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A Method for Rapid Determination of Lactose1,2
A Method for Rapid Determination of Lactose ~,2 F. F. FEITOSA TELES, C H E R Y L K. Y O U N G , A N D J. W. STULL Nutrition and Food Science Department The University of Arizona Tucson 85721 ABSTRACT
p h o s p h a t e dehydrogenase and triphosphopyridine nucleotide (TPM or NADP) serves as an indicator, and the TPNH formed is measured by the increase of absorption at 340 nm (15). Fluorimetry also is used following enzymatic hydrolysis of the disaccharide (9). These methods have characteristics requiring extensive time, sophisticated equipment, expensive or corrosive reagents, and considerable glassware or appreciable analytic skill. Our technique was developed in consideration of many of the disadvantages described above.
A simple, precise, and accurate colorimetric method for estimation of lactose in fluid milk and whey is described. The color development is based on the combined action of phenol, sodium hydroxide, picric acid, and sodium bisulfite with lactose. The method can be completed in less than 30 rain. There was little difference between results by the method and the A.O.A.C. procedure. This method has advantages over other methods which r e q u i r e extensive time, sophisticated equipment, expensive or corrosive reagents, considerable glassware, or appreciable analytic skill.
M A T E R I A L S A N D METHODS
INTRODUCTION
Most of the various methods for analysis of lactose in fluid products such as milk and whey are based on the reduction of cupric sulfate to water insoluble cuprous oxide. The latter then is determined gravimetrically by direct weighing (4), by titration with sodium thiosulfate (1, 2, 6, 7, 8, 10), or by electrolytic deposition from nitric acid solutions (11, 16). The cuprous oxide so determined is transferred to a lactose equivalent with the aid of the Hammond table for calculating lactose alone or in the presence of sucrose (4). Lactose also can be determined polarimetrically after deproteinization with mercuric iodide (4). Other methods are based on colorimetric reactions (5, 12, 13, 14, 18). In addition, enzymatic procedures based on the combined action of/3-galactosidase in the presence of hexokinase and adenosine triphosphate (ATP) are available. The oxidation o f the glueose-6-phosphate with glucose-6-
Samples were milk or whey, uniformly mixed at room temperature (ca. 20 C). Reagents were 4.5% barium hydroxide, kept in well-stoppered container, 5% zinc sulfate, and Teles' reagent (17) (Stock solutions in distilled water: 1% phenol (stable), 5% NaOH (stable), 1% picric acid (stable), and 1% sodium bisulfite, analytical grade, 2-day shelf life). For a working solution (2-day shelf life) of Teles' reagent mix in the following order: 1 vol phenol, 2 vol NaOH, 2 vol picric acid, and 1 vol sodium bisulfite solutions, mixing well after each addition. Keep the working solution in a dark container. Equipment consisted of centrifuge tubes, 16 x 100 mm or equivalent; blood sugar tubes, Folin, graduated at 12.5 and 25 ml; spectrophotometer equipped with 10 mm ID glass cuvettes; and a centrifuge, to operate at a minimum of 1000 rpm, at room temperature. PROCEDURE Dilution
Transfer 2.0 ml milk or whey to a 100-ml volumetric flask and dilute to volume with distilled water. Mix well.
Received August 28, 1977.
1Journal paper 2781 of the Arizona Agriculture Experiment Station. ZSupported in part by Grant AID/la-145 (Brazil
Colorimetry
Contract).
1978 J Dairy Sci 61:506--508
506
To a 2.5-mi aliquot of the diluted sample in a centrifuge tube, add .2 ml 5% zinc sulfate
TECHNICAL NOTE
507
TABLE 1. Comparison of lactose in milk and whey by the AOAC method and by Teles' reagent. Milk lactose (%)
Whey lactose (%)
Replication no.
