Effect of phenylalanine treatment on chilling tolerance and biochemical attributes of grape during postharvest cold storage

Abstract

BACKGROUND:

phenylalanine treatment affect on chilling tolerance during postharvest cold storage.

OBJECTIVE:

This work aimed to investigate exogenous application of phenylalanine (Phe; 0, 15 and 30 mM) on berry phenolic compounds, quality and chilling tolerance of ‘Red Sahebi’ grape during 60 days at 1°C.

METHODS:

Every 15 days (storage durations were 0, 15, 30, 45, and 60 days), approximately 150 gr of grape bunches were sampled randomly from cool chamber for following physicochemical and microbial analysis.

RESULTS:

Phe-treated grape at 30 mM maintained higher titratable acid, total sensory score, total soluble solid, soluble sugars, organic acids, and antioxidant capacity compared to control grapes. At the end of storage time, 30 mM Phe-treated grapes showed higher flavonols (11% myricetin, 20% quercetin, and 23% kaempferol), flavanols (22% catechin, 34% epicatechin, and 40% epigallocatechin) and anthocyanins (42% malvidin-3-O-glucoside, 30% delphinidin-3-O-glucoside, 25% cyanidin-3-O-glucoside, and 23% pelargonidin-3-O-glucoside) compared to control samples due to lower polyphenol oxidase but higher antioxidant enzyme activities. Also, 30 mM Phe was found to be effective for berry trans-resveratrol and phenolic acid preservation, displayed less fungal decay. The efficiency of Phe on chilling tolerance was monitored by lower rachis browning, electrolyte leakage, and malondialdehyde but higher abscisic acid content.

CONCLUSION:

Phenylalanine application retained higher TA, TSS, and vitamin C, antioxidant capacity and total sensory score in treated fruits and alleviates chilling injury of table grape during cold storage.

Keywords

Anthocyanins flavonols organic acids phenylalanine soluble sugars table grape

1 Introduction

Grapes are one of the rich sources of phenolic compounds among fresh fruits. Phenolic compounds contribute significantly to sensory characteristics because of their role in color, aroma, and flavor determination of grapes [1]. Moreover, phenolic compounds have been broadly studied due to their potential beneficial effects on human health such as cardioprotective effects and anti-inflammatory, anti-carcinogenic, and anti-microbial activities [2]. The phenolic compounds of fruits such as flavonoids (flavonols, flavanols and anthocyanins), stilbenes (resveratrol), and phenolic acids (derived from hydroxycinnamic acid and hydroxybenzoic acids) are synthesized from phenylalanine (Phe) as a precursor of phenylpropanoid pathway through de-amination of Phe to cinnamic acid by the activities of phenylalanine ammonia-lyase (PAL) and other enzymes [3, 4]. However, fresh grapes are perishable commodities with low shelf life due to loss of firmness, berry shedding, rachis drying and browning, berry wrinkled, water loss, and fungal decay [5, 6]. Also, during long storage, there is the possibility of losses in polyphenols and anthocyanins by the activities of polyphenol oxidase (PPO) and peroxidase [7]. These physiological and pathological disorders can negatively affect the appearance of the fruits and their internal compounds, including organic acids, soluble sugars, and phenolic compounds. Therefore, the application of proper post-harvest techniques to reduce fruits losses and maintain their quality during long storage is very important.

The storage of grapes under low-temperature conditions is one of the post-harvest management approaches that can be used for long-term maintenance of these fruits but this technique does not completely prevent the decline in content of phenolics, organic acids, or other quality-related compounds during a prolonged storage. Moreover, storage at low temperatures may cause chilling injury, which is manifested by berry skin and pulp browning, skin pitting, and rachis browning [8]. Ultrastructurally, cold stress induces reactive oxygen species (ROS) accumulation in damaged fruits, causes an increase in peroxidation of biomembranes lipids and consequently electrolyte leakage rate [9]. These changes provide condition for gray mold (Botrytis cinerea) to grow rapidly even under low temperatures and contaminate the berries. One common way to control the gray mold is to use a sulfur-releasing pad or sulfur dioxide (SO₂) fumigation in storage. However, SO₂ has undesirable effects such as berry skin discoloration or bleaching and change the berry flavor. Moreover, sulfur residue on berry skin can be dangerous to human health and has also adverse effects on the environment [6, 10]. As a result, an alternative health-friendly method is needed to improve the postharvest-related characteristics of stored grapes.

Until now, various treatments such as the use of natural and artificial compounds as well as physical treatments have been used to control or reduce horticultural crops postharvest losses [6 , 11]. Recently, biologically active natural compounds have been used as a proper alternative in preserving fruit quality during storage. Among these compounds, exogenous Phe has been employed as a beneficial technique for maintaining postharvest quality and enhancing chilling tolerance in some horticultural crops such as tomato [12] and plume [13] mainly through increasing superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX) activity. Based on literature Aghdam et al. [12]; Sogvar et al. [13], there are few reports regarding the effects of Phe pretreatment on chilling tolerance and postharvest quality retention of some fruit crops. However, the effect of exogenous Phe on individual phenolic acid, flavonols, flavan-3-ols, anthocyanin, soluble sugars, and organic acid during postharvest life remained unclear, especially in grapes. Recently, it has been reported that foliar application of urea and Phe cooperate in biosynthesis of phenolic compounds grape berries [14] and its role in metabolism of flavonoids was demonstrated via in situ C-13 labeling and liquid chromatography detection [15]. To our knowledge, this is the first study to consider the impact of Phe on preserving the antioxidant properties and the profile of individual phenolic acids, flavonoids, anthocyanidins, organic acids and soluble sugars of grape during postharvest cold storage. Our hypothesis is that pretreatment of Phe can affect positively berry internal biochemical compounds and protect them from chilling injury and fungal decay during cold storage. Therefore, the objective of this work was to study the effect of postharvest application of Phe on grape qualitative parameters, biochemical characteristics, antioxidant properties, decay rate, and chilling tolerance during storage at 1°C for 60 days.

2 Materials and methods

2.1 Fruits and Phe and cold storage treatments

Grapes (Vitis vinifera L. cv ‘Red Sahebi’) were harvested during cool hours of day at the commercial maturity stage with TSS = 17° Brix from an experimental vineyard at Research Institute for Grape and Raisin (lat. 34° 30' N, long. 48° 85' E, alt. 1766 m), Malayer University, Iran, and the experiment was conducted in the Horticultural Research laboratory in October 2019. Grape bunches were selected based on uniformity in size, maturity, and color, and the absence of physical damage or fungal infection. Phenylalanine was purchased from Sigma-Aldrich Company, Germany. Other chemicals used in this study were analytical grade (>99%) and all solvents were HPLC grade.

After sorting and grading, the grape bunches were divided into three equal groups. 1st group was dipped in distilled water and named as a control sample, 2nd group was immersed in 15 mM Phe and the 3rd group in 30 mM Phe solution for 15 min at 23°C. After drying under ambient temperature of laboratory, the grape bunches (0.8 Kg) were packed in polyethylene plastic containers (dimensions 21 cm×14 cm×8 cm) and stored for 60 days in a cool chamber with temperature and relative humidity of 1±0.5°C and 85%, respectively. Every 15 days (storage durations were 0, 15, 30, 45, and 60 days), approximately 150 gr of grape bunches were sampled randomly from cool chamber for following physicochemical and microbial analysis.

2.2 Total soluble solid (TSS), titratable acidity (TA), pH, Vitamin C and sensory attributes

Berry TSS was determined with a digital refractometer (Atago, Japan). For TA assay, 10 ml of berry fresh juice was tittered with 0.1 N NaOH to pH 8.1, and the result was expressed as a percentage of tartaric acid. Also, berry juice pH was quantified by a pH meter (Atago, Japan; [16, 17]. For sensory attributes evaluation, three grape clusters per treatment were chosen by five panelists according to the method of Deng et al. [18]. Vitamin C (ascorbic acid) was measured according to Arya [19] by titration of starch solution with potassium iodide.

