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Fractionation and Analysis of the High Density Fraction of Egg Yolk1
Fractionation and Analysis of the High Density Fraction of Egg Yolk1 K. N. KUAN, 2 R. JAMBUNATHAN, M. M. SIMLOT AND R. E. CLEGG Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506 (Received for publication October 21, 1972)
POULTRY SCIENCE 52: 1358-1363,
INTRODUCTION
I
N previous reports Clegg and Simlot (1968) described the preparation of four components from the high-density fraction of egg yolk. One of the components was a high-phosphorus, fat-free substance; the other three were dextranprecipitated substances, which were shown to contain lipid by a lipid staining technique (Clegg and Simlot, 1968). The dextran-precipitating technique was not suitable for large scale preparations; therefore, in work reported here, those three components were prepared by a combination of ultra-centrifuge and chromatographic techniques. The four purified fractions were analyzed and the analyses were compared with past reports. MATERIALS AND METHODS
Fraction IIIB, obtained by ultracentrifugation (Clegg and Simlot, 1968), was dialyzed for two days against 0.067 M bicarbonate buffer, pH 8.85, containing 0.1 M NaCl. A solution containing 6 to 8 grams of that sample was layered on top 1 Contribution No. 145, Department of Biochemistry, Kansas Agricultural Experiment Station. Manhattan, Kansas. 2 A portion of a dissertation in partial fulfillment of the requirements for the Ph.D. degree.
1973
of a preparative DEAE (diethylaminoethyl)-cellulose column (5 X 50 cm.) which had been equilibrated previously with the same buffer at 4°C. A stepwise pH gradient consisting of 0.067 M bicarbonate buffer of pH 8.85, 9.50, 9.70, and 9.80, each containing 0.1 M NaCl, was used to elute proteins from the column. This step is a modification of Jambunathan's method (1969) for separating fraction IIIB into three fractions using the same bicarbonate buffer, but using pH gradients of 8.85, 9.50, and 9.80. Twenty ml. of eluate were collected at a flow rate of about two ml. per minute. Solutions collected were read at 280 nm. with a Coleman autoset spectrophotometer. When an optical density reading of less than 0.05 was obtained, the buffer was changed. Accordingly, four fractions designated as 8.85, 9.50, 9.70, and 9.80, corresponding to the pH of buffer used for elution, were obtained. The eluates from each buffer were pooled, concentrated by biodryex,* dialyzed against doubly distilled water, and lyophilized. Total nitrogen was determined by the Kjeldahl method (Niederl and Niederl, 1946); total phosphorus, by the method of * Cellulosa fiber obtained from L6vdalens Industri Aktie Bolag, Central-palatset, Tegelbacken, 11152 Stockholm, Sweden
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ABSTRA CT Fraction IIIB (Clegg and Simlot, 1968), obtained by ultracentrifugation of a high-density fraction of egg yolk in 10% aqueous sodium chloride solution, was separated into four fractions by stepwise pH gradient elution from DEAE ion-exchange cellulose. The homogeneity of each fraction was tested by moving-boundary electrophoresis. The four fractions, 8.85, 9.50, 9.70, and 9.80, contained 0.99%, 1.25%, 2.33%, and 10.09% phosphorus, respectively, on an ash and moisture-free basis. The amino acid composition of each fraction also was determined. Fractions 8.85 and 9.50 were strikingly similar, while fraction 9.70 differed from the others in amino acid composition. Fraction 9.80 contained more serine than any other amino acid.
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H I G H D E N S I T Y F R A C T I O N OF E G G Y O L K
T h e sialic acid determination was based on the method of Svennerholm as described by Spiro (1966). Before the colorimetric determination, hexosamine was liberated b y acid hydrolysis as described by Johansen et ah (1960) and Spiro (1962). Colorimetric estimation of hexosamine was based essentially on the ElsonMorgan method as used by Rimington
70
(1940) and Winzler (1955). Protein sample was hydrolyzed b y 6 N HCl in sealed, evacuated ampoules a t 110°C. for 20 hours and the hydrolyzate was analyzed using a Beckman model 120C Amino Acid Analyzer; 0.067 M bicarbonate buffer, p H 9.80, was used for moving-boundary electrophoresis. RESULTS AND DISCUSSION Stepwise p H elution of fraction I I I B from the D E A E column, using 0.067 M bicarbonate buffer containing 0.1 M NaCl, gave four major peaks (Figure 1). Fraction 8.85 could not be used for electrophoretic work because it precipitated upon dialysis against p H 9.80 bicarbonate buffer. Fractions 9.50 and 9.80, dialyzed against p H 9.80 bicarbonate buffer, each gave a single peak in the ascending and
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120
130
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150
160
170
Number of Fractions
FIG. 1. Stepwise pH elution of fraction IIIB from DEAE cellulose column. Each point represents the average absorbance value obtained from three tubes, each having 20 ml. of eluate. Starting buffer was 0.067 M bicarbonate buffer, pH 8.85, containing 0.1 M NaCl. a. Introduction of 0.067 M bicarbonate buffer, pH 9.50, containing 0.1 M NaCl. b. Introduction of 0.067 M bicarbonate buffer, pH 9.70, containing 0.1 M NaCl. c. Introduction of 0.067 M bicarbonate buffer, pH 9.80, containing 0.1 M NaCl.
