Renato Amadò

Dityrosine: In vitro production and characterization

Publisher Summary This chapter describes two methods for the preparation of dityrosine by peroxidase oxidation of tyrosine and N -acetyltyrosine, respectively. Using tyrosine as starting material in the production of dityrosine by the means of the peroxidase–hydrogen peroxide system, the yield of dityrosine was relatively small, higher oxidation products such as trimer and tetramer being the main products of the oxidation. Furthermore, the isolation of pure dityrosine from the incubation mixture proved to be difficult. Therefore N-acetyltyrosine is suggested as the starting material for the in vitro production of dityrosine. This derivative has the advantage of being highly water- and ethanol-soluble, which allows the production of larger amounts of the dimer in one batch. The identification of dityrosine is best done on the basis of its characteristic fluorescence. Dityrosine present in proteins can be identified after hydrolysis by amino acid analysis. The dimer behaves like a peptide and gives a relatively broad peak that elutes between phenylalanine and histidine. Newer methods for the identification of dityrosine include chromatography on a diethylaminoethanol (DEAE)-cellulose column and high-performance liquid chromatography.

Influence of different types of thermal treatment on the chemical composition and physical properties of wheat bran

The purpose of the present investigation was to submit commercially available wheat bran to various thermal treatments and to measure the resulting effects on the functional properties. Water uptake, water-binding capacity, rheological behaviour in the Farinograph and oil absorption of boiled, steam-cooked, autoclaved, roast, micronised and extruded bran were determined. The water uptake of the modified samples was increased, compared with that of the untreated bran. Only boiling affected the water-binding capacity significantly. The largest effects of the thermal modifications were rheological changes as observed in the Farinograph. Doughs, consisting of 80% patent flour and 20% wheat bran, showed a diminished maximum resistance and a prolonged mixing time compared with a standard dough with long patent flour. Wet heat treatments altered the surface of the wheat bran, resulting in a change in the oil absorption capacity.

Effect of galactose content on the solution and interaction properties of guar and carob galactomannans

Abstract Guar galactomannan has been modified by treatment with an α- d -galactosidase A preparation from lucerne seeds. This enzyme was purified by affinity chromatography on N -ϵ-aminocaproyl-α- d -galactopyranosylamine linked to Sepharose 4B, had a high activity towards galactomannans, and was completely devoid of β- d -mannanase. On incubation for 2 h, this enzyme removed ⪢ 75% of the galactose from guar galactomannan with no concurrent decrease in viscosity. Eventual decrease in viscosity was associated with the formation of insoluble, mannan-type precipitates. This phenomenon, although directly related to the galactose content of the galactomannan, was also time-dependent. The limiting viscosity numbers calculated for the “mannan backbones” of α- D -galactosidase-treated, guar galactomannan having galactose-mannose ratios of 38:62 to 15:85 were the same. Modified, guar galactomannan (at 0.4% w/v) having a galactose-mannose ratio of 20:80, or less, forms a gel on storage at 4° over several weeks. Also, gel particles form when solutions of these galactomannans are passed through a freeze-thaw cycle. Samples containing ⪡ 10% of galactose rapidly precipitate from solution even at 30°. The interaction of guar galactomannan with xanthan is greatly increased by removal of galactose residues. Samples having galactose-mannose ratios of ~19:81 interact with xanthan to essentially the same degree as carob galactomannan (Gal/Man = 23:77).

Determination of carbohydrates in fruit juices by capillary electrophoresis and high-performance liquid chromatography

