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Light-induced oxidation in sliced Havarti cheese packaged in modified atmosphere
International Dairy Journal 10 (2000) 95}103
Light-induced oxidation in sliced Havarti cheese packaged in modi"ed atmosphere Dorthe Kristensen , Vibeke Orlien , Grith Mortensen, Per Brockho! , Leif H. Skibsted * Food Chemistry, Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Arla Foods amba, Innovation & Environment, Roerdrumvej 2, DK-8220 Brabrand, Denmark Department of Mathematics and Physics, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Received 13 December 1999; accepted 28 March 2000
Abstract Sliced Havarti cheese, stored at 53C for up to 21 days in retail packages with an atmosphere of 25% CO and 75% N with an initial 0.4% O exposed to light (1000 lx) or protected against light, showed an increase in redness, no signi"cant change in lightness and a tendency of decrease in yellowness measured as the aH, ¸H and bH tristimulus colour parameters, respectively. Light exposure decreased the ribo#avin content signi"cantly. The tendency of formation of free radicals in the cheese, determined in freeze-dried samples by ESR spectroscopy as an early event in lipid oxidation, decreased during the initial stages of storage to reach a minimum after four days, most rapidly in cheeses exposed to light. Whereas the lack of ability to resist lipid oxidation could not be detected in the peroxide value, sensory evaluation of odour and taste gave a clear indication of the role of light in promoting oxidation. Principal component analysis moreover showed a high correlation of the individual sensory characteristics. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Cheese; Light-induced oxidation; Modi"ed atmosphere; Sensory evaluation; Ribo#avin
1. Introduction Light is known to initiate oxidation processes resulting in discoloration and development of o!-#avours especially related to the lipid phase of foods (BekboK let, 1990). Dairy products are often exposed to light during retail storage and display and such exposure also a!ects the quality of cheese packaged in transparent "lms or plastic containers. Especially the combined action of light and oxygen was found to be harmful to Cheddar cheese wrapped in "lms with high oxygen transmission rate, as determined by the thiobarbituric acid method for detection of secondary lipid oxidation products (Hong, Wendor! & Bradley Jr., 1995a). However, protection was obtained when oxygen was excluded from Parmigiano Reggiano cheese by vacuum packaging, as no increase in lipid oxidation could be detected for storage even at
* Corresponding author. Tel.: #45-35-28-32-21; fax: #45-35-2833-44. E-mail address: [email protected] (L.H. Skibsted).
room temperature (Severini, Bressa, Romani & Dalla Rosa, 1998). For shorter storage periods lipid oxidation initiated by light exposure may be di$cult to detect by sensory evaluation, as concluded for Cheddar cheese following 14 days of display (Deger & Ashoor, 1987). However, lipid oxidation still seems to be initiated. In the search for methods to detect these early events of oxidation that may be useful for prediction of shelf-life, electron spin resonance (ESR) spectroscopy has recently been applied to processed cheese (Kristensen & Skibsted, 1999). Following storage for only 11 days it could be concluded, from signi"cant di!erences in the formation of free radicals in the product, that light rather than temperature was the important factor for the formation of radicals, which notably are precursors of peroxides as primary lipid oxidation products. Accordingly, ESR can also be explored as a method for detection of oxidative changes in cheeses with shorter shelf-life than processed cheese in which ribo#avin may play a role as photosensitizer (Deger & Ashoor, 1987). Ribo#avin is known as a sensitizer of both protein and lipid photooxidation in milk resulting in the &burnt
0958-6946/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 0 ) 0 0 0 2 8 - 5
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feather' and &cardboard' o!-#avour, respectively (BekboK let, 1990). However, the role of ribo#avin as a photosensitizer in cheeses and dairy spreads is less clear (Hansen & Skibsted, 2000), as is the importance of photodegradation of ribo#avin in such products, resulting in loss of this important nutrient. For many cheeses ribo#avin together with carotenoids are important for the colour, and a further perspective of the light-induced oxidation is the e!ect of discoloration on consumer acceptability of such products. For some products no change in appearance could be observed following retail display (Deger & Ashoor, 1987), while, other products like Cheddar cheese, with or without Annatto added as colorant, demonstrated a decrease in yellowness during storage exposed to #uorescent light, and especially when packaging "lm with a high oxygen transmission rate was used (Hong, Wendor! & Bradley Jr., 1995a, b). The varying results obtained for di!erent cheeses indicate that light sensitivity should be investigated for individual products, and that especially early indications of oxidation processes should be correlated with subsequent changes in appearance and sensory characteristics. Due to its mild #avour, rindless Havarti cheese is a rather sensitive product for which we have embarked on such investigation combining ESR spectroscopy and other analytical methods with sensory evaluation.
2. Materials and methods 2.1. Packaging and storage of cheese Sliced Havarti cheeses 55#(34% fat) were obtained from Arla Foods amba (Viby, Denmark) as part of its standard production. The slices were round with a diameter of 9 cm and a thickness of 3 mm. Each sample (200 g sliced cheese) corresponding to approximately 10}12 slices was packaged in conventional packaging materials consisting of a thermoformed transparent dome made of polyester, a thermoformed burgundy coloured polystyrene base, and with a polyester barrier layer. The oxygen transmission rate (OTR) of the dome was determined to 0.034 cm per package (24 h, 233C, 0/50% RH) determined by Teknologisk Institut, Taastrup, Denmark, according to standard method (ASTM F 1307). The OTR of the barrier layer was 60 cm per m (24 h, 233C, 5/95% RH) according to the manufacturer (Danisco #exible, Lyngby, Denmark). No labels were attached to the packages. The light transmission of the dome was determined to be between 200 and 800 nm using a Cintra 40 spectrometer (CBC Scienti"c Equipment Pty Ltd, Victoria, Australia) equipped with an integrating sphere detector. The light transmittance of the dome is shown in Fig. 1. The transparent dome was a good barrier to wavelengths shorter than 320 nm.
Fig. 1. Transmission spectrum of the cheese dome.
