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RESEARCH ARTICLE

Optimizing pulsed electric field and high-power ultrasound treatments to preserve anthocyanin stability and physicochemical quality in stored strawberry juice

Anica Bebek Markovinović1, Višnja Stulić1, Predrag Putnik2*, Tibor Janči1, Branimir Pavlić3, Sanja Milošević3, Zoran Herceg1, Amin Mousavi Khaneghah4,5, Danijela Bursać Kovačević1

1Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia;

2Department of Food Technology, University North, Koprivnica, Croatia;

3Faculty of Technology, University of Novi Sad, Novi Sad, Serbia;

4Faculty of Biotechnologies (BioTech), ITMO University, Saint Petersburg, Russia;

5Halal Research Center of IRI, Iran Food and Drug Administration, Ministry of Health and Medical Education, Tehran, Iran

Abstract

This study presents a novel approach to preserving the quality of strawberry juice through the innovative combination (hurdle) of pulsed electric field (PEF) and high-power ultrasound (HPU) treatments. The objective was to evaluate the impact of various PEF (30 kV/cm, 100 Hz, 1.5-4.5 min) and HPU (25% amplitude, 50% pulse, 2.5-7.5 min) treatments on anthocyanin stability, color, and physicochemical properties (conductivity, browning index, dissolved oxygen, and hydroxymethylfurfural content) during 7-day storage at 4°C. Our findings reveal that storage significantly influenced anthocyanin content, physicochemical properties, and color. The combined PEF (3 min) and HPU (2.5-7 min) treatments markedly enhanced anthocyanin stability compared to the control samples. Importantly, this combined non-thermal treatment approach significantly affected all studied parameters except for hydroxymethylfurfural content. Optimal anthocyanin content was achieved with a PEF treatment of 2.19 min and an HPU treatment of 7.48 min over 7 days of storage, while minimal color changes were observed with PEF (3.14 min) + HPU (7.50 min). This study demonstrates the potential of combining PEF and HPU as a feasible and effective strategy for processing functional strawberry juices, ensuring anthocyanin stability and desirable physicochemical properties.

Key words: browning index, conductivity, high-power ultrasound, hydroxymethylfurfural, non-thermal hurdle technologies, optimization, oxygen content, pulsed electric field

*Corresponding Author: Predrag Putnik, Department of Food Technology, University North, Trg dr. Žarka Dolinara 1, 48000 Koprivnica, Croatia. Email: pputnik@alumni.uconn.edu

Academic Editor: Ismail Eş, PhD., Institute of Biomedical Engineering, Old Road Campus Research Building, University of Oxford, Headington, Oxford OX3 7DQ, UK

Received: 18 September 2024; Accepted: 3 January 2025; Published: 29 January 2025

DOI: 10.15586/qas.v17i1.1521

© 2025 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)

Introduction

As with different types of berries (Zhang et al., 2023), the strawberry (Fragaria × ananassa Duch.) is also a popular fruit due to its high nutritional (e.g. polyphenolic) and commercial values (Warner et al., 2021). Many studies on the antioxidant, anti-inflammatory, antihypertensive, and antiproliferative properties of strawberries have shown that strawberry products (e.g., juices) have significant bioactive potential because of the high content of polyphenols (Cosme et al., 2022). The attractive red color of strawberries is attributed to anthocyanins, among their most critical polyphenolic compounds. The anthocyanin content in strawberries depends on numerous factors, such as cultivar, ripeness, growing, storage conditions (Bebek Markovinović et al., 2023; Crecente-Campo et al., 2012). Due to their high thermolability, they rapidly degrade during processing and thus lose their color and bioactive properties. Numerous factors have been identified that affect strawberry quality (Bebek Markovinović et al., 2024a), hence special attention must be dedicated to its protection during processing to obtain a high-quality final product (Enaru et al., 2021).

The most sensitive step in the production of strawberry juice, which significantly impacts the final product’s quality, is pasteurization, as heat treatments usually lead to the deterioration of the food’s nutritional, sensory, and phytochemical properties. Heat treatment of juice can lead to browning, the formation of hydroxymethylfurfural (HMF), color changes, and the loss of bioactive compounds (Galanakis, 2021). However, major changes in juice quality can also occur during storage (Chen et al., 2020). The simultaneous degradation of anthocyanins, the fading of the red color, and the formation of brown pigments due to enzymatic and/or non-enzymatic reactions lead to undesirable color changes. The brown color of pasteurized strawberry juice during storage is mostly caused by non-enzymatic processes such as Maillard reactions and acid-catalyzed sugar degradation, as heat treatment inhibits the activity of quality-degrading enzymes (Buvé et al., 2018).

Consumers today prefer affordable fruit juices with qualities as close to fresh fruits (e.g., fresh or cold-pressed juices). Such products should have excellent nutritional and functional qualities and a long shelf life (Martins et al., 2019; Yildiz et al., 2021) while replacing synthetic additives with natural alternatives from plants (Gladikostić et al., 2023). Fruit production technology is steadily being transformed with non-thermal alternatives, not only for pasteurization but also for other operations, for example, homogenization, such as emulsification of pectins (Gharibzahedi et al., 2019).

Recently, the application of hurdle technology, i.e., the simultaneous application of several technologies in lower intensity treatments than if each were applied alone, has attracted much attention. This concept offers numerous advantages in terms of the final quality of the product during storage and, at the same time, good preservation of the nutritional, biological, and sensory properties of the product, which is why it has great potential for sustainable industrial application (Dixit et al., 2018; Djekić et al., 2023).

