The Effect of Freezing, Frozen Storage and Thawing on the Strawberry Fruit Composition

ABSTRACT

Strawberries intended for processing are usually frozen to withstand storage and distribution. However, freezing and thawing can cause the degradation of bioactive compounds. The aim of this study was to determine the optimal conditions for frozen storage and thawing to minimize any loss of nutrients prior processing. Different freezing treatments (−20°C, shock-freezing with liquid nitrogen, −80 °C) up to 6 months of storage and thawing treatments (4°C, room temperature, 37°C, microwave) were compared based on the content of organic acids, sugars, and phenolic compounds as well as the activity of peroxidase and polyphenol oxidase. During frozen storage at −20°C over 6 months, the sucrose content decreased by 59%, but only a 17% loss of ascorbic acid was observed, and no significant loss of anthocyanins was detected. However, more significant changes in composition were detected after thawing. When thawed at 4°C for 24 h, ascorbic acid content decreased by 32%, anthocyanin content by 30%, and sucrose content by 66%. Based on our study, the frozen storage caused smaller changes in the fruit’s composition than thawing. From the thawing procedures, the microwave thawing showed minimal changes in the composition of strawberries.

KEYWORDS:

• Anthocyanins
• ascorbic acid
• enzyme activity
• freezing
• phenolic compounds
• strawberry

Introduction

Strawberries are one of the most important berry fruits used for industrial food processing and are used for producing smoothies, drinks and desserts (Holzwarth et al., 2012; Sinir et al., 2018). Due to the seasonality of the supply of strawberries and the short period of shelf-life of fresh strawberries, strawberries for processing are distributed as frozen, and there is a need for large-scale production of frozen strawberries (Pukszta, 2016), but freezing and consequent thawing may affect the texture, flavor, appearance, color and nutrients, which influences their nutritional and sensorial quality (Skrede, 2019).

To date, different results have been reported on the effect of freezing on nutrients such as vitamin C, anthocyanins, and other phenolic compounds. In general, vitamin C content decreases during frozen storage and thawing mainly due to the enzymatic activity, but some losses could also be attributed to drip loss (Bulut et al., 2018; Holzwarth et al., 2012; Sahari et al., 2004). As for anthocyanins and other phenolic compounds, both an increase and a decrease in the content were reported during frozen storage (Bulut et al., 2018; Kamiloglu, 2019; Ngo et al., 2007; Oszmiański et al., 2009; Sahari et al., 2004). Additionally, a significant loss of anthocyanins was reported during thawing, but the degradation differed among the individual anthocyanins, and no effect of thawing was observed on the content of non-anthocyanin phenolic compounds (Holzwarth et al., 2012). On top of the changes in the composition, the activity of enzymes such as peroxidase and polyphenol oxidase is important, especially in strawberries intended for processing, as these enzymes are involved in the degradation of phenolic compounds, including anthocyanins (Chisari et al., 2007) and could therefore affect the color stability of the final product. Both the content of phenolic compounds and ascorbic acid content affect the nutritional quality and the color stability of the processed product.

Additionally, freezing also affects the taste of the fruit (Hancock, 2020), which is determined by the content of sugars and organic acids. Changes in the composition of sugars during frozen storage were previously reported (Castro et al., 2002; Moraga et al., 2006), mainly affecting the sucrose content and a decrease in sucrose content has also been previously reported after thawing (Skrede, 1983). However, the fruit’s acidity can also change during frozen storage, as reported by Sahari et al. (2004). All these changes can ultimately affect the overall quality of the fruit or the processed products. Therefore, it is necessary to understand these changes and provide a comprehensive study of the changes on the level of the individual compounds to understand their stability during freezing and thawing.

The aim of this study was to determine which procedure for freezing, frozen storage and thawing that has minimal effects on the composition of strawberries. The content of ascorbic acid, other organic acids, sugars, anthocyanins and other phenolic compounds was analyzed using a high-performance liquid chromatography (HPLC) and the individual phenolic compounds were identified using mass spectrometer so that the changes in the content of individual compounds could be determined. As part of the analysis, peroxidase and polyphenol oxidase activity was also measured, as these enzymes are involved in enzymatic browning in strawberries.

Materials and Methods

Plant Material and Sample Preparation

The strawberry fruit of Fragaria × ananassa Duch. (cultivar ˈCapriˈ) was used for analyses. The fruit was grown in Pesje in SE Slovenia (latitude 45°56’26’’ N, longitude 15°33’11’’ E) at an open field production plantation under an integrated production system equipped with a drip irrigation system. The everbearing cultivar ˈCapriˈ selected for this study was planted in the autumn of 2020. The fruit was collected on 28th October 2021 and the fruit was sorted, unripe and overripe fruit was eliminated, and only strawberry fruit of a similar size was used for the study. The collected fruit was frozen according to the procedures further described below on the same day.

Freezing Procedure and Storage

The strawberry fruit was frozen using four different treatments – directly to −20°C, shock-frozen using liquid nitrogen and then stored at -20°C, pre-frozen at -80°C for 24 h and then stored at -20°C and directly to -80°C. The samples were stored up to 6 months (24 weeks). For extractions, the strawberries were crushed and homogenized in mortar when still frozen to avoid any effect of thawing on the results. Only samples stored at -80°C were left for 15 min at room temperature to allow homogenization. Five replicates were prepared for each extraction and each replicate was pooled from at least five fruits.

Thawing Procedure

For the thawing treatments, fruit frozen and stored for 4 weeks at -20°C was used. Four thawing treatments were defined for this study based on a previous work by Holzwarth et al. (2012). Fruits were thawed until the center of the fruit reached the outside temperature, which was in a fridge at 4°C after 24 h, at room temperature (24 °C) after 6 h, in the oven at 37°C after 2 h. Additionally, we included also thawing in the microwave (600 W) for 5 min. For each thawing procedure, approximately 125 g of fruit was used for each replicate. For the thawing procedure, both the solid part and drip loss were mixed for the extraction, since for fruit puree used for the production of juices and nectars should be obtained from the edible part of the whole or peeled fruit without removing the juice (CODEX STAN 247–2005, 2005) Five replicates from each thawing procedure were prepared for each extraction, and each replicate was pooled from at least five fruits.

