CN111735891A - 家禽源的特征性胶原肽及在胶原水解物和其制品检测中的应用 - Google Patents

家禽源的特征性胶原肽及在胶原水解物和其制品检测中的应用 Download PDF

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CN111735891A
CN111735891A CN202010329408.9A CN202010329408A CN111735891A CN 111735891 A CN111735891 A CN 111735891A CN 202010329408 A CN202010329408 A CN 202010329408A CN 111735891 A CN111735891 A CN 111735891A
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poultry
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collagen peptide
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黄雅钦
郭尚伟
邓贵雅
刘秀芳
蒋伟
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Foshan Win Cosmetic Co ltd
Beijing University of Chemical Technology
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Beijing University of Chemical Technology
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Abstract

家禽源的特征性胶原肽及在胶原水解物和其制品检测中的应用，属于检测领域。特征性胶原肽氨基酸序列Gly‑Leu‑Val‑Gly‑Glu‑Hyp‑Gly‑Pro‑Ala‑Gly‑Ala‑Lys、Gly‑Glu‑Gly‑Gly‑Pro‑Ala‑Gly‑Pro‑Ala‑Gly‑Pro‑Ala‑Gly‑Ala‑Arg和Val‑Gly‑Pro‑Ile‑Gly‑Pro‑Ala‑Gly‑Asn‑Arg，通过检测一条或多条特征性胶原肽，确定是否含有家禽源性成分。通过对样品中的蛋白成分进行胰酶酶切，使特征性胶原肽游离出来，通过液质联用仪检测。该方法特征性强，灵敏度高，可对胶原水解物及其制品中的家禽源性成分进行测定。

Description

家禽源的特征性胶原肽及在胶原水解物和其制品检测中的 应用
技术领域
本发明涉及3个家禽源的特征性胶原肽及在胶原水解物和其制品检测中的应用，属于医药与食品检测领域。
背景技术
胶原在动物体内含量非常丰富，主要存在于在动物的骨、皮、筋和腱等结缔组织中。明胶是胶原的降解物，是由动物的骨、皮、筋和腱等为原料经适度水解而制得，具有优良的理化性能，广泛应用于食品、医药等诸多领域。明胶绝大部分是由猪、牛和羊的皮或骨为原材料，但由于家禽和牲畜的疾病可能带来的安全风险、清真和宗教信仰不同，对明胶进行动物源性成分溯源具有重要意义。
明胶中主要成分为胶原蛋白多肽，可利用液质联用仪检测多肽来进行动物源性成分的溯源，然而由于多肽在同一物种中存在突变等现象，选定的多肽能否作为溯源的特征性胶原肽，关系到溯源的成功与否。而对家禽源性成分溯源的特征性胶原肽的选择，不但要克服基因突变带来的影响，而且还需在鸡、鸭、鹅等家禽中含有，而其他动物中不含有。选择家禽中不突变的特征性胶原肽，成为家禽源性成分溯源的关键。直接采用液质联用仪对样品进行检测分析，无法克服上述困难。从基因入手，分析寻找特征性胶原肽，是一条可行的路径。
发明内容
本发明针对上述问题，提供了家禽源的特征性胶原肽及在胶原水解物和其制品中家禽源性成分的检测方法，该方法操作简单，特征性强，灵敏度高，可用于胶原水解物及其制品中家禽源性成分的溯源鉴定和含量测定。
本发明的技术方案如下：
