Method for extracting pectic polysaccharide rich in RG-I based on deep eutectic solvent containing cellulase

Abstract

The invention discloses a method for extracting pectic polysaccharide rich in RG-I based on a water-containing deep eutectic solvent containing cellulase, which comprises the following steps: S1, mixing purified plant cell walls with the deep eutectic solvent containing cellulase, stirring and heating to obtain a solid-liquid mixture; s2, centrifuging the solid-liquid mixture to obtain a first supernatant and solid residues; s3, adding water into the solid residues again, centrifuging after heating and oscillating, collecting a supernatant water solution, and repeating for five times; and S4, combining the supernatant water solutions, adding alcohols, centrifuging, collecting precipitates, and carrying out decolorization treatment. The choline chloride / propylene glycol deep eutectic solvent and the cellulase are adopted to form a synergistic extraction system, the cellulase precisely degrades a cellulose skeleton, choline chloride / propylene glycol and exposed RG-I fully interact, selective release and enrichment of the RG-I structural domain are realized, the conditions are mild, high yield and low solvent residue are ensured, the process is environment-friendly, and the method is suitable for industrial production. The pectic polysaccharide product with high RG-I content can be efficiently prepared, and the method has industrial application potential.

Description

Technical Field

[0001] This invention relates to the field of biomass extraction and food processing technology, specifically a method for extracting pectin polysaccharides rich in RG-I type using a deep eutectic solvent containing cellulase. Background Technology

[0002] Pectin is a complex acidic polysaccharide widely found in plant cell walls, typically composed of homogalacturonan (HG) and rhamnogalacturonan-I (RG-I) domains. Among these, RG-I pectin, due to its highly branched neutral sugar side chain structure, exhibits superior bioactivity compared to traditional HG pectin in areas such as regulating gut microbiota, immune modulation, anti-inflammation, and biomaterial construction, and has attracted widespread attention in the food and biomedical fields in recent years.

[0003] Current industrial and laboratory pectin extraction methods primarily rely on acid extraction. This method, typically conducted under strong acid and high temperature conditions, achieves high total pectin extraction rates. However, the intense acid hydrolysis environment easily leads to the breakage of rhamnose-galacturonic acid bonds in the RG-I backbone, while also damaging its side chain structure. The resulting pectin is predominantly composed of HG domains with low RG-I content, failing to meet the application requirements for high-value-added RG-I type pectin. Furthermore, acid extraction processes also suffer from high energy consumption, severe equipment corrosion, and environmental pollution.

[0004] Enzymatic extraction, by introducing cellulase, hemicellulase, or pectin-related enzymes to degrade cell wall structures, can alleviate the problem of pectin structure damage to some extent. However, single-enzyme methods are often limited by substrate accessibility, especially the dense network structure formed by cellulose-hemicellulose-pectin in plant cell walls, which makes it difficult for enzymes to fully reach the RG-I region, resulting in limited RG-I release efficiency. Furthermore, enzymatic extraction has a long cycle and high cost, making it difficult to achieve both high efficiency and selectivity.

[0005] In recent years, deep eutectic solvents (DES) have been used as a novel green solvent for the extraction and pretreatment of polysaccharides from plant cell walls. Existing techniques have reported the use of DES to disrupt the hydrogen bond network in plant cell walls, thereby improving polysaccharide release efficiency. However, current research on DES extraction of pectin mainly focuses on increasing the total pectin yield, especially acidic DES systems. While these systems have a strong ability to deconstruct cell walls, they may still adversely affect the RG-I domain, hindering the selective enrichment of RG-I.

[0006] On the other hand, the technical approach of introducing bioenzymes into aqueous DES systems, enabling DES to simultaneously serve as a cell wall regulation medium and an enzyme reaction medium, is still immature. In particular, effective process design is lacking in achieving the targeted release of the RG-I domain from plant cell walls. Furthermore, the presence of non-structural components such as polyphenols and starch in plant cell walls further interferes with pectin extraction and structure retention, a problem that current technologies have not adequately addressed.

[0007] Therefore, there is an urgent need for a novel extraction method that can synergistically regulate cell wall structure and achieve selective release and enrichment of RG-I type pectin under mild and green conditions. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for extracting pectin polysaccharides rich in RG-I type using a deep eutectic solvent containing cellulase.

[0009] The objective of this invention is achieved through the following technical solution: On the one hand, a method for extracting pectin polysaccharides rich in RG-I type using an aqueous eutectic solvent containing cellulase includes the following steps: S1. Take the purified plant cell wall and mix it with the eutectic solvent containing cellulase, stir and heat to obtain a solid-liquid mixture; S2. Centrifuge the solid-liquid mixture to obtain a first supernatant and solid residue; S3. Add water to the solid residue, heat and shake, then centrifuge, collect the supernatant aqueous solution, repeat five times to obtain multiple supernatant aqueous solutions; S4. Combine the supernatant aqueous solutions obtained from the previous steps, add ethanol, centrifuge to collect the precipitate, and decolorize.

