# Impact of conventional and non-conventional processing technologies on phenolic compounds and polysaccharides in strawberry nectar: A comparative study of stability

S Rincon, Claire Dufour, H Murray, M Gössinger, H Zia, A Moreno, D Pavón-Vargas, L Cattani, A Puzović, M Mikulic-Petkovsek, et al.

► To cite this version:

S Rincon, Claire Dufour, H Murray, M Gössinger, H Zia, et al. Impact of conventional and non-conventional processing technologies on phenolic compounds and polysaccharides in strawberry nectar: A comparative study of stability. Food Chemistry, 2026, 505, 10.1016/j.foodchem.2026.148127. hal-05478176

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# Impact of conventional and non-conventional processing technologies on phenolic compounds and polysaccharides in strawberry nectar: A comparative study of stability

S. Rincon<sup>a</sup>, C. Dufour<sup>a</sup>, H. Murray<sup>b</sup>, M. Gössinger<sup>b</sup>, H. Zia<sup>c</sup>, A. Moreno<sup>d</sup>, D. Pavón-Vargas<sup>d,e</sup>, L. Cattani<sup>d</sup>, A. Puzović<sup>f</sup>, M. Mikulic-Petkovsek<sup>f</sup>, A. Slatnar<sup>f</sup>, H. Halbwirth<sup>g</sup>, O. Dangles<sup>a</sup>, C. Le Bourvellec<sup>a,\*</sup>

## ARTICLE INFO

### Keywords:

Redberry

Juice processing

Pasteurisation

Ohmic heating

High pressure processing

Pulsed electric fields

Polyphenol

Cell wall

## ABSTRACT

Thermal treatments extend the shelf life of processed products however, they may modify their qualities. This study examines the effects of processing and storage (4 °C during 60 days) on phenolic compounds and cell walls in strawberry nectar. Under microbial inactivation standardization, conventional thermal treatment (TT) was compared to Ohmic Heating (OH), High Pressure Processing (HPP), and Pulsed Electric Field (PEF). HPP resulted in the least colour variation after processing. After storage, colour decreased significantly after PEF, while TT preserved colour most effectively. Immediately after processing HPP-and OH- samples exhibited the highest total phenolic compounds (no reduction and a 4% reduction). However, after 60 days of storage TT better preserved phenolic compounds (a 7% reduction). Proanthocyanidins and anthocyanins degraded over time (a 14% (HPP-samples) and 42% (PEF-samples) reduction respectively). Cell wall components such as neutral sugars and galacturonic acid uniformly degraded after storage. Pectin degree of methylation significantly decreased after storage especially after PEF and HPP treatments which probably failed to achieve complete enzyme inactivation. Storage had a more significant impact on nectar quality attributes than processing.

## 1. Introduction

The development of taste and healthy food products with a low environmental impact is a major challenge of food processing (Sanchez-Siles et al., 2019). Fruit juices and nectars appeal consumers because of their prolonged shelf life, flavour variety and nutritional benefits (Sanchez-Moreno et al., 2009). Nectars can be prepared using two main approaches, depending on whether pressing and filtration are involved. Nectars are produced from fruit purees by adding water, with or without sugars, honey, and/or syrups (Shahidi & Alasalvar, 2016). This method

avoids pressing and filtration, which helps preserve phenolic compounds that would otherwise be lost in the pomace (Brahem et al., 2017). By skipping these steps, the nutrient profile is diluted but retains beneficial compounds bound to cell walls. Alternatively, nectars can be also made from concentrated juices or freshly squeezed juices, which involve pressing and filtration. The resulting juice is then diluted with water to achieve the desired juice content, depending on the specific fruit or vegetable used.

Strawberry products are becoming increasingly popular due to their attractive colour, aroma, and sweet taste (Giampieri et al., 2012; Li

**Abbreviations:** TT, Thermal Treatment; OH, Ohmic Heating; HPP, High Pressure Processing; PEF, Pulsed Electric Field; HIPEf, High Intensity Pulsed Electric Field; mDP, Mean degree of polymerisation; PAC, Proanthocyanidins; PPO, Polyphenol Oxidase; POD, Peroxidase; PME, Pectin methyl esterase; DM, Degree of methylation; GalU, Galacturonic Acid.

* Corresponding author at: INRAE, UMR408 SQPOV «Safety and Quality of Plant Products», 228 route de l'aérodrome CS, 40509 F-84914 Avignon cedex 9, France.
E-mail address: carine.le-bourvellec@inrae.fr (C. Le Bourvellec).

<footnote><sup>a</sup> INRAE, Avignon Université, UMR SQPOV, F-84000 Avignon, France</footnote>

<footnote><sup>b</sup> Department of fruit Processing, Federal College and Institute for Viticulture and Pomology, A-3400 Klosterneuburg, Austria</footnote>

<footnote><sup>c</sup> GfL Gesellschaft für Lebensmittel-Forschung mbH, Landgrafenstrasse 16, 10787 Berlin, Germany</footnote>

<footnote><sup>d</sup> University of Parma, Viale delle Scienze 181/A, 43124 Parma, Italy</footnote>

<footnote><sup>e</sup> CFT S.P.A, Via Paradina, 94/a, 43122 Parma, Italy</footnote>

<footnote><sup>f</sup> Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Kongresni trg 12, 1000 Ljubljana, Slovenia</footnote>

<footnote><sup>g</sup> Institute of Chemical, Environmental & Bioscience Engineering, Technische Universität Wien, Vienna 1060, Austria</footnote>
et al., 2020). However, strawberries are prone to microbial growth despite their low pH (3.0-4.5) (Odriozola-Serrano et al., 2008). Hence, thermal processing is commonly used to inactivate microorganisms and inhibit enzymes; which can also trigger the degradation of heat-sensitive phenolic compounds (Patras et al., 2009). This can lead to colour loss, primarily due to anthocyanin degradation and the concomitant generation of brown and yellow products (Dangles, 2024). However, according to EU food law nectar producer cannot legally use artificial colorants that are commonly used to other products, such as strawberry juice drink, to hinder colour loss. Likewise, thermal processing may alter the structure of pectins in nectar, thereby modifying their functionalities (Liu et al., 2020) and their potential interactions with other molecules (i. e. proanthocyanidins) (Le Bourvellec et al., 2009). Furthermore, the structure of pectin may undergo modifications during processing and storage due to the residual activity of pectin-related enzymes -such as pectin methylesterase (PME) and polygalacturonase (PG)- which can alter pectin macromolecular properties in the final product. (Christiaens et al., 2016).

Therefore, novel non-thermal food processing technologies such as High-Pressure Processing (HPP) (Cao et al., 2012; Marszalek et al., 2017), Pulsed Electric Field (PEF) (Odriozola-Serrano et al., 2009; Yildiz et al., 2021b), and Ohmic Heating (OH) (Junqua et al., 2021; Mercali et al., 2015) have been explored and increasingly implemented in the food industry. The stability of anthocyanins and other flavonoids under mild preservation technologies, such as PEF and HPP, has been investigated and compared to thermal treatment (TT) in various plant-based systems. HPP has shown positive effects on anthocyanin stability in strawberry pulp and purée (Cao et al., 2011; Terefe et al., 2013), whereas PEF has minimal effects on phenolic compounds in strawberry juice. The degradation of cell wall components has been reviewed by Liu et al. (2020), regarding conventional TT, Ultrasound (US), HPP, and combined methods, noting the effects on cloud stability, texture, rheological and nutritional properties. HPP induced texture loss in carrots (Trejo Araya et al., 2007) and pumpkin (Paciulli et al., 2019) due to rapid pressure increase. It also increased the viscosity and emulsifying properties of mango pulp due to change in pectin structure such as modification of galacturonic acid content, degree of methylation and molecular weight, which are pH-dependent (Liu et al., 2020). Pectin depolymerisation by HPP depends on branching and molecular weight (Zhang et al., 2022). These observations suggest that each food matrix, and therefore pectin structure, responds differently to each treatment. Moreover, pectin alteration may have affect pectin-bound polyphenols, particularly proanthocyanidins (Bautista-Ortín et al., 2016; Brahem et al., 2017; Ertan et al., 2020; Le Bourvellec & Renard, 2012; Liu et al., 2020; Osete-Alcaraz et al., 2019; Watrelot et al., 2014) and anthocyanins (Erşan et al., 2022; Fernandes et al., 2021; Koh et al., 2020).

Fruit juices are highly susceptible to spoilage during storage, due to microbial, chemical, physical, and enzymatic factors, unless processing is properly handled (Bates, Morris, & Crandall, 2001). Studies have examined changes in phenolic compounds in strawberry products during storage following the application of non-conventional technologies (Aaby et al., 2018; Marszalek et al., 2017; Terefe et al., 2013; Yildiz et al., 2021a). Whatever the method used, storage adversely affected phenolic compound content, particularly at high temperatures (Teleszko et al., 2016). However, research on the impact of processing and storage on cell wall polysaccharides is limited. Studies on strawberries are even scarce and usually restricted to untreated fruits. A recent study on blueberries indicated that low temperature storage prevented the solubilisation of galacturonic acid. Additionally, the activities of four cell wall-degrading enzymes increased and subsequently decreased during storage, with cold storage further inhibiting these enzymes (Chen et al., 2015).

This study aims to outline and compare the effects of non-conventional technologies (OH, PEF and HPP) vs. conventional thermal treatments (TT), for the pasteurisation of strawberry nectar under equivalent microbial inactivation using a targeted 5-log reduction of E

coli. The analysis focused on the major phenolic compounds in strawberries (anthocyanins and proanthocyanidins) and on cell wall polysaccharides. Additionally, the impacts of the different processes are compared during storage at 4 °C after 30 and 60 days. This study provides new data on cell wall modification after processing and storage, and its consequences on organoleptic properties, such as colour, which is a key to consumers' purchase decision.

## 2. Material and methods

### 2.1. Standards and chemicals

Ethanol and acetone were from Fisher Scientific (Strasbourg, France). Acetonitrile of HPLC grade was obtained from VWR International (Radnor, USA). Hexane, methanol, hydrochloric acid and acetic acid were from Merck (Darmstadt, Germany). Sugar standards (arabinose, fucose, galactose, xylose, mannose and rhamnose) were from Fluka (Buchs, Switzerland). Formic acid, NaBH₄, N-methylimidazole, acetic anhydride, lignin alkali, (+)-catechin, (-)-epicatechin, quercetin, kaempferol, caffeic acid, p-coumaric acid, acetyl chloride, menthofuran, inositol, galacturonic acid, citric acid, were provided from Sigma-Aldrich (Saint Quentin Fallavier, France). Methanol-d₃ was from Acros Organics (Geel, Belgium). Anthocyanin standards (pelargonidin-3-O-glucoside, cyanidin-3-O-galactoside) were provided by Extrasynthèse (Lyon, France).