Wave-lengths of visible light were increasingly transmitted between 320 and 400 nm; at the latter wavelength more than 80% of the light was transmitted. Cheeses were packaged in a modi"ed atmosphere consisting of 25% CO and 75% N at a local dairy plant, and the initial residual oxygen was found to be 0.4% in the packages. The headspace of each sample was approximately 90 cm. The sliced cheeses were matured at 53C for one month in retail packages in dark storage prior to storage at 53C in a display counter under conditions similar to those in a retail store. Samples were exposed to light from Philips TLD 18/83 18 W #uorescent tubes with a light intensity of 1000 lx. Half of the packages were covered with black plastic bags in order to protect the cheeses from light. Chemical and colour measurements were performed after 0, 1, 2, 3, 4, 7, 11 and 21 days of storage, each day withdrawing two packages stored in the dark and two packages exposed to #uorescent light. Only the top slices of the packages were used for analysis. Sensory evaluations were performed after 0, 4, 11 and 21 days of storage using 15 samples stored in the dark and 15 samples exposed to #uorescent light at each time of evaluation. 2.2. Gas composition and dry matter Before the cheese domes were opened for further analyses, gas composition was analysed using a Gasspace gas analyser (Systech Instruments Ltd, Thame, UK) by penetrating a needle through the package. The dry matter of the cheese (2 g) was determined by drying for 4 h at 1053C on day 0 and 21. The results reported are average of measurements on two di!erent packages of cheese exposed to the same treatment. 2.3. Colour Surface colour of the top slice in the cheese package was measured by a Minolta tristimulus Chromometer CR-300 (Minolta Camera Co. Ltd, Osaka 541, Japan) using CIELAB ¸HaHbH values. The chromometer was standardized with a white standard plate. The results
D. Kristensen et al. / International Dairy Journal 10 (2000) 95}103
reported are average of "ve measurements on each of the two di!erent cheeses exposed to the same treatment. 2.4. Formation of free radicals Electron spin resonance (ESR) spectroscopy measurements were used to monitor formation of radicals in the Havarti cheese based on the procedure previously described (Kristensen & Skibsted, 1999): two samples stored in the dark and two samples exposed to #uorescent light were frozen (!503C, 15 h) and subsequently freeze-dried overnight (p&0.1 mbar, 24 h). Karl Fischer titration (Mettler DL18, Schwerzenbach, Switzerland) was used to determine water content in the freeze-dried samples by incubating samples in methanol ((0.005% H O, Merck, Darmstadt, Germany) for water extraction for 24 h. The freeze-dried samples were crushed in a mortar, poured into cylindrical 710-SQ quartz ESR tubes with an inner diameter of 4 mm (Wilmad Glass Co., Buena, NJ, USA), and weighed (approx. 0.260 g of sample accurate weight at a height of 4.5 cm in the glass tube) prior to measuring. Three tubes were prepared from each of the four freeze-dried samples, resulting in 2;3 samples from cheese stored in light and 2;3 samples stored in the dark. The ESR measurements were carried out with a Jerol FR30 spectrometer (Jerol, Tokyo, Japan). Typical instrument parameters used were as follows: microwave power 4 mW; centre "eld 333.718 mT; sweep width 5 mT; sweep time 8 min; modulation width 0.4 mT; time constant 1 s. The relative signal intensity was measured using a manganese internal standard. The relative heights of the signals per gram of dry matter were calculated, correcting for water content (Karl Fischer titration). The g-value of the recorded spectra was determined using a low pitch standard with a g-value of 2.0028. Sample preparation and measuring times were less than 15 min. The presented results are average of triple measurements on two di!erent cheeses exposed to the same treatment. 2.5. Lipid oxidation For peroxide determination, lipid was extracted from 2.5 g of cheese using a chloroform/methanol solution (2 : 1 v/v) following homogenisation with an Ultra Turrax homogenizer (Jankel & Kunkel IKA-Labortechnik, D-7813 Staufen, Germany). Ten mL of 1 mM CaCl was added to the homogenate which was subsequently shook vigorously for 15 s. The resulting mixture was centrifuged for 30 min, and the chloroform phase was transferred to an evaporation #ask. Thirty mL chloroform was added to the remaining aqueous phase, and the mixture was again homogenised and centrifuged, and the two extracts were pooled. The chloroform phase was dried o! using a vacuum evaporator, and the lipid was dissolved in chloroform and stored at !803C prior to analysis. The
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peroxide values were determined by a method based on an IDF standard (IDF standard 74 A: 1991, anhydrous milk fat, determination of peroxide value) entailing reaction of peroxides with iron(II) chloride and ammonium thiocyanate, followed by absorbance measurements at 500 nm after reaction for exactly 5 min, recording the red iron(III) thiocyanate complex. The peroxide values were obtained using a standard curve based on H O . The presented values are average of duplicate measurements on two di!erent cheeses exposed to the same treatment. 2.6. Riboyavin The ribo#avin content in the cheese was measured by the #uorometric method established by the Association of O$cial Analytical Chemists using an Aminco Bowman series 2 luminescence spectrometer (SLM-Aminco, Urbana, IL, USA). The presented values are average of duplicate measurements on two di!erent cheeses exposed to the same treatment. 2.7. Sensory evaluation Descriptive sensory evaluations were carried out using the top slice of each package cut into four and placed in 200 mL neutral plastic beakers marked with random 3-digit numbers. The samples were stored at 53C until 45 min prior to tasting, and subsequently placed at 14$43C until evaluation (IDF standard 99A: 1987, sensory evaluation of dairy products). The samples were served to the trained eight-member sensory panel in a randomised order, using the software program Fizz (Biosystemes, Couternon, France) to prevent carry-over and exhaustion e!ects. Evaluations were carried out in an evaluation room complying with ISO standard 8589 (1988) and ASTM STP 913 (1986). During training sessions, the panel developed the following descriptors for the sensory attributes for odour and taste of the cheese samples: sweet, buttery, rancid and sour (acidious). A sample stored in the dark was used for calibration of assessors. A scale of 15 cm (from zero"not detectable to 15"pronounced) was used. 2.8. Statistical analysis The results from measurements of gas composition, colour, radical, peroxides and ribo#avin were subjected to mixed model analysis of variance including the main e!ects and interaction of the factors, storage time and treatment (light exposure), using SAS version 6.12 software (SAS Institute Inc., Cary, NC, USA). Storage time was included as a co-variate with linear e!ect. The model included the factor &day', as well as the interaction between days and light exposure as random factors. These e!ects were included to account for various laboratory variations in the measurements. In the analysis of
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Fig. 2. Surface colour (CIELAB ¸HaHbH values) in sliced Havarti cheese during chill storage (53C) in display cabinets under continuous light exposure (*) or in the dark (䊏). Bar denotes the standard deviation.
radicals logarithms were used. Each sensory attribute was subjected to mixed model analysis of variance, in which the main e!ect of assessors and the interaction between storage time, treatment and assessors, were considered random. In this way the e!ects of storage time, treatment, and their interaction were tested versus an error term consisting of exactly the storage time-treatment-assessor interaction term. For these analyses the storage time is considered a class variable. The average sensory scores were also subjected to principal components analysis. Again the SAS system was used.
