CN107177007A - A kind of preparation method of Auricularia polysaccharide - Google Patents
A kind of preparation method of Auricularia polysaccharide Download PDFInfo
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- CN107177007A CN107177007A CN201710561131.0A CN201710561131A CN107177007A CN 107177007 A CN107177007 A CN 107177007A CN 201710561131 A CN201710561131 A CN 201710561131A CN 107177007 A CN107177007 A CN 107177007A
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- auricularia polysaccharide
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
The invention discloses a kind of preparation method of Auricularia polysaccharide, belong to edible fungus active Component Extraction field.The preparation method is dissolved after agaric is crushed, pH is adjusted after being digested successively with pectase, cellulase and alpha amylase, neutral proteinase is recycled to be digested, carried out after the completion of enzymolysis ultrasonically treated, extraction processing is carried out after ultrasonically treated, clear liquid is separated after extraction, is concentrated, is dried after alcohol precipitation and obtains Auricularia polysaccharide.The present invention considers extraction process cycle, ecological protection, energy consumption, production cost, Product Activity and the factor such as technique is complicated and simple, the preparation method provided have efficiently, crushing effect is good, high income, greatly strengthen the hypolipidemic activity of extract.The present invention prepares the method high income of black fungus extract up to more than 45%, and the lipid-lowering effect of gained Blackfungus polyhexose is greatly enhanced, and blood fat indices reach the difference pole level of signifiance (p<0.01) existing and customary preparation methods, are considerably better than.
Description
Technical field
The invention belongs to edible fungus active Component Extraction field；Specifically related to a kind of preparation method of Auricularia polysaccharide.
Background technology
Black fungus (Auricularia auricula) is the famous dietotherapeutic fungi of China, with higher nutrition and
Medical value, in three provinces in the northeast of China, yield is larger and quality is good, and industry development is rapid.Black fungus has reducing blood lipid, antithrombotic, anti-spoke
Penetrate, anti-mutation, anti-oxidant, anti-aging, it is hypoglycemic, improve immunity, the bioactivity such as anticancer.In recent years, people pass through research
Clear and definite hypertension, high fat of blood are the high main causes of cardiovascular and cerebrovascular disease, can directly result in cerebral infarction, fat
The cardiovascular and cerebrovascular diseases such as liver, cholelithiasis and atherosclerosis coronary cardiopathy, and cardiovascular and cerebrovascular disease is that the mankind are strong
First big killer of health.The current treatment to high fat of blood is based on Western medicine, and side effect is big, therefore develops evident in efficacy, side effect
Low is of great significance with food or the medicine tool puted prevention first.Complicated component in compound preparation for reducing blood fat is also or more
Or rare certain side effect.Therefore developing the real health products or medicine for having blood fat reducing function and having no toxic side effect has
Particularly significant meaning and broad prospect of application.Therefore, black fungus active material (particularly Blackfungus polyhexose) is pushed to industrialization
Production tool is of great significance.
In order to effectively carry out the extraction of black fungus active component and ensure its pharmacological activity, it is necessary to assure extract and purified
Cheng Kexue, prevents from destroying the structure of active component.At present for black fungus active material particularly Polyose extraction and isolating and purifying
And the research in terms of activity is more, up to the present, has been established for hot water extraction, acidity extraction, alkalinity extraction, microwave and carry
Take, ultrasonic wave extraction, enzymatic isolation method extract etc. all multi-methods, the excellent of metrics evaluation extracting method is typically used as using recovery rate and yield
It is bad, although what is had has carried out inhibiting cancer, improved immunity, reducing blood lipid, anti-oxidant isoreactivity experimental study, not yet set up with spy
Fixed active extracting method or the appraisement system of technology as evaluation index.Because the extraction and separation of black fungus active site are pure
The final purpose of change is application, and the optimum extraction technology for obtaining the strong black fungus active component of given activity is that it eats as function
The important prerequisite of product, pharmaceutical raw material source and product development.
