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Part II Goa University M - 3908 QUALITY CONTROL OF FOOD : QUALITY CONTROL OF FOOD In general, the quality control is the maintenance of quality at levels and tolerance acceptable to the buyer, minimizing the cost for the vender. But, from the scientific angle, the overall quality refers to technological, physical, chemical, microbiological, nutritional and sensory parameters to achieve the wholesome food. Microbiological Contamination : Microbiological Contamination It has been stated that microbiological contaminated food is perhaps the most prevalent health problem in the contemporary world. For safe food microbiological criteria should be established and freedom from pathogenic microorganisms must be ensured, including the raw materials, ingredients and finished products at any stage of production/processing. Accordingly the microbiological examination of the foods products has to be adopted widely. The microbiological criteria must be applied to define the distinction between acceptable and unacceptable foods. Consuming old, used, residual, fermented, spoiled, and contaminated, toxic and bacterial infested food causes food poisoning Why newer methods? : Why newer methods? Traditional microbiological detection and identification methods for food borne pathogens are time consuming and laborious to perform, and are increasingly unable to meet the demands for rapid food testing Rapid method is generally characterized as a test giving quicker results than the standard accepted method of isolation and biochemical and/or serological identification. IMS or DMS : IMS or DMS Ion mobility spectrometry (IMS) differential mobility spectrometers(DMS) Developed in the 1970th, ion mobility spectrometry has at first mainly been used for military purpose, e.g. the detection of chemical warfare agents, explosives and illegal drugs. Slide 6: IMS or DMS (cont…) : IMS or DMS (cont…) A drift gas (mostly synthetic air or nitrogen) is introduced via the drift region into the ionisation chamber of an ion mobility spectrometer most commonly by a radioactive source (e.g.63Ni). Those ions are called reactant ions. When analyte molecules enter the ionisation chamber, the reactant ions transfer their charge to the analyte to form ions, thus resulting in a decrease of the reactant ion peak and an increase of one or more analyte ion peaks. The ions are moved in a weak external electrical field in direction towards the detector (Faraday-plate) and normally against a drift gas flow in the opposite direction. The ions collide with the drift gas molecules depending on their shape, charge and molecular weight, thereby leading to a constant resulting velocity. By this reason, they reach the detector in the ideal case totally separated depending on their mobility in the available drift gas and generate ion spectra characteristic for the analyte. This enables the identification of the analytes and—using the signal area as a measure—their quantification by comparison with a calibration carried out earlier. IMS or DMS (cont…) : IMS or DMS (cont…) The method enables the identification and quantification of analytes with high sensitivity (ngl-1 range). The selectivity can even be increased - as necessary for the analyses of complex mixtures - using pre-separation techniques such as gas chromatography or multi-capillary columns (MCC). No pre-concentration of the sample is necessary. Those characteristics of the method are preserved even in air with up to a 100% relative humidity rate Slide 9: The peaks are well known and described in detail. Concentration profiles are available and stored in data bases. A comparison of a present measurement with the spectra stored deliver the nature of the analyte and the actual concentration. Slide 10: Various applications - in breath analysis, food control, medical applications and the detection of biomarkers or metabolites, detection of traces of substances in water and process analysis in general become more and more common. (MCC-IMS) is described for the detection of volatile organic compounds from microbes and the MCC-IMS is also used for characterization of metabolic activity of growing Escherichia coli. Slide 11: The method is suitable for application in the field of food quality and safety -including storage, process and quality control as well as the characterization of food stuffs Electronic-nose : Electronic-nose “An e-nose is an instrument which comprises an array of electronic chemical sensors with partial specificity or broad-band chemical selectivities and an appropriate pattern recognition system, capable of recognizing simple or complex odours.” (Gardner and Bartlett’s 1994) This feature is the key property on which electronic noses found their working principle. Their main application concerns the classification, discrimination and recognition of chemical patterns occurring in various kinds of samples. Typical examples are found in food analysis, in which, often, foods are classified according to categories such as freshness and edibility. The basic principle of an electronic nose is that the chemical patterns occurring in a certain environment are ‘translated’ by the sensors into a response pattern, whose features are linked to the selectivities of each sensor; generally, the response pattern is a nonlinear combination of all the components forming the chemical