Quantification of the interactions between beta-lactoglobulin and pectin through capillary electrophoresis analysis

Biopolymer interactions have many potential applications in pharmaceutical, cosmetic, nutraceutical, and functional food industries. Attractive interactions between proteins and polysaccharides can lead to the formation of complexes. Binding parameters of beta-lactoglobulin (beta-lg)/pectin complexes were determined using frontal analysis continuous capillary electrophoresis and the overlapping binding site model. At pH 4, approximately 23 beta-lg molecules were cooperatively complexed on low-methoxyl pectin, where each beta-lg molecule covered an average of 12 galacturonic acid residues. The calculated binding constant was 1431 M(-1). The interactions between pectin and four selected peptides located on the outer surface of the beta-lg were investigated in order to identify which part of the protein was likely to interact with the pectin. The peptide beta-lg 132-148, which corresponds to the alpha-helix zone, and the peptides beta-lg 76-83, 41-60, and 1-14 would be involved in the interaction with the pectin.     

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.     

Cationic antimicrobial (ε-polylysine)-anionic polysaccharide (pectin) interactions: influence of polymer charge on physical stability and antimicrobial efficacy

The cationic biopolymer ε-polylysine (ε-PL) is a potent food-grade antimicrobial that is highly effective against a range of food pathogens and spoilage organisms. In compositionally complex systems such as foods and beverages, cationic ε-PL molecules may associate with anionic substances, leading to increased turbidity, sediment formation, and reduced antimicrobial activity. This study therefore characterized the interactions between cationic ε-PL and anionic pectins with different degrees of esterification (DE) and then investigated the influence of these interactions on the antimicrobial efficacy of ε-PL. The nature of the interactions was characterized using isothermal titration calorimetry (ITC), microelectrophoresis (ME), and turbidity measurements. High (DE 61%), medium (DE 51%), and low (DE 42%) methoxyl pectins interacted with ε-PL molecules through electrostatic forces, forming either soluble or insoluble complexes with various electrical charges, depending on the relative mass ratio of pectin and ε-PL. The interaction of pectin with ε-PL increased as the negative charge density on the pectin molecules increased, that is, with decreasing DE. The antimicrobial efficacy of ε-PL against two acid-resistant spoilage yeasts (Zygosaccharomyces bailii and Saccharomyces cerevisiae) decreased progressively in the presence of increasing levels of all three pectins. Nevertheless, the low DE pectin decreased the antimicrobial efficacy of ε-PL much more dramatically, likely due to strong electrostatic binding of ε-PL onto low DE pectin molecules reducing its interaction with anionic microbe surfaces. This study provides knowledge that will facilitate the rational application of ε-PL as an antimicrobial in complex food systems.     

Isothermal titration calorimetry study of pectin-ionic surfactant interactions

Isothermal titration calorimetry (ITC) was used to measure enthalpy changes resulting from injection of anionic (sodium dodecyl sulfate, SDS) or cationic (dodecyl trimethylammonium bromide, DTAB) surfactants into aqueous 1 wt % pectin solutions (30, 60, or 90% methoxylated). In the absence of pectin, the critical micelle concentrations (cmc) determined by ITC were 14.7 mM for DTAB and 7.7 mM for SDS. Binding of DTAB to pectin was endothermic and was attributed to electrostatic attraction between the cationic surfactant and anionic biopolymer. Binding of SDS to pectin was exothermic and was attributed to hydrophobic interactions. Pectin reduced the cmc of SDS, probably because of long-range electrostatic repulsion between the molecules. Above a particular concentration, which depended on pectin and surfactant type, both ionic surfactants promoted pectin aggregation (monitored by turbidity increase). This study demonstrates the potential of ITC for providing valuable information about interactions between polysaccharides and amphiphiles.     

Pressure-induced conformational changes of beta-lactoglobulin by variable-pressure Fourier transform infrared spectroscopy.

Pressure-induced conformational changes in D(2)O solutions of the two genetic variants of beta-lactoglobulin A (beta-lg A) and beta-lactoglobulin B (beta-lg B) and an equal mixture of both variants (beta-lg A+B) were studied by employing variable-pressure Fourier transform infrared (VP-FTIR) spectroscopy. Changes in the secondary structure of beta-lg A were observed at lower pressure compared to beta-lg B, indicating that beta-lg A had a more flexible structure. During the decompression cycle beta-lg A showed protein aggregation, accompanied by an increase in alpha-helical conformation. The changes in the secondary structure of beta-lg B with the pressure were minor and for the most part reversible. Upon decompression no aggregation in beta-lg B was observed. Increasing the pressure from 0.01 to 12.0 kbar of a solution containing beta-lg A+B resulted in substantial broadening of all major amide I bands. This effect was partially reversed by decreasing the hydrostatic pressure. beta-lg A+B underwent less aggregate formation than beta-lg A, possibly as a result of protein-protein interactions between beta-lg A and beta-lg B. Hence, it is likely that the functional or biological attributes of beta-lg proteins may be affected in different ways by hydrostatic pressure.     

