WO2022217573A1 - 一种具有高rg-i结构含量果胶的制备方法 - Google Patents

一种具有高rg-i结构含量果胶的制备方法 Download PDF

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WO2022217573A1
WO2022217573A1 PCT/CN2021/087691 CN2021087691W WO2022217573A1 WO 2022217573 A1 WO2022217573 A1 WO 2022217573A1 CN 2021087691 W CN2021087691 W CN 2021087691W WO 2022217573 A1 WO2022217573 A1 WO 2022217573A1
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pectin
solution
ethanol
supernatant
extraction
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PCT/CN2021/087691
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陈士国
侯志强
叶兴乾
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浙江大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof

Definitions

  • the invention relates to a preparation method of pectin with high RG-I structure content.
  • Pectin is the most abundant water-soluble dietary fiber in plant-derived foods such as fruits and vegetables. Its structure mainly includes homogalacturonan (HG), rhamnogalacturonan I (RG-I), rhamnogalacturonan Lacturonan II and other domains. Commercial pectin is mainly used as a gel, thickener and stabilizer in food systems, and its product quality control is mainly based on its high-sugar system to form a high-elastic gel, requiring pectin to be mainly HG (uronic acid content >65%).
  • RG-I is a "hairy region" domain in pectin with ⁇ -1,5-arabinan, ⁇ -1,4-galactan, and arabinohalogen linked to the rhamnogalacturonic acid backbone. Abundant neutral sugar side chain structures such as lactan. RG-I pectin is widely found in citrus, apple, pumpkin, carrot, okra and other fruits and vegetables. Among the by-products of citrus, sugar beet and other bulk fruit and vegetable processing, its proportion even exceeds that of traditional HG-type commodity pectin.
  • RG-I pectin has not only been reported to have a large number of unique physiological functions superior to traditional HG pectin, such as regulating intestinal flora, inhibiting cancer, regulating chronic metabolic syndrome, regulating immune function, etc., and more literatures. It shows that it can form gel in sugar-free or low-sugar system and has a more prominent thickening and stabilizing effect, indicating its potential as a new functional food additive and a new type of prebiotic.
  • the object of the present invention is to aim at the deficiencies of the prior art, and to provide a preparation method of pectin with high RG-I structure content.
  • the hydrolysis of the pectin side chain in citrus powder was carried out, and the RG-I structure of pectin and its arabinose and galactose side chains were preserved. After alcohol precipitation, washing and drying, pectin rich in RG-I domain was obtained.
  • the problems of long time and low extraction rate of traditional solution extraction are effectively solved, and the pectin product rich in RG-I type domain prepared by the method of the present invention is more beneficial to human health.
  • the preparation method of pectin with high RG-I structure content provided by the present invention comprises the following steps:
  • step 3 adding an acidic solution to adjust the pH of the supernatant obtained in step 2) to 6-7, then adding 95wt% ethanol for precipitation for 12-24 hours, the volume of 95wt% ethanol added is 2-4 times the volume of the supernatant; After the precipitation is over, filter and retain the precipitate;
  • step 4) The precipitate obtained in step 3) is washed with 95 wt% ethanol and dried to obtain pectin with high RG-I structure content, and the RG-I structure content is 72.12-74.51%.
  • the alkaline solution is an aqueous solution of sodium hydroxide or an aqueous solution of potassium hydroxide.
  • the acidic solution is a hydrochloric acid solution.
  • the polysaccharide needs to be washed 2-3 times with 95% ethanol, so that the polysaccharide with good properties is obtained after drying.
  • the extraction conditions of pectin polysaccharide are controlled in alkaline conditions, so that the polygalactose (HG) structure is hydrolyzed, the RG-I structure and the side chain of the pectin neutral sugar are retained to the greatest extent, and the ultra-high pressure treatment can accelerate the ⁇ -degradation
  • the occurrence of the reaction improves the extraction efficiency of pectin, and the final product obtained is pectin with high RG-I structure content, good emulsifying potential and biological activity such as antioxidant. No toxic and harmful chemical reagents are added in the whole extraction process, and it has the advantages of natural greenness, high efficiency and strong practicality.
