Effect of gamma irradiation and storage on lutein and zeaxanthin in liquid, frozen and dried egg yolk samples

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Effect of gamma irradiation and storage on lutein and zeaxanthin in liquid, frozen and dried egg yolk samples

Mine Uygun-Sarıbay a, Ece Ergun a , Turhan Köseoğlu a

a _{TAEA, Sarayköy Nuclear Research and Training Center, Kazan, 06983, Ankara, TURKEY}

Abstract

The aim of this study was to monitor the effects of gamma irradiation and storage on the content of lutein and zeaxanthin in egg yolk samples. Liquid, frozen and dried egg samples were subjected to gamma irradiation doses of 0, 1, 2 and 3 kGy followed by storage of liquid samples at +4±1 °C for 21 days, frozen samples at -18±1 °C and dried samples at room temperature for 1 year. The xanthophyll concentrations were determined by high-performance liquid chromatography-diode array detector (HPLC-DAD). It was observed that concentrations of both lutein and zeaxanthin were decreased significantly (P < 0.05) after irradiation and during storage. The mechanism for radiation-induced degradation was proposed as radical formation which initiate chain reactions. It was suggested that during storage active radical species and oxygen caused the degradation.

Keywords: Egg yolk; gamma irradiation; lutein; zeaxanthin; storage

1. Introduction

Food irradiation is a process of treating food in order to eliminate food-borne pathogens, make it safer to eat and have a longer shelf life. Food can be irradiated by exposing it to the gamma rays of a radioisotope (one that is widely used is cobalt-60), energetic electrons from partical accelerators, and X-rays. These are suitable source of ionizing energy for the food irradiation applications because they have enough energy to penetrate a considerable thickness of food products. An extensive review of science related to microbiological safety indicates that irradiation is an effective solution to the problem of microbial contamination. Irradiation is accepted as a food safety process by independent health organizations and

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regulatory agencies around the world [1]. At present, specific applications of food irradiation are approved by national legislations in over 55 countries worldwide [2].

Egg is known to be an excellent source of high-quality proteins, lipids, vitamins and minerals. Besides the direct consumption, egg is used as ingredient in foods due to the functional properties of the yolk and white such as flavor, color, foaming, emulsifying, binding, thickening and leavening. However, eggs are responsible for causing food-born illnesses when contaminated by different microorganisms, among which Salmonella enteritidis is most significant. The processes such as rapid cooling, washing with antimicrobial solutions and heat pasteurization can be used in order to reduce external and internal contamination of egg and egg-containing products.

Ionizing radiation at medium doses can also reduce or eliminate pathogens in eggs. FDA has approved irradiation of eggs at doses up to 3 kGy. However, it is important to examine the changes in chemical and sensory characteristics. In order to investigate the effect of irradiation, radiation induced changes in chemical, physicochemical and functional properties such as pH, viscosity, color, texture, foaming ability, emulsion capacity and total carotenoid of egg samples have been determined by several reseachers [3-13]. However, no study has been directly examined the effect of irradiation on lutein and zeaxanthin, the most abundant xanthophylls found in egg yolk and responsible for the yellow color. These xanthophylls are the two major components of the macular pigment of the retina and have dual functions; to act as powerful antioxidants and to help protect eyes against damage due to ultraviolet radiation from the sun [14]. They are not produced in the body and must be consumed through diet. The yolks of chicken eggs involve different amounts of lutein and zeaxanthin due to the genetic variations and husbandry conditions [15].

