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Inactivation of mushroom polyphenoloxidase in model systems exposed to high-pressure carbon dioxide
Accepted Manuscript Title: Inactivation of mushroom polyphenoloxidase in model systems exposed to high-pressure carbon dioxide Author: Lara Manzocco Alexandra Ignat Fabio Valoppi Krystel Rita Burrafato Giovanna Lippe Sara Spilimbergo Maria Cristina Nicoli PII: DOI: Reference:
S0896-8446(15)30082-6 http://dx.doi.org/doi:10.1016/j.supflu.2015.07.029 SUPFLU 3405
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
11-5-2015 22-7-2015 23-7-2015
Please cite this article as: L. Manzocco, A. Ignat, F. Valoppi, K.R. Burrafato, G. Lippe, S. Spilimbergo, M.C. Nicoli, Inactivation of mushroom polyphenoloxidase in model systems exposed to high-pressure carbon dioxide, The Journal of Supercritical Fluids (2015), http://dx.doi.org/10.1016/j.supflu.2015.07.029 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.
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Inactivation of mushroom polyphenoloxidase in model systems exposed to high-pressure
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carbon dioxide
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Lara Manzocco*, Alexandra Ignat, Fabio Valoppi, Krystel Rita Burrafato, Giovanna Lippe, Sara
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Spilimbergo§, Maria Cristina Nicoli
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Università di Udine, Dipartimento di Scienze degli Alimenti, Via Sondrio 2/A, 33100 Udine, Italy
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*Corresponding author. Tel.: +39 0432 558152; Fax: +39 0432 558130
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Università di Padova, Dipartimento di Ingegneria Industriale, Via Marzolo 9, 35131 Padova, Italy
E-mail address: [email protected]
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Keywords: dense-phase CO2; tyrosinase; inactivation kinetics; SDS-PAGE
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Highlights:
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HP-CO2 treatments quickly inactivated mushroom polyphenoloxidase in model systems
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Pressure sensitivity (zP ) of enzyme inactivation was circa 5.5 MPa at 20 and 35 °C
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At 20 and 35 °C, activation volume V was about -1000 cm3 mol-1
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HP-CO2 did not change the electrophoretic pattern of polyphenoloxidase
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Abstract
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An aqueous solution containing mushroom polyphenoxidase was exposed at 20, 35 and 45 °C for
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up to 15 min to CO2 at increasing pressure up to 18 MPa. Samples were analysed for residual
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enzymatic activity and SDS-PAGE patterns. At 20 and 35 °C, HP-CO2 allowed non-thermal and
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irreversible inactivation of polyphenoloxidase with decimal reduction time (DP) that decreased
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when pressure increased. At 45 °C, complete inactivation was achieved in less than 0.2 min at all
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CO2 pressures. The pressure sensitivity parameter (zP) of inactivation rate resulted similar at 20 and Page 1 of 22
35 °C (5.4 and 5.5 MPa, respectively). Accordingly, temperature increase from 20 to 35 °C caused
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minor modifications of activation volume ( V) (circa -1000 cm3 mol-1) but almost doubled the pre-
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exponential factor of the Eyring equation. SDS-PAGE data showed that polyphenoloxidase
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inactivation followed mechanisms other than those associated with thermal inactivation and
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proceeded with no structural changes, probably implying the formation of bindings between
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molecular CO2 and enzyme terminal groups.
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Introduction
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High pressure carbon dioxide (HP-CO2), also known as dense phase carbon dioxide (DP-CO2) has
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been proposed as a promising non-thermal alternative to traditional pasteurisation methods. During
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the treatment, food is in contact with pressurised CO 2 at temperature/pressure conditions that may
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be below or above the critical point (31.1 °C, 7.38 MPa). Typical CO2 pressure is generally within 4
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and 30 MPa, rarely exceeding 50 MPa. Temperature is generally between 20 and 50 °C, low enough
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to avoid negative quality effects associated with traditional pasteurisation.
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Significant lethal effects of pressurised CO2 on different microorganisms has been demonstrated not
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only in juices but also in solid foods such as meat and fish derivatives dried ingredients and fresh-
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cut produce [3-10]. In particular, up to 5 Log reductions were reported in the literature depending
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on the treatment conditions and the food matrix [11]. While liquid food treatment seems not to
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impair the product fresh-likelihood, application of this technology to the preservation of solid foods
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could adversely affect product quality.
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HP-CO2 has also been shown to inactivate enzymes responsible for food quality decay. The extent
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of inactivation depends on the nature of the enzyme and is strongly affected by CO 2 pressure,
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temperature and treatment time. Zhou et al. [12] completely inactivated pectin methylesterase in
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carrot extract upon exposure for 15 min to 15 MPa CO2 pressure at 55 °C. By contrast, more than
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75 min were required to inactivate the same enzyme in peach extract. In the case of horseradish
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peroxidise and soybean lipoxygenase, only 90% inactivation was observed at 30 MPa [13,14].
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Polyphenoloxidase from different fruit juices and seafood has also been demonstrated to be HP-CO2
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sensitive [15-17]. Xu et al. [3] completely inactivated polyphenoloxidase in apple juice after 10 min
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at 22 MPa and 60 °C. In the case of melon juice, complete inactivation of polyphenoloxidase was
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not achieved even increasing CO2 pressure [18]. Besides processing parameters, the composition of
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the matrix surrounding the enzyme and its compartmentalisation could also affect sensitivity to HP-
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CO2. For instance, as compared to enzymes within plant and animal tissues, higher inactivation was
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generally observed when the crude extracts were treated [8,12,17,19].
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The mechanism of enzyme inactivation by HP-CO2 is hypothesised to be the result of local pH
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lowering [20]. According to other authors, pressurised CO2 could cause changes in the conformation
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of the secondary structure of the enzyme [21]. To this regard, HP-CO2 inactivation of different
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enzymes was observed in concomitance with a decrease in the residual α-helix content of the
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proteins, analogously to thermal induced inactivation [13,22]. Despite these findings, the effect of
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HP-CO2 on the activity and structure of food enzymes is still under study.
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The present paper was addressed to investigate enzymatic inactivation kinetics following HP-CO2
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treatment of model system. To this aim, polyphenoloxidase was chosen as an example of enzyme
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that impairs food quality. An aqueous solution containing mushroom polyphenoxidase was exposed
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at 20, 35 and 45 °C for up to 15 min to CO2 at increasing pressures up to 18 MPa. Samples were
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analysed for residual enzymatic activity. Inactivation rate constants were used to estimate decimal
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reduction parameters (DP and zP) as well as activation volume (V) and frequency factor (lnkatm)
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by the Eyring equation [23]. Finally, to compare changes induced by pressurised CO 2 and
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conventional thermal treatment on polyphenoloxidase, SDS-PAGE separation patterns were also
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obtained.
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2. Material and methods
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2.1 Polyphenoloxidase solution
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Mushroom tyrosinase (polyphenoloxidase) (T3824 50KU, Sigma, St. Louis, MO, USA), purity
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>98%, from Agaricus bisporus was used. Polyphenoloxidase solution was prepared diluting the
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enzyme in 0.1 M potassium phosphate buffer pH 7.0 (Carlo Erba, Milano, Italy).
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Polyphenoloxidase initial activity on 1.5×10−3 M L-Dopa (Carlo Erba, Milano, Italy) was 0.004 Abs
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min-1.
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2.2 High-pressure CO2 treatments
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Figure 1 shows a schematic representation of the apparatus used for high-pressure CO2 treatments.
