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Influences of storage time and temperature on the xanthophyll content of freeze-dried egg yolk
Food Chemistry 124 (2011) 1343–1348
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Influences of storage time and temperature on the xanthophyll content of freeze-dried egg yolk Michael Wenzel a,⇑, Ingrid Seuss-Baum a, Elmar Schlich b a b
Department of Food Technology, University of Applied Sciences Fulda, Marquardstraße 35, 36039 Fulda, Germany Department of Process Engineering in Food and Servicing Business, Justus Liebig University Giessen, Stephanstraße 24, 35390 Gießen, Germany
a r t i c l e
i n f o
Article history: Received 8 February 2010 Received in revised form 13 June 2010 Accepted 26 July 2010
Keywords: Egg yolk Pasteurisation Storage Xanthophylls Freeze-drying
a b s t r a c t The influences of storage time, temperature (18 or +20 °C, both in the dark), and prior pasteurisation on the xanthophyll content of freeze-dried egg yolk were investigated. After six months of storage, the synthetic xanthophylls all-E-canthaxanthin and b-apo-8-carotenoic acid ethyl ester showed considerably higher stability (with losses of 19–34%) than did the natural pigments all-E-lutein and all-E-zeaxanthin (losses of 59–69%). At all stages of storage, the xanthophyll contents of unpasteurised and previously pasteurised samples did not differ significantly, and no obvious influence of storage temperature was observed. With respect to xanthophyll content, the results suggest that there is no necessity for lowtemperature storage of freeze-dried egg yolk. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Egg yolks contain substantial concentrations of highly bioavailable carotenoids, especially the xanthophylls lutein and zeaxanthin (Handelman, Nightingale, Lichtenstein, Schaefer, & Blumberg, 1999). Because consumers associate intense colours with high food quality, xanthophylls have been used to enrich poultry feed (Christensen, 1983). In the European Union (EU), the law permits the addition of eight xanthophylls possessing varying functional groups and carbon-chain lengths (C30–C40) to the feed of poultry and laying hens in amounts of up to 80 mg/kg of feedstuff (EC, 2003b). These substances are the natural xanthophylls capsanthin (C40), b-cryptoxanthin (C40), lutein (C40), zeaxanthin (C40), and the synthetic xanthophylls b-apo-80 -carotenal (C30), b-apo-80 -carotenoic acid ethyl ester (C30), canthaxanthin (C40), and citranaxanthin (C33). The amounts of these carotenoids in the egg yolk can differ enormously and depend on husbandry conditions and genetic variation in the hens (Schlatterer & Breithaupt, 2006). In the EU, commercial eggs are classified according to their rearing method, resulting in four different classes (0, ecological; 1, free range; 2, barn; 3, cage) as well as classifications for size (S, <53 g; M, 53 to <63 g; L, 63 to <73 g; XL, P73 g) and grade (A extra; A; B) (EC, 2003a).
⇑ Corresponding author. Tel.: +49 6221 894540. E-mail address: [email protected] (M. Wenzel). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.085
For microbiological and economic reasons, in the bakery and pasta industries, eggs from class 3 (cage) are commonly processed to produce pasteurised liquid egg yolk or spray-dried egg-yolk powder. As a result, the xanthophyll content of the final product depends on the amount of egg yolk and the processing. There are various methods for the identification of carotenoids in fat-free foodstuffs using liquid chromatography–mass spectrometry (LC–MS) analysis (see, e.g., Breithaupt & Schwack, 2000; De Jesus Ornelas-Paz, Yahia, & Gardea-Bejar, 2007; De Rosso & Mercadante, 2007), but to date, only a few studies have been conducted concerning the determination of xanthophylls of foods rich in fat, e.g., eggs (Kang, Kim, Cho, Yim, & Kim, 2003; Liu, Zhang, Peng, Wang, & Zhang, 2004; Schlatterer & Breithaupt, 2006). The influence of storage conditions on the carotenoid contents of intermediate products and foodstuffs, especially tomato products, juices and fish, is well-known, with a number of studies reported in the literature (Lin & Chen, 2005; Meléndez-Martínez, Vicario, & Heredia, 2003; Sánchez-Moreno, Plaza, De Ancos, & Cano, ´ Connor, Sheehy, Buckley, & FitzGerald, 1998; 2003; Sheehan, O Shi & Chen, 1997; Tang & Chen, 2000). For eggs, the only available findings concern the influence of freeze-drying on the functional properties of egg yolk (Jaekel, Dautel, & Ternes, 2008), the influence of spray-drying conditions on the functionality of dried whole egg (Franke & Kießling, 2002), the influence of household cooking (Schlatterer & Breithaupt, 2006), and the stability of polyunsaturated fatty acids (PUFAs) during processing and storage of egg-yolk powder (Galobart, Barroeta, Baucells, Cortinas, & Guardiola, 2001; Guardiola, Codony, Manich, Rafecas, & Boatella, 1995). To the best
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of our knowledge, however, there have been no storage studies considering the influence of time, temperature, and prior pasteurisation on the content of xanthophylls in freeze-dried egg yolk. This study was conducted to obtain extensive information about the retention of xanthophyll concentrations in freeze-dried egg yolks with high dry-matter contents (97.7–99.2%) stored for up to six months. Accordingly, a fast and economical high pressure liquid chromatography (HPLC)-method was developed whereby peak identification was supported by liquid chromatography– atmospheric pressure chemical ionisation mass spectrometry [LC–(APCI)MS] analysis. 2. Materials and methods 2.1. Chemicals and samples Tert-butyl methyl ether (TBME) and methanol (MEOH) were purchased from LGC Promochem (Wesel, Germany); formic acid was purchased from Merck (Darmstadt, Germany). High-purity water was prepared with an Arium 611UV water-purification system (Sartorius, Goettingen, Germany). Before use, all solvents were degassed by an ultrasonic treatment. Canthaxanthin (purity = 98.0%) and b-apo-80 -carotenoic acid ethyl ester (purity = 96.0%) were obtained from CaroteNature (Lupsingen, Switzerland). Lutein (purity = 98.8%) and zeaxanthin (purity = 93.2%) were purchased from LGC Promochem (Wesel, Germany). Two batches of eggs (n = 80 each) from a single egg-producing factory of husbandry class 1 (size M, quality A) were purchased from a local supermarket. Analyses were always performed more than 14 days before the expiration date. Eggs were cracked and the yolks manually separated from the egg white under yellow light. Egg yolks of each batch were pooled and homogenised (60 s, speed level 1, KitchenAid Professional, USA). Half of the homogenised masses were immediately pasteurised (61.5 °C, 3.5 min, Lab Reactor LR-A 1000, IKA, Germany), while the remaining half of the sample masses were nitrogen-purged and temporarily