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Enzymic analysis AusAID CARD Project 008/07VIE Workshop 4, FCRI February 2009 Les Copeland University of Sydney Outline  Principles of analysis  Enzymology  Enzymes in analysis Quality  Suitability of a product for a particular purpose  Properties that have usefulness – processing, nutrition, consumption  Influenced by – genetics and environment (G x E) – processing and testing technology  Quality is different from safety – freedom from contaminants  Quality is assessed by comparing results of chemical and physical analyses with functional tests relevant to the specific end use Measuring quality  Objective analyses – chemical composition – physical and chemical characteristics of components – nutritional, anti-nutritional and toxic constituents – digestibility  Subjective evaluation – colour, taste, odour, appearance, size, shape, texture, firmness – important in guiding consumer selection of foods Analytical methods Chemical analyses  Specific constituents measured  Usually well defined and accurate  Difficult to relate to chemical properties to functionality Functional tests  Practical and relevant to applications  Measure collective properties of complex materials  Difficult to relate cause of properties and effect Types of samples analyzed  Raw materials – to determine that a delivery conforms to standards (composition, contaminants) – to assure consistency of supply – to evaluate new suppliers  Process control samples to monitor operations  Finished materials to determine that product meets standards and claims  Complaint samples  Competitors’ samples Stages of a typical analytical procedure  Sampling  Sample preparation  Measuring  Data processing  Verifying the procedure  Appropriateness and cost of methods – protein determination Main sources of analytical errors Most common sources of sampling errors  Uneven distribution of measured constituents in foods – contaminants, fat in meat, proteins in sweetpotato  Lack of randomness - where and when sample is taken  Many sampling protocols are used – random, stratified, systematic, judgment  An analytical result can never be better than the sample Sample preparation  Sample is ground, milled or blended – need to avoid of heat generation (loss of moisture and volatiles, chemical reactions)  Enzymes are inactivated to prevent loss of labile compounds – heating, drying, 80% ethanol, protein denaturants, … – amylases for starch determination  Sample is prepared in a way that does not cause loss of the component being analyzed – addition of reducing agent (eg, ascorbic acid) to prevent oxidations (eg, unsaturated fatty acids)  Chemical knowledge of the analyte is essential Choice of many methods for measuring stage  Gravimetric (by weight)  Volumetric (titration)  Spectrometric (spectral properties, chromogenic reagents)  Chromatographic (HPLC, GC)  Electrophoretic (PAGE, CE)  Enzymic  Immunochemical (ELISA) [...]... germination 7 Lipases and lipoxygenases cause off flavours in dairy products and flour Lipases Lipase Triglyceride Lipoxygenases Monoglyceride + free fatty acids End-point analysis - the analyte is made the limiting part of the analysis Example: determination of glucose (or O2) using glucose oxidase/peroxidase (GOPOD) – highly specific for glucose; can be performed in presence of other sugars ... Bioavailability - example 2 Crab meat was found to contain 1.4 - 13 ppm of Cd, which is above limits of an average acceptable daily intake of Cd of 20 - 60 ppb Should crab meat be removed from sale? Analysis of the bioavailability of Cd in crab meat showed that only 2-3% of the metal was available from the gastrointestinal tract, which gives the food an acceptable margin of safety (assuming the consumer’s... GMOs is common Enzymes used in processing are not usually pure – concentrate of a culture supernatant – typically contain other enzymes (side activities) High purity usually required for enzymes used in analysis Comparing enzyme preparations from different suppliers is a common problem in industrial enzymology – no internationally agreed and industrially relevant units – manufacturers tend to define their... Plants, animals, environmental Clinical Activity of an enzyme is measured as an indicator of the quality or condition of a material Enzyme is used to convert an analyte to a measurable product (end-point analysis) – selectivity and sensitivity of enzymes is used to measure biological molecules α-Amylase activity is measured to assess suitability of cereal grains for end use Quality of wheat flour for baking... matrix – pH and redox conditions – adsorption, complexing and precipitation reactions Bioavailability - example 1 Turkeys were fed a carefully formulated, nutritionally balanced diet based on soybean meal; analysis showed the diet to contain more than sufficient amounts of all essential trace metals However, the turkeys grew poorly Why? Zn was bound to proteins in the soy meal and was not available to the . Enzymic analysis AusAID CARD Project 008/07VIE Workshop 4, FCRI February 2009 Les Copeland University of Sydney Outline  Principles of analysis  Enzymology  Enzymes in analysis . properties, chromogenic reagents)  Chromatographic (HPLC, GC)  Electrophoretic (PAGE, CE)  Enzymic  Immunochemical (ELISA) Definitions  Limit of detection – the minimum amount that can. 1 Turkeys were fed a carefully formulated, nutritionally balanced diet based on soybean meal; analysis showed the diet to contain more than sufficient amounts of all essential trace metals.