Changes in antioxidant and biochemical activities in castor oil-coated Capsicum annuum L. during postharvest storage

Changes in antioxidant and biochemical activities in castor oil-coated Capsicum annuum L. during... This study, for the first time, evaluates the efficiency of castor oil when used as an external coating on Capsicum annuum L., to increase postharvest storage-life at 4 ± 1 °C. The castor oil-coated fruits were successfully stored for 36 days, while the non-coated fruits could only sustain for 18 days. Throughout the storage period (at 9-day intervals), different antioxidants and biochemical assays (allied with storage) such as titratable acidity, ascorbic acid content, ferrous ion chelating activity, reduc- ing power, DPPH scavenging activity, hydroxyl radical scavenging activity, total phenolic content, total sugar estimation, and enzymatic study of polyphenol oxidase and pectate lyase, were assessed. During storage, the castor oil-coated fruits showed a substantial decrease in titratable acidity, ascorbic acid content, total phenolic content, including antioxidant activities such as reducing power and DPPH activity; however, an increase in ferrous ion chelating activity, total soluble sugar content, polyphenol oxidase activity and initial pectate lyase activity was observed, in contrast to that of the non-coated fruits. The application of castor oil proved to be effective in delaying the ripening process of fruits during storage. Keywords Antioxidant activity · Castor oil · Green chilli · Postharvest · Storage-life Introduction Capsicum annuum L. (green chilli), an important member of Solanaceae family, is mainly grown as a cash crop owing to its distinctly pungent non-climacteric fruits, enriched with vitamins and minerals. Inspite of having such high * Jitendriya Panigrahi nutritional values it has limited storage-life (Panigrahi et al. jitenp2001@gmail.com 2017), which is a major problem for fruits and vegetable * Saikat Gantait growers. Following its harvest, the fruits undergo gradual saikatgantait@yahoo.com deterioration due to desiccation, oxidative reactions, micro- Mansi Patel bial growth, and other biochemical changes. Such rapid maansipatel1512@gmail.com deterioration in the form of softening, wrinkling, wilting Niyati Patel and decaying of the harvested produce during storage and niyaa_001@yahoo.com transportation, fail to provide the anticipated profit, con- Bhumi Gheewala sumer-acceptance or to even meet the actual cultivation cost. bhumigheewala93@gmail.com Softening of fruits is directly linked with water loss during storage (Lownds et al. 1994; Rao and Shivashankara 2015). Department of Biotechnology, Shri A. N. Patel Post Herein, external coating(s), acting as partial barrier Graduate Institute of Science and Research, Anand, Gujarat 388001, India to water vapour, gases and restricting rapid metabolism, might be helpful to preserve the texture, quality and exter- All India Coordinated Research Project on Groundnut, Directorate of Research, Bidhan Chandra Krishi nal appearance of harversted fruits, and in due course may Viswavidyalaya, Nadia, Kalyani, West Bengal 741235, India also improve their storage-life, significantly influencing the Department of Genetics and Plant Breeding, Faculty functions of storage-associated biochemicals and antioxi- of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, dants (Baldwin 1994; Li et al. 2017; Panigrahi et al. 2017). Nadia, Mohanpur, West Bengal 741252, India Vol.:(0123456789) 1 3 280 Page 2 of 8 3 Biotech (2018) 8:280 External coatings comprise hydrophobics (lipids or waxes), Materials and methods hydrocolloids (polysaccharides or proteins) or a combination of both the compounds (composite coatings). The coating Collection, preparation and castor oil coating composition determines the barrier properties of the material of fruits with respect to water vapour, oxygen, carbon dioxide and lipid transfer in food systems (Guilbert et al. 1996). Even Mature C. annuum fruits (var. New Mexico Chile) were col- though hydrophilic coatings function as excellent carbon lected from the local market. The collected fruits were thor- dioxide and oil barriers and provide strength and structural oughly washed with tap water before dipping in chlorine water integrity to the fruits, however, they are not at all effective for 30 s. After soaking up the adhered water using filter papers, moisture barriers and can create adverse anaerobic condi- the fruits were drenched in castor oil for 5 s and then wrapped tions (McHugh and Krochta 1994). Hydrophobic lipid or in aluminium foil, prior to their storage in common refrigerator oil coatings have good water vapour barrier properties, due at 4 ± 1 °C. Throughout the storage period (for 36 days with to their low polarity (Guilbert et al. 1996). Usually, climac- the intervals of 9 days), the following antioxidants and bio- teric fruits like green peppers do not show any detectable chemical assays were performed. changes in respiration rates during storage (Conforti and Zinck 2002). However, it was recorded that when cucumber Titratable acidity (following Chen et al. 1986) and bell pepper were exogenously coated with chitosan, the rate of respiration noticably reduced (Ghaouth et al. 1991). The fruit pulps weighing 5  g were macerated with 50  ml Furthermore, Wong et al. (1994) reported that when cut distilled water and centrifuged at 5000 rpm for 10 min. The apple pieces were treated with multilayer coatings, it aided supernatant (homogenate) solution was titrated to measure the in maintaining the internal carbon dioxide concentrations. titratable acidity, using 0.1N NaOH up to a pH level of 8.1. Castor oil is an important vegetable oil with a rich source of ricinoleic acid, a mono-unsaturated, 18-carbon fatty acid. Ascorbic acid content (following Ranganna 1986) Ricinoleic acid amid all the other fatty acids, is exceptional since it includes a hydroxyl group on its twelfth carbon; it Ten milliliter of homogenate was mixed with equal volume of is for the virtue of this functional group that ricinoleic acid 20% meta phosphoric acid, and collected in a 100 ml volumet- (and castor oil) is more polar than the other fats (Thomas ric flask to adjust the volume with distilled water. The solution 2012). Castor oil proved to be efficient with its greater was titrated with the standard 2, 6-dichlorophenol indophenol resistance to increased CO and reduced O in the internal dyes. The ascorbic acid content of each sample was measured 2 2 environ of fruits, in comparison to that of the essential oils by the equation: Ascorbic acid (mg/100 ml homogenate, i.e. or mineral