Occurrence of Heterocyclic Amines

in Muscle Foods and Mitigation with

Antioxidants

J. Scott Smith, Kanithaporn Puangsombat, Terry Houser, Melvin Hunt, Prini Gadgil, Riba Ameri

Animal Sciences and Industry

and Food Safety Consortium

Heterocyclic Amines

• Heterocyclic amines (HCAs) are mutagenic and

carcinogenic compounds that are present at ppb

levels in muscle foods cooked at high temperature.

• HCAs form via Maillard reaction with creati(ni)ne,

amino acids and sugars originally found in muscle foods.

• To date, more than 20 HCAs have been isolated from

different cooked muscle foods.

International Agency for Research on Cancer (IARC - WHO)

Classifies 8 of known HCAs (MeIQ, MeIQx, PhIP, AαC, MeAαC,Trp-2, Glu-p-1) as possible human carcinogens and IQ as a probable human carcinogen.

USA National Toxicology Program (Health and Human Services)

11th Report on Carcinogens:

Classifies 4 HCAs (IQ, MeIQ, MeIQx, and PhIP) as "reasonably anticipated to be human carcinogens."

HCAs and Cancer

HCAs and Cancers

Epidemiological Studies – various results

The high intake of well-done meat and high

exposure to meat carcinogens, particularly HCAs,

may increase the risk of human cancers.

• Colorectal cancer

• Breast cancer

• Prostate cancer

• Pancreatic cancer

Zheng and Lee (2009).

Heterocyclic Amines- types

• Aminoimidazole-azaarenes (AIAs) – Polar

• Produced by moderately high heating – typical

cooking/frying. Most studied group.

• Amino-carbolines (ACs) – Polar

• Pyrolysis of amino acids and proteins at temperature

above 575 ºF (300 ºC) higher temperatures

• Harman and Norharman – Carbolines - Nonpolar – not

mutagenic

• May still be toxic

Common HCAs in Foods

N R

N

N

NH2

CH3IQ R = H

MeIQ R = CH3

R1

R2 N

N

NH2

R

R R1 R2

IQx H H H

MeIQx H H CH3

DiMeIQx CH3 H CH3

N N

N

NH2

CH3

PhIP

(Murkovic 2004)

CHO

CHOH

CHOH

R

CH

CHOH

CHOH

R

NR CH NHR

CH OH

Carbohydrate Schiff Base

Glycol-aldehydealkylimine (enol type)

N

N

H

H

H

H

R

R

CH NR

CH2 OH

CH O

CH O

CH NR

CH O

Glyoxal

-RNH2

+H2O Glyoxalmono-alkylimine

Glycol-aldehydealkylimine

Dialkyl-dihydropyrazine

NH2HN

NCH3HOOCCondensation

Creatine

-e +e

N

+

Pyridine radical

N

NCH3

NH2

O

Creatinine Dialkyl-pyrazineradical

N

NCH3

N

NH2

N

NCH3

N

NH2

CH3 N

NCH3

N

NH2

H3C

N

NCH3

N

NH2

CH3

H3C

HCHOCH3CHO HCHO

CH3CHO

IQ MeIQ MeIQx 4,8-DiMeIQx

Reverse-aldolreaction

+RNH2

-H2O

N

N

+

R

R

sugar

amino acids

Creati(in)ne

Pyridine

radicals

Pyrazine

radicals

aldehyde

Formation

mechanism

of HCAs

Vitaglione and Fogliano (2004)

via Maillard rection

antioxidant antioxidant

7

PhIP similar

mechanism

• Meat and fish products cooked by frying, grilling

and broiling (especially in the surface part)

PhIP > MeIQx > DiMeIQx > IQ, MeIQ

• Pan residues

• Grill scrapings

(nd – 50 ng/g) (nd – 8 ng/g) (nd – 6 ng/g) (nd – 4 ng/g)

HCAs in Foods

(Knize and Felton 2005)

• Cooking time and temperature

• Cooking methods – frying, broiling, grilling

• Concentration of precursors

(creatine/creatinine, amino acid, sugar)

• Moisture content - low

• Lipid content - lower

• Meat characteristics

(animal species, muscle type, aging, meat quality)

