Chapter 20
Analytical Methodology in Biotechnology: An Overview 1
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Gary R. Takeoka , Akio Kobayashi , and Roy Teranishi 1
Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany,CA94710 Laboratory of Food Chemistry, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112, Japan
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For every advance in scientific knowledge, there must a development of a method or technique to uncover new information. Biotechnology is the use of biological agents to provide us with foods and flavors with better quality and/or higher yields. In order to follow changes brought about by biotechnology, we must have better methods of evaluating these changes for food quality or for toxicity aspects. In this section, various methods for isolation, identification and characterization of food constituents altered by biotechnology are discussed. Michael Faraday in 1830 (7) said, "Chemistry is necessarily an experimental science; its conclusions are drawn from data, and its principles supported by evidence derived from facts. A constant appeal to facts, therefore, is necessary; and yet so small, comparatively, is the number of these presented to us spontaneously by Nature, that were we to bound our knowledge by them, it would extend but to a very small distance, and in that limited state be exceedingly uncertain in its nature. To supply the deficiency, new facts have been created by experiment, the contrivance and hand of the philosopher having been employed in their production and variance." Chemistry is still an experimental science. New reactions must be devised and experimental methods extended and improved, or new ones invented, to provide new data. Modern biotechnology is a much more recent area of investigation and therefore demands even more innovation to accumulate new facts by experimentation. If modern technology is to help produce more and better foods, chemistry must be utilized to increase acceptability of such new foods. Recent developments in analytical methodology have enabled flavor chemists to make considerable advances in recent years (2). Methods developments have progressed to the point that ripening of fruit can be followed quantitatively as the fruit progresses from green to ripe. Compounds contributing the green aroma decrease in concentration while compounds contributing to the ripe fruit aroma increase as nectarines go from green, green-red, shipping ripe, to full ripe (5). Maximum aroma 0097-6156/96/0637-0216$15.00/0 © 1996 American Chemical Society
In Biotechnology for Improved Foods and Flavors; Takeoka, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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of apples is reached approximately two weeks after harvest, and the drop in aroma has been followed as apples are stored (4). One of the first sensory analysis of apple aroma was done with Red Delicious apple essence (5, 6), but now sensory evaluation utilizing aroma values of different varieties of apples has been studied (7). Currently, there is much interest in flavor research in determining what compounds are actually responsible for the characteristic flavor of a food. Researchers have utilized odor unit values (compound concentration/odor threshold) to determine the contribution of individual constituents to the overall flavor of a food. For example, citrus peel oil aroma quality has been characterized using logarithmic odor unit values (8). Tamura et al. (this volume) discuss the suitability of the detection threshold and the recognition threshold in determining the limited odor unit for characterizing citrus aroma quality. Advantages and disadvantages of simultaneous distillation and extraction and dynamic headspace sampling methods for isolating volatiles from foods are compared by Buttery and Ling (this volume). A high flow dynamic headspace sampling technique with "closed loop stripping" was found to be most practical, rapid, and comprehensive for studying seasonal development of new cultivars of com and other food crops. This technique combined with the addition of excess sodium sulfate permitted the quantitaive analysis of very water soluble and polar flavor compounds such as 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol®) and 3-hydroxy-2-methyl4-pyrone (maltol). This method will lead to the identification of additional highly water soluble odorants which are not effectively isolated by conventional sample preparation methods. Accurate quantitative data are a prerequisite for determining the contribution of individual constituents to the overall food flavor. A stable isotope dilution assay is an accurate method for quantitation of flavor constituents which are unstable, inefficiently extracted, and/or which occur at trace concentrations. Grosch and co workers (9, 10, 11, 12) have effectively used this method to quantitate key flavor compounds in a variety of foods. In this method deuterated compounds with only a slight mass difference to the constituents of interest are synthesized and used as internal standards. The food sample is spiked with the deuterated internal standards and the volatiles are isolated. Since the chemical and physical properties such as volatility, reactivity and chromatographic behavior of the analyte and its deuterated analogue are nearly identical, these internal standards are ideal for correcting for any losses during sample preparation. Stable isotope-dilution mass spectrometry has been used by Allen (this volume) to follow the concentration of methoxypyrazines in berry maturity and vine growing conditions of Sauvignon blanc and Cabernet Sauvignon grapes. Biochemical pathways of the origin of pyrazines has been suggested from the observations of the abundance of the pyrazines determined by this stable isotopedilution mass spectrometry method. Study of precursors (13, 14, 15) and how enzymatic action releases characteristic volatiles from precursor compounds has progressed only because of the advance in methods of handling glycosides. Various modem countercurrent chromatographic techniques (16) have been successfully employed in the isolation of flavor precursors such as glycosides (17, 18, 19). The biotechnological possibilities, perspectives and limitations, from the knowledge of the pathways of the biogeneration of important norisoprenoid compounds, such as P-damascenone and the theaspiranes,
In Biotechnology for Improved Foods and Flavors; Takeoka, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS
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are discussed by Winterhalter (this volume). Tremendous progress in the chirospecific analysis of these desirable flavor compounds has been achieved, primarily due to the development of modified cyclodextrin capillary columns (20) as well as advances in multidimensional gas chromatography (MDGC) coupled with mass spectrometry (21). Liquid chromatographic-thermospray mass spectrometric analysis of crude plant extracts containing phenolic and terpene glycosides has been described by Hostettman and co-workers (22). The development of atmospheric pressure chemical ionization and electrospray ionization, liquid chromatography coupled with standard mass spectrometry and with tandem mass spectrometry, has opened further dimensions in the field of bio-organic analysis. These tools now provide elucidation of structures of a variety of flavor precursors and will influence flavor biotechnology in many ways. Importantly, these analyses can be performed on very small samples such as a single strawberry. Analytical methods for non-volatile flavor compounds have been developed. To extend understanding of flavor development during fruit ripening, these methods have been used to study changes in total soluble solids during the ripening of melons grown under hydroponic conditions (Wyllie et al., this volume). One of the first steps in the safety assessment of genetically modified foods is compositional analysis. A major interest in genetically modified potatoes is the concentration of the naturally occurring alkaloids, which are toxic if concentrations exceed certain limits. A broad spectrum of genetically modified potatoes, from greenhouse experiments to field trials, has been screened for steroidal glycoalkaloids for safety considerations (Engel et al., this volume). Methods developed for studying the safety aspects of genetically modified foods have been discussed (23). Processes of interest to food and flavor industries are: sterilization of juices without adverse effects, such as those caused by hydrolyses of various food constituents, esterification and transesterification, rearrangements, isomerizations, condensations, etc. The microwave oven is found in many homes because of the convenience of rapid heating by this method. Advantages of microwave technology are: rapid heating and quenching, minimal temperature gradients, elimination of wall effects, etc. Continuous and batchwise laboratory-scale microwave reactors developed for controlled heating under pressure are described by Strauss and Trainor (this volume).