3. Results 3.1. Gas composition and water content Cheese is respiring during ripening and storage, and the balance between such processes and the oxygen transmission of the packaging material is important for the development of the packaging atmosphere. Thus, a high oxygen transmission rate may result in a signi"cant amount of oxygen in the headspace of the packaging containers available for oxidative reactions. The headspace of the cheese packages was analysed for CO and O , and no notable di!erence was found in the CO and O contents comparing the cheeses stored exposed to light and in the dark. Furthermore, composition did not change during storage. The contents of CO and
O were 36$2% and 0.023$0.013%, respectively, throughout the storage period. On the other hand, there was a signi"cant decrease in the O level observed for both treatments during the initial ripening period. The dry matter of the cheese was 61.7$0.6% on day 0, then increasing to 65.6$0.8% and 64.3$1.3% for samples stored under light and in the dark, respectively, on day 21, indicating that any in#uence of light exposure on the dry matter content was not statistically signi"cant. 3.2. Colour The colour of stored cheeses exposed to continuous light or stored in the dark was followed as CIELAB ¸H (white}black component), aH (red}green component) and bH (yellow}blue component). The exposure to light resulted in a slight, but signi"cant increase in redness (aH component) compared to cheese stored in the dark (Fig. 2). For the yellow and white components no signi"cant change was observed for the cheeses either exposed to light or protected against light, although a tendency of decreasing yellowness was notable. 3.3. Formation of free radicals Radicals could be detected in all the freeze-dried samples of cheese by ESR spectroscopy. The ESR spectra were broad and without structure and comparable with spectra recorded for freeze-dried processed cheese
D. Kristensen et al. / International Dairy Journal 10 (2000) 95}103
Fig. 3. Relative concentration of free radicals in freeze-dried Havarti cheese following chill (53C) storage of sliced Havarti cheese in display cabinets under continuous light exposure (*) or in the dark (䊏). Bar denotes the standard deviation.
presently reported (Kristensen & Skibsted, 1999) with a g-value of 2.0045, which is comparable to the g-value of 2.0046 found for processed cheese. The concentration of radicals in the freeze-dried cheese, given as the relative height of the ESR-signal normalised to 1 for day 0, as a measure of the tendency of formation of radicals in the cheese during storage is depicted in Fig. 3. A rapid decrease in the tendency of formation of radicals is noted at an early stage in the storage period for both sets of storage conditions, i.e. light exposure or storage in the dark. Notably, the highest tendency of formation of radicals was detected in cheeses stored in the dark, indicating a higher rate of transformation of radicals into non-radicals in cheeses exposed to light. During the following days a further decrease in the radical concentration was noted for the samples stored in the dark, and after four days of storage both samples reached the same low tendency of radical formation that became constant during the remaining part of the storage period. Based on statistical analyses of log-transformed data, a signi"cant interaction was found between time and light exposure (P"0.0631). 3.4. Lipid oxidation The progression in lipid oxidation was monitored by the peroxide value as an analysis of primary oxidation products. As directly seen from Fig. 4, the peroxide values were found to be rather variable, with values between 0.5 and 1.25 mEq O /kg lipid for both treat ments during the storage period, and the statistical analysis revealed no signi"cant di!erence between cheeses stored under light or in the dark. The variation found for
99
Fig. 4. Peroxide value in sliced Havarti cheese during chill storage (53C) in display cabinets under continuous light exposure (*) or in the dark (䊏). Bar denotes the standard deviation.
Fig. 5. The ribo#avin content in sliced Havarti cheese during chill storage (53C) in display cabinets under continuous light exposure (*) or in the dark (䊏). Bar denotes the standard deviation.
the peroxide value of the samples was probably due to the natural variations between the cheeses. 3.5. Riboyavin The ribo#avin content in cheeses stored in display cabinets exposed to light or protected against light is presented in Fig. 5. Continuous exposure to light resulted in a signi"cant decrease in ribo#avin content compared to the non-exposed cheeses. However, ribo#avin was not degraded instantaneously, and a signi"cant decrease in ribo#avin content in cheese exposed to light was "rst observed after storage for 11 days. On day 21, 40% of the ribo#avin originally present was retained in the top cheese slice in the package exposed to light.
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Fig. 6. Pro"ling wheel of odour (o) and taste (t) characteristic of sliced Havarti cheese during chill storage (53C) in display cabinets under continuous light exposure (thin line) or in the dark (bold line). Evaluations on day 0, 4, 11 and 21.
3.6. Sensory evaluation The results of the sensory evaluations after various times are illustrated as sensory attributes in Fig. 6. For all attributes other than sour odour there was a signi"cant treatment e!ect, i.e. e!ect of light exposure, and for all attributes other than sweet and sour odour and sour taste, a signi"cant storage time e!ect was noted, see Table 1. None of the eight sensory attributes exhibited signi"cant interactions between storage time and treatment (P-values '0.05). The analyses were performed in such a way that this was only a test for non-parallel time developments from day 4 to day 21. Since on day 0 the treatment had not started yet, a treatment di!erence will induce a non-parallel pattern from day 0 to day 4, which is not a true interaction. For the attributes showing signi"cant storage time e!ects, each combination of time point was compared by a t-test (in the mixed model), see Table 2. For sweet taste the e!ect happens only in the beginning of the storage, whereas for buttery and rancid odour and taste, signi"cant di!erences are evident between day 21 and days 4 and 11.
Table 1 P-values for the tests of treatment (light exposure) and storage time di!erences. A low P-value means a highly signi"cant di!erence. The test for treatment e!ect compares the average levels of the two treatments on day 4, 11 and 21. The storage time e!ect includes the day 0 level Sensory attribute
P-values of treatment
P-values of storage time
Sweet odour Buttery odour Rancid odour Sour odour Sweet taste Buttery taste Rancid taste Sour taste
0.0017 0.0011 0.0139 0.0820 0.0001 0.0001 0.0001 0.0040
0.0689 0.0008 0.0021 0.4060 0.0086 0.0001 0.0001 0.1233
To summarise the sensory information, a principal components analysis (PCA) was performed on the 8;8 dimensional co-variance matrix of the average scores for each combination of storage time and treatment. All eight sensory attributes appear to be highly associated with each other, as shown by the fact that 95.6% of the total variation was explained by the "rst component.
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Table 2 Signi"cances from each combination of time point of the sensory attributes showing storage time e!ects Sensory attribute
Pairwise comparison of signi"cant di!erence between di!erent days 21 vs. 0
Buttery odour Rancid odour Sweet taste Buttery taste Rancid taste
11 vs. 0
4 vs. 0
21 vs. 4
21 vs. 11
11 vs. 4
NS
NS NS NS NS NS
NS NS NS
NS
P(0.05. P(0.01. P(0.001. NS: non-signi"cant.
Table 3 Loadings of the sensory variables on principal component PC1. Since 95.6% of the variation could be accommodated in the "rst-principal component, this one-dimensional presentation is valid
Fig. 7. Scores of the "rst-principal component of the in#uence of treatment and storage time under continuous light exposure (*) or in the dark (䊏).