2.3 Antioxidant capacity (DPPH and FRAP)

The antioxidant capacity of grape berry was performed based on DPPH (1, 1-diphenyl-2- picrylhydrazyl) scavenging method Bozin et al. [20]. Also, the ferric reducing ability of plasma (FRAP) of grape berry samples was measured according to the method described by Benzie and Strain [21].

2.4 Activities of antioxidant enzymes

The activities of superoxide dismutase (SOD; [22]), catalase (CAT; [23]), guaiacol peroxidase (GPX; [24]), and ascorbate peroxidase (APX; [25]) of all grape samples after cold storage were measured by spectrophotometer (Spekol 2000, Analytic Jena, Germany).

2.5 Phenolic compounds (non-flavonoids and flavonoids)

Grape berry phenolic compounds, including 1. non-flavonoid (i. phenolic acids (gallic acid, caffeic acid, ferulic acid, and p-coumaric acid), ii. resveratrol) and 2. flavonoids (i. flavonoids (flavonols: myricetin, quercetin and kaempferol), ii. flavan-3-ols (catechin, epicatechin, epigallocatechin), iii. some anthocyanidins (delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, malvidin-3-O-glucoside, pelargonidin-3-O-glucoside) were analysed in current study (Peonidin-3-O-glucoside and petunidin-3-O-glucoside were not measured in grape samples). For analysis of delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, malvidin-3-O-glucoside, pelargonidin-3-O-glucoside, ground powders of entire berries (3 g) of each treatment were boiled in 0.1 N HCL for 25–30 min. The filtrate was then separated with ethyl acetate and dissolved in water and the portion insoluble in water dissolved in 80% methanol and filtered through a Millex HA 0.45μm filter (Milipore Crop.), before injection to a HPLC pump at 518 nm wavelength.

For other berry phenolic compounds measurement, an amount of 1 g of finally ground sample was suspended in 30 ml of methanol and the mixture was covered to prevent evaporation. The sample was stirred for 30 min at room temperature. Later, it was sonicated in a water bath for 20 min at room temperature and homogenate subsequently filtered. The obtained solutions were separated with ethyl acetate, which soluble and insoluble portions dissolved in water and 80% methanol, respectively. Samples were filtered again through a Millex HA 0.45μm filter (Millipore Crop.) before injection to a HPLC pump (Crystal 200 series, Unicam, Cambridge, UK) under room temperature [26]. Separations were done with a UV-Vis detector, 4.6×250 mm, 5μm ODS column (HiChrom, USA) at 254 nm wavelength with potassium di-hydrogen phosphate and acetonitrile (80:20, v: v) at a flow rate of 1 mL min^–1 as mobile phase. Stock solutions of the standard acids (E. Merck) were prepared in a concentration of 1 g 100 mL^–1 in pure methanol [27].

2.6 Polyphenol oxidase activity

Polyphenol oxidase activity was measured through sodium hydrogen phosphate and citrate monohydrate as McIlvaine buffer for extraction and then its activity was measured at 420 nm based on Halpin and Lee [28].

2.7 Organic acids (tartaric acid and malic acid)

Liquid chromatography (LC) equipped with a LC-15C series pumping system (Shimadzu) with an auto-sampler (SIL-10AF) and a SPD-15C series UV double wavelength detector (Shimadzu) was used for tartaric acid and malic acid measurement at 210 nm [29]. Ultrapure acetonitrile (5%) and 0.01 mol L^–1 potassium dihydrogen phosphate (95%, pH 2.7) with a flow rate of 0.8 mL /min were used as solvents (mobile phase).

2.8 Soluble sugars (glucose, fructose and sucrose)

Berry samples (0.5 g) were powdered, dissolved in 10 ml ethanol (80%), and centrifuged at 8000 rpm for 15 min at 4°C. After filtration, 10μL was injected into HPLC pump (ATI Unicam, UK) equipped with a SPD UV –Vis detector and a Spherisorb ODS-2 Column (0.3μm, 150 mm×4.6 mm i.d.) with 500 mM sodium citrate (pH 5.5) and ultrapure acetonitrile (1:99, V/V) as the mobile phase at a flow rate of 0.1 ml^–1 min [17, 30]. The concentrations of each soluble sugar were determined based on standard solution calibration and expressed as μmol g^–1 fresh weight (FW).

2.9 Abscisic acid (ABA)

Endogenous ABA analysed using a Crystal 200 series HPLC pomp (ATI Unicam, Cambridge, UK) equipped with a UV-Vis detector (SPD Philips, Cambridge, UK) and a Diamonsic-C₁₈ Column (5μm, 250 mm×4.6 mm i.d.; Berkshire, UK) with 20–75% methanol in 1% acetic acid (v/v) as mobile phase and a flow rate of 1.2 mL min^–1 [31].

2.10 Membrane electrolyte leakage, lipid peroxidation and chilling injury (CI)

For membrane permeability measurement, relative electrolyte leakage (EL) rate of cold-stored samples was assayed using a digital EC-meter (Atago, Japan) according to the method of Deng et al. [18]. Membrane stability of berries was expressed by malondialdehyde (MDA) production rate as a lipid peroxidation end-product based on the methods of Heath and Packer [32]. Chilling injury (CI) index was individually assessed in fruits (12 grape bunches in each treatment) with a 4-grade level based on the percentage of berry skin and flesh affected by CI symptoms (berry desiccation, browning, and pitting) and classified as follow: 0 (bunches with no berry CI symptoms), 1 (bunches with less than 25% CI symptoms), 2 (bunches with 26–50% CI symptoms) and 3 (bunches with more than 51% CI symptoms). Chilling injury index (%) was calculated using the following formula:

CI index (%) = [Σ(CI grade level)×(number of bunches with the corresponding grade level)] / [(total number of bunches×highest level)]×100

2.11 Weight loss, rachis browning and decay incident

The percentage of cluster weight loss in each pack was calculated by dividing the weight of the clusters after storage into their initial weight before storage [16]. Cluster rachis browning index in the replication was measured visually based on a 1–5 scale with references to pictures [16]. Fungal decay assessment (predominantly gray mold) was examined every 15 days of cold storage followed by 4 days of shelf life at 24±1.0°C according to Asgarian et al. [8].

2.12 Experimental design and statistical analysis

This experiment consisted of a factorial arrangement (2 factors including Phe at 3 levels of 0, 15, and 30 mM and storage duration at 5 durations of 0, 15, 30, 45, and 60 days) of variables using a completely randomized design with three replications per treatment. Data analysis of variance (ANOVA) were established by GLM procedures of SAS (9.2), and Duncan’s multiple range test at a probability level of 1% was used for mean comparisons.

3 Results and discussion

3.1 Berry TSS, TA, pH, vitamin C, and sensory attributes

The mean data for table grape quality parameters including pH, TA, TSS, and vitamin C are shown in Table 1. According to the results, a significant difference was found between Phe-treated grape and control grape samples for any parameters (Table 1). Exogenous Phe had an impact on pH, TA, TSS, and vitamin C of grape berries. At the end of storage time, the berry pH, TA, TSS, and vitamin C of Phe-treated samples were retained 2.6%, 9.3%, 6.1%, and 7.5% compared to the control samples, respectively (Table 1).