AOAC
Teles
AOAC
Teles
1 2 3 4 5 6
5.29 5.27 5.24 5.47 5.33 5.27
5.34 5.26 5.34 5.34 5.34 5.34
4.35 4.45 4.48 4.45 4.52 4.48
4.34 4.34 4.48 4.48 4.34 4.42
SD
5.31 a .083
5.33 a .032
4.46 b .058
4.40 b .069
t = .013 (P<.05)
t = .021 (P<.05)
a'bAverage values with the same superscripts are not statistically different (P<.05).
s o l u t i o n f o l l o w e d by .2 ml 4.5% barium h y d r o x i d e . Centrifuge 15 to 30 s at 2 5 0 0 rpm, or 1 min at 1000 rpm. Transfer 1.0 ml of the clear supernatant to a Folin blood sugar tube, add 2.5 ml of Teles' reagent, and stopper tightly with a dry rubber stopper. Immerse the b o t t o m of the tube, 4 to 6 cm, in a violently boiling water bath for exactly 6 min. Cool i m m e d i a t e l y in cold tap water. Bring the v o l u m e to 12.5 or 25 ml with distilled water depending on the lactose c o n t e n t of the sample. Invert the tubes six times to mix. R e a d the absorbance at 520 n m against a similarly treated reagent blank in which 2.5 ml water substitutes for the sample. C o m p a r e the results with a standard solution of dry lactose in distilled water (1 m g / m l ) or dissolve 1.052 g o f l a c t o s e - H 2 0 to 1000 ml with distilled water (shelf life one w e e k if u n d e r refrigeration), and proceed with the c o l o r i m e t r y as for the sample. Calculations
Lactose (mg/ml) = absorbance of sample x 50/absorbance of standard or use a standard curve. RESULTS A N D DISCUSSION
The results were statistically comparable (P<.05) w i t h the "Official Final A c t i o n Polarimetric M e t h o d " (4) for h o m o g e n i z e d w h o l e milk and cottage cheese w h e y f r o m c o m m e r c i a l plants in Arizona (Table 1). The averages were c o m p a r e d by the t test (3). The values represent a typical replicated analysis of single samples of milk and whey. The usable Journal of Dairy Science Vol. 61, No. 4, 1978
range of d e t e c t i o n of the m e t h o d is .2 to 2 mg of lactose. Sucrose, ethanol, or lead acetate do n o t i n t e r f e r e with d e t e r m i n a t i o n , but reducing substances do. For example, the m e t h o d is n o t applicable for lactose d e t e r m i n a t i o n in frozen dairy desserts or o t h e r formulations containing corn syrup products. The developed color is stable, or fades s l o w l y a n d p r o p o r t i o n a l l y . Accurate absorbancies can be read up to 12 h after color development. The entire p r o c e d u r e can be c o m p l e t e d in less than 30 min, and standard deviations are usually .08 or less. In a d d i t i o n to being relatively inexpensive, the reagents can be prepared with distilled water with no need for deionization or o t h e r water retreatment. The detergent action of the Teles' reagent allows effective cleaning of the Folin tubes by simple rinsing f o u r times with distilled water. The m e t h o d will allow use of a flow-through cuvette.
REFERENCES
1 Association of Official Agricultural Chemists (A.O.A.C.). 1928. Sugar and sugar products. Changes in the official and tentative methods of analysis made at the forty-fourth annual convention. JAOC 12:38. 2 Association of Official Agricultural Chemists (A.O.A.C.). 1934. Changes in the official and tentative methods of analysis made at the fiftieth annual convention. JAOC 18: 83. 3 Bauer, E. L. 1971. A statistical manual for chemists. 2rid ed. Academic Press, New York. 4 Association of Official Analytical Chemists. 1975. Official methods of analysis. W. Horwitz, ed. 12th
508
TELES El" AL.