stored in a cooling chamber at +2 °C. After pasteurisation, all of the samples were immediately processed in the freeze-dryer. Before freeze-drying in the laboratory lyophilizer (beta 2–16, Christ, Germany), the pure egg-yolk material had to be quick-frozen (30 min) at 40 °C in a lab freezer. During the drying procedures, all of the equipment was covered by foil, impermeable to light, to protect the xanthophylls in the egg yolks. Immediately after drying, aliquots were procured for LC–MS analysis. The main amounts of the sample materials were carefully placed in plastic bags (approximately 2 g of egg-yolk powder per bag), nitrogen-purged, and vacuumsealed. One half of these sample materials was stored at 18 °C in a freezer, and the other half at +20 °C in an air-conditioned room. After 2, 4, 8, and 26 weeks of storage in the dark the samples were analysed again. The dry-matter contents were determined in duplicate for all the samples to ensure correct quantification results. All the LC–MS analyses were performed in triplicate with three injections each. 2.2. Extraction of xanthophylls Aliquots (0.5 g) of fresh and processed egg yolks were placed directly into glass centrifuge tubes (40 ml). After adding 13 ml of methanol, the tubes were covered with ParafilmÒ to avoid solvent evaporation and immediately treated with ultrasound (60 s) to improve extraction (Mason, Paniwnyk, & Lorimer, 1996). In several advance tests, a single ultrasound-assisted extraction (UAE) of xanthophylls from egg yolk with pure methanol showed a statistically comparable (fresh egg yolk) and significantly higher (dried egg yolk) extraction efficiency from the same source material than a
double extraction with pure methanol without ultrasound. A significantly higher extraction efficiency of the single UAE with methanol from fresh and dried egg yolk was also observed when comparing single UAE with methanol and double UAE with methanol/tert-butyl methyl ether [2:1 v:v]. The residue from fresh and dried egg yolks, after a single UAE with methanol, was colourless, so it was concluded that extraction was complete. To assist extraction, the samples were homogenised using an Ultra-Turrax T25 (IKA, Staufen, Germany) at 9500 rpm (60 s). After homogenisation, the stirring unit of the Ultra-Turrax was carefully washed twice with 1 ml of methanol each. ParafilmÒ was again used to cover the glass tubes and, after an incubation time of 20 min, the samples were centrifuged (Labofuge 400R, Thermo Scientific, Karlsruhe, Germany) at 4500 rpm (5 min, 20 °C). Aliquots of the supernatants were directly placed in HPLC vials using a plastic syringe (10 ml) and a membrane filter (0.45 lm). 2.3. HPLC and LC/MS analyses To analyse the xanthophylls, a liquid chromatography system, consisting of a Waters Alliance 2695 (Eschborn, Germany) with a Model 996 photodiode array detector and coupled to a micromass Quattro LC mass-spectrometer (Manchester, UK), was used. For separation, a Stability 100 C30 (250 4.6 mm i.d.) endcapped analytical column (Dr. Maisch HPLC GmbH, Ammerbuch–Entringen, Germany) filled with a 5 lm phase material, including an accompanying precolumn (10 4.0 mm i.d.), was used and held at 30 °C. The mobile phase consisted of methanol/purified water/formic acid [994:5:1 v/v/v, (A)] and tert-butyl methyl ether/methanol [93:7 v/v, (B)], using isocratic elution (93:7 A:B, 20 min, 1.1 ml/ min, 40 ll). (APCI)MS was operated in the positive mode, the APCI source was heated to 150 °C, and the APCI probe was held at 450 °C. Corona voltage was set to 3.1 kV, the cone to 35.0 V, the extractor to 3.0 V, and the Rangefinder (RF) lens to 0.1 V. Nitrogen was used as a nebulizer and desolvation gas at 150 and 800 l/h, respectively. Mass spectra were acquired over a scanning range of m/z 400–600 (scan time 1.0 s, inter-scan delay 0.1 s). UV–Vis spectra were recorded from 220 to 600 nm with a resolution of 1.2 nm; quantification of xanthophylls was applied at a wavelength of 450 nm according to Schlatterer & Breithaupt (2006) and Tang & Chen (2000). Data were processed using MassLynx 4.0 software. 2.4. Calibration and validation Stock solutions of the xanthophylls all-E-lutein, all-E-zeaxanthin, canthaxanthin, and b-apo-80 -carotenoic acid ethyl ester were prepared under yellow light using ultrasound treatment. Calibration was performed using dilutions in the range 0.125–1.00 lg xanthophyll/ml. Calibration graphs were constructed by plotting the appropriate peak heights (450 nm, AUs) against the concentrations (lg/ml) due to slightly overlapping peaks. Coefficients of determination were always higher than 0.996. Limits of quantification (LOQ) and determination (LOD) were calculated from the respective calibration graphs, using signal/noise-ratios of 10:1 and 3:1 (Kromidas & Kuss, 2008, Table 1). To determine recovery rates of free xanthophylls, homogenised fresh egg-yolk samples low in synthetic xanthophylls (class 2, grade A) were individually spiked with aliquots (500 ll each) of stock solutions of lutein (c = 0.5 lg/ml of methanol), zeaxanthin (c = 2.6 lg/ml methanol), canthaxanthin (c = 1.7 lg/ml of methanol), and b-apo-80 -carotenoic acid ethyl ester (c = 2.4 lg/ml methanol). After spiking, the samples were extracted and analysed by HPLC-DAD. Recovery rates were calculated (Kromidas, 2000) with the following results (n = 9 each): 95.3% (lutein), 95.0% (zeaxanthin
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M. Wenzel et al. / Food Chemistry 124 (2011) 1343–1348 Table 1 Limits of quantification (LOQ) and determination (LOD) and calibration graphs used for quantification of xanthophylls in fresh and processed egg yolks by HPLC-DAD. Xanthophyll a
Lutein Zeaxanthina Canthaxanthina b-Apo-80 -carotenoic acid ethyl ester a
Calibration range (lg/ml)
LOD (lg/l)
LOQ (lg/l)
Calibration graphs c (lg/ml)
0.125–0.75 0.125–0.75 0.10–0.80 0.25–1.00
13.9 10.1 13.4 13.3
46.4 33.7 44.7 44.3
[height [height [height [height
(mAUs)–321.88]/19,368 (mAUs)+73.68]/14,695 (mAUs)+120.63]/10,777 (mAUs)–26.50]/13,743
The same calibration graph was used for the calculation of dedicated isomers.
Table 2 Spectroscopic and LC–(APCI)MS data used for identification of xanthophylls in fresh and processed egg yolks. Xanthophylla
(1) (2) (3) (4) (5) (6) (7) (8) a b c
All-E-lutein All-E-zeaxanthin 13-Z-lutein 13-Z-zeaxanthin 9-Z-canthaxanthin All-E-canthaxanthin Z-isomer canthaxanthin b-Apo-80 -carotenoic acid ethyl ester
VIS maximab I
II
III
420 420 417 416 363 – – –
443 446 441 442 478 478 478c 445
474 475 472 473 – – – –
Q-ratio
RT (min)
Main ions (m/z ; intensity)
III/II 0.61 III/II 0.23 III/II 0.30 III/II 0.11 I/II 0.23 – – –
6.42 6.72 7.13 7.76 9.82 10.36 11.18 15.71
569 569 569 569 565 565 565 461
(78%) [M + H]+, 551 (100%) [M + H –H20]+ (100%) (100%) (100%) (100%) (100%) (100%)c (100%)
Shown in elution order. Determined in the HPLC eluents. Weak signal.
