Stabilization of galactose oxidase by high hydrostatic pressure: Insights on the role of cavities size.

High hydrostatic pressure stabilized galactose oxidase (GaOx) at 70.0-80.0°C against thermal inactivation. The pseudo-first-order rate constant of inactivation kinact decreased by a factor of 8 at 80°C and by a factor of 44 at 72.5°C. The most pronounced effect of pressure was at the lowest studied temperature of 70.0°C with an activation volume of inactivation ΔV‡ of 78.8 cm3 mol-1. The optimal pressure against thermal inactivation was between 200 and 300 MPa. Unlike other enzymes, as temperature increased the ΔV‡ of inactivation decreased, and as pressure increased the activation energy of inactivation Eai increased. Combining the results for GaOx with earlier research on the pressure-induced stabilization of other enzymes suggests that ΔV‡ of inactivation correlates with the total molar volume of cavities larger than ~100 Å3 in enzyme monomers for enzymes near the optimal pH and whose thermal unfolding is not accompanied by oligomer dissociation.

Increased activity of alcohol oxidase at high hydrostatic pressure.

Alcohol oxidase (AOx) from P. pastoris has potential applications in the production of carbonyl compounds and for the detection and quantification of alcohols. However, AOx's poor stability and low activity have hindered its practical application. There are two fractions of AOx in P. pastoris with different thermal stability. High hydrostatic pressure (HHP) increased the activity of the labile (L) + resistant (R) combined fractions but not of the R fraction alone. The activity of the L + R fractions increased 2.4-fold at 160 MPa and 30 °C compared to the activity at 0.1 MPa. At higher temperatures, the increase in activity with pressure was greater due to the combined stabilization and activation effects. The reaction rate of the R fraction at 50 °C was 17.9 ± 3.6 or 17.7 ± 0.8 µM min-1 at 80 or 160 MPa, respectively, and was not significantly different from the activity of the L + R fractions under the same conditions (18.4 ± 2.7 µM min-1). The activation energy of the R fraction was not significantly different between 80 MPa (41.5 ± 10.5 kJ mol-1) and 160 MPa (43.8 ± 7.8 kJ mol-1). The combined increase in the stability of the R fraction at HHP enables the use of the enzyme at 50 °C with little loss of activity and an increased catalytic rate.

Pectinmethylesterase affects the flow behavior of orange pulp.

In the orange juice industry, pulp (ruptured juice sacs) is separated from the juice after extraction and pasteurized separately before blending back with juice or sold for other food applications. However, pulp is not always pasteurized immediately after extraction and the flow behavior is affected by endogenous pectinmethylesterase (PME). This is particularly important because of the high power required to pump orange pulp in industrial pasteurizers. This study characterized the effect of PME on the flow behavior of citrus pulp during storage at 4 °C using rotational rheometry and during pulp storage at 22 °C using capillary rheometry. PME activity was 0.011 ± 0.001 PEU for the pulp used in rotational rheometry and 0.030 ± 0.002 PEU for the pulp used in capillary rheometry. Rotational rheometry was used to assess the effect of PME on the Power Law parameters at shear rates ( γ ̇ ) below those that cause slip. There were no significant differences among storage time on the flow behavior index (n). However, the consistency coefficient (K) increased with storage time. Significant differences were found after 12-hr storage increasing from 120 ± 30 Pa·sn to 160 ± 15 Pa·sn (16.7%) after 24 hr. Capillary rheomtery was used to assess the effect of PME on the pressure drop in a flow system. Significant differences were found at or after 12-hr storage. The mean pressure drop increased by 34% after 24 hr, for a flow rate of 50 × 10-6 m3 /s (0.8 GPM) in an 8.9-m long (29.2 ft), 0.022-m (1-in) diameter pipe. PRACTICAL APPLICATION: Design and optimization of processing equipment and industrial handling systems of orange pulp require detailed knowledge of its rheological (flow) properties. Citrus pulp is rich in pectinmethylesterase, an enzyme that causes thickening of the product when not inactivated during pasteurization. Understanding how fast PME affects the flow properties of orange pulp is important for citrus processors to decide how long pulp can be stored before pasteurizing it.

FEAST of biosensors: Food, environmental and agricultural sensing technologies (FEAST) in North America.

