ELSEVIER

Int. Datry Journal 7 (1997) 331-339 q 1997 Elsevier Science Ltd

All rights reserved. Printed in Great Britain

PII: SO9SS-6946(97)00016-Z 0958~6946/97 $17.00+0.00

Effect of Heat Treatment, Water Activity and Storage Temperature on the Oxidative Stability of

Whole Milk Powder

Henrik Stapelfeldt a, Bo R. Nielsen” and Leif H. Skibsted””

“Department qf Dairy and Food Science, Royal Veterinar!! and Agricultural University, Rolighedswj 30, DK-1958 Frederiksberg C, Denmark

(Received 7 October 1996; accepted 6 April 1997)

ABSTRACT

The oxidative status of high-heat, medium-heat and low-heat whole milk powder was investigated at moderately accelerated storage conditions, with exposure to atmospheric air at 25 or 45°C and at three water activities (0.11, 0.23 and 0.33 at 25°C and 0. II, 0.17 and 0.31 at 45°C) for 2 months using: (i) electron spin resonance spectrometry for measurement of the level of free radicals, (ii) determination of thiobarbituric acid reactive substances (TBARS) as a measurement of secondary lipid oxidation products, (iii) size-exclusion HPLC for measurement of fluorescent protein polymerization products, and (iv) sensory evaluation. Lipid oxidation was affected greatly by storage temperature, with a maximum level of free radicals being detected after 47 days at 45°C and with the highest level in low-heat powder, irrespective of water activity. The sensory quality dropped to an unacceptable level for low-heat powder within 33 days of storage, as confirmed by measurement of TBARS, and the increasing TBARS value was parallelled by a decrease of ‘free’ thiol groups to an unmeasurable level in low-heat powder, in contrast to medium- and high- heat powders, in which the initial level of free thiol groups was only reduced by one-third after 63 days of storage. In contrast to common beliefs, initial powder quality was retained best at water activities between 0.11 and 0.23, where powders showed no significant differences in sensory quality, irrespective of preheat treatment. No difference in sensory score was found for storage at different water activities at 25°C whereas storage at water activity 0.31 at 45°C markedly affected all powders. This effect was ascribed to a combination of increased autoxidation rate and increased Maillard reaction rate giving rise to protein polymerization, in effect decreasing the powder solubility, as often seen during storage under tropical conditions. 13: 1997 Elsevier Science Ltd. All rights reserved

Keywords: whole milk powder; heat treatment; water activity; oxidative stability; storage temperature

INTRODUCTION

The preheat treatment of milk prior to the manufacture of milk powder is the major factor controlling the oxidative stability of the product, since heat treatment at high temperatures, apart from increasing the microbial safety, delays the onset of oxidized flavour, which is the limiting factor for the storage of milk powder (Rotkiewicz et al., 1979; Binder et al., 1981; Ipsen and Hansen, 1988; Baldwin et al., 1991). The increased antioxidative capacity obtained by increased heat treatment has been ascribed to protein unfolding which exposes thiol groups, especially in /I- lactoglobulin (Walstra and Jenness, 1984). The thiols (R-SH) can, due to the rather low binding energy of the S-H bond, act as hydrogen atom donors and inactivate reactive oxygen species such as lipid alkoxy and peroxy radicals, while the resulting thiyl radicals (RS.) yield inactive disulphides in chain-terminating reactions (see Huxtable, 1986).

Another important factor influencing the rate of

*Corresponding author.

autoxidation in milk powder, although less investigated, is the water activity, and Labuza et al. (1970) and Quast et al. (1972) have, based on investigations in model systems, reported reaction rate minima at a, 0.30 and 0.35, respectively. Since water activity is governed by final moisture content, milk powder structure and storage conditions, a better knowledge of the effect of water activity on autoxidation should enable milk powder to be dried to an optimal water activity in order to yield maximum oxidative stability and also minimal rate of browning processes. Notably, results obtained with milk powder deviate considerably from those obtained in model systems, and Loncin et al. (1968) found that autoxidation in unspecified milk powder, as measured by peroxide values, is stimulated by water activity below 0.11 and unaffected by water activities between this value and 0.75, while Kacyn et al. (1983) found autoxidation to increase from ‘dry’ to a water activity of 0.55 in non-fat milk powder/methyl linoleate.

