T F L Effect of methoxy-esters distribution pattern on the rheological properties of pectin-calcium gels: Toward understanding pectin structure-function relations Doungla E. Ngouémazong 1 , Thomas Duvetter 1 , Ilse Fraeye 1 , Ann Van Loey 1 , Paula Moldenaers 2 , Marc Hendrickx 1 1 Laboratory of Food Technology and Leuven Food Science and Nutrition Research Centre (LFoRCe),Department of Microbial and Molecular Systems (M 2 S), Katholieke Universiteit Leuven (K.U.Leuven); Kasteelpark Arenberg 22 box 2457, 3001 Leuven, Belgium. 2 Division of Applied Rheology and Polymer Technology, K.U.Leuven, Willem de Croylaan 46 , box 2423, 3001 Leuven, Belgium INTRODUCTION Partially demethoxylated pectin (PDP) PDP-Ca 2+ network (gel) Pectin structure - function relation ? Pectin structural features : DM and PM (PM as DB abs , indicative for non-methoxylated GalA (NMG) residues present in blocks, thus available for JZ formation) Rheological characteristics (gel strength) of cured pectin-Ca 2+ gels ? Methyl group Galacturonic acid (GalA) DM: Degree of methoxylation DB abs : Absolute degree of blockiness MATERIALS AND METHODS Citrus pectin (DM ~94%; DB abs < d.l.) Chemical (NaOH) saponification (pH 11.0) Plant (Carrot) PME (pH 7.0) Fungal (A. aculea- tus) PME (pH 4.5) Demethoxylation PME: Pectinmethylesterase A.: Aspergillus Structural characterization of PDPs: DM and DB abs DB abs estimation: (1) Endo-PG digestion of PDPs and release of NMG (2) Chromatographic determination of NMG as mono-, di- and tri-GalA P-PDP F-PDP C-PDP 50°C µls PDP solution (2%(w/v); 50°C; pH 6.0) µls CaCl 2 solution (30°C) Rheological characterization of PDP-Ca 2+ gels: gel strength (G’) of cured gels Curing: 50°C for 10 min 50°C - 20°C @ 0.5°C/min 20°C for 5 hours Lowering Peltier hood Peltier controlled Physica MCR 501 rheometer (on anti-vibration table) RESULTS AND DISCUSSION  Structural characterization of partially demethoxylated pectins (PDPs) Δ DM & PM R = 2[Ca 2+ ] / [COO - ] → [Ca 2+ ] varies with DM d.l.: detection limit PG: Polygalacturonase PM: Pattern of methoxylation DM = (mole / mole ) x 100 DB abs = (Total NMG released / Total GalA) x 100  Effect of demethoxylation on DB abs  Effect of demethoxylation on proportions of mono-, di- and tri-GalA released 0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100% DBabs DM C-pectins F-pectins P-pectins Maximum DBabs (R² = 0.999) (R² = 0.998) (R² = 0.986)  Rheological characterisation of PDP-calcium gels (R = 2.0) Ca 2+  Effect of degree and pattern of methoxylation on G’  Effect of pattern of methoxylation on gel strength (G’)  Each de-esterification method resulted in ≠ relation between DM and DB abs indicating ≠ methoxy-ester distribution pattern. Occurrence of ≠ number and/or size of NMG blocks at similar DM.  C-pectins (completely random distribution of NMG residues/blocks) revealed an exponential relation (approximated by 3 rd order polynomial) between DM and DB abs .  As the distribution pattern of NMG became more blocky, the relation gradually shifted toward linearity.  Actually, a linear relation is theorized between DM and DB abs of ideally (theoretical) blocky pectins, where virtually, all NMG are released by endo-PG as mono-, di- or tri- GalA (implying total NMG released ≈ total NMG residues of pectin), thereby resulting in DB abs ≈ 100%-DM.  PDPs displayed ≠ proportions of mono-, di- and tri-GalA at early stages of de-esterification.  From a certain critical DB abs value (~20%, ~30%, and ~30%) corresponding to DM values of ~35%, ~40% and ~60% in C-, F- and P-pectins respectively, all PDPs showed similar proportions of mono-di-tri-GalA, with tri- GalA higher in proportion than the rest. This suggested the occurrence of large NMG blocks in these PDPs From critical DB abs , all PDPs show some similarities in distribution of NMG blocks over the entire polymer In gel networks, high G’ related to high number of cross- link points (junction zones (JZ) for pectin-Ca 2+ gels) per polymer chain.  