Journal of Food Engineering xxx (2011) xxx–xxx
Contents lists available at ScienceDirect
Journal of Food Engineering
journal homepage: www.elsevier.com/locate/jfoodeng
Effects of high pressure CO2 treatments on microflora, enzymes and some
quality attributes of apple juice
Zenghui Xu a,b,c,1, Liyun Zhang a,b,c,1, Yongtao Wang a,b,c, Xiufang Bi a,b,c, Roman Buckow d, Xiaojun Liao a,b,c,⇑
a
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
Key Laboratory of Fruits and Vegetables Processing of Ministry of Agriculture, China
c
Research Center for Fruit and Vegetable Processing Engineering of Ministry of Education, China
d
Food Science Australia, A Joint Venture of CSIRO and the Victorian Government, 671 Sneydes Road, Werribee, Vic. 3030, Australia
b
a r t i c l e
i n f o
Article history:
Received 26 September 2010
Received in revised form 16 January 2011
Accepted 18 January 2011
Available online xxxx
Keywords:
Continuous high pressure CO2
Microflora
Polyphenol oxidase
Pectin methylesterase
Quality
Cloudy apple juice
a b s t r a c t
The inactivation of microorganisms and enzymes of cloudy apple juice, and some physico-chemical properties of the juice were investigated after continuous high pressure carbon dioxide (HPCD) at 25 MPa and
43 °C, for 2 min and at 22 MPa and 60 °C for 3, 5 and 10 min, respectively. The coliform bacteria were
completely inactivated in all the cases. Total aerobic bacteria was reduced by 3.72 log cycles, pectin
methylesterase was reduced by 54.3% and polyphenol oxidase was completely inactivated after 10 min
treatment at 22 MPa and 60 °C. The yeasts and molds were completely inactivated and the turbidity
increased at 22 MPa and 60 °C regardless of time, while the L and a value reduced, but browning degree
did not change. The particle size distributions of the juice changed after HPCD but were regained as treatment time was prolonged. The pH was reduced at 22 MPa and 60 °C for 3 or 5 min.
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction
The cloudy apple juice has a growing share in the current market due to its sensory and nutritional qualities, which meets the
consumers’ demand for fresh-like products with little or no degradation of nutritional and organoleptic properties (Iyidögan and
Bayındırlı, 2004; Özöglu and Bayındırlı, 2002). The main problem
with cloudy apple juice during production and storage is the colour
and cloud stability. The discolouring of cloudy apple juice is a result of enzymatic browning, which involves the action of polyphenol oxidase (PPO, EC1. 14. 18. 1) catalyzing oxidation of phenolic
compounds (Joslyn and Ponting, 1951). The cloud stability of cloudy juices is closely related to the pectin methylesterase (PME, EC 3.
1. 1. 11) activity. Undesired clarification is strongly influenced by
demethylation of pectin by PME yielding acidic pectin with a lower
degree of esterification (Assis et al., 2001; Cameron et al., 1994).
Traditionally, thermal processes are applied in fruit juice processing to ensure its safety and to inactivate indigenous enzymes that
can cause undesirable quality change. However, heat-sensitive
⇑ Corresponding author at: College of Food Science and Nutritional Engineering,
China Agricultural University, Beijing 100083, China. Tel.: +86 10 62737434 602;
fax: +86 10 62737434 604.
E-mail address: [email protected] (X. Liao).
1
Were equally the first authors.
nutrients and volatile compounds may be negatively affected by
thermal processing.
High pressure carbon dioxide (HPCD) processing has been rapidly developing over the past decades as a novel, non-thermal pasteurization method for liquid food pasteurization. It has the ability
to inactivate different microorganisms without exposing foods to
adverse effects of heat and can retain their fresh-like physical,
nutritional, and sensory qualities (Damar and Balaban, 2006). Its
efficacy on at least 12 gram-positive bacteria, 10 gram-negative
bacteria, 8 bacterial spores, and 8 fungi as filament or spores, has
been studied over the last few years (Zhang et al., 2006). HPCD
has also been proven effective to inactivate many indigenous fruit
enzymes, including PPO (Pozo-Insfran et al., 2007; Gui et al., 2007;
Chen et al., 1992; Liu et al., 2008), Peroxidase (Liu et al., 2008),
Lypoxygenase (Liao et al., 2009) and PME (Arreola et al., 1991; Park
et al., 2002; Kincal et al., 2006; Zhi et al., 2008; Zhou et al., 2009a).
However, there is only a limited number of published studies on
the effects of HPCD on the quality of foods (Damar and Balaban,
2006), or the change of particle size distribution (PSD) of cloudy
juice. Zhou et al. (2009b) investigated the effect of HPCD (batch
system) on the PSD of carrot juice, concluding that its change depends on the treatment time. It was reported that the particle size
was affected by the soluble solids concentration, which was attributed to conformational changes in juice particles aggregates,
simultaneously with a reduction in particle solvatation (Benitez
et al., 2007). According to Genovese and Lozano (2000), cloudy
0260-8774/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2011.01.020
Please cite this article in press as: Xu, Z., et al. Effects of high pressure CO2 treatments on microflora, enzymes and some quality attributes of apple juice.