家禽源性成分的特征性胶原肽及在胶原水解物和其制品检测中的应用，所述的胶原水解物包括明胶，基于家禽的胶原蛋白序列中包含特征性胶原肽，所述特征性胶原肽其氨基酸序列为Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys或/和Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg或/和Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg，其中Hyp为4羟基脯氨酸或3羟基脯氨酸。它是从牛、猪、羊、鸡、鸭、鹅中COL1A2蛋白的mRNA出发，找出鸡、鸭、鹅家禽的稳定存在的特征mRNA序列，翻译成蛋白序列后利用液质联用技术检测，最终选定的特征性胶原肽。
该特征性胶原肽在胶原水解物及其制品检测中的应用，包括以下步骤：
(1)待检测样品用胰蛋白酶进行酶切，或提取待检测样品中的蛋白成分用胰蛋白酶进行酶切；
(2)放入液质联用仪，并以特征性胶原肽或包含所述特征性胶原肽的胰蛋白酶酶切后的鸡、鸭、鹅明胶中的一种或几种明胶作为对照品，选择特征性胶原肽的母离子及其子离子进行监测；如果检出该离子的保留时间与对照品一致，且其子离子与对照品的子离子一致，则所述胶原水解物或其制品含有家禽源性成分，家禽源性成分含量可由对照品与检测样品中的峰面积来计算；如果没有与对照品保留时间一致的该离子，则不含有家禽源性成分。
上述在明胶是由动物的皮、骨或鳞为原料经水解所制得的明胶，明胶制品是原料中含有明胶的食品、保健品或药品。
所述的家禽选自鸡、鸭、鹅中的一种两种或三种。
采用本发明的方法，能够快速检测胶原水解物及其制品中是否含有家禽源性成分。本发明从基因角度寻找对应的肽，尽管动物中存在基因突变导致蛋白序列差异，但本发明的特征性胶原肽的基因在家禽中保守性强，且为家禽特有，通过该特征性胶原肽，可对胶原水解物及其制品中家禽源性成分进行溯源检测。
本发明可通过从基因角度提供一种或几种特征性胶原肽，检测其中一条或多条特征性胶原肽，来确定是否含有家禽源性成分。具体地，通过对样品中的蛋白成分进行胰酶酶切，使特征性胶原肽游离出来，然后通过液质联用仪检测是否含有该特征性胶原肽，来判定是否含有家禽源性成分。该方法具有特征性强，准确度高，灵敏度高，操作简单，可对胶原水解物及其制品中的家禽源性成分进行测定。
附图说明
图1是鸭明胶中特征性胶原肽Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys的子离子扫描质谱图(母离子m/z535.1，子离子扫描范围m/z200～1200)；
图2是鸭明胶中特征性胶原肽Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg的子离子扫描质谱图(母离子m/z611.6，子离子扫描范围m/z200～1300)；
图3是鸭明胶中特征性胶原肽Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg的子离子扫描质谱图(母离子m/z469.5，子离子扫描范围m/z200～1000)；
图4是以不同含量鸡明胶对应的特征性胶原肽的质谱检测峰面积(m/z535.1→613.5)的关系曲线图；
图5是鸡明胶及明胶制品(以QQ糖为例)在SRM扫描模式下选择离子对m/z535.1→613.5、800.3监测的质谱图。
图6是鸡、鸭、鹅、牛、猪、羊的COL1A2蛋白的部分mRNA序列及对应的比较。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1
1材料和试剂
材料：不同动物来源的明胶，分别来源于鸡、鸭、鹅、牛、猪、羊，分别从鸡皮、鸭皮、鹅皮、牛皮、猪皮、羊皮提取获得。
试剂：碳酸氢铵(分析纯)、胰蛋白酶(序列纯，购自中国药品生物制品检定研究院)。
2特征性胶原肽的筛选与确定