The hypolipidemic activity composition Study of current black fungus focuses primarily upon polysaccharide, and for the normal of Blackfungus polyhexose extraction
The shortcoming for advising aqueous extraction-alcohol precipitation technology is as follows：1. yield is relatively low, and cost is higher, and Blackfungus polyhexose extract yield is generally 4~
9%, and mostly laboratory stage.High temperature causes the hypolipidemic activity of active ingredient to reduce when 2. due to extraction, limits its extensive
Using.
In addition, the extracting method of existing Blackfungus polyhexose typically carries out digesting simultaneously using complex enzyme, or in enzymolysis
While carry out assisted extraction using the technology such as ultrasonic wave.For example with cellulase, three kinds of enzymes of pectase and papain
Complex enzyme is made, then is extracted in combination with ultrasonic wave.Although this extracting method results in higher recovery rate, but obtains
The active effect of the Blackfungus polyhexose extract obtained can be under some influence.
Meanwhile, the mostly cake mass for the Blackfungus polyhexose extract that existing extracting method is obtained is, it is necessary to be crushed
Processing, its extract it is water-soluble poor, grown the time required to dissolving, insoluble residue is more.
The content of the invention
To solve in the prior art that Blackfungus polyhexose recovery rate is relatively low, in extraction process active material power loss of tests it is serious,
The technical problems such as the poorly water-soluble of product are obtained, the invention provides a kind of preparation method and application of Auricularia polysaccharide, are taken
Technical scheme it is as follows：
A kind of preparation method of Auricularia polysaccharide of the present invention is carried out in the steps below：
Step 1: black fungus powder is added into citric acid-sodium citrate buffer, mix, obtain black fungus suspension；
Step 2: then adding pectinase enzymatic hydrolysis, enzymolysis liquid A is obtained；
Step 3: adding cellulase degradation, enzymolysis liquid B is obtained；
Step 4: then adding enzymolyzing alpha-amylase, enzymolysis liquid C is obtained；
Step 5: regulation pH value adds neutral protease enzymolysis to neutrality thereafter；
Step 6: after heating up again, ultrasonically treated, added water extraction, and supernatant is taken after centrifugation, concentrate, dried after alcohol precipitation, obtained
Blackfungus polyhexose.
Further limit, black fungus powder described in step one is that the mesh sieve series of 160 mesh~1000 is crossed after black fungus is crushed
, the concentration of the citric acid-sodium citrate buffer is 0.1mol/L, and pH value is 4.8~5.2, citric acid-lemon
Sour sodium cushioning liquid consumption is 40 times~60 times of black fungus powder volume.
Further limit, the addition of pectase described in step 2 is 1%~6% (m/v) of black fungus suspension,
Enzyme activity is 15000U/g, and hydrolysis temperature is 45 DEG C~55 DEG C, and enzymolysis time is 60min~120min.
Further limit, the addition of cellulase described in step 3 is enzymolysis liquid A 1%~3% (m/v)；Enzyme
Vigor is 15000U/g, and hydrolysis temperature is 45 DEG C~55 DEG C, and enzymolysis time is 60min~120min.
Further limit, the addition of alpha-amylase described in step 4 is enzymolysis liquid B 1%~2% (m/v), enzyme
It is 50 DEG C to solve temperature, and enzymolysis time is 60min.
Further limit, the addition of neutral proteinase described in step 5 is enzymolysis liquid C 1%~3% (m/v)；
Enzyme activity is 15000U/g, and hydrolysis temperature is 50 DEG C, and enzymolysis time is 90min.
Further limit, 80 DEG C~90 DEG C be warming up in step 6, ultrasonically treated ultrasonic power is 300W~800W,
The time of ultrasound is 20min~40min.
Further limit, flooding processing is the distilled water for adding 20% volume, stirring and leaching, during extraction in step 6
Between be 2.5h, extraction temperature be 80 DEG C~90 DEG C, during extraction mixing speed be 30r/min.
Further limit, concentration is to be concentrated under reduced pressure in step 6.