[0010] Specifically, the method for extracting plant cell walls described in S1 includes the following steps: S11. Plant cell walls are treated with organic solvents to remove phenolic substances and reducing sugars, resulting in pre-treated cell walls; S12. The pre-treated cell wall is treated with amylase to remove starch, resulting in an enzymatically hydrolyzed cell wall; S13. The enzymatically hydrolyzed cell wall is subjected to dialysis to remove small molecule impurities and obtain the purified plant cell wall; S14. Freeze-dry the purified plant cell walls, pulverize and sieve them, and store them in a sealed container at -20°C; Specifically, healthy plants with uniform maturity are selected, cut into small pieces, and homogenized using a juicer. The resulting homogenate is filtered through four layers of sterile gauze to separate the liquid and solid phases. The filtered plant cell walls are collected and placed in a 50°C forced-air drying oven for continuous drying until constant weight. Preferably, the plant selection in this application includes apples; Preferably, in S11, the plant includes an apple, the organic solvent includes ethanol, and the mass ratio of the plant cell wall to the volume of the organic solvent is 1:10. Preferably, in S12, the amylase includes α-amylase, and the mass ratio of the pre-treated cell wall to the amylase is 25:1; Preferably, in S13, the enzymatically hydrolyzed cell wall is placed in a dialysis bag with a molecular weight cutoff of 8000-14000 Da; Preferably, in S14, the purified plant cell wall is pulverized and then passed through a 60-mesh sieve; Specifically, the aqueous eutectic solvent is composed of choline chloride and 1,2-propanediol, wherein the molar ratio of choline chloride to 1,2-propanediol is 1:2. Specifically, in the aqueous eutectic solvent, the mass percentage of water is 20% to 30%. In this study, a deep eutectic solvent composed of ChCl:PG was prepared with a molar ratio of 1:2. The residual water content in pure DES was determined by Karl Fischer titration. To investigate the effect of hydration, pure DES was mixed with an appropriate amount of ultrapure water to prepare DES with a water content of 23.57 wt%. The molar ratio (n) of ChCl / PG / H2O was 1:2:n, where n = 5. Specifically, the amount of cellulase added is 0.30-0.40g per gram of dry weight of plant cell wall material, and the enzyme activity of the cellulase is 15000-20000 U; Ideally, in S1, the heating temperature is 45℃~55℃ and the time is 2~3 h; Ideally, in S2, the centrifugation speed is 4000 g / min and the centrifugation time is 20 min; Ideally, in S3, the centrifugation speed is 4000 g / min and the centrifugation time is 20 min; Ideally, in S4, the centrifugation speed is 4000 g / min and the centrifugation time is 20 min; Specifically, in S3, the temperature of the heating oscillation is 70℃~90℃, and the heating oscillation time is 10 min; Specifically, in S4, pectin polysaccharides are recovered from the supernatant using an alcohol precipitation method, wherein the alcohols include ethanol, and the volume ratio of the alcohols to the supernatant aqueous solution is 4:1.

[0011] On the other hand, the RG-I type pectin polysaccharide obtained by the method of deep eutectic solvent extraction with cellulase is also described. Specifically, the RG-I type pectin polysaccharide includes: fucose 1.40±0.06 mol; arabinose 24.50±2.45 mol; rhamnose 12.51±0.94 mol; galactose 31.76±2.27 mol; glucose 2.89±0.74 mol; xylose 1.28±0.60 mol; mannose 0.92±0.51 mol; and galacturonic acid 24.74±1.13 mol.

[0012] The beneficial effects of this invention are: (1) This application uses an alcoholic eutectic solvent composed of choline chloride / propylene glycol and controls its specific water content. While reducing the viscosity of the system and enhancing the mass transfer performance, it provides a suitable reaction environment for cellulase, which is conducive to achieving the gentle deconstruction of plant cell walls. (2) In this application, cellulase is introduced into the aqueous DES system for synergistic pretreatment. By weakening the hydrogen bond network of the cell wall through DES and selectively degrading the cellulose skeleton by cellulase, the interaction between DES and exposed RG-I is enhanced, and the RG-I domain is preferentially released from the cell wall complex structure, thereby increasing the enrichment of RG-I type pectin. (3) This application avoids the damage to the main chain and side chain structure of RG-I under strong acid conditions, which is conducive to maintaining the structural integrity and functional properties of RG-I; (4) This application achieves effective dissolution of RG-I type pectin by combining DES and enzyme co-pretreatment with hot water extraction, while reducing the risk of DES residue and facilitating subsequent separation and purification. (5) This application uses plant cell walls treated to remove polyphenols and starch as raw materials, which reduces the interference of non-target components on the extraction process and is conducive to obtaining pectin polysaccharide products with high purity, clear structural characteristics and high RG-I content; (6) The overall process conditions of this application are mild and environmentally friendly, reducing the use of chemical reagents and waste liquid discharge, and have good industrial application prospects and high added value potential. Attached Figure Description

