CN107141369B - A kind of preparation method of modified pectin - Google Patents

Abstract

The invention belongs to bioactive polysaccharide preparation technical fields, disclose a kind of preparation method of modified pectin.The preparation method is that: pectin is add to deionized water, is mixed, ultrasonic disperse, stirring and dissolving is uniform, obtains pectin solution；It is 7~12 with the pH value that alkali adjusts pectin solution, hydrogen peroxide is then added dropwise, under ultraviolet light, is stirred to react at room temperature；It is adjusted with acid reaction solution pH after the reaction was completed to neutrality, is then added in ethanol-water solution, stirs, standing sedimentation, filter；Solid product is washed, it is dry after purify, obtain modified pectin.The present invention uses ultraviolet catalytic-hydrogen peroxide oxidation method of modifying, the degradation of energy one step realization pectin and de- ester, has the advantages that the reaction time is short, pollution-free, product recoveries are high, low energy consumption.

Description

A kind of preparation method of modified pectin

technical field

The invention belongs to the technical field of bioactive polysaccharide preparation, and in particular relates to a preparation method of modified pectin.

Background technique

Pectin is a class of macromolecular acidic polysaccharides with complex structure, which widely exists in nature, especially in the cell walls of land plants. Almost all plants contain pectin, citrus, lemon, grapefruit and other peels contain about 30% pectin, which is the most abundant source of pectin. The FAO/WHO Joint Committee on Food Additives recommends citrus pectin as a safe natural food additive and does not limit its daily intake. In 2006, the world's annual consumption of pectin was about 45 million kilograms, and it was increasing year by year. At present, pectin has been widely used in food, cosmetics, chemical industry, textile, medicine and other fields, and has played an important role in improving people's life. In recent years, the biological activity of pectin has attracted widespread attention. As a dietary fiber, pectin can reduce serum cholesterol and blood sugar levels, stimulate phage cells and macrophages, proliferate splenocytes, resist complement activity, and inhibit hyaluronic acid. Various physiological and nutritional effects such as release of acidase and histamine, endotoxin-induced inflammatory response, and prevention of cancer occurrence and metastasis.

Due to the large molecular weight of natural pectin, it is not easy to be digested and absorbed, thus limiting its biological functions and other applications. Therefore, the research on the modification of pectin has become a hot topic in recent years. Currently, GCS-100 and Pectasol-C, clinical anti-cancer products that have attracted much attention, are both Modified Pectin (MP), but their preparation technology is still protected by US patents. The modification of pectin is a structural modification method whose main purpose is to reduce the molecular weight and the degree of esterification. By reducing the molecular weight and esterification degree of pectin modification, pectin can be absorbed in the blood and small intestine, and the retention time in the body can be increased. More and more studies have confirmed that modified pectin in the human body can cause anti-tumor metastasis by recognizing galectin-3 (Gal-3) expressed on the surface of tumors.

The methods for modifying pectin include: chemical method, enzymatic method and physical method. Chemical modification is the most commonly used modification method of citrus pectin, including alkaline deesterification, amidation deesterification, acid deesterification and acid degradation of pectin molecular weight. On the one hand, chemical modification has the problem of environmental pollution caused by the extensive use of strong acid and strong alkali; on the other hand, the chemical process is complicated, the reaction conditions are harsh, and the degradation efficiency of pectin molecular weight is low. The enzymatic method uses pectinase to reduce the molecular weight of pectin and the degree of esterification by pectin methylesterase, which has the advantages of good specificity and mild reaction conditions, but the enzyme used is expensive. Physical methods include high-temperature, high-pressure treatment or mechanical shearing. The physical method has a simple process, but the product has poor stability and consumes a lot of energy. Related patents on modified pectin: pectin modification method and its application (CN200680020291.4), using heat treatment method; modified beet pectin and its application (CN201080004412.2), using high-pressure modification; A method for preparing pectin oligosaccharides by dynamic high-pressure micro-jet combined with acid method (CN201210012614.2); preparation process and anti-tumor application of high bioavailability modified pectin (CN201110200381.4); a modified pectin Preparation process and its anti-tumor effect (CN201410698032.3); a preparation method of pectin acid oligosaccharides (CN200710059738.5); preparation method and application of pectin enzymatic hydrolyzate with hangover and anti-drunk functions (CN201210406190. 8); enzymatic preparation method of low molecular weight pectin (CN201310380333.7); the above patents all adopt enzymatic method to prepare modified pectin or low molecular weight pectin.

