Effects of Peach Cultivar on Enzymatic Browning Following Cell

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Effects of Peach Cultivar on Enzymatic Browning Following Cell Damage from High Pressure Processing Chukwan Techakanon, Thomas M. Gradziel, and Diane Marie Barrett J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01879 • Publication Date (Web): 14 Sep 2016 Downloaded from http://pubs.acs.org on September 15, 2016

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Journal of Agricultural and Food Chemistry

Effects of Peach Cultivar on Enzymatic Browning Following Cell Damage from High Pressure Processing Chukwan Techakanona,b, Thomas M. Gradzielc, Diane M. Barretta,* a

Department of Food Science and Technology, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States

b

Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani Campus, 31 Makham Tia, Muang Surat Thani, Suratthanee 84000, Thailand c

Department of Pomology, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States

*Corresponding author:[email protected]

ACS Paragon Plus Environment


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Abstract Peach cultivars contribute to unique product characteristics and may affect the degree of

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browning after high pressure processing (HPP). Nine peach cultivars were subjected to HPP

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at 0, 100 and 400 MPa for 10 min. Proton nuclear magnetic resonance (1H-NMR)

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relaxometry, light microscopy, color, polyphenol oxidase (PPO) activity and total phenols

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were evaluated. The development of enzymatic browning during refrigerated storage

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occurred due to damage during HPP that triggered loss of cell integrity, allowing substrates to

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interact with enzymes. Increasing pressure levels resulted in greater damage, as determined

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by shifts in transverse relaxation time (T2) and by light micrographs. Discoloration was

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triggered by membrane de-compartmentalization but limited by PPO activity, which was

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found to correlate to cultivar harvest time (early, mid and late season). Outcomes from the

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microstructure, 1H-NMR and PPO activity evaluation were an effective means of determining

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membrane de-compartmentalization and allowed for prediction of browning scenarios.

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Key words: High pressure, peach cultivar, 1H-NMR, cell integrity, enzymatic browning


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1. Introduction Peaches are one of the most popular summer fruits consumed in the U.S. and are well adapted to

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California production environments. As they have a relatively short available season, high

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pressure processing (HPP) can be an alternative method for preservation of these high quality

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fruits that are rich in nutrients, have a desirable texture and a natural flavor. HPP is a novel

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advanced process being extensively studied because of its ability to create products that retain

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their fresh-like attributes, while inducing destruction of microorganisms and inactivating the

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enzymes that cause deterioration of quality.1-3 A recent study reported that the market value of

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high-pressure preserved products had reached more than $3 billion, and among these, fruit and

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vegetable products ranked as the largest product group.4

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Peach cultivar plays a crucial role in final product characteristics, e.g. color, taste, texture and

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flavor, especially after undergoing high pressure treatment. During HPP treatment, elevated

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pressures have been reported to induce loss of membrane permeability in plant-based materials.5

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Discoloration in HPP-treated peaches occurs as a consequence of the process, and the hypothesis

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is that cell integrity is the primary determining factor affecting enzymatic browning reactions in

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refrigerated and stored high pressure processed peaches. After loss of fruit cell integrity, the

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enzyme polyphenoloxidase (PPO), initially located in the plastids, and the phenolic substrates,

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initially located in the vacuole, are allowed to interact, creating brown products. The

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discoloration of fruits following HPP has been explored extensively in several commodities, e.g.

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apple purée2, avocado purée6, banana purée7, green beans8, guacamole9, and pears10. This is the

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first study to correlate loss of cell integrity to enzymatic browning reactions in different peach

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cultivars after high pressure treatment.

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In order to quantitatively follow changes in cell integrity resulting from HPP application, nuclear

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magnetic resonance (NMR) and light microscopy were employed. NMR water proton



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relaxometry is a tool that can be used to detect physiological changes in the various water

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compartments in a plant tissue. This method has undergone continuous development and has

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now been applied in a wide range of plant studies, in particular for quantification of cell

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membrane damage.5,11 The degree of membrane damage can be determined by observing

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changes in water proton relaxation behavior (T2) of the vacuolar, cytoplasm and cell wall

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compartments. The proton spin–spin (T2) relaxation time is related to properties of water in

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different locations in the tissue, water content, and the interaction of water with

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macromolecules.12 Since the browning reaction is a consequence of membrane rupture, T2

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relaxation time of the vacuolar compartment can be used in predicting the browning scenario of a

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product.

