Effect of Milk Fat on the Stability of Vitamin A in Ultra-High Temperature Milk

Effect of Milk Fat on the Stability of Vitamin A in Ultra-High Temperature Milk BETTY L. T. LAU, Y. K A K U D A , and D. R. A R N O T T Department of Food Science U niversity of Guelph Guelph, Ontario, Canada NIG 2W1

ABSTRACT

The stability o f vitamin A in ultra-high temperature milks with .15, 2.92, 6.16, and 9.7% fat during storage at 26°C was studied over 3 wk. Milks were fortified with synthetic retinyl palmitate to a final concentration of approximately 120 /ag retinol equivalent/100 ml milk. The four milk samples were ultra-high temperature processed and packaged in 100 ml sterile milk dilution bottles and stored in the dark at 26°C for 3 wk. Vitamin A concentrations decreased rapidly during the first 2 wk of storage then stabilized. Degradation rates during the first 2 wk were linear and varied inversely with the fat content in the milk (the more fat, the slower the rate of degradation). Final vitamin A concentrations at the end of 3 wk of storage were higher in milk with high fat and closely corresponded to the native vitamin A concentrations present in milk prior to fortification, The results indicate a possible protective effect due to fat or a difference in stability between native and synthetic vitamin A. INTRODUCTION

The advent of ultra-high temperature (UHT) processing of milk has evoked a great deal o f research in the area of milk quality. Higher processing temperatures and longer storage times (at room temperature) associated with UHTprocessed milk have raised concerns about the retention of labile nutrients and, in particular, the stability of vitamin A. Ford et al. (7), Burton et al. (3), and Gorner and Uherova (8) found insignificant losses of vitamin A during processing and subsequent storage. Ferretti et

Received January 24, 1986. 1986

J Dairy Sci 69:2052-2059

al. (6) reported storage losses but no processing loss, whereas Lembke et al. (10) reported both processing and storage losses. In a recent study by McCarthy et al. (unpublished results), concentrations of vitamin A in four batches of commercially prepared UHT milk (2% fat) decreased significantly over 15-wk when stored at 22°C. In all four batches, vitamin A decreased rapidly at first, then stabilized at approximately 15 /ag retinol/100 ml of milk. Dissolved oxygen content was monitored throughout the study, and a small but significant relationship between dissolved oxygen and the degradation o f vitamin A was observed. Le Maguer and Jackson (9) reported losses of vitamin A o f up to 7.5% in UHT milk (2% fat) after 12 wk of storage at 20°C and 36.9 to 38.5% for 28 wk. They postulated that the initial lag phase could have been caused by the effective scavenging of residual oxygen by sulfhydryl groups formed as a result of ~-lactoglobulin denaturation. These authors also observed a doubling of the vitamin A degradation when storage temperatures increased from 20 to 35°C. Cox et al. (4) studied the stability of vitamin A in pasteurized whole and skim milk. They found less vitamin A degradation in whole milk than in skim milk and suggested that natural antioxidants such as tocopherols in the milk fat improved the stability of vitamin A. Thompson and Erdody (12) showed that the vitamin A added as a supplement to whole milk was more susceptible to photodegradation than native vitamin A. deMan (5) also observed a difference in vitamin A stability in milks with three different amounts of fat. Samples with added vitamin A (skim and 2% milk) underwent greater vitamin A degradation than unfortified whole milk. These results, however, cannot be explained solely on the basis of differing stability between native and added vitamin A, because 32% of

2052

STABILITY OF VITAMIN A the native vitamin A in whole milk also was destroyed. These results suggest that the stability of vitamin A in milk is related in some way to fat concentration. This study was undertaken to investigate the relationship between concentration of milk fat and the rate of oxidation of vitamin A in UHT milk. MATERIALS A N D METHODS Preparation of Milk Samples