We review the challenges and opportunities for biosensor research in North America aimed to accelerate translational research. We call for platform approaches based on: i) tools that can support interoperability between food, environment and agriculture, ii) open-source tools for analytics, iii) algorithms used for data and information arbitrage, and iv) use-inspired sensor design. We summarize select mobile devices and phone-based biosensors that couple analytical systems with biosensors for improving decision support. Over 100 biosensors developed by labs in North America were analyzed, including lab-based and portable devices. The results of this literature review show that nearly one quarter of the manuscripts focused on fundamental platform development or material characterization. Among the biosensors analyzed for food (post-harvest) or environmental applications, most devices were based on optical transduction (whether a lab assay or portable device). Most biosensors for agricultural applications were based on electrochemical transduction and few utilized a mobile platform. Presently, the FEAST of biosensors has produced a wealth of opportunity but faces a famine of actionable information without a platform for analytics.

Combined rotational and capillary rheomtery to determine slip coefficients and other rheological properties of orange pulp.

The characterization of the rheological properties of orange pulp under typical processing temperatures is needed for the design and optimization of orange pulp processing systems. The flow of orange pulp produced slip at shear rates at ∼1 to 5 s-1 . Rotational rheometry revealed that the flow behavior of orange pulp before slip occurrence followed the Power Law model for concentrations of ∼500 to 800 g/L at 4 to 80 °C. The consistency coefficient (K) ranged from 33 to 234 Pa·sn and the flow behavior index n ranged from 0.18 to 0.24. Both, K and n decreased with temperature. While K fitted well an Arrhenius-like model, n best fitted a linear model. As concentration increased K increased linearly, while n was not significantly (P > 0.05) affected. The flow without slip was calculated using the Power Law parameters from rotational rheometry and the wall shear stress (σw ) from capillary rheometry for the experimental flow rates. This allowed calculating the corrected slip coefficient ßc and obviated the need for pipes with multiple diameters. ßc decreased by one order of magnitude when temperature increased from 4 to 50 °C when σw was 0.1 kPa. The effect was exacerbated with increased flow rate. Similarly, ßc increased by about one order of magnitude when pulp concentration increased from ∼550 to 850 g/L at 80 °C. The increase in ßc with temperature indicated that the effect of temperature in the consistency of the bulk was different from its effect on the consistency of the liquid phase near the pipe wall. PRACTICAL APPLICATION: Design and optimization of processes equipment and industrial handling systems of orange pulp require detailed knowledge of their rheological (flow) properties. Citrus pulp like fruit pastes and purees produce less friction than one would anticipate when they flow because the liquid fraction acts as a lubricant. This study presents an original method for such characterization and shows that wall slip is greatly affected by temperature and concentration.

Continuous flow system for biofilm formation using controlled concentrations of Pseudomonas putida from chicken carcass and coupled to electrochemical impedance detection.

Most studies dealing with monitoring the dynamics of biofilm formation use microbial suspensions at high concentrations. These conditions do not always represent food or water distribution systems. A continuous flow system capable of controlling the concentration of the microbial suspension stream from 104 to 106 CFU ml-1 is reported. Pseudomonas putida biofilms formed using 100-fold, 1,000-fold or 10,000-fold diluted bacterial suspensions were monitored in-line by electrochemical impedance spectroscopy (EIS) and total plate counts. Randles equivalent circuit model and a modified Randles model with biofilm elements were used to fit the EIS data. In Randles equivalent circuit, the charge transfer resistance decreased as the biofilm formed. The log colony counts of the biofilm correlated to the charge transfer resistance. In the biofilm model, the biofilm resistance and the double layer capacitance decreased as the biofilm formed. The log colony counts of the biofilm correlated to the biofilm resistance.

Catalytic activity and stabilization of phenyl-modified glucose oxidase at high hydrostatic pressure.

