8/12/2019 food flavor review http://slidepdf.com/reader/full/food-flavor-review 1/24 FOOD BIOTECHNOLOGY, (2&3), 167-190 (1994) ENZYMES AND FOOD IKLAVOR - A REVIEW j$&sten'* and A. ópez-Munguía2 ORSTOM. nstitut Français de Recherche Scientifique pur le Ddveloppement en Coo- #ration. Cicer6n 609, Col. LosMorales. Mexico DR , CP 11530, MEXICO. Instituto de Biotecnologfa. Universidad Nacional Aut6noma de MBxico. Apdo Postal 510-3, Cuernavaca, Mor., CP 62271, MEXICO. ABSTRACT The use of enzymes in flavor generation in food technology is reviewed. In the first part, important products derived from natural macromolecules present in foods such as ats, proteins,nucleic acids and flavorprecursors are discussed in terms of the enzymes involved in thereactions and therelation ofthe products with flavor. Enzymes that are used toeli- minate natural or process induced off-flavors are.dsodiscussed. In the second part, the useof enzymes for the direct syn- thesis of flavoring compounds is presented. WTRODUCTION Flavor compounds synthesis by biotechnological processes plays nowadays an increasing role in the food industry. This is the result, among otherthings, of scientific advances in biological pro- cesses,making use of microorganisms or enzymes as an alternative o chemical synthesis, combined with recent developments in analytical techniques such as HPLC, GC, IR or mass spectrometry (Knorr 1987). This can be evidenced by the great number of reviews related to flavorpublish edin he last twelve years covering a broad area: Schindler and Schmid (1982), Kempler (1983), Sharpell (1985), Gatfield (1986, 1988), Crouzet (1989). Welsh et hL(1989), Herráiz (1990), Cheetham (1991,1993), Janssens et aL(1992), Gutierrez and Revah.(1993); or concerning specific topics: li- pases characteristics (Borgström and Brockman 1984) and their industrial applications (Macrae 1983,West 1988), enzymes involved in-the cheese flavor biosynthesis (Kinsella and Hwang 1976, Law 1984, Seitz 1990), enzymes affecting the flavor of citrus products (J3ruemmer e? al. 1977) or tea (Jain and Take0 1984), enzymatic aroma genesis in food (Schwimmer 1981) or biocatalysts in the natural generation of flavor (Schreier 1985) to give only a fewexamples.Thereis still more potential for this area in biotechnology since liquid cultures of plant cells may also be used as a technique to * corresponding author Copyright 1994 by Marcel Dekkcr Inc. 167
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ORSTOM. nstitut Français de Recherche Scientifiquepur le Ddveloppement en Coo-#ration. Cicer6n 609, Col. LosMorales. Mexico DR , CP 11530,MEXICO.
Instituto d e Biotecnologfa. Universidad Nacional Aut6noma de MBxico. Apdo Postal510-3,Cuernavac a, Mor., CP62271,MEXICO.
ABSTRACT
The use of enzymes in flavor generation in food technology is reviewed. In the first part, im portant products derivedfrom natural macromolecules present in foods suchas ats, proteins, nucleic acids and flavorp recursors arediscussed interms of the enzymes involved in thereactions and therelation ofthe products with flavor. Enzymes that are used toeli-minate natural or process induced off-flavors are.dsodiscussed.In the second part, the useof enzymes for the direct syn-thesis of flavoring compounds is presented.
WTRODUCTION
Flavor compounds synthesis by biotechnological processes plays nowadays an increasing role
in the food industry. This is the result, among other things, of scientific advances in biological pro-
cesses, making use of microorganisms or enzymesas an alternative o chemical synthesis, combined
with recent developments in analytical techniques such as HPLC, GC, IR or mass spectrometry
(Knorr1987).Thiscanbeevidencedby the great number of reviews related to flavorpublishedin he
last twelve years covering a broad area: Schindler and Schmid (1982), Kempler (1983), Sharpell
synthesize a wide array of chemicals (Whitaker and Evans 1987, Knorr eral.1990). Research in this
area for new products and bio-processes is also enhanced by a growing market and an increasing
public concern for the total wholesomeness and chemical safety of food ingredients (Basset,l990).