and canthaxanthin), and 95.4% (b-apo-80 -carotenoic acid ethyl ester). Quantitative data are presented as means ± S D Analysis of variance (ANOVA) and determination of Pearson’s product-moment correlation were performed on the obtained data using Microsoft Excel XP software and R (version 2.10.1). The significant statistical level was set to p < 0.05. 3. Results and discussion 3.1. Identification of xanthophylls in the standard solutions and eggyolk samples In addition to the unambiguous retention times of the xanthophylls and their UV–Vis spectra, detected masses obtained by
LC–MS were used to support peak assignments. In particular, the identity of (Z)-isomers not found in the standard solutions could be assigned because of their mass. The mass traces were extracted from the total ion chromatograms (TIC) and were considered sufficient, although no cleanup of the egg-yolk samples was performed beyond the xanthophyll-extraction process. The data set used for identification of the xanthophylls present in standard solutions and egg-yolk samples is shown in Table 2. Eight substances of relevant quantities in the sample materials were obtained. A representative HPLC chromatogram is depicted in Fig. 1. All-E-lutein (1), 13-Z-lutein (2), all-E-zeaxanthin (3), 13-Zzeaxanthin (4), 9-Z-canthaxanthin (5), all-E-canthaxanthin (6), and b-apo-80 -carotenoic acid ethyl ester (8) were clearly identified by their characteristic retention times, masses and, in addition, by their spectral fine structures (Q-ratios of III/II and I/II). The masses
Fig. 1. LC–(APCI)MS analysis of a freeze-dried egg-yolk extract. The bottom trace (A) corresponds to detection at 450 nm (DAD). Traces B and C correspond to DAD signals (450 nm) from calibration solutions of canthaxanthin and zeaxanthin. Peak numbers correspond to the assignments given in Table 2.
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Table 3 Concentrations of xanthophylls in freeze-dried egg yolks directly after processing from two egg batches of husbandry class 1, presented as mean values ± standard deviation (micrograms per 100 g of egg yolk, n = 9 each). Xanthophyll
Unpasteurised (egg batch 1)
Pasteurised (egg batch 1)
Unpasteurised (egg batch 2)
Pasteurised (egg batch 2)
All-E-lutein All-E-zeaxanthin 13-Z-lutein 13-Z-zeaxanthin 9-Z-canthaxanthin All-E-canthaxanthin Z-isomer canthaxanthin b-apo-80 -carotenoic acid ethyl ester Total xanthophylls
1015 ± 11 792 ± 13 71 ± 4 148 ± 4 454 ± 6 1233 ± 13 73 ± 4 326 ± 7 4112 ± 44
1005 ± 10 781 ± 30 75 ± 4 154 ± 6 478 ± 14 1229 ± 39 72 ± 6 325 ± 13 4119 ± 110
749 ± 26 473 ± 24 68 ± 5 82 ± 5 405 ± 17 1136 ± 50 62 ± 3 301 ± 20 3277 ± 134
692 ± 29 429 ± 23 68 ± 3 77 ± 2 395 ± 25 1053 ± 41 59 ± 3 271 ± 9 3044 ± 117
used for identification were 569 Da ( 1–4), 565 Da ( 5–7), and 461 Da (8), i.e., quasimolecular ions; strong mass signals of dedicated molecular ions [M+] could also be observed. Because the spectral data and masses were almost equal to (6), it was concluded that substance (7) might represent a (Z)-isomer of canthaxanthin. Furthermore, in every HPLC chromatogram, about six peaks, indicating the presence of minor components were observed (see Fig. 1), but identification and quantification of these substances failed because the standard solutions gave no corresponding peaks and their UV–Vis and mass signals were not distinctive. 3.2. Quantification of xanthophylls in freeze-dried egg-yolk samples In this study, the extraction of xanthophylls was accomplished with pure (slightly nonpolar) methanol, assisted by an ultrasound treatment. The use of ultrasound treatment in facilitating the extraction of carotenoids from foods has been widely acknowledged (Mason et al., 1996; Sun, Xu, & Godber, 2006) but must be applied carefully because degradation and isomerization effects are possible (Zhao et al., 2006). The calibration graphs, LOQs, and LODs of substances determined in this study are shown in Table 1. All-E-canthaxanthin was the predominant xanthophyll present in the two batches of freeze-dried egg yolk made from fresh commercial eggs from husbandry class 1, followed by all-E-lutein, all-Ezeaxanthin, 9-Z-canthaxanthin, and b-apo-80 -carotenoic acid ethyl ester. Xanthophyll contents of batch 1 were slightly higher than those of batch 2, but both showed a very similar distribution pattern, indicating that the hens’ feed was of the same quality (see Table 3). The observed distribution and amounts of xanthophylls detected in eggs from husbandry class 1 are in agreement with those reported in prior studies (Majchrzak & Elmadfa, 1997; Schlatterer & Breithaupt, 2006) and in accordance with legal regulations (EC, 2008, 2003b). Initial absolute xanthophyll contents of both egg batches, shown in Table 3, were set to 100% each and the relative changes during storage at 18 °C and +20 °C were calculated. For generalisation, the relative changes in the xanthophyll contents of both batches are summarised. Percentage changes in the contents of all-E-lutein, all-E-zeaxanthin, all-E-canthaxanthin, and total xanthophylls during storage are presented as mean values (n = 18) in Figs. 2 and 3 (j 18 °C, not pasteurised; h +20 °C, not pasteurised; N 18 °C, pasteurised; D +20 °C, pasteurised), whereby p-values were always lower than 0.001. The contents of all-E-lutein (losses of 65–69%), all-E-zeaxanthin (losses of 59–62%), 13-Z-lutein (losses of 46–53%), 13-Z-zeaxanthin (losses of 50–60%), 9-Z-canthaxanthin (losses of 14–15%), all-Ecanthaxanthin (losses of 19–25%), and b-apo-80 -carotenoic acid ethyl ester (losses of 29–34%) in freeze-dried egg yolk fell continuously over the storage period of six months, following an exponential function, whereby almost all losses occurred during the first four weeks of storage time. Coefficients of determination of the
regression curves for the data of Figs. 2 and 3 were always higher than 0.70. This progressive loss is consistent with results of other xanthophyll-storage studies (Lin & Chen, 2005; Shi & Chen, 1997). In contrast, the content of the Z-isomer of canthaxanthin (7) proved quite stable (relative changes from 7% to +11%). The results of this study also suggest that natural pigments, such as all-E-lutein and all-E-zeaxanthin, are more liable to an intense degradation in the egg matrix than are the synthetic xanthophylls allE-canthaxanthin and b-apo-80 -carotenoic acid ethyl ester. In this context, the findings of prior studies, which emphasised the relatively high stability of all-E-canthaxanthin during a longer storage period in a fatty matrix (Sheehan et al., 1998) and a low degradation rate of b-apo-80 -carotenoic acid ethyl ester during the boiling
Fig. 2. Percentage changes in the contents of all-E-lutein and all-E-zeaxanthin in freeze-dried egg yolk during 26 weeks of storage at 18 °C and +20 °C in the dark (n = 18) and according equations of regression.
