Stability of Flavonoid, Carotenoid, Soluble Sugar and Vitamin C in ‘Cara Cara’ Juice during Storage

In view of understanding the stability of sterilized ‘Cara Cara’ juice during storage, the changes of specific quality parameters (flavonoid, carotenoid, vitamin C, soluble sugar and antioxidant activities) of ‘Cara Cara’ juice were systematically investigated over the course of 16 weeks in storage at 4, 20, 30 and 40 °C. Total flavonoid and carotenoid indexes showed slight degradation at each temperature, while vitamin C and soluble sugar degraded intensively, especially at 40 °C storage with a great amount of HMF (5-hydroxymethylfurfural) accumulated. There were 29 carotenoids detected during storage, including carotenes and carotenoid esters. Carotenes were kept stable, while the degradations of carotenoid esters were fitted by biexponential function. Carotenoid ester group 2 contained epoxy structures that quickly decreased in the first four weeks at all storage temperatures, while the ester group 1 (belonged to β-cryptoxanthin ester) was degraded gradually. The 13- or 15-cis-lycopene, isomerized from all-(trans)-lycopene, increased with storage time at each temperature. Total flavonoid and carotenoid indexes in stored ‘Cara Cara’ juice were positively correlated with hydrophilic and lipophilic antioxidant abilities.


Introduction
Citrus juice possesses an attractive natural color, with a sweet and sour taste, making it popular with food consumers around the world [1]. Intake of citrus juice is confirmed to be effective for prevention of human chronic-degenerative diseases [2,3], and micronutrients of carotenoids, flavonoids and ascorbic acid are responsible for the physiological function of citrus juice [4,5]. Orange cv. 'Cara Cara', a bud mutation of navel orange (Citrus. sinensis L. Osbeck) originating in Venezuela in the 1980s, displays an attractive bright red color due to the accumulation of lycopene [6], and it has been widely planted in China [7]. Changes of food sensorial and nutritional quality during storage limits the date of food consumption. 'Cara Cara' juice products have not been commercially available in China, and the nutritional changes of 'Cara Cara' juice during storage have not been investigated.
Citrus juice products are usually exposed to various temperatures in the food supply chain. It is necessary to investigate the changes of carotenoid, flavonoid and ascorbic acid during storage at different temperatures, since these components play an important role in the healthy function of citrus juice. Rapisarda reported that the flavanone in sweet orange juice decreased about 50% after storage at 4 • C for 20 days [8], while Klimczak found that the flavanone in commercial pure orange juice was rather stable during storage with only minor changes observed [9]. Apigenin-6,8-di-C-glucoside, narirutin-4'-O-glucoside, narirutin, hesperidin and didymi were confirmed as typical flavonoids in (19,360× g, 4 min) to obtain the liquid phase. After extraction of the residue twice, all the liquid phases were combined, and then washed by separatory funnel to collect the non-polar supernatants. The obtained supernatant was evaporated to dryness, then re-dissolved by methyl tert-butyl ether and filtered (0.22 µm polytetrafluoroethylene filter) for the analysis of carotenoids.

Extraction of Flavonoid from 'Cara Cara' Juice
Flavonoids in 'Cara Cara' juice were extracted based on our previous study, with minor modification [15]. Briefly, 'Cara Cara' juice (1 mL) was homogenized with the extract solvent (85% aqueous ethanol containing 0.1% HCl, 4 mL) by an ultrasonic cleaner (KQ-500E, Kun Shan Ultrasound Instrument Co., Jiangsu, China) at 40 kHz for 30 min. The mixture was centrifuged (9680× g for 5 min) and filtered through 0.22 µm PTFE filter for further analysis.

