CN106117389A - Extract from Semen avenae nudae grain and the method for purification beta glucan - Google Patents
Extract from Semen avenae nudae grain and the method for purification beta glucan Download PDFInfo
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- CN106117389A CN106117389A CN201610712035.7A CN201610712035A CN106117389A CN 106117389 A CN106117389 A CN 106117389A CN 201610712035 A CN201610712035 A CN 201610712035A CN 106117389 A CN106117389 A CN 106117389A
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- beta glucan
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
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Abstract
The present invention discloses a kind of extraction from Semen avenae nudae grain and the method for purification beta glucan, described method is with Semen avenae nudae grain as raw material, through grinding, sieve, enzymolysis, precipitate with ethanol, vacuum lyophilization, defat obtain Raw samples, afford purification of samples via filler cellulose DE 32 and S 400 the most successively after its dissolving.Described method can the effective highly purified beta glucan of isolated from Semen avenae nudae grain, in its Raw samples, the extraction ratio of beta glucan is up to 31.1%, and purity is up to 98.86%.
Description
Technical field
The invention belongs to field of food, be specifically related to a kind of extraction from Semen avenae nudae grain and the method for purification beta glucan.
Background technology
Beta glucan (β-glucan) is a kind of linear without branch's viscous polysaccharide, and its different carbon head glycosidic bond connected mode is β
Type, is formed by connecting β-D-gluco-pyranose units by β-(1 → 3) and β-(1 → 4) glycosidic bond, is distributed widely in animals and plants
And microbial world.Beta glucan chemical constitution in corn is similar, but in molecule shared by β-(1 → 3) and β-(1 → 4) glycosidic bond
Ratio there are differences.The water solublity of beta glucan is relevant with the ratio of both glycosidic bonds.It is said that in general, water solublity beta glucan
The ratio of middle β-(1 → 4) glycosidic bond and β-(1 → 3) glycosidic bond is 2.3:1, rather than in water solublity beta glucan this ratio is relatively
High.According to report, beta glucan has: 1) reduces serum cholesterol, prevents cardiovascular disease；2) regulation blood sugar level, prevention
Diabetes；3) promote proliferation of intestinal probiotics, prevent intestinal cancer；4) regulating the effects such as body's immunity, 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.
Having scholar to point out, the beta glucan content in Semen avenae nudae is generally higher than the barley variety of other area plantation of the world.Cause
This beta glucan is also considered as in Semen avenae nudae the nutrient substance the closest with health and long-lived relation.About beta glucan
Extraction, the Extraction solvent of report has hot water, cold water, acid, alkali, dimethyl sulfoxide etc., Extracting temperature from room temperature to 100 DEG C,
Material-water ratio (m/V) between 1:10 to 1:50, extraction time 30min to 22h.Use for reference experts and scholars β-Portugal in corn is gathered
The research method of sugar, we optimize extracting method and technique thereof, success from Semen avenae nudae grain isolated and purified obtain highly purified β-
Glucosan.
Summary of the invention
Present invention aims to the deficiencies in the prior art, it is provided that a kind of extraction and purification β-Portugal from Semen avenae nudae grain
The method of polysaccharide.With Semen avenae nudae grain as raw material, through grinding, sieving, enzymolysis, precipitate with ethanol, vacuum lyophilization, defat obtain slightly
Sample preparation product, it affords purification of samples via filler cellulose DE-32 and S-400 after dissolving the most successively.Described method
Can the effective highly purified beta glucan of isolated from Semen avenae nudae grain, can be used for its structure elucidation, physicochemical property is identified and raw
Thing activity research etc..
For achieving the above object, the present invention uses below scheme:
The present invention relates to a kind of extraction from Semen avenae nudae grain and the method for purification beta glucan, comprise the following steps:
A1, pretreatment of raw material: cross 0.4mm screen cloth after being ground by the Semen avenae nudae grain selected, obtain powder sample；
A2, enzymolysis: powder sample is added distilled water and dissolves, be sequentially added into Thermostable α-Amylase, papain, saccharifying
Enzyme carries out enzymolysis, is centrifuged by enzymolysis solution, obtains supernatant a；
A3, precipitate with ethanol: in supernatant a, drip dehydrated alcohol, centrifugal after standing overnight, obtain precipitate with ethanol precipitate；
A4, vacuum lyophilization: precipitate with ethanol precipitate distilled water redissolves, 60～90 DEG C of water-bath evaporation and concentration, gained concentrates
At liquid-20 DEG C after freezing, vacuum lyophilization 24～48h, obtain pressed powder；
A5, defat: pressed powder is extracted under 60～90 DEG C of water bath condition repeatedly 6～8h；Then dry at 30～50 DEG C
Do and i.e. obtain Raw samples；
A6, eluting: take Raw samples and add after distilled water dissolves centrifugal, obtain supernatant b, supernatant b is first washed through DE-32
De-, then after S-400 eluting, i.e. can get high-purity beta-glucosan.