The combined use of non-thermal technologies such as pulsed electric field (PEF), high hydrostatic pressure (HHP), high-power ultrasound (HPU), and cold plasma (CP) in juice processing is being extensively investigated (Putnik et al., 2020). However, the combined use of PEF and HPU in processing strawberry juice, a very demanding fruit matrix, has not yet been sufficiently researched. PEF and HPU were chosen for this study due to their complementary mechanisms and proven potential to enhance the stability of bioactive compounds. PEF uses short bursts of high voltage to disrupt cell membranes through electroporation, facilitating the extraction of intracellular compounds (Graybill & Davalos, 2020). HPU, on the other hand, generates cavitation bubbles that collapse and produce intense shear forces, improving mass transfer and extraction efficiency (Barba et al., 2020). Evidence suggests that combining these two technologies is expected to synergistically enhance anthocyanin stability, reduce browning, inactivate microorganisms and enzymes, and maintain desirable physicochemical properties in strawberry juice (Guerrero-Beltrán & Welti-Chanes, 2016).

Therefore, in this study, the combined effects of PEF (30 kV/cm, 100 Hz) for 1.5, 3.0, and 4.5 min and HPU (amplitude 25%, pulse 50%) for 2.5, 5.0, and 7.5 min on color changes, physicochemical properties (oxygen content, conductivity, browning index, and HMF content), and anthocyanins in strawberry juice during 7 days of storage at 4°C were investigated using chemometrics. The aim was to investigate the quality of juices treated as a “fresh-like” product that would interest consumers. Finally, optimization was performed to determine the best processing parameters for combined PEF and HPU treatments with the highest anthocyanin yield, the least color change, and browning, in addition to the lowest content of HMF.

Materials and Methods

Production of strawberry juices

The strawberries (Fragaria × ananassa Duch.) cv. ‘Albion’ used for juice preparation were purchased from Jagodar HB d.o.o. (Donja Lomnica, Croatia). After transportation to the laboratory, the fruits were washed, stalks were removed, then dried, and stored at -18°C. The pH of the raw material was 3.23, the soluble solids content (SSC) was 9.85%, and the total acidity was 0.89%. The day before the experiment, the fruits were thawed overnight and then processed into juice using a Kuvings B6000 (VerVita d.o.o., Zagreb, Croatia) cold press juicer (240 W, 60 rpm) with a filter diameter of 0.2 mm.

Pulsed electric field (PEF) and high-power ultrasound (HPU) treatment of strawberry juices

Considering the optimized process parameters for the PEF and HPU technologies determined in previous studies (Bebek Markovinović et al., 2022a; Bebek Markovinović et al., 2022c), the experimental design for the combined treatment of PEF followed by HPU was established (Table 1).

Table 1. Experimental design.

Sample ID PEF exposure (min) HPU exposure (min) Storage (days)
1 1.5 2.5 0
2 1.5 5 0
3 1.5 7.5 0
4 1.5 2.5 7
5 1.5 5 7
6 1.5 7.5 7
7 3 2.5 0
8 3 5 0
9 3 7.5 0
10 3 2.5 7
11 3 5 7
12 3 7.5 7
13 4.5 2.5 0
14 4.5 5 0
15 4.5 7.5 0
16 4.5 2.5 7
17 4.5 5 7
18 4.5 7.5 7

PEF treatments were performed using an HVG60/1 PEF device (Impel d.o.o., Zagreb, Croatia), as previously reported (Bebek Markovinović et al., 2022c). A 200 mL batch treatment chamber was equipped with two parallel stainless-steel electrodes, each with a diameter of 68 mm and a distance of 25 mm. The electric field strength was set to 30 kV/cm, with a pulse frequency of 100 Hz and a constant pulse width of 1 µs.

Immediately after the PEF treatment, the samples were subjected to HPU treatment. The Hielscher UP400St High Power Sonicator, 400 W, 24 Hz, with DN22 (surface area 546 mm2 and diameter 2.2 cm) titanium sonotrode (Hielscher Ultrasonics GmbH, Germany) was used as previously described (Bebek Markovinović et al., 2022a). The juice samples were treated with an amplitude of 25% and a pulse of 50%. A 200 mL of the sample was treated in a glass beaker immersed in a cold-water bath with ice to minimize the impact of temperature during the treatment. During the treatment, the sonotrode was positioned at the center of the glass beaker and immersed to a depth of 2.5 cm in the sample. The temperature of the treated juices was monitored before and after PEF and HPU treatment using the PCE-777 thermometer (PCE-Instruments, UK). The average temperature of the samples before and after all treatments did not exceed 19.55°C. Therefore, the effect of temperature on all observed dependent variables was not considered in this study. After the treatments, one batch of juices was analyzed, while the other batch was stored at 4°C for 7 days and then analyzed. All juices were stored in sterile, securely sealed glass bottles (250 mL), filled to the top, in a dark place.

Determination of electrical conductivity

The electrical conductivity of the samples was measured in duplicate using a HI-2030-Edge conductivity meter (Hanna Instruments, USA). The results were expressed in µS/cm of the juice sample.

Determination of dissolved oxygen

The dissolved oxygen of the samples was measured in duplicate using a SevenGo Duo Pro SG68-FK2 device (Mettler-Toledo GmbH, Greifensee, Switzerland). The results were expressed in mg/L of the juice sample.

Instrumental color measurements

Color measurements for all trials were conducted using a Konica Minolta Spectrophotometer (CM-700d, Konica Minolta, Japan) equipped with a D65 light source, 10° standard observer, and a target mask with an 8 mm aperture, plate, and open cone. A 20 mL of sample was pipetted into an optical glass cell CR-A504 (Konica Minolta, Japan), placed onto a plate of the target mask, and covered with a zero calibration box CM-A182 (Konica Minolta, Japan) to avoid the influence of ambient light. Colorimetric variables (L*, a *, b*) were measured and color change (∆E*ab), chroma (C*), and hue (H*) were calculated as (Bursać Kovačević et al., 2016):

ΔE*abΔL*2+Δa*2+Δb*2 (1)
C*=a*2+b*2 (2)
H*=tan1b*a* (3)

where all ΔL*2, Δa*2 and Δb*2 were calculated in reference to the untreated samples. All measurements were done in triplicate.

