CN103304678B - A kind of method utilizing multiplex-enzyme extraction Alfalfa Polysaccharide - Google Patents
A kind of method utilizing multiplex-enzyme extraction Alfalfa Polysaccharide Download PDFInfo
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- CN103304678B CN103304678B CN201310226122.8A CN201310226122A CN103304678B CN 103304678 B CN103304678 B CN 103304678B CN 201310226122 A CN201310226122 A CN 201310226122A CN 103304678 B CN103304678 B CN 103304678B
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Abstract
The present invention provides one to utilize the method that compound enzyme (cellulase, papain and pectase) extracts Alfalfa Polysaccharide, it is possible to reduce the Extracting temperature of Alfalfa Polysaccharide, improves the extraction ratio of Alfalfa Polysaccharide, is suitable for promoting the use of.The present invention determines the trial stretch of the factor affecting Alfalfa Polysaccharide extraction ratio by single factor experiment, orthogonal test is used to determine the optimum proportioning of compound enzyme, then Central Composite test is used to combine the relational expression that the response surface (RSM) meets with a response between value and influence factor, and the level of influence factor when being calculated maximum response, so that it is determined that the optimum extraction condition of multiplex-enzyme extraction Alfalfa Polysaccharide, i.e. cellulase 2.5% (weight), papain 2.0% (weight), pectase 3.0% (weight), Extracting temperature 52.7 DEG C, extract pH value 3.87, extract solid-to-liquid ratio 78.91, 2.73 hours extraction times.Under the conditions of empirical tests obtains this, the extraction ratio of Alfalfa Polysaccharide is more than 5.0%.
Description
Technical field
The present invention relates to the extracting method of a kind of active components of plants polysaccharide, utilize cellulase, papain and pectase to extract more particularly to one
The method of Alfalfa Polysaccharide.
Background technology
Alfalfa Polysaccharide (Alfalfa Polysaccharides) is the water soluble polysaccharide extracted from alfalfa stem and leaf, has multiple biological activity.A large amount of examinations
Verifying bright in a certain amount of dosage range, Alfalfa Polysaccharide can promote the growth of poultry, improves the immunity of poultry body, oxidation resistance and resists
Sick power.Additionally, Alfalfa Polysaccharide also has certain curative effect effect to treatment hypercholesterolemia, hyperglycemia, arteriosclerosis and myocardial infarction, more attach most importance to
Want is that body is had no side effect by Alfalfa Polysaccharide.Based on above advantage, health product and additive based on Alfalfa Polysaccharide will become new type of health food
Product and important a member of feed additive, thus improve people's life and promote the healthy aquaculture of animal.
Herba Medicaginis, as a kind of perennial high-quality draft leguminous forage, have the laudatory title of " King of Pasture ", extensively plants in countries in the world, and resource is the abundantest,
Production for Alfalfa Polysaccharide provides abundance and cheap raw material.But, the extraction process of Alfalfa Polysaccharide is not perfect, and current extracting method is main
For hot water alcohol deposition method, by continual heating, making plant cell wall soften and rupture, polysaccharide overflows.The method not only operating time is long, extracts
Rate is low, and continual high-temperature heating can destroy polysaccharide structures, reduces the biological function of polysaccharide, and therefore extraction process needs improvement badly.
Summary of the invention
It is an object of the invention to provide the method that one utilizes compound enzyme (cellulase, papain, pectase) to extract Alfalfa Polysaccharide, according to plant
The compositing characteristic of cell wall, utilizes cellulase, papain, pectase to make alfalfa cell wall splitting decomposition, contributes to polysaccharide and leaches.The party
Method can not only reduce temperature used during Polyose extraction, shortens extraction time, saves the energy, and can significantly improve Polyose extraction efficiency, is suitable for
Application.
The technical scheme that the present invention provides, it is characterised in that comprise the following steps that:
1, the alfalfa meal air-dried or dry is pulverized, cross 40 mesh sieves, apparatus,Soxhlet's utilizes petroleum ether or ether carry out ungrease treatment, until on
Clear liquid transparent clear, then 40 DEG C of drying obtain defat grass meal；
2, cellulase, papain and the pectase that the method uses is bought in Chemical Reagent Co., Ltd., Sinopharm Group, enzyme respectively 15000U/g alive,
6000U/mg、50U/g。
3, extracting degreasing grass meal, based on grass meal weight, adds 1.5-2.5% cellulase, 1.0-3.0% papain, 1.0-3.0% pectase, then
Add the disodium hydrogen phosphate-citrate buffer solution (pH=3.0-5.0) of 60-80 times of grass meal weight；
4, being placed in thermostat water bath, temperature is increased to 40-60 DEG C, keeps constant temperature later, extracts 2.0-3.0h, stirs every 20min during this period
Once, act on vacuum pump sucking filtration after terminating, collect filtrate, obtain polysaccharide extraction liquid；
5, polysaccharide extraction liquid boiling 5-10min, enzyme denaturing is lived；
6, polysaccharide extraction liquid Rotary Evaporators is concentrated at 60 DEG C the 1/5 of original volume, is subsequently adding the dehydrated alcohol of 4 times of volumes, under the conditions of 4 DEG C
Precipitation 12h；
7, by precipitate with ethanol solution centrifugal 10min under 4000rpm/min, lower sediment, i.e. Alfalfa Polysaccharide are collected；
8, dissolve polysaccharide precipitation, utilize phend-sulphuric acid to measure polyoses content under 490nm.
