CN104231103A - Flammulina velutipes polysaccharide as well as preparation method and application thereof - Google Patents
Flammulina velutipes polysaccharide as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses flammulina velutipes polysaccharide as well as a preparation method and an application thereof. The flammulina velutipes polysaccharide is characterized in that the preparation method comprises steps as follows: polysaccharide in flammulina velutipes is extracted by water firstly, polysaccharide in the flammulina velutipes is then extracted by an alkali solution, and polysaccharide extracted by water and polysaccharide extracted by the alkali solution are combined. The raw material is the flammulina velutipes sold in the market, the flammulina velutipes polysaccharide yield is high, and the polysaccharide has higher antioxidant activity; the polysaccharide has high capability of clearing free radical and high reduction capability, and accordingly, the polysaccharide can be applied to antioxidant, anti-tumor and immunity-boosting foods and drugs.
Description
Technical field
The invention belongs to food processing technology field, relate to a kind of polysaccharide and preparation and application thereof, particularly relating to a kind of take needle mushroom as polysaccharide and the preparation and application thereof of raw material.
Background technology
Needle mushroom is the medicine-food two-purpose bacterium of cultivation of a kind of autumn and winter, sliding tender, the stem of its cap elongated tender and crisp, shape is beautiful, delicious, there is higher nutritive value and pharmaceutical use.Needle mushroom contains a kind of polysaccharide of important high biological activity.Research finds that flammulina velutipes has the multiple biological activitys such as antitumor, anti-oxidant, anticancer, strengthening immunity.In addition, needle mushroom is as natural phant, and it there is no negative interaction to human body.So the biological activity of exploitation needle mushroom, improves its range of application and using value, has wide market.
In recent years about the research report of the extracting method of flammulina velutipes has a lot.Adopt microwave―assisted extraction, draw best flammulina velutipes extraction process (Yu TOOLING etc. by single factor experiment and optimization of orthogonal test, microwave―assisted extraction extracts the technical study of flammulina velutipes, Jilin agricultural, 2010,250 (12): 75-79).Adopt Papain ferment treatment, orthogonal test is carried out to enzyme amount, operative temperature, optimum pH, action time, draw the optimum process condition (Liang Min etc. of Extracting Flammulina Velutipes Polysaccharide by Enzymes, the research of Extracting Flammulina Velutipes Polysaccharide by Enzymes, food research and development, 2012,33 (11): 100-102).Papoid and cellulase Combined Processing is utilized to extract flammulina velutipes from needle mushroom, by experiment of single factor and orthogonal experiment studying enzyme consumption, hydrolysis temperature, pH, enzymolysis time on the impact (Liang Min etc. of flammulina velutipes extraction yield, combined-enzyme method extracts flammulina velutipes and spectroscopic analysis, Agriculture In Hubei Province science and technology, 2012,51 (6): 1210-1213).Adopt ultrasonic-assisted extraction method, by single factor test and orthogonal test, (Ma Yinfei etc. are studied on the extraction yield impact on flammulina velutipes of Extracting temperature, extraction time and extraction power, the technical study of ultrasonic-assisted extraction flammulina velutipes, food engineering, 2012,4:105-109).Water extraction and alcohol precipitation method is adopted to extract flammulina velutipes, draw optimum extraction process, and the anti-oxidant activity of flammulina velutipes is evaluated, result shows, flammulina velutipes has good anti-oxidant activity (Ye Min, the extraction of flammulina velutipes and the research of scavenging hydroxyl activity, Bijie institute journal in vitro, 2011,29 (4): 90-94).The extraction of polysaccharide or preparation method affect its anti-oxidant activity (Wang Yanli etc., ultrasound parameter on the impact of bighead atractylodes rhizome polysaccharide anti-oxidative activity, biological processing [J], 2012,10 (1): 7-12; Lixia ZHANG etc., Different Extraction Method on the impact of Fuscoporia obliqua polysaccharide anti-oxidant activity, Agriculture of Anhui science [J], 2012,40 (10): 5870-5872).
