A method for promoting dissolution of RG-I pectin based on polysaccharide interaction

Abstract

The present invention discloses a RG-I pectin dissolution-promoting method based on polysaccharide interaction, and relates to the field of biotechnology. The method comprises the following steps: after adding an aqueous solution of negatively charged polysaccharides to an RG-I pectin extract and mixing evenly, adjusting the pH of the mixed system to 3-7, and then performing an alcohol precipitation treatment to obtain a precipitate; after washing the precipitate, drying and crushing it to obtain a polysaccharide-pectin mixture; the RG-I pectin extract is obtained by extracting orange peel powder with dilute hydrochloric acid. The present invention utilizes negatively charged polysaccharides and RG-I pectin to blend and interact before heat drying treatment, thereby enhancing the anti-aggregation ability of the entire system, and then achieving the purpose of promoting the dissolution of heat-dried RG-I pectin, and has good industrial application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for promoting the dissolution of RG-I pectin based on polysaccharide interaction. Background Art

[0002] Pectin is a type of anionic heteropolysaccharide widely found in the cell walls of higher plants, accounting for about 2-35% of the composition of plant cell walls. It is the most abundant water-soluble dietary fiber in nature. The main structural domains of pectin include homogalacturonan (HG), rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), in addition to a small amount of substituted galacturonan. RG-I pectin is widely distributed in nature, and most terrestrial plants are rich in RG-I pectin. In recent years, RG-I pectin has attracted widespread attention due to its excellent physiological activity and great health benefits, such as anti-cancer, anti-inflammatory, anti-obesity, antioxidant, and immunomodulatory effects.

[0003] The preparation process of RG-I pectin usually includes several steps, including extraction, pH adjustment, alcohol precipitation separation and drying. Among them, heat treatment is an inevitable unit operation in the drying process due to its low price and easy operation, such as hot air drying, spray drying, etc. The difference between RG-I pectin and commercial pectin mainly lies in its rich side chain structure, so the RG-I region is also called the "hair region" on the pectin chain. During the heat drying process, the chains of RG-I pectin are entangled and aggregated with each other, and thermal aggregation is prone to occur, which in turn causes the problem of poor solubility. The current application of RG-I pectin is generally based on the dissolved RG-I pectin aqueous solution system, so solubility is an important prerequisite and performance indicator for its application.

[0004] There have been some reports on the interaction of polysaccharide blends, but they generally focus on improving the functional properties of polysaccharides, such as blending gelatin with chitosan and konjac glucomannan to give modified gelatin better viscosity and stability (Chinese patent CN103980717A); blending pullulan, sodium alginate and propylene glycol alginate to prepare ternary polysaccharides to improve their stability under high temperature and high humidity conditions (Chinese patent CN105131345A). Preparation of composite hydrogels with excellent performance, such as blending cellulose nanofibers with sodium alginate, the prepared composite hydrogel has both the good flexibility of cellulose nanofibers and the biological activity of sodium alginate, improving the medicinal properties of the drug (Chinese patent CN114699385A); blending alginate with gellan gum to prepare W / O / W double network emulsion gel, its surfactant-free characteristics and better stability make it suitable for use as a fat substitute (Chinese patent CN117281251A). Preparation of high-performance degradable composite membranes, such as mixing chitosan, β-cyclodextrin and sodium benzoate, improves the functional properties of the composite membrane and reduces the production cost of the composite membrane (Chinese patent CN101538371). The current blending technology is generally used to improve the functional properties of polysaccharides, and there is no report on the use of blending technology in the preparation process for the purpose of improving the solubility of the product. Summary of the invention

[0005] The purpose of the present invention is to provide a method for promoting the dissolution of RG-I pectin based on polysaccharide interaction to solve the problems existing in the above-mentioned prior art. The present invention utilizes negatively charged polysaccharides and RG-I pectin to mix and interact before heat drying treatment to enhance the anti-aggregation ability of the entire system, thereby achieving the purpose of promoting the dissolution of heat-dried RG-I pectin, and has good industrial application prospects.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for promoting dissolution of RG-I pectin based on polysaccharide interaction, comprising the following steps:

[0008] After adding the negatively charged polysaccharide aqueous solution to the RG-I pectin extract and mixing evenly, the pH of the mixed system is adjusted to 3-7, and then alcohol precipitation is performed to obtain a precipitate;

[0009] The precipitate is washed, dried and crushed to obtain a polysaccharide-pectin mixture;

[0010] The RG-I pectin extract is obtained by extracting orange peel powder using dilute hydrochloric acid.