Milk samples with .15, 3, 6, and 10% fat were prepared by mixing skim milk with cream (approximately 35% fat). Raw cream and skim milk were obtained 1 d prior to processing from a local dairy. For practical reasons the samples were processed on 2 consecutive d; thus, two batches of skim milk and cream were used. The first batch was used to prepare the 3 and 10% samples and the second batch was used for the .15% (skim) and 6% samples. The correct mixing ratios were calculated from knowledge of the fat content of each batch of cream and skim determined by infrared analysis at the Central Milk Testing Laboratory in Guelph. After UHT processing, bottled samples were analyzed by infrared analysis to determine the exact fat concentration: .15, 2.92, 6.I6, and 9.70% fat. Raw cream was also assayed for its native vitamin A content to calculate the amount of synthetic vitamin A required to bring the total

2053

in each milk sample up to 120//g retinol equivalenffl00 ml or 400 IU/100 ml. Synthetic vitamin A (retinyl palmitate in the beadlet form, Palma-Sperse Type 250-S, Hoffmann-La Roche Ltd., Brampton, Ontario)was prepared as a concentrated dispersion in water 2 h prior to processing and kept at 4°C in the dark until added to the milk samples. Just prior to UHT processing, the amount of skim milk and cream needed to give the correct fat concentration was mixed together. Preheating and mixing times are given in Table 1. Attempts were made to have all initial vitamin A concentration at 120 /ag retinol equivalent/ 100 ml by supplementation of natural vitamin A with synthetic retinyl palmitate. However, initial analyses taken 2 h after processing showed that the actual values ranged from 110 to 124 #g/100 ml. Ultra-High Temperature Processing and Packaging

Milk samples were processed with an indirect tubular type UHT unit manufactured by Cherry Burrell Corporation, Chicago, IL, Model No-Bac Unitherm IV. Processing conditions are outlined in Table 1. The UHT-processed milk was collected manually in 100 ml sterile milk dilution bottles under a laminar air flow hood. Special precautions were observed to avoid contamination. Milk dilution bottles were loosely capped, wrapped in brown paper, and sterilized by auto-

TABLE 1. Processing conditions for uhra-high temperature (UHT) milk. Process parameters Preheating and mixing times Temperature of the milk Agitation time prior to addition of vitamin A Agitation time after the addition of vitamin A UHT Processing Temperature of the first stage heater Temperature of the second stage heater Holding time Initial cooling (with cold city water) Homogenizer pressure Final cooling (with chill water)

Conditions

4oOc 15 min 5 min 104°C 143°C 4s 40°C 10,343 kPa1 25°C

i Homogenizer pressure for 9.7% milk was 13,790 kPa. Journal of Dairy Science Vol. 69, No. 8, 1986

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LAU ET AL.

claving at 103.4 kPa and 121°C for 15 min. The paper was removed under the laminar air flow hood just prior to filling. The bottles were filled to three-fourths full, hand capped, wrapped with aluminum foil to protect against light, and placed in a 26°C incubator (60 bottles per run). Dilution bottles were not airtight, allowing oxygen to equilibrate with the milk. Samples were assayed daily during the first 2 wk of storage and then every other day for the 3rd wk. A minimum of two bottles per fat level per time interval were analyzed for vitamin A and microbial contamination. Each bottle represented one experimental unit.

peak from 2.2 min to approximately 6 min. The volume of sample injected was reduced from 100 to 40 /41 to prevent overloading the column with fat. Cleanup for the column was also changed. Instead of a 25:75 (diethyl ether:hexane) mixture, a 50:50 mixture was used. The column was purged for 15 min at 4 ml/min with the 50:50 mixture to remove adsorbed fat. Follow-

(o)

Microbiological Test

To check for microbial contamination, every bottle was visually examined for curdling. If curdling was absent, each bottle was shaken vigorously 20 times to homogenize the contents, and two 1.0-ml samples were removed with a sterile pipette for standard plate count (SPC) as outlined in Dairy Microbiological Tests, Publication 22, Ontario Department of Agriculture. Samples were plated without dilutlon and incubated at 32 C for 48 h. Another 2-ml sample was taken from the same bottle for vitamin A determination. Vitamin A results were accepted only if the plates from SPC showed no microbial growth after the specified incubation period. All samples were handled under a laminar airflow system. •

