The impact of cumin essential oil on cold storedradish tubers

Notable biological compounds in radish, made it as one of the most popular crops in the raw vegetables global market. However, storing it under low temperature conditions is associated with browning and taste changing. The present research aimed to evaluate the effects of different concentrations of cumin essential oil (0, 1.56, 3.13, 6.25, 12.5 and 25 ppm) and the storage period (0, 3, 6 and 9 days) on antioxidant parameters of radish tubers under low temperature conditions. The results indicated declining trends in the L* and a* values, beside ascending trend in b* value after nine days of storage. However, over the storage period of tubers, these parameters increased in cumin essential oil treated tubers. According to our findings, the application of cumin essential oil increased protein content, but reduced the malondialde­ hyde content, polyphenol oxidase and peroxidase activities. The cold­stored radishes received the most effective treatment of cumin essential oil at the concentration of 12.5 and 25 ppm.

The global marketing of radish has improved due to be included in easytoprepare foods especially in the northern European countries like Holland and Germany (Salerno et al., 2005). However, the storage of radish under ambient conditions leads to shrinking and hence failing its marketing (Luegno and Calbo, 2001).
Low temperature conditions are commonly used in the food industry to maintain the quality of horti cultural products in storage, reduce the respiration rate and delay metabolic processes (Patel et al., 2016). But lipid peroxidation, the most important deteriorating factor during low temperature storage, affects the nutritional value and sensory evaluation of food products (Liang et al., 2020), i.e radish lose up to 5% of its weight and 43% of its nutritional con tent after exposure to low temperature (10°C) for 10 days (Del Aguila et al., 2006).
The antioxidants are essential compounds that prevent or delay the lipid peroxidation (Khalid et al., 2016). Food industries use artificial antioxidants such as BHA (Butylated hydroxylanisole), BHT (Butylated hydroxyltoluene), TBHQ (Tertiarybutyl hydro quinone), and PG (Propyl gallate) to extend the shelf life of coldstored products. However, the side effects of these synthetic components have caused con sumers to be concerned (Andre´ et al., 2010;Liang et al., 2020) and drawn the attention of researchers to safe alternatives such as natural derived products. Plants essential oils (EOs) are a rich source of antioxi dants which reduce the production of reactive oxy gen species (ROS) or scavenge the formed ROS (Khalid et al., 2016).
Browning and taste changing are the main disor ders which influence the quality of radishes under low temperatures (Del Aguila et al., 2008;Ramachandran et al., 2013). Using antioxidant com pounds, such as citric acid and ascorbic acid (Del Aguila et al., 2006;Lee et al., 2007) or covering the tuber with chitosan (Ramachandran et al., 2013) have reduced the browning intensity under cold stor age conditions. Packed radishes can also be stored for six days under low temperatures with no brown ing symptoms (Nicola et al., 2004;Ayub et al., 2013).
Despite several reports on the antimicrobial char acteristics of cumin EO (Thippeswamy and Naidu, 2005; Gachkar et al., 2007; Milan et al., 2008Dua et al., 2012), there is no research on its potential to serve as an antioxidant with ROS scavenging capacity, specifically in applying on coldstored products. Therefore, the present research aimed to evaluate the effects of different concentrations of cumin EO, as an organic antioxidant compound, on the bio chemical and antioxidant parameters of stored radishes under low temperature conditions.

Plant material preparation
Radish (Raphanus sativus L. var 'Cherry Belle') seeds were planted (October 1 st 2019) in the research greenhouse (RH=50%, temperature: 26 ± 1°C day /20± 0.5°C night, and 50% shade) at the University of Hormozgan (53° 33ʹ E 28° 30ʹ N, 10 m), Iran. The plas tic pots (25×18 cm 2 ) were filled by a media mixture (soilsandsilt in 1:1:1 ratio). Daily drip irrigation sys tem was used for all experimental units. The tubers were harvested 40 days later. The experiment was repeated next months (November 1 st 2019 December 10 th 2019). The radish tubers were then transferred to the laboratory. After wards, well formed and uniform tubers (1.5×1.5 cm 2 ) which were healthy, smooth, firm and free from decay, damage or cracks were selected, washed and dried.