oils. Castor oil coating significantly delayed the 10 g fruit pulp) = (Titre × dye factor × volume made up × 100)/ softening and retained the flavour in fruits (Baldwin 1994). (volume taken for titration × sample weight). Owing to such property, the castor oil coating on seeds of edible pulse or oilseed crops is being used traditionally, to Total phenolic content (following Singleton et al. extend the storage life since long, in the southern districts 1998) of Gujarat State, India (Parmar and Jain 2016). There are several reports on postharvest storage-life of C. annuum To collect the polyphenol, 1 g of homogenised fruit was mixed published till date that include semperfresh edible coating well with 10 ml acidic methanol and kept at 4 °C. The solution (Özden and Bayindirli 2002), shellac-based surface coating was then filtered through ordinary filter paper. Next, 150 µl of (Chitravathi et al. 2014), essential oil (cinnamon) coating the above filtrate was mixed with 350 µl of distilled water and (Ali et al. 2014), and gibberellic acid coating (Panigrahi later on 2.5 ml Folin Ciocalteu reagent and 2 ml 7.5% (w/v) et al. 2017). sodium carbonate were added in it. The solution was then kept However, there is no report that addresses the efficacy in a shaker in the dark for 2 h. The samples were measured at of castor oil as a coating to slow down the postharvest rip- 765 nm with a UV spectrophotometer with gallic acid as a ening process till date. In this study, for the first time, we standard. The results were expressed as mg GAE (gallic acid evaluated the influence of castor oil as an external coating extract)/g extract (fruit). on different storage-associated biochemical and antioxidant activities with the aim to extend the postharvest storage-life Total soluble sugar content (following Thimmaiah of C. annuum L. 2006) Hundred milligrams of homogenised fruits were mixed with 5 ml of 0.25 N HCl and incubated for a period of 3 h. The 1 3 3 Biotech (2018) 8:280 Page 3 of 8 280 mix was then cooled and neutralized by solid N a CO and bromothymol blue and 0.83 ml distilled water. The mixture 2 3 the solution volume was adjusted to 100 ml with distilled was incubated at 25 °C in a water bath. The initial absorb- water. The solution was centrifuged at 2800 g. Next, 1 ml ance was measured at 620 nm against blank (water). The of phenol solution was added in each aliquot. This was fol- enzymatic reaction was commenced with the addition of lowed by the addition of 3 ml of 95% H SO and incubation 100  µl enzyme solution and the successive absorbances 2 4 for another 20 min in boiling water bath at 25–30 °C. The were recorded at 620 nm up to 80 s at the intervals of 20 s. absorbance was recorded at 490 nm for final estimation. The unit activity was measured as the quantity of enzyme resulted in 0.01 variation in absorbance. DPPH radical scavenging activity assay (following Shimada et al. 1992) Polyphenol oxidase activity assay (following Deng et al. 2009) A 200 µl of homogenate was mixed with 2.5 ml of metha- nolic solution of DPPH and incubated for 30 min in dark. One gram of fruit pulp was homogenized with 10 ml of The optical density of the solutions was measured at 517 nm, 0.05 M potassium phosphate buffer (pH 6.8) and centrifuged considering methanol with 200 µl of deionized water (to at 7000 g for 15 min. The supernatant (homogenate) was replace homogenate) as blank. treated as enzyme extract. To track the polyphenol oxidase activity, 0.2 ml of the enzymatic extract was reacted with a Ferrous ion chelating activity (following Shimada mixture of 3 ml phosphate buffer and 1 ml 0.02 M catechol. et al. 1992) Next, the absorbance values were recorded at 398 nm at an interval of 2 min for final estimation. The results were 2+ The assessment of ferrous (Fe ) ion chelating activity was expressed with the increase in absorbance in every min from carried out considering ethylene diamine tetra acetic acid each ml of enzyme solution. (EDTA) as the control and calculated in terms of regression in absorbance ratio in the presence of polysaccharide and Statistical analysis 2+ expressed as millimolal (mm) of chelted Fe ion. All the 10 assessments were arranged in a completely rand- Hydroxyl radical scavenging activity assay (following omized design and carried out in three replications. Twenty Kaur and Halliwell 1994) samples were used in each replication. Uncoated samples were considered as control. The collected data were statisti- The hydroxyl radicals were obtained by a Fenton reaction cally analysed by one-way analysis of variance and presented 3+ (Fe-ascorbate-EDTA- H O system) and the scavenging as mean ± standard error that were then compared with each 2 2 activity was measured following deoxyribose method. other using Tukey’s test at P < 0.05 with the aid of SPSS (version 11, SPSS Inc. Chicago, USA) software. Reducing power activity assay (following Benzie and Strain 1996) Results and discussion A 100 µl of homogenate was mixed with 100 µl of 0.2M potassium phosphate buffer containing 1% potassium fer - Titratable acidity ricyanide. Following incubation at 50 °C for 20 min, 100 µl of 10% TCA, along with 300 µl of distilled water were added This study shows the potential of castor oil coating in reduc- in the mixture. Finally, 60 µl of 1% ferric chloride was added ing the postharvest losses of storage-life of C. annuum. and the mixture was incubated for 10 min. Then absorb- Although the titratable acidity showed a decreasing trend ance was recorded at 700 nm and the recorded absorbance in both non-coated and coated fruits with the passage of strength was interpreted as reducing power activity. storage period, yet at the same time it was better maintained in coated fruits (Fig.  1a). The coated fruits displayed an Pectate lyase activity assay (following Moran et al. extended storage-life up to 36 days, which was significantly 1968) longer as compared to the non-coated fruits having a limited storage period of 18 days only. On the other note, the decline For the enzyme extraction, 1 g of fruits were homogenized in titratable acidity value was observed to be instantane- with 15 ml cold NaCl (8.8%) and centrifuged at 13,500 g ous in comparison to the steady reduction in case of coated for 10 min. The supernatant (homogenate) was collected fruits. The probable reason of such outcome might be attrib- and the pH was adjusted at 7.5 with NaOH. To assess the uted to lesser metabolic activities (Özden and Bayindirli pectate lyase activity, 2 ml of pectin was mixed with 0.15 ml 2002) and delay in consumption of citric acids (Yaman and 1 3 280 Page 4 of 8 3 Biotech (2018) 8:280 1 3 3 