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Z. Xu et al. / Journal of Food Engineering xxx (2011) xxx–xxx
apple juice is a colloidal dispersion where the dispersed matter is
mainly formed by cellular tissue comminuted during fruit
processing.
Most studies dealing with HPCD have applied the technology
batch-wise. There are only a few studies that used continuous
HPCD. Since a continuous system allows the flow of both CO2
and liquid food to go through the system, it was effective in killing
pathogens and spoilage bacteria in a short time (Damar and
Balaban, 2006; Kincal et al., 2005; Lecky and Balaban, 2005; Yagiz
et al., 2005). Parton et al. (2007) proved continuous HPCD as an
effective way for the pasteurization of natural grape must and tomato paste. Kincal et al. (2005) showed that the natural microfloras in orange juice significantly decreased from 102 to 103 CFU/mL
to <10 CFU/mL following continuous HPCD at room temperature.
Studies also showed that the quality of fruit juice was well maintained after continuous HPCD treatment (Kincal et al., 2006; Lecky
and Balaban, 2005; Yagiz et al., 2005). Yagiz et al. (2005) found that
continuous HPCD enhanced cloudiness of mandarin juice up to
38.4%, increased its L and b value, and decreased its a value without
changing its pH value and the content of total soluble solids (TSS).
The purpose of this work was to investigate the effectiveness of
continuous HPCD processing of cloudy apple juice on its natural
microorganisms flora, as well as PPO and PME activity. At the same
time the change of colour, particle size distribution, turbidity, viscosity, and other physico-chemical properties of the juice due to
continuous HPCD was studied.
2. Materials and methods
2.1. Preparation of cloudy apple juice
Apples (Fuji, Yantai, Shandong, China) were purchased from the
local market in Beijing (China) and stored in a cold warehouse at an
average temperature of 6 °C. The apples were washed, diced and
squeezed with a screw juice extractor (T6G7, Zhejiang Light Industry Machinery Plant, Zhejiang, China) and were then filtered with 4
layers of cheesecloth. To prevent the browning of the juice, 0.7‰ Lascorbic acid (Aoboxing Biotechnical Co., Beijing, China) was added
to the freshly squeezed juice. Because the valves and pipelines in
the HPCD system might clog if the juice was very rich in pulp, all
the juice was continuously centrifuged in 9720 rpm (Westfallia
centrifuge, Germany) after being filtrated and was then mixed to
ensure its uniformity. The juice was later evenly divided into different sterilized containers, each with volume of 10 L, and stored
at the cold warehouse until the experiment started.
2.2. Continuous HPCD system
A pilot-scale continuous HPCD system constructed by China
Agriculture University is presented in Fig. 1. Firstly, the CO2 (purity
99.5%, Beijing Jing Cheng Co.) is cooled below 4 °C. Then liquid CO2
and juice sample are simultaneously pumped to the preheating
vessels and then to the reactor by plunger-type variable speed
pumps (Pumps 1 and 2, Fig. 1) in different flow rates. Blended in
the entrance of the reactor, the CO2/juice mixture is to flow
through the holding tube (i.d. 9 mm 10 m) for HPCD treatment.
Installed within the holding tube are 2 thermocouples that measure the temperature of the mixture at the middle and end of the
holding tube. The reactor is maintained at constant temperature
and pressure which is manually controlled by the back pressure
regulator. The final section of the system consists of 2 separators
for depressurization and separation of carbon dioxide and juice.
After processing, the treated juice is collected with aseptic manipulation in a clean bench. The separated CO2 passes through a tube
and is either vented to the air or recycled to the chiller for cycling
treatment. The frequency of the pumps, temperatures of the reactor and two preheating vessels can also be set up and recorded by
the computer system. Since there is no monitor in this system for
recording the holding time in the reactor, the holding time is recorded manually as below:
Holding time ¼ T 0 Tt
ð1Þ
where T0 means the time from the fluids being pumped to the fluids
flowing out of the separator 1. Tt means the time from the fluids
being pumped to the CO2/juice mixture reaching the entrance of
the reactor. The frequencies of the two pumps are adjusted, respectively to reach the desirable holding time. The frequencies of Pumps
1 and 2 are 9 Hz and 15 Hz for 2 min (at 5 MPa, 43 °C), respectively,
and are both 14 Hz, 8 Hz and 6 Hz for 3, 5 and 10 min (at 22 MPa,
60 °C), respectively.
2.3. HPCD processing
2.3.1. Untreated juice as a control
Freshly squeezed juice which was pumped through the HPCD
system without CO2 or heating under atmospheric pressure was
used as the control to test its natural microorganisms, enzymes
and other quality parameters.
2.3.2. Processing of cloudy apple juice using HPCD
Effects of HPCD on natural microorganisms, PPO, PME and related qualities of cloudy apple juice were discussed firstly at
43 °C for 2 min, under 10 MPa and 25 MPa, respectively. Then, effects of HPCD on apple juice at 60 °C and 22 MPa for longer holding
time from 3 min to 10 min were studied. Finally the treated juices
were stored at 6 °C or frozen at 80 °C for further analysis. The
microbial inactivation and quality changes of apple juice were
studied immediately after HPCD. The PPO and PME activity was
measured after being thawed from the frozen sample in the following days after treatment.
