Int. Dairy Journal 2 (1992) 363-371

Effect of E-Carotene on Vitamin A Light Stability in Fortified Milk

M. Zahar,* D. E. Smith* & J. J. Warthesen

Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Avenue, St Paul, Minnesota 55108, USA

(Received 30 October 1991, revised version accepted 8 February 1992)

ABSTRACT

The effect of incorporation of [3-carotene into the carrier system on the light stability of all-trans retinyl palm#ate in fortified skim milk was investigated. Skim milk was fortified with all-trans retinyl palmitate using corn oil with and without added [3-carotene as the vitamin carrier. Two levels of [3-carotene were used, one similar to, the other half the concentration of vitamin A in the carrier system. After homogenization and pasteurization, fortified milk samples were exposed to fluorescent light (1614 Ix) at 4°C in glass tubes.

Less light degradation of a//-trans retinyl palmitate occurred in samples containing added O-carotene in the carrier systems compared to samples without added [3-carotene. The [3-carotene content of fortified milk samples showed only a slight decrease over the four-day period of exposure.

*Present address: Department de Chimie-Biochimie Alimentaire, Institut Agronomique et Veterinaire Hassan II, B.P. 6202 Rabat-lnstituts, Rabat, Morocco. *To whom correspondence should be addressed. Published as Paper No. 19557 of the contribution series of the Minnesota Agricultural Experiment Station based on research conducted under Project 18-24, supported by University of Minnesota Computer Center and National Dairy Promotion and Research Board.

363 Int. Dairy Journal 0958-6946/92/$05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Ireland

364 M. Zahar, D. E. Smith, J J. Warthesen

INTRODUCTION

Fortification of low-fat milk with vitamin A is mandatory in the USA. Milk is fortified by adding a concentrated oil solution of vitamins A (retinyl palmitate) and D. The added vitamin A is more light sensitive than the native vitamin A (Thompson & Erdody, 1974; deMan, 1981; Senyk & Shipe, 1981; Bartholomew & Ogden, 1990).

Several factors have been investigated for their effect on light-induced degradation of vitamin A in milk. These factors include type of packaging material (Hedrick & Glass, 1975; deMan, 1978; White & Bulthaus, 1982; Nelson & Cathcart, 1984), intensity and wavelength distribution of the light sources in retail stores (Sattar et al., 1977; Nelson & Cathcart, 1984), compositional factors such as fat level (deMan, 1981; Senyk & Shipe, 1981; Gaylord et al., 1986; Zahar et al., 1986) and non-fat milk solids (Gaylord et al., 1986). Other factors include composition and level of the vitamin A carrier system (Zahar et al., 1986, 1987), presence of antioxidant (Berge et al., 1987), and effect of emulsifiers (Bartholomew & Ogden, 1990).

Protective effect offl-carotene on light induced destruction of vitamin A in chloroform was studied by Sattar et al. (1977). Beta-carotene at concentrations <1/~g/ml had no effect on photolysis of vitamin A. However, fl-carotene concentration of 2-5-7.5/~g/ml significantly protected vitamin A at a level of 2-5/~g/ml. Such an inhibitory effect has been ascribed to an overall reduction of incident light energy by fl-carotene or by its light-induced decomposition products. In a previous work Zahar (1985) studied the effect of fl-carotene on the degradation of all-trans retinyl palmitate in corn oil exposed to light. Less degradation ofal l- trans retinyl palmitate occurred in corn oil system samples with added fl-carotene (5/.tg/g oil) as compared to corn oil samples without added fl-carotene. However, the rate constants were not significantly different (P > 0-05).

In milk fat, Sattar etal . (1977) found that the loss offl-carotene was caused by wavelengths <465 nm while vitamin A was primarily destroyed by wavelengths <415 nm. Longer wavelength, 465-750 nm, had little or no effect on fl-carotene. Sinha (1963) observed that all-trans fl-carotene irradiated with fluorescent light for 3 and 6 h showed an increased absorption between 300 and 380 nm and a consequent decrease in absorption in the visible region.

Interest in carotenoids (and vitamin A) has increased recently because of their possible association with the process and progress of carcino- genesis. Therefore, the addition of fl-carotene along with vitamin A in milk fortification may have several advantages.

[3-Carotene protective effect 365

The objective of this work was to investigate the protective effect of /3-carotene on the light-induced degradation ofall-trans retinyl palmitate in fortified milk.

