Mulberry leaf polyferose as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of polyferose, and particularly relates to mulberry leaf polyferose as well as a preparation method and application thereof. The preparation method comprises the following steps: taking mulberry leaves as a raw material, preparing mulberry leaf polysaccharide by adopting enzymolysis, concentration and ammonium sulfate deproteinization methods, adding potassium dihydrogen phosphate and water into the mulberry leaf polysaccharide for dissolving, adding a pH regulator to regulate the pH value to be 6-10, stirring, slowly adding a 30% ferric sulfate solution and a 20% potassium hydroxide solution until brownish red precipitate appears, reacting for 15-30 minutes, carrying out suction filtration, and drying to obtain the mulberry leaf polysaccharide. Adding ethanol into the filtrate, drying the obtained precipitate to obtain mulberry leaf polyferose powder, adding water to prepare a solution, dialyzing to remove salt, adding ethanol, and freeze-drying the precipitate to obtain a pure mulberry leaf polyferose product. The content of iron in the mulberry leaf polysaccharide iron is 3.65%-8.74%, and when the mulberry leaf polysaccharide iron is used in piglet breeding, the content of iron in piglet serum can be increased, and the liver metabolism and oxidation resistance of piglets are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of polysaccharide iron, and in particular relates to mulberry leaf polysaccharide iron and a preparation method and application thereof. Background Art

[0002] Iron has many important physiological functions in animals, which are essential for their health and survival. The following are the main functions of iron in animals: ① Oxygen transport: Iron is the main component of hemoglobin, which helps oxygen transport in the blood and provides necessary oxygen to various parts of the organism. ② DNA synthesis: Iron participates in the synthesis process of DNA and is essential for cell division and growth. ③ Electron transport: Iron is a component of many electron transport chains and participates in energy metabolism and cellular respiration. ④ Material metabolism: Iron participates in material metabolism in the body, and as an active ingredient of many enzymes, it catalyzes various biochemical reactions. ⑤ Immune function: Iron has an important effect on the immune function of animals, and iron deficiency can lead to decreased immune function. ⑥ Physiological defense: Iron participates in the physiological defense mechanism of animals and has a bactericidal effect. In addition, iron is also involved in the formation of transferrin, which, in addition to carrying iron, also has the function of preventing the body from contracting diseases. Since newborn animals, especially piglets, obtain limited iron from their mothers, it is very necessary to supplement piglets with iron. During cell culture, ensuring that the culture medium contains an appropriate amount of iron is key to maintaining cell growth and function. The iron concentration needs to be moderate. Too little can lead to restricted cell growth, while too much can cause problems such as oxidative stress.

[0003] my country is the largest mulberry planting country. Mulberry leaves are the main product of mulberry trees, accounting for 64% of the aboveground production. Therefore, mulberry leaves have great germplasm resource advantages in my country. Since ancient times, mulberry cultivation in my country has been mainly used for traditional sericulture. The use of mulberry leaves in sericulture accounts for only a small part, and a large amount of surplus mulberry leaves causes resource waste. Therefore, developing the application value of mulberry leaves and exploring their economic value are urgent issues to be solved. The rich nutritional value of mulberry leaves has attracted more and more industrial attention, and the development and utilization of mulberry leaves has become diversified. Mulberry leaf polysaccharides have the effects of antioxidant, improving immune function, and improving intestinal microbial flora. They can effectively improve the overall growth performance of livestock and poultry and reduce mortality. Polysaccharide-iron complex is a complex of polysaccharides and iron elements formed by the co-heating method of biopolysaccharides and iron ions. When used as an iron supplement, it not only has a stable structure, good solubility, and low gastrointestinal irritation, but also has the advantages of strong iron chelating ability and wide bioavailability. While supplementing iron, it can also promote blood circulation, lower blood sugar, and regulate blood pressure. Therefore, it is necessary to design a method for preparing an iron supplement using mulberry leaves as raw materials, which can reduce the cost of the iron supplement while also having the function of polysaccharides. Summary of the invention

[0004] In view of the problems and shortcomings in the prior art, the present invention aims to provide a mulberry leaf polysaccharide iron and a preparation method and application thereof.

[0005] In order to achieve the purpose of the present invention, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a method for preparing mulberry leaf polysaccharide iron, comprising the following steps:

[0007] (1) washing, airing, drying and crushing mulberry leaves, adding 20 to 40 times the mass of water, and then adding a composite enzyme preparation of 0.05% of the total mass of mulberry leaves and water, performing enzymatic extraction at 40° C. to 60° C. for 24 hours, inactivating the enzyme, filtering, and obtaining a mulberry leaf polysaccharide water extract;

[0008] (2) concentrating the mulberry leaf polysaccharide water extract in step (1) to 1 / 2 volume to obtain a concentrated solution, adding methanol, standing at 4° C. for 10 to 24 hours, and centrifuging to obtain a precipitate as a crude extract of mulberry leaf polysaccharide;

[0009] (3) adding water to the crude extract of mulberry leaf polysaccharide in step (2) to form a crude extract solution of mulberry leaf polysaccharide, adding 40% ammonium sulfate to shake and deproteinize until no precipitation occurs, centrifuging, and washing the precipitate with ethanol, acetone, and ether, respectively, to obtain a pure mulberry leaf polysaccharide;

