CN111638279A - 一种测定余甘子氨基酸组成及含量的方法 - Google Patents
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Abstract
本发明公开了一种测定余甘子氨基酸组成及含量的方法,包括如下步骤:精确称取余甘子果实干粉100mg‑200mg,将样品送至聚四氟乙烯微波水解罐底部,样品水解盐酸的添加样品中精确加入6mol/L HCl 8ml,轻轻摇匀,静置,吹入氮气,约20‑30s,氮气压力宜在0.1左右,置于常规水解管中,加入6mol/L HCl 8ml,摇匀静置,同样吹入氮气,封管,置于恒温烘箱中110℃条件下水解22h‑24h;按照设定好的微波水解程序进行样品水解,消解完毕后,冷却至室温,混匀,开管,定容至50ml,0.45μm滤膜过滤;吸取水解液0.5ml置于5ml EP管中,温度60℃,置于氮吹仪插孔中赶酸,距离液面0.5cm,加入1‑2ml样品稀释液pH为2.2,使氨基酸浓度为50‑200nmol/L为宜,震荡混匀,用0.22μm滤膜过滤,供上机测定;本发明提高了余甘子氨基酸测定的精度和效率。
Description
技术领域
本发明属于食品检测技术领域,具体涉及到一种测定余甘子氨基酸组成及含量的方法。
背景技术
余甘子(PhyllanthusemblicaL.),俗称滇橄榄,大戟科叶下珠属,是一种十分独特的分布于干热河谷地区的资源植物。原产印度、巴基斯坦等地。目前为中国和印度分布面积最大,产量最高。我国余甘子资源相当丰富,大面积栽培以福建、广西较多,野生资源以云南最为丰富。余甘子营养丰富,风味独特,药食两用,含有多种对人体有益的微量元素,富含维生素、酚类、黄酮、多糖和氨基酸等化学物质,具有抗氧化、抗炎、抗肿瘤、抗病毒、抗衰老、降血压、降糖、降脂等药理保健作用,开发利用价值极高。
据研究,地球上近150万种动物、30万种植株和20万种微生物本质只有一个,那就是“生命是蛋白质的存在方式,这种存在方式的实质就是这些蛋白体化学成分的不断自我更新”。而蛋白体的化学成分就是组成蛋白的基本单元--氨基酸,而“不断自我更新”就是氨基酸不断的重新排列、组合。因此,对氨基酸组成及含量的分析检测具有重要意义。
相比于国外,我国余甘子氨基酸的开发和利用研究却相对较少,因此,余甘子氨基酸组成及含量的研究具有较好应用前景。目前,有关余甘子氨基酸分析的研究不多,大部分参照国标(GB/T5009.124—2003)测定食品中氨基酸含量,采用恒温烘箱进行常规酸水解,水解过程复杂,试剂用量大,控温不准确,费时费力,水解不彻底,安全隐患大。但是,目前没有利用微波水解进行样品前处理,然后用氨基酸分析仪测定余甘子氨基酸的一整套方法,且现有方法测定的余甘子氨基酸精确度和精密度均较差,重复性不高。
发明内容
本发明的目的在于样品预处理过程复杂、样品水解不彻底、试剂用量大、控温不准确、水解过程费时费力、存在安全隐患、环境污染的问题,提供一种适宜的高效精确测定余甘子氨基酸的方法,提高氨基酸测定的精度和效率。
为达到上述目的,本发明采用了下列技术方案:
一种测定余甘子氨基酸组成及含量的方法,包括如下步骤:
1、余甘子果实干粉的制备
选取健康完好的余甘子果实,洗净,去核,烘干,进行粉碎,过筛,得到余甘子果实干粉;
2、称取样品
精确称取所述的余甘子果实干粉100mg-200mg,采用297mm×40mm大小的纸槽将样品送至聚四氟乙烯微波水解罐底部,样品称量太小,氨基酸峰面积太小;样品称量太大,氨基酸浓度太高,出峰易秃顶;
3、样品水解盐酸的添加
上述样品中精确加入6mol/L HCl 8ml,轻轻摇匀,静置,吹入氮气,约20-30s,氮气压力宜在0.1左右,以免吹出液体,拧紧消解罐,确保完全密封。同时,将上述样品置于常规水解管中,加入6mol/L HCl 8ml,轻轻摇匀,静置,同样吹入氮气,封管,置于恒温烘箱中110℃条件下水解22h-24h;
4、微波水解程序设定
按照设定好的微波水解程序进行样品水解,消解完毕后,冷却至室温,混匀,开管,定容至50ml,0.45μm滤膜过滤;
5、水解液赶酸
