Effects of pre-storage ascorbic and salicylic acids ...

Vibrational Spectroscopy 115 (2021) 103269

Available online 24 May 20210924-2031/© 2021 Elsevier B.V. All rights reserved.

Effects of pre-storage ascorbic and salicylic acids treatments on the enzymatic browning and nutritional quality of dried fig: Combined use of biochemical and ATR-FTIR analyses

Lahcen Hssaini a,*, Rachida Ouaabou b, Jamal Charafi a, Rachid Razouk a, Karim Houmanat a, Ahmed Irchad a

a National Institute for Agricultural Research (INRA), BO 578, Meknes, Morocco b Faculty of Science Semlalia, Cadi Ayyad University, B.P. 2390, Marrakesh, 40000, Morocco

A R T I C L E I N F O

Keywords: Enzymatic browning Polyphenol oxidase ATR-FTIR Ascorbic acid Salicylic acid

A B S T R A C T

The effect of ascorbic acid (AA) and salicylic acid (SA) used separately at the following concentrations 0.1, 0.2 and 0.3 %, on enzymatic browning and quality of “Nabout’’ dried fig during 6 weeks-storage at 4 ◦C within sealed polyethylene terephthalate (PET) bags, was investigated. The results showed that AA and SA at higher concentrations, particularly at 0.3 %, lowered the browning index compared to the control by 55 and 54 % respectively. The same dose suppressed samples polyphenoloxidase (PPO) to 75 and 80 %, respectively, compared to other concentrations. Both treatments showed a significant impact (p < 0.05) on dried figs quality by lowering the total phenols and antioxidant capacity loss during storage period. This trend was significantly important when the concentration applied was maximum (0.3 %). The same pattern was observed using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) technique, that showed a decreasing absorbance and integrated intensity around the vibration region of 1175− 940 cm− 1. This tendency was also depressed with the increase of anti-browning agents’ concentration. High concentrations of AA (0.3 %) and SA (0.3 %), separately, minimized the overlapping in this region, showing high integrated intensities compared to other concentrations. In conclusion, the pre-storage application either of AA or SA at 0.3 % on dried figs could be appropriate to delay enzymatic browning and quality loss and therefore to extend their shelf-life.

1. Introduction

Fig (Ficus carica L.) is an important crop worldwide, since they are naturally rich in various beneficial metabolites such fibers, sugars, phenols, carotenoids and vitamins [1,2]. It is harvested at its full ripening stage, with a high-water content level, which is generally greater than 75 %. Keeping this highly perishable commodity fresh, has always been the best way to maintain its health nutritional value. However, such a decision implies high-costs, since it requires low tem-peratures, which are difficult to maintain throughout the distribution chain [3]. Instead, many alternatives have been practiced since cen-turies ago such as salting, dehydration and fermentation [4]. Drying is one of fig shelf-life extending technics that is still being widely practiced until today. During this process, fig undergoes several changes leading to their senescence and deterioration such as biochemical reactions and microbial growth. Some of these changes continue even after drying,

such as undesirable browning reactions. This process is the cause of reduction in quality that alters the color, flavor, texture and results in nutrients loss. Changes in appearance affect consumers’ sensory evalu-ation of dried fig because browning is often related with decay [5]. The surface browning is the major cause for fig deterioration during and after drying, as it is rich in polyphenols and highly susceptible to enzymatic browning [6]. It has been reported that enzymatic browning is among the biggest issues in fruit storage and processing and is behind more than 50 % of fruit industry losses [7]. This phenomenon is mainly attributed to the polyphenoloxidases (PPOs) which catalyze the con-version of polyphenols to quinones [8]. Once this reaction has taken place, the reactive quinones can polymerize spontaneously and produce melanins (brown pigments), which are responsible for brown color formation [9]. Most techniques aiming to control this browning process are either inhibiting PPOs activity or converting quinones to colorless materials. Sulfites are well-known anti-browning agents and used to be

* Corresponding author. E-mail address: [email protected] (L. Hssaini).

