EVALUATION OF MALONALDEHYDE AS A MARKER OF OXIDATIVE RANCIDITY IN MEAT PRODUCTS

FEREIDOON SHAHIDI and CAROLINE HONG

Department of Biochemistry Memorial University of Newfoundland

St. John's, Newfoundland, A I B 3x9, Canada

Received for Publication July 19, 1990 Accepted for Publication February 15, 1991

ABSTRACT

QuantiJication of malonaldehyde as a marker of lipid oxidation in comminuted meat systems, with or without additives, by a distillation and an extraction procedure was investigated. Each method had its own benefits and drawbacks. The extraction procedure generally afforded more realistic results. However, presence of colored additives such as the cooked cured-meat pigment, as well as dispersed lipids in the extracts, interfered with accurate determination of the colored chromogen between malonaldehyde and the 2-thiobarbituric acid (TBA) reagent. On the other hand, the distillation procedure afforded higher TBA values as compared to the extraction method since decomposition of labile hydrope- roxides occurred. Addition of antioxidants to the mixtures prior to distillation proved beneficial in some cases. In the presence of residual nitrite, addition of sulfanilamide to the mixture prior to distillation prevented underestimation of malonaldehyde presumably by inhibiting its nitrosation.

INTRODUCTION

Kohn and Liversedge (1944) were the first to use the 2-thiobarbituric acid (TBA) test for evaluation of the oxidative state of meats. They observed that animal tissues, upon aerobic incubation, gave rise to a pink-colored compound when combined with 2-thiobarbituric acid (TBA) reagent. During autoxidation of polyunsaturated fatty acids of lipids, malonaldehyde (MA) is produced. This secondary oxidation product is highly reactive and remains bound to other food ingredients. An acid/heat treatment of food would presumably release the bound malonaldehyde (Tarladgis et al. 1960).

Journal of Food Biochemistry 15 (1991) 97-105. All Rights Reserved. 0 Copyright 199I by Food & Nutrition Press, Inc., Trumbull, Connecticut 97

98 FEREIDOON SHAHIDI and CAROLINE HONG

'1

Malonaldehyde is the principle substance reacting with the TBA reagent as shown in Fig. 1 (Nair and Turner 1984). The pink-colored chromogen produced has an absorption maximum at 532 nm. Spectrophotometric determination of this complex is the method usually employed for quantification of malonaldehyde and other TBA reactive substances. In addition to MA, 2,4-alkadienals, and to a lesser extent, 2-alkenals also produce a pink-colored pigment which absorbs at 532 nm (Marcuse and Johansson 1973).

The TBA test may be performed directly on a food product (Wills 1965), and this may be followed by extraction of the colored pigment into butanol or butanol- pyridine mixture (Placer er al. 1966; Uchiyama and Mihara 1978; Ohkawa er al. 1979). The test may also be carried out on an aliquot of an acid extract of food (usually 7.5 to 28% trichloroacetic acid (TCA) solution; Siu and Draper 1978) or on a portion of a steam distillate of the sample under investigation (Tarladgis er al. 1960, 1964). The distillation procedure developed by Tarladgis et al. (1960) is the method frequently used and the TCA extraction procedure has also been used by many researchers.

The TBA number, which expresses lipid oxidation and defined as mg of malonaldehyde per kg of sample (Sinnhuber and Yu 1958), is calculated by multiplying the absorbance of the TBA-MA complex at 532 nm by a constant. This constant is obtained by the use of standard precursors of malonaldehyde

Halonaldehyde 2-Thiobarbituric Acid TBA-.YA Adduct

FIG. I . ADDUCT FORMATION BETWEEN MALONALDEHYDE AND THE 2-THIOBARBITURIC ACID REAGENT

MALONALDEHYDE AS A MARKER 99

such as lt1,3,3-tetramethoxypropane (TMP) or 1,1,3,3-tetraethoxypropane (TEP).

Several modified procedures have been reported to improve the standard method of Tarladgis et al. (1960). These include coupling of the procedure with an HPLC method, in place of colorimetry or extraction of the TBA-MA into an organic solvent, to avoid possible interference from other colored impurities (Kakuda et al. 1981).

This paper examines the benefits and drawbacks of each of the two major methods employed in the determination of the TBA numbers of meat products, mainly the distillation method and the extractiodfiltration procedure. Effect of minor components present in the meat or added to the samples prior to or during quantification of TBARS will also be presented.

