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Determination of Phosphorus in Milk Lipids
552
JOURNAL
OF
of u n f r a e t i o n a t e d milk proteins suitable for eleetrophoretie analysis. No changes in umbilities or relative concentrations of electrophoretic components at p H 8.7 and 0.1 ~, could be attributed to this m a n n e r of sample preparation. A question is raised as to the identity of a small peak observed in the electrophoretie patterns of homogenized whole milk which moved with a slightly higher mobility than a-casein. J. TOBIAS~ R. M. SS~F ~ Department of Food Technology, University of Illinois, Urbana. ~Present address: Foremost Dairies, Inc., San Francisco, California.
DETERMINATION
DAIRY
SCIENCE
REFERENCES (1) REINEI~, M., AND FENICI~EL, R. L. Dialysis of Protein Solutions for Electrophoresis. Sc/ence, 108: 164. 1948. (2) S~I~E, C. M., AND PATTON, S. Preparation of Milk Fat. I I L Properties of Butteroils Prepared by the Use of Surface Active Agents. J. Dairy Sol., 36: 516. 1953. (3) TISELIUS, A. A New Apparatus for Electrophoretie Analysis of Colloidal Mixtures. Trans. Faraday Soc., 33: 524. 1937. (4) TOBIAS, J., WHITNEY, R. M., AND TRACY, P. H. Eleetrophoresis of Milk Proteins. I. Preparation of Samples of Skimmilk for Electrophoretic Studies. J. Dairy Sci., 35: 1026.
1952. (5) WAah~E~, R. C. Separation of a- and fl-Casein. J. Am. Chem. Soc., 65: 1725. 1944.
OF PHOSPHORUS
Investigation of nfilk lipids often requires determination of the phosphorus in residues f r o m extracts such as those obtained by the Moj o n n i e r procedure or f r o m chromatographic eluates. Colorimetric procedures generally are pref e r r e d to titrimetric or g r a v i m e t r i c methods, as being faster and better adapted to analyzing small amounts of material. I n our laboratory we have used a modification of the method of H a r r i s and P o p a t (2) f o r determining phosphorus in cottonseed lipids. The method employs perchloric acid for sample digestion and p-methylaminophenol sulfate (elon) as a reducing agent for color development. Both of these steps are p e r f o r m e d in the same test tube, to avoid t r a n s f e r losses. I n developing their method, H a r r i s and P o p a t investigated the effect of a number of variables on the digestion of lipid samples and on color development with elon. They stated that the oxidation and digestion procedure can be completed within 5 rain. However, they did not rep o r t any data showing the effect of varying the time of digestion, although they emphasized that the final clarification step in the digestion procedure must be complete. Furthermore, no data were presented on the recovery of organic phosphorus. W h e n various individuals in our labo r a t o r y used H a r r i s and P o p a t ' s original procedure to determine phosphorus in ndlk lipids and in a variety of phospholipid samples, the results often were erratic. Subsequent investigation has shown that results are more consistent and accurate when a longer digestion period is employed. The modified procedure is described below. Samples are p r e p a r e d by p i p e t t i n g volumes of lipid solutions of known concentrations into 35-ml. P y r e x test tubes calibrated at 20.0 nil. and containing two glass beads. Solvents are evaporated with a water bath at 70 ° C. and 1.0 ml. of 72% perchlorie acid is added to each tube. To avoid explosion hazard, the lipid weight should not exceed 75 rag. f o r this amount of