Statistical Analysis
All the experiments were conducted in triplicate, and the data were presented as mean ± standard deviation of triplicate independent experiments. One-way analysis of variance (ANOVA) was applied to compare the means, and the differences between the means were analyzed by Duncan's multiple range tests at a significance level of 0.05. Correlation analysis of the matrix was analyzed by Pearson correlation coefficient (t-test). All statistical analyses were processed by IBM SPSS Statistics version 20.0. Carotenoid compounds were quantified in 'Cara Cara' juice during the 16 weeks of storage at different temperatures. The data were arranged to have carotenoid components at different temperatures as objects (rows) and storage weeks as variables (columns) and processed by principal component analysis (XLSTAT 2016, Addinsoft, New York, NY, USA). The results were presented with graphs plotting the projections of the units onto the components, and the loadings of the variables. Correlation between variables was evaluated by Pearson's correlation coefficient [20].

Changes of Flavonoids in 'Cara Cara' Juice
Based on our previous study [10,15], flavonoids were confirmed to be the dominant phenolic compounds in 'Cara Cara' juice, including apigenin-6,8-di-C-glucoside, narirutin-4'-O-glucoside, narirutin, hesperidin and didymin. The content changes of individual flavonoids are shown in Table 1. Compared with a previous study [21], the contents of narirutin and hesperidin reported in this study were relatively higher. Each individual flavonoid was not significantly changed during storage at 4 • C. Didymin and narirutin were stable with no significant decrease observed at all storage temperatures, while other flavonoids (apigenin-6,8-di-C-glucoside, narirutin-4'-O-glucoside, hesperidin) were significantly degraded during storage at 20, 30 and 40 • C. The degradation of flavonoids in fruit juice was probably associated with the peroxidase activity, which might not be completely inactive by sterilization [22]. HMF eluted with flavonoids on UPLC was only detected at 40 • C storage, with a slow accumulation in the first 12 weeks (y = 0.006x + 0.012, R 2 = 0.778) and a rapid increase was found in the last four weeks (y = 0.071x + 0.833, R 2 = 0.871), with the final content reaching 285.74 µg/mL. HMF is generated from the decomposition of vitamin C or sugar degradation, and it is typically used to evaluate the deterioration severity of juice [23,24].

Carotenoid Composition
Citrus was reported as a natural carotenoids source [25].
A total of 29 peaks were detected by HPLC-DAD (Figure 1), and they were identified according to our previous studies [7,15].
HMF eluted with flavonoids on UPLC was only detected at 40 °C storage, with a slow accumulation in the first 12 weeks (y = 0.006x + 0.012, R 2 = 0.778) and a rapid increase was found in the last four weeks (y = 0.071x + 0.833, R 2 = 0.871), with the final content reaching 285.74 μg/mL. HMF is generated from the decomposition of vitamin C or sugar degradation, and it is typically used to evaluate the deterioration severity of juice [23,24].

Carotenoid Degradation
The total carotenoid index in the sterilized 'Cara Cara' juice before storage was 309.06 ± 11.28 µg/mL, and the dominate compound was phytoene (69.78%), followed by total phytofluene (19.98%), carotenoid esters (4.15%), β-carotene (3.39%), lycopene (1.69%), and others (1.01%). The total carotenoids showed a declining trend at all storage temperatures, but their degradations did not reach a significant level. Carotenes of phytoene, β-carotene and lycopene were kept stable during storage, while all-trans-phytofluenes and cis-phytofluenes were irregularly fluctuated. Matrix protection might have been responsible for the stability of the carotenes in 'Cara Cara' juice [15,27,28]. Carotenoid ester, especially ester group 2, decreased dramatically during storage, and this might be related to their unstable xanthophyl structure which could be easily isomerized and degraded. The content changes of ester group 1 and ester group 2 in 'Cara Cara' juice are presented in Figure 2. Their degradation was fitted by biexponential function (Equation (1)) and the detailed kinetic parameters are presented in the Supplementary Materials ( Figures S1 and S2).
y t , is the carotenoid concentration at real time; y ∞, is the theoretical concentration of carotenoid at infinite time. A 1 and A 2 represent the pre-exponential factors; α and β, are the observed rate constants for fast and slow degradation. Biexponential degradation of carotenoid indicated both irreversible (degraded into volatiles or epoxides) and reversible (isomerization) degradation were involved, and this degradation form was also observed during thermal treatment of carotenoid juices [15,29]. Ester group 2 was quickly decreased in the first four weeks at all storage temperatures (Figure 2), while ester group 1 was degraded gradually. Therefore, the storage time of 'Cara Cara' juice could be estimated by combining the degrading rates of ester group 1 and ester group 2 at each temperature, and this issue will be explored in our future research.