[0010] (4) Add potassium dihydrogen phosphate and water to the pure mulberry leaf polysaccharide product of step (3) to dissolve it, then add a pH adjuster to adjust the pH to 6-10, stir at 40°C-80°C and 1000 r / min for 10 min, slowly add 30% ferric sulfate solution and 20% potassium hydroxide solution until a brown-red precipitate appears, react for 15-30 min, filter with suction, add 3-4 times the volume of ethanol to the filtrate, let it stand, centrifuge, dry the obtained precipitate to obtain a crude powder of mulberry leaf polysaccharide iron, add water to prepare a solution, dialyze to remove salt, then add 3-4 times the volume of ethanol, let it stand, centrifuge, and freeze-dry the precipitate to obtain a pure mulberry leaf polysaccharide iron product.

[0011] Preferably, in step (4), the mass ratio of pure mulberry leaf polysaccharide, potassium dihydrogen phosphate and water is (1-4):(1-5):24.

[0012] Preferably, the pH adjuster in step (4) is one or more of dipotassium hydrogen phosphate, sodium hydroxide, and citric acid.

[0013] Preferably, the standing condition in step (4) is 12 to 18 hours at 4° C., and the dialysis desalination time is 24 to 36 hours.

[0014] Preferably, the complex enzyme preparation in step (1) consists of cellulase, β-glucanase and pectinase in a mass ratio of 2:1:1.

[0015] Preferably, the mass ratio of the concentrated solution in step (2) to methanol is (3-5):1.

[0016] Preferably, in step (3), the dosage ratio of ammonium sulfate to the crude extract of mulberry leaf polysaccharide solution is (150-243) g:1L.

[0017] Preferably, the centrifugation conditions of step (2), step (3) and step (4) are all 6000-8000 r / min for 5-10 min.

[0018] Preferably, the deproteinization by oscillation in step (3) is performed for 10 to 15 minutes each time.

[0019] The second aspect of the present invention provides a mulberry leaf polysaccharide iron prepared by the method described in the first aspect of the present invention.

[0020] The third aspect of the present invention provides an application of the mulberry leaf polysaccharide iron described in the second aspect of the present invention in a multifunctional iron supplement for animals.

[0021] Preferably, the method of using the mulberry leaf polysaccharide iron of the present invention is to add it to animal feed in an amount of 0.01% to 0.1%.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The present invention provides a method for preparing mulberry leaf polysaccharide iron, which comprises lysing plant cell walls by a composite enzyme preparation to release more polysaccharides in the plant cells, and removing impurities such as protein by concentration and ammonium sulfate deproteinization, thereby greatly improving the purity of mulberry leaf polysaccharide, and better complexing with iron sulfate to increase the iron content in mulberry leaf polysaccharide iron. The iron content in mulberry leaf polysaccharide iron prepared by the method of the present invention is 3.65% to 8.74%.

[0024] (2) The mulberry leaf polysaccharide iron of the present invention is added to the basic feed at a dosage of 0.01% to 0.1% in piglet breeding. Compared with piglets not fed with mulberry leaf polysaccharide iron, it can increase the iron content in the piglet's serum and improve the piglet's liver metabolism and antioxidant capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention is a process flow chart for preparing mulberry leaf polysaccharide iron;

[0026] Figure 2 This is a graph showing the iron content of mulberry leaf polysaccharide iron prepared in Examples 1 to 5 of the present invention and Comparative Examples 1 to 2. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail by way of embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] Example 1

[0029] A method for preparing mulberry leaf polysaccharide iron, the preparation process is as follows Figure 1 As shown, the following steps are included:

[0030] (1) After washing and naturally drying the mulberry leaves, place them in an oven at 60° C. and dry them for 1 hour. Then, use a blender with blades to crush the mulberry leaves, send them to a traditional Chinese medicine extraction tank via a conveyor belt, add 20 times the weight of water, and then add 0.05% of a composite enzyme preparation composed of cellulase, β-glucanase and pectinase. Extract at 40° C. for 24 hours. After the enzymolysis is completed, boil and keep boiling for 5 minutes to inactivate the enzyme, filter, and obtain a mulberry leaf polysaccharide water extract;

[0031] (2) using a spherical vacuum low-temperature concentrator to concentrate the mulberry leaf polysaccharide water extract prepared in step (1) to 1 / 2 of its volume to obtain a concentrated solution, adding methanol at a mass ratio of 3:1 between the concentrated solution and methanol, standing at 4° C. for 15 h, and centrifuging at a speed of 8000 r / min for 10 min to obtain a precipitate as a crude extract of mulberry leaf polysaccharide;

[0032] (3) adding deionized water to the crude extract of mulberry leaf polysaccharide prepared in step (2) to dissolve it, to obtain a mulberry leaf polysaccharide solution, adding 342 g of ammonium sulfate with a concentration of 40% per liter of mulberry leaf polysaccharide solution to shake and deproteinize, shaking for 10 minutes each time until no precipitation occurs, centrifuging at a speed of 8000 r / min for 10 minutes, and washing the precipitate with ethanol, acetone, and ether respectively to obtain a white powdery pure mulberry leaf polysaccharide;

