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Understanding the effect of Pulsed Electric Fields on multilayered solid plant foods: Bunching onions (Allium fistulosum) as a model system
Accepted Manuscript Understanding the effect of Pulsed Electric Fields on multilayered solid plant foods: Bunching onions (Allium fistulosum) as a model system
Tingting Liu, David John Burritt, Indrawati Oey PII: DOI: Reference:
S0963-9969(18)30891-3 https://doi.org/10.1016/j.foodres.2018.11.006 FRIN 8069
To appear in:
Food Research International
Received date: Revised date: Accepted date:
17 June 2018 28 October 2018 3 November 2018
Please cite this article as: Tingting Liu, David John Burritt, Indrawati Oey , Understanding the effect of Pulsed Electric Fields on multilayered solid plant foods: Bunching onions (Allium fistulosum) as a model system. Frin (2018), https://doi.org/10.1016/ j.foodres.2018.11.006
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ACCEPTED MANUSCRIPT
Understanding the effect of Pulsed Electric Fields on multilayered solid plant foods: bunching onions (Allium fistulosum) as a model system
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Tingting Liu1,2,3 , David John Burritt2 , and Indrawati Oey1,3*
Department of Food Science, University of Otago, PO Box 56, Dunedin 9054, New Zealand
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Department of Botany, University of Otago, PO Box 56, Dunedin 9054, New Zealand
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Riddet Institute, Palmerston North, New Zealand
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*Corresponding author:
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Professor Dr. Indrawati Oey
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Department of Food Science, University of Otago
PO BOX 56, Dunedin 9054, New Zealand
Phone: +64-3-479-8735, Fax: +64-3-479-7567
Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract
While it is well known that the nature of the applied electric field and the heterogeneity of the tissue can influence the impact of PEF treatment on the plant tissues found in plant-based foods, few studies have investigated the influence of PEF on plant structures that are made up of
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multiple structurally similar organs. The aim of this study was to understand the effect of pulsed electric fields (PEF), at different electric field strengths (0, 0.3, 0.7 and 1.2 kV/cm) and specific
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energy (7, 21 and 52 kJ/kg), on a multilayered plant material, with bunching onion bulb tissues
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being used as a model system. The present study found that carbohydrates leakage was an
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appropriate index to assess PEF induced damage and that plasmolysis of epidermal cells was a good indicator of plasma membrane integrity after PEF. In addition, electric field strength had a
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greater impact on the cell integrity than specific energy applied. While other studies have shown that different cell types have different sensitivities to PEF, using plasmolysis as an
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indicator of cell damage, this study clearly showed that the same PEF treatment conditions had a greater effect on the epidermal cells of the outer scales compared to the inner scales. Hence,
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while different plant cell types vary in their sensitivities to PEF the spatial location of the same cell type within a complex plant material made up of multiple similar organs, i.e. an onion bulb,
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can also influence how cells respond to the PEF treatment. Despite PEF induced disruption at the cellular level being detected by carbohydrate leakage, the epidermal cell plasmolysis test and by cryo-scanning electromicroscopy (cryo-SEM), no gross structural changes at the organ level were observed using cryo-SEM or fluorescence microscopy. This study also reports for the first time that PEF treatment can enhance fructan leakage from onion bulbs, which means that PEF treatments have the potential to manipulate the fructan contents of some plant-based foods.
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ACCEPTED MANUSCRIPT Key words: plant structure; electric field strength; bunching onion; plasmolysis; fructans;
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microscopic level.
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ACCEPTED MANUSCRIPT 1. Introduction Pulsed electric field (PEF) processing applies very short pulses (μs) of high electric field strength to a product placed between two electrodes, which can lead to the permeabilization of cell membranes. The use of PEF technology to treat solid materials such as potato tubers
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(Faridnia, Burritt, Bremer, & Oey, 2015) and carrots (Leong, Richter, Knorr, & Oey, 2014) has shown a great potential to reduce the energy required for subsequent food processing, e.g. the
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cutting and frying steps required for the production of low fat sweet potato chips (Liu et al.,
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2017).
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Studies to date have shown that the responses of solid, complex and inhomogeneous plant
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materials, such as onion sections (Fincan & Dejmek, 2002), whole potatoes (Faridnia et al., 2015) and whole sweet potatoes (Liu et al., 2017), to PEF are very variable and the effects on different plant organs/tissues are often uneven and difficult to predict. Plant organs are made up
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of different cell and tissue types with varying physical, chemical, electrical and topological
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properties, all of which can influence both their conductivity and sensitivity to PEF induced electroporation. Thus, the homogeneity of the cells and tissues that make up solid plant foods is
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one of the major factors responsible for the uneven distribution of PEF effects.
However, unlike root or tuberous vegetables (such as carrots, potatoes or sweet potatoes), onion bulbs are made up of multiple layers of fleshy scales (modified leaves), with each scale having the same tissue organisation. While the arrangement of the scales is generally consistent between onion bulbs, depending on their position (e.g. inner vs outer layers) the scales themselves can differ in size and to some extent physiological properties. Hence, the combination of a consistent multilayered structural pattern with only minimal inhomogeneity between the layers of scales makes onions an interesting model system to study the influence of 4
ACCEPTED MANUSCRIPT electric fields on complex plant structures of relevance to food science. The main objective of the present study was to understand whether the same tissue types respond the same way to quasi- homogeneous electric fields irrespective of their spatial position, e.g. inner vs outer scales of the bunching onion bulb.
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An issue, when studying the influence of PEF on complex plant tissues, is variability between samples/specimens, which can often make identifying the subtle impacts of PEF on plant tissues
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difficult. Considerable variability in bulb size and developmental stage can be found in
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commercial batches of onion (Allium cepa L) bulbs and postharvest storage conditions can greatly influence various aspects of their metabolism, especially carbohydrate metabolism. For
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these reasons instead of using commercially available onion bulbs we used, as our model
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system, bunching onions (Allium fistulosum) grown under controlled conditions were PEF treated immediately after harvest in order to minimize variability. The edible bases of bunching
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onions are structurally very similar to commercial onion bulbs and when grown under consistent
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conditions are structurally quite uniform.
Electrolyte leakage, measured as a change in conductivity, is often used to measure PEF induced disruption of plant materials. However, the use of electrolyte leakage to assess PEF induced
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damage in onion bulbs has been questioned, as only 20% of total solute leakage from onion bulbs is electrolytes, with the remaining 80% being carbohydrates (Palta, Levitt, & Stadelmann, 1977). Also, 85% of the electrolytes leaking from PEF treated onion bulbs diffuses out of living cells (Palta et al., 1977) and so electrolyte leakage might not be a good indicator of irreversible cell damage in onion bulbs (Murray, Cape, & Fowler, 1989; Asavasanti, Ersus, Ristenpart, Stroeve, & Barrett, 2010; Faridnia et al., 2015). Hence in the present study we used, in addition to electrolyte leakage/conductivity, the release of soluble carbohydrates including fructans, and a plasmolysis susceptibility test to assess PEF- induced cellular damage to bunching onion bases. 5
ACCEPTED MANUSCRIPT Carbohydrates make up 65% to 80% of the dry weight of onion bulbs (Shiomi, Benkeblia, & Onodera, 2005), with fructans being one of the major carbohydrates present. Breakdown of the cell membrane could result in the release of soluble carbohydrates, including fructans, so leakage and these molecules rather than electrolytes could be a better measure of cell membrane damage for PEF treated onion bulbs, and so far we investigated carbohydrate leakage as a means
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to assess PEF induced cellular damage. Microscopy techniques, including fluorescence
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induced changes at the cell, tissue and organ (scale) levels.
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microscope and cryo-scanning electron microscope (cryo-SEM) were also used to visualise PEF
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2.1 Chemicals, reagents and equipment
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2. Materials and methods
Sodium dihydrogen phosphate (NaH2 PO4 ), disodium phosphate (Na2 HPO 4 ), phenol, sulfuric
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acid (98%) and sodium chloride (NaCl) were purchased from BDH chemicals (Poole, UK). 4Hydroxybenzhydrazide (PABAH), and sulphuric acid (>98%) were purchased from Sigma
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Aldrich (St. Louis, USA). Distilled water was used in this study, unless indicated.
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The equipment used in this research includes ELCRACK® HVP 5 PEF system (German Institute of Food Technologies, Quakenbruck, Germany), oscilloscope (Model UT2025C, UniTrend Group Ltd, China), handheld conductometer (LF-STAR, R. Mathäus, Germany), temperature/conductivity meter (CyberScan CON 11, Eutech Instruments, Singapore), scalpel blade (Heinz, Hamburg, Germany), microscope slide (Sail brand, China), cover slip (ESCO, New Jersey, USA), light microscope (Model CH-2, Olympus Optical, Japan), iPhone camera (iPhone 6 plus S, Apple Inc., Cupertino, California), stereomicroscope (Olympus SZX16, T7 Tokyo, Japan), fluorescence excitation light source (X-Cite®, series 120Q, New York, USA),
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ACCEPTED MANUSCRIPT digital microscope camera (Olympus DP73, Tokyo, Japan), JEOL JSM 6700F field emission scanning electron microscope (JEOL Ltd, Tokyo, Japan), Gatan Alto 2500 cryo-preparation chamber/ cryo-stage (Gatan Inc, Pleanton, California, USA) and cryogrinder (IKA®A11B, Rawang, Malaysia).
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2.2 Bunching onions sample
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White (Allium fistulosum, cv. Ishikura) and red bunching onions (Allium fistulosum, cv. Red
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bunching) seeds were planted between October and November 2015 in the Department of Botany’s experimental garden (University of Otago, Dunedin, New Zealand). White bunching
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onions with diameters of 10 mm ± 1 mm and red bunching onions with diameters of 14 mm ± 2 mm were harvested in February 2016. Immediately after harvest any dirt on the surface of the
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onions was rinsed off under cold running water (8°C ± 2 °C), blotted dry using a hand towel,
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and any dry outer scales were removed. Onions were PEF treated on the day of harvest.
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2.3 Pulsed electric field treatments
PEF treatments were performed in a batch treatment configuration. The treatment chamber
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consisted of two parallel stainless-steel electrodes with a distance of 80 mm and had a dimension of 100 mm length × 80 mm width × 50 mm depth (total volume of 400 mL). The pulse shape (square wave bipolar, pulse width of 20 µs) was monitored on- line using an oscilloscope. The pulse frequency used was 50 Hz. The specific energy input was calculated based on Eq. (1).
Specific energy input, Wspec (kJ/kg) =
(1)
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ACCEPTED MANUSCRIPT p is the pulse energy, n is the number of pulses applied (dimensionless), and W is the total weight of sample (onion and buffer).
