Interactions between bovine beta-lactoglobulin and peptides under different physicochemical conditions

The aim of this study was to determine if peptides could interact with beta-lactoglobulin (beta-LG) and what the physicochemical conditions promoting their interaction with the protein are. The binding of negatively charged (beta-LG 125-135 and 130-135), positively charged (beta-LG 69-83 and 146-149), and hydrophobic (alphaS1-CN 23-34 and beta-LG 102-105, both bioactive peptides) peptides to bovine beta-LG was determined using an ultrafiltration method under different physicochemical conditions: pH 3.0, 6.8, and 8.0; buffers of 0.05 and 0.1 M; 4, 25, and 40 degrees C; beta-LG/peptide ratios of 1:5 and 1:10. At pH 3.0, none of the peptides interacted with beta-LG at any temperature, buffer molarity, or beta-LG/peptide ratio probably due to electrostatic repulsions between the highly protonated species. At pH 6.8 and 8.0, charged peptides beta-LG 130-135, 69-83, and 146-149 bound to beta-LG under some physicochemical conditions, possibly by nonspecific binding. However, both hydrophobic peptides probably bind to the inner cavity (beta-barrel) of beta-LG, provoking the release of materials absorbing at 214 nm. Given the known biological activities of the hydrophobic peptides used in this study (opioid and ACE-inhibitory activities), their binding to beta-LG may be relevant to a better understanding of the physiological function of the protein.     

基于前表面荧光光谱鉴别新鲜与冻融大黄鱼

为研究反复冻融对水产品品质的影响,通过理化方法检测了不同冻融次数处理对大黄鱼解冻损失、pH 值、色泽、硫代巴比妥酸值、羰基含量等指标的影响,并采用前表面荧光光谱结合主成分分析（principal component analysis,PCA）和 Fisher 线性判别分析法（Fisher linear discriminant analysis,FLDA）对不同冻融次数的大黄鱼进行区分。结果显示随着冻融次数增加,大黄鱼的解冻损失显著增加（P<0.05）;pH 值呈先上升后下降的趋势;L*（亮度）值、b*（黄度）值均有不同程度的增加（P<0.05）,a*（红度）值下降（P<0.05）;羰基含量和硫代巴比妥酸反应物值（thiobarbituric acid reactive substances,TBARS）增加（P<0.05）,反复冻融导致大黄鱼的品质下降。色氨酸和烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide,NADH）的荧光光谱分别结合 PCA 和 FLDA 对不同冻融处理组进行分析,结果表明 FLDA 识别效果优于 PCA。通过 FLDA 建立了新鲜大黄鱼与冻融大黄鱼荧光光谱判别模型,发现色氨酸原始判别的准确率和交叉验证的准确率分别为68.3%和66.7%,NADH 原始判别的准确率和交叉验证的准确率均达到100%。由此可见,利用 NADH荧光光谱结合化学计量分析可以鉴别不同冻融处理的大黄鱼。研究结果为水产品新鲜度的快速评价提供参考。     

Interactions of vitamin D3 with bovine beta-lactoglobulin A and beta-casein

It is of nutritional significance to fortify processed dairy products (e.g., cheese, yogurt, and ice cream) with vitamin D3; however, the inherent complexity of these foods may influence the stability and bioavailability of this nutrient. In the present study, the interactions of vitamin D3 with beta-lactoglobulin A and beta-casein were investigated under various environmental conditions (i.e., pH and ionic strength) using fluorescence and circular dichroism spectroscopic techniques. The results indicated that vitamin D3 was bound to both beta-lactoglobulin A and beta-casein depending on the solution conditions. The apparent dissociation constants ranged from 0.02 to 0.29 microM for beta-lactoglobulin A, whereas the beta-casein apparent dissociation constants ranged from 0.06 to 0.26 microM. The apparent mole ratios were also comparable, i.e., 0.51-2.04 and 1.16-2.05 mol of vitamin D3 were bound per mole of beta-lactoglobulin A and beta-casein, respectively. It was concluded that these interactions may strongly influence vitamin D3 stability and, hence, bioavailability in processed dairy products.     