Table 1
Berry quality parameters, total sensory score and antioxidant capacity (DPPH and FRAP) of ‘Red Sahebi’ grape in response to postharvest treatment of phenylalanine during cold storage

Treatments pH Titratable Total soluble Vit. C Total DPPH FRAP

acid solid (mg/ 100 g sensory (%) (μmol Fe II)

(%) (°Brix) DW) score

P₁D₀ 3.10±0.06f 0.76±0.01a 18.00±0.50g 8.87±0.01a 30±0.4a 41.80±0.91a 26.10±0.06a

P₂D₀ 3.10±0.06f 0.76±0.01a 18.00±0.50g 8.87±0.01a 30±0.6a 41.80±0.91a 26.10±0.06a

P₃D₀ 3.10±0.06f 0.76±0.01a 18.00±0.50g 8.87±0.01a 30±0.6a 41.80±0.91a 26.10±0.06a

P₁D₁ 3.48±0.01de 0.61±0.01c 19.98±0.87f 7.46±0.09b 26±0.3c 33.09±0.79d 20.10±1.10d

P₂D₁ 3.56±0.07de 0.64±0.01b 20.86±0.83ef 7.77±0.07b 27±0.7bc 36.50±0.68b 24.20±0.06b

P₃D₁ 3.40±0.06e 0.65±0.01b 21.73±0.12de 7.78±0.04b 28±0.5b 37.85±0.42b 26.60±0.06a

P₁D₂ 3.63±0.07bc 0.52±0.01e 23.16±0.02bc 6.07±0.17d 21±0.5e 30.00±0.04fg 17.10±0.52f

P₂D₂ 3.66±0.07bc 0.55±0.02de 23.20±0.00bc 6.96±0.23c 24±0.9d 30.81±0.71de 22.70±0.06c

P₃D₂ 3.47±0.02de 0.55±0.01d 23.28±0.09bc 6.92±0.23c 26±0.9c 31.29±0.60d 24.20±0.06b

P₁D₃ 3.69±0.01bc 0.43±0.01g 22.54±0.46dc 5.27±0.12e 19±0.8f 25.83±0.43h 13.50±0.23g

P₂D₃ 3.60±0.06cd 0.44±0.01g 23.35±0.03bc 5.82±0.05d 21±0.8e 29.18±0.36e 19.00±0.00e

P₃D₃ 3.63±0.03bcd 0.47±0.01f 23.50±0.09bc 5.87±0.06d 24±0.9d 29.28±0.31e 23.80±0.06b

P₁D₄ 3.80±0.06b 0.39±0.01h 23.67±0.59bc 4.72±0.01f 16±0.9g 14.57±0.02k 12.10±0.40h

P₂D₄ 4.10±0.00a 0.41±0.00gh 24.46±0.13ab 5.01±0.04ef 18±0.6f 16.69±0.01j 17.25±0.32f

P₃D₄ 3.70±0.10bc 0.43±0.01g 25.20±0.29a 5.10±0.09e 20±0.8ef 18.18±0.02i 19.50±0.06de

Treatments	pH	Titratable	Total soluble	Vit. C	Total	DPPH	FRAP
P₁D₀	3.10±0.06f	0.76±0.01a	18.00±0.50g	8.87±0.01a	30±0.4a	41.80±0.91a	26.10±0.06a
P₂D₀	3.10±0.06f	0.76±0.01a	18.00±0.50g	8.87±0.01a	30±0.6a	41.80±0.91a	26.10±0.06a
P₃D₀	3.10±0.06f	0.76±0.01a	18.00±0.50g	8.87±0.01a	30±0.6a	41.80±0.91a	26.10±0.06a
P₁D₁	3.48±0.01de	0.61±0.01c	19.98±0.87f	7.46±0.09b	26±0.3c	33.09±0.79d	20.10±1.10d
P₂D₁	3.56±0.07de	0.64±0.01b	20.86±0.83ef	7.77±0.07b	27±0.7bc	36.50±0.68b	24.20±0.06b
P₃D₁	3.40±0.06e	0.65±0.01b	21.73±0.12de	7.78±0.04b	28±0.5b	37.85±0.42b	26.60±0.06a
P₁D₂	3.63±0.07bc	0.52±0.01e	23.16±0.02bc	6.07±0.17d	21±0.5e	30.00±0.04fg	17.10±0.52f
P₂D₂	3.66±0.07bc	0.55±0.02de	23.20±0.00bc	6.96±0.23c	24±0.9d	30.81±0.71de	22.70±0.06c
P₃D₂	3.47±0.02de	0.55±0.01d	23.28±0.09bc	6.92±0.23c	26±0.9c	31.29±0.60d	24.20±0.06b
P₁D₃	3.69±0.01bc	0.43±0.01g	22.54±0.46dc	5.27±0.12e	19±0.8f	25.83±0.43h	13.50±0.23g
P₂D₃	3.60±0.06cd	0.44±0.01g	23.35±0.03bc	5.82±0.05d	21±0.8e	29.18±0.36e	19.00±0.00e
P₃D₃	3.63±0.03bcd	0.47±0.01f	23.50±0.09bc	5.87±0.06d	24±0.9d	29.28±0.31e	23.80±0.06b
P₁D₄	3.80±0.06b	0.39±0.01h	23.67±0.59bc	4.72±0.01f	16±0.9g	14.57±0.02k	12.10±0.40h
P₂D₄	4.10±0.00a	0.41±0.00gh	24.46±0.13ab	5.01±0.04ef	18±0.6f	16.69±0.01j	17.25±0.32f
P₃D₄	3.70±0.10bc	0.43±0.01g	25.20±0.29a	5.10±0.09e	20±0.8ef	18.18±0.02i	19.50±0.06de

In each column, means with same letters are not significant (P≤0.01) statistically based on Duncan’s multiple range test. Means are average of three replications±standard errors. P₁; 0 mM phenylalanine or control; P₂; 15 mM phenylalanine and P₃; 30 mM phenylalanine. D₀ (day 0); D₁ (15th day); D₂ (30th day); D₃ (45th day) and D₄ (60th day).

Berry TSS, TA, and vitamin C are the most important parameters used to evaluate the quality of fruit and reducing these materials decreases the quality and marketability of the table grape [6]. Based on the results, 30 mM Phe treatment caused greater retention in berry TSS and vitamin C content during storage than control fruits (Table 1) because Phe can modulate these compound catabolism due to postharvest respiration in horticultural crops [33]. The results of this study were consistent with those obtained by Sogvar et al. [13].

Regarding sensory attributes, a significant difference was found between control and Phe-treated grape samples during storage durations (Table 1). At the end of storage time, the total sensory score of Phe-treated grapes increased by 20% in compared to the control grape samples (Table 1). This may arise from higher TSS/TA, organic acids, soluble sugars, and also higher accumulation of phenolic compounds in Phe-treated grape samples in the current study. Phenolic compounds in grapes are important in determining the color, taste, and storage quality of fruit [17]. As recently reviewed by Luo et al. [34] for Muscadine grape, these phenolic compounds are considered to have several valuable effects on human health, in relation to their antioxidant properties.

3.2 Berry antioxidant capacity (DPPH and FRAP)

Changes in the antioxidant capacity as measured by DPPH and FRAP methods in grapes during cold storage durations are shown in Table 1. Berry antioxidant capacity decreased in control grape and Phe-treated grape samples from the beginning to the end of the storage. However, Phe treatment had a positive effect on antioxidant capacity retaining in treated grapes during cold storage durations (Table 1). During storage time, the berry DPPH scavenging capacity of 30 mM Phe-treated samples did not differ significantly with grape treated with the lower dose of Phe (15 Mm). However, regarding FRAP, grape treated with Phe showed higher scavenging capacity in respect to other treatments (Table 1). At the end of storage time, the berry DPPH and FRAP scavenging capacity of 30 mM Phe-treated samples were 20%, and 38% more than control grape samples, respectively (Table 1). High antioxidant activity reduced free radicals and oxidative damage [17, 34] and increased the storage of grape products [6]. According to many authors, antioxidant capacity of grapes, results mainly from phenolics, particularly flavonoids. Some researchers found a strong correlation between antioxidant capacities and phenolic compounds such as flavonoids and non-flavonoids of grape berry [8, 17]. In current work, Phe application increased antioxidant activity in treated grape berries. In agreement with our results, exogenous application of Phe improved postharvest antioxidant capacity on some horticultural commodities such as tomato and plum [12, 13].