Ascorbic Acid Extraction and Determination

Ascorbic acid extraction was done using 2.5 g of the fruit sample and 5 ml of 3% metaphosphoric acid, as described by Simkova et al. (2023). Ascorbic acid content was determined using the Vanquish HPLC system (ThermoScientific, USA) with the column Rezex ROA-Organic acid H + 8% (150 mm × 7.8 mm; Phenomenex, California, USA) with 4 mM sulfuric acid in bi-distilled water (pH 2) as mobile phase at the same conditions as described by Simkova et al. (2023). The sample response was measured using UV detector at 245 nm. Sample chromatogram can be found in Supplementary Material S1: Figure S1–1. An external standard from Sigma-Aldrich (Steinheim, Germany) was used for identification and quantification of the results.

Sugars and Organic Acids Extraction and Determination

The extraction of organic acids and sugars was done using 1 g of sample and 5 ml of bi-distilled water following the method described by Simkova et al. (2023). Organic acids were analyzed using the Vanquish HPLC system (ThermoScientific, USA) with the column Rezex ROA-Organic acid H + 8% (150 mm x 7.8 mm; Phenomenex, California, USA) and 4 mM sulfuric acid in bi-distilled water (pH 2) as mobile phase coupled with UV detector measuring at 210 nm. Organic acids results were quantified using external standards for citric, malic and fumaric acids from Fluka Chemie (Buchs, Switzerland) and shikimic acid from Sigma-Aldrich (Steinheim, Germany). Sample chromatogram can be found in Supplementary material S1. Figure S1–2.

Individual sugars were analyzed using the Vanquish HPLC system (ThermoScientific, USA) with the column Rezex RCM-monosaccharide Ca + 2% (300 mm x 7.8 mm; Phenomenex, California, USA) and bi-distilled water as mobile phase coupled with A refractive index (RI) detector. All other conditions for the HPLC analysis and quantification of sugars and organic acids were the same as described by Simkova et al. (2023). Individual sugars results were quantified using external standards for fructose, glucose and sucrose (Fluka Chemie GmBH, Buchs, Switzerland). Sample chromatogram can be found in Supplementary material S1: Figure S1–3.

Phenolics Extraction and Determination

The phenolics extraction was done using 3 g of the fruit sample was extracted with 6 ml of 80% methanol acidified with formic acid (3%) followed by ultrasonic bath according to Simkova et al. Phenolic compound composition was analyzed by Dionex UltiMate 3000 HPLC (Thermo Scientific, USA) system with the column Gemini C18 (150 × 4.6 mm, 3 µm; Phenomenex, Torrance, USA). The mobile phases used in the system were 3% acetonitrile and 0.1% formic acid in bi-distilled water (v/v/v) as mobile phase A and 3% bi-distilled water and 0.1% formic acid in acetonitrile (v/v/v) as mobile phase B. The gradient of the mobile phases was 5% solvent B from 0 to 15 min, 5%-20% B from 15 to 20 min, 20%-30% B from 20 to 30 min, 30%-90% B from 30 to 35 min, 90%-100% B from 35 to 45 min and then 100%-5% solvent B from 45 to 50 min. The response of samples was measured at 280, 350 and 530 nm using the same conditions described by Simkova et al. (2023).

The phenolic compound identification was based on previous identification using mass spectrometry on the same cultivar in previous work (Simkova et al., 2023). The quantification of the phenolic compounds was done by calculation according to a corresponding external standard or structurally similar compound. The content was expressed as an equivalent of this compound.

Enzyme Activity Measurements

The enzyme extraction and assays followed the procedure described by Simkova et al. (2023) with some minor modifications in the extraction procedure described below.

Sample (1 g) was mixed in mortar with 0.5 g Polyclar and 0.5 g sand and 4 ml extraction buffer (0.01 M TRIS, 0.007 M EDTA and 0.01 M Borax). The mixture was vortexed for 30 s and then centrifuged for 10 min at 10 000 rpm at 4°C (Eppendorf Centrifuge 5810 R, Germany). The extract (400 µl) was washed through Sephadex G-25 gel columns before measurement. The measurement of the assays was performed on a Genesys 10S UV-Vis Spectrometer (Thermo-Scientific, USA), and data were collected by VISIONlite software. The peroxidase (POD) assay used o-dianisidine as a substrate and the polyphenol oxidase (PPO) assay used pyrocatechol as a substrate at the same concentrations and ratios as described by Simkova et al. (2023). The PPO assay consisted of the sample (130 μL) with 300 µL of McIlvaine buffer (0.1 M Na₂HPO₄ at pH 6.5) and 170 μL of 0.2 M pyrocatechol solution and was measured for 20 min at 410 nm. The POD assay consisted of the sample (100 μl) with 1000 µL of H₂O₂ -KPi buffer (pH 6.5) and 10 μl of 0.04 M o-dianisidine solution in methanol and was measured for 20 min at 460 nm.

The protein content was measured using the Bradford method following the protocol defined by Kruger (1994). The enzyme activity is presented as the change of absorbance per minute and protein content in the enzyme assay (A/min/mg protein).

Statistical Analysis

The data were statistically analyzed in R×644.1.2 using the package Rcmdr. The data were expressed as means ± standard error. Significant differences in the data were determined by one-way variance analysis (ANOVA) with Duncan’s tests, separately for freezing treatments, storage time and thawing treatments. A significant difference was considered at p < .05.

