Effects of protein and peptide addition on lipid oxidation in powder model system

The effect of protein and peptide addition on the oxidation of eicosapentaenoic acid ethyl ester (EPE) encapsulated by maltodextrin (MD) was investigated. The encapsulated lipid (powder lipid) was prepared in two steps, i.e., mixing of EPE with MD solutions (+/- protein and peptides) to produce emulsions and freeze-drying of the resultant emulsions. EPE oxidation in MD powder progressed more rapidly in the humid state [relative humidity (RH) = 70%] than in the dry state (RH = 10%). The addition of soy protein, soy peptide, and gelatin peptides improved the oxidation stability of EPE encapsulated by MD, and the inhibition of lipid oxidation by the protein and the peptides was more dramatic in the humid state. Especially, the oxidation of EPE was almost perfectly suppressed when the lipid was encapsulated with MD + soy peptide during storage in the humid state for 7 days. Several physical properties such as the lipid particle size of the emulsions, the fraction of nonencapsulated lipids, scanning electron microscopy images of powder lipids, and the mobility of the MD matrix were investigated to find the modification of encapsulation behavior by the addition of the protein and peptides, but no significant change was observed. On the other hand, the protein and peptides exhibited a strong radical scavenging activity in the powder systems as well as in the solution systems. These results suggest that a chemical mechanism such as radical scavenging ability plays an important role in the suppression of EPE oxidation in MD powder by soy proteins, soy peptides, and gelatin peptides.     

Antioxidant mechanisms of enzymatic hydrolysates of beta-lactoglobulin in food lipid dispersions

The antioxidant activities of aqueous phase beta-lactoglobulin (beta-Lg) and its chymotryptic hydrolysates (CTH) were compared in this study. Proteins and peptides have been shown to inhibit lipid oxidation reactions in oil-in-water emulsions; however, a more fundamental understanding of the antioxidant activity of these compounds in dispersed food lipid systems is lacking. CTH was more effective than an equivalent concentration of beta-Lg in retarding lipid oxidation reactions when dispersed in the continuous phase of Brij-stabilized oil-in-water emulsions (pH 7). Furthermore, it was observed that CTH had higher peroxyl radical scavenging and iron-binding values than beta-Lg. Liquid chromatography-mass spectrometry (LC-MS) was used to measure the rate of oxidation of three oxidatively labile amino acid residues (Tyr, Met, and Phe) in certain CTH peptide fragments. Significant oxidation of specific Tyr and Met residues present in two separate 12 amino acid peptide fragments was observed in the days preceding lipid oxidation (39 and 55% of Tyr and Met were oxidized, respectively, by day 4 of the study); however, no significant oxidation of the Phe residue present in a specific 14 amino acid peptide fragment could be observed during the same time period. These data could suggest that Met and Tyr residues are capable of scavenging radical species and have the potential to improve the oxidative stability dispersed food lipids.     

Amino acid and protein scavenging of radicals generated by iron/hydroperoxide system: an electron spin resonance spin trapping study

Reduction of free radicals generated by Fe(II)/cumene-hydroperoxide (CumOOH) by amino acids (Gly, Cys, Met, His, and Trp) and proteins (bovine serum albumin (BSA), beta-lactoglobulin, and lactoferrin) was followed by electron spin resonance spectroscopy using alpha-phenyl-N-tert-butylnitrone (PBN), 2-methyl-2-nitrosopropane (MNP), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin traps. The radical species detected were mostly carbon-centered radicals from CumOOH fragmentation (methyl/*H3 and ethyl/*H2CH3), although carbon-centered radicals originated from amino acids could be formed in the presence of Cys, Met, His, or Trp. All proteins and amino acids, except Cys, were effective at inhibiting generation of radicals from the Fe(II)/CumOOH system. Trp was the amino acid with the highest antiradical activity, followed by His > Gly approximately Met. Lactoferrin was the protein showing the most efficient inhibition of radical formation from the Fe(II)/CumOOH system, and BSA and beta-lactoglobulin were not significantly different in their antiradical activities. These results suggest that proteins with higher inhibitory activity on lipid oxidation promoted by transition metal catalytic decomposition of hydroperoxides should be those with elevated metal-chelating and radical-scavenging properties as well as low concentration and accessibility of reducing groups from amino acids capable of activating metals, such as sulfhydryl groups.     