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Liquid carbon dioxide (purity 99.995%, Sapio, Monza, Italy) was cooled down to 4 °C using a F34-
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ED chiller (C; Julabo, Milano, Itlay) after been filtered with a 15 μm filter (B; Ham-Let, Milano,
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Italy). Subsequently, CO2 was pressurised with a Orlita MhS 35/10 diaphragm pump (D; ProMinent
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Italiana S.r.l., Bolzano, Italy) into two identical stainless steel cylinders (E and F, internal volume
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150 mL) with a screwed cap, previously filled with 10 mL of polyphenoloxidase aqueous solution.
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The vessels connected in parallel, were immersed in a water bath connected to a CB 8-30e
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thermostatic bath (H; Heto, Allerød, Denmark) that ensured maintenance of constant temperature.
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Before starting the pressurisation, the temperature of the sample was allowed to reach the
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equilibrium. The time needed to reach the desired temperature (20, 35 or 45 °C) was lower than 3
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min. Once the desired temperature was reached, less than 2 min were needed to reach the target
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pressure. As a consequence, the desired conditions of temperature and pressure were reached in less
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than 5 min. As an example, supplementary material 1 shows the temperature and pressure profile of
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the treatment carried out at 35 °C and 12 MPa for 2 min. After reaching the desired pressure (6, 8,
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10, 12, 15 and 18 MPa), the pump was switched off and valves connected to each vessel (V2 and
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V3) were tightly closed. In order to carefully control operating parameters such as pressure and
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temperature during experiments, two thermocouples (TT1 and TT2) connected to a digital data
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logger (G) and a manometer (PT3) were used. The mixing between samples and CO2 inside the
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vessels was assured by two magnetic stirrers (MS1 and MS2) (Microstirrer, Velp Scientifica,
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Usmate, Italy) placed below each vessel. After increasing treatment time up to 15 min, vessels were
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depressurised opening V2 and V3 valves, the outlet metering (V4) and micrometric (V5) valves. The
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valves V4 and V5 were heated in a water bath connected to a thermostatic bath (H) to prevent
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freezing during decompression. In order to assure an adequate heat exchange in both water baths,
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two small water pumps (P1 and P2) were used. In all experiments, depressurisation was completed
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within 3 min and the outlet flow was controlled using a PFM 750 digital flowmeter (I) (SMC Italia
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S.p.A., Milano, Italy). Additional control samples were prepared by treating the enzyme solution in
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the vessels at atmospheric pressure (0.1 MPa) and thus at CO 2 partial pressure equal to 0.0039 MPa.
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2.3 Sample storage
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Aliquots of 1 mL of polyphenoloxidase solution were introduced in Eppendorf® vials of 1.5 mL
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capacity and stored for up to 7 days at 4 °C in a refrigerated cell. At increasing time during storage,
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samples were removed from the refrigerator, equilibrated at 22 °C and submitted to the analysis.
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2.4 Temperature, pH
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Temperature and pH were respectively assessed by a thermocouple probe (Hanna Instruments,
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Tersid s.r.l., Milano, Italy) and a Mettler Toledo 355 pH-meter (Lou Analyzer, Halstead, Essex,
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England).
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2.5 Polyphenoloxidase activity
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The polyphenoloxidase activity was assayed spectrophotometrically (Shimadzu UV-2501PC, UV-
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Vis recording spectrophotometer, Shimadzu Corporation, Kyoto, Japan) at 25 °C according to the
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methodology of Kahn [24]. The reaction was started by the addition of 20 µL of polyphenoloxidase
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solution to 2 mL of 0.1 M potassium phosphate buffer pH 7 and 1.5 10-3 M L-Dopa (Carlo Erba,
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Milano, Italy). The absorbance at 420 nm was monitored each minute for 10 min. The changes in
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absorbance per min were calculated by linear regression, applying the pseudo zero order kinetic
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model. The eventual final stationary phase was excluded from regression data. One arbitrary unit of
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polyphenoloxidase was defined as the amount of enzyme that produced an increase in absorbance at
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420 nm of 0.001 Abs min-1 under the testing conditions. Polyphenoloxidase residual activity was
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calculated as the percentage ratio between enzymatic activity of the treated solution and that of the
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control treated at atmospheric pressure (0.1 MPa).
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2.6 SDS-polyacrylamide gel electrophoresis
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Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed
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according to Laemmli [25]. Aliquots of polyphenoloxidase in the aqueous solution were added with
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sample buffer containing 0.2% bromophenol blue, 0.5 M Tris-HCl, pH 6.8, 10% (w/v) SDS, 50%
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(v/v) glycerol and 25% (v/v) β-mercaptoethanol. The samples were loaded on a 13% (w/v)
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polyacrylamide separating gel containing 40% (v/v) acrylamide and 2% (v/v) bis-acrylamide cross
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linker, mounted on a Mini 2-D (Bio-Rad, Richmond, CA, USA) vertical electrophoresis cell. The
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electrophoretic separation were obtained by running the samples at 15 mA with standard proteins of
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known molecular weight (Precision Plus Protein™ Dual Color Standards, Bio-Rad, Segrate, Italy)
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were used to determine the apparent molecular mass of the sample bands. An additional reference
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sample, obtained by thermal inactivation of polyphenoloxidase was also prepared. In particular, 10
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mL enzyme solution were placed into 50 mL capacity glass tube and heated in a water bath (IKA-
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Werke, Staufen, Germany) at 70 °C for 5 min.
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The gels were subsequently stained with NOVEX ® Colloidal Blue Staining Kit (Invitrogen,
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Carlsbad, CA, USA) for one night and then destained in bi-distilled water. Gels were scanned and
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analysed with the ImageQuant TL Image Analysis Software (Amersham Bioscience Inc.,
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Piscataway, NJ, USA).
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2.7 Data analysis
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Treatments were carried out at least in duplicate and analyses were performed on at least triplicated
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samples. Data were expressed as mean ± S.D. Apparent inactivation rate constants of
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polyphenoloxidase were analysed by using a conventional first-order equation:
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(1)
Where A is the residual activity of polyphenoloxidase (%) at time t (min) and k is the inactivation
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rate constant (min-1). The value of k was obtained by regressing the natural logarithm of A vs. t. The
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inactivation rate constant (k) was then derived from the slope of the obtained line.
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The kinetic parameter DP was obtained using procedures analogous to that employed in thermal
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death time studies. In particular DP is the decimal reduction time, i.e. the treatment time needed for
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90% enzyme activity reduction at a given pressure and temperature. DP was computed as:
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(2)
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The pressure increase needed for a 90% reduction of the DP value is computed as zp (MPa). The
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value of zp was obtained by regressing the decimal logarithm of DP versus pressure (P):
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(3) The zP was then derived as the negative reciprocal slope of the regression line.
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The pressure dependence of the inactivation rate constants (k) was expressed by the activation
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volume (V, cm3/mol), according to the Eyring equation [26]:
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(4)
where P is pressure (MPa), katm is the inactivation rate constant at ambient pressure Patm (0.1 MPa),
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R is the gas constant (8.31 cm3 MPa K-1 mol-1) and T is temperature (K). (V) was estimated from
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the slope of the line obtained by regressing the natural logarithm of k vs P.
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Goodness-of-fit was evaluated by means of the determination coefficients (R 2) and p values
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(Statistica for Windows, ver. 5.1, Statsoft Inc., Tulsa, OK, USA, 1997).
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3. Results and discussion
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Figure 2 shows the residual polyphenoloxidase activity of an aqueous solution containing 4 U of
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mushroom polyphenoloxidase as a function of treatment time at different CO 2 pressure at 20 °C.
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Independently on CO2 pressure, samples were always subjected to CO2 under subcritical conditions.
