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Phase transitions during high pressure treatment of frozen carrot juice and influence on Escherichia coli inactivation
Accepted Manuscript Phase transitions during high pressure treatment of frozen carrot juice and influence on Escherichia coli inactivation Songming Zhu, Chunfang Wang, Hosahalli S. Ramaswamy, Yong Yu PII:
S0023-6438(17)30022-1
DOI:
10.1016/j.lwt.2017.01.022
Reference:
YFSTL 5974
To appear in:
LWT - Food Science and Technology
Received Date: 2 August 2016 Revised Date:
1 December 2016
Accepted Date: 8 January 2017
Please cite this article as: Zhu, S., Wang, C., Ramaswamy, H.S., Yu, Y., Phase transitions during high pressure treatment of frozen carrot juice and influence on Escherichia coli inactivation, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.01.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Phase transitions during high pressure treatment of frozen carrot juice and
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influence on Escherichia coli inactivation
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Songming Zhu a,b, Chunfang Wang a,b, Hosahalli S. Ramaswamy c, Yong Yu a,b,*
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a
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Road, Hangzhou 310058, China
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b
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Ministry of Agriculture, 866 Yuhangtang Road, Hangzhou 310058, China
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c
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Canada
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College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang
Key Laboratory of Equipment and Informatization in Environment Controlled Agriculture,
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Department of Food Science, McGill University, St-Anne-de-Bellevue, QC H9X 3V9,
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*
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E-mail address: [email protected] (Yong Yu)
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Corresponding author. Tel.: +86 571 88982181; fax: +86 571 88982181.
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Abstract
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Influence of high pressure (HP) treatment (200-400 MPa; 0-10 min) on phase
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transition behavior of frozen carrot juice and the resulting influence on the
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inactivation kinetics of Escherichia coli ATCC 25922 were evaluated. Experiments
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were carried out in a specially designed container to prevent heat exchange from the
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environment, except for the compression heating and decompression cooling. Solid to
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solid and solid to liquid transitions were recognized during HP treatment. Transition
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to Ice-III was observed from the temperature-pressure profiles when the application
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pressure was >350 MPa. Inactivation of E. coli in frozen carrot juice followed the first
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order kinetics with D values between 2.62 to 2.12 min at 300 to 400 MPa pressure
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levels, much shorter than those observed in unfrozen carrot juice. The combination of
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frozen state, phase transition status and pressure level likely contributed to the better
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inactivation of E. coli in frozen carrot juice.
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Keywords: High pressure; Frozen carrot juice; Phase transition
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1. Introduction High pressure (HP) processing is a promising alternative to thermal processing
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for extending the shelf-life and improving the safety of food products (Bari, Ukuku,
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Mori, Kawamoto, & Yamamoto, 2007; Picouet, Sárraga, Cofán, Belletti, & Dolors,
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2015; Ramaswamy, 2011; Vervoort et al., 2012). Studies have confirmed that HP
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processing can effectively inactivate vegetative cells of pathogenic microorganisms
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(Ramaswamy, Zaman, & Smith, 2008) without changing the sensory and nutritional
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properties of the food (Dede , Alpas, & Bayındırlı, 2007; Oey , Lille, Loey, &
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Hendrickx, 2008; Picouet et al., 2015). HP processing does not affect the low
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molecular and covalently bound food compounds, such as vitamins, flavoring agents,
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etc., and hence HP processing can provide “fresh-like” carrot juice with better sensory
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properties for long refrigerated storage (Picouet et al., 2015).
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HP effects on microorganisms depend on several factors, but the effects of
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pressure level and time have been studied most often. A number of studies have
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demonstrated that the combination of pressure and elevated temperatures have
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synergist acceleration of microbial destruction and enzyme inactivation kinetics
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(Ramaswamy, Riahi, & Idziak, 2003). Temperature effect on pressure destruction
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kinetics of microbial spores has also been shown to be significant when the
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temperatures are elevated and approached the lethal levels (Shao, Ramaswamy, &
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Zhu, 2007). HP effects on destruction kinetics have also been recognized to depend on
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ACCEPTED MANUSCRIPT product properties such as pH, soluble solid concentrations, composition (fat, protein
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and carbohydrate content) (Ramaswamy, Jin, & Zhu, 2009). In some cases, protection
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effect of pressure on thermal destruction kinetics at elevated temperature levels in the
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lethal range has been reported (Shao et al., 2007). This has been reported to be due to
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the increase in temperature and pressure causing opposite effects on volume
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expansion; with temperature contributing to volume increase while pressure reversing
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that effect. For similar reasons, some studies have shown that at sub-lethal levels, the
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pressure destruction effects are elevated at lower temperatures (Su et al., 2014).
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There are far fewer studies in HP processing research that use the combination of
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pressure and refrigerated conditions. Research related to the use of subzero (frozen)
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temperatures on inactivation kinetics are even more scarce (Luscher, Balasa, Fröhling,
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Ananta, & Knorr, 2004; Picart, Dumay, Guiraud, & Cheftel, 2005). Su et al., (2014)
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reported a combination or interaction effect of pressure and subzero temperature for
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the inactivation of microorganisms in food. Phase transitions of Ice-I / Ice-III by
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pressurizing frozen systems above 200 MPa was shown to be responsible for bacterial
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destruction (Fernández et al., 2007). However, there is little information on the
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evaluation of temperature profiles and phase transition in frozen samples under HP
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processing conditions, especially using a real food matrix.
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Fruit and vegetables are important components of a healthy diet. Their daily
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consumption in adequate quantities could help prevent several major diseases (Picouet
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et al., 2016), such as heart problem, cancer, diabetes and obesity, as well as the
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prevention and alleviation of several micronutrient deficiencies. Among common
ACCEPTED MANUSCRIPT fruits and vegetables, carrots are high in fibers, carotenoids, vitamins C and E, and
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phenolics such as p-coumaric, chlorogenic, and caffeic acids (Alasalvar, Grigor,
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Zhang, Quantick, & Shahidi, 2001). However, the low-acid condition in carrot juice is
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conducive to the growth of pathogenic microorganisms (Patterson, McKay, Connolly,
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& Linton, 2012). So storage of raw, unprocessed carrot juice may also lead to
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microbiological safety problems and shortened shelf-life. Therefore, extending the
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shelf-life using mild processing technologies that minimally affect the sensory and
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nutritional properties will be of interest for the food industry. Van Opstal et al. (2005)
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reported that pressure inactivation of Escherichia coli MG1655 was significantly
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lower in carrot juice than in buffer (150-600 MPa, 5-45 oC). Pilavtepe-Çelik et al.
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(2009) reported that carrot juice even had a protective effect on E. coli.
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Therefore, the objective of this study was to evaluate the phase transition
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behavior of frozen carrot juice during HP treatment and to extend the work of Su et al.
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(2014) on E. coli inactivation kinetics in frozen carrot juice. A specially designed
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container was used to isolate the sample and to achieve the ice phase transition under
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HP at room temperatures. This helped to overcome a serious practical problem of
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maintaining the entire pressure chamber at subzero temperatures. Maintaining subzero
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temperatures in commercial HP vessels is not only impractical but also be very
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expensive and energy intensive. Inactivation kinetic of E. coli was used for comparing
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the HP treatment effectiveness in frozen vs unfrozen carrot juice.
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2. Materials and methods
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2.1. Escherichia coli strain and culture preparation
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ACCEPTED MANUSCRIPT The culture of E. coli ATCC 25922 (CGMCC 1.2385) was obtained from China
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General Microbiological Culture Collection Center (CGMCC, Beijing, China). A
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fresh culture was prepared every 2 weeks to ensure their viability. To prepare the
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inoculation stock, several loops of isolated colonies of stock culture were transferred
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to 50 mL sterile nutrient broth (Sinopharm Chemical Reagent Co., Ltd. Shanghai,
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China) in 100 mL Erlenmeyer flasks and incubated at 37 oC for 24 h with agitation
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(150 rpm). Several loops of the incubated broth were then transferred to another 50
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mL sterile nutrient broth and incubated at 37 oC for 24 h incubation. Following this,
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30 mL incubated broth was aseptically transferred into a 50 mL sterilized centrifuge
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tube and centrifuged at 3200 g for 5 min at 20 oC (5810R, Eppendorf AG, Germany).
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The cell pellet obtained was re-suspended in 20 mL nutrient broth and enumerated
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using the pouring plate method on a brain heart infusion agar plate (BHIA)
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(Hangzhou Tianhe Microorganism Reagent Co., Ltd., Hangzhou, China), and
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incubated at 37 oC for 48 h and counted. The E coli selective media was not used in
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this study since a pure culture of E. coli was used and in order to obtain counts of both
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surviving and injured cells (Ramaswamy et al., 2003). The initial population of E. coli
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in the inoculated culture stock was approximately 108 colony forming units
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(CFU)/mL.
