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The objectives of this study were to elucidate the effects of degree of methyl esterification (DM) and charge distribution of pectin on the stability of emulsions and to analyze bioaccessibility of curcumin incorporated in emulsions stabilized by pectins. Three commercial pectins, CP72 (DM72), CP50 (DM50), and CP7 (DM7), were used. MP50 (DM50) with consecutive demethylesterified galacturonic acid residues was prepared from CP72 via demethylesterification to induce different charge distributions. Emulsions containing curcumin were prepared and were stored for 30 days. The CP72 and CP50 emulsions remained relatively stable for 30 days. However, MP50 and CP7 were less effective at forming stable emulsions. When the pectin emulsions passed through each phase of the simulated gastrointestinal tract (GIT), the CP72 and CP50 emulsions retained their initial droplet structures after in vitro mouth and gastric digestion, whereas the MP50 and CP7 emulsions exhibited gel-like clusters, although the gel-like formation of MP50 was distinct from that observed in CP7. MP50 emulsion showed a high degree of final lipid digestion and high bioaccessibility of curcumin while CP72 emulsion displayed a low degree of final lipid digestion. CP50 exhibited low bioaccessibility of curcumin, which might have been contributed by its fast lipid digestion profiles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Florida citrus production has declined 75% due to Huanglongbing (HLB), a disease caused by the pathogenic bacterium Candidatus Liberibacter asiaticus (CLas). Methods to combat CLas are costly and only partially effective. The cross-compatible species Poncirus trifoliata and some of its hybrids are known to be highly tolerant to CLas, and thus can potentially serve as an alternative feedstock for many citrus products. To further investigate the commercial potential of citrus hybrids, three citrus hybrids, US-802, US-897, and US-942, were studied for their potential as feedstocks for citrus co-products using steam explosion (STEX) followed by water extraction. Up to 93% of sugars were recovered. US-897 and US-942 have similar volatile profiles to that of the commercial citrus fruit types and as much as 85% of these volatiles could be recovered. Approximately 80% of the pectic hydrocolloids present in all three hybrids could be obtained in water washes of STEX material. Of the phenolics identified, the flavanone glycosides, i.e., naringin, neohesperidin, and poncirin were the most abundant quantitatively in these hybrids. The ability to extract a large percentage of these compounds, along with their inherent values, make US-802, US-897, and US-942 potentially viable feedstock sources for citrus co-products in the current HLB-blighted environment.

Culled whole grapefruit (WG) and grapefruit juice processing residues (GP) are currently incorporated into low-cost animal feed. If individual chemical components found within these side streams could be recovered as high-value coproducts, this would improve the overall value of the grapefruit crop. In this study, pectic hydrocolloids, sugars, volatiles, phenolics, and flavonoids were extracted from Star Ruby, Rio Red, and Ruby Red GP and WG using a continuous pilot scale steam explosion system. Up to 97% of grapefruit juice oils and peel oils could be volatilized and contained 87-94% d -limonene. The recovery of pectin, as determined by galacturonic acid content, was between 2.06 and 2.72 g 100 g -1 . Of the phenolics and flavonoids analyzed in this study, narirutin and naringin were extracted in the amounts of up to 10,000 and 67,000 μg g -1 , respectively., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dorado, Cameron, Manthey, Bai and Ferguson.)

The objective of this research was to evaluate the use of a controlled-release carvacrol powder to delay storage decay and maintain the safety of blueberries. The controlled-release carvacrol powder was a microcapsule of carvacrol (11% ( w / w ) active carvacrol) surrounded by a pectin/sodium alginate matrix. The microcapsules were packed in an air-permeable pouch, and then attached to the top of a clamshell filled with blueberries. The blueberries, inoculated with Escherichia coli or Colletotrichum acutatum , or non-inoculated control, were monitored for microbial growth and quality for 10 days at 10 °C and 5 days at 20 °C. Three treatments were compared: controlled-release microencapsulated carvacrol, non-encapsulated carvacrol, and control. The results showed that both the microencapsulated carvacrol and the non-encapsulated carvacrol treatments significantly reduced the populations of yeast and mold, and of E. coli and mesophilic aerobic bacteria. The microencapsulated carvacrol treated berries retained better quality due to significantly lower weight loss than control after 10 days at 10 °C. Sensory panelists found that the microencapsulated carvacrol berries had significantly higher overall blueberry flavor and lower discernible off-flavor in comparison with the non-encapsulated treatment after 3 days at 20 °C. The fruit internal quality, including total soluble solids content (SSC), and titratable acidity (TA), was not significantly affected by any treatment. These results indicate that pectin/sodium alginate controlled-release microencapsulated carvacrol can be used for the preservation of blueberries or other small fruit.

