Fractionation, separation, and identification of antioxidative peptides in potato protein hydrolysate that enhance oxidative stability of soybean oil emulsions

Abstract: The objective of the study was to identify the active peptides responsible for the antioxidant activity of potato protein hydrolysate (PPH). PPH was fractionated using ammonium sulfate precipitation; the efficacy of different fractions for scavenging 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^{+ •}) radicals and inhibiting lipid oxidation (hexanal, TBARS) in soybean oil-in-water emulsions was investigated. Of all fractions, the fraction precipitated by 50% saturated ammonium sulfate (P50) exhibited the strongest ABTS^{+ •} scavenging activity and antioxidant activity. Active peptides based on the ABTS^{+ •} scavenging assay were isolated and purified by RP-HPLC and ultra performance liquid chromatography. Amino acid sequencing by tandem mass spectrometry identified Ser-Ser-Glu-Phe-Thr-Tyr and Ile-Tyr-Leu-Gly-Gln in P50 to be the dominant peptides that matched the sequences in metallocarboxypeptidase inhibitor and lipoxygenase 1, respectively.     

Effect of concentrated flaxseed protein on the stability and rheological properties of soybean oil-in-water emulsions

Flaxseed protein concentrate containing-mucilage (FPCCM) was used to stabilize soybean oil-in-water emulsions. The effects of FPCCM concentration (0.5, 1.0, 1.5% w/v) and oil-phase volume fraction (5, 10, 20% v/v) on emulsion stability and rheological properties of the soybean oil-in-water emulsions were investigated. Z-average diameter, zeta-potential, creaming index and rheological properties of emulsions were determined. The result showed that FPCCM concentration significantly affected zeta-potential, creaming rate and emulsion viscosity. The increasing of FPCCM concentration led to a more negative charged droplet and a lower creaming rate. Oil-phase volume fraction significantly affected Z-average diameter, rheological properties, creaming index and creaming rate. With the increase of oil-phase volume fraction, both Z-average diameter and emulsion viscosity increased, while creaming index and creaming rate decreased. The rheological curve suggested that the emulsions were shear-thinning non-Newtonian fluids.     

大豆蛋白水解产物性能的研究

对大豆蛋白水解产物的性能进行了研究。结果表明,其水解产物的黏度显著降低;具有良好的钙包溶性;在水解时间3~4 h时乳化稳定性最强;麦芽糊精、脱脂奶粉、乳清粉、蔗糖酯对大豆分离蛋白水解产物的乳化稳定性有增加作用。     

Antioxidant activity and functional properties of porcine plasma protein hydrolysate as influenced by the degree of hydrolysis

Antioxidant activity and functional properties of porcine blood plasma protein hydrolysates (PPH) prepared with Alcalase at 6.2%, 12.7% and 17.6% of degree of hydrolysis (DH) were investigated. The PPH showed stronger radical-scavenging ability and possessed stronger Cu²⁺-chelation ability and a reducing power compared to non-hydrolysed plasma protein (P < 0.05). The antioxidant activity of PPH, indicated by thiobarbituric acid-reactive substance (TBARS) values in a liposome-oxidising system, increased with increasing DH (P < 0.05). The Alcalase hydrolysis increased protein solubility from its original 68.46–81.79% (non-hydrolysed) to 82.95–94.94% (hydrolysed) over a broad pH range (3.0–8.0). However, hydrolysis decreased surface hydrophobicity and suppressed emulsifying and foaming capacity of the plasma protein. To identify antioxidant peptide, PPH was subjected to ultrafiltration, ion-exchange chromatography and reverse-phase high performance liquid chromatography (RP-HPLC), and the amino acid sequences of isolated peptides were determined by liquid chromatography/tendem mass spectrometry (LC–MS/MS). The peptide with the strongest antioxidant activity had the amino acid sequence of His-Asn-Gly-Asn. The results indicated that PPH could be used as a novel antioxidant but may be of limited utility as an emulsifying or foaming agent.     

