Combined effect of high pressure carbon dioxide and mild heat treatment on overall quality parameters of watermelon juice

Innovative Food Science and Emerging Technologies 13 (2012) 112–119

Contents lists available at SciVerse ScienceDirect

Innovative Food Science and Emerging Technologies journal homepage: www.elsevier.com/locate/ifset

Combined effect of high pressure carbon dioxide and mild heat treatment on overall quality parameters of watermelon juice Ye Liu a,⁎, Xiaosong Hu b, Xiaoyan Zhao c, Huanlu Song a a b c

School of Food and Chemical Engineering, Beijing Technology and Business University, Beijing 100048, China College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China

a r t i c l e

i n f o

Article history: Received 23 July 2011 Accepted 1 November 2011 Editor Proof Receive Date 30 November 2011 Keywords: High pressure carbon dioxide Mild heat Watermelon juice Quality parameters

a b s t r a c t The aim of this study was to investigate the combined effect of high pressure carbon dioxide (HPCD) and mild heat treatment on the overall quality of watermelon juice. The residual activity of polyphenoloxidase (PPO), peroxidase (POD), and pectin methylesterase (PME) decreased with pressure and treatment time after HPCD and heat treatment. The total color difference (△E) value was greater than 3.5, suggesting the significant change of color. Browning degree (BD) decreased with pressure and treatment time; pH and lycopene content of HPCD-treated juice slightly decreased; cloudiness and titratable acidity (TA) increased; and viscosity of 10 and 20 MPa treated juice at 31.62 1 s− 1 did not change. While after control treatment, the residual activity of enzymes and BD decreased slightly; cloudiness decreased greatly; pH, TA, lycopene content, and color were stable. HPCD and mild heat treatment inactivated enzyme activities drastically which affected the shelf life and quality of watermelon juice greatly during storage. And it increased cloudy stability that was the important parameter to influence appearance of juice. Overall, HPCD treatment has greater advantages to maintain the quality of watermelon juice. Industrial relevance: The application of high pressure carbon dioxide (HPCD) processing on several food products has already been proven to be successful for pasteurization. HPCD processing to inactivate endogenous enzymes and retain quality of food needs to be further studied. Studies dealing with the combination effect of pressure and mild heat conditions on enzyme activities and quality parameters are relevant to understand the prospects for watermelon juice processing. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Watermelon contains some phenolic, vitamin C and a large amount of lycopene which contributes to the red color (Gil, Aguayo, & Kaer, 2006; Perkins-Veazie, Collins, Pair, & Roberts, 2001). The pigments, vitamins and minerals can provide potential health benefits and reduce incidence of coronary heart disease and cancer by the consumption of watermelon juice (Edwards et al., 2003; Fraser & Bramley, 2004; Giovannucci, 2002). However, pathogenic microorganisms may grow in watermelon juice without processing due to the low acidity and high water activity of this fruit. Hence, pasteurization is the necessary process in the production of watermelon juice. Thermal treatment is the traditional effective method to destroy pathogenic microorganisms in foods (Lambert, 2003; Mann, Kiefer, & Leuenberger, 2001; Zanoni, Pagliarini, Giovanelli, & Lavelli, 2003). However, watermelon is a kind

Abbreviation: HPCD, High Pressure Carbon Dioxide; PPO, Polyphenoloxidase; POD, Peroxidase; PME, Pectin METHYLESTERASE; △E, Total color difference; BD, Browning degree; TA, Titratable acidity; TSS, Total soluble solid; ANOVA, Analysis of variance. ⁎ Corresponding author. Tel.: + 86 10 68985378. E-mail address: [email protected] (Y. Liu). 1466-8564/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2011.11.001

of thermo-sensitive fruit whose organoleptic and nutritional properties will be destroyed by heat inevitably. For this reason, high pressure carbon dioxide (HPCD) as novel non-thermal process is applied to the investigations of fruit juice processing recently. And it is proved to be able to inactivate pathogenic microorganisms without significantly destroying thermo-sensitive nutrients, texture, color, and flavor of foods (Gui et al., 2007; Zhou, Wang, Hu, Wu, & Liao, 2009). HPCD can inactivate microorganisms by lowering pH of cell, inducing precipitation of intracellular carbonate calcium and magnesium, disrupting cell, modifying cell membrane and extracting cellular components (Hong & Pyun, 1999, 2001; Isenschmid, Marison, & Stockar, 1995; Lin, Yang, & Chen, 1993; Spilimbergo, Elvassore, & Bertucco, 2003). The combination of CO2 and high pressure had a significant interaction and survival of L. plantarum ATCC 8014 was decreased (Corwin & Shellhammer, 2002). The inactivation of E. coli in cloudy apple juice significantly increased when increasing the exposure time with CO2 at 20 MPa and 37 °C or at 30 MPa and 42 °C (Liao, Hu, Liao, Chen, & Wu, 2007). The inactivation kinetics of S. typhimurium in model solutions of brain heart infusion broth and physiological saline were investigated under HPCD from 1.51 to 7.56 MPa at 35 °C, which showed that the inactivation rates increased

Y. Liu et al. / Innovative Food Science and Emerging Technologies 13 (2012) 112–119

with pressure, suspending medium and exposure time (Erkmen & Karaman, 2001). Meanwhile, HPCD can inactivate enzyme activity by changing the conformation of enzyme protein (Chen, Balaban, Wei, Marshall, & Hsu, 1992; Gui et al., 2006a). More inactivation of PPO in cloudy apple juice could be caused by pressure, temperature, and treatment time increasing by HPCD treatment and the maximum reduction of PPO activity reached more than 60% at 30 MPa and 55 °C for 60 min (Gui et al., 2007). HPCD treatment could inactivate PPO from frozen red raspberry effectively and the minimum of PPO residual rate was 36.6% under 30 MPa and 55 °C for 60 min (Liu et al., 2010). However, there have been relatively few studies, which monitor the physical, chemical and sensory quality of liquid foods following HPCD treatment (Garcia-Gonzalez et al., 2007). Studies on orange juice showed that HPCD treatment could improve some physical and nutritional quality such as cloudiness, stability, color, and titratable acidity (Arreola et al., 1991; Kincal et al., 2006). Aroma of HPCD treated beer was not significantly different from fresh beer after one month storage at 1.67 °C, but heat-treated beer changed significantly in taste and aroma (Folkes, 2004). △E of cloudy apple juice after HPCD treatment was significantly reduced by enhancing the pressure level and was significantly less than that of the control throughout storage time (Gui et al., 2006b). HPCD enhanced cloudiness up to 38.4%, increased lightness and yellowness, decreased redness, and did not change pH, °Brix of mandarin juice (Damar & Balaban, 2005). The browning degree and pH of HPCD-treated carrot juice decreased, and the cloudiness, titratable acidity, and particle size increased significantly (P b 0.05) (Zhou et al., 2009). From the previous studies, mild heat was always applied with HPCD treatment because both methods alone could not inactivate enzymes effectively (Gui et al., 2007; Liu et al., 2008; Liu et al., 2010). While mild heat had little effect on the quality parameters, such as hunter parameters, hydroxymethylfurfural, brown pigment of pineapple juice, and the content of α- and β-carotene of carrot juice (Kim, Park, Cho, & Park, 2001; Rattanathanalerk, Chiewchan, & Srichumpoung, 2005). Similar results were presented from the preexperiments. To the best of our knowledge, there is no report on the application of HPCD treatment on watermelon juice. Hence, this study focused on the combined effect of HPCD and mild heat treatment on the activity of endogenous enzymes and quality of watermelon juice.