Lutein and zeaxanthin are both dihydroxy-carotenoids having conjugated trans double bonds with the ionone ring systems being substituted at both the 3 and 3' carbon (Fig. 1). In zeaxanthin, the ionone rings are both β types and the β-ionone ring double bond is found between the C5 and C6 carbons so that all double bonds are conjugated with the polyene chain. On the other hand, lutein has both a β-ionone ring and an ε-ionone ring. The ε-ionone ring has a C4-C5 double bond and an allylic 3'-hydroxyl group [16]. These structural properties play an important role in the oxidation and degradation of these xanthophylls. Recent studies have been indicated that carotenoids are susceptible to ionizing radiation, but this effect has been investigated by total carotenoid analysis and color measurements [4, 6,

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10, 17] . None of these studies directly monitor the radiation-induced degradation of lutein and zeaxanthin, and discuss the degradation mechanism. The main objective of this study is to identify the effect of gamma irradiation on the content of lutein and zeaxanthin present in commercial liquid, frozen and dried egg yolks by high-performance liquid chromatography-diode array dedector (HPLC-DAD). The effect of storage on these xanthophylls is also examined. In addition, mechanisms are proposed for the degradation of lutein and zeaxanthin.

2. Experimental

2.1. Chemicals and standards

Tert-butyl methyl ether (TBME), methanol, ethyl acetate, petroleum ether and triethyl amine

were purchased from Merck (Darmstadt, Germany). High-purity water was prepared with a Milli-Q Gradient water-purification system (Millipore, Eschborn, Germany). Before HPLC analysis, all solvents were degassed by ultrasonic treatment (Bandelin Sonorex, Berlin, Germany). Pure Lutein (Rotichrom®) and zeaxanthin (Rotichrom®) standards were purchased from Carl Roth (Karlsruhe, Germany).

2.2. Samples, irradiation and storage

Liquid and dried egg yolk samples were supplied from AB Foods Inc. (Balıkesir, Turkey). A certain amount of liquid egg samples had been frozen immediately at -18±1 °C in a freezer (Beko 3400 CF, Turkey). All egg samples (liquid, frozen and dried) were exposed to ionizing radiation using tote-box type irradiator equipped with 60_{Co gamma source (SVST-1 category} IV) at Turkish Atomic Energy Authority, Sarayköy Nuclear Research and Training Center (Ankara, Turkey). Irradiation was performed at doses of 0, 1, 2 and 3 kGy at ambient temperature and atmosphere without any gas introduction. Irradiated liquid egg samples were stored at +4±1 °C for 21 days and analyses were performed on day 0, 7, 14, 21. Frozen and dried egg samples were stored for 1 year at -18±1 °C and at room temperature, respectively. Analyses of these samples were carried out at 0, 3, 6, 9, 12 months. Sample containers were covered with aluminum foil during storage in darkness in order to avoid photooxidation. The dose distribution was measured using dichromate dosimeter (Harwell Gammachrome,UK).

2.3. HPLC analysis of lutein and zeaxanthin

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published methods, with minor modifications [18,19]. Aliquots (1 g) of irradiated and non-irradiated liquid, frozen and dried egg yolks were replaced directly into a separating funnel. A ternary solvent mixture (methanol/ethyl acetate/petroleum ether, 1:1:1, v/v/v) was added and shaken gently. The supernatant was collected in a round-bottom flask. This procedure was repeated until the sample became colorless. The mixture was evaporated to dryness on a rotary evaporator at 30 °C. The residue was then dissolved in 5 ml of tert-butyl methyl ether /methanol mixture (1:1, v/v), filtered through a 0.45 μm membrane filter and filled in amber USP Type I vial. The samples were immediately analysed using HPLC-DAD system consisting of Waters Alliance 2695 (Eschborn, Germany) separation module with Waters 2996 photodiode array dedector. The analytical scale separation was developed on a C30 column (5 μm, 250mm × 4.6 mm, YMC Europa GmbH, Dinslaken Wilmington, NC) and column was kept at 35 °C. The mobile phases were methanol/purified water/ triethyl amine (90/10/0.1, v/v/v, eluent A) and tert-butyl methyl ether/methanol/purified water/triethyl amine (90/6/4/0.1, v/v/v/v, eluent B). The gradient procedure, at flow rate of 1.0 mL/min, was as follows: (1) start at 93.5% eluent A and 6.5% eluent B (2) a 34-min linear gradient to 100% eluent B (3) a 4-min linear gradient back to 93.5% eluent A and 6.5% eluent B (4) a 5-min hold at 93.5% eluent A and 6.5% eluent B.

