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Applications of Oxidoreductases in Foods
Colja LaaneDSM Food Specialties, Delft, The Netherlands
Yvonne BruggemanUnilever Research, Vlaardingen, The Netherlands
Chris WinkelQuest International, Naarden, The Netherlands
I. INTRODUCTION
Oxidative and reductive processes play an important
role in foods. They influence not only the taste but also
the texture, appearance, shelf life, nutritional value,
and process tolerance of food products. Both enzy-
matic and nonenzymatic redox processes are involved.
In some cases redox processes lead to undesirable
effects such as off-flavor, reduced shelf life, or texturalproblems. In other cases, they contribute in a positive
way to the final aroma, an improved texture, a more
desirable appearance, or an increased shelf life.
Controlling the redox behavior in food systems during
all stages of processing and storage is therefore of
utmost importance.
Up until now redox reactions in foods were con-
trolled mainly by carefully selecting raw materials, by
adapting process conditions, by adding chemicals or
antioxidants, or by designing air-tight packaging mate-
rials. As yet, little attention has been paid to tailor
redox reactions in foods by the addition of oxidore-ductases or by changing the profile or content of oxi-
doreductases in food raw materials by genetic tools.
Presumably, the major bottleneck for the application
of oxidoreductases to foods is that economically effi-
cient enzyme production is, with a few exceptions, still
not feasible. In addition, public concerns about the us
of recombinant enzymes in food products is slowing
down their market introduction.
In this chapter the current and potential usage o
oxidoreductases in controlling the taste, texture
appearance (i.e., color), shelf life, and the nutritiona
value of food products will be discussed. Increasingly
redox enzymes are being used for biosensor applica
tions in food systems. This topic will not be discussedin this chapter, however. Table 1 lists the most impor
tant redox enzymes and their functions in food sys
tems. Most attention will be paid to the marke
segments bakery, beverages, and dairy and to oxidases
since relatively little is known about role and applica
tions of reductases in food systems. Furthermore, th
emphasis will be on added enzymes and not o
enzymes already present in the food product constitu
ents. For detailed information on the properties of th
individual oxidoreductases the reader is referred to in
Sec. IIA of this book.
II. TASTE
Many oxidoreductases play a role by influencing th
taste profile of food products. They are involved in th
in vivo/in situ biogenesis of desirable aroma compo
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Table 1 Overview of the Main (Potential) Applications of Oxidoreductases in Food Products
Enzymes Taste Texture Appearance Shelf life
Lipoxygenases (LOX) In situ and in vitro
(off)-flavor
production
Dough extensibility
and strength
Flour bleaching
Alcohol oxidases and
dehydrogenases
(AO, ADH)
In situ and in vitro
flavor production
(ILOX)
AO plus catala
removal
Sulfhydryl oxidases
(SOX)
Removal of cooked
flavor in UHT-milk
Dough strengthening in
combination with
POX
Peroxidases (POX,
LPO)
In vitro flavor
production and
debittering
Crosslinking
biopolymers
Assisting PPO in color
formation
LPO, antimicr
(Poly)phenol oxidases
(PPO)
Debittering of coffee,
cacao, and olives
(Dis)coloration; in vitro
production of colors
Laccase, O2 re
Carbohydrate oxidases
(GOX, HOX,
PYROX)
GOX, mild acid
production
Dough strengthening
via H2O2; thickening
agent
GOX plus cata
removal
Ascorbic acid oxidase
(AAO)
Browning AAO plus cata
removal
Xanthine oxidases (XO) Antimicrobial
Superoxide dismutases
(SOD)
Removal react
species with
Catalases Removal exces
Cholesterol
oxidoreductases
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nents, in the in vitro production of flavoring topnotes,
and in the endogenous formation of off-flavors. The
main redox enzymes are discussed below.
A. Lipoxygenases
Lipoxygenase (LOX, EC 1.13.11.12; see Chapter 43,
for detailed information), formerly known as lipoxi-dase or carotene oxidase, is an iron-containing
dioxygenase which catalyzes the oxidation of polyun-
saturated fatty acids containing cis, cis-1, 4-pentadiene
groups (linoleic, linolenic, and arachidonic acids) to
the corresponding conjugated cis, trans dienoic mono-
hydroperoxides. In addition LOX also accepts a rela-
tively broad range of phenolic compounds as
substrates (1) and is capable of oxidizing other sub-
strates than the actual substrate. This process is
known as co-oxidation and includes compounds like
carotenoids and polyphenols (2).
