Variations in Non-enzymatic Antioxidants in Hisar Arun ( Local ) and Kashi Vishesh ( Hybrid ) Cultivars of Tomato Fruits Treated with H 2 O 2 during storage periods

Alterations in non-enzymatic antioxidants of tomato fruits of two cultivars (Kashi Vishesh: a local & Hisar Arun: a hybrid) during their storage were studied. Tomatoes, harvested at mature green, breaker, and mature red stages were treated with 1% and 4% solution of H2O2 and then kept at storage temperatures 5°C,10°C, and 15°C. Antioxidant activity, ascorbic acid, carotenoids, Lycopene and total phenol content were measured after every 7-day interval up to a total storage duration of 21 days. The recorded non-enzymatic characteristics shown an increase upto 14 days and then started declining irrespective of storage temperature and concentration of H2O2 treatment and maximum change was seen at15°C and 1% H2O2. On the other hand, lycopene content increased asymptotically at all maturity stages and at all storage temperatures which is suggestive of slowing ripening process. In conclusion, local cultivar, harvested at mature green stage, showed slowest increase rate in antioxidants activities when treated with 1% H2O2 and stored at 5°C. keywords: Antioxidant Activity, Carotenoids Lycopene, Total Phenol and Tomato.


INTROdUCTION
Tomato (Lycopersicum esculentum) belongs to a night shade family which is an adaptable vegetable and considered to be extensively consumed produce (Ahmed et al., 2012)  1 .On one hand, tomato fruits are consumed in fresh state while processed products are also commonly produced.Additionally, the nutritional content offered by tomatoes appeals for its induction in dietary habit leading to healthy life style (Uthairatanakij et al., 2017) 2 .Further, functional characteristics of tomato fruits undeniably allow for the devastating epidemiological sign leading to reduction in the risk of chronic disease for instance cancer and cardiovascular disease (Sgherri et al., 2008)  3 .
The defensive act of tomato fruits is characteristically ascribed to antioxidant compounds ascorbic acid, carotenoids including lycopene and beta-carotene, pro-vitamin A, flavonoids, phenolics and vitamin E (Odriozola-Serrano et al., 2008; Mehdizadeh et al., 2013) 4,5 .Tomato is a climacteric fruit, in which, the process of respiration can occur even after harvesting.In the course of ripening fruits experiences a sequences of structural, physiological and biochemical changes which are characterized by depletion of chlorophyll, softening of fruit, and rise in respiration rate, ethylene production and synthesis of sugars, lycopene and acids (Joshi et al., 2017)  6 which are subject to be controlled during pre-and post-harvest by means of chemical treatment as well as developing customized storage conditions with the aim to enhance their shelf life.
The physical and chemical processes in the course of ripening of tomatoes have been extensively studies for extending shelf life of tomato fruits.The specific information on the actual stage of harvest and consequence of ripening action on antioxidant capacity and antioxidant content were not available (Bayoumi, 2008)  7 .In this context, Hydrogen peroxide (H 2 O 2 ) has been approved as a GRAS (Generally regarded as safe) treatment by USA, as an alternative to currently used chemicals in postharvest treatments (Rodrigues et al., 2012)  8 .Owing to the less toxicity and safe decomposition products; hydrogen peroxide which is a strong oxidizing agent recommended as a substitute for decontamination of fruits and vegetables (Alexandre  et al., 2012; Loredo et al., 2013)  9,10 .An extensive variation in transmittable biological agents ranging as of spores of bacteria, vegetative cells, protozoa and their cysts, fungi, viruses and even prions have been inactivated by hydrogen peroxide.(Malik et    13 .Therefore, in this paper, we present an in-depth investigation of the evolution of non-enzymatic characteristics of tomatoes when treated with H 2 O 2 .

Sample Preparation
The experiment was conducted in the research laboratory of SHUATS, Allahabad.Two cultivars of tomato fruit (L.esculentum) namely Hisar arun, (a Local variety) and Kashi Vishesh, (a hybrid variety) were har vested from the experimental field at different maturity stages i.e.Mature Green, Breaker and Mature red; fruits were then graded according to shape, size, color and appearance.Fruits were then rinsed with tap water and dipped in an aqueous solution of 1% sodium hypochlorite for 1 min.for surface sterilization.After surface sterilization, fruits were dipped in 1% and 4% Hydrogen peroxide (H 2 O 2 ) solution for 20 min.next, fruits were stored in suitable plastic box for 21 days at a temperature of 5°C, 10°C and 15°C.The analysis was done for an interval of every 7 days.Over-ripened tomatoes of different treatments with the passage of time during storage were excluded from the trial.As following, we present the specific examinations made in the present investigation.