Before and after each experiment, the HPCD system was rinsed
by hot water and pasteurized with 75% ethanol in continuous flow
for more than 10 min. All the experiments were repeated in
triplicate.
2.4. Microbiological enumeration
To detect the viable cells of total aerobic bacteria (TAB) and
yeasts and molds (Y&M) in apple juice, the pour-plating method
was used. Treated and untreated samples were serially diluted
with sterile 0.85% NaCl solution and 1.0 mL of diluted (or nondiluted) samples was plated into duplicated plates of appropriate
agar. The Nutrient Agar (NA, Beijing Aoboxing Biological Technology Co. Ltd., Beijing, China) was used for detecting the viable cells
of the TAB and the plates were incubated at 37 °C for 48 ± 2 h. The
Rose Bengal Agar (RBA, Beijing Aoboxing Biological Technology Co.
Ltd., Beijing, China) was used for detecting the viable cells of the
Y&M, the plates were incubated at 28 °C for 3–5 days. After incubation, plates of 30–300 CFU for TAB, 10–150 CFU for Y&M were chosen for enumeration. Log N was calculated to determine the
inactivation effect, where N represented the number of microorganisms colonies of the untreated or treated samples.
Most probable number (MPN) method was used to detect the
coliforms in apple juice (Suwansonthichai and Rengpipat, 2003).
In the pre-fermentation test, samples were diluted into three
appropriate series, and 1.0 mL of each diluted (or non-dilute) samples was inoculated in triplicate tubes of 10 mL lauryl sulfate tryptose (LST) broth (Beijing Luqiao Biological Technology Co. Ltd.,
Beijing, China). Inoculated tubes were incubated at 37 °C for
48 ± 2 h. In the later re-fermentation test, loopfuls of samples from
inoculated LST broth, which were positive for turbidity and gas for-
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Z. Xu et al. / Journal of Food Engineering xxx (2011) xxx–xxx
Fig. 1. Schematic of continuous high pressure CO2 system. T1, T2: temperature indicators for the out-let of preheating vessel; T3: temperature indicator for the out-let of the
reactor; P1, P2: pressure indicator for the CO2 tank and out-let of the chiller; P3: pressure indicator for the reactor.
mation, were inoculated in tubes of 10 mL brilliant green lactose
bile (BGLB) broth(Beijing Luqiao Biological Technology Co. Ltd.,
Beijing, China). After incubated at 37 °C for 48 ± 2 h, the positive
tubes with colour and turbidity changes and gas formation were
counted. Results of the test were reported as the MPN per 100 mL
of sample.
2.5. Enzyme activity determination
2.5.1. PPO assay
The activity of PPO was assayed by a spectrophotometric method (Weemaes et al., 1997) with some modifications. Catechol was
chosen as the substrate, and 0.05 M catechol substrate solution
was prepared with 0.05 M phosphate buffer (pH 6.0). The juice
was centrifuged in 9000g for 15 min (GL-166-A, Shanghai Anting
Scientific Equipment Factory, Shanghai, China) and the supernatant was taken as tested sample. All samples were analyzed by
adding 500 lL centrifuged juice into 2.5 mL substrate solution.
The increase in absorbance at 420 nm was monitored at intervals
of 0.1 s1 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 30 ± 0.1 °C and
an in-built electromagnetic stirring to mix up the substrate and
juice. Prior to measurement, a pre-equilibrium of the substrate
solution and the juice at 30 °C by the peltier thermo-statted cell
holder was 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:
Residual activity ¼
PPO specific activity after HPCD treatment
PPO specific activity in the control
100%
ð2Þ
2.5.2. 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 was 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 was measured for the solution’s pH to return to 7.5. PME
activity (A) expressed in pectin methylesterase units (PMEU) was
calculated by the following:
A¼
½NaOHV NaOH
0
V tsample
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 t0 was the time (in minutes)
needed for pH to return to 7.5 after the addition of NaOH
Residual activityðRAÞ ¼
A
100%
A0
where A0 represented the mean activity of PME in the control, A represented the mean activity of PME after HPCD treatment.
2.6. Determination of colour changes
2.6.1. Colour assessment
Colour assessment was conducted at room temperature with a
colour difference meter (SC-80, Kangguang Co., Beijing, China) in
the reflectance mode. The L, a, and b values of cloudy apple juice
were measured. DE was calculated as below:
DE ¼ ½ðLt L0 Þ2 þ ðat a0 Þ2 þ ðbt b0 Þ2 1=2
where the Lt, at and bt meant the L, a and b value of cloudy apple
juice after HPCD treatment, respectively. And the L0, a0 and b0
meant the L, a and b value of the control, respectively.
2.6.2. Determination of browning degree (BD)
The BD of cloudy apple juice was analyzed with a spectrophotometric method described by Roig et al. (1999). Cloudy apple juice
Please cite this article in press as: Xu, Z., et al. Effects of high pressure CO2 treatments on microflora, enzymes and some quality attributes of apple juice.