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Fiske and Subbarow (Hawk et ah, 1947). T r y p t o p h a n analysis was of the intact protein by the procedure of Spies and Chambers (1949) or by a slightly modified procedure of Hernandez and Bates (1969) using sodium phosphate instead of sodium acetate buffer. Amide ammonia was determine b y the method of Wilcox (1967). Total hexose was determined by the phenolsulfuric method as described by Dubois et ah (1956).
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K. N. KUAN, R. JAMBTJNATHAN, M. M. SIMLOT AND R. E. CLEGG TABLE 1.—Nitrogen, phosphorus, ash, moisture, and protein contents o.f indicated fractions*
Fraction
Nitrogen %
Phosphorus %
Moisture %
'Ash %
Nitrogen % corrected for ash and moisture
8.85 9.50 9.70 9.80
13.43 + 0.08 12.23 + 0.28 11.00 + 0.76 9.41+0.56
0.94 + 0.04 1.12 + 0.08 1.96+0.29 6.12 + 0.16
4.37 + 0.08 4.38 + 0.08 6.35 + 0.89 11.25 + 0.88
1.25 + 0.19 6.30 + 0.87 9.63+1.70 27.98+0.41
14.23 + 0.08 13.65 + 0.31 13.08 + 0.77 15.50+1.10
Phosphorus % corrected for ash and moisture 0.99 + 1.25 + 2.33 + 10.09 +
0.04 0.09 0.34 0.39
Protein % (corrected nitrogen value X6.25) 88.94 85.31 81.75 96.88
* Average of three to six determinations.
positions of fractions 8.85 and 9.50 were strikingly similar. When results were expressed as g. moles of amino acid residue per 105 grams of protein, as shown in the third column, 14 (aspartic acid, threonine, proline, glycine, half-cystine, methionine, isoleucine, leucine, phenylalanine, lysine, tryptophan, hexosamine, sialic acid, and hexose) of the 23 constituents showed excellent agreement. Of the remaining 9, only 5 (serine, glutamic acid, amide ammonia, arginine, and phosphorus) differed by more than 5 residues, and all were higher in the 9.50 fraction than in fraction 8.85. The amino acid composition of frac-
TABLE 2.—Results from fraction 8.85 analyses
TABLE 3.—Results from fraction 9.50 analyses
Constituents
Grams per 100 g. protein
Nitrogen as% of total nitrogen
G. moles residue per 106 g. protein
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Amide ammonia Arginine Tryptophan* Hexosamine Sialic acid Hexose Phosphorus
8.88 4.60 6.72 11.67 5.27 2.92 5.40 2.03 6.79 3.26 5.91 9.44 5.34 4.65 7.14 2.46 1.38 7.64 1.74 0.66 0.19 2.16 1.12
6.57 3.08 6.30 7.81 4.50 3.83 5.97 0.83 5.71 2.15 4.44 7.08 2.90 2.76 9.63 4.69 7.97 17.27 1.69 0.41 0.06
66.7 38.6 64.0 79.3 45.8 38.9 60.7 18.0 58.0 21.9 45.1 71.9 29.5 28.1 48.8 15.8 81.2 43.9 8.5 3.7 0.6 12.0 36.2
Determined by method of Spies and Chambers.
Constituents
Grams per 100 g. protein
Nitrogen as% of total nitrogen
G. moles residue per 106 g. protein
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Amide ammonia Arginine Tryptophan* Hexosamine Sialic acid Hexose Phosphorus
8.81 4.68 7.71 12.80 5.40 2.98 5.78 1.30 6.42 3.15 5.90 9.16 4.47 4.30 7.24 2.91 1.48 8.76 1.83 0.74 0.36 2.48 1.46
6.79 4.03 7.53 8.93 4.82 4.07 6.66 0.55 5.62 2.16 4.62 7.17 2.50 2.67 10.17 5.75 8.92 20.64 1.84 0.42 0.11
66.2 39.3 73.4 87.0 46.9 39.6 64.9 10.8 54.8 21.1 45.0 69.8 24.7 26.0 49.5 18.7 87.0 50.3 9.0 4.2 1.2 13.8 47.4
"Determined by method of Spies and Chambers.
