Investigation into the interaction between resveratrol and whey proteins using fluorescence spectroscopy

Fluorescence spectroscopy was used to investigate the interaction between resveratrol and whey proteins. The whey proteins examined were lactoferrin, holo‐lactoferrin, apo‐lactoferrin, whey protein isolate (WPI) and the β‐lactoglobulin‐ and α‐lactalbumin‐rich fractions of WPI. Both an analytical‐grade and food‐grade resveratrol were examined. In all the systems studied, it was found that resveratrol interacted with the whey proteins to form a 1:1 complex. The binding constant, K_s, for the protein–resveratrol complex for all the proteins examined varied from 1.7 × 10⁴ to 1.2 × 10⁵ m ^?1. Furthermore, the interaction between the whey proteins and resveratrol did not affect the secondary structure of the proteins.     

The thermal denaturation of the total whey protein in reconstituted whole milk

The kinetic parameters for thermal denaturation of the total whey proteins in whole milk were determined. Denaturation was a second‐order reaction, and an Arrhenius plot showed a change in slope at ~85 °C. At 70–85 °C, the activation energy, enthalpy and entropy were in the range expected for denaturation processes, whereas at 85–115 °C, these parameters were typical for chemical reactions such as aggregation. Equations to predict the denaturation after heating were developed and tested on a range of independently prepared milk samples. There was a good agreement between the predicted and the experimentally determined denaturation levels.     

Thermal modifications of structure and codenaturation of α‐lactalbumin and β‐lactoglobulin induce changes of solubility and susceptibility to proteases

Study of heat denaturation of major whey proteins (β‐lactoglobulin or α‐lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other co‐denaturation partner, and of existence of a previous thermal pretreatment (industrial whey). α‐Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at ⪈85°C. After its denaturation, α‐lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys‐X and Arg‐X bonds). Heating over 85°C of β‐lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co‐denaturation of α‐lactalbumin with β‐lactoglobulin increased their aggregation and resulted in complete exposure of β‐lactoglobulin peptic cleavage sites and partial unveiling of α‐lactalbumin tryptic cleavage sites. The exposure of α‐lactalbumin tryptic cleavage sites was slightly enhanced when the α‐lactalbumin/β‐lactoglobulin mixture was heated at pH 7.5. Co‐denaturation of fresh whey by heating at 95°C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by β‐mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.     

α‐Lactalbumin solubilisation from a precipitated whey protein concentrates fraction: pH and calcium concentration effects

The selective precipitation of α‐lactalbumin (α‐La) is the basis for one of the possible methods in whey protein fractionation. Calcium concentration, type of acid added and pH play important roles in α‐La precipitation and on the following resolubilisation. Two washing steps are enough for quantitative removal of β‐lactoglobulin entrapped in the precipitate. α‐La losses are minimised during washing steps (5%) when NaCl is used as washing agent. The most important parameter to control during the resolubilisation step is pH, the maximum amount of the initial re‐dissolved α‐La being 76% when the pH is adjusted to 7.5, CaCl2 concentration is 0.2 m and prior precipitation is carried out adding citric acid. Addition of CaCl2 is not necessary to dissolve α‐La because of the fact that there is enough calcium in the precipitate to join all α‐La; however, its presence improves the solubilisation yield (66% vs. 75%).     

Low‐field NMR: A tool for studying protein aggregation

Low‐field nuclear magnetic resonance (NMR) spin–spin relaxation (T₂) measurements were used to study the denaturation and aggregation of β‐lactoglobulin (β‐LG) solutions of varying concentrations (1–80 g L^?1) as they were heated at temperatures ranging from ambient up to 90 °C. For concentrations of 1–10 g L^?1, the T₂ of β‐LG solutions did not change, even after heating to 90 °C. A decrease in T₂ was only observed when solutions having higher concentrations (20–80 g L^?1) were heated. Circular dichroism (CD) spectroscopy and fluorescence tests using the dye 1‐anilino‐8‐naphthalene sulfonate (ANS) on 0.2 and 1 g L^?1 solutions, respectively, indicated there were changes in the protein's secondary and tertiary conformations when the β‐LG solutions reached 70 °C and above. In addition, dynamic light scattering (DLS) showed that protein aggregation occurred only at concentrations above 10 g L^?1 and for heating at 70 °C and above. The hydrodynamic radius increased as T₂ decreased. When excess 2‐mercaptoethanol was added, the changes in both T₂ and the hydrodynamic radius followed the same trend for all β‐LG protein concentrations between 1 and 40 g L^?1. These observations led to the conclusion that the changes in T₂ were due to protein aggregation, not protein unfolding. Copyright © 2007 Society of Chemical Industry     