[0033] (4) adding the pure mulberry leaf polysaccharide prepared in step (3), potassium dihydrogen phosphate and water in a mass ratio of 1:1:24 into a reactor to dissolve, then adding citric acid to adjust the pH to 6, stirring at 60°C and 1000 r / min for 10 min, slowly adding 30% ferric sulfate solution and 20% potassium hydroxide solution until a brown-red precipitate appears, and after the solution reacts for 30 min, filtering with filter paper, taking the filtrate, adding 3 times the volume of ethanol, standing at 4°C for 12 h, centrifuging for 10 min, and placing the precipitate under natural conditions to dry to obtain mulberry leaf polysaccharide iron powder; dissolving the mulberry leaf polysaccharide iron powder with an appropriate amount of distilled water, and after it is fully dissolved, placing the resulting solution in a dialysis bag for desalting, then adding 3 times the volume of ethanol, standing at 4°C for 12 h, centrifuging for 10 min, and rotating at 8000 r / min, and freeze-drying the precipitate to obtain pure mulberry leaf polysaccharide iron.

[0034] Example 2

[0035] A method for preparing mulberry leaf polysaccharide iron comprises the following steps:

[0036] (1) After washing and naturally drying the mulberry leaves, place them in a 60°C oven and dry them for 1 hour. Then, use a blender with blades to crush the mulberry leaves, send them to a traditional Chinese medicine extraction tank via a conveyor belt, add 20 times the weight of water, and then add 0.05% of a composite enzyme preparation, extract at 40°C for 24 hours, boil and keep boiling for 5 minutes after the enzymolysis is completed, inactivate the enzyme, and filter to obtain a mulberry leaf polysaccharide water extract;

[0037] (2) using a spherical vacuum low-temperature concentrator to concentrate the mulberry leaf polysaccharide water extract prepared in step (1) to 1 / 2 of its volume to obtain a concentrated solution, adding methanol at a mass ratio of 3:1 between the concentrated solution and methanol, standing at 4° C. for 15 h, and centrifuging at a speed of 8000 r / min for 10 min to obtain a precipitate as a crude extract of mulberry leaf polysaccharide;

[0038] (3) adding deionized water to the crude extract of mulberry leaf polysaccharide prepared in step (2) to dissolve it, to obtain a mulberry leaf polysaccharide solution, adding 342 g of ammonium sulfate with a concentration of 40% per liter of mulberry leaf polysaccharide solution to shake and deproteinize, shaking for 10 minutes each time until no precipitation occurs, centrifuging at a speed of 8000 r / min for 10 minutes, and washing the precipitate with ethanol, acetone, and ether respectively to obtain a white powdery pure mulberry leaf polysaccharide;

[0039] (4) adding the pure mulberry leaf polysaccharide prepared in step (3), potassium dihydrogen phosphate and water in a mass ratio of 1:1:24 into a reactor to dissolve, then adding potassium dihydrogen phosphate to adjust the pH to 7, stirring at 60°C and 1000 r / min for 10 min, slowly adding 30% ferric sulfate solution and 20% potassium hydroxide solution until a brown-red precipitate appears, and after the solution reacts for 30 min, filtering with filter paper, taking the filtrate, adding 3 times the volume of ethanol, standing at 4°C for 12 h, centrifuging for 10 min, and placing the precipitate under natural conditions to dry to obtain mulberry leaf polysaccharide iron powder; dissolving the mulberry leaf polysaccharide iron powder with an appropriate amount of distilled water, and after it is fully dissolved, placing the resulting solution in a dialysis bag for desalting, then adding 3 times the volume of ethanol, standing at 4°C for 12 h, centrifuging for 10 min, and rotating at 8000 r / min, and freeze-drying the precipitate to obtain pure mulberry leaf polysaccharide iron.

[0040] Example 3

[0041] A method for preparing mulberry leaf polysaccharide iron comprises the following steps:

[0042] (1) After washing and naturally drying the mulberry leaves, place them in a 60°C oven and dry them for 1 hour. Then, use a blender with blades to crush the mulberry leaves, send them to a traditional Chinese medicine extraction tank via a conveyor belt, add 20 times the weight of water, and then add 0.05% of a composite enzyme preparation, extract at 40°C for 24 hours, boil and keep boiling for 5 minutes after the enzymolysis is completed, inactivate the enzyme, and filter to obtain a mulberry leaf polysaccharide water extract;

[0043] (2) using a spherical vacuum low-temperature concentrator to concentrate the mulberry leaf polysaccharide water extract prepared in step (1) to 1 / 2 of its volume to obtain a concentrated solution, adding methanol at a mass ratio of 3:1 between the concentrated solution and methanol, standing at 4° C. for 15 h, and centrifuging at a speed of 8000 r / min for 10 min to obtain a precipitate as a crude extract of mulberry leaf polysaccharide;

[0044] (3) adding deionized water to the crude extract of mulberry leaf polysaccharide prepared in step (2) to dissolve it, to obtain a mulberry leaf polysaccharide solution, adding 342 g of ammonium sulfate with a concentration of 40% per liter of mulberry leaf polysaccharide solution to shake and deproteinize, shaking for 10 minutes each time until no precipitation occurs, centrifuging at a speed of 8000 r / min for 10 minutes, and washing the precipitate with ethanol, acetone, and ether respectively to obtain a white powdery pure mulberry leaf polysaccharide;

