Study On The Antioxidation Effects Of Soybean Protein Hydrolysates And Tyrosine-containing Dipeptides In Heterogenous Systems

Lipids are one of the main components in foods,but susceptible to active free radicals-,metal ions-,enzymes-,and light-induced oxidation.Excessive lipid oxidation not only leads to the abnormal function of biofilm and the polymerization or cross-linking of protein molecules,but also seriously reduces the quality of foods.It is essential to study lipid oxidation and its inhibition mechanism,due to the increasing requirement of health and nutrition.Soybean protein isolate(SPI)can be hydrolyzed by enzymatic method to small-molecule peptides,which have high safety,strong antioxidant activity and amphipathy,and can be used as antioxidant and emulsifier in food system.In view of the fact that most of the food systems are heterogeneous systems,a simplified model was constructed in this paper to simulate food systems:Liposomes formed by phospholipids dispersing in water and self-assembling into a double-layer membrane structure and finally encapsulating the water phase.Free radical inducers were used to induce the oxidation of liposomes to simulate the lipid oxidation in the heterogeneous food system,so as to study the antioxidant effect of peptides.In this paper,soybean protein isolate hydrolysates(SPIH)were produced from SPI by several commercial proteases.We systematically studied the antioxidation effects of SPIH in liposomes,and then focused on the relationship between structures of five Tyr-containing dipeptides and their antioxidation effects in liposomes,aiming to provide theoretical reference for the application of peptides as effective antioxidants and emulsifiers in food heterogeneous system.The main contents and conclusions are as follows:(1)SPIH with different characteristics were prepared from SPI by pepsin(Pep),trypsin(Try)and alcalase(Alc)alone or combined with flavourzyme(Fla).The antioxidation effects of SPIH in liposomes were investigated by comparing with their antioxidant activities in homogeneous(aqueous phase)systems.The result showed that SPIH were rich in fractions with molecular weight<5 k Da(the content was more than 52.73%,the highest was 93.73%),with the fraction with molecular weight<1 k Da having the highest amount.The ABTS and ORAC radical scavenging activity of SPIH,and their rate of inhibition on lipid oxidation(K_I)were significantly correlated with their degree of hydrolysis(DH)(P<0.05),indicating that the increase of enzymatic hydrolysis degree was conducive to the enhancement of SPIH activity.The combined hydrolysis could effectively increase DH and the content of free hydrophobic amino acids in SPIH and significantly influence their reducing power and Fe²⁺chelating capacity.The antioxidant activity of SPIH in liposomes was significantly correlated with their ROO·or RO·scavenging activity and hydrogen atom/electron providing capacity in homogeneous system(P<0.05).SPIH with higher K_I were found more helpful to inhibit the increase of liposomal average size,so as to maintain the systematic stability.(2)SPIH(PF,TF and AF)with higher antioxidant activity which prepared by combined hydrolysis of Pep,Try or Alc with Fla from SPI were added to liposomes,and interactions between SPIH and liposomes were investigated using techniques including synchronous fluorescence,circular dichroism and Fourier transform infrared spectroscopy(FTIR),as well as their effects on the secondary structure of SPIH,liposomal membrane structure and oxidation stability of liposomes.The result showed that SPIH inhibited lipid oxidation in a dose-dependent manner(best dose:0.50 mg/m L;best SPIH:AF).The presence of SPIH in liposomes was realized via electrostatic force,hydrophobic effect and hydrogen bonding,causing enhanced liposomal rigidity,and AF had more affinity with phospholipid membranes,so that its adsorption to the lipid-water interface were more effectively and it can exert a better effect on inhibiting lipid oxidation.SPIH-liposomes interactions favored high hydrophobicity,?-structure and disordered structure in SPIH.Tyr and Trp in SPIH played important roles in interactions and stabilizing systematic stability.TF(0.50 mg/m L)was more effective in maintaining the systematic stability by suppressing the increase of liposomal average size.(3)AF with the highest antioxidant activity was added at different concentrations to liposomes by two loading methods separately(addition before lipid film hydration or after liposomes formation),and their effects on the antioxidant efficiency of AF,AF-liposomes interactions and the systematic stability were explored.The result showed that the loading methods and concentrations of AF directly influenced K_I:Taking 0.096 mg/m L as the cut-off point,K_I was higher when added after liposomes formation at low concentrations,while the situation reversed at higher concentrations of AF.The loading methods and concentrations of AF affected strongly the electrostatic interactions but weakly the hydrophobic interactions,while the hydrogen bond interactions were more significant at higher AF concentrations.More effective adsorption of AF onto liposomal membrane occurred when it was added at 0.50mg/m L before lipid film hydration or added at lower concentrations(<0.192 mg/m L)after liposomes formation.In maintaining the systematic stability,the addition of AF before lipid film hydration helped more in inhibiting the increase of liposomal average size,while the addition of AF(>0.096 mg/m L)after liposomes formation functioned more effectively in increasing the absolute value of liposomal surface charge.(4)Tyr plays an important role in the interaction between SPIH and phospholipid membrane,and it contains both hydrophilic group(-OH)and hydrophobic benzene ring.Thus,five Tyr-containing dipeptides(YY,YF,FY,YA and YS)were designed,and their antioxidant activities in homogeneous(aqueous phase)systems were compared to explore the relationship between their structure and antioxidant activities in liposomes under oxidation induced by hydrophilic(AAPH)or lipophilic(AMVN)radical initiator,as well as their interactions with phospholipid membranes and effects on systematic stability.The result showed that YY displayed the highest activity in both aqueous phase and liposomes,while the location of Tyr at the N-/C-terminus being a crucial factor to the antioxidant activities of dipeptides.Compared with YF,FY exhibited a higher activity in ORAC assay but a lower activity in ABTS assay.The antioxidant activity of Tyr-containing dipeptides in liposomes under oxidation induced by AAPH was significantly correlated with their electron donating abilities and ORAC radical scavenging abilities in homogeneous systems(P<0.05),and the K_I of Tyr-containing dipeptides in liposomes under oxidation induced by AMVN was significantly higher than that in liposomes under oxidation induced by AAPH.The stability of liposomes under oxidation induced by AMVN was higher than that under oxidation induced by AAPH during the oxidation process.Among all Tyr-containing dipeptides,YY functioned most effectively to maintain the systematic stability.