Impact of moderate intensity pulsed electric field (MIPEF) treatment on lycopene content and lycopene in vitrobioaccessibility of whole tomato

In this study, the impact of moderate intensity pulsed electric field (MIPEF) treatment parameters on enhancing the lycopene content and lycopene in vitro bioaccessibility was investigated. In the first experiment, tomato fruits were treated at 0.4, 1 and 2 kV/cm using 1 or 10 monopolar pulses of 4 μs at a frequency of 0.1 Hz and analysed 24 h after holding period at 4 0 C while in the second experiment, the influence of treatmentintensity (0, 4, 80 and 320 μs) and holding period (0, 24 and 48 h) on lycopene contents and lycopene in vitro bioaccessibility was evaluated. Fresh sample, without MIPEF treatment, showed the lowest lycopene content (23.27±2.13 μg/g FW) and the lowest lycopene bioaccessibility (8.1±1.70%), while all MIPEF treatments (4 or 40 μs) at different electric field strength (0.4-2.0 kV/cm) enhanced the lycopene content and lycopene bioaccessibility. The results of second experiment revealed that the highest duration treatment (320 μs) showed the maximum lycopene enhancement immediately after the treatment (57.98±4.48μg/g) and decreased thereafter. All the treatments except 320 μs sample enhanced the lycopene invitrobioaccessibility after 24 h after holding period and decreased thereafter. The maximum total lycopene bioaccessibility content (9.6%) of whole tomato was achieved by a 4 μs (at 1 kV/cm) treatment after a 24 h holding period.


INTRODUCTION
Interest in the bioaccessibility of vitamins and other food components (i.e., carotenoids) has greatly increased for different reasons, including the existence of undernourished populations worldwide and groups at risk of developing micronutrient deficiencies. Among micronutrients available in fruits and vegetables, lycopene is an important carotenoid which has shown the epidemiological evidence suggesting protective effects against several diseases such as cancer, cardiovascular diseases, cataracts and neural tube defects (Campbell et al., 2004). Tomato is a popular fruit crop which contains significant amount of lycopene. However, studies have indicated the on low lycopene bioaccessibility of tomato products (Stahl and Sies, 1992;Svelander et al., 2010). Therefore, it is a challenge to investigate 1 Institute for Global Food Security, Queen's University Belfast, Belfast, Northern Ireland, UK.
2 School of Agriculture and Food Science, University College Dublin, Ireland * Author of correspondence : lasanthijayathunge@yahoo.com optimum processing conditions that can result in maximum overall lycopene bioaccessibility and bioavailability.
Pulsed electric field (PEF) treatment is a one of the widely investigated technologies during last few decades among different non-thermal processing technologies (Rasoet al., 1998;Nguyen and Mittal, 2007;Odriozola-Serrano et al., 2007;2008a,b;Mosqueda-Melgaret al., 2012) as an alternative to thermal processing. In addition to processing, there has been increasing interest in the use of moderate intensity pulsed electric field (MIPEF) technology due to its potential to induce non-thermal permeabilisation and stress reaction at cellular level in the plant (Soliva-Fortunyet al., 2009). This permeabilisation and stress reactions have been reported to be beneficial in enhancing and stimulating total polyphenolic and carotenoid content in plants (Balasaet al., 2006;Toepflet al., 2006;Vallverdu-Queraltet al., 2013). However, no information is available concerning the effect of MIPEF on lycopene bioaccessibility of tomato fruits or their products.
Hence, this study was conducted to study the impact of MIPEF treatment parameters in changing the lycopene content and in vitro lycopene bioaccessibility of tomato fruit.

Materials
Tomato fruits of Pitenza (origin-Spain) variety at fully ripe stage (peel colour-fully red, TSS content-5 o Brix) were purchased in several batches from a local store in Northern Ireland (UK) between December 2014 and July 2015. In total, 60 kg of tomatoes were purchased in six batches and graded before processing (mean weight, 85±5 g and mean circumference, 15±1 cm); odd shaped and sized tomatoes were excluded. All chemicals were purchased from Sigma-Aldrich (Dorset, UK). Packaging materials (polyethylene/polyamide film, 200 µm thickness) were obtained from Scobie&Junor (Mallusk, UK).