Further limit, alcohol precipitation is that high-speed homogenization processing is carried out with the ethanol of 95% (volume) in step 6, ethanol is used
Measure as 3 times of the volume of concentrate；Described homogenate (homogeneous) process：95% ethanol is placed in more than -20 DEG C of refrigerator 4h, added dense
In contracting liquid, refiner rotating speed 10000-15000rpm is homogenized 30s, repeats homogenate 3-4 times；Polysaccharide is fully contacted with ethanol, so that
The comparison for making glycocalix ethanol precipitation fully, solves polysaccharide alcohol precipitation process and disperses uneven, and easily polysaccharide dissolves caused by caking
The bad technical barrier of property.
The present invention considers that extraction process cycle, ecological protection, energy consumption, production cost, Product Activity and technique are complicated and simple
Etc. factor, the preparation method provided have efficiently, crushing effect is good, high income, the reducing blood lipid that greatly strengthen extract is lived
Property.Conbined enzymolysis combining ultrasonic ripple method provided by the present invention prepares the method yield of black fungus extract up to 48.2%,
And use HPLC methods detection gained polysaccharide sample in 90% be 3000Da~10000Da for mean molecule quantity micromolecular polysaccharide,
10% is the macromolecular polysaccharide that mean molecule quantity is more than 70000Da.The effect for reducing blood fat effect of gained Blackfungus polyhexose is through animal
Experimental verification is considerably better than Blackfungus polyhexose prepared by existing and commonsense method.The present invention is exploitation with northeast black fungus extract
Based on blood fat-reducing product carry shelves upgrading lay the foundation, available for reducing blood lipid class health food or medicine, including solid beverage, liquid
Body beverage, soft capsule, hard shell capsules, electuary, tablet etc..
Preparation method provided by the present invention, carries out stepwise discretization using certain enzyme, reuses what ultrasonic wave was extracted
Technique, has used and has been digested simultaneously with complex enzyme in the prior art, the difference that other assisted extraction means such as ultrasonic wave are acted on simultaneously
Method.For solve how high efficiency extraction Blackfungus polyhexose, keep extract activity technical problem there is provided a kind of different think of
Road.
Meanwhile, enzymolysis causes viscosity reduction, and the homogenized of alcohol precipitation step makes polysaccharide good dispersion, therefore adds black wood
The water solubility of fungus polysaccharides, hence it is evident that better than existing method.
The present invention prepares the method high income of black fungus extract up to more than 45%, while the drop blood of gained Blackfungus polyhexose
Fat effect is greatly enhanced, and blood fat indices reach the difference pole level of signifiance (p<0.01), it is considerably better than existing and conventional system
Preparation Method.
Brief description of the drawings
Fig. 1 is that the method for embodiment 1 extracts polysaccharide HPLC collection of illustrative plates；
Fig. 2 is that conventional hot water's extraction method extracts polysaccharide HPLC collection of illustrative plates.
Embodiment
Embodiment 1：A kind of preparation method of Auricularia polysaccharide in present embodiment is carried out in the steps below：
Step 1: the citric acid-sodium citrate buffering that the concentration for being 5.0 by black fungus powder addition pH value is 0.1mol/L is molten
Liquid, above-mentioned cushioning liquid consumption is 40 times of black fungus powder volume, is stirred, and obtains black fungus suspension；
Wherein, the black fungus powder is to be crossed after black fungus is crushed made from 800 mesh sieves；
Step 2: then adding the pectase that enzyme activity is 15000U/g, the addition of pectase is black fungus suspension
4% (m/v), 90min is digested under 50 DEG C of water-baths, obtains enzymolysis liquid A；
Step 3: adding the cellulase that enzyme activity is 15000U/g, the addition of cellulase is enzymolysis liquid A's
2% (m/v), 90min is digested under 50 DEG C of water-baths, obtains enzymolysis liquid B；
Step 4: then adding the alpha-amylase that enzyme activity is 15000U/g, the addition of alpha-amylase is enzymolysis liquid B's
1.5% (m/v), 60min is digested under 50 DEG C of water-baths, obtains enzymolysis liquid C；
Step 5: regulation pH value adds the neutral proteinase that enzyme activity is 15000U/g, neutral proteinase to neutrality thereafter
Addition is enzymolysis liquid C 2% (m/v), and 90min is digested under 50 DEG C of water-baths；
Step 6: 80 DEG C after heating up again, the ultrasonically treated 30min under conditions of ultrasonic power 596W, plus 20% volume
Distilled water, temperature be 86 DEG C, mixing speed be to extract 2.5h, centrifugation under the conditions of 30r/min after take supernatant, be concentrated under reduced pressure on
Solid content is 3%~4% in clear liquid, and the ethanol for adding 3 times of volumes 95% carries out alcohol precipitation using high-speed homogenization processing, 80
2h~4h is dried at DEG C, Blackfungus polyhexose is obtained；
(homogeneous) process is homogenized wherein described in step 6：95% ethanol is placed in -20 DEG C of refrigerator 4h, concentrate is added
In, refiner rotating speed 12000rpm is homogenized 30s, repeats homogenate 3-4 times.
