羊肉产地指纹图谱溯源技术研究
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摘要
食品产地溯源体系是保障食品安全、保护地理标志性产品及特色产品、增强消费者信心的有力措施之一。同位素比率、矿物元素含量及近红外光谱分析被认为是产地溯源的有效技术之一。为探讨不同指纹图谱技术对中国羊肉产地溯源的可行性，本研究通过在内蒙古自治区锡林郭勒盟、阿拉善盟和呼伦贝尔市，以及山东省菏泽市和重庆市5个羊肉主产区采集羊肉、羊颈毛、饲料及饮用水样品，利用稳定性同位素比率质谱仪（IRMS）测定δ13C、δ15N、δ2H值，利用电感耦合等离子质谱仪（ICP-MS）测定羊肉样品中的Be、Na、Al、Ca、Mg、K、V、Cr、Mn、Fe、Co、Ni、Cu、Zn、As、Se、Ag、Sb、Ba、Tl、Pb、Th、U、Cd和Mo25种矿物元素的含量，利用近红外分析仪（NIRS）测定羊肉样品的近红外光谱特征，并结合方差分析、主成分分析、聚类分析、判别分析等数理统计方法，系统研究不同地域来源羊组织中的同位素组成、矿物元素含量、近红外光谱指纹的特征及其差异，它们对地域的判别效果，以及与地域环境(饲料、饮水、土壤等)的关系。
为了明确动物组织中的同位素、矿物元素和近红外光谱指纹溯源信息变化机理，进一步确证上述指纹技术对羊肉产地溯源的有效性和可靠性，设计了羊的人为迁徙饲喂模型试验。通过分析同一地域不同饲喂期和不同地地域迁徙喂养的羊的肌肉、心脏、肝脏、肺脏和毛发中的稳定性碳、氮、氢同位素组成，57种矿物元素含量，以及近红外光谱特征及差异，探讨羊组织器官中的各溯源指标随地域、饲喂期、饲料组成等改变的变化规律，比较羊不同组织器官中同位素组成、矿物元素含量及近红外光谱指纹的差异及相关关系，并建立羊肉产地溯源的有效指标体系。研究得到以下主要结论：
(1)不同地域来源羊组织中的同位素比率、矿物元素含量及近红外光谱指纹均有显著差异，具有其各自的指纹特征。单一指标体系与指标组合体系对羊肉产地的整体正确判别率均在85%以上，表明它们均是羊肉产地溯源的有效方法。其中同位素和矿物元素组合指标溯源效果优于单一的同位素和矿物元素溯源技术。不同方法建立的溯源指标体系依次为：稳定性同位素：δ13C、δ15N、δ2H指标组合；矿物元素：Be、Cr、Mn、Fe、Cu、Zn、As、Sb、V、Ba、Ni和Na；同位素和矿物元素结合：δ2H、δ15N、V、Cr、Mn、Tl和Zn。
(2)羊组织器官中碳、氮、氢同位素在地域和饲喂期间均有显著性差异，并且受地域的影响远大于饲喂期和饲喂期×地域交互作用的影响，证实了碳、氮、氢同位素是羊肉产地溯源的有效指标，且利用稳定性同位素对羊肉产地溯源时，还应考虑季节变化的影响。
(3)羊不同组织器官中同位素的转化速率不同，肝脏、肺脏、心脏等内脏器官中碳、氮、氢同位素的转化速率快于肌肉和毛发。因此可根据饲喂时间选择合适的组织作为溯源研究对象。羊不同组织器官中同位素组成存在差异。除毛发高于肝脏外，其余组织器官中碳同位素组成差异不显著；除心脏高于脱脂肌肉外，其余组织器官中氮同位素组成差异不显著；氢同位素组成在羊各组织器官中的大小顺序依次为：毛发、肝脏>心脏、肺脏>肌肉组织。不同组织器官之间碳、氮、氢同位素存在相关关系。
(4)羊组织器官在地域间存在差异显著的元素。肌肉中为Li、Ni、Mo、Cs、Se、Cu、Rb、As、Zn、Mn和Fe，心脏中为Be、In、Sb、Dy、Li、Sc、Cs、Se、Sn、Rb和As，肝脏中为Y、Nb、Sb、La、Ce、Pr、Nd、Sm、Eu、Gd、Tb、Dy、Ho、Er、Yb、Th、Li、Cs、Se、Rb、Mn和K，肺脏中为Cd、Cs、Rb、Ti、Be、In、Sb、La、Ce、Pr、Nd、Gd、Tb、Dy、Ho、U、Li、Sc、Ni、Mo、Pb、Bi、Se、Sn、Zn、K和Mg。羊组织器官在同一地域不同饲喂期间也存在差异不显著的矿物元素。这些元素在牧区太仆寺旗较少，而在农区杨凌较多。在地域间差异显著，饲喂期间差异不显著的元素可用于羊肉矿物元素产地溯源。
(5)羊不同组织器官中矿物元素富集特征不同，筛选出的产地溯源参考指标也有所不同，肌肉中为Tb、Rb、Mo、Fe；心脏中为Be、Gd、Lu、Rb、Cs、As；肝脏中为Eu、Ce、Dy、Yb、Cs；肺脏中为Be、In、La、Gd、Ho、Th、Sc、Pb、Bi、Rb、Cs。其中Rb和Cs是羊各组织器官的的共有溯源参考指标。
(6)地域和饲喂期对羊组织器官的近红外光谱均具有显著影响，表明在进行近红外光谱产地溯源时需充分考虑饲喂期的影响。此外，羊肌肉、心脏、肝脏和肺脏等组织器官近红外光谱的地域、饲喂期差异类似，均可作为近红外光谱产地溯源的研究材料。
(7)方差分析、主成分分析、聚类分析及判别分析等统计方法是羊肉产地溯源指标筛选及判别模型建立不可缺少的数学工具。
Food geographical origin traceability system is a powerful approach to ensure foodsafety, protect geographical indication and strengthen consumers’ confidence. Analyses ofstable isotope ratio, mineral element content and near infrared reflectance spectroscopy areconsidered as effective techniques for food geographical origin traceability. The objective ofthis paper was to investigate the potential of these fingerprint techniques for geographicalorigin traceability of lamb meat in China. Lamb meat, wool, their feeds and drinking wateroriginating from five major lamb meat producing regions of China including Alxa League,Xilin Gol League and Hulunbuir City in Inner Mongolia, Heze City in