This means that all observations may be summarised by studying the scores of the "rst-principal component, see Fig. 7, together with the loadings for the "rst component, see Table 3. From the loadings, it appears that sweet and buttery odour and taste go together (are positively correlated) and subsequently negatively related to rancid and sour odour and taste (and those four are positively correlated). Fig. 7 shows a decrease in the "rst-principal component, which corresponds to a decrease in sweet and buttery and an increase in the rancid and sour attributes from day 0 to day 4, i.e. a decrease in the PC-score corresponds to a decrease for those variables with positive loadings and an increase for those with negative loadings and vice versa. The product of the scores and loadings expresses the magnitude of the change. In all cases the change is most apparent for light. From day 4 to day 11 there is generally no di!erence. From day 11 to day 21 a continuation of the initial decreasing/increasing structure is noted. However, now the change is the same for both treatments. 4. Discussion Oxidative changes in cheeses are initiated by the formation of free radicals, the precursors for the
Sensory attribute
Loadings
Sweet odour Buttery odour Rancid odour Sour odour Sweet taste Buttery taste Rancid taste Sour taste
0.26 0.41 !0.28 !0.18 0.36 0.49 !0.44 !0.31
hydroperoxides, which are normally regarded as primary oxidation products and accordingly used to predict oxidative stability of dairy products. Free radicals may be formed enzymatically, by transition metals catalysis, or as a result of exposure to light (Skibsted, 2000). Prediction of the sensory quality of the sliced Havarti cheese stored in a modi"ed atmosphere exposed to light or protected against light is clearly not possible using the peroxide value, as no di!erence is seen in the product protected against light and the product exposed to light (Fig. 4). However, the product exposed to light developed o!-#avours more signi"cantly than the product protected against light. Accordingly, oxidation is initiated by light, and the photosensitizer ribo#avin is partly degraded during sensitization. In contrast to the peroxide value, the tendency of formation of radicals of the cheese measured as the ESR signal of freeze-dried samples showed the di!erences between the cheese slices exposed to light and the protected slices (Fig. 3). The further decrease in the level of free radical in the cheese exposed to light is followed by a further decrease in the ribo#avin content (Fig. 5); consequently, these two analyses should be adapted for the prediction of oxidative stability of products like the Havarti cheese stored under varying
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conditions. The decrease in the tendency of formation of radicals may seem surprising when comparing to results obtained with milk powder (Stapelfeldt, Nielsen & Skibsted, 1997) which exhibited a high level of free radicals correlating with #avour deterioration. However, in the cheese, the capacity of initiation of chain reactions leading to secondary oxidation products apparently needs light to lead to the secondary oxidation products. Following light exposure, the capacity developed during the ripening of the cheese seems to be depleted resulting in lower ESR signals than was the case for products stored in the dark. The novel use of ESR spectra should certainly be explored further, since this method requires only minimal sample preparation and is easily automated. Peroxide value was also constant and at a low level between 0 and 0.2 mEq/kg in vacuum-packaged Parmigiano Reggiano cheese during storage at !25, 2 and 253C for 120 days (Severini et al., 1998). Such inhibition of lipid oxidation seems to be related to the low oxygen content in the package, as a critical level of available oxygen has been identi"ed for oxidation in cheese (Riddet, Whitehead, Robertson & Harkness, 1961). Objective colour measurement revealed that only minor colour changes were observed, and only the a-values changed signi"cantly and only for light exposed samples. The stable value of the bH-parameter may again be related to the low oxygen transmission rate of the package in the present study in contrast to the packaging material employed in the study of Cheddar (Hong et al., 1995a) although the light-exposed cheeses showed a tendency of decreasing yellowness. The light intensity may also be of importance (Deger & Ashoor, 1987), as the colour of cheese may fade more signi"cantly at light intensities higher than 1600 lx. Hence, the light intensity employed in the present study may be useful for practical display as it does not cause a signi"cant lowering of bH-values. Ribo#avin was degraded in the Havarti cheese; a result in agreement with the "ndings for Cheddar cheese packaged in transparent polyethylene wrapping during storage at 5}103C for 12 days (Deger & Ashoor, 1987). The retention of ribo#avin in Cheddar cheese with a thickness of 0.6 cm was found to be 76, 61 and 56% with light intensities of 538, 1614 and 5380 lx, respectively, showing that photodegradation of ribo#avin does not depend linearly on the light intensity (Deger & Ashoor, 1987). Sensory characteristics of the Havarti cheese were negatively in#uenced as the intensity of rancid and sour odour and taste increased with exposure to light. The di!erences between light and dark samples were evident already at the "rst evaluation time, e.g. after four days of light exposure. This di!erence was maintained throughout the experiment. It is noteworthy that light-induced degradation of ribo#avin could not be correlated with the sensory response to light, as the former was noted initially after 11 days of storage, whereas the sensory
characteristics were irretrievably impaired after a few days. 5. Conclusions The sensory attributes of sliced Havarti cheese stored under light exposure in an almost oxygen free atmosphere changed signi"cantly during the storage period. However, it appeared that the changes in the sensory attributes for cheeses exposed to light and cheeses stored in the dark were parallel to each other throughout the storage period. The colour characteristics of the cheese were only slightly impaired by light exposure. Ribo#avin was degraded under the continuous light exposure, but the degradation was not correlated with the sensory response to light. However, no direct conclusions could be made regarding the e!ect of light on lipid oxidation in the product as measured by the peroxide value. The relative high tendency of formation of radicals in the product after the initial ripening period decreased rapidly during the "rst days of the experiment and light accelerated this process. This demonstrates that ESR spectroscopy provides new information about the early events in oxidation processes in cheese. The ESR results are correlated with the sensory changes in the product; this relationship could not be found when applying the classical analysis for lipid hydroperoxides. Acknowledgements Pia Moesgaard Poulsen and Lars Ma nsson are thanked for excellent technical assistance. The work was part of a collaboration project between Arla Foods amba, Innovation & Environment, Brabrand, Denmark; Institute of Biochemistry and Nutrition, Technical University of Denmark; and Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Frederiksberg, sponsored by the Danish Ministry of Food, Agriculture and Fisheries. References BekboK let, M. (1990). Light e!ects on food. Journal of Food Protection, 53, 430}440. Deger, D., & Ashoor, S. H. (1987). Light-induced changes in taste, appearance, odour, and ribo#avin content of cheese. Journal of Dairy Science, 70, 1371}1376. Hansen, E., & Skibsted, L. H. (2000). Light-induced oxidative changes in a model dairy spread. Wavelength dependence of quantum yields and inner-"lter protection by b-carotene. Journal of Agricultural and Food Chemistry, submitted for publication. Hong, C. M., Wendor!, W. L., & Bradley Jr., R. L. (1995a). E!ects of packaging and lightning on pink discoloration and lipid oxidation of annatto-colored cheeses. Journal of Dairy Science, 78, 1896}1902. Hong, C. M., Wendor!, W. L., & Bradley Jr., R. L. (1995b). Factors a!ecting light-induced pink discoloration of annatto-colored cheese. Journal of Food Science, 60, 94}97.
D. Kristensen et al. / International Dairy Journal 10 (2000) 95}103 Kristensen, D., & Skibsted, L. H. (1999). Comparison of three methods based on electron spin resonance spectrometry for evaluation of oxidative stability of processed cheese. Journal of Agricultural and Food Chemistry, 47, 3099}3104. Riddet, W., Whitehead, H. R., Robertson, P. S., & Harkness, W. L. (1961). Fat oxidation in Cheddar cheese. Journal of Dairy Research, 28, 139}149. Severini, C., Bressa, F., Romani, S., & Dalla Rosa, M. (1998). Physical and chemical changes in vacuum packaged Parmigiano Reggiano
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cheese during storage at 25, 2 and !253C. Journal of Food Quality, 21, 355}367. Skibsted, L. H. (2000). IDF Bulletin, in press. Stapelfeldt, H., Nielsen, B. R., & Skibsted, L. H. (1997). Towards use of electron spin resonance spectrometry in quality control of milk powder. Correlation between sensory score of instant whole milk powders and concentration of free radicals and 2thiobarbituric acid reactive substances. Milchwissenschaft, 52, 682}685.