Stability of spray-dried tuna oil emulsions encapsulated with two-layered interfacial membranes

omega-3 Fatty acids have numerous health benefits, but their addition to foods is limited by oxidative rancidity. Spray-drying tuna oil-in-water emulsion droplets with a coating of lecithin and chitosan multilayer system could produce emulsion droplet interfacial membranes that are cationic and thick, both factors that can help control lipid oxidation. Physicochemical and oxidative stability of the spray-dried emulsions were determined as a function of storage temperature and relative humidity (RH). The combination of ethylenediaminetetraacetic acid (EDTA) and mixed tocopherols was able to increase the oxidative stability of dried emulsions. Lipid oxidation was more rapid during storage at low relative humidity (11% and 33% compared to 52% RH). At high moisture, physical modifications in the sample were observed, including reduced dispersibility and formation of brown pigments. Sugar crystallization or Maillard products produced at the higher humidities may have inhibited oxidation. Overall, spray-dried tuna oil-in-water emulsions stabilized by lecithin-chitosan membranes were more oxidatively stable than bulk oils and thus have excellent potential as an omega-3 fatty acid ingredient for functional foods.     

海马水解蛋白的氨基酸组成与抗氧化能力的关系

为了探讨大海马蛋白质的高效利用方法,本试验将脱脂大海马粉经碱性蛋白酶和胰蛋白酶联合消解后,酶解液(PH)经超滤膜分离为5组分(PH-Ⅰ、PH-Ⅱ、PH-Ⅲ、PH-Ⅳ、PH-Ⅴ),结合各组分的氨基酸组成,回归分析了各组分的自由基清除能力与氨基酸组成间的相关关系。结果表明,酶解液经超滤膜分离的5组分中PH-Ⅴ(<2 500 Da)抗氧化能力较强,清除DPPH自由基能力为34.2%±0.1%,对超氧阴离子的清除能力为29.2%±0.1%,Fe³⁺还原力为0.28%±0.1%。海马酶解多肽各组成中疏水性氨基酸含量低于亲水性氨基酸,但疏水性氨基酸对自由基的清除能力起着决定性作用,极性氨基酸和非极性氨基酸可通过协同作用来增强海马多肽清除自由基的能力,且每个单位极性氨基酸的抗氧能力是非极性氨基酸的1.2倍,每个单位芳香族氨基酸的抗氧化能力是脂肪族氨基酸的2倍。本研究不仅制备了一类具有开发价值的抗氧化多肽,并且分析了多肽抗氧化能力与氨基酸组成的相关关系,为大海马的高值化利用提供了理论依据。     

Antioxidant activity of cysteine, tryptophan, and methionine residues in continuous phase beta-lactoglobulin in oil-in-water emulsions

Proteins dispersed in the continuous phase of oil-in-water emulsions are capable of inhibiting lipid oxidation reactions. The antioxidant activity of these proteins is thought to encompass both free radical scavenging by amino acid residues and chelation of prooxidative transition metals; however, the precise mechanism by which this occurs remains unclear. In this study, the oxidative stability of cysteine, tryptophan, and methionine residues in continuous phase beta-lactoglobulin (beta-Lg) in a Brij-stabilized menhaden oil-in-water emulsion was determined. The presence of low concentrations of continuous phase beta-Lg (250 and 750 microg/mL) significantly inhibited lipid oxidation as determined by lipid hydroperoxides and thiobarbituric acid reactive substances analysis. It was observed that cysteine oxidized before tryptophan in beta-Lg, and both residues oxidized before lipid oxidation could be detected. No oxidation of the methionine residues of beta-Lg was observed despite its reported high oxidative susceptibility. It is conceivable that surface exposure of amino acid residues greatly affects their oxidation kinetics, which may explain why some residues are preferentially oxidized relative to others. Further elucidation of the mechanisms governing free radical scavenging of amino acids could lead to more effective applications of proteins as antioxidants within oil-in-water food emulsions.     

