苦荞凝集素的稳定性及体外消化性

The Stability and in Vitro Digestibility of Lectin from Tartary Buckwheat (Fagopyrum tataricum)

目的:从苦荞种子中分离得到一种凝集素(tartary buckwheat lectin,TBL),研究其酸碱、热稳定性及体外消化性。方法:采用酸性缓冲液浸提脱脂荞麦粉及阴离子交换层析纯化凝集素,TBL分别经沸水浴加热不同时间、不同pH处理、模仿胃液、模仿胰液消化后,采用SDS-PAGE及灰度扫描分析,研究其稳定性及体外消化性。结果表明:选用pH4.7浸提缓冲液可简化提取工艺,采用一步层析可得到电泳纯TBL。沸水浴加热60 min时,TBL仍可保留50%以上。在pH4～12条件处理30 min后,BTL的保留率均在80%以上。体外消化实验表明,TBL对人工模拟胃液有一定抗性,降解一半TBL所需时间为10 min,而TBL在人工模拟肠液中很难消化,即使作用时间为50 min时,TBL也未发生明显降解。将TBL沸水浴加热30 min后再进行体外消化实验,发现在模拟肠液及模拟胃液中,仅处理10 min时,SDS-PAGE结果中TBL蛋白条带完全消失,表明被TBL被消化酶完全降解。结论:天然TBL具有良好的稳定性,耐热、耐酸碱、耐胰蛋白酶消化,在胃蛋白酶液中降解也较为缓慢。预加热处理可明显提高TBL在模拟胃液及模拟肠液中的消化率。

Purpose :To purify lectin from tartary buckwheat(TBL)seeds and investigate the stability and digestibility of TBL in vitro.Methods:Acid buffer was used to extract TBL from the defatted buckwheat flour and anion exchange chromatography was applied to purify TBL.The effects of heat treatment,pH,the digestibility of TBL in simulated gastric fluid (SGF)and simulated intestinal fluid (SIF)were studied,using kinetic densitometric analysis for SDS-PAGE.Results :Extraction with acid buffer simplified the purification protocol,as TBL was separated to electrophoretic purity by only one step of chromatography.After heated in boil water for 60min,more than half of TBL still remained.Furthermore,no obvious degradation was observed after TBL was placed in different buffers ranging from pH4 to 12 for 30min.It was found that TBL was reasonably stable in SGF with a half-time of digestion of 10min.In SIF it was extremely stable.Even after 50min of digestion there was no discernible degradation.But the susceptibility of TBL to hydrolysis by proteases was markedly increased by preheating.The preheated protein was totally degraded in both SGF and SIF after being digested for only 10min and the protein band disappeared by SDS -PAGE.Conclusion:TBL was stable when exposed to heat and over a wide pH range.Moreover,no trypsin digestion was observed and hydrolysis upon exposure to pepsin was limited.However,preheating could obviously increase the digestibility of BTI in SGF and SIF.