Extraction, characterisation and properties of polysaccharides from novel sources

In many foods, pectins are used as thickeners, gelling agents, texturizers, emulsifiers and stabilizers; these functional properties rely on their physio-chemical properties and therefore on their extraction and growing conditions or the pectin source including genetic variants. Pectins were isolated using aqueous extraction at pH 6.0 from okra pods of different genetic varieties and from different geographical origin. Furthermore, pectins from pumpkin were extracted at different pHs, times and temperatures. An isolation protocol and a statistical experimental approach were designed to extract pectin and to study the influence of a number of factors on the physicochemical properties of pectins from natural sources. After the successful isolation full characterization via an array of analytical techniques was carried out. By high-performance anion exchange chromatography HPAEC and nuclear magnetic resonance (NMR), it was determined that the carbohydrates were most likely pectin based which is due to the presence of galacturonic acid. Also, extracted polysaccharides were assessed using size exclusion chromatography (SEC). An Fourier Transform infrared spectroscopy (FT-IR) method was developed to measure the degree of esterification (DE) of pectin samples. The properties and stability of the resulting pectin were examined by means of ζ-potential measurements and capillary viscometry, as pectin extracted from both pumpkin and okra were negatively charged polyelectrolytes. NMR spectroscopy was used to further elucidate the chemical structure of the pectin. An ultraviolet visible (UV-Vis) spectrophotometer was also applied to determine the acetyl content, protein content, uronic acid content and the free radical content for antioxidant activity. The present investigation for okra and pumpkin pectins show that: okra genotypes had pectin yields of 11.3–14.6% while the pumpkin pectin produced in quantities between 2.8% -8.0% of alcohol insoluble residue (AIR). The greatest total amount of pumpkin pectin yield 8.0% on a dry basis was found for 1h of extraction and at 80oC, pH 2for pumpkin pectin and pH is the most important factor in the extraction of pumpkin pectins. Total polysaccharide and protein content for okra pectins ranging from 58% –70.1% and 8.0 –15.1% respectively, whereas pumpkin varied from 30.9 to 72.2% total polysaccharides and 1.8–14.3% protein content. Galacturonic acid was the main component for okra and pumpkin samples and both had similar range between 43.7 to 72.6% mol % and 41.7 to 72.6% mol % galacturonic acid respectively, where is it important to note that for commercial purposes pectins are required to be at least 65% galacturonic acid. The degree of esterification, calculated using Infra-red spectroscopy (IR), the highest value was 96% for pumpkin pectin whilst for okra pectin was 39%. The degree of acetylation for both was high and in the same range varied from 30.3% to 76.2%. Monosaccharide composition analysis showed that both pectins extracted contained particularly, rhamnose, arabinose, galactose, glucose and xylose. The intrinsic viscosities were high for both polysaccharide values solutions. This was attributed to the okra polysaccharides having a relatively large molecular mass which was further supported by using SEC. Furthermore, most of the zeta potentials obtained in this study for okra and pumpkin pectins were highly negative. 1H NMR spectroscopy confirmed the presence of uronic acids in the free and methyl ester forms. The antioxidant activity for both pectins was estimated using 1,1-diphenyl-2-picrylhydrazl and hydroxyl radical assays. A new combined hydrodynamic approach was used to estimate the conformation and flexibility of the pectin solution. In terms of the persistence length, Lp of the equivalent worm-like chain model, the chain flexibility was then investigated. Equivalent radii and ratios of radii from solution properties were the two main factors that were used as indicators for macromolecular conformation, shape, and flexibility. In addition, both the average number and length of side chains on a pectin molecule was estimated using the physicochemical data coupled with the conformational data. For the first time (to our knowledge) this data from the conformational analysis was combined with structural information from the chemical analysis of pectins to obtain information on the degree and the length of branching in the “hairy” RG-I regions. Principal component analysis (PCA) was used for both pectins to show global differences similarities between samples using all the primary data determined.