The experimental design
The factorial experiment was a completely ran domized design with six replications (10 samples per each experimental unit). The factors were cumin EO concentrations (0, 1.56, 3.13, 6.25, 12.5 and 25 ppm) and the storage period (0, 3, 6 and 9 days). The con trol (day 0) measurement was done before applying the treatments. The cumin EO was prepared from Zardband Pharmaceuticals industry, Tehran, Iran. The phytochemical and microbiological properties of the EO were described by Zarband Company (Table 1). The Cumin oil was obtained by steam distillation of seed. Ground seed were sieved and then subjected to water distillation using a Clevenger apparatus (3 hours) (Beis et al., 2000).
The cumin EO was diluted with distilled water (0, 1.56, 3.13, 6.25, 12.5 and 25 ppm) and the radishes were immersed at 20°C for 10 min. Then all radishes were airdried under room temperature for 1 h, put in polyethylene plastic containers (10 tubers per each container) and placed in the cold storage (5°C, 95% RH). The tubers were selected for the following mea surements at 0, 3, 6 and 9 days of storage.

Color parameters
Tuber skin color was measured using a colorime ter (KonicaCR400 Minolta, Japan) under reflected light in CIE L*a*b* system, where L* expressed color lightness from 0 (black) to 100 (white), a*defined the proportion of red (+a) to green (a), and b* repre sented the proportion of yellow (+b) to blue (b). The average of six records was considered for every color parameter.

Malondialdehyde content
According to Heath and Packer (1968), 0.5 g of the tuber was homogenized in 5 ml of 1% Trichloroacetic acid (TCA), centrifuged (10000 rpm, 5 min) and the supernatant (250 μl) was mixed with 1 ml of Malondialdehyde (MDA) solution containing 20% TCA and 5% TBA (Thiobarbituric acid). It was then incu bated in a hot water bath (95°C) for 30 min, immedi ately cooled and recentrifuged (10000 rpm, 10 min). The absorbance of the sample was measured at 532 and 600 nm (using a Cecil CE2501 spectrophotome ter). The MDA content was calculated using the equation (1). MDA (mg g 1 FW) = [(A 532 A 600 ) ×W×1000]/116 (1) Where A 532 and A 600 are the sample absorptions at 530 and 600 nm, respectively, W is the sample weight (mg) and 116 is dilution factor

Total phenol content
The total phenol content was determined by the FolinCiocalteu procedure (Spanos and Wrolstad, 1990). Tuber tissue (0.5 g) was homogenized with 10 ml of 80% methanol. The mixture was centrifuged (10000 rpm, for 10 min). Then 10 µl of the super natant, 490 µl of distilled water and 500 µl of Folin Ciocalteu reagent were mixed and incubated under dark ambient conditions (24±1°C) for 3 min. Then 500 µl of sodium carbonate (1%) was added and the mix tures were reincubated under the same conditions for 30 min. The absorbance was measured at 750 nm using a Cecil CE2501 spectrophotometer and the phenol content was expressed in µg gallic acid g 1 FW, using a gallic acid (00.1 mg ml 1 ) standard curve.

Antioxidant activity (DPPH)
The DPPH (2,2Diphenyl1Picrylhydrazyl) assay was done according to Singleton et al. (1999) procedure. Briefly, 0.1 g of tuber tissue was powdered in liquid nitrogen and then 100 ml of 96% ethanol was added. After centrifuging (at 3500 rpm, 5 min), 950 µl of 0.1 N DPPH was added to 50 µl of each sample and stirred immediately. Each sample was then kept in ambient dark conditions for 30 min. Finally, the absorption of the extract was measured at 517 nm. The antioxidant activity was evaluated using equation (2).

Protein content
Tuber tissue (0.1 g) was homogenized in 1 ml of 50mM sodium phosphate buffer (containing 129.18 mM NAH 2 PO 4 , 383.96 mM NA 2 HPO 4 , 12.66 mM EDTA, pH=7). The homogenates were then centrifuged (10000 rpm, 4°C for 10 min). The Bradford solution (1 ml) was added to the supernatant (50 μl) and the absorbance was measured at 595 nm. The protein content was evaluated according to the standards curve of Bovine serum albumin (BSA) and expressed in mg g 1 fresh weight (Bradford, 1976).

