Detection of Pork in Processed M eat: Exp erim ental
Com parison of M ethodology
W a j i h N . S a w a y a , ° T . S a e e d , ° M . M a m e e s h , ~ E . E 1 - R a y e s , °
A . H u s a i n , ° S. A l P & H . A b d u l R a h m a n °
° Ku w ait Inst i tute for Scientif ic Research, PO Box 24885,
13109 Safa t, Ku wai t
b Biochemis try Dep ar tment , K uw ai t Univers i ty , PO Box 5969,
13060 Safa t, Ku wai t
(Received 5 May 1989; rev i sed vers ion rece ived and accepted
28 September 1989)
A B S T R A C T
F o u r d i f fe r e n t m e t h o d s w e r e c o m p a r e d t o d e t e c t p o r k i n p r o c e s s e d m e a t s .
These inc luded: ana lys i s o f m ea t f a t by h igh per form ance l iqu id chroma-
t o g ra p h y ( H P L C ) f o r t ri gl yc e ri de s ( T G s ) a n d b y g a s c h ro m a t o g r ap h y
( G C ) f o r f a t t y a cid s; a n d a n a ly s is o f m e a t p r o t e in s b y e n z y m e - l in k e d
i m m u n o s o r b e n t a s s a y ( E L I S A ) , a n d o f o ph id in e d i p ep ti de s b y H P L C .
L o w le ve ls o f p o r k i n p r o c e s s e d m e a t s c a n b e d e t e c t e d b y e i t h e r f a t o r
p r o t e i n a na ly sis ; f a t a n a l y si s c a n a l s o b e u s e d f o r a l l f o o d p r o d u c t s t h a t
con ta in po rk fa t . TG ana lys i s is m ore re liab le than fa t t y ac id ana lys i s u s ing
C 20 :2 as a marker ; however , t he G C m e tho d i s s imp ler , f a s t e r an d requ ire s
l e ss s a m p l e p r e p a ra t io n . B o t h T G a n d G C m e t h o d s c a n d e t e c t l ev e ls a s l o w a s
2 p o r k in p r o c e s s e d m e a t .
In the EL IS A t echn ique , c rude prepara t ions o f sheep-an t ip ig an t i se rum can
d e t e c t lo w le v e ls ( 2 ) o f p o r k i n b e e f o r m u t t o n s a m p l e s h e a t e d a t 7 0 , 1 00
an d 120°C. The a na lys i s o f oph id ine d ipep tides can a l so de tec t l ow l eve ls
( 2 - 5 ) o f p o r k i n h e a t e d /p r o c e s s e d m e a t s ; h ow e v er , t h is m e t h o d w a s n o t
t e s t ed fo r d i f fe rences in sex , b reed, d i e t and m usc le t ype .
I N T R O D U C T I O N
T h e d e t e c t i o n o f p o r k i n v a r i o u s f o o d p r o d u c t s , p a r t ic u l a r ly i n c o o k e d o r
p r o c e s s e d m e a t , h a s b e e n a n i m p o r t a n t s u b j ec t o f s t u d y i n m a n y c o u n t ri e s,
e spec ia l ly where religious laws pro hibi t the c onsum ption o f p o r k p r o d u c t s .
201
Food Chemistry 0308-8146/90/$03.50 © 19 90 Elsevier Science Publishers Ltd , En gland .
Printed in Great Br i ta in
 
202 W a j i h N . S a w a y a et al.
M ost o f the research work repor ted on th i s subjec t has concen t ra ted on the
ana lys is o f po rk fa t an d the iden t i f ica t ion of po rk pro te ins .
Po rk fa t is uniqu e in i ts pecul iar fa t ty acid dis t r ibut ion an d t r iglycer ide
(TG) com posi t ion. Since the fa t ty acid 11,14 eicosadien oic acid (C20:2) was
repor ted t o be p re sen t i n po rk f a t an d absen t in o the r com m only consumed
m eats an d fa ts (Saeed e t a l . 1986), i ts presence in m eat prod ucts was u sed as
an ind ica tor for the presence o f pork . How ever , the presence of C20:2 was
recent ly repor ted in some beef and mut ton samples (F i res tone , 1988) .
Rugraf f and Kar lesk ind (1983) separa ted sa tura ted TG s and obta ine d 2-
m onog lycer ides using panc reat ic lipase, an d used the r at io o f palmit ic acid in
the sa tura ted TG s a nd the 2-monoglycer ides to de tec t pork in mea t . This
un ique compos i t i on o f in t a c t po rk TG s was u ti li zed fo r t he de t ec t ion o f po rk
in processed meats (Sawaya & Saeed, 1988) . In co ntra st to oth er an im al fa ts ,
po rk T G s are m ost ly es teri fied b y saturated fa t ty acids a t the C-2 p osi t ion.
Fo r exam ple, ab ou t 8 0% o f tota l palm it ic acid conte nt is es teri fied at the C -2
pos i t ion in po rk (Bradford e t a l . 1965) , bu t on ly 1 5-27% in beef, lam b an d
deer . Accord ing to the n um ber o fsa tu ra ted (S) or unsa tura ted (U) fa t ty ac ids
in the TG molecules, TG s are classified int o fou r types: $3, S2U, SU2 an d U3.
Tw o types , S2U an d SU2 can ex is t in two i somet r ic forms , g iv ing SUS and
SSU, and UU S and US U respective ly . Ch ack o and Perk ins (1965) repor ted
tha t po rk f a t con ta in s 38% SSU, 41% USU , 1% SUS and 7% UU S. In o the r
an imal fa t s (Bradford
e t a l .
1965) , the T G com pos i t ion was d i s t inc t ly
different: 9-14% SSU, 13--38% SUS a nd 2 6-3 8% UU S. The character is t ic
c o m p o s i t i o n o f f a t T G s a n d t h e u s e o f h i g h p e r f o r m a n c e l i q u i d
ch ro m a tog raph y (HPLC) in t he l as t ten yea r s fo r s epa ra t i on and
ident i f icat ion o f t he TG s o f na tu ra l f a ts and o i ls (P l a tt ne r e t a l . 1977; H erslo f
e t a l . 1979; Herslof , 1981; Plat tner , 1981) hav e bee n useful in d etect ing po rk
in m eat p roduc ts .
