Method for enriching and extracting RG-I pectin from citrus

Abstract

The invention relates to the technical field of pectin preparation, and discloses a method for enriching and extracting RG-I pectin from citrus, which comprises the following steps: treating citrus fruit tissues at 35-40 DEG C for 15-60 days, extracting water-soluble pectin, and purifying to obtain a product rich in RG-I pectin. According to the method, the improvement of the content of RG-I pectin in a citrus raw material is combined with an extraction process, and a pectin component rich in an RG-I region is obtained from water-soluble polysaccharide of granulated juice sacs through elution of a 0.05 mol / L NaCl solution and a 0.1 mol / L NaCl solution by anion exchange gel chromatography. The method can be used for raw material selection, pretreatment and extraction of industrial production of RG-I pectin.

Description

Technical Field

[0001] The invention relates to the technical field of pectin preparation, in particular to a method for enriching and extracting RG-I type pectin from citrus. Background Art

[0002] Citrus fruits are rich in pectin and are currently an important source of industrial pectin. Pectin generally contains a homogalacturonan (HG) domain and a rhamnogalacturonan-I (RGⅠ) domain. The former is a linear polymer formed by D-galacturonic acid connected by α-1,4-glycosidic bonds, and the latter is composed of [→4)-α-D-galacturonic acid-(1→2)-α-L-rhamnose (1→] repeating units, with side chains of arabinan, galactan and / or arabinogalactan, connected to the O-4 site of the main chain rhamnose residue. In order to obtain pectin of uniform quality in commercial production, high temperature and strong acid extraction conditions are usually used to treat the raw materials. The commercial pectin obtained is usually dominated by the HG domain (>65%), and the RG-Ⅰ domain is destroyed under these conditions.

[0003] In recent years, a large number of studies have shown that multi-branched pectin rich in RG-I has better biological activities such as intestinal probiotics, immune regulation, anti-cancer metastasis, antioxidant and regulation of chronic metabolic diseases. Therefore, the extraction method of RG-I pectin has also attracted attention. The extraction methods of RG-I-rich pectin reported so far include: acid-base continuous extraction, enzyme-assisted extraction, ultrasound-assisted alkaline extraction, pressure ultrasonic extraction, etc.

[0004] A Chinese invention patent (publication number: CN107629135B, publication date: 2020.05.12) discloses a method for extracting RG-I rich pectin polysaccharides. The method comprises the following steps: mixing citrus peel powder with a dilute hydrochloric acid solution at low temperature to destroy the cell wall of the citrus peel, filtering and mixing the retentate with a sodium hydroxide solution for low-temperature extraction, adding ethanol precipitation to the filtered solution, washing the obtained polysaccharide with ethanol and drying it to obtain the above-mentioned pectin polysaccharide. Compared with the traditional pectin polysaccharide extraction method, the method described in the invention reduces the hydrolysis of the pectin side chain by controlling the extraction temperature. The extracted pectin polysaccharide is rich in RG-I type domains and has high biological activity in preventing cancer, cardiovascular diseases, etc. At the same time, since the extraction process is carried out at room temperature, energy can be greatly saved.

[0005] A Chinese invention patent (publication number: CN118221845A, publication date: 2024.06.21) discloses a preparation method for extracting RG-I type pectin from pomelo peel. Pomelo pectin is extracted by combining a low-concentration alkaline solution with dielectric barrier discharge plasma technology. Compared with the existing technology, the concentration of the sodium hydroxide alkaline solution in the inventive method is up to 2.4g / L, which is much lower than the ordinary concentration and is green and environmentally friendly. Under the premise of chemical methods, plasma technology can effectively improve the extraction rate of pectin. The extracted RG-I type pectin has a high extraction rate and a stable RG-I domain content with high purity, which is suitable for extracting pectin biomacromolecules.

[0006] However, there are many varieties of citrus fruits, and the content of RG-I pectin varies greatly between different varieties and different tissue parts. Therefore, in addition to improving the extraction process, using fruits rich in RG-I pectin as raw materials is of great significance for improving the yield and simplifying the extraction process. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides a method for increasing the content of RG-I pectin in citrus raw materials and combining it with an extraction process, firstly inducing the synthesis of RG-I pectin in the raw materials by temperature, and then extracting and collecting components rich in RG-I pectin. The method can be used for raw material selection, pretreatment and extraction of industrial production of RG-I pectin.

