RESEARCH PAPERS Photodegradation of Riboflavin in Milks Exposed to Fluorescent Light C. A L L E N a n d O . W. PARKS Eastern Regional Research Center I Philadelphia, PA 19118 ABSTRACT
riboflavin in milk. A recently developed procedure for quantitating riboflavin content in fluid milks lends itself readily to this type of investigation and forms the basis of this study. Objectives were 1) to determine if riboflavin is photodegraded prior to development of an off-flavor in whole milk exposed to light, 2) to compare reaction rates of whole and skim milk, and 3) to determine the reaction order of photodegradation of riboflavin in fluid milks.
Photodegradation of riboflavin in 50-ml samples of fluid milks exposed to fluorescent light (2690 lux) followed first-order reaction kinetics, and the reaction rate (s -1) averaged 1.86 x 10 -s in skim milk and 1.47 x 10 -s in whole milk. Additional evidence led us to conclude that photodegradation of riboflavin proceeds prior to the appearance of a light-induced off-flavor.
MATERIALS AND METHODS INTRODUCTION
Increasing emphasis on the nutritional aspects of foods has renewed interest in photochemical reactions in milk from exposure to light. Hence, major emphasis has been placed on the photodegradation of riboflavin since milk contributes 40 to 50% of the total dietary riboflavin in the United States and many other western nations (5). The extent of riboflavin photodegradation in fluid milk depends on numerous factors including the wavelength of light, the intensity of light, exposure time, protective effect of the milk container, surface area of milk in relation to volume in the package, and temperature of the product (4, 8). Whereas factors influencing the extent of photodegradation have been investigated extensively (8), kinetics of the photochemical reaction in milk have had scant attention. Singh et al. (9) on limited data indicated that "riboflavin degradation in milk can be best described by assuming a first order reaction." From a practical standpoint their studies emphasized the utility of kinetic studies in selecting optimum storage conditions for preventing photodegradation of
Received March 8, 1979. 1Agricultural Research, Science and Education Administration, US Department of Agriculture. Reference to brand or firm name does not constitute endorsement by the US Department of Agriculture over others of a similar nature not mentioned. 1979 J Dairy Science 62:1377-1379
Fifty milliliter samples of commercial milk in 20 x 2.5 cm pyrex screw cap culture tubes were exposed to fluorescent light (2690 lx) for 1 to 32 h at 2.5 C in a walk-in cooler. The light source consisted of two 15 watt cool white fluorescent bulbs (Westinghouse F15T8/CW) 2 positioned 15.25 cm above the sample tubes which were placed in a horizontal position. Following exposure, the samples and a distilled water control were passed through (flow rate 2.5 ml/min) individual columns of Amberlite XAD-4 prepared in the following manner: 4.0 g of resin in redistilled methanol were added to a 53 x .8 cm glass buret which was fitted with a teflon stopcock and plugged with glass wool. The resin was washed consecutively with 75-ml portions of methanol, acetone, and distilled water at a flow rate of 2.5 ml/min, with the solvent level kept above the resin at all times. A plug of glass wool was added to the top of the resin before the sample was introduced. Following passage of the sample through the column, the resin was washed free of unabsorbed sample with 75 ml of distilled water, and the absorbed constituents were eluted with 30 ml of methanol. The methanol effluent was evaporated to dryness in a 8.5 x 2.3 cm screw cap vial. Silicic Acid Chromatography
Isolation of riboflavin from other X A S 4 absorbed materials, including lumichrome, was accomplished by silicic acid chromatography in
1 377
1378
ALLEN AND PARKS