Antioxidant activity
Total antioxidant activity was assayed by % scavenging of the DPPH free radicals as the method mentioned by Yen and Duh (1994)14.
DPPH solution (0.004% w/v) was prepared in 95% methanol.The crude extracts were mixed with 95% methanol to prepare solution of known concentration as 20μg/ml, 40μg/ml, 60μg/ml, 80μg/ml and 100μg/ ml respectively in five test tubes.Freshly prepared DPPH solution (0.004% w/v) was added in each of these test tubes and after 10 min.the absorbance was taken at 517 nm wavelength.Ascorbic acid was used as a reference standard and dissolved in distilled water to make the stock solution with the same concentration (10mg/100ml or 100μg/ml) of extracts.Control sample was prepared containing the same volume without any extract.

Ascorbic Acid
Ascorbic acid was estimated using the method described in AOAC (1984) 15 .The 1.0 g dried and finely powdered sample were dissolved with 10 ml of 0.4% oxalic acid in water and centrifuged at 8000 rpm.Supernatant was used to test the content of Ascorbic Acid. 1 ml aliquots of the supernatant were maintained to 3.0 ml by 0.4% oxalic acid followed by the addition of 7.0 ml of 2, 6-dichlorophenol indophenol dye solution.The test mixture was properly mixed and its absorbance was recorded immediately at 518 nm.The amount of ascorbic acid was estimated by comparing with a standard curve drawn under identical experimental conditions.

Carotenoids
Total carotenoids in the plant tissues were estimated according to the method by Jensen  (1978) 16.One gram of each sample were extracted with 80% methanol and centrifuged.The supernatants were concentrated to dryness.The residues thus obtained were dissolved in 15 ml of diethyl ether and after addition of 15 ml of 10% methanolic KOH, the mixture was washed with 5% ice-cold saline water to remove alkali.The collective saline washings were extracted with ether (3:15 v/v).The ether extract from both were mixed together followed by washing with cold water till alkali free.The alkali free ether extract was dried over anhydrous Na 2 SO 4 for two hours in the dark.The ether extracts were filtered and its absorbance was measured at wavelength 450 nm (λmax) by using ether as blank.

Lycopene
Lycopene was determined through method adopted in Sadasivam and Manickam (1992) 17 .1.0g of tomato sample, as weighed into a conical flask, was transferred into a volumetric flask and filled with distilled water to reach 100 ml mark.Next, it undergone proper mixing and then transferred into a separating funnel in which 25 ml of petroleum ether was also added.It was shaking vigorously for about 15 minutes.The aqueous layer was run off and the absorbance of petroleum ether layer was recorded at 505 nm.

Total Phenol
Quantitative estimation of total phenol was done by the method described in Ragazzi and Veronese (1973) 18 .The 10 mg plant extract was dissolved in 10 ml of 50% MeOH: H 2 O (1:1), overnight at the room temperature.Subsequently in a volume of 1.0 ml of the aforementioned solution, 1.0 ml of Folin's Reagent (1N) and 2.0 ml of Na 2 CO 3 (20%) were added.The test mixture was mixed properly on cyclomixer, and then and maintained to 25 ml with water which is then kept at room temperature for 30 minutes.The absorbance of test mixture was measured at wavelength 725 nm on Varian Cary 50 Spectrophotometer.Graphs prepared using the standardized gallic acid solution of different concentrations and total phenol content have been expressed in mg/100 g material.

Statistical analysis
Each treatment had three replicates and all experiments were run at least twice, revealing similar results.All the data were collected and analyzed by multifactor ANOVA with SPSS 11.0 for windows.Significant effect was assessed at 5% (p ≤ 0.05) level of significance and the mean was separated using least significant difference (LSD) procedure.

Total Phenol
Total phenol content was affected by H 2 O 2 concentration, temperature, maturity stages and cultivars (Fig 1. and Table 1).Similar to antioxidant activity total phenol content increases upto 14 days of storage and then it starts decline upto 21 days.Hybrid variety & lower concentration (1%) of H 2 O 2 shows better treatments in terms of shelf life as the rate of change were very slow as compared to higher concentration (4%) & local varieties in all maturity stages at all the three varying temperature.The synthesis of phenylalanine ammonialyase (PAL) and hydroxycinnamoyl quinate transferase (HQT) enzymes is greatly assisted by the reduced storage temperature leading to increased total phenolics (Macheix et al., 1990; Toor et al., 2006)  19,20 .Additionally, the possibility that during storage of fruits, some compounds could be formed and react with the Folin-Ciocalteu's reagent and significantly enhance the phenolic content (Kallithraka et al.,  2009)  21 can also be accounted for increase in the total phenolics.Change in total phenol content during storage is a temperature dependent i.e. at 5°C temperature content were increase slowly followed by 10°C and 15°C during entire analysis.Initially content was high in mature green fruits but the rate of change was high in mature red stages followed by breaker and mature green.

