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Amount and Fatty Acid Composition of Phospholipids in Broiler Neck Tissues1
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ROBERT K. RINGER
changes associated with aging in the BBB turkey. Poultry Sci. 38: 395-397. Ringer, R. K., H. S. Weiss and P. D. Sturkie, 1955. Effect of sex and age on blood pressure in the duck and pigeon. Am. J. Physiol. 183: 141-143. Ringer, R. K., P. D. Sturkie and H. S. Weiss, 1957. The role of the gonads in the control of
blood pressure in chickens. Am. J. Physiol. 190: 54-56. Sturkie, P. D., H. S. Weiss and R. K. Ringer, 1953. Effects of age on blood pressure in the chicken. Am. J. Physiol. 174: 405-407. Sturkie, P. D., 1965. Avian Physiology, Comstock Publishing Associates 2nd ed. 766 pages.
Amount and Fatty Acid Composition of Phospholipids in Broiler Neck Tissues1
(Received for publication March 18, 1968)
"O ECENTLY, boning machines have -^ *- been developed which will give high yield of edible product from raw chicken necks. Such product, because of physical characteristics and content of soluble protein, has been shown to be of use in emulsion meats. Neck tissues have been shown to contain salt-soluble protein which had unusually high fat binding characteristics as compared to other poultry tissues in a model system (May and Hudspeth, 1961; and Hudspeth and May, 1967). Maurer and Baker (1966) observed the same high emulsifying capacity of chicken neck tissue and attributed it to their high collagen content. However, Franzen and May (1968) found that phospholipid extracted from chicken neck tissue, significantly increased the amount of corn oil bound by a given quantity of salt soluble protein when over 250 mg. was added to the model system as a substitute for an equal quantity of corn oil. Since poultry neck tissues are being used in emulsion meats in some quantity, and 1 University of Georgia College of Agriculture Experiment Stations, Journal Series Paper Number 80, College Station, Athens, Ga. 30601
conceivably might be used in much greater amounts, and since phospholipid content influences such use, it was deemed advisable to learn more about the phospholipid content of neck as compared to other poultry tissues. Other studies (Peng and Dugan, 1965; and Marion and Woodroof, 1965) have reported on phospholipid content of chicken tissues other than necks. Thus, this study was conducted to determine the amount of phospholipid in various chicken neck tissues and to ascertain their fatty acid composition. EXPERIMENTAL METHODS
Source and treatment of samples. Necks from six broilers were obtained from a commercial source for this study. Since broilers are remarkably uniform, and are marketed within a narrow age range, they vary little in total lipid composition (Hebert and Branson, 1956) justifying the small initial sample size. Four of the necks were dissected into the following tissue components: skin; connective, adipose, other tissues; muscle; and bone. Connective, adipose, and other tissue was obtained by scraping the internal skin area and removing any non-muscle substances from
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R. W. FRANZEN, JR., H. M. EDWARDS, JR. AND K. N. MAY Departments of Food Science and Poultry Science, University of Georgia, Athens, Georgia 30601
PHOSPHOLIPIDS IN NECKS
before the plates were introduced. The major phospholipids separated by this procedure were sphingomyelin and lysophosphatidylcholine (S), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE). The developed plates were sprayed with a solution of 200 mg. 2', 7' dichlorofluroescein in 100 ml. 95 percent ethanol and observed under a UV light. The spots were outlined while the plate was under the UV light with a syringe needle, and they were then scraped from the plate. The scraping of individual phospholipids from four spots of each sample, from each side of the plate, were obtained as duplicates. The phospholipids were then removed from the Silica gel by transferring the plate scrapings to a centrifuge tube and then extracting with 10 ml. of 5:4:1, v/v/v chloroform: methanol: distilled water, and this mixture was then stirred (a single phase system). To this mixture was added 2.4 ml. distilled water and the solution was again stirred (a two phase system). The tubes were centrifuged at 2,200 RPM and the upper water-methanol layer was siphoned off. Most of the Silica gel was spun to the bottom leaving a clear layer of the phospholipids in chloroform. An aliquot was taken from this layer and analyzed for phosphorous. This extraction procedure has been tested by one of the authors (HME) on several occasions utilizing both P analyses and 32P counting as criteria of recovery and has been found to give 95%—105% recovery of phospholipids placed on the plate. Fatty acid determinations: The fatty acid content of the four phospholipid fractions obtained by TLC was determined by gas-liquid chromatography. The scrapings from the TLC plate were refluxed for two and one-half hours in 25 ml. of 5 percent H 2 S0 4 in methanol v/v, on a hot plate to form the methyl esters of the fatty acids.
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the outer section of the muscular area. Similar tissues from two necks were combined to give more representative samples. The two remaining whole necks were then totally combined. Samples were also taken of a commercially deboned neck product for investigation. The weights of the neck and dissected tissues were recorded and from these, tissue composition was calculated. The tissues were homogenized in an omni-mixer and aliquots taken for chemical analysis. Gross chemical analyses. Duplicate samples were analyzed for percent moisture, ash and protein (A.O.A.C, 1955). The lipid of each sample was extracted by the method of Folch et al. (1957), and the amount present determined by weight. These lipid extracts were analyzed for phosphorous, employing the procedure described by Chen et al. (1956). The quantity of phospholipid in the tissue lipid extract was determined by multiplying the phosphorous values by 25 (based on phospholipids containing approximately 4% phosphorous) and the neutral lipid was obtained by subtracting the phospholipid from the total lipid. Phospholipid fractionation and identification. The phospholipids were separated by thin-layer chromatography (TLC) on glass plates 20 X 20 cm. coated with 1.0 mm. thick Silica gel H. After pouring, the plates were activated by air drying for 12 hours followed by heating at 170°C. for 30 minutes. Each plate was spotted with 8 spots containing approximately 1 mg. of lipid for the high neutral lipid low phospholipid tissues, eg. skin, and 0.15 mg. of lipid for the low neutral lipid high phospholipid tissues, eg. muscle. The plates were developed with a solution consisting of 100:50:8:4 ml; v/v/v/v, chloroform: methanol:glacial acetic acid:distilled water, and this solution was allowed to equilibrate with the blotting paper lining for one hour
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FEANZEN, JR., H. M.
Apparatus Gas carrier Column length Stationary phase Supporting solid Temperature
Flow rate Detector
F & M model 810 Nitrogen 72 X VA inch 20% diethylene glycol succinate Chromasorb W 80-100 mesh Column 20O°C. Detector 250°C. Injector 260°C. 30 ml./minute Flame ionization
Fatty acids were identified by comparison with standards from the Hormel Institute, National Heart Institute, and by comparing the unknown peaks to the semi-log plots of retention time vs. carbon number of that of the known fatty acid methyl esters. Quantitative results agreed with the fatty acid standards' stated composition data, with a relative error less than 2% for major components ( > 10% of total mixture), and less than 6% for minor components ( < 10% of total mixture). Hydrogenation of unknown fatty acids was also performed in efforts to identify them positively.
N.
