Effect of low thermal pasteurization in combination with carvacrol on color, antioxidant capacity, phenolic and vitamin C contents of fruit juices

Abstract Mild thermal treatment in combination with natural antimicrobials has been described as an alternative to conventional pasteurization to ensure fruit juices safety. However, to the best of our knowledge, no study has been undertaken to evaluate what could be its effect on their color and nutritional value. This study therefore aimed at assessing how a low thermal pasteurization in combination with carvacrol could affect these parameters, with orange, pineapple, and watermelon juices as selected fruit juices. The experimental design used had levels ranging from 50 to 90°C, 0 to 60 μl/L, and 0 to 40 min for temperature, concentration of carvacrol supplemented, and treatment length, respectively. The only supplementation of fruit juices with carvacrol did not affect their color. In comparison with high thermal pasteurization (>70°C), a combined treatment at mild temperatures (50–70°C) better preserved their color, antioxidant capacity (AOC), and vitamin C content, and increased their total phenolic content (TPC). Globally, carvacrol supplementation had a positive impact on the TPC of thermally treated juices and increased the AOC of treated watermelon juice, which was the lowest of the three fruit juices. Mild heat treatment in combination with natural antimicrobials like carvacrol is therefore an alternative to limit the negative effects of conventional pasteurization on fruit juices quality.

Carvacrol (5-isopropyl-2-methylphenol) is the major component of Origanum spp essential oil. It is a GRAS compound known for its strong and wide antimicrobial spectrum and classified as a natural economical food preservative (Nostro & Papalia, 2012;Ramos et al., 2013;Sánchez, Aznar, & Sánchez, 2015). Kamdem et al. (2011) and Ait-Ouazzou et al. (2013) have observed that mild thermal treatment of fruit juices in combination with this compound could be a method to ensure safety against Listeria monocytogenes 56 LY and Escherichia coli O157:H7. This study aimed at assessing how such combined treatment could affect visual color and nutritional value of some fruit juices.

| Fruit juices production
Oranges (Citrus sinensis), pineapples (Ananas comosus), and watermelons (Citrullus vulgaris) were purchased from a local supermarket of Gembloux (Belgium). The juices were extracted using a fruit juice extractor (Kenwood A900 Centrifuge, Tokyo, Japan) and passed through a filter with a pore diameter of 280 μm. The pH and Brix values of the produced juices were measured using a portable pH meter ProfiLine pH 3210 Set 4 (WTW GmbH, Weilheim, Germany) and an Atago DBX-55 refractometer (Atago Co. Ltd., Tokyo, Japan), respectively.

| Experimental design
A central composite design (CCD), reinforced at its center and edges with three variables at five equidistant levels, was implemented.
Hue angle value, AOC, total phenolic content (TPC), and vitamin C or ascorbic acid content (AAC) of the juices after treatment were measured to assess the effects of treatment temperature, supplemented concentration of carvacrol (natural, 99%, Food Grade, Sigma-Aldrich, St. Louis, MO, USA), and duration of the thermal treatment. The tested levels of temperature (50-90°C) and length of treatment (0-40 min) were chosen to cover those generally tested in combination with natural antimicrobials and those conventionally practiced in fruit juice industries. The concentrations of carvacrol to be tested (0-60 μl/L) were chosen based on a preliminary sensorial analysis (Tchuenchieu et al., 2018). Each of the 27 runs of the CCD was performed in triplicate. The effect of the treatment on the AAC was assessed only with orange juice as this juice is known to have a higher content compared to the two other fruit juices.
Besides, the change in color and the variation of AOC and TPC of juices induced by the only carvacrol supplementation at the tested concentrations were also assessed.

| Thermal treatment
Thermal treatment was performed as described in a previous study (Ngang et al., 2014). It consisted of vials introduced in a thermostatically controlled water bath with fixed temperature to attain the desired temperature in the juice samples. For each tested condition, vials containing 9.9 ml of juice were supplemented with 0.1 ml of an ethanolic solution of carvacrol. The samples were then heated, and length of treatment measured from the moment the juice reached the desired temperature. (Generally, it took about 5 min to reach this temperature.) The samples were immediately cooled in a cryogenic solution (−10°C) once the treatment length was reached.

| Color measurement
The hue angle values of the juice samples, which characterize their visual color, were measured using a ColorFlex EZ colorimeter (Hunter Associates Laboratory Inc., VA, USA). The instrument was calibrated and used according to the manufacturer's recommendations.

| Measurement of antioxidant capacity
Antioxidant capacity (AOC) was measured using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method . Samples were diluted fourfold with methanol. Then, 0.2 ml of the diluted sample was added to 7.8 ml of a 0.03 g/L methanolic solution of DPPH (Sigma-Aldrich). The obtained mixture was then shaken on a vortexer and incubated in the dark for 30 min before reading the absorbance at 515 nm with a UV-2401 spectrophotometer (Shimadzu, Kyoto, Japan). Trolox (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) from Sigma-Aldrich was used as standard. The calibration curve was plotted using concentrations from 0 to 2 mmol/L. The AOC of samples was expressed as mmol/L TEAC (Trolox Equivalent Antioxidant Capacity).