Onions were harvested as detailed above, trimmed to a length of 71 mm ± 1 mm (approximately 3 mm of roots and 68 mm of shoot) and placed in the PEF chamber perpendicular to the
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electrodes or parallel to the electric current (Figure 2) to minimize the interruption of electric current delivery passing through different layers and tissues. The average conductivity of
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bunching onions used was 1.4±0.3 mS/cm, measured using a handheld conductometer. The PEF
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chamber was then filled with phosphate buffer (10 mM, pH 7), with a matching conductivity of 1.4 mS/cm. Large observable air bubbles in the buffer and on the outer surface of onion were
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manually removed by taking both the sample and the buffer out from the PEF chamber and then
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slowly putting them back inside the chamber. The total weight of each bunching onion and the buffer solution was measured and standardized throughout the study.
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The PEF treatment conditions were labelled as the level of electric filed strength (first code) and
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specific energy (second code), e.g. a PEF treatment at electric field strength of 0.3 kV/cm (L) with specific energy of 7 kJ/kg (L) was named ‘LL”. The PEF parameters are listed in Table 1, with three electric field strengths being applied, 0.3 kV/cm (first code, L), 0.7 kV/cm (first code,
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M), and 1.2 kV/cm (first code, H) and specific energies of 7 (second code, L), 21 (second code, M) and 52 kJ/kg (second code, H). The high specific energy of 52 kJ/kg (second code, H) was conducted only at an electric field strength of 1.2 kV/cm, the aim being to ensure maximum damage. These electric field strength and specific energy combinations were selected based on the finding of previous studies. Ersus & Barrett (2010) and Fincan & Dejmek (2002) reported that an electric field strength of 0.3 kV/cm was the critical electric field strength to rupture the plasma membrane of onion cells. In addition, electric field strength of 0.7 kV/cm and 1.2 kV/cm have been reported to cause partial and complete cellular damages, respectively, on solid plant 8
ACCEPTED MANUSCRIPT materials (Faridnia et al, 2015; Liu et al, 2017). The temperature and electrical conductivity of the buffer were measured before and after PEF treatment using a temperature/conductivity meter. The initial temperature of the phosphate buffer was 20 ± 1°C. After PEF treatment, the temperature increase was less than 1 °C, except for HH which was 3 °C. After PEF treatment,
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each onion was removed from the chamber and subjected to further analysis.
2.4 Evaluation of the influence of PEF on the stability of the plasma membranes of
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epidermal cells of red bunching onions
To evaluate the stability of the plasma membrane after PEF treatment, plasmolysis test based on
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the semipermeable properties of intact cell plasma membrane was used. The selective permeability of intact cell makes the protoplast able to retract in hypertonic solutions, causing
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the detachment of plasma membrane from the rigid cell wall (Lang, Sassmann, Schmidt, & Komis, 2014). However, damaged plasma membrane will lose its selective permeability,
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resulting in no plasmolysis when soaking in the hypertonic solution.
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Immediately after PEF treatment, onions were removed from the treatment buffer. The epidermal tissues of scales/leaves 1, 2, and 3 of the red onions were sliced using a scalpel blade
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(Figure 2) and the sections (0.3 to 0.5mm thick) were placed on a microscope slide thick and covered using a cover slip (22 X 22mm). The tissue sections were observed immediately using a light microscope and images were taken, through one eyepiece, using an iPhone camera. A drop of hypertonic solution (4M sodium chloride solution) was then added to the section to induce plasmolysis. After 30 seconds images were taken to observe plasmolysis. Exposure for longer than 1 min to the plasmolysis solution led to the loss of cellular pigments in the PEF treated onion epidermal tissues (ML, MM, HL, HM, HH) due to irreversible cell membrane damage. Three red bunching onions were used at each treatment level and due to the lack of red pigment
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ACCEPTED MANUSCRIPT in the epidermal cells of the inner scales/leaves only the first 3 scales/leaves were used in this study (Figure 1 and 2). 2.5. Structural analysis of bunching onions using fluorescence microscopy
After PEF treatment, the onions were soaked in the PEF treatment buffer for 2 hrs to allow the
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PEF induced changes to develop before imaging. Transverse onion sections were obtained using
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a razor blade and placed on a microscope slide (26 X 76mm, 1mm-1.2 mm thick). The tissue sections were observed under a stereomicroscope fitted with a fluorescence excitation light
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source. Tissue sections were observed using ultraviolet excitation (ex 330-385nm, em 420nm
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and images were captured online using a Peltier cooled, digital microscope camera and CellSense software (Version 1.7, Olympus, Tokyo, Japan). Constant exposure settings were
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used throughout the study.
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2.6. Microstructure analysis of white bunching onion using Cryo-SEM
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Due to the size limit of sample subjected to Cryo-SEM analysis, it is impossible to mount the whole cross section of the red bunching onion. Thus, only white bunching onions were used for the present study. After PEF treatment, the onions were soaked as described above and
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transverse tissue sections (10mm in diameter and 1.5 to 2mm thick) were sliced from 1 cm above the basal plate. Sections were washed with the same buffer as used for PEF treatment, to remove the surface debris, and then attached to aluminium discs with Tissue Tek OCT. The samples were immediately frozen using liquid nitrogen and stored in liquid nitrogen until they were imaged. For imaging, the samples were transferred to a cryo-preparation chamber/ cryostage and maintained at a constant temperature of -135°C. The samples were sublimed at -85°C
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ACCEPTED MANUSCRIPT for 2-5 minutes to remove ice build-up and then viewed using a field emission scanning electron microscope.
2.7. Analysis of s welling/shrinkage, total soluble carbohydrate leakage, and fructans leakage of white and red bunching onions after PEF treatment.
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Untreated, ML (0.7kV/cm and 7.14 kJ/kg), HM (1.2 kV/cm and 21 kJ/kg) and HH (1.2 kV/cm
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and 52 kJ/kg) treated samples were used for the swelling and leakage tests. For each cultivar,
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three onion shoots were fitted in the same PEF chamber as shown in Figure 2. Three replicates with a total of 9 onion shoots for each PEF treatment condition were used for each onion
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cultivar. Since the PEF treatment buffer used did not have any effect on the epidermal cells of untreated onions (see Figure 3, before plasmolysis), hence phosphate buffer (10 mM, pH 7) was
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used as soaking buffer for the leaching study to avoid any changes occurred by changing the
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solution.
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2.7.1 Swelling/shrinkage index of white and red bunching onions
The initial weight of the onion sections (W0 ) was measured before PEF treatment. After PEF
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treatment the onions were transferred to a 500 ml polystyrene container (LabServe ®, Australia) containing phosphate buffer (solid/liquid ratio of 1:15 w/w) and the temperature was maintained at 25°C. A preliminary study showed that the changes in onion weight was minimal after a soaking period of 4.5 hrs (data not presented). Hence, after soaking period of 4.5 hrs the onions were removed from the buffer and any surface water was removed using a paper towel. The final weight of each onion (Wf ) was measured and a swelling/shrinkage index that represents a percentage gain/loss in weight (SIw) was calculated using equation (2).
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ACCEPTED MANUSCRIPT (%) swelling/shrinkage index (SIw) =
(2)
2.7.2 Evaluation of total soluble carbohydrate and fructan leakage into PEF processing medium
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To determine the effect of PEF treatments on carbohydrate leakage, (1) the kinetics of total carbohydrate leakage during soaking (4.5 hours) was followed by taking 1 ml samples during
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soaking as detailed below, and (2) total fructan leakage was measured after soaking for 4.5 hrs.
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After PEF treatment, untreated and PEF treated onions were soaked in phosphate buffer as described in section 2.4.1. During the soaking period, 1 ml of the buffer was periodically
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transferred to 1.5 ml centrifuge tubes (Labcon, Hannover, Germany) for up to 4.5 hrs. The
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interval between sampling was 20 min for the first hour and then 30 min for a total of 4.5 hrs. The sampled solution was stored at -18 °C until further analysis. The total amount of solution
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taken from the polystyrene container was 10 ml. The remaining soaking buffer was subjected to the fructan leakage assay. The conductivity changes during the soaking period were recorded
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using a temperature/conductivity meter. Afterwards, the onions were removed from the container, frozen in liquid nitrogen and stored at -80°C until further analysis.
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The total carbohydrates leakage (TCL) at time t during soaking was determined in the buffer samples taken using the phenol-sulphuric acid assay according to Dubois, Gilles, Hamilton, Rebers, and Smith (1956), with values adjusted for the change in volume due to sample removal. Due to the large biological variation between onion samples, the total carbohydrates for each onion sample was measured, which includes the total carbohydrates in the soaking buffer (TCL) and in the solid matrix (TCsolid ) after soaking, and used as the denominator to calculate the % total carbohydrates leakage (% TS) at time t during soaking, To analyse the TCsolid , onions after soaking (stored in the -80 °C freezer) were then removed and ground into 12
ACCEPTED MANUSCRIPT fine frozen powder with liquid nitrogen using a cryogrinder. The frozen onion powder (0.5 g) was mixed with 15 ml of phosphate buffer (10mM, pH 7) in a 15 ml falcon tube (LabServe®, Australia) and maintained at 25°C for 2 hrs for total carbohydrate extraction (TCsolid ). The tube was then centrifuged at 2000 g for 20 minutes at 4 °C. The supernatant was collected and subjected to total carbohydrates analysis. The total carbohydrate content in the onion after
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soaking was expressed as TC. The % leakage of total carbohydrates (% TS) were calculated as
× 100
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%TS =
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shown in Eq. (3).
(3)
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Where TCL is the total carbohydrates leaked into the buffer during the soaking period, and
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TCsolid is the extractable carbohydrates left in the onion after soaking.
To compare the kinetics of carbohydrate leakage, the experimental data points were fitted using
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a growth-saturation model (Asavasanti et al., 2010) as described in Equation 4.
y=
(4)
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Where y is the % of total carbohydrate leakage % TS and t is the leakage time. In this model, a is the asymptote that represents the maximum percentage of sugar leakage and b is the time taken to reach 50% of the maximum percentage of sugar leakage. An equation with a low b value will approach the maximum % carbohydrate leakage more rapidly.
To determine the fructans leakage, the buffer solution after 4.5 hrs soaking was subjected to fructan analysis using a fructan assay kit (K-FRUC 03/14, Megazyme, Wicklow, Ireland). The value was expressed as mg fructan per gram of onion.
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ACCEPTED MANUSCRIPT 2.8. Statistical analysis
Data was analysed by using both SAS 9.4 (SAS Institute Inc., 2013) and R (version 3.2.2., 2015) software. A one-way analysis of variance was used to study the swelling index and total fructans leakage using R (version 3.2.2., 2015). Significant difference (p < 0.05) for the
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experimental data was determined using Tukey’s post hoc comparison test. The estimation of kinetic parameters, a and b values at each treatment level were conducted using nonlinear
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regression analysis (SAS 9.4, 2013). The model parameters of each replicate were estimated
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using nonlinear regression analysis (SAS 9.4, 2013) and then analysed using Tukey’s comparison test (p < 0.05). The quality of the fitting between the experimental data and the
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predicted data to estimate the aforementioned kinetic parameters was evaluated based on visual
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inspection of the fitting, residual analysis and coefficient of determination as corrected r2 based
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on Eq (5) (Van Loey, Indrawati, Smout, & Hendrickx, 2002).