ed. Association o f Official Analytical Chemists, Washington, DC. 5 Benham, G. H. and J. E. Despaul. 1948. Glucose-A direct colorimetric m e t h o d for determining carbohydrates. Anal. Chem. 20:933. 6 Bright, H. A. 1937. Use o f arsenious oxide in the standardization o f solutions of potassium permanganate. J. Res. Nat. Bur. Stds. 19:691. 7 Foote, H. W. and J. E. Vance. 1935. A modified iodimetric m e t h o d o f determining copper. J. Amer. Chem. Soc. 57:845. 8 Fowler, R. M. and H. A. Bright. 1935. Standardization of permanganate solutions with sodium oxalate. J. Res. Nat. Bur. Stds. 15:493. 9 Guilbaut, G. G. 1976. H a n d b o o k o f e n z y m a t i c m e t h o d s o f analysis. Marcel Dekker, Inc., New York. 10 H a m m o c k , E. W. a n d E. H. Swift. 1949. Iodometric determination of copper. Effect of thiocyanate on end point and use of sulfate-hydrogen sulfate buffers. Anal. Chem. 21:975. 11 Jackson, R. F. 1940. Report on sugars and sugar products. JAOAC. 23: 558. 12 Marier, J. R. and M. Boulet. 1959. Direct analysis
Journal of Dairy Science Vol. 61, No. 4, 1978
13
14
15
16
17
18
of lactose in milk and serum. J. Dairy Sci. 42:1390. Nickerson, T. A., I. F. Vujicic, and A. V. Lin. 1976. Colorimetric estimation of lactose and its hydrolytic products. J. Dairy Sci. Potter, F. E. 1950. A colorimetric m e t h o d for the quantitative determination of the degree of lactose hydrolysis. J. Dairy Sci. 33:803. Reikhel, F . J. 1965. Lactose. Methods of enzymatic analysis. H. U. Bergrneyer, ed. 2rid Rev. Verlag Chemie, Academic Press, New York. Scherrer, J. A., R. K. Bell, and W. D. Mogerman. 1939. Electroanalytical determination of copper and lead in nitric acid solutions containing small a m o u n t s of hydrochloric acid. J. Res. Nat. Bur. Stds. 22:697. Silveira, A. J., F. F. Teles and J. W. Stull. 1978. A rapid technique for total nonstructural carbohydrate determination. J. Agr. and Food Chem. (In press) Tauber, H. and I. S. KleJner. 1932. A m e t h o d for the determination of monosacchar~des and its application to blood analysis. J. Biol. Chem. 99: 249.
p h o s p h a t e dehydrogenase and triphosphopyridine nucleotide (TPM or NADP) serves as an indicator, and the TPNH formed is measured by the increase of absorption at 340 nm (15). Fluorimetry also is used following enzymatic hydrolysis of the disaccharide (9). These methods have characteristics requiring extensive time, sophisticated equipment, expensive or corrosive reagents, and considerable glassware or appreciable analytic skill. Our technique was developed in consideration of many of the disadvantages described above.
A simple, precise, and accurate colorimetric method for estimation of lactose in fluid milk and whey is described. The color development is based on the combined action of phenol, sodium hydroxide, picric acid, and sodium bisulfite with lactose. The method can be completed in less than 30 rain. There was little difference between results by the method and the A.O.A.C. procedure. This method has advantages over other methods which r e q u i r e extensive time, sophisticated equipment, expensive or corrosive reagents, considerable glassware, or appreciable analytic skill.