[0045] (4) adding the pure mulberry leaf polysaccharide prepared in step (3), potassium dihydrogen phosphate and water in a mass ratio of 1:1:24 into a reactor to dissolve, then adding potassium dihydrogen phosphate and sodium hydroxide to adjust the pH to 8, stirring at 60°C and 1000 r / min for 10 min, slowly adding 30% ferric sulfate solution and 20% potassium hydroxide solution until a brown-red precipitate appears, and after the solution reacts for 30 min, filtering with filter paper, taking the filtrate, adding 3 times the volume of ethanol, standing at 4°C for 12 h, centrifuging for 10 min, and placing the precipitate under natural conditions to dry to obtain mulberry leaf polysaccharide iron powder; dissolving the mulberry leaf polysaccharide iron powder with an appropriate amount of distilled water, and after it is fully dissolved, placing the resulting solution in a dialysis bag for desalting, then adding 3 times the volume of ethanol, standing at 4°C for 12 h, centrifuging for 10 min, and rotating at 8000 r / min, and freeze-drying the precipitate to obtain pure mulberry leaf polysaccharide iron.

[0046] Example 4

[0047] A method for preparing mulberry leaf polysaccharide iron comprises the following steps:

[0048] (1) After washing and naturally drying the mulberry leaves, place them in a 60°C oven and dry them for 1 hour. Then, use a blender with blades to crush the mulberry leaves, send them to a traditional Chinese medicine extraction tank via a conveyor belt, add 20 times the weight of water, and then add 0.05% of a composite enzyme preparation, extract at 40°C for 24 hours, boil and keep boiling for 5 minutes after the enzymolysis is completed, inactivate the enzyme, and filter to obtain a mulberry leaf polysaccharide water extract;

[0049] (2) using a spherical vacuum low-temperature concentrator to concentrate the mulberry leaf polysaccharide water extract prepared in step (1) to 1 / 2 of its volume to obtain a concentrated solution, adding methanol at a mass ratio of 3:1 between the concentrated solution and methanol, standing at 4° C. for 15 h, and centrifuging at a speed of 8000 r / min for 10 min to obtain a precipitate as a crude extract of mulberry leaf polysaccharide;

[0050] (3) adding deionized water to the crude extract of mulberry leaf polysaccharide prepared in step (2) to dissolve it, to obtain a mulberry leaf polysaccharide solution, adding 342 g of ammonium sulfate with a concentration of 40% per liter of mulberry leaf polysaccharide solution to shake and deproteinize, shaking for 10 minutes each time until no precipitation occurs, centrifuging at a speed of 8000 r / min for 10 minutes, and washing the precipitate with ethanol, acetone, and ether respectively to obtain a white powdery pure mulberry leaf polysaccharide;

[0051] (4) adding the pure mulberry leaf polysaccharide prepared in step (3), potassium dihydrogen phosphate and water in a mass ratio of 1:1:24 into a reactor to dissolve, then adding potassium dihydrogen phosphate and sodium hydroxide to adjust the pH to 9, stirring at 60°C and 1000 r / min for 10 min, slowly adding 30% ferric sulfate solution and 20% potassium hydroxide solution until a brown-red precipitate appears, and after the solution reacts for 30 min, filtering with filter paper, taking the filtrate, adding 3 times the volume of ethanol, standing at 4°C for 12 h, centrifuging for 10 min, and placing the precipitate under natural conditions to dry to obtain mulberry leaf polysaccharide iron powder; dissolving the mulberry leaf polysaccharide iron powder with an appropriate amount of distilled water, and after it is fully dissolved, placing the resulting solution in a dialysis bag for desalting, then adding 3 times the volume of ethanol, standing at 4°C for 12 h, centrifuging for 10 min, and rotating at 8000 r / min, and freeze-drying the precipitate to obtain pure mulberry leaf polysaccharide iron.

[0052] Example 5

[0053] A method for preparing mulberry leaf polysaccharide iron comprises the following steps:

[0054] (1) After washing and naturally drying the mulberry leaves, place them in a 60°C oven and dry them for 1 hour. Then, use a blender with blades to crush the mulberry leaves, send them to a traditional Chinese medicine extraction tank via a conveyor belt, add 20 times the weight of water, and then add 0.05% of a composite enzyme preparation, extract at 40°C for 24 hours, boil and keep boiling for 5 minutes after the enzymolysis is completed, inactivate the enzyme, and filter to obtain a mulberry leaf polysaccharide water extract;

[0055] (2) using a spherical vacuum low-temperature concentrator to concentrate the mulberry leaf polysaccharide water extract prepared in step (1) to 1 / 2 of its volume to obtain a concentrated solution, adding methanol at a mass ratio of 3:1 between the concentrated solution and methanol, standing at 4° C. for 15 h, and centrifuging at a speed of 8000 r / min for 10 min to obtain a precipitate as a crude extract of mulberry leaf polysaccharide;

