CN106977592A - A kind of method for isolating and purifying Agricus blazei fructification activated protein - Google Patents
A kind of method for isolating and purifying Agricus blazei fructification activated protein Download PDFInfo
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- CN106977592A CN106977592A CN201610026939.4A CN201610026939A CN106977592A CN 106977592 A CN106977592 A CN 106977592A CN 201610026939 A CN201610026939 A CN 201610026939A CN 106977592 A CN106977592 A CN 106977592A
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/375—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Basidiomycetes
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Abstract
The invention discloses a kind of method for isolating and purifying Agricus blazei fructification activated protein, belong to biological technical field, be adapted to large-scale production.Cellulase and pectinase enzymatic hydrolysis are added after dry Agricus blazei fructification powder degreasing, are extracted with ultrasonic assistant；Hot water extraction optimizes processing to protein extraction technique；Agricus blazei fructification activated protein of saltouing is precipitated with saturated ammonium sulfate solution, vacuum freeze drying obtains Agricus blazei fructification crude protein；Using Agricus blazei fructification albumen is separated at the beginning of ion-exchange chromatography, eluent is collected；The Agricus blazei fructification albumen after separation is further purified using sephadex, the higher Agricus blazei fructification activated protein of purity is obtained.Agricus blazei fructification activated protein molecular weight is determined using SDS-PAGE.Agricus blazei fructification activated protein prepared by the present invention has obvious bioactivity.
Description
Technical field
The present invention relates to health food processing technique field, more particularly to one kind isolates and purifies Agricus blazei fructification activity
The method of albumen.
Background technology
Agricus blazei（Agaricus Blazei Murill）Also known as Brazilian mushroom, Bai Shi mushrooms, little Song mushrooms, it is a kind of rare
It is leaved for development be to want dual-purpose fungi, originate in the ground such as Brazil, the Peru of North American southern.Agricus blazei in reducing blood lipid, hypotensive, control
Have in terms for the treatment of diabetes and be of miraculous efficacy, " last food on the earth " is once referred to as by Japan, medicinal and health value
By the very big concern of medical science, pharmacy and cuisines circle.
Agricus blazei cap is tender, and stem is crisp, and mouthfeel is fabulous, and the pure fresh perfume (or spice) of taste, edibility is quite high.Agricus blazei rich in protein,
Sugar, vitamin, dietary fiber, mineral matter, aliphatic acid and trace element etc..New fresh sporophore moisture 85% ~ 87%,
Containing crude protein 40% ~ 50%, sugar 38% ~ 45%, cellulosic 6% ~ 8%, coarse ash 5% ~ 7%, fat 3% ~ 4% in dry mushroom；
In addition, amino acid content enriches very much, higher than other edible mushrooms；Also containing multivitamin and abundant trace element.Ji Song
Fine and soft protein composition includes 18 kinds of amino acid, and 8 kinds of essential amino acids of human body are complete, contain multivitamin and ergot steroid
Alcohol.
Agricus blazei is a kind of edible and medicinal fungi for having market development value.Agricus blazei contains abundant protein and many
Sugar, with high nutritive value and medical value, is mainly used in field of medicaments and health food industry.Agricus blazei contains many
Kind of physiological activator, including polysaccharide, activated protein, steroids, agglutinin and nucleic acid etc., with regulation immunity, it is antitumor,
The medicinal efficacies such as antibacterial.
The research to Agricus blazei mostly concentrates on liquid fermentation process, cultivation, separation of polysaccharides purifying aspect at present, to Ji Song
The research of fine and soft albumen is less.On the one hand, edible fungus protein content is high, and fat content is low, and nutritive value is better than plant
Albumen and animal protein, edible fungus protein is the food between vegetable protein and animal protein under normal circumstances, be can be good at
Demand of the mankind to nutrition is met, is referred to as " body-building food ".On the other hand, after Agaricus Blazei Murrill polysaccharide is extracted, in lower sizing material also
Containing substantial amounts of protein, research shows, Agricus blazei albumen has significant antitumaous effect.Therefore carry out at present to Agricus blazei
The research of entity activated protein, the separation purifying technique of the Agricus blazei fructification activated protein of Erecting and improving, for improving Ji Song
Fine and soft value-added content of product has great importance.
The realization of the separation purifying technique of Agricus blazei fructification activated protein, improves the utilization rate of Agricus blazei product, is
The industrialized production of Agricus blazei fructification activated protein provides technical parameter and theoretical direction, is Agricus blazei fructification active component
Full-scale development research provide direction.
The content of the invention
The purpose of the present invention is to isolate and purify a kind of activated protein, and provides a kind of Agricus blazei fructification isolated and purified and live
The method of property albumen.
To realize that the technical scheme that the purpose of the present invention is used is as follows.