### 2.2. Strawberry nectar

The strawberry puree was purchased from SVZ International BV (Breda, The Netherlands). Considering the large quantities needed for treatment trials, it was not possible to obtain strawberry puree from a single variety of strawberries due to limited availability. Hence, the puree came from a blend of three different strawberry Spanish varieties. The strawberry puree sorted into 18 kg leakproof cardboard boxes and transported under frozen storage (−20 °C) to the respective industrial partners for pilot-scale trials. For nectar formulation, the strawberry puree was thawed at room temperature until it was soft enough to be blended. The strawberry nectar was prepared before each trial by mixing 40% strawberry puree with water. Sucrose and citric acid were added to achieve a Brix value of 12 and an acidity of 5 g/kg, respectively.

#### 2.2.1. Pilot-scale trials

Pilot-scale trials Pilot-scale trials were conducted as described by Pavón et al. (2025). Thermal treatment trials were done at CFT SpA in Parma, Italy. HPP treatment trials were done at HPP Italia in Tavversetolo, Italy. OH treatment trials were done at CTCPA in Avignon, France and PEF treatment trials were done at ELEA in Quakenbrück,

<table><caption>**Table 1**
Processing conditions used for each processing technology.</caption><thead><tr><td>Processing Technology</td><td>Processed quantity (L)</td><td>Processing parameters</td></tr></thead><tbody><tr><td>TT</td><td>400</td><td>Temperature: 72 °C<br>Holding time: 117 s</td></tr><tr><td>HPP</td><td>50</td><td>Applied pressure: 600 MPa<br>Holding time: 5 min</td></tr><tr><td>OH</td><td>400</td><td>Temperature: 72 °C<br>Holding time: 117 s<br>Frequency: 25 kHz<br>Field strength: 80 V/cm</td></tr><tr><td>PEF</td><td>70</td><td>Field strength: 20 kV/cm<br>Specific Energy: 100 kJ/Kg<br>Preheating Temp: 40 °C<br>Treatment time: 109 µs</td></tr></tbody></table>
Germany (Table 1). The processing conditions (Table 1) were determined to ensure equivalent processing conditions to achieve a 5-log reduction of the reference microorganism in the strawberry nectar which was an ATCC 8739 strain of E. coli. The processing conditions for TT and OH treatments were determined based on the thermal resistance parameters for the reference microorganism as determined previously (Gędas et al., 2024). The conditions for PEF and HPP were initially estimated using available literature (Mendes-Oliveira et al., 2020; Usaga et al., 2021) and then further optimised using a trial-and-error approach to achieve 5-log reduction of the reference pathogen. The processing conditions for each technology were validated for a 5-log reduction at the pilot scale using a surrogate matrix corresponding to a solution of sucrose with water which brix degree and acidity were adjusted to 12° with sucrose and 5 g/kg of citric acid to mimic nectar parameters (Gędas et al., 2024). The treated strawberry nectars were aseptically packed. A bag of untreated strawberry nectar also collected for each processing technology on the respective trial day to serve as a control for analysis. The samples were stored at -18 °C until further transport.

Temperature values were only recorded for conventional thermal processing technologies, such as thermal treatment and ohmic heating, i. e., 72 °C for 117 s (Table 1). For non-conventional (non-thermal) technologies, temperature was not determined, as these processes do not rely on heat transfer. However, for high-pressure processing (HPP), the temperature increased by only 3 °C per 100 MPa during compression. Given that the target pressure of 600 MPa was reached in approximately 100 s, the final temperature rise was limited to 18 °C, resulting in a maximum temperature of 38 °C as before strawberry nectar, was preheated to 20 °C. For pulse electric fields (PEF), the inlet temperature of the strawberry nectar was maintained at +40 °C using a coil heat exchanger.

### 2.2.2. Sample preparation and storage

All samples from the four treatments were delivered together in frozen form. An untreated nectar was used as control for each technology. Upon arrival, the samples were thawed and then gently crushed. Analyses of phenolic compounds and cell wall polysaccharides were conducted simultaneously across all treatments. For the shelf-life study, the thawed samples were stored at +4 °C in the dark and analysed at 30- and 60-day intervals. At each specific storage stage (i.e., 0-, 30-, and 60-days of storage), samples were frozen, freeze-dried, and stored at -20 °C until analysis.

### 2.2.3. Colour measurement

CIELAB parameters L* (lightness), a* (red-green), and b* (yellow-blue) were measured by a Minolta CM-5 spectrophotometer (spectrophotometric method, D65, 30 mm 10°, reflection measurement, gloss excluded, Minolta, Osaka, Japan). C* (Chroma) and h° (hue angle) were calculated (Kammerer et al., 2007).

The total colour difference ΔE was calculated and the colour change was categorised as previously published (Zia et al., 2024). Briefly, the colour differences ΔE was used in order to estimate how human eye perceives the colour difference between samples, considering 5 as the threshold above which the colour difference is considered significant and visible. ΔE was calculated for each sample between before and after treatment (i.e. untreated vs treated) and between 0-day after treatment and each specific storage stage (0-day vs 30-day and 0-day vs 60-day after storage). ΔE is defined as the arithmetic distance between the coordinates of two samples.

Acceptance Factor (AF), was used as a colour parameter in nectars as previously reported (Geossinger et al., 2009). Briefly, AF is calculated using eq. 1, and is defined as the ratio of the a*colour parameter to the hue angle (h°) (eq. 1) and is used to predict whether the colour will be accepted from the consumers or not (excellent [AF > 0.7], acceptable [AF > 0.4] or not acceptable [AF < 0.4]). AF can quantify the likelihood of consumers accepting the colour of nectar and can be used to track the degradation of nectar's colour after processing and during storage

(Murray et al., 2023). Nectar colour was measured before and after treatment, then after 30 and 60 days of storage at 4 °C.

$$AF = a^*/h \qquad (1)$$

### 2.2.4. Preparation of cell walls

Alcohol-Insoluble Solids (AIS) were prepared from strawberry freeze-dried powder according to the literature (Renard, 2005). Briefly, strawberry powder was dissolved in boiling 70% ethanol (15 mL g⁻¹). Samples were cooled and mixed overnight at 4 °C. Solids were recovered on a G3 crucible filter (pore size from 16 to 40 µm with a capacity of 50 mL and a diameter of 35 mm). This process was repeated until the filtrates were sugar-free (phenol – H₂SO₄ test, Dubois et al., 1956). AIS were washed three times with aqueous acetone (60, 80, 100%), then dried overnight in an oven at 40 °C. Experiments were carried out in triplicate with batches of 5 g of freeze-dried strawberry.

### 2.2.5. Cell wall chemical composition

#### 2.2.5.1. Hydrolysis.
In order to determine cellulosic glucose, neutral sugars included non-cellulosic glucose (NCGlc) and cellulosic glucose (CGlc) were submitted to hydrolysis with two options.

For cellulosic glucose analysis, approximately 10 mg of AIS were pre-hydrolysed with 72% aqueous H₂SO₄ (250 µL, 13 M) for 1 h at room temperature (Saeman et al., 1954), and then diluted to 1 M H₂SO₄ by the addition of water and an internal standard (inositol).

For non-cellulosic glucose analysis, no prehydrolysis was carried out, samples were dissolved in 1 M sulfuric acid in water with internal standard (inositol).

All samples, with or without prehydrolysis, were placed in oven at 100 °C for 3 h for hydrolysis.

#### 2.2.5.2. Neutral sugar composition.
Neutral sugars were converted to alditol acetates for analysis (Englyst et al., 1982). Briefly, after hydrolysis, samples were cooled to room temperature. A 1 mL aliquot of each sample was neutralized using 32% NH₄OH to achieve a pH > 9. Freshly prepared 3 M NH₄OH containing 100 mg/mL NaBH₄ was added, and the mixture was agitated and incubated for 1 h at room temperature. The reaction was quenched with 0.1 mL glacial acetic acid, vortexed.

For acetylation, 300 µL of the neutralized solution was combined with 200 µL N-methyl-imidazole and 3 mL acetic anhydride, and incubated for 30 min at room temperature. Neutral sugars were extracted into the organic phase by adding ultrapure water and 3 mL CH₂Cl₂, followed by vigorous mixing. The aqueous phase was removed, and the organic phase was washed three times. The organic phase was dried, concentrated to 0.5 mL under nitrogen, and transferred to a GC vial for analysis.

The samples were injected in a GC-FID GC-2010-Pro (Shimadzu, Kyoto, Japan) equipped with a capillary column (30 m × 0.25 mm i.d. coated with DB225 MS, 0.25 µm film thickness). Split mode (1:15 ratio); injector and detector temperatures = 250 °C and 280 °C, respectively; H₂ as carrier gas at 45 cm/s; column flow = 1.3 mL/min; oven temperature = 220 °C (isothermal).

#### 2.2.5.3. Uronic acid content.
Uronic acids were measured by the meta-hydroxyl-diphenyl assay (Blumenkrantz & Asboe-Hansen, 1973) after Saeman hydrolysis. Briefly, samples or standards (0.5 mL) were mixed with 3 mL of a 0.0125 M solution of tetraborate in concentrated sulfuric acid and incubated in a water bath at 80 °C for 10 min. The reaction was stopped by cooling the tubes in an ice-water bath. Fifty microliters of MHDPP reagent were added to samples and standards. As carbohydrates produce a pinkish chromogen with sulfuric acid/tetraborate at 100°C, a blank sample was run without addition of the reagent, which was replaced by 50 µL of N/8 NaOH. The tubes were shaken and, within 10
min, absorbance was measured at 520 nm using a UV-visible spectro-
photometer (V-530 Jasco, Tokyo, Japan), The absorbance of the blank
sample was subtracted from the total absorbance. Uronic acid contents
were calculated against a calibration curve with galacturonic acid as an
external standard (r² = 0.9976).

**2.2.5.4. Degree of methylation.** Methanol was determined in AIS by Headspace-GC-MS after saponification (Renard & Ginies, 2009). Samples of 10 mg were suspended in 4 ml of distilled water then saponified by addition of 1 mL of 1 M KOH containing CD₃OH (7 µmol mL⁻¹) as internal standard, and incubating for 2 h at room temperature. Calibration curve was prepared with methanol in the range 0.6–6 µmol mL⁻¹ with CD₃OH as internal standard at 1.4 µmol mL⁻¹ as above.

