CN101358224B - Extraction method of hericium erinaceus polysaccharide - Google Patents
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Abstract
The invention provides a hericium erinaceus polysaccharide extraction method with high product yield and good purity. The method comprises that: hericium erinaceus dried powder is added with water, is extracted by 8 to 20 KHz ultrasonic under the temperature of 50 to 80 DEG C for 0.5 to 2.0 hours, and is filtered; the filtrate is hydrated orderly by sufficient cellulose, pectinase and papain, finally is added with hydrogen peroxide to be stirred in heat preservation under the temperature of 30 to 50 DEG C for 0.5 to 1 hour, and is hyperfiltrated by a hyperltration membrane; the components with the molecular weight of more than 10000 dalton are collected, frozen and dried to obtain the hericium erinaceus polysaccharide. The hericium erinaceus polysaccharide extraction method has the advantages that 1. the prepared product has high purity; wherein, the content of Beta-dextran is as high as more than 40 percent and the biological activity is high, and the hericium erinaceus polysaccharide yield can reach 12.2 percent; 2. the hydrogen peroxide is adopted to decolorize, the conditions are mild, the polysaccharide structure is difficult to damage and the by-product is water, the method is green and environment-friendly, and coincides with the production requirements of the health care food.
Description
(1) technical field
The present invention relates to a kind of extracting method of hericium erinaceum polysaccharide.
(2) background technology
Hericium erinaceus (Bull. Ex Fr.) Pers. (Hericium erinaceus) has another name called hedgehog hydnum, hedgehog hydnum mushroom, Hericium erinaceus (Bull. Ex Fr.) Pers., hedgehog bacterium etc., is a kind of macro fungi, belongs to Basidiomycetes, Aphyllophorales, hedgehog fungus section, hedgehog hydnum genus, because of its sporophore shape is gained the name as the monkey head.The polysaccharide that from hericium erinaceus fruiting body, extracts be main chain by β-1,3 glycosidic link, side chain is the dextran mixture that β-1,6 glycosidic link connects, molecular weight is between 8000～40000.A large amount of medical science and pharmaceutical researches show, this hericium erinaceum polysaccharide has physiological functions such as improving immunizing power, antitumor, anti-ageing, reducing blood-fat, all effective in cure to diseases such as the canceration of Digestive tract, ulcer, antral gastritis, chronic gastritiss, and can improve the immunological competence of human body.Therefore, medicine and healthcare products that Hericium erinaceus polysaccharide is made are loved by the people, and DEVELOPMENT PROSPECT is wide.
The extracting method of hericium erinaceum polysaccharide commonly used has alkaline extraction, sour formulation, salt formulation to also have the hot water extraction at present.Diluted acid, diluted alkaline method, reagent dosage is few, and instrument is conventional instrument, and therefore operation is simple.But under diluted acid, the diluted alkaline condition, easily make the fracture of polysaccharide generation glycosidic link, part polysaccharide generation hydrolysis and the extraction yield of polysaccharide is reduced, and also foreign matter content is also many, and certain scope of application is arranged.Liu Zhen Lam patent of invention " process for refinement and purification of fungus polysaccharide " has been pointed out purification fungus polysaccharide method (200610012682.3), but at hericium erinaceum polysaccharide, itself existing makes the darker phenol type compound of color be connected with sugar chain, difficulty is removed, and also there is the interference of biomacromolecules such as Mierocrystalline cellulose, pectin, problems such as the easy obstruction of ultrafiltration fenestra.Therefore, also there is the further defective of raising of needs such as product color, efficient, purity in the hericium erinaceum polysaccharide of method acquisition thus.
(3) summary of the invention
The present invention seeks to optimize and improve the extraction process of hericium erinaceum polysaccharide, a kind of product yield height, hericium erinaceum polysaccharide extracting method that purity is good are provided.