LOX is one of those endogenous enzymes that playan important role in both flavor generation and off-
flavor formation in virtually all food products derived
from plant raw materials. Depending on the final con-
centration and the type of food product the same fla-
vor component can be a desired aroma component or
an off-flavor at higher concentrations. For example,
C6-aldehydes and alcohols derived by the LOX-cata-
lyzed oxidation of (poly)unsaturated fatty acids have,
in most cases, a positive effect on the aroma profile
(e.g., wines and juices), but in other beverages, have
an undesired flavor effect (e.g., beer). Likewise, endo-
genous LOX is known to generate carbonyl com-pounds in dough systems and hence influences bread
flavor (3).
The formation of C6 compounds requires the
sequential action of four enzymes of which two are
redox enzymes, namely an acylhydrolase, a lipoxygen-
ase, a hydroperoxide lyase, and a yeast-derived alcohol
dehydrogenase (4). Typically, off-flavor formation is
prevented by using crop variants deficient in LOX iso-
enzymes, either by screening or by genetic tools (5) or
by controlling the oxygen levels during processing (6),
as well as by removing the malodorous oxidation pro-
ducts afterwards. The deodorization of off-flavors canbe achieved by physical techniques such as adsorption,
or enzymatically by converting the undesired alde-
hydes (e.g., with aldehyde dehydrogenase/oxidase), or
alcohols (e.g., with alcohol oxidase) into their corre-
sponding less flavorful carboxylic acid. The use of
redox enzymes for this purpose has been claimed for
several products such as margarine, cream, fish oil,
noodles, cooked rice, and soybean products (7, 8).
LOX is also used for the in vitro production o
several natural topnote flavors, which are mainl
added to beverages and dairy products to tailor the
flavor profile (9, 10). Typical examples include the con
version of polyunsaturated fatty acids into variou
short to medium-chain aldehydes/alcohols (13), o
into S(-)--decalactone (butter flavor; 12). Well
known fatty acidderived flavoring aldehydes/alcoholinclude the above mentioned C5 and C6 compounds
as well as (E2, E6)-nonadienal (cucumber), 1-octen-3
one (field mushroom), (Z5)-octadien-3-one (geranium
leaves), (E3, E5)-undecatriene (blasamic), and (E3, Z5
Z8)-undecatetraene (seaweed). Depending on th
degree of unsaturation and the regioselectivity of th
lipoxygenase, different hydroperoxy compounds ar
formed, from which the above-mentioned compound
can be derived by subsequent enzymatic reactions. Fo
(Z3)-hexenol, linolenic acid is used as a substrate, whil
for most of the other aldehydes/alcohols higher unsa
turated fatty acids are required. The production o(Z3)-hexenol has recently been commercialized usin
plant homogenates (e.g., alfalfa sprouts, green pep
pers) which are relatively rich in hydroperoxide lyase
the enzyme required to split the hydroperoxide fatt
acid into smaller fragments. The generated (Z3)-hexe
nal was converted into (Z3)-hexenol using the reduc
tive enzymatic power of bakers yeast (13). A ver
elegant approach has been taken recently b
Givaudan-Roure. To unify all three enzymes involved
in the formation of (Z3)-hexenol, they have cloned and
overexpressed both soybean LOX and the hydroper
oxide lyase from banana in bakers yeast. In this waythey have developed a single-step process which pro
duces (Z3)-hexenol in relatively high yield (14).
For the production of lactones a different strategy
has to be followed after the formation of the linolei
acid hydroperoxide. It involves the fermentative -oxi
dation of the hydroperoxide intermediate by the yeas
Pichia etchellsii, and the subsequent cyclization of 5
hydroxydecanoic acid to the corresponding S(-)--dec
alactone (10).
As shown by Quest International (1), lipoxygenase
also accept a relatively broad spectrum of phenoli
compounds as substrates. Of interest to the flavoindustry are the LOX-catalyzed conversions of isoeu
genol and coniferyl benzoate from Siam resin int
vanillin. At present the commercialization of these bio
transformations is hampered by the fact that isoeu
genol is not readily available and that conifery
benzoate is difficult to handle in a reactor.
Other well-known reactions of LOX include the co
oxidation reaction of carotenoids to yield, amon
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others, -ionone (2, 15). More recently is the genera-
tion of a toasted or cookie-like flavor in starchy mate-
rials by a combination of LOX and an -amylase (16).
