Small-molecular low-esterification-degree pectin as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of pectin extraction, and particularly relates to micromolecular low-esterification-degree pectin as well as a preparation method and application thereof. The small-molecule low-esterification-degree pectin is prepared by adding a specific amount of pectinesterase and polygalacturonase, and the small-molecule low-esterification-degree pectin which is extracted and prepared from a sunflower disc through a biological enzymolysis method and has a weight-losing effect has an excellent weight-losing effect and can effectively reduce fat accumulation in the abdominal cavity and the liver.

Description

Technical Field

[0001] The present invention belongs to the technical field of pectin extraction, and more specifically, relates to small molecule low-esterified pectin and its preparation method and application. Background Art

[0002] Natural pectin is an acidic polysaccharide substance in plants, which is mainly composed of galacturonic acid, and is a white to light yellow powder with a slightly sour taste and a bad smell. Currently, high molecular pectin with a molecular weight above 100,000 Da is commonly used in the food processing industry, such as gelling agents, thickeners, stabilizers, suspending agents, emulsifiers, etc. Since natural pectin is a long-chain carbohydrate of polysaccharide with poor water solubility, and the application of high molecular pectin in biological activities is limited, it is widely used in the food industry.

[0003] In recent years, with many researchers discovering that small molecules with a molecular weight range of 5000 - 30000 Da or low-esterified pectin with an esterification degree below 50% have good biological activities in inhibiting tumors, reducing the toxic and side effects of radiotherapy and chemotherapy, lowering blood sugar and blood lipids, enhancing immunity, removing heavy metals, relieving alcohol intoxication and beautifying the skin, the market demand for small molecule and low-esterified modified pectin has gradually increased. However, the source of naturally extracted small molecule low-esterified pectin is single and its content in plants is small, so there are more and more reports on the preparation methods of small molecule low-esterified pectin. Currently, the commonly used methods include alkaline solution catalytic de-esterification method, acidified ethanol de-esterification method, and amidation method. However, these preparation methods are not easy to control the degree of reaction, and the chemical reagents used will also produce various harmful substances, affecting the application range and performance of the products. Summary of the Invention

[0004] The purpose of the present invention is to provide small molecule low-esterified pectin and its preparation method and application.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides a preparation method of small molecule low-esterified pectin, comprising the following steps:

[0007] S1. Mix sunflower disk micropowder with cellulase solution, react and then filter to collect the filtrate, and add anhydrous ethanol to the filtrate to precipitate sunflower disk pectin;

[0008] S2. Mix sunflower disk pectin with water, adjust the pH to acidic, add pectin esterase, after enzymatic hydrolysis reaction, adjust the pH to neutral, and add polygalacturonase for enzymatic hydrolysis reaction to obtain a pectin solution, and dialyze and freeze-dry the pectin solution to obtain small molecule low-esterified pectin;

[0009] The mass-volume ratio of the sunflower disk pectin, water, pectin esterase, and polygalacturonase is 0.5-1.5 g: 20-30 ml: 0.05-0.15 ml: 0.5-1 g.

[0010] The present invention controls the degree of pectin esterification by adding pectin esterase, controls the molecular weight of pectin by adding polygalacturonase, and combines a specific amount of pectin esterase and polygalacturonase to prepare a small molecule pectin with low degree of esterification. This small molecule pectin with low degree of esterification extracted from sunflower disks by enzymatic hydrolysis method has excellent weight loss effect.

[0011] Furthermore, the concentration of the cellulase solution is 0.008-0.024 g / ml.

[0012] Furthermore, the mass-volume ratio of the sunflower disk fine powder to the cellulase solution is 1-2 g: 5-50 ml.

[0013] Furthermore, the conditions for the mixed reaction are 30-40 °C and 1-4 h.

[0014] Furthermore, the volume ratio of the filtrate to absolute ethanol is 0.5-1.5: 1-10.

[0015] Furthermore, the enzymatic hydrolysis reaction conditions are enzymatic hydrolysis at 30-40 °C for 1-4 h.

[0016] The present invention also provides a small molecule pectin with low degree of esterification, which is prepared by the above preparation method.

[0017] The present invention also provides the application of the small molecule pectin with low degree of esterification in the preparation of drugs for treating fatty liver.

[0018] The present invention also provides the application of the small molecule pectin with low degree of esterification in the preparation of weight loss products.