[0008] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: A method for enriching and extracting RG-I type pectin from citrus, the method comprising the following steps: 1) Enrichment of RG-I pectin: Place fresh citrus fruit tissue at 35-40°C for 15-60 days to increase the content of RG-I pectin in the citrus raw material; 2) Extraction of alcohol-insoluble solids: Dry the citrus fruit tissue and grind it into powder. Use alcohol to extract and remove the alcohol-soluble substances, leaving the alcohol-insoluble solids. 3) Extraction of water-soluble pectin: After drying the alcohol-insoluble solids, dissolve them in boiling water to extract the water-soluble pectin, precipitate the water extract with alcohol, and dry the precipitate to obtain a crude water-soluble pectin extract; 4) Purification: The crude water-soluble pectin extract is subjected to anion exchange gel chromatography to obtain a component rich in RG-I pectin, and a product rich in RG-I pectin is obtained by desalting through dialysis and drying.

[0009] Preferably, the drying in step (2) and step (4) is performed by vacuum freeze drying.

[0010] Preferably, the extraction of the alcohol-insoluble solids comprises the following steps: 1) Add 95% ethanol to citrus fruit powder and perform ultrasonic-assisted water bath extraction at room temperature to remove alcohol-soluble substances; repeat three times; 2) Collect the residue and continue to add 95% ethanol to it, heat to boiling, use a condensation reflux device to continuously extract and remove alcohol-soluble matter, and dry the residue at 60-70°C overnight to obtain alcohol-insoluble solids.

[0011] Among them, the solid-liquid ratio of citrus fruit powder or residue and 95% ethanol is 1:10 (w / v).

[0012] Among them, ultrasonic-assisted water bath extraction takes 20-40 min; condensation reflux device continues extraction for 2-4 hours.

[0013] Preferably, the extraction of the water-soluble pectin specifically comprises the following steps: 1) adding distilled water containing 2% w / v sodium thimerosal to the alcohol-insoluble solid powder, extracting water-soluble pectin by ultrasonic-assisted water bath at room temperature, collecting the supernatant, and obtaining a crude water-soluble pectin extract; 2) The crude water-soluble pectin extract was concentrated in vacuo to 1 / 5 of its original volume, and 4 volumes (v / v) of 95% ethanol were added for precipitation overnight. The precipitate was collected and washed repeatedly with anhydrous ethanol for 3 times, and dried at 60-70°C overnight to obtain water-soluble pectin.

[0014] The solid-liquid ratio of the alcohol-insoluble solid powder to distilled water is 1:10 (w / v).

[0015] Among them, ultrasound-assisted water bath extraction was performed for 3-5h.

[0016] Preferably, a preparation method for obtaining a component rich in RG-I pectin using anion exchange gel chromatography specifically comprises the following steps: the ion exchange chromatography column uses DEAE-agarose gel, and is eluted successively with distilled water, 0.05 mol / L NaCl solution, and 0.1 mol / L NaCl solution, with each eluent eluting 3-4 column volumes, and the elution flow rate is set to 1 / 85-1 / 75 column volume of eluent per minute.

[0017] Preferably, the dialysis desalination uses a 6000 Da to 8000 Da dialysis bag to dialyze the eluate for desalination.

[0018] The invention induces the synthesis of RG-I pectin in citrus by temperature, obtains citrus juice cells in a granulated state after 15-60 days of treatment, and then extracts and collects components rich in RG-I pectin. Among them, the proportion of RG-I regions of gels and slightly granulated juice cells eluted with 0.05 mol / L NaCl solution and 0.1 mol / L NaCl solution is significantly increased. The method can be used for raw material selection, pretreatment and extraction of industrial production of RG-I pectin. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the WSF content of citrus juice cells in different states.

