Oligosaccharides, process for their preparation and use in aquaculture feed

Abstract

The application provides oligosaccharide, a preparation method thereof and application thereof in aquatic feed, and belongs to the technical field of plant polysaccharide. The mulberry polysaccharide is sequentially subjected to pectinase enzymolysis, H2O2-Vc degradation and microwave degradation, the degradation product obtained is subjected to sulfation modification, and sulfated mulberry oligosaccharide is obtained. The sulfated mulberry oligosaccharide has the effects of improving the antioxidant capacity of aquatic animals, improving the antiviral property of aquatic animals, improving the immune capacity of aquatic animals and improving the growth performance of aquatic animals, and can be used as a feed additive in aquatic breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant polysaccharide technology, specifically relating to an oligosaccharide, its preparation method, and its application in aquatic feed. Background Technology

[0002] Mulberries are a common fruit, slightly sweet and delicious, with extremely high nutritional value, containing abundant vitamins, minerals, mulberry polysaccharides, and amino acids. Mulberry polysaccharides, in particular, offer numerous beneficial effects on human health. Reports indicate that mulberry polysaccharides lower blood lipids, reducing cholesterol and triglyceride levels in the blood and decreasing the risk of atherosclerosis. They also stimulate the body's immune system, playing an anti-tumor role. Furthermore, mulberry polysaccharides have a strong immune-enhancing effect, strengthening disease resistance. They also possess strong antioxidant properties, effectively scavenging free radicals, which is significant for the prevention and treatment of various degenerative diseases. In addition, mulberry polysaccharides protect the liver and promote gastrointestinal health, offering multiple benefits for maintaining overall health.

[0003] Mulberry oligosaccharides are small-molecule sugar structures obtained by further degradation of mulberry polysaccharides. Existing technologies report the use of mulberry oligosaccharides to produce prebiotic products for maintaining human health. However, there are currently no reports on the use of sulfated mulberry oligosaccharides for antiviral purposes. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing sulfated mulberry oligosaccharides, wherein the sulfated mulberry oligosaccharides obtained by multi-step degradation and sulfation modification by protease, H2O2-Vc and microwave have antiviral activity.

[0005] This invention provides a method for preparing sulfated mulberry oligosaccharides, comprising the following steps:

[0006] Mulberry polysaccharides were sequentially subjected to pectinase hydrolysis, H2O2-Vc degradation, and microwave degradation to obtain degradation products;

[0007] The degradation products were modified by sulfation to obtain sulfated mulberry oligosaccharides.

[0008] Preferably, the final concentration of pectinase during pectinase hydrolysis is 5-10 g / L.

[0009] Preferably, the pectinase hydrolysis temperature is 40–60°C, and the pectinase hydrolysis time is 25–35 min.

[0010] Preferably, during the H2O2-Vc degradation process, the final concentration of H2O2 is 1% to 5%, and the final concentration of Vc is 10% to 15%.

[0011] Preferably, the H2O2-Vc degradation temperature is 48-52℃; the H2O2-Vc degradation time is 8-12 min.

[0012] Preferably, during the microwave degradation, the microwave power is 600-800W and the microwave treatment time is 8-12 minutes.

[0013] Preferably, the sulfation modification method involves reacting the degradation product with sulfur trioxide-pyridine in a water bath.

[0014] The final concentration of sulfur trioxide-pyridine is 2% to 5%; the temperature of the water bath reaction is 78 to 82°C; and the time of the water bath reaction is 1 to 1.5 hours.

[0015] This invention provides sulfated mulberry oligosaccharides prepared by the aforementioned method, wherein the molecular weight of the sulfated mulberry oligosaccharides is 3000 Da to 5000 Da; the sulfated mulberry oligosaccharides include the following monosaccharides: glucose, xylose, and galactose; the molar ratio of glucose, xylose, and galactose is 1:(1-2):(6-8); the sulfonic acid groups in the sulfated mulberry oligosaccharides replace the hydroxyl groups of the monosaccharides; the degree of substitution of the sulfonic acid groups is 1.251.

[0016] The present invention provides an animal feed comprising the sulfated mulberry oligosaccharide and a base feed.

