CN106317260A - Method for extraction from highland barley grain and purification of araboxylan - Google Patents
Method for extraction from highland barley grain and purification of araboxylan Download PDFInfo
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0057—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Xylans, i.e. xylosaccharide, e.g. arabinoxylan, arabinofuronan, pentosans; (beta-1,3)(beta-1,4)-D-Xylans, e.g. rhodymenans; Hemicellulose; Derivatives thereof
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Abstract
The invention discloses a method for extraction from highland barley grain and purification of araboxylan. For the method, based on highland barley grain as the raw material, the crude sample is obtained through grinding, sieving, enzymolysis, alkaline extraction, dialysis, alcohol precipitation, vacuum freeze drying and degreasing. After dissolution, the purified sample is obtained after elution successively through the fillers cellulose DE- 32 and S- 400. The method can effectively separate the highland barley grain for high-purity araboxylan. In the crude sample, the extraction rate of araboxylan is 13. 8% and the purity are 96. 08%.
Description
Technical field
The present invention relates to a kind of method of isolated and purified araboxylan from Semen avenae nudae, be specifically related to a kind of from Semen avenae nudae paddy
Extract in Li and the method for purification araboxylan.
Background technology
Araboxylan (Arabinoxylans, AX) is the heteropolysaccharide in a kind of non-starch source, mainly by xylose and I
Uncle's sugar is constituted, and is widely present in nature particularly cereal crops.According to its water soluble nature, araboxylan is divided into again
Water solublity and water-insoluble two class.Comparatively speaking, soluble arabinoxylan structure is relatively easy, and content also ratio is relatively low；
Water-insoluble araboxylan structure is relative complex, and intramolecular and intermolecular cross-linking degree are higher.Internal and experiment in vitro table
Bright, araboxylan has the merits such as increase feces volume, blood sugar lowering, cholesterol reducing, antioxidation, regulation immunity even antitumor
Effect, its using value is high, application prospect is extensive.
Semen avenae nudae is grown in the area, plateau of severe cold anoxia, is topmost carbohydrate in the middle of Tibetan people diet every day
Source, has prominent medicinal health care function it is considered to be one of long-lived key factor of Tibetan people.Severe cold, anoxia, dry
The special environments such as dry, strong illumination make Semen avenae nudae obtain extremely strong resistance, create the abundantest secondary metabolite.
Semen avenae nudae also because of its trophic component comprehensively and unique, food therapy value is high and is increasingly favored by the common people.Scholar is had to point out, in Semen avenae nudae
Beta glucan content be generally higher than the world other area plantation barley variety.Therefore during beta glucan is also considered as Semen avenae nudae
The nutrient substance the closest with health and long-lived relation.But, araboxylan in cereal crops in the last few years
Physiological function is seen in report the most more and more and is affirmed.
Research to araboxylan is focused primarily on the cereal crops such as Semen Tritici aestivi, Fructus Hordei Vulgaris, Semen Maydis, and its extracting method is also led
The combination of water law to be divided into, alkaline process, enzyme process and these methods.Both at home and abroad in the hull-less barley one of hull-less barley (Semen avenae nudae be)
The research of araboxylan is relatively fewer.
Summary of the invention
It is an object of the invention to overcome above-mentioned the deficiencies in the prior art, it is provided that a kind of extraction from Semen avenae nudae grain
Method with purification araboxylan.Described method with Semen avenae nudae grain as raw material, through grinding, sieving, enzymolysis, alkali liquor molten
Solution, dialysis, precipitate with ethanol, vacuum lyophilization, defat obtain Raw samples, the most successively via filler cellulose after its dissolving
DE-32 and S-400 affords purification of samples.Described method can the effective highly purified Arab of isolated from Semen avenae nudae grain
Xylan, can be used for its structure elucidation, physicochemical property qualification and bioactivity research etc..