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Z. Xu et al. / Journal of Food Engineering xxx (2011) xxx–xxx
was centrifuged with a refrigerated Centrifuge (GL-166-A,
Shanghai Anting Scientific Equipment Factory, Shanghai, China)
at 9000g at 4 °C for 30 min, and then passed through a 0.45 lm
cellulose nitrate membrane (Beijing Bomex Co., Beijing, China).
The BD was determined by measuring the A (absorbance at
420 nm) value by a UV-762 spectrophotometer (T6, PG General,
Beijing, China) with a cell of 1 cm path length at ambient temperature (20 ± 1 °C).
2.7. Turbidity determination
The turbidity of cloudy apple juice was measured at room temperature with the method proposed by Reiter et al. (2003). A digital
photoelectrical turbidimeter (WGZ-200, Shanke Instrument Factory, Shanghai, China) with 5 mm cuvette at colour correction
mode was applied. Cloudy apple juice was diluted in 1/20 with distilled water. Turbidity was expressed in nephelometric turbidity
units (NTU).
2.8. Determination of PSD
PSD of juice was determined by a LS 230 particle size analyzer
(Beckman Coulter, Inc., Florida, USA). A laser light with a wavelength of 750 nm was applied in the system, to measure particles
from 0.4 to 2000 lm by light diffraction. Fourier optics collected
the diffracted light and the PSD was calculated by Fraunhofer
model.
Firstly, distilled water from a tank was pumped into a sample
cell at the speed of approximately 8 L/min until the cell was full.
The juice was added into the cell using a pipette and mixed with
distilled water. Particles in juice were dispersed and suspended
in distilled water. The measurement started when the obscuration
percentage increased from 0 to 10%. Data obtained were analyzed
by software LS v3.29. Volume mean diameter D[4,3] and surface
mean diameter D[3,2] were determined for all samples.
2.9. Some physico-chemical properties
2.9.1. Viscosity determination
The viscosity of cloudy apple juice was determined by a rotational viscometer (Visco Basic Puls L, FungiLab S.A., Barcelona,
Spain) in LCP mode at 100 rpm. The temperature was kept at
30 °C by constant-temperature water bath circulation device
(HX-1050, Beijing De-Tianyou Technology Development Co., Ltd,
Beijing, China).
2.9.2. Determination of total soluble solids (TSS)
The TSS of cloudy apple juice was determined as °Brix at ambient temperature (20 ± 1 °C) by WAY-2S digital Abbe Refraction meter (Shanghai Precision and Scientific Instrument Co., Shanghai,
China).
2.9.3. pH Determination
The pH was measured at 25 °C using digital Thermo Orion 555A
pH meter (Thermo Fisher Scientific Inc., MA, USA). 10-mL cloudy
apple juice was inserted with a pH electrode (Thermo Orion Ross
9103BN, MA, USA) and pH was recorded after stabilization.
2.10. Statistical analysis
Analysis of variance (ANOVA) was conducted 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 triplicate.
3. Results and discussions
3.1. Inactivation of natural microorganisms
High temperatures stimulate the diffusivity of CO2, and increase
the fluidity of the bacterial cell membrane, which enhances penetration easier (Lin et al., 1993; Hong et al., 1997, 1999). Several
studies have proved that CO2 at supercritical state (>31 °C and
>7.34 MPa) attained higher inactivation efficacy (Lin et al., 1992;
Shimoda et al., 1998; Isenschmid et al., 1995; Ishikawa et al.,
1997). With low viscosity and better diffusivity and dissolving
power, it is more effective for supercritical CO2 to penetrate into
cells, extract essential substances from cells or membranes, and
to disorder the conformation of cytoplasmic membranes and
organelles, resulting in the disruption of biological system in the
cell (Shimoda et al., 1998; Tomasula et al., 2003). Therefore, HPCD
experiments were carried out at temperatures higher than ambient
temperature in this study.
The inactivation of the TAB, Y&M and coliforms in cloudy apple
juice by HPCD is illustrated in Table 1. The coliform bacteria in
cloudy apple juice were very sensitive to HPCD and were completely inactivated in all the cases. 1.30 and 2.32 log cycles reduction of the TAB and Y&M was attained at 10 MPa and 43 °C for
2 min, whereas it was about 3.03 and 2.61 log cycles reduction at
25 MPa and 43 °C for 2 min, indicating the important role that
the pressure plays for inactivation of the TAB and Y&M. Similar results were reported in previously studies (Hong et al., 1999;
Ballestra et al., 1996; Dillow et al., 1999; Erkmen and Karaman,
2001; Lin et al., 1992; Spilimbergo et al., 2003; Liao et al., 2007).