From a total world market of 6 billion dollars for the flavor and fragrance industry in 1990, food fla-
vors account for25%, ith about 5%annual growth rate (Cheetham 1991). Flavor sales were esti-
mated tB be aboutU S$675 millions in 1991and are expected to reachUS$376 millions in the Euro-
pean Community (Cheetham 1993). Most of the major companies producing aromas are carrying
out research programs for developing the biotechnological production of such compounds (Dziezak
1986a). It must be alsopointed out that a specific policy concerning labelling of processed foods
containing natural compounds has been issued in several countries (e.g. in the EC with the directive
.published in July 1988 in theOfficial Journal of the European Community, Spinnler 1989).
On the other hand, the food industry has been strongly influenced by the increasing public awa-reness of the nutritional characteristics of their diet and, in particular,ofthe additives used in the food
industry.This is shown, not only by the high number ofindustrial products low in fat, sodium, caffei-
ne or cholesterol, but also n the displacement of saccharine by aspartame and the search for natural
colorants or alternative antioxidants and preservatives.
Enzymes asbiocatalysts offer a wide variety of possibilities for food flavor production: their
specificity, whether applied via whole-cell or cell-free systems enable the production of certain
chemicals difficult to synthesize; their stereoselectivity is an important advantage for the food
industry where aspecific optical conformation may be associated to flavorproperties. Enzymes may
alsobe used directly as food additives, not only toproduce or liberateflavor from precursors, but also
to correct off-flavors caused by specific compounds, naturally occurring or produced during proces-
sing (Bigelis,1992). Whitaker (1990) presents in a broad review on the prospective of enzymes in
food technology in general. Enzymes involved in flavors may also be endogenous, inherent to the
food or may come frommicrobial sources, added intentionally to foodsor coming from contamina-
tion. In Table 1,the main objectives for the use of enzymes in flavor technology are presented.
In this article, different ways in which enzymes are related to flavor are reviewed, presenting
examples of actual research in this area as well as potential applications. In some cases, processes
arid reactions known for two decades-are mentioned and updated with recent advances such as who-
le-cell biocatalysts or reaction in organic media.
~- . _
ENZYMATIC MODIFICATIONS OF MACROMOLECULES
Almost all macromolecules present in foods have an impact on flavor when hydrolyzed. There
is a wide variety ofenzymes available for-thehydrolysis ofproteins, starch and othercarbohydrates,
fats and nucleic acids (Shahani e t al.1976). so there are enormous alternatives for their
transformation or for the development of processes having asamain objective the production of fla-
voring compounds.
1. Fats.
There &e many examples in the food industry where the main flavor properties are derived
from far. As proof of the interest of scientists and companies in the applicaticn of lipolytic enzymes
TABLE -Enzyme technology related to food flavor.. dditives to enhance orproduce flavor from precursors.. iocatalysts in processes for flavor production.. dditives in flavor extraction processes from natural raw materials..Activation of endogenous enzymes to induce reactions leading to flavor production.. nactivation ofendo genou s enzymes to avoid off-flavor generation..Use ofenzvm es.for the elimination of off-flavors.
.
as a tool to improve cheese manufacture, one can refer to the reviews of Arnold et al. 1975), Kilara
(1985a) and Dziezak (1986b).The basic and first step of the process is the lipase-catalyzed hydroly-
sis of glycerides. In some cases, the free fatty acids released are converted to the flavoring com-
pounds by microorganisms (e.g. in the case of cheeses). Fatty acid profiles required for particular
flavors are obtained with various lipases: short chain fatty acids (C4) will develop a sharp, tangy
flavor tending towards rancid notes, while intermediate chain fatty acids (like C12) are associated
with a soapy flavor (Nelson 1972). This isa problem for example in the piña colada beverage were
the C12 fatty acid of he coconut isreleased by the thermostable lipase of the pineapple producing a
strong soapy off-flavor in the cannedbeverage (Heath and Reineccius 1986). On the other hand, lar-
gest fatty acids (up to C12 are known to do not make a significant contibution to flavor.
