Preparation and application of black fungus enzyme extraction polysaccharide

Abstract

The invention discloses preparation and application of black fungus enzyme extraction polysaccharide. The preparation method comprises the following steps: S1, drying black fungus, grinding, sieving, and collecting to obtain black fungus superfine powder; s2, the black fungus superfine powder is subjected to degreasing, enzyme extraction, alcohol precipitation and drying, and the black fungus enzyme extraction polysaccharide is obtained. The black fungus enzyme extraction polysaccharide reacts with a complexing agent to prepare the black fungus enzyme extraction polysaccharide-iron compound which is used for preparing iron supplementing soft sweets. The black fungus enzyme-extracted polysaccharide iron-supplementing soft sweets are prepared by taking black fungus as a raw material, so that the product types of iron-supplementing and blood-enriching medicines and health-care functional products in China are enriched, and the existing market gap in China is supplemented.

Description

Technical Field

[0001] The invention relates to the technical field of health food, in particular to the preparation and application of black fungus enzyme-extracted polysaccharide. Background Art

[0002] Iron deficiency anemia affects the body's oxygen metabolism, energy metabolism, hematopoiesis and other life activities. It has always been one of the malnutrition diseases that people have been committed to conquering. In recent years, functional soft candies have become a mainstream trend in the development of soft candies. Functional soft candies are mainly based on gelling agents and syrups, with the addition of functional raw materials and various additives. After multiple processes, gel candies of different shapes and tastes are obtained. Functional soft candies can use the good flavor of the soft candies themselves to solve the problem of poor taste of certain raw materials and improve patient compliance with medication. Most of the iron-supplementing soft candies currently sold on the market have a low iron content of about 1 mg / g. There is still a rusty taste in the mouth and the price is relatively high. In clinical practice, FeSO4 is often used to supplement insufficient iron intake, but Fe 2 + It is easy to produce endogenous free radicals, which lead to lipid peroxidation of cell membranes and damage to cell membranes. Existing oral iron supplements often cause side effects such as upper abdominal pain, diarrhea and constipation. Therefore, it is necessary to study new iron supplements without side effects.

[0003] Black fungus, also known as black vegetable, fungus, cloud fungus, belongs to the Auriculariaceae family, Auricularia genus, is a precious colloid fungus for both medicine and food in my country, and is also recognized as a health food in the world. It is mainly distributed in Heilongjiang, Jilin, Hubei, Yunnan, Sichuan, Guizhou, Hunan and other provinces in my country. Black fungus is not only smooth and delicious, but also rich in nutrition, and enjoys the reputation of "meat in vegetarians" and "king of vegetarians". Black fungus contains protein, fat, carbohydrates, cellulose, and thiamine, riboflavin, niacin, carotene, calcium, phosphorus, iron and other vitamins and minerals, among which iron is the most abundant. Black fungus is one of the foods with the highest iron content among all kinds of foods. Every 100 grams of black fungus contains 98 mg of iron, which is about 5 times higher than the highest iron content in animal foods, pork liver, and 30 times higher than spinach, which has the highest iron content among green leafy vegetables. Black fungus enzyme-extracted polysaccharide is a polysaccharide extracted and separated from black fungus, with a content of up to 65%. As a biological response effector, black fungus enzyme-extracted polysaccharide has cell protection effects such as anti-coagulation, anti-tumor, and anti-inflammation. It also has multiple physiological functions such as lowering blood lipids, blood sugar, blood viscosity, cholesterol, as well as anti-diabetes, anti-aging, and anti-radiation. At the same time, it can also promote the biosynthesis of nucleic acids and proteins and prevent and treat a variety of geriatric diseases. For people who want to prevent cardiovascular and cerebrovascular diseases and pursue health, appropriate supplementation of black fungus enzyme-extracted polysaccharide is beneficial to people's health. Summary of the invention

[0004] In view of the above problems existing in the prior art, the present invention provides a preparation and application of black fungus enzyme-extracted polysaccharide. The present invention uses black fungus as a raw material to prepare black fungus enzyme-extracted polysaccharide iron-supplementing soft candy, which can not only enrich the black fungus series products, improve the utilization rate of black fungus resources, reduce the environmental pollution pressure of the black fungus industry, and extend the black fungus deep processing industry chain, but also can provide a scientific basis for the industrial development and application of medicines and health care products with anti-anemia activity using black fungus enzyme-extracted polysaccharide iron complex as an auxiliary material, which is conducive to enriching the product categories of iron-supplementing and blood-supplementing medicines and health care products in my country and supplementing the existing market gap in my country.

[0005] The technical solution of the present invention is as follows:

[0006] A method for preparing black fungus enzyme-extracted polysaccharide comprises the following steps:

[0007] S1, drying, grinding, sieving and collecting the black fungus to obtain black fungus ultrafine powder;

[0008] S2. Defatting, enzyme extraction, alcohol precipitation and drying the ultrafine black fungus powder to obtain black fungus enzyme-extracted polysaccharide.

[0009] In one embodiment of the present invention, in S1, the black fungus is ground by a grinder and then passed through a 200-mesh sieve, and the powder is then ground by an ultra-high-speed ultrafine pulverizer to obtain ultrafine black fungus powder.