Previously, we showed the weight average molecular weight ( M w ) and intrinsic viscosity ([ ƞ ]) of pectic hydrocolloids recovered from steam-exploded citrus peel were low, suggesting fragmentation due to process temperature and/or time-at-temperature. We have tested this hypothesis on a commercial lime pectin peel, washed to remove soluble sugars and dried for stabilization, using a static steam explosion system. We examined temperatures of 120-150°C at 1-3 min hold times. Galacturonic acid recovery and M w ranged from 22% to 82% and 142-214 kDa, respectively. Recovery of most major pectic sugars increased concomitantly with galacturonic acid as temperature and time-at-temperature increased. [ ƞ ] ranged from 1.75 to 6.83 dl/g. The degree of methylesterification ranged from 66.5% to 72.1%. Tan (δ) (Loss modulus/Storage modulus; G″/G') values of sugar-acid gels for 120-140°C treatments were <1.0. Ideal optimization analysis, where time, [ ƞ ], and percent recovery were maximized, identified processing conditions that favor either increased [ ƞ ] or percent recovery. The results presented here support our hypothesis that temperature and time-at-temperature affect M w and [ η ] of the recovered pectic hydrocolloids. These results also demonstrate that manipulating either temperature or time-at-temperature enables the production of structurally varied populations of pectic hydrocolloids. Based on optimization analysis, commercially viable values of [ ƞ ] can be obtained while recovering approximately 50% of the pectic hydrocolloids., (© 2021 The Authors. Food Science & Nutrition published by Wiley Periodicals LLC.)

The objectives of this research were to microencapsulate tangeretin, and to evaluate the basic characteristics of the microcapsule products. Tangeretin is a polymethoxyflavone (PMF) which has been revealed to possess various health benefits and is abundant in tangerine and other citrus peels. Microencapsulation technology is widely employed in the food and pharmaceutical industries to exploit functional ingredients, cells, and enzymes. Spray drying is a frequently applied microencapsulation method because of its low cost and technical requirements. In this research, tangeretin dissolved at different concentrations in bergamot oil was microencapsulated in a citrus pectin/sodium alginate matrix. The resulting microcapsule powder showed promising physical and structural properties. The retention efficiency of tangeretin was greater at a concentration of 2.0% (98.92%) than at 0.2% (71.05%), probably due to the higher temperature of the emulsion during the homogenizing and spray-drying processes. Encapsulation efficiency was reduced with increased concentration of tangeretin. Our results indicate that tangeretin could be successfully encapsulated within a citrus pectin/sodium alginate matrix using bergamot oil as a carrier.

Hemagglutination inhibition (HAI) assay is a simple method quantifying relative binding activities of glycan-lectin interactions. Currently, interpretation of HAI data remains a manual task depending on visual observation. In this study we developed a digital data reading method for HAI assay by using the area scanning function of a microplate reader. This was based on galectin-3-induced hemagglutination inhibition assay. OD values showed a four-parameter logistic correlation with concentrations of galectin-3 inhibitors (R 2  ≥ 0.97), and IC 50 values were obtained from the curve fitting. The method provides an objective and robust data interpretation for HAI assays conducted with chicken erythrocytes., (Copyright © 2019. Published by Elsevier Inc.)