| Measurement of total phenolic content
Total phenolic content (TPC) of the samples was determined by the Folin-Ciocalteu method (Medina, 2011). Samples were first diluted fourfold with distilled water, and a volume of 0.5 ml of the diluted juice was then added to 4.3 ml of distilled water and 0.2 ml of Folin-Ciocalteu 2 N (Sigma-Aldrich). After vortexing, 0.5 ml of a 20% Na 2 CO 3 solution and 4.5 ml of distilled water were added. The mixture was then vortexed again and incubated in the dark for 1 hr at room temperature before reading the absorbance at 725 nm. Gallic acid (Sigma-Aldrich) was used as standard. The calibration curve was plotted using concentrations from 0 to 500 mg/L. The TPC of samples was expressed as mg/L gallic acid equivalent (mg/L GAE).

| Measurement of ascorbic acid content
Ascorbic acid content (AAC) was measured by polarography using a 626 Polarecord equipped with a 663 VA stand from Metrohm (Herisau, Switzerland). The electrode system consisted of a glass carbon auxiliary electrode, a dropping mercury electrode, and a silver chloride reference electrode (Ag/AgCl/KCl 3 mol/L). We added 10 ml of an acetate buffer solution (acetic acid-sodium acetate 0.5 N) to 10 ml of the juice sample. The mixture was poured into the polarographic vessel and deaerated with nitrogen gas for 5 min before analysis. The potential was ramped from +0.31 V to −0.15 V with a natural mercury drop. The calibration curve was plotted with L(+)-ascorbic acid (VWR, Fontenay-sous-Bois, France) concentrations from 0 to 200 mg/L.

| Statistical analysis
Statistical analyses were performed using Statistica.10 software of Statsoft (Dell, TX, USA). A one-way ANOVA-Bonferroni post hoc test (homogeneous group) was performed to evaluate significant differences between the mean values obtained. Besides, an ANOVA-effect estimates analysis was also performed on the CCD results to assess the effect of the tested variables on each response studied.
As noticed with ethanolic solutions (Figure 2), the AOC of the tested concentrations of carvacrol (0-60 μl/L) was almost null, while their TPC was rising with the concentration supplemented.
Supplementation of this compound to fruit juices up to 60 μl/L did not significantly affect their color ( Figure 3). In fact, whatever the fruit juice, the hue angle values observed in the presence of carvacrol were similar to those of nonsupplemented samples. Table 1 shows the hue angle values and change in the AOC of fruit juices after the different treatments. As revealed by the statistical analysis of these data (Table 2) The assessment of the AOC of nontreated juices showed that pineapple juice (4.38 ± 0.77 mmol/L TEAC) had an AOC two and ten times higher than the ones of orange (2.29 ± 0.68 mol/L TEAC) and watermelon (0.42 ± 0.14 mmol/L TEAC) juices, respectively. From data obtained after heat treatment for this nutritional parameter, the statistical analysis generated a tendency explaining 88% of the ones obtained with watermelon juice (R 2 = .88), but only 51% and 29% of those obtained with pineapple and orange juices, respectively. The temperature of treatment had a significant linear negative effect on this AOC of the three fruit juices. This could be appreciated through the decrease observed with the rise of temperature. Besides, a quadratic negative effect was also observed with orange and pineapple juices. In fact, for these two latter juices, the effect of temperature on AOC was relatively small between 50 and 70°C, while AOC decreased almost linearly with temperature above 80°C ( Figure 5).

| Effect of combined treatment on the color and bioactive compounds of fruit juices
Carvacrol had a significant effect on the AOC only in treated watermelon juice, and this was linear and positive. The higher the level of carvacrol in watermelon juice, the higher its AOC. The length of the treatment significantly affected the AOC of juices with the exception of orange juice. A quadratic positive effect was observed with pineapple juice. Indeed, almost no change in AOC was noticed in this juice at the beginning of treatments, a stability that was followed by a little increase with time. In watermelon juice, treatment duration had a significant linear positive effect on this parameter. The higher the length of treatment, the higher its AOC, especially between 50 and 70°C. A negative interaction between time and temperature was also noticed in this juice.
Data on the change in total phenolic and ascorbic acid contents of fruit juices after the different treatments are presented in Table 3, and the associated statistical analysis is in Table 4.
TPC of nontreated orange (967 ± 139 mg/L GAE) and pineapple (995 ± 72 mg/L GAE) juices was almost similar but clearly higher than the one of watermelon juice (276 ± 31 mg/L GAE). From  the CCD results, it came out that the temperature of treatment had a significant effect on fruit juices TPC. A linear positive effect was observed in the case of orange juice, but negative in the case of pineapple and watermelon juices. This could be appreciated through the increase in TPC with the rise of temperature for orange juice, and rather a decrease with the two other juices.
Carvacrol supplementation had a linear positive significant effect on the TPC of the three juices. This was particularly obvious at high temperature for orange juice. Besides, a significant positive interaction with temperature was noticed in this juice. On the contrary, it is a negative interaction between carvacrol and temperature that was observed in watermelon juice. Indeed, the increase in temperature rather tended to reduce the positive impact of carvacrol ( Figure 6). A significant effect of length of treatment was noticed only with watermelon juice, and this was quadratic. This could be seen through the rise of the TPC in samples treated for 10 and 20 min, an increase that was followed by a gradual reduction with time (samples treated for 30 and 40 min).
Concerning AAC (vitamin C content), temperature of treatment had a significant linear and quadratic negative effect on this parameter (R 2 = .95). The rise of temperature led to a loss of the AAC that was higher at 80 and 90°C, than between 50 and 70°C. Time also had a significant negative effect, the losses increasing with the length of treatment. The statistical analysis also revealed a negative interaction between treatment duration and temperature. In fact, the negative impact of the length of treatment on juice AAC was more important above 80°C than below (Figure 7). No significant effect of carvacrol was noticed on this nutritional parameter.