Calibration was performed in the range of 0.5-10 μg/ml using dilutions of the respective stock solutions of lutein and zeaxanthin in methanol. Calibration graphs were constructed by plotting the respective peak areas (450 nm, mAUs) against the concentrations (μg/ml). Coefficients of determination were always higher than 0.997.

Peak identification was based on comperisons with retention time of lutein and zeaxanthin standards. Quantification was carried out by integrating peak areas in the HPLC chromatograms. All HPLC analyses were performed in triplicate.

2.4. Statistical analysis

Quantitative data were presented as means ± SD of at least triplicate experiments. Means were compared according to Duncan’s multiple-range test. The significant statistical level was set to P <0.05.

3. Results and discussion

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The application of ionizing radiation on eggs in order to reduce and elimate Salmonella can cause changes in lutein and zeaxanthin content. The amount of lutein and zeaxanthin loss due to the irradiation is affected by several factors, including dose, temperature, presence of oxygen, and sample type. Therefore, to investigate this effect, liquid, frozen and dried egg yolk samples were irradiated at doses of 0, 1, 2 and 3 kGy, and lutein and zeaxanthin concentrations of these samples were determined using HPLC-DAD. Representative HPLC chromatograms are shown in Fig. 2. Satisfactory separation was obtained and proper identification and quantification for lutein (retention time = 15.5 min) and zeaxanthin (retention time = 16.8 min) was carried out. Table 1 and 2 summarize the amounts of lutein and zeaxanthin in irradiated and non-irradiated egg yolk samples and the effect of storage. The amounts of lutein and zeaxanthin were found to be 4.45 ± 0.08 and 2.18 ± 0.07 μg/g of non-irradiated liquid egg yolk, respectively. However, xanthophyll contents in liquid sample were decreased significantly with increasing irradiation dose (at 3 kGy, 2.17 ± 0.06 μg/g for lutein and 1.02 ± 0.03 μg/g for zeaxanthin, P < 0.05). It was observed that after irradiation at dose of 3 kGy, 51 % of lutein and 53 % of zeaxanthin were degraded in liquid egg yolk samples. On the other hand, radiation-induced degradation of lutein and zeaxanthin in frozen and dried egg yolk samples was much less than in liquid samples. In frozen egg yolk samples, the amounts of lutein and zeaxanthin were decreased from 5.96 ± 0.08 μg/g to 5.08 ± 0.08 μg/g (15 % degraded) and from 1.37 ± 0.10 μg/g to 1.11 ± 0.05 μg/g (19 % degraded), respectively. In dried egg yolk samples, the results indicated that 23 % of lutein and 25 % of zeaxanthin were degraded after irradiation at dose of 3 kGy.

Losses of xanthophylls can be explained by two mechanisms commonly called direct and indirect effects of radiation on molecules. Direct effect could be responsible for the losses of lutein and zeaxanthin in dried samples. The energy absorbed from ionizing radiation gives rise to formation of ions and excited xanthophylls (Eq. 1) which may decompose into radicals. Therefore, the initial reaction mechanism could be the ion dissociation giving a radical (Eq. 2), ion-molecule reaction (Eq. 3), germinate recombination of ions (Eq. 4), dissipation of excitation energy without reaction (Eq. 5) dissociation of excited molecules to radical products (Eq. 6) and recombination of radicals (Eq. 7) [20].

6 XH  XH+ , e- , XH* (1) XH+ X + _{+ H} (3)  (2) X + + XH _XH+ _{+ X}  XH+ + e- XH* (4) XH* XH (5) XH* _{X + H (6)}   X + H XH (7)

X· and H· radicals are formed through homolytic cleavage of the weakest bonds. Previous report [21] indicates that for zeaxanthin C4-H, C4'-H bonds in the β-ionone rings have the lowest dissociation energy. However, for lutein, C6'-H bond in the ε-ionone ring is the weakest bond. The reason could be the conjugated C=C double bonds which decreases the dissociation energy of the allylic hydrogen. On the other hand, hydrogen can easily be abstracted from a saturated carbon atom in a position allylic to the polyene chain [22]. However, the bond dissociation energy of 9a and 13a positions is approximately 0.6 eV higher than the lowest bond dissociation energy reported in the paper [21].