Inclusion complex of conjugated linoleic acid (CLA) with cyclodextrins

Conjugated linoleic acid (CLA) inclusion complexes with alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), and gamma-cyclodextrin (gamma-CD) (designated CLA/CDs inclusion complexes) were prepared to determine the mole ratio of CLA complexed with CDs and the oxidative stability of CLA in the CLA/CDs inclusion complexes. When measured by GC, (1)H NMR, and T(1) value analyses, 1 mole of CLA was complexed with 5 mol of alpha-CD, 4 mol of beta-CD, and 2 mol of gamma-CD. The oxidation of CLA induced at 35 degrees C for 80 h was completely prevented by the formation of CLA/CDs inclusion complexes.     

Stabilization of oil-in-water emulsions by beta-lactoglobulin-polyethylene glycol conjugates

The disulfide bonds of beta-lactoglobulin (beta-lg) were modified by oxidative sulfitolysis to generate beta-lgSO(3). The native protein (beta-lg) and the modified protein (beta-lgSO(3)) were conjugated to activated polyethylene glycol (PEG) to generate beta-lgPEG and beta-lgSO(3)PEG, respectively. Oil-in-water (o/w) emulsions containing 1% beta-lg or beta-lg conjugates were prepared at pH 2.8, 5.0, and 7.0. Emulsion droplet diameters and zeta potentials were measured. For the same emulsifier, emulsion droplet diameters decreased when emulsion pH increased. Zeta potentials of emulsion droplets increased with pH for beta-lg and beta-lgSO(3). Zeta potentials of beta-lgPEG and beta-lgSO(3)PEG approached zero, suggesting that the protein molecule was covered by PEG chains. Accelerated and 7-day storage stabilities at 21 degrees C of the emulsions were monitored. The emulsifying activity index (EAI) of beta-lgPEG was not significantly different from the EAI of beta-lg. The EAI of beta-lg was enhanced following sulfitolysis of beta-lactoglobulin. The emulsifying activity increased more when the oxidatively modified protein was conjugated to polyethylene glycol. Emulsions made with beta-lgSO(3)PEG were more stable than emulsions made with beta-lg, beta-lgPEG, or beta-lgSO(3) under accelerated stability study and for 7 days at 21 degrees C. The stability of o/w emulsions stabilized with beta-lgSO(3)PEG increased because individual droplets were better protected, against protein bridging or coalescence, by the thick adsorbed protein-PEG layer.     

Fibril assemblies in aqueous whey protein mixtures

Fibril formation in mixtures of whey proteins upon heating at pH 2 was investigated. Fibrils were found to coexist with other structures, such as spherulites. These spherulites consist of radially oriented fibrils. At total protein concentrations above 6 wt %, transparent gels were formed. Changing the ratio between the various whey proteins did not affect this gelation concentration as long as beta-lactoglobulin (beta-lg) was present, suggesting that beta-lg was dominant in the gelation. Pure alpha-lactalbumin and pure bovine serum albumin did not form fibrils, nor did they gel upon heating at pH 2 and 80 degrees C for up to 10 h. They did however induce a decrease in the beta-lg concentration needed for gel formation upon heating at pH 2. Our results suggest that beta-lg is the only fibril forming protein at the conditions used and that no mixed fibrils are formed.     

Formation and stabilization of antimicrobial delivery systems based on electrostatic complexes of cationic-non-ionic mixed micelles and anionic polysaccharides

