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Spectrophotometric Method for Determination of Heat-Activated Sulfhydryl Groups of Skimmilk1
TECHNICAL
NOTES
Spectrophotometric Method for Determination of Heat-Activated Sulfhydryl Groups of Skimmilk' Abstract
By suitable modification, adaptation was made of the 5,5'-dithiobis (2-nitrobenzoic acid) reagent method for the spectrophotometric determination of activated sulfhydryl ( - - S H ) groups of heated skimmilk. The modifications consisted of using a nitrogen-purged system, controlling pH, adding ammonium sulfate, filtering, and determining absorbance of the filtrate at 412 mlz. A standard cysteine-HC1 curve was prepared in a similar manner. The modified procedure yielded results consistent with other cmTently used methods. The activation of - - S H groups in heated skimmilk, as determined by the modified procedure, followed the kinetics of a first order reaction with reaction rate constants (kn, l._l) of 0.078, 0.142, 0.384, and 0.805 for 75, 80, 85, and 90 C, respectively.
8.0, and 4 ml of distilled water were added. The lots were agitated and two grains of ammonium sulfate added. The samples were again shaken vigorously and the contents filtered 1"2 I'(~ -.i
E 0"8 _¢2
'~ 0'6 Q
•
o 0.4 ^
'0454
0",
Many of the current procedures used for the determination of heat-activated sulfhydryl ( - - S H ) groups of milk are lengthy, complicated, or have only limited application to milk systems. Several investigators (4, 5) have used a sulfhydryl-disulfide interchange reagent, 5,5'dithiobis (2-nitrobenzoic acid), DTNB, for the estimation of unmasked - - S H groups in nonmilk systems. Lyster (9) applied the DTNB reagent method to milk and determined the degree of yellow color development in the heated samples by visual comparision with specially prepared standards of known free - - S H concentration by the use of a Lovibond Comparator and reflected
f .
0
.
2
.
.
4
|
i
i
I
6 8 Cysteine.HCI
I
I
_
I0
xloTM
Fro. 1. Statistically calculated eysteine-HCl standard curve by the DTNB procedure. s 7 x~ 6
light. This p a p e r reports on modifications and improvements in the DTNB procedure which permit quantitative determination of - - S H values of heated milks by direct spectrophotometric means. Five-milliliter quantities of raw skimmilk ( p H 6.7) contained in screw-cap test tubes were purged with nitrogen gas for 10 sec and then tightly stoppered. One lot served as the unheated control and the remaining lots were heated in a thermostatically controlled water bath, cooled immediately, and treated with 0.1 ml of" a DTNB solution [39.37 mg of 5,5"dithiobis (2-nitrobenzoic acid) in 10 ml of 0.1 phosphate buffer of p i t 7.0]. The yellow color was developed for 3 rain at room temperature, then 1 ml of 0.1 ~ phosphate buffer, p H Article 5:67. Department of Dairy Technology, The Ohio State University. This investigation was supported by Public Health Services Research Grant EF-00]80 from the Division of Environmental Engineering and Food Protection.
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.× s
~ 2i
I
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,~
|
|
0
2
t2
io
i,
ie
~4
I
i
i
!
i
22
26
TIME IN MINUTES AT
6
I0
14
18
75°C
AT
80" G
'AT
85°G
AT
90'PC
~z 6b i0
3
FIG. 2. Rate of free --SH group formation in skimmilk following heat treatment. 217
218
JOUI%NAL O F D A I R Y S C I E N C E
through Whatman 42 filter paper. The absorbance of the filtrate was read at a wavelength of 412 mtt in a Hitachi Perkin-Elmer Model 139 Spectrophotometer with a digital readout attachment. Preliminary studies had indicated that maximum absorbance of the filtrate was at 412 mt~. The blank tube contained all the reagents, excepting that 5 ml of 0.1 M phosphate buffer, p H 6.7, was substituted for the skimmilk. For the cysteine-HC1 standard curve, dilutions were in p H 6.7 phosphate buffer; further treatments, including the addition of ammonium sulfate, followed the standard procedure. By application of statistics (1), the plot of absorbance at 412 m/x versus eysteine-HC1 in moles (Fig. 1) yielded the best fit straight line with a calculated slope of: :Y =
(0.9488) (10~)Z -- 0.0434.
