Mutagenesis vol. 19 no. 6 pp. 431--439, 2004
doi:10.1093/mutage/geh053
REVIEW
Prevention of mutagen formation in heated meats and model systems
Kiyomi Kikugawa1
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1
Horinouchi, Hachioji, Tokyo 192-0392, Japan
Possible means for preventing mutagen formation in
cooked meats and in heated model systems are described.
One way to reduce mutagenicity in cooked meats is to
control cooking temperature, time and method. Another
way is to increase water content or to avoid loss of water
in meats during cooking. Addition of an excessive amount
of reducing sugars to meats before cooking is effective in
minimizing mutagen formation, which may be due to
suppression of generation of the pyrazine cation radical
Maillard intermediate of heterocyclic amines. Addition of
a small amount of ascorbate or erythorbate is also effective,
which may be the result of scavenging the intermediary
pyrazine cation radical.
Introduction
Heterocyclic amine (HCA) mutagens are produced in a wide
variety of cooked or processed meats and fish and are known
to be carcinogenic in rodents and considered to be probable
human carcinogens (Sugimura and Sato, 1983; Layton et al.,
1995; Skog et al., 1998). Epidemiological studies have
demonstrated that the risks of colorectal adenoma (Sinha
et al., 1999, 2001) and lung cancer (Sinha et al., 2000) are
increased by the intake of well-done or grilled meats containing HCA mutagens. It is an important objective to reduce
or minimize mutagen formation due to HCAs in cooked or
processed meats and fish. Here, possible means for preventing or minimizing the mutagenicity of cooked meats due to
HCAs are briefly reviewed.
Mechanisms of mutagen formation in heat-dried meats
and model systems
It was found that addition of sugars and creatine/creatinine
to ground beef increased the mutagenicity of cooked beef
and that the precursors of HCAs were sugars, amino acids
and creatine/creatinine (J€agerstadt et al., 1983). The precursors of HCA mutagens in meats have been extensively
studied (J€agerstad et al., 1984; Negishi et al., 1984;
Reutersward et al., 1987; Skog et al., 1998). A dramatic
increase in mutagenicity was observed on adding creatine to
a recipe combining pork, beef, veal, starch and milk (Nes,
1986; Becher et al., 1988).
The heating temperature was shown to be important as an
increase in temperature had a marked effect on overall mutagenicity of foods (Knize et al., 1985; Felton and Knize, 1991).
1
2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)
is a common mutagenic HCA found in a wide variety of fried
or heated meats (Negishi et al., 1991) and fish (Kikugawa and
Kato, 1987b).
Creatine or one of 15 amino acids was mixed with minced
pork before broiling at 200 C (Overvik et al., 1989). Addition of 5% (w/w) creatine increased the overall mutagenicity
of crust, pan residue and aerosol 4-fold. Amino acid addition
(1% w/w) increased the overall mutagenicity between 1.5
(lysine) and 43 times (threonine). In most cases the mutagenicity profiles on reversed phase HPLC separation of the
crust and pan residues were changed by amino acid addition.
Serine, threonine, phenylalanine, alanine, leucine and tyrosine were all shown to give rise to one of the known HCAs,
MeIQx, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)
or 2-amino-1,5,6-trimethylimidazo[4,5-b]pyridine (TMIP). Water
was not a prerequisite for mutagen formation in meat. MeIQx,
2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx)
and PhIP were shown to be present in dry-heated meat juice.
It was concluded that creatine and free amino acids were the
main reactants in the mutagen-forming reactions that occur during frying of meat.
Mixtures of glucose, amino acids and creatinine simulating the composition of six different kind of meats (beef,
chicken breast, chicken thigh, turkey breast, pork and fish)
were dry-heated to simulate the formation of HCAs in meat.
The presence of 16 HCAs was investigated in the model
systems and in the six meats and their corresponding meat
drippings to determine the importance of meat composition
to HCA formation (Pais et al., 1999). Nine HCAs, 2-amino3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) MeIQx, 4,8-DiMeIQx, PhIP,
2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx), 2-amino(1,6-dimethylfuro[3,2-e]imidazo[4,5-b])pyridine (IFP), 2amino-1,6-dimethylimidazo[4,5-b]pyridine (DMIP) and TMIP
were found to be present at concentrations >0.1 ng/g in some
of the model systems and in some of the meats of pan residues.