Biotech (2018) 8:280 Page 5 of 8 280 ◂Fig. 1 Influence of castor oil coating on titratable acidity, ascor - when compared to the non-coated ones. A comparable trend bic acid content, total phenolic content, total soluble sugar content, in total phenolic content during postharvest storage of gib- DPPH radical scavenging activity and ferrous ion chelating activity berellic acid coated Capsicum annuum was most recently in Capsicum annuum L. during cold storage. Mean columns with dif- reported by Panigrahi et al. (2017). Phenolic compounds ferent letters are significant at P < 0.05 based on one-way analysis of variance followed by Tukey test. *Represents unavailability of infor- are potential antioxidants and free radical scavengers. In mation due to postharvest deterioration of fruits addition, phenolic compounds deal with the growth and reproduction, simultaneously protecting the fruits against Bayindirli 2001). Citric acid is the prime substrate for res- predators and pathogens (Bravo 2009). piration, wherein a decrease in acidity level and a rise in pH level are coupled with highly respiring fruits (Panigrahi et al. Total soluble sugar content 2017). However, the decline in titratable acidity is a vital event during ripening, since it turns the fruits less acidic or With the extension of storage period, the quantity of total sour (Valero and Serrano 2010). Following the harvest of soluble sugar tended to increase in both the castor oil- fruits, the respiration increases with a decrease in citric acid coated and non-coated fruits. Interestingly, the degree of and other intermediate products of TCA cycle. The coat- this increase in total soluble sugar content was significantly ing on fruit surface might have hindered the sudden rise in higher in non-coated fruits at the very early stage (9 days) of respiration and consequent postharvest maturation, which storage; however, the coated fruits surpassed the content of was also reported earlier by Yaman and Bayindirli (2001). total soluble sugar in its later stages of storage (18–36 days). The highest sugar content was observed to be ~ 272.6 mg/g Ascorbic acid content following 36 days of storage in coated fruits. On the con- trary, the non-coated fruits registered significantly lower The ascorbic acid contents in both castor oil-coated and non- quantities (~ 242.0 mg/g) of sugar even at 18 days of stor- coated fruits showed gradual decline (but with significant age, following which the fruits perished (Fig. 1d). As the difference in between) throughout the storage period. Cas- fruits proceeded towards ripening, the macromolecules tor oil coating secured the significantly higher ascorbic acid degraded into micromolecules in order to be used up rap- content (~ 9.476 mg/10 g fruit) at the same storage dura- idly. In contrast of that, in the present study, the mature fruits tion (18 days) than the non-coated ones (~ 3.500 mg/10 g under storage multiplied the total soluble sugar content and fruit) (Fig. 1b). Similar trend in variation of ascorbic acid thus maintained their quality for a longer period, that could content during postharvest storage was also observed in kin- be highly qualified for consumer acceptance. The present now (Kumar et al. 2000). It has been well established that, results of change in total soluble sugar content corroborates ascorbic acid level varies with genotypic variations, pre- previous studies on the use of coatings in tomato (Beckles harvest climatic conditions, level of maturity and postharvest 2012). handling methods (Plaza et al. 2006). Ascorbic acid is gener- ally degraded by oxidative process, which is accelerated in DPPH radical scavenging activity the presence of light, oxygen and enzymes like peroxidase (Plaza et al. 2006). Similarly, during the storage of orange DPPH radical scavenging activity is recognized based on juice it was observed that the vitamin C content was affected the power of DPPH (2,2-diphenyl-1-picrylhydrazyl), an by the storage conditions, packing, and processing (Ayhan unchanging free radical, to bleach out the presence of anti- et al. 2001; Polydera et al. 2003). oxidants. The castor oil coating on fruits scavenged the DPPH radicals by higher percentages (~ 3.037%) as com- Total phenolic content pared to non-coated ones (~ 2.250%), at the same time it improved the storage-life of fruits up to 36 days (Fig. 1e). Total phenolic content showed an inversely proportional pat- The DPPH radical is basically reduced to form DPPH-H tern in both the non-coated and castor oil-coated fruits in and the said reduction is done by polysaccharide extract. connection with the storage-duration, yet the quantity was A positive correlation of postharvest ripening process and maintained better in the coated ones (Fig. 1c). Immediately DPPH activity was observed in Lycium barbarum fruits (Li after harvest, the phenolic content starts to decline both in et al. 2007). But, initial increase and gradual decrease in the non-coated and coated fruits. The total phenolic content at DPPH activity was observed in fresh cut pears and tomato the commencement of storage (at 0 day) was recorded to (Oms-Oliu et al. 2008). It is noteworthy to mention that there be the highest (~ 0.558 mg GAE/g fruit). It sustained sig- are also several other factors influencing DPPH activities nificantly longer in coated fruits up to a postharvest storage such as environmental and genetic backgrounds, methodol- period of 36 days. It was observed that the coated fruits ogy of harvest and postharvest storage conditions (Dumas maintained the total phenolic content for a longer period et al. 2003). 1 3 280 Page 6 of 8 3 Biotech (2018) 8:280 hence can act as primary and secondary antioxidants. Here, Ferrous ion chelating activity during the assessment method, the transformation of the yel- low coloured solution to a blue one confirmed the effect on Ferrous ions are most efficient pro-oxidants in biological systems, since their interaction with hydrogen peroxide leads the reducing power of each compound (Dave 2009). to formation of highly reactive hydroxyl radicals. The fer- rous ion chelating activity of coated and non-coated fruits Pectate lyase activity under postharvest storage is shown in Fig. 1f. Significant differences in the trends of increasing ferrous ion chelat- Pectic enzymes play the key role to soften the tissues of fruits and vegetables during the ripening process; and it is ing activity in coated and non-coated fruits were observed during storage. Ferrous ion chelating activity gradually also during this course that changes in the elements of cell wall and esterification of pectin is typically observed. In