113

2. Materials and methods 2.1. Preparation of watermelon juice Watermelons (Jingling) were harvested maturely in Yanqing farm. The watermelons were washed, peeled, and crushed with a blender (Philips, HR2860, Zhuhai, China). The homogenous solution was filtrated by gauzes. The filtrate was subjected to the following treatments. 2.2. HPCD process system As shown in Fig. 1, the stainless steel pressure vessel with a volume of 850 mL was designed to withstand a pressure of 50 MPa. The vessel temperature was maintained by a THYS-15 thermostatic bath (Ningbo Tianheng Instrument Factory, Zhejiang, China). An XMTA-7512 temperature controller (Yuyao Temperature Meter Factory, Zhejiang, China) was used to monitor the temperature with two thermocouples. One thermocouple was fixed in the vessel lid to monitor the CO2 temperature in the upper part of the vessel and the other was placed at the middle wall of the vessel to monitor the temperature of watermelon juice in the vessel. A 2TD plunger pump (Huaan Supercritical Fluids Extraction Co. Ltd., Jiangsu, China) with a maximum pressure of 50 MPa and a maximum flow rate 50 L h − 1 was used to pressurize the vessel. A DBY-300 pressure transducer (Shanxi Qingming Electronic Group Corporation, Shanxi, China) was fixed in the vessel lid to monitor the vessel pressure. All the data of temperature and pressure were displayed on a control panel. All parts of the system exposed to high pressure were made of stainless steel. The vessel had gas-tight connections to the gas inlet and outlet. The vessel lid could be sealed by screws during HPCD processing. Commercially available CO2 of 99.5% purity was purchased from Beijing JingCheng Co. (Beijing, China), and was passed through an active carbon filter before entering the pressure vessel. 2.3. HPCD treatment of watermelon juice After being rinsed and sanitized, the pressure vessel was heated to the required temperature. For each experiment, aliquot of 25 mL watermelon juice was placed in a 50 mL plastic tube without the cap and

Fig. 1. Diagram of high pressure carbon dioxide processing equipment.

114

Y. Liu et al. / Innovative Food Science and Emerging Technologies 13 (2012) 112–119

then placed in the HPCD vessel which had been preheated to the experimental temperature. Then CO2 was fed over 5–10 min until the pressure reached the experimental level. The sample was held at constant pressure and temperature during treatment. At the end of treatment, the vessel was slowly depressurized over a period of 10 min. After treatment, the juice was removed and immediately cooled in an ice bath for further analysis. The pressure of HPCD treatment was 10, 20, and 30 MPa at 50 °C, and the treatment time was 5, 15, 30, 45 and 60 min at each pressure. 2.4. Control treatment of watermelon juice

2.8. Determination of pH The pH value of the sample was measured at 25 °C with a Thermo Orion 868 pH meter (Thermo Fisher Scientific, Inc., MA, U.S.A), which was calibrated with pH 4.0 and 7.0 buffer. 2.9. Determination of titratable acidity (TA) 10 mL watermelon juice was titrated using standardized 0.02 M NaOH to the phenolphthalein end point (pH = 8.1) (Rodrigo et al., 2003).

For each experiment, aliquot of 25 mL watermelon juice was placed in a 50 mL plastic tube without the cap and then placed in the HPCD vessel which had been preheated to the experimental temperature. Without pressurizing, the juice only treated by the same temperature and treatment time as HPCD treatment.

2.10. Determination of browning degree (BD)

2.5. Thermal treatment of watermelon juice

TSS of watermelon juice was determined as Brix using a PAL-α portable digital Refractometer (ATAGO, Japan) at 25 °C (Wang et al., 2006).

Watermelon juice was placed in a stainless steel cup (50 mL) and heated until the centre of juice reached to 95 °C (determined by electronic temperature meter immerging in juice centre) with a short come-up time in a water bath, held for 1 min. The juice after thermal treatment was immediately cooled to 15 °C in an ice water bath for further analysis. 2.6. Determination of PPO and POD activity The watermelon juice was stirred with 0.01 g mL − 1 polyvinyl polypyrrolidone (Sigma-Aldrich Co., Shanghai, China) and 0.01 g mL− 1 Triton X-100 at 4 °C for 1 h, and centrifuged at 10,000 ×g (Sigma, 3–18 sartorius, Germany) and 4 °C for 20 min. The supernate was prepared for the assay of PPO and POD activity. PPO activity was assayed according to the method described by Duangmal and Apenten (1999) with minor modifications. Aliquot of the supernate (0.6 mL) was mixed with 2.4 mL substrate solution [100 mM catechol in 100 mM citrate buffer (pH 5.4)] and measured at 420 nm (Unico, UV-3802, Shanghai, China). The initial velocity of each reaction was calculated from the linear portion of the absorbance–time curve and taken as the enzymatic activity. One unit of PPO activity was defined as the change in absorbance of 0.001 min− 1 at 420 nm and per milliliter of watermelon juice. POD activity was assayed according to the method described by Onsa, Saari, Selamat, and Baker (2004) with minor modifications. Aliquot of the supernate (0.05 mL) was mixed with 2.7 mL, pH 6.0, 200 mM phosphate buffer; 0.2 mL guaiacol and 0.05 mL hydrogen peroxide. The absorbance of the mixture was measured at 470 nm (Unico, UV-3802, Shanghai, China). The initial velocity of each reaction was calculated from the linear portion of the absorbance–time curve and taken as the enzymatic activity. One unit of POD activity was defined as the change in absorbance of 0.001 min − 1 at 470 nm and per milliliter of watermelon juice. The residual activities of PPO and POD were calculated according to Eq. 1.