3.3 Berry antioxidant activities

The activities of grape berry antioxidant enzymes (SOD, CAT, GPX, and APX) were affected significantly (P≤0.01) by Phe treatments and cold storage durations (Fig. 1A-D). Berry SOD, GPX, and APX activities increased on the 15th day then a decreasing trend were observed to the end of storage durations in Phe-treated grape samples. However, in control non-Phe-treated samples, SOD, GPX, and APX activities decreased from the beginning to the end of storage durations (Fig. 1A, C and D). Berry CAT activity decreased from the beginning to the end of storage in all grape samples (Fig. 1B). Interestingly, exogenous Phe had an impact on all SOD, CAT, GPX, and APX, increased their activities at higher levels in treated grapes during cold storage durations. For example, at the end of storage time, the activities of SOD, CAT, GPX, and APX of Phe-treated samples increased 43.9%, 23.8%, 57.3%, and 37.5 % more than control samples, respectively (Fig. 1). Under low temperature, due to the production of ROS, the activities of antioxidant enzymes are triggered [33]. Indeed, during the cold storage period, the products prevent chilling stress-induced oxidative stress by enzymatic and non-enzymatic antioxidant systems. In the present study, 30 mM Phe-treated fruits had higher activities of antioxidant enzymes (SOD, CAR, GPX, and APX) compared to control grapes, confirming the involvement of this amino acid in stimulation of ROS-scavenging enzymes which has been associated with reduced in membranes lipid peroxidation and electrolyte leakage. Exogenous application of Phe in some fruits and vegetables has led to an increase in the enzymatic antioxidant system in these products [12, 13].

Fig. 1

Effect of postharvest treatment with phenylalanine on SOD (A), CAT (B), GPX (C) and APX (D) activity of ‘Red Sahebi’ grape cultivar during storage at 1°C for 60 day. In each column, means with same letters are not significant (P≤0.01) statistically based on Duncan’s multiple range test. Means are average of three replications±standard errors.

3.4 Berry flavonoids

Flavonoids compounds including flavonols, flavanols (Table 2) and some anthocyanidins (delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, malvidin-3-O-glucoside, pelargonidin-3-O-glucoside; Table 3) of ‘Red Sahebi’ grape were affected significantly (P≤0.01) by Phe treatments and cold storage durations.

Table 2
Berry individual flavonols and flavanols (μg/g) and total flavonoids of ‘Red Sahebi’ grape in response to postharvest treatment with phenylalanine during cold storage

Treatments Flavonols Flavanols Total

(μg g^–1 berry) (μg g^–1 berry) flavonoids

Kaempferol Quercetin Myricetin Catechin Epicatechin Epigallocatechin (mg g^–1 berry)

P₁D₀ 2.73±0.01cd 16.92±0.01a 2.96±0.01e 11.52±0.00a 3.50±0.00a 3.25±0.00a 0.32±0.04c

P₂D₀ 2.73±0.01cd 16.92±0.01a 2.96±0.01e 11.52±0.00a 3.50±0.00a 3.25±0.00a 0.32±0.04c

P₃D₀ 2.73±0.01cd 16.92±0.01a 2.96±0.01e 11.52±0.00a 3.50±0.00a 3.25±0.00a 0.32±0.04c

P₁D₁ 2.42±0.05e 13.53±0.16f 3.11±0.07d 9.65±0.15d 2.36±0.06de 2.40±0.04c 0.37±0.07bc

P₂D₁ 2.83±0.01b 15.22±0.01d 3.66±0.01c 10.68±0.01c 2.95±0.01c 2.83±0.02b 0.45±0.05b

P₃D₁ 2.94±0.03a 15.74±0.01c 3.84±0.01b 11.01±0.01b 3.17±0.01b 3.14±0.00a 0.47±0.01b

P₁D₂ 2.22±0.05f 14.83±0.22d 2.83±0.04f 7.80±0.24g 1.55±0.09g 1.23±0.05e 0.18±0.03de

P₂D₂ 2.67±0.01d 16.34±0.01b 3.86±0.01b 9.48±0.01d 2.11±0.01f 1.93±0.06d 0.58±0.02a

P₃D₂ 2.91±0.02a 16.81±0.01a 4.22±0.01a 10.45±0.01c 2.43±0.10d 2.37±0.05c 0.58±0.03a

P₁D₃ 2.13±0.04g 13.75±0.46f 2.63±0.09g 7.33±0.33h 1.32±0.07h 0.93±0.03g 0.11±0.00e

P₂D₃ 2.19±0.02fg 14.32±0.01e 3.81±0.01b 8.66±0.33f 1.63±0.04g 1.35±0.04e 0.20±0.04de

P₃D₃ 2.80±0.00bc 14.39±0.02e 4.19±0.00a 9.00±0.58e 2.22±0.01ef 1.84±0.04d 0.27±0.03cd

P₁D₄ 2.05±0.02h 10.14±0.14h 2.63±0.03g 6.56±0.11g 1.10±0.03i 0.82±0.03g 0.12±0.01e

P₂D₄ 2.15±0.01fg 12.34±0.16g 2.92±0.06e 7.03±0.17i 1.30±0.14h 1.03±0.03f 0.14±0.01e

P₃D₄ 2.67±0.01d 12.72±0.01g 2.96±0.01e 8.42±0.01f 1.67±0.01g 1.37±0.05e 0.18±0.03de

Treatments	Flavonols	Flavanols	Total
P₁D₀	2.73±0.01cd	16.92±0.01a	2.96±0.01e	11.52±0.00a	3.50±0.00a	3.25±0.00a	0.32±0.04c
P₂D₀	2.73±0.01cd	16.92±0.01a	2.96±0.01e	11.52±0.00a	3.50±0.00a	3.25±0.00a	0.32±0.04c
P₃D₀	2.73±0.01cd	16.92±0.01a	2.96±0.01e	11.52±0.00a	3.50±0.00a	3.25±0.00a	0.32±0.04c
P₁D₁	2.42±0.05e	13.53±0.16f	3.11±0.07d	9.65±0.15d	2.36±0.06de	2.40±0.04c	0.37±0.07bc
P₂D₁	2.83±0.01b	15.22±0.01d	3.66±0.01c	10.68±0.01c	2.95±0.01c	2.83±0.02b	0.45±0.05b
P₃D₁	2.94±0.03a	15.74±0.01c	3.84±0.01b	11.01±0.01b	3.17±0.01b	3.14±0.00a	0.47±0.01b
P₁D₂	2.22±0.05f	14.83±0.22d	2.83±0.04f	7.80±0.24g	1.55±0.09g	1.23±0.05e	0.18±0.03de
P₂D₂	2.67±0.01d	16.34±0.01b	3.86±0.01b	9.48±0.01d	2.11±0.01f	1.93±0.06d	0.58±0.02a
P₃D₂	2.91±0.02a	16.81±0.01a	4.22±0.01a	10.45±0.01c	2.43±0.10d	2.37±0.05c	0.58±0.03a
P₁D₃	2.13±0.04g	13.75±0.46f	2.63±0.09g	7.33±0.33h	1.32±0.07h	0.93±0.03g	0.11±0.00e
P₂D₃	2.19±0.02fg	14.32±0.01e	3.81±0.01b	8.66±0.33f	1.63±0.04g	1.35±0.04e	0.20±0.04de
P₃D₃	2.80±0.00bc	14.39±0.02e	4.19±0.00a	9.00±0.58e	2.22±0.01ef	1.84±0.04d	0.27±0.03cd
P₁D₄	2.05±0.02h	10.14±0.14h	2.63±0.03g	6.56±0.11g	1.10±0.03i	0.82±0.03g	0.12±0.01e
P₂D₄	2.15±0.01fg	12.34±0.16g	2.92±0.06e	7.03±0.17i	1.30±0.14h	1.03±0.03f	0.14±0.01e
P₃D₄	2.67±0.01d	12.72±0.01g	2.96±0.01e	8.42±0.01f	1.67±0.01g	1.37±0.05e	0.18±0.03de

Table 3

Some individual anthocyanins (mg/g), total anthocyanins (mg/100 g) and organic acids (mg/100 g) of ‘Red Sahebi’ grape in response to postharvest treatment with phenylalanine during cold storage