[0010] In one embodiment of the present invention, in S2, the degreasing is performed using petroleum ether as a solvent, and the mass volume ratio of the black fungus superfine powder to the petroleum ether is 1:40-50 g / mL.

[0011] In one embodiment of the present invention, in S2, the degreasing conditions are immersion at 55-65°C for 90-120 min, extraction at 65-75°C for 120-150 min, and recovery at 75-80°C for 15-30 min.

[0012] In one embodiment of the present invention, in S2, the enzyme extraction is to add deionized water to the defatted black fungus superfine powder at a ratio of 1:70-90, add 4-6 μL / g of high temperature resistant α-amylase to the above solution, and bathe in 80°C water for 2h; the water bath temperature is adjusted to 60°C, and 4-6 μL / g of papain is added to the black fungus superfine powder, and bathe in 80°C water for 2h; inactivate the enzyme in 100°C water bath for 20min, add 30-50 μL / g of black fungus superfine powder of saccharifying enzyme after cooling, and bathe in 60°C water for 2h; inactivate the enzyme in 100°C water bath for 20min, and bathe in 60°C water bath for 4h after cooling to obtain an enzymolyte; then adjust the pH to 3, add 100-300U / g of black fungus superfine powder of pectinase and 500-700U / g of black fungus superfine powder of cellulase, and inactivate the enzyme in 100°C water bath for 20min after keeping warm at 60°C for 3h. After extraction, centrifuge and collect the supernatant.

[0013] In one embodiment of the present invention, alcohol precipitation is to concentrate the supernatant obtained by centrifugation after enzymatic hydrolysis to 1 / 10-1 / 20 of the original volume in a water bath at 60-80°C, add 3-4 times the volume of ethanol dropwise while stirring after cooling, and let stand for one day. Centrifuge and collect the precipitate.

[0014] In one embodiment of the present invention, drying is vacuum freeze drying of the precipitate obtained by alcohol precipitation and centrifugation to obtain black fungus enzyme-extracted polysaccharide. The vacuum freeze drying is performed after freezing at -80°C for one day, and the conditions are -60°C, 6 hours; -35°C, 15 hours; -20°C, 15 hours; 25°C, 48 hours.

[0015] The invention discloses an application of polysaccharide extracted from black fungus by enzyme, which is used for preparing a product for resisting iron deficiency anemia.

[0016] In one embodiment of the present invention, the anti-iron deficiency anemia product is a food, a health food or a medicine.

[0017] In one embodiment of the present invention, the black fungus enzyme-extracted polysaccharide is made into one of beverages, biscuits, soft candies, capsules, granules, pills and tablets.

[0018] In one embodiment of the present invention, black fungus enzyme-extracted polysaccharide reacts with a complexing agent to prepare a black fungus enzyme-extracted polysaccharide-iron complex for use in preparing iron-supplementing soft candy.

[0019] An iron-supplementing soft candy containing the black fungus enzyme-extracted polysaccharide is prepared from 40-60wt% of a black fungus enzyme-extracted polysaccharide-iron complex, 10-20wt% of gelatin, 20-40wt% of xylitol and 1-5wt% of citric acid.

[0020] In one embodiment of the present invention, the iron-supplementing soft candy is made of 50 wt % black fungus enzyme-extracted polysaccharide-iron complex, 15 wt % gelatin, 30 wt % xylitol, and 5 wt % citric acid.

[0021] A method for preparing the above-mentioned polysaccharide iron-supplementing soft candy comprises the following steps:

[0022] The black fungus enzyme-extracted polysaccharide and the complexing agent are dissolved in water at a ratio of 8-12:1, heated in a water bath to 50-90° C., and then slowly dripped with 10-30wt% sodium hydroxide solution and the pH of the solution is adjusted to 7-12; the complexing agent is one or more of trisodium citrate, sodium alginate, and sodium tartrate;

[0023] Then, an aqueous solution of a trivalent iron compound (0.1-10 M) is added dropwise while maintaining the pH value of the solution constant. When reddish brown insoluble matter appears in the solution, the addition of the sample is stopped, and heating in a water bath is continued. After the reaction is completed, the solution is centrifuged while hot, filtered, and the supernatant is obtained to obtain a mixed solution, and anhydrous ethanol is added for alcohol precipitation. After centrifugation, filtration, washing, and drying, a black fungus enzyme-extracted polysaccharide-iron complex is obtained;

[0024] 40-60wt% black fungus enzyme-extracted polysaccharide iron complex and 20-40wt% xylitol are added to the gelatin solution, vortexed evenly, and a 50wt% citric acid aqueous solution is slowly added dropwise, so that the gelatin addition amount is 10-20wt% and the citric acid is 1-5wt%, respectively. After stirring evenly, the mixture is molded, cooled, and the shaped soft candy is taken out and individually packaged in a heat-sealed bag to obtain the soft candy with iron supplement effect.

[0025] The black fungus enzyme-extracted polysaccharide iron-supplementing soft candy of the present invention weighs 2-4g per piece, and the iron content can reach 10mg / g. The obtained iron-supplementing soft candy has good sensory senses, is sticky and refreshing, has no bad smell, and has broad commercial prospects.

[0026] The black fungus enzyme-extracted polysaccharide iron-supplementing soft candy of the invention has high iron content, good iron dissolution performance, easily acceptable taste, and no toxic side effects, thus providing a new choice for the development of iron-supplementing health products for resisting iron-deficiency anemia.

