WO2017173638A1 - 角鲨烯作为橄榄油和油茶籽油鉴别标记物的建立方法 - Google Patents

角鲨烯作为橄榄油和油茶籽油鉴别标记物的建立方法 Download PDF

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WO2017173638A1
WO2017173638A1 PCT/CN2016/078743 CN2016078743W WO2017173638A1 WO 2017173638 A1 WO2017173638 A1 WO 2017173638A1 CN 2016078743 W CN2016078743 W CN 2016078743W WO 2017173638 A1 WO2017173638 A1 WO 2017173638A1
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squalene
oil
seed oil
camellia seed
olive oil
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PCT/CN2016/078743
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French (fr)
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沈立荣
翟量
辛晓璇
祝洪刚
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浙江大学
浙江常发粮油食品有限公司
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Priority to PCT/CN2016/078743 priority Critical patent/WO2017173638A1/zh
Publication of WO2017173638A1 publication Critical patent/WO2017173638A1/zh

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/88Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

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  • the invention relates to the field of oil and fat detection in food detection.
  • the invention relates to a gas chromatography detection method and a criterion for determining the squalene content as the basis for the identification of olive oil and camellia seed oil.
  • Olive oil is an ancient oil species native to the Mediterranean. It has many effects such as reducing the occurrence of cardiovascular and cerebrovascular diseases, improving the function of the digestive system, anti-cancer, anti-aging, promoting blood circulation and nervous system development. The world has long been recognized as “liquid gold” and “the queen of vegetable oil”.
  • Camellia seed oil is an ancient Chinese oil extracted from the seeds of Camellia oleifera. Its chemical properties, especially fatty acid composition, are very similar to olive oil. Because it has the functions of preventing cardiovascular disease, anti-oxidation and scavenging free radicals, it also has high nutritional value.
  • Camellia seed oil is known as "Oriental Olive Oil” and "Longevity Oil”.
  • Camellia oleifera China is the country of origin of Camellia oleifera and is the world's largest producer of Camellia oleifera. It is well received by domestic consumers. Due to limited production, prices have been rising in recent years. Olive oil in the domestic market is mainly imported from Europe. Olive oil is divided into two types: pour oil and solvent-derived pomace oil. Usually, olive oil is pressed as edible oil, and the price is high. Pomace oil is generally used as industrial oil for cosmetics and daily chemical products and feed. The price is not high. Even much lower than domestically produced camellia seed oil. In the past, the camellia seed oil that appeared in the Chinese market was generally the lower price of palm oil.
  • a patent relating to the identification of camellia seed oil such as a simple method for identifying the authenticity of camellia seed oil (application number CN201010217382.5), according to the description of the patent application, using the carcass component contained in camellia seed oil, the component and the color developing agent
  • the principle of color development can be used to distinguish other vegetable oils that do not contain carcasses.
  • the disadvantage of this method is that olive oil and other carcasses are contained.
  • the vegetable oil is unusable and the color reaction is not precise enough to allow for quantitative analysis.
  • the principle used is the fatty acid composition of Camellia oleifera seed oil, such as Yan Xiaoli and Xu Wei in the Qualitative Identification of Adulterated Camellia Seed Oil-Gas Chromatography (Food Engineering) In the 12th issue of 2011), the disadvantage of using this type of principle is that it is difficult to distinguish olive oil with similar fatty acid composition of camellia seed oil. Zhou Jianping and Guo Hua in the "Quantitative Determination Method of Camellia Seed Oil", “China Oils and Fats", 2003, Vol. 28, No. 2, pp.
  • a method for establishing squalene as an identification marker for olive oil and camellia seed oil comprising the steps of:
  • the squalene content in the olive oil and the camellia seed oil in the olive oil and the camellia seed oil are respectively 0.15 ⁇ 0.07 mg/g and 6.72 ⁇ 2.05 mg/g, p ⁇ 0.01.
  • the concentration of the potassium hydroxide-ethanol solution in the step (4) is 2 mol/L, the addition amount is 5 mL, the saponification temperature is 85 ° C, and the saponification time is 60 min.
  • the centrifugal speed was 2000 r/min, and the centrifugation time was 5 min.