We consider it timely to reinvestigate the relationship between heat treatment, water activity, storage temperature and the oxidative stability of milk powder,

331

332 H. Stapelfeldt et al.

and have embarked on a series of accelerated storage experiments with milk powder kept under different conditions. Rather than relying on a single analytical method such as measurement of thiobarbituric acid reactive substances (TBARS), we have applied several new techniques for measurement of different stages of oxidation in milk powder, together with sensory evaluation. These techniques include electron spin resonance (ESR) spectrometry, for measurement of formation of free radicals in the early stages of oxidation, and detection of fluorescent oxidation products associated with the whey proteins, which is supplemented by size-exclusion chromatography for detection of protein polymerization induced by exposure to oxo-compounds originating from lipid oxidation and non-enzymic browning. We report here on the development of chemical changes during short- term storage of milk powder using moderately elevated temperature conditions and exposure to air in order to accelerate the oxidative processes in milk powder. Moderately accelerated temperature conditions were carefully chosen since high-temperature conditions will affect the proteins and change their conformation and antioxidative properties. The use of accelerated oxidation tests at temperatures around lOO”C, as in certain widely used commercial instruments, has recently been criticized and must be considered of limited value in predicting storage stability at ambient conditions due to dramatic changes in the food protein matrix and due to non-Arrhenius behaviour of radical scavenging reactions, especially of the tocopherols (Frankel, 1993). In the second part of this study (Nielsen et al., 1997) we report the result of an investigation of a possible prediction of storage stability of milk powder from very early measurements using the physico-chemical methods characterized in the present study, as an alternative to the standard accelerated oxidation tests.

dry matter in a five-stage NIRO falling-film evaporator. The concentrate was homogenized at 35 kgcm-* and preheated to 72°C in a scraped-surface evaporator prior to spray drying (rotary atomizer at 14,000 rpm, air inlet temperature 2OO”C, outlet temperature 77°C) in a NIRO two-stage unit. The resulting milk powder with moisture content of approximately 6% ex-chamber was dried to the final moisture content in two combined vibrodized fluid beds. The gross composition of the milk powder, analysed using methods described in Sorensen et al. (1978), is given in Table 1.

Experimental design

MATERIALS AND METHODS

The storage stability of the milk powders was investigated during storage for 9 weeks in a complete 3 x 3 x 2x6 factor experiment combining the three types of milk powders (low-heat, medium-heat and high- heat) with three water activities (0.11, 0.23/0.17 and 0.33/0.31) and two storage temperatures (25°C and 45°C) and 6 days of analysis during the storage period. Results from a preliminary short-term experiment with the same design as the main experiment, but with storage for only 10 days and 4 days of analysis, was used in the planning of the main experiment. However, only the ESR result from the short-term experiment is reported. The milk powder in the main experiment was analysed at day zero (the day the milk powder was exposed to air) and then from day 33 and once a week onwards, as significant developments in the variables describing secondary oxidation and protein damage were expected after a storage period of this length (based on the result from the short-term experiment). A 4-way analysis of variance was carried out on each of the variables separately, using the GLM procedure of the SAS version 6.08 software (SAS Institute, Cary, NC). The 4-factor interaction was used as the error term since no repeated runs were made. The P-values stated in the text are the probabilities that the main effect or interaction term in question is zero.

Manufacture of powder Powder storage

Whole milk powder was manufactured in the month of July on an industrial plant (MD Foods AKAFA, DK-9230 Svenstrup J, Denmark) from bulk milk. Standardization of fat content was done by mixing the appropriate volumes of raw milk and pasteurized (72”C/2Os) skim milk prior to evaporation. Low-heat powder was manufactured from heat-treated (73”C/20 s) standardized milk which was subsequently concentrated to 46% dry matter in a four-stage Van der Ploegh falling-film evaporator. The concentrate was homogenized at 35 kgcm-* and preheated to 72°C in a scraped-surface evaporator prior to spray drying (nozzle atomizer at 220 bar, air inlet temperature 200°C outlet temperature 62°C) in a NIRO multi-stage unit (NIRO A/S, DK-2860 Soborg, Denmark). The resulting milk powder with moisture content of approximately 6% ex-chamber was dried to the final moisture content in a stationary fluid bed connected to a vibrodized fluid bed. Medium-heat and high-heat powders were manufactured from standardized milk given heat treatments of 8O”C/2Os and 88”C/2Os, respectively, and subsequently concentrated to 46%

The freshly manufactured milk powder was packed in 400-g cans under a 70% N2/30% CO* gas mixture. On the first day of the experiment, the seals were broken, and the milk powder (100 g per portion) was placed in 250-ml round-bottom flasks in desiccators, together with saturated aqueous solutions of LiCl, potassium acetate, and MgC12 as water activity buffers (Stokes and Robinson, 1949). Maintenance of constant

Table 1. Gross Composition of Milk Powder

Low-heat Medium- High-heat heat

Protein, % 27.2 21.2 26.9 Fat, % 25.9 26.3 26.1 Free fat, % 1.8 1.7 4.1 Moisture, % 2.8 2.5 3.8 Ash, % 5.3 5.4 5.6 WPNI” I.0 4.5 1.3

a Whey protein nitrogen index (mg undenatured whey protein per g powder).