Light-induced oxidation in sliced Havarti cheese packaged in modi"ed atmosphere Dorthe Kristensen , Vibeke Orlien , Grith Mortensen, Per Brockho! , Leif H. Skibsted * Food Chemistry, Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Arla Foods amba, Innovation & Environment, Roerdrumvej 2, DK-8220 Brabrand, Denmark Department of Mathematics and Physics, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Received 13 December 1999; accepted 28 March 2000
Abstract Sliced Havarti cheese, stored at 53C for up to 21 days in retail packages with an atmosphere of 25% CO and 75% N with an initial 0.4% O exposed to light (1000 lx) or protected against light, showed an increase in redness, no signi"cant change in lightness and a tendency of decrease in yellowness measured as the aH, ¸H and bH tristimulus colour parameters, respectively. Light exposure decreased the ribo#avin content signi"cantly. The tendency of formation of free radicals in the cheese, determined in freeze-dried samples by ESR spectroscopy as an early event in lipid oxidation, decreased during the initial stages of storage to reach a minimum after four days, most rapidly in cheeses exposed to light. Whereas the lack of ability to resist lipid oxidation could not be detected in the peroxide value, sensory evaluation of odour and taste gave a clear indication of the role of light in promoting oxidation. Principal component analysis moreover showed a high correlation of the individual sensory characteristics. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Cheese; Light-induced oxidation; Modi"ed atmosphere; Sensory evaluation; Ribo#avin
1. Introduction Light is known to initiate oxidation processes resulting in discoloration and development of o!-#avours especially related to the lipid phase of foods (BekboK let, 1990). Dairy products are often exposed to light during retail storage and display and such exposure also a!ects the quality of cheese packaged in transparent "lms or plastic containers. Especially the combined action of light and oxygen was found to be harmful to Cheddar cheese wrapped in "lms with high oxygen transmission rate, as determined by the thiobarbituric acid method for detection of secondary lipid oxidation products (Hong, Wendor! & Bradley Jr., 1995a). However, protection was obtained when oxygen was excluded from Parmigiano Reggiano cheese by vacuum packaging, as no increase in lipid oxidation could be detected for storage even at
* Corresponding author. Tel.: #45-35-28-32-21; fax: #45-35-2833-44. E-mail address: [email protected] (L.H. Skibsted).
room temperature (Severini, Bressa, Romani & Dalla Rosa, 1998). For shorter storage periods lipid oxidation initiated by light exposure may be di$cult to detect by sensory evaluation, as concluded for Cheddar cheese following 14 days of display (Deger & Ashoor, 1987). However, lipid oxidation still seems to be initiated. In the search for methods to detect these early events of oxidation that may be useful for prediction of shelf-life, electron spin resonance (ESR) spectroscopy has recently been applied to processed cheese (Kristensen & Skibsted, 1999). Following storage for only 11 days it could be concluded, from signi"cant di!erences in the formation of free radicals in the product, that light rather than temperature was the important factor for the formation of radicals, which notably are precursors of peroxides as primary lipid oxidation products. Accordingly, ESR can also be explored as a method for detection of oxidative changes in cheeses with shorter shelf-life than processed cheese in which ribo#avin may play a role as photosensitizer (Deger & Ashoor, 1987). Ribo#avin is known as a sensitizer of both protein and lipid photooxidation in milk resulting in the &burnt
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feather' and &cardboard' o!-#avour, respectively (BekboK let, 1990). However, the role of ribo#avin as a photosensitizer in cheeses and dairy spreads is less clear (Hansen & Skibsted, 2000), as is the importance of photodegradation of ribo#avin in such products, resulting in loss of this important nutrient. For many cheeses ribo#avin together with carotenoids are important for the colour, and a further perspective of the light-induced oxidation is the e!ect of discoloration on consumer acceptability of such products. For some products no change in appearance could be observed following retail display (Deger & Ashoor, 1987), while, other products like Cheddar cheese, with or without Annatto added as colorant, demonstrated a decrease in yellowness during storage exposed to #uorescent light, and especially when packaging "lm with a high oxygen transmission rate was used (Hong, Wendor! & Bradley Jr., 1995a, b). The varying results obtained for di!erent cheeses indicate that light sensitivity should be investigated for individual products, and that especially early indications of oxidation processes should be correlated with subsequent changes in appearance and sensory characteristics. Due to its mild #avour, rindless Havarti cheese is a rather sensitive product for which we have embarked on such investigation combining ESR spectroscopy and other analytical methods with sensory evaluation.
2. Materials and methods 2.1. Packaging and storage of cheese Sliced Havarti cheeses 55#(34% fat) were obtained from Arla Foods amba (Viby, Denmark) as part of its standard production. The slices were round with a diameter of 9 cm and a thickness of 3 mm. Each sample (200 g sliced cheese) corresponding to approximately 10}12 slices was packaged in conventional packaging materials consisting of a thermoformed transparent dome made of polyester, a thermoformed burgundy coloured polystyrene base, and with a polyester barrier layer. The oxygen transmission rate (OTR) of the dome was determined to 0.034 cm per package (24 h, 233C, 0/50% RH) determined by Teknologisk Institut, Taastrup, Denmark, according to standard method (ASTM F 1307). The OTR of the barrier layer was 60 cm per m (24 h, 233C, 5/95% RH) according to the manufacturer (Danisco #exible, Lyngby, Denmark). No labels were attached to the packages. The light transmission of the dome was determined to be between 200 and 800 nm using a Cintra 40 spectrometer (CBC Scienti"c Equipment Pty Ltd, Victoria, Australia) equipped with an integrating sphere detector. The light transmittance of the dome is shown in Fig. 1. The transparent dome was a good barrier to wavelengths shorter than 320 nm.
Fig. 1. Transmission spectrum of the cheese dome.
