Molecular changes of phenolic-protein interactions in isolated proteins from flaxseed and soybean using Native-PAGE, SDS-PAGE, RP-HPLC, and ESI-MS analysis.

The effects of protein-phenolic interactions on the molecular characteristics of soybean and flaxseed proteins were investigated. Proteins were isolated from soybean and flaxseed using isoelectric precipitation, followed by extraction of free and bound phenolics. The effects of elimination of the phenolic compounds on molecular characteristics of the protein isolates were studied using reversed phase-high performance liquid chromatography (RP-HPLC), Native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and electron spray ionization-mass spectrometry (ESI-MS). The Native-PAGE fractions from isolated proteins from full-fat flaxseed and soybean revealed that protein migration was affected by removal of bound phenolics. SDS-PAGE from full-fat and defatted protein isolates of flaxseed and soybean revealed that the removal of bound phenolics affected the molecular characteristics of protein subunits. Soybean protein isolates had protein-phenolic interactions through acidic and basic subunits. RP-HPLC and ESI-MS showed that the removal of bound and free phenolics had only minor effects on the molecular characteristics of isolated proteins from defatted and full-fat soybean. With respect to isolated proteins from flaxseed, the removal of bound phenolics showed little effect on the electrophoretic behavior of the proteins or the protein subunits. PRACTICAL APPLICATIONS: Phenolic-protein and phenolic-lipid-protein interactions may affect the nutritional, physicochemical, and functional properties of isolated proteins from food in flaxseed and soybean.

Occurrence, types, properties and interactions of phenolic compounds with other food constituents in oil-bearing plants.

Phenolic phytochemicals have become of interest due to their therapeutic potential, particularly with regards to their anti-cancer, anti-inflammatory, hypolipidemic, and hypoglycemic properties. An evolving area of research involving phenolics in foods and their products pertains to the functional, biological, and nutritional consequences resulting from the binding between certain phenolic compounds and the macronutrient and micronutrient constituents of foods. The goal of this review is to provide a summary of studies investigating endogenous phenolic interactions with major components in food systems, including carbohydrates, proteins, lipids, minerals and vitamins, with a focus on the phenolic compounds and nutrients in oil-bearing plants. Another major objective is to provide a comprehensive overview of the chemical nature of phenolic interactions with food constituents that could affect the quality, nutritional and functional properties of foods. Such information can assist in the discovery and optimization of specific phenolic complexes in plant-based foods that could be utilized towards various applications in the food, nutraceutical and pharmaceutical industries.

A review of phenolic compounds in oil-bearing plants: Distribution, identification and occurrence of phenolic compounds.

Over the last two decades, separation, identification and measurement of the total and individual content of phenolic compounds has been widely investigated. Recently, the presence of a wide range of phenolic compounds in oil-bearing plants has been shown to contribute to their therapeutic properties, including anti-cancer, anti-viral, anti-oxidant, hypoglycemic, hypo-lipidemic, and anti-inflammatory activities. Phenolics in oil-bearing plants are now recognized as important minor food components due to several organoleptic and health properties, and they are used as food or sources of food ingredients. Variations in the content of phenolics in oil-bearing plants have largely been attributed to several factors, including the cultivation, time of harvest and soil types. A number of authors have suggested that the presence phenolics in extracted proteins, carbohydrates and oils may contribute to objectionable off flavors The objective of this study was to review the distribution, identification and occurrence of free and bound phenolic compounds in oil-bearing plants.

Interactions of caseins with phenolic acids found in chocolate.

To investigate the interactions between caseins and phenolic acids, such as the ones present in chocolate, casein was incubated with protocatechuic acid or p-coumaric acid at 55°C. In addition, casein was isolated from chocolate and the phenolic compounds within these caseins were quantified. Electrophoresis results revealed that casein-phenolic interactions were induced by incubation; minor aggregation of casein subunits was observed after incubation of casein with protocatechuic acid. Minor aggregation of casein isolated from milk chocolate was also observed. In vitro hydrolysis of casein control, casein-protocatechuic acid, casein-p-coumaric acid, caseins isolated from milk chocolate and white chocolate using trypsin showed degree of hydrolysis of 19.3, 18.6, 17.7, 10.4 and 17.8% respectively. The presence of protocatechuic acid and p-coumaric acid in the model system and the presence of phenolic compounds in milk chocolate, in addition to the structural changes occurring during processing, affected the peptide profiles of casein hydrolysates.