Several m ethods have been used for spec ia t ion of f resh meat , inc lud ing
imm unodi f fus ion (Swakt & W ilks , 1982; Do bers te in & Greue l , 1982; Shaw
e t a l . 1983), e lectrophoresis (Slat tery & Sinclair , 1983; Glesso n e t a l . 1983)
and enzyme- l inked imm uno sorbe nt assay (ELISA) (Kan g 'e the & Pa t te rson ,
1982; W hi t take r e t a L 1983; Jones & Pa t te rson , 1986). Spec ia t ion of coo ked
m eat i s m ore d if ficu lt because the dur a t ion and temp era ture o fhe a t in g a ffec t
the pro tein s t ructure an d species-specif ic ant igenic dete rm inan ts (M urak im i
e t a l . 1983; K atsub e & Imaizu m i, 1968); how ever , repo rts in the l i teratu re
ind ica te the presence of therm os tab le an t igens in an im al ti ssues. Ha yde n
(1981), u t i lized adre nal heat-s tab le ant igen s fo r species iden t i f icat ion o f
coo ked sausage (core tem perature , 71°C), us ing aga r gel imm unod iffusion.
K ing (1984) , ana lyzed the t he rmos tab l e enzyme adeny la t e k inase
(EC 2 .7 .4 .3) in p ork , rena tura ted wi th 6M urea or 6M guanid ine h ydro-
ch lor ide , us ing enzyme s ta in ing o f i soe lec t rofocus ing ge ls. Kang 'e the
e t a l .
Detection of pork in processed meat 203
(1986) and Kang ' e the and Ga thu m a (1987) u sed the rmos t ab le musc le
an t igens (TMA) for spec ia t ion of meat p roduc ts so ld in Kenya ' s re ta i l
marke t s an d o f au toc l aved mea t s ample s by imm unod i f fu s ion and enzyme
im m uno assay (EIA), respectively. How ever , these ant igens are presen t in low
conc ent ra t ions and a re no t h ighly monospec i fic .
An alysis of histidine d ipeptides ( ,8-alanyl-t.-histidine, carno sine; p-alany l-
c-m ethylhis t idine, anser ine; ~-alanyl- t . -3-m ethylhis t idine, balenine) has
been used for m eat spe c ia t ion (Carnegie
e t a l .
1982) . Tinb ergen an d Slum p
(1976) showed tha t ch icken could be iden ti fied in lunch eon m eats by ana lys is
of anser ine and carnos ine . Carnegie e t a l . (1982) showed th a t the an ser ine-
ba len ine ra t io is usefu l in com par ing t inned ham s. O lsman and S lump (1981)
rev iewed the problem and conc luded tha t the h i s t id ine d ipept ides could be
useful for the ident i f icat ion o f me at species used in mea t produ cts . Carnegie
e t a l .
(1984) used this m eth od to es t imate pig con tent in different processed
m eats; however, the use of pep tide ratios fo r species identification s limited by
large v ariatio ns am on g different muscles in the same a nim al (Crush, 1970;
T a n a k i e t a L 1977) an d by sm all differences between closely relate d species.
Al th oug h severa l m ethods hav e been repor ted for d e tec t ion of pork fa t o r
po rk m eat in m eat pro ducts , there is s t il l a n eed fo r re l iable sensit ive an d
s imple m ethods to de tec t low percentages of po rk in hea t -processed meat .
This inves t iga tion was u nde r taken to explore such metho ds and to eva lua te
them by ana lys is o f mixed mea ts , f a ts and hea t ed mea t p roduc t s o f kno wn
compos i t i on .
M A T E R I A L S A N D M E T H O D S
Collect ion o f samples
Authen t i c s amples o f po rk f rom an ima l s r a is ed und e r con t ro l led cond i ti ons
were ob ta ined f rom the Danish Meat Research Ins t i tu te (Roskl ide ,
Denmark ) . Samples o f bee f and mu t ton were ob t a ined f rom the loca l
marke t .
P r o d u c t i o n o f a n t i sp e c i e s /a n t is e r a
Po rk m eat ex t rac t (an t igen) was p repared f rom represen ta t ive samples of
lean pork . The m eat sample was h om ogenized wi th 5% sa l ine (1:1 v /v) and
autoc laved a t 120°C for 30 min . Th e h om oge nate was cooled , a llowed to
se tt le an d then f il te red 0V ha tm an f il te r paper N o. 42) . Tw o sheep were
imm unized by in jec t ing 2 m l o f po rk an t igen (au toc laved and f il te red meat
ex t rac t ) in Freund ' s comple te ad juva nt (3 ml o fm ea t ex t rac t to 5 ml Freu nd ' s
 
204 Wajih N Sawayaet al.
Booster doses were given every other week using the antigen in Freund's
incomplete adjuvant. The animals were bled first after 4 weeks and
thereafter every 2 weeks. The collected blood was centrifuged at 1500g and
the serum was then stored in 2-3 ml portions at -20°C.
Sampling
Laboratory prepared samples (standards) were made from either pure fat or
lean meat (i.e. without visible fat). Single-species meat samples were minced
separately, combined in different proportions and mixed thoroughly before
processing. Commercially prepared meat samples in tinned cans (size U4,
300 x 207, weight approximately 270 g/tin), containing pure pork luncheon
meat, pure beef sausage or a mixture of 0, 1 and 5% pork/beef sausage mix
were processed by the Institute of Food Research, Bristol Laboratory, and
the Campden Food Preservation Research Association (Chipping Camp-
den, Gloucestershire, UK). The retort processing met a Fo 16 value, to give
reasonable protection against thermophilic spoilage (total process time was
80 min). These samples were used as representative of commercially canned
meat (unknowns) in testing different methods of determining the content
percentage of pork.
Sam ple preparation
H P L C ana ly sis o f TGs
One to two gram samples were extracted with 15 ml chloroform/methanol
1:2 v/v. The combined filtrate was evaporated to dryness under vacuum by a
rotary evaporator and stored under nitrogen in a deep freeze. TGs were
separated from other lipids by column chromatography on acid-washed
florisil (Carrol, 1976), using a chromatographic column (1 5 x 20 cm) packed
with 30 g of acid-treated florisil in hexane as slurry. The flow rate was
adjusted to 1 ml/min. The sample (100mg) was introduced as a hexane
solution at the top of the column and was washed with 20 ml hexane
followed by 20 ml 5 % diethyl ether in hexane. The TGs were then eluted with
20ml 15% ether in hexane.