The oxidative degradation of polysaccharides by hydrogen peroxide is based on the process of hydrogen peroxide attacking the glycosidic bonds in the sugar chains, which undergoes a reduction reaction to generate hydroxyl radicals, and the generated hydroxyl radicals start more redox reactions, making the sugar chains Oxidation and reduction lead to the breakage of sugar chains and produce low-weight sugar substances, thereby achieving the degradation of polysaccharides. However, depending on hydrogen peroxide alone, the reaction efficiency is low, the oxidation process is uncontrollable, and there are many by-products. Degradation of chitosan, the degradation efficiency is not high; there are studies to improve the oxidation efficiency through the synergistic effect of various oxidants, such as Dr. Hydrogen + vitamin C oxidation system. There are also documents that use physical fields to strengthen the process of hydrogen peroxide oxidative degradation of polysaccharides, such as the document: Ultrasound-assisted hydrogen peroxide oxidative degradation to prepare relatively low molecular weight Eucheuma polysaccharides (Ma Xiajun, etc., Chinese Journal of Marine Medicine, 2005, 24:10- 13). Microwave-assisted hydrochloric acid/hydrogen peroxide degradation of Enteromorpha polysaccharides and its antioxidant activity (Duan Ke et al., Food Science and Technology, 2015: 142-147), microwave radiation can promote the dissociation of hydrochloric acid and hydrogen peroxide, thereby reducing the amount of hydrochloric acid used, Improve the utilization rate of hydrogen peroxide. In addition, microwaves can reduce the activation energy of the reaction, significantly increase the degradation rate, and greatly shorten the reaction time.

Contents of the invention

In view of the above shortcomings and deficiencies in the prior art, the object of the present invention is to provide a method for preparing modified pectin.

The object of the invention is achieved through the following technical solutions:

A preparation method of modified pectin, comprising the following preparation steps:

(1) adding pectin to deionized water, mixing, ultrasonically dispersing, stirring and dissolving evenly, to obtain a pectin solution;

(2) Adjust the pH value of the pectin solution to 7-12 with alkali, then add hydrogen peroxide dropwise, and stir and react at room temperature under ultraviolet light irradiation;

(3) Adjust the pH of the reaction solution in step (2) to neutral with an acid, then add it to an ethanol-water solution, stir, settle, and filter; the solid product is washed, dried, and purified to obtain modified pectin.

The pectin described in the step (1) is a high-ester pectin with a degree of esterification greater than 50%, and the source of the pectin is citrus, apple, lemon or grapefruit, preferably citrus pectin.

The mass volume ratio of pectin and deionized water described in step (1) is (1-10): 1 mg/ml; preferably (3-8): 1 mg/ml.

The ultrasonic dispersion described in step (1) uses a probe-type ultrasonic instrument with an ultrasonic power of 200W and an ultrasonic time of 10-20 minutes. During the ultrasonic process, ice-water mixture is used for cooling.

The alkali described in step (2) is preferably NaOH solution.

The hydrogen peroxide in the step (2) is a hydrogen peroxide aqueous solution with a volume fraction of 30%, and the consumption of the hydrogen peroxide aqueous solution is 50-500 μL/g pectin.

Preferably, the power of the ultraviolet light in step (2) is 500w, and the wavelength is 365nm.

The stirring speed of the stirring reaction in step (2) is 80-200 rpm, and the stirring reaction time is 1-7 h.

After the stirring reaction is completed, the method for terminating the reaction is to turn off the UV light source, or cool down at 4°C+turn off the UV light source (the latter is suitable for reactions where the amount of hydrogen peroxide aqueous solution exceeds 300 μl/g pectin).

Preferably, the acid described in step (3) refers to 3mol/L hydrochloric acid.

Preferably, in the ethanol-water solution described in step (3), the volume percentage of ethanol is 70%-90%.

Preferably, the washing described in step (3) refers to washing with ethanol.

Preferably, the purification process in step (3) is: dissolving the dried product in water, membrane dialysis for desalting, concentration and drying. The drying is one of vacuum drying, spray drying and freeze drying; the dialysis refers to dialysis with a dialysis membrane with a molecular weight cut off of 800Da.