  • the invention discloses a preparation method of pectin with high RG-I structure content.
  • the method is to mix citrus powder with alkali at normal temperature and then use ultra-high pressure treatment, which greatly prevents the pectin side chain in the citrus powder.
  • the hydrolysis of pectin retains the pectin RG-I structure and its arabinose and galactose side chains. After alcohol precipitation, washing and drying, pectin rich in RG-I domain can be obtained.
  • Pectin monosaccharide composition was analyzed by high performance anion exchange chromatography.
  • the pectin polysaccharide sample (about 2 mg) was dissolved, 4 mol/L trifluoroacetic acid was added, hydrolyzed at 110 °C for 6 h, and then 1 ml of methanol was added, and dried under nitrogen for three times at 50 °C.
  • the dried sample was dissolved in 6 mL of deionized water and passed through a 0.22 ⁇ m water film before being injected.
  • High performance anion liquid chromatography conditions were: Thermo Fisher ICS-5000+ instrument equipped with Carbopac guard column (4 ⁇ 50mm, Dionex) and Carbopac PA1 analytical column (4 ⁇ 250mm, Dionex), and the temperature was 30°C. Elution procedure: 18 mM NaOH for the first 15 min, and a mixture containing 18 mM NaOH and 100 mM NaOAc for the last 35 min.
  • the molecular weight of pectin was determined by high performance gel filtration chromatography-differential detection-multi-angle laser light scattering combined technique.
  • Pectin polysaccharide was dissolved in deionized water to make a solution with a concentration of 5 mg/mL, passed through a 0.22 ⁇ m water film, and then 100 ⁇ L was injected into the SEC-MALLS-RI (Wyatt Dawn Heleos-II, USA) system for determination.
  • SEC-MALLS-RI Wangt Dawn Heleos-II, USA
  • a Shodex SB-806HQ column Showa Denko KK, Japan
  • the mobile phase was 0.15M sodium chloride solution
  • the flow rate was 0.5 mL/min.
  • Pectin monosaccharide composition was analyzed by high performance anion exchange chromatography.
  • the pectin polysaccharide sample (about 2 mg) was dissolved, 4 mol/L trifluoroacetic acid was added, hydrolyzed at 110 °C for 6 h, methanol was added, and nitrogen was blown dry three times at 50 °C.
  • the dried sample was dissolved in 6 mL of deionized water and passed through a 0.22 ⁇ m water film before being injected.
  • High performance anion liquid chromatography conditions were: Thermo Fisher ICS-5000+ instrument equipped with Carbopac guard column (4 ⁇ 50mm, Dionex) and Carbopac PA1 analytical column (4 ⁇ 250mm, Dionex), and the temperature was 30°C. Elution procedure: 18 mM NaOH for the first 15 min, and a mixture of 18 mM NaOH and 100 mM NaOAc for the last 35 min.
  • the molecular weight of pectin was determined by high performance gel filtration chromatography-differential detection-multi-angle laser light scattering combined technique.
  • Pectin polysaccharide was dissolved in deionized water to make a solution with a concentration of 5 mg/mL, passed through a 0.22 ⁇ m water film, and then 100 ⁇ L was injected into the SEC-MALLS-RI (Wyatt Dawn Heleos-II, USA) system for determination.
  • SEC-MALLS-RI Wangt Dawn Heleos-II, USA
  • a Shodex SB-806HQ column Showa Denko KK, Japan
  • the mobile phase was 0.15M sodium chloride solution
  • the flow rate was 0.5 mL/min.
  • Pectin monosaccharide composition was analyzed by high performance anion exchange chromatography.
  • the pectin polysaccharide sample (about 2 mg) was dissolved, 4 mol/L trifluoroacetic acid was added, hydrolyzed at 110 °C for 6 h, methanol was added, and nitrogen was blown dry three times at 50 °C.
  • the dried sample was dissolved in 6 mL of deionized water and passed through a 0.22 ⁇ m water film before being injected.
