CN117304361A - Antiviral sulfated mulberry leaf oligosaccharide and preparation method, animal feed and application thereof - Google Patents
Antiviral sulfated mulberry leaf oligosaccharide and preparation method, animal feed and application thereof Download PDFInfo
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- CN117304361A CN117304361A CN202311394125.2A CN202311394125A CN117304361A CN 117304361 A CN117304361 A CN 117304361A CN 202311394125 A CN202311394125 A CN 202311394125A CN 117304361 A CN117304361 A CN 117304361A
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- sulfated
- mulberry leaf
- oligosaccharide
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- leaf oligosaccharide
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- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Birds (AREA)
- Marine Sciences & Fisheries (AREA)
- Insects & Arthropods (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Fodder In General (AREA)
Abstract
The invention provides antiviral sulfated mulberry leaf oligosaccharide, a preparation method thereof, animal feed and application, and belongs to the technical field of oligosaccharides. The invention carries out microwave degradation and H on mulberry leaf polysaccharide 2 O 2 And (3) Vc degradation, and carrying out sulfation modification on degradation products to obtain sulfated mulberry leaf oligosaccharide. Experiments prove that the sulfated mulberry leaf oligosaccharide can promote the growth of micropterus salmoides when being added into feeds for aquaculture, improve the utilization rate of the feeds, the oxidation resistance, the lipid metabolism and the immune function and improve the resistance to virus infection.
Description
Technical Field
The invention belongs to the technical field of oligosaccharides, and particularly relates to an antiviral sulfated mulberry leaf oligosaccharide, a preparation method thereof, animal feed and application.
Background
The largemouth weever (Micropterus salmoides) is one of the most important freshwater economic fishes in China and has important culture value. The cultured micropterus salmoides not only provides a large amount of high-quality animal proteins for China, but also brings good economic benefit. However, as the cultivation scale is enlarged and the cultivation density is continuously increased, the cultivation environment is gradually deteriorated, the disease problem of the micropterus salmoides is also more and more prominent, great loss is brought to economy, and the sustainable development of the industry is severely restricted. In order to prevent, treat and control diseases, therapeutic agents such as antibiotics and chemicals are often used in aquaculture, however, excessive use of these therapeutic agents may cause drug residues, which adversely affects the environment, human and animal safety. At present, china has forbidden to add antibiotics into feed, and 'anti-scratch resistance' has become a trend. Therefore, the development of environmentally friendly and safe antibiotic alternatives is a hotspot in industry research. Among them, functional additives having preventive effects such as prebiotics and plant extracts have been the focus of research.
Prebiotics are organic substances that are not directly digested and absorbed by the host, but which selectively promote the growth or increase the activity of a few beneficial bacteria in the colon, thereby improving the health of the host. Along with the continuous and deep research on the functions and action mechanisms of the prebiotics, the prebiotics are widely applied to the livestock and poultry animal breeding of chickens, pigs, cattle, sheep and the like. Since prebiotics can improve the health of aquaculture animals, promote growth and improve immunity, etc., they have been increasingly used in aquaculture since the nineties of the last century. The prebiotics which can be used as feed additives at present are mainly oligosaccharides made of monosaccharide units such as fructose, galactose, glucose or xylose, etc., such as commercial fructo-oligosaccharides, galacto-oligosaccharides, isomalto-oligosaccharides, etc. Currently commercialized oligosaccharides are generally chemically synthesized, single in component, and less studied for functional oligosaccharides of vegetable origin.
The mulberry leaf polysaccharide (Mulberry leafpolysaccharide, MLP) is a natural active ingredient extracted from mulberry leaves, and has various functions of resisting oxidation, resisting bacteria, promoting probiotics, reducing blood sugar, stimulating immunity and the like. Researches show that the addition of MLP in daily ration can improve intestinal microbial flora of weaned pigs, reduce diarrhea rate and improve growth performance of early weaned pigs. In addition, MLP can obviously promote secretion of IL-2, IFN-gamma and sIgA in jejunum and trachea of chicken, promote generation of IgA in cecum and tonsil, and strengthen immune function of intestinal tract and tracheal mucosa. It is generally believed that the degradation of polysaccharides to small molecular weight oligosaccharides by physical, chemical and biological means can enhance their biological activity. The MLP can be subjected to enzymolysis to obtain mulberry leaf oligosaccharide (Mulberry leafoligosaccharides, MLO). Research shows that compared with mulberry leaf polysaccharide, mulberry leaf oligosaccharide has higher probiotic activity and antioxidant capacity. In addition, MLO can smoothly pass through the digestive system to reach the colon without being degraded, and the MLO has stronger activity in promoting the growth of probiotics and improving the capability of the probiotics to produce short-chain fatty acid. However, the application research of MLO as feed additive in antiviral of aquatic animals has not been reported so far.