[0020] Figure 2 The contents of DEAE-Sepharose elution components in WSF of citrus juice cells in different states (A: DEAE-Sepharose anion exchange elution curve of citrus juice cells WSF with different granulation degrees; B: percentage of content of different polysaccharide elution components in WSF).

[0021] Figure 3 The monosaccharide composition (A) and RG-I pectin content (B) of each elution fraction of citrus juice cells in different states.

[0022] Figure 4 FT-IR spectra of the eluted components of citrus juice cells in different states.

[0023] Figure 5A-5F These are the NMR spectra of the eluted components of citrus juice cells in different states.

[0024] Figure 6 This is a flow chart of the preparation of RG-I type pectin in Example 1. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1: Preparation of RG-I-rich pectin from grapefruit juice cells 1) Test methods 1. Juice cell sample preparation: Peel off the normal juice cells (transparent) of mature pomelo fruits, freeze them quickly with liquid nitrogen, and then perform vacuum freeze-drying. Take them out after 3-5 days, grind them into sample powder with a crusher, and store them in a -20℃ refrigerator.

[0027] 2. Extraction and determination of alcohol insoluble solids (AIR): Mix the above sample powder with 95% ethanol at a solid-liquid ratio of 1:10 (w / v), perform ultrasonic-assisted water bath extraction at room temperature for 30 min, remove the supernatant by vacuum filtration, collect the residue, add 95% ethanol solution to it again at a solid-liquid ratio of 1:10 (w / v) for ultrasonic-assisted extraction, repeat the above operation, collect the residue and repeat the extraction once. Install a condensation reflux device, add 95% ethanol to the residue at a solid-liquid ratio of 1:10 (w / v), boil it and then heat and reflux for extraction for 3 h. After cooling to room temperature, perform vacuum filtration, collect the solid residue, put it in a 60°C oven to dry overnight, and weigh the AIR.

[0028] 3. Extraction and determination of water-soluble pectin: Weigh AIR powder, add distilled water (containing 2% w / v sodium thimerosal) at a solid-liquid ratio of 1:100 (w / v) and mix well. Perform ultrasonic-assisted water bath extraction at room temperature for 4 h, collect the supernatant, and obtain a crude water-soluble pectin extract (water soluble fraction, WSF) solution. Take 0.1 mL of WSF solution, add 0.6 mL of concentrated H2SO4 (containing 0.48% sodium tetraborate), hydrolyze at room temperature for 12 h, add 10 µL of 0.15% m-hydroxybiphenyl (dissolved in 0.5% NaOH) solution, mix well, and let stand for 40 min. Take 200 µL of the reaction product and add it to a 96-well plate. Use a microplate reader (BIO-RAD, USA) to detect the absorbance at 530 nm. Galacturonic acid (GalAc) is used as a standard curve for quantification of pectin content (mg / g dry weight).

[0029] 4. Ethanol precipitation: Vacuum concentrate the WSF solution to 1 / 5 of the original volume, add 95% ethanol solution at a ratio of 1:4 (v / v), shake evenly and let stand overnight at room temperature. Filter the flocculent suspension and wash it repeatedly with anhydrous ethanol for 3 times, place it in a 65℃ oven to dry overnight to obtain the WSF solid extract. Weigh it, seal it with a sealing strip, and store it in a -20℃ refrigerator.

[0030] 5. Ion exchange chromatography purification and determination of pectin: ① Column loading: Fix the cleaned glass column vertically on the iron frame, connect the constant flow pump, and check the air tightness. Open the lower outlet switch, stir the treated filler with a glass rod, and pour it into the column along the inner wall of the glass column. At this time, the lower outlet switch of the column drains water while adding the stirred filler to the upper outlet, so that the filler settles continuously and evenly, gradually forming a uniform chromatography column. When it reaches about 2 cm from the top of the column, immediately close the lower outlet to allow the filler to settle.

[0031] ②Balance: Cover the filler with a 2-3 cm liquid layer, turn on the constant flow pump, and balance with distilled water at a flow rate of 0.5 mL / min overnight.