HCA concentrations were affected by precursor composition
in the model system and the same nine HCAs formed in the
meat and in the model system, showing that the model system
is a good surrogate for the reaction conditions occurring in
meats during cooking.
A mixture of glucose, glycine and creatinine in
diethyleneglycol--water generated imidazoquinoxaline-type
HCAs, including MeIQx. A pathway including condensation
of the pyrazine cation radical (Namiki and Hayashi, 1975)
generated in the Maillard reaction of sugar/amino acid with
creatine/creatinine has been suggested for HCA formation
(Pearson et al., 1992; Milic et al., 1993) and participation
of the pyrazine cation radical (Figure 1) in the formation of
imidazoquinoxaline-type HCAs has been demonstrated (Kato
Tel: 181 426 76 4503; Fax: 181 426 76 4508; Email: [email protected]
Mutagenesis vol. 19 no. 6 ß UK Environmental Mutagen Society 2004; all rights reserved.
431
K.Kikugawa
Fig. 1. Pyrazine cation radical with aN 5 0.81 mT, aH-aro 5 0.30 mT and
aH-side 5 0.40 mT.
et al., 1996; Kikugawa, 1999). Electron spin resonance
(ESR) studies showed that the characteristic multiline signals
of the pyrazine cation radical appeared in a model system
composed of 0.2 M glucose and 0.4 M glycine in diethyleneglycol--water heated at 120 C for 5 min and generation
of this radical was found to be a prerequisite for the formation of imidazoquinoxaline-type HCA mutagens, including
MeIQx, in subsequent condensation with creatinine heated
for 2 h. Considering the involvement of the pyrazine cation
radical in mutagen formation, preventing its generation or
scavenging it in the early stages of the Maillard reaction
may lead to prevention of HCA formation in the subsequent
condensation with creatinine.
Minimizing mutagen formation in cooked meats by
controlling heating temperature and time
Heating temperature, heating time and heating method greatly
affected HCA formation in the model systems and in cooked
meats. Earlier studies showed that HCA formation was controlled in part by surface temperature and the length of time
a high temperature was maintained (Spingarn et al., 1980). One
method of lowering the surface temperature was to mix ground
meat with soy proteins or with other components that retain
water. Thus, satisfactory cooking of an item, such as a soyburger, could be achieved at a lower maximum temperature and,
therefore, with decreased production of HCAs (Wang et al.,
1982). Mutagenicity in lean pork meat fried at 250 C was
increased by the addition of fat, probably due to more efficient
heat transfer from the frying pan to the meat (Nilsson et al.,
1986). The effect of cooking time on the mutagenicity of
the crust, pan residue and smoke from pan-broiled pork patties
was studied (Berg et al., 1990). Meat was broiled at 200 C for
various times between 2 and 10 min. Broiled meat was
reheated up to 5 times at 200 C, each time to a centre temperature of 70 C. Reheating was performed in a microwave oven
for 2 min and in an electric oven at 200 C for 10 min. In
addition, broiled patties were kept warm at 60 C in an incubator for up to 9 h. Mutagenicity increased rapidly in all fractions
except the volatile phase over the first 6 min of cooking, after
which time only a slight increase was seen. At cooking times
54 min no mutagenicity was detected in the smoke. Reheating
or keeping the meat warm had very little effect on the mutagenicity of the meat. Hence, during pan broiling at 200 C the
major part of the mutagenicity was formed during the first 6 min
of cooking. Reheating the meat or keeping it warm did not
significantly affect the mutagenicity.
432
Microwaving meat for 1--1.5 min separated the juice appearing at the bottom of the dish from the meat itself. When the meat
was cooked the resulting mutagenicity was ~50-fold lower than
that in meat cooked without microwaving. When the juice was
added back to the meat and recooked, the mutagenicity was
restored (Taylor et al., 1986). This reduction in mutagenicity
may be due to removal of water-soluble HCA precursors in the
juice on microwaving. Pretreatment of beef patties by microwaving reduced the mutagenicity of cooked hamburgers, which
may be due to the loss of HCA precursors (Felton et al., 1994).