大豆油乳状液体系氧化稳定性影响因素的研究

研究了乳状液体系中不饱和脂肪酸的氧化机制，重点探讨了乳化剂种类、用量、pH和EDTA对大豆油乳化体系（O／W）氧化稳定性的影响。结果显示：乳化剂种类和pH对于乳状液体系的氧化稳定性有显著影响，对于阴离子乳化剂SDS稳定的乳状液，pH4．0的氧化速率比pH7．0和9．0快得多；对于非离子乳化剂Tween20稳定的乳状液，pH的影响不显著；对于阳离子乳化剂CTAB稳定的乳状液，随着pH的升高，氧化速率变快；同时在pH4．0条件下，氧化速率SDS〉CTAB〉Tween20，而在pH7．0和9．0条件下，氧化速率CTAB〉SDS≌Tween20。乳状液体系中自带的微量金属离子对于体系也有相当大的影响，随着金属离子螯合剂EDTA浓度的增加，其乳状液氧化速率显著降低。乳化剂用量也会影响体系的氧化稳定性，随着乳化剂用量的增加，乳状液的氧化稳定性变差，这种稳定性的弱化是由于粒径变小、油滴表面积增大引起的。     

Design of interfacial films to control lipid oxidation in oil-in-water emulsions

In oil-in-water (O/W) emulsions, the droplets covered by native proteins are more prone to oxidation than droplets covered by surfactants. We attempted in this work to improve the barrier properties of protein-stabilized interfacial layers by controlled modifications of their composition and structure. Native bovine β-lactoglobulin (BLG) or β-casein (BCN), partially aggregated BLG and mixtures of the proteins with dilauroyl phosphatidylcholine (DLPC) were used to prepare emulsions and reconstituted Langmuir–Blodgett films. Lipid oxidation in the emulsions, as evaluated from oxygen uptake and formation of conjugated dienes, propanal and hexanal was roughly unmodified with aggregated BLG and DLPC–BLG mixtures and even favored with DLPC–BCN mixtures. The reconstituted phospholipid/protein interfacial layers presented interfacial heterogeneity evidenced by atomic force microscopy (AFM). This indicates that the structural homogeneity of the interface could be a key factor in controlling lipid oxidation.     

Ability of flaxseed and soybean protein concentrates to stabilize oil-in-water emulsions

The ability of flaxseed protein concentrate (FPC) to stabilize soybean oil-in-water emulsion was compared with that of soybean protein concentrate (SPC). The stability of emulsions increased with increase in protein concentration. The FPC-stabilized emulsions had smaller droplet size and higher surface charge, but worse stability at the same protein concentration compared to SPC-stabilized emulsions. Oil-in-water emulsions stabilized by both proteins were diluted and compared at different pH values (3–7), ionic strength (0–200 mM NaCl) and thermal treatment regimes (25–95 °C for 20 min). Considerable emulsion droplet flocculation occurred around iso-electric point of both proteins: FPC (pH 4.2) and SPC (pH 4.5). FPC and SPC-stabilized emulsions remained relatively stable against droplet aggregation and creaming at NaCl concentration below 100 and 50 mM, respectively. The emulsions stabilized by both proteins were fairly stable within these thermal processing regimes. FPC appears to be less effective as an emulsifier compared to SPC due to its lower emulsion viscosity. Hence, FPC could be more effective in emulsions that are fairly viscous.     

Oxidative stability of oil-in-water emulsions stabilised with protein or surfactant emulsifiers in various oxidation conditions

Many studies have investigated the effect of emulsifiers on the oxidative stability of oil-in-water (O/W) emulsions. A better oxidative stability of surfactant-stabilised O/W emulsions as compared to protein-stabilised emulsions has been recently shown in conditions when the major part of the emulsifier is adsorbed at the oil-water interface and oxidation is induced by iron−ethylenediaminetetraacetic acid (EDTA) complex. In this work, the contribution of the interfacial layer to the oxidation of emulsified lipids is investigated under various incubation conditions, involving different oxidation mechanisms. O/W emulsions were formulated at pH 6.7 with limited amounts of emulsifiers in the aqueous phase. Emulsions were incubated either at 33 °C without initiator at 25 °C in the presence of iron/ascorbate, metmyoglobin or 2,2′-azobis(2-amidinopropane)-dihydrochloride (AAPH). Oxygen uptake and volatile compound formation confirmed that protein-stabilised emulsions are less oxidatively stable than Tween 20-stabilised ones. This work also shows complex oxidative interrelationships between oxidation initiator and certain proteins, such as β-casein and bovine serum albumin.     