Higher pressure enhances CO2 solubility to facilitate its contact
with and penetration into cells (Hong et al., 1997). The inactivation
of the TAB and Y&M induced by HPCD at 22 MPa and 60 °C was,
therefore, studied as a function of time from 3 min to 10 min in this
study. The TAB significantly decreased with the increasing of the
treatment time. Chen et al. (2010) reported the effect of time on
microorganisms under HPCD treatment at 35 MPa and 55 °C, concluding that long treatment times resulted in significant TAB
reduction. The Y&M were completely inactivated by HPCD at
22 MPa and 60 °C, regardless of treatment time, in this study, suggesting that HPCD brought about better inactivation on Y&M than
on the TAB at 60 °C. This observation was in accordance with findings from some previous studies. Pozo-Insfran et al. (2006) showed
that compared with TAB, Y&M were inactivated at a significantly
higher rate under identical processing conditions of HPCD. Liao
et al. (2010) also observed that the Y&M in cloudy apple juice were
more susceptible to a batch HPCD treatment than TAB. Microbial
inactivation was highly dependent on the type of microorganisms
presented in the food matrix due to distinct microbial cell microstructure. The inactivation of microorganisms in cloudy apple juice
without CO2 at 22 MPa and 60 °C for 10 min was investigated as
the heat control (data not shown in the Table 1). 3.07 and 4.3 log
cycles reduction were attained for TAB and Y&M, respectively,
which were less than the HPCD group. Liao et al. (2010) study
showed HPCD increased the susceptibility of natural microorganisms to temperature and enhanced their microbial inactivation.
3.2. Inactivation of PPO and PME in cloudy apple juice
The residual activity (RA) of PPO and PME in cloudy apple juice
after HPCD treatment is shown in Fig. 2. The RA of PPO in cloudy apple juice was 47.29% when the juice was subjected to HPCD at
25 MPa and 43 °C for 2 min, which was very similar to the case at
22 MPa and 60 °C for 3 min. Moreover, the activity of PPO in cloudy
apple juice reduced significantly with an increasing treatment time
at 22 MPa and 60 °C. Complete inactivation of the enzyme was
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Table 1
Microbial inactivation in cloudy apple juice by HPCD.
Treatment conditions
10 MPa,
25 MPa,
22 MPa,
22 MPa,
22 MPa,
43 °C, 2 min
43 °C, 2 min
60 °C, 3 min
60 °C, 5 min
60 °C,10 min
Aerobic bacteria (Log N)
Yeasts and molds (Log N)
Coliforms (MPN/100 mL)
Control
HPCD
Control
HPCD
Control
HPCD
4.78 ± 0.10
4.45 ± 0.01
5.56 ± 0.04
5.20 ± 0.01
5.07 ± 0.01
3.48 ± 0.08
1.42 ± 0.03
2.32 ± 0.09
1.77 ± 0.13
1.35 ± 0.01
4.24 ± 0.01
4.39 ± 0.16
4.17 ± 0.08
4.19 ± 0.03
4.77 ± 0.04
1.92 ± 0.54
1.78 ± 0.05
ND
ND
ND
2.4 103
2.4 103
2.4 103
4.6 103
4.6 103
ND
ND
ND
ND
ND
All data were the means ± SD, n = 3. ND = not detectable.
1.2
PPO
PME
1.1
1.0
Residual Activity
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
A
B
C
D
Fig. 2. Residual activity of PPO and PME in cloudy apple juice by HPCD (A: 25 MPa,
43 °C, 2 min, B: 22 MPa, 60 °C, 3 min; C: 22 MPa, 60 °C, 5 min; D: 22 MPa, 60 °C,
10 min). All data were the means ± SD, n = 3.
found when the treatment time was 10 min. The effects of HPCD
treatment temperature and time on PPO activity in juice or matrices
have been previously studied. For example, Gui et al. (2007) showed
that higher temperature and longer treatment time resulted in
higher inactivation of PPO. The highest enzyme reduction in cloudy
apple juice was greater than 60% when applying batch HPCD at
30 MPa and 55 °C for 60 min. Liu et al. (2008) reported that the
activity of PPO in red beet extract also decreased as HPCD temperature and time increased. For example, the PPO original activity was
reduced by 95% with a batch HPCD at 22.5 MPa and 55 °C for 60 min.
Chen et al. (1992) reported in their study, that purified Florida lobster, brown shrimp and potato PPOs activities declined as treatment
time proceeded with a batch HPCD at 5.8 MPa and 43 °C. Park et al.
(2002) found that a batch HPCD treatment at 4.9 MPa and 5 °C for
10 min was responsible for 61% reduction of PPO activity in carrot
juice. HPCD treatment at 30 °C and 27.6 MPa for 6.25 min resulted
in a 75% decrease in PPO activity in muscadine juice with a batch
system (Pozo-Insfran et al., 2007). None of these studies reported
complete inactivation of PPO by HPCD. Different inactivation levels
of PPO could be attributed to different HPCD systems, treatment
conditions, PPO sources and cultivars. For example, PPO from the
potato was more resistant to HPCD than that from the spiny lobster
and shrimp (Chen et al., 1992). In this study, PPO was completely
inactivated by continuous HPCD at 22 MPa and 60 °C for 10 min,
whereas in Gui’s study (Gui et al., 2007), the maximal reduction of
PPO from the same apple cultivar was more than 60% with a batch
HPCD at 30 MPa and 55 °C for 60 min. Although the HPCD conditions in this study were less intense, the difference in inactivating
PPO was possibly attributed to the higher efficiency of continuous
HPCD system than that of a batch HPCD system.