Factors affecting HCA formation

Cooking Methods

cooking conditions

rela

tiv

e l

ev

els

of

HC

AS

Very low levels

or non-detected

Low levels

High levels

Boiling, Deep fat-frying

Roasting, Baking

Grilling,

Broiling,

Pan-frying

Effect of cooking methods on HCA formation in cooked meat products (Chen and Chiu 1998)

0

5

10

15

20

25

30

2 4 6 8 10

Cooking time per side (min)

HC

As

(n

g/g

be

ef)

temp 230 ºC

(446 ºF)

temp 190 °C

(374 °F)

150 °C

(302 °F)

HCAs (PHIP) in fried beef patties, cooked at different

temperatures/times (modified from: Thomson, 1999)

time > 4 min

Cooking Methods

• Modifying cooking methods

- cook at lower temperature/time

- microwave precooking

• Addition of antioxidants

- synthetic antioxidant (BHA, BHT, PG, TBHQ)

- natural antioxidant polyphenolics from a wide variety

of sources (fruit/plant and their extracts)

• Improving water-holding capacity

- water-binding compounds (carbohydrate, phosphate)

Inhibition of HCAs

Heterocyclic Amine Content

in Commercial Ready to Eat

Meat Products

Abstract presented at IFT Meeting 2009

Publication: Puangsombat K, Gadgil P, Houser TA, Hunt MC, Smith JS.

Heterocyclic amine content in commercial ready to eat

meat products . Meat Science 2011

Supported by the American Meat Institute Foundation and National Pork Board

Objective

To estimate the amount of HCAs in

commonly consumed RTE meat products

• Hot dog beef

• Hot dog beef-pork-turkey

• Deli roast beef

• Deli ham

• Deli turkey

• Pepperoni

• Fully cooked bacon

• Rotisserie chicken

Hot dog beef, Hot dog beef-pork turkey

Wrapped in a paper towel and heated in a microwave for 35 s

Fully-cooked bacon

Heated in microwave for 30 s

Pepperoni (from frozen pizza)

A frozen pizza heated in an oven at 400 ºF for 23 min

Analyzed as obtained

chicken - skin and meat were separated

Sample Preparation

followed the package directions

HCA (

ng/g)

0

2

4

6

8

10

12

14

16

18

20

hotdogbeef

hotdogcombo

deli roastbeef

deli ham deli turkey pepperoni bacon chickenmeat

chickenskin

PhIP

DiMeIQx

MeIQx

IQx

IQ

deli ham

deli turkey

pepperoni

S.D. ± 18.63

b b

a

a

ab

b b b b

16

Conclusion

• HCA levels in RTE meat products are very low (less

than 1 ng/g), except for rotisserie chicken skin.

- Low-temperature manufacturing process (165-180 ºF)

- Presence of ingredients and additives that inhibit HCA

formation (e.g. salt, phosphate, spices)

• Consumption of RTE meat products contributes very

little to HCA intake.

Abstract presented in part at IFT Meeting 2010

Currently under review for publication

Supported by the American Meat Institute Foundation and National Pork Board

Heterocyclic Amine Content

in Cooked Meat Products

Objective

To determine the HCA content in cooked meat

products prepared by various cooking methods

that are preferred by U.S. meat consumers.

Previous studies but products have changed over

the years

Choice of meat samples in this study >>

Exponent Inc. conducted Internet-based survey of

U.S. consumers‟ preference for method of cooking

and degree of doneness of meat and fish.

Cooking conditions Sample Types/cuts of

meat

Cooking

method

Cooking

temperature

Desire internal

temperature

Beef Top loin Frying 400 ºF 135 ºF for medium-rare

160 ºF for well-done

Top loin Oven-broiling 450 ºF 135 ºF for medium-rare

160 ºF for well-done

Pork Top loin Frying 400 ºF 160 ºF

Bacon Frying 342 ºF

3 min/side

*cook 3 slices each time

Chicken Breast with and

without skin

Frying 400 ºF

160 ºF

Fish Catfish, Salmon,

Tilapia

Frying 400 ºF

145 ºF

Oven-baking 350 ºF 145 ºF

Results HCA levels Cooked meat products Total HCAs

(ng/g)

Low

( < 5 ng/g)

Medium-rare fried beef

Fried chicken with skin

2.73

3.13

Intermediate

(5 -10 ng/g)

Well-done broiled beef

Fried chicken without skin #1 (9.57 g/day)

Baked fish

Well-done fried beef #2 (8.52 ng/g)

6.04

7.06

8.32

8.92

High

( > 10 ng/g)

Fried fish

Fried pork #3 (2.89 g/day)

Fried bacon #4 (1.39 g/day)

11.57

13.91

17.91

# 1-4: Most frequently consumed meat items in U.S. (National Health and Nutrition Examination Survey 2003-2006).

Conclusions

• HCA content in cooked meat depends on type of

meat, cooking methods, and cooking

time/temperature.

