Influence of oxygen-containing sulfur flavor molecules on the stability of β-carotene under UVA irradiation

Article abstract of DOI:10.3390/molecules24020318

The influence of 11 kinds of oxygen-containing sulfur flavor molecules was examined on β-carotene stability under UVA irradiation in ethanol system. Both the effects of sulfides on dynamic degradation of β-carotene and the relation between structure and effect were investigated. The oxidation products of β-carotene accelerated by sulfides under UVA irradiation were also identified. The results indicated that the disulfides had more obvious accelerative effects on the photodegradation of β-carotene than mono sulfides. The degradation of β-carotene after methyl (2-methyl-3-furyl) disulfide (MMFDS), methyl furfuryl disulfide (MFDS) and bis(2-methyl-3-furyl) disulfide (BMFDS) exposure followed first-order kinetics. Furan-containing sulfides such as MMFDS and BMFDS showed more pronounced accelerative effects than their corresponding isomers. The oxidation products were identified as 13-cis-β-carotene, 9,13-di-cis-β-carotene and all-trans-5,6-epoxy-β-carotene. These results suggest that both the sulfur atom numbers and the furan group in oxygen-containing sulfides play a critical role in the photooxidation of β-carotene.

Full text of DOI:10.3390/molecules24020318

Article
Influence of Oxygen-Containing Sulfur Flavor
Molecules on the Stability of β-Carotene under UVA
Irradiation
Gong-Liang Zhang 1, Hong-Yan Wu 2, Ying Liang 2, Jie Song 1, Wei-Qi Gan 1
and Hong-Man Hou 1,*
1
School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China;
zgl_mp@163.com (G.-L.Z.); sj101521@163.com (J.S.); 17317138996@163.com (W.-Q.G.)
2
Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan;
wuhongyan1908@hotmail.com (H.-Y.W.); dlliangying@163.com (Y.L.)
* Correspondence: houhongman2011@hotmail.com; Tel: +86-411-86322020
Academic Editor: Lillian Barros
Received: 28 December 2018; Accepted: 13 January 2019; Published: 16 January 2019
Abstract: The influence of 11 kinds of oxygen-containing sulfur flavor molecules was examined on
β-carotene stability under UVA irradiation in ethanol system. Both the effects of sulfides on
dynamic degradation of β-carotene and the relation between structure and effect were
investigated. The oxidation products of β-carotene accelerated by sulfides under UVA irradiation
were also identified. The results indicated that the disulfides had more obvious accelerative effects
on the photodegradation of β-carotene than mono sulfides. The degradation of β-carotene after
methyl (2-methyl-3-furyl) disulfide (MMFDS), methyl furfuryl disulfide (MFDS) and bis(2-methyl-
3-furyl) disulfide (BMFDS) exposure followed first-order kinetics. Furan-containing sulfides such
as MMFDS and BMFDS showed more pronounced accelerative effects than their corresponding
isomers. The oxidation products were identified as 13-cis-β-carotene, 9,13-di-cis-β-carotene and all-
trans-5,6-epoxy-β-carotene. These results suggest that both the sulfur atom numbers and the furan
group in oxygen-containing sulfides play a critical role in the photooxidation of β-carotene.
Keywords: oxygen-containing sulfur flavor molecules; β-carotene; bis(2-methyl-3-furyl) disulfide
(BMFDS); oxidation products
1. Introduction
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 [7,8] 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 [9,10]. 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
Molecules2019, 24, 318; doi:10.3390/molecules24020318
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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)-, (13Z)- 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(Me2S3) and dimethyl tetrasulfide(Me2S4) [21]. Furthermore, β-carotene combined with
Me2S4 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 onβ-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.
2. Results
2.1. The Effects of Oxygen-Containing Sulfur Flavor Molecules onβ-Carotene Degradation under UVA
Irradiation
The structures of 11 kinds of oxygen-containing sulfur flavor molecules are shown in Table1.
Most of the sulfides contain furan or furfuryl group, such as 2-methyl-3-(methylthio) furan (MMTF)
and methyl furfuryl disulfide (MFDS). Some sulfides contain different numbers of sulfur atoms but
have the same side chain groups, such as MMTF and methyl (2-methyl-3-furyl) disulfide (MMFDS),
difurfuryl sulfide (DFS) and difurfuryl disulfide (DFDS). Furthermore, it is worth noting the
existence of isomers, such as bis(2-methyl-3-furyl) disulfide (BMFDS) and DFDS, MMFDS and
MFDS.
The effects of 11 kinds of oxygen-containing sulfur flavor molecules assisted by UVA
irradiation onβ-carotene stability are shown in Figure1A. After irradiating under UVA for 60 min in
which the light intensity was 2.5 mW/cm2, the contents of β-carotene in all groups showed a
reducing trend. To gain more knowledge about the correlation between the structure of coexistent
sulfides and the degradation ratios of β-carotene, the remaining amounts of β-carotene were
compared according to the structural characteristics of coexistent sulfides. The amounts of β-
carotene treated with MMFDS and BMFDS were dramatically decreased by approximately 96.05
and 99.70%, respectively (p < 0.05). Likewise, the remaining amount of β-carotene in the presence of
MFDS declined approximately 43.64%. These findings proved the fact that natural sulfur substance
may affectβ-carotene stability.
2.2. The Effects of Furan-Containing Sulfides onβ-Carotene Degradation under UVA Irradiation