[0032] ③ Loading: Dissolve semi-pure pectin with a certain amount of distilled water to make the sample concentration 25 mg / mL. Then put the column liquid level just level with the filler, and avoid the liquid level being lower than the filler surface. Slowly add 4 mL of semi-pure pectin solution along the glass rod, without stirring the column filler, and then put the liquid level level with the filler surface. In the same way, add buffer to 2 to 3 cm above the filler surface.

[0033] ④ Elution: Use distilled water, 0.05, 0.1, 0.2, 0.3, 0.4 mol / L NaCl in sequence (3 column volumes per gradient wash, 120 mL), flow rate 0.5 mL / min. Collect 4 mL of eluate into one tube, a total of 180 tubes.

[0034] ⑤ Sugar content determination: 5% phenol solution and 98% concentrated sulfuric acid were mixed at a ratio of 1:5 (v / v) and cooled in an ice water bath; 40 µL of the sample or standard solution diluted to an appropriate concentration with distilled water was taken and mixed with 120 µL of the above phenol sulfuric acid solution, and the absorbance at 490 nm was measured with a Synergy H1 microplate reader (Biotek, USA) after being kept at 100 °C for 30 min. The standard curve was made with 10, 20, 30, 40, 50, 60, 70, and 80 μg / mL glucose solutions, and the regression equation y = 419.0x -21.01, R² = 0.999 (x is the glucose concentration, y is OD490 nm). The total sugar content of the sample is expressed in mg glucose / g. Three replicates were set for each sample.

[0035] ⑥ Protein content determination: Determine the total protein content in the polysaccharide according to the instructions of the Bradford protein quantification kit. Measure the UV-visible spectrum of the sample aqueous solution (1 mg / mL) at a wavelength of 570 nm on a microplate reader.

[0036] ⑦ Dialysis: Combine the main sugar-containing tubes, concentrate them by rotary distillation at 45 ℃, add them into a dialysis bag (6000 kDa) and dialyze them with distilled water for 48 h to completely remove NaCl. Concentrate the dialyzed samples by vacuum rotary evaporator and then freeze-dry them in vacuum to obtain the pectin components (the elution components of distilled water, 0.05 mol / L NaCl, 0.11 mol / L NaCl, and 0.2 mol / L NaCl are WA, 0.5 S, 1 S, and 2S, respectively). Seal and store at -20 ℃ for future use.

[0037] 6. Structural analysis: ① Analysis of monosaccharide composition: 1 mg of polysaccharide sample was hydrolyzed with 1 mL of 2 mol / L trifluoroacetic acid (TFA) at 120 °C for 2 h under sealed conditions, and the solvent was removed using a vacuum rotary evaporator at 30 °C. The residue was rinsed several times with methanol to remove excess TFA. Finally, the residue was dissolved in 100 μL ddH2O for further derivatization. Each sample was repeated 3 times. For pre-column derivatization, 100 μL of sample or 10 mg / mL monosaccharide standard solution was mixed with 120 μL of 0.5 mol / L 1-phenyl-3-methyl-5-pyrazolone methanol solution and 100 μL of 0.3 mol / L NaOH solution and incubated at 70 °C for 1 h. Then 100 μL of 0.3 mol / L HCl solution was added to neutralize the NaOH. Finally, 1 mL of dichloromethane was added, the mixture was shaken vigorously and centrifuged for 5 min. The supernatant was filtered through a 0.22 μm membrane, and 10 μL of the resulting solution was taken for HPLC analysis. HPLC analysis of monosaccharide content was performed using a Waters 2695-2996 HPLC system (Waters, Milford, MA, USA) with a Sunfire C18 analytical column (4.6 × 250 mm, Waters, USA) at a column temperature of 25 °C. The mobile phase consisted of 0.1 mol / L KH2PO4 buffer (pH 6.7) (eluent A) and acetonitrile (eluent B). The gradient program was as follows: 0-30 min, 18% B; 30-60 min, 18-25% B; 60-65 min, 25-18% B. The flow rate of the mobile phase was 1 mL / min, and the compounds were detected at 245 nm. The monosaccharide species were identified by comparing the retention time with the standard, and the monosaccharide standard curve was used for quantification.