IN MILK
LIPIDS
perchloric acid. Oxidation is initiated by adding two drops of nitric acid immediately before gently heating esch tube (held in a test-tube holder) by hand over a micro-burner. The reaction mixture should be agitated continuously until f o a m i n g subsides and it boils evenly. Under these conditions, the rate of oxidation can be controlled readily. Then tile mixture is heated more strongly until it becomes clear and essenTABLE 1 Effect of digestion time on determination of lipid phosphorus Sample Milk Lipid A
Milk Lipid E
Diges- AbsorWeight riCh time bance
Phosphorus
(rag.) 10.4 10.4 10.4 10.4 10.4 10.4 10.4 4.16 6.24 8.32 2.64 2.64 2.64 2.64 2.64 2.64 2.64
0.235 0.258 0.267 0.267 0.275 0.271 0.267 0.107 0.162 0.221 0.370 0.505 0.550 0.555 0.559 0.553 0.558
(%) 0.357 0.392 0.406 0.406 0.418 0.412 0.406 0.406 0.410 0.410 2.22 3.03 3.30 3.33 3.35 3.32 3.35
0.407 0.224 0.266 0.291 0.242 0.482 0.244 0.494 0.488
3.08 3.37 4.00 4.38 4.43 4.40 4.46 4.52 4.46
(rain.) 0 6 9 12 15 18 20 20 '20 20 0 6 9 12 15 18 21
Dimyristoyl
lecithin "~
2.10 1.05 1.05 1.05 0.86 1.73 0.86 1.73 1.73
0 1 2 3 9 9 12 15 21
'~LaMotte Chemical Products Co., Chestertown, Maryland.
TECHNICAL NOTES
tially colorless, and white fumes of perchloric acid appear. After the above oxidation, two nmre drops of nitric acid are added to each tube. Then they are placed in a micro-Kjeldahl digestion rack and the samples digested for 20 rain. with sufficient heat to boil them gently without appreciable loss of perchloric acid. After cooling and diluting with about 15 ml. of water, each sample is mixed with 1.0 nil. of 5% ammonimn nmlybdate solution. Then 2.0 ml. of elon solution (0.5 g. p-methylaminophenol sulfate, 12.5 g. sodimn bisulfite, and 2.4 g. sodium sulfite made up to 100 mh) is added and the mixture diluted to 20.0 ml. The absorbance (optical density) at 820 Int~ of each sample is read against a blank 15 rain. after the elon addition, and the percentage of phosphorus is obtained from a standard curve in the usual way. Table ] illustrates the effect of ti~m of digestion on lipid phosphorus recovery. The samples required at least 12 min. of digestion after the initial major oxidation steps. On the basis of these results, and subsequent experience with samples ranging up to 60 rag., the standard digestion period of 20 rain. was adopted. The results for the pure synthetic L-a-(dimyristoyl)-
A CURD FIRMNESS
553
lecithin check with the theoretical value of 4.46%. This sample contained 4.40 and 4.41% phosphorus (gravimetric method), according to the analysis submitted by the supplier. Note: Because lipids are comparatively difficult to digest, perchloric acid often has been used for this purpose, despite the necessity of taking proper precautions to avoid explosive conditions (1, 2). I n the above procedure, the initial oxidation steps should be carried out behind a safety-glass shield in a fume hood and the analyst should wear protective rubber gloves and a face-shield. L. M. S~ITE R. R. LowRY E. L. JACK Department of Dairy Industry, University of Califol~nia, Davis REFERENCES (1) FISHEI% SCIE~NTIFIC' CO~[PANY. Manual of Lab-
oratory Safety. Bull. FS 201. Pittsburgh, Pa. 1956. (2) ttARIClS, W. D., AND POPAT, P. Determination of the Phosphorus Content of Lipids. J. Am. Oil Chemists' Soc., 31: ]24. 1954.
