CN109001359A - 一种金针菇多糖二维指纹图谱的构建方法 - Google Patents
一种金针菇多糖二维指纹图谱的构建方法 Download PDFInfo
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Abstract
本发明涉及一种金针菇多糖二维指纹图谱的构建方法,属于食用菌、功能食品及其制品的指纹图谱技术领域。本发明包括金针菇多糖的提取,对提取的金针菇多糖进行红外光谱分析和对其酸水解产物PMP衍生化进行HPLC单糖组成分析,构建了金针菇多糖的红外光谱标准指纹图谱和HPLC标准指纹图谱,根据红外光谱和HPLC色谱共有特征峰的相对峰面积,应用主成分分析进行金针菇多糖的识别检测。本发明方法具有操作方便、稳定性好、准确率高等优点,构建的金针菇多糖二维指纹图谱可对未知来源的金针菇多糖进行质量控制,比一维指纹图谱可信度更高,为食用菌的质量检测提供了一种新型简易的鉴定方法。
Description
技术领域
本发明属于食用菌、功能食品原料及其制品的指纹图谱技术领域,具体涉及一种利用金针菇多糖二维指纹图谱对其进行质量控制的方法。
背景技术
金针菇别名冬菇、朴蕈、构菌等,属真菌门、担子菌亚门、层菌纲、伞菌目、口蘑科、金钱菌属。广泛分布于中国、日本、俄罗斯、欧洲、北美洲、澳大利亚等地。在我国,金针菇栽培历史十分悠久,2017年中国金针菇年产量已超过300万吨,是世界上金菇针菇产量最多的国家。
金针菇所含的金针菇多糖具有多种生物活性,但由于复杂的提取过程及各种其他因素的影响,金针菇多糖的质量参差不齐,金针菇多糖产品的真伪良莠难以鉴别。金针菇多糖产品相关产业及消费人群需要一种有效的金针菇多糖产品质量的鉴别方法。然而,目前对于金针菇类产品质量相关标准的研究还相对较少,没有形成统一的标准,未确立科学有效的检测方法。
发明内容
本发明的目的是建立一种金针菇多糖二维指纹图谱的构建方法,并能通过二维指纹图谱对金针菇多糖进行质量控制,为金针菇多糖的质量控制提供一种快速有效的方法。
本发明是通过以下步骤实现的:
1.金针菇多糖二维指纹图谱的构建:
(1)金针菇多糖的提取:将新鲜的金针菇于50℃热风循环烘箱干燥,然后经粉碎机粉碎制得金针菇粉。称取一定量的金针菇粉,按照料液比1:25在80℃热水浴条件下浸提4 h,8000 r/min条件下离心10min,上清液加入Sevag试剂(正丁醇:三氯甲烷=1:4,v/v),10000r/min条件下离心15min除去蛋白,向其中加入剩余上清液体积4倍的无水乙醇沉淀过夜,离心后取沉淀用3500Da孔径规格透析袋,4℃透析72h,真空浓缩后冷冻干燥,备用;
(2)金针菇多糖红外光谱分析及其标准指纹图谱的构建:将20批不同产地、不同颜色和不同栽培方式的金针菇多糖烘干至恒重,称取3-5 mg的多糖样品与与100-200 mg溴化钾在玛瑙研钵中混合研磨后压片,在4000cm-1-400cm-1波长范围,2cm-1分辨率条件下扫描32次,得到20种金针菇多糖的红外光谱图,建立金针菇多糖的红外光谱标准指纹图谱;
(3)金针菇多糖HPLC单糖组成分析及其标准指纹图谱的建立:吸取100 μL浓度为4 mg/mL的多糖溶液于具塞试管中,加入100 μL 4 M三氟乙酸溶液,混匀后用N2密封,置于110 ℃烘箱中水解120 min,冷却至室温后加入200 μL甲醇,用N2吹干以除去残留的三氟乙酸,重复4次,将干燥的样品用100 μL超纯水溶解;取100 μL多糖水解液与等体积100 μL 0.6 M氢氧化钠溶液混合,吸取50 μL的混合溶液,加入50 μL 0.5 M的PMP甲醇溶液混匀,于70 ℃下反应100 min后冷却至室温,用50 μL 0.3 M盐酸溶液中和,加入超纯水补齐至1 mL,然后加入1mL的三氯甲烷,涡旋静置分层后弃去有机相,重复此步骤3次,吸取水相过0.45 μm微孔滤膜过滤后进行分析,得到金针菇多糖HPLC标准指纹图谱。