For achieving the above object, the present invention uses below scheme:
The present invention relates to a kind of extract and the method for purification araboxylan from Semen avenae nudae grain, described method include with
Lower step:
S1, enzymolysis: take Semen avenae nudae grain powder sample and add distilled water dissolving, be sequentially added into α-amylase, papain, sugar
Changing enzyme and carry out enzymolysis, enzymolysis solution is centrifuged；
S2, alkali carry: enzymolysis precipitate adds alkali liquor and dissolves, and regulation pH value is to 6.5～6.8, centrifugal；
S3, precipitate with ethanol: alkali puts on 2～3 times of volume dehydrated alcohol of dropping in clear liquid, stands overnight, centrifugal；
S4, vacuum lyophilization: precipitate with ethanol precipitate concentrates after redissolving with distilled water, vacuum lyophilization, obtains pressed powder；
S5, defat: with normal hexane as solvent, carry out surname extraction to pressed powder under 60～90 DEG C of water bath condition, low
Temperature dries to obtain Raw samples；
S6, DE-32 eluting: take Raw samples and add distilled water dissolving, centrifugal；Supernatant is via DE-32 eluting；Phenol-sulfur
Acid system has selected sugar test tube, and merging filtrate, 60～90 DEG C of water-bath evaporation and concentration obtain concentrated solution；
S7, S-400 eluting: concentrated solution is via S-400 eluting；Phend-sulphuric acid has selected sugar test tube, and sequentially determining is respectively arranged with
The sugar composition of filtrate and purity in sugar test tube, merge that single sugar is identical and purity > filtrate of 90%, and by single sugar not
Same separate collection；60～90 DEG C of water-bath evaporation and concentration, vacuum lyophilization obtains purification of samples powder, according to the liquid phase of purification of samples
Chromatograph testing result and Monosaccharide analysis experiment obtain araboxylan.
Preferably, the preparation of described Semen avenae nudae grain powder sample includes: sort without normal Semen avenae nudae grain such as pathological changes, after grinding
Cross 0.4～0.6mm screen cloth, obtain described powder sample.
Preferably, in step S1, Semen avenae nudae grain powder sample is 10g: 60～90mL with the amount ratio of distilled water.
Preferably, in step S1, be sequentially added into α-amylase, papain, saccharifying enzyme carry out enzymolysis particularly as follows: add
50～100 μ L Thermostable α-Amylase, 80～100 DEG C of water-baths 1～2h；Bath temperature is adjusted to 60～80 DEG C, adds 10～20mg
Papain, water-bath 1～2h；100 DEG C of water-bath enzyme denaturing 10～20min, add 100～500 μ L saccharifying enzyme, 60～80 after cooling
DEG C water-bath 1～2h；100 DEG C of water-bath enzyme denaturing 10～20min, 60～90 DEG C of water-baths 2～4h after cooling.
Preferably, in step S1, described centrifugal condition is: 3000～3500 turns, 10～15min.
Preferably, in step S2, enzymolysis precipitate adds 60～90mL 0.1～1.0mol/LNaOH solution dissolves, 60～90
DEG C water-bath 2～4h；Dense HCl solution alkali tune extract pH value is to 6.5～6.8, centrifugal.
Preferably, in step S2, described centrifugal condition is: 3000～3500 turns, 10～15min.
Preferably, in step S3, it is that the alkali after dialysis puts on clear liquid that described alkali puts on clear liquid.
Preferably, described dialysis includes: alkali is put on clear liquid and is transferred to bag filter (44mm bag filter, MW:3000) and by it
Being placed in clear water, every 8～12h change water once, change water totally 2～3 times, and it is 1: 500～1000 that alkali puts on the ratio of clear liquid and clear water.
Preferably, in step S3, described centrifugal condition is: 3000～3500 turns, 10～15min.
Preferably, in step S4, precipitate with ethanol precipitate redissolves with 60～90mL distilled water, and 60～90 DEG C of water-bath evaporation and concentration are extremely
About 5～10mL；After-20 DEG C of freezings, vacuum lyophilization 24～48h.
Preferably, in step S5, described surname extraction is extracting 6～8h repeatedly；The temperature of described oven drying at low temperature be 30～
50℃。
Preferably, in step S6, during eluting, 2～3mL/ pipes are used to collect 60～100 pipes.