As shown in Fig. 2, PME activity was increased after HPCD treatment at 25 MPa and 43 °C for 2 min. To our best knowledge, no
study has reported on HPCD-induced activation of PME in cloudy
apple juice. However, the activation of PME in cloudy apple juice
by thermal processing (70 °C, 50–100 s) has been previously reported (Krapfenbauer et al., 2006; Denes et al., 2000). The assumed
reason lies in the fragmentation of cell wall, which might possibly
occur during HPCD processing. The fragmentation could favor enzyme accessibility and thus increase the apparent PME activity
since PME is ionically-bounded to the cell wall. Thus, more studies
are needed to investigate the effect of mild HPCD treatment on
PME. When the temperature was increased to 60 °C, PME activity
was slightly reduced. After 5 min HPCD treatment, the PME activity
in cloudy apple juice was reduced significantly, and after 10 min it
was reduced by 58.3%. Similar to PPO inactivation, the results also
showed that temperature and time played an important role in
HPCD inactivation of PME. Zhi et al. (2008) reported that better
inactivation of apple PME with an increase of temperature from
35 to 55 °C at 30 MPa and the maximal reduction of apple PME
activity (in Tris-chloride buffer, pH 7.5) was 94.57% at 30 MPa
and 55 °C for 60 min. Zhou et al. (2009a) also showed that the
activity of carrot and peach PME was reduced as the treatment
time extended. Several other studies also studied the inactivation
of PME in juices by HPCD treatment. Kincal et al. (2006) showed
that the highest inactivation (46.3%) of PME in orange juice was
achieved when the pressure was 107 MPa and no heat was applied
with a continuous HPCD. Park et al. (2002) reported that the RA of
PME in carrot juice was about 40% after a batch HPCD treatment at
4.90 MPa and 5 °C for 10 min. Arreola et al. (1991) observed that a
batch HPCD at 29 MPa and 50 °C for 4 h resulted in complete inactivation of PME in orange juice.
A complete inactivation of PPO in this study was found while
the maximum reduction of PME was only 58.30%, indicating that
PPO is more susceptible to HPCD than PME. This might be attributed to their structural difference. PPO is assumed to have 3 or 4
subunits (Van Gelder et al., 1997; Heimdal et al., 1994) with a
molecular weight of 46 kDa (Janovitz-Klapp et al., 1989) whereas
PME has one subunit (D’Avino et al., 2003) with a molecular weight
of 36 kDa (Macdonald and Evans, 1996). In general, the larger an
enzyme and the more complex its structure is, the more susceptible it is to high temperature (Yang et al., 2004). Similarly, it could
be reasoned that the greater sensitivity of PPO to HPCD treatment
than PME is caused by the difference in its structure. Furthermore,
the inactivation of PPO and PME induced by HPCD could also result
from the alterations in secondary and tertiary structures (Park
et al., 2002; Zhou et al., 2009a).
3.3. Change of colour in cloudy apple juice
Table 2 shows the colour change of cloudy apple juice after continuous HPCD treatment. The L value of cloudy apple juice exhibited no alteration after 2 min HPCD at 25 MPa and 43 °C, but it
was significantly reduced after HPCD treatment at 60 °C regardless
of the treatment time. The a value of cloudy apple juice significantly decreased after all treatments, indicating that the apple
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Z. Xu et al. / Journal of Food Engineering xxx (2011) xxx–xxx
Table 2
Change of color in cloudy apple juice induced by HPCD.
Treatment conditions
Color parameters
BD
L
25 MPa,
22 MPa,
22 MPa,
22 MPa,
43 °C,
60 °C,
60 °C,
60 °C,
2 min
3 min
5 min
10 min
a
b
DE
Control
HPCD
Control
HPCD
Control
HPCD
53.48 ± 0.15
51.28 ± 0.21a
51.93 ± 0.45a
51.57 ± 0.24a
53.51 ± 0.49
50.75 ± 0.16b
51.14 ± 0.41b
50.65 ± 0.08b
0.80 ± 0.04a
0.70 ± 0.08a
0.73 ± 0.09a
0.60 ± 0.11a
0.55 ± 0.12b
0.36 ± 0.14b
0.07 ± 0.19b
0.01 ± 0.06b
14.39 ± 0.39a
18.06 ± 0.26
17.22 ± 0.82
16.41 ± 0.57
13.72 ± 0.11b
18.05 ± 0.58
17.21 ± 1.41
16.16 ± 0.49
0.72
1.18
1.12
1.13
Control
HPCD
0.21 ± 0.01b
0.35 ± 0.02
0.25 ± 0.01
0.30 ± 0.03
0.26 ± 0.01a
0.30 ± 0.03
0.29 ± 0.04
0.33 ± 0.02
All data were the means ± SD, n = 3.
Different letters represented a significant difference within the same treatment.
a,b
respectively. After HPCD treatment, the PSDs changed as the volume number of the larger peak decreased and that of the small
peak increased. However, the PSDs were gradually regained as
the treatment time was prolonged. In Table 3, the change of
D[4,3] (volume mean diameter) and D[3,2] (surface mean diameter) of cloudy apple juice after HPCD is demonstrated. It indicated
an increase after 3 min and 5 min and then a decrease after 10 min.
