Comparative Study On Cloudy Apple Juice Qualities From Apple Slices

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Innovative Food Science and Emerging Technologies 11 (2010) 91–97 Contents lists available at ScienceDirect Innovative Food Science and Emerging Technologies j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i f s e t Comparative study on cloudy apple juice qualities from apple slices treated by high pressure carbon dioxide and mild heat Shuang Niu 1 , Zenghui Xu 1, Yudan Fang 1, Liyun Zhang, Yingjie Yang, Xiaojun Liao ⁎, Xiaosong Hu College of Food Science and Nutritional Engineering, China Agricultural University, Key Laboratory of Fruit and Vegetable Processing, Ministry of Agriculture, Engineering Research Center for Fruit and Vegetable Processing, Ministry of Education, Beijing 100083, China a r t i c l e i n f o Article history: Received 21 February 2009 Accepted 13 September 2009 Editor Proof Receive Date 9 October 2009 Keywords: Cloudy apple juice Apple slices High pressure carbon dioxide Mild heat Polyphenol oxidase Pectin methylesterase a b s t r a c t Qualities of cloudy apple juices from apple slices treated by high pressure carbon dioxide (HPCD) and mild heat (MH) were evaluated. Temperatures were from 25 to 65 °C, time 20 min, and pressure 20 MPa. Polyphenol oxidase (PPO) was completely inactivated by HPCD and its minimal residual activity (RA) by MH at 65 °C was 38.6%. RA of pectin methylesterase (PME) with HPCD was significantly lower than MH and its minimum was 18%. L value of cloudy apple juice from HPCD-treated apple slices was significantly greater than that from MH-treated apple slices, however, b value, browning degree (BD) and turbidity were lower. And no differences in a value, total soluble solids, pH and conductivity were observed. After 7-day storage at 4 °C, HPCD caused no BD alteration but a significant turbidity loss. MH increased BD at 55 and 65 °C, and led to turbidity loss from 35 to 65 °C. The turbidity was not well related to RA of PME. Industrial relevance: Cloudy apple juice is one of the popular fruit juices, and it requires strict processing treatment conditions to protect its quality, especially to prevent enzymatic browning and cloud loss. HPCD is one promising novel non-thermal technique and is likely to replace or partially substitute thermal processes. This study analyzed the effect of HPCD as a pretreatment means on qualities of cloudy apple juice, including inactivating enzymes which are crucial to quality control. Available data provided in this study will benefit the fruit juice industry. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction Apple juice is commonly considered to be a clear juice, but there is a growing market for natural cloudy apple juice (Braun, 2003). Nagel (1992) describes a cloudy apple juice as a light, whitish yellow juice showing definite cloudiness, which shows no sedimentation, is full bodied and juicy, but has no astringent or bitter taste. The main problem with cloudy apple juice production is the assurance of color and cloud stability (Genovses, Elustondo & Lozano, 1997), which are related to enzyme activities. The discoloring of cloudy apple juice results from enzymatic browning, which is caused by the action of polyphenol oxidase (PPO, EC1. 14. 18. 1) catalyzing oxidation of phenolic compounds (Joslyn & Ponting, 1951). The control of endogenous pectin methylesterase (PME, EC 3. 1. 1. 11) activity is crucial for the cloud stability of cloudy juices (Assis Lima & Oliviera, 2001). Undesired clarification is strongly influenced by demethylation of pectin by PME yielding acidic pectin with a lower degree of esterification, which can cross-link with polyvalent cations such as Ca2+ to form insoluble pectate precipitates, or becomes a target for pectin-degrading polygalacturonases (PG) (Cameron, Niedz & Groh- ⁎ Corresponding author. Tel.: +86 10 62737434 602; fax: +86 10 62737434 604. E-mail address: [email protected] (X. Liao). 1 They were equally the first authors. 