versus t were prepared from the data, using the mathematical treatment suggested by Daniels and Alberty (3), a straight line was obtained at each temperature and several holding times, indicating that the unmasking of the - - S H groups in skimmilk upon heat treatment followed the kinetics of a first-order reaction. The first-order reaction rate constants (km;~-l) for 75, 80, 85, and 90 C were 0.078, 0.142, 0.384, and 0.805, respectively. The reaction rate constant at 75 C approximated the reported (8) value of 0.0875/nfin for the activation of sulfhydryl groups of crystalline /3-1actoglobulin at 74.6 C, as ascertained by the 8-iodosobenzoate method. Figure 4 presents results obtained when milk 32
.lg
Application of the modified procedure was made to heated skimmilks held at four different temperatures, ranging from a total time of 3 min at 90C to 60 rain at 75C (Fig. 2). From the nature of the curves and the data obtained, it is evident that the modified method measures heat-activated - - S H groups and generally corroborates the work of others (2, 6, 7, 10) by revealing that the concentration of these groups increases with heating temperatures and holding times for the conditions used. When kinetic plots (Fig. 3) of In(a/a-x)
• 28
-"
o 2o -I-
16
7-~^-' 0"8049X-I- 3.324
/
/
I
12
E ¢o
5I
24
.Q.
o .I-
8
'
4
=0"5856X + 0"916
,Is TEMPERATURE
(°C)
FIG. 4. --SH values of sklmmilk held for 15 mla at several different temperatures.
o1': ¢:
-0"142 X -I" 0-194
/
o
J
-0"078X-0.085
C~.
0
~'
i
'4
i
i
i
i
i
. . . . . ae
8 12 16 20 24 HEATING T I M E - MINUTES
FIo. 3. Kinetics of free --SE group formation in skimmitk upon heat treatment. J. DAII%Y SOIEIRC ~. V O L . 51, NO. 2
was heated at various temperatures at 15 min holding time. The liberation of free - - S H groups was almost negligible below 70 C and increased rapidly above this temperature. However, at 121 C, there was a marked decrease in the - - S H values. Lyster (9) with the DTNB method also observed a similar rapid decrease in - - S H values, but at a temperature of 85 C and above. I n general, the results obtained with the modified DTNB method for milk reveal that: a) a relationship exists between the heat intensity applied and the - - S H values, b) the kinetics of the relationship were of the first order, and e) the method demonstrates the normal trend in active - - S H group formation when milk is exposed for an extended period at a high temperature. This treatment results in a peak of - - S H groups, followed by a marked
T E C H N I C A L NOTES
decrease as the heating time is prolonged. The curves obtained were similar to those reported by Townley and Gould (10) for tt:S production and by Boyd and Gould (2) for active - - S H concentration by the use of the thiamine disulfide method. I n conclusion, the proposed modifications of the DTNB method for the quantitative estimation o£ - - S H values of heated milk by speetrophotometric means were found to yield results consistent with conventionally used procedures. The principal advantages of the method are: a) ease of manipulation, b) speed of analysis, and c) straightforwardness. M. KOKA E. M. MIKOLAJCIK and
(3) (4) (5)
(6)
(7) (8)
I. A. GOULD
Department of Dairy Technology The Ohio State University Columbus References
(9)
(10)