Authentication of the botanical and geographical origin of honey by front-face fluorescence spectroscopy

Front-face fluorescence spectroscopy, directly applied on honey samples, was used for the authentication of 11 unifloral and polyfloral honey types (n = 371 samples) previously classified using traditional methods such as chemical, pollen, and sensory analysis. Excitation spectra (220-400 nm) were recorded with the emission measured at 420 nm. In addition, emission spectra were recorded between 290 and 500 nm (excitation at 270 nm) as well as between 330 and 550 nm (excitation at 310 nm). A total of four different spectral data sets were considered for data analysis. Chemometric evaluation of the spectra included principal component analysis and linear discriminant analysis; the error rates of the discriminant models were calculated by using Bayes' theorem. They ranged from <0.1% (polyfloral and chestnut honeys) to 9.9% (fir honeydew honey) by using single spectral data sets and from <0.1% (metcalfa honeydew, polyfloral, and chestnut honeys) to 7.5% (lime honey) by combining two data sets. This study indicates that front-face fluorescence spectroscopy is a promising technique for the authentication of the botanical origin of honey and may also be useful for the determination of the geographical origin within the same unifloral honey type.     

Heat treatment of beta-lactoglobulin: structural changes studied by partitioning and fluorescence

Functional properties of whey protein concentrates (WPC) are primarily dependent on the degree of denaturation of beta-lactoglobulin (beta-LG), the major globular whey protein. Irreversible modifications in the tertiary structure and association state of beta-LG after heat treatment were studied by partition in aqueous two-phase systems and fluorescence quenching. Partitioning of preheated beta-LG in two-phase systems containing 5% (w/w) poly(ethylene glycol) and 7% (w/w) dextran, between pH 6.0 and7.0, are appropriately related with the intensity of heat treatment. An increase in the partition coefficient of beta-LG was observed with increasing temperature of heat treatment. On the other hand, fluorescence quenching of beta-LG by acrylamide was used to study the conformational flexibility of the protein at pH values between 4. 0 and 9.0. The values of bimolecular quenching rate constant (k(q)) obtained showed that beta-LG appears to be more flexible at high pH values, while at low pH the protein assumes a more compact form. The efficiency of acrylamide quenching on preheated beta-LG was substantially more pronounced than for the untreated protein. This difference can be ascribed to the presence of unfolded monomers and aggregates of denatured molecules formed after heat treatment, whose tryptophanyl residues are more exposed to the solvent. In conclusion, the results suggest that partition studies in aqueous two-phase systems and fluorescence quenching are very useful tools to detect changes in conformation and aggregation of beta-LG induced by heat treatment.     

Authentication of the botanical origin of honey by front-face fluorescence spectroscopy. A preliminary study

The potential of front-face fluorescence spectroscopy for the authentication of unifloral and polyfloral honey types (n = 57 samples) previously classified using traditional methods such as chemical, pollen, and sensory analysis was evaluated. Emission spectra were recorded between 280 and 480 nm (excit: 250 nm), 305 and 500 nm (excit: 290 nm), and 380 and 600 nm (excit: 373 nm) directly on honey samples. In addition, excitation spectra (290-440 nm) were recorded with the emission measured at 450 nm. A total of four different spectral data sets were considered for data analysis. After normalization of the spectra, chemometric evaluation of the spectral data was carried out using principal component analysis (PCA) and linear discriminant analysis (LDA). The rate of correct classification ranged from 36% to 100% by using single spectral data sets (250, 290, 373, 450 nm) and from 73% to 100% by combining these four data sets. For alpine polyfloral honey and the unifloral varieties investigated (acacia, alpine rose, honeydew, chestnut, and rape), correct classification ranged from 96% to 100%. This preliminary study indicates that front-face fluorescence spectroscopy is a promising technique for the authentication of the botanical origin of honey. It is nondestructive, rapid, easy to use, and inexpensive. The use of additional excitation wavelengths between 320 and 440 nm could increase the correct classification of the less characteristic fluorescent varieties.     