Results

Ascorbic Acid and Organic Acids Content

The ascorbic acid content was lower in the fresh strawberries (0.27 mg/g) than in the frozen strawberries (0.40–0.46 mg/g) (Table 1). Among the freezing treatments, strawberries stored at -80°C or strawberries pre-frozen at -80°C before storage at -20°C showed higher ascorbic acid content than strawberries frozen at -20°C and strawberries shock-frozen with liquid nitrogen. After thawing, the ascorbic content was in the range of 0.32 to 0.54 mg/g, with the highest content recorded in the strawberries thawed in the microwave. The ascorbic acid content decreased in frozen strawberries stored at -20°C for 6 months compared to 4 weeks, but the content decreased only by 17%.

Table 1. Content of ascorbic acid and other organic acids (mg/g fresh weight) in fresh, frozen and thawed strawberries.

	Freezing conditions	Storage time	Thawing conditions	Organic acid content (mg/g fresh weight)
				Ascorbic	Citric	Malic	Shikimic	Fumaric	Total
Freezing treatments	fresh fruit	−	−	0.27 ± 0.01 c	6.96 ± 0.56 b	2.97 ± 0.04 ab	0.018 ± 0.002 c	0.010 ± 0.001 b	10.2 ± 0.6 c
	−20 °C	6 months	−	0.40 ± 0.01 b	8.45 ± 0.31 a	2.86 ± 0.07 b	0.028 ± 0.001 a	0.013 ± 0.000 a	11.7 ± 0.4 b
	liquid N2 => −20 °C	6 months	−	0.42 ± 0.01 b	9.11 ± 0.23 a	3.12 ± 0.05 a	0.026 ± 0.001 ab	0.012 ± 0.000 a	12.7 ± 0.3 ab
	−80°C => −20 °C	6 months	−	0.46 ± 0.01 a	9.49 ± 0.22 a	3.17 ± 0.10 a	0.025 ± 0.001 ab	0.013 ± 0.000 a	13.1 ± 0.3 a
	−80 °C	6 months	−	0.45 ± 0.02 a	9.35 ± 0.42 a	2.86 ± 0.11 b	0.024 ± 0.001 b	0.012 ± 0.000 a	12.7 ± 0.5 ab
Storage time	fresh fruit	−	−	0.27 ± 0.01 c	6.96 ± 0.56 c	2.97 ± 0.04 b	0.018 ± 0.002 c	0.010 ± 0.001 c	10.2 ± 0.6 b
	−20°C	4 weeks	−	0.48 ± 0.02 a	10.45 ± 0.79 a	3.90 ± 0.20 a	0.035 ± 0.003 a	0.014 ± 0.001 a	14.9 ± 1.0 a
	−20°C	3 months	−	0.45 ± 0.01 a	8.79 ± 0.38 b	3.09 ± 0.05 b	0.026 ± 0.001 b	0.012 ± 0.000 bc	12.0 ± 0.4 b
	−20°C	6 months	−	0.40 ± 0.01 b	8.45 ± 0.31 bc	2.86 ± 0.07 b	0.028 ± 0.001 b	0.013 ± 0.000 ab	11.7 ± 0.4 b
Thawing treatments	−20 °C	4 weeks	−	0.48 ± 0.02 ab	10.45 ± 0.79 a	3.90 ± 0.20 a	0.035 ± 0.003 a	0.014 ± 0.001 ab	14.9 ± 1.0 a
		4 weeks	at 4°C for 24 h	0.32 ± 0.01 c	9.23 ± 0.60 a	3.28 ± 0.31 b	0.028 ± 0.003 b	0.013 ± 0.002 b	12.9 ± 0.9 a
		4 weeks	at 24°C for 6 h	0.47 ± 0.02 b	9.15 ± 0.61 a	3.10 ± 0.22 b	0.025 ± 0.002 b	0.012 ± 0.001 b	12.7 ± 0.8 a
		4 weeks	at 37°C for 2 h	0.49 ± 0.03 ab	9.50 ± 0.36 a	3.23 ± 0.15 b	0.024 ± 0.001 b	0.013 ± 0.001 b	13.3 ± 0.5 a
		4 weeks	microwave	0.54 ± 0.02 a	9.88 ± 0.14 a	3.98 ± 0.09 a	0.022 ± 0.000 b	0.017 ± 0.000 a	14.4 ± 0.2 a

Different lowercase letters indicate statistically significant differences among the different freezing treatments, storage time and thawing treatments (ANOVA, p < .05).

The total organic acid content was detected lower in the fresh fruit than in the frozen fruit (Table 1). The content decreased during storage for 3 months at -20°C by 20% when compared to the content after 4 weeks of storage, but the content did not decrease significantly after the 3-month storage. The freezing treatments showed comparable total organic acids content between 11.7–13.1 mg/g with only small differences. After thawing, no significant difference was detected among the treatments.

The citric acid showed a lower content in fresh fruit than in the fruit frozen for 4 weeks at -20°C. The citric acid content was comparable between the different freezing treatments as well as between the thawing treatments. The malic acid content did not show many significant differences between the treatments. Among the freezing treatments, the malic acid showed a slightly higher content when shock-frozen with liquid nitrogen and when pre-frozen at -80°C for 24 h. During storage, the content was higher in the fruit frozen for 4 weeks at -20°C, and among the thawing treatments, the content was higher in the fruit thawed in the microwave. However, all the treatments showed comparable malic acid content in the range of 2.86 and 3.28 mg/g. The shikimic acid content was the lowest in the fresh fruit and the highest in the fruit stored for 4 weeks at -20°C. The shikimic acid content did not show many significant differences among the thawing treatments and freezing treatments. The shikimic acid decreased only during storage from 0.035 mg/g at 4 weeks of storage to 0.028 mg/g at 6 months of storage. The fumaric acid content also showed the lowest content in fresh fruit and during storage the content decreased only by 10% after 6 months. After thawing, a higher content of fumaric acid was detected in the fruit thawed by microwave. The fumaric acid content after the other thawing treatments was comparable among each other, and additionally, no significant differences were found among the freezing treatments.