Residual activity ¼

enzyme activity treated with HPCD  100% enzyme activity before HPCD

ð1Þ

BD was evaluated using a spectrophotometric method described by Roig, Bello, and Rivera (1999). 2.11. Determination of total soluble solid (TSS)

2.12. Determination of lycopene content Lycopene content was measured spectrophotometrically using the methods of Sadler, Davis, and Dezman (1990) with modifications. And lycopene content was calculated with the standard linear curve: Y = 0.0154 + 0.244X (R 2 = 0.998) of lycopene solution in hexane in concentrations from 0.5 to 5 μg mL − 1. 2.13. Determination of color Color assessment was conducted at 25 °C using a Color Difference Meter (Konica Minolta CR-200, Japan) in the reflectance mode. Hunter L⁎-, a⁎-, and b⁎-values of juice was measured and the total color difference (△E) was calculated as:

ΔE ¼

h




 2

L −L0

i

  2  2 1=2 þ a −a0 þ b −b0

ð2Þ

where L⁎ — lightness of treated juice, L⁎0 — lightness of the control, a⁎ — redness of treated juice, b⁎ — yellowness of treated juice, a⁎0 — redness of the control, and b⁎0 — yellowness of the control. 2.14. Determination of total phenolics content Total phenolic in the watermelon juice was determined with Folin–Ciocalteu reagent by the method of Singleton and Rossi (1965) using pyrogallol as a standard. 2.15. Determination of cloudiness The cloudiness of watermelon juice was determined using a 1900C portable turbidimeter (HACH, U.S.A.), and was reported as Nephelometric Turbidity Units (NTU) (Reiter, Stuparic, Neidhart, & Carle, 2003). Watermelon juice diluted with distilled water (1:15, V/V) was measured in a 20 mL quartz cuvette. 2.16. Determination of viscosity

2.7. Determination of PME activity The activity of PME was measured at pH 7.5 and 30 °C according to the method proposed by Kimball (1991).

The viscosity of watermelon juice was determined using an AR1500 rheometer (TA Instruments, Waters Co., Ltd., Surrey, Great Britain). 1 mL of watermelon juice was applied at each measurement with 25 °C controlled by circulating water in a thermostatic system.

Y. Liu et al. / Innovative Food Science and Emerging Technologies 13 (2012) 112–119

Control 10 MPa 20 MPa 30 MPa

120

Analysis of variance (ANOVA) and Duncan's multiple range tests were carried out by using the software SAS (SAS Institute Inc., Cary, NC, U.S.A.). The ANOVA test was performed for all experimental run, to determine the significance at 95% confidence interval. All experiments were performed in triplicates. 3. Results and discussion 3.1. Effect of HPCD and mild heat treatment on PPO, POD and PME activity of watermelon juice

POD residual activity (%)

2.17. Statistical analysis

100

80

60

40

20 0

120

Control 10 MPa 20 MPa 30 MPa

100 80 60 40 20

10

20

30

40

50

60

Time (min) Fig. 3. Inactivation of POD of watermelon juice by HPCD treatment of different pressures at 50 °C. Error bars represent the standard deviation.

isoenzyme of PME might be so large that it could not be inactivated during 0–60 min completely. However, the inactivation of PME in apple by HPCD treatment was fitted according to a two-fraction model which contained a fast inactivation period followed by a decelerated decay. And the residual activity of PME dropped to about 6% after HPCD treatment of 30 MPa and 55 °C for 60 min (Zhi, Zhang, Hu, Wu, & Liao, 2008). The residual activities of PPO and POD after thermal of 95 °C for 1 min were 49.1% and 82.9%, respectively, and that of PME was 85.7%. From Figs. 2, 3, and 4, the residual activities of PPO, POD, and PME after control treatment were all higher than that of HPCD treatment (except for control and 10 MPa at 45 min in Fig. 3), which were 67.9%, 83.2%, and 86.3% after mild heat treatment of 50 °C for 60 min, respectively. The combined effect of HPCD and mild heat treatment on the activities of PPO, POD, and PME was better than control and thermal treatments under the experiment conditions of this study. But it was difficult to compare the inactivation capability of combined and thermal treatments because of the different treatment time. For the cost consideration, treatment time could be shorten to reach the similar inactivation effect with traditional thermal treatment of juice (95 °C for 1 min). 3.2. Effect of HPCD and mild heat treatment on pH of watermelon juice The combined effect of HPCD and mild heat treatment on pH of watermelon juice is shown in Table 1. The initial pH of juice was 5.83. After HPCD treatment of 50 °C, the pH of watermelon juice decreased significantly from 5.83 to 5.46–5.65 (P b 0.05). This result was in agreement with the investigations of carrot juice, apple juice, Control 10 MPa 20 MPa 30 MPa

120

PME residual activity (%)

The combined effect of HPCD and mild heat treatment on PPO and POD residual activity of watermelon juice is shown in Figs. 2 and 3, respectively. The residual activities of both enzymes decreased significantly with treatment time and pressure during the first 5 min (P b 0.05). For PPO residual activity, it decreased fast to slowly with treatment time. This might be due to two isoenzymes existing in PPO of watermelon juice. One isoenzyme was sensitive to pressure and the other was stable. When pressure was applied, the liable one could be inactivated easily but the stable one could not. The similar inactivation effect was shown in PPOs of lobster, shrimp and potato by HPCD treatment (Chen et al., 1992). However, the different phenomena are presented in studies on PPOs of apple juice, red beet, and table grape, in which residual activities of PPOs decreased linearly by HPCD treatment (Fortea, López-Miranda, Serrano-Martínez, Carreño, & Núñez-Delicado, 2009; Gui et al., 2007; Liu et al., 2008). The discrepancy among these investigations resulted from the different sources of enzymes, which led to the difference of inactivation effect. While POD residual activity decreased linearly generally if ignoring 10 MPa for 45 min and 20 MPa for 5 min. It presented the similar tendency with POD residual activity in red beet (Liu et al., 2008). At each treatment time, the residual activity of PPO and POD decreased to 4.2% and 42.1% at 30 MPa and 50 °C for 60 min with the pressure generally. The residual activity of PPO in cloudy apple juice was about 40% by HPCD treatment of 30 MPa and 55 °C for 60 min (Gui et al., 2007). HPCD treatment at 37.5 MPa for POD and 22.5 MPa for PPO (55 °C for 60 min) in red beet extract resulted in their residual activities dropping to approximately 14% and 5%, respectively (Liu et al., 2008). The combined effect of HPCD and mild heat treatment on residual activity of PME of watermelon juice is shown in Fig. 3. It also decreased with treatment time and pressure except 10 MPa for 30 and 45 min. In general, the residual activity of PME decreased linearly during 0–60 min by HPCD treatment. And the minimum of PME residual activity was 14.5% at 30 MPa and 50 °C for 60 min. This result might be due to two possibilities. Firstly, PME in watermelon juice might have only one existing form; secondly, the proportion of liable

PPO residual activity (%)

115

100 80 60 40 20

0 0 0

10

20

30

40

50

60

Time (min) Fig. 2. Inactivation of PPO of watermelon juice by HPCD treatment of different pressures at 50 °C. Error bars represent the standard deviation.