The yield of the Blackfungus polyhexose of the present embodiment method is 48.2%.
Using the purity of Phenol sulfuric acid procedure Detection and Extraction polysaccharide, it is Y=to obtain regression equation by Standard glucose solution
0.6953X-0.0275, glucose standard curve equation of linear regression regression coefficient R>0.999.The extracted black fungus of detection is more
Sugared purity is 37.8%.
The present embodiment method is extracted into polysaccharide HPLC collection of illustrative plates (Fig. 1) and conventional hot water's extraction method extraction polysaccharide HPLC collection of illustrative plates
(Fig. 2) is contrasted, it is known that, the molecular weight of the method for embodiment 1 is substantially reduced.
Comparative example 1, difference from Example 1 is：Pectase, cellulase and alpha-amylase are together added to black
Enzymolysis processing is carried out in agaric suspension, hydrolysis temperature is 50 DEG C, and enzymolysis time is 90min, other steps and parameter and implementation
Example 1 is identical.
The yield of the Blackfungus polyhexose of the method for comparative example 1 is 33.8%.
Comparative example 2, difference from Example 1 is：Cellulase degradation is first carried out, then carries out pectinase enzymatic hydrolysis, most
Carry out enzymolyzing alpha-amylase and neutral protease enzymolysis successively afterwards, other steps and parameter are same as Example 1.
The yield of the Blackfungus polyhexose of the method for comparative example 2 is 27.5%.
Comparative example 3, difference from Example 1 is：Enzymolysis process synchronizes enzymolysis using complex enzyme, is used
Complex enzyme be by cellulase, pectase and papain according to weight ratio be cellulase：Pectase：Papain=
5:4:2 ratio carries out the complex enzyme of mixing acquisition, and the addition of enzyme is 4.5%, and hydrolysis temperature is 45 DEG C, and enzymolysis time is
140min, other steps and parameter are same as Example 1.
The yield of the Blackfungus polyhexose of the method for comparative example 3 is 17.3%.
The difference from Example 1 of comparative example 4 is：Enzymolysis process synchronizes enzymolysis using complex enzyme, is used
Complex enzyme be by cellulase, pectase and papain according to weight ratio be cellulase：Pectase：Papain=
5:4:2 ratio carries out the complex enzyme of mixing acquisition, and the addition of enzyme is 4.5%, and hydrolysis temperature is 45 DEG C, and enzymolysis time is
140min.While enzymolysis, extracted using ultrasonic wave, the frequency of ultrasonic wave is 20kHz, and ultrasonic power is 20W, other
Step and parameter are same as Example 1.
The yield of the Blackfungus polyhexose of the method for comparative example 4 is 19.7%.
Comparative example 5：The technical scheme taken and existing patent：A kind of integrated extraction technique of Auricularia polysaccharide, application number
Carried out for the scheme of the embodiment 2 described in 201110326114.1 specifications, difference is Auricularia extract 1 and agaric
Extract 2 does not have packaging to weigh after mixing.
The combined extraction method of the Auricularia polysaccharide is：
1st, agaric dries fructification, picks out impurity, weighs inventory, is eluriated with water and removes silt, then adds 20 times of amounts
Water, is heated to 50 degree and soaks 8 hours.Go round and round a millstone and starch post.
2nd, with fiberizer by the immersion fully agaric of expansion and water together defibrination, slurries are uniform.Turn over filter post.