Reference to the literature
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Disclosure of Invention
Therefore, the invention aims to provide sulfated mulberry leaf oligosaccharide which has obvious antiviral activity and improves the toxicity-eliminating survival rate of the micropterus salmoides.
The invention provides an antiviral sulfated mulberry leaf oligosaccharide, which comprises the following monosaccharides: glucuronic acid, rhamnose and mannose;
the molar ratio of glucuronic acid to rhamnose to mannose is 2 (3-4) (4-7);
in the monosaccharide, sulfonic groups replace hydroxyl groups in the monosaccharide, and the substitution degree of the polysaccharide is 1.107-1.126;
the molecular weight of the sulfated mulberry leaf oligosaccharide is 2000Da to 4000Da.
Preferably, the molar ratio of glucuronic acid, rhamnose and mannose is 2:3:4.
Preferably, the polysaccharide has a degree of substitution of 1.126.
The invention provides a preparation method of antiviral sulfated mulberry leaf oligosaccharide, which comprises the following steps:
microwave degrading and H-degrading folium Mori polysaccharide 2 O 2 Vc degradation to obtain degradation products;
and (3) carrying out sulfation modification on the degradation product to obtain the sulfated mulberry leaf oligosaccharide.
Preferably, when the microwaves are degraded, the power of the microwaves comprises 500-750W, and the treatment time of the microwaves is 20-30 min; the concentration of the mulberry leaf polysaccharide is 10-100 mg/mL;
at said H 2 O 2 When Vc is degraded, the final volume concentration of hydrogen peroxide is 0.5-3%; the final concentration of Vc is 5% -10%; the H is 2 O 2 The degradation temperature of Vc is 50-55 ℃ and the degradation time is 10-12 min;
the sulfation modification method is that the degradation product and sulfur trioxide-pyridine are subjected to water bath reaction; the final concentration of the sulfur trioxide-pyridine is 10mg/ml; the temperature of the water bath reaction is 90-95 ℃, and the time of the water bath reaction is 1-1.5 h.
The invention provides application of the antiviral sulfated mulberry leaf oligosaccharide or the sulfated mulberry leaf oligosaccharide obtained by the preparation method in aquaculture.
Preferably, the aquaculture comprises the following applications:
1) Improving the antiviral property of aquatic animals;
2) Improving the antioxidation capability of aquatic animals;
3) Improving the immunity of aquatic animals;
4) Improving the feed utilization rate of aquatic animals;
5) Improving lipid metabolism of aquatic animal.
Preferably, the virus comprises largemouth weever iridovirus (LMBV);
the aquatic product comprises micropterus salmoides.
The invention provides aquatic animal feed, which comprises the antiviral sulfated mulberry leaf oligosaccharide or the sulfated mulberry leaf oligosaccharide obtained by the preparation method and basic feed.
Preferably, the antiviral sulfated mulberry leaf oligosaccharide accounts for 0.5-1.2% of the mass of the basic feed.
The invention provides an antiviral sulfated mulberry leaf oligosaccharide, which comprises the following monosaccharides: glucuronic acid, rhamnose and mannose; the molar ratio of glucuronic acid to rhamnose to mannose is 2:93-4) (4-7); in the monosaccharide, sulfonic groups replace hydroxyl groups in the monosaccharide, and the substitution degree of the polysaccharide is 1.107-1.126; the molecular weight of the sulfated mulberry leaf oligosaccharide is 2000Da to 4000Da. The sulfated mulberry leaf oligosaccharide is used as a feed additive to be added into basic feed for application and aquaculture, the influence of the sulfated mulberry leaf oligosaccharide on the LMBV virus resistance of the micropterus salmoides is studied, and the result shows that after the feed containing the sulfated mulberry leaf oligosaccharide is fed, the survival rate of the micropterus salmoides after the toxicity attack is more than 70%, and the survival rate of a blank control group is only 25%, so that the sulfated mulberry leaf oligosaccharide can obviously improve the survival rate of the micropterus salmoides under LMBV infection, and the sulfated mulberry leaf oligosaccharide has antiviral activity. In addition, the influence of the sulfated mulberry leaf oligosaccharide on the growth performance, the antioxidant capacity, the glycolipid metabolism, the liver health, the intestinal flora and the like of the largehead jewfish is also researched, and the result shows that the sulfated mulberry leaf oligosaccharide has the activities of promoting the growth of the largehead jewfish, improving the utilization rate of feed, improving the antioxidant capacity, the lipid metabolism, the immune function and the like of aquatic products. The sulfated mulberry leaf oligosaccharide provided by the invention provides a theoretical basis for reasonable application of MLO in the compound feed of micropterus salmoides, and enriches the biological functions of the oligosaccharide.