(5)
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Corrected r2 = 1-
Where m is the sum of observations, SSQReg is sum of squares of the model, SSQTotal is sum of
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squares of total observations and j is sum of parameters.
3. Results and discussion
Changes in conductivity after PEF treatments were determined to assess the PEF- induced cellular membrane damage. It was found that when the electric field strength was increased from 0.3 kV/cm to 1.2 kV/cm the electrolyte leakage measured using conductivity change before and after PEF treatments were not significantly different from untreated onions after 4.5 hours
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ACCEPTED MANUSCRIPT incubation (p value of 0.11 and 0.30 respectively for white and red bunching onions). This result demonstrates that monitoring the changes in the conductivity of PEF treatment medium, which is currently a standard method for determining the occurrence of PEF induced damage, is not suitable for carbohydrate rich plant organs such as bunching onions. Therefore, various
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measurements were used to assess the PEF-induced cellular damages in bunching onions.
3.1 Use of plasmolysis test to study the functionality of epidermal cells in different onion
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layers after PEF treatment
The epidermal layers of each onion scale are relatively homogenous (Figure 3) and easy to
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separate from the underlying tissue, therefore the impact of PEF on epidermal cells located in different layers can help to understand the importance of spatial location (inner and outer scales)
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with respect to the impacts of PEF in a multi- layered plant system. The integrity and functionality of the plasma membranes of onion scale epidermal cells after PEF treatment was
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evaluated using a plasmolysis test. If the plasma membranes of epidermal cells lose their ability
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to act as semipermeable barriers, due to PEF induced damage, water and solutes will be able to move freely into and out of the cells and plasmolysis (cytosolic shrinking due water loss) will
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not be observed when cells incubated in a hypertonic solution.
Figure 3 shows light microscopy images of the epidermal cells of the outer three red bunching onion scales before and after plasmolysis. Plasmolysis can be easily observed in these cells without staining due to the presence of red anthocyanin pigments. Most of the epidermal cells in the first outer scale leaves of PEF treated onions had damaged plasma membranes, even at the lowest PEF intensity (LL, 0.3 kV/cm and 7 kJ/kg) (Figure 3). An electric field strength of 0.3 kV/cm has been reported as the critical electric field strength to rupture the plasma membrane of onion cells in other studies (Ersus & Barrett, 2010; Fincan & Dejmek, 2002). Thus, the
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ACCEPTED MANUSCRIPT epidermal cell of the outermost (first) scales are likely to have been exposed to a local electric field strength close to 0.3 kV/cm. In contrast, there was almost no plasma membrane damage apparent in the epidermal cells of the second outermost scale of onions treated at 0.3 kV/cm (LL and LM), which indicates a local electric field strength of <0.3 kV/cm. Furthermore, some epidermal cells in the third outermost scale were not affected even when treated at medium
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electric field strength of 0.7 kV/cm (ML). Overall, PEF induced plasma membrane damage to
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epidermal cells, at low and medium electric field strengths, became less in the scales closer to the inner core (Figure 3). However, the epidermal cells of the inner scales were susceptible to
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plasma membrane damage at higher electric field strengths (Figure 3).
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Based on the authors knowledge, the present study is the first to show that the distribution of the
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PEF effect is layer rather than cell/tissue type dependent in a largely intact, living, multilayered plant structure, under quasi-homogenous electric field treatments. Increasing electric field
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strength at the same energy led to more PEF induced cell membrane damage than increasing specific energy input. However, when the same amount of specific energy was applied,
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increasing the electric field strength from 0.3 to 0.7 kV/cm increased plasma membrane damage to epidermal cells in the three outermost scales. Increasing the PEF electric field strength (<1.2
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kV/cm) could cause more cell damage with less energy consumption.
The present study has shown that for some multilayered plant materials epidermal cell plasmolysis can be potentially be used to assess both PEF-induced plasma membrane damage and to evaluate the distribution of PEF effect throughout the plant structure. In addition, this technique could be used to test the reversibility of cellular membrane damage after PEF. The limitation of the plasmolysis test, as used in the present study, is that PEF-induced plasma membrane damage can only be assessed in epidermal cells and not the cells that make up other tissues. Hence, to further understand the influence of PEF on our multilayered model system, 16
ACCEPTED MANUSCRIPT fluorescence and scanning electron microscopy were further used to assess changes at the tissue and whole organ levels.
3.2 Fluorescence and cryo-SEM microscopy assessment of PEF induced changes in onions
Plant tissues contains many fluorescent molecules that can exhibit autofluorescence under
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fluorescence microscopy, using ultraviolet, violet, or blue light
(Willemse, 1988).
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Autofluorescent molecules are commonly found in the cell walls, chloroplasts and vacuoles of
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plant cells and include lignin, cutin, suberin in cell walls, and NAD(P)H, pterins, and flavins in the protoplast (Roshchina, 2012). While it is often difficult to relate autofluorescence to a
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specific molecule or a group of molecules, autofluorescence can be used to examine structural changes which occurred after PEF treatment without the need for tissue staining. Figure 4A
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shows examples of autofluorescence images of PEF treated (HH) and untreated onion sections, with the fluorescence, under the ultraviolet excitation, more pronounced in the epidermal and
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vascular tissues possibly due to the presence of cells with thicker walls than cells found in the
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other tissues. Additionally, the cells in the epidermal layer are smaller relative to the cells of ground tissue and have a higher ratio of cell wall material to cytosol (Taylor, Taylor, & Krings, 2009), which could result in greater fluorescence. In the present study no apparent changes in
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the distribution of tissue fluorescence were observed after PEF treatment of onions and even though cell damage did occur (see Sections 3.1 above and 3.3. below), even the high PEF treatment did not cause any gross changes to structural organisation within individual scales.
Further investigation of any structural alterations caused by PEF was conducted using cryoSEM. Examples of PEF (HH) treated and untreated white onions evaluated using cryo-SEM are shown in Figure 4B. In the untreated onions the cells that make up the upper regions of each scale can be clearly seen and the epidermal and underlying ground tissues are well organized.
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ACCEPTED MANUSCRIPT The gaps between the scales are generally uniform and the scales are well defined and separated (Figure 4B). The apparent gaps separating individual scales could influence the flow of electric current during PEF treatment and in part play a role in the scale positional effects observed with respect to epidermal cell damage (section 3.1).
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Following PEF (HH) treatment while individual scales can still be distinguished, individual tissues could not be clearly observed and the gaps between the scales were much less uniform
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and not as clearly defined, as seen in the untreated onions. While this was likely, at least in part,
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to be due to a loss of cell integrity within the tissues and possibly tissue swelling/shrinkage, the cut surfaces also appeared to be obscured due to being covered with considerable qualities of
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polysaccharides, e.g. fructans. To investigate these results further, tissue swelling and
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polysaccharide leakage following PEF treatment were investigated.
3.3 The influence of PEF on the tissue swelling/s hrinkage and total soluble carbohydrates
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leakage
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PEF treated onions appeared more transparent than untreated onions after prolonged soaking in PEF buffer (data not show), which could be attributed to the loss of cellular components or
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water uptake. Therefore, changes in weight were used to generate a swelling/shrinkage index after soaking in PEF buffer for 4.5 hours. Based upon the swelling indices of both the white and red onions (Table 2), it was concluded that PEF treatment combined with soaking significantly reduced sample fresh weights (indicated by a negative swelling index). This was attributed to the loss of cellular content, due to PEF- induced cell membrane rupture. In contrast, untreated bunching onions, both white and red, showed a significant increase in fresh weight (p<0.05), probably due to water uptake from the use of a hypotonic PEF processing buffer.
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ACCEPTED MANUSCRIPT For PEF treated white onions, increasing the PEF intensity (electric field strength and specific energy) did not result in a greater weight loss, while for PEF treated red onions increasing the PEF intensity resulted in increased weight loss (Table 1.). In other words, white onions are less sensitive than red onions to PEF intensity and increasing PEF intensities (>1.2 kV/cm or > 52 kJ/kg) could lead to a significant increase in the weight loss for red, but not white onions. The
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responsible for the weight losses observed in PEF treated onions.
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leakage of the cellular content (e.g. electrolyte, carbohydrates) into the PEF buffer might be
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In the present study, carbohydrate leakage after PEF treatment was shown to be a promising method to study PEF induced damages in onions (Figure 5). The kinetics of total carbohydrate
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leakage were used to investigate the relationship between the percentage of total carbohydrate
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lost and leaching time. Total carbohydrate loss followed an asymptotic curve and the relationship could be described using a growth saturation model (Figure 5). As expected, the
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percentages of total carbohydrate leakage for untreated white and red onions were the lowest. At any time during the soaking period, the % total carbohydrate leakage increased with increasing
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PEF intensity. This carbohydrate leakage could explain the decrease in weight of the onions after PEF treatment, described above.
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The estimated values of a (%, represent the maximum % of total carbohydrates leakage) and b (min) are shown in Table 2. For both white and red onions, higher electric field strengths resulted in a significantly higher a value (p<0.05). Increasing the specific energy level, at the high electric field strength of 1.2 kV/cm, did not increase the a value of white onions, but did increase the a value of red onions, suggesting that increasing energy input at 1.2 kV/cm led to more cellular damage for red onions. The time required to reach 50% of the maximum % total carbohydrate leakage, varied between PEF treated onions, but was not significantly different. Similarly, Gonzalez, Anthon, and Barrett (2010) found no statistical differences between the 19
ACCEPTED MANUSCRIPT half time of ion leakage from high pressure and thermal processed onion tissues when using the same kinetic model. Hence, the rate of leaching was not affected by PEF treatment. Overall, the kinetics of carbohydrate leakage indicate that increasing PEF intensity increases the degree of cellular damage. These results show that in plant materials where electrolyte leakage is not a
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good indicator of PEF induced damage, carbohydrate leakage could be used instead.
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3.4 PEF induced fructan leakage
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The PEF- induced carbohydrate leakage results also demonstrate the potential for using PEF to manipulate the content of soluble carbohydrates in plant-based foods. Zhenzhou, Olivier, Nabil,
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& Eugene (2012) reported that PEF could be used to extract inulin, a linear beta (2-->1) fructan, from chicory. Onions contain large quantities of fructans, which are polymers of fructose
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molecules, as a major carbohydrate storage form (Valluru & Van Den Ende, 2008). Fructans are synthesised in the vacuoles of plant cells and can also be found in the cytoplasm and in the
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extracellular/apoplastic space. However, fructans in the extracellular space are o ften associated
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with the plasma membrane, by H-bonding with the phosphate groups of the membrane lipids, and it is difficult for them to move out of intact plant cells/tissues (Valluru & Van Den Ende,
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2008).
As part of the human diet fructans are valuable carbohydrates and can function as prebiotics promote the activity or growth of beneficial microorganisms in the human gut (Franco-Robles & López, 2015; Rolim, 2015), but some people are fructan intolerant. Consumption of fructans by people that are fructan intolerant can promote gastrointestinal disorders, such as bloating and irritable bowel syndrome (IBS) (Fedewa & Rao, 2014).