[0027] The beneficial technical effects of the present invention are:

[0028] The present invention uses enzyme extraction to extract black fungus enzyme-extracted polysaccharide from black fungus, which is both a medicine and food, as the main raw material. The black fungus material source is rich and the polysaccharide content is rich, which can reach about 65%, which is helpful for popularization and application in industrial production.

[0029] The present invention adopts black fungus enzyme-extracted polysaccharide as a glycoside, reacts it with a trivalent iron compound to prepare a black fungus enzyme-extracted polysaccharide-iron complex, and effectively improves the iron content of the prepared black fungus enzyme-extracted polysaccharide-iron complex by optimizing the process conditions, thereby further enhancing the biological activity of the black fungus enzyme-extracted polysaccharide-iron complex;

[0030] The black fungus enzyme-extracted polysaccharide-iron complex prepared by the present invention has good dissolution performance, small side effects, high stability, good solubility, and is easier to be absorbed by the body compared with traditional iron supplements. In addition, the black fungus enzyme-extracted polysaccharide-iron complex has antioxidant activity in vitro, and has strong antioxidant activity against DPPH and ABTS free radicals;

[0031] The preparation process of the invention is simple and the raw material source is abundant, and the invention can be used for preventing or treating iron deficiency anemia, thereby providing a new choice for a novel iron supplement. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The iron release amount of the black fungus enzyme-extracted polysaccharide-iron complex in the simulated gastrointestinal tract in vitro obtained in Example 8;

[0033] Figure 2 The scavenging rates of hydroxyl radicals (A), superoxide anion radicals (B), ABTS radicals (C), and DPPH radicals (D) of black fungus enzyme-extracted polysaccharides, black fungus enzyme-extracted polysaccharide iron complexes, and vitamin C;

[0034] Figure 3 Effects of black fungus enzyme-extracted polysaccharide-iron complex (A), gelatin addition (B), xylitol (C) and citric acid (D) addition on sensory scores. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0036] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0037] Example 1

[0038] A method for preparing black fungus enzyme-extracted polysaccharide comprises the following steps:

[0039] (1) After fully drying the black fungus, first grind it into powder using a grinder, pass it through a 200-mesh sieve, and then grind it using an ultra-high-speed ultra-fine grinder to obtain ultra-fine black fungus powder;

[0040] (2) The black fungus ultrafine powder is defatted, enzyme-extracted, alcohol-precipitated and dried to obtain black fungus enzyme-extracted polysaccharide.

[0041] Degreasing: Use petroleum ether as solvent for degreasing, the ratio of black fungus ultrafine powder to petroleum ether is 1g:40mL. The degreasing conditions are soaking at 65℃ for 120min, extraction at 75℃ for 150min, and recovery at 80℃ for 30min.

[0042] Enzyme extraction: The extraction process is to add deionized water to the defatted black fungus superfine powder at a ratio of 1:80, add 5 μL / g of high temperature resistant α-amylase to the above solution, and bathe at 80°C for 2 hours; adjust the water bath temperature to 60°C, add 5 μL / g of papain to the black fungus superfine powder, and bathe at 80°C for 2 hours; inactivate the enzyme at 100°C for 20 minutes, add 40 μL / g of saccharifying enzyme to the black fungus superfine powder after cooling, and bathe at 60°C for 2 hours; inactivate the enzyme at 100°C for 20 minutes, and bathe at 60°C for 4 hours after cooling to obtain an enzymolysis solution; then adjust the pH to 3, add 200U / g of pectinase and 600U / g of cellulase to the black fungus superfine powder, keep warm at 60°C for 3 hours, and then bathe at 100°C for 20 minutes to inactivate the enzyme. Centrifuge after the extraction is completed and collect the supernatant.

[0043] Alcohol precipitation: The collected supernatant was concentrated to 1 / 10 of the original volume in a water bath at 80°C. After cooling, 4 times the volume of ethanol was added dropwise while stirring. The solution was allowed to stand for one day, centrifuged, and the precipitate was collected. The precipitate was frozen at -80°C for one day, and vacuum freeze-dried to obtain black fungus enzyme-extracted polysaccharides. The vacuum freeze-drying conditions were -60°C, 6 hours; -35°C, 15 hours; -20°C, 15 hours; 25°C, 48 hours, in sequence.

[0044] Example 2

[0045] A method for preparing black fungus enzyme-extracted polysaccharide comprises the following steps:

[0046] (1) After fully drying the black fungus, first grind it into powder using a grinder, pass it through a 200-mesh sieve, and then grind it using an ultra-high-speed ultra-fine grinder to obtain ultra-fine black fungus powder;

[0047] (2) The black fungus ultrafine powder is defatted, enzyme-extracted, alcohol-precipitated and dried to obtain black fungus enzyme-extracted polysaccharide. Degreasing: petroleum ether is used as solvent for degreasing, and the ratio of black fungus ultrafine powder to petroleum ether is 1g:40mL. The degreasing conditions are soaking at 65℃ for 120min, extraction at 75℃ for 150min, and recovery at 80℃ for 30min.

