98.
PHOSPHOLIPIDS I N REAT H E T H O D S O F i S O 1 A T l O N A N D I N F L U E N C E OM F E A T C H A R A C T E R I S T I C S
L. R.
DUGAN
Introduction
The growing r e a l i z a t i o n of t h e influence of minor components
on system p r o p e r t i e s and t h e advances i n s o p h i s t i c a t i o n and s e n s i t i v i t y
of measurement of molecular moieties has drawn a t t e n t i o n t o a r e a s which
long have been neglected i n food systems. One a r e a of t h i s nature i s
t h a t of t h e phospholipids i n food systems and e s p e c i a l l y i n foods of
animal o r i g i n . The presence of phospholipids has been recognized f o r
many years and more r e c e n t l y t h e i r r o l e as s t r u c t u r a l and f u n c t i o n a l
components of c e l l s and membranes has been a s u b j e c t of d e t a i l e d i n v e s t i gation. Phospholipids have been used a s e m u l s i f i e r s , a s c r y s t a l modifiers,
and a s a n t i o x i d a n t s i n various food a p p l i c a t i o n s . Phospholipids i n muscles
have a number of p h y s i o l o g i c a l functions i n t h e l i v e animal but t h e i r
importance i n t h e q u a l i t y of f r e s h and processed meats i s s t i l l r e l a t i v e l y
ob s cure.
I s o l a t i o n of Fhospholipids
-
E x t r a c t i o n A major f a c t o r i n t h e i s o l a t i o n of phospholipids r e s i d e s i n
t h e e x t r a c t i o n procedures. The phospholipids a r e bound t o p r o t e i n s i n a
manner which d e f i e s e x t r a c t i o n by conventionally used f a t s o l v e n t s which
e x t r a c t e s s e n t i a l l y o n l y t h e n e u t r a l l i q u i d s . Most e x t r a c t i o n methods f o r
t o t a l l i p i d s follow t h e p r a c t i c e of Folch, e t a l . ( 6 ) or some v a r i a t i o n of
t h i s procedure. This involves hoxogenization of t h e t i s s u e w i t h 2:l-CHClz:
MeOH
and washing t h e e x t r a c t by a d d i t i o n t o it of 0.2 i t s volume of
water o r appropriate s a l t s o l u t i o n . The r e s u l t i n g mixture s e p a r a t e s i n t o
two phases upon standing, The lower phase i s t h e t o t a l l i p i d e x t r a c t .
I n t h e washing procedure, t h e system of t i s s u e e x t r a c t p l u s water contains
CHC13, MeOH, and water i n t h e proportions 8:4:3 (V:V) a s c r i t i c a l proport i o n s which must be kept constant. Variations on t h i s procedure have been
employed f o r f i s h Bligh and Dyer (2) and muscle t i s s u e s Ostrander and
Dugan (13). The a l c o h o l e f f e c t i v e l y r e l e a s e s t h e p r o t e i n and t h e chloroform t h e n provides t h e solvent f o r e f f e c t i v e removal of t h e l i p i d from
t h e system.
--
Separation - Most methods of s e p a r a t i o n of phospholipids, e i t h e r a s a c l a s s
or a s i n d i v i d u a l phospholipid components, depend on some means of s e l e c t i v e
adsorption. Choudhury and Arnold ( 4 ) were able t o determine t h e n e u t r a l o i l
content of vegetable o i l s a s follows: shake f o r 1 0 min. i n a 125 m l e r l e n meyer f l a s k 2-3 g crude o i l , 25 g s i l i c i c a c i d and 50 m l CHC13. A f t e r
f i l t e r i n g through a s i n t e r e d g l a s s funnel under vacuum, t h e s i l i c i c a c i d i s
washed with f i v e 50-ml lots CHC13. The combined f i l t r a t e and washings a r e
f r e e d from CHCIS on a water b a t h and t h e residue heated t o constant weight
a t 105OC i n a vacuum oven or under PITz.
The phospholipids a r e determined
PY washing t h e s i l i c i c a c i d w i t h MeOH, evaporating t h e MeOH and weighing
t h e residue. They found phospholipid content t o be 6.25$ i n soybean o i l ,
5.83% i n corn o i l and 3.25% i n cottonseed o i l .
