Effect of bioprocessing of wheat bran in wholemeal wheat breads on the colonic SCFA production in vitro and postprandial plasma concentrations in men.

The health benefits of whole grain consumption can be partly attributed to the inclusion of the bran or outer-layers of the grain rich in dietary fibre. Fibre is fermented in the colon, leading to the production of beneficial metabolites, such as short-chain fatty acids (SCFA). The effect of five different types of bread on the SCFA production was studied in an in vitro model of human colon. Additionally, the postprandial effects of two selected breads on the SCFA plasma concentrations were investigated in men. A higher in vitro production of butyrate was induced by wholemeal wheat bread with bioprocessed bran than by native bran. The increase in butyrate seemed to be in exchange for propionate, whilst the total SCFA production remained similar. However, differences between the two breads in the postprandial butyrate concentrations could not be detected in peripheral blood of men, probably due to an effective utilisation by colonocytes.

Bioprocessing of wheat bran in whole wheat bread increases the bioavailability of phenolic acids in men and exerts antiinflammatory effects ex vivo.

Whole grain consumption has been linked to a lower risk of metabolic syndrome, which is normally associated with a low-grade chronic inflammation. The benefits of whole grain are in part related to the inclusion of the bran, rich in phenolic acids and fiber. However, the phenols are poorly bioaccessible from the cereal matrix. The aim of the present study was to investigate the effect of bioprocessing of the bran in whole wheat bread on the bioavailability of phenolic acids, the postprandial plasma antioxidant capacity, and ex vivo antiinflammatory properties. After consumption of a low phenolic acid diet for 3 d and overnight fasting, 8 healthy men consumed 300 g of whole wheat bread containing native bran (control bread) or bioprocessed bran (bioprocessed bread) in a cross-over design. Urine and blood samples were collected for 24 h to analyze the phenolic acids and metabolites. Trolox equivalent antioxidant capacity was measured in plasma. Cytokines were measured in blood after ex vivo stimulation with LPS. The bioavailabilities of ferulic acid, vanillic acid, sinapic acid, and 3,4-dimethoxybenzoic acid from the bioprocessed bread were 2- to 3-fold those from the control bread. Phenylpropionic acid and 3-hydroxyphenylpropionic acid were the main colonic metabolites of the nonbioaccessible phenols. The ratios of pro-:antiinflammatory cytokines were significantly lower in LPS-stimulated blood after the consumption of the bioprocessed bread. In conclusion, bioprocessing can remarkably increase the bioavailability of phenolic acids and their circulating metabolites, compounds which have immunomodulatory effects ex vivo.

Crosslinking food proteins for improved functionality.

Different possibilities for protein crosslinking are examined in this review, with special emphasis on enzymatic crosslinking and its impact on food structure. Among potential enzymes for protein crosslinking are transglutaminase (TG) and various oxidative enzymes. Crosslinking enzymes can be applied in cereal, dairy, meat, and fish processing to improve the texture of the product. Most of the current commercial applications are based on TG. The reaction mechanisms of the crosslinking enzymes differ, which in turn results in different technological properties.

Extensive dry ball milling of wheat and rye bran leads to in situ production of arabinoxylan oligosaccharides through nanoscale fragmentation.

