Emilia Selinheimo

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.

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.

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 °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.

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.

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.

Wheat bran AX properties and choice of xylanase affect enzymic production of wheat bran-derived arabinoxylan-oligosaccharides.

ABSTRACTWheat bran-derived arabinoxylan-oligosaccharides (AXOS) recently have been shown to potentially exert prebiotic effects. In this study, 15 bran samples obtained by milling different wheat cultivars were treated with xylanases from Hypocrea jecorina (XHJ), Aspergillus aculeatus (XAA), and Pseudoalteromonas haloplanktis (XPH) to assess the effect of bran source and xylanase properties on the AXOS yield and structure. The total arabinoxylan (AX) extraction yield was higher with XHJ (8.2–10.7%) and XAA (8.2–10.8%) than with XPH (6.9–9.5%). Irrespective of the enzyme, a significant negative correlation was observed between extraction yield and arabinose to xylose (A/X) ratio of bran AX (r = –0.7), but not between yield and bran AX level. The A/X ratio of the extracted material was 0.27–0.34 for all bran samples and all enzymes, which combined with yield data and microscopic analysis, indicated primary hydrolysis of aleurone and nucellar epidermis AX. The average degree of polymerization (avDP) of the e...

Using crosslinking enzymes to improve textural and other properties of food

Publisher Summary This chapter explains that food texture plays a major role in food product quality. The rheological properties of a food are determined by the number of weak and strong physical interactions and the permanent covalent bonds, crosslinks, present in the food matrix. Covalent crosslinks make the major contribution to the firmness of food matrices. Crosslinks can be introduced to a food matrix by chemical, enzymatic, and physical means as reviewed by Munindar P. Singh (1991) and Scott N. Gerrard (2002) . Enzymatic crosslinking of food biopolymers is an attractive option owing to the specificity of enzymes and mild reaction conditions. Both food proteins and carbohydrates can be crosslinked by enzymes. Sensory perception of texture plays an important role in different types of foods. The significance of food texture has further been increased with the trend toward low fat products and “natural” additive-free products. Enzymes provide specific and natural means for tailoring food structure. By the use of enzymes, it will be possible to transform inherently available food components into functional ingredients during food processing and manufacturing. Enzymatic crosslinking in the food matrix can occur via proteins or certain carbohydrates. The type of enzyme used affects the chemistry of the crosslink formed and subsequently the structure of the biopolymer network of the food product.

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.

Anti-inflammatory effect of breads in relation to the bioavailability of phenolic compounds

Phenolic compounds (PC) are food-derived bioactive compounds contained in the daily diet. PC have been claimed to help prevent cancer, degenerative diseases and chronic and acute inflammation. A possible mechanism of anti-inflammatory action is by their effect on the redox sensitive transcription factor nuclear factor kappaB, inducible nitric oxide synthase expression and cyclooxygenase inhibition. The anti-inflammatory effects of PC are evident in vitro using high concentrations; however, physiological concentrations are dependent on their bioavailability from the food source, which is often limited. Cereal products contribute substantially to the total intake of PC. Most of the PC in the cereal grain are concentrated in the outermost part, the bran. Therefore, wholemeal breads containing either native or bioprocessed bran were tested for their post-prandial effects on an inflammatory response ex vivo. The objective of this investigation was to evaluate the anti-inflammatory effect of PC in relation to their bioavailability from a natural dietary source, such as bread. The study design was: crossover, with 8 healthy men undergoing 3-days low phenolic diet prior to intervention and at least 1 week of wash-out period between the two intervention days. Blood was drawn before bread ingestion (t0 or baseline), and at 1 h 15 min, 6 h and 12 h after ingestion. Whole blood was incubated with LPS (1 ng/ml) for 24 h and TNF-a, IL-6, IL-1b and IL-10 were determined. The PC quantified in plasma were: ferulic acid, vanillic acid, p-coumaric acid, sinapic acid and their colonic metabolites: phenylpropionic acids, phenylacetic acids, and benzoic acids with different grades of hydroxylation. The ratio of pro-/anti-inflammatory cytokines at 1 h 15 min, 6 h and 12 h after bread ingestion was lower than baseline (t0). The bread with bioprocessed bran showed a stronger effect than the one with native bran, which could be associated with an increase in the bioavailability of PC by the bioprocessing.