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2.2. Sample preparation
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Fresh carrots were purchased from Wal-Mart store near Zhejiang University
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(Hangzhou, China). The carrots were peeled and squeezed with a juice extractor
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(JYZ-B550, Joyoung Co., Ltd., Hangzhou, China), and the juice was then centrifuged
ACCEPTED MANUSCRIPT at 3200 g for 10 min to remove the suspended solids. The supernatant was filtered
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through a 0.23-mm-pore-diameter filter. The clear carrot juice had a pH of 6.5±0.3
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(FE 20, Mettler-Toledo, Shanghai, China). The juice was subjected to a thermal
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treatment, in a thermostatically controlled water bath (DKS-224, Zhongxin Medical
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Instrument Co., Ltd., Jiaxing, China) at 65 oC for 30 min. Microbial enumeration on
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BHI agar in heat treated samples returned negative counts.
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The prepared carrot juice was transferred aseptically into sterile bags (110 mm ×
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185 mm, BagLight PolySilk, Interscience, Paris, France) (about 150 mL for each bag)
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and sealed using a heat sealer (FS-300, Yongkang Teli Packing Machinery Co., Ltd.,
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China). The bags were frozen stored in a freezer (BC/BD-103HA, Haier, China) at -20
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C for subsequent handling.
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Carrot juice was fully thawed at 4 oC for ~4 h prior to inoculation of E. coli
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culture. After thawing, carrot juice was aseptically transferred to a sterile beaker. The
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previously prepared E. coli pellet was re-suspended in 20 mL carrot juice and then
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added to the rest of carrot juice in a beaker. After stirring at 500 rpm (RCT BS25, IKA,
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Staufen, Germany) for 1 min, the inoculated carrot juice was aseptically transferred to
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a sterile 2.0 mL cryogenic vial (430659, Corning Inc., USA), up to the brim and
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closed with sterile screw cap leaving no headspace to avoid possible cracking during
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HP treatment.
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2.3. Insulated container
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The cryogenic vials were individually vacuum packed in two layers of
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polyethylene bags and divided into two batches. One batch of the cryogenic vials were
ACCEPTED MANUSCRIPT then loaded into a specially designed plastic container consisting of a compressed
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sponge as previous study used (Su et al., 2014) and slightly modified, as shown in Fig.
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1. A K-type thermocouple (OMEGA Engineering, Stamford, CT, USA) was installed
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into the container for temperature recording using a data logger (34970A, Agilent
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Technologies GMBH, Germany). Four cryogenic vials were placed in one container
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with one of them used for recording sample temperature during HP treatment. The
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plastic container was filled water to completely soak the sponge and frozen at -20 oC
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for 24 h, and were secured by vacuum-packing in a flexible thermo-stable PA/PE
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pouches (30 cm × 42 cm) prior to HP treatment (details shown in Fig. 1). Samples of
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E. coli inoculated carrot juice frozen stored at -20 oC, prepared in a similar manner
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but without HP treatment, were used as control. The second batch was not frozen but
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stored at 4 oC and treated likewise for providing HP inactivation data under unfrozen
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conditions.
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2.4. High pressure equipment
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HP treatments were carried out in a laboratory-scale HP equipment (UHPF-750,
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Baotou Kefa High Pressure Technology Co., Ltd., China) with a maximum chamber
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capacity of 5 L. The high pressure unit was connected to a data logger for temperature
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and pressure (current signal) recoding during HP treatment. Water was used as the
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pressure-transmitting medium. The pressure vessel was maintained at room
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temperature (~20 oC) and with some added ice if the temperature was above 20 oC
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before pressure treatment. The near room temperature setup was used so that the
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process can be replicated in other commercial HP equipment using the special
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attachment. The pressure come-up rate was about 200 MPa/min and the
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depressurization time was less than 10 s. Data were recorded at one second interval.
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2.5. HP treatment The frozen samples carrot juice were subjected to HP treatment at selected
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pressure levels (200, 250, 300, 350 and 400 MPa) for selected treatment times (0, 1, 3,
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5, 7.5 and 10 min holding time). Unfrozen samples were only treated at 300, 350, and
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400 MPa for holding times 0, 1, 3, 5, 7.5 and 10 min. The pressure holding times
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mentioned did not include the pressure come-up time (1-2 min) and pressure release
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time (~10 s). After HP treatment of frozen samples, the special plastic container
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holding the test vials were observed to make sure water soaked sponge remained
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largely in the frozen state (melting extending no more than 7 mm on the sides) and
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then immersed in running water (room temperature) for 2 h to complete thawing.
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Treated sample vials (thawed vials of frozen and vials of unfrozen) were held at 4 oC
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(maximum 4 h) prior to microbial enumeration. Due to adiabatic heating, the
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temperature of the pressure transmission medium (water) is expected to increase
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about 3 oC per 100 MPa on an average. However, because of addition of some ice, the
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water temperature during the treatment remained between 23 and 30 oC.
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2.6. Enumeration of E. coli
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The sample vials were aseptically opened and the carrot juice was aseptically
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transferred to a sterile tube, followed by a serial dilution in 1 g/L peptone water
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(Sinopharm Chemical Reagent Co., Ltd, Shanghai, China). Surviving cells of E. coli
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were enumerated on BHIA using pour plate method. Colonies of survived E. coli were
ACCEPTED MANUSCRIPT counted after aerobic incubation at 37 oC for 48 h. Initial counts of E. coli in carrot
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juice were obtained likewise from untreated control samples both at 4 and -20 oC to
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differentiate the effect of freezing on microbial reduction.
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2.7. Kinetic analysis
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The pressure inactivation kinetics of E. coli during pressure holding time was
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analyzed based on a first order reaction rate (Shao et al., 2007) indicating a
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logarithmic order of death, and expressed as: N t log10 t = − D N0
(1)
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where Nt is the number of E. coli survivors (CFU mL-1) after HP hold time
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treatment time of t (min), and N0 is the initial number of E. coli (CFU mL-1) in frozen
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samples at -20 oC without HP treatment. D value (min) is the decimal reduction time
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of E. coli which implies treatment holding time in minutes at constant pressure for 90 %
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destruction of the existing microbial population. D values were obtained from the
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linear regression slope of log10(Nt/N0) versus t as shown in Eq. (2) or time taken to
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traverse one logarithmic cycle:
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(2)
For pressure resistance (pressure sensitivity) analysis, Zp values were used (Shao
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et al., 2007). Zp value represented the pressure range between which the D value
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change by a decimal factor and can be determined as the negative reciprocal of the
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slope of log10 (D) versus pressure curve.
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Statistical analysis was performed using one-way analysis of variance (ANOVA).
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Duncan’s test (P < 0.05) was applied to compare the differences in average values by
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3. Results and discussion
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3.1. Temperature and phase transition in frozen carrot juice during HP
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treatment
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Fig. 2 illustrates the transient temperatures in frozen carrot juice during HP
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treatment for a holding time of 1 min at different pressure levels (Fig. 2a, c, e, g, i)
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and temperature-pressure profiles of frozen carrot juice superimposed on the phase
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diagram of water (Bridgman, 1912) (Fig. 2b, d, f, h, j), respectively. Pressure
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increased in a stepwise fashion and the duration of t1-t2 depended on the final pressure.
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During pressure treatment 400 MPa, come up time was 2 min and shorter times were
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required proportionally at other pressure levels. The interval t2-t3 was 60 s in all plots.
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During decompression, the pressure decreased (t3-t4) rapidly within 10 s. Table 1
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shows the temperatures achieved during HP treatment of frozen carrot juice. It can be
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observed that the temperature was kept well below the conventional freezing point
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during HP freezing,
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The intensifier used for generating high pressure was a batch type unit which
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generated a stepwise ladder-like pressurization build up. Hence the ladder-like
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pressure and temperature change was visible during the compression. Carrot juice
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samples contained in the special container were taken out from a freezer at -20 oC.
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The temperature rose slightly during the preparation, vacuum packing, connecting of
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thermocouples, installation of sample container into the pressure chamber and the
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quick filling of water into the chamber. During compression, the temperature still rose
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ACCEPTED MANUSCRIPT at first, as the heat of compression and the heat input from pressure chamber exceeded
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the heat uptake of melting ice crystals (endothermic reaction) in the sponge
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surrounding the vials. Partial thawing occurred and the melting of ice crystals
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increased in the sponge. However, the heat of compression and the heat input from
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pressure chamber were insufficient to fully melt ice in the sponge and hence help to
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protect the frozen carrot juice in the test vials. The temperature decreased upon further
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compression due to lowering of the freezing point and approached the melting line of
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carrot juice (water melting line shown in Fig. 2). Heat of compression and latent heat
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in this phase were in equilibrium, and solid-liquid transition was initiated. When the
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temperature was -10 oC or -5 oC at the beginning (Fig. 2c, g), the temperature could
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decrease even during compression and approached the melting line. Because the
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temperature was still lower than the freezing point at the process pressure, the results
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indicated that the special container with water soaked frozen sponge had enough heat
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sink and insulation to protect the sample from heat gain and hold them under frozen
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state.
