Impact of ozonation process on the antioxidant status in blackcurrant Ribes nigrum L. fruit

Abstract

BACKGROUND:

Ozonation is promising method for prolonging the postharvest shelf life as well as decrease of microbiological contamination and pesticide residue in soft fruit. In view of the fact, that ozone is a substance that can induce the reactive oxygen species generation in plant cells, it is necessary to determine the effect of ozone treatment on the antioxidant status in fruit.

OBJECTIVE:

The purpose of this study was to investigate the impact of ozonation on the antioxidant status in blackcurrant fruit.

METHODS:

Blackcurrants were exposed to ozone at the concentration of 16 mg L^–1 and 160 mg L^–1 for 5, 15 and 30 min. Changes in antioxidant activity, total phenolic content and activity of selected antioxidant enzymes after ozonation were analyzed.

RESULTS:

It was found, that under proposed conditions the activity of enzymes involved in scavenging of reactive oxygen species (quaiacol peroxidase, ascorbate peroxidase and catalase) increased with ozone concentration and treatment time. In addition, the ozone treatment activated the phenylalanine ammonia-lyase, whose an activity showed a strong correlation with total phenolic content and antioxidant activity of blackcurrants.

CONCLUSIONS:

Ozonation process activated a protective mechanisms against occurrence of oxidative stress in blackcurrant fruit.

Keywords

Antioxidant blackcurrant fruit ozone stress response

1 Introduction

It is well-known that a properly designed diet providing adequate portions of fruit and vegetables is an important way to prevent many civilization diseases. Especially berry fruit, including blackcurrants Ribes nigrum L., are characterized by a wide range of substances with documented antioxidant properties, mainly flavonoids, phenolic acids and vitamin C. It has been proven that these compounds help protect the human body against oxidative stress, which contributes to cancer, cardiovascular and neurodegenerative diseases [9 , 28]. Furthermore, phenolic compounds extracted from blackcurrants exhibit an anticancer activity inhibiting the multiplication and growth of cancer cells by inducing apoptosis in them [19]. In addition, anthocyanins, especially derivatives of cyanidin and delphinidin are used in the treatment of eye diseases [15, 16].

Fruit after harvest still show the characteristics of living organisms where life processes occur, especially breathing, transpiration and maturation. Over time they lead to loss of individual biological and sensory characteristics such as sugars, proteins, acidity, vitamins, polyphenols, weight and firmness. In addition, improper fruit storage conditions contribute to the growth of microorganisms, mainly molds, which cause an accelerated spoilage of fruit. If the berry fruit do not meet the microbiological safety standards, they cannot be sent to trade and food processing. Therefore, the alternative methods for inhibiting fruit quality deterioration processes and growth of microorganisms are sought [21].

Ozone is an agent with strong antibacterial and antifungal properties. Scientific reports show many benefits from the use of ozone in gas phase in prolonging shelf-life of berry fruit. Ozone effectively reduces a level of mold, yeast and bacteria in fruit and oxidizes ethylene, produced during the storage, which is responsible for accelerated maturation and aging of the fruit. Furthermore, a protective effect of ozone in relation to bioactive substances i.e. polyphenols and vitamins, was proven. Research have shown that the content of polyphenols and antioxidant activity in ozonated fruit were significantly higher than in non-ozonated fruit [7 , 21]. In addition, ozone treatment reduces the level of pesticide residues in plant materials [1]. It should be pointed, that ozonation process does not cause an additional contamination of products compared to traditional chemical preservatives, because, ozone in contact with treated material it decomposes into molecular oxygen. Therefore, ozonation technology could be successfully used in organic fruit production [20].