[0017] This invention provides the application of the sulfated mulberry oligosaccharide in at least one of the following aspects in aquaculture:

[0018] 1) Enhance the antioxidant capacity of aquatic animals;

[0019] 2) Improve the antiviral resistance of aquatic animals;

[0020] 3) Improve the immunity of aquatic animals;

[0021] 4) Improve the growth performance of aquatic animals.

[0022] This invention provides a method for preparing sulfated mulberry oligosaccharides, comprising the following steps: sequentially subjecting mulberry polysaccharides to pectinase hydrolysis, H2O2-Vc degradation, and microwave degradation to obtain degradation products; and subjecting the degradation products to sulfation modification to obtain sulfated mulberry oligosaccharides. The sulfated mulberry oligosaccharides prepared by this invention not only possess antiviral activity but also antioxidant capacity, enhancing the immunity and promoting the growth of fed animals, and can provide a new approach to animal husbandry as a feed additive. Detailed Implementation

[0023] This invention provides a method for preparing sulfated mulberry oligosaccharides, comprising the following steps:

[0024] Mulberry polysaccharides were sequentially subjected to pectinase hydrolysis, H2O2-Vc degradation, and microwave degradation to obtain degradation products;

[0025] The degradation products were modified by sulfation to obtain sulfated mulberry oligosaccharides.

[0026] This invention involves sequentially subjecting mulberry polysaccharides to pectinase hydrolysis, H2O2-Vc degradation, and microwave degradation to obtain degradation products.

[0027] The present invention does not impose any particular limitation on the preparation method of the mulberry polysaccharide, and any preparation method of mulberry polysaccharide known in the art can be used, such as water extraction and alcohol precipitation.

[0028] In this invention, the final concentration of pectinase during enzymatic hydrolysis is preferably 5-10 g / L, more preferably 8 g / L. The hydrolysis temperature is preferably 40-60°C, more preferably 50°C; the hydrolysis time is preferably 25-35 min, more preferably 30 min. The function of the pectinase is to enzymatically hydrolyze the pectin on the surface of mulberry, allowing the mulberry polysaccharides to be fully released.

[0029] In this invention, during the H2O2-Vc degradation, the final concentration of H2O2 is preferably 1% to 5%, more preferably 2%; the final concentration of Vc is preferably 10% to 15%, more preferably 13%. The degradation temperature of H2O2-Vc is preferably 48 to 52°C, more preferably 50°C; the degradation time of H2O2-Vc is preferably 8 to 12 minutes, more preferably 10 minutes. The preferred function of H2O2-Vc degradation is to degrade mulberry polysaccharides into mulberry oligosaccharides.

[0030] In this invention, during microwave degradation, the microwave power is preferably 600-800W, more preferably 650W. The microwave treatment time is preferably 8-12 minutes, more preferably 10 minutes. The purpose of the microwave degradation is to further degrade the mulberry oligosaccharides prepared by H2O2-Vc under microwave irradiation.

[0031] 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 2%–5%, more preferably 3.5%; the temperature of the water bath reaction is preferably 78–82°C, more preferably 80°C; and the reaction time is preferably 1–1.5 h, more preferably 1 h.

[0032] This invention provides sulfated mulberry oligosaccharides prepared by the aforementioned method, wherein the molecular weight of the sulfated mulberry oligosaccharides is 3000 Da to 5000 Da; the sulfated mulberry oligosaccharides include the following monosaccharides: glucose, xylose, and galactose; the molar ratio of glucose, xylose, and galactose is 1:(1-2):(6-8); the sulfonic acid groups in the sulfated mulberry oligosaccharides replace the hydroxyl groups of the monosaccharides; the degree of substitution of the sulfonic acid groups is 1.251.

[0033] The present invention provides an animal feed comprising the sulfated mulberry oligosaccharide and a base feed.

[0034] In this invention, the mass percentage of sulfated mulberry oligosaccharides in the basic feed is preferably 0.5% to 2%, more preferably 1%.

[0035] This invention provides the application of the sulfated mulberry oligosaccharide in at least one of the following aspects in aquaculture:

[0036] 1) Enhance the antioxidant capacity of aquatic animals;

[0037] 2) Improve the antiviral resistance of aquatic animals;

[0038] 3) Improve the immunity of aquatic animals;

[0039] 4) Improve the growth performance of aquatic animals.