Determination of theEffect of Electric Field Strength (EFS)
MIPEF treatments were conducted in batch mode using a laboratory scale PEF unit (C-tech Innovations Ltd, Capenhurst, UK) located in University College Dublin. A stainless steel parallel treatment chamber with 8 cm gap was used. A tomato fruit was placed in the treatment chamber and filled with tap water. Tomato fruits were treated at 0.4, 1 and 2 kV/cm using 1 or 10 monopolar pulses of 4 μs at a frequency of 0.1 Hz, according to Vallverdu-Queraltet al. (2013). MIPEF-treated fruits were collected and immediately refrigerated at 4 o C for 24 h. Fresh untreated tomatoes were also stored separately at 4 o C for 24 h. Each treatment consisted of three replicates and six fruits were included within each replicate. The whole experiment was conducted within two days using independent batches. Samples were stored at -80 o C after the 24 h holding period until analysis commenced. Total lycopene content and lycopene invitrobioaccessibility was calculated for all treatments after homogenisation with T25 Ultra-Turrax homogeniser (IKA ® Werke GmbH, Germany) and suitable EFS was selected for further experiments.

Determination of theEffect of Treatment Duration and Holding Period
Whole ripe tomatoes were subjected to 0, 1, 20, 80 mono-polar pulses (4 μs, frequency of 0.1 Hz, EFS 1 kV/cm), which is equivalent to 0, 4, 80 and 320 μs treatment duration respectively, using a laboratory scale PEF unit as described in the above section. MIPEF treated and untreated fruits were collected and immediately refrigerated at 4 o C for 48 h. Each treatment consisted of three replicates with six fruits per replicate and the whole experiment was conducted on two separate days as independent batches. Samples were withdrawn after 0, 24 and 48 h and subjected to analysis. Samples were stored at -80 o C until analysis andtotal lycopene content and lycopene invitrobioaccessibility were evaluated.

Quality Analysis Quantification of Total Lycopene Content of Tomatoes
Lycopene content of freeze dried tomato extracts was determined colorimetrically using the method described by Sadler et al. (1990). Lycopene was extracted from freeze dried samples using hexane:ethanol:acetone, 2:1:1 (0.05/50 ml) mixture. The hexane phase, containing lycopene, was separated from the polar phase and absorbance of the lycopene extract was measured at 472 nm using UV/vis spectrophotometer (JENWAY 6305, UK) against the hexane blank. Concentration of lycopene was calculated using the following equation: Where, C is the lycopene concentration (µg/ml), Ais the absorbance at 472 nm, E % 1 cm is the extinction coefficient (3450 for lycopene in hexane) at a path length of1 cm.

Determination of InvitroBioaccessibilityLycopene
Tomato samples (2.5 g) were subjected to a simulated human gastric and small intestinal digestion based on the method described by Garrett et al. (1999) andHedrenet al. (2002) with minor modifications (Colleet al., 2010;Aneseet al., 2013) to determine the in vitro bioaccessibility of lycopene. All steps were carried out under protection from light using amber tubes and vials.
Gastric digestion simulation: A NaCl/ascorbic acid solution (2.5. ml) (0.9% NaCl, 1% ascorbic acid in water) and a stomach electrolyte (2.5 ml, 0.30% NaCl, 0.11% KCl, 0.15% CaCl 2 .2H 2 O, 0.05% KHPO 4 , 0.07% MgCl 2 .6H 2 O in water) were added to 2.5 g of sample. The pH of this mixture was adjusted to 4 ± 0.05 (with 1M HCl) before the addition of pepsin solution (0.52% porcine pepsin in stomach electrolyte). Subsequently, the headspace of the tubes was flushed with nitrogen and the mixture was incubated for 30 min at 37 °C while shaking. Before continuing the incubation for 30 min, the mixture was acidified to pH 2 ± 0.05 (with 1M HCl) and the headspace was flushed again with nitrogen. The adjustment of the pH in two steps mimicked the gradual drop of the gastric pH after the intake of a meal.
Small intestinal digestion simulation: The pH of the partially digested tomato pulp was raised to pH 6.9 ± 0.05 (with 1M NaOH) and a mixture of pancreatin, lipase, and bile salts (1.5 ml, 0.4% porcine pancreatin, 0.2% porcine pancreas lipase, 2.5% bile extract, 0.5% pyrogallol, 1% α-tocopherol in water) was added. Finally, the headspace of the sample was flushed with nitrogen and the sample was incubated for 2 h at 37 °C.
After incubation, the samples were centrifuged (SORVALL Legend RT, Woburn, Germany) at 5000 g for 1 h to remove the non-digested particles. The lycopene concentration in the supernatant was then measured using the spectrophotometric and HPLC methods described below. The lycopene bioaccessibility of a sample is reported as the ratio (%) of the in vitro bioaccessible lycopene content to the corresponding lycopene content of the sample.