MATERIALS AND METHODS

Materials

All chemicals and reagents used in these studies were reagent-grade, and were used without further purification. The cooked cured-meat pigment was prepared as described previously (Shahidi and Pegg 1988).

The meat, pork loin obtained from Newfoundland Farm Products, St. John’s, Newfoundland, was deboned and trimmed of most of its surface fat. It was then ground twice using an Oster meat grinder. Additives, along with 20% (w/w) of distilled water, were added to meat samples prior to cooking. In all cases, the mixtures were thoroughly mixed to obtain homogeneous samples. The addition levels of different additives are given in corresponding tables.

Homogenized meat samples were cooked in glass containers in a thermostated water bath at 80°C for 40 min to reach an internal temperature of 75°C. After cooling to room temperature, they were homogenized for 20 s in a Waring Blendor and were stored in plastic bags at 4°C until used.

The TBA numbers of meat samples were determined by a distillation and/or an extraction procedure. The distillation procedure was essentially that of Tar- ladgis et al. (1960) with minor modifications as described elsewhere (Shahidi et al. 1987). A 10 g meat sample was placed into a 500-mL round-bottom flask containing 97.5 mL distilled water and 2.5 mL 4 N HCI, along with a few drops of Dow Antifoam A and several glass beads. For some cured meats, sulfanilamide was added to the mixture as described elsewhere (Shahidi 1989a). The mixture was then heated for approximately 20 min to collect 50 mL of distillate. The distillation unit was equipped with a 30-cm fractionating column. A 5 mL aliquot of the distillate was pipetted into a 50 mL vial containing 5 mL of 0.02 M aqueous solution of 2-thiobarbituric acid reagent. The vial was then capped and

100 FEREIDOON SHAHIDI and CAROLINE HONG

heated in a boiling water bath for 35 min to obtain the TBA-MA chromogen. After cooling the vial in cold water to room temperature, the absorbance of the complex was read at 532 nm in a DU-8 spectrophotometer. Using 1,1,3,3- tetramethoxypropane (TMP) as a standard, a conversion factor of 8.1 was ob- tained for converting the TBA-MA absorbance readings to TBA numbers.

For the extraction procedure, a 2 g meat sample was placed into a vial along with 5 mL of a 10% (wh) trichloroacetic acid solution. The mixture was vortexed for 2 min and then 5 mL of 0.02 M aqueous solution of 2-thiobarbituric acid was added and the mixture vortexed again for 2 min. The mixture was then centrifuged and the resultant supernatant was pipetted off for color development and measurement of the TBA-MA absorbance reading. Using a TMP standard, a factor of 3.6 was obtained for converting the absorbance units to TBA numbers.

In some experiments an antioxidant or a sequesterant was added to meat samples prior to distillation/extraction. The level of addition of these additives is given in tables where they appear. All experiments were performed in triplicate with means and standard deviations reported. Analysis of variance was conducted according to Snedecor and Cochran (1980).

RESULTS AND DISCUSSIONS

Table 1 summarizes the results for the TBA numbers of meat systems prepared by addition of certain additives such as butylated hydroxyanisole (BHA), tert- butylhydroquinone (TBHQ), sodium tripolyphosphate (STPP), disodium salt of ethylenediaminetetraacetic acid (Na,EDTA), sodium nitrite (NaNO,) and the preformed cooked cured-meat pigment (CCMP) prior to cooking. These additives showed strong inhibitory effects against the development of lipid oxidation in the cooked meats (Table I). The results indicate the following trends: (a) the TBA numbers of meat systems determined by the distillation method were gen- erally higher than those obtained by the extraction procedure; (b) storage of the meat samples at 4°C for nearly 3 weeks resulted in a substantial increase in the TBA numbers; (c) presence of sodium nitrite in the systems gave results which varied in different directions dependent on the nitrite concentration when sulf- anilamide was added to the mixture prior to distillation (i.e., at 25 ppm nitrite level, the values became smaller while at 150 ppm nitrite concentration, the values became larger); (d) addition to meat of CCMP, the pigment responsible for imparting the cured color to meats (Shahidi 1989b; Shahidi and Pegg 1991 ), gave substantially higher TBA values when the extraction procedure, as com- pared to the distillation method, was employed. However, the opposite was true when meats were stored for nearly 3 weeks; and (e) addition of TBHQ to the meat system containing CCMP gave results which were always higher when using extraction, as compared to distillation, procedure.

TABL

E 1.

