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
], and micronutrients of carotenoids, flavonoids and ascorbic acid are responsible for the physiological function of citrus juice [4
]. 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
-glucoside, narirutin, hesperidin and didymi were confirmed as typical flavonoids in ‘Cara Cara’ juice [10
]. There is little information on the influence of storage temperature and duration on flavonoid content in ‘Cara Cara’ juice.
Due to the complex composition of carotenoids in oranges, the saponification procedure has typically been applied to simplify the analysis, by transferring esterified carotenoids into free cartoenoids [11
]. The stability of carotenoids can be influenced by temperature, time, and the availability of light and oxygen [13
]. Previous studies have mainly focused on the degradation of free carotenoids during storage [12
], while the change of carotenoid esters in citrus juice during storage has not been reported. There have been 19 carotenoid esters inferred in ‘Cara Cara’ fruit, with the 9-cis-violaxanthin ester confirmed as the dominant component [7
], and esterified β-cryptoxanthin considered to be the most stable ester during thermal treatment [15
]. The change of free and esterified cartoenoids in ‘Cara Cara’ juice needs to be further explored.
Non-enzymatic browning, frequently observed in citrus juice, plays an important role in the color, flavor and nutritional quality of the stored citrus juice [1
]. Non-enzymatic browning is also strongly connected to the degradation of ascorbic acid and sugar, with HMF (5-hydroxymethylfurfural) detected as the indicator [1
]. A previous report confirmed that vitamin C in citrus juice was affected by the storage temperature and duration [9
]. However, changes of flavonoid, carotenoids, vitamin C and sugar in ‘Cara Cara’ juice during storage at different temperatures have been not investigated, especially in terms of carotenoid esters.
We, therefore, carried out a pilot study to investigate the changes of carotenoid, flavonoid and vitamin C in ‘Cara Cara’ juice during 16 weeks of storage at different temperatures. In addition, the lipophilic and hydrophilic antioxidant abilities of stored ‘Cara Cara’ juice were analyzed.
2. Materials and Methods
2.1. Sample Preparation
‘Cara Cara’ juice (15.3 °Brix, pH 3.70, titratable acidity 0.93%) was obtained from commercial matured fruit of Orange cv. Cara Cara, harvested from the Fujian province of China in December 2017. The fresh ‘Cara Cara’ fruit (50 kg) was immediately peeled and squeezed with a fruit extruder. Half of the crude juice was stored at −80 °C and half was directly subjected to a rapid thermal sterilization. Specifically, ‘Cara Cara’ juice was boiled in a stainless-steel container with an electronic thermometer (F1, invisible, Guangdong, China) monitoring the internal temperature of the ‘Cara Cara’ juice (98 °C, 16 s). The sterilized juice was immediately filled into glass bottles (50 mL). These bottles and their caps were disinfected before use. After cooling, the juice was stored at 4, 20, 30 and 40 °C for 16 weeks without lights. At each sampling time, three bottles of juice were taken and frozen at −80 °C until use.
2.2. Chemicals and Reagents
The standards of narirutin, hesperidin, didymin, lycopene, β-carotene, fructose, glucose and sucrose were acquired from Yuanye Bio-Technology Co., Ltd (Shanghai, China). Phytoene and violaxanthin were purchased from CaroteNature (Lupsingen, Switzerland) and Sigma (St. Louis, MO, USA), respectively. ABTS+ (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Yuanye Bio-Technology Co., Ltd (Shanghai, China). High performance liquid chromatography purity solvents, including methyl tert-butyl ether (MTBE), methanol and hexane were obtained from Thermo Fisher Scientific (Leicestershire, UK). Other analytical grade chemicals, such as ethanol, hexane and sodium hydroxide were bought from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
2.3. Extraction of Carotenoid from ‘Cara Cara’ Juice
The extraction of carotenoids from ‘Cara Cara’ juice was performed according to our previous study [15
]. Briefly, ‘Cara Cara’ juice (10 mL) was homogenized with 10 mL ethanol/hexane (4:3, v
, 0.1% butylated hydroxytoluene) by stirring at 700 rpm for 0.5 h. The mixture was subsequently centrifuged (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.
2.4. 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.
2.5. Antioxidant Assays
Based on a previous study [16
], the antioxidant abilities of hydrophilic and lipophilic extracts in ‘Cara Cara’ juice were measured by DPPH and ABTS+
assays. The DPPH activity was evaluated as previously described [10
], and the final results were expressed as μmol ascorbic acid equivalent (AAE) per milliliter ‘Cara Cara’ juice (μmol AAE/mL, y
+ 0.0438, R2
= 0.9902). The ABTS+
assay was conducted according to the existing protocol [17
], while the final results were expressed as μmol Trolox equivalent (TE) per milliliter ‘Cara Cara’ juice (μmol TE/mL, y
+ 0.076, R2
= 0.9807). The hydrophilic and lipophilic capacities of stored ‘Cara Cara’ juice were calculated every four weeks (0, 4, 8, 12, 16 weeks).
2.6. Analysis of Carotenoids and Flavonoids in ‘Cara Cara’ Juice
The analysis of carotenoids was performed on HPLC (2695 system, Waters Corp., Milford, MA, USA) using a C30
reversed phase column (250 × 4.6 mm, 5 μm; YMC, Inc. Wilmington, NC, USA) and flavonoids were separated on the Waters Acquity UPLC system (Waters Corp., Milford, MA, USA) with a BEH C18
column (100 mm × 2.1 mm, 1.7 μm). Their chromatographic separation, identification and quantification procedures were conducted based on our previous study [15
]. The content of flavonoids in ‘Cara Cara’ juice during storage were analyzed every four weeks (0, 4, 8, 12, 16 weeks), and the sampling times for carotenoids quantification in stored ‘Cara Cara’ juice were set at 0, 2, 4, 6, 8, 12 and 16 weeks.
2.7. Ascorbic Acid Measurement
The ascorbic acid was determined by the titration method, using 2,6-dichlorophenolindophenol dye [18
2.8. Sugar Measurement
Modified from a previous study [19
], the soluble sugar in ‘Cara Cara’ juice was determined by HPLC (2695 system, Waters Corp., Milford, MA, USA) with 2414 refractive index detector (Waters, Milford, MA, USA), and an inertsil NH2
column (250 × 4.6 mm, 5 μm; Dikma Technologies Inc., Beijing, China) was used for sugar separation. Zinic acetate solution (21.9%, 0.1 mL) and potassium ferrocyanide solution (10.6%, 0.1 mL) were added in ‘Cara Cara’ juice (1 mL) to precipitated proteins. Afterwards, distilled water was added to 2 mL, and centrifuged to obtain the supernatant for HPLC analysis. Mobile phase was acetonitrile/water (75/25) and the HPLC operating conditions were set as: injection volume 20 μL; column temperature 40 °C; detector temperature 40 °C; flow rate 1 mL/min. The contents of sugar in ‘Cara Cara’ juice during storage were detected every two weeks (0, 2, 4, 6, 8, 10, 12, 14, 16 weeks).
2.9. 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
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.