Preben Rasmussen

Microstructure and rheological behaviour of alginate/pectin mixed gels

Abstract The synergistic interaction between alginate and pectin was systematically investigated using samples of different chemical compositions. Pectin samples with high and low degrees of esterification (DE) and amidated pectin (LA) were mixed with alginate of high and low M/G (mannuronic acid/guluronic acid) ratio. The microstructure of the gels was characterised by TEM (transmission electron microscopy) and the rheological properties by dynamic oscillatory measurements. The TEM images of the mixed gels revealed a coarse, strand-like network with pores in the range of microns, independent of the ratio and the composition of the samples. A comparison with the microstructure of a pure pectin gel showed that the pectin network was composed of thinner strands and smaller pore sizes than the mixed network. The strongest synergism was found between alginate with low M/G ratio and pectin with a high DE. These gels show the highest G ′ (storage modulus) and the fastest kinetics of gel formation. Lower G ′ and slower kinetics were found for gels based on alginate with a high M/G ratio and pectin with a low DE, or LA pectin. The nature of the pectin sample affected the network density and the strand characteristics. In contrast, no influence was found of the alginate sample. Gels based on pectin with a high DE showed a dense network composed of highly branched strands, whereas the LA-pectin based gels showed a sparse, open network, composed of long, straight strands. A relation close to 1:1 for low-G alginate and pectin with a high DE resulted in gels with the highest G ′. In contrast, for LA-pectin based gels, the highest G ′ was found for mixtures of alginate dominant ratios. For the overall network properties, the homogeneity in the microstructure decreased with alginate content, independent of the pectin sample.

Pectin methyl esterase from orange fruit: characterization and localization by in-situ hybridization and immunohistochemistry

Abstract. Pectin methyl esterase (PME) from orange (Citrus sinensis L.) fruit peels has been purified by ammonium sulphate precipitation, and ion-exchange and gel-filtration chromatography. Characterization of the enzyme revealed a 36-kDa protein with an isoelectric point >9, a pH optimum at 7 and temperature optimum at 50 °C. The substrate specificity and kinetic experiments showed that the affinity of PME for pectin was highly dependent on the degree of esterification (DE) of the pectin, with Km values of 0.7 mg ml-1 for pectin with a DE of 70% and 17 mg ml-1 for pectin with a DE of 25%. The sequences of the NH2-terminal end of digested peptides from the mature protein were obtained. A DNA fragment of 501 bp was cloned by polymerase chain reaction amplification using degenerate primers and was further used for screening of a cDNA library. Two cDNA clones were isolated encoding PMEs of 584 amino acids and 362 amino acids, respectively, including a putative signal peptide. The deduced amino acid sequence showed full identity to the sequenced peptides. Polyclonal antibodies raised against orange peel PME were used for immunohistochemistry. The main localization of PMEs was in the outer cell layers of the juice vesicles, in the outer cell layers of the lamellae between the segments and in the inner cell layers of the albedo in the peel. In-situ hybridization showed that the mRNA is very abundant in the fruit and was found in the same cell layers as the native enzyme. A very intensive staining for PME mRNA was also seen in the core and in the flavedo close to the oil glands.