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Observation under a light microscope is a useful complement to 1H-NMR for determining cell

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integrity of peach samples following HPP. In this experiment, cell integrity was observed under a

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light microscope using the vacuole staining method developed by Admon et al. (1980)13. Neutral

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red (NR) dye diffuses across the tonoplast membrane and ionizes in the acidic environment of

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the vacuole, appearing as an intense red color. Once cell membrane damage occurs, the vacuoles

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lose their integrity, thus resulting in the dye spreading throughout the cell and therefore

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appearing less intense in red color. The use of this dye allows for discriminating intact vacuoles

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from damaged, non-intact ones. The number and color intensity of intact vacuoles represents the

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level of damage to the cells. Thus the comparison of micrographs of samples from different

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peach cultivars processed using different pressure treatments can be correlated to the degree of

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de-compartmentalization.

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The objectives of this study were to determine the effect of peach cultivar on cell integrity after

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high pressure processing at different pressure levels (0, 100 and 400), using NMR and


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microscopic studies, and to correlate that to the development of brown color, as affected by PPO

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activity and total phenols content. Phenotypic differences in peach cultivars, such as texture and

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browning susceptibility, may be useful in defining which cultivars are best suited for

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preservation by HPP.

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2. Materials and Methods

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2.1 Raw Materials

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The clingstone type peach cultivars, Carson, Andross, Ross, Evert, Dr.Davis, Late Ross, Halford,

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Lilleland and the freestone type, Summerset, were harvested by hand from Foundation Plant

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Services, at the University of California, Davis, CA. All cultivars were of the same variant

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(clingstone non-melting variant) except for Summerset, which was a freestone melting type.

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Peaches were harvested at 157-164 days after bloom and then sorted to a firmness of 35-40 N

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using a TA-XT2 Texture Analyzer (Stable Micro Systems Ltd., Surrey, UK) and stored at 4 °C

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RH 84% for approximately two days until processing. In each replicate, 3 fruits of the same

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cultivar were hand peeled, sliced into approximately 3 cm thickness and placed into polyethylene

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bags (4 mil vacuum pouches, Ultrasource, Missouri, USA). Each bag contained 3 peach slices of

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the same cultivar, 1 from each of the 3 different fruits, and separate bags were created for each

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analysis method. In addition, approximately 2 mL of peach extracts (see description below) were

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vacuum packed in polyethylene bags. The samples were kept at ambient temperature (22 ± 2°C)

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for 30 min after packaging prior to high pressure treatment. On each of three replicate days of

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processing, each peach cultivar was processed at each of two pressures (100 and 400 MPa). The

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control treatment was an unprocessed, sliced peach samples in a vacuum package (approximately

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0 MPa). The same fruit was analyzed for all parameters, e.g. difference in lightness, the T2



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relaxation time using NMR, PPO activity, total phenols and determination of viable cells using

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light microscopy.

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2.2 Experimental Design

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Nine peach cultivars were used in this study and each was high pressure treated at 100 and 400

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MPa. Non-HPP treated samples (0 MPa) were used as controls. The HPP processing for this

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entire experiment was three distinct times, which occurred on three separate days. On each

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replicate processing day, three fruit per cultivar were used. Each fruit was sliced into 3 cm thick

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slices, on the two sides of the pit, and then sub-divided into 5 parts. Each of the 5 parts was used

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for a different analytical measurement, e.g. color, NMR (paramagnetic analysis of protons), PPO

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activity, total phenols and light microscopy. Prior to HPP treatment, slices were packaged

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according to analytical method, so only one package needed to be opened prior to each analytical

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method. The entire process design was repeated 3 separate times, therefore, in total nine samples

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per cultivar were analyzed for each of the five analytical parameters.

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2.3 High pressure processing (HPP)

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The packaged samples were processed at 100 and 400 MPa for 10 min in a high pressure

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processing unit model QFP 2L-700 (Avure Technologies Inc, Kent, WA, USA). The high

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pressure unit uses water as a pressurizing medium and has a maximum pressure level at 600 MPa.

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The initial temperature in the chamber (Ti) was around 23 °C. The maximum temperature in the

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high pressure chamber was dependent on the set pressure, which for 100 and 400 MPa was 25

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and 33°C, respectively.

