Thermal degradation kinetics of all-trans and cis-carotenoids in a light-induced model system

Accepted Manuscript Thermal degradation kinetics of all-trans and cis-carotenoids in a light-induced model system Ya-dong Xiao, Wu-yang Huang, Da-jing Li, Jiang-feng Song, Chun-quan Liu, Qiu-yu Wei, Min Zhang, Qiu-ming Yang PII: DOI: Reference:

S0308-8146(17)31089-0 http://dx.doi.org/10.1016/j.foodchem.2017.06.107 FOCH 21329

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

18 November 2016 19 June 2017 20 June 2017

Please cite this article as: Xiao, Y-d., Huang, W-y., Li, D-j., Song, J-f., Liu, C-q., Wei, Q-y., Zhang, M., Yang, Qm., Thermal degradation kinetics of all-trans and cis-carotenoids in a light-induced model system, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.06.107

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Thermal degradation kinetics of all-trans and cis-carotenoids in a light-induced model system Ya-dong Xiaoa, # , Wu-yang Huanga, # , Da-jing Lia, * , Jiang-feng Songa, Chun-quan Liua, Qiu-yu Weia, Min Zhangb, Qiu-ming Yanga, c a

Institute of Farm Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu,

210014, China; b

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122,

China； c

College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, 210095,

China.

#

*

Both authors contributed equally to this work.

Corresponding author.

Tel.: +86 25 84391255; Fax: +86 25 84391570. E-mail address: [email protected] (D. J. Li), or [email protected] (Y. D. Xiao)

1

Abstract: Thermal degradation kinetics of lutein, zeaxanthin, β-cryptoxanthin, β-carotene was studied at 25, 35, and 45 °C in a model system. Qualitative and quantitative analyses of all-trans- and cis-carotenoids were conducted using HPLC-DAD-MS technologies. Kinetic and thermodynamic parameters were calculated by non-linear regression. A total of 29 geometrical isomers and four oxidation products were detected, including all-trans-, keto compounds, mono-cis- and di-cis-isomers. Degradations of all-trans-lutein, zeaxanthin, β-cryptoxanthin, and β-carotene were described by a first-order kinetic model, with the order of rate constants as kβ-carotene > kβ-cryptoxanthin > klutein > kzeaxanthin. Activation energies of zeaxanthin, lutein, β-cryptoxanthin, and β-carotene were 65.6, 38.9, 33.9, and 8.6 kJ/moL, respectively. Cis-carotenoids also followed with the first-order kinetic model, but they did not show a defined sequence of degradation rate constants and activation energies at different temperatures. A possible degradation pathway of four carotenoids was identified to better understand the mechanism of carotenoid degradation. Keywords: all-trans- and cis-carotenoids, HPLC-DAD-MS, degradation kinetics, degradation rate constant, activation energy, degradation mechanism

Chemical compounds studied in this article: Lutein (PubChem CID: 5281243); Zeaxanthin (PubChem CID: 5280899); β-cryptoxanthin (PubChem CID: 5281235); β-carotene (PubChem CID: 5280489); 15-cis-beta-carotene (PubChem CID: 12305639); 13-cis-beta-carotene (PubChem CID: 10256668); 9-cis-beta-carotene (PubChem CID: 9828626); 13-apo-beta-carotenone (PubChem CID: 5363697); cis-beta-cryptoxanthin (PubChem CID: 101088204)

2

Introduction Lutein, zeaxanthin, β-cryptoxanthin, and β-carotene (Fig. 1), belonging to carotenoids family, are commonly found in vegetables, fruits, and algae. Scientists have worked on them for a long time. Among them, xanthophylls (lutein and zeaxanthin) were shown to be human macular pigments (Bone, Landrum, & Tarsis, 1985). Numerous epidemiological studies showed that regular consumption of foods rich in lutein and zeaxanthin can prevent age-related macular degeneration (AMD), cataracts, and other chronic eye diseases (Hankinson et al., 1992; Lyle et al., 1999; Schalch et al., 2007). Being effective vitamin A precursors, β-carotene and β-cryptoxanthin can scavenge free radicals and strengthen the human immune system (Abnet et al., 2003; Wu, Han, Riaz, Wang, Cai, & Yang, 2013). Biological functions of carotenoids in combination with their attractive colors have led to an increasing use as dietary supplements and additives, especially for lutein, zeaxanthin, and β-carotene (Humayoun Akhtar & Bryan, 2008). However, due to their highly unsaturated structures, carotenoids are prone to oxidation, isomerization, and degradation, which can be caused by exposure to heat, light, oxygen, catalysts, and other factors, being highly complex both in model systems and in food matrices. Pesek & Warthesen (1988) found that photo-degradation of β-carotene could generate cis-β-carotene, and be fitted by first-order kinetics in an aqueous model system. Chen & Huang (1998) got the same results from oven-heating, reflux-heating, and (non)-iodine-catalyzed photo-degradation, while 13-cis, 15-cis, 9-cis, and 13,15-di-cis-β-carotene were detected in three systems. Degradation of lutein, β-carotene, and β-cryptoxanthin also followed with the first-order kinetics in oil or oil model systems (Achir et al., 2010; Aparicio-Ruiz et al., 2011; Henry et al., 1998; Sampaio et al., 2013), just as that of β-carotene and β-cryptoxanthin in pulp and juices (Ahmed, Shivhare, & Sandhu, 2002; Dutta et al., 2006; Dhuique-Mayer et al., 2007; Saxena, Maity, Raju, & Bawa, 2012). However, β-carotene and 3