Reactive oxygen spices (ROS) are produced in plant cells as a side products of biochemical reactions in cell respiration, photosynthesis and photorespiration. The ROS generation in plant is intensified by the presence of pesticides, heavy metals and strong temperature and humidity fluctuations in environment. Due to the fact that excess of ROS in cell is toxic, the plant has developed an appropriate protective mechanisms, such as antioxidant enzymes i.e. superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase. In addition, low molecular weight of antioxidants (glutathione, ascorbic acid, polyphenols) are also able to decrease the ROS concentration to a safe level. Ozone is a substance that can induce the reactive oxygen species generation in plant cells. Therefore, it is necessary to determine the influence of ozone treatment on the ROS defense mechanisms in berries [30, 31].

The purpose of the study was to determine the impact of ozonation process on the antioxidant potential of blackcurrant fruit. Changes in antioxidant activity, total phenolic content and activity of key enzymes involved in the response on oxidative stress in plants i.e. phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), catalase (CAT, EC 1.11.1.6.), guaiacol peroxidase (GPOX, EC 1.11.1.7), ascorbate peroxidase (APOX, EC 1.11.1.11) after ozonation were analyzed.

2 Materials and methods

2.1 Reagents and devices

2.1.1 Reagents

Analyses were conducted using reagents produced by Chempur (Piekary Śląskie, Poland): disodium hydrogen phosphate (≥99%), sodium dihydrogen phosphate dihydrate (≥99%), boric acid (99.5%), sodium tetraborate (99%), Folin-Ciocalteu reagent, sodium carbonate (99.8%), sodium hydroxide (98.8%), L-ascorbic acid (≥99%), potassium persulfate (≥99%), hydrogen peroxide (30%); produced by POCH (Gliwice, Poland): EDTA-disodium salt dihydrate (99%), sodium chloride (99%), methanol (99.8%), sodium acetate, hydrochloric acid (35–38%); and produced by Sigma-Aldrich (Darmstadt, Germany): quercetin (≥95%), DPPH (2,2^′-diphenyl-1-picrylhydrazyl, ≥95%), ABTS (2,2^′-azino-bis(3-ethylobenzothiazoline-6-sulphonic acid, ≥98%), gallic acid (≥98%), L-phenylalanine (≥98%), guaiacol, 2-mercaptoethanol (≥99%), polyvinylpolypyrrolidone (≥98%, Sigma-Aldrich), phenylmethylsulfonyl fluoride (≥99%, Sigma-Aldrich), Triton X-100 (Sigma-Aldrich).

2.1.2 Devices

Ozone generator TS-30 (Ozone Solution Inc. Hull, IA, USA), Ozone analyzer, 106-M (2B Technologies), UV-Vis absorption spectrophotometer (Metash), laboratory centrifuge with cooling (MPW, Warsaw-Poland), homogenizer (Virtis), pH meter (Elmetron, Zabrze-Poland).

2.2 Fruit material

The research material was fresh fruit of blackcurrant Ribes nigrum L. purchased from a local producer, immediately after harvest. The fruit was fully coloured, mature and ready for consumption, without signs of mold infestation or mechanical damage.

2.3 Ozonation procedure

To ozonation utilized a device presented on Fig. 1. Blackcurrants in an amount of 500 g were evenly distributed on the perforated shelf of ozonation chamber. Blackcurrants were exposed to ozone at the concentration of 16 mg L^–1 and 160 mg L^–1 (gas flow rate: 2 m³ h^–1), for 5, 15 and 30 min. Ozone concentration was analyzed at the inlet of reactor chamber using Ozone Analyzer UV-106 M. The non-ozonated blackcurrants were a control sample. After ozonation, blackcurrants were stored for 1 day at 7°C (humidity: 90–95%) and then passed to analysis.

Fig.1

Scheme of the ozonation devices: 1-pump, 2-ozone generator, 3-the layer of fruit, 4-ozonation chamber, 5-cyclone.

The ozonation procedure was designed, based on the patent applications [5, 6] and the available research [1, 7, 20, 21].

The experiment was performed in triplicate.

2.4 Biochemical analysis

2.4.1 Extraction of antioxidant compounds from fruit

Blackcurrants (5 g) were homogenized in 50 mL of methanol-distilled water mixture (1 : 1 v/v). The homogenate was shaken for 30 min (150 rpm) and clarified by centrifugation at 5,000g for 10 min. The obtained supernatant was used to determine the total polyphenols content and antioxidant activity.