[0011] Furthermore, the pH of the dilute hydrochloric acid is 1-3.

[0012] Furthermore, the mass ratio of RG-I pectin in the RG-I pectin extract to the negatively charged polysaccharide is 1:1.

[0013] Furthermore, the negatively charged polysaccharide is HG pectin, κ-carrageenan or ι-carrageenan.

[0014] Preferably, the negatively charged polysaccharide is iota-carrageenan.

[0015] Furthermore, the mass ratio of the orange peel powder to the dilute hydrochloric acid is 1:10-20.

[0016] Furthermore, in the aqueous solution of the negatively charged polysaccharide, the mass volume ratio of the solute to the solvent is 0.5 to 5:100.

[0017] Furthermore, the specific step of adding the aqueous solution of negatively charged polysaccharides to the RG-I pectin extract includes: firstly adding 40-60% of the aqueous solution of negatively charged polysaccharides dropwise into the RG-I pectin extract, while constantly stirring to achieve sufficient premixing, and then adding the remaining aqueous solution of negatively charged polysaccharides after mixing and stirring evenly.

[0018] Furthermore, the alcohol precipitation treatment is carried out using anhydrous ethanol, and the added volume of the anhydrous ethanol is 3 to 5 times that of the mixed system.

[0019] The present invention also provides a polysaccharide-pectin mixture prepared according to the above method.

[0020] The present invention discloses the following technical effects:

[0021] The present invention has found that the presence of neutral sugar side chains in RG-I pectin will cause the pectin chain to lack sufficient charge, resulting in small intermolecular electrostatic repulsion and weak electrostatic repulsion, which leads to entanglement and aggregation between chains of RG-I pectin during the heat drying process, resulting in thermal aggregation, and then causing the problem of poor redissolubility. Based on this discovery, the present invention utilizes other polysaccharides with different charge properties to blend and interact with RG-I pectin before heat drying treatment, thereby enhancing the anti-aggregation ability of the entire system, thereby achieving the purpose of promoting the dissolution of heat-dried RG-I pectin, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1Schematic diagram of the insoluble precipitate after re-dissolving and centrifugation of the samples prepared in Examples 2 to 4 and Comparative Examples 1 and 2;

[0024] Figure 2 The data bar graph of the bulk density of the samples prepared in Examples 2 to 4 and Comparative Examples 1 and 2;

[0025] Figure 3 These are microscopic images of samples prepared in Examples 2 to 4 and Comparative Examples 1 and 2; wherein (A) is RG-I; (B) is CS-PC; (C) is HG-PC; (D) is κ-C-PC; and (E) is ι-C-PC. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1

[0032] RG-I Pectin Extraction:

[0033] Take 10g of crushed orange peel powder, add dilute hydrochloric acid with pH=1 (dilute hydrochloric acid with pH=3 can be used at most), control the material-liquid mass ratio to be 1:20 (a material-liquid ratio of 1:10-20 can achieve the same effect), and react with magnetic stirring at 40°C (temperature range can be 20°C-60°C) for 1h (time range can be 0.5-1.5h). After the reaction is completed, cool to room temperature, filter out the supernatant with filter cloth, and obtain RG-I pectin extract.

[0034] Take 10mL of RG-I pectin extract, add 4 times (3 to 5 times can achieve the same effect) volume of anhydrous ethanol, precipitate for 2h (1 to 12h can achieve the same effect), filter out the precipitate with filter cloth, wash the precipitate with 70% ethanol, filter it again, place it in an oven at 60°C and dry it for 1h, then weigh it to get the weight of RG-I pectin. After calculation, the RG-I pectin content in the RG-I pectin extract is 0.5g / 100mL.

[0035] Example 2

[0036] A method for preparing a polysaccharide-pectin mixture (HG Pectin Compound, HG-PC), the specific steps are as follows:

[0037] (1) Take 0.5g HG pectin and dissolve it in 50mL deionized water, stir it magnetically at 30℃ until it is completely dissolved to obtain HG pectin solution (when the mass volume ratio of solute to solvent is in the range of 0.5 to 5:100, the same effect can be achieved). Place a beaker containing 100mL RG-I pectin extract (prepared in Example 1) on a magnetic stirrer, heat it to 40℃ (30-40℃ can achieve the same effect) and keep it warm. First, add 50% (40-60% can achieve the same effect) of HG pectin solution dropwise into the RG-I pectin extract, and stir it continuously to achieve full premixing. After mixing, add the remaining HG pectin solution and continue stirring. After the mixed system is completely stirred, adjust the pH to 4 (pH 3-7 can achieve the same effect), add 4 times (3-5 times) the volume of anhydrous ethanol of the mixed system, and precipitate for 2h (1-12h can achieve the same effect), and filter out the precipitate with a filter cloth.