Potato protein isolates: recovery and characterization of their properties.

An imitation of industrial potato fruit juice (PFJ) was prepared, using Canadian variety of potatoes, and was characterized of being composed of 22.9% patatin, 53.3% protease inhibitors, and 23.7% high MW proteins. To isolate potato proteins from PFJ, several extraction techniques were explored including thermal/acidic combination, acidic, FeCl3, MnCl2, ethanol and (NH4)2SO4 precipitations, and carboxymethyl cellulose complexation. (NH4)2SO4 precipitation led to the highest yield (98.6%) and to the recovery of protein isolates enriched in patatin with high resolubility. FeCl3 precipitation resulted in the highest purification factor (6.2) and isolates with the lowest relative proportion of high MW proteins (<4.6%); however, its optimal isolate showed a wide minimum solubility pH range of 3.0-6.0. FeCl3 and MnCl2 were identified as the best precipitating agents for the enrichment of isolates with >15kDa protease inhibitors. Trypsin inhibiting activities of protease inhibitors were highly preserved upon protein isolation than the chymotrypsin ones. Acidic-based protein isolate showed the highest specific lipid acyl hydrolase activity of patatin towards o-nitrophenyl butyrate, whereas FeCl3-based one exhibited the highest activity towards 4-nitrophenyl laurate.

Effect of phenolic compound removal on rheological, thermal and physico-chemical properties of soybean and flaxseed proteins.

This study aimed to investigate the effect of removal of phenolics on physico-chemical properties of protein isolates obtained from flaxseed and soybean. Proteins were isolated (I) from full-fat (F) and defatted (D) soybean (s) and flaxseed (f) using isoelectric precipitation. Free and bound phenolics were removed from the protein isolates. Thermal and gelation properties of protein isolates before and after removal of phenolics were investigated. Protein isolates from defatted soybean after removal of free and bound phenolics were showed a decrease in thermal stability of glycinin. For protein isolate from full-fat soybean, the results showed the removal of free phenolics increase thermal stability of glycinin with increase water holding capacity (WHC) and produce more viscous and less elastic gels as compared to protein isolate after removal of bound phenolics. Removal of free and bound phenolics from flaxseed protein isolates decrease thermal stability, WHC and viscoelastic properties as compared to protein isolate after removal of free phenolics.

Distribution, antioxidant and characterisation of phenolic compounds in soybeans, flaxseed and olives.

The distribution of free and bound phenolic compounds present in soybean, flaxseed and olive were investigated. The phenolic compounds were fractionated on the basis on their solubility characteristics in water, alcohol, dilute base and dilute acid. Reversed phase high pressure liquid chromatography (RP-HPLC) and mass spectrometry (MS) were used for identification of individual components of phenolic compounds. Antioxidant activity (AA%) of free and bound phenolic compounds was measured using the linoleic acid/ß-carotene assay. The water-soluble phenolic compound fractions represented 68-81%, 50-72% and 46-56% of the total phenolic compounds measured in full-fat soybean, olive and flaxseed, respectively. Methanolic extraction of free phenolic compounds without heat, solubilised 21-56%, 42-62% and 34-51% of the total phenolic compounds measured in soybean, olive and flaxseed, respectively; methanol extraction of free phenolic compounds with heat solubilised a further 24-34%, 31-37% and 36-37% of phenolic compounds from soybean, olive and flaxseed, respectively. Further dilute alkali and dilute acid solubilised the remaining 10-40%, 1-21% and 12-29% of the total phenolic compounds from soybean, olive and flaxseed, respectively. Results indicated that the full-fat meals of soybean, flaxseed and olive showed higher antioxidant activity compared to defatted meals. RP-HPLC and LC-MS/MS profil1 for soybean, flaxseed and olive indicate two classes of phenolic compounds designated as free and bound phenolic compounds.

Mutation studies in the active site of ß-glycosidase from Pyrococcus furiosus DSM 3638.

