Carotenoids are natural pigments of the isoprenoid family, commonly biosynthesized in fruits and vegetables [1
], presenting potential physiological benefits, such as antioxidants in food and pro-vitamin A activity [2
]. As one of the most commonly used carotenoids, β-carotene is expected to be conducive to health because of its valuable nutritional properties and antioxidant capacities, which confer on this compound an important role in lowering the risk of cataracts [3
], inhibiting age-related macular degeneration [4
], and enhancing the prevention of cardiovascular diseases [5
However, due to its poor water solubility [6
] and low bioavailability [7
] during food processing and storage, widespread applications of β-carotene in food matrices normally suffer considerable challenges. Moreover, the restriction of β-carotene utilization as a nutritional ingredient in the food industry is currently also attributed to the existence of numerous unsaturated groups, resulting in high vulnerability to degradation reaction when exposed to light, heat and other external factors [9
]. It has been reported that strong illumination can influence the stability of β-carotene extracted from palm oil, unveiling the formation of cis isomers [11
]. The in-depth study carried out by Ayu et al. [12
], who investigated interactive influence of tocopherols, tocotrienols, and β-carotene in the process of photooxidation of red palm oil, suggesting that the degradation of β-carotene easily occurs under the irradiation of light. Apart from that, a comparison of β-carotene degradation under different UV stresses was conducted by Chen et al. [13
], which showed that the longer the wavelength applied, the faster the degradation rate.
Several reports have also focused on the effects of chemical substances and their stability. The presence of 1,4-dimethylnaphthalene-1,4-endoperoxide and lycopene had the potential to induce the generation of (9Z
)- and (15Z
)-β-carotene, which was associated with the formation of singlet oxygen [14
]. Lewis acids, including titanium tetrachloride and ferric chloride, can catalyze the degradation of β-carotene to form an intermediate radical carbocation [15
Currently, more than 300 sulfides have been registered as Generally Recognized as Safe (GRAS) substances with various threshold limits, making them the critical food flavors [16
]. Biological functions including antithrombotic [17
], antimicrobial [18
], anticancer [19
], and anti-inflammatory activities [20
] in combination with their attractive odor characteristics such as garlic, onion, meat and nut flavors, have increased their feasibility of acting as food additives. In our previous studies, we discovered that dimethyl sulfides exerted apoptosis-inducing effects in leukemia cell lines via the generation of reactive oxygen species, especially for dimethyl trisulfide(Me2
) and dimethyl tetrasulfide(Me2
]. Furthermore, β-carotene combined with Me2
under UVA irradiation presented a synergistic action in inhibiting the viability of HL-60 cells viability, and elevating caspase-3 levels [22
], mostly like probably raising the possibility of the reaction between sulfides and β-carotene assisted by UVA.
In this study, we selected 11 kinds of oxygen-containing sulfur flavor molecules, commonly used in the food industry, as experimental materials to examine their influence on β-carotene stability under UVA irradiation. Moreover, both the dynamic analysis of β-carotene degradation and the structural effects of sulfides that accelerated the degradation of β-carotene were investigated to provide a clearer and better comprehension of their acceleration effects. Furthermore, the oxidation products of β-carotene under UVA irradiation were also analyzed in order to elucidate its degradation mechanism.
β-Carotene was susceptible to be affected when exposed to external factors, consistent with previous reports, which considered that light can exert influence on β-carotene degradation [27
]. In this study, sulfides have also showed to be involved in interfering the stability of β-carotene, which agreed with the report of Wei et al., implying that the stability of β-carotene can be significantly influenced when chitosan-(−)-epigallocatechin-3-gallate conjugates on β-carotene emulsions covered by sodium caseinate [30
]. Moreover, in agreement with previous studies, both the number of sulfur atoms and the type of side group can affect the accelerated degradation of β-carotene under UVA irradiation [24
]. It has been reported that the coexistence of disulfides can remarkably decrease the residual ratios of β-carotene to approximately 51.8–69.1%, while the presence of mono sulfides did not show obvious accelerating effects compared to the absence of mono sulfides [24
β-Carotene degradation upon exposure to MMFDS, BMFDS and MFDS followed first-order kinetics, consistent with the kinetic model in dichloromethane system [23
]. On the basis of the previous report which focused on the phenomenon of the existence of isomers [32
], we also studied the accelerated effects of side groups among these sulfides on the degradation of β-carotene. Although MMFDS and MFDS both possess the same molecular formula (C6
), MMFDS showed a stronger accelerated degradation effect than MFDS. It is presumably because there is a methyl group and a furan group on the side of the disulfide bond in MMFDS, while a methyl group and a furfuryl group exist on the side of the disulfide bond in MFDS. For DFDS and its corresponding isomer BMFDS. Similarly, there is a furan group on both ends of the disulfide bond in BMFDS, while there is a furfuryl group on each side of the disulfide bond in DFDS. Their different abilities to promote the degradation of β-carotene can be related to the existence of various side groups. These results may account for the fact that furan-containing sulfur flavor molecules (MMFDS and BMFDS) showed a much more remarkable acceleration effect on the degradation of β-carotene than furfuryl-containing sulfur flavor molecules (MFDS and DFDS, respectively). Therefore, the number of sulfur atoms and the furan group in oxygen-containing sulfur flavor molecules may play a critical role in the accelerated degradation of β-carotene under UVA irradiation in ethanol system.