[0048] Enzyme extraction: The extraction process is to add deionized water to the defatted black fungus powder at a ratio of 1:80, add 5 μL / g of high temperature resistant α-amylase to the above solution, and bathe at 80°C for 2 hours; adjust the bath temperature to 60°C, add 5 μL / g of papain to the black fungus powder, and bathe at 80°C for 2 hours; inactivate the enzyme at 100°C for 20 minutes, add 40 μL / g of saccharifying enzyme after cooling, and bathe at 60°C for 2 hours; inactivate the enzyme at 100°C for 20 minutes, and bathe at 60°C for 4 hours after cooling to obtain an enzymatic solution; then adjust the pH to 3, add 200U / g of pectinase and 600U / g of cellulase to the black fungus powder, keep warm at 60°C for 3 hours, and inactivate the enzyme at 100°C for 20 minutes. Centrifuge after the extraction is completed and collect the supernatant.

[0049] Alcohol precipitation: The collected supernatant was concentrated to 1 / 10 of the original volume in a water bath at 80°C. After cooling, 4 times the volume of ethanol was added dropwise while stirring. The solution was allowed to stand for one day, centrifuged, and the precipitate was collected. The precipitate was frozen at -80°C for one day, and vacuum freeze-dried to obtain black fungus enzyme-extracted polysaccharides. The vacuum freeze-drying conditions were -60°C, 6 hours; -35°C, 15 hours; -20°C, 15 hours; 25°C, 48 hours, in sequence.

[0050] Example 3

[0051] A method for preparing black fungus water-extracted polysaccharide comprises the following steps:

[0052] (1) After the black fungus is fully dried, it is first ground into powder using a grinder and passed through a 200-mesh sieve to obtain black fungus powder;

[0053] (2) The black fungus powder is defatted, water-extracted, alcohol-precipitated and dried to obtain black fungus water-extracted polysaccharides.

[0054] Degreasing: Use petroleum ether as solvent for degreasing, the ratio of black fungus powder to petroleum ether is 1g:40mL. The degreasing conditions are soaking at 65℃ for 120min, extraction at 75℃ for 150min, and recovery at 80℃ for 30min.

[0055] Water extraction: The extraction process is to add deionized water to the defatted black fungus powder at a ratio of 1:80, and repeat the extraction twice in a water bath at 80°C for 3 hours. After the extraction is completed, centrifuge and collect the supernatant.

[0056] Alcohol precipitation: The collected supernatant was concentrated to 1 / 10 of the original volume in a water bath at 80°C. After cooling, 4 times the volume of ethanol was added dropwise while stirring. The solution was allowed to stand for one day, centrifuged, and the precipitate was collected. The precipitate was frozen at -80°C for one day, and vacuum freeze-dried to obtain black fungus enzyme-extracted polysaccharides. The vacuum freeze-drying conditions were -60°C, 6 hours; -35°C, 15 hours; -20°C, 15 hours; 25°C, 48 hours, in sequence.

[0057] Performance Testing

[0058] Determination of polysaccharide content in black fungus

[0059] Weigh 10 mg of the black fungus enzyme-extracted polysaccharide obtained in Example 1-2 and the black fungus water-extracted polysaccharide obtained in Example 3, add 10 mL of distilled water to dissolve, then transfer 1 mL of the polysaccharide solution, inject 1.0 mL of phenol solution (5%) and 5.0 mL of concentrated sulfuric acid in sequence, carefully shake and mix, bathe in 25°C water for half an hour, and use an ultraviolet spectrophotometer to detect the sample absorbance at a wavelength of 490 nm.

[0060]

[0061] In the formula: w is the polysaccharide content in the black fungus polysaccharide sample (%); A is the absorbance value of the sample; a and b are the coefficient values ​​in the linear equation of the standard working curve; v is the volume of the black fungus polysaccharide solution after dilution (mL); m is the mass of the weighed black fungus polysaccharide (mg).

[0062] Table 1

[0063] Sample No. Polysaccharide yield (%) Polysaccharide content (%) Example 1 41.6 77.4 Example 2 36.5 73.5 Example 3 18.2 56.3

[0064] As shown in Table 1, compared with hot water extraction, compound enzyme extraction can significantly increase the yield of black fungus polysaccharides, and ultrafine grinding can further increase the yield of polysaccharides. In addition, the purity of polysaccharides in black fungus ultrafine powder is also greatly increased after compound enzyme extraction.

[0065] Embodiment 4-6

[0066] A method for preparing a black fungus polysaccharide-iron complex comprises the following steps:

[0067] (1) Weigh 5 g of the black fungus enzyme-extracted polysaccharide obtained in Example 1-2 and 5 g of the black fungus water-extracted polysaccharide obtained in Example 3 and 0.5 g of trisodium citrate, dissolve them in 60 mL of water, and heat them in a 70° C. water bath while stirring continuously.

[0068] (2) Slowly drip 20% NaOH solution and adjust the pH of the solution to 9, then add 2M FeCl3 solution dropwise, and continuously adjust the pH of the solution to maintain it at pH = 9. When reddish brown insoluble matter appears in the solution, stop adding the sample and continue heating in a water bath at 70°C for 1 hour.

[0069] (3) After the reaction is completed, the solution is centrifuged at 8000 r / min for 10 minutes while hot, the supernatant is collected, and anhydrous ethanol is added to obtain a mixed solution until the anhydrous ethanol accounts for 80% of the total volume of the mixed solution. The solution is placed at 4°C for alcohol precipitation overnight. After alcohol precipitation overnight, the solution is centrifuged at 8000 r / min for 10 minutes, and the precipitate is collected. The precipitate is washed three times with anhydrous ethanol and then freeze-dried to obtain a freeze-dried powder of black fungus polysaccharide iron complex.