Determination of browning index (BI) and content of hydroxymethylfurfural (HMF)

The BI was determined according to the method described in the literature (Cohen et al., 1998). Briefly, 5 mL of 96% ethanol was added to 5 g of strawberry juice, and the mixture was centrifuged at 6500 rpm for 10 min. The supernatant was separated from the precipitate, and one part was used to determine the BI, while the other part was used to determine HMF by spectrophotometry.

To determine the BI, the absorbance was measured at 420 nm with distilled water as blank. The BI was calculated using the following formula:

BI=A420 nmDF (4)

where DF is the dilution factor, and A420 nm is the measured absorbance at 420 nm.

The HMF content was determined using a method described in the literature (Cohen et al., 1998). Briefly, 2 mL of the supernatant (adequately diluted with distilled water), 2 mL of 12% trichloroacetic acid, and 2 mL of 0.025 M thiobarbituric acid were mixed in a test tube and incubated at 40°C for 50 min. The contents of the test tubes were then cooled for a few minutes with a stream of cold tap water, and the absorbance was measured at 443 nm. A blank sample was prepared similarly, but distilled water was used instead of the supernatant. All measurements were performed in duplicate. The results of the HMF content were calculated using a calibration curve prepared from standard solutions of different HMF concentrations (2.5, 5, 7.5, 10, 15 and 20 mg/L). A brief protocol for the determination of HMF involves mixing 2 mL of the standard solution with 2 mL of 12% trichloroacetic acid and 2 mL of 0.025 M thiobarbituric acid. The mixture is vortexed briefly and incubated at 40 °C for 50 minutes in a thermostatic bath. After incubation, the sample is rapidly cooled using cold tap water, and the absorbance is measured at 443 nm. A blank is prepared similarly, substituting distilled water for the standard solution. Results are expressed as mg HMF/L in the sample.

Determination of monomeric anthocyanins

The determination of monomeric anthocyanins in strawberry juice extracts was carried out using an ultrasound-assisted extraction method. A 5 g sample of strawberry juice was mixed with 20 mL of 1% formic acid in 80% methanol (v/v). The mixture was treated in an ultrasonic bath (DT 514 H Sonorex Digitec, 13.5 L, 860 W, 40 kHz, Bandelin Electronic, Berlin, Germany) at 50 °C for 15 min. Following the extraction, the solution was filtered, and the supernatant was transferred to a 25 mL volumetric flask, and diluted with the extraction solvent. The extracts were stored at −18 °C under an inert gas atmosphere until analysis (Bebek Markovinović et al., 2024b). The method described in the literature was used to determine monomeric anthocyanins (AOAC, 1990). In brief, 1 mL of the extract and 4 mL of buffer pH 1 (potassium chloride buffer, 0.025 M) were pipetted into a test tube, while 1 mL of the same extract was mixed with 4 mL of buffer pH 4.5 (sodium acetate buffer, 0.4 M) in a second test tube. After 20 min, absorbance was measured at 520 nm and 700 nm on an LLG-uniSPEC 2 spectrophotometer (Lab Logistics Group GmbH, Meckenheim, Germany), using deionized water as a blank. All measurements were performed in parallel, and the concentration was expressed as pelargonidin-3-glucoside equivalent (Pg-3-G) (mg/100 mL) according to the equation:

A×MW×DF×103ε×1 (5)

where are:

A= (A520nm – A700nm)pH=1,0 - (A520nm – A700nm)pH=4,5

MW - molecular weight for pelargonidin-3-glucoside

DF - dilution factor

ε = molar absorption extinction coefficient

l = thickness of the cuvette.

Statistical analysis

Experiments were designed as full factorial randomized designs (n=40). Dependent variables were the content of monomeric anthocyanins, electrical conductivity, browning index, content of hydroxymethylfurfural, CIELab variables, and dissolved oxygen in juice. Independent variables included: exposure to PEF (1.5, 3.0, 4.5 min), exposure to HPU (2.5, 5.0, 7.5 min), and length of storage (0 and 7 days). Descriptive statistics were used to assess the basic information about the experimental dataset. Differences in treatments (continuous variables) were tested by multivariate analysis of variance (MANOVA). Exploratory hierarchical Ward’s cluster analysis was used to measure standardized similarities in samples. Nonparametric analysis employed the Kruskal–Wallis test. The significance level for rejection of a null hypothesis in all tests were α ≤ 0.05. Linear regression was employed to build and compare mathematical models. The significance level for all tests was α ≤ 0.05, all variance inflation factors were ≤ 5. Only statistically significant predictors were retained in the models (p ≤ 0.05). Analyses were performed with IBM SPSS Statistics (v.24), and the experimental design was performed by Statgraphics Centurion® (StatPoint Technologies, Inc, VA, USA).

Results

Chemometric evaluation of the influence of processing on the physicochemical properties, anthocyanins, and color stability of strawberry juices during storage

An exploratory hierarchical Ward’s cluster analysis was performed to determine which samples in the data set had similar standardized investigated properties (Figure 1). Samples treated with PEF for 3.0-4.5 min and HPU 2.5-7.5 min on day 0 of storage were most similar to controls (untreated samples) on day 0. Interestingly, after 7 days of storage, the control samples exhibited similar properties to those treated with PEF for 1.5-3.0 min and those treated with PEF for 1.5-3.0 min and HPU for 2.5-5.0 min. Since the stability of bioactive compounds, especially anthocyanins, depends on the duration of treatment, prolonged treatment is not conducive to the preservation of these compounds (Maza et al., 2020). When comparing the results of the control samples with the most similarly treated samples, it was found that the intensification treatments had a similar effect on storage (0 vs. 7 days) in terms of the duration of the extension treatment. This was observed in the additional analysis below.

Figure 1. Results of the hierarchal cluster analysis of the averaged and standardized samples.

Table 2 shows the numerical values of the significance of the Kruskal–Wallis test between the treated and the control samples. The treated samples differed from the control samples with respect to conductivity and the color parameters a*, b*, C*, H*, and ΔE*ab. The median values of these results are shown graphically in Figure 2.

Table 2. Kruskal–Wallis test statistics for samples treated by combined technology vs. untreated samples.