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HP-CO2 treatments promoted a significant decrease in the activity of the enzyme. In particular,
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while the polyphenoloxidase control sample, treated at 20 °C under environmental pressure (0.1
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MPa), did not show significant changes in activity, exposure for increasing time to pressurised CO 2
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strongly decreased this parameter. Enzyme inactivation by pressurised CO 2 has already been
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reported for polyphenoloxidase from different origins [13,17,21]. However, in those cases, much
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more intense treatments were required. For instance, the complete inactivation of shrimp
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polyphenoloxidase was only achieved in the 4-25 MPa pressure range by carrying out prolonged
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treatments at 37 °C (> 30 min) [17]. In addition, treatments carried out at 35 °C at 8 MPa for up to
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30 min were not able to decrease the activity of apple polyphenoloxidase [13,27]. This goal was
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only reached when apple polyphenoloxidase was submitted to longer treatments and/or higher
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temperatures [3]. Data shown in Figure 2 indicate that mushroom polyphenoloxidase was
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completely inactivated at room temperature within few minutes of treatment by selecting the proper
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pressure conditions. The higher susceptibility to pressurised CO 2, as compared to that reported from
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the same enzymes extracted or included in food matrices other than mushroom tissue, is likely to be
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attributable to the enzyme origin. The latter is well known to affect the sensitivity of enzymes to
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different inactivation treatments, including those based on high pressure [28]. In addition, the
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sensitivity of the enzyme could also be affected by its environmental conditions during the
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treatment. To this regard, Zhou et al. [12] observed that the inactivation of pectin methylesterase in
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a buffer was enhanced as compared to the original enzyme in carrot and peach juices.
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When polyphenoloxidase was exposed to HP-CO2 at 35 °C, inactivation was observed in even
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shorter times (Figure 3). Samples were always under supercritical conditions of CO2 with the only
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exception of those exposed to 6 MPa. It can be noted that no abrupt change in enzyme inactivation
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seemed to be associated with the transition from subcritical to supercritical condition. The effect of
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temperature on enzyme inactivation was further confirmed by additional trials carried out at 45 °C.
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The latter showed that exposure to the minimum pressure tested (6 MPa, subcritical conditions) for
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just 30 s was sufficient to completely inactivating the enzyme. It is noteworthy that the control
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sample, treated at the same conditions of temperature and time but at atmospheric pressure (0.1
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MPa), presented a residual polyphenoloxidase activity approaching 90%. These data demonstrate
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the existence of a negative relation between polyphenoloxidase activity and the increase in both
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CO2 pressure and temperature at least under the experimental conditions here tested. Antagonistic
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effects of pressure and temperature on polyphenoloxidase activity have actually been reported by
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the application of hydrostatic pressure at temperature higher than circa 60 °C [26].
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Based on data shown in Figures 2 and 3, the apparent inactivation rate constants of
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polyphenoloxidase were computed (Table 1). The residual activity of polyphenoloxidase, expressed
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in natural logarithmic values, had good relationships (R2>0.77, p<0.05) with the treatment time. DP
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values were thus derived from the rate constants, using procedures analogous to that employed in
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thermal death time studies (Table 1).
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In particular, DP was defined as the treatment time needed for 90% enzyme activity reduction at a
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given pressure. For instance, the treatment at 6 MPa and 20 °C leaded to a tenfold decrease of
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activity in circa 18 min. At the same temperature, this goal was achieved at 12 MPa in circa 3 min.
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On the other hand, keeping constant CO2 pressure at 6 MPa, inactivation was achieved at 35 or 45
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°C in 3.7 or less than 0.2 min, respectively. It is noteworthy that, when the enzyme resulted
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completely inactivated following the shortest treatment considered in the experimental plan (0.2
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min), the rate constant was not determinable. However, in such cases, the DP value can be assumed
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as shorter than 0.2 min. These low DP values are in agreement with the higher susceptibility of
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mushroom polyphenoloxidase to HP-CO2 treatments, as compared to that from other origin. To this
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regard, Gui et al. [13] reported polyphenoloxidase in cloudy apple juice to be inactivated in circa
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220 min upon exposure at 35 °C to 30 MPa. Similarly, Zhang [17] reported Pacific white shrimp
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polyphenoloxidase to exert a Dp of circa 30 min at 15 MPa and 37 °C.
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Dp values relevant to experiments carried out at 20 and 35 °C (Table 1) were used to calculate the
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parameter zP, describing the sensitivity of polyphenoloxidase to pressurised CO2. The decimal
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logarithmic values of DP resulted well correlated with pressure for treatments carried out at 20 and
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35 °C (Figure 4). The existence of a good correlation also in the case of treatments carried out at 35
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°C (subcritical at 6 MPa, supercritical at higher pressure) seems to confirm that enzyme inactivation
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is more affected by the overall processing conditions (time, temperature and pressure) rather than
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by the transition from sub to supercritical CO2 condition. The zP value, which represents the
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pressure range within which the DP changes tenfold, was calculated as the negative reciprocal slope
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of the regression line (Figure 4). The pressure sensitivity parameter zP resulted circa 5.5 MPa,
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independently on whether the treatments were carried out at 20 or 35 °C. This indicates that an
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increase in pressure of 5.5 MPa is necessary to get a 90 % decrease in DP at both temperatures.
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The effect of CO2 pressure on polyphenoloxidase inactivation was also expresses through the
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activation volume (V ) concept. Activation volumes (V) were computed from the slope plots of
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the natural logarithm of enzyme activity as a function of pressure (Figure 5).
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According to the transition state theory, the activation volume is a measure of the volume difference
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between the initial reactants and the activated complex at the transition state [23]. Data reported in
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Figure 5 show that polyphenoloxidase inactivation is characterized by negative V values with
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high absolute value. This indicates that the increase in pressure strongly favoured the denaturation
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of the enzymatic protein. The values of activation volume for mushroom polyphenoloxidase
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resulted one order of magnitude lower than those reported in the literature for the same enzyme in
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apple (-94.3 cm3 mol-1 at 55 °C) or shrimps (-120.9 cm3 mol-1 at 37 °C). This result indicate that
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polyphenoloxidase in these food matrices is less susceptible to CO 2 pressure variation than that
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extracted from mushrooms. In addition, activation volume assumed almost equal values (circa -
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1000 cm3 mol-1), regardless of the temperature experienced by the samples during the treatment.
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This is in agreement with the similarity in zp values (Figure 4) and indicates that the increase in
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temperature from 20 to 35 °C only reduces the time required for the inactivation of the enzyme (Dp,
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Table 1) but has no effects on its sensitivity to the increase in CO 2 pressure. By contrast,
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temperature would affect the pre-exponential or frequency factor (lnkatm) that determines how often
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the protein is properly oriented in order to undergo structural modifications on chemical reactions
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leading to denaturation. To this regard, it can be observed that rising the temperature from 20 to 35
259
°C increased the frequency factor by more than two orders of magnitude (Figure 5), making the
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reaction significantly quicker.
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The occurrence of almost identical z p and V is quite unusual in the literature relevant to the effect
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of pressure on enzymes. For instance, Polydera et al. [29] reported activation volume for pectin
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methylesterase inactivation by high hydrostatic pressure to be affected by temperature according to
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Page 11 of 22
a linear function. In that case, the higher the temperature, the higher the activation volume,
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indicating that enzyme inactivation rate was less susceptible to the increase in pressure when
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temperature was increased. Weemaes et al. [26] reported similar data with reference to avocado
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polyphenoloxidase treated in a wide interval of hydrostatic pressure and temperature. However, in
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this case, a temperature range was identified in which the activation volume could be considered
269
almost constant, similarly to the result obtained by exposing mushroom polyphenoloxidase to HP-
270
CO2 treatments (Figure 5).