[0070] Performance Testing

[0071] Determination of iron content in black fungus polysaccharide-iron complex samples

[0072] 10 mg of the black fungus polysaccharide-iron complex prepared in Example 4-6 was weighed separately, dissolved in 20 mL of distilled water to prepare a 0.5 mg / mL black fungus polysaccharide-iron complex solution, 1 mL of hydroxylamine hydrochloride solution (10%) and 2.5 mL of o-phenanthroline solution (0.1%) were added respectively, and then 5 mL of glacial acetic acid-sodium acetate buffer solution (pH = 4.5) was added, and placed in a water bath at 30°C for 15 min, and the absorbance was detected at a wavelength of 510 nm using a UV spectrophotometer.

[0073]

[0074] In the formula: I is the iron content of the black fungus polysaccharide iron complex (%); A is the absorbance value measured by the o-phenanthroline method; V1 is the volume of the black fungus polysaccharide iron complex after dissolution (mL); V2 is the volume of the black fungus polysaccharide iron complex aqueous solution transferred for detection (mL); m is the mass of the weighed black fungus polysaccharide iron complex (mg).

[0075] Table 2

[0076] Sample No. Iron content (%) Example 4 65.4％ Example 5 52.7％ Example 6 41.3％

[0077] The molecular structures of polysaccharides obtained by different extraction methods are different. The present invention greatly improves the yield and purity of black fungus polysaccharides by combining ultrafine grinding and complex enzyme extraction. After the hydrolysis of the complex enzyme, more hydroxyl groups on the black fungus polysaccharide chains are exposed, increasing the complexation sites between the polysaccharide and the iron element. Therefore, the iron content in the black fungus enzyme-extracted polysaccharide-iron complex is significantly improved than that in the black fungus water-extracted polysaccharide-iron complex.

[0078] Example 7-9 uses black fungus enzyme-extracted polysaccharide as a variable.

[0079] A method for preparing a black fungus enzyme-extracted polysaccharide-iron complex comprises the following steps:

[0080] Weigh 0.5g, 2g, and 4g of the black fungus enzyme-extracted polysaccharide obtained in Example 1 and 0.5g of trisodium citrate respectively, dissolve in 60mL of water, place in a 70°C water bath and heat in a water bath with constant stirring. Slowly drip 20% NaOH solution and adjust the solution pH to 9, then add 2M FeCl3 solution dropwise, and continuously adjust the solution pH to maintain the solution at pH = 9. When reddish-brown insoluble matter appears in the solution, stop adding the sample and continue heating in a water bath at 70°C for 1h. After the reaction is completed, take the solution and centrifuge it while hot at 8000r / min for 10min, collect the supernatant, obtain a mixed solution, add anhydrous ethanol until anhydrous ethanol accounts for 80% of the total volume of the mixed solution, and place the solution at 4°C for alcohol precipitation overnight. After alcohol precipitation overnight, the solution is centrifuged at 8000r / min for 10min, and the precipitate is collected. The precipitate is washed three times with anhydrous ethanol and freeze-dried to obtain a black fungus enzyme-extracted polysaccharide-iron complex freeze-dried powder.

[0081] Example 10-11: FeCl3 as variable

[0082] Weigh 2g of black fungus enzyme-extracted polysaccharide obtained in Example 1 and 0.5g of trisodium citrate and dissolve them in 60mL of water, place in a 70°C water bath and heat in a water bath with constant stirring. Slowly drip 20% NaOH solution and adjust the solution pH to 9, then add 0.1M and 10M FeCl3 solutions dropwise respectively, and constantly adjust the pH of the solution to maintain the solution at pH = 9. When reddish-brown insoluble matter appears in the solution, stop adding the sample and continue heating in a water bath at 70°C for 1h. After the reaction is completed, take the solution and centrifuge it while hot at 8000r / min for 10min, collect the supernatant, obtain a mixed solution and add anhydrous ethanol until anhydrous ethanol accounts for 80% of the total volume of the mixed solution, and the solution is placed at 4°C for alcohol precipitation overnight. After alcohol precipitation overnight, the solution is centrifuged at 8000r / min for 10min, and the precipitate is collected. The precipitate is washed three times with anhydrous ethanol and freeze-dried to obtain a black fungus enzyme-extracted polysaccharide-iron complex freeze-dried powder.

[0083] Example 12-13 takes reaction temperature as variable

[0084] Weigh 2g of black fungus enzyme-extracted polysaccharide obtained in Example 1 and 0.5g of trisodium citrate and dissolve them in 60mL of water, place them in 50℃ and 90℃ water baths for heating and stirring. Slowly drip 20% NaOH solution and adjust the pH of the solution to 9, then add 2M FeCl3 solution dropwise, and constantly adjust the pH of the solution to maintain the solution at pH=9. When reddish-brown insoluble matter appears in the solution, stop adding the sample and continue heating in a water bath at 70℃ for 1h. After the reaction is completed, take the solution and centrifuge it while hot at 8000r / min for 10min, collect the supernatant, obtain a mixed solution and add anhydrous ethanol until anhydrous ethanol accounts for 80% of the total volume of the mixed solution, and the solution is placed at 4℃ for alcohol precipitation overnight. After alcohol precipitation overnight, the solution is centrifuged at 8000r / min for 10min, and the precipitate is collected. The precipitate is washed three times with anhydrous ethanol and freeze-dried to obtain a freeze-dried powder of black fungus enzyme-extracted polysaccharide-iron complex.

