By: Dr.

Satrijo Saloko

Courtesy of Aisha
 Acrylamide
Polycyclic aromatic
Maillard reaction product amine
Toxicant from food N-nitrosamines
processing
Polycyclic aromatic
hydrocarbons Benzo[a]pyrene
Source :
 Charred or smoked meat and fish
 Cereals
 Flour
 Vegetables
 Fruits
 Water, soil, dust
 Cigarette smoke
 Vegetables oil

-
 the largest class of chemical compounds, containing two or more
fused aromatic rings made up of carbon and hydrogen atoms.
PAHs are formed in incomplete combustion processes which
occur whenever wood, coal or oil are burnt.
 Result of the environmental pollution but also as a consequence of
some thermal treatments
 Food processing (smoking, drying, roasting, baking or frying)
produces PAHs in certain levels
 Formed mainly from carbohydrates cooked at hight temp in the
absence of oxygen.
 Broiling meat in hot ceramic or charcoal briquettes  the melted
fat will come into contact with very hot surface.
 PAHs then produced in the ensuing reactions, PAHs rise with
resulting cooking fumes and are deposited on the meat.
 Polycyclic aromatic hydrocarbon (PAH)
that is a byproduct of incomplete
combustion or burning of organic (carbon-
containing) items
 The most commonly known carcinogenic
PAH.
 Heating starch at :
370-390 0C =0.7 ppb
650 0C =17 ppb
 Many cooking processes: 370—390 0C
 The surface temperature of baking bread
may approach 400 0C and deep fat frying is
400 - 600 0C,
 10 mg of BaP 3x a week: skin tumors
100 mg of BaP in 10 weeks : stomach cancers
 Benzo[a]pyrene is not mutagenic and carcinogenic by itself, but must be
first converted to active metabolites.
 Metabolic conversion:
- A cytochrome’ P450-mediated oxidation produce a 7,8-epoxide.
- The 7,8- epoxide undergoes an epoxide hydrolase-mediated hydration,
produce the 7,8-diol
- 7,8-diol further oxidized by cytochrome P450, produces diolepoxide.
- Diolepoxide: highly mutagenic without metabolic activation and is also
highly carcinogenic at the site of administration.
- The benzo[a]pyrene diolepoxide can react with various components in
the cells, including DNA
Case History: Benzopyrene from meat on a barbeque

Natural molecules can

become dangerous during
food preparation!
 Conventional method:  Modern method:
- Direct smoking (smoke - Indirect smoking (using
developed in the external smoke generator).
smoking chamber, - Used in modern industrialized
traditionally in kilns
smokehouses) - Operated automatically under
carefully controlled conditions
- Smoke can be washed from
particles before coming into
contact with the food.
 Mean content of 15 PAHs in meat products industrially smoked, μg/kg

PAHs Ham Cooked cured loin Medium ground

sausages
External Internal External part Internal External Internal
part part part part part
Cyclopenta[c,d]pyrene 1.62 0.53 0.07 n.d 11.39 7.07
Benzo[a]anthracene 8.88 0.41 6.48 0.38 8.78 1.29
Chrysene 6.29 0.56 4.46 0.48 6.63 0.55
5-metylchrysene 1.72 0.28 2.73 0.30 2.06 0.36
Benzo[j]fluoroanthene 0.31 n.d 0.31 n.d 1.2 n.d
Benzo[a]pyrene 0.37 0.28 0.30 0.28 0.89 0.32

Ciecierska, Marta. 2007. INFLUENCE OF SMOKING PROCESS ON POLYCYCLIC AROMATIC HYDROCARBONS’ CONTENT
IN MEAT PRODUCTS* . Acta Sci. Pol., Technol. Aliment. 6(4) 2007, 17-28
 Mean content of 15 PAHs in meat products traditionally smoked, μg/kg

PAHs Ham Cooked cured loin Medium ground

sausages
External Internal External part Internal External Internal
part part part part part
Cyclopenta[c,d]pyrene 3.26 0.49 1.27 n.d 12.17 7.78
Benzo[a]anthracene 6.74 0.40 9.25 0.22 5.86 1.51
Chrysene 6.20 0.68 9.12 0.42 5.07 0.57
5-metylchrysene 1.28 0.28 2.98 0.31 1.39 0.38
Benzo[j]fluoroanthene 0.59 n.d 0.43 n.d 0.83 n.d
Benzo[a]pyrene 0.43 0.27 0.37 0.29 0.40 0.31

Ciecierska, Marta. 2007. INFLUENCE OF SMOKING PROCESS ON POLYCYCLIC AROMATIC HYDROCARBONS’ CONTENT
IN MEAT PRODUCTS* . Acta Sci. Pol., Technol. Aliment. 6(4) 2007, 17-28
 1. Traditional method of smoking affected the higher contamination PAHs
than industrial proceess.
2. Industrial smoking process influenced higher PAHs content only in case of
exteriors of medium-ground sausages.
3. For all products smoked using both methods, interiors had a significantly
lower PAHs contamination than exteriors of the same products.
4. Benzo[a]pyrene’s content was much lower than maximum tolerable
limit of 5 μg·kg-1, which was set for smoked meat products in
Commission Regulation (EC) No. 208/2005. Therefore industrial and
even traditional smoking of meat is a safe process.