For ozonolysis, 10 mg TGs were dissolved in 5 ml hexane in a centrifuge
tube. The tube was cooled in a mixture of dry ice/acetone, and ozone from a
microozonizer was allowed to pass through the solution at 200 ml/min for
15 min, until the iodine/starch indicator solution changed colour. The tube
was then flushed with nitrogen to purge unreacted ozone. The ozonide was
cleaved to the aldehyde by adding 2-3 mg Lindle catalyst and stirring the
solution under hydrogen for about 30 min.
 
Detection o f p ork in processed mea t 205
a l d e h y d e s o l u t io n a n d 1 0 m g p - n i t ro b e n z y l o x a m i n e h y d r o c h l o r i d e (P N B A )
and 100/zl pyr id ine were added . T he tub e w as capped and hea ted fo r 1 h a t
50°C. W hen the reac t ion was comple te , the pyr id ine was eva pora ted and the
sample d i s so lved in methy lene ch lor ide and washed twice wi th wa te r
(2 x 3 m l).
G L a n a ly sis o f f a t t y a c i d
A 1-2 g sample was ex t rac ted w i th 20m l ch lo roform /m ethan ol (2 :1 ) in an
ul t raso nic b ath for 20 min. The dissolved fa t was f i l tered and the f i l t ra te
evapo ra ted to d ryness . The ex t rac ted fa t w as d i s so lved in 1 m l hexane , and
250 # l 0 .1N m ethano l ic K O H was added to e ffec t t r anses te r i fi ca t ion . The v ia l
was sha ken fo r 10 m in and the l ayers were a l lowed to separa te . An a l iquo t
(1 # l) f rom the hexane l aye r was in jec ted in to a gas chrom atograph .
Histidine dipeptides
Pure pork , beef and sheep and d i ffe ren t mix tures o f po rk /be ef (P /B) and
pork / sheep (P /S) were made f rom ground musc le t i s sues . His t id ine
d ipep t ides were ex t rac ted f rom 3 g mea t samples w i th 30 ml 0 .9% sa l ine and
120 m l 8 5-sulfosalicyl ic ac id . The m ixture was hom oge nized us ing a t i ssue
hom ogenize r fo r 2 m in and the hom ogena tes were cen t ri fuged a t 9000g a t
r o o m t e m p e r a t u r e f o r I h . T h e s u p e r n a t a n t f r a c ti o n s c o n t a i n i n g t h e
d ipep t ides were f il te red th ro ug h a M i l l ipore p re - f il te r ( type AW ) and f il te r
( type GS , 0 22/zm diam eter pore) .
Enzyme-linked immunosorbent assays
Sam ples o f pure pork , beef, sheep and d i ffe ren t mix tures o f P /B and P /S were
p r e p a r e d i n t h e l a b o r a t o r y b y c o - h o m o g e n i z a t io n i n a n e q u a l v o l u m e o f 1 :1
sal ine (0 .85 g/100 ml H20 ) of the f ine ly mixed pu re m ea t samples which had
been au toc laved a t 120°C for 30 min , fo llowed by cen t r i fug ing a t 47 000g fo r
30 ra in a t 4°C. The sup erna tan t was f i lt e red th rough W ha tm an No. 42 f il te r
paper and s to red f rozen in 3 m l a liquo ts . Var ious ex t rac t ion con d i t ions o f
the commerc ia l ly -canned mea t samples , were t e s ted to ob ta in the h ighes t
recovery o f pork musc le an t igens . The fo l lowing procedure was found to
g ive op t im al resul ts , and was thu s used fo r ex t rac t ion . A 10 g sample o fm ea t
was h om ogenized in 90 ml h o t (90-95°C) sa l ine (3%) a nd t rans fe r red to a
hea t - res i s t an t g lass bo t t le , which w as sealed and then au toc laved fo r 30 m in
a t 121°C; a f t e r coo ling , the m ea t m ix ture in the bo t t l e w as rehomogenized ,
cen t r i fuged a t 10000g fo r 15min and the superna tan t f i l t e red th rough
Whatman f i l t e r No . 3 .
 
Analytical procedure
T G s a n a l y s i s
HPLC analysis of the TGs was carded out using a Shimadzu LC 4A HPLC
instrument equipped with gradient capability and a variable UV detector.
The S2U fraction was analyzed using a silica C is (octadecylsilica) reversed
phase column, 25 cm long and 5 mm in diameter, packed with 5/tin particles.
Data were acquired and processed using a Shimadzu CR-3A with floppy
disk drive and monitor. Derived TGs of different pork samples were
analyzed under isocratic conditions using acetonitrile/methylene chloride
(90:10) as the mobile phase. All other samples were run using a gradient
elution. The gradient program was as follows: 5% CH2C12 in acetonitrile for
the first 10 min after which the gradient was programmed to 27% CH2CI 2
for 5 min and this composition held for 25 min. The flow rate for the mobile
phase was 1.5 ml/min. The wavelength used for the UV detector was 254 nm.
F a t t y a c i d a n a ly s is
Fatty acids were analyzed using a gas chromatograph equipped with an inlet
splitter for the capillary column and a flame ionization detector as reported
by Saeed
(1986) with some modification. The FFAP capillary column
was replaced with an OV-225 (25 cmx 0.25 mm) column since the FFAP
stationary phase is generally less stable than OV-225 and bleeds quite
rapidly, resulting in loss of column efficiency unless compensated for by a
longer column (50 m). A hexane layer (1/A) was injected into the column with
a split ratio of 1:30. Nitrogen carrier gas flow through the column was
adjusted to 2ml/min. Column temperature, initially 140°C, was pro-
grammed to reach 210°C at 2°C/min. The final temperature was held for
20 rain. Injection port temperature was kept at 250°C and the detector at
260°C. Fatty acids were identified by comparing their retention times with
those of the standard. Quantification was based on area normalization.
H i s t i d i n e d i p e p t i d e a n a l y s i s
The dipeptides, in 5/zl of extract, were separated on a Whatman Partisil-10-
SCX column with a lithium formate buffer containing 0.2M lithium
hydroxide titrated to pH 2.9 with formic acid (Carnegie
e t a l .