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According to the temperature-pressure profiles superimposed on the phase
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diagram of water in Fig. 2 (b, d, f, h, j), the metastable Ice-I region was positioned
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above 220 MPa. This pressure was little higher than 207 MPa reported by Su et al.
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(2014) for E. coli suspension. On the other hand, the phase transition point of pure
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water is 210 MPa. Hence there existed some small differences which were probably
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due to differences in the nature of the support medium (water vs carrot juice). In Fig.
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2 (g and i), a sudden increase in sample temperature (-35 to -19 oC) was observed
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ACCEPTED MANUSCRIPT during compression (highlighted in a gray circle). A sudden pressure decrease (349 to
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334 MPa) was also observed at 400 MPa (highlighted in a dark gray circle in Fig. 2i).
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These results indicated the transition to Ice-III, including metastable Ice-I to Ice-III
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(endothermic reaction) and water re-crystallization to Ice-III (exothermic reaction).
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Because Ice-III (1.14 g/cm3) has a higher density than water (1 g/cm3) and Ice-I (0.92
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g/cm3), the volume change contributes to a change in pressure. The temperature
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increase might be caused by the exothermic transformation of some liquid water to
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Ice-III (Van Buggenhout, Grauwet, Van Loey, & Hendrickx, 2007).
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After compression, the temperatures at 200, 250, 300, 350 and 400 MPa
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remained at -19, -21, -27, -18 and -18 oC, respectively, with a slight increase. Since
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the surrounding temperature was always higher than the sample container, it is likely
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that thawing continued (endothermic reaction) in surrounding sponge space in the
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container during the holding phase while keeping the sample to stay at lower
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temperature. When the pressure was released, the phase transition from Ice-III to Ice-I
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and then to liquid water resulted at 350 and 400 MPa (exothermic reaction). At
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pressures lower than 350 MPa, phase transition of metastable Ice-I to Ice-I and water
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seemed to occur (exothermic reaction).
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Super cooling was caused during the decompression, so partial pressure shift
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freezing also occurred (exothermic reaction). The sample temperature decrease as a
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result of expansion cooling was only observed at 350 and 400 MPa during
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decompression. During depressurization, the increase or decrease of sample
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temperature depended on the combined effect of expansion cooling, the amount of
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melted water converted to Ice-I (an exothermic reaction) and the amount of Ice-III
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converted to Ice-I (also an exothermic reaction) (Van Buggenhout, Messagie, Van der
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Plancken, & Hendrickx, 2006). At higher pressures (above ~220 MPa), a solid-solid transition could be induced
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as the theoretical Ice-I and Ice-III phase transition line of pure water was exceeded
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upon compression (Van Buggenhout et al., 2007). Similar phase transition of Ice-I to
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Ice-III has been reported at pressures above 250 MPa (Su et al., 2014; Van
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Buggenhout et al., 2007), but the sudden changes in pressure and temperature were
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not similar. Again this might due to the differences in the media used: microbial
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culture (335 MPa, -35 to -18 oC), carrot (280 MPa, -36 to -30 oC) and carrot juice
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(349 MPa, -35 to -19 oC). Fernández et al. (2007) reported that phase diagram for beef
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meat showed the same phase transition regions as water, but shifted to lower
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temperature and different pressure values.
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3.2. High pressure inactivation of E. coli in unfrozen carrot juice
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The average initial concentration of E. coli in carrot juice before freezing was 7.8
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× 107 CFU/mL. After frozen storage for 24 h, E. coli count in carrot juice was reduced
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slightly to 7.65 × 107 CFU/mL. The initial concentration of E. coli in carrot juice
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samples varied between 107 and 108 CFU/mL and around were normalized to 107
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CFU/mL (by dividing all counts by their actual initial count and then multiplying by
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107). This facilitates better presentation of survival curves for comparison purposes
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with all curves starting from the same point. The normalization process only results in
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a small intercept shift and will not affect the slope, and therefore D values remain the
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same. Fig. 3 shows the survivor curves of E. coli in the unfrozen carrot juice, and Table
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2 shows the computed kinetic parameters of E. coli for HP treatment. The extent of
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microbial inactivation at 300 and 350 MPa during the 10 min holding time was small
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resulting in calculated D values of 28.5 and 9.2 min, respectively. It improved a little
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better at 400 MPa, with a calculated D value of 5.3 min. The range of holding times
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(0-10 min) was low but kept the same as for HP treatment of frozen samples to
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provide baseline data for comparison.
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The extent of HP inactivation of E. coli in unfrozen carrot juice were similar to
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those reported by other researchers (Bari et al., 2007; Patterson et al., 2012;
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Pilavtepe-Çelik et al., 2009; Van Opstal et al., 2005). D values ranging from 38 to 2
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min were reported by Van Opstal et al. (2005) for E. coli MG1655 inactivation after
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HP treatment at 300-400 MPa in the temperature range 23-40 oC. Su et al. (2014)
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reported slightly lower D values for the same strain of E. coli suspended in water. A
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carrot juice matrix was used in this study and the use of real food matrix could have
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been the reason for the observed higher values. Others have also reported that cells in
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food matrix are more resistant than in buffer solutions (Pilavtepe-Çelik et al., 2009;
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Alpas, Lee, Bozoglu, & Kaletunç, 2003).
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3.3. High pressure inactivation of E. coli in frozen carrot juice
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Fig. 4 shows the logarithmic survivor curves of E. coli in frozen carrot juice
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subjected to HP treatment at 200 to 400 MPa for 0 to 10 min holding time. The E. coli
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inactivation in frozen samples also followed the first order rate kinetics during the
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pressure holding time (R2>0.95). Table 3 shows the D values and other kinetic
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parameters of E. coli in frozen carrot juice treated by HP. The reduction trends in E. coli in frozen carrot juice were similar to those in
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unfrozen carrot juice samples - survivors decreased with increasing pressure and
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treating time, only by a greater margin (both first order rate). The survival lines were
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steeper at higher pressures indicating that the associated D values decreased at higher
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pressures.
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The E. coli count reduction at 200 MPa after pressure treatment for 0 and 10 min
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were 0.42 and 3.00 log10CFU/mL, while they increased to 2.00 and 6.80
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log10CFU/mL at 400 MPa, respectively. The count reduction was far more in frozen
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carrot juice than in unfrozen samples. For example, after 10 min treatment at 400
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MPa, the reduction of E. coli count in unfrozen samples was 1.87 log10CFU/mL, but
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6.80 log10CFU/mL in frozen samples. The calculated D value (5.32 min) of E. coli at
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400 MPa in unfrozen samples was longer than the D values in frozen samples
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obtained even at much lower pressures of 200 and 250 MPa (4.03 and 3.97 min,
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respectively. The D value of frozen carrot juice at 400 MPa (2.12 min) found in this
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study was close to Van Opstal et al. (2005) reported value of 2.0 min of E. coli
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MG1655 at 400 MPa, 40 oC in freshly extracted carrot juice; but they found the D
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values to be longer at lower temperatures (5-30 oC). The D value of frozen carrot
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juice at 300 MPa (2.62 min) was consistent with Su et al. (2014) of frozen E. coli
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suspension (2.59 min). The frozen state therefore contributed to a remarkable
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enhancement of E. coli inactivation in carrot juice.
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ACCEPTED MANUSCRIPT From the D value data, the computed pressure sensitivity parameter (Zp value)
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was 613 MPa much higher than the 196 MPa reported by Su et al. (2014). It is clear
335
that the pressure sensitivity was affected significantly when carrot juice was treated in
336
the frozen state. The nature of the phase transition, pressure and sample state could be
337
the reasons for this observed remarkable difference in the computed Zp value.
338
Although the D value data were combined for the purpose of computation of Zp value,
339
it can be seen from Fig. 3 that the HP inactivation rate of E. coli at the two lower
340
pressure levels (200, 250 MPa) were distinctly different from those at the two higher
341
levels (300-400 MPa). At 200 and 250 MPa the associated D values were 4.03 and
342
3.97 min, respectively, while at 300, 350 and 400 MPa they were 2.62, 2.21 and 2.12
343
min, respectively, indicating a break in the continuity pattern. Therefore, although the
344
inactivation rate increased with pressure level (consistent with general understanding),
345
a pattern difference was also apparent. This is probably due to the re-crystallization of
346
Ice-III and phase transition from metastable Ice-I to Ice-III during compression and
347
Ice-III to Ice-I and/or water during decompression, when the pressure was above 250
348
MPa. It has been reported earlier that phase transitions may lead to mechanical
349
disintegration of cells (Edebo & Hedén, 1960) and the inactivation would be caused
350
by a mechanical effect related to the Ice-I to Ice-III phase transition (Luscher et al.,
351
2004; Fernández et al., 2007). The crystal formation and phase transition therefore
352
could play important roles in the HP in inactivation on E. coli in frozen medium.
