CN106905440B - A kind of method that tremella polysaccharides extract - Google Patents
A kind of method that tremella polysaccharides extract Download PDFInfo
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Abstract
The invention discloses the new methods that a kind of tremella polysaccharides extract: the tremella dry product of selection, is pulverized, after ethanol decolorization removal of impurities, is extracted using water at low temperature, obtain the polysaccharide leaching liquor and cold water-insoluble tremella residue of cold water solubles；After the polysaccharide leaching liquor concentration of cold water solubles, sevage reagent is added and removes deproteinized, quaternary ammonium salt solution is added to complex-precipitation, sediment is dissolved in sodium chloride solution, it removes insoluble matter and obtains water soluble polymer amount tremella polysaccharides after concentration, alcohol precipitation, centrifugation, washing and drying, it can make natural moisture preserving material or substitution hyaluronic acid, be used for cosmetics and biomedicine field；Cold water-insoluble tremella residue still contains a large amount of hot water soluble polysaccharide and insoluble colloid polysaccharide and obtains powdered tremella residue after dry and crushing, can make the additive of health food, functional food or ordinary food.The present invention optimizes cold water solubles and the insoluble polysaccharide of cold water using ultramicro grinding and low temperature flooding combination technique.
Description
Technical field
The present invention relates to a kind of extracting method of edible fungi polysaccharide, the method that especially a kind of tremella polysaccharides extract belongs to
Edible mushroom deep process technology field.
Background technique
Tremella also known as tremella, tremella etc. are a kind of dual-purpose of drug and food bacterium full of nutrition, economic value is high, have enriching yin,
Moistening lung, nourishing the stomach are promoted the production of body fluid and other effects, belong to rare tonic in ancient times.Tremella polysaccharides are the main actives of tremella, are not only had
The physiological actions such as excellent raising human immunologic function, antiviral and antitumor, antiulcer, reducing blood lipid, hypoglycemic, norcholesterol,
And water-retaining property with super strength and good film forming, it applies as natural moisturizer and is widely weighed in cosmetic field
Depending on.Currently, most of tremella available on the market is crude tremella dry product, nutritional ingredient utilization rate is low, and product is attached
Value added small, the economic value and market potential of tremella can not embody.Therefore, tremella resource how is rationally utilized, research tremella is more
The highly effective extraction method of sugar is health food, functional food, cosmetics, biology doctor using the technological means of more economical material benefit
The industries such as medicine provide the tremella polysaccharides of high-quality, are the developing direction of tremella intensive processing, have a vast market foreground.
Tremella polysaccharides are made of as a kind of high-molecular compound, main chain mannose with α-(1-3) glucosides key connection, main
The 2 of chain are connected with the side chain of the residues such as glucose, xylose, fucose and common uronic acid composition on 4,6, are a kind of molecules
Structure is complicated and huge glucide.Currently, the research of edible fungi polysaccharide and the report utilized are increasing, the biology of polysaccharide
Active and moistening effect has very big correlation with its molecular size range, and such as: (1) molecular weight has in 3~5kDa or so polysaccharide
Reduce the effect of blood glucose；(2) molecular weight polysaccharide between 10~100kDa, has the function of anti-inflammatory；(3) molecular weight is in 30kDa
The above polysaccharide has antineoplastic action；(4) molecular weight polysaccharide between 1,000~5,000kDa has superpower water-retaining property
Can, but its most important condition to play a role is dissolved in water.Therefore, the tremella polysaccharides of suitable molecular weight are selected and improve tremella polysaccharides
Water solubility can increase the bioactivity and moistening effect of tremella polysaccharides, led in particular for cosmetics and biological medicine
Domain.