  • High performance anion liquid chromatography conditions were: Thermo Fisher ICS-5000+ instrument equipped with Carbopac guard column (4 ⁇ 50mm, Dionex) and Carbopac PA1 analytical column (4 ⁇ 250mm, Dionex), and the temperature was 30°C. Elution procedure: 18 mM NaOH for the first 15 min, and a mixture of 18 mM NaOH and 100 mM NaOAc for the last 35 min.
  • the molecular weight of pectin was determined by high performance gel filtration chromatography-differential detection-multi-angle laser light scattering combined technique.
  • Pectin polysaccharide was dissolved in deionized water to make a solution with a concentration of 5 mg/mL, passed through a 0.22 ⁇ m water film, and then 100 ⁇ L was injected into the SEC-MALLS-RI (Wyatt Dawn Heleos-II, USA) system for determination.
  • SEC-MALLS-RI Wangt Dawn Heleos-II, USA
  • a Shodex SB-806 HQ column Showa Denko KK, Japan
  • the mobile phase was 0.15M sodium chloride solution
  • the flow rate was 0.5 mL/min.
  • Pectin monosaccharide composition was analyzed by high performance anion exchange chromatography.
  • the pectin polysaccharide sample (about 2 mg) was dissolved, 4 mol/L trifluoroacetic acid was added, hydrolyzed at 110 °C for 6 h, methanol was added, and nitrogen was blown dry three times at 50 °C.
  • the dried sample was dissolved in 6 mL of deionized water and passed through a 0.22 ⁇ m water film before being injected.
  • High performance anion liquid chromatography conditions were: Thermo Fisher ICS-5000+ instrument equipped with Carbopac guard column (4 ⁇ 50mm, Dionex) and Carbopac PA1 analytical column (4 ⁇ 250mm, Dionex), and the temperature was 30°C. Elution procedure: 18 mM NaOH for the first 15 min, and a mixture containing 18 mM NaOH and 100 mM NaOAc for the last 35 min.
  • the molecular weight of pectin was determined by high performance gel filtration chromatography-differential detection-multi-angle laser light scattering combined technique.
  • Pectin polysaccharide was dissolved in deionized water to make a solution with a concentration of 5 mg/mL, passed through a 0.22 ⁇ m water film, and then 100 ⁇ L was injected into the SEC-MALLS-RI (Wyatt Dawn Heleos-II, USA) system for determination.
  • SEC-MALLS-RI Wangt Dawn Heleos-II, USA
  • a Shodex SB-806HQ column Showa Denko KK, Japan
  • the mobile phase was 0.15M sodium chloride solution
  • the flow rate was 0.5 mL/min.
  • the extraction rate of pectin extracted by the method examples 1 and 2 was as high as 32-34%, while the extraction rate of pectin in Comparative Example 1 was only 26%, indicating that the ultra-high pressure auxiliary alkaline solution effectively promoted the extraction of pectin; and It can be seen from Table 1-3 that the galacturonic acid content of the polysaccharides extracted by ultra-high pressure assisted alkali extraction is 22.87-23.82%, but it is rich in arabinose content (39.62-41.81%), indicating that the main It is composed of neutral sugar, the ratio of rhamnose to galacturonic acid is 0.25-0.30, and the content of RG-I structure is as high as 72.12-74.51%, indicating that it is mainly composed of RG-I structure, which can be seen from the monosaccharide analysis. It is a pectin polysaccharide rich in RG-I structure, and the obtained pectin has a high degree of branching and a large molecular weight.