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https://doi.org/10.1016/j.vibspec.2021.103269 Received 23 February 2021; Received in revised form 25 April 2021; Accepted 18 May 2021

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widely applied in food processing to prevent browning pigmentation. However, their use was restricted due to their negative effect on con-sumer health [10]. This prompted a research of more healthy and safe anti-browning agents. Therefore, various healthier anti-browning agents have gained significant attention recently, among them are ascorbic and salicylic acids; inhibit PPOs activity either by lowering or increasing the pH or inactivation of the enzyme by chelating the copper at its active site to inhibit the browning reaction [11]. Physical treatments, such as heat and irradiation have also been tested to prevent fruit browning [12,13]. However, they can potentially alter organoleptic attributes and nutrient level of the final product [14].

Ascorbic acid (vitamin C) is among least toxic antibrowning agent used in the food industry, because of its conservatory effects attributed to the reduction of the reactive quinones to the original colorless diphenols [15]. Some reports recommend its use in concentrations in the range of 0.1 and 0.3 % [10,13,15]. Salicylic acid (SA) is a phenolic compound, which is harmless to human health, is reported for its effect in lowering enzymatic browning through a competitive inhibition mechanism of SA on PPO activity [16]. It was also reported for its role in induction of plant defense against various biotic and abiotic stresses forms [16,17]. In the study of Zhou et al. [5], the effect of SA was investigated on fresh-cut Chinese chestnut (Castanea mollissima) using concentrations between 0.1 and 0.5 %. The effect was significantly important with higher concentrations. Similar results were reported in several studies that used the abovementioned anti-browning agents [9, 14,16].

To date, several studies have been carried out to find several unharmful strategies to prevent food enzymatic browning. These stra-tegies were mainly assayed using biochemical markers, such as PPO activity, polyphenols kinetic and browning index. However, very few studies have investigated this phenomenon by combining these markers to the Fourier Transform Infrared Spectroscopy (FTIR) using chemo-metric methods. This highly sensitive and non-destructive method pro-vides different levels of molecular information regarding molecular structures. It measures predominantly the vibrations of bands within functional groups and provides a specific infrared spectrum, which is the biochemical fingerprint that provides information about molecular composition [18]. The present study investigated the effects of ascorbic and salicylic acids on enzymatic browning and PPO activity in 315 dried figs. It combines traditional methods such as monitoring of polyphenol oxidase (PPO) activity with UV spectrophotometry, quantifying the total phenolic content (TPC) using the Folin-Ciocalteu micro-method, and assessing the antioxidant activity with DPPH and ABTS assays, with more a rapid but more technical method such as ATR-FTIR to assess the effect of the two anti-browning agents on improving the shelf life of dried figs.

2. Materials and methods

2.1. Samples preparation

Fresh figs (Ficus carica L.) belonging to the local variety “Nabout” were brought from a commercial orchard in a village of Taounat in Northern Morocco in the last decade of August 2018 (350 figs). Selected fruits had uniform size and maturity, with no diseases and visual blemishes. Initial moisture content was determined using the oven dried method as described by AOAC and was noted to be 78 ± 1 % wet base (w.b.).

2.2. Fruit pretreatment

Prior to drying in a hot air dryer at a temperature of 80 ◦C and a velocity of 300 m3/h, figs were dipped for 5 min in distilled water (control) or in the following concentrations of 0.1, 0.2 and 0.3 % (w/v) of either ascorbic acid or salicylic acid as described by Ali et al. [15] and Zhou et al. [5], respectively. The drying was stopped at a moisture

content of 25 % according to the dried fig commercial quality standards developed by the United Nations Economic Commission for Europe (UNECE STANDARD DDP-15). All dried samples, with the same level of moisture, were packaged and sealed in polyethylene terephthalate (PET) bags (size: 17 × 12 cm L/W; permeability: 50–100 and 245.83–408.64 cm3.μm/m2.h. atm for O2 and CO2, respectively; permeability to water vapor: 16.25–21.25 g.μm/m2.h) to serve as replicates. Twenty-one bags each containing fifteen fruits were prepared for each treatment. Samples were arranged in a complete randomized design, with one fruit per replication for each treatment, and stored at 4 ◦C for six weeks. At the end of the storage period, water activity (aw) of dried samples were 0.41, 0.4 and 0.38 for dried samples treated with salicylic acid and ascorbic acid and control, respectively, using calibrated electric hygrometer (HygroLab, Rotronic, Bassersdorf, Switzerland). All measurements were performed in triplicate.