B. Alcohol Oxidases and Dehydrogenases
In general, aldehydes are more potent flavor com-pounds than their alcoholic counterparts. Hence, alco-
hol oxidases are interesting enzymes for the in vitro
production of flavoring preparations which, among
others, can be applied in beverages. Typical examples
include the use of methanol oxidase from Pichia,
Hansenula, and Candida (17) for the production of
natural acetaldehyde from ethanol. This enzyme is
induced during growth on methanol. At the end of
the logarithmic growth phase cells are harvested and
incubated with ethanol. In this way concentrations of
$ 1:5% natural acetaldehyde can be achieved, which
can be concentrated further to the desired applicationlevel. From yeast to yeast the substrate specificity of
the alcohol oxidase is different. Hence, this procedure
can also be used to convert other alcohols, such as
hexenol and other long-chain alcohols, to their corre-
sponding aldehyde (10, 18).
As alternatives to alcohol oxidases the correspond-
ing dehydrogenases could in principle be used (19). A
severe drawback, however, is that these dehydro-
genases require the expensive cofactor NAD(P)
instead of (cheap) oxygen as an electron acceptor.
Although various sophisticated NAD(P) cofactor
regenerating systems have been designed and substan-tial cost reductions have been realized in this way, it is
evident that in commercial applications oxidases are
preferred over their dehydrogenase counterparts. The
use of dehydrogenases for food purposes seems to be
restricted to whole-cell conversions.
Vanillyl alcohol oxidase (VAO) from Penicillium
simplicissimum is a special type of alcohol oxidase.
Recently, it has been shown that this stable, flavin-
containing enzyme has a very broad substrate specifi-
city and readily converts para-substituted phenols into
interesting flavor precursors or flavoring compounds
(2022). Apart from natural vanillin and coniferylalcohol, different vinylphenols (e.g., para-vinylguaia-
col) and allylphenols can be produced from cheap
raw materials and oxygen as an electron acceptor.
VAO can also be used for generation of flavor building
blocks. To that end a natural mix of phenolic com-
pounds could be treated with VAO to enrich foods
or flavor preparations with a range of vinylic/allylic
and aldehydic substances.
C. Sulfhydryl Oxidases
Sulfhydryl oxidase (SOX, no EC number assigned
Chapter 41 for more details) catalyzes the formatio
disulfide bonds from (protein) thiols. SOX is an Fe
containing glycoprotein and has been detected
bovine, human, goat, pig, rabbit, and rat milks (
The enzyme is capable of oxidizing the sulfhygroups of cysteine and glutathione, as well as milk
teins to their corresponding disulfides using molec
oxygen as electron acceptor (24). In practical app
tions bovine milk SOX may be added to UHT mi
reduce cooked flavors. Patents (see Refs. in 24) for
application have been issued. The enzyme has
immobilized on porous glass, and its effectivene
ameliorating the cooked flavor has been demonstr
on a pilot scale using immobilized enzyme column
D. Peroxidases
Peroxidases (POX, EC 1.11.1.7; see Chapters 28 an
for more details) occur widely in nature and is
general name for a group of both highly specific
nonspecific enzymes which use hydrogen pero
instead of oxygen as an electron accep
Peroxidases, especially the heme-containing ones,
catalyze a large number of different reactions inclu
sulfoxidation, N-demethylation, oxidation, and hy
xylation and hence are of potential interest in the
duction of specific flavoring topnotes. A re
example involves the demethylation of methy
methylanthranilate (ex Citrus) to monomethylantnilate (25). The latter compound is an important
note flavor in Concord grapes. Soybean, horserad
and microperoxidases were found to be convenient
alysts for this reaction. In addition POX gives rise
fresh flavor profile when added to tomato paste (2
E. Polyphenol Oxidases
Polyphenol oxidases (PPO) are a group of sev
enzymes. Different activities can be found within
group: tyrosinase (monophenol monooxygenase,
1.14.18.1; see Chapter 39) converting a phenol incatechol group; 1,2-diphenol oxidase or catechol
dase (EC 1.10.3.1) converting catechol into an o-
none, and laccase (EC 1.10.3.2; see Chapter
converting a 1,4-diphenol into p-quinone. Usually
first two activities are linked, as catechol is much m
readily oxidized than a phenol. Most enzymes
catalyze 1,4-diphenol oxidation also act on 1,2-di
nols.
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The bitter taste in many food products is often the
result of the presence of polyphenolic compounds such
as guaiacols. PPOs are capable of oxidizing these com-
pounds and hence can be applied to reduce bitterness.
At present the debittering of coffee beans (27), Adzuki
beans (28), and cacao beans (29) by PPOs have been
claimed. Another application involves the use of laccase
to debittering olives by treating stoned, chopped olivesin the presence of air at increased temperatures (30).
