Kinetic Stability of Lutein in Freeze-Dried Sweet Corn Powder Stored under Different Conditions

Abstract

The powder of freeze-dried sweet corn was stored under different conditions at 4, 20 and 37°C for 12 weeks, and the stability of lutein and its isomers during storage were studied. It was found that total lutein content declined depending on storage conditions and the kind of lutein isomer. The highest residual of all-trans-lutein was found stored at 4°C with vacuum and dark. Under the storage of air and light at 37°C, major isomers 9-cis-lutein, 9′-cis-lutein, 13′-cis-lutein and 13-cis-lutein showed significant increases by 35.0, 50.0, 38.4 and 37.1%, respectively, of its original content after 12 weeks of storage, which indicated isomerization might take place during storage. The kinetics of all-trans-lutein degradation under different conditions followed first order kinetics well, the apparent rate constant values were lowest under vacuum and dark and highest under air and light at each storage temperature, confirming potential detrimental effects of oxygen and light on lutein loss.

Introduction

Lutein, one of the important carotenoids, has gathered increasing attention due to their association with eye health. (Huck et al., 2000; Johnson, 2004). It is mostly found in fruits, vegetables, grains, and eggs (Perry et al., 2009). Lutein plays an important role in preventing cataracts and age-related macular degeneration (AMD), which is the leading cause of blindness among the elderly. Lutein acts as a blue-light filter and protects the underlying tissues from phototoxic damage (Ma, et al., 2012). Studies have also shown lutein in human plasma has antioxidative function such as the scavenging of free radicals and singlet oxygen and thus reducing the risk of certain cancers (Handelman, 2001; Schunemann et al., 2002).

Sweet corn (Zea mays L.) contains significant amounts of lutein and other carotenoids and is becoming a popular vegetable in the Chinese diet (Scott and Eldridge, 2005; Niu et al., 2011). Freeze-drying process of sweet corn is used to increase its shelf life with a minimal loss of nutrients, which may be a promising technique to avoid amounts of lutein decreased via heat degradation, several researchers have analyzed nutrient content in freeze-dried plant materials (Siriamornpun et al., 2012; Zhang et al., 2009; Kao et al., 2011), however few reports have evaluated the lutein content in processed corn under different storage conditions.

Naturally, lutein is usually present in their all-trans-configuration, except for processing of food such as drying, heating, and cooking that affecting all-trans-lutein partially converted into their cis-isomers (Bengtsson et al., 2008; Aparicio-Ruiz et al., 2011; Kao et al., 2012). Furthermore, storage time and storage conditions, such as oxygen, temperature and light, facilitate the formation of cis-isomers (Tang and Chen, 2000; Çinar, 2004). The nutritional consequences of cis/trans isomerization could be changed in bioavailability and physiological activity (Jabeen, et al., 2013). Literature data also suggest that each lutein shows an individual pattern of absorption, plasma transport, and metabolism (Parker, 1996). Investigating the effects of processing on lutein stability is therefore of great interest.

In view of these facts, a study was planned to determine effects of various storage conditions on the stability of lutein isomers in freeze-dried sweet corn kernel powder, and kinetics of all-trans-lutein degradation during storage was also conducted.

Materials and Methods

Materials    Frozen sweet corn kernels were obtained from Engineering Research Center for Agricultural Products Processing, National Agricultural Science and Technology Innovation Center in East China. Uniform shape and size samples without any physical damage were selected. They were weighed and lyophilized (FD-1A-50, Beijing Boyikang Laboratory Instruments Co., Ltd, China) at −50°C (vacuum degree of 15 Pa) until constant weight was obtained with 2.6% water content. After complete dryness, the kernel samples were ground to a powder using a grinder (FW100, Tianjin Taisite Instrument Co., Ltd, China) and sieved using 40 standard meshes to ensure symmetry of particle size.