β-cryptoxanthin degradation in water or organic solvents by oxygen was best described by zero-order kinetics (Henry et al., 2000; Kanasawud & Crouzet, 1990). The thermal degradation of lutein, zeaxanthin, and β-cryptoxanthin in juice was described by a second-order kinetic model (Hadjal, Dhuique-Mayer, Madani, Dornier, & Achir, 2013). Cis-isomers were also detected in these model systems or food matrix. External environment has a great influence on the isomerization and degradation of carotenoids. Only Cremer (1955) and Galwey (1977) considered heterogeneous catalysis and compensation effects on interfering with carotenoids degradation. However, different structures of carotenoids might influence their stability, the effects of position and numbers of unsaturated double bonds as well as cis-carotenoids degradation and the presence or absence of conjugated double bonds and hydroxyl groups are unknown up to date. Trans-cis-isomerization and degradation rate of carotenoids depend on environmental conditions such as organic solvent, illumination, catalyst, temperature, pH, dissolved oxygen content et al.. The isomerization of carotenoids has been studied in an iodine-catalyzed photo-isomerization model system (Aman et al., 2005; Chen & Huang, 1998). Some mono-cis- and di-cis-isomers of all-trans-lutein and β-cryptoxanthin have been identified in the same model system by Li et al.(2014, 2015). In order to better understand the degradation kinetics of carotenoids and their cis-isomers in model systems, it is necessary to determine a possible degradation pathway for major carotenoids, which is caused by oxidation and isomerization. The objectives of current research were to study: (1) the degradation kinetics of all-trans-carotenoids during heating at different temperatures with iodine and light, (2) the degradation kinetics of cis-carotenoids generated in the model system during heating at different temperatures, and (3) the possible degradation pathway of all-trans-carotenoids in the model system with iodine and light 4

at different temperatures. The results would clearly identify influencing factors (iodine ions, illumination, or temperature) and help reveal the degradation mechanism of carotenoids. 2. Materials and Methods 2.1. Materials All-trans-lutein (purity ≥ 97%), zeaxanthin (purity ≥ 95%), β-cryptoxanthin (purity ≥ 97%), and β-carotene (purity ≥ 95%) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Analytical reagent grade hexane, iodine, sodium sulfate, and sodium thiosulfate were obtained from Nanjing Chemical Reagent Co., Ltd. (Nanjing, Jiangsu, China). HPLC grade methyl tert-butyl ether (MTBE) and methanol (MeOH) were purchased from Tedia Co., Inc. (Fairfield, OH, USA). 2.2. Sample preparation The iodine-catalyzed photoisomerization of all-trans-lutein, zeaxanthin, β-cryptoxanthin, and β-carotene into an equilibrium mixture of isomers was performed according to the method of Aman et al. (2005). Briefly, all-trans-carotenoids were dissolved in hexane, and 1 mL of this solution was transferred into a tube, followed by the addition of hexane (2 mL) and a solution of iodine in hexane (1 mL, c = 1.6 µg/mL, final iodine concentration ca. 1-2 wt.% with respect to carotenoids). The tubes containing all-trans-lutein were illuminated by a fluorescent lamp for 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, and 10 h at 25, 35, and 45 °C, and all-trans-zeaxanthin was exposed to the light for 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 9, and 11 h, while the corresponding exposure times were 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, and 8 h for all-trans-β-cryptoxanthin and 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h for all-trans-β-carotene, respectively. The illumination intensity (1800 lux) was measured by a digital illuminometer (TES-1334A, TES Electrical Electronic Co., Taiwan, China). After being washed twice with sodium thiosulfate, the 5

solution was transferred into a tube and blown dry with nitrogen (MD200-1, Hangzhou Aosheng Instruments Co., Ltd., Zhejiang, China). Samples were stored in an ultra-low-temperature refrigerator (-80 °C) for subsequent HPLC analysis. 2.3. Carotenoid analysis after iodine-catalyzed photo-isomerization 2.3.1. HPLC analysis The Agilent 1200 Series system (Agilent Technologies Deutschland GmbH, Santa Clara, California, USA) was used for carotenoid analysis, consisting of a G1322A degasser, a manual injector with a 20-µL loop, a G1311A pump, a G1316A temperature-controlled column oven, and a G1315B diode array detector (DAD) with a computer workstation. Separation was carried out on a YMC-C30 column (250 × 4.6 mm I.D. (inner diameter), 5 µm dP (Particle diameter) (YMC CO., Ltd., Kyoto, Japan). For separation of lutein, β-cryptoxanthin, β-carotene, and their cis-isomers, a linear gradient of 95% MeOH/MTBE/water (70/25/5, v/v/v, solvent A) and 5% MeOH/MTBE/water (10/85/5, v/v/v, solvent B) was used, with a flow rate of 0.6 mL/min at 25 °C of the column temperature, and 20 µL of the injection volume. A different linear gradient of water/MTBE/MeOH (5/15/80, v/v/v, solvent A) and MTBE/MeOH (10/1, v/v, solvent B) were used for the separation of all-trans-zeaxanthin and its cis-isomers at 1.0 mL/min of the flow rate. 2.3.2. Mass-spectrometric (MS) analysis MS analysis was performed by a 6530 Q TOF-MS spectrometer. Mass spectra were recorded in a positive ion mode for m/z 80-1000 of the mass range, with nitrogen drying gas at 5.0 L/min of the flow rate and an APCI carrier gas at 20 psi of the pressure. Corona discharge voltage was optimized to 2500 V at 4 µA current. 2.4. Kinetic study 6