Preferably, in step S6, during DE-32 eluting, flow velocity is 0.1～1.0mL/min, flowing be followed successively by mutually distilled water and
The NaCl solution of 0.5～1mol/L；(60～100 pipe) filtrate of distilled water eluting discards, (the 60～100 of NaCl solution eluting
Pipe) filtrate retention；The specification of chromatographic column is: 2.6cm x 30cm.
Preferably, in step S6, after merging filtrate, in distilled water, dialysis 36～48h, every 8～12h changes water once, thoroughly
Analysis liquid is 1: 10～20 with the ratio of distilled water；60～90 DEG C of water-bath evaporation and concentration are to about 1～5mL.
Preferably, in step S6, Raw samples is 0.1g: 1～2mL with the amount ratio of distilled water.
Preferably, in step S7, during eluting, 2～3mL/ pipes are used to collect 60～100 pipes.
Preferably, in step S7, during S-400 eluting, flow velocity is 0.1～1.0mL/min, and flowing is distilled water mutually；Chromatography
The specification of post is: 1.6cm x 100cm.
Preferably, in step S7, the high performance liquid chromatograph sequentially determining being equipped with Composition distribution is used to be respectively arranged with sugar examination
In pipe, the sugar of filtrate forms and purity.
Preferably, during the detection of described high performance liquid chromatograph, Composition distribution model is Waters 2414, and sugar column type number is
SUGAR KS-805 Composition distribution temperature is 30 DEG C, and column temperature is 30～60 DEG C；Flowing is distilled water mutually, flow velocity
It is 0.5～1.0mL/min.
Preferably, in step S7,60～90 DEG C of water-bath evaporation and concentration are to about 3～5mL；After-20 DEG C of freezings, carry out vacuum cold
Lyophilizing dry 24～48h.
The guiding theory of the present invention is separated off water solublity beta glucan exactly, the most isolated and purified obtains high-purity
Araboxylan is used for follow-up study.
Compared with prior art, there is advantages that
1, the enzymolysis stage fully removes the macromole impurity such as starch, protein, then be separated off water solublity beta glucan and
Retain water-insoluble non-starch polysaccharides(nsp) composition, drastically increase purity and the extraction ratio of araboxylan；
2, after defatting step is placed in enzymolysis step, it is greatly enhanced defat efficiency and saves the time, reduce material resources consumption；
3, ion-exchange cellulose DE-32 is used to go the small-molecule substances such as depigmentation, glucose resin S-400 to separate difference
The unknown polysaccharide component of molecular weight, efficiency is high, effective；
4, by optimizing extracting method and technique thereof, success is isolated and purified from Semen avenae nudae grain obtains highly purified Arab
Xylan.
Accompanying drawing explanation
By the detailed description non-limiting example made with reference to the following drawings of reading, the further feature of the present invention,
Purpose and advantage will become more apparent upon:
Fig. 1 is the flow chart of araboxylan component in isolated and purified Semen avenae nudae grain；
Fig. 2 is to measure the gas chromatogram of monosaccharide composition in unknown polysaccharide component 1.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in detail.Following example will assist in those skilled in the art
It is further appreciated by the present invention, but limits the present invention the most in any form.It should be pointed out that, to those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make certain adjustments and improvements.These broadly fall into the guarantor of the present invention
Protect scope.
Embodiment 1
The present embodiment relates to a kind of extraction from Semen avenae nudae grain and the method for purification araboxylan, its flow chart such as figure
Shown in 1, specifically comprise the following steps that
Sort some without normal Semen avenae nudae grain such as pathological changes, grind, cross 0.4mm screen cloth；Weigh 10g powdered samples and add 90mL
Distilled water dissolves, and adds 50 μ L Thermostable α-Amylase, 80 DEG C of water-bath 1h；Bath temperature is adjusted to 60 DEG C, adds 15mg Fructus Chaenomelis egg
White enzyme, water-bath 1h；100 DEG C of water-bath enzyme denaturing 10min, add 400 μ L saccharifying enzyme, 60 DEG C of water-bath 1h after cooling；100 DEG C of water-bath enzyme denaturing
10min, cools down rear 86 DEG C of water-bath 4h；Enzymolysis solution 3000 leaves heart 15min, and supernatant is retained, and precipitate is standby.