The technical solution used in the present invention is:
A kind of extracting method of hericium erinaceum polysaccharide, described method comprises:
(1) gets dry Hericium erinaceus (Bull. Ex Fr.) Pers. (can obtain through natural air drying), pulverize, cross 50～120 mesh sieves, get the Hericium erinaceus (Bull. Ex Fr.) Pers. dried powder by new fresh Hericium erinaceus;
(2) the Hericium erinaceus (Bull. Ex Fr.) Pers. dried powder adds the distilled water of 5～35 times of quality, and 50～80 ℃ following to 8～20KHz ultrasonic extraction 0.5h～2.0h, filters, and gets filtrate 1; Filter residue can repeat the ultrasonic extraction operation, and filtrate is carried out next step extraction after merging;
(3) regulating filtrate 1pH is 3～6, add the capacity cellulase, 30～60 ℃ of hydrolysis 1.0h～4.0h, it is 3～6 that centrifuging and taking filtrate 2 is transferred pH, adds the capacity polygalacturonase, 30～60 ℃ of hydrolysis 0.5h～1.5h, it is 3～6 that centrifuging and taking filtrate 3 is transferred pH, adds the capacity papoid, 30～60 ℃ of hydrolysis 1.0h～2.0h, centrifugal, get filtrate 4;
(4) regulating filtrate 4pH is 8～9, and adding volume is 20～40% hydrogen peroxide of 0.5～1.0 times of filtrate 4 volume, in 30～50 ℃ of insulated and stirred 0.5h～1h, the ultra-filtration membrane ultrafiltration, collect the above component of molecular weight 10000 dalton, lyophilize obtains hericium erinaceum polysaccharide.
Described cellulase can adopt commercial commercial enzyme, and consumption is advisable with capacity, and preferred, enzymic activity is that the cellulase quality consumption of 10000～20000u/g is 0.5～1.0 times of filtrate 1 quality.
Described polygalacturonase can adopt commercial commercial enzyme, and consumption is advisable with capacity, and preferred, enzymic activity is that the polygalacturonase quality consumption of 15000～25000u/g is 1.0～1.5 times of filtrate 2 quality.
Described papoid can adopt commercial commercial enzyme, and consumption is advisable with capacity, and enzymic activity is that the papoid quality consumption of 800000～1000000u/g is 0.5～1.5 times of filtrate 3 quality.
Concrete, described method is as follows:
(1) gets dry Hericium erinaceus (Bull. Ex Fr.) Pers., fully pulverize, cross 50～120 mesh sieves, get the Hericium erinaceus (Bull. Ex Fr.) Pers. dried powder;
(2) the Hericium erinaceus (Bull. Ex Fr.) Pers. dried powder adds the distilled water of 10～25 times of quality, 50～60 ℃ down with 10～15KHz ultrasonic extraction 0.5h～2.0h, filter, get filtrate 1-1, filter residue adds the distilled water of 10～25 times of quality, and 50～60 ℃ down with 10～15KHz ultrasonic extraction 0.5h～2.0h, filter, get filtrate 1-2, merging filtrate 1-1 and filtrate 1-2 get filtrate 1;
(3) pH of adjusting filtrate 1 is 3.5～4.0, adding quality is the cellulase of the 15000u/g of 0.6～0.9 times of filtrate 1 quality, 40～45 ℃ of hydrolysis 1.0h～2.0h, it is 4.0～4.5 that centrifuging and taking filtrate 2 is transferred pH, adding quality is the polygalacturonase of the 20000u/g of 1.0～1.2 times of filtrate 2 quality, 45～50 ℃ of hydrolysis 1.0h～1.5h, it is 4.0～4.5 that centrifuging and taking filtrate 3 is transferred pH, adding quality is the papoid of the 800000u/g of 0.8～1.2 times of filtrate 3 quality, 45～50 ℃ of hydrolysis 1.2h～1.5h, centrifugal, get filtrate 4;
(4) regulating filtrate 4pH is 8～9, and adding volume is 30% hydrogen peroxide of 0.5～1.0 times of filtrate 4 volume, in 40～45 ℃ of insulated and stirred 0.5h～1h, polysulfone membrane ultrafiltration with 5k, collect the above component of molecular weight 10000 dalton, lyophilize obtains hericium erinaceum polysaccharide.
Beneficial effect of the present invention is mainly reflected in: 1, obtained product purity height, and wherein beta-glucan content can be up to more than 40%, the biological activity height; 2, contain the interference of impurity such as Mierocrystalline cellulose, pectin, protein at hericium erinaceus fruiting body itself, screening is applicable to this cellulase, polygalacturonase, papoid reaction system, impurity is fully removed, Hericium erinaceus (Bull. Ex Fr.) Pers. Crude polysaccharides yield can reach 12.2%, extracts yield than traditional hot water extraction Crude polysaccharides and has improved nearly 67.4%; 3, adopt the hydrogen peroxide decolouring, mild condition, not fragile polysaccharide structures, and by product is water, environmental protection meets the health care food production requirement; 4, adopt polysulfone membrane, the tangential flow filtration technology is applied in the preparation of hericium erinaceum polysaccharide, the filtering while also to the polysulfone membrane surface erosion, make the film surface can not form impurity macromole gel coat, thereby guarantee to stablize, ultrasiltrated rate fast, shorten the process time greatly, be convenient to suitability for industrialized production.