The beef patties received microwave treatment for various
times before frying. Microwave pretreatment for 0, 1, 1.5, 2 or
3 min before frying at either 200 or 250 C for 6 min per side
reduced HCA precursors (creatine, creatinine, amino acids and
glucose), water and fat by up to 30% and resulted in a decrease
in mutagenicity of up to 95%. The sum of four HCAs, MeIQx,
IQ, 4,8-DiMeIQx and PhIP, decreased 3- to 9-fold compared
with non-microwaved beef patties fried under identical conditions. Hence, microwaving meat is effective in reducing the
mutagenicity of cooked meat. However, much of the taste of
the meat may be removed by this procedure.
Turning over beef patties repeatedly at lower temperatures
reduced the mutagenicity of cooked hamburgers (Salmon et al.,
2000).
Chemical analysis of foods has shown that flame grilling can
form both polycyclic aromatic hydrocarbon and HCA mutagens and that frying forms predominantly HCA mutagens
(Knize et al., 1999). Pan-roasted beef showed lower mutagenicity after various degrees of cooking than beef cooked over
charcoal. The high mutagenicity of barbecued beef was due to
the formation of more HCAs because of rapid and direct heating of the surface of the meat to a high temperature (Gu et al.,
2001). Seasoning decreased the mutagenicity of pan-roasted
beef but increased the mutagenicity of barbecued beef with
decreased kinds of HCAs, probably due to a change in HCA
precursors or, alternatively, to the formation of other mutagens
(Gu et al., 2001). Seasoning was also reported to increase
mutagenic activity in chicken and hamburger patties charcoaled at 220 C (Tikkanen et al., 1996).
These studies demonstrate that understanding the cooking or
processing conditions that lead to the formation of mutagens
can give rise to methods to greatly reduce their occurrence in
cooked or processed foods.
Avoiding loss of water minimizes mutagenicity of heated
meats and model systems
A high water content in beef extract prevents mutagen formation (Taylor et al., 1986). Decreasing the water content of
the model system and loss of water from fish increased the
mutagenicity after heating (Kikugawa and Kato, 1987a). When
mixtures of glucose, glycine and creatinine in diethyleneglycol--water or dioxane--water with varying water contents were
heated in sealed tubes at 120 C for 2 h, the overall mutagenicity produced in the mixtures decreased as the water content
increased (Figure 2), indicating that a higher water content
in the model systems suppressed the formation of mutagenic
compounds. Decreased mutagenicity with a higher water
content may be due to disappearance of the intermediary
pyrazine cation radical, because heating of glucose and glycine
in water alone did not give the appropriate ESR signals
whereas heating glucose and glycine in diethyleneglycol--water
gave intense signals for the radical (Kato et al., 1996). Bonito,
Prevention of mutagen formation in heated meats
tunny and mackerel gradually generated mutagenicity during
heating at 100--110 C in air over a period of ~50 h, which was
mainly due to MeIQx formation (Kikugawa and Kato, 1987a).
Loss in weight and mutagenicity were followed over time
under these conditions (Figure 3). The mutagenicity of fish
increased after 460% weight loss, indicating that loss of
water is a driver for the decrease in mutagenicity. The development of overall mutagenicity in the fish heated at 100--110 C
for 24 h under air and steam was compared (Table I). Loss of
weight of each fish on heating under steam was one-tenth that on
heating in air and the overall mutagenicity of each meat heated
under steam was less than one-fifth of that heated in air. These
results indicate that formation of mutagenicity in fish depends
not only on heating temperature and time but also on loss of
water. Hence, avoiding loss of water during cooking is effective
in minimizing the mutagenicity of meats. Boiling meats does
not induce mutagenicity and roasting or frying meats under
conditions of lowered water loss can minimize mutagenicity.
Fig. 2. Effects of water content on mutagen formation in the model system.