Treatments	Malvidin-	Delphinidin-	Cyanidin-	Pelargonidin-	Total	Tartaric	Malic
	3-O-	3-O-	3-O-	3-O-	anthocyanins	acid	acid
	glucoside	glucoside	glucoside	glucoside
P₁D₀	4.48±0.01a	5.97±0.01a	4.11±0.06b	5.94±0.02a	6.17±0.38a	177.1±0.0a	39.66±1.23a
P₂D₀	4.48±0.01a	5.97±0.01a	4.11±0.06b	5.94±0.02a	6.17±0.38a	177.1±0.0a	39.66±1.23a
P₃D₀	4.48±0.01a	5.97±0.01a	4.11±0.06b	5.94±0.02a	6.17±0.38a	177.1±0.0a	39.66±1.23a
P₁D₁	2.49±0.05h	4.40±0.07d	2.17±0.05g	4.66±0.08e	5.70±0.21ab	145.2±0.3c	33.30±0.40c
P₂D₁	3.43±0.02d	5.07±0.01c	3.38±0.00d	5.43±0.01c	6.07±0.30a	157.6±0.3b	37.00±0.58b
P₃D₁	3.57±0.01c	5.35±0.03b	3.64±0.01c	5.71±0.01b	6.03±0.31c	159.7±1.3b	39.46±0.23a
P₁D₂	3.03±0.06f	3.18±0.06j	2.87±0.06e	3.81±0.07h	4.98±0.07bc	114.3±1.4e	24.00±0.40f
P₂D₂	3.62±0.01c	3.94±0.01f	4.21±0.01b	4.74±0.01e	5.13±0.24bc	130.6±0.3d	29.33±0.17de
P₃D₂	3.82±0.01b	4.24±0.01e	4.48±0.01a	4.94±0.01d	5.20±0.21bc	143.6±1.4c	30.26±0.13d
P₁D₃	1.88±0.06i	2.72±0.06k	1.72±0.07j	3.21±0.09i	3.75±0.17d	93.4±1.1g	22.76±0.39f
P₂D₃	3.14±0.01e	3.61±0.01h	2.63±0.02f	4.40±0.07f	4.82±0.05c	129.9±0.5d	28.20±0.35e
P₃D₃	3.15±0.01e	3.82±0.01g	3.47±0.01d	4.34±0.01f	4.76±0.11c	130.3±1.1d	30.30±0.00d
P₁D₄	1.81±0.05i	2.39±0.06l	1.51±0.05k	3.17±0.08i	3.60±0.20d	91.5±1.04g	17.70±0.35g
P₂D₄	2.91±0.06g	3.11±0.07j	1.88±0.06i	3.93±0.07h	4.76±0.06c	110.7±1.4f	27.90±0.40e
P₃D₄	3.15±0.01e	3.40±0.00i	2.02±0.01h	4.14±0.01g	4.72±0.05c	114.8±1.0e	28.16±0.22e

Individual flavonols including quercetin, myricetin, and kaempferol as three predominant grape flavonols were affected significantly (P≤0.01) by Phe treatments and cold storage durations. The berry quercetin, myricetin, and kaempferol contents decreased simultaneously with advancement in storage durations in control and Phe-treated fruits (Table 2). However, this flavonols contents were found to be higher in Phe-treated grape samples compared to control grape samples (Table 2). At the end of storage time, berry kaempferol and myricetin contents of 30 mM Phe-treated grapes were retained 23.2% and 11.2% more than control grape samples, respectively (Table 2). Regarding berry quercetin, the highest contents on the 15th and 30th days were related to those grapes treated with Phe at 30 mM, however, in storage durations of 45 and 60 days, no significant differences were found between grapes treated with 15 and 30 mM Phe (Table 2). Flavonols are a group of colorless flavonoids that accumulate mainly in the skin of the berry. More than 90% of flavonols in colored-skin grapes are related to quercetin, myricetin, and kaempferol [4]. In the present study, the contents of these three important flavonols in grapes treated with Phe were higher than control grapes. Increased flavonols contents in response to foliar application of Phe and urea in grapes has been reported, which confirm the findings of the present study [14].

Individual flavanols (flavan-3-ols), including catechin, epicatechin, and epigallocatechin as three main grape flavanols were affected significantly (P≤0.01) by Phe treatments and cold storage durations (Table 2). The berry flavanols contents decreased concurrently with progress in storage durations in control and Phe-treated fruits (Table 2). However, catechin, epicatechin, and epigallocatechin were found to be higher in Phe-treated grape samples especially at 30 mM compared to control grape samples (Table 2). For example, at the end of storage time, the berry catechin, epicatechin, and epigallocatechin of 30 mM Phe-treated samples were preserved 22 %, 34%, and 40 % more than control grape samples, respectively (Table 2).

Flavanols (flavan-3-ols) are synthesized in large quantities in the skin, pulp, and seed of berries. Among flavanols, catechin and epicatechin are the two most important flavanols in grapes, and gallocatechin and epigallocatechin present in lower proportions [4]. In the present study, exogenous Phe led to an increase in flavanols in Phe-treated fruits compared to control fruits, indicating the role of this amino acid in the biosynthesis or retention of catechin, epicatechin, and other flavanols. In a study by Portu et al. [14], foliar application of Phe in Tempranillo grapes increased biosynthesis and accumulation of berry flavanols of Phe-treated vines. However, in the present study, it seems that Phe has led to the preservation of these flavonoids mainly through preventing or lowering the catabolism of flavanols than their biosynthesis, which of course needs further investigation. Although the effect of postharvest Phe application on grape phenolic acid and individual flavonoids have not been studied until this work, some authors have documented the effectiveness of the foliar application of Phe in berry phenolic compounds enhancement of Tempranillo grapevine during growing season [14] which is line with current study results.

Delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, malvidin-3-O-glucoside, pelargonidin-3-O-glucoside were affected significantly (P≤0.01) by Phe treatments and cold storage durations (Table 3). Berry anthocyanidins contents decreased concomitant with progress in storage durations in all fruits, however, their contents were found to be higher in Phe-treated grape samples compared to control grape samples (Table 3). Exogenous Phe at 30 mM was found to be effective in berry Delphinidin-3-O-glucoside, cyanidin-3-O-glucoside and pelargonidin-3-O-glucoside retention compared to 15 mM Phe during all storage durations. Regarding malvidin-3-O-glucoside, no significant differences were found between grapes treated with 15 and 30 mM Phe except for 60th day which its content was higher in grapes treated with Phe at 30 mM than 15 mM (Table 3). At the end of storage time, the berry malvidin-3-O-glucoside, delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, and pelargonidin-3-O-glucoside of 30 mM Phe-treated samples were preserved 42.5%, 29.7%, 25.2%, and 23.4% more than control grape samples, respectively (Table 3).

Anthocyanins as the end-product of the phenylpropanoid pathway are formed by the condensation of sugars [4]. In the present study, cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, malvidin-3-O-glucoside, and pelargonidin 3-O-glucoside were measured in all grape samples during storage. Pelargonidin 3-O-glucoside is present in small amounts in Vitis vinifera grape varieties [35], but interestingly, this anthocyanidin was detected in both control and Phe-treated ‘Red Sahebi’ grape samples, which is consistent with previous reports [8 , 37]. This may be related to cultivar genetics background, vineyard site, viticultural operations, harvesting time and also cold storage conditions [8 , 17]. It should be noted that ‘Red Sahebi’ is a seeded late -ripening red grape cultivar [8] which usually harvested in the second week of October in Malayer, Iran. The presence of pelargonidin in this cultivar may be arise from low temperature (with an average of 16ºC) near harvest time and altitude of vineyard situation (in term of intensity and quality of light i.e. UV radiation), which, of course, requires further investigation.

In current work, the contents of all measured anthocyanidins were maintained at higher levels in Phe-treated fruits with respect to control fruits during storage. This is the first report of Phe-induced anthocyanidin in grapes during postharvest cold storage. However, the positive effect of Phe on anthocyanin biosynthesis in foliar-sprayed grapevine with Phe during the growing season was reported by Purto et al. [14] which confirm the current study results. Anthocyanins are one of the most important phenolic compounds affecting fruit quality due to their antioxidant properties and also these phenolic compounds can protect the fruits against biotic and abiotic stresses [17]. Alongside their nutritional values, the higher accumulation of anthocyanins in fruits and vegetables during cold storage causes an increase in cold tolerance [38]. Considering the role of Phe as one of the main precursors of the phenylpropanoid pathway, as the main pathway for the biosynthesis of phenolic compounds such as anthocyanins [4], it seems that the exogenous application of this amino acid affects the biosynthesis of various phenolic compounds leading to the regulation of its content during cold storage course, however, the measuring of enzymes involving in the biosynthesis pathway of anthocyanins can improve our outlook to this issue.