[0040] In this invention, the aquatic animal preferably includes the largemouth bass. The virus preferably includes an iridovirus.

[0041] In this embodiment of the invention, aquatic animals were fed a diet containing sulfated mulberry oligosaccharides, and the effects on aquaculture were analyzed. The results showed that, compared with the control group, the diet containing sulfated mulberry oligosaccharides effectively improved the growth performance (including weight gain and feed conversion ratio) of largemouth bass, while also enhancing their immunity and antioxidant activity. Finally, the results of the virus challenge experiment showed that after 14 days of challenge, the survival rate of the control group was 40%, while the survival rate of the sulfated mulberry oligosaccharide group in the embodiments was above 90%, indicating that sulfated mulberry oligosaccharides have the effect of improving the virus resistance of aquatic animals.

[0042] The following detailed description, in conjunction with embodiments, illustrates an oligosaccharide, its preparation method, and its application in aquatic feed provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] Mulberry polysaccharide: After crushing mulberries, pass them through a 20-mesh sieve, extract with water at 70℃ for 5 hours, concentrate the liquid to 1 / 3, precipitate with 3 times 95% ethanol for 48 hours, recover the ethanol, and collect the precipitate to obtain mulberry polysaccharide;

[0045] Concentration determination and dilution: Polysaccharide concentration was determined by the phenol-sulfuric acid method, and appropriately diluted to 50 mg / mL;

[0046] Pectinase hydrolysis: Take 1L of polysaccharide liquid, add 5-10g of pectinase, and hydrolyze at 50℃ for 30min. H2O2-Vc degradation: Add 20% H2O2 to make the final concentration of H2O2 2%, stir at 50℃ for 10min; then add 5mol / L Vc to make the final concentration of Vc 13%, and stir at 50℃ for 10min.

[0047] Microwave degradation: Add water of equal mass and microwave treatment at 650W for 10 minutes;

[0048] Sulfation modification: Sulfur trioxide-pyridine was added to a final concentration of 3.5%, and the mixture was incubated in a water bath at 80°C for 1 hour, then cooled to room temperature with ice water. 5 mol / L NaOH was added to adjust the pH to 7, and the solution was dialyzed through a 3000 Da dialysis bag for 24 hours. The precipitate was then collected by centrifugation.

[0049] Freeze-drying: Sulfated mulberry oligosaccharides were freeze-dried to obtain sulfated mulberry oligosaccharides.

[0050] Mulberry oligosaccharides: Sulfated mulberry oligosaccharides were obtained by sulfur trioxide-pyridine sulfation modification. The polysaccharide substitution degree was 1.251, and the monosaccharide composition consisted of glucose, xylose and galactose in a molar ratio of 1:1:6.

[0051] Example 2

[0052] Mulberry polysaccharide: After crushing mulberries, pass them through a 20-mesh sieve, extract with water at 70℃ for 5 hours, concentrate the liquid to 1 / 3, precipitate with 3 times 95% ethanol for 48 hours, recover the ethanol, and collect the precipitate to obtain mulberry polysaccharide;

[0053] Concentration determination and dilution: Polysaccharide concentration was determined using the phenol-sulfuric acid method, and appropriately diluted to 75 mg / mL;

[0054] Pectinase hydrolysis: Take 1L of polysaccharide liquid, add 5-10g of pectinase, and hydrolyze at 50℃ for 30min. H2O2-Vc degradation: Add 20% H2O2 to make the final concentration of H2O2 3%, stir at 50℃ for 10min; then add 5mol / L Vc to make the final concentration of Vc 10.5%, and stir at 50℃ for 10min.

[0055] Microwave degradation: Add an equal amount of water and microwave at 600W for 10 minutes.

[0056] Sulfation modification: Sulfur trioxide-pyridine was added to a final concentration of 2.5%, and the mixture was incubated in a water bath at 78°C for 1.3 h, then cooled to room temperature with ice water. 5 mol / L NaOH was added to adjust the pH to 7, and the mixture was desalted using a 3000 Da dialysis bag for 24 h. The precipitate was then collected by centrifugation.