(c6 CIO)isimportant in fat flavor development: pancreatic lipases are adequate for short chain fatty
acids,Aspergillusand Candida spp. for a wide range of sizes and Penicillium roqueforti for buty-
the liberationof free fatty acids. Cheddar cheese has alsobeen studied (Arbigeet al. 1986): afteriso-
lating a very active lipase fromAspergiZZus oryzae, these authors canied out the accelerated ageingof the cheese by adding a combination of alipase and aprotease, obtaining a balance between flavor
development and body breakdown in areduced time. El-Sodaetal. 1992), by using freeze-shocked
mutant strains of Lactobacillus casei as a source of lipase, could shorten the ripening time of the
Egyptian Ras cheese, minimizing the development of bitterness. As another example, Davide and
Foley (198 1), ried to improve sensorial properties (appearence, flavor and texture) of Cheddar type
cheese withcoconut oilas asubstituteformilkfat by addingcommerciallipasepreparations. Experi-
ments showed it was not feasible. As a conclusion, full flavored cheeses of different types may be
obtained, with a specific fatty acid profile, depending on the enzyme used (Godfrey and Hawkins
1991, Kim Ha and Lindsay 1993).
Lipases have also been used for the modification of animal fats and tallows.Asan example, the
production of aromas from raw material such as butter has been proposed (Seitz 1990). In 1984, a
process for producing an aroma rich fat phase from butter was patented: a mixture of P.roqueforti
cells and pancreatic lipase was used on a pilot scale (Kunzer al.1984). Another process for this pur-
pose employed extra and intra cellular enzymes of Lactobacillus plantarum (Reimerdes 1984).
(1991) applied this process to the hydrolysis of milk fat in reversed micelles stabilized by lecithin.
They optimized this technique for parameters such as temperature, pH and molar ratio of water to
surfactantand found that enzyme activity could be improved with increasingenzyme and surfactant
concentrations.Garcia t al. 1991) carried out a selective ipolysis of glycerides from butteroil with
anAspergillus nigerlipase in order to obtain a pleasant flavor enhancement (ieleasing preferably
butyric acid). Luck and Hagg (1991) discussed the influence of parameters such as pH, enzyme
concentration and temperature on the kinetics of lipolysis in an enzymatichicrobiological process
for the production of cheese flavor from a butter emulsion. Finally, it is nteresting to point out that
fungal lipase produced in solid state fermentation displayed3.3 times greater activity than in sub-
merged culture and could be applied to hydrolyze oliveoilor for a simple control of flavor profile of
lipolyzed milk (Chen and McGill 1992).
Although lipases and cheeses are the most common enzymes and substrates, respectively, in
relation to enzymatic flavor production from fats, there are other examples: active soya flour is
added as a source of lipoxygenase- acting by hyper oxidation of linoleic acid and other polyunsatu-
red lipids - to bleach and to improve the volarde composition of bread. It has been found that the
concentrations of hexanal, hexanol, l-penten-3 -01,l-pentano1 and 2-heptanone are increased uponthe addition of soya flour (Luning et al.1991 Addo etal. 1993). Fatty acids are oxidized for the pro-
duction of "green" flavor components, the so-called "leaf aldehydes" and "leafalcohols", which may
be obtainedenzymatically through lipoxygenase and hydroperoxide yase. However,thisarea isstill
far from practical applicationsdue to the lack of availability of these enzymes (Gatfield 1988).
Josephson and Lindsay (1986) reported that lipoxygenase could be employed successfully in
the generation of fresh fish aroma, liberating alcohols and carbonyls from polyunsatured fatty acids.
They also pointed out that plant-derived lipoxygenases may be potentiallyused to restore thisaroma
The development of soy sauce fermentation, centuries ago, is probably one of the first proces-
ses where traditional biotechnology had a stronger impact on flavor than in preservation. More
recent developments of protein hydrolysates from vëgetals, soy bean, wheat or yeasts are
'specifically related to the production of flavor and flavor enhancers (Kilara 1985b). Although the
most important commercial product -yeast extraçt - is produced by autolysis, which involves the
activation of degradativeenzymes nherently present in the yeasç (Dziezak 1987,Mermelstein 1989,
Nagodawithana 1992), when proteases ike papain are used, glutamic acid may be obtained as a free
amino acid : ts perception in food is the main factor influencing flavor. Cysteineisalso important in
the developmentofmeat flavor due toits participation in the Maillard reaction (Tyrrelll990, Grosch
and Zeiler-Hilgart 1992); methionine, eucine and isoleucine (in that order) are the next most reacti-ve (Weir 1986). Different proteases have been proposed for the production of flavorings from pro-
tein hydrolysates: an immobilized protease from Penicillium duponti for the hydrolysis of soy pro-
tein; pronase for casein hydrolysis; pepsin andrenin or pea protein and reconstituted skimmed milk
respectively; pepsin for cotton seed and a variety of proteases for the protein from faba bean, as re-
viewed by Weir (1986).