O.xidatiw stability of milk powder 333

water activity was achieved by placing a battery-driven fan in the desiccators, and fast equilibration of water activity in the milk powder was secured by evacuating the desiccators to the vapour pressure of the saturated salt solutions and letting air in after 24 h. Hereafter, the desiccators and their contents were stored in a thermostat for 62 days at 25~1°C (water activity of 0.11, 0.23 and 0.33) and at 451tl”C (water activity of 0.11, 0.17 and 0.3 1) (see Greenspan, 1977).

Sensory evaluation

Milk was reconstituted by dissolving 13 g 100 mL-’ water at 40°C and keeping it at this temperature for 30min prior to cooling to 13°C at which temperature samples were presented to a three-member expert panel. Samples were presented in odourless plastic glasses and evaluated on a O&15 point scale at which 0 is ‘unacceptable’ and 15 is ‘excellent’. Scores equal to or higher than 10 indicate an acceptable sample whereas lower scores indicate defective samples; for scores lower than 10, the panellists were asked to support their score by an appropriate description of the defect.

Thiobarbituric acid reactive substances (TBARS)

TBARS were measured by the method of King (1962) and the results expressed as absorbance at 450mm and 532 mm (A4s0 and A& as measured on an HP 8452A Diode Array Spectrophotometer (Hewlett Packard, Palo Alto, California, USA).

Reactive SH groups

The content of reactive SH groups was measured using Ellman’s reagent (5,5’-dithiobis(2-nitrobenzoic acid), Merck, Darmstadt, Germany) according to the method of Koka et al. (1968) as modified by Stapelfeldt et 01. (1997). The results are expressed as absorbance at 412 nm, A4r2.

Electron spin resonance spectrometry

Milk powder was transfered to a cylindrical 710-SQ quartz ESR tube with 4mm inner diameter (Wilmad Glass Co., Buena, NJ, USA) and the tube was tapped a few times against the table. ESR experiments were carried out using an ECS 106 spectrometer (Bruker Analytische Messtechnik GmbH, Karlsruhe, Germany) equipped with an ER 4103 TM cylindrical mode X- band resonator. The result was expressed as the peak- to-peak height of the broad ESR signal, divided by the peak-to-peak height of a reference signal from a strong pitch sample, and divided by the density of the milk powder in the tube (gem-‘). Typical instrument parameters used were: sweep width 100 Gauss, microwave power 0.632 mW, modulation frequency IO0 kHz, modulation amplitude 8 Gauss, receiver gain 2x lo’, time constant 20.48 ms, conversion time X1.92ms, and total scan time 84s.

Colour measurements

The surface colour was measured with a tristimulus calorimeter (Hunterlab D25 with a D25 M sensory

head; Hunter Associates Laboratory Inc., Reston, VA, USA), calibrated against a white tile (L = 90.7, a = -0.9 and b = -0.1). The tristimulus redness parameter, Hunter u (+j-), represents red/green, the yellowness parameter, Hunter b (+ /-), represents yellow/blue, and the brightness parameter, Hunter L (+/-), represents white/black. The milk powder samples were measured in a Petri dish (11 g in a flattened, non- transparent layer, diameter 7cm, covered with black tape on the outside rim to avoid stray light) and the colour of lO.OmL milk samples was measured in a similar Petri dish.

Fluorescent oxidation products

The content of fluorescent oxidation products associated with the serum proteins (Stapelfeldt and Skibsted, 1994) was measured at 25°C with an SLM48000S spectrofluorometer (SLM Instruments Inc., Urbana, IL, USA) using 350nm excitation wavelength and measuring the fluorescence intensity at 425 nm relative to O.lOOmgL- of quinine sulphate in 0.5 M H$O+ The serum phase was obtained by slow addition of 0.10 M HCl to 10.00 mL milk to yield a pH of 4.7. The turbid solution was diluted to 100.0 mL and filtered through S&S 589 filter paper (Schleicher & Schuell, Dassel, Germany) after 30min standing, to obtain the serum phase.