TEST FOR COTTAGE CHEESE 1
An objective test to measure the firmness of cottage cheese curd is needed for research and manufacturing control. The Lundstedt cheese curd meter (2), which requires about 400 g. of curd for testing, is especially useful as a control in the manufacturing process, but lacks sensitivity when applied to finished curd. The purpose of this paper is to describe a test for curd firmness that uses only a small amount of curd, is sensitive enough for use on finished curd, and is simple enough to use for control purposes during manufacturing. Equipment and method of performing the curd firmness test. The apparatus for measuring curd firmness is' shown in "Figure I. It is a modification of the Cherry-Burrell curd-tension meter (1). The slotted container holds the curd into which the wire cutter is forced by the driving mechanism. The resistance offered by the curd is registered in grams on the 500-g. dietetic scale. The curd container is a slotted cylinder made from 2-in. stainless steel piping which is closed at the bottom by soldering to it a circular, flat piece of stainless steel. A 5-lb. weight for pressing the curd into the cylindrical container, and the slotted container itself, are shown in Figure 2. The weight is solid stainless steel machined to fit snugly, yet to slide freely into the container when it is placed on top of the curd. z Approved for publication by the Director of the Wisconsin Agricultural Experiment Station.
The wire cutter is a U-shaped frame with a 3-in. length of 0.016-in. stainless steel wire stretched tautly from tip-to-tip of the U-frame. The base of the [J is joined by a vertical bolt to the driving mechanism. The base of the (J is extended to form an arm which moves smoothly and securely in a vertical guiding slot on the fixed frame of the driving mechanism; this guide prevents the wire cutter from twisting and keeps the wire from touching the slotted container when force is applied. The driving mechanism forces the wire cutter downward at a rate of 2 in. in 15 sec. The test is made by packing curd into the cylindrical slotted container with a spoon. It is packed firndy with the spoon, after each spoonful is added, to minimize pockets of air or liquid between curd particles. When the container is full, the 5-lb. weight is placed on the curd and balanced there, by hand if necessary, for approximately 10 sec., to pack the curd uniformly. The slots in the container must be closed during this pressing interval, to prevent the escape of curd; two fiat pieces of stainless steel held lightly against the slots serve this purpose. At the same time, free whey or cream may escape without loss of curd. The container of curd is then positioned on the dietetic scale under the wire cutter so that the cutter can be forced downward into the curd without touching the sides of the slotted container. Irregularities of consistency in the packed curd cause some ttuctuations of
JOURNAL
OF
of u n f r a e t i o n a t e d milk proteins suitable for eleetrophoretie analysis. No changes in umbilities or relative concentrations of electrophoretic components at p H 8.7 and 0.1 ~, could be attributed to this m a n n e r of sample preparation. A question is raised as to the identity of a small peak observed in the electrophoretie patterns of homogenized whole milk which moved with a slightly higher mobility than a-casein. J. TOBIAS~ R. M. SS~F ~ Department of Food Technology, University of Illinois, Urbana. ~Present address: Foremost Dairies, Inc., San Francisco, California.
DETERMINATION
DAIRY
SCIENCE
REFERENCES (1) REINEI~, M., AND FENICI~EL, R. L. Dialysis of Protein Solutions for Electrophoresis. Sc/ence, 108: 164. 1948. (2) S~I~E, C. M., AND PATTON, S. Preparation of Milk Fat. I I L Properties of Butteroils Prepared by the Use of Surface Active Agents. J. Dairy Sol., 36: 516. 1953. (3) TISELIUS, A. A New Apparatus for Electrophoretie Analysis of Colloidal Mixtures. Trans. Faraday Soc., 33: 524. 1937. (4) TOBIAS, J., WHITNEY, R. M., AND TRACY, P. H. Eleetrophoresis of Milk Proteins. I. Preparation of Samples of Skimmilk for Electrophoretic Studies. J. Dairy Sci., 35: 1026.
1952. (5) WAah~E~, R. C. Separation of a- and fl-Casein. J. Am. Chem. Soc., 65: 1725. 1944.