Interactions between beta-lactoglobulin and pectins during in vitro gastric hydrolysis

This paper deals with the influence of different levels of three pectins, low-methylated pectin (LMP), high-methylated pectin (HMP), and low-methylated and amidated pectin (LMA), on the in vitro gastric hydrolysis of beta-lactoglobulin (beta-lg). Proteolysis by pepsin consisted of a 2-h progressive reduction of pH. A turbidity measurement of beta-lg-pectin mixtures was carried out during the proteolysis. The influence of pectins on pepsin enzymatic activity was also evaluated. beta-Lg was resistant to peptic digestion. The presence of each of the three pectins at a concentration of 50 wt % increased the N release at all pH values considered, despite a significant inhibition of the pepsin enzymatic activity with the pectins. The turbidity of beta-lg solutions during proteolysis was reduced by the addition of pectins, because of the formation of electrostatic complexes between this protein and pectins. The increase of N release could be a false positive result due to the difficulty of precipitating protein by trichloroacetic acid because of the formation of electrostatic complexes demonstrated by the decrease of turbidity.     

Interactions between beta-lactoglobulin and aroma compounds: different binding behaviors as a function of ligand structure

Interactions between beta-lactoglobulin (BLG) in its monomeric form and a wide range of aroma compounds were investigated by Fourier transform infrared (FT-IR) and 2D nuclear magnetic resonance (NMR) spectroscopies. A screening of the ligands was carried out by FT-IR through the amide I region changes of BLG upon binding. The location of two binding sites was determined by 2D NMR from the study of 10 selected ligands with different structures. All of the data suggest at least two binding behaviors as a function of the chemical class, the hydrophobicity, or the structure of the ligands. The binding of the elongated aroma compounds, such as 2-nonanone or ethyl pentanoate, within the central cavity involves residues located at the entrance of the calyx and Trp19. The binding onto the protein surface of aroma compounds that have or adopt a compact structure occurs in a site located between strand beta-G, alpha helix, and strand beta-I.     

Reactivity of bovine whey proteins, peptides, and amino acids toward triplet riboflavin as studied by laser flash photolysis

The reaction between the triplet excited state of riboflavin and amino acids, peptides, and bovine whey proteins was investigated in aqueous solution in the pH range from 4 to 9 at 24 degrees C using nanosecond laser flash photolysis. Only tyrosine and tryptophan (and their peptides) were found to compete with oxygen in quenching the triplet state of riboflavin in aqueous solution, with second-order rate constants close to the diffusion limit, 1.75 x 10(9) and 1.40 x 10(9) L mol(-1) s(-1) for tyrosine and tryptophan, respectively, with beta-lactoglobulin and bovine serum albumin having comparable rate constants of 3.62 x 10(8) and 2.25 x 10(8) L mol(-1) s(-1), respectively. Tyrosine, tryptophan, and their peptides react with the photoexcited triplet state of riboflavin by electron transfer from the tyrosine and tryptophan moieties followed by a fast protonation of the resulting riboflavin anion rather than by direct H-atom abstraction, which could be monitored by time-resolved transient absorption spectroscopy as a decay of triplet riboflavin followed by a rise in riboflavin anion radical absorption. For cysteine- and thiol-containing peptides, second-order rate constants depend strongly on pH, for cysteine corresponding to pKaRSH = 8.35. H-atom abstraction seems to operate at low pH, which with rising pH gradually is replaced by electron transfer from the thiol anion. From the pH dependence of the second-order rate constant, the respective values for the H-atom abstraction (k = 1.64 x 10(6) L mol(-1) s(-1)) and for the electron transfer (k = 1.20 x 10(9) L mol(-1) s(-1)) were determined.     

Effect of heat treatment on bovine beta-lactoglobulin A, B, and C explored using thiol availability and fluorescence.