3.5 Berry non-flavonoid compounds and PPO activity

The results of berry non-flavonoid phenolic compounds, including phenolic acids, resveratrol, and total phenol in response to Phe and cold storage treatments are shown in Table 4.

Table 4
Berry non-flavonoid compounds (μg/g), total phenol (mg/g) and polyphenol oxidase (PPO; U/g) enzyme of ‘Red Sahebi’ grape in response to postharvest treatment with phenylalanine during cold storage

Treatments Gallic Caffeic P-coumaric Ferulic trans- Total PPO

acid acid acid acid Resveratrol phenol activity

P₁D₀ 8.24±0.00a 3.45±0.00a 13.93±0.01a 11.46±0.21a 21.52±0.01a 2.73±0.3a 2.96±0.08g

P₂D₀ 8.24±0.00a 3.45±0.00a 13.93±0.01a 11.46±0.21a 21.52±0.01a 2.73±0.3a 2.96±0.08g

P₃D₀ 8.24±0.00a 3.45±0.00a 13.93±0.01a 11.46±0.21a 21.52±0.01a 2.73±0.3a 2.96±0.08g

P₁D₁ 6.82±0.19c 2.48±0.06c 12.11±0.14d 9.23±0.10e 18.15±0.17d 2.28±0.1c 3.40±0.06e

P₂D₁ 7.86±0.01b 3.12±0.01b 12.95±0.01c 10.56±0.01c 19.20±0.01c 2.42±0.3b 3.27±0.13f

P₃D₁ 8.13±0.01ab 3.18±0.01b 13.23±0.01b 10.97±0.01b 20.54±0.01b 2.56±0.3b 3.25±0.07f

P₁D₂ 5.54±0.17ef 1.23±0.05gh 9.41±0.17g 7.34±0.18g 14.16±0.17i 1.90±0. 4e 4.36±0.00b

P₂D₂ 6.25±0.01d 1.68±0.01e 9.72±0.01ef 9.38±0.01e 15.63±0.01f 2.18±0.2d 3.92±0.03d

P₃D₂ 6.43±0.01d 1.83±0.01d 9.97±0.01e 9.76±0.01d 19.95±0.01e 2.35±0.2bc 3.46±0.04e

P₁D₃ 4.65±0.10g 1.12±0.06i 7.63±0.14i 6.34±0.18h 13.13±0.19k 1.69±0.4f 4.78±0.00a

P₂D₃ 5.71±0.18e 1.50±0.05f 9.47±0.13fg 7.62±0.01g 14.62±0.02h 2.06±0.3e 4.12±0.01c

P₃D₃ 6.15±0.03d 1.63±0.03e 9.95±0.01e 8.05±0.10f 15.51±0.04f 2.22±0.5d 3.80±0.05d

P₁D₄ 4.40±0.10g 0.92±0.02j 6.63±0.14j 5.62±0.11i 11.74±0.14l 1.12±0.1h 4.89±0.00a

P₂D₄ 5.31±0.15f 1.22±0.04hi 8.53±0.08h 6.36±0.14h 13.50±0.22j 1.52±0.4g 4.33±0.09b

P₃D₄ 5.34±0.17f 1.32±0.01g 8.63±0.14h 6.40±0.10h 14.95±0.01g 1.80±0.2ef 3.90±0.00d

Treatments	Gallic	Caffeic	P-coumaric	Ferulic	trans-	Total	PPO
P₁D₀	8.24±0.00a	3.45±0.00a	13.93±0.01a	11.46±0.21a	21.52±0.01a	2.73±0.3a	2.96±0.08g
P₂D₀	8.24±0.00a	3.45±0.00a	13.93±0.01a	11.46±0.21a	21.52±0.01a	2.73±0.3a	2.96±0.08g
P₃D₀	8.24±0.00a	3.45±0.00a	13.93±0.01a	11.46±0.21a	21.52±0.01a	2.73±0.3a	2.96±0.08g
P₁D₁	6.82±0.19c	2.48±0.06c	12.11±0.14d	9.23±0.10e	18.15±0.17d	2.28±0.1c	3.40±0.06e
P₂D₁	7.86±0.01b	3.12±0.01b	12.95±0.01c	10.56±0.01c	19.20±0.01c	2.42±0.3b	3.27±0.13f
P₃D₁	8.13±0.01ab	3.18±0.01b	13.23±0.01b	10.97±0.01b	20.54±0.01b	2.56±0.3b	3.25±0.07f
P₁D₂	5.54±0.17ef	1.23±0.05gh	9.41±0.17g	7.34±0.18g	14.16±0.17i	1.90±0. 4e	4.36±0.00b
P₂D₂	6.25±0.01d	1.68±0.01e	9.72±0.01ef	9.38±0.01e	15.63±0.01f	2.18±0.2d	3.92±0.03d
P₃D₂	6.43±0.01d	1.83±0.01d	9.97±0.01e	9.76±0.01d	19.95±0.01e	2.35±0.2bc	3.46±0.04e
P₁D₃	4.65±0.10g	1.12±0.06i	7.63±0.14i	6.34±0.18h	13.13±0.19k	1.69±0.4f	4.78±0.00a
P₂D₃	5.71±0.18e	1.50±0.05f	9.47±0.13fg	7.62±0.01g	14.62±0.02h	2.06±0.3e	4.12±0.01c
P₃D₃	6.15±0.03d	1.63±0.03e	9.95±0.01e	8.05±0.10f	15.51±0.04f	2.22±0.5d	3.80±0.05d
P₁D₄	4.40±0.10g	0.92±0.02j	6.63±0.14j	5.62±0.11i	11.74±0.14l	1.12±0.1h	4.89±0.00a
P₂D₄	5.31±0.15f	1.22±0.04hi	8.53±0.08h	6.36±0.14h	13.50±0.22j	1.52±0.4g	4.33±0.09b
P₃D₄	5.34±0.17f	1.32±0.01g	8.63±0.14h	6.40±0.10h	14.95±0.01g	1.80±0.2ef	3.90±0.00d

In each column, means with same letters are not significant (P≤0.01) statistically based on Duncan’s multiple range test. Means are average of three replications±standard errors. P₁; 0 mM phenylalanine or control; P₂; 15 mM Phenylalanine and P₃; 30 mM Phenylalanine. D₀ (day 0); D₁ (15th day); D₂ (30th day); D₃ (45th day) and D₄ (60th day).

Gallic acid as a main hydroxybenzoic acid in grapes was affected significantly (P≤0.01) by Phe treatments and cold storage durations. Berry gallic acid content decreased concomitantly with progress in storage durations in both control and Phe-treated fruits (Table 4). However, gallic acid was found to be higher in Phe-treated grape samples compared to control grape samples. There were no significant differences between the Phe-treated fruits at 15 and 30 mM for gallic acid content during storage durations except for 45th day which Phe at 30 mM showed higher gallic acid content (Table 4).

The contents of main hydroxycinnamic acids in grape berry, including p-coumaric acid, caffeic acid, and ferulic acid were affected significantly (P≤0.01) by Phe treatments and cold storage durations (Table 4). Berry hydroxycinnamic acids contents decreased concomitant with progress in storage durations in all fruits, however, their contents were found to be higher in Phe-treated grape samples compared to control grape samples (Table 4). Exogenous Phe at 30 mM was found to be effective in berry p-coumaric acid, caffeic acid, and ferulic acid retention compared to 15 mM during all storage durations except for 60th day which did not significantly differ with a lower dose of Phe for p-coumaric acid and ferulic acid (Table 4).

Resveratrol as a main stilbene in grapes and also total phenol contents were affected significantly (P≤0.01) by Phe treatments and cold storage durations. Berry trans-resveratrol and total phenol content decreased simultaneously with advancement in storage durations in control and Phe-treated fruits (Table 4). However, this stilbene and total phenol contents were found to be higher in Phe-treated grape samples especially at 30 mM compared to control grape samples. At the end of storage time, the berry trans-resveratrol and total phenol content of 30 mM Phe-treated grape were preserved 19.5% and 37.7% more than control grape samples, respectively (Table 4).