2.3. Browning index determination

Changes in dried figs color as affected by different treatments (con-trol included) were measured using Minolta CM-700 colorimeter (Min-olta Camera Co., Osaka, Japan), standardized with white and black calibration. All measurements were obtained from two randomized spots located on opposite sides of the equatorial region of the fruit. The mean of the two measurements was considered as one replicate. Fifteen replications (one fruit per replicate) per sample were considered. The color was studied in the CIEL*a*b* color space with illuminant D65, SCI mode and an observer angle of 10◦. Low reflectance glass (Minolta CR- A51/1829-752) was placed between the samples and the equipment. The CIEL*a*b* coordinates determined were: lightness (L*), redness (a*, coordinate red/green), and yellowness (b*, coordinate yellow-blue). The browning index was calculated using the formula described Palou et al. (1999).

BI =100 (X − 0.31)

0.172

Where, X = (a * + 1.75 L *) / (5.645 L * + a * - 0.3012b *)

2.4. Polyphenol oxidase (PPO) activity

Enzyme extraction and PPO activity determination were performed as described by Zhou et al. [5]. In brief, 20 g of dried fig were homog-enized in 100 mL of citrate phosphate buffer (0.05 M, pH 5.6,) using an IKA T-18 Basic Ultra-Turrax homogenizer (IKA Werke GmbH & Co., Staufen, Germany). The mixture was centrifuged for 30 min at 0 ◦C and 10,000 g; and the supernatant was then supplemented with 100 mL of the same buffer. The PPO activity was evaluated by adding 0.1 M catechol (1 mL) as substrate to a mixture of 0.1 M citrate phosphate buffer (pH 5.6, 3 mL) to the enzyme extract solution (0.5 mL). The change in absorbance at 420 nm was measured every 30 s for 3 min using UV-1700 Shimadzu, Japan spectrophotometer. Results were rep-resented as a specific activity, defined as increase in absorbance per minute in 1 mg enzyme of reaction mixture (ΔA420/min/mg).

2.5. Determination of total phenolic content (TPC)

Phenolic extraction was performed on the powder of lyophilized fruits as described by Xie and Bolling [19]. Total phenols were quanti-fied using Folin-Ciocalteu micro-method on dried fig methanolic ex-tracts as described by Singleton et al. [20]. The analysis was performed in triplicate (one fruit per replicate) and the results were expressed as mg of gallic acid equivalent (GAE) per 100 g.

2.6. Antioxidant activity

The antioxidant activity was assessed as the free radical scavenging

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activity using two assays DPPH and ABTS in methanolic solution [MeOH/water; 80/20 %; v:v; + 1% HCl v:v]. The DPPH (radical 2,2- diphenyl-1-picrylhydrazyl) method was performed as described by Brand-Williams et al. [21], while the ABTS [2, 2-azinobis-(3-ethylbenzothiazoline- 6-sulphonic acid)] assay carried out as described by Re et al. [22]. All assays were performed in triplicate.

Antioxidant activity was calculated using the following equation:

I (%)

(Acontrol − Asample

)

Acontrolx100

2.7. FTIR spectroscopy

The Fourier transform infrared (FTIR) spectroscopy measurements were performed using Bruker Vertex 70 FTIR spectrometer equipped with ATR accessory (Bruker Optics Inc., Ettlingen, Germany), in the region of 4000–450 cm− 1 with 4 cm− 1 spectral resolution. For each FTIR spectrum, three scans were averaged and the infrared (IR) spectrum corresponded to the accumulation of 128 scans. Measurement was performed by applying methanolic extracts of each sample (50 μL) to the germanium crystal, and spread it using the tip of the pipette. Prior to sample measurements, a background spectrum was collected from an empty germanium crystal surface and automatically subtracted from the

Fig. 1. Effects of different concentrations of ascorbic (AA) and salicylic acid (SA) on the browning index of dried over time (six weeks). Values are given as means ±standard deviations (n = 3 fruits).

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spectra of the sample. The crystal cell was cleaned between spectral collections using a methanol rinse and soft tissue, and then dried.