Interface characterization and aging of bovine serum albumin stabilized oil-in-water emulsions as revealed by front-surface fluorescence

This paper is devoted to the application of front-surface fluorescence to the study of aging and oxidation of oil-in-water emulsions. Emulsions with two oil droplet sizes were stabilized with bovine serum albumin (BSA) and stored at 37 or 47 degrees C. Lipid oxidation was demonstrated by measurement of hydroperoxides and headspace pentane. Front-surface fluorescence spectra (excitation wavelength = 355 nm) revealed gradual formation of oxidized lipid-protein adducts during the 4 weeks of storage. Fluorescence (excitation = 290 nm) of BSA tryptophanyl residues (Trp) declined during the first day of aging and then decreased slightly and linearly. Fourth-derivative Trp spectra exhibited peaks at 316 and 332 nm. Their evolution indicated that the ratio of Trp in hydrophobic environments to total Trp increased in small droplet emulsions. This suggests that, during lipid oxidation, the adsorbed and nonadsorbed protein underwent various degrees of Trp degradations, polymerization, and aggregation. Thus, front-surface fluorescence makes it possible to evaluate, noninvasively, protein modification and lipid oxidation end-products during processing and storage of food emulsions.     

Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry

Two series of combinatorial tripeptide libraries were constructed, based on an antioxidative peptide isolated from a soybean protein hydrolysate. One was a library of 108 peptides containing either His or Tyr residues. Another was a library of 114 peptides related to Pro-His-His, which had been identified as an active core of the antioxidative peptide. The antioxidative properties of these libraries were examined by several methods, such as the antioxidative activity against the peroxidation of linoleic acid, the reducing activity, the radical scavenging activity, and the peroxynitrite scavenging activity. Two Tyr-containg tripeptides showed higher activities than those of two His-containing tripeptides in the peroxidation of linoleic acid. Tyr-His-Tyr showed a strong synergistic effects with phenolic antioxidants. However, the tripeptide had only marginal reducing activity and a moderate peroxynitrite scavenging activity. Cysteine-containing tripeptides showed the strong peroxynitrite scavenging activity. Change of either the N-terminus or C-terminus of Pro-His-His to other amino acid residues did not significantly alter their antioxidative activity. Tripeptides containing Trp or Tyr residues at the C-terminus had strong radical scavenging activities, but very weak peroxynitrite scavenging activity. The present results allow us to understand why protein digests have such a variety of antioxidative properties.     

Chemical and antioxidant properties of casein peptide and its glucose Maillard reaction products in fish oil-in-water emulsions

Maillard reaction products (MRPs) were prepared by reacting casein peptides with different concentrations of glucose at 80 °C for up to 12 h. The chemical properties of MRPs and their effects on lipid oxidation in fish oil-in-water emulsions were investigated. Increasing browning development and absorbance in 294 nm in the MRPs caused an increase in DPPH radical scavenging, but a decrease in iron chelation, which could be related to the loss of free amino groups in the peptides. The MRPs produced with longer reaction time or higher glucose concentrations were less effective in inhibiting lipid oxidation in emulsions at pH 7.0 compared to casein peptides alone. However, the antioxidant activity of MRPs in emulsions at pH 3.0 was not decreased by prolonged heating. The bitterness of MRPs was less than that of the original casein peptides, and bitterness decreased with increasing heating time and glucose concentrations. Therefore, the Maillard reaction was a potential method to reduce the bitterness of casein peptides while not strongly decreasing their antioxidant activity.     