[0085] Example 14-15: pH of solution as variable

[0086] Weigh 2g of black fungus enzyme-extracted polysaccharide obtained in Example 1 and 0.5g of trisodium citrate and dissolve them in 60mL of water, place them in a 70°C water bath and heat them in a water bath with constant stirring. Slowly drip 10% and 30% NaOH solution respectively and adjust the pH of the solution to 7 and 12; then add 2M FeCl3 solution dropwise, and constantly adjust the pH of the solution to maintain the pH of the solution. When reddish-brown insoluble matter appears in the solution, stop adding the sample and continue heating in a water bath at 70°C for 1h. After the reaction is completed, take the solution and centrifuge it while hot at 8000r / min for 10min, collect the supernatant, obtain a mixed solution, add anhydrous ethanol until anhydrous ethanol accounts for 80% of the total volume of the mixed solution, and place the solution at 4°C for alcohol precipitation overnight. After alcohol precipitation overnight, the solution was centrifuged at 8000r / min for 10min, and the precipitate was collected. The precipitate was washed three times with anhydrous ethanol and freeze-dried to obtain a freeze-dried powder of black fungus enzyme-extracted polysaccharide-iron complex.

[0087] Performance Testing

[0088] The black fungus enzyme-extracted polysaccharide-iron complex obtained in Examples 7-15 was tested as follows.

[0089] 1. Test of iron release of black fungus enzyme-extracted polysaccharide-iron complex in simulated gastrointestinal tract in vitro

[0090] A 0.5 mg / mL black fungus enzyme-extracted polysaccharide-iron complex solution was prepared as the sample solution. 5.0 M HCl was added to adjust the pH of the sample solution to 2.0, and 0.85 mL of artificial gastric juice was added to the sample solution. The sample was stirred at 37 ° C for 2 h using a constant temperature magnetic stirrer. After the gastric juice reaction was completed, 1.0 M NaHCO3 solution was added to adjust the pH of the sample solution to 6, and 4.25 mL of artificial intestinal juice was added. The pH of the sample solution was adjusted to 7 with 1.0 M NaOH, and 120 mM NaCl and 5 mM KCl were added to 13.4 mL. Then, the mixture was stirred at 37 ° C for 3 h. Throughout the reaction, the stirring speed was 150 r / min, and 1.5 mL of sample was taken out every 20 min. The sample solution was filtered through a 0.22 μm filter membrane and centrifuged at 4000 r / min for 20 min. The iron content in the supernatant of the sample solution was determined by o-phenanthroline spectrophotometry and recorded in Table 3.

[0091] Table 3

[0092]

[0093]

[0094] From the data analysis in Table 3, it can be seen that the iron ion content in the black fungus enzyme-extracted polysaccharide-iron complex prepared by the preparation method proposed in the present invention generally reaches more than 55%, indicating that the black fungus enzyme-extracted polysaccharide-iron complex prepared by the preparation method proposed in the present invention has a significant advantage in iron loading, and due to the easy absorbability of black fungus enzyme-extracted polysaccharides and their own pharmacological effects, the black fungus enzyme-extracted polysaccharide-iron complex as an iron supplement not only has high bioavailability, but also has additional positive effects.

[0095] Example 8 Auricularia auricula enzyme-extracted polysaccharide-iron complex was prepared, and the iron release was measured in artificial gastric juice (pH=2.0) and artificial intestinal juice (pH=7.0) for 120 min. Figure 1 As shown, the results show that the black fungus enzyme-extracted polysaccharide-iron complex has a good release rate in the human body. In simulated gastric juice, the black fungus enzyme-extracted polysaccharide-iron complex is gradually released within 120min, and the release amount gradually increases with time. In simulated intestinal fluid, although the amount of free iron in the solution decreases compared to that in gastric juice, its release amount also increases with time. This study shows that the black fungus enzyme-extracted polysaccharide-iron complex of the present invention can resist the gastric acid environment well, slowly release iron ions, and reduce the side effects such as gastrointestinal discomfort and peroxidative stress caused by the rapid release of a large amount of iron ions.

[0096] 2. Antioxidant activity test

[0097] Example 8 Preparation of black fungus enzyme-extracted polysaccharide iron complex, black fungus enzyme-extracted polysaccharide, black fungus enzyme-extracted polysaccharide iron complex and vitamin C solution with concentrations of 0, 0.0625, 0.125, 0.25, 0.5, 1 mg / mL were prepared with deionized water, and their scavenging activities of DPPH free radicals, ABTS free radicals, hydroxyl free radicals and superoxide anion free radicals were detected according to the following method. Figure 2 Data analysis shows that, with vitamin C as the positive control, black fungus enzyme-extracted polysaccharides and black fungus enzyme-extracted polysaccharide-iron complexes both have certain antioxidant activity. The antioxidant activity of black fungus enzyme-extracted polysaccharides is generally higher than that of black fungus enzyme-extracted polysaccharide-iron complexes. Black fungus enzyme-extracted polysaccharides and black fungus enzyme-extracted polysaccharide-iron complexes have better antioxidant activity against DPPH free radicals, ABTS free radicals and hydroxyl free radicals. In the determination of superoxide anion free radical scavenging rate, the scavenging rate of black fungus enzyme-extracted polysaccharide-iron complexes was low.

