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Rapid assessment of food safety via novel at-line measurements

Deliverables

The result: The overall product is a simple, low-cost batch of methods for the determination of chlorophenol and chloroanisole compounds in liquid food matrices. The product has been developed from the point of providing measurement solutions end-users in the wine industry, where chlorophenol and chloroanisole contamination of wine products is a particular problem. However, the solutions developed are wider than this example scenario, in that the product may also serve as a measurement tool for assessing target analyte levels in other liquidic food products, such as fruit juices and potable water, and with further development, any foodstuff that can be applied to the assay in liquidic form. The assay procedure is compatible with food processing operations that have small, simple laboratories attached, as is the case in, for example, the wine industry. The assay is split into 2 distinct phases: sample preparation and immunochemical analysis, using the well-known technique of Enzyme Linked Immunosorbent Assay (ELISA). Sample preparation methods have been developed for both the chlorophenols and chloroanisoles based on either simple solvent extraction procedures, or by the use of simple stream distillation processes coupled to solid-phase extraction. These methods are simple to apply, requiring low-cost instrumentation and minimal operator training. Such sample preparation procedures are required to simplify the food matrix (wines for example are extremely complex materials) and to pre-concentrate the target analytes sufficiently to allow their subsequent detection by the ELISA method. Chlorophenols can impart unpalatable tastes and odours to wines at low part per million/upper part per trillion levels, whilst chloroanisoles have even lower sensory thresholds. Sample preparation methods have been developed for wines, bottle washings, packaging materials and corks, thereby offering the end-user means of conducting traceability studies to determine the source of the chlorophenol/ chloroanisole contamination. The ELISA process itself is simple to perform and is amenable to multi-samples throughput the process is reliant on only very cheap, simple readily available instrumentation and can be performed by an operator with minimal training. Results can be readily interpreted allowing appropriate remedial processes to be immediately implemented. The benefit of the process over existing methods resides in cost, simplicity and the fact that the assay may be performed in simple, low-level equipped laboratories. The method is complimentary to the current GC-MS methods, in that the immunochemical systems can be used for screening purposes allowing data to be rapidly obtained. Suspect samples may be sent to a centralised facility for confirmatory analysis using validated standard instrumentation methods.
The result: The product offered here is a raft of sample preparation protocols that can be used for the preparation of liquidic samples with respect to the ubiquitous environmental contaminant chlorophenol (the chlorophenol family consists of a number of congeners, based on the number and positioning of chlorine atoms around the central phenolic structure) and its methylated group of sister compounds, the chloroanisoles. Chlorophenols arise from a number of sources, including their usage as fungicides, as wood preservatives and due to the conversion of phenols by chlorine-treated water supplies. Chlorophenols are considered toxic at high concentrations, and impart unpalatable tastes and odours to food products at even very low concentrations. Chloroanisoles arise from the fungal methylation of chlorophenols and, again as extremely unpalatable compounds, present a particular problem to the wine industry. Foodsafe has developed a number of high quality sample preparation methods that require very simple protocols and low-cost instrumentation to allow even low-level equipped laboratories to perform sample extraction procedures on a routine basis with minimally trained personnel. The aim of the sample preparation methods are to reduce the complexity of the sample matrix with respect to potential interferents and to pre-concentrate the target analytes to allow ultra-low levels of detection, typically in the part per trillion range with respect to the chlorophenol contaminants.
Chloroanisole contaminants in cork that give rise to the characteristic "corked” wine taint are largely associated with contamination by 2,4,6,trichlorophenol (TCA). In mature wines there is considerable evidence that TCA contamination arises largely from chlorophenolic contaminants in cork. Chloroanisoles are known to be produced by the methylation of chlorophenols by chemical and microbiological processes on storage. These chemical changes are very slow and it may take many years for the impact of chlorophenol contaminantion to be experienced in the final product. In the wine industry cork taint has been shown to be a major problem and is a significant contributor to production and sales losses due to contaminated wine. In view of this cork production methods will require significant improvements to production and quality control procedures to enable the production of taint free material that is acceptable in a modern competitive marketplace. Methods have been developed in Foodsafe to allow the determination of the chlorophenol and chloroanisole content of cork samples. The developed procedures are suitable for the analysis of chlorophenol contaminants in a laboratory location where the use and disposal of small quantities of flammable solvents is not permitted. Two sample extraction procedures are offered, both compatible with the use of the high sensitivity ELISA procedure developed under Foodsafe: Extraction method 1: PRESCREENING METHOD FOR THE EXTRACTION OF CHLOROPHENOLIC CONTAMINANTS FROM CORK SAMPLES, in which a large number of cork samples are cut and ground and amalgamated to give an overall screen with respect to chlorophenol levels. Extraction method 2: QUALITY CONTROL OF CHLOROPHENOLIC CONTAMINANTS IN CORK SAMPLES in which a single cork sample is extracted without ant cutting. In both cases, samples of cork are extracted using alkaline detergent and purified using steam distillation into dilute sodium carbonate. The pH-adjusted extracts are then tested for chlorophenols using the FOODSAFE- high sensitivity ELISA procedure. FOODSAFE- high sensitivity ELISA procedure: This ELISA assay is based on competitive antibody binding between the antigen (PCP) and a synthetic antigen-enzyme conjugate produced by chemically bonding horseradish peroxidase to a tetrachloro-hydroxy-phenyl (HRP-conjugate) moiety. The retained HRP conjugate is determined using the fluorescent substrate Pierce, QuantaBlu (TM) with fluorescence detection.