Sugars Content

The total sugar content was higher in the fruits frozen for 4 weeks at -20°C than in the fresh fruit (Table 2). The total sugar content significantly decreased during storage at -20°C for 3 months by 17% and during storage for 6 months by 28%. The total sugar content decreased also during thawing at 4°C for 24 h and at 24°C for 6 h by 18% and 9%, respectively. However, there was no significant difference in total sugar content between the frozen fruit stored for 6 months using different freezing treatments.

Table 2. Individual and total sugars content (mg/g fresh weight) in fresh, frozen and thawed strawberries.

	Freezing conditions	Storage time	Thawing conditions	Sugar content (mg/g fresh weight)
				Sucrose	Glucose	Fructose	Total
Freezing treatments	fresh fruit	-	-	21.3 ± 1.9 a	21.6 ± 1.0 b	22.9 ± 0.8 b	65.9 ± 3.5 a
	-20 °C	6 months	-	9.6 ± 0.4 b	24.0 ± 0.2 a	25.8 ± 0.3 a	59.5 ± 0.6 b
	liquid N2 => -20 °C	6 months	-	10.2 ± 1.0 b	23.9 ± 0.7 a	25.7 ± 0.9 a	59.8 ± 1.2 b
	-80°C => -20 °C	6 months	-	11.0 ± 1.1 b	21.1 ± 0.7 b	22.8 ± 0.8 b	54.9 ± 1.0 b
	-80 °C	6 months	-	12.2 ± 0.5 b	17.4 ± 0.5 c	24.9 ± 0.8 ab	54.5 ± 0.9 b
Storage time	fresh fruit	-	-	21.3 ± 1.9 a	21.6 ± 1.0 b	22.9 ± 0.8 c	65.9 ± 3.5 bc
	-20°C	4 weeks	-	23.6 ± 1.1 a	29.2 ± 0.9 a	30.6 ± 0.9 a	83.5 ± 2.8 a
	-20°C	3 months	-	17.1 ± 0.4 b	27.2 ± 1.3 a	25.2 ± 0.9 b	69.5 ± 1.3 b
	-20°C	6 months	-	9.6 ± 0.4 c	24.0 ± 0.2 b	25.8 ± 0.3 b	59.5 ± 0.6 c
Thawing treatments	-20 °C	4 weeks	-	23.6 ± 1.1 b	29.2 ± 0.9 ab	30.6 ± 0.9 ab	83.5 ± 2.8 ab
		4 weeks	at 4°C for 24 h	8.0 ± 0.3 d	29.1 ± 1.8 ab	31.0 ± 1.9 a	68.2 ± 4.0 c
		4 weeks	at 24°C for 6 h	14.8 ± 0.8 c	29.7 ± 1.1 a	31.3 ± 1.2 a	75.8 ± 2.6 bc
		4 weeks	at 37°C for 2 h	23.1 ± 1.2 b	28.5 ± 1.3 ab	29.9 ± 1.2 ab	81.5 ± 3.3 ab
		4 weeks	microwave	33.9 ± 0.9 a	25.7 ± 0.6 b	26.8 ± 0.6 b	86.5 ± 2.1 a

Different lowercase letters indicate statistically significant differences among the different freezing treatments, storage time and thawing treatments (ANOVA, p < .05).

Among the freezing treatments, the glucose was significantly lower when frozen at -80°C and when pre-frozen at -80°C and stored at -20°C. The fructose content was only significantly lower in fruit pre-frozen at -80°C and stored at -20°C compared to the other fruit stored at -20°C. Among the thawing treatments, the glucose and fructose content showed no difference after thawing compared to the frozen fruit. However, the sucrose content differed significantly between the different thawing treatments. Fruit defrosted by microwave showed even higher sucrose content (33.9 mg/g), but the fruit thawed for 24 h at 4°C and 6 h at 20°C decreased by 66% and 37% compared to frozen fruit, respectively. The fruit thawed at 37°C for 2 h showed comparable sucrose content as the frozen fruit (-20°C for 4 weeks). There was no significant difference between the fresh fruit and frozen fruit stored for 4 weeks (between 21.3 and 23.6 mg/g). Furthermore, the sucrose content decreased from 4 weeks to 3 months by 28% and to 6 months of storage by 59%. The results showed no significant difference in the sucrose content between the freezing treatments.

Phenolics Content

As presented in Table 3, the lowest total phenolic content among the freezing treatments was observed in the fresh fruit (163 mg/kg). Most freezing and storage time treatments showed similar total phenolics content with only a few significant differences. Additionally, the fruit thawed by microwave showed no significant difference in total phenolics content compared to the frozen fruit (4 weeks). However, the fruit thawed by other treatments shows a significant decrease in total phenolics content. The content has decreased by 28–30% compared to the frozen fruit (4 weeks).

Table 3. Total phenolics content and phenolics content per group (mg/kg fresh weight) in fresh, frozen and thawed strawberries.