  • the column used in the step (11) was HP-5MS (30m ⁇ 0.25mm ⁇ 0.25 ⁇ m), and the instrument was an Agilent 7890A gas chromatograph.
  • the method, further gas chromatography conditions are: carrier gas: high purity nitrogen; inlet temperature: 250 ° C; detector FID temperature 270 ° C; column temperature 100 ° C, 2 min, 15 ° C / min temperature to 290 ° C, 5 min
  • carrier gas high purity nitrogen
  • inlet temperature 250 ° C
  • detector FID temperature 270 ° C detector FID temperature 270 ° C
  • column temperature 100 ° C, 2 min, 15 ° C / min temperature to 290 ° C, 5 min
  • the injection volume is 1 ⁇ L, and the split ratio is 50:1.
  • the use of the squalene as an identification marker for olive oil and camellia seed oil comprises the steps of: taking a sample from the olive oil or camellia seed oil to be tested for high-temperature saponification, and extracting squalene with petroleum ether, The peak area of squalene in the sample to be tested is detected by gas chromatography, and the peak area is substituted into the standard curve to obtain the squalene content of the sample to be tested; the squalene of the sample to be tested is compared with the reference olive oil or The species or authenticity of the olive oil or camellia seed oil to be tested is determined by comparing the squalene content in the camellia seed oil.
  • the present invention uses squalene as a marker to identify olive oil and camellia seed oil, and the method is simple, and generally can be carried out in a laboratory having a gas chromatograph;
  • the method has good repeatability and reliable results.
  • the recoveries of spiked standards are 100%-106.3%, and the relative standard deviations are 0.13%-0.81%.
  • the method has a small sample amount, which greatly reduces the loss of the sample and has high detection efficiency
  • This method can provide a great reference for the uniform standard for the identification of olive oil and camellia seed oil.
  • the method directly extracts the oil from the raw material, thereby greatly avoiding the influence of the squalene content and the artificial addition factor during the processing.
  • Figure 1 is a standard curve of squalene
  • Figure 2 is a difference analysis of the content of squalene between camellia seed oil and olive oil
  • Figure 3 is a GC-MS spectrum of extracted camellia seed oil
  • Figure 4 is a structural formula of squalene extracted from camellia seed oil
  • Figure 5 is a GC-MS spectrum of freshly squeezed olive oil
  • Figure 6 is a structural formula of squalene in freshly squeezed olive oil
  • Figure 7 is a GC-MS spectrum of squalene standard
  • Figure 8 is a squalene standard structure.
  • the invention demonstrates the content of squalene in a variety of self-raised and commercially available olive oil and camellia seed oil, and provides a method and application for the identification of squalene as an identification marker for olive oil and camellia seed oil.
  • the invention adopts a chemical method, and uses a relatively common gas chromatograph as a detecting instrument, and can separate squalene well by a simple pretreatment process, and can directly judge the result by comparing the peak areas, and establishes the result by the external standard method.
  • the standard curve can accurately calculate the concentration or content of squalene in the sample, so as to accurately quantify the olive oil and camellia seed oil.
  • the method has good repeatability and the data is intuitive and reliable.
  • Camellia seed oil is extracted by solvent method
  • extraction solvent is n-hexane
  • olive oil is extracted by physical pressing method.
  • Spectral collection and processing qualitatively compared with the standard retention time, that is, qualitative according to the relative retention value; using external standard method to quantify, that is, the standard curve is established by the peak area of different concentrations of squalene standard sample, which will be tested The peak area of the sample is brought into the standard curve to calculate the corresponding concentration.
  • Gas chromatographic conditions chromatographic conditions: HP-5MS (30m ⁇ 0.25mm ⁇ 0.25 ⁇ m) capillary column; carrier gas: high purity nitrogen; inlet temperature: 250 ° C; detector (FID) temperature 270 ° C; The temperature was 100 ° C (2 min), and the temperature was raised to 290 ° C (5 min) at 15 ° C / min; the injection volume was 1 ⁇ L, and the split ratio was 50:1.
  • GC-MS was used to compare the peak time, fragmentation and chemical structure of squalene and standard squalene in the sample.
