Stability of Juice Beverages as Affected by Pectinmethylesterase

Chapter 11

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Stability of Juice Beverages as Affected by Pectinmethylesterase L . Wicker Department of Food Science and Technology, Physical Properties Group, University of Georgia, Athens, GA 30602

Orange juice concentrates are often used as a base in juice blends with other fruit juice beverages as well as milk drinks. Enrichment of juices and other beverages with minerals in addition to the use of concentrates from citrus, kiwi, mango, passion fruit and other fruits provide a ready source of tailored juice beverages that are high in phytochemicals; however, mixed juices or juice blends are often not stable upon blending or storage. Pectinmethylesterase (PME) is responsible for blockwise de-esterification of pectin and is implicated in clarification of citrus juices and juice-containing beverages by initiating the formation of calcium reactive pectic acid. Since cloud instability or clarification is a major defect affecting the appearance of juices and concentrates, identification of the causative factors relevant to the juice and nutritional beverage industry is appropriate.

© 2004 American Chemical Society In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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134 Pectinmethylesterase (PME) occurs naturally in all plant tissues. In citrus, disruption of the cell wall matrix releases PME, enhances the de-esterification of pectin, the formation of insoluble, large molecular weight calcium pectate aggregates, and "cloud" loss (clarification) (1,2). Several forms of PME have been separated and characterized from lemons (3) and navel oranges (4). Other thermolabile (TL-PME) and thermostable (TS-PME) PME isozymes have been identified (J) that differ in low pH tolerance, hydrophobicity, secondary structure, clarification rates (6-10) and gelling characteristics of pectin (5). Different physico-chemical properties of PME are implicated in varied rates of clarification. In spite of the ostensible role of PME in quality of juices, no direct correlation between PME, degree of de-esterification (%DE) of pectin, and clarification of juices has been reported. Citrus juice is a colloidal dispersion of sugars, cellular organelles, membranes, chromatophores, oils,flavonoids,and cell wallfragments;however, citrus cloud has not been described in colloidal terms. Citrus juice cloud is typically described by compositional and particle size analysis. After commercial juice extraction andfinishing,large particulate material composed of juice sacs sediment and this substance is termed settling pulp. The material that remains suspended is the citrus juice cloud, which has a particle size of less than 2 μιη (//). Cloud analysis yields an approximate composition of 52% protein, 4.5% pectin, 25% lipid, 2% hemicellulose, and 1.5% cellulose (12-15). PME modification of pectin plays a major role in cloud stability of not only citrus juices, but also juice containing beverages, fortified sports drinks, and acidified milk drinks. Further, it offers the potential to prepare tailored pectins of specific total charge and unique charge distribution properties. The functional performance of pectins as a stabilizer of colloidal dispersions can be better predicted and controlled with more complete information on the impact of PME activity. To prepare protein fortified drinks such as acidified milk drinks, sports drinks, and juice beverages, stabilizers like pectin should prevent sedimentation and serum separation, imparting a smooth, creamy mouthfeel. In addition, the stabilizer should moderate particle size of dispersions and tolerate process treatments, such as pasteurization. In general, pectins that are good stabilizers of acidified drinks have a high molecular weight and low net charge. High ester pectins that are calcium sensitive and anchor pectin to the protein surface have the best stabilizing properties and may be obtained by chromatographic separation of desirable pectins (16) or by enzymatic modification of native pectins (17). Modified pectins likely have different physical properties depending on the extent of modification and the length of de-esterified blocks. The distribution of esterification plays an important role in pectin interactions with cations. In this chapter, information and research that indicates PME activity is influenced by extrinsic factors, including cations, non-PME protein and PME-

In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

135 pectin complexes are provided. Clarification of citrus juices and juice containing beverages, as well as the instability of pectin stabilized drinks is attributed to pectin interactions. Factors that influence PME activity, the role of PME in modification of pectin charge and stability of dispersions, are emphasized.


