CN109682894A - 一种饮料中索马甜的检测方法 - Google Patents
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Abstract
本发明涉及一种饮料中索马甜的检测方法,特别涉及一种利用超高效液相色谱法和SDS‑PAGE法对饮料中索马甜进行分析的方法;超高效液相色谱法是将饮料进行超滤处理后,选择大孔径的色谱填料作为分离介质,以DAD为检测器,进行液相色谱分析,实现定性定量检测;SDS‑PAGE法采用12.5%的分离胶,以考马斯亮蓝染色,能够实现对索马甜的定性及半定量分析,其灵敏度满足标准限值使用要求,为进一步分析验证提供了途径。本发明的优点在于:本发明提供了饮料中索马甜的分析方法,灵敏度满足对索马甜的限量要求,其中色谱法和电泳法能够相互佐证,实现对样品的确证。
Description
技术领域
本发明属于食物的理化检验技术领域,特别涉及一种饮料中索马甜的检测方法。
背景技术
索马甜(也译作嗦吗甜、莎马甜,Thaumatin),是从天然植物Thaurnatococcusdaniellii的果实中提取出的超甜蛋白质,与其它植物来源具有类似特性的蛋白质如莫内林(Monellin)、仙茅蛋白(库克灵,Curculin)、马槟榔蛋白(Mabinlin)、奇果蛋白(Miraculin)、培它丁(Pentadin)、布拉齐因(Brazzein)、 Neoculin等一起被称之为甜蛋白。相对于传统的碳水化合物糖类或化学合成甜味剂,这些天然甜蛋白一般具有甜度高、热量低、口感好、安全无毒等优点,同时,还具有改善食品风味、可降解为人体所需的各种氨基酸以及不会引起血糖升高、蛀牙等特点,被认为非常具有发展前景的食品添加剂。研究发现,索马甜属于碱性蛋白质,其等电点介于11.5~12.5之间,主要成分有2种,索马甜I为207个氨基酸以直链状结合的化合物,分子量22209;索马甜II为由198个氨基酸结合而成,分子量22293的蛋白质。索马甜的甜度是等质量蔗糖的3000倍,其甜感在pH2~10范围内、 100℃以下加热(或100℃以上的超高温瞬时杀菌)性能稳定,与糖类甜味剂一起使用时,具有协同效应和改善风味作用。
目前,索马甜作为甜味剂、增味剂,在欧美国家和日本具有一定的使用规模,国内使用相对较少,根据我国现行的《食品安全国家标准食品添加剂使用标准》(GB 2760-2014),索马甜可以作为甜味剂在冷冻饮品(食用冰除外)、加工坚果与籽类、焙烤食品、餐桌甜味料、饮料类(包装饮用水除外)食品中限量使用,其最大允许使用量均为0.025g/kg。
然而,国内尚无针对索马甜的检测方法标准,对其检测方法的报道也非常少,仅见利用高效液相色谱-蒸发光检测器针对包括索马甜在内的甜味剂检测方法的研究。
发明内容
本发明要解决的技术问题是提供一种饮料索马甜的检测方法,利用超高效液相色谱法和SDS-PAGE法对饮料中的索马甜进行定性、定量分析,具有准确性高的优点。
为解决上述技术问题,本发明的技术方案为:一种饮料中索马甜的检测方法,其创新点在于:通过超高效液相色谱法或SDS-PAGE法对饮料中索马甜进行分析检测,对于样品中的疑似检测结果,通过超高效液相色谱法和SDS-PAGE法的检测结果进行互相印证,确定样品结果;其中,
所述超高效液相色谱法的检测步骤,具体如下:
(1)样品处理:将饮料摇匀,取2.5mL或对应质量移入到25 mL容量瓶中,用水定容,在4℃下以8000rpm的转速离心10min,取上清液15mL到超滤管中进行超滤处理,轻轻冲洗滤膜,并将截留液定容至3mL,进行色谱分析前用0.45μm滤头过滤;