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ACCEPTED MANUSCRIPT As shown in Table 2, untreated onion shows minimal fructan leakage compared to PEF treated onions. PEF treatment of onions resulted in the leakage of fructans from the onion tissues into the soaking medium. Increasing electric field strengths and specific energy resulted in a significant increase in fructan leakage for both white (F(3,8) =25.5, p<0.0005) and red (F(3,8) =102.6, p<0.0005) onions (Table 2). When the electric field strength was increased from 0.7
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kV/cm to 1.2 kV/cm, fructan leakage increased, with fructan leakage almost doubling (Table 2).
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Previous study (Culbertson, Kreider, Greenwood, & Cooke, 2010) showed that PEF treatment of a single onion bulb scale with electric field strength over 0.33 kV/cm achieved 92 % cell
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ruptures. However in the present study, an electric field strength of 0.7 kV/cm caused only
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partial damage in largely intact bunching onion bases. The results of the present study could be at least in part attributed to the multilayered nature of the onions we used (Figure 4B), which
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may interrupt the smooth delivery of electric current to the inner scales and therefore result in
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only partial cellular damage at an electric field strength of 0.7 kV/cm.
When PEF energy is increased from 21 kJ/kg (HM) to 52 kJ/kg (HH) at the same electric field
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strength (1.2 kV/cm), no significant increase (p<0.05) in the leakage of fructans was observed for white onions and only a minor increase was observed for red onions. This shows the
release.
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importance of using appropriate PEF parameters when trying to achieve maximal fructan
In the present study we explored various techniques to assess PEF- induced damage in a multilayered carbohydrate rich plant material, bunching onions. We also found that PEF is an effective technique to facilitate the extraction fructans from onions, which has not been previously reported. From a food perspective this finding is important for two reasons. Firstly, extracted fructans could be used as a food supplement or functional ingredient to enhance the nutritional value of other foods or as a prebiotic to promote gut health. Since PEF enhanced the 21
ACCEPTED MANUSCRIPT release of fructans from onions, it could also potentially be used to produce low fructan onionbased food products, which could provide some the benefits associated with having onions in the diet for people who are fructan intolerant.
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4. Conclusions
This study has shown that in a semi- intact, multilayered plant material the spatial location of the
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same cell type is an important factor influencing the PEF induced damage. The influence of PEF
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treatment on the same cell type decreased from the outer to inner scales, indicating that the local electric field strength could decrease from the outer to inner scales. This study has also shown
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that carbohydrate leakage can be used as a technique to assess PEF induced damage in plant
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material, where electrolyte leakage/conductivity changes is not suitable. Based upon the results presented it can be concluded that the PEF induced damage is electric field strength dependent for bunching onions, i.e. increasing the electric field strength will increase plasma membrane
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damage and cytosolic leakage. In addition, the present study demonstrated the potential of PEF
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to enhance the extraction of oligosaccharides, e.g. fructans, from onions. Further research is required to optimise the PEF processing parameters (i.e. high electric field strength at low specific energies) in order to achieve maximum fructan extraction from bunching onions with
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low energy consumption.
Acknowledgement
This research was supported by the Riddet Institute, a New Zealand Centre of Research Excellence, funded by the Tertiary Education Commission. The authors also thank Riddet Institute CoRE for providing PhD scholarship to Tingting Liu.
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ACCEPTED MANUSCRIPT References: Asavasanti, S., Ersus, S., Ristenpart, W., Stroeve, P., & Barrett, D. M. (2010). Critical electric field strengths of onion tissues treated by pulsed electric fields. Journal of Food Science, 75(7), E433-43. Culbertson, J. Y., Kreider, R. B., Greenwood, M., & Cooke, M. (2010). Effects of Beta-alanine on muscle carnosine and exercise performance: A review of the current literature. Nutrients, 2(1), 75–98.
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Dubois, M., Gilles, K., Hamilton, J., Rebers, P., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350–356.
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Ersus, S., & Barrett, D. M. (2010). Determination of membrane integrity in onion tissues treated by pulsed electric fields: Use of microscopic images and ion leakage measurements. Innovative Food Science and Emerging Technologies, 11(4), 598–603.
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Faridnia, F., Burritt, D. J., Bremer, P. J., & Oey, I. (2015). Innovative approach to determine the effect of pulsed electric fields on the microstructure of whole potato tubers: Use of cell viability, microscopic images and ionic leakage measurements. Food Research International, 77, 556–564. Fedewa, A., & Rao, S. S. C. (2014). Dietary fructose intolerance, fructan intolerance and FODMAPs. Current Gastroenterology Reports, 16(1), A8.
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Fincan, M., & Dejmek, P. (2002). In situ visualization of the effect of a pulsed electric field on plant tissue. Journal of Food Engineering, 55(3), 223–230.
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Franco-Robles, E., & López, M. G. (2015). Implication of fructans in health: immunomodulatory and antioxidant mechanisms. The Scientific World Journal, Article ID 289267, 15 pages.
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Gonzalez, M. E., Anthon, G. E., & Barrett, D. M. (2010). Onion cells after high pressure and thermal processing: Comparison of membrane integrity changes using different analytical methods and impact on tissue texture. Journal of Food Science, 75(7), 426–432. Lang, I., Sassmann, S., Schmidt, B., & Komis, G. (2014). Plasmolysis: loss of turgor and beyond. Plants, 3(4), 583–593.
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Leong, S. Y., Richter, L. K., Knorr, D., & Oey, I. (2014). Feasibility of using pulsed electric field processing to inactivate enzymes and reduce the cutting force of carrot (Daucus carota var. Nantes). Innovative Food Science and Emerging Technologies, 26, 159–167. Liu, T., Dodds, E., Leong, S. Y., Eyres, G. T., Burritt, D. J., & Oey, I. (2017). Effect of pulsed electric fields on the structure and frying quality of “kumara” sweet potato tubers. Innovative Food Science & Emerging Technologies, 245, 890–898. Loey, A. Van, Oey, I., Smout, C., & Hendrickx, M. (2002). Inactivation of enzymes: from experimental design to kinetic modeling. In Handbook of Food Enzymology (pp. 49–58). New York: Marcel Dekker. Murray, M. B., Cape, J. N., & Fowler, D. (1989). Quantification of frost damage in plant tissues by rates of electrolyte leakage. New Phytologist, 113(3), 307–311. Ouyang, X., Van Voorthuysen, T., Toorop, P., & Hilhorst, H. (2002). Seed vigor, aging, and osmopriming affect anion and sugar Leakage during imbibition of Maize (Zea mays L.)
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ACCEPTED MANUSCRIPT Caryopses. International Journal of Plant Sciences, 163(1), 107–112. Palta, J. P., Levitt, J., & Stadelmann, E. J. (1977). Freezing injury in onion bulb cells. Plant Physiology, 60(3), 398–401. Rolim, P. M. (2015). Development of prebiotic food products and health benefits. Food Science and Technology (Campinas), 35(1), 3–10. Roshchina, V. V. (2012). Vital Autofluorescence: Application to the Study of Plant Living Cells. International Jurnal of Spectroscopy, 2012, 1–14.
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Shiomi, N., Benkeblia, N., & Onodera, S. (2005). The metabolism of the fructooligosaccharides in onion bulbs: a comprehensive review. Journal of Applied Glycoscience, 52, 121–127.
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Taylor, T. N., Taylor, E. L., & Krings, M. (2009). 7 - Introduction to Vascular Plant Morphology and Anatomy. In T. N. Taylor, E. L. Taylor, & M. Krings (Eds.), Paleobotany (2nd ed., pp. 201–222). London: Academic Press.
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Valluru, R., & Van Den Ende, W. (2008). Plant fructans in stress environments: Emerging concepts and future prospects. Journal of Experimental Botany, 59(11), 2905–2916.
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Willemse, M. T. . (1988). Cell wall autofluorescence. In A. Chesson & E. R. ORSKOV (Eds.), Physio-chemical characterization of plant residues for industrial and feed use (pp. 50–57). London, UK: Elsevier science.
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Zhenzhou, Z., Olivier, B., Nabil, G., & Eugene, V. (2012). Pilot scale inulin extraction from chicory roots assisted by pulsed electric fields. International Journal of Food Science & Technology, 47(7), 1361–1368.
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Tables Table 1. PEF parameters used in the current study PEF parameters
Sample code LL
LM
ML
MM
0.3±0.00
0.7±0.00
0.7±0.00
1.20±0.07
I R
HL
HM
HH
C S
1.20±0.06
1.20±0.01
7.14±0.08
21.14±0.15
52.20±0.01
3.6
10.8
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6.01±0.00
10.20±0.00
8.82±0.97
9.30±0.10
43.5±0.2
73.3±0.1
85.3±4.1
86.4±7.5
260.5±0.7
749.0±0.0
767.1±7.0
792.1±6.7
4.4±0.0
4.4±0.0
13.3±0.1
13.2±0.1
13.3±0.0
50
50
50
50
50
Field strength (kV/cm)
0.3±0.00
Specific energy (kJ/kg)
7.14±0.00 21.01±0.01
7.14±0.05
21.10±0.01
Treatment Time (ms)
120
10.8
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Pulse voltage (kV)
2.00±0.00 2.05±0.07
5.53±0.14
Pulse current (A)
11.2±0.4
11.7±0.9
47.0±1.3
Pulse power (kW)
22.5±0.7
23.5±0.7
246.3±5
A M
Pulse energy (J)
0.4±0.0
0.4±0.0
Frequency (Hz)
50
50
350
T P
T P
D E
E C
C A
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ACCEPTED MANUSCRIPT Table 2. The swelling/shrinkage index, total fructan extracted from bunching onions (white and red) after soaking in hypotonic buffer for 4.5 hrs and the estimated kinetic parameters of carbohydrates leakage Swelling index (%)
Fructan (mg/g)
% Total carbohydrates leakage
T P
Model fit: y= a×t/(b+t) Treatment
White
Red
White
Red (a*) (%)
Untreated
5.69±1.19 a
4.58±0.46 a
0.03±0.03 c
0.07±0.01
ML
-1.44±0.42 b
-0.92±0.98 b
0.94±0.21 b
HM
-1.50±0.88 b
-2.60±1.31 c
HH
-1.83±0.92 b
-3.38±1.56
One-way ANOVA a,b, c….
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White d
(b) (min)
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(a*) (%)
(b) (min)
1.80±0.58 c
193.00±81.70
3.01±0.20 c
18.62±3.03 b
100.14±37.31b
1.75±0.48 a
5.10± 0.73 b
29.42±4.68 a
150.47±27.12
b
27.97±0.93
d
1.76±0.19 a
5.74 ± 0.43a
28.15±5.00 ab
101.06±26.76
b
36.96± 2.24 a
F(3,20)=123.3, F(3,20)=79, P<0.0005 P<0.0005
F( 3,8)=25.5, p<0.0005
F( 3,8)=102.6, F( 3, 8) = 68.38; F( 3, 8) = 8.9; F( 3, 8) = 504.1; F( 3, 8) = 0.546; p<0.0005 P<0.005 P<0.05 P<0.0005 P>0.05
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A M
T P
a
Red
1.74±0.12 d
132.90±20.80
19.03±0.86 c
106.50±11.97
b
112.30±7.41 131.10±17.82
Different letters in the same column indicate significant differences (p<0.05) according to Tukey’s HSD multiple comparison test.