鸡、鸭、鹅等动物的胶原产品主要是Ⅰ型胶原，包括COL1A1和COL1A2蛋白，并且具有一定的保守性，我们可以从COL1A2蛋白中筛选出一段肽作为家禽源的特征性胶原肽。为了从基因中筛选出用于检测的稳定的特征性胶原肽，应满足以下条件：(a)与该段肽所对应mRNA序列相邻的三个碱基，翻译后应为精氨酸或赖氨酸，且该段肽所对应mRNA序列中第1～3个碱基，翻译后不应为脯氨酸；(b)该段肽所对应的mRNA序列中，碱基C、G的含量应大于60％；(c)该段肽上至少有一个氨基酸对应的mRNA符合以下任一条件：该氨基酸对应的密码子的三个碱基中，若第一个碱基若为C或G，则相应地在牛、猪或羊中第一个碱基应为G或C；若第二个碱基为A或T，则相应地在牛、猪或羊中第二个碱基应为T或A；若第二个碱基为C或T，则相应地在牛、猪或羊中第二个碱基应为T或C；(d)利用质谱检测筛选出来的该段肽是否有翻译后修饰(即：是否有脯氨酸发生羟基化)，如果没有翻译后修饰则筛选出来的该段肽即为特征性胶原肽；如果有翻译后修饰则翻译后修饰的多肽即为特征性胶原肽；(e)通过质谱检测应没有其他干扰信号。为此我们进行了如下实验：
(1)特征性胶原肽的初筛
a)特征性胶原肽Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys的初筛
将鸡、鸭、鹅、牛、猪、羊的COL1A2蛋白对应的mRNA序列进行比对，部分序列如图6所示(注：mRNA由细胞核内的DNA转录来的，有时候也用DNA链上的碱基顺序来表示mRNA上的序列)。其中图6(a)线框中鸡、鸭、鹅的COL1A2蛋白的mRNA中均含有“GGA CTT GTT GGT GAACCA GGC CCT GCT GGT GCC AAG”，该序列满足上述条件(a)、(b)、(c)。该序列翻译后，对应的多肽为“Gly-Leu-Val-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Lys”。为确定多肽Gly-Leu-Val-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Lys是否为家禽(鸡、鸭、鹅)的特征性胶原肽，需进一步确定是否有翻译后修饰(即：是否有脯氨酸发生羟基化)。
b)特征性胶原肽Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg的初筛。
将鸡、鸭、鹅、牛、猪、羊的COL1A2蛋白对应的mRNA序列进行比对，部分序列如图6所示(注：mRNA由细胞核内的DNA转录来的，有时候也用DNA链上的碱基顺序来表示mRNA上的序列)。其中图6(c)线框中鸡的mRNA中含有“GGT GAA GGA GGT CCT GCT GGT CCC GCT GGTCCT GCT GGT GCC CGT”；鸭、鹅中含有“GGT GAA GGA GGT CCT GCT GGT CCT GCC GGT CCTGCT GGT GCC CGT”，两个序列满足上述条件(a)、(b)、(c)。该两个序列翻译后，对应的多肽均为“Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg”。为确定多肽Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg是否为家禽(鸡、鸭、鹅)的特征性胶原肽，需进一步确定是否有翻译后修饰(即：是否有脯氨酸发生羟基化)。
c)特征性胶原肽Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg的初筛将鸡、鸭、鹅、牛、猪、羊的COL1A2蛋白对应的mRNA序列进行比对，部分序列如图6所示(注：mRNA由细胞核内的DNA转录来的，有时候也用DNA链上的碱基顺序来表示mRNA上的序列)。其中图6(b)线框中鸡的COL1A2蛋白的mRNA中含有“GTT GGG CCA ATC GGT CCA GCT GGT AAT AGA”，鸭的含有“GTT GGA CCA ATT GGT CCA GCT GGC AAC AGA”，鹅的含有“GTT GGA CCA ATT GGTCCA GCT GGC AAT AGA”，三个序列满足上述条件(a)、(b)、(c)。该三个序列翻译后，对应的多肽为“Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg”。为确定多肽Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg是否为家禽(鸡、鸭、鹅)的特征性胶原肽，需进一步确定是否有翻译后修饰(即：是否有脯氨酸发生羟基化)，因此进行了质谱检测试验来确定是否有翻译后修饰。
(2)特征性胶原肽的确定(质谱检测试验)