III. TEXTURE
The use of oxidoreductases in texturizing food is based
on their ability to crosslink proteins and/or polysac-
charides. This property is of particular interest to
improve the texture of dough and paste products (soft-
ness, volume, elasticity, crunchiness) or dairy products
(mouth feel, appearance), and of fish and meat pastes.
Basically, enzymatic crosslinking can occur via tworoutes:
1. Indirect via enzymatically produced hydrogen
peroxide (e.g., glucose oxidase, hexose oxidase, ascor-
bic acid oxidase), or via the enzymatic production of
radicals (e.g., peroxidase, lipoxygenase).
2. Direct via the oxidation of functional groups in
the protein. Examples include the linkage of tyrosine
and ferulic acid residues by tyrosinase and laccases,
linkages of lysine residues by lysyl oxidase, or the for-
mation of disulfide bridges by sulfhydryl oxidase.
An alternative, nonredox procedure for direct cross-
linking is being explored using transglutaminases (seeChapter 51).
These enzymatic routes are being explored to re-
place chemical oxidizers, such as dehydroascorbic
acid and potassium bromate. Replacement of the non-
specific chemical compounds by redox enzymes could
have the benefit of more specific and better-controlled
oxidation processes. Other disadvantages of the chemi-
cal substances are related to safety and labeling issues.
One of the major challenges in the baking industry is to
find a good replacer for potassium bromate, which is a
difficult task since bromate is still more effective and
cheaper than the enzymatic alternative. The mostimportant texturizing redox enzymes will be discussed
below.
A. Lipoxygenases
A rich source of LOX is soybeans. Soybean LOX con-
tains three distinct lipoxygenase isoenzymes, desig-
nated as L1, L2, and L3. These isoenzymes have
been isolated from seeds of commercial cultivars and
have been well characterized (31, 32). Recent studie
indicate that L2 has the greatest effect among LOX
isoenzymes on dough extensibility and strength (33
and is also mostly responsible for the production o
undesirable aroma compounds in bread doughs (34
see Sec. II, Chapter 43). For L3 an increase in foamin
activity has been reported, as well as an overaimprovement in breadmaking quality of wheat flou
(35). The bakery yeast Saccharomyces cerevisiae also
contains LOX. Recently, this enzyme was partiall
purified (36), but its potential, if any, on breadmakin
remains to be established.
Nowadays, it is feasible to change the profile and
content of LOX (iso)enzymes in plants either by clas
sical means (5), or potentially by genetic modification
For example, by appropriate crosses, near-isogeni
soybean seeds have been developed that lack eithe
isoenzymes L1 and L3, or isoenzymes L2 and L3
These LOX-minus mutants still grow well in the field(37). In principle, transgenic plants lacking or overex
pressing one or more LOX isoenzymes could be con
structed and tailored to specific applications (38). To
that end the heterologous expression of one or more
soybean LOX isoenzymes in wheat could be of interest
B. Sulfhydryl Oxidases
The action of SOX (see Sec. II, Chapter 41, for genera
information) may be the same as those of chemica
oxidizing agents, provided the formation of disulfid
bonds is the primary mode by which these agents function. In extensive testing (39, 40) it was found tha
SOX alone has no influence on loaf volume, doug
strength, or mixing tolerance. Also, relatively high con
centrations using recombinant SOX from Aspergillu
awamori did not have any significant positive effec
(40). One reason could be that SOX has only a limited
affinity for thiol groups in gluten proteins and as
result its application in the food industry seems to b
limited to the removal of small off-flavor molecule
(see Sec. II, Chapter 41).
C. Peroxidases
For detailed information about POX see Section II
Chapters 28 and 29. Wheat flour contains a peroxidas
that can crosslink phenolic constituents such as feruli
acid and vanillic acid. However, the pH optimum o
the wheat enzyme is 4.5, and in the pH range of 56 o
wheat doughs the activity is considerably lower than a
pH 4.5 (41). Although the dough-improving effect o
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peroxidase is well documented, the reactions involved
in the dough are only poorly understood.
Model studies with peroxidase, glutathione, and
cysteine indicate that sulfhydryl and/or lysyl groups
may be involved (42). Peroxidase-catalyzed reactions
result in a decreased amount of lysine recovered from
proteins after acid hydrolysis. Peroxidases, or the qui-
nones formed by the enzyme, oxidatively deaminatelysyl residues to form lysylaldehydes, resulting in the
formation of protein polymers, as was revealed by gel
filtration. These aggregates could not be dissociated by
detergents, which indicates that covalent bonds were
formed. In addition, the dough-strengthening effect of
peroxidases is also ascribed to the crosslinking of car-
bohydrates and the coupling of carbohydrates to pro-
teins. The gelation of wheat pentosans is due to the
oxidative dimerization of ferulic acid moieties, which
are covalently linked to pentosans (43).