The assay of protease, catalase, peroxidase and polyphenol oxidase activities
The radish tuber (0.5 g) was powdered with liquid nitrogen and mixed with 1ml of the extraction solu tion (containing 100 ml of 50 mM phosphate buffer, 1.27 mM of EDTA and 4Mm of PVP). The mixture was then centrifuged (1000 rpm, 15 min). Afterwards the supernatant, as an enzyme extract, was used for determining the activity of the following enzymes (Dhindsa et al., 1981).
The protease activity was determined using a pro cedure defined by Homaei and Samari (2017). Briefly, 50 μl of the enzyme extract was mixed with 350 μl of 50mM sodium phosphate buffer (pH=7.5) and then 800 μl of 1% casein was added. The mixture was incu bated for 10 min at ambient temperature. Then 400 μl of 10% TCA was added and the mixture was again reincubated at ambient temperature for 20 min. Finally, the samples were centrifuged (10000 rpm, 10 min) and the absorption at 280 nm was measured. The coefficient of excitation was 26.40 mM 1 cm 1 .
To assay the catalase activity, 50 μl of the enzyme extract was mixed with 1 ml of the catalase reaction solution (containing 50 mM phosphate buffer with pH=7 and 15 mM H 2 O 2 ). Then the absorption was measured at 240 nm and the coefficient of excitation was 39.4 mM 1 cm 1 (Dhindsa et al., 1981).
To determine the peroxidase activity, 33 ml of the enzyme extract was mixed with 1 ml of peroxidase reaction solution (containing 13 mM guaiacol, 5 mM H 2 O 2 and 50 mM phosphatepotassium buffer with pH= 7). The sample absorption was then measured at 470 nm and the coefficient of excitation was 26.6 mM 1 cm 1 (Chance and Maehly, 1995).
In order to assay the polyphenol oxidase (PPO) activity, 100 μl of the enzyme extract was mixed with 1 ml of pyrogallol reaction solution (containing 2.5 ml of 50 mM potassium phosphate buffer and 200 μl of 0.2 M pyrogallol). The sample absorbance was then measured at 280 nm and the coefficient of excitation was 26.4 mM 1 cm 1 (Kar and Mishra, 1976).

Data analysis
The statistical analysis was done using SAS (ver sion 9.1.3) (SAS Institute Inc. Cary, NC, USA, 1990). The ShapiroWilks test confirmed the data normality (procedure: PROC UNIVARIATE, SAS). The Multivariate Analysis of Variance was performed related to the observation period and cumin levels, both of which were considered as independent vari ables (procedure: PROC GLM, SAS). Pillai's trace test confirmed the variance homogeneity (procedure: PROC GLM, SAS). Tukey's test was used in order to compare the mean values (procedure: Files, Sedit, Factor, Range, P<0.01, MSTATC). Excel 2013 was used to draw the figures. The presented mean values are the average of two growing seasons.

Color parameters
The results indicated that all color parameters were influenced during the observation period. A declining trend was observed in the L* value after nine days of storage. The highest L* value (35.1±0.56) was observed on day 0 (in control plants) and the lowest value (23.19±0.97) on 9 days (in control plant). However, over the storage period of tubers, this factor increased in cumin EO treated tubers. Increase of 17.43 % and 17.32% in L* value in the 9 days of treatment was the results of 12.5 and 25 ppm cumin EO application, respectively (Fig. 1A). The results indicated that over the storage period, the a* trend was declining. The highest (30.32±0.75) and the lowest (19.35±0.88) values were observed on the 0 and 9 days, respectively, in the control after being under cold storage. But, increasing the concen tration of cumin EO in each measurement period (3, 6 and 9 days), led to an increase in a* value. Based on the results, the highest value (29.42% increment) occurred at 25 ppm cumin EO on 3 days (Fig. 1B).
According to our results the b* value showed an ascending trend and rose from 10.5±0.65 on day 0 to 16.87±0.59 on 9 days of 25 ppm EO treatment. Moreover, increase in the concentration of cumin EO made significant increments in b* value (8.37%, 3.43% and 12.64%, respectively on 3, 6 and 9 days) (Fig. 1C).