For GC we used the GC-MS (5977 Agilent, Palo Alto, USA) with capillary column Cp_wax_52cb 30 m × 0.32 mm × 0.5 µm (Varian, Inc., Palo Alto, USA) equipped with auto sampler AOC5000. The sealed vial was placed at 50 °C for 15 min and then 0.5 mL of head-space was injected in split injector (ratio 1:10). GC conditions were: helium as gas carrier at 45 cm/s, oven temperature isothermal at 40 °C. Mass detector conditions were: electronic impact ionisation mode (70 eV), temperature of source 200 °C with data collected using SIM for selected ions (m/z 31; 32; 35) at 5 scans/s. Methanol concentrations were calculated by plotting the peak area ratio (normal to deuterated forms) for ion pairs (m/z 31/35) over range of concentration ratios (CH₃OH/CD₃OH). The ion at m/z 35 is the molecular ion for CD₃OH and highly specific for this compound. The ion at m/z 31 (CD₃O⁺) was preferred to the molecular ion at m/z 32 for CH₃OH as it gave better signal/noise ratio. The degree of methylation (DM) was calculated as molar ratio of methanol to uronic acid.

**2.2.6. Extraction of phenolic compounds**

Phenolic compounds were analysed by HPLC-DAD before ('crude
extract') and after the acidic depolymerisation of proanthocyanidins in
the presence of menthofuran (furanolysis). Proanthocyanidins were thus
characterised by furanolysis to determine their subunit composition, the
average degree of polymerisation (mDP) and content.

HPLC-DAD analyses of crude methanolic extracts (not submitted to furanolysis) were performed to separatively assays and quantify mono-meric flavan-3-ols naturally present in the samples as after furanolysis, no distinction can be made between native catechins and catechins coming from the terminal units of proanthocyanidins.

Furanolysis and crude methanolic extracts were performed on the
freeze-dried strawberry powder as described in (Rincon et al., 2025). All
extractions were performed in triplicate.

**2.2.6.1. Identification of phenolic compounds by UHPLC/DAD/ESI-MS².**
The identification of phenolic compounds was done according to
(Rincon et al., 2025). HPLC coupled to ElectroSpray Ionisation Mass
Spectrometry (UHPLC/DAD/ESI-MS²) analysis was performed on an
Acquity Ultra performance LC (UPLC) apparatus from Waters (Milford,
MA, USA), equipped with a photodiode array detector coupled with a
Bruker Daltonics (Bremen, Germany) HCT ultra ion trap mass spec-
trometer with an electrospray ionisation source. Separations were ach-
ieved using a Luna Omega Polar C18 column (50 mm × 2.1 mm × 3 µm
column, Phenomenex, Torrance, USA) operated at 30 °C. The mobile
phase consisted of water/formic acid (98,2, mL/mL) (eluent A) and
acetonitrile (eluent B). The flow rate was 1 mL/min. The elution pro-
gram was as follows: 3–9% B (0–5 min); 9–16% B (5–15 min); 16–50% B
(15–45 min); 50–90% B (45–48 min); 90–90% B (48–52 min). Ten µL of
samples (“furanolysis” and “crude” extracts) were injected. For poly-
phenol characterisation, a capillary voltage of 2 kV was used in the
negative ion mode. N2 was used as drying and nebulizing gas with a flow
rate of 12 L/min. The desolvation temperature was set at 365 °C and the
nebulisation pressure at 0.4 MPa. The ion trap was operated in the
Ultrascan mode from m/z 100 to 1000. For anthocyanin

characterisation, a capillary voltage of 1.8 kV was used in the positive
ion mode under the same conditions.

The characterisation of phenolic compounds in strawberries involved a tentative identification process utilizing UV spectra, retention time, molecular weight, and MS/MS fragmentation patterns, alongside comparison with existing literature and standards (Supplementary material S1).

**2.2.6.2. Quantification of polyphenols by HPLC-DAD.** Phenolic compounds were quantified by HPLC-DAD (Shimadzu, Kyoto, Japan) and separations were achieved as described above. Analyses of phenolic compounds were carried out with or without furanolysis as described in sections 2.7 and 2.8. Separations were achieved using a Luna Omega Polar C18 column (50 mm × 2.1 mm × 3 µm column, Phenomenex, Torrance, USA) operated at 30 °C. The mobile phase consisted of water/formic acid (98,2, mL/mL) (eluent A) and acetonitrile (eluent B). The flow rate was 1 mL/min. The elution program was as follows: 3–9% B (0–5 min); 9–16% B (5–15 min); 16–50% B (15–45 min); 50–90% B (45–48 min); 90–90% B (48–52 min). 20 µL of samples (“furanolysis” and “crude” extracts) were injected.

The most prevalent compounds were quantified in the UV-visible region by chromatographic analysis conducted at wavelengths of 280 nm (flavanols), 320 nm (phenolic acids), 360 nm (flavonols), and 520 nm (anthocyanins). Individual compounds were quantified in mg/100 g of fresh weight (FW) by comparison with external standards: (+)-catechin and (−)-epicatechin for flavanols, caffeic and *p*-coumaric acid for phenolic acids, quercetin and kaempferol for flavanols, and pelargoniin-3-O-glucoside and cyanidin-3-O-glucoside for anthocyanins (r² for all phenolic compounds >0.99). The mDP of proanthocyanidins was calculated as depicted in (Rincon et al., 2025).

**2.2.7. Statistical analysis**

Data are reported as the mean ± pooled standard deviation (Box
et al., 1978). Statistical analysis was performed using two-way ANOVA
to determine statistical significance on both treatment and storage time
using the statistical software program XLSTAT package of Microsoft
Excel (Version 2023.3.1.1416).

**3. Results and discussion**

**3.1.1. Cell wall characterisation of untreated nectar**

Following freeze-drying, the dry matter content and Alcohol Insol-
uble Solids (AIS) in nectar were quantified (Table 2). The dry matter
exhibited a mean value of 12.2% (g/g FW of nectar), while the AIS yield
showed a mean of 2.9% (g/g DW). Comparable formulations of nectar
with 40% strawberry puree content reported a mean dry matter of
15.2% and an AIS yield of 3.6% (g/g DW) (Schieber et al., 2005).

Galacturonic acid was the main component in all cell walls (mean content of 277 mg/g AIS, Table 2), accounting for 56% of cell wall composition, followed by cellulosic glucose (mean content of 123 mg/g AIS). Regarding the neutral sugars, galactose (mean content of 41 mg/g AIS), xylose (mean content of 27 mg/g AIS), and arabinose (mean content of 22 mg/g AIS) predominated. Arabinose and galactose belong to the pectin side chains, while xylose, together with non-cellulosic glucose and fucose, are present in hemicelluloses (Kosmala et al., 2013). This indicates high presence of pectins, cellulose and hemi-celluloses in strawberry cell wall, in agreement with previous studies (Broxterman & Schols, 2018; Rincon et al., 2025).