MAY
Individual fatty acids were calculated as a percent of the total fatty acids in the sample. This was done by triangulation; measuring the area by multiplying height of a peak by width of one-half height. Throughout this study, the lipid extracts were stored under nitrogen in glass stoppered flasks, and in a freezer, to prevent oxidation of the fatty acids. There was never a time lapse of more than one and one-half hours from the time when the plates were spotted to the time when the fatty acids were methylated. RESULTS AND DISCUSSION Broiler neck was found to contain 18.2% skin; 20.6% connective, adipose, and other tissue; 28.1% muscle; 20.9% bone; with 12.2% being unaccounted for. The unaccounted weight loss during dissection was probably caused by both evaporation of moisture, since tissue separation is a tedious and time consuming operation, and also because of losses of small pieces of tissue. The overall chemical composition (wet weight basis) of the tissues studied is described in Table 1. The tissues which exhibited relatively low moisture levels contained large lipid and low protein percentages when compared to those which contained high amounts of moisture. The whole neck was found to be similar in composition to the commercial deboned neck, except for ash, which was higher in the whole neck since bone was present. Neck tissue lipids are divided into major TABLE 1.—Composition (wet basis) of broiler neck tissues Tissue Skin Connective and Adipose Bone Muscle Whole Neck Comm. Deboned Neck
Water Protein
(%) (%) 61.5 50.0 59.9 75.8 69.0 71.5
14.7 8.9 20.2 18.4 14.5 12.4
Ash Total Lipid Total
(%) (%)
(%)
.6 22.0 98.8 .6 37.5 97.0 12.9 6.7 99.7 .8 6.2 101.2 3.1 15.4 102.0 .9 15.0 99.8
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After addition of water to the reaction mixture, the methyl esters were extracted with 30-60 B.P. petroleum ether, dried with anhydrous sodium sulfate, filtered and dried to a small volume under nitrogen. The methyl esters were then spotted on small 10 X 8.5 cm. glass thin layer plates coated with 0.5 mm. Silica gel G. The plates were developed in 50 percent heptane, 50 percent benzene v/v and were sprayed as previously described. The methyl ester band which was compared with a standard, was scraped from the plate and the scrapings were added to a column containing sand, supported by cotton. The methyl esters were eluted by a mixture of diethyl ether and petroleum ether (1:9, v/v). The methyl esters, after concentration to a small volume (approx. 1 |xl.) under a brisk flow of nitrogen, were injected into the GLC using the following conditions:
EDWARDS, JR., AND K.
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PHOSPHOLIPIDS IN NECKS
TABLE 2.—Lipid composition of broiler neck tissues
Tissue Skin Connective and adipose Bone Muscle Whole Neck Comm. Deboned Neck
Total Phospholipids of the total Lipids lipids
Neutral lipids of the total lipids
(%>
(%)
22.0
2.5
97.5
37.5 6.7 6.2 15.4
1.2 12.6 17.1 4.7
98.8 87.4 82.9 95.3
15.0
3.8
96.2
(%)
TABLE 3.—Phospholipid fractions of total phospholipid in broiler neck tissue Phospholipid fractions8 Tissue
Skin Connective and adipose Bone Muscle Whole Neck Comm. Deboned
Other phospholipids
S.
P.C.
P.S.
P.E.
21.4
37.1
11.3
22.0
8.2
16.5 10.6 9.0 7.8 6.8
41.9 39.5 43.2 30.3 39.3
9.2 8.7 5.8 9.5 1.8
23.9 32.2 21.4 24.4 20.2
8.6 9.1 20.7 28.0 31.9
(%) (%) (%) (%)
(%)
a S. = sphingomyelin and lysophosphatidylcholine; P.C. = phosphatidylcholine; P.S. =phospnatidylserine; P.E. =phosphatidylethanolamine.
cent total lipid divided into 57.6 percent neutral lipid and 39.1 percent phospholipid. Variation in values found for neck muscle in this study and other tissues reported by Katz et al. (1966) and Peng and Dugan (1965) would indicate differences among individual muscles of the carcass or possibly composition differences due to diet. Individual phospholipids described as a percent of the total phospholipids present are compared in Table 3. Phosphatidylcholine (PC) represented the greatest amounts with phosphatidylethanolamine (PE) being second. Large amounts of phospholipids in the muscle and the other tissues where muscle was present did not appear in the four individual fractions measured. This large amount of "other phospholipids" appeared as a large long spot on the thin layer plates between the lysophosphatidylcholine and the origin. The separation between this spot and the lysophosphatidylcholine was not at all clean. Thus, in those cases where the muscle was part of the sample the area of the plate marked and analyzed as sphingomyelin and lysophosphatidylcholine (S) could have contained an amount of some other phospholipid and that area below the lysophosphatidylcholine that was analyzed as part of the "other lipids" could have contained some lysophosphatidylcholine.
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classes in Table 2. Muscle and bone tissues were found to contain the largest percentage of phospholipids as a percent of total lipids in the tissues studied. The total lipid composition of broiler neck skin was found to be 2.5 and 97.5 percent phospholipids and neutral lipids, respectively. These results were similar to those reported by Katz et al. (1966). They found broiler skin lipids to contain 2.0 and 98 percent phospholipids and neutral lipids, respectively. However, Marion and Woodroof (1965) reported that broiler skin phospholipids were 8.1 percent of the total lipids. The connective, adipose, and other tissue in the present study was found to contain 1.2 percent phospholipid and 98.8 percent neutral lipid of the total lipids. These results are comparable to those of broiler depot fat reported by Katz et al. (1966). They reported 0.9 percent phospholipid, and 99.1 percent neutral lipid as percentages of the total depot lipid. The total lipid in connective and adipose tissue in the present study was 37.5 percent, while depot fat has been reported by Katz et al. (1966) to contain 60-80 percent lipid. Broiler neck muscle (dark meat) was found to contain 6.2 percent total lipid, with 17.1 percent being phospholipid, and 82.9 percent being neutral lipid. Katz et al. (1966) reported dark meat to contain 2.5 percent total lipids which consisted of 21 percent phospholipid and 79 percent neutral lipid. Peng and Dugan (1965) also working with chicken dark meat (thigh muscle) found 1.9 per-
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FRANZEN, JR., H.
M. EDWARDS, JR., AND K.
TABLE 4.—Fatty acid composition of the sphingomyelin and lysophosphatidylcholine fraction of broiler neck tissues Neck Tissue Fatty* Acid Skin
(%)
(%)
1.8 0.2
! - Kb
29.1
41.0
2.6 0.3 0 9.2
6.2 2.1 3.3 0.6 0.3 0.9 1.5 0 1.0 0 0
Whole Neck.
(%)
Commercial Deboned Neck
(%)
1.4 tr
1.2 tr
0.9 0.1
0.5 0.1
23.4
25.7
23.1
26.5
35.1
48.3
14.5 38.0
18.6 31.9
16.5 26.8
28.5 11.3
7
tr
1.8 0 0 9.8
6.5 1.6 3.3 1.2 0.5 0 0.9 1.1 0 0 0
2.9 0.4 0.3
6.9 1.2 3.4 1.9 0 0.3 0 2.3 0 0 0
3.0 0.6 0.1
9.8 3.0 3.5 0.8 tr 0 0 4.4 0 tr tr
1.1 0.4 0
9.9 2.0 4.6 0 0 2.0 3.1 3.7 0 1.6 0.9
1.5 1.0 0
6.1 2.6 4.3 tr 0 0 4.4 4.6 0 0 0
* Carbon number: number of double bonds. tr = trace.