PCA Analysis
PCA was investigated to understand the segregation and correlation among carotenoid compounds in 'Cara Cara' juice at all storage temperatures. According to the PCA results, three principal components were obtained to account for the total variance. PC1 and PC2 accounted for 69.02% and 15.77% of the total variance, respectively. Carotenoid compounds, decreased with the storage time (Table S1), were sorted in the same group and they were strongly and positively correlated with PC1 ( Figure 3A). Other compounds including 13-or 15-cis-lycopenes (4, 20, 30, 40 ℃), cis-phytofluenes (4, 20, 30, 40 °C), ester groups 3 (4, 20, 30, 40 °C) and ζ-carotenes (30, 40 °C) were classified into the other two groups. The 13-or 15-cis-lycopene, derived from all-(trans)-lycopene by isomerization, was increased with storage time at each temperature, and the contents of 13-or 15-cislycopenes (4, 20, 30, 40 °C) were strongly and negatively correlated with PC1. The contents of cisphytofluene, ester group 3 and ζ-carotene were irregularly changed in 'Cara Cara' juice during the overall storage period, and their contents at 4, 20, 30, 40 °C were sorted into different groups. Wibowo et al. proved that ζ-carotene increased during juice storage at different temperatures, while Cortés presented the opposite view [12,30]. In this study, the increase of ζ-carotene was just observed at 40 °C.
As presented in the PCA score plot ( Figure 3B), the carotenoid profiles of 'Cara Cara' juice stored at different times were clearly divided into four groups. The sterilized juice at 0 week was grouped in the lower right quadrant, showing a positive correlation with PC1 and a negative correlation with PC2. Similarly, the other three groups (2 and 4 weeks, 6 and 8 weeks, 12 and 16 weeks) were distributed in the different quadrants, indicating that they have different correlations with PCs. Storage time of each group (2 and 4 weeks, 6 and 8 weeks, 12 and 16 weeks) had a similar impact on the change of carotenoid.

PCA Analysis
PCA was investigated to understand the segregation and correlation among carotenoid compounds in 'Cara Cara' juice at all storage temperatures. According to the PCA results, three principal components were obtained to account for the total variance. PC1 and PC2 accounted for 69.02% and 15.77% of the total variance, respectively. Carotenoid compounds, decreased with the storage time (Table S1), were sorted in the same group and they were strongly and positively correlated with PC1 ( Figure 3A). Other compounds including 13-or 15-cis-lycopenes (4, 20, 30, 40°C), cis-phytofluenes (4, 20, 30, 40 • C), ester groups 3 (4, 20, 30, 40 • C) and ζ-carotenes (30, 40 • C) were classified into the other two groups. The 13-or 15-cis-lycopene, derived from all-(trans)-lycopene by isomerization, was increased with storage time at each temperature, and the contents of 13-or 15-cis-lycopenes (4, 20, 30, 40 • C) were strongly and negatively correlated with PC1. The contents of cis-phytofluene, ester group 3 and ζ-carotene were irregularly changed in 'Cara Cara' juice during the overall storage period, and their contents at 4, 20, 30, 40 • C were sorted into different groups. Wibowo et al. proved that ζ-carotene increased during juice storage at different temperatures, while Cortés presented the opposite view [12,30]. In this study, the increase of ζ-carotene was just observed at 40 • C.
As presented in the PCA score plot ( Figure 3B), the carotenoid profiles of 'Cara Cara' juice stored at different times were clearly divided into four groups. The sterilized juice at 0 week was grouped in the lower right quadrant, showing a positive correlation with PC1 and a negative correlation with PC2. Similarly, the other three groups (2 and 4 weeks, 6 and 8 weeks, 12 and 16 weeks) were distributed in the different quadrants, indicating that they have different correlations with PCs. Storage time of each group (2 and 4 weeks, 6 and 8 weeks, 12 and 16 weeks) had a similar impact on the change of carotenoid.  Note: mut for mutatoxanthin, zea for zeaxanthin, β-cry for β-cryptoxanthin, ζ-car for ζ-carotene, βcar for β-carotene, cis-lyc for 13-or 15-cis-lycopene, lyc for lycopene, pto for phytoene, pfl for phytofluene, cis-pfl for cis-phytofluene, ester 1 for ester group 1, ester 2 for ester group 2, ester 3 for ester group 3. PC1and PC2 represent the first principal component and the second principal component, respectively. mut for mutatoxanthin, zea for zeaxanthin, β-cry for β-cryptoxanthin, ζ-car for ζ-carotene, β-car for β-carotene, cis-lyc for 13-or 15-cis-lycopene, lyc for lycopene, pto for phytoene, pfl for phytofluene, cis-pfl for cis-phytofluene, ester 1 for ester group 1, ester 2 for ester group 2, ester 3 for ester group 3. PC1and PC2 represent the first principal component and the second principal component, respectively.