the following sequence o f steps: Step I. 1.5 g of silicic acid (Mallinckrodt 100-mesh), previously washed (6) and v a c u u m oven dried (65 C and .064 atm for 16 h), was slurried in m e t h y l e n e chloride and added to a 15 × 1.0 cm c h r o m a t o g r a p h i c c o l u m n plugged with glass wool. The silicic acid was washed with 15 ml o f m e t h y l e n e chloride, and a small wad o f glass w o o l was added to the top of the column. Step 2. One-tenth milliliter of m e t h a n o l was added to the sample vial followed by 9.9 ml of m e t h y l e n e chloride. The c o n t e n t s were m i x e d t h o r o u g h l y and added to the silicic acid column. Following percolation of the sample, the silicic acid was eluted with 10 ml of 1% methanol in m e t h y l e n e chloride. Step 3. The sample vial was rinsed with t w o 2.0-ml portions of 2% m e t h a n o l in m e t h y l e n e chloride. The solution was added to the column, and the silicic acid was eluted with 25 ml o f 2% m e t h a n o l in m e t h y l e n e chloride. Step 4. The sample vial was rinsed with two 2.0-ml portions of 7.5% m e t h a n o l in m e t h y l e n e chloride. The rinses were added to the c o l u m n and the silicic acid was eluted with 20 ml o f 7.5% m e t h a n o l in m e t h y l e n e chloride. Step 5. The sample vial was rinsed with t w o 2.0-ml portions of 10% m e t h a n o l in m e t h y l e n e chloride. The rinses were added to the column, and riboflavin was eluted with 40 ml of 10% m e t h a n o l in m e t h y l e n e chloride. Quantitative Determination
The riboflavin fraction was evaporated to dryness under a stream of nitrogen in an 8.5 × 2.3 cm screw cap vial. The dried sample was dissolved in 3 ml of distilled water by gentle
shaking and warming the solution in a water bath at 40 C. The sample was cooled to r o o m temperature, and the a m o u n t of riboflavin was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y at 445 nm, with a molar absorptivity of 11,800. Preliminary studies on k n o w n c o n c e n t r a t i o n s of riboflavin showed an 80% recovery following X A D 4 extraction and silicic acid chromatography. Therefore, the concentrations det e r m i n e d were adjusted accordingly. All experimental procedures for the isolation and quantitation o f riboflavin f r o m exposed and u n e x p o s e d samples were w i t h o u t direct lighting. RESULTS A N D DISCUSSION
The nanamoles of riboflavin remaining in 50-ml samples of w h o l e and skim milks exposed to fluorescent light for 1 to 32 h under the conditions of this study are in Table 1. Photodegradation was kinetically o f a first-order t y p e and could be expressed m a t h e m a t i c a l l y by kt = O O in a /a, where k is the rate constant, a the initial concentration, and a equals the n m of riboflavin remaining at t i m e t. By this e q u a t i o n the slope of a straight line, by least squares, corresponding to the reaction rate was obtained. Figure 1 illustrates the c o n f o r m i t y of the e x p e r i m e n t a l data of whole milk sample 1 (r = .99) and skim milk sample 1 (r = .98) to the e q u a t i o n . The reaction rates for all samples are in Table 2. The role of riboflavin in t h e p h o t o c h e m i c a l reactions leading to off-flavors in fluid milk is well established (8). Storgards and Ljungren had concluded (10) t h a t skim milk was more readily susceptible than high fat fluid products to off-flavors. It was n o t u n e x p e c t e d , therefore,
TABLE 1. Effect of time on riboflavin concentration in fluid milks exposed to 2690 Ix of fluorescent light. nM Riboflavin/50 ml fluid milk Milk sample Whole 1 2 3 4 5 Skim 1 2 3
0
1
2
160.6 149.7 159.0 156.7 131.4 138.3 137.2 166.2
149.5 144.9 150.3 149.4 122.6 123.1 126.0 163.1
146.3 144.9 143.9 133.5 117.8 118.4 124.5 155.1
Journal of Dairy Science Vol. 62, No. 9, 1979
Exposure time (h) 4 8 135.4 132.2 118.1 108.3 92.6 97.9
99.2 136.1
113.0 104.5 105.9 92.3 67.0 58.3 75.5 61.1
16 65.7 74.7 59.0 49.7 44.9 35.9 58.0 43.7
24
32
.... .... .... 35.4 30.6 20.21 .... ....
29.3 35.6 29.8 25.8 22.9 15.4 18.1 19.9
RIBOFLAVIN PHOTODEGRADATION
2.5
2.5
WHOLM EILK
2.0
TABLE 2. Specific reaction rates of riboflavin photodegradation in fluid milks.