Sequence alignments and homology modeling of Pyrococcus furiosus thermostable glycosidase (PFTG) showed that the residue 150 is conserved as tryptophan in ß-glycosidase and in other related enzymes such as ß- mannosidase and ß-galactosidase. To elucidate the relationship between the substrate size and geometric shape of the catalytic site of thermophilic ß-glycosidase and category of PFTG, the Q77, Q150 and D206 located at the interface of the dimer were replaced with Trp and Asn. Also, to confirm the role of active sites of PFTG, the Q77R/Q150W double mutant was created through subcloning. Temperature and pH optima of both mutants and native enzyme were same at 100 °C and pH 5.0 in sodium citrate buffer, respectively. The catalytic efficiencies (k(cat)/K(m)) of the mutants on synthetic and natural substrates by Isothermal Titration Calorimetry were slightly changed, but indicated the characteristics of ß-glycosidase activity. Kinetic parameters of the mutant enzymes indicated that they possess characteristics of both ß- galactosidase and ß-mannosidase activities. Although the mutant enzymes showed similar substrate specificities compared to the recombinant enzyme, they had more affinity (K(m)) to substrates with low turnover number (k(cat)).

Overexpression and characterization of a novel transgalactosylic and hydrolytic ß-galactosidase from a human isolate Bifidobacterium breve B24.

After the complete gene of a ß-galactosidase from human isolate Bifidobacterium breve B24 was isolated by PCR and overexpressed in E. coli, the recombinant ß-galactosidase was purified to homogeneity and characterized for the glycoside transferase (GT) and glycoside hydrolase (GH) activities on lactose. One complete ORF encoding 691 amino acids (2,076 bp) was the structural gene, LacA (galA) of the ß-gal gene. The recombinant enzyme shown by activity staining and gel-filtration chromatography was composed of a homodimer of 75 kDa with a total molecular mass of 150 kDa. The K(m) value for lactose (95.58 mM) was 52.5-fold higher than the corresponding K(m) values for the synthetic substrate ONPG (1.82 mM). This enzyme with the optimum of pH 7.0 and 45°C could synthesize approximately 42.00% of GOS from 1M of lactose. About 97.00% of lactose in milk was also quickly hydrolyzed by this enzyme (50 units) at 45°C for 5h to produce 46.30% of glucose, 46.60% of galactose and 7.10% of GOS. The results suggest that this recombinant ß-galactosidase derived from a human isolate B. breve B24 may be suitable for both the hydrolysis and synthesis of galacto-oligosaccharides (GOS) in milk and lactose processing.

Expression and characterization of an extremely thermostable ß-glycosidase (mannosidase) from the hyperthermophilic archaeon Pyrococcus furiosus DSM3638.

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF0356) similar to the enzymes in glycoside hydrolase family 1. This ß-glycosidase, designated PFTG (P. furiosus thermostable glycosidase), was cloned and expressed in Escherichia coli. The expressed enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The gene was composed of 1,452 bp encoding 483 amino acids for a protein with a predicted molecular mass of 56,326 Da. The temperature and pH optima were 100°C and 5.0 in sodium citrate buffer, respectively. The substrate specificity of PFTG suggests that it possesses characteristics of both ß-galactosidase and ß-mannosidase activities. However, through kinetic studies by ITC (Isothermal Titration Colorimetry) which is very sensitive method for enzyme kinetics, PF0356 enzyme revealed the highest catalytic efficiency toward p-nitrophenyl-ß-d-mannopyranoside (3.02 k(cat)/K(m)) and mannobiose (4.32 k(cat)/K(m)). The enzyme showed transglycosylation and transgalactosylation activities toward cellobiose, lactose and mannooligosaccharides that could produce GOS (galactooligosaccharides) and MOS (maltooligosaccharides). This novel hyperthermostable ß-glycosidase may be useful for food and pharmaceutical applications.

Optimisation and characterisation of various extraction conditions of phenolic compounds and antioxidant activity in olive seeds.