Since the samples were irradiated at low dose values, molecules could not be damaged so severely, small amounts of radical could be formed, and recombination of these radicals might be the main reaction mechanism rather than diffusion from the spurs into the bulk. However, irradiation was carried out at ambient atmosphere (in the presence of oxygen). Thus the most probable route is the reaction of oxygen with the radical species. According to this reaction both X· and H· radicals are converted into peroxyl radicals (Eq. 8 and 9).

 X + O₂ XOO (8)  H + O₂ (9)   _HOO

Peroxyl transients can attack polyene chain which is susceptible to radical attack due to the delocalisation of π-electrons. After that, the stable final products (epoxides and carbonyls) can be produced[23]. The proposed mechanisms are given in Eq. 10-16.

7  XOO + XH XO₂XH ₍₁₀₎ XO₂XH _{XO + epoxide} ₍₁₁₎ XO + XH _XOXH ₍₁₂₎ XOXH _{+ O} 2 XOXHO2 (13) XOXHO₂ _{+ XH XOXHO} 2XH (14)

XOXHO₂XH _XOXHO_{+ epoxide (15)}

XOXHO _{X + 2 carbonyls} ₍₁₆₎

However, in aqueous samples another effect called ‘indirect effect’ should be considered. When an aqueous system is irradiated, water absorbs large fraction of the radiation energy and very reactive species namely hydroxyl and hydrogen radicals, hydrated electrons and molecular products (hydrogen and hydrogen peroxide) are formed [20]. These radical species could react with lutein and zeaxanthin and cause degradation. Hydrogen radicals, hydrated electrons acts as reducing agents in air-free media, but in the presence of oxygen these species are converted into peroxyl radicals (Eq. 9 and 17).

e_sol + O₂ O₂ (17)

.

Therefore, peroxy and hydroxyl radicals act as oxidizing transients. However, hydroxyl radicals are more powerful oxidants than peroxyl radicals and participate in a number of rections such as addition, electron transfer and H-atom abstraction reactions. Hydroxyl radicals can readily attack to the double bonds of the xanthophylls and generate a radical site on the molecule. The resulting radical adducts can also scavenge oxygen. However, these peroxyl transients are rather unsable and can undergo hydrolysis forming various final products (Eq. 18 and 19).

XH + OH  ₍₁₈₎ XHOH + O₂   _{XOH + HO} 2 (19) XHOH OOXOH

Hydroxyl transients can also react with lutein and zeaxanthin abstracting a hydrogen yielding a free radical of xanthophylls (Eq. 20). It is easier for xanthophylls to give hydrogen to hydroxyl radicals having a high reduction potential than to alkyl peroxy radicals [24].

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XH + OH _{X + H}

2O

 ₍₂₀₎

.

Xanthophyll radicals can add oxygen (Eq. 8) and further reactions (Eq. 10-16) can take place. In dried egg yolk samples, direct effect of ionizing radiation could be the main mechanism for the degradation of lutein and zeaxanthin. On the other hand, in aqueous samples (liquid and frozen egg yolks) xanthophyll destruction is expected to be more effective due to the radiolysis products of water. However, in frozen samples, radicals diffuse much more slowly due to the lower temperature. They are trapped in the frozen material, tend to recombine to form the original substances rather than diffuse through the food and react with other components. Therefore, loss of lutein and zeaxanthin was reduced when egg samples were irradiated in frozen state. Eventually, reduction in the amounts of lutein and zeaxanthin in liquid egg yolk samples were much greater than in dried and frozen samples (Fig. 3).