Abstract A capillary zone electrophoresis (CZE) method with indirect UV detection was adapted for the routine determination of carbohydrates in a variety of fruit juices. The method was optimized with respect to the effect of buffer pH, temperature and capillary length. Potassium sorbate was chosen as the background electrolyte and chromophore for UV detection at 256 nm. Optimum separation conditions were found with a buffer of a pH of 12.2–12.3 and a subambient temperature of 15°C. The optimized CZE method was compared with a routine method for the determination of sugars in fruit juices, a high-performance anion-exchange chromatographic method with pulsed amperometric detection (HPAEC-PAD), with respect to separation efficiency, sensitivity, linearity and repeatability. The CZE method showed a 10–20-fold increase in separation efficiency compared with the HPAEC-PAD method, but the amperometric detection in the latter proved to result in detection limits of 2–3 orders of magnitude lower than those obtained by indirect UV detection. Both methods showed good linearity in the investigated concentration ranges and good repeatability for migration times and peak areas. CZE with commercial instrumentation was applied to the routine determination of carbohydrates in fruit juices such as orange, apple and grape juice. The quantitative CZE results with internal calibration showed no significant differences from those for the HPAEC-PAD reference method. It was demonstrated that capillary electrophoresis (CE) can be applied in a routine food testing laboratory. The method is simple, inexpensive and easy to implement and will further broaden the application range of CE in food analysis.

Pectic substances isolated from apple cellulosic residue: structural characterisation of a new type of rhamnogalacturonan I

Abstract The cellulosic residue (CR) of ripe apples (variety ‘Glockenapfel’) was analysed. Uronic acid in the CR indicated the presence of pectic substances. The objectives of the work were to isolate and characterise the CR associated pectic substances and to find possible cross-links between the two cell wall polymers. Prior to characterisation enzymatic degradation of the cellulose was necessary. The degradation products were removed by ultrafiltration and the enzyme resistant material submitted to linkage analysis to obtain information about the structure of the CR-pectin. A model of the CR-pectin consisting of five units was developed: a highly ramified rhamnogalacturonan I backbone containing an equal number of galacturonic acid (1,4-GalA p ) and rhamnose (sum of 1,2-Rha p and 1,2,4-Rha p ) residues was found. About 80% of the rhamnose residues was origin of side chains. Attached to the backbone was a xylogalacturonan side chain, containing 1,4-GalA p , 1,3,4-Gal p , 1,4-Xyl p and T-Xyl p . Highly ramified arabinans, with a 1,3-linked backbone, made up more than half of the neutral sugar residues. Two galactose containing units were found: a 1,4-linked galactan with few 1,3- and 1,6-linked short side chains (located near to the backbone) and a short strictly linear 1,4-galactooligomer. The postulated model was confirmed by further degradation of the CR with pectin specific enzymes. An interaction between cellulose and CR-pectin is suggested to occur through the galactan side chains.

Occurrence of acrylamide in selected foods and mitigation options

Acrylamide reduction in certain food products is an important issue for both the food industry and academic research institutions. The present paper summarises past and current research on the occurrence and reduction of acrylamide in potatoes, bakery products, almonds, olives and dried fruit. In potatoes, the control of reducing sugars, process temperature and moisture is imperative to limit acrylamide formation. In bakery products, free asparagine and the type of baking agent largely determine acrylamide formation and present the starting points for reduction. The application of asparaginase is promising in this respect because it acts only on the key precursor, asparagine, whereby the product character remains unchanged. The baking agent NH4HCO3 promotes acrylamide formation in sweet bakery but its replacement by NaHCO3 effectively decreases acrylamide concentrations. Temperature and free asparagine are the key factors for acrylamide formation in roasted almonds. Olives and dried fruit may contain acrylamide and large amounts of acrylamide can be formed upon heating these products, a phenomenon which needs further investigation.

In vitro digestibility and colonic fermentability of aleurone isolated from wheat bran

Two aleurone preparations, isolated from wheat bran, with different levels of tissue purity, were compared to the wheat bran starting material. Chemical composition, in vitro digestibility and in vitro fermentability were investigated and microscopy was used to visualize morphological changes. Arabinoxylans constituted the main part of the dietary fibre fraction. However, the degree of branching was found to be lower in arabinoxylans from aleurone than in that from original bran. Due to the high fibre content, in vitro digestibility was rather low but digestible compounds were largely eliminated. In vitro fermentability using fresh human faecal material turned out to be higher in aleurone than in wheat bran. Moreover, the degree of purity of the aleurone preparations influenced the amount of fermentation products. Proportions of the main short chain fatty acids were very similar in all samples, with ratios of propionate and butyrate slightly above average compared to other dietary fibre sources. Arabinoxylans from aleurone were virtually completely degraded within 8 h, whereas with wheat bran, substantial amounts were still present after 24 h. Moreover, increasing arabinose:xylose ratios during fermentation suggested a better fermentability of arabinoxylans with a lower branching degree. Microscopic investigations confirmed the better fermentability of aleurone compared to wheat bran. In addition, a specific breakdown pattern of aleurone cell walls could be observed.