pattern. The Electronic NOSE (Neotronics Olfactory Sensing Equipment) It mimics the human olfactory sensory system Slide 13: Photo of the electronic nose having gas sensors. Computer Controlled Electronic Nose System Slide 15: Electronic Noses include three major parts: a sample delivery system, a detection system, a computing system. The sample delivery system enables the generation of the headspace (volatile compounds) of a sample, which is the fraction analyzed. The system then injects this headspace into the detection system of the electronic nose. The detection system, which consists of a sensor set, is the “reactive” part of the instrument. When in contact with volatile compounds, the sensors react, which means they experience a change of electrical properties. Each sensor is sensitive to all volatile molecules but each in their specific way. Most electronic noses use sensor arrays that react to volatile compounds on contact: the adsorption of volatile compounds on the sensor surface causes a physical change of the sensor. A specific response is recorded by the electronic interface transforming the signal into a digital value. Recorded data are then computed based on statistical models. The more commonly used sensors include metal oxide semiconductors (MOS), conducting polymers (CP), quartz crystal microbalance, surface acoustic wave (SAW), and field effect transistors (MOSFET). In recent years, other types of electronic noses have been developed that utilize mass spectrometry or ultra fast gas chromatography as a detection system. The computing system works to combine the responses of all of the sensors, which represents the input for the data treatment. This part of the instrument performs global fingerprint analysis and provides results and representations that can be easily interpreted. Analysis techniques : Analysis techniques A number of analysis techniques could be used for the application, for example graphical representation of the individual sensor outputs with time; polar plots or spider plots; statistical techniques; offset polar or difference plots; neural networks. The electronic nose results can be correlated to those obtained from other techniques ( GC, GC/MS). Applications : Applications The electronic nose finds wide applications in the food industry. It is used to detect the bacterial growth on foods such as meat and fresh vegetables. It can be used to test the freshness of fish. It is used in the process control of cheese, sausage, beer, and bread manufacture. Milk and dairy products to detect off-flavors. Other applications include Identification of spilled chemicals. Quality classification of stored grain for aroma (also aroma of toxin or spores like Aspergillus). Identification of source and quality of coffee, Monitoring of roasting process, and so on. Applications : Applications In R&D laboratories for: Formulation or reformulation of products Benchmarking with competitive products Shelf life and stability studies Selection of raw materials Packaging interaction effects Simplification of consumer preference test In Quality Control laboratories for: Conformity of raw materials, intermediate and final products Batch to batch consistency Detection of contamination, spoilage, adulteration Origin or vendor selection Monitoring of storage conditions. In process and production departments for: Managing raw material variability Comparison with a reference product Measurement and comparison of the effects of manufacturing process on products Following-up cleaning in place process efficiency Scale-up monitoring Cleaning in place monitoring. Advantages : Advantages The advantages of the electronic nose can be attributed to its rapidity, objectivity, versatility, non requirement for the sample to be pretreatment, easy to use etc. Immunochemical methods : Immunochemical methods First, the antibodies themselves are highly specific for the antigen (analyte), and second, either the antigen, the antibody, or an antiglobulin may be conjugated to an enzyme that produces an intensely colored or fluorescent product in the presence of the enzyme substrate to enhance the detectability of the analyte in an amplification step. ELISA test have also been applied to detect the presence of toxin produced by staphylococcus (SEA-SEE) in less than 0.5mg/100g sample. Also other toxins of E.coli, Clostridium, Salmonella, Shiga – toxin Spectinomycin is an antibiotic isolated from the fermentation broth of Streptomyces spectabilis. Mason et al. (1961) Spectinomycin is labeled for use as a feed additive in the poultry industry; however, significant “off-label” use occurs in the treatment of cattle for bovine respiratory disease (shipping fever) and consequently spectinomycin enters the food supply in an unregulated manner. . EIA (Enzyme Immunoassay) : EIA (Enzyme Immunoassay) Microorganisms are often characterized and identified by the presence of unique protein carbohydrate markers (antigens) located within the body or the flagella of the cell. Detection of these unique antigens has been a cornerstone of diagnostic microbiology for many years. In recent years, EIA using monoclonal antibodies have made available rapid and consistent microbiological detection systems. The most widely used systems employ a sandwich technique using antibody attached to a polystyrene matrix to which the sample is added. Post incubation, a second antibody, which