[0038] ② Fourier transform infrared spectroscopy (FT-IR): 1 mg of freeze-dried polysaccharide was mixed with KBr powder, ground, and then pressed into 1 mm pieces. The spectra were collected on a Nicolet iS10 Fourier transform infrared spectrometer (Thermo Fisher Scientific, USA) (range 4000 - 400 cm -1 ), with a resolution of 4 cm -1 , the number of scans is 32.

[0039] ③ Nuclear magnetic resonance (NMR): The polysaccharide was dissolved in heavy water (D2O) with a sample concentration of 40 mg / mL. A JEOL JNM-ECZ500R / M1 500 MHz NMR spectrometer (JEOL, Japan) was used to collect 1 H spectrum and 13 C spectrum. 1 The H spectrum scan times are 256 times.13 The number of C spectrum scans was 10240. Chemical shifts are expressed in δ ppm.

[0040] 2) Test results AIR is a general term for all alcohol-insoluble compounds, mainly composed of cell wall components such as pectin, cellulose, hemicellulose, and lignin. In normal pomelo juice cells, the yield of AIR is 21.18%. The yield of WSF extracted from AIR is 53.90%. The contents of WA, 0.5S, 1S, and 2S in normal pomelo juice cells are 0.66 mg / g DW, 0.19 mg / g DW, 1.75 mg / g DW, and 3.66 mg / g DW, respectively. Figure 2 ).

[0041] In WA, the proportions of mannose (Man), rhamnose (Rha), glucuronic acid (GluAc), galacturonic acid (GalAc), glucose (Glu), galactose (Gal), xylose (Xyl), and arabinose (Ara) were 9.28%, 1.45%, 2.00%, 4.27%, 24.78%, 35.62%, 2.31%, and 20.29%, respectively. In 0.5S, the proportions of Man, Rha, GluAc, GalAc, Glu, Gal, Xyl, and Ara were 3.29%, 3.66%, 1.11%, 45.93%, 11.84%, 22.21%, 1.39%, and 10.56%, respectively. In 1S, the proportions of Man, Rha, GluAc, GalAc, Glu, Gal, Xyl, and Ara were 1.57%, 0.94%, 2.07%, 61.95%, 4.25%, 16.73%, 1.58%, and 10.91%, respectively. In 2S, the proportions of Man, Rha, GluAc, GalAc, Glu, Gal, Xyl, and Ara were 0.47%, 4.62%, 1.53%, 81.70%, 2.94%, 4.93%, 0.80%, and 3.00%, respectively. The RG-I region is composed of repeating units of [→4)-α-D -GalpA-(1 → 2)-α-L-Rhap(1 → ], with side chains of galactan, arabinan, and / or arabinogalactan attached to the O-4 position of Rha. The proportion of RG-I can be relatively quantified by the sum of 2Rha mol % + Gal mol % + Ara mol %. The degree of RG-I branching can be expressed by the molar ratio of (Gal + Ara) / Rha. In WA, 0.5S, 1S, and 2S, the proportions of RG-I are 58.81%, 40.09%, 29.52%, and 17.17%, respectively, and the degree of RG-I branching is 38.57%, 8.96%, 29.48%, and 1.72%, respectively. Figure 3 ).

[0042] Since the polysaccharide content in WA was less than 50%, it did not meet the requirements of FTIR and NMR analysis, so WA was not subjected to FTIR and NMR analysis. The results of FT-IR analysis showed that 0.5S, 1S, and 2S all showed characteristic peaks at 3405 cm⁻¹, 1637 cm⁻¹, 1407 cm⁻¹, and 2935 cm⁻¹, which were attributed to the hydroxyl stretching vibration of polysaccharides, the asymmetric stretching vibration of non-ester carbonyl C=O, the variable angle vibration of CH, and the stretching vibration of CH, respectively. They also showed CO stretching vibration at 1262 cm⁻¹ and COC vibration at 1147 cm⁻¹. The ester carbonyl C=O asymmetric stretching vibration peak shared by 0.5S, 1S, and 2S at 1745 cm⁻¹ indicated the presence of esterified carboxyl groups. The peaks at 832 cm⁻¹ and 920 cm⁻¹ indicated the presence of α-glycosidic bonds and β-glycosidic bonds. The peak at 762 cm⁻¹ indicates the presence of D-glucose pyranose ring. The peak at 638 cm⁻¹ indicates the presence of out-of-plane vibration of OH ( Figure 4 ).