Recently, a study has been described showing the
effect of different peroxidases on the baking perfor-mance of German Kaiser rolls (41). Deliberately
sticky doughs were prepared in order to demonstrate
the effects of added peroxidases. Especially the addi-
tion of soybean POX led to strengthening of wheat
doughs as determined by the stability of the dough
after shaking the dough for 1 min in a laboratory
shaker. This improving effect was observed at both
short and long fermentation times. Dough volume
increased after application of these peroxidases.
Compared with (GOX), the results of peroxidases are
better or at least equal in terms of stability and volume.
In bread dough, peroxidases seem to act without theextra addition of hydrogen peroxide in spite of their
peroxide dependence. This may indicate that peroxide
is present in dough at sufficient amounts, or that it is
generated as a result of the peroxidase reaction. In this
way, substrate radicals formed by peroxidase can react
with oxygen to form hydrogen peroxide. When this
reaction is occurring, catalytic amounts of peroxide
present in dough are sufficient to get the cycle started,
and may explain why no hydrogen peroxide has to be
added together with a peroxidase. An alternative
explanation is that wheat contains endogenous oxi-
dases, which are active enough to produce some hydro-gen peroxide in situ. Indeed, the addition of hydrogen
peroxideproducing enzymes such as GOX has a ben-
eficial effect on baking (4446).
In other systems than doughs, POX has been
claimed as a thickening and stabilizing agent in, for
example, ice creams, deserts, sauces, and jams and jel-
lies (47). There is also a recent patent application (48)
on the POX-catalyzed gelling of hemicelluloses to form
gels or viscous media for application as fat or gel
replacer, as well as for flavor delivery, coating, or g
ing.
D. Glucose Oxidases
Glucose oxidase (GOX, EC 1.1.3.4; see Chapter 30
detailed information) catalyzes the conversion of cose into the mild-tasting gluconic acid via glucon
lactone and hydrogen peroxide. GOX complies
the FAO/WHO and GRAS requirements for
grade enzymes and is one of the few commerc
available oxidases from Aspergillus niger
Penicillium strains at relatively low costs. Especi
the generation of hydrogen peroxide is believe
give the antiweakening effect in bread doughs
In a recent study by Hilhorst et al. (49) the effec
GOX on Dutch rusk dough was dough stiffening
a clear loss in extensibility. This made the overall e
quite undesirable. For comparison, they also teperoxidase (see Sec. III.C above). This enzyme ga
dough-stiffening effect without loss in extensib
The authors explain the difference by the fact
hydrogen peroxide from the GOX-catalyzed reac
oxidizes randomly and links the gluten network
the arabinoxylan network, whereas the peroxidase
increases the amount of crosslinks in the arabinox
fraction without affecting the gluten network or
coupling between the networks.
It has been found that synergistic effects occur w
using a combination of oxidative enzymes like
combination of GOX and SOX (50). Furthermthe dough has an increased stability.
E. Hexose Oxidases
A newcomer in the field of redox enzymes for ba
products is hexose oxidase (HOX, EC 1.1.3.5) from
seaweeds. This glycosylated flavoprotein is relate
GOX but has a broader substrate specificity tow
hexose sugars, including oligomers. Like GOX
acts on the C1-position of the sugar moiety
Recently, the enzyme from Chondrus crispus has isolated, cloned, and overexpressed in several reco
nant organisms such as Pichia pastoris, Saccharom
cerevisiae, and E. coli (51, 52). However, the pro
tion levels of active enzyme are still poor at
moment and have to be improved. For this rea
and because C. crispus has a long standing tradi
as an edible organism, efforts have been mad
develop a large-scale production method with the
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of carrageenase, which reduces the viscosity of the
crude extract (51).
For HOX similar applications can be envisaged as
for GOX (see above). In particular, its application in
bakery products is foreseen (53), since HOX is able to
generate more H2O2 owing to its broader substrate
specificity, and hence should be more effective than
GOX in dough strengthening. Likewise, combinationsof HOX and H2O2-consuming enzymes, such as POX,
may be envisaged.
Another enzyme with similar properties as HOX,
isolated from Acremonium strictum T1, has been
reported in the literature (54). The enzyme is called
an oligosaccharide oxidase and is able to oxidize sev-
eral di- and oligosaccharides at the anomeric site,
yielding the corresponding di- and oligobionic acids.