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3.4. Pressure-pulse inactivation of E. coli in frozen carrot juice
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The inactivation effect of pressure pulse on E. coli in frozen carrot juice is shown
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pressure levels from 200 to 400 MPa (data not shown). However, the pressure pulse
357
effect was clearly noticeable in frozen carrot juice samples which was low at 200 and
358
250 MPa, but significantly increased to 0.85, 1.41 and 2.00 logarithmic cycle
359
reductions at 300, 350 and 400 MPa, respectively. It was consistent with the D values
360
pattern between 200-250 MPa and 300-400 MPa discussed earlier. Therefore these
361
differences could also be ascribed to the frozen state of carrot juice and phase
362
transition during HP, especially the Ice-I/Ice-III transition above 300 MPa. Bari et al.
363
(2007) reported that repeating a 10 min high pressure treatment cycle with a 5 sec
364
pause could achieve extra reduction. It is possible that repeating HP pulse treatment
365
likewise would get more reduction as demonstrated in some previous studies with
366
multiple pressure pulse inactivation (Basak and Ramaswamy, 2011).
367
4. Conclusions
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The transient time temperature data obtained under pressure processing
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conditions involving frozen carrot juice revealed that the ice structure could be
370
predominantly of type Ice-III or Ice-I. Solid-solid and solid-liquid transitions were
371
apparent during compression and decompression. E. coli inactivation in frozen carrot
372
juice followed the first order rate kinetics after accounting for a pulse effect
373
inactivation. The high pressure inactivation of E. coli in frozen samples was far better
374
than in unfrozen samples, and was enhanced by pressure level and holding time. The
375
phase transition, pressure level and sample state contributed to the better inactivation
376
of E. coli in frozen carrot juice, especially the Ice-I/Ice-III transitions at pressure >
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378
potential for commercial application. Further, the special insulator attachment
379
provides opportunity for accomplishing HP inactivation in the frozen state using HP
380
equipment operating at room temperatures. However, further studies are needed to
381
evaluate the stability and the influence on the food quality and properties and the use
382
of these processes might be limited due to the frozen state.
Acknowledgements
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This work was supported by the Key Program of Natural Science Foundation of
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Zhejiang Province (Grant No. LZ14C200002) and the National Natural Science
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Foundation of China (Grant No. 31171779).
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Alasalvar, C., Grigor, J. M ., Zhang, D., Quantick, P. C., & Shahidi, F. (2001).
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Comparison of volatiles, phenolics, sugars, antioxidant vitamins, and sensory
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pressure sensitivity of Staphylococcus aureus and Escherichia coli O157:H7 by
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Bari, M. L., Ukuku, D. O., Mori, M., Kawamoto, S., & Yamamoto, K. (2007). Effect
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of high-pressure treatment on survival of Escherichia coli O157:H7 population
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in tomato juice. Journal of Food Agriculture & Environment, 5(1), 111-115.
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Basak, S., & Ramaswamy, H. S. (2001). Pulsed high pressure inactivation of pectin
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methyl esterase in single strength and concentrated orange juices. Canadian
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Biosystems Engineering, 43(3), 25-29. Bridgman, P. W. (1912). Proc.Am. Acad.Arts Sci. XLVIl(13),439-558
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Dede, S., Alpas, H., & Bayındırlı, A. (2007). High hydrostatic pressure treatment and
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storage of carrot and tomato juices: Antioxidant activity and microbial safety.
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Journal of the Science of Food and Agriculture, 87(5), 773-782.
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Edebo, L., & Hedén, C. G. (1960). Disruption of frozen bacteria as a consequence of
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changes in the crystal structure of ice. Journal of Biochemistry, Microbiology
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and Technological Engineering, 2(1), 113-120.
Fernández, P. P., Sanz, P. D., Molina-García, A. D., Otero, L., Guignon, B., &
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Vaudagna, S. R. (2007). Conventional freezing plus high pressure–low
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temperature treatment: Physical properties, microbial quality and storage
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stability of beef meat. Meat Science, 77(4), 616-625.
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Luscher, C., Balasa, A., Fröhling, A., Ananta, E., & Knorr, D. (2004). Effect of
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high-pressure-induced Ice I-to-Ice III phase transitions on inactivation of Listeria
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innocua in frozen suspension. Applied & Environmental Microbiology, 70(7),
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4021-9.
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Oey, I., Lille, M., Loey, A. V., & Hendrickx, M. (2008). Effect of high-pressure
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processing on colour, texture and flavour of fruit- and vegetable-based food
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products: a review. Trends in Food Science & Technology, 19(6), 320-328.
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Patterson, M. F., McKay, A. M., Connolly, M., & Linton, M. (2012). The effect of
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high hydrostatic pressure on the microbiological quality and safety of carrot juice
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during refrigerated storage. Food Microbiology, 30(1), 205-212.
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Picouet, P. A., Hurtado, A., Jofré, A., Bañon, S., Ros, J., & Dolors Guàrdia, M.
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(2016). Effects of thermal and high-pressure treatments on the microbiological,
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nutritional and sensory quality of a multi-fruit smoothie. Food and Bioprocess Technology, 9(7), 1219-1232.
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Picouet, P. A., Sárraga, C., Cofán, S., Belletti, N., & Dolors Guàrdia, M. (2015).
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Effects of thermal and high-pressure treatments on the carotene content,
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microbiological safety and sensory properties of acidified and of non-acidified
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carrot juice. LWT - Food Science and Technology, 62(1), 920-926.
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Picart, L., Dumay, E., Guiraud, J. P., & Cheftel, J. C. (2005). Combined high
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pressure-subzero temperature processing of smoked salmon mince: phase
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transition phenomena and inactivation of Listeria innocua. Journal of Food
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Engineering, 68(1), 43–56. Pilavtepe-Çelik, M., Buzrul, S., Alpas, H., & Bozoğlu, F. (2009). Development of a
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new mathematical model for inactivation of Escherichia coli O157:H7 and
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Staphylococcus aureus by high hydrostatic pressure in carrot juice and peptone
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water. Journal of Food Engineering, 90(3), 388-394.
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Ramaswamy, H. S. (2011). High pressure sterilization of foods. Food Engineering
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Interfaces, Food Engineering Series, J.M. Aguilera et al. (eds.), DOI
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10.1007/978-1-4419-7475-4_15, # Springer Science & Business Media, LLC. Pp
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Ramaswamy, H. S., Jin, H., & Zhu, S. (2009). Effects of fat, casein and lactose on
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high-pressure destruction of Escherichia coli K12 (ATCC-29055) in milk. Food
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and Bioproducts Processing, 87(C1),1-6.
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Ramaswamy, H. S., Riahi, E., & Idziak, E. (2003). High-pressure destruction kinetics
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of E. coli (29055) in apple juice. Journal of Food Science, 68, 1750-1756. Ramaswamy, H. S., Zaman, S. U., & Smith, J. P. (2008). High pressure destruction
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kinetics of Escherichia coli (O157:H7) and Listeria monocytogenes (Scott A) in
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a fish slurry. Journal of Food Engineering, 87(1), 99-106.
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Shao, Y., Ramaswamy, H. S., & Zhu, S. (2007). High pressure destruction kinetics of
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spoilage and pathogenic bacteria in raw milk cheese. Journal of Food Process
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Engineering, 30(3), 357-374.
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Silva, F.V.M., Tan, E.K., & Farid, M. (2012). Bacterial spore inactivation at 45-65 oC
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using high pressure processing: Study of Alicyclobacillus acidoterrestris in
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orange juice. Food Microbiology, 32(1), 206-11.
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Su, G., Yu, Y., Ramaswamy, H. S., Hu, F., Xu, M., & Zhu, S. (2014). Kinetics of
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Escherichia coli inactivation in frozen aqueous suspensions by high pressure and
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its application to frozen chicken meat. Journal of Food Engineering, 142, 23-30.
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Van Buggenhout, S., Grauwet, T., Van Loey, A., & Hendrickx, M. (2007). Effect of
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high-pressure induced ice I/ice III-transition on the texture and microstructure of
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fresh and pretreated carrots and strawberries. Food Research International,
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40(10), 1276-1285. Van Buggenhout, S., Messagie, I., Van der Plancken, I., & Hendrickx, M. (2006).
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Influence of high-pressure–low-temperature treatments on fruit and vegetable
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quality related enzymes. European Food Research and Technology, 223(4),
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475-485.