The authors do not have any explanation for the low level of phosphatidylcholine (PC) present in the whole neck sample or the low level of phosphatidylserine (PS) in the commercial deboned neck. The fatty acid composition of phospholipid fractions of the various tissues is preTABLE 5.—Fatty acid composition of the phosphatidylcholine fraction of broiler neck tissues Neck Tissue
14:0 14:1 16:0 16:1 16:2 17:0 18:0 18:1 18:2 20:0 18:3 20:2 20:3 20:4 22:1 24:0 24:1 24:2 22:5 22:6 24:5
(%)
Commercial Deboned Neck
0.4 0.1
0.4 0.1
0.4 0.1
33.3
25.4
34.5
42.6
12.7 31.4 13.6
19.1 19.4 24.8
15.4 19.5 18.2
19.4 19.2 12.1
(%)
Conn., adipose other
2.4 0.1
1.1 0.2
0.5 0.1
28.4
33.6
14.6 31.5 14.6 b
11.1 30.9 14.2
Fatty Acid* Skin
2.6 0.6 0.1
tr tr tr 0 6.5 0 0 0 0 0 0 0
(%)
2.2 0.4 0.1
tr tr tr 0.4 5.6 0 0.3 0 0 0 0 0
Bone Muscle (%) (/o)
3.0 0.5 0.1
tr 0.1 tr tr 4.2 0.3 tr tr 0 0 0 0
1.5 0.4 0
0.2 tr 0.4 0.6 6.5 0 1.2 tr 0.1 0 tr 0
Whole Neck
1.1 0.4 0
0 0 0.4 0.8 7.4 0 1.3 0 0 tr 0.5 0
*b Carbon number: number of double bonds. tr=trace.
(%)
1.9 0.3 0
0.2 0.3 0.3 0.7 2.2 0 0.4 0 0 0 0 0
MAY
sented in Tables 4-7. These data indicate that the fatty acid composition of a particular phospholipid follows the same general composition pattern regardless of the tissue from which it originated. This is particularly true when one compares the fatty acid composition of the various phospholipids from skin or connective and adipose tissues. The fatty acid composition of the muscle and bone phospholipids usually varied at least in the composition of one or two acids. For instance, with respect to sphingomyelin (S) and PS the skin and connective tissue samples were very similar, while the bone and muscle samples showed much more stearic acid and lower levels of oleic acid. The muscle sample also contained greater amounts of linoleic acid and linolenic acid. The commercial deboned neck samples showed very high levels of palmitic and stearic acid in the S and lysophosphatidylcholine fraction, with a correspondingly low level of oleic acid. The fatty acid composition of the PC from skin, connective tissue, and bone samples were similar. However, the muscle PC contained much higher levels of stearic and linoleic acid and lower levels of oleic acid. TABLE 6.—Fatty acid composition of the phosphatidylserine fraction of broiler neck tissues Neck Tissue Fatty Acid* Skin
(%)
Conn., adipose & Bone Muscle other (%) (%)
Whole Neck
(%)
(%)
(%)
14:0 14:1 14:2 16:0 16:1 16:2 17:0 18:0 18:1 18:2 20:0 18:3 20:2 20:3 20:4 22:1 24:0 24:1 22:6
Commercial Deboned Neck
2.0 0.1 0
2.4 0.1 0
2.0 0.1 0.2
1.7 tr 0
0.7 0.1 0
0.7 tr 0
11.7
13.3
13.7
13.1
10.9
11.5
18.3 54.6
16.6 54.9
15.8 • 22.9 55.0 43.1
28.3 38.7
33.8 31.0
1.2 0 0.1
6.0 0.2 trb 0 0 5.6 0 0 0 0
1.8 0 0
6.3 tr 0 0 .0 3.7 0 0.4 0 0.6
2.1 0 0.4 6.7 0.7 0.9 0 0 1.7 0.7 0 0 0
2.2 0.4 0.1
7.3 tr tr 0 0 7.6 1.1 0 0.6 0
1.5 0 0
6.5 0.9 0.8 0 0
10.2 0 1.6 0 0
*b Carbon number: number of double bonds. tr=trace.
1.3 0.4 0
8.6 1.2 1.9 0.6 1.8 4.1 0 3.2 0 0
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14:0 14:1 16:0 16:1 16:2 17:0 18:0 18:1 18:2 20:0 20:4 22:1 22:2 20:5 24:0 24:1 22:4 22:5 24:5
Conn., adipose & Bone Muscle other (%) (%)
N.
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PHOSPHOLIPIDS IN NECKS
TABLE 7.—Fatty acid composition of the phosphatidylethanolamine fraction of broiler neck tissues Neck Tissue Fatty Add" Skin
(%) 14:0 14:1 14:2 16:0 16:1 16:2 17:0 18:0 18:1 18:2 20:0 18:3 20:2 20:3 20:4 22:1 22:2 20:5 24:0 24:1 24:2 22:4 22:6 24:? 24:5
Conn., Muscle adipose & Bone (%) other (%)
tr^ 0
10.0
10.9 1.1 0 0
1.0 tr tr 9.7 2.1 0.3 0.2
12.9 46.0
15.0 44.6
13.4 43.4
6.5 0.2 tr 0 0
17.2 0 0 0 3.1 0.3 0 tr 0.6 0 0
(%)
(%) 1.3 0 0
1.3 0.1 0.2
Whole Neck
7.6 0.2 0.3 0 0.6
12.9 0 0 0 4.2 0 0 tr 0.8 0 0
6.3 0.2 2.5 0.5 0
12.2 0.6 tr 0.4 3.7 1.4 0 0.6 1.7 0 0
0.4 tr 0.1 6.9 0.7 0.3 0.1
0.8 0.2 0 9.6 1.5 0.4 0
27.4 17.8
19.9 25.0
8.2 0.3 0.2 0.2 0.9
22.4 0.5 0.1 0 4.5 3.1 2.4 0 3.1 0 0.3
8.3 0.5 1.6 0 0.7
21.9 0 0 0 5.2 0.8 0 0 1.2 2.6 0
*b Carbon number: number of double bonds. tr=trace.
Commerdal Deboned Neck
(%) 0.4 0.1 0
10.2 1.5 0.3 0
26.8 20.1 10.9 0.5 3.4 0.8 2.7
14.4 0 tr 0 4.5 0.4 0.5 0 2.6 0 0
TABLE 8.—Results of Duncan's multiple range test of fatty acid composition of phospholipids in chicken neck*
16:0 PE
PS
S
PC
9.4
13.0
25.3
30.2
S
PC
PE
PS
13.0
14.4
17.2
18.4
PC
PE
S
PS
28.3
38.0
39.8
51.9
PS
PE
S
PC
6.6
7.2
7.4
16.8
S
PS
PC
PE
3.4
4.7
5.7
16.3
18:0
18:1
18:2
20:4
» Phospholipids connected by lines are not significantly different ( 1 % level).
