Impaginato 89 Adv. Hort. Sci., 2022 36(2): 89­96 DOI: 10.36253/ahsc­12403 Postharvest application of calcium chloride and 1­methylcyclopropene for quality conservation on organic ripe fig M.B.D. Tofanelli 1 (*), F.L. Cuquel 1, J.W. de O. D’Angelo 2, J.G.S. Medeiros 3 1 Department of Plant Science, Federal University of Paraná, 80350‐050 Curitiba‐PR, Paraná, Brazil. 2 National Rural Apprenticeship Service, 80010‐010 Curitiba‐PR, Paraná, Brazil. 3 Federal Institute of Education, 68740‐970 Castanhal, Pará, Brazil. Key words: Brown Turkey fig, Ficus carica, fruit conservation, shelf life. Abstract: The postharvest phase is an important step in the fruit production chain. Fig is an especially perishable fruit, which has encouraged researchers to study the effects of various substances on the postharvest life of this commodi­ ty. The objective of the present work was to evaluate the effects of calcium chloride (CaCl2) and 1­methylcyclopropene (1­MCP) on the postharvest quality of the ‘Roxo­de­Valinhos’ fig cultivar. This study aimed to verify the effects of applying a 4% solution of CaCl2 and a 1% solution of 1­MCP to figs and evaluat­ ing at four different storage times (0, 2, 4, and 6 days). The results showed that a 4% solution of CaCl2 promoted better firmness, and when CaCl2 at 4% solution was applied in combination with 1­MCP at 10 μg l­1, the maturation index increased. In contrast, the 1­MCP treatment alone did not improve the posthar­ vest quality of ‘Roxo­de­Valinhos’ ripe fig. We conclude that application of 4% solution of CaCl2 and 1­MCP at 10 μg l­1 promote firmness and increase matura­ tion index of ‘Roxo­deValinhos’ figs. 1. Introduction Fig (Ficus carica L.) is an important crop globally, particularly in Southeast Europe, the Middle East, North Africa, and the Americas, where the USA and Brazil are highlighted for fig production (Rosianski et al., 2016; Uzundumlu et al., 2018; Paolucci et al., 2020). In Brazil, 19.6 tonnes were harvested from 2.114 hectares in 2020 (IBGE, 2022), where fig trees are cultivated mainly in the southeast and south regions where ripe figs are harvested for consumption and unripe figs for industrializa­ tion (Tofanelli et al., 2018). Although fig tree cultivation has the potential to escalate, some pro­ duction bottlenecks have inhibited this expansion, such as lack of com­ mercial cultivars, unfamiliarly about ripe fig as excellent fruit for food from consumers, relative high price of ripe figs in the market, inefficient fresh ripe fig distribution and difficult to conserve quality of ripe figs after (*) Corresponding author: mbrasildt@ufpr.br Citation: TOFANELLI M.B.D., CUQUEL F.L., DE O. D’ANGELO J.W., MEDEIROS J.G.S., 2022 ­ Postharvest appli‐ cation of calcium chloride and 1‐methylcyclopro‐ pene for quality conservation on organic ripe fig. ­ Adv. Hort. Sci., 36(2): 89­96. Copyright: © 2022 Tofanelli M.B.D., Cuquel F.L., de O. D’Angelo J.W., Medeiros J.G.S. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 7 December 2021 Accepted for publication 4 February 2022 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-12403 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2022 36(2): 89­96 90 harvested. Thus, one of the principal challenges is the high perishability of ripe fig after it has been harvest­ ed (Mirshekari et al., 2020), especially in Brazil where the main cultivar used is ‘Roxo­de­Valinhos’, also known as ‘Brown Turkey’, which produces highly­per­ ishable fruit (Ferraz et al., 2020). ‘Roxo­de­Valinhos’ figs have a very short postharvest life of 1 to 2 days when stored under environmental conditions of mar­ ket shelf, or 4 to 10 days under refrigerated storage (Lakshmi et al., 2018). Several studies have been conducted on the effects of postharvest treatments on figs to extend their storage duration. The majority of research has focused on substances such as calcium chloride (CaCl2), 1­methylcyclopropene (1­MCP), fungicides, and sodium hypochlorite (Irfan, 2013; Song et al., 2019; Jusoh et al., 2020). CaCl2 has been applied to fruits both in preharvest and postharvest to promote quality conservation (LI et al., 2014). This nutrient has been considered the most consequential for fruit quality promotion through its contributions to decreased softening, browning, and senescence of fruits, thereby extend­ ing their postharvest life (Suriati et al., 2021). 1­MCP has an unsaturated cyclic olefin that acts as a com­ petitive ethylene antagonist and blocks ethylene receptors, consequently inhibiting maturation processes such as ethylene production, respiration rate, browning, and softening, and extending the shelf life of fruits (Sozzi et al., 2005; Watkins, 2008; Freiman et al., 2012). The response of CaCl2 or 1­MCP application on fruits varies according to the application form, fruit species, cultivar, maturation, and ripening stage of the fruit (Sozzi et al., 2005; Watkins, 2006; Irfan, 2013). Currently, there are few reports applying CaCl2 and 1­MCP to figs to evaluate their effects on fruit quality. Therefore, the objective of this study was to eval­ uate the effects of the application of CaCl2 and 1­MCP on quality of fresh ripe fig ‘Roxo­de­Valinhos’ after harvested to extend their shelf life. 2. Materials and Methods Plant materials The experiment was conducted at the Federal University of Paraná State. Figs were harvested from plants grown in the orchard located in the Canguiri Experimental Farm (Pinhais County, Paraná State, Brazil, 25°26ʹ S and 49°16ʹ W, 947 m above sea level), considered Cfb by the Köppen climate classification, with annual maximum