| 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.
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