ANT COND BIHMF L* a* b* C* H* ∆E*ab O2
Chi-Square 3.09 7.83 0.462.94 3.59 7.81 7.32 7.81 4.30 7.81 0.00
df 1 1 11 1 1 1 1 1 1 1
Sig. 0.08 ≤ 0.01* 0.500.09 0.06 ≤ 0.01* ≤ 0.01* ≤ 0.01* 0.04* ≤ 0.01* 0.97

*The Kruskal–Wallis test is significant at p ≤ 0.05. ANT-Monomeric Anthocyanins (mg/100 g); COND-Conductivity (µS/cm-); BI-Browning index; HMF-Hydroxymethylfurfural (mg/L); L*-lightness; a*, b*-CIELab coordinates; C*-chroma; H*-hue; ΔE*ab-color change; O2-dissolved oxygen in juice (mg/L).

Figure 2. Median values of monomeric anthocyanins, physicochemical and color parameters in untreated vs. treated juice samples.

In general, the control samples differed from the treated samples, as they had lower median values for conductivity and all higher values for the CIELab parameters, except ΔE*ab, which was lower (Figure 2). Some literature reports suggest that the treated samples have a higher conductivity than the untreated ones, considering that the electrical conductivity of the sample increases with the degree of damage to its tissue due to the disintegration of the cell membrane by the phenomena of electroporation and cavitation during PEF and HPU treatment (Lebovka et al., 2002). However, other findings have shown that the electric field influences the electrical conductivity. Therefore, the results obtained are in agreement with those of (Cserhalmi et al., 2006) who found significantly higher conductivity values in treated grapefruit and orange juices than untreated ones. In addition, the control samples had higher median values of the color parameters a*, b*, C*, and H* than the treated samples, suggesting that the combination of PEF and HPU technologies significantly influenced juice color. However, this influence did not result in the degradation of anthocyanins, the formation of HMF, or an increase in BI, as no statistically significant differences were found between the control and treated samples regarding these parameters. Other variables did not differ significantly from the control samples.

The influence of pulsed electric field (PEF) in combination with high-power ultrasound (HPU) on the physicochemical properties of strawberry juices during storage

The results of the effects of storage, PEF, and HPU treatments on the conductivity, dissolved oxygen, browning index, and HMF content in strawberry juices are shown in Table 3. Storage significantly affected all parameters studied, with an increase in conductivity, browning index, and HMF content observed after 7 days of storage, while dissolved oxygen was lower at 0 days of storage. Increased conductivity in treated juices during storage was previously explained by increased extraction of intracellular contents during processing, resulting in mechanical damage to cell membranes (Lebovka et al., 2001). The browning index and HMF content increased significantly at the end of storage compared to the initial values. This was expected due to the formation of HMF in the stored fruit juices, which could be associated with various degradation reactions, such as non-enzymatic browning. The reduced amount of dissolved oxygen in the juices during storage could be result of oxidation, as shown by the results of storage-induced changes in color parameters (Enaru et al., 2021).

Table 3. Influence of storage, PEF, and HPU treatments on the conductivity, dissolved oxygen, browning index, and HMF content in strawberry juices.

Variables n COND O2 BI HMF
Storage p0.01† p0.01t p0.01† p < 0.01t
0 days 18 2733.4±20.7b 7.69±0.08a 1.51±0.01b 4.43±0.04b
7 days 18 3302.4±20.7a 5.84±0.08b 1.61±0.01a 4.79±0.04a
Average 36 3017.9±20.7 6.77±0.08 1.56±0.01 4.61±0.04
PEF p0.01† p0.01† p = 0.66* p = 0.33*
1.5 min 12 2725.2±25.3c 7.05±0.10a 1.57±0.01a 4.63±0.04a
3 min 12 3054.1±25.3b 6.87±0.10a 1.56±0.01a 4.64±0.04a
4.5 min 12 3274.6±25.3a 6.38±0.10a 1.56±0.01a 4.55±0.04a
Average 36 3018.0±25.3 6.77±0.10 1.56±0.01 4.61±0.04
HPU p0.01† p0.01† p0.01† p = 0.29*
2.5 min 12 2754.2±25.3c 6.96±0.10a 1.57±0.01a 4.63±0.04a
5 min 12 2987.1±25.3b 6.86±0.10a 1.59±0.01a 4.65±0.04a
7.5 min 12 3312.6±25.3a 6.47±0.10b 1.52±0.01b 4.55±0.04a
Average 36 3018.0±25.3 6.76±0.10 1.56±0.01 4.61±0.04
PEF + HPU p0.01t† p = 0.23‡ p0.01† p = 0.06*
1.5 min + 2.5 min 12 2174.8±13.9c 7.14±0.2a 1.64±0.02a 4.77±0.07a
1.5 min + 5 min 12 2838.3±13.9b 7.27±0.2a 1.59±0.02a 4.64±0.07a
1.5 min + 7.5 min 12 3162.5±13.9a 6.73±0.2a 1.47±0.02b 4.49±0.07a
Average 36 2725.2±43.9 7.05±0.21 1.57±0.02 4.63±0.07
PEF + HPU p0.01† p = 0.42* p0.01† p = 0.07*
3 min + 2.5 min 12 3156.5±35.5a 7.02±0.14a 1.60±0.01a 4.68±0.08a
3 min+5 min 12 2740.5±35.5b 6.75±0.14a 1.60±0.01a 4.77±0.08a
3 min+7.5 min 12 3265.2±35.5a 6.85±0.14a 1.47±0.01b 4.47±0.08a
Average 36 3054.1±35.5 6.87±0.14 1.56±0.01 4.64±0.08
PEF+HPU p0.01† p0.01† p0.01† p = 0.17*
4.5 min+2.5 min 12 2931.3±43.9b 6.74±0.15a 1.48±0.02b 4.44±0.09a
4.5 min+5 min 12 3382.5±43.9a 6.57±0.15a 1.58±0.02a 4.53±0.09a
4.5 min+7.5 min 12 3510.0±43.9a 5.85±0.15b 1.61±0.02a 4.70±0.09a
Average 36 3274.6±43.9 6.39±0.15 1.56±0.02 4.56±0.09

The results are expressed as mean ± standard error. Values represented with different letters are statistically different at p ≤ 0.05;

significant factor in multifactor analysis.

not significant factor in multifactor analysis.