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated at temperatures between about 40 and 94 degrees C for 10 min, cooled, and analyzed using Trp fluorescence and extrinsic fluorescence spectra of the probe 1,8-anilinonaphthalene sulfonate (ANS). Thiol availabilities using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were determined using a separate set of samples. The normalized ANS fluorescence emission intensity and the thiol availability results showed a 1:1 relationship with the loss of nativelike but not SDS-monomeric protein, as determined by PAGE analysis. The normalized Trp emission intensity results did not show a comparable 1:1 relationship with the loss of nativelike protein, indicating that the Trp intensity arose from consequential disulfide bond reorganization and not the initial unfolding reaction. The results were also analyzed in terms of two-state models, and the midpoint temperatures (T(mid)) for the proteins were generally beta-Lg C > beta-Lg A > beta-Lg B, and the slopes at the midpoint temperatures for the A variant were generally less than those for the B and C variants indicating that beta-Lg A may denature by a different mechanism from that of beta-Lg B or beta-Lg C. The T(mid) parameters derived from the ANS fluorescence intensity results were similar to those for thiol availability and both were lower than the T(mid) values for Trp emission intensity showing that creation of an ANS binding site on a beta-Lg molecule was linked to the irreversible exposure of a thiol group and the loss of native beta-Lg but preceded the decrease in Trp(61) fluorescence quenching. These results for the differences between the behavior of the A and B or the C variants involved the creation of a destabilizing cavity by the Val(118)Ala (A --> B) substitution and the changed charge distribution within the CD loop caused by the Asp(64)Gly (A --> B) substitution.     

Effect of gamma-sterilization and autoclaving on soil organic matter structure as studied by solid state NMR, UV and fluorescence spectroscopy

Sterilized soil is often used, for example in degradation studies, sorption experiments, microbiological tests and plant test systems, to distinguish between microbial processes and abiotic reactions. The most commonly used technique for sterilization is autoclaving of the soil. Another technique is irradiation with high‐level gamma radiation (γ‐radiation). One major drawback of sterilization procedures is the possible alteration of the structure of soil components, for example the organic matter. A change in the chemical structure of the soil organic matter can cause different reactions in the above‐mentioned experiments and hence interfere with the aim of clearly distinguishing between biotic and abiotic processes. Two soils (Gleyic Cambisol and Orthic Luvisol) were sterilized by two γ‐irradiation procedures (4 kGy hour^?1 for 9 hours and 1.3 kGy hour^?1 for 27 hours) and repeated autoclaving at 121°C. Gentle physical aggregate fractionation of the sterilized soils revealed a decrease in the aggregation of the soil, which was reflected in an increase of the clay fraction. Subsequent analysis of the aqueous phase revealed much more dissolved organic matter (DOM) in the γ‐sterilized and autoclaved soils than in the untreated soils. Ultraviolet (UV) and fluorescence spectra of the DOM showed a decrease in the aromaticity and polycondensation of the dissolved organic carbon (DOC). ¹³C cross‐polarization/magic‐angle spinning nuclear magnetic resonance (¹³C‐CP/MAS NMR) spectra of the unfractionated soils and their respective soil fractions before and after sterilization showed that the most important change occurred in the carbohydrate and N‐alkyl region, the main components of microorganisms. In general, the impact of the sterilization method was stronger for autoclaving. The γ‐sterilized soils and fractions displayed both fewer and smaller changes in the soil organic matter.     

Binding of the pepper alkaloid piperine to bovine beta-lactoglobulin: circular dichroism spectroscopy and molecular modeling study

The pepper alkaloid piperine is a nontoxic, natural dietary compound with a broad range of physiological activity. The present work is the first demonstration of its interaction with a mammalian protein. Circular dichroism (CD) spectroscopy was used to reveal and analyze the binding of piperine to a lipocalin protein. Induced CD spectra measured in pH 7.7 phosphate buffer at 37 degrees C demonstrated reversible, non-covalent association of piperine with bovine beta-lactoglobulin (BLG), the major whey protein in milk. The binding parameters (K(a) approximately 8 x 10(4) M(-1), n = 0.8) determined from the CD titration data showed no significant differences between the piperine binding properties of the two main genetic variants of BLG (A and B). The vanishing extrinsic CD signal obtained upon acidification of the piperine-BLG sample solution (Tanford transition) suggested that the ligand binds in the central hydrophobic cavity of the beta-barrel. The cavity binding concept was further supported by a CD displacement experiment using palmitic acid, the well-known hydrophobic ligand of BLG. Molecular docking calculations showed that piperine can be efficiently accommodated within the calyx of BLG. Additional molecular modeling calculations indicated that the beta-barrel of human tear lipocalin, human serum retinol binding protein, and human neutrophil gelatinase associated lipocalin might also accommodate a piperine molecule.     