Glucose oxidase (GOx) was modified by attaching phenyl groups to either carboxyl or amino side chains on the enzyme. High hydrostatic pressure (HHP) stabilized the aniline-, and benzoate-modified GOx at 69.1-80 °C compared to atmospheric pressure. At 240 MPa and 80.0 °C, the first order rate constant of inactivation kinact. of aniline-modified GOx was 20 × 10-2 min-1, or 3.7 times smaller than for the native GOx, while the kinact for benzoate-modified GOx was 26 × 10-2 min-1, or 2.8 times smaller than for the native GOx at the same temperature. Furthermore, at 240 MPa and 80.0 °C, the kinact of the aniline-modified GOx was 69 times smaller than the kinact of native GOx (1530 × 10-2 min-1) at 0.1 MPa and 80.0 °C. Similar results were obtained for benzoate-modified GOx. At each temperature in this study (25-69.1 °C), the catalytic activity of the native, aniline-, or benzoate-modified GOx increased with HHP, and reached a maximum at around 180 MPa. At 180 MPa and 69.1 °C, aniline-modified GOx produced the fastest catalytic rate, followed by benzoate-modified GOx, and then native GOx. An increase in temperature increased the activation volume of the reaction. Similarly, the activation energy increased with pressure. The combination of HHP and hydrophobic modification made GOx more thermostable and increased the effect of temperature in enzyme activity.

Increased thermal stability of a glucose oxidase biosensor under high hydrostatic pressure.

We report the effects of high hydrostatic pressure (HHP), immobilization in electrochemically generated poly-o-phenylenediamine nano-films, and reticulation with glutaraldehyde on the thermal stability of glucose oxidase (GOx). The pseudo-first-order rate constant of inactivation of immobilized GOx inactivated at 70 °C and atmospheric pressure was 20.6 times smaller than that of GOx in solution under the same conditions. Immobilized GOx inactivated at 70 °C and 180 MPa was 87.6 times more stable than GOx in solution inactivated at 70 °C and atmospheric pressure. However, applying high pressure during electropolymerization or cross-linking with glutaraldehyde only had minor influences on GOx thermal stability. The stabilizing effect of HHP was not retained upon depressurization.

GC-MS study of changes in polar/mid-polar and volatile compounds in Persian lime (Citrus latifolia) during fruit growth.

BACKGROUND: Citrus fruits possess a high content of bioactive compounds whose changes during fruit maturation have not been studied in depth. Fruits were sampled from week 1, after fruit onset (7 days after flowering), to week 14. Volatile compounds isolated by headspace-solid-phase microextraction and polar extracts from all samples were analyzed by gas chromatography-mass spectrometry. RESULTS: The relative abundance of 107 identified metabolites allowed differences among samples at different stages of fruit growth to be established. Principal component analysis showed a clear discrimination among samples, and analysis of variance revealed significant differences in 94 out of the 107 metabolites. Among total volatiles, monoterpenes increased their relative abundance from 86% to 94% during fruit growth, d-limonene, γ-terpinene and ß-pinene being the most abundant; conversely, sesquiterpenes decreased from 11.5% to 2.8%, ß-bisabolene and α-bergamotene being the most concentrated. Sugars, in general, exhibited a gradual increase in abundance, reaching a maximum between weeks 9 and 12. Citric and malic acids, representing approximately 90% of the total identified carboxylic acids, reached a maximum concentration at commercial maturity (week 14). CONCLUSION: Of the 107 tentatively identified metabolites during Persian lime growth, sugars, carboxylic acids, and volatiles were those that experienced more significant changes and more clearly created differences among fruit growth stages. © 2018 Society of Chemical Industry.

Stability and Stabilization of Enzyme Biosensors: The Key to Successful Application and Commercialization.

Fifty-five years have passed and more than 100,000 articles have been published since the first report of an electrochemical enzyme biosensor. However, very few biosensors have reached practical application and commercialization. The bulk of the research effort has been on increasing sensitivity and selectivity. In contrast, the number of publications dealing with stability or stabilization of enzyme biosensors is very small. Here, we critically review enzyme stabilization strategies as well as the progress that has been done in the past 20 years with respect to enzyme biosensor stabilization. Glucose oxidase, lactate oxidase, alcohol oxidase, and xanthine oxidase are the focus of this review because of their potential applications in food. The inconsistency in reporting biosensor stability was identified as a critical hurdle to research progress in this area. Fundamental questions that remain unanswered are outlined.

Effects of high hydrostatic pressure or hydrophobic modification on thermal stability of xanthine oxidase.

