RGI pectin dissolution promoting preparation method based on pH regulation and control

Abstract

The invention provides an RGI pectin dissolution promoting preparation method based on pH regulation and control. The technical prejudice that the extracting solution is adjusted to be neutral before alcohol precipitation in the original common means is overcome, the pH value of the extracting solution before alcohol precipitation is adjusted to be 1-3 so as to influence the structural form of the RGI pectin, the anti-aggregation ability of the whole system is enhanced, the purpose of promoting dissolution of the hot-dried RG-I pectin is achieved, and the method has good industrial application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing RGI pectin based on pH regulation. Background Art

[0002] Pectin is an acidic heteropolysaccharide widely found in the primary cell walls and middle lamella of plants, accounting for up to 30% of the cell wall. Its structure is mainly composed of three domains: homogalacturonan (HG), rhamnogalacturonan I (RGI), and rhamnogalacturonan II (RGII), with smaller amounts of xylogalacturonan (XG) and apigalacturonan (AG) regions. RGI pectin is widely used in the food industry due to its excellent gelling, thickening, and emulsifying properties, particularly in the production of jellies, jams, and juices. Furthermore, RGI pectin possesses antioxidant and immunomodulatory activities, making it an ideal ingredient in health foods and nutritional supplements. In recent years, with in-depth research into its functions and properties, the application areas of RG-I pectin have continued to expand.

[0003] The preparation process of RGI pectin generally includes extraction (low-temperature acid extraction), pH adjustment to neutral, alcohol precipitation, and drying. Among drying methods, heat treatment is widely used in the industrial production of pectin due to its low cost and low technical requirements. However, RGI pectin prepared by heat drying exhibits a unique thermal aggregation phenomenon, leading to a sharp decrease in the solubility of the finished pectin and affecting its full utilization. Therefore, a simple and easy method is urgently needed to inhibit or improve the thermal aggregation phenomenon of RGI pectin to achieve solubility promotion.

[0004] Currently, commonly used pectin extraction methods include acid extraction, alkali extraction, and enzymatic extraction. Improvement of solubility often employs physical methods (micro-refining, adding solubilizers, etc.), degradation methods, and derivatization methods. However, different methods have limitations such as damaging polysaccharide structures, limited solubilization effects, and method specificity, restricting large-scale application. Previous experiments have found that the pH value of the extraction solution during alcohol precipitation significantly affects the structure, thermal aggregation state, and solubility of the finally dried RGI pectin, and that alcohol precipitation at unconventional acidic pH conditions significantly improves its solubility. Therefore, this invention innovatively uses the pH of alcohol precipitation to regulate the thermal aggregation of RGI pectin, aiming to achieve highly efficient solubilization under simple operating conditions. Summary of the Invention

[0005] To address the problem of poor solubility of pectin extracts in existing technologies, this invention provides a pH-controlled RGI pectin solubilization preparation method. In extraction processes, alcohol precipitation is a commonly used technique for separating macromolecules. Its principle is to use ethanol to reduce the polarity of the solution, causing macromolecules such as polysaccharides and proteins to precipitate. Traditionally, it is customary to adjust the extract to neutral or near-neutral (pH ≈ 4-7) before alcohol precipitation. This operation is usually performed before the concentration step to avoid damage to the structure of the active ingredients due to the acidic environment during concentration heating; even without concentration, the pH still needs to be adjusted to this range before alcohol precipitation to improve the precipitation yield. Failure to adjust the pH may lead to denaturation of the active ingredients or incomplete precipitation, affecting extraction efficiency and quality.

[0006] This invention overcomes the technical bias of adjusting the extract to neutral pH before alcohol precipitation in conventional methods. By adjusting the pH of the extract to 1-3 before alcohol precipitation to control the structural morphology of RG-I pectin, and combining this with a low-temperature alcohol precipitation strategy to reduce the Brownian motion intensity of pectin molecules, decrease the frequency of molecular collisions, and avoid disordered aggregation, the synergistic effect of low pH and low temperature significantly inhibits strong interactions such as hydrophobic association and hydrogen bonding between pectin molecules, enhancing the system's anti-aggregation ability. Ultimately, this achieves a solubilizing effect on heat-dried RG-I pectin, making the dried product easier to depolymerize in water, with significantly improved swelling rate and solubility. Maintaining high yield while ensuring good solubility of the pectin extract demonstrates promising prospects for industrial application.

[0007] Specifically, it includes the following steps: (1) Orange peel powder was extracted using dilute hydrochloric acid with pH=1. The mass ratio of orange peel powder to dilute hydrochloric acid was 1:10~20. The extraction temperature was 40℃ and the extraction time was 1.5 h. RGI pectin extract was obtained. (2) The pH of the extract was adjusted to 1-3 using sodium hydroxide, followed by alcohol precipitation and filtration to obtain the precipitate; (3) After washing the precipitate with ethanol, it is dried and crushed to obtain pectin samples with different ethanol precipitation pH.