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
A. to the hericium erinaceus fruiting body pre-treatment, get fresh Hericium erinaceus (Bull. Ex Fr.) Pers.,, after fully pulverizing, cross 60 mesh sieves its natural air drying.
B. the raw material after will sieving adds the distilled water of 10 times of quality, stirs and heats up gradually, and the ultrasonic wave with 10KHz in 60 ℃ of scopes is carried out the ultrasonic extraction of 1.5h to it, filters, and filter residue repeats above-mentioned steps, extracts once merging filtrate again.
C. regulate filtrate pH4.0, add earlier 0.8 times of quality cellulase (the enzyme 15000U/g that lives, Wuxi City snow plum zymin Science and Technology Ltd., down with), controlled temperature is at 40 ℃, hydrolysis 1.5h, frozen centrifugation discards residue; Re-adjustment filtrate pH4.0, continue to add in the filtrate 1.0 times of quality polygalacturonase (the enzyme 20000U/g that lives, the sharp magnificent zymin in Tianjin factory, down with), controlled temperature is at 45 ℃, hydrolysis 1.0h, frozen centrifugation discards residue; Last re-adjustment filtrate pH4.0, add 0.8 times of quality papoid (the enzyme 800000U/g that lives, Wuxi City snow plum zymin Science and Technology Ltd., down with), controlled temperature is at 50 ℃, hydrolysis 1.5h, frozen centrifugation is collected filtrate.
D. regulating pH is 8, adds 30% hydrogen peroxide of 0.6 times of volume then gradually, stirs, and control filtrate temperature is at 40 ℃, and insulation 30min shoals to color.
E. adopt the cross-flow ultrafiltration technology, use ultra-filtration membrane to carry out ultrafiltration, use the polysulfone membrane (Beijing Trihigh Membrane Technology Co., Ltd., down together) of 5k, pressure is no more than 0.4MPa, and filtrate is divided into two portions, collects the above part of 10000 dalton.
F. the filtrate that operation steps e is obtained is carried out lyophilize, promptly obtains required hericium erinaceum polysaccharide, and through carrying out sealed barrier package (soft packaging, bottled) after the weighing automatically, vanning is at last put in storage.
The detection of polysaccharide content:
Accurately take by weighing hericium erinaceum polysaccharide 10mg and place the 100ml beaker, add the 80ml redistilled water and boil 1 hour postcooling, add the redistilled water constant volume again to 100ml to room temperature.Measure 1.0ml and move in the cuvette, add 5.0% and heavily steam phenol 2.0ml, add vitriol oil 5.0ml again, shake up the back room temperature and place 20min, measure absorbancy at wavelength 490nm place.
The hericium erinaceum polysaccharide of preparation is a pale yellow powder, easily moisture absorption, and soluble in water, content is 43%, and meets following index:
1. heavy metal index: Pb＜1.5ppm As＜1.0ppm Hg＜0.3ppm.
2. sanitary index: bacterial count＜1000/g intestinal bacteria, the mite that lives must not detect, fungi count＜35/g.
Embodiment 2:
A. to the hericium erinaceus fruiting body pre-treatment, get fresh Hericium erinaceus (Bull. Ex Fr.) Pers.,, after fully pulverizing, cross 100 mesh sieves its natural air drying.
B. the raw material after will sieving adds the distilled water of 20 times of quality, stirs and heats up gradually, and the ultrasonic wave with 15KHz in 50 ℃ of scopes is carried out the ultrasonic extraction of 0.8h to it, filters, and filter residue repeats above-mentioned steps, extracts once merging filtrate again.
C. regulate filtrate pH4.0, add earlier the cellulase of 0.6 times of quality, controlled temperature is at 45 ℃, hydrolysis 1.0h, and frozen centrifugation discards residue; Re-adjustment filtrate pH4.5 continues to add the polygalacturonase of 1.0 times of quality in the filtrate, and controlled temperature is at 50 ℃, hydrolysis 1.0h, and frozen centrifugation discards residue; Last re-adjustment filtrate pH4.5 adds the papoid of 1.2 times of quality, and controlled temperature is at 45 ℃, hydrolysis 1.2h, and frozen centrifugation is collected filtrate.