Several studies have investigated the order of kinetics on the degradation of β-carotene in different model systems under various conditions. It also followed a first order reaction under ambient storage, ultraviolet radiation and even heat treatments [13
]. The photodegradation of β-carotene treated by BMFDS in ethanol system followed first-order kinetics, which agreed with previous studies carried out in food model systems such as carrots [33
], oil/carrot emulsion system [34
], oil model systems [10
] and pulp or juices [36
]. Ferreira et al. observed a first-order reaction for β-carotene degradation in a low-moisture and aqueous model system, as well as in lyophilized guava under different processing and storage conditions [38
In addition, our results were in good agreement with Li et al. who summarized that the trans
isomerization of carotenoids can be generated via contacting with acids, thermal treatment or light [39
]. It has been a long time since 13-cis
-β-carotene was recognized as one of the main cis
forms of β-carotene in food [40
]. Chen et al. even analyzed it by different processing means, including over-heating and (non)-iodine-catalyzed photodegradation [41
]. In addition, 9,13-di-cis
-β-carotene was also confirmed as a common β-carotene degradation product according to Glaser et al. [42
]. Moreover, our founding was consistent with Handelman et al., who had detected 5,6-epoxide of β-carotene through utilizing HPLC with mass analysis [43
]. Similarly, Zeb identified all-trans
-5,6-epoxy-β-carotene by an HPLC system and single ion monitoring mass spectrometry as well [44
4. Materials and Methods
4.1. Materials and Chemicals
Eleven kinds of oxygen-containing sulfur flavors and β-carotene were obtained from Sigma-Aldrich (St. Louis, MO, USA). The structures of these sulfides are presented in Table 1
. Methanol and methyl tert
-butyl ether (MTBE)were procured from Damao Chemical factory (Tianjin, China) and Fisher Scientific (Pittsburgh, PA, USA), respectively. The other chemicals and reagents were of analytical grade.
4.2. Preparation of the Model Systems
For the preparation of the ethanol model system, 1 mg β-carotene was dissolved in 15 mL ethanol according to Onsekizoglu et al. [27
] with minor modifications. The β-carotene solution was prepared daily and kept in the dark at 4 °C before use. Stock solutions of 11 kinds of sulfides were prepared in ethanol at a concentration of 10 mM and kept at 4 °C prior to use.
4.3. Kinetic Analysis of β-Carotene Degradation
The working solutions of β-carotene were transferred into quartz cuvettes, followed by the addition of 10 μL sulfur flavors. The control was performed with 10 μL ethanol. Then, the mixture was treated by UVA light (2.5 mW/cm2) with the aim of assessing the degradation kinetics of β-carotene treated with sulfide. The degradation of β-carotene was measured immediately in a UV-1750 spectrophotometer (Shimadzu, Tokyo, Japan) at the wavelength of 450 nm for 60 min, which was monitored every 10 min. All measurements were performed in triplicate and data are expressed as mean of three independent experiments.
4.4. Degradation Kinetics Modeling
The trial-and-error procedure was carried out in accordance with the integral method outlined by Sánchezet al. [45
] to determine the reaction order of theβ-carotene degradation. Different order models can be represented as follows:
In these formulas, c (μM) is thereactantconcentrationat a given time, c0 (μM) is the initial reactant concentration, k (min−1) is the degradation rate constant, and t (min) is the treatment time.
4.5. Analysis of β-Carotene Treated with UVA Irradiation and BMFDS
The working solutions of β-carotene were transferred into quartz cuvettes, followed by the addition of 10 μL BMFDS. The control was performed with 10 μL ethanol. Then, the mixture containing β-carotene and BMFDS was placed under a UV lamp (Shimadzu, Japan) with an intensity of 2.5 mW/cm2 for 5 min, followed by drying completely under a nitrogen stream. The residue was redissolved in 0.1 mL MTBE before use.
The further analysis was carried out and relative parameters were applied according to Santos et al. [46
]. Briefly, once redissolved, the solution was passed through a 0.22 μm filter, followed by the injection into an HPLC-DAD-APCI-MS system (Agilent, Santa Clara, CA, USA) for closer analysis. A YMC C30
column (250 × 4.6 mm, 5 μm) and gradient mobile phase of methanol-MTBE-water (85:15:5, v
) and MTBE (100%) were used for β-carotene detection.
4.6. Determination of Degradation Products by Raman Spectroscopy
The Raman spectra of the degradation products were recorded on a Raman spectrometer (Bruker Instruments Inc., Bill-erica, MA, USA). The wave number was in the range of 400–4000 cm−1 using the 785 nm as the excitation line. The power was 10 Mw while the integration time was 20 s.
4.7. Statistical Analysis
All the data were expressed as the mean ± SD or mean and subjected to the Student’s t-test for statistical analysis. Statistical significance was considered at a p < 0.05.