High galacturonic acid content indicated predominant pectin con-
tent with an average degree of methylation of 20% (Table 2), contrasting
with the 60–70% reported in strawberries (Lefever et al., 2004; Rincon
et al., 2025) suggesting significant modification of pectin methylation
<table><caption>**Table 2**
Dry matter (g100 /g fresh weight) and Alcohol-Insoluble Solids yields (mg/g dry weight), neutral sugars, galacturonic acid, methanol mg/g of AIS) and pectin characteristics, and ANOVA results of the different treatments of strawberry nectar.</caption><thead><tr><th>Technology</th><th>Days</th><th>Dry matter</th><th>AIS</th><th>Rha</th><th>Fuc</th><th>Ara</th><th>Xyl</th><th>Man</th><th>Gal</th><th>NCGlc</th><th>CGlc</th><th>Gal A</th><th>MeOH</th><th>Total</th><th>DM (%)</th><th>Gal A/Rha</th><th>(Ara + Gal)/Rha</th><th>Ara/Gal</th></tr></thead><tbody><tr><td>HPP</td><td>0</td><td>12.5</td><td>0.35</td><td>7</td><td>34</td><td>21</td><td>30</td><td>11</td><td>41</td><td>5</td><td>142</td><td>233</td><td>9</td><td>501.</td><td>20.2</td><td>29.0.8</td><td>9.0</td><td>0.6</td></tr><tr><td>OH</td><td>0</td><td>11.9</td><td>0.35</td><td>12</td><td>5</td><td>34</td><td>37.</td><td>21</td><td>58</td><td>17</td><td>193</td><td>267</td><td>8</td><td>620</td><td>17.6</td><td>18.9</td><td>7.7</td><td>0.6</td></tr><tr><td>PEF</td><td>0</td><td>12.2</td><td>0.40</td><td>4</td><td>1</td><td>17</td><td>22</td><td>8</td><td>32</td><td>11</td><td>73</td><td>354</td><td>15</td><td>536</td><td>24.7</td><td>71.9</td><td>11.4</td><td>0.5</td></tr><tr><td>TT</td><td>0</td><td>12.1</td><td>0.37</td><td>4</td><td>2</td><td>15</td><td>20</td><td>6</td><td>29</td><td>5</td><td>78</td><td>270</td><td>8</td><td>437</td><td>15.3</td><td>52.2</td><td>9.9</td><td>0.6</td></tr><tr><td>Pooled SD</td><td></td><td>0.7</td><td>0.22</td><td>0.4</td><td>0.4</td><td>1</td><td>2</td><td>1</td><td>3</td><td>1</td><td>12</td><td>22</td><td>1</td><td>28</td><td>1.8</td><td>4.3</td><td>0.9</td><td>0.0</td></tr><tr><td>F-Value and Significance Control</td><td></td><td>0</td><td>1</td><td>79***</td><td>17***</td><td>40***</td><td>27***</td><td>47***</td><td>17***</td><td>70***</td><td>24***</td><td>6*</td><td>52***</td><td>10**</td><td>5*</td><td>3</td><td>31***</td><td>3</td></tr><tr><td>HPP</td><td>1</td><td>12.5</td><td>0.37</td><td>7</td><td>3</td><td>24</td><td>35</td><td>12</td><td>47</td><td>5</td><td>167</td><td>299</td><td>8</td><td>605</td><td>14.9</td><td>37.8</td><td>10.3</td><td>0.5</td></tr><tr><td>OH</td><td>1</td><td>12.9</td><td>0.37</td><td>12</td><td>5</td><td>34</td><td>36</td><td>21</td><td>60</td><td>18</td><td>184</td><td>319</td><td>12</td><td>701</td><td>21.0</td><td>23.2</td><td>8.0</td><td>0.6</td></tr><tr><td>PEF</td><td>1</td><td>12.1</td><td>0.39</td><td>4</td><td>23</td><td>14</td><td>23</td><td>6</td><td>29</td><td>6</td><td>85</td><td>277.</td><td>16</td><td>461</td><td>31.7</td><td>61.1</td><td>10.9</td><td>0.5</td></tr><tr><td>TT</td><td>1</td><td>14.7</td><td>0.37</td><td>13</td><td>4</td><td>33</td><td>24</td><td>9</td><td>57</td><td>14</td><td>70</td><td>384</td><td>14</td><td>620</td><td>20.2</td><td>24.9</td><td>6.8</td><td>0.6</td></tr><tr><td>Pooled SD</td><td></td><td>0.6</td><td>0.03</td><td>0.4</td><td>0.4</td><td>1.5</td><td>1.0</td><td>1</td><td>3</td><td>1</td><td>12</td><td>17</td><td>0.5</td><td>27</td><td>1.3</td><td>3.5</td><td>0.7</td><td>0.0</td></tr><tr><td>F-Value and Significance Tech*Process</td><td></td><td>2</td><td>1</td><td>38***</td><td>4***</td><td>12***</td><td>1</td><td>2</td><td>7**</td><td>44***</td><td>1</td><td>11***</td><td>27***</td><td>8**</td><td>9***</td><td>3</td><td>9**</td><td>1</td></tr><tr><td>HPP</td><td>30</td><td>11.9</td><td>0.25</td><td>12</td><td>0</td><td>19</td><td>33</td><td>0</td><td>50</td><td>2</td><td>134</td><td>332</td><td>2</td><td>584</td><td>3.3</td><td>33.3</td><td>6.7</td><td>0.5</td></tr><tr><td>OH</td><td>30</td><td>12.7</td><td>0.31</td><td>10</td><td>4</td><td>31</td><td>35</td><td>20</td><td>60</td><td>20</td><td>185</td><td>348</td><td>13</td><td>728</td><td>21.9</td><td>28.6</td><td>8.7</td><td>0.6</td></tr><tr><td>PEF</td><td>30</td><td>15.3</td><td>0.46</td><td>6</td><td>4</td><td>19</td><td>23</td><td>7</td><td>33</td><td>13</td><td>88</td><td>340</td><td>9</td><td>541</td><td>15.0</td><td>50.4</td><td>8.3</td><td>0.6</td></tr><tr><td>TT</td><td>30</td><td>12.5</td><td>0.40</td><td>5</td><td>1</td><td>13</td><td>17</td><td>5</td><td>29</td><td>10</td><td>77</td><td>369</td><td>12</td><td>540</td><td>19.9</td><td>61.8</td><td>8.5</td><td>0.5</td></tr><tr><td>HPP</td><td>60</td><td>12.1</td><td>0.35</td><td>7</td><td>4</td><td>24</td><td>29</td><td>9</td><td>39</td><td>13</td><td>92</td><td>282</td><td>2</td><td>499</td><td>3.6</td><td>33.6</td><td>8.3</td><td>0.6</td></tr><tr><td>OH</td><td>60</td><td>12.2</td><td>0.38</td><td>4</td><td>1</td><td>12</td><td>16</td><td>8</td><td>31</td><td>11</td><td>108</td><td>375</td><td>13</td><td>579</td><td>19.3</td><td>82.2</td><td>10.8</td><td>0.4</td></tr><tr><td>PEF</td><td>60</td><td>16.3</td><td>0.47</td><td>6</td><td>3</td><td>19</td><td>20</td><td>12</td><td>36</td><td>10</td><td>80</td><td>299</td><td>4</td><td>488</td><td>6.8</td><td>43.8</td><td>9.3</td><td>0.6</td></tr><tr><td>TT</td><td>60</td><td>14.7</td><td>0.43</td><td>7</td><td>3</td><td>22</td><td>27</td><td>7</td><td>33</td><td>11</td><td>98</td><td>345</td><td>13</td><td>565</td><td>20.4</td><td>37.6</td><td>7.0</td><td>0.7</td></tr><tr><td>Pooled SD</td><td></td><td>0.7</td><td>0.03</td><td>1</td><td>1</td><td>2</td><td>2</td><td>1</td><td>5</td><td>1</td><td>12</td><td>14</td><td>0.3</td><td>25</td><td>0.7</td><td>6.3</td><td>0.8</td><td>0.5</td></tr><tr><td>F-Value and Significance Tech*Storage</td><td></td><td>4**</td><td>4**</td><td>7***</td><td>12***</td><td>17***</td><td>10***</td><td>21***</td><td>5**</td><td>26***</td><td>5**</td><td>3*</td><td>81***</td><td>4*</td><td>96***</td><td>4**</td><td>15***</td><td>3*</td></tr></tbody></table>

TT: Thermal Treatment, HPP: High-Pressure Processing, OH: Ohmic Heating, PEF: Pulse Electric Field, AIS: Alcohol Insoluble Solids, Rha: rhamnose, Fuc: fucose, Ara: arabinose, Xyl: xylose, Man: mannose, Gal: galactose, Glc: glucose, GalU: Galacturonic acid, CGlc: glucose from cellulose, NCGlc: non-cellulosic glucose, MeOH: methanol, DM: degree of methylation. Ratios are calculated using the yields of neutral sugar and expressed in mol %. The ratio between different sugars can contribute to understand information on polymer nature. The ratios of GalU/Rha, (Ara + Gal)/Rha and Ara/Gal are characteristic for pectin backbone homogalacturonan vs rhamnogalacturonan contribution, the branching of RG-I and the proportion of arabinose vs galactose, respectively. Pooled SD: pooled standard deviation (degree of freedom: 28). Fisher's value, $p \le 0.05: *, p \le 0.01: **, p \le 0.001: ***$. Processing values are presented relative to the control, while storage values are relative to the processed nectar.
degree during processing into puree and nectar. The pectin linearity (GalU/Rha ratio) averaged 43.3, and the rhamnogalacturonan-I branching ((Gal + Ara)/Rha ratio) was 9.5. The side chains' arabinose and galactose proportions (Ara/Gal ratio) averaged 0.6. These ratios suggested that untreated strawberry nectar pectins were linear with side chain of galactose predominance, characteristics expected after processes such as thawing, mashing, and mixing, compared to the more branched arabinose-rich pectins found in unprocessed fruit (Rincon et al., 2025).

Significant differences were observed in the cell wall composition and characteristics of the untreated strawberry nectars depending on the processing conditions. These differences could be linked to changes occurring during transportation and thawing before treatment, influenced by the processing capacity of each device, as shown in Table 1. Notably, the time required to thaw 400 L of puree for TT or OH treatment was considerably longer than the time needed for 50 or 70 L in HPP and PEF, potentially leading to the reactivation or enhancement of enzymatic activity.

### 3.1.2. Effects of processing on cell wall

The impact of processing on strawberry nectar cell wall was evaluated in comparison to untreated nectar, which was previously characterised for each technology tested in this study.

#### 3.1.2.1. Dry matter content and cell wall yields.
Across all treatments, no significant changes in AIS yields were observed, however, TT treatment led to an increase of the dry matter by 21% (Table 2). Thermal processing could induce water evaporation due to elevated temperatures, leading to a relative increase in the dry matter content of the product. Additionally, thermal treatments may promote structural changes in the food matrix, such as cell wall disruption, which could further enhance water loss and contribute to the observed increase in dry matter even if it was not significant.

#### 3.1.2.2. Cell wall composition.
Thermal treatment (TT) significantly increased galacturonic acid content by 42% (*p* < 0.0001; Table 2). OH and HPP showed non-significant increases of 20 and 28%, respectively, whilst PEF led to a galacturonic acid decrease of 22% (Table 2). Unexpectedly, TT led to significant increases in total content of neutral sugars, specially rhamnose and arabinose. These changes could additionally be linked to the observed rise in dry matter. However, these results contrasts with the expected effects from processing, characterised by the reduction of pectin's neutral side chains composed of arabinose and galactose, and increase in hemicellulosic and cellulosic material (xylose and cellulosic glucose) (Fraeye et al., 2007; Liu et al., 2020). Effects from TT may be related to a higher disruption of cells, following a detachment of pectin middle lamella from cellulose fibers, as reported previously after TT (70 °C – 40 min) (Fraeye et al., 2010). Similar behaviour was observed in different pectic fractions from carrot treated by TT (100 °C – up to 120 min), with marked increases in rhamnose, arabinose and galactose (Sila et al., 2006). The opposite results observed after PEF treatment, may be due to its distinct electroporation mechanism, which could have enhanced release and contact of substrates with enzymes, such as PME and PG, which were not completely inactivated after processing (Icier, 2012).

The DM increased in a significant way only after PEF processing from 24.7% to 31.7% (Table 2). On the contrary, HPP resulted in a decrease from 20.2% to 14.9% (Table 2). Pectin methylation degree is mostly controlled by the enzymatic activity of PME (Broxterman & Schols, 2018), which is known to be thermic and pressure-stable (Ly-Nguyen et al., 2002). While comprehensive data on the effects of pressure and temperature on strawberry's PME are limited, studies in model systems have shown that PME activity is the highest at pressures between 200 and 300 MPa and temperatures of 50–55 °C (Fraeye et al., 2007). Thus, the decrease in the DM after HPP is likely due to the PME activity which

was not completely inactivated after processing or activated depending on HPP conditions (Chakraborty et al., 2014). This could also be connected to the enhanced extraction of pectin fractions with lower methylation degree or a decrease in the total content of galacturonic acid due to PG activity following PME catalysis (Brummell & Harpster, 2001). The reduction in galacturonic acid might be attributed to elevated temperatures and mildly acidic pH (around 3.4), conditions that could lead to acid hydrolysis, especially in low-methylated pectins (<20% average) (Fraeye et al., 2007). None of the technologies showed a significant effect in the molar ratios representing the linear nature of pectin in the cell wall and its side chains composition.

### 3.1.3. Effect of storage on cell wall

#### 3.1.3.1. Dry matter and cell wall yields.
No significant differences on the dry matter content depending on storage time were observed (Table 2). However, storage led to a reduction of AIS content which could be attributed to enzyme reactivation especially for HPP treatment (Chakraborty et al., 2014).

