Protein-polyphenol Complexes Research Articles

Influence of polyphenols on soil nitrogen mineralization through the formation of bound protein in tropical montane forests of Mount Kinabalu, Borneo

We investigated the role of polyphenols in soil solution on N dynamics in tropical montane forests, in which soil N mineralization was typically low. We compared two sites on Mount Kinabalu, Borneo, which contrast in soil age (the Tertiary versus Quaternary period). The older site had a much thicker soil organic matter and reduced N availability. Soil solution was collected from twenty lysimeters that were installed at two depths (10 cm and 50 cm) at each of ten random locations at each site, and was fractionated into hydrophobic and hydrophilic fractions. The hydrophobic fraction was hydrolyzed to determine the concentration of bound N as amino acids. The mean concentration of phenolics was five times greater in soil solution of the upper horizon at the older N-poor site compared to the younger N-rich site. The mean concentration of hydrolyzed amino acids was twice greater in the upper horizon at the older N-poor site than that at the younger N-rich site; phenolics and amino acids were significantly greater under conifers than under non-conifers within the older site. DOC:DON ratio in soil solution was wider at the older N-poorer site (56) than at the younger N-richer site (34), reflecting the lower quality of litter for the former. We suggest that more protein–polyphenol complexes (PPC) in soil solution are formed at the older site and consequently the recalcitrance of N adds to the soil infertility. The dominance of conifer trees exacerbates the formation of PPC.

Nanocomplex formation between β-lactoglobulin or caseinomacropeptide and green tea polyphenols: Impact on protein gelation and polyphenols antiproliferative activity

The development of milk functional foods including health-promoting green tea polyphenols represents a challenge for the food industry since the formation of protein–polyphenol complexes may affect both protein technological properties and polyphenols biological activity. The present work aimed at the characterization of complexes formed between green tea polyphenols and either β-lactoglobulin (β-lg) or caseinomacropeptide (CMP), as well as to evaluate how this complexation may impact on protein gelation and polyphenol antiproliferative activity against tumor cell lines.Particle size and charge of protein–polyphenol complexes depend on protein nature and pH. At pH 6 they had the smallest size and were soluble. The presence of polyphenols accelerated the gelation of both β-lg and CMP, and mainly affected viscoelasticity of β-lg gels. Polyphenol complexation by proteins did not inhibit its anti-proliferative activity. Moreover, they exerted a better performance on some particular tumor cell lines.

Capillary electrophoresis methods for the determination of covalent polyphenol–protein complexes

The bioactivities and bioavailability of plant polyphenols including proanthocyanidins and other catechin derivatives may be affected by covalent reaction between polyphenol and proteins. Both processing conditions and gastrointestinal conditions may promote formation of covalent complexes for polyphenol-rich foods and beverages such as wine. Little is known about covalent reactions between proteins and tannin, because suitable methods for quantitating covalent complexes have not been developed. We established capillary electrophoresis methods that can be used to distinguish free protein from covalently bound protein-polyphenol complexes and to monitor polyphenol oxidation products. The methods are developed using the model protein bovine serum albumin and the representative polyphenol (-)epigallocatechin gallate. By pairing capillaries with different diameters with appropriate alkaline borate buffers, we are able to optimize resolution of either the protein-polyphenol complexes or the polyphenol oxidation products. This analytical method, coupled with purification of the covalent complexes by diethylaminoethyl cellulose chromatography, should facilitate characterization of covalent complexes in polyphenol-rich foods and beverages such as wine.

Honey melanoidins: Analysis of the compositions of the high molecular weight melanoidins exhibiting radical-scavenging activity

Size-exclusion chromatography (SEC) and activity-guided fractionation of honeys allowed the isolation of high molecular weight brown compounds, ranging in size from 66 to 235kDa that exhibited peroxyl radical-scavenging activity. Their concentrations, antioxidant activity and degree of browning increased after heat-treatment of honeys, suggesting that they represent melanoidins. Chemical analysis of melanoidins demonstrated the presence of proteins, polyphenols and oligosaccharides. Heat-treatment caused an increased incorporation of phenolics into high molecular weight melanoidins and drastically decreased the protein content in these fractions with a concomitant appearance of high molecular weight protein–polyphenol complexes of reduced solubility. LC–ESI–MS demonstrated the presence of oligosaccharide moieties, supporting the postulated origin of melanoidins. The changes in the phenolic content of melanoidins from heated honeys were strongly correlated with their oxygen radical absorbance capacity (ORAC) values (R=0.75, p<0.0001), indicating that polyphenols contribute to the antioxidant activity of melanoidins. In summary, honey melanoidins are multi-component polymers consisting of protein–polyphenol–oligosaccharide complexes. A direct interaction between polyphenols and melanoidins resulted in a loss or gain of function for melanoidin antioxidant activity.