Lauric arginate (LAE) is a cationic surfactant that is of great interest to the food industry because of its strong antimicrobial activity. However, its application within foods and beverages is currently restricted because of its limited solubility in aqueous solutions and its bitter taste, which have been associated with its cationic nature. This study examines whether electrostatic complexes between cationic LAE mixed micelles and anionic polysaccharides could be used to improve LAE functionality. Two types of pectin (high and low methoxyl) were titrated into buffer solutions containing either LAE micelles or LAE/Tween 20 mixed micelles (pH 3.5, 50 mM citrate buffer). The electrical characteristics of the micelles or micelle/pectin complexes were assessed by microelectrophoresis measurements, while their stability to aggregation was evaluated by light scattering measurements. LAE micelle/pectin complexes formed large aggregates that rapidly sedimented. On the other hand, mixed micelle/pectin complexes (1:1 LAE/Tween 20, w/w) were stable to aggregation and formed clear solutions. The electrical charge of mixed micelles changed from +8 to -15 mV when the pectin concentration was increased (0.00-0.05 wt %), indicating an electrostatic interaction between anionic pectin molecules and cationic micelles. Lower concentrations of low methoxyl pectin were required (0.01 wt %) to change the net charge of mixed micelles from positive to negative than high methoxyl pectin (0.025 wt %). Our results suggest that the addition of pectin to mixed LAE/Tween 20 micelles leads to the formation of electrostatic complexes that may be useful as functional ingredients in food and other products.     

Interactions of a cationic antimicrobial (ε-polylysine) with an anionic biopolymer (pectin): an isothermal titration calorimetry, microelectrophoresis, and turbidity study

ε-Polylysine (ε-PL) is a food-grade cationic antimicrobial that is highly effective against a range of food pathogens and spoilage organisms. In compositionally complex environments, like those found in most foods and beverages, the antimicrobial activity of cationic ε-PL is likely to be impacted by its interactions with anionic components. The purpose of this study was to characterize the interactions between cationic ε-polylysine and an anionic biopolymer (high methoxyl pectin, HMP) using isothermal titration calorimetry (ITC), microelectrophoresis (ME), and turbidity measurements. ITC and ME measurements indicated that ε-PL bound to pectin, while turbidity measurements indicated that the complexes formed could be either soluble or insoluble depending on solution composition. Ionic strength and pH were also shown to affect the interactions significantly, highlighting their electrostatic origin. This study demonstrates that ε-PL can form either soluble or insoluble complexes with anionic biopolymers depending on the composition of the system. Our study provides basic knowledge that will facilitate the more rational application of ε-PL in complex food systems.     

Impact of electrostatic interactions on formation and stability of emulsions containing oil droplets coated by beta-lactoglobulin-pectin complexes

Interfacial protein-polysaccharide complexes can be used to improve the physical stability of oil-in-water emulsions. The purpose of this study was to examine the impact of ionic strength on the formation and stability of oil-in-water emulsions containing polysaccharide-protein-coated droplets. Emulsions were prepared that contained 0.1 wt % corn oil, 0.05 wt % beta-lactoglobulin, and 0.02 wt % pectin at pH 7. The emulsions were then adjusted to pH 4 to promote electrostatic deposition of the pectin molecules onto the surfaces of the protein-coated droplets. The salt concentration of the aqueous phase (0 or 50 mM NaCl) was adjusted either before or after deposition of the pectin molecules onto the droplet surfaces. We found that stable emulsions containing polysaccharide-protein-coated droplets could be formed when the salt was added after pectin adsorption but not when it was added before pectin adsorption. This phenomenon was attributed to the ability of NaCl to promote droplet flocculation in the protein-coated droplets so that the pectin molecules adsorbed onto the surfaces of flocs rather than individual droplets when salt was added before pectin adsorption. We also found that polysaccharide-protein-coated droplets had a much improved stability to salt-induced flocculation than protein-coated droplets with the same droplet charge (zeta-potential). Theoretical predictions indicated that this was due to the ability of the adsorbed polysaccharide layer to strongly diminish the van der Waals attraction between the droplets.     

Heat-induced aggregation of beta-lactoglobulin as a function of pH.

The effect of pH in the range 6.0-8.0 on the denaturation and aggregation of beta-lactoglobulin (beta-lg) was investigated. Results were interpreted in terms of the reaction scheme for the denaturation and aggregation of beta-lg proposed by Roefs and De Kruif (Eur. J. Biochem. 1994, 226, 883-889). The rate of conversion of native beta-lg increased strongly at higher pH values, whereas the molecular mass of the aggregates decreased strongly. In the pH range 6.4-8.0 aggregates were formed mainly by intermolecular disulfide bonds, but even at pH 6.0, thiol/disulfide exchange reactions were involved, although to a lesser extent. The time course of the exposure of the thiol group in native beta-lg upon heating and the subsequent disappearance of this group through the formation of disulfide-linked aggregates was investigated by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) and varied strongly with pH. These observations could be used, in combination with the reaction steps of the reaction scheme, to describe qualitatively the strongly pH-dependent isothermal calorimetry curves, measured at 65 degrees C.     