After proteolysis it is possible to further enhance the flavor by treatment with a glutaminase
(Yasuyuki et al. 1989). The flavor enhancing glutaminase enzyme increased the level of glutamic : '
acid by a factor of 2.6 in a mash ofKoji wheat treated withBacillus subtìlìsand Aspergillusoryzae ,
proteases.
A succesful approach in this context is that of "cascade hydrolysis". It consists oftwoorthree
successive enzymatic hydrolysis steps starting from an alkaline protease allowing the pH to fall or
maintaining i t constant. The final steps may include peptidases to hydrolyze fragments that will
otherwise give bittemess to the product. Acid hydrolysis results in products such as mono and ,
dichloro compounds that have recently given rise to concem (Godfrey 1990).
Peptidases and proteases may also be used in cheese making processes (Kilara and Iya 1985).
Muir et a1.(1992), reported that enhancement of the level of degradative enzymes in reduced-fat
cheese, by addition to curd of an attenuated starter culture rich in peptidase and protease, resulted in
significant improvements in both intensity of Cheddar flavor and in the mouth-coating character. In
areview, Femandez-García (1986) discussed thenew trends in the accelerating process of cheese ri-
pening. It appears that not only lipases may be helpful, but alsoß-galactosidase and proteases. If the
use of a lactase preparation displayedan improvement in sensorial properties of cheese, proteasesmustbe employedcarefully because of the possible induction of bitter taste due to the release of hy-
drophobic residues (Femandez-García et al. 1988).Thisresearch group also demonstrated hat neu-
tral bacterial protease could highly increase the amount of non-protein nitrogen in two kindsof Spa-
nish cheese and therefore contribute to the accelerationof the ripening of such cheeses (Femandez-
García etal. 1990,1993). Ithas alsobeen reported that bacterial methioninase may be helpful in the
generation of aroma when added to unripened Cheddar cheese by promoting the transformation of
sulphurcontaining amino acids into methanethiol, one of the constituentsofthe typical aroma of that
"earthy", "stalky" and "tobacco".Wines onsidered of higher quality wereratedhigher n these non-berry attributes. The use of this technique lead Francis et aL(1992) to similar conclusions with Se-
millon, Chardonnay and SauvignonBlancgrapes.Enzymatic or acid hydrolysiswas ound toenhan-
ce intensity of the attributes "honey", "tea", "lime" or "floral" present in the neutral wine.
5. Eliminationof off-flavorsin foods.
If some enzymes, like ipoxygenasesinsoybeans, lipases in dairy products or proteases n aged
cheeses, have been reported to be associated with off-flavor generation (Heath and Reineccius
1986), here is alsoan ncreasing number of enzymes proposed to eliminateoff-flavors naturally oc-
curing in foods or producedasa consequence of food processing. Some examples date back decades.
This is the case of the use of amicrobial naringinase (a fungal enzyme withrhamnosidase and ß-glu-
cosidase activities) to debitterize grapefruit uice or grapefruit concentrate (Chase 1974).This en-
zyme hydrolyzes the naringin (responsible or the bitter taste) to prunin and rhamnose. There is evi-
dence that thisprocess could be achieved by immobilizing the enzyme on porous glass (Manjón et
al.1985). Another interesting example is given by Hasegawa and Maier (1983). These authors used
a limonoate dehydrogenase fromAcinetobucter to oxidize limonin, a bitter compound of grapefruit
and orange to non-bitter limonoate A-ring lactone and they showed that the process could be greatly
improved by immobilizing the enzyme in acrylamide gel. In the field of alcoholic beverages, the re-
moval of diacetyl from beer has been studied by Tolls etal. 1970). They used a bacterial diacetyl re-
ductase to reduce diacetyl (responsible of unpleasant butter-like note) to2,3 butylene glycol but, al-
though the process was successful, it presented the drawback of requiring great amounts of NADH
cofactor. A different approach exists in the addition of acetolactate decarboxylase to the ferqenta-tion broth at the end of the process. This enzyme allows the direct transformation of a-acetolactic
acid to acetoin avoiding its spontaneous conversion to diacetyl (Cochet 1988). In a more recent re-
port, the gene coding forthisenzyme has already been cloned in Saccharomycescerevisiueused for
brewing, reducing the levelofdiacetyltoless than 0.01ppm(Scott 1989). However, the brewing in-
dustry is very conservative,soit isdifficult to predict if thiskindof strain will reachcommercializa-
tion stage.
