CN106893001B - A kind of preparation method of the Ultra-low molecular weight pectin rich in RG-I - Google Patents
A kind of preparation method of the Ultra-low molecular weight pectin rich in RG-I Download PDFInfo
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- CN106893001B CN106893001B CN201710097564.5A CN201710097564A CN106893001B CN 106893001 B CN106893001 B CN 106893001B CN 201710097564 A CN201710097564 A CN 201710097564A CN 106893001 B CN106893001 B CN 106893001B
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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
- C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
Abstract
The preparation method of the invention discloses a kind of Ultra-low molecular weight pectin rich in RG-I changes method by ultrasound-enhanced oxidation reaction, promotes the Fe generated in reaction system3+It is reduced to Fe2+, strengthen Fe2+It is reacted again with hydrogen peroxide, generates hydroxyl radical free radical, caused pectin sugar chain oxidation scission, and the galacturonic acid in Idiotype attack pectin, form the low molecular weight pectin for being rich in RG-I structural domain.The weight average molecular weight of gained pectin is mostly the Ultra-low molecular weight pectin for being rich in RG-I in 5-10kDa, esterification degree decline, product, and antioxidant activity is significantly higher than protopectin, and comprehensive utilization ratio improves.
Description
Technical field
The preparation method of the present invention relates to a kind of Ultra-low molecular weight pectin rich in RG-I, in particular to it is a kind of to utilize ultrasound
Assisted oxidation processing green, the quick method for preparing Ultra-low molecular weight pectin belong to polysaccharide degradation field.
Background technique
Mainly by D- galacturonic acid through a kind of acid heteroglycan of α-Isosorbide-5-Nitrae glucosides key connection, side chain contains pectin
The neutral sugars such as a large amount of rhamnose, arabinose and galactolipin.Pectin mainly includes equal polygalacturonic acid glycan (HG), sandlwood
Three structural domains of galacturonic acid glycan I (RG-I) and phammogalacturonane II (RG-II), are widely present in plant
In, it in apple, citrus, guava, floral disc of sunflower and sweet potato dregs, and is considered as a kind of dietary fiber of solubility, tool
Having reduces cholesterol, prevention diabetes, prevents fat and maintain the different physiological roles such as intestinal health.But pectin molecule amount is too
Greatly, it is not easy to be digested absorption, therefore limits its absorption in small intestine, influence the performance of its physiological activity.And it is modified super
Low molecular weight pectin dissolubility is good, is easily absorbed, and RG-I content increases, and can show stronger bioactivity, therefore open
The method for sending out depolymerized pectin novel, prepares modified low molecular weight pectin and is of great significance.
The method of pectin degrading mainly includes biodegrade, mechanical degradation and chemical degradation at present.Enzymic reaction temperature
With, specificity height, but severe reaction conditions, higher cost.Physical degradation methods mainly have ultrasonotomography, radiation degradation, microwave drop
Solution etc., radiation method degradation efficiency highest, but molecular structure is destroyed greatly, and safety has dispute, it is restricted its application；
Microwave degradation, which is also easy to produce high temperature, influences catabolite bioactivity；Ultrasonotomography is small to the destruction of lytic activity structure, but it is degraded
Ability is limited, and pectin degrading to 30kDa or so cannot still can only be absorbed by the body.Chemical degradation mainly includes acid drop at present
Solution and alkaline degradation, but since strong acid, highly basic etc. is added, easily cause environmental pollution, and reaction rate is slower, and it is general at high temperature into
Row reaction, therefore influence to destroy larger, reduction bioactivity on structure.
Summary of the invention
It is an object of the invention to improve at present preparation RG-I type Ultra-low molecular weight pectin technology, provide it is a kind of green,
The preparation method of safety, quickly the Ultra-low molecular weight pectin rich in RG-I.
Realize the main feature of production technology of the present invention the following steps are included:
(1) distilled water is taken, appropriate HCl solution adjustment pH is added between 3-5, FeCl is added2·4H2O、FeSO4·
7H2O、Cu(CH3COO)2·H2O or CuSO4·5H2O is configured to the solution A that concentration of metal ions is 0.5-5mmol/L；
(2) pectin is taken to be dissolved in solution A, so that the concentration of pectin is 2-10mg/mL, the concussion of vortex oscillation instrument keeps its complete
Fully dissolved；
(3) 5mol/LH is added in the pectin solution that step 2 is prepared2O2Solution makes metal ion and hydrogen peroxide mole
Concentration ratio is 1:20, is transferred in 0-50 DEG C of environment and is ultrasonically treated, ultrasound intensity 1.5-20W/mL.