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Abstract

一种具有高RG-Ⅰ结构含量果胶的制备方法，包括将柑橘皮粉与碱性溶液在低温下进行混合，然后进行超高压处理，压力200-600MPa，时间为1-20min。所述方法将果胶多糖提取条件控制在碱性条件，使得聚半乳糖（HG）结构水解，最大程度保留了RG-Ⅰ结构以及果胶中性糖侧链。终产物主要是富含半乳糖侧链的RG-Ⅰ结构域果胶多糖，提取率在30%以上，有效解决了传统提取方法中提取率低、耗时长的问题，经所述方法制得的产品，分子量大，具有较好的乳化潜力。

Description

一种具有高RG-I结构含量果胶的制备方法 技术领域
本发明涉及一种具有高RG-I结构含量果胶的制备方法。
背景技术
果胶是果蔬等植物源食物中含量最高的水溶性膳食纤维，其结构主要包括同型半乳糖醛酸聚糖(HG)、鼠李半乳糖醛酸聚糖I(RG-I)、鼠李半乳糖醛酸聚糖II等结构域。商品果胶在食品体系中主要作为凝胶、增稠和稳定剂大量使用，其产品质量控制主要基于其高糖体系形成高弹性凝胶的需求，要求果胶以HG为主(糖醛酸含量>65％)。然而，近年来随着膳食观念的提升，食品工业对传统高糖果胶凝胶体系的需求下降，果胶健康功能需求受到空前重视，果蔬中大量存在的其他功能型果胶结构域因此成为新的关注热点。
RG-I是果胶中的“多毛区”结构域，其鼠李聚半乳糖醛酸主干上连接了α-1,5-阿拉伯聚糖、β-1,4-半乳聚糖以及阿拉伯半乳聚糖等丰富的中性糖侧链结构。RG-I型果胶广泛存在于柑橘、苹果、南瓜、胡萝卜、秋葵等果蔬中。在柑橘、甜菜等大宗果蔬加工副产物中，其占比甚至超过传统HG型商品果胶。近五年来，RG-I型果胶不仅被报道具有大量优于传统HG果胶的独特生理功能，如调节肠道菌群、抑制癌症、调节慢性代谢综合征、调节免疫功能等，更有文献表明其可以在无糖或者低糖体系形成凝胶并具有更突出的增稠稳定效果，预示其作为新型功能型食品添加剂、新型益生元的潜力。
相对于活性研究的火热，目前针对RG-I型果胶的提取技术仍极为匮乏，特别是长期以高HG结构为目标的商品果胶生产，忽视了RG-I果胶的大量存在并致其丢失。目前所采用的传统溶液提取也仍存在耗时长、提取率低、成本高等问题，需继续拓展针对RG-I果胶的绿色高效提取技术。
发明内容
本发明目的在于针对现有技术的不足，提供一种具有高RG-I结构含量果胶的制备方法，该方法是将柑橘粉在常温下与碱混合后再使用超高压处理，极大的防止了柑橘粉中果胶侧链的水解，保留了果胶RG-I结构及其阿拉伯糖和半乳糖侧链，酒精沉淀、洗涤烘干后可得富含RG-I型结构域果胶。有效解决了传统溶液提取耗时长、提取率低的问题，经本发明方法制得的富含RG-I型结构域果胶产品更有益于人体健康。
本发明所提供的具有高RG-I结构含量果胶的制备方法，包括下述步骤：
1)将柑橘皮烘干粉碎后，与碱性溶液按照料液比1g:20-40mL混合得到混合液；所述碱性溶液中，碱的含量为0.05-1wt％；
2)对混合液进行超高压处理，其中，处理压力为：200-600MPa，时间为：1-20min，处理温度为：0-40℃；过滤并保留上清液；
3)加入酸性溶液调节步骤2)中得到的上清液的pH至6-7，然后加入95wt％乙醇沉淀12-24小时，95wt％乙醇的加入体积是上清液体积的2-4倍；沉淀结束后，过滤保留沉淀物；
4)使用95wt％乙醇洗涤步骤3)中得到的沉淀物并干燥后得到高RG-I结构含量果胶，RG-I结构含量为72.12-74.51％。
进一步地，所述步骤1)中，碱性溶液为氢氧化钠的水溶液或氢氧化钾的水溶液。
进一步地，所述步骤3)中，，酸性溶液为盐酸溶液。
进一步地，所述步骤4)中，需将多糖用95％乙醇洗涤2-3次，使得烘干后得到性状良好的多糖。