The preparation of tremella polysaccharides first extracts Thick many candies from Tremella fructification generally using the method extracted and refined, then
Purification is carried out to the Thick many candies of acquisition.Traditional extraction process is Hot water extraction, is mentioned primarily directed to exocellular polysaccharide
Take, and the extracted amount of cell wall polysaccharide be it is very limited, that there is recovery rates is low, time-consuming, causes the problems such as wasting of resources.Closely
Nian Lai, researcher is using zymohydrolysis extracting method, ultrasonic extraction, microwave loss mechanisms, ultramicro grinding method and Subcritical Water Extraction
The emerging technologies such as method all improve the recovery rate of tremella polysaccharides to a certain extent.Such as: Chinese patent (application number
201410546381.3) a kind of production method that tremella polysaccharides are extracted using Subcritical Water Extraction-hot water extraction two-step method, the party
The method first step uses the Subcritical Water Extraction tremella polysaccharides of lower solid-to-liquid ratio, has good infiltration and dissolution using subcritical water
Ability makes cell wall polysaccharide come into full contact with and dissolve out with subcritical water, and second step is using sufficiently molten compared with the deionized water of high solid-liquid ratio
Solution and leaching tremella polysaccharides, while there is very high thermal energy using subcritical water, the extraction temperature of heating deionized water improves silver
The extraction yield of fungus polysaccharides.The side of a kind of tremella intracellular polyse separation of Chinese patent (application number 201410796766.5), purifying
Method, this method obtain tremella polysaccharides crude extract using enzymatic hydrolysis, ultrasonic wave extraction, ethanol precipitation, tremella polysaccharides crude extract using
The tremella polysaccharides that ultrasonic wave dissolution, D101 resin column chromatography, DEAE celluosic resin are separated, purified；Chinese patent (application
Number 201510336334.0) preparation method of tremella polysaccharides, this method using pulverize, ultrasonic wave auxiliary hot water extracts, second
Alcohol precipitating, Sevage reagent take off albumen, hydrogen peroxide for decoloration, freeze-drying, obtain required tremella polysaccharides.However, tremella polysaccharides
Molecular size range and its it is water-soluble have biggish relationship with its physicochemical property and bioactivity, tremella polysaccharides extraction and
There are no by due attention in subtractive process.Since the molecular weight distribution of tremella polysaccharides is very wide, even if using identical silver
The difference of ear raw material, extraction or refining methd, physicochemical property and bioactivity can also generate biggish difference, directly influence
Yield, quality and the application value of tremella polysaccharides.
Summary of the invention
The purpose of the present invention is overcoming the deficiencies of the prior art and provide a kind of method that tremella polysaccharides extract, i.e., with ultra micro
The tremella of crushing is that raw material is extracted the tremella polysaccharides of cold water solubles, further isolated and purified, obtained using water at low temperature microwave leaching technology
Water soluble polymer amount tremella polysaccharides, be used for cosmetics and biomedicine field, pulverize and low temperature flooding after
Tremella residue, still contain a large amount of hot water soluble polysaccharide and insoluble colloid polysaccharide, be used as health food, functional food or common
The additive of food plays their use effect to the maximum extent, realizes the high efficiency extraction and comprehensive utilization of tremella, improves silver
The economic benefit and utility value of ear deep processing.
The technical solution of the present invention is as follows: a kind of method that tremella polysaccharides extract, it is as follows that the method comprising the steps of:
(1) it pulverizes: selecting clean disease-free tremella dry product, after coarse powder first is made, then continued with micronizer
It crushes, obtains tremella Ultramicro-powder；
(2) decoloration removal of impurities: by tremella Ultramicro-powder obtained by step (1), being added 70%wt~100%wt ethyl alcohol and extracted,
Filtering removes polyphenol, fat, pigment small molecular weight impurity, obtains the tremella Ultramicro-powder of decoloration removal of impurities；
(3) low temperature extracts: by tremella Ultramicro-powder obtained by step (2), deionized water is added and carries out low temperature extraction, extraction temperature
≤ 40 DEG C, centrifugation obtains the polysaccharide leaching liquor and cold water-insoluble tremella residue of cold water solubles；
(4) deproteinization: polysaccharide leaching liquor obtained by step (3) is concentrated, and sevage reagent is added in concentrate, is filled
Divide stirring, stratification collects upper layer polysaccharide solution；
(5) quaternary ammonium salt solution purification: is added to complex-precipitation, sediment dissolution in polysaccharide solution obtained by step (4)
In sodium chloride solution, insoluble matter is removed, supernatant is concentrated into polysaccharide and obtains after alcohol precipitation, centrifugation, washing, drying close to saturation
To water soluble polymer amount tremella polysaccharides；
(6) residue utilizes: by tremella residue obtained by step (3), dry and crushing obtains powdered tremella residue.