In such scheme, ethanol used is dehydrated alcohol.In order to calculate polysaccharide extract rate the most accurately, when extracting with sucking filtration, used container steams
Distilled water is washed 3 times, is incorporated to polysaccharide solution.The most retortable recovery of dehydrated alcohol used in above step gives over to use next time.
The present invention passes through test of many times, with Alfalfa Polysaccharide extraction ratio as index, utilizes single factor experiment to determine the extraction temperature when polysaccharide extract rate maximum successively
Degree, extraction pH value, solid-liquid ratio, extraction time, cellulase ratio, papain ratio and pectase ratio.Then, by orthogonal examination
Test and determine cellulase, papain and the optimum proportioning of pectase ratio.By response surface experiments (central composite design) optimize Extracting temperature,
Extract pH value, solid-liquid ratio, four factors of extraction time, so that it is determined that the optimum extraction condition of Alfalfa Polysaccharide, for exploitation and the life of Alfalfa Polysaccharide
Produce and foundation is provided.
The invention has the beneficial effects as follows, utilize compound enzyme (cellulase, papain, pectase) that plant cell wall is carried out splitting decomposition, it is easy to
Polysaccharide leaches, thus the extraction efficiency of polysaccharide is greatly improved, and reduces the Polyose extraction time simultaneously.Additionally, temperature used when reducing extraction,
Save the input of energy.Easy to operation, it is suitable for large-scale production.May advantageously facilitate Alfalfa Polysaccharide as health product and feed additive
Exploitation application.
Accompanying drawing explanation
Hereinafter, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:
Fig. 1 is Y=f (X1, X2) response surface solid scattergram (X3=70, X4=2.5h)；
Fig. 2 is Y=f (X1, X2) contour map (X3=70, X4=2.5h)；
Fig. 3 is Y=f (X1, X3) response surface solid scattergram (X2=4.0, X4=2.5h)；
Fig. 4 is Y=f (X1, X3) contour map (X2=4.0, X4=2.5h)；
Fig. 5 is Y=f (X1, X4) response surface solid scattergram (X2=4.0, X3=70)；
Fig. 6 is Y=f (X1, X4) contour map (X2=4.0, X3=70)；
Fig. 7 is Y=f (X2, X3) response surface solid scattergram (X1=50 DEG C, X4=2.5h)；
Fig. 8 is Y=f (X2, X3) contour map (X1=50 DEG C, X4=2.5h)；
Fig. 9 is Y=f (X2, X4) response surface solid scattergram (X1=50 DEG C, X3=70)；
Figure 10 is Y=f (X2, X4) contour map (X1=50 DEG C, X3=70)；
Figure 11 is Y=f (X3, X4) response surface solid scattergram (X1=50 DEG C, X2=4.0)；
Figure 12 is Y=f (X3, X4) contour map (X1=50 DEG C, X2=4.0)；
In Fig. 1 to Figure 12, X1Represent Extracting temperature (DEG C), X2Represent and extract pH value, X3Represent solid-liquid ratio, X4Represent extraction time (h), Y table
Show polysaccharide extract rate.
Detailed description of the invention
1, the alfalfa meal air-dried or dry is pulverized, cross 40 mesh sieves, apparatus,Soxhlet's utilizes petroleum ether or ether carry out ungrease treatment, until on
Clear liquid transparent clear, then 40 DEG C of drying obtain defat grass meal；
2, cellulase, papain and the pectase that the method uses is bought in traditional Chinese medicines group chemical reagent Beijing company limited, and enzyme is lived and is respectively
15000U/g、6000U/mg、50U/g。
3, extracting degreasing grass meal, based on grass meal weight, adds 2.5% cellulase, 2.0% papain, 3.0% pectase, is subsequently adding 70 times of grass meals
Disodium hydrogen phosphate-the citrate buffer solution (pH=3.87) of weight；
4, being placed in thermostat water bath, temperature is increased to 52.7 DEG C, keeps constant temperature later, extracts 2.73h, stirs once every 20min during this period,
With vacuum pump sucking filtration after effect, collect filtrate, obtain polysaccharide extraction liquid；
5, polysaccharide extraction liquid boiling 6min, enzyme denaturing is lived；
6, polysaccharide extraction liquid Rotary Evaporators is concentrated at 60 DEG C the 1/5 of original volume, is subsequently adding the dehydrated alcohol of 4 times of volumes, under the conditions of 4 DEG C
Precipitation 12h；
7, by precipitate with ethanol solution centrifugal 10min under 4000rpm/min, lower sediment is collected；
8, distilled water dissolves polysaccharide precipitation, utilizes phend-sulphuric acid to measure polyoses content under 490nm more than 5.0%.