2.4.2 Total phenolic content assay

To 50μL of blackcurrant extract, 950μL of methanol-distilled water mixture (1 : 1 v/v), 50μL of Folin-Ciocalteu reagent diluted with distilled water (1 : 1 v/v) and 100μL of 7% Na₂CO₃ solution was added. The reaction mixture was incubated in darkness for 30 min and then, absorbance was measured at the wavelength λ = 750nm. The obtained results were expressed as equivalent of gallic acid (g) per kg of fresh fruit [20].

2.4.3 ABTS^•+ radical scavenging activity assay

To 1 mL of ABTS^•+ radical solution, 10μL of blackcurrant extract was added. After 15 min of incubation in darkness, the absorbance of the solutions was measured at λ = 734nm. Quercetin (0–500μM concentration) was used for calibration and the results were expressed as g of quercetin equivalent per kg of fresh fruit [21].

2.4.4 DPPH^• – radical scavenging activity assay

To 1 mL of DPPH^• radical solution, 30μL of blackcurrant extract was added. After 15 min. of incubation in darkness, absorbance was measured at λ = 515nm. Quercetin (0–250μM concentration) was used for calibration and the results were expressed as g of quercetin equivalent per kg of fresh fruit [21].

2.4.5 Measurement of phenylananine ammonia lyase (PAL) activity

The PAL enzyme was extracted from blackcurrants using 0.1 M boric acid-borax buffer solution (pH 8.8) containing 1 mM EDTA, 20 mM mercaptoethanol, 1% (w/v) polyvinylpolypyrrolidone (PVPP) and 0.05% (w/v) triton X-100. The ratio of buffer volume to fruit sample mass was 5 : 1. After centrifugation (30,000 g, at 4 °C for 30 min) the supernatant was used for determination of enzymatic activity [31].

To 0.5 mL of PAL extract, 1 mL of 0.2 M sodium borate buffer (pH 8.8) and 0.5 mL of 50 mM L-phenylalanine as a substrate was added. The mixture was incubated at 35 °C for 1 h. The reaction was terminated by adding 0.1 mL of 6 M HCl.

One unit of PAL activity was defined as the amount of enzyme extracted from 1 g of fresh fruit, needed to catalyze the formation of 1μmol trans-cinnamic acid in a 1 cm cuvette within 1 hour (molar coefficient of trans-cinnamic acid at λ = 280nm, ɛ= 17 400 M cm^–1).

2.4.6 Measurement of guaiacol peroxidase (GPOX), catalase (CAT) and ascorbate peroxidase (APOX) activity

Blackcurrants (5 g) were homogenized with 10 mL of 50 mM sodium phosphate buffer (pH 7.0) containing 2% (w/v) PVPP, 0.1% (w/v) triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 30.000·g for 30 min, at 4 °C. The supernatant was collected for determination of GPOX, CAT, APOX activity.

GPOX activity was analyzed according to the method presented in Zhou et al. (2014) with some modifications. The assay mixture (1 mL) contained 250μL of enzyme extract, 250μL of 50 mM sodium phosphate buffer, 50μL of 40 mM guaiacol, and 30μL of 0.5 M H₂O₂. The mixture was incubated at 35 °C for 30 min. One unit of GPOX activity was defined as the amount of enzyme extracted from 1 g of fresh fruit, causing a 0.01 increase in the absorption at 470 nm within one minute.

The reaction mixture for determining CAT consisted of 0.95 mL of 15 mM H₂O₂ prepared from 50 mM sodium phosphate buffer (pH 7.0) and 50μL of diluted enzyme extract (50 times). The mixture was incubated at 35 °C for 1 h. One unit of CAT activity was defined as the amount of enzyme extracted from 1 g of fresh fruit, causing a 0.01 decrease in the absorption at 280 nm within one minute.