1466-8564/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2009.09.002 mann, 1994; Ly Nguyen, Van Loey, Smout, Verlent, Duvetter & Hendrickx, 2003; Damar & Balaban, 2006). Traditionally, thermal process has been used to ensure the safety of food against pathogenic microorganisms. Thermal energy, however, inevitably leads to destruction of heat sensitive nutrients, texture, color, and flavor (Balny, Hayashi, Heremans & Masson, 1992). On the other hand, high pressure carbon dioxide (HPCD) has been used as one of nonthermal preservation techniques, without exposing foods to adverse effects of heat and retaining their fresh-like physical, nutritional, and sensory qualities(Damar & Balaban, 2006). Many studies demonstrated that HPCD treatments can effectively inactivate microorganisms in both artificial broths and real food products at moderate pressure and temperature (Garcia-Gonzalez et al., 2007). Meanwhile, the effect of HPCD on inactivation of PPO (Chen, Balaban, Wei, Marshall & Hsu, 1992; PozoInsfran, Balaban & Talcott, 2007; Gui et al., 2007; Liu et al., 2008) and PME (Arreola et al., 1991; Park, Lee, & Park, 2002; Kincal et al., 2006; Zhi, Zhang, Hu, Wu & Liao, 2008; Zhou, Wu, et al., 2009) were reported. Based on these studies, it is reasoned that HPCD technique may be regarded as a pretreatment of fruit or vegetable slices for juice processing. To our knowledge, HPCD is directly applied to juice or buffer systems. There is no investigation on effect of HPCD-pretreated fruit slices on juice up to date. In our preliminary study, PPO could be completely inactivated in apple slices and the quality of cloudy apple juice extracted from HPCD-treated slices was better than that of the juice HPCD directly processed. There was no finding that HPCD could 92 S. Niu et al. / Innovative Food Science and Emerging Technologies 11 (2010) 91–97 2.5. Storage study completely inactivate PPO in other literatures up to date. Gui et al. (2007) showed that PPO could only be inactivated up to 61.5% in HPCDtreated cloudy apple juice. Moreover, there are a limited number of published studies regarding the effects of HPCD on the quality of foods (Damar & Balaban, 2006). Therefore, it is worthwhile investigating the quality change of cloudy apple juice from HPCD-pretreated slices. The purpose of this study is to analyze the effect of HPCD as a pretreatment on the juice qualities, including inactivation of PPO and PME, and other quality indicators such as color, turbidity and browning, and to make a comparison with mild heat (MH). To study the effect of HPCD and MH on cloudy apple juice after 7day storage at 4 °C in a refrigerator, the alteration of the visual color, BD and turbidity of cloudy apple juice was analyzed. One group of 10 mL plastic test tubes (Beijing Bomex Co., Beijing, China) was halffilled with cloudy apple juice for BD analysis and visual color images were taken using a digital camera (EX-S600, Casio electronic Inc., China). The other group of 7 ml plastic test tubes was fully-filled with cloudy apple juice for turbidity analysis. 2. Materials and methods 2.6. Methods 2.1. Preparation of cloudy apple juice 2.6.1. PPO activity assay The activity of PPO was assayed by a spectrophotometric method (Sánchez-Ferrer Bru, Cabanes & Garcia-Carmona, 1988) with some modifications. Catechol was chosen as the substrate, and 0.1 M catechol substrate solution was prepared with 0.1 M phosphate buffer (pH 6.5). The assay was performed for all samples by adding 100 μL juice into 2.9 mL substrate solution. The increase in absorbance at 420 nm was monitored at intervals of 0.1 s− 1 immediately after incubation with a Cary 50 spectrophotometer (Varian Co. Ltd., California, USA), which was equipped with a peltier thermo-statted cell holder, a water pump (Varian Co. Ltd., California, USA) to keep temperature at 20 ± 0.1 °C and an in-built electromagnetic stirring to mix up the substrate and juice. Prior to measurement, a preequilibrium at 20 °C of the substrate solution as well as the juice by the peltier thermo-statted cell holder is obtained. The slope of the very first linear part of the reaction curve was taken as the PPO specific activity (Abs/min). The RA of PPO was estimated with the following equation. Apples (Fuji) at commercial maturity were purchased from a local market. After being washed, apples were cut into about 35× 25× 5 mm slices, then immersed in about 0.2% L-ascorbic acid solution (Aoboxing biotechnical Co, Beijing, China) to avoid undesirable enzymatic browning during the processing and drained quickly. For the control group, 300 g apple slices in each experiment were weighted by an EY 300 A electronic balance (A and D Co., Ltd, Tokyo, Japan), sealed in a plastic bag and then left for 20 min at room temperature (about 20 °C). For MH or HPCD groups, 300 g apple slices