（1）Cellulase and pectinase enzymatic hydrolysis cell wall constituent, control are added after dry Agricus blazei fructification powder degreasing
50 DEG C of hydrolysis temperature, enzyme addition is 2%, and enzymolysis time is 3h.
（2）Solution after above-mentioned enzymolysis is subjected to ultrasonic assistant extraction, ultrasonic power is 250W, and sonication times are
35min。
（3）By above-mentioned by enzymolysis and the Agricus blazei fructification protein powder of ultrasonication, using Hot water extraction pair
Influence the factor of extraction effect（Extraction temperature, extraction time, solid-liquid ratio, extraction time）Optimize processing.
（4）Ammonium sulfate is added into Agricus blazei protein extract to different saturation degrees, 4 DEG C stand the 24h that saltouts, from
The heart, sediment is dissolved with PBS phosphate solutions, is freezed after dialysis, is produced Agricus blazei fructification albumen crude product.
（5）Agricus blazei fructification activated protein is separated with ion-exchange chromatography, is followed the trail of and surveyed using Coomassie brilliant blue G250
It is fixed, collect eluent.
（6）Agricus blazei fructification activated protein is further purified using sephadex, dialysis desalting, vacuum refrigeration is done
It is dry, obtain the higher Agricus blazei fructification activated protein of purity.Agricus blazei fructification activated protein molecular weight is surveyed with SDS-PAGE.
The step of optimizing Agricus blazei fructification activated protein using Hot water extraction is as follows：Experiment determine enzymolysis time,
On the basis of enzyme addition, ultrasonic power and sonication times, by digesting the Agricus blazei fructification powder with ultrasonication
End, to extraction temperature, extraction time, solid-liquid ratio, extraction time carry out the level of four factor three orthogonal experiment optimize, investigate it is each because
Influence of the element to recovery rate.
The step of using saturated ammonium sulphate Agricus blazei fructification activated protein, is as follows：Into Agricus blazei protein extract
Ammonium sulfate is added to different saturation degrees, 4 DEG C of standing 12h, centrifugation dissolves sediment with PBS phosphate solutions, surveys extinction
Value, determines the most suitable saturation degree of ammonium sulfate.H is added while stirring2O2Solution is dialysed to color fade, vacuum refrigeration
Dry, obtain Agricus blazei fructification crude protein.
Using as follows the step of Agricus blazei fructification activated protein is separated at the beginning of DEAE-52 ion-exchange chromatographies：Use phosphoric acid
Sodium buffer solution（pH6.0）Chromatographic column is balanced, by Agricus blazei protein crude extract upper prop, after adsorption equilibrium, with sodium phosphate buffer point
Step elution, flow velocity is 0.5mL/min.Measure is tracked using Coomassie brilliant blue G250, received finally according to gradient elution curve
Collection merges eluent.
The step of purifying Agricus blazei fructification activated protein using Sephadex G150 sephadexes is as follows：Will
Distilled water immersion 2-3 d under Sephadex G150 normal temperature, adjust pH to 6, by Agricus blazei egg with 50mmol/L sodium phosphate buffers
After white just separating liquid upper prop, adsorption equilibrium, stepwise elution is carried out with sodium phosphate buffer, flow velocity is 0.5mL/min.Using examining horse
This light blue G250 is tracked measure, merges eluent finally according to gradient elution curve.
Compared with prior art, the beneficial effects of the invention are as follows：
1st, the present invention is to Agricus blazei fructification powder pre-treating isolating and purifying the method for Agricus blazei fructification activated protein
When, extracted using cellulase and pectinase enzymatic hydrolysis Agricus blazei cell wall constituent, and using ultrasonic assistant, be conducive to improving a Ji
The recovery rate of matsutake albumen.This method is efficient, easy, economical, be adapted to industrial-scale production；
2nd, the present invention extracts Agricus blazei fructification activated protein using the method for hot water extraction, it is to avoid the method extracted using alkali
The proteinaceous nutrient ingredients from lossing caused, reduces the generation of coloring matter, and the work of Agricus blazei fructification is remained to greatest extent
The activity of property albumen；
3rd, the present invention realize first Agricus blazei fructification activated protein is extracted and purifying process research, protein after purification
Content, up to 88.56%, is that Agricus blazei fructification activated protein large-scale production from now on and functional study are applied and provide theoretical base
Plinth.
Brief description of the drawings
Fig. 1 is separation Agricus blazei fructification activated protein at the beginning of DEAE-52 ion-exchange chromatographies（Pillar 20* used
100mm）；
Fig. 2 is SephadexG-150 sephadexes purifying Agricus blazei fructification activated protein（Pillar 15*650mm used）.