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.     

Interactions between methyl ketones and beta-lactoglobulin: sensory analysis, headspace analysis, and mathematical modeling

Interaction of flavor compounds with proteins is known to have an influence on the release of flavor from food. Hydrophobic interactions were found between beta-lactoglobulin and methyl ketones; the affinity constant increases by increasing the hydrophobic chain. Addition of beta-lactoglobulin (0.5 and 1%) to aroma solutions (12.5, 50, and 100 microL L(-)(1)) of three methyl ketones induces a significant decrease in odor intensity. The chosen methyl ketones were 2-heptanone (K(b) = 330), 2-octanone (K(b) = 950), and 2-nonanone (K(b) = 2440). The release of these flavor compounds (50 microL L(-)(1)) was studied by static headspace in water solution (50 mM NaCl, pH 3) with different concentrations of beta-lactoglobulin (0, 0.5, 1, 2, 3, and 4%). Increasing the concentration of protein increases the retention of volatiles, and this effect is greatest for 2-nonanone, the compound with the highest affinity constant, and lowest for 2-heptanone. A mathematical model previously developed to describe flavor release from aqueous solutions containing flavor-binding polymers (Harrison, M.; Hills, B. P. J. Agric. Food Chem. 1997, 45, 1883-1890) was used to interpret the data. The model assumes that the polymer-flavor interaction is reversible and the rate-limiting step for release is the transfer of volatiles across the macroscopic gas-liquid interface. This model was used to predict the equilibrium partitioning properties and the rate of release of the three methyl ketones. Increasing the affinity constant leads to decreased release rates and a lower final headspace aroma concentration.     

Front-face fluorescence spectroscopy as a rapid and nondestructive tool for differentiating various cereal products: a preliminary investigation

The potential of intrinsic fluorescence spectroscopy was investigated for differentiating between processed grains (flours, pasta, and semolinas) of different wheat cereal products. A total of 59 samples (e.g., 20 complete Kamut, semicomplete Kamut, and soft wheat flours, 28 pasta, and 11 semolinas manufactured from complete Kamut, semicomplete Kamut, and hard wheat flours) were analyzed by front-face fluorescence spectroscopy. Tryptophan fluorescence spectra were scanned between 305 and 400 nm on samples following excitation at 290 nm. The principal component analysis (PCA) performed on flour spectra clearly differentiated complete Kamut and semicomplete Kamut samples from those produced from complete and semicomplete soft wheat flours. The PCA performed on pasta spectra discriminated samples manufactured from complete Kamut and complete hard wheat flours from those made with semicomplete Kamut and semicomplete hard wheat flours. The best discrimination was obtained from tryptophan spectra recorded on semolinas since the four groups were well discriminated. Correct classification amounting to 61.9% was obtained for pasta spectra. A better classification was obtained for flour and semolina spectra since correct classification amounted to 86.7% and 87.9%, respectively. Front-face fluorescence spectroscopy has the potential to be a rapid, low-cost, and efficient method for the authentication of cereal products.     

Binding between bixin and whey protein at pH 7.4 studied by spectroscopy and isothermal titration calorimetry

Bixin is the major coloring component of annatto used in manufacturing colored cheeses, but its presence in liquid whey causes undesirable quality of the recovered whey protein ingredients. The objective of this work was to study molecular binding between bixin and three major whey proteins (β-lactoglobulin, α-lactalbumin, and bovine serum albumin) at pH 7.4 using UV-vis absorption spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and circular dichroism. These complementary techniques illustrated that the binding is a spontaneous complexation process mainly driven by hydrophobic interactions. The complexation is favored at a lower temperature and a higher ionic strength. At a lower temperature, the binding is entropy-driven, while it changes to an enthalpy-driven process at higher temperatures. The binding also increases the percentage of unordered secondary structures of proteins. Findings from this work can be used to develop whey protein recovery processes for minimizing residual annatto content in whey protein ingredients.     