1984). The
column was kept at 40°C at a flow rate of 0-7 ml/min from a Shimadzu LC-
6A pump. The eluate from the column was mixed with o-phthalaldehyde
(OPA) reagent (Nakamura e t a l . 1979) delivered at a rate of 1.2 ml/min with
another Shimadzu LC-6A pump. OPA reagent was prepared by dissolving
 
  etect ion o f por k in processed mea t
207
30% Br ij 35 ( t rade nam e fo r a comm erc ia l ly ava il ab le ser ies o fpo ly ox ye th y-
lene a lcohols ) , 2 -0ml mercap toe thanol and 10ml methanol con ta in ing
500 m g O PA. Deion ized d i s t il l ed w ate r was ad ded to m ake 1 l itr e. Af te r
t h o r o u g h l y p u r g i n g w i t h n i tr o g e n , t h e O P A r e a g e n t re m a i n e d s t ab l e f o r a t
l e as t 2 w e ek s a t r o o m t e m p e r a t u r e i n t h e d a r k . T h e O P A r e a g e n t w a s m i x e d
wi th the co lum n e lua te as a pos t -co lum n der iva t ion reag en t in a m ix ing coi l.
T h e d e r i v at iv e s t h u s f o r m e d w e r e d e te c te d b y a S h i m a d z u R F - 5 3 0 H P L C
f luorescence de tec to r . Th e exc i t a t ion wave leng th was 340 nm and em iss ion
w a v e l e n g t h w a s s e t a t 4 5 0 n m . T h e d e t e c t o r o u t p u t w a s r e c o r d e d a n d
p r o ce s se d u s i n g a S h i m a d z u i n t e g r a t o r ( M o d e l C R - 3 A ). Q u a n t i t a t io n w a s
d o n e u s i n g a n e x t e r n a l s t a n d a r d s o l u t i o n c o n t a i n i n g 3 1 2 - 5 p p m e a c h o f
ca rnos ine , anse r ine and ba len ine ( the ba len ine w as a g i f t f rom D r J . Wol ff ,
N a t i o n a l I n s t it u t e o f H e a l t h , B e t h e sd a , M D , U S A ).
Competitive E L I S A
Sheep a nt ip ig ant ise ra (300/~1) w as pip et ted in to 24 m l of ph osp ha te buffer
sa l ine p lus 0 .2% Tw een 20 (PBST) co n ta in ing 2 ml e ach of beef , sheep and
horse mea t ex t rac t s (hea ted a t 120°C for 30ra in and ex t rac ted wi th 5%
sal ine ). T he m ix ture w as sh ake n g en t ly fo r 1 h a t 20-25°C ( room
tempera tu re ) . O pt im al b lock ing cond i t ions were es tab l i shed by p re l iminary
checkerboard fo rm at m ic ro t i tr a t ion assays accord ing to Jones and Pa t te r son
(1986) . The e ffec ts o f he te ro logou s t rea tm ent and an t ib od y d i lu t ion were
eva lua ted by m ix ing a l iquo ts o f the sheep-an t ip ig an t ise ra , se r i al ly d i lu ted in
PBS T, wi th equa l vo lum es o f the th ree he te ro logous m ea t ex t rac t s (250 #g ,
310/~g and 1 m g/m l f rom each o f beef, sheep and horse) in the same d i luen t
and incub a t ing as above before app l ica t ion to the well s o f the E LIS A p la te
precoa ted wi th the va r ious mea t ex t rac t s .
A n t i g e n , a n t i b o d y a n d c o n j u g a t e w o r k i n g d i l u ti o n s f o r e a c h a n t i g e n w e r e
de te rm ined o n a checkerb oard t i t r a t ion . T i t ra t io n curves o f an t igen versus
a n t i b o d y c o n c e n t r a t i o n s w e re p e r f o r m e d t o d e t e r m i n e o p t i m a l c o n d i t i o n s
f o r t h e E L I S A m e t h o d , i n c l u d i n g p H , i n c u b a t i o n t im e f o r s u b s t r a te e n z y m e
reac tion , subs t ra te se lec t ion and conc en t ra t ion and IgG op t im al d ilu t ions .
T h e c o m p e t it iv e E L I S A m e t h o d w a s u s e d t o d e t ec t d if fe r en t P / B o r P / S
mix tures . P ig m usc le ex t rac t (180 /z l) , r aw an d /o r hea ted a t d i f fe ren t
temperatures and di lu ted 1:100 wi th PBS (phosphate buffer sa l ine , 0-15M,
pH 7 2 ), was ad ded to an EL ISA m icrop la te (96 h igh-b ind ing and f la t -
b o t t o m w e l l p l at es , N u n c , D e n m a r k ) a n d i n c u b a t e d a t 4 5 ° C f o r 4 0 m i n
(an t igen coa ting) . Th e p la te was then w ashed th ree to four t imes wi th PBS T
and dr ied by t app ing on c lean paper towels . Sheep-an t ip ig an t i se rum was
di luted w i th block ing buffer (1:100) and incu bate d for 1 h a t 4°C. Ser ia l
 
208 Wafih N. Sawaya et al.
species were ad de d in du plica te to eac h w ell (90/~1). Th en 90/zl o f sheep-
an t ip ig an t i se rum in b lock ing buffer , was added to each wel l. The p la te was
shaken for 20min (Dyna tech , Microp la te Shaker ) , washed th ree to four
t imes wi th PBST an d dr ied by t app ing o n c lean paper towels . Di lu ted go a t
immunoglobul ins (1 :1000 in PBST) , HRP-conjuga ted (horse rad i sh
perox idase-con juga ted , D ako pa t t , Den ma rk) , was added to a l l we ll s excep t
one b lan k wel l. Th e p la te was shaken fo r 20 min , washed th ree to fou r t imes
wi th d i s ti ll ed w ate r an d d r ied by t app ing on c lean pape r towels .
Az ino-b i s so lu t ion (ABTS) (az ino-b i s -d i , 3 -e thy l benzy l th iazo l ine
s u l p h ur i c ac id d i a m m o n i u m s o lu t io n ) w a s p r e p a r e d b y a d d i n g 2 m l A B T S
s t o c k s o l u t i o n ( 1 5 m g A B T S / m l ) t o 5 0 # 1 6 % H 2 0 2 i n 2 5 m l c i t r a t e
ph osp hate buffer 0 .15 M, pH 5 0 an d 180/~1 wa s ad ded to each wel l. Th e pla te
was shaken fo r 20 m in un t i l the co lour w as c lea r g reen . The a bsorban ce was
m e a s u r e d i n e a c h m i c r o w e l l b y a m i c r o E L I S A p l a t e r e a d e r ( M R - 7 0
Dy natech) a t 405 tam. The reac t ion was s topped by a dd ing 150/~ l 2 .5% N aF
to a l l we ll s. Low co lo ur in tens i ty ind ica ted pos i t ive response an d h igh co lo ur
density, negative response.