Background: Citrus pre-harvest fruit drop, caused by huanglongbing infection, has increased dramatically concomitant with declining tree health and crop harvest size. This loss of harvestable fruit is damaging to both growers and juice processors. Recovering and converting this fruit to alternative value added products would benefit the citrus industry. Therefore, we have explored the potential of using this fruit as a feedstock in our newly developed pilot scale continuous steam explosion process., Results: Whole fruits were converted to steam-exploded biomass using a continuous pilot scale process. The sugar composition of raw fruit and steam-exploded biomass was determined. Recovered pectic hydrocolloids and phenolic compounds were characterized. Pectic hydrocolloids comprised 78 g kg -1 of the dry material in the dropped fruit. Following the steam explosion process almost all of the pectic hydrocolloids were recoverable with a water wash. They could be functionalized in situ or separated from the milieu. Additionally, approximately 40% of the polymethoxylated flavones, 10% of the flavanone glycosides, 85% of the limonoids and almost 100% of hydroxycinnamates were simultaneously recovered., Conclusion: The continuous steam explosion of pre-harvest dropped citrus fruit provides an enhanced, environmentally friendly method for the release and recovery of valuable coproducts from wasted biomass. Published 2017. This article is a U.S. Government work and is in the public domain in the USA., (Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

Florida "Valencia" oranges have a wide harvest window, covering four months after first reaching the commercial maturity. However, the influence of harvest time on juice flavor chemicals is not well documented, with the exception of sugars and acids. Therefore, we investigated the major flavor chemicals, volatile (aroma), non-volatile (taste) and mouth feel attributes, in the two harvest seasons (March to June in 2007 and February to May in 2012). Bitter limonoid compounds, limonin and nomilin, decreased gradually. Out of a total of 94 volatiles, 32 increased, 47 peaked mid to late season, and 15 decreased. Juice insoluble solids and pectin content increased over the season; however, pectin methylesterase activity remained unchanged. Fruit harvested in the earlier months had lower flavor quality. Juice from later harvests had a higher sugar/acid ratio with less bitterness, while, many important aroma compounds occurred at the highest concentrations in the middle to late season, but occurred at lower concentrations at the end of the season. The results provide information to the orange juice processing industry for selection of optimal harvest time and for setting of precise blending strategy.

Despite the longstanding importance of the thermally tolerant pectin methylesterase (TT-PME) activity in citrus juice processing and product quality, the unequivocal identification of the protein and its corresponding gene has remained elusive. TT-PME was purified from sweet orange [ Citrus sinensis (L.) Osbeck] finisher pulp (8.0 mg/1.3 kg tissue) with an improved purification scheme that provided 20-fold increased enzyme yield over previous results. Structural characterization of electrophoretically pure TT-PME by MALDI-TOF MS determined molecular masses of approximately 47900 and 53000 Da for two principal glycoisoforms. De novo sequences generated from tryptic peptides by MALDI-TOF/TOF MS matched multiple anonymous Citrus EST cDNA accessions. The complete tt-pme cDNA (1710 base pair) was cloned from a fruit mRNA library using RT- and RLM-RACE PCR. Citrus TT-PME is a novel isoform that showed higher sequence identity with the multiply glycosylated kiwifruit PME than to previously described Citrus thermally labile PME isoforms.