The average grain diameter of tremella Ultramicro-powder described in step (1) >=600 mesh.
The mass volume ratio of tremella Ultramicro-powder described in step (2) and ethyl alcohol is 1:3~10, extraction time 2~for 24 hours,
Extracting times 1~3 time.
Tremella Ultramicro-powder described in step (3) and the mass volume ratio of deionized water are 1:40~100, extraction time 4
~48h, extracting times 1~3 time.
Sevage reagent described in step (4) is chloroform: n-butanol volume ratio is 4~5:1 mixed liquor, and additional amount is
1/4~3/4 times of the polysaccharide volume of the concentrated liquid discards middle protein layer, recycles lower layer sevage reagent, and it is molten to collect upper layer polysaccharide
Liquid, repetitive operation 2-4 times.
Quaternary ammonium salt described in step (5) is cetylpyridinium chloride, brocide or cetyl three
One of methyl bromide ammonium.
The concentration of the quaternary ammonium salt solution is 1%wt~10%wt.
Concentration of sodium chloride solution described in step (5) is 0.4~2mol/L, and it is molten that sediment is dissolved in >=40 DEG C of sodium chloride
It is dissociated in liquid.
Alcohol precipitation described in step (5), centrifugation, washing, it is dry be polysaccharide solution be added the 95%wt of 2~4 times of volumes~
100%wt ethyl alcohol, stratification are centrifuged, after precipitates washed with EtOH 1~2 time, vacuum drying or freeze-drying.
The utility model has the advantages that
1, the present invention optimizes cold water solubles and the insoluble polysaccharide of cold water using ultramicro grinding and low temperature flooding combined technology
Application field, cold water solubles polysaccharide molecular weight is mainly distributed on 2, between 000~4,000kDa, as natural moisture preserving material or
Hyaluronic acid is substituted, is used for cosmetics and biomedicine field, the insoluble polysaccharide molecular weight of cold water is mainly distributed on 200~
Between 500kDa, as the additive of health food, functional food or ordinary food, makes the best use of everything, play it to the maximum extent
Use effect.
2, Tremella fructification of the present invention be concentrated by ultramicros grinding, ethanol decolorization removal of impurities, low temperature flooding, leaching liquor,
After Sevage reagent takes off albumen, quaternary salt deposit purification, ethanol precipitation, solvent washing and vacuum drying, obtained high water solubility
Molecular weight tremella polysaccharides, extracting factor is mild, the structure of survivable polysaccharide, transparent as natural moisture preserving material or substitution
Matter acid, meets the quality requirement of medicine and cosmetic field, is a kind of tremella intensive processing product of high added value.
3, for Tremella fructification of the present invention after ultramicro grinding, ethanol decolorization removal of impurities, low temperature flooding, obtained tremella is residual
Slag still contains a large amount of hot water soluble polysaccharide and insoluble colloid polysaccharide, and by pulverizing, cell wall is broken, it is easier to
It digested, absorb and utilizes by human body, there is certain health-care efficacy and nutritive value, do not use poisonous and hazardous chemical solution
Agent meets food production requirement, the additive as health food, functional food or ordinary food, realizes efficiently mentioning for tremella
It takes and comprehensively utilizes, improve the economic benefit and utility value of tremella deep processing.
