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norisoprenoids

glucosidases from Aspergillus niger or A. oryzae. Some of these fungal hydrolases are distinguished by a superior activity at elevated processing temperature and at high concentrations of ethanol; their application to wine should enhance the flavor.

Looking at the second field of application, rhamnosidase could be the target of a genetic approach. This enzyme is present as an impurity in commercial pectinases. Its action on Citrus wastes containing bitter glycosides results in the liberation of a glucose/rhamnose mixture. Glucose oxidase from Gluconobacter suboxydans is then used subsequently to remove any remaining glucose. The residual pure rham-nose is an important precursor of the widely used flavor, 2,5-dimethyl-4-hydroxy-2,3-dihydrofuran-3-one (Fur-aneol) by reacting the methylpentose with an amino acid. The product resulting from the oxidation of glucose, 5-keto-gluconic acid, may serve as a precursor of the savory flavor 4-hydroxy-5-methyl-3(2ff)-furanone(26).

The third approach is exemplified by the treatment of extracted vanilla pods with an exo-enzyme mixture of A. niger that led to the additional formation of flavor (33). This idea has been transferred to other flavor-bearing plant sources with a rigid morphology, such as Iris rhizom. The addition of enzymes for the removal or prevention of off-flavors, for example, the enzymatic debittering of Citrus products, may also be interpreted as an indirect action of enzymes on the final sensory quality of the food product.

Classical Fermented Food

Numerous enzymes of various microorganisms must act together in a well-balanced mode to bring about the typical sensory characteristics of the complex substrates of fermented food. In an approach to rationalize the traditional technology, enzyme and substrate mixtures, or purified lipases and proteases together with defined substrates, such as butter oil, are used. Various microbial lipases preferably cleave the ester bond between glycerol and short- to medium-chain fatty acids. The liberated free fatty acids are the sensory backbone of many dairy flavors. Butter and cheese flavor are now generated by the action of lipases or combinations of enzymes on curd or milk fractions, and some of the flavor-enriched products are traded as "Enzyme Modified Cheese."

Some meat products, such as salami-type sausages, were also described to benefit from added enzymes: lipase, protease, and collagenase enzymes improved taste and accelerated ripening (34-36). Proteolytic, lipolytic, and further biopolymer degrading activities can be isolated from yeast cultures and combined with the inactivated producer cells to obtain food flavors with savory notes. The claimed gain in flavor intensity is seldomly quantified, which makes the success of certain bioprocesses difficult to assess. If, in a food-type substrate, the addition of a single enzyme caused a fortified flavor, the traditionally used starter culture strain should benefit from a transmission of the corresponding gene or from the use of gene dosage effects. Several recent reviews deal with rDNA techniques for the elucidation and improvement of relevant properties of starter cultures (eg, 37,38), but their application to improve the flavor formation properties is still somewhat neglected (39).

Concerted Enzymatic Production of Flavor

Lipase and esterase preparations catalyze reverse hydrolysis (esterification, lactonization) and transesterification reactions in one-phase, two-phase, microemulsion, and even in supercritical fluid systems (40). Elegant methods perform the reaction in one of the substrates as the solvent and apply chemical or physical sinks to trap the reaction water. Although essential for the hydration of the enzyme, water is detrimental in excess and will lead to incomplete conversion. Various techniques for water limitation such as the use of molecular sieve, distillation, condensation on a cold surface, and reduced pressure were described. Flavor applications included bacterial, fungal, and mammalian sources of enzyme for the production of chiral aliphatic esters, terpenol esters, and lactones with fruity, flowery, nutty, and creamy odors (Table 3). Similarly, proteases were used for the production of aspartam, a dipeptide sweetener, and for peptides possessing umami or sodium glutamate-like flavors (44). Carbohydrate esters may find an industrial use as surfactants to stabilize emulsion-type composed Citrus flavors, for example, in nonalcoholic beverages (45).

Vice versa, the selective hydrolysis of racemic amides or esters yields chiral amines, alcohols, or acids. Since the early seventies important industrial applications of hydrolases are the enzymatic resolution of racemic amino acids, and the asymmetric hydrolysis of menthyl esters to produce optically pure L-menthol (candies, sweets, toothpaste, tobacco, ointments, etc), one of the bulk compounds of the flavor industry. More than 50 lipases of different origin have been characterized with respect to their efficiency and selectivity. Only tertiary alcohols and branched-chain and benzoic acids were usually excluded from the wide range of substrates. Subjects of recent work were the effects of solvent, pH, temperature, surfactants, water content, immobilization of the enzyme, pretreatment of the solid support, mass transfer-reaction interactions, isozymes, and chemical modification of the enzyme. Few researchers have been interested in the unambiguous identification of the catalytically active constituent of the often crude industrial enzyme mixtures. A closer look at commercial porcine pancreas extract showed that the N-terminal end of the active protein was homologous to a known cholesterol esterase sequence (47). This enzyme

Table 3. Compounds from Enzymatic Hydrolysis or Reverse Hydrolysis

Substrates

Products

Ref.

Racemic carboxylic

Chiral alcohols

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