MATERIALS AND METHODS

Carrier system

Corn oil was used as the carrier of all-trans retinyl palmitate and /3-carotene (type 5 and 1, respectively, Sigma Chemical Co., St Louis, MO). Corn oil, free of antioxidants, (Hain Pure Food Co., Inc., Los Angeles, CA) was purchased commercially. All-trans retinyl palmitate was added to the oil in sufficient quantity to provide approximately 2000 IU/liter after fortification of milk using 0.1% (vol./vol.) carrier system. Beta-carotene was added at two levels to give approximately 1000 and 2000 IU/liter of milk. A vitamin A fortified sample with no added /3-carotene was also produced.

Preparation of milk samples

Skim milk was prepared by separation from whole milk obtained from the University of Minnesota dairy herd. Carrier systems were added to 15 kg skim milk batches at a level of0-1% (vol./wt) carrier in milk. Milk batches were heated to about 50°C before homogenization (one stage) at 17.24 MPa (2500psi) using a Manton Gaulin Homogenizer (model 12583 MF 12A-8DSM, Everett, MA). Milk batches were then immediately HTST pasteurized (72°C, 15 s). Approximately 2 liters of pasteurized milk from each batch were collected for the study.

Light exposure

Milk samples fortified with all-trans retinyl palmitate with and without added/3-carotene were exposed in glass tubes to fluorescent light inside a light chamber. At the surface of the tube the intensity of the light was approximately 1614 lx (150 ft-c). Glass tubes were 1 cm in diameter and contained a volume of 8 ml. Tubes were completely filled to eliminate any interference from air. Light chambers (aquariums painted with flat black paint) were fitted with overhead fluorescent lights (General Electric Cool White No. F15T8-CW), and a rheostat was used to adjust light intensity as measured by a type 214 General Electric light meter. Sample tubes, closed with Teflon-lined screw caps, were laid horizontally, without agitation. Light chambers and samples were maintained at 4°C

366 M. Zahar, D. E. Smith, J. J. Warthesen

in a cold room. Samples were removed at 12, 24, 48, 72, and 96 h of exposure and analyzed for all-trans retinyl palmitate and r-carotene content.

Quantification of retinyl paimitate and fl-carotene

Extraction and HPLC analysis of retinyl palmitate and r-carotene from fortified milk samples were performed as described by Thompson et al. (1980). Quantification of all-trans retinyl palmitate and r-carotene was accomplished by comparing the peak heights of the samples to the peak heights of a standard solution of all-trans retinyl palmitate or r-carotene.

Statistical analysis

The entire experiment was repeated three times. Duplicate tubes of the same initial sample were used for each time of exposure. The design of the experiment was a split plot with duplicate samples, where the three replications of the experiment were blocks. ANOVA was used to determine the effect ofaddedfl-carotene, time of light exposure, and their interactions. The last term in the ANOVA table, subsampling, was used to evaluate the error due to the differences among the duplicate tubes. This term was found to be small when compared to error term of the whole plot.

Linear regression was used to determine the slope or rate constant of the retention ofall-trans retinyl palmitate in light-exposed samples. Log~0 values of the dependent variable (all-trans retinyl palmitate content) were used to determine the rate constant, since the semi-logarithmic relationship was found to fit better (higher P values) than the linear relationship.

To account for the variability in the level of retinyl palmitate or r-carotene at time zero within replicated experiments, ANOVA was conducted using % retention (A/Ao, i.e. amount at a given time of exposure/initial amount), while the rate constant was calculated using amounts (IU/liter of milk) and a regression model with dummy variables as described by Weisberg (1985).

All statistical analyses were tested at a 5% level of significance.

RESULTS AND DISCUSSION

Figure 1 shows per cent retention of added all-trans retinyl palmitate during exposure to light of samples with and without r-carotene. Two

fl-Carotene protective effect 367

120

n

l oo

8o T

~ 60 g

40

~ 20 I I I I

24 48 72 96

Time of exposure (hrs)

Fig. 1. Effect of added B-carotene on the retention ofall-trans retinyl palmitate (R.P.) in fortified skim milk exposed to light intensity of 1614 lx (150 ft-c) at 4°C. Samples without B-carotene (O) and with fl-carotene at level s imilar (&) or ha l f (O) the concentration of

vitamin A in IU per unit volume.

levels of fl-carotene were tested, one similar to, and the other half the concentration of vitamin A in IU per unit volume. Calculated rate constants (k) for loss ofall-trans retinyl palmitate are reported in Table 1. The k-values were calculated considering a first-order degradation of all-trans retinyl palmitate by light. Less degradation of all-trans retinyl palmitate occurred in samples with added/3-carotene in the vitamin A carrier system as compared to samples without added fl-carotene.