[0038] (2) The precipitate obtained in step (1) was washed with 70% ethanol, filtered again, and dried in an oven at 60° C. for 1 h. After drying, the precipitate was ground into powder to obtain a polysaccharide-pectin mixture (HG-PC) powder.

[0039] Preparation method of HG pectin: using citrus as raw material, extracting with acid at pH=1.5 (extraction temperature above 70°C) for more than 1h, filtering, alcohol precipitation and drying to obtain the pectin.

[0040] Compared with Comparative Example 1, the polysaccharide-pectin mixture obtained in this example is more fluffy ( Figure 2 ), and its structure is relatively loose from a microscopic perspective ( Figure 3 ), which increased the solubility of RG-I pectin in the polysaccharide-pectin mixture to 54.88%, and the amount of insoluble precipitate after centrifugation was also reduced ( Figure 1 ).

[0041] Example 3

[0042] A method for preparing a polysaccharide-pectin mixture (κ-Carrageenan Pectin Compound, κ-C-PC), the specific steps are as follows:

[0043] (1) Take 0.5g of κ-carrageenan and dissolve it in 50mL of deionized water, stir it magnetically at 80℃ until it is completely dissolved, and obtain a κ-carrageenan solution (when the mass volume ratio of solute to solvent is in the range of 0.5 to 5:100, the same effect can be achieved). Place a beaker containing 100mL of RG-I pectin extract (prepared in Example 1) on a magnetic stirrer, heat it to 40℃ (30-40℃ can achieve the same effect) and keep it warm. First, add 50% (40-60% can achieve the same effect) of κ-carrageenan solution dropwise to the RG-I pectin extract, and stir it continuously to achieve full premixing. After mixing, add the remaining κ-carrageenan solution and continue stirring. After the mixed system is completely stirred, adjust the pH to 4 (pH 3-7 can achieve the same effect), add 4 times (3-5 times) the volume of anhydrous ethanol of the mixed system, and precipitate for 2h (1-12h can achieve the same effect), and filter out the precipitate with a filter cloth.

[0044] (2) The precipitate obtained in step (1) was washed with 70% ethanol, filtered again and dried in an oven at 60° C. for 1 h. After drying, the precipitate was ground into powder to obtain a polysaccharide-pectin mixture (κ-C-PC) powder.

[0045] Compared with Comparative Example 1, the polysaccharide-pectin mixture obtained in this example is more fluffy ( Figure 2 ), and its structure is relatively loose from a microscopic perspective ( Figure 3 ), which increased the solubility of RG-I pectin in the polysaccharide-pectin mixture to 59.07%, and the amount of insoluble precipitate after centrifugation was also reduced ( Figure 1 ).

[0046] Example 4

[0047] A method for preparing a polysaccharide-pectin mixture (ι-Carrageenan Pectin Compound, ι-C-PC), the specific steps are as follows:

[0048] (1) Take 0.5g ι-carrageenan and dissolve it in 50mL deionized water. Stir it magnetically at 80℃ until it is completely dissolved to obtain ι-carrageenan solution (when the mass volume ratio of solute to solvent is in the range of 0.5 to 5:100, the same effect can be achieved). Place a beaker containing 100mL RG-I pectin extract (prepared in Example 1) on a magnetic stirrer, heat it to 40℃ (30-40℃ can achieve the same effect) and keep it warm. First, add 50% (40-60% can achieve the same effect) of ι-carrageenan solution dropwise to the RG-I pectin extract, and stir it continuously to achieve full premixing. After mixing, add the remaining ι-carrageenan and continue stirring. After the mixed system is completely stirred, adjust the pH to 4 (pH 3-7 can achieve the same effect), add 4 times (3-5 times) the volume of anhydrous ethanol of the mixed system, and precipitate for 2h (1-12h can achieve the same effect), and filter out the precipitate with a filter cloth.

[0049] (2) The precipitate obtained in step (1) was washed with 70% ethanol, filtered again and dried in an oven at 60° C. for 1 h. After drying, the precipitate was ground into powder to obtain a polysaccharide-pectin mixture (ι-C-PC) powder.