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tent over six months of dark storage depended mostly on the distribution of natural and synthetic pigments in the egg yolk and, to a much lesser degree, on storage temperature. The arithmetically averaged percentage losses of the natural xanthophylls, all-E-lutein and all-E-zeaxanthin, reached a total of about two-thirds, and the lost of the synthetic xanthophylls all-E-canthaxanthin and b-apo80 -carotenoic acid ethyl ester totaled about one-fifth to one-third. Moreover, knowledge of xanthophyll concentrations in dried egg yolks, at all stages of storage, will help to enable estimations of dietary xanthophyll intake from foods formulated with processed egg yolk, such as pasta and pastries. References
Fig. 3. Percentage changes in the contents of all-E-cantha-xanthin and total xanthophylls in freeze-dried egg yolk during 26 weeks of storage at 18 °C and +20 °C in the dark (n = 18) and according equations of regression.
of eggs (Schlatterer & Breithaupt, 2006), were confirmed. In contrast to the observation that, in some cases (e.g., lutein and zeaxanthin), samples stored at ambient temperature showed even slightly higher xanthophyll contents, freeze-dried egg yolk stored at +20 °C showed no significant difference in xanthophyll content compared to freeze-dried egg yolk stored at 18 °C (see Figs. 2 and 3). The effect of greater xanthophyll retention at higher temperatures during storage has been reported previously (Xianquan, Shi, Kakuda, & Yueming, 2005) and was explained by an improved oxygen solubility at lower temperatures, which may contribute to a higher oxidation rate. The results of this study support the conclusion that freeze-dried egg yolks do not need to be stored at low temperatures because the putative protective influence on xanthophyll retention was refuted. Thus, the advice of egg-yolk-powder manufacturers to store their products at ambient temperature was confirmed. 4. Conclusions It must be emphasised that stability data, based on the egg yolk selected here as a model system, may not be generally transferred to comparable food matrices or even other egg batches without intensive investigation, because they inevitably contain variable amounts of bioactive compounds, such as antioxidants. Based on the outcome of this study, the retention of total xanthophyll con-
Breithaupt, D. E., & Schwack, W. (2000). Determination of free and bound carotenoids in paprika (Capsicum annuum L.) by LC/MS. European Food Research and Technology, 211, 52–55. Christensen, C. M. (1983). Effects of colour on aroma, flavor and texture judgments of foods. Journal of Food Science, 48, 787–790. Commission Regulation (EC) 2295/2003 of 23 December 2003 introducing detailed rules for implementing Council Regulation (EEC) 1907/90 on certain marketing standards for eggs (2003a). Art. 5-7, Art. 13, Annex II. Commission Regulation (EC) 775/2008 of 4 August 2008 establishing maximum residue limits for the feed additive canthaxanthin in addition to the conditions provided for in Directive 2003/7/EC. Community register of feed additives pursuant to Regulation (EC) 1831/2003 (2003b), released 17 November 2009 (http://ec.europa.eu/comm/food/food/ animalnutrition/feedadditives/registeradditives_en.htm); Regulation (EC) 1831/2003 of the European Parliament and of the council of 22 September 2003 on additives for use in animal nutrition. De Jesus Ornelas-Paz, J., Yahia, E. M., & Gardea-Bejar, A. (2007). Identification and quantification of xanthophyll esters, carotenes, and tocopherols in the fruit of seven mexican mango cultivars by liquid chromatography-atmospheric pressure chemical ionization-time-of-flight mass spectrometry [LC-(APCI+)MS]. Journal of Agricultural and Food Chemistry, 55, 6628–6635. De Rosso, V. V., & Mercadante, A. Z. (2007). Identification and quantification of carotenoids, by HPLC-PDA-MS/MS, from amazonian fruits. Journal of Agricultural and Food Chemistry, 55, 5062–5072. Franke, K., & Kießling, M. (2002). Influence of spray drying conditions on functionality of dried whole egg. Journal of the Science of Food and Agriculture, 82, 1837–1841. Galobart, J., Barroeta, A. C., Baucells, M. D., Cortinas, L., & Guardiola, F. (2001). Ftocopherol transfer efficiency and lipid oxidation in fresh and spray-dried eggs enriched with x3-polyunsaturated fatty acids. Poultry Science, 80, 1496–1505. Guardiola, F., Codony, R., Manich, A., Rafecas, M., & Boatella, J. (1995). Stability of polyunsaturated fatty acids in egg powder processed and stored under various conditions. Journal of Agricultural and Food Chemistry, 43, 2254–2259. Handelman, G. J., Nightingale, Z. D., Lichtenstein, A. H., Schaefer, E. J., & Blumberg, J. B. (1999). Lutein and zeaxanthin concentrations in plasma after dietary supplementation with egg yolk. American Journal of Clinical Nutrition, 70, 247–251. Jaekel, T., Dautel, K., & Ternes, W. (2008). Preserving functional properties of hen’s egg yolk during freeze-drying. Journal of Food Engineering, 87, 522–526. Kang, D. K., Kim, S. I., Cho, C. H., Yim, Y. H., & Kim, H. S. (2003). Use of lycopene, an antioxidant carotenoid, in laying hens for egg yolk pigmentation. AsianAustralian Journal of Animal Sciences, 16, 1799–1803. Kromidas, S. (2000). Validierung in der analytik. Weinheim, Germany: Wiley-VCH. Kromidas, S., & Kuss, H.-J. (2008). Chromatogramme richtig integrieren und bewertenEin Praxishandbuch für die HPLC und GC. Weinheim, Germany: Wiley-VCH. Lin, C. H., & Chen, B. H. (2005). Stability of carotenoids in tomato juice during storage. Food Chemistry, 90, 837–846. Liu, L., Zhang, M., Peng, G., Wang, H., & Zhang, S. (2004). Separation and identification of red pigments in natural red yolk of duck’s eggs by HPLC– MS–MS. Chinese Journal of Chromatography, 22, 197–201. Majchrzak, D., & Elmadfa, I. (1997). Carotinoid- und retinolgehalt in hühnereiern. Fett/Lipid, 99, 365–368. Mason, T. J., Paniwnyk, L., & Lorimer, J. P. (1996). The uses of ultrasound in food technology. Ultrasonics Sonochemistry, 3, S253–S260. Meléndez-Martínez, A. J., Vicario, I. M., & Heredia, F. J. (2003). A routine highperformance liquid chromatography method for carotenoid determination in ultrafrozen orange juices. Journal of Agricultural and Food Chemistry, 51, 4219–4224. Sánchez-Moreno, C., Plaza, L., De Ancos, B., & Cano, M. P. (2003). Vitamin C, provitamin A carotenoids, and other carotenoids in high-pressurized orange juice during refrigerator storage. Journal of Agricultural and Food Chemistry, 51, 647–653. Schlatterer, J., & Breithaupt, D. E. (2006). Xanthophylls in commercial egg yolks: Quantification and identification by HPLC and LC–(APCI)MS using a C30 phase. Journal of Agricultural and Food Chemistry, 54, 2267–2273. Sheehan, E. M., Connor, T. P., Sheehy, P. J. A., Buckley, D. J., & FitzGerald, R. (1998). Stability of astaxanthin and canthaxanthin in raw and smoked Atlantic salmon (Salmo salar) during frozen storage. Food Chemistry, 63, 313–317.
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Shi, X.-M., & Chen, F. (1997). Stability of lutein under various storage conditions. Nahrung, 41, 38–41. Sun, T., Xu, Z., & Godber, J. S. (2006). Ultrasound assisted extraction in quantifying lutein from chicken liver using high-performance liquid chromatography. Journal of Chromatography B, 830, 158–160. Tang, Y. C., & Chen, B. H. (2000). Pigment change of freeze-dried carotenoid powder during storage. Food Chemistry, 69, 11–17.