2.0
1.5 / 1.0
I.C I
.5
5
0
I
0
I
10
I
I 20
I
I
30
I
0 Ii
EXPOSURE
1 379
110
L t l
20
310
I
Milk sample
Reaction rate k (s -1 )
Whole 1 2 3 4 5 Skim 1 2 3
1.50 × 1.28 X 1.47 × 1.58 × 1.52 × 1.90× 1.75 X 1.92 X
10 -s 10 -5 10 -s 10 -s 10 -s 10 -s 10 -s 10 -s
TIME, HOURS
Figure 1. First order reaction plot of riboflavin in whole and skim milk exposed to 2690 lux of fluorescent light.
that the reaction rates for the photodegradation of riboflavin in the skim milk samples were greater. T h e lower r e a c t i o n rate o n w h o l e milk sample 2, a l t h o u g h n o t readily e x p l a i n e d , m a y reflect d i f f e r e n c e s in processing c o n d i t i o n s (10), l e n g t h o f storage p r i o r t o e x p o s u r e (2), a n d / o r a h i g h e r fat c o n t e n t relative t o t h e o t h e r w h o l e milk samples. R e a c t i o n rates do n o t reflect r e a c t i o n rates o b t a i n a b l e o n r e p r e s e n t a t i v e samples o f m i l k e x p o s e d u n d e r similar light c o n d i t i o n s in c o m m e r c i a l milk c o n t a i n e r s . Previous studies (1) d e m o n s t r a t e d t h a t light o f w a v e l e n g t h 4 3 6 n m p e n e t r a t e s w h o l e a n d skim milk t o an effective d e p t h o f 1.0 a n d 1.2 cm. Hence, t h e p h o t o d e g r a d a t i o n of r i b o f l a v i n in u n d i s t u r b e d milk samples w o u l d b e c o n f i n e d to t h a t p o r t i o n of t h e milk in relatively close p r o x i m i t y to t h e light source. T h e e x p e r i m e n t a l results o n r e p r e s e n t a t i v e samples o f milk e x p o s e d to light in c o m m e r c i a l milk c o n t a i n e r s , nevertheless, s h o u l d fit t h e e q u a t i o n for first-order kinetics. In this respect, an analysis of t h e d a t a in Hansen et al. (3) on r e p r e s e n t a t i v e samples of w h o l e milk e x p o s e d to 2 1 5 2 lx of f l u o r e s c e n t light in .95-liter p o l y e t h y l e n e containers revealed t h a t t h e p h o t o d e g r a d a t i o n o f riboflavin in t h a t s t u d y c o n f o r m e d to first-order k i n e t i c s a n d k(s "1 ) = 5.0 × 10 -7. Hansen et al. (3) suggested t h a t r i b o f l a v i n was n o t p h o t o d e g r a d e d p r i o r to t h e o n s e t of off-flavors in fluid w h o l e milk b e c a u s e t h e y d e t e c t e d n o changes in t h e r i b o f l a v i n c o n t e n t of fluid milks at t h e t i m e of flavor d e v e l o p m e n t . Flavor studies o n t h e w h o l e milk samples
revealed t h a t w h e r e a s off-flavors were n o t o b v i o u s p r i o r to 4 h o f e x p o s u r e , a decrease in t h e riboflavin c o n t e n t a n d q u a l i t a t i v e d e t e c t i o n of l u m i c h r o m e (7) was d e t e r m i n e d readily f o l l o w i n g 2 h o f e x p o s u r e t o f l u o r e s c e n t light. These o b s e r v a t i o n s , in a d d i t i o n to t h e e v i d e n c e s h o w i n g t h a t t h e p h o t o d e g r a d a t i o n o f riboflavin conforms to first-order r e a c t i o n kinetics, w i t h o u t an a p p a r e n t i n d u c t i o n period, led us t o c o n c l u d e t h a t r i b o f l a v i n is p h o t o d e g r a d e d prior to a p p e a r a n c e of a n off-flavor.