[0050] Compared with Examples 2 to 3 and Comparative Example 1, the polysaccharide-pectin mixture obtained in this example is the most fluffy ( Figure 2 ), and its structure is the loosest at a microscopic level ( Figure 3 ), which makes the solubility of RG-I pectin in the polysaccharide-pectin mixture increase the most, to 87.71%, and the amount of insoluble precipitate after centrifugation is the least ( Figure 1 ).

[0051] Comparative Example 1

[0052] A method for preparing pure RG-I pectin, the specific steps are as follows:

[0053] (1) Take 10 g of crushed orange peel powder, add dilute hydrochloric acid with pH = 1, control the mass ratio of material to liquid to be 1:20, and react at 40°C with magnetic stirring for 1 h. After the reaction is completed, cool to room temperature, filter out the supernatant with filter cloth to obtain RG-I pectin extract. Adjust the pH of the extract to 4, add 4 times the volume of anhydrous ethanol, precipitate for 2 h, and filter out the precipitate with filter cloth.

[0054] (2) The precipitate was washed with 70% ethanol, filtered again, and dried in an oven at 60° C. for 1 h. After drying, the precipitate was ground into powder to obtain RG-I pectin powder.

[0055] The pure RG-I pectin obtained in this comparative example has poor powder bulkiness due to thermal aggregation ( Figure 2 ), and its structure is also denser from a microscopic perspective ( Figure 3), the solubility index was poor, at 33.32%, and the amount of insoluble precipitate after centrifugation was large ( Figure 1 ).

[0056] Comparative Example 2

[0057] A method for preparing a polysaccharide-pectin mixture (Chitosan Pectin Compound, CS-PC), the specific steps are as follows:

[0058] (1) 0.5 g of chitosan was dissolved in 50 mL of acetic acid solution of pH 2, and magnetically stirred at 80° C. until completely dissolved to obtain a chitosan solution. A beaker containing 100 mL of RG-I pectin extract (prepared in Example 1) was placed on a magnetic stirrer, heated to 40° C. for heat preservation, 50% of the chitosan solution was first added dropwise to the RG-I pectin extract, and stirred continuously to achieve sufficient premixing. After mixing, the remaining chitosan solution was added and continued to stir. After the mixed system was completely stirred, the pH was adjusted to 4, and 4 times the volume of anhydrous ethanol of the mixed system was added, and the mixture was precipitated for 2 hours, and the precipitate was filtered out using a filter cloth.

[0059] (2) The precipitate obtained in step (1) was washed with 70% ethanol, filtered again, and dried in an oven at 60° C. for 1 h. After being completely dried, the precipitate was ground into powder using a grinder to obtain a polysaccharide-pectin mixture (CS-PC) powder.

[0060] Compared with Examples 2 to 4 and Comparative Example 1, the polysaccharide-pectin mixture obtained in this comparative example has the worst fluffiness ( Figure 2 ), its structure is also the most dense at a microscopic perspective, showing a flat sheet structure ( Figure 3 ), which caused the solubility of RG-I pectin in the polysaccharide-pectin mixture to drop significantly to -46.36%, and the amount of insoluble precipitate after centrifugation was the largest ( Figure 1 ).

[0061] Results Analysis

[0062] Compared with Comparative Example 1 without adding polysaccharide, the solubility rate of RG-I pectin in Comparative Example 2 dropped sharply. At the same time, since the polysaccharide could not be completely precipitated during the alcohol precipitation process, the actual mass of the polysaccharide in the mixture could not be obtained when the solubility rate was finally calculated, resulting in a negative result. However, Examples 2 to 4 are three negatively charged polysaccharides, which all have a solubilizing effect on heat-dried RG-I pectin. The solubilizing effect is related to the charge density of the polysaccharide. The greater the charge density of the polysaccharide, the better the solubilizing effect. The solubilizing effect of Example 4 is the best.

[0063] Figure 1The schematic diagram is that 0.05 g of samples prepared in Examples 2 to 4 and Comparative Examples 1 and 2 were taken, dissolved in 10 mL of water, shaken for 5 minutes and then centrifuged, wherein the precipitate remaining at the bottom of the tube is a poorly soluble precipitate, and the statistical results are detailed in Table 1. The precipitate at the bottom of the tube in Comparative Examples 1 and 2 is greater than that in Examples 2 to 4, and the amount of precipitate decreases as the solubility rate increases, and the amount of precipitate in Example 4 is the least.