吸取水解液0.5ml置于5mlEP管中,温度60℃,置于氮吹仪插孔中赶酸,氮气压力不易过大,距离液面0.5cm,以不吹出液体为宜。蒸发至干,必要时加少许去离子水,重复蒸干1-2次;
6、上机样品制备
上述样品中加入1-2ml样品稀释液pH为2.2,使氨基酸浓度为50-200nmol/L为宜,震荡混匀,用0.22μm滤膜过滤,供上机测定;
7、标准曲线的制作
将17种氨基酸标样,即2.5umol/L,胱氨酸减半,稀释25倍至100nmol/L供上机测定,分别进样5ul、10ul、20ul、50ul,制作标准曲线。检测条件为色谱柱:LCAK06/Na;柱温:58℃-74℃梯度控温;洗脱泵0.45ml/min,衍生泵0.25ml/min;检测波长:570nm+440nm;反应器温度130℃;45分钟标准水解时间;
8、样品检测
将上述样品依次进样50ul,测定峰面积,根据标准曲线计算余甘子干粉中氨基酸组成及含量;
9、加标回收实验
取已知含量的余甘子果实干粉样品,精密加入一定量的混合氨基酸标准品溶液,测定其回收率;
10、重复性实验
分别吸取样品1、2、3各50ul,利用氨基酸分析仪进行分析,计算测定结果。
有益效果:与现有技术相比,本发明具有如下优点:
1、样品预处理过程简单;2、样品水解彻底;3、节约试剂用量;4、控温准确;5、水解过程安全省时,提高效率;6、操作安全,环境污染小。
具体实施方式
为了便于本领域技术人员的理解,下面结合实施例对本发明作进一步的说明,实施方式提及的内容并非对本发明的限定。
实施例
本实施例提供了一种测定余甘子氨基酸组成及含量的方法,其特征在于,包括如下步骤:
1、余甘子果实干粉的制备
选取健康完好的余甘子果实,洗净,去核,烘干,进行粉碎,过筛,得到余甘子果实干粉;
2、称取样品
精确称取所述的余甘子果实干粉100mg-200mg,采用297mm×40mm大小的纸槽将样品送至聚四氟乙烯微波水解罐底部,样品称量太小,氨基酸峰面积太小;样品称量太大,氨基酸浓度太高,出峰易秃顶;
3、样品水解盐酸的添加
上述样品中精确加入6mol/L HCl 8ml,轻轻摇匀,静置,吹入氮气,约20-30s,氮气压力宜在0.1左右,以免吹出液体,拧紧消解罐,确保完全密封。同时,将上述样品置于常规水解管中,加入6mol/L HCl 8ml,轻轻摇匀,静置,同样吹入氮气,封管,置于恒温烘箱中110℃条件下水解22h-24h;
4、微波水解程序设定
按照设定好的微波水解程序进行样品水解,消解完毕后,冷却至室温,混匀,开管,定容至50ml,0.45μm滤膜过滤;
微波水解程序的初始设定如表1所示,
表1微波水解程序
最大功率/W | 爬升时间/min | 温度/℃ | 保持时间/min |
1800 | 15 | 160 | 30 |
1800 | 冷却20min | — | — |
5、水解液赶酸
吸取水解液0.5ml置于5mlEP管中,温度60℃,置于氮吹仪插孔中赶酸,氮气压力不易过大,距离液面0.5cm,以不吹出液体为宜。蒸发至干,必要时加少许去离子水,重复蒸干1-2次;
6、上机样品制备
上述样品中加入1-2ml样品稀释液pH为2.2,使氨基酸浓度为50-200nmol/L为宜,震荡混匀,用0.22μm滤膜过滤,供上机测定;
7、标准曲线的制作
将17种氨基酸标样,即2.5umol/L,胱氨酸减半,稀释25倍至100nmol/L供上机测定,分别进样5ul、10ul、20ul、50ul,制作标准曲线。
检测条件为色谱柱:
LCAK06/Na;柱温:
58℃-74℃梯度控温;
洗脱泵0.45ml/min,
衍生泵0.25ml/min;
检测波长:570nm+440nm;
反应器温度130℃;
45分钟标准水解时间;
8、样品检测
将上述样品依次进样50ul,测定峰面积,根据标准曲线计算余甘子干粉中氨基酸组成及含量;见表2所示。
9、加标回收实验
取已知含量的余甘子果实干粉样品,精密加入一定量的混合氨基酸标准品溶液,测定其回收率;
10、重复性实验
分别吸取样品1、2、3各50ul,利用氨基酸分析仪进行分析,计算测定结果,见表3。
