【作者】 张欣；

【导师】 熊幼翎；

【作者基本信息】 江南大学 ， 食品科学， 2014， 博士

【摘要】 酶法水解蛋白可以提高蛋白的可消化性，并获得完整蛋白所不具备的一些生理活性，如抗氧化活性等。然而，在水包油（O/W）乳状液体系中，水解蛋白因大部分分布于水相环境，无法有效作用于氧化反应最为集中的界面相和油相环境，因此，需要在其它抗氧化剂的辅助作用下才能获得理想的抗氧化效果。本文采用低致敏且具有清除游离基能力的水解豌豆蛋白（PPH）为原料，考察了常用的脂溶性食品配料甘草提取物（LE）与其在乳化体系（脂质体体系和乳状液体系）中联合抑制脂肪氧化的作用，并深入探讨了二者的协同抗氧化机理。为改善PPH的乳化性能并获得更为稳定的协同抗氧化效果，采用糖基化反应对水解蛋白进行共价修饰，获得了乳化性能明显改善的新型水解豌豆蛋白产品。随后，进一步考察了甘草提取物对水解蛋白糖基化反应的影响及其与高乳化活性的糖基化水解豌豆蛋白在乳化体系中的协同抗氧化效应。本研究首先考察了7种常用蛋白酶（胰蛋白酶、胰凝乳蛋白酶、木瓜蛋白酶、胃蛋白酶、碱性蛋白酶、风味蛋白酶和复合蛋白酶）对豌豆蛋白的酶解作用，并对各水解蛋白的抗氧化性及乳化性进行测定。结果表明，所有水解蛋白的清除游离基能力都较完整蛋白有显著提高，但是乳化能力明显下降。风味蛋白酶和复合蛋白酶制备的水解蛋白具有最强的抑制脂质体氧化的能力和相对较好的乳化能力。酶解前对豌豆蛋白的加热处理（90C，5min）有助于改善水解蛋白的抗氧化能力和乳化能力。接着，在脂质体体系中考察了水溶性PPH与脂溶性LE对脂肪氧化的联合抑制作用，以每升脂质体悬浊液所含硫代巴比妥酸反应物（TBARS）的量表示脂肪氧化的程度。结果表明，大多数PPHs，尤其是加热豌豆蛋白与风味蛋白酶和复合蛋白酶的酶解产物Fla-PPH和Pro-PPH，能够与LE产生协同增效的抑制脂肪氧化的作用。这主要与PPH富含可电离氨基酸以及疏水性N末端、LE中富含抗氧化多酚物质有关。通过对抑制TBARS能力最强的多肽组分进一步分离纯化和质谱检测，鉴定出10种抗氧化肽结构，其中LQEEDNVISQ、ITPERTLQLQDLDIFVN和LANRDDNEDLVGVL与豌豆vicilin序列相匹配。另外，PPH能够在脂质体表面形成自组装的网状结构，保护了脂质体不被氧化物质破坏。LE则通过与豌豆多肽发生非共价相互作用强化了网状结构的厚度和致密程度，进一步提高了脂质体对外界氧化胁迫的抵抗能力。所以，PPH与LE协同增效的抗氧化作用是二者化学机制和物理机制共同作用的结果。基于以上结果，进一步考察了PPH与LE在O/W乳状液体系中对脂肪氧化的联合抑制作用。将前面得到的抑制脂质体氧化能力较强的Fla-PPH和Pro-PPH以及LE添加到Tween20为辅助乳化剂的大豆油O/W乳状液（油含量10%，pH7.0）中，采用37C自动氧化，定期测定乳状液贮藏过程中过氧化值（POV）和丙二醛反应物（TBARS）的生成量。结果表明，Fla-PPH和Pro-PPH能够有效地抑制乳状液体系中脂肪氧化的进程。电镜结果表明，PPH能够在乳化液滴表面形成了一层保护膜，从而减弱氧化物对油脂的氧化胁迫。LE显著提高了两种PPH抑制脂肪氧化的作用（协同抗氧化作用）。原因是二者的联合使用有效清除了水相和油相中的自由基，并且降低了乳化液滴的表面电势和平均粒径，促进了厚实且致密的界面保护层的形成，大大改善了界面层对外界氧化胁迫的抵抗能力。该结果进一步证明了PPH与LE的协同抗氧化效应是化学机制和物理机制共同作用的结果。芹糖甘草苷、芹糖异甘草苷、光甘草素和18β-甘草次酸是LE中主要分布在油水界面的多酚化合物，这些化合物很有可能是与PPH产生协同抗氧化效应的主要成分。虽然，PPH与LE能够在乳化体系中产生协同增效的抗氧化作用，但是PPH自身的乳化性能较差，无法获得稳定的乳化体系，因而限制了PPH作为多功效食品组分在生产加工中的应用以及PPH与LE的协同抗氧化功效在乳化产品中的发挥与应用。因此，本文采用温和的美拉德反应对PPH进行糖基化改性以获得具有较高乳化活性的水解蛋白，再进一步考察LE与高乳化活性的糖基化水解豌豆蛋白在乳化体系中的协同抗氧化作用。结果表明，PPH与葡萄糖、麦芽糊精和葡聚糖在pH7.0、60C保温48h后生成了以初期产物希夫碱为主要产物的美拉德反应共聚物。其中，葡聚糖，虽然反应活性低于葡萄糖和麦芽糊精，但显著改善了PPH的乳化能力，并基本保留了PPH自身的抗氧化活性。所有共聚物中，Pro-PPH与葡聚糖形成的共聚物的乳化能力改善最多。因此，可利用葡聚糖参与的美拉德反应改善水解豌豆蛋白的乳化性能，从而制备兼具良好乳化性和抗氧化性的新型水解豌豆蛋白产品。随后，将乳化能力改善最多的Pro-PPH–葡聚糖共聚物添加到大豆油O/W乳状液体系中，进一步检验其对乳化油滴的稳定作用及对外界环境胁迫的抵抗能力。结果表明，该共聚物具有良好的乳化性能以及抑制油脂氧化的能力。电镜结果表明，这与其具有高度交联的空间网状结构密切相关。此交联结构不仅有助于在乳化液滴表面形成坚实的保护层，也增大了体系的粘度，因此，有效限制了乳化液滴的碰撞、聚集和合并以及过氧化产物的扩散和转移。另外，此交联结构也降低了PPH与外界不良环境因素（热、酸碱等）的接触及相应的变性应激。Pro-PPH凝胶分离组分中分子量较大的PF1和PF2组分具有最强的美拉德反应活性。Pro-PPH与糖的糖基化反应容易发生在肽链的端基，检测到的糖基化肽段包括豌豆蛋白11S中的IISPPEK和LSIISPPEK及7S中的KLP、NVIVK和LSPGDVF。最后，探讨了LE对Pro-PPH与葡聚糖糖基化反应的影响及其与Pro-PPH–葡聚糖共聚物在乳化体系中的联合抗氧化效果。结果表明，LE具有促进Pro-PPH糖基化改性的作用，可作为桥梁媒介提高Pro-PPH与葡聚糖的反应活性，进一步改善Pro-PPH–葡聚糖共聚物的乳化性能。LE与Pro-PPH–葡聚糖共聚物依然具有协同增效的抑制脂肪氧化的作用。可见，无论糖基化反应前或后加入甘草提取物，都将对乳化体系的稳定性（物理稳定性以及化学稳定性）产生积极的改善作用。总之，水解豌豆蛋白与甘草提取物能够在乳化体系中产生协同增效的抑制脂肪氧化的作用。水解豌豆蛋白的糖基化改性对二者协同作用影响不大。所以，合理配比水解豌豆蛋白与甘草提取物，可用于开发具有强抗氧化效力的组合抗氧化剂产品，用以最大限度地改善乳状液产品的化学及物理稳定性，开辟豌豆蛋白与甘草提取物新的应用价值和领域，为食品工业创造更大的价值。更多还原

【Abstract】 Enzymatic hydrolysis of protein can not only improve the digestibility, but also thebioactivities, especially the antioxidant activity. However, due to size reduction and increasedcharges, peptides are distributed preferentially in the aqueous phase, therefore more effectivein the aqueous phase than in the lipid phase where hydroperoxides and peroxide radicals aregenerated. Hence, other antioxidants were required to work with protein hydrolysates toobtain ideal inhibitory effect on lipid oxidation in oil-in-water (O/W) emulsions. In this study,radical-scavenging pea protein hydrolysate (PPH) with low-allergenicity was used toinvestigate its cooperative inhibitory effect with lipophilic licorice extract (LE) on lipidoxidation in O/W emulsions. The mechanism of synergistic antioxidative