Background: Fresh orange juice is perceived to be more wholesome than processed juice. Fresh juice may have nutrients and phytonutrients that differ from pasteurized or processed juice., Results: 'Hamlin' and 'Valencia' oranges were extracted using a commercial food service juicer, pasteurized or not, resulting in fresh-commercial juice (FCJ) or pasteurized FCJ (FCPJ) for comparison with pasteurized processed juice (PPJ) in 2009, and gently hand-squeezed 'Valencia' juice (HSJ) in 2010 for nutrient and phytonutrient content. Regardless of pasteurization, FCJ/FCPJ contained 25-49% lower insoluble solids than the PPJ, while in HSJ the insoluble solids content was between that of FCJ and PPJ. The major orange juice flavonoid glycosides were twofold higher in PPJ than in FCJ/FCPJ and HSJ, indicating that the extraction and finishing process led to more peel tissue in the juice than fresh juice extraction methods. The total phenolic content (TPC) in the juices followed a similar pattern to the flavonoid glycoside content. The polymethoxylated flavones (PMFs), associated with peel oil, occurred at the highest levels in the FCJ/FCPJ and lowest in HSJ. Limonoids and alkaloids occurred at higher levels in PPJ and HSJ than in FCJ/FCPJ., Conclusion: The high peel oil content of FCJ/FCPJ resulted in higher PMF levels compared to PPJ and HSJ, while flavonoid glycosides, limonoids and alkaloids, which occur at high concentrations in the inner peel albedo, occurred at higher concentrations in PPJ., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Background: In Florida, 90% of citrus is processed into juice, leaving large amounts of peel waste that could be processed into food ingredients such as pectins for other applications. Pectins of low degree of methylation (DM < 50) have important functional properties for use in foods and pharmaceuticals. Thus determination of DM is important, but DM analyses are difficult to perform and assays can have interference due to the presence of salts or neutral sugars., Results: A chromatography method has been developed for determination of DM of food pectin using alkaline hydrolysis followed by pectin digestion with a commercial pectinase. Analysis was performed by ion exclusion chromatography (IEC) to obtain methanol (MeOH) concentrations and by anion exchange chromatography (AEC) to obtain galacturonic acid (GalA) concentrations. Data were compared with values obtained using other DM analysis procedures. For example, a 55-75 DM pectin assayed as 55.5, 59.6, 60.1 and 57.0 DM by IEC, AEC, colorimetric and conductivity detection methods respectively., Conclusion: The chromatography procedure described in this paper provides an alternative method for determination of DM of food pectins for accurate and efficient analysis when confronted with limited quantities or large numbers of samples., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Fresh orange juice is perceived to be more wholesome than processed juice. Fresh juice may have flavor and nutrients that differ from pasteurized or processed juice., Results: 'Hamlin' and 'Valencia' oranges were extracted using a commercial food service juicer, pasteurized or not, resulting in fresh-commercial juice (FCJ) or pasteurized juice (FCPJ) for comparison with pasteurized processed juice (PPJ) in 2009, and gently hand-squeezed 'Valencia' juice (HSJ) in 2010 for quality attributes. There was higher peel oil, lower pectin content, and less cloud loss in FCJ/FCPJ compared to PPJ and HSJ regardless of pasteurization. Titratable acidity was generally higher and the ratio of solids to acids lower in FCJ/FCPJ or HSJ compared to PPJ. FCJ/FCPJ had generally higher levels of most aroma volatiles than did PPJ and, overall, the highest esters and terpenes, while methanol and ethanol levels were highest in HSJ. For sensory evaluation, FCJ/FCPJ had more peel oil and PPJ more cooked flavor than other samples, while 'Valencia' HSJ was preferred over the other juices., Conclusion: High peel oil content and thermo-pasteurization process decreased cloud loss of orange juice. Extraction and finishing processes rather than pasteurization or oil content were major factors in influencing orange juice flavor quality., (Copyright © 2012 Society of Chemical Industry.)

We purified a Carica papaya pectin methylesterase (CpL-PME; EC 3.1.1.11) from a commercial papain preparation. This CpL-PME was separated from the abundant cysteine endopeptidases activities using sequential hydrophobic interaction and cation-exchange chromatographies and then purified by affinity chromatography using Sepharose-immobilized kiwi PME inhibitor protein to obtain a single electrophoretically homogeneous protein. The enzyme was purified 92-fold with 38% yield, providing a specific activity of 1200 U/mg. The molecular weight was determined to be 35,135 by MALDI-TOF-MS in linear mode. MALDI-TOF-MS peptide mass fingerprinting following trypsin digestion indicated CpL-PME represents a novel Carica PME isoform. The CpL-PME required salt for activity, and it showed a broad activity range (pH 6-9) and moderate thermostability (optimum ca. 70°C). A calcium-insensitive methylated lime pectin treated with CpL-PME to reduce degree of methylesterification by 6% converted the substrate to high calcium sensitivity, indicating a processive mode of action. These properties support further research to apply CpL-PME to tailor pectin nanostructure., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The hydrolytic and oxidative stability of L-(+)-ascorbic acid (AA) into plasticized pectin films were separately studied in view of preserving vitamin C activity and/or to achieve localized antioxidant activity at pharmaceutical and food interfaces. Films were made with each one of the enzymatically tailored pectins (50%, 70%, and 80% DM; Cameron et al. Carbohydr. Polym.2008, 71, 287-299) or commercial high methoxyl pectin (HMP; 72% DM). Since AA stability was dependent on water availability in the network, pectin nanostructure affected the AA kinetics. Higher AA retention and lower browning rates were achieved in HMP films, and calcium presence in them stabilized AA because of higher water immobilization. Air storage did not change AA decay and browning rates in HMP films, but they significantly increased in Ca-HMP films. It was concluded that the ability of the polymeric network to immobilize water seems to be the main factor to consider in order to succeed in retaining AA into film materials.