OF PHOSPHORUS
Investigation of nfilk lipids often requires determination of the phosphorus in residues f r o m extracts such as those obtained by the Moj o n n i e r procedure or f r o m chromatographic eluates. Colorimetric procedures generally are pref e r r e d to titrimetric or g r a v i m e t r i c methods, as being faster and better adapted to analyzing small amounts of material. I n our laboratory we have used a modification of the method of H a r r i s and P o p a t (2) f o r determining phosphorus in cottonseed lipids. The method employs perchloric acid for sample digestion and p-methylaminophenol sulfate (elon) as a reducing agent for color development. Both of these steps are p e r f o r m e d in the same test tube, to avoid t r a n s f e r losses. I n developing their method, H a r r i s and P o p a t investigated the effect of a number of variables on the digestion of lipid samples and on color development with elon. They stated that the oxidation and digestion procedure can be completed within 5 rain. However, they did not rep o r t any data showing the effect of varying the time of digestion, although they emphasized that the final clarification step in the digestion procedure must be complete. Furthermore, no data were presented on the recovery of organic phosphorus. W h e n various individuals in our labo r a t o r y used H a r r i s and P o p a t ' s original procedure to determine phosphorus in ndlk lipids and in a variety of phospholipid samples, the results often were erratic. Subsequent investigation has shown that results are more consistent and accurate when a longer digestion period is employed. The modified procedure is described below. Samples are p r e p a r e d by p i p e t t i n g volumes of lipid solutions of known concentrations into 35-ml. P y r e x test tubes calibrated at 20.0 nil. and containing two glass beads. Solvents are evaporated with a water bath at 70 ° C. and 1.0 ml. of 72% perchlorie acid is added to each tube. To avoid explosion hazard, the lipid weight should not exceed 75 rag. f o r this amount of
IN MILK
LIPIDS
perchloric acid. Oxidation is initiated by adding two drops of nitric acid immediately before gently heating esch tube (held in a test-tube holder) by hand over a micro-burner. The reaction mixture should be agitated continuously until f o a m i n g subsides and it boils evenly. Under these conditions, the rate of oxidation can be controlled readily. Then tile mixture is heated more strongly until it becomes clear and essenTABLE 1 Effect of digestion time on determination of lipid phosphorus Sample Milk Lipid A
Milk Lipid E
Diges- AbsorWeight riCh time bance
Phosphorus
(rag.) 10.4 10.4 10.4 10.4 10.4 10.4 10.4 4.16 6.24 8.32 2.64 2.64 2.64 2.64 2.64 2.64 2.64
0.235 0.258 0.267 0.267 0.275 0.271 0.267 0.107 0.162 0.221 0.370 0.505 0.550 0.555 0.559 0.553 0.558
(%) 0.357 0.392 0.406 0.406 0.418 0.412 0.406 0.406 0.410 0.410 2.22 3.03 3.30 3.33 3.35 3.32 3.35
0.407 0.224 0.266 0.291 0.242 0.482 0.244 0.494 0.488
3.08 3.37 4.00 4.38 4.43 4.40 4.46 4.52 4.46
(rain.) 0 6 9 12 15 18 20 20 '20 20 0 6 9 12 15 18 21
Dimyristoyl
lecithin "~
2.10 1.05 1.05 1.05 0.86 1.73 0.86 1.73 1.73
0 1 2 3 9 9 12 15 21
'~LaMotte Chemical Products Co., Chestertown, Maryland.
