• Bioactivity of beta-lactoglobulin and alpha-lactalbumin-Technological implications for processing

      Chatterton, Dereck E.W.; Smithers, Geoffrey; Roupas, Peter; Brodkorb, Andre (Elsevier, 2006-08-17)
      The dairy industry faces new technological challenges in order to exploit and maintain some of the bioactive properties of dairy components throughout processing. This review outlines these issues with respect to the two major whey proteins β-lactoglobulin (β-lg) and α-lactalbumin (α-la). Biological activities of both the intact proteins, and peptides derived from the proteins, are discussed e.g. inhibition of angiotensin-converting enzyme (ACE), anti-microbial activity, anti-carcinogenic activity, hypocholesterolemic effect, metabolic and physiological effects. The levels necessary to provide beneficial effects and, if available, evidence from clinical trials are reported. Developments in the purification and enrichment of the proteins are discussed, and the technological implications of industrial processing on the bio-activity of the proteins are examined. The supplementation of infant formulas with α-lactalbumin enriched whey proteins is also discussed in light of its potentially improved bioactive properties.
    • Complexes between linoleate and native or aggregated β-lactoglobulin: Interaction parameters and in vitro cytotoxic effect.

      Le Maux, Solene; Bouhallab, Said; Giblin, Linda; Brodkorb, Andre; Croguennec, Thomas (Elsevier, 2013-11)
      Iron is essential for human health, but it sometimes causes an unpleasant taste, rusty colour and a decrease in the stability of food products. Previously, we found that ethanol-treated yeast (ETY) cells could remove iron from wine and juice, and reduce the fishy aftertaste induced by iron in wine–seafood pairings. However, the mechanism of iron sorption by ETY cells is undefined; thus, there is no indicator that can be used to estimate the iron sorption capacity of these cells. In this study, we showed that cell wall components are not mainly associated with iron sorption by investigating ETY cells with the cell wall removed. Moreover, plasma membrane permeability was correlated with the iron sorbing capacity of the cells. Microscopic analysis showed that iron accumulated within ETY cells. Proteinase-treated ETY cells had no iron sorbing capacity. On the basis of these results, we conclude that intracellular proteins are involved in iron sorption by ETY cells.
    • Cytotoxic Complexes of Sodium Oleate with β-Lactoglobulin

      Liskova, Kamila; Auty, Mark; Chaurin, Valerie; Min, Soyoung; Mok, K. Hun; O'Brien, Nora; Kelly, Alan L.; Brodkorb, Andre (Wiley VCH-Verlag GmbH & Co., 2011-08-19)
      A complex of α-lactalbumin and oleic acid has previously been shown to induce apoptosis in cancer cells in a number of in vitro and in vivo trials. This complex is called HAMLET or BAMLET, depending on the origin of α-la (human/bovine alpha-lactalbumin made lethal to tumour cells). In the current study, it was shown that bovine β-lactoglobulin (β-lg), upon binding sodium oleate (NaOle), the salt of oleic acid, also acquires cytotoxicity towards tumour cells (human monocytic cells U937), analogously to HAMLET/BAMLET complexes. The properties of the complex were characterized using FIR spectroscopy, HPLC and SDS-PAGE. It was shown that the level of covalent oligomerization (dimers and trimers) of β-lg increased with increasing the molar ratio of sodium oleate NaOle:β-lg in the preparation procedure. At the same time, increasing the molar ratio of NaOle:β-lg increased the cytotoxicity of the complex. The increase in cytotoxicity appeared to be dependent on the amount of bound NaOle in the complex, but not on the content of multimeric forms of β-lg. The NaOle/β-lg complex also showed similarity with BAMLET in penetrating the cell membrane and co-localizing with the cell nucleus. Furthermore, DNA fragmentation studies suggested that tumour cells (U937) treated with the complex died by apoptosis, as in the case of BAMLET, and healthy cells appeared to be less affected by treatment, as shown with model rat adrenal pheochromocytoma cells PC12. In conclusion, β-lg and NaOle can form complexes with apoptosis-inducing qualities comparable to those of BAMLET.
    • The cytotoxicity of fatty acid/α-lactalbumin complexes depends on the amount and type of fatty acid