[0013] Figure 1 This is a flowchart illustrating the method for extracting RG-I type pectin polysaccharides using a cellulase-containing eutectic solvent in this application. Figure 2 The images shown are atomic force microscopy images of the pectin polysaccharides prepared in Example 3, Comparative Example 1, and Comparative Example 2 in Experimental Example 3 (the top image shows the surface morphology of the extracted pectin corresponding to the three preparation methods ChCl / PG&E-P, ChCl / PG-P, and EP; the bottom image shows the three-dimensional imaging images corresponding to the three preparation methods ChCl / PG&E-P, ChCl / PG-P, and EP). Figure 3 Fourier transform infrared spectra of pectin polysaccharides prepared in Example 3, Comparative Example 1, and Comparative Example 2 in Experimental Example 4. Figure 3 A is the infrared spectrum; Figure 3 B is the ¹H NMR spectrum; Figure 3 C represents the molecular weight diagram; Figure 3 D represents the apparent viscosity diagram. Detailed Implementation

[0014] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.

[0015] Example 1: Preparation of plant cell walls The plant used in this embodiment is apple, and the specific steps for making apple cell walls include: 1. Raw material pretreatment: Select healthy Fuji apples with uniform ripeness, wash them and cut them into small pieces, and use a juicer to homogenize the apple pieces; 2. Preliminary separation: The homogenized product is filtered through four layers of sterile gauze to separate the liquid phase and solid phase, and the filter residue, i.e., the coarse apple cell wall material, is collected. 3. Drying: Place the coarse apple cell wall material in a 50℃ forced-air drying oven and dry until constant weight; 4. Removal of phenols and reducing sugars: Take 10 g of dried crude material, add 95% ethanol solution at a material-to-liquid ratio of 1:10 (w / v), stir continuously in a 40℃ water bath for 12 hours, centrifuge at 4000 g / min for 15 minutes, collect the precipitate and dry it; 5. Starch removal: Add α-amylase to the dried precipitate obtained in the previous step, with an enzyme to substrate mass ratio of 1:25 (w / w), and add deionized water at a material-to-liquid ratio of 1:10 (w / v). Perform enzymatic hydrolysis at 50°C for 4 hours. 6. Dialysis purification: Transfer all the enzymatically hydrolyzed mixture to a pre-treated dialysis bag with a molecular weight cutoff of 8000-14000 Da. Completely immerse the dialysis bag in a large amount (more than 100 times the total volume) of deionized water and dialyze at 4°C for 48 hours, changing the external dialysis solution every 6-8 hours during the process. 7. Post-processing and storage: After dialysis, the contents of the dialysis bag are collected and freeze-dried. The dried product is then processed by a pulverizer and passed through a 60-mesh sieve to obtain purified apple cell wall powder. The powder is then placed in a sealed bag and stored at -20°C for later use.

[0016] Example 2 Preparation of Aqueous DES The preparation method of water-containing DES without enzymes includes the following specific steps: 1. Raw material pretreatment: Choline chloride (ChCl) and 1,2-propanediol (PG) were dried to constant weight in a vacuum drying oven at 40℃ for later use; 2. Synthesis of pure deep eutectic solvent: Dry ChCl and PG are mixed in a molar ratio of 1:2, and the mixture is continuously stirred and heated at 80°C until a homogeneous, clear and transparent liquid is formed, indicating that a deep eutectic solvent (DES) has been formed. The prepared pure DES is transferred to a desiccator and sealed and stored at room temperature. 3. Preparation of aqueous eutectic solvent: The residual water content of the pure DES was determined by Karl Fischer titration. To obtain DES with a specific water content, a calculated mass of ultrapure water was added to the pure DES and mixed thoroughly to prepare aqueous DES with a water content of 23.57 Wt%. The molar ratio of each component in the aqueous DES was calculated to be ChCl / PG:H2O = 1:2:5.

[0017] The preparation method of aqueous DES-enzyme includes the following specific steps: 1. Raw material pretreatment: Choline chloride (ChCl) and 1,2-propanediol (PG) were dried to constant weight in a vacuum drying oven at 40℃ for later use; 2. Synthesis of pure deep eutectic solvent: Dry ChCl and PG are mixed in a molar ratio of 1:2, and the mixture is continuously stirred and heated at 80°C until a homogeneous, clear and transparent liquid is formed, indicating that a deep eutectic solvent (DES) has been formed. The prepared pure DES is transferred to a desiccator and sealed and stored at room temperature. 3. Preparation of aqueous eutectic solvent: The residual water content of the pure DES was determined by Karl Fischer titration. To obtain DES with a specific water content, a calculated mass of ultrapure water was added to the pure DES and mixed thoroughly to prepare aqueous DES with a water content of 23.57 Wt%. The molar ratio of each component in the aqueous DES was calculated to be ChCl / PG:H2O = 1:2:5. 4. Preparation of enzyme-containing extractant: Measure 125 mL of the above-mentioned ChCl / PG-based aqueous DES with a water content of 23.57 wt%, add 0.37 g of cellulase to it, place the mixture on a magnetic stirrer, and stir at room temperature for 15 minutes to ensure that the cellulase is fully dissolved and uniformly dispersed in the solvent, thus obtaining the desired aqueous DES-enzyme composite extractant.