ences were found for all of the fatty acids except stearic acid. These differences were consistent in every tissue observed. The S and PC always contained larger amounts of palmitic acid than PS or PE. The PC, except for one instance, was always lower in oleic acid than any of the other phospholipids studied, while PS was always highest. The PC also contained more linoleic acid than any of the other phospholipids studied. Arachidonic acid was always found to be more abundant in PE than in any of the other phospholipids examined. Glende and Cornatzer (1966) studied the fatty acid composition of phospholipids from the mitochondria of rat liver, kidney, heart, and spleen. They found that palmitate and linoleate were higher in PC than in PE, whereas PE exhibited larger amounts of stearate and arachidonate than PC. Macfarlane et al. (1960) found that the cephalin fraction of rat liver mitochondria
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The PC from commercial deboned neck contained very high levels of palmitic acid and stearic acid. There was very little difference in the fatty acid composition of PS from the various tissues. The sample from bone was low in arachidonic acid. The fatty acid composition of the PE from muscle was different from the other tissues. It contained a much greater amount of stearic acid and arachidonic acid and a lower level of oleic acid. The sample from commercial deboned neck more closely resembled muscle than any other tissue, however, it did not have the very high level of arachidonic acid that muscle PE contained. In view of the general similarity in fatty acid composition of a specific phospholipid regardless of source, average values were calculated for the major fatty acids and the data analyzed to show any difference in fatty acid composition between phospholipids (Table 8). Highly significant differ-
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R. W. FRANZEN, JR., H. M. EDWARDS, JR., AND K. N. MAY
percent was reported by Katz et al. (1966). The oleic acid (18:1) content of neck skin phospholipids was found to be 43.2 percent, which was extremely high when compared to values of 14.6 percent and 19.5 percent reported for broiler skin phospholipids by Marion and Woodroof (1965), and Katz et al. (1966), respectively. The linoleic acid (18:2) and arachidonic acid (20:4) content of neck skin phospholipid were found to be 8.3 percent and 8.2 percent, respectively. These values were lower than those reported by either Marion and Woodroof (1965), and Katz et al. (1966) of 14.8 percent and 11.4 percent, respectively. Peng and Dugan (1965) reported similar overall results for broiler dark and white meat phospholipid fatty acid composition as those for broiler neck muscle phospholipid fatty acids. They found the PE to contain stearic acid as a predominant fatty acid. Oleic acid was reported as being the predominant fatty acid in both PS and S, while palmitic acid was dominant in PC. Similar results were found for neck muscle (Tables 4-7), although differences in percentages of the fatty acids reported were apparent. SUMMARY A study was designed to determine the phospholipid composition of various tissues of broiler necks. Four component tissues dissected from broiler necks and a sample of commercially deboned chicken neck meat were analyzed for moisture, lipid, protein and ash content. The lipid fractions were further examined for percentage neutral lipid and phospholipid composition. The total phospholipids were fractioned further into individual phospholipids by thin-layer chromatography. Muscle and bone tissues contained rel-
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and microsomes contained larger amounts of arachidonate and stearate than lecithin from the same subcellular particles, while lecithin contained a greater amount of linoleate than did the cephalins. Gray and Macfarlane (1961), demonstrated that cephalin from pigeon breast muscle and the spleen, lung, and kidney of swine, contained higher levels of stearic and arachidonic acids as compared to lecithin, while lecithin was found to contain more palmitic and linoleic acids than cephalin. Kates and James (1961) reported that PE from fowl blood cells contained a greater quantity of stearic acid and smaller quantity of palmitic acid than did PC. When the individual phospholipids from the separated broiler neck tissues were compared for stearic acid (18:0) content, no significant differences were observed. Several characteristic patterns were observed in all the tissues for PS. Phosphatidylserine contained a significantly higher amount of oleic acid (18:1) when compared to PC. This relationship was also found by Peng and Dugan (1965) in chicken breast muscle. Also, PS could be grouped with PE with regards to its content of palmitic acid (16:0) but would not be grouped with PE as regards its content of arachidonic acid (20:4). The average percent of palmitic acid (16:0) in the four phospholipids studied from broiler neck skin was found to be 19.7 which is in good agreement with reports of Marion and Woodroof (1965) who found 22.4 percent of 16:0 in broiler skin phospholipids and Katz et al. (1966) who reported 22.1 percent of 16:0 in broiler skin phospholipids. Stearic acid (18:0) was found to comprise an average of 13.7 percent of the fatty acids in the neck skin tissue phospholipids studied. Marion and Woodroof (1965) found 18.7 percent 18:0 in broiler skin phospholipids, while 11.3
PHOSPHOLIPIDS IN NECKS
REFERENCES Association of Official Agricultural Chemists, 1960. Official Methods of Analysis. 9th E. A.O.A.C., Washington, D. C. Chen, P. S., Jr., T. Y. Toriborg and H. Warner, 1956. Microdetermination of phosphorous. Anal. Chem. 28: 1756-1758. Folch, J., M. Lees and G. H. Sloane-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226:497-509. Franzen, R. W., Jr., and K. N. May, 1968. Effect of phospholipids and fatty acid composition of oil on emulsifying capacity of soluble protein of chicken. Poultry Sci. 47 : 623-625. Glende, E. A., Jr., and W. E. Cornatzer, 1966. The phospholipid fatty acid composition of liver, kidney, heart, and spleen mitochondria from
rats of various age groups. Biochem. Biophys. Acta. 125:310-318. Gray, G. M., and M. G. Macfarlane, 1961. Composition of phospholipids of rabbit, pigeon and trout muscle and various pig tissues. Biochem. J. 8 1 : 480-488. Hebert, B. A., and C. C. Brunson, 1956. The effect of sex hormones on the fat and moisture content of broiler carcasses. Poultry Sci. 35: 1147. Hudspeth, J. P., and K. N. May, 1967. A study of the emulsifying capacity of soluble proteins of poultry meat. 1. Light and dark meat tissues of turkeys, hens and broilers, and dark meat tissues of ducks. Food Technol. 2 1 : 1141-1142. Kates, M., and A. T. James, 1961. Phosphatide components of fowl blood cells. Biochem. Biophys. Acta, 50: 478-488. Katz, M. A., L. R. Dugan, Jr. and L. E. Dawson, 1966. Fatty acids in neutral lipids and phospholipids from chicken tissues. J. Food Sci. 3 1 : 717-720. Macfarlane, M. G., G. M. Gray and L. W. Wheeldon, 1960. Fatty acid composition from subcellular particles of rat liver. Biochem. J. 77: 626-631. Marion, J. E., and J. G. Woodroof, 1965. Lipid fractions of chicken broiler tissues and their fatty acid composition. J. Food Sci. 30: 38-43. Maurer, A. J., and R. C. Baker, 1966. The relationship between collagen content and emulsifying capacity of poultry meat. Poultry Sci. 4 5 : 1317-1321. May, K. N., and J. P. Hudspeth, 1966. A study of emulsifying capacity of soluble protein from various poultry meats. 13th World's Poultry Congress, Keiv, U.S.S.R., p. 61. Peng, C. Y., and L. R. Dugan, 1965. Composition and structure of phospholipids in chicken muscle tissue. J. Am. Oil Chem. Soc. 42: 533-536.
NEWS AND NOTES (continued from page 1578) of American Poultry Industries, Chicago, Illinois; Travel—Dr. A. W. Jasper, American Agricultural Marketing Association, Chicago, Illinois, and M. C. Small, National Turkey Federation, Mount Morris, Illinois; Special Tours—Harry Drews, Lincoln, Nebraska; Finance—L. A. Watt, Watt Publishing Company, Mount Morris, Illinois; and Information and Publicity—Alexander Gordeuk, Merck Sharpe and Dohme Research Laboratories, Rah-
way, New Jersey. The Chairman of the TJ.S.D.A. Committee has not been named. The Official Travel Agency for the Congress and all related tours will be the International Travel Service, Inc., 36 South Wabash Avenue, Chicago, Illinois. The World's Poultry Congress and Exhibition will be held during the second week in September, 1970, but the exact dates have not been announced.
(continued on page 1630)
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atively larger quantities of phospholipids than did any of the other tissues studied. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were found in relatively greater amounts than phosphatidylserine (PS) and sphingomyelin (S). The fatty acid content of the individual phospholipids was determined by gas-liquid chromatography. Palmitic acid (16:0) was found to be significantly higher in S and PC than in PE or PS. Oleic acid (18:1) was significantly higher in PS than in PC. Linoleic acid (18:2) was found to be present in significantly greater amounts in PC when compared to the other phospholipids studied.