The effect of high hydrostatic pressure (HHP) on the kinetics of thermal inactivation of xanthine oxidase (XOx) from bovine milk was studied. Inactivation of XOx followed pseudo-first-order kinetics at 0.1-300MPa and 55.0-70.0°C. High pressure up to at least 300MPa stabilized XOx at all the studied temperatures. The highest stabilization effect of HHP on XOx was at 200-300MPa at 55.0 and 58.6°C, and at 250-300MPa at 62.3-70.0°C. The stability of XOx increased 9.5 times at 300MPa and 70.0°C compared to atmospheric pressure at the same temperature. The activation energy of inactivation of XOx decreased with pressure and was 1.9 times less at 300MPa (97.0±8.2kJmol-1) than at 0.1MPa (181.7±12.1kJmol-1). High pressure decreased the dependence of the rate constant of inactivation to temperature effects compared to atmospheric pressure. The stabilizing effect of HHP on XOx was highest at 70.0°C where the activation volume of inactivation of XOx was 28.9±2.9cm3mol-1. A second approach to try to increase XOx stability involved hydrophobic modification using aniline or benzoate. However, the thermal stability of XOx remained unaffected after 8-14 modifications of carboxyl side groups per XOx monomer with aniline, or 12-17 modifications of amino side groups per XOx monomer with benzoate.

Glucose oxidase stabilization against thermal inactivation using high hydrostatic pressure and hydrophobic modification.

High hydrostatic pressure (HHP) stabilized glucose oxidase (GOx) against thermal inactivation. The apparent first-order kinetics of inactivation of GOx were investigated at 0.1-300 MPa and 58.8-80.0°C. At 240 MPa and 74.5°C, GOx inactivated at a rate 50 times slower than at atmospheric pressure at the same temperature. The apparent activation energy of inactivation at 300 MPa was 281.0 ± 17.4 kJ mol-1 or 1.3-fold smaller than for the inactivation at atmospheric pressure (378.1 ± 25.6 kJ mol-1 ). The stabilizing effect of HHP was greatest at 74.5°C, where the activation volume of 57.0 ± 12.0 cm3 mol-1 was highest compared to all other studied temperatures. Positive apparent activation volumes for all the treatment temperatures confirmed that HHP favors GOx stabilization. A second approach to increase GOx stability involved crosslinking with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and either aniline or benzoate. The modified enzyme remained fully active with only slight increases in KM (1.3-1.9-fold increases for aniline and benzoate modification, respectively). The thermal stability of GOx increased by 8°C with aniline modification, while it decreased by 0.9°C upon modification with benzoate. Biotechnol. Bioeng. 2017;114: 516-525. © 2016 Wiley Periodicals, Inc.

Nonthermal inactivation of soy (Glycine max Sp.) lipoxygenase by pulsed ultraviolet light.

This study investigated pulsed ultraviolet (PUV) illumination at different distances from the PUV source on soybean lipoxygenase (LOX) (0.4 mg/mL in 0.01 M Tris-HCl buffer, pH 9) activity. Samples (5 mL) were illuminated for 1, 2, 4, 8, and 16 s at 3 distances 6, 8.5, and 11 cm from the PUV lamp's quartz window. The temperature of 33.5 ± 1.8°C was observed for the highest treatment time of 16 s at the shortest distance of 6 cm, and resulted in a 3.5 log reduction (99.95%) in initial LOX activity. Illumination time and distance from the lamp significantly (P ≤ 0.05) affected LOX inactivation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on treated LOX samples and further protein profile for treated LOX filtrate (≤10 kDa), was analyzed by reverse phase high-performance liquid chromatography (RP-HPLC). The protein profile analysis revealed that LOX protein degradation was influenced significantly (P ≤ 0.05) by PUV illumination time.

An HPLC-MS characterization of the changes in sweet orange leaf metabolite profile following infection by the bacterial pathogen Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) presumably caused by Candidatus Liberibacter asiaticus (CLas) threatens the commercial U.S. citrus crop of an annual value of $3 billion. The earliest shift in metabolite profiles of leaves from greenhouse-grown sweet orange trees infected with Clas, and of healthy leaves, was characterized by HPLC-MS concurrently with PCR testing for the presence of Clas bacteria and observation of disease symptoms. Twenty, 8-month-old 'Valencia' and 'Hamlin' trees were grafted with budwood from PCR-positive HLB source trees. Five graft-inoculated trees of each variety and three control trees were sampled biweekly and analyzed by HPLC-MS and PCR. Thirteen weeks after inoculation, Clas was detected in newly growing flushes in 33% and 55% of the inoculated 'Hamlin' and 'Valencia' trees, respectively. Inoculated trees remained asymptomatic in the first 20 weeks, but developed symptoms 30 weeks after grafting. No significant differences in the leaf metabolite profiles were detected in Clas-infected trees 23 weeks after inoculation. However, 27 weeks after inoculation, differences in metabolite profiles between control leaves and those of Clas-infected trees were evident. Affected compounds were identified with authentic standards or structurally classified by their UV and mass spectra. Included among these compounds are flavonoid glycosides, polymethoxylated flavones, and hydroxycinnamates. Four structurally related hydroxycinnamate compounds increased more than 10-fold in leaves from 'Hamlin' and 'Valencia' sweet orange trees in response to Clas infection. Possible roles of these hydroxycinnamates as plant defense compounds against the Clas infection are discussed.