[0018] Example 3 Extraction of pectin polysaccharides rich in RG-I type using an aqueous eutectic solvent containing cellulase ( ChCl / PG&E-P ） The specific steps for extracting RG-I type pectin polysaccharides from apple cell walls prepared in Example 1 and aqueous DES-enzyme prepared in Example 2 include: 1. ChCl / PG / H2O pretreatment: 5 g of apple cell wall was mixed with 125 ml of ChCl / PG / H2O solution and heated with magnetic stirring at 50 °C for 2 h. Then, the supernatant and solid residue were separated by centrifugation at 4000 g / min for 20 min. 2. Hot water extraction: Collect the pretreated precipitate, add 125 ml of ultrapure water to the precipitate, shake at 80℃ for 10 min, centrifuge at 4000 g / min for 15 min, and collect the supernatant solution. Repeat this process five times. 3. Alcohol precipitation: The combined supernatant solution was precipitated overnight with four times its volume of ethanol. The extracted pectin was centrifuged at 4000 g / min for 15 min and collected, then washed three times with ethanol for decolorization. 4. Reconstitution and freeze-drying: Dissolve pectin in ultrapure water and transfer it to a pre-treated dialysis bag with a molecular weight cutoff of 8000-14000 Da. Completely immerse the dialysis bag in a large amount (more than 100 times its total volume) of deionized water and dialyze at 4°C for 48 hours. Replace the external dialysis solution every 6-8 hours during the process. Collect the pectin solution in the dialysis bag and freeze-dry it.

[0019] Comparative Example 1: Extraction of pectin polysaccharides rich in RG-I type using an aqueous eutectic solvent without cellulase ( ChCl / PG-P ） This comparative example uses apple cell walls prepared in Example 1 and water-containing DES (without cellulase) prepared in Example 2. The specific steps include: 1. Pretreatment: Mix 5 g of apple cell wall with 125 ml of aqueous DES, heat with magnetic stirring at 50 °C for 2 h, and then centrifuge at 4000 g / min for 20 min to separate the supernatant and solid residue. 2. Hot water extraction: Collect the pretreated precipitate, add 125 ml of ultrapure water to the precipitate, shake at 80℃ for 10 min, centrifuge at 4000 g / min for 15 min, and collect the supernatant solution. Repeat this process five times. 3. Alcohol precipitation: The combined supernatant solution was precipitated overnight with four times its volume of ethanol. The extracted pectin was centrifuged at 4000 g / min for 15 min and collected, then washed three times with ethanol for decolorization. 4. Reconstitution and freeze-drying: Finally, dissolve the pectin in ultrapure water and transfer it to a pre-treated dialysis bag with a molecular weight cutoff of 8000-14000 Da. Completely immerse the dialysis bag in a large amount (more than 100 times the total volume) of deionized water and dialyze at 4°C for 48 hours. Replace the external dialysis solution every 6-8 hours during the process. Collect the pectin solution in the dialysis bag and freeze-dry it to obtain the pectin sample.

[0020] Comparative Example 2: Extraction with phosphate buffer solution ( EP ） The difference between this comparative example and Example 3 is that a phosphate-buffered enzyme solution was used to extract pectin. The specific steps include: 1. Pretreatment: Mix 5 g of apple cell wall with 125 ml of phosphate buffer solution, heat with magnetic stirring at 50 °C for 2 h, and then centrifuge at 4000 g / min for 20 min to separate the supernatant and solid residue. 2. Alcohol precipitation: Collect the supernatant and precipitate overnight with four times its volume of ethanol. Centrifuge the extracted pectin at 4000 g / min for 15 min, collect, and then wash three times with ethanol for decolorization. 3. Reconstitution and freeze-drying: Dissolve pectin in ultrapure water and transfer it to a pretreated dialysis bag with a molecular weight cutoff of 8000-14000 Da. Completely immerse the dialysis bag in a large amount (more than 100 times the total volume) of deionized water and dialyze at 4°C for 48 hours. Replace the external dialysis solution every 6-8 hours during the process. Collect the pectin solution in the dialysis bag and freeze-dry it to obtain the pectin sample E-AP.

[0021] Experimental Example 1: Determination of Chemical Composition of Pectin Polysaccharides The chemical composition of the pectin polysaccharides prepared by the preparation methods of ChCl / PG&E-P, ChCl / PG-P, and EP in Examples 3, 1, and 2 was determined.