in each experiment were packed in a sealed plastic bag for MH treatment or nylon bag for HPCD treatment, respectively. After MH or HPCD treatments, apple slices were placed into a pulper (Beimei electrical appliance Co., Zhuhai, China), followed by adding 30 g 0.1% L-ascorbic acid solution and then pulped for 1 min. Apple pulp was packed by a 4 layer cheese cloth and juiced manually, then cloudy apple juice was centrifuged in 543 ×g for 5 min (GL-166-A, Shanghai Anting Scientific Equipment Factory, Shanghai, China). After the resulting cloudy apple juice was accomplished, the indices of qualities were determined immediately. Residual activity = PPO specific activity after HPCD or MH treatment PPO specific activity in the control ð1Þ 2.2. HPCD treatment system The diagram of HPCD system was described by Liao, Hu, Chen, Wu and Liao (2007). 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 processing HPCD processing was conducted using 20 MPa at 25, 35, 45, 55 and 65 °C for 20 min, respectively. For each experiment, 300 g apple slices packed in a nylon bag were placed in the reactor vessel which had been preheated to the experimental temperature. After the equilibrium temperature, it was pressurized by CO2 for about 2.5 min until the pressure reached 20 MPa, then apple slices in the vessel were held at constant pressure and temperature during HPCD treatment. After 20 min, the vessel was slowly depressurized in 1.5–2 min, the temperature reduction of apple slices was about 8–12 °C due to Joule–Thomson cooling effect. Following HPCD treatment, apple slices were processed immediately into cloudy apple juice. 2.4. MH processing To examine the effect of temperatures alone under atmospheric pressure on cloudy apple juice quality, MH processing was conducted at 25, 35, 45, 55 and 65 °C for 20 min, respectively. Apple slices placed in a sealed package were heated in a water bath at these fixed temperatures. The treatment time was recorded when temperature inside apple slices measured by digital thermometer (JM222, Living-Jinming Ltd., China) reached the desired one. Following MH treatment, apple slices were processed immediately into cloudy apple juice. 2.6.2. PME assay The activity of PME was measured at pH 7.5 and 30 °C according to the method proposed by Kimball (1991), which was based on carboxyl group titration. 5 mL cloudy apple juice was mixed with 20 mL of 1% apple pectin (DE 70–75%, Andre Co., Shandong, China) containing 0.1 M NaCl at 30 °C and incubated at 30 °C. The mixture solution was adjusted to pH 7.0 with 2.0 N NaOH, and then the pH of the solution was readjusted to pH 7.5 with 0.05 N NaOH. After the pH reached 7.5, 0.05 mL of 0.05 N NaOH was added. The time required for the solution's pH to return to 7.5 was measured. PME activity (A) expressed in pectin methylesterase units (PMEU) was calculated by the following. A= ½NaOHVNaOH Vsample t ′ ð2Þ where [NaOH] was NaOH concentration (0.05 N), VNaOH was the volume of NaOH used (0.05 mL), Vsample was the volume of sample used(=5 mL cloudy apple juice), and t′ was the time (in minutes) needed for pH to return to 7.5 after the addition of NaOH Residual Activity ðRAÞ = A A0 ð3Þ where A0 represented the mean activity of PME in the control, and A represented the mean activity of PME after HPCD or MH treatment. S. Niu et al. / Innovative Food Science and Emerging Technologies 11 (2010) 91–97 93 2.6.3. Juice yield measurement The weight of cloudy apple juice was measured after being pressed. Juice yield was expressed as followed. Juice Yield ð%Þ = W−W′ × 100% W0 ð4Þ where W was weight of cloudy apple juice; W′ was weight of 0.1% L-ascorbic acid solution added when pulping (equal to 30 g), W0 was weight of AS. 2.6.4. Color assessment Color assessment was conducted at an ambient temperature (20 ± 1 °C) using a Color Difference Meter (SC-80, Kangguang Co., Beijing, China) in the reflectance mode. Hunter L, a, and b values of cloudy apple juice were measured. 