Kinetic experimental data were presented in a dimensionless form using CA and CA,0 variables, where CA was the carotenoid concentration at time t and CA,0 was the initial carotenoid concentration. Degradation kinetics of lutein, zeaxanthin, β-cryptoxanthin, β-carotene, and their cis-isomers were described by the following equations: (1) (2)

(3)

where k was the reaction rate constant, and t was the length of heating time. Equations (1), (2), and (3) corresponded to zero-order, first-order, and second-order kinetics, respectively. The reaction rate constants were based on linear plots of (CA – CA,0) vs. t, ln(CA/CA,0) vs. t, and (1/CA – 1/CA,0) vs. t. The kinetic reaction order was determined by comparison of correlation coefficients at the experimental temperature T (298.15, 308.15, and 318.15 K). The effect of temperature on the reaction rate constant was calculated by the Arrhenius equation to determine the activation energy:

(4)

The logarithmic form of the above equation was written as:

(5)

where k was the reaction rate constant at the reference temperature T, Ea was the activation energy (kJ/moL), R was the universal gas constant (8.314 J/(mol·K)), T was the absolute temperature (K), and A was the pre-exponential factor (h–1). Ea was estimated based on linear regression analysis of the plot of lnki vs. 1/Ti (i = 25, 35, and 45 °C). 7

‡

According to the activated complex theory, the activation enthalpy (∆H ) was determined by the following equation:

(6)

where k was the reaction rate constant at the reference temperature T, R was the universal gas ‡

constant (8.314 J/(mol·K)), kb was the Boltzmann constant, and h was the Planck constant. ∆H was calculated based on the linear regression analysis of plots of ln (ki /Ti) versus 1/Ti. 2.5. Statistical analysis

The carotenoids were quantified using calibration curves for all-trans-lutein, zeaxanthin, β-cryptoxanthin, and β-carotene with ten concentration levels (0.05, 0.1, 0.2, 0.4, 0.8, 2, 4, 6, 8, and 10 µg/mL). The content of cis-carotenoids was calculated using the curves for the corresponding all-trans-carotenoids. Each experiment was replicated at least twice, and SAS V8 software was used for variance analysis. All figures were plotted using Origin 9.0. 3. Results and Discussions 3.1. Separation and identification of carotenoids and their isomers As shown in Table 1, 29 geometrical isomers and four oxidation products were identified and separated using the HPLC-DAD method. They were based on the retention time, DAD spectrum, MS fragmentation, and comparison with previous reports. Assignments of the cis-isomers of carotenoids were done on the following principles (Rodriguez-Amaya et al., 2004; Hui et al., 2005). Firstly, the cis-isomers have typical “cis peak” at 330-340 nm of the UV-visible spectra. Secondly, the maximum absorption of the mono-cis isomers and di-cis isomers is hypsochromically shifted 4-6 nm and 8-12 nm comparing to all-trans configuration. Thirdly, the larger the polarity, the earlier the elution. Identifications of some isomers and oxidation products have been reported in the literature. 13-cis- and 8

9-cis-isomers of lutein and zeaxanthin have been identified by HPLC-DAD-APCI-MS and nuclear magnetic resonance (NMR) spectroscopy (Aman et al., 2005). Godoy et al. (1990) have analyzed epoxides of β-cryptoxanthin using MS and NMR techniques. 5,6-epoxy-β-cryptoxanthin has been detected in cashew apple processed products and Amazonian fruits (Rosso et al., 2007; Zepka et al., 2009). Fragment at m/z 221, characteristic of an epoxy substituent in β-ring with a hydroxyl group, was found (de Rosso et al., 2007). 15-cis-carotene eluting before 13-cis- and 9-cis-carotene, 13,15-di-ciseluting before 15-cis-, 9,13-di-cis- eluting between 13-cis- and all-trans-, and 9,9-di-cis- eluting between all-trans- and 9-cis-β-carotene have been observed so far (Kao et al., 2012; Strohschein et al., 1997; Glaser et al., 2003; Mortensen, 2005). For lutein, ten cis-isomers have been detected, namely 13,15-, 9,15-, 9,13-, 9,9’-di-cis-lutein and 15-, 13/13’-, 9-, 9’-, 7-mono-cis-lutein, and all-trans-zeaxanthin. Three common mono-cis-isomers and a