Therefore, those skilled in the art is devoted to develop a kind of flammulina velutipes preparation method that can improve productive rate and strengthen polysaccharide anti-oxidative activity and reducing power.
Summary of the invention
Because the above-mentioned defect of prior art, technical problem to be solved by this invention is to provide a kind of method preparing flammulina velutipes, and its obtained flammulina velutipes yield is higher and have strong anti-oxidant activity.
For achieving the above object, the invention provides a kind of flammulina velutipes, the preparation of described flammulina velutipes adopts water extraction and alkali to put forward the mode combined and extracts, namely first water extraction is adopted, obtain par-tial polysaccharide extracting solution, then alkali is carried out to filter residue carry, obtain another part polysaccharide extraction liquid, above-mentioned two portions extracting solution is merged to concentrate and obtains final product
In the food that flammulina velutipes prepared by the present invention can be applicable to anti-oxidant, antitumor and strengthening immunity and medicine.
The concrete preparation method of flammulina velutipes of the present invention comprises the following steps:
One, mix: needle mushroom removal of impurities is cleaned, adds distilled water, smash at a high speed with sander, obtain mixed solution;
Two, water extraction: in a heated condition, carries out magnetic agitation to the mixed solution in step one, and by mixed solution filter-cloth filtering after for some time, the filtrate obtained is Aqueous extracts, alkali is carried out in filter residue taking-up and carries;
Three, alkali is carried: in the filter residue of step 2, add NaOH solution, carries out magnetic agitation in a heated condition, and with filter-cloth filtering after for some time, the filtrate obtained is alkali extract;
Four, dialyse: in the alkali extract of step 3, add hydrochloric acid, adjust solution ph to 7, then dialyse with dialysis tubing;
Five, concentrated: the alkali extract in the Aqueous extracts in combining step two and step 4 after dialysis, heating is concentrated, obtains concentrated solution;
Six, precipitate: in the concentrated solution of step 5, add ethanol, hold over night, to remove deproteinize;
Seven, rinse: by centrifugal for the concentrated solution obtained after hold over night in step 6, removes supernatant liquid, precipitates with low boiling point organic solvent rinse;
Eight, dry: the precipitation after step 7 rinse to be dried up, obtains flammulina velutipes.
Preferably, the needle mushroom in step one is commercially available needle mushroom, and its water content is 8% by weight; The weight adding distilled water is 20-50 times of needle mushroom weight; Sander used is domestic type or use for laboratory type.
Preferably, the Heating temperature in step 2 and step 3 is 80-100 DEG C, and churning time is 2-3 hour; Filter cloth used is 200 orders or more.
Preferably, the concentration adding NaOH solution in step 3 is 0.5-1mol/L, and the weight added is 20-40 times of filter residue weight.
Preferably, the hydrochloric acid added in step 4 be concentrated hydrochloric acid dilution after dilute hydrochloric acid solution, its preferably concentration be 6mol/L; The molecular weight cut-off that the dialysis tubing specification used in dialysis procedure is 3500Da, dialysis time is 24 hours.
Preferably, the concentration process of step 5 carries out on the constant temperature heating device of adjustable temps, and Heating temperature is 80-100 DEG C; The concentrated solution volume finally obtained be condensate precursor long-pending 1/20.
Preferably, the amount adding ethanol in step 6 is greater than 80% for making the concentration of volume percent of ethanol in final mixed solution.
Preferably, centrifugal in step 7 rotating speed is 4000rpm; Rinse low boiling point organic solvent used is one or more in acetone, ethanol and ether.
Present invention employs above technical scheme, there is following advantage and beneficial effect:
1, the invention provides a kind of preparation method of flammulina velutipes, compared to existing customary preparation methods, the polysaccharide of gained has higher yield;
2, the flammulina velutipes of gained of the present invention, compared to existing customary preparation methods, has higher anti-oxidant activity;
3, the flammulina velutipes of gained of the present invention, compared to existing customary preparation methods, has stronger reducing power.