MDA, phenol contents and antioxidant activity
The MDA and phenol contents, along with the antioxidant activity, were significantly affected by the EO levels and storage duration. During the nine days of radish storage, the MDA, total phenol content and the antioxidant activity showed ascending trend. Furthermore, the cumin EO treatment resulted in a significant reduction in MDA and phenol contents ( Figs. 2A, B).
According to our findings, malondialdehyde gen erally increased as a result of storage period extent. The MDA content of the first assessment, day 0, (0.75±0.16 mg g 1 fresh weight) rose to 3.29±0.15 mg g 1 fresh weight in the last assessment (9 days of stor age). The cumin EOtreated tubers had 0.76±0.14 mg g 1 fresh weight MDA content at 25 ppm on 9 days. However, in each assessment period, a declining trend in malondialdehyde content was a result of increment in the concentration of cumin EO. The highest amount of MDA was related to the control treatment value on the 9 days (3.29±0.15 mg g 1 fresh weight) and the lowest value (0.55±0.14 mg g 1 fresh weight) was recorded at the 25 ppm cumin EO on the 3 days ( Fig. 2A).
The results indicated that the total phenol con tent showed an increasing trend over the storage period. The phenol content rose from 1243±116.19 μg of gallic acid g 1 fresh weight, in the first observa tion (day 0) to 4613.05±258.90 μg of gallic acid g 1 fresh weight on the last day of storage. Although, the total phenol content showed a declining trend, with increasing the concentration of cumin EO. The lowest content of total phenol was observed at 25 ppm cumin EO on the 3 days (2024.03±212.84 μg of gallic acid g 1 fresh weight). (Fig. 2B).
Regarding the antioxidant activity, over the dura tion of storage period and also with increasing the concentration of cumin EO, an ascending trend was observed compared to the control. The value of antioxidant activity on day 0 (21.2±2.49%) for control plants, improved significantly on days 3 (54.98±3.62%), 6 (57.19±3.33%) and 9 (75.24±1.41%). The 25 ppm cumin treatedtubers showed a same trend, which was reached from 84.37±2.41% on 3 days to 95.18±3.07% on 9 days (Fig. 2C).

Protein content and enzyme activities
The effect of low temperature storage on the radishes caused in the reduction in the protein con tent along with an increase in the activity of pro tease. Different levels of cumin EO in each observa tion period had a significant effect on both traits (Figs. 3A, B). The results indicated that the protein content decreased over the storage period of radish. The protein content in the control treatment on the day 0 measurement was 27.1±0.33 mg g 1 fresh weight and reached to 15.48±0.94 mg g 1 on the 9 days. The highest content of protein was related to 1.56 ppm cumin EO (26.78±0.33 mg g 1 fresh weight) on the 3 days and the lowest content was related to 3.13 ppm cumin EO (26.4±0.32 mg g 1 fresh weight). On the 6 and 9 days of storage, the highest value was occurred in 25 ppm cumin EO (25.29±1.22 and 19.40±1.32 mg g 1 fresh weight increment) (Fig. 3A).
The protease activity on day 0 (1.1±0.16 μmol min 1 g 1 fresh weight) had an ascending trend during 9 days of cold storage. Also increasing the concentra tions of cumin EO (6.25±0.11 ppm and more), increased the protease activity. This trait was varied from 1.35±0.15 to 2.53±0.20 μmol min 1 g 1 fresh weight on the 3 days and from 4.93±0.11 to 6.93±0.14 μmol min 1 g 1 fresh weight on the 6 days, as a result of different cumin EO levels. However, on the 9 days of storage, the protease activity varied from 9.55±0.11 to 11.9±0.15 μmol min 1 g 1 fresh weight as a result of different EO concentrations (Fig.  3B).
There were enhancements in the activities of radish antioxidant enzymes (Figs. 4A, B, C). According to figure 4A, a weak increment was observed in the catalase activity, by increasing the storage days until 6 days. Nonetheless, the first measurement did not differ much than the second measurement. However, on 9 days, the highest activity was observed in all concentrations compared to the control. Different concentrations of cumin EO showed various patterns on different days. On 3 days, the lowest catalase activity was observed in the control (106.34±12.39 μmol min 1 g 1 fresh weight) and the highest was at 1.56 ppm cumin EO (132.51±22.27 μmol min 1 g 1 fresh weight). The highest enzyme activity on the 6 days was at 3.13 ppm (209.40±18.38 μmol min 1 g 1 fresh weight) and the lowest was at 6.25 ppm (202.27±17.46 μmol min 1 g 1 fresh weight). On 9 days, the trend was completely declining, as the highest enzyme activity was in the control (1013.45±19.26 μmol min 1 g 1 fresh weight) and the lowest was observed at 25 ppm cumin EO (602.72±19.07 μmol min 1 g 1 fresh weight) (Fig. 4A).
Peroxidase activity increased over the storage period and changed from 78.23±2.4 μmol min 1 g 1 fresh weight on day 0 to 178.94±4.39 μmol min 1 g 1 fresh weight in the last observation (9 days). In addi tion, increasing in the EO concentration reduced the activity of this enzyme. Accordingly, the lowest val ues (29.81±4.73, 45.90±4.42 and 61.41±3.76 μmol min 1 g 1 fresh weight) of the peroxidase activity were observed at 25 ppm cumin EO on the 3, 6 and 9 days, respectively and the highest activity was in the con trol (113.80±4.60, 149.79±3.72 and 178.94±4.39 μmol min 1 g 1 fresh weight, respectively) on 3, 6 and 9 days, respectively (Fig. 4B).
The activity of polyphenol oxidase enzyme increased over the storage days (from day 0 to 9 days) at zero and 1.56 ppm; as the highest activity (25.31±0.73 μmol min 1 g 1 fresh weight) was observed in cumin EOfree tubers on 9 days. Changes in the activity of polyphenol oxidase on 3 and 6 days showed similar trends. Its activity on day 0 (12.76±0.45 μmol min 1 g 1 fresh weight) rose to 25.31±0.73 μmol min 1 g 1 fresh weight on the 9 days. In the cumin EO treated tubers, the PPO activity indi cated a declining trend, over time and the lowest activity (8.44±0.49 μmol min 1 g 1 fresh weight) was observed at 25 ppm, on 6 days (Fig. 4C).