[0022] The total phenol content of the sample was determined using the Folin-Ciocalteu method. 250 μL of Folin-Ciocalteu reagent (2 M) was added to 500 μL of a 1 mg / mL pectin aqueous solution. After mixing, the mixture was allowed to stand for 2 min. Then, 750 μL of 20% Na2CO3 was added, and the volume was adjusted to 5 mL with ultrapure water. After incubation at room temperature for 1 h, the absorbance of the supernatant was read at 760 nm. A standard curve was prepared using gallic acid as a standard to calculate the total phenol content in the sample.

[0023] Protein content was determined by the Bradford method, in which 200 µL of pectin solution (1 mg / mL) was mixed with 1 mL of Coomassie brilliant blue solution, incubated in the dark for 10 min, and the absorbance was measured at 595 nm. The protein content in the sample was calculated using the bovine serum albumin standard curve.

[0024] The total sugar content in the sample was determined using the phenol-sulfuric acid method. 100 μL of 6% phenol solution was added to 200 μL of pectin sample solution (1 mg / mL), and after thorough mixing, 500 μL of concentrated sulfuric acid was added. The mixture was vortexed to ensure complete mixing, and after reacting at 90℃ for 10 min, the absorbance was measured at 490 nm. An equal volume of ultrapure water was used as a blank in place of the sample solution, and a standard curve was constructed using glucose as the standard. The total sugar content of the sample was calculated using the standard curve.

[0025] Experimental Example 2: Determination of Monosaccharide Composition of Pectin Polysaccharides The pectin polysaccharides prepared in Examples 3, 1, and 2 were analyzed for monosaccharide composition using high-performance liquid chromatography (HPLC). 1 mL of a 6.0 mg / mL pectin solution was hydrolyzed with 1.0 mL of 4.0 mol / L trifluoroacetic acid in a 95°C water bath for 12 h. The hydrolyzed pectin sample was then transferred to a rotary evaporator, and 1 mL of methanol was added in four portions. The mixture was then evaporated under vacuum until dry to remove the trifluoroacetic acid, and finally diluted to 1 mL with ultrapure water. 50 μL of the polysaccharide hydrolysate or monosaccharide standard solution, 100 μL of 0.6 mol / L NaOH solution, and 100 μL of 0.5 mol / L PMP methanol solution were thoroughly mixed and incubated at 70°C in the dark for 100 min. After the reaction, an appropriate amount of 0.3 mol / L HCl solution was added for neutralization, and the mixture was diluted to 1 mL with ultrapure water. Extraction was then performed with chloroform until the solution sample became clear. Finally, the sample was filtered through a 0.22 μm organic filter membrane for high-performance liquid chromatography (HPLC) analysis. Chromatographic conditions: injection volume 20 µL; ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm, id 5 µm); mobile phase a mixture of 0.1 mol / L phosphate buffer (pH=6.7) and acetonitrile (83:17, v / v); flow rate 1.0 mL / min; DAD wavelength 245 nm. The monosaccharide standard solution included galacturonic acid, galactose, arabinose, fucose, rhamnose, glucose, mannose, and xylose. The types of monosaccharides in pectin were determined by peak time, and the molar ratio of monosaccharides in pectin was calculated using the area normalization method based on the peak area and concentration of the standards.

[0026] Experimental Example 3: Atomic Force Microscopy Analysis of Pectin Polysaccharides The morphology of pectin was characterized using atomic force microscopy (AFM). Three pectin polysaccharide samples (30 μg / mL, 20 μL) were pipetted onto freshly peeled mica substrates and dried at room temperature. Subsequently, AFM images were obtained using the tapping mode, and the images were analyzed using Nanoscope Analysis 1.8 software. Figure 2 ).

[0027] Experimental Example 4: Infrared Spectroscopic Analysis of Pectin The mixture of pectin and potassium bromide was ground at a ratio of 1:100 (w / w), and Fourier transform infrared spectroscopy was used to analyze the mixture at a wavelength of 4000-4000 cm⁻¹. -1 The region was scanned to obtain an infrared spectrum, and each peak in the spectrum was analyzed. Figure 2 ).

[0028] The degree of esterification (DE) is measured at 1700-1750 cm⁻¹. -1 (Esterified aldehyde) and 1600-1630 cm -1 Based on (free aldehyde acid), calculate according to formula (1): (1) Where A 1700-1750 This indicates that the FTIR spectrum is between 1700 and 1750 cm⁻¹. -1 Peak area at point A 1600-1630 1600-1630cm -1 The peak area.

[0029] Experimental Example 5: Determination of the degree of acetylation of pectin polysaccharides Take 1 mL of pectin solution (5 mg / mL) and add 5 mL of HCl (0.1 mol / L) and 5 mL of NaOH (0.1 mol / L) sequentially. Mix well and let stand for 20 min. Then add 5 mL of HCl (2 mol / L) to neutralize. After 15 min, add 10 mL of FeCl3 solution (0.37 mol / L). Dilute with deionized water and measure the absorbance at 500 nm. Plot a calibration curve using 5-O-acetyl-β-D-glucose standard and calculate the degree of acetylation according to formula (2): (2) Where 43.04 is the molecular weight of acetyl and 176.12 is the molecular weight of non-esterified dehydrated galacturonic acid.