[0098] Examples 16-18

[0099] The black fungus enzyme-extracted polysaccharide iron complex, gelatin, xylitol and citric acid prepared in Example 8 were selected as raw materials. The addition ratio range of each raw material was determined by a single factor experiment, and the specific operation method is as follows. This embodiment provides the preparation of the iron-supplementing soft candy of the present invention, with the black fungus enzyme-extracted polysaccharide iron complex as a variable, and screened by sensory scoring: soak the gelatin with water, put it in a constant temperature water bath and stir to dissolve it, then weigh 4g, 5g, 6g of the black fungus enzyme-extracted polysaccharide iron complex and 3g of xylitol respectively and add them to the gelatin solution, vortex to make it evenly dispersed in the gelatin, and slowly drip 50% citric acid aqueous solution to ensure that the amount of gelatin added is 15% of the total system and the citric acid is 5%. After stirring evenly, injection molding and cooling, after taking out the shaped soft candy, use heat-sealed bags to individually package it to obtain soft candy with iron supplementation effect. By Figure 3 A shows that when the addition amount of black fungus enzyme-extracted polysaccharide-iron complex is 5g (50%), the sensory score is the highest.

[0100] Examples 19-21

[0101] This example provides the preparation of the iron-supplementing soft candy of the present invention, with the amount of gelatin added as a variable, and screening by sensory scoring:

[0102] Soak the gelatin in water, put it in a 70℃ constant temperature water bath and stir to dissolve it, weigh 5g of the black fungus enzyme-extracted polysaccharide iron complex prepared in Example 8 and 3g of xylitol, add them to the gelatin solution, vortex evenly, slowly add 50% citric acid aqueous solution, so that the gelatin addition amount is 10%, 15%, 20%, and citric acid is 5%. After stirring evenly, inject into the mold, cool, take out the shaped soft candy, and use heat-sealed bags to package it independently to obtain soft candy with iron supplement effect. Figure 3B shows that when the gelatin addition is 15%, the sensory score is the highest.

[0103] Examples 22-24

[0104] This example provides the preparation of the iron-supplementing soft candy of the present invention, with the amount of xylitol added as a variable, and screened by sensory scoring: soak gelatin in water, put it in a 70°C constant temperature water bath and stir to dissolve it, weigh 5g of the black fungus enzyme-extracted polysaccharide iron complex prepared in Example 8, 2g, 3g, and 4g of xylitol, add them to the gelatin solution, vortex evenly, and slowly drip 50% citric acid aqueous solution so that the amount of gelatin added is 15% and the citric acid is 5%. After stirring evenly, inject into the mold, cool, take out the shaped soft candy, and use heat-sealed bags to package it independently to obtain soft candy with iron supplement effect. Figure 3 C shows that when the amount of xylitol added is 3g (30%), the sensory score is the highest.

[0105] Examples 25-27

[0106] This embodiment provides the preparation of the iron-supplementing soft candy of the present invention, with the amount of citric acid added as a variable, and screened by sensory scoring: soak gelatin in water, put it in a 70°C constant temperature water bath and stir to dissolve it, weigh 5g of the black fungus enzyme-extracted polysaccharide iron complex and 3g of xylitol prepared in Example 8, add them to the gelatin solution, vortex evenly, and slowly drip 50% citric acid aqueous solution, so that the amount of gelatin added is 15%, and the amounts of citric acid added are 1%, 2%, and 5%, respectively. After stirring evenly, inject into the mold, cool, take out the shaped soft candy, and use heat-sealed bags to individually package it to obtain soft candy with iron supplement effect. Figure 3 D shows that when the addition amount of citric acid is 5%, the sensory score is the highest.

[0107] Embodiment 28

[0108] This embodiment provides the preparation of the iron-supplementing soft candy of the present invention, wherein the optimal addition amount of the black fungus enzyme-extracted polysaccharide iron complex, gelatin, xylitol, and citric acid is screened by sensory scoring; the orthogonal test adopts the L9 (34) orthogonal table, and the orthogonal test is performed with the addition amount of the black fungus enzyme-extracted polysaccharide iron complex, gelatin, xylitol, and citric acid, respectively. The raw material orthogonal test factors and level design are shown in Table 4.