Drawings
FIG. 1 is a graph showing the effect of MLO on the levels of inflammation-associated gene tables; and (3) injection: the values with different letters differ significantly (P <0.05 (mean±sem, n=3);
FIG. 2 is a graph showing the effect of MLO on the levels of glycolipid metabolism-related genes; and (3) injection: the values with different letters differ significantly (P <0.05 (mean±sem, n=3);
FIG. 3 is the effect of MLO on liver histology (H & E staining) after 80 days on micropterus salmoides.
FIG. 4 shows the result of MLO vs. Lateolabrax latissimus anti-LMBV survival curve analysis.
Detailed Description
The invention provides an antiviral sulfated mulberry leaf oligosaccharide, which comprises the following monosaccharides: glucuronic acid, rhamnose and mannose;
the molar ratio of glucuronic acid to rhamnose to mannose is 2:3-4:4-7;
in the monosaccharide, sulfonic groups replace hydroxyl groups in the monosaccharide, and the substitution degree of the polysaccharide is 1.107-1.126;
the molecular weight of the sulfated mulberry leaf oligosaccharide is 2000Da to 4000Da.
In the present invention, the molar ratio of glucuronic acid, rhamnose and mannose is preferably 2:3:4. The polysaccharide preferably has a degree of substitution of 1.126.
The invention provides a preparation method of antiviral sulfated mulberry leaf oligosaccharide, which comprises the following steps:
microwave degrading and H-degrading folium Mori polysaccharide 2 O 2 Vc degradation to obtain degradation products;
and (3) carrying out sulfation modification on the degradation product to obtain the sulfated mulberry leaf oligosaccharide.
The invention carries out microwave degradation and H on mulberry leaf polysaccharide 2 O 2 Vc degradation to give degradation products.
The source of the mulberry leaf polysaccharide is not particularly limited, and the mulberry leaf polysaccharide is prepared by adopting an extraction method of the mulberry leaf polysaccharide which is well known in the art or purchased through a commercial way. The extraction method of the mulberry leaf polysaccharide is preferably completed by adopting a water extraction and alcohol precipitation method. After the mulberry leaf polysaccharide is obtained, concentration measurement is preferably performed. The concentration determination method is preferably a phenol sulfuric acid method.
In the invention, when the microwaves are degraded, the power of the microwaves preferably comprises 500-750W, and the treatment time of the microwaves is preferably 20-30 min; the concentration of the mulberry leaf polysaccharide is preferably 10 to 100mg/mL, more preferably 20 to 80mg/mL, still more preferably 30 to 70mg/mL, and most preferably 50mg/mL. The microwave degradation is favorable for degrading the mulberry polysaccharide into the mulberry oligosaccharide under the action of microwaves, and simultaneously reduces the viscosity of a reaction system.
In the present invention, in the above H 2 O 2 The final volume concentration of hydrogen peroxide upon Vc degradation is preferably 0.5% to 3%, more preferably 1%; the final concentration of Vc is preferably 5-10%, more preferably 6%. The H is 2 O 2 The temperature of Vc degradation is preferably 50-55 ℃, and the degradation time is preferably 10-12 min. The H is 2 O 2 Vc degradation is favorable for mulberry leaf oligosaccharide prepared by microwave degradation in H 2 O 2 -re-degradation under Vc.
In the present invention, the method of sulfation modification is preferably a water bath reaction of the degradation product and sulfur trioxide-pyridine; the final concentration of sulfur trioxide-pyridine is preferably 10mg/ml; the temperature of the water bath reaction is preferably 90-92 ℃, and the time of the water bath reaction is preferably 1-1.5 h. After the water bath reaction is finished, the pH value of the reaction product is preferably adjusted to be neutral, the salt is removed, and the solid phase substance is separated. The method of desalting is preferably dialysis. The dialysis bag for dialysis preferably has a molecular weight cut-off of 2000Da. The dialysis time is preferably 48 hours. The method of separating the solid phase material is preferably centrifugation. The rotational speed of the centrifugation is preferably 8000 Xg to 13000 Xg, and most preferably 10000 Xg. After the solid phase material is obtained, freeze drying is preferably performed.
Based on the activity of the sulfated mulberry leaf oligosaccharide, the invention provides aquatic animal feed, which comprises the antiviral sulfated mulberry leaf oligosaccharide or the sulfated mulberry leaf oligosaccharide obtained by the preparation method and basic feed.