Wave-lengths of visible light were increasingly transmitted between 320 and 400 nm; at the latter wavelength more than 80% of the light was transmitted. Cheeses were packaged in a modi"ed atmosphere consisting of 25% CO and 75% N at a local dairy plant, and the initial residual oxygen was found to be 0.4% in the packages. The headspace of each sample was approximately 90 cm. The sliced cheeses were matured at 53C for one month in retail packages in dark storage prior to storage at 53C in a display counter under conditions similar to those in a retail store. Samples were exposed to light from Philips TLD 18/83 18 W #uorescent tubes with a light intensity of 1000 lx. Half of the packages were covered with black plastic bags in order to protect the cheeses from light. Chemical and colour measurements were performed after 0, 1, 2, 3, 4, 7, 11 and 21 days of storage, each day withdrawing two packages stored in the dark and two packages exposed to #uorescent light. Only the top slices of the packages were used for analysis. Sensory evaluations were performed after 0, 4, 11 and 21 days of storage using 15 samples stored in the dark and 15 samples exposed to #uorescent light at each time of evaluation. 2.2. Gas composition and dry matter Before the cheese domes were opened for further analyses, gas composition was analysed using a Gasspace gas analyser (Systech Instruments Ltd, Thame, UK) by penetrating a needle through the package. The dry matter of the cheese (2 g) was determined by drying for 4 h at 1053C on day 0 and 21. The results reported are average of measurements on two di!erent packages of cheese exposed to the same treatment. 2.3. Colour Surface colour of the top slice in the cheese package was measured by a Minolta tristimulus Chromometer CR-300 (Minolta Camera Co. Ltd, Osaka 541, Japan) using CIELAB ¸HaHbH values. The chromometer was standardized with a white standard plate. The results
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reported are average of "ve measurements on each of the two di!erent cheeses exposed to the same treatment. 2.4. Formation of free radicals Electron spin resonance (ESR) spectroscopy measurements were used to monitor formation of radicals in the Havarti cheese based on the procedure previously described (Kristensen & Skibsted, 1999): two samples stored in the dark and two samples exposed to #uorescent light were frozen (!503C, 15 h) and subsequently freeze-dried overnight (p&0.1 mbar, 24 h). Karl Fischer titration (Mettler DL18, Schwerzenbach, Switzerland) was used to determine water content in the freeze-dried samples by incubating samples in methanol ((0.005% H O, Merck, Darmstadt, Germany) for water extraction for 24 h. The freeze-dried samples were crushed in a mortar, poured into cylindrical 710-SQ quartz ESR tubes with an inner diameter of 4 mm (Wilmad Glass Co., Buena, NJ, USA), and weighed (approx. 0.260 g of sample accurate weight at a height of 4.5 cm in the glass tube) prior to measuring. Three tubes were prepared from each of the four freeze-dried samples, resulting in 2;3 samples from cheese stored in light and 2;3 samples stored in the dark. The ESR measurements were carried out with a Jerol FR30 spectrometer (Jerol, Tokyo, Japan). Typical instrument parameters used were as follows: microwave power 4 mW; centre "eld 333.718 mT; sweep width 5 mT; sweep time 8 min; modulation width 0.4 mT; time constant 1 s. The relative signal intensity was measured using a manganese internal standard. The relative heights of the signals per gram of dry matter were calculated, correcting for water content (Karl Fischer titration). The g-value of the recorded spectra was determined using a low pitch standard with a g-value of 2.0028. Sample preparation and measuring times were less than 15 min. The presented results are average of triple measurements on two di!erent cheeses exposed to the same treatment. 2.5. Lipid oxidation For peroxide determination, lipid was extracted from 2.5 g of cheese using a chloroform/methanol solution (2 : 1 v/v) following homogenisation with an Ultra Turrax homogenizer (Jankel & Kunkel IKA-Labortechnik, D-7813 Staufen, Germany). Ten mL of 1 mM CaCl was added to the homogenate which was subsequently shook vigorously for 15 s. The resulting mixture was centrifuged for 30 min, and the chloroform phase was transferred to an evaporation #ask. Thirty mL chloroform was added to the remaining aqueous phase, and the mixture was again homogenised and centrifuged, and the two extracts were pooled. The chloroform phase was dried o! using a vacuum evaporator, and the lipid was dissolved in chloroform and stored at !803C prior to analysis. The
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peroxide values were determined by a method based on an IDF standard (IDF standard 74 A: 1991, anhydrous milk fat, determination of peroxide value) entailing reaction of peroxides with iron(II) chloride and ammonium thiocyanate, followed by absorbance measurements at 500 nm after reaction for exactly 5 min, recording the red iron(III) thiocyanate complex. The peroxide values were obtained using a standard curve based on H O . The presented values are average of duplicate measurements on two di!erent cheeses exposed to the same treatment. 2.6. Riboyavin The ribo#avin content in the cheese was measured by the #uorometric method established by the Association of O$cial Analytical Chemists using an Aminco Bowman series 2 luminescence spectrometer (SLM-Aminco, Urbana, IL, USA). The presented values are average of duplicate measurements on two di!erent cheeses exposed to the same treatment. 2.7. Sensory evaluation Descriptive sensory evaluations were carried out using the top slice of each package cut into four and placed in 200 mL neutral plastic beakers marked with random 3-digit numbers. The samples were stored at 53C until 45 min prior to tasting, and subsequently placed at 14$43C until evaluation (IDF standard 99A: 1987, sensory evaluation of dairy products). The samples were served to the trained eight-member sensory panel in a randomised order, using the software program Fizz (Biosystemes, Couternon, France) to prevent carry-over and exhaustion e!ects. Evaluations were carried out in an evaluation room complying with ISO standard 8589 (1988) and ASTM STP 913 (1986). During training sessions, the panel developed the following descriptors for the sensory attributes for odour and taste of the cheese samples: sweet, buttery, rancid and sour (acidious). A sample stored in the dark was used for calibration of assessors. A scale of 15 cm (from zero"not detectable to 15"pronounced) was used. 2.8. Statistical analysis The results from measurements of gas composition, colour, radical, peroxides and ribo#avin were subjected to mixed model analysis of variance including the main e!ects and interaction of the factors, storage time and treatment (light exposure), using SAS version 6.12 software (SAS Institute Inc., Cary, NC, USA). Storage time was included as a co-variate with linear e!ect. The model included the factor &day', as well as the interaction between days and light exposure as random factors. These e!ects were included to account for various laboratory variations in the measurements. In the analysis of
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Fig. 2. Surface colour (CIELAB ¸HaHbH values) in sliced Havarti cheese during chill storage (53C) in display cabinets under continuous light exposure (*) or in the dark (䊏). Bar denotes the standard deviation.
radicals logarithms were used. Each sensory attribute was subjected to mixed model analysis of variance, in which the main e!ect of assessors and the interaction between storage time, treatment and assessors, were considered random. In this way the e!ects of storage time, treatment, and their interaction were tested versus an error term consisting of exactly the storage time-treatment-assessor interaction term. For these analyses the storage time is considered a class variable. The average sensory scores were also subjected to principal components analysis. Again the SAS system was used.
3. Results 3.1. Gas composition and water content Cheese is respiring during ripening and storage, and the balance between such processes and the oxygen transmission of the packaging material is important for the development of the packaging atmosphere. Thus, a high oxygen transmission rate may result in a signi"cant amount of oxygen in the headspace of the packaging containers available for oxidative reactions. The headspace of the cheese packages was analysed for CO and O , and no notable di!erence was found in the CO and O contents comparing the cheeses stored exposed to light and in the dark. Furthermore, composition did not change during storage. The contents of CO and
O were 36$2% and 0.023$0.013%, respectively, throughout the storage period. On the other hand, there was a signi"cant decrease in the O level observed for both treatments during the initial ripening period. The dry matter of the cheese was 61.7$0.6% on day 0, then increasing to 65.6$0.8% and 64.3$1.3% for samples stored under light and in the dark, respectively, on day 21, indicating that any in#uence of light exposure on the dry matter content was not statistically signi"cant. 3.2. Colour The colour of stored cheeses exposed to continuous light or stored in the dark was followed as CIELAB ¸H (white}black component), aH (red}green component) and bH (yellow}blue component). The exposure to light resulted in a slight, but signi"cant increase in redness (aH component) compared to cheese stored in the dark (Fig. 2). For the yellow and white components no signi"cant change was observed for the cheeses either exposed to light or protected against light, although a tendency of decreasing yellowness was notable. 3.3. Formation of free radicals Radicals could be detected in all the freeze-dried samples of cheese by ESR spectroscopy. The ESR spectra were broad and without structure and comparable with spectra recorded for freeze-dried processed cheese
D. Kristensen et al. / International Dairy Journal 10 (2000) 95}103
Fig. 3. Relative concentration of free radicals in freeze-dried Havarti cheese following chill (53C) storage of sliced Havarti cheese in display cabinets under continuous light exposure (*) or in the dark (䊏). Bar denotes the standard deviation.