[0030] Experimental Example 6: Determination of the molecular weight of pectin polysaccharides The molecular weight (Mw) and dispersion index (Mw / Mn) of pectin were determined using high performance size exclusion chromatography (HPSEC-MALLS-RID) with a refractive index detector and a multi-angle laser scattering spectrometer. The pectin concentration was approximately 1.0 mg / mL, and the injection volume was 100 μL. The chromatographic column was a Shodex OHpak SB-806M HQ (300 mm × 8.0 mm, id) tandem with a Shodex OHpak SB-G 6B (50 mm × 6.0 mm) gel column. The column temperature was set to 35℃, the mobile phase was 0.9% NaCl aqueous solution, and the flow rate was set to 0.5 mL / min. The data were analyzed using Astra software.

[0031] The specific yield, chemical composition, and monosaccharide composition of pectin polysaccharides are shown in Table 1.

[0032] Table 1. Yield, chemical composition, and monosaccharide composition of pectin polysaccharides Experimental Example 1 systematically describes the structural features of EP, ChCl / PG-P, and ChCl / PG&E-P. First, the chemical composition shows that polysaccharides are the main components of the three polymers, with ChCl / PG&E-P (88.23 mg / 100 mg) being the most abundant. -1 The highest total sugar content was found in ChCl / PG-P (80.54 mg / 100 mg). -1 Finally, the EP (75.96 mg 100 mg) -1 (See Table 1). The total protein content of EP, ChCl / PG-P, and ChCl / PG&P ranged from 3.56 mg to 100 mg. -1 Up to 10.72 mg 100 mg -1 Besides the arabinogalactan protein known to be present in apples, the higher total protein content in EP may also originate from cellulase co-precipitated with polysaccharides via ethanol, and contains 0.15-0.59 mg / 100 mg of [protein / protein content missing]. -1 Compared to commercial pectin containing phenolic compounds, the total phenolic content in ChCl / PG&E-P and ChCl / PG-P was 0.30 mg and 100 mg, respectively. -1 and 0.47 mg 100 mg -1 The total phenol content in EP was significantly higher than that in ChCl / PG&E-P and ChCl / PG-P ( p< 0.05), the ability of DES to recover phenolic compounds from various plant cell walls has been widely confirmed. Using ChCl / PG / CE or ChCl / PG to extract some phenolic compounds from apple cell walls resulted in lower total phenol content in ChCl / PG&E-P and ChCl / PG-P.

[0033] In Experiment 2, the monosaccharide composition of all polysaccharide components was determined, and a total of eight sugars were detected (see Table 1). Among the different monosaccharides, the monosaccharides related to pectin, including galacturonic acid (GalA), rhamnose (Rha), arabinose (Ara), and galactose (Gal), accounted for 85.30%, 89.16%, and 93.51% of the total content in EP, ChCl / PG-P, and ChCl / PG&E-P, respectively. Pectin, the main polysaccharide, was effectively isolated from the apple cell wall using the ChCl / PG / CE-assisted water extraction method. As for other monosaccharides, mannose (Fuc) and xylose (Xyl) not only attached to the main chains of rhamnose galactan II and xylose galactan but also formed the side chains of xylan.

[0034] Mannuronic acid (Man) originates from the dextran / cellulose backbone. Although CE was used to hydrolyze cellulose in the EP and ChCl / PG&E-P groups, the cellulose oligosaccharides generated by hydrolysis during polysaccharide precipitation and removal via dialysis were excluded. Therefore, glucose (Glc) in this study mainly originated from hemicellulose, a conclusion confirmed by the presence of glucose (Glc) found in ChCl / PG-P. Compared with EP and ChCl / PG-P, trace amounts of hemicellulose were extracted from ChCl / PG&E-P. The total content of Fuc, Xyl, Man, and Glc in EP was 14.7 mol%, while that in ChCl / PG-P was 10.84 mol% and that in ChCl / PG&E-P was 6.49 mol%. Since the HG / RG-Ⅰ ratio in all pectin components was less than 1, pectin rich in RG-Ⅰ domains was extracted. Compared with EP, pretreatment of apple cell walls with ChCl / PG or ChCl / PG / CE significantly increased the content of RG-Ⅰ extracted with water, with the highest content in ChCl / PG&E-P at 81.28 mol%. p <0.05, Table 1), in addition to the RG I main chain, the increase in RG I content in ChCl / PG-P and ChCl / PG&E-P is also due to their rich side chain structure. Compared with EP and ChCl / PG-P, the Ara and Gal contents in ChCl / PG&E-P are significantly increased ( p < 0.05).