Antioxidant properties of protein hydrolysate from Douchi by membrane ultrafiltration

Protein extracted from Douchi was hydrolyzed by alcalase based on a single-factor experiment and response surface methodology was employed to optimize hydrolysis conditions. The optimum conditions were determined to be: 63℃ extraction temperature, 1.4% quantities of enzyme and substrate, and 1.7 h extraction time. The optimal protein hydrolysate was subsequently subjected to ultrafiltration, and the fraction of molecular weight 10–50 kDa was identified as the most potent contributor to antioxidant activity. Amino acid analysis indicated that acidic amino acids were the most abundant in all fractions of the protein hydrolysate. Infrared spectroscopy showed there were no significant differences in secondary structure between fractions.     

Interfacial properties of deamidated wheat protein in relation to its ability to stabilise oil-in-water emulsions

Isolated wheat protein (IWP) is an acidic deamidated wheat protein. The deamidation process enhances the protein solubility at pHs greater than 6, and therefore its potential ability to act as a food emulsifier. The interfacial properties and the mechanism by which this protein stabilises oil-in-water emulsions were investigated by measuring the protein's absorbed layer thickness on latex particles, its interfacial rheology, and the colloidal and thermal stability of IWP stabilised emulsions. IWP forms a relatively thick interfacial layer of 18 nm upon adsorption onto latex beads, suggesting that the protein adsorbed with the long axis perpendicular to the surface, i.e. end-on, at a full protein coverage. The interfacial rheology measurement showed that IWP formed a relatively weak fluid-like interface. Similar to other protein emulsifiers, the colloidal stability of IWP emulsions is provided largely through electrostatic repulsion. Although IWP emulsions were sensitive to salt induced flocculation, the presence of excess protein in the aqueous phase (e.g. 4 wt%) was able to reduce the effect of salt screening (50 mM CaCl₂) on a 25 wt% oil-in-water emulsion completely. The emulsions underwent minimal coalescence when droplets were in close contact, e.g. flocculated, because the interfacial layer of IWP provides a barrier to droplet coalescence, even in high salt environments. IWP emulsions were resistant to thermal treatment with no changes in particle size observed when the emulsions were heated (up to 90 °C for 20 min) in the absence or the presence of 150 mM NaCl. The heat stability of IWP emulsions is thought to arise from the structure of IWP at the interface. A lack of free cysteines combined with few hydrophobic regions meant that there were minimal interactions between protein molecules adsorbed onto the same droplet or on neighbouring droplets. The unique interfacial properties of IWP, e.g. its physical layer thickness and the structure provide enhanced stability for emulsions against coalescence and heating.     

Antioxidant and free radical-scavenging activities of chickpea protein hydrolysate (CPH)

Chickpea protein hydrolysate (CPH) was fractionated by gel filtration on Sephadex G-25. The antioxidant and free radical-scavenging activities of four CPH fractions (Fra.I, Fra.II, Fra.III, and Fra.IV) were measured using reducing power, inhibition of linoleic acid autoxidation, and 1,1-diphenyl-2- pycrylhydrazyl (DPPH)/superoxide/hydroxyl radical-scavenging assay. The antioxidant activity of Fra.IV (81.13%) was closer to that of α-tocopherol (83.66%) but lower than that of BHT (99.71%) in the linoleic acid oxidation system. Amino acid analyses showed that Fra.IV with the strongest antioxidant activity also had the highest total hydrophobic amino acids content (38.94% THAA) and hydrophobicity (125.62 kcal/mol amino acid residue) compared with the other three fractions. The molecular weight distribution of Fra.IV was found to vary from 200 to 3000 Da.     

Influence of chitosan concentration on the stability,microstructure and rheological properties of O/W emulsions formulated with high-oleic sunflower oil and potato protein