• Controlling cooking time and temperature can

minimize the HCA formation.

The Use of Antioxidants and

Marinades to Inhibition of

Heterocyclic Amines

Abstract presented in part at IFT Meeting 2007

Funded in part by Mitsubishi Chemical

Puangsombat K, JS Smith. 2010. Inhibition of heterocyclic amine formation in

beef patties by ethanolic extracts of rosemary. Journal of Food Science

75(2): T40-47.

Smith JS, Ameri, F., and P. Gadgil. 2008. Effect of Marinades on the Formation

of Heterocyclic Amines in Beef Steaks. J. Food Sci. 73:1854-18591.

• Wide variety of research on the role of antioxidant

rich plant sources inhibition of HCA formation.

• Tea, chocolate, wine, fruits, and spices have been

studied. – All are rich polyphenolics which can block

the reactions via several mechanisms

• Rosemary/oregano extracts are frequently use

because easy to extract polyphenolics from volatiles

• Currently used in fresh and processed/frozen

products, often labeled as „natural flavor”

Introduction

• Add antioxidant/extract directly to product (ground beef), apply to surface, inject or marinade

• Cook on thermostat-controlled electric grill at 375-400 °F for various times

• Samples grind and analyze via HPLC

• Analyze the spice/extracts for antioxidant levels by HPLC

How - Methods

Objectives

• To investigate the influence of commercial marinades containing natural antioxidants on the reduction of HCAs on grilled beef steaks.

• Finding the correlation of the antioxidative components in spices and HCAs formation.

• Marinated steaks were treated with each of 3 marinades separately as labeled for one hour at 4 C before grilling.

• Controls and base formulations

• Meat samples (eye round steak) were grilled in a thermostat-controlled Teflon coated electric grill at 400 ºF (204 ºC) for 5 minutes each side.

• 2-4 mm thickness of steaks removed for analysis.

Materials & Methods

Marinades

Base Ingredients Spices Composition of liquids to formulate

marinade

Marinade #1 salt, sugar, caramel color thyme, red pepper,

black pepper,

allspice, rosemary,

chives

1/4 cup water, 2 tbs soybean oil, 2

tbs vinegar

Marinade #2 salt, sugar maltodextrin, , silicon

dioxide

paprika, red

pepper, oregano,

thyme, black

pepper, garlic,

onion,

2 tbs water, 1/4 cup soybean oil, 2

tbs vinegar,

Marinade#3 salt, sugar maltodextrin,

modified corn starch, whey

solids, soy protein, wheat gluten,

calcium silicate

oregano, basil,

garlic, onion,

jalapeno pepper,

parsley, red pepper

1/3 cup water,3 tbs soybean 3 tbs

vinegar,

HCAs in unmarinated and marinated steaks

-10

10

30

50

70

90

110

130

150

Blank Marinated # 1 Marinated # 2 Marinated # 3

MeIQ MeIQx PhIp Harman Norharman

Perc

ent

of contr

ols

Level of antioxidants in different marinades

0

5

10

15

20

25

30

35

40

45

Co

nc

en

tra

tio

n (

pp

m)

Marinade # 1 Marinade # 2 Marinade # 3

Rosmarinic Acid Carnosol Carnosic acid

Enhancement and Commercial

Products

Supported by the American Meat Institute Foundation and National Pork Board

Summary

Enhancement of fresh meat - addition of salt and

phosphate (7-15%), significant reduction in HCA

levels

• Pork 50 % reduction

• Commercially enhanced and marinated products:

• With 12-30% enhancement see 30-60 % reductions

• Some is clearly tied to water retention but also

to presence of antioxidants (spices)

Reciprocation

• Where do we go from here?

• Revised products with more enhancement?

• Add more antioxidants?

• Promote antioxidants in meat products.

• How to deal with “bad” press? Do we

ignore or should there be more discussion

and education?