Lipid oxidation in corn oil-in-water emulsions stabilized by casein,whey protein isolate,and soy protein isolate

Proteins can be used to produce cationic oil-in-water emulsion droplets at pH 3.0 that have high oxidative stability. This research investigated differences in the physical properties and oxidative stability of corn oil-in-water emulsions stabilized by casein, whey protein isolate (WPI), or soy protein isolate (SPI) at pH 3.0. Emulsions were prepared with 5% corn oil and 0.2-1.5% protein. Physically stable, monomodal emulsions were prepared with 1.5% casein, 1.0 or 1.5% SPI, and > or =0.5% WPI. The oxidative stability of the different protein-stabilized emulsions was in the order of casein > WPI > SPI as determined by monitoring both lipid hydroperoxide and headspace hexanal formation. The degree of positive charge on the protein-stabilized emulsion droplets was not the only factor involved in the inhibition of lipid oxidation because the charge of the emulsion droplets (WPI > casein > or = SPI) did not parallel oxidative stability. Other potential reasons for differences in oxidative stability of the protein-stabilized emulsions include differences in interfacial film thickness, protein chelating properties, and differences in free radical scavenging amino acids. This research shows that differences can be seen in the oxidative stability of protein-stabilized emulsions; however, further research is needed to determine the mechanisms for these differences.     

ACE-inhibitory and radical-scavenging activity of peptides derived from beta-lactoglobulin f(19-25). Interactions with ascorbic acid

In this work, the angiotensin-converting enzyme (ACE)-inhibitory and radical-scavenging activities of the beta-lactoglobulin (beta-Lg)-derived peptides WY f(19-20), WYS f(19-21), WYSL f(19-22), WYSLA f(19-23), WYSLAM f(19-24), and WYSLAMA f(19-25) have been determined. The ACE-inhibitory activity (IC50) varied from 38.3 to 90.4 microM, with the exception of WYS (>500 microM). All beta-Lg-derived peptides also exhibited radical-scavenging activity (oxygen radical absorbance capacity (ORAC) values ranged from 4.45 to 7.67 micromol Trolox equivalents/micromol of peptide). The presence and position of amino acids Trp, Tyr, and Met were proposed to be responsible for the antioxidant activity. The equimolar amino acid mixtures of all the peptides showed ORAC values lower than those of the corresponding peptides, indicating that the peptidic bond or the structural conformation had a positive influence on this activity. Finally, positive antioxidant effects of WYS, WYSL, and WYLA with ascorbic acid were observed, whereas WY and WYSLAM showed negative effects, both cases for different molar ratio mixtures. These results should be taken into account in the development of new food ingredients on the basis of peptides from beta-Lg.     

Antioxidant potential of hydroxycinnamic acid glycoside esters

Hydroxycinnamic acids are natural antioxidants found in fruits, vegetables, and cereals. In this study, the antioxidant activity of various types of hydroxycinnamoyl glycoside esters that mimic the structure of polymeric carbohydrates was studied in different model systems prone to oxidation, namely, liposomes and emulsions. In addition, radical scavenging activity against the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was tested. It was found that the esterification in the primary hydroxyl group of the glycoside resulted in the improved radical scavenging activity of both sinapoyl and feruloyl glycosides compared to conjugation to the secondary hydroxyl group. Increased activity was also observed, particularly in the case of feruloyl glucosides in inhibiting the oxidation of liposomes emulsions. The results showed that sinapic and ferulic acid glycoside esters were as effective or more efficient antioxidants than their free forms. In conclusion, the strength of their antioxidant effect depends on the nature of conjugation.     