is specific for the organism and has been tagged with an enzyme, is added. The addition of enzyme substrate to the mixture completes the EIA. The presence of the specific organism results in a colorimetric change in the enzyme substrate, which may be observed visually or with a spectrophotometer. Most EIA are very specific but lack sensitivity. Normal sensitivity is in the range of 106 org/ml. Biosensors: Applications for Dairy Food Industry : Biosensors: Applications for Dairy Food Industry Biosensors are defined as indicators of biological compounds that can be as simple as temperature-sensitive point or as complex as DNA-RNA probes Biosensors provide sensitive, miniaturized systems that can be used to detect unwanted microbial activity or the presence of a biologically active compound, such as glucose or a pesticide in food. Immunodiagnostics and enzyme biosensors are two of the leading technologies that have had the greatest impact on the food industry. Slide 24: Two of the most commonly used definitions of biosensor are given: 1. A biosensor may be a device or instrument comprising a biological sensing element coupled to a transducer . According to this, examples of biological sensing elements include enzymes, organelles, antibodies, whole cells, DNA, and tissue. 2. A biosensor may be a self-contained analytical system that responds directly and selectively to biologically important species that is, a device or system that detects a biological event. Clearly, the term biosensor is used in diverse ways, but, generally, a biosensor should respond selectively, continuously, rapidly, specifically, and ideally without added reagent to biological events. Enzyme biosensors may respond as quickly as 30s, but 15 min is acceptable for immunosensors. All methods have the same objective, to reduce the time required for the detection of a biological event. TYPES OF BIOSENSORSNucleic Acid Probes : TYPES OF BIOSENSORSNucleic Acid Probes The principle of selective detection is based on the detection of a unique sequence of nucleic acid bases through hybridization A DNA probe is added to DNA or RNA from an unknown sample. If the probe hybridizes (combines) with the unknown nucleic acid because of pairing of complementary base recognition, detection and identification are possible. This hybridization is usually visualized through color development combining enzyme immunoassay (EIA) technology with the biosystem. Commercially, biosensing nucleic acid probes exist for the detection of Salmonella,Listeria, Escherichia coli, and Staphylococcus aureus. Slide 26: Conductance Bioluminescence Enzyme Sensors- potentiometric, amperometric, electrochemical, optoelectric, calorimetric, and piezoelectric Basically, all enzyme sensors work by immobilization of the enzyme system onto a transducer. As the sensor comes in contact with the substrate, the resultant by-product of the enzymatic reaction produces an electric signal, which is measured by the transducer. Enzyme-based biosensor with penicillinase immobilized onto the surface of a pH-sensitive transistor is able to detect penicillin in milk as it flows from truck to dairy. Immunosensors: Biosensors using antibodies as receptors are often referred to as immunosensors. Microbial Sensors Microbial sensors are defined as microorganisms that are associated with a transducer. Electrochemical microbial sensors usually detect respiratory activity of the microorganisms. E.g. The ammonia gas sensor which sensor uses nitrifying bacteria as the detector immobilized on an oxygen electrode. As the bacteria oxidize the ammonia, oxygen is consumed in direct proportion to the amount of ammonia substrate. Electrochemical biosensors : Electrochemical biosensors The interaction of the analyte with the bioreceptor is designed to produce an effect measured by the transducer, which converts the information into a measurable effect, for example, an electrical signal There are four major types of transducers: electrochemical ,mass, optical, and thermal. The application of nanomaterials in biosensor fabrication allows the use of many new signal transduction technologies in their manufacture. Because of their size, nanosensors, nanoprobes, and other nanosystems are revolutionizing the fields of chemical and biological analysis. Electrochemical biosensors have been reported for analyzing food and beverages , for detection of GMO content in food , for measuring the freshness of food , etc. Slide 28: In recent years, solid electrodes of gold, platinum, silver, nickel, copper, various doped or undoped forms of carbon, dimensionally stable anions, etc., have replaced the conventional mercury electrodes because of their toxicity. These materials can be either bare or chemically modified for improved selectivity, sensitivity, and stability, mostly by using polymers of varied characteristics. The principle of electrochemical sensors is that the electroactive analyte is oxidized or reduced on the working electrode surface, which is subjected to some predefined pattern of fixed or varying potential, and the variation on electron fluxes leads to the generation of an electrochemical signal, which is measured by the electrochemical detector The two most important subclasses of electrochemical sensors include the voltammetric and potentiometric biosensors. Flow Cytometer in Microbiological Food QC : Flow Cytometer in Microbiological Food