  • GC-MS chromatographic conditions were the same as GC, mass spectrometry conditions were: solvent delay 3.5 min; ion source: EI; ion source temperature: 230 ° C; Sub-energy 70 eV; MSD transmission line temperature: 250 ° C; squalene qualifier ions m / z 69.1 and 221.0.
  • a standard solution of squalene having a concentration of 10 mg/L to 700 mg/L was prepared using n-hexane as a solvent.
  • the squalene concentration (mg/L) is plotted on the abscissa, and the corresponding peak area is plotted on the ordinate as a standard curve (Fig. 1).
  • the sample squalene peak area is substituted into the regression equation to calculate the sample concentration, and then the concentration is substituted into the following formula, and the corresponding content ( ⁇ g/g) can be converted:
  • X squalene content in the original sample oil, in milligrams per gram (mg/g);
  • Cs The content of squalene in the injection solution is calculated from the standard curve of the squalene peak area in the sample, and the unit is milligrams per liter (mg/L);
  • V total volume of the injection solution, in microliters ( ⁇ L);
  • Example 2 Sample collection and determination of squalene content
  • Camellia seed oil and olive oil were detected in this example (Table 1).
  • the Camellia seeds numbered 1-21 and the olive fruits numbered 23-26 were provided by the Institute of Subtropical Forestry of the Chinese Academy of Forestry.
  • the camellia seed was derived from Jinhua and Jiangxi in Zhejiang Province, and the olive fruit was from Qingchuan, Sichuan.
  • the fresh olive fruit is crushed, the core is taken out, a small amount of water is added, the mixture is fully pulverized, stirred, and centrifuged, and the supernatant is freshly squeezed olive oil.
  • the tea seed oil is obtained by steaming and drying the supernatant containing the oil tea seed oil to a constant weight.