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By comparing the values of V for polyphenoloxidase inactivation by HP-CO2 treatments (Figure
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5) to those estimated by applying high hydrostatic pressure only [26], it can be observed that the
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latter were associated with higher V values (-14/-36 cm3 mol-1). As previously suggested [17, 21,
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30], this result confirms that the activity of polyphenoloxidase is susceptible not only to the
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variation of pressure but also to the molecular effects of pressurised CO2.
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Polyphenoloxidase samples showing no residual activity after the treatments were also analysed for
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activity after storage for 7 days at 4 °C. The enzyme resulted unable to recover its activity upon
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storage. This result is in agreement with literature evidence showing the capability of pressurised
279
CO2 in irreversibly decomposing the -helix of the protein structure [21]. To better understand the
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nature of the polyphenoloxidase modification induced by pressurised CO 2, the attention was
281
focused on samples that underwent enzyme inactivation by exposure for 1 min at 20 °C at 6, 12 and
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18 MPa. SDS-PAGE was performed on polyphenoloxidase samples treated at increasing pressure
283
by using β-mercaptoethanol as a reducing agent (Supplementary material 2). Exposure of
284
polyphenoloxidase to pressurised CO2, despite decreasing its residual activity, did not promote
285
changes in the electrophoretic pattern as compared to that of the unpressurised enzyme (0.1 MPa).
286
In fact, control and polyphenoloxidase treated samples showed one clear band of molecular weight
287
of about 48 kDa [31-33]. In other words, the treatments did not induce evident fragmentation and/or
288
aggregation of the polyphenoloxidase subunits. This result is consistent with data obtained by
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HPLC gel permeation analysis of polyphenoloxidase submitted to pressurised CO 2 at 22 °C [34]
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and with indications relevant to the effect of this physical treatment on other enzymes [14]. To
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verify whether the mechanism of polyphenoloxidase inactivation was different from that associated
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with thermal inactivation, an additional sample, with no residual activity, was prepared by
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maintaining the polyphenoloxidase solution at 70 °C for 5 min (Supplementary material 2).
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Thermal inactivation of polyphenoloxidase was associated with the smearing of the band at the top
295
of the lane, suggesting the presence of high molecular weight protein aggregates. Heating would
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thus promote enzyme inactivation by mechanisms other than those promoted by HP-CO2 treatments.
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In particular, the exposure to HP-CO2 could promote local pH lowering, possibly resulting in
298
enzyme inactivation [2,20]. In the present study, the pH of the treated sample resulted similar to that
299
of the untreated one (7.0). However, due to plant constraints, sample pH was only analysed after
300
depressurisation, not allowing to appreciate pH modifications during CO 2 solubilisation in the
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treatment vessel. It was also hypothesised that HP-CO2 exposure could involve minor
302
conformational modifications which would be however able to reduce enzyme functionality [21].
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To this regard, it can be hypothesised that inactivation of polyphenoloxidase by HP-CO2 treatments
304
could involve the formation of carbamino groups between CO 2 and N-terminus groups of the
305
enzyme, with a mechanism similar to that observed in haemoglobin [35].
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Conclusions
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HP-CO2 treatments allowed quick, non-thermal and irreversible inactivation of polyphenoloxidase
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in aqueous model systems. The complete inactivation of the enzyme was achieved within minutes
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of treatment even at room temperature by selecting the proper CO 2 pressure. Kinetic analysis of the
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pressure and temperature induced inactivation demonstrated that the increase in temperature
313
reduced inactivation time but did not affect enzyme sensitivity to the pressure variation.
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Polyphenoloxidase inactivation by HP-CO2 was not associated with changes in electrophoretic
315
pattern and occurred following mechanisms other than those of thermal treatment.
Page 13 of 22
316
Being CO2 relatively inert, cheap, non-toxic and recyclable, the results acquired provide additional
317
evidence of HP-CO2 potential as an efficacious and sustainable non-thermal blanching technology.
318
Acknowledgement
320
This research was supported by Fondazioni in Rete per la Ricerca Agroalimentare, “AGER -
321
Agroalimentare e Ricerca”. Project 2010 2370.
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Chemistry 75 (2001) 317-324.
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Tyrosinase, J Biological Chemistry 245 (1970) 1613-1625.
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Mooibroek, C. Soler-Rivas, Cloning, expression and characterisation of two tyrosinase cDNAs
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two new polyphenol oxidase cDNAs from Agaricus bisporus. Biotechnology Letters 32 (2010)
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417 418
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419
Fig.1. Schematic representation of the high-pressure CO2 apparatus.
420 421
Fig. 2. Polyphenoloxidase activity in an aqueous solution as a function of exposure time to
422
increasing CO2 pressure at 20 °C. Control was treated at environmental pressure (0.1 MPa).
ip t
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Fig. 3. Polyphenoloxidase activity in an aqueous solution as a function of exposure time to
425
increasing CO2 pressure at 35 °C. Control was treated at environmental pressure (0.1 MPa).
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424
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426
Fig. 4. DP values of polyphenoloxidase inactivation by HP-CO2 treatments carried out at increasing
428
pressure at 20 and 35 °C. zP values and relevant determination coefficients are also shown.
an
427
429
Fig. 5. Logarithmic values of polyphenoloxidase apparent inactivation rate constants (k) as a
431
function of HP-CO2 treatments carried out at increasing pressure at 20 and 35 °C. Activation
432
volume (V), frequency factor (lnkatm) and relevant determination coefficients are also shown.
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430
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434 435
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433
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441
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440
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437
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A: CO2 tank PT: manometers V: valves B: CO2 filter C: chiller D: pump E, F: vessels TT: thermocouple G: digital temperature data logger H: thermostatic bath I: digital flowmeter L: volatiles trap P: water recirculation pump
442
Page 19 of 22
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443
445
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Page 20 of 22
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20
0.1
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Table 1 Polyphenoloxidase apparent inactivation rate constants (k), relevant determination coefficient and DP values of HP-CO2 treatments carried out at increasing pressure at 20, 35 and 45 °C. Temperature Pressure (MPa) k (min-1) R2 DP (min) (°C) 0.0014
0.90
1645.1
0.1264
0.95
18.2
8.0
0.4050
0.91
5.7
12.0
0.7272
0.96
3.1
15.0
2.6593
0.99
0.8
18.0
4.0829
0.99
0.6
0.1
0.0518
0.83
44.5
6.0
0.6235
0.77
3.7
8.0
3.0714
0.85
0.7
12.0
6.6378
0.89
0.3
15.0
*
<0.2
18.0
*
<0.2
0.1
0.0723
6.0
*
ce pt
6.0
35
45
Ac
447 448 449
0.80
31.8 <0.2
Page 21 of 22
450 451
8.0
*
<0.2
12.0
*
<0.2
15.0
*
<0.2
18.0
*
<0.2
* The enzyme resulted completely inactivated after the shortest treatment time (0.2 min). HP-CO2 treatments quickly inactivated mushroom polyphenoloxidase in model systems
453
Pressure sensitivity (zP ) of enzyme inactivation was circa 5.5 MPa at 20 and 35 °C
454
At 20 and 35 °C, activation volume V was about -1000 cm3 mol-1
455
HP-CO2 did not change the molecular mass of the enzyme subunits
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456
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Page 22 of 22
S0896-8446(15)30082-6 http://dx.doi.org/doi:10.1016/j.supflu.2015.07.029 SUPFLU 3405
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
11-5-2015 22-7-2015 23-7-2015
Please cite this article as: L. Manzocco, A. Ignat, F. Valoppi, K.R. Burrafato, G. Lippe, S. Spilimbergo, M.C. Nicoli, Inactivation of mushroom polyphenoloxidase in model systems exposed to high-pressure carbon dioxide, The Journal of Supercritical Fluids (2015), http://dx.doi.org/10.1016/j.supflu.2015.07.029 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.