The Changes of Soluble Sugars in 'Cara Cara' Juice
Changes of soluble sugar are shown in Table 2. The total soluble sugars were gradually decreased with the improved temperature and prolonged storage. The total soluble sugars were decreased by 1.39%, 1.63%, 9.33% and 31.68% respectively when the juice was stored at 4, 20, 30 and 40 • C. It was reported that fructose, glucose and sucrose were greatly degraded in grapefruit juice during its storage at 37 • C for 16 weeks [19], and the loss of soluble sugars might be related to browning reactions. In the present study, sucrose in 'Cara Cara' juice was minorly hydrolyzed at 4, 20 and 30 • C, but it was completely degraded at 40 • C, with fructose and glucose being increased. It was shown that the hydrolysis of sucrose was followed by pseudo first-order reaction, and the hydrolysis progress was correlated with acid concentration and storage time [31]. The increased content of glucose and fructose in 'Cara Cara' juice stored at 40 • C was not stoichiometrically in line with the hydrolyzed sucrose ( Table 2), indicating that hydrolyzate might be partly engaged in Maillard reactions [19].

The Changes of Vitamin C in 'Cara Cara' Juice
The percent retention of vitamin C in 'Cara Cara' juice during storage is shown in Figure 4, and vitamin C retention decreased with prolonged storage and increased temperature. No significant loss of vitamin C was detected in the 'Cara Cara' juice stored at 4 • C for 16 weeks. The concentration of vitamin C decreased by 23.93%, 35% and 69.58% respectively when 'Cara Cara' juice was stored at 20, 30 and 40 • C for 16 weeks. The degradation of vitamin C in citrus juice has been widely studied, and the degradation mode has been fitted to the first-order reaction [32,33]. A similar result was found in our study. The presence of vitamin C in citrus juice could protect carotenoids from oxidation, and a lower loss of carotenoid was confirmed in vitamin C fortified juice [34].

The Changes of Vitamin C in 'Cara Cara' Juice
The percent retention of vitamin C in 'Cara Cara' juice during storage is shown in Figure 4, and vitamin C retention decreased with prolonged storage and increased temperature. No significant loss of vitamin C was detected in the 'Cara Cara' juice stored at 4 °C for 16 weeks. The concentration of vitamin C decreased by 23.93%, 35% and 69.58% respectively when 'Cara Cara' juice was stored at 20, 30 and 40 °C for 16 weeks. The degradation of vitamin C in citrus juice has been widely studied, and the degradation mode has been fitted to the first-order reaction [32,33]. A similar result was found in our study. The presence of vitamin C in citrus juice could protect carotenoids from oxidation, and a lower loss of carotenoid was confirmed in vitamin C fortified juice [34].