QC The dynamics of the growth, survival and biochemical activity of microorganisms in food are the result of stress reactions (such as change in genome, proteome) in response to the changing physical and chemical conditions in the food microenvironment (e.g. the pH gradients, oxygen, water activity, salt, and temperature). Slide 30: Specific detection of pathogenic strains can be achieved by Flow cytometry using immunofluorescence techniques, which allow microorganism detection at the single-cell level. It can be used for food samples, but requires prior isolation of the target organism to generate antibodies. The use of specific antibodies directed against pathogens such as certain strains of Escherichia coli, Salmonella, Pseudomonas and other cell types allowed their cytometric detection with sensitivity and specificity. The detection of resistant forms of microorganisms has been achieved cytometrically (eg. Endospores of Bacillus) Also Nucleic Acid can be stained and detected. FCM is used in milk quality control, in brewing, etc Detection of specific food contaminants can be achieved by FISH coupled with FCM (FLOW–FISH). Slide 31: The advantage of FCM is that it can also differentiate VBNC bacteria from healthy cultivable cells. It has ability to detect microorganisms at relatively low concentrations in a short time. Multiple labelling allows the detection of different organisms or different stages in the same sample. PCR Applications : PCR Applications PCR analysis of a food includes the following steps: Isolation of DNA from the food, Amplification of the target sequences by PCR, Separation of the amplification products by agarose gel electrophoresis Estimation of their fragment size by comparison with a DNA molecular mass marker after staining with ethidium bromide and Finally a verification of the PCR results by specific cleavage of the amplification products by restriction endonuclease or the more time-consuming, Southern Blot Alternatively amplification products may be verified by direct sequencing or a second PCR. Slide 33: A very convenient approach is to perform PCR amplification and verification in one single run by using a target-specific fluorescent-labelled oligonucleotide probe in a real-time PCR system. Real-time PCR requires more expensive laboratory equipment, but allows the gel free product detection without the need to open the PCR tubes after amplification again. This approach is therefore less time-consuming and labour-intensive. It implies a lower risk of contamination and there is no need to use mutagenic staining dyes such as ethidium bromide. With real-time PCR also highly accurate quantitative results can be obtained. Slide 34: Typically, 25-40 amplification cycles are run to produce a detectable quantity of copies of the template DNA fragment. The sampling plan determines how representative the result of the analytical procedure is, whereas quality and quantity of the analytes may vary depending on sample preparation. The higher the degree of heterogeneity, the more critical will be the choice of the appropriate sampling plan, because the sample has to be representative for the material to be analyzed. Slide 35: Quality and yield of the isolated DNA are two critical factors in DNA preparations for PCR analysis. With the exception of grains, fruits and other raw materials, the products to be analyzed such as food samples, contain material that has undergone varying levels of processing, including physical, chemical and enzymatic treatment that influences the quality and amount of the DNA. Mechanical treatment results in fragmentation of DNA, heat treatment in DNA degradation, low pH in increased chemical hydrolysis and denaturation of DNA and enzymatic treatment may result in an enzymatic hydrolysis or modification of the DNA. Moreover processing may lead to a complete degradation or removal of the DNA. Failures in extracting detectable DNA levels have so far been reported for soybean sauce, margarine, sugar, refined oils. Slide 36: Two different DNA isolation protocols with plenty of variations are used for DNA extraction from complex food matrices: The classical protocol for DNA isolation is based on an incubation of the samples in the presence of a detergent such as Cetyl Trimethyl Ammonium Bromide (CTAB) or sodium dodecyl sulfate (SDS) and a treatment with organic solvents such as chloroform and phenol, respectively, followed by precipitation of DNA with isopropanol or ethanol. The second protocol is based on commercially available DNA-binding silica column resins as ready to use kits. Slide 37: There are many PCR assays that have been described for food pathogens such as Salmonella, E. coli O157, L. monocytogenes, Campylobacter, and several others are commercially available (FDA,AOAC) PCR detects toxin producing S. aureus, Vibrio cholerae, Clostridium botulinum, C. perfringens, Bacillus cereus, and E. coli by amplifying specific genes that code for toxin. DNA Microarray : DNA Microarray DNA microarrays consist of multiple-specific oligonucleotides or PCR probes spotted mechanically on to a glass microchip in a lattice-type configuration. Target nucleic acid, which may be either PCR products, genomic DNA, or RNA, cDNA is then applied to the microarray and hybridization detected by a fluorescent label incorporated directly into the target nucleic acid. The fluorescence pattern is then recorded