Discussion and Conclusions
Excess production of ROS and oxidative burst, under cold conditions, elicit some plant physiological reactions. The formed ROS interact with cellular com ponents, trigger cascade of oxidative responses, per oxidase lipids, degrade proteins, inactivate enzymes and damage the DNA (Mittler, 2002). Exposure to cold conditions causes the discoloration of radish and production of cracks on its edible parts (Abdel, 2016). In addition, low temperatures influence carbohydrate accumulation in radish roots, accelerate plant growth rates and increase the shoot/root biomass ratio (Sirtautas et al., 2011).
Color is an important factor in the consumer's acceptance of foods. The L*, a* and b* values describe color parameters of food products (WalkowiakTomczak et al., 2008). The color of storedfruits and vegetables mostly changes and turns darker over time. HernandezMunoz etal. (2008) reported a decrease in the values of color indices of cold storedstrawberry fruits. In the pre sent study, there was a significant reduction in L* and a* values, despite an increase in b*of radish tubers through storage period, which was related to a reduction in water content and product browning (Hassani etal., 2012). The impact of organic compounds on the color char acteristics of horticultural products has been report ed previously (RaybaudiMassilia etal., 2008;Asghari Marjanlo et al., 2009). Clove EO prevented browning of grapes by preventing water loss (MartinezRomero et al., 2007). According to our findings, treatment with different cumin EO concentrations caused differ ent color values in the radishes.
Cold disturbs the electron transfer chain in mito chondria and chloroplasts. This happens by an excess production of ROS and causes oxidative damage to the membrane, thereby accelerates the lipid peroxi dation and MDA over production (Larkindale and Huang, 2004). The results of this study showed a sig nificant increase in the malondialdehyde content during the nine days of cold storage. Antioxidant compounds inhibit free radical's generation, inter rupt its functions or lead to ROS destruction (Melo et al., 2005;Srivsatava et al., 2011). The cinnamon EO has reportedly prevented the membrane lipids per oxidation in peaches (MonteroPrado et al., 2011). Our findings regarding the coldstored radishes treat ed with cumin EO, confirm this finding.
In this work, the antioxidant capacity of cold stored radishes was assayed using the scavenging ability of DPPH radicals, which is used widely for eval uating the radical scavenging effects of chemical and organic materials. DPPH functions by absorbing elec trons or hydrogens, thereby becomes a stable mole cule (Sagar and Sing, 2011). The results indicated that the scavenging capacity of cumintreated tubers was significantly higher than those of the control, proving that cumin improved the scavenging ability in radish tubers. According to our results, cumin EO displayed a dosedependent manner in the scavenging of free radicals, as previously reported by Dua et al. (2012). Similarly, the antioxidant properties of peppermint and clove oils can be attributed to the ability of free radical scavenging (Tripathi and Dubey, 2004).
Polyphenol compounds naturally show an ability to scavenge ROS. They are considered to be the effective nonenzymatic antioxidants (Ma et al., 2011; Zrig et al., 2011. The application of organic compounds was report edly capable in improving the phenol content of cold stored mangoes (Wang and Lin, 2000;Razzaq et al., 2015). This can occur through changes in both polyphenol oxidase (PPO) and phenylalanine ammo nialyase (PAL) activities (Sun et al., 2010;Deng et al., 2015). In our work, keeping the radish tubers in cold storage conditions made an increase in their phenol content. The application of cumin EO improved the antioxidant capacity and gradually decreased the phenol content.