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ROBERT K. RINGER
changes associated with aging in the BBB turkey. Poultry Sci. 38: 395-397. Ringer, R. K., H. S. Weiss and P. D. Sturkie, 1955. Effect of sex and age on blood pressure in the duck and pigeon. Am. J. Physiol. 183: 141-143. Ringer, R. K., P. D. Sturkie and H. S. Weiss, 1957. The role of the gonads in the control of
blood pressure in chickens. Am. J. Physiol. 190: 54-56. Sturkie, P. D., H. S. Weiss and R. K. Ringer, 1953. Effects of age on blood pressure in the chicken. Am. J. Physiol. 174: 405-407. Sturkie, P. D., 1965. Avian Physiology, Comstock Publishing Associates 2nd ed. 766 pages.
Amount and Fatty Acid Composition of Phospholipids in Broiler Neck Tissues1
(Received for publication March 18, 1968)
"O ECENTLY, boning machines have -^ *- been developed which will give high yield of edible product from raw chicken necks. Such product, because of physical characteristics and content of soluble protein, has been shown to be of use in emulsion meats. Neck tissues have been shown to contain salt-soluble protein which had unusually high fat binding characteristics as compared to other poultry tissues in a model system (May and Hudspeth, 1961; and Hudspeth and May, 1967). Maurer and Baker (1966) observed the same high emulsifying capacity of chicken neck tissue and attributed it to their high collagen content. However, Franzen and May (1968) found that phospholipid extracted from chicken neck tissue, significantly increased the amount of corn oil bound by a given quantity of salt soluble protein when over 250 mg. was added to the model system as a substitute for an equal quantity of corn oil. Since poultry neck tissues are being used in emulsion meats in some quantity, and 1 University of Georgia College of Agriculture Experiment Stations, Journal Series Paper Number 80, College Station, Athens, Ga. 30601
conceivably might be used in much greater amounts, and since phospholipid content influences such use, it was deemed advisable to learn more about the phospholipid content of neck as compared to other poultry tissues. Other studies (Peng and Dugan, 1965; and Marion and Woodroof, 1965) have reported on phospholipid content of chicken tissues other than necks. Thus, this study was conducted to determine the amount of phospholipid in various chicken neck tissues and to ascertain their fatty acid composition. EXPERIMENTAL METHODS
Source and treatment of samples. Necks from six broilers were obtained from a commercial source for this study. Since broilers are remarkably uniform, and are marketed within a narrow age range, they vary little in total lipid composition (Hebert and Branson, 1956) justifying the small initial sample size. Four of the necks were dissected into the following tissue components: skin; connective, adipose, other tissues; muscle; and bone. Connective, adipose, and other tissue was obtained by scraping the internal skin area and removing any non-muscle substances from
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R. W. FRANZEN, JR., H. M. EDWARDS, JR. AND K. N. MAY Departments of Food Science and Poultry Science, University of Georgia, Athens, Georgia 30601
PHOSPHOLIPIDS IN NECKS
before the plates were introduced. The major phospholipids separated by this procedure were sphingomyelin and lysophosphatidylcholine (S), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE). The developed plates were sprayed with a solution of 200 mg. 2', 7' dichlorofluroescein in 100 ml. 95 percent ethanol and observed under a UV light. The spots were outlined while the plate was under the UV light with a syringe needle, and they were then scraped from the plate. The scraping of individual phospholipids from four spots of each sample, from each side of the plate, were obtained as duplicates. The phospholipids were then removed from the Silica gel by transferring the plate scrapings to a centrifuge tube and then extracting with 10 ml. of 5:4:1, v/v/v chloroform: methanol: distilled water, and this mixture was then stirred (a single phase system). To this mixture was added 2.4 ml. distilled water and the solution was again stirred (a two phase system). The tubes were centrifuged at 2,200 RPM and the upper water-methanol layer was siphoned off. Most of the Silica gel was spun to the bottom leaving a clear layer of the phospholipids in chloroform. An aliquot was taken from this layer and analyzed for phosphorous. This extraction procedure has been tested by one of the authors (HME) on several occasions utilizing both P analyses and 32P counting as criteria of recovery and has been found to give 95%—105% recovery of phospholipids placed on the plate. Fatty acid determinations: The fatty acid content of the four phospholipid fractions obtained by TLC was determined by gas-liquid chromatography. The scrapings from the TLC plate were refluxed for two and one-half hours in 25 ml. of 5 percent H 2 S0 4 in methanol v/v, on a hot plate to form the methyl esters of the fatty acids.
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the outer section of the muscular area. Similar tissues from two necks were combined to give more representative samples. The two remaining whole necks were then totally combined. Samples were also taken of a commercially deboned neck product for investigation. The weights of the neck and dissected tissues were recorded and from these, tissue composition was calculated. The tissues were homogenized in an omni-mixer and aliquots taken for chemical analysis. Gross chemical analyses. Duplicate samples were analyzed for percent moisture, ash and protein (A.O.A.C, 1955). The lipid of each sample was extracted by the method of Folch et al. (1957), and the amount present determined by weight. These lipid extracts were analyzed for phosphorous, employing the procedure described by Chen et al. (1956). The quantity of phospholipid in the tissue lipid extract was determined by multiplying the phosphorous values by 25 (based on phospholipids containing approximately 4% phosphorous) and the neutral lipid was obtained by subtracting the phospholipid from the total lipid. Phospholipid fractionation and identification. The phospholipids were separated by thin-layer chromatography (TLC) on glass plates 20 X 20 cm. coated with 1.0 mm. thick Silica gel H. After pouring, the plates were activated by air drying for 12 hours followed by heating at 170°C. for 30 minutes. Each plate was spotted with 8 spots containing approximately 1 mg. of lipid for the high neutral lipid low phospholipid tissues, eg. skin, and 0.15 mg. of lipid for the low neutral lipid high phospholipid tissues, eg. muscle. The plates were developed with a solution consisting of 100:50:8:4 ml; v/v/v/v, chloroform: methanol:glacial acetic acid:distilled water, and this solution was allowed to equilibrate with the blotting paper lining for one hour
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FEANZEN, JR., H. M.
Apparatus Gas carrier Column length Stationary phase Supporting solid Temperature
Flow rate Detector
F & M model 810 Nitrogen 72 X VA inch 20% diethylene glycol succinate Chromasorb W 80-100 mesh Column 20O°C. Detector 250°C. Injector 260°C. 30 ml./minute Flame ionization
Fatty acids were identified by comparison with standards from the Hormel Institute, National Heart Institute, and by comparing the unknown peaks to the semi-log plots of retention time vs. carbon number of that of the known fatty acid methyl esters. Quantitative results agreed with the fatty acid standards' stated composition data, with a relative error less than 2% for major components ( > 10% of total mixture), and less than 6% for minor components ( < 10% of total mixture). Hydrogenation of unknown fatty acids was also performed in efforts to identify them positively.
N.