TECHNICAL NOTES
tially colorless, and white fumes of perchloric acid appear. After the above oxidation, two nmre drops of nitric acid are added to each tube. Then they are placed in a micro-Kjeldahl digestion rack and the samples digested for 20 rain. with sufficient heat to boil them gently without appreciable loss of perchloric acid. After cooling and diluting with about 15 ml. of water, each sample is mixed with 1.0 nil. of 5% ammonimn nmlybdate solution. Then 2.0 ml. of elon solution (0.5 g. p-methylaminophenol sulfate, 12.5 g. sodimn bisulfite, and 2.4 g. sodium sulfite made up to 100 mh) is added and the mixture diluted to 20.0 ml. The absorbance (optical density) at 820 Int~ of each sample is read against a blank 15 rain. after the elon addition, and the percentage of phosphorus is obtained from a standard curve in the usual way. Table ] illustrates the effect of ti~m of digestion on lipid phosphorus recovery. The samples required at least 12 min. of digestion after the initial major oxidation steps. On the basis of these results, and subsequent experience with samples ranging up to 60 rag., the standard digestion period of 20 rain. was adopted. The results for the pure synthetic L-a-(dimyristoyl)-
A CURD FIRMNESS
553
lecithin check with the theoretical value of 4.46%. This sample contained 4.40 and 4.41% phosphorus (gravimetric method), according to the analysis submitted by the supplier. Note: Because lipids are comparatively difficult to digest, perchloric acid often has been used for this purpose, despite the necessity of taking proper precautions to avoid explosive conditions (1, 2). I n the above procedure, the initial oxidation steps should be carried out behind a safety-glass shield in a fume hood and the analyst should wear protective rubber gloves and a face-shield. L. M. S~ITE R. R. LowRY E. L. JACK Department of Dairy Industry, University of Califol~nia, Davis REFERENCES (1) FISHEI% SCIE~NTIFIC' CO~[PANY. Manual of Lab-
oratory Safety. Bull. FS 201. Pittsburgh, Pa. 1956. (2) ttARIClS, W. D., AND POPAT, P. Determination of the Phosphorus Content of Lipids. J. Am. Oil Chemists' Soc., 31: ]24. 1954.
TEST FOR COTTAGE CHEESE 1
An objective test to measure the firmness of cottage cheese curd is needed for research and manufacturing control. The Lundstedt cheese curd meter (2), which requires about 400 g. of curd for testing, is especially useful as a control in the manufacturing process, but lacks sensitivity when applied to finished curd. The purpose of this paper is to describe a test for curd firmness that uses only a small amount of curd, is sensitive enough for use on finished curd, and is simple enough to use for control purposes during manufacturing. Equipment and method of performing the curd firmness test. The apparatus for measuring curd firmness is' shown in "Figure I. It is a modification of the Cherry-Burrell curd-tension meter (1). The slotted container holds the curd into which the wire cutter is forced by the driving mechanism. The resistance offered by the curd is registered in grams on the 500-g. dietetic scale. The curd container is a slotted cylinder made from 2-in. stainless steel piping which is closed at the bottom by soldering to it a circular, flat piece of stainless steel. A 5-lb. weight for pressing the curd into the cylindrical container, and the slotted container itself, are shown in Figure 2. The weight is solid stainless steel machined to fit snugly, yet to slide freely into the container when it is placed on top of the curd. z Approved for publication by the Director of the Wisconsin Agricultural Experiment Station.
The wire cutter is a U-shaped frame with a 3-in. length of 0.016-in. stainless steel wire stretched tautly from tip-to-tip of the U-frame. The base of the [J is joined by a vertical bolt to the driving mechanism. The base of the (J is extended to form an arm which moves smoothly and securely in a vertical guiding slot on the fixed frame of the driving mechanism; this guide prevents the wire cutter from twisting and keeps the wire from touching the slotted container when force is applied. The driving mechanism forces the wire cutter downward at a rate of 2 in. in 15 sec. The test is made by packing curd into the cylindrical slotted container with a spoon. It is packed firndy with the spoon, after each spoonful is added, to minimize pockets of air or liquid between curd particles. When the container is full, the 5-lb. weight is placed on the curd and balanced there, by hand if necessary, for approximately 10 sec., to pack the curd uniformly. The slots in the container must be closed during this pressing interval, to prevent the escape of curd; two fiat pieces of stainless steel held lightly against the slots serve this purpose. At the same time, free whey or cream may escape without loss of curd. The container of curd is then positioned on the dietetic scale under the wire cutter so that the cutter can be forced downward into the curd without touching the sides of the slotted container. Irregularities of consistency in the packed curd cause some ttuctuations of