperature during the culture period was 22.4–29.4℃, dissolved oxygen ≥5.0mg / L, nitrite content ≤0.05mg / L, ammonia nitrogen content ≤0.2mg / L, pH 7.0–8.2, and natural light cycle.

[0119] 2. Sample Collection

[0120] After the experiment, fish were fasted for 24 hours. Nine fish were randomly selected from each rearing tank, anesthetized with MS-222, and their body length and weight were measured. They were then dissected, and their viscera and liver were removed and weighed. The body weight, body length, and the weights of the viscera and liver were recorded for calculating final weight (WG), condition factor (CF), visceral-to-body ratio (VSI), and hepatic-to-body ratio (HIS). Six fish were randomly selected from each rearing tank, and blood was drawn from the tail vein using a 1 mL syringe. After standing at room temperature for 2 hours, the blood was centrifuged (3000 rpm, 10 min, 4℃), and the supernatant was collected and stored at -20℃ for serum biochemistry and enzyme activity analysis. Three fish were randomly selected from each rearing tank, and their livers and intestines were dissected, flash-frozen in liquid nitrogen, and stored at -20℃ for enzyme activity assays. Three fish were randomly selected from each rearing tank, and their liver tissue was dissected, flash-frozen in liquid nitrogen, and stored at -80℃ for gene expression analysis.

[0121] 3. Biochemical indicator testing

[0122] 3.1 Serum Biochemical Indicators Determination: Serum samples stored at -20℃ were thawed on ice, and then the levels of low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), total cholesterol (T-CHO), triglycerides (TG), reduced glutathione (GSH), malondialdehyde (MDA), total antioxidant capacity (T-AOC), and blood glucose (GLU) were determined using a kit (Nanjing Jiancheng Bioengineering Institute). Similarly, the enzyme activities of alkaline phosphatase (AKP), total superoxide dismutase (T-SOD), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were determined using a kit (Nanjing Jiancheng Bioengineering Institute). The specific experimental methods were described in the kit instructions.

[0123] 3.2 Liver index determination: Liver tissue stored at -80℃ was thawed on ice. The tissue was accurately weighed and added to 9 volumes of physiological saline at a weight (g):volume (mL) ratio of 1:9. The tissue was mechanically homogenized under ice-water bath conditions and centrifuged at 4℃ and 2500 r / min for 10 min. The supernatant was collected for determination of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), and malondialdehyde (MDA). The determination methods were based on the instructions of the kit produced by Nanjing Jiancheng Bioengineering Institute.

[0124] 3.3 Intestinal enzyme activity assay: Intestinal tissue stored at -80℃ was thawed on ice. The tissue was accurately weighed and added to 9 volumes of physiological saline at a weight (g):volume (mL) ratio of 1:9. The tissue was mechanically homogenized under ice-water bath conditions and centrifuged at 4℃ and 2500 r / min for 10 min. The supernatant was collected, and the activities of amylase (AMS), lipase (LPS), and trypsin (TRY) in the intestine were determined using a kit (Nanjing Jiancheng Bioengineering Institute). The specific method was described in the kit instructions.

[0125] 4. Calculation formula

[0126] Survival rate (SR, %) = (Number of last digits / Number of initial digits) × 100% (Formula I)

[0127] Weight gain rate (WGR, %) = [Final average weight (g) - Initial average weight (g)] / Initial average weight (g) × 100% Formula II

[0128] Specific growth rate (SGR, % / d) = 100 × [ln(final average weight + mortality weight) - ln(initial average weight)] / number of days of rearing Formula III

[0129] Feeding rate FI (%) / d = 100 × total weight of feed consumed / [(total weight of initial fish + total weight of final fish) / 2] / number of days (Formula IV)

[0130] Feed conversion ratio (FCR) = Feed amount / [Final average weight (g) - Initial average weight (g)] (Formula V)

[0131] Conditionness (CF, g / cm³) 3 = Body mass (g) / Body length 3 (cm) 3 Formula VI Liver-to-body weight ratio (HSI, %) = Liver mass (g) / Body mass (g) × 100% (Formula VII) Visceral-to-body ratio (HVI, %) = Visceral mass (g) / Body mass (g) × 100% Formula III.

[0132] 5. Observation of liver and intestinal tissue sections

[0133] Three fish were randomly selected from each rearing tank for dissection. The intestines were separated, surface fat was removed, and a 1cm length of the midgut was harvested. The liver was also separated, and a 0.5cm length was harvested from the same location on the liver. 3 Liver tissue was fixed with paraformaldehyde fixative for observation of intestinal and liver tissue sections. The paraformaldehyde-fixed intestinal and liver tissues were embedded in paraffin to prepare paraffin sections with a thickness of 6 μm, which were then stained with hematoxylin and eosin (HE). The sections were scanned and photographed using a panoramic digital slide scanner (PANNORAMIC-1000, 3DHISTECH). Tissue images were extracted using CaseViewer 2.2 (3DHISTECH) software, and Image-Pro Plus 6.0 (Media Cybemetics) analysis software was used to measure intestinal villus height, villus width, and muscle layer thickness, with four sets of values ​​measured for each section.