Embodiment
1st, Agricus blazei fructification will be dried to crush, and will cross degreasing after 60 mesh sieves, add cellulase and pectinase enzymatic hydrolysis cell
Wall composition, enzyme addition is 2%, 50 DEG C of hydrolysis temperature, and enzymolysis time is 3h.
2nd, the solution after above-mentioned enzymolysis is subjected to ultrasonic assistant extraction, ultrasonic power is 250W, and sonication times are
35min。
3rd, on the basis of experiment determines enzymolysis time, enzyme addition, ultrasonic power, sonication times, using hot water
Extraction, the optimization processing of protein extraction is carried out to the Agricus blazei fructification by enzymolysis and ultrasonication, and extraction temperature is
90 DEG C, extraction time is 2.5h, and solid-liquid ratio is 1:2, extraction time is 2 times, on this condition Agricus blazei fructification activated protein
Recovery rate is 38.78%.
4th, 12 parts of 5mL Agricus blazei protein extracts are taken, respectively plus (NH4) 2SO4 to saturation degree be 10%, 20%, 25%,
30%th, 35%, 40%, 45%, 50%, 55%, 60%, 75% and 100%, 4 DEG C stand the 24h that saltouts, 3000r/min centrifugations
30min, collects albumen precipitation, plus a small amount of PBS is allowed to dissolve, and inserts dialysis membrane, plus 10mmol/L pH7.0 phosphate buffers, puts
In 4 DEG C of refrigerators, dialyse 24h, as vacuum freeze drying, Agricus blazei fructification activated protein crude product.Ammonium sulfate is determined
Saturation degree is 80%.
5th, using separation Agricus blazei fructification activated protein at the beginning of DEAE-52 ion-exchange chromatographies：Use 50mmol/L phosphoric acid
Sodium buffer solution（pH6.0）Balance DEAE-52 chromatographic columns（20×100mm）, untill post trickle pH is 6.0, by 5mL
After Agricus blazei protein crude extract upper prop, adsorption equilibrium, salinity is used to delay for 0,0.1,0.2,0.3,0.5mo1/L sodium phosphates successively
Fliud flushing carries out stepwise elution, and flow velocity is 0.5mL/min.Measure is tracked using Coomassie brilliant blue G250, according to gradient elution
Curve, which is collected, merges eluent.Isolated one obvious eluting peak and three small peaks, and be named as successively ABMP-1,
ABMP-2, ABMP-3, ABMP-4, collect ABMP-2 eluting peaks, are further isolated and purified.
6th, Agricus blazei fructification activated protein is purified using SephadexG-150 sephadexes：Weigh 3.5g
Distilled water immersion 2-3 d are used under Sephadex G150, normal temperature, are balanced with 50mmol/L sodium phosphate buffers (pH6.0)
SephadexG150 sephadexes（15×650mm）, untill post trickle pH is 6.0, by 5mL Agricus blazei albumen
It is that 0.3 sodium phosphate buffer carries out stepwise elution with salinity, flow velocity is 0.5mL/ after first separating liquid upper prop, adsorption equilibrium
min.Measure is tracked using Coomassie brilliant blue G250, is collected according to gradient elution curve and merges eluent.By gel column
Chromatography, the eluting peak isolates two eluting peaks, ABMP-21, ABMP-22 is named as successively again.The two eluting peaks are collected, thoroughly
Desalination is analysed, vacuum freeze drying obtains the Agricus blazei fructification activated protein powder of relatively purifying, and protein content is after purification
88.56%。
7th, using polyacrylamide gel electrophoresis（SDS-PAGE）Determine Agricus blazei fructification activated protein molecular weight：Sample
The mg/mL of protein concentration 10, with the separation gel of 13% concentration, Agricus blazei protein protomer molecular mass is analyzed by SDSPAGE,
Main protein molecular weight is 85.3 kD.
Present invention comprehensive utilization combined-enzyme method synergistic supersonic wave method, Hot water extraction, ion-exchange chromatography, glucan coagulate
Plastic column chromatography, Freeze Drying Technique and SDS-PAGE methods, the Agricus blazei fructification activated protein purity of preparation are higher.