[0057] Freeze-drying: Sulfated mulberry oligosaccharides were freeze-dried to obtain sulfated mulberry oligosaccharides.

[0058] Mulberry oligosaccharides: Sulfated mulberry oligosaccharides were obtained by sulfur trioxide-pyridine sulfation modification. The polysaccharide substitution degree was 1.213, and the monosaccharide composition consisted of glucose, xylose and galactose in a molar ratio of 1:1.2:6.8.

[0059] Example 3

[0060] Mulberry polysaccharide: After crushing mulberries, pass them through a 20-mesh sieve, extract with water at 70℃ for 5 hours, concentrate the liquid to 1 / 3, precipitate with 3 times 95% ethanol for 48 hours, recover the ethanol, and collect the precipitate to obtain mulberry polysaccharide;

[0061] Concentration determination and dilution: Polysaccharide concentration was determined using the phenol-sulfuric acid method, and appropriately diluted to 65 mg / mL;

[0062] Pectinase hydrolysis: Take 1L of polysaccharide liquid, add 5-10g of pectinase, and hydrolyze at 50℃ for 30min. H2O2-Vc degradation: Add 20% H2O2 to make the final concentration of H2O2 2.5%, stir at 50℃ for 10min; then add 5mol / L Vc to make the final concentration of Vc 14%, and stir at 50℃ for 10min.

[0063] Microwave degradation: Add water of equal mass and microwave treatment at 750W for 10 minutes;

[0064] Sulfation modification: Sulfur trioxide-pyridine was added to a final concentration of 5%, and the mixture was incubated in a water bath at 82°C for 1.4 h, then cooled to room temperature with ice water. 5 mol / L NaOH was added to adjust the pH to 7, and the solution was dialyzed through a 3000 Da dialysis bag for 24 h. The precipitate was then collected by centrifugation.

[0065] Freeze-drying: Sulfated mulberry oligosaccharides were freeze-dried to obtain sulfated mulberry oligosaccharides.

[0066] Mulberry oligosaccharides: Sulfated mulberry oligosaccharides were obtained by sulfur trioxide-pyridine sulfation modification. The polysaccharide substitution degree was 1.202, and the monosaccharide composition consisted of glucose, xylose and galactose in a molar ratio of 1:1.8:6.2.

[0067] Comparative Example 1: Modified from Example 1: The pectinase hydrolysis step was omitted.

[0068] Mulberry polysaccharide: After crushing mulberries, pass them through a 20-mesh sieve, extract with water at 70℃ for 5 hours, concentrate the liquid to 1 / 3, precipitate with 3 times 95% ethanol for 48 hours, recover the ethanol, and collect the precipitate to obtain mulberry polysaccharide;

[0069] Concentration determination and dilution: Polysaccharide concentration was determined by the phenol-sulfuric acid method, and appropriately diluted to 50 mg / mL;

[0070] H2O2-Vc degradation: Add 20% H2O2 to make the final concentration of H2O2 2%, stir at 50℃ for 10 min; then add 5 mol / L Vc to make the final concentration of Vc 13%, and stir at 50℃ for 10 min.

[0071] Microwave degradation: Add water of equal mass and microwave treatment at 650W for 10 minutes;

[0072] Sulfation modification: Sulfur trioxide-pyridine was added to a final concentration of 3.5%, and the mixture was incubated in a water bath at 80°C for 1 hour, then cooled to room temperature with ice water. 5 mol / L NaOH was added to adjust the pH to 7, and the solution was dialyzed through a 3000 Da dialysis bag for 24 hours. The precipitate was then collected by centrifugation.

[0073] Freeze-drying: Sulfated mulberry oligosaccharides were freeze-dried to obtain sulfated mulberry oligosaccharides.

[0074] Mulberry oligosaccharides: Sulfated mulberry oligosaccharides were obtained by sulfur trioxide-pyridine sulfation modification. The polysaccharide substitution degree was 1.041, and the monosaccharide composition consisted of glucose, xylose and galactose in a molar ratio of 1:4:2.

[0075] Comparative Example 2: Modified from Example 1: Vc is degraded first, followed by H2O2 degradation.