1
Inactivation of mushroom polyphenoloxidase in model systems exposed to high-pressure
2
carbon dioxide
3
Lara Manzocco*, Alexandra Ignat, Fabio Valoppi, Krystel Rita Burrafato, Giovanna Lippe, Sara
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Spilimbergo§, Maria Cristina Nicoli
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Università di Udine, Dipartimento di Scienze degli Alimenti, Via Sondrio 2/A, 33100 Udine, Italy
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§
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*Corresponding author. Tel.: +39 0432 558152; Fax: +39 0432 558130
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Università di Padova, Dipartimento di Ingegneria Industriale, Via Marzolo 9, 35131 Padova, Italy
E-mail address: [email protected]
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Keywords: dense-phase CO2; tyrosinase; inactivation kinetics; SDS-PAGE
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Highlights:
15
HP-CO2 treatments quickly inactivated mushroom polyphenoloxidase in model systems
16
Pressure sensitivity (zP ) of enzyme inactivation was circa 5.5 MPa at 20 and 35 °C
17
At 20 and 35 °C, activation volume V was about -1000 cm3 mol-1
18
HP-CO2 did not change the electrophoretic pattern of polyphenoloxidase
19
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Abstract
21
An aqueous solution containing mushroom polyphenoxidase was exposed at 20, 35 and 45 °C for
22
up to 15 min to CO2 at increasing pressure up to 18 MPa. Samples were analysed for residual
23
enzymatic activity and SDS-PAGE patterns. At 20 and 35 °C, HP-CO2 allowed non-thermal and
24
irreversible inactivation of polyphenoloxidase with decimal reduction time (DP) that decreased
25
when pressure increased. At 45 °C, complete inactivation was achieved in less than 0.2 min at all
26
CO2 pressures. The pressure sensitivity parameter (zP) of inactivation rate resulted similar at 20 and Page 1 of 22
35 °C (5.4 and 5.5 MPa, respectively). Accordingly, temperature increase from 20 to 35 °C caused
28
minor modifications of activation volume ( V) (circa -1000 cm3 mol-1) but almost doubled the pre-
29
exponential factor of the Eyring equation. SDS-PAGE data showed that polyphenoloxidase
30
inactivation followed mechanisms other than those associated with thermal inactivation and
31
proceeded with no structural changes, probably implying the formation of bindings between
32
molecular CO2 and enzyme terminal groups.
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Page 2 of 22
34 35
Introduction
36
High pressure carbon dioxide (HP-CO2), also known as dense phase carbon dioxide (DP-CO2) has
38
been proposed as a promising non-thermal alternative to traditional pasteurisation methods. During
39
the treatment, food is in contact with pressurised CO 2 at temperature/pressure conditions that may
40
be below or above the critical point (31.1 °C, 7.38 MPa). Typical CO2 pressure is generally within 4
41
and 30 MPa, rarely exceeding 50 MPa. Temperature is generally between 20 and 50 °C, low enough
42
to avoid negative quality effects associated with traditional pasteurisation.
43
Significant lethal effects of pressurised CO2 on different microorganisms has been demonstrated not
44
only in juices but also in solid foods such as meat and fish derivatives dried ingredients and fresh-
45
cut produce [3-10]. In particular, up to 5 Log reductions were reported in the literature depending
46
on the treatment conditions and the food matrix [11]. While liquid food treatment seems not to
47
impair the product fresh-likelihood, application of this technology to the preservation of solid foods
48
could adversely affect product quality.
49
HP-CO2 has also been shown to inactivate enzymes responsible for food quality decay. The extent
50
of inactivation depends on the nature of the enzyme and is strongly affected by CO 2 pressure,
51
temperature and treatment time. Zhou et al. [12] completely inactivated pectin methylesterase in
52
carrot extract upon exposure for 15 min to 15 MPa CO2 pressure at 55 °C. By contrast, more than
53
75 min were required to inactivate the same enzyme in peach extract. In the case of horseradish
54
peroxidise and soybean lipoxygenase, only 90% inactivation was observed at 30 MPa [13,14].
55
Polyphenoloxidase from different fruit juices and seafood has also been demonstrated to be HP-CO2
56
sensitive [15-17]. Xu et al. [3] completely inactivated polyphenoloxidase in apple juice after 10 min
57
at 22 MPa and 60 °C. In the case of melon juice, complete inactivation of polyphenoloxidase was
58
not achieved even increasing CO2 pressure [18]. Besides processing parameters, the composition of
59
the matrix surrounding the enzyme and its compartmentalisation could also affect sensitivity to HP-
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Page 3 of 22
CO2. For instance, as compared to enzymes within plant and animal tissues, higher inactivation was
61
generally observed when the crude extracts were treated [8,12,17,19].
62
The mechanism of enzyme inactivation by HP-CO2 is hypothesised to be the result of local pH
63
lowering [20]. According to other authors, pressurised CO2 could cause changes in the conformation
64
of the secondary structure of the enzyme [21]. To this regard, HP-CO2 inactivation of different
65
enzymes was observed in concomitance with a decrease in the residual α-helix content of the
66
proteins, analogously to thermal induced inactivation [13,22]. Despite these findings, the effect of
67
HP-CO2 on the activity and structure of food enzymes is still under study.
68
The present paper was addressed to investigate enzymatic inactivation kinetics following HP-CO2
69
treatment of model system. To this aim, polyphenoloxidase was chosen as an example of enzyme
70
that impairs food quality. An aqueous solution containing mushroom polyphenoxidase was exposed
71
at 20, 35 and 45 °C for up to 15 min to CO2 at increasing pressures up to 18 MPa. Samples were
72
analysed for residual enzymatic activity. Inactivation rate constants were used to estimate decimal
73
reduction parameters (DP and zP) as well as activation volume (V) and frequency factor (lnkatm)
74
by the Eyring equation [23]. Finally, to compare changes induced by pressurised CO 2 and
75
conventional thermal treatment on polyphenoloxidase, SDS-PAGE separation patterns were also
76
obtained.
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2. Material and methods
79
2.1 Polyphenoloxidase solution
80
Mushroom tyrosinase (polyphenoloxidase) (T3824 50KU, Sigma, St. Louis, MO, USA), purity
81
>98%, from Agaricus bisporus was used. Polyphenoloxidase solution was prepared diluting the
82
enzyme in 0.1 M potassium phosphate buffer pH 7.0 (Carlo Erba, Milano, Italy).
83
Polyphenoloxidase initial activity on 1.5×10−3 M L-Dopa (Carlo Erba, Milano, Italy) was 0.004 Abs
84
min-1.
85
2.2 High-pressure CO2 treatments
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Figure 1 shows a schematic representation of the apparatus used for high-pressure CO2 treatments.
87
Liquid carbon dioxide (purity 99.995%, Sapio, Monza, Italy) was cooled down to 4 °C using a F34-
88
ED chiller (C; Julabo, Milano, Itlay) after been filtered with a 15 μm filter (B; Ham-Let, Milano,
89
Italy). Subsequently, CO2 was pressurised with a Orlita MhS 35/10 diaphragm pump (D; ProMinent
90
Italiana S.r.l., Bolzano, Italy) into two identical stainless steel cylinders (E and F, internal volume
91
150 mL) with a screwed cap, previously filled with 10 mL of polyphenoloxidase aqueous solution.