0

10

20

30

40

50

60

Time (min) Fig. 4. Inactivation of PME of watermelon juice by HPCD treatment of different pressures at 50 °C. Error bars represent the standard deviation.

116

Y. Liu et al. / Innovative Food Science and Emerging Technologies 13 (2012) 112–119

Table 1 Effect of HPCD treatment of different pressures at 50 °C on the quality parameters of watermelon juice. Pressure (MPa)

Time (min)

pH

TSS (°Brix)

L

A

b

△E

Total phenolic content (μg mL− 1)

Viscosity (mPa·s)

15 30 45 60 15 30 45 60 15 30 45 60 15 30 45 60

5.83 ± 0.12a 5.85 ± 0.08a 5.85 ± 0.05a 5.82 ± 0.05a 5.83 ± 0.07a 5.52 ± 0.10a 5.60 ± 0.11a 5.56 ± 0.06a 5.45 ± 0.10a 5.54 ± 0.18a 5.57 ± 0.13a 5.52 ± 0.16a 5.54 ± 0.07 a 5.51 ± 0.05a 5.58 ± 0.07a 5.60 ± 0.13a 5.51 ± 0.20a

9.55 ± 0.63a 9.53 ± 0.60a 9.56 ± 0.37a 9.61 ± 0.68a 9.54 ± 0.60a 9.16 ± 0.81a 9.81 ± 0.50a 9.90 ± 0.92a 9.23 ± 0.55a 9.87 ± 0.31a 9.19 ± 0.66a 9.18 ± 0.85a 9.93 ± 0.60 a 9.25 ± 0.21a 9.19 ± 0.25a 9.68 ± 0.17a 9.10 ± 0.45a

26.37 ± 0.35e 26.53 ± 0.67e 24.96 ± 0.25f 24.95 ± 0.32f 24.82 ± 0.17fg 29.21 ± 0.28abc 28.68 ± 0.57cd 28.84 ± 0.52bcd 28.97 ± 0.19bcd 28.83 ± 0.34bcd 29.50 ± 0.41ab 29.79 ± 0.08a 29.23 ± 0.11 abc 28.40 ± 0.19d 29.20 ± 0.16abc 29.24 ± 0.13abc 29.18 ± 0.18abc

18.80 ± 1.05a 18.83 ± 0.65a 18.62 ± 0.51a 17.59 ± 0.66cd 17.30 ± 0.72cd 16.22 ± 0.50cd 15.48 ± 0.11d 15.02 ± 0.26d 14.90 ± 0.35cd 16.26 ± 0.28b 17.44 ± 0.66cd 15.68 ± 0.21d 14.86 ± 0.28 bc 16.01 ± 0.04cd 15.56 ± 0.40cd 14.98 ± 0.02d 15.03 ± 0.14d

6.61 ± 0.33f 6.52 ± 0.09f 6.46 ± 0.23fg 6.24 ± 0.10fg 6.09 ± 0.08fg 7.42 ± 0.36bc 8.14 ± 0.00bcd 7.87 ± 0.30abc 8.23 ± 0.06bc 6.98 ± 0.11ef 8.83 ± 0.43a 6.83 ± 0.07ef 8.49 ± 0.18 cd 7.40 ± 0.11de 7.30 ± 0.28de 7.87 ± 0.11bcd 7.75 ± 0.07cd

0.00 0.18 1.43 1.90 2.22 3.91 4.32 4.68 4.95 3.55 4.06 4.62 5.22 3.53 4.35 4.94 4.84

16.17 ± 0.49a 16.17 ± 0.52a 16.15 ± 0.30a 16.19 ± 0.45a 16.16 ± 0.51a 16.37 ± 0.85a 16.06 ± 0.38a 16.02 ± 0.51a 16.09 ± 0.15a 16.09 ± 0.45a 16.37 ± 0.43a 16.16 ± 0.39a 16.28 ± 0.80 a 16.00 ± 0.29a 16.46 ± 0.27a 16.37 ± 0.51a 16.18 ± 0.33a

1.57 ± 0.09b 1.57 ± 0.05b 1.59 ± 0.05b 1.59 ± 0.11b 1.60 ± 0.07b 1.53 ± 0.05b 1.52 ± 0.04b 1.53 ± 0.11b 1.57 ± 0.08b 1.53 ± 0.05b 1.61 ± 0.05b 1.63 ± 0.07b 1.60 ± 0.05 b 1.82 ± 0.05a 1.93 ± 0.08a 1.89 ± 0.07a 1.86 ± 0.02a

Untreated Control

10

20

30

Value are means, n = 3. The different letters within the same column represent a significant difference (P b 0.05).

was relevant to CO2 solubility in watermelon juice. Except for 15 min, TA after treatment of 30 MPa was all higher than that of 10 MPa at other treatment times (P b 0.05), which attributed to more CO2 dissolving into the juice. TA and pH presented the different variation trends. It was because carbonic acid dissociated to H + ions could induce pH lowering. From the difference, we might conclude that CO2 existed as carbonic acid without being dissociated. Moreover, TA of watermelon juice treated by thermal of 95 °C for 1 min and control treatment also did not change significantly (P b 0.05). Similar results were also obtained in previous studies (Gui et al., 2006a; Kincal et al., 2006; Zhou et al., 2009). 3.4. Effect of HPCD and mild heat treatment on BD of watermelon juice The influence of the combination of HPCD and mild heat treatment on BD of watermelon juice is shown in Fig. 6. The initial BD of juice was 1.102. BD of the HPCD-treated watermelon juice decreased significantly (P b 0.05). BD of watermelon juice after HPCD and mild heat treatment decreased with pressure and treatment time increasing which dropped to 0.444 at 50 °C and 30 MPa for 60 min. Similarly, it decreased after the control treatment and reached to 0.876 at 50 °C for 60 min. In addition, BD of watermelon juice treated by thermal of 95 °C for 1 min was 0.791. Hence, the combination of HPCD and mild heat treatment was a more effective method to control browning of watermelon juice than the control and thermal treatment. Similarly, HPCD treatment resulted in a significant reduction of BD in cloudy apple juice during the storage as compared to the control (Gui et al., 1.2 1.0

