食品科学 ›› 2017, Vol. 38 ›› Issue (11): 122-127.doi: 10.7506/spkx1002-6630-201711020

• 基础研究 • 上一篇    下一篇

棉籽抗菌活性肽的分离纯化及鉴定

韩晓燕,包郁明,辛凤姣,黄 颖,Christophe BLECKER,戴小枫   

  1. 1.中国农业科学院农产品加工研究所,农业部农产品加工重点实验室,北京 100193;2.列日大学食品学院,比利时 列日 999013
  • 出版日期:2017-06-15 发布日期:2017-06-19

Purification and Identification of Antibacterial Peptides from Cottonseed Protein Isolate Hydrolysates

HAN Xiaoyan, BAO Yuming, XIN Fengjiao, HUANG Ying, Christophe BLECKER, DAI Xiaofeng   

  1. 1. Key Laboratory of Agro-Products Processing, Ministry of Agriculture, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2. Department of Food Science, University of Liège, Liège 999013, Belgium
  • Online:2017-06-15 Published:2017-06-19

摘要: 通过体外模拟消化系统对棉籽分离蛋白(cottonseed protein isolate,CPI)进行酶解,得到具有抗菌活性的酶解产物,并采用超滤(ultrafiltration,UF)、阴离子交换色谱(anion exchange chromatography,AEC)、半制备高效液相色谱(semi-preparation high performance liquid chromatography,semi-P-HPLC)分离技术对棉籽抗菌活性肽进行分离纯化,用电喷雾串联质谱(electrospray ionization-tandem mass spectrometry,ESI-MS/MS)鉴定棉籽抗菌肽的氨基酸序列。在抗菌活性肽分离纯化过程中,用UF对具有抗菌活性的CPI酶解产物进行分离,得到3 个组分U-Ⅰ~U-Ⅲ。抗菌活性检测表明U-Ⅲ的抗菌能力最强;用AEC分离U-Ⅲ得到3 个组分QF-Ⅰ~QF-Ⅲ,其中QF-Ⅱ抗菌能力最强;进一步采用semi-P-HPLC分离QF-Ⅱ得到4 个组分PF-Ⅰ~PF-Ⅳ,其中PF-Ⅲ的抗菌能力最强,经HPLC检测为单一峰,ESI-MS/MS检测分析得到该肽的氨基酸序列为ISGLIYEETR(Ile-Ser-Gly-Leu-Ile-Tyr-Glu-Glu-Thr-Arg)。

关键词: 棉籽分离蛋白酶解产物, 抗菌活性肽, 分离纯化, 氨基酸序列

Abstract: Cottonseed protein isolate (CPI) was digested in vitro to prepare hydrolysates with antibacterial activity. Ultrafiltration (UF), anion exchange chromatography (AEC), and semi preparative high performance liquid chromatography (semi-P-HPLC) were used to isolate and purify antibacterial peptides from CPI hydrolysates, and the purified peptides were sequenced by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). During the purification process, CPI hydrolysates were separated into three fractions: U-Ⅰ, U-Ⅲ and U-III by UF and the obtained U-Ⅲ, with higher antibacterial ability, was further separated into QF-Ⅰ, QF-Ⅱ and QF-Ⅲ by AEC. After filtration, QF-Ⅱ, which showed higher antibacterial ability, was further fractionated using semi-P-HPLC into four subfractions: PF-Ⅰ, PF-Ⅱ, PF-Ⅲ and PF-Ⅳ, among which, PF-Ⅲ was determined to have the highest antibacterial activity. The purity of PF-Ⅲ showed a single peak in HPLC. Finally, by ESI-MS/MS, and the amino acid sequence of the peptide was identified as ISGLIYEETR (Ile-Ser-Gly-Leu-Ile-Tyr-Glu-Glu-Thr-Arg).

Key words: cottonseed protein isolate hydrolysates, peptide with antibacterial activity, isolation and purification, amino acid sequence

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