The reaction mixture (1 mL) for determining APOX activity consisted of 300μL 0.5 mM of ascorbic acid, 50μL of 0.1 mM EDTA-2Na, 100μL of 0.1 mM H₂O₂, 300μL of 50 mM sodium phosphate buffer (pH 7.0), and 250μL of enzyme extract. The mixture was incubated at 35 °C, for 1 h. One unit of APOX activity was defined as the amount of enzyme extracted from 1 g of fresh fruit, causing a 0.01 decrease in the absorption at 290 nm within one minute [30, 31].

2.4.7 Statistical analysis

Data are presented as mean±SD from least 3 independent experiments. Statistical analysis was performed using the STATISTICA 13.0 software. The statistical significance of differences was estimated using two-way analysis of variance (ANOVA) and the Tukey test (α= 0.05).

3 Results

3.1 Total polyphenols content and antioxidant activity

Various biological activities of berry fruit are associated with the presence of phenolic compounds and their antioxidant properties. Changes in total polyphenols content and antioxidant activity against ABTS^•+ and DPPH^• of blackcurrant after ozonation are presented on the Fig. 2.

Fig.2

The impact of ozonation process on antioxidant activity against DPPH^• (A) and ABTS^•+ (B) radicals and total polyphenols content (C) in blackcurrant fruit. Mean values with the same lower case are not statistically significant according to the T-Tukey test (α= 0.05).

The two-way analysis of variance (ANOVA) showed that ozone concentration in ozonation chamber and time of process had a significant effect on the total phenolic content and antioxidant activity of blackcurrants (p < 0.05). After 5 min of ozonation with ozone at the concentration of 160 mg L^–1, total phenolic content (TPC) and antioxidant activity (AA) increased by ∼29%, ∼33% (DPPH^•) and ∼29% (ABTS^•+) respectively, compared to the initial value. In turn, the use of gaseous ozone at the concentration of 16 mg L^–1 caused a significant increase in AA only after 30 min. of ozonation (p < 0.05).

3.2 Phenylalanine ammonia-lyase activity

The changes in PAL activity in blackcurrants during ozonation are shown in Fig. 3. Research showed, that PAL activity in ozonated fruit was significantly higher than in non-ozonated fruit (p < 0.05). The highest activity of this enzyme was observed in fruit treated with ozone for 5 min, with gaseous ozone at the concentration of 160 mg L^–1. After 5 min of ozonation, PAL activity was ∼134% higher than in control sample. In subsequent time points, the PAL activity in ozonated fruit showed a decreasing tendency.

Fig.3

The impact of ozonation process on the activity of phenylalanine ammonia-lyase (PAL). Mean values with the same lower case are not statistically significant according to the T-Tukey test (α= 0.05).

3.3 Ascorbate peroxidase, guaiacol peroxidase and catalase activity

As shown in Fig. 4, APOX and GPOX activity increased with ozonation time. However, the activity of these enzymes in fruit treated with ozone at the concentration of 160 mg L^–1 was several times higher than 16 mg L^–1. After 30 min of ozonation, the activity of APOX and GPOX increased by ∼19.2 and ∼6.1 times in fruit treated with ozone at concentration of 160 mg L^–1, while in fruit ozonated with 16 mg L^–1, only 8.2 and 2.6 times, compared to the initial value.

Fig.4

The impact of ozonation process on the activity of catalase (A), ascorbate peroxidase (B) and guaiacol peroxidase (C). Mean values with the same lower case are not statistically significant according to the T-Tukey test (α= 0.05).

The CAT activity increased sharply to 5 min of ozonation. Finally, the catalase activity increased by ∼20% (16 mg L^–1) and ∼24% (160 mg L^–1) compared to the control sample.