TA ( mg L-1 )

and horseradish peroxide solution (Gui et al., 2006a; Gui et al., 2007; Zhou et al., 2009). The decrease of pH was caused by CO2 dissolving into juices or solutions, which further dissociated to bicarbonate, carbonate and H + ions. Hence, no change in the pH was observed in muscadine grape juice after the removal of CO2 by vacuum during the last stage of dense phase CO2 processing (Pozo-Insfran, Balaban, & Talcott, 2007). And pH did not change significantly in orange juice after HPCD treatment (Kincal et al., 2006). It was because the dissociation constants of carbonic acid and bicarbonate were pKa = 6.57 and pKa = 10.62, the low pH in the original orange juice resulted in the carbonic acid formed by CO2 dissolving into the juice difficultly being dissociated into H + ions (Damar & Balaban, 2006). Although pH of watermelon juice decreased after HPCD treatment, the slight variation did not affect the quality of the juice. pH of watermelon juice treated by thermal of 95 °C for 1 min was 5.85, which had no significant difference with the control. Similarly, it did not change after the control treatment of 50 °C. In addition, pH was an important factor associated with the enzyme inactivation. The residual activity of apple PME not subjected to HPCD lost about 21.2% of its original activity after pH adjustment from 7.5 to 5.0 (Zhi et al., 2008). Alternatively, the activity of purified PME (citrate–phosphate buffer, 6 mM) which optimum pH was 7.5 from apples (Golden Delicious) at pH 4.0 remained only 1% (Denès, Baron, & Drilleau, 2000). It was due to the fact that enzyme-bound arginine can easily interact with CO2 to form a bicarbonate–protein complex under a lower pH environment, which resulted in the loss of enzyme activity (Weder, 1984; Weder & Bokor, 1992). In addition, CO2 treatment resulted in changes in the secondary structures of the PPO by spectropolarimetric analysis (Chen et al., 1992). The reduction of horseradish peroxidase activity caused by supercritical CO2 treatment was related to the changes in the secondary and tertiary structures (Gui et al., 2006a). Similarly, the combined effect of HPCD and mild heat treatment to enzymes of watermelon juice leaded to the structure change of protein, which contributed to the inactivation of PPO, POD and PME.

0.8

Control 10 MPa 20 MPa 30 MPa

0.6 0.4

3.3. Effect of HPCD and mild heat treatment on TA of watermelon juice 0.2

The influence of the combination of HPCD and mild heat treatment on TA of watermelon juice is shown in Fig. 5. The initial TA of juice was 0.69 mg L − 1. After HPCD treatment, the TA of watermelon juice increased significantly to 0.80–1.05 mg L − 1 (P b 0.05). TA includes all the free acid in watermelon juice. Carbonic acid derived from CO2 dissolving into the juice induced the increase of TA, which

0.0 -10

0

10

20

30

40

50

60

70

Time (min) Fig. 5. Effect of HPCD treatment of different pressures at 50 °C on TA of watermelon juice. Error bars represent the standard deviation.

Y. Liu et al. / Innovative Food Science and Emerging Technologies 13 (2012) 112–119

1.2 1.0

Control 10 MPa 20 MPa 30 MPa

0.6 0.4 0.2 0.0 -10

0

10

20

30

40

50

60

70

3.7. Effect of HPCD and mild heat treatment on cloudiness of watermelon juice

Time (min) Fig. 6. Effect of HPCD treatment of different pressures at 50 °C on BD of watermelon juice. Error bars represent the standard deviation.

2006b). The BD of carrot juices after HPCD treatment all decreased significantly in contrast with the control (Zhou et al., 2009). The browning of watermelon juice happened at the low temperature, which implied that it might be induced by enzymes. PPO and POD in watermelon juice was closely related to the browning. HPCD treatment could inactivate PPO and POD activity effectively, and their residual activity decreased with pressure and treatment time increasing, which was consistent with BD variation. Hence, the combination of HPCD and mild heat treatment restrain browning of watermelon juice by inactivating PPO and POD activity. 3.5. Effect of HPCD and mild heat treatment on TSS of watermelon juice The influence of the combination of HPCD and mild heat treatment on TSS of watermelon juice is shown in Table 1. The initial TSS value of juice was 9.55 °Brix. After HPCD treatment, TSS showed fluctuations, but did not change significantly (P > 0.05). Similarly, TSS of watermelon juice treated by the thermal of 95 °C for 1 min and control treatment also unchanged significantly (P>0.05). Hence, HPCD, thermal and control treatment all had no effect on carbohydrate (e.g. glucose, fructose, and sucrose et al.) which were the main composition of TSS of watermelon juice. Similarly, HPCD (10, 20, and 30 MPa; 10, 30, 45, and 60 min; 25 °C) and heat treatment (90 °C for 1 min) also did not change TSS of carrot juice (Zhou et al., 2009). 3.6. Effect of HPCD and mild heat treatment on the lycopene content of watermelon juice The combined effect of HPCD and mild heat treatment on the lycopene content of watermelon juice is shown in Fig. 7. The initial lycopene content of juice was 62.56 μg mL− 1, it slightly decreased (P b 0.05) after HPCD treatment, but it did not present a certain regularity with

Lycopene content ( ug mL-1)

increasing pressure level and treatment time. On one hand, supercritical carbon dioxide could extract lycopene from food system, lycopene was carried by high pressure carbon dioxide and escaped from watermelon juice when HPCD treatment ended, which contributed to decrease of lycopene content (Ollanketo, Hartonen, Riekkola, Holm, & Hiltunen, 2001). On the other hand, the oxidative degradation of lycopene into aroma volatiles (e.g. noncyclic norisoprenoids and geranial) during HPCD treatment also resulted in the decrease (Park, Lee, & Park, 2002). In addition, lycopene content of juice treated by the control and thermal treatment of 95 °C for 1 min did not change significantly (P > 0.05).

