| Whey protein has drawn widely attention in food industry for its high nutritional value and excellent functional properties. Some studies suggested that the Maillard reaction products (MRPs) formed from whey protein and sugars have a better antioxidatant activity. Compared with synthetic antioxidants, natural antioxidant has a higher safety. Recently, the most of studies about the antioxidant activity of MRPs focus on the reaction between proteins and reducing sugar, and few studies involve the antioxidant activity of MRPs from whey protein hydrolysates and reducing sugar.In this study, Maillard reaction products of whey protein peptide and reducing sugar (WPP-MRPs) were obtained by the reaction between alcalase hydrolysates products of whey protein and glucose. First, the preparation conditions of WPP-MRPs were determined. Its structures after separation and purification were analyzed, and its antioxidant activity in vitro and effect on inducing tumor cell apoptosis were evaluated. Finally, the influences of the hydroxyl radical-generating system (HRGS) on the structure and function of WPP-MRPs were studied. The main results are as follows:1. The optimum preparation conditions of WPP-MRPs were established by single factor experiment and response surface optimization design, and the reducing power were used as analyzing index. The optimum preparation parameters to obtain the highest reducing power were8%glucose concentration at94?for3.2h, and the reducing power (A700nm) was0.94±0.05. During reaction, the pH value in solution constantly decreased (P<0.05), the browning degree deepened (P<0.05), fluorescence intensity experienced the trend of rising at first and then falling (P<0.05), and the free amino acid gradually decreased (P<0.05).2. The antioxidant activities of WPP-MRPs were evaluated, and butyl hydroxy anisole (BHA;0.1mg/mL) and ascorbic acid (Vc;0.1mg/mL) were used as control. The results showed when the concentration of WPP-MRPs was at40mg/mL, the reducing power, hydroxyl radical scavenging rate, ABTS radical scavenging rate, and Cu2+chelating ability were0.39±0.04,99.37±2.01%,81.58±1.33%, and40.47±2.15%, respectively. Compared with0.1mg/mL Vc and0.1mg/mL BHA,40mg/mL WPP-MRPs exhibited a significant higher hydroxyl radical scavenging ability and a stronger metal (Cu2+, Fe2+) chelating ability (P<0.05), while its reducing power was lower than Vc (P<0.05), and has no significantly difference compare to BHA. The inhibition of WPP-MRPs to TB ARS (thiobarbituric acid-reactive substances) was also lower than BHA and higher than Vc.3. The antioxidant stabilities of WPP-MRPs were studied by evaluating the effects of temperature, pH, light condition, metal ions and H2O2on the antioxidant activity. The results showed that heating has a little effect on the antioxidant activity of WPP-MRPs. The total reducing power, hydroxyl radical scavenging rate and the ABTS radical scavenging rate of WPP-MRPs had no significant changes when WPP-MRPs were heated at50?110?(P>0.05). The antioxidant activity of WPP-MRPs at slightly alkaline condition was stronger than that at slightly acidic or neutral condition, and the total reducing power, hydroxyl radical scavenging rate and the ABTS radical scavenging rate of WPP-MRPs were the highest at pH8.0(P<0.05). The WPP-MRPs were placed under the condition of outdoor natural light, indoor natural light and avoid light for25d. The results showed that the antioxidant activity of WPP-MRPs had significantly decreased at outdoor natural light and the decreasing order was:outdoor natural light> indoor natural light> avoid light (P<0.05). Metal ions, such as Na+, K+, Mg2+, Ca2+and Zn2+, had few effects on the total reducing power, the hydroxyl radical and the ABTS radical scavenging ability of WPP-MRPs (P>0.05). However, the metal ions such as Cu2+, Fe2+, Fe3+and the oxidant H2O2could largely reduce the antioxidant activity of WPP-MRPs (P<0.05). So, WPP-MRPs should be processed at conditions of slightly alkaline, stored at dark, and avoided contact copper and iron utensils and H2O2in order to keep its good antioxidant.4. WPP-MRPs were classified through the hollow fiber ultrafiltration membrane, and part I (molecular weight<0.8kD), ?