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*
a: maximu m % electrolyte leakage can be reached after a prolonged soaking period; b : is the time required to reach 50% of t he maximu m percentage of electrolyte leakage, also called half time
C A
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ACCEPTED MANUSCRIPT Caption of Figures
Figure 1. Transverse view of white (left) and red (right) bunching onion shoots.
Figure 2. Experiment flow chart of this study
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Figure 3. Representative light microscope images showing the results before (left) and after
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(right) plasmolysis treatment on epidermal cells from the scale leaf of PEF treated (electric
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field strength 0.3, 0.7 and 1.2 kV/cm; specific energies 7, 21, and 52 kJ/kg) and untreated red bunching onion. S-1, 2, 3 represent the first, second, third outer scale leaf respectively.
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Symbol “√” underneath the microscopic images indicates the occurrence of plasmolysis.
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Magnifications 400x.
Figure 4. Representative micrographs of untreated and PEF treated (HH: 1.2 kV/cm, 52
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kJ/kg) bunching onions. A: the fluorescent micrographs showing the fluorescent transverse view of red (a) and white (b) onions under blue violet and ultra violet excitation. B: The SEM
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micrographs showing the microstructure of transverse view of white bunching onion.
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Figure 5. Fitting of growth saturation model and the experimental % of total soluble carbohydrates leakage in the PEF buffer: white bunching onion (A); red bunching onion (B).
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ACCEPTED MANUSCRIPT Research Highlights:
Electric field strength had a greater impact on cell membrane permeability than specific energy input.
The effect of PEF treatment on epidermal cells increased from the outer to inner scale
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leaves The effect of PEF on the same tissue type depended on the location of the tissue
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Increasing the electric field strength increased fructan leakage.
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within the same bulb.
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Tingting Liu, David John Burritt, Indrawati Oey PII: DOI: Reference:
S0963-9969(18)30891-3 https://doi.org/10.1016/j.foodres.2018.11.006 FRIN 8069
To appear in:
Food Research International
Received date: Revised date: Accepted date:
17 June 2018 28 October 2018 3 November 2018
Please cite this article as: Tingting Liu, David John Burritt, Indrawati Oey , Understanding the effect of Pulsed Electric Fields on multilayered solid plant foods: Bunching onions (Allium fistulosum) as a model system. Frin (2018), https://doi.org/10.1016/ j.foodres.2018.11.006
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ACCEPTED MANUSCRIPT
Understanding the effect of Pulsed Electric Fields on multilayered solid plant foods: bunching onions (Allium fistulosum) as a model system
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Tingting Liu1,2,3 , David John Burritt2 , and Indrawati Oey1,3*
Department of Food Science, University of Otago, PO Box 56, Dunedin 9054, New Zealand
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Department of Botany, University of Otago, PO Box 56, Dunedin 9054, New Zealand
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Riddet Institute, Palmerston North, New Zealand
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*Corresponding author:
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Professor Dr. Indrawati Oey
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Department of Food Science, University of Otago
PO BOX 56, Dunedin 9054, New Zealand
Phone: +64-3-479-8735, Fax: +64-3-479-7567
Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract
While it is well known that the nature of the applied electric field and the heterogeneity of the tissue can influence the impact of PEF treatment on the plant tissues found in plant-based foods, few studies have investigated the influence of PEF on plant structures that are made up of
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multiple structurally similar organs. The aim of this study was to understand the effect of pulsed electric fields (PEF), at different electric field strengths (0, 0.3, 0.7 and 1.2 kV/cm) and specific
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energy (7, 21 and 52 kJ/kg), on a multilayered plant material, with bunching onion bulb tissues
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being used as a model system. The present study found that carbohydrates leakage was an
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appropriate index to assess PEF induced damage and that plasmolysis of epidermal cells was a good indicator of plasma membrane integrity after PEF. In addition, electric field strength had a
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greater impact on the cell integrity than specific energy applied. While other studies have shown that different cell types have different sensitivities to PEF, using plasmolysis as an
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indicator of cell damage, this study clearly showed that the same PEF treatment conditions had a greater effect on the epidermal cells of the outer scales compared to the inner scales. Hence,
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while different plant cell types vary in their sensitivities to PEF the spatial location of the same cell type within a complex plant material made up of multiple similar organs, i.e. an onion bulb,
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can also influence how cells respond to the PEF treatment. Despite PEF induced disruption at the cellular level being detected by carbohydrate leakage, the epidermal cell plasmolysis test and by cryo-scanning electromicroscopy (cryo-SEM), no gross structural changes at the organ level were observed using cryo-SEM or fluorescence microscopy. This study also reports for the first time that PEF treatment can enhance fructan leakage from onion bulbs, which means that PEF treatments have the potential to manipulate the fructan contents of some plant-based foods.
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ACCEPTED MANUSCRIPT Key words: plant structure; electric field strength; bunching onion; plasmolysis; fructans;
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microscopic level.
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ACCEPTED MANUSCRIPT 1. Introduction Pulsed electric field (PEF) processing applies very short pulses (μs) of high electric field strength to a product placed between two electrodes, which can lead to the permeabilization of cell membranes. The use of PEF technology to treat solid materials such as potato tubers
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(Faridnia, Burritt, Bremer, & Oey, 2015) and carrots (Leong, Richter, Knorr, & Oey, 2014) has shown a great potential to reduce the energy required for subsequent food processing, e.g. the
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cutting and frying steps required for the production of low fat sweet potato chips (Liu et al.,
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2017).
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Studies to date have shown that the responses of solid, complex and inhomogeneous plant
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materials, such as onion sections (Fincan & Dejmek, 2002), whole potatoes (Faridnia et al., 2015) and whole sweet potatoes (Liu et al., 2017), to PEF are very variable and the effects on different plant organs/tissues are often uneven and difficult to predict. Plant organs are made up
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of different cell and tissue types with varying physical, chemical, electrical and topological
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properties, all of which can influence both their conductivity and sensitivity to PEF induced electroporation. Thus, the homogeneity of the cells and tissues that make up solid plant foods is
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one of the major factors responsible for the uneven distribution of PEF effects.
However, unlike root or tuberous vegetables (such as carrots, potatoes or sweet potatoes), onion bulbs are made up of multiple layers of fleshy scales (modified leaves), with each scale having the same tissue organisation. While the arrangement of the scales is generally consistent between onion bulbs, depending on their position (e.g. inner vs outer layers) the scales themselves can differ in size and to some extent physiological properties. Hence, the combination of a consistent multilayered structural pattern with only minimal inhomogeneity between the layers of scales makes onions an interesting model system to study the influence of 4
ACCEPTED MANUSCRIPT electric fields on complex plant structures of relevance to food science. The main objective of the present study was to understand whether the same tissue types respond the same way to quasi- homogeneous electric fields irrespective of their spatial position, e.g. inner vs outer scales of the bunching onion bulb.
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An issue, when studying the influence of PEF on complex plant tissues, is variability between samples/specimens, which can often make identifying the subtle impacts of PEF on plant tissues
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difficult. Considerable variability in bulb size and developmental stage can be found in
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commercial batches of onion (Allium cepa L) bulbs and postharvest storage conditions can greatly influence various aspects of their metabolism, especially carbohydrate metabolism. For
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these reasons instead of using commercially available onion bulbs we used, as our model
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system, bunching onions (Allium fistulosum) grown under controlled conditions were PEF treated immediately after harvest in order to minimize variability. The edible bases of bunching
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onions are structurally very similar to commercial onion bulbs and when grown under consistent
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conditions are structurally quite uniform.
Electrolyte leakage, measured as a change in conductivity, is often used to measure PEF induced disruption of plant materials. However, the use of electrolyte leakage to assess PEF induced
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damage in onion bulbs has been questioned, as only 20% of total solute leakage from onion bulbs is electrolytes, with the remaining 80% being carbohydrates (Palta, Levitt, & Stadelmann, 1977). Also, 85% of the electrolytes leaking from PEF treated onion bulbs diffuses out of living cells (Palta et al., 1977) and so electrolyte leakage might not be a good indicator of irreversible cell damage in onion bulbs (Murray, Cape, & Fowler, 1989; Asavasanti, Ersus, Ristenpart, Stroeve, & Barrett, 2010; Faridnia et al., 2015). Hence in the present study we used, in addition to electrolyte leakage/conductivity, the release of soluble carbohydrates including fructans, and a plasmolysis susceptibility test to assess PEF- induced cellular damage to bunching onion bases. 5
ACCEPTED MANUSCRIPT Carbohydrates make up 65% to 80% of the dry weight of onion bulbs (Shiomi, Benkeblia, & Onodera, 2005), with fructans being one of the major carbohydrates present. Breakdown of the cell membrane could result in the release of soluble carbohydrates, including fructans, so leakage and these molecules rather than electrolytes could be a better measure of cell membrane damage for PEF treated onion bulbs, and so far we investigated carbohydrate leakage as a means
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to assess PEF induced cellular damage. Microscopy techniques, including fluorescence
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induced changes at the cell, tissue and organ (scale) levels.
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microscope and cryo-scanning electron microscope (cryo-SEM) were also used to visualise PEF
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2.1 Chemicals, reagents and equipment
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2. Materials and methods
Sodium dihydrogen phosphate (NaH2 PO4 ), disodium phosphate (Na2 HPO 4 ), phenol, sulfuric
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acid (98%) and sodium chloride (NaCl) were purchased from BDH chemicals (Poole, UK). 4Hydroxybenzhydrazide (PABAH), and sulphuric acid (>98%) were purchased from Sigma
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Aldrich (St. Louis, USA). Distilled water was used in this study, unless indicated.
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The equipment used in this research includes ELCRACK® HVP 5 PEF system (German Institute of Food Technologies, Quakenbruck, Germany), oscilloscope (Model UT2025C, UniTrend Group Ltd, China), handheld conductometer (LF-STAR, R. Mathäus, Germany), temperature/conductivity meter (CyberScan CON 11, Eutech Instruments, Singapore), scalpel blade (Heinz, Hamburg, Germany), microscope slide (Sail brand, China), cover slip (ESCO, New Jersey, USA), light microscope (Model CH-2, Olympus Optical, Japan), iPhone camera (iPhone 6 plus S, Apple Inc., Cupertino, California), stereomicroscope (Olympus SZX16, T7 Tokyo, Japan), fluorescence excitation light source (X-Cite®, series 120Q, New York, USA),
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ACCEPTED MANUSCRIPT digital microscope camera (Olympus DP73, Tokyo, Japan), JEOL JSM 6700F field emission scanning electron microscope (JEOL Ltd, Tokyo, Japan), Gatan Alto 2500 cryo-preparation chamber/ cryo-stage (Gatan Inc, Pleanton, California, USA) and cryogrinder (IKA®A11B, Rawang, Malaysia).