99.
A number of i n v e s t i g a t o r s have applied t h i s procedure t o
determination of phospholipids o r t o concentrating them p r i o r t o separat i o n i n t o i n d i v i d u a l components. Kuchmak and Dugan (11)reported t h a t
phospholipids from pork muscle were w e l l separated i n t h i s manner b u t
t h a t some r e s i d u a l n e u t r a l f a t s remained w i t h t h e phospholipids.
Hornstein, e t a l . ( 9 ) made t h e same observation. They modified t h e
p o l a r i t y of t h e solvent and with a mixture of chloroform-hexane-diethyl
e t h e r ( 2 : l : l ) were a b l e t o e f f e c t a q u a n t i t a t i v e s e p a r a t i o n of
phospholipids.
--
Separation i n t o i n d i v i d u a l phospholipids has been accomplished
by s i l i c i c a c i d column chromatography and by t h i n - l a y e r chromatography.
Column chromatography w i t h s i l i c i c acid seldom gives clean-cut s e p a r a t i o n
of components. The cephalins, phosphatidyl ethanolamine and phosphatidyl
s e r i n e , f r e q u e n t l y e l u t e t o g e t h e r and l e c i t h i n overlaps w i t h l y s o l e c i t h i n
and o c c a s i o n a l l y with sphingomyelin. Kuchmak and Dugan ( 1 2 ) used s i l i c i c
a c i d and separated t h e cephalins on a s i l i c i c a c i d - s i l i c a t e column
et a l . (15).
described by Rouser, Rouser, et a l . ( 1 6 ) e s t a b l i s h e d a l i p i d s e p a r a t i o n scheme based
on DEAE Cellulose which permitted e l u t i o n of l e c i t h i n , sphingomyelin,
ceramide, cerebroside, l y s o l e c i t h i n , s t e r o l e s t e r s and g l y c e r i d e s a s a
group. Phosphatidyl ethanolamine e l u t e d a s a s e p a r a t e f r a c t i o n and
phosphatidyl s e r i n e e l u t e d w i t h t h e gangliosides a s shown i n Table 1.
Table 1.
E l u t i o n of Lipids from DEAE Cellulose (Acetate Form)”
Substance
Solvent
1. Lecithin, sphingomyelin, ceramide,
cerebroside, c h o l e s t e r o l , l y s o l e c i t h i n ,
s t e r o l e s t e r s , and g l y c e r i d e s
2.
Phosphatidyl ethanolamine
CHC13/MeOH
7/1
CHCl,/MeOH
7/3
3, Water s o l u b l e n o n l i p i d s ( sugars,
amino a c i d s , purines, s a l t )
MeOH
4.
Uncharacterized components and f r e e
f a t t y acids
3/1
CHC13/glacial CH3COOH
containing 0.001 M CH3COOK
5.
Phosphatidyl s e r i n e and ganglioside
G l a c i a l CH3COOH
6.
Cerebroside s u l f a t e , i n o s i t o l
phosphatide and c a r d i o l i p i n
CHCL/MeOH
4/1
containing 10& / l i t e r
concentrated aqueous
NH40H
*From Rouser e t a l . ( 1 6 )
7
-
100.
They t h e n separated t h e components of t h e f i r s t f r a c t i o n from
t h e DEAE Cellulose column on a S i l i c i c Acid-Silicate-Water column as
shown i n Table 2 ,
Table 2.
E l u t i o n from a S i l i c i c Acid-Silicate-Water Column*
Solvent
Sub stance
CHC13
Cholesterol
CHC13/MeOH, 19/1
Ceramide
CHC13/MeOH,
4/1 + 0.5% H20
Cerebrosides
CHC13/MeOH,
4/1 + 1.5% H20
Lecithin
CHCl3/Me0H,
4/1 + 2.0% H20
Sphingomyelin
MeOH + 2% H20
Lysolecithin
*From Rouser, et a l . (16)
Hornstein e t a l . (10) separated beef and pork l i p i d s i n t o f o u r
f r a c t i o n s by s i l i c i c a c i d chromatography using successive 300-ml p o r t i o n s
of CHC13/MeOH
20/1, CHC13/MeOH
1/1, and MeOH. They reported t h a t t h e
f i r s t two f r a c t i o n s consisted mainly of n e u t r a l l i p i d s , t h e t h i r d f r a c t i o n contained t h e cephalins, and t h e f o u r t h contained l e c i t h i n and
sphingomyelin. The range of components i n beef and pork l i p i d s a r e given
i n Table 3.