(0.8KD<molecular weight<5kD), and III (molecular weight>5kD) were obtained. Part ? had the best reducing power (P<0.05) and was used for gel filtration chromatography purification. The optimal purification condition for preparing WM (WPP-MRPs II)was determined by single factor experiment by using Sephadex G-25column (1.0×50cm) as the purification medium and the distilled as the eluant. Three components with different molecular weight ranges, including WM1(2781-7437D), WM2(813-2781D), and WM3(54-813D) were obtained after separation and purification under the conditions of buffer flow rate at0.2mL/min and the injection volume at1.5mL. The results showed that WM2had better antioxidant activity (P <0.05). When the concentration of WM2was40mg/mL, its reducing power (A700nm) was0.68±0.04; the hydroxyl radical scavenging rate was99.95±2.70%; the ABTS radical scavenging rate was88.63±3.04%; Cu2+chelating ability was45.41±1.12%; Fe2+chelating ability was11.28±0.05%; and TBARS was0.83±0.06mg/L.5. The structure analysis showed that the total amino acid composition of the WPP-MRPs experienced significant changes. Compared with that of WPP, lysine content declined significantly and decreased by0.86%, and the contents of arginine, histidine and proline experienced slightly declining as well (P<0.05). Fourior transform infrared spectroscopy (FTIR) analysis and circular dichroism (CD) indicated that saccharide conjugated with WPP covalently, which made the content of a-helical and ?-turn in the secondary structure of MRPs decreased (P<0.05), but that of (3-sheet and random structure increased (P<0.05). This revealed that Maillard reaction had a certain effect on the secondary structure of WPP. It made the transition from order to disorder. The results of circular dichroism (CD) spectra correspond with that of FTIR in spite of only slight difference in data. Fluorescence spectrum showed that a new peak value occurred between420nm and430nm, indicating that a kind of new substance with fluorescence property emerged in the MRPs. The scan results of ultraviolet absorption spectrum suggested that Maillard reaction increased the chromophore content.6. The inhibition of WM2on cervical cancer cells (Hela) and colon cancer cells (Ht-29) were studied, the result indicated that WM2inhibited the proliferation of Ht-29and Hela cells dependent on dose and time. The IC50value of Hela cells were39.12mg/mL?35.91mg/mL, respectively, through interacting about12hours and24hours, and the figures of Ht-29cell's IC50were35.80mg/mL and31.94mg/mL. The cell expressed typically apoptosis characteristic such as cell shrinkage, formation of cytoplasmic filaments, condensation of nuclear chromatin, and loss of cell adherent capacity after interacting with WM2by observing the cell structure by the inverted microscope. Furthermore, flow cytometry results further illustrated the WM2induced Ht-29cell apoptosis. Correlation analysis between WM2antioxidant activity and Ht-29inhibition rate showed the correlation coefficient of cancer cell inhibition rate with hydroxy clearance rate was0.91(P<0.05), and the correlation coefficient with ABTS radical scavenging was0.90(P<0.05), and the correlation coefficient with reducing power was0.94(P<0.05).7. The effects of HRGS on WM2structure and function of WM2were studied. The results indicated that the dimer of L-tyrosine of WM2after oxidation exhibited a significant increase with increased H2O2concentration and oxidation time (P<0.05). Active thiol and free amino were both apparently declined (P<0.05). The oxidation made the structure of WM2change, which impacted its emulsifying function. Studies demonstrated that the emulsifying property and stability of WM2rose a little after O.lmmol/L of H2O2oxidation for1h (P>0.05), but the emulsifying property and stability of WM2significantly decreased with ascending concentrations of H2O2and extending the oxidation time (P<0.05).In conclusion, WPP-MRPs and their separation and purification products possess strong antioxidant activity and appropriate stability. Therefore, they can be extensively utilized in foods and health-care products. Besides, this research provides basis and ideas for expanding whey protein modification and developing natural and efficient antioxidants as well. |
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