(1) Bliss, C. I. 1967. Statistics iu Biology. Vol. 1. McGraw-Hill, New York. (2) Boyd, E. N., and Gould, I. A. 1957. Volatile and Nonvolatile Sulfhydryl Content of Heat-
219
ed Milk and Milk Products. J. Dairy Sci., 40 : 1294. Da~dels, F., and Alberty, 1~. A. 1961. Physiaal Chemistry. 2rid ed. John Wiley & Sons, New York. Ellmazl, G. L. 1959. Tissue Sulfhydry] Groups. Arch. Biochem. Biophys., 82:70. Fernandez Diez, M. J., Osuga, D. T., and Feeney, i~. F. 1964. The Sulfhydryls of Avian Ovalbumins, Bovine fl-Lactoglobulin, and Bovine Serum Albumin. Arch. Biochem. Biophys., 107:449. Harland, H. A., Coulter, S. T., and Jenness, R. 1949. Some Factors Influencing the Reducing Systems in Dry Whole Milk. J. Dairy Sci., 32:334. Jenness, R. 1954. Milk Proteins. Effects of Heat Treatment on Serum Proteins. J. Agr. Food Chem., 2:75. Larson, B. L., and Jenness, R. ]952. Characterization of the Sulfhydryl Groups and the Kinetics of the Heat Denaturation of Crystalline fl-Lactoglobulin. J. Amer. Chemists" Soc., 74:3090. Lyster, R. L. J. 1964. The Free and Masked Sulphydryl Groups of Heated Milk and Milk Powder and a New Method for Their Determination. J. Dairy Res., 31:41. Town]ey, !~. C., and Gould, I. A. 1943. A Quantitative Study of Heat Labile Sulfides of Milk. I. Method of Determination a~d the Influence of Temperature and Time. J. Dairy Sci., 26:689.
Effect of Direct Steam Heating and Vacuum Treatment on Levels of Pesticide Residues in Milk Abstract
Milks containing added DDT (p,p'-), heptachlor, lindaae, and dieldrin were passed through commercial steam distillation--vacuum processing equipment. This process, developed to expel volatile offflavors, removed the following percentages of pesticides: dieldrin, 3.0; heptachlor, 0; lindane, 23.8; and DDT (p,p'-), 8.4. Feeds containing low levels of chlorinated hydrocarbon insecticides are sources of residues in the milks of lactating cows (1, 6). Recently, an F D A official, commenting on the new tolerance for DDT and its analogs in dairy products, mentioned that this insecticide is so widely distributed in the environment that it is impractical to prevent exposure of dairy animals to it (3). Any process or manufacturing operation which removes or reduces residue concentrations in foods should be an important influence in food marketing. Some research interest is evident in this area (5, 7). While the effects of some milk manufacturing procedures on residue concentrations have been studied (7), the influence of commercial, off-flavor removal pro-
cessing has not been reported. The volatilities of chlorinated hydrocarbon insecticides at elevated temperatures prompted a study of the effect of direct steam heating and vacuum processing on the concentrations of four pesticides in milk. Materials and Methods Pesticide solutions. Purified p,p'-DDT [1,l,l-triehloro -2~2:di- (p-chlorophenyl) ethane], dieldrin (1,2,3,4,10,10-hexachloro -6,7 - epoxy - 1,4,4a, 5,6,7,8,8a -octahydro -1,4 - 5,8-dimethanonaphthalene), heptaehlor (1,4,5,6,7,8,8-heptachlor -3a,4,7,Ta - tetrahydro -4,7-methanoindene), and lindane (7-isomer of 1,2,3,4,5,6 hexachlorocyc]ohexane) were studied in experiments at the 0.5-ppm level on a product basis. Each was initially dissolved in a small amount of ethanol, mixed with approximately 3.7 liters of homogenized milk, and diluted with 190 liters of water. Direct steam injection and vacuum treatment.