Antioxidant activity
In this present study, the antioxidant activity of tomato fruits during storage treated with H 2 O 2 , was analyzed using DPPH radical scavenging assay, which is based on electron transfer reactions providing a scale of antioxidant reducing capacity.The percentage difference of the antioxidant activity, plotted in Fig. 2 (absolute values listed in Table 2) of tomato fruits of both the cultivars were found increased till 14 days of storage and then it starts decreasing.This trend is unanimously seen for all the storage temperatures, maturity stages and concentration level of H 2 O 2 .However, difference in increment was seen to be higher when fruits were stored upto 7 days during entire analysis.15°C storage temperature resulted comparatively higher activities of antioxidant than 5°C and 10°C temperatures and displayed much more activities when treated with 4 % H 2 O 2 .The increment in the antioxidant activity during progressive stages of ripening, and storage, may be caused due to the deposition of total phenolics and carotenoids (Toor  et al., 2006; Bhandari and Lee, 2016) 22,23 However, in contrast to our results, the investigation of the antioxidant and total phenolic content in the H 2 O 2 treated fresh-cut tomatoes, made by Kim et al.,  (2007)  24 , revealed a declining trend.This may be attributed to the fact that their study is subject to oxidation, and its use in lignin formation, led by post-harvest damage made during cutting the tomatoes which is not the case in our investigation.Pinelo et al., (2005)  25 suggested the promotional tendency of poly phenols in the synthesis of polymerized compounds to be the cause of increase in antioxidant activity.Further, the decrease in antioxidant activity is the consequence of polymerization exceeding a critical value, which leads to enhanced molecular complexity and steric hindrance disrupting hydroxyl groups' reaction with the DPPH radicals (Piljac-Zegarac et al., 2009)  26 .The local variety fruits (Kashi Vishesh) having a good source of antioxidants compared to hybrid variety (Hisar Arun), also responded remarkably to all the studied treatment supplements.The percent increment of antioxidant activity was found to be maximum in mature green stage in both local (Kashi Vishesh) as well as hybrid variety (Hisar Arun) fruits during storage.In particular, antioxidant activity of tomatoes harvested at mature green stage is found to be higher followed by breaker and mature red fruits for the entire analysis in both varieties.Further, increased concentration of H 2 O 2 led to increase in the antioxidant activity during storage along with increasing temperatures.The activity in tomato fruits was achieved highest when treated with 4% H 2 O 2 , stored for 14 days at temperature 15°C but slow rate of increment was perceived in matured red fruits under these storage condition.

Ascorbic Acid
The evolution in ascorbic acid (AA) content were recorded during the storage period upto 21 days.It was observed that AA content (percentage difference in Fig. 3 and absolute values in Table 3) were strongly dependent on the maturity stages, temperature and H 2 O 2 concentration.The present studies revealed that ascorbic acid rose upto 14 days of storage at all maturity stages; at each storage temperatures and H 2 O 2 concentrations after that it starts declining at all storage conditions.Arthur et al., (2015) 27 suggested the respiration and transpiration physiological process to be inducing the reduction in ascorbic acid content.Rate of increment was found directly proportional to the concentration of H 2 O 2 but major change was recorded in mature green fruits.However, temperature also played important role in the synthesis of Ascorbic acid i.e. at low temperature rate of change were slow and at higher temperature, the change was higher during storage periods in all stages.Positive effect of increase in the storage temperature on ascorbic acid synthesis may be an indication of active ripening process (Sammi and Masud, 2007) 28 while its decrement is indicative of senescent fruit.The ascorbic acid change in fruits of local variety treated with 1 % H 2 O 2 kept at 15°C after 14 days of storage were found almost similar at 4 % H 2 O 2 stored at temperature 5°C.Chemical H 2 O 2 @4% accelerated upto 30% increase in AA while at low concentration (@1%) displayed increment only upto 15% in mature green fruits at 14 days stored at the temperature 15°C.Whereas, only 6% and 4% increment were found respectively in mature red and breaker at same condition in local variety.In particular, it is noted that the rate of change of ascorbic acid is slower in hybrid variety when compared to local variety in all the applied treatments.