presently reported (Kristensen & Skibsted, 1999) with a g-value of 2.0045, which is comparable to the g-value of 2.0046 found for processed cheese. The concentration of radicals in the freeze-dried cheese, given as the relative height of the ESR-signal normalised to 1 for day 0, as a measure of the tendency of formation of radicals in the cheese during storage is depicted in Fig. 3. A rapid decrease in the tendency of formation of radicals is noted at an early stage in the storage period for both sets of storage conditions, i.e. light exposure or storage in the dark. Notably, the highest tendency of formation of radicals was detected in cheeses stored in the dark, indicating a higher rate of transformation of radicals into non-radicals in cheeses exposed to light. During the following days a further decrease in the radical concentration was noted for the samples stored in the dark, and after four days of storage both samples reached the same low tendency of radical formation that became constant during the remaining part of the storage period. Based on statistical analyses of log-transformed data, a signi"cant interaction was found between time and light exposure (P"0.0631). 3.4. Lipid oxidation The progression in lipid oxidation was monitored by the peroxide value as an analysis of primary oxidation products. As directly seen from Fig. 4, the peroxide values were found to be rather variable, with values between 0.5 and 1.25 mEq O /kg lipid for both treat ments during the storage period, and the statistical analysis revealed no signi"cant di!erence between cheeses stored under light or in the dark. The variation found for
99
Fig. 4. Peroxide value in sliced Havarti cheese during chill storage (53C) in display cabinets under continuous light exposure (*) or in the dark (䊏). Bar denotes the standard deviation.
Fig. 5. The ribo#avin content in sliced Havarti cheese during chill storage (53C) in display cabinets under continuous light exposure (*) or in the dark (䊏). Bar denotes the standard deviation.
the peroxide value of the samples was probably due to the natural variations between the cheeses. 3.5. Riboyavin The ribo#avin content in cheeses stored in display cabinets exposed to light or protected against light is presented in Fig. 5. Continuous exposure to light resulted in a signi"cant decrease in ribo#avin content compared to the non-exposed cheeses. However, ribo#avin was not degraded instantaneously, and a signi"cant decrease in ribo#avin content in cheese exposed to light was "rst observed after storage for 11 days. On day 21, 40% of the ribo#avin originally present was retained in the top cheese slice in the package exposed to light.
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Fig. 6. Pro"ling wheel of odour (o) and taste (t) characteristic of sliced Havarti cheese during chill storage (53C) in display cabinets under continuous light exposure (thin line) or in the dark (bold line). Evaluations on day 0, 4, 11 and 21.
3.6. Sensory evaluation The results of the sensory evaluations after various times are illustrated as sensory attributes in Fig. 6. For all attributes other than sour odour there was a signi"cant treatment e!ect, i.e. e!ect of light exposure, and for all attributes other than sweet and sour odour and sour taste, a signi"cant storage time e!ect was noted, see Table 1. None of the eight sensory attributes exhibited signi"cant interactions between storage time and treatment (P-values '0.05). The analyses were performed in such a way that this was only a test for non-parallel time developments from day 4 to day 21. Since on day 0 the treatment had not started yet, a treatment di!erence will induce a non-parallel pattern from day 0 to day 4, which is not a true interaction. For the attributes showing signi"cant storage time e!ects, each combination of time point was compared by a t-test (in the mixed model), see Table 2. For sweet taste the e!ect happens only in the beginning of the storage, whereas for buttery and rancid odour and taste, signi"cant di!erences are evident between day 21 and days 4 and 11.
Table 1 P-values for the tests of treatment (light exposure) and storage time di!erences. A low P-value means a highly signi"cant di!erence. The test for treatment e!ect compares the average levels of the two treatments on day 4, 11 and 21. The storage time e!ect includes the day 0 level Sensory attribute
P-values of treatment
P-values of storage time
Sweet odour Buttery odour Rancid odour Sour odour Sweet taste Buttery taste Rancid taste Sour taste
0.0017 0.0011 0.0139 0.0820 0.0001 0.0001 0.0001 0.0040
0.0689 0.0008 0.0021 0.4060 0.0086 0.0001 0.0001 0.1233
To summarise the sensory information, a principal components analysis (PCA) was performed on the 8;8 dimensional co-variance matrix of the average scores for each combination of storage time and treatment. All eight sensory attributes appear to be highly associated with each other, as shown by the fact that 95.6% of the total variation was explained by the "rst component.
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Table 2 Signi"cances from each combination of time point of the sensory attributes showing storage time e!ects Sensory attribute
Pairwise comparison of signi"cant di!erence between di!erent days 21 vs. 0
Buttery odour Rancid odour Sweet taste Buttery taste Rancid taste
11 vs. 0
4 vs. 0
21 vs. 4
21 vs. 11
11 vs. 4
NS
NS NS NS NS NS
NS NS NS
NS
P(0.05. P(0.01. P(0.001. NS: non-signi"cant.
Table 3 Loadings of the sensory variables on principal component PC1. Since 95.6% of the variation could be accommodated in the "rst-principal component, this one-dimensional presentation is valid
Fig. 7. Scores of the "rst-principal component of the in#uence of treatment and storage time under continuous light exposure (*) or in the dark (䊏).