2.8. Chemometric analysis

The experimental design consists of three-factor model that are treatments (AA and SA) x applied dose (0, 0.1, 0.2, 0.3) x storage period (0–6 weeks). The data were subjected to Three-way analysis of variance (ANOVA) to assess the effects of treatments, their respective

concentrations and storage duration besides their interactions. Differ-ences among IR intensities were determined by the least significance difference (LSD) which was computed at 5% the probability level to estimate the significant differences among the means for applied doses of each treatment. As it tends to have the most general applicability, Wilks’s lambda (λ) was used to evaluate the significance of each inter-action level. Data were tested for normality and homogeneity of vari-ance prior to each analysis to ensure the validity of statistical analysis using SPSS software v22.

Fig. 2. Variation of enzymatic specific activities of polyphenol oxidase (PPO) in dried figs over time (six weeks) (AA: ascorbic acid; SA: salicylic acid). Values are given as means ± standard deviations (n = 3 fruits).

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Regarding FTIR analysis, the standard normal variate (SNV) and multiplicative scattering correction (MSC) were first carried out to correct multiplicative interferences [23]. Then, the raw FTIR spectra were corrected by the extended Multibounce attenuated total reflection (ATR) correction procedure using Essential FTIR software (version 3.50.183) (angle of incidence = 45◦; number of ATR reflection = 1; mean refractive index of sample = 1.5; maximum interaction = 50; 1.8 mm crystal surface). Corrected spectra within phenols vibration region (1175− 940 cm− 1) were plotted using the OriginPro software v 8.5 (OriginLab Corporation Inc.) in order to show differences in absorbance among samples.

3. Results and discussion

3.1. Treatments effect on browning Index (BI)

The instant enzymatic browning is the main factor that restricts the selling of dried figs. Therefore, BI is considered as an important index of the browning process and surface decay.

The BI of the control samples increased from 10 to 60 after the six- weeks storage period, while that of the AA and SA treated samples showed a low browning rate, which varied depending on the treatment and following the applied concentrations (Fig. 1). Generally, treated figs

Fig. 3. Effect of different concentrations of ascorbic (AA) and salicylic acid (SA) on Total phenolic content (TPC) of dried figs. Vertical bars show standard error of the means and data are mean of three replicates.

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maintained low browning index during the whole storage time. It is noteworthy that BI remained almost unchanged during the first week of storage. After that, treatment doses showed significant impact on dried fig browning rate, that was negatively correlated to the treatment dose. Thus, over all, the samples treated with a concentration of 0.3 % showed low BI compared to 0.1 and 0.2 %, particularly for SA. At the end of the storage time, the BI of samples treated with 0.3 % of AA and SA was 34.25 and 33.64, which represent 55 and 54 % of that of the control, respectively (Fig. 1.). The results showed that AA and SA delayed the browning reaction on the surface of dried fig during the cold storage time, and the effects were influenced by their respective concentrations. Both treatments showed the same effects, with no remarkable differ-ences between them. Similar results were showed by Zhou et al. [5], who reported that SA has lowered BI of fresh-cut Chinese chestnut during six days storage. Zhang et al. [24] has also observed that melatonin treat-ment inhibited the postharvest browning of litchi fruit. The same results were found by Ali et al. [16], using cysteine, ascorbic acid and citric acid as PPO inhibitors and anti-browning agents on lettuce-head fresh-cut. The authors stressed that high concentrations of ascorbic acid (>1.5 %) reduced the quinone instantly formed to the catechol (original sub-strate), whereas it showed a competitive mechanism at lower concen-trations. The browning index has been proven to be the best indirect way to investigate the enzymatic activity in foods, since it provides a valu-able information of color deterioration, which represents the purity of brown color, indicating where enzymatic browning process takes place

as a conversion of polyphenols to quinones [16].