Claims (6)
1. the method utilizing multiplex-enzyme extraction Alfalfa Polysaccharide, the method utilizes cellulase, Fructus Chaenomelis
Protease, pectase process alfalfa meal simultaneously and make alfalfa cell wall splitting decomposition and simultaneously with phosphoric acid hydrogen
Disodium-citrate buffer solution extraction obtains polysaccharide extraction liquid, then obtains precipitate with ethanol solution by ethanol precipitation,
Alfalfa Polysaccharide is obtained after Li Xin；
Wherein, based on grass meal weight, the enzyme cellulase additional proportion for 15000U/g alive is 1.5%
-2.5%, enzyme live for the papain additional proportion of 6000U/mg be 1.0%-3.0%, enzyme work be
The pectase additional proportion of 50U/g is 1.0%-3.0%；
Wherein, extraction temperature is 40～60 DEG C, keeps constant temperature.
Method the most according to claim 1, it is characterised in that at phosphoric acid described in leach step
The addition of disodium hydrogen-citrate buffer solution is 60-80 times of grass meal weight.
Method the most according to claim 2, it is characterised in that disodium hydrogen phosphate-citric acid delays
The pH rushing liquid is 3.0-5.0.
Method the most according to claim 1, it is characterised in that extraction time is 2.0-3.0h.
Method the most according to claim 4, it is characterised in that by polysaccharide extraction liquid with rotating steaming
Send out instrument at 60 DEG C, be concentrated into the 1/5 of original volume, be subsequently adding the dehydrated alcohol of 4 times of volumes, 4 DEG C of bars
12h is precipitated under part.
Method the most according to claim 5, it is characterised in that precipitate with ethanol solution is existed
Under 4000rpm/min, centrifugal 10min, collects lower sediment.
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103980371B (en) * | 2014-04-22 | 2016-06-08 | 中国农业科学院北京畜牧兽医研究所 | The Preparation method and use of Alfalfa Polysaccharide |
| CN104082820A (en) * | 2014-06-10 | 2014-10-08 | 秋实草业有限公司 | Hypoglycemic soluble granules containing alfalfa |
| CN105524186B (en) * | 2016-02-21 | 2018-01-30 | 张桂娟 | A kind of preparation method of Aralia mandshurica bud polysaccharide |
| CN108191989A (en) * | 2017-12-29 | 2018-06-22 | 昭通学院 | A kind of method of combined-enzyme method extraction kidney tea polysaccharide |
| CN110790846B (en) * | 2019-10-14 | 2022-03-04 | 沈阳化工大学 | Preparation of biologically active alfalfa stem and leaf polysaccharide and selenized modified polysaccharide thereof |
| LU101969B1 (en) | 2020-08-05 | 2022-02-07 | Luxembourg Inst Science & Tech List | Method of extraction of a galactomannan gum from alfalfa seeds |
| CN113854212A (en) * | 2021-10-29 | 2021-12-31 | 海南归耘田农业科技有限公司 | Directional cultivation method for crayfish seedlings |
Citations (2)
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| CN102212144A (en) * | 2011-04-15 | 2011-10-12 | 沈阳农业大学 | Method for preparing pure polysaccharose from alfalfa hay |
| JP5172095B2 (en) * | 2006-02-14 | 2013-03-27 | ユーシーシー上島珈琲株式会社 | Method for producing polysaccharides from coffee beans and / or coffee extraction residue |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5172095B2 (en) * | 2006-02-14 | 2013-03-27 | ユーシーシー上島珈琲株式会社 | Method for producing polysaccharides from coffee beans and / or coffee extraction residue |
| CN102212144A (en) * | 2011-04-15 | 2011-10-12 | 沈阳农业大学 | Method for preparing pure polysaccharose from alfalfa hay |
Non-Patent Citations (1)
| Title |
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| "苜蓿多糖提取工艺研究";杨丽娥等;《上海交通大学学报（农业科学版）》;20021230;第20卷;第6-10、18页 * |
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