(2)色谱条件:Agilent 1290超高效液相色谱仪;色谱柱: 300SB-C18,RRHD 1.8μm,100mm×2.1mm(i,d);流动相:乙腈、0.1%三氟乙酸水溶液;流速:0.3mL/min;进样量:20μL;柱温:25℃。梯度洗脱:0-3min,乙腈10%-0.1%三氟乙酸水溶液90%;3-15min,乙腈(10%-100%),0.1%三氟乙酸水溶液(90%-0%);15.1-20min,乙腈(100%);20.1-25min,乙腈10%-0.1%三氟乙酸水溶液90%;检测器:DAD,检测波长278.2nm,同时进行190-400nm波长扫描;
(3)标准曲线的制作:将索马甜分别用水稀释到浓度为10 mg/L、25mg/L、50mg/L、100mg/L、200mg/L、500mg/L系列标准溶液,进行分析测定,以278.2nm下色谱峰面积与相应浓度绘制标准曲线,获得标准曲线回归方程;
(4)样品测定:将处理后的待测样品按照上述(2)所述色谱条件进行分析,以保留时间定性,或借助光谱图进行辅助定性;并根据待测样品278.2nm下峰面积和样品稀释因子计算其含量;
(5)检出限和定量限:将不含索马甜的饮料稀释10倍,加入索马甜标准溶液,使其浓度在10mg/L,按照(1)所述方法进行超滤处理,重复测定20次,以结果标准偏差的3倍计算出方法的检出限,以结果标准偏差的l0倍计算出定量限;
(6)回收率和精密度:分别向不同类型饮料样品中添加25 mg/L、50mg/L、250mg/L不同浓度的索马甜,每个加标样品按照所建立的方法重复测定6次,计算回收率和精密度;
所述SDS-PAGE法的检测步骤,具体如下:
(1)电泳条件:分离胶浓度12.0%,上样量60μL,电泳条件 135V、50min,考马斯亮蓝染色,脱色液脱色;使用蛋白标样作为参照,蛋白标样分子量覆盖20kD~24kD的区间;
(2)样品处理:分别取一定体积的索马甜标准溶液,按体积比1:5 的比例加入5X蛋白上样缓冲液,沸水热处理3~5min;
(3)将索马甜标准品用水稀释到浓度为10mg/L、25mg/L、50 mg/L、100mg/L、200mg/L、500mg/L、1000mg/L系列标准溶液,分别取一定体积的索马甜标准溶液和待测样品,按上述(2)进行处理,按(1)进行SDS-PAGE分析;
(4)通过比较索马甜标准溶液及待测样品中SDS-PAGE图上条带大小、深浅,判定待测样品中索马甜含量范围。
进一步地,所述超高效液相色谱法的检测步骤中,样品处理时,碳酸类饮料在饮料摇匀前,超声处理4~6min。
本发明的优点在于:本发明饮料索马甜的检测方法,基于色谱法和电泳法建立饮料中索马甜的分析测试方法,其中所用色谱法以DAD为检测器较之通用型检测器具有选择性,可以起到一定的定性作用,此外,通过SDS-PAGE与超高效液相色谱法相互印证,对于较为复杂的食品基质,能够获得更为可靠的结果。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1为本发明实施例1中索马甜标准溶液超高效液相色谱图。
图2为本发明实施例1中碳酸饮料加标样品色谱图。
图3为本发明实施例2中茶饮料中索马甜加标色谱图。
图4为本发明实施例3中索马甜标准溶液SDS-PAGE图。
图5为本发明实施例3中加标样品SDS-PAGE图。
其中;图4中的M表示蛋白标样,1~7分别代表浓度为10mg/L、 25mg/L、50mg/L、100mg/L、250mg/L、500mg/L以及1000mg/L的索马甜标准溶液,图5中的M表示蛋白标样,1~7分别代表为不同饮料加标样品。
具体实施方式
下面的实施例可以使本专业的技术人员更全面地理解本发明,但并不因此将本发明限制在所述的实施例范围之中。
实施例1
本实施例提供了利用超高效液相色谱法分析碳酸饮料中索马甜的案例,具体步骤如下:
(1)色谱条件:Agilent 1290超高效液相色谱仪;色谱柱: 300SB-C18,RRHD 1.8μm,100mm×2.1mm(i,d);流动相:乙腈、0.1%三氟乙酸水溶液;流速:0.3mL/min;进样量:20μL;柱温:25℃。梯度洗脱:0-3min,乙腈10%-0.1%三氟乙酸水溶液90%;3-15min,乙腈(10%-100%),0.1%三氟乙酸水溶液(90%-0%);15.1-20min,乙腈(100%);20.1-25min,乙腈10%-0.1%三氟乙酸水溶液90%;检测器:DAD,检测波长278.2nm,同时进行190-400nm波长扫描;
(2)标准曲线的制作:将索马甜分别用水稀释到浓度为10 mg/L、25mg/L、50mg/L、100mg/L、200mg/L、500mg/L系列标准溶液,利用(1)描述的超高效液相色谱方法进行分析测定,以色谱峰面积与相应浓度绘制标准曲线,获得标准曲线回归方程;索马甜标准溶液超高效液相色谱图如图1所示,标准曲线方程为 Y=3.62568X-4704715,所得线性相关系数为0.9983;
(3)样品处理:将加标碳酸饮料样品超声5min除去二氧化碳,摇匀,取2.5mL移入到25mL容量瓶中,用水定容,在4℃下以8000 rpm的转速离心10min,取上清液15mL到超滤管中进行超滤处理,轻轻冲洗滤膜,并将截留液定容至3mL,进行色谱分析前用0.45μm 滤头过滤;利用(1)描述的超高效液相色谱方法进行分析测定,结果如图2所示;计算其含量为204.6mg/L,回收率102.3%。
实施例2
本实施例提供了利用超高效液相色谱法分析茶饮料中索马甜的案例,具体步骤如下:
(1)色谱条件:Agilent 1290超高效液相色谱仪;色谱柱: 300SB-C18,RRHD 1.8μm,100mm×2.1mm(i,d);流动相:乙腈、0.1%三氟乙酸水溶液;流速:0.3mL/min;进样量:20μL;柱温:25℃。梯度洗脱:0-3min,乙腈10%-0.1%三氟乙酸水溶液90%;3-15min,乙腈(10%-100%),0.1%三氟乙酸水溶液(90%-0%);15.1-20min,乙腈(100%);20.1-25min,乙腈10%-0.1%三氟乙酸水溶液90%;检测器:DAD,检测波长278.2nm,同时进行190-400nm波长扫描;
(2)标准曲线的制作:将索马甜分别用水稀释到浓度为10 mg/L、25mg/L、50mg/L、100mg/L、200mg/L、500mg/L系列标准溶液,利用(1)描述的超高效液相色谱方法进行分析测定,以色谱峰面积与相应浓度绘制标准曲线,获得标准曲线回归方程;索马甜标准溶液超高效液相色谱图如图1所示,标准曲线方程为 Y=3.62568X-4704715,所得线性相关系数为0.9983;
(3)样品处理:将加标茶饮料样摇匀,取2.5mL移入到25mL 容量瓶中,用水定容,在4℃下以8000rpm的转速离心10min,取上清液15mL到超滤管中进行超滤处理,轻轻冲洗滤膜,并将截留液定容至3mL,进行色谱分析前用0.45μm滤头过滤;利用(1)描述的超高效液相色谱方法进行分析测定,结果如图3所示;计算其含量为 46.9mg/L,回收率93.8%。
实施例3
本实施例提供了利用SDS-PAGE法分析索马甜的案例,具体步骤如下:
(1)样品处理:将索马甜标准品用水稀释到浓度为10mg/L、25 mg/L、50mg/L、100mg/L、200mg/L、500mg/L、1000mg/L系列标准溶液,分别取80μL的索马甜标准溶液和加标待测样品,加入20μ L 5X蛋白上样缓冲液,沸水热处理5min;
(2)SDS-PAGE电泳:采用浓度12.0%的分离胶进行电泳,索马甜标准溶液及待测样品上样量60μL,电泳条件135V、50min,考马斯亮蓝染色,脱色液脱色;使用蛋白标样作为参照,蛋白标样分子量范围为14.4kD~98kD,上样量10μL;其中,索马甜标准溶液SDS-PAGE结果如图4所示,加标样品SDS-PAGE结果如图5所示;由图中可以看出,含量为10mg/L的索马甜在SDS-PAGE中显示微弱条带,含量达到25mg/L条带已较为清晰明显,能够满足GB 2760-2014中最大允许使用量(0.025g/kg)的分析要求。