,

|

|

O

(b)

Vitamin A Analysis

Vitamin A was analyzed with the high performance liquid chromatography (HPLC) m e t h o d developed by Thompson et al. (13) with minor modifications. The solvent composition was changed from 49:49:2 (wet hexane: dry hexane:diethyl ether) to 49:49:1. Wet hexane was prepared by 50 ml of water added to 1 1 of hexane, mixed, and allowed to stand for 24 h before use. This change in solvent composition resulted in better peak resolution but required longer analysis time. A sample of commercial retinyl palmitate was separated into three peaks instead of two with this modified solvent (Figure 1). The first eluted peak was the cis isomer, the second smaller peak was unknown, and the third peak was the major alltrans isomer. The change in solvent composition increased the retention time of the trans Journal of Dairy Science Vol. 69, No. 8, 1986

0

2

RETENTION

4

6

TIME

8

(rain)

Figure 1. High performance liquid chromatography chromatograms of standard retinyl palmitate: (a) solvent composition 49:49:2, dry hexane:wet hexane: diethyl ether (13). (b) solvent composition 49:49:1, dry hexane:wet hexane:diethyl ether.

STABILITY OF VITAMIN A ing cleanup, the column was reequilibrated with the normal solvent system for 15 to 20 rain at a flow rate of 4 ml/min. High performance liquid chromatography grade hexane and spectroscopic grade diethyl ether were purchased from Fisher Chemical Company. All solvents were filtered through a .4-pM Millipore filter (Millipore Corporation, Bedford, MA). Absolute ethanol was purchased from Consolidated Alcohol, Toronto. The HPLC system (Water Associates: Milford, MA) consisted of: model 6000A solvent delivery system, a septumless U6K injector, and model 440 absorbance detector. The integrating system consisted of Spectra Physics model SP4000 central processor, model SP4020 data integrator, and model SP4050 printer/plotter. A Lichrosorb Si-100, 5-/1 particle silica column (4.6 m m x 25 cm) was purchased from Brownlee Labs, Santa Clara, CA. Solvent flow rate was 2.0 ml/min. Absorbance measurements were recorded at 313 nm at a sensitivity o f .02 absorbance units full scale and at a chart speed of 1.0 cm/min. A standard curve was prepared daily and consisted of four points. The standard vitamin A solution was prepared with approximately 5 to 6 mg of pure retinyl palmitate (1,710,000 USP units/g, Type IV) from Sigma Chemical Corporation, St. Louis, MO dissolved in 10 ml hexane in a 15-ml stoppered glass tube wrapped with aluminum foil to prevent exposure to light. This stock solution was further diluted by transferring 100 /al into another 10 ml of hexane. This second stock solution was used to prepare a series of four standard solutions with concentrations ranging from .2 to 1 /lg/ml. Absorbance of each of these vitamin A solutions was measured at 325 nm (Beckman Model 35 Spectrophotometer, Irvine, CA), and the corresponding concentration was calculated as the equivalent amount of retinol using E = 1830 (1%, 1 cm) (13). The calculation is based on the assumption that EMAX = 52,200 for retinyl esters. The standard curve was obtained by injecting 40 ~tl of each of the four solutions and regressing the t r a n s peak height on their corresponding concentration. All vitamin A concentrations reported are in micrograms of allt r a n s retinol/100 ml unless otherwise specified. Statistical analysis was performed using the general linear model procedure developed by

2055

Statistical Analysis System (SAS Institute Inc., SAS Circle, Cary, NC). RESULTS AND DISCUSSION