Ciecierska, Marta. 2007. INFLUENCE OF SMOKING PROCESS ON POLYCYCLIC AROMATIC HYDROCARBONS’ CONTENT
IN MEAT PRODUCTS* . Acta Sci. Pol., Technol. Aliment. 6(4) 2007, 17-28
 1912, L. C. Maillard hypothesized reaction of
brown pigments and polymers produced from the
reaction of amino group of amino acid and carbonyl
group of sugar.
 Nonenzymatic browning reaction:
 Reaction of amines and carbonyl under heat
treatment  in vivo and biological systems
damage
 Reaction products: brown color, characteristic
roasted or smoky odors, pro- and antioxidants,
mutagens, carcinogens.
 Occurs between reducing sugars and amines
at high temperatures
 Produces flavor-aroma
 Produces color
 Produces antioxidant products
 Produces toxic products
 Destroys nutrients (lysine)
 Melanoidins
Ammonia Brown colour
Amine
Phospholipids
Amino acids
Proteins HEAT
Amino Carbonyl
Interaction Volatile compounds
Aldehydes
Ketones
(Amadori product)
Carbonyl
Reducing sugars
Polysaccharides
Carbonyls
Oxidised lipids
Esters
Amide (acrylamide)
Heterocyclic compounds
  Source: produced during the cooking of protein-rich food; beef,
chicken, broiled seafood, hamburger,
 Mutagens , esp on the surface layers where most pyrolysates are found.
Imidazoquinoline Heating a mixture of creatine/creatinine, amino
(IQ)-type acids, and sugar

PAA Cyclization of creatinine to form the imidazole

moiety due to heat treatment

Pyrolysis products formed from

Non-IQ type
tryptophan
 Revertants of TA98
Food 2500C 3000C 4000C
Beef 178 11400
Chicken 661 15120
Egg 121 4750
Hairtail 849 12320
Eel 309 6540
Squid, dried 269 8000 4490
Skipjack tuna, dried 1,220 24300 6200
Seaweed, nori 260 3040
The IQ-Type

Abbreviation Z R1 R2 R3
IQ C H H H
MeIQ C Me H H
MeIQx N H H Me
4,8-DiMeIQx N Me H Me
The non-IQ-Type
 Trp-P-1 and Trp-P-2 are toxic to animals
 LD50 of Trp-P-1: 200 mg/kg in mice, 380 mg/kg in hamsters, 100 mg/kg in
rats. Over LD50 died in 1h.
 IQ and MeIQX in Beef extracts  metabolically converted to active
mutagens by liver tissue through N-oxidation and O-acetylation  produce
highly reactive metabolites that for, DNA adducts
 Food with Trp-P-1 or Trp-P-2  tumor and hepatoma in mice
 Highly potent mutagens and carcinogens
PAA Found in Revertants /µg
MeIQ Broiled sardine 661,000
IQ Fried beef 433,000
MeIQx Fried beef 1435,000
Trp-P-1 Broiled sardine 104,000
Glu-P-1 Glutamic acid pyrolysates 49,000
Trp-P-1 Broiled sardine, broiled beef 39,000
Glu-P-2 Broiled, dried cuttlefish 1,900
Aflatoxin B1 Corn 6,000
Benzo[a]pyrene Broiled beef 320
 Formed from the interaction of nitrite and secondary, tertiary amines.
 Found in wide variety of foods
 A significant source of nitrite: cured meat
 Nitrite ions in curing meat: antimicrobial (inhibit the growth of C. botulinum,
produces appealing red color to meats from nitrosomyoglobin and
nitrosylhemoglobin pigments, gives a desirable cured flavor to meat products
 Permissible levels of nitrite in cured foods :vary, 10 to 200 ppm
 nitrite in humans : from reduction of dietary nitrate to nitrite by bacteria in
the mouth and in the intestinal tract.
 Nitrate in the relatively in high levels (1000—3000 ppm (cabbage,
cauliflower, carrots, celery, and spinach)
 pH dependent (3.4)
 Weakly basic amines are more rapid
 Several anions, such as halogens and thiocyanate, promote the nitrosation
process
 High temperature food heating , i.e bacon
 Diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) occur in the
gastric juice of experimental animals and humans fed diets containing amines
and nitrite
 Heating of nitrite-treated foods  produce nitrosamines.
 Cured meats have all been shown to contain nitrosamines, the higher levels
appearing in cured meats that have been subjected to relatively high heating.
 In Norway in 1962, epidemic of food poisoning in sheep, extremely high levels
of nitrosamines were detected in herring meal treated with nitrite as a
preservative. The sheep suffered severe liver disease and many of them died.
 formation of nitrosamine in nitrite-treated fish: dependent on the temperature of
preparation following the addition of nitrite.
 Refrigerated fish treated with nitrite had no more nitrosamine than fresh fish
treated with nitrite,
 heat treatment of fish increased the rate of nitrosamine formation following
addition of nitrite  due to increased concentrations of secondary amines
resulting from protein degradation during the heating process.
meat Nitrosamines Level (ppb)
Smoked sausages Dimethylnitrosamines <6
Diethylnitrosamines <6
frankfuters Dimethylnitrosamines 11-84
salami Dimethylnitrosamines 1-4
Fried bacon Dimethylnitrosamines 1-40
Nitrosoproline 1-40
 Over 100 food substances assayed, approximately 80% were shown to be
carcinogenic in at least one animal species.
 Dimethyl- and diethylnitrosamine are two of the most potent carcinogens
 Administration of dimethylnitrosamine at 50 ppm in the diet produces
malignant liver tumors in rats in 26—40 weeks. Higher doses produce kidney
tumors.
 As the dose of diethylnitrosamine is reduced below 0.5 mg/kg,. No clear
threshold dose for carcinogenicity of nitrosamines in the diet has yet been
established.
 Addition of reducing agent (erythsorbate or ascorbate) to the
curing mix to reduce or eliminate nitrosamine formation
 Reduce food processing temperature
 Minimize cured meat consumption
 First report, Sweden, April 2002 Food Amount (ppb)
 Found in a wide range of foods,
including dietary staples Baby food 17-130
• Potato products French fries 70-1036
• Breakfast cereal Potato chips 117-2764
• Coffee Cereals 47-266
• Bakery products
• Snack foods
Bread and bakery 10-354
Snack foods 111-1168
coffe 275-351
Chocolate products 45-909
Dried foods 11-1184
 Formed from asparagine and glucose via Maillard
reaction.
 Acrylamide Formation (heating at
1800C, 30 min)
▪ Asparagine 0.99 µg/g
▪ Asparagine + glucose 1200 µg/g
▪ Asparagine + Potato starch + glucose 9270 µg/g
 Asparagine and carbonyl compound (glucose,
glyceraldehide) play an important role in
acrylamide formation in cooked foods.
 Effectiveness of Amino Acids and
Dextrose to Form Acrylamide
Model System
Amino acid fry
+ Reducing sugar
Variety of ingredients
Potato Starch + Water Measure Acrylamide