本发明的有益效果在于：
本发明将果胶多糖提取条件控制在碱性条件，使得聚半乳糖(HG)结构水解，最大程度保留了RG-I结构以及果胶中性糖侧链，同时超高压处理可以加速β-降解反应的发生，提高果胶提取效率，所得终产物是高RG-I结构含量果胶，较好的乳化潜力和抗氧化等生物活性。整个提取过程中无有毒有害化学试剂的添加，具有天然绿色、效率高、实践性强等优点。
具体实施方式
本发明公开了一种具有高RG-I结构含量果胶的制备方法，该方法是将柑橘粉在常温下与碱混合后再使用超高压处理，极大的防止了柑橘粉中果胶侧链的水解，保留了果胶RG-I结构及其阿拉伯糖和半乳糖侧链，酒精沉淀、洗涤烘干后可得富含RG-I型结构域果胶。下面，结合具体实施例对本发明作进一步解释说明。
实施例1
将柑橘皮烘干粉碎后，取2g柑橘粉与0.05wt％氢氧化钠溶液按照料液比1g:40mL混合；对混合液进行超高压处理，处理压力为：500MPa，时间为：10min，处理温度为：20℃。过滤并保留上清液；加入盐酸溶液调节步骤(2)中得到的上清液的pH至7，然后加入95％乙醇沉淀12小时，95wt％乙醇的加入体积是上清液体积的2倍。沉淀结束后，过滤保留沉淀物；使用95wt％乙醇洗涤步骤(3)中得到的沉淀物3次并干燥后，得到0.68g果胶多糖。
采用高效阴离子交换色谱法对果胶单糖组成分析进行分析。将果胶多糖样品(约2毫克)溶解，加入4mol/L三氟乙酸，110℃水解6h后加入1ml甲醇，50℃下氮气吹干三次。干燥后的样品溶于6mL去离子水中，过0.22μm水膜后待进样。高效阴离子液相色谱条件为：装配有Carbopac guard柱(4×50mm,Dionex)和Carbopac PA1 analytical柱(4×250mm，Dionex)的Thermo Fisher ICS-5000+仪器，温度为30℃。洗脱程序：前15min为18mM的NaOH，后35min为含有18mM NaOH和100mM NaOAc的混合液。
采用高效凝胶过滤色谱-示差检测-多角度激光光散射联用技术测定果胶的分子量。果胶多糖溶于去离子水中制成浓度为5mg/mL的溶液，过0.22μm水膜，然后将100μL进样到SEC-MALLS-RI(Wyatt Dawn Heleos-II,USA)系统中进行测定。使用Shodex SB-806HQ柱(Showa Denko KK,Japan)，流动相为0.15M氯化钠溶液，流速0.5mL/min。
表1超高压(500MPa/10min)提取柑橘果胶的单糖组成及分子量
项目 结果
提取率(％) 34.0
鼠李糖(mol％) 6.89
阿拉伯糖(mol％) 41.81
半乳糖(mol％) 18.92
葡萄糖(mol％) 6.45
半乳糖醛酸(mol％) 22.87
鼠李糖/半乳糖醛酸 0.30
RG-I(％) 74.51
Mw(kDa) 528.8
Mn(kDa) 256.7
实施例2
将柑橘皮烘干粉碎后，取2g柑橘粉与0.05wt％氢氧化钠溶液按照料液比1g:40mL混合；对混合液进行超高压处理，处理压力为：300MPa，时间为：10min，处理温度为：20℃。过滤并保留上清液；加入盐酸溶液调节步骤(2)中得到的上清液的pH至7，然后加入95wt％乙醇沉淀12小时，95wt％乙醇的加入体积是上清液体积的2 倍。沉淀结束后，过滤保留沉淀物；使用95％乙醇洗涤步骤(3)中得到的沉淀物3次并干燥后，得到0.65g果胶多糖。
采用高效阴离子交换色谱法对果胶单糖组成分析进行分析。将果胶多糖样品(约2毫克)溶解，加入4mol/L三氟乙酸，110℃水解6h后加入甲醇，50℃下氮气吹干三次。干燥后的样品溶于6mL去离子水中，过0.22μm水膜后待进样。高效阴离子液相色谱条件为：装配有Carbopac guard柱(4×50mm,Dionex)和Carbopac PA1 analytical柱(4×250mm，Dionex)的Thermo Fisher ICS-5000+仪器，温度为30℃。洗脱程序：前15min为18mM的NaOH，后35min含有18mM NaOH和100mM NaOAc的混合液。