3.2. Effect of storage on lutein and zeaxanthin

In this study, the effect of storage on amount of lutein and zeaxanthin in irradiated and non-irradiated egg yolk samples was also monitored. The results indicated that amount of lutein significantly (P < 0.05) decreased for both irradiated and non-irradiated liquid samples (Table 1, Fig. 4a). It was observed that in non-irradiated liquid sample, 28 % of lutein was degraded during a 21 day storage period. In irradiated liquid samples, loss of lutein after storage was 36 %, 40 % and 38 % at dose of 1, 2 and 3 kGy, respectively. On the other hand, percentages of lutein loss obtained in irradiated and non-irradiated dried samples were 46 %, 47 %, 49 % and 64 % at dose of 0, 1, 2 and 3 kGy, respectively (Table 1, Fig. 4c). However, lutein was degraded much less in frozen samples (20 %, 21 %, 18 % and 16 % at dose of 0, 1, 2 and 3 kGy, respectively) after the same storage period (Table 1, Fig. 4b). The results indicated that minimum zeaxanthin degradation also occurred in frozen samples after storage. It was observed that in frozen samples during a 12 month storage period, 13 %, 7 %, 11 % and 14 % of zeaxanthin was degraded at dose of 0, 1, 2 and 3 kGy, respectively (Table 1, Fig. 5b). However, after the same storage period, losses of zeaxanthin in dried samples at dose of 0, 1, 2 and 3 kGy were found to be 24 %, 18 %, 21 %, 23 %, respectively (Table 1, Fig. 5c). On the other hand, percentages of zeaxanthin loss obtained in

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irradiated and non-irradiated liquid samples were 25 %, 35 %, 33%, 36 % (Table 1, Fig. 5a).

Degredation of non-irradiated lutein and zeaxanthin during storage was due to the oxidation of these xanthophylls under the influence of atmospheric oxygen. The efficiency of oxidative degradation is affected by the light conditions, temperature, oxygen availability, moisture content [25]. Oxygen and light exposure during storage were prevented by properly sealed dark packaging and keeping the samples in the dark so that only the oxygen present in the container can have caused the oxidation. In order to monitor the oxidative degradation, samples were stored without purging with nitrogen or any inert gas. Therefore, the oxidative degradation of non-irradiated samples was mainly affected by the concentration of oxygen in the container, temperature and moisture content. The influence of water activity on oxidation is studied by several reseachers. It has been evidenced that the lower water activity increases the β-carotene degradation [26, 27], cholesterol oxidation [28]. Therefore, non-irradiated dried samples (low water activity) were much more degraded than non-irradiated frozen samples over a 12 month period. Furthermore, thermal process and storage at higher temperature (room temperature) can cause the all-trans-isomer to the cis-isomer in dried samples. It is supposed that isomerisation could be the first step of oxidation, leading to a diradical of carotenoids which can easily be attacked by oxygen on either side of the cis bond. This radical attack followed by homolytic internal substitution gives the epoxides, apocarotenones and apocarotenals [29]. Therefore, lower temperature (-18±1 °C in a freezer) may have helped to reduce the degradation of lutein and zeaxanthin during 12 months of storage of non-irradiated frozen samples. However, degradation of non-irradiated liquid samples were also high despite the short storage period and high water content. High-water content may increase the rate of oxidation by enhancing the mobility of reactants and the solubility of oxygen in foods.

Same results were obtained for irradiated liquid, frozen and dried samples. The least degradation occurred in frozen samples. In irradiated samples, besides the effects mentioned above, active radicals which do not undergo recombination during the initial storage period can play an important role in the degradation of lutein and zeaxanthin. It was observed that this effect was much more remarkable for liquid samples due to the short storage period and active products of water radiolysis. After the 21 day storage period, degradation of lutein and zeaxanthin was more effective in irradiated liquid samples than in non-irradiated liquid

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3. Conclusions

Lutein and zeaxanthin belong to the xanthophyll family of carotenoids and are the two major components found in egg yolks. They are considered as powerful antioxidants due to their singlet oxygen quenching abilities. In addition, they are efficient in scavenging free radicals. Therefore, they are important nutrients for human that need to be consumed to reduce the potential damage in the cells. However, application ionizing radiation on eggs in order to reduce the microorganisms causes degradation of these xanthophylls. Thus, it is important to monitor and identify the effect of ionizing radiation on these molecules.