COND, conductivity (µS/cm); O2-oxygen content (mg/L); BI, browning index; HMF, hydroxymethylfurfural (mg/L).

The results also show that the processing time for the PEF and HPU treatments significantly influences the conductivity, with higher values achieved with longer processing times. An exception is the treatment with PEF (3 min) + HPU (2.5 min), where significantly higher conductivity values were obtained, as well as the treatment with the longest processing time (PEF 3 min + HPU 7.5 min). A longer treatment time for both technologies and their combination leads to a more effective electroporation of the cell membrane and, consequently, an easier efflux of cell material into the extracellular space, which may be associated with osmotic flow and a redistribution of moisture within the sample (Lebovka et al., 2001). The results obtained are consistent with the findings of Zou & Jiang (2016), where conductivity values increased with the increasing duration of ultrasonic treatment of carrot juice.

Oxygen in juices can lead to negative changes in anthocyanin content due to oxidation and can also negatively affect color stability (Buvé et al., 2018). Storage significantly affected oxygen reduction in the juice samples, which is consistent with previous reports (Solomon et al., 1995). In most cases, longer PEF, HPU, and PEF+HPU treatments reduced oxygen content in the samples. PEF treatment allows easier removal of air from the tissue due to the electroporation and improved permeability of cell membranes (Trusinska et al., 2023). Ultrasound can also be successfully used to degas juices and other fluids (Khan et al., 2020). Dissolved gasses, such as oxygen, can act as nuclei and form bubbles, which float to the surface and can be removed from the juice, i.e., the treated liquid (Zenker et al., 2003). The results obtained show that lower dissolved oxygen concentrations were observed at shorter PEF (1.5 and 3 min) and HPU (2.5 and 5 min) processing times. Interestingly, when the technologies were combined, the processing time had no significant effect on the dissolved oxygen content, except for the longest processing times where the minimum dissolved oxygen was observed at PEF 4.5 min + HPU 7.5 min. This confirms that the synergistic effect of PEF and HPU technologies is most effective in reducing dissolved oxygen at longer processing times, allowing greater stability of strawberry juices during storage.

Color plays an important role for consumers when selecting strawberry products, and the development of undesirable browning was determined by the browning index. The storage of strawberry juices significantly influenced the development of brown coloration; stored juices had higher BI values than non-stored juices. Brown coloration can be caused by factors such as storage temperature, light, oxygen, pH, metals, and enzymes (Holzwarth et al., 2011; Holzwarth et al., 2012). As shown in Table 4, dissolved oxygen decreased during storage, making oxidation processes less likely. The most likely cause of the development of an undesirable brown color is enzymatic. Since samples were treated with non-thermal technologies, this excluded the use of high temperatures in the pre-treatment that are commonly used for enzymatic inactivation in juice production. Hence, it is likely that active enzymes caused these undesirable changes. This has already been confirmed in a study where thermal treatment (75°C/20 min) had the strongest impact on the inactivation of polyphenol oxidase compared to PEF and thermosonification in orange juice (Sulaiman et al., 2016). In addition, ultrasonic treatment can cause an increase in polyphenol oxidase activity (Bi et al., 2015). Considering the influence of different treatment times, BI was found to be more pronounced in juices treated with shorter treatment times. The only exception was an increase in BI during the longest treatment with a combination of PEF (4.5 min) + HPU (7.5 min). These results suggest that there is a limit beyond which prolonged treatment time with the combination of PEF+HPU technologies will cause the loss of red color, which could be related to the degradation of anthocyanins.

Table 4. Influence of storage, PEF and HPU treatments on the anthocyanins and color parameters.