In the present study, the contents of phenolics, including flavonoid and non-flavonoid compounds showed a decreasing trend during the cold storage periods. However, the contents of these groups of secondary metabolites were significantly higher in grapes treated with Phe, especially at 30 mM which can be attributed with changes occurring in phenol metabolism in treated grapes during storage periods. In addition to biosynthesis through phenylpropanoid pathway, the regulation or turnover of phenolics in plants may take place through three important reactions, including 1. interconversions in the biosynthetic sequence, 2. catabolism and the production of primary metabolites, and 3. oxidative polymerization reactions [3, 4]. Phenolic degradation in grapes may be associated with enzymatic activities especially, PPO as the main agent responsible for the degradation of phenolic compounds in plants because of the naturally synthesized of this enzyme by fruit [7].

Although the effects of postharvest application of Phe on grape phenolic acids and individual flavonoids have not been studied yet, some authors have documented the effectiveness of the foliar application of Phe in berry phenolic compounds enhancement of Tempranillo grapevine [14] which is in line with current study results. Due to grapes are non-climacteric fruits, therefore the higher contents of phenolic compounds of Phe-treated fruits may be arise from reduced catabolism and oxidation reactions as documented by lower PPO activity of treated fruits in current study. However, more detailed studies regarding genes involved in the biosynthesis of phenolic compounds as well as enzymes involved in the biosynthetic pathways of these compounds are necessary to assess the metabolism and catabolism of these compounds during storage in response to exogenous application of Phe.

Polyphenol oxidase activity also increased in both control and Phe-treated fruit throughout the storage periods, reached the maximum value at the end of cold storage (Table 4). However, PPO activity was lower in Phe-treated fruit than in control fruit during cold storage. At the end of storage, the fruit treated with Phe at 30 mM, showed 20% lower PPO activity than control fruits (Table 4). However, in the present study, the activity of PPO was found to be higher in control grapes than Phe-treated grapes, indicating the involvement of this amino acid in preventing the reduction of various phenolic compounds during storage. On the other hand, our results showed the higher activities of antioxidant enzymes (SOD, CAT, GPX, and APX) and antioxidant capacity (DPPH, FRAP) in fruits treated with Phe than control fruits. This finding indicates a decrease in free oxygen radicals in Phe -treated fruits and therefore a decrease in the oxidation of various molecules, representing the higher preservation of phenolic compounds by reducing the destructive process including catabolism or enzymatic and non-enzymatic oxidation. Enzymatic browning is one of the major disorders in grapes. Berry skin browning is caused by enzymatic oxidation such as polyphenol oxidase, which is a serious post-harvest problem in grapes [39]. In plum, PPO activity increased due to cold injury when fruit stored at 0°C [39]. Therefore, low PPO activity in Phe-treated fruits appears to be associated with lower cold injury and rachis browning occurrence. Sogvar et al. [13] reported that treatment of plums with Phe reduced PPO activity during storage at 1°C, which confirms the results of the present study.

3.6 Berry organic acids (tartaric acid and malic acid)

Changes in berry tartaric acid and malic acid as two predominant organics acids in grapes during cold storage durations are shown in Table 3. Berry tartaric acid and malic acid contents decreased in control and Phe-treated grape samples from the beginning to the end of the storage. However, Phe treatment had a positive effect on organic acids and increases their contents in treated grapes during cold storage durations. For example, on the 60th day, the berry tartaric acid and malic acid of 30 mM Phe-treated samples were preserved 20.3% and 37.1% more than control samples, respectively (Table 3). Many organic acids such as citric acid, malic acid, and tartaric acid are present in fruits, and most fruits are dominated by one or two organic acids while the amount of other acids is low [33]. Organic acids can also maintain fruit quality and protect fruits from abiotic stresses. The decrease in the content of organic acids in the present study maybe is due to the participation of organic acids in the respiration process, which also leads to an increase in sugar concentration. The preserved organic acids in grapes treated with 30 mM Phe may arise from keeping the respiration rate down.

3.7 Berry soluble sugars (fructose, glucose and sucrose)

The results of berry soluble sugars including glucose, fructose, and sucrose contents during of 60 days cold storage are shown in Fig. 2. All soluble sugars were affected by Phe treatments and storage durations in both control and Phe-treated grape samples (Fig. 2).

Fig. 2

Effect of postharvest treatment with phenylalanine on glucose (A), fructose (B),sucrose (C) and abscisic acid (ABA) (D) content of ‘Red Sahebi’ grape cultivar during storage at 1°C for 60 day. In each column, means with same letters are not significant (P≤0.01) statistically based on Duncan’s multiple range test. Means are average of three replications±standard errors.

Berry glucose and fructose contents increased on the 15th day of storage, then decreased on the 30th, 45th and 60th days of storage in control grape and Phe-treated grape samples (Fig. 2A and B). Regarding berry sucrose content, an increase in this disaccharide was monitored on the 15th day then a decreasing trend observed to the end of storage durations in Phe-treated grape samples. However, in control non-Phe-treated samples, sucrose content decreased from the beginning to the end of storage durations (Fig. 2C). Interestingly, exogenous Phe had an impact on all soluble sugars and increased their content in treated grapes during cold storage durations. For example, at the end of storage time, the berry glucose, fructose, and sucrose of Phe-treated samples were retained 21.8%, 24.3%, and 23.2 % more than control samples, respectively (Fig. 2). Sugars are one of the most important sources of energy that contribute to the quality and taste of fruit [17]. Sugar metabolism is essential to help antioxidant compounds for protecting fruit cells from cold and oxidative stress [40].

3.8 Berry abscisic acid

Based on the results, ABA content increased gradually in Phe-treated and control grape samples on the 15th day of storage and then decreased to some extent until the end of storage time (Fig. 2D). However, the berry ABA content always was higher in Phe-treated grape samples. For example, on the 15th day and 60th day of storage, berry ABA content of Phe-treated at 30 mM increased 37% and 21% compared to control samples, respectively (Fig. 2D). Abscisic acid, as a signaling molecule, has been reported to be effective for maintaining tolerance to cold injury by activating antioxidant enzymes [8, 9] (Karimi et al., 2016; Asgarian et al., 2022). The higher chilling tolerance observed in the current study was linked with higher berry ABA content in Phe treated samples.

3.9 Berry membrane EL, MDA and CI

The results for EL, MDA, and CI indices are shown in Table 5. There were significant differences between the Phe-treated and control bunches for EL, MDA, and CI parameters. During cold storage periods the membranes health indices and CI increased in all samples, however, these indices were found to be lower in Phe-treated grapes compared to control grapes. At the end of storage (60th day), the EL, MDA, and CI of 30 mM Phe-treated grape samples were 23.4%, 32.5%, and 34.9% lower than control non-Phe-treated grapes, respectively (Table 5). Electrolyte leakage and MDA indices had been used to evaluate membrane integrity in fruits and vegetables in response to cold stress [33]. Reduction in berry EL, MDA, and CI in 30 mM Phe-treated grapes compared to control grapes may arise from the higher activities of antioxidant enzymes (SOD, CAT, GPX, and APX) and higher accumulation of different phenolic compounds and also higher content of ABA in these fruits. The effect of Phe on individual flavonoids and ABA contents of cold storage fruits such as grapes has not been studied to date, however, the effect of this amino acid on maintaining the stability and integrity of the membrane of fruits Phe has been reported in previous studies [12, 13]. Phenylpropanoid pathway products such as phenols have antioxidant properties [3] which can alleviate the chilling- induced oxidative stress in fruits during low temperature storage periods [9]. Therefore, the application of treatments that can reduce the degradation, oxidation, and polymerization of phenolic compounds can also preserve these secondary metabolites and increase cold tolerance of cold-stored fruits. The ability of Phe to stimulate the activities of antioxidant enzymes (SOD, CAT, GPX, and APX) and to retain various phenolic compounds, including flavonoids and resveratrol in treated fruits is important in terms of nutritional value for human health.