Suppressive effect of saturated acyl L-ascorbate on the oxidation of linoleic acid encapsulated with maltodextrin or gum arabic by spray-drying

6-O-Palmitoyl L-ascorbate was added to linoleic acid at various molar ratios of the ascorbate to the acid, the mixtures were emulsified with a maltodextrin or gum arabic solution, and the emulsions were spray-dried to produce microcapsules. At higher molar ratios, the oil droplets in the emulsions were smaller, and the oxidative stabilities of the encapsulated linoleic acid were higher for both the maltodextrin- and gum arabic-based microcapsules. 6-O-Capryloyl, caproyl, and lauroyl L-ascorbates, which were synthesized through lipase-catalyzed condensation in acetone, were also used for the microencapsulation of linoleic acid. Except for capryloyl L-ascorbate, the addition of a saturated acyl ascorbate, especially caproyl ascorbate, to linoleic acid was effective for preparing oil droplets of small particle diameter and for suppressing the oxidation of the encapsulated linoleic acid.     

Caseins and casein hydrolysates. 2. Antioxidative properties and relevance to lipoxygenase inhibition

The antioxidant activity of caseins and casein-derived peptides was evaluated by using three free radical producing reactions-the lipoxygenase- and AAPH-catalyzed oxidation of linoleic acid and the hemoglobin-catalyzed oxidation of linoleic acid hydroperoxide. Caseins and casein-derived peptides were able to inhibit enzymatic and nonenzymatic lipid peroxidation, suggesting they were preferred targets for the free radical intermediates. The antioxidative feature was not lost with the dephosphorylation or the proteolysis of the proteins. The fractionation of the tryptic beta-casein digest yielded peptides with antioxidant activity. A structure-function relationship between the amino acid sequence and the antioxidant capacity and effectiveness is proposed. In addition, indirect evidence suggested that the trapping of free radicals by the proteins/peptides was accompanied by the oxidation of proteins/peptides, according to a sequence-specific mechanism.     

Lipid oxidation in emulsions as affected by charge status of antioxidants and emulsion droplets

The influence of charge status of both lipid emulsion droplets and phenolic antioxidants on lipid oxidation rates was evaluated using anionic sodium dodecyl sulfate (SDS) and nonionic polyoxyethylene 10 lauryl ether (Brij)-stabilized emulsion droplets and the structurally similar phenolic antioxidants gallamide, methyl gallate, and gallic acid. In nonionic, Brij-stabilized salmon oil emulsions at pH 7.0, gallyol derivatives (5 and 500 microM) inhibited lipid oxidation with methyl gallate > gallamide > gallic acid. In the Brij-stabilized salmon oil emulsions at pH 3.0, low concentrations of the galloyl derivatives were prooxidative or ineffective while high concentrations were antioxidative. In SDS-stabilized salmon oil emulsions, oxidation rates were faster and the galloyl derivatives were less effective compared to the Brij-stabilized emulsions. Differences in antioxidant activity were related to differences in the ability of the galloyl derivatives to partition into emulsion droplets and to increase the prooxidant activity of iron at low pH.     

Antioxidative activity of urate in bovine milk

The antioxidative effects of urate on peroxidase-induced protein oxidation and light-induced riboflavin degradation and lipid oxidation in whole milk were studied. In addition, experiments using ascorbate were conducted to directly compare the antioxidative activity of urate and ascorbate. The presence of urate and/or ascorbate (10-30 mg/L) lowered peroxidase-induced formation of dityrosine by 44-96% in unpasteurized whole milk. No synergistic effect of urate and ascorbate on peroxidase-induced dityrosine formation was registered, but merely an additive effect. Light exposure of pasteurized whole milk showed that ascorbate was oxidized at the expense of urate, which indicated ascorbate-mediated recycling of the urate radical. Moreover, both urate and ascorbate (30 mg/L) retarded light-induced lipid oxidation in pasteurized whole milk as measured by formation of lipid hydroperoxides with urate being the most effective (28% reduction in lipid hydroperoxides) compared with ascorbate (14%). Finally, addition of urate or ascorbate (300 mg/L) to pasteurized whole milk showed a slight protective effect against light-induced degradation of riboflavin with urate being the most effective.     