Variables n ANT L* a* b* C* H* ∆E*ab
Storage p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01†
0 days 18 17.72±0.04b 31.48±0.01a 18.64±0.03a 7.96±0.01a 20.27±0.03a 23.08±0.04a 2.85±0.06b
7 days 18 18.81±0.04a 30.87±0.01b 17.77±0.03b 7.16±0.01b 19.16±0.03b 21.95±0.04b 4.11±0.06a
Average 36 18.27±0.04 31.18±0.01 18.21±0.03 7.56±0.01 19.72±0.03 22.52±0.04 3.48±0.06
PEF p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01†
1.5 min 12 18.49±0.05b 30.78±0.01c 17.76±0.03c 7.21±0.01c 19.17±0.03c 22.09±0.05c 4.13±0.07a
3 min 12 18.93±0.05a 31.24±0.01b 18.29±0.03b 7.66±0.01b 19.83±0.03b 22.66±0.05a 3.34±0.07b
4.5 min 12 17.37±0.05c 31.50±0.01a 18.57±0.03a 7.82±0.01a 20.16±0.03a 22.80±0.05b 2.96±0.07c
Average 36 18.26±0.05 31.17±0.01 18.21±0.03 7.56±0.01 19.72±0.03 22.52±0.05 3.48±0.07
HPU p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p = 0.02t p ≤ 0.01†
2.5 min 12 18.12±0.05b 31.17±0.01b 18.39±0.03a 7.62±0.01a 19.91±0.03a 22.48±0.05b 3.30±0.07b
5 min 12 19.03±0.05a 31.10±0.01c 18.02±0.03c 7.46±0.01b 19.50±0.03c 22.43±0.05b 3.71±0.07a
7.5 min 12 17.64±0.05c 31.25±0.01a 18.21±0.03b 7.61±0.01a 19.74±0.03b 22.63±0.05a 3.43±0.07b
Average 36 18.26±0.05 31.17±0.01 18.21±0.03 7.56±0.01 19.72±0.03 22.51±0.05 3.48±0.07
PEF+HPU p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01†
1.5 min+2.5 min 12 19.01±0.06a 30.91±0.02a 18.34±0.04a 7.47±0.02a 19.80±0.04a 22.14±0.04a 3.50±0.14b
1.5 min+5 min 12 18.51±0.06b 30.64±0.02c 17.36±0.04c 6.98±0.02c 18.71±0.04c 21.89±0.04b 4.61±0.14a
1.5 min+7.5 min 12 17.96±0.06c 30.80±0.02b 17.58±0.04b 7.18±0.02b 18.99±0.04b 22.22±0.04a 4.28±0.14a
Average 36 18.49±0.06 30.78±0.02 17.76±0.04 7.21±0.02 19.17±0.04 22.08±0.04 4.13±0.14
PEF+HPU p = 0.05t p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01†
3 min+2.5 min 12 18.71±0.10b 31.01±0.04b 17.92±0.03a 7.38±0.02b 19.38±0.04b 22.34±0.04b 3.84±0.10a
3 min+5 min 12 19.17±0.10a 31.01±0.04b 17.81±0.03b 7.38±0.02b 19.28±0.04b 22.45±0.04b 3.93±0.10a
3 min+7.5 min 12 18.90±0.10a,b 31.71±0.04a 19.14±0.03c 8.22±0.02a 20.83±0.04a 23.18±0.04a 2.26±0.10b
Average 36 18.93±0.10 31.24±0.04 18.29±0.03 7.66±0.02 19.83±0.04 22.66±0.08 3.34±0.10
PEF+HPU p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01† p ≤ 0.01†
4.5 min+2.5 min 12 16.64±0.08b 31.60±0.02a 18.91±0.09a 8.02±0.02a 20.55±0.08b 22.96±0.13a 2.55±0.13b
4.5 min+5 min 12 19.42±0.08a 31.65±0.02a 18.88±0.09a 8.02±0.02a 20.52±0.08b 22.96±0.13a 2.59±0.13b
4.5 min+7.5 min 12 16.05±0.08c 31.25±0.02b 17.93±0.09b 7.42±0.02b 19.40±0.08a 22.49±0.13b 3.74±0.13a
Average 36 17.37±0.08 31.50±0.02 18.57±0.09 7.82±0.02 20.16±0.08 22.80±0.13 2.96±0.13

The results are expressed as mean ± standard error. Values represented with different letters are statistically different at p ≤ 0.05;

significant factor in multifactor analysis;

not significant factor in multifactor analysis.

ANT, monomeric anthocyanins (mg/100 g); L*, lightness; a*, b*, CIELab coordinates; C*, chroma; H*, hue; ΔE*ab, color change.

When examining the HMF content, the results show that only storage significantly influences the formation of HMF, with a higher concentration found in juices stored for 7 days compared to those stored for 0 days. These results align well with various studies that reported a proportional increase in HMF content during storage and when exposed to higher temperature (Singh & Sharma, 2017). The PEF and HPU technologies with different treatment times, had no significant effect on the HMF content of the treated juice samples. The findings are consistent with literature data that PEF technology had no effect on HMF formation in orange juice (Agcam et al., 2016), in sour cherry juice (Akdemir Evrendilek et al., 2021) and in apple juice (Akdemir Evrendilek et al., 2016). Compared to those stored for 0 days. These results align well with various studies that reported a proportional increase in HMF content during storage and when exposed to higher temperatures (Singh & Sharma, 2017). The PEF and HPU technologies, with different treatment times, had no significant effect on the HMF content of the treated juice samples. These findings are consistent with literature data, which indicate that PEF technology had no effect on HMF formation in orange juice (Agcam et al., 2016), sour cherry juice (Akdemir Evrendilek et al., 2021), and apple juice (Akdemir Evrendilek et al., 2016). This trend could be related to the high retention of ascorbic acid in juices, which, if not oxidized, cannot provide reactive carbonyl groups that can act as precursors for non-enzymatic browning reactions (Akdemir Evrendilek et al., 2016).

The influence of pulsed electric field (PEF) in combination with high-power ultrasound (HPU) and on the anthocyanins and color stability of strawberry juices during storage

The average anthocyanin content of the treated strawberry juices (17.37 ± 0.08 mg/100 mL) is consistent with previous results (Bebek Markovinović et al., 2022a; Bebek Markovinović et al., 2022b) (Table 4). The storage of strawberry juice led to a significant increase (6.1%) in the anthocyanin content. The same trend was observed in an earlier study (Bebek Markovinović et al., 2022c), in which strawberry juices treated with PEF during a 7-day storage period showed a 7.7% increase in anthocyanin content. The positive effect of storage on anthocyanin content can be explained by its subsequent extraction, i.e., the release of intracellular compounds from damaged cells during storage.

When considering the influence of processing time during PEF treatment on the stability of anthocyanins, the greatest stability was observed at a medium PEF treatment time of 3 minutes. In contrast, anthocyanins were preserved to a lesser extent at both short and long PEF treatment times, indicating an inflection point in the data. Literature reports that the content of anthocyanins in strawberry juices depends significantly on the duration of the PEF treatment and the strength of the electric field, with shorter treatments and higher electric field strengths leading to greater preservation of anthocyanins (Odriozola-Serrano et al., 2008).

In contrast, in the case of HPU, a treatment duration of 5 min resulted in the highest anthocyanin stability, while a treatment duration of 7.5 minutes resulted in the lowest anthocyanin stability. This is consistent with previous results, where a significant effect of treatment duration was found on the reduction of anthocyanin content in HPU-treated strawberry juices (Tiwari et al., 2008). The increase in anthocyanin content with shorter treatment times could be explained by the improved extraction of pigments from the suspended pulp.

The combined PEF and HPU treatments at different durations significantly influenced the stability of the anthocyanins. With shorter exposures to PEF (1.5 min) and HPU (2.5 min), the stability of the anthocyanins was better. However, as the processing time in PEF technology increased (3 min and 4.5 min), longer processing times in HPU technology (5 min and 7.5 min) were required for higher stability of the anthocyanins. This confirms the synergistic influence of the combination of PEF and HPU on the stability of the anthocyanins. Additionally, the combination of 4.5 min PEF + 5 min HPU showed the best effect on the stability of the anthocyanins.