[0035] The arabinose side chain decomposes rapidly under mild acidic conditions. While RG I-rich pectin can be recovered via CE extraction, the arabinose content in EP is significantly reduced due to the acidity of the phosphate buffer solution (pH 4.8). The degree of branching, calculated based on the ratio of the sum of arabinose and galactose to rhamnose, roughly reflects the ratio of the content to the length of the side chain on the rhamnose residues. ChCl / PG&E-P exhibits the highest degree of branching, indicating the presence of more / longer RG I side chains. As shown in Table 1, EP shows the highest degree of acetylation at 2.82%, followed by ChCl / PG-P at 1.79%, and finally ChCl / PG&E-P at 1.43%. Since the degree of methyl esterification in all RG I-rich pectins is less than 50%, they are classified as low-methoxyl (LM) pectins.

[0036] Depend on Figure 2 It can be seen that the two-dimensional surface morphology of ChCl / PG&E-P exhibits uniform dispersion, high coverage, and a network-like appearance. The height of the oligomeric molecular structure ranges from 0.2 to 1.9 nm, with clearly visible single-stranded polysaccharide chains and branched structures. ChCl / PG&E-P has a high total sugar content and a high proportion of pectin characteristic monosaccharides, which is conducive to the formation of a dense and highly covered network structure. Due to the high content of RG-I in ChCl / PG&E-P, it acts as the main chain to help cross-link between molecules. At the same time, the high content of arabinose and galactose forms a three-dimensional network structure through entanglement and connection. The two-dimensional surface morphology of ChCl / PG-P shows a chain-like dispersion with low coverage, and the height of the oligomeric molecular structure ranges from 0.0 to 1.7 nm. The low density of branched structures (nm) suggests that the molecular chains may adsorb onto the substrate in a more extended or monolayered manner. While the total sugar content and proportion of pectin characteristic monosaccharides in ChCl / PG-P are higher than in EP, they are lower than in ChCl / PG&E-P, resulting in relatively limited adsorption and coverage density on the substrate surface. Due to the relatively insufficient abundance and branching of its side chains, its structural ability to form complex networks through side chain entanglement is directly limited, resulting in a dispersed chain morphology and fewer cross-linking nodes as shown in the figure. EP, in the two-dimensional surface morphology diagram, shows a discrete spherical or granular aggregate dispersion with low substrate coverage. No continuous chain or network structure was observed, nor was there a clear branching morphology; the oligomer height is distributed between 1.9 and 9.3. Within a relatively wide range of nm, it is significantly higher than the other two groups of samples. EP has the lowest total sugar content and the lowest proportion of pectin characteristic monosaccharides. EP has a low arabinose content, which makes its branching degree the lowest among the three. The reduction and degradation of side chains directly weakens the ability of molecular chains to form extended networks or stable branches through side chain entanglement, thus prompting molecular chains to fold, curl up or form spherical or granular aggregates through other interactions.

[0037] like Figure 3 As shown, through Fourier transform infrared spectroscopy, nuclear magnetic resonance hydrogen spectroscopy, molecular weight and rheological analysis, the differences between different pectin components were further revealed at the levels of molecular bonding, fine structure and macroscopic properties, such as... Figure 3 As shown in A, all samples exhibited characteristic absorption peaks at 3400 cm⁻¹, 2922 cm⁻¹, 1749 cm⁻¹, 1640 cm⁻¹, 1105 cm⁻¹, and 1060 cm⁻¹, corresponding to the stretching vibrations of OH bonds, CH bonds, C=O bonds, carboxyl groups, and COC and CO of sugar rings, respectively. Among them, the absorption peak intensities were highest for ChCl / PG&E-P, followed by ChCl / PG-P, and highest for EP, indicating that ChCl / PG&E-P has higher chemical purity and a more complete molecular structure, with the most abundant hydroxyl groups, hydrocarbon skeletons, and glycosidic bonds. Figure 3 B is the ¹H NMR spectrum. The three samples, including EP, ChCl / PG-P, and ChCl / PG&E-P, show a series of typical sugar ring proton signals in the 3.0-5.5 ppm region, including labeled peaks such as Ara-H1, GalA-H1, GalA-H5, GalA-H4, GalA-H3, Gal-H4, GalA-H2, and Gal-H2. At the same time, dense peak groups related to polysaccharide or polyol backbones can be seen in the 3.6-3.7 ppm region. Overall, it is indicated that all three samples retain the structural features dominated by pectin polysaccharide backbones. However, ChCl / PG&E-P has a higher signal-to-noise ratio and sharper peaks at each characteristic peak. Compared with EP, choline-related characteristic signals were found in ChCl / PG&E-P and ChCl / PG-P. Meanwhile, characteristic peaks of branched Rha-H6 and unbranched Rha-H6 were found in all pectin samples, with higher intensity in ChCl / PG&E-P, confirming the presence of more RG-I type pectin polysaccharides in ChCl / PG&E-P. Compared to EP, ChCl / PG&E-P and ChCl / PG-P have higher molecular weights ( Figure 3 C). Furthermore, the Mw / Mn values ​​of ChCl / PG&E-P and ChCl / PG-P are 1.38 and 1.25, respectively. A value closer to 1 indicates a narrow molecular weight distribution and uniform structure, suggesting that DES-assisted extraction can yield more structurally regular products; such as Figure 3 The apparent viscosity of all three systems shown in D decreases with increasing shear rate, exhibiting shear-thinning behavior. ChCl / PG-P has the highest viscosity in the low shear region and the decrease is more significant, followed by ChCl / PG&E-P, while EP has the lowest overall viscosity and is closer to that of a dilute solution. Using the method of this application significantly enhances the viscosity and structural strength of the system.