Specific embodiment
The present invention will be further described in detail below with reference to the embodiments, but they are not limitation of the present invention.
Determination of polysaccharide of the invention is measured using phenol-sulfuric acid and colorimetric method, and polysaccharide molecular weight measurement is seeped using gel
Saturating chromatography (GPC) measurement.
Embodiment 1
Clean disease-free tremella dry product is selected, is placed in common grinder and is crushed to 40 mesh, then pulverized with air-flow
Machine continues to be crushed to 800 mesh, obtains tremella Ultramicro-powder.Tremella Ultramicro-powder 20g is weighed, 90% ethanol solution of 200mL is added, often
Temperature extraction 12h, filtering discard the leaching liquor containing impurity such as polyphenol, fat, pigments, tremella residue add 2000mL go from
Sub- water, room temperature extract for 24 hours, coarse filtration, and 10,000r/min centrifugation 10min obtain tremella residue and leaching liquor.Leaching liquor decompression is dense
It is reduced to the concentrate that polysaccharide concentration is 5%, is added 3/4 volume sevage reagent (chloroform: n-butanol=4:1), is sufficiently stirred, it is quiet
Layering is set, middle protein layer is discarded, lower layer sevage reagent is recycled, repetitive operation 3 times, collects upper layer polysaccharide solution.Polysaccharide is molten
2% cetylpyridinium chloride solution is added to precipitating completely in liquid, and stratification, 10,000r/min centrifugation 10min, it is heavy to collect
2mol/L sodium chloride solution is added in starch, and under the conditions of 50 DEG C after stirring dissociation, 10,000r/min centrifugation 10min are removed not
Molten object, supernatant is concentrated under reduced pressure into polysaccharide close to saturation, is slowly added to dehydrated alcohol, stirring while adding, until ethanol content reaches
70%, stratification, 10,000r/min centrifugation 10min remove supernatant, collect polysaccharide precipitation object, wash 2 with dehydrated alcohol
Secondary, vacuum freeze drying is ground, and the high molecular weight tremella polysaccharides of 1.96g white powder are made, and polyoses content is
92.2%, polysaccharide yield 9.0%.Tremella residue drains, and 50 DEG C of vacuum drying, pulverizer crushes to get the powdered silver of 13.8g
Gluten elasticity, enhancing gluten moisture holding capacity can be improved as the additive of Flour product in ear residue, polyoses content 24.2%.
Embodiment 2
1 tremella Ultramicro-powder 100kg of embodiment is weighed, 80% ethanol solution of 400L is added, stirring at normal temperature extracts 4h, mistake
Filter, residue repeat extraction 1 time, discard the leaching liquor containing impurity such as polyphenol, fat, pigments, residue adds 5000L deionization
Water, stirring at normal temperature extract 12h, and filter-cloth filtering sets the separation of 5000r/min centrifuge, and residue repeats extraction 1 time, it is residual to obtain tremella
Slag and leaching liquor.Sevage reagent (the chloroform: just of 1/4 volume is added in the concentrate that leaching liquor nanofiltration is 5% to polysaccharide concentration
Butanol=4:1), it is sufficiently stirred, stratification, discards middle protein layer, recycle lower layer sevage reagent, repetitive operation 4 times, receive
Collect upper layer polysaccharide solution.1% cetyl trimethylammonium bromide solution is added to precipitating completely in polysaccharide solution, and stratification is set
The separation of 5000r/min centrifuge, collects sediment, and 1mol/L sodium chloride solution is added and sets under the conditions of 40 DEG C after stirring and dissolving
The separation of 5000r/min centrifuge removes insoluble matter, and supernatant nanofiltration is concentrated into polysaccharide close to saturation, is slowly added to 95% ethyl alcohol,
Stirring while adding, until precipitating is completely, stratification sets the separation of 5000r/min centrifuge, removes supernatant, collects polysaccharide
Sediment is washed 1 time with 95% ethyl alcohol and dehydrated alcohol respectively, and vacuum freeze drying is ground, and 10.12kg white is made
Powdered high molecular weight tremella polysaccharides, polyoses content 92.8%, polysaccharide yield 9.4%.Tremella residue drains, vacuum is dry
It is dry, pulverize and sieve to get the powdered tremella residue of 68.5kg, polyoses content 25.2%, according to beverage factory formula and technique
It is required that add suitable auxiliary material, such as milk powder, white granulated sugar, acid and stabilizer auxiliary material, through mixing, homogeneous, filling and sterilization,
After cooling, the tremella protein beverage with nutrition and health care effect can be made into.