M A T E R I A L S A N D METHODS
INTRODUCTION
Most of the various methods for analysis of lactose in fluid products such as milk and whey are based on the reduction of cupric sulfate to water insoluble cuprous oxide. The latter then is determined gravimetrically by direct weighing (4), by titration with sodium thiosulfate (1, 2, 6, 7, 8, 10), or by electrolytic deposition from nitric acid solutions (11, 16). The cuprous oxide so determined is transferred to a lactose equivalent with the aid of the Hammond table for calculating lactose alone or in the presence of sucrose (4). Lactose also can be determined polarimetrically after deproteinization with mercuric iodide (4). Other methods are based on colorimetric reactions (5, 12, 13, 14, 18). In addition, enzymatic procedures based on the combined action of/3-galactosidase in the presence of hexokinase and adenosine triphosphate (ATP) are available. The oxidation o f the glueose-6-phosphate with glucose-6-
Samples were milk or whey, uniformly mixed at room temperature (ca. 20 C). Reagents were 4.5% barium hydroxide, kept in well-stoppered container, 5% zinc sulfate, and Teles' reagent (17) (Stock solutions in distilled water: 1% phenol (stable), 5% NaOH (stable), 1% picric acid (stable), and 1% sodium bisulfite, analytical grade, 2-day shelf life). For a working solution (2-day shelf life) of Teles' reagent mix in the following order: 1 vol phenol, 2 vol NaOH, 2 vol picric acid, and 1 vol sodium bisulfite solutions, mixing well after each addition. Keep the working solution in a dark container. Equipment consisted of centrifuge tubes, 16 x 100 mm or equivalent; blood sugar tubes, Folin, graduated at 12.5 and 25 ml; spectrophotometer equipped with 10 mm ID glass cuvettes; and a centrifuge, to operate at a minimum of 1000 rpm, at room temperature. PROCEDURE Dilution
Transfer 2.0 ml milk or whey to a 100-ml volumetric flask and dilute to volume with distilled water. Mix well.
Received August 28, 1977.
1Journal paper 2781 of the Arizona Agriculture Experiment Station. ZSupported in part by Grant AID/la-145 (Brazil
Colorimetry
Contract).
1978 J Dairy Sci 61:506--508
506
To a 2.5-mi aliquot of the diluted sample in a centrifuge tube, add .2 ml 5% zinc sulfate
TECHNICAL NOTE
507
TABLE 1. Comparison of lactose in milk and whey by the AOAC method and by Teles' reagent. Milk lactose (%)
Whey lactose (%)
Replication no.
AOAC
Teles
AOAC
Teles
1 2 3 4 5 6
5.29 5.27 5.24 5.47 5.33 5.27
5.34 5.26 5.34 5.34 5.34 5.34
4.35 4.45 4.48 4.45 4.52 4.48
4.34 4.34 4.48 4.48 4.34 4.42
SD
5.31 a .083
5.33 a .032
4.46 b .058
4.40 b .069
t = .013 (P<.05)
t = .021 (P<.05)
a'bAverage values with the same superscripts are not statistically different (P<.05).
s o l u t i o n f o l l o w e d by .2 ml 4.5% barium h y d r o x i d e . Centrifuge 15 to 30 s at 2 5 0 0 rpm, or 1 min at 1000 rpm. Transfer 1.0 ml of the clear supernatant to a Folin blood sugar tube, add 2.5 ml of Teles' reagent, and stopper tightly with a dry rubber stopper. Immerse the b o t t o m of the tube, 4 to 6 cm, in a violently boiling water bath for exactly 6 min. Cool i m m e d i a t e l y in cold tap water. Bring the v o l u m e to 12.5 or 25 ml with distilled water depending on the lactose c o n t e n t of the sample. Invert the tubes six times to mix. R e a d the absorbance at 520 n m against a similarly treated reagent blank in which 2.5 ml water substitutes for the sample. C o m p a r e the results with a standard solution of dry lactose in distilled water (1 m g / m l ) or dissolve 1.052 g o f l a c t o s e - H 2 0 to 1000 ml with distilled water (shelf life one w e e k if u n d e r refrigeration), and proceed with the c o l o r i m e t r y as for the sample. Calculations
Lactose (mg/ml) = absorbance of sample x 50/absorbance of standard or use a standard curve. RESULTS A N D DISCUSSION
The results were statistically comparable (P<.05) w i t h the "Official Final A c t i o n Polarimetric M e t h o d " (4) for h o m o g e n i z e d w h o l e milk and cottage cheese w h e y f r o m c o m m e r c i a l plants in Arizona (Table 1). The averages were c o m p a r e d by the t test (3). The values represent a typical replicated analysis of single samples of milk and whey. The usable Journal of Dairy Science Vol. 61, No. 4, 1978
range of d e t e c t i o n of the m e t h o d is .2 to 2 mg of lactose. Sucrose, ethanol, or lead acetate do n o t i n t e r f e r e with d e t e r m i n a t i o n , but reducing substances do. For example, the m e t h o d is n o t applicable for lactose d e t e r m i n a t i o n in frozen dairy desserts or o t h e r formulations containing corn syrup products. The developed color is stable, or fades s l o w l y a n d p r o p o r t i o n a l l y . Accurate absorbancies can be read up to 12 h after color development. The entire p r o c e d u r e can be c o m p l e t e d in less than 30 min, and standard deviations are usually .08 or less. In a d d i t i o n to being relatively inexpensive, the reagents can be prepared with distilled water with no need for deionization or o t h e r water retreatment. The detergent action of the Teles' reagent allows effective cleaning of the Folin tubes by simple rinsing f o u r times with distilled water. The m e t h o d will allow use of a flow-through cuvette.