	Freezing conditions	Storage time	Thawing conditions	Phenolics content (mg/kg fresh weight)
				Anthocyanins*	Hydroxycinnamic acid der.	Flavanols	Hydroxybenzoic acid der.	Flavonols	Total
Freezing treatments	fresh fruit	-	-	113 ± 20 d	22.1 ± 4.4 c	4.1 ± 0.7 b	12.8 ± 2.3 d	10.7 ± 1.6 c	163 ± 28 c
	-20°C	6 months	-	319 ± 24 ab	64.5 ± 1.0 a	14.3 ± 1.0 a	42.6 ± 2.3 a	27.0 ± 3.0 ab	467 ± 27 a
	liquid N2 => -20 °C	6 months	-	280 ± 7 bc	59.3 ± 2.4 ab	13.6 ± 1.7 a	33.7 ± 2.1 b	31.0 ± 1.5 a	418 ± 10 ab
	-80°C => -20 °C	6 months	-	263 ± 16 c	64.3 ± 3.1 a	12.4 ± 0.5 a	33.1 ± 1.3 b	21.6 ± 2.2 b	394 ± 20 b
	-80 °C	6 months	-	356 ± 6 a	53.2 ± 1.1 b	13.9 ± 1.4 a	23.9 ± 0.8 c	28.7 ± 1.7 a	475 ± 8 a
Storage time	fresh fruit	-	-	113 ± 20 b	22.1 ± 4.4 c	4.1 ± 0.7 b	12.8 ± 2.3 c	10.7 ± 1.6 b	163 ± 28 b
	-20°C	4 weeks	-	326 ± 14 a	48.2 ± 3.4 b	13.8 ± 0.8 a	34.0 ± 2.6 b	33.7 ± 2.6 a	456 ± 21 a
	-20°C	3 months	-	320 ± 5 a	67.1 ± 1.9 a	14.1 ± 1.1 a	34.2 ± 1.3 b	28.6 ± 0.5 a	464 ± 7 a
	-20°C	6 months	-	319 ± 24 a	64.5 ± 1.0 a	14.3 ± 1.0 a	42.6 ± 2.3 a	27.0 ± 3.0 a	467 ± 27 a
Thawing treatments	-20°C	4 weeks	-	326 ± 14 a	48.2 ± 3.4 ab	13.8 ± 0.8 b	34.0 ± 2.6 ab	33.7 ± 2.6 a	456 ± 21 a
		4 weeks	at 4°C for 24 h	229 ± 16 b	36.1 ± 3.2 c	13.6 ± 1.6 b	34.1 ± 3.9 ab	15.2 ± 1.3 c	328 ± 25 b
		4 weeks	at 24°C for 6 h	215 ± 10 b	37.0 ± 1.4 c	9.4 ± 0.5 c	26.3 ± 1.4 b	22.5 ± 1.4 b	310 ± 13 b
		4 weeks	at 37°C for 2 h	219 ± 16 b	41.7 ± 3.3 bc	11.1 ± 1.1 bc	29.6 ± 2.5 b	19.5 ± 2.2 bc	320 ± 25 b
		4 weeks	microwave	291 ± 13 a	55.9 ± 2.6 a	21.7 ± 0.8 a	38.0 ± 0.7 a	31.2 ± 0.8 a	438 ± 17 a

*The total anthocyanins content was calculated as a sum of the contents of all individual anthocyanins identified by the mass spectrometry. Der., derivatives. Different lowercase letters indicate statistically significant differences among the different freezing treatments, storage time and thawing treatments (ANOVA, p < .05).

Similar differences were also observed in the content of different non-anthocyanin phenolic groups. For all groups, the content was lower in fresh fruit than in the fruit frozen for 6 months. The fruit thawed by microwave or at 37°C for 2 h did not show a significant difference in the content of the groups of phenolic compounds except for an increase in the flavanols content after microwave thawing and a significant decrease in flavonols content (by 42%) in fruit thawed at 37°C for 2 h. Flavanols content showed a significant decrease after thawing only when thawed at 24°C. Hydroxycinnamic acid derivatives content showed a significant decrease in fruit thawed at 4°C and 24°C, and the decrease ranged between 23 and 25%. The hydroxybenzoic acid derivatives content did not show any significant changes after thawing compared to the frozen fruit. However, the flavonols content decreased after all thawing treatments (except for microwave), ranging between 33% and 55%. The contents of all individual phenolic compounds of these groups of compounds are available in Supplementary Table S2.

The total anthocyanin content was detected higher in the frozen fruit treated with different freezing treatments than in the fresh fruit. There was no significant difference between the frozen fruit stored at -20°C for 4 weeks and 6 months. However, there were differences among the freezing treatments, where frozen fruit stored at -80°C had higher anthocyanin content than the fruit pre-frozen at -80°C and the fruit shock-frozen by liquid nitrogen and then stored at -20°C. As for the thawing treatments, the fruit thawed in the microwave had a comparable anthocyanin content as the frozen fruit. However, the fruit thawed using the other conditions showed a decrease in the total anthocyanin content by 30–34%.

Similar differences were also observed in the content of the individual anthocyanins (Table 4). For all the individual anthocyanins, the content was detected lower in the fresh fruit compared to the frozen fruit (stored for 4 weeks). As for the thawing treatments, fruits thawed in the microwave did not show a significant decrease in the content of most of the individual anthocyanins except for pelargonidin-3-O-acetylglucoside, where the content decreased by 31%. This anthocyanin also showed a significant decrease during storage at -20°C as the content decreased by 39% after 6 months. Among the freezing treatments, fruit stored at -80°C showed the highest content of pelargonidin-3-O-acetylglucoside. On the other hand, pelargonidin-3-(6˝malonyl)glucoside content did not significantly decrease after the thawing or the storage. The content of all other anthocyanins decreased after thawing (except for microwave thawing). While the content of pelargonidin-3-O-glucoside, pelargonidin-3-O-rutinoside, and 5-pyranopelargonidin-3-glucoside did not significantly differ among the thawing treatments at 4°C, 24°C and 37°C, there were significant differences in the cyanidin-3-O-glucoside and pelargonidin-3-O-acetylglucoside content. The cyanidin-3-O-glucoside content decreased the most at 4°C for 24 h (by 48%), and the pelargonidin-3-O-acetylglucoside decreased the most at 24°C for 6 h (by 57%) and at 37°C for 2 h (by 51%).

Table 4. Individual anthocyanin content (mg/kg fresh weight) in fresh, frozen and thawed strawberries.