TABLE I Rate Constants k (h-~) ofall-trans Retinyl Palmitate Degradation in Skim Milk Fortified Using Vitamin A Carde r with and without fl-Carotene and Exposed

to Light at 1614 Ix (150 ft-c) at 4°C a

Added [3-carotene (IU/liter milk) k(h -1) SE b

None 0.00326 ~ 0-00027 1,000 0-00261 b 0.00022 2,000 0-00247 b 0.00013

a'bWithin the same column, values with different superscripts are different (P < 0"05). aCarrier system added at level 0-1% (vol./wt) to give approximately 2000 IU vitamin A/li ter milk. bSE = Standard error of the calculated k-value.

368 M. Zahar, D. E. Smith, J. J. Warthesen

TABLE 2 ANOVA for the Effect of Replication (R), Addition offl-Carotene (C), and Time of Light

Exposure (T) on the Loss of all-trans Retinyl Palmitate in Fortified Milk

Line Term DF ~ SS" M S ~ F-value p-value

1 R 2 403-53 201 '76 3"87 0' 116 2 C 2 3590"60 1795"30 34"39 0-003 3 Error I 4 208"80 52"20 4 T 4 10236-00 2559"00 83'82 0.000 5 CxT 8 116'09 14"51 0'48 0'862 6 Error 2 24 732"66 30-53 7 Error 3 45 248-44 5"52

"DF = Degrees of freedom. SS = Sum of squares, MS = Mean squared.

Analysis of variance (Table 2) indicated a significant effect (P < 0.05) for the presence of/J-carotene, at both levels studied, on the loss of all-trans retinyl palmitate by light-induced degradation in the fortified skim milk samples.

The protective effect of/j-carotene on light-induced degradation of all-trans retinyl palmitate shown in this study may partially account for the enhanced stability to light of the native vitamin A when compared to the added vitamin A as observed by several authors (Thompson & Erdody, 1974; deMan, 1981; Senyk & Shipe, 1981; Bartholomew & Ogden, 1990). In milk fat, besides the naturally present vitamin A, some /j-carotene is also present. Renner et al. (1989) listed approximate values of 10-13 and 22--32pg of carotene per 100 ml of winter and summer milk, respectively. In this study negligible amounts of/j-carotene were detected in unfortified skim milk. The presence of/j-carotene in butter oil may also contribute, to some extent, to the reduced light-induced degradation of all-trans retinyl palmitate when using butter oil as a carrier system for vitamin A compared to corn or peanut oil (Zahar et al., 1986, 1987).

Figure 2 shows the per cent retention of added/j-carotene during exposure of the fortified skim milk samples to fluorescent light. Analysis of variance of the data (Table 3) showed no significant variation of /j-carotene content with time over the period of light exposure.

All- trans retinyl palmitate was degraded more rapidly than/j-carotene during light exposure. Similar results were obtained in chloroform (Sattar et al., 1977) and milk fat (Krukovsky et al., 1943; Sattar et al., 1976)

r-Carotene protective effect 369

120

2 100

"5 ¢ . -

.9 a)

n-"

60

40

20 I 418 i I 0 24 72 96

Time of exposure (hrs)

Fig, 2, Retention offl-carotene in fortified skim milk exposed to light intensity of 1614 Ix (150 ft-c) at 4°C. Initial levels: (0 ) fl-carotene alone at 2000 IU/liter milk: (©) 1000 and

( 1 ) 2000 IU offl-carotene/l i ter milk fortified with vitamin A (2000 IU/liter).

TABLE 3 ANOVA for the Effect of Replication (R), Concentrat ion (C), and Time of Light Exposure

(T) on the Loss off l -Carotene in Fortified Milk

Line Term DF <' SS" MS<' F-value p-value

1 R 2 292.08 146,04 0"82 0-503 2 C 2 179'61 89"81 0"50 0"638 3 Error 1 4 712-93 178"23 4 T 4 251 "55 62-89 0" 15 0'962 5 CxT 8 454-97 56"87 0'13 0.997 6 Error 2 24 3948"90 426'62 7 Error 3 45 1464"50 32.54

"See Table 2.

systems exposed to light. Sattar et al. (1977) explained their results by the limited availability of oxygen in tubes of r-carotene. In the present experiment, the reduced variation in the r-carotene content observed over the period of light exposure may be due to the fact that there was less degradation of r-carotene or that isomerization of all-trans r-carotene could not be detected. Normal-phase HPLC used for r-carotene quantification does not permit the separation of the different isomers of r-carotene. During light exposure, r-carotene is subjected to a group of competing reactions. Pesek & Warthesen (1990) reported that photo-

370 M. Zahar, D. E. Smith. J. J. Warthesen

isomerizat ion reactions compete with photodegrada t ion reactions, and the p r e dominan t reaction will depend upon temperature, light intensity, and the presence of addi t ional catalysts.