Initially, only two bottles per fat concentration per day were analyzed for vitamin A. However, on the 5th d of storage, one of the bottles with 2.92% fat showed unexpectedly low vitamin A. Because of this, the 2.92% samples were subsequently analyzed using three bottles per day until the end o f the 2nd wk of storage. The reason for the deviation is not known. After 2 wk of incubation, rates of vitamin A degradation slowed substantially in all samples, allowing the analysis interval to be increased to every 2nd d. There were four vitamin A values from milk with 2.92% fat that showed unexplained deviations from the rest. The data were statistically analyzed with and without these values at the 5% significance and the results are shown in Table 2. The R-square were .94 and .93, respectively. From the mean squares of the parameters, with the exception of the quadratic term of fat (fat square), all parameters in both sets of data had a significant effect on the vitamin A in the milk. Closer examination of the fat square term in the corrected set of data revealed that although the analysis showed significance, the value was much smaller than those of the other parameters, indicating a relatively small effect. It was therefore decided that the vitamin A values for d 5, 7, 8, and 9 in the 2.92% fat sample could be discarded, as they were unexpectedly lower than the rest of the data and had little effect on the analysis. Figure 2 shows the plots of the regression equations of vitamin A on days of storage in each milk from the corrected set of data as well as their 95% confidence limits. Figure 2B shows the four omitted data points lying outside the 95% confidence limits. Statistical results in Table 2 show that storage time has the greatest effect on the decrease of vitamin A. Closer examination of the coefficients of the parameters in the regression equation (Figure 2) reveals that if all other parameters were held constant and days increased by one unit, the entire term would decrease by 11.7 units, indicating that the longer the storage time, the greater the vitamin A degradation. When examining the fat term, a

Journal of Dairy Science Vol. 69, No. 8, 1986

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L A U ET AL.

TABLE 2. Effect o f fat and storage time on vitamin A ~ degradation in ultra-high temperature milk stored at 26°C for 3 wk. Source

df

Mean square ~

Mean square ~

Fat Storage time Fat square Storage time square Storage time X fat Error Total

1 1 1 1 1 62 67

4257.95* 71,594.05" 135.93 7959.00* 1149.28" 90.00

3549.18" 74,556.97* 429.08* 6595.96* 1369.62" 97.97

Based o n m e a n o f duplicate or triplicate determinations. Original data, R ~ = .94. ~ Corrected to e x c l u d e d 5, 7, 8, and 9 in milk with 2.92% fat, R ~ = .93. *P<.05.

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Figure 2. Degradation of vitamin A in ultra-high temperature milk with A) .15%, B) 2.92%, C) 6.16%, and D) 9.70% fat during storage at 26°C. • V i t a m i n A c o n t e n t (experimental), -- predicted vitamin A, --- 95% confidence limits. Predicted values were obtained from the regression equation using vitamin A c o n t e n t from all four fat concentrations. Y = 1 3 3 . 2 6 + 1 . 7 5 X a - 1 1 . 6 9 X 2 - .20X 12 + .26X22 + . 2 0 X , X 2 where Y = predicted vitamin A, Xt = percent fat in milk, X 2 = storage time, Xt 2 = fat square, X22 = storage time square, and X~X~ = fat × storage. Journal o f Dairy Science

Vol, 69, No. 8, 1986

STABILITY OF VITAMIN A

2057

TABLE 3. Percent decrease in vitamin A in ultra-high temperature milk with four concentrations of fat after 13 and 21 d of storage at 26°C. % Fat in milk Storage

.15

2.92

6.16

9.70

After 13 d After 21 d

82.71 89.95

78.81 84.62

68.11 69.95

58.07 61.86

one unit increase in percent fat (holding all other parameters constant) would cause a 1.75 unit increase in this term, implying that a higher percent fat in milk slows degradation of vitamin A. The day square term was also significant and accounts for the curvature in the vitamin A plots. The plots also show that after the initial lag period, most of the degradation of vitamin A t o o k place within the first 2 wk. Table 3 presents the percent decrease in vitamin A after 2 wk and at the end of the storage study. At the completion of the storage study, both the skim and 2.92% fat samples showed greater vitamin A degradation than the other two milks. It appears that the vitamin A in the milks with the higher fat contents was more stable. During the first 2 d o f storage, very little degradation of vitamin A was detected, but a strong cooked flavor in the milk was noticed b y the author when the bottles were opened. The cooked flavor disappeared by the 3rd or 4th d. Cooked flavor is characteristic of UHT milk and is caused by exposure o f sulfhydryt groups following denaturation of whey proteins. Rate of oxidation of these sulfhydryl groups is dependent on the availability o f oxygen and the storage temperature. In this study, it appeared that during the first few days of storage, the predominant reaction in the UHT milk was the oxidation of sulfhydryl groups with very little oxidation of vitamin A. This conclusion was also expressed by Le Maguer and Jackson (9). Another possible reason for the apparent stability o f vitamin A during the first few days o f storage could be the presence of antioxidants in the beadlets added by the manufacturer to protect the vitamin from oxidation. The lag phase also could represent the time required to build up reactive intermediates sufficient for the autoxidation of vitamin A as suggested by