Xianquan, S., Shi, J., Kakuda, Y., & Yueming, J. (2005). Stability of lycopene during food processing and storage. Journal of Medicinal Food, 8, 413–422. Zhao, L., Zhao, G., Chen, F., Wang, Z., Wu, J., & Hu, X. (2006). Different effects of microwave and ultrasound on the stability of (all-E)-astaxanthin. Journal of Agricultural and Food Chemistry, 54, 8346–8351.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Influences of storage time and temperature on the xanthophyll content of freeze-dried egg yolk Michael Wenzel a,⇑, Ingrid Seuss-Baum a, Elmar Schlich b a b
Department of Food Technology, University of Applied Sciences Fulda, Marquardstraße 35, 36039 Fulda, Germany Department of Process Engineering in Food and Servicing Business, Justus Liebig University Giessen, Stephanstraße 24, 35390 Gießen, Germany
a r t i c l e
i n f o
Article history: Received 8 February 2010 Received in revised form 13 June 2010 Accepted 26 July 2010
Keywords: Egg yolk Pasteurisation Storage Xanthophylls Freeze-drying
a b s t r a c t The influences of storage time, temperature (18 or +20 °C, both in the dark), and prior pasteurisation on the xanthophyll content of freeze-dried egg yolk were investigated. After six months of storage, the synthetic xanthophylls all-E-canthaxanthin and b-apo-8-carotenoic acid ethyl ester showed considerably higher stability (with losses of 19–34%) than did the natural pigments all-E-lutein and all-E-zeaxanthin (losses of 59–69%). At all stages of storage, the xanthophyll contents of unpasteurised and previously pasteurised samples did not differ significantly, and no obvious influence of storage temperature was observed. With respect to xanthophyll content, the results suggest that there is no necessity for lowtemperature storage of freeze-dried egg yolk. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Egg yolks contain substantial concentrations of highly bioavailable carotenoids, especially the xanthophylls lutein and zeaxanthin (Handelman, Nightingale, Lichtenstein, Schaefer, & Blumberg, 1999). Because consumers associate intense colours with high food quality, xanthophylls have been used to enrich poultry feed (Christensen, 1983). In the European Union (EU), the law permits the addition of eight xanthophylls possessing varying functional groups and carbon-chain lengths (C30–C40) to the feed of poultry and laying hens in amounts of up to 80 mg/kg of feedstuff (EC, 2003b). These substances are the natural xanthophylls capsanthin (C40), b-cryptoxanthin (C40), lutein (C40), zeaxanthin (C40), and the synthetic xanthophylls b-apo-80 -carotenal (C30), b-apo-80 -carotenoic acid ethyl ester (C30), canthaxanthin (C40), and citranaxanthin (C33). The amounts of these carotenoids in the egg yolk can differ enormously and depend on husbandry conditions and genetic variation in the hens (Schlatterer & Breithaupt, 2006). In the EU, commercial eggs are classified according to their rearing method, resulting in four different classes (0, ecological; 1, free range; 2, barn; 3, cage) as well as classifications for size (S, <53 g; M, 53 to <63 g; L, 63 to <73 g; XL, P73 g) and grade (A extra; A; B) (EC, 2003a).
⇑ Corresponding author. Tel.: +49 6221 894540. E-mail address: [email protected] (M. Wenzel). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.085
For microbiological and economic reasons, in the bakery and pasta industries, eggs from class 3 (cage) are commonly processed to produce pasteurised liquid egg yolk or spray-dried egg-yolk powder. As a result, the xanthophyll content of the final product depends on the amount of egg yolk and the processing. There are various methods for the identification of carotenoids in fat-free foodstuffs using liquid chromatography–mass spectrometry (LC–MS) analysis (see, e.g., Breithaupt & Schwack, 2000; De Jesus Ornelas-Paz, Yahia, & Gardea-Bejar, 2007; De Rosso & Mercadante, 2007), but to date, only a few studies have been conducted concerning the determination of xanthophylls of foods rich in fat, e.g., eggs (Kang, Kim, Cho, Yim, & Kim, 2003; Liu, Zhang, Peng, Wang, & Zhang, 2004; Schlatterer & Breithaupt, 2006). The influence of storage conditions on the carotenoid contents of intermediate products and foodstuffs, especially tomato products, juices and fish, is well-known, with a number of studies reported in the literature (Lin & Chen, 2005; Meléndez-Martínez, Vicario, & Heredia, 2003; Sánchez-Moreno, Plaza, De Ancos, & Cano, ´ Connor, Sheehy, Buckley, & FitzGerald, 1998; 2003; Sheehan, O Shi & Chen, 1997; Tang & Chen, 2000). For eggs, the only available findings concern the influence of freeze-drying on the functional properties of egg yolk (Jaekel, Dautel, & Ternes, 2008), the influence of spray-drying conditions on the functionality of dried whole egg (Franke & Kießling, 2002), the influence of household cooking (Schlatterer & Breithaupt, 2006), and the stability of polyunsaturated fatty acids (PUFAs) during processing and storage of egg-yolk powder (Galobart, Barroeta, Baucells, Cortinas, & Guardiola, 2001; Guardiola, Codony, Manich, Rafecas, & Boatella, 1995). To the best
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of our knowledge, however, there have been no storage studies considering the influence of time, temperature, and prior pasteurisation on the content of xanthophylls in freeze-dried egg yolk. This study was conducted to obtain extensive information about the retention of xanthophyll concentrations in freeze-dried egg yolks with high dry-matter contents (97.7–99.2%) stored for up to six months. Accordingly, a fast and economical high pressure liquid chromatography (HPLC)-method was developed whereby peak identification was supported by liquid chromatography– atmospheric pressure chemical ionisation mass spectrometry [LC–(APCI)MS] analysis. 2. Materials and methods 2.1. Chemicals and samples Tert-butyl methyl ether (TBME) and methanol (MEOH) were purchased from LGC Promochem (Wesel, Germany); formic acid was purchased from Merck (Darmstadt, Germany). High-purity water was prepared with an Arium 611UV water-purification system (Sartorius, Goettingen, Germany). Before use, all solvents were degassed by an ultrasonic treatment. Canthaxanthin (purity = 98.0%) and b-apo-80 -carotenoic acid ethyl ester (purity = 96.0%) were obtained from CaroteNature (Lupsingen, Switzerland). Lutein (purity = 98.8%) and zeaxanthin (purity = 93.2%) were purchased from LGC Promochem (Wesel, Germany). Two batches of eggs (n = 80 each) from a single egg-producing factory of husbandry class 1 (size M, quality A) were purchased from a local supermarket. Analyses were always performed more than 14 days before the expiration date. Eggs were cracked and the yolks manually separated from the egg white under yellow light. Egg yolks of each batch were pooled and homogenised (60 s, speed level 1, KitchenAid Professional, USA). Half of the homogenised masses were immediately pasteurised (61.5 °C, 3.5 min, Lab Reactor LR-A 