【作者】 程宇；

【导师】 熊幼翎；

【作者基本信息】 江南大学 ， 食品科学， 2010， 博士

【摘要】 水包油(O／W)乳状液是广泛存在于食品中的两相分散体系。因油脂氧化而导致食品品质下降也是这类食品普遍存在的问题。已经有研究表明在乳状液中加入具有抗氧化活性的蛋白水解物可以抑制乳状液中油脂氧化。但是具有抗氧活性的蛋白水解物在乳状液中的分布及其对乳状液氧化稳定性影响的机理尚未得到阐明。为了更好的设计乳状液抗氧化的策略来提高产品氧化稳定性,有必要对具有抗氧活性的蛋白水解物在乳状液中的分布与油脂氧化抑制作用之间关系的机理进行研究。同时,马铃薯蛋白作为马铃薯淀粉工业的副产物,由于功能性质差而附加值较低。通过的酶解的手段改善其功能性质,将其开发成具有抗氧化活性的蛋白水解物则可以拓宽其使用范围。因此,本论文的研究对于进一步阐明具有抗氧化活性的蛋白水解物在乳状液中的抗氧化机理有着重要的学术意义。同时,对提高马铃薯蛋白的应用价值和产品附加值有一定的现实意义。论文首先研究了马铃薯蛋白水解物对O/W乳状液乳化稳定性及氧化稳定性的影响。采用Acalase水解马铃薯蛋白,制备得到具有抗氧化活性的马铃薯蛋白水解物,将蛋白水解物加入到Tween 20为乳化剂的大豆油O/W乳状液(油含量10%,pH7.0)中,采用烘箱氧化法(37℃),通过定期测定乳状液在氧化过程(0-14天)中过氧化值(POV)和丙二醛反应物(TBARS)的变化,考察马铃薯蛋白水解物了对乳状液氧化稳定性的影响。结果表明对于以Tween 20和马铃薯蛋白水解物混合物为乳化剂制备得到的乳状液,在37℃下贮藏7天后,和未添加马铃薯蛋白水解物的样品相比,马铃薯蛋白水解物对乳状液中POV和TBARS的抑制率最高分别可达到53.4%和70.8%(P<0.05)。马铃薯蛋白水解物不论是在乳状液均质前加入还是在乳状液均质后加入,都能起到了抑制乳状液中油脂氧化的作用,但是抑制机制不同,马铃薯蛋白水解物在均质前加入可以更好抑制过氧化物的形成,而马铃薯蛋白水解物均质后加入则可以更好抑制丙二醛类物质的形成。上述结果暗示了马铃薯蛋白水解物在乳状液中的抗氧化机制与其在乳状液的分布有关。接着进一步研究了马铃薯蛋白水解物的分子量和极性对于乳状液氧化稳定性的影响。通过凝胶色谱分离和硫酸铵沉淀两种方法对马铃薯蛋白水解物进行了分级,分别得到了不同分子量和不同极性的马铃薯蛋白水解物组分。通过测定ABTS+’自由基清除能力和金属离子螯合能力分析了这些组分的抗氧化性质。同时,以正己醛和TRARS为指标,采用烘箱氧化法(37℃)考察了具有不同分子量和不同极性的马铃薯蛋白水解物组分对Tween 20为乳化剂制备得到的大豆油O/W乳状液(油含量10%,pH7.0)氧化稳定性的影响。结果表明,不同性质的马铃薯蛋白水解物组分的自由基清除能力与其在乳状液中抗氧化能力有不一致性,凝胶色谱分离得到的低分子量组分Peak 3(分子量低于1043Da)和50%硫酸铵沉淀得到的组分P50表现出了相对较好的自由基清除活性和抑制油脂氧化能力。然后采用制备型液相色谱对Peak 3和P50进行了进一步纯化,对纯化得到的组分中ABTS+·自由基清除能力的组分采用超高液相色谱串联质谱联用技术(UPLC-MS/MS)对其中的活性多肽结构进行鉴定,并将鉴定得到的多肽与NCBI数据库中马铃薯蛋白的序列进行了相似性匹配。结果表明,从组分Peak 3中分离得到四个与马铃薯蛋白中主要蛋白Patatin序列片段相匹配的多肽,分别为Thr-Tyr, Tyr-Phe-Glu, Tyr-Ser-Thr-Ala和Asn-Tyr-Lys-Gln-Met。从组分P50鉴定得到了两个与马铃薯蛋白序列相匹配的多肽,分别为Ser-Ser-Glu-Phe-Thr-Tyr和Ile-Tyr-Leu-Gly-Gln。这两个多肽序列分别存在与马铃薯蛋白metallocarboxypeptidase inhibitor和lipoxygenase 1中。通过制备型液相色谱对从组分Peak 3中分离得到的组分P3F5进行了进一步纯化,利用UPLC-MS/MS确定了存在于Patatin中的三肽Tyr-Phe-Glu为高活性的抗氧化肽。同时,采用离心法分析了马铃薯蛋白水解物在乳状液中的分布,结果表明有8-15%的马铃薯蛋白水解物分布在了乳状液的界面上。随着乳状液的pH从3调到7,乳状液的ζ-电位有了明显的增加(P<0.05)。通过常规透射电镜对乳状液进行表征,结果表明乳状液中油脂氧化的抑制和分布在水相中乳状液颗粒周围的马铃薯蛋白水解物的化学作用和物理屏蔽机制有关。利用原子力显微镜的高度成像模式和相位成像模式对乳状液形貌进行的表征,结果表明加入马铃薯蛋白水解物作为辅乳化剂的乳状液中油脂可以得到更好地分散。而且,和Tween 20为单一乳化剂制备的乳状液相比,分布在脂肪球界面膜上的马铃薯蛋白水解物会改善界面的状况。通过激光共聚焦扫描显微镜和冷冻透射电镜对乳状液脂肪球界面膜进行了进一步的表征。从微观结构上直观地证明了马铃薯蛋白水解物在界面膜上的分布,在超微观结构上显示了界面膜的形态为非连续分布的棒状结构。最后,通过离心分离结合有机溶剂提取的方法得到了界面上的多肽。采用G15凝胶色谱对这部分多肽进行了进一步分离,按时间(2 min)收集并得到了一系列组分。通过固相萃取柱脱除组分中的Tween 20后,采用超高液相色谱与飞行时间质谱联用技术(UPLC-Q-TOF-MS)对其中部分ABTS+·自由基清除能力相对较好的组分中的多肽进行了分离鉴定。对得到的多肽的性质分析表明,得到的多肽是由2-7个氨基酸组成的短肽,分子量分布在200-800之间。亮氨酸／异亮氨酸、脯氨酸、赖氨酸和精氨酸在这些多肽中出现的概率较高。通过多肽的序列分析多肽的亲疏水性较氨基酸组成分析的方法更合理。以上的结果暗示马铃薯蛋白水解物在O/W乳状液中的抗氧化机制和马铃薯蛋白水解物水解物在油水界面形成的物理屏障效应有关,即分布在乳化油脂颗粒油水界面上的多肽形成的空间阻碍和静电束缚作用以及覆盖在乳化油脂颗粒周围的多肽网络结构形成的物理屏障的阻隔作用。更多还原