MAY
Individual fatty acids were calculated as a percent of the total fatty acids in the sample. This was done by triangulation; measuring the area by multiplying height of a peak by width of one-half height. Throughout this study, the lipid extracts were stored under nitrogen in glass stoppered flasks, and in a freezer, to prevent oxidation of the fatty acids. There was never a time lapse of more than one and one-half hours from the time when the plates were spotted to the time when the fatty acids were methylated. RESULTS AND DISCUSSION Broiler neck was found to contain 18.2% skin; 20.6% connective, adipose, and other tissue; 28.1% muscle; 20.9% bone; with 12.2% being unaccounted for. The unaccounted weight loss during dissection was probably caused by both evaporation of moisture, since tissue separation is a tedious and time consuming operation, and also because of losses of small pieces of tissue. The overall chemical composition (wet weight basis) of the tissues studied is described in Table 1. The tissues which exhibited relatively low moisture levels contained large lipid and low protein percentages when compared to those which contained high amounts of moisture. The whole neck was found to be similar in composition to the commercial deboned neck, except for ash, which was higher in the whole neck since bone was present. Neck tissue lipids are divided into major TABLE 1.—Composition (wet basis) of broiler neck tissues Tissue Skin Connective and Adipose Bone Muscle Whole Neck Comm. Deboned Neck
Water Protein
(%) (%) 61.5 50.0 59.9 75.8 69.0 71.5
14.7 8.9 20.2 18.4 14.5 12.4
Ash Total Lipid Total
(%) (%)
(%)
.6 22.0 98.8 .6 37.5 97.0 12.9 6.7 99.7 .8 6.2 101.2 3.1 15.4 102.0 .9 15.0 99.8
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After addition of water to the reaction mixture, the methyl esters were extracted with 30-60 B.P. petroleum ether, dried with anhydrous sodium sulfate, filtered and dried to a small volume under nitrogen. The methyl esters were then spotted on small 10 X 8.5 cm. glass thin layer plates coated with 0.5 mm. Silica gel G. The plates were developed in 50 percent heptane, 50 percent benzene v/v and were sprayed as previously described. The methyl ester band which was compared with a standard, was scraped from the plate and the scrapings were added to a column containing sand, supported by cotton. The methyl esters were eluted by a mixture of diethyl ether and petroleum ether (1:9, v/v). The methyl esters, after concentration to a small volume (approx. 1 |xl.) under a brisk flow of nitrogen, were injected into the GLC using the following conditions:
EDWARDS, JR., AND K.
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PHOSPHOLIPIDS IN NECKS
TABLE 2.—Lipid composition of broiler neck tissues
Tissue Skin Connective and adipose Bone Muscle Whole Neck Comm. Deboned Neck
Total Phospholipids of the total Lipids lipids
Neutral lipids of the total lipids
(%>
(%)
22.0
2.5
97.5
37.5 6.7 6.2 15.4
1.2 12.6 17.1 4.7
98.8 87.4 82.9 95.3
15.0
3.8
96.2
(%)
TABLE 3.—Phospholipid fractions of total phospholipid in broiler neck tissue Phospholipid fractions8 Tissue
Skin Connective and adipose Bone Muscle Whole Neck Comm. Deboned
Other phospholipids
S.
P.C.
P.S.
P.E.
21.4
37.1
11.3
22.0
8.2
16.5 10.6 9.0 7.8 6.8
41.9 39.5 43.2 30.3 39.3
9.2 8.7 5.8 9.5 1.8
23.9 32.2 21.4 24.4 20.2
8.6 9.1 20.7 28.0 31.9
(%) (%) (%) (%)
(%)
a S. = sphingomyelin and lysophosphatidylcholine; P.C. = phosphatidylcholine; P.S. =phospnatidylserine; P.E. =phosphatidylethanolamine.
cent total lipid divided into 57.6 percent neutral lipid and 39.1 percent phospholipid. Variation in values found for neck muscle in this study and other tissues reported by Katz et al. (1966) and Peng and Dugan (1965) would indicate differences among individual muscles of the carcass or possibly composition differences due to diet. Individual phospholipids described as a percent of the total phospholipids present are compared in Table 3. Phosphatidylcholine (PC) represented the greatest amounts with phosphatidylethanolamine (PE) being second. Large amounts of phospholipids in the muscle and the other tissues where muscle was present did not appear in the four individual fractions measured. This large amount of "other phospholipids" appeared as a large long spot on the thin layer plates between the lysophosphatidylcholine and the origin. The separation between this spot and the lysophosphatidylcholine was not at all clean. Thus, in those cases where the muscle was part of the sample the area of the plate marked and analyzed as sphingomyelin and lysophosphatidylcholine (S) could have contained an amount of some other phospholipid and that area below the lysophosphatidylcholine that was analyzed as part of the "other lipids" could have contained some lysophosphatidylcholine.
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classes in Table 2. Muscle and bone tissues were found to contain the largest percentage of phospholipids as a percent of total lipids in the tissues studied. The total lipid composition of broiler neck skin was found to be 2.5 and 97.5 percent phospholipids and neutral lipids, respectively. These results were similar to those reported by Katz et al. (1966). They found broiler skin lipids to contain 2.0 and 98 percent phospholipids and neutral lipids, respectively. However, Marion and Woodroof (1965) reported that broiler skin phospholipids were 8.1 percent of the total lipids. The connective, adipose, and other tissue in the present study was found to contain 1.2 percent phospholipid and 98.8 percent neutral lipid of the total lipids. These results are comparable to those of broiler depot fat reported by Katz et al. (1966). They reported 0.9 percent phospholipid, and 99.1 percent neutral lipid as percentages of the total depot lipid. The total lipid in connective and adipose tissue in the present study was 37.5 percent, while depot fat has been reported by Katz et al. (1966) to contain 60-80 percent lipid. Broiler neck muscle (dark meat) was found to contain 6.2 percent total lipid, with 17.1 percent being phospholipid, and 82.9 percent being neutral lipid. Katz et al. (1966) reported dark meat to contain 2.5 percent total lipids which consisted of 21 percent phospholipid and 79 percent neutral lipid. Peng and Dugan (1965) also working with chicken dark meat (thigh muscle) found 1.9 per-
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FRANZEN, JR., H.
M. EDWARDS, JR., AND K.
TABLE 4.—Fatty acid composition of the sphingomyelin and lysophosphatidylcholine fraction of broiler neck tissues Neck Tissue Fatty* Acid Skin
(%)
(%)
1.8 0.2
! - Kb
29.1
41.0
2.6 0.3 0 9.2
6.2 2.1 3.3 0.6 0.3 0.9 1.5 0 1.0 0 0
Whole Neck.
(%)
Commercial Deboned Neck
(%)
1.4 tr
1.2 tr
0.9 0.1
0.5 0.1
23.4
25.7
23.1
26.5
35.1
48.3
14.5 38.0
18.6 31.9
16.5 26.8
28.5 11.3
7
tr
1.8 0 0 9.8
6.5 1.6 3.3 1.2 0.5 0 0.9 1.1 0 0 0
2.9 0.4 0.3
6.9 1.2 3.4 1.9 0 0.3 0 2.3 0 0 0
3.0 0.6 0.1
9.8 3.0 3.5 0.8 tr 0 0 4.4 0 tr tr
1.1 0.4 0
9.9 2.0 4.6 0 0 2.0 3.1 3.7 0 1.6 0.9
1.5 1.0 0
6.1 2.6 4.3 tr 0 0 4.4 4.6 0 0 0
* Carbon number: number of double bonds. tr = trace.