	Freezing conditions	Storage time	Thawing conditions	Anthocyanin content (mg/kg fresh weight)
				cy-3-O-glc	plg-3-O-glc	plgn-3-O-rut	plg-3-(6˝malonyl)glc	5-pyranoplg-3-glc	plg-3-O-acetylglc
Freezing treatments	fresh fruit	−	−	6.4 ± 0.2 c	98 ± 18 c	6.3 ± 1.1 c	0.24 ± 0.04 c	0.16 ± 0.04 c	1.92 ± 0.27 c
	-20 °C	6 months	−	10.3 ± 1.3 a	281 ± 20 b	22.2 ± 1.7 a	0.67 ± 0.06 a	0.62 ± 0.06 a	3.85 ± 0.37 b
	liquid N2 => -20 °C	6 months	-	10.6 ± 0.4 a	247 ± 6 b	18.0 ± 0.3 b	0.42 ± 0.03 b	0.48 ± 0.02 b	3.63 ± 0.04 b
	-80°C => -20 °C	6 months	−	7.4 ± 0.9 bc	234 ± 14 b	17.4 ± 1.0 b	0.51 ± 0.03 b	0.45 ± 0.02 b	3.78 ± 0.20 b
	−80 °C	6 months	-	8.9 ± 0.4 ab	317 ± 5 a	22.1 ± 0.2 a	0.48 ± 0.02 b	0.42 ± 0.01 b	6.46 ± 0.17 a
Storage time	fresh fruit	−	−	6.4 ± 0.2 b	98 ± 18 b	6.3 ± 1.1 b	0.24 ± 0.04 b	0.16 ± 0.04 b	1.92 ± 0.27 c
	-20 °C	4 weeks	−	10.2 ± 1.0 a	291 ± 11 a	17.9 ± 1.8 a	0.62 ± 0.15 a	0.46 ± 0.07 a	6.36 ± 0.28 a
	-20°C	3 months	-	10.3 ± 0.4 a	286 ± 4 a	20.0 ± 1.0 a	0.64 ± 0.02 a	0.59 ± 0.02 a	3.19 ± 0.22 b
	−20 °C	6 months	-	10.3 ± 1.3 a	281 ± 20 a	22.2 ± 1.7 a	0.67 ± 0.06 a	0.62 ± 0.06 a	3.85 ± 0.37 b
Thawing treatments	−20 °C	4 weeks	-	10.2 ± 1.0 a	291 ± 11 a	17.9 ± 1.8 a	0.62 ± 0.15 a	0.46 ± 0.07 a	6.36 ± 0.28 a
		4 weeks	at 4°C for 24 h	5.3 ± 0.2 c	205 ± 15 b	13.1 ± 0.8 b	0.53 ± 0.08 a	0.26 ± 0.05 c	4.11 ± 0.26 b
		4 weeks	at 24°C for 6 h	7.6 ± 0.6 b	191 ± 9 b	12.6 ± 0.8 b	0.42 ± 0.03 a	0.28 ± 0.02 bc	2.76 ± 0.09 c
		4 weeks	at 37°C for 2 h	6.4 ± 0.5 bc	196 ± 15 b	12.8 ± 1.0 b	0.47 ± 0.06 a	0.22 ± 0.02 c	3.14 ± 0.20 c
		4 weeks	microwave	10.8 ± 0.2 a	260 ± 12 a	15.3 ± 0.9 ab	0.56 ± 0.12 a	0.38 ± 0.02 ab	4.38 ± 0.21 b

Cy, cyanidin; plg, pelargonidin; glc, glucoside. Different lowercase letters indicate statistically significant differences among the different freezing treatments, storage time and thawing treatments (ANOVA, p < .05).

Enzyme Activity

The peroxidase activity (POD) showed comparable values among the freezing treatments, and the activity did not significantly differ between storage for 4 weeks, 3 months and 6 months (Table 5). As for the polyphenol oxidase activity (PPO), the activity was higher after 3 months and 6 months of storage compared to 4 weeks of storage, and among the freezing treatments, the highest activity was detected in fruit stored at -80°C. Although there were some significant differences, the activity among the freezing treatments and storage times for both enzymes was within a similar range. There were higher differences in the activity of these enzymes among the thawing procedures, where the enzyme activity significantly increased compared to the frozen fruit, except for fruit thawed in the microwave, where no enzyme activity was detected. Both for POD and PPO, the highest activity per protein content was in the fruit thawed at 24°C.

Table 5. Peroxidase (POD) and polyphenol oxidase (PPO) activity in fresh, frozen and thawed strawberries.

	Freezing conditions	Storage time	Thawing conditions	POD	PPO
				(A/min/mg protein)	(A/min/mg protein)
Freezing treatments	fresh fruit	−	−	0.86 ± 0.10 c	0.28 ± 0.02 c
	-20°C	6 months	-	1.09 ± 0.09 bc	0.33 ± 0.02 bc
	liquid N2 => -20 °C	6 months	−	1.29 ± 0.03 ab	0.28 ± 0.00 c
	-80°C => -20 °C	6 months	-	1.55 ± 0.11 a	0.34 ± 0.01 b
	−80 °C	6 months	-	1.07 ± 0.24 bc	0.40 ± 0.02 a
Storage time	fresh fruit	-	-	0.86 ± 0.10 b	0.28 ± 0.02 ab
	-20°C	4 weeks	−	1.17 ± 0.08 a	0.23 ± 0.02 b
	-20°C	3 months	−	1.05 ± 0.07 ab	0.32 ± 0.01 a
	-20°C	6 months	-	1.09 ± 0.09 ab	0.33 ± 0.02 a
Thawing treatments	-20°C	4 weeks	−	1.17 ± 0.08 c	0.23 ± 0.02 c
		4 weeks	at 4°C for 24 h	2.69 ± 0.12 b	0.48 ± 0.03 bc
		4 weeks	at 24°C for 6 h	6.58 ± 0.16 a	2.14 ± 0.12 a
		4 weeks	at 37°C for 2 h	2.16 ± 0.30 b	0.71 ± 0.13 b
		4 weeks	microwave	nd	nd

Different lowercase letters indicate statistically significant differences among the different freezing treatments, storage time and thawing treatments (ANOVA, p < .05).