2.6.5. Determination of browning degree (BD) The BD of cloudy apple juice was analyzed using a spectrophotometric method described by Roig Bello, Rivera and Kennedy (1999). Cloudy apple juice was centrifuged with a refrigerated Centrifuge (GL166-A, Shanghai Anting Scientific Equipment Factory, Shanghai, China) at 9000 ×g at 4 °C for 30 min, then passed through a 0.45 μm cellulose nitrate membrane (Beijing Bomex Co., Beijing, China). The BD was determined by measuring the A (absorbance at 420 nm) value using a UV-762 spectrophotometer (T6, PG General, Beijing, China) at an ambient temperature (20 ± 1 °C) with a 1 cm pathlength cell. 2.6.6. Turbidity determination The turbidity of cloudy apple juice was measured at an ambient temperature (20 ± 1 °C) using the method proposed by Reiter, Stuparic, Neidhart and Carle (2003) with a digital photoelectrical turbidimeter (WGZ-200, Shanke Instrument Factory, Shanghai, China) using 5 mm cuvette at color correction mode. Cloudy apple juice was diluted in 1/20 with distilled water. Turbidity was expressed in nephelometric turbidity units (NTU). 2.6.7. Determination of total soluble solids (TSS) The TSS of cloudy apple juice was determined as oBrix at an ambient temperature (20 ± 1 °C) using WAY-2S digital Abbe Refraction meter (Shanghai Precision and Scientific Instrument Co., Shanghai, China). 2.6.8. pH determination The pH measurement was carried out at an ambient temperature (20 ± 1 °C) using digital Thermo Orion 555 A pH meter (Thermo Fisher Scientific Inc, MA, USA). The meter was calibrated with commercial buffer solutions at pH 6.8 and 4.0. 10 mL cloudy apple juice was inserted with a pH electrode (Thermo Orion Ross 9103BN, MA, USA) and pH was recorded after stabilization. Fig. 1. RA of PPO in cloudy apple juice from HPCD- and MH-treated apple slices as a function of treatment temperature for 20 min. All data were the means ± SD, n = 4. 3. Result and discussion 3.1. Inactivation of HPCD and MH on PPO and PME in cloudy apple juice The RA of PPO and PME in cloudy apple juice was monitored and shown in Figs. 1 and 2. As compared with the control cloudy apple juice, PPO in the cloudy apple juice from MH-treated apple slices was activated at 25, 35 and 45 °C and the RA of PPO increased by 21.8–50.8%, but the RA of PPO significantly decreased by 33.6% at 55 °C and 61.4% at 65 °C. The activation and inactivation of PPO depending on temperatures were reported in earlier studies. Soysal (2008) found that apple PPO in phosphate buffer was activated by approximately 10% and 5% at 45 and 55 °C for 20 min, respectively. In higher plants, PPO occurs in various isoforms such as immature, mature latent and active forms (Seo, Sharma & Sharma, 2003).The observed increase in activity of apple PPO by heating could be due to a releasing of latent PPO (Yemenicioglu, Ozkan & Cemeroglu, 1997; Soysal, 2008). However, Gui et al. (2007) found a decrease of PPO activity in MH-treated cloudy apple juice as increasing temperatures from 35 to 55 °C. Rapeanu, Van Loey, Smout and Hendricks (2006) reported that approximately 90% of PPO original activity in grape must still exist before 50 °C while 50% of PPO activity was lost at 60 °C. Varietal difference in enzyme stability is a well-known phenomenon, the activity and properties of PPO depended on the source, variety and environmental and physicochemical conditions such 2.6.9. Conductivity determination The conductivity of cloudy apple juice was measured at an ambient temperature (20 ± 1 °C) by a conductivity meter (EC 215, Hanna Instrument Inc., Rhode Island, USA). The meter was calibrated with conductivity calibration solution (HI7039, Hanna Instrument Hungary Kft., Hungary) to 5.0 ms/cm. 2.7. Statistical analysis Analysis of variance (ANOVA and paired t tests were accomplished with the software Microcal Origin 7.5 (Microcal Software, Inc., Northampton, USA). They were performed for all experimental run, to determine the significance at 95% confidence. All experiments were performed in quadruplicate. Fig. 2. RA of PME in cloudy apple juice from HPCD- and MH-treated apple slices as a function of treatment temperature for 20 min. All data were the means ± SD, n = 4. 94 S. Niu et al. / Innovative Food Science and Emerging Technologies 11 (2010) 91–97 as pH and temperature (Soysal, 2008). As shown in Fig. 1, the RA of PPO in cloudy apple juice from HPCD-treated apple slices was not found, indicating that PPO in apple slices was completely inactivated by HPCD independently of the temperatures applied. Changes in PPO activity by HPCD could be attributed to many causes such as pH lowering, conformational changes of PPO secondary structure and inhibitory effect of molecular CO2 (Damar & Balaban, 2006). Chen et al. (1992) investigated changes in PPO activity associated with different high pressure CO2 conditions and concluded that pressure-induced inactivation was