Size-exclusion chromatography of tea tannins and intercepting potentials of peptides for the inhibition of trypsin-caseinolytic activity by tea tannins

Molecular-weight distribution and characterization of tea tannin were investigated by high-performance liquid chromatography and the equivalent preparative exclusion gel chromatography using Sephadex G-25. The characteristics of the fractions were studied regarding the amounts of terminal catechin, sugar, and gallic acid, the color reaction of the Folin-Chiocalteu reagent, the UV absorbance, and the inhibition activity for the trypsin-caseinolytic activity per weight. Furthermore, we investigated the intercepting activities of the inhibition by the amino acids, peptides, their analogues, poly(ethylene glycol)s (PEGs), and histatin 5 using the inhibition of trypsin-caseinolytic activity by tea. Arg, Lys, and their peptides had strong intercepting activities for the inhibition, but only a weak activity was detected in the Pro peptides or gelatin-like peptides of (Pro-Pro-Gly)(n) (n = 5 or 10). The guanidyl group of Arg and the amino methylene group of Lys were important for the intercepting activity, but the activity was weakly dependent upon the peptide bond formation. The intercepting activity of the peptides or PEG exponentially increased with the number of polymerizations. Histatin 5 did not have a remarkably strong intercepting activity considering the peptide length. The activity of the synthetic histatin 5 in which all of the Lys and Arg were substituted by Ala was at the same level as histatin 5.     

Role of continuous phase protein on the oxidative stability of fish oil-in-water emulsions

Whey protein isolate (WPI), soy protein isolate (SPI), and sodium caseinate (CAS) can inhibit lipid oxidation when they produce a positive charge at the interface of emulsion droplets. However, when proteins are used to stabilize oil-in-water emulsions, only a fraction of them actually absorb to the emulsion droplets, with the rest remaining in the continuous phase. The impact of these continuous phase proteins on the oxidative stability of protein-stabilized emulsions is not well understood. WPI-stabilized menhaden oil-in-water emulsions were prepared by high-pressure homogenization. In some experiments WPI was removed from the continuous phase of the emulsions through repeated centrifugation and resuspension of the emulsion droplets (washed emulsion). Unwashed emulsions were more oxidatively stable than washed emulsions at pH 7.0, suggesting that continuous phase proteins were antioxidative. The oxidative stability of emulsions containing different kinds of protein in the continuous phase decreased in the order SPI > CAS > WPI, as determined by both hydroperoxide and headspace propanal formation. Iron-binding studies showed that the chelating ability of the proteins decreased in the order CAS > SPI > WPI. The free sulfhydryls of both WPI and SPI were involved in their antioxidant activity. This research shows that continuous phase proteins could be an effective means of protecting omega-3 fatty acids from oxidative deterioration.     

Spray-dried multilayered emulsions as a delivery method for omega-3 fatty acids into food systems

Emulsion can be produced with electrostatic layer-by-layer deposition technologies to have cationic, thick multilayer interfacial membranes that are effective at inhibiting the oxidation of omega-3 fatty acids. This study investigated the stability of spray-dried multilayer emulsion upon reconstitution into an aqueous system. The primary (lecithin) and multilayered secondary emulsions (lecithin and chitosan) were spray-dried with corn syrup solids (1-20 wt %). The lecithin-chitosan multilayer interfacial membrane remained intact on the emulsion droplets upon reconstitution into an aqueous system. Reconstituted secondary (lecithin-chitosan) emulsions were more oxidatively stable than reconstituted primary (lecithin) emulsions. A minimum of 5 wt % corn syrup solids was needed to microencapsulate the secondary emulsion droplets. Maximum oxidative stability of both the powder and the reconstituted secondary emulsions was observed in samples with 5% and 20% corn syrup solids. Addition of EDTA (25 microM) inhibited oxidation of reconstituted primary and secondary emulsions. These studies suggest that a microencapsulated multilayered emulsion system could be used as a delivery system for omega-3 fatty acids in functional foods.