Storage at 4°C for 7 days significantly reduced the lightness (L*), suggesting that storage contributed to the darkening of the juices. Considering that the BI increased significantly and the HMF content decreased during storage (Table 4), the changes in L* values can most likely be attributed to the previously mentioned enzymatic activity (Bi et al., 2015; Sulaiman et al., 2016). Furthermore, these results are consistent with the findings of Tiwari et al. (2009), who also observed decreased L* values in orange juice after 7 days of storage at 10°C

By extending the PEF treatment duration, the L* value increases significantly. In contrast, a different trend was observed when extending the duration of the single HPU treatment. Extending the HPU treatment up to 5 min resulted in a significant decrease in L*, while extending the treatment up to 7.5 min caused an increase in L*. Similar findings were previously reported, where L* increased with ultrasound treatment of carrot juice up to a 20 min treatment duration, after which L* stagnated (Zou & Jiang, 2016). In the combination of PEF (1.5 min) and HPU treatments, an increase in HPU treatment duration resulted in a significant decrease in L* up to 5 min. In contrast, further treatment led to a significant increase, but not beyond the initial value. Additionally, a different trend was observed with PEF (3 min) and HPU treatment. No change in L* was observed when the HPU treatment time was extended up to 5 min, after which L* increased significantly. The increase in L* could be caused by the degradation of unstable particles during ultrasonic treatment, which affects the changes in color parameters (Tiwari et al., 2009). In contrast to these results, the same trend was observed with the combination of PEF (4.5 min) and HPU treatment up to 5 min. After that, the opposite trend was observed, with the L* value decreasing significantly as the duration increased to 7.5 min. This could be related to the sample being exposed to prolonged treatment, resulting in degradation and a reduction in anthocyanin content, with the anthocyanin value being significantly reduced after the PEF (4.5 min) + HPU (7.5 min) treatment.

The a* and b* values follow almost the same trend with respect to storage and the technologies used. Storage led to a significant decrease in both a* and b* values. These results are consistent with the color parameters of canned strawberries with added black carrot concentrate during the same storage period (Kammerer et al., 2006). With increasing PEF treatment duration, there was a significant increase in both a* and b* values. In contrast, the individual HPU treatment showed a significant decrease in a* and b* values when the treatment duration was increased from 2.5 to 5 min. However, a further extension of the treatment to 7.5 min increased both a* and b* values. The combination of PEF (1.5 and 3 min) and HPU treatment showed the same trend as HPU treatment alone, with an increase in duration (the exception being PEF at 3 min) and HPU, with an increase in treatment time from 2.5 to 5 min. In the PEF (4.5 min) and HPU treatment, there was a significant decrease in the a* value with increasing HPU treatment duration. In contrast, the b* value remained unchanged with an increase in duration from 2.5 to 5 min and decreased significantly with further treatment (7.5 min). Color changes during sonication could be attributed to the influence of cavitation, which causes physical, chemical, and biological reactions that lead to the cleavage of certain particles, such as enzymes (Sala et al., 1995).

Values of C* (chroma) and H* (hue) follow almost the same trend with respect to storage conditions and the application of different PEF and HPU treatment times, both individually and in combination. Storing the juices at 4°C for 7 days had a significant effect on the reduction of both C* and H* values. These results are consistent with previous reports (Kammerer et al., 2006), where a decrease in these values was observed in canned strawberries after 4 weeks of storage. Increasing the PEF treatment duration significantly affected the increase in both values (C* and H*). In contrast, increasing the HPU treatment time from 2.5 to 5 min decreased both values, while further treatment caused a significant increase in these values. The combination of PEF (1.5 min) and HPU treatment showed the same trend in C* and H* values as HPU treatment alone. PEF (3 min) and HPU did not affect C* and H* levels when the HPU treatment time was increased up to 5 min, after which there was an increase in these two values. In contrast, PEF (4.5 min) and HPU treatment above 5 min showed a decrease in these values. These results are partially consistent with previous reports on colorimetric parameters in red and yellow watermelon juice sonicated for 4, 8, 12, and 16 min (Yıkmış, 2020). The trends are similar, and the differences can be attributed to the varying treatment times and the intensity of the device.

ΔE*ab, i.e. the overall color difference between the untreated and treated samples, was evaluated to determine whether the PEF and HPU treatments produce a color change that the consumer can perceive. The mean values for ΔE*ab ranged from 2.96±0.13 to 4.13±0.14. These were not large or very significant differences, hence the treated juices may be sensory acceptable in terms of color. However, when ΔE*ab = 1.5–3.0, the color change is noticeable, and when ΔE*ab = 3.0–6.0, the difference is considerable (Chen, 2008). Storage significantly affected the ΔE*ab value, which was significantly higher in stored samples. An increase in ΔE* value after 4 weeks of storage of canned strawberries was also observed (Kammerer et al., 2006). The PEF treatment reduced the ΔE*ab, while the application of the HPU treatment showed the opposite trend. Extending HPU treatment from 2.5 to 5 min led to an increase in ΔE*ab, while further treatment led to a decrease. The same trend was observed with the combination of PEF (1.5 min) and HPU treatment. PEF (3 min) and HPU did not affect ΔE*ab values when the HPU treatment time was increased to 5 min, after which there was an increase in ΔE*ab value. In contrast, when using PEF (4.5 min) and HPU treatment for 5 min of the other treatment technology, a decrease in ΔE*ab value was observed. Overall, the values of ΔE*ab were below 6, which indicates that the differences in color were acceptable, i.e., appreciable (Chen, 2008).