caused by changes in enzyme secondary structure. The inactivation of horseradish peroxidase (Gui, Wang, et al., 2006), PME (Zhou, Wu, et al., 2009), and lipoxygenase (Liao et al., 2009) by HPCD was related to the change of secondary and/or tertiary structure. To our knowledge, the inactivation of PPO in HPCD-treated juices, buffers and extracts is previously reported. Chen et al. (1992) found that purified Florida spiny lobster, brown shrimp, and potato PPOs after HPCD at 5.8 MPa and 43 °C for 1 min retained only 2, 22, and 45% of the original activity, respectively. PPO in carrot juice lost 61% of its original activity after HPCD at 4.9 MPa and 5 °C for 10 min (Park et al., 2002). Pozo-Insfran et al. (2007) reported that HPCD at 27.6 MPa and 30 °C for 6.25 min leads to 75% reduction in PPO activity in muscadine grape juice. Gui et al. (2007) found that the maximum reduction of PPO activity in cloudy apple juice reached more than 60% by HPCD at 30 MPa and 55 °C for 60 min. Liu et al. (2008) observed that HPCD at 22.5 MPa and 55 °C for 60 min resulted in an approximate 95% reduction of PPO activity in red beet extract. The inactivation level of PPO in these studies is affected by PPO sources, maturity, matrix and HPCD treatment conditions. PPO from potato was more resistant to HPCD than that from spiny lobster and shrimp (Chen et al., 1992). Gui et al. (2007) proposed that higher temperature, higher pressure and longer treatment time during HPCD result in more reduction of apple PPO activity. Pozo-Insfran et al. (2007) also found that the level of CO2 was the primary variable influencing PPO activity when setting the fixed temperature and residence time. In this study, the total inactivation of PPO in apple slices even if using mild HPCD processing conditions suggested that HPCD may perform better in inactivating PPO in apple slices. The RA of PME in cloudy apple juice from HPCD- and MH-treated apple slices was shown in Fig. 2. The RA of PME in cloudy apple juice from MH-treated apple slices exhibited some fluctuations at different temperatures, its maximal loss was 31% at 65 °C, indicating PME was rather stable to the experimental temperatures in this study. This observation was in accordance with earlier reports. Zhi et al. (2008) found the maximum reduction of the RA of crude apple PME in 20 mM Tris–chloride buffer (pH 7.5) was less than 20% at 55 °C for 60 min. Assis, Lima and Faria Oliveria (2000) observed that the crude PME from acerola (borate–acetate buffer, pH 8.3) was stable at 50 °C with 10% loss of activity for 100 min and needed 110 min for its inactivation at 98 °C. HPCD significantly reduced the RA of PME and the reduction increased with increasing temperatures. The minimal RA of PME in cloudy apple juice from HPCD-treated apple slices was 18% at 65 °C. The paired t test indicated that the RA of PME in cloudy apple juice from HPCD-treated apple slices was significantly lower than that from MH-treated apple slices. Earlier investigations show that HPCD could inactivate PME in juice or buffer systems. PME in orange juice could be completely inactivated under HPCD conditions of 29 MPa and 50 °C for 4 h (Arreola et al., 1991). Park et al. (2002) find the minimum RA of PME in carrot juice is approximately 40% by 4.90 MPa HPCD. Kincal et al. (2006) also reported that PME in orange juice could lose up to 56% of its original activity at 72 MPa for 10 min by continuous HPCD. Zhi et al. (2008) reported that the loss of the RA of crude apple PME in 20 mM Tris–chloride buffer (pH 7.5) was 94.57% by HPCD at 30 MPa and 55 °C for 60 min. Similarly, PME matrix, sources, fruit maturity and HPCD treatment conditions may also account for different inactivation level in these literatures. Zhou, Zhang, et al. (2009) found the RA of PME in peach juice was significantly greater than that in 20 mM Tris– chloride buffer (pH 7.5) under HPCD treatment conditions of 8 MPa for 200 min, 15 MPa for 210 min and 22 MPa for 180 min, which indicated the original PME in the juice was less inactivated by HPCD. Sampedro, Rodrigo and Hendrickx (2008) also reported higher stability of PME in an orange juice-milk beverage than purified PME under treatment of 5 min in a temperature range from 45 to 90 °C. Arreola et al. (1991) showed that orange PME was completely inactivated under HPCD conditions of 29 MPa, and 50 °C for 4 h. Kincal et al. (2006) observed only a reduction of orange PME of 