Probing protein-tannin interactions by isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) has been applied to investigate protein-tannin interactions. Two hydrolyzable tannins were studied, namely myrabolan and tara tannins, for their interaction with bovine serum albumin (BSA), a model globular protein, and gelatin, a model proline-rich random coil protein. Calorimetry data indicate that protein-tannin interaction mechanisms are dependent upon the nature of the protein involved. Tannins apparently interact nonspecifically with the globular BSA, leading to binding saturation at estimated tannin/BSA molar ratios of 48:1 for tara- and 178:1 for myrabolan tannins. Tannins bind to the random coil protein gelatin by a two-stage mechanism. The energetics of the first stage show evidence for cooperative binding of tannins to the protein, while the second stage indicates gradual saturation of binding sites as observed for interaction with BSA. The structure and flexibility of the tannins themselves alters the stoichiometry of the interaction, but does not appear to have any significant affect on the overall binding mechanism observed. This study demonstrates the potential of ITC for providing an insight into the nature of protein-tannin interactions.     

Effects of salt concentration on formation and dissociation of beta-lactoglobulin/pectin complexes

The formation and dissociation of beta-lactoglobulin/pectin complexes at various sodium chloride concentrations (CNaCl) have been studied by turbidimetric titration. An increase of CNaCl up to 0.1 M shifts the critical pHphi1, which designates the formation of beta-lactoglobulin/pectin coacervates, to higher pH values, whereas further increase of CNaCl from 0.1 to 0.8 M decreases pHphi1 values. These salt effects can be explained in terms of a salt-enhanced effect at lower salt concentrations or a salt-reduced effect at higher salt concentrations, respectively. On the other hand, the value of pHphi2, which corresponds to the dissociation of beta-lactoglobulin/pectin coacervates, tends to have smaller pH values when CNaCl increases from 0.1 to 0.3 M. No observable pHphi2 values are found at CNaCl higher than 0.3 M. The disappearance of pHphi2 is mainly attributed to the strong self-aggregation capability of beta-lactoglobulin at higher CNaCl. The aggregation of beta-lactoglobulin at high CNaCl is reversible, as suggested by the atomic force microscopy results.     

Origin and thermodynamic properties of the instability of synthetic azo colorants in gum arabic solutions

The instability of some industrially important synthetic azo colorants, including sunset yellow, azorubine, and allura red, toward gum arabic in aqueous solution has been a long-standing problem for the beverage and confectionery industries. Precipitation of these colorants causes the deterioration of product appearance and properties. This work examines the origin and nature of the problem by analysis of the precipitate and thermodynamic studies of gum arabic-colorant interactions using isothermal titration calorimetry (ITC). The presence of divalent alkaline earth metals in gum arabic samples, that is, calcium and magnesium, is shown to be responsible for the precipitation of the azo colorants. There is no direct interaction between gum arabic and the colorant molecules, and the precipitate is formed likely due to the mediation/bridging by the divalent cations. The thermodynamic knowledge gained from the ITC studies, for example, binding affinity, stoichiometry, and enthalpy, enables interpretation of many industrial observations.     

Effect of different dextrose equivalent of maltodextrin on the interactions with anionic surfactant in an isothermal titration calorimetry study

Isothermal titration calorimetry (ITC) was used to study interactions between an anionic surfactant (sodium dodecyl sulfate, SDS) and maltodextrins with different dextrose equivalents (DE) in a buffer solution (pH 7.0, 10 mM NaCl, 20 mM Trizma, 30.0 degrees C). The interaction between SDS and maltodextrin was exothermic, which was attributed to incorporation of the hydrocarbon tail of the surfactant into a helical coil formed by the maltodextrin molecules. ITC measurements indicated that the number of SDS molecules bound per gram of maltodextrin increased with decreasing maltodextrin DE, i.e., increasing molecular weight. It was proposed that SDS only binds to maltodextrin molecules that have a DE greater than 10 glucose units.     

Characterization of the temperature activation of pectin methylesterase in green beans and tomatoes

Low-temperature blanching of vegetables activates the enzyme pectin methylesterase (PME), which demethylates cell wall pectins and improves tissue firmness. This temperature activation of PME has been investigated by measuring the formation of methanol in intact tissue of green beans and tomatoes. Rates of methanol formation at temperatures of 35-65 degrees C were obtained by measuring the release of methanol from thin slices of tomato pericarp or green bean pod material. Activation energies of 112 and 97 kJ mol(-1) were calculated for PME activity in green beans and tomatoes, respectively. These activation energies indicate that the rate of pectin demethylation at 65 degrees C will be nearly 100 times that at 25 degrees C. PME activity was also determined titrimetrically using a solubilized form of the enzyme and purified pectin at temperatures from 30 to 60 degrees C. Under these conditions, much lower activation energies of 37 and 35 kJ mol(-1) were obtained for green beans and tomatoes, respectively. Methanol accumulation during heating of whole intact green beans was also determined and yielded an activation energy similar to that obtained with sliced beans. Whole green beans held at room temperature did not accumulate any methanol, but sliced or homogenized beans did. If whole beans were first heated to 45 degrees C and then cooled, methanol accumulation was observed at room temperature. These results indicate that two factors contribute to the observed high rate of pectin de-esterification during low-temperature blanching: (1) An irreversible change, causing PME to become active, occurs by heating to > or = 45 degrees C. (2) The high activation energy for pectin de-esterification means that the rate of de-esterification increases substantially with increasing temperature.     