[0038] In summary, the extraction method profoundly affects the multilevel structure and functional properties of apple pectin. Compared with the traditional acid extraction method, the method of hot water extraction assisted by pretreatment with ChCl / PG containing cellulase in this application can more efficiently and completely obtain pectin polysaccharides from the cell wall. This method successfully enriched the highly branched RG-I domain, obtaining a high molecular weight, narrowly distributed and structurally uniform pectin component. Atomic force microscopy directly revealed the corresponding nanoscale continuous three-dimensional network morphology, while ¹H NMR and FTIR spectroscopy confirmed its structural integrity and high purity at the molecular level. It also has a high apparent viscosity. Therefore, the method of this application can extract pectin polysaccharides rich in RG-I type.

[0039] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A method for extracting pectin polysaccharides rich in RG-I type using an aqueous eutectic solvent containing cellulase, characterized in that, Includes the following steps: S1. Take the purified plant cell wall and mix it with the eutectic solvent containing cellulase, stir and heat to obtain a solid-liquid mixture; S2. Centrifuge the solid-liquid mixture to obtain a first supernatant and solid residue; S3. Add water to the solid residue, heat and shake, then centrifuge, collect the supernatant aqueous solution, repeat five times to obtain multiple supernatant aqueous solutions; S4. Combine the supernatant aqueous solutions from the previous steps, add alcohol, centrifuge, collect the precipitate, and decolorize.

2. The method according to claim 1, characterized in that, The method for extracting plant cell walls described in S1 includes the following steps: S11. Plant cell walls are treated with organic solvents to remove phenolic substances and reducing sugars, resulting in pre-treated cell walls; S12. The pre-treated cell wall is treated with amylase to remove starch, resulting in an enzymatically hydrolyzed cell wall; S13. The enzymatically hydrolyzed cell wall is subjected to dialysis to remove small molecule impurities and obtain the purified plant cell wall; S14. Freeze-dry the purified plant cell walls, pulverize and sieve them, and store them in a sealed container at -20°C.

3. The method according to claim 2, characterized in that, In S11, the plant includes an apple, the organic solvent includes ethanol, and the mass ratio of the plant cell wall to the volume of the organic solvent is 1:

10. And / or, in S12, the amylase includes α-amylase, and the mass ratio of the pre-treated cell wall to the amylase is 25:1; And / or, in S13, the enzymatically hydrolyzed cell wall is placed in a dialysis bag with a molecular weight cutoff of 8000-14000 Da; And / or, in S14, the purified plant cell wall is pulverized and passed through a 60-mesh sieve.

4. The method according to claim 1, characterized in that, The aqueous eutectic solvent is composed of choline chloride and 1,2-propanediol, wherein the molar ratio of choline chloride to 1,2-propanediol is 1:

2. In the aqueous eutectic solvent, the mass percentage of water is 20% to 30%.

5. The method according to claim 1, characterized in that, The amount of cellulase added is 0.30~0.40 g per gram of dry weight of plant cell wall material, and the enzyme activity of the cellulase is 15000~20000 U; And / or, in S1, the heating temperature is 45℃~55℃, and the time is 2~3 h.

6. The method according to claim 1, characterized in that, In S2, the centrifugation speed is 4000 g / min, and the centrifugation time is 20 min; And / or, in S3, the centrifugation speed is 4000 g / min and the centrifugation time is 20 min; And / or, in S4, the centrifugation speed is 4000 g / min and the centrifugation time is 20 min.

7. The method according to claim 1, characterized in that, In S3, the temperature of the heating oscillation is 70℃~90℃; And / or, the heating oscillation time is 10 min.

8. The method according to claim 1, characterized in that, In S4, pectin polysaccharides are recovered from the supernatant aqueous solution by alcohol precipitation, wherein the alcohol includes ethanol, and the volume ratio of the alcohol to the supernatant is 4:

1.

9. The RG-I type pectin polysaccharide obtained by the method according to any one of claims 1-8.

10. The pectin polysaccharide rich in RG-I type according to claim 9, characterized in that, The monosaccharide components of the RG-I type pectin polysaccharide-rich product include: Fucose 1.40 ± 0.06 mol% Arabinose 24.50±2.45 mol% Rhamnose 12.51 ± 0.94 mol% Galactose 31.76 ± 2.27 mol% Glucose 2.89 ± 0.74 mol% Xylose 1.28 ± 0.60 mol% Mannose 0.92±0.51 mol% Galacturonic acid 24.74±1.13 mol%.

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