  • the peak area of squalene detected by gas chromatography after pretreatment is substituted into a standard curve to obtain the concentration of squalene in the sample, and then substituted into the following formula.
  • the corresponding content ( ⁇ g/g) can be converted: where: X: squalene content in the sample oil, in milligrams per gram (mg/g); Cs: concentration of squalene in the injection solution, from the sample
  • the area of the squalene peak is calculated by substituting the standard curve in milligrams per liter (mg/L); V: the total volume of the injection solution in microliters ( ⁇ L); m: the sample volume of the sample to be tested in the pretreatment, unit For grams (g).
  • Example 3 GC-MS comparison of squalene and standard in samples

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Abstract

一种角鲨烯作为橄榄油和油茶籽油鉴别标记物的建立方法,首先取角鲨烯标准品配成不同浓度,用气相色谱仪检测得到不同浓度的峰面积,经计算得到峰面积与浓度关系的标准曲线和计算公式;然后分别从油茶籽、橄榄果提取的油茶籽油和橄榄油,或商品油取微量样品进行高温皂化处理,用石油醚萃取角鲨烯,用气相色谱仪检测得到样品中角鲨烯的峰面积,得到各种样品的角鲨烯含量;最后,对所有油茶籽油、橄榄油的角鲨烯含量做差异统计分析,得出油茶籽油、橄榄油的角鲨烯含量。

Description

角鲨烯作为橄榄油和油茶籽油鉴别标记物的建立方法 技术领域
本发明涉及食品检测中油脂检测领域,特别地,本发明涉及一种用角鲨烯含量作为橄榄油和油茶籽油鉴别依据的气相色谱检测方法及判别标准。
背景技术
橄榄油是一种原产于地中海的古老油种,具有减少心脑血管疾病发生、改善人体消化系统功能、抗癌、防衰老、促进血液循环和神经系统发育等多种功效,其营养价值在世界早已得到公认,被誉为“液体黄金”,“植物油皇后”。油茶籽油是从山茶科油茶树种子中提取的中国本土古老油种,其化学性质,尤其是脂肪酸组成与橄榄油非常相似。因其具有预防心血管疾病、抗氧化清除自由基等功效也具有很高的营养价值,油茶籽油被誉为“东方橄榄油”、“长寿油”。
中国是油茶的原产地,是世界上最大的油茶籽油生产国,深受国内消费者欢迎,因产量有限,近年来价格处于上涨趋势。国内市场的橄榄油主要从欧洲进口。橄榄油分为压油和溶剂提取的果渣油两类,通常压榨橄榄油作为食用油,价格较高,而果渣油一般作为工业用油,用于化妆品和日化用品、饲料,价格不高,甚至大大低于国产油茶籽油。过去中国市场出现的油茶籽油一般是其价格较低的棕榈油等。随着食用油检测中气相色谱仪的普遍采用,通过脂肪酸分布和含量,用其常用食用油冒充油茶籽油已很容易鉴别,近年来为此不法商人开始用脂肪酸分布和比例与油茶籽油相似的进口劣质果渣橄榄油冒充油茶籽油,在浙江省已查处多起用果渣橄榄油冒充油茶籽油的重大案件。据2012年8月8日衢州日报报道,该市质监部门曾针对用橄榄果渣油冒充油茶籽油的违法失信行为开展了专项治理。安徽省霍山县专项整治油茶籽油活动中也曾将“油橄榄果渣油”冒充油茶籽油作为查处内容。但迄今尚未见可有效鉴别橄榄油冒充油茶籽油的现成检测方法。