Complexation between ferric oxide particles and pectins with degree of methylation 50% but having ordered or random arrangement of free carboxyl groups is investigated by electric light scattering and electrophoresis. The influence of charge distribution in pectin chain on the electrical properties of oppositely charged oxide particles and stability of their suspensions is examined as a function of pectin concentration. Although the difference in charge density of pectin samples is ~5%, we found small but measurable difference in the behavior of both oxide/pectin complexes. This is attributed to condensation of counterions near the chains of pectin with ordered distribution of charges, leading to a decrease in the effective charge density and to a corresponding decrease in the contour length of the adsorbing pectin chains. Two parameters are sensitive to the conformation of the adsorbed chains in suspensions, stabilized by pectin adsorption (at particle charge reversal). The electro-optical effect is higher for the complex with less charged pectin, which is explained with larger amount of chains, adsorbed in more coiled conformation than the chains of pectin with random distribution of free carboxyl groups. The addition of small amounts of CaCl(2) has no significant influence on the thickness of the layer from the less charged pectin, in agreement with a more compact conformation of the chains in this adsorbed layer. In contrast, the thickness of the layer from pectin with random distribution of charged groups decreases with increasing concentration of CaCl(2), indicating a more loose structure of this layer.

Methyl ester distribution in pectin homogalacturonan has a major influence on functionality. Enzymatic engineering of the pectin nanostructure for tailoring functionality can expand the role of pectin as a food-formulating agent and the use of in situ modification in prepared foods. We report on the mode of action of a unique citrus thermally tolerant pectin methylesterase (TT-PME) and the nanostructural modifications that it produces. The enzyme was used to produce a controlled demethylesterification series from a model homogalacturonan. Oligogalacturonides released from the resulting demethylesterified blocks introduced by TT-PME using a limited endopolygalacturonase digestion were separated and quantified by high-pressure anion-exchange chromatography (HPAEC) coupled to an evaporative light-scattering detector (ELSD). The results were consistent with the predictions of a numerical simulation, which assumed a multiple-attack mechanism and a degree of processivity ∼10, at both pH 4.5 and 7.5. The average demethylesterified block size (0.6-2.8 nm) and number of average-sized blocks per molecule (0.8-1.9) differed, depending upon pH of the enzyme treatment. The mode of action of this enzyme and consequent nanostructural modifications of pectin differ from a previously characterized citrus salt-independent pectin methylesterase (SI-PME).

The multiple forms of the enzyme pectin methylesterase (PME) present in citrus fruit tissues vary in activity toward juice cloud-associated pectin substrates and, thus, in their impact on juice cloud stability and product quality. Because the proteins responsible for individual PME activities are rarely identified by structural properties or correlated to specific PME genes, matrix-assisted laser desorption-ionization with tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) was investigated as a direct means to unequivocally identify the thermolabile (TL-) PME isoforms isolated from sweet orange [ Citrus sinensis (L.) Osbeck] fruit tissue. Affinity-purified TL-PME preparations were separated by SDS-PAGE prior to trypsin digestion and analyzed by MS for peptide mass fingerprinting. The two major PME isoforms accumulated in citrus fruit matched existing accessions in the SwissProt database. Although similar in size by SDS-PAGE, isoform-specific peptide ion signatures easily distinguished the two PMEs.