(4) after being ultrasonically treated, sodium hydrogensulfite is added, terminates reaction；Then it is replaced by cation exchange resin column molten
Cation in liquid；
(5) 4000r/min centrifugation is carried out to step 4 treated solution, takes supernatant, adjust supernatant pH to 5-8；
(6) by the bag filter of step 5 treated supernatant is transferred to 500Da, flowing water is dialysed for 24 hours, and pure water is dialysed 12h,
After place it in vacuum refrigerating machine and be lyophilized, obtain the Ultra-low molecular weight pectin rich in RG-I.
Further, in the step 2, the concentration of pectin is 5mg/mL.
Further, in the step 3, the environment temperature of ultrasonic treatment is 30 DEG C, ultrasound intensity 3-5W/mL
Further, in the step 5, supernatant pH preferably 6.
The beneficial effects of the present invention are:
(1) present invention is provides a kind of green, safety, fast degradation method, yield rich in RG-I Ultra-low molecular weight pectin
It can reach 65% or more, products obtained therefrom purity is higher, quality is stable, at low cost.
(2) degradation technique is simple, and the reaction time is short, and resin used is reusable, and process conditions are environment friendly and pollution-free.
(3) weight average molecular weight of present invention gained pectin is in 5-10kDa, and esterification degree decline, product is mostly to be rich in RG-I's
Ultra-low molecular weight pectin, antioxidant activity are significantly higher than protopectin, and comprehensive utilization ratio improves.
Specific embodiment
The present invention promotes the Fe generated in reaction system (such as formula 1) by ultrasound-enhanced oxidation reaction3+It is reduced to Fe2 +, strengthen Fe2+It is reacted again with hydrogen peroxide, generates hydroxyl radical free radical, cause pectin sugar chain oxidation scission, and Idiotype is attacked
Galacturonic acid in pectin forms the low molecular weight pectin for being rich in RGI structural domain.
Fe2++H2O2→Fe3++·OH+OH-
Below with reference to specifically example is applied, the present invention is further elaborated.
Embodiment 1:
(1) 25mL distilled water is taken, 3mol/L HCl solution is added, is 3.0 with pH meter adjustment pH, FeCl is added2·4H2O,
It is configured to 0.5mmol/L Fe2+Solution A；
(2) it takes 60mg pectin (Sigma company) to be dissolved in 25ml solution A, after completely dissolution, it is (interior to be transferred to ultraphonic pipe
Diameter 3cm) in；
(3) it places it in 30 DEG C of thermostats, ultrasonic probe is placed under liquid level at 1.5cm and is ultrasonically treated, simultaneously
50 μ L 5mol/LH are added2O2Solution sets ultrasound intensity 1.5W/mL, is ultrasonically treated 40min；
(4) excessive sodium hydrogensulfite is added in degradation solution, restores extra hydrogen peroxide, to terminate reaction；
(5) step 4 treated solution is passed through into cation exchange resin column, coutroi velocity 0.5mL/min, so that positive
Ion exchange resin replaces metal cation therein；
(6) the solution 4000r/min after displacement is centrifuged, takes supernatant, adjust pH 6.0；
(7) above-mentioned solution is transferred to the bag filter flowing water dialysis of 500Da for 24 hours, pure water is dialysed 12h, after place it in it is true
It is lyophilized in empty refrigerator；Pectin sample is taken to carry out molecular weight and monosaccharide composition measuring, the results are shown in Table 1, after processing, molecular weight
6.32kDa is down to by 448.26kDa, pectin RG-I domain ratio increases, mainly based on RG-I structure.
The degradation of table 1 front and back pectin monosaccharide composition
The present embodiment passes through the degradation for having quickly completed pectin for 40 minutes, as can be seen from the table, the pectin after degradation
In oligosaccharides, galactose content is substantially reduced, and Rha/GalA ratio rises to 0.55 from 0.13, illustrate RG-I structural domain accounting point
Cloth increases, and (Gal+Ara)/Rha represents RG-I side chain lengths, and side chain lengths slightly decline, but relative to RG-I structural domain accounting
With galactonic acid acid content, change smaller.In addition, GalA/ (Fuc+Rha+GlcA+Ara+Gla+Xyl) ratio will from 2.20
To 0.56, illustrate that the pectin linearity is remarkably decreased, i.e., branch degree increases, and can deduce combination of ultrasound oxidation drop from information above
Solution, mainly acts on the domain HG in pectin, causes the reduction of the domain HG content, and RG-I structure preferably retains, and is rich in prepare
RG-I pectin.