现行中国国标《GB11765-2003油茶籽油》中没有关于对脂肪酸组成类似的橄榄油的鉴别方法,现有公开的专利申请中有利用物理检测手段鉴别橄榄油品种及橄榄油品质的内容,如采用拉曼光谱特征峰信号强度比值的橄榄油快速检测方法(申请号200810106530.9),但它所用掺假油脂中并未提及油茶籽油和橄榄油的鉴别,且所需仪器价格昂贵,不易操作,数据分析繁琐。有关于油茶籽油鉴别的专利,如一种鉴别油茶籽油真伪的简捷方法(申请号CN201010217382.5),根据该专利申请描述,利用油茶籽油中含甾体成分,该成分与显色剂可显色的原理来区别其他不含甾体的植物油,这种方法的缺点在于橄榄油和其他含有甾体成分 的植物油无法使用,并且显色反应不够精确,无法做到定量分析。文献和一些标准中有涉及到油茶籽油掺伪鉴别方法,所用的原理是油茶籽油脂肪酸组成,如严晓丽、徐昕在《掺假油茶籽油定性鉴别-气相色谱法》(《食品工程》2011年12期),中运用该类原理的方法其缺点在于难以将与油茶籽油脂肪酸组成相似的橄榄油区别开来。周建平和郭华在《油茶籽油定量测定方法研究》《中国油脂》2003年第28卷第2期55-57页采用了醋酸酐-浓硫酸显色发鉴别油茶籽油中有色络合物方法,但其化学原理可能是与残留的茶皂素期反应,但我们进行验证发现该法对对纯度较高的油茶籽油效果不好,重复性不好。
钟冬莲等的“油茶籽油中角鲨烯含量的气相色谱法测定”(《分析试验室》2011年第30卷第11期104-106页)中有关于油茶籽油中角鲨烯含量的测定,耿树香等的“不同品种及成熟度橄榄油中角鲨烯的检测分析|”(《中国粮油学报》2013年第28卷第2期123-129页)中有关于橄榄油中角鲨烯含量的测定,但这两篇论文只涉及角鲨烯检测的方法学论证,没有油脂鉴别相关方面研究内容。龙正海、王道平“油茶籽油与橄榄油化学成分研究”(《中国粮油学报》2008年第23卷第2期121-123页)报道橄榄油中角鲨烯含量明显高于油茶籽油,但其所采用样品均为商品,分别只有1个样品量,没有采用从原果提取油品的可靠方法,没有普遍性和代表性意义,更没有做方法学验证。
发明内容
为了克服现有技术的不足,本发明的目的在于提供一种角鲨烯作为橄榄油和油茶籽油鉴别标记物的建立方法。
本发明的目的是通过以下技术方案实现的。
一种角鲨烯作为橄榄油和油茶籽油鉴别标记物的建立方法,包括以下步骤:
(1)取角鲨烯标准品,用色谱级正己烷配制成梯度溶液,通过GC测定,建立标准曲线;
(2)将橄榄果破碎,用物理压榨法提取橄榄油;通过化学溶剂法提取油茶籽油;
(3)取微量标准橄榄油或者油茶籽油;橄榄油的取样量为5mg,油茶籽油为100mg;
(4)加入氢氧化钾-乙醇溶液对样品进行高温皂化处理;
(5)冷却至室温,加入饱和氯化钠溶液后用石油醚萃取样品中的角鲨烯;
(6)静置分层,分离上清液后加入纯水,洗至中性;
(7)静置分层,分离上清液后加入少量无水硫酸钠;
(8)震荡后离心,取上清液并吹干;
(9)加入有机溶剂复溶后过膜;所述有机溶剂为正己烷,过0.45μm的有机膜;
(10)氮气吹干后加入正己烷复溶后进GC进行检测;
(11)将气相色谱图上角鲨烯峰面积带入标准曲线计算出角鲨烯含量;
(12)对油茶籽油和橄榄油角鲨烯含量进行差异性分析;
(13)利用GC-MS对样品中角鲨烯和标准品角鲨烯的出峰时间、碎片图和化学结构比对。
步骤(11)中所述标准曲线得出的线性回归方程:y=0.3662x-1.0791,R2=0.9999。
所述步骤(11)中,橄榄油、油茶籽油中橄榄油和油茶籽油中角鲨烯含量分别为0.15±0.07mg/g和6.72±2.05mg/g,p<0.01。
所述步骤(4)中氢氧化钾-乙醇溶液浓度为2mol/L,添加量为5mL,皂化温度为85℃,皂化时间为60min。
所述步骤(8)中,离心速度为2000r/min,离心时间为5min。
所述步骤(11)中用所用色谱柱为HP-5MS(30m×0.25mm×0.25μm),仪器为Agilent7890A型气相色谱仪器。
所述的方法,进一步气相色谱条件为:载气:高纯氮气;进样口温度:250℃;检测器FID温度270℃;柱温100℃,2min,15℃/min升温至290℃,5min;进样量1μL,分流比50:1。