and minimum temperatures of approximately 24°C and 11°C, respectively, and annual average rainfall of 1,500 mm. Fig trees used were 5­year­old Roxo­de­Valinhos cultivars, also known as ‘Brown Turkey’. The first har­ vest was done when plants were 2 years old. Plants were cultivated in an organic plantation system, sub­ jected to heavy annual pruning, and spaced at 1×2.5 m. Fertilization was done using cattle manure at 60 L per plant annually, separated into three 60 days intervals beginning in August. Phytosanitary manage­ ment was performed by spraying a 2% lime sulfur solution after pruning and a 0.2% bordeaux mixture (biweekly in rainy period and monthly in dry period) from the vegetative and production stages of the fig trees to two weeks prior to harvest, mainly to pre­ vent rust (Cerotelium fici). To control pests, such as tree borer (Azochis gripusalis) and borer beetle (Coleobogaster cyanitarsis), a 1% solution of oil extracted from neem (Azadirachta indica A. Juss) was sprayed on plants when these pests were detected damaging the fig trees. Mechanical weeding in crop rows and mowing between rows were used to con­ trol weeds, integrated with black oat (Avena strigosa L.) planted in July in 2012 and 2013. Fruit materials Figs were hand­harvested on April 1, 2014. Overall, this period is the late season for harvest of ripe fig in Brazil. Fruits were harvested when they showed at least a 50% change to their skin color, from green to reddish brown at stage 6 (Freiman et al., 2012) by visual evaluation. This maturation stage is usually better accepted by Brazilian market. After harvesting, figs were immediately transported to the Postharvest Laboratory and located in the same experimental farm for experimental proceedings. Figs were divided into lots containing 8 figs each lot for storage during 0, 2, 4, and 6 days, which were then placed in a paper box commonly used in ripe fig mar­ kets. Postharvest treatments After figs were harvested, they were treated with calcium chloride (CaCl2) (Oliveira Junior et al., 2018) and 1­methylcyclopropene (1­MCP) (Tofanelli et al., 2018). Half of the selected figs were dipped in the Tofanelli et al. ‐ Application of CaCl2 and 1‐MCP for ripe fig quality 91 solution of CaCl2 at 4%, and the other half were dipped in distilled water and immersed for 15 min. After that, figs were taken from the treatments to dry their surface by natural air­drying under the labo­ ratory bench for 30 min. After drying, figs were placed in a specific com­ mercial paper box that fit 8 figs each. The packed figs were placed into a plastic container (70 L) when they were ready to be treated with 1­MCP. SmartFreshÒ powder (0.14% active ingredient) was used to pre­ pare the 1­MCP solution. This powder was measured with a precision balance and dissolved in distilled water to create a concentration of 10 μg l­1 inside the container, where figs were kept after treatment for 24 h. Untreated figs (control) were compounded only with distilled water and stored under the same con­ ditions. Thus, 1­MCP powder was introduced into a syringe and supplemented with water until a specific volume was reached. The 1­MCP solution and control were injected into the respective plastic containers, which were immediately blocked. Therefore, both untreated figs and those treated with CaCl2 were also treated with 1­MCP or the control (distilled water), resulting in a combination of 2 levels of CaCl2 (0 and 4%) vs. 2 concentrations of 1­MCP (0 and 10 μg l­1). After 1­MCP treatment, packed figs were removed from boxes and stored at 4±1°C and 90­95% RH for 6 days, when they were removed to proce­ dure analyses. Fruit quality parameters Fig quality conservation was evaluated using firm­ ness, total soluble solids (TSS) concentration, titrat­ able acidity (TA), maturation index (MI), and weight loss. The samples were withdrawn from the refrigera­ tor and 2 figs per replicate were analyzed every two days. Fruit­peel firmness was determined on the equator of the fig using a manual penetrometer (PTR­ 100) with a 7.9­mm­diameter tip and expressed in terms of lb force. Fruit juice was extracted from the stored figs using a centrifuge in order to assess TSS and TA. TSS was measured with a handheld refractometer using a drop of juice and expressed as a percentage while TA was expressed as % citric acid and measured using 100 ml of the solution (10 ml of juice + 90 ml distilled water) that was immediately titrated with 0.1 N NaOH using 3 drops of phenolphthalein as an indica­ tor. MI was determined by dividing TSS by TA (TSS/TA ratio). Weight loss of stored figs was obtained using the relative weight variation (%) of 2 figs per replicate during a storage period of 6 days. The figs were weighed at harvest time and at every storage period of 2 days, and weight loss was determined. Fig weights were assessed using an electronic balance. Experimental design Three replicates of samples with 8 figs per repli­ cate for each treatment were used for physical and chemical quality analyses; 2 figs were analyzed immediately after 1­MCP proceedings and the other 6 figs were stored. Four storage periods (0, 2, 4, and 6 days) were used to evaluate postharvest quality conservation. Theses samples of figs were random­ ized selected avoiding to select damage figs by pests and mechanical injury. The experimental design was completely random­ ized. Treatments included the application or absence of 1­MCP (10 μg l­1) combined with the application or absence of CaCl2 (4%) and with no storage (0) and three storage times (2, 4, and 6 days). Thus, the experiment was