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The vessels connected in parallel, were immersed in a water bath connected to a CB 8-30e
93
thermostatic bath (H; Heto, Allerød, Denmark) that ensured maintenance of constant temperature.
94
Before starting the pressurisation, the temperature of the sample was allowed to reach the
95
equilibrium. The time needed to reach the desired temperature (20, 35 or 45 °C) was lower than 3
96
min. Once the desired temperature was reached, less than 2 min were needed to reach the target
97
pressure. As a consequence, the desired conditions of temperature and pressure were reached in less
98
than 5 min. As an example, supplementary material 1 shows the temperature and pressure profile of
99
the treatment carried out at 35 °C and 12 MPa for 2 min. After reaching the desired pressure (6, 8,
100
10, 12, 15 and 18 MPa), the pump was switched off and valves connected to each vessel (V2 and
101
V3) were tightly closed. In order to carefully control operating parameters such as pressure and
102
temperature during experiments, two thermocouples (TT1 and TT2) connected to a digital data
103
logger (G) and a manometer (PT3) were used. The mixing between samples and CO2 inside the
104
vessels was assured by two magnetic stirrers (MS1 and MS2) (Microstirrer, Velp Scientifica,
105
Usmate, Italy) placed below each vessel. After increasing treatment time up to 15 min, vessels were
106
depressurised opening V2 and V3 valves, the outlet metering (V4) and micrometric (V5) valves. The
107
valves V4 and V5 were heated in a water bath connected to a thermostatic bath (H) to prevent
108
freezing during decompression. In order to assure an adequate heat exchange in both water baths,
109
two small water pumps (P1 and P2) were used. In all experiments, depressurisation was completed
110
within 3 min and the outlet flow was controlled using a PFM 750 digital flowmeter (I) (SMC Italia
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Page 5 of 22
S.p.A., Milano, Italy). Additional control samples were prepared by treating the enzyme solution in
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the vessels at atmospheric pressure (0.1 MPa) and thus at CO 2 partial pressure equal to 0.0039 MPa.
113
2.3 Sample storage
114
Aliquots of 1 mL of polyphenoloxidase solution were introduced in Eppendorf® vials of 1.5 mL
115
capacity and stored for up to 7 days at 4 °C in a refrigerated cell. At increasing time during storage,
116
samples were removed from the refrigerator, equilibrated at 22 °C and submitted to the analysis.
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2.4 Temperature, pH
118
Temperature and pH were respectively assessed by a thermocouple probe (Hanna Instruments,
119
Tersid s.r.l., Milano, Italy) and a Mettler Toledo 355 pH-meter (Lou Analyzer, Halstead, Essex,
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England).
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2.5 Polyphenoloxidase activity
122
The polyphenoloxidase activity was assayed spectrophotometrically (Shimadzu UV-2501PC, UV-
123
Vis recording spectrophotometer, Shimadzu Corporation, Kyoto, Japan) at 25 °C according to the
124
methodology of Kahn [24]. The reaction was started by the addition of 20 µL of polyphenoloxidase
125
solution to 2 mL of 0.1 M potassium phosphate buffer pH 7 and 1.5 10-3 M L-Dopa (Carlo Erba,
126
Milano, Italy). The absorbance at 420 nm was monitored each minute for 10 min. The changes in
127
absorbance per min were calculated by linear regression, applying the pseudo zero order kinetic
128
model. The eventual final stationary phase was excluded from regression data. One arbitrary unit of
129
polyphenoloxidase was defined as the amount of enzyme that produced an increase in absorbance at
130
420 nm of 0.001 Abs min-1 under the testing conditions. Polyphenoloxidase residual activity was
131
calculated as the percentage ratio between enzymatic activity of the treated solution and that of the
132
control treated at atmospheric pressure (0.1 MPa).
133
2.6 SDS-polyacrylamide gel electrophoresis
134
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed
135
according to Laemmli [25]. Aliquots of polyphenoloxidase in the aqueous solution were added with
136
sample buffer containing 0.2% bromophenol blue, 0.5 M Tris-HCl, pH 6.8, 10% (w/v) SDS, 50%
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Page 6 of 22
(v/v) glycerol and 25% (v/v) β-mercaptoethanol. The samples were loaded on a 13% (w/v)
138
polyacrylamide separating gel containing 40% (v/v) acrylamide and 2% (v/v) bis-acrylamide cross
139
linker, mounted on a Mini 2-D (Bio-Rad, Richmond, CA, USA) vertical electrophoresis cell. The
140
electrophoretic separation were obtained by running the samples at 15 mA with standard proteins of
141
known molecular weight (Precision Plus Protein™ Dual Color Standards, Bio-Rad, Segrate, Italy)
142
were used to determine the apparent molecular mass of the sample bands. An additional reference
143
sample, obtained by thermal inactivation of polyphenoloxidase was also prepared. In particular, 10
144
mL enzyme solution were placed into 50 mL capacity glass tube and heated in a water bath (IKA-
145
Werke, Staufen, Germany) at 70 °C for 5 min.
146
The gels were subsequently stained with NOVEX ® Colloidal Blue Staining Kit (Invitrogen,
147
Carlsbad, CA, USA) for one night and then destained in bi-distilled water. Gels were scanned and
148
analysed with the ImageQuant TL Image Analysis Software (Amersham Bioscience Inc.,
149
Piscataway, NJ, USA).
150
2.7 Data analysis
151
Treatments were carried out at least in duplicate and analyses were performed on at least triplicated
152
samples. Data were expressed as mean ± S.D. Apparent inactivation rate constants of
153
polyphenoloxidase were analysed by using a conventional first-order equation:
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(1)
Where A is the residual activity of polyphenoloxidase (%) at time t (min) and k is the inactivation
156
rate constant (min-1). The value of k was obtained by regressing the natural logarithm of A vs. t. The
157
inactivation rate constant (k) was then derived from the slope of the obtained line.
158
The kinetic parameter DP was obtained using procedures analogous to that employed in thermal
159
death time studies. In particular DP is the decimal reduction time, i.e. the treatment time needed for
160
90% enzyme activity reduction at a given pressure and temperature. DP was computed as:
161
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155
(2)
Page 7 of 22
162
The pressure increase needed for a 90% reduction of the DP value is computed as zp (MPa). The
163
value of zp was obtained by regressing the decimal logarithm of DP versus pressure (P):
164
(3) The zP was then derived as the negative reciprocal slope of the regression line.
166
The pressure dependence of the inactivation rate constants (k) was expressed by the activation
167
volume (V, cm3/mol), according to the Eyring equation [26]:
cr
168
ip t
165
(4)
where P is pressure (MPa), katm is the inactivation rate constant at ambient pressure Patm (0.1 MPa),
170
R is the gas constant (8.31 cm3 MPa K-1 mol-1) and T is temperature (K). (V) was estimated from
171
the slope of the line obtained by regressing the natural logarithm of k vs P.
172
Goodness-of-fit was evaluated by means of the determination coefficients (R 2) and p values
173
(Statistica for Windows, ver. 5.1, Statsoft Inc., Tulsa, OK, USA, 1997).
M
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us
169
175
3. Results and discussion
ce pt
176
ed
174
Figure 2 shows the residual polyphenoloxidase activity of an aqueous solution containing 4 U of
178
mushroom polyphenoloxidase as a function of treatment time at different CO 2 pressure at 20 °C.
179
Independently on CO2 pressure, samples were always subjected to CO2 under subcritical conditions.