T

BA

NU

MBE

RS

OF

MEA

T SY

STEM

S D

ETER

MIN

ED B

Y A

DIS

TILL

ATI

ON

OR

EXTR

AC

TIO

N M

ETH

OD

AFT

ER 1

AN

D

20 D

AY

S O

F ST

OR

AG

E A

T 4°

C

i -!!a

>: 20-3avs

No.

Ad

dit

i'J+

s Gist i

1 1 a

t io

n

Ex t

r sc L i o

n

Dist

illa

tion

Ex

trac

tion

No a

dd

iciv

e

BHA

(20

0 p

pm)

TBH

Q

(20

0 p

pm)

STPP

(3

00

0 p

pm)

Na2E

DTA

(50

0 p

pm)

NaN0

2 (2

5 p

pm)

NaN0

2 (1

50 p

pm)

CCM

P (1

2 p

pm)

3+

8

3.1

7 5

0

.05

au

(2

.98

2 0

.08

)bw

0.1

2 2

0.0

1cy

0.1

0 2

O.O

ldW

0.0

9 5

O.O

lcu

0.15

5 0

.02

eU

1.14 5

0.0

2fu

(1.08

2 0.01)gY

0.0

1 5 O

.OO

hY

(0.2

9 2

O.O

1)iY

0.3

6 5

0.0

2J"

1.e

; k o

.05

d~

1

0.2

2 2

0.

1~

__

_ (9

.80

2 O

.lO

)bx

0.15 5

0.0

2b

X

0.41

i 0

.02

cv

0.18 2

O.O

lCx

0.2

0

0.0

3&

0.1

2 2

O.O

ldu

0.7

1 2

0.0

2e

X

0.2

0 5

O

.Olc

Y

0.41

5 O

.OlC

x

1.3

8 5 O

.Ole

x 3

.40

i 0

.05

'v

0.2

8 2

0.0

3'"

0.1

0 2

O.O

lhv

1.0

9 ?

0

.02

gx

8.3

2 &

0.0

5jy

___

(3.2

8 2

O.0

3)gx

___

(0.3

6 2

0.0

2)i

y

5.3

3 2

0.0

3a

2

0.3

7

& 0

.03

h

0.2

5 &

0.0

2cv

0.4

8 2

O.O

l+

0.4

1 2

O.O

lex

2.9

3 2

0.0

2f2

0.2

6 2

O.O

lCx

4.0

9

0.02

gz

___

___

_--

0.1

6 5

O.O

leu

0.3

3 2

O.O

lha

0.1

2 &

0.01"

0.3

4 2

O.O

lbx

'Sym

bols

are: b

utyl

ated

hyd

roxy

aniso

le,

BH

A; t

ert-b

utyl

hydr

oqui

none

, TBH

Q; s

odiu

m tr

ipol

ypho

spha

te, S

TP

P d

isodi

um e

thyl

en-

diam

inet

etra

acet

ic ac

id s

alt,

Na,

EDTA

; co

oked

cure

d-m

eat p

igm

ent,

CC

MP.

Val

ues i

n pa

rent

hese

s ar

e fo

r sa

mpl

es co

ntai

ning

add

ed

sulfa

nila

mid

e. M

eans

in t

he s

ame

colu

mn

(a-j)

or

in t

he s

ame

row

(w

-z)

with

the

sam

e su

pers

crip

t are

not

diff

eren

t (P

> 0

.05)

.

102 FEREIDOON SHAHIDI and CAROLINE HONG

The above results may be interpreted in the following manner. Breakdown of labile hydroperoxides during the distillation process is responsible for larger TBA values when this method was employed. As expected, storage of meat for an extended period of time results in production of a larger amount of malon- aldehyde in most of the systems. For nitrite-treated samples, apart from the antioxidant effect of nitrite, nitrosation of malonaldehyde by the residual nitrite present in the systems was responsible for their underestimation. However, addition of sulfanilamide (Zipser and Watts 1962) resulted in a noticeable increase in the TBA values when meats were cured with 150 ppm of sodium nitrite. Sulfanilamide reacted with nitrite molecules, resulting in better quantification of the malonaldehyde in the meat sample. For meat systems devoid of nitrite or treated with 25 ppm nitrite, addition of sulfanilamide resulted in a decrease in the TBA numbers. Formation of adducts between sulfanilamide and the malon- aldehyde have been reported to be responsible for this observation (Shahidi et al. 1987; Shahidi ef al. 1991).