a)取0.05g明胶样品于25ml量瓶中，加少量1％NH4HCO3溶液浸泡10min，60℃加热使其溶解，用1％NH4HCO3溶液稀释至刻度，摇匀，微孔滤膜过滤，精密量取续滤液0.2ml加入2mg/ml的胰蛋白酶溶液20μl，37℃恒温酶解12h。
b)分别取5μl的鸡、鸭、鹅的明胶的酶解溶液放入液质联用仪，检测多肽是否有羟基化修饰。液相条件：C18反相色谱柱(2.1mm×100mm，1.8μm)，流动相A为0.1％甲酸溶液，流动相B为乙腈，流速0.3ml/min；梯度洗脱：0～25min，98％→80％流动相A，2％→20％流动相B。质谱条件：电喷雾正离子模式(ESI+)，对于多肽Gly-Leu-Val-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Lys分别选择m/z527.1和m/z535.1为母离子进行子离子全扫描，对于Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg分别选择m/z611.6和m/z619.6为母离子进行子离子全扫描，对于Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg分别选择m/z469.5和m/z477.5为母离子进行子离子全扫描。结果：以m/z535.1为母离子的子离子全扫描的质谱图与多肽Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys相符(其中鸭明胶的见图1)，表明此特征性胶原肽中有一个氨基酸发生了羟基化修饰；以m/z611.6为母离子的子离子全扫描的质谱图与多肽Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg相符(其中鸭明胶的见图2)，表明此特征性胶原肽中没有氨基酸被羟基化修饰；以m/z469.5为母离子的子离子全扫描的质谱图与多肽Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg相符(其中鸭明胶的见图3)，表明此特征性胶原肽中没有氨基酸被羟基化修饰。
c)取5μl各明胶样品的酶解溶液放入液质联用仪检测。液相条件：C18反相色谱柱(2.1mm×100mm，1.8μm)，流动相A为0.1％甲酸溶液，流动相B为乙腈，流速0.3ml/min；梯度洗脱：0～25min，98％→80％流动相A，2％→20％流动相B。质谱条件：电喷雾正离子模式(ESI+)选择进行SRM检测，分别选择m/z535.1→613.5、800.3，m/z611.6→696.3、753.2，m/z469.5→571.5、684.5作为检测离子对。结果：只有鸡、鸭、鹅的明胶中检出相应的离子峰，其他均没有检出。
综上，多肽Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys、Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg和Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg可作为家禽源性成分的特征性胶原肽。
实施例2
1材料和试剂
材料：混合明胶样品(包括：含5％鸡明胶的猪明胶、10％鸡明胶的猪明胶、20％鸡明胶的猪明胶、40％鸡明胶的猪明胶、80％鸡明胶的猪明胶，纯鸡明胶)由实施例1中的明胶精确混合制成。
试剂：碳酸氢铵(分析纯)、胰蛋白酶(序列纯，购自中国药品生物制品检定研究院)。
2检测方法
(1)取0.05g明胶样品于25ml量瓶中，加少量1％NH4HCO3溶液浸泡10min，60℃加热使其溶解，用1％NH4HCO3溶液稀释至刻度，摇匀，微孔滤膜过滤，精密量取续滤液0.2ml加入2mg/ml的胰蛋白酶溶液20μl，37℃恒温酶解12h。
(2)取5μl酶解溶液放入液质联用仪检测。液相条件：C18反相色谱柱(2.1mm×100mm，1.8μm)，流动相A为0.1％甲酸溶液，流动相B为乙腈，流速0.3ml/min；梯度洗脱：0～25min，98％→80％流动相A，2％→20％流动相B。质谱条件：电喷雾正离子模式(ESI+)选择进行SRM检测，选择m/z535.1→613.5、800.3作为检测离子对。