[0056] (3) adding deionized water to the crude extract of mulberry leaf polysaccharide prepared in step (2) to dissolve it, to obtain a mulberry leaf polysaccharide solution, adding 342 g of ammonium sulfate with a concentration of 40% per liter of mulberry leaf polysaccharide solution to shake and deproteinize, shaking for 10 minutes each time until no precipitation occurs, centrifuging at a speed of 8000 r / min for 10 minutes, and washing the precipitate with ethanol, acetone, and ether respectively to obtain a white powdery pure mulberry leaf polysaccharide;

[0057] (4) adding the pure mulberry leaf polysaccharide prepared in step (3), potassium dihydrogen phosphate and water in a mass ratio of 1:1:24 into a reactor to dissolve, then adding potassium dihydrogen phosphate and sodium hydroxide to adjust the pH to 10, stirring at 60°C and 1000 r / min for 10 min, slowly adding 30% ferric sulfate solution and 20% potassium hydroxide solution until a brown-red precipitate appears, and after the solution reacts for 30 min, filtering with filter paper, taking the filtrate, adding 3 times the volume of ethanol, standing at 4°C for 12 h, centrifuging for 10 min, and placing the precipitate under natural conditions to dry to obtain mulberry leaf polysaccharide iron powder; dissolving the mulberry leaf polysaccharide iron powder with an appropriate amount of distilled water, and after it is fully dissolved, placing the resulting solution in a dialysis bag for desalting, then adding 3 times the volume of ethanol, standing at 4°C for 12 h, centrifuging for 10 min, and rotating at 8000 r / min, and freeze-drying the precipitate to obtain pure mulberry leaf polysaccharide iron.

[0058] Comparative Example 1

[0059] A method for preparing mulberry leaf polysaccharide iron comprises the following steps:

[0060] (1) After washing and naturally drying the mulberry leaves, the leaves were placed in a 60°C oven for 1 hour, and then crushed to pass a 60-mesh sieve to obtain mulberry leaf powder, which was extracted with petroleum ether and ethanol for 3 hours to remove impurities such as fat-soluble and flavonoids, and after the solvent was volatilized at room temperature, the leaves were placed in a 50°C oven for drying; 30 times the weight of water was added, and the leaves were placed in a constant temperature water bath for leaching at 80°C for 3 hours, and the leaves were centrifuged at a speed of 8000 r / min for 15 minutes. The supernatant was a mulberry leaf polysaccharide extract, and the extract was performed twice, filtered, and the filtrate was combined to obtain a mulberry leaf polysaccharide water extract;

[0061] (2) using a spherical vacuum low-temperature concentrator to concentrate the mulberry leaf polysaccharide water extract prepared in step (1) to 1 / 2 of its volume to obtain a concentrate, adding methanol at a mass ratio of 3:1 between the concentrate and methanol, standing at 4° C. for 15 h, and centrifuging at a speed of 8000 r / min for 10 min to obtain a precipitate as a crude extract of mulberry leaf polysaccharide;

[0062] (3) Deionized water is added to the crude extract of mulberry leaf polysaccharide prepared in step (2) to dissolve it, thereby obtaining mulberry leaf polysaccharide liquid; 342 g of 40% ammonium sulfate is added per liter of mulberry leaf polysaccharide liquid for deproteinization by oscillation; each oscillation is performed for 10 min until no precipitation occurs; the mixture is centrifuged at a speed of 8000 r / min for 10 min; the precipitate is then washed with ethanol, acetone, and ether, respectively, to obtain a pure white powder of mulberry leaf polysaccharide.

[0063] (4) adding the pure mulberry leaf polysaccharide prepared in step (3), potassium dihydrogen phosphate and water in a mass ratio of 1:1:24 into a reactor to dissolve, then adding citric acid to adjust the pH to 6, stirring at 60°C and 1000 r / min for 10 min, slowly adding 30% ferric sulfate solution and 20% potassium hydroxide solution until a brown-red precipitate appears, and after the solution reacts for 30 min, filtering with filter paper, taking the filtrate, adding 3 times the volume of ethanol, standing at 4°C for 12 h, centrifuging for 10 min, and placing the precipitate under natural conditions to dry to obtain mulberry leaf polysaccharide iron powder; dissolving the mulberry leaf polysaccharide iron powder with an appropriate amount of distilled water, and after it is fully dissolved, placing the resulting solution in a dialysis bag for desalting, then adding 3 times the volume of ethanol, standing at 4°C for 12 h, centrifuging for 10 min, and rotating at 8000 r / min, and freeze-drying the precipitate to obtain pure mulberry leaf polysaccharide iron.