[0043] In the 0.5S and 1S components, the normal juice cell sample is mainly composed of 1,4-6MeGalA, and its NMR spectrum is dominated by peaks at 100.547, 68.137, 79.262, 70.695, 170.92 and 52.971, which are respectively attributed to C1, C2 / C3, C4, C5, C6 and –OCH3 at the C6 position of 1,4-6MeGalA (Figure 5).

[0044] Example 2: Comparison of RG-I pectin content in different raw materials 1) Test methods 1. Juice cell sample preparation: mature pomelo fruit was used as the experimental material. The juice cells of the fruit were peeled off and graded according to the state of the juice cells: healthy juice cells (H), normal and transparent, juice flows out after cutting; gelatinization stage juice cells (G1), swollen but still transparent, the outer skin of the juice cells and the inside can be peeled off, and the inside is gelatinous; slightly granulated juice cells (G2), the juice cells swell and appear turbid white; severely granulated juice cells (G3), the juice cells swell and even twist, harden, and appear turbid white or yellow. Rapidly freeze with liquid nitrogen and then vacuum freeze-dry, take out after 3-5 days, use a crusher to grind into sample powder and store in a -20℃ refrigerator.

[0045] 2. The methods of extraction and determination of alcohol-insoluble solids (AIR), extraction and determination of water-soluble pectin, ethanol precipitation, ion exchange chromatography purification and determination of pectin, and structural analysis were the same as those in Example 1.

[0046] 2) Test results The AIR yields of normal, gel, slightly granulated and severely granulated samples were 21.18%, 23.57%, 29.41% and 33.71%, respectively. The WSF yields from AIR were 53.90%, 55.21%, 55.09% and 53.18%, respectively. The WSF contents per gram of juice cell samples extracted from normal, gel, slightly granulated and severely granulated samples were 114.13 mg / g DW, 130.13 mg / g DW, 162.00 mg / g DW and 185.74 mg / g DW, respectively. Figure 1 ).

[0047] The quantitative results of the purified water-soluble pectin extracts from juice cells showed that the pectin content showed an overall increasing trend with the increase in the degree of granulation. The experimental results showed that the yields of purified pectin (WA+0.5S+1S+2S) in samples H, G1, G2 and G3 were 3.66 mg / g DW, 3.97 mg / g DW, 5.90 mg / g DW and 10.03 mg / g DW, respectively. Among them, the contents of WA, 0.5S, 1S, and 2S in normal juice cells were 0.66 mg / g DW, 0.19 mg / g DW, 1.75 mg / g DW, and 3.66 mg / gDW, respectively; the contents of WA, 0.5S, 1S, and 2S in G1 juice cells were 0.50 mg / g DW, 0.44 mg / g DW, 1.01 mg / g DW, and 2.02 mg / g DW, respectively; the contents of WA, 0.5S, 1S, and 2S in G2 juice cells were 0.97 mg / g DW, 0.47 mg / g DW, 1.35 mg / g DW, and 3.11 mg / gDW, respectively; the contents of WA, 0.5S, 1S, and 2S in G3 juice cells were 1.58 mg / g DW, 0.80 mg / g DW, 2.93 mg / g DW, and 4.72 mg / gDW, respectively. Figure 2 ).