Bhattacharya et al. (1) and Budowski and Bondi (2). Because virtually all the degradation t o o k place b y the end of the 2nd wk, the data were divided into two sets; the first set consisted of data for the first 2 wk and the second set consisted of data for the last week of the study. Data from the first 14 d were statistically analyzed at 5% significance. The R-square was .92 (Table 4). There was a significant relationship between the fat present and vitamin A. Figure 3 shows the predicted vitamin A values in milk during the first 2 wk of storage. F r o m the slope of the lines, it can be seen that the higher the fat content, the slower the rate of vitamin A degradation. By the 3rd wk of storage, the rate of vitamin A degradation in all samples slowed considerably. In particular, samples with 6.16 and 9.7% fat showed little change during this period. Similar results were observed by McCarthy et al. (unpublished results) with commercially prepared 2% milk. In their study, the stability

TABLE 4. Effect of percent fat and storage time on vitamin A1 degradation in ultra-high temperature milk during the first 2 wk of storage at 26°C. Source of variation

df

Mean square

Fat Storage time Fat X storage time Residue (error) Total

1 1 1 49 52

1127.52" 53,736.29* 842.79 101.19

1Based on mean of duplicate and triplicate determinations. *P<.05. Journal of Dairy Science Vol. 69, No. 8, 1986

2058

LAU ET AL.

of vitamin A in two replicates of fortified and nonfortified milk was compared. Although there was a large difference in the initial vitamin A content (15 /ag retinol/100 ml milk for unfortified versus 63 /lg/100 ml for fortified), all final concentrations ranged between 14 to 18/lg/100 ml after 15 wk. The major loss of vitamin A, however, occurred during the first 10 wk of storage. Very little decrease was observed between the 10th and 15th wk. deMan (5) and Thompson and Erdody (12) observed that a portion of the vitamin A in milk appeared to be stable to photodegradation. deMan (5) found that as the fat amount increased from skim to 2% to whole milk, the amount of vitamin A remaining after 48 h exposure to light also increased. It was suggested that the naturally present vitamin A was more stable to photodegradation than the added synthetic form. In the present study, the amount of native vitamin A in each milk sample was estimated

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from the results obtained from the initial assays performed on the raw cream. The final content of vitamin A after 3 wk of storage could then be compared with these estimates. The results show that the final content of vitamin A with 2.92, 6.16, and 9.7% fat agree closely with the native vitamin A (Table 5). Vitamin A in the 9.7% fat sample, however, had fallen slightly below the native vitamin A content, indicating some degradation of native vitamin A. In the case of skim milk with essentially no native vitamin A, the final content was the lowest of the four samples, and although the rate of degradation had slowed, further losses seem possible. From these results, it appears that the difference in the rate of degradation could be associated with the difference in the amount of native vitamin A in milk. If this is the case, then there must be some difference in stability between native and synthetic vitamin A. However, in this experiment, it was not possible to determine which form of the vitamin was being degraded. It is known that natural vitamin A in milk is associated with the fat globules (11); thus, an increase in milk fat will lead to an increase in natural vitamin A content. If the natural vitamin A is localized in the fat globules, then it may be protected from oxygen and thus appear more stable than the synthetic vitamin A. The synthetic vitamin A being dispersed in the serum phase could be preferentially oxidized due to greater contact with oxygen while the natural vitamin A would remain unaffected.