The antiviral sulfated mulberry leaf oligosaccharide accounts for 0.5-1.2% of the mass of the basic feed in the invention.
The invention provides application of the antiviral sulfated mulberry leaf oligosaccharide or the sulfated mulberry leaf oligosaccharide obtained by the preparation method in aquaculture.
In the present invention, the aquaculture preferably comprises the use of:
1) Improving the antiviral property of aquatic animals;
2) Improving the antioxidation capability of aquatic animals
3) Improving the immunity of aquatic animals;
4) Improving the feed utilization rate of aquatic animals;
5) Improving lipid metabolism of aquatic animal.
In the present invention, the virus preferably comprises the megapterus iridovirus (LMBV). The aquatic product preferably comprises micropterus salmoides, more preferably micropterus salmoides. In the embodiment of the invention, the sulfated mulberry leaf oligosaccharide is respectively used for preparing aquatic feeds (MLOL and MLOH) in two different addition amounts, and the resistance of the sulfated mulberry leaf oligosaccharide to viruses of aquatic animals is studied. The results show that the blank group (largehead weever fed with only the basic feed with sulfated mulberry leaf oligosaccharide) started to die at 3d challenge, the low dose group (MLOL) died at day 4, and the high dose group (MLOH) died at day 6; meanwhile, the death numbers are not changed after 14 days of virus attack, the survival rate of the control group is 25%, the survival rate of the MLOL group is 65%, and the survival rate of the MLOH group is 70%. The relative protection of the MLOL treated group was 42.86% and the MLOH treated group was 50% compared to the control group. Therefore, the sulfated mulberry leaf oligosaccharide is beneficial to reducing the death rate of the micropterus salmoides caused by toxicity attack, and shows that the sulfated mulberry leaf oligosaccharide has antiviral activity.
In the embodiment of the invention, the influence of the sulfated mulberry leaf oligosaccharide on the growth performance of the micropterus salmoides is also studied, and the result shows that the sulfated mulberry leaf oligosaccharide can reduce the feed coefficient and the dirty body ratio of the micropterus salmoides, and the high dose group (MLOH) also obviously reduces the liver body ratio.
In the embodiment of the invention, the influence of sulfated mulberry leaf oligosaccharide on the blood biochemical index of the micropterus salmoides is also studied, and the result shows that the sulfated mulberry leaf oligosaccharide is favorable for reducing the triglyceride and blood sugar content in blood and reducing the enzyme activity of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase. The influence of sulfated mulberry leaf oligosaccharide on liver tissues of the largehead perches is also studied, and the sulfated mulberry leaf oligosaccharide is favorable for improving cavitation and cell nucleus displacement conditions of liver tissue cells, and has complete liver cell structure and clear outline. This indicates that the sulfated mulberry leaf oligosaccharide can significantly improve liver health of micropterus salmoides.
In the embodiment of the invention, the influence of the sulfated mulberry leaf oligosaccharide on the antioxidant capacity of the micropterus salmoides is also studied, and the result shows that the sulfated mulberry leaf oligosaccharide can effectively improve the enzyme activity of the total superoxide dismutase, improve the GSH content and reduce the malondialdehyde content, so that the sulfated mulberry leaf oligosaccharide can effectively improve the antioxidant capacity of the blood and liver of the micropterus salmoides.
In the embodiment of the invention, the influence of the sulfated mulberry leaf oligosaccharide on the inflammatory reaction of the micropterus salmoides is also studied, and the result shows that the sulfated mulberry leaf oligosaccharide can effectively improve the expression of anti-inflammatory factors and reduce the expression of pro-inflammatory factors, and the sulfated mulberry leaf oligosaccharide has an inhibition effect on the inflammatory reaction of the micropterus salmoides.
In the embodiment of the invention, the sulfated mulberry leaf oligosaccharide has the effect on the liver glycolipid metabolism of the micropterus salmoides, and the result shows that the sulfated mulberry leaf oligosaccharide improves the expression level of glycolytic genes and the expression level of lipid metabolism related genes, thereby indicating that the sulfated mulberry leaf oligosaccharide can obviously improve the glycolipid metabolism function of the liver of the micropterus salmoides.