di-cis-isomer

have

been

separated

for

zeaxanthin,

corresponding

to

9/9’-,

15/15’-,

13/13’-mono-cis-zeaxanthin and 9,13-di-cis-zeaxanthin. For β-cryptoxanthin, two keto compounds, two di-cis-isomers, and five mono-cis-isomers have been identified as β,β-caroten-3-one, 5,6-epoxy-β,β-caroten-3-one, 13,15- and 9,9’-di-cis-β-cryptoxanthin, and 15-, 13-, 13’-, 9-, 9’-mono-cis-β-cryptoxanthin. For β-carotene, besides the common 15-cis-, 13-cis, and 9-cis-β-carotene, we have also obtained 13-apo-β-carotenone, 5,6-epoxy-β-carotene, and 9,13-di-cis-β-carotene. The thermal isomerization and oxidative degradation of all-trans-β-carotene are assumed to occur simultaneously in this model system. Indeed, for β-carotene, only isomerization reaction was found at the chosen timescale at 120 °C in vegetaline (one kind of oil). It implies that oxidative degradation reaction probably requires a longer heating time to make cis-isomers to be potential precursors of cleavage products (Caris-Veyrat, 2009; 9

Mordi et al., 1993). We observed simultaneous isomerization and oxidative degradation at a lower temperature for the iodine/light model system in this study, resulting from the effects of iodine, light, or both. The exact reasons are unclear at present. We are going to make a deep and thorough exploration on this phenomenon in our future research. 3.2. Quantitative analysis of all-trans- and cis-carotenoids

Figure 2 shows lutein, zeaxanthin, β-cryptoxanthin, and β-carotene content changes at 25, 35, and 45 °C heating. All-trans-carotenoids content decreased with the increase of heating time length and temperature. After heating for 8 h at 25, 35, and 45 °C, all-trans-lutein were degraded by 82.1, 96.9, and 97.3%, respectively, while the corresponding values for all-trans-zeaxanthin were 48.1, 77.5, and 90.7%. These facts indicated that degradation of lutein was faster than that of zeaxanthin, especially at lower temperatures. The fractions of degraded β-cryptoxanthin were 98.1, 99.3, and 100% after 25, 35 and 45 °C heating for six hours, respectively. This revealed that β-cryptoxanthin was more sensitive to heating degradation than lutein and zeaxanthin were in the model system. After heating for 2.5 hours at 25, 35, and 45 °C, 93.0, 95.7, and 97.6% of β-carotene were lost, respectively. No all-trans-β-carotene was detected after heating for 4-6 hours in all treatments. Among the carotenoids studied, β-carotene exhibited the fastest degradation rate. It was in agreement with the results of Achir, Aparicio-Ruiz and Henry et al., who reported that β-carotene was more susceptible to degradation than lutein and β-cryptoxanthin in oil model systems (Achir et al., 2010; Aparicio-Ruiz et al., 2011; Henry et al., 1998). Fig. 2 also displays the content of cis-carotenoids in the model system during heating. The content of cis-carotenoids initially rose, but subsequently dropped with the increases of heating time length and temperature. Cis-lutein reached its maximum content after heating for two hours at 25 °C, half an hour 10

at 35 °C, and one hour at 45 °C. After seven-hour heating, the loss of cis-lutein was 48.9, 98.0, and 79.5% at 25, 35, and 45 °C, respectively. These results indicated that there were different formations and degradation rates of cis-lutein at different heating temperatures, and a high temperature would accelerate cis-lutein formation and degradation. Cis-zeaxanthin reached its maximum content after one-hour heating at 25 and 35 °C, while 81.0 and 100% were lost after six-hour heating. Its highest content occurred at 45 °C heating for half an hour, and it lost by 100% after six-hour heating. In contrast to all-trans-zeaxanthin, cis-zeaxanthin was less stable than cis-lutein. The content of cis-β-cryptoxanthin was unstable at 25 °C heating, with an increase followed by a decrease and a new increase. Six-hour heating at 25, 35, and 45 °C resulted in a loss of 97.6, 97.7, and 100% of cis-β-cryptoxanthin, respectively. In contrast to other cis-carotenoids, the content of cis-β-carotene in the standard of all-trans-β-carotene was higher, probably since β-carotene underwent isomerization during storage and pre-treatment. The highest content of cis-β-carotene occurred at 25, 35, and 45 °C heating for half an hour, while a complete degradation was at 25 °C heating for four-hour and at 35 and 45 °C heating for three-hour, respectively. Therefore, cis-β-carotene is the most sensitive to heating among cis-carotenoids in this study. All-trans- and cis-carotenoids exhibit different stability sequences in the model system at 25, 35, and 45 °C heating. Moreover, the content of cis-isomers gradually increases until reaching an equilibrium state of the maximum concentration during a given time interval, with a subsequent disruption by isomerization. It demonstrated that the cis-isomers of carotenoids existed in the total degradation reaction of carotenoids. It would be necessary to carry out a further study on simultaneous degradation of all-trans- and cis-carotenoids. Whereas major reason for different initial contents of all-trans-carotenoids at these temperatures in Fig. 2 might be their origin from various sample batches. 11