Table 5
Berry weight loss, rachis browning, chilling injury, membrane health indices (EL and MDA) and decay incident of ‘Red Sahebi’ grape in response to postharvest treatment of phenylalanine during cold storage

Treatments Weight loss Rachis Chilling injury Electrolyte Malondialdehyde Decay

(%) browning (%) leakage (μmol g^–1 FW) incident

(%) (%) (%)

P₁D₀ 0.00±0.00e 1.00±0.00c 0.00±0.00l 25.7±0.00j 1.08±0.01g 0.00±0.0j

P₂D₀ 0.00±0.00e 1.00±0.00c 0.00±0.00l 24.8±0.00j 1.08±0.01g 0.00±0.0j

P₃D₀ 0.00±0.00e 1.00±0.00c 0.00±0.00l 25.2±0.00j 1.08±0.01g 0.00±0.0j

P₁D₁ 0.38±0.09d 1.33±0.33c 5.00±0.00j 39.5±0.09h 1.18±0.00g 3.54±0.8i

P₂D₁ 0.36±0.10d 1.16±0.17c 2.00±0.00k 35.2±1.04i 1.10±0.01g 0.00±0.0j

P₃D₁ 0.25±0.00d 1.00±0.00c 2.00±0.00k 32.5±0.09i 1.09±0.02g 0.00±0.0j

P₁D₂ 0.38±0.09d 1.33±0.33c 13.35±0.08g 48.0±0.58f 1.52±0.09e 14.4±1.9g

P₂D₂ 0.37±0.08d 1.33±0.33c 10.95±0.19h 45.6±0.12g 1.20±0.01f 7.18±1.4h

P₃D₂ 0.30±0.12d 1.16±0.17c 8.09±0.10i 41.3±0.12h 1.20±0.03f 4.69±1.3i

P₁D₃ 0.91±0.16b 3.33±0.33a 27.38±1.06d 59.3±0.06c 1.91±0.02bc 27.9±2.1c

P₂D₃ 0.70±0.02c 2.33±0.33b 19.00±1.29e 55.2±1.04d 1.88±0.04c 18.0±1.2e

P₃D₃ 0.44±0.02d 1.83±0.44bc 15.56±2.31f 52.5±0.09e 1.73±0.07d 13.6±0.9f

P₁D₄ 1.20±0.03a 3.33±0.33a 42.58±0.46a 74.5±0.12a 2.47±0.59a 41.3±1.9a

P₂D₄ 0.97±0.04b 2.33±0.33b 32.80±0.97b 63.0±0.58b 2.09±0.06b 30.8±1.5b

P₃D₄ 0.70±0.06c 1.83±0.44bc 27.73±1.86 c 57.1±0.12c 1.85±0.08c 24.4±2.3d

Treatments	Weight loss	Rachis	Chilling injury	Electrolyte	Malondialdehyde	Decay
P₁D₀	0.00±0.00e	1.00±0.00c	0.00±0.00l	25.7±0.00j	1.08±0.01g	0.00±0.0j
P₂D₀	0.00±0.00e	1.00±0.00c	0.00±0.00l	24.8±0.00j	1.08±0.01g	0.00±0.0j
P₃D₀	0.00±0.00e	1.00±0.00c	0.00±0.00l	25.2±0.00j	1.08±0.01g	0.00±0.0j
P₁D₁	0.38±0.09d	1.33±0.33c	5.00±0.00j	39.5±0.09h	1.18±0.00g	3.54±0.8i
P₂D₁	0.36±0.10d	1.16±0.17c	2.00±0.00k	35.2±1.04i	1.10±0.01g	0.00±0.0j
P₃D₁	0.25±0.00d	1.00±0.00c	2.00±0.00k	32.5±0.09i	1.09±0.02g	0.00±0.0j
P₁D₂	0.38±0.09d	1.33±0.33c	13.35±0.08g	48.0±0.58f	1.52±0.09e	14.4±1.9g
P₂D₂	0.37±0.08d	1.33±0.33c	10.95±0.19h	45.6±0.12g	1.20±0.01f	7.18±1.4h
P₃D₂	0.30±0.12d	1.16±0.17c	8.09±0.10i	41.3±0.12h	1.20±0.03f	4.69±1.3i
P₁D₃	0.91±0.16b	3.33±0.33a	27.38±1.06d	59.3±0.06c	1.91±0.02bc	27.9±2.1c
P₂D₃	0.70±0.02c	2.33±0.33b	19.00±1.29e	55.2±1.04d	1.88±0.04c	18.0±1.2e
P₃D₃	0.44±0.02d	1.83±0.44bc	15.56±2.31f	52.5±0.09e	1.73±0.07d	13.6±0.9f
P₁D₄	1.20±0.03a	3.33±0.33a	42.58±0.46a	74.5±0.12a	2.47±0.59a	41.3±1.9a
P₂D₄	0.97±0.04b	2.33±0.33b	32.80±0.97b	63.0±0.58b	2.09±0.06b	30.8±1.5b
P₃D₄	0.70±0.06c	1.83±0.44bc	27.73±1.86 c	57.1±0.12c	1.85±0.08c	24.4±2.3d

3.10 Weight loss, rachis browning and fungal decay

The result of bunches’ weight loss, rachis browning, and fungal decay are shown in Table 5. Based on the results from the beginning to the 30th day of storage, there was no significant difference between the Phe-treated grapes and control grapes for weight loss and rachis browning percentage (Table 5). However, on the 45th day and 60th day of storage durations, the weight loss and rachis browning were significantly lower in Phe-treated grape samples compared to control samples (Table 5). On the 60th day of storage, the weight loss and rachis browning rate of 30 mM Phe-treated grape bunches were 32% and 45% lower than control samples, respectively (Table 5). Weight loss of fruits during storage is due to evaporation of water from the fruit surface and depletion of fruit reserves as a result of respiration [11]. Also, Sogvar et al. [13] showed that Phe has an effective role in preventing plum fruit weight loss. Rachis browning is a common marketing problem for table grapes. A lower rate of rachis browning was observed in Phe-treated grapes in our experiment (Table 5) which may arise from lower PPO activity and higher antioxidant enzyme activities in these fruits.

Data analysis revealed that exogenous Phe had an impact on grape berries’ fungal decay incident. Indeed, Phe-treated grape samples, especially at 30 mM, displayed less fungal decay incident (41%) during cold storage durations compared to control grape samples (Table 5). Higher accumulation of phenolic compounds, especially resveratrol, in Phe-treated fruits can reduce or inhibit the growth of microorganisms in this fruit. Resveratrol as the main phytoalexins can stimulate endogenous resistance mechanisms against the pathogen [17]. The positive effect of resveratrol as a phytoalexins on reduction in decay produced by B. cinerea infection in the surface of grape berry was reported by Asgarian et al. [8] confirming the results of the current study.

4 Conclusion

The Phe- treated fruits (30 mM) had a high activity of antioxidant enzymes such as SOD, CAT, GPX, and APX during storage for 60 days. Phenylalanine caused a considerable retain in grape berries phenolic acids compared to control samples after the 60th day. Moreover, flavonols, flavan-3-ols and anthocyanidins (cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, malvidin-3-O-glucoside, and pelargonidin-3-O-glucoside) and non-flavonoids contents including phenolic acids and trans-resveratrol increased in fruits treated with Phe at 30 mM owing to decreased polyphenol oxidase activity. At the end of storage, treatment of the ‘Red Sahebi’ grape with Phe reduced the cold symptoms, MDA, EL, and fungal decay due to the higher activities of antioxidant enzymes including GPX, CAT, SOD, and APX. According to the findings of this study, it can be concluded that Phe creates a strong antioxidant system, protecting fruits against oxidative stress and ROS and improving the fruit’s defense system. The higher preservation of phenolic acids and flavonoids especially in Phe-treated grape berries during cold storage period is important nutritionally due to their potential beneficial effects on human health.

Footnotes

Acknowledgments

Funding was provided by Malayer University, Iran (R. Karimi; Grant no. 84.5–310). The authors gratefully acknowledge the Malayer University.

Funding

The authors report no funding.

Conflict of interest

The authors have no conflict of interest to report.

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