PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
Enzymatic Modification of Pectin to Produce Tailored Functional Stabilizer
L. Wicker and Y. Kim
Dept. Food Science and Technology, University of Georgia, Athens, GA, USA
lwicker@uga.edu
Abstract
Isozymes of citrus pectinmethylesterase (PME) de-esterified pectin from 73% degree of
esterification (DE) to ~61% DE. The modified pectins retained molecular weight, were calcium sensitive
and formed gels. Zeta potential for surface charge and interactions with milk proteins indicated that the
charge-modified pectins were unique. Modified pectins with tailored functionality may enhance utilization
of pectins from novel sources.
Keywords: de-esterification, charge distribution, protein interactions, stabilizer
Main Text
Pectins are important structural polysaccharides in the cell walls of many plants, which are of
considerable interest as gelling or stabilizing agent in the food industry (1). The degree of methyl
esterification (DE) implies a specific gelling mechanism. Besides the methoxy content, the distribution
pattern of free and esterified carboxyl groups and the length of unsubstituted galacturonan backbone
affect gelling. Chemical de-esterification is a random de-esterification process that results in decreased
molecular weight due to de-polymerization of pectin backbone by β-elimination. However, enzymatic deesterification
results in a blockwise distribution and undesired depolymerization of pectins are reduced (2;
3). Plant pectinmethylesterase can create a calcium sensitive pectin (CSP) in which HMP can gel in the
presence of Ca without the addition of sucrose as long as blocks of de-esterified pectin are present (4).
Willats et al. (5) found that the degree and pattern of methyl-esterification affects the elasticity of the gels
as well as their response to compressive strain. Ralet et al. (6) also showed that the blocks formed by
plant-PME treatment allowed blocks to form calcium-pectinate precipitates for high DE even though these
blocks were not long enough to induce abnormal polyelectrolyte behaviour. The objective of this research
was to characterize the calcium sensitivity of Valencia PME modified pectin by direct measure of viscous
and gelling properties in the presence of CaCl2. In addition, the effect of calcium on the surface charge of
the modified pectins was estimated by measuring the ζ-potential. This research has application to the
development of tailored pectins for specific functional properties using PMEs of known mechanism of
action.
Materials and Methods
Valencia PMEs were isolated as previously developed (Fig. 1) and used to de-esterify high methoxyl
pectin (73% DE) at 30°C to a target 63% DE. Pectin was precipitated and washed in ethanol and stored
at -20°C until use. Modified pectins were denoted at O-Pec, B-Pec, and U-Pec for original pectin, first
modified pectin and second modified pectin. Analytical ion exchange chromatography (IEX) was used to
estimate charge distribution using a 5 mL Q column (Bio Rad, Hercules, CA, USA), equilibrated in 0.5M
acetate, pH 5.0, and then eluted through the gradient of 0.5 to 1.3 M acetate buffer, pH 5.0. Molecular
weights (Mw) were determined using an HPSEC-multi angle light scattering system consisting of a
Waters P515 pump with an in-line degasser (Waters, Milford, MA) and two in-line filters (0.22 and 0.10
mm pore size, Millipore, Bedford, MA). Measurement of ζ- potential was performed using a Particle Size
Analyzer adding the BI-Zeta option (90 Plus, Brookhaven Inst., Holtsville, NY) with a 50 mV diode laser
(90 angle) and a BI-9000AT correlator. Gelling was evaluated after a 500 mM CaCl2 solution was added
to 2% pectin dispersion at 60°C to a final concentration of 35 mM. The mixture was immediately mixed
by a vortex, stored 24 h at 4°C. After the gels were tempered to room temperature, the texture profile
was measured (TA-XT2i, Texture Technologies Corp, Scarsdale, NY, fitted with a 25 kg load cell). The
particle size distribution was determined by laser diffraction using a Mastersizer S, (Malvern Instruments,
MA).