The influence of the combination of HPCD and mild heat treatment on cloudiness of watermelon juice is shown in Fig. 8. The initial cloudiness of juice was 49.85 NTU. After HPCD treatment, the cloudiness of juice increased with pressure (P b 0.05), and increased with treatment time under 20 and 30 MPa. Firstly, PME inactivation could stabilize the system of juice and maintain the cloudiness. Secondly, it was attributed to the homogenization of high pressure on pectin in watermelon juice. High pressure and long treatment time could diminish pellets, which stabilized the juice system. Thirdly, portions of proteins have been found to be soluble and coagulable and to be involved with cloud instability. It was presented to occur through binding of heat-coagulated soluble proteins to colloidal cloud constituents which affected by pH (Shomer, 1988). Hence, HPCD treatment can improve stability of watermelon juice. Conversely, the cloudiness of juice treated by thermal of 95 °C for 1 min was 32.59 NTU, which decreased significantly (P b 0.05). And it decreased with treatment time and dropped to 27.32 NTU at 60 min (P b 0.05). It was because that lycopene aggregated and precipitated at the bottom of juice after high temperature treatment. Thus, thermal and control treatments impacted the organoleptic quality and reduced the stability of watermelon juice. 3.8. Effect of HPCD and mild heat treatment on the color of watermelon juice The influence of HPCD treatment on color of watermelon juice is shown in Table 1. The initial L⁎-, a⁎-, and b⁎-values of juice were 26.37, 18.80, and 6.61, respectively. After HPCD treatment, L⁎- and b⁎-values increased, a⁎-value decreased (P b 0.05). However, L⁎-, a⁎-, and b⁎-values did not show a certain regularity with pressure and treatment time increasing. BD decrease after HPCD treatment induced L⁎-value increase. Lycopene is the main source of red color of watermelon (Perkins-Veazie et al., 2001). HPCD treatment reducing lycopene content of watermelon juice resulted in a⁎-value decrease

70

160

60

140

50

Control 10 MPa 20 MPa 30 MPa

40 30 20 10 0 -10

Cloudiness (NTU)

BD

0.8

117

120 100

Control 10 MPa 20 MPa 30 MPa

80 60 40 20

0

10

20

30

40

50

60

70

Time (min) Fig. 7. Effect of HPCD treatment of different pressures at 50 °C on lycopene content of watermelon juice. Error bars represent the standard deviation.

0 -10

0

10

20

30

40

50

60

70

Time (min) Fig. 8. Effect of HPCD treatment of different pressures at 50 °C on cloudiness of watermelon juice. Error bars represent the standard deviation.

Y. Liu et al. / Innovative Food Science and Emerging Technologies 13 (2012) 112–119

and b⁎-value increase. Results showed that the regularity of a⁎-value with pressure and treatment time is in accordance with that of lycopene content. BD of juice decreased with pressure and treatment time, but L⁎-value had no similar regularity, which implied that BD was not the only factor to affect L⁎-value. L⁎-value of carrot and orange juice increased and a⁎-value decreased significantly after HPCD treatment (Kincal et al., 2006; Park et al., 2002). In general, L⁎-value presented a certain relationship with cloudiness because reflected light was affected by cloudiness under reflectance mode. However, L⁎-value had no correlation with cloudiness in this study, suggesting that L⁎-value was even more influenced by lycopene loss after HPCD treatment. For the b⁎-value, it rose in carrot juice but exhibited no significant change in orange juice (Kincal et al., 2006; Zhou et al., 2009). In addition, L⁎-, a⁎-, and b⁎-values of watermelon juice treated by thermal of 95 °C for 1 min was 27.25, 18.73, and 6.77, respectively. Compared to the untreated juice, L⁎-value increased (P b 0.05), a⁎-, and b⁎-values did not change (P > 0.05). However, L⁎-, a⁎-, and b⁎-value all slightly decreased after control treatment. The decrease of L⁎-value might be due to the great residual activity of PPO and POD which could lead to browning of juice, and the decrease of a⁎- and b⁎-values could be contributed by oxidative cleavage of carotene after prolonged mild heat treatment. △E of watermelon juice was calculated according to Eq. 2. A noticeable difference can be visualized between two colors when they differ by △E > 2–3.5 (Krapfenbauer, Kinner, Gossinger, Schonlechner, & Berghofer, 2006). The △E values of thermal and all HPCD treated juices were greater than 2, indicating that thermal and HPCD led to a visible color difference of watermelon juice. It was similar to the results of the effect of heat and HPCD treatment on the color of carrot juice (Zhou et al., 2009). However, △E values of control treated juices were all smaller than 3.5, suggesting that control treatment could maintain the color of watermelon juice better. 3.9. Effect of HPCD and mild heat treatment on total phenolic content of watermelon juice The influence of the combination of HPCD and mild heat treatment on total phenolic content of watermelon juice is shown in Table 1. The initial total phenolic content of juice was 16.17 μg mL− 1. After HPCD treatment, total phenolic content did not change significantly with pressure and treatment time (P > 0.05). Supercritical CO2 can extract total phenolic from the food system, but the solubility of phenolic in CO2 is so low that it is difficult to extract phenolic from watermelon juice using CO2 alone (Van Leer & Paulaitis, 1980). Hence, HPCD has little effect on total phenolic content of watermelon juice without cosolvent. Meanwhile, total phenolic content of juice treated by thermal of 95 °C for 1 min was 16.15 μg mL− 1, which had no significant difference with that of the control (P > 0.05). Similar result was shown in the control treatment. 3.10. Effect of HPCD and mild heat treatment on viscosity of watermelon juice The influence of HPCD treatment on viscosity of watermelon juice is shown in Fig. 9 and Table 1. It was shown in Fig. 9 that the viscosity of the control, thermal treatment, and HPCD of 10, 20, and 30 MPa for 60 min treated juice changed with a shear rate of 0.1–100 s − 1. Usually, the shear rate of human tongue while drinking was between 30 and40 s − 1 approximately (Huang, Fang, & Chen, 2011). So the viscosity at 31.62 s − 1 of HPCD treatment of different pressures and treatment times at 50 °C was recorded and listed in Table 1. The initial viscosity of juice was 1.57 mPa·s. The viscosity of juice did not change significantly after HPCD treatment of 10 and 20 MPa (P > 0.05), while it increased after treatment of 30 MPa (P b 0.05). And the viscosity among different treatment times under the same pressure had no significant difference (P > 0.05). Similarly, it did not change significantly

1.000 CK Thermal 10MPa 20MPa 30MPa

0.1000

viscosity (Pa.s)

118

0.01000

1.000E-3

1.000E-4 0.01000

0.1000

1.000

10.00

100.0

1000

shear rate (1/s) Fig. 9. Effect of thermal and HPCD treatment of different pressures on viscosity of watermelon juice.