R E S U L T S A N D D I S C U S S I O N
Fo ur d if fe ren t m ethods fo r de tec t ing po rk in beef were eva lua ted fo r
a c c u r a c y a n d p r e c i s i o n : H P L C a n a l y s i s o f f a t T G s , H P L C a n a l y s i s o f
h i s t id ine d ipep t ides , f a t ty ac id (C20:2) ana lys i s and ELISA. Resu l t s o f
labora to ry p repared samples ( s t andards ) were compared wi th resu l t s f rom
c o m m e r c ia l ly m a n u f a c t u r e d s a m p l e s c o n t a i n i n g c a n n e d b e e f sa u sa g e , p o r k
luncheon mea t and d i f fe ren t mix tures o f P /B . These samples con ta ined
d i f fe ren t p ropor t ions o f P /B and were l abe l l ed as fo l lows : A = pure beef
(12 1% fat) , A1 =0 5% po rk (12-0% fat ), A 5 = 1% po rk (12.3% fat ),
A10 = 10% p ork (10 .7% fat ), A30 = 5% po rk (10 .7% fa t) and A20 po rk
luncheon meat (17.8% fat ) .
HPLC analys is of fat TGs (S2U fract ion)
H P L C a n a l y si s w a s p e r fo r m e d o n l a b o r a t o r y - p r e p a r e d s a m p l e s o f le a n
pork, beef and P/B m ixtures (0 , 1, 3 , 5 , 10, 30 an d 50% pork) an d
commercia l ly canned P/B samples (0 , 0-5 , 1 .0 , 5-0 , 10 and 100% pork) . A
t y p i ca l H P L C c h r o m a t o g r a m ( sa m p l e A 3 0) i s s h o w n i n F i g . 1. T h e S S U / S U S
ra t io was de te rmined fo r beef, sheep , po rk and P /B and P /S m ix tures (Saeed
e t a l .
1989). S ince pork con ta ins m os t ly the S SU i somer , bu t n o s ign i f ican t
 
I
Fig. 1. HP LC analysis of samp le A30 triglycerides.
d i ffe ren t ca rbon equ iva len t s (CE), where CE i s the to ta l ca rbo n num bers in
the T G mo lecule ( i .e . 39, 41, 43 and 45) , wi l l vary w i th th e d i f fer ing
p r o p o r ti o n s o f S S U o f th e se c a rb o n s i n p o r k T G s . T h e S S U / S U S r a ti o o f
C E 4 3 w o u ld c h a n g e m o r e s i g n i fi c a nt ly t h a n t h a t o f t h e C E 4 1 w h e n p o r k i s
a d d e d t o b ee f. T h e S S U / S U S r a t i o f o r C E 4 5 w o u l d n o t c h a n g e m u c h s in c e
the SS U o f th i s ca rbo n equ iva len t is p resen t in pork in smal l am ounts . Th e
ra t io o f to ta l CE 41 and CE 43 fo r SSU/SUS i somers would a l so re f l ec t
add i t ion o f pork .
In the ana lys i s o f f a t TGs in the commerc ia l ly p repared samples
(unknowns) , the S SU /SU S ra t ios fo r C E 41, CE 43 , and the to ta l o f
CE 41 + CE 43 (Tab le 1) m atched those o f P /B , and hence were iden t if i ed as
pork in beef. Al l r a t ios were inc reased by inc reas ing the am oun ts o f por k in
the mix tures . Th is was observed even wi th smal l add i t ion s o f po rk ind ica t ing
t h a t a s l o w a s 2 % o f p o r k w a s d e t e c t a b l e b y t h i s m e t h o d . W h e n t h e
SS U /SU S ra t ios fo r di ffe ren t CE s were p lo t t ed aga in s t pe rcen tages o f P /B
(Fig. 2) , and regress ion analys is wa s performe d, th e genera l form o f the
eq ua t ion was l inear of the f i rs t degree: y = a x b . T h e co rre lat i on coefficient
(R) wa s 0-99 for a l l CE s (me an of f ive repl icate analyses) indicat in g a hig h
 
210 W a j ih N S a w a y a et al.
TABLE 1
SSU/SUS Ratios of Unknown Samples
S a m p l e n o P o r k m e a t C E 4 1 C E 4 3 T o t a l ~ E s t i m a t e d
( ) p o r k f a t ( )
A 0 0.70 0 38 0-40 < 1
AI 0-5 0-71 0-42 0.46 - 1
A5 1 0-75 0.44 0.54 3--4
AI0 10 0.76 0 61 0.66 7-8
A20 100 ND ND ND 100
A30 5 0.75 0.46 0.56 4
Lean beef 0 0-68 0.38 0-43 --
Lean sheep 0 0.85 0.65 0-70 --
* Total = SSU (CE41 + CE43)/SUS (CE41 + CE43).
ND, not determined since SUS was too low.
the SSU/SU S ratios of the fat TGs in commercially canne d meat (Table 1)
were statistically com pare d with those of the la borat ory prepared samples
using the t-test, there was no significant difference at the P > 0.05 level,
indicating that the SSU/SUS ratio of all CEs o f the TGs can be used to detect
low levels of pork in canne d me at mixtures, with the highest value being that
of CE 43 followed by CE 43 + 41 and CE 41.
1.0~
0.9~
0.80
~ ' ~ ~ ~ ° ~ ° ~ ~ . . . ~ . . . . . ~ - ' ~
1 2 3 4 5 8 7 8 g
% pork iA b w f
Fig. 2. SSU/SUS ratios of pork/beef mixtures.