A mixture of 0.2 M glucose, 0.4 M glycine and 0.4 M creatinine in
diethyleneglycol--water (open circle) or dioxane--water (filled circle) was heated
in a sealed tube at 120 C for 2 h. The mutagens in the solution were extracted by
the blue cotton method and mutagenicity (His 1 revertants) was assayed on
Salmonella typhimurium TA98 with S9 mix (Kikugawa and Kato, 1987a).
Controlling sugar content reduces mutagenicity of
heated meats and model systems
It has been shown that the addition of reducing sugars such as
glucose and fructose is effective in inhibiting HCA formation
in cooked beef patties, resulting in a reduction in mutagenicity
(Skog and J€agerstad, 1990, 1991; Skog et al., 1992). Although
sugar is viewed as a major contributor to HCA formation, the
addition of sugars to ground beef patties at levels ranging from
2 to 4% reduced HCA formation and overall mutagenicity of
cooked ground meat. It has been thought that the addition of
reducing sugars to meat beyond the optimum needed for the
formation of HCAs results in the formation of Maillard reaction products that inhibit HCA formation.
The present author investigated whether sugar content
affects generation of the intermediary pyrazine cation radical
in the early stages of the Maillard reaction and the formation
of imidazoquinoxaline-type HCAs. When mixtures of 0--0.8 M
glucose and 0.8 M glycine in polyethyleneglycol--water were
heated at 120 C for 5 min the intensities of the pyrazine cation
radical ESR signals were highest in the mixture with glucose
at 0.4 M (Figure 4) (Kikugawa et al., 1999). When mixtures of
0.025--0.8 M glucose, 0.4 M glycine and 0.4 M creatinine
in diethyleneglycol--water were heated at 120 C for 2 h, the
mixture with glucose at 0.4 M gave the highest overall mutagenicity (Figure 5) (Kato et al., 1998). Hence, both pyrazine
cation radical generation in mixtures of glucose and glycine
and overall mutagenicity formation in mixtures of glucose,
glycine and creatinine depend on the glucose concentration.
The optimal glucose:glycine mixture was 1:2, suggesting that
mutagen formation was reduced at higher glucose ratios.
The effect of adding reducing sugars to ground beef on the
overall mutagenicity of cooked hamburgers was also examined
(Figure 6) (Kato et al., 2000b). The initial reducing sugar content
of ground beef was 0.07% (w/w). Addition of 0.08% (w/w)
glucose to ground beef (total glucose content 0.15% w/w)
increased the mutagenicity ~2-fold, but addition of 40.67%
(w/w) glucose to ground beef (total glucose content 0.74%
w/w) decreased the mutagenicity by 50% (Figure 6A).
Addition of other reducing sugars, such as fructose and
lactose, to ground beef gave rise to similar sugar-dependent
mutagen formation (Figure 6B and C). However, addition of a
Fig. 3. Time courses of loss in weight of fish and mutagen formation. A piece (40--50 g) of bonito (A), tunny (B) or mackerel (C) was heated in a
stainless steel box in air at 100--110 C. The mutagens were extracted by the blue cotton method and mutagenicity (His 1 revertants) was assayed on
Salmonella typhimurium TA98 with S9 mix. The mutagenicity is presented on the basis of weight dried at 120 C for 2 h (Kikugawa and Kato, 1987a).
433
K.Kikugawa
Table I. Effect of heating conditions on mutagen formation in fish
Meat
Bonito
Tunny
Mackerel
Heated at 100--110 C
for 24 h in air
Heated at 110 C
for 24 h in steam
Weight
loss (%)
His 1
revertants (per g)
Weight
loss (%)
His 1
revertants (per g)
69.6
73.3
65.1
612
391
227
5.6
7.3
4.1
74
69
23
A piece of each meat (40--50 g) was heated in a stainless steel box in
air at 100--110 C or in an autoclave under steam at 100 C for 24 h.