Glucose oxidase can be helpful in removing tracesof dissolved oxygen which cause oxidative
rancidity and removing glucose which leads, inturn, o enzymatic browning in processed foods.As
an example, Takenawa et aL(1990) patented a process in which this enzyme is added to hydrated
soybeans in order to reduce the development of oxidation in the preparation of soya milk. Thisen-
zyme has also beenused in processed eggs, dehydrated potatoes andmayonnaise Godfrey and Rei-
chelt 1983).
Another example of enzymatic off-flavor removal is the use of sulphydryloxidase in ultrahigh
temperature ilk. This enzyme oxidizes sulphydryl or thiol groups to disulfides, the former
found that hexanal was converted to either hexanol or hexanoic acid under the action of ADH, alde-
hyde dehydrogenase and the NADH oxidase enzymes. Theyalsoreported its possible application to
reduce off-flavor in deffatted soy milk, since the system was used during sixty cycles without any
loss in enzyme activity. The ADH has also been studieddirectly in foods. Matobaer al. 1990) repor-
tedthe reduction of n-hexanal to n-hexanol in soybean extracts with best results usi@NADH asco-
factor. Hildebrand (1992) reported also that genetic engineering could be an interesting solution t o
indirectly eliminate some off-flavors from food: or example by altering the gene coding for the li-
poxygenases system synthesis thereby stopping the lipoxygenase-catalyzed peroxidation of fatty
acids reponsible for the formation of hexanal.
Finally, an indirect participation of enzymes in the eliminationof off-flavors s the increasing
use of cyclodextrins (CD) in the food industry. Thesearecyclic glucose oligosaccharides containing
6,7or 8 glucose units, prgduced by the enzyme cyclodextrin glycosyl transferasefrom starch. CDs
have been successfully used in the removal of the off-flavor due to naringin and bitter peptides (Psze-
Zola 1988, Korpella etal 1989).
ENZYMATIC SYNTHESISOFFLAVOR COMPOUNDS
One of the important focusses in the flavor ndustry is the biosynthesis, solation and purifica-
tion of individual active chemicalsusing enzymes. Themany advantages of enzymes in biosynthesis
are widely recognized, considering their specificity(enantio and stereo selectivity), he use of mild
reaction conditions and their availability(Sicsic1987). There are parallel efforts in the enzyme re-
search to develop new enzymes through protein engineering (Haas 1984, Leuchtenberger 1992),
new ways of stabilization by immobilization or chemical modification (Shamieral.1989),cofactorregeneration process in redox reactions (Duine 1991 .new strategies suchasextractive bioconver-
sions (Andersson and Hahn-Hagerdal1990) and new properties and specificitiesby the modifica-
tion of the reaction medium (Klibanov 1986,Zaks and Klibanov 1988). In the following section we
will review some examples of flavor compounds that maybe roduced by enzymatic processes, fol-
lowing the already mentioned trends in enzyme technology.
1.Lipases and esterases.
A great deal of research has been directed towards the use of lipid related enzymes - lipases
mainly- (Welsh etal.1989,Yamane 1991) forinvitroflavor synthesis.InFigure 1,the reactions in
this field catalyzed by lipasesareshown. Moreover, special’interest as.been given to ushg organic
solvents as eaction mediabecausein hese conditions, hese hydrolytic enzymes work preferably in
the synthetic way (Klibanov 1986).Recently, Vulfson (1993) reviewed the application of this tech-
nique for food ingredient production.