D. regulating pH is 8, adds 30% hydrogen peroxide of 1.0 times of volumes then gradually, stirs, and control filtrate temperature is at 45 ℃, and insulation 30min shoals to color.
E. adopt the cross-flow ultrafiltration technology, use ultra-filtration membrane to carry out ultrafiltration, especially use the polysulfone membrane of 5k, pressure is no more than 0.4MPa, and filtrate is divided into two portions, collects the above part of 10000 dalton.
F. the filtrate that operation steps e is obtained is carried out lyophilize, promptly obtains required hericium erinaceum polysaccharide, and through carrying out sealed barrier package (soft packaging, bottled) after the weighing automatically, vanning is at last put in storage.
The detection of polysaccharide content:
Accurately take by weighing hericium erinaceum polysaccharide 10mg and place the 100ml beaker, add the 80ml redistilled water and boil 1 hour postcooling, add the redistilled water constant volume again to 100ml to room temperature.Measure 1.0ml and move in the cuvette, add 5.0% and heavily steam phenol 2.0ml, add vitriol oil 5.0ml again, shake up the back room temperature and place 20min, measure absorbancy at wavelength 490nm place.
The hericium erinaceum polysaccharide of preparation is a pale yellow powder, easily moisture absorption, and soluble in water, content is 45%, and meets following index:
1. heavy metal index: Pb＜1.5ppm As＜1.0ppm Hg＜0.3ppm.
2. sanitary index: bacterial count＜1000/g intestinal bacteria, the mite that lives must not detect, fungi count＜35/g.
Embodiment 3:
A. to the hericium erinaceus fruiting body pre-treatment, get fresh Hericium erinaceus (Bull. Ex Fr.) Pers.,, after fully pulverizing, cross 80 mesh sieves its natural air drying.
B. the raw material after will sieving adds the distilled water of 25 times of quality, stirs and heats up gradually, and the ultrasonic wave with 15KHz in 60 ℃ of scopes is carried out the ultrasonic extraction of 0.8h to it, filters, and filter residue repeats above-mentioned steps, extracts once merging filtrate again.
C. regulate filtrate pH3.5, add earlier the cellulase of 0.9 times of quality, controlled temperature is at 45 ℃, hydrolysis 2.0h, and frozen centrifugation discards residue; Re-adjustment filtrate pH4.5 continues to add the polygalacturonase of 1.2 times of quality in the filtrate, and controlled temperature is at 45 ℃, hydrolysis 1.0h, and frozen centrifugation discards residue; Last re-adjustment filtrate pH4.0 adds the papoid of 1.0 times of quality, and controlled temperature is at 45 ℃, hydrolysis 1.5h, and frozen centrifugation is collected filtrate.
D. regulating pH is 8, adds 30% hydrogen peroxide of 0.8 times of volume then gradually, stirs, and control filtrate temperature is at 45 ℃, and insulation 30min shoals to color.
E. adopt the cross-flow ultrafiltration technology, use ultra-filtration membrane to carry out ultrafiltration, especially use the polysulfone membrane of 5k, pressure is no more than 0.4MPa, and filtrate is divided into two portions, collects the above part of 10000 dalton.
F. the filtrate that operation steps e is obtained is carried out lyophilize, promptly obtains required hericium erinaceum polysaccharide, and through carrying out sealed barrier package (soft packaging, bottled) after the weighing automatically, vanning is at last put in storage.
The detection of polysaccharide content:
Accurately take by weighing hericium erinaceum polysaccharide 10mg and place the 100ml beaker, add the 80ml redistilled water and boil 1 hour postcooling, add the redistilled water constant volume again to 100ml to room temperature.Measure 1.0ml and move in the cuvette, add 5.0% and heavily steam phenol 2.0ml, add vitriol oil 5.0ml again, shake up the back room temperature and place 20min, measure absorbancy at wavelength 490nm place.
The hericium erinaceum polysaccharide of preparation is a pale yellow powder, easily moisture absorption, and soluble in water, content is 47%, and meets following index:
1. heavy metal index: Pb＜1.5ppm As＜1.0ppm Hg＜0.3ppm.
2. sanitary index: bacterial count＜1000/g intestinal bacteria, the mite that lives must not detect, fungi count＜30/g.