Degradation mechanism and the proportions of reactive species depend on the irradiation conditions and nature of the material being irradiated [20]. Therefore, three types of egg samples (liquid, frozen and dried) were irradiated at dose of 0, 1, 2 and 3 kGy at ambient conditions. After each irradiation, concentrations of lutein and zeaxanthin were determined by HPLC-DAD. It was observed that with increasing doses, lutein and zeaxanthin contents of egg samples were decreased significantly (P < 0.05). It was suggested that absorption of radiation energy gave rise to radical formation and in the presence of oxygen these radical species were converted into peroxyl transients and stable final products (epoxides and carbonyls). The greatest losses of lutein and zeaxanthin were found in liquid samples due to the radiolysis products of water. However, in frozen samples, chemical changes were reduced due to the lower mobility of free radicals.

Effect of storage on both irradiated and non-irradiated samples were also monitored. During the storage period of non-irradiated samples, lutein and zeaxanthin were degraded due to the interaction of xanthophylls and oxygen. However, the degradation rate of lutein and zeaxanthin in irradiated liquid samples was found more than in non-irradiated liquid samples. It was attributed to life time and activity of radical species.

Acknowledgement

Authors wish to express their gratitude to Turkish Atomic Energy Authority ( TAEK-A3.H1.P1.01), which financially supported this work.

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Figure Captions

Figure 1.The structures of lutein and zeaxanthin.

Figure 2. Representative HPLC-DAD chromatogram of (a) lutein calibration solution, (b) zeaxanthin calibration solution, (c) non-irradiated egg yolk extract, (d) irradiated egg yolk extract.

Figure 3. Effect of irradiation on the amount of a) lutein b) zeaxanthin in liquid, frozen and dried egg yolk samples.

Figure 4. Effect of storage on the amount of lutein in a) liquid, b)frozen and c) dried egg yolk

samples.

Figure 5. Effect of storage on the amount of zeaxanthin in a) liquid, b)frozen and c) dried egg

yolk samples.

Table Captions

Table 1. Lutein and zeaxanthin in irradiated and non-irradiated liquid, frozen and dried egg

yolk samples during storage. Mean value and standard error (n = 3).

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Figure 2. Representative HPLC-DAD chromatogram of (a) lutein calibration solution, (b) zeaxanthin calibration solution, (c) non-irradiated egg yolk extract, (d) irradiated egg yolk extract.

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Figure 3. Effect of irradiation on the amount of a) lutein b) zeaxanthin in liquid, frozen and dried egg yolk samples.

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Figure 4. Effect of storage on the amount of lutein in a) liquid, b)frozen and c) dried egg yolk

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Figure 5. Effect of storage on the amount of zeaxanthin in a) liquid, b)frozen and c) dried egg

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Table 1. Lutein and zeaxanthin in irradiated and non-irradiated liquid, frozen and dried egg

yolk samples during storage. Mean value and standard error (n = 3).