Lutein standard was purchased from Sigma Chemical Co. (St. Louis, MO, USA). HPLC-grade methanol and methyl tertiary butyl ether (MTBE) were from TEDIA (Fairfiled, OH, USA). All other chemicals in the investigation were of analytical grade from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

Sample preparation and storage    Aliquots of freeze-dried powder were stored at 4, 20 and 37°C under four conditions: (a) vacuum and dark, (b) air and dark, (c) vacuum and light, and (d) air and light. The samples were packaged with air or vacuum in polyethylene bags (5 g per bag) and stored in the dark or light (the light intensity of the incandescent measured about 1600 lux) at selected temperatures 4, 20, 37°C for 12 weeks, respectively. At the time interval of 2 weeks, the powders were removed from storage and collected for HPLC analysis of lutein.

Extraction of lutein    Lutein was extracted from freeze-dried sweet corn by adopting a method described by Inbaraj et al. (2006). In short, 3 g of the freeze-dried powder was treated with a 30 mL mixture of hexane-ethanol-acetone-toluene (10:6:7:7, v/v) in a 100 mL volumetric flask and shaken for 1 h. To the contents, 2 mL of 40% methanolic KOH was added for saponification at 25°C in the dark under nitrogen gas for 16 h. After saponification, 30 mL of hexane was added for partition of carotenoids, shaken for 1 min and 10% sodium sulfate solution was added and diluted to volume. The mixture was allowed to stand until two phases separated clearly. The upper layer containing lutein was collected, evaporated to dryness, redissolved in 10 mL methanol, and filtered through a 0.45 µm membrane filter for HPLC analysis. The whole extraction procedure was carried out under dimmed light and nitrogen gas was flushed into vials to avoid isomerization or degradation of lutein.

Determination of lutein isomers    Lutein isomers were performed according to the revised method of Aman et al. (2005). The chromatographic analysis was performed using an analytical scale C₃₀ reversed phase column (250 mm × 4.6 mm i.d.) with a particle size of 5 µm (YMC, Wilmington, MA, USA). Eluent A consisted of methanol/ MTBE/water (92: 4:4, v /v), eluent B was prepared by mixing MTBE/methanol/water (90: 6:4, v/v). Separation was performed at a column temperature of 25°C using a linear gradient from 100% A to 6% B within 80 min at a flow rate of 1 mL/min. Aliquots of 20 µL were used for HPLC.

Kinetics analysis    The loss of all-trans-lutein in freeze-dried sweet corn kernels during storage was calculated by using the standard equation for a first-order reaction model (Labuza, 1984), given below

  

Where C, the concentration at time t; C₀, the concentration at time zero; k, the first-order rate constant; t, the storage time (week).

Statistical analysis    The mean values and the standard deviation were calculated from the data obtained with triplicate trials. Kinetics data were analyzed by regression analysis using Microsoft Excel 2003 and Origin 7.0.

Results and Discussion

Changes of all-trans-lutein during storage    Though storage conditions such as temperature, oxygen levels, and humidity had all been reported to affect the stability of carotenes in other foods during storage (Wenzel et al., 2011; Nhung et al., 2010; Lin and Chen, 2005), very limited number of studies on the lutein stability of freeze-dried sweet corn were reported in the literature, so changes of all-trans-lutein in freeze-dried sweet corn during various storages were investigated (Fig.1). Figure 1a showed the contents of all-trans-lutein in freeze-dried sweet corn packaged with vacuum during storage in the dark. The initial all-trans-lutein content in freeze-dried powder was 20.14 µg/g, after 12 weeks storage, all-trans-lutein contents of the vacuum-packaged samples at 4, 20, 37°C under dark decreased to 19.30, 18.39, 16.02 µg/g, respectively, it was observed that all-trans-lutein decreased with increasing temperature as expected and retention of all-trans-lutein (%) in those samples was 95.83, 91.31, 79.54, respectively; however, those samples with air-packaged had the less all-trans-lutein (%) retention of 92.50, 80.98, 70.51, respectively, the presence of oxygen should be responsible for the degradation process (Fig. 1b).

Fig. 1.

Lutein degradation in freeze-dried sweet corn during storage, a) vacuum packaged, dark; b)air packaged, dark; c) vacuum packaged, light; d)air package, light. ▪ 4°C, •20°C, ▴37°C.