Kinetic studies of the thermal inactivation of plasmin in acid or sweet whey

The thermal behaviour of the milk alkaline proteinase, plasmin, was studied in acid and sweet (rennet) whey; indigenous (bovine) plasmin was studied in the former system, but endogenous porcine plasmin was added in the latter, due to the very low levels of residual plasmin. The inactivation of plasmin in both systems followed first-order inactivation kinetics, which was consistent with previous observations of plasmin inactivation in milk and model milk systems. The thermal inactivation of plasmin in acid whey (D_{90 °C}=108±29 min, z=24.5±1.2 °C) was much slower than in the sweet whey system (D_{90 °C}=0.021±0.006 min, z=7.3±0.3 °C). Similarly, denaturation of β-lactoglobulin (β-lg) followed a first-order inactivation profile and this protein was also more heat stable in acid whey (D_{90 °C}=86±76 min, z=13.7±1.5 °C) than sweet whey (D_{90 °C}=0.81±0.29 min, z=9.1±0.5 °C). While it is possible that the increased heat stability of plasmin in acid whey is due to reduced sulphydryl/disulphide interchange reactions between plasmin and β-lg, it also appears that structural changes in the plasmin molecule were a significant contributory effect on the thermal stability of plasmin in this system. Increasing the pH of acid whey decreased the heat stability of plasmin. However, adjusting the pH of sweet whey had little effect on the heat stability of plasmin. Overall, severe heat treatments may be required to ensure inactivation of the enzyme in acid whey, but a balance is required between reducing the activity of plasmin and maintaining the functionality of whey proteins as food ingredients.     

Crude extract of Fuzhuan brick tea ameliorates DSS‐induced colitis in mice

Inflammatory bowel disease (IBD) is highly prevalent worldwide and includes ulcerative colitis (UC) and Crohn's disease. It is a high incidence rate disease all over the world and an inducement of colon cancer. The aim of this study was to investigate the potential protective effect of Fuzhuan brick tea (FBT) against colitis induced by dextran sulphate sodium (DSS) in mice. ICR mice were administered FBT orally for 7 days before drinking 3% DSS (w/v). The FBT significantly attenuated the symptoms of colitis including diarrhoea, rectal bleeding and loss of body weight. FBT reduced the shortening of colon length and alleviated the histopathological damages. The myeloperoxidase activity, nitric oxide and malondialdehyde level in colon tissues were also significantly decreased by FBT. Besides, FBT treatment obviously suppressed the expression of the inflammatory cytokines such as TNF‐α, IL‐1β and IFN‐γ. Our results provide a safe and efficient method for preventing and treating colitis.     

Emulsifying Properties of Legume Proteins Compared to β‐Lactoglobulin and Tween 20 and the Volatile Release from Oil‐in‐Water Emulsions

The emulsifying properties of plant legume protein isolates (soy, pea, and lupin) were compared to a milk whey protein, β‐lactoglobulin (β‐lg), and a nonionic surfactant (Tween 20). The protein fractional composition was characterized using sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The following emulsion properties were measured: particle diameter, shear surface ζ‐potential, interfacial tension (IT), and creaming velocity. The effect of protein preheat treatment (90 °C for 10 min) on the emulsifying behavior and the release of selected volatile organic compounds (VOCs) from emulsions under oral conditions was also investigated in real time using proton transfer reaction‐mass spectrometry. The legume proteins showed comparable results to β‐lg and Tween 20, forming stable, negatively charged emulsions with particle diameter d_3,2 < 0.4 μm, and maintained stability over 50 d. The relatively lower stability of lupin emulsions was significantly correlated with the low protein surface hydrophobicity and IT of the emulsion. After heating the proteins, the droplet size of pea and lupin emulsions decreased. The VOC release profile was similar between the protein‐stabilized emulsions, and greater retention was observed for Tween 20‐stabilized emulsions. This study demonstrates the potential application of legume proteins as alternative emulsifiers to milk proteins in emulsion products.     