[0048] From the monosaccharide ratio, it can be seen that the monosaccharide composition of the water-soluble pectin component gradually changes with the increase of granulation degree. In the WA component, the ratio of GalAc and Xyl continues to increase, while the ratio of Ara, Gal and Man tends to decrease. The change trends of the 0.5S and 1S components are similar to each other and opposite to the change trend of the WA component, that is, with the increase of granulation degree, the ratio of GalAc decreases, while the ratio of Rha, Ara, Gal and Man continues to increase. In G2 and G3 juice cells, the sum of neutral monosaccharides accounts for more than 50%. In the 2S component, the ratio of GalAc and Rha decreases slightly, while the ratio of Ara, Gal and Man continues to increase with the increase of granulation degree. With the increase of granulation degree, the ratio of RG-I region (molar percentage of 2Rha+Ara+Gal) increases significantly, especially the 0.5S and 1S components, which increase from 40.09% and 29.52% in healthy juice cells to 78.54% and 84.64% in G3 juice cells, respectively. Correspondingly, the proportion of HG region (molar percentage of GalA-Rha) decreased as granulation progressed. In G2 and G3 juice cells, the (Ara+Gal) / Rha ratios of 0.5S, 1S, and 2S also increased, indicating that the RG-I side chain in the granulated juice cells was elongated. Therefore, the RG-I region of the water-soluble polysaccharide and the length of the arabinogalactan side chain in the granulated juice cells increased ( Figure 3 ).

[0049] From the NMR results, it can be seen that compared with normal juice cells, the polysaccharide structure in granulated juice cells has changed significantly, especially in the 0.5S and 1S components. In the 0.5S component, the normal juice cell sample is mainly composed of 6-O-methyl-1,4-galacturonic acid (1,4-6MeGalA), 13The peaks 100.547, 68.137, 79.262, 70.695, 170.92 and 52.971 in the C spectrum were respectively assigned to C1, C2 / C3, C4, C5, C6 and –OCH3 of C6 in 1,4-6MeGalA. With the increase of the degree of granulation, the intensity of these 1,4-6MeGalA signal peaks decreased, while the relative abundance of 1,4-GalA (1,4-galacturonic acid) increased, and the peaks 99.548, 68.833, 79.56, 71.118 and 175.287 corresponding to C1, C2 / C3, C4, C5 and C6 were significantly increased to detectable levels in G2 samples, and further increased in G3 samples. In addition, the peak signals of 1,2,4-Rha (1,2,4-rhamnose), t-Ara (arabinose terminal group), and 1,4-Gal (1,4-galactose) increased in G2 and dominated in G3 samples (Figure 5), indicating the increase of RG-I region and branched side chains, which is consistent with the results of monosaccharide molar ratio determination by high performance liquid chromatography (HPLC) (Supplementary Figure 5). Figure 4 ). The variation pattern of the 1S component in different granulated juice cells was similar to that of the 0.5S component (Figure 5). In contrast, the 2S component was dominated by 1,4-6MeGalA in both normal juice cells and granulated juice cells. In granulated juice cells, the signal of 1,4-6MeGalA decreased slightly, while the signal of 1,4-GalA increased slightly (Figure 5).

[0050] Example 3: Method for increasing the content of RG-I pectin in citrus raw materials 1) Test methods 1. Fresh fruits were packed in PE bags and placed in a 35°C environment. The juice cells of the fruits were peeled off on the 7th, 15th, 30th, 45th and 60th days, and the fruits were quickly frozen with liquid nitrogen and then vacuum-freeze-dried. After 3-5 days, the fruits were taken out and ground into sample powder using a crusher and stored in a -20°C refrigerator.

[0051] 2. The methods of extraction and determination of alcohol-insoluble solids (AIR), extraction and determination of water-soluble pectin, ethanol precipitation, ion exchange chromatography purification and determination of pectin, and structural analysis were the same as those in Example 1.

[0052] 2) Test results With the extension of treatment time at 35℃, the healthy juice cells (H) in normal and transparent state gradually expanded and loosened, the gelatinous degree inside the juice cells continued to deepen, the transparency gradually decreased and gradually turned white, the contact between the juice cells and the white cortex became loose, and finally the juice cells expanded or even twisted, hardened, and appeared turbid white or yellow, and the hardness continued to increase, showing a typical change from the granulation G1 stage to the G3 stage. The AIR yield also increased significantly with the extension of treatment time. The AIR content on the 7th, 15th, 30th, 45th and 60th days was 137.34 mg / g DW, 232.94 mg / g DW, 280.00 mg / gDW, 330.89 mg / g DW, and 514.49 mg / g DW, respectively. The WSF contents in AIR were 221.27 mg / g DW, 343.23 mg / gDW, 325.18 mg / g DW, 364.11 mg / g DW and 409.32 mg / g DW, respectively.