#### 3.1.3.2. Cell wall composition.
The impact of storage on strawberry nectar cell wall composition was evaluated after 30 and 60 days (Table 2). While a minimal but significant effect (*p* < 0.05) was observed in galacturonic acid content after 60 days, this effect technology-dependent. OH and PEF treatments resulted in an increase in GalA, whereas TT and HPP treatment led to a decrease. Regarding neutral sugars composition, samples treated with OH exhibited a significant decrease (*p* < 0.001) during storage, especially in rhamnose and fucose content, with 66% and 82% decrease respectively after 60 days (Table 2).

The linearity of pectin, expressed as GalU/Rha ratio, exhibited significant differences after OH, shifting in ratio from 23 to 80 over 60 days. This change was consistent with the previously observed marked reduction in neutral sugars (Table 2). In contrast, GalU/Rha and Ara/Gal ratios from PEF samples exhibited significant changes after storage. The GalU/Rha ratio decreased from 61 to 20, indicating a shift towards less linear pectins. In contrast, the Ara/Gal ratio increased from 0.6 to 3.5, indicating a substantial rise in arabinose content within the lateral chains, suggesting a higher proportion of arabinan relative to galactan (Table 2).

Marked reductions after OH treatment could be related to the mechanisms behind this technology, i.e. both thermal and non-thermal effects. This hybrid effect is based on the temperature rise in the product due to its electrical resistance, in addition to the mild cell electroporation due to the applied electric current (Shao et al., 2021). Moreover, it has been reported that enzyme activity can be modulated by processing conditions, i.e. the electric field strength, frequency and temperature used in OH treatments (Samaranayake & Sastry, 2016). The authors show an increase of even 23% in PG activity of tomato homogenate. Hence, specific conditions could enhance the degradation of cell wall in strawberry, which is primary attributed to pectin depolymerisation and solubilisation after rhamnogalacturonan I side chain losses, i.e. galactan and arabinan side chain, through β-galactanase and α-arabinofuranosidase activities (Ramos et al., 2018; Rosli et al., 2004). Thus, enzymatic activity may fluctuates depending on the thermal and non-thermal conditions of OH, probably enhanced by the major release and contact of substrates with activated enzymes if temperature conditions are not optimal (Icier, 2012).

Following TT and OH mechanisms, it seems that the “thermal” mechanisms led to a major effect towards cell wall integrity, as it has been observed previously in other fruits as plum (Kosmala et al., 2013), bilberry and black currant pulp and juice (Hilz et al., 2005) and apple juice (Scaman et al., 2004). Temperature generally promotes neutral sugar losses after cleavage of their bonds in the RG-I (Einhorn-Stoll et al., 2014). Under TT, remaining enzymatic activity is not a common cause of cell wall components degradation. However, some have reported the
increase of synthesizing enzymes whose activities are higher in the latter part of storage (Scaman et al., 2004), which could explain the release of pectins rich in arabinose and galactose, leading to an increase after quantification.

Both methanol contents and DM were significantly reduced with storage after HPP and PEF treatments (p < 0.0001), whilst remaining stable in OH and TT treated nectars (Table 2). DM decreased from 14.9% to 3.6% and from 31.7% to 6.8% after storage for HPP and PEF treatment respectively. For OH and TT, DM were closed to 20%. As mentioned before, PME catalyses the de-esterification of pectins and is highly pressure resistant (Ly-Nguyen et al., 2002), explaining the marked decrease in HPP and the much higher DM stability in OH and TT. This may indicate a resistance to inactivation through PEF electroporation mechanisms. In addition, PEF, due to membrane electroporation, may have favoured binding between PME and pectins allowing their de-esterification, explaining the DM decrease.

### 3.1.4. Characterisation of phenolic compounds in untreated nectar

Four major phenolic groups, i.e., phenolic acids, flavan-3-ols (catechin monomers and proanthocyanidins), flavonols and anthocyanins, with a total of twelve individual compounds, were identified and quantified in strawberry nectars (Table 3).

Proanthocyanidins (PAC) were the main phenolic compounds in nectar composed of 40% fruit, with an average content of 25.0 mg/100 g FW (Table 3). In other works, PACs were quantified in fresh strawberry juice with concentrations ranging between 21.2 and 43.5 mg/100 mL FW after multiple thermal steps (thawing, thermomix, pasteurisation) (Teleszko et al., 2016). PAC content ranged between 15.9 and 23.5 mg/100 mg FW (Oszmiański & Wojdyło, 2009) in diluted purée (50:50) after 5 min at 90 °C.

PAC mean degree of polymerisation (mDP) was 9.1 and they were mainly composed of (–)-epicatechin (56% of total units), both as extension and terminal units, followed by (+)-catechin (20%) and (–)-epiafzelechin (13%) as extension units. As previously reported, terminal units were only composed of (+)-catechin (11%). This percentage was slightly higher than the one previously found in fresh strawberries (7.4%) (Rincon et al., 2025), probably due to the variety and processing (Buendía et al., 2010; Salazar-Orbea et al., 2023).

PAC were followed by anthocyanins (average content 11.2 mg/100 g FW), phenolic acids (1.7 mg/100 g FW), flavonols (1.6 mg/100 g FW) and finally flavan-3-ol monomers (1.2 mg/100 g FW). These contents were within the usual range in strawberry (Oszmiański & Wojdyło, 2009) but in contrast with other reports (Aaby et al., 2012; Määttä-Riihinen et al., 2004). Differences could be attributed to extraction methods, which often underestimate total PACs (Kajdžanoska et al., 2011), but also to the varieties used.

Among, anthocyanins, pelargonidin-3-O-glucoside (79.8%), pelargonidin-3-O-6-malonylglucoside (11.2%) and pelargonidin-3-O-rutinoside (6.2%) were predominant. Minor anthocyanins included cyanidin-3-O-glucoside (1.8%) and cyanidin-3-O-malonylglucoside (0.3%). Despite the prevalence of different kaempferol derivatives, quercetin-3-O-glucuronide was the main flavonol, accounting for 68.5%. The principal phenolic acid was *p*-coumaroyl hexose. All these components were identified as predominant in fresh strawberries and purees (Salazar-Orbea et al., 2023).

Significant differences were identified among the controls of each technology tested concerning the content of pelargonidin-3-O-glucoside, quercetin-3-O-glucuronide, total flavonols and flavan-3-ol monomer (Table 3). Moreover, PAC structure was also different among controls (Table 4) with a higher mDP with a concomitant modification of constitutive units for PEF nectar than other samples. The different freezing and thawing times depending on the puree volumes implemented in each technology (Table 1) could led to differences in phenolic compound availability and stability. Freezing-thawing steps disrupt cells leading to membrane degradation, thus affecting the extractability of phenolic compounds like anthocyanins (Tomaz et al., 2018) and enzyme

activity leading to phenolic compound degradation. In general, PPO is the main enzyme responsible for this oxidative mechanism, with catechin as a primary substrate, which would explain the significant differences observed in flavan-3-ol monomer contents. Moreover, PPO could also be involved in anthocyanin degradation by a coupled oxidation-reduction reaction involving also hydroxycinnamic acids, (Cheynier et al., 1988; Cheynier & Ricardo da Silva, 1991; Sarni-Manchado et al., 1997; Kader et al., 1997, 2006).

### 3.1.5. Effects of processing on phenolic compounds

#### 3.1.5.1. Flavan-3-ols.
Flavan-3-ols were composed of (+)-catechin (as a monomer) and PAC (Table 3). Nor (+)-catechin nor PAC contents were modified after processing. However, OH and TT led to a significant increase (p < 0.05) on (+)-catechin as extension unit with concomitant decrease of epiafzelechin, which was significant (p < 0.05) only after TT (Table 4).

#### 3.1.5.2. Anthocyanins.
The treated nectar exhibited total anthocyanin contents between 10.3 (OH) and 12.3 (HPP) mg/100 g FW (Table 3). This agreed with data from Teleszko et al., 2016 in cloudy juices (90 °C–2 min), but were slightly lower than content reported by Holzwarth et al., 2012, after treating strawberry puree at 60, 75, and 90 °C for a holding time of 3 min.

Unlike our observations, where no significant changes in total or individual anthocyanin contents were detected across processing technologies (Table 3), published studies report conflicting outcomes. Some authors observed increases in anthocyanin levels following HPP, TT, and PEF treatments (Stübler et al., 2020; Yildiz et al., 2021b), with gains of up to 15%. Conversely, other studies documented significant reductions, particularly after thermal treatments (e.g., 25% loss at 95 °C for 1 min, Teribia et al., 2021; 43% loss at 90 °C for 15 min, Marszałek et al., 2017). A third group of findings aligns with our results, showing no significant impact of HPP (100–400 MPa) on anthocyanin content (Bodelón et al. (2013). Additionally, the effects of PEF appear highly parameter-dependent: Odriozola-Serrano et al., 2008 reported both decreases and increases in anthocyanins depending on pulse duration and frequency. These discrepancies underscore the influence of specific processing conditions and matrix characteristics on anthocyanin stability.

The differences observed between our study and others could be due to food matrix specificity, i.e. the presence of copigments, such as phenolic acids and flavonols, known to modulate the stability of anthocyanins through non-covalent interactions, mainly π-stacking interactions that protect flavylium cation from nucleophilic attack from water (Dangles, 2024). Polysaccharides through hydrophobic contacts (π-π stacking and van der Waals forces) can also protect anthocyanins but with far less effective way. However, cell wall encapsulation is a promising effect, attributed to the macromolecular properties of pectins (Gençdağ et al., 2022; Oszmiański & Wojdyło, 2009). These effects are modulated by cultivars as their copigment content (Aaby et al., 2012; Buendía et al., 2010) and cell wall components (Lefever et al., 2004) widely varies.