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descending limb, with the peak for fraction 9.80 somewhat asymmetrical. Fraction 9.70 had a major peak and a minor band moving ahead of the major component. Nitrogen, phosphorus, moisture, and ash contents for these fractions are given in Table 1. Fraction 9.80 contained the highest percentage of phosphorus and 96.88% protein. The other fractions, 8.85, 9.50, and 9.70, contained 88.94%, 85.31%, and 81.75% protein, respectively. Compositions of fractions 8.85, 9.50, 9.70, and 9.80 are shown in Tables 2, 3, 4, and 5, respectively. The amino acid com-
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HIGH DENSTTY FRACTION OF EGG YOLK TABLE 4.—Results from fraction 9.70* analyses Grams per 100 g. protein
Nitrogen as% of total nitrogen
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Amide ammonia Arginine Tryptophan 11 Phosphorus
8.94 4.33 11.18 11.96 4.01 2.63 5.05 0.86 5.32 2.45 4.66 7.50 3.51 3.33 7.02 3.12 1.06 8.34 0.26 2.85
7.19 3.89 11.39 8.71 3.73 3.75 6.07 0.76 4.86 1.76 3.80 6.12 2.07 2.16 10.28 6.47 6.66 20.52 0.28
G. moles residue per 10* g. protein 67.2 36.3 106.4 81.3 34.8 35.1 56.7 7.0 45.4 16.4 35.5 57.2 19.4 20.2 48.0 20.1 62.3 47.9 1.3 92.2
Constituents
Grams per 100 g. protein
Nitrogen as% of total nitrogen
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Amide ammonia Arginine Tryptophan 11 Phsophorus
5.73 1.78 27.52 5.98 1.34 1.37 2.23 0 1.41 0.46 0.85 1.53 0.50 1.01 7.15 4.93 1.00 6.49 0.23 10.41
3.89 1.35 23.67 3.68 1.05 1.65 2.26 0 1.08 0.28 0.58 1.05 0.25 0.55 8.84 8.62 5.29 13.47 0.21
G. moles residue per 10* g. protein 43.0 14.9 261.7 40.7 11.6 18.2 25.0 0 12.0 3.1 6.5 11.7 2.7 6.1 48.9 31.8 58.5 37.2 1.1 336.7
a No carbohydrate analysis was performed on this fraction. b Determined by modified procedure of Hernandez and Bates.
a No carbohydrate analysis was performed on this fraction. b Determined by modified procedure of Hernandez and Bates.
tion 9.70 differed from the other fractions. Phosphorus content was greater in fraction 9.70 than in fractions 8.85 or 9.50, but far less than in fraction 9.80. As none of the three fractions, 8.85, 9.50, and 9.70, used for amino acid analysis was extracted for lipid, none has been compared with values reported for a- or /3-vitellin. Fraction 9.80 is a unique phosphoprotein; it contained 27.52% serine and 10.41% phosphorus. No correction was made for possible losses of serine during acid hydrolysis. While the acidic and neutral amino acids present were at levels similar to those found in many other proteins, neutral amino acids were scarce. Amino acid distribution was roughly 36% threonine and serine, 14% dicarboxylic acids, 23% basic amino acids, and 12% nonpolar amino acids. No half-cystine was found in the hydrolyzate. Both similarities and differences were found when the amino acid composition
of fraction 9.80 was compared with compositions published for phosvitin (Table 6). Our values and those reported by Allerton and Perlmann (1965) and by Taborsky and Allende (1962) agreed quite well. Except for aspartic acid, glutamic acid, proline, valine, leucine, amide ammonia, and arginine, which are somewhat higher in our data, the amino acids agree within experimental error. Our serine was low because we are reporting uncorrected values. Phosphorus content of fraction 9.80 was in better agreement with that reported by Allerton and Perlmann (1965) than with that reported by Taborsky and Allende (1962). Both Lewis et al. (1950) and Taborsky and Allende (1962) used the Mecham and Olcott method (1949), while Allerton and Perlmann (1965) used the Joubert and Cook method (1958) for preparing phosvitin. The similarity of fraction 9.80's phosphorus content with contents that previous workers reported
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Constituents
TABLE 5.—Results from fraction 9.80"- analyses
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K. N. KUAN, R. JAMBUNATHAN, M. M. SIMLOT AND R. E. CLEGG TABLE 6.—Amino acid analyses of phosvitin compared g. moles residue/10s g. protein
Constituents
Fraction 9.80
a b 0 d
Value Value Value Value
corrected corrected corrected corrected
for for for for
Taborsky and Allende
Lewis el al.
Mecham and Olcott
39 14 334-355" 37 9 15 22 0 9 2 5 7 2 5 47 29 47 32 2 339 12.35 10.40 9 15
36 12 372b 32 9 16 20 0 6 0 4 7 7 7 45 30
33 12 310° 23 9 21 17
32 trace1 310 23 9 21
25% destruction 32% destruction 10% destruction 10% destruction
31 3 323 11.90 9.90
0 9 2 4 8 1 4 40 31 28 11.90 9.70
2-3 8 < 1 4 40 31 60 28 3 310 11.90 9.70
during hydrolysis. during hydrolysis. during hydrolysis. during hydrolysis.