10
  etection of pork in processed meat 211
The HP LC ana lysi s o f th e T Gs can t hus be u t il iz ed fo r s amples con ta in ing
m eat and fat. Since fat is no t s ignif icant ly affected d uring processing, the
m etho d appl ies to f resh as well as p rocessed meats . Th e m ethod perm i ts the
de t ec ti on o f 2% po rk i n bee f and 3% pork i n m u t ton , and has been
invest igated for dif ferent var iables such as age, sex, muscle type an d diet . Th e
m ajor d i sadvantage is the lengthy and ted ious sample prepara t ion , a ba tch
of s ix samples requi ring a t l east 3 days . The m ethod i s no t appl icab le to fa ts
tha t h ave been chem ica l ly modi f ied , e .g . hyd rogen a ted fat s. T he reproduci-
b i li ty o f H PL C da ta cou ld be poo r i f con t am ina t ion o f the s ample du r ing the
lengthy prepara t ion i s no t care fu l ly avoided .
HPLC analysis of histidine dipeptides
HP LC ana lyses of h i s tid ine d ipept ides ex t rac ted f rom autoc laved mix tures
o f l abo ra to ry -p repa red l ean P /B (1 -50% pork ) and o f comm erc ia l ly -canned
m eat samples conta in in g lean bee f (35%), po rk (58%) an d P /B (35%)
m ixtures (0 , 0 .5 , 1, 5 , 10 an d 100% pork ) were perfo rm ed (Fig. 3), an d the
ra t ios ca lcu la ted of ca rnos ine -anser ine (C/A) an d ba len ine -anser ine (B/A)
Tables 2 an d ). S ince beef and po rk have s imi la r va lues for carnos ine an d
l
I / /
i
i
A20, histidine dipeptides.
Dipcpt ide Rat ios in Lab ora tory-P repared (Standard)
Pork /Bee f Mix tu re s
P o r k / b e e f ( ) C / A B / A
0 5 1 3 0 - 0 3 1
I 5 - 1 0 0 - 0 4 6
2 5 . 1 9 0 0 6 8
5 5 - 2 0 0 . 1 3 5
I 0 5 1 6 0 . 2 6 7
3 0 5 . 2 6 0 . 8 2 8
5 0 5 . 2 4 1 . 5 2 0
I 0 0 5 . 2 4 4 - 0 0 0
anser ine , the C /A ra t io was o f no va lue in de tec t ing the add i t ion o f pork to
beef . Both p ork and beef a re re la t ive ly h igh in ca rnos ine an d low in anse r ine ,
w hich is the m ajor d ipept ide in sheep. Balenine , however , i s uniq uely high in
pork. These differences are ref lected in the C /A an d B/A ra t ios for d i fferent
m e a t s . T h e B / A r a t i o f o r p o r k i s h i g h c o m p a r e d w i t h o t h e r c o m m o n l y
consumed mea ts and can be used as an ind ica to r fo r pork . The C/A ra t io ,
a l thou gh n o t use fu l fo r de tec t ing pork , cou ld ind ica te the p resence o f mea t s
for wh ich th is value is low, e .g . sheep, chicken, kang aro o and rab bi t
(Carneg ie e t a l . 1982) . How ever , there is a l ine ar re la t ionsh ip between B/A
ra t io and percen tage o f pork in the l abora to ry -prepared samples (Tab le 2),
and the add i t ion o f on ly 1% pork resu l ted in a s ign if i can t inc rease in B /A
rat io . Reg ress ion analys is indicated a h igh corre la t ion (R = > 0-99) between
B/A va lue and percen tage o f pork .
The B/A ra t ios o f the comm erc ia lly canned samples A, A1 , AS, A10 , A20
and A30 (Tab le 3 ), were s t a t i s t i ca lly com pared wi th those o f the l ab ora to ry -
pre pa red sam ples (Table 2) using the t-test . Th ere w as a significan t difference
between the two a t the 1% and 5% levels . This d i f ference could be due to
musc le source o r age o f the an im als (Carneg ie e t a l . 1982). O ther factors th at
m ay a ffec t the concen t ra t ion o fd ipe p t ides in mea t sam ples a re b reed , sex and
d iet . The resu l t s ob ta ined wi th s t anda rd samples p repared in the l abora to ry
ind ica te tha t h i s t id ine d ipep t ides cou ld be used to de tec t low level s o f po rk in
processed m ea ts. How ever , the re li ab i l ity and scope o f th i s metho d depends
on d e te rm in ing the ef fect o f the sources o f va r iab i l i ty on the concen t ra t ion o f
his t id ine dipept ides in beef , sheep and pork.
HPLC ana lys i s o f h i s t id ine d ipep t ides fo r pork de tec t ion i s a re la t ive ly
s imple p rocess. Sam ple p repara t ion i s s imple and rap id , t ak ing an average o f
90 min . H P L C ana lys i s is a l so fas t (10 min) . A de tec t ion l eve l o f 5% por k in
 
213
Sa m pl e Actual pork C/A B/A Estimated
( ) pork lean ( )
AI0 10 5-57 0.073 Approx. 3
A20 100 15 62 0-770 Approx. 30
A30 5 4.54 0.066 Approx. 2-5
ND, not determined since B was too low.
the de tec t ion l imi t m ay be fu r the r reduced . The m ethod , however , requ i res a
com pl ica ted se tup fo r H P L C ana lys i s (pos t -co lum n der iva tion) .
Fatty acid analysis
A t y p i c a l g a s c h r o m a t o g r a m o f t h e f a t t y a c id m e t h y l es te rs o f o n e o f t h e
com m erc ia lly canne d samples (A10) i s show n in F ig . 4 , and the fa t ty ac id
com pos i t ion o f the s ix samples p resen ted in Ta b le 4 . E icosad ieno ic ac id w as
detected in f ive of the samples , indicat ing the presence of pork. I t was
t o d e t e r m i n e lo w l e v e l s o f p o r k m i x e d w i t h o t h e r m e a t s s in c e t h e C 2 0 : 2
con ten t in pork m ea t i s va r iab le . How ever , s ince the m ethod was repor ted to
de tec t a s li t tl e a s 1% pork in beef and m ut to n mix tures, the p resence o f
C 2 0 : 2 i s a p o s i ti v e i n d i c a t o r o f > 1 % p o r k i n th e s a m p l e. U n k n o w n s a m p l e s
were ana lyzed fo r C20:2 , and the resu l t s com pared wi th those ob ta ined b y
Saeed
(1986).