[0076] Mulberry polysaccharide: After crushing mulberries, pass them through a 20-mesh sieve, extract with water at 70℃ for 5 hours, concentrate the liquid to 1 / 3, precipitate with 3 times 95% ethanol for 48 hours, recover the ethanol, and collect the precipitate to obtain mulberry polysaccharide;

[0077] Concentration determination and dilution: Polysaccharide concentration was determined by the phenol-sulfuric acid method, and appropriately diluted to 50 mg / mL;

[0078] Pectinase hydrolysis: Take 1L of polysaccharide liquid, add 5-10g of pectinase, and hydrolyze at 50℃ for 30min. Vc-H2O2 degradation: Add 5mol / L of Vc to make the final concentration of Vc 13%, stir at 50℃ for 10min; then add 20% H2O2 to make the final concentration of H2O2 2%, stir at 50℃ for 10min.

[0079] Microwave degradation: Add water of equal mass and microwave treatment at 650W for 10 minutes;

[0080] Sulfation modification: Sulfur trioxide-pyridine was added to a final concentration of 3.5%, and the mixture was incubated in a water bath at 80°C for 1 hour, then cooled to room temperature with ice water. 5 mol / L NaOH was added to adjust the pH to 7, and the solution was dialyzed through a 3000 Da dialysis bag for 24 hours. The precipitate was then collected by centrifugation.

[0081] Freeze-drying: Sulfated mulberry oligosaccharides were freeze-dried to obtain sulfated mulberry oligosaccharides.

[0082] Mulberry oligosaccharides: Sulfated mulberry oligosaccharides were obtained by sulfur trioxide-pyridine sulfation modification. The polysaccharide substitution degree was 1.075, and the monosaccharide composition consisted of glucose, xylose and galactose in a molar ratio of 1:6:1.5.

[0083] Comparative Example 3: Modified from Example 1: Microwave degradation followed by H2O2-Vc degradation.

[0084] Mulberry polysaccharide: After crushing mulberries, pass them through a 20-mesh sieve, extract with water at 70℃ for 5 hours, concentrate the liquid to 1 / 3, precipitate with 3 times 95% ethanol for 48 hours, recover the ethanol, and collect the precipitate to obtain mulberry polysaccharide;

[0085] Concentration determination and dilution: Polysaccharide concentration was determined by the phenol-sulfuric acid method, and appropriately diluted to 50 mg / mL;

[0086] Pectinase hydrolysis: Take 1L of polysaccharide liquid, add 5-10g of pectinase, and hydrolyze at 50℃ for 30min.

[0087] Microwave degradation: Add water of equal mass and microwave treatment at 650W for 10 minutes;

[0088] H2O2-Vc degradation: Add 20% H2O2 to make the final concentration of H2O2 2%, stir at 50℃ for 10 min; then add 5 mol / L Vc to make the final concentration of Vc 13%, and stir at 50℃ for 10 min.

[0089] Sulfation modification: Sulfur trioxide-pyridine was added to a final concentration of 3.5%, and the mixture was incubated in a water bath at 80°C for 1 hour, then cooled to room temperature with ice water. 5 mol / L NaOH was added to adjust the pH to 7, and the solution was dialyzed through a 3000 Da dialysis bag for 24 hours. The precipitate was then collected by centrifugation.

[0090] Freeze-drying: Sulfated mulberry oligosaccharides were freeze-dried to obtain sulfated mulberry oligosaccharides.

[0091] Mulberry oligosaccharides: Sulfated mulberry oligosaccharides were obtained by sulfur trioxide-pyridine sulfation modification. The polysaccharide substitution degree was 1.021, and the monosaccharide composition consisted of glucose, xylose and galactose in a molar ratio of 1:3:2.

[0092] Comparative Example 4: Modified from Example 1: The sulfuric acid modification step was omitted.