The antiviral sulfated mulberry leaf oligosaccharide, the preparation method thereof, the animal feed and the application thereof provided in the present invention are described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation method of sulfated mulberry leaf oligosaccharide
1. Mulberry leaf polysaccharide: pulverizing folium Mori, extracting with 70deg.C water for 5 hr, concentrating to 1/3, precipitating with 3 times of 95% ethanol for 48 hr, collecting precipitate to obtain folium Mori polysaccharide
2. Concentration was measured and diluted: the concentration of polysaccharide is measured by phenol sulfuric acid method, and is properly diluted to 70mg/mL
3. And (3) microwave degradation: taking 100mL polysaccharide liquid, and treating 500W with microwave for 20min
4.H 2 O 2 Vc degradation: 1mL of 30% H was added 2 O 2 Stirring at 50deg.C for 10min; adding 1mL of 4mol/L Vc, stirring at 50deg.C for 10min
5. Sulfating modification: 1g of sulfur trioxide-pyridine was added, water-bath was carried out at 90℃for 1 hour, and ice water was cooled to room temperature. Adding 5mol/L NaOH, adjusting pH to 7, desalting with 2000Da dialysis bag for 48 hr, centrifuging, and collecting precipitate
6. And (3) freeze drying: and freeze-drying the sulfated and modified mulberry leaf oligosaccharide to obtain the sulfated mulberry leaf oligosaccharide.
The substitution degree of the polysaccharide of the sulfonic acid group is determined by adopting a barium chloride gelatin method, and the monosaccharide type and proportion of the sulfated mulberry leaf oligosaccharide are determined by adopting a GC-MS.
The results showed that the polysaccharide has a degree of substitution of 1.126 and the monosaccharide composition consists of glucuronic acid, rhamnose and mannose in a molar ratio of 2:3:4.
Example 2
Preparation method of animal feed
The sulfated mulberry leaf oligosaccharide prepared in example 1 was weighed into a commercial feed (formula and nutrition level are shown in Table) of California bass (Jie Dafeed Co., south sea area of Buddha, china Buddha) as a basal feed to prepare feed (MLOH) containing 0.5% MLO feed (MLOL) and 1% MLO, respectively, in mass concentration.
Table 1 basal diet formulation and nutrient levels
1 CK,basal diet；MLOL,supplemented with 0.5％MLO；MLOH,supplemented with1.0％MLO.
2) Each kilogram of vitamin premix contains: A66,666,666.7IU, 400,000,000IU, 1g, 2 g, 15 g, 25 g, 65 g, 121 g, 20g, 10g, 1g, 20g, 200g, 700g of defatted rice bran.
3) Each kilogram of mineral premix comprises: cuCO 3 4 g，FeC 6 H 5 O 7 15 g，MgO 26g，MnSO 4 5 g，KCl 250g，ZnSO 4 50g, naCl 50g and zeolite powder 600g.
Example 3
Application of sulfated mulberry leaf oligosaccharide in aquaculture
1. Experimental method
1. Feeding management
The largehead jewfish used in the test is purchased from Guangdong Liang aquatic products industry Co., ltd, and after the experimental fish is transported back to Guangzhou, the experimental fish is temporarily cultured in an indoor circulating water culture system of the Guangdong agricultural academy of sciences of silkworm industry and agricultural product processing institute for 2 weeks, and the experimental fish is fed with basic feed every day during the temporary culture period. Before the formal test, stopping feeding for 24 hours, selecting 450 tail juvenile fish with an average initial weight of 26.89+/-1.16 g, randomly dividing the juvenile fish into 9 cultivation barrels with the diameter of 75cm (the volume is 350L), randomly dividing the 9 cultivation barrels into 3 groups of which the number is 3, and repeating each group. The two test groups were fed with basal feed containing 0.5% MLO (MLOL) and 1% MLO (MLOH), respectively, and the control group (CK) was fed with basal feed. In the formal test, the fish is fed 2 times a day (09:00 and 16:30), the feeding amount is 2-4% of the weight of the fish, and the test period is 80 days. The indoor circulating water culture system consists of a microfiltration system, a biochemical reaction system, a protein separator, an ultraviolet disinfection device and a culture barrel, wherein a culture water source is tap water after disinfection and aeration, water is changed 3 times per week during a test period, the water change amount is 1/4, the water temperature during the culture period is 22.4-29.4 ℃, dissolved oxygen is more than or equal to 5.0mg/L, nitrite content is less than or equal to 0.05mg/L, ammonia nitrogen content is less than or equal to 0.2mg/L, pH is 7.0-8.2, and the natural illumination period is realized.
2. Sample collection
After the end of the experiment, 9 fish were randomly selected from each cultivation tank, body length and body weight were measured after anesthesia with MS-222, and then dissected, viscera, liver were taken and weighed, and body weight, body length, viscera and liver weight were recorded for calculation of end Weight (WG), fullness (CF), visceral volume ratio (VSI) and hepatic volume ratio (HIS). 6 fish were randomly picked up from each cultivation barrel, blood was drawn from the tail vein using a 1mL syringe, and centrifuged (3000 r/min,10min,4 ℃) after standing at normal temperature for 2 hours, and the supernatant was taken and stored at-20℃for serum biochemical and enzyme activity analysis. 3 fish were randomly selected from each cultivation barrel, and liver and intestinal tract were obtained after dissection, and stored at-20deg.C after quick freezing with liquid nitrogen for enzyme activity measurement. 3 fish were randomly selected from each cultivation barrel, liver tissue was isolated after dissection, and stored at-80 ℃ after liquid nitrogen flash freezing for gene expression analysis.