Results: MARKET STUDY REPORT on wine sector: The main objective of the survey was to know the appraisals and demands of potential end-users of immunosensor devices as tools for the rapid at-line assessment of the toxicity of components (chlorophenols and chloroanisoles) within the process supply chain. The winemaking sector was chosen as representative of the foodstuffs industry, since it would be one of the main recipients of the biosensor. This market study includes the following topics: business outlook of food diagnostic, immunoassay technologies, and wine sector situation. Regarding the potential end-user of immunodiagnostic tools developed in this project the market study includes also the innovation strategy of wine industries, diagnostic of control equipment in wineries, appraisal of development of sensors in wine sector, description of end-user requirements with respect to the operational capabilities of immunodiagnostic devices to determinate chlorophenols and chloroanisols and the predisposition towards purchase of the immunodiagnostic.
The methodology offered here are simple preparation protocols that can be used for the preparation of wine and cork samples before determination of chlorophenols and chloroansioles by gas chromatography. The aim of the simple preparation methods is to extraction and concentration of chlorophenols and chloroanisoles in wine/corks simples to allow ultra-low levels of detection, typically in the part per trillion range. CHLOROPHENOLS, particularly commercial preparations of pentachlorophenol (PCP) have been used as general biocides and wood preservatives for more than 50 years and are presently widespread in the environment. In addition to industrial production and usage, chlorophenols are produced from naturally occurring phenols as a result of chlorine bleaching of wood pulp in the paper industry and through the chlorination of domestic water supplies and swimming pools. CHLOROANISOLES are not used industrially, but it has been established that they are formed from the chorophenols due to fungal methylation. Many Aspergillus and Penicillium moulds are capable of methylating chlorophenols. Food contamination by chlorophenols and chloroanisoles has been well reported and documented. These compounds have been found in a multitude of food products: wine, canned carbonated beverages, reconstituted fruit juice, poultry, bulk chocolate, modified flour, canned fruit, cocoa powder, packaged biscuits, chilled meat and so on. Contamination with chlorophenols and chloroanisoles at concentrations below 10-3mg/kg is of toxicological
The results: This study includes the identification of critical points within the wine processing operation with regard to the introduction, processing and fate of the hazardous fungicide residue targets. The identification of critical control points where exits a chemical contamination hazards (appearance and presence of chlorophenols and/or chloroanisoles) have been determinate in conjunction with appropriate preventive to minimise or eliminate this hazard. Moreover, contains a "Good Manufacturing Practices", recommendation for the wine industry in order to minimise or eliminate the hazard of presence of toxic compounds in processing operation and to have traceability of foreign materials in wineries. These recommendations could be applied at different levels: environment and facilities; production (raw materials, equipment, winemaking, packaging operations) and storage and distribution. The recommendations will improve the safety and hygiene aspects of wine production, reduce the risk and minimise the number and extent of exposures to hazards (as the contamination by chlorophenols and chloroanisoles), thereby increasing the overall safety of wines for human consumption. The adoption of Good Manufacturing practice will facilitate the development and introduction of quality programs, such as Hazard Analysis Critical Control Point (HACCP) based food safety programs. Such an approach is expected to allow the evaluation of contaminants in raw materials prior to their integration into the manufacturing process, aiding the traceability of toxic compounds within the overall operation.
Instrumentation development for a "FOODSAFE ANALYSER" was initiated by creating a block diagram incorporating the significant analogue and digital requirements of the instrumentation. To ensure the maximum speed and performance of the microprocessor the assembly language was chosen for the software development. For the same reasons, the direct memory access mode (DMA) microprocessor ADC operation was used. In order to broaden the functional characteristics of the instrument there are two algorithms of working signal generation were implemented: staircase, and differential pulse techniques. For the purposes of visualisation of electrochemical measurements and their comparison with the measurement from standard industrial equipment the PC software using Delphi 6 programming system was developed. Development of amperometric measurement mode and integration prototype of stand alone instrument with microfluidic system. As a part of instrumentation activities the amperometric measurement mode and means for integration with immunosensor microfluidic system has been introduced into developing prototype of the standalone instrument "FOODSAFE ANALYSER". This enhancement of instrumentation capabilities demanded of hardware modernisation (microcontroller port extending, using additional microcontroller ADC input and DAC output) and development of special software. PRELIMINARY TESTING OF SYSTEM WITH ELECTROCHEMICAL IMMUNOSENSOR DEVICE Amperometric electrochemical data sets involving carbon electrodes were used to define suitable signal processing algorithms, which will be of generic interest to biosensors based on amperometric measurements, NMRC will continue verification of these methods as further amperometric data sets become available.
The development of specific immunochemical methods that offer high sensitivity, high sample throughput whilst being low cost and simple to operate will aid traceability of toxic agents and hence reduce the level of toxic components within processed food products. The applicability of this approach was assessed using toxic chlorophenolic fungicides and their chloroanisole breakdown products that were oxic contaminants of many food products at the part per billion levels. The present study focused on the development of monoclonal antibodies against penta-chlorophenol(PCP) and penta-chloroanisole (PCA) and its application to immunoassay procedures in the wine industry. The project was specifically target wine, fruit juice and potable water food production processes since the target compounds were commonly identified in such matrices due to fungicide treatment of raw materials. Monoclonal antibodies were produced using established biological techniques at Diaclone. Chlorophenol (PCP) and chloroanisole (PCA) fungicide derivatives were conjugated to carrier proteins. Immunogens PCP-KLH and PCA-BSA were injected into mice. Monoclonal antibodies were obtained against PCP called B-L36 and B-P31 and against PCA named B-A46, B-G40 and B-R28. Using the mAb B-L36, an indirect competitive enzyme immunoassay (IEA) was developed for measurement of PCP. During preliminary studies, false positive diagnosis due to interference within the complex wine samples was obtained. To improve the correlation between gas chromatography (GC) and ELISA methods, the development of a highly efficient selective sample purification procedure to remove unrelated background matrix components has been reported.