The principles of the present invention are as follows: firstly, hydrogen peroxide generates free radicals under specific conditions, and free radicals degrade pectin glycosidic bonds, causing pectin to produce a modification in which the molecular weight is reduced. Therefore, pectin degradation is a free radical oxidative degradation reaction; Second, deesterification of pectin occurs under neutral or alkaline conditions to realize the deesterification of pectin; third, ultraviolet light irradiation accelerates the generation of free radicals and improves the degradation efficiency of pectin; fourth, ultraviolet light Light irradiation accelerates the deesterification reaction of pectin. Therefore, the present invention can achieve the dual purposes of pectin degradation and deesterification through a one-step process, and realize the efficient preparation of modified pectin.

Compared with the prior art, the preparation method of the present invention has the following advantages and beneficial effects:

(1) The average molecular weight of the modified pectin obtained by the method of the present invention is 20,000-200,000 Da, the degree of esterification is 17-25%, the yield is greater than 82%, and the content of uronic acid is greater than 64%. It shows that the present invention is an economical and efficient pectin modification method. The obtained modified pectin has good application potential in the fields of medicine, health food and the like.

(2) Compared with the existing chemical method, enzymatic method and physical method, the method of the present invention has short reaction time, no pollution, high product recovery rate and low energy consumption.

(3) The prior art cannot realize the degradation of pectin molecular weight and the two degradation targets of pectin deesterification simultaneously, or the problem of deesterification is not considered; and the preparation method of the present invention realizes the degradation and deesterification of pectin by one-step method Ester two modified targets.

(4) The method of the present invention is different from the existing polysaccharide oxidative degradation technology. The existing polysaccharide oxidative degradation technology adopts the traditional hydrogen peroxide degradation method, and the amount of hydrogen peroxide is large, which increases the difficulty of separation and purification of degradation products. The degradation temperature is relatively high, which is likely to cause discoloration of the product, affect the appearance of the product, and have poor stability and repeatability. However, the amount of hydrogen peroxide in the method of the present invention is greatly reduced, there is no hydrogen peroxide residue, and there are few side reactions.

(5) The method of the present invention is different from the existing polysaccharide oxidation degradation technology. Although the existing polysaccharide oxidation technology also considers the application of physical means to strengthen the hydrogen peroxide oxidation degradation polysaccharide, but because the mechanism of action of each physical means is different, Its effect is relatively limited. The invention adopts the ultraviolet light irradiation method to greatly shorten the reaction time, and the degradation degree of the molecular weight of the product and the degree of esterification is completely controllable.

Description of drawings

Fig. 1 is a process flow chart of the preparation method of modified pectin in the embodiment of the present invention.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1

Weigh 100mg of citrus pectin powder, add 20ml of deionized water, oscillate and mix evenly, insert the ultrasonic probe into the solution 2cm below, set the ultrasonic power to 200W, and ultrasonic time to 10min. Cool the pectin solution, and after the ultrasonic wave is over, continue magnetic stirring for 8 hours to fully dissolve the pectin to obtain a pectin solution; adjust the pH of the pectin solution to 12 with 0.5N NaOH solution to obtain an alkaline pectin solution; Add 20 μL of hydrogen peroxide dropwise to the glue solution, turn on the high-pressure mercury lamp, under the irradiation of 365nm ultraviolet light, stir and react at room temperature at 100rpm for 6h, then turn off the ultraviolet lamp, terminate the reaction, and obtain a degraded pectin solution (alkaline); use 0.3N HCl adjusted the reaction solution pH=7 to obtain a degraded pectin mixed solution (neutral); the resulting mixed solution was poured into 60 ml of ethanol-water solution with a volume percentage of 80% (V/V), stirred, and left to settle for 30 min. Filtrate under reduced pressure, and the resulting filtrate is distilled under reduced pressure to reclaim ethanol; the filter residue is washed with ethanol twice, dried in vacuum, and dissolved in 10ml of deionized water to obtain a degraded pectin solution. After dialysis in water for 3 days, the modified pectin solution after the dialysis was concentrated and vacuum-dried to obtain the modified pectin with a product yield of 87%. The process flow diagram of the above preparation method is shown in Figure 1.

The average molecular weight of the citrus pectin raw material before modification in this embodiment is 1225171, the molecular weight distribution width coefficient Mw/Mn=6.7, the degree of esterification is 72%, and the content of uronic acid is 75%; the modified pectin Mw216 obtained by the above method, 00Da, molecular weight distribution breadth coefficient Mw/Mn=2.2, esterification degree 19%, uronic acid content 72%. The modified pectin prepared by the above method has low degree of esterification and low molecular weight, and the distribution of molecular weight is narrower.