Shandong Provinceand Chongqing City were collected for the analysis. δ13C, δ15N and δ2H values of thesesamples were determined by isotope ratio mass spectrometry(IRMS), the contents of Al、Ca、V、Cr、Mn、Fe、Co、Ni、Cu、Zn、As、Se、Ag、Sb、Ba、Tl、Pb、Th、U、Mg、K、Cd and Mo in lamb meat were measured by inductively coupled plasma mass spectrometry(ICP-MS), and near infrared spectra of lamb meat were scanned using NIR spectrometer.Multivariate statistical methods including ANOVA, principal component analysis (PCA),cluster analysis (CA), and linear discriminant analysis (LDA) were used for data analysis. Thestudy mainly analyzed the characteristics and differences of stable isotopes, element contentsand NIR fingerprints in lamb tissues from above different regions, discrimination power ofthese techniques for lamb meat geographical origin assignment, and the relationship of stableisotopes, element contents and NIR fingerprints in lamb tissues with their feedingenvironment.
Lamb transfer-feeding model experiment was designed to investigate the changemechanisms of traceability information of stable isotopes, element contents and NIR in lambtissues, and to further confirm the validity and stability of these techniques for tracing lambmeat geographical origin. Carbon, nitrogen and hydrogen isotope compositions, contents of57elements, and NIR fingerprints in muscle, heart, liver, lung and wool of lamb that weretransfer-fed in different feeding periods at the same region and in different regions weredetermined, and their changes with the variations of origin, feeding periods and feeding typewere analyzed. In addition, the differences of these indicators in different organs and tissues of lamb were compared, and the suitable indicators for geographical origin traceability wereestablished. The main conclusions were as follows:
(1) There were significant differences of stable isotopes, element contents and NIR inlamb tissues according to their geographical origin. Each region had its unique fingerprint.The total correct classification rate over85%was obtained according to the geographicalorigin using any of these techniques, which indicated the feasiablity and validity of thesemethods for tracing the lamb meat origin. The combination of stable isotopes and mineralelements notablely improved the correct classification rate and had a better traceability effecton lamb meat origin than those single methods. The indicator system of each method for lambmeat origin traceability was as follow: the combination of δ13C、 δ15N and δ2H values forstable isotope analysis, the contents of Be、Cr、Mn、Fe、Cu、Zn、As、Sb、V、Ba、Ni andNa for mineral element analysis, and δ2H、δ15N、V、Cr、Mn、Tl and Zn for analysis of stableisotopes combined with mineral elements.