Enzymolysis precipitate adds 83mL 0.5mol/LNaOH solution and dissolves, 86 DEG C of water-bath 4h；Dense HCl solution alkali tune extract pH
Value is to 6.5-6.8, and 3000 leave heart 15min；Precipitate discards, and supernatant is transferred to bag filter and is placed in clear water, often
12h changes water once, changes water totally 2 times；Supernatant after dialysis is slowly added dropwise 3 times of anhydrous second of volume under conditions of magnetic agitation
Alcohol, stands overnight；3000 leave heart 15min, and alcohol deposit fluid discards, and precipitate redissolves with appropriate distilled water, and 90 DEG C of water-bath evaporations are dense
It is reduced to about 10mL；-20 DEG C of freezings, vacuum lyophilization 24h, the pressed powder obtained is placed in Soxhlet extraction device, with just
Hexane makees solvent, repeatedly extracts 6-8h under 90 DEG C of water bath condition；40 DEG C of oven drying at low temperatures i.e. obtain Raw samples.
Taking 0.5g Raw samples and add distilled water dissolving, 3000 leave heart 15min；Supernatant is via DE-32 eluting, and flow phase
It is respectively distilled water and the NaCl solution of 1mol/L, under the conditions of flow velocity 0.2mL/min, presses 3mL/ pipe collect 100 pipes, Qi Zhongzheng
100 pipe filtrates of distilled water eluting are discarded；Phend-sulphuric acid has selected sugar test tube, merging filtrate；Dialyse in distilled water 36h,
Every 12h changes water once；90 DEG C of water-bath evaporation and concentration are to about 2mL.
Concentrated solution is again by S-400 eluting, and flowing is distilled water mutually, presses 3mL/ pipe and collect under the conditions of flow velocity 0.2mL/min
100 pipes；Phend-sulphuric acid has selected sugar test tube, and high performance liquid chromatograph (is equipped with Composition distribution Waters 2414, sugar post
SUGAR KS-805 Testing conditions: detector temperature 30 DEG C, column temperature 35 DEG C, flowing is distilled water mutually, and flow velocity is
The sugar composition of filtrate and purity during 1mL/min) sequentially determining is respectively arranged with sugar test tube, merge that single sugar is identical and purity > 90%
Filtrate, and by single sugar difference separate collection；90 DEG C of water-bath evaporation and concentration are to about 5mL；-20 DEG C of freezings, vacuum freezing is done
Dry 24h i.e. obtains purification of samples powder.
According to liquid chromatographic detection result understand this example isolated and purified go out two kinds of unknown polysaccharide components 1 and 2, its purity is respectively
Being 96.08% and 97.23%, the extraction ratio in Raw samples is respectively 13.8% and 14.1%；Fig. 2 is for measuring unknown polysaccharide group
Divide the gas chromatogram of monosaccharide composition in 1, the gas chromatographic detection result of Fig. 2 understand unknown polysaccharide component 1 main by me
Uncle's sugar and xylose form, wherein arabinose: xylose: the ratio of glucose is 28.9: 24.4: 1, for araboxylan.Do
During Monosaccharide analysis, find unknown polysaccharide component 1 and the component 2 of same quality, the arabinose in component 2 and the content ratio of xylose
Component 1 much smaller 25 times of component 2 (component 1 be approximately).
Embodiment 2
Embodiment 2 is with the difference of embodiment 1: the elution flow rate of DE-32 with S-400 is different, i.e. DE-in embodiment 2
The elution flow rate of 32 and S-400 is 0.5mL/min.Other operation is with embodiment 1.