Optimization pulsed electric field (PEF) and high-power ultrasound (HPU) processing parameters

In order to obtain products with the best nutritional and/or biological value, as well as the best physicochemical properties, it is important to optimize the process parameters of the technologies. Table 5 shows the optimal parameters for the PEF and HPU treatments that led to maximum anthocyanin yield and minimum changes in color, conductivity, BI, and HMF content. As mentioned above, longer storage favors the content of anthocyanins, with a yield of 20.29 mg/100 g of these polyphenols on the 7th day of storage, achieved with PEF and HPU treatment durations of 2.19 and 7.48 minutes, respectively. On the other hand, if the combined technology aims to minimize conductivity and browning, the samples should not be stored at all (i.e., storage should be 0 days), and the duration of the PEF treatment should be relatively short, no longer than 1.5 minutes. The optimal values for the lowest conductivity (2156.7 µS/cm) and browning index (1.13) should be achieved with 2.5 and 7.5 minutes of HPU treatment, respectively. The final color change was achieved with 3.14 minutes of PEF and 7.5 minutes of HPU.

Table 5. Optimal processing parameters for the combination of PEF and HPU technologies to achieve maximum monomeric anthocyanin content and minimum changes in color, conductivity, browning index, and HMF content.

Parameter ANT COND BI HMF ∆E*ab
Storage (days) 7.00 0.00 0.00 7.00 0.00
PEF treatment (min) 2.19 1.50 1.50 3.77 3.14
HPU treatment (min) 7.48 2.50 7.50 2.50 7.50
Optimum 20.29 2156.7 1.13 4.17 1.53

ANT, monomeric anthocyanins (mg/100 g); COND, conductivity (µS/cm); BI, browning index; HMF, hydroxymethylfurfural (mg/L); ΔE*ab, color change. PEF, pulsed electric field (30 kV/cm, 100 Hz); HPU, high-power ultrasound (amplitude 25%, pulse 50%).

Based on the parameters studied, and if PEF was used as the initial treatment followed by HPU in this hurdle concept, it was more practical to shorten the PEF treatment and prolong the HPU treatment to achieve optimal nutritional value and the least adverse effects on the samples. There is no data in the literature regarding the optimization of the parameters for the combination of PEF and HPU technologies for anthocyanin preservation. In a previous study, the authors compared the yield of anthocyanins from blueberry by-products using PEF treatment (5-30 kV/cm, 1000-3000 Hz) and ultrasonic treatment (125 W, 60 min, 40°C). It was shown that PEF treatment is significantly more effective and requires a shorter treatment time and lower temperatures than sonication (Zhou et al., 2015). These results are consistent with our findings, where the optimization of the parameters of the combined PEF and HPU technologies showed that a shorter PEF treatment than HPU was required to achieve the best anthocyanin yield and the least changes in color and HMF content.

Conclusions

This work aimed to investigate the influence of pulsed electric field (PEF) and high-power ultrasound (HPU) treatment at different operating times on anthocyanin content, color parameters, conductivity, dissolved oxygen, HMF content, and browning index in strawberry juices stored at 4°C for 7 days using a chemometric approach. First, the untreated (controls) samples were compared with the treated samples (PEF + HPU), and it was found that they differed in terms of conductivity and color parameters. Conductivity was higher in the treated samples than in the control samples, as was the overall color change ΔE*ab. A storage time of 7 days at 4°C had a significant effect on the increase in anthocyanin concentration, conductivity, browning index, HMF content, and total color change ΔE*ab, while the opposite was true for the dissolved oxygen and CIEL*a*b* parameters. The highest anthocyanin stability was observed in the samples treated with PEF (3 min) and HPU technology (2.5, 5, and 7.5 min). An increase in treatment time led to increased conductivity without any effect on the formation of hydroxymethylfurfural. The order and length of exposure mattered in the sense that shorter PEF treatments followed by HPU led to decreased browning, while the inverse treatment (i.e., the most intensive PEF treatment followed by HPU) increased it. PEF and HPU treatments showed good effects in reducing dissolved oxygen in the juices. Moreover, the color changes of the treated samples ranged from noticeable to considerable, which was still within the acceptable limits. All these results indicate that the PEF and HPU technologies are suitable for the production of strawberry-based functional juices as they help to preserve their biological and nutritional value and properties.

The optimal parameters for combining PEF and HPU treatments to obtain strawberry juice with the highest yield of anthocyanins were a short 2.19 min for PEF treatment followed by a 7.48 min HPU treatment during a 7-day storage period. In general, shorter PEF treatment times and longer HPU treatment times favored better anthocyanins yield and minimal color changes, browning index, and hydroxymethylfurfural formation. In summary, the combination of PEF and HPU technologies has the potential to preserve the nutritional and biological value of strawberry juices, prevent the formation of hydroxymethylfurfural, and preserve the physicochemical properties of the native product.

Author Contributions

Conceptualization, D.B.K., P.P., A.M.K.; methodology, D.B.K., V.S., T.J., and B.P.; software, P.P.; validation, A.B.M., S.M., B.P., and Z.H..; formal analysis, A.B.M., V.S., T.J., B.P., and S.M.; investigation, A.B.M., V.S., T.J., B.P., and S.M.; resources, D.B.K. and Z.H. ; data curation, P.P.; writing—original draft preparation, A.B.M., D.B.K., and. P.P.; writing—review and editing, V.S., T.J., B.P., S.M., Z.H., and A.M.K.; visualization, A.B.M., P.P., S.M., B.P. and T.J.; supervision, D.B.K.; project administration, D.B.K. and Z.H.; funding acquisition, D.B.K. and A.M.K. All authors have read and agreed to the published version of the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Funding

This research was funded by the Croatian Science Foundation through the funding of the Hurdle Technology and 3D Printing for Sustainable Fruit Juice Processing and Preservation project [IP-2019-04-2105]; Republic of Croatia Ministry of Science and Education through the European Regional Development Fund through the project “Equipping the semi-industrial practice for the development of new food technologies” [KK.01.1.1.02.0001]; and the “Young Researchers’ Career Development Project—Training of Doctoral Students” of the Croatian Science Foundation [DOK-2020–01].

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