      Brinkman, Christel Rothe; Brodkorb, Andre; Thiel, Steffen; Kehoe, Joseph James (Wiley, 2013-04-17)
      Complexes of the milk protein, α-lactalbumin, and the fatty acid, oleic acid, have previously been shown to be cytotoxic. Complexes of α-lactalbumin and five different fatty acids (vaccenic, linoleic, palmitoleic, stearic, and elaidic acid) were prepared and compared to those formed with oleic acid. All complexes were cytotoxic to human promyelocytic leukemia-derived (HL-60) cells but to different degrees depending on the fatty acid. The amount of fatty acid per α-lactalbumin molecule was found to correlate with the cytotoxicity; the higher the number of fatty acids per protein, the more cytotoxic the complex. Importantly, all the tested fatty acids were also found to be cytotoxic on their own in a concentration dependent manner. The cytotoxic effect of complexes between α-lactalbumin and linoleic acid, vaccenic acid, or oleic acid was further investigated using flow cytometry and found to induce cell death resembling apoptosis on Jurkat cells. Practical applications: Cytotoxic complexes of α-lactalbumin and several different fatty acids could be produced. The cytotoxicity of all the variants is similar to that previously determined for α-lactalbumin/oleic acid complexes.
    • Dairy food structures influence the rates of nutrient digestion through different in vitro gastric behaviour

      Mulet-Cabero, Ana-Isabel; Rigby, Neil M.; Brodkorb, Andre; Mackie, Alan (Elsevier, 2016-12-31)
      The purpose of this study was to investigate in vitro the extent to which specific food structures alter gastric behaviour and could therefore impact on nutrient delivery and digestion in the small intestine. Results obtained from a specifically developed gastric digestion model, were compared to results from a previous human study on the same foods. The semi-dynamic model could simulate the main gastric dynamics including gradual acidification, lipolysis, proteolysis and emptying. Two dairy-based foods with the same caloric content but different structure were studied. The semi-solid meal comprised a mixture of cheese and yogurt and the liquid meal was an oil in water emulsion stabilised by milk proteins. Our findings showed similar gastric behaviour to that seen previously in vivo. Gastric behaviour was affected by the initial structure with creaming and sedimentation observed in the case of liquid and semi-solid samples, respectively. Lipid and protein digestion profiles showed clear differences in the amount of nutrients reaching the simulated small intestine and, consequently, the likely bioaccessibility after digestion. The semi-solid sample generated higher nutrient released into the small intestine at an early stage of digestion whereas nutrient accessibility from liquid sample was delayed due to the formation of a cream layer in the gastric phase. This shows the strong effect of the matrix on gastric behaviour, proteolysis and lipolysis, which explains the differences in physiological responses seen previously with these systems in terms of fullness and satiety.
    • Determination of exposed sulphydryl groups in heated β-lactoglobulin A using IAEDANS and mass spectrometry

      Kehoe, Joseph James; Brodkorb, Andre; Molle, Daniel; Yokoyama, Emilie; Famelart, Marie-Helene; Bouhallab, Said; Morris, Edwin R; Croguennec, Thomas (American Chemical Society, 2007-07-25)
      This paper takes a new approach to determining which sulfhydryl groups are exposed during the heat denaturation of bovine β-lactoglobulin A. The sulfhydryl groups exposed after heating were blocked with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS). The results show that IAEDANS is a suitable blocking agent, and its absorbance at 336 nm enabled the quantification of exposed sulfhydryl groups in a mixture of protein species by gel permeation chromatography. Combined with the specific fragmentation of bound IAEDANS by matrix-assisted laser desorption ionization (MALDI) MS/MS in negative ionization mode, this facilitated the identification of peptides that contained blocked cysteines after enzymatic digestion of the protein. During MALDI MS/MS of the peptides, in positive ionization mode, the IAEDANS molecule remained bound to the cysteines, making it possible to identify exactly which cysteine had been exposed after heating. In β-lactoglobulin A it was found that cysteine 66 and cysteine 160 were predominantly exposed regardless of the length of exposure to heat.
    • Enzymatic Hydrolysis of Heat-induced Aggregates of Whey Protein Isolate