increased in both the coated and non-coated fruits, but it persisted more in the coated fruits and lasted for 36 days our present study, it was observed that the activity of pec- tate lyase enzyme increased in the beginning of the storage that was substantially (18 days) for a longer duration as com- pared to the non-coated ones. The role of iron as a transition period ~ 0.249 (U/mg proteins) but declined till the end of the storage; this activity lasted (~ 0.095 U/mg proteins) for metal creates free radicals from peroxides by the Fenton reaction and creates several diseases (Halliwell and Gut- 36 days in the coated fruits (Fig. 2c) and conversely for 18 + 2 days in case of the non-coated fruits. Interestingly, pectate teridge 1990). Fe is also used in lipid peroxidation and + 2 such reduction of F e concentration during Fenton’s reac- lyase enzyme activity diminished (~ 0.149 U/mg proteins) at a faster rate up to 18 days of storage period in the non- tion can avoid oxidative damage (Singh and Rajini 2004). coated fruits. However, the limited activity was observed in all the coated fruits in contrast to that of the non-coated Hydroxyl radical scavenging activity ones, which suggested that castor oil contributed to the for- mation of a layer over the fruits that might have decreased With the advancement of storage period, the hydroxyl radi- cal scavenging activities of both the castor oil-coated and the availability of CO and O and supported natural inhibi- 2 2 tion of fruit senescence by restricting the functions of cell non-coated fruits were recorded to increase consistantly. However, the coated fruits sustained the 36-day storage wall degenerating enzymes. Such restriction in the ripen- ing mechanism through regulated pectate lyase activity was period with the highest hydroxyl radical scavenging activ- ity value of ~ 2.324% as compared to the non-coated fruits reported in green bell peppers using hydrocolloid-lipid coat- ing (Conforti and Zinck 2002). (~ 2.02%), that thrived only up to a 18-day postharvest stor- age period (Fig. 2a). The most important active oxygen spe- Polyphenol oxidase activity cies that cause lipid oxidation and biological damage are hydroxyl radicals (Gutteridge 1984). In our study, castor oil In the present experiment, the polyphenol oxidase value dur- coating proved to be efficient in minimizing the hydroxyl radical development with enhanced scavenging activity. ing initiation of storage was ~ 0.0533 (U/100 mg fruit pulp) (Fig. 2d). The polyphenol oxidase activity showed an initial Similar trend of such increasing hydroxyl radical scavenging activity in gibberellic acid coated C. annuum L. was reported increase and gradual significant decrease towards the end of the storage period, both in non-coated as well as castor oil- by Panigrahi et al. (2017). coated fruits. However, the polyphenol oxidase activity com- paratively faced greater inhibition in all the coated fruits. Reducing power activity Such initial increase and gradual inhibition of polyphenol oxidase activity was reported during cold storage of nitric The decreased absorbance of reaction mixture represents a drop in reducing power. The reducing power activity of cas- oxide treated banana. Polyphenol oxidase is a terminal oxi- dase existing in plants that catalyses oxidation of phenolics tor oil-coated fruits was recorded to incline at initial stage (9 days) and decline in successive days of storage that by and resulting in tissue browning of fruits and vegetables (Zhang and Quantick 1997). by extended up to 36 days (Fig. 2b). For non-coated fruits such activity displayed a continuous declining trend. This suggests the direct contribution of antioxidant activity put forward by the castor oil coating during the extended storage Conclusion of fruits, resisting the aging process. Such initial incline and late decline of the reducing power activity was also reported The application of castor oil as an external coating on C. annuum fruits resulted in restricted metabolic activities, regu- by Sim and Sil (2008) in Capsicum pericarp. Reducing power serves as an important evidence of antioxidant activ- lated functions of associated biochemicals and antioxidants during cold storage, delaying the ageing process as a result. ity. Compounds with reducing power are electron donors and 1 3 3 Biotech (2018) 8:280 Page 7 of 8 280 Fig. 2 Influence of castor oil coating on hydroxyl radical scaveng- Mean columns with different letters are significant at P < 0.05 based ing activity, reducing power activity, pectate lyase activity and poly- on one-way analysis of variance followed by Tukey test. *Represents phenol oxidase activity in Capsicum annuum L. during cold storage. unavailability of information due to postharvest deterioration of fruits Funding This research did not obtain any specific grant from funding Eventually, this has prolonged the storage-life of C. annuum agencies in the public, commercial, or not-for-profit sectors. up to 36 days. Till date, castor oil is traditionally used as a preservative during storage of food grains (viz. seeds of rice, Compliance with ethical standards pulses, oilseeds etc.) only. Based on the results we recorded in this study, it can be suggested that the described method Conflict of interest On behalf of all authors, the corresponding author can be used by farmers to preserve other fruits and vegetables states that there is no conflict of interest. for a longer period, since it is non-hazardous, biodegradable and of low-cost in comparison to other chemical coatings. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat iveco mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- Authors’ contribution JP, SG—Conceived the research idea and tion, and reproduction in any medium, provided you give appropriate designed the experiments; MP, NP, BG—Executed the experiments; JP, credit to the original author(s) and the source, provide a link to the SG—Performed statistical analysis, interpreted the results and wrote Creative Commons license, and indicate if changes were made. the manuscript. 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Changes in antioxidant and biochemical activities in castor oil-coated Capsicum annuum L. during postharvest storage

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Springer Journals
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Copyright © 2018 by The Author(s)
Subject
Chemistry; Biotechnology; Agriculture; Cancer Research; Bioinformatics; Stem Cells; Biomaterials
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2190-572X
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2190-5738
D.O.I.