3.2. Impact of various inhibitors on the PPO activity 2

The variation of PPO activity in dried figs over time are shown in Fig. 2. The increase in the specific activity of PPO was significantly less in AA, and SA treated dried figs than in control. After six weeks storage period, these treatments had significantly reduced the specific activity of PPO compared to control (p ≤ 0.05). The control samples specific PPO activity increased gradually during storage. Thus, it increased by 200 % after three-weeks storage, and then subsequently increased to more than three time of the initial value after a six-weeks storage period. Without inhibitors, the PPO activity displayed an exponential rate before it showed low increase starting from the end of the third week. Treated samples showed increasing PPO activity during the first week storage, before it decreased substantially till the end of the storage period. The treated figs specific activity of PPO decreased to 17 and 25 % of that of the beginning of the treatment following the concentrations of 0.1 and 0.2 % AA. The same treatment at 0.3 %, displayed a similar decreasing rate by lowering the initial PPO activity by 75 %. Which means that the specific activity of PPO was decreased more than 15 and 13 times of that of the control following treatment with 0.1 % and 0.2, and 0.3 % AA, respectively (Fig. 2).

At low SA concentration, the specific PPO activity at the sixth week was decreased to 7.5 % of that of the first week, which is equivalent to 15

Fig. 4. Effect of different concentrations of ascorbic (AA) and salicylic acid (SA) on free radical scavenging activity (DPPH and ABTS) of dried figs. Vertical bars show standard error of the means and data are mean of three replicates.

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times lower that of the control samples after treatment. A high decrease of the specific PPO activity was observed at 0.2 and 0.3 % of SA, where the activity was decreased to 17 and 12 %, respectively at the end of each treatment, and therefore the PPO activity was lowered 20–27 times of that of the control samples (Fig. 2). Enzyme activities in both AA and SA- treated figs were significantly lower than those in untreated fruit during storage period. The general inhibition of PPO activities by the anti-browning agents would lead to reduced contact between both en-zymes and substrates. Nevertheless, even both treatments showed similar kinetics during the storage period with similar patterns in lowering the PPO specific activity particularly at high concentrations (0.2 an 0.3 %), salicylic acid revealed high PPO activity lowering (80 %), while ascorbic acid lowering level was 75 %, compared to the control. The low increase of PPO after the first week-storage for all treatments and concentrations may be due to a synergistic effect of SA with some polyphenols present in the crude enzyme, which possibly affect the binding of AA and SA in the PPO active site, and then allow the con-version of polyphenols to quinones. According to Zhou et al. [5], hydrogen bonds formed between oxygen atoms of the antibrowning agent and hydrogen atoms of phenols may be the main factor leading to the inactivation of the PPO. Indeed, with the increasing of the treat-ments’ concentration, the inhibition effect of AA and SA on PPO activity progressively became effective. This phenomenon has been reported for other browning inhibitors, such as sodium chlorite [25], cysteine and citric acid [16], chlorine dioxide [14] and chitosan [26]. Several studies have shown that both AA and SA are a competitive inhibitors of PPO, that inhibit the conversion of polyphenols to quinones, thus the spon-taneous polymerization of quinones to melanin which result in brown-ing is hindered. [5,6,9,14,16].

3.3. Effect on total phenolic content (TPC)

As storage time increased, TPC radical substantially declined in both control and treated dried figs (Fig. 3). During first week-storage TPC increased, then substantially decreased till the end of storage period. Throughout the experiment, the control samples exhibited a very low TPC, with a sharp decrease over the three first weeks, before it remained relatively constant at a low level during the two consecutive weeks, then finally the curve shows relatively a low decrease rate. In the control samples, after six weeks, the TPC decreased to 17 % of that of the

beginning of storage period. Similar to their specific activity of PPO, the treated samples displayed a substantial increase of TPC during the first week storage before it started to decrease significantly till the sixth week of the storage period. This peak was remarkable in samples treated by AA, where the TPC was generally about 139.5 mg GAE/100 g, compared to those treated by SA (120 mg GAE/100 g). This is probably due to the synergetic effect between antibrowning agents with the phenolic com-pounds contained in the samples, which leaded to the suppression of free radical formation and dissipation of free radicals by the two used anti- browning agents (Sikora and Swieca, 2018). It could be also attributed to the excess quantity of AA and SA by dipping treatment since both AA and SA interfere with Folin-Ciocalteu reagent. This phenomenon was also demonstrated by Saito and Kawabata [27]. The reason behind the higher levels of TPC in the sixth week particularly at higher dose (0.3 %) in the case of SA compared to AA may be explained by their effect on the specific activity of PPO. Hence, at 0.3 % SA suppressed up to 80 % of PPO, while AA lowering the initial PPO activity by 75 %.