after control treatment (P > 0.05). After thermal treatment of 95 °C for 1 min, the viscosity of watermelon juice was 3.39 mPa·s, which was higher than that of the control and HPCD treated juice significantly (P b 0.05). Hence, HPCD has little effect on the viscosity of watermelon juice as compared to thermal treatment. 4. Conclusions The results showed the effect of HPCD and mild heat treatment on quality of watermelon juice as compared to the control and thermal treatments. The activity of PPO, POD, and PME decreased with pressure and treatment time after HPCD and mild treatment. BD decreased with pressure and treatment time significantly; pH value and lycopene content of HPCD-treated watermelon juice slightly decreased; the cloudiness and TA increased with pressure and treatment time; the viscosity of 30 MPa at 31.62 1 s − 1 increased significantly as compared to that of control and HPCD treated juice of 10 and 20 MPa; TSS and total phenolic content were stable; L⁎and b⁎-values increased, a⁎-value decreased, △E was higher than 3.5, which implied that color changed significantly. From the results, the watermelon juice after HPCD treatment of 30 MPa and 60 min could obtain the best quality. However, out of cost consideration, HPCD treatment of 30 MPa and 15 min was suggested to be applied to watermelon juice processing. The quality parameters such as pH, lycopene content, and color after control treatment were stable, but BD and residual activities of PPO, POD, and PME were far higher than HPCD treatment which could affect the shelf life and quality of watermelon juice greatly during storage. Cloud stability decreased after control treatment which was an important parameter to influence appearance of juice. Overall, HPCD treatment has greater advantages to maintain the quality of watermelon juice in our study. Acknowledgement The authors gratefully acknowledge the System of Modern Agricultural Industry Technology and National High Technology Research & Development Program of China (2007AA100405). References Arreola, A. G., Balaban, M. O., Marshall, M. R., Replow, A. J., Wei, C. I., & Cornell, J. A. (1991). Supercritical carbon dioxide effects on some quality attributes of single strength orange juice. Journal of Food Science, 56, 1030–1033.

Y. Liu et al. / Innovative Food Science and Emerging Technologies 13 (2012) 112–119 Chen, J. S., Balaban, M., Wei, C. -i., Marshall, M. R., & Hsu, W. Y. (1992). Inactivation of polyphenol oxidase by high-pressure carbon dioxide. Journal of Agricultural and Food Chemistry, 40, 2345–2349. Corwin, H., & Shellhammer, T. H. (2002). Combined carbon dioxide and high pressure inactivation of pectin methylesterase, polyphenol oxidase, Lactobacillus plantarum and Escherichia coli. Journal of Food Science, 67, 697–701. Damar, S., & Balaban, M. O. (2005). Cold pasteurization of coconut water with a dense phase CO2 system. IFT Annual Meeting Book of Abstracts (pp. 1). Institute of Food Technologists: New Orleans, La. Damar, S., & Balaban, M. O. (2006). Review of dense phase CO2 technology: Microbial and enzyme inactivation, and effects on food quality. Journal of Food Science, 71, 1–11. Denès, J. M., Baron, A., & Drilleau, J. F. (2000). Purification, properties and heat inactivation of pectin methylesterase from apple (cv Golden Delicious). Journal of the Science of Food and Agriculture, 80, 1503–1509. Duangmal, K., & Apenten, R. K. O. (1999). A comparative study of polyphenoloxidases from taro (Colocasia esculenta) and potato (Solanum tuberosum var. Romano). Food Chemistry, 64, 351–359. Edwards, A. J., Vinyard, B. T., Wiley, E. R., Brown, E. D., Collins, J. K., Perkins-Veazie, P., Baker, R. A., & Clevidence, B. A. (2003). Consumption of watermelon juice increases plasma concentrations of lycopene and β-carotene in humans. Nutrition and Metabolism, 133, 1043–1050. Erkmen, O., & Karaman, H. (2001). Kinetic studies on the high pressure carbon dioxide inactivation of Salmonella typhimurium. Journal of Food Engineering, 50, 25–28. Folkes, G. (2004). Pasteurization of beer by a continuous dense-phase CO2 system [PhD dissertation]. Gainesville, Florida: Univiersity of Florida. 110 p. Fortea, M. I., López-Miranda, S., Serrano-Martínez, A., Carreño, J., & Núñez-Delicado, E. (2009). Kinetic characterisation and thermal inactivation study of polyphenol oxidase and peroxidase from table grape (Crimson Seedless). Food Chemistry, 113, 1008–1014. Fraser, P. D., & Bramley, P. M. (2004). The biosynthesis and nutritional uses of carotenoids. Progress in Lipid Research, 43, 228–265. Garcia-Gonzalez, L., Geeraerd, A. H., Spilimbergo, S., Elst, K., Van Ginneken, L., Debevere, J., Van Impe, J. F., & Devlieghere, F. (2007). High pressure carbon dioxide inactivation of microorganisms in foods: The past, the present and the future. International Journal of Food Microbiology, 117, 1–28. Gil, M. I., Aguayo, E., & Kaer, A. A. (2006). Quality changes and nutrient retention in fresh-cut versus whole fruits during storage. Journal of Agricultural and Food Chemistry, 54, 4284–4296. Giovannucci, E. (2002). A review of epidemiologic studies of tomatoes, lycopene, and prostate cancer. Experimental Biology and Medicine, 227, 852–859. Gui, F. Q., Chen, F., Wu, J. H., Wang, Z. F., Liao, X. J., & Hu, X. S. (2006). Inactivation and structural change of horseradish peroxidase treated with supercritical carbon dioxide. Food Chemistry, 97, 480–489. Gui, F. Q., Wu, J. H., Chen, F., Liao, X. J., Hu, X. S., Zhang, Z. H., & Wang, Z. F. (2006). Change of polyphenol oxidase activity, color, and browning degree during storage of cloudy apple juice treated by supercritical carbon dioxide. European Food Research and Technology, 223, 427–432. Gui, F. Q., Wu, J. H., Chen, F., Liao, X. J., Hu, X. S., Zhang, Z. H., & Wang, Z. F. (2007). Inactivation of polyphenol oxidases in cloudy apple juice exposed to supercritical carbon dioxide. Food Chemistry, 100, 1678–1685. Hong, S. I., & Pyun, Y. R. (1999). Inactivation kinetics of Lactobacillus plantarum by high pressure CO2. Journal of Food Science, 64(4), 728–733. Hong, S. I., & Pyun, Y. R. (2001). Membrane damage and enzyme inactivation of Lactobacillus plantarum by high pressure CO2 treatment. International Journal of Food Microbiology, 63, 19–28. Huang, J. Q., Fang, T., & Chen, J. Q. (2011). Sterilization process and rheological properties of canned abalone with sauce. Food Science, 32, 67–71. Isenschmid, A., Marison, I. W., & Stockar, U. V. (1995). The influence of pressure and temperature of compressed CO2 on the survival of yeast cells. Journal of Biotechnology, 39, 229–237. Kim, Y. S., Park, S. J., Cho, Y. H., & Park, J. (2001). Effect of combined treatment of high hydrostatic pressure and mild heat on the quality of carrot juice. Journal of Food Science, 66, 1355–1360. Kimball, D. A. (1991). Juice cloud, color of citrus juices and citrus microbiology. Citrus processing-Quality control and technology (pp. 117–125). New York: Van Nostrand Reinhold. Kincal, D., Hill, W. S., Balaban, M., Portier, K. M., Sims, C. A., Wei, C. I., & Marshall, M. R. (2006). A continuous high-pressure carbon dioxide system for cloud and quality retention in orange juice. Journal of Food Science, 71, 338–344. Krapfenbauer, G., Kinner, M., Gossinger, M., Schonlechner, R., & Berghofer, E. (2006). Effect of thermal treatment on the quality of cloudy apple juice. Journal of Agriculture and Food Chemistry, 54, 5453–5460. Lambert, R. J. W. (2003). A model for the thermal inactivation of micro-organisms. Journal of Applied Microbiology, 95, 500–507.