3. Biochemical index detection
3.1 serum biochemical index determination: serum samples stored at-20 ℃ were thawed on ice, and then the enzyme activities of low density lipoprotein (LDL-C), high lipoprotein (HDL-C), total cholesterol (T-CHO), triglyceride (TG), reduced Glutathione (GSH), malondialdehyde (MDA), total antioxidant capacity (T-AOC) and blood Glucose (GLU) were determined using a kit (institute of biological engineering, south-kyo) as well as alkaline phosphatase (AKP), total superoxide dismutase (T-SOD), glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST), with specific experimental methods referring to the kit instructions.
3.2 liver index determination: thawing liver tissue stored at-80 ℃ on ice, accurately weighing the tissue according to the weight (g): volume (mL) =1: 9, adding 9 volumes of physiological saline, mechanically homogenizing under ice water bath condition, centrifuging at 4deg.C and 2500r/min for 10min, taking supernatant, and measuring total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD) and Malondialdehyde (MDA) indexes, wherein the measuring method refers to the kit instruction book produced by Nanjing's biological engineering research.
3.3 intestinal enzyme activity assay: thawing intestinal tissues stored at-80 ℃ on ice, and accurately weighing the tissues according to the weight (g): volume (mL) =1: 9, adding 9 volumes of physiological saline, mechanically homogenizing under ice water bath condition, centrifuging at 4deg.C and 2500r/min for 10min, collecting supernatant, and measuring the activities of Amylase (AMS), lipase (LPS) and Trypsin (TRY) in intestinal tract by using a kit (Nanjing institute of biological engineering, nanjing), wherein the specific method is described in the specification of the kit.
4. Calculation formula
Survival rate (SR,%) =last mantissa/first mantissa×100% formula I
Weight gain ratio (WGR,%) = [ final homogeneous mass (g) -initial average mass (g) ]/initial average mass (g) ×100% formula II
Specific growth rate (SGR,%/d) =100× [ ln (final average body weight+dead body weight) -ln initial average body weight ]/days of feeding formula III
Feeding rate FI (%/d) =100×total weight of feed fed/[ (total weight of initial fish+total weight of final fish)/2 ]/day formula IV
Feed Coefficient (FCR) =feeding amount/[ last homogeneous amount (g) -initial homogeneous mass (g) ] formula V
Full of fertilizer (CF, g/cm) 3 ) Body length (g)/body length 3 (cm) 3 ) Formula VI
Liver volume ratio (HSI,%) =liver mass (g)/body mass (g) ×100% formula VII
Dirty body ratio (HVI,%) =visceral mass (g)/body mass (g) ×100% formula III.
5. Liver and intestinal tissue section observation
3 fish is randomly selected from each cultivation barrel for dissection, intestinal tract is separated, surface fat is removed, midgut with length of about 1cm is taken, liver is separated, and the same part of liver is taken about 0.5cm 3 Is fixed by paraformaldehyde fixing solution respectively and is used for observing intestinal tracts and liver tissue sections. Paraffin embedding is carried out on intestinal canal and liver tissues fixed by paraformaldehyde solution, paraffin sections are manufactured, the thickness of the sections is 6 mu m, and hematoxylin-eosin (HE) staining is carried out. The slices were scanned using a panoramic slice digital scanner (PANNORAMIC-1000,3DHISTECH), the tissue photographs of the slices were taken using caseviewer2.2 (3 DHISTECH) software, and then the intestinal villus height, villus width and muscle layer thickness were measured using Image-Pro Plus 6.0 (Media Cybemetics) analysis software, respectively, with 4 sets of values measured for each slice.