4 Discussion

Ozonation is promising method for prolonging postharvest shelf life as well as decrease of microbiological contamination and pesticide residue in soft fruit [19]. In our previous study we found, that ozonation of raspberry and blueberry effectively reduced the microbial contamination and the loss of antioxidants, including phenolic compounds during storage. Furthermore, ozonated fruit characterized by more intense colour than non-ozonated fruit, due to the higher content of athocyanins [20, 21]. Antos et al. (2013) concluded that ozone treatment reduced the pesticide residue (Thiram) in blackcurrants by ∼40%, using gaseous ozone at the concentration of 16 mg L^–1. In view of the fact, that ozone is a substance that can induce the reactive oxygen species generation in plant cells, it is necessary to verify the effect of ozone treatment on the antioxidant status of blackcurrants [1, 30]. In order to determine the changes in antioxidant status in blackcurrant fruit after ozonation process, ozone concentration was increased from 16 mg L^–1 to 160 mg L^–1 and process was conducted for 5, 15 and 30 min.

Our research showed, that time of process and ozone concentration in ozonation chamber had a significant impact on the total phenolic content (TPC) and antioxidant activity of blackcurrants. The use of gaseous ozone at the concentration of 160 mg L^–1 caused an increase in phenolic content in fruit by ∼29% after 5 min of ozonation. In turn, ozonation with ozone at the concentration of 16 mg L^–1 caused a significantly increase in TPC only after 30 min. Furthermore, TPC was strongly correlated with total antioxidant activity of fruit (tab. 1). Similar observations were also reported by Ali et al. (2014), who investigated the effect of ozone treatment on the stability of antioxidant compounds in papaya fruit during storage. They showed, that the fruit ozonated with an ozone dose of 1.5–3.5 mg L^–1, for 95 h, before a 10 days long storage had higher values of antioxidant capacity by 0.03–30.9% in comparison to control fruit. The positive effects of ozone in relation to antioxidant compounds were also noted in research described by Barth et al (2009), Yeoh et al. (2014) and Botondi et al. (2015).

Table 1
Values of Pearson’s linear correlation between the obtained results (α= 0.05)

Pearson correlation DPPH^• ABTS^•+ TPC PAL CAT APOX GPOX

coefficients (p < 0.05)