10.1007/s13205-018-1284-1
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Abstract

This study, for the first time, evaluates the efficiency of castor oil when used as an external coating on Capsicum annuum L., to increase postharvest storage-life at 4 ± 1 °C. The castor oil-coated fruits were successfully stored for 36 days, while the non-coated fruits could only sustain for 18 days. Throughout the storage period (at 9-day intervals), different antioxidants and biochemical assays (allied with storage) such as titratable acidity, ascorbic acid content, ferrous ion chelating activity, reduc- ing power, DPPH scavenging activity, hydroxyl radical scavenging activity, total phenolic content, total sugar estimation, and enzymatic study of polyphenol oxidase and pectate lyase, were assessed. During storage, the castor oil-coated fruits showed a substantial decrease in titratable acidity, ascorbic acid content, total phenolic content, including antioxidant activities such as reducing power and DPPH activity; however, an increase in ferrous ion chelating activity, total soluble sugar content, polyphenol oxidase activity and initial pectate lyase activity was observed, in contrast to that of the non-coated fruits. The application of castor oil proved to be effective in delaying the ripening process of fruits during storage. Keywords Antioxidant activity · Castor oil · Green chilli · Postharvest · Storage-life Introduction Capsicum annuum L. (green chilli), an important member of Solanaceae family, is mainly grown as a cash crop owing to its distinctly pungent non-climacteric fruits, enriched with vitamins and minerals. Inspite of having such high * Jitendriya Panigrahi nutritional values it has limited storage-life (Panigrahi et al. jitenp2001@gmail.com 2017), which is a major problem for fruits and vegetable * Saikat Gantait growers. Following its harvest, the fruits undergo gradual saikatgantait@yahoo.com deterioration due to desiccation, oxidative reactions, micro- Mansi Patel bial growth, and other biochemical changes. Such rapid maansipatel1512@gmail.com deterioration in the form of softening, wrinkling, wilting Niyati Patel and decaying of the harvested produce during storage and niyaa_001@yahoo.com transportation, fail to provide the anticipated profit, con- Bhumi Gheewala sumer-acceptance or to even meet the actual cultivation cost. bhumigheewala93@gmail.com Softening of fruits is directly linked with water loss during storage (Lownds et al. 1994; Rao and Shivashankara 2015). Department of Biotechnology, Shri A. N. Patel Post Herein, external coating(s), acting as partial barrier Graduate Institute of Science and Research, Anand, Gujarat 388001, India to water vapour, gases and restricting rapid metabolism, might be helpful to preserve the texture, quality and exter- All India Coordinated Research Project on Groundnut, Directorate of Research, Bidhan Chandra Krishi nal appearance of harversted fruits, and in due course may Viswavidyalaya, Nadia, Kalyani, West Bengal 741235, India also improve their storage-life, significantly influencing the Department of Genetics and Plant Breeding, Faculty functions of storage-associated biochemicals and antioxi- of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, dants (Baldwin 1994; Li et al. 2017; Panigrahi et al. 2017). Nadia, Mohanpur, West Bengal 741252, India Vol.:(0123456789) 1 3 280 Page 2 of 8 3 Biotech (2018) 8:280 External coatings comprise hydrophobics (lipids or waxes), Materials and methods hydrocolloids (polysaccharides or proteins) or a combination of both the compounds (composite coatings). The coating Collection, preparation and castor oil coating composition determines the barrier properties of the material of fruits with respect to water vapour, oxygen, carbon dioxide and lipid transfer in food systems (Guilbert et al. 1996). Even Mature C. annuum fruits (var. New Mexico Chile) were col- though hydrophilic coatings function as excellent carbon lected from the local market. The collected fruits were thor- dioxide and oil barriers and provide strength and structural oughly washed with tap water before dipping in chlorine water integrity to the fruits, however, they are not at all effective for 30 s. After soaking up the adhered water using filter papers, moisture barriers and can create adverse anaerobic condi- the fruits were drenched in castor oil for 5 s and then wrapped tions (McHugh and Krochta 1994). Hydrophobic lipid or in aluminium foil, prior to their storage in common refrigerator oil coatings have good water vapour barrier properties, due at 4 ± 1 °C. Throughout the storage period (for 36 days with to their low polarity (Guilbert et al. 1996). Usually, climac- the intervals of 9 days), the following antioxidants and bio- teric fruits like green peppers do not show any detectable chemical assays were performed. changes in respiration rates during storage (Conforti and Zinck 2002). However, it was recorded that when cucumber Titratable acidity (following Chen et al. 1986) and bell pepper were exogenously coated with chitosan, the rate of respiration noticably reduced (Ghaouth et al. 1991). The fruit pulps weighing 5  g were macerated with 50  ml Furthermore, Wong et al. (1994) reported that when cut distilled water and centrifuged at 5000 rpm for 10 min. The apple pieces were treated with multilayer coatings, it aided supernatant (homogenate) solution was titrated to measure the in maintaining the internal carbon dioxide concentrations. titratable acidity, using 0.1N NaOH up to a pH level of 8.1. Castor oil is an important vegetable oil with a rich source of ricinoleic acid, a mono-unsaturated, 18-carbon fatty acid. Ascorbic acid content (following Ranganna 1986) Ricinoleic acid amid all the other fatty acids, is exceptional since it includes a hydroxyl group on its twelfth carbon; it Ten milliliter of homogenate was mixed with equal volume of is for the virtue of this functional group that ricinoleic acid 20% meta phosphoric acid, and collected in a 100 ml volumet- (and castor oil) is more polar than the other fats (Thomas ric flask to adjust the volume with distilled water. The solution 2012). Castor oil proved to be efficient with its greater was titrated with the standard 2, 6-dichlorophenol indophenol resistance to increased CO and reduced O in the internal dyes. The ascorbic acid content of each sample was measured 2 2 environ of fruits, in comparison to that of the essential oils by the equation: Ascorbic acid (mg/100 ml homogenate, i.e. or