【Abstract】 Oil-in-water (O/W) emulsions are common biphasic dispersions that exist 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 incorporated. Recent studies have shown that many protein hydrolysates or mixed peptides can act as antioxidants 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. Potato protein, a by-product of the starch industry, has poor functional properties due to the harsh dehydration conditions employed for protein recovery. Limited hydrolysis is able to increase the solubility and related functionality of potato protein, and possibly its antioxidant potential.The aim of the present research was to unravel the molecular mechanisms by which potato peptides inhibit lipid oxidation in emulsified foods. The ultimate goal was to improve both the economical and nutritional values of potato protein.In Experiment 1, the efficacy of a previously developed antioxidative potato protein hydrolysate (PPH) for the stabilization of oil droplets and inhibition of lipid.oxidation in soybean O/W emulsions was investigated. Emulsions (10% lipid, pH 7.0) with PPH-coated oil droplets were found less (P< 0.05) stable than those produced with Tween 20. However, the presence of PPH, whether added before or after homogenization with Tween 20, retarded emulsion oxidation, showing reduced formation of peroxides up to 53.4% and malonaldehyde-equivalent substances (TBARS) up to 70.8% after 7-d storage at 37℃(P< 0.05), when compared with PPH-free emulsions.In Experiment 2, antioxidative PPH was fractionated using gel filtration and ammonium sulfate precipitation. The efficacy of different fractions for inhibiting lipid oxidation in soybean O/W emulsions and neutralizing 2,2’-azinobis (3-ethylbenzothiszoline-6-sulfonic acid) (ABTS+·) radicals was studied. Low-molecular-weight fraction Peak 3 (<1043 Da) from gel filtration and the fraction precipitated by 50% saturated ammonium sulfate exhibited the strongest antioxidant activity and radical scavenging activity.In Experiment 3, active peptides present in different fractions of PPH based on the ABTS+·scavenging assay were isolated and purified by preparetive HPLC, and the amino acid sequences were determined by LC-MS/MS. Of various peptides sequenced, Asn-Tyr-Lys-Gln-Met, Thr-Tyr, Tyr-Ser-Thr-Ala, and Tyr-Phe-Glu were identified to be the prominent peptides in Peak 3 of