-
-
Table 3,
Lipids of Beef and Pork Phscle (%)*
Lipid
Non-phosphorus
Beef
Pork
-
2-4
5-7
Phospholipid
0.8-1.0
0.7-0.9
Cephalin
40-45
40-45
Lec it h i n
40-45
40-45
Sphingomyelin
10-15
10-15
5-10
5-10
Protein
--
*From Hornstein e t , a l . (10)
101.
--
--
Hidaka e t a l . ( 8 ) used t h e procedure of Hornstein e t a l .
t o s e p a r a t e t h e l i p i d s of s e v e r a l muscles of Japanese Black C a t t l e .
of t h e i r r e s u l t s a r e shown i n Table 4.
(10)
Some
Table 4.
Percentage of Lipid Fractions i n T o t a l Lipids from
Muscles of Japanese Black C a t t l e *
Total
Muscle
Lipid
Fraction 1
(Triglycerides )
Fraction 2
( Cephalins )
Fraction 3
(Lecithins
and Sphingomyelins )
Denatured
Proteins
Fore sha'nk
100
84.85
4.65
6.87
1.79
Hind shank
100
81.92
4.90
8.01
1.81
Tender Loin
100
8%.84
3.80
4.98
1.32
Loin
100
90.55
2.87
4.60
1.11
*From Hidaka, e t a l . ( 8 )
Kuchmak and Dugan (11)used t h e scheme shown i n Table 5 for
s e p a r a t i n g t h e l i p i d s of pork muscle i n t o 6 f r a c t i o n s
Table 5.
Scheme f o r Chromatographic Separation on S i l i c i c Acid*
Eluant
Fraction
I
CHC13
Volume
2
-Components
Nonphospholipids
CH3COC%
113
I11
10%MeOH i n CHC13
15%MeOH i n CHC13
1
X m l
Cerebrosides ( ? )
IV
20% MeOH i n CHC13
25% MeOH i n CHCl3
1
Xml
Cephalins
V
35% MeOH i n CHC13
4
Lecithins
Me OH
2
Sphingomyelins
I1
VI
*From Kuchmak and Dugan (11)
P r i n c i p a l l y nonphospholipids
102
.
The phospholipids found i n various muscles of t h e hog c a r c a s s a r e
shown i n Table 6.
Table 6.
Percentage of Each Phospholipid Type i n t h e
Phospholipids of Hog Muscle Tissue*
phospholipid
Belly
Carcass Location
Loin
Ha
~
Phosphatidyl ethanolamine
Phosphatidyl s e r i n e
Lecithin
Sphingomyelin
-.
Rib
32.8
34.2
33.3
28.4
4.7
7.8
4.7
2.5
58.6
54.7
60.8
63.0
3.9
3.3
1.2
6.1
*From Kuchmak and Dugan (11)
L e c i t h i n and phosphatidyl ethanolamine a r e t h e dominant
phospholipids and a l l phospholipids vary from muscle t o muscle i n t h e carcass.
A technique applied r e c e n t l y i n our l a b o r a t o r y uses a multibore
column packed w i t h s i l i c i c a c i d , This has given c l e a n d i s c r e t e s e p a r a t i o n
of i n d i v i d u a l phospholipids. The apparent overlap of l e c i t h i n , obtained
by us and o t h e r s , w i t h a long t a i l i n g of minute q u a n t i t i e s of l y s o l e c i t h i n
may be explained by t h e observation of Camejo ( 3 ) t h a t l e c i t h i n i n contact
w i t h methanol on c e r t a i n s i l i c i c a c i d p r e p a r a t i o n s may undergo l i m i t e d
hydrolysis and y i e l d l y s o l e c i t h i n p l u s some f r e e f a t t y a c i d and methyl e s t e r s .