3.3. Kinetic study 3.3.1. Determination of kinetic reaction order Table 2 presents correlation coefficients (R2) of different kinetic models at 25, 35, and 45 °C for all-trans-lutein, zeaxanthin, β-cryptoxanthin, β-carotene, and their cis-isomers. Comparison of R2 values for three reactions at each temperature indicated that the degradation of all-trans-lutein, β-cryptoxanthin, and β-carotene was best described by the first-order kinetic model. It accorded with the report of Aparicio-Ruiz et al. (2011) for thermal degradation of lutein, β-carotene, and β-cryptoxanthin in virgin olive oil by the first-order kinetic mechanism. Hadjal et al. (2013), however, thought that lutein and β-cryptoxanthin degradations were best described by the second-order kinetic model in real food or simulated systems. In addition, the degradations of all-trans-β-carotene and β-cryptoxanthin were also found by ozone, oxygen, or nitrogen followed zero-order kinetic models (El-Tinay & Chichester, 1970; Henry et al., 2000; Minguez-Mosquera & Jaren-Galan, 1995). Carotenoids degradation was closely related to environment factors including experimental and testing environmental conditions. In this study, all-trans-zeaxanthin degradation was fitted by both the first-order and the second-order models (Table 2). Likewise, cis-carotenoids degradation was also best fitted by the first-order model. The differences between all-trans- and cis-carotenoids lied in the isoprene unit configuration at 15-, 13-, 9-, or other positions, which might exhibit no significant effect on the kinetic degradation. Despite of the R2 values for the three models, the zero-order model was best suitable for cis-zeaxanthin, but the first-order model was selected because of its well predicting the final cis-zeaxanthin content. These novel findings, especially for cis-zeaxanthin, cis-β-cryptoxanthin, and cis-β-carotene, have provided us with a theoretical basis for a further study on cis-carotenoids.

12

3.3.2. Determination of kinetic and thermodynamic degradation parameters According to the kinetic equations for all-trans- and cis-carotenoids, the corresponding reaction rate constants (k), half-life periods (t1/2), D values (time required for 90% degradation), activation ‡

energies (Ea), and enthalpies (∆H ) were estimated consequently (Table 3). The degradation rates of lutein, zeaxanthin, β-cryptoxanthin, and β-carotene gradually went up with the same increasing order of all temperatures, namely kβ-carotene > kβ-cryptoxanthin > klutein > kzeaxanthin. Among the carotenoids, the strongest activation energy was observed for zeaxanthin (65.6 kJ/moL), followed by lutein (38.9 kJ/moL) and β-cryptoxanthin (33.9 kJ/moL), while that for β-carotene (8.6 kJ/moL) was weakest. These results were different from those of Hadjal et al. (2013), who reported free lutein, zeaxanthin, and β-cryptoxanthin activation energies of 60.1, 65.1, and 48.7 kJ/moL in a neutral model system; 98.3, 105, and 96.0 kJ/moL in an acid model system; and 65.0, 132, and 62.3 kJ/moL in a real juice system, respectively. However, the stability of these carotenoids in this study was consistent with the above reference. Activation enthalpy of carotenoids appeared the same order as the activation energy (Table 3). All-trans-zeaxanthin was the most stable carotenoid in our model system, whereas all-trans-β-carotene was the least one. Despite showing the same order of degradation rates, all-trans-carotenoids, cis-carotenoids exhibited a different relationship with kinetic and thermodynamic parameters. The sequence of degradation rate constant was kcis-β-carotene > kcis-β-cryptoxanthin > kcis-zeaxanthin > kcis-lutein at 25 and 35 °C, but kcis-β-cryptoxanthin > kcis-β-carotene > kcis-zeaxanthin > kcis-lutein at 45 °C. At a low temperature, cis-β-carotene was the least stable compound in the model system, while at a higher temperature, cis-β-cryptoxanthin showed a slightly larger degradation rate than cis-β-carotene did. It means that cis-β-cryptoxanthin was more sensitive to heating than the other cis-carotenoids in this study, especially at a high temperature. 13

In contrast to all-trans-carotenoids, degradation process of cis-zeaxanthin was faster than that of cis-lutein, revealing that isomerization of the all-trans-zeaxanthin structure tended to instability. The order of activation energy was Ea

cis-β-cryptoxanthin

> Ea

cis-β-carotene

> Ea

cis-lutein

> Ea

cis-zeaxanthin

with a

respective value of 53.7, 24.9, 24.0, and 18.5 kJ/moL, respectively. Cis-β-cryptoxanthin had the strongest activation energy, and cis-zeaxanthin was the weakest one. Theoretically, reaction rate constant should be negatively correlated with its activation energy. Due to the limited number of reports on the degradation kinetics of cis-carotenoids, satisfactory explanation is currently unavailable. In conclusion, carotenoids degradation is strongly dependent on their structures and reaction media. Few kinetic parameters are currently available. Our results might provide a valuable reference for further studies and promote cis-carotenoid research. Future experiments are required to gain a more insight into molecular reactivity over a broad temperature range and in real food matrices. In section 3.1.1, the first-order kinetic model was chosen for the degradation of all-trans- and cis-zeaxanthin. By comparing the actual time length required for 50 and 90% degradation of all-transand cis-zeaxanthin with theoretically predicted values, it has been recognized that the degradation of these carotenoids was best fitted by the first-order model. 3.4. Kinetic mechanism for the degradation of four carotenoids The degradation kinetics of carotenoids was generally studied without considering intermediate reactions, with only few explorations for a probable degradation pathway with cis-isomers as intermediates. Aparicio-Ruiz (2011) showed that lutein exhibited a different degradation kinetic mechanism because the isomerization of all-trans lutein and the degradation of 13-cis-lutein and 9-cis-lutein included in virgin olive oils during heating. Thus, the degradation kinetic mechanism of other all-trans-carotenoids should be proposed to include mono-cis-, di-cis-isomers and other oxidation 14