      O'Loughlin, Ian B.; Murray, Brian A.; Kelly, Philip M.; Fitzgerald, Richard J.; Brodkorb, Andre (American Chemical Society, 2012-04-26)
      The effects of heat induced denaturation and subsequent aggregation of Whey Protein Isolate (WPI) solutions on the rate of enzymatic hydrolysis was investigated. Denaturation of whey proteins was monitored by reversed-phase and size exclusion HPLC and observed by native- and SDS-PAGE. Treated and un-treated WPI solutions (100 g L-1 protein) were hydrolysed to a target degree of hydrolysis (DH) of 5 % with Corolase® PP. Aggregate formation was monitored using light microscopy, with size distribution determined by particle size. Viscosity and surface hydrophobicity exhibited large increases with heat-treatment and the major protein components in WPI showed differences in their rates of aggregation. Results revealed an increased rate of hydrolysis of protein solutions, which were subjected to a pre-hydrolysis heattreatment. Light and Confocal Laser Scanning Microscopy (CLSM) images illustrated the optical clarification of the solution, weakening of the gel network and disintegration of aggregates indicative of hydrolysis. Comparison of samples where there was a heat-treatment prior to hydrolysis and a control non-treated hydrolysis reaction, revealed significant differences in the time to reach 5 %DH (P < 0.001). The heat-treatments ≥ 75 ºC for 5 min produced significantly (P < 0.001) more rapid reactions than the other 5 heat-treatments and the control un-treated reaction. The viscosity, surface hydrophobicity, and insolubility of the heat-treated WPI solutions subsequently declined upon their hydrolysis. The extensive aggregation in some heattreated solutions was postulated to relate to the congruent increased rate of hydrolysis. This study demonstrated that prior thermal treatment of ≥ 75 ºC for 5 min can accelerate the enzymatic hydrolysis reaction of WPI with Corolase® PP.
    • The Formation of an Anti-Cancer Complex Under Simulated Gastric Conditions

      Sullivan, Louise M.; Mok, K.Hun; Brodkorb, Andre (Springer-Verlag, 2013-05-01)
      A potent anti-cancer complex has previously been formed from two major components of milk. Human/bovine α-lactalbumin made lethal to tumour cells (H/BAMLET) is a protein–fatty acid complex that has been produced using the whey protein α-lactalbumin (α-LA) and the fatty acid oleic acid (OA). It was shown that it possesses selective anti-tumour and anti-microbial activity, which was first identified in acidic fractions of human breast milk. The aim of this study was to determine whether the two components would form a bioactive complex during simulated gastric (GI) transit. Results showed that a complex consisting of α-LA and OA is formed as the protein unfolds under acidic conditions and subsequently refolds upon pH increase. Analysis of this complex using Nuclear Magnetic Resonance and Fourier Transform Infra-Red (FTIR) spectroscopies estimated a stoichiometry of 4.1 and 4.4 oleic acids per mole of protein, respectively. FTIR and fluorescence spectroscopies showed that the structure was similar to that of BAMLET. Cytotoxicity testing against cancer cell line U937 cells showed that the complex had an LC50 value of 14.08 μM compared to 9.15 μM for BAMLET. These findings suggest that a BAMLET-like complex may be formed under the tested in vitro GI conditions.
    • Formation of non-native β-lactoglobulin during heat-induced denaturation