Phospholipid E f f e c t on Meat C h a r a c t e r i s t i c s
Composition e f f e c t s - The amount of phospholipid i n t o t a l l i p i d of meats
i s small and t h e e f f e c t on meat c h a r a c t e r i s t i c s must be derived from t h e
strong c o n t r i b u t i o n of unique c h a r a c t e r i s t i c s of t h e phospholipid moieties..
The f a c t o r s which a r e unique a r e t h e presence of e s t e r i f i e d phosphoric
a c i d , a nitrogenous moiety i n most phospholipids, t h e r e l a t i v e l y high uns a t u r a t i o n of t h e f a t t y a c i d s , and t h e close a s s o c i a t i o n with p r o t e i n s .
The a s s o c i a t i o n w i t h p r o t e i n determines i n l a r g e measure t h e
d i f f i c u l t y encountered i n e x t r a c t i n g phospholipids. This binding prevents
d i r e c t e x t r a c t i o n w i t h chloroform and even extends t o f r e e z e - d r i e d meats.
G i a m and Dugan ( 7 ) found a marked d i f f e r e n c e i n t h e f a t t y acid composition.
of ' f r e e ' l i p i d s , t h o s e which may be e x t r a c t e d by o r d i n a r y f a t solvents,
and 'bound' l i p i d s , those which r e q u i r e treatment w i t h methanol p r i o r t o
or concurrent w i t h t h e use of chloroform a s e x t r a c t i n g s o l v e n t . Repres e n t a t i v e d a t a f o r pork, beef, and lamb a r e shown i n Table 7.
103.
Table 7.
F a t t y Acid Composition of "Free" and "€bund" Lipids of
Freeze-dried Raw Pork, Lamb, and Beef
of t o t a l f a t t y a c i d s ) . *
(4
Raw Pork
Fatty
acid
Raw Lamb
Raw Beef
Free
-
Bound
Free
-
Bound
Free
-
8
Tr
0.8
Tr
Tr
Tr
Tr
10
Tr
Tr
Tr
Tr
Tr
Tr
12
Tr
Tr
Tr
Tr
Tr
Tr
12:l
Tr
1.0
Tr
Tr
Tr
0.6
14
2.3
1.2
4.7
0.9
4.8
1.0
14:l
Tr
0.7
1.0
0.6
2.0
0.8
18.7
16.7
22.8
14.0
20.2
14.5
8.5
2.1
5 .O
2.9
9.4
4.2
18
13.2
13.1
16.9
10.6
19.3
10.3
18:l
35.1
15.9
38.7
23.3
34.0
23.3
18:2
12.1
29.5
5.1
24.6
4.4
21.1
18:3
2.6
1.1
1.9
2.7
1.9
3.3
21.1
0.9
0.9
Tr
1.0
Tr
0.6
22
Tr
1.8
Tr
0.8
Tr
2.2
20:4
1.1
11.5
Tr
8.8
Tr
9.0
X
2 .o
0.9
1.0
3.6
1.0
4.0
24:l
Tr
1.3
Tr
1.1
Tr
0.6
Y
0.7
0.5
0.5
2.8
0.8
3 .O
Z
1.2
Tr
0.8
1.8
1.0
1.1
16
16 :1
*From Giam and Dugan ( 7 )
Bound
104.
The d i f f e r e n c e s noted a r e p a r t i c u l a r l y marked f o r l i n o l e i c and
arachidonic a c i d s , both of which a r e found i n e l e v a t e d concentrations i n
phospholipids.
Kuchmak and Dugan ( 1 2 ) examined t h e f a t t y a c i d composition and
t h e d i s t r i b u t i o n of t h e f a t t y a c i d s i n t h e phospholipids from various
pork muscles. Marked d i f f e r e n c e s i n composition were noted f o r t h e
various carcass l o c a t i o n s . The f a t t y a c i d s of t h e phosphatidylethanolamine
f r a c t i o n a r e shown i n Table 8.