RNA extraction and real-time quantitative fluorescent quantitative PCR
Liver tissue stored at-80 ℃ was thawed on ice, total mRNA was extracted from liver tissue samples using a total animal tissue RNA extraction kit (tengen), treated with an 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), and then reverse transcribed into cDNA using a tengen kit (china).
mRNA expression of the relevant genes in the liver of micropterus salmoides was determined by real-time fluorescent quantitative PCR. The core fragments of all genes were from the RNA-seq database. Quantitative real-time PCR was performed using a Bio-Rad MiniOption TM real-time fluorescent quantitative PCR detection system (Bio-Rad, U.S.A.), wherein the reaction system (10 μl): 1. Mu.l each of the upstream and downstream primers, 1. Mu.l of cDNA template, 5. Mu.l of SYBR green colorqPCR MasterMix (TIANGEN, beijin) and ddH 2 O2. Mu.l. The reaction procedure: pre-denaturation at 95℃for 3min, then denaturation at 95℃for 10s, annealing at 60℃for 30s, extension at 72℃for 1min,40 cycles. 3 biological replicates were performed for each experiment, 3 technical replicates were performed for each biological replicate, and 2 was used with beta-actin as reference gene -△△Ct The method calculates the mRNA relative expression amount of the target gene.
TABLE 2 primers for RT-qPCR amplification
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7. Toxicity test
After the test, 25 micropterus salmoides are randomly taken from the test group and the control group, and 4 multiplied by 10 is injected into the abdominal cavity 5 TCID 50 LMBV cytotoxic supernatant, test fish is placed in a 100cm multiplied by 60cm water tank, aeration is carried out continuously, and the water temperature is 20-25 ℃. Feeding was maintained daily for 21 days post infection and the test fish were observed for mortality and recorded.
8. Statistical analysis of data
Experimental data are expressed as mean ± Standard Error (SEM), single factor analysis of variance (One-WayANOVA) using IBM SPSS Statistics 26.0.0 software, multiple comparison tests with significant differences between groups using Duncan, P <0.05 indicating significant differences.
2. Results
1. Influence of MLO (methyl alcohol) addition in feed on growth performance of micropterus salmoides
As can be seen from table 3, while there was no significant difference between WGR, SGR, FI and CF of micropterus salmoides in the MLOL and MLOH groups (P > 0.05), FCR and HVI were significantly reduced (P < 0.05) compared to the CK group. In addition, MLOH group HSI was significantly reduced (P < 0.05).
TABLE 3 influence of MLO addition in feed on growth performance of Lateolabrax
The same row of data shoulder marks have no letter or the same letter indicates that the difference is not significant (P > 0.05), and the different lower case letters indicate that the difference is significant (P < 0.05), as follows.
2. Influence of MLO (methyl alcohol) addition in feed on serum biochemical index of micropterus salmoides
As is clear from Table 4, the MLOL and MLOH groups showed significantly reduced TG and GLU levels (P < 0.05) and ALT and AST enzyme activities (P < 0.05) compared to the CK group. The TC, LDL-C, HDL-C contents of the MLOL and MLOH groups were not significantly different (P > 0.05) compared to the CK group.
TABLE 4 influence of Mulberry leaf oligosaccharide addition in feed on serum Biochemical index of Lateolabrax
3. Influence of MLO (methyl alcohol) addition in feed on antioxidant capacity of micropterus salmoides
As can be seen from Table 5, the activity of T-SOD was significantly increased (P < 0.05) in serum of MLOL and MLOH groups compared to CK group, GSH content was significantly higher than that of CK group (P < 0.05), and MDA content was significantly lower than that of CK group (P < 0.05). In addition, the addition of MLO has no significant effect (P > 0.05) on T-AOC in serum of each group, which indicates that the addition of MLO can significantly improve the antioxidant capacity of the blood of micropterus salmoides.
The activity of T-SOD of liver tissues of MLOL and MLOH groups is obviously improved (P < 0.05), T-AOC is obviously improved (P < 0.05), the content of GSH is obviously higher than that of CK group (P < 0.05), but the content of MDA is obviously reduced (P < 0.05), which proves that the antioxidant capacity of the liver of micropterus salmoides can be obviously improved by adding MLO.
TABLE 5 influence of addition of Mulberry leaf oligosaccharide to liver health of Lateolabrax
4. Influence of MLO (methyl alcohol) addition in feed on liver inflammation related gene expression of micropterus salmoides
The results of detecting the expression level of genes related to oxidation resistance and inflammation of the liver of the micropterus salmoides show that compared with the CK group, the MLO added in the feed obviously reduces the expression of pro-inflammatory factors (tnf-alpha, il-8 and nf-kappa b) of the MLOL group and the MLOH group, and obviously improves the expression of anti-inflammatory factors (il-10 and tgf-beta) (figure 1), which shows that the immune function of the liver of the micropterus salmoides can be obviously improved by adding the MLO.
5. Influence of MLO (methyl alcohol) addition in feed on liver glycolipid metabolism related gene expression of micropterus salmoides
The result of detecting the expression level of liver lipid metabolism related genes of micropterus salmoides shows that the addition of MLO obviously improves the expression level (P < 0.05) of glycolytic genes pfk and gk; compared with the CK group, the expression levels of lipid metabolism related genes acc, ppr-alpha and cpt-1 in the MLOL and MLOH groups are significantly improved (P < 0.05) (FIG. 2), which shows that the addition of MLO can significantly improve the glycolipid metabolism function of the liver of micropterus salmoides.