[0093] Mulberry polysaccharide: After crushing mulberries, pass them through a 20-mesh sieve, extract with water at 70℃ for 5 hours, concentrate the liquid to 1 / 3, precipitate with 3 times 95% ethanol for 48 hours, recover the ethanol, and collect the precipitate to obtain mulberry polysaccharide;

[0094] Concentration determination and dilution: Polysaccharide concentration was determined by the phenol-sulfuric acid method, and appropriately diluted to 50 mg / mL;

[0095] Pectinase hydrolysis: Take 1L of polysaccharide liquid, add 5-10g of pectinase, and hydrolyze at 50℃ for 30min. H2O2-Vc degradation: Add 20% H2O2 to make the final concentration of H2O2 2%, stir at 50℃ for 10min; then add 5mol / L Vc to make the final concentration of Vc 13%, and stir at 50℃ for 10min.

[0096] Microwave degradation: Add water of equal mass and microwave treatment at 650W for 10 minutes;

[0097] Freeze-drying: Mulberry oligosaccharides degraded by microwaves were freeze-dried to obtain sulfated mulberry oligosaccharides.

[0098] Mulberry oligosaccharides: Sulfated mulberry oligosaccharides were obtained by sulfur trioxide-pyridine sulfation modification. The polysaccharide substitution degree was 1.055, and the monosaccharide composition consisted of glucose, xylose and galactose in a molar ratio of 1:6.7:1.3.

[0099] Comparative Example 5: Modified from Example 1: first sulfation modification, then H2O2-Vc degradation and microwave degradation.

[0100] Mulberry polysaccharide: After crushing mulberries, pass them through a 20-mesh sieve, extract with water at 70℃ for 5 hours, concentrate the liquid to 1 / 3, precipitate with 3 times 95% ethanol for 48 hours, recover the ethanol, and collect the precipitate to obtain mulberry polysaccharide;

[0101] Concentration determination and dilution: Polysaccharide concentration was determined by the phenol-sulfuric acid method, and appropriately diluted to 50 mg / mL;

[0102] Pectinase hydrolysis: Take 1L of polysaccharide liquid, add 5-10g of pectinase, and hydrolyze at 50℃ for 30min.

[0103] Sulfation modification: Sulfur trioxide-pyridine was added to a final concentration of 3.5%, and the mixture was incubated in a water bath at 80°C for 1 hour, then cooled to room temperature with ice water. 5 mol / L NaOH was added to adjust the pH to 7, and the solution was dialyzed through a 3000 Da dialysis bag for 24 hours. The precipitate was then collected by centrifugation.

[0104] H2O2-Vc degradation: Add 20% H2O2 to make the final concentration of H2O2 2%, stir at 50℃ for 10 min; then add 5 mol / L Vc to make the final concentration of Vc 13%, and stir at 50℃ for 10 min.

[0105] Microwave degradation: Add water of equal mass and microwave treatment at 650W for 10 minutes;

[0106] Freeze-drying: Mulberry oligosaccharides degraded by microwaves were freeze-dried to obtain sulfated mulberry oligosaccharides.

[0107] Mulberry oligosaccharides: Sulfated mulberry oligosaccharides were obtained by sulfur trioxide-pyridine sulfation modification. The degree of polysaccharide substitution was 1.062, and the monosaccharide composition consisted of glucose, xylose and galactose in a molar ratio of 1:8:3.

[0108] Example 4

[0109] Feed containing 1% sulfated mulberry oligosaccharides

[0110] Commercial California bass feed (Foshan Nanhai District Jieda Feed Co., Ltd., Foshan, China) was used as the base feed. The sulfated mulberry oligosaccharides prepared in the examples and comparative examples were accurately weighed at an addition amount of 1% of the base feed and thoroughly mixed with the base feed to obtain feeds containing 1% sulfated mulberry oligosaccharides.

[0111] Example 5

[0112] The effects of sulfated mulberry oligosaccharides on aquaculture

[0113] 1. Experimental Design

[0114] First, a trial rearing with a basic feed was conducted for 14 days. Then, robust and uniformly sized largemouth bass fry were selected and randomly divided into 3 groups, with 3 replicates per group and 50 fish per replicate. The formal experiment began, with a trial period of 80 days. Largemouth bass raised on a diet containing 1% sulfated mulberry oligosaccharides (Examples 1-3 and Comparative Examples 1-3 and Comparative Example 5) or mulberry oligosaccharides (Comparative Example 4) served as the experimental group, while those raised on a basic feed served as the control group. The daily feeding rate was 1%-2% of body weight. After the rearing period, the total weight of the fish was measured, and the following indicators were determined using a kit (Nanjing Jiancheng Bioengineering Institute): total protein, albumin, serum lysozyme, immunoglobulin M, immunoglobulin G, alkaline phosphatase, acid phosphatase, nitric oxide, superoxide dismutase, malondialdehyde, and total antioxidant capacity.