[0064] Comparative Example 2

[0065] A method for preparing mulberry leaf polysaccharide iron comprises the following steps:

[0066] (1) After washing and naturally drying the mulberry leaves, place them in an oven at 60° C. for 1 hour, then crush the mulberry leaves with a blender with blades and send them to a traditional Chinese medicine extraction tank via a conveyor belt, add 20 times the weight of water, and then add 0.05% of a composite enzyme preparation composed of cellulase, β-glucanase and pectinase, extract at 40° C. for 24 hours, boil and keep boiling for 5 minutes after the enzymolysis is completed, inactivate the enzyme, filter, and obtain a mulberry leaf polysaccharide water extract;

[0067] (2) using a spherical vacuum low-temperature concentrator to concentrate the mulberry leaf polysaccharide water extract prepared in step (1) to 1 / 2 volume to obtain a concentrated solution, adding citric acid to adjust the pH to 6, stirring at 60°C and a speed of 1000 r / min for 10 min, slowly adding 30% ferric sulfate solution and 20% potassium hydroxide solution until a brown-red precipitate appears, after the solution is fully reacted for 30 min, filtering with filter paper, taking the filtrate, adding 3 times the volume of ethanol, placing at 4°C for 12 h, centrifuging for 10 min, and placing the precipitate under natural conditions to dry to obtain mulberry leaf polysaccharide iron powder; dissolving the mulberry leaf polysaccharide iron powder with an appropriate amount of distilled water, after fully dissolving, placing the resulting solution in a dialysis bag for desalting, then adding 3 times the volume of ethanol, placing at 4°C for 12 h, centrifuging for 10 min, rotating at 8000 r / min, and freeze-drying the precipitate to obtain a pure mulberry leaf polysaccharide iron product.

[0068] Example 6 Determination of iron content in mulberry leaf polysaccharide iron

[0069] Pipette 0, 1.00, 2.00, 4.00, 6.00, 8.00, 10.00mL of standard iron solution (10μg / mL) into a 50mL volumetric flask, add 1.0mL of 10% hydroxylamine chloride solution respectively, mix evenly, let stand for 5min, add 2.0mL of 0.15% 1.10-phenanthroline, 5.0mL of 1.0mol / L sodium acetate solution, add distilled water to the scale, mix evenly, let stand for 20min to develop color, measure the A value at 510nm, draw a standard curve with the mass concentration of iron as the horizontal coordinate and the absorbance as the vertical coordinate, and the regression equation is: Y=0.1947X+0.0017, R 2 =0.9967. Weigh 10 mg of mulberry leaf polysaccharide iron sample and dissolve it in 20 ml of deionized water to prepare a sample solution. Pipette 5 mL from it and transfer it to a 25 mL volumetric flask. Perform the same operation according to the standard curve method, substitute the measured results into the standard curve, and calculate the iron content in mulberry leaf polysaccharide iron. Iron content / %=C×V×M×n×1000 / m×100%. Wherein: C—mass concentration of iron in mulberry leaf polysaccharide iron (μg / ml); V—volume of mulberry leaf polysaccharide iron solution (ml); M—molar mass of iron atoms; m—mass of mulberry leaf polysaccharide iron sample (mg); n—dilution multiple of the sample.

[0070] The results of the determination of the iron content in mulberry leaf polysaccharide iron in Examples 1 to 5 and Comparative Examples 1 to 2 are as follows Figure 2 As shown, when the pH value is 6 in Example 1, the iron content in the corresponding mulberry leaf polysaccharide iron is the highest, which is 8.74%; in Examples 2 to 4, as the pH value increases, the iron content in mulberry leaf polysaccharide iron gradually decreases; when the pH value is 9 in Example 4, the iron content in mulberry leaf polysaccharide iron is the lowest, which is 3.65%; the iron content in mulberry leaf polysaccharide iron in Example 5 is slightly increased compared with Example 4. The iron content of mulberry leaf polysaccharide iron prepared by extracting mulberry leaf polysaccharides with organic solvents in Comparative Example 1 is lower than the iron content of mulberry leaf polysaccharide iron prepared by enzymatic hydrolysis of mulberry leaves in Example 1. The iron content of mulberry leaf polysaccharide iron in Comparative Example 2 after mulberry leaves without protein removal and chelation with iron sulfate is lower than the iron content of mulberry leaf polysaccharide iron after protein removal in Example 5.

[0071] Example 7 Mulberry leaf polysaccharide iron application test

[0072] 1. Experimental Grouping

[0073] The experiment selected 240 healthy 40-day-old weaned Duroc×Chang×Dachshund triple hybrid piglets with an initial body weight of (9.52±1.60) kg and randomly divided them into 4 groups, 15 in each group, with 4 replicates. The control group was fed only with a basic diet, the experimental group 1 was fed with a basic diet supplemented with 0.01% of the mulberry leaf polysaccharide iron prepared in Example 1, the experimental group 2 was fed with a basic diet supplemented with 0.05% of the mulberry leaf polysaccharide iron prepared in Example 1, and the experimental group 3 was fed with a basic diet supplemented with 0.1% of the mulberry leaf polysaccharide iron prepared in Example 1 (all in terms of iron), and the experimental period was 28 days.

[0074] 2. Index determination

[0075] 2.1 Serum iron content

[0076] The serum iron content was detected using the Nanjing Jiancheng Bioengineering Institute kit (A039-1-1).

[0077] 2.2 Serum biochemical indexes

[0078] Serum biochemical indices, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were detected using HITEC 7100 fully automatic biochemical analyzer (Hitachi High-Tech International Trading Co., Ltd.).

[0079] 2.3 Serum antioxidant index

[0080] After the serum samples were thawed on ice, the contents of glutathione peroxidase (GSH-Px), peroxidase (CAT), reduced glutathione (GSH) and malondialdehyde (MDA) in the serum samples of each experimental group were detected according to the instructions of the kit (Nanjing Jiancheng Bioengineering Institute).