Further studies on the role of water in R67 dihydrofolate reductase.

Previous osmotic pressure studies of two nonhomologous dihydrofolate reductase (DHFR) enzymes found tighter binding of the nicotinamide adenine dinucleotide phosphate cofactor upon addition of neutral osmolytes. This result is consistent with water release accompanying binding. In contrast, osmotic stress studies found weaker binding of the dihydrofolate (DHF) substrate for both type I and type II DHFRs in the presence of osmolytes; this observation can be explained if dihydrofolate interacts with osmolytes and shifts the equilibrium from the enzyme-bound state toward the unbound substrate. Nuclear magnetic resonance experiments support this hypothesis, finding that osmolytes interact with dihydrofolate. To consider binding without added osmolytes, a high-pressure approach was used. In this study, the type II enzyme, R67 DHFR, was subjected to high hydrostatic pressure (HHP). Both enzyme activity and fluorescence measurements find the protein tolerates pressures up to 200 MPa. Binding of the cofactor to R67 DHFR weakens with increasing pressure, and a positive association volume of 11.4 ± 0.5 cm(3)/mol was measured. Additionally, an activation volume of 3.3 ± 0.5 cm(3)/mol describing k(cat)/K(m(DHF)) was determined from progress curve analysis. Results from these HHP experiments suggest water release accompanies binding of both the cofactor and DHF to R67 DHFR. In an additional set of experiments, isothermal titration calorimetry studies in H2O and D2O find that water reorganization dominates the enthalpy associated with binding of DHF to R67 DHFR·NADP(+), while no obvious effects occur for cofactor binding. The combined results indicate that water plays an active role in ligand binding to R67 DHFR.

Aroma characterization of tangerine hybrids by gas-chromatography-olfactometry and sensory evaluation.

BACKGROUND: Tangerines have a distinct flavor among citrus fruit. However, information on tangerine volatiles remains limited. Volatile compounds from a breeding population of tangerines were earlier identified by gas chromatography-mass spectrometry. In this study, five hybrids with a distinct volatile profile were analyzed by gas-chromatography-olfactometry (GC-O) and descriptive sensory analysis. RESULTS: Forty-nine aroma active compounds were found in a consensus by GC-O. Aldehydes were the most important group with odor activity, as well as monoterpenes, esters, alcohols and ketones. 1,8-Cineole, ß-myrcene, (E,E)-2,4-nonadienal, hexanal, ethyl-2-methylbutanoate, and linalool were perceived with high intensity in most samples. Two 'Clementine' × 'Minneola' and one 'Fortune' × 'Murcott' hybrids with tangerine, sulfury and woody/spicy flavors had aroma active compounds with terpeney, fatty/vegetable and metallic/rubber descriptors. A tangerine with 'Valencia' orange in its parentage had a characteristic orange flavor, which could be explained by esters and ketones, high in fruity and floral odor intensities. A hybrid of unknown origin had a distinct fruity-non-citrus and pumpkin/fatty flavor; that sample had the lowest amount of aroma-active volatiles, with the least compounds with terpeney odors. CONCLUSION: There was no one compound characteristic of tangerine flavor. Nevertheless, each sample sensory characteristic could be explained by a set of aroma-active volatile compounds.

GC-MS metabolomic differentiation of selected citrus varieties with different sensitivity to citrus huanglongbing.

Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The rapid identification of tolerant varieties is considered a critical step towards controlling HLB. GC-MS metabolite profiles were used to differentiate HLB-tolerant citrus varieties 'Poncirus trifoliata' (TR) and 'Carrizo citrange' (CAR) from HLB-sensitive varieties 'Madam Vinous sweet orange' (MV) and 'Duncan' grapefruit (DG). PCR analyses revealed that MV was the most sensitive variety followed by DG and the tolerant varieties CAR and TR. Metabolomic multivariate analysis allowed classification of the cultivars in apparent agreement with PCR results. Higher levels of the amino acids l-proline, l-serine, and l-aspartic acid, as well as the organic acids butanedioic and tetradecanoic acid, and accumulation of galactose in healthy plants were characteristic of the most sensitive variety MV when compared to all other varieties. Only galactose was significantly higher in DG when compared to the tolerant varieties TR and CAR. The tolerant varieties showed higher levels of l-glycine and mannose when compared to sensitive varieties MV and DG. Profiling of the sensitive varieties MV and DG over a 20-week period after inoculation of those with the HLB-containing material revealed strong responses of metabolites to HLB infection that differed from the response of the tolerant varieties. Significant changes of l-threonine level in the leaves from old mature flushes and l-serine, l-threonine, scyllo-inositol, hexadecanoic acid, and mannose in the leaves from young developing flushes were observed in MV. Significant changes in myo-inositol in old flushes and l-proline, indole, and xylose in new flushes were observed in DG.

GC-MS based metabolomics for rapid simultaneous detection of Escherichia coli O157:H7, Salmonella Typhimurium, Salmonella Muenchen, and Salmonella Hartford in ground beef and chicken.

A metabolomic-based method for rapid detection of Escherichia coli O157:H7, Salmonella Hartford, Salmonella Typhimurium, and Salmonella Muenchen in nonselective media was developed. All pathogenic bacteria were grown in tryptic soy broth (TSB) at 37 °C followed by metabolite quantification at 2-h intervals for 24 h. Results were compared with the metabolite profiles similarly obtained with E. coli K12, Pseudomonas aeruginosa, Staphylococcus aureus, Saccharomyces cereviseae, and Aspergillus oryzae grown individually or as a cocktail under the same conditions. Principal component analysis (PCAS) discriminated pathogenic microorganisms grown in TSB. Metabolites responsible of PCAS classification were dextrose, cadaverine, the aminoacids L-histidine, glycine, and L-tyrosine, as well as the volatiles 1-octanol, 1-propanol, 1butanol, 2-ethyl-1-hexanol, and 2,5-dimethyl-pyrazine. Partial least square (PLS) models based on the overall metabolite profile of each bacteria were able to detect the presence of Escherichia coli O157:H7 and Salmonella spp. at levels of approximately 7 ± 2 CFU/25 g of ground beef and chicken within 18 h.

Kinetics of lipase catalyzed isoamyl acetate synthesis at high hydrostatic pressure in hexane.

Lipase-catalyzed synthesis of isoamyl acetate in hexane at 10-250 MPa at 80 degrees C and 1-100 MPa at 40 degrees C resulted in activation volumes of -12.9 +/- 1.7 and -21.6 +/- 2.9 cm(3) mol(-1), respectively. Increasing pressure from 10 to 200 MPa resulted in approximately 10-fold increase in V(max) at both 40 and 80 degrees C. Pressure increased the K(m) from 2.4 +/- 0.004 to 38 +/- 0.78 mM at 40 degrees C. In contrast, at 80 degrees C the pressure did not affect the K(m).

Untargeted metabolite analysis of healthy and Huanglongbing-infected orange leaves by CE-DAD.

Huanglongbing (HLB) is considered the most destructive bacterial citrus disease worldwide. Early detection of HLB is crucial for minimizing its spread. CE was used for the discovery of potential biomarkers for HLB. Optimization of extraction and separation allowed resolving 24 compounds of which 6 were present in significantly higher (p<0.05) concentrations in HLB-infected samples collected monthly for 6 months during the 2007-2008 season. Three of these compounds were identified by mobility and UV spectra as hesperidin, naringenin, and quercetin with mean increase in concentration of 154, 555, and 467%, respectively, above that in healthy leaves. Results support the potential of CE-DAD for untargeted plant metabolomic analysis. CZE, NACE, and MEEKC were compared for metabolic differentiation of healthy and HLB-infected citrus leaves. CZE in a semi-aqueous BGE solution consisting of 8.5 mM of sodium borate (pH 9.3), 15% ACN, and 9% 1-butanol yielded the best peak separation with detection at 190 nm.