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2.4 Nuclear magnetic resonance (NMR) relaxometry

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Following HPP, each sample slice was cut into a cylindrical piece using a cork borer with a 15

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mm diameter and 15 mm height. The cylindrical piece was blotted dry then placed into a covered


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NMR tube, which was placed in a plastic sample holder of the NMR unit. Measurements of

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NMR relaxometry were performed using an NMR spectrometer (Aspect AI, Industrial Area

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Havel Modi’in, Shoham, Israel) with a magnetic field of 1.02 T and frequency of 43.5 MHz. The

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Carr-Purcell-Meiboom-Gill sequence with an echo time of 0.5 ms and 15000 echoes was used to

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obtain T2 relaxation decay curve. T2 spectrum inversion was performed by Non-negative Least

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Square using Prospa (Magritek, Wellington, New Zealand).

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2.5 Light Microscopy study

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Section preparation: After high pressure treatment, 2 pieces with a rectangular shape of

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approximately 1.0×0.5×0.3 cm were obtained from each sliced sample and placed in a sample

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holder. Peach specimens approximately 200 µm in thickness were obtained using a Vibratome

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1000 Plus (The Vibratome Co., St. Louis, Mo., U.S.A.) before being submerged for 2 h in the

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staining solution. The solution was prepared using 0.5% neutral red in an acetone stock solution

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filtered twice with Whatman paper # 1, and diluted to 0.04% in 0.55 M mannitol – 0.01 M

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HEPES (N-[2-hydroxyethyl] piperazine-N’-[2-ethane-sulfonic acid]) buffer, pH 7.8. Peach

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sections were rinsed for 0.5 h in the 0.55M mannitol−0.01M HEPES buffer solution before being

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mounted on a microscope slide and covered with a coverslip.

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Microscopic observation: Stained specimens were observed at 40x magnification using a light

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microscope (Olympus System Microscope, Model BHS, Shinjuku-Ku, Tokyo, Japan). Color

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photomicrographs were captured by a digital color camera (Olympus MicroFire, Olympus,

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Tokyo, Japan) attached to the microscope using Olympus software (Olympus America, Melville,

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N.Y., U.S.A.).

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Image processing and analysis: Micrographs were analyzed using Image J (NIH, U.S.A.), an

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image processing software. Fifteen micrographs were randomly selected from 3 replicates for



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each peach cultivar following different treatments: control (0 MPa) and HPP at 100 and 400 MPa.

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A cell counter plug-in developed by de Vos (2008)14 was used to semi-quantify the number of

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viable cells, which were distinguished by smooth pink to red stained cells, while inviable cells

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appear in a rough membrane without dye retained.

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2.6 Degree of browning (Difference in lightness)

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The degree of browning of peach samples was determined as the difference in lightness (DL*),

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which is the difference between initial lightness and the lightness of the sample after storage at

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4°C for 2 weeks. The lightness was monitored using a Minolta CR-400 colorimeter (Minolta

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camera Co, Ltd., Japan) with a beam diameter of 11 mm and a viewing angle of 0°. The values

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were expressed by the CIE L*a*b* system. A white tile used for calibration has L* = 96.88,

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a* = 0.02, b* = 2.05.

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2.7 Partial purification of peach polyphenol oxidase extracts

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The method for protein partial purification using Triton X-114 by Espin et al. (1995)15 was used

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with some modifications to obtain peach extracts. A peach sample (200 g) was homogenized

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with 100 mL of cold solution of 0.1 M sodium phosphate dibasic anhydrous (Fisher, Fair lawn,

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N.J., USA) (pH 7.3), 6% (w/v) Triton X-114 (Sigma Aldrich, St.Louis, M.O., USA) and 20 mM

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EDTA (Fisher, Fair lawn, N.J., USA) for 2 min. The mixture was refrigerated for 60 min at 4 °C

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then centrifuged with a Sorvall-RC5 (E. I. DuPont Co, Wilmington, Del) at 4 °C and 28,373.6 ×g

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for 45 min. The supernatant was mixed with 8% (w/v) of the surfactant Triton X-114 before it

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was incubated in a 40°C water bath for 15 min, after which the change to opaque yellowish was

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observed. After centrifugation at 578.6 ×g for 10 min at 25°C, a detergent-rich phase was

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discarded, and the clear supernatant was collected. A second phase partitioning step with 8%

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(w/v) Triton X-114 was performed, then it was incubated again in a 40 °C water bath for 15 min.


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The enzyme extract was collected from the supernatant after centrifuged at 578.6 ×g for 10 min

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at 25°C and was stored at -10°C until used.