isomers. In this study, the complex in degradation of lutein, zeaxanthin, β-cryptoxanthin, and β-carotene produced not only several mono-cis-isomers, but also di-cis-isomers and oxidations products. Therefore, a probable kinetic mechanism was proposed for the degradation of four trans-carotenoids based on the individual quantitative data for cis- and trans-carotenoids. As shown in Fig. 3, the degradation of all-trans-lutein was quite complex. It was involved in two parallel reactions for formation of 13/13’-cis-lutein and 9/9’-cis-lutein, three reactions for formation of 9,13-di-cis-lutein and 9,9’-di-cis-lutein, and five concurrent reactions of four-cis-lutein and all-trans-lutein for colorless products. Whereas thermal degradation of all-trans-zeaxanthin consisted of three mono-cis-isomers and one di-cis-isomer, with consecutive transformations of all-trans-zeaxanthin into four-cis-zeaxanthin for colorless products. In contrast to the former two carotenoids, epoxidized and cleavage products were generated by the thermal degradation of all-trans-β-cryptoxanthin and β-carotene with more complicated degradation mechanisms (Fig. 3). Based on these results, it was suggested that lutein and zeaxanthin exhibited analogous degradation kinetic mechanisms, just the same as β-cryptoxanthin and β-carotene. Obviously, the thermal stability and degradation mechanism were significantly affected by geometric configuration. In general, the decreasing number of coplanar conjugated double bonds and the presence of hydroxyl groups in carotenoids decreased their reactivities in radical-scavenging reactions, implying that hydroxy-carotenoids were more stable than carotenes. Von Eular and Moore confirmed that β-ring carotenoids are precursors of vitamin A. As shown in Fig. 1, β-cryptoxanthin and β-carotene contain one and two β-rings, respectively, meaning that the pro-vitamin A activity of β-carotene is higher than that of β-cryptoxanthin. In addition to three 15

mono-cis-isomers and one epoxidized product, the thermal degradation of all-trans-β-carotene generated 13-apo-β-carotenone in the model system. A reasonable explanation would be that β-cryptoxanthin differed from β-carotene due to the presence of a hydroxyl group at the C-3 position. Miller (1996) found that the carotenoid stability toward radical cations was significantly reduced when each β-ring contained one hydroxyl group. Thus, β-carotene was less stable than β-cryptoxanthin, with a more complex degradation mechanism. For lutein and zeaxanthin, both sides of rings are hydroxyl-substituted, and the former exhibits rings hydroxyl-substituted on both sides (ε- and β-rings). Therefore, zeaxanthin has more coplanar conjugated double bonds than lutein and has a higher stability. 4. Conclusion The degradation of lutein, zeaxanthin, β-cryptoxanthin, and β-carotene in the model system was best fitted by the first-order kinetics, with the rate constants being correlated negatively with activation energy. All-trans-β-carotene was the most susceptible compound to the degradation, while zeaxanthin was the most stable one in this model system. Cis-carotenoids also displayed the first-order of degradation kinetics, without an obvious order of the degradation rate constants at different temperatures. A probable degradation pathway for these carotenoids was identified on the isolated compounds. These novel results can help us better understand the degradation of carotenoids. A further effort will be made to examine the effect of cis-carotenoids on the degradation kinetic mechanism of trans-carotenoids during the whole degradation and isomerization reaction in model systems and real food matrices.

Acknowledgment The authors thank the financial support from Jiangsu Science and Technology Program for the key 16

research and development of modern agriculture [Project No. BE2016363] Conflict of interest statement: Ya-dong Xiao, Wu-yang Wang, Da-jing Li, Jiang-feng Song, Chun-quan Liu, Qiu-yu Wei, Min Zhang, Qiu-ming Yang and others have no conflict of interest for this manuscript.

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21

Table 1. Identification data for all-trans carotenoids, their geometrical isomers and oxidation products RT

All-trans carotenoids, geometrical

(min)

isomers, oxidation products

9.08

Compound

Lutein

λ (nm)