effect waselucidated. Saccharides with various lengths (glucose, maltodextrin, and dextran) wereintroduced into pea peptides via Maillard reaction to try to improve the emulsifying propertiesof PPHs in emulsions. The effect of LE on glycosylation of PPH as well as the cooperativeeffect of glycated PPH and LE on lipid oxidation in O/W emulsions were also studied.Pea protein isolates (PPIs) with or without heating (90C,5min) were hydrolyzed usingseven proteases (trypsin, chymotrypsin, papain, pepsin, Alcalase, Flavourzyme and Protamex)to produce pea protein hydrolysates (PPHs). Antioxidant activity and emulsifying property ofPPHs were assessed. Results showed all PPHs exhibited stronger radical scavenging activitythan intact proteins, but weaker emulsifying property. Those prepared with Flavourzymeand Protamex were most effective in inhibiting lipid oxidation in a liposomal system, and alsoshowed relatively good emulsifying property. Overall, PPHs from heated PPIs exhibited betterantioxidant activity and emulsifying property than those from native PPIs.The cooperative effect of PPHs in combination with LE for the inhibition of lipidoxidation in a liposomal model system was assessed. Degree of oxidation was expressed asformation of TBA reactive substances (TBARS) per liter of liposome suspension. Resultsshowed that most PPHs, especially those prepared from heated protein with Flavourzyme andProtamex (Fla-PPH and Pro-PPH) exhibited remarkable synergistic inhibitory effects with LE.The synergism was attributed to the abundant charged amino acid side chain groups and thedominating presence of hydrophobic N-terminus of constituting peptides in the proteinhydrolysates and the preponderance of phenolic compounds in licorice extract. Afterseparation and purification of peptide fractions that exhibited excellent TBARS inhibition,10peptides were identified, of which, LQEEDNVISQ, ITPERTLQLQDLDIFVN andLANRDDNEDLVGVL matched the sequence of pea vicilin. While self-assembled networksformed around lipid droplets appeared to be a prevalent physical mechanism by whichpeptides diminished the potency of oxidants, the peptides’ association with phenolic and otheractive components from licorice extract afforded a more compact and rigid protective network,providing liposomes with a stronger defense against oxidative stress. Both chemical andphysical mechanisms exerted by PPH and LE were responsible for the antioxidativeprotection.The cooperative inhibitory effect of PPH with LE on lipid oxidation in an O/W emulsionwas further determined. Fla-PPH and Pro-PPH, which exhibited the strongest inhibition on liposome oxidation, and LE were subjected to soybean O/W emulsions (oil phase10%, pH7)co-stabilized by Tween20. The suppression of lipid oxidation (POV and TBARS) wasassessed during storage at37°C. Results showed that both PPHs significantly retardedoxidation of the emulsions. Electron microscopy revealed an interfacial peptidyl membranearound oil droplets which afforded steric restrictions to oxidation initiators. When LE wasalso used in emulsion preparation, a remarkable synergistic oxidation inhibition was observedwith both PPHs. The combination of PPH and LE, on one hand, allowed the efficient removalof radicals in both aqueous and lipid phases; on the other hand, reduced the surface charge andparticle size of oil droplets, thereby facilitating the formation of thick and rigid interfacialmembrane which offered superior defense against oxidative stress. This result furtherconfirmed that both chemical and physical