Preferably, in step A2, described enzymolysis specifically use following methods: powder sample upon dissolution adds resistance to
High-temperatureα-amylase, 80～100 DEG C of water-baths 1～2h；Bath temperature is adjusted to 60～80 DEG C, add papain, water-bath 1～
2h；100 DEG C of water-bath enzyme denaturing 10～20min, add saccharifying enzyme, 60～80 DEG C of water-baths 1～2h after cooling；100 DEG C of water-bath enzyme denaturing 10
～20min, after cooling, 60～90 DEG C of water-baths 2～4h, obtain enzymolysis solution.
Preferably, described solid sample is in terms of 10g, and Thermostable α-Amylase, papain, the addition of saccharifying enzyme are divided
It is not 50～100 μ L, 10～20mg, 100～500 μ L.
Preferably, in step A3, described supernatant a is 1:2～3 with the volume ratio of dehydrated alcohol.
Preferably, described centrifugal condition is: rotating speed 3000rmp, centrifugal 15min.
Preferably, in step A5, the solvent of described extracting is normal hexane.
Preferably, described when DE-32 eluting, flow velocity is 0.1～1.0mL/min, and flowing is distilled water mutually；Chromatographic column
Specification is: 2.6cm x 30cm.
Preferably, described when S-400 eluting, flow velocity is 0.1～1.0mL/min, and flowing is distilled water mutually；Chromatographic column
Specification is: 1.6cm x 100cm.
Preferably, described supernatant b is the filtrate of the single sugar of separate collection after S-400 eluting, by containing beta glucan
Filtrate is through 60～90 DEG C of water-bath evaporation and concentration to about 5mL；After-20 DEG C of freezings, vacuum lyophilization 24～48h i.e. obtains purification
Beta glucan.
Preferably, the sugar purity of the filtrate of described single sugar uses high performance liquid chromatograph to measure, the sugared post of employing
Model is SUGAR KS-805Detector 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.
Compared with prior art, the present invention has a following beneficial effect:
1. the enzymolysis stage fully removes the macromole impurity such as starch, protein, is separated off water-insoluble non-starch polysaccharides(nsp)
And retain soluble non-starch polysaccharide composition, drastically increase purity and the extraction ratio of beta glucan；
2., after defatting step is placed in enzymolysis step, it is greatly enhanced defat efficiency and saves the time, reduce material resources consumption；
3. use ion-exchange cellulose DE-32 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.
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 beta glucan in isolated and purified Semen avenae nudae grain；
Fig. 2 is that embodiment 1 measures the gas chromatogram of monosaccharide composition in beta glucan filtrate.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.Following example will assist in the technology of this area
Personnel are further appreciated by the present invention, but limit the present invention the most in any form.It should be pointed out that, the ordinary skill to this area
For personnel, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement.These broadly fall into the present invention
Protection domain.
Embodiment 1:
Present embodiments provide and a kind of extract and the method for purification beta glucan from Semen avenae nudae grain, flow process as it is shown in figure 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 precipitate is retained, and supernatant is standby.
Supernatant is slowly added dropwise 3 times of volume dehydrated alcohol under conditions of magnetic agitation, stands overnight；3000 leave the heart
15min, alcohol deposit fluid discards, and precipitate redissolves with appropriate distilled water, 90 DEG C of water-bath evaporation and concentration to about 10mL；-20 DEG C of freezings, very
Vacuum freecing-dry 24h, is placed in the pressed powder obtained in Soxhlet extraction device, makees solvent with normal hexane, 90 DEG C of water bath condition
Under repeatedly extract 6-8h；40 DEG C of oven drying at low temperatures i.e. obtain Raw samples, the crude polysaccharides powder that i.e. enzymolysis post processing obtains.
Taking 0.5g Raw samples and add distilled water dissolving, 3000 leave heart 15min；Supernatant is via DE-32 eluting, and flow phase
Under the conditions of flow velocity 0.2mL/min, press 3mL/ pipe for distilled water and collect 100 pipes；Phenol sulfuric acid procedure has selected sugar test tube, merges
Filtrate；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；Phenol sulfuric acid procedure has selected sugar test tube, uses gas chromatograph for determination to be respectively arranged with the sugar composition of sugar test tube kind greenery,
High performance liquid chromatograph is used (to be equipped with Composition distribution Waters 2414, sugar post SUGAR KS-805Detector bar
Part: detector temperature 30 DEG C, column temperature 35 DEG C, flowing be distilled water mutually, and flow velocity is 1mL/min) sequentially determining is respectively arranged with in sugared test tube
The sugar purity of filtrate, merges that single sugar is identical and purity > filtrate of 90%, and by single sugar difference separate collection；Institute
The beta glucan filtrate obtained, then through 90 DEG C of water-bath evaporation and concentration to about 5mL；-20 DEG C of freezings, vacuum lyophilization 24h and get final product
The beta glucan of purification.