[0064] Table 1 Redissolution centrifugal precipitation of samples prepared in Examples 2 to 4 and Comparative Examples 1 and 2

[0065] sample Mass of reconstituted centrifugal precipitate (g) RG-I 0.03 CS-PC 0.042 HG-PC 0.018 κ-C-PC 0.015 ι-C-PC 0.003

[0066] Figure 2 The data bar graph of the sample bulk density prepared in Examples 2 to 4 and Comparative Examples 1 and 2 is as follows: Figure 2 As shown, the order of bulk density is Comparative Example 2, Comparative Example 1, Example 2, Example 3, and Example 4, with Comparative Example 2 having the largest bulk density and Example 4 having the smallest bulk density. As a macroscopic index, bulk density reflects the fluffiness of the sample powder, and can reflect the degree of aggregation of the sample at a microscopic perspective to a certain extent. The larger the bulk density, the worse the sample fluffiness and the stronger the degree of aggregation of the sample, that is, the degree of aggregation of the sample in Comparative Example 2 is the strongest, and the degree of aggregation of the sample in Example 4 is the weakest, which is consistent with the solubility rate data.

[0067] Figure 3 The microscopic images of the samples prepared in Examples 2 to 4 and Comparative Examples 1 and 2 are as follows: Figure 3 As shown, Comparative Example 1 has an uneven banded structure with many irregular cavities inside. In Comparative Example 2, due to the electrostatic attraction between RG-I pectin and chitosan, CS-PC presents a flat sheet structure. This makes the dissolution area of ​​CS-PC in contact with water molecules smaller, and the solubility is worse than that of RG-I pectin. The negatively charged polysaccharides in Examples 2 to 4 can enable the polysaccharide-pectin mixture to form a network structure with different pore sizes. Compared with Examples 2 and 3, Example 4 has a smaller pore size and a larger pore density, which may be due to the fact that ι-carrageenan has a larger charge density and can provide greater electrostatic repulsion. In addition, the microstructures of different samples are consistent with the conclusions of solubility and bulk density.

[0068] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for promoting dissolution of RG-I pectin based on polysaccharide interaction, characterized in that: The following steps are involved: After adding the negatively charged polysaccharide aqueous solution to the RG-I pectin extract and mixing evenly, the pH of the mixed system is adjusted to 3-7, and then alcohol precipitation is performed to obtain a precipitate; The precipitate is washed, dried and crushed to obtain a polysaccharide-pectin mixture; The RG-I pectin extract is obtained by extracting orange peel powder using dilute hydrochloric acid.

2. The RG-I pectin dissolution promoting method according to claim 1, characterized in that: The pH of the dilute hydrochloric acid is 1-3.

3. The RG-I pectin dissolution promoting method according to claim 1, characterized in that: The mass ratio of the RG-I pectin in the RG-I pectin extract to the negatively charged polysaccharide is 1:

1.

4. The RG-I pectin dissolution promoting method according to claim 1, characterized in that: The negatively charged polysaccharide is HG pectin, kappa-carrageenan or iota-carrageenan.

5. The RG-I pectin dissolution promoting method according to claim 1, characterized in that: The negatively charged polysaccharide is iota-carrageenan.

6. The RG-I pectin dissolution promoting method according to claim 1, characterized in that: The mass ratio of the orange peel powder to the dilute hydrochloric acid is 1:10-20.

7. The RG-I pectin dissolution promoting method according to claim 1, characterized in that: In the aqueous solution of negatively charged polysaccharides, the mass volume ratio of solute to solvent is (0.5~5) g:100 mL.

8. The RG-I pectin dissolution promoting method according to claim 1, characterized in that: The specific steps of adding the aqueous solution of negatively charged polysaccharides to the RG-I pectin extract include: firstly adding 40-60% of the aqueous solution of negatively charged polysaccharides dropwise to the RG-I pectin extract while continuously stirring to achieve sufficient premixing, and then adding the remaining aqueous solution of negatively charged polysaccharides after mixing and stirring evenly.

9. The RG-I pectin dissolution promoting method according to claim 1, characterized in that: The alcohol precipitation treatment is carried out using anhydrous ethanol, and the added volume of the anhydrous ethanol is 3 to 5 times that of the mixed system.

10. A polysaccharide-pectin mixture prepared according to the method according to any one of claims 1 to 9.

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