This means that all observations may be summarised by studying the scores of the "rst-principal component, see Fig. 7, together with the loadings for the "rst component, see Table 3. From the loadings, it appears that sweet and buttery odour and taste go together (are positively correlated) and subsequently negatively related to rancid and sour odour and taste (and those four are positively correlated). Fig. 7 shows a decrease in the "rst-principal component, which corresponds to a decrease in sweet and buttery and an increase in the rancid and sour attributes from day 0 to day 4, i.e. a decrease in the PC-score corresponds to a decrease for those variables with positive loadings and an increase for those with negative loadings and vice versa. The product of the scores and loadings expresses the magnitude of the change. In all cases the change is most apparent for light. From day 4 to day 11 there is generally no di!erence. From day 11 to day 21 a continuation of the initial decreasing/increasing structure is noted. However, now the change is the same for both treatments. 4. Discussion Oxidative changes in cheeses are initiated by the formation of free radicals, the precursors for the
Sensory attribute
Loadings
Sweet odour Buttery odour Rancid odour Sour odour Sweet taste Buttery taste Rancid taste Sour taste
0.26 0.41 !0.28 !0.18 0.36 0.49 !0.44 !0.31
hydroperoxides, which are normally regarded as primary oxidation products and accordingly used to predict oxidative stability of dairy products. Free radicals may be formed enzymatically, by transition metals catalysis, or as a result of exposure to light (Skibsted, 2000). Prediction of the sensory quality of the sliced Havarti cheese stored in a modi"ed atmosphere exposed to light or protected against light is clearly not possible using the peroxide value, as no di!erence is seen in the product protected against light and the product exposed to light (Fig. 4). However, the product exposed to light developed o!-#avours more signi"cantly than the product protected against light. Accordingly, oxidation is initiated by light, and the photosensitizer ribo#avin is partly degraded during sensitization. In contrast to the peroxide value, the tendency of formation of radicals of the cheese measured as the ESR signal of freeze-dried samples showed the di!erences between the cheese slices exposed to light and the protected slices (Fig. 3). The further decrease in the level of free radical in the cheese exposed to light is followed by a further decrease in the ribo#avin content (Fig. 5); consequently, these two analyses should be adapted for the prediction of oxidative stability of products like the Havarti cheese stored under varying
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conditions. The decrease in the tendency of formation of radicals may seem surprising when comparing to results obtained with milk powder (Stapelfeldt, Nielsen & Skibsted, 1997) which exhibited a high level of free radicals correlating with #avour deterioration. However, in the cheese, the capacity of initiation of chain reactions leading to secondary oxidation products apparently needs light to lead to the secondary oxidation products. Following light exposure, the capacity developed during the ripening of the cheese seems to be depleted resulting in lower ESR signals than was the case for products stored in the dark. The novel use of ESR spectra should certainly be explored further, since this method requires only minimal sample preparation and is easily automated. Peroxide value was also constant and at a low level between 0 and 0.2 mEq/kg in vacuum-packaged Parmigiano Reggiano cheese during storage at !25, 2 and 253C for 120 days (Severini et al., 1998). Such inhibition of lipid oxidation seems to be related to the low oxygen content in the package, as a critical level of available oxygen has been identi"ed for oxidation in cheese (Riddet, Whitehead, Robertson & Harkness, 1961). Objective colour measurement revealed that only minor colour changes were observed, and only the a-values changed signi"cantly and only for light exposed samples. The stable value of the bH-parameter may again be related to the low oxygen transmission rate of the package in the present study in contrast to the packaging material employed in the study of Cheddar (Hong et al., 1995a) although the light-exposed cheeses showed a tendency of decreasing yellowness. The light intensity may also be of importance (Deger & Ashoor, 1987), as the colour of cheese may fade more signi"cantly at light intensities higher than 1600 lx. Hence, the light intensity employed in the present study may be useful for practical display as it does not cause a signi"cant lowering of bH-values. Ribo#avin was degraded in the Havarti cheese; a result in agreement with the "ndings for Cheddar cheese packaged in transparent polyethylene wrapping during storage at 5}103C for 12 days (Deger & Ashoor, 1987). The retention of ribo#avin in Cheddar cheese with a thickness of 0.6 cm was found to be 76, 61 and 56% with light intensities of 538, 1614 and 5380 lx, respectively, showing that photodegradation of ribo#avin does not depend linearly on the light intensity (Deger & Ashoor, 1987). Sensory characteristics of the Havarti cheese were negatively in#uenced as the intensity of rancid and sour odour and taste increased with exposure to light. The di!erences between light and dark samples were evident already at the "rst evaluation time, e.g. after four days of light exposure. This di!erence was maintained throughout the experiment. It is noteworthy that light-induced degradation of ribo#avin could not be correlated with the sensory response to light, as the former was noted initially after 11 days of storage, whereas the sensory
characteristics were irretrievably impaired after a few days. 5. Conclusions The sensory attributes of sliced Havarti cheese stored under light exposure in an almost oxygen free atmosphere changed signi"cantly during the storage period. However, it appeared that the changes in the sensory attributes for cheeses exposed to light and cheeses stored in the dark were parallel to each other throughout the storage period. The colour characteristics of the cheese were only slightly impaired by light exposure. Ribo#avin was degraded under the continuous light exposure, but the degradation was not correlated with the sensory response to light. However, no direct conclusions could be made regarding the e!ect of light on lipid oxidation in the product as measured by the peroxide value. The relative high tendency of formation of radicals in the product after the initial ripening period decreased rapidly during the "rst days of the experiment and light accelerated this process. This demonstrates that ESR spectroscopy provides new information about the early events in oxidation processes in cheese. The ESR results are correlated with the sensory changes in the product; this relationship could not be found when applying the classical analysis for lipid hydroperoxides. Acknowledgements Pia Moesgaard Poulsen and Lars Ma nsson are thanked for excellent technical assistance. The work was part of a collaboration project between Arla Foods amba, Innovation & Environment, Brabrand, Denmark; Institute of Biochemistry and Nutrition, Technical University of Denmark; and Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Frederiksberg, sponsored by the Danish Ministry of Food, Agriculture and Fisheries. References BekboK let, M. (1990). Light e!ects on food. Journal of Food Protection, 53, 430}440. Deger, D., & Ashoor, S. H. (1987). Light-induced changes in taste, appearance, odour, and ribo#avin content of cheese. Journal of Dairy Science, 70, 1371}1376. Hansen, E., & Skibsted, L. H. (2000). Light-induced oxidative changes in a model dairy spread. Wavelength dependence of quantum yields and inner-"lter protection by b-carotene. Journal of Agricultural and Food Chemistry, submitted for publication. Hong, C. M., Wendor!, W. L., & Bradley Jr., R. L. (1995a). E!ects of packaging and lightning on pink discoloration and lipid oxidation of annatto-colored cheeses. Journal of Dairy Science, 78, 1896}1902. Hong, C. M., Wendor!, W. L., & Bradley Jr., R. L. (1995b). Factors a!ecting light-induced pink discoloration of annatto-colored cheese. Journal of Food Science, 60, 94}97.
D. Kristensen et al. / International Dairy Journal 10 (2000) 95}103 Kristensen, D., & Skibsted, L. H. (1999). Comparison of three methods based on electron spin resonance spectrometry for evaluation of oxidative stability of processed cheese. Journal of Agricultural and Food Chemistry, 47, 3099}3104. Riddet, W., Whitehead, H. R., Robertson, P. S., & Harkness, W. L. (1961). Fat oxidation in Cheddar cheese. Journal of Dairy Research, 28, 139}149. Severini, C., Bressa, F., Romani, S., & Dalla Rosa, M. (1998). Physical and chemical changes in vacuum packaged Parmigiano Reggiano
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cheese during storage at 25, 2 and !253C. Journal of Food Quality, 21, 355}367. Skibsted, L. H. (2000). IDF Bulletin, in press. Stapelfeldt, H., Nielsen, B. R., & Skibsted, L. H. (1997). Towards use of electron spin resonance spectrometry in quality control of milk powder. Correlation between sensory score of instant whole milk powders and concentration of free radicals and 2thiobarbituric acid reactive substances. Milchwissenschaft, 52, 682}685.