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2.8 Polyphenol oxidase (PPO) assay

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PPO activity was assayed using the spectrophotometric method described by Espín et al.

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(1995)15 with some modifications. This method uses 3-methyl-2-benzothiazolinone (MBTH) as a

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chromogenic coupling agent, which reacts with the quinone products obtained from the oxidation

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of di-phenol with PPO. Assays began by adding 10 µL of peach polyphenol oxidase extract to

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1.0 mL of a medium containing 0.6 mL of 100 mM acetate buffer (pH 5.5), 0.2 mL of 2.5 mM

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MBTH (Sigma Aldrich, St.Louis, M.O., USA) and 0.2 mL of 25.0 mM dihydroxyhydrocinnamic

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acid (Sigma Aldrich, St.Louis, M.O., USA). The adduct formed from this reaction appeared to be

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reddish in color. PPO activity was determined by an increase in absorbance at 500 nm using UV

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spectrophotometry (UV2101PC, Shimadzu Scientific Instruments Inc., Columbia, MD, USA).

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The activity calculation was performed using the following equation, where Abs (0) is the initial

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absorbance and Abs (1) is absorbance at the end of linearity.

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PPO Activity (units/mL) = Abs(1)-Abs(0) /min· mL of juice

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2.9 Analysis of total phenols

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Analysis of total phenols in peach samples was performed by the Folin-Ciocalteu method

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described by Waterhouse (2002)16 with some modifications. The analysis measures the total

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concentration of phenolic hydroxyl groups in plant extracts based on reduction of the reagent

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(Folin-Ciocalteu reagent). The formation of a blue complex product can be measured at 760 nm

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and gallic acid (Arcos Organics, Geel, Belgium) in a range of 0–500 mg/L is used for calibration

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of the standard curve. A 20 g peach sample was homogenized with 30 mL deionized water, then

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6.4 g of the homogenate was blended with 27.6 mL of 76% (v/v) aqueous acetone for 2 min. The



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solution was placed in a shaker for 10 min for further mixing, after which the cell wall particles

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were separated using a centrifuge (Centra CL2 tabletop centrifuge, IEC, Needham, MA, USA) at

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578.6 ×g for 10 min. One mL of the supernatant was vortexed with 0.36 mL of 2N Folin reagent

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(Sigma Aldrich, Buchs, Switzerland) and allowed to stand for 5 min. After this, 7.5% (w/v)

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sodium carbonate (6 mL) was added to the solution, which was mixed well before adding 2.64

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mL of deionized water. The solution was homogenized again and incubated in a 50°C water bath

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for 5 min. After the solution was cooled down to room temperature for 1 h, the absorbance was

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monitored at 760 nm. The results were expressed as gallic acid equivalents (GAE)/fresh weight

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of peaches (g).

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2.10 Statistical Analysis

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This experiment was carried out in three replicate processing runs which occurred on three

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separate days. The effect of peach cultivar on the T2 relaxation time, % water, % viable cells,

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difference in lightness, PPO activity of the intact fruit, PPO activity of the extract and total

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phenols content were analyzed using Analysis of Variance for each cultivar and processing

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pressure level. Fisher’s least significant difference (LSD) test was used to compare means of

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each peach cultivar processed at different pressure levels at p< 0.05 (SAS version 9.4, Cary,

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N.C., U.S.A.). The plots present the mean with its standard deviation for each determination.

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3. Results and Discussion

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3.1 Effect of peach cultivar on cell integrity following HPP as measured using NMR relaxometry

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1

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various water compartments in a plant tissue. The shift in spin-spin or T2 relaxation time from

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CPMG pulse sequence correlates to changes in the properties of water, the interaction of water

H-NMR relaxometry is a non-destructive measurement that probes physiological changes of the


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with macromolecules, and the permeability of the compartments.17 In the present study, T2

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relaxation peaks of peach samples from different cultivars showed a similar spectrum of three

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distinct compartments (Figure 1). According to previous research on apple parenchyma tissue,

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the three compartments have been assigned to the vacuole (the highest peak and longest T2),

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cytoplasm (second compartment), and cell wall (the shortest T2).17 Figure 2 illustrates that most

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of the cultivars showed the same pattern, with an increasing T2 in the vacuolar compartment after

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100 MPa, which was only significant (p

Effects of Peach Cultivar on Enzymatic Browning Following Cell

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