More than 90% of oranges in Florida are processed, and since Huanglongbing (HLB) disease has been rumored to affect fruit flavor, chemical and physical analyses were conducted on fruit and juice from healthy (Las -) and diseased (Las +) trees on three juice processing varieties over two seasons, and in some cases several harvests. Fruit, both asymptomatic and symptomatic for the disease, were used, and fresh squeezed and processed/pasteurized juices were evaluated. Fruit and juice characteristics measured included color, size, solids, acids, sugars, aroma volatiles, ascorbic acid, secondary metabolites, pectin, pectin-demethylating enzymes, and juice cloud. Results showed that asymptomatic fruit from symptomatic trees were similar to healthy fruit for many of the quality factors measured, but that juice from asymptomatic and especially symptomatic fruits were often higher in the bitter compounds limonin and nomilin. However, values were generally below reported taste threshold levels, and only symptomatic fruit seemed likely to cause flavor problems. There was variation due to harvest date, which was often greater than that due to disease. It is likely that the detrimental flavor attributes of symptomatic fruit (which often drop off the tree) will be largely diluted in commercial juice blends that include juice from fruit of several varieties, locations, and seasons.

Approximately 1 million metric tons of grapefruit were processed in the 2003/04 season resulting in 500,000 metric tons of peel waste. Grapefruit peel waste is usually dried, pelletized, and sold as a low-value cattle feed. This study tested different loadings of commercial cellulase and pectinase enzymes and pH levels to hydrolyze grapefruit peel waste to produce sugars. Pectinase and cellulase loadings of 0, 1, 2, 5, and 10mgprotein/g peel dry matter were tested at 45 degrees C. Hydrolyses were supplemented with 2.1mg beta-glucosidase protein/g peel dry matter. Five mg pectinase/g peel dry matter and 2mgcellulase/g peel dry matter were the lowest loadings to yield the most glucose. Optimum pH was 4.8. Cellulose, pectin, and hemicellulose in grapefruit peel waste can be hydrolyzed by pectinase and cellulase enzymes to monomer sugars, which can then be used by microorganisms to produce ethanol and other fermentation products.

A simulation methodology for predicting the time-course of enzymatic digestions is described. The model is based solely on the enzyme's subsite architecture and concomitant binding energies. This allows subsite binding energies to be used to predict the evolution of the relative amounts of different products during the digestion of arbitrary mixtures of oligomeric or polymeric substrates. The methodology has been specifically demonstrated by studying the fragmentation of a population of oligogalacturonides of varying degrees of polymerization, when digested by endo-polygalacturonase II (endo-PG II) from Aspergillus niger.

The thermally tolerant pectin methylesterase (TT-PME) was isolated as a monocomponent enzyme from sweet orange fruit (Citrus sinensis var. Valencia). It was also isolated from flower and vegetative tissue. The apparent molecular weight of fruit TT-PME was 40800 by SDS-PAGE and the isoelectric point estimated as pI 9.31 by IEF-PAGE. MALDI-TOF MS identified no tryptic-peptide ions from TT-PME characteristic of previously described citrus PMEs. TT-PME did not absolutely require supplemented salt for activity, but salt activation and pH-dependent activity patterns were intermediate to those of thermolabile PMEs. Treatment of non-calcium-sensitive pectin with TT-PME (reducing the degree of methylesterification by 6%) increased the calcium-sensitive pectin ratio from 0.01 to 0.90, indicating a blockwise mode of action. TT-PME produced a significantly lower end-point degree of methylesterification at pH 7.5 than at pH 4.5. Extensive de-esterification with TT-PME did not reduce the pectin molecular weight or z-average radius of gyration, as determined by HPSEC.