Ester synthesis by means of lipase is &interesting altemative considering that there aremany
well known flavor esters in the natural aroma of fruits, traditionally obtained by extractionor by
chemical synthesis. The enzymatic synthesis of more than 50esters has been described to date by
Welsh etp_I,( 989). Some of the more recent examples are reported in.Table 3.
out that hydration of immobilized C. cylindr ce lipase decreased the final ester concentration, al-though the hydrated enzyme wasmore stable after repeated use. In terms of strategy, microaqueous
or biphasic water/solvent systems are preferable to reverse micelles as shown by Boaeix et
aL(1992) with butyl butyrate synthesis as a model.
described (Omata etal.1981). Moreover, these authors showed that the catalytic activity of the cellswas enhanced because of its entrapment n polyurethane resin gels (estimated half life of more than
55days)yieldingL-mentholoflOO opticalpurity.Theesterificationcanbecarriedoutdirectlyin a
specific process to produce L-menthol-5-phenyl valerate using Candida Cylindracea lipase adsor-
bed onto celite and entrapped in polyurethane (Cheetham 1991). The same selective esterification
may be carried out for the resolution of DL-citronellol (Cheetham 1991) and DL-bomeol mixtures
with esterase of Trichudenna (Oritani and Yamashita 1976).
Enantiomericsecondary alcohols are present in fruits such asblackberry, com, coconut or ba-
nana. As an altemative oute to synthesize hese compounds, lipase enantioselectiveesterifications
have been studied n thelast few years. Janssen etal.( 1991)have shown it was possible to apply this
technique successfullyfor the resolution of secondary alcohols by transesterification n alkyl carbo-
xylatesassolvent. They pointedout that addition of amolecularsieve o the reaction mixture greatly
improved he reaction yield for theresolution of 1-phenylethanol. Another examp e is given by Ger-
lach etal.( 1989)who prepared aliphatics and phenyl alkanols with high chemical and optical yields,
outlining the importance of experimental conditions such as temperature, chain length of the sub-
strate and enzyme immobilization.
Lipase ofMucorspecies has been shown to catalyze the lactonization reaction of 15-hydroxy-
pentadecanoic and 16-hydroxyhexadecanoic acids to the corresponding macrocyclic lactones
(Antczaketal. 1991).Thereaction was achieved in an organic mediumcontaining ether and'ioluene
at80 Cand aconversionyieldofover30%wasreached.The yieldofthisreaction can beincreasedif
free water contentismaintained at0.083% and if sugar alcohols such aserythritol, arabitol or sorbi-
tol are added before lyophilization of the enzyme (Yamane et al. 1990).
2. Oxidoreductases.
Oxidoreductases play major roles in determining the quality of certain food products (Whita-
ker 1984). Nevertheless, very few of them have been used for industrial applications, mainly due to
the difficulties found in the economical production and/or regeneration of cofactors-involved n the
process. There are, however, some examples indicating the potential application of oxidoreducta-
ses, regenerating the cofactor by a second enzymatic reaction, mainly in a membrane reactor where
the cofactor may be retained.
2.1 Alcohol dehvdroeenase (ADH).
Thisenzyme canbeextractedfromhorse liver, plants like the grape (Molina etal.1986, 1987)
or produced by microorganisms.I t s able to workinboth directions, for reducing ketones to alde-
hydes, aldehydes to alcohols or oxidizing alcohols to the corresponding aldehydes. Initially, possi-
ble applications of the ADHwere reported by Ericksson (1975). The relation between the intercon-
version yield of aldehyde to alcohol and the sensorial note obtained was studied, showing that ADH
added with NADH to milk containing polyunsaturated fats could reduce the amountof aldehydes,
particularly hexanal (Erickssoner al.1977). A few years later, Tamaki and Hama (1982) purified
and characterized the ADH from bakers' yeast. This enzyme was found to be active for the oxidation
of a wide range of aldehydes to their corresponding carboxylic acids.