56% of its original activity at 72 MPa for 10 min by continuous HPCD. Clearly, PPO in apple slices was more susceptible to HPCD than PME in this study. PPO in higher plant was assumed to have 3 or 4 subunits (Van Gelder, Flurkey & Wichers, 1997; Heimdal, Larsen & Poll, 1994) and apple PPO had a molecular weight of 46 kDa (Janovitz-Klapp, Richard & Nicolas, 1989). Apple PME has a molecular mass of 36 kDa (Macdonald & Evans, 1996) with one subunit (D' Avino, Camardella, Christensen, Giovane & Servillo, 2003). In general, the larger an enzyme and the more complex its structure, the more susceptible it is to high temperature (Yang, Li & Zhang, 2004). Similarly, it could be reasoned that the difference in their structure caused the greater sensitivity of PPO to HPCD treatment. 3.2. Effect of HPCD and MH on the visual color, color parameters and BD in cloudy apple juice Fig. 3 showed the visual color of cloudy apple juice half-filled in 7 mL test tubes after 7-day storage at 4 °C. The initial color of cloudy apple juice from the control, MH- and HPCD-treated apple slices was whitish yellow and light. This observation was similar to what Nagel (1992) proposed. As shown in Fig. 3A and B, the cloudy apple juice from HPCD-treated apple slices in initial day was less yellow and lighter than that from MH-treated apple slices, which resulted from prevention of the enzymatic browning in the cloudy apple juice from HPCD-treated apple slices. After 7-day storage at 4 °C, the initial whitish yellow color of cloudy apple juice from the control and MHtreated apple slices turned brown in Fig. 3C, indicating that the enzymatic browning happened in these juices due to the presence of O2 in the upper half of the test tubes. But the cloudy apple juice from HPCD-treated apple slices showed no browning in Fig. 3D. In the 7th day, the more whitish appearance of cloudy apple juice from HPCDtreated apple slices could be attributed to the occurrence of precipitation of unstable particles, meanwhile no enzymatic browning happened in the absence of PPO. Meanwhile, the color parameters of the cloudy apple juice fullyfilled in test tubes in initial day were analyzed as shown in Fig. 4. As compared to the control, the L value of the cloudy apple juice from HPCD-treated apple slices exhibited no increase except at 35 °C and that from MH-treated apple slices showed a significant decrease from 45 to 65 °C, and the paired t test indicated that there was a significant difference between the L value of cloudy apple juice from HPCD- and MH-treated apple slices. Gui, Wu, et al. (2006) reported that an insignificant increase in L value of apple juice occurred after HPCD, but Arreola et al. (1991) and Kincal et al. (2006) found that HPCD led lighter orange juices than untreated orange juices. Furthermore, the paired t test showed the BD of the cloudy apple juice from MH-treated apple slices was significantly higher than that from HPCD-treated apple slices in initial day (shown in Table 1). As compared to the control, the BD of the cloudy apple juice from MH-treated apple slices except at 45 °C exhibited no difference. HPCD decreased the BD of the cloudy apple juice significantly. And there was no difference in the BD among the cloudy apple juice from HPCD-treated apple slices, indicating that the decrease in the BD of the cloudy apple juice was independent of temperatures under HPCD conditions in this study. The alterations of the BD of the cloudy apple juice from HPCD- and MH-treated apple slices closely corresponded to those of the L value in Fig. 4A. After 7-day storage at 4 °C, the BD of the cloudy apple juice S. Niu et al. / Innovative Food Science and Emerging Technologies 11 (2010) 91–97 95 Fig. 3. Visual color image of cloudy apple juice half-filled in test tubes during the 7-day storage at 4 °C (A: MH in initial day; B: HPCD in initial day; C: MH in 7th day; and D: HPCD in 7th day). from HPCD-treated apple slices showed no difference but that from MH-treated apple slices showed a significant increase at 55 and 65 °C. All these observations confirmed that a complete inactivation of PPO in apple slices subjected to HPCD was attained and the enzymatic browning reaction in apple juices was prevented. As shown in Fig. 4B, the a value of the cloudy apple juice from HPCD-treated apple slices showed an increasing tendency, but the paired t test also indicated that there was no significant difference between