REFERENCES 1 Burgess, W. H., and B. L. Herrington. 1955. The diffuse reflection of light by milk. J. Dairy Sci. 38:250. 2 Dunkley, W. L., J. D. Franklin, and R. M. Pangborn. 1962. Effects of fluorescent light on flavor, ascorbic acid, and riboflavin in milk. J. Food Technol. 16:112. 3 Hansen, A. P., L. G. Turner, and L. W. Aurand. 1975. Fluorescent light activated flavor in milk. J. Milk and Food Technol. 38:388. 4 Hedrick, T. I., and L. Glass. 1975. Chemica/ changes in milk during exposure to fluorescent light. J. Milk and Food Technol. 38:129. 5 Marston, R., and B. Friend. 1971. National food situation. NFS 158:25. 6 McCarthy, R. D., and A. H. Duthie. 1962. A rapid quantitative method for the separation of free fatty acids from other lipids. J. Lipid Res. 3:117. 7 Parks, O. W., and C. Allen. 1977. Photodegradation of riboflavin to lumichrome in milk exposed to sunlight. J. Dairy Sci. 60:1038. 8 Sattar, A., and J. M. deMan. 1975. Photooxidation of milk and milk products: A review. Crit. Rev. Food Sci. and Nutr. 1 : 13. 9 Singh, R. P., D. R. Heldman, and J. R. Kirk. 1975. Kinetic analysis of light-induced riboflavin loss in whole milk. J. Food Sci. 40:164. 10 Storgards, T., and B. Ljungren. 1962. Some observations on the formation of light-induced oxidized flavor. Milchwissenschaft 17: 406. Journal of Dairy Science Vol. 62, No. 9, 1979
riboflavin in milk. A recently developed procedure for quantitating riboflavin content in fluid milks lends itself readily to this type of investigation and forms the basis of this study. Objectives were 1) to determine if riboflavin is photodegraded prior to development of an off-flavor in whole milk exposed to light, 2) to compare reaction rates of whole and skim milk, and 3) to determine the reaction order of photodegradation of riboflavin in fluid milks.
Photodegradation of riboflavin in 50-ml samples of fluid milks exposed to fluorescent light (2690 lux) followed first-order reaction kinetics, and the reaction rate (s -1) averaged 1.86 x 10 -s in skim milk and 1.47 x 10 -s in whole milk. Additional evidence led us to conclude that photodegradation of riboflavin proceeds prior to the appearance of a light-induced off-flavor.
MATERIALS AND METHODS INTRODUCTION
Increasing emphasis on the nutritional aspects of foods has renewed interest in photochemical reactions in milk from exposure to light. Hence, major emphasis has been placed on the photodegradation of riboflavin since milk contributes 40 to 50% of the total dietary riboflavin in the United States and many other western nations (5). The extent of riboflavin photodegradation in fluid milk depends on numerous factors including the wavelength of light, the intensity of light, exposure time, protective effect of the milk container, surface area of milk in relation to volume in the package, and temperature of the product (4, 8). Whereas factors influencing the extent of photodegradation have been investigated extensively (8), kinetics of the photochemical reaction in milk have had scant attention. Singh et al. (9) on limited data indicated that "riboflavin degradation in milk can be best described by assuming a first order reaction." From a practical standpoint their studies emphasized the utility of kinetic studies in selecting optimum storage conditions for preventing photodegradation of
Received March 8, 1979. 1Agricultural Research, Science and Education Administration, US Department of Agriculture. Reference to brand or firm name does not constitute endorsement by the US Department of Agriculture over others of a similar nature not mentioned. 1979 J Dairy Science 62:1377-1379
Fifty milliliter samples of commercial milk in 20 x 2.5 cm pyrex screw cap culture tubes were exposed to fluorescent light (2690 lx) for 1 to 32 h at 2.5 C in a walk-in cooler. The light source consisted of two 15 watt cool white fluorescent bulbs (Westinghouse F15T8/CW) 2 positioned 15.25 cm above the sample tubes which were placed in a horizontal position. Following exposure, the samples and a distilled water control were passed through (flow rate 2.5 ml/min) individual columns of Amberlite XAD-4 prepared in the following manner: 4.0 g of resin in redistilled methanol were added to a 53 x .8 cm glass buret which was fitted with a teflon stopcock and plugged with glass wool. The resin was washed consecutively with 75-ml portions of methanol, acetone, and distilled water at a flow rate of 2.5 ml/min, with the solvent level kept above the resin at all times. A plug of glass wool was added to the top of the resin before the sample was introduced. Following passage of the sample through the column, the resin was washed free of unabsorbed sample with 75 ml of distilled water, and the absorbed constituents were eluted with 30 ml of methanol. The methanol effluent was evaporated to dryness in a 8.5 x 2.3 cm screw cap vial. Silicic Acid Chromatography