1000, IKA, Germany), while the remaining half of the sample masses were nitrogen-purged and temporarily stored in a cooling chamber at +2 °C. After pasteurisation, all of the samples were immediately processed in the freeze-dryer. Before freeze-drying in the laboratory lyophilizer (beta 2–16, Christ, Germany), the pure egg-yolk material had to be quick-frozen (30 min) at 40 °C in a lab freezer. During the drying procedures, all of the equipment was covered by foil, impermeable to light, to protect the xanthophylls in the egg yolks. Immediately after drying, aliquots were procured for LC–MS analysis. The main amounts of the sample materials were carefully placed in plastic bags (approximately 2 g of egg-yolk powder per bag), nitrogen-purged, and vacuumsealed. One half of these sample materials was stored at 18 °C in a freezer, and the other half at +20 °C in an air-conditioned room. After 2, 4, 8, and 26 weeks of storage in the dark the samples were analysed again. The dry-matter contents were determined in duplicate for all the samples to ensure correct quantification results. All the LC–MS analyses were performed in triplicate with three injections each. 2.2. Extraction of xanthophylls Aliquots (0.5 g) of fresh and processed egg yolks were placed directly into glass centrifuge tubes (40 ml). After adding 13 ml of methanol, the tubes were covered with ParafilmÒ to avoid solvent evaporation and immediately treated with ultrasound (60 s) to improve extraction (Mason, Paniwnyk, & Lorimer, 1996). In several advance tests, a single ultrasound-assisted extraction (UAE) of xanthophylls from egg yolk with pure methanol showed a statistically comparable (fresh egg yolk) and significantly higher (dried egg yolk) extraction efficiency from the same source material than a
double extraction with pure methanol without ultrasound. A significantly higher extraction efficiency of the single UAE with methanol from fresh and dried egg yolk was also observed when comparing single UAE with methanol and double UAE with methanol/tert-butyl methyl ether [2:1 v:v]. The residue from fresh and dried egg yolks, after a single UAE with methanol, was colourless, so it was concluded that extraction was complete. To assist extraction, the samples were homogenised using an Ultra-Turrax T25 (IKA, Staufen, Germany) at 9500 rpm (60 s). After homogenisation, the stirring unit of the Ultra-Turrax was carefully washed twice with 1 ml of methanol each. ParafilmÒ was again used to cover the glass tubes and, after an incubation time of 20 min, the samples were centrifuged (Labofuge 400R, Thermo Scientific, Karlsruhe, Germany) at 4500 rpm (5 min, 20 °C). Aliquots of the supernatants were directly placed in HPLC vials using a plastic syringe (10 ml) and a membrane filter (0.45 lm). 2.3. HPLC and LC/MS analyses To analyse the xanthophylls, a liquid chromatography system, consisting of a Waters Alliance 2695 (Eschborn, Germany) with a Model 996 photodiode array detector and coupled to a micromass Quattro LC mass-spectrometer (Manchester, UK), was used. For separation, a Stability 100 C30 (250 4.6 mm i.d.) endcapped analytical column (Dr. Maisch HPLC GmbH, Ammerbuch–Entringen, Germany) filled with a 5 lm phase material, including an accompanying precolumn (10 4.0 mm i.d.), was used and held at 30 °C. The mobile phase consisted of methanol/purified water/formic acid [994:5:1 v/v/v, (A)] and tert-butyl methyl ether/methanol [93:7 v/v, (B)], using isocratic elution (93:7 A:B, 20 min, 1.1 ml/ min, 40 ll). (APCI)MS was operated in the positive mode, the APCI source was heated to 150 °C, and the APCI probe was held at 450 °C. Corona voltage was set to 3.1 kV, the cone to 35.0 V, the extractor to 3.0 V, and the Rangefinder (RF) lens to 0.1 V. Nitrogen was used as a nebulizer and desolvation gas at 150 and 800 l/h, respectively. Mass spectra were acquired over a scanning range of m/z 400–600 (scan time 1.0 s, inter-scan delay 0.1 s). UV–Vis spectra were recorded from 220 to 600 nm with a resolution of 1.2 nm; quantification of xanthophylls was applied at a wavelength of 450 nm according to Schlatterer & Breithaupt (2006) and Tang & Chen (2000). Data were processed using MassLynx 4.0 software. 2.4. Calibration and validation Stock solutions of the xanthophylls all-E-lutein, all-E-zeaxanthin, canthaxanthin, and b-apo-80 -carotenoic acid ethyl ester were prepared under yellow light using ultrasound treatment. Calibration was performed using dilutions in the range 0.125–1.00 lg xanthophyll/ml. Calibration graphs were constructed by plotting the appropriate peak heights (450 nm, AUs) against the concentrations (lg/ml) due to slightly overlapping peaks. Coefficients of determination were always higher than 0.996. Limits of quantification (LOQ) and determination (LOD) were calculated from the respective calibration graphs, using signal/noise-ratios of 10:1 and 3:1 (Kromidas & Kuss, 2008, Table 1). To determine recovery rates of free xanthophylls, homogenised fresh egg-yolk samples low in synthetic xanthophylls (class 2, grade A) were individually spiked with aliquots (500 ll each) of stock solutions of lutein (c = 0.5 lg/ml of methanol), zeaxanthin (c = 2.6 lg/ml methanol), canthaxanthin (c = 1.7 lg/ml of methanol), and b-apo-80 -carotenoic acid ethyl ester (c = 2.4 lg/ml methanol). After spiking, the samples were extracted and analysed by HPLC-DAD. Recovery rates were calculated (Kromidas, 2000) with the following results (n = 9 each): 95.3% (lutein), 95.0% (zeaxanthin
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M. Wenzel et al. / Food Chemistry 124 (2011) 1343–1348 Table 1 Limits of quantification (LOQ) and determination (LOD) and calibration graphs used for quantification of xanthophylls in fresh and processed egg yolks by HPLC-DAD. Xanthophyll a
Lutein Zeaxanthina Canthaxanthina b-Apo-80 -carotenoic acid ethyl ester a
Calibration range (lg/ml)
LOD (lg/l)
LOQ (lg/l)
Calibration graphs c (lg/ml)
0.125–0.75 0.125–0.75 0.10–0.80 0.25–1.00
13.9 10.1 13.4 13.3
46.4 33.7 44.7 44.3
[height [height [height [height
(mAUs)–321.88]/19,368 (mAUs)+73.68]/14,695 (mAUs)+120.63]/10,777 (mAUs)–26.50]/13,743
The same calibration graph was used for the calculation of dedicated isomers.
Table 2 Spectroscopic and LC–(APCI)MS data used for identification of xanthophylls in fresh and processed egg yolks. Xanthophylla
(1) (2) (3) (4) (5) (6) (7) (8) a b c
All-E-lutein All-E-zeaxanthin 13-Z-lutein 13-Z-zeaxanthin 9-Z-canthaxanthin All-E-canthaxanthin Z-isomer canthaxanthin b-Apo-80 -carotenoic acid ethyl ester
VIS maximab I
II
III
420 420 417 416 363 – – –
443 446 441 442 478 478 478c 445
474 475 472 473 – – – –
Q-ratio
RT (min)
Main ions (m/z ; intensity)
III/II 0.61 III/II 0.23 III/II 0.30 III/II 0.11 I/II 0.23 – – –
6.42 6.72 7.13 7.76 9.82 10.36 11.18 15.71
569 569 569 569 565 565 565 461
(78%) [M + H]+, 551 (100%) [M + H –H20]+ (100%) (100%) (100%) (100%) (100%) (100%)c (100%)
Shown in elution order. Determined in the HPLC eluents. Weak signal.