| D ISCUSS I ON
Color is one of the most important quality attributes of fruit juices.
This color of orange juice has been correlated to its carotenoid pigment contents and is generally used as an index of this carotenoid content (Meléndez-Martínez et al., 2011;Vikram et al., 2005;Wibowo et al., 2015). Sánchez-Moreno et al. (2005) observed that thermal treatment of orange juice at 70°C for 30 s and 90°C for 1 min had no impact on carotenoid contents. As reported by Gama and de Sylos (2007), carotenoids are generally stable to heat treatment such as blanching, cooking, and canning. These authors found that total carotenoid pigment content loss in Valencia orange juice was not significant after a thermal pasteurization at temperatures between 95 and 105°C for 10 s. If certain carotenoid compounds were negatively affected by this treatment (violaxanthin and lutein), this was not the case for other compounds such as β-carotene, αcarotene, and β-cryptoxanthin. In the case of pineapple juice, we noticed that temperature and length of treatment had a significant effect on visual color, but this was very weak. Thermal treatment of pineapple juice at high temperature has been reported as leading to changes in color due to nonenzymatic browning through Maillard TA B L E 3 Change in the total phenolic and ascorbic acid contents of fruit juices after the different treatments reaction, pigment destruction, and sugar caramelization. By treating this juice between 55 and 95°C, Rattanathanalerk, Chiewchan, and Srichumpoung (2005) observed a linear increase in browning indicators (hydroxymethylfurfural and brown pigments) with temperature and a reduction in total carotenoids (pigments found in pineapple).
In contrast to the two other juices, the color of watermelon juice changed more remarkably under the effect of the combined treatment. The rise of temperature and length of treatment significantly increased its hue angle value, which means a decoloration of the juice naturally red to a more yellowish color. Sharma, Kaur, Oberoi, and Sogi (2008) had already observed, by treating watermelon juice between 50 and 90°C up to 5 hr, that the degradation of total carotenoids, lycopene, and color was following a first-order kinetic.  (Gardner et al., 2000). A correlation can therefore be made between the high decrease in AOC and the AAC losses observed at 90°C. The AOC of pineapple juice has rather been reported to be mainly associated with its TPC (Almeida et al., 2011), vitamin C conferring only 5% of this capacity (Gardner et al., 2000).
The significant negative effect of temperature noticed on the AOC of this juice could therefore be correlated to the significant negative effect on its TPC. Concerning watermelon, it is known as containing very low vitamin C and TPCs. As noticed in this study, AOC of nontreated watermelon juice was the lowest of the three fruit juices (100%) produced. Oms-Oliu et al. (2009) observed that AOC of watermelon juice was linked to its lycopene content. The high reduction in this compound under the effect of the thermal treatment (appreciable through the change in juice color) may therefore explain the decrease in its AOC at high temperatures. A link can also be established between the increase in TPC and the increase in AOC observed in this juice at mild temperatures.
The supplementation of fruit juices with carvacrol had a positive effect on their TPC after thermal treatment as it is a phenolic compound (5-isopropyl-2-methylphenol). At the tested concentrations, this supplementation did not have an impact on AOC of pineapple and orange juices, but on the one of thermally treated watermelon juice where the initial AOC was very low. This suggests, considering the almost null AOC of the tested concentrations, that carvacrol may interact during the thermal treatment with some fruit juices components leading to the formation of compounds with an AOC.
This was more appreciable in watermelon juice as it had an almost null AOC.

| CON CLUS IONS
Supplementation of fruit juices with natural antimicrobials like carvacrol followed by a mild thermal treatment may definitely be an effective alternative to conventional pasteurization in order to ensure their safety while preserving their color and nutritional value.
However, the application of such combined treatment must take into account the nature of the fruit juice to be treated as their properties or characteristics are not the same.

ACK N OWLED G EM ENT
The authors thank Wallonie-Bruxelles International who through the IN.WBI doctoral fellowship program has supported this research.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.