indicated that stepwise pH elution of fraction II1B from DEAE ion-exchange columns is an alternate method for preparing phosvitin. REFERENCES Allerton, S. E., and G. E. Perlmann, 1965. Chemical characterization of the phosphoprotein phosvitin. J. Biol. Chem. 240:3892-3898. Clegg, R. E., and M. M. Simlot, 1968. Preparation of an electrophoretically homogeneous phosphoprotein from egg yolk. Poultry Sci. 47: 617-623. Dubois, M., K. A. GiUes, J. K. Hamilton, P. A. Rebers and F. Smith, 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350-356. Hawk, P. B., B. L. Oser and W. H. Summerson, 1947. Practical Physiological Chemistry. Blakiston Company, Philadelphia. Hernandez, H. H., and L. S. Bates, 1969. A modified method for rapid tryptophan analysis of maize. Centro Internacional De Mejoramiento De Maiz y Trigo, Mexico, Research Bulletin No. 13: 1-7. Jambunathan, R., 1969. Ph.D. Dissertation. Kansas
State University, Manhattan, Kansas. Isolation and characterization of egg yolk granule proteins. Johansen, P. G., R. D. Marshall and A. Neuberger, 1960. Carbohydrates in protein. The hexose, hexosamine, acetyl and amide-nitrogen content of hen's-egg albumin. Biochem. J. 77: 239-247. Joubert, F. J., and W. H. Cook, 1958. Preparation and characterization of phosvitin from hen egg yolk. Can. J. Biochem. Physiol. 36: 399-408. Lewis, J. C , N . S. Snell, D. J. Hirschmann and H. Fraenkel-Conrat, 1950. Amino acid composition of egg proteins. J. Biol. Chem. 186: 23-35. Mecham, D. K., and H. S. Olcott, 1949. Phosvitin, the principal phosphoprotein of egg yolk. J. Am. Chem. Soc. 71:3670-3679. Niederl, J. B., and V. Niederl, 1946. Organic Quantitative Microanalysis, 2nd edition. John Wiley and Sons, New York. Rimington, C , 1940. Seromucoid and the bound carbohydrate of the serum proteins. Biochem. J. 34:931-940. Spies, J. R., and D. C. Chambers, 1949. Chemical determination of tryptophan in proteins. Anal. Chem. 21:1249-1266.
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43 15 262 41 12 18 25 0 12 3 6 12 3 6 49 32 58 37 1 337 15.50 10.41
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Amide ammonia Arginine Tryptophan P03 Nitrogen % Phosphorus % Hexosamine Hexose
Allerton and Perlmann
HIGH DENSITY FRACTION or EGG YOLK Spiro, R. G., 1962. Studies on the monosaccharide sequence of the serum glycoprotein fetuin. J. Biol. Chem. 237:646-652. Spiro, R. G., 1966. Analysis of sugars found in glycoproteins. In: Methods in Enzymology, vol. VIII. Eds. S. P. Colowick and N. O. Kaplan. Academic Press, N. Y. pp. 3-26. Taborsky, G., and C. C. Allende, 1962. A rearrangement in the structure of phosvitin. Biochemistry,
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1:406-411. Wilcox, P. E., 1967. Determination of amide residues by chemical methods. In: Methods in Enzymology, vol. X I . Eds. S. P . Colowick and N . O. Kaplan. Academic Press, N. Y. pp. 63-76. Winzler, R. J., 1955. Determination of serum glycoproteins. In: Methods of Biochemical Analysis, vol. II. Ed. D. Glick. Interscience Publishers, Inc., N. Y. pp. 279-311.
G. SCHNELL, K. R. NATH, J. M. DARFLER, D. V. VADEHRA AND R. C. BAKER Department of PoiAtry Science, Cornell University, Ithaca, New York 14850 (Received for publication October 23, 1972) ABSTRACT Frankfurters were made from laboratory prepared mechanically deboned poultry meat (MDPM) using backs and necks. They were compared with frankfurters made from commercially made puree (mechanically deboned meat) and hand deboned carcass meat. Variables studied were (1) the effect of grind and screen sizes used in the production of the MDPM; (2) the effect of the percent of added skin in the formula; and (3) the effect of selected additives. Parameters used for evaluation included organoleptic scores, emulsion stability and shear press values. The smaller the screen size, the more tender, better flavored and more acceptable were the end products. Hand deboned carcass produced the firmest frankfurter. Flavor and acceptability of frankfurters from both hand deboned carcasses and commercial puree were not significantly different as compared to the product made with MDPM from the smallest screen (0.05 cm.). Increasing the skin added to the formula increased the fat content, increased organoleptic tenderness and viscosity and decreased emulsion stability. The addition of 3 % sodium caseinate, 3 % acid whey or 0.5% Kena had very little effect on the organoleptic qualities of the frankfurters. Kena markedly decreased emulsion viscosity, whereas, sodium caseinate increased it. POULTRY SCIENCE 52: 1363-1369,1973
INTRODUCTION
O
NE of the most economical sources of animal protein is mechanically deboned poultry meat (MDPM). Since the advent of several types of deboning machines, the production of this type of material from backs and necks of fryers, turkey racks and even spent layer carcasses, has increased enormously. With the availability of the product, information is needed on its composition and use in further processing. A considerable amount of basic infor-
mation can be found in the literature on mechanically deboned poultry meat. Vadehra and Baker (1970) and Grunden et al. (1972) have examined its chemical and physical characteristics. Ostovar et al. (1971) examined its microbiological quality. Essary and Ritchey (1968) have determined the amino acid composition and Maxon and Marion (1970), the lipid content of the product. Effect of variables encountered in the production of the material have been studied, such as the influence of the skin
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Physical and Functional Properties of Mechanically Deboned Poultry Meat as Used in the Manufacture of Frankfurters
POULTRY SCIENCE 52: 1358-1363,
INTRODUCTION
I
N previous reports Clegg and Simlot (1968) described the preparation of four components from the high-density fraction of egg yolk. One of the components was a high-phosphorus, fat-free substance; the other three were dextranprecipitated substances, which were shown to contain lipid by a lipid staining technique (Clegg and Simlot, 1968). The dextran-precipitating technique was not suitable for large scale preparations; therefore, in work reported here, those three components were prepared by a combination of ultra-centrifuge and chromatographic techniques. The four purified fractions were analyzed and the analyses were compared with past reports. MATERIALS AND METHODS
Fraction IIIB, obtained by ultracentrifugation (Clegg and Simlot, 1968), was dialyzed for two days against 0.067 M bicarbonate buffer, pH 8.85, containing 0.1 M NaCl. A solution containing 6 to 8 grams of that sample was layered on top 1 Contribution No. 145, Department of Biochemistry, Kansas Agricultural Experiment Station. Manhattan, Kansas. 2 A portion of a dissertation in partial fulfillment of the requirements for the Ph.D. degree.