This observation was in agreement with Zhou et al. (2009b)
study. Precipitation caused by pH shift is characteristic of proteins
(Roig et al., 1999). As shown in Table 4, the pH of HPCD-treated
juices declined after HPCD treatment at 22 MPa and 60 °C due to
the dissociation of carbonic acid, which is formed by CO2 dissolution into the juices. The HPCD-induced increase of the average particle size of the juice was possibly a result of the acid-induced
protein coagulation, while the decrease in particle size of HPCDtreated juices after longer treatment time was probably attributed
to the HPCD-induced homogenization effect (Zhou et al., 2009b).
Longer treatment resulted in a stronger homogenization effect of
the HPCD treatment. Therefore, it was assumed that the alteration
of the PSD for HPCD-treated juices was an interaction between the
acid-induced protein coagulation and the HPCD-induced homogenization, which was affected by the pH change of the juice. After a
short HPCD treatment (3 and 5 min), the pH was reduced and the
acid-induced protein coagulation seemed to dominate, while the
HPCD-induced homogenization effect alternated for a longer treatment (10 min) as the pH remained unchanged. On the other hand,
the increase of the average particle size of HPCD-treated cloudy apple juice at 25 MPa, 43 °C, and 2 min (shown in Table 3) was higher
than that treated at 22 MPa, 60 °C, and 3 min, although the treatment time of the former was shorter and pH stayed unchanged.
This might be attributed to the finding that a higher temperature
(60 °C compared to 43 °C) helped to accelerate the HPCD homogenization effect. In addition, the different ratio of CO2 to the juice,
caused by the different the flows/frequencies of the pumps, was
probably another influential factor which needed further
investigation.
juice lost some redness after HPCD. The b value did not change except at 25 MPa and 43 °C for 2 min. Although the colour difference
DE of cloudy apple juice increased when the treatment temperature slightly increased from 43 to 60 °C, it showed no significant
change for different treatment times at 22 MPa and 60 °C
(DE < 2). BD of cloudy apple juice increased significantly with
HPCD at 25 MPa and 43 °C for 2 min, and changed insignificantly
after HPCD at 22 MPa and 60 °C, which was in accordance with
the inactivation of PPO.
Other studies measured colour change of fruit juice after HPCD
treatment as well. Gui et al. (2006) found that the L, a and b value
of cloudy apple juice remained the same after HPCD treatments
(55 °C, 8–30 MPa, 60 min). However, in Kincal et al. (2006) study,
the L value of orange juice significantly increased, the a value significantly decreased, and the b value exhibited no change after continuous HPCD treatment (room temperature, 38, 72, and 107 MPa
for 10 min). Arreola et al. (1991) also found that the HPCD-treated
orange juice was brighter, less red and more yellow than the untreated sample. Zhou et al. (2009b) found that the L value of HPCD
(25 °C, 10–30 MPa, 15–60 min) treated carrot juices increased significantly, the a value increased with the increasing of the treatment time, while the b value of HPCD-treated juice did not
change. The difference on the change of L, a and b value between
these previous studies and ours was probably attributed to the different investigated subjects and/or treatment conditions (such as
temperature).
3.4. Change of PSD of cloudy apple juice
Fig. 3 illustrates the PSDs of cloudy apple juice as a function of
the treatment time with HPCD at 22 MPa and 60 °C. The PSD of
HPCD-treated juice at 25 MPa and 43 °C for 2 min was not discussed due to the difficult comparison upon the trilaminated effects of time, temperature and pressure. The PSDs of untreated
cloudy apple juice showed two peaks, which were characterized
by a large peak and small peak at approximately 1 and 7 lm,
5
A
6
Control
HPCD
2
1
4
3
2
1
0
0
0
2
4
6
8
Particle Diameter (µm)
10
12
C
Control
HPCD
5
Volume (%)
Volume (%)
3
6
Control
HPCD
5
4
Volume (%)
B
4
3
2
1
0
1
2
3
4
5
6
7
8
Particle Diameter (µm)
9 10 11
0
0
1
2
3
4
5
6
7
8
Particle Diameter (µm)
Fig. 3. Volume change of PSDs in cloudy apple juice as a function of treatment time induced by HPCD (A: 22 MPa, 60 °C, 3 min; B: 22 MPa, 60 °C, 5 min; C: 22 MPa, 60 °C,
10 min). The PSD of HPCD-treated cloudy apple juice at 25 MPa, 43 °C, and 2 min was not included. All data were the means, n = 3.
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Table 3
Change of particle size in cloudy apple juice induced by HPCD.
Treatment conditions
25 MPa,
22 MPa,
22 MPa,
22 MPa,
43 °C,
60 °C,
60 °C,
60 °C,
D[4,3]
2 min
3 min
5 min
10 min
D[3,2]
Control
HPCD
Control
HPCD
4.684 ± 1.945b
1.802 ± 0.046b
1.615 ± 0.055
2.132 ± 0.029
6.791 ± 0.531a
2.832 ± 0.320a
1.776 ± 0.193
2.039 ± 0.107
1.469 ± 0.143b
1.081 ± 0.007b
1.034 ± 0.016
1.207 ± 0.021b
1.989 ± 0.070a
1.333 ± 0.126a
1.060 ± 0.057
1.086 ± 0.036a
All data were the means ± SD, n = 3.
a,b
Different letters represented a significant difference within the same treatment.