[0019] The present invention has the following beneficial effects:

[0020] The present invention uses the enzymatic hydrolysis method, controls the degree of reaction esterification and molecular weight by controlling the amount of enzyme solution, combines a specific amount of pectin esterase and polygalacturonase to prepare a small molecule pectin with low degree of esterification, reduces the use of chemical reagents, and the prepared small molecule pectin with low degree of esterification has excellent weight loss effect and can be developed and applied as a new type of weight loss product. Description of the Drawings

[0021] Figure 1 It is a graph of different degrees of esterification of small molecule pectin with low degree of esterification with different amounts of pectin esterase added.

[0022] Figure 2 It is a graph of different molecular weights of small molecule pectin with low degree of esterification with different amounts of polygalacturonase added.

[0023] Figure 3 It is a graph of the body weight change of an obese mouse model, where A is the graph of body weight change and B is the graph of body weight growth rate.

[0024] Figure 4 It is a morphological diagram of the peritoneal fat of mice treated with different pectins.

[0025] Figure 5 It is a morphological diagram of the liver of mice treated with different pectins. Specific implementation manners

[0026] The present invention will be described in detail below with reference to specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0027] Example 1: Preparation method of small molecule low-esterified pectin.

[0028] The sunflower disk is crushed to obtain sunflower disk micropowder with a size of 80 mesh. The sunflower disk micropowder is mixed and reacted with a cellulase solution with a concentration of 0.01 g / ml. The mass-volume ratio of the micropowder to the cellulase solution is 1.5 g: 25 ml. The reaction temperature is 45 °C and the reaction time is 3 h. After the reaction, the filtrate is collected by filtration. Absolute ethanol is added to the filtrate to precipitate the pectin. The precipitate is collected and dried to obtain sunflower disk pectin.

[0029] Dissolve 2 g of sunflower disk pectin in 50 ml of deionized water, adjust the pH to 4 with citric acid and 1 M sodium hydroxide solution, then add 0.1 ml of pectin esterase, mix well and enzymatically hydrolyze at 35 °C for 3.5 h. Then adjust the pH to 6 with citric acid and 1 M sodium hydroxide solution, and add 0.8 g of polygalacturonase and enzymatically hydrolyze at 35 °C for 2 h. After dialysis for 24 h with a dialysis membrane (Beijing Bio-Atlas Technology Co., Ltd., MD4403), it is freeze-dried to obtain small molecule low-esterified pectin.

[0030] Verification Example 1: Influence of different enzyme dosages on the preparation effect of small molecule low-esterified pectin.

[0031] 1. Influence of different amounts of pectin esterase: In order to verify the influence of the amount of pectin esterase on the preparation of small molecule low-esterified pectin, the following experimental settings are carried out: Add 200 μL, 150 μL, 100 μL, 50 μL, 10 μL, 1 μL of pectin esterase for enzymatic hydrolysis, and the remaining steps are the same as in Example 1. The degree of esterification is measured by nuclear magnetic resonance hydrogen spectrum (NMH-R). The curve of enzyme addition amount and degree of esterification is fitted by polynomial.

[0032] 2. Effects of different amounts of polygalacturonase: To verify the effects of the amount of polygalacturonase on the preparation of low-esterified pectin with small molecules, the following experimental settings were carried out: 1.2 g, 0.8 g, 0.4 g, 0.2 g, and 0.1 g of polygalacturonase were added for enzymatic hydrolysis, and the remaining steps were the same as in Example 1. The molecular weight of the product was determined using a high-performance size exclusion chromatography-multi-angle laser light scattering-refractive index detector combined system. A polynomial was used to fit the curve of enzyme addition amount versus molecular weight.

[0033] 3. Experimental results: The experimental results are shown in Table 1, Table 2, Figure 1 and Figure 2 As shown, in this study, 0.8 g of polygalacturonase and 100 μL of pectin methylesterase were selected to enzymatically hydrolyze pectin to prepare low-esterified pectin with small molecules. It was detected that the molecular weight of the low-esterified pectin with small molecules of the present invention was less than 23,000 Da, and the degree of esterification was less than 3%.

[0034] Table 1: Degree of esterification of pectin obtained by enzymatic hydrolysis with different amounts of pectin methylesterase

[0035] Enzyme addition amount μL 200 150 100 50 10 1 Degree of esterification % 0.42 1.38 2.97 18.59 33.05 46.15

[0036] Table 2: Molecular weight of pectin obtained by enzymatic hydrolysis with different amounts of polygalacturonase

[0037] Enzyme addition amount g 1.2 0.8 0.4 0.2 0.1 Molecular weight Da 16701 22734 117585 243214 327417