A tempering vat was connected to an ARO-VAC instrument (Cherry-Burrell Corporation) to adjust the milk to 71 C before passage through the steam distillation-vacuum process. The J. DAn%Y SCIENCE x~7"OL.51, NO. 2
NOTES
Spectrophotometric Method for Determination of Heat-Activated Sulfhydryl Groups of Skimmilk' Abstract
By suitable modification, adaptation was made of the 5,5'-dithiobis (2-nitrobenzoic acid) reagent method for the spectrophotometric determination of activated sulfhydryl ( - - S H ) groups of heated skimmilk. The modifications consisted of using a nitrogen-purged system, controlling pH, adding ammonium sulfate, filtering, and determining absorbance of the filtrate at 412 mlz. A standard cysteine-HC1 curve was prepared in a similar manner. The modified procedure yielded results consistent with other cmTently used methods. The activation of - - S H groups in heated skimmilk, as determined by the modified procedure, followed the kinetics of a first order reaction with reaction rate constants (kn, l._l) of 0.078, 0.142, 0.384, and 0.805 for 75, 80, 85, and 90 C, respectively.
8.0, and 4 ml of distilled water were added. The lots were agitated and two grains of ammonium sulfate added. The samples were again shaken vigorously and the contents filtered 1"2 I'(~ -.i
E 0"8 _¢2
'~ 0'6 Q
•
o 0.4 ^
'0454
0",
Many of the current procedures used for the determination of heat-activated sulfhydryl ( - - S H ) groups of milk are lengthy, complicated, or have only limited application to milk systems. Several investigators (4, 5) have used a sulfhydryl-disulfide interchange reagent, 5,5'dithiobis (2-nitrobenzoic acid), DTNB, for the estimation of unmasked - - S H groups in nonmilk systems. Lyster (9) applied the DTNB reagent method to milk and determined the degree of yellow color development in the heated samples by visual comparision with specially prepared standards of known free - - S H concentration by the use of a Lovibond Comparator and reflected
f .
0
.
2
.
.
4
|
i
i
I
6 8 Cysteine.HCI
I
I
_
I0
xloTM
Fro. 1. Statistically calculated eysteine-HCl standard curve by the DTNB procedure. s 7 x~ 6
light. This p a p e r reports on modifications and improvements in the DTNB procedure which permit quantitative determination of - - S H values of heated milks by direct spectrophotometric means. Five-milliliter quantities of raw skimmilk ( p H 6.7) contained in screw-cap test tubes were purged with nitrogen gas for 10 sec and then tightly stoppered. One lot served as the unheated control and the remaining lots were heated in a thermostatically controlled water bath, cooled immediately, and treated with 0.1 ml of" a DTNB solution [39.37 mg of 5,5"dithiobis (2-nitrobenzoic acid) in 10 ml of 0.1 phosphate buffer of p i t 7.0]. The yellow color was developed for 3 rain at room temperature, then 1 ml of 0.1 ~ phosphate buffer, p H Article 5:67. Department of Dairy Technology, The Ohio State University. This investigation was supported by Public Health Services Research Grant EF-00]80 from the Division of Environmental Engineering and Food Protection.
~_ 5 ~ 4' o
.× s
~ 2i
I
o~
,~
|
|
0
2
t2
io
i,
ie
~4
I
i
i
!
i
22
26
TIME IN MINUTES AT
6
I0
14
18
75°C
AT
80" G
'AT
85°G
AT
90'PC
~z 6b i0
3
FIG. 2. Rate of free --SH group formation in skimmilk following heat treatment. 217
218
JOUI%NAL O F D A I R Y S C I E N C E
through Whatman 42 filter paper. The absorbance of the filtrate was read at a wavelength of 412 mtt in a Hitachi Perkin-Elmer Model 139 Spectrophotometer with a digital readout attachment. Preliminary studies had indicated that maximum absorbance of the filtrate was at 412 mt~. The blank tube contained all the reagents, excepting that 5 ml of 0.1 M phosphate buffer, p H 6.7, was substituted for the skimmilk. For the cysteine-HC1 standard curve, dilutions were in p H 6.7 phosphate buffer; further treatments, including the addition of ammonium sulfate, followed the standard procedure. By application of statistics (1), the plot of absorbance at 412 m/x versus eysteine-HC1 in moles (Fig. 1) yielded the best fit straight line with a calculated slope of: :Y =
(0.9488) (10~)Z -- 0.0434.