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Van Opstal, I., Vanmuysen, S. C. M., Wuytack, E. Y., Masschalck, B., & Michiels, C.
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W. (2005). Inactivation of Escherichia coli by high hydrostatic pressure at
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different temperatures in buffer and carrot juice. International Journal of Food
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Microbiology, 98(2), 179-191.
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Vervoort, L., Plancken, I. V. D., Grauwet, T., Verlinde, P., Matser, A., Hendrickx, M.,
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& Loey, A. V. (2012). Thermal versus high pressure processing of carrots: A
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comparative pilot-scale study on equivalent basis. Innovative Food Science &
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Emerging Technologies, 15(1), 1-13.
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Figure Captions.
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Fig. 1. Schematic cross-section and details of the insulated container for holding test
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samples (the red color material is the sponge covering the vials).
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Fig. 2. Temperature changes with pressure of frozen carrot juice samples during HP
482
treatment holding for 1 min under different pressures (a, c, e, g, i) and
483
temperature-pressure profiles superimposed on the phase diagram of water (b, d, f, h,
484
j).
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Fig. 3. E. coli survivors in unfrozen carrot juice samples after different high pressure
486
(300 MPa (○), 350 MPa (△), 400 MPa (◊)) and different holding time.
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Fig. 4. E. coli survivors in frozen carrot juice samples after different high pressure
488
(200 MPa (□), 250 MPa (▽)300 MPa (○), 350 MPa (△), 400 MPa (◊)) and different
489
holding time.
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ACCEPTED MANUSCRIPT Table 1 Temperatures achieved at the end of pressure come up time and holding time (n=3). Temperature (oC) Pressure (MPa) 200
-20.1±1.1a
-19.2±1.7
250
-22. 6±1.3
300
-27.5±0.3
350
-18.2±0.2
400
-17.6±0.8
-21.7±1.5
-26.6±0.0 -18.2±0.2
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Mean values±standard deviation
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End of holding 1 min
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ACCEPTED MANUSCRIPT Table 2 Inactivation kinetics of E. coli in unfrozen carrot juice under high pressure. D value (min)
R2Adj
Zp value (MPa)
R2Adj
300
28.53±5.27 a
0.850
139±28
0.922
350
9.20±1.03
0.940
400
5.32±1.06
0.827
Standard error (SE)
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ACCEPTED MANUSCRIPT Table 3 Inactivation kinetics of E. coli in frozen carrot juice under high pressure. D value (min)
R2Adj
Zp value (MPa)
R2Adj
200
4.03±0.35a
0.963
613±118
0.866
250
3.97±0.39
0.954
300
2.62±0.19
0.974
350
2.21±0.10
0.991
400
2.12±0.08
0.994
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SE - standard error
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ACCEPTED MANUSCRIPT Table 4 Pulse effect of high pressure on E. coli in frozen carrot juice. Mean values±standard deviation (n = 3). Reduction (log10CFU/mL) 0.42±0.11a
250
0.53±0.05a
300
0.85±0.09b
350
1.41±0.35c
400
2.00±0.24d
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Pressure (MPa)
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Different letters (a, b, c, d) indicate significant difference (p < 0.05) between treatments.
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Highlights Phase transition in frozen carrot juice was evaluated under HP at room temperature.
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Transition to Ice III was observed at pressures above 350 MPa. Special container was used to maintain frozen conditions during treatment. Enhanced inactivation of E. coli was demonstrated in frozen carrot juice.
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Substrate state, phase transition and pressure level influenced inactivation.
S0023-6438(17)30022-1
DOI:
10.1016/j.lwt.2017.01.022
Reference:
YFSTL 5974
To appear in:
LWT - Food Science and Technology
Received Date: 2 August 2016 Revised Date:
1 December 2016
Accepted Date: 8 January 2017
Please cite this article as: Zhu, S., Wang, C., Ramaswamy, H.S., Yu, Y., Phase transitions during high pressure treatment of frozen carrot juice and influence on Escherichia coli inactivation, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.01.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Phase transitions during high pressure treatment of frozen carrot juice and
1 2
influence on Escherichia coli inactivation
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Songming Zhu a,b, Chunfang Wang a,b, Hosahalli S. Ramaswamy c, Yong Yu a,b,*
4
a
5
Road, Hangzhou 310058, China
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b
7
Ministry of Agriculture, 866 Yuhangtang Road, Hangzhou 310058, China
8
c
9
Canada
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College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang
Key Laboratory of Equipment and Informatization in Environment Controlled Agriculture,
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Department of Food Science, McGill University, St-Anne-de-Bellevue, QC H9X 3V9,
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*
11
E-mail address: [email protected] (Yong Yu)
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Corresponding author. Tel.: +86 571 88982181; fax: +86 571 88982181.
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Abstract
14
Influence of high pressure (HP) treatment (200-400 MPa; 0-10 min) on phase
15
transition behavior of frozen carrot juice and the resulting influence on the
16
inactivation kinetics of Escherichia coli ATCC 25922 were evaluated. Experiments
17
were carried out in a specially designed container to prevent heat exchange from the
18
environment, except for the compression heating and decompression cooling. Solid to
19
solid and solid to liquid transitions were recognized during HP treatment. Transition
20
to Ice-III was observed from the temperature-pressure profiles when the application
21
pressure was >350 MPa. Inactivation of E. coli in frozen carrot juice followed the first
22
order kinetics with D values between 2.62 to 2.12 min at 300 to 400 MPa pressure
23
levels, much shorter than those observed in unfrozen carrot juice. The combination of
24
frozen state, phase transition status and pressure level likely contributed to the better
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inactivation of E. coli in frozen carrot juice.
26
Keywords: High pressure; Frozen carrot juice; Phase transition
27
1. Introduction High pressure (HP) processing is a promising alternative to thermal processing
29
for extending the shelf-life and improving the safety of food products (Bari, Ukuku,
30
Mori, Kawamoto, & Yamamoto, 2007; Picouet, Sárraga, Cofán, Belletti, & Dolors,
31
2015; Ramaswamy, 2011; Vervoort et al., 2012). Studies have confirmed that HP
32
processing can effectively inactivate vegetative cells of pathogenic microorganisms
33
(Ramaswamy, Zaman, & Smith, 2008) without changing the sensory and nutritional
34
properties of the food (Dede , Alpas, & Bayındırlı, 2007; Oey , Lille, Loey, &
35
Hendrickx, 2008; Picouet et al., 2015). HP processing does not affect the low
36
molecular and covalently bound food compounds, such as vitamins, flavoring agents,
37
etc., and hence HP processing can provide “fresh-like” carrot juice with better sensory
38
properties for long refrigerated storage (Picouet et al., 2015).
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HP effects on microorganisms depend on several factors, but the effects of
40
pressure level and time have been studied most often. A number of studies have
41
demonstrated that the combination of pressure and elevated temperatures have
42
synergist acceleration of microbial destruction and enzyme inactivation kinetics
43
(Ramaswamy, Riahi, & Idziak, 2003). Temperature effect on pressure destruction
44
kinetics of microbial spores has also been shown to be significant when the
45
temperatures are elevated and approached the lethal levels (Shao, Ramaswamy, &
46
Zhu, 2007). HP effects on destruction kinetics have also been recognized to depend on
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ACCEPTED MANUSCRIPT product properties such as pH, soluble solid concentrations, composition (fat, protein
48
and carbohydrate content) (Ramaswamy, Jin, & Zhu, 2009). In some cases, protection
49
effect of pressure on thermal destruction kinetics at elevated temperature levels in the
50
lethal range has been reported (Shao et al., 2007). This has been reported to be due to
51
the increase in temperature and pressure causing opposite effects on volume
52
expansion; with temperature contributing to volume increase while pressure reversing
53
that effect. For similar reasons, some studies have shown that at sub-lethal levels, the
54
pressure destruction effects are elevated at lower temperatures (Su et al., 2014).
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There are far fewer studies in HP processing research that use the combination of
56
pressure and refrigerated conditions. Research related to the use of subzero (frozen)
57
temperatures on inactivation kinetics are even more scarce (Luscher, Balasa, Fröhling,
58
Ananta, & Knorr, 2004; Picart, Dumay, Guiraud, & Cheftel, 2005). Su et al., (2014)
59
reported a combination or interaction effect of pressure and subzero temperature for
60
the inactivation of microorganisms in food. Phase transitions of Ice-I / Ice-III by
61
pressurizing frozen systems above 200 MPa was shown to be responsible for bacterial
62
destruction (Fernández et al., 2007). However, there is little information on the
63
evaluation of temperature profiles and phase transition in frozen samples under HP
64
processing conditions, especially using a real food matrix.