According to liquid chromatographic detection result understand this example the most isolated and purified go out two kinds of unknown polysaccharide components 1 and 2, its purity is divided
Being not 95.25% and 95.38%, the extraction ratio in Raw samples is respectively 2.7% and 4.9%；Tie according to gas chromatographic detection
Fruit understands unknown polysaccharide component 1 and is mainly made up of arabinose and xylose, wherein arabinose: xylose: the ratio of glucose is
22.4: 19.7: 1, for araboxylan.When doing Monosaccharide analysis, find unknown polysaccharide component 1 and component 2, the group of same quality
Arabinose and the content of xylose in points 2 more much smaller than component 1 25 times of component 2 (component 1 be approximately).
Comparative example 1
Comparative example 1 is with the difference of embodiment 1: the eluting order of DE-32 with S-400 is different, i.e. first uses in comparative example 1
S-400 eluting uses DE-32 eluting again.Other operation is with embodiment 1.
Understand this example according to liquid chromatography results and isolated and purified can not obtain the single unknown polysaccharide that purity is high, Raw samples
The extraction ratio of middle mixing polysaccharide is 8.9%, does not remake Monosaccharide analysis.
Comparative example 2
Comparative example 2 is with the difference of embodiment 1: enzymolysis final stage precipitate is different with the choice of supernatant, the most right
Ratio 2 discards precipitate and retains supernatant.In addition to this example is except omitting alkali liquor and dissolve the step for, other operation is with embodiment 1.
According to liquid chromatography results understand this example isolated and purified go out a kind of unknown polysaccharide, its purity is 98.86%, rough sample
Extraction ratio in product is 31.1%；Understand unknown polysaccharide according to gas chromatographic detection result to be mainly made up of glucose, Qi Zhongpu
Grape sugar: arabinose: xylose=39.8: 1: 1.4, (wherein araboxylan component accounts for polysaccharide should to be mainly beta glucan
The 6% of total amount).
Table 1 is araboxylan (AX) purity, extraction ratio and mixing polysaccharide extraction ratio contrast table
| Embodiment 1 | Embodiment 2 | Comparative example 1 | Comparative example 2 | |
| Unknown polysaccharide extract rate | 27.9% | 7.6% | 8.9% | 31.1% |
| AX extraction ratio | 13.8% | 2.7% | 0 | 0 |
| AX purity | 96.08% | 95.25% | -- | -- |
Above the specific embodiment of the present invention is described.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformation or amendment within the scope of the claims, this not shadow
Ring the flesh and blood of the present invention.
Claims (10)
1. one kind is extracted and the method for purification araboxylan from Semen avenae nudae grain, it is characterised in that described method include with
Lower step:
S1, enzymolysis: take Semen avenae nudae grain powder sample and add distilled water dissolving, be sequentially added into α-amylase, papain, saccharifying enzyme
Carrying out enzymolysis, enzymolysis solution is centrifuged；
S2, alkali carry: enzymolysis precipitate adds alkali liquor and dissolves, and regulation pH value is to 6.5～6.8, centrifugal；
S3, precipitate with ethanol: alkali puts on 2～3 times of volume dehydrated alcohol of dropping in clear liquid, stands overnight, centrifugal；
S4, vacuum lyophilization: precipitate with ethanol precipitate concentrates after redissolving with distilled water, vacuum lyophilization, obtains pressed powder；
S5, defat: with normal hexane as solvent, carry out surname extraction under 60～90 DEG C of water bath condition to pressed powder, and low temperature dries
Do to obtain Raw samples；
S6, DE-32 eluting: take Raw samples and add distilled water dissolving, centrifugal；Supernatant is via DE-32 eluting；Phenol one sulfuric acid process
Having selected sugar test tube, merging filtrate, 60～90 DEG C of water-bath evaporation and concentration obtain concentrated solution；
S7, S-400 eluting: concentrated solution is via S-400 eluting；Phenol one sulfuric acid process has selected sugar test tube, and sequentially determining is respectively arranged with sugar
The sugar composition of filtrate and purity in test tube, merge that single sugar is identical and the filtrate of purity ＞ 90%；60～90 DEG C of water-baths are steamed
Sending out and concentrate, vacuum lyophilization obtains purification of samples powder, according to liquid chromatographic detection result and the Monosaccharide analysis of purification of samples
Experiment obtains araboxylan.