3rd, press filtering machine is first filled into 80 mesh steel meshes, then extruding filtering changes the extruding filtering of 150 mesh steel meshes, separate filter residue
And filtrate, filtrate decompression concentration, filter residue turn extraction post.
3.1 filtrate decompressions are concentrated：
Vavuum pump and circulating water pump are opened, when vacuum rises to 0.04MPa, extract solution is added extremely into evaporator
Liquid level continues to vacuumize in the middle part of lower visor, when vacuum is to 0.08MPa, opens steam cock, and control vacuum exists
Between 0.08-0.09MPa, between 65-85 DEG C of temperature, liquid level is kept in the range of visor.
When liquid concentration to proportion is 1.60 (60 DEG C), quick filtration (mesh of aperture 80) turns vacuum drying post.
The vacuum drying of 3.2 Auricularia polysaccharides
During the vacuum drying of Auricularia polysaccharide, vacuum pressure control is 0.09-0.10Mpa, and temperature is 75 degree, is dried to moisture
Weight percentage be less than 6%, take around 11 hours.
3.3, by dried Auricularia polysaccharide, are crushed to more than 80 mesh with 100 eye mesh screen pulverizers, obtain Auricularia polysaccharide 1,
Extract yield is 35%, and the weight percentage of polysaccharide is the 10% (spirit with reference to described in Chinese Pharmacopoeia 2010 edition one after testing
Sesame or the polysaccharide detection method changed under sealwort are tested), turn mixing post.
4th, the enzymolysis ultrasonic wave extraction of agaric filter residue
In agaric filter residue input extractor, add water 50 times and measure, ultrasonic frequency 20kHz, ultrasonic power 20W, enzymolysis temperature
45 DEG C of degree, pH value 4.5, time 150min, enzyme concentration is 3%, and enzyme is the mixing of cellulase, pectase and papain
Enzyme, the part by weight of three is 5: 4: 2, and enzymolysis terminal is warming up to 100 degree, and the enzyme that goes out is extracted 1 hour, filters extract solution.
The vacuum concentration of 4.1 agaric filter residue extract solutions
Vavuum pump and circulating water pump are opened, when vacuum rises to 0.04MPa, extract solution is added extremely into evaporator
Liquid level continues to vacuumize in the middle part of lower visor, when vacuum is to 0.08MPa, opens steam cock, and control vacuum exists
Between 0.08-0.09MPa, between 65-85 DEG C of temperature, liquid level is kept in the range of visor.
When liquid concentration to proportion is 1.50 or so (60 DEG C), quick filtration (mesh of aperture 80), filtrate puts spray drying
In basin, in case spray drying is used.
4.2 spray drying, EAT control is at 175 ± 3 DEG C, and leaving air temp is controlled at 75 ± 3 DEG C.Collect Auricularia polysaccharide
Extract, obtains Auricularia extract 2, i.e. Auricularia polysaccharide 2.It is fitted into PE bags, extract yield is 10% or so, after testing polysaccharide
Weight percentage is 15% (ganoderma lucidum or the sealwort lower polysaccharide detection method with reference to described in Chinese Pharmacopoeia 2010 edition one
Test).
5th, batch mixed, by Auricularia polysaccharide 1 and Auricularia polysaccharide 2, puts into mixer, all well mixed, after testing black wood
The yield of fungus polysaccharides is 39.3%.
Comparative example 6 uses traditional water extraction, i.e., extracted according to procedure below：Pretreatment of raw material agaric → agaric leaching
Bubble (40 times of water, 50 DEG C of immersion 8h) → → hot bath (86 DEG C, 2.5h) → cooling → 10000r/min is centrifuged → takes supernatant plus 3
Times ethanol of volume 95% → stand overnight → 10000r/min centrifugations → Thick many candies；The yield of Blackfungus polyhexose is after testing
9.23%.