80

ACKNOWLEDGMENTS

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TABLE 5. Comparison between amounts of natural vitamin A and vitamin A remaining after 3 wk of storage for milks with .15, 2.92, 6.16, and 9.70% fat.

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All-trans retinol in milk

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Final vitamin A (after 3 wk)

.15 2.92 6.16 9.70

0 19.43 35.69 59.77

12.21 19.14 39.36 42.07

14

STORAGE

Figure 3. Predicted vitamin A in ultra-high temperature milk with a) .15, b) 2.92, 6.16, and 9.70% fat during the first 2 wk of storage at 26°C. Regression equation: Y = 132.25 - .66X 1 - 8.77X~ + .26X~Xz where Y = predicted vitamin A, X I = percent fat in milk, Xz = storage time (days), X~X~ = fat × storage time, and R 2 = .92. Journal of Dairy Science Vol. 69. No. 8, 1986

% Fat in milk

Natural vitamin A (by calculation)

STABILITY OF VITAMIN A

received f r o m t h e National Sciences and Engineering Council o f Canada and t h e O n t a r i o Ministry o f A g r i c u l t u r e a n d F o o d .

REFERENCES

1 Bhattacharya, S., N. K. Chowdhury, and U. P. Basu. 1954. Studies of vitamin A in solution. Part VI. Stability in relation to concentration and chemical forms of vitamin A and solvents. J. Ind. Chem. Soc. 31:231. 2 Budowski, P., and A. Bondi. 1960. Autoxidation of carotene and vitamin A. Influence of fat and antioxidants. Arch. Biochem. Biophys. 89:66. 3 Burton, H., J. E. Ford, A. G. Perkin, J.W.G. Porter, K. J. Scott, S. Y. Thompson, J. Toothill, and J. D. Edwards-Webb. 1970. Comparison of milks processed by the direct and indirect methods of ultrahigh-temperature sterilization. IV. The vitamin composition of milks sterilized by different processes. J. Dairy Res. 37:529. 4 Cox, D. H., S. T. Coulter, and W. O. Lundberg. 1957. Effect of nordihydroguaiaretic acid (NDGA) and other factors on stability of added vitamin A in dry and fluid milks. J. Dairy Sci. 40:564. 5 deMan, J. M. 1981. Light-induced destruction of vitamin A in milk. J. Dairy Sci. 64:2031. 6 Ferretti, L., M. E. Lelli, C. Miuccio, and C. Ragni.

2059

1970. Variazioni quantitative di alcune vitamine nel latte. U.H.T. durante la corservazione. Quad. Nutr. 30:124. 7 Ford, J. E., J.W.G. Porter, S. Y. Thompson, J. Toothill, and J. Edwards-Webb. 1969. Effects of ultra-high-temperature (UHT) processing and of subsequent storage on the vitamin content of milk. J. Dairy Res. 36:447. 8 Gorner, F., and R. Uherova. 1980. Retention yon einigen vitaminen wahrend der ultrahocherhitzung yon milch. Die Nahrung. 24:713. 9 Le Maguer, I., and H. Jackson. 1983. Stability of vitamin A in pasteurized and ultra-high temperature processed milks. J. Dairy Sci. 66:2452. 10 Lembke, V. A., H. Frahm, and K. H. Wegener. 1968. Ernahrungspbysiologische untersuchungen zur ultrahocherhitzung der milch. Kiel. Milchwirtseh. Forschungsber. 20:331. 11 Mulder, H., and P. Walstra. 1974. The fat in milk. Page 27 in The milk fat globule. Emulsion science as applied to milk products and comparable foods. Universities Press, Belfast, Northern Ireland. 12 Thompson, J. N., and P. Erdody. 1974. Destruction by light of vitamin A added to milk. J. Can. Inst. Food Sci. Technol. 7:157. 13 Thompson, J. N., G. Hatina, and W. B. Maxwell. 1980. High performance liquid chromatographic determination of vitamin A in margarine, milk, partially skimmed milk and skimmed milk. J. Assoc. Offic. Anal. Chem. 63:894.

Journal of Dairy Science Vol. 69, No. 8, 1986