The mutagens were extracted by the blue cotton method and mutagenicity
(His 1 revertants) was assayed on Salmonella typhimurium TA98 with
S9 mix. The mutagenicity is presented on the basis of weight dried at
120 C for 2 h (Kikugawa and Kato, 1987a).
It was found that addition of foods that contain reducing
sugars to ground beef affects the mutagenicity of cooked
hamburgers. Addition of wine containing reducing sugars to
ground beef increased the mutagenicity of the cooked hamburger (Kato et al., 2000b). When a brand of red wine
containing reducing sugars at 0.10% (w/v) or a brand of
white wine containing reducing sugars at 0.13% (w/v) was
added to ground beef at 33% (v/w), the total reducing sugar
content of the ground beef was close to the optimum for the
mutagen formation and mutagenicity of the cooked hamburger increased 2-fold. Addition of brandy (containing no
sugars) to ground beef had no effect on mutagen formation
in cooked hamburgers.
In Japan, as elsewhere, onion is added to ground beef when
preparing homemade hamburgers. This cooking style was
found to be a beneficial way to reduce hamburger mutagenicity
(Kato et al., 1998). Onions obtained in Japan contain ~7.6%
(w/w) reducing sugars and addition of 33% (w/w) onion to
ground beef raised the sugar content well above the optimal
ratio. The resulting mutagenicity of the cooked hamburger,
estimated on the basis of weight of ground beef, was about
one-fifth of that cooked without onion.
Addition of a large amount of corn starch to ground beef
(1.5% w/w) also reduced the overall mutagenicity of cooked
hamburgers (Kato et al., 1998). The effects of different oligosaccharides, fructooligosaccharides, galactooligosaccharides
and isomaltooligosaccharides and inulin on HCA formation
and overall mutagenicity in fried ground patties were evaluated
(Shin et al., 2003a). The addition of a large amount of saccharides to ground beef (1.5%, w/w) inhibited total HCA formation
by 46--54%. It also reduced overall mutagenicity by 48--59%.
The studies confirmed that saccharides have the potential to
reduce HCA formation in cooked beef patties, although the
reasons for this effect are not known.
Antioxidants and reductones reduce the mutagenicity of
heated meats and model systems
Fig. 4. ESR spectra of a mixture of 0--0.8 M glucose and 0.4 M glycine in
polyethyleneglycol--water (6:4 v/v) heated at 120 C for 5 min (Kikugawa
et al., 1999).
non-reducing sugar, sucrose, at the concentrations studied did
not affect mutagen formation (Figure 6D). Hence, a glucose
content in ground beef of 40.7% (w/w) can reduce the overall
mutagenicity of cooked hamburgers.
434
Food additives that minimize the mutagenicity of model systems and cooked meats have been investigated. Addition of
tryptophan, indole derivatives and proline (Jones and
Weisburger, 1988a,b,c) to the model system or to beef reduced
mutagen formation. The mechanism for this inhibition has not
yet been elucidated. Phenolic antioxidants, butylated hydroxyanisole (BHA) and chlorogenic acid (Wang et al., 1982;
Weisburger, 1991) and EDTA (Barnes and Weisburger,
1984) prevent mutagen formation in beef, while ferrous ion
enhances formation (Barnes and Weisburger, 1984). A contribution of lipid peroxidation to the mutagen formation has been
suggested and prevention of lipid peroxidation by antioxidants
was thought to be effective in reducing mutagen formation.
Defatted glandless cottonseed flour inhibits mutagen formation
(Rhee et al., 1987). Diallyl disulfide and dipropyl disulfide,
minor components in onion, were shown to prevent HCA
formation in boiled pork juice (Tsai et al., 1996). Garlic-related
sulfur compounds were also inhibitory (Shin et al., 2002a,b).
Concentrations of HCAs in and overall mutagenicity of fried
ground beef patties can be reduced by the addition of substances such as the whole cherry tissue (Britt et al., 1998),
antioxidant spices (Murkovic et al., 1998), vitamin E (Balogh
et al., 2000) and unifloral honeys (Shin et al., 2003b).
The effect of fresh made virgin olive oil containing a high
level of dihydroxyphenylethanol derivatives on the formation
Prevention of mutagen formation in heated meats
Fig. 5. Mutagenicity of a mixture of glucose, glycine and creatinine in diethyleneglycol--water (8:2 v/v) heated at 120 C for 2 h. (A) Varying concentrations
of glucose, 0.4 M glycine and 0.4 M creatinine. (B) Varying concentrations of glycine, 0.2 M glucose and 0.4 M creatinine. (C) Varying concentrations of
creatinine, 0.2 M glucose and 0.4 M glycine. The mutagens in the solution were extracted by the blue cotton method and mutagenicity was assayed on
Salmonella typhimurium TA98 with S9 mix (Kato et al., 1998).