119

Liao, H. M., Hu, X. S., Liao, X. J., Chen, F., & Wu, J. H. (2007). Inactivation of Escherichia coli inoculated into cloudy apple juice exposed to dense phase carbon dioxide. International Journal of Food Microbiology, 118, 126–131. Lin, H. M., Yang, Z. Y., & Chen, L. F. (1993). Inactivation of Leuconostoc dextranicum with CO2 under pressure. Chemical Engineering Journal and the Biochemical Engineering Journal, 52, 29–34. Liu, X., Gao, Y. X., Peng, X. T., Yang, B., Xu, H. G., & Zhao, J. (2008). Inactivation of peroxidase and polyphenol oxidase in red beet (Beta vulgaris L.) extract with high pressure carbon dioxide. Innovative Food Science and Emerging Technologies, 9, 24–31. Liu, Y., Zhang, C., Zhao, X. Y., Ma, Y., Li, W., Liao, X. J., & Hu, X. S. (2010). Inactivation of polyphenol oxidase from frozen red raspberry (Rubus idaeus L.) by high pressure carbon dioxide treatment. International Journal of Food Science and Technology, 45, 800–806. Mann, A., Kiefer, M., & Leuenberger, H. (2001). Thermal sterilization of heat-sensitive products using high-temperature short-time sterilization. Journal of Pharmaceutical Sciences, 90, 275–287. Ollanketo, M., Hartonen, K., Riekkola, M. L., Holm, Y., & Hiltunen, R. (2001). Supercritical carbon dioxide extraction of lycopene in tomato skins. European Food Research and Technology, 212, 561–565. Onsa, G. H., Saari, N. B., Selamat, B., & Baker, J. (2004). Purification and characterization of membrane-bound peroxidases form Metroxylon sagu. Food Chemistry, 85, 365–376. Park, S. J., Lee, J. I., & Park, J. (2002). Effects of a combined process of high-pressure carbon dioxide and high hydrostatic pressure on the quality of carrot juice. Journal of Food Science, 67, 1827–1834. Perkins-Veazie, P., Collins, J. K., Pair, S. D., & Roberts, W. (2001). Lycopene content differs among red-fleshed watermelon cultivars. Journal of the Science of Food and Agriculture, 81, 983–987. Pozo-Insfran, D. d., Balaban, M. O., & Talcott, S. T. (2007). Inactivation of polyphenol oxidase in muscadine grape juice by dense phase-CO2 processing. Food Research International, 40, 894–899. Rattanathanalerk, M., Chiewchan, N., & Srichumpoung, W. (2005). Effect of thermal processing on the quality loss of pineapple juice. Journal of Food Engineering, 66, 259–265. Reiter, M., Stuparic, M., Neidhart, S., & Carle, R. (2003). The role of process technology in carrot juice cloud stability. Swiss Society of Food Science and Technology, 36, 165–172. Rodrigo, D., Arranz, J. I., Koch, S., Frigola, A., Rodrigo, M. C., Esteve, M. J., et al. (2003). Physicochemical characteristics and quality of refrigerated Spanish orange–carrot juices and influence of storage conditions. Journal of Food Science, 68, 2111–2116. Roig, M. G., Bello, J. F., & Rivera, Z. S. (1999). Studies on the occurrence of nonenzymatic browning during storage of citrus juice. Food Research International, 32, 609–619. Sadler, G., Davis, J., & Dezman, D. (1990). Rapid extraction of lycopene and β-carotene from reconstituted tomato paste and pink grapefruit homogenates. Journal of Food Science, 55, 1460–1465. Shomer, I. (1988). Protein self-encapsulation: a mechanism involved with colloidal flocculation in citrus fruit extract. Journal of the Science of Food and Agriculture, 42, 55–65. Singleton, V. L., & Rossi, J. A., Jr. (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16, 144–158. Spilimbergo, S., Elvassore, N., & Bertucco, A. (2003). Inactivation of microorganisms by supercritical CO2 in a semi-continuous process. Italian Journal of Food Science, 1(15), 115–124. Van Leer, R. A., & Paulaitis, M. E. (1980). Solubilities of phenol and chlorinated phenols in supercritical carbon dioxide. Journal of Chemical and Engineering Data, 25(3), 257–259. Wang, H. Y., Hu, X. S., Chen, F., Wu, J. H., Zhang, Z. H., & Liao, X. J. (2006). Kinetic analysis of non-enzymatic browning in carrot juice concentrate during storage. European Food Research and Technology, 223, 282–289. Weder, J. K. P. (1984). Studies on proteins and amino acids exposed to supercritical carbon dioxide extraction conditions. Food Chemistry, 15, 175–190. Weder, J. K. P., & Bokor, M. V. (1992). Effect of supercritical carbon dioxide on arginine. Food Chemistry, 44, 287–290. Zanoni, B., Pagliarini, E., Giovanelli, G., & Lavelli, V. (2003). Modelling the effects of thermal sterilization on the quality of tomato puree. Journal of Food Engineering, 56, 203–206. Zhi, X., Zhang, Y., Hu, X. S., Wu, J. H., & Liao, X. J. (2008). Inactivation of apple pectin methylesterase induced by dense phase carbon dioxide. Journal of Agricultural and Food Chemistry, 56(13), 5394–5400. Zhou, L. Y., Wang, Y. Y., Hu, X. S., Wu, J. H., & Liao, X. J. (2009). Effect of high pressure carbon dioxide on the quality of carrot juice. Innovative Food Science and Emerging Technologies, 10, 321–327.