表2氨基酸含量检测结果
氨基酸 | 酸水解/% | 微波水解/% | 回收率/% |
天门冬氨酸Asp | 0.22 | 0.23 | 101.5 |
苏氨酸Thr | 0.07 | 0.07 | 99.5 |
丝氨酸Ser | 0.09 | 0.09 | 105.3 |
谷氨酸Glu | 1.02 | 1.03 | 100.8 |
脯氨酸Pro | 0.29 | 0.30 | 104.3 |
甘氨酸Gly | 0.08 | 0.10 | 99.6 |
丙氨酸Ala | 0.15 | 0.16 | 97.5 |
胱氨酸Cys | 0.01 | 0.01 | 97.6 |
缬氨酸Val | 0.14 | 0.14 | 95.6 |
甲硫氨酸Met | - | 0.01 | 90.5 |
异亮氨酸Ile | 0.07 | 0.07 | 101.4 |
亮氨酸Leu | 0.12 | 0.12 | 98.5 |
酪氨酸Tyr | 0.06 | 0.06 | 105.6 |
苯丙氨酸Phe | 0.07 | 0.08 | 110.5 |
组氨酸His | 0.23 | 0.23 | 106.3 |
赖氨酸Lys | 0.10 | 0.10 | 102.3 |
精氨酸Arg | 0.13 | 0.14 | 99.9 |
总含量 | 2.85 | 2.93 |
表3不同水解方法重复性实验结果比较
样品序号 | 微波水解法/% | 常规水解法/% |
1 | 2.93 | 2.85 |
2 | 2.98 | 2.89 |
3 | 2.85 | 2.75 |
通过表3对比所示,微波水解法的实验结果显示:样品预处理过程简单;样品水解彻底;节约试剂用量控温准确;水解过程安全省时可提高效率,并且操作安全,环境污染小。
本文中所描述的具体实施例仅仅是对本发明精神作举例说明。本发明所属技术领域的技术人员可以对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代,但并不会偏离本发明的精神或者超越所附权利要求书所定义的范围。
Claims (1)
1.一种测定余甘子氨基酸组成及含量的方法,其特征在于,包括如下步骤:
1、余甘子果实干粉的制备
选取健康完好的余甘子果实,洗净,去核,烘干,进行粉碎,过筛,得到余甘子果实干粉;
2、称取样品
精确称取所述的余甘子果实干粉100mg-200mg,采用297mm×40mm大小的纸槽将样品送至聚四氟乙烯微波水解罐底部,
3、样品水解盐酸的添加
上述样品中精确加入6mol/L HCl 8ml,轻轻摇匀,静置,吹入氮气,约20-30s,氮气压力宜在0.1左右,以免吹出液体,拧紧消解罐,确保完全密封,同时将上述样品置于常规水解管中,加入6mol/L HCl 8ml,轻轻摇匀,静置,同样吹入氮气,封管,置于恒温烘箱中110℃条件下水解22h-24h;
4、微波水解程序设定
按照设定好的微波水解程序进行样品水解,消解完毕后,冷却至室温,混匀,开管,定容至50ml,0.45μm滤膜过滤;
5、水解液赶酸
吸取水解液0.5ml置于5mlEP管中,温度60℃,置于氮吹仪插孔中赶酸,距离液面0.5cm,以不吹出液体为宜,蒸发至干,必要时加少许去离子水,重复蒸干1-2次;
6、上机样品制备
上述样品中加入1-2ml样品稀释液pH为2.2,使氨基酸浓度为50-200nmol/L为宜,震荡混匀,用0.22μm滤膜过滤,供上机测定;
7、标准曲线的制作
将17种氨基酸标样,即2.5umol/L,胱氨酸减半,稀释25倍至100nmol/L供上机测定,分别进样5ul、10ul、20ul、50ul,制作标准曲线;
8、样品检测
将上述样品依次进样50ul,测定峰面积,根据标准曲线计算余甘子干粉中氨基酸组成及含量;
9、加标回收实验
取已知含量的余甘子果实干粉样品,精密加入一定量的混合氨基酸标准品溶液,测定其回收率;
10、重复性实验
分别吸取样品1、2、3各50ul,利用氨基酸分析仪进行分析,计算测定结果。
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