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Fruit and vegetables are important components of a healthy diet. Their daily
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consumption in adequate quantities could help prevent several major diseases (Picouet
67
et al., 2016), such as heart problem, cancer, diabetes and obesity, as well as the
68
prevention and alleviation of several micronutrient deficiencies. Among common
ACCEPTED MANUSCRIPT fruits and vegetables, carrots are high in fibers, carotenoids, vitamins C and E, and
70
phenolics such as p-coumaric, chlorogenic, and caffeic acids (Alasalvar, Grigor,
71
Zhang, Quantick, & Shahidi, 2001). However, the low-acid condition in carrot juice is
72
conducive to the growth of pathogenic microorganisms (Patterson, McKay, Connolly,
73
& Linton, 2012). So storage of raw, unprocessed carrot juice may also lead to
74
microbiological safety problems and shortened shelf-life. Therefore, extending the
75
shelf-life using mild processing technologies that minimally affect the sensory and
76
nutritional properties will be of interest for the food industry. Van Opstal et al. (2005)
77
reported that pressure inactivation of Escherichia coli MG1655 was significantly
78
lower in carrot juice than in buffer (150-600 MPa, 5-45 oC). Pilavtepe-Çelik et al.
79
(2009) reported that carrot juice even had a protective effect on E. coli.
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Therefore, the objective of this study was to evaluate the phase transition
81
behavior of frozen carrot juice during HP treatment and to extend the work of Su et al.
82
(2014) on E. coli inactivation kinetics in frozen carrot juice. A specially designed
83
container was used to isolate the sample and to achieve the ice phase transition under
84
HP at room temperatures. This helped to overcome a serious practical problem of
85
maintaining the entire pressure chamber at subzero temperatures. Maintaining subzero
86
temperatures in commercial HP vessels is not only impractical but also be very
87
expensive and energy intensive. Inactivation kinetic of E. coli was used for comparing
88
the HP treatment effectiveness in frozen vs unfrozen carrot juice.
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2. Materials and methods
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2.1. Escherichia coli strain and culture preparation
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92
General Microbiological Culture Collection Center (CGMCC, Beijing, China). A
93
fresh culture was prepared every 2 weeks to ensure their viability. To prepare the
94
inoculation stock, several loops of isolated colonies of stock culture were transferred
95
to 50 mL sterile nutrient broth (Sinopharm Chemical Reagent Co., Ltd. Shanghai,
96
China) in 100 mL Erlenmeyer flasks and incubated at 37 oC for 24 h with agitation
97
(150 rpm). Several loops of the incubated broth were then transferred to another 50
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mL sterile nutrient broth and incubated at 37 oC for 24 h incubation. Following this,
99
30 mL incubated broth was aseptically transferred into a 50 mL sterilized centrifuge
100
tube and centrifuged at 3200 g for 5 min at 20 oC (5810R, Eppendorf AG, Germany).
101
The cell pellet obtained was re-suspended in 20 mL nutrient broth and enumerated
102
using the pouring plate method on a brain heart infusion agar plate (BHIA)
103
(Hangzhou Tianhe Microorganism Reagent Co., Ltd., Hangzhou, China), and
104
incubated at 37 oC for 48 h and counted. The E coli selective media was not used in
105
this study since a pure culture of E. coli was used and in order to obtain counts of both
106
surviving and injured cells (Ramaswamy et al., 2003). The initial population of E. coli
107
in the inoculated culture stock was approximately 108 colony forming units
108
(CFU)/mL.
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2.2. Sample preparation
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Fresh carrots were purchased from Wal-Mart store near Zhejiang University
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(Hangzhou, China). The carrots were peeled and squeezed with a juice extractor
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(JYZ-B550, Joyoung Co., Ltd., Hangzhou, China), and the juice was then centrifuged
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114
through a 0.23-mm-pore-diameter filter. The clear carrot juice had a pH of 6.5±0.3
115
(FE 20, Mettler-Toledo, Shanghai, China). The juice was subjected to a thermal
116
treatment, in a thermostatically controlled water bath (DKS-224, Zhongxin Medical
117
Instrument Co., Ltd., Jiaxing, China) at 65 oC for 30 min. Microbial enumeration on
118
BHI agar in heat treated samples returned negative counts.
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The prepared carrot juice was transferred aseptically into sterile bags (110 mm ×
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185 mm, BagLight PolySilk, Interscience, Paris, France) (about 150 mL for each bag)
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and sealed using a heat sealer (FS-300, Yongkang Teli Packing Machinery Co., Ltd.,
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China). The bags were frozen stored in a freezer (BC/BD-103HA, Haier, China) at -20
123
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C for subsequent handling.
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Carrot juice was fully thawed at 4 oC for ~4 h prior to inoculation of E. coli
125
culture. After thawing, carrot juice was aseptically transferred to a sterile beaker. The
126
previously prepared E. coli pellet was re-suspended in 20 mL carrot juice and then
127
added to the rest of carrot juice in a beaker. After stirring at 500 rpm (RCT BS25, IKA,
128
Staufen, Germany) for 1 min, the inoculated carrot juice was aseptically transferred to
129
a sterile 2.0 mL cryogenic vial (430659, Corning Inc., USA), up to the brim and
130
closed with sterile screw cap leaving no headspace to avoid possible cracking during
131
HP treatment.
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2.3. Insulated container
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The cryogenic vials were individually vacuum packed in two layers of
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polyethylene bags and divided into two batches. One batch of the cryogenic vials were
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sponge as previous study used (Su et al., 2014) and slightly modified, as shown in Fig.
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1. A K-type thermocouple (OMEGA Engineering, Stamford, CT, USA) was installed
138
into the container for temperature recording using a data logger (34970A, Agilent
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Technologies GMBH, Germany). Four cryogenic vials were placed in one container
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with one of them used for recording sample temperature during HP treatment. The
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plastic container was filled water to completely soak the sponge and frozen at -20 oC
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for 24 h, and were secured by vacuum-packing in a flexible thermo-stable PA/PE
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pouches (30 cm × 42 cm) prior to HP treatment (details shown in Fig. 1). Samples of
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E. coli inoculated carrot juice frozen stored at -20 oC, prepared in a similar manner
145
but without HP treatment, were used as control. The second batch was not frozen but
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stored at 4 oC and treated likewise for providing HP inactivation data under unfrozen
147
conditions.
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2.4. High pressure equipment
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HP treatments were carried out in a laboratory-scale HP equipment (UHPF-750,
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Baotou Kefa High Pressure Technology Co., Ltd., China) with a maximum chamber
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capacity of 5 L. The high pressure unit was connected to a data logger for temperature
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and pressure (current signal) recoding during HP treatment. Water was used as the
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pressure-transmitting medium. The pressure vessel was maintained at room
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temperature (~20 oC) and with some added ice if the temperature was above 20 oC
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before pressure treatment. The near room temperature setup was used so that the
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process can be replicated in other commercial HP equipment using the special
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attachment. The pressure come-up rate was about 200 MPa/min and the
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depressurization time was less than 10 s. Data were recorded at one second interval.
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2.5. HP treatment The frozen samples carrot juice were subjected to HP treatment at selected
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pressure levels (200, 250, 300, 350 and 400 MPa) for selected treatment times (0, 1, 3,
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5, 7.5 and 10 min holding time). Unfrozen samples were only treated at 300, 350, and
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400 MPa for holding times 0, 1, 3, 5, 7.5 and 10 min. The pressure holding times
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mentioned did not include the pressure come-up time (1-2 min) and pressure release
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time (~10 s). After HP treatment of frozen samples, the special plastic container
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holding the test vials were observed to make sure water soaked sponge remained
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largely in the frozen state (melting extending no more than 7 mm on the sides) and
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then immersed in running water (room temperature) for 2 h to complete thawing.
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Treated sample vials (thawed vials of frozen and vials of unfrozen) were held at 4 oC
170
(maximum 4 h) prior to microbial enumeration. Due to adiabatic heating, the
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temperature of the pressure transmission medium (water) is expected to increase
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about 3 oC per 100 MPa on an average. However, because of addition of some ice, the
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water temperature during the treatment remained between 23 and 30 oC.
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2.6. Enumeration of E. coli
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The sample vials were aseptically opened and the carrot juice was aseptically
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transferred to a sterile tube, followed by a serial dilution in 1 g/L peptone water
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(Sinopharm Chemical Reagent Co., Ltd, Shanghai, China). Surviving cells of E. coli
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were enumerated on BHIA using pour plate method. Colonies of survived E. coli were
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juice were obtained likewise from untreated control samples both at 4 and -20 oC to
181
differentiate the effect of freezing on microbial reduction.