The authors do not have any explanation for the low level of phosphatidylcholine (PC) present in the whole neck sample or the low level of phosphatidylserine (PS) in the commercial deboned neck. The fatty acid composition of phospholipid fractions of the various tissues is preTABLE 5.—Fatty acid composition of the phosphatidylcholine fraction of broiler neck tissues Neck Tissue
14:0 14:1 16:0 16:1 16:2 17:0 18:0 18:1 18:2 20:0 18:3 20:2 20:3 20:4 22:1 24:0 24:1 24:2 22:5 22:6 24:5
(%)
Commercial Deboned Neck
0.4 0.1
0.4 0.1
0.4 0.1
33.3
25.4
34.5
42.6
12.7 31.4 13.6
19.1 19.4 24.8
15.4 19.5 18.2
19.4 19.2 12.1
(%)
Conn., adipose other
2.4 0.1
1.1 0.2
0.5 0.1
28.4
33.6
14.6 31.5 14.6 b
11.1 30.9 14.2
Fatty Acid* Skin
2.6 0.6 0.1
tr tr tr 0 6.5 0 0 0 0 0 0 0
(%)
2.2 0.4 0.1
tr tr tr 0.4 5.6 0 0.3 0 0 0 0 0
Bone Muscle (%) (/o)
3.0 0.5 0.1
tr 0.1 tr tr 4.2 0.3 tr tr 0 0 0 0
1.5 0.4 0
0.2 tr 0.4 0.6 6.5 0 1.2 tr 0.1 0 tr 0
Whole Neck
1.1 0.4 0
0 0 0.4 0.8 7.4 0 1.3 0 0 tr 0.5 0
*b Carbon number: number of double bonds. tr=trace.
(%)
1.9 0.3 0
0.2 0.3 0.3 0.7 2.2 0 0.4 0 0 0 0 0
MAY
sented in Tables 4-7. These data indicate that the fatty acid composition of a particular phospholipid follows the same general composition pattern regardless of the tissue from which it originated. This is particularly true when one compares the fatty acid composition of the various phospholipids from skin or connective and adipose tissues. The fatty acid composition of the muscle and bone phospholipids usually varied at least in the composition of one or two acids. For instance, with respect to sphingomyelin (S) and PS the skin and connective tissue samples were very similar, while the bone and muscle samples showed much more stearic acid and lower levels of oleic acid. The muscle sample also contained greater amounts of linoleic acid and linolenic acid. The commercial deboned neck samples showed very high levels of palmitic and stearic acid in the S and lysophosphatidylcholine fraction, with a correspondingly low level of oleic acid. The fatty acid composition of the PC from skin, connective tissue, and bone samples were similar. However, the muscle PC contained much higher levels of stearic and linoleic acid and lower levels of oleic acid. TABLE 6.—Fatty acid composition of the phosphatidylserine fraction of broiler neck tissues Neck Tissue Fatty Acid* Skin
(%)
Conn., adipose & Bone Muscle other (%) (%)
Whole Neck
(%)
(%)
(%)
14:0 14:1 14:2 16:0 16:1 16:2 17:0 18:0 18:1 18:2 20:0 18:3 20:2 20:3 20:4 22:1 24:0 24:1 22:6
Commercial Deboned Neck
2.0 0.1 0
2.4 0.1 0
2.0 0.1 0.2
1.7 tr 0
0.7 0.1 0
0.7 tr 0
11.7
13.3
13.7
13.1
10.9
11.5
18.3 54.6
16.6 54.9
15.8 • 22.9 55.0 43.1
28.3 38.7
33.8 31.0
1.2 0 0.1
6.0 0.2 trb 0 0 5.6 0 0 0 0
1.8 0 0
6.3 tr 0 0 .0 3.7 0 0.4 0 0.6
2.1 0 0.4 6.7 0.7 0.9 0 0 1.7 0.7 0 0 0
2.2 0.4 0.1
7.3 tr tr 0 0 7.6 1.1 0 0.6 0
1.5 0 0
6.5 0.9 0.8 0 0
10.2 0 1.6 0 0
*b Carbon number: number of double bonds. tr=trace.
1.3 0.4 0
8.6 1.2 1.9 0.6 1.8 4.1 0 3.2 0 0
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14:0 14:1 16:0 16:1 16:2 17:0 18:0 18:1 18:2 20:0 20:4 22:1 22:2 20:5 24:0 24:1 22:4 22:5 24:5
Conn., adipose & Bone Muscle other (%) (%)
N.
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PHOSPHOLIPIDS IN NECKS
TABLE 7.—Fatty acid composition of the phosphatidylethanolamine fraction of broiler neck tissues Neck Tissue Fatty Add" Skin
(%) 14:0 14:1 14:2 16:0 16:1 16:2 17:0 18:0 18:1 18:2 20:0 18:3 20:2 20:3 20:4 22:1 22:2 20:5 24:0 24:1 24:2 22:4 22:6 24:? 24:5
Conn., Muscle adipose & Bone (%) other (%)
tr^ 0
10.0
10.9 1.1 0 0
1.0 tr tr 9.7 2.1 0.3 0.2
12.9 46.0
15.0 44.6
13.4 43.4
6.5 0.2 tr 0 0
17.2 0 0 0 3.1 0.3 0 tr 0.6 0 0
(%)
(%) 1.3 0 0
1.3 0.1 0.2
Whole Neck
7.6 0.2 0.3 0 0.6
12.9 0 0 0 4.2 0 0 tr 0.8 0 0
6.3 0.2 2.5 0.5 0
12.2 0.6 tr 0.4 3.7 1.4 0 0.6 1.7 0 0
0.4 tr 0.1 6.9 0.7 0.3 0.1
0.8 0.2 0 9.6 1.5 0.4 0
27.4 17.8
19.9 25.0
8.2 0.3 0.2 0.2 0.9
22.4 0.5 0.1 0 4.5 3.1 2.4 0 3.1 0 0.3
8.3 0.5 1.6 0 0.7
21.9 0 0 0 5.2 0.8 0 0 1.2 2.6 0
*b Carbon number: number of double bonds. tr=trace.
Commerdal Deboned Neck
(%) 0.4 0.1 0
10.2 1.5 0.3 0
26.8 20.1 10.9 0.5 3.4 0.8 2.7
14.4 0 tr 0 4.5 0.4 0.5 0 2.6 0 0
TABLE 8.—Results of Duncan's multiple range test of fatty acid composition of phospholipids in chicken neck*
16:0 PE
PS
S
PC
9.4
13.0
25.3
30.2
S
PC
PE
PS
13.0
14.4
17.2
18.4
PC
PE
S
PS
28.3
38.0
39.8
51.9
PS
PE
S
PC
6.6
7.2
7.4
16.8
S
PS
PC
PE
3.4
4.7
5.7
16.3
18:0
18:1
18:2
20:4
» Phospholipids connected by lines are not significantly different ( 1 % level).