Low treatments concentrations showed a relatively quick decrease in TPC. Thus, at 0.1 % of AA and SA treatments, TPC decreased to 30 and 33 % of the initial value after six weeks-storage, whereas, it was reduced to 13 and 16 % after treatment with 0.3 %, respectively. It is noteworthy that TPC were maintained in superior levels throughout the storage period in SA (0.3 %) treated samples than AA using the same concen-tration. These results can be explained by the fact that the antibrowning agents inhibit the oxidation of phenolic compounds during storage time. They indicate that AA and SA treatments had beneficial effects in maintaining the total phenolic content of dried figs, which is in agree-ment with several previous reports [6,9,14,16].

The reduced phenolics oxidation eventually helps in reducing enzymatic browning during postharvest storage [16]. Hence, a higher TPC in AA and SA treated samples was probably associated to the affect of the binding of these antibrowning agents in the PPO active site, and then prevent the conversion of polyphenols to quinones. This type of findings suggested that the used treatments suppressed the browning process in dried figs by inhibiting the oxidation of its phenolic compounds.

3.4. Effect on free radical scavenging activity (FRSA)

The FRSA of dried fig samples with different treatment conditions

Table 1 Test of between-subject effects (3-way ANOVA) on dried figs stored at 4 ◦C for six weeks.

Interactions Variables df Mean Square F p-value Wilks’ Lambda Value Effect p-value.

T * C * S

DPPH 10 54.97 7.34 p<0.001

0.194 p<0.001 ABTS 10 52.91 1.27 .023 PPT 10 1760.43 2.18 .041 PPO 10 3.69 3.02 .006 BI 10 10.67 3.40 .014

T * C

DPPH 2 71.55 9.55 p<0.001

0.491 p<0.001 ABTS 2 100.68 2.42 .097 PPT 2 1297.42 2.18 .09 PPO 2 7.82 0.88 .209 BI 2 16.02 .60 .548

C * S

DPPH 10 54.52 7.27 p<0.001

0.128 p<0.001 ABTS 10 224.42 5.40 p<0.001 PPT 10 2016.32 2.50 .020 PPO 10 3.90 3.19 .004 BI 10 12.26 1.46 .030

T * S

DPPH 5 8.62 1.15 .041

0.357 p<0.001 ABTS 5 600.84 14.48 p<0.001 PPT 5 681.74 1.84 .020 PPO 5 2.18 1.79 .042 BI 5 13.02 1.49 .032

T: Treatment; C: Concentration; S: Storage period. Sig.: significance. DPPH: radical 2,2-diphenyl-1-picrylhydrazy; ABTS : 2,2-azinobis-(3-ethylbenzothiazoline- 6-sulphonic acid)]; PPT: total phenolic content; PPO: polyphenoloxidase; IB:browning index.

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was evaluated using DPPH and ABTS assays to assess their antioxidant capacity kinetics during storage (Fig. 4). For both assays, FRSA has substantially declined in similar pattern to TPC in both control and treated dried figs throughout the storage period, with a typical increase during the first week-storage. The decrease in the antioxidant activity was significantly less important in AA, and SA treated dried figs than in control.

DPHH scavenging activity was significantly decreasing in the AA and SA treated dried figs from week 2–6 (p ≤ 0.05), which was in agreement with the previous study on fresh figs treated with chitosan (1%) and ascorbic acid (1.5 %) during 9 days [26]. The same results were reported by Zhang et al. [28] in litchi fruit as influenced by apple polyphenols. The authors stressed a similar suppression in DPPH radical-scavenging activity as storage time increased. The ABTS radical scavenging

activity kinetic (Fig. 4) was the same as DPPH. The ABTS radical scav-enging activity has significantly decreased in both AA and SA treated dried figs after the first week till the end of storage period (P < 0.05). The results displayed consistent trend with total phenolic content, which suggest that the FRSA declines due to accelerated senescence during storage [29]. These findings suggested that AA and SA delay the FRSA decrease, thus both treatments could maintain higher antioxidant ac-tivity compared to the control samples, and it was probably related with the same effect they had on TPC kinetics during storage period. The mechanism of AA and SA treatments in reducing dried figs browning were able to lower FRSA decrease, which was proved by the DPPH and ABTS radical scavenging kinetics, therefore, inhibiting the process of phenolic compounds convert into quinones. These results were in agreement with Tareen et al. [16], Zheng et al. [6] and Ali et al. [29].