[0008] Furthermore, the mass concentration of the sodium hydroxide solution is 1-4%, and the pH of the extract is preferably adjusted to 2-3.

[0009] Furthermore, the alcohol precipitation treatment is carried out using anhydrous ethanol, and the volume of anhydrous ethanol added is 3 to 5 times that of the RGI pectin extract after the addition of sodium hydroxide; the alcohol precipitation temperature is 0-4℃.

[0010] Furthermore, the drying method in step 3 is to dry in an oven at 60°C for 1 hour.

[0011] The pH of the system before alcohol precipitation affects the structural morphology of RGI pectin. Increased pH increases the deprotonation of carboxyl groups in pectin, thereby affecting hydrogen bonding with water molecules and reducing hydration. This exposes more hydrophobic groups, leading to pectin chain aggregation dominated by hydrophobic interactions. Furthermore, it promotes hydrogen bonding between pectin molecules, inducing self-aggregation and entanglement, thus affecting the aggregation state and solubility of dried pectin. Based on this discovery, this invention regulates the pectin chain morphology by adjusting the pH of the RGI pectin extract to an unconventional acidic level before alcohol precipitation, thereby enhancing the anti-aggregation ability of the entire system. This simple and efficient method promotes the solubility of heat-dried RGI pectin, demonstrating promising industrial application prospects.

[0012] The beneficial effects of the present invention are: The pH of the extract before alcohol precipitation was adjusted to 1-3 to affect the structural morphology of RGI pectin, thereby enhancing the anti-aggregation ability of the entire system and promoting the solubility of heat-dried RG-I pectin. The resulting RGI pectin powder had a solubility of 83.08% and a yield of not less than 4.80%, which is significantly higher than that of RGI pectin powder prepared by conventional methods. Attached Figure Description

[0013] Figure 1 Scanning electron microscope images of the samples prepared in Examples 1-3 and Comparative Examples 1-4; Figure 2 The images show dynamic observations of the dissolution process of the samples prepared in Examples 1-3 and Comparative Examples 1-4. Figure 3 The images show the physical morphology of the samples from Examples 1-3 and Comparative Examples 1-4 during the alcohol precipitation process. Detailed Implementation

[0014] The following examples are provided to further illustrate the present invention and are intended to explain the invention, not to limit its scope. Unless otherwise specified, all figures are expressed in parts by weight and weight percentages.

[0015] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0016] The embodiments of the present invention will be further described below with reference to several examples.

[0017] It should be understood that the described embodiments are merely some, not all, of the embodiments in this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0018] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0019] Example 1 A method for preparing RGI pectin (pH=1) by alcohol precipitation, the specific steps of which are as follows: (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 1:10, and stir magnetically at 40℃ for 1.5 h. After the reaction is completed, cool to room temperature, filter the supernatant with a filter cloth to obtain RGI pectin extract, add 3 times the volume of anhydrous ethanol to precipitate at 1℃ for 2 h, and filter the precipitate with a filter cloth.

[0020] (2) The above precipitate was washed once with anhydrous ethanol, filtered again, and then dried in an oven at 60°C for 1 h. After drying, it was ground into powder to obtain RGI pectin powder with ethanol precipitation pH = 1.

[0021] Compared to Comparative Examples 1-4, the pectin obtained in this example is more fluffy (Table 1), and its structure is also more porous under a microscopic perspective. Figure 1 The soluble content of pectin was 83.08% (Table 1), and the yield was 4.80%. Figure 3 ).

[0022] Example 2 A method for preparing RGI pectin (pH=2) by alcohol precipitation, the specific steps of which are as follows: (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 stir magnetically at 40℃ for 1.5 h. After the reaction is completed, cool to room temperature, filter the supernatant with a filter cloth to obtain RGI pectin extract. Adjust the pH of the extract to 2 with 4% sodium hydroxide solution, add 3 times the volume of anhydrous ethanol and precipitate at 4℃ for 2 h, and filter the precipitate with a filter cloth.

[0023] (2) The above precipitate was washed with anhydrous ethanol, filtered again, and then dried in an oven at 60°C for 1 h. After drying, it was ground into powder to obtain RGI pectin powder with ethanol precipitation pH = 2.

[0024] Compared to Comparative Examples 1-4, the pectin obtained in this example is more fluffy (Table 1), and its structure is also more porous under a microscopic perspective. Figure 1 The soluble content of pectin was 80.44% (Table 1), and the yield was 5.05%. Figure 3 ).