Comparative example 7 uses ultrasonic method, i.e., extracted according to procedure below：Pretreatment of raw material agaric → agaric immersion (40
Times water, 50 DEG C of immersion 8h) → ultrasonic wave extraction (parameter is same as Example 1) → hot bath (86 DEG C, 2.5h) → cooling →
10000r/min is centrifuged, and → taking supernatant plus 3 times of ethanol of volume 95% → stands overnight → 10000r/min centrifugations → Thick many candies；
The yield of Blackfungus polyhexose is 14.83% after testing.
Blackfungus polyhexose carries out mouse auxiliary lipid-lowering test checking invention effect.
1. experimental animal and rearing conditions
Male SD rat is provided by Shanghai Slac Experimental Animal Co., Ltd., and production licence number is SCXK
(Shanghai) 2012-0002, animal quality certification number is 0214188, SPF grades, and the weight of animals is 160-180g when buying.Drinking water is to go out
Two grades of ultra-pure waters of bacterium, drinking water quality meets National Standard of the People's Republic of China《Standards for drinking water quality》(GB5749-
2006) regulation.Experimental animal room is SYXK (Zhejiang) 2015-0008, feeding environment using credit number：Temperature range 20~25
DEG C, RH range 40~70%.Adapted to 6 days in Animal House environment before rat test.
Blood lipids index detection kit：
T-CHOL determines kit (enzymatic reagent method)：Manufacturer：Roche, article No.：03039773-190, lot number：
115474-01.Triglyceride determination kit (enzymatic reagent method)：Manufacturer：Roche, article No.：20767107-322, lot number：
122155-01.HDL-C determines kit：Manufacturer：Roche, article No.：04399803-190, lot number：
615016-01.LDL-C determines kit：Manufacturer：Roche, article No.：03038866-322, lot number：
614869-01。
The proving test of model is analyzed with the software processings of Design-Expert 8.0.Reducing blood lipid zoopery institute total
According to this mean+SD () represent, variance analysis and statistical test are using Sun Ruiyuan etc.：In DAS statistical softwares
Multigroup mean analysis.Homogeneity test of variance first is carried out to data, if variance is neat, overall ratio is carried out using one-way analysis of variance
Compared with being counted with the comparative approach two-by-two of mean between multiple dosage groups and a control group mean；To abnormal or variance not
Neat data are used rank test instead and counted.P<0.05 is considered as notable in 0.05 level difference；P<0.01 is considered as
0.01 level difference is notable.
2. detection process
Experiment sets experimental group, model control group and blank control group.Blank control group gives maintenance feed, model control group
And experimental group gives model feed.
Modeling：Experiment is using in state of State Food and Drug Administration food medicine prison [2012] No. 107 literary annexes 6 of guarantorization
Combined hyperlipidemia familial animal model.In feeding 20d, the equal non-fasting intraocular corner of the eyes blood sampling of blank control group, animal pattern group rat,
Serum is separated after blood sampling, serum TC, TG, HDL-C, LDL-C level are determined with automatic clinical chemistry analyzer.It will be made according to TC levels
The successful rat of mould is randomly divided into experimental group and model control group, every group 10, each sample group and model control group ratio after packet
Compared with TC, TG, LDL-C, HDL-C difference, there are no significant.
Given the test agent is given：Experimental group rat oral gavage gives corresponding sample liquid and purified water, once a day, continuously
60d.During experiment, blank control group gives maintenance feed, and model control group and each experimental group give model feed.
Indexs measure：In giving high lipid food 60d, the blood sampling of the rat non-fasting intraocular corner of the eyes separates serum after blood sampling, with full-automatic
Biochemical Analyzer determines serum TC, TG, HDL-C, LDL-C level.