Sample Storage Lutein (μg g

-1 _{sample)/irradiation dose (kGy)}

0 1 2 3 Liquid egg yolk 0 x _4.45a _{± 0.08 4.26}b _{± 0.07 3.28}def _{± 0.10 2.17}h _{± 0.06} 7x _3.80c _{± 0.07 3.42}d_{± 0.08 2.19}h _{± 0.02 1.66}j _{± 0.04} 14x _3.36de _{± 0.04 3.26}ef _{± 0.06 2.10}h _{± 0.07 1.51}k _{± 0.06} 21x _3.21f _{± 0.08 2.71}g _{± 0.03 1.96}i _{± 0.04 1.34}l _{± 0.03} Frozen egg yolk 0 y _5.96a _{± 0.08 5.78}a _{± 0.05 5.41}bc _{± 0.11 5.08}de _{± 0.08} 3y _5.81a _{± 0.07 5.68}ab _{± 0.07 4.98}de _{± 0.04 4.86}def _{± 0.04} 6y _5.13cd _{± 0.08 4.76}efg _{± 0.06 4.52}ch _{± 0.04 4.37}h _{± 0.03} 9y _4.95de _{± 0.07 4.61}de _{± 0.07 4.42}h _{± 0.06 4.50}gh _{± 0.07} 12y _4.77efg _{± 0.05 4.59}fgh _{± 0.06 4.44}ch _{± 0.03 4.26}h _{± 0.07} Dried egg yolk 0 z _8.84a _{± 0.12 8.33}b _{± 0.08 7.23}d _{± 0.07 6.84}e _{± 0.06} 3z _7.64c _{± 0.09 6.27}g _{± 0.07 4.84}j _{± 0.06 3.71}m _{± 0.05} 6z _6.32f _{± 0.06 5.65}h _{± 0.05 4.39}k _{± 0.04 3.06}n _{± 0.03} 9z _5.60h _{± 0.08 4.89}ı _{± 0.07 4.09}l _{± 0.05 2.86}o _{± 0.04} 12z _4.76j _{± 0.05 4.38}k _{± 0.04 3.64}m _{± 0.02 2.47}p _{± 0.07}

Sample Storage Zeaxanthin (μg g

-1 _{sample)/irradiation dose (kGy)}

0 1 2 3 Liquid egg yolk 0 x _2.18a _{± 0.07 1.98}b _{± 0.08 1.25}cde _{± 0.06 1.02}de _{± 0.03} 7x _1.88c _{± 0.09 1.65}bc _{± 0.06 1.01}de _{± 0.04 0.89}de _{± 0.04} 14x _1.69bc _{± 0.06 1.45}bcd _{± 0.09 0.92}de _{± 0.03 0.77}e _{± 0,04} 21x _1.64bc _{± 0.05 1.29}cde _{± 0.11 0.84}de _{± 0.07 0.65}f _{± 0,05} Frozen egg yolk 0y _1.37ab _{± 0.10 1.23}abc _{± 0.03 1.19}bcd _{± 0.02 1.11}cdef _{± 0.05} 3y _1.38a _{± 0.08 1.22}abc _{± 0.07 1.08}cdef _{± 0.10 0.98}ef _{± 0.08}

6y _1.33ab _{± 0.03 1.20}abc _{± 0.06 1.10}cdef _{± 0.09 1.01}def _{± 0.07}

9y _1.23abc _{± 0.06 1.21}abc _{± 0.10 1.11}cdef _{± 0.07 0.95}f _{± 0.13}

12y _1.20abc _{± 0.08 1.14}cde _{± 0.07 1.06}cdef _{± 0,07 0.97}ef _{± 0.08}

Dried egg

yolk 0

z _1.91a _{± 0.07 1.80}ab _{± 0.04 1.64}bcd _{± 0.06 1.43}fgh _{± 0.13}

3z _1.80ab _{± 0.10 1.66}bc _{± 0.03 1.54}cdefg _{± 0.05 1.29}hij _{± 0.08}

6z _1.63cde _{± 0.08 1.60}cdef _{± 0.03 1.44}fgh _{± 0.03 1.26}ijk _{± 0.08}

9z _1.53cdefg _{± 0.03 1.62}cde _{± 0.07 1.42}ghi _{± 0.08 1.18}jk _{± 0.11}

12z _1.46efgh _{± 0.05 1.47}defg _{± 0.04 1.30}hij _{± 0.11 1.10}k _{± 0.03} x _{Day, 4 ± 1 °C}

y _{Month, -18 ± 1 °C} z _{Month, room temperature}