[0038] Example 2: Verification of pectin efficacy

[0039] 1. Experimental method: Kunming mice were induced to become obese by feeding them a high-fat diet. Fifty Kunming mice (female, 2 months old) were divided into 5 groups: a control group (n = 10), a model group (n = 10), a commercially available low-esterified pectin group (n = 10) ( Pectin LM-101AS), a commercially available high-esterified pectin group (n = 10) ( PectinYM-115-H), and a low-esterified pectin with small molecules group (n = 10). The mice in the control group were fed with a normal diet, and the mice in each group except the control group were fed with a high-fat diet (60% fat). The mice in the model group were only fed with the high-fat diet. The mice in the commercially available low-esterified pectin group were intragastrically administered 200 mg / kg of the commercially available low-esterified pectin daily. The mice in the commercially available high-esterified pectin group were intragastrically administered 200 mg / kg of the commercially available high-esterified pectin daily. The mice in the low-esterified pectin with small molecules group were intragastrically administered 200 mg / kg of the low-esterified pectin with small molecules prepared in Example 1 daily. The experiment lasted for 8 weeks, and the body weight of the mice was monitored once a week. At the end of the experiment, the abdominal fat accumulation and liver morphology of the mice were observed, and pathological examinations of the mouse liver were performed using HE-stained sections.

[0040] 2. Experimental results: As Figure 3 shown, after feeding with high-fat diet, the body weight of mice increased significantly. After intragastric administration of pectin with different molecular weights to mice, the weight gain of mice slowed down. Among them, the body weight of mice treated with low-esterified pectin with small molecules was lower than that of other pectin treatment groups, indicating that the prepared low-esterified pectin with small molecules has a good weight loss effect. As Figure 4 shown, the abdominal fat of mice treated with low-esterified pectin with small molecules was less than that of the commercially available low-esterified pectin group, indicating that the prepared low-esterified pectin with small molecules has a certain effect on alleviating obesity. As Figure 5 shown, observing the livers of mice in each group, it was found that the appearance of the liver tissue in the model group was significantly pale, the edges were blunt and round, and the volume was enlarged with a greasy feeling, which was significantly improved after pectin intragastric administration. In order to observe the changes of microscopic cells in the liver, HE staining sections were used. It was found that a large number of lipid vacuoles appeared in the livers of mice in the model group, and some cell nuclei were squeezed to one side, accompanied by cell atrophy and edema. However, lipid droplets were basically invisible in the low-esterified pectin with small molecules group, while there were still a small number of lesions in the commercially available low-esterified pectin group and the commercially available high-esterified pectin group, indicating that the prepared low-esterified pectin with small molecules has a certain protective effect on fatty liver caused by obesity.

[0041] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To prevent redundancy, the preferred embodiments of the present invention are described.

[0042] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0043] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A preparation method of a small molecule pectin with low esterification degree, characterized in that, It includes the following steps: S1. Mix sunflower disk micropowder with cellulase solution, react and then filter to collect the filtrate. Add absolute ethanol to the filtrate for precipitation to obtain sunflower disk pectin; S2. Mix the sunflower disk pectin with water, adjust the pH to acidic, add pectin esterase, after enzymatic hydrolysis reaction, adjust the pH to neutral, and add polygalacturonase for enzymatic hydrolysis reaction to obtain a pectin solution. Dialyze and freeze-dry the pectin solution to obtain low-esterified pectin with small molecules; The mass-volume ratio of the sunflower disk pectin, water, pectin esterase and polygalacturonase is 0.5 - 1.5 g: 20 - 30 ml: 0.05 - 0.15 ml: 0.5 - 1 g.

2. The preparation method of a small molecule pectin with low esterification degree according to claim 1, characterized in that The concentration of the cellulase solution is 0.008 - 0.024 g / ml.

3. The preparation method of a small molecule pectin with low esterification degree according to claim 1, characterized in that, The mass-volume ratio of the sunflower disk micropowder to the cellulase solution is 1 - 2 g: 5 - 50 ml.

4. The preparation method of a small molecule pectin with low esterification degree according to claim 1, characterized in that, The conditions for the mixing reaction are 30 - 40 °C and 1 - 4 h.

5. The preparation method of a small molecule pectin with low esterification degree according to claim 1, characterized in that, The volume ratio of the filtrate to absolute ethanol is 0.5 - 1.5: 1 - 10.

6. The preparation method of a small molecule pectin with low esterification degree according to claim 1, characterized in that, The conditions for the enzymatic hydrolysis reaction are enzymatic hydrolysis at 30 - 40 °C for 1 - 4 h.

7. A small molecule pectin with low degree of esterification, characterized in that, Prepared by using the preparation method described in any one of claims 1 - 6.

8. Use of the low-esterified pectin with small molecules described in claim 7 in the preparation of drugs for treating fatty liver.

9. Use of the low-esterified pectin with small molecules described in claim 7 in the preparation of weight loss products.

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