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The order of the reaction with respect to the photodegradation of β-carotene was acquired
according to Equation (2) to examine the changes of β-carotene concentration with time after UVA
irradiation.
Table 1. Structure of eleven oxygen-containing sulfur flavor molecules.
FEMA
Name
Abbreviation
MMFDS
MFDS
MMTP
MMTF
MTPD
EMTP
EFTP
Structure
Number
S
3573
Methyl (2-Methyl-3-furyl) disulfide
Methyl furfuryl disulfide
S
O
3362
2720
3949
2747
3343
3674
3161
3238
3146
3259
S
S
S
O
O
Methyl 3-methylthiopropionate
2-Methyl-3-(methylthio) furan
3-(Methylthio) propionaldehyde
Ethyl 3-methylthiopropionate
Ethyl 3-(furfurylthio) propionate
Furfuryl isopropyl sulfide
Difurfuryl sulfide
O
S
O
O
S
O
O
S
S
O
O
O
S
S
FIS
O
O
O
DFS
O
S
S
Difurfuryl disulfide
DFDS
O
S
S
Bis (2-methyl-3-furyl) disulfide
BMFDS
O
O
FEMA: Flavor and Extract Manufacturers Association of the United States.
Figure 1. The effect of oxygen-containing sulfides on the degradation of β-carotene in ethanol under
UVA irradiation. β-Carotene was treated with various oxygen-containing sulfur flavor molecules
under UVA irradiation for 60 min (A) or within 60 min (B) in ethanol system. The residual β-
carotene (A) and the first-order kinetics curve of β-carotene degradation (B) were determined. The

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bar results are expressed as means ± SD from three independent replicates. Different small letters
show significant differences (p < 0.05).
As presented in Figure 1B, β-carotene degradation upon exposure to MMFDS, BMFDS and
MFDS followed first-order kinetics, consistent with the kinetic model in dichloromethane system
[31]. The corresponding kinetic parameters are listed in Table 2. It can be seen that the presence of
BMFDS in ethanol significantly improved the k and shortened the t1/2 of β-carotene degradation,
compared with the other two sulfides. The k and t1/2 were 0.131 min−1and 5.29 min for BMFDS,
while they were 0.0633 min−1 and 10.95 min for MMFDS and 0.0095 min−1 and 72.96 min for MFDS,
respectively. Furthermore, it should also be noticed that these three kinds of sulfides (MMFDS,
BMFDS and MFDS) having obviously promoting effects on the degradation of β-carotene contain at
least two sulfur atoms, which is consistent with the previous report [23]. These results suggest that
sulfides with more sulfur atoms might trigger a stronger chemical effect onβ-carotene stability in
ethanol model system.
Table 2. Degradation kinetics parameters of β-carotene in the presence of BMFDS, MMFDS, MFDS
under UVA irradiation in ethanol system.
Sulfides K(min−1)
R2
t1/2(min)
72.96
10.95
5.29
MFDS
MMFDS
BMFDS
0.0095
0.0633
0.131
0.9959
0.9356
0.9720
BMFDS: Bis (2-methyl-3-furyl) disulfide, MMFDS: Methyl (2-Methyl-3-furyl) disulfide, MFDS:
Methyl furfuryl disulfide.
2.3. Kinetics of β-Carotene Degradation Treated with BMFDS under UVA Irradiation
To further clarify the acceleration effect of BMFDS, the degradation kinetics parameters of β-
carotene under UVA irradiation were determined. As shown in Table 3, the photodegradation of β-
carotene treated by BMFDS in an ethanol system followed first-order kinetics. In terms of the rate
constant k, the degradation degree of β-carotene treated with BMFDS was much higher,
approximately 156 times than that in the control group. Therefore, our findings demonstrated that
β-carotene degradation was followed first-order kinetics after treatment with BMFDS.
Table 3. Degradation kinetics parametersβ-carotene in the presence of BMFDS under UVA
irradiation.
R Zero-order (c) R First-Order (ln c) R Second-Order (1/c) k (min−1)
R2
Control
BMFDS
0.9468
0.6376
0.9512
0.9766
0.9554
0.9141
0.0012
0.1879
0.9785
0.9537
c is the concentration of reactant.
2.4. The Analysis of Photooxidation Products of β-Carotene Treated with BMFDS under UVA Irradiation
According to the remarkable acceleration effect of BMFDS, HPLC-DAD-APCI-MS combined
with Raman spectroscopy was applied to make a preliminary identification about the oxidation
products of β-carotene, given their low contents and rather complex process of products collection.
The chromatographic and spectral data of the oxidation products of β-carotene by HPLC-DAD-
ACPI-MS are shown in Figure 2 and Table 4. The chromatographic peaks of leading β-carotene
oxidation products had the same peak time in both the experimental group and the control group.
Therefore, it was supposed that β-carotene treated with or without BMFDS under UVA irradiation
had the same oxidation products (Figure 2A). In comparison to the retention time of a standard
product (24.322 min) (Table 4), peak 3 was confirmed as all-trans-β-carotene (24.319 min) (Figure
3A). The UV spectra data are depicted in Figure 2B, and among the four obvious peaks, peak 2 and