mechanisms exerted by PPH and LE wereresponsible for the antioxidative protection. Liquiritin apioside, neolicuroside, glabrene and18β-glycyrrhetic acid were the predominant compounds in LE partitioning at the oil–waterinterface and were likely responsible for the strong antioxidant activity of LE in O/Wemulsions and the notable synergism when coupled with PPHs.Although PPH exerted synergistic antioxidative effects with LE in O/W emulsions, thepoor emulsifying property hindered PPH from making stable emulsions and high-performancesynergistic antioxidative effects with LE in these emulsions. Hence, Maillard-typeglycosylation was employed to improve the emulsifying propery of PPH. Results showed thatinitial stage products Schiff base was generated when incubated PPHs and saccharides(glucose, maltodextrin, and dextran) in aqueous solutions at pH7.0,60C for48h. Despitethe low degree of glycation relative to glucose and maltodextrin, dextran exhibited the highestefficacy on improving emulsifying property and maintaining the antioxidant potential of PPHs.Pro-PPH–dextran conjugate showed the highest improvement in emulsifying capacity.Therefore, controlled Maillard reaction with dextran, can be used as an effective tool toimprove the emulsifying property of antioxidative pea protein hydrolysates.Next, apply Pro-PPH–dextran conjugates into soybean O/W emulsions to furthercharacterize their emulsifying property. Results showed that Pro-PPH–dextran conjugateswere effective emulsifiers and offered superior defense against oxidative stress. This can beattributed to their highly interactive and crosslinked network structure, which facilitated theformation of thick and rigid interfacial membrane and the improvement of viscosity. As aresult, the collision or aggregation of oil droplets was restricted, and the diffusion and transferof oxidative substrates were also hindered. Meanwhile, the stabilization of structure andfunction of PPHs in these emulsions towards environmental stresses were also strengthened.PF1and PF2, the first two largest gel filtration fractions of Pro-PPH, showed the highestMaillard reaction reactivity. N-termini of peptide chains were more accessible to saccharides.MS/MS analyses detected the glycated peptides IISPPEK and LSIISPPEK from pea protein11S, and KLP, NVIVK and LSPGDVF from pea protein7S.Ultimately, study the effect of LE on glycosylation of PPH as well as the cooperativeinhibitory effect of Pro-PPH–dextran conjugates with LE on lipid oxidation in O/W emulsions.Results showed that LE promoted the covalent conjugation between Pro-PPH and dextran. It can act as a bridge to enhance the reactivity between Pro-PPH and dextran, thus furtherimproving the emulsifying property of Pro-PPH–dextran conjugates. On the other hand,synergistic antioxidative effects were still observed on Pro-PPH–dextran conjugates and LE inO/W emulsions. Hence, the addition of LE either before of after glycosylation of PPH, canbring positive improvements to the properties of emulsion-type food products containing PPH.Overall, pea protein hydrolysates when used in combination with licorice extract, canproduce a synergistic inhibition on lipid oxidation in O/W emulsions. Glycosylation of PPHshowed little affect on its synergism with LE, but enhaced the reliability of synergism inemulsions. Hence, enzymatic pea protein hydrolysates and licorice extract can be appliedtogether to develop superior antioxidant ingredient and to maximize the stability of emulsion-type food products, both physically and chemically.更多还原