3. experimental result
After testing, gained testing result is as shown in table 1：
The Auricularia polysaccharide extract of table 1 to hyperlipidemia ratses lipid (N=10) mmolL-1
Note：Compared with blank groupaP<0.01,bP<0.05；Compared with model group,cP<0.01,dP<0.05。
As known from Table 1, in the Blackfungus polyhexose enzymatic isolation method extraction process in the present invention enzyme-added species and enzyme-added order for
The hypolipidemic activity of extract is most important, such as the T-CHOL of embodiment 1, triglycerides, HDL-C and
LDL-C content is with the poor heteropole of model group significantly (p<0.01), and enzyme class is constant, simply become mixed
Conjunction is handled, then lipid-lowering effect is substantially reduced, such as comparative example 1；It is first to carry out and the enzymolysis of comparative example 2 order is different from embodiment 1
Cellulase degradation, then pectinase enzymatic hydrolysis are carried out, enzymolyzing alpha-amylase and neutral protease enzymolysis are finally carried out successively, then it is produced
The hypolipidemic activity of thing is also reduced；Comparative example 3 and 4 is the complex enzyme being made up of cellulase, pectase and papain,
Although comparative example 4 also passes through ultrasonication, the yield of the two and the hypolipidemic activity of product are not as the effect of the present invention
It is really good；In comparative example 5, although yield is higher, but its hypolipidemic activity can not show a candle to effect of the present invention；And traditional water extraction and super
The hypolipidemic activity that sonic method obtains polysaccharide is general, be not as good as effect of the present invention.Therefore find that the present invention is not by repeatedly contrast
The polysaccharide of high yield is only obtained, and with extremely strong hypolipidemic activity, the present invention is with fabulous application value in this regard.
Dissolubility to the Auricularia polysaccharide extract prepared by embodiment 1 and comparative example 1-6 is determined, measure side
Method is：
Extract dissolution time is determined：100mg samples are weighed, deionized water 50mL are added, with certain rotating speed in constant temperature
Stir, recorded since stirring to the time (s) required for the whole dispersing and dissolvings of powder agglomates on magnetic stirring apparatus.
The determination of residual amount after extract dissolving：Accurate weighing sample 100mg, is placed in small beaker, adds deionized water extremely
With magnetic stirrer 10min under 50mL, room temperature condition, fully dissolving is allowed to.Solution is transferred in centrifuge tube completely,
3000r/min centrifuges 10min, takes precipitation, and 105 DEG C of drying are to constant, and this is undissolved residual quantity.
Dissolubility determines (meltage)：1 subtract residual quantity percentage then be meltage percentage.
Measurement result such as table 2 shows.
The dissolubility of the Auricularia polysaccharide extract of table 2
| Dissolubility (is evaluated, meltage %) | Dissolution time (s) | Residual quantity (%) | |
| Embodiment 1 | It is good, 99.9 | 55 | 0.10 |
| Comparative example 1 | Typically, 94.22 | 250 | 5.78 |
| Comparative example 2 | Typically, 94.11 | 265 | 5.89 |
| Comparative example 3 | Typically, 94.48 | 260 | 5.52 |
| Comparative example 4 | Typically, 93.20 | 265 | 6.80 |
| Comparative example 5 | Typically, 94.44 | 235 | 5.56 |
| Comparative example 6 | Difference, 91.68 | 360 | 9.32 |
From table 2, the dissolubility for the black fungus extract that patent of the present invention is obtained is greatly enhanced, and dissolution time shortens 5
More than times.This using so that the molecular weight of product is reduced by the enzymolysis order of science and alpha-amylase that be due to several enzymes,
Dissolubility is greatly enhanced, and makes polysaccharide alcohol precipitation process scattered evenly with cold ethanol homogenized step during alcohol precipitation, is directly obtained
Powdered polysaccharide, polysaccharide dissolubility is good, is more beneficial for development and application.
Embodiment 2：The present embodiment difference from Example 1 is：The hydrolysis temperature of neutral proteinase is 50 DEG C, other
The hydrolysis temperature of two kinds of enzymes is 45 DEG C, and enzymolysis time is 120min, and other steps and parameter are same as Example 1.After testing,
The yield of the Blackfungus polyhexose of acquisition is 48.1%, and dissolubility is 99.79%.
Embodiment 3：The present embodiment difference from Example 1 is：Present embodiments provide for a kind of Blackfungus polyhexose
Extracting method, the technical scheme difference from Example 1 taken is：The hydrolysis temperature of neutral proteinase is 50 DEG C,
The hydrolysis temperature of other two kinds of enzymes is 55 DEG C, and enzymolysis time is 120min, and other steps and parameter are same as Example 1.Through
Detection, the yield of the Blackfungus polyhexose of acquisition is 47.8%, and dissolubility is 99.38%.
