Biotechnology for Improved Foods and Flavors - ACS Publications

Due to public controversy about recombinant DNA techniques, the flavor industry has been ... is the production of enzymes from genetically modified or...
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Chapter 11

Generation of Flavors by Microorganisms and Enzymes: An Overview Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 7, 2015 | http://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0637.ch011

Karl-Heinz Engel and Irmgard Holing Lehrstuhl für Allgemeine Lebensmitteltechnologie, Technische Universität München, D-85350 Freising-Weihenstephan, Germany

The generation of individual flavor compounds or complex flavor mixtures by the use of microorganisms and enzymes, the topic of subsequent symposium contributions, is summarized. The two basic approaches, de novo-synthesis in the course of microbial fermen­ tations and biotransformations of suitable precursors are outlined. The increased compositional and structural knowledge of the substrates/precursors needed and the tailor-made design of the microorganisms/enzymes employed are presented as bases for strategies to optimize the biogeneration of flavors.

Fermentation is one of the original traditional biotechnological methods for preservation of foods. This primary purpose is frequently accompanied by the formation of typical flavors. For centuries, the microorganisms and enzymes involved have been employed almost unwittingly. Modern biotechnology makes use of the increasing knowledge of the underlying scientific principles and is starting to exploit the advantages offered by biocatalysts in a more specific way. The incorporation of biotechnological steps in the manufacture of high fructose corn syrup, the production of sweeteners, organic acids, vitamins or amino acids has been well established (7). Due to the advances in microbial fermentation and enzyme technology, individual flavor compounds or complex flavor mixtures, examples of low-volume but high-value products, are increasingly becoming targets for production on an industrial scale (2). The exploitation of such techniques is especially attractive, because flavors or flavor compounds obtained via biotech­ nology are considered to be natural by regulatory authorities in many countries, as long as certain conditions, such as the natural origin of the raw material, have been met. Biotechnological processes leading toflavorproduction can be divided into two major groups: dert0V0-synthesisin the course of microbial fermentation (3) and biotransformations/bioconversions of suitable precursors either by microorganisms or by enzymes (4,5). Recent research developments in these areas are described in the subsequent symposium contributions.

0097-6156/96/0637-0120$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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11. ENGEL & ROLING Generation of Flavors by Microorganisms & Enzymes 121 Non-volatile precursors. An essential prerequisite for optimum flavor generation by microorganisms or enzymes is detailed knowledge of the composition and the availability of the substrates/precursors needed. Flavor precursors have been objects of intensive studies (6,7). A class for which tremendous progress in knowledge of composition and distribution has been achieved recently is the glycoconjugated precursors. Structures of numerous non-volatile, flavorless glycosides of monoterpenes, Aitfrisoprenoids, and shikimic acid metabolites present in fruits, wines, and some vegetable products have been elucidated (8). Aroma liberation can result from either acid- or enzyme-catalyzed hydrolysis. The application of suitable hydrolytic enzymes and enzyme preparations, respectively, has been reported (9). Knowledge acquired of the precursors available in certain fruits, combined with the possibilities offered by modern enzyme technology, e.g. the tailoring of specific biocatalysts, will open new dimensions to influence the release of bound aroma compounds. Another class of compounds well known as non-volatile flavor precursors are unsaturated fatty acids. The lipoxygenase-catalyzed biogeneration of aroma active C and C aldehydes and alcohols from C polyunsaturated fatty acids is an important mechanism well studied in plant systems (10,11). An analogous process, the oxygenase-catalyzed conversion of fatty acids to oxylipins by diverse marine life, such as algae, has emerged as an exciting new trend (12). The biogeneration of flavor compounds from these structurally unique oxylipins might reveal a source of unexplored metabolic activity. 6

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Biotransformations/Bioconversions. The efficiency of microbial de novo synthesis of aroma compounds can be increased by offering suitable precursors which serve as starting points for biotransformations (single step reactions) or bioconversions (multi-step reactions) yielding the desired products. Natural sources containing the required precursors in high amounts can be used directly as substrates. An exemplary approach is the fermentative production of (R)-Y-decalactone from castor oil by Candida lipolytica. This process makes use of the fact that ricinoleic acid, the precursor metabolized via 0-oxidation, represents 90% of the triglyceride fatty acids of castor oil (13). Alternatively, single compounds isolated from abundant natural sources, e.g. terpenes from essential oils or the above mentioned C aldehydes and alcohols from plant tissues, can be subjected to highly specific microbially catalyzed reaction sequences. 6

Enzymes. The use of enzymes is an integral part of many important processes in food production. Hydrolytic enzymes especially are employed on an industrial scale, mainly because no costly regeneration of cofactors is required, in contrast to oxidoreductases. The release of specific fatty acid profiles by lipases in the course of cheese manufacture, or the cleavage by proteases of peptide fragments in protein hydrolyzates that otherwise will cause bitterness are examples for the impact of enzyme-catalyzed reactions on the final flavor of foods (5). The outstanding features of enzymatic reactions, e.g. high substrate specificity even in complex matrices, high reaction specificity, mild reaction conditions and reduction in waste product formation, are also of importance in the synthesis of single flavor compounds. Two additional factors have boosted the

In Biotechnology for Improved Foods and Flavors; Takeoka, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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122

BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS

application of enzyme technology in the synthesis of flavor substances: (i) the stability of enzymes (lipases, proteases) in organic solvents allows the catalysis of reactions which are not feasible in aqueous medium, e.g. esterifications, transesterifications and lactonizations, thus providing access to a broad spectrum of important volatiles (5); and (ii) the increasing knowledge of the influence of absolute configuration on the flavor properties of chiral compounds and analytical progress in the determination of naturally occurring enantiomeric compositions, which have increased the need for biocatalyzed reactions resulting in the "correct" enantiomer. Enzyme-catalyzed biotransformations of prochiral substrates as well as kinetic resolutions of racemic precursors can also be applied (14,15). Recombinant DNA techniques. Mutagenesis and selection techniques based on classical bacteriological and genetic methods are common procedures to optimize and standardize microorganisms used in food fermentations. Recombinant DNA techniques offer the potential of altering the properties of microorganisms more precisely in terms of production efficiency, product quality, safety, and diversity (16). A broad spectrum of recombinant microorganisms is available for industrial and agricultural applications (17). Recombinant DNA techniques are applied in such traditional areas as sake and beer brewing (18). Aflavor-relatedexample is the reduction of the amount of diacetyl, one of the major off-flavors in beer. The construction of a brewer's yeast containing a bacterial acetolactate carboxylase gene has been described. This yeast has the ability to convert acetolactate, the precursor of diacetyl, to acetoin which has no impact on beer flavor (19,20). Due to public controversy about recombinant DNA techniques, the flavor industry has been reluctant to make use of genetically modified organisms, but such methods will definitely become more common in the future. An area at the forefront of commercial applications of genetic engineering is the production of enzymes from genetically modified organisms. The milkclotting protease, chymosin, has been the first food ingredient produced via recombinant DNA techniques to be cleared for food use (21). The use of designed enzymes adjusted to specific process requirements will also provide new possibilities in the field of flavors. Literature Cited 1. 2. 3. 4. 5. 6.

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Cheetham, P.S.J. Chemistry & Industry 1995, 265-268. Berger, R . G . Aroma Biotechnology, Springer Verlag: Berlin, Germany, 1995. Mizutani, S.; Hasegawa, T. Perfumer & Flavorist 1990, 15, 265-268. Gatfield, I.L. Perfumer & Flavorist 1995, 20, 5-14. Christen, P.; Lopez-Munguia, A . Food Biotechnology 1994, 8, 167-190. Flavor Precursors: Thermal and Enzymatic Conversions; Teranishi, R.; Takeoka, G.R.; Güntert, M., Eds.; A C S Symposium Series 490; American Chemical Society: Washington, D . C . , 1992. Progress in Flavour Precursor Studies; Schreier, P.; Winterhalter, P., Eds.; Allured: Carol Stream, Illinois, 1993.

In Biotechnology for Improved Foods and Flavors; Takeoka, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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Generation of Flavors by Microorganisms & Enzymes

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Williams, P.J.; Sefton, M . A . ; Marinos, V . A . In Recent Developments in Flavor and Fragrance Chemistry; Hopp, R.; Mori, K . , Eds.; V C H Verlagsgesellschaft: Weinheim, Germany, 1993. Gunata, Z , ; Dugelay, J . ; Sapis, J . C . ; Baumes, R.; Bayonove, C . In Progress in Flavour Precursor Studies; Schreier, P.; Winterhalter, P., Eds.; Allured: Carol Stream, Illinois, 1993, pp 219-234. Hatanaka, A . Phytochemistry 1993, 34, 1201-1218. Winterhalter, P.; Schreier, P. In Flavor Science; Acree, T . E . ; Teranishi, R., Eds.; American Chemical Society: Washington, D . C . , 1993, pp 225-258. Gerwick, W . H . Biochim. Biophys. Acta 1994, 1211, 243-255. Gatfield, I . L . , Sommer, H . In Recent Developments in Flavor and Fragrance Chemistry; Hopp, R.; Mori, K . , Eds.; V C H Verlagsgesellschaft: Weinheim, Germany, 1993, pp 291-304. Schreier, P. In Progress in Flavour Precursor Studies; Schreier, P.; Winterhalter, P., Eds.; Allured: Carol Stream, Illinois, 1993, pp 45-61. Engel, K . - H . In Flavor Precursors: Thermal and Enzymatic Conversions; Teranishi, R.; Takeoka, G.R.; Güntert, M., Eds.; A C S Symposium Series 490; American Chemical Society: Washington, D . C . , 1992, pp 21-31. Geisen. R.; Stander, L.; Leistner, L . Food Biotechnol. 1990, 4, 497-504. Recombinant Microbes for Industrial and Agricultural Applications; Muraoka, Y . ; Imanaka, I.; Eds.; Marcel Dekker: New York, New York, 1993. Biotechnology & Genetic Engineering Reviews; Tombs, M . P . , E d . ; Intercept: Andover, 1991. Vogel, J . ; Wackerbauer, K . ; Stahl, U . In Genetically Modified Foods: Safety Aspects; Engel, K . - H . ; Takeoka, G.R.; Teranishi, R., Eds.; A C S Symposium Series 605; Americal Chemical Society: Washington, D . C . , 1995, pp 160-170. Takahashi, R.; Kawasaki, M . ; Sone, H . ; Yamano, S. In Genetically Modified Foods: Safety Aspects; Engel, K . - H . ; Takeoka, G.R.; Teranishi, R., Eds.; A C S Symposium Series 605; Americal Chemical Society: Washington, D . C . , 1995, pp 171-180. Flamm E . L . Bio/Technology 1994, 12, 152-155.

In Biotechnology for Improved Foods and Flavors; Takeoka, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.