Proteolysis,protein distribution and stability of UHT milk during storage at room temperature

Proteolytic degradation and distribution of caseins and whey proteins between the soluble and colloidal phases were studied in six batches of commercial UHT milk (three skim and three whole milks) during storage at 25 ± 2 °C. For that purpose, at 30 day intervals, milk samples were ultracentrifuged and the pellets and supernatants analysed by capillary electrophoresis and SDS‐PAGE. Samples were also visually examined for signs of gelation. Extensive proteolytic degradation of the micellar fractions and severe changes in the electrophoretic pattern of the proteins present in the serum fractions were observed in all the batches. A higher proportion of denatured whey proteins not attached to the micelle surface was found in the skim milk samples as compared with the whole milk samples that could provide less resistance against gelation. In addition to β‐Lg, para‐κ‐casein was also found in the serum fraction. A high proteolytic activity against κ‐casein could be responsible for the hydrolysis of serum‐liberated κ‐casein or could have enhanced the liberation of β‐Lg–para‐κ‐casein complexes through proteolysis of micellar κ‐casein. © 1999 Society of Chemical Industry     

Characterisation of whey protein isolate–gum tragacanth electrostatic interactions in aqueous solutions

The main objectives of this study were to measure molecular parameters of gum tragacanth by GPC‐MALLS system and investigate the complexation behaviour of whey protein isolate/gum tragacanth mixed dispersions (0.5 wt% total biopolymer concentration) as a function of pH (7.00–2.00) and the biopolymer mixing ratio (r = 0.1–10) using spectrophotometric, zeta potential and precipitate yield determination methods. GPC‐MALLS revealed that gum tragacanth contains relatively heterogeneous particles with high weight‐average and number‐average (M_w = 7.74 × 10⁵ g mol^?1 and M_n = 3.87 × 10⁵ g mol^?1) molecular mass and high dispersity index (~2.04 ± 0.3). Results of complexation displayed that as the biopolymer mixing ratio increases, the net neutrality shifts to the higher pHs. The critical values associated with the complex structure formation were found at r = 2 in which the charge density of the mixture was near zero at a wide range of pH (3.0–4.0). However, the highest precipitate yield achieved in pH 3.4.     

Oxidatively induced chemical changes and interactions of mixed myosin, β‐lactoglobulin and soy 7S globulin

The effects of oxidation on the chemical characteristics of myosin, β‐lactoglobulin and soy 7S globulin were investigated in a free radical‐generating system (FeCl₃/H₂O₂/ascorbate). Oxidised myosin exhibited a marked increase (>10‐fold) in carbonyls, a small increase in amines and conversion of some free sulphhydryls to disulphide bonds. Oxidised β‐lactoglobulin and 7S globulin also showed a major increase in the carbonyl content (five‐ and threefold respectively), but other chemical changes were relatively minor. In the oxidised myosin/β‐lactoglobulin composites, some cross‐linked aggregates were formed. Aggregation was also evidenced in the myosin/7S globulin composites exposed to the oxidising agents. The results indicated that oxidation enhanced interactions of the non‐muscle proteins with myosin apparently through the modification of amino acid side chains. These physicochemical changes may influence the functionality of processed muscle foods. © 2000 Society of Chemical Industry     

A Simple and Fast Detection Method for Bovine Milk Residues in Foods: A 2‐Site Monoclonal Antibody Immunochromatography Assay