[0134] 6. RNA extraction and real-time quantitative PCR

[0135] Liver tissue stored at -80°C was thawed on ice, and total mRNA was extracted from the liver tissue samples using the Animal Tissue Total RNA Extraction Kit (TIANGEN). The samples were treated with RNA-free DNase reagent to remove DNA contaminants, and the quality of RNA extraction was assessed using a spectrophotometer (ND-2000, Nano-Drop Technologies, Wilmington, USA). The RNA was then reverse transcribed into cDNA using the TIANGEN kit (China).

[0136] The mRNA expression of relevant genes in the liver of largemouth bass was determined by real-time quantitative PCR. Core fragments of all genes were obtained from RNA-seq databases. Quantitative real-time PCR was performed using a Bio-Rad MiniOption™ real-time quantitative PCR detection system (Bio-Rad, USA). The reaction mixture (10 μl) consisted of 1 μl each of forward and reverse primers, 1 μl of cDNA template, 5 μl of SYBR green color qPCR MasterMix (TIANGEN, Beijing), and 2 μl of ddH2O. The reaction program was: pre-denaturation at 95℃ for 3 min, followed by denaturation at 95℃ for 10 s, annealing at 60℃ for 30 s, and extension at 72℃ for 1 min, for 40 cycles. Each experiment was performed in triplicate (biological replicates), and each biological replicate was performed in triplicate (technical replicates). β-actin was used as a reference gene. -△△Ct The method calculates the relative mRNA expression level of the target gene.

[0137] Table 2 Primers used for RT-qPCR amplification

[0138]

[0139]

[0140] 7. Challenge Test

[0141] After the experiment, 25 largemouth bass were randomly selected from each of the experimental and control groups and injected intraperitoneally with 4×10 5 TCID 50 LMBV cytotoxic supernatant was collected, and the experimental fish were placed in a 100cm×60cm×60cm tank with continuous aeration at a water temperature of 20–25℃. The fish were fed daily for 21 days post-infection, and mortality was observed and recorded.

[0142] 8. Data Statistical Analysis

[0143] Experimental data are expressed as mean ± standard error (SEM). One-way ANOVA was performed using IBM SPSS Statistics 26.0 software. Duncan's multiple comparison test was used to determine the significance of differences between groups. P < 0.05 was considered significant.

[0144] II. Results

[0145] 1. Effects of MLO Addition to Feed on Growth Performance of Largemouth Bass

[0146] As shown in Table 3, compared with the CK group, although there were no significant differences in WGR, SGR, FI, and CF in the MLOL and MLOH groups (P>0.05), FCR and HVI were significantly decreased (P<0.05). In addition, HSI was significantly decreased in the MLOH group (P<0.05).

[0147] Table 3. Effects of MLO supplementation in feed on growth performance of largemouth bass

[0148]

[0149] Data with no letter or the same letter in the same column indicate no significant difference (P>0.05), while different lowercase letters indicate significant difference (P<0.05). The same applies to the table below.

[0150] 2. Effects of MLO Addition to Feed on Serum Biochemical Parameters of Largemouth Bass

[0151] As shown in Table 4, compared with the CK group, the TG and GLU contents in the MLOL and MLOH groups were significantly decreased (P < 0.05), and the ALT and AST enzyme activities were significantly decreased (P < 0.05). There were no significant differences in the TC, LDL-C, and HDL-C contents between the MLOL and MLOH groups and the CK group (P > 0.05).

[0152] Table 4. Effects of mulberry leaf oligosaccharide supplementation in feed on serum biochemical parameters of largemouth bass.

[0153]

[0154]

[0155] 3. Effects of MLO supplementation in feed on the antioxidant capacity of largemouth bass

[0156] Table 5 shows that, compared with the CK group, the serum T-SOD activity in the MLOL and MLOH groups was significantly increased (P < 0.05), the GSH content was significantly higher than that in the CK group (P < 0.05), and the MDA content was significantly lower than that in the CK group (P < 0.05). Furthermore, the addition of MLO had no significant effect on serum T-AOC in any group (P > 0.05), indicating that the addition of MLO can significantly improve the antioxidant capacity of largemouth bass blood.

[0157] The activity of T-SOD in the liver tissue of the MLOL and MLOH groups was significantly increased (P<0.05), the activity of T-AOC was significantly increased (P<0.05), and the content of GSH was significantly higher than that of the CK group (P<0.05), but the content of MDA was significantly decreased (P<0.05), indicating that the addition of MLO can significantly improve the antioxidant capacity of largemouth bass liver.

[0158] Table 5. Effects of feed supplementation with mulberry leaf oligosaccharides on liver health of largemouth bass.

[0159]

[0160] 4. Effects of MLO supplementation in feed on the expression of liver inflammation-related genes in largemouth bass.