#### 3.1.5.3. Flavonols.
No significant effects from processing were observed on flavonols, regarding the technology (Table 3). Nevertheless, HPP led to a 10% increase in flavonols, whereas the other three processes led to a significant decrease in their contents, especially after OH (–8%). Salazar-Orbea et al., 2023 reported a 59% decrease in total flavonols after TT on strawberry purée (90 °C–30 s). Flavonol losses after processing in strawberry products could be due to oxidative reactions (Häkkinen et al., 2000). In contrast, other works reported an increase in flavonol contents in nectars after TT, PEF and HPP (Stübler et al., 2020). High flavonol stability has been reported in strawberry juices treated with TT (90 °C–30 and 60 s) (Odriozola-Serrano et al., 2008). Differences might be related to processing, as blanching, pectinase treatment and pressing
<table><caption>**Table 3**
Phenolic contents (mg/100 g FW) and ANOVA results depending on processing technology and storage time.</caption><thead><tr><th>Technology</th><th>Days</th><th>Catechin</th><th>PAC</th><th>pCoum Hex</th><th>Kpf3Hex</th><th>Q 3GlcU</th><th>Kpf 3Coum Hex</th><th>Kpf 3GlcU</th><th>Kpf 3MalGlc</th><th>Flvs</th><th>Cy3 Glc</th><th>Cy3Mal Glc</th><th>Plg3 Glc</th><th>Plg3 Rut</th><th>Plg3MalGlc</th><th>ACNs</th></tr></thead><tbody><tr><td>TT</td><td>0</td><td>1.09</td><td>24.9</td><td>1.64</td><td>0.20</td><td>0.93</td><td>0.00</td><td>0.23</td><td>0.09</td><td>1.46</td><td>0.21</td><td>0.03</td><td>8.60</td><td>0.81</td><td>1.33</td><td>10.97</td></tr><tr><td>HPP</td><td>0</td><td>1.01</td><td>23.6</td><td>1.61</td><td>0.19</td><td>0.84</td><td>0.01</td><td>0.23</td><td>0.09</td><td>1.35</td><td>0.24</td><td>0.03</td><td>9.19</td><td>0.82</td><td>1.40</td><td>11.67</td></tr><tr><td>OH</td><td>0</td><td>1.87</td><td>25.0</td><td>1.64</td><td>0.19</td><td>1.72</td><td>0.00</td><td>0.12</td><td>0.05</td><td>2.08</td><td>0.13</td><td>0.00</td><td>7.76</td><td>0.59</td><td>0.80</td><td>9.28</td></tr><tr><td>PEF</td><td>0</td><td>1.41</td><td>26.7</td><td>1.80</td><td>0.21</td><td>0.90</td><td>0.00</td><td>0.26</td><td>0.10</td><td>1.48</td><td>0.24</td><td>0.07</td><td>10.01</td><td>0.90</td><td>1.55</td><td>12.78</td></tr><tr><td>Pooled SD</td><td></td><td>0.07</td><td>2.4</td><td>0.11</td><td>0.01</td><td>0.08</td><td>0.00</td><td>0.03</td><td>0.01</td><td>0.12</td><td>0.04</td><td>0.02</td><td>0.58</td><td>0.05</td><td>0.23</td><td>0.80</td></tr><tr><td>F-Value and Significance Control</td><td></td><td>30***</td><td>0</td><td>0</td><td>1</td><td>29***</td><td>1</td><td>3</td><td>3</td><td>8**</td><td>2</td><td>4</td><td>3*</td><td>7</td><td>2</td><td>3</td></tr><tr><td>TT</td><td>1</td><td>1.11</td><td>22.8</td><td>1.88</td><td>0.20</td><td>0.90</td><td>0.00</td><td>0.23</td><td>0.09</td><td>1.43</td><td>0.22</td><td>0.00</td><td>9.15</td><td>0.80</td><td>1.31</td><td>11.49</td></tr><tr><td>HPP</td><td>1</td><td>1.09</td><td>24.4</td><td>1.71</td><td>0.21</td><td>0.92</td><td>0.01</td><td>0.25</td><td>0.10</td><td>1.49</td><td>0.17</td><td>0.02</td><td>9.82</td><td>0.90</td><td>1.42</td><td>12.33</td></tr><tr><td>OH</td><td>1</td><td>1.97</td><td>24.1</td><td>1.90</td><td>0.20</td><td>1.53</td><td>0.01</td><td>0.13</td><td>0.05</td><td>1.91</td><td>0.00</td><td>0.00</td><td>8.37</td><td>0.43</td><td>1.45</td><td>10.25</td></tr><tr><td>PEF</td><td>1</td><td>1.37</td><td>25.2</td><td>1.78</td><td>0.21</td><td>0.89</td><td>0.00</td><td>0.24</td><td>0.10</td><td>1.45</td><td>0.00</td><td>0.04</td><td>9.62</td><td>0.55</td><td>1.49</td><td>11.71</td></tr><tr><td>Pooled SD</td><td></td><td>0.08</td><td>1.7</td><td>0.15</td><td>0.02</td><td>0.08</td><td>0.00</td><td>0.04</td><td>0.01</td><td>0.13</td><td>0.04</td><td>0.01</td><td>0.75</td><td>0.14</td><td>0.21</td><td>0.97</td></tr><tr><td>F-Value and Significance Tech*Process</td><td></td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>1</td><td>1</td><td>0</td></tr><tr><td>TT</td><td>30</td><td>1.12</td><td>20.3</td><td>2.02</td><td>0.21</td><td>1.19</td><td>0.00</td><td>0.27</td><td>0.10</td><td>1.78</td><td>0.00</td><td>0.03</td><td>8.69</td><td>0.80</td><td>1.23</td><td>10.75</td></tr><tr><td>HPP</td><td>30</td><td>0.66</td><td>23.3</td><td>1.32</td><td>0.17</td><td>1.14</td><td>0.00</td><td>0.23</td><td>0.08</td><td>1.63</td><td>0.04</td><td>0.01</td><td>6.35</td><td>0.58</td><td>0.85</td><td>7.83</td></tr><tr><td>OH</td><td>30</td><td>1.55</td><td>19.7</td><td>1.88</td><td>0.19</td><td>1.52</td><td>0.06</td><td>0.14</td><td>0.07</td><td>1.98</td><td>0.11</td><td>0.00</td><td>6.61</td><td>0.56</td><td>0.95</td><td>8.23</td></tr><tr><td>PEF</td><td>30</td><td>0.66</td><td>22.1</td><td>1.47</td><td>0.17</td><td>0.86</td><td>0.00</td><td>0.21</td><td>0.08</td><td>1.33</td><td>0.00</td><td>0.03</td><td>6.68</td><td>0.62</td><td>0.99</td><td>8.31</td></tr><tr><td>TT</td><td>60</td><td>1.13</td><td>23.7</td><td>1.87</td><td>0.22</td><td>1.22</td><td>0.00</td><td>0.25</td><td>0.10</td><td>1.79</td><td>0.00</td><td>0.02</td><td>7.30</td><td>0.69</td><td>1.02</td><td>9.03</td></tr><tr><td>HPP</td><td>60</td><td>0.78</td><td>20.3</td><td>1.36</td><td>0.20</td><td>1.33</td><td>0.00</td><td>0.27</td><td>0.09</td><td>1.89</td><td>0.00</td><td>0.02</td><td>6.39</td><td>0.63</td><td>0.79</td><td>7.82</td></tr><tr><td>OH</td><td>60</td><td>1.14</td><td>22.3</td><td>1.72</td><td>0.17</td><td>1.65</td><td>0.00</td><td>0.13</td><td>0.07</td><td>2.02</td><td>0.09</td><td>0.00</td><td>5.69</td><td>0.32</td><td>0.50</td><td>6.61</td></tr><tr><td>PEF</td><td>60</td><td>0.94</td><td>25.7</td><td>1.58</td><td>0.21</td><td>1.40</td><td>0.00</td><td>0.25</td><td>0.08</td><td>1.95</td><td>0.00</td><td>0.00</td><td>5.46</td><td>0.66</td><td>0.71</td><td>6.83</td></tr><tr><td>Pooled SD</td><td></td><td>0.10</td><td>1.3</td><td>0.15</td><td>0.02</td><td>0.11</td><td>0.02</td><td>0.04</td><td>0.01</td><td>0.16</td><td>0.04</td><td>0.02</td><td>0.71</td><td>0.13</td><td>0.18</td><td>0.91</td></tr><tr><td>F-Value and Significance Tech*Storage</td><td></td><td>5**</td><td>2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>1</td><td>1</td><td>5**</td><td>1</td><td>1</td><td>1</td><td>1</td><td>19***</td></tr></tbody></table>

TT: Thermal Treatment, HPP: High-Pressure Processing, OH: Ohmic Heating, PEF: Pulse Electric Field, PAC: Proanthocyanidins, pCoumHex: p-Coumaroylhexose, Kpf3Hex: Kaempferol-3-O-Hexose, Q3GlcU: Quercetin-3-O-glucuronide, Kpf3CoumHex: Kaempferol-3-O-p-coumaroylhexoside, Kpf3GlcU: Kaempferol-3-O-glucuronide, Kpf3MalGlc: Kaempferol-3-O-malonylglucoside, Cy3Glc: Cyanidin-3-O-glucoside, Cy3MalGlc: Cyanidin-3-O-malonylglucoside, Plg3Glc: Pelargonidin-3-O-glucoside, Plg3Rut: Pelargonidin-3-O-rutinoside, Plg3MalGlc: Pelargonidin-3-O-malonylglucoside, Pooled SD: pooled standard deviation (degree of freedom: 32). Fisher's value, $p \le 0.05$: *, $p \le 0.01$: **, $p \le 0.001$: ***. Processing values are presented relative to the control, while storage values are relative to the processed nectar.
<caption>**Table 4**</caption>

Mean degree of polymerisation and proportion of constitutive units (%) of strawberry proanthocyanidins after furanolysis in strawberry nectar and ANOVA results depending on processing technology and storage time.