versus t were prepared from the data, using the mathematical treatment suggested by Daniels and Alberty (3), a straight line was obtained at each temperature and several holding times, indicating that the unmasking of the - - S H groups in skimmilk upon heat treatment followed the kinetics of a first-order reaction. The first-order reaction rate constants (km;~-l) for 75, 80, 85, and 90 C were 0.078, 0.142, 0.384, and 0.805, respectively. The reaction rate constant at 75 C approximated the reported (8) value of 0.0875/nfin for the activation of sulfhydryl groups of crystalline /3-1actoglobulin at 74.6 C, as ascertained by the 8-iodosobenzoate method. Figure 4 presents results obtained when milk 32
.lg
Application of the modified procedure was made to heated skimmilks held at four different temperatures, ranging from a total time of 3 min at 90C to 60 rain at 75C (Fig. 2). From the nature of the curves and the data obtained, it is evident that the modified method measures heat-activated - - S H groups and generally corroborates the work of others (2, 6, 7, 10) by revealing that the concentration of these groups increases with heating temperatures and holding times for the conditions used. When kinetic plots (Fig. 3) of In(a/a-x)
• 28
-"
o 2o -I-
16
7-~^-' 0"8049X-I- 3.324
/
/
I
12
E ¢o
5I
24
.Q.
o .I-
8
'
4
=0"5856X + 0"916
,Is TEMPERATURE
(°C)
FIG. 4. --SH values of sklmmilk held for 15 mla at several different temperatures.
o1': ¢:
-0"142 X -I" 0-194
/
o
J
-0"078X-0.085
C~.
0
~'
i
'4
i
i
i
i
i
. . . . . ae
8 12 16 20 24 HEATING T I M E - MINUTES
FIo. 3. Kinetics of free --SE group formation in skimmitk upon heat treatment. J. DAII%Y SOIEIRC ~. V O L . 51, NO. 2
was heated at various temperatures at 15 min holding time. The liberation of free - - S H groups was almost negligible below 70 C and increased rapidly above this temperature. However, at 121 C, there was a marked decrease in the - - S H values. Lyster (9) with the DTNB method also observed a similar rapid decrease in - - S H values, but at a temperature of 85 C and above. I n general, the results obtained with the modified DTNB method for milk reveal that: a) a relationship exists between the heat intensity applied and the - - S H values, b) the kinetics of the relationship were of the first order, and e) the method demonstrates the normal trend in active - - S H group formation when milk is exposed for an extended period at a high temperature. This treatment results in a peak of - - S H groups, followed by a marked
T E C H N I C A L NOTES
decrease as the heating time is prolonged. The curves obtained were similar to those reported by Townley and Gould (10) for tt:S production and by Boyd and Gould (2) for active - - S H concentration by the use of the thiamine disulfide method. I n conclusion, the proposed modifications of the DTNB method for the quantitative estimation o£ - - S H values of heated milk by speetrophotometric means were found to yield results consistent with conventionally used procedures. The principal advantages of the method are: a) ease of manipulation, b) speed of analysis, and c) straightforwardness. M. KOKA E. M. MIKOLAJCIK and
(3) (4) (5)
(6)
(7) (8)
I. A. GOULD
Department of Dairy Technology The Ohio State University Columbus References
(9)
(10)
(1) Bliss, C. I. 1967. Statistics iu Biology. Vol. 1. McGraw-Hill, New York. (2) Boyd, E. N., and Gould, I. A. 1957. Volatile and Nonvolatile Sulfhydryl Content of Heat-