180
HP-CO2 treatments promoted a significant decrease in the activity of the enzyme. In particular,
181
while the polyphenoloxidase control sample, treated at 20 °C under environmental pressure (0.1
182
MPa), did not show significant changes in activity, exposure for increasing time to pressurised CO 2
183
strongly decreased this parameter. Enzyme inactivation by pressurised CO 2 has already been
184
reported for polyphenoloxidase from different origins [13,17,21]. However, in those cases, much
185
more intense treatments were required. For instance, the complete inactivation of shrimp
186
polyphenoloxidase was only achieved in the 4-25 MPa pressure range by carrying out prolonged
Ac
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Page 8 of 22
treatments at 37 °C (> 30 min) [17]. In addition, treatments carried out at 35 °C at 8 MPa for up to
188
30 min were not able to decrease the activity of apple polyphenoloxidase [13,27]. This goal was
189
only reached when apple polyphenoloxidase was submitted to longer treatments and/or higher
190
temperatures [3]. Data shown in Figure 2 indicate that mushroom polyphenoloxidase was
191
completely inactivated at room temperature within few minutes of treatment by selecting the proper
192
pressure conditions. The higher susceptibility to pressurised CO 2, as compared to that reported from
193
the same enzymes extracted or included in food matrices other than mushroom tissue, is likely to be
194
attributable to the enzyme origin. The latter is well known to affect the sensitivity of enzymes to
195
different inactivation treatments, including those based on high pressure [28]. In addition, the
196
sensitivity of the enzyme could also be affected by its environmental conditions during the
197
treatment. To this regard, Zhou et al. [12] observed that the inactivation of pectin methylesterase in
198
a buffer was enhanced as compared to the original enzyme in carrot and peach juices.
199
When polyphenoloxidase was exposed to HP-CO2 at 35 °C, inactivation was observed in even
200
shorter times (Figure 3). Samples were always under supercritical conditions of CO2 with the only
201
exception of those exposed to 6 MPa. It can be noted that no abrupt change in enzyme inactivation
202
seemed to be associated with the transition from subcritical to supercritical condition. The effect of
203
temperature on enzyme inactivation was further confirmed by additional trials carried out at 45 °C.
204
The latter showed that exposure to the minimum pressure tested (6 MPa, subcritical conditions) for
205
just 30 s was sufficient to completely inactivating the enzyme. It is noteworthy that the control
206
sample, treated at the same conditions of temperature and time but at atmospheric pressure (0.1
207
MPa), presented a residual polyphenoloxidase activity approaching 90%. These data demonstrate
208
the existence of a negative relation between polyphenoloxidase activity and the increase in both
209
CO2 pressure and temperature at least under the experimental conditions here tested. Antagonistic
210
effects of pressure and temperature on polyphenoloxidase activity have actually been reported by
211
the application of hydrostatic pressure at temperature higher than circa 60 °C [26].
Ac
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187
Page 9 of 22
Based on data shown in Figures 2 and 3, the apparent inactivation rate constants of
213
polyphenoloxidase were computed (Table 1). The residual activity of polyphenoloxidase, expressed
214
in natural logarithmic values, had good relationships (R2>0.77, p<0.05) with the treatment time. DP
215
values were thus derived from the rate constants, using procedures analogous to that employed in
216
thermal death time studies (Table 1).
217
In particular, DP was defined as the treatment time needed for 90% enzyme activity reduction at a
218
given pressure. For instance, the treatment at 6 MPa and 20 °C leaded to a tenfold decrease of
219
activity in circa 18 min. At the same temperature, this goal was achieved at 12 MPa in circa 3 min.
220
On the other hand, keeping constant CO2 pressure at 6 MPa, inactivation was achieved at 35 or 45
221
°C in 3.7 or less than 0.2 min, respectively. It is noteworthy that, when the enzyme resulted
222
completely inactivated following the shortest treatment considered in the experimental plan (0.2
223
min), the rate constant was not determinable. However, in such cases, the DP value can be assumed
224
as shorter than 0.2 min. These low DP values are in agreement with the higher susceptibility of
225
mushroom polyphenoloxidase to HP-CO2 treatments, as compared to that from other origin. To this
226
regard, Gui et al. [13] reported polyphenoloxidase in cloudy apple juice to be inactivated in circa
227
220 min upon exposure at 35 °C to 30 MPa. Similarly, Zhang [17] reported Pacific white shrimp
228
polyphenoloxidase to exert a Dp of circa 30 min at 15 MPa and 37 °C.
229
Dp values relevant to experiments carried out at 20 and 35 °C (Table 1) were used to calculate the
230
parameter zP, describing the sensitivity of polyphenoloxidase to pressurised CO2. The decimal
231
logarithmic values of DP resulted well correlated with pressure for treatments carried out at 20 and
232
35 °C (Figure 4). The existence of a good correlation also in the case of treatments carried out at 35
233
°C (subcritical at 6 MPa, supercritical at higher pressure) seems to confirm that enzyme inactivation
234
is more affected by the overall processing conditions (time, temperature and pressure) rather than
235
by the transition from sub to supercritical CO2 condition. The zP value, which represents the
236
pressure range within which the DP changes tenfold, was calculated as the negative reciprocal slope
237
of the regression line (Figure 4). The pressure sensitivity parameter zP resulted circa 5.5 MPa,
Ac
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Page 10 of 22
independently on whether the treatments were carried out at 20 or 35 °C. This indicates that an
239
increase in pressure of 5.5 MPa is necessary to get a 90 % decrease in DP at both temperatures.
240
The effect of CO2 pressure on polyphenoloxidase inactivation was also expresses through the
241
activation volume (V ) concept. Activation volumes (V) were computed from the slope plots of
242
the natural logarithm of enzyme activity as a function of pressure (Figure 5).
243
According to the transition state theory, the activation volume is a measure of the volume difference
244
between the initial reactants and the activated complex at the transition state [23]. Data reported in
245
Figure 5 show that polyphenoloxidase inactivation is characterized by negative V values with
246
high absolute value. This indicates that the increase in pressure strongly favoured the denaturation
247
of the enzymatic protein. The values of activation volume for mushroom polyphenoloxidase
248
resulted one order of magnitude lower than those reported in the literature for the same enzyme in
249
apple (-94.3 cm3 mol-1 at 55 °C) or shrimps (-120.9 cm3 mol-1 at 37 °C). This result indicate that
250
polyphenoloxidase in these food matrices is less susceptible to CO 2 pressure variation than that
251
extracted from mushrooms. In addition, activation volume assumed almost equal values (circa -
252
1000 cm3 mol-1), regardless of the temperature experienced by the samples during the treatment.
253
This is in agreement with the similarity in zp values (Figure 4) and indicates that the increase in
254
temperature from 20 to 35 °C only reduces the time required for the inactivation of the enzyme (Dp,
255
Table 1) but has no effects on its sensitivity to the increase in CO 2 pressure. By contrast,
256
temperature would affect the pre-exponential or frequency factor (lnkatm) that determines how often
257
the protein is properly oriented in order to undergo structural modifications on chemical reactions
258
leading to denaturation. To this regard, it can be observed that rising the temperature from 20 to 35
259
°C increased the frequency factor by more than two orders of magnitude (Figure 5), making the
260
reaction significantly quicker.
261
The occurrence of almost identical z p and V is quite unusual in the literature relevant to the effect
262
of pressure on enzymes. For instance, Polydera et al. [29] reported activation volume for pectin
263
methylesterase inactivation by high hydrostatic pressure to be affected by temperature according to
Ac
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238
Page 11 of 22
a linear function. In that case, the higher the temperature, the higher the activation volume,
265
indicating that enzyme inactivation rate was less susceptible to the increase in pressure when
266
temperature was increased. Weemaes et al. [26] reported similar data with reference to avocado
267
polyphenoloxidase treated in a wide interval of hydrostatic pressure and temperature. However, in
268
this case, a temperature range was identified in which the activation volume could be considered
269
almost constant, similarly to the result obtained by exposing mushroom polyphenoloxidase to HP-
270
CO2 treatments (Figure 5).