This study investigated the potential of ball milling as a dry process for in situ production of arabinoxylan oligosaccharides (AXOS) in arabinoxylan (AX)-rich wheat and rye bran. An extensive lab-scale ball mill treatment (120 h, 50% jar volume capacity) increased the wheat bran water-extractable arabinoxylan (WE-AX) level from 4% (untreated bran) to 61% of the wheat bran AX. Extractable AX fragments had an arabinose/xylose ratio (A/X ratio) of 0.72 and a molecular mass (MM) of 15 kDa. Ball milling of rye bran gave rise to similar results, with the A/X ratio of the extractable AX being considerably lower (0.51). Optimization of the ball mill treatment by varying the degree of filling of the milling jar permitted us to obtain equally high WE-AX levels (>70%) in wheat and rye bran within a 24 h ball-milling period. Ball milling at optimal conditions (24 h, 16% jar volume capacity) yielded wheat bran AXOS, with an A/X ratio of 0.65 and a MM of 6 kDa. Ball milling (24 h, 50% jar volume capacity) of pericarp-enriched wheat bran increased the WE-AX level from 1% (untreated pericarp) to 63%. The extractable material had a high A/X ratio (0.97) and a low MM (5 kDa). Fluorescence microscopy revealed that the extensive ball mill treatment led to the almost complete disappearance of discernible tissue structures in the ball-milled material, indicating bran particle size reductions down to the nanoscale level. It further visualized the aggregation of the treated material. These results show that AXOS can be produced in situ from wheat or rye bran in a single-stage dry milling process, rendering a wet extraction step redundant. The higher A/X ratio of the obtained AXOS than of enzymically produced wheat bran-derived AXOS offers perspectives for the production of a wide range of AXOS structures. Moreover, ball milling makes upgrading of the low-value pericarp layer feasible.

Bioprocessing of wheat bran improves in vitro bioaccessibility and colonic metabolism of phenolic compounds.

Ferulic acid (FA) is the most abundant phenolic compound in wheat grain, mainly located in the bran. However, its bioaccessibility from the bran matrix is extremely low. Different bioprocessing techniques involving fermentation or enzymatic and fermentation treatments of wheat bran were developed aiming at improving the bioaccessibility of phenolic compounds in bran-containing breads. The bioaccessibility of ferulic acid, p-coumaric acid, and sinapic acid was assessed with an in vitro model of upper gastrointestinal tract (TIM-1). Colonic metabolism of the phenolic compounds in the nonbioaccessible fraction of the breads was studied with an in vitro model of human colon (TIM-2). The most effective treatment was the combination of enzymes and fermentation that increased the bioaccessibility of FA from 1.1% to 5.5%. The major colonic metabolites were 3-(3-hydroxyphenyl)propionic acid and 3-phenylpropionic acid. Bran bioprocessing increases the bioaccessibility of phenolic compounds as well as the colonic end metabolite 3-phenylpropionic acid.

Formation of protein-oligosaccharide conjugates by laccase and tyrosinase.

Proteins and certain carbohydrates contain phenolic moieties, which are potential sites for modification of the function of the biopolymers. In this study, the capability of two different fungal oxidative enzymes, laccase from Trametes hirsuta (ThL) and tyrosinase from Trichoderma reesei (TrT), to catalyze formation of hetero-cross-linking between tyrosine side chains of alpha-casein and phenolic acids of hydrolyzed oat spelt xylan (hOSX) was studied. Formation of reaction products was followed by size exclusion chromatography (SEC), fluorescence spectroscopy, and SDS-PAGE, using specific staining methods for proteins and protein-carbohydrate conjugates. ThL and TrT were observed to differ significantly in their ability to catalyze the formation of protein-carbohydrate conjugates or the linking of the small molecular weight phenolic compounds to alpha-casein. The efficiency of these enzymes to directly cross-link protein also differed notably. TrT was able to cross-link alpha-casein more efficiently than ThL. ThL-catalyzed casein cross-linking was significantly enhanced by ferulic acid, p-coumaric acid, and also hOSX. The main reaction products by ThL appeared to be phenolic acid-bridged alpha-caseins. Indications of hetero-cross-link formation between alpha-casein and hOSX by both oxidative enzymes could be visualized by glycoprotein-specific staining in the SDS-PAGE analysis, although ThL was observed to be more effective in the heteroconjugate formation than TrT.

Oxidation of peptides and proteins by Trichoderma reesei and Agaricus bisporus tyrosinases.