m/z

13,15-di-cis-lutein

330,432,460

551.4[M+H-18]+

9.30

9,15-di-cis-lutein

332,430,456

551.4[M+H-18]+

9.97

15-cis-lutein

328,438,466

551.4[M+H-18]+

10.33

9,13-di-cis-lutein

332,418,434, 458

551.4[M+H-18]+

330,418,438,

551.4[M+H-18]+,

10.81

13/13’-cis-lutein 466

533.4[M+H-18-18]+ 551.4[M+H-18]+,

11.25

all-trans lutein

422,444,472

533.4[M+H-18-18]+ 569.4[M+H]+,

12.15

all-trans zeaxanthin

418,450,478

551.4[M+H-18]+, 533.4[M+H-18-18]+ 551.4[M+H-18]+,

12.62

9,9’-di-cis-lutein

13.38

9-cis-lutein

14.61

15.09

Zeaxanthin

10.21

10.79

11.19

410,434,462

533.4[M+H-18-18]+

332,418,440,

551.4[M+H-18]+,

466

533.4[M+H-18-18]+

330,422,440,

551.4[M+H-18]+,

468

533.4[M+H-18-18]+

328,420,442,

551.4[M+H-18]+,

462

533.4[M+H-18-18]+

336,416,440,

569.4[M+H]+,

466

551.4[M+H-18]+

337,420,445,

569.4[M+H]+,

472

551.4[M+H-18]+

336,422,444,

569.4[M+H]+,

468

551.4[M+H-18]+

9’-cis-lutein

7-cis-lutein

9,13-di-cis-zeaxanthin

15/15’-cis-zeaxanthin

13/13’-cis-zeaxanthin

569.4[M+H]+, 11.73

all-trans zeaxanthin

14.38

9/9’-cis-zeaxanthin

428,450,478

551.4[M+H-18]+

340,422,446,

569.4[M+H]+,

468

551.4[M+H-18]+ 551.4[M+H]+,

340,420,451, β-cryptoxanthin

11.47

β,β-caroten-3-one 480

533.4[M+H-20]+, 459.4[M+H-94]+ 567.4[M+H]+,

5,6-epoxy-β,β-caroten-

337,420,446,

551.4[M+H+1-2],

3-one

474

477.4[M+H-76]+,

332,414,438,

553.4[M+H]+,

466

535.4[M+H-18]+

12.29

221 13.39

13,15-di-cis-β-cryptoxanthin

22

14.31

14.81

15.00

16.15

16.80

17.63

18.15 β-carotene

6.38

336,418,446,

553.4[M+H]+,

466

535.4[M+H-18]+

338,424,444,

553.4[M+H]+,

468

535.4[M+H-18]+

338,424,444,

553.4[M+H]+,

468

535.4[M+H-18]+

338,428,450,

553.4[M+H]+,

478

535.4[M+H-18]+

338,420,440,

553.4[M+H]+,

468

535.4[M+H-18]+

342,424,446,

553.4[M+H]+,

474

535.4[M+H-18]+

342,422,448,

553.4[M+H]+,

472

535.4[M+H-18]+

15-cis-β-cryptoxanthin

13-cis-β-cryptoxanthin

13’-cis-β-cryptoxanthin

all-trans β-cryptoxanthin

9,9’-di-cis-β-cryptoxanthin

9-cis-β-cryptoxanthin

9’-cis-β-cryptoxanthin 13-apo-β-carotenone

259,215 535.4[M+H-18]+

15.90

5,6-epoxy-β-carotene

420,446,474

461.4[M+H-92]+, 205.4 537.4[M+H]+,

18.63

15-cis-β-carotene

408,446,474

19.23

13-cis-β-carotene

422,444,472

19.83

9,13-di-cis-β-carotene

412,438,468

20.64

all-trans β-carotene

424,452,478

21.68

9-cis-β-carotene

424,448,474

444.4[M-92]+ 537.4[M+H]+, 444.4[M-92]+ 537.4[M+H]+, 444.4[M-92]+ 537.4[M+H]+, 444.4[M-92]+ 537.4[M+H]+,

23

444.4[M-92]+

Table 2. Reaction rate constant (k) and correlation of coefficient (R2) of three kinetic models at 25, 35, 45℃ of all-trans and total cis-carotenoids in a model system Compounda

Lutein

Zeaxanthin

β-cryptoxanthin

β-carotene

Cis-lutein(D)

Cis-zeaxanthin(D)

Cis-β-cryptoxanthin(D)

Cis-β-carotene(D)

k/(h-1) (R2)

Temperature(℃) Zero-order reaction

The 1 -order reaction

The 2nd -order reaction

25

0.9917(0.7984)

0.1672(0.9338)

0.0329(0.8893)

35

0.9697(0.8896)

0.3454(0.9274)

0.4802(0.6461)

45

2.4346(0.6592)

0.4462(0.9197)

0.0976(0.6152)

25

0.6250(0.7662)

0.0623(0.7901)

0.0099(0.7925)

35

1.3088(0.7424)

0.1974(0.8562)

0.0419(0.9318)

45

1.5505(0.6023)

0.3263(0.7938)

0.1495(0.8535)

25

1.1126(0.7159)

0.5509(0.8925)

0.5810(0.5932)

35

1.3655(0.7621)

0.7260(0.9805)

1.4922(0.7583)

45

3.7654(0.4709)

1.3063(0.9566)

8.2516(0.7455)

25

5.4396(0.7021)

1.1223(0.9723)

0.4306(0.7573)

35

4.1735(0.7416)

1.2781(0.9940)

0.5141(0.8734)

45

4.4683(0.6710)

1.3964(0.9834)

0.7001(0.7322)

25

0.3126(0.8308)

0.1679(0.9135)

0.1020(0.8688)

35

0.4292(0.8726)

0.2968(0.9048)

1.5469(0.7313)

45

0.7789(0.6450)

0.3067(0.7350)

0.1420(0.5354)