A detailed comparison between HOX and oligosac-
charide oxidase performance in dough applications
has not been made yet.
F. Pyranose Oxidases
Pyranose oxidase (PYROX, EC 1.1.3.10; see Refs.
5557, 59 for more detailed information) catalyzes
the oxidation of several monosaccharides at the C2-
position and is therefore different from GOX, which
oxidizes glucose at the C1-position. While GOX is very
specific, PYROX is able to catalyze other substrates
such as maltose and pentoses (e.g., xylose). As a result,
the PYROX oxidation product of glucose is 2-keto-
glucose and not gluconic acid. At present, this enzyme
has been purified from several white rot fungi and aBacidiomycetous fungus (5557). PYROX has also
been reported to show significant activity toward D-
glucono-1,5-lactone, which is produced by the GOX-
catalyzed oxidation of glucose (58). So if PYROX is
combined with GOX, there will be more substrate
available for PYROX, thereby prolonging the activity
of PYROX and enhancing the total amount of hydro-
gen peroxide produced (59). The claimed effects in
baking are gluten strengthening, reduced dough sticki-
ness, and increased volume and crumb structure for
bread (59).
IV. APPEARANCE/COLOR
Apart from texture, the appearance of food products is
determined to a large extent by their color. Several
redox enzymes are known to influence the color of
foodstuffs. The most important ones will be discussed
below.
A. Lipoxygenases
It is interesting to note that the use of soybean lipox
ygenase was described in the 1930s as a means t
bleach the flour in preparation of white bread. Mor
recent experiments have shown that carotenoids pre
sent in wheat flour are destroyed by co-oxidation
Wheat flour itself contains little LOX activity, buLOX is abundantly present in, for example, soybeans
To that end wheat flour is often fortified with up to
0.5% enzyme-active soy flour (34, 60). Other applica
tions of LOX include the bleaching of noodles, whey
products, rice, and wheat bran (6164).
B. (Poly)phenol Oxidases and Peroxidases
PPOs (see Sec. II, Chapter 39 for general information
play an important role in the browning of fresh fruit
and vegetables (65, 66), in the coloring and flavoring o
tea (67, 68), and in improving the quality of coffee (69)A major concern in the food industry is to preven
the development of enzymatic browning prior to th
processing of fruits and vegetables (70). This is accom
plished by removing oxygen or by inhibiting PPO
activity using inhibitors such as metal chelating agents
inorganic ions (e.g., halide anions), benzoic acid and
some substituted cinnamic acids, reducing agents (e.g
L-cysteine, glutathione, sulfite, SO2, ascorbic acid)
small natural peptides, and combinations thereo
(68). In contrast, during the fermentation of black
tea PPO is used to initiate browning by oxidation o
polyphenolic substances such as catechins to theaflavins.
The quality of tea, based on sensory evaluation o
color and bitterness, has been correlated with tota
theaflavin content (71). Theaflavins, thearubigins, and
caffeine are all essential ingredients in high-qualit
teas. The addition of microbial laccases and/or perox
idases to green tea has shown that no higher theaflavin
levels can be obtained than with endogenous PPO
Addition of exogenous laccase/peroxidase to black
tea, however, did yield a very significant increase in
the color intensity of the tea (72).
Polyphenol oxidases also play an important role incoffee processing (69). The activity of PPO in green
coffee beans has been consistently related to the qualit
of the coffee beverage. The exact role of PPO in coco
beans is less well understood. There are, however, indi
cations that it plays a role in browning during th
curing of the cocoa beans (73).
In cereals, PPO is responsible for the darkening o
the breadcrumb, particularly in whole-grain and ry
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breads. The rate of browning is greatest in sourdough
baking. Excessive discoloration can be prevented by
the addition of sodium metabisulfite or ascorbic acid
(41). Exogenous PPO is also reported to have a posi-
tive influence on the rheological properties of bread
(74).
PPO can also be used for the in vitro production of
colors. For example, Pruidze (75) reported the produc-tion of a whole range of natural colors by treating
waste streams of beet and tea with PPO.
Furthermore, it was claimed that safflower pigments
become darker red when safflower petals were sprayed
with a dilute laccase solution (76).
The role of endogenous POX in (dis)coloration is
not fully understood. It seems that the oxidative role of
peroxidases is useful in assisting PPOs in oxidizing the
polyphenols and ultimately contributing toward the
color and flavor of tea. The same seems to be true
for coffee and cocoa.