mineral oils. Castor oil coating significantly delayed the 10 g fruit pulp) = (Titre × dye factor × volume made up × 100)/ softening and retained the flavour in fruits (Baldwin 1994). (volume taken for titration × sample weight). Owing to such property, the castor oil coating on seeds of edible pulse or oilseed crops is being used traditionally, to Total phenolic content (following Singleton et al. extend the storage life since long, in the southern districts 1998) of Gujarat State, India (Parmar and Jain 2016). There are several reports on postharvest storage-life of C. annuum To collect the polyphenol, 1 g of homogenised fruit was mixed published till date that include semperfresh edible coating well with 10 ml acidic methanol and kept at 4 °C. The solution (Özden and Bayindirli 2002), shellac-based surface coating was then filtered through ordinary filter paper. Next, 150 µl of (Chitravathi et al. 2014), essential oil (cinnamon) coating the above filtrate was mixed with 350 µl of distilled water and (Ali et al. 2014), and gibberellic acid coating (Panigrahi later on 2.5 ml Folin Ciocalteu reagent and 2 ml 7.5% (w/v) et al. 2017). sodium carbonate were added in it. The solution was then kept However, there is no report that addresses the efficacy in a shaker in the dark for 2 h. The samples were measured at of castor oil as a coating to slow down the postharvest rip- 765 nm with a UV spectrophotometer with gallic acid as a ening process till date. In this study, for the first time, we standard. The results were expressed as mg GAE (gallic acid evaluated the influence of castor oil as an external coating extract)/g extract (fruit). on different storage-associated biochemical and antioxidant activities with the aim to extend the postharvest storage-life Total soluble sugar content (following Thimmaiah of C. annuum L. 2006) Hundred milligrams of homogenised fruits were mixed with 5 ml of 0.25 N HCl and incubated for a period of 3 h. The 1 3 3 Biotech (2018) 8:280 Page 3 of 8 280 mix was then cooled and neutralized by solid N a CO and bromothymol blue and 0.83 ml distilled water. The mixture 2 3 the solution volume was adjusted to 100 ml with distilled was incubated at 25 °C in a water bath. The initial absorb- water. The solution was centrifuged at 2800 g. Next, 1 ml ance was measured at 620 nm against blank (water). The of phenol solution was added in each aliquot. This was fol- enzymatic reaction was commenced with the addition of lowed by the addition of 3 ml of 95% H SO and incubation 100  µl enzyme solution and the successive absorbances 2 4 for another 20 min in boiling water bath at 25–30 °C. The were recorded at 620 nm up to 80 s at the intervals of 20 s. absorbance was recorded at 490 nm for final estimation. The unit activity was measured as the quantity of enzyme resulted in 0.01 variation in absorbance. DPPH radical scavenging activity assay (following Shimada et al. 1992) Polyphenol oxidase activity assay (following Deng et al. 2009) A 200 µl of homogenate was mixed with 2.5 ml of metha- nolic solution of DPPH and incubated for 30 min in dark. One gram of fruit pulp was homogenized with 10 ml of The optical density of the solutions was measured at 517 nm, 0.05 M potassium phosphate buffer (pH 6.8) and centrifuged considering methanol with 200 µl of deionized water (to at 7000 g for 15 min. The supernatant (homogenate) was replace homogenate) as blank. treated as enzyme extract. To track the polyphenol oxidase activity, 0.2 ml of the enzymatic extract was reacted with a Ferrous ion chelating activity (following Shimada mixture of 3 ml phosphate buffer and 1 ml 0.02 M catechol. et al. 1992) Next, the absorbance values were recorded at 398 nm at an interval of 2 min for final estimation. The results were 2+ The assessment of ferrous (Fe ) ion chelating activity was expressed with the increase in absorbance in every min from carried out considering ethylene diamine tetra acetic acid each ml of enzyme solution. (EDTA) as the control and calculated in terms of regression in absorbance ratio in the presence of polysaccharide and Statistical analysis 2+ expressed as millimolal (mm) of chelted Fe ion. All the 10 assessments were arranged in a completely rand- Hydroxyl radical scavenging activity assay (following omized design and carried out in three replications. Twenty Kaur and Halliwell 1994) samples were used in each replication. Uncoated samples were considered as control. The collected data were statisti- The hydroxyl radicals were obtained by a Fenton reaction cally analysed by one-way analysis of variance and presented 3+ (Fe-ascorbate-EDTA- H O system) and the scavenging as mean ± standard error that were then compared with each 2 2 activity was measured following deoxyribose method. other using Tukey’s test at P < 0.05 with the aid of SPSS (version 11, SPSS Inc. Chicago, USA) software. Reducing power activity assay (following Benzie and Strain 1996) Results and discussion A 100 µl of homogenate was mixed with 100 µl of 0.2M potassium phosphate buffer containing 1% potassium fer - Titratable acidity ricyanide. Following incubation at 50 °C for 20 min, 100 µl of 10% TCA, along with 300 µl of distilled water were added This study shows the potential of castor oil coating in reduc- in the mixture. Finally, 60 µl of 1% ferric chloride was added ing the postharvest losses of storage-life of C. annuum. and the mixture was incubated for 10 min. Then absorb- Although the titratable acidity showed a decreasing trend ance was recorded at 700 nm and the recorded absorbance in both non-coated and coated fruits with the passage of strength was interpreted as reducing power activity. storage period, yet at the same time it was better maintained in coated fruits (Fig.  1a). The coated fruits displayed an Pectate lyase activity assay (following Moran et al. extended storage-life up to 36 days, which was significantly 1968) longer as compared to the non-coated fruits having a limited storage period of 18 days only. On the other note, the decline For the enzyme extraction, 1 g of fruits were homogenized in titratable acidity value was observed to be instantane- with 15 ml cold NaCl (8.8%) and centrifuged at 13,500 g ous in comparison to the steady reduction in case of coated for 10 min. The supernatant (homogenate) was collected fruits. The probable reason of such outcome might be attrib- and the pH was adjusted at 7.5 with NaOH. To assess the uted to lesser metabolic activities (Özden and Bayindirli pectate lyase activity, 2 ml