The capability of a novel tyrosinase from Trichoderma reesei (TrTyr) to catalyse the oxidation and oxidative cross-linking of l-tyrosine (l-Y) and tyrosine side-chains in GYG and EGVYVHPV peptides, in bovine serum albumin (BSA) and beta-casein proteins as well as in proteinaceous wool fibres was studied by oxygen consumption measurement, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reverse phase high-performance liquid chromatography (RP-HPLC), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and fluorescence microscopy. TrTyr was compared to the well-characterised tyrosinase from Agaricus bisporus (AbTyr) in terms of oxidation and cross-linking. According to the results obtained TrTyr was capable of cross-linking peptides and proteins more efficiently than AbTyr. However, the size and three-dimensional structure of the proteinaceous substrates proved to be crucial for the success of the enzymatic catalysis. Random coil beta-casein could be cross-linked by TrTyr already in three hours, but large and compact BSA was not cross-linked even in 24h. TrTyr could also be used to incorporate a diphenolic compound, l-dihydroxyphenyl alanine (l-dopa), into protein fibres whereas incorporation of a monophenol, l-Y was less efficient. Thus TrTyr is a potential tool for protein cross-linking and/or modification.

Comparison of the characteristics of fungal and plant tyrosinases.

Enzymatic crosslinking provides valuable means for modifying functionality and structural properties of different polymers. Tyrosinases catalyze the hydroxylation of various monophenols to the corresponding o-diphenols, and the subsequent oxidation of o-diphenols to the corresponding quinones, which are highly reactive and can further undergo non-enzymatic reactions to produce mixed melanins and heterogeneous polymers. Tyrosinases are also capable of oxidizing protein- and peptide-bound tyrosyl residues, resulting in the formation of inter- and intra-molecular crosslinks. Tyrosinases from apple (AT), potato (PT), the white rot fungus Pycnoporus sanguineus (PsT), the filamentous fungus Trichoderma reesei (TrT) and the edible mushroom Agaricus bisporus (AbT) were compared for their biochemical characteristics. The enzymes showed different features in terms of substrate specificity, stereo-specificity, inhibition, and ability to crosslink the model protein, alpha-casein. All enzymes were found to produce identical semiquinone radicals from the substrates as analyzed by electron spin resonance spectroscopy. The result suggests similar reaction mechanism between the tyrosinases. PsT enzyme had the highest monophenolase/diphenolase ratio for the oxidation of monophenolic L-tyrosine and diphenolic L-dopa, although the tyrosinases generally had noticeably lower activity on monophenols than on di- or triphenols. The activity of AT and PT on tyrosine was particularly low, which largely explains the poor crosslinking ability of the model protein alpha-casein by these enzymes. AbT oxidized peptide-bound tyrosine, but was not able to crosslink alpha-casein. Conversely, the activity of PsT on model peptides was relatively low, although the enzyme could crosslink alpha-casein. In the reaction conditions studied, TrT showed the best ability to crosslink alpha-casein. TrT also had the highest activity on most of the tested monophenols, and showed noticeable short lag periods prior to the oxidation.

Elucidating the mechanism of laccase and tyrosinase in wheat bread making.

Cross-linking enzymes generate covalent bonds in and between food biopolymers. These enzymes are interesting tools for tailoring dough and bread structures, as the characteristics of the biopolymers significantly determine the viscoelastic and fracture properties of dough and bread. In this study, the influence of oxidative cross-linking enzymes, tyrosinase from the filamentous fungus Trichoderma reesei and laccase from the white rot fungus Trametes hirsuta, on dough and bread were examined. Oxidation of low molecular weight phenolic model compounds of flour, cross-linking of gluten proteins, dough rheology, and bread making were characterized during or after the enzymatic treatments. In the dough and bread experiments, laccase and tyrosinase were also studied in combination with xylanase. Of the model compounds tyrosine, p-coumaric acid, caffeic acid, ferulic acid, and Gly-Leu-Tyr tripeptide, tyrosinase oxidized all except ferulic acid. Laccase was able to oxidize each of the studied compounds. The phenolic acids were notably better substrates for laccase than l-tyrosine. When the ability of the enzymes to cross-link isolated gliadin and glutenin proteins was studied by the SDS-PAGE analysis, tyrosinase was found to cross-link the gliadin proteins effectively, whereas polymerization of the gliadins by laccase was observed only when a high enzyme dosage and prolonged incubation were used. Examination of large deformation rheology of dough showed that both laccase and tyrosinase made doughs harder and less extensible, and the effects increased as a function of the enzyme dosage. In bread making, interestingly, the pore size of the breads baked with tyrosinase turned out to be remarkably larger and more irregular when compared to that of the other breads. Nevertheless, both of the oxidative enzymes were found to soften the bread crumb and increase the volume of breads, and the best results were achieved in combination with xylanase.