1973
of a preparative DEAE (diethylaminoethyl)-cellulose column (5 X 50 cm.) which had been equilibrated previously with the same buffer at 4°C. A stepwise pH gradient consisting of 0.067 M bicarbonate buffer of pH 8.85, 9.50, 9.70, and 9.80, each containing 0.1 M NaCl, was used to elute proteins from the column. This step is a modification of Jambunathan's method (1969) for separating fraction IIIB into three fractions using the same bicarbonate buffer, but using pH gradients of 8.85, 9.50, and 9.80. Twenty ml. of eluate were collected at a flow rate of about two ml. per minute. Solutions collected were read at 280 nm. with a Coleman autoset spectrophotometer. When an optical density reading of less than 0.05 was obtained, the buffer was changed. Accordingly, four fractions designated as 8.85, 9.50, 9.70, and 9.80, corresponding to the pH of buffer used for elution, were obtained. The eluates from each buffer were pooled, concentrated by biodryex,* dialyzed against doubly distilled water, and lyophilized. Total nitrogen was determined by the Kjeldahl method (Niederl and Niederl, 1946); total phosphorus, by the method of * Cellulosa fiber obtained from L6vdalens Industri Aktie Bolag, Central-palatset, Tegelbacken, 11152 Stockholm, Sweden
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ABSTRA CT Fraction IIIB (Clegg and Simlot, 1968), obtained by ultracentrifugation of a high-density fraction of egg yolk in 10% aqueous sodium chloride solution, was separated into four fractions by stepwise pH gradient elution from DEAE ion-exchange cellulose. The homogeneity of each fraction was tested by moving-boundary electrophoresis. The four fractions, 8.85, 9.50, 9.70, and 9.80, contained 0.99%, 1.25%, 2.33%, and 10.09% phosphorus, respectively, on an ash and moisture-free basis. The amino acid composition of each fraction also was determined. Fractions 8.85 and 9.50 were strikingly similar, while fraction 9.70 differed from the others in amino acid composition. Fraction 9.80 contained more serine than any other amino acid.
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H I G H D E N S I T Y F R A C T I O N OF E G G Y O L K
T h e sialic acid determination was based on the method of Svennerholm as described by Spiro (1966). Before the colorimetric determination, hexosamine was liberated b y acid hydrolysis as described by Johansen et ah (1960) and Spiro (1962). Colorimetric estimation of hexosamine was based essentially on the ElsonMorgan method as used by Rimington
70
(1940) and Winzler (1955). Protein sample was hydrolyzed b y 6 N HCl in sealed, evacuated ampoules a t 110°C. for 20 hours and the hydrolyzate was analyzed using a Beckman model 120C Amino Acid Analyzer; 0.067 M bicarbonate buffer, p H 9.80, was used for moving-boundary electrophoresis. RESULTS AND DISCUSSION Stepwise p H elution of fraction I I I B from the D E A E column, using 0.067 M bicarbonate buffer containing 0.1 M NaCl, gave four major peaks (Figure 1). Fraction 8.85 could not be used for electrophoretic work because it precipitated upon dialysis against p H 9.80 bicarbonate buffer. Fractions 9.50 and 9.80, dialyzed against p H 9.80 bicarbonate buffer, each gave a single peak in the ascending and
80
90
100
110
120
130
HO
150
160
170
Number of Fractions
FIG. 1. Stepwise pH elution of fraction IIIB from DEAE cellulose column. Each point represents the average absorbance value obtained from three tubes, each having 20 ml. of eluate. Starting buffer was 0.067 M bicarbonate buffer, pH 8.85, containing 0.1 M NaCl. a. Introduction of 0.067 M bicarbonate buffer, pH 9.50, containing 0.1 M NaCl. b. Introduction of 0.067 M bicarbonate buffer, pH 9.70, containing 0.1 M NaCl. c. Introduction of 0.067 M bicarbonate buffer, pH 9.80, containing 0.1 M NaCl.