C. Ascorbic Acid Oxidases
Ascorbic acid oxidase (E.C.1.10.3.3; see Ref. 77 for
detailed information) plays a role in beverages such
as lemon and grapefruit juices, where it is responsible
for the initiation of browning and loss of vitamin C
activity during storage (77). The extent of browning
can be minimized by steam blanching or by the exclu-
sion of oxygen. The rate of ascorbic acid oxidation
increases markedly in the presence of metallic ions,
especially copper and iron. Hence, food processed in
tin cans and processing equipment should be copperfree. While the loss of ascorbic acid cannot be pre-
vented completely, it can be reduced to a minimal
level during processing.
V. SHELF LIFE
The application of redox enzymes for improving the
shelf life of food products includes mainly enzymes
which are capable of removing oxygen or reactive oxy-
gen species (e.g., H2O2 and superoxide anion), as well
as enzymes that are able to generate antimicrobialagents. In this way the stability of foods can be
increased significantly with respect to taste, appear-
ance, and microbial spoilage.
A. Lactoperoxidases
Lactoperoxidase (LPO; see Ref. 78 and Chapters 19
and 20 for detailed information) is the most prominent
enzyme in bovine milk, where it is found in concen
tions around 30 mg/L. It is a glycoprotein with a sin
covalently bound heme group (78). Lactoperoxi
requires hydrogen peroxide and thiocyanate (SC
for antibacterial activity. All three components
referred to as the LP system. The growth inhibi
effect of the LP system is mediated by the genera
of SCN
oxidation products, mainly hypothiocyaions (OSCN), which attack sulfhydryl groups of
metabolic enzymes of the microorgani
Mammalian cells are not affected by the LP sys
Only 1020 ppm of lactoperoxidase is required fo
effective system. The cofactor requirements are
very low: 1025 ppm for thiocyanate and 1015
for H2O2. Without the enzyme H2O2 is also bacte
dal, but at much higher concentrations: 300900
(79). Therefore LPO is often applied in combina
with H2O2-generating enzymes. From a toxicolog
point of view, the levels of the cofactors in the
system as well as the oxidation products are repoto be harmless.
The envisaged applications of the LP system
food products (e.g., liquid milk, cheese, meat,
and poultry products, and functional foods),
and veterinarian products (e.g., milk replacers,
antidiarrhea and antimastitis preparations), and de
products (7880). Typically, applications have
claimed for Lactobacillus fermented milk prod
(81), pickled foods (82), fish products (LPO in com
nation with GOX; 83), and white mold cheeses suc
Camembert (84). Also of interest is the effect of L
on yogurt. By adding LPO to yogurt the excessive production of lactic acid bacteria in the yogurt is
pressed (85). These applications are becoming wi
reach now that it is possible to isolate lactoperoxi
from milk with high purity on an industrial scale
Currently, LPO is already commercially availab
relatively low costs.
B. Xanthine Oxidases
Xanthine oxidase (XO, EC 1.2.3.2; see Chapter
and 42 for detailed information) is widely distrib
in animals, plants, and microorganisms. It catathe oxidation of hypoxanthine to xanthine
xanthine into uric acid. In addition XO is able to
dize a wide range of purines, aldehydes, and pterid
with concomitant reduction of O2 to H2O2. U
certain conditions XO also produces the highly r
tive superoxide anion. Bovine milk is very rich in
($ 35mg=L; 86). XO has been implicated in the ox
tive deterioration of milk and dairy products via
yright 2003 by Marcel Dekker, Inc. All Rights Reserved.
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production of superoxide anion during oxidation of its
substrates (87). However, Bruder et al. (88) found no
evidence to support a role for native bovine milk XO in
lipid oxidation. The results of Weihrauch (89) seem to
indicate that in the presence of purines, XO activity
generates H2O2 for the lactoperoxidase system in
milk, making it a bactericidal or bacteriostatic agent
in milk (23).
C. Superoxide Dismutases and Catalases
Superoxide dismutase (SOD; EC 1.15.1.1) and catalase
(EC 1.11.1.6; see Chapters 27 and 38 for detailed infor-
mation) are present in milk and are able to remove
reactive oxygen species generated by other (bio)chem-
ical processes (90). SOD catalyzes the reduction of
superoxide anion, as produced, for example, by XO,
to H2O2 and O2. In turn, catalase is able to neutralize
H2O2 to water and oxygen. A low level of exogenousSOD, coupled with catalase, is a very effective antiox-
idant in dairy products (91).
Recently, SOD has been shown to protect beer
against free radical damage (92). Obviously, the
commercial feasibility of SOD as an antioxidant
depends on cost, particularly compared to chemical
antioxidants, if permitted. As far as is known, SOD is
not used commercially as an antioxidant in food
systems.