The ingredient declaration on food labels assumes paramount importance in the protection of food‐allergic consumers. China has not implemented Food allergen labeling. A gold immunochromatography assay (GICA) was developed using 2 monoclonal antibodies (mAb) against the milk allergen β‐lactoglobulin in this study. The GICA was specific for pure milk samples with a sensitivity of 0.2 ng/mL. Milk protein traces extracted from 110 food products were detected by this method. The labels of 106 were confirmed by our GICA method: 57 food samples originally labeled as containing milk were positive for β‐lactoglobulin and 49 food samples labeled as not containing milk were negative for β‐lactoglobulin. However, 3 food samples falsely labeled as containing milk were found to contain no β‐lactoglobulin whereas 1 food sample labeled as not containing milk actually contained β‐lactoglobulin. First, these negatives could be because of the addition of a casein fraction. Second, some countries demand that food manufacturers label all ingredients derived from milk as “containing milk” even though the ingredients contain no detectable milk protein by any method. Our GICA method could thus provide a fast and simple method for semiquantitatation of β‐lactoglobulin in foods. Practical Application: The present method provides a fast, simple, semiquantitative method for the determination of milk allergens in foods.     

Polyelectrolyte screening effects on the dissolution of whey protein gels at high pH conditions

The literature reports an optimum NaOH concentration for the alkaline cleaning of whey deposits or gels; at NaOH concentrations higher than this optimum, cleaning proceeds much more slowly. Although this phenomenon is of great importance in the cleaning of dairy equipment, no conclusive physical explanation has yet been presented. In this study, we present strong evidence that the dissolution rate is affected by the equilibrium-swelling ratio in β-lactoglobulin (βLg) gels. The swelling ratio is greatly reduced in the presence of salts due to the polyelectrolyte screening effect of the cations. This has been observed in free-swelling βLg gels using gravimetrical analysis and in the uniaxial swelling of WPC gel deposits using fluid dynamic gauging. At high dissolution pH (>13.3), the high Na⁺ concentration reduces swelling in spite of the high surface charge of the protein. It is proposed that the reduction of the free volume inside the gel impedes the transport of the protein aggregates out of the NaOH penetration zone. We have also observed that the final dissolution rate of gels pre-soaked in 1 M NaOH or NaCl is similar, despite the difference in pH, and much lower than for untreated gels: the high Na⁺ concentration in the soaked gels hinders swelling, inhibiting the disentanglement of the protein clusters regardless of the high pH.     

The initial stage of high‐pressure induced β‐lactoglobulin aggregation: the long‐run simulation

Research objective of this study was to clarify how the initial stage of high‐pressure induced aggregation of β‐lactoglobulin takes place. For this purpose a special simulation method was developed. Distinctive features of this approach are: the lowest resolution model of protein particles, the local interaction potential and the abandonment of the continuous simulation of particle trajectories. Relatively short molecular dynamics trajectories are used only in order to find the average time step between the collisions. Results of particle collisions that occur with some probability, are separately (‘statically’) modelled using a random variable. This allows the analysis of the process which takes place within 10²–10³ s real‐time, with an existing probability of successful collision of 10^?10–10^?11. Modelling results confirm that at the initial stage of aggregation of 0.5–2% solutions with a neutral pH‐value only dimers as well as trimers arise due to SH/S–S interaction. In addition it was shown that aggregation follows the general principles of the reaction‐limited aggregation of colloids. The proposed approach could further be used in research projects examining the aggregation of β‐lactoglobulin or similar systems.     

Conditions Governing Food Protein Amyloid Fibril Formation. Part II: Milk and Legume Proteins

Both intrinsic and extrinsic factors impact amyloid formation of food proteins. We here review the impact of various conditions and food constituents on amyloid fibrillation of milk and legume proteins. Much less is known about casein and legume protein amyloid‐like fibril formation than about that of whey proteins such as β‐lactoglobulin, α‐lactalbumin, and bovine serum albumin. Proteins of both sources are often studied after heating under strong acidic (pH < 3) conditions. The latter induces changes in protein conformation and often peptide hydrolysis. At higher pH values, alcohols, chaotropic and/or reducing agents induce the conformational changes required to enhance fibrillation. Different types of food proteins can impact each other's fibrillation. Also, the presence of other food constituents can enhance or reduce it. No general conclusions on the mechanisms or impact of different food constituents on food proteins can be made. Optimal conditions for AF formation, that is, heating for several days at low pH, are rare in food processing. However, this does not exclude the possibility of AF formation in food products. For example, slow cooking of hydrolyzed proteins may enhance it. Future research should focus on the prevalence of AFs in complex food systems or model systems relevant for food processing.