以上显示和描述了本发明的基本原理和主要特征以及本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (2)
1.一种饮料中索马甜的检测方法,其特征在于:通过超高效液相色谱法或SDS-PAGE法对饮料中索马甜进行分析检测,对于样品中的疑似检测结果,通过超高效液相色谱法和SDS-PAGE法的检测结果进行互相印证,确定样品结果;其中,
所述超高效液相色谱法的检测步骤,具体如下:
(1)样品处理:将饮料摇匀,取2.5mL或对应质量移入到25mL容量瓶中,用水定容,在4℃下以8000rpm的转速离心10min,取上清液15mL到超滤管中进行超滤处理,轻轻冲洗滤膜,并将截留液定容至3mL,进行色谱分析前用0.45μm滤头过滤;
(2)色谱条件:Agilent 1290超高效液相色谱仪;色谱柱:300SB-C18,RRHD 1.8μm,100mm×2.1mm(i,d);流动相:乙腈、0.1%三氟乙酸水溶液;流速:0.3mL/min;进样量:20μL;柱温:25℃。梯度洗脱:0-3min,乙腈10%-0.1%三氟乙酸水溶液90%;3-15min,乙腈(10%-100%),0.1%三氟乙酸水溶液(90%-0%);15.1-20min,乙腈(100%);20.1-25min,乙腈10%-0.1%三氟乙酸水溶液90%;检测器:DAD,检测波长278.2nm,同时进行190-400nm波长扫描;
(3)标准曲线的制作:将索马甜分别用水稀释到浓度为10mg/L、25mg/L、50mg/L、100mg/L、200mg/L、500mg/L系列标准溶液,进行分析测定,以278.2nm下色谱峰面积与相应浓度绘制标准曲线,获得标准曲线回归方程;
(4)样品测定:将处理后的待测样品按照上述(2)所述色谱条件进行分析,以保留时间定性,或借助光谱图进行辅助定性;并根据待测样品278.2nm下峰面积和样品稀释因子计算其含量;
(5)检出限和定量限:将不含索马甜的饮料稀释10倍,加入索马甜标准溶液,使其浓度在10mg/L,按照(1)所述方法进行超滤处理,重复测定20次,以结果标准偏差的3倍计算出方法的检出限,以结果标准偏差的l0倍计算出定量限;
(6)回收率和精密度:分别向不同类型饮料样品中添加25mg/L、50mg/L、250mg/L不同浓度的索马甜,每个加标样品按照所建立的方法重复测定6次,计算回收率和精密度;
所述SDS-PAGE法的检测步骤,具体如下:
(1)电泳条件:分离胶浓度12.0%,上样量60μL,电泳条件135V、50min,考马斯亮蓝染色,脱色液脱色;使用蛋白标样作为参照,蛋白标样分子量覆盖20kD~24kD的区间;
(2)样品处理:分别取一定体积的索马甜标准溶液,按体积比1:5的比例加入5X蛋白上样缓冲液,沸水热处理3~5min;
(3)将索马甜标准品用水稀释到浓度为10mg/L、25mg/L、50mg/L、100mg/L、200mg/L、500mg/L、1000mg/L系列标准溶液,分别取一定体积的索马甜标准溶液和待测样品,按上述(2)进行处理,按(1)进行SDS-PAGE分析;
(4)通过比较索马甜标准溶液及待测样品中SDS-PAGE图上条带大小、深浅,判定待测样品中索马甜含量范围。
2.根据权利要求1所述的饮料中索马甜的检测方法,其特征在于:所述超高效液相色谱法的检测步骤中,样品处理时,碳酸类饮料在饮料摇匀前,超声处理4~6min。
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