Recognition characters in peptide–polyphenol complex formation

Dietary polyphenols have received attention for their anti-oxidative, anti-carcinogenic and anti-neurodegenerative effects. Polyphenols bind to proteins leading to the formation of soluble or insoluble protein–polyphenol complexes which could significantly influence their biological activities. NMR and molecular modeling studies were performed to investigate the influence of the bulk, flexibility and hydrophobicity of polyphenols on the association with bradykinin, the peptide model. Our results show that the strength of the interactions could be positively correlated with polyphenol hydrophobicity and a comparison between pentagalloylglucose and vescalagin indicated that flexibility might play a positive role in the interaction with peptides and proteins.

Characterization of protein–polyphenol interactions

Dietary polyphenols have received attention for their biologically significant functions as antioxidants, anticarcinogens or antimutagens, which have led to their recognition as potential nutraceuticals. Polyphenols also characteristically possess a significant binding affinity for proteins, which can lead to the formation of soluble and insoluble protein–polyphenol complexes. Questions remain concerning whether and to what extent the protein–polyphenol interaction influences functionality. For example, is the formation of protein–polyphenol complexes an obstacle to the nutritional bioavailability of either species? This article discusses the development of suitable methodologies to investigate the physicochemical basis of protein–polyphenol interactions and the influence of structure–activity relationships on binding affinities.

Beer clarification by cross-flow microfiltration — effect of surface hydrodynamics and reversed membrane morphology

The separation characteristics in beer microfiltration is based on a fine balanced retention of particles (yeast cells, debris, and coagulated protein–polyphenol complexes) and transmission of dissolved macromolecules (proteins, carbohydrates, colour and flavour compounds) to achieve beer clarification whilst preserving the ‘wholesome’ quality of the filtered beer. The required porous transmission of large amount of macromolecular species led to an unavoidable and complex membrane fouling in terms of fouling constituents, formation and structure, which are the major obstacles to obtaining an economically viable flux and consistent permeate quality. This paper describes an experimental study of beer clarification by cross-flow microfiltration with the aim of increasing a low permeation rate to an economically acceptable level by employing and optimising some flux-enhancement techniques. These include, (i) modification of bulk feed flow hydrodynamic conditions by superimposing a helical-flow pattern and a two-way reversing flow pulsation; (ii) application of an automated high frequency backflush programme with controllable frequency and backpulse strength; and (iii) filtration at reversed membrane morphology to allow the open substructure of the asymmetric ceramic membrane (Ceramem) to face the feed flow and thereby a reversed transmembrane pressure (TMP) gradient. The increased bulk-flow turbulence generated by flow pulsation was found disappointingly ineffective, and the superimposed helical flow pattern was only marginally better. The backflush technique was a more effective method and application of an optimised backflush programme at normal cross-flow conditions achieved 410% flux increase. The most dramatic flux improvement was achieved when the filtration was carried out with high frequency backflush at a reversed membrane mounting configuration. The author attributed the large flux increase to a mechanism of reversible ‘particle holding and releasing’ by the open sub-structure of the membrane under the influence of high frequency backflush, which greatly reduced fouling in the critical membrane phase.

Quantitative determination of fouling layer composition in the microfiltration of beer

Abstract Experiments to determine fouling layer composition have been conducted on three different beers: (i) a standard lager; (ii) a commercial lager; and (iii) a standard ale. Fresh Millipore membranes, nominal pore size 0.45 μm, were fouled by applying a transmembrane pressure of 0.7 barg. at 0°C in a stirred cell. Each beer was tested both pre- and post-centrifugation. In each experiment, the flux decline data was obtained and then the fouling layer was removed using a sonicator. The fouling layers were freeze-dried and re-suspended in 0.1 M imidazole. They were then analysed for carbohydrate, polyphenols and protein. Unfiltered samples of each beer, both pre- and post-centrifugation were also analysed using the same procedure. The flux decline data show that the presence of yeast enhances flux. Furthermore, the final fluxes obtained for Beer 2, the commercial beer, were an order of magnitude greater than those obtained for the non-commercial beers. Analysis of the fouling layers reveals that for all beers, polyphenol is present in disproportionately greater quantities than are present in the unfiltered beer, while carbohydrate is present in disproportionately smaller amounts. The data for Beers 2 and 3 also suggest that protein is present in disproportionately greater quantities. It is further concluded that, for all beers, the fouling layer consists of both protein–polyphenol complexes and carbohydrate gels.