the gel filtration-separated PPH, which matched sequences in papatin, a main protien component in potato. Likewise, Ser-Ser-Glu-Phe-Thr-Tyr and Ile-Tyr-Leu-Gly-Gln were identified to be key peptides present in P50 of saturated ammonium sulfate-separated PPH; they matched with the sequences in metallocarboxy-peptidase inhibitor and lipoxygenase 1, respectively. In Experiment 4, the distribution of peptides in antioxidative PPH at the interface of soybean O/W emulsions was determined. In the emulsions stabilized by Tween 20 as the primary emulsifier and PPH as the secondary emulsifier,8-15% of PPH was distributed at the interface. Adjustment of the pH from 3 to 7 markedly increased (P< 0.05)ζ-potential of such emulsions. Several biophysical methods, including conventional TEM, AFM, CLSM and Cryo-TEM, were used for emulsion and emulsion droplet membrane representation. When PPH was incorporated into the emulsions as secondary emulsifier, dispersions of oil droplets in the emulsion were better than the control according to the results of AFM. Moreover, PPH distributed in the interfacial membrane improved the integiry of the interface. On the other hand, CLSM images demonstrated the existence of PPH peptides in the interfacial membrane that were directly ancherd in the microstructure of emulsions. Also, Cryo-TEM illustrated the morphology of the interfacial membrane as a non-continuous short fibril structure in the ultra-structure of emulsions.Finally, peptides which partitioned in the interfacial membrane were recovered by centrifugation and separated and purified using G15 gel filtration, or collected using solid phase extraction. Selected peptides were subjected to UPLC-Q-TOF-MS for sequence identification after removal of Tween 20. The peptides identified had molecular weights of 200-800 Da comprising of 2-7 amino acids. Leu (lle), Pro, Lys and Arg were most abundant amino acids in these peptides.The overall results demonstrate for the first time that inhibition of lipid oxidation by PPH in soybean O/W emulsions is due to both chemical and physical actions. Peptide adsorption on the surface of oil droplets and partitioning in the emulsion interface play an important role in inhibiting lipid oxidation. These steric hindrance and possibly electrostatic and shielding effects are complemented by the radical scavenging activity of peptides thereby jointly contributing to the stabilization of the soybean O/W emulsion system.更多还原