25

0.4891(0.7347)

0.3151(0.7442)

0.4268(0.7922)

35

1.1500(0.9118)

0.5063(0.8879)

0.2372(0.8653)

45

1.3128(0.9264)

0.5016(0.9036)

0.2067(0.7933)

25

1.0665(0.7988)

0.6512(0.8729)

1.0305(0.6122)

35

0.7012(0.9043)

0.6798(0.9688)

1.8890(0.7668)

45

2.9190(0.5851)

2.5755(0.9423)

13.2940(0.8555)

25

3.2671(0.8610)

0.8697(0.8856)

0.2856(0.8489)

35

3.9880(0.8646)

1.2432(0.8788)

0.5770(0.6934)

45

3.9960(0.8919)

1.5963(0.9435)

1.4502(0.6521)

a: Those with a D in parentheses represent degradation.

24

st

Table 3. Kinetic and thermodynamic parameters of four carotenoids and their total cis-isomers during degradation Compounda

Lutein

Zeaxanthin

β-cryptoxanthin

β-carotene

Cis-lutein(D)

Cis-zeaxanthin(D)

Temperature

k(h-1 )

t1/2

2

D

(℃ ℃)

(R )

(h)

(h)

25

0.1672(0.9338)

4.1456

13.7714

35

0.3454(0.9274)

2.0068

6.6664

45

0.4462(0.9197)

1.5534

5.1604

25

0.0623(0.7901)

11.1260

36.9596

35

0.1974(0.8562)

3.5114

11.6646

45

0.3263(0.7938)

2.1243

7.0567

25

0.5509(0.8925)

1.2582

4.1797

35

0.7260(0.9805)

0.9547

3.1716

45

1.3063(0.9566)

0.5306

1.7627

25

1.1223(0.9723)

0.6176

2.0517

35

1.2781(0.9940)

0.5423

1.8016

45

1.3964(0.9834)

0.4964

1.6489

25

0.1679(0.9135)

4.1283

13.7140

35

0.2968(0.9496)

2.3354

7.7580

45

0.3067(0.8984)

2.2600

7.5076

25

0.3151(0.7442)

2.1998

7.3015

35

0.5063(0.8879)

1.3690

4.5479

45

0.5106(0.8513)

1.3819

4.5905

25

0.6512(0.8729)

1.0644

3.5359

35

0.6798(0.9668)

1.0196

3.3872

45

2.5755(0.9423)

0.2691

0.8940

Ea(kJ/mol) 2

‡

∆H‡ (kJ/mol)

(R )

(R2)

38.8958(0.9385)

36.3357(0.9298)

65.5539(0.9588)

62.9946(0.9554)

33.9036(0.9506)

31.3443(0.9431)

8.6324(0.9918)

6.0720(0.9827)

23.9784(0.8508)

21.4185(0.7672)

18.5319(0.7513)

15.9720(0.6908)

53.6494(0.7575)

51.0895(0.7394)

24.9112(0.9900)

22.3514(0.9874)

Cis-β-cryptoxanthin (D)

Cis-β-carotene(D)

25

0.8497(0.8856)

0.8158

2.7099

35

1.2432(0.8788)

0.5576

1.8521

45

1.5963(0.9435)

0.4342

1.4425

a: Those with a D in parentheses represent degradation.

25

1. The degradation of carotenoids was best described by a first-order kinetic model. 2. The relationship of degradation rate for cis-carotenoids was obtained. 3. A possible degradation mechanism of four all-trans-carotenoids was identified.

26

β-Carotene（C40H56）

HO

β-Cryptoxanthin（C40H56O） OH

HO

Zeaxanthin（C40H56O2） OH

HO

Lutein（C40H56O2）

Fig.1 Stereostructures of all-trans carotenoids

Fig.2. Concentration changes of all-trans- and cis-carotenoids during heating at different temperatures (Those with solid and hollow icons are trans- and cis- carotenoids, respectively)

13/13’-cislutein

k1 all-trans lutein

k1

k4 k5

k2

13/13’-ciszeaxanthin

k7

9/9’-cislutein k6 k3

9,13-di-cislutein 9,9’-di-cislutein

k8

all-trans zeaxanthin

k9

9/9’-ciszeaxanthin

k2 k3

k7 k5 k6

9,13-di-ciszeaxanthin

15/15’-ciszeaxanthin

k10

k1 all-trans β-cryptoxanthin

k2 k3 k4

13/13’-cisβ-cryptoxanthin 9/9’-cisβ-cryptoxanthin

k9 k10

k4 colorless

5,6-epoxyβ,β-carotene-3-one 15-cisβ-cryptoxanthin

k8

colorless

k9 k6 k7 k8

13,15-di-cisβ-cryptoxanthin

k10

9,9’-di-cisβ-cryptoxanthin

k11

k5

k1 all-trans β-carotene

k2 k3 k4 k5

β-apo-13-carotene 5,6-epoxy-β-carotene 15-cis-β-carotene 13-cisβ-carotene k7 9,13-di-cisk8 9-cisβ-carotene β-carotene

k9 k10

k11 k12 k14

k13

k6

colorless colorless

Fig.3. Kinetic mechanisms for thermal degradation pathway of all-trans carotenoids in the model systems