Embodiment 2:
(1) 100mL distilled water is taken, 3mol/L HCl solution is added, is 4.0 with pH meter adjustment pH, FeCl is added2·4H2O,
It is configured to 2mmol/L Fe2+Solution A；
(2) it takes 500mg pectin (Sigma company) to be dissolved in 100ml solution A, after completely dissolution, is transferred to ultraphonic pipe
In (internal diameter 6cm)；
(3) it places it in 30 DEG C of thermostats, ultrasonic probe is placed under liquid level at 1.5cm and is ultrasonically treated, simultaneously
800 μ L high concentration H are added2O2Solution sets ultrasound intensity 4.0W/mL, is ultrasonically treated 30min；
(4) excessive sodium hydrogensulfite is added in degradation solution, restores extra hydrogen peroxide, to terminate reaction；
(5) step 4 treated solution is passed through into cation exchange resin column, coutroi velocity 1.0mL/min, so that positive
Ion exchange resin replaces metal cation therein；
(6) the solution 4000r/min after displacement is centrifuged, takes supernatant, adjust pH 6.0；
(7) above-mentioned solution is transferred to the bag filter flowing water dialysis of 500Da for 24 hours, pure water is dialysed 12h, after place it in it is true
It is lyophilized in empty refrigerator；Pectin sample is taken to carry out molecular weight and monosaccharide composition measuring, the results are shown in Table 2, after processing, molecular weight
7.29kDa is down to by 448.26kDa, pectin RG-I domain ratio increases, mainly based on RG-I structure.
The degradation of table 2 front and back pectin monosaccharide composition
The present embodiment passes through the degradation for having quickly completed pectin for 30 minutes, as can be seen from the table, the pectin after degradation
In oligosaccharides, galactose content is substantially reduced, and Rha/GalA ratio is increased to 0.53 from 0.13, illustrates RG-I structural domain accounting point
Cloth dramatically increases, and (Gal+Ara)/Rha represents RG-I side chain lengths, and side chain lengths slightly decline, but relative to RG-I structural domain
Accounting and galactonic acid acid content change smaller.In addition, GalA/ (Fuc+Rha+GlcA+Ara+Gla+Xyl) ratio from
2.20 near 0.58, illustrate that the pectin linearity declines, i.e., branch degree increases, and can deduce combination of ultrasound oxidation from information above
Degradation, mainly acts on the domain HG in pectin, causes the reduction of the domain HG content, and RG-I structure preferably retains, to prepare richness
Pectin containing RG-I.
Embodiment 3:
(1) 1000mL distilled water is taken, 3mol/L HCl solution is added, is 5.0 with pH meter adjustment pH, FeCl is added2·
4H2O is configured to 5mmol/L Fe2+Solution A；
(2) it takes 10g pectin (Sigma company) to be dissolved in 1000ml solution A, after completely dissolution, is transferred in ultrasonic tank；
(3) it places it in 40 DEG C of thermostats, and 20mL high concentration H is added2O2Solution sets ultrasound intensity 4.0W/mL,
It is ultrasonically treated 30min；
(4) excessive sodium hydrogensulfite is added in degradation solution, restores extra hydrogen peroxide, to terminate reaction；
(5) step 4 treated solution is passed through into cation exchange resin column, coutroi velocity 2.0mL/min, so that positive
Ion exchange resin replaces metal cation therein；
(6) the solution 4000r/min after displacement is centrifuged, takes supernatant, adjust pH 6.0；
(7) above-mentioned solution is transferred to the bag filter flowing water dialysis of 500Da for 24 hours, pure water is dialysed 12h, after place it in it is true
It is lyophilized in empty refrigerator；Pectin sample is taken to carry out molecular weight and monosaccharide composition measuring, the results are shown in Table 2, after processing, molecular weight
2.14kDa is down to by 448.26kDa, pectin RG-I domain ratio increases, mainly based on RG-I structure.