Example 2

Weigh 200mg of citrus pectin powder, add 20ml of deionized water, oscillate and mix well, insert the ultrasonic probe into the solution 2cm below, set the ultrasonic power to 200W, and ultrasonic time to 15min. Cool the pectin solution, and after the ultrasonic wave is over, continue magnetic stirring for 7 hours to fully dissolve the pectin to obtain a pectin solution; adjust the pH of the pectin solution to 12 with 0.5N NaOH solution to obtain an alkaline pectin solution; Add 30 μl of hydrogen peroxide dropwise to the glue solution, turn on the high-pressure mercury lamp, under the irradiation of 365nm ultraviolet light, stir the reaction at room temperature at 100rpm for 5h, then turn off the ultraviolet lamp, terminate the reaction, and obtain the degraded pectin solution (alkaline); use 0.3N HCl adjusted the reaction solution pH=7 to obtain a degraded pectin mixed solution (neutral); the resulting mixed solution was poured into 60 ml of ethanol-water solution with a volume percentage of 80% (V/V), stirred, and left to settle for 30 min. Filtrate under reduced pressure, and the resulting filtrate is distilled under reduced pressure to reclaim ethanol; the filter residue is washed with ethanol twice, dried in vacuum, and dissolved in 10ml of deionized water to obtain a degraded pectin solution. After dialysis in water for 3 days, the modified pectin solution after the dialysis was concentrated and vacuum-dried to obtain the modified pectin with a product yield of 92%. The process flow diagram of the above preparation method is shown in Figure 1.

The average molecular weight of the citrus pectin raw material before modification in this embodiment is 1225171, the molecular weight distribution width coefficient Mw/Mn=6.7, the degree of esterification is 72%, and the content of uronic acid is 75%; the modified pectin obtained by the above method is Mw65295Da, molecular weight The distribution width coefficient Mw/Mn=3.2, the degree of esterification is 23%, and the content of uronic acid is 71.4%. The modified pectin prepared by the above method is low-ester pectin, and the molecular weight distribution is narrower and more uniform than that of raw materials.

Example 3

Weigh 200mg of citrus pectin powder, add 20ml of deionized water, oscillate and mix well, insert the ultrasonic probe into the solution 2cm below, set the ultrasonic power to 200W, and ultrasonic time to 15min. Cool the pectin solution, and after the ultrasonic wave is over, continue magnetic stirring for 7 hours to fully dissolve the pectin to obtain a pectin solution; adjust the pH of the pectin solution to 12 with 0.5N NaOH solution to obtain an alkaline pectin solution; Add 30 μl of hydrogen peroxide dropwise to the glue solution, turn on the high-pressure mercury lamp, and under the irradiation of 365nm ultraviolet light, stir the reaction at a speed of 100 rpm at room temperature for 7 hours, then turn off the ultraviolet lamp, terminate the reaction, and obtain a degraded pectin solution (alkaline); use 0.3N HCl adjusted the reaction solution pH=7 to obtain a degraded pectin mixed solution (neutral); the resulting mixed solution was poured into 60 ml of ethanol-water solution with a volume percentage of 80% (V/V), stirred, and left to settle for 30 min. Filtrate under reduced pressure, and the resulting filtrate is distilled under reduced pressure to reclaim ethanol; the filter residue is washed with ethanol twice, dried in vacuum, and dissolved in 10ml of deionized water to obtain a degraded pectin solution. After 3 days of dialysis in water, the modified pectin solution after the dialysis was concentrated and vacuum-dried to obtain the modified pectin with a yield of 91.6%. The process flow diagram of the above preparation method is shown in Figure 1.

The average molecular weight of the citrus pectin raw material before modification in this embodiment is 1225171, the molecular weight distribution width coefficient Mw/Mn=6.7, the degree of esterification is 72%, and the content of uronic acid is 75%; the modified pectin obtained by the above method is Mw46285Da, molecular weight The distribution width coefficient Mw/Mn=2.8, the degree of esterification is 25%, and the content of uronic acid is 69%. The modified pectin prepared by the above method is a low-molecular-weight low-ester pectin, which has a lower molecular weight distribution coefficient and narrower molecular weight distribution than raw materials.