[0109] Table 4

[0110]

[0111] Table 5

[0112]

[0113] It can be seen from Tables 4 and 5 that the order of influence of each factor is the addition amount of black fungus enzyme-extracted polysaccharide iron complex (B)> gelatin addition amount (A)> citric acid addition amount (D)> xylitol addition amount (C). The best combinations A2, B2, C2, and D2 can be obtained through the K value, that is, the addition amount of gelatin is 15%, the addition amount of black fungus enzyme-extracted polysaccharide iron complex is 50%, the addition amount of xylitol is 20%, and the addition amount of citric acid is 5%. The addition amount of black fungus enzyme-extracted polysaccharide iron complex has a significant effect on the sensory score of soft candy. The optimal formula of soft candy was obtained by orthogonal experiment, and the obtained black fungus enzyme-extracted polysaccharide iron-supplementing soft candy has good iron dissolution performance, easy to accept taste, and no toxic side effects. The iron content of the black fungus enzyme-extracted polysaccharide iron-supplementing soft candy prepared under this formula condition is as high as 10 mg / g, which is 10 times that of the commonly available commercially available iron-supplementing soft candy; it is the patent CN 118303499 A with glycerol as Fe 2+ The chelation-formed metal complex is 7.5 times that of the soft candy prepared by the present invention, and the ferric chloride is selected as the raw material, which avoids Fe 2+ Side effects on the gastrointestinal digestive system.

[0114] Comparative Examples 1-3

[0115] This comparative example provides a preparation of a ferric chloride emulsion, and ferric chloride is added to glycerol in a ratio of 1:20, 1:30, and 1:40 to prepare a ferric chloride emulsion, and the black fungus enzyme-extracted polysaccharide iron complex is replaced in the same proportion, and then iron-supplementing soft candies are prepared according to the addition amount of gelatin of 15%, the addition amount of black fungus enzyme-extracted polysaccharide iron complex of 50%, the addition amount of xylitol of 20%, and the addition amount of citric acid of 5%. The taste and flavor differences of ferric chloride soft candies, commercially available iron-supplementing soft candies, and black fungus enzyme-extracted polysaccharide iron-supplementing soft candies are compared by sensory scoring. The results are shown in Table 6, indicating that the taste and flavor of the soft candies of the present invention are the same as those of commercially available products, and the iron content is higher than that of commercially available products. The black fungus enzyme-extracted polysaccharide iron complex can slowly release iron ions in the stomach and intestines, reducing adverse reactions such as gastrointestinal discomfort and peroxidative stress reactions caused by the rapid release of a large amount of iron ions, and are novel products with excellent anti-iron deficiency anemia effects. The preparation process of the invention is simple and the raw material source is abundant, and the invention can be used for preventing or treating iron deficiency anemia, thereby providing a new choice for a novel iron supplement.

[0116] Table 6

[0117] Sample name Sensory Rating Ferric chloride soft candy (1:20) 80 Ferric chloride soft candy (1:30) 91 Ferric chloride soft candy (1:40) 84 Commercially available iron supplement gummies 93 Black Fungus Enzyme Extract Polysaccharide Iron Supplement Soft Candy 93

[0118] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing polysaccharides extracted from black fungus by enzyme extraction, characterized in that: The steps include: S1, drying, grinding, sieving and collecting the black fungus to obtain black fungus ultrafine powder; S2. Defatting, enzyme extraction, alcohol precipitation and drying the ultrafine black fungus powder to obtain black fungus enzyme-extracted polysaccharide.

2. The preparation method according to claim 1, characterized in that: In S1, the black fungus is ground by a grinder and then passed through a 200-mesh sieve, and the powder is further ground by an ultra-high-speed ultra-fine pulverizer to obtain ultra-fine black fungus powder.

3. The preparation method according to claim 1, characterized in that: In S2, petroleum ether is used as a solvent for degreasing, and the mass volume ratio of the black fungus ultrafine powder to the petroleum ether is 1:40-50 g / mL.

4. The preparation method according to claim 1, characterized in that: In S2, the enzyme extraction is to add deionized water to the defatted black fungus superfine powder at a ratio of 1:70-90, add 4-6 μL / g of high temperature resistant α-amylase to the above solution, and bathe in 80°C water for 2 hours; adjust the water bath temperature to 60°C, add 4-6 μL / g of papain to the black fungus superfine powder, and bathe in 80°C water for 2 hours; inactivate the enzyme in 100°C water bath for 20 minutes, add 30-50 μL / g of saccharifying enzyme to the black fungus superfine powder after cooling, and bathe in 60°C water for 2 hours; inactivate the enzyme in 100°C water bath for 20 minutes, and bathe in 60°C water bath for 4 hours after cooling to obtain an enzymolysis solution; then adjust the pH to 3, add 100-300U / g of pectinase and 500-700U / g of cellulase to the black fungus superfine powder, keep warm at 60°C for 3 hours, and then inactivate the enzyme in 100°C water bath for 20 minutes. After extraction, centrifuge and collect the supernatant.

5. The preparation method according to claim 1, characterized in that: In S2, alcohol precipitation is to precipitate the supernatant obtained by centrifugation after enzymatic hydrolysis in ethanol.

6. A black fungus enzyme-extracted polysaccharide obtained by the preparation method according to any one of claims 1 to 5.

7. An application of the black fungus enzyme-extracted polysaccharide according to claim 6, characterized in that: Used to prepare anti-iron deficiency anemia products.

8. The use according to claim 7, characterized in that: Anti-iron deficiency anemia products are foods, health foods or medicines.

9. The use according to claim 7, characterized in that: The polysaccharide extracted from black fungus enzyme is made into beverage, biscuit, soft candy, capsule, granule, pill and tablet.

10. The use according to claim 7, characterized in that: The black fungus enzyme-extracted polysaccharide reacts with a chelating agent to prepare a black fungus enzyme-extracted polysaccharide-iron complex, which is used to prepare iron-supplementing soft candy.

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