步骤(3)中,HPLC色谱条件为:ZORBAX Eclipse XDB-C18色谱柱,4.6 mm×250 mm,5 μm;柱温:30℃;流速:1 mL/min;流动相:0.1 M磷酸盐(pH 6.7)缓冲液/乙腈,体积比为83:17;检测波长:250 nm;进样体积:20 μL。
金针菇多糖红外光谱标准指纹图谱有9个特征共同峰,分别为3383.24 cm-1、2932.73 cm-1、2138.10 cm-1、1647.48 cm-1、1422.57 cm-1、1248.20 cm-1、1078.69 cm-1、889.75 cm-1、573.52 cm-1。
金针菇多糖HPLC标准指纹图谱有8个特征共有峰,对应单糖分别为甘露糖、氨基葡萄糖、核糖、鼠李糖、葡萄糖、半乳糖、木糖、岩藻糖,8个共有特征峰相对保留时间的相对标准偏差RSD均小于2%,即:
1号峰平均相对保留时间为0.481,RSD为0.21%;
2号峰平均相对保留时间为0.598,RSD为0.13%;
3号峰平均相对保留时间为0.632,RSD为0.17%;
4号峰平均相对保留时间为0.675,RSD为0.14%;
5号峰平均相对保留时间为0.000,RSD为0;
6号峰平均相对保留时间为1.143,RSD为0.09%;
7号峰平均相对保留时间为1.227,RSD为0.12%;
8号峰平均相对保留时间为1.494,RSD为0.08%。
2.主成分分析
主成分分析(PCA)是一种基于将原始变量转化为不相关的新特征变量的线性组合,并将样本方差最大化以反映数据内在可变性的数据降维建模方法。红外光谱图中由于不同样品颗粒不均一造成的光散射等物理扰动,采用SNV光谱预处理方法平滑原始光谱和消除基线的漂移后再进行主成分分析;HPLC图谱中以8个共有峰面积数据组成的矩阵进行主成分分析。
本发明的有益效果:
(1)本发明通过构建金针菇多糖的二维指纹图谱,包括红外光谱标准指纹图谱和高效液相色谱标准指纹图谱,对未知来源金针菇多糖进行全面质量控制,以及保障了金针菇多糖类产品的质量安全;
(2)本发明方法提取的金针菇多糖纯度较高,既避免了传统分离纯化多糖方法的繁琐,又在一定程度上保证了用于建立指纹图谱金针菇多糖的质量,增加了金针菇多糖标准指纹图谱的可靠性;
(3)本发明方法具有精密度高、重复性好、操作简单稳定的优点,涉及不同品种、不同产地和不同栽培方式的新鲜金针菇,原料来源广泛,可以作为一种快速鉴别金针菇多糖的方法应用于市场监管,对规范金针菇多糖市场具有重要意义;
(4)相比于其他多糖类标准指纹图谱方法的建立,本发明方法运用了主成分分析,可应用于金针菇多糖的真伪识别检测,更加快速直观地鉴别了金针菇多糖,同时提高了该方法的准确率。
附图说明
图1为20种金针菇多糖红外光谱图;
图2为金针菇多糖红外光谱标准指纹图谱;
图3为金针菇多糖红外光谱标准指纹图谱的PCA分析图;
图4为12种单糖混合标准品PMP衍生物的HPLC图谱;
图5为20种金针菇多糖的HPLC图谱;
图6为金针菇多糖HPLC标准指纹图谱;
图7为金针菇多糖HPLC标准指纹图谱的PCA分析图。
具体实施方式
下面结合实施例对本发明做进一步说明,下述实施例仅用于说明本发明而非对本发明的限制。
实施例1基于金针菇多糖二维指纹图谱PCA分析的金针菇多糖的识别检测
金针菇样品:20个品种分别来自江苏、山东、福建、四川、广东、河北等不同产地。
1.金针菇多糖的提取
将新鲜的金针菇于50 ℃热风循环烘箱干燥,然后经粉碎机粉碎制得金针菇粉。称取一定量的金针菇粉,按照料液比1:25在80 ℃热水浴条件下浸提4 h,8000 r/min条件下离心10 min,上清液加入Sevag试剂(正丁醇:三氯甲烷=1:4,v/v),10000 r/min条件下离心15min除去蛋白,向其中加入剩余上清液体积4倍的无水乙醇沉淀过夜,离心后取沉淀用3500Da孔径规格透析袋,4 ℃透析72 h,真空浓缩后冷冻干燥,备用。
2.金针菇多糖红外光谱分析及其标准指纹图谱的建立