Evaluation of Rapid Colloidal Stabilization with Polyvinylpolypyrrolidone (PVPP)

Several colorimetric, chromatographic, and nephelometric tests were compared as tools for monitoring the rapid (<3 days) colloidal stabilization of lager beers by a combination of cold filtration at −1°C and treatments with polyvinylpolypyrrolidone (PVPP). No significant changes in composition were detected during storage of beer for seven days at −1°C prior to filtration on a bench scale. The effects of dosage on a pilot-scale with different amounts of recoverable PVPP (10–50 g/hl) or single-use PVPP (10–20 g/hl) on total polyphenols, total flavanols, simple flavanoids, protein sensitivity, alcohol-cooling haze, and tannoids were compared with concomitant changes in colloidal stability, as revealed by accelerated aging at both 37 and 60°C. Close correlations were found between the results of the haze forcing tests and all tests other than the tannoid measurement made on freshly bottled beers. Although decreases in the contents of the simple flavanoids related most closely to improvements in colloidal stability, the alcohol-cooling test was also a useful indicator of stabilization effectiveness. The colloidal stabilities of beers aged at 37°C were related to both the amounts of simple flavanoids that were oxidized during aging and also to the contents of tannoids in the aged beers. Treatment with recoverable PVPP at the highest rate effectively stabilized the sample beer by adsorbing both the simple flavanoid precursors of chill haze and the tannoid components of protein-polyphenol complexes.

STUDIES ON HAZE PRECURSORS IN BEERS AND WORTS

Detailed studies have been carried out on the fractionation of worts and beers on Sephadex and Bio-gel columns with high degrees of cross-linking. On G.25 Sephadex, the excluded fraction contains much of the total protein in the form of complexes with polyphenols bound in such a way that they do not interact with the column material. Continued elution with aqueous solvents gives many low molecular weight wort components and in addition some protein-polyphenol complexes which are retarded by relatively weak adsorption on the column. These compounds have been further studied by rechromatography in urea to dissociate the complexes and after reaction with excess flavonoid. Since the adsorbed compounds interact with the column through hydrogen bonding of active phenolic bonds, it may be that these are also the compounds which cross-link with other protein complexes in the beer to build up haze particles. Molybdate and alkali can be used subsequently to elute the free tannin and flavonoid molecules which are strongly absorbed.

Tobacco polyphenols. IV. A complex polyphenol in flue-cured tobacco

A complex polyphenol isolated from flue-cured tobacco comprises equimolecular amounts of the major tobacco polyphenols, rutin, chlorogenic acid, and scopolin. Hydrolysis studies suggest that these constituents are linked together with covalent bonds and show that the glucoside bond of scopolin is essential to the stability of the compound. It is dialyzable and hence of low molecular weight. No amino acids are detectable in acid hydrolyzates indicating the absence of proteins or peptides, in contrast to the nondialyzable protein-polyphenol complexes of air-cured tobaccos.

An Investigation of the interactions between milk proteins and tea polyphenols

Electrophoretic methods have been used to investigate the interactions in solution between coloured tea polyphenols and milk proteins. It has been found that: 1.When a tea infusion is mixed with milk in the proportions but not the overall concentration obtaining in a cup of tea with milk, the coloured tea polyphenols interact mainly with the α-casein complex and the β-casein of the milk to form soluble casein-polyphenol complexes. β-Lactoglobulin and α-lactalbumin, the main whey proteins, appear to be unaffected by the polyphenols in the presence of the casein at the concentrations used in this investigation.2.In the absence of the casein both α-lactalbumin and β-lactoglobulin interact with the coloured tea polyphenols, forming either soluble or insoluble protein-polyphenol complexes, the type formed depending on the relative and overall proportions of protein and polyphenol present in the mixture.3.Paper electrophoresis of tea infusion in borate buffer (pH 8.5) and in molybdate buffer (pH 4.9) is a useful method for the comparison of tea infusions and of polyphenol fractions isolated from tea.4.Paper electrophoresis of tea infusion with added casein in molybdate buffer indicates that the casein interacts selectively with some of the coloured tea polyphenols in the presence of the molybdate complexing agent.5.Membrane filter electrophoresis in phosphate buffer (pH 6.7) and 7 M with respect to urea indicates that the milk protein/tea polyphenol interactions are at least initiated by the formation of hydrogen bonds.