<table><thead><tr><th rowspan="2">Technology</th><th rowspan="2">Days</th><th rowspan="2">DP</th><th>Terminal unit</th><th colspan="3">Extension unit</th></tr><tr><th>(+)-Catechin</th><th>(+)-Catechin</th><th>(-)-Epicatechin</th><th>(-)-Epiafzelechin</th></tr></thead><tbody><tr><td>TT</td><td>0</td><td>9.6</td><td>10.4</td><td>19.2</td><td>56.6</td><td>13.8</td></tr><tr><td>HPP</td><td>0</td><td>8.8</td><td>11.4</td><td>19.3</td><td>56.3</td><td>13.0</td></tr><tr><td>OH</td><td>0</td><td>7.7</td><td>13.1</td><td>21.2</td><td>53.6</td><td>12.2</td></tr><tr><td>PEF</td><td>0</td><td>10.4</td><td>9.6</td><td>20.0</td><td>58.7</td><td>11.7</td></tr><tr><td>Pooled SD</td><td></td><td>0.1</td><td>0.2</td><td>0.1</td><td>0.3</td><td>0.1</td></tr><tr><td>F-Value and Significance</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Control</strong></td><td></td><td>79***</td><td>69***</td><td>46***</td><td>40***</td><td>45***</td></tr><tr><td>TT</td><td>1</td><td>8.7</td><td>11.7</td><td>19.9</td><td>55.9</td><td>12.5</td></tr><tr><td>HPP</td><td>1</td><td>9.4</td><td>10.7</td><td>19.4</td><td>56.6</td><td>13.4</td></tr><tr><td>OH</td><td>1</td><td>8.4</td><td>12.0</td><td>22.0</td><td>54.2</td><td>11.9</td></tr><tr><td>PEF</td><td>1</td><td>10.1</td><td>9.9</td><td>19.9</td><td>58.3</td><td>11.9</td></tr><tr><td>Pooled SD</td><td></td><td>0.4</td><td>0.5</td><td>0.2</td><td>0.5</td><td>0.2</td></tr><tr><td>F-Value and Significance</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Tech * Process</strong></td><td></td><td>2</td><td>2</td><td>4*</td><td>1</td><td>6**</td></tr><tr><td>TT</td><td>30</td><td>10.8</td><td>9.3</td><td>19.0</td><td>55.4</td><td>16.3</td></tr><tr><td>HPP</td><td>30</td><td>11.2</td><td>8.9</td><td>17.5</td><td>55.2</td><td>18.4</td></tr><tr><td>OH</td><td>30</td><td>8.6</td><td>11.7</td><td>21.8</td><td>52.7</td><td>13.8</td></tr><tr><td>PEF</td><td>30</td><td>11.2</td><td>9.0</td><td>19.0</td><td>57.6</td><td>14.4</td></tr><tr><td>TT</td><td>60</td><td>10.2</td><td>9.9</td><td>17.9</td><td>52.0</td><td>20.3</td></tr><tr><td>HPP</td><td>60</td><td>13.8</td><td>7.4</td><td>16.5</td><td>53.7</td><td>22.5</td></tr><tr><td>OH</td><td>60</td><td>9.8</td><td>10.2</td><td>19.7</td><td>51.2</td><td>18.8</td></tr><tr><td>PEF</td><td>60</td><td>10.5</td><td>9.8</td><td>18.5</td><td>55.1</td><td>16.6</td></tr><tr><td>Pooled SD</td><td></td><td>0.7</td><td>0.6</td><td>0.2</td><td>0.7</td><td>0.6</td></tr><tr><td>F-Value and Significance</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Tech * Storage</strong></td><td></td><td>2</td><td>2</td><td>10***</td><td>1</td><td>6***</td></tr></tbody></table>

TT: Thermal Treatment, HPP: High-Pressure Processing, OH: Ohmic Heating, PEF: Pulse Electric Field. DP: mean degree of polymerisation of proanthocyanidins. Pooled SD: pooled standard deviation (degree of freedom: 32). Fisher's value, $p \le 0.05$: *, $p \le 0.01$: **, $p \le 0.001$: ***, Processing values are presented relative to the control, while storage values are relative to the processed nectar.

were included in juice production, possibly affecting the extractability of phenolic compounds. In our processes, no additional thermal step was included, and pressing was not necessary with nectar, which might explain the minor losses compared with the literature.

3.1.5.4. *p-Coumaroyl hexose.* Processing did not show a significant effect on the content of *p*-coumaroyl hexose (Table 3). In contrast with Odriozola-Serrano et al. (2008) who observed decreased after HIPEF and TT (90 °C–30 and 60 s). Likewise, Salazar-Orbea et al. (2023) reported a 15% decrease in hydroxycinnamic acids (*p*-coumaroyl and feruloyl hexosides) after TT treatment (90 °C–30 s): Differences on these studies could be due to higher temperatures used during TT and the use of HIPEF instead of conventional PEF.

### 3.1.6. Effect of storage on phenolic compounds

3.1.6.1. *Flavan-3-ols.* Regardless of technology, storage decreased proanthocyanidin contents after 60 days without significant effects (Table 3). Nevertheless, storage significantly increased proanthocyanidin mDP on HPP treated nectars (Table 4) probably due to a degradation of short-chain structure. Furthermore, their composition underwent significant modifications, especially HPP (Table 4). Hence, storage led to a significant increase ($p < 0.0001$) on (–)-epiafzelechin as extension unit in all technologies, with a maximum increase of 68% after HPP and a concomitant decrease on (+)-catechin as extension units during storage. For (–)-epicatechin as extension unit, a significant decrease was only observed after TT. Moreover, storage led to a decrease in (+) catechin as terminal unit after HPP treatment which could be linked with proanthocyanidin's DP increase.

The particular effect of HPP on the proanthocyanidins suggested a degradation of low molecular weight oligomers, which could be due to particular effects of high pressure as inadequate pressures that may lead

to partial enzyme inactivation or enzyme reactivation during storage (Chakraborty et al., 2014). Enzymes like PPOs catalyse the conversion of o-diphenols into o-quinones. Proanthocyanidins are not substrate of PPO (Le Bourvellec et al., 2004) however they could be oxidized by a coupled oxidoreduction reaction with catechin o-quinone (Cheynier et al., 1988; Cheynier & Ricardo da Silva, 1991; Kader et al., 1997, 2006; Salazar-Orbea et al., 2023). Moreover, (+)-catechin as extension unit decrease could be due to their hydroxylation patterns, as only catechins bear an oxidizable o-diphenol group compare to epiafzelechin and could be oxidable by this mechanism. Hence, propelargonidins were mainly resistant to storage, compare to procyanidins, probably due to their low susceptibility to oxidation mechanism.

Reports on proanthocyanidins stability after processing are ambiguous and scarce. Teleszko et al., 2016 reported an increase in proanthocyanidins after 6-months storage at 4 °C of thermal treated (90 °C–2 min) cloudy strawberry juices. Opposite results were reported in purée, and clear and cloudy juices, with a general decrease in proanthocyanidin in all products after 6 months of storage at 4 °C or 30 °C (Oszmianski & Wojdyło 2009).

3.1.6.2. *Anthocyanins.* Total anthocyanin contents significantly decrease in PEF treated nectar after 60 days with a marked loss of –42%. Not significant losses were observed after OH (–36%) and HPP (–37%) (Table 3). This is in accordance with previous studies showing that anthocyanins of strawberry purée degraded after TT and HPP, with losses up to 75 and 80% after 3 months at 4 °C (Terefe et al., 2013). The analysis of anthocyanins in strawberry purée and juice after TT (85 °C–2 min) and HPP (400–600 MPa – 1.5 or 3 min) showed minimal effects from processing, but a marked decrease after 2 months of storage at 6 °C, particularly after HPP at 400 MPa (Aaby et al., 2018). In a recent study, after 42 days at 4 °C, the anthocyanins content decreased after TT, HPP and PEF with respectively 25%, 19% and 21% less anthocyanins
compared to production day (Yildiz et al., 2021a). The effect of storage (4 °C–56 days) of PEF- and HPP-treated blueberry juice showed significant differences only for PEF with a 16% decrease in total ACN content (Barba et al., 2012).

The degradation mechanism of anthocyanins during storage, though not fully understood, could be attributed to both enzymatic and non-enzymatic reactions (Oliveira et al., 2011). The differences in anthocyanin retention depending on the different processes could be related to differing extents of inactivation of the oxidative enzymes under the processing conditions. Both enzymatic oxidation and thermal degradation of anthocyanins involve the same steps, i.e., hydrolysis of the sugar moiety with aglycone formation, conversion of the flavylium nucleus into the trans-chalcone (hydration and subsequent isomerisation steps), final breakdown of the trans-chalcone aglycone into phenolic acids and aldehydes (Patras et al., 2010). However, the sequence of events may be different. Cleavage of the glycosyl bonds may come first in the presence of hydrolytic enzymes, while being rate-limiting in their absence (Sadilova et al., 2006).

In general, residual PPO activities after processing have been suggested to cause colour deterioration in strawberry products during storage (Aaby et al., 2018; Gössinger et al., 2009; Holzwarth et al., 2012). Aaby et al., 2018 estimated that PPO activity was only reduced by 24% after TT (85 °C – 2 min) and 30% after HPP (600 MPa), supporting studies reporting the high heat and pressure resistance of strawberry PPO (Cao et al., 2011; Marszałek et al., 2016). Moreover, after 49 days, a doubling in PPO activity (suggesting reversibility in PPO inactivation) was observed in HPP purée, vs. a constant decline in TT purée (Terefe et al., 2014).

### 3.1.6.3. Flavonols.
During storage, flavonol contents increased in TT (25%), HPP (27%), and PEF (34%) samples, whereas almost no change was observed in OH samples (Table 3). Specifically, storage had a significant effect on quercetin-3-O-glucuronide, with a 58% increase after 60 days in PEF nectar, followed by 44% and 36% increases in HPP and TT nectars, respectively. By contrast, minimal changes in contents of quercetin and kaempferol derivatives were observed (independently of the matrix) after six months of storage of strawberry products (Oszmiański & Wojdyło, 2009). A 30% increase in flavonols, mainly quercetin-3-O-glucuronide, during cold storage, was also observed in strawberry products (Häkkinen et al., 2000; Teleszko et al., 2016). Similar results were reported for chokeberry juices stored at 25 °C, with quercetin derivatives increasing by 8% and 15% after 30 and 60 days of storage, respectively, and total flavonols decreasing only after 4 months of storage at ambient temperature (Wilkes et al., 2014). The increase observed could be due to the disruption of cell wall polysaccharides after both conventional and non-conventional treatments, thus allowing enhanced flavonol extraction. However, after 56 days of storage at 4 °C, strawberry juice having undergone heat treatment (90 °C–60s) or HIPEF, showed significant decrease in kaempferol with both processes, and in quercetin with HIPEF only (Odriozola-Serrano et al., 2008).

### 3.1.6.4. p-Coumaroyl hexose.
In contrast to anthocyanins, levels of p-coumaroyl hexose remained largely stable during storage (Table 3). Similar observations were made in chokeberry juices during 3 months of storage at 25 °C (Wilkes et al., 2014). Moreover, p-coumaric acid in strawberry juices was also found to be relatively stable over 56 days of storage following HIPEF, whereas degradation was observed in thermally-processed juices (90 °C–60 s) (Odriozola-Serrano et al., 2008). These changes depend on cultivar selection and type of product (Oszmiański & Wojdyło, 2009), where cloudy juice and puree showed slightly higher content in p-coumaroyl hexose after 6 months, compared with clear juice.

### 3.1.7. Colour stability during processing and storage

#### 3.1.7.1. Processing effect on colour stability.
The total colour change (ΔE), which corresponds to difference between the treated and untreated strawberry nectar, varied significantly across treatments, ranging from 0.5 for HPP to 7.2 for TT (Table 5). The colour change for HPP (ΔEp = 0.50) was significantly lower than the other processing technologies, and remained below the perceptual threshold of ΔEp = 5, indicative a visually non-significant difference (Cherif et al., 2024). Hence, among the tested processing technologies, HPP treatment resulted in the least colour change. However, no significant changes in total or individual anthocyanin contents were detected across treatments (Table 3). While anthocyanins are the primary contributors to nectar colour, our results revealed discrepancies between total colour change between the treated and the untreated strawberry nectar (CIE-Lab) and anthocyanin content (HPLC-DAD). Several factors may contribute to these discrepancies.