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.     

Heat-induced redistribution of disulfide bonds in milk proteins. 2. Disulfide bonding patterns between bovine beta-lactoglobulin and kappa-casein

Heat treatment of milk causes the heat-denaturable whey proteins to aggregate with kappa-casein (kappa-CN) via thiol-disulfide bond interchange reactions. The particular disulfide bonds that are important in the aggregates are uncertain, although Cys(121) of beta-lactoglobulin (beta-LG) has been implicated. The reaction at 60 degrees C between beta-LG A and an activated kappa-CN formed small disulfide-bonded aggregates. The tryptic peptides from this model system included a peptide with a disulfide bond between a Cys residue in the triple-Cys peptide [beta-LG(102-124)] and kappa-CN Cys(88) and others between kappa-CN Cys(88) or kappa-CN Cys(11) and beta-LG Cys(160). Only the latter two novel disulfide bonds were identified in heated (90 degrees C/20 min) milk. Application of computational search tools, notably MS2Assign and SearchXLinks, to the mass spectrometry (MS) and collision-induced dissociation (CID)-MS data was very valuable for identifying possible disulfide-bonded peptides. In two instances, peptides with measured masses of 4275.07 and 2312.07 were tentatively assigned to beta-LG(102-135):kappa-CN(11-13) and beta-LG A(61-69):kappa-CN(87-97), respectively. However, sequencing using the CID-MS data demonstrated that they were, in fact, beta-LG(1-40) and beta-LG(41-60), respectively. This study supports the notion that reversible intramolecular disulfide-bond interchange precedes the intermolecular interchange reactions.     

Production of lactoferricin and other cationic peptides from food grade bovine lactoferrin with various iron saturation levels

Purification of lactoferricin (Lfcin), a cationic antimicrobial peptide, was achieved by peptic digestion of food grade bovine lactoferrin (LF) followed by fractionation on an industrial grade cation exchange resin with stepwise salt gradient elution. The digest and eluted fractions were partially characterized by MALDI-ToF MS and N-terminal sequencing. A fraction eluted using phosphate buffer with 2.0 M NaCl contained predominantly two peptides with masses of 3196 and 3124 Da, which corresponded to the 26- and 25-amino acid peptides FKCRR WQWRM KKLGA PSITC VRRAF (A), containing the Lfcin sequence. Putative sequences of cationic peptides in other eluted fractions included FKNKS RSFQ, WRMKK LGAPS ITCVR RA, and GAPSI TCVRR AFALE CIRAI AEKKA. The iron saturation level of LF had no effect on the production of Lfcin. Nevertheless, the digestion of LF containing lower iron content led to the production of a higher quantity of low molecular weight cationic peptides. A two-step process using industrial grade cation exchange resin led to 35% recovery of Lfcin and also produced other cationic peptides with potential bioactive properties.     

Preparation of antioxidant enzymatic hydrolysates from alpha-lactalbumin and beta-lactoglobulin. Identification of active peptides by HPLC-MS/MS

We have investigated the antioxidant activity of hydrolysates from whey proteins bovine alpha-lactalbumin (alpha-La) and beta-lactoglobulin A (beta-Lg A) by commercial proteases (pepsin, trypsin, chymotrypsin, thermolysin, and Corolase PP). Corolase PP was the most appropriate enzyme to obtain antioxidant hydrolysates from alpha-La and beta-Lg A (ORAC-FL values of 2.315 and 2.151 micromol of Trolox equivalent/mg of protein, respectively). A total of 42 peptide fragments were identified by HPLC-MS/MS in the beta-Lg A hydrolysate by Corolase PP. One of the sequences (Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile) possessed radical scavenging (ORAC-FL value of 2.621 micromol of Trolox equivalent/micromol of peptide) higher than that of butylated hydroxyanisole (BHA). Our results suggest that whey protein hydrolysates could be suitable as natural ingredients in enhancing antioxidant properties of functional foods and in preventing oxidation reaction in food processing.