Relatively concentrated (40 wt%) O/W emulsions formulated with high-oleic sunflower oil as disperse phase, potato protein isolate as emulsifier and chitosan as stabiliser were prepared by rotor–stator/high-pressure valve/rotor–stator homogenization. The influence of chitosan concentration on the physical stability of emulsions was studied in (0.25–1) wt% range by visual inspection, rheological and microstructural techniques. Steady shear flow curves were sensitive to the occurrence of creaming upon the rise of zero-shear viscosity values. The effect of increasing concentration of chitosan on the zero-shear viscosity turned out to be dependent on emulsion ageing and always resulted in a stepwise increase of the critical shear rate for the onset of shear thinning flow. The critical oscillatory shear stress for the onset of non-linear viscoelastic behaviour was more sensitive than the critical shear rate to detect creaming in emulsions. Mechanical spectra are definitely demonstrated to be the most powerful tool to detect not only creaming but also oil droplet flocculation on account of changes in the plateau relaxation zone. CSLM micrographs supported the interpretation of dynamic viscoelastic results, especially when flocculation as well as coalescence took place. Cryo-SEM micrographs evidenced the formation of increasingly denser protein–polysaccharide networks with chitosan concentration and the fact that the latter governs the microstructure of the emulsion when reaches 1 wt% concentration promoting enhanced physical stability.     

Production and properties of casein hydrolysate microencapsulated by spray drying with soybean protein isolate

The aim of this work was to encapsulate casein hydrolysate by spray drying with soybean protein isolate (SPI) as wall material to attenuate the bitter taste of that product. Two treatments were prepared: both with 12 g/100 g solids and containing either two proportions of SPI: hydrolysate (70:30 and 80:20), called M1 and M2, respectively. The samples were evaluated for morphological characteristics (SEM), particle size, hygroscopicity, solubility, hydrophobicity, thermal behavior and bitter taste with a trained sensory panel using a paired-comparison test (non-encapsulated samples vs. encapsulated samples). Microcapsules had a continuous wall, many concavities, and no porosity. Treatments M1 and M2 presented average particle sizes of 11.32 and 9.18 μm, respectively. The wall material and/or the microencapsulation raised the hygroscopicity of the hydrolysate since the free hydrolysate had hygroscopicity of 53 g of water/100 g of solids and M1 and M2 had 106.99 and 102.19 g of water/100 g of solids, respectively. However, the hydrophobicity decreases, the absence of a peak in encapsulated hydrolysates, and the results of the panel sensory test considering the encapsulated samples less bitter (p < 0.05) than the non-encapsulated, showed that spray drying with SPI was an efficient method for microencapsulation and attenuation of the bitter taste of the casein hydrolysate.     

Phytosterol oxidation in oil-in-water emulsions and bulk oil

Dietary plants sterols (phytosterols) have been shown to lower plasma cholesterol level in humans. Since phytosterols may protect against coronary heart diseases, they are being incorporated into functional foods. However, phytosterols are susceptible to oxidative degradation. The purpose of this study was to evaluate the formation of phytosterols oxidation products (POPs) in oil-in-water emulsions and bulk corn oil. The extent of lipid oxidation was monitored by measuring the lipid hydroperoxides and hexanal, whereas 7-keto derivatives of phytosterols were determined by gas chromatography to follow sterol oxidation. A higher POPs level and formation rate was found in the oil-in-water (o/w) emulsion than in the bulk oil. Interfacial tension measurements showed that phytosterols had a high degree of surface activity, which would allow them to migrate to the oil–water interface of the emulsion droplets where oxidative stress is high.     

Physicochemical properties of whey protein isolate stabilized oil-in-water emulsions when mixed with flaxseed gum at neutral pH

Concentrations ranging from 0% to 0.33% (w/v) of gum (Emerson and McDuff) were added to the emulsions at pH 7. Particle size distribution, viscosity, ζ-potential, microstructure, and phase separation kinetics of the emulsions were observed. Both polysaccharides and protein coated droplets are negatively charged at this pH, as shown by ζ-potential measurements. At all the concentrations tested, the addition of gum did not affect significantly (p < 0.05) the apparent diameter of the emulsion droplets. At low concentrations (gum  0.075% (w/v)), no visual phase separation was observed and the emulsion showed a Newtonian behaviour. However, at concentrations above the critical concentration of gum, depletion flocculation occurred: when 0.1 flaxseed gum was present, there was visual phase separation over time and the emulsion exhibited shear-thinning behaviour. These results demonstrate that flaxseed gum is a non-interacting polysaccharide at neutral pH; it could then be employed to strengthen the nutritional value of some milk-based drinks, but at limited concentrations.     