Isolation of riboflavin from other X A S 4 absorbed materials, including lumichrome, was accomplished by silicic acid chromatography in
1 377
1378
ALLEN AND PARKS
the following sequence o f steps: Step I. 1.5 g of silicic acid (Mallinckrodt 100-mesh), previously washed (6) and v a c u u m oven dried (65 C and .064 atm for 16 h), was slurried in m e t h y l e n e chloride and added to a 15 × 1.0 cm c h r o m a t o g r a p h i c c o l u m n plugged with glass wool. The silicic acid was washed with 15 ml o f m e t h y l e n e chloride, and a small wad o f glass w o o l was added to the top of the column. Step 2. One-tenth milliliter of m e t h a n o l was added to the sample vial followed by 9.9 ml of m e t h y l e n e chloride. The c o n t e n t s were m i x e d t h o r o u g h l y and added to the silicic acid column. Following percolation of the sample, the silicic acid was eluted with 10 ml of 1% methanol in m e t h y l e n e chloride. Step 3. The sample vial was rinsed with t w o 2.0-ml portions of 2% m e t h a n o l in m e t h y l e n e chloride. The solution was added to the column, and the silicic acid was eluted with 25 ml o f 2% m e t h a n o l in m e t h y l e n e chloride. Step 4. The sample vial was rinsed with two 2.0-ml portions of 7.5% m e t h a n o l in m e t h y l e n e chloride. The rinses were added to the c o l u m n and the silicic acid was eluted with 20 ml o f 7.5% m e t h a n o l in m e t h y l e n e chloride. Step 5. The sample vial was rinsed with t w o 2.0-ml portions of 10% m e t h a n o l in m e t h y l e n e chloride. The rinses were added to the column, and riboflavin was eluted with 40 ml of 10% m e t h a n o l in m e t h y l e n e chloride. Quantitative Determination
The riboflavin fraction was evaporated to dryness under a stream of nitrogen in an 8.5 × 2.3 cm screw cap vial. The dried sample was dissolved in 3 ml of distilled water by gentle
shaking and warming the solution in a water bath at 40 C. The sample was cooled to r o o m temperature, and the a m o u n t of riboflavin was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y at 445 nm, with a molar absorptivity of 11,800. Preliminary studies on k n o w n c o n c e n t r a t i o n s of riboflavin showed an 80% recovery following X A D 4 extraction and silicic acid chromatography. Therefore, the concentrations det e r m i n e d were adjusted accordingly. All experimental procedures for the isolation and quantitation o f riboflavin f r o m exposed and u n e x p o s e d samples were w i t h o u t direct lighting. RESULTS A N D DISCUSSION
The nanamoles of riboflavin remaining in 50-ml samples of w h o l e and skim milks exposed to fluorescent light for 1 to 32 h under the conditions of this study are in Table 1. Photodegradation was kinetically o f a first-order t y p e and could be expressed m a t h e m a t i c a l l y by kt = O O in a /a, where k is the rate constant, a the initial concentration, and a equals the n m of riboflavin remaining at t i m e t. By this e q u a t i o n the slope of a straight line, by least squares, corresponding to the reaction rate was obtained. Figure 1 illustrates the c o n f o r m i t y of the e x p e r i m e n t a l data of whole milk sample 1 (r = .99) and skim milk sample 1 (r = .98) to the e q u a t i o n . The reaction rates for all samples are in Table 2. The role of riboflavin in t h e p h o t o c h e m i c a l reactions leading to off-flavors in fluid milk is well established (8). Storgards and Ljungren had concluded (10) t h a t skim milk was more readily susceptible than high fat fluid products to off-flavors. It was n o t u n e x p e c t e d , therefore,
TABLE 1. Effect of time on riboflavin concentration in fluid milks exposed to 2690 Ix of fluorescent light. nM Riboflavin/50 ml fluid milk Milk sample Whole 1 2 3 4 5 Skim 1 2 3
0
1
2
160.6 149.7 159.0 156.7 131.4 138.3 137.2 166.2
149.5 144.9 150.3 149.4 122.6 123.1 126.0 163.1
146.3 144.9 143.9 133.5 117.8 118.4 124.5 155.1
Journal of Dairy Science Vol. 62, No. 9, 1979
Exposure time (h) 4 8 135.4 132.2 118.1 108.3 92.6 97.9
99.2 136.1
113.0 104.5 105.9 92.3 67.0 58.3 75.5 61.1
16 65.7 74.7 59.0 49.7 44.9 35.9 58.0 43.7
24
32
.... .... .... 35.4 30.6 20.21 .... ....