(2) Carbon, nitrogen and hydrogen isotope ratios in lamb tissues and organs hadsignificant differences between different regions and between different feeding periods. Theywere more affected by origins than feeding periods and their interaction. This result confirmedthe validity of stable carbon, nitrogen and hydrogen isotopes for lamb meat origin traceabilty,and seasonal variation was also should be considered.
(3) There were different turnover rates of stable isotopes in lamb tissues and organs. Theturnover of carbon, nitrogen and hydrogen isotopes in in internal tissues such as liver, lungand heart were faster than those in defatted muscle and wool. It’s implied that the suitabletissues can be chosen for lamb meat origin according to the feeding time. There were alsosignificant differences of stable isotopes between the different tissues. The carbon isotope intissues had no significant difference except for its higer existance in wool than in liver. Thenitrogen isotope in tissues had no significant difference except for its higer existance in heartthan in defatted muscle. The order of hydrogen isotope in tissues from high to low were wooland liver> heart and lung> defatted muscle. There were correlations of carbon, nitrogen andhydrogen between the different tissues despite their differences.
(4) The distinct element fingerprint features were found in lamb tissues and organs fromdifferent regions, with Li、Ni、Mo、Cs、Se、Cu、Rb、As、Zn、Mn and Fe in defatted muscle,Be、In、Sb、Dy、Li、Sc、Cs、Se、Sn、Rb and As in heart, Y、Nb、Sb、La、Ce、Pr、Nd、Sm、Eu、Gd、Tb、Dy、Ho、Er、Yb、Th、Li、Cs、Se、Rb、Mn and K in liver, andCd、Cs、Rb、Ti、Be、In、Sb、La、Ce、Pr、Nd、Gd、Tb、Dy、Ho、U、Li、Sc、Ni、Mo、Pb、Bi、Se、Sn、Zn、K and Mg in lung. Elments with no significant difference were alsoexisted in lamb tissues and organs from the different feeding periods within the same origin, which were relatively less in Taipusiqi than those in Yangling Zone. The element that hadsignificant difference between the regions but no significant difference between feedingperiods can be used as tracing indicators.
(5) The element fingerprints in different tissues and organs had distinct characteristics,resulting in different referred indictors for origin traceability, with Tb、Rb、Mo and Fe formuscle, Be、Gd、Lu、Rb、Cs and As for heart, Eu、Ce、Dy、Yb and Cs for liver, Be、In、La、Gd、Ho、Th、Sc、Pb、Bi、Rb and Cs for lung. Rb and Cs were the common tracingindicators for all tissues and organs.
(6) There were both significant differences of NIR fingerprints in lamb tissues andorgans between their origins and between their feeding periods, which indicated that theseasonal variation should be considered when using NIR fingerprint for origin traceablity. Inaddition, NIR fingerprints of lamb muscle, heart, liver and lung had similar differencesaccording to different origins and different feeding period, which domenstrated that all thesetissues can be used for lamb meat origin traceability.
(7) The statistics methods including variance analysis, principal component analysis,cluster analysis and discriminant analysis were essential tools for selecting the indicators andestablishing the discriminant models in lamb meat origin traceability.