Size-exclusion HPLC

Quantitative changes in whey proteins were analysed on the serum phase from analysis of fluorescent oxidation products by size-exclusion chromatography. A Tosohaas TSK-GEL G3000SWXL column (i.d. 7.8 x 300 mm; Tosohaas, Stuttgart, Germany) mounted with a TSK SWXL guard column (i.d. 6x40mm) was used for the separation. The sample injection volume was 2OpL and the column was eluted at l.OOmL mini’ with 50mM phosphate buffer, pH 6.8, 1=0.16, fed by an LKB 2150 HPLC pump with an LKB 2152 HPLC controller (LKB Products AB, Stockholm-Bromma 1, Sweden). An LKB 2152 HPLC Uvicord was used for detection at 280mm, and the HPLC peaks were integrated by a Peak Master I Chromatography Data System (Harley Systems, Princes Risborough, UK). /I- Lactoglobulin and cc-lactalbumin were tentatively identified from retention times of the pure proteins obtained from Sigma (St Louis, MO, USA).

RESULTS AND DISCUSSION

The formation of free radicals is the initial step in lipid oxidation. Abstraction of hydrogen atoms from allylic carbon atoms in unsaturated lipids generates a lipid radical and breaks the spin barrier for reaction between ground state oxygen (- 02) and the lipids. The content of free radicals in solid foods is accordingly expected to correlate with the oxidative stability, as each free radical has the potential to initiate the chain reactions of the autoxidation cycle. The potential of ESR spectroscopy to predict shelf-life of foods susceptible to oxidation was demonstrated in the short- term storage experiment. During a IO-day storage

334 H. Stapelfeldt et al.

period, the content of free radicals increased and could be attributed solely to the main effects of time, heat treatment, storage temperature and water activity (Fig. 1).

From the results of the preliminary experiment, it was expected that the long-term stability would be influenced negatively by a low preheat intensity to the milk, a high storage temperature and a high water activity during storage. While the effect of preheat treatment and storage was in qualitative agreement with earlier findings, the effect of water activity should be noted, especially since these findings were further substantiated by the techniques used to follow different stages of oxidation in the main experiment.

Free radicals

The average content of free radicals in powders increased during storage and, as may be seen from Fig. 2, the initial rate of formation of free radicals was highest at 45°C giving rise to significantly higher (P < 0.001) contents than in powders stored at 25°C.

The content of free radicals at 45°C increased to more than twice the initial content, to a maximum at 47 days of storage, after which it declined toward the end of the storage period. The appearance of a

5.8 , I

5.7 -- WC

5.24 : : : : : : : : : 1

0 20 40 80 80 100 Days

Fig. 1. Main effects of heat treatment and storage condi- tions on free radical content in milk powder as determined by electron spin resonance spectrometry during storage for 10 days. (A) Storage time. (B) Heat treatment. (C) Storage tem- perature. (D) Water activity.

5 16j-‘I’

1,,,/,,/1,1

0 7 14 21 26 35 42 49 56 63

Fig. 2. Time course of free radical formation in milk pow- der, as measured by electron spin resonance spectrometry, during storage at 25°C and 45X, respectively (pooled data for all water activities and all powders).

maximum in free radical concentration reflects conditions at which the generation of free radicals balances the conversion of radicals to non-radical products, e.g. breakdown of oxy radicals to secondary lipid oxidation products and combination of protein- based radicals. The content of free radicals is seen to increase steadily at 25°C to a level 50% higher than the initial content, and the lower overall reaction rate at this lower temperature obviously prevents the maximum from being reached within the storage time used in the current experiment. Free radicals are precursors of peroxides, and the observed time dependency is in accord with the results of Van Mil and Jans (1991) who, for whole milk powder stored in bags with 2 1% 02, found a maximum peroxide value at 18 months at 35°C and 24-27 months at 27°C while the maximum peroxide value was not reached within a 36-month storage period at 20°C.

A significant interaction (PC 0.01) between heat treatment and storage temperature was found. The free radical content differed for all heat classes at 25°C and was approximately 20% higher at 45°C at which temperature the content of free radicals was not significantly different between medium-heat and high- heat powders (Table 2A). The highest content of free radicals was found in powders stored at 45°C (Table 2B). The highest content was found in powder stored at water activity 0.33/0.31 and the lowest at water activity 0.11, but as seen from Table 3, the radical content in powder stored at water activities 0.11 and 0.23/O. 17 was significantly (P < 0.001) dependent on storage temperature.

There was also a significant interaction between water activity and heat treatment (PC O.OOl), and between water activity and storage temperature (P<O.OOl). As may be seen from Fig. 3, the concentration of free radicals was highest for the low- heat powder, independently of water activity, and for all powders the highest content was found at water activity 0.33/0.31.