[0115] The calculation formulas involved are as follows:

[0116] Survival rate (SR, %) = (Number of last digits / Number of initial digits) × 100% Formula I

[0117] Weight gain rate (WGR, %) = [Final average weight (g) - Initial average weight (g)] / Initial average weight (g) × 100% Formula II

[0118] Feed conversion ratio (FCR) = Feeding amount / [Final average weight (g) - Initial average weight (g)] Formula III

[0119] 2. Antiviral Experiment: Healthy largemouth bass were selected and fed with ordinary feed (control group) and feed prepared with oligosaccharides obtained in the examples and comparative examples (experimental group) for 2 months, followed by an iridovirus challenge experiment. Fish mortality was observed and recorded several days after challenge.

[0120] 3. Results

[0121] As shown in Table 1, when the oligosaccharide prepared in the example was added to the feed, the weight gain rate and feed conversion ratio of the largemouth bass did not change significantly compared with the control group. This proves that the modified sulfated mulberry oligosaccharide is safe and usable for fish growth. The weight gain rate of the control group was significantly lower and the feed conversion ratio was higher, indicating that the control group had a certain impact on fish growth.

[0122] Table 1 Effects of sulfated mulberry oligosaccharides on the growth performance of largemouth bass

[0123] index Weight gain rate / % Feed conversion ratio control group 546.80±9.86 1.27±0.15 Example 1 553.33±8.45 1.25±0.14 Example 2 549.56±7.36 1.28±0.25 Example 3 545.56±5.63 1.28±0.36 Comparative Example 1 429.56±4.52 1.48±0.58 Comparative Example 2 428.56±4.96 1.49±0.61 Comparative Example 3 443.56±5.02 1.52±0.52 Comparative Example 4 459.56±5.12 1.48±0.49 Comparative Example 5 439.56±4.96 1.50±0.68

[0124] The total protein content in serum can reflect the body's immune status; a higher content indicates better immunity. Albumin promotes the transport of nutrients and immune regulation. Lysozyme activity is also an important indicator for evaluating the body's immunity; lysozyme can dissolve peptidoglycan in bacterial cell walls, thereby resisting foreign pathogens. Immunoglobulin M and immunoglobulin G can enhance the animal's disease resistance; higher serum levels can help resist disease.

[0125] Alkaline phosphatase and acid phosphatase are important lysosomal enzymes in fish, participating in various metabolic activities and breaking down foreign particles, reflecting the body's non-specific immune capacity. Nitric oxide participates in physiological activities such as blood circulation and nerve transmission, but it can also combine with superoxide anion free radicals to generate peroxynitrite, damaging cells and causing disease. Under normal physiological conditions, appropriate amounts of nitric oxide have a protective effect on the body; however, in inflammatory states, the body produces large amounts of nitric oxide, leading to tissue damage.

[0126] Table 2 shows that the sulfated mulberry oligosaccharide in the feeding example significantly increased the levels of total protein, albumin, serum lysozyme, immunoglobulin M, and immunoglobulin G in the serum of largemouth bass, all indicators related to immunity. Table 3 shows that alkaline phosphatase and acid phosphatase levels increased, while nitric oxide levels decreased in the intestines of largemouth bass. By measuring the immune indicators in the blood and intestines, it can be concluded that adding the sulfated mulberry oligosaccharide prepared in the example to the feed improves the immunity of largemouth bass.

[0127] Table 2. Effects of sulfated mulberry oligosaccharides on serum immunogenicity of largemouth bass.

[0128]

[0129]

[0130] Table 3. Effects of sulfated mulberry oligosaccharides on intestinal immunity of largemouth bass.