of pectin was mixed with 0.15 ml 2002) and delay in consumption of citric acids (Yaman and 1 3 280 Page 4 of 8 3 Biotech (2018) 8:280 1 3 3 Biotech (2018) 8:280 Page 5 of 8 280 ◂Fig. 1 Influence of castor oil coating on titratable acidity, ascor - when compared to the non-coated ones. A comparable trend bic acid content, total phenolic content, total soluble sugar content, in total phenolic content during postharvest storage of gib- DPPH radical scavenging activity and ferrous ion chelating activity berellic acid coated Capsicum annuum was most recently in Capsicum annuum L. during cold storage. Mean columns with dif- reported by Panigrahi et al. (2017). Phenolic compounds ferent letters are significant at P < 0.05 based on one-way analysis of variance followed by Tukey test. *Represents unavailability of infor- are potential antioxidants and free radical scavengers. In mation due to postharvest deterioration of fruits addition, phenolic compounds deal with the growth and reproduction, simultaneously protecting the fruits against Bayindirli 2001). Citric acid is the prime substrate for res- predators and pathogens (Bravo 2009). piration, wherein a decrease in acidity level and a rise in pH level are coupled with highly respiring fruits (Panigrahi et al. Total soluble sugar content 2017). However, the decline in titratable acidity is a vital event during ripening, since it turns the fruits less acidic or With the extension of storage period, the quantity of total sour (Valero and Serrano 2010). Following the harvest of soluble sugar tended to increase in both the castor oil- fruits, the respiration increases with a decrease in citric acid coated and non-coated fruits. Interestingly, the degree of and other intermediate products of TCA cycle. The coat- this increase in total soluble sugar content was significantly ing on fruit surface might have hindered the sudden rise in higher in non-coated fruits at the very early stage (9 days) of respiration and consequent postharvest maturation, which storage; however, the coated fruits surpassed the content of was also reported earlier by Yaman and Bayindirli (2001). total soluble sugar in its later stages of storage (18–36 days). The highest sugar content was observed to be ~ 272.6 mg/g Ascorbic acid content following 36 days of storage in coated fruits. On the con- trary, the non-coated fruits registered significantly lower The ascorbic acid contents in both castor oil-coated and non- quantities (~ 242.0 mg/g) of sugar even at 18 days of stor- coated fruits showed gradual decline (but with significant age, following which the fruits perished (Fig. 1d). As the difference in between) throughout the storage period. Cas- fruits proceeded towards ripening, the macromolecules tor oil coating secured the significantly higher ascorbic acid degraded into micromolecules in order to be used up rap- content (~ 9.476 mg/10 g fruit) at the same storage dura- idly. In contrast of that, in the present study, the mature fruits tion (18 days) than the non-coated ones (~ 3.500 mg/10 g under storage multiplied the total soluble sugar content and fruit) (Fig. 1b). Similar trend in variation of ascorbic acid thus maintained their quality for a longer period, that could content during postharvest storage was also observed in kin- be highly qualified for consumer acceptance. The present now (Kumar et al. 2000). It has been well established that, results of change in total soluble sugar content corroborates ascorbic acid level varies with genotypic variations, pre- previous studies on the use of coatings in tomato (Beckles harvest climatic conditions, level of maturity and postharvest 2012). handling methods (Plaza et al. 2006). Ascorbic acid is gener- ally degraded by oxidative process, which is accelerated in DPPH radical scavenging activity the presence of light, oxygen and enzymes like peroxidase (Plaza et al. 2006). Similarly, during the storage of orange DPPH radical scavenging activity is recognized based on juice it was observed that the vitamin C content was affected the power of DPPH (2,2-diphenyl-1-picrylhydrazyl), an by the storage conditions, packing, and processing (Ayhan unchanging free radical, to bleach out the presence of anti- et al. 2001; Polydera et al. 2003). oxidants. The castor oil coating on fruits scavenged the DPPH radicals by higher percentages (~ 3.037%) as com- Total phenolic content pared to non-coated ones (~ 2.250%), at the same time it improved the storage-life of fruits up to 36 days (Fig. 1e). Total phenolic content showed an inversely proportional pat- The DPPH radical is basically reduced to form DPPH-H tern in both the non-coated and castor oil-coated fruits in and the said reduction is done by polysaccharide extract. connection with the storage-duration, yet the quantity was A positive correlation of postharvest ripening process and maintained better in the coated ones (Fig. 1c). Immediately DPPH activity was observed in Lycium barbarum fruits (Li after harvest, the phenolic content starts to decline both in et al. 2007). But, initial increase and gradual decrease in the non-coated and coated fruits. The total phenolic content at DPPH activity was observed in fresh cut pears and tomato the commencement of storage (at 0 day) was recorded to (Oms-Oliu et al. 2008). It is noteworthy to mention that there be the highest (~ 0.558 mg GAE/g fruit). It sustained sig- are also several other factors influencing DPPH activities nificantly longer in coated fruits up to a postharvest storage such as environmental and genetic backgrounds, methodol- period of 36 days. It was observed that the coated fruits ogy of harvest and postharvest storage conditions (Dumas maintained the total phenolic content for a longer period et al. 2003). 