Acetaldehyde plays asignificant role in the flavor of certain fruits ike orange and foods like yo-
gurt. Raymond (1984) patented an enzymatic procedure employing ADH to prdduce acetaldehyde
from ethanol present in orange essence, in which the cofactor NAD is regenerated by use of flavin
mononucleotide (FMN)in a light-catalyzedprocess with oxygen and catalase. Three other different
methods for the production of flavor aldehydes were tested by Legoy et aL(1985). These authors
studied the conversion of geraniol to geranial n a biphasic system (waterhexane) taking advantage
of the difference in solubility of each compound in the organic phase.This system avoids the end
product inhibitory effect allowing conversion yields close o50%. t has been applied successfully to
the production of citronellal, hexanal and 3-phenyl propanal. BowenetaL 1986)used asimilarpro-
cess to study the conversion of cinnamaldehyde to cinnamyl alcohol with an immobilized yeast
ADH.Reducing enzymes (specifically secondary ADH) of acetic acid bacteria were found to trans-
form a great number of ketones to their corresponding alcohols.Best results were found with the en-
zymatic system ofGluconobacteroxydansandAcetobacter acetiireducing 12 ketones to S)- alco-
holswith an enantiomeric excessofmore than 94% (Adlercreutz 1991) while the redox-enzymatic
system of Gluconobacter subuxydanswas found to be able to convert glucose in 5-ketogluconic
acid, which is then convertedby controlledheating into amonomethyl furanone, astrongly meat-fla-
vor molecule (Cheetham 1991).
Braun and Olson (1986) attained he controlledproduction of acetic acid, 3-methyl-butanal and
3-methyl-butanol with cofactor recycling (NAD-NADH) with the reducing enzyme system of Glii-
conobacteroxydansand Streptococcus lactis.The alcohol dehydrogenase and aldehyde dehydro-
genase of G. oxydanswere shown to be able to regenerate NADH and to produce acetic acid fromethanol while the enzymatic system ofS. l c h transaminase, decarboxylase and ADH) was found
to synthesize the aldehyde (with malty flavor and the alcohol from leucine as precursor).
2.2 Alcohol oxidase.
Benzaldehyde is themainmolecule in the cherry fruit flavor and is enzymatically fo.rmed when
the seeds or pits of fruits suchasalmonds, apricots, peaches orapplesarecmshed. The biotechnolo-
gical production of this compound by enzymic oxidation with alcohol oxidase has been studied in
the past few years (Cheetham 1993). Duff andMusay (1989), showeditwaspossible ousethisuni-
directionalenzyme (only oxidation pathway) to produce benzaldehyde from benzyl alcohol. They
developed a two phase whole-cell process, withPichiapastoris, a methylotrophic yeast, using cata-
lase to transform the denaturing H202 produced. The same authors (1990), demonstrated that it was
possible to extend this application to the oxidation of c2 c6aliphatics alcohols to their correspon-
ding aldehydes. Nevertheless,Nikolova and Ward (1992), showed that the whole-cell process gave
higher rates of conversion than cell-free extracts with Saccharomyces cerevisiae. As a novel
concept in bioprocess engineering, Banana et aL(1989) studied the enzymatic oxidation of ethanol
in the gaseous phase with twomicrobial alcohol oxidases.The authors demonstrated he importance
of water activity on both enzyme thermostabilityand activity.- -
In astrategy of cofactorregeneration by means of coupling two enzymatic reactions, a process
has been developed by the Denki Kagaku Kogyo company to produce gluconic acid, an acidulant,
from glucose using glucose dehydrogenase requiringNADP+, the cofactor s reduced again with the
sihe substrate, glucose, but transformed by the enzyme aldose reductase to sorbitol: the resulting
concentrated syrup has sweet and acid taste (Fukui 1990).
2.4 Other oxidoreductases.
- A dehydrogenase fromPseudomonasputidahas been found to be able to convert L-menthone
into L-menthol (Nakajimaetal.1978). Thisfinding canbe applied to the bioconversion of the essen-
tial oil (containing nearly only L-menthone) of the plantMentha piperita inits immature stage in or-der toincreask the proportion of L-menthol, the active flavoring compound. Mitsui Company has re-
cently reported a similar process using cells of Cellulomonas urbata.In both cases, there is a need
for a cofactor regeneration system. In 1979, Takahashi etal.isolated an aldehyde oxidase from bo-
vine liver and applied it for the reduction of the green beany taste in soybean. The reaction was car-
ried out under aerobic conditions with dissolved oxygen as the electron acceptor, without the need of
cofactor. It is assumed that the loss in the intensity of green note was due to the oxidation of n-penta-
nal and n-hexanal.