[0053] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be apparent to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will conform to the widest range consistent with the principles and novelties disclosed herein.

Claims

1. A method for enriching and extracting RG-I type pectin from citrus, characterized in that: The method comprises the following steps: 1) Enrichment of RG-I pectin: Place fresh citrus fruit tissue at 35-40°C for 15-60 days to increase the content of RG-I pectin in the citrus raw material; 2) Extraction of alcohol-insoluble solids: Dry the citrus fruit tissue and grind it into powder. Use alcohol to extract and remove the alcohol-soluble substances, leaving the alcohol-insoluble solids. 3) Extraction of water-soluble pectin: After drying the alcohol-insoluble solids, dissolve them in boiling water to extract the water-soluble pectin. After the water extract is precipitated with alcohol, the precipitate is taken and dried to obtain a crude water-soluble pectin extract; 4) Purification: The crude water-soluble pectin extract is subjected to anion exchange gel chromatography to obtain a component rich in RG-I pectin, and a product rich in RG-I pectin is obtained by desalting through dialysis and drying.

2. The method for enriching and extracting RG-I type pectin from citrus according to claim 1, characterized in that: The drying in step (2) and step (4) is carried out by vacuum freeze drying.

3. The method for enriching and extracting RG-I type pectin from citrus according to claim 1, characterized in that: The extraction of the alcohol-insoluble solids specifically comprises the following steps: 1) Add 95% ethanol to citrus fruit powder and perform ultrasonic-assisted water bath extraction at room temperature to remove alcohol-soluble substances; repeat three times; 2) Collect the residue and continue to add 95% ethanol to it, heat to boiling, use a condensation reflux device to continuously extract and remove alcohol-soluble matter, and dry the residue at 60-70°C overnight to obtain alcohol-insoluble solids.

4. The method for enriching and extracting RG-I type pectin from citrus according to claim 3, characterized in that: The solid-liquid ratio of citrus fruit powder or residue to 95% ethanol is 1:10 (w / v).

5. The method for enriching and extracting RG-I type pectin from citrus according to claim 1, characterized in that: Ultrasonic-assisted water bath extraction for 20-40 min; condensation reflux device for continuous extraction for 2-4 hours.

6. The method for enriching and extracting RG-I pectin from citrus according to claim 1, characterized in that: The extraction of the water-soluble pectin specifically comprises the following steps: 1) adding distilled water containing 2% w / v sodium thimerosal to the alcohol-insoluble solid powder, extracting water-soluble pectin by ultrasonic-assisted water bath at room temperature, collecting the supernatant, and obtaining a crude water-soluble pectin extract; 2) The crude water-soluble pectin extract was concentrated in vacuo to 1 / 5 of its original volume, and 4 volumes (v / v) of 95% ethanol were added for precipitation overnight. The precipitate was collected and washed repeatedly with anhydrous ethanol for 3 times, and dried at 60-70°C overnight to obtain water-soluble pectin.

7. The method for enriching and extracting RG-I pectin from citrus according to claim 6, characterized in that: The solid-liquid ratio of the alcohol-insoluble solid powder to distilled water is 1:10 (w / v).

8. The method for enriching and extracting RG-I pectin from citrus according to claim 6, characterized in that: Ultrasonic-assisted water bath extraction for 3-5h.

9. The method for enriching and extracting RG-I pectin from citrus according to claim 1, characterized in that: The invention discloses a method for preparing a component rich in RG-I pectin by using anion exchange gel chromatography, which specifically comprises the following steps: the ion exchange chromatography column adopts DEAE-agarose gel, and is eluted with distilled water, 0.05 mol / L NaCl solution, and 0.1 mol / L NaCl solution in sequence, with each eluent eluting for 3-4 column volumes, and the elution flow rate is set to 1 / 85-1 / 75 column volume of eluent per minute.

10. The method for enriching and extracting RG-I pectin from citrus according to claim 1, characterized in that: Dialysis Desalination The eluate was dialyzed to desalinate using 6000 Da to 8000 Da dialysis bags.

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