DPPH ^• 16 mg L^–1 1.00 0.63 0.59 0.49^ns 0.76 0.91 0.80

160 mg L^–1 1.00 0.98 0.99 0.99 0.95 0.74 0.67

ABTS^•+ 16 mg L^–1 0.63 1.00 0.34^ns 0.04^ns 0.88 0.82 0.97

160 mg L^–1 0.98 1.00 0.99 0.95 0.86 0.58 0.50^ns

TPC 16 mg L^–1 0.59 0.34^ns 1.00 0.93 0.13^ns –0.38^ns –0.26^ns

160 mg L^–1 0.99 0.99 1.00 0.92 0.92 0.68 0.59

PAL 16 mg L^–1 0.49^ns 0.04^ns 0.93 1.00 0.47^ns –0.10^ns 0.09^ns

160 mg L^–1 0.99 0.95 0.92 1.00 0.70 0.35 0.22^ns

CAT 16 mg L^–1 0.76 0.88 0.13^ns 0.47^ns 1.00 0.77 0.92

160 mg L^–1 0.95 0.86 0.92 0.70 1.00 0.91 0.84

APOX 16 mg L^–1 0.91 0.82 –0.38^ns –0.10^ns 0.77 1.00 0.92

160 mg L^–1 0.74 0.58 0.68 0.35 0.91 1.00 0.96

GPOX 16 mg L^–1 0.80 0.97 –0.26^ns 0.09^ns 0.92 0.92 1.00

160 mg L^–1 0.67 0.50^ns 0.59 0.22^ns 0.84 0.96 1.00

Pearson correlation	DPPH^•	ABTS^•+	TPC	PAL	CAT	APOX	GPOX
DPPH ^•	16 mg L^–1	1.00	0.63	0.59	0.49^ns	0.76	0.91	0.80
	160 mg L^–1	1.00	0.98	0.99	0.99	0.95	0.74	0.67
ABTS^•+	16 mg L^–1	0.63	1.00	0.34^ns	0.04^ns	0.88	0.82	0.97
	160 mg L^–1	0.98	1.00	0.99	0.95	0.86	0.58	0.50^ns
TPC	16 mg L^–1	0.59	0.34^ns	1.00	0.93	0.13^ns	–0.38^ns	–0.26^ns
	160 mg L^–1	0.99	0.99	1.00	0.92	0.92	0.68	0.59
PAL	16 mg L^–1	0.49^ns	0.04^ns	0.93	1.00	0.47^ns	–0.10^ns	0.09^ns
	160 mg L^–1	0.99	0.95	0.92	1.00	0.70	0.35	0.22^ns
CAT	16 mg L^–1	0.76	0.88	0.13^ns	0.47^ns	1.00	0.77	0.92
	160 mg L^–1	0.95	0.86	0.92	0.70	1.00	0.91	0.84
APOX	16 mg L^–1	0.91	0.82	–0.38^ns	–0.10^ns	0.77	1.00	0.92
	160 mg L^–1	0.74	0.58	0.68	0.35	0.91	1.00	0.96
GPOX	16 mg L^–1	0.80	0.97	–0.26^ns	0.09^ns	0.92	0.92	1.00
	160 mg L^–1	0.67	0.50^ns	0.59	0.22^ns	0.84	0.96	1.00

^*ns – not significant (α= 0.05).

According to the literature, the effect of ozone treatment on the level of antioxidant compounds is associated with process conditions i.e. ozone concentration, time of process and gas flow rate, as well as previously used technological processes in raw materials. In research of Tiwari et. al (2012) and Torres et al. (2012) on the effect of ozonation on the quality of fruit juices, it was described a strong degradation of phenolic compounds after one-time exposure to ozone. Authors found, that the anthocyanins content and antioxidant activity in ozonated juice decreased with increase of ozone concentration and time of ozonation. This was explained by the authors by means of the course of ozonolysis reaction, which occurs gradually easy in a media deprived of the majority of fruit matrix which has a protective effect on phenolic compounds. It should be pointed that mass exchange between gas liquid and gas solid system is totally different.

Phenylalanine ammonia-lyase (PAL, E.C. 4.3.1.5) plays an important role in biosynthesis of phenolic compounds in plants. PAL catalyzes the deamination of L-phenylalanine to trans-cinnamic acid, which can be transformed into flavonoids, phenolic acids or anthocyanins. The high activity of PAL is observed in the cells of plants undergoing various stress factors such as, strong temperature and humidity fluctuations, UV radiation. Furthermore, many scientific reports show that PAL activity in plant is closely correlated with phenolic compounds content [26]. Christopolous and Tsantili (2016) investigated the effect of application of PAL inhibitors on the polyphenols content in fresh cold stressed walnut kernels. The authors showed, that the total polyphenol content was definitely lower in walnuts treated with inhibitors of PAL, than in control sample. In addition, they observed that the PAL activity in walnut kernels exposed to cold stress is induced at the stage of gene expression.

In presented research, it was found that PAL activity in blackcurrants changed with ozonation conditions. The ozonation of fruit with ozone at the concentration of 16 and 160 mg L^–1, for 5 min. caused a significant increase in enzyme activity compared to the control sample, in turn, after this time PAL activity showed a downward trend. In addition, PAL activity was strongly correlated with TPC and antioxidant activity of fruit. It can be assumed that the changes in the activity of PAL after ozone treatment, may be relevant in shaping of the phenolic compounds levels and, finally, with the total antioxidant activity of blackcurrants (Table 1). Sachadyn-Król et al. (2016) investigated the effect of ozone treatment (ozone concentration: 2 mg mL^–1) for 1 and 3 hours on the flavonoids content and PAL activity in pepper fruit. The authors found that the levels of majority flavonoids, in particular those of quercetin glycosides content was higher in ozonated fruit than control sample after 20 days of cold storage. However, total phenolic content, antioxidant activity and PAL activity were lower than in non-ozonated fruit.