1 3 280 Page 6 of 8 3 Biotech (2018) 8:280 hence can act as primary and secondary antioxidants. Here, Ferrous ion chelating activity during the assessment method, the transformation of the yel- low coloured solution to a blue one confirmed the effect on Ferrous ions are most efficient pro-oxidants in biological systems, since their interaction with hydrogen peroxide leads the reducing power of each compound (Dave 2009). to formation of highly reactive hydroxyl radicals. The fer- rous ion chelating activity of coated and non-coated fruits Pectate lyase activity under postharvest storage is shown in Fig. 1f. Significant differences in the trends of increasing ferrous ion chelat- Pectic enzymes play the key role to soften the tissues of fruits and vegetables during the ripening process; and it is ing activity in coated and non-coated fruits were observed during storage. Ferrous ion chelating activity gradually also during this course that changes in the elements of cell wall and esterification of pectin is typically observed. In increased in both the coated and non-coated fruits, but it persisted more in the coated fruits and lasted for 36 days our present study, it was observed that the activity of pec- tate lyase enzyme increased in the beginning of the storage that was substantially (18 days) for a longer duration as com- pared to the non-coated ones. The role of iron as a transition period ~ 0.249 (U/mg proteins) but declined till the end of the storage; this activity lasted (~ 0.095 U/mg proteins) for metal creates free radicals from peroxides by the Fenton reaction and creates several diseases (Halliwell and Gut- 36 days in the coated fruits (Fig. 2c) and conversely for 18 + 2 days in case of the non-coated fruits. Interestingly, pectate teridge 1990). Fe is also used in lipid peroxidation and + 2 such reduction of F e concentration during Fenton’s reac- lyase enzyme activity diminished (~ 0.149 U/mg proteins) at a faster rate up to 18 days of storage period in the non- tion can avoid oxidative damage (Singh and Rajini 2004). coated fruits. However, the limited activity was observed in all the coated fruits in contrast to that of the non-coated Hydroxyl radical scavenging activity ones, which suggested that castor oil contributed to the for- mation of a layer over the fruits that might have decreased With the advancement of storage period, the hydroxyl radi- cal scavenging activities of both the castor oil-coated and the availability of CO and O and supported natural inhibi- 2 2 tion of fruit senescence by restricting the functions of cell non-coated fruits were recorded to increase consistantly. However, the coated fruits sustained the 36-day storage wall degenerating enzymes. Such restriction in the ripen- ing mechanism through regulated pectate lyase activity was period with the highest hydroxyl radical scavenging activ- ity value of ~ 2.324% as compared to the non-coated fruits reported in green bell peppers using hydrocolloid-lipid coat- ing (Conforti and Zinck 2002). (~ 2.02%), that thrived only up to a 18-day postharvest stor- age period (Fig. 2a). The most important active oxygen spe- Polyphenol oxidase activity cies that cause lipid oxidation and biological damage are hydroxyl radicals (Gutteridge 1984). In our study, castor oil In the present experiment, the polyphenol oxidase value dur- coating proved to be efficient in minimizing the hydroxyl radical development with enhanced scavenging activity. ing initiation of storage was ~ 0.0533 (U/100 mg fruit pulp) (Fig. 2d). The polyphenol oxidase activity showed an initial Similar trend of such increasing hydroxyl radical scavenging activity in gibberellic acid coated C. annuum L. was reported increase and gradual significant decrease towards the end of the storage period, both in non-coated as well as castor oil- by Panigrahi et al. (2017). coated fruits. However, the polyphenol oxidase activity com- paratively faced greater inhibition in all the coated fruits. Reducing power activity Such initial increase and gradual inhibition of polyphenol oxidase activity was reported during cold storage of nitric The decreased absorbance of reaction mixture represents a drop in reducing power. The reducing power activity of cas- oxide treated banana. Polyphenol oxidase is a terminal oxi- dase existing in plants that catalyses oxidation of phenolics tor oil-coated fruits was recorded to incline at initial stage (9 days) and decline in successive days of storage that by and resulting in tissue browning of fruits and vegetables (Zhang and Quantick 1997). by extended up to 36 days (Fig. 2b). For non-coated fruits such activity displayed a continuous declining trend. This suggests the direct contribution of antioxidant activity put forward by the castor oil coating during the extended storage Conclusion of fruits, resisting the aging process. Such initial incline and late decline of the reducing power activity was also reported The application of castor oil as an external coating on C. annuum fruits resulted in restricted metabolic activities, regu- by Sim and Sil (2008) in Capsicum pericarp. Reducing power serves as an important evidence of antioxidant activ- lated functions of associated biochemicals and antioxidants during cold storage, delaying the ageing process as a result. ity. Compounds with reducing power are electron donors and 1 3 3 Biotech (2018) 8:280 Page 7 of 8 280 Fig. 2 Influence of castor oil coating on hydroxyl radical scaveng- Mean columns with different letters are significant at P < 0.05 based ing activity, reducing power activity, pectate lyase activity and poly- on one-way analysis of variance followed by Tukey test. *Represents phenol oxidase activity in Capsicum annuum L. during cold storage. unavailability of information due to postharvest deterioration of fruits Funding This research did not obtain any specific grant from funding Eventually, this has prolonged the storage-life of C. annuum agencies in the public, commercial, or not-for-profit sectors. up to 36 days. Till date, castor oil is traditionally used as a preservative during storage of food grains (viz. seeds of rice, Compliance with ethical standards pulses, oilseeds etc.) only. Based on the results we recorded in this study, it can be suggested that the described method Conflict of interest On behalf of all authors, the corresponding author can be used by farmers to preserve other fruits and vegetables states that there is no conflict of interest. for a longer period, since it is non-hazardous, biodegradable and of low-cost in comparison to other chemical coatings. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat iveco mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- Authors’ contribution JP, SG—Conceived the research idea and tion, and reproduction in any medium, provided you give appropriate designed the experiments; MP, NP, BG—Executed the experiments; JP, credit to the original author(s) and the source, provide a link to the SG—Performed statistical analysis, interpreted the results and wrote Creative Commons license, and indicate if changes were made. the manuscript. 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3 BiotechSpringer Journals

Published: Jun 1, 2018

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