2. extraction and the method for purification araboxylan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that step
In rapid S1, Semen avenae nudae grain powder sample is 10g: 60～90mL with the amount ratio of distilled water.
3. extraction and the method for purification araboxylan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that step
In rapid S1, be sequentially added into α-amylase, papain, saccharifying enzyme carry out enzymolysis particularly as follows: add the 50～100 high temperature resistant α of μ L-
Amylase, 80～100 DEG C of water-baths 1～2h；Bath temperature is adjusted to 60～80 DEG C, add 10～20mg papains, water-bath 1～
2h；100 DEG C of water-bath enzyme denaturing 10～20min, add 100～500 μ L saccharifying enzyme, 60～80 DEG C of water-baths 1～2h after cooling；100℃
Water-bath enzyme denaturing 10～20min, 60～90 DEG C of water-baths 2～4h after cooling.
4. extraction and the method for purification araboxylan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that step
In rapid S2, enzymolysis precipitate adds 60～90mL0.1～1.0mol/LNaOH solution dissolves, 60～90 DEG C of water-baths 2～4h；Dense HCl
Solution alkali tune extract pH value is to 6.5～6.8, centrifugal.
5. extraction and the method for purification araboxylan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that step
In rapid S3, it is that the alkali after dialysis puts on clear liquid that described alkali puts on clear liquid.
6. extraction and the method for purification araboxylan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that step
In rapid S4, precipitate with ethanol precipitate redissolves with 60～90mL distilled water, and 60～90 DEG C of water-bath evaporation and concentration are to about 5～10mL；-20 DEG C cold
After freezing, vacuum lyophilization 24～48h.
7. extraction and the method for purification araboxylan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that step
In rapid S5, described surname extraction is extracting 6～8h repeatedly；The temperature of described oven drying at low temperature is 30～50 DEG C.
8. extraction and the method for purification araboxylan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that step
In rapid S6, during DE-32 eluting, flow velocity is 0.1～1.0mL/min, and flowing is followed successively by distilled water and 0.5～1.0mol/L mutually
NaCl solution；The filtrate of distilled water eluting discards, and the filtrate of NaCl solution eluting is retained；The specification of chromatographic column is: 2.6cm x
30cm。
9. extraction and the method for purification araboxylan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that step
In rapid S7, during S-400 eluting, flow velocity is 0.1～1.0mL/min, and flowing is distilled water mutually；The specification of chromatographic column is: 1.6cm x
100cm。
10. extraction and the method for purification araboxylan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that
In step S7, the high performance liquid chromatograph sequentially determining being equipped with Composition distribution is used to be respectively arranged with the sugar group of filtrate in sugar test tube
Become and purity；During the detection of described high performance liquid chromatograph, Composition distribution model is Waters 2414, and sugar column type number is SUGAR
KS-805 Composition distribution temperature is 30 DEG C, and column temperature is 30～60 DEG C；Flowing be distilled water mutually, flow velocity be 0.5～
1.0mL/min。
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Cited By (6)
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| CN106674382A (en) * | 2017-01-13 | 2017-05-17 | 河南工业大学 | Method for extracting high-purity xylan from pawpaw fruits |
| CN106800609A (en) * | 2017-02-24 | 2017-06-06 | 天津挑战博德生物技术有限公司 | A kind of method that xylan is extracted from wheat bran |
| CN109164175A (en) * | 2018-07-04 | 2019-01-08 | 中国农业科学院北京畜牧兽医研究所 | A kind of feed non-starch polysaccharide component analysis method |
| CN111378055A (en) * | 2020-03-26 | 2020-07-07 | 南昌大学 | Method for continuously extracting and preparing non-starch polysaccharide from highland barley |
| CN114874344A (en) * | 2022-04-01 | 2022-08-09 | 上海交通大学 | Preparation method and application of alkali-extracted polysaccharide of highland barley tender leaves |
| CN115073621A (en) * | 2022-06-10 | 2022-09-20 | 江苏科技大学 | Preparation method of arabinoxylan |
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