一种所述角鲨烯作为橄榄油和油茶籽油鉴别标记物的应用,包括以下步骤:从待测的橄榄油或油茶籽油中取样品进行高温皂化处理,用石油醚萃取角鲨烯,用气相色谱仪检测得到待测样品中角鲨烯的峰面积,将峰面积代入所述的标准曲线,得到待测样品的角鲨烯含量;将待测样品的角鲨烯与参考橄榄油或参考油茶籽油中的角鲨烯含量相比较,判断待测的橄榄油或油茶籽油的种属来源或者真伪。
本发明的有益效果是:
(1)本发明以角鲨烯为标记物,对橄榄油和油茶籽油进行鉴别,方法简便,一般具有气相色谱仪的实验室都可以进行;
(2)该方法重复性好,结果可靠,加标回收率100%-106.3%,相对标准差在0.13%-0.81%。
(3)该方法取样量少,极大的减少了样品的损耗,检测效率高;
(4)该方法能对目前没有鉴别橄榄油和油茶籽油的统一标准提供极大参考;
(5)该方法直接从原料中提取油脂,极大避免了加工过程中对角鲨烯含量的影响和人为添加的因素。
附图说明
下面结合具体实施例和附图,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明范围。
图1是角鲨烯标准曲线图谱;
图2是油茶籽油与橄榄油角鲨烯含量差异分析;
图3是提取油茶籽油GC-MS图谱;
图4是提取油茶籽油中角鲨烯结构式;
图5是鲜榨橄榄油GC-MS图谱;
图6是鲜榨橄榄油中角鲨烯结构式;
图7是角鲨烯标样GC-MS图谱;
图8是角鲨烯标样结构式。
具体实施方式
本发明论证了多种自提和市售橄榄油及油茶籽油中角鲨烯的含量,提供了一种角鲨烯作为橄榄油和油茶籽油鉴别标记物的建立方法及应用。本发明采用化学方法,以较为常用的气相色谱仪作为检测仪器,通过简单的前处理过程,能够较好的分离角鲨烯,通过峰面积的比较可直接做出结果判断,通过外标法建立的标准曲线则可对样品中的角鲨烯浓度或含量进行精确计算,从而对橄榄油和油茶籽油做出准确的定量鉴别,方法重复性好,数据直观可靠。
1、取角鲨烯标准品,配制成梯度溶液,用气相色谱仪器检测,通过浓度和峰面积的关系得到标准曲线和浓度计算公式。
2、从油茶籽和橄榄果子中提取油茶籽油和橄榄油,油茶籽油采用溶剂法提取,提取溶剂为正己烷,橄榄油采用物理压榨法提取。
3、取微量(茶油为100mg,橄榄油为5mg)待测样品于30mL试管中,加入5mL的2mol/L KOH-乙醇溶液;置于85℃水浴锅中皂化1h后移出冷却;加入5mL饱和NaCl溶液,再加入4mL石油醚;充分震荡后静置分层,取上清液于试管中,加入4mL纯水水洗至中性,取上层液体于另一试管中,并加入少量无水硫酸钠;震荡后以2000r/min离心5min;转移上清液于新试管中,氮吹至无液体;加入2mL正己烷(分析纯)充分溶解后过过膜(ф13*0.45μm有机滤膜);再次氮吹至干,100μL正己烷(色谱纯)复溶后进行气相色谱检测。检测步骤如下:
4、气相色谱分析:用Agilent 7890A型气相色谱仪测定脂肪酸,用安捷伦化学工作站采集图谱,并处理分析所采集的谱图数据。色谱峰采用角鲨烯标样来进行鉴定。定量方式采用面积法,定量方法为外标法。
5、谱图采集处理:采用与标准品保留时间对照的方法定性,即根据相对保留值定性;采用外标法定量,即通过不同浓度角鲨烯标准样品的峰面积建立标准曲线,将待测样品峰面积带入标准曲线可计算出相应浓度。
6、气相色谱分析条件:色谱条件:HP-5MS(30m×0.25mm×0.25μm)毛细管柱;载气:高纯氮气;进样口温度:250℃;检测器(FID)温度270℃;柱温100℃(2min),15℃/min升温至290℃(5min);进样量1μL,分流比50:1。
7、利用GC-MS对样品中角鲨烯和标准品角鲨烯的出峰时间、碎片图和化学结构比对。GC-MS色谱条件和GC相同,质谱条件为:溶剂延迟3.5min;离子源:EI;离子源温度:230℃;电 子能量70eV;MSD传输线温度:250℃;角鲨烯定性离子m/z69.1和221.0。
实施例一:建立标准曲线
以正己烷为溶剂,配制浓度为10mg/L-700mg/L的角鲨烯标准溶液。以角鲨烯浓度(mg/L)为横坐标,相应的峰面积为纵坐标做标准曲线(图1),角鲨烯的线性回归方程:y=0.3662x-1.0791(R2=0.9999)。最低检出限(LOD):以基线噪音的3倍所对应的浓度为标准,本方法为2.38μg/mL。
检测具体样品时,将样品中角鲨烯峰面积代入回归方程可计算得到样品浓度,再将浓度代入以下计算公式,可换算得到相应的含量(μg/g):