Embodiment 3：The present embodiment difference from Example 1 is：Ultrasonically treated condition is：Ultrasonic power 300W, place
Reason time 40min, other steps and parameter are same as Example 1.After testing, the yield of the Blackfungus polyhexose of acquisition is
47.9%, dissolubility is 99.67%.
Claims (10)
1. a kind of preparation method of Auricularia polysaccharide, it is characterised in that the preparation method of Auricularia polysaccharide is carried out in the steps below：
Step 1: black fungus powder is added into citric acid-sodium citrate buffer, mix, obtain black fungus suspension；
Step 2: then adding pectinase enzymatic hydrolysis, enzymolysis liquid A is obtained；
Step 3: adding cellulase degradation, enzymolysis liquid B is obtained；
Step 4: then adding enzymolyzing alpha-amylase, enzymolysis liquid C is obtained；
Step 5: regulation pH value adds neutral protease enzymolysis to neutrality thereafter；
Step 6: after heating up again, ultrasonically treated, added water extraction, and supernatant is taken after centrifugation, concentrate, dried after alcohol precipitation, obtain black wood
Fungus polysaccharides.
2. a kind of preparation method of Auricularia polysaccharide according to claim 1, it is characterised in that black fungus powder described in step one
It is to be crossed after black fungus is crushed made from the mesh sieve of 160 mesh~1000, the concentration of the citric acid-sodium citrate buffer is
0.1mol/L, and pH value is 4.8~5.2, citric acid-sodium citrate buffer consumption is black fungus powder volume 40 times~60
Times.
3. a kind of preparation method of Auricularia polysaccharide according to claim 1, it is characterised in that pectase described in step 2
Addition is 1%~6% (m/v) of black fungus suspension, and enzyme activity is 15000U/g, hydrolysis temperature is 45 DEG C~55 DEG C, enzyme
The solution time is 60min~120min.
4. a kind of preparation method of Auricularia polysaccharide according to claim 1, it is characterised in that cellulase described in step 3
Addition be enzymolysis liquid A 1%~3% (m/v)；Enzyme activity is 15000U/g, and hydrolysis temperature is 45 DEG C~55 DEG C, during enzymolysis
Between be 60min~120min.
5. a kind of preparation method of Auricularia polysaccharide according to claim 1, it is characterised in that alphalise starch described in step 4
The addition of enzyme is enzymolysis liquid B 1%~2% (m/v), and hydrolysis temperature is 50 DEG C, and enzymolysis time is 60min.
6. a kind of preparation method of Auricularia polysaccharide according to claim 1, it is characterised in that neutral egg described in step 5
1%~3% (m/v) that the addition of white enzyme is enzymolysis liquid C；Enzyme activity is 15000U/g, and hydrolysis temperature is 50 DEG C, enzymolysis time
For 90min.
7. the preparation method of a kind of Auricularia polysaccharide according to claim 1, it is characterised in that 80 DEG C are warming up in step 6
~90 DEG C, ultrasonically treated ultrasonic power is 300W~800W, and the ultrasonic time is 20min~40min.
8. the preparation method of a kind of Auricularia polysaccharide according to claim 1, it is characterised in that flooding is handled in step 6
It is the distilled water for adding 20% volume, stirring and leaching, extraction time is 2.5h, extraction temperature is 80 DEG C~90 DEG C, is stirred during extraction
Speed is mixed for 30r/min.
9. the preparation method of a kind of Auricularia polysaccharide according to claim 1, it is characterised in that concentration is decompression in step 6
Concentration.
10. the preparation method of a kind of Auricularia polysaccharide according to claim 1, it is characterised in that alcohol precipitation is to use in step 6
The ethanol of 95% (volume) carries out high-speed homogenization processing, and ethanol consumption is 3 times of the volume of concentrate；Described homogenate (homogeneous) mistake
Journey：95% ethanol is placed in more than -20 DEG C of refrigerator 4h, added in concentrate, refiner rotating speed 10000-15000rpm, homogenate
30s, repeats homogenate 3-4 times.
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