Table 8.
F a t t y Acid Composition of Phosphatidylethanolamine
from Porcine Muscles (Mole $)*
Fatty
Acid
1o:o
12:o
13:O
a
14:O
b
15:O
16:O
16:l
17:O
18:O
18:l
18:2
18:3
2O:l
21:o
20:4
22:o
22 :1
Yuscle Source
-
Belly
Ham
Loin
Ribs
2.0
1.7
0.8
0.3
0.6
0.8
2.3
3.1
1.1
0.8
17.9
8.7
36.6
1.4
1.4
3.3
17.4
2.9
2.7
5.0
2.4
0.9
0.4
0.7
0.5
0.8
7.9
3 .O
3.1
1.5
0.7
1.1
0.9
2.0
6.6
1.9
1.7
32.8
14.5
20.3
...
...
...
...
1.6
1.3
0.9
5.7
1.5
1.8
30.6
12.7
28.2
1.1
1.2
28.9
14.2
24.7
1.7
1.0
5.3
1.0
1.0
3.1
0.5
3.0
2.2
2.3
...
...
...
3.4
e..
2.3
2.1
2.3
+From Kuchmak and Dugan ( 1 2 )
E s p e c i a l l y marked variakions were noted i n t h e amounts of s t e a r i c ,
l i n o l e i c , and arachidonic a c i d s . The arachidonic a c i d content of t h e
b e l l y muscle was more t h a n t h r e e times g r e a t e r t h a n t h a t of t h e ham muscle.
Allen, e t a l . (1)determined t h e comparative l i p i d composition of
t h r e e porcine muscles: t h e diaphragm, L. d o r s i , and Psoas major. I n
e v a l u a t i n g t h e d i f f e r e n c e s between composition of n e u t r a l l i p i d s and of
t o t a l l i p i d e x t r a c t s , t h e y r e l a t e d d i f f e r e n c e s i n s t e a r i c and o l e i c a c i d s
t o an i n f e r r e d cephalin o r l e c i t h i n content. They noted f u r t h e r an
apparent g r e a t e r u n s a t u r a t i o n i n non-neutral l i p i d s from psoas major and
I
-
105.
suggested t h a t l i p i d s f r o m t h i s muscle might become rancid more quickly
t h a n those of t h e o t h e r two muscles.
Peng and Dugan (14)showed t h a t t h e dark meat of chicken cont a i n e d p r o p o r t i o n a l l y more l i n o l e i c a c i d and arachidonic a c i d i n given
phospholipids t h a n were found i n t h e same phospholipids of white meat.
-
Oxidative and o t h e r q u a l i t y e f f e c t s
Some of t h e q u a l i t y f a c t o r s
r e l a t e d t o phospholipids a r e only i n f e r r e d while o t h e r s may be d i r e c t l y
a t t r i b u t e d t o t h e phospholipids.
a l . ( 1 0 ) observed s t r o n g f i s h y odors from
Hornstein, . et heating of cephalins from pork o r beef and a diminished f i s h y odor
a s s o c i a t e d w i t h an aroma suggestive of l i v e r when t h e l e c i t h i n s of pork
o r beef were heated. Both t o t a l l i p i d and phospholipid f r a c t i o n s from
pork o r beef became r a n c i d quickly when exposed t o a i r . They concluded
t h a t phospholipids p o s s i b l y contributed t o poor f l a v o r s i n excessively
l e a n meat. Marked darkening of t h e phospholipids accompanied t h e development of r a n c i d i t y .
Younathan and Watts ( 1 7 ) compared t h e development of r a n c i d i t y
They noted t h a t TM
values of t h e t i s s u e s were many f a c t o r s g r e a t e r t h a n i n e x t r a c t e d f a t .
When t h e l i p i d s were examined, it was found t h a t t h e TBA of n e u t r a l l i p i d s
was much l e s s t h a n t h a t of a t o t a l l i p i d e x t r a c t or of t h e f r a c t i o n which
t h e y r e f e r r e d t o a s phospho- o r p r o t e o l i p i d s as i n Table 9.
i n e x t r a c t e d f a t w i t h t h a t i n whole t i s s u e from pork.