Example 4

Weigh 60mg of citrus pectin powder, add 20ml of deionized water, oscillate and mix well, insert the ultrasonic probe into the solution 2cm below, set the ultrasonic power to 200W, and ultrasonic time for 5min. During the ultrasonic process, use the ice-water mixture Cool the pectin solution, and after the ultrasonic wave is over, continue magnetic stirring for 8 hours to fully dissolve the pectin to obtain a pectin solution; adjust the pH of the pectin solution to 12 with 0.5N NaOH solution to obtain an alkaline pectin solution; Add 15 μl hydrogen peroxide dropwise to the glue solution, turn on the high-pressure mercury lamp, under the irradiation of ultraviolet light of 365nm, stir the reaction at room temperature at 100rpm for 5h, then turn off the ultraviolet lamp, terminate the reaction, and obtain the degraded pectin solution (alkaline); use 0.3N HCl adjusted the reaction solution pH=7 to obtain a degraded pectin mixed solution (neutral); the resulting mixed solution was poured into 60 ml of ethanol-water solution with a volume percentage of 80% (V/V), stirred, and left to settle for 30 min. Filtrate under reduced pressure, and the resulting filtrate is distilled under reduced pressure to reclaim ethanol; the filter residue is washed with ethanol twice, dried in vacuum, and dissolved in 10ml of deionized water to obtain a degraded pectin solution. After 3 days of dialysis in water, the modified pectin solution after the dialysis was concentrated and vacuum-dried to obtain the modified pectin with a product yield of 82%. The process flow diagram of the above preparation method is shown in Figure 1.

The average molecular weight of the citrus pectin raw material before modification in this embodiment is 1225171, the molecular weight distribution width coefficient Mw/Mn=6.7, the degree of esterification is 72%, and the content of uronic acid is 75%; the modified pectin obtained by the above method is Mw17739Da, molecular weight The distribution width coefficient Mw/Mn=2.0, the degree of esterification is 17%, and the content of uronic acid is 64%. The modified pectin prepared by the above method is low-ester and low-molecular-weight pectin, and compared with raw materials, the molecular weight distribution is more uniform.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (9)

1. a preparation method of modified pectin, is characterized in that comprising following preparation steps: (1) adding pectin to deionized water, mixing, ultrasonically dispersing, stirring and dissolving evenly, to obtain a pectin solution; (2) Adjust the pH value of the pectin solution to 7-12 with alkali, then add hydrogen peroxide dropwise, and stir and react at room temperature under ultraviolet light irradiation; (3) adjusting the pH of the reaction solution in step (2) to neutral with an acid, then adding it to an ethanol-water solution, stirring, settling, and filtering; the solid product is purified after washing and drying to obtain modified pectin; The ultrasonic dispersion described in step (1) uses a probe-type ultrasonic instrument with an ultrasonic power of 200W and an ultrasonic time of 10-20 minutes. During the ultrasonic process, ice-water mixture is used for cooling. 2. the preparation method of a kind of modified pectin according to claim 1 is characterized in that: the pectin described in step (1) is the high-ester pectin that degree of esterification is greater than 50%, and pectin source is Citrus, apple, lemon or grapefruit. 3. the preparation method of a kind of modified pectin according to claim 1, is characterized in that: the mass volume ratio of pectin described in step (1) and deionized water is (1～10):1mg/ ml. 4. the preparation method of a kind of modified pectin according to claim 1 is characterized in that: the hydrogen peroxide described in step (2) is the hydrogen peroxide aqueous solution that volume fraction is 30%, and the consumption of hydrogen peroxide aqueous solution is 50～500 μ L/ g pectin. 5. the preparation method of a kind of modified pectin according to claim 1, is characterized in that: the power of ultraviolet light described in step (2) is 500w, and wavelength is 365nm. The preparation method of a kind of modified pectin according to claim 1, characterized in that: the stirring speed of the stirring reaction in the step (2) is 80-200rpm, and the stirring reaction time is 1-7h. 7. the preparation method of a kind of modified pectin according to claim 1 is characterized in that: the alkali described in step (2) refers to NaOH solution; The acid described in step (3) refers to 3mol/ L of hydrochloric acid. 8. The preparation method of a kind of modified pectin according to claim 1, characterized in that: in the ethanol-water solution described in step (3), the volume percentage of ethanol is 70% to 90%. 9. the preparation method of a kind of modified pectin according to claim 1 is characterized in that: the washing described in step (3) refers to washing with ethanol; The process of described purification is: the product after drying Dissolving in water, membrane dialysis for desalination, concentration, and drying; the drying is one of vacuum drying, spray drying, and freeze drying; the dialysis refers to dialysis with a dialysis membrane with a molecular weight cut-off of 800Da.