为了明确动物组织中的同位素、矿物元素和近红外光谱指纹溯源信息变化机理，进一步确证上述指纹技术对羊肉产地溯源的有效性和可靠性，设计了羊的人为迁徙饲喂模型试验。通过分析同一地域不同饲喂期和不同地地域迁徙喂养的羊的肌肉、心脏、肝脏、肺脏和毛发中的稳定性碳、氮、氢同位素组成，57种矿物元素含量，以及近红外光谱特征及差异，探讨羊组织器官中的各溯源指标随地域、饲喂期、饲料组成等改变的变化规律，比较羊不同组织器官中同位素组成、矿物元素含量及近红外光谱指纹的差异及相关关系，并建立羊肉产地溯源的有效指标体系。研究得到以下主要结论：
(1)不同地域来源羊组织中的同位素比率、矿物元素含量及近红外光谱指纹均有显著差异，具有其各自的指纹特征。单一指标体系与指标组合体系对羊肉产地的整体正确判别率均在85%以上，表明它们均是羊肉产地溯源的有效方法。其中同位素和矿物元素组合指标溯源效果优于单一的同位素和矿物元素溯源技术。不同方法建立的溯源指标体系依次为：稳定性同位素：δ13C、δ15N、δ2H指标组合；矿物元素：Be、Cr、Mn、Fe、Cu、Zn、As、Sb、V、Ba、Ni和Na；同位素和矿物元素结合：δ2H、δ15N、V、Cr、Mn、Tl和Zn。
(2)羊组织器官中碳、氮、氢同位素在地域和饲喂期间均有显著性差异，并且受地域的影响远大于饲喂期和饲喂期×地域交互作用的影响，证实了碳、氮、氢同位素是羊肉产地溯源的有效指标，且利用稳定性同位素对羊肉产地溯源时，还应考虑季节变化的影响。
(3)羊不同组织器官中同位素的转化速率不同，肝脏、肺脏、心脏等内脏器官中碳、氮、氢同位素的转化速率快于肌肉和毛发。因此可根据饲喂时间选择合适的组织作为溯源研究对象。羊不同组织器官中同位素组成存在差异。除毛发高于肝脏外，其余组织器官中碳同位素组成差异不显著；除心脏高于脱脂肌肉外，其余组织器官中氮同位素组成差异不显著；氢同位素组成在羊各组织器官中的大小顺序依次为：毛发、肝脏>心脏、肺脏>肌肉组织。不同组织器官之间碳、氮、氢同位素存在相关关系。
(4)羊组织器官在地域间存在差异显著的元素。肌肉中为Li、Ni、Mo、Cs、Se、Cu、Rb、As、Zn、Mn和Fe，心脏中为Be、In、Sb、Dy、Li、Sc、Cs、Se、Sn、Rb和As，肝脏中为Y、Nb、Sb、La、Ce、Pr、Nd、Sm、Eu、Gd、Tb、Dy、Ho、Er、Yb、Th、Li、Cs、Se、Rb、Mn和K，肺脏中为Cd、Cs、Rb、Ti、Be、In、Sb、La、Ce、Pr、Nd、Gd、Tb、Dy、Ho、U、Li、Sc、Ni、Mo、Pb、Bi、Se、Sn、Zn、K和Mg。羊组织器官在同一地域不同饲喂期间也存在差异不显著的矿物元素。这些元素在牧区太仆寺旗较少，而在农区杨凌较多。在地域间差异显著，饲喂期间差异不显著的元素可用于羊肉矿物元素产地溯源。
(5)羊不同组织器官中矿物元素富集特征不同，筛选出的产地溯源参考指标也有所不同，肌肉中为Tb、Rb、Mo、Fe；心脏中为Be、Gd、Lu、Rb、Cs、As；肝脏中为Eu、Ce、Dy、Yb、Cs；肺脏中为Be、In、La、Gd、Ho、Th、Sc、Pb、Bi、Rb、Cs。其中Rb和Cs是羊各组织器官的的共有溯源参考指标。
(6)地域和饲喂期对羊组织器官的近红外光谱均具有显著影响，表明在进行近红外光谱产地溯源时需充分考虑饲喂期的影响。此外，羊肌肉、心脏、肝脏和肺脏等组织器官近红外光谱的地域、饲喂期差异类似，均可作为近红外光谱产地溯源的研究材料。
(7)方差分析、主成分分析、聚类分析及判别分析等统计方法是羊肉产地溯源指标筛选及判别模型建立不可缺少的数学工具。
Food geographical origin traceability system is a powerful approach to ensure foodsafety, protect geographical indication and strengthen consumers’ confidence. Analyses ofstable isotope ratio, mineral element content and near infrared reflectance spectroscopy areconsidered as effective techniques for food geographical origin traceability. The objective ofthis paper was to investigate the potential of these fingerprint techniques for geographicalorigin traceability of lamb meat in China. Lamb meat, wool, their feeds and drinking wateroriginating from five major lamb meat producing regions of China including Alxa League,Xilin Gol League and Hulunbuir City in Inner Mongolia, Heze City in Shandong Provinceand Chongqing City were collected for the analysis. δ13C, δ15N and δ2H values of thesesamples were determined by isotope ratio mass spectrometry(IRMS), the contents of Al、Ca、V、Cr、Mn、Fe、Co、Ni、Cu、Zn、As、Se、Ag、Sb、Ba、Tl、Pb、Th、U、Mg、K、Cd and Mo in lamb meat were measured by inductively coupled plasma mass spectrometry(ICP-MS), and near infrared spectra of lamb meat were scanned using NIR spectrometer.Multivariate statistical methods including ANOVA, principal component analysis (PCA),cluster analysis (CA), and linear discriminant analysis (LDA) were used for data analysis. Thestudy mainly analyzed the characteristics and differences of stable isotopes, element contentsand NIR fingerprints in lamb tissues from above different regions, discrimination power ofthese techniques for lamb meat geographical origin assignment, and the relationship of stableisotopes, element contents and NIR fingerprints in lamb tissues with their feedingenvironment.