Sugar composition according to gas chromatograph for determination beta glucan filtrate is as in figure 2 it is shown, contain substantial amounts of in this filtrate
Glucose.Further, using β-1, glucose is identified by 3 glucosan detection kit, and its result shows in this filtrate
Glucose be beta glucan.The purity understanding beta glucan according to liquid chromatographic detection result is 98.68%, obtains after enzymolysis
The extraction ratio of beta glucan that obtains after eluting of Raw samples be 31.1%.
Embodiment 2:
Present embodiments provide a kind of extraction from Semen avenae nudae grain and the method for purification beta glucan, with the side of embodiment 1
Method is essentially identical, and the elution flow rate differing only in DE-32 with S-400 is different, i.e. the eluting of DE-32 and S-400 in embodiment 2
Flow velocity is 0.5mL/min.
The present embodiment collect beta glucan filtrate according to gas chromatograph for determination its sugar composition understand, in this filtrate
Containing substantial amounts of glucose.Further, using β-1, glucose is identified by 3 glucosan detection kit, its result table
Glucose in this filtrate bright is beta glucan.Pure according to beta glucan prepared by the present embodiment knowable to liquid chromatographic detection result
Degree is 97.63%, and the extraction ratio of the beta glucan that the Raw samples obtained after enzymolysis obtains after eluting is 11.6%.With enforcement
Example 1 is compared, the purity of the present embodiment and embodiment 1 quite, but the extraction ratio of beta glucan that Raw samples obtains after eluting
Decline, this be due to elution flow rate slow when, each component the unknown polysaccharide press the size of molecular weight slowly via eluting outflow,
At this moment separating effect is relatively good；The when that elution speed being fast, each component the unknown polysaccharide eluting flows out and overlap, separating effect occurs
Being deteriorated, extraction ratio reduces the most naturally.
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.
The method using this comparative example can not isolate the sugar of one-component, and the polysaccharide that i.e. prepared by the present invention is many for mixing
Sugar, it is impossible to isolated and purified go out beta glucan.Raw samples extraction ratio of the mixing polysaccharide of gained after eluting is 27.6%.
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 beta glucan from Semen avenae nudae grain, it is characterised in that comprise the following steps:
A1, pretreatment of raw material: cross 0.4mm screen cloth after being ground by the Semen avenae nudae grain selected, obtain powder sample；
A2, enzymolysis: powder sample adds distilled water and dissolves, be sequentially added into Thermostable α-Amylase, papain, saccharifying enzyme enter
Row enzymolysis, is centrifuged enzymolysis solution, obtains supernatant a；
A3, precipitate with ethanol: in supernatant a, drip dehydrated alcohol, centrifugal after standing overnight, obtain precipitate with ethanol precipitate；
A4, vacuum lyophilization: precipitate with ethanol precipitate distilled water redissolves, 60～90 DEG C of water-bath evaporation and concentration, gained concentrated solution-20
At DEG C after freezing, vacuum lyophilization 24～48h, obtain pressed powder；
A5, defat: pressed powder is extracted under 60～90 DEG C of water bath condition repeatedly 6～8h；Then dry i.e. at 30～50 DEG C
Obtain Raw samples；
A6, eluting: take Raw samples and add after distilled water dissolves centrifugal, obtain supernatant b, by supernatant b first through DE-32 eluting, then
High-purity beta-glucosan is i.e. can get after S-400 eluting.
2. extraction and the method for purification beta glucan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that step A2
In, described enzymolysis specifically use following methods: powder sample upon dissolution adds Thermostable α-Amylase, 80～100
DEG C water-bath 1～2h；Bath temperature is adjusted to 60～80 DEG C, adds papain, water-bath 1～2h；100 DEG C of water-bath enzyme denaturing 10～
20min, adds saccharifying enzyme, 60～80 DEG C of water-baths 1～2h after cooling；100 DEG C of water-bath enzyme denaturing 10～20min, after cooling 60～90
DEG C water-bath 2～4h, obtains enzymolysis solution.