C O N C L U S I O N S

Addi t ion of r -caro tene with all-trans retinyl palmitate in the carrier system had a protective effect on its photodegrada t ion in fortified skim milk. All- trans retinyl palmitate was degraded faster than r -caro tene during light exposure of the fortified skim milk samples. The/3-carotene content of milk samples was almost constant over the 4-day period of light exposure. Such protection of all-trans retinyl palmitate by r -caro tene could be utilized by the dairy industry to minimize photodegrada t ion of added vi tamin A in the fortification of fluid milk.

R E F E R E N C E S

Bartholomew, B. P. & Ogden, L. V. (1990). Effect of emulsifiers and fortification methods on light stability of vitamin A in milk. J. Dairy Sci.. 73, 1485-8.

Berge, C., Smith, D. E. & Warthesen, J. J. (1987). Effect of two antioxidants on the light stability of vitamin A in 2% fat milk. J. Dairy Sci., 70 (Suppl. 1), 60.

deMan, J. M. (1978). Possibilities of light-induced quality loss of milk. J. Inst. Can. Sci. Technol. Aliment., !1, 152-7.

deMan, J. M. (1981). Light-induced destruction of vitamin A in milk.J. Dairy Sci., 64, 2031-2.

Gaylord, A. M., Warthesen, J. J. & Smith, D. E. (1986). Influence of milk fat, milk solids, and light intensity on the light stability of vitamin A and riboflavin in lowfat milk. J. Dairy Sci., 69, 2779-84.

Hedrick, T. I. & Glass, L. (1975). Chemical changes in milk during exposure to fluorescent light. J. Milk Food TechnoL, 38, 129-34.

Krukovsky, V. N., Ellis, G. H. & Percy, W. P. (1943). The oxidation of vitamin A and carotene in milkfat. J. Dairy Sci., 26, 869-75.

Nelson, K. H. & Cathcart, W. M. (1984). Transmission of light through pigmented polyethylene milk bottles. J. Food Protect., 47, 346-52.

Pesek, C. A. & Warthesen, J. J. (1990). Kinetic model for photoisomerization and concomitant photodegradation of r-carotene. J. Agric. Food Chem., 38, 1313-15.

Renner, E., Schaafsma, G. & Scott, K. J. (1989). Micronutrients in milk. In Micronutrients in Milk and Milk-based Food Products, ed. E. Renner. Elsevier Applied Science Publishers, p. 40.

Sattar, A., deMan, J. M. & Alexander, J. C. (1976). Stability of edible oils and fats to fluorescent light irradiation. J. Am. Oil Chem. Soc., 53, 473-7.

~-Carotene protective effect 371

Sattar, A., deMan, J. M. & Alexander, J. C. (1977). Wavelength effect on light- induced decomposition of vitamin A and/3-carotene in solutions and milk fat. J. lnst. Can. Sci. Technol. Aliment., 10, 56-60.

Senyk, G. F, & Shipe, W. F. (1981). Protecting your milk from nutrient losses. Dairy Field, 3, 81-5.

Sinha, S. P. (1963). The effect of fluorescent light on the vitamin A and /3-carotene content of milk. Intern. J. Vit. Res.. 33, 262-7.

Thompson, J. N. & Erdody, P. (1974). Destruction of light of vitamin A added to milk. J. Inst. Can. Sci. Technol. Aliment., 7, 157-8.

Thompson, J. N., Hatina, G. & Maxwell, W. B. (1980). High performance liquid chromatographic determination of vitamin A in margarine, milk, partially skimmed milk, skimmed milk. J. Assoc. Off Anal. Chem.. 63, 894-8.

Weisberg, S. (1985). Model building I: defining new variables. In Applied Linear Regression. John Wiley and Sons, New York.

White, C. H. & Bulthaus, M. (1982). Light activated flavor in milk. J. Dairy Sci.. 65, 489-94.

Zahar, M. (1985). Effect of vitamin A carrier on the light stability of vitamin A in fortified lowfat milks. MS thesis, University of Minnesota, St Paul, USA.

Zahar, M., Smith, D. E. & Warthesen, J. J. (1986). Effect of carrier type and amount on vitamin A light degradation in fortified and skim milks. J. Dairy Sci., 69, 2038-44.

Zahar, M., Smith, D. E. & Warthesen, J. J. (1987). Factors related to the light stability of vitamin A in various carriers. J. Dairy Sci., 70, 13-19.