      Kehoe, Joseph James; Wang, Lizhe; Morris, Edwin R; Brodkorb, Andre (Springer, 2011-12-01)
      A mechanism describing the denaturation and aggregation behavior during heat-treatment of pure β-lactoglobulin and β-lactoglobulin in whey protein isolate (WPI) under selected conditions (20 to 90 gL−1 in water at pH 7.0, 78 °C) is presented. A combination of reversed-phase and gel permeation chromatography was used to study the disappearance of native β-lactoglobulin and the formation of non-native intermediates in the aggregation process. The mean reaction order for pure β-lactoglobulin and β-lactoglobulin in WPI were the same, 1.4. While the rate of β-lactoglobulin denaturation was greater in WPI there was less aggregation compared to that of pure β-lactoglobulin. More of the β-lactoglobulin in WPI remained in a non-native monomer intermediate state after 30 min of heating. After an initial lag period, during which non-native monomers appeared, aggregates formed and rapidly reached a plateau in terms of their size. These aggregates were visualized using atomic force microscopy. There was no significant effect of protein concentration on either aggregate size or the number of exposed sulfhydryls in the heated solutions.
    • Heat-induced Maillard reaction of the tripeptide IPP and ribose: Structural characterization and implication on bioactivity

      Jiang, Zhanmei; Rai, Dilip K; O'Connor, Paula M.; Brodkorb, Andre (Elsevier, 2012-09-28)
      Maillard reaction products (MRPs) were prepared from aqueous model mixtures containing 60 g L− 1 ribose and 30 g L− 1 of the bioactive tripeptide IPP (Ile-Pro-Pro), heated at 98 °C. MRP and associated reactions with changes in IPP were observed within one hour of heat-treatment. The pH of MRPs decreased significantly during the heat treatment of IPP–ribose mixtures from 9.0 to 7.6 after one hour. The amino group content, IPP and ribose concentration decreased significantly during heat treatment. The fluorescence intensity of the IPP–ribose MRPs reached the maximum within 2 h. Modification of the UV/vis spectra for IPP–ribose MRPs was mainly due to a condensation reaction of IPP with ribose. Compounds with molecular weight between 300 and 650 Da were dominant while compounds smaller than 250 Da were also produced during the reactions, as characterized by size exclusion chromatography. Mass spectrometry revealed that IPP was conjugated to ribose at the N-terminal (m/z of 458.3) upon heat-treatment. The presence of ribose also promoted peptide degradation to dehydrated IP (m/z of 211.1). IPP–ribose MRPs lost the known angiotensin-I-converting enzyme (ACE) inhibitory activity of IPP; however, strong antioxidant properties were detected.
    • The influence of bovine serum albumin on β-lactoglobulin denaturation, aggregation and gelation

      Kehoe, Joseph James; Morris, Edwin R; Brodkorb, Andre (Elsevier, 2006-11-22)
      The effect of bovine serum albumin (BSA) on the heat-induced denaturation, aggregation and subsequent acid-induced gelation of β-lactoglobulin (β-lg) was investigated in this work. Changes in the denaturation kinetics of β-lg during heating at 78 °C were determined by monitoring the disappearance of the native protein by reverse-phase chromatography. Replacing β-lg with increasing amounts of BSA, while keeping the total protein concentration constant at 5% (w/w), significantly increased the denaturation rate of β-lg from 2.57±0.30×10−3(g L−1)(1−n)s−1 to 5.07±0.72×10−3(g L−1)(1−n)s−1 (β-lg: BSA ratio of 3:1 w/w). The reaction order for β-lg was 1.40±0.09. Partial replacement of β-lg with BSA (β-lg: BSA ratio of 3:1 w/w) significantly increased the reaction order to 1.67±0.13. Heat-induced aggregates between β-lg and BSA were studied by dynamic light scattering, two-dimensional electrophoresis and size exclusion chromatography. The partial replacement of β-lg with BSA significantly changed the gelling properties of the acid-induced gels. A rapid rate of acidification resulted in a significant decrease, while a slow acidification rate resulted in a significant increase in gel strength. Size exclusion chromatography demonstrated that intermolecular disulphide bond formation occurred during both heat-induced denaturation/aggregation and subsequent acid-induced gelation. Results clearly indicate that BSA contributed to the formation of these disulphide bonds.
    • Interactions between sodium oleate and α-lactalbumin: the effect of temperature and concentration on complex formation