These findings on the effect of water activity are in accord with the results from the short-term experiment (Fig. 1). Since free radicals are precursors for hydroperoxides, our results are, assuming a quantitative conversion to hydroperoxides, different

Table 2. Mean Content of Free Radicals (arbitrary units), as Measured by Electron Spin Resonance Spectroscopy, in Milk Powder at Different Storage Temperatures. (A) In Powders of Different Heat Classes (Pooled Data for all Water Activities and All Storage Times). (B) At Different Water Activities

(Pooled Data for All Heat Classes and All Storage Times)

(A) Preheat treatment

High-heat Low-heat Medium- heat

25°C 6.70 10.10 7.67 45°C 10.29 12.37 10.17

(B) Water activity

0.11 0.22/0.17 0.33/0.31

25°C 6.57 7.01 11.61 45°C 10.19 10.92 11.58

Oxidative stability of milk powder 335

Table 3. Selected Analyses of Milk Powders (Pooled Data for All Heat Classes and Storage Times) Stored at Different Water Activities and Temperatures. (A) Average Content of Thiobarbituric Acid Reactive Substances, as Measured by the Absorbance of the TBA-Aldehyde Adduct at 450 nm. (B) Average Hunter h-value (+ I- Represents Yellow/Blue). (C) Average Hunter L-value ( + /- represents white/black). (D) Average Content of Fluorescent Products (Excitation at 350 nm, Emission at 425 nm) Associated

with Milk Serum Proteins, Expressed as Fluorescence Intensity Relative to 0.1 ppm Quinine in 0.5 M H2S04

Water activity

Temperature (“C) 0.11 0.22/o. 17 0.33/0.31

(A) 25 0.053 0.062 0.059 45 0.136 0.133 0.223

(B) 25 13.85 13.78 13.75 45 14.21 14.01 14.97

(C) 25 74.95 75.27 74.84 45 74.56 75.11 73.24

(D) 25 0.630 0.629 0.635 45 0.678 0.669 0.806

I-

--- ~w

Low-heat

Medrum-heat *__-___ ~-

,,,.A ,. .’

Y’ High-heat

--. ~~ ..~ 0 11 0.22 0.33

a,

Fig. 3. Mean content of free radicals, as measured by elec- tron spin resonance spectrometry, in low-heat, medium-heat, and high-heat powder at different water activities (pooled data for both storage temperatures).

from those reported by Van Mil and Jans (1991) who, for 3 years’ storage of whole milk powder, found the peroxide value to increase when initial moisture content was reduced from 3.0 to 2.4%. As seen from Table 2B and Fig. 3, the content of free radicals was, apart from water activity, highly affected by storage temperature, which could explain the observed difference, at least in part. Moreover, Van Mil and Jans (1991) found different peroxide values in whole milk powder with the same WPNI, suggesting that specific protein-protein interactions and powder structure may play a role in the oxidative stability of milk powder in relation to water content/water activity.

Secondary lipid oxidation products

Lipid hydroperoxides are primary lipid oxidation products and have, together with the free radicals which precede them, practically no impact on the sensory properties. In contrast, the secondary lipid oxidation products, such as the aldehydes, formed by cleavage of the lipid hydroperoxides, have a profound impact on both sensory and functional properties.

The measurement of thiobarbituric acid reactive substances (TBARS), once thought to be specific for the secondary lipid oxidation product malondialdehyde, is the most commonly used method for evaluating the oxidative status of foods and

biological systems (Slater, 1984). Traditionally, the absorbance at 532nm of the pink pigment formed in the reaction is measured, even though only a slight amount of this pigment is formed in milk products due to their low content of polyunsaturated lipids. This led Patton and Kurtz (1955) and Jennings et al. (1955) to propose measurement of the more intensively absorbing yellow pigment at 450 nm for dairy products. Since TBARS is a measure of the formation of secondary oxidation products, e.g. carbonyls, being responsible for the sensory impact of lipid oxidation (Hall and Andersson, 1985), this method has been found to correlate well with sensory quality (Stapelfeldt et al., 1992). Secondary lipid oxidation products were accordingly evaluated by the TBARS method both as the pink (TBA532) and yellow (TBA450) reaction products. The absorbance of the yellow product was approximately 10 times higher than that of the pink, and as these analyses gave rise to similar qualitative conclusions, TBA450 was consequently preferred. In accordance with storage temperature effects on formation of free radical precursors, TBARS increased steadily during storage of milk powders at 45°C while TBARS only increased slightly at 25°C (Fig. 4).