and canthaxanthin), and 95.4% (b-apo-80 -carotenoic acid ethyl ester). Quantitative data are presented as means ± S D Analysis of variance (ANOVA) and determination of Pearson’s product-moment correlation were performed on the obtained data using Microsoft Excel XP software and R (version 2.10.1). The significant statistical level was set to p < 0.05. 3. Results and discussion 3.1. Identification of xanthophylls in the standard solutions and eggyolk samples In addition to the unambiguous retention times of the xanthophylls and their UV–Vis spectra, detected masses obtained by
LC–MS were used to support peak assignments. In particular, the identity of (Z)-isomers not found in the standard solutions could be assigned because of their mass. The mass traces were extracted from the total ion chromatograms (TIC) and were considered sufficient, although no cleanup of the egg-yolk samples was performed beyond the xanthophyll-extraction process. The data set used for identification of the xanthophylls present in standard solutions and egg-yolk samples is shown in Table 2. Eight substances of relevant quantities in the sample materials were obtained. A representative HPLC chromatogram is depicted in Fig. 1. All-E-lutein (1), 13-Z-lutein (2), all-E-zeaxanthin (3), 13-Zzeaxanthin (4), 9-Z-canthaxanthin (5), all-E-canthaxanthin (6), and b-apo-80 -carotenoic acid ethyl ester (8) were clearly identified by their characteristic retention times, masses and, in addition, by their spectral fine structures (Q-ratios of III/II and I/II). The masses
Fig. 1. LC–(APCI)MS analysis of a freeze-dried egg-yolk extract. The bottom trace (A) corresponds to detection at 450 nm (DAD). Traces B and C correspond to DAD signals (450 nm) from calibration solutions of canthaxanthin and zeaxanthin. Peak numbers correspond to the assignments given in Table 2.
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Table 3 Concentrations of xanthophylls in freeze-dried egg yolks directly after processing from two egg batches of husbandry class 1, presented as mean values ± standard deviation (micrograms per 100 g of egg yolk, n = 9 each). Xanthophyll
Unpasteurised (egg batch 1)
Pasteurised (egg batch 1)
Unpasteurised (egg batch 2)
Pasteurised (egg batch 2)
All-E-lutein All-E-zeaxanthin 13-Z-lutein 13-Z-zeaxanthin 9-Z-canthaxanthin All-E-canthaxanthin Z-isomer canthaxanthin b-apo-80 -carotenoic acid ethyl ester Total xanthophylls
1015 ± 11 792 ± 13 71 ± 4 148 ± 4 454 ± 6 1233 ± 13 73 ± 4 326 ± 7 4112 ± 44
1005 ± 10 781 ± 30 75 ± 4 154 ± 6 478 ± 14 1229 ± 39 72 ± 6 325 ± 13 4119 ± 110
749 ± 26 473 ± 24 68 ± 5 82 ± 5 405 ± 17 1136 ± 50 62 ± 3 301 ± 20 3277 ± 134
692 ± 29 429 ± 23 68 ± 3 77 ± 2 395 ± 25 1053 ± 41 59 ± 3 271 ± 9 3044 ± 117
used for identification were 569 Da ( 1–4), 565 Da ( 5–7), and 461 Da (8), i.e., quasimolecular ions; strong mass signals of dedicated molecular ions [M+] could also be observed. Because the spectral data and masses were almost equal to (6), it was concluded that substance (7) might represent a (Z)-isomer of canthaxanthin. Furthermore, in every HPLC chromatogram, about six peaks, indicating the presence of minor components were observed (see Fig. 1), but identification and quantification of these substances failed because the standard solutions gave no corresponding peaks and their UV–Vis and mass signals were not distinctive. 3.2. Quantification of xanthophylls in freeze-dried egg-yolk samples In this study, the extraction of xanthophylls was accomplished with pure (slightly nonpolar) methanol, assisted by an ultrasound treatment. The use of ultrasound treatment in facilitating the extraction of carotenoids from foods has been widely acknowledged (Mason et al., 1996; Sun, Xu, & Godber, 2006) but must be applied carefully because degradation and isomerization effects are possible (Zhao et al., 2006). The calibration graphs, LOQs, and LODs of substances determined in this study are shown in Table 1. All-E-canthaxanthin was the predominant xanthophyll present in the two batches of freeze-dried egg yolk made from fresh commercial eggs from husbandry class 1, followed by all-E-lutein, all-Ezeaxanthin, 9-Z-canthaxanthin, and b-apo-80 -carotenoic acid ethyl ester. Xanthophyll contents of batch 1 were slightly higher than those of batch 2, but both showed a very similar distribution pattern, indicating that the hens’ feed was of the same quality (see Table 3). The observed distribution and amounts of xanthophylls detected in eggs from husbandry class 1 are in agreement with those reported in prior studies (Majchrzak & Elmadfa, 1997; Schlatterer & Breithaupt, 2006) and in accordance with legal regulations (EC, 2008, 2003b). Initial absolute xanthophyll contents of both egg batches, shown in Table 3, were set to 100% each and the relative changes during storage at 18 °C and +20 °C were calculated. For generalisation, the relative changes in the xanthophyll contents of both batches are summarised. Percentage changes in the contents of all-E-lutein, all-E-zeaxanthin, all-E-canthaxanthin, and total xanthophylls during storage are presented as mean values (n = 18) in Figs. 2 and 3 (j 18 °C, not pasteurised; h +20 °C, not pasteurised; N 18 °C, pasteurised; D +20 °C, pasteurised), whereby p-values were always lower than 0.001. The contents of all-E-lutein (losses of 65–69%), all-E-zeaxanthin (losses of 59–62%), 13-Z-lutein (losses of 46–53%), 13-Z-zeaxanthin (losses of 50–60%), 9-Z-canthaxanthin (losses of 14–15%), all-Ecanthaxanthin (losses of 19–25%), and b-apo-80 -carotenoic acid ethyl ester (losses of 29–34%) in freeze-dried egg yolk fell continuously over the storage period of six months, following an exponential function, whereby almost all losses occurred during the first four weeks of storage time. Coefficients of determination of the
regression curves for the data of Figs. 2 and 3 were always higher than 0.70. This progressive loss is consistent with results of other xanthophyll-storage studies (Lin & Chen, 2005; Shi & Chen, 1997). In contrast, the content of the Z-isomer of canthaxanthin (7) proved quite stable (relative changes from 7% to +11%). The results of this study also suggest that natural pigments, such as all-E-lutein and all-E-zeaxanthin, are more liable to an intense degradation in the egg matrix than are the synthetic xanthophylls allE-canthaxanthin and b-apo-80 -carotenoic acid ethyl ester. In this context, the findings of prior studies, which emphasised the relatively high stability of all-E-canthaxanthin during a longer storage period in a fatty matrix (Sheehan et al., 1998) and a low degradation rate of b-apo-80 -carotenoic acid ethyl ester during the boiling
Fig. 2. Percentage changes in the contents of all-E-lutein and all-E-zeaxanthin in freeze-dried egg yolk during 26 weeks of storage at 18 °C and +20 °C in the dark (n = 18) and according equations of regression.