Anthocyanins often form complexes with copigments (e.g., flavonoids, phenolic acids) or other matrix components (e.g., polysaccharides), which stabilize their coloured forms. Processing could disrupt these interactions, leading to colour fading while anthocyanin concentration remains unchanged. Moreover, the presence of suspended particles in nectars can interfere with colorimetric measurements through turbidity or light scattering, leading to an underestimation of colour, whereas anthocyanin-specific analyses (i.e., HPLC-DAD) remains unaffected by these matrix effects. The minimal colour change observed after HPP may be linked to structural modifications in proanthocyanidins, specifically an increased proportion of (–)-epiafzelechin as an extension unit compared to other methods. This shift suggests a higher ratio of propelargonidins relative to procyanidins, a composition previously correlated with enhanced colour stability in strawberry nectar (Rincón et al., 2025).

These findings highlight the need for complementary analytical approaches to accurately assess pigment stability and to avoid inconsistency between anthocyanin and colour results.

#### 3.1.7.2. Storage effect on colour stability.
The colour change (ΔE) after

<table><caption>Table 5</caption><caption>Effect of processing and storage in the total colour change of strawberry nectars.</caption><thead><tr><th>Technology</th><th>Days</th><th>Colour change ΔE</th><th>Acceptance factor ΔF</th></tr></thead><tbody><tr><td>HPP</td><td>0</td><td>0.0</td><td>0.820</td></tr><tr><td>OH</td><td>0</td><td>0.0</td><td>0.795</td></tr><tr><td>PEF</td><td>0</td><td>0.0</td><td>0.808</td></tr><tr><td>TT</td><td>0</td><td>0.0</td><td>0.861</td></tr><tr><td>Pooled SD</td><td></td><td></td><td>0.003</td></tr><tr><td>F-Value and Significance</td><td></td><td></td><td>121***</td></tr><tr><td>HPP</td><td>1</td><td>0.5</td><td>0.846</td></tr><tr><td>OH</td><td>1</td><td>7.0</td><td>0.824</td></tr><tr><td>PEF</td><td>1</td><td>6.7</td><td>0.841</td></tr><tr><td>TT</td><td>1</td><td>7.2</td><td>0.856</td></tr><tr><td>Pooled SD</td><td></td><td>0.088</td><td>0.002</td></tr><tr><td>F-Value and Significance</td><td></td><td>7370***</td><td>163***</td></tr><tr><td>HPP</td><td>30</td><td>2.7</td><td>0.885</td></tr><tr><td>OH</td><td>30</td><td>2.2</td><td>0.876</td></tr><tr><td>PEF</td><td>30</td><td>2.8</td><td>0.883</td></tr><tr><td>TT</td><td>30</td><td>3.4</td><td>0.885</td></tr><tr><td>HPP</td><td>60</td><td>4.3</td><td>0.859</td></tr><tr><td>OH</td><td>60</td><td>3.4</td><td>0.819</td></tr><tr><td>PEF</td><td>60</td><td>5.6</td><td>0.835</td></tr><tr><td>TT</td><td>60</td><td>2.2</td><td>0.913</td></tr><tr><td>Pooled SD</td><td></td><td>0.316</td><td>0.004</td></tr><tr><td>F-Value and Significance</td><td></td><td>68***</td><td>137***</td></tr></tbody></table>

In the table, different letters in columns indicate significant differences between values (p < 0.05); Processing values are presented relative to the control, while storage values are relative to the processed nectar.
30 days (ΔEs₃₀) and 60 days (ΔEs₆₀) was measured during storage at 4 °C (Table 5) and compared to colour of treated nectar (Day 1). No significant differences after 30 days between treatments were observed indicating comparable rates of colour change during this period. Colour degradation increased after 60 days, as indicated by the high values of ΔEs₆₀ for all treatments, except for TT. After 60 days, TT samples had the lowest ΔEs₆₀, followed by OH nectar, indicating higher colour stability, in contrast to HPP and PEF which exhibited the highest ΔEs₆₀, exceeding perceptual threshold (ΔEp > 5) (Cherif et al., 2024). These findings align with those of previous studies, indicating that TT treatment is better than HPP treatment for long term colour stability in strawberry products (Aaby et al., 2018).

The inactivation of enzymes through high-pressure processing (HPP) and pulsed electric fields (PEF) involves distinct yet complementary mechanisms. HPP disrupts the native structure of enzymes by unfolding their tertiary and quaternary conformations, typically at pressures below 700 MPa. This leads to alterations in the active site, which modify enzymatic activity rather than achieving complete inactivation (Chakraborty et al., 2014). PEF, on the other hand, modifies the secondary and tertiary structural linkages of enzymes through charge, dipole, or induced dipole interactions. The extent of these modifications depends on electric field strength, pulse width, temperature, and treatment medium (Min et al., 2007). For both technologies, these mechanisms could result in partial inactivation, which may explain the colour changes observed during nectar storage.

The differential values of ΔEs₆₀ between thermal treatment/ohmic heating (TT/OH) and high-pressure processing/pulsed electric fields (HPP/PEF) were accompanied by modification in cell wall structure, further supporting their role in colour stability as previously observed (Rincon et al., 2025). Notably, ΔEs₆₀ in OH treated samples were comparable to those in TT-treated samples but significantly lower than those in PEF-treated samples. OH treatment-induced substantial changes in cell wall structure. OH-treated nectars exhibited the highest GalU/Rha ratio, suggesting a reduced contribution of the pectic hairy region. Additionally, the side chain composition shifted significantly, with the Ara/Gal ratio decreasing from 0.8 to 0.4, indicating a loss of arabinose relative to galactose. These structural modifications are particularly relevant as galactan side chains in pectins have been linked with to enhance colour stability. Their linear and flexible structure facilitates interaction with anthocyanins (Rincon et al., 2025) unlike of globular arabinins which have shown to reduce pectin affinity for phenolic compounds (Watrelot et al., 2014).

Although ΔE reflects absolute changes in CIELAB components, Gössinger et al. (2009) proposed assessing consumer colour acceptability through the acceptance factor (AF). AF has been used to evaluate effects of factors on colour preservation of strawberry nectars (Murray et al., 2023; Rincon et al., 2025). AF values exceeded the minimum of 0.4 across samples (Table 5) suggesting that colour changes did not negatively affect consumer perception, as all preservation technologies maintained the fresh-like colour of strawberry nectar expected by consumers during eight weeks of storage at 4 °C.

## 4. Conclusions

Following HPP, OH, and PEF treatments, the cell wall matrix remained unchanged, in contrast to the effects observed with TT. Conversely, storage significantly disrupted cell wall components irrespective to the technology employed, showing no specificity towards neutral sugars or galacturonic acid. Similarly, substantial modifications were noted in the DM, particularly after 60 days of storage post HPP and PEF treatments, suggesting a demethylation process resulting from partial inactivation or even reactivation of pectin methylesterase (PME).

Regarding phenolic compounds, total PAC content was influenced by storage without considerable effect from processing. However, constitutive units and mDP significantly changed due storage effects, with marked increase in (–)-epiafzelechin, and decrease in (+)-catechin as

extension units suggesting mechanisms altering PAC structure rather than total content. The levels of anthocyanins were mainly affected by storage, except for cyanidin-3-O-(6-O-malonyl) glucoside and pelargonicidin-3-O-rutinoside, which were slightly affected by processing. Thereby, the rate and extent of degradation depend on intrinsic and extrinsic factors. Firstly, anthocyanin's structure, depending on their hydroxylation and glycosylation patterns. Second, the type of product is a key during processing (purées, clear juices, cloudy juices, jams and nectars). For instance, food matrix rich in cell wall components (i.e. puree), limits phenolics interactions with oxidative / hydrolytic enzymes, and with transition metal traces susceptible to initiate autoxidation (Hartmann et al., 2008). However, purees might also encapsulate more O₂ than clear juices, a phenomenon that could mitigate protection, especially with anthocyanins (Cao et al., 2012).

Although TT and OH initially caused more colour change than PEF and HPP due to high temperatures, they allowed a long-term colour retention, potentially due to enhanced inactivation of enzymes. Moreover, TT was the only process to present less colour change during the second month of storage. According to our dataset, storage had a significant impact on both phenolic compounds and polysaccharides, especially for technologies that did not allow complete enzyme inactivation.

Based on the observed data, HPP and PEF should be recommended for short-term storage of strawberry nectar. These non-thermal technologies induce minimal initial colour changes (e.g., ΔE = 0.5 for HPP) and preserve anthocyanin content, making them ideal for products with a shorter shelf life or immediate consumption. In contrast, OH and TT could be better suited for long-term storage. Despite causing greater initial colour modifications due to thermal effects, these methods provide superior long-term colour retention, likely due to effective enzyme inactivation.

### CRediT authorship contribution statement

S. Rincon: Writing – original draft, Investigation, Formal analysis, Data curation. C. Dufour: Writing – review & editing, Supervision, Investigation, Formal analysis, Conceptualization. H. Murray: Methodology, Investigation, Conceptualization. M. Gössinger: Supervision, Resources, Methodology, Funding acquisition, Conceptualization. H. Zia: Methodology, Investigation, Conceptualization. A. Moreno: Methodology, Investigation, Conceptualization. D. Pavón-Vargas: Methodology, Investigation, Conceptualization. L. Cattani: Supervision, Resources, Methodology, Funding acquisition, Conceptualization. A. Puzović: Methodology, Investigation, Conceptualization. M. Mikulic-Petkovsek: Supervision, Resources, Methodology, Funding acquisition, Conceptualization. A. Slatnar: Validation, Supervision, Methodology, Funding acquisition, Conceptualization. H. Halbwirth: Validation, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. O. Dangles: Writing – review & editing, Supervision, Investigation, Formal analysis, Conceptualization. C. Le Bourvellec: Writing – review & editing, Validation, Supervision, Resources, Project administration, Investigation, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 956257. Recognition to all HistabJuice partners: CTCPA, ELEA, HPP Italia and CFT, that allowed the pilot scale
processing of nectars necessary for the study. Thanks to Caroline Garcia, Claire Demenitroux and Marielle Boge (IRAE, UMR408 SQPOV) for their technical help.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2026.148127.

## Data availability

Data will be made available on request.

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