C2 0:2 w as no t de tec ted in sample A (Table 4 ), ind ica t ing a l a rd con ten t o f
less than 1%, w hich is the de tec t ion l imi t fo r th i s m ethod . S amp le A1 showed
l i t t l e C 2 0 : 2 , i n d i c a t i n g t h a t t h e a m o u n t o f p o r k p r e s e n t w a s n e a r t h e
de tec t ion l imi t . The exac t amount o f pork cannot be re l i ab ly de te rmined
f r o m t h e v a lu e o f C 2 0 : 2 a l o n e s in c e C 2 0 : 2 c o n t e n t c a n v a r y c o n s id e r a b l y in
p u r e p o r k ( H u b b a r d & P o c k l i n g t o n , 1 96 8). T h e c o m p o s i t io n o f t h e r e st o f
the fa t ty ac ids was s imi la r to tha t o f sample A. Samples A5 and A10
c o n t a i n e d s l i g h tl y m o r e C 2 0 : 2 t h a n s a m p l e A a n d w e r e e s ti m a t e d t o c o n t a i n
2- 5% pork fa t. Sam ple A20 was s ign i fi can t ly d i ffe ren t f rom the o ther
samples . F i r s t , i t con ta ined more C20:2 than tha t r epor ted by Saeed e t a l .
(1986) fo r pure pork . Secondly , i t s overa l l f a t ty ac id com pos i t ion was s im i la r
to tha t o f pure pork fa t. Thus , sam ple A20 was es t imated to con ta in a t l eas t
 
g
w
o
i
14:0 \ / / C10:2
ime
(rain)
:20:1
0:2
8 3 2
F i g . 4 . G a s c h r o m a t o g r a m o f f a tt y a c i d ( m e t h y l e s te r s) o f s a m p l e A I 0 .
that it contained higher amounts of C20:2. The pork fat content of this
sample was estimated at about 10%.
In general, the gas chromatographic method using eicosadienoic acid is
rapid. Sample preparation after modification s simple and can be completed
within 1.5 h since chromatographic analysis takes about 40 rain and the total
time required for fatty acid analysis is about 2 h. The method has the
advantages of being straightforward, since it uses the presence or absence of
C20: 2, and sensitive, detecting as little as 1% pork content. Since the method
is based on fat analysis, it is applicable to fresh and processed meat and other
fat-containing samples.
The method was found to detect as little as 1% pork in model beef and
mutton mixtures; the presence of C20:2 could be considered as a positive
indicator of greater than 1% pork fat in the sample. However, the method
cannot accurately determine pork concentration since the eicosadienoicacid
 
T A B L E 4
Fatty Acid Composition of Com mercial Meat Samples
Fatty acid A AI A5 AIO A20 A30
C I4 :0 3.45 2-17 3 20 2.98 1.19 2.89
C14:1 0-67 0-30 0-76 0-63 ND 0-64
C1 5:0 0-71 0-58 0 70 0.56 ND 0.56
C 16 :0 2 4 .8 0 2 4 . 6 7 2 6 - 1 0 2 5 . 2 0 1 9 8 0 2 5-2 9
C16:1 2.85 2.53 3 29 3.70 1.90 3 72
C17 :0 1-94 1.35 1.18 1 20 0-45 0-95
C18:0 + C18 :1 51 .30 57 .3 2 51 .3 0 54 .5 5 50 -5 0 54-00
C1 8:2 5.54 2.88 3 99 5 .6 0 17 .37 4.65
C19 :0 0-23 1.10 1-17 0.67 0-24 0-57
C20:0 ND 0.60 0-28 0.20 0.15 0.20
C20:1 0.47 0-78 0.40 0-72 1-28 0-69
C2 0:2 ND 0-06 0-15 0.16 0-97 0-22
Estimated pork fat < 1% 1% 2-4% 2-5% 100 % 10%
ND , not detected, i.e. less than 0.1%.
M o r e o v e r , F i r e s t o n e (1 98 8) re c e n t ly r e p o r t e d t h e p r e s e n c e o f C 2 0 : 2 i n t h e
t is s u es o f a n i m a l s o t h e r t h a n p ig , c a s t i n g d o u b t o n i ts r e li a b il it y a s t h e s o l e
i n d i c a t o r o f l o w l ev e ls o f p o r k i n p r o c e s s e d m e a t .
Enzyme-linke d immunosorbent assays EL IS A )
L o w l e v el s o f P / B ( 2, 3 a n d 4 % ) , t h a t w e r e n o t a v a il a b l e c o m m e r c i a l l y w e r e
p r e p a r e d b y m i x i n g c o m m e r c ia l ly c a n n e d p u r e p o r k l u n c h e o n m e a t a n d b e e f
s a u sa g e , e it h e r b y m i x i n g a p p r o p r i a t e v o l u m e s o f p o r k a n d b e e f e x t ra c t s
( v / v ) , o r b y m i x i n g t h e t w o p u r e m e a t s i n d e f i n e d p r o p o r t i o n s b y w e i g h t
( w /w ) a n d t h e n e x t r a c ti n g t h e m i x t u re . W h e n c o m p e t i ti v e E L I S A t es ts w e r e
p e r f o r m e d o n d i f f e re n t v / v s a m p l e s , th e r e w a s a d i f f e re n c e o f 0 .5 2 2 O D
( D O D ) b etw e en p u re be e f ( O D = 0 . 8 2 4 _ 0 . 0 9 7 ) a n d 2 % P /B ( O D =
0 3 0 9 _ 0 0 34 8). H o w e v e r , f o r 1 % P / B , t h e D O D w a s 0 .4 1 w h i c h is l a r g e
e n o u g h t o d e t e c t t h e p r e s e n c e o f p o r k . S i m i l a r re s u l ts w e r e o b t a i n e d w i t h
d i ff e r en t p e r c e n t a g e s o f P /B p r e p a r e d o n a w t / w t b a si s, w h e r e a 0 .5 4 O D
d if fe r en c e w a s o b t a i n e d b e t w e e n p u r e b e e f a n d 2 % P /B . T h e O D d if fe r en c e
w a s o n l y 0 . 25 a t t h e 1 % l ev e l, i n d i c a t i n g t h a t i t is m o r e d i f f i c u lt t o d e t e c t l e ss
t h a n 2 % p o r k o n a w / w b a si s. T h e d i f f er e n t d e t e c t i o n l i m i ts o f s a m p l e s
p r e p a r e d b y v / v a n d w / w m e t h o d s , c o u l d b e d u e t o t h e h i g h f a t c o n t e n t o f th e
m e a t m i x t u r e (5 0 % ) , w h i c h i s u s u a ll y r e m o v e d b y c e n t r i f u g i n g b e f o r e t h e v / v
 
al.