the a value of the cloudy apple juice from HPCD-treated apple slices and that from MH-treated apple slices. Kincal (2000) showed a similar trend in the a value of orange juice after HPCD, and Gui, Wu, et al. (2006) reported that the a value of HPCD-treated apple slices remained almost constant. As compared to the control, the b value of the cloudy apple juice from HPCD-treated apple slices decreased significantly at 35 and 55 °C and from MH-treated apple slices insignificantly increased in Fig. 4C. As indicated by the paired t test, the b value of the cloudy apple juice from HPCD-treated apple slices was significantly less than that from MH-treated apple slices. Table 1 BD and turbidity of cloudy apple juice from HPCD- and MH-treated apple slices during the 7-day storage at 4 °C. Fig. 4. Change of the color parameters of cloudy apple juice fully-filled in test tubes in initial day from HPCD- and MH- treated apple slices as a function of treatment temperature for 20 min. (A) L value, (B) b value, (C) a value. (□) Control; (▲) 20 MPa HPCD; (▽) atmospheric pressure-MH. All data are the means ± SD, n = 4. Treatment conditions BD In initial day In 7th day Turbidity (NTU) In initial day In 7th day Control 20 °C 25 °C 35 °C MH 45 °C 55 °C 65 °C 25 °C 35 °C HPCD 45 °C 55 °C 65 °C 0.433 ± 0.051Ba 0.587 ± 0.104AB 0.441 ± 0.068AB 0.776 ± 0.093A 0.444 ± 0.107AB 0.402 ± 0.042AB 0.153 ± 0.054b 0.137 ± 0.024b 0.169 ± 0.022b 0.227 ± 0.078b 0.160 ± 0.060b 0.965 ± 0.239a 1.481 ± 0.607 1.091 ± 0.400 1.580 ± 0.387 1.578 ± 0.240 1.372 ± 0.028 0.179 ± 0.128b 0.088 ± 0.001b 0.165 ± 0.044b 0.123 ± 0.025b 0.116 ± 0.016b 63.50±11.79BCb 62.21 ± 6.82C 69.33 ± 4.75BC 74.46 ± 6.12BC 82.00 ± 9.67AB 96.50 ± 8.89A 64.58 ± 7.23b 60.00 ± 4.01b 64.75 ± 7.11b 70.71 ± 5.44ab 87.12 ± 10.41a 53.29 ± 7.18 53.46 ± 6.63 55.74 ± 7.42 58.62 ± 4.54 58.09 ± 7.91 65.25 ± 6.34 46.04 ± 6.68 42.16 ± 2.72 45.49 ± 4.14 46.62 ± 5.44 51.97 ± 11.43 Values were means ± SD of four measurements, n = 4. a,b,c, or A,B Different letters represented a significant difference within the same MH or HPCD column. 96 S. Niu et al. / Innovative Food Science and Emerging Technologies 11 (2010) 91–97 Gui, Wu, et al. (2006) showed the b value of apple juice remained almost constant after HPCD, but Kincal et al. (2006) reported that the b value of orange juice increased after HPCD. 3.3. Effect of HPCD and MH on the turbidity in cloudy apple juice Table 1 also showed the turbidity of the cloudy apple juice from HPCD- and MH-treated apple slices. The turbidity of the cloudy apple juice from HPCD- and MH-treated apple slices exhibited an increasing tendency as temperature increased, especially those at 65 °C from both treatment statistically higher than that of the control. These results indicated that higher temperature increased the turbidity of the cloudy apple juice, which was possibly attributed to more dissolution of substances such as pectin in apple cells into juices and partial inactivation of PME in apple slices in Fig. 2. The turbidity of the cloudy apple juice from HPCD-treated apple slices was significantly lower than that from MHtreated apple slices although the RA of PME in the cloudy apple juice from HPCD-treated apple slices was significantly lower in Fig. 2. After 7-day storage at 4 °C, there was a significant turbidity loss in all the cloudy apple juice except the control and cloudy apple juice from MH-treated apple slices at 25 °C. The greatest loss of cloudy apple juice turbidity was 40.35% with HPCD at 65 °C even if the RA of PME in the cloudy apple juice was lowest in Fig. 2, while only 16.08% loss was achieved for the control. Theoretically, the juice turbidity is well related to the RA of PME in juices, the lower RA of PME was conducive to the stability of the turbidity. However, the turbidity of the cloudy apple juice from HPCD-treated apple slices was not well related to the RA of PME in the cloudy apple juice in the study, this contradiction was possibly due to a non-enzymatic mode causing turbidity loss of the cloudy apple juice from HPCD-treated apple slices. For example, HPCD induced the denaturation of acid-sensitive proteins in apple slices, and then the denatured proteins were removed by centrifuging in the processing of cloudy apple juice, which reduced the turbidity of the cloudy apple juice from HPCD-treated apple slices. Park et al. (2002) also proposed that HPCD affected the cloud of carrot juice in a non-enzymatic way since the cloud of carrot juice decreased after HPCD while the lowest PME activity was achieved. However, the cloud of orange juice was enhanced by 1.27–4.01 times after HPCD (Arreola et al., 1991). Kincal et al. (2006) also found the cloud increased up to 846% in orange juice treated by HPCD, where it was hypothesized that depressurization of the system led to homogenization of orange juice causing smaller particles of the juice colloid. 