Carotenoids
Figure 4 shows the percentage difference in the carotenoid content (Table 4 contains absolute values), recorded every 7 days within the total storage duration of 21 days.We witnessed changes in carotenoid content to increase until 14 days after which the same started declining.The rate of change was maximum (21%; T2) in mature red fruits of hybrid variety after 14 days of storage treated with 1 % H 2 O 2 which was kept at 15°C while minimum (T3; 3%) were in mature green fruits of local variety after 14 days of storage when treated with 4% H 2 O 2 and kept at 5°C.The changes were higher in hybrid variety and at lower concentration of H 2 O 2 .In fruits treated with 1% H 2 O 2 , changes were measured up to 22 % which is the maximum value for mature red fruits of hybrid variety.From T3 and T4, we determined that the change in carotenoid content got doubled in for increased H 2 O 2 concentration but the rate of change was slower at low temperature (5°C).The investigation of Yumbya et al., (2014) 29 revealed similar trend in the carotenoids, however, in mangoes and passions fruits.During storage, in the sequence of ripening process of tomatoes, decomposition of chlorophyll occurs which results in subsequent release of carotenoids (Joyce et al., 2016) 30 leading to increased carotenoids as revealed in our investigation.

Lycopene
Change in lycopene content during storage in two cultivars of tomato fruits treated with H 2 O 2 ,  stored at different temperatures were evaluated as shown in Fig. 5 and enlisted in    34,35,31 .Concentration of H 2 O 2 is directly proportional to the change in the lycopene content.From Fig. 5, it can be noted clearly that the rate got doubled in T4 when compared with that in T2 while in case of T3, the rate of increment was slow, probably due to low storage temperature.On the other hand, the storage temperature has shown a dominating effect in the form of significant reduction (by 50%) in the lycopene content for both the cases of H 2 O 2 concentrations.Thus lower temperature can help in depressing the increment in the lycopene content.Further, the change was higher in local variety than hybrid variety.Also, the lycopene content increased faster in fruits harvested at mature green stage.

CONCLUSION
Tomato is used as an integral part of human diet.Being a climacteric fruit, tomato is prone to irreversible changes leading to reduction in its shelf life.Therefore, in this paper, we investigate the variation of non-enzymatic antioxidant content of the two most commonly consumed Indian tomato cultivars namely Hisar arun (a Local variety), and Kashi Vishesh (a Kashi Vishesh variety) which were harvested at different maturity stages namely Mature green, Breaker, and Mature red, when treated with varied concentration (1% and 4 %) of H 2 O 2 and stored at various temperature 5°C, 10°C and 15°C.In particular, we record Antioxidant activity, Ascorbic acid, Lycopene, Carotenoid, and Total Phenol contents every 7 days and until 21 days of complete storage time.Our investigation revealed the aforementioned contents to be increasing till 14 days after which they show a decreasing trend.It is of note that the rate of increase in the carotenoid content is found to be high for the mature red stage in comparison to the mature green stage.Summarily, we found tomato fruits, harvested at mature green stage, retained significantly higher amount of Ascorbic Acid after 14 days of storage compared to fruit harvested at the breaker and red stage.Additionally, our investigations are suggestive of 1% H 2 O 2 treatment to be the most effective in terms of offering a definitive control in the rate of evolution of non-enzymatic antioxidants of the tomatoes such as lycopene and carotenoids.Further, although Hisar arun (Local) variety is found to retain more nutritional content than Kashi Vishesh (hybrid) variety, latter shows higher shelf life.

Fig. 2 .
Fig. 2. Percentage difference in Antioxidant Activity for 21 days of investigation of tomato fruits stored at different temperature and H 2 O 2 concentration.The color code and line style were kept similar to that used in Figure 1

Fig. 3 .
Fig. 3. Percentage difference in Ascorbic Acid content for 21 days of investigation of tomato fruits stored at different temperature and H 2 O 2 concentration.The color code and line style were kept similar to that used in Figure 1

Fig. 4 .
Fig. 4. Percentage difference in Carotenoids content for 21 days of investigation of tomato fruits stored at different temperature and H 2 O 2 concentration.The color code and line style were kept similar to that used in Figure 1 al., 2012; Delgado et al., 2012; Loredo et al., 2013) 11,12,10.Hydrogen peroxide (H 2 O 2 ) can have a lethal or inhibitory effect on microorganisms, depending on the pH, temperature and other environmental factors(Augspole et al., 2017)

Table 4 : Carotenoids content (mg/100g FW) during storage in tomato fruits treated with hydrogen peroxide
23,33.The transformation of chloroplast in chromoplast during the ripening process of tomatoes (Bhandari and Lee, 2016; Dibbisa et al., 2016)23,33can be understood to cause the increase in lycopene content. Furthmore, progression in the enzyme activity associated with phytoene synthase I is also attributed to contribute to the synthesis of lycopene content during the storage and ripening process(Ronen et al., 1999; Paul and peter, 2004; Ilahy  et al., 2011)