The present invention is not it is pointed out that for those skilled in the art, departing from original of the invention
On the premise of reason, some improvements and modifications can also be made, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (5)
1. a kind of method for isolating and purifying Agricus blazei fructification activated protein, it is characterised in that comprise the steps：
（1）Cellulase and pectinase enzymatic hydrolysis cell wall constituent, controlled enzymatic hydrolysis are added after dry Agricus blazei fructification powder degreasing
Temperature 50 C, enzyme addition is 2%, and enzymolysis time is 3h；
（2）Solution after above-mentioned enzymolysis is subjected to ultrasonic assistant extraction, ultrasonic power is 250W, and sonication times are
35min；
（3）By enzymolysis and the Agricus blazei fructification protein powder of ultrasonication hot water extraction is carried out by above-mentioned, using orthogonal
Assay optimization has the factor significantly affected to extraction process；
（4）Agricus blazei fructification activated protein of saltouing is precipitated with ammonium sulfate saturated solution, it is 80% to determine its saturation degree, and pH is adjusted
To 3.5, vacuum freeze drying obtains Agricus blazei fructification crude protein；
（5）Agricus blazei fructification crude protein is separated using DEAE-52 ion-exchange chromatographies, followed the trail of using Coomassie brilliant blue G250
Determine, collect eluent；
（6）Agricus blazei fructification crude protein, dialysis desalting, vacuum are further purified using SephadexG-150 sephadexes
Freeze-drying, obtains the higher Agricus blazei fructification activated protein of purity；
（7）Agricus blazei fructification activated protein molecular weight is determined using SDS-PAGE.
2. the method for isolating and purifying Agricus blazei fructification activated protein according to right 1, it is characterised in that use orthogonal examination
The step of testing the extraction process of optimization Agricus blazei fructification activated protein is as follows：In experiment determination enzymolysis time, enzyme addition, surpass
On the basis of acoustic power, sonication times, the water of four factor three is carried out to extraction temperature, extraction time, solid-liquid ratio, extraction time
Flat optimization of orthogonal test, analyzes the influence of each factors on extraction rate.
3. the method for isolating and purifying Agricus blazei fructification activated protein according to right 2, it is characterised in that use saturation sulphur
It is as follows that sour ammonium precipitates the step of saltouing Agricus blazei fructification activated protein：Ammonium sulfate is added into Agricus blazei protein extract
To different saturation degrees, 4 DEG C stand the 24h that saltouts, and centrifugation is dissolved sediment with PBS phosphate solutions, freezed after dialysis, it is determined that
The most suitable saturation degree of ammonium sulfate.
4. the method for isolating and purifying Agricus blazei fructification activated protein according to right 3, it is characterised in that use DEAE-
The step of Agricus blazei fructification activated protein is separated at the beginning of 52 ion-exchange chromatographies is as follows：Balanced and chromatographed with sodium phosphate buffer
Post, by Agricus blazei protein crude extract upper prop, after adsorption equilibrium, uses sodium phosphate buffer stepwise elution, flow velocity is 0.5mL/min；
Measure is tracked using Coomassie brilliant blue G250, is collected according to gradient elution curve and merges eluent.
5. the method for isolating and purifying Agricus blazei fructification activated protein according to right 4, it is characterised in that use
The step of Sephadex G150 sephadexes purify Agricus blazei fructification activated protein is as follows：Balanced with sodium phosphate buffer
After SephadexG150 sephadex columns, adsorption equilibrium, stepwise elution is carried out with sodium phosphate buffer, flow velocity is 0.5mL/
min；Measure is tracked using Coomassie brilliant blue G250, is collected according to gradient elution curve and merges eluent.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109336951A (en) * | 2018-10-30 | 2019-02-15 | 北华大学 | Agricus blazei polypeptide and its preparation method and application |
| CN113087762A (en) * | 2021-05-17 | 2021-07-09 | 吉林农业大学 | Preparation method of protein-derived antioxidant hydrolyzed peptide of tricholoma matsutake fruiting body |
| CN113969302A (en) * | 2021-11-09 | 2022-01-25 | 四川旅游学院 | Preparation process of tricholoma matsutake protein peptide |
Citations (1)
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| CN104431995A (en) * | 2015-01-14 | 2015-03-25 | 佟立君 | Preparation method of fresh tricholoma matsutake active material |
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2016
- 2016-01-18 CN CN201610026939.4A patent/CN106977592A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104431995A (en) * | 2015-01-14 | 2015-03-25 | 佟立君 | Preparation method of fresh tricholoma matsutake active material |
Non-Patent Citations (1)
| Title |
|---|
| 王振等: "姬松茸蛋白制备及分析研究", 《现代农业科技》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109336951A (en) * | 2018-10-30 | 2019-02-15 | 北华大学 | Agricus blazei polypeptide and its preparation method and application |
| CN113087762A (en) * | 2021-05-17 | 2021-07-09 | 吉林农业大学 | Preparation method of protein-derived antioxidant hydrolyzed peptide of tricholoma matsutake fruiting body |
| CN113087762B (en) * | 2021-05-17 | 2024-05-31 | 吉林农业大学 | Preparation method of tricholoma matsutake fruiting body protein source antioxidant hydrolysis peptide |
| CN113969302A (en) * | 2021-11-09 | 2022-01-25 | 四川旅游学院 | Preparation process of tricholoma matsutake protein peptide |
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