3.4. Effect of HPCD and MH on juice yield, TSS, pH and conductivity of cloudy apple juice Table 2 showed the change of juice yield, TSS, pH and conductivity of the cloudy apple juice from HPCD- and MH-treated apple slices. As compared to the control, there was no difference in juice yield after HPCD and MH apart from MH at 65 °C. Similarly, no significant difference in TSS, pH and conductivity of apple juices was observed after HPCD and MH treatment. Kincal (2000) noticed no significant change in pH and TSS of HPCD-treated orange juice. Yagiz, Lim and Balaban (2005) also found similar trend in HPCD-treated mandarin juice. Although papers have reported that HPCD, as one of the non-thermal technology with cell membrane permeabilizing features, could improve juice yield by pressure/concentration gradient (Knorr, 2003), we found that juice yield decreased after HPCD treatment. Such discrepancy may be possibly attributed to the unavoidable loss in our study since small amount of free juice by pressure effect remained in the pipes and/or vessel of HPCD equipment that we hardly collect. On the other hand, statistical analysis showed there was insignificant difference in juice yield among the MH and HPCD-treated samples, except MH at 65 °C, as compared to the control. What is more, TSS of cloudy apple juice, an important index reflecting apple juice characteristic did not significantly change after both treatments. We assumed that the difference of enzymes levels, BD, and turbidity etc were much related to the effect of MH or HPCD treatment, rather than to the difference of juice yield. Therefore, the effect of difference of juice yield on BD etc. could be neglected. 4. Conclusion HPCD completely inactivated PPO and significantly reduced the activity of PME in apple slices. It also inhibited the browning in cloudy apple juice with no impact on juice yield, TSS, pH, and conductivity of cloudy apple juice. However, HPCD increased the turbidity of cloudy apple juice at 65 °C. Therefore, HPCD was possibly regarded as a pretreatment means in fruit and vegetable juice processing. Cloudy apple juice from HPCD-treated slices did not exhibit enzymatic browning but experienced cloud loss during the 7-day storage time. This presented both chance and challenge for HPCD application in juice industry. Further studies are still needed to investigate the effect of HPCD on PME in apple slices and its relation to cloud stability. HPCD parameters are also needed to improve to enhance the effect, especially PME inactivation, of HPCD on fruit slices. Acknowledgement The research work was jointly supported by the Undergraduate Research Plan of China Agricultural University, the Project 30571297 of National Natural Science Foundation of China, and Project 2006BAD05A02 of the Science and Technology Support in the Eleventh Five Plan of China. References Table 2 Some physicochemical indices of cloudy apple juice from HPCD- and MH-treated apple slices. Treatment conditions Control MH HPCD 20 °C 25 °C 35 °C 45 °C 55 °C 65 °C 25 °C 35 °C 45 °C 55 °C 65 °C Juice yield (%) TSS (°Brix) pH Conductivity (10− 1 S/m) 65.27 ± 1.71a 64.47 ± 4.41ab 61.50 ± 0.62ab 61.50 ± 5.97ab 59.10 ± 1.59ab 56.47 ± 1.04b 57.33 ± 2.37ab 59.50 ± 3.90ab 58.50 ± 0.44ab 57.33 ± 1.33ab 58.40 ± 0.96ab 9.43 ± 0.32 9.77 ± 0.31 9.47 ± 0.15 10.17 ± 0.21 9.37 ± 0.70 9.53 ± 0.67 9.40 ± 0.52 9.40 ± 0.17 9.73 ± 0.15 9.33 ± 0.50 9.47 ± 0.32 3.61 ± 0.08 3.64 ± 0.06 3.65 ± 0.06 3.69 ± 0.05 3.64 ± 0.07 3.69 ± 0.12 3.64 ± 0.03 3.61 ± 0.07 3.61 ± 0.04 3.61 ± 0.04 3.63 ± 0.11 1.88 ± 0.17 1.89 ± 0.11 1.91 ± 0.12 1.87 ± 0.16 1.90 ± 0.20 1.91 ± 0.23 1.88 ± 0.12 1.91 ± 0.11 1.90 ± 0.13 1.97 ± 0.14 1.94 ± 0.14 Values were means ± SD of four measurements, n = 4. a,b,c Different letters represented a significant difference within the same column. Arreola, A. G., Balaban, M. O., Marshall, M. R., Peplow, A. J., Wei, C. I., & Cornell, J. A. (1991). 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