Vacuum-packaged freeze-dried samples stored at 20°C dark and light lost their all-trans-lutein 8.69% and 20.85% during 12 weeks storage (Fig. 1c). While air-packaged freeze-dried samples stored at 37°C, the content of all-trans-lutein was found to decrease with increasing illumination time, and the residual concentration was 5.99 µg/g after 12 weeks exposure to light, which amounted to a 70.3 % loss (Fig. 1d). This result showed that the higher the storage temperature, the faster all-trans-lutein degraded, a more pronounced degradation of all-trans-lutein occurred during light storage than dark storage.

Degradation kinetics of all-trans-lutein    First-order reactions were, by far, the most common and best studied in the destruction of pigments during processing and storage, there had been studies reported that lutein destruction followed a first-order reaction (Bechoff et al., 2010; Chen et al., 1996). Thermal kinetic values of all-trans-lutein in freeze-dried sweet corn during different storage, were shown in Table 1, the apparent reaction rate constant (k, week⁻¹) increased with an increase in storage temperature from 4 to 37°C under different storage conditions. The k values were lowest under vacuum and dark and highest under air and light at each storage temperature, confirming the potential detrimental effects of oxygen and light on all-trans-lutein loss.

Table 1. Thermal kinetic values of all-trans-lutein in freeze-dried sweet corn during storage

Storage	Rate constant, k(week−1)	Determination coefficient, R2	Activation energy, Ea(kJ/mol)	Q10
vacuum & dark
4°C	0.071	0.989	32.40 ± 0.25	1.57 ± 0.08
20°C	0.136	0.965
37°C	0.318	0.928
air & dark
4°C	0.132	0.985	28.64 ± 0.32	1.49 ± 0.18
20°C	0.327	0.994
37°C	0.494	0.987
vacuum & light
4°C	0.166	0.970	26.76 ± 0.28	1.46 ± 0.11
20°C	0.342	0.992
37°C	0.571	0.989
air & light
4°C	0.519	0.942	18.88 ± 0.41	1.30 ± 0.05
20°C	0.842	0.994
37°C	1.241	0.987

Temperature dependence of all-trans-lutein degradation was determined by using the Arrhenius equation

  

Where k, rate constant; k₀, pre-exponential factor; E_a, activation energy (kJ mol⁻¹); R, gas constant (8.3145 × 10⁻³kJ mol⁻¹K⁻¹); T, absolute temperature in K.

An Arrhenius plot between natural logarithm of k values versus 1/T followed an approximate linear relationship as shown in Fig. 2. From the distribution of the points, it could be seen that the plots were approximately linear (R² = 0.96 ∼ 0.99), confirming the reaction of all-tran-lutein degradation to be first order. Activation energies (Table1) were calculated by using Arrhenius plots of all-trans-lutein degradation in freeze-dried sweet corn and found higher under vacuum and dark, air and dark, vacuum and light than that of air and light. The lower activation energy was probably because of an autocatalytic reaction and a decreased stability might occur. No information is available in the literature on the activation energy of all-trans-lutein loss during under different conditions, only Sharma and Maguer (1996) reported activation energy values from 19.87 to 27.74 kJ/mol while studying lycopene degradation in tomato pulp solids stored at −20 ∼ 25°C.

Fig. 2.

An Arrhenius plot between natural logarithm of apparent reaction rate constant (k, week⁻¹) versus 1/T for all-trans-lutein loss during storage of freeze-dried sweet corn under vacuum & dark (▪, R² = 0.9916); air & dark (▴, R² = 0.9613); vacuum & light (•, R² = 0.9931); air & light (▾, R² = 0.9977)

Temperature quotient (Q₁₀) values were also calculated for the temperature ranges of 4 ∼ 37°C varied from 1.30 to 1.57 under different storage conditions. According to these values, the least effect of temperature rise on all-trans-lutein degradation was observed in freeze-dried sweet corn.