Statistical Analysis
Significance of the results and statistical differences were analysed using the SPSS (IBM, UK) statistical package. Data were analysed by two-way analysis of variance (ANOVA) procedure. Duncan multiple range test (DNMRT) was employed to determine the differences among treatment means, with a level of significance of P<0.05.

Effect of Electrical Field Strength (EFS) on Total Lycopene Content and Lycopene In VitroBioaccessibility of Tomato Fruits
The total lycopene content of fresh and MIPEF treated fruits (undigested) and digesta (digested) of corresponding treatments, analysed by spectrophotometric method and calculated percentage in vitrobioaccessibility is given in Table 1. The fresh sample (without PEF treatment) showed the lowest lycopene content (23.27±2.13 µg/g FW) and were within the range described in the literature (Svelanderet al., 2010). Similarly, retention of lycopene content in digested untreated samples was 1.88±0.29 µg/g FW, resulting in 8.1±1.70% bioaccessibility. Investigations of the in vitro bioaccessibility of lycopene from tomato are very limited, but values reported for the digesta are fairly low and in the range of 2-13% of total content (Stahl and Sies, 1992;Gartner et al., 1997;Svelanderet al., 2010).
All MIPEF treatments (4 or 40 µs) at different electric field strengths (0.4-2.0 kV/cm) enhanced the lycopene concentration, and 40 µs treatment at 1 kV/cm EFS showed 50% lycopene enhancement in comparison to fresh control samples. The lycopene concentration in digesta of MIPEF treated fruits significantly increased in comparison to untreated fruits and EFS of 1 kV/cm, 4 µs treatment showed the highest lycopene content (3.37±0.12 µg/g FW). The results show that MIPEF treatment is able to increase production and availability for digestion of lycopene in plant tissues. Vallverdu-Queralt (2013) also reported that application of 1.2 kV/cm EFS and 20 µs (5 pulses) treatment duration enhances the carotenoid content of tomatoes, including lycopene in comparison to 0.4 and 2 kV/cm EFS levels. Values are means ± standard deviation. Values within each column followed by the same letter are not significantly different at p<0.05.
It is already known that MIPEF processing may activate enzymes needed for carotenoid and phenol synthesis and stimulate the production of secondary metabolites of different fruits and vegetables (Cunningham and Gantt, 1998). For instance, an improvement (51-67%) of β-carotene extraction efficiency of carrot juice (Knorr et al., 1994) and an increase (13-28%) of total phenolic content of grape juice (Balasa et al., 2006) has been reported. According to Heinz et al. (2003), critical EFS needed to induce membrane permeabilisation is dependent on cell geometry and size, and a dose of 1-2 kV/cm of EFS is required for plant cells (cell size 40-200 mm). However, high intensity treatments also have been proven effective in cell membrane permeabilisation and total polyphenol extractability in grape marc (Balasaet al., 2006).
Here, the highest in vitrobioaccessibilities of 13.15±1.08 and 12.46±1.98% were achieved with treatments of 2 kV/cm, 40 µs and 1 kV/cm, 4 µs, respectively (Table 5.1). The treatments were not significantly different although using 1kV/cm would be more economical in comparison to 2 kV/cm. Hence, the EFS of 1 kV/cm was chosen to perform other experiments since it yielded high lycopene extraction both in digested and undigested samples.