Comparative example 3
The preparation method of Chinese patent (application number 201510336334.0) tremella polysaccharides: select embodiment 1 clean disease-free
Harmful tremella dry product is placed in common grinder and is crushed to 80 mesh, then is continued to be crushed to 200 mesh with micronizer, obtains silver
Ear Ultramicro-powder.Tremella Ultramicro-powder 50g is weighed, deionized water 3000mL is added, stirs evenly, in ultrasound works frequency 40KHz item
Interval 2 times (every 0.5 hour ultrasound 0.5h) of ultrasound under part, then continuously 4h is extracted in shaking under the conditions of 100 DEG C, sets 10,000r/
The separation of min centrifuge is added 95% ethyl alcohol of 4 times of volumes, is stirred with glass bar after taking supernatant, rotary evaporation to be concentrated in vacuo
It mixes, sets precipitates overnight in 4 DEG C of refrigerators, supernatant is abandoned in the separation of 10,000r/min centrifuges, with 95% ethanol washing precipitating one
It is secondary, deionized water dissolving is added, adds isometric Sevage reagent (chloroform: n-butanol=4:1), is sufficiently stirred, stands and divides
Layer discards middle protein layer, recycles lower layer sevage reagent, repetitive operation 4 times, collects upper layer polysaccharide solution, rotary evaporation is true
Sky concentration, concentrate are decolourized with peroxide passivation, and flowing water dialysis 12h, vacuum freeze drying obtains 15.5g pale yellow powder shape
Tremella polysaccharides, polyoses content 78.4%, polysaccharide yield 24.3%.
Polyoses content is measured using phenol-sulfuric acid and colorimetric method and gel permeation chromatography (GPC) measures polysaccharide molecular weight, it is real
The water soluble polymer amount tremella polysaccharides that example 1 and embodiment 2 refine, polyoses content >=92% are applied, molecular weight is mainly distributed on
Between 2,000~4,000kDa, and the polyoses content of tremella residue is 25% or so, and molecular weight is mainly distributed on 200~
Between 500kDa.The tremella polysaccharides that comparative example 3 refines, polyoses content is about 78%, molecular weight mainly by 2,000~4,
Two section compositions of 000kDa and 200~500kDa.
Claims (9)
1. a kind of method that tremella polysaccharides extract, which is characterized in that comprise the following steps that
Step (1) pulverizes: selecting clean disease-free tremella dry product, after coarse powder first is made, then is continued with micronizer
It crushes, obtains tremella Ultramicro-powder；
Step (2) decoloration removal of impurities: by tremella Ultramicro-powder obtained by step (1), being added 70%wt ~ 100%wt ethyl alcohol and extracted, mistake
Filter removes polyphenol, fat, pigment small molecular weight impurity, obtains the tremella Ultramicro-powder of decoloration removal of impurities；
The extraction of step (3) low temperature: by tremella Ultramicro-powder obtained by step (2), deionized water is added and carries out low temperature extraction, extraction temperature
≤ 40 DEG C, centrifugation obtains the polysaccharide leaching liquor and cold water-insoluble tremella residue of cold water solubles；
Step (4) deproteinization: polysaccharide leaching liquor obtained by step (3) is concentrated, and sevage reagent is added in concentrate, sufficiently
Stirring, stratification collect upper layer polysaccharide solution；
Step (5) purification: quaternary ammonium salt solution is added to complex-precipitation, sediment dissolution in polysaccharide solution obtained by step (4)
In sodium chloride solution, insoluble matter is removed, supernatant is concentrated into polysaccharide and obtains after alcohol precipitation, centrifugation, washing, drying close to saturation
To water soluble polymer amount tremella polysaccharides；
Step (6) residue utilizes: by tremella residue obtained by step (3), dry and crushing obtains powdered tremella residue.