      Kehoe, Joseph James; Brodkorb, Andre (Elsevier, 2012-09-26)
      Complexes of α-lactalbumin and oleic acid have previously been shown to be cytotoxic to cancer cells. In this study oleic acid is replaced by the more soluble sodium oleate and complexes of α-lactalbumin and sodium oleate are formed. Dynamic light scattering results showed that there was a small linear increase in the particle size of α-lactalbumin when it was titrated with sodium oleate. The fluorescence spectra of α-lactalbumin showed a linear increase in the emission maximum when sodium oleate was added up to a molar ratio of 8–11 oleate molecules per α-lactalbumin. Differential scanning calorimetry results show that the thermal unfolding of α-lactalbumin is altered by the presence of the sodium oleate. There is a decrease in size of the endothermic peak of apo α-lactalbumin when sodium oleate is added. The temperature at which unfolding occurred decreased for both apo and holo α-lactalbumin. FTIR measurements showed no significant effect of sodium oleate in the amide I region of the α-lactalbumin spectrum indicating the presence of oleate has little or no effect on the secondary structure of α-lactalbumin. The interactions between α-lactalbumin and sodium oleate/oleic acid are pH dependent, turbidity and dynamic light scattering measurements showed that the association between the two was optimal between pH 6.0 and 8.0. The results obtained here suggest that α-lactalbumin can bind at least a 20 fold molar excess of oleate, most likely in a non-specific manner.
    • Isolation and characterisation of κ-casein/whey protein particles from heated milk protein concentrate and role of κ-casein in whey protein aggregation

      Gaspard, Sophie J.; Auty, Mark A.E.; Kelly, Alan L.; O'Mahony, James A.; Brodkorb, Andre (Elsevier, 2017-06-12)
      Milk protein concentrate (79% protein) reconstituted at 13.5% (w/v) protein was heated (90 °C, 25 min, pH 7.2) with or without added calcium chloride. After fractionation of the casein and whey protein aggregates by fast protein liquid chromatography, the heat stability (90 °C, up to 1 h) of the fractions (0.25%, w/v, protein) was assessed. The heat-induced aggregates were composed of whey protein and casein, in whey protein:casein ratios ranging from 1:0.5 to 1:9. The heat stability was positively correlated with the casein concentration in the samples. The samples containing the highest proportion of caseins were the most heat-stable, and close to 100% (w/w) of the aggregates were recovered post-heat treatment in the supernatant of such samples (centrifugation for 30 min at 10,000 × g). κ-Casein appeared to act as a chaperone controlling the aggregation of whey proteins, and this effect was stronger in the presence of αS- and β-casein.
    • New Insights into Cell Encapsulation and the Role of Proteins During Flow Cytometry

      Doherty, Sinead B.; Brodkorb, Andre (Intech, 2012-06-13)
      Modern approaches to science tend to follow divergent paths. On one hand, instruments and technologies are developed to capture as much information as possible with the need for complex data analysis to identify problematic issues. On the other hand, formulation focused, minimalistic approaches that gather only the most pertinent data for specific questions also represent a powerful methodology. This chapter will provide many examples of the latter by integrating Flow Cytometry (FACS - Fluorescence-Activated Cell Sorting) technology with high throughput screening (HTS) of encapsulation systems with extensive utility of one-dimensional (1-D) imaging for protein localisation. In this regard, less information is acquired from each cell, data files will be more manageable, easier to analyse and throughput screening will be significantly enhanced beyond traditional HTS analysis, irrespective of the protein concentration present in the background or delivery media.
    • Pilot-scale Production of Hydrolysates with Altered Bio-functionalities based on Thermally-denatured Whey Protein Isolate