以鸡明胶的浓度为横坐标(X)，以峰面积(m/z535.1→613.5)为纵坐标(Y)，进行曲线绘制。结果见图4，标准曲线Y＝585507X-17438，线性相关系数R2＝0.9946，线性良好。可见该法可用于鸡皮源性成分的含量检测。
实施例3
1材料和试剂
材料：鸡明胶、加入牛明胶的QQ糖(进行加入自制)、加入鸡明胶的QQ糖(进行加入自制)；
试剂：碳酸氢铵(分析纯)、胰蛋白酶(序列纯，购自中国药品生物制品检定研究院)。
2检测方法
(1)取0.05～0.25g的鸡明胶、加入牛明胶的QQ糖、加入鸡明胶的QQ糖，分别置于25ml量瓶中，加少量1％NH4HCO3溶液浸泡10min，60℃加热使其溶解，用1％NH4HCO3溶液稀释至刻度，摇匀，微孔滤膜过滤，精密量取续滤液0.2ml加入2mg/ml的胰蛋白酶溶液20μl，37℃恒温酶解12h。
(2)取5μl酶解溶液放入液质联用仪检测。液相条件：C18反相色谱柱(2.1mm×100mm，1.8μm)，流动相A为0.1％甲酸溶液，流动相B为乙腈，流速0.3ml/min；梯度洗脱：0～25min，98％→80％流动相A，2％→20％流动相B。质谱条件：电喷雾正离子模式(ESI+)选择进行SRM检测，选择m/z535.1→613.5、800.3作为检测离子对。
结果见图5，10.6min处鸡明胶和加入鸡明胶的QQ糖检出相应的离子峰，加入牛明胶的QQ糖没有检出，可见该法能够特异性检测明胶制品中的家禽源性成分。
上述实施例2和3替换为鸭、鹅，可得到同样的效果。
序列表
<110>北京化工大学
<120>家禽源的特征性胶原肽及在胶原水解物和其制品检测中的应用
<160>3
<210>1
<211>12
<212>PRT
<400>1
Gly Leu Val Gly Glu Hyp Gly Pro Ala Gly Ala Lys.
1 5 10
<210>2
<211>15
<212>PRT
<400>2
Gly Glu Gly Gly Pro Ala Gly Pro Ala Gly Pro Ala Gly Ala Arg.
1 5 10 15
<210>3
<211>15
<212>PRT
<400>3
Val Gly Pro Ile Gly Pro Ala Gly Asn Arg.
1 5 10

Claims (6)

1.家禽源的特征性胶原肽，其特征在于，所述特征性胶原肽氨基酸序列为Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys或/和Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg或/和Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg，其中Hyp为4羟基脯氨酸或3羟基脯氨酸。
2.权利要求1所述的家禽源性成分的特征性胶原肽，其特征在于，特征性胶原肽是从牛、猪、羊、鸡、鸭、鹅中COL1A2蛋白的mRNA出发，找出鸡、鸭、鹅家禽的稳定存在的特征mRNA序列，翻译成蛋白序列后利用液质联用技术检测，最终选定的特征性胶原肽。
3.权利要求1所述的家禽源性成分的特征性胶原肽的应用，用于家禽源性成分胶原水解物和其制品的检测，用于溯源鉴定和含量测定。
4.权利要求3所述的应用，其特征在于，具体方法包括以下步骤：
(1)待检测样品用胰蛋白酶进行酶切，或提取待检测样品中的蛋白成分用胰蛋白酶进行酶切；
(2)放入液质联用仪，并以特征性胶原肽或包含所述特征性胶原肽的胰蛋白酶酶切后的鸡、鸭、鹅明胶中的一种或几种明胶作为对照品，选择特征性胶原肽的母离子及其子离子进行监测；如果检出该离子的保留时间与对照品一致，且其子离子与对照品的子离子一致，则所述胶原水解物或其制品含有家禽源性成分，家禽源性成分含量可由对照品与检测样品中的峰面积来计算；如果没有与对照品保留时间一致的该离子，则不含有家禽源性成分。
5.权利要求3所述的应用，明胶是由动物的皮、骨或鳞为原料经水解所制得的明胶，明胶制品是原料中含有明胶的食品、保健品或药品。
6.权利要求3所述的应用，所述的家禽选自鸡、鸭、鹅中的一种、两种或三种。
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