Fig. 5. Typical ATR6FTIR spectra of dried fig samples (example of 0.1 % treatment) measured in the region of 450–4000 cm− 1.

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3.5. Interactions between experimental design factors

3-way ANOVA was performed to assess the different interaction levels on the dependent variables. All measured variables were signifi-cantly affected by the interaction between treatment, concentration and storage time (p ≤ 0.05). The higher effect was shown in the PPO and BI, where this interaction was highly significant (p = 0.006 and p = 0.014, respectively). The full model was statistically significant and had a great effect on the browning enzymatic and quality factors of dried figs, since it displayed a low Wilks’s λ, which was about 0.19 (Table 1). Second

level interactions between design experimental factors also showed significant effects (p ≤ 0.05, with low Wilks’s λ values) over all vari-ables, except BI and PPO, particularly, which haven’t been impacted (p = ≤ 0.548 and p = ≤ 0.209, respectively) by the interaction between treatment and concentration, without considering the storage time factor. This is may be obvious since these two variables are also depending on the storage period to take effect, while PPT and FRSA (DPPH and ABTS) could display a significant effect due to synergetic effect between phenolic compounds contained in the samples and the applied antibrowning agents (Table 1).

Fig. 6. ATR-FTIR spectra in the region of 1775-940 cm− 1 as a function of storage period and treatment dose. Each sample IR represent the mean of 3 spectra.

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3.6. ATR-FTIR spectral analysis

Fourier transform infrared spectroscopy (FTIR) spectra of different samples and treatments are shown in the Fig. 5. The spectra observed between 4000 and 450 cm− 1 showed five absorption regions: 3700− 3000, 3000− 2800, 1800− 1500, 1500− 1175, 1175− 940 cm− 1. The first region is assigned to OH-stretching vibrations arising from hydrogen bonding in cellulose. The main vibration of this region was visible at 3336 cm− 1, and it is assigned to intra-molecular hydrogen bonding between C(3)OH⋯O(5) and C(6)O⋯O(2)H) [30,31]. The absorbance at the region 3000− 2800 cm− 1 is most likely assigned to C–H, O–H and NH3, which may be referred to carbohydrates, car-boxylic acids, free amino acids [32,33]. The bands at 1800–1500 cm− 1

appeared as a small shoulder in the FTIR spectra, and were assigned to O–H in-plane deformation, elongation of C––O of ester type carboxylic and C––O stretching [34,35]. The absorption bands in the region of 1500− 1175 cm− 1 correspond possibly to phosphodiester groups. This band is probably a result of several weak peaks that could not be differentiated among analyzed extracts. According to several studies, this band usually includes, among others, a vibration around 1392 cm− 1, that is most likely assigned to carbohydrates or amino acids side chain [36], the 1315 cm− 1 vibration, which is associated to CH2 rocking [30],

and a vibration approximately at 1155 cm− 1, which is a result of C–O stretching [36]. Finally, the vibrations in the region of 1175− 940 cm− 1

marks small peak at 1086 cm− 1 probably assigned to C–OH group and a sharp peak around 1044 cm− 1 attributed to the stretches C–C and C–O in the carbohydrate structure and C–O in the phenol (Fig. 5). The first band (1086 cm− 1) appears as a small shoulder and most probably a conjugation of the C–O–C stretching vibration of PPO [37]. The effect of anti-browning agents used is particularly occurred in this region, since it is often related to phenols, subject of the spontaneous conversion of phenols to quinones, which are responsible for browning color.

