Randall G. Cameron

FRACTIONATION AND PRETREATMENT OF ORANGE PEEL BY DILUTE ACID HYDROLYSIS

Abstract Solubilization and depolymerization of carbohydrates by treatment of orange peel with dilute (0·06 and 0·5%) sulfuric acid at 100, 120 and 140°C has been investigated. The acid treatments solubilized a large portion of total carbohydrates in orange peel. However, only soluble sugars and sugars derived from hydrolysis of hemicelluloses were efficiently released by the treatment with hot dilute sulfuric acid. Cellulose and segments of pectin containing galacturonic acid units were very resistant to acid-catalyzed hydrolysis. The treatment with dilute sulfuric acid had a positive effect on the rate of subsequent enzymatic hydrolysis of orange peel by a mixture of cellulolytic and pectinolytic enzymes.

Effect of Liberibacter Infection (Huanglongbing Disease) of Citrus on Orange Fruit Physiology and Fruit/Fruit Juice Quality: Chemical and Physical Analyses

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.

Enzymatic modification of a model homogalacturonan with the thermally tolerant pectin methylesterase from Citrus: 1. Nanostructural characterization, enzyme mode of action, and effect of pH.

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).

Fermentation of galacturonic acid and pectin-rich materials to ethanol by genetically modified strains of Erwinia

Evaluation of the four ethanologenic constructs of bacteria in the genus Erwinia indicates that two strains E. chrysanthemi EC16 and E. carotovora SR38 show promise for development of direct hydrolysis and fermentation of pectin-rich substrates to mixtures of ethanol and acetate. Both strains fermented glucose to ethanol in nearly theoretical yields, but produced mainly acetate and ethanol by fermentation of D-galacturonic acid. Both strains depolymerized citrus pectin, polygalacturonic acid and polysaccharides in citrus peel and converted resulting sugars to carbon dioxide, acetate, ethanol and lesser amounts of formate and succinate.

Fermentation of sugars in orange peel hydrolysates to ethanol by recombinant Escherichia coli KO11

The conversion of monosaccharides in organe peel hydrolysates to ethanol by recombinantEscherichia coli KO11 has been investigated in pH-controlled batch fermentations at 32 and 37°C. pH values and concentration of peel hydrolysate were varied to determine approximate optimal conditions and limitations of these fermentations. Very high yields of ethanol were achieved by this microorganism at reasonable ethanol concentrations (28–48 g/L). The pH range between 5.8 and 6.2 appears to be optimal. The microorganism can convert all major monosaccharides in organe peel hydrolysates to ethanol and to smaller amounts of acetic and lactic acids. Acetic acid is coproduced in equimolar amounts with ethanol by catabolism of salts of galacturonic acid.

Acid-catalyzed hydrolysis of hesperidin at elevated temperatures ☆

Dilute sulfuric acid was used as a catalyst for hydrolysis of hesperidin suspensions in water at temperatures ranging from 25 to 180 degrees C. Significant acceleration of the reaction was observed at 120 degrees C and higher temperatures. This increase in the rate of hydrolysis can be attributed to increased solubilization of hesperidin in water at higher temperatures. Partial hydrolysis of hesperidin at 140 degrees C was used for the preparations of hesperetin-7-glucoside, which has a value in the synthesis of dihydrochalcone sweeteners. Simple separation of hesperetin and hesperetin-7-glucoside by extraction with dry acetone or lower alcohols has been developed.

Development of a Valencia Orange Pectin Methylesterase for Generating Novel Pectin Products

We are interested in developing enzyme technologies for the preparation of novel specialty pectins and pectic materials from agricultural processing residues with new uses beyond current food applications. Enzymes can be used to modify efficiently and selectively pectin structure and to thereby change dependent functional properties. Recent work in our laboratories has focused on plant pectin methylesterases (PMEs), particularly those present in Valencia orange fruit. Our approach is to: 1) prepare highly pure individual isoenzymes, 2) correlate structural identity with biochemical and functional properties, 3) then apply the enzymes to well-defined pectin substrates, and 4) determine the structural and physical properties of the enzymatically modified pectins. We present here our progress to characterize the major salt-independent isoenzyme purified from Valencia orange peel and use it specifically to modify methylester patterns in citrus pectin.

Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation.

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.

Separation and Characterization of a Salt-Dependent Pectin Methylesterase from Citrus sinensis Var. Valencia Fruit Tissue

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.

Fermentation of orange peel hydrolysates by ethanologenic Escherichia coli : Effects of nutritional supplements

Orange peel, an abundant byproduct of the citrus processing industry, is converted to a mixture of glucose, galacturonic acid, fructose, arabinose, galactose, and xylose by hydrolysis with mixed pectinase and cellulase enzymes. All these sugars can be fermented to ethanol or ethanol and acetic acid by the recombinant bacteriumEscherichia coli KO11. The fermentation efficiency is improved by the addition of yeast extract, tryptone, mixed amino acids, corn steep liquor, or, by proteolytic digestion of endogenous proteins. Batch fermentations of supplemented peel hydrolysate containing 111 g/L of initial total sugars produced 35–38 g/L of ethanol in 48–72 h and a 75–85% yield.Orange peel, an abundant byproduct of the citrus processing industry, is converted to a mixture of glucose, galacturonic acid, fructose, arabinose, galactose, and xylose by hydrolysis with mixed pectinase and cellulase enzymes. All these sugars can be fermented to ethanol or ethanol and acetic acid by the recombinant bacterium Escherichia coli KO11. The fermentation efficiency is improved by the addition of yeast extract, tryptone, mixed amino acids, corn steep liquor, or by proteolytic digestion of endogenous proteins. Batch fermentations of supplemented peel hydrolysate containing 111 g/L of initial total sugars produced 35-38 g/L of ethanol in 48-72 h and a 75-85% yield.