Catalase is used for the cold-sterilization of milk in
regions lacking refrigeration and could in principle be
applied in developed countries for the treatment ofcheese milk (90). Good sources for catalase are beef,
liver, Aspergillus niger, and sweet potato. There is
also interest in using immobilized catalase reactors
for milk pasteurization or for glucose oxidasecata-
lase reactions (93). Besides the removal of reactive
oxygen species by SOD and catalase, other enzymes
can be applied to remove the less but still reactive
oxygen. Typical examples include glucose oxidase,
D-amino acid oxidase, alcohol oxidase, and ascorbic
acid oxidase. The disadvantage of these oxidases is
that they produce H2O2, which by itself is a powerful
oxidant. Catalase can be added to remove H2O2, butthen oxygen is produced again. More recently, poly-
phenol oxidases such as laccase have been proposed
as a deoxygenation tool for beer (94) and juices (95,
96). These enzymes have the advantage that they do
not produce H2O2, and thus the combination with
catalase is not necessary. As a result PPOs allow a
more efficient oxygen removal.
VI. NUTRITIONAL VALUE
Redox enzymes can have both pro- as well as antinu
tritional effects. For example, lipoxygenase, apart from
all its other functions in food products (see Table 1), i
involved in the oxidative destruction of liposolubl
vitamins (provitamin A) and essential fatty acids (3)
Likewise, ascorbic acid oxidase has an antinutritionaeffect since it oxidizes vitamin C (97).
Increasingly, redox enzymes are being claimed t
improve the healthiness of especially beverages
For example, peroxidase and catalase have been
claimed for the removal of unhealthy hydrogen per
oxide in coffee and tea (98). Other examples includ
the use of cholesterol oxidase, epicholesterol dehydro
genase and cholesterol reductase to lower the choles
terol level in foods such as meat, fish, milk, and egg
products (99101).
VII. CONCLUDING REMARKS
Compared to the usage of hydrolytic enzymes such a
proteases, carbohydrates, and lipases, the application
of oxidoreductases as a tool to improve the processa
bility and quality of food products is still in its infancy
Like hydrolytic enzymes, oxidoreductases are capabl
of tailoring the taste, texture, appearance, shelf life
nutritional value, and process tolerance of foods and
the properties of all major food constituents (e.g., pro
teins, carbohydrates, oils, fats, flavors; see Table 1). In
both cases, they can exert a positive as well as a negative effect on the food quality parameters, which can
be reduced or eliminated by careful selection of the raw
materials, by properly controlling the process condi
tion, by the addition of counteracting ingredients
and by genetic tools. Likewise, hydrolytic and redox
active enzymes often have more than one effect. A
typical redox example is the multifunctional enzym
lipoxygenase, which can be applied to influence eithe
taste, texture, appearance, and/or nutritional value o
food products. The key difference resides in the type
of reactions they catalyze. As a result, redox enzyme
can be combined with hydrolytic enzymes in a varietyof food products to create improved or even new func
tionalities, which cannot be realized by either one o
them.
At present, major bottlenecks for the large-scal
application of oxidoreductases include their limited
availability, their safety status (not GRAS), their sta
bility, and the fact that they often initiate radical reac
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tions, which propagate via redox-active food constitu-
ents such as metal ions and quinones and hence are
difficult to control. In addition, many oxidoreductases
require expensive cofactors for catalysis. Despite these
disadvantages over hydrolytic enzymes, certain oxidor-
eductases are increasingly finding a home in food pro-
cessing. The first generation (see Table 1) includes
enzymes which are cofactor independent and thriveon oxygen (e.g., oxidases) and hydrogen peroxide
(e.g., peroxidases). Owing to advances in recombinant
DNA technology the low-cost, large-scale production
of these enzymes in GRAS host microorganisms is
becoming within reach, and the tools are ready to tai-
lor redox enzymes to specific needs by site-directed
mutagenesis and directed evolution. Also feasible will
be the in planta overexpression of desired redox
enzymes in food raw materials and the deletion of
undesirable redox traits. The usage of cofactor-depen-
dent oxidative enzymes seems tentatively to be con-
fined to whole-cell systems, in which the cofactor canbe regenerated, and hence to the in vitro as well as the
in situ production of functional food ingredients such
as flavors. The same seems to be true for reductases,
since most if not all require reducing equivalents in the
form of a small protein, hydrogen, and/or NAD(P)H.
Clearly, the usage of oxidoreductases in food pro-
cessing is emerging. The benefits and limitations are
becoming known, which allows a promising and
focused search for new opportunities.
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