Impact of free fatty acid concentration and structure on lipid oxidation in oil-in-water emulsions

Free fatty acids are strong prooxidants in both bulk and emulsified oils. Addition of oleic acid to an oil-in-water emulsions increased lipid hydroperoxide and hexanal formation at free fatty acid concentrations as low as 0.1% of the lipid. The prooxidant effect of free fatty acids was dependent on fatty acid type with lipid oxidation rates being in the order of linolenic < linoleic < oleic. There were no significant differences in lipid oxidation rates when free fatty acid isomers with cis or trans double bonds were compared. The prooxidant activity of the free fatty acids was postulated to be due to their ability to attract prooxidant metals as well as co-oxidise the triacylglycerol in the oil. Overall, these results show that the oxidative stability of oil-in-water emulsions is strongly linked to both the concentration and type of free fatty acids present.     

Heat-induced changes in oil-in-water emulsions stabilized with soy protein isolate

The physicochemical properties of soy proteins stabilized oil-in-water emulsions were studied after heating at two different temperatures, 75 and 95 °C. The effect of changing the order of the process (heating the solution before emulsification, or heating the emulsion) was also studied. The heating temperatures were chosen as they are known to selectively cause denaturation of the two major proteins present in the soy protein isolate: β-conglycinin and glycinin. The thermal transitions observed for soy proteins adsorbed at the interface were different from those measured in protein solutions, suggesting that some changes occur in the structure of the soy proteins upon adsorption on the oil droplet. Heating induces aggregation of the oil droplets, as shown by an increase of the particle size and the bulk viscosity of the emulsions, with a more prominent effect after heating at 95 °C. Transmission electron microscopy observations clearly demonstrate that heating induces the formation of large protein aggregates at the interface. In addition, the composition of the protein present at the interface changes depending on the order of heating and homogenization. While heating the solutions before emulsification results in all the protein subunits to be present at the interface in an aggregated form, when heating is applied after emulsification, a portion of the α and the α′ subunit of β-conglycinin as well as the acidic subunits of glycinin remain unadsorbed.     

Antioxidative properties of protein hydrolysate from defatted peanut kernels treated with esperase

The aim of this study was to facilitate development of natural antioxidants from defatted peanut kernels. Protein hydrolysates obtained from defatted peanut kernels with esperase treatment for 2 h exhibited higher antioxidative activity toward linolenic acid peroxidation than other proteases (including neutrase, pepsin, protease A and protease N). The esperase hydrolysate of peanut protein (EHPP) was further separated with 3 and 5 kDa molecular cut-off membranes to determine the influence of molecular weight. EHPP with molecular weight 3∼5 kDa showed higher relative antioxidative activity, DPPH radical scavenging activity and metal chelating activity than that with molecular weight lower than 3 kDa or higher than 5 kDa. The fraction was further purified by ion exchange chromatography and high-performance liquid chromatography; the final peanut peptides exhibited higher relative antioxidative activity (27.5) than ascorbic acid (9.5). This study reported for the first time that protein hydrolysates from defatted peanut kernels possess antioxidative activity.     

Fluorescence spectroscopy as a novel approach for the assessment of myofibrillar protein oxidation in oil-in-water emulsions

The oxidation of oil-in-water emulsions (37 °C/10 days) containing increasing levels of myofibrillar proteins (MP) (0.5%, 1% and 2% on the basis of lipid content) was investigated. Protein oxidation was assessed by measuring the loss of natural tryptophan fluorescence and the increase in fluorescent protein oxidation products (FP) using fluorescence spectroscopy. Lipid oxidation was simultaneously analysed by measuring the increase of conjugated dienes (CD) and hexanal. The oxidative degradation of tryptophan residues occurred as an early event in MP oxidation whereas FP formed later as secondary protein oxidation products. The shift of the tryptophan maximum fluorescence emission wavelength during oxidation provided information about the location of oxidising tryptophan residues. Emulsions with higher concentrations of MP displayed higher tryptophan fluorescence and yielded a higher amount of FP. MP acted as inhibitors of lipid oxidation because emulsions with higher MP contents contained lower levels of CD and hexanal. Significant negative correlations were found between tryptophan fluorescence and CD, reflecting the timely interaction between primary lipid oxidation products and protein oxidation. Both spectrophotometric techniques were useful although the loss of tryptophan fluorescence is more reliable since it is a specific measurement which is not affected by the presence of other fluorescent protein oxidation products.