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2.2 Bunching onions sample
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White (Allium fistulosum, cv. Ishikura) and red bunching onions (Allium fistulosum, cv. Red
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bunching) seeds were planted between October and November 2015 in the Department of Botany’s experimental garden (University of Otago, Dunedin, New Zealand). White bunching
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onions with diameters of 10 mm ± 1 mm and red bunching onions with diameters of 14 mm ± 2 mm were harvested in February 2016. Immediately after harvest any dirt on the surface of the
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onions was rinsed off under cold running water (8°C ± 2 °C), blotted dry using a hand towel,
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and any dry outer scales were removed. Onions were PEF treated on the day of harvest.
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2.3 Pulsed electric field treatments
PEF treatments were performed in a batch treatment configuration. The treatment chamber
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consisted of two parallel stainless-steel electrodes with a distance of 80 mm and had a dimension of 100 mm length × 80 mm width × 50 mm depth (total volume of 400 mL). The pulse shape (square wave bipolar, pulse width of 20 µs) was monitored on- line using an oscilloscope. The pulse frequency used was 50 Hz. The specific energy input was calculated based on Eq. (1).
Specific energy input, Wspec (kJ/kg) =
(1)
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ACCEPTED MANUSCRIPT p is the pulse energy, n is the number of pulses applied (dimensionless), and W is the total weight of sample (onion and buffer).
Onions were harvested as detailed above, trimmed to a length of 71 mm ± 1 mm (approximately 3 mm of roots and 68 mm of shoot) and placed in the PEF chamber perpendicular to the
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electrodes or parallel to the electric current (Figure 2) to minimize the interruption of electric current delivery passing through different layers and tissues. The average conductivity of
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bunching onions used was 1.4±0.3 mS/cm, measured using a handheld conductometer. The PEF
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chamber was then filled with phosphate buffer (10 mM, pH 7), with a matching conductivity of 1.4 mS/cm. Large observable air bubbles in the buffer and on the outer surface of onion were
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manually removed by taking both the sample and the buffer out from the PEF chamber and then
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slowly putting them back inside the chamber. The total weight of each bunching onion and the buffer solution was measured and standardized throughout the study.
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The PEF treatment conditions were labelled as the level of electric filed strength (first code) and
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specific energy (second code), e.g. a PEF treatment at electric field strength of 0.3 kV/cm (L) with specific energy of 7 kJ/kg (L) was named ‘LL”. The PEF parameters are listed in Table 1, with three electric field strengths being applied, 0.3 kV/cm (first code, L), 0.7 kV/cm (first code,
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M), and 1.2 kV/cm (first code, H) and specific energies of 7 (second code, L), 21 (second code, M) and 52 kJ/kg (second code, H). The high specific energy of 52 kJ/kg (second code, H) was conducted only at an electric field strength of 1.2 kV/cm, the aim being to ensure maximum damage. These electric field strength and specific energy combinations were selected based on the finding of previous studies. Ersus & Barrett (2010) and Fincan & Dejmek (2002) reported that an electric field strength of 0.3 kV/cm was the critical electric field strength to rupture the plasma membrane of onion cells. In addition, electric field strength of 0.7 kV/cm and 1.2 kV/cm have been reported to cause partial and complete cellular damages, respectively, on solid plant 8
ACCEPTED MANUSCRIPT materials (Faridnia et al, 2015; Liu et al, 2017). The temperature and electrical conductivity of the buffer were measured before and after PEF treatment using a temperature/conductivity meter. The initial temperature of the phosphate buffer was 20 ± 1°C. After PEF treatment, the temperature increase was less than 1 °C, except for HH which was 3 °C. After PEF treatment,
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each onion was removed from the chamber and subjected to further analysis.
2.4 Evaluation of the influence of PEF on the stability of the plasma membranes of
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epidermal cells of red bunching onions
To evaluate the stability of the plasma membrane after PEF treatment, plasmolysis test based on
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the semipermeable properties of intact cell plasma membrane was used. The selective permeability of intact cell makes the protoplast able to retract in hypertonic solutions, causing
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the detachment of plasma membrane from the rigid cell wall (Lang, Sassmann, Schmidt, & Komis, 2014). However, damaged plasma membrane will lose its selective permeability,
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resulting in no plasmolysis when soaking in the hypertonic solution.
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Immediately after PEF treatment, onions were removed from the treatment buffer. The epidermal tissues of scales/leaves 1, 2, and 3 of the red onions were sliced using a scalpel blade
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(Figure 2) and the sections (0.3 to 0.5mm thick) were placed on a microscope slide thick and covered using a cover slip (22 X 22mm). The tissue sections were observed immediately using a light microscope and images were taken, through one eyepiece, using an iPhone camera. A drop of hypertonic solution (4M sodium chloride solution) was then added to the section to induce plasmolysis. After 30 seconds images were taken to observe plasmolysis. Exposure for longer than 1 min to the plasmolysis solution led to the loss of cellular pigments in the PEF treated onion epidermal tissues (ML, MM, HL, HM, HH) due to irreversible cell membrane damage. Three red bunching onions were used at each treatment level and due to the lack of red pigment
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ACCEPTED MANUSCRIPT in the epidermal cells of the inner scales/leaves only the first 3 scales/leaves were used in this study (Figure 1 and 2). 2.5. Structural analysis of bunching onions using fluorescence microscopy
After PEF treatment, the onions were soaked in the PEF treatment buffer for 2 hrs to allow the
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PEF induced changes to develop before imaging. Transverse onion sections were obtained using
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a razor blade and placed on a microscope slide (26 X 76mm, 1mm-1.2 mm thick). The tissue sections were observed under a stereomicroscope fitted with a fluorescence excitation light
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source. Tissue sections were observed using ultraviolet excitation (ex 330-385nm, em 420nm
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and images were captured online using a Peltier cooled, digital microscope camera and CellSense software (Version 1.7, Olympus, Tokyo, Japan). Constant exposure settings were
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used throughout the study.
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2.6. Microstructure analysis of white bunching onion using Cryo-SEM
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Due to the size limit of sample subjected to Cryo-SEM analysis, it is impossible to mount the whole cross section of the red bunching onion. Thus, only white bunching onions were used for the present study. After PEF treatment, the onions were soaked as described above and
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transverse tissue sections (10mm in diameter and 1.5 to 2mm thick) were sliced from 1 cm above the basal plate. Sections were washed with the same buffer as used for PEF treatment, to remove the surface debris, and then attached to aluminium discs with Tissue Tek OCT. The samples were immediately frozen using liquid nitrogen and stored in liquid nitrogen until they were imaged. For imaging, the samples were transferred to a cryo-preparation chamber/ cryostage and maintained at a constant temperature of -135°C. The samples were sublimed at -85°C
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ACCEPTED MANUSCRIPT for 2-5 minutes to remove ice build-up and then viewed using a field emission scanning electron microscope.
2.7. Analysis of s welling/shrinkage, total soluble carbohydrate leakage, and fructans leakage of white and red bunching onions after PEF treatment.
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Untreated, ML (0.7kV/cm and 7.14 kJ/kg), HM (1.2 kV/cm and 21 kJ/kg) and HH (1.2 kV/cm
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and 52 kJ/kg) treated samples were used for the swelling and leakage tests. For each cultivar,
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three onion shoots were fitted in the same PEF chamber as shown in Figure 2. Three replicates with a total of 9 onion shoots for each PEF treatment condition were used for each onion
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cultivar. Since the PEF treatment buffer used did not have any effect on the epidermal cells of untreated onions (see Figure 3, before plasmolysis), hence phosphate buffer (10 mM, pH 7) was
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used as soaking buffer for the leaching study to avoid any changes occurred by changing the
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solution.
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2.7.1 Swelling/shrinkage index of white and red bunching onions
The initial weight of the onion sections (W0 ) was measured before PEF treatment. After PEF
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treatment the onions were transferred to a 500 ml polystyrene container (LabServe ®, Australia) containing phosphate buffer (solid/liquid ratio of 1:15 w/w) and the temperature was maintained at 25°C. A preliminary study showed that the changes in onion weight was minimal after a soaking period of 4.5 hrs (data not presented). Hence, after soaking period of 4.5 hrs the onions were removed from the buffer and any surface water was removed using a paper towel. The final weight of each onion (Wf ) was measured and a swelling/shrinkage index that represents a percentage gain/loss in weight (SIw) was calculated using equation (2).
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ACCEPTED MANUSCRIPT (%) swelling/shrinkage index (SIw) =
(2)
2.7.2 Evaluation of total soluble carbohydrate and fructan leakage into PEF processing medium
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To determine the effect of PEF treatments on carbohydrate leakage, (1) the kinetics of total carbohydrate leakage during soaking (4.5 hours) was followed by taking 1 ml samples during
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soaking as detailed below, and (2) total fructan leakage was measured after soaking for 4.5 hrs.
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After PEF treatment, untreated and PEF treated onions were soaked in phosphate buffer as described in section 2.4.1. During the soaking period, 1 ml of the buffer was periodically
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transferred to 1.5 ml centrifuge tubes (Labcon, Hannover, Germany) for up to 4.5 hrs. The
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interval between sampling was 20 min for the first hour and then 30 min for a total of 4.5 hrs. The sampled solution was stored at -18 °C until further analysis. The total amount of solution
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taken from the polystyrene container was 10 ml. The remaining soaking buffer was subjected to the fructan leakage assay. The conductivity changes during the soaking period were recorded
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using a temperature/conductivity meter. Afterwards, the onions were removed from the container, frozen in liquid nitrogen and stored at -80°C until further analysis.
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The total carbohydrates leakage (TCL) at time t during soaking was determined in the buffer samples taken using the phenol-sulphuric acid assay according to Dubois, Gilles, Hamilton, Rebers, and Smith (1956), with values adjusted for the change in volume due to sample removal. Due to the large biological variation between onion samples, the total carbohydrates for each onion sample was measured, which includes the total carbohydrates in the soaking buffer (TCL) and in the solid matrix (TCsolid ) after soaking, and used as the denominator to calculate the % total carbohydrates leakage (% TS) at time t during soaking, To analyse the TCsolid , onions after soaking (stored in the -80 °C freezer) were then removed and ground into 12
ACCEPTED MANUSCRIPT fine frozen powder with liquid nitrogen using a cryogrinder. The frozen onion powder (0.5 g) was mixed with 15 ml of phosphate buffer (10mM, pH 7) in a 15 ml falcon tube (LabServe®, Australia) and maintained at 25°C for 2 hrs for total carbohydrate extraction (TCsolid ). The tube was then centrifuged at 2000 g for 20 minutes at 4 °C. The supernatant was collected and subjected to total carbohydrates analysis. The total carbohydrate content in the onion after
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soaking was expressed as TC. The % leakage of total carbohydrates (% TS) were calculated as
× 100
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%TS =
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shown in Eq. (3).
(3)
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Where TCL is the total carbohydrates leaked into the buffer during the soaking period, and
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TCsolid is the extractable carbohydrates left in the onion after soaking.