采用高效凝胶过滤色谱-示差检测-多角度激光光散射联用技术测定果胶的分子量。果胶多糖溶于去离子水中制成浓度为5mg/mL的溶液，过0.22μm水膜，然后将100μL进样到SEC-MALLS-RI(Wyatt Dawn Heleos-II,USA)系统中进行测定。使用Shodex SB-806HQ柱(Showa Denko KK,Japan)，流动相为0.15M氯化钠溶液，流速0.5mL/min。
表2超高压(300MPa/10min)提取柑橘果胶的单糖组成及分子量
项目 结果
提取率(％) 32.4
鼠李糖(mol％) 5.92
阿拉伯糖(mol％) 39.62
半乳糖(mol％) 20.66
葡萄糖(mol％) 6.73
半乳糖醛酸(mol％) 23.82
鼠李糖/半乳糖醛酸 0.25
RG-I(％) 72.12
Mw(kDa) 536.2
Mn(kDa) 272.4
实施例3
将柑橘皮烘干粉碎后，取2g柑橘粉与1wt％氢氧化钠溶液按照料液比1g:20mL混合；对混合液进行超高压处理，处理压力为：200MPa，时间为：20min，处理温度为：0℃。过滤并保留上清液；加入盐酸溶液调节步骤(2)中得到的上清液的pH至6，然后加入95wt％乙醇沉淀12小时，95wt％乙醇的加入体积是上清液体积的4倍。沉 淀结束后，过滤保留沉淀物；使用95wt％乙醇洗涤步骤(3)中得到的沉淀物3次并干燥后，得到0.64g果胶多糖。
采用高效阴离子交换色谱法对果胶单糖组成分析进行分析。将果胶多糖样品(约2毫克)溶解，加入4mol/L三氟乙酸，110℃水解6h后加入甲醇，50℃下氮气吹干三次。干燥后的样品溶于6mL去离子水中，过0.22μm水膜后待进样。高效阴离子液相色谱条件为：装配有Carbopac guard柱(4×50mm,Dionex)和Carbopac PA1 analytical柱(4×250mm，Dionex)的Thermo Fisher ICS-5000+仪器，温度为30℃。洗脱程序：前15min为18mM的NaOH，后35min含有18mM NaOH和100mM NaOAc的混合液。
采用高效凝胶过滤色谱-示差检测-多角度激光光散射联用技术测定果胶的分子量。果胶多糖溶于去离子水中制成浓度为5mg/mL的溶液，过0.22μm水膜，然后将100μL进样到SEC-MALLS-RI(Wyatt Dawn Heleos-II,USA)系统中进行测定。使用Shodex SB-806 HQ柱(Showa Denko KK,Japan)，流动相为0.15M氯化钠溶液，流速0.5mL/min。
表3超高压(200MPa/20min)提取柑橘果胶的单糖组成及分子量
项目 结果
提取率(％) 31.8
鼠李糖(mol％) 6.25
阿拉伯糖(mol％) 38.35
半乳糖(mol％) 19.48
葡萄糖(mol％) 6.41
半乳糖醛酸(mol％) 25.58
鼠李糖/半乳糖醛酸 0.24
RG-I(％) 70.33
Mw(kDa) 586.3
Mn(kDa) 236.2
对比例1
将柑橘皮烘干粉碎后，取2g柑橘粉与0.05wt％氢氧化钠溶液按照料液比1g:40mL混合；对混合液过滤并保留上清液；加入盐酸溶液调节步骤(2)中得到的上清液的pH至7，然后加入95wt％乙醇沉淀12小时，95wt％乙醇的加入体积是上清液体积的2倍。 沉淀结束后，过滤保留沉淀物；使用95wt％乙醇洗涤步骤(3)中得到的沉淀物3次并干燥后，得到0.52果胶多糖。