Characterization of the surface-active components of sugar beet pectin and the hydrodynamic thickness of the adsorbed pectin layer

The fraction of sugar beet pectin (SBP) adsorbed onto limonene oil droplets during emulsification has been isolated, and its chemical and physicochemical characteristics have been determined. While the SBP sample itself was found to contain 2.67 and 1.06% protein and ferulic acid, respectively, the adsorbed fraction contained 11.10% protein and 2.16% ferulic acid. The adsorbed fraction was also found to have a higher degree of acetylation, notably at the C2 position on the galacturonic acid residues, and was also found to contain a higher proportion of neutral sugars, which are present in the ramified side chains of the pectin molecules. The thickness of the layer of SBP adsorbed onto polystyrene latex particles was studied by dynamic light scattering and was found to increase with increasing surface coverage. It was found to have a value of approximately 140 nm at plateau coverage, which closely corresponded to the hydrodynamic diameter of the pectin chains. The adsorbed layer thickness was found to be sensitive to pH and the presence of electrolyte. The thickness at a surface coverage of approximately 20 mg/m(2) in the absence of electrolyte at pH approximately 4 was 107 nm and at pH 8.8 was 70 nm, while at pH approximately 4 in the presence of 10 mM NaCl the thickness was found to be 70 nm. It was concluded that the SBP molecules form multilayers at the surface due to electrostatic interaction between the positively charged protein moieties and the galacturonic acid residues. The removal of calcium from the SBP had no effect on the adsorbed layer thickness; hence, multilayer formation due to calcium ion cross-linking was considered unlikely.     

Enterocyte and M-cell transport of native and heat-denatured bovine beta-lactoglobulin: significance of heat denaturation

The three-dimensional structure, digestibility, and immunological properties of bovine beta-lactoglobulin (beta-lg) are modified by heat treatments used in processing of liquid milk products. Because it is not known if such treatments also modify the intestinal transport properties of beta-lg, the transport of native and heat-denatured bovine beta-lg was investigated in experimental cell models using Caco-2 cells and M cells. Transport of beta-lg labeled with a fluorescent marker was followed with fluorometric measurements, electrophoretic analyses, and fluorescence microscopy. The data show that both cell types transported native beta-lg more efficiently than they did heat-denatured beta-lg. In addition, M cells transported native beta-lg more than Caco-2 cells. Transport of native and heat-denatured beta-lg was transcellular. The electrophoretic data also suggest that heat-denatured beta-lg may have degraded more than native beta-lg during the transport.     

镉安全水稻材料根系细胞壁果胶对镉的响应特征

【目的】研究镉(Cd)处理下水稻根系细胞壁果胶对Cd胁迫的响应,进一步深化Cd安全水稻材料根系细胞壁Cd的固持机制。【方法】以Cd安全水稻材料D62B为研究对象,普通材料Luhui17为对照进行水培试验。设4个Cd质量浓度处理：0 mg/L (CK)、0.5 mg/L (Cd0.5)、1.0 mg/L (Cd1)、2.0 mg/L (Cd2)。在水稻分蘖期采集根系样品,分析细胞壁多糖中果胶、半纤维1、半纤维2以及残渣部分的Cd含量,测定果胶糖醛酸含量、果胶酯化度、果胶甲酯酶(PME)活性、根系过氧化氢(H₂O₂)含量以及细胞壁过氧化物酶(POD)活性,进而分析根系细胞壁果胶对Cd的响应特征。【结果】1) Cd胁迫下,D62B和Luhui17根系细胞壁果胶合成增加,根系细胞壁低酯化和高酯化果胶糖醛酸含量均表现为D62B高于Luhui17。Cd处理下D62B根系细胞壁低酯化和高酯化果胶糖醛酸含量较对照分别增加了13.21%～71.82%和22.10%～64.27%,Luhui17分别增加了24.14%～137.86%和13.12%～41.26%。...