These results are in accordance with the findings of Tuohy et al. (1981) and of Ipsen and Hansen (1988), of whom the latter found the TBA value to increase with

25°C

-4

Fig. 4. Average content of thiobarbituric acid reactive sub- stances of milk powder during storage at 25°C and 45”C, re- spectively, as measured by the absorbance of the TBA- aldehyde adduct at 450nm (pooled data for all water activ- ities and all powders).

336 H. Stapelfeldt et al.

storage temperature and to be doubled after 70 weeks of storage at 30°C compared to 20°C irrespective of heat treatment measured as WPNI.

The content of TBARS in milk powders stored at different water activities was almost identical for

25°C storage, while the increase in TBARS at 45°C could mainly be ascribed to a significantly higher (PC 0.001) content of TBARS in milk powder stored at a, 0.33/0.31 (Table 3A). This increase for a, 0.33/

0.31 may be due to major changes in the powder structure, leaving the lipids more accessible to oxygen and catalysts than at lower water activities. Surprisingly, no significant difference was found for the TBARS content of powders given different heat treatment, although the content in high-heat powders tended to be lower than in medium and low-heat powders (data not shown).

Colour changes

The onset of Maillard reactions spoils the appearance as well as the flavour of milk powder. Traditionally, the degree of Maillard reaction is measured as the content of hydroxymethyl furfural (HMF), but in this study the browning of milk powder was followed directly by tristimulus calorimetry on the powder surface and on reconstituted milk. The milk powder turned yellower during storage, as shown for measurements of the Hunter b-value in Fig. 5.

Only a small increase in Hunter b-value with storage temperature was found for milk powders stored at the lower water activities, whereas the colour change was more profound for milk powder stored at high a, , which turned more yellow at higher temperatures, as may be seen from Table 3B. The direct tristimulus calorimetric measurement provides results in agreement with the results reported by Lea and Hannan (1949) who found the rate of reaction of non-enzymatic browning in milk powder to increase with increasing a, up to 0.60.7. Not surprisingly, the non-enzymic browning, as measured directly from powder colour, was favoured by the high storage temperature, in accordance with the results of Ipsen and Hansen (1988) who found a significant increase in HMF content only at storage temperatures higher than 30°C and also with the results of Kieseker and Clarke

I IF-- F n 7 vv 2 14.4 - a,

I136-

1111111111~~llliLyyl,","~,',,,,1"",,~,','LLJ~ o 7 14 21 28 35 42 49 56 63

Days

Fig. 5. Average. Hunter b-value (+ /- represents yellow/ blue) of all milk powders during storage (pooled data for all water activities, all powders and both storage temperatures).

(1984), who found changes in HMF content to be more pronounced for high-heat powders stored at 40°C.

Interestingly, this interaction between water activity and rate of browning could not be observed by means of the b-value measured for the reconstituted milk, but only for the L-value, which showed significant (P < 0.00 1) loss of lightness of reconstituted milk when stored at a, 0.3310.31, with only minor differences between powders stored at a, 0.11 and 0.22/0.17, as illustrated in Table 3C. The insignificant change in Hunter b-value for the reconstituted milk is probably due to dilution making the changes too small to be distinguished by the calorimeter used.

Fluorescent oxidation products

Carbonyls formed as secondary oxidation products or from Maillard reactions, are known to react with amino groups in proteins forming fluorescent conjugates (see Kikugawa, 1986). At 25°C the relative fluorescence intensity was similar in all powders, but it increased at 45°C most notably (P<O.OOl) in milk powder stored at high water activity (Table 3D).

The measurement of fluorescence intensity provided results in agreement with the results from the lysine analysis of Kieseker and Clarke (1984) and Van Mil and Jans (199 I), who observed a loss of available lysine during storage of non-fat milk powder and whole milk powder, respectively, at high storage temperature. Van Mil and Jans (1991) found the effect of initial water content on decreases of lysine content to be insignificant, but this discrepancy may be caused by the narrow interval (2.43.0%) used in their study. Milk powders stored at a, 0.33/0.31 (above a water activity buffer, see Sec. 2) gradually turned sticky, and since microbial growth by even the most xerophilic moulds is limited to a, >0.60 (Troller and Christian, 1978) this stickiness and other effects observed were obviously of a chemical nature. The concomitant development of fluorescent products reflects an increase in crosslinking of whey proteins, which may explain the frequently observed decrease in solubility during storage of milk powder (Ipsen and Hansen, 1988; Kieseker and Clarke, 1984; Tuohy et al., 1981). Moreover, attempts to measure reactive SH groups failed at the end of the storage period for all powders stored at 45°C and a, 0.33/0.31 due to an incomplete salting-out of the caseins upon addition of (NH&S04. This change in protein solubility likewise indicates more general changes in protein structure, resulting from reaction of proteins with carbonyls originating from both Maillard reactions and lipid oxidation. The decrease in free radical content after passing through a maximum, as shown in Fig. 2, could also indicate recombination of protein-based radicals to yield polymers. A substantial degree of polymerization of the whey proteins with time was furthermore confirmed by size-exclusion HPLC of these powders (Fig. 6) in agreement with previous findings in a model system (Stapelfeldt and Skibsted, 1994) and in support of the observed insolubility arising during long-term storage.