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Fiske and Subbarow (Hawk et ah, 1947). T r y p t o p h a n analysis was of the intact protein by the procedure of Spies and Chambers (1949) or by a slightly modified procedure of Hernandez and Bates (1969) using sodium phosphate instead of sodium acetate buffer. Amide ammonia was determine b y the method of Wilcox (1967). Total hexose was determined by the phenolsulfuric method as described by Dubois et ah (1956).
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K. N. KUAN, R. JAMBTJNATHAN, M. M. SIMLOT AND R. E. CLEGG TABLE 1.—Nitrogen, phosphorus, ash, moisture, and protein contents o.f indicated fractions*
Fraction
Nitrogen %
Phosphorus %
Moisture %
'Ash %
Nitrogen % corrected for ash and moisture
8.85 9.50 9.70 9.80
13.43 + 0.08 12.23 + 0.28 11.00 + 0.76 9.41+0.56
0.94 + 0.04 1.12 + 0.08 1.96+0.29 6.12 + 0.16
4.37 + 0.08 4.38 + 0.08 6.35 + 0.89 11.25 + 0.88
1.25 + 0.19 6.30 + 0.87 9.63+1.70 27.98+0.41
14.23 + 0.08 13.65 + 0.31 13.08 + 0.77 15.50+1.10
Phosphorus % corrected for ash and moisture 0.99 + 1.25 + 2.33 + 10.09 +
0.04 0.09 0.34 0.39
Protein % (corrected nitrogen value X6.25) 88.94 85.31 81.75 96.88
* Average of three to six determinations.
positions of fractions 8.85 and 9.50 were strikingly similar. When results were expressed as g. moles of amino acid residue per 105 grams of protein, as shown in the third column, 14 (aspartic acid, threonine, proline, glycine, half-cystine, methionine, isoleucine, leucine, phenylalanine, lysine, tryptophan, hexosamine, sialic acid, and hexose) of the 23 constituents showed excellent agreement. Of the remaining 9, only 5 (serine, glutamic acid, amide ammonia, arginine, and phosphorus) differed by more than 5 residues, and all were higher in the 9.50 fraction than in fraction 8.85. The amino acid composition of frac-
TABLE 2.—Results from fraction 8.85 analyses
TABLE 3.—Results from fraction 9.50 analyses
Constituents
Grams per 100 g. protein
Nitrogen as% of total nitrogen
G. moles residue per 106 g. protein
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Amide ammonia Arginine Tryptophan* Hexosamine Sialic acid Hexose Phosphorus
8.88 4.60 6.72 11.67 5.27 2.92 5.40 2.03 6.79 3.26 5.91 9.44 5.34 4.65 7.14 2.46 1.38 7.64 1.74 0.66 0.19 2.16 1.12
6.57 3.08 6.30 7.81 4.50 3.83 5.97 0.83 5.71 2.15 4.44 7.08 2.90 2.76 9.63 4.69 7.97 17.27 1.69 0.41 0.06
66.7 38.6 64.0 79.3 45.8 38.9 60.7 18.0 58.0 21.9 45.1 71.9 29.5 28.1 48.8 15.8 81.2 43.9 8.5 3.7 0.6 12.0 36.2
Determined by method of Spies and Chambers.
Constituents
Grams per 100 g. protein
Nitrogen as% of total nitrogen
G. moles residue per 106 g. protein
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Amide ammonia Arginine Tryptophan* Hexosamine Sialic acid Hexose Phosphorus
8.81 4.68 7.71 12.80 5.40 2.98 5.78 1.30 6.42 3.15 5.90 9.16 4.47 4.30 7.24 2.91 1.48 8.76 1.83 0.74 0.36 2.48 1.46
6.79 4.03 7.53 8.93 4.82 4.07 6.66 0.55 5.62 2.16 4.62 7.17 2.50 2.67 10.17 5.75 8.92 20.64 1.84 0.42 0.11
66.2 39.3 73.4 87.0 46.9 39.6 64.9 10.8 54.8 21.1 45.0 69.8 24.7 26.0 49.5 18.7 87.0 50.3 9.0 4.2 1.2 13.8 47.4
"Determined by method of Spies and Chambers.