The degradation of table 3 front and back pectin monosaccharide composition
The present embodiment passes through the degradation for having quickly completed pectin for 30 minutes, as can be seen from the table, the pectin after degradation
In oligosaccharides, galactose content is substantially reduced, and Rha/GalA ratio is increased to 0.61 from 0.13, illustrates RG-I structural domain accounting point
Cloth dramatically increases, and (Gal+Ara)/Rha represents RG-I side chain lengths, and side chain lengths slightly decline, but relative to RG-I structural domain
Accounting and galactonic acid acid content change smaller.In addition, GalA/ (Fuc+Rha+GlcA+Ara+Gla+Xyl) ratio from
2.20 near 0.53, illustrate that the pectin linearity declines, i.e., branch degree increases, and can deduce combination of ultrasound oxidation from information above
Degradation, mainly acts on the domain HG in pectin, causes the reduction of the domain HG content, and RG-I structure preferably retains, to prepare richness
Pectin containing RG-I.
Claims (4)
1. a kind of preparation method of the Ultra-low molecular weight pectin rich in RG-I, which comprises the following steps:
(1) distilled water is taken, appropriate HCl solution adjustment pH is added between 3-5, FeCl is added2·4H2O、FeSO4·7H2O、Cu
(CH3COO)2·H2O or CuSO4·5H2O is configured to the solution A that concentration of metal ions is 0.5-5mmol/L；
(2) pectin is taken to be dissolved in solution A, so that the concentration of pectin is 2-10mg/mL, the concussion of vortex oscillation instrument keeps it completely molten
Solution；
(3) 5mol/LH is added in the pectin solution that step (2) are prepared2O2Solution keeps metal ion dense with hydrogen peroxide mole
Degree ratio is 1:20, is transferred in 0-50 DEG C of environment and is ultrasonically treated, ultrasound intensity 1.5-20W/mL, in ultrasonic procedure, is produced
Raw hydroxyl radical free radical, causes pectin sugar chain oxidation scission, and the galacturonic acid in specific attack pectin；
(4) after being ultrasonically treated, sodium hydrogensulfite is added, terminates reaction；Then by cation exchange resin column substitutional solution
Cation；
(5) 4000r/min centrifugation is carried out to step (4) treated solution, takes supernatant, adjust supernatant pH to 5-8；
(6) by the bag filter of step (5) treated supernatant is transferred to 500Da, flowing water is dialysed for 24 hours, and pure water is dialysed 12h, after
It places it in vacuum refrigerating machine and is lyophilized, obtain the Ultra-low molecular weight pectin rich in RG-I.
2. preparation method according to claim 1, which is characterized in that in the step (2), the concentration of pectin is 5mg/
mL。
3. preparation method according to claim 1, which is characterized in that in the step (3), the environment temperature of ultrasonic treatment
It is 30 DEG C, ultrasound intensity 3-5W/mL.
4. preparation method according to claim 1, which is characterized in that in the step (5), supernatant pH is 6.
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| WO2019061018A1 (en) * | 2017-09-26 | 2019-04-04 | 浙江大学 | Method for extracting pectin rich in rg-i |
| CN109400756B (en) * | 2018-11-26 | 2021-02-09 | 广州大学 | Preparation method for preparing agar oligosaccharide by hydroxyl radical reaction system |
| US11485799B2 (en) * | 2019-12-03 | 2022-11-01 | Zhejiang University | Ultrasonic-assisted method of extracting a pectin rich in RG-I |
| US11952436B2 (en) * | 2019-12-03 | 2024-04-09 | Zhejiang University | Ultrasound and pressure assisted method for extracting pectin rich in RG-I |
| CN111920705A (en) * | 2020-08-24 | 2020-11-13 | 钟卫倩 | Application of V' RG-I in cosmetics |
| WO2022217573A1 (en) * | 2021-04-16 | 2022-10-20 | 浙江大学 | Preparation method for pectin having high rg-i structure content |
| CN115181194B (en) * | 2022-06-21 | 2023-07-07 | 浙江大学 | Quick purification degradation and structure analysis method of pectin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104530255A (en) * | 2014-12-25 | 2015-04-22 | 浙江大学 | Method for preparing low-methoxy pectin by ultrasonic assisted acid process |
| CN104987433A (en) * | 2015-07-23 | 2015-10-21 | 中国科学院西北高原生物研究所 | Preparation method of RG-I type lycium barbarum pectin with anti-aging activity |
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| EP1153933B1 (en) * | 2000-05-12 | 2005-08-31 | Seiko Epson Corporation | Process for the manufacture of polyuronic acids |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104530255A (en) * | 2014-12-25 | 2015-04-22 | 浙江大学 | Method for preparing low-methoxy pectin by ultrasonic assisted acid process |
| CN104987433A (en) * | 2015-07-23 | 2015-10-21 | 中国科学院西北高原生物研究所 | Preparation method of RG-I type lycium barbarum pectin with anti-aging activity |
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