271
By comparing the values of V for polyphenoloxidase inactivation by HP-CO2 treatments (Figure
272
5) to those estimated by applying high hydrostatic pressure only [26], it can be observed that the
273
latter were associated with higher V values (-14/-36 cm3 mol-1). As previously suggested [17, 21,
274
30], this result confirms that the activity of polyphenoloxidase is susceptible not only to the
275
variation of pressure but also to the molecular effects of pressurised CO2.
276
Polyphenoloxidase samples showing no residual activity after the treatments were also analysed for
277
activity after storage for 7 days at 4 °C. The enzyme resulted unable to recover its activity upon
278
storage. This result is in agreement with literature evidence showing the capability of pressurised
279
CO2 in irreversibly decomposing the -helix of the protein structure [21]. To better understand the
280
nature of the polyphenoloxidase modification induced by pressurised CO 2, the attention was
281
focused on samples that underwent enzyme inactivation by exposure for 1 min at 20 °C at 6, 12 and
282
18 MPa. SDS-PAGE was performed on polyphenoloxidase samples treated at increasing pressure
283
by using β-mercaptoethanol as a reducing agent (Supplementary material 2). Exposure of
284
polyphenoloxidase to pressurised CO2, despite decreasing its residual activity, did not promote
285
changes in the electrophoretic pattern as compared to that of the unpressurised enzyme (0.1 MPa).
286
In fact, control and polyphenoloxidase treated samples showed one clear band of molecular weight
287
of about 48 kDa [31-33]. In other words, the treatments did not induce evident fragmentation and/or
288
aggregation of the polyphenoloxidase subunits. This result is consistent with data obtained by
289
HPLC gel permeation analysis of polyphenoloxidase submitted to pressurised CO 2 at 22 °C [34]
Ac
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Page 12 of 22
and with indications relevant to the effect of this physical treatment on other enzymes [14]. To
291
verify whether the mechanism of polyphenoloxidase inactivation was different from that associated
292
with thermal inactivation, an additional sample, with no residual activity, was prepared by
293
maintaining the polyphenoloxidase solution at 70 °C for 5 min (Supplementary material 2).
294
Thermal inactivation of polyphenoloxidase was associated with the smearing of the band at the top
295
of the lane, suggesting the presence of high molecular weight protein aggregates. Heating would
296
thus promote enzyme inactivation by mechanisms other than those promoted by HP-CO2 treatments.
297
In particular, the exposure to HP-CO2 could promote local pH lowering, possibly resulting in
298
enzyme inactivation [2,20]. In the present study, the pH of the treated sample resulted similar to that
299
of the untreated one (7.0). However, due to plant constraints, sample pH was only analysed after
300
depressurisation, not allowing to appreciate pH modifications during CO 2 solubilisation in the
301
treatment vessel. It was also hypothesised that HP-CO2 exposure could involve minor
302
conformational modifications which would be however able to reduce enzyme functionality [21].
303
To this regard, it can be hypothesised that inactivation of polyphenoloxidase by HP-CO2 treatments
304
could involve the formation of carbamino groups between CO 2 and N-terminus groups of the
305
enzyme, with a mechanism similar to that observed in haemoglobin [35].
306
308
Conclusions
Ac
307
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290
309
HP-CO2 treatments allowed quick, non-thermal and irreversible inactivation of polyphenoloxidase
310
in aqueous model systems. The complete inactivation of the enzyme was achieved within minutes
311
of treatment even at room temperature by selecting the proper CO 2 pressure. Kinetic analysis of the
312
pressure and temperature induced inactivation demonstrated that the increase in temperature
313
reduced inactivation time but did not affect enzyme sensitivity to the pressure variation.
314
Polyphenoloxidase inactivation by HP-CO2 was not associated with changes in electrophoretic
315
pattern and occurred following mechanisms other than those of thermal treatment.
Page 13 of 22
316
Being CO2 relatively inert, cheap, non-toxic and recyclable, the results acquired provide additional
317
evidence of HP-CO2 potential as an efficacious and sustainable non-thermal blanching technology.
318
Acknowledgement
320
This research was supported by Fondazioni in Rete per la Ricerca Agroalimentare, “AGER -
321
Agroalimentare e Ricerca”. Project 2010 2370.
ip t
319
cr
322
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417 418
Page 17 of 22
419
Fig.1. Schematic representation of the high-pressure CO2 apparatus.
420 421
Fig. 2. Polyphenoloxidase activity in an aqueous solution as a function of exposure time to
422
increasing CO2 pressure at 20 °C. Control was treated at environmental pressure (0.1 MPa).
ip t
423
Fig. 3. Polyphenoloxidase activity in an aqueous solution as a function of exposure time to
425
increasing CO2 pressure at 35 °C. Control was treated at environmental pressure (0.1 MPa).
cr
424
us
426
Fig. 4. DP values of polyphenoloxidase inactivation by HP-CO2 treatments carried out at increasing
428
pressure at 20 and 35 °C. zP values and relevant determination coefficients are also shown.
an
427
429
Fig. 5. Logarithmic values of polyphenoloxidase apparent inactivation rate constants (k) as a
431
function of HP-CO2 treatments carried out at increasing pressure at 20 and 35 °C. Activation
432
volume (V), frequency factor (lnkatm) and relevant determination coefficients are also shown.
ed
M
430
Ac
434 435
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433
Page 18 of 22
ip t 439
an
Ac
441
ce pt
440
us
cr 438
M
437
ed
436
A: CO2 tank PT: manometers V: valves B: CO2 filter C: chiller D: pump E, F: vessels TT: thermocouple G: digital temperature data logger H: thermostatic bath I: digital flowmeter L: volatiles trap P: water recirculation pump
442
Page 19 of 22
ip t cr us
443
445
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an
444
Page 20 of 22
ip t cr us an
446
20
0.1
ed
M
Table 1 Polyphenoloxidase apparent inactivation rate constants (k), relevant determination coefficient and DP values of HP-CO2 treatments carried out at increasing pressure at 20, 35 and 45 °C. Temperature Pressure (MPa) k (min-1) R2 DP (min) (°C) 0.0014
0.90
1645.1
0.1264
0.95
18.2
8.0
0.4050
0.91
5.7
12.0
0.7272
0.96
3.1
15.0
2.6593
0.99
0.8
18.0
4.0829
0.99
0.6
0.1
0.0518
0.83
44.5
6.0
0.6235
0.77
3.7
8.0
3.0714
0.85
0.7
12.0
6.6378
0.89
0.3
15.0
*
<0.2
18.0
*
<0.2
0.1
0.0723
6.0
*
ce pt
6.0
35
45
Ac
447 448 449
0.80
31.8 <0.2
Page 21 of 22
450 451
8.0
*
<0.2
12.0
*
<0.2
15.0
*
<0.2
18.0
*
<0.2
* The enzyme resulted completely inactivated after the shortest treatment time (0.2 min). HP-CO2 treatments quickly inactivated mushroom polyphenoloxidase in model systems
453
Pressure sensitivity (zP ) of enzyme inactivation was circa 5.5 MPa at 20 and 35 °C
454
At 20 and 35 °C, activation volume V was about -1000 cm3 mol-1
455
HP-CO2 did not change the molecular mass of the enzyme subunits
us
cr
ip t
452
456
Ac
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ed
M
an
457
Page 22 of 22