 Acrylamide Formation
– Potato starch <50 ppb
– Potato starch + dextrose <50 ppb
– Potato starch + asparagine 117 ppb
– Potato starch + dextrose + asparagine 9270 ppb
Other Amino Acids
–Alanine <50 ppb Arginine <50 ppb
–Aspartic A. <50 ppb Cysteine <50 ppb
–Lysine <50 ppb Methionine <50 ppb
–Threonine <50 ppb Valine <50 ppb
–Glutamine 156 ppb Asparagine 9270 ppb
 Amino Acid Composition in Potatoes

Approximately 50% of amino acids are in the free state

(not incorporated into protein).

Asparagine is roughly half of the free amino acid content.

 Amide moiety
NH2
C O
CH2 H2O CO2
NH2 CH COOH
Carbonyl
Asparagine Schiff’s base Acrylamide
(glucose)
 3H2O NH3
From amino acid
(O)

2H2O Acrylic acid

Lipid Glycerol Acrolein .NH 2 Acrylamide

From amino acid

Radical
 Ingestion of heat-treated starch-rich foods (potato chips and
french fries)
 Acute toxicity :LD50 in rats: 159 mg/kg -300 mg/kg body weight
 Classified by the US EPA ( as a B2 (probable human carcinogen)
IARC (international Agency for research on
cancer) as a 2B (possible human carcinogen)
ACGIH (American Conference of Industrial
Hygiene) as A3 (confirmed animal carcinogen with unknown
relevance to human
Acrylamide - toxicology

 Proven neurotoxic compound in animals and in humans

 Effects range from drowsiness to incoordination,
hallucinations, confusion, abnormal sensation, muscle
weakness, incoordination
 Genotoxic compound with the potential to affect the
germinal cells thus leading to hereditary changes
 Causing cancer in laboratory animals (rats)
 Studies in humans (e.g. 8000 workers in China) which
were positive on neurotoxicity failed to prove
relationship with cancer in humans (too small
numbers ?)
POTATO PRODUCTS
 Acrylamide levels
increased with degree of
browning 45 µg/kg 76 µg/kg 262 µg/kg

 Brown color as measured

by “L” and “a” values
correlated highly with
acrylamide levels 516 µg/kg 866 µg/kg 1512 µg/kg
 Same acrylamide levels
found in French fries with
micrograms acrylamide/kg

10000

micrograms acrylamide/kg
similar degree of
10000
R2 = 0.8551
French fries

R2 = 0.8558
1000

French fries
browning (fried French
1000

100
100

fries) 10
45 55 65 75
10
5 10 15 20
"L" value
"a" value
 Food should not be cooked excessively (for too long or at too high temp)
 Maintain cooking at temp under 1500C, no preferable flavor chemicals
 FDA: Eat a balanced diet, choose a variety of foods that are low fat and rich
in high-fiber grains, fruits, and veggies