Okamoto and Hayashi (1985) found no ’ major changes in lysine content and degree of browning during 1 month of storage at 40°C in milk powders for a, less than 0.23, but found major changes to occur at

Oxidative stability gf’milk powder 331

3l Day 63 2’

2 E ‘I Polymers

0 I--L__- t

AA

Day 33 2

2' E 1

o r __m_m---_._____

3. a-La Day 1

2

Z' E 1

._~>__-_d- .,, 1

0 120 240 360 480 600 720 840

Retention time (s)

Fig. 6. Size-exclusion HPLC of the whey fraction of low- heat powders stored at 45°C and a, 0.31. Prior to storage only /I-lactoglobulin (8-Lg) and cy-lactalbumin (Q-La) was de- tected in the chromatogram, but during storage protein poly- mers gradually appeared.

a, higher than 0.57, i.e. browning, loss of lysine, decreased digestibility and decreased solubility. It may thus be concluded that increased mobility of reactants at the higher water activity may be more important for these condensation reactions than the mass action law effect from the removal of water as one of the reaction products.

Sensory quality

Under standard storage conditions, whole milk powders given an appropriate preheat treatment display excellent storage stabilities when packed with inert gases, and even for high-heat milk powders bagged with atmospheric air, sensory scores do not decrease until 9-12 months of storage at 37°C (Van Mil and Jans, 1991). The sensory quality in the present study was clearly affected by an increase in storage temperature, giving rise to off-flavours from autoxidation (cardboard-like) and Maillard reaction products (caramel-like) in the reconstituted milk. The sensory quality of the powders was significantly affected by storage temperature and also by exposure to the atmosphere, since the major decrease in quality occurred within the first 33 days of storage (Fig. 7).

At 25°C the sensory quality was unaffected by water activity, whereas the sensory quality was dramatically lowered (P < 0.001) for powders stored at high water activity at 45°C (Table 4).

The overall decrease in sensory score was mainly caused by quality defects in the low-heat powder, whereas the sensory quality of medium-heat and high- heat powders was more stable (Fig. 8).

The observed pattern in quality deterioration can be

understood in terms of competing autoxidation and non-enzymatic browning, since Maillard reaction products are known to possess antioxidative properties. From the data of Ipsen and Hansen (1988), an apparent energy of activation of 55 kJmol-’ for the autoxidation of milk powders with WPNI 3.G5.7mg undenatured protein per gram powder can be calculated, whereas the apparent energy of activation for lysineealdehyde condensation has been found to be 90 kJ mol-’ (Stapelfeldt and Skibsted, 1996). The significant difference in energy of activation yields quite different temperature dependencies for the two

I

1.1 I rrr_.-LI.LII u .I_LLd 0 7 14 21 28 35 42 49 56 63

Days

Fig. 7. Time course of average sensory score (15 is perfect, 0 is unacceptable) for reconstituted milk made from milk pow- ders during storage at different temperatures (pooled data for all water activities and all powders).

Table 4. Average Sensory Score (15 is perfect, 0 is unacceptable) for Reconstituted Milk Made from Milk Powders (pooled data for all heat classes and storage times), During Storage at Different Storage Temperatures and Water

Activities

Temperature

(“C) 0.11

Water activity

0.22/0.17 0.33/0.31

25 9.33 9.35 9.43 45 8.65 8.49 5.38

6

5 i__L-_L_L_., ,,I_,, ,,I ,, l,_____i 0 7 14 21 28 35 42 49 56 63

Days

Fig. 8. Average sensory score (15 is perfect, 0 is unaccepta- ble) of reconstituted milk made from milk powders of differ- ent heat classes during storage (pooled data from different water activities and both storage temperatures).

H. Stapelfeldt et al.

interdependent types of reaction, and non-enzymic browning will consequently be strongly favoured with increase of storage temperature.