Claims (3)
1. the extracting method of a hericium erinaceum polysaccharide, described method comprises:
(1) gets dry Hericium erinaceus (Bull. Ex Fr.) Pers., pulverize, cross 50～120 mesh sieves, get the Hericium erinaceus (Bull. Ex Fr.) Pers. dried powder;
(2) the Hericium erinaceus (Bull. Ex Fr.) Pers. dried powder adds the distilled water of 5～35 times of quality, and 50～80 ℃ following to 8～20KHz ultrasonic extraction 0.5h～2.0h, filters, and gets filtrate 1;
(3) regulating filtrate 1pH is 3～6, add the capacity cellulase, 30～60 ℃ of hydrolysis 1.0h～4.0h, it is 3～6 that centrifuging and taking filtrate 2 is transferred pH, adds the capacity polygalacturonase, 30～60 ℃ of hydrolysis 0.5h～1.5h, it is 3～6 that centrifuging and taking filtrate 3 is transferred pH, adds the capacity papoid, 30～60 ℃ of hydrolysis 1.0h～2.0h, centrifugal, get filtrate 4; Described cellulose enzyme activity is 10000～20000u/g, and described cellulase quality consumption is 0.5～1.0 times of filtrate 1 quality; Described polygalacturonase enzymic activity is 15000～25000u/g, and described polygalacturonase quality consumption is 1.0～1.5 times of filtrate 2 quality; Described papoid enzymic activity is 800000～1000000u/g, and described papoid quality consumption is 0.5～1.5 times of filtrate 3 quality;
(4) regulating filtrate 4pH is 8～9, and adding volume is 20～40% hydrogen peroxide of 0.5～1.0 times of filtrate 4 volume, in 30～50 ℃ of insulated and stirred 0.5h～1h, the ultra-filtration membrane ultrafiltration, collect the above component of molecular weight 10000 dalton, lyophilize obtains hericium erinaceum polysaccharide.
2. the method for claim 1, the extraction that it is characterized in that described step (2) is to repeat to extract for 2 times: the Hericium erinaceus (Bull. Ex Fr.) Pers. dried powder adds the distilled water of 5～35 times of quality, 50～80 ℃ down with 8～20KHz ultrasonic extraction 0.5h～2.0h, filter, get filtrate 1-1, filter residue continues to repeat to extract with 8～20KHz ultrasonic wave down in 50～80 ℃ with the distilled water of 5～35 times of quality, filters, get filtrate 1-2, merging filtrate 1-1 and filtrate 1-2 are filtrate 1.
3. the method for claim 1 is characterized in that described method is as follows:
(1) gets dry Hericium erinaceus (Bull. Ex Fr.) Pers., pulverize, cross 50～120 mesh sieves, get the Hericium erinaceus (Bull. Ex Fr.) Pers. dried powder;
(2) the Hericium erinaceus (Bull. Ex Fr.) Pers. dried powder adds the distilled water of 10～25 times of quality, 50～60 ℃ down with 10～15KHz ultrasonic extraction 0.5h～2.0h, filter, get filtrate 1-1, filter residue adds the distilled water of 10～25 times of quality, and 50～60 ℃ down with 10～15KHz ultrasonic extraction 0.5h～2.0h, filter, get filtrate 1-2, merging filtrate 1-1 and filtrate 1-2 get filtrate 1;
(3) pH of adjusting filtrate 1 is 3.5～4.0, the adding quality is that the enzymic activity of 0.6～0.9 times of filtrate 1 quality is the cellulase of 15000u/g, 40～45 ℃ of hydrolysis 1.0h～2.0h, it is 4.0～4.5 that centrifuging and taking filtrate 2 is transferred pH, the adding quality is that the enzymic activity of 1.0～1.2 times of filtrate 2 quality is the polygalacturonase of 20000u/g, 45～50 ℃ of hydrolysis 1.0h～1.5h, it is 4.0～4.5 that centrifuging and taking filtrate 3 is transferred pH, the adding quality is that the enzymic activity of 0.8～1.2 times of filtrate 3 quality is the papoid of 800000u/g, 45～50 ℃ of hydrolysis 1.2h～1.5h, centrifugal, get filtrate 4;
(4) regulating filtrate 4pH is 8～9, and adding volume is 30% hydrogen peroxide of 0.5～1.0 times of filtrate 4 volume, in 40～45 ℃ of insulated and stirred 0.5h～1h, polysulfone membrane ultrafiltration with 5k, collect the above component of molecular weight 10000 dalton, lyophilize obtains hericium erinaceum polysaccharide.
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