Table 4
Change of some physical chemical properties in cloudy apple juice induced by HPCD.
Treatment conditions
25 MPa,
22 MPa,
22 MPa,
22 MPa,
43 °C,
60 °C,
60 °C,
60 °C,
2 min
3 min
5 min
10 min
Viscosity (CP)
TSS (°Brix)
pH
Turbidity(NTU)
Control
HPCD
Control
HPCD
Control
HPCD
Control
HPCD
1.41 ± 0.02
1.42 ± 0.02
1.39 ± 0.05
1.34 ± 0.01
1.43 ± 0.02
1.40 ± 0.00
1.38 ± 0.03
1.32 ± 0.01
8.1 ± 0.1
10.3 ± 0.1
10.2 ± 0.1
10.3 ± 0.2
8.0 ± 0.1
10.4 ± 0.0
10.1 ± 0.0
10.3 ± 0.1
4.21 ± 0.01
4.07 ± 0.02a
4.30 ± 0.09a
4.23 ± 0.00
4.22 ± 0.02
3.98 ± 0.02b
4.20 ± 0.06b
4.25 ± 0.02
47 ± 1
49 ± 0b
47 ± 0b
52 ± 0b
43 ± 2
60 ± 2a
57 ± 2a
57 ± 2a
All data were the means ± SD, n = 3.
a,b
Different letters represented a significant difference within the same treatment.
3.5. Change of turbidity, viscosity, TSS and pH of cloudy apple juice
The change of turbidity of cloudy apple juice after HPCD treatment is shown in Table 4. The turbidity of cloudy apple juice after
HPCD treatment at 25 MPa and 43 °C for 2 min did not change,
whereas it increased with HPCD at 22 MPa and 60 °C regardless
of treatment time. However, the particle size increased under these
conditions, apart from the one unchanged at 22 MPa and 60 °C for
10 min. It seems that the turbidity is not closely related to the particle size. Mc Clements (1999) found that the turbidity of dispersions depended on the concentration, size, and relative refractive
index of their particles. It is therefore reasoned that the treatment
conditions in this study probably favors the effect of any of these
factors on the turbidity. Kincal et al. (2006) suggested that the increase of turbidity could be attributed to the decrease of particles
of the juice colloid due to homogenization, which thereby increased cloud stability to PME attack. However, the PSD of HPCDtreated juice was not reported in this study.
It was found that the cloud destabilization process and the
cloud loss were caused by PME (Castaldo et al., 1997). However, active PME in cloudy apple juice after HPCD treatment was also observed in this study (Fig. 2) and the RA of PME in cloudy apple juice
was at least 41.70% after HPCD. Kincal et al. (2006) also found that
the cloudiness of orange juice was increased by up to 846% of its
original value with continuous HPCD at 38 MPa, despite active
PME still presented. Therefore, the turbidity of cloudy apple juice
is not necessarily associated with the RA of PME.
Table 4 also shows the change of some physico-chemical properties of cloudy apple juice induced by continuous HPCD. No significant differences in viscosity and TSS were observed. This was in
accordance to previous studies (Arreola et al., 1991; Kincal et al.,
2006; Yagiz et al., 2005). However, Zhou et al. (2009b) showed that
the viscosity of juices increased significantly. In this study, the pH
of cloudy apple juice was reduced significantly after HPCD treatment at 22 MPa and 60 °C for both 3 and 5 min. The decline of
pH in HPCD-treated juices is caused by the dissociation of carbonic
acid formed by the CO2 dissolution into the juices. A higher temperature and/or higher pressure may accelerate this process by
enhancing the diffusivity of CO2 into juice (Lin et al., 1993; Hong
et al., 1997), but a longer treatment time possibly retards this process. As discussed in the PSD section above, this observation was in
accordance with what was found for the PSD change. However,
most studies indicated that HPCD treatment did not cause a detectable pH change in orange juices (Arreola et al., 1991; Kincal et al.,
2006), except for Park et al. (2002) and Zhou et al. (2009b), which
reported a final pH drop in the carrot juice.
4. Conclusions
Microorganisms and native enzymes in cloudy apple juice
showed enhanced inactivation with increasing treatment temperature, pressure and time when subjected to continuous HPCD processing. The extend of colour (such as BD) was related to the level
of PPO inactivation. However, no correlation of the juice’s turbidity
was found with the change of RA of PME, or particle size distribution of the juice. The pH of the juice was reduced significantly after
HPCD treatments for 3 and 5 min at 60 °C and 22 MPa, which
seemed to influence the PSD of cloudy apple juice. This study also
showed that the decrease of the juice’s pH is higher at elevated
temperature (e.g. 60 °C), which possibly led to higher protein coagulation, while a longer treatment time may retard these effects by
enhancing pressure-induced homogenization. The quality characteristic of continuous HPCD-treated cloudy apple juice was good
but further studies are needed on storage stability and sensory
attributes are needed to prove its acceptance by consumers.
Acknowledgement
This research work was supported by Project No. 2006BAD05A02 of the Science and Technology Support in the Eleventh Five
Plan of China.
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