InvitroBioaccssibility of Tomato Fruits
Lycopene content in fresh tomatoes remained stable during 48 h holding period without significant differences at p<0.05 (Fig.1). In contrast to fresh tomatoes, lycopene concentrations were enhanced after the MIPEF treatment of tomato. Moreover, lycopene concentrations of 4µs and 80 µs treatments further increased with extension of the holding period up to 24 and 48 h (Fig.1) which is in line with the literature (Soliva-Fortunyet al., 2009;Vallverdu-Queraltet al., 2013). The highest duration treatment (320 µs) showed the maximum lycopene enhancement immediately after the treatment (57.98±4.48 µg/g FW) and decreased thereafter. This might be due to lethal damage to cells due to irreversible loss of cell membrane permeability properties (Zimmermann et al., 1974;Aronsson et al., 2001). Similar decreases in lycopene contents were reported by Vallverdu-Queralt et al (2013) for MIPEF treated (2 kV/cm, 120 µs) tomato 24 h after the treatment.
According to two-way ANOVA, total lycopene contents of fresh tomatoes (both undigested and digested) were not significanly different until 24 h and showed significant increase (32.35 ±µg/g) after 48 h. Tomatoes treated with 4 µs showed significant increase in total lycopene content 48 h (undigested) and 24 h (digested) after treatment. On the other hand, 80 µs treated samples (undigested) showed non-significant changes throughout the the holding period, while the longest duration treatment demonstrated significant reduction in total lycopene (both undigested and digested) content from 0 to 48 h. A B

C D
The effect of treatment duration and holding period (at 4 o C) on lycopene invitro bioaccessibility of whole tomato is shown in Fig.2. Release and subsequent absorption of lycopene from raw tomato is low and Svelanderet al. (2010) reported that 5-13% bioaccessibility for crushed fresh tomatoes. Similarly, the lycopene invitro bioaccessibility of untreated samples were in the range of 7.7-8.6 % and remained non-signifiamt during the 48 h holding period, which agrees with the litertaure. For the treated samples, the lowest duration treatment (4 µs) showed non-significant increment while rest of the treatments (80 and 320 µs) decreased the invitro bioaccessibility immediately after the treatment (Figure 2). Results show that with an increasing MIPEF intensity, the lycopene invitrobioaccessibility was significantly reduced (P < 0.05). The lycopene bioaccessibilities investigated after 24 h holding period were 8.60, 9.64, 7.59 and 7.11% for fresh, 4, 80 and 320 µs MIPEF treatments, respectively and all the levels were enhanced after 24 h holding period in comparison to the value investigated immediately after the treatment except for the highest duration treatment (320 µs). However, the lycopene bioaccessibility of all treatments reduced drastically 48 h after the treatment irrespective of the treatment intensity.This decrement might suggest that lycopene became entrapped by process induced barriers, thus hindering its subsequent incorporation into micelles.
No systematic studies, neither in vivo orin vitro, that report lycopene bioaccessibility in tomato as affected by MIPEF technology seem to exist currently. Studies conducted on thermal processing (Palmeroet al., 2014), in red tomatoes, reported that the lycopene bioaccessibility is mostly depend on the barrier properties of the chromoplast. Therefore, this structural barrier would be an important target tobe modified upon any processing treatments applied.

CONCLUSIONS
The results of this study clearly demonstrate the benefit of MIPEF as a treatment to enhance the lycopene bioaccessibility of whole tomato. Total carotenoid and lycopene contents could be enhanced by increasing the treatment duration (from 4-320 µs, EFS-1kV/cm) due to cell