This study was conducted to optimise the extraction conditions of phenolic compounds to evaluate antioxidant extraction parameters and to identify the major free and bound phenolic compounds in olive seeds. The results obtained using methanol as an extraction solvent for olive seeds indicated that the optimised total phenolic content and antioxidant activity were obtained at an extraction time of 12 h, an extraction temperature of 70°C and an extraction cycle of three stages. The correlation coefficient between total phenolic compounds and antioxidant activities was positive (R² = 0.83). The major finding is that the predominant phenolic compounds in olive seeds were present in free form. However, a small percentage of the bound phenolic compounds was found in olive seeds compared to that of the free phenolic compounds. This study recommends that olive seeds with optimised extraction conditions (i.e. optimised correlation between phenolic compound contents and antioxidant activities) can be used as potential food additive candidates in functional, nutraceutical and pharmaceutical industries.

Effect of limited solid-state glycation on the conformation of lysozyme by ESI-MSMS peptide mapping and molecular modeling.

Although protein glycation has been implicated in the alteration of protein functionality, both in vivo (in biological systems) and in vitro (in food systems), the effect of the protein-bound glycan moiety on the structure/conformation of proteins that result in the modification of functionality is not clear. In this article, we report a study of the conformational changes of glycated lysozyme using LC-ESI-MSMS peptide mapping, and molecular modeling. A comparison of the RP-HPLC of the tryptic digests of unglycated and glycated lysozyme showed markedly different chromatographic profiles. Analysis of the peptide composition of the chromatographic fractions of the tryptic digests revealed that glycation of lysozyme resulted in the modification of its conformation. Glycation-induced changes in the conformation of lysozyme resulted in the exposure of its active site region to increased proteolytic activity of trypsin. Molecular simulation of triglycated lysozyme also showed that limited glycation of lysozyme caused reorientation of the active site residues (Arg 45, Arg 68, Asn 44, and Trp 62) and increased solvent accessibility into the active site region of the protein. The results of the modeling experiment corroborated the results of the RP-HPLC and ESI-MSMS peptide mapping.

Charge state distribution and hydrogen/deuterium exchange of alpha-lactalbumin and beta-lactoglobulin preparations by electrospray ionization mass spectrometry.

Charge state distribution (CSD) and hydrogen/deuterium (H/D) exchange of preparations of alpha-lactalbumin (alpha-Lac) and beta-lactoglobulin (beta-Lg) were investigated using electrospray ionization mass spectrometry (ESI-MS). Storage of alpha-Lac at pH 3 resulted in substantial changes in its CSD, with the emergence of new ion species and shifts toward higher charge state, indicating less stable conformation. ESI spectra of alpha-Lac kept at pH 5.5 for 4 days showed stable conformation; however, extending the storage period resulted in substantial changes in CSD and a decrease in the stability of holo-alpha-Lac (Ca(2+)-bound form). In comparison to apo-alpha-Lac, the relative intensity of holo-alpha-Lac was higher at pH 6.8 but lower at pH 8 during the storage period. beta-Lg showed stable CSD at pH 3, substantial changes at pH 5.5, and minor changes at pH 6.8 and 8 during storage. The H/D exchange results demonstrate that the conformation of holo-alpha-Lac was more stable than that of apo-alpha-Lac and that the conformation of beta-Lg variant B was more stable than that of the beta-Lg variant A. Kinetics of H/D exchange indicated that alpha-Lac and beta-Lg fractions obtained from whey protein preparations have the same or improved conformational stabilities compared to those of alpha-Lac and beta-Lg standards. The presence of four or more hexose residues in alpha-Lac enhanced its conformational stability; the presence of two hexose residues in beta-Lg resulted in a less stable conformation.

Protein-lipid interactions in food systems: a review.

Proteins and lipids, both individually or as complexes, play important functional roles in foods. Since the 1970s food scientists have devoted attention to the nature of these interactions and particularly to their effects on functional characteristics of protein-based foods. Previously, most of the published work was devoted to the biochemical aspects of protein-lipid interactions in biological systems. This article reviews the protein-lipid interactions of both naturally occurring protein-lipid complexes and protein-lipid complexes formed by induced interactions in foods and food products. The physicochemical characteristics of known protein-lipid complexes, the nature of binding which results in formation of these complexes and the effect of the interactions on food functionality are reviewed.