不同模式超声预处理对莲子蛋白酶解物及其结构的影响

超声波参数中,超声频率是影响蛋白酶解的主要因素。因此,本实验以不同模式超声频率下得到的莲子蛋白酶解物为研究对象,首先对不同模式超声进行优化,以血管紧张素转化酶(angiotensin converting enzyme,ACE)抑制率和水解度为指标,得出了不同模式下的最优频率参数:单频率超声20 kHz、双频率组合20/35 kHz、三频率超声组合20/35/50 kHz。然后采用表面疏水性、氨基酸分析及内源性荧光光谱等表征不同模式的超声预处理对莲子蛋白酶解物及结构特征影响。结果显示:随着超声频率组合增多,表面疏水性越来越大,疏水性氨基酸含量增多,主要是由于超声预处理使蛋白结构展开,使最初被掩埋在蛋白分子内部的疏水基团暴露。通过荧光光谱发现,超声预处理提高了莲子蛋白的荧光强度,且荧光强度顺序为三频双频单频;同时,莲子蛋白酶解物λmax发生轻微的红移现象。上述结果对于莲子蛋白酶解及ACE抑制肽的制备至关重要。

Effects of Ultrasonic Pretreatment with Different Working Modes on Enzymatic Hydrolysis and Structural Characteristics of Lotus Seed Protein

Ultrasonic frequency is one of the major ultrasonic parameters that affects protein enzymatic hydrolysis. In this study, we therefore investigated the effect of ultrasonic pretreatment with different working modes (single-, dual- and triplefrequency ultrasonic) on the degree of papain-catalyzed hydrolysis of lotus seed protein and the angiotensin converting enzyme (ACE) inhibitory activity of the resulting hydolsates. Twenty, 20/35 and 20/35/50 kHz were found to be the optimal conditions for single-, dual- and triple-frequency ultrasonic treatment, respectively. Moreover, the effect of ultrasonic pretreatment with different working modes on the surface hydrophobicity and amino acid composition of the enzymatic hydrolysates and we also characterized their structure by intrinsic fluorescence spectroscopy. The results showed that the surface hydrophobicity and the number of hydrophobic amino acids with the increase of ultrasonic frequency, primarily because ultrasonic treatment resulted in protein unfolding and consequently exposure of hydrophobic groups buried within the protein molecule. Furthermore, ultrasonic treatment increased the fluorescence intensity of lotus seed protein and the highest fluorescence intensity was obtained with triple-frequency mode followed by dual- and single-frequency modes. The maximum absorption wavelength of the enzymatic hydrolysates showed a slight red shift. The above findings are of great significance for studying the enzymatic hydrolysis of lotus seed protein to prepare ACE inhibitory peptides.