将20批不同产地、不同颜色和不同栽培方式的金针菇多糖烘干至恒重,称取溴化钾粉末,称取3-5 mg的多糖样品与与100-200 mg溴化钾在玛瑙研钵中混合研磨后压片,在4000cm-1-400 cm-1波长范围,2 cm-1分辨率条件下扫描32次,得到20种金针菇多糖的红外光谱图,建立金针菇多糖的红外光谱标准指纹图谱。
比较20个不同品种金针菇多糖红外光谱图,确定其共有9个特征共同峰,分别为分别为3383.24 cm-1、2932.73 cm-1、2138.10 cm-1、1647.48 cm-1、1422.57 cm-1、1248.20 cm-1、1078.69 cm-1、889.75 cm-1、573.52 cm-1。
3.金针菇多糖HPLC单糖组成分析及其标准指纹图谱的建立
吸取100 μL浓度为4 mg/mL的多糖溶液于具塞试管中,加入100 μL 4 M三氟乙酸溶液,混匀后用N2密封,置于110 ℃烘箱中水解120 min,冷却至室温后加入200 μL甲醇,用N2吹干以除去残留的三氟乙酸,重复4次,将干燥的样品用100 μL超纯水溶解;取100 μL多糖水解液与等体积100 μL 0.6 M氢氧化钠溶液混合,吸取50 μL的混合溶液,加入50 μL 0.5 M的PMP甲醇溶液混匀,于70 ℃下反应100 min后冷却至室温,用50 μL 0.3 M盐酸溶液中和,加入超纯水补齐至1 mL,然后加入1 mL的三氯甲烷,涡旋静置分层后弃去有机相,重复此步骤3次,吸取水相过0.45 μm微孔滤膜过滤后进行分析,高效液相色谱条件为:ZORBAX EclipseXDB-C18色谱柱,4.6 mm×250 mm,5 μm;柱温:30℃;流速:1 mL/min;流动相:0.1 M磷酸盐(pH 6.7)缓冲液/乙腈,体积比为83:17;检测波长:250 nm;进样体积:20 μL,得到金针菇多糖高效液相色谱标准指纹图谱。
4.金针菇多糖高效液相色谱标准指纹图谱的共有峰信息
通过比较20种金针菇多糖高效液相色谱图,确定有8个特征共有峰,对应单糖分别为甘露糖、氨基葡萄糖、核糖、鼠李糖、葡萄糖、半乳糖、木糖、岩藻糖,以5号峰的保留时间和峰面积为基准,计算其余特征峰的相对保留时间和相对峰面积,见表1。
表1金针菇多糖HPLC指纹图谱的共有峰信息
5.指纹图谱方法重复性验证
取1号金针菇样品,进行提取、酸水解和衍生化的前处理后,连续进样5次,相对保留时间和相对峰面积的标准偏差RSD均小于3%,说明方法精密度高;
取1号金针菇样品共5份,进行提取、酸水解和衍生化的前处理后进样,相对保留时间和相对峰面积的标准偏差RSD均小于5%,说明方法稳定性好;
取1号金针菇进行提取、酸水解和衍生化的前处理后,分别放置0、2 h、4 h、8 h、16 h、24 h、48 h后进样,相对保留时间和相对峰面积的标准偏差RSD均小于5%,说明方法具有良好的重现性。
6.主成分分析
主成分分析(PCA)是一种基于将原始变量转化为不相关的新特征变量的线性组合,并将样本方差最大化以反映数据内在可变性的数据降维建模方法。红外光谱图中不同样品颗粒不均一造成的光散射等物理扰动,采用SNV光谱预处理方法平滑原始光谱和消除基线的漂移后进行主成分分析(见附图3),其中编号1-20为20批不同来源金针菇提取的多糖,编号21-23为3种未知真伪的金针菇多糖用于验证,由主成分得分图可知,20批金针菇多糖差异很小,21号金针菇多糖与这20批金针菇多糖差异很小,然而22号和23号与其差异很大,由此可以判断21号为真实来源金针菇多糖,22号和23号为伪劣金针菇多糖,经检验该结果准确。在HPLC图谱中以8个共有峰面积数据组成的矩阵进行主成分分析(见附图6),虽然不同来源的金针菇多糖有一定的差异,但仍能区分出22和23号伪劣金针菇多糖。
Claims (7)
1.一种金针菇多糖二维指纹图谱的构建方法,其特征在于,包括如下步骤:
(1)将新鲜金针菇子实体于50℃热风循环烘箱干燥,然后粉碎至80目,用于提取金针菇多糖;
(2)对得到的金针菇多糖进行红外光谱分析和对其酸水解产物PMP衍生化进行HPLC单糖组成分析,构建金针菇多糖的红外标准指纹图谱和HPLC标准指纹图谱;
