Enyzme Stabilization and Rapid Methods for Citrus and Fruit Juice Quality

NON-TECHNICAL SUMMARY: Assuring the production and quality of US agricultural commodities, processed foods and beverages is vital to the country's security and market competitiveness. Fresh and processed foods need to be safe as well as nutritious and good tasting. Maximizing sensory attributes and nutritional value while retaining fresh-like quality and ensuring safety are requirements for all food processors eager to conquer diverse emerging markets. <P>

The goals of this project are (I) to carry out exploratory research on stabilization and activation of citrus and other food enzymes and (II) to develop new and improved methods for plant and food pathogen detection, quality assurance of food and beverage products. Pectic enzymes are used for viscosity reduction and yield increase in the fruit juice industry. Lipases are used in the production of natural flavors. Stabilization and reuse of enzymes has the potential to decrease production costs and increase productivity. The effects of high hydrostatic pressure (HHP) on enzyme activity will be characterized by applying HHP to pectic enzymes and lipases at different temperatrues. Faster and more accurate and automated quality methods are required in the food industry. <P>This research will focus on developing novel sensors, biosensors or rapid assays to replace the current assays for pectinesterase and oil content in juice. Physical, biochemical and electrochemical strategies will be used. We also expect to develop biosensors for indirect rapid detection of food pathogens. Citrus Huanglongbing (HLB)is one of the most threatening citrus diseases in the world and it is gravely affecting Florida's industry. Rapid in-field diagnosis of the disease can help reducing its spread. Knowing the changes in metabolites present in infected trees can help understanding the mechanisms of infection. In this research we will focus on identifying biomarkers for rapid detection of citrus HLB. <P>Based on these biomarkers, we expect to develop portable sensors or biosensors for rapid, in-field diagnosis of HLB. Outcomes. a) Improved understanding of the effects of HHP on enzyme catalysis and structure. b) Incorporation of research findings into two graduate courses taught by Dr. Reyes De Corcuera: Citrus Processing Technology and Food Kinetics. c) Quality assurance laboratories are expected to save time and improve product quality by implementing a faster PME activity method for fruit juices. d) A faster and more sensitive method to determine oil in juice is expected to reduce processing costs to citrus juice and oil processors by reducing assay time and providing feed-back process control and more accurate quality control. e) In-field determination of titratable acidity that citrus growers can readily and inexpensively adopt at harvesting and increase crop value. f) Rapid methods for Salmonella and E. coli O157:H7 detection in foods reduce assay time and minimize the likelyhood that contaminated or under processed foods reach the consumer, thus, minimizes foodborne disease outbreaks. g)In-field diagnosis of HLB is expected to help citrus growers mitigate the spread of this disease

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APPROACH: <BR>I Enzyme Stabilization. Thermal stability of the enzyme will be determined when incubated in the absence of substrate for various amounts of time at different temperature-pressure combinations. Activity will be assayed ex-situ after incubation using spectrophotometry, GC or HPLC. Once the range of temperatures and pressures under which the enzyme is stable has been determined, the enzyme will be exposed to the substrate and the activity will be measured. In-situ determination of enzyme activity will be done by absorbance and fluorescence spectroscopy will be used in a sapphire window HHP cell instrumented with a fiber optic spectrophotometer. Dr. Reyes De Corcuera (Food Engineering) will focus on enzyme kinetics and modeling, Dr Goodrich (Food Safety and Quality) will focus on process validation and Dr. Rouseff (Flavor Chemistry) on chemical analysis. <BR> II Bioelectrochemical Methods. Pectin esterase activity will be measured. A methanol biosensor will be fabricated by immobilizing alcohol oxidase in an electrochemically generated polymer as described elsewhere (Reyes-De-Corcuera et al., 2005a). The biosensor will be optimized and tested first on a model system with pectin solutions and pectin esterase from citrus and then on orange juice. Effects of electroactive interfering species will be assessed. Pectin esterase activity will be assayed at pH in the range of 7.0 to 8.5. Preliminary research on the development of ultramicrobiosensors to measure glucose and sucrose fluxes in citrus cells has been carried out. Glucose ultramicrobiosensors will be inserted in citrus vacuoles previously captured with a micropipette. Different concentrations of glucose will be added to the surroundings of the cell. Glucose uptake will be monitored continuously. Exploratory research will be carried out to determine the effects of oil concentration in orange juice (0 to 0.1% v/v oil) on its dielectric properties at frequencies from 1 to 2000 MHz. containing . All determinations will be compared to the conventional method. Drs. Reyes De Corcuera and Goodrich will work on the validation of the proposed prototype. A prototype sensor consisting of two platinum flags separated by a plastic divider has been built. A pH probe is placed adjacent to the cathode where hydroxide ions are produced. The dynamics of the prototype sensor will be characterized and compared to a conventional titration. The effects of hydrolysis potential and temperature will be characterized. <BR><BR>Metabolomics. Changes in metabolite profiles in 'Valencia' orange leaves caused by water stress, girdling and zinc deficiency will be characterized by HPLC-MS, GC-MS and CE and compared to those produced by HLB and other plant stresses (girdling, water stress etc). This work will be done by Drs. Brlanski, (Plant Pathology), Wang (Microbiology), and Reyes De Corcuera The Metabolome of Salmonella, E-coli O157:H7 and mixed cultures will be analyzed by HPLC-MS and GC-MS. Dr. Danyluk will supervise microbiology tasks