The lethal impact of oxidative stress on biological organisms is correlated to the destruction of proteins and the inactivation of enzymes (Srivsatava et al., 2011). In the present study, the cold stored radishes displayed a visible decrease in protein content and an increase in antioxidant enzyme activities. The antioxi dant enzymes act as agents that trigger detoxification at low temperatures (Mittler, 2002) and convert free radicals to the compounds that eventually release water and oxygen (Zheng and Tian, 2006 (Wang, 1995). Catalase converts free radicals to oxygen and water (Zheng and Tian, 2006). Our results of increases in catalase, peroxidase and PPO activities in cold stored radishes, confirmed the previous researches.
Essential oils can be considered as agents that aid the antioxidant defense system, reduce the release of radicals, prevent the destruction of cells and thus increase stress tolerance of plants (Holley and Patel, 2005). The usage of carvacrol and anethole in rasp berry causes a reduction in antioxidant enzyme activ ity (Jin et al., 2012). Lipoxygenase activity is also reported to be reduced in cinnamontreated peaches (MonteroPrado et al., 2011).
Microbiology limit test confirmed that bacteria, mold and yeasts in our essential oil sample can be ignored. Also it has no Salmonellae neither Escherichia.coli. The density of cumin essential oil is 0.90 (Lewis, 1999). The pale yellow color and strong odor of our essential oil sample was similar to stan dard reference. Cumin seeds contain flavonoids (β pinene, pcymene, cumin aldehyde and cuminyl alco hol) are recognized to have antioxidant activity and scavenging capacity of the superoxide anion (Sowbhagya 2013). The refractive index of an essen tial oil is a unique number that designates how the oil responds to and bends light. Essentially, it is a measurement that tests how the speed of light is altered when passing through the oil. The refractive index of our essential oil sample (1.48) and cumin aldehyde content (36.33%) were close to which reported earlier (1.49 and 45%, respectively) (Fahlbusch et al., 2005). Cumin aldehyde is a con stituent of the essential oils of eucalyptus, myrrh and cumin (Morshedi et al., 2015). βpinene is a monoter pene, an organic compound found in cumin essential oil (Li and Jiang, 2004). The presence of polyphenolic compounds in cumin EO inhibits protein oxidation and enzyme inactivation (Melo et al., 2005). According to our findings, cumin EO improved the antioxidant system of tubers so caused in decreased catalase, peroxidase and PPO activities.
The market for chilledfresh products has a dra matic rise in the recent decade, encouraged mostly by the consumer request for nutritious, fresh and additivefree products. Hence, the food industry has to respond with novel preservation, storage and han dling strategies. In our study, the storage of green househarvested radishes at low temperatures declined the L* and a*values, and protein content during nineday of storage, but increased the b* value, phenol content, antioxidant capacity, MDA level, PPO, peroxidase, catalase and protease activi ties. The application of cumin EO reduced the MDA content, polyphenol oxidase and peroxidase activi ties. Finally, coldstored radishes received the most effective treatment of cumin EO at the concentration of 12.5 and 25 ppm.