2. the method that a kind of tremella polysaccharides according to claim 1 extract, it is characterised in that: silver as described in step (1)
The average grain diameter of ear Ultramicro-powder >=600 mesh.
3. the method that a kind of tremella polysaccharides according to claim 1 extract, it is characterised in that: silver described in step (2)
The mass volume ratio of ear Ultramicro-powder and ethyl alcohol is 1:3 ~ 10, extraction time 2 ~ for 24 hours, extracting times 1 ~ 3 time.
4. the method that a kind of tremella polysaccharides according to claim 1 extract, it is characterised in that: silver described in step (3)
Ear Ultramicro-powder and the mass volume ratio of deionized water are 1:40 ~ 100,4 ~ 48h of extraction time, extracting times 1 ~ 3 time.
5. the method that a kind of tremella polysaccharides according to claim 1 extract, it is characterised in that: described in step (4)
Sevage reagent is chloroform: n-butanol volume ratio is 4 ~ 5:1 mixed liquor, and additional amount is 1/4 ~ 3/4 times of the polysaccharide volume of the concentrated liquid,
Middle protein layer is discarded, lower layer sevage reagent is recycled, collects upper layer polysaccharide solution, repetitive operation 2-4 times.
6. the method that a kind of tremella polysaccharides according to claim 1 extract, it is characterised in that: season described in step (5)
Ammonium salt is one of cetylpyridinium chloride, brocide or cetyl trimethylammonium bromide.
7. the method that a kind of tremella polysaccharides according to claim 1 extract, it is characterised in that: the quaternary ammonium salt solution
Concentration is 1%wt ~ 10%wt.
8. the method that a kind of tremella polysaccharides according to claim 1 extract, it is characterised in that: chlorine described in step (5)
Change sodium solution concentration is 0.4 ~ 2mol/L, and sediment is dissolved in >=40 DEG C of sodium chloride solutions and is dissociated.
9. the method that a kind of tremella polysaccharides according to claim 1 extract, it is characterised in that: alcohol described in step (5)
Heavy, centrifugation, washing, dry 95%wt ~ 100%wt ethyl alcohol that 2 ~ 4 times of volumes are added for polysaccharide solution, stratification are centrifuged, precipitating
Object is with after ethanol washing 1 ~ 2 time, vacuum drying or freeze-drying.
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| WO2020245809A3 (en) * | 2019-06-07 | 2021-01-14 | Vivatis Pharma Gmbh | Identification and selection of a plant starting material of a plant chondroitin sulfate and hyaluronic acid, and transformation of such plant starting material to obtain ingredients for use in foods, supplements, medical devices or drugs |
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| CN107459585B (en) * | 2017-08-30 | 2019-11-19 | 华熙生物科技股份有限公司 | A kind of production method of low molecular weight tremella polysaccharides |
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| CN112442136A (en) * | 2019-09-03 | 2021-03-05 | 广东乐尔康生物科技股份有限公司 | Method for extracting functional components from tremella |
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| CN116462778B (en) * | 2023-04-17 | 2024-02-23 | 浙江天草生物科技股份有限公司 | Extraction method of tremella polysaccharide |
| CN116574200A (en) * | 2023-06-05 | 2023-08-11 | 上海蜂花日用品有限公司 | Tremella polysaccharide extraction and recovery method and device |
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