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descending limb, with the peak for fraction 9.80 somewhat asymmetrical. Fraction 9.70 had a major peak and a minor band moving ahead of the major component. Nitrogen, phosphorus, moisture, and ash contents for these fractions are given in Table 1. Fraction 9.80 contained the highest percentage of phosphorus and 96.88% protein. The other fractions, 8.85, 9.50, and 9.70, contained 88.94%, 85.31%, and 81.75% protein, respectively. Compositions of fractions 8.85, 9.50, 9.70, and 9.80 are shown in Tables 2, 3, 4, and 5, respectively. The amino acid com-
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HIGH DENSTTY FRACTION OF EGG YOLK TABLE 4.—Results from fraction 9.70* analyses Grams per 100 g. protein
Nitrogen as% of total nitrogen
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Amide ammonia Arginine Tryptophan 11 Phosphorus
8.94 4.33 11.18 11.96 4.01 2.63 5.05 0.86 5.32 2.45 4.66 7.50 3.51 3.33 7.02 3.12 1.06 8.34 0.26 2.85
7.19 3.89 11.39 8.71 3.73 3.75 6.07 0.76 4.86 1.76 3.80 6.12 2.07 2.16 10.28 6.47 6.66 20.52 0.28
G. moles residue per 10* g. protein 67.2 36.3 106.4 81.3 34.8 35.1 56.7 7.0 45.4 16.4 35.5 57.2 19.4 20.2 48.0 20.1 62.3 47.9 1.3 92.2
Constituents
Grams per 100 g. protein
Nitrogen as% of total nitrogen
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Amide ammonia Arginine Tryptophan 11 Phsophorus
5.73 1.78 27.52 5.98 1.34 1.37 2.23 0 1.41 0.46 0.85 1.53 0.50 1.01 7.15 4.93 1.00 6.49 0.23 10.41
3.89 1.35 23.67 3.68 1.05 1.65 2.26 0 1.08 0.28 0.58 1.05 0.25 0.55 8.84 8.62 5.29 13.47 0.21
G. moles residue per 10* g. protein 43.0 14.9 261.7 40.7 11.6 18.2 25.0 0 12.0 3.1 6.5 11.7 2.7 6.1 48.9 31.8 58.5 37.2 1.1 336.7
a No carbohydrate analysis was performed on this fraction. b Determined by modified procedure of Hernandez and Bates.
a No carbohydrate analysis was performed on this fraction. b Determined by modified procedure of Hernandez and Bates.
tion 9.70 differed from the other fractions. Phosphorus content was greater in fraction 9.70 than in fractions 8.85 or 9.50, but far less than in fraction 9.80. As none of the three fractions, 8.85, 9.50, and 9.70, used for amino acid analysis was extracted for lipid, none has been compared with values reported for a- or /3-vitellin. Fraction 9.80 is a unique phosphoprotein; it contained 27.52% serine and 10.41% phosphorus. No correction was made for possible losses of serine during acid hydrolysis. While the acidic and neutral amino acids present were at levels similar to those found in many other proteins, neutral amino acids were scarce. Amino acid distribution was roughly 36% threonine and serine, 14% dicarboxylic acids, 23% basic amino acids, and 12% nonpolar amino acids. No half-cystine was found in the hydrolyzate. Both similarities and differences were found when the amino acid composition
of fraction 9.80 was compared with compositions published for phosvitin (Table 6). Our values and those reported by Allerton and Perlmann (1965) and by Taborsky and Allende (1962) agreed quite well. Except for aspartic acid, glutamic acid, proline, valine, leucine, amide ammonia, and arginine, which are somewhat higher in our data, the amino acids agree within experimental error. Our serine was low because we are reporting uncorrected values. Phosphorus content of fraction 9.80 was in better agreement with that reported by Allerton and Perlmann (1965) than with that reported by Taborsky and Allende (1962). Both Lewis et al. (1950) and Taborsky and Allende (1962) used the Mecham and Olcott method (1949), while Allerton and Perlmann (1965) used the Joubert and Cook method (1958) for preparing phosvitin. The similarity of fraction 9.80's phosphorus content with contents that previous workers reported
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Constituents
TABLE 5.—Results from fraction 9.80"- analyses
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K. N. KUAN, R. JAMBUNATHAN, M. M. SIMLOT AND R. E. CLEGG TABLE 6.—Amino acid analyses of phosvitin compared g. moles residue/10s g. protein
Constituents
Fraction 9.80
a b 0 d
Value Value Value Value
corrected corrected corrected corrected
for for for for
Taborsky and Allende
Lewis el al.
Mecham and Olcott
39 14 334-355" 37 9 15 22 0 9 2 5 7 2 5 47 29 47 32 2 339 12.35 10.40 9 15
36 12 372b 32 9 16 20 0 6 0 4 7 7 7 45 30
33 12 310° 23 9 21 17
32 trace1 310 23 9 21
25% destruction 32% destruction 10% destruction 10% destruction
31 3 323 11.90 9.90
0 9 2 4 8 1 4 40 31 28 11.90 9.70
2-3 8 < 1 4 40 31 60 28 3 310 11.90 9.70
during hydrolysis. during hydrolysis. during hydrolysis. during hydrolysis.