      O'Loughlin, Ian B.; Murray, Brian A.; FitzGerald, Richard J.; Brodkorb, Andre; Kelly, Philip M. (Elsevier, 2013-08-13)
      Whey protein isolate (WPI) solutions (100 g L−1 protein) were subjected to a heat-treatment of 80 °C for 10 min. Unheated and heat-treated WPI solutions were hydrolysed with Corolase® PP at pilot-scale to either 5 or 10% degree of hydrolysis (DH). Hydrolysates were subsequently processed via cascade membrane fractionation using 0.14 μm, and 30, 10, 5 and 1 kDa cut-off membranes. The compositional and molecular mass distribution profiles of the substrate hydrolysates and membrane processed fractions were determined. Whole and fractionated hydrolysates were assayed for both angiotensin-I-converting enzyme (ACE) inhibitory activity and ferrous chelating capabilities. A strong positive correlation (P < 0.01) was established between the average molecular mass of the test samples and the concentration needed to chelate 50% of the iron (CC50) in solution. The lowest ACE inhibition concentration (IC50 = 0.23 g L−1 protein) was determined for the 1 kDa permeate of the heat-treated 10% DH hydrolysate
    • A Simple Method for the Purification of Nisin

      Gough, Ronan; Gomez-Sala, Beatriz; O'Connor, Paula M.; Rea, Mary C.; Miao, Song; Hill, Colin; Brodkorb, Andre (Springer, 2017-05-29)
      Nisin, an antimicrobial peptide showing activity against a broad range of Gram-positive bacteria, is widely used as a food preservative and has potential as a therapeutic for a range of infectious diseases. Here, we present a simple purification method, based on a salting-out approach, which can produce a powder containing ∼33% nisin, from a nisin-producing culture in a whey permeate-based medium. This process removes over 99% of the lactic acid, NaCl, lactose and non-nisin proteins from the cell-free culture supernatant. The approach can also enrich a commonly used commercial nisin preparation over 30-fold to a purity of ∼58%. These are higher purities than comparable published methods. The simplicity of this approach facilitates its use in research and also its scale-up.
    • Simulated gastrointestinal digestion of nisin and interaction between nisin and bile

      Gough, Ronan; O'Connor, Paula M.; Rea, Mary C.; Gomez-Sal, Beatriz; Miao, Song; Hill, Colin; Brodkorb, Andre (Elsevier, 2017-08-14)
      Nisin, an antimicrobial peptide showing activity against many Gram positive bacteria, is widely used as a food preservative. The simulated gastrointestinal digestion of nisin (variant A) was studied using the in vitro INFOGEST digestion method. Following oral, gastric and small intestinal digestion, there was no intact nisin in the system and the nisin was primarily digested by pancreatin. After digestion, six nisin fragments (1–11, 1–12, 1–20, 1–21, 1–29 and 1–32) were identified by reversed phase high performance liquid chromatography and mass spectroscopy and four of these nisin fragments (1–20, 1–21, 1–29 and 1–32) demonstrated low antibacterial activity against Lactococcus lactis HP in agar diffusion activity assays. Additionally, it was observed that bile salts form a complex with nisin. This was examined by atomic force microscopy, turbidity and dynamic light scattering, which showed that this interaction resulted in significantly larger bile salt micelles. The presence of bile salts at physiological levels significantly altered the relative amounts of the nisin fragments 1–12, 1–20 and 1–29 produced during an in vitro digestion. This study highlights the importance of including bile in simulated digestions of antimicrobial peptides in order to obtain a more accurate simulation of the in vivo digestion products and their activity.
    • Stabilising effect of α-lactalbumin on concentrated infant milk formula emulsions heat treated pre- or post-homogenisation