[0025] Example 3 A method for preparing RGI pectin (pH=3) by alcohol precipitation, the specific steps of which are as follows: (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 stir magnetically at 40℃ for 1.5 h. After the reaction is completed, cool to room temperature, filter the supernatant with a filter cloth to obtain RGI pectin extract. Adjust the pH of the extract to 3 with 4% sodium hydroxide solution, add 3 times the volume of anhydrous ethanol, precipitate at 4℃ for 2 h, and filter the precipitate with a filter cloth.

[0026] (2) The above precipitate was washed with anhydrous ethanol, filtered again, and then dried in an oven at 60°C for 1 h. After drying, it was ground into powder to obtain RGI pectin powder with ethanol precipitation pH = 3.

[0027] Compared to Comparative Examples 1-4, the pectin obtained in this example is more fluffy (Table 1), and its structure is also more porous under a microscopic perspective. Figure 1 The soluble content of pectin was 77.51% (Table 1), and the yield was 5.78%. Figure 3 ).

[0028] Comparative Example 1 A method for preparing RGI pectin (pH=4) by alcohol precipitation, the specific steps of which are as follows: (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 stir magnetically at 40℃ for 1.5 h. After the reaction is completed, cool to room temperature, filter the supernatant with a filter cloth to obtain RGI pectin extract. Adjust the pH of the extract to 4 with 4% sodium hydroxide solution, add 3 times the volume of anhydrous ethanol, precipitate at 4℃ for 2 h, and filter the precipitate with a filter cloth.

[0029] (2) The above precipitate was washed with anhydrous ethanol, filtered again, and then dried in an oven at 60°C for 1 h. After drying, it was ground into powder to obtain RGI pectin powder with ethanol precipitation pH = 4.

[0030] Compared to Examples 1-3, the pectin obtained in this example showed a significant decrease in fluffiness and a reduction in solubility to 39.82% (Table 1), but the degree of aggregation was still lower than that of Comparative Examples 2-4, with a yield of 6.45%. Figure 3 ).

[0031] Comparative Example 2 A method for preparing RGI pectin (pH=5) by alcohol precipitation, the specific steps of which are as follows: (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 stir magnetically at 40℃ for 1.5 h. After the reaction is completed, cool to room temperature, filter the supernatant with a filter cloth to obtain RGI pectin extract. Adjust the pH of the extract to 5 with 4% sodium hydroxide solution, add 3 times the volume of anhydrous ethanol and precipitate at 4℃ for 2 h, and filter the precipitate with a filter cloth.

[0032] (2) The above precipitate was washed with anhydrous ethanol, filtered again, and then dried in an oven at 60°C for 1 h. After drying, it was ground into powder to obtain RGI pectin powder with ethanol precipitation pH = 5.

[0033] Comparative Example 3 A method for preparing RGI pectin (pH=6) by alcohol precipitation, the specific steps of which are as follows: (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 stir magnetically at 40℃ for 1.5 h. After the reaction is completed, cool to room temperature, filter the supernatant with a filter cloth to obtain RGI pectin extract. Adjust the pH of the extract to 6 with 4% sodium hydroxide solution, add 3 times the volume of anhydrous ethanol and precipitate at 4℃ for 2 h, then filter the precipitate with a filter cloth.

[0034] (2) The above precipitate was washed with anhydrous ethanol, filtered again, and then dried in an oven at 60°C for 1 h. After drying, it was ground into powder to obtain RGI pectin powder with ethanol precipitation pH = 6.

[0035] Comparative Example 4 A method for preparing RGI pectin (pH=7) by alcohol precipitation, the specific steps of which are as follows: (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 stir magnetically at 40℃ for 1.5 h. After the reaction is completed, cool to room temperature, filter the supernatant with a filter cloth to obtain RGI pectin extract. Adjust the pH of the extract to 7 with 4% sodium hydroxide solution, add 3 times the volume of anhydrous ethanol, precipitate at 4℃ for 2 h, and filter the precipitate with a filter cloth.

[0036] (2) The above precipitate was washed with anhydrous ethanol, filtered again, and then dried in an oven at 60°C for 1 h. After drying, it was ground into powder to obtain RGI pectin powder with ethanol precipitation pH = 7.

[0037] Comparative Example 5 The only difference from Example 3 is that the alcohol precipitation temperature is room temperature.

[0038] Table 1 Compared to Examples 1-3, the pectin aggregation degree obtained in Comparative Examples 2-4 was significantly enhanced, and the solubility decreased to about 30% (Table 1), with yields of 6.89%, 7.41%, and 7.89%, respectively. Figure 3 ).