Fig. 6. Mutagenicity of cooked hamburger prepared by addition of glucose (A), fructose (B), lactose (C) or sucrose (D) to ground beef originally containing 0.07%
(w/w) reducing sugars. The beef was heated at 200 C for 20 min. The mutagens were extracted by the blue cotton method and mutagenicity was assayed
on Salmonella typhimurium TA98 with S9 mix (Kato et al., 2000b).
of HCAs in an aqueous model system composed of glucose,
glycine and creatinine was examined (Monti et al., 2001).
Virgin olive oil inhibited mutagen formation by between 30
and 50% compared with control 1-year-old oil. The inhibition
of HCA formation was verified using phenolic compounds
extracted from virgin olive oil.
We looked for scavengers of the pyrazine cation radical
formed in the early stages of the Maillard reaction of glucose
and glycine in diethyleneglycol--water or polyethyleneglycol-water. The results are summarized in Table II. The antioxidants
BHA, sesamol and epigallocatechin gallate (EGCG), most
abundant polyphenolic in green tea, were effective at relatively
high concentrations (Kato et al., 1996). Thiol compounds,
including cysteine (CySH) and N-acetylcysteine (NAC)
(Kikugawa et al., 1999), and reductones, including ascorbate
and erythorbate (Kikugawa et al., 2000a,b), were effective
radical scavengers at relatively lower concentrations. The
scavenging effects of ascorbate and erythorbate are shown in
Figure 7. Unsaturated fatty acids were also effective radical
scavengers (Kikugawa et al., 1999). The reductones generated
in the Maillard reaction, 4-hydroxy-3-(2H)-furanones, promote
formation of the pyrazine cation radical by interaction with
glycine (Kikugawa et al., 2000b).
Scavengers of the pyrazine cation radical were tested as
inhibitors of mutagen formation in the model system composed
of glucose, glycine and creatinine in diethyleneglycol--water
(Table II). The antioxidants BHA, propylgallate (PG), sesamol
and EGCG were effective in reducing the overall mutagenicity
at relatively high concentrations (Kato et al., 1996). This
observation is consistent with earlier ones showing that a
wide variety of flavonoids, including EGCG, inhibit HCA
formation in the model system (Weisburger et al., 1994;
Oguri et al., 1998). Thiol compounds and unsaturated fatty
acids also inhibit mutagen formation in the model system
435
K.Kikugawa
Table II. Effect of antioxidants and reductones on the generation of pyrazine cation radical and mutagen formation
Antioxidant/reductone
ESR signals of the
model system (%) (mM)a
Mutageniciy of the
model system (%) (mM)b
Mutagenicity of
cooked hamburger (%)c
None
Phenolic antioxidants
BHA
Sesamol
EGCG
100
100
100
Thiol compounds
CySH
NAC
Reductones
Ascorbate
Erythorbate
Maillard reductone
4-Hydroxy-3(2H)-furanones
0 (100)
0 (100)
0 (100)
20 (100)
40 (100)
40 (100)
97 (0.33)
10 (10)
0 (10)
30 (50)
100 (0.33)
94 (0.33)
0 (50)
0 (50)
60 (50)
60 (50)
65 (0.33)
60 (0.33)
200(20)
100 (100)
a
The model system was composed of 0.2 M glucose and 0.4 M glycine in diethyleneglycol--water (8:2 v/v) or in polyethyleneglycol--water (6:4 v/v) heated
at 120 C for 5 min.
b
The model system was composed of 0.2 M glucose, 0.4 M glycine and 0.4 M creatinine in diethyleneglycol--water (8:2 v/v) heated at 120 C for 2 h.
c
Hamburger prepared by heating ground beef at 200 C for 20 min (Kikugawa et al., 2000a).