219
ed Milk and Milk Products. J. Dairy Sci., 40 : 1294. Da~dels, F., and Alberty, 1~. A. 1961. Physiaal Chemistry. 2rid ed. John Wiley & Sons, New York. Ellmazl, G. L. 1959. Tissue Sulfhydry] Groups. Arch. Biochem. Biophys., 82:70. Fernandez Diez, M. J., Osuga, D. T., and Feeney, i~. F. 1964. The Sulfhydryls of Avian Ovalbumins, Bovine fl-Lactoglobulin, and Bovine Serum Albumin. Arch. Biochem. Biophys., 107:449. Harland, H. A., Coulter, S. T., and Jenness, R. 1949. Some Factors Influencing the Reducing Systems in Dry Whole Milk. J. Dairy Sci., 32:334. Jenness, R. 1954. Milk Proteins. Effects of Heat Treatment on Serum Proteins. J. Agr. Food Chem., 2:75. Larson, B. L., and Jenness, R. ]952. Characterization of the Sulfhydryl Groups and the Kinetics of the Heat Denaturation of Crystalline fl-Lactoglobulin. J. Amer. Chemists" Soc., 74:3090. Lyster, R. L. J. 1964. The Free and Masked Sulphydryl Groups of Heated Milk and Milk Powder and a New Method for Their Determination. J. Dairy Res., 31:41. Town]ey, !~. C., and Gould, I. A. 1943. A Quantitative Study of Heat Labile Sulfides of Milk. I. Method of Determination a~d the Influence of Temperature and Time. J. Dairy Sci., 26:689.
Effect of Direct Steam Heating and Vacuum Treatment on Levels of Pesticide Residues in Milk Abstract
Milks containing added DDT (p,p'-), heptachlor, lindaae, and dieldrin were passed through commercial steam distillation--vacuum processing equipment. This process, developed to expel volatile offflavors, removed the following percentages of pesticides: dieldrin, 3.0; heptachlor, 0; lindane, 23.8; and DDT (p,p'-), 8.4. Feeds containing low levels of chlorinated hydrocarbon insecticides are sources of residues in the milks of lactating cows (1, 6). Recently, an F D A official, commenting on the new tolerance for DDT and its analogs in dairy products, mentioned that this insecticide is so widely distributed in the environment that it is impractical to prevent exposure of dairy animals to it (3). Any process or manufacturing operation which removes or reduces residue concentrations in foods should be an important influence in food marketing. Some research interest is evident in this area (5, 7). While the effects of some milk manufacturing procedures on residue concentrations have been studied (7), the influence of commercial, off-flavor removal pro-
cessing has not been reported. The volatilities of chlorinated hydrocarbon insecticides at elevated temperatures prompted a study of the effect of direct steam heating and vacuum processing on the concentrations of four pesticides in milk. Materials and Methods Pesticide solutions. Purified p,p'-DDT [1,l,l-triehloro -2~2:di- (p-chlorophenyl) ethane], dieldrin (1,2,3,4,10,10-hexachloro -6,7 - epoxy - 1,4,4a, 5,6,7,8,8a -octahydro -1,4 - 5,8-dimethanonaphthalene), heptaehlor (1,4,5,6,7,8,8-heptachlor -3a,4,7,Ta - tetrahydro -4,7-methanoindene), and lindane (7-isomer of 1,2,3,4,5,6 hexachlorocyc]ohexane) were studied in experiments at the 0.5-ppm level on a product basis. Each was initially dissolved in a small amount of ethanol, mixed with approximately 3.7 liters of homogenized milk, and diluted with 190 liters of water. Direct steam injection and vacuum treatment.
A tempering vat was connected to an ARO-VAC instrument (Cherry-Burrell Corporation) to adjust the milk to 71 C before passage through the steam distillation-vacuum process. The J. DAn%Y SCIENCE x~7"OL.51, NO. 2