In C- and F-PDP gels, a two step increase of G’ was observed with increasing DB abs . The initial pronounced increase (DB abs < critical DB abs ) was mostly related to increase in number of JZ (mainly increase in umber of NMG blocks per PDP chain) at early de-esterification while the slow increasing step was mostly attributed to an increase in the size of the JZ (mainly increase in size of NMG blocks later during de-esterification). PDPs with DB abs < critical DB abs generate gels predominantly characterized by high number of shorter JZ, while PDPs with DB abs ≥ critical DB abs yield networks consisting of few but longer JZ. In C- and F-pectin gels, high G’ is either related to high number of JZ or/and longer JZ per pectin chain  P-PDP gels showed a more gradual increase of G’ with increasing DB abs . This indicates that the mechanism inducing an increase in G’ is rather similar in these gels. In P-pectin gels, increase in G’ was related not only to the increase in the number / size of JZ per chains (DB abs < critical Db abs ) but also to the number of Ca 2+ dimerized pectin chains in the network. PDPs with DB abs < critical DB abs generate gels predominantly characterized by shorter JZ and few Ca 2+ dimerized chains, while PDPs with DB abs ≥ critical DB abs yield networks consisting of longer JZ and more Ca 2+ dimerized chains In P-pectin gels, high G’ is either related to high number or/and larger size of JZ per pectin chain or/and high number of Ca 2+ dimerized chains in the network.  A combined effect of degree and pattern of methoxylation revealed C-, F- and P-pectin gels having quite comparable strength at rather similar DM.  At similar DM, PDPs carry NMG blocks but while P-PDPs have larger blocks on few chains, many (if not all) F- and C-PDP chains carry shorter and shortest NMG blocks respectively. The large size of JZ seems to fully compensate for the presence of few Ca 2+ dimerized pectin chains in P-pectin gel networks, so that the net effect of all parameters (number & size of JZ per chain and number of dimerized chains) becomes rather comparable at similar DM. CONCLUSION  Increase in the number of NMG blocks per PDP chain, thus increase in the number of junction zones per chain in corresponding Ca 2+ gels, results in an increase of gel strength.  Increase in the size of NMG blocks per PDP chain, thus increase in the size of junction zones in corresponding Ca 2+ gels, equally results in an increase of gel strength.  Increase in the number of demethoxylated chains, thus increase in the number of dimerized chains in the corresponding Ca 2+ gel also results in an increase of gel strength. In concentrated pectin-Ca 2+ gels saturated with Ca 2+ , DM (rather than PM) plays a major role in controlling gel strength. REFERENCES • Daas, P.; Voragen, A. G. J.; Schols, H. (2000). Carbohydrate Research, 326 (2), 120-129. • Doungla N.E., Vandebril, S., Duvetter T., Van Loey A., Moldenaers P., Hendrickx, P. (2009). Quick and reliable method of preparation of strong homogeneous calcium-pectin gels. Proceedings 5 th ISFRS, 734-735. • Fraeye, I.; Doungla, N.E.; Duvetter, T.; Moldenaers, P. ; Van Loey, A. ; Hendrickx, M. (2009). Food Hydrocolloids, 23 (8), 2069-2077. ACKNOWLEDGMENT This research has been supported by the Research Council of K.U.Leuven e-mail: eugenie.doungla@biw.kuleuven.be Rhamnogalacturonan II Rhamnogalacturonan I Homogalacturonan 1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5 0,0 0,2 0,4 0,6 0,8 1,0 0,0 0,2 0,4 0,6 0,8 G' (Pa) DB abs DM P-pectin gels F-pectin gels C-pectin gels 1E+0 1E+1 1E+2 1E+3 1E+4 1E+5 0% 20% 40% 60% 80% 100% G' (Pa) DBabs P-pectins F-pectins C-pectins Junction zone (JZ) STEP I STEP II 0 20 40 60 80 0% 20% 40% 60% 80% 100% Mono- di- tri-GalA (%) DBabs Mono Di Tri C-pectins DM~35% 0% 20% 40% 60% 80% 100% DBabs Mono Di Tri F-pectins DM~40% 0% 20% 40% 60% 80% 100% DBabs Mono Di Tri P-pectins DM~60%