REFERENCES
1 Association of Official Agricultural Chemists (A.O.A.C.). 1928. Sugar and sugar products. Changes in the official and tentative methods of analysis made at the forty-fourth annual convention. JAOC 12:38. 2 Association of Official Agricultural Chemists (A.O.A.C.). 1934. Changes in the official and tentative methods of analysis made at the fiftieth annual convention. JAOC 18: 83. 3 Bauer, E. L. 1971. A statistical manual for chemists. 2rid ed. Academic Press, New York. 4 Association of Official Analytical Chemists. 1975. Official methods of analysis. W. Horwitz, ed. 12th
508
TELES El" AL.
ed. Association o f Official Analytical Chemists, Washington, DC. 5 Benham, G. H. and J. E. Despaul. 1948. Glucose-A direct colorimetric m e t h o d for determining carbohydrates. Anal. Chem. 20:933. 6 Bright, H. A. 1937. Use o f arsenious oxide in the standardization o f solutions of potassium permanganate. J. Res. Nat. Bur. Stds. 19:691. 7 Foote, H. W. and J. E. Vance. 1935. A modified iodimetric m e t h o d o f determining copper. J. Amer. Chem. Soc. 57:845. 8 Fowler, R. M. and H. A. Bright. 1935. Standardization of permanganate solutions with sodium oxalate. J. Res. Nat. Bur. Stds. 15:493. 9 Guilbaut, G. G. 1976. H a n d b o o k o f e n z y m a t i c m e t h o d s o f analysis. Marcel Dekker, Inc., New York. 10 H a m m o c k , E. W. a n d E. H. Swift. 1949. Iodometric determination of copper. Effect of thiocyanate on end point and use of sulfate-hydrogen sulfate buffers. Anal. Chem. 21:975. 11 Jackson, R. F. 1940. Report on sugars and sugar products. JAOAC. 23: 558. 12 Marier, J. R. and M. Boulet. 1959. Direct analysis
Journal of Dairy Science Vol. 61, No. 4, 1978
13
14
15
16
17
18
of lactose in milk and serum. J. Dairy Sci. 42:1390. Nickerson, T. A., I. F. Vujicic, and A. V. Lin. 1976. Colorimetric estimation of lactose and its hydrolytic products. J. Dairy Sci. Potter, F. E. 1950. A colorimetric m e t h o d for the quantitative determination of the degree of lactose hydrolysis. J. Dairy Sci. 33:803. Reikhel, F . J. 1965. Lactose. Methods of enzymatic analysis. H. U. Bergrneyer, ed. 2rid Rev. Verlag Chemie, Academic Press, New York. Scherrer, J. A., R. K. Bell, and W. D. Mogerman. 1939. Electroanalytical determination of copper and lead in nitric acid solutions containing small a m o u n t s of hydrochloric acid. J. Res. Nat. Bur. Stds. 22:697. Silveira, A. J., F. F. Teles and J. W. Stull. 1978. A rapid technique for total nonstructural carbohydrate determination. J. Agr. and Food Chem. (In press) Tauber, H. and I. S. KleJner. 1932. A m e t h o d for the determination of monosacchar~des and its application to blood analysis. J. Biol. Chem. 99: 249.