      Buggy, Aoife K.; McManus, Jenifer J.; Brodkorb, Andre; McCarthy, Noel; Fenelon, Mark A. (Springer, 2016-11-22)
      Protein type and/or heat treatment pre- or post-homogenisation can affect the physical stability of infant formulations during manufacture. Previous research has described the use of α-lactalbumin addition in infant formulae, but has not demonstrated the effect of heating pre- or post-emulsion formulation during processing. The objective of this study was to evaluate the effect of both of these parameters. Three batches of model 1st-stage infant formula containing differing whey protein ratios (60:40 whey: casein with α-lactalbumin content 12, 30 or 48% of total protein) were prepared. Each batch was split; one half receiving heat treatment pre-homogenisation and the second half homogenised and then heat treated. Emulsion stability was determined by size exclusion chromatography, SDS-PAGE, particle size and viscosity measurements. There was a significant (P < 0.05) reduction in the formation of large soluble aggregates upon increasing α-lac concentration in emulsions heat treated either before or after homogenisation. Heat treatment of formulations post-homogenisation resulted in a higher (P < 0.05) D.v09 within the particle size distribution; increasing α-lactalbumin concentration to 30 or 48% significantly (P < 0.05) reduced the D.v09 within the particle size distribution in these emulsions. The viscosity of concentrates (55 % total solids) containing the 12% α-lactalbumin, heat treated post-homogenisation, was significantly greater (P < 0.05) than the equivalent emulsion heat treated pre-homogenisation; increasing the α-lactalbumin concentration to 30 or 48% significantly (P < 0.05) reduced viscosity. When the α-lactalbumin content was increased to 48% as a percentage of the total protein, heating before or after emulsion formation had no effect on concentrate viscosity. The findings demonstrate the importance of thermal denaturation/aggregation of whey proteins (and in particular, the ratio of α-lactalbumin to β-lactoglobulin) prior to homogenisation of infant formula emulsions.
    • Structure and antioxidant activity of Maillard reaction products from α-lactalbumin and β-lactoglobulin with ribose in an aqueous model system

      Jiang, Zhanmei; Brodkorb, Andre (Elsevier, 2012-02-11)
      Maillard reaction products (MRPs) were prepared from aqueous model mixtures containing 3% (w/w) ribose and 3% (w/w) of the dairy proteins α-lactalbumin (α-LA) or β-lactoglobulin (β-LG), heated at 95 °C, for up to 5 h. The pH of MRPs decreased significantly during heat treatment of α-LA-Ribose and β-LG-Ribose mixtures from 8.4 to 5.3. The amino group content in MRPs, derived from the α-LA-Ribose and β-LG-Ribose model system, was decreased noticeably during the first hour and did not change thereafter. The loss of free ribose in MRPs was higher for β-LG-Ribose than for α-LA-Ribose. During the Maillard reaction, the concentration of native and non-native α-LA, or β-LG, decreased and the formation of aggregates was observed. Fluorescence intensity of the β-LG-Ribose MRPs reached maximum within 1 h, compared to 2 h for α-LA-Ribose MRPs. Meanwhile, modification of the UV/vis absorption spectra for α-LA and β-LG was mainly due to a condensation reaction with ribose. Dynamic light scattering showed a significant increase in the particle size of the MRPs. Size exclusion chromatography of MRPs revealed the production of both high and low molecular weight material. Electrophoresis of MRPs indicated polymerization of α-LA and β-LG monomers via inter-molecular disulfide bridge, but also via other covelant bonds. MRPs from α-LA-Ribose and β-LG-Ribose exhibited increased antioxidant activities, therefore theses MRPs may be used as natural antioxidants in food products.
    • Tryptophan-Mediated Denaturation of β-Lactoglobulin A by UV Irradiation

      Kehoe, Joseph James; Remondetto, Gabriel E; Subirade, Muriel; Morris, Edwin R; Brodkorb, Andre (American Chemical Society, 2008-06-04)
      β-Lactoglobulin A, a genetic variant of one of the main whey proteins, was irradiated at 295 nm for 24 h. After irradiation, 18% of the protein was denatured (determined by reverse-phase chromatography). The fluorescence spectrum of the irradiated protein was red-shifted compared to that of the native protein, indicating a change in protein folding. Sulfhydryl groups, which are buried in native β-lactoglobulin, were exposed following irradiation and became available for quantification using the Ellman assay. The quantity of exposed sulfhydryls increased, but the number of total sulfhydryl groups decreased. Gel permeation chromatography showed that some protein aggregation occurred during irradiation. Fourier transform infrared (FTIR) spectroscopy of irradiated β-lactoglobulin revealed changes in the secondary structure, comparable to that of early events during heat-induced denaturation. There was evidence for some photo-oxidation of tryptophan.