3. extraction and the method for purification beta glucan from Semen avenae nudae grain as claimed in claim 1 or 2, it is characterised in that institute
State solid sample in terms of 10g, Thermostable α-Amylase, papain, the addition of saccharifying enzyme be respectively 50～100 μ L, 10
～20mg, 100～500 μ L.
4. extraction and the method for purification beta glucan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that step A3
In, described supernatant a is 1:2～3 with the volume ratio of dehydrated alcohol.
5. as claimed in claim 1 extract from Semen avenae nudae grain and the method for purification beta glucan, it is characterised in that described from
The condition of the heart is: rotating speed 3000rmp, centrifugal 15min.
6. extraction and the method for purification beta glucan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that step A5
In, the solvent of described extracting is normal hexane.
7. extraction and the method for purification beta glucan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that described warp
During DE-32 eluting, flow velocity is 0.1～1.0mL/min, and flowing is distilled water mutually；The specification of chromatographic column is: 2.6cmx 30cm.
8. extraction and the method for purification beta glucan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that described warp
During S-400 eluting, flow velocity is 0.1～1.0mL/min, and flowing is distilled water mutually；The specification of chromatographic column is: 1.6cmx 100cm.
9. extraction and the method for purification beta glucan from Semen avenae nudae grain as claimed in claim 1, it is characterised in that on described
Clear liquid b is the filtrate of the single sugar of separate collection after S-400 eluting, the filtrate containing beta glucan is evaporated through 60～90 DEG C of water-baths
It is concentrated into about 5mL；After-20 DEG C of freezings, vacuum lyophilization 24～48h i.e. obtains the beta glucan of purification.
10. extraction and the method for purification beta glucan from Semen avenae nudae grain as claimed in claim 9, it is characterised in that described
The sugar purity of the filtrate of single sugar uses high performance liquid chromatograph to measure, and the sugared column type number of employing is SUGARKS-805Detector temperature is 30 DEG C, and column temperature is 30～60 DEG C；Flowing is distilled water mutually, and flow velocity is 0.5～1.0mL/min.
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Cited By (8)
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| CN109134695A (en) * | 2018-08-08 | 2019-01-04 | 劲牌生物医药有限公司 | A kind of alcohol-soluble beta glucan extract and preparation method thereof, application and health liquor |
| CN109182013A (en) * | 2018-08-08 | 2019-01-11 | 劲牌生物医药有限公司 | Improve the method and health liquor of health liquor ingredient and colour stability |
| CN109206535A (en) * | 2018-10-08 | 2019-01-15 | 江苏沿江地区农业科学研究所 | A method of preparing high-purity beta glucan from wheat wheat bran |
| CN109824796A (en) * | 2019-02-01 | 2019-05-31 | 四川省食品发酵工业研究设计院 | The extracting and developing purification process of beta-glucosan from oat bran |
| CN110229246A (en) * | 2019-06-06 | 2019-09-13 | 北京市营养源研究所 | The preparation method of beta glucan in a kind of highland barley vinasse |
| CN110897082A (en) * | 2019-11-27 | 2020-03-24 | 上海交通大学 | Preparation method of highland barley nutritional rice based on thin-layer coating technology |
| CN111410701A (en) * | 2020-05-12 | 2020-07-14 | 劲牌有限公司 | Highland barley β -glucan and purification process thereof |
| CN115725004A (en) * | 2022-11-23 | 2023-03-03 | 上海交通大学 | Preparation method and application of highland barley tender leaf enzyme-extracted polysaccharide |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109134695A (en) * | 2018-08-08 | 2019-01-04 | 劲牌生物医药有限公司 | A kind of alcohol-soluble beta glucan extract and preparation method thereof, application and health liquor |
| CN109182013A (en) * | 2018-08-08 | 2019-01-11 | 劲牌生物医药有限公司 | Improve the method and health liquor of health liquor ingredient and colour stability |
| CN109134695B (en) * | 2018-08-08 | 2021-06-08 | 劲牌生物医药有限公司 | Alcohol-soluble beta-glucan extract, preparation method and application thereof, and health-care wine |
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| CN110897082A (en) * | 2019-11-27 | 2020-03-24 | 上海交通大学 | Preparation method of highland barley nutritional rice based on thin-layer coating technology |
| CN111410701A (en) * | 2020-05-12 | 2020-07-14 | 劲牌有限公司 | Highland barley β -glucan and purification process thereof |
| CN115725004A (en) * | 2022-11-23 | 2023-03-03 | 上海交通大学 | Preparation method and application of highland barley tender leaf enzyme-extracted polysaccharide |
| CN115725004B (en) * | 2022-11-23 | 2023-10-31 | 上海交通大学 | Preparation method and application of highland barley tender leaf enzyme extracted polysaccharide |
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