Table 9.
Rancidity i n Pork Lipid Fractions*
W
t Lipid/g Tissue
Lipid F r a c t i o n
Rancid
Sample
Antioxidant
Treated Sample
TBA Values/g Tissue
Rancid
Sample
Antioxidant
Treated Sample
g
g
T o t a l Lipids
.272
.289
0.65
0.13
Neutral f a t
,246
.263
0.20
0.14
Phospho- or
Proteolipids
,026
.026
0.59
0.06
*From Younathan and Watts ( 1 7 )
Similar observations were made by Zipser, e t a l .
i n v e s t i g a t i o n of l i p i d s from oxidizing mullet.
(18) an
E l Gharbawi and Dugan ( 5 ) studied t h e s t a b i l i t y of nitrogenous
compounds and l i p i d s during s t o r a g e of f r e e z e - d r i e d beef i n cans w i t h
varying oxygen-nitrogen r a t i o s . The f a t t y acid composition of t h e n e u t r a l
l i p i d and t h e phospholipid f r a c t i o n s changed w i t h o x i d a t i o n during s t o r a g e
106.
according t o t h e oxygen content of t h e can atmosphere. The o x i d a t i o n of
t h e t i s s u e l i p i d s appeared t o occur i n two s t a g e s w i t h t h e phospholipids
oxidizing f i r s t . Loss of t h e unsaturated f a t t y a c i d s i n t h e phospholipids
was more pronounced t h a n i n t h e n e u t r a l f a t . Associated w i t h t h e l i p i d
changes were decreased s o l u b l e p r o t e i n and non-protein nitrogen, decrease
of f r e e amino groups and l o s s of rehydration capacity.
The r e l a t i o n of composition of phospholipids t o changes i n t h e
meat from which t h e s e a r e derived may be d i r e c t e d t o t h e observation of
Kuchmak and Dugan ( 1 2 ) t h a t t h e l i n o l e i c a c i d content of l e c i t h i n from
pork b e l l y muscle was markedly g r e a t e r t h a n t h e l i n o l e i c content of
l e c i t h i n i n t h e o t h e r muscles studied. The l i n o l e i c content of phosphat i d y l ethanolamine from b e l l y muscle i s a l s o g r e a t e r t h a n t h a t i n t h e
o t h e r muscles s t u d i e d b u t t h e most dramatic d i f f e r e n c e l i e s i n t h e
arachidonic a c i d content of t h i s phospholipid. With t h e higher l e v e l of
polyunsaturated f a t t y a c i d s i n t h e phospholipids of b e l l y muscle, it
seems reasonable t o s p e c u l a t e t h a t t h e tendency t o s t a l i n g and r a n c i d i t y
i n s l i c e d bacon may c o r r e l a t e q u i t e w e l l w i t h t h e s e compositional f a c t o r s .
The r o l e of o t h e r f a c t o r s a r e not w e l l e s t a b l i s h e d a s y e t .
Among t h e s e a r e t h e f u n c t i o n of t h e nitrogenous moiety as well a s t h a t of
t h e phosphoric a c i d p o r t i o n of t h e molecule a s r e g u l a t o r s of o x i d a t i v e
processes. The presence of a s u b s t a n t i a l q u a n t i t y of plasmalogen
a s s o c i a t e d w i t h t h e comparable d i a c y l phosphoglyceride r a i s e s a number of
i n t e r e s t i n g i s s u e s . Our c u r r e n t program i s concerned both with plasmalogens and w i t h o x i d a t i v e mechanisms of t h e phosphoglycerides.
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DR. CRAIG: Thank you D r . Dugan f o r a most i n t e r e s t i n g discussion
of t h e phospholipids i n meat. We will hold t h e discussion of t h i s paper
and t h e other two papers u n t i l t h e end of t h e program. The t i t l e of t h e
i s "The I s o l a t i o n and
second paper, t o be presented by D r . VanderbJal,
Characterization of Pork S k e l e t a l Muscle Sarcolemma." I ' m happy t o present
t o you D r . VanderWal t o present t h i s t o p i c t o you.