A high-performance size-exclusion chromatography-evaporative light scattering detector method was used to separate, detect and quantify galacturonic acid (GA) oligomers. In 40 mM acetic acid GA monomer, dimer and trimer could be separated with baseline resolution but polygalacturonic acid (PGA) precipitated and could not be eluted from the column. An NH4OAc, pH 3.7, buffer was developed as the eluent which separated GA oligomers as well as PGA and pectin without precipitation. Linear calibration curves for mono-, di- and tri-GA were produced with this buffer which could be used to estimate masses of tetra-, penta- and hexa-GA, as well as 19mer and 20mer.

A pectin methylesterase (PME) from sweet orange fruit rag tissue, which does not destabilize citrus juice cloud, has been characterized. It is a salt-dependent PME (type II) and exhibits optimal activity between 0.1 and 0.2 M NaCl at pH 7.5. The pH optimum shifted to a more alkaline range as the salt molarity decreased (pH 8.5-9.5 at 50 mM NaCl). It has an apparent molecular mass of 32.4 kDa as determined by gel filtration chromatography, an apparent molecular mass of 33.5 kDa as determined by denaturing electrophoresis, and a pI of 10.1 and exhibits a single activity band after isoelectric focusing (IEF). It has a K(m) of 0.0487 mg/mL and a V(max) of 4.2378 nkat/mg of protein on 59% DE citrus pectin. Deblocking the N-terminus revealed a partial peptide composed of SVTPNV. De-esterification of non-calcium-sensitive pectin by 6.5% increased the calcium-sensitive pectin ratio (CSPR) from 0.045 +/- 0.011 to 0.829 +/- 0.033 but had little, if any, effect on pectin molecular weight. These properties indicate this enzyme will be useful for studying the PME mode of action as it relates to juice cloud destabilization.

The pectin methylesterase (PME; EC 3.1.1.11) present in a commercial orange peel enzyme preparation was characterized to establish its identity among the multiple PME isozymes present in Valencia orange (Citrus sinensis L.) peel. We show the commercial enzyme corresponds to the major peak 2 PME previously separated by heparin-Sepharose chromatography (Cameron et al., J. Food Sci. 1998, 63, 253). Both PMEs have comparable elution profiles on cation-exchange and hydrophobic-interaction perfusion chromatography columns, molecular weights (ca. 34 kDa) and pI (pH 9.2), and biochemical properties, including a broad pH activity range and activity in the absence of added cations. An identical partial amino terminal peptide sequence was also obtained for the PMEs, which further demonstrated a structural identity with other plant PMEs. The biochemical and structural properties readily distinguish this Valencia orange PME from salt-dependent isozymes and further suggest that it is an ortholog to the salt-independent fruit-specific isozyme of tomato. This work provides a well-defined, enzymatically homogeneous, salt-independent (type 1) plant PME isozyme that is suitable for studying details of the enzyme's mode of action and for use in modifying methylester patterns for studying the structure-functional property relationships in pectin.

A commercial high-methoxy citrus pectin was treated with a purified salt-independent pectin methylesterase (PME) isozyme isolated from Valencia orange peel to prepare a series of deesterified pectins. A series of alkali-deesterified pectins was also prepared at pH 10 under conditions permitting beta-elimination. Analysis of these pectins using high-performance size exclusion chromatography (HPSEC) with on-line multiangle laser light-scattering, differential viscometer, and refractive index (RI) detectors revealed no reduction in weight-average molecular weight (M(w); 150000) in the PME-treated pectin series, whereas a 16% reduction in intrinsic viscosity (IV) occurred below a degree of esterification (DE) of 47%. In contrast, alkali deesterification rapidly reduced both M(w) and IV to less than half of that observed for untreated pectin. PME treatment of a non-calcium-sensitive citrus pectin introduced calcium sensitivity with only a 6% reduction in the DE. Triad blocks of unesterified galacturonic acid were observed in (1)H nuclear magnetic resonance spectra of this calcium-sensitive pectin (CSP). These results demonstrate that the orange salt-independent PME isozyme utilizes a blockwise mode of action. This is the first report of the preparation of a CSP by PME treatment without significant loss of the pectin's M(w) due to depolymerization.