As it is difficult to visualize to impact of each treatment on the samples absorbances, the infrared spectra (IR) of each treatment were plotted in the region of 1775− 940 cm− 1, which includes the region of phenols vibration (1175− 940 cm− 1) (Fig. 6). The absorbance decreases gradually as storage time increased, which is in concordance with the above results. In both treatments, the IR bands overlapping becomes less remarkable as the concentration of each treatment increased. This feature is notably observed in SA treated samples. Integrated intensity at 1175− 940 cm− 1, which is attributed to the phenols’ vibration, was calculated using Essantial FTIR software and plotted for each treatment (Fig. 7). In all samples, the integrated intensities showed a decreasing trend with the advancement of storage time indicating the decrease in samples phenolic compounds. This trend was inversed with the increase of each treatment dose. Thus, integrated intensities decrease was low-ered as the concentration of the antibrowning agents increased. Even being a qualitative method, FTIR spectroscopy was useful for charac-terizing changes in dried samples and the effect of AA and SA treatments and their corresponding concentrations. Several studies reported the worth of FTIR method in the screen of the distribution of numerous biomolecules in several food samples including fruits, vegetables or beverages e.g. biosynthesis of silver nanoparticles in figs [38], olive pulp cell-wall polysaccharides [39], authentication of pomegranate juice concentrate [40] and discrimination of bovine, porcine and fish gelatins [18]. In the study conducted by Baltacıoglu et al. [41], the FTIR tech-nique gave satisfactory results in investigating the changes in structure and activity of PPO during thermal treatment in the temperature range of 25–80 C. In our study, the FTIR results were, generally consistent to those of TPC, FRSA, BI and PPO kinetics, which displayed a significant impact of AA and SA treatments on lowering BI and PPO activity, besides maintaining the TPC and FRSA in higher level compared to the control samples.

4. Conclusion

Enzymatic browning is limitative factor of dried figs quality and marketability. This study demonstrated that the polyphenol oxidase activity, an enzyme associated to browning of dried figs, may be repressed using ascorbic acid or salicylic acid. The postharvest treatment with those antibrowning agents limits enzymatic browning of dried figs via PPO competitive mechanism, which lowered the quality loss of samples. This pattern was more important as their concentrations in-crease, as well as their synergetic effect with samples phenolic com-pounds. 3-way ANOVA showed a highly significant effect of the full model on all studied variables. ATR-FTIR spectroscopy technique was used to investigate the change in the samples’ phenolic extracts during storage period. The results were consistent with previous ones, since the absorbance showed a deceasing rate in the region of phenols vibration (1175− 940 cm− 1), that was lowered as the antibrowning agents’ con-centration decreases. High AA and SA concentrations also showed a low overlapping level, which allowed to clearly compare the absorbance band of each treatment. Integrated intensities observed at this vibration region, also showed low decreasing pattern in high inhibitors concen-trations. Both treatments revealed similar effect, with a slight superi-ority of salicylic acid, that lowered the specific activity of PPO by 80 % compared to 75 % exhibited by ascorbic acid. Similarly, the same doses applied lowered the BI of samples by 55 and 54 % of that of the control,

Fig. 7. Integrated intensity of the 1175-940 cm− 1 vibration (phenols vibration) as a function of storage period and treatment dose showing that the decrease of the intensity was lowered by the decrease of treatment dose. (AA: ascorbic acid; SA: salicylic acid). LSD values display the significant effect of treatments and their respective doses at p ≤ .05.

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respectively, which showed a slight superiority of SA compared to AA effect. However, the antioxidant activities were slightly higher at the end of storage in figs treated by AA compared to SA, which is apparently due to the synergetic effect with total phenols of samples, that was important in fig AA-treated compared to SA-treatment. Taking into ac-count these results, this study suggests the use of SA, that showed a slight superiority in its effect to lower enzymatic browning in dried figs, since both applied treatments are harmless to human health, easy to use and do not significantly differ in their prices. The study provides interesting and promising results on the separate use of ascorbic and salicylic acids as a unharmful anti-browning agent for inhibition of dried fig enzymatic browning and at the same time lowering their antioxidant quality loss.

Statement

Lahcen Hssaini: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization and Project administration

Rachida Ouaabou: Formal analysis and Investigation Rachid Razouk, Jamal Charafi: Visualisation and Investigation Karim Houmanat,Ahmed Irchad: Visualisation, Data Curation

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

Declaration of Competing Interest

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

Acknowledgements

FTIR analyses were performed in the Analysis and Characterization Center (Centre d’analyse et de caracterisation) of Faculty of Science Semlalia, Cadi Ayyad University, Marrakesh, Morocco

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