[0039] The pH of alcohol precipitation is closely related to the solubility of RGI pectin. Compared with conventional alcohol precipitation at pH 4-7, the solubility of RGI pectin in Examples 1-3 was improved to varying degrees at different alcohol precipitation pH. The degree of improvement was pH-dependent, generally showing a trend of higher solubility of RGI pectin as the pH of alcohol precipitation decreased. The solubility showed a jump between pH 3 and 4, while the variation was not significant within the pH range of 1-3 or 4-7. Example 1 showed the best solubility improvement. Furthermore, within the pH range of 1-7, the pectin yield was positively correlated with the alcohol precipitation pH. Considering both pectin solubility and yield / cost, an alcohol precipitation pH of 2-3 was chosen as the optimal condition.

[0040] Furthermore, as shown in Table 1, the order of bulk density is as follows: Comparative Example 4, Comparative Example 3, Comparative Example 2, Comparative Example 1, Example 3, Example 2, and Example 1. Comparative Example 4 has the highest bulk density, while Example 1 has the lowest. Bulk density, as a macroscopic indicator, reflects the fluffiness of the sample powder and can, to some extent, reflect the degree of aggregation of the sample at a microscopic level. A higher bulk density indicates poorer fluffiness and stronger aggregation; that is, the sample in Comparative Example 4 exhibits the strongest aggregation, while the sample in Example 1 exhibits the weakest aggregation, further supporting the results regarding solubility.

[0041] Figure 1 The images show scanning electron microscope (SEM) images of the samples prepared in Examples 1-3 and Comparative Examples 1-4. The samples with high solubility obtained at low alcohol precipitation pH exhibit a dense honeycomb network structure, while the pectin chains show a filamentous structure. This results in a larger specific surface area for pectin to contact water molecules when dissolved in water, and stronger interaction between the hydrophilic groups and water molecules, i.e., enhanced solubility. However, as the pH increases, the pectin morphology gradually transforms into a fragmented structure, resulting in a smaller dissolution area for the sample in contact with water molecules and poorer solubility.

[0042] Figure 2 These are dynamic observation diagrams of the dissolution process of the samples prepared in Examples 1-3 and Comparative Examples 1-4. Figure 2 It can be observed that samples with high solubility exhibit finer powder characteristics without the addition of distilled water, while showing a faster dissolution rate and better overall dissolution effect after the addition of distilled water. Among them, the hydration of Example 1 is significantly stronger, the dynamic change rate of the sample powder when dissolving in water is faster, and most areas are completely dissolved within 60 seconds.

[0043] Figure 3These are morphological images of the samples from Examples 1-3 and Comparative Examples 1-4 during the alcohol precipitation process. According to... Figure 3 It is evident that the morphology of the alcohol precipitation system varies significantly at different pH levels. Between pH 1 and 3, the sample floats on the surface and exhibits a porous gel-like structure. However, when the pH is adjusted to 4, the sample precipitates at the bottom as particles or flocculent precipitates, which corresponds to the change in solubility to some extent. Furthermore, the dried sample images show a trend where a more fluffy sample indicates a higher solubility, representing a lower degree of aggregation.

[0044] The above embodiments describe in detail the structure, features, and effects of the present invention. The above description is only a preferred embodiment of the present invention. Any changes made in accordance with the concept of the present invention, or equivalent embodiments modified to have equivalent changes, shall still fall within the scope of protection of the present invention if they do not exceed the scope covered by the specification.

Claims

1. A method for preparing RGI pectin based on pH regulation, characterized in that, The following steps are involved: (1) Orange peel powder was extracted using dilute hydrochloric acid at pH=1 to obtain RGI pectin extract; (2) The pH of the extract was adjusted to 1-3 using sodium hydroxide, followed by alcohol precipitation and filtration to obtain the precipitate; (3) After washing the precipitate with ethanol, it is dried and crushed to obtain a soluble pectin sample.

2. The method according to claim 1, characterized in that, The mass ratio of orange peel powder to dilute hydrochloric acid is 1:10~20.

3. The method according to claim 1, characterized in that, The extraction temperature is 40℃.

4. The method according to claim 1, characterized in that, The extraction time is 1.5 hours.

5. The method according to claim 1, characterized in that, The mass concentration of the sodium hydroxide solution is 1-4%.

6. The method according to claim 1, characterized in that, The alcohol precipitation process is carried out using anhydrous ethanol, and the volume of anhydrous ethanol added is 3 to 5 times that of the RGI pectin extract after the addition of sodium hydroxide; the alcohol precipitation temperature is 0-4℃.

7. The method according to claim 1, characterized in that, Step 3 involves drying in an oven at 60°C for 1 hour.

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