indicated that stepwise pH elution of fraction II1B from DEAE ion-exchange columns is an alternate method for preparing phosvitin. REFERENCES Allerton, S. E., and G. E. Perlmann, 1965. Chemical characterization of the phosphoprotein phosvitin. J. Biol. Chem. 240:3892-3898. Clegg, R. E., and M. M. Simlot, 1968. Preparation of an electrophoretically homogeneous phosphoprotein from egg yolk. Poultry Sci. 47: 617-623. Dubois, M., K. A. GiUes, J. K. Hamilton, P. A. Rebers and F. Smith, 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350-356. Hawk, P. B., B. L. Oser and W. H. Summerson, 1947. Practical Physiological Chemistry. Blakiston Company, Philadelphia. Hernandez, H. H., and L. S. Bates, 1969. A modified method for rapid tryptophan analysis of maize. Centro Internacional De Mejoramiento De Maiz y Trigo, Mexico, Research Bulletin No. 13: 1-7. Jambunathan, R., 1969. Ph.D. Dissertation. Kansas
State University, Manhattan, Kansas. Isolation and characterization of egg yolk granule proteins. Johansen, P. G., R. D. Marshall and A. Neuberger, 1960. Carbohydrates in protein. The hexose, hexosamine, acetyl and amide-nitrogen content of hen's-egg albumin. Biochem. J. 77: 239-247. Joubert, F. J., and W. H. Cook, 1958. Preparation and characterization of phosvitin from hen egg yolk. Can. J. Biochem. Physiol. 36: 399-408. Lewis, J. C , N . S. Snell, D. J. Hirschmann and H. Fraenkel-Conrat, 1950. Amino acid composition of egg proteins. J. Biol. Chem. 186: 23-35. Mecham, D. K., and H. S. Olcott, 1949. Phosvitin, the principal phosphoprotein of egg yolk. J. Am. Chem. Soc. 71:3670-3679. Niederl, J. B., and V. Niederl, 1946. Organic Quantitative Microanalysis, 2nd edition. John Wiley and Sons, New York. Rimington, C , 1940. Seromucoid and the bound carbohydrate of the serum proteins. Biochem. J. 34:931-940. Spies, J. R., and D. C. Chambers, 1949. Chemical determination of tryptophan in proteins. Anal. Chem. 21:1249-1266.
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43 15 262 41 12 18 25 0 12 3 6 12 3 6 49 32 58 37 1 337 15.50 10.41
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Amide ammonia Arginine Tryptophan P03 Nitrogen % Phosphorus % Hexosamine Hexose
Allerton and Perlmann
HIGH DENSITY FRACTION or EGG YOLK Spiro, R. G., 1962. Studies on the monosaccharide sequence of the serum glycoprotein fetuin. J. Biol. Chem. 237:646-652. Spiro, R. G., 1966. Analysis of sugars found in glycoproteins. In: Methods in Enzymology, vol. VIII. Eds. S. P. Colowick and N. O. Kaplan. Academic Press, N. Y. pp. 3-26. Taborsky, G., and C. C. Allende, 1962. A rearrangement in the structure of phosvitin. Biochemistry,
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1:406-411. Wilcox, P. E., 1967. Determination of amide residues by chemical methods. In: Methods in Enzymology, vol. X I . Eds. S. P . Colowick and N . O. Kaplan. Academic Press, N. Y. pp. 63-76. Winzler, R. J., 1955. Determination of serum glycoproteins. In: Methods of Biochemical Analysis, vol. II. Ed. D. Glick. Interscience Publishers, Inc., N. Y. pp. 279-311.
G. SCHNELL, K. R. NATH, J. M. DARFLER, D. V. VADEHRA AND R. C. BAKER Department of PoiAtry Science, Cornell University, Ithaca, New York 14850 (Received for publication October 23, 1972) ABSTRACT Frankfurters were made from laboratory prepared mechanically deboned poultry meat (MDPM) using backs and necks. They were compared with frankfurters made from commercially made puree (mechanically deboned meat) and hand deboned carcass meat. Variables studied were (1) the effect of grind and screen sizes used in the production of the MDPM; (2) the effect of the percent of added skin in the formula; and (3) the effect of selected additives. Parameters used for evaluation included organoleptic scores, emulsion stability and shear press values. The smaller the screen size, the more tender, better flavored and more acceptable were the end products. Hand deboned carcass produced the firmest frankfurter. Flavor and acceptability of frankfurters from both hand deboned carcasses and commercial puree were not significantly different as compared to the product made with MDPM from the smallest screen (0.05 cm.). Increasing the skin added to the formula increased the fat content, increased organoleptic tenderness and viscosity and decreased emulsion stability. The addition of 3 % sodium caseinate, 3 % acid whey or 0.5% Kena had very little effect on the organoleptic qualities of the frankfurters. Kena markedly decreased emulsion viscosity, whereas, sodium caseinate increased it. POULTRY SCIENCE 52: 1363-1369,1973
INTRODUCTION
O
NE of the most economical sources of animal protein is mechanically deboned poultry meat (MDPM). Since the advent of several types of deboning machines, the production of this type of material from backs and necks of fryers, turkey racks and even spent layer carcasses, has increased enormously. With the availability of the product, information is needed on its composition and use in further processing. A considerable amount of basic infor-
mation can be found in the literature on mechanically deboned poultry meat. Vadehra and Baker (1970) and Grunden et al. (1972) have examined its chemical and physical characteristics. Ostovar et al. (1971) examined its microbiological quality. Essary and Ritchey (1968) have determined the amino acid composition and Maxon and Marion (1970), the lipid content of the product. Effect of variables encountered in the production of the material have been studied, such as the influence of the skin
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Physical and Functional Properties of Mechanically Deboned Poultry Meat as Used in the Manufacture of Frankfurters