采用高效阴离子交换色谱法对果胶单糖组成分析进行分析。将果胶多糖样品(约2毫克)溶解，加入4mol/L三氟乙酸，110℃水解6h后加入甲醇，50℃下氮气吹干三次。干燥后的样品溶于6mL去离子水中，过0.22μm水膜后待进样。高效阴离子液相色谱条件为：装配有Carbopac guard柱(4×50mm,Dionex)和Carbopac PA1 analytical柱(4×250mm，Dionex)的Thermo Fisher ICS-5000+仪器，温度为30℃。洗脱程序：前15min为18mM的NaOH，后35min为含有18mM NaOH和100mM NaOAc的混合液。
采用高效凝胶过滤色谱-示差检测-多角度激光光散射联用技术测定果胶的分子量。果胶多糖溶于去离子水中制成浓度为5mg/mL的溶液，过0.22μm水膜，然后将100μL进样到SEC-MALLS-RI(Wyatt Dawn Heleos-II,USA)系统中进行测定。使用Shodex SB-806HQ柱(Showa Denko KK,Japan)，流动相为0.15M氯化钠溶液，流速0.5mL/min。
表4碱提取柑橘果胶的单糖组成及分子量
项目 结果
提取率(％) 26.0
鼠李糖(mol％) 5.76
阿拉伯糖(mol％) 39.03
半乳糖(mol％) 20.12
葡萄糖(mol％) 6.47
半乳糖醛酸(mol％) 25.44
鼠李糖/半乳糖醛酸 0.23
RG-I(％) 59.53
Mw(kDa) 673.4
Mn(kDa) 366.8
结论：
首先，使用本方法实例1和2提取的果胶提取率高达32-34％，而对比例1果胶提取率仅为26％，说明超高压辅助碱性溶液有效促进了果胶的提取；而从表1-3可以看出，超高压辅助碱提取所提取的多糖的半乳糖醛酸含量为22.87-23.82％，但含有丰富的阿拉伯糖含量(39.62-41.81％)，说明所提取多糖的主要由中性糖组成，鼠李糖与半乳糖醛酸的比值为0.25-0.30，RG-I结构含量高达72.12-74.51％，说明主要由RG-I结构组成，从单糖分析中可以看出这是一种富含RG-I结构的果胶多糖，而且所得到的果胶分支程 度较高，分子量较大。
最后还需说明，以上说明对本发明而言只是说明性的，而非限制性的，本领域普通技术人员理解，在不脱离权利要求所限定的保护范围的情况下，可作出的任何修改、变化或等效，都将落入本发明的保护范围之内。

Claims (4)

  1. 一种具有高RG-I结构含量果胶的制备方法，其特征在于，包括下述步骤：
    1)将柑橘皮烘干粉碎后，与碱性溶液按照料液比1g:20-40mL混合得到混合液；所述碱性溶液中，碱的含量为0.05-1wt％；
    2)对混合液进行超高压处理，其中，处理压力为：200-600MPa，时间为：1-20min，处理温度为：0-40℃；过滤并保留上清液；
    3)加入酸性溶液调节步骤2)中得到的上清液的pH至6-7，然后加入95wt％乙醇沉淀12-24小时，95wt％乙醇的加入体积是上清液体积的2-4倍；沉淀结束后，过滤保留沉淀物；
    4)使用95wt％乙醇洗涤步骤3)中得到的沉淀物并干燥后得到高RG-I结构含量果胶，RG-I结构含量为72.12-74.51％。
  2. 根据权利要求1所述的方法，其特征在于：所述步骤1)中，碱性溶液为氢氧化钠的水溶液或氢氧化钾的水溶液。
  3. 根据权利要求1-3中任一项所述的方法，其特征在于：所述步骤3)中，酸性溶液为盐酸溶液。
  4. 根据权利要求1-3中任一项所述的方法，其特征在于：所述步骤4)中，需将多糖用95wt％乙醇洗涤2-3次，使得烘干后得到性状良好的多糖。
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