Literature Cited 1.
Faraday, M . Chemical Manipulation; 2nd edition; John Murray: London, 1830;
2.
Teranishi, R. In Flavor Chemistry: Trends and Developments; Teranishi, R.; Buttery, R. G . ; Shahidi, F., Eds.; American Chemical Society Symposium Series No. 388; American Chemical Society: Washington, D C , 1989; pp 1-6. Engel, K.-H.; Ramming, D. W.; Flath, R. A.; Teranishi, R. J. Agric. Food Chem. 1988, 36, 1003-1006. Dirinck, P.; De Pooter, H . ; Schamp, N . In Flavor Chemistry: Trends and Developments; Teranishi, R.; Buttery, R. G.; Shahidi, F., Eds.; A C S Symposium Series No. 388; American Chemical Society: Washington, D C , 1989; pp 23-34.
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Flath, R. A.; Black, D. R.; Guadagni, D. G.; McFadden, W. H.; Schultz, T. H . J. Agric. Food Chem. 1967, 15, 29-35. Guadagni, D. G . In Correlation of Subjective-Objective Methods in the Study of Odors and Taste; Special Technical Publication No. 440; American Society for Testing and Materials: Philadelphia, PA, 1968; pp 36-48. Petersen, M . A.; Poll, L . In Aroma: Perception, Formation, Evaluation; Rothe, M . , Kruse, H.-P., Eds.; Deutsches Institut fur Ernährungsforschung, Potsdam -Rehbrücke, 1995, pp 533-543. Tamura, H.; Yang, R.-H.; Sugisawa, H . In Bioactive Volatile Compounds from Plants; Teranishi, R.; Buttery, R.G.; Sugisawa, H . , Eds.; A C S Symposium Series No. 525; American Chemical Society: Washington, D C , 1993, pp 121136. Schieberle, P.; Grosch, W. J. Agric. Food Chem. 1987, 35, 252-257. Guth, H.; Grosch, W. Lebensm.-Wiss. u.-Technol. 1990, 23, 513-522. Sen, A.; Laskawy, G.; Schieberle, P.; Grosch, W. J. Agric. Food Chem. 1991, 39, 757-759. Schieberle, P.; Gassenmaier, K.; Guth, H.; Sen, A.; Grosch, W. Lebensm.-Wiss. u.-Technol. 1993, 26, 347-356. Williams, P. J.; Sefton, M . A.; Wilson, B. In Flavor Chemistry: Trends and Developments; Teranishi, R.; Buttery, R. G.; Shahidi, F., Eds.; A C S Symposium Series No. 388; American Chemical Society: Washington, D C , 1989; pp 35-48. Flavor Precursors: Thermal and Enzymatic Conversions; Teranishi, R.; Takeoka, G . R.; Guentert, M . , Eds.; A C S Symposium Series 490; American Chemical Society: Washington, D C , 1992, 258 pp. Progress in Flavour Precursor Studies; Schreier, P.; Winterhalter, P., Eds.; Allured: Carol Stream, IL, 1993, 507 pp. Conway, W.D. Countercurrent Chromatography: Apparatus, Theory & Applications; V C H Publishers, Inc.: New York , N Y , 1990. Humpf, H.-U.; Schreier, P. J. Agric. Food Chem. 1992, 40, 1898-1901. Krammer, G.E.; Buttery, R.G.; Takeoka, G.R. In: Fruit Flavors: Biogenesis, Characterization and Authentication; Rouseff, R.L.; Leahy, M . M . , Eds.; A C S Symposium Series 596; American Chemical Society: Washington, D C , 1995; pp 164-181. Skouroumounis, G.; Winterhalter, P. J. Agric. Food Chem. 1994, 42, 10681072. Koenig, W . A . Gas Chromatographic Enantiomer Separation with Modified Cyclodextrins; Huethig: Heidelberg, 1992, 168 pp. Full, G.; Winterhalter, P.; Schmidt, G.; Herion, P.; Schreier, P. J. High Resol. Chromatogr. 1993, 16, 642-644. Wolfender, J.L.; Maillard, M . ; Hostettmann, K. J. Chromatogr. 1993, 647, 183190. Genetically Modified Foods: Safety Aspects; Engel, K.-H.; Takeoka, G.; Teranishi, R., Eds.; A C S Symposium Series 605, American Chemical Society: Washington, D C , 1995, 243 pp.
In Biotechnology for Improved Foods and Flavors; Takeoka, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