Materials and Methods

Source of Material PME was extracted from citrus (Valencia orange or Marsh grapefruit (MGF)) using 0.25M Tris-Cl-, 0.1M NaCl, pH 8.0 (1:4) (18) or 1 M NaCl without pH adjustment (19). The PME extracts were chromatographed on HiTrap SP, Hi-Trap Heparin Sepharose, and ConA (Amersham-Pharmacia Biotech, Piscataway, NJ.) according to established procedures (20). The alkaline extraction procedure extracts TL-PME and TS-PME in Valencia and MGF. The endogenous pH extraction procedure is selective for TS-PME from Marsh grapefruit pulp but is not selective for TS-PME extracted from Valencia (Wicker, unpublished results). Protein content was estimated by the dye binding method (21). PME activity was determined by titration at pH 7.5 in 1% pectin, 0.1M NaCl at 30°C and was expressed as microequivalents of ester hydrolyzed per min.

Modified pectins and interaction with milk proteins A high methoxyl, calcium reactive pectin was obtained from Copenhagen Pectin A/S (Copenhagen, Denmark). It had 92% uronic acid, estimated MW of 244,700, and 73% degree of esterification (DE). Sodium casemate was prepared by acid precipitation from Nilac skim milk powder (NIZO, Kernhemseweg, the Netherlands) and high β-lactoglobulin; whey protein isolate was obtained from Amor Proteins (St. Brice enCogles, France). The protein dispersions were acidified to pH 4.0 with glucono-delta-lactone and homogenized with pectin to a final concentration of 0.1, 0.25 or 0.35% pectin. The supernatant was collected after separation in a microfiige centrifuge and analyzed for MW with a Viscotek 250 Triple Detection (RI, viscosity, and light scattering (Oss, Netherlands) with Biogel TSK 60-40-30XL columns in series, using 0.2M citrate, pH 4.0.


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Results and Discussion The lack of correlation between PME activity and clarification presents ambiguity in the PME model for juice clarification. When clarification occurred in pasteurized juices, it was attributed to a variety of factors, including a priori PME activity, residual TS-PME activity, improper inactivation of PME in pulp of pulp added juices, and PME isozyme activity. The amount of PME or TSPME detectable in juice was not well correlated with quality indicators of juice such as percentage of settling pulp, Brix, pH, and titratable acidity, among others. (22). Versteeg et al. (5) suggested that juice clarification at 4°C is primarily due to TS-PME, thus subsequent research was directed towards quantifying, characterizing, and inactivating the TS-PME. A study by Cameron et al. (23) confirmed that heated extracts of juice, peel, and rag (100% TS-PME) induced the most rapid clarification. However, their results clearly indicated that % thermostable activity could not be used solely to estimate propensity to clarification, since tissue specific differences were observed. Recent studies that have partially purified PME and tested for juice clarification are summarized in Table I. The total units of activity (microequivalents of ester hydrolyzed per min) per mL of extract or specific activity (microequivalents of ester hydrolyzed per min per mg of protein) are reported if provided, and the time of change in transmittance was estimated from the data presented. Cameron et al (23) presented strong evidence that insoluble PME extracted from juices, which was only 5.6% thennostable, clarified juice at equal rates to soluble PME that was extracted from pulp and heated (100% thermostable). They also observed that the source (juice, rag, or peel) and solubility of PME influenced PME induced clarification. At 4°C, insoluble PME extracted from juice (DP) or soluble PME from peel (DS) clarified juice in the shortest period of time. In a later study, Cameron et al (24) showed that less than 1.5U PME per mL of fresh juice resulted in a decrease in absorbance within a week and related it to peel residue in the juice of "hard extracted" juice. Regardless of extraction method, PME activity estimated at juice pH (4.5) was a better predictor of clarification than PME activity estimated near the pH optimum (7.5). This suggests that the mechanism of de-esterification in juice may be different as reported for the mechanism of de-esterification of commercial pectins by apple PME at pH 4.5 and 7.5 (25). Some of the reported PME isozymes are likely to be PME-pectin complexes (3,26) that influence isoelectric focusing and ion exchange separation of PMEs, detectable activity, thermostability, and rates of clarification. PME can be solubilized from an inactive pectin complex (27,28) and activate apparent PME activity. At higher concentrations, cations inhibit PME by competitive displacement. In addition, the effect on PME activity is cation specific (29-32). Calcium chloride has a broad concentration effect on activity (Fig 1A) unlike



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In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. 4

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Table I. Source, total activity, specific activity and onset of clarification reported for citrus juices, stored at 4°C Activity Source Specific Clarification Reference activity Valencia orange U^L 5U/mL juice Cameron et al U/Hgpro 1997 OD

Stability of Juice Beverages as Affected by Pectinmethylesterase

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