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peak 4 were identified tentatively as 13-cis-β-carotene [24] and 9,13-di-cis-β-carotene [25],
respectively (Figure 3C,D) according to the spectral characteristics and Q-ratios (Table 4), which
were stipulated as the absorbance ratio of the middle main absorption peak to the cis peak.
Figure 2. The products of β-carotene treated with BMFDS under UVA irradiation. HPLC
chromatograms (A) and UV-Vis spectra (B) of β-carotene oxidation products induced by BMFDS
treatment for 5 min in ethanol under UVA irradiation. The blue line in HPLC chromatograms is

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referred to standard β-carotene. Peak identification for (A) and (B): Peak 1, all-trans-5,6-expoxy-β-
carotene; Peak 2, 13-cis-β-carotene; Peak 3, all-trans-β-carotene; Peak 4, 9,13-di-cis-β-carotene. Raman
spectra (C) at different wave number corresponded to β-carotene under UVA irradiation for 5 min
with BMFDS (a) and without BMFDS (b), respectively.
Table 4. Product identification of β-carotene treated with BMFDS under UVA irradiation.
λ (nm)
Q-Ratio
Retention
Molecular Ion,
Formula
Peak
Isomer
Time (min)
Found
Reported
Found
Reported
All-trans-5,6-expoxy-
1
19.467
553, C18H57O
446
444
β-carotene
2
3
4
21.953
24.319
25.399
537, C18H57
537, C18H57
537, C18H57
13-cis-β-carotene
All-trans-β-carotene
9,13-di-cis-β-carotene
340 446
451
340 445
455
0.41
0.16
0.41
0.16
350 446
335 439
Figure 3. The structure of β-carotene and its oxidation products induced by BMFDS under UVA
irradiation. (A) all-trans-β-carotene; (B) all-trans-5,6-epoxy-β-carotene; (C) 13-cis-β-carotene; (D)
9,13-di-cis-β-carotene.
The Raman spectra at various wavenumber (cm−1) of β-carotene in ethanol system treated with
or without BMFDS under UVA irradiation are displayed in Figure2C. By comparison of Figure 2C
(a) and Figure 2C (b), both υ1 (–C=C–) at 1520.67 cm−1 and υ2 (–C=C–C=C–) at 1155.48 cm−1 can be
observed. However, the distinction existed in the appearance of a new polar function group (C–O)
presented in Figure 2C (a). Combined with its m/z of 553 (Table 4), this new vibration peak was

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identified tentatively as all-trans-5,6-epoxy-β-carotene (Figure 3B). As would have been expected,
mono-epoxide is susceptible to generate from different sorts of carotenoids.
3. Discussion
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 [26–28].
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 [29]. 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[23,30]. 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 [23].
β-Carotene degradation upon exposure to MMFDS, BMFDS and MFDS followed first-order
kinetics, consistent with the kinetic model in dichloromethane system [31]. 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 (C6H8OS2), 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 [10,35] and pulp or juices [36,37]. 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-
cis 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

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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. [26] 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:
c − c0 = −kt
ln c/c0 = −kt
1/c − 1/c0 = kt
(1)
(2)
(3)
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 × 4.6mm, 5 μm) and gradient mobile phase of methanol-MTBE-
water (85:15:5, v/v/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.

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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.
5. Conclusions
Overall, through applying the results we obtained and drawing upon the information
provided by other studies, it is supposed that the oxidation products of β-carotene treated with
BMFDS under UVA irradiation in ethanol system might include 13-cis-β-carotene, 9,13-di-cis-β-
carotene and all-trans-5,6-epoxy-β-carotene. Our results might shed new light on the accelerative
effect of BMFDS on the photodegradation of β-carotene. More insights into the mechanism
involved in degradation of oxidation products of BMFDS-treated β-carotene should be further
studied further.
Author Contributions: methodology, Y.L. and W.-Q.G. formal analysis, J.S. data curation, Y.L. and J.S.;
writing—original draft preparation, G.-L.Z. and H.-Y. W.; writing—review and editing, G.-L.Z. and H.-M.H.;
supervision, H.-M.H.; funding acquisition, G.-L.Z. All authors discussed the results and approved the final
manuscript.
Funding: This research was funded by The National Natural Science Foundation of China, grant numbers
[31571888 and 31201419].
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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