Be described further below with reference to the technique effect of accompanying drawing to design of the present invention, concrete steps and generation, to understand object of the present invention, characteristic sum effect fully.
Accompanying drawing explanation
Fig. 1 is the yield comparison diagram of the polysaccharide that two preferred embodiments of the present invention and comparative example obtain;
Fig. 2 is the ORAC anti-oxidant activity comparison diagram of the polysaccharide that two preferred embodiments of the present invention and comparative example obtain, and wherein TE is trolox equivalent;
Fig. 3 is the FRAP reducing activity comparison diagram of the polysaccharide that two preferred embodiments of the present invention and comparative example obtain, and wherein TE is trolox equivalent.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
embodiment 1
One, mix: commercially available needle mushroom removal of impurities is cleaned, adds 20 times of weight distilled water, smash at a high speed with sander, obtain mixed solution;
Two, water extraction: under Heating temperature is 100 DEG C of conditions, magnetic agitation is carried out to mixed solution in step one, water extraction 2 hours, then by mixed solution filter-cloth filtering, obtain filtrate and be Aqueous extracts, alkali is carried out in filter residue taking-up and carries;
Three, alkali is carried: the NaOH solution (0.5mol/L) adding 20 times of weight in the filter residue of step 2, under Heating temperature is 100 DEG C of conditions, carry out magnetic agitation, with filter-cloth filtering after alkali carries 2 hours, obtains filtrate and is alkali extract;
Four, dialyse: in the alkali extract of step 3, add hydrochloric acid (6mol/L), regulator solution pH value to 7, then dialyse 24 hours;
Five, concentrated: the alkali extract in the Aqueous extracts in combining step two and step 4 after dialysis, is concentrated into 1/20 of original volume, obtains concentrated solution under Heating temperature is 80 DEG C of conditions;
Six, precipitate: in the concentrated solution of step 5, add ethanol, make the concentration of volume percent mixing rear ethanol final be greater than 80%, hold over night;
Seven, rinse: by centrifugal for the concentrated solution of hold over night in step 6 rear removal supernatant liquid, in succession by acetone and ether rinse precipitation;
Eight, dry: the precipitation after step 7 rinse to be dried up, weighs, and carries out anti-oxidant activity evaluation.
embodiment 2
One, mix: commercially available needle mushroom removal of impurities is cleaned, adds the distilled water of 50 times of weight, smash at a high speed with sander, obtain mixed solution;
Two, water extraction: under Heating temperature is 80 DEG C of conditions, magnetic agitation is carried out to mixed solution in step one, water extraction 3 hours, then by mixed solution filter-cloth filtering, obtain filtrate and be Aqueous extracts, alkali is carried out in filter residue taking-up and carries;
Three, alkali is carried: the NaOH solution (1mol/L) adding 40 times of weight in the filter residue of step 2, is to carry out magnetic agitation under the condition of 80 DEG C in Heating temperature, with filter-cloth filtering after alkali carries 3 hours, obtains filtrate and is alkali extract;
Four, dialyse: in the alkali extract of step 3, add hydrochloric acid (6mol/L), regulator solution pH value to 7, then dialyse 24 hours;
Five, concentrated: the alkali extract in the Aqueous extracts in combining step two and step 4 after dialysis, is concentrated into 1/20 of original volume, obtains concentrated solution under Heating temperature is 100 DEG C of conditions;
Six, precipitate: in the concentrated solution of step 5, add ethanol, make the concentration of volume percent mixing rear ethanol final be greater than 80%, hold over night;
Seven, rinse: by centrifugal for the concentrated solution of hold over night in step 6 rear removal supernatant liquid, precipitates twice with dehydrated alcohol rinse;
Eight, dry: the precipitation after step 7 rinse to be dried up, weighs, and carries out anti-oxidant activity evaluation.