Fig. 7. ESR spectra of mixtures of 0.2 M glucose and 0.4 M glycine in diethyleneglycol--water (8:2 v/v) in the presence of ascorbate or erythorbate at the indicated
concentration heated at 120 C for 5 min. Two signals are due to the monodehydroascorbyl (or erythorbyl) radical with aH 50.18 mT (Kikugawa et al., 2000b).
(Kikugawa et al., 1999). Ascorbate and erythorbate inhibit
mutagen formation, but the Maillard intermediates
4-hydroxy-3-(2H )-furanones were not inhibitory (Kikugawa
et al., 2000b). A possible pathway for generation of the pyrazine cation radical and formation of imidazoquinoxaline-type
HCAs and the effects of antioxidants and reductones are shown
in Figure 8.
We attempted to reduce the mutagenicity of smoked-anddried bonito (katsuobushi in Japanese), a common seasoning
in Japan. When a small piece of bonito meat (~20 g) was
boiled in a 0.5% EGCG solution or in a 5% green tea extract
436
and subsequently heated in air at 100 C for 24 h, the
mutagenicity of the heated-and-dried bonito meat was
reduced to 550% (Kato et al., 1996). Decreasing the mutagenicity of katusuobushi by boiling bonito meat in green tea
extract was attempted. A large piece (~500 g) of bonito meat
was boiled in 2.5 and 5% (w/v) green tea extract, then
smoked-and-dried in the usual manner of katsuobushi
processing. The mutagenicty of katsuobushi was only
slightly decreased, thus there was a limit to the use of
green tea extract to decrease the mutagenicity of katsuobushi
(Kato and Kikugawa, 1999).
Prevention of mutagen formation in heated meats
Fig. 8. Possible pathway for the generation of pyrazine cation radical and imidazoquinoxaline-type mutagens and the effects of antioxidants and reductones.
effective in reducing the mutagenicity of cooked hamburger
by 50% (Figure 9) (Kato et al., 2000a). The taste and smell of
the hamburger were not affected by cooking with ascorbate
and erythorbate at these doses.
Antioxidants and reductones are effective in reducing overall mutagenicity due to HCA formation in the heated model
system and cooked meat through scavenging the intermediary
pyrazine cation radical in the early stages of the Maillard
reaction. However, the potencies of the antioxidants and reductones differ by compound. Ascorbate and erythorbate are the
most potent scavengers of the radical and the most potent
inhibitors of HCA formation at practicable low doses.
Conclusion
Fig. 9. Mutagenicity of cooked hamburger prepared by addition of ascorbate
(filled circle) and erythorbate (filled triangle). Ascorbate or erythorbate was
added to 3 g of ground beef at the indicated amount and the beef was heated at
200 C for 20 min. The mutagens were extracted by the blue cotton method
and mutagenicity was assayed on Salmonella typhimurium TA98 with S9
mix (Kato et al., 2000a).
We then tried to reduce the mutagenicity of cooked hamburger by the introduction of antioxidants or reductones to ground
beef (Table II). EGCG, CySH and NAC at doses lower than
0.33% (w/w) were not effective in reducing mutagen formation
(Kato et al., 2000a), whereas tea polyphenols have been shown
to prevent mutagen formation in cooked meat (Weisburger
et al., 2002). Ascorbate and erythorbate at 0.33% (w/w) were
In this review various methods for minimizing mutagenicity
derived from HCA formation in heated model systems and
cooked meats have been described. Because the temperature,
time and cooking method greatly affect mutagen formation,
controlling these factors can reduce the mutagenic activity.
Avoiding loss of water from model systems and meats is
another way to reduce mutagen formation. Inhibition of the
formation of the intermediary pyrazine cation radical in the
early stages of the Maillard reaction or scavenging the radical
is also effective in reducing mutagenicity. Thus, addition of
an excess amount of reducing sugars or of a small amount of
ascorbate or erythorbate to meats before cooking is effective in
minimizing mutagen formation.
References
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inhibition of heterocyclic aromatic amines in fried ground beef patties.
Food Chem. Toxicol., 38, 395--401.
437
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Received on March 15, 2004; revised on August 9, 2004;
accepted on August 19, 2004
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