6. Influence of MLO (methyl alcohol) addition in feed on liver tissue morphology of micropterus salmoides
The results of slicing observation on the liver tissue morphology of the largemouth bass show that the liver cells of the CK group have obvious cavitation and the cell nucleus has displacement, the cell cavitation and cell nucleus displacement conditions of the MLOL group and the MLOH group are obviously improved, the liver cell structure is complete, and the contour is clear (figure 3), so that the liver health of the largemouth bass can be obviously improved by adding the MLO.
Effect of MLO on Lateolabrax japonicus resistance to LMBV
The in vitro anti-LMBV activity measurement result of the MLO shows that death starts to occur 3d after the toxicity attack of the largemouth bass in the control group, the death starts on the 4 th day of the MLOL group, the death starts on the 6 th day of the MLOH group, and compared with the control group, the infection death time of the largemouth bass in the MLO treatment group is obviously delayed. The death number of each group is not changed after 14 days of toxin attack. Wherein, the survival rate of the control group is 25%, the survival rate of the MLOL group is 65%, and the survival rate of the MLOH group is 70%. The relative protection of the MLOL treated group compared to the control group was 42.86% and that of the MLOH treated group was 50% (FIG. 4). The Log-rank (Mantel-Cox) test shows that MLO significantly improves the survival rate of micropterus salmoides (P < 0.05).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. An antiviral sulfated mulberry leaf oligosaccharide comprising monosaccharides of the following classes: glucuronic acid, rhamnose and mannose;
the molar ratio of glucuronic acid to rhamnose to mannose is 2 (3-4) (4-7);
in the monosaccharide, sulfonic groups replace hydroxyl groups in the monosaccharide, and the substitution degree of the polysaccharide is 1.107-1.126;
the molecular weight of the sulfated mulberry leaf oligosaccharide is 2000Da to 4000Da.
2. An antiviral sulfated mulberry leaf oligosaccharide according to claim 1, wherein the molar ratio of glucuronic acid, rhamnose and mannose is 2:3:4.
3. An antiviral sulfated mulberry leaf oligosaccharide according to claim 1, wherein the polysaccharide has a degree of substitution of 1.126.
4. A method for preparing an antiviral sulfated mulberry leaf oligosaccharide according to any one of claims 1 to 3, comprising the steps of:
microwave degrading and H-degrading folium Mori polysaccharide 2 O 2 Vc degradation to obtain degradation products;
and (3) carrying out sulfation modification on the degradation product to obtain the sulfated mulberry leaf oligosaccharide.
5. The method according to claim 4, wherein the power of the microwave is 500-750W and the treatment time of the microwave is 20-30 min; the concentration of the mulberry leaf polysaccharide is 10-100 mg/mL;
at said H 2 O 2 When Vc is degraded, the final volume concentration of hydrogen peroxide is 0.5-3%; the final concentration of Vc is 5% -10%; the H is 2 O 2 The degradation temperature of Vc is 50-55 ℃ and the degradation time is 10-12 min;
the sulfation modification method is that the degradation product and sulfur trioxide-pyridine are subjected to water bath reaction; the final concentration of the sulfur trioxide-pyridine is 10mg/ml; the temperature of the water bath reaction is 90-95 ℃, and the time of the water bath reaction is 1-1.5 h.
6. Use of an antiviral sulfated mulberry leaf oligosaccharide according to any one of claims 1 to 3 or a sulfated mulberry leaf oligosaccharide obtained by a process according to claim 4 or 5 in aquaculture.
7. The use according to claim 6, wherein the aquaculture comprises the use of:
1) Improving the antiviral property of aquatic animals;
2) Improving the antioxidation capability of aquatic animals;
3) Improving the immunity of aquatic animals;
4) Improving the feed utilization rate of aquatic animals;
5) Improving lipid metabolism of aquatic animal.
8. The use according to claim 6 or 7, wherein the virus comprises a largemouth bass iridovirus;
the aquatic product comprises micropterus salmoides.
9. An aquatic animal feed product comprising the antiviral sulfated mulberry oligosaccharide according to any one of claims 1 to 3 or the sulfated mulberry oligosaccharide obtained by the production method according to claim 4 or 5 and a basic feed.
10. An aquatic animal feed product according to claim 9, wherein the antiviral sulfated mulberry leaf oligosaccharide comprises 0.5% to 1.2% by mass of the base feed product.
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