界面马铃薯多肽改善水包油乳状液化学稳定性的物理化学机制研究

Oil-in-water emulsions are common biphasic dispersions that exists in food products.Oxidation is a common problem for this type of food emulsions.To improve the oxidative stability of O/W emulsions, various antioxidants are incorparated. Recent studies have shown that many protein hydrolysates or mixed peptides can act as antixidants to inhibit lipid oxidation in emulsion systems.However, the mechanisms by which peptides curtail lipid oxidation in emulsified foods have not been clearly elucidated. Understanding peptides' mode of action in a two-phase emulsion system would aid in the development of emulsion formulations and processing strategies to maximize food product stability and nutritive value. The presence of potato protein hydrolysate (PPH) in soybean O/W emulsions has been shown to improve the oxidative stability of emulsions. We hypothesize that peptides from PPH that partition in the interface of the emulsion droplets play an important role in enhancing the emulsion physicochemical stability. The present study was to elucidate the peptides adsorbed in the interface of emulsion droplets and structural characteristics of the interfacial membrane. The efficacy of a antioxidative potato protein hydrolysates for the stabilization of oil droplets and inhibition of lipid oxidation in soybean O/W emulsions is investigated, following by determining the peptides partitioning at the interface and zeta-potential of the soybean O/W emulsions. Biophysical methods, including atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM), and cryogenic transmission electron microscopy (Cryo-TEM), were used for emulsion and emulsion droplet membrane representation. Peptides partitioning in the interface were liberated by centrifugation and fractionated by gel filtration. After the removal of Tween 20 using solid phase extraction,all fractions were tested for radical-scavenging activity. Those showing strong activity were subjected to UPLC followed by Q-TOF-MS for individual peptide sequence identification. Electrochemical method was established to investigate redox at interface. The role of peptides adsorbed on the surface of oil droplets or partitioning in the emulsion interface is illustrated according to the overall result.

水包油乳状液是广泛存在于食品中的两相分散体系。脂肪尤其是不饱和脂肪的 氧化是食品乳状液体系中普遍存在的问题。添加抗氧化剂及设计合理油水界面膜被认为是抑制乳状液中油脂氧化比较有效的手段。抗氧化肽是一种公认的较为有前景的潜在天然抗氧化剂，然而其在食品乳状液体系中的应用研究不多，其在乳状液体系中的抗氧化机制还不明确。本课题将基于抗氧化肽的马铃薯蛋白水解物作为辅乳化剂用于Tween 20 为主乳化剂的大豆油水包油乳状液体系。研究了抗氧化肽在乳状液中的分布，并重点分析界面上多肽的抗氧化作用。通过多种生物物理手段联合表征乳状液界面膜和界面多肽来阐明界面多肽空间阻隔物理作用。利用固相萃取前处理结合色谱及质谱技术对界面多肽进行鉴定来阐明界面多肽的化学作用。拟建立电化学方法分析食品乳状液界面的氧化还原特性来进一步阐明界面多肽的化学作用。拟从微观结构和物质基础两方面上阐明界面上多肽抑制乳状液油脂氧化的机理。

本课题研究了马铃薯蛋白水解物（PPH）在乳状液中的抗氧化作用，并通过对乳状液界面组成及性质的分析阐明了界面上多肽提高乳状液氧化稳定性的作用机理，具体研究结果如下：首先，以脂肪氧化产物TBARS为指标考察了不同水解时间（0.5、1、6h）PPH对吐温20为主乳化剂的乳状液氧化稳定性的影响。结果表明不同水解时间PPH都可以提高乳状液氧化稳定性。其中，1h PPH抑制乳状液脂肪氧化的能力最强。其次，通过zeta电位和透射电镜分析了乳状液的表面电势和乳状液的微观结构。然后，利用离心法定量分析了PPH在乳状液中的分布，并利用凝胶排阻色谱分析了界面和水相中多肽的分子量分布。结果表明PPH的加入改变了乳状液的表面电势及界面膜厚度。尽管界面上PPH分布的量差异较小（p>0.05），1h PPH制备乳状液界面上多肽的平均分子量高于0.5和6h PPH制备乳状液界面上多肽的平均分子量。再次，采用G15凝胶色谱对1hPPH制备乳状液中的界面多肽进行分离，并利用RP-HPLC分析组分的极性大小，利用UPLC-TOF-MS对ABTS+自由基清除能力较强的组分中的抗氧化肽结构进行鉴定。同时，通过超滤、凝胶色谱、制备液相色谱手段对1hPPH中自由基清除能力较高组分进一步分离纯化，并用UPLC-MS/MS对抗氧化肽进行了结构鉴定。结果表明1hPPH分布在界面上多肽中的低分子量组分极性较强，而高分子量组分则极性较弱。小分子组分的ABTS+自由基清除能力则高于大分子组分。鉴定得到的界面抗氧化肽与1h马铃薯蛋白水解物中的抗氧化肽并未有重合，并且只有SFY与FY两个肽有部分匹配。最后，利用原子力显微镜、激光共聚焦扫描显微镜、冷冻透射电子显微镜等物理手段对1hPPH在界面上的分布进行了分析。结果表明多肽在界面上的分布可能存在两种形式，即界面的直接分布和界面表面层的复合吸附。这些在界面上直接分布的不同分子量的多肽形成的界面膜呈现非连续不同长度的短纤维状形态。研究表明马铃薯蛋白水解物在O/W乳状液中的抗氧化机制与其改变了乳状液油水界面的性质和组成有关。油水界面上分布的大分子多肽形成了阻碍氧化因子侵袭的空间物理屏障，而抗氧化能力较强的小分子多肽则形成了阻碍氧化因子侵袭的化学屏障，从而阻碍并延缓了自由基及过渡金属离子对脂肪的作用。