182
2.7. Kinetic analysis
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The pressure inactivation kinetics of E. coli during pressure holding time was
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analyzed based on a first order reaction rate (Shao et al., 2007) indicating a
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logarithmic order of death, and expressed as: N t log10 t = − D N0
(1)
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where Nt is the number of E. coli survivors (CFU mL-1) after HP hold time
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treatment time of t (min), and N0 is the initial number of E. coli (CFU mL-1) in frozen
189
samples at -20 oC without HP treatment. D value (min) is the decimal reduction time
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of E. coli which implies treatment holding time in minutes at constant pressure for 90 %
191
destruction of the existing microbial population. D values were obtained from the
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linear regression slope of log10(Nt/N0) versus t as shown in Eq. (2) or time taken to
193
traverse one logarithmic cycle:
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(2)
For pressure resistance (pressure sensitivity) analysis, Zp values were used (Shao
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et al., 2007). Zp value represented the pressure range between which the D value
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change by a decimal factor and can be determined as the negative reciprocal of the
198
slope of log10 (D) versus pressure curve.
199
Statistical analysis was performed using one-way analysis of variance (ANOVA).
200
Duncan’s test (P < 0.05) was applied to compare the differences in average values by
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3. Results and discussion
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3.1. Temperature and phase transition in frozen carrot juice during HP
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treatment
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Fig. 2 illustrates the transient temperatures in frozen carrot juice during HP
206
treatment for a holding time of 1 min at different pressure levels (Fig. 2a, c, e, g, i)
207
and temperature-pressure profiles of frozen carrot juice superimposed on the phase
208
diagram of water (Bridgman, 1912) (Fig. 2b, d, f, h, j), respectively. Pressure
209
increased in a stepwise fashion and the duration of t1-t2 depended on the final pressure.
210
During pressure treatment 400 MPa, come up time was 2 min and shorter times were
211
required proportionally at other pressure levels. The interval t2-t3 was 60 s in all plots.
212
During decompression, the pressure decreased (t3-t4) rapidly within 10 s. Table 1
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shows the temperatures achieved during HP treatment of frozen carrot juice. It can be
214
observed that the temperature was kept well below the conventional freezing point
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during HP freezing,
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The intensifier used for generating high pressure was a batch type unit which
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generated a stepwise ladder-like pressurization build up. Hence the ladder-like
218
pressure and temperature change was visible during the compression. Carrot juice
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samples contained in the special container were taken out from a freezer at -20 oC.
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The temperature rose slightly during the preparation, vacuum packing, connecting of
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thermocouples, installation of sample container into the pressure chamber and the
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quick filling of water into the chamber. During compression, the temperature still rose
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the heat uptake of melting ice crystals (endothermic reaction) in the sponge
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surrounding the vials. Partial thawing occurred and the melting of ice crystals
226
increased in the sponge. However, the heat of compression and the heat input from
227
pressure chamber were insufficient to fully melt ice in the sponge and hence help to
228
protect the frozen carrot juice in the test vials. The temperature decreased upon further
229
compression due to lowering of the freezing point and approached the melting line of
230
carrot juice (water melting line shown in Fig. 2). Heat of compression and latent heat
231
in this phase were in equilibrium, and solid-liquid transition was initiated. When the
232
temperature was -10 oC or -5 oC at the beginning (Fig. 2c, g), the temperature could
233
decrease even during compression and approached the melting line. Because the
234
temperature was still lower than the freezing point at the process pressure, the results
235
indicated that the special container with water soaked frozen sponge had enough heat
236
sink and insulation to protect the sample from heat gain and hold them under frozen
237
state.
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According to the temperature-pressure profiles superimposed on the phase
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diagram of water in Fig. 2 (b, d, f, h, j), the metastable Ice-I region was positioned
240
above 220 MPa. This pressure was little higher than 207 MPa reported by Su et al.
241
(2014) for E. coli suspension. On the other hand, the phase transition point of pure
242
water is 210 MPa. Hence there existed some small differences which were probably
243
due to differences in the nature of the support medium (water vs carrot juice). In Fig.
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2 (g and i), a sudden increase in sample temperature (-35 to -19 oC) was observed
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ACCEPTED MANUSCRIPT during compression (highlighted in a gray circle). A sudden pressure decrease (349 to
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334 MPa) was also observed at 400 MPa (highlighted in a dark gray circle in Fig. 2i).
247
These results indicated the transition to Ice-III, including metastable Ice-I to Ice-III
248
(endothermic reaction) and water re-crystallization to Ice-III (exothermic reaction).
249
Because Ice-III (1.14 g/cm3) has a higher density than water (1 g/cm3) and Ice-I (0.92
250
g/cm3), the volume change contributes to a change in pressure. The temperature
251
increase might be caused by the exothermic transformation of some liquid water to
252
Ice-III (Van Buggenhout, Grauwet, Van Loey, & Hendrickx, 2007).
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After compression, the temperatures at 200, 250, 300, 350 and 400 MPa
254
remained at -19, -21, -27, -18 and -18 oC, respectively, with a slight increase. Since
255
the surrounding temperature was always higher than the sample container, it is likely
256
that thawing continued (endothermic reaction) in surrounding sponge space in the
257
container during the holding phase while keeping the sample to stay at lower
258
temperature. When the pressure was released, the phase transition from Ice-III to Ice-I
259
and then to liquid water resulted at 350 and 400 MPa (exothermic reaction). At
260
pressures lower than 350 MPa, phase transition of metastable Ice-I to Ice-I and water
261
seemed to occur (exothermic reaction).
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Super cooling was caused during the decompression, so partial pressure shift
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freezing also occurred (exothermic reaction). The sample temperature decrease as a
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result of expansion cooling was only observed at 350 and 400 MPa during
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decompression. During depressurization, the increase or decrease of sample
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temperature depended on the combined effect of expansion cooling, the amount of
ACCEPTED MANUSCRIPT 267
melted water converted to Ice-I (an exothermic reaction) and the amount of Ice-III
268
converted to Ice-I (also an exothermic reaction) (Van Buggenhout, Messagie, Van der
269
Plancken, & Hendrickx, 2006). At higher pressures (above ~220 MPa), a solid-solid transition could be induced
271
as the theoretical Ice-I and Ice-III phase transition line of pure water was exceeded
272
upon compression (Van Buggenhout et al., 2007). Similar phase transition of Ice-I to
273
Ice-III has been reported at pressures above 250 MPa (Su et al., 2014; Van
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Buggenhout et al., 2007), but the sudden changes in pressure and temperature were
275
not similar. Again this might due to the differences in the media used: microbial
276
culture (335 MPa, -35 to -18 oC), carrot (280 MPa, -36 to -30 oC) and carrot juice
277
(349 MPa, -35 to -19 oC). Fernández et al. (2007) reported that phase diagram for beef
278
meat showed the same phase transition regions as water, but shifted to lower
279
temperature and different pressure values.
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3.2. High pressure inactivation of E. coli in unfrozen carrot juice
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The average initial concentration of E. coli in carrot juice before freezing was 7.8
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× 107 CFU/mL. After frozen storage for 24 h, E. coli count in carrot juice was reduced
283
slightly to 7.65 × 107 CFU/mL. The initial concentration of E. coli in carrot juice
284
samples varied between 107 and 108 CFU/mL and around were normalized to 107
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CFU/mL (by dividing all counts by their actual initial count and then multiplying by
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107). This facilitates better presentation of survival curves for comparison purposes
287
with all curves starting from the same point. The normalization process only results in
288
a small intercept shift and will not affect the slope, and therefore D values remain the
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same. Fig. 3 shows the survivor curves of E. coli in the unfrozen carrot juice, and Table
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2 shows the computed kinetic parameters of E. coli for HP treatment. The extent of
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microbial inactivation at 300 and 350 MPa during the 10 min holding time was small
293
resulting in calculated D values of 28.5 and 9.2 min, respectively. It improved a little
294
better at 400 MPa, with a calculated D value of 5.3 min. The range of holding times
295
(0-10 min) was low but kept the same as for HP treatment of frozen samples to
296
provide baseline data for comparison.
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The extent of HP inactivation of E. coli in unfrozen carrot juice were similar to
298
those reported by other researchers (Bari et al., 2007; Patterson et al., 2012;
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Pilavtepe-Çelik et al., 2009; Van Opstal et al., 2005). D values ranging from 38 to 2
300
min were reported by Van Opstal et al. (2005) for E. coli MG1655 inactivation after
301
HP treatment at 300-400 MPa in the temperature range 23-40 oC. Su et al. (2014)
302
reported slightly lower D values for the same strain of E. coli suspended in water. A
303
carrot juice matrix was used in this study and the use of real food matrix could have
304
been the reason for the observed higher values. Others have also reported that cells in
305
food matrix are more resistant than in buffer solutions (Pilavtepe-Çelik et al., 2009;
306
Alpas, Lee, Bozoglu, & Kaletunç, 2003).