Expression of the Trichoderma reesei tyrosinase 2 in Pichia pastoris: isotopic labeling and physicochemical characterization.

Trichoderma reesei tyrosinase TYR2 has been demonstrated to be able to oxidize various phenolic compounds and also peptide and protein bound tyrosine, and thus is of great interest for different biotechnological applications. In order to understand the reaction mechanism of the enzyme it would be essential to solve its three dimensional structure. Pichia pastoris is a suitable expression system for the production of recombinant enzymes for NMR studies and therefore we expressed TYR2 in this host. As a result of extensive optimization, the production yield of active histidine tagged tyrosinase purified from P. pastoris shake flask cultures was increased from 2.5 to 24 mg/L. Correct copper concentration in the growth medium was critical for the expression of this copper containing enzyme. Our analysis showed that TYR2 expressed in P. pastoris is post-translationally modified; the C-terminal domain of 153 amino acids of the protein is proteolytically cleaved off from the catalytic domain and the only potential N-glycosylation site is glycosylated. The activities of TYR2 expressed in P. pastoris and T. reesei on diphenolic L-dopa and monophenolic L-tyrosine were rather similar. The TYR2 expressed in P. pastoris showed the same physicochemical properties in CD and unfolding assays as the native TYR2 enzyme. Uniform isotopic (15)N-labeling of TYR2 was carried out with (15)NH(4)SO(4) in minimal medium to assess the suitability of the expression system for investigation by NMR spectroscopy.

Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei.

A homology search of the genome database of the filamentous fungus Trichoderma reesei identified a new T. reesei tyrosinase gene tyr2, encoding a protein with a putative signal sequence. The gene was overexpressed in the native host under the strong cbh1 promoter, and the tyrosinase enzyme was secreted into the culture supernatant. This is the first report on a secreted fungal tyrosinase. Expression of TYR2 in T. reesei resulted in good yields, corresponding to approximately 0.3 and 1 g.L(-1) tyrosinase in shake flask cultures and laboratory-scale batch fermentation, respectively. T. reesei TYR2 was purified with a three-step purification procedure, consisting of desalting by gel filtration, cation exchange chromatography and size exclusion chromatography. The purified TYR2 protein had a significantly lower molecular mass (43.2 kDa) than that calculated from the putative amino acid sequence (61.151 kDa). According to N-terminal and C-terminal structural analyses by fragmentation, chromatography, MS and peptide sequencing, the mature protein is processed from the C-terminus by a cleavage of a peptide fragment of about 20 kDa. The T. reesei TYR2 polypeptide chain was found to be glycosylated at its only potential N-glycosylation site, with a glycan consisting of two N-acetylglucosamines and five mannoses. Also, low amounts of shorter glycan forms were detected at this site. T. reesei TYR2 showed the highest activity and stability within a neutral and alkaline pH range, having an optimum at pH 9. T. reesei tyrosinase retained its activity well at 30 degrees C, whereas at higher temperatures the enzyme started to lose its activity relatively quickly. T. reesei TYR2 was active on both l-tyrosine and l-dopa, and it showed broad substrate specificity.