Discussion

Freezing and thawing are procedures commonly used for preserving and handling strawberries intended for further processing in the industry, but they are also used for handling samples for scientific purposes. Freezing is very often used to preserve samples before further analysis. While in industry, the fruit is usually directly frozen or frozen using IQF, for scientific purposes, the fruit can often be frozen in extreme conditions (-80 °C). Further more prior to processing and preparation of samples, the fruit is thawed. However, freezing and thawing can affect the composition of the fruit and consequently affect the quality of the fruit (Pukszta, 2016).Therefore, this study explores freezing and thawing procedures used for fruit processing or fruit sample preparation and the effect on the fruit’s composition.

During the freezing, the structure of the fruit changes and the formation of crystals causes cellular disruption (De Ancos et al., 2007). The cell disruption can then enhance the extraction of the compounds from the fruit, which is confirmed by our results as most metabolites showed a significant increase in their content after freezing except for sucrose. This trend has been previously reported for phenolic compounds in strawberries (Bulut et al., 2018), but also in other fruits, such as raspberries (González et al., 2003) and blackberries (Veberic et al., 2014). Additionally, Bulut et al. (2018) have also reported an increase in ascorbic acid content from fresh fruit to frozen fruit, which has been also observed in our study. This shows that the cell disruption aids the release of not only phenolics but also primary metabolites. Since both primary and secondary metabolites are located in the vacuole (Liu et al., 2023), and the disruption during freezing helps to release all these metabolites, the content of the detected metabolites (except for sucrose) increased after freezing. This can also improve the compounds’ bioavailability, as previously shown by Kamiloglu (2019), where the bioavailability of anthocyanins increased after freezing. However, the cell disruption also allows reactions between enzymes and their corresponding substrates (Tomás-Barberán and Espín, 2001), and these enzymatic activities can lead to changes in the composition.

Some of such changes can also involve the sugars content, which can affect the taste perception of the fruit (Schwieterman et al., 2014) and freezing alters the fruit taste, including the sweetness (Hancock, 2020). Similar to previous studies (Castro et al., 2002; Moraga et al., 2006), the sugar content decreased during frozen storage, mainly affecting the sucrose content. This can be attributed to the enzyme activity, namely the invertase activity, which hydrolyzes sucrose (Basson et al., 2010). However, sucrose serves as a precursor for other metabolic pathways and a substrate for other enzymes. Since the content of glucose and fructose did not increase as the sucrose content decreased, the loss of sucrose cannot be solely attributed to the invertase activity. Additionally, the activity of these enzymes can affect the sugar content during the thawing process observed in our study. A decrease in sucrose content has been previously reported (Skrede, 1983), where the sucrose content decreased by 70% when thawed at 4°C. This agrees with our study, where the most pronounced loss in the sucrose content was observed after thawing at 4°C for 24 h, followed by the thawing at 24°C for 6 h. These results suggest that the loss depends more on the thawing time than the temperature. However, the loss of sucrose in the fruit can be compensated during further processing since it is allowed to add sugar during the production of products such as strawberry nectar or strawberry jam (Council of the European Union, 2001; Haffner, 2002; Murray et al., 2023), but it can increase the cost since more sugar needs to be added to achieve the optimal level of sweetness.

Apart from sugars, the organoleptic perception of the fruit depends on the organic acid content, but they also play a role in stabilizing the pigments of the fruit – anthocyanins (Newerli-Guz et al., 2023). In a previous study (Sahari et al., 2004), a significant decrease in acidity during frozen storage was reported at all studied temperatures (-12°C, -18°C and -24 °C), which is in line with our results where a significant decrease in total organic acids content was observed after storage at -20°C. Differences in the organic acids content can affect the pH of the fruit, and an increase was reported after freezing in strawberries (Sahari et al., 2004) and in apples (Chassagne-Berces et al., 2010). Such changes in pH could then affect not only the taste but also the stability of anthocyanins.

Out of the detected organic acids, ascorbic acid plays a significant role since it also serves as a vitamin in human nutrition. However, ascorbic acid is very susceptible to heat, light and oxygen, and additionally, ascorbate oxidases can contribute to ascorbic acid degradation in fruits and vegetables (Davey et al., 2000). It has been previously reported (Sahari et al., 2004) that the highest decrease in ascorbic acid content was observed for strawberries stored at -12°C, compared to storage at lower temperatures. This is similar to our case, where a decrease in ascorbic acid content was observed in frozen fruit stored at -20°C, and no effect was observed in fruits stored at -80°C. However, the decrease was only by 17% from 4 weeks to 6 months of storage, which is much lower than what was reported by Bulut et al. (2018), where the ascorbic acid decreased by more than 40% in frozen fruit stored at -27°C for 13 weeks. This suggests that there can be differences in how different strawberry cultivars respond to freezing, which was previously observed by Castro et al. (2002), where a significant difference in loss of ascorbic acid after frozen storage was observed between two strawberry cultivars. These differences could be due to differences in the enzyme activity, namely the ascorbic acid oxidase, which is responsible for the oxidation of ascorbic acid (Reid and Barrett, 2005). Moreover, ascorbic acid can serve as an anti-browning agent counteracting the enzymatic degradation of phenolic compounds by reducing the o-quinones back to their phenolic substrates (Oms-Oliu et al., 2010; Suttirak and Manurakchinakorn, 2011) but ascorbic acid degrades in this process. Additionally, the ascorbic acid content can also be affected by the thawing procedure. According to Holzwarth et al. (2012), the most pronounced losses during thawing occurred at 4°C and the least loss was reported for strawberries that were thawed in a microwave oven. This is also in agreement with our study, where the highest decrease was observed at 4°C and similar to the previous study, the content decreased by approximately 30%. This decrease could be attributed to the extended exposure to oxygen during thawing at 4°C (24 h).