Presence of CCMP in freshly cooked meat systems (day l), gave rise to larger TBA values by the extraction procedure. This was due to the extraction of this pigment into the TCA solution and its interference with the determinations. However, as the samples were stored and rancidity developed, interference from CCMP was overshadowed by production of large amounts of malonaldehyde in the systems and also due to further breakdown of labile hydroperoxides during the distillation process. Presence of an antioxidant such as TBHQ in the system prevented the development of rancidity and thus the trend remained unchanged (Table 1).

A further scrutiny of the results shows that for samples treated with strong antioxidants such as BHA and TBHQ (Table l ) , or powerful chelators such as EDTA, somewhat larger TBA numbers were obtained when an extraction pro- cedure was employed. This is due to the dispersion of some lipid particles in the aqueous extracts, and perhaps also due to dissolution of some proteins, thus causing turbidity. Turbidity of the extraction solutions results in artificially high TBA values.

In another set of experiments the effect on the TBA values of addition of antioxidants/sequesterants prior to the distillation or extraction process of the samples was monitored. Results summarized in Table 2 indicate that the addition of antioxidants had a slight effect in lowering the TBA values by the distillation procedure. However, little effect was observed when the extraction process was employed. Presence of Na,EDTA, alone or in combination with an antioxidant, resulted in a slight increase in the TBA numbers. This may be due to the fact that iron in the systems prior to its release would be kept by EDTA in its more powerful pro-oxidant ferrous state rather than being converted to its less potent pro-oxidant ferric state. The pro-oxidant activity of ferrous and ferric ions, as

TAB

LE 2

. EFFECT O

F ANTIOXIDANT/SEQUESTERANT AD

DED

TO

MEA

TS P

RIO

R TO

TB

A D

ETER

MIN

ATI

ON

BY

A D

ISTI

LLA

- TI

ON

OR

AN

EX

TRA

CTI

ON

MET

HO

D'

I

Cont

rol

Trea

ted

Cont

rol

Trea

ted

Z i? Fi

Dist

illa

tion

Ex

trac

tion

No

. An

tiox

idan

t/

Sequ

este

rant

t

1 2 3 4 5 6 7 8 9 10

BHA

(50

0 p

pm)

7.3

7 2

0.0

8aU

BH

T (500 p

pm)

8.8

0 2

0.O

Sbu

Etho

xyqu

in

(20

0 p

pm)

6.7

3 5

0.0

3c

y

PG

(50

0 p

pm)

4.3

2

2 0

.02

* Na

ZEDT

A (5

00

ppm

) 4

.32

5 O

.OSd

Y Te

nox

I1

(20

0 p

pm)

11

.46

2 O

.lO

eY

Teno

x A

(2

00

ppm

) 8

.80

5 0

.10~

1+

5

7.3

7 2

0.1

2aY

Fe(II)

(10

PPm

) 3

.20

5 0

.05

fy

Fe(I

I1)

(10

ppm

) 3

.20

2 O

.0S

fy

7.0

0 2

0.0

3a

X

8.3

9 2

O.l

Obx

6

.12

2

0.09

" 3

.92

2

0.0

8d

X

4.5

2 5 0

.06

eX

1

0.4

3 2

O.l

ZfX

7.4

0

0.0S

hU

4.9

3

2 0.

04"

4.7

0 2

0.0

5jX

7.7

1 2

0.10

9~

f

<

4.9

2

2 0.

02'v

5

.48

2 0

.02b

v 4

.96

2 0

.05'

v 5

.42

2

0.0

Zb

2

8 3

.28

2

0.O

Zcv

-

% 2

.92

2 0

.05*

3

.33

2

0.0

2-

>

7.5

2 2

0.0

5e

v 7

.43

2 0.03d2

I

3.3

4 2

0.0

2cz

-

'Sym

bols

are

foo

tnot

ed to

Tab

le 1

. M

eans

in t

he s

ame

colu

mn

(a-j)

or

in t

he s

ame

row

(x-

z)

with

a c

omm

on s

uper

scrip

t are

not

di

ffer

ent (P >

0.0

5).

104 FEREIDOON SHAHIDI and CAROLINE HONG

quantified by their effect on the TBA number of meats, are given in Table 2. Furthermore, EDTA-Fe(I1) may cause decomposition of hydroperoxides, thus giving rise to artifically high TBA values.

ACKNOWLEDGMENTS

This work was financially supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada.

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