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tent over six months of dark storage depended mostly on the distribution of natural and synthetic pigments in the egg yolk and, to a much lesser degree, on storage temperature. The arithmetically averaged percentage losses of the natural xanthophylls, all-E-lutein and all-E-zeaxanthin, reached a total of about two-thirds, and the lost of the synthetic xanthophylls all-E-canthaxanthin and b-apo80 -carotenoic acid ethyl ester totaled about one-fifth to one-third. Moreover, knowledge of xanthophyll concentrations in dried egg yolks, at all stages of storage, will help to enable estimations of dietary xanthophyll intake from foods formulated with processed egg yolk, such as pasta and pastries. References
Fig. 3. Percentage changes in the contents of all-E-cantha-xanthin and total xanthophylls in freeze-dried egg yolk during 26 weeks of storage at 18 °C and +20 °C in the dark (n = 18) and according equations of regression.
of eggs (Schlatterer & Breithaupt, 2006), were confirmed. In contrast to the observation that, in some cases (e.g., lutein and zeaxanthin), samples stored at ambient temperature showed even slightly higher xanthophyll contents, freeze-dried egg yolk stored at +20 °C showed no significant difference in xanthophyll content compared to freeze-dried egg yolk stored at 18 °C (see Figs. 2 and 3). The effect of greater xanthophyll retention at higher temperatures during storage has been reported previously (Xianquan, Shi, Kakuda, & Yueming, 2005) and was explained by an improved oxygen solubility at lower temperatures, which may contribute to a higher oxidation rate. The results of this study support the conclusion that freeze-dried egg yolks do not need to be stored at low temperatures because the putative protective influence on xanthophyll retention was refuted. Thus, the advice of egg-yolk-powder manufacturers to store their products at ambient temperature was confirmed. 4. Conclusions It must be emphasised that stability data, based on the egg yolk selected here as a model system, may not be generally transferred to comparable food matrices or even other egg batches without intensive investigation, because they inevitably contain variable amounts of bioactive compounds, such as antioxidants. Based on the outcome of this study, the retention of total xanthophyll con-
Breithaupt, D. E., & Schwack, W. (2000). Determination of free and bound carotenoids in paprika (Capsicum annuum L.) by LC/MS. European Food Research and Technology, 211, 52–55. Christensen, C. M. (1983). Effects of colour on aroma, flavor and texture judgments of foods. Journal of Food Science, 48, 787–790. Commission Regulation (EC) 2295/2003 of 23 December 2003 introducing detailed rules for implementing Council Regulation (EEC) 1907/90 on certain marketing standards for eggs (2003a). Art. 5-7, Art. 13, Annex II. Commission Regulation (EC) 775/2008 of 4 August 2008 establishing maximum residue limits for the feed additive canthaxanthin in addition to the conditions provided for in Directive 2003/7/EC. Community register of feed additives pursuant to Regulation (EC) 1831/2003 (2003b), released 17 November 2009 (http://ec.europa.eu/comm/food/food/ animalnutrition/feedadditives/registeradditives_en.htm); Regulation (EC) 1831/2003 of the European Parliament and of the council of 22 September 2003 on additives for use in animal nutrition. De Jesus Ornelas-Paz, J., Yahia, E. M., & Gardea-Bejar, A. (2007). Identification and quantification of xanthophyll esters, carotenes, and tocopherols in the fruit of seven mexican mango cultivars by liquid chromatography-atmospheric pressure chemical ionization-time-of-flight mass spectrometry [LC-(APCI+)MS]. Journal of Agricultural and Food Chemistry, 55, 6628–6635. De Rosso, V. V., & Mercadante, A. Z. (2007). Identification and quantification of carotenoids, by HPLC-PDA-MS/MS, from amazonian fruits. Journal of Agricultural and Food Chemistry, 55, 5062–5072. Franke, K., & Kießling, M. (2002). Influence of spray drying conditions on functionality of dried whole egg. Journal of the Science of Food and Agriculture, 82, 1837–1841. Galobart, J., Barroeta, A. C., Baucells, M. D., Cortinas, L., & Guardiola, F. (2001). Ftocopherol transfer efficiency and lipid oxidation in fresh and spray-dried eggs enriched with x3-polyunsaturated fatty acids. Poultry Science, 80, 1496–1505. Guardiola, F., Codony, R., Manich, A., Rafecas, M., & Boatella, J. (1995). Stability of polyunsaturated fatty acids in egg powder processed and stored under various conditions. Journal of Agricultural and Food Chemistry, 43, 2254–2259. Handelman, G. J., Nightingale, Z. D., Lichtenstein, A. H., Schaefer, E. J., & Blumberg, J. B. (1999). Lutein and zeaxanthin concentrations in plasma after dietary supplementation with egg yolk. American Journal of Clinical Nutrition, 70, 247–251. Jaekel, T., Dautel, K., & Ternes, W. (2008). Preserving functional properties of hen’s egg yolk during freeze-drying. Journal of Food Engineering, 87, 522–526. Kang, D. K., Kim, S. I., Cho, C. H., Yim, Y. H., & Kim, H. S. (2003). Use of lycopene, an antioxidant carotenoid, in laying hens for egg yolk pigmentation. AsianAustralian Journal of Animal Sciences, 16, 1799–1803. Kromidas, S. (2000). Validierung in der analytik. Weinheim, Germany: Wiley-VCH. Kromidas, S., & Kuss, H.-J. (2008). Chromatogramme richtig integrieren und bewertenEin Praxishandbuch für die HPLC und GC. Weinheim, Germany: Wiley-VCH. Lin, C. H., & Chen, B. H. (2005). Stability of carotenoids in tomato juice during storage. Food Chemistry, 90, 837–846. Liu, L., Zhang, M., Peng, G., Wang, H., & Zhang, S. (2004). Separation and identification of red pigments in natural red yolk of duck’s eggs by HPLC– MS–MS. Chinese Journal of Chromatography, 22, 197–201. Majchrzak, D., & Elmadfa, I. (1997). Carotinoid- und retinolgehalt in hühnereiern. Fett/Lipid, 99, 365–368. Mason, T. J., Paniwnyk, L., & Lorimer, J. P. (1996). The uses of ultrasound in food technology. Ultrasonics Sonochemistry, 3, S253–S260. Meléndez-Martínez, A. J., Vicario, I. M., & Heredia, F. J. (2003). A routine highperformance liquid chromatography method for carotenoid determination in ultrafrozen orange juices. Journal of Agricultural and Food Chemistry, 51, 4219–4224. Sánchez-Moreno, C., Plaza, L., De Ancos, B., & Cano, M. P. (2003). Vitamin C, provitamin A carotenoids, and other carotenoids in high-pressurized orange juice during refrigerator storage. Journal of Agricultural and Food Chemistry, 51, 647–653. Schlatterer, J., & Breithaupt, D. E. (2006). Xanthophylls in commercial egg yolks: Quantification and identification by HPLC and LC–(APCI)MS using a C30 phase. Journal of Agricultural and Food Chemistry, 54, 2267–2273. Sheehan, E. M., Connor, T. P., Sheehy, P. J. A., Buckley, D. J., & FitzGerald, R. (1998). Stability of astaxanthin and canthaxanthin in raw and smoked Atlantic salmon (Salmo salar) during frozen storage. Food Chemistry, 63, 313–317.
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Shi, X.-M., & Chen, F. (1997). Stability of lutein under various storage conditions. Nahrung, 41, 38–41. Sun, T., Xu, Z., & Godber, J. S. (2006). Ultrasound assisted extraction in quantifying lutein from chicken liver using high-performance liquid chromatography. Journal of Chromatography B, 830, 158–160. Tang, Y. C., & Chen, B. H. (2000). Pigment change of freeze-dried carotenoid powder during storage. Food Chemistry, 69, 11–17.
Xianquan, S., Shi, J., Kakuda, Y., & Yueming, J. (2005). Stability of lycopene during food processing and storage. Journal of Medicinal Food, 8, 413–422. Zhao, L., Zhao, G., Chen, F., Wang, Z., Wu, J., & Hu, X. (2006). Different effects of microwave and ultrasound on the stability of (all-E)-astaxanthin. Journal of Agricultural and Food Chemistry, 54, 8346–8351.