[0161] The expression levels of genes related to antioxidant and inflammation in the liver of largemouth bass were detected. The results showed that, compared with the control group, dietary supplementation with MLO significantly reduced the expression of pro-inflammatory factors (TNF-α, IL-8, and NF-κB) and significantly increased the expression of anti-inflammatory factors (IL-10 and TGF-β) in both the MLOL and MLOH groups. Figure 1 This indicates that adding MLO can significantly improve the immune function of the liver of largemouth bass.

[0162] 5. Effects of MLO supplementation in feed on the expression of genes related to liver glycolipid metabolism in largemouth bass.

[0163] The expression levels of genes related to lipid metabolism in the liver of largemouth bass were detected. The results showed that the addition of MLO significantly increased the expression levels of glycolysis genes pfk and gk (P<0.05); compared with the CK group, the expression levels of lipid metabolism-related genes acc, ppar-α, and cpt-1 were significantly increased in the MLOL and MLOH groups (P<0.05). Figure 2 This indicates that adding MLO can significantly improve the glycolipid metabolism function of the liver of largemouth bass.

[0164] 6. Effects of MLO Addition to Feed on Liver Morphology of Largemouth Bass

[0165] The liver tissue morphology of largemouth bass was observed by section examination. The results showed that the liver cells in the CK group had obvious vacuolation and nuclear displacement, while the MLOL and MLOH groups showed significant improvement in cell vacuolation and nuclear displacement, and the liver cells had intact structure and clear outlines. Figure 3 This indicates that adding MLO can significantly improve the liver health of largemouth bass.

[0166] 7. Effect of MLO on resistance to LMBV in largemouth bass

[0167] The results of in vitro anti-LMBV activity assays using MLO showed that mortality in the control group of largemouth bass began 3 days after challenge, mortality in the MLOL group began on day 4, and mortality in the MLOH group began on day 6. Compared with the control group, the mortality time of largemouth bass infected with MLO was significantly delayed. The number of deaths in each group remained unchanged 14 days after challenge. The survival rate was 25% in the control group, 65% in the MLOL group, and 70% in the MLOH group. Compared with the control group, the relative protection rate was 42.86% in the MLOL group and 50% in the MLOH group. Figure 4 Log-rank (Mantel-Cox) test showed that MLO significantly improved the survival rate of largemouth bass (P<0.05).

[0168] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antiviral sulfated mulberry leaf oligosaccharide, characterized in that, The monosaccharides consist of glucuronic acid, rhamnose and mannose; The molar ratio of the glucuronic acid, rhamnose and mannose is 2:(3-4):(4-7); In the monosaccharides, the sulfonic acid group substitutes the hydroxyl group in the monosaccharides, and the polysaccharide substitution degree is 1.107-1.126; The molecular weight of the sulfated mulberry leaf oligosaccharide is 2000 Da-4000 Da.

2. The antiviral sulfated mulberry leaf oligosaccharide according to claim 1, characterized by, The molar ratio of the glucuronic acid, rhamnose and mannose is 2:3:

4.

3. The antiviral sulfated mulberry leaf oligosaccharide according to claim 1, characterized in that, The polysaccharide substitution degree is 1.

126.

4. The method for preparing the antiviral sulfated mulberry leaf oligosaccharide according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: The mulberry leaf polysaccharide is subjected to microwave degradation and H2O2-Vc degradation to obtain a degradation product; The degradation product is subjected to sulfation modification to obtain a sulfated mulberry leaf oligosaccharide.

5. The preparation method according to claim 4, characterized in that, In the microwave degradation, the power of the microwave is 500-750 W, and the microwave treatment time is 20-30 min; the concentration of the mulberry leaf polysaccharide is 10-100 mg / mL; In the H2O2-Vc degradation, the final volume concentration of hydrogen peroxide is 0.5%-3%; the final concentration of Vc is 5%-10%; the H2O2-Vc degradation temperature is 50-55 ℃, and the degradation time is 10-12 min; The sulfation modification method is to subject the degradation product and sulfur trioxide-pyridine to water bath reaction; the final concentration of the sulfur trioxide-pyridine is 10 mg / ml; the water bath reaction temperature is 90-95 ℃, and the water bath reaction time is 1-1.5 h.

6. The application of the antiviral sulfated mulberry leaf oligosaccharide in claims 1-3 or the sulfated mulberry leaf oligosaccharide obtained by the preparation method in claims 4 or 5 in the preparation of aquatic animal feed, wherein the aquatic animal is a large mouth bass; The aquatic animal feed comprises the following applications: 1) improving the antiviral property of the aquatic animal; the virus is a large mouth bass iridovirus; 2) improving the antioxidant capacity of the aquatic animal; 3) improving the immune capacity of the aquatic animal; 4) improving the feed utilization rate of the aquatic animal; 5) improving the lipid metabolism capacity of the aquatic animal.

7. An aquaculture animal feed, characterized in that, The antiviral sulfated mulberry leaf oligosaccharide in claims 1-3 or the sulfated mulberry leaf oligosaccharide obtained by the preparation method in claims 4 or 5 and a basic feed.

8. The aquatic animal feed of claim 7, wherein, The mass percentage of the antiviral sulfated mulberry leaf oligosaccharide in the basic feed is 0.5%-1.2%.

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