and analyzed using a scanner. Potentially thousands of oligonucleotides or probes can be spotted on to a single microarray slide offering the opportunity not only of detecting a broad range of pathogens simultaneously with high specificity, but also of providing detailed genetic characterization or genotyping of specific foodborne pathogens. Slide 40: Several microarrays have been developed for food pathogens, which can simultaneously detects and genotypes organisms such as (EHEC), Salmonella, Shigella, different species of Listeria. PFGE : PFGE PFGE is a restriction-based typing method that is considered by many to be the “gold standard” molecular typing method for bacteria. Here DNA fragments are separated under conditions where there is incremental switch of the polarity of the electric field in the running apparatus. This electrophoretic approach allows for the resolution of DNA fragments up to 800 kb in size. When DNA is restricted with a restriction enzyme, PFGE provides a DNA “fingerprint” that reflects the DNA sequence of the entire bacterial genome. Slide 42: A bacterial suspension is prepared with an optimal cell concentration mixed with molten agarose and cast into plug molds The embedded cells are treated with detergents and/or enzymes, such as sarcosine and proteinase K, to lyse the cells and release the DNA The plug is thoroughly washed to remove cellular debris and treated with a rare-cutting restriction enzyme Following enzyme treatment, the plugs are inserted into an agarose gel and the restriction fragments separated under conditions of switching polarity electrophoresis pattern of DNA separation is visualized staining ethidium bromide The banding pattern from one isolate can be compared with those of other isolates and information about the relatedness of the strains can be detected. Slide 43: Standardized criterion is developed to determine the genetic relatedness of isolates based on their PFGE banding patterns. Recently, additional standardized criteria have been developed for PFGE analysis of heterogeneous foodborne pathogens that allows for better resolution of pathogen relatedness. This standardization of technique has allowed PFGE to become a widely accepted method for comparing the genetic identity of bacteria PFGE typing has demonstrated a high level of reproducibility for foodborne pathogens. Because all of the DNA extraction and restriction enzyme digestion occurs within the agarose plugs, the free DNA is not pipetted, thus the banding patterns generated are related to restriction enzyme digestion, not nonspecific mechanical shearing. Slide 44: Advantages of using PFGE PFGE is a universal method that may be used for subtyping of bacteria with small modifications dependent on the bacterial species investigated. Usually, only the choice of the restriction enzyme and conditions for electrophoresis need to be optimized for each species. DNA restriction patterns generated by PFGE are stable and reproducible at the intra- and inter-laboratory levels when the method is highly standardized. Due to its relative simplicity, high discriminatory power and reproducibility, PFGE has become the gold standard for bacterial subtyping. Limitations of PFGE Time consuming Requires a high-level of skills The DNA fragments are separated by their size, not by their sequence Fragments with the same size may represent different parts of the genome Observed differences in PFGE patterns may be difficult to interpret Some strains are untypable by PFGE Magnetic separation-applications : Magnetic separation-applications Anti Salmonella magnetic beads Mattingly (1984) separated Salmonella from food and fecal mater using myeloma protein and hybrid antibody (for Oantigen), conjugated to a polycarbonate- coated metal bead. Beads capture Salmonella cells, wash the beads, transfer into impedance system. Food sample like brie, milk, yogurt, meat and vegetables can be tested. Slide 46: Detection of E. coli Problem is in the isolation of pathogenic strain from nonpathogenic strain. Magnetic beads coated with monoclonal antibodies to K88 antigen specifically detects K88 positive E. coli from mixed sample. E. Coli O157:H7, antibody against 0, H antigen are coated on magnetic particles. Slide 47: Detection Listeria monosytogens Skjerve et al. (1990) coated magnetic beads with monoclonal antibodies against L. monocytogens flagella and separated immunomagnetically. Immunomagnetic separation of S. aureus was carried out by Johne et al. (1989) using magnetic beads coated with polyclonal antibodies which detected the protein A REFERENCE : REFERENCE http://www.worldfoodscience.org/cms/?pid=1003869 Ralf Greiner and Ursula Konietzny Modern Molecular Methods (PCR) in Food Control: GMO, Pathogens, Species Identification, Allergens Yousef Haik et al.; MagneticTechniques for Rapid Detectionof Pathogens Springer Science+Business Media, LLC 2008 Jaume Comas-Riu, Núria Rius Flow cytometry applications in the food industry; J Ind Microbiol Biotechnol (2009) 36:999–1011 Corrado Di Natale et al.; Electronic-nose modelling and data analysis using a self-organizing map; Meas. Sci. Technol. 8 (1997) 1236–1243. Slide 49: Mike Stones, Electronic nose sniffs out food aroma quality, Dec-2009. Corrado Di Natale et al.; Electronic nose analysis of urine samples containing blood; Physiol. Meas. 20 (1999) 377–384. You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.