29.3 35.6 29.8 25.8 22.9 15.4 18.1 19.9
RIBOFLAVIN PHOTODEGRADATION
2.5
2.5
WHOLM EILK
2.0
TABLE 2. Specific reaction rates of riboflavin photodegradation in fluid milks.
2.0
1.5 / 1.0
I.C I
.5
5
0
I
0
I
10
I
I 20
I
I
30
I
0 Ii
EXPOSURE
1 379
110
L t l
20
310
I
Milk sample
Reaction rate k (s -1 )
Whole 1 2 3 4 5 Skim 1 2 3
1.50 × 1.28 X 1.47 × 1.58 × 1.52 × 1.90× 1.75 X 1.92 X
10 -s 10 -5 10 -s 10 -s 10 -s 10 -s 10 -s 10 -s
TIME, HOURS
Figure 1. First order reaction plot of riboflavin in whole and skim milk exposed to 2690 lux of fluorescent light.
that the reaction rates for the photodegradation of riboflavin in the skim milk samples were greater. T h e lower r e a c t i o n rate o n w h o l e milk sample 2, a l t h o u g h n o t readily e x p l a i n e d , m a y reflect d i f f e r e n c e s in processing c o n d i t i o n s (10), l e n g t h o f storage p r i o r t o e x p o s u r e (2), a n d / o r a h i g h e r fat c o n t e n t relative t o t h e o t h e r w h o l e milk samples. R e a c t i o n rates do n o t reflect r e a c t i o n rates o b t a i n a b l e o n r e p r e s e n t a t i v e samples o f m i l k e x p o s e d u n d e r similar light c o n d i t i o n s in c o m m e r c i a l milk c o n t a i n e r s . Previous studies (1) d e m o n s t r a t e d t h a t light o f w a v e l e n g t h 4 3 6 n m p e n e t r a t e s w h o l e a n d skim milk t o an effective d e p t h o f 1.0 a n d 1.2 cm. Hence, t h e p h o t o d e g r a d a t i o n of r i b o f l a v i n in u n d i s t u r b e d milk samples w o u l d b e c o n f i n e d to t h a t p o r t i o n of t h e milk in relatively close p r o x i m i t y to t h e light source. T h e e x p e r i m e n t a l results o n r e p r e s e n t a t i v e samples o f milk e x p o s e d to light in c o m m e r c i a l milk c o n t a i n e r s , nevertheless, s h o u l d fit t h e e q u a t i o n for first-order kinetics. In this respect, an analysis of t h e d a t a in Hansen et al. (3) on r e p r e s e n t a t i v e samples of w h o l e milk e x p o s e d to 2 1 5 2 lx of f l u o r e s c e n t light in .95-liter p o l y e t h y l e n e containers revealed t h a t t h e p h o t o d e g r a d a t i o n o f riboflavin in t h a t s t u d y c o n f o r m e d to first-order k i n e t i c s a n d k(s "1 ) = 5.0 × 10 -7. Hansen et al. (3) suggested t h a t r i b o f l a v i n was n o t p h o t o d e g r a d e d p r i o r to t h e o n s e t of off-flavors in fluid w h o l e milk b e c a u s e t h e y d e t e c t e d n o changes in t h e r i b o f l a v i n c o n t e n t of fluid milks at t h e t i m e of flavor d e v e l o p m e n t . Flavor studies o n t h e w h o l e milk samples
revealed t h a t w h e r e a s off-flavors were n o t o b v i o u s p r i o r to 4 h o f e x p o s u r e , a decrease in t h e riboflavin c o n t e n t a n d q u a l i t a t i v e d e t e c t i o n of l u m i c h r o m e (7) was d e t e r m i n e d readily f o l l o w i n g 2 h o f e x p o s u r e t o f l u o r e s c e n t light. These o b s e r v a t i o n s , in a d d i t i o n to t h e e v i d e n c e s h o w i n g t h a t t h e p h o t o d e g r a d a t i o n o f riboflavin conforms to first-order r e a c t i o n kinetics, w i t h o u t an a p p a r e n t i n d u c t i o n period, led us t o c o n c l u d e t h a t r i b o f l a v i n is p h o t o d e g r a d e d prior to a p p e a r a n c e of a n off-flavor.
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