comparative example
One, mix: commercially available needle mushroom removal of impurities is cleaned, adds 20 times of weight distilled water, smash at a high speed with sander, obtain mixed solution;
Two, first time water extraction: be under the condition of 100 DEG C in Heating temperature, magnetic agitation carried out to the mixed solution in step one, water extraction 3 hours, then by mixed solution filter-cloth filtering, the Aqueous extracts obtained is retained, by further for filter residue water extraction;
Three, second time water extraction: the distilled water adding 20 times of weight in the filter residue of step 2, be carry out magnetic agitation under the condition of 100 DEG C in Heating temperature, water extraction 3 hours, then uses filter-cloth filtering, is retained by the Aqueous extracts obtained;
Four, concentrated: the Aqueous extracts in combining step two and step 3, is concentrated into 1/20 of original volume, obtains concentrated solution under Heating temperature is 80 DEG C of conditions;
Five, precipitate: in the concentrated solution of step 4, add ethanol, and make the final volume percentage concentration of ethanol be greater than 80%, hold over night;
Six, rinse: by the concentrated solution centrifugal segregation supernatant liquid after the hold over night in step 5, in succession by acetone and ether rinse precipitation;
Seven, dry: the precipitation after step 6 rinse to be dried up, weighs, and carries out anti-oxidant activity evaluation.
evaluation experimental
Embodiment 1, embodiment 2 and comparative example gained flammulina velutipes are carried out anti-oxidant ORAC evaluation respectively.
Concrete evaluation method is as follows: add 300 μ L water at 96 hole enzyme plate (black) most peripherals.25 μ L samples are added successively, 150 μ L 4x10 in metering orifice
-6mM Fluress.Enzyme plate is put into microplate reader, at 37 DEG C after preheating 30min, adds 25 μ L AAPH and start fluorescent quenching reaction.Its empty group replaces sample with 25 μ L damping fluids, and control group replaces sample with the Trolox solution of 25 μ L different concns.Sample parallel measures three times.Result represents ORAC value using Trolox content as equivalent.Microplate reader measuring condition arranges as follows: excitation wavelength 485nm, emission wavelength 528nm; After adding AAPH, amplitude 1mm, concussion 8S.After reaction starts, measuring cycle index is 120, and loop cycle is 1min.
Embodiment 1, embodiment 2 and comparative example gained flammulina velutipes are carried out anti-oxidant FRAP evaluation respectively.
Concrete evaluation method is as follows: the FeCl measuring the acetate buffer solution (pH3.6) of 50mL, the TPTZ hydrochloric acid soln (10mmol/L) of 5ml and 5ml respectively
3the aqueous solution (20mmol/L), namely obtains FRAP reagent after mixing, and it is for subsequent use to be placed in 37 DEG C of water-baths; Parallel Trolox solution and the flammulina velutipes solution adding 50,100,200,300,400,500 μm of ol/L of 150 μ L respectively in each test tube, blank group adds the DMSO of equivalent; The FRAP reagent adding 2.85mL in each test tube, preserve at 37 DEG C, is placed in dark place 30min; The light absorption value of each solution is measured under 593nm.
result with compare
As shown in Figure 1, the polysaccharide yield of embodiment 1 and embodiment 2 is respectively 3.14% and 4.19% (w/w), and comparative example is 1.74% (w/w).As can be seen here, the preparation method inventing the flammulina velutipes provided can improve the productive rate of flammulina velutipes.
As shown in Figure 2, the polysaccharide ORAC value that embodiment 2 obtains is the highest, and be 4.7 μm of ol TE/mg, namely the activity of scavenging free radicals is best, and being secondly embodiment 1, is 3.2 μm of ol TE/mg, and the polysaccharide anti-oxidative value that comparative example obtains is minimum, is only 0.5 μm of ol TE/mg.