Phenolic compounds content is an important factor in determining the quality of strawberries. A major group of phenolic compounds in strawberries, anthocyanins serve as the pigments, and their content affects the color stability of strawberry products, such as juice or purees. Additionally, phenolic compounds also contribute to nutritional quality by contributing to lowering the risk of chronic diseases such as cancer or cardiovascular diseases (Afrin et al., 2016; Cassidy et al., 2013; da Silva Pinto et al., 2010). Both increase and decrease in the content of phenolic compounds in strawberries during frozen storage were previously reported (Bulut et al., 2018; Oszmiański et al., 2009; Salazar-Orbea et al., 2023). However, in our study, the phenolics content remained stable during the frozen storage. But the stability of different phenolic compounds can differ, as previously reported by Salazar-Orbea et al. (2023), where the content of ellagic acid derivatives (hydroxybenzoic acid derivatives) increased and in the flavonols content decreased after storage at -20°C for 6 months, same as in our study. However, our study showed that the hydroxybenzoic acid derivatives content was more sensitive to lower temperatures, such as -80°C, where the content decreased. Additionally, the content of hydroxycinnamic acid derivatives and flavanols showed as stable during the frozen storage. This shows that differences in the phenolic profile can determine the effect of freezing and frozen storage on the retention of these compounds.

Although freezing did not show a strong negative effect on the phenolics content, some of the thawing procedures showed a significant loss of the phenolics content. Although in a previous study (Holzwarth et al., 2012), no significant effect on the non-anthocyanin phenolic content was observed during thawing, our results showed a significant decrease in the total phenolics content after thawing in all cases except for microwave thawing. This suggests that there could be differences in the enzymatic activity between cultivars. Out of all the thawing procedures, fruit thawed at 24°C showed a significant decrease in the content of all groups of phenolic compounds, which can be explained by the high activity of peroxidase (POD) and polyphenol oxidase (PPO) at this temperature.

The major group of phenolic compounds present in strawberries are anthocyanins, but temperature, pH, light, oxygen and ascorbic acid highly affect the half-life of anthocyanins. The stability of anthocyanins during freezing and thawing depends on their structure as well as the enzymatic activity, mainly peroxidase (POD) and polyphenol oxidase (PPO). Previous studies have shown different effects of freezing on strawberries: a reduction (Sahari et al., 2004), no effect (Kamiloglu, 2019), and an increase (Ngo et al., 2007). Our results showed that all thawing treatments, except for microwave thawing, had a negative effect on the content of anthocyanins. However, as observed in our study, different anthocyanins can show different levels of degradation during thawing. Similarly, as in the previous study by Holzwarth et al. (2012), the pelargonidin-3-O-malonylglucoside content was not significantly affected by the thawing, which was attributed to the acylation of the sugar moiety. This demonstrates that each cultivar can show different levels of pigment degradation depending on their anthocyanin profile.

As mentioned above, the content of anthocyanins and other phenolics compounds also depends on the enzymatic activity of peroxidase (POD) and polyphenol oxidase (PPO). While freezing and frozen storage had a small effect on the activity of POD and PPO, the thawing conditions significantly affected their activity. As previously reported (Chisari et al., 2007), the activity of POD and PPO can vary depending on the temperature, and the enzymes can exhibit enzymatic activity even at lower temperatures. Our results suggest that the enzyme activity of POD and PPO increases during thawing with an optimal temperature close to the room temperature. A particular case is microwave thawing, where no enzymatic activity was detected, which could be caused by the deactivation of the enzymes by the microwave treatment (Bulhões Bezerra Cavalcante et al., 2021).

Conclusion

Our study showed that both frozen storage and the thawing procedure can significantly affect the composition of strawberry fruit and, consequently, the quality of the fruit. An extended frozen storage (up to 6 months) had a negative effect mainly on sugar and organic acid content (including ascorbic acid content) and, in some cases, on the anthocyanin content, which would consequently negatively affect the taste of the fruit. Although there were slight differences in the content of anthocyanins and ascorbic acid between the storage at different temperatures, the differences are minor and would not justify the increased costs of storage at lower temperatures, such as -80°C. Furthermore, our study showed that the thawing procedure significantly affected the content of sugars, organic acids, phenolic compounds, anthocyanins, and enzymatic activity. From all the thawing procedures covered by this study, microwave thawing showed as the best thawing treatment. On the other hand, thawing for an extended time at 4°C or 24°C is discouraged as it causes significant differences in the composition of the fruit and can negatively affect its quality. However, further study on the combination of different freezing and thawing treatments is needed to understand the interactions between these two factors in order to identify the right treatment of the fruit for further processing or for sample preparation.

Data Set

Research dataset can be accessed here: http://hdl.handle.net/20.500.12556/RUL-156238.

CRediT author statement

Kristyna Simkova: conceptualization, formal analysis, investigation, data curation, writing – original draft, visualization; Robert Veberic: writing – review and editing; Metka Hudina: resources, writing – review and editing, funding acquisition; Mariana Cecilia Grohar: investigation; Massimiliano Pelacci: investigation; Jerneja Jakopic: conceptualization, methodology, validation, supervision, writing – review and editing.

Disclosure Statement

No potential conflict of interest was reported by the author(s).

Supplementary Material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15538362.2024.2355920

Additional information

Funding

This work was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 956257; and the Slovenian Research and Innovation Agency (ARIS) within the research program Horticulture (P4-0013).