Lamb transfer-feeding model experiment was designed to investigate the changemechanisms of traceability information of stable isotopes, element contents and NIR in lambtissues, and to further confirm the validity and stability of these techniques for tracing lambmeat geographical origin. Carbon, nitrogen and hydrogen isotope compositions, contents of57elements, and NIR fingerprints in muscle, heart, liver, lung and wool of lamb that weretransfer-fed in different feeding periods at the same region and in different regions weredetermined, and their changes with the variations of origin, feeding periods and feeding typewere analyzed. In addition, the differences of these indicators in different organs and tissues of lamb were compared, and the suitable indicators for geographical origin traceability wereestablished. The main conclusions were as follows:
(1) There were significant differences of stable isotopes, element contents and NIR inlamb tissues according to their geographical origin. Each region had its unique fingerprint.The total correct classification rate over85%was obtained according to the geographicalorigin using any of these techniques, which indicated the feasiablity and validity of thesemethods for tracing the lamb meat origin. The combination of stable isotopes and mineralelements notablely improved the correct classification rate and had a better traceability effecton lamb meat origin than those single methods. The indicator system of each method for lambmeat origin traceability was as follow: the combination of δ13C、 δ15N and δ2H values forstable isotope analysis, the contents of Be、Cr、Mn、Fe、Cu、Zn、As、Sb、V、Ba、Ni andNa for mineral element analysis, and δ2H、δ15N、V、Cr、Mn、Tl and Zn for analysis of stableisotopes combined with mineral elements.
(2) Carbon, nitrogen and hydrogen isotope ratios in lamb tissues and organs hadsignificant differences between different regions and between different feeding periods. Theywere more affected by origins than feeding periods and their interaction. This result confirmedthe validity of stable carbon, nitrogen and hydrogen isotopes for lamb meat origin traceabilty,and seasonal variation was also should be considered.
(3) There were different turnover rates of stable isotopes in lamb tissues and organs. Theturnover of carbon, nitrogen and hydrogen isotopes in in internal tissues such as liver, lungand heart were faster than those in defatted muscle and wool. It’s implied that the suitabletissues can be chosen for lamb meat origin according to the feeding time. There were alsosignificant differences of stable isotopes between the different tissues. The carbon isotope intissues had no significant difference except for its higer existance in wool than in liver. Thenitrogen isotope in tissues had no significant difference except for its higer existance in heartthan in defatted muscle. The order of hydrogen isotope in tissues from high to low were wooland liver> heart and lung> defatted muscle. There were correlations of carbon, nitrogen andhydrogen between the different tissues despite their differences.
(4) The distinct element fingerprint features were found in lamb tissues and organs fromdifferent regions, with Li、Ni、Mo、Cs、Se、Cu、Rb、As、Zn、Mn and Fe in defatted muscle,Be、In、Sb、Dy、Li、Sc、Cs、Se、Sn、Rb and As in heart, Y、Nb、Sb、La、Ce、Pr、Nd、Sm、Eu、Gd、Tb、Dy、Ho、Er、Yb、Th、Li、Cs、Se、Rb、Mn and K in liver, andCd、Cs、Rb、Ti、Be、In、Sb、La、Ce、Pr、Nd、Gd、Tb、Dy、Ho、U、Li、Sc、Ni、Mo、Pb、Bi、Se、Sn、Zn、K and Mg in lung. Elments with no significant difference were alsoexisted in lamb tissues and organs from the different feeding periods within the same origin, which were relatively less in Taipusiqi than those in Yangling Zone. The element that hadsignificant difference between the regions but no significant difference between feedingperiods can be used as tracing indicators.
(5) The element fingerprints in different tissues and organs had distinct characteristics,resulting in different referred indictors for origin traceability, with Tb、Rb、Mo and Fe formuscle, Be、Gd、Lu、Rb、Cs and As for heart, Eu、Ce、Dy、Yb and Cs for liver, Be、In、La、Gd、Ho、Th、Sc、Pb、Bi、Rb and Cs for lung. Rb and Cs were the common tracingindicators for all tissues and organs.
(6) There were both significant differences of NIR fingerprints in lamb tissues andorgans between their origins and between their feeding periods, which indicated that theseasonal variation should be considered when using NIR fingerprint for origin traceablity. Inaddition, NIR fingerprints of lamb muscle, heart, liver and lung had similar differencesaccording to different origins and different feeding period, which domenstrated that all thesetissues can be used for lamb meat origin traceability.
(7) The statistics methods including variance analysis, principal component analysis,cluster analysis and discriminant analysis were essential tools for selecting the indicators andestablishing the discriminant models in lamb meat origin traceability.
引文
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