(3)将得到的红外光谱经SNV光谱预处理后,运用主成分分析进行判别;对得到的HPLC图谱的共有峰面积数据运用主成分分析进行判别。
2.根据权利要求1所述的金针菇多糖二维指纹图谱的构建方法,其特征在于,金针菇多糖的提取步骤包括:称取一定量的金针菇粉,按照料液比1:25在80℃热水浴条件下浸提4h,8000 r/min条件下离心10min;上清液中加入Sevag试剂(正丁醇:三氯甲烷=1:4,v/v),10000r/min条件下离心15min除去蛋白,向其中加入剩余上清液体积4倍的无水乙醇沉淀过夜,离心后取沉淀用3500Da孔径规格透析袋,4℃透析72h,真空浓缩后冷冻干燥,备用。
3.根据权利要求1所述的金针菇多糖二维指纹图谱的构建方法,其特征在于,包括如下步骤:
(1)金针菇多糖红外光谱标准指纹图谱的构建
称取3-5mg的多糖样品与100-200mg溴化钾在玛瑙研钵中混合研磨后压片,在4000cm-1-400cm-1波长范围,2cm-1分辨率条件下扫描32次,根据不同品种金针菇多糖的傅里叶变换红外光谱图,得到金针菇多糖红外光谱的标准指纹图谱;
(2)金针菇多糖HPLC标准指纹图谱的构建
吸取100μL浓度为4 mg/mL的多糖溶液于具塞试管中,加入100μL 4 M三氟乙酸溶液,混匀后用N2密封,置于110 ℃烘箱中水解120 min,冷却至室温后加入200 μL甲醇,用N2吹干以除去残留的三氟乙酸,重复4次,将干燥的样品用100μL超纯水溶解;取100μL多糖水解液与等体积100 μL0.6 M氢氧化钠溶液混合,吸取50μL的混合溶液,加入50μL0.5 M的PMP甲醇溶液混匀,于70℃下反应100min后冷却至室温,用50μL0.3 M盐酸溶液中和,加入超纯水补齐至1mL,然后加入1 mL的三氯甲烷,涡旋静置分层后弃去有机相,重复此步骤3次,吸取水相过0.45μm微孔滤膜过滤后进行分析,得到金针菇多糖的HPLC标准指纹图谱。
4.根据权利要求3所述的金针菇多糖二维指纹图谱的构建方法,其特征在于,步骤(2)中,HPLC色谱条件为:ZORBAXEclipseXDB-C18色谱柱,4.6mm×250 mm,5μm;柱温:30℃;流速:1 mL/min;流动相:0.1 M磷酸盐(pH 6.7)缓冲液/乙腈,体积比为83:17;检测波长:250nm;进样体积:20μL。
5.根据权利要求1所述的金针菇多糖二维指纹图谱的构建方法,其特征在于,所述金针菇多糖红外光谱标准指纹图谱有9个特征共同峰,分别为3383.24 cm-1、2932.73 cm-1、2138.10 cm-1、1647.48 cm-1、1422.57 cm-1、1248.20 cm-1、1078.69 cm-1、889.75 cm-1、573.52 cm-1。
6.根据权利要求1所述的金针菇多糖二维指纹图谱的构建方法,其特征在于,所述金针菇多糖HPLC标准指纹图谱有8个特征共有峰,对应单糖分别为甘露糖、氨基葡萄糖、核糖、鼠李糖、葡萄糖、半乳糖、木糖、岩藻糖,8个共有特征峰相对保留时间的相对标准偏差RSD均小于2%,即:
1号峰平均相对保留时间为0.481,RSD为0.21%;
2号峰平均相对保留时间为0.598,RSD为0.13%;
3号峰平均相对保留时间为0.632,RSD为0.17%;
4号峰平均相对保留时间为0.675,RSD为0.14%;
5号峰平均相对保留时间为0.000,RSD为0;
6号峰平均相对保留时间为1.143,RSD为0.09%;
7号峰平均相对保留时间为1.227,RSD为0.12%;
8号峰平均相对保留时间为1.494,RSD为0.08%。
7.根据权利要求1所述的金针菇多糖二维指纹图谱的构建方法,其特征在于,所述红外光谱的主成分分析方法,以光谱图谱经SNV光谱预处理后进行主成分分析;所述HPLC图谱的主成分分析方法,以8个特征共同峰的相对峰面积的矩阵进行主成分分析。
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