To compare the kinetics of carbohydrate leakage, the experimental data points were fitted using
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a growth-saturation model (Asavasanti et al., 2010) as described in Equation 4.
y=
(4)
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Where y is the % of total carbohydrate leakage % TS and t is the leakage time. In this model, a is the asymptote that represents the maximum percentage of sugar leakage and b is the time taken to reach 50% of the maximum percentage of sugar leakage. An equation with a low b value will approach the maximum % carbohydrate leakage more rapidly.
To determine the fructans leakage, the buffer solution after 4.5 hrs soaking was subjected to fructan analysis using a fructan assay kit (K-FRUC 03/14, Megazyme, Wicklow, Ireland). The value was expressed as mg fructan per gram of onion.
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ACCEPTED MANUSCRIPT 2.8. Statistical analysis
Data was analysed by using both SAS 9.4 (SAS Institute Inc., 2013) and R (version 3.2.2., 2015) software. A one-way analysis of variance was used to study the swelling index and total fructans leakage using R (version 3.2.2., 2015). Significant difference (p < 0.05) for the
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experimental data was determined using Tukey’s post hoc comparison test. The estimation of kinetic parameters, a and b values at each treatment level were conducted using nonlinear
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regression analysis (SAS 9.4, 2013). The model parameters of each replicate were estimated
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using nonlinear regression analysis (SAS 9.4, 2013) and then analysed using Tukey’s comparison test (p < 0.05). The quality of the fitting between the experimental data and the
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predicted data to estimate the aforementioned kinetic parameters was evaluated based on visual
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inspection of the fitting, residual analysis and coefficient of determination as corrected r2 based
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on Eq (5) (Van Loey, Indrawati, Smout, & Hendrickx, 2002).
(5)
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Corrected r2 = 1-
Where m is the sum of observations, SSQReg is sum of squares of the model, SSQTotal is sum of
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squares of total observations and j is sum of parameters.
3. Results and discussion
Changes in conductivity after PEF treatments were determined to assess the PEF- induced cellular membrane damage. It was found that when the electric field strength was increased from 0.3 kV/cm to 1.2 kV/cm the electrolyte leakage measured using conductivity change before and after PEF treatments were not significantly different from untreated onions after 4.5 hours
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ACCEPTED MANUSCRIPT incubation (p value of 0.11 and 0.30 respectively for white and red bunching onions). This result demonstrates that monitoring the changes in the conductivity of PEF treatment medium, which is currently a standard method for determining the occurrence of PEF induced damage, is not suitable for carbohydrate rich plant organs such as bunching onions. Therefore, various
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measurements were used to assess the PEF-induced cellular damages in bunching onions.
3.1 Use of plasmolysis test to study the functionality of epidermal cells in different onion
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layers after PEF treatment
The epidermal layers of each onion scale are relatively homogenous (Figure 3) and easy to
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separate from the underlying tissue, therefore the impact of PEF on epidermal cells located in different layers can help to understand the importance of spatial location (inner and outer scales)
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with respect to the impacts of PEF in a multi- layered plant system. The integrity and functionality of the plasma membranes of onion scale epidermal cells after PEF treatment was
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evaluated using a plasmolysis test. If the plasma membranes of epidermal cells lose their ability
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to act as semipermeable barriers, due to PEF induced damage, water and solutes will be able to move freely into and out of the cells and plasmolysis (cytosolic shrinking due water loss) will
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not be observed when cells incubated in a hypertonic solution.
Figure 3 shows light microscopy images of the epidermal cells of the outer three red bunching onion scales before and after plasmolysis. Plasmolysis can be easily observed in these cells without staining due to the presence of red anthocyanin pigments. Most of the epidermal cells in the first outer scale leaves of PEF treated onions had damaged plasma membranes, even at the lowest PEF intensity (LL, 0.3 kV/cm and 7 kJ/kg) (Figure 3). An electric field strength of 0.3 kV/cm has been reported as the critical electric field strength to rupture the plasma membrane of onion cells in other studies (Ersus & Barrett, 2010; Fincan & Dejmek, 2002). Thus, the
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ACCEPTED MANUSCRIPT epidermal cell of the outermost (first) scales are likely to have been exposed to a local electric field strength close to 0.3 kV/cm. In contrast, there was almost no plasma membrane damage apparent in the epidermal cells of the second outermost scale of onions treated at 0.3 kV/cm (LL and LM), which indicates a local electric field strength of <0.3 kV/cm. Furthermore, some epidermal cells in the third outermost scale were not affected even when treated at medium
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electric field strength of 0.7 kV/cm (ML). Overall, PEF induced plasma membrane damage to
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epidermal cells, at low and medium electric field strengths, became less in the scales closer to the inner core (Figure 3). However, the epidermal cells of the inner scales were susceptible to
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plasma membrane damage at higher electric field strengths (Figure 3).
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Based on the authors knowledge, the present study is the first to show that the distribution of the
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PEF effect is layer rather than cell/tissue type dependent in a largely intact, living, multilayered plant structure, under quasi-homogenous electric field treatments. Increasing electric field
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strength at the same energy led to more PEF induced cell membrane damage than increasing specific energy input. However, when the same amount of specific energy was applied,
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increasing the electric field strength from 0.3 to 0.7 kV/cm increased plasma membrane damage to epidermal cells in the three outermost scales. Increasing the PEF electric field strength (<1.2
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kV/cm) could cause more cell damage with less energy consumption.
The present study has shown that for some multilayered plant materials epidermal cell plasmolysis can be potentially be used to assess both PEF-induced plasma membrane damage and to evaluate the distribution of PEF effect throughout the plant structure. In addition, this technique could be used to test the reversibility of cellular membrane damage after PEF. The limitation of the plasmolysis test, as used in the present study, is that PEF-induced plasma membrane damage can only be assessed in epidermal cells and not the cells that make up other tissues. Hence, to further understand the influence of PEF on our multilayered model system, 16
ACCEPTED MANUSCRIPT fluorescence and scanning electron microscopy were further used to assess changes at the tissue and whole organ levels.
3.2 Fluorescence and cryo-SEM microscopy assessment of PEF induced changes in onions
Plant tissues contains many fluorescent molecules that can exhibit autofluorescence under
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fluorescence microscopy, using ultraviolet, violet, or blue light
(Willemse, 1988).
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Autofluorescent molecules are commonly found in the cell walls, chloroplasts and vacuoles of
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plant cells and include lignin, cutin, suberin in cell walls, and NAD(P)H, pterins, and flavins in the protoplast (Roshchina, 2012). While it is often difficult to relate autofluorescence to a
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specific molecule or a group of molecules, autofluorescence can be used to examine structural changes which occurred after PEF treatment without the need for tissue staining. Figure 4A
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shows examples of autofluorescence images of PEF treated (HH) and untreated onion sections, with the fluorescence, under the ultraviolet excitation, more pronounced in the epidermal and
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vascular tissues possibly due to the presence of cells with thicker walls than cells found in the
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other tissues. Additionally, the cells in the epidermal layer are smaller relative to the cells of ground tissue and have a higher ratio of cell wall material to cytosol (Taylor, Taylor, & Krings, 2009), which could result in greater fluorescence. In the present study no apparent changes in
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the distribution of tissue fluorescence were observed after PEF treatment of onions and even though cell damage did occur (see Sections 3.1 above and 3.3. below), even the high PEF treatment did not cause any gross changes to structural organisation within individual scales.
Further investigation of any structural alterations caused by PEF was conducted using cryoSEM. Examples of PEF (HH) treated and untreated white onions evaluated using cryo-SEM are shown in Figure 4B. In the untreated onions the cells that make up the upper regions of each scale can be clearly seen and the epidermal and underlying ground tissues are well organized.
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ACCEPTED MANUSCRIPT The gaps between the scales are generally uniform and the scales are well defined and separated (Figure 4B). The apparent gaps separating individual scales could influence the flow of electric current during PEF treatment and in part play a role in the scale positional effects observed with respect to epidermal cell damage (section 3.1).
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Following PEF (HH) treatment while individual scales can still be distinguished, individual tissues could not be clearly observed and the gaps between the scales were much less uniform
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and not as clearly defined, as seen in the untreated onions. While this was likely, at least in part,
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to be due to a loss of cell integrity within the tissues and possibly tissue swelling/shrinkage, the cut surfaces also appeared to be obscured due to being covered with considerable qualities of
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polysaccharides, e.g. fructans. To investigate these results further, tissue swelling and
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polysaccharide leakage following PEF treatment were investigated.
3.3 The influence of PEF on the tissue swelling/s hrinkage and total soluble carbohydrates
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leakage
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PEF treated onions appeared more transparent than untreated onions after prolonged soaking in PEF buffer (data not show), which could be attributed to the loss of cellular components or
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water uptake. Therefore, changes in weight were used to generate a swelling/shrinkage index after soaking in PEF buffer for 4.5 hours. Based upon the swelling indices of both the white and red onions (Table 2), it was concluded that PEF treatment combined with soaking significantly reduced sample fresh weights (indicated by a negative swelling index). This was attributed to the loss of cellular content, due to PEF- induced cell membrane rupture. In contrast, untreated bunching onions, both white and red, showed a significant increase in fresh weight (p<0.05), probably due to water uptake from the use of a hypotonic PEF processing buffer.
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ACCEPTED MANUSCRIPT For PEF treated white onions, increasing the PEF intensity (electric field strength and specific energy) did not result in a greater weight loss, while for PEF treated red onions increasing the PEF intensity resulted in increased weight loss (Table 1.). In other words, white onions are less sensitive than red onions to PEF intensity and increasing PEF intensities (>1.2 kV/cm or > 52 kJ/kg) could lead to a significant increase in the weight loss for red, but not white onions. The
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responsible for the weight losses observed in PEF treated onions.
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leakage of the cellular content (e.g. electrolyte, carbohydrates) into the PEF buffer might be
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In the present study, carbohydrate leakage after PEF treatment was shown to be a promising method to study PEF induced damages in onions (Figure 5). The kinetics of total carbohydrate
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leakage were used to investigate the relationship between the percentage of total carbohydrate
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lost and leaching time. Total carbohydrate loss followed an asymptotic curve and the relationship could be described using a growth saturation model (Figure 5). As expected, the
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percentages of total carbohydrate leakage for untreated white and red onions were the lowest. At any time during the soaking period, the % total carbohydrate leakage increased with increasing
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PEF intensity. This carbohydrate leakage could explain the decrease in weight of the onions after PEF treatment, described above.
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The estimated values of a (%, represent the maximum % of total carbohydrates leakage) and b (min) are shown in Table 2. For both white and red onions, higher electric field strengths resulted in a significantly higher a value (p<0.05). Increasing the specific energy level, at the high electric field strength of 1.2 kV/cm, did not increase the a value of white onions, but did increase the a value of red onions, suggesting that increasing energy input at 1.2 kV/cm led to more cellular damage for red onions. The time required to reach 50% of the maximum % total carbohydrate leakage, varied between PEF treated onions, but was not significantly different. Similarly, Gonzalez, Anthon, and Barrett (2010) found no statistical differences between the 19
ACCEPTED MANUSCRIPT half time of ion leakage from high pressure and thermal processed onion tissues when using the same kinetic model. Hence, the rate of leaching was not affected by PEF treatment. Overall, the kinetics of carbohydrate leakage indicate that increasing PEF intensity increases the degree of cellular damage. These results show that in plant materials where electrolyte leakage is not a
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good indicator of PEF induced damage, carbohydrate leakage could be used instead.