Figure PCTCN2016078743-appb-000001
式中:
X:原样品油中角鲨烯含量,单位为毫克每克(mg/g);
Cs:进样溶液中角鲨烯含量,由样中角鲨烯峰面积代入标准曲线计算所得,单位为毫克每升(mg/L);
V:进样溶液总体积,单位为微升(μL);
m:前处理时待测样品取样量,单位为克(g)。
实施例二:样品采集及角鲨烯含量测定
1、油茶籽及橄榄果采集及油脂提取
本实施例中所检测的共有30种油茶籽油和橄榄油(表1)。其中编号1-21的油茶籽和编号23-26的橄榄果由中国林业科学研究院亚热带林业研究所提供,油茶籽来源于浙江金华和江西,橄榄果来源于四川青川。
表1待检测样品明细
Figure PCTCN2016078743-appb-000002
将鲜橄榄果粉碎后取出果核,加入少量水,充分粉碎并搅拌后离心,上清液即为鲜榨橄榄油。取适量油茶籽仁置于50℃烘箱中一定时间。从烘箱中取出后冷却至室温,精确称量烘干后的油茶籽仁6g,用剪刀将油茶籽仁剪碎置于玻璃管中,按料液比1:5g/mL加入正己烷,在水浴温度50℃条件下超声辅助萃取30min后离心分层,取上清。将含有油茶籽油的上清液旋蒸后烘干至恒重后即得油茶籽油。
2、样品前处理
取微量(茶油为100mg,橄榄油为5mg)待测样品于30mL试管中,加入5mL的2mol/L  KOH-乙醇(5.6g氢氧化钾,50mL无水乙醇)溶液;置于85℃水浴锅中皂化1h后移出冷却;加入5mL饱和NaCl溶液,再加入4mL石油醚;充分震荡后静置分层,取上清液于试管中,加入4mL纯水水洗至中性,取上层液体于另一试管中,并加入少量无水硫酸钠;震荡后以2000r/min离心5min;转移上清液于新试管中,氮吹至无液体;加入2mL正己烷(分析纯)充分溶解后过过膜(0.45μm有机滤膜);再次氮吹至干,100μL正己烷(色谱纯)复溶后进样。
3、仪器及条件
仪器型号:Agilent 7890A;色谱条件:HP-5MS(30m×0.25mm×0.25μm)毛细管柱;载气:高纯氮气;进样口温度:250℃;检测器(FID)温度270℃;柱温100℃(2min),15℃/min升温至290℃(5min);进样量1μL,分流比50:1。
4、检测结果
将前处理后通过气相色谱检测出的角鲨烯的峰面积代入标准曲线,得到样品中的角鲨烯浓度,再代入以下计算公式,
Figure PCTCN2016078743-appb-000003
可换算得到相应的含量(μg/g):式中:X:样品油中角鲨烯含量,单位为毫克每克(mg/g);Cs:进样溶液中角鲨烯浓度,由样品中角鲨烯峰面积代入标准曲线计算所得,单位为毫克每升(mg/L);V:进样溶液总体积,单位为微升(μL);m:前处理时待测样品取样量,单位为克(g)。
24种油茶籽油中角鲨烯含量的检测结果如表2所示,6种橄榄油中角鲨烯含量的检测结果如表3所示。
表2各种油茶籽油角鲨烯含量测定结果
Figure PCTCN2016078743-appb-000004
表3各种橄榄油的角鲨烯含量测定结果
Figure PCTCN2016078743-appb-000005
5、橄榄油和油茶籽油中角鲨烯含量分析
将24种油茶籽油和6种橄榄油中角鲨烯含量进行对比(图2),结合表2和表3可见橄 榄油和油茶籽油的角鲨烯含量分别为6.72±2.05mg/g和0.15±0.07mg/g,橄榄油的角鲨烯含量是油茶籽油的44.8倍,具有显著性差异(p<0.01),此结果表明角鲨烯是一种有效的鉴别橄榄油和油茶籽油的标记物。
实施例二:角鲨烯加标回收
取100mg油茶籽油,按具体实施方法中的描述进行样品前处理,每个样品重复5次。同时取100mg油茶籽油样品两份,分别添加含有5μg和10μg的角鲨烯标准溶液进行加标回收实验。方法精密度与回收率见表4。结果标明该方法回收率在100%-106.3%之间,相对标准偏差在0.13%-0.81%之间。
表4油茶籽油中加标回收实验结果
Figure PCTCN2016078743-appb-000006
实施例三:GC-MS对样品中角鲨烯和标准品比对
1、样品前处理