Reactive SH groups

The milk powders could clearly be differentiated with respect to WPNI (Table 1) but this index was not parallelled in the number of reactive SH groups as measured by Ellman’s reagent. The initial content of reactive SH groups was almost identical in high-heat and medium-heat powders (absorbance readings at 412 nm were 0.32 and 0.31, respectively), being four times higher than in the low-heat powder (0.08). During the first 33 days of storage, the content of reactive SH groups in all three types of powders was reduced by approximately the same absolute amount (0.08), corresponding to the onset of lipid oxidation in the low-heat powder (compare with Figs 3 and 8). In the low-heat powder, the content of reactive SH groups was reduced to a non-detectable level, in contrast with that of high-heat and medium-heat powders which still possessed a protective level that remained almost unchanged during the next 28 days of storage. As noted above, milk powders stored at 45°C and a, 0.331 0.31 turned sticky at the end of the period, thereby hampering the analysis. The statistical analysis was consequently confined to data based on the content of reactive SH groups at a, 0.11 and 0.23/O. 17, which did not differ significantly during storage. The content of reactive SH groups was slightly higher (PC 0.05) in powder stored at 45°C. The results give a good indication of the forced storage conditions, since the decrease in content of reactive SH groups corresponds to the average decrease observed for 12 months’ storage of whole milk powder in bags at 30°C (Baldwin and Ackland, 1991).

CONCLUSIONS

The shelf-life of whole milk powder clearly depends on the preheat treatment of the milk, the water activity of the’ product and the storage temperature. For all storage conditions used, the low-heat milk powder demonstrated the lowest storage stability, as it was subject to severe oxidative changes and non-enzymic browning. During accelerated storage at 45°C and with full access to atmospheric oxygen, the medium-heat and the high-heat milk powders were less affected than the low-heat powder. To the best of our knowledge, this is the first study of the effect of controlled water activity on the storage stability of whole milk powder, and in the range of water activities investigated (0.1 l- 0.33), the rate of autoxidation increased steadily with increasing water activity, i.e. a possible minimum of autoxidation corresponds to a water activity lower than 0.11. Notably, the generalization of Labuza (1971) regarding foods does not seem to hold for milk powder. However, it is known that milk powder with an extremely low moisture content is susceptible to oxidation, because the powder particles disintegrate, thereby increasing the amount of lipid exposed to air. In this context a further complication at higher temperatures and high water activity is that non- enzymic browning is promoted, and this affects the

sensory evaluation and results in browning products with antioxidative properties.

As a result, the combination of high temperature and high water activity should not be used for accelerated storage of whole milk powder with the sole purpose of evaluation of oxidative stability, but is a combination worth trying for assessment of overall milk powder resistance to tropical storage conditions, as these promote both autoxidation, non-enzymic browning and changes of functional properties such as solubility. The initiation of autoxidation is clearly forced, since a maximum of free radicals was observed by ESR spectroscopy at day 47 for storage at 45”C, as an average of all powders. The high free radical content, not seen at ambient temperature, is indicative of a high oxidation activity and can be compared with the maximum in peroxide value observed at 18 months’ storage at 35°C by Van Mil and Jans (1991). Likewise, the sensory score of the high-heat powder was affected after 33 days, which is comparable to similar changes after 12 months in the study of Van Mil and Jans (1991). Notably, this comparison shows that the processes of oxidative deterioration are accelerated by a factor of ten for a 10” C increase in temperature. Since the apparent energy of activation is only 55 kJmol_‘, as calculated from data of Ipsen and Hansen (1988), this corresponds to slightly more than a two-fold increase in reaction rate for a 10” C increase in temperature, and the forced oxidative deterioration cannot be explained as a temperature effect on autoxidation alone. The accessibility of oxygen is therefore clearly rate-limiting for the powder stored in commercial bags but not under air-accelerated storage conditions. Future research into a standard accelerated test for evaluation of shelf-life of milk powders with different preheat treatments should accordingly focus on identification of the rate-limiting step under different conditions in relation to the reaction mechanisms for autoxidation and protein damage; such work is in progress.

ACKNOWLEDGEMENTS

The research was performed as part of a cooperation project between the KVL Centre for Food Research, NIRO A/S and MD Foods AKAFA, and was sponsored by the Danish Ministry of Agriculture, the Danish Ministry of Industry and the Danish Ministry of Education as part of the F0TEK Programme. The spectrofluorometer was made available through a grant from the Danish Technical Research Council, the Danish Agricultural and Veterinary Research Council and Direktplr Ib Hknriksens Fond. The authors are grateful to Laurette Sosniecki, Astrid Beth Hansen and Karl Georgsen for excellent technical assistance.

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