Development of a model for quality assessment of tomatoes and apricots

Abstract The quality of tomato and apricot cultivars was assessed by sensory evaluation, consumer tests and instrumental methods. The overall sensory appreciation of tomatoes was mainly reflected by attributes such as “sweetness”, “aroma”, “juiciness” and “firmness”. Hedonic classification of samples allowed to significantly improve the correlation between instrumental data and consumer appreciation. Instrumental measurements were focused on total sugar content (°Brix) and total amount of volatile compounds and texture (firmness). For tomatoes the CAR/PDMS fibre and for apricots the PDMS fibre were used for the analysis of total volatiles. The results obtained with tomatoes and apricots confirmed the applicability of the quality assessment model developed for evaluation of the quality of strawberries.

Separation of 8-aminonapthalene-1,3,6-trisulfonic acid-labelled neutral and sialylated N-linked complex oligosaccharides by capillary electrophoresis

Complex oligosaccharides, both neutral and sialylated, were derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and separated by capillary electrophoresis. The derivatization reaction was carried out in a total reaction volume of 2 microliters. The separated peaks were detected by laser-induced fluorescence detection using the 325-nm line of a He-Cd laser. Concentration and mass detection limits of 5 x 10(-8) M and 500 amol, respectively, could be achieved. The limiting step for higher sensitivity is not the detector performance, however, but the chemistry with a derivatization limit of 2.5 x 10(-6) M. Two labelling protocols were established, one with overnight reaction at 40 degrees C and the other with a 2.5-h derivatization time at 80 degrees C. Neutral oligosaccharides could be labelled with either protocol. However, sialylated oligosaccharides hydrolysed when labeled at 80 degrees C. Low nanomole to picomole amounts of oligomannose-type and complex-type oligosaccharide mixtures were derivatized and separated in less than 8 min with excellent resolution using a phosphate background electrolyte at pH 2.5. The linear relationship between the electrophoretic mobility and the charge-to-mass ratios of the ANTS conjugates was used for peak assignment. Further, the influence of the three-dimensional structure of the complex oligosaccharides on their migration behaviour is discussed. The suitability of the ANTS derivatization and the subsequent separation for the analysis of complex oligosaccharide patterns is demonstrated with oligosaccharide libraries derived from ovalbumin and bovine fetuin. For peak assignment the patterns are compared with those of the oligomannose and the complex-type oligosaccharide mixtures.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the pectic substances of apples during development and postharvest ripening: Part 2: Analysis of the pectic fractions

Pectic material was extracted from the alcohol-insoluble residues of apples during growth on the tree and at different stages of ripening during postharvest storage. Three pectic fractions, soluble in the chelating agent trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA) in cold dilute sodium carbonate and in sodium carbonate at room temperature, were isolated. The galacturonic acid, neutral sugar and starch content of the fractions were determined as well as the degrees of methyl esterification and acetylation of the CDTA-soluble fraction. In addition, the molecular weight distribution was examined by gel filtration chromatography. The high galacturonic acid content of the CDTA-soluble fraction was consistent with the polysaccharide being essentially linear and originating in the middle lamella. The sodium carbonate-soluble pectin had a higher rhamnose to galacturonic acid ratio, indicating that it was more highly branched. Galactose and arabinose residues were lost in the polymers from all three fractions during ripening, with the greatest loss occurring from the sodium carbonate-soluble fractions. At the same time, the pectins became less polydisperse and shifted towards higher molecular weights. It seems that during development there is a dynamic turnover with new pectic polymers, which contain fewer neutral sugars, but are of high molecular weight, appearing in the middle lamella and primary cell wall.