[0081] 3. Test results

[0082] (1) Serum iron content

[0083] On the 14th and 28th days of the experiment, six piglets were randomly selected from each group to collect blood from the jugular vein to detect the serum iron content of the piglets. The results are shown in Table 1.

[0084] Table 1 Effects of feeding different doses of mulberry leaf polysaccharide iron on serum iron content in 14d and 28d piglets

[0085] Test items Control group Experimental Group 1 Experimental Group 2 Experimental Group 3 14d serum iron content (μmol / L) 29.77±1.15 40.32±3.22 41.59±2.56 38.73±4.23 Serum iron content at 28 days (μmol / L) 46.32±1.58 38.52±3.25 36.78±4.74 37.54±4.52

[0086] As can be seen from Table 1, the serum iron content of piglets in test groups 1 to 3 and 14 days was higher than that in the control group, indicating that the mulberry leaf polysaccharide iron of the present invention can quickly enter the blood and increase the iron content in the blood. At 28 days, the serum iron content of piglets in test groups 1 to 3 was lower than that in the control group, indicating that not only the supplemented mulberry leaf polysaccharide iron was efficiently absorbed, but also the body's absorption of iron in the blood was promoted, greatly improving the efficiency of iron absorption and utilization in piglets. The serum iron content at 14 days in test group 2 was higher than that in test group 1 and test group 3, and the serum iron content at 28 days was lower than that in test group 1 and test group 3. It can be seen that when the addition amount of mulberry leaf polysaccharide iron in the diet is 0.05%, mulberry leaf polysaccharide iron has the best iron supplementation effect on piglets.

[0087] (2) Serum biochemical indicators

[0088] On the 14th and 28th days of the experiment, blood was collected from the jugular vein of 6 piglets randomly selected from each group to test the serum biochemical indices of the piglets. The results are shown in Table 2.

[0089] Table 2 Effects of feeding different doses of mulberry leaf polysaccharide iron on serum biochemical parameters of 14d and 28d piglets

[0090]

[0091] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are enzymes that play an important role in the normal physiological activities of the liver. ALT and AST play a role in transamination in the process of catalyzing the reaction of α-ketoglutarate and aspartate to produce glutamate and oxaloacetate, and in the process of α-ketoglutarate and alanine to produce glutamate and pyruvate. Liver damage in piglets will cause an increase in AST and ALT, and their content will decrease after the damage is relieved. The liver is an important place for iron metabolism and iron storage, and AST and ALT are important indicators reflecting liver function.

[0092] As shown in Table 2, at 14 days, the AST and ALT contents in the test groups 1 to 3 were lower than those in the serum of the control group; at 28 days, the AST and ALT contents in the test groups 1 to 3 were lower than those in the serum of the control group; indicating that the inorganic iron content in the diet stimulates liver metabolism and causes liver oxidative damage. After adding the present invention, organic iron is absorbed by the body through the polysaccharide channel without liver metabolism. At the same time, the released polysaccharide can also alleviate liver oxidative damage and reduce the AST and ALT contents in the serum of piglets. It can be seen that when the addition amount of mulberry leaf polysaccharide iron in the diet is 0.05%, mulberry leaf polysaccharide iron has the best effect on alleviating liver damage in piglets.

[0093] (3) Serum antioxidant index

[0094] On the 14th and 28th days of the experiment, blood was collected from the jugular vein of 6 piglets randomly selected from each group to detect the piglet serum antioxidant indexes. The results are shown in Table 3.

[0095] Table 3 Effects of feeding different doses of mulberry leaf polysaccharide iron on serum antioxidant indexes of 14d and 28d piglets

[0096]

[0097] The biological function of catalase (CAT) is to promote the decomposition of hydrogen peroxide in cells so that it will not further produce highly toxic hydroxyl free radicals, thereby protecting the function of the antioxidant enzyme system. The higher its content, the more it indicates that the body is in a state of oxidative stress. Malondialdehyde (MDA) is a common indicator for measuring oxidative stress. High levels of MDA indicate increased oxidative stress, which may cause damage to biological macromolecules such as cell membranes. In severe cases, cell damage and disease risks increase. Reduced glutathione (GSH) is one of the most important antioxidant molecules in the body, and its content in cells is very high. GSH can neutralize free radicals and oxidizing substances in the body and protect cells from oxidative damage. GSH is also an important component of the internal circulation system of cells. Reduced glutathione (GSH) plays an important role in the biochemical defense system in the body and has many physiological functions. Its main physiological function is to remove free radicals in the body. As an important antioxidant in the body, it protects the sulfhydryl groups in many proteins and enzymes. The lower its content, the more it indicates that the body is in a redox state. Glutathione peroxidase (GSH-Px) is a peroxide decomposing enzyme that can reduce toxic peroxides into non-toxic hydroxyl compounds, thereby protecting the structure and function of cell membranes from being disturbed and damaged by peroxides. The lower its content, the more the body is in a redox state.