The Changes of Antioxidants in 'Cara Cara' Juice
The antioxidants in 'Cara Cara' juice were evaluated in hydrophilic and lipophilic fractions, which represented the antioxidant ability of flavonoids and carotenoids, respectively. It was reported that lipophilic fractions usually displayed much lower antioxidant ability than the hydrophilic fraction in common fruits and vegetables [16,35]. A similar phenomenon was also found in our present study, which might be attributed to the higher content of total flavonoid index than that of the total carotenoid index ( Table 1 and Table S1). The changes in both hydrophilic and lipophilic antioxidant abilities in 'Cara Cara' juice during the 16 weeks of storage at different temperatures are shown in Table 3.

The Changes of Antioxidants in 'Cara Cara' Juice
The antioxidants in 'Cara Cara' juice were evaluated in hydrophilic and lipophilic fractions, which represented the antioxidant ability of flavonoids and carotenoids, respectively. It was reported that lipophilic fractions usually displayed much lower antioxidant ability than the hydrophilic fraction in common fruits and vegetables [16,35]. A similar phenomenon was also found in our present study, which might be attributed to the higher content of total flavonoid index than that of the total carotenoid index ( Table 1 and Table S1). The changes in both hydrophilic and lipophilic antioxidant abilities in 'Cara Cara' juice during the 16 weeks of storage at different temperatures are shown in Table 3. A correlation between flavonoid compositions and antioxidant activity in hydrophilic extracts from 'Cara Cara' juice was explored. A positive correlation was found between the total flavonoid index and hydrophilic antioxidant ability (Table 4), in accordance with a previous study [36]. Each individual flavonoid was significantly and positively correlated with tested antioxidant activities at p < 0.01 level (Table 4). Therefore, flavonoid compounds in 'Cara Cara' juice were the important contributions to the hydrophilic antioxidant ability. Correlations between carotenoid compositions and lipophilic antioxidant activities of 'Cara Cara' juice are presented in Table 5. Consisting of zeaxanthin, β-cryptoxanthin, lycopene, phytoene, phytofluene, ester group 1, ester group 2 and the total carotenoid index were significantly and positively correlated with the tested bioactivities at p < 0.01. β-carotene correlated with ABTS + capacity and DPPH scavenging at p < 0.01 level and p < 0.05 level, respectively. ζ-carotene, 13-or 15-cis-lycopene, cis-phytofluene and ester group 3 were negatively correlated with the tested bioactivities. The result suggested that zeaxanthin, β-cryptoxanthin, lycopene, phytoene, phytofluene, ester group 1 and ester group 2 were contributors to the lipophilic antioxidant ability of 'Cara Cara' juice.

Conclusions
The micronutrients in 'Cara Cara' juice were investigated during storage at 4, 20, 30 and 40 • C for a period of 16 weeks. Total flavonoid and carotenoid indexes showed slight degradation at each temperature, while vitamin C and soluble sugar degraded intensively, especially at 40 • C storage. Although the total carotenoids were stable at each storage temperature, most carotenoid esters were significantly degraded and fitted by biexponential function. Specifically, the ester group 2 with epoxy structures quickly decreased in the first four weeks at all storage temperatures, while the ester group 1 (belonged to β-cryptoxanthin ester) degraded gradually. The combined degrading rates of the two type of esters might be further applied to estimate the storage time of 'Cara Cara' juice. Total flavonoid and carotenoid indexes in stored 'Cara Cara' juice were positively correlated with hydrophilic and lipophilic antioxidant abilities. This study provided information on changes of flavonoid, carotenoids, vitamin C and sugar in 'Cara Cara' juice during storage at moderate and elevated temperatures, which might be useful for the quality prediction of 'Cara Cara' juice during storage.
Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/8/9/417/s1, Figure S1: Biexponential fitting with eq 1 of the experimental data obtained for the degradation of ester group 1 in Cara Cara juice during 16 weeks storage at 4 • C (A), 20 • C (B), 30 • C (C) and 40 • C (D), respectively. Figure S2: Biexponential fitting with eq 1 of the experimental data obtained for the degradation of ester group 2 in Cara Cara juice during 16 weeks storage at 4 • C (A), 20 • C (B), 30 • C (C) and 40 • C (D), respectively. Table S1: Changes in the content of carotenoids (µg/mL) in 'Cara Cara' juice during 16 weeks of storage at different temperature.