ences were found for all of the fatty acids except stearic acid. These differences were consistent in every tissue observed. The S and PC always contained larger amounts of palmitic acid than PS or PE. The PC, except for one instance, was always lower in oleic acid than any of the other phospholipids studied, while PS was always highest. The PC also contained more linoleic acid than any of the other phospholipids studied. Arachidonic acid was always found to be more abundant in PE than in any of the other phospholipids examined. Glende and Cornatzer (1966) studied the fatty acid composition of phospholipids from the mitochondria of rat liver, kidney, heart, and spleen. They found that palmitate and linoleate were higher in PC than in PE, whereas PE exhibited larger amounts of stearate and arachidonate than PC. Macfarlane et al. (1960) found that the cephalin fraction of rat liver mitochondria
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The PC from commercial deboned neck contained very high levels of palmitic acid and stearic acid. There was very little difference in the fatty acid composition of PS from the various tissues. The sample from bone was low in arachidonic acid. The fatty acid composition of the PE from muscle was different from the other tissues. It contained a much greater amount of stearic acid and arachidonic acid and a lower level of oleic acid. The sample from commercial deboned neck more closely resembled muscle than any other tissue, however, it did not have the very high level of arachidonic acid that muscle PE contained. In view of the general similarity in fatty acid composition of a specific phospholipid regardless of source, average values were calculated for the major fatty acids and the data analyzed to show any difference in fatty acid composition between phospholipids (Table 8). Highly significant differ-
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R. W. FRANZEN, JR., H. M. EDWARDS, JR., AND K. N. MAY
percent was reported by Katz et al. (1966). The oleic acid (18:1) content of neck skin phospholipids was found to be 43.2 percent, which was extremely high when compared to values of 14.6 percent and 19.5 percent reported for broiler skin phospholipids by Marion and Woodroof (1965), and Katz et al. (1966), respectively. The linoleic acid (18:2) and arachidonic acid (20:4) content of neck skin phospholipid were found to be 8.3 percent and 8.2 percent, respectively. These values were lower than those reported by either Marion and Woodroof (1965), and Katz et al. (1966) of 14.8 percent and 11.4 percent, respectively. Peng and Dugan (1965) reported similar overall results for broiler dark and white meat phospholipid fatty acid composition as those for broiler neck muscle phospholipid fatty acids. They found the PE to contain stearic acid as a predominant fatty acid. Oleic acid was reported as being the predominant fatty acid in both PS and S, while palmitic acid was dominant in PC. Similar results were found for neck muscle (Tables 4-7), although differences in percentages of the fatty acids reported were apparent. SUMMARY A study was designed to determine the phospholipid composition of various tissues of broiler necks. Four component tissues dissected from broiler necks and a sample of commercially deboned chicken neck meat were analyzed for moisture, lipid, protein and ash content. The lipid fractions were further examined for percentage neutral lipid and phospholipid composition. The total phospholipids were fractioned further into individual phospholipids by thin-layer chromatography. Muscle and bone tissues contained rel-
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and microsomes contained larger amounts of arachidonate and stearate than lecithin from the same subcellular particles, while lecithin contained a greater amount of linoleate than did the cephalins. Gray and Macfarlane (1961), demonstrated that cephalin from pigeon breast muscle and the spleen, lung, and kidney of swine, contained higher levels of stearic and arachidonic acids as compared to lecithin, while lecithin was found to contain more palmitic and linoleic acids than cephalin. Kates and James (1961) reported that PE from fowl blood cells contained a greater quantity of stearic acid and smaller quantity of palmitic acid than did PC. When the individual phospholipids from the separated broiler neck tissues were compared for stearic acid (18:0) content, no significant differences were observed. Several characteristic patterns were observed in all the tissues for PS. Phosphatidylserine contained a significantly higher amount of oleic acid (18:1) when compared to PC. This relationship was also found by Peng and Dugan (1965) in chicken breast muscle. Also, PS could be grouped with PE with regards to its content of palmitic acid (16:0) but would not be grouped with PE as regards its content of arachidonic acid (20:4). The average percent of palmitic acid (16:0) in the four phospholipids studied from broiler neck skin was found to be 19.7 which is in good agreement with reports of Marion and Woodroof (1965) who found 22.4 percent of 16:0 in broiler skin phospholipids and Katz et al. (1966) who reported 22.1 percent of 16:0 in broiler skin phospholipids. Stearic acid (18:0) was found to comprise an average of 13.7 percent of the fatty acids in the neck skin tissue phospholipids studied. Marion and Woodroof (1965) found 18.7 percent 18:0 in broiler skin phospholipids, while 11.3
PHOSPHOLIPIDS IN NECKS
REFERENCES Association of Official Agricultural Chemists, 1960. Official Methods of Analysis. 9th E. A.O.A.C., Washington, D. C. Chen, P. S., Jr., T. Y. Toriborg and H. Warner, 1956. Microdetermination of phosphorous. Anal. Chem. 28: 1756-1758. Folch, J., M. Lees and G. H. Sloane-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226:497-509. Franzen, R. W., Jr., and K. N. May, 1968. Effect of phospholipids and fatty acid composition of oil on emulsifying capacity of soluble protein of chicken. Poultry Sci. 47 : 623-625. Glende, E. A., Jr., and W. E. Cornatzer, 1966. The phospholipid fatty acid composition of liver, kidney, heart, and spleen mitochondria from
rats of various age groups. Biochem. Biophys. Acta. 125:310-318. Gray, G. M., and M. G. Macfarlane, 1961. Composition of phospholipids of rabbit, pigeon and trout muscle and various pig tissues. Biochem. J. 8 1 : 480-488. Hebert, B. A., and C. C. Brunson, 1956. The effect of sex hormones on the fat and moisture content of broiler carcasses. Poultry Sci. 35: 1147. Hudspeth, J. P., and K. N. May, 1967. A study of the emulsifying capacity of soluble proteins of poultry meat. 1. Light and dark meat tissues of turkeys, hens and broilers, and dark meat tissues of ducks. Food Technol. 2 1 : 1141-1142. Kates, M., and A. T. James, 1961. Phosphatide components of fowl blood cells. Biochem. Biophys. Acta, 50: 478-488. Katz, M. A., L. R. Dugan, Jr. and L. E. Dawson, 1966. Fatty acids in neutral lipids and phospholipids from chicken tissues. J. Food Sci. 3 1 : 717-720. Macfarlane, M. G., G. M. Gray and L. W. Wheeldon, 1960. Fatty acid composition from subcellular particles of rat liver. Biochem. J. 77: 626-631. Marion, J. E., and J. G. Woodroof, 1965. Lipid fractions of chicken broiler tissues and their fatty acid composition. J. Food Sci. 30: 38-43. Maurer, A. J., and R. C. Baker, 1966. The relationship between collagen content and emulsifying capacity of poultry meat. Poultry Sci. 4 5 : 1317-1321. May, K. N., and J. P. Hudspeth, 1966. A study of emulsifying capacity of soluble protein from various poultry meats. 13th World's Poultry Congress, Keiv, U.S.S.R., p. 61. Peng, C. Y., and L. R. Dugan, 1965. Composition and structure of phospholipids in chicken muscle tissue. J. Am. Oil Chem. Soc. 42: 533-536.
NEWS AND NOTES (continued from page 1578) of American Poultry Industries, Chicago, Illinois; Travel—Dr. A. W. Jasper, American Agricultural Marketing Association, Chicago, Illinois, and M. C. Small, National Turkey Federation, Mount Morris, Illinois; Special Tours—Harry Drews, Lincoln, Nebraska; Finance—L. A. Watt, Watt Publishing Company, Mount Morris, Illinois; and Information and Publicity—Alexander Gordeuk, Merck Sharpe and Dohme Research Laboratories, Rah-
way, New Jersey. The Chairman of the TJ.S.D.A. Committee has not been named. The Official Travel Agency for the Congress and all related tours will be the International Travel Service, Inc., 36 South Wabash Avenue, Chicago, Illinois. The World's Poultry Congress and Exhibition will be held during the second week in September, 1970, but the exact dates have not been announced.
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atively larger quantities of phospholipids than did any of the other tissues studied. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were found in relatively greater amounts than phosphatidylserine (PS) and sphingomyelin (S). The fatty acid content of the individual phospholipids was determined by gas-liquid chromatography. Palmitic acid (16:0) was found to be significantly higher in S and PC than in PE or PS. Oleic acid (18:1) was significantly higher in PS than in PC. Linoleic acid (18:2) was found to be present in significantly greater amounts in PC when compared to the other phospholipids studied.
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