Phenolic compounds, aside of high antioxidant activity, they are responsible for sensory properties of berry fruit. Phenolic acids, in particular chlorogenic acid, as well as tannins shape a taste of berries, while anthocyanins are the main group of phenolics responsible for fruit colour. It should be assumed that ozonation process under proposed conditions caused an increase of the content of compounds shaping a sensory attributes. Therefore, in the future, it is planning to define an influence of ozonation on the phenolics profile and sensory properties of blackcurrant under proposed process conditions.

Plants have evolved an efficient antioxidant defense system that can prevent the accumulation of reactive oxygen species. This defense system involves low molecular-weight lipid-soluble (β-carotene, tocopherols) and water-soluble (glutathione, ascorbic acid, phenolic compounds) antioxidants, as well as antioxidant enzymes, such as: catalase (CAT) quaiacol peroxidase (GPOX) and ascorbate peroxidase (APOX), which are involved in reduction of hydrogen peroxide in cells [29, 30].

In our research, the activity of CAT, GPOX and APOX showed upward trend with the increase of ozone concentration and time of process (p < 0.05). According to literature, the level of hydrogen peroxide in cells is closely correlated with the activity of antioxidant enzymes. Therefore, the increase of antioxidant enzymes after ozonation could be caused by the generation of H₂O₂ in plant cells as a result of degradation of ozone in fruit or an enzymatic transformation of other ROS i.e. O₂^-•, OH^• to H₂O₂, whose generation is stimulated by ozone. In research of Chernicova et al. (2000), the effect of ozone treatment (2μL L^–1 of ozone, 7 h, for 5 day in week) on the selected oxidative stress markers in two varieties of soybeans (Essex and Forrest) was investigated. The authors noted that the activity of GPOX in ozonated soybean was higher than in control sample by ∼39% (Essex) and ∼62% (Forrest) in turn, the activity of cytosolic APOX isolated from ozonated plants was higher by ∼16 and ∼33%, respectively.

5 Conclusions

Ozonation process activated a protective mechanisms against occurrence of oxidative stress in blackcurrant fruit. The activity of key enzymes involved in the scavenging of reactive oxygen species (guaiacol peroxidase, ascorbate peroxdidase, catalase) increased with ozone concentration in ozonation chamber and process time. In addition, the ozone treatment activated the phenylalanine ammonia-lyase, whose the activity showed a strongly correlation with total phenolic content and antioxidant activity of fruit. Based on this and our previous research the ozone concentration should be in the range of dozen mg L^–1 (16 mg L^–1) and process should be conducted for 30 min. These conditions are enough for fruit sanitation process or decreasing pesticide residue in berry fruit. It should be pointed that ozonated blackcurrant fruit in this conditions are still in good shape. The presented research give the opportunity to conduct further research on the use of ozonation technology in blackcurrant processing.

Conflict of interest

The authors have no conflict of interest to report.

Funding

Financial support of Rzeszow University for research. PB/KChiTZ/2017.

Footnotes

Acknowledgments

The authors have no acknowledgments.

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Impact of ozonation process on the antioxidant status in blackcurrant Ribes nigrum L. fruit

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1 Introduction

2 Materials and methods

2.1 Reagents and devices

2.1.1 Reagents

2.1.2 Devices

2.2 Fruit material

2.3 Ozonation procedure

2.4.1 Extraction of antioxidant compounds from fruit

2.4.2 Total phenolic content assay

2.4.3 ABTS•+ radical scavenging activity assay

2.4.4 DPPH• – radical scavenging activity assay

2.4.5 Measurement of phenylananine ammonia lyase (PAL) activity

2.4.6 Measurement of guaiacol peroxidase (GPOX), catalase (CAT) and ascorbate peroxidase (APOX) activity

2.4.7 Statistical analysis

3 Results

3.1 Total polyphenols content and antioxidant activity

Conflict of interest

Funding

Footnotes

Acknowledgments

References

2.4.3 ABTS^•+ radical scavenging activity assay

2.4.4 DPPH^• – radical scavenging activity assay