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3.4 PEF induced fructan leakage
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The PEF- induced carbohydrate leakage results also demonstrate the potential for using PEF to manipulate the content of soluble carbohydrates in plant-based foods. Zhenzhou, Olivier, Nabil,
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& Eugene (2012) reported that PEF could be used to extract inulin, a linear beta (2-->1) fructan, from chicory. Onions contain large quantities of fructans, which are polymers of fructose
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molecules, as a major carbohydrate storage form (Valluru & Van Den Ende, 2008). Fructans are synthesised in the vacuoles of plant cells and can also be found in the cytoplasm and in the
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extracellular/apoplastic space. However, fructans in the extracellular space are o ften associated
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with the plasma membrane, by H-bonding with the phosphate groups of the membrane lipids, and it is difficult for them to move out of intact plant cells/tissues (Valluru & Van Den Ende,
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2008).
As part of the human diet fructans are valuable carbohydrates and can function as prebiotics promote the activity or growth of beneficial microorganisms in the human gut (Franco-Robles & López, 2015; Rolim, 2015), but some people are fructan intolerant. Consumption of fructans by people that are fructan intolerant can promote gastrointestinal disorders, such as bloating and irritable bowel syndrome (IBS) (Fedewa & Rao, 2014).
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ACCEPTED MANUSCRIPT As shown in Table 2, untreated onion shows minimal fructan leakage compared to PEF treated onions. PEF treatment of onions resulted in the leakage of fructans from the onion tissues into the soaking medium. Increasing electric field strengths and specific energy resulted in a significant increase in fructan leakage for both white (F(3,8) =25.5, p<0.0005) and red (F(3,8) =102.6, p<0.0005) onions (Table 2). When the electric field strength was increased from 0.7
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kV/cm to 1.2 kV/cm, fructan leakage increased, with fructan leakage almost doubling (Table 2).
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Previous study (Culbertson, Kreider, Greenwood, & Cooke, 2010) showed that PEF treatment of a single onion bulb scale with electric field strength over 0.33 kV/cm achieved 92 % cell
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ruptures. However in the present study, an electric field strength of 0.7 kV/cm caused only
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partial damage in largely intact bunching onion bases. The results of the present study could be at least in part attributed to the multilayered nature of the onions we used (Figure 4B), which
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may interrupt the smooth delivery of electric current to the inner scales and therefore result in
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only partial cellular damage at an electric field strength of 0.7 kV/cm.
When PEF energy is increased from 21 kJ/kg (HM) to 52 kJ/kg (HH) at the same electric field
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strength (1.2 kV/cm), no significant increase (p<0.05) in the leakage of fructans was observed for white onions and only a minor increase was observed for red onions. This shows the
release.
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importance of using appropriate PEF parameters when trying to achieve maximal fructan
In the present study we explored various techniques to assess PEF- induced damage in a multilayered carbohydrate rich plant material, bunching onions. We also found that PEF is an effective technique to facilitate the extraction fructans from onions, which has not been previously reported. From a food perspective this finding is important for two reasons. Firstly, extracted fructans could be used as a food supplement or functional ingredient to enhance the nutritional value of other foods or as a prebiotic to promote gut health. Since PEF enhanced the 21
ACCEPTED MANUSCRIPT release of fructans from onions, it could also potentially be used to produce low fructan onionbased food products, which could provide some the benefits associated with having onions in the diet for people who are fructan intolerant.
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4. Conclusions
This study has shown that in a semi- intact, multilayered plant material the spatial location of the
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same cell type is an important factor influencing the PEF induced damage. The influence of PEF
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treatment on the same cell type decreased from the outer to inner scales, indicating that the local electric field strength could decrease from the outer to inner scales. This study has also shown
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that carbohydrate leakage can be used as a technique to assess PEF induced damage in plant
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material, where electrolyte leakage/conductivity changes is not suitable. Based upon the results presented it can be concluded that the PEF induced damage is electric field strength dependent for bunching onions, i.e. increasing the electric field strength will increase plasma membrane
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damage and cytosolic leakage. In addition, the present study demonstrated the potential of PEF
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to enhance the extraction of oligosaccharides, e.g. fructans, from onions. Further research is required to optimise the PEF processing parameters (i.e. high electric field strength at low specific energies) in order to achieve maximum fructan extraction from bunching onions with
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low energy consumption.
Acknowledgement
This research was supported by the Riddet Institute, a New Zealand Centre of Research Excellence, funded by the Tertiary Education Commission. The authors also thank Riddet Institute CoRE for providing PhD scholarship to Tingting Liu.
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ACCEPTED MANUSCRIPT References: Asavasanti, S., Ersus, S., Ristenpart, W., Stroeve, P., & Barrett, D. M. (2010). Critical electric field strengths of onion tissues treated by pulsed electric fields. Journal of Food Science, 75(7), E433-43. Culbertson, J. Y., Kreider, R. B., Greenwood, M., & Cooke, M. (2010). Effects of Beta-alanine on muscle carnosine and exercise performance: A review of the current literature. Nutrients, 2(1), 75–98.
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Franco-Robles, E., & López, M. G. (2015). Implication of fructans in health: immunomodulatory and antioxidant mechanisms. The Scientific World Journal, Article ID 289267, 15 pages.
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ACCEPTED MANUSCRIPT Caryopses. International Journal of Plant Sciences, 163(1), 107–112. Palta, J. P., Levitt, J., & Stadelmann, E. J. (1977). Freezing injury in onion bulb cells. Plant Physiology, 60(3), 398–401. Rolim, P. M. (2015). Development of prebiotic food products and health benefits. Food Science and Technology (Campinas), 35(1), 3–10. Roshchina, V. V. (2012). Vital Autofluorescence: Application to the Study of Plant Living Cells. International Jurnal of Spectroscopy, 2012, 1–14.
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Valluru, R., & Van Den Ende, W. (2008). Plant fructans in stress environments: Emerging concepts and future prospects. Journal of Experimental Botany, 59(11), 2905–2916.
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Willemse, M. T. . (1988). Cell wall autofluorescence. In A. Chesson & E. R. ORSKOV (Eds.), Physio-chemical characterization of plant residues for industrial and feed use (pp. 50–57). London, UK: Elsevier science.
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Tables Table 1. PEF parameters used in the current study PEF parameters
Sample code LL
LM
ML
MM
0.3±0.00
0.7±0.00
0.7±0.00
1.20±0.07
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HL
HM
HH
C S
1.20±0.06
1.20±0.01
7.14±0.08
21.14±0.15
52.20±0.01
3.6
10.8
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6.01±0.00
10.20±0.00
8.82±0.97
9.30±0.10
43.5±0.2
73.3±0.1
85.3±4.1
86.4±7.5
260.5±0.7
749.0±0.0
767.1±7.0
792.1±6.7
4.4±0.0
4.4±0.0
13.3±0.1
13.2±0.1
13.3±0.0
50
50
50
50
50
Field strength (kV/cm)
0.3±0.00
Specific energy (kJ/kg)
7.14±0.00 21.01±0.01
7.14±0.05
21.10±0.01
Treatment Time (ms)
120
10.8
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Pulse voltage (kV)
2.00±0.00 2.05±0.07
5.53±0.14
Pulse current (A)
11.2±0.4
11.7±0.9
47.0±1.3
Pulse power (kW)
22.5±0.7
23.5±0.7
246.3±5
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Pulse energy (J)
0.4±0.0
0.4±0.0
Frequency (Hz)
50
50
350
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T P
D E
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C A
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Fructan (mg/g)
% Total carbohydrates leakage
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Model fit: y= a×t/(b+t) Treatment
White
Red
White
Red (a*) (%)
Untreated
5.69±1.19 a
4.58±0.46 a
0.03±0.03 c
0.07±0.01
ML
-1.44±0.42 b
-0.92±0.98 b
0.94±0.21 b
HM
-1.50±0.88 b
-2.60±1.31 c
HH
-1.83±0.92 b
-3.38±1.56
One-way ANOVA a,b, c….
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White d
(b) (min)
C S
(a*) (%)
(b) (min)
1.80±0.58 c
193.00±81.70
3.01±0.20 c
18.62±3.03 b
100.14±37.31b
1.75±0.48 a
5.10± 0.73 b
29.42±4.68 a
150.47±27.12
b
27.97±0.93
d
1.76±0.19 a
5.74 ± 0.43a
28.15±5.00 ab
101.06±26.76
b
36.96± 2.24 a
F(3,20)=123.3, F(3,20)=79, P<0.0005 P<0.0005
F( 3,8)=25.5, p<0.0005
F( 3,8)=102.6, F( 3, 8) = 68.38; F( 3, 8) = 8.9; F( 3, 8) = 504.1; F( 3, 8) = 0.546; p<0.0005 P<0.005 P<0.05 P<0.0005 P>0.05
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A M
T P
a
Red
1.74±0.12 d
132.90±20.80
19.03±0.86 c
106.50±11.97
b
112.30±7.41 131.10±17.82
Different letters in the same column indicate significant differences (p<0.05) according to Tukey’s HSD multiple comparison test.
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a: maximu m % electrolyte leakage can be reached after a prolonged soaking period; b : is the time required to reach 50% of t he maximu m percentage of electrolyte leakage, also called half time
C A
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ACCEPTED MANUSCRIPT Caption of Figures
Figure 1. Transverse view of white (left) and red (right) bunching onion shoots.
Figure 2. Experiment flow chart of this study
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Figure 3. Representative light microscope images showing the results before (left) and after
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(right) plasmolysis treatment on epidermal cells from the scale leaf of PEF treated (electric
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field strength 0.3, 0.7 and 1.2 kV/cm; specific energies 7, 21, and 52 kJ/kg) and untreated red bunching onion. S-1, 2, 3 represent the first, second, third outer scale leaf respectively.
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Symbol “√” underneath the microscopic images indicates the occurrence of plasmolysis.
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Magnifications 400x.
Figure 4. Representative micrographs of untreated and PEF treated (HH: 1.2 kV/cm, 52
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kJ/kg) bunching onions. A: the fluorescent micrographs showing the fluorescent transverse view of red (a) and white (b) onions under blue violet and ultra violet excitation. B: The SEM
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micrographs showing the microstructure of transverse view of white bunching onion.
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Figure 5. Fitting of growth saturation model and the experimental % of total soluble carbohydrates leakage in the PEF buffer: white bunching onion (A); red bunching onion (B).
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ACCEPTED MANUSCRIPT Research Highlights:
Electric field strength had a greater impact on cell membrane permeability than specific energy input.
The effect of PEF treatment on epidermal cells increased from the outer to inner scale
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leaves The effect of PEF on the same tissue type depended on the location of the tissue
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Increasing the electric field strength increased fructan leakage.
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within the same bulb.
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