[0131]

[0132] The antioxidant capacity of an organism can be used to evaluate the health of fish. Excessive free radicals lead to the peroxidation of unsaturated fatty acids in cell membranes, which are eventually broken down into malondialdehyde (MDA) in the fish's body. Superoxide dismutase (SOD), MDA, and total antioxidant capacity in the liver and serum are important indicators for evaluating the body's antioxidant capacity. Higher SOD activity and total antioxidant capacity, as well as lower MDA content, reflect that the fish has a high antioxidant capacity and strong disease resistance.

[0133] Table 4 reflects the effect of sulfated mulberry oligosaccharides on the antioxidant capacity of largemouth bass liver. Adding the sulfated mulberry oligosaccharides prepared in the examples to the feed can increase the activity of superoxide dismutase (SOD) in the liver and reduce the content of malondialdehyde (MDA) in the liver. Table 5 shows that adding the sulfated mulberry oligosaccharides prepared in the examples to the feed can increase serum SOD activity and total antioxidant capacity, and reduce MDA content. By measuring the antioxidant indicators of the liver and blood, it can be concluded that feeding largemouth bass with the sulfated mulberry oligosaccharides prepared in the examples improves their antioxidant capacity, which is superior to the oligosaccharides prepared in the comparative example.

[0134] Table 4. Effects of sulfated mulberry oligosaccharides on the antioxidant capacity of largemouth bass liver.

[0135] index Superoxide dismutase (U / mgprot) Malondialdehyde (nmol / mgprot) Comparison 14.75±1.23 1.05±0.04 Example 1 21.67±1.17 0.51±0.04 Example 2 18.16±1.21 0.68±0.06 Example 3 19.24±1.36 0.71±0.23 Comparative Example 1 10.52±1.02 2.54±0.31 Comparative Example 2 9.85±0.98 3.12±0.27 Comparative Example 3 10.14±0.95 2.98±0.24 Comparative Example 4 8.52±0.94 3.02±0.42 Comparative Example 5 9.34±0.65 3.05±0.24

[0136] Table 5 Effects of sulfated mulberry oligosaccharides on the antioxidant capacity of largemouth bass serum

[0137]

[0138] The survival rate curves of the largemouth bass after being challenged with iridovirus are shown in Table 6. At the end of the experimental period, the survival rate of the control group was 40%; the survival rate of the sulfated mulberry oligosaccharide group in the example was higher than 90%, and its survival rate was significantly higher than that of the control group and the comparative group.

[0139] Table 6 Survival rate of largemouth bass

[0140] index Survival rate (%) Comparison 40 Example 1 96 Example 2 94 Example 3 96 Comparative Example 1 20 Comparative Example 2 24 Comparative Example 3 22 Comparative Example 4 24 Comparative Example 5 20

[0141] 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. The application of a sulfated mulberry oligosaccharide in at least one of the following aspects in the preparation of aquatic feed: 1) Enhance the antioxidant capacity of aquatic animals; 2) Enhance the antiviral resistance of aquatic animals; 3) Enhance the immune capacity of aquatic animals; The aquatic animal in question is the largemouth bass; The virus in question is an iridovirus; The method for preparing the sulfated mulberry oligosaccharide includes the following steps: Mulberry polysaccharides were sequentially subjected to pectinase hydrolysis, H2O2-Vc degradation, and microwave degradation to obtain degradation products; The degradation products were modified by sulfation to obtain sulfated mulberry oligosaccharides.

2. The application according to claim 1, characterized in that, During the pectinase hydrolysis, the final concentration of pectinase is 5–10 g / L.

3. The application according to claim 1, characterized in that, The pectinase hydrolysis temperature is 40–60°C; the pectinase hydrolysis time is 25–35 min.

4. The application according to claim 1, characterized in that, During the degradation of H2O2-Vc, the final concentration of H2O2 is 1% to 5%; the final concentration of Vc is 10% to 15%.

5. The application according to claim 1, characterized in that, The H2O2-Vc degradation temperature is 48–52℃; the H2O2-Vc degradation time is 8–12 min.

6. The application according to claim 1, characterized in that, During the microwave degradation process, the microwave power is 600–800W, and the microwave treatment time is 8–12 minutes.

7. The application according to claim 1, characterized in that, 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 2% to 5%; the temperature of the water bath reaction is 78 to 82°C; and the time of the water bath reaction is 1 to 1.5 hours.

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