As shown in Figure 3, the flammulina velutipes FRAP reduction value that embodiment 1 and embodiment 2 obtain is 12.5 and 18.4 μm of ol TE/mg, all high than comparative example (6.1 μm of ol TE/mg), prove that the polysaccharide that embodiment 1 and embodiment 2 obtain has better reductibility.As can be seen here, the preparation method of the flammulina velutipes provided is provided, not only increases the free radical scavenging effect of flammulina velutipes and strengthen its reducing power.
More than describe preferred embodiment of the present invention in detail.Should be appreciated that those of ordinary skill in the art just design according to the present invention can make many modifications and variations without the need to creative work.Therefore, all technician in the art, all should by the determined protection domain of claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.
Claims (10)
1. a flammulina velutipes, it is characterized in that, the preparation of described flammulina velutipes adopts water extraction and alkali to put forward the mode combined and extracts: first adopt water extraction, obtain par-tial polysaccharide extracting solution, carry out alkali to filter residue again to carry, obtain another part polysaccharide extraction liquid, obtain final product by concentrated for above-mentioned two portions extracting solution merging.
2. flammulina velutipes according to claim 1, is characterized in that, its preparation method comprises the following steps:
One, mix: needle mushroom removal of impurities is cleaned, adds distilled water, smash at a high speed with sander, obtain mixed solution;
Two, water extraction: in a heated condition, carries out magnetic agitation to the mixed solution in step one, and by mixed solution filter-cloth filtering after for some time, the filtrate obtained is Aqueous extracts, alkali is carried out in filter residue taking-up and carries;
Three, alkali is carried: in the filter residue of step 2, add NaOH solution, carries out magnetic agitation in a heated condition, and with filter-cloth filtering after for some time, the filtrate obtained is alkali extract;
Four, dialyse: in the alkali extract of step 3, add hydrochloric acid, adjust solution ph to 7, then dialyse with dialysis tubing;
Five, concentrated: the alkali extract in the Aqueous extracts in combining step two and step 4 after dialysis, heating is concentrated, obtains concentrated solution;
Six, precipitate: in the concentrated solution of step 5, add ethanol, hold over night, to remove deproteinize;
Seven, rinse: by centrifugal for the concentrated solution obtained after hold over night in step 6, removes supernatant liquid, precipitates with low boiling point organic solvent rinse;
Eight, dry: the precipitation after step 7 rinse to be dried up, obtains flammulina velutipes.
3. flammulina velutipes according to claim 2, is characterized in that, by weight, its water content is 5% to the needle mushroom in step one, and the weight adding distilled water is 20-40 times of needle mushroom weight.
4. flammulina velutipes according to claim 2, is characterized in that, the Heating temperature in step 2 and step 3 is 80-100 DEG C, and churning time is 2-3 hour.
5. flammulina velutipes according to claim 2, is characterized in that, described in step 2 and step 3, filter cloth is 200 orders or more; The dialysis tubing specification that uses of dialysing described in the step 4 molecular weight cut-off that is 3500Da, dialysis time is 24 hours.
6. flammulina velutipes according to claim 2, is characterized in that, the concentration of the described NaOH solution added in step 3 is 0.5-1mol/L, and the weight added is 20-40 times of filter residue weight.
7. flammulina velutipes according to claim 2, is characterized in that, the amount adding ethanol described in step 6 is greater than 80% for making the final volume percentage concentration of ethanol in mixed solution.
8. the application of the flammulina velutipes according to claim 1-7 any one in antioxidant food and antioxidant drug.
9. the application of the flammulina velutipes according to claim 1-7 any one in Anticancer Food and antitumor medicine.
10. a preparation method for flammulina velutipes, is characterized in that, comprises step: water extraction and alkali are carried; Described water extraction refers to the polysaccharide in water extraction needle mushroom, and described alkali carries the polysaccharide referred to in alkaline solution extraction needle mushroom.
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