Ionones and other related compounds including ß-ionone and ß-damasconearevery important
aroma compounds in tobacco and many fruits, formed by enzymatic degradation of carotenoids.
Sode et aL(1989) have been able to cany out the microbial conversion of ß-ionone using A. niger
cells in organic solvents. However, using enzymes such as polyphenoloxidase, it has been possible
to obtain small amounts of ß-ionone, when acting on substrates ike palm oil rich in carotenoids
(Therre, 1990).
In the field of lactones, Maguire et al. 1991) achieved the reaction of lactonization of the lino-
leic acid employing an immobilized soybean lipoxygenase leading to acl macrocyclic lactone as a
product. Willets et al. 1991), obtained the transformation of endo-bicyclo heptan-2-01s and endo-
bicyclo hept-2-en-6-01 into their corresponding lactones using a coupled enzyme system (dehydro-
genase from Themoanaerobium brockii and monooxygenase fromAcinetobacter calcoaceticus)
with coupled regeneration of NADPH+/NkDP+ cofactor.
As an indirect positive influence on the development of flavor in wine, the action of glucose
oxidase to remove any remaining glucose, transforming it in gluconic acid maybecited (Heresztyn
1987).
Using formate dehydrogenase as he second enzyme for regeneration of NAD, the company
Genzyme produces a wide range of optically active a-hydroxy acids via lactate dehydrogenase as
first enzyme (Dunnet al.1991).In he case of flavor compounds, several enzymes display potential
applications. Magee et al. 1981) produced diacetyl (responsible for butter flavor) and acetoin by
multienzymatic system consisting ofmilk fat coated microencapsulated cell free extracts of Strep-
tococcus lactis. These authors assumed that diacetyl was synthesizedthrough the oxidative path-
way involvinglacetyl-CoA, nd acetoin through the a-acetolactate route, both of them by means of
oxidative enzymes.
3. Other enzymes
The major groups of enzymes involved in the productionof flavors are hydrolases or oxidore-
ductases. Nevertheless, other types of biocatalysts canberegarded aspotentialy interesting.This is
the caseof a lyase: the a-terpineol dehydratase catalyzes the reaction of limonene (a by-product of
the citrus ndustry)conversion o a-terpineol, monoterpeneof great interest.This enzyme has been
isolated from Pseudomonas gladioli by Cadwallader er aL(1992). These authors showed that the
isolate was composed of2proteins with apparent molecular weight of 94,500 and 206,500 daltons.
Almosnino and Belin (199 1) solated an enzyme systemfrom apple pomace, able to produce C6-Ciovolatile aldehydes fromlinoleic acid through the action of two main enzymes: lipoxygenasewhich is
reponsible of the formation of hydroperoxides from unsaturatedfatty acids and hydroperoxide yase
which ensures the generation of the volatile aldehydes. They found that the amount of aroma com-
pound could be significantly increased by adding SO2 or vitamin C in the reaction medium.
CONCLUSIONS
Research n the areas of food technology and biotechnologyhas been influenced by the increa-
sing demand of consumers for nutritious ood and natural food ingredients. In this review, the impact
of enzyme technology on flavorhas been examined. Itmust be pointed out that this review mainly
concernsreports in the scientific and technological iterature and shows he trends in research on fla-
vorrelated enzymes. It is helpful1 to add that most of the actual researchis far from the appkation to
the flavor industry. Some potential applications are mentioned and they stillneed further research
and to show economical feasability in order to reach the commercial scale. Other problems encoun-
tered are the production capacity and the enzymes stability. Genetic and protein engineering may
contribute to overcome some of these drawbacks but it is mportant to consider the development
costs including eventual toxicological studies and market sizes.Also, some sectors of the food in-
dustry are regarded as traditional and might not beopened soeasily to the introduction ofnew pro-
ducts or processes.
However, some advantages of enzymatic-cátalyzed synthesis (mild temperature, pressure and
pH conditions,high enantioor regiospecificity, ackof contaminatingby-products) are attractive for
the production offlavorcompounds.Itmaybeconcludedthat theuseofenzymes as additives forfla-
vor production and/or modification may become an important part of enzyme technology as shown
by some recent developments. The better understandingof flavor generation in foods aswell as the
advances n the use of enzymes in non-aqueous media alsqoffersa great potential for this activity in
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