To check i f EL ISA resu l ts fo r com merc ia l ly canned samples agreed wi th
those ob ta ined f rom labora to ry-prep ared P /B s tandards , s t a t is t i ca l ana lys i s
us ing the t - t e s t was per fo rmed on the DOD va lues o f bo th . The resu l t s
show ed n o s ignif icant d if ference a t th e 1 and 5% levels between the
e x p e r i m e n t a l ( u n k n o w n s a m p l e s ) a n d t h e s t a n d a r d D O D v a l u e s a t a l l
pe rcen tages o f P /B tes ted (1 -10%) , ind ica t ing tha t the sheep-an t ip ig
an t i se rum produced aga ins t au toca lved whole -p ig musc le ex t rac t s can be
u s ed t o d e t e c t l ow p e r c e n t ag e s o f P / B i n u n k n o w n c a n n e d / p r oc e s s ed m e a t
m ix tures (Fig. 5 ). I t was a l so demo ns t ra ted tha t the same an t i se rum can be
used to de tec t low percen tages (2%) of pork in l abora to ry-p repared m ea t
mix tures tha t hav e been hea ted a t 70 and 100°C for 30m in (Sawaya e t a l .
1990).
These resu l t s conf i rm the p resence o f hea t - s tab le an t igens in an imal
t is sues , p a r t i cu la r ly m usc le (Hay den , 1981; K ang 'e the e t a l . ]985, 1986;
K ang 'e the & G athu m a, 1987). Th e successfu l use o f unpur i fi ed ex t rac t s o f
m ea t o r m ea t m ix tures as an t igens fo r the p rod uc t ion o f an t i se ra is r epor ted
here in fo r the f i r s t t ime , bu t was p rev ious ly sugges ted by Pa t t e r son and
Spencer (1985) . The p roposed method e l imina tes complex pur i f i ca t ion
procedures and has the abi l i ty to discr iminate between close species .
M oreover , the use of a com plem enta ry species for the required ant isera , e .g .
an t ip ig an t isera b eing ra ised in sheep, i s more effective tha n us ing rabbi ts , as
prev ious ly repor ted by P a t t e r son and Spencer (1985), and by Jones in 1986
0 , T ~ -
" " - C o m m e r c i a l
i i I J i
2 3 4 $ 6 7 8 9 1 0
P / 6 ( ~ )
 
217
(p r iva te comm unica t ion). In fac t, r abb i t an t i -p ig an t i se rum produced to the
sam e ant ige n used in sheep (muscle ex tract autoc laved a t 120°C for 30 min)
showed h igh c ross - reac t iv ity w i th the he te ro logous species and cou ld no t be
used to de tec t low percen tages o f P /B or P /S even a f te r t r ea tment wi th a
b lock ing buf fe r . The advan tages o f ELISA are the s imple p repara t ion
procedure , the shor t t ime requ i red to ob ta in resu l t s and the low cos t o f
a n a ly s is . T h e d i s a d v a n t a g e o f t h e i m m u n o a s s a y m e t h o d i s t h a t i t c a n o n l y
d e t e ct p o r k p r o t e i n s a n d n o t p o r k f a t. M o r e o v e r, i m m u n o c h e m i c a l m e t h o d s
are sub jec t to b io log ica l va r iab i l i ty requ i r ing f requen t s t andard iza t ion .
In summary , the resu l t s o f t es t ing a l imi ted number o f commerc ia l ly -
prepared canned mea t samples ind ica te tha t pork can be de tec ted in
processed m ea ts by fa t a s we l l a s p ro te in -based de tec t ion methods . Th e fa t
m ethod i s m ore un iversa l , i .e . i t can be used fo r the de tec t ion o f bo th m ea t o r
f at . H P L C a n a l y s i s o f T G s i s m o r e r e li a bl e t h a n G C f a t t y ac i d a n a l y si s u s i n g
C20 :2 as a marke r , pa r t i cu la r ly since C2 0:2 was recen t ly repor ted in an imal
t is sue o ther th an p ig (F i res tone , 1988) ; a l so the va r ia t ion in C20:2 con te n t
betwee n di f ferent mu scles is large.
I f the sample con ta ins mea t , then e i the r p ro te in -based method i s
p re fe rab le to e i the r fa t-based method . How ever , the imm uno assay meth od i s
eas ie r and c heaper to use than the de te rm ina t ion o f oph id ine d ipep tides , and
requ i res l ess sample p r epara t ion and overa l l time . The oph id ine d ipep t ide
m ethod , a l t ho ug h fa i r ly re liable , has ye t to be tes ted for d if ferent va r iables
such as breed, m uscle type, sex and die t .
I n c o n c lu s io n , w e r e c o m m e n d t h e i m m u n o a s s a y m e t h o d f o r th e d e t e c ti o n
of pork in p rocessed mea ts and the oph id ine d ipep t ide method as a
complementa ry a l t e rna t ive . I f the sample con ta ins on ly fa t , then we
r e c o m m e n d t h e C 2 0 : 2 m e t h o d f o r p r e l i m i n a r y s c r e e n i n g a n d t h e H P L C
a n a l y s is o f t h e T G s f o r c o n f i rm a t i o n .
A C K N O W L E D G E M E N T S
T h i s p r o j e c t w a s p a r t i a l l y f u n d e d b y t h e K u w a i t F o u n d a t i o n f o r t h e
Advancement o f Sc iences (KFAS) , whose f inanc ia l and admin is t ra t ive
sup por t i s g ra te fu l ly acknowledged .
The au thors thank Mrs S . Jones (AFRC Ins t i tu te , Br i s to l , England) fo r
her adv ice and gu idance , pa r t i cu la r ly he r recommenda t ion on an t igen
e x t ra c t io n : M r B . Y a s i n ( U n i v e r s it y o f K u w a i t , A n i m a l R e s o u r c es C e n t e r)
f o r m a i n t a i n i n g t h e r a b b i ts ; a n d D r M . A . R a z z a q u e ( K I S R ) f o r d o n a t i n g t h e
s h e e p fo r p r o d u c t i o n o f a n ti b o d i e s; a n d D r M . K h a n ( K I S R ) f o r s t a ti s ti c a l
 
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