Cis-lutein formation and accumulation during storage    Table 2 showed the concentrations of lutein cis isomers in freeze-dried sweet corn after 12 weeks of storage under different storage conditions. All the cis isomers of lutein showed an inconsistent change, the rapid decrease in all-trans-lutein was accompanying to isomer formation (Fig. 1). For instance, prior to storage, the total amount of lutein was found to contain 89.4% all-trans-lutein, 1.8% 9-cis-lutein, 1.3% 9′- cis-lutein, 3.8% 13-cis -lutein, and 3.7% 13′-cis-lutein. During light storage, cis isomers of lutein in air-packaged freeze-dried sweet corn showed an increase trend under each storage temperature, and following a 12-week storage period, the level of 13-cis-lutein increased by 0.33 µg/g at 37°C, which was higher than dark storage. This result implied that light energy could be more destructive to all-trans-lutein. While cis isomers of lutein with vacuum showed a declined trend under each storage temperature, the level of 13-cis-lutein decreased by 0.09 µg/g at 37°C, which was higher than dark storage. These results indicated that there might some differences in predominant formation and degradation of lutein isomers under different conditions. The decreased level of cis-lutein was probably because of the further conversion to another cis form of lutein through intermediate all-trans-lutein (Lin and Chen, 2005; Tang and Chen, 2000).

Table 2. Contents of lutein cis isomers in freeze-dried sweet corn after 12 weeks of storage under different storage conditions

storage	cis-lutein (µg/g)
	9-cis	9′-cis	13-cis	13′-cis
initial	0.40 ± 0.08	0.28 ± 0.06	0.86 ± 0.07	0.81 ± 0.05
vacuum & dark
4°C	0.40 ± 0.01	0.27 ± 0.03	0.87 ± 0.05	0.80 ± 0.04
20°C	0.39 ± 0.02	0.27 ± 0.06	0.83 ± 0.03	0.79 ± 0.02
37°C	0.36 ± 0.02	0.23 ± 0.01	0.82 ± 0.03	0.75 ± 0.04
air & dark
4°C	0.41 ± 0.03	0.28 ± 0.02	0.88 ± 0.07	0.82 ± 0.06
20°C	0.42 ± 0.02	0.30 ± 0.03	0.91 ± 0.03	0.88 ± 0.07
37°C	0.44 ± 0.04	0.32 ± 0.05	0.95 ± 0.08	0.89 ± 0.03
air & light
4°C	0.43 ± 0.08	0.29 ± 0.04	0.92 ± 0.06	0.85 ± 0.02
20°C	0.48 ± 0.05	0.33 ± 0.05	1.02 ± 0.04	0.93 ± 0.05
37°C	0.54 ± 0.04	0.42 ± 0.04	1.19 ± 0.05	1.11 ± 0.03
vacuum & light
4°C	0.39 ± 0.02	0.26 ± 0.03	0.84 ± 0.03	0.77 ± 0.03
20°C	0.37 ± 0.03	0.24 ± 0.04	0.80 ± 0.05	0.75 ± 0.02
37°C	0.33 ± 0.03	0.19 ± 0.07	0.77 ± 0.06	0.67 ± 0.01

Compared to air storage, the degradation of all cis forms of lutein in vacuum-packaged sweet corn preceded move slowly, and showed certain decrease, because the exposure of powder to atmospheric oxygen is excluded. Just like β-carotene, it had been well established that both the isomerization and degradation of lutein could proceed simultaneously, and which reaction dominated should depend on heating temperature, illumination intensity and presence of catalyst (Subagio et al., 1998).

Conclusions

The all-trans-lutein content loss increased with the exposure of freeze-dried sweet corn powder to air, light and high storage temperature. The kinetics of all-trans-lutein degradation followed a first order reaction at 4, 20 and 37°C under different storage conditions. With increasing temperature and storage time, the degradation was dominated over isomerizations, the all-trans-lutein degraded more quickly during air and light storage, thus, higher stability of lutein was achieved using lower temperature and shorter-duration storage under vacuum and dark; it is noteworthy that the results shown in this study should be identical to that in a real food system because of the presence of complex components of the freeze-dried sweet corn powder. This information is useful and could be considered when freeze-dried sweet corn powder is developed and utilized as a food supplement.

Acknowledgements This research was financially supported by the Fundamental Research Funds for the Central Universities (Grant No. DL10BA12) and Natural Science Fund in Jiangsu Province (Grant No. BK2010467).

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