取微量(茶油为100mg,橄榄油为5mg)待测样品于30mL试管中,加入5mL的2mol/L KOH-乙醇(5.6g氢氧化钾,50mL无水乙醇)溶液;置于85℃水浴锅中皂化1h后移出冷却;加入5mL饱和NaCl溶液,再加入4mL石油醚;充分震荡后静置分层,取上清液于试管中,加入4mL纯水水洗至中性,取上层液体于另一试管中,并加入少量无水硫酸钠;震荡后以2000r/min离心5min;转移上清液于新试管中,氮吹至无液体;加入2mL正己烷(分析纯)充分溶解后过过膜(0.45μm有机滤膜);再次氮吹至干,100μL正己烷(色谱纯)复溶后进样。
2、仪器条件
仪器型号:Agilent 6890B;色谱条件:HP-5MS UI(30m×0.25mm×0.25μm)超高惰性毛细管柱;载气:高纯氦气;进样口温度:250℃;柱温100℃(2min),15℃/min升温至290℃(5min);进样量1μL。质谱条件:溶剂延迟3.5min;离子源:EI;离子源温度:230℃;电子能量70eV;MSD传输线温度:250℃;角鲨烯定性离子m/z 69.1和221.0。
3、结果分析
油茶籽油和橄榄油经过前处理后,样品通过GC-MS检测(如图3和图5),与质谱库中数据对比后相似度最高的物质为角鲨烯。直接用角鲨烯标样进入GC-MS检测(如图7),发现保留时间完全吻合,角鲨烯标准品的质谱图见图8,样品中角鲨烯的质谱图见图4和图6。由以上可见样品中角鲨烯和标准品角鲨烯的保留时间、化学结构式和离子碎片图一致。

Claims (8)

  1. 一种角鲨烯作为橄榄油和油茶籽油鉴别标记物的建立方法,其特征在于,包括以下步骤:
    (1)取角鲨烯标准品,用色谱级正己烷配制成梯度溶液,通过GC测定,建立标准曲线;
    (2)将橄榄果破碎,用物理压榨法提取橄榄油;通过化学溶剂法提取油茶籽油;
    (3)取微量标准橄榄油或者油茶籽油;橄榄油的取样量为5mg,油茶籽油为100mg;
    (4)加入氢氧化钾-乙醇溶液对样品进行高温皂化处理;
    (5)冷却至室温,加入饱和氯化钠溶液后用石油醚萃取样品中的角鲨烯;
    (6)静置分层,分离上清液后加入纯水,洗至中性;
    (7)静置分层,分离上清液后加入少量无水硫酸钠;
    (8)震荡后离心,取上清液并吹干;
    (9)加入有机溶剂复溶后过膜;所述有机溶剂为正己烷,过0.45μm的有机膜;
    (10)氮气吹干后加入正己烷复溶后进GC进行检测;
    (11)将气相色谱图上角鲨烯峰面积带入标准曲线计算出角鲨烯含量;
    (12)对油茶籽油和橄榄油角鲨烯含量进行差异性分析;
    (13)利用GC-MS对样品中角鲨烯和标准品角鲨烯的出峰时间、碎片图和化学结构比对。
  2. 如权利要求1所述的方法,其特征在于,步骤(11)中所述标准曲线得出的线性回归方程:y=0.3662x-1.0791,R2=0.9999。
  3. 如权利要求1所述的方法,其特征在于,所述步骤(11)中,橄榄油、油茶籽油中橄榄油和油茶籽油中角鲨烯含量含量分别为0.15±0.07mg/g和6.72±2.05mg/g,p<0.01。
  4. 如权利要求1所述的方法,其特征在于,所述步骤(4)中氢氧化钾-乙醇溶液浓度为2mol/L,添加量为5mL,皂化温度为85℃,皂化时间为60min。
  5. 如权利要求1所述的方法,其特征在于,所述步骤(8)中,离心速度为2000r/min,离心时间为5min。
  6. 如权利要求1所述的方法,其特征在于,所述步骤(11)中用所用色谱柱为HP-5MS,仪器为Agilent 7890A型气相色谱仪器。
  7. 如权利要求6所述的方法,其特征在于,进一步所述气相色谱条件为:载气:高纯氮气;进样口温度:250℃;检测器FID温度270℃;柱温100℃,2min,15℃/min升温至290℃,5min;进样量1μL,分流比50:1。
  8. 一种如权利要求1所述角鲨烯作为橄榄油和油茶籽油鉴别标记物的应用,其特征在于,包括以下步骤:从待测的橄榄油或油茶籽油中取样品进行高温皂化处理,用石油醚萃取角鲨烯,用气相色谱仪检测得到待测样品中角鲨烯的峰面积,将峰面积代入所述的标准曲线,得到待测样品的角鲨烯含量;将待测样品的角鲨烯与参考橄榄油或参考油茶籽油中的角鲨烯含量相比较,判断待测的橄榄油或油茶籽油的种属来源或者真伪。
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