307
3.3. High pressure inactivation of E. coli in frozen carrot juice
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Fig. 4 shows the logarithmic survivor curves of E. coli in frozen carrot juice
309
subjected to HP treatment at 200 to 400 MPa for 0 to 10 min holding time. The E. coli
310
inactivation in frozen samples also followed the first order rate kinetics during the
ACCEPTED MANUSCRIPT 311
pressure holding time (R2>0.95). Table 3 shows the D values and other kinetic
312
parameters of E. coli in frozen carrot juice treated by HP. The reduction trends in E. coli in frozen carrot juice were similar to those in
314
unfrozen carrot juice samples - survivors decreased with increasing pressure and
315
treating time, only by a greater margin (both first order rate). The survival lines were
316
steeper at higher pressures indicating that the associated D values decreased at higher
317
pressures.
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The E. coli count reduction at 200 MPa after pressure treatment for 0 and 10 min
319
were 0.42 and 3.00 log10CFU/mL, while they increased to 2.00 and 6.80
320
log10CFU/mL at 400 MPa, respectively. The count reduction was far more in frozen
321
carrot juice than in unfrozen samples. For example, after 10 min treatment at 400
322
MPa, the reduction of E. coli count in unfrozen samples was 1.87 log10CFU/mL, but
323
6.80 log10CFU/mL in frozen samples. The calculated D value (5.32 min) of E. coli at
324
400 MPa in unfrozen samples was longer than the D values in frozen samples
325
obtained even at much lower pressures of 200 and 250 MPa (4.03 and 3.97 min,
326
respectively. The D value of frozen carrot juice at 400 MPa (2.12 min) found in this
327
study was close to Van Opstal et al. (2005) reported value of 2.0 min of E. coli
328
MG1655 at 400 MPa, 40 oC in freshly extracted carrot juice; but they found the D
329
values to be longer at lower temperatures (5-30 oC). The D value of frozen carrot
330
juice at 300 MPa (2.62 min) was consistent with Su et al. (2014) of frozen E. coli
331
suspension (2.59 min). The frozen state therefore contributed to a remarkable
332
enhancement of E. coli inactivation in carrot juice.
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ACCEPTED MANUSCRIPT From the D value data, the computed pressure sensitivity parameter (Zp value)
334
was 613 MPa much higher than the 196 MPa reported by Su et al. (2014). It is clear
335
that the pressure sensitivity was affected significantly when carrot juice was treated in
336
the frozen state. The nature of the phase transition, pressure and sample state could be
337
the reasons for this observed remarkable difference in the computed Zp value.
338
Although the D value data were combined for the purpose of computation of Zp value,
339
it can be seen from Fig. 3 that the HP inactivation rate of E. coli at the two lower
340
pressure levels (200, 250 MPa) were distinctly different from those at the two higher
341
levels (300-400 MPa). At 200 and 250 MPa the associated D values were 4.03 and
342
3.97 min, respectively, while at 300, 350 and 400 MPa they were 2.62, 2.21 and 2.12
343
min, respectively, indicating a break in the continuity pattern. Therefore, although the
344
inactivation rate increased with pressure level (consistent with general understanding),
345
a pattern difference was also apparent. This is probably due to the re-crystallization of
346
Ice-III and phase transition from metastable Ice-I to Ice-III during compression and
347
Ice-III to Ice-I and/or water during decompression, when the pressure was above 250
348
MPa. It has been reported earlier that phase transitions may lead to mechanical
349
disintegration of cells (Edebo & Hedén, 1960) and the inactivation would be caused
350
by a mechanical effect related to the Ice-I to Ice-III phase transition (Luscher et al.,
351
2004; Fernández et al., 2007). The crystal formation and phase transition therefore
352
could play important roles in the HP in inactivation on E. coli in frozen medium.
353
3.4. Pressure-pulse inactivation of E. coli in frozen carrot juice
354
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The inactivation effect of pressure pulse on E. coli in frozen carrot juice is shown
ACCEPTED MANUSCRIPT in Table 4. For unfrozen samples, the pressure pulse had almost no effect at any of the
356
pressure levels from 200 to 400 MPa (data not shown). However, the pressure pulse
357
effect was clearly noticeable in frozen carrot juice samples which was low at 200 and
358
250 MPa, but significantly increased to 0.85, 1.41 and 2.00 logarithmic cycle
359
reductions at 300, 350 and 400 MPa, respectively. It was consistent with the D values
360
pattern between 200-250 MPa and 300-400 MPa discussed earlier. Therefore these
361
differences could also be ascribed to the frozen state of carrot juice and phase
362
transition during HP, especially the Ice-I/Ice-III transition above 300 MPa. Bari et al.
363
(2007) reported that repeating a 10 min high pressure treatment cycle with a 5 sec
364
pause could achieve extra reduction. It is possible that repeating HP pulse treatment
365
likewise would get more reduction as demonstrated in some previous studies with
366
multiple pressure pulse inactivation (Basak and Ramaswamy, 2011).
367
4. Conclusions
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The transient time temperature data obtained under pressure processing
369
conditions involving frozen carrot juice revealed that the ice structure could be
370
predominantly of type Ice-III or Ice-I. Solid-solid and solid-liquid transitions were
371
apparent during compression and decompression. E. coli inactivation in frozen carrot
372
juice followed the first order rate kinetics after accounting for a pulse effect
373
inactivation. The high pressure inactivation of E. coli in frozen samples was far better
374
than in unfrozen samples, and was enhanced by pressure level and holding time. The
375
phase transition, pressure level and sample state contributed to the better inactivation
376
of E. coli in frozen carrot juice, especially the Ice-I/Ice-III transitions at pressure >
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378
potential for commercial application. Further, the special insulator attachment
379
provides opportunity for accomplishing HP inactivation in the frozen state using HP
380
equipment operating at room temperatures. However, further studies are needed to
381
evaluate the stability and the influence on the food quality and properties and the use
382
of these processes might be limited due to the frozen state.
Acknowledgements
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This work was supported by the Key Program of Natural Science Foundation of
386
Zhejiang Province (Grant No. LZ14C200002) and the National Natural Science
387
Foundation of China (Grant No. 31171779).
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Vervoort, L., Plancken, I. V. D., Grauwet, T., Verlinde, P., Matser, A., Hendrickx, M.,
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& Loey, A. V. (2012). Thermal versus high pressure processing of carrots: A
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comparative pilot-scale study on equivalent basis. Innovative Food Science &
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Emerging Technologies, 15(1), 1-13.
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Figure Captions.
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Fig. 1. Schematic cross-section and details of the insulated container for holding test
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samples (the red color material is the sponge covering the vials).
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Fig. 2. Temperature changes with pressure of frozen carrot juice samples during HP
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treatment holding for 1 min under different pressures (a, c, e, g, i) and
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temperature-pressure profiles superimposed on the phase diagram of water (b, d, f, h,
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j).
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Fig. 3. E. coli survivors in unfrozen carrot juice samples after different high pressure
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(300 MPa (○), 350 MPa (△), 400 MPa (◊)) and different holding time.
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Fig. 4. E. coli survivors in frozen carrot juice samples after different high pressure
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(200 MPa (□), 250 MPa (▽)300 MPa (○), 350 MPa (△), 400 MPa (◊)) and different
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holding time.
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ACCEPTED MANUSCRIPT Table 1 Temperatures achieved at the end of pressure come up time and holding time (n=3). Temperature (oC) Pressure (MPa) 200
-20.1±1.1a
-19.2±1.7
250
-22. 6±1.3
300
-27.5±0.3
350
-18.2±0.2
400
-17.6±0.8
-21.7±1.5
-26.6±0.0 -18.2±0.2
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Mean values±standard deviation
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End of holding 1 min
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R2Adj
Zp value (MPa)
R2Adj
300
28.53±5.27 a
0.850
139±28
0.922
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9.20±1.03
0.940
400
5.32±1.06
0.827
Standard error (SE)
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ACCEPTED MANUSCRIPT Table 3 Inactivation kinetics of E. coli in frozen carrot juice under high pressure. D value (min)
R2Adj
Zp value (MPa)
R2Adj
200
4.03±0.35a
0.963
613±118
0.866
250
3.97±0.39
0.954
300
2.62±0.19
0.974
350
2.21±0.10
0.991
400
2.12±0.08
0.994
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SE - standard error
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ACCEPTED MANUSCRIPT Table 4 Pulse effect of high pressure on E. coli in frozen carrot juice. Mean values±standard deviation (n = 3). Reduction (log10CFU/mL) 0.42±0.11a
250
0.53±0.05a
300
0.85±0.09b
350
1.41±0.35c
400
2.00±0.24d
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Different letters (a, b, c, d) indicate significant difference (p < 0.05) between treatments.
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Highlights Phase transition in frozen carrot juice was evaluated under HP at room temperature.
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Transition to Ice III was observed at pressures above 350 MPa. Special container was used to maintain frozen conditions during treatment. Enhanced inactivation of E. coli was demonstrated in frozen carrot juice.
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Substrate state, phase transition and pressure level influenced inactivation.