[0098] As shown in Table 3, at 14 days, the GSH-Px and GSH contents in the serum of piglets in the experimental groups 1 to 3 were higher than those in the serum of the control group; the CAT and MDA contents in the serum of piglets in the experimental groups 1 to 3 were lower than those in the serum of the control group, indicating that the use of mulberry leaf polysaccharide iron enhanced the antioxidant capacity of piglets. In the experimental groups 1 to 3, the GSH-Px and GSH contents in the serum of piglets in the experimental group 2 were higher than those in the serum of the experimental groups 1 and 3; the CAT and MDA contents in the serum of the experimental group 2 were lower than those in the serum of the experimental groups 1 and 3. Overall, the antioxidant capacity of piglets in the experimental group 2 was the strongest. It can be seen that when the addition amount of mulberry leaf polysaccharide iron in the feed is 0.05%, mulberry leaf polysaccharide iron has the strongest effect on improving the antioxidant capacity of piglets.

[0099] At 28 days, the GSH-Px and GSH contents in the serum of piglets in the experimental groups 1 to 3 were higher than those in the serum of the control group; the CAT and MDA contents in the serum of piglets in the experimental groups 1 to 3 were lower than those in the serum of the control group, indicating that the use of mulberry leaf polysaccharide iron enhanced the antioxidant capacity of piglets. In the experimental groups 1 to 3, the GSH-Px and GSH contents in the serum of piglets in the experimental group 2 were higher than those in the serum of piglets in the experimental groups 1 and 3; the CAT and MDA contents in the serum of piglets in the experimental group 2 were lower than those in the serum of piglets in the experimental groups 1 and 3. Overall, the antioxidant capacity of piglets in the experimental group 2 was the strongest. It can be seen that when the addition amount of mulberry leaf polysaccharide iron in the feed is 0.05%, mulberry leaf polysaccharide iron has the strongest effect on improving the antioxidant capacity of piglets.

[0100] The above test results show that the mulberry leaf polysaccharide iron prepared by the present invention increases the iron content in the serum of piglets, enhances the metabolic function of the piglet liver and the antioxidant capacity of the body, and improves the immunity of the piglets. It is a multifunctional iron supplement and has the best effect when added to the feed at a rate of 0.05%.

[0101] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. A method for preparing mulberry leaf polysaccharide iron, characterized in that: The following steps are involved: (1) Wash, air-dry, and crush the mulberry leaves, add 20 to 40 times the mass of water, and then add a composite enzyme preparation at a total mass of 0.05% of the mulberry leaves and water, perform enzymatic extraction at 40°C to 60°C for 24 hours, inactivate the enzyme, and filter to obtain a mulberry leaf polysaccharide water extract; (2) Concentrating the mulberry leaf polysaccharide water extract in step (1) to 1 / 2 volume to obtain a concentrated solution, adding methanol, standing at 4° C. for 10 to 24 hours, and centrifuging to obtain a precipitate as a crude extract of mulberry leaf polysaccharide; (3) adding water to the crude extract of mulberry leaf polysaccharide obtained in step (2) to form a crude extract solution of mulberry leaf polysaccharide, adding 40% ammonium sulfate to shake and deproteinize until no precipitation occurs, centrifuging, and washing the precipitate with ethanol, acetone, and ether, respectively, to obtain a pure mulberry leaf polysaccharide; (4) Add potassium dihydrogen phosphate and water to the pure mulberry leaf polysaccharide product of step (3) to dissolve, then add a pH adjuster to adjust the pH to 6-10, stir at 40°C-80°C and 1000 r / min for 10 min, slowly add 30% ferric sulfate solution and 20% potassium hydroxide solution until a brown-red precipitate appears, react for 15-30 min, filter with suction, add 3-4 times the volume of ethanol to the filtrate, let stand, centrifuge, dry the obtained precipitate to obtain a crude powder of mulberry leaf polysaccharide iron, add water to prepare a solution, dialyze to remove salt, then add 3-4 times the volume of ethanol, let stand, centrifuge, and freeze-dry the obtained precipitate to obtain a pure mulberry leaf polysaccharide iron product.

2. The preparation method according to claim 1, characterized in that: In step (4), the mass ratio of pure mulberry leaf polysaccharide, potassium dihydrogen phosphate and water is (1-4): (1-5):

24.

3. The preparation method according to claim 2, characterized in that: The pH regulator in step (4) is one or more of dipotassium hydrogen phosphate, sodium hydroxide, and citric acid.

4. The preparation method according to claim 3, characterized in that: The standing condition of step (4) is 12 to 18 hours at 4°C, and the dialysis desalination time is 24 to 36 hours.

5. The preparation method according to claim 1, characterized in that: The composite enzyme preparation in step (1) is composed of cellulase, β-glucanase and pectinase in a mass ratio of 2:1:

1.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the concentrated solution in step (2) to methanol is (3-5):

1.

7. The preparation method according to claim 6, characterized in that: In step (3), the dosage ratio of ammonium sulfate to the crude extract of mulberry leaf polysaccharide solution is (150-243) g:1L.

8. The preparation method according to claim 6, characterized in that: The centrifugation conditions of step (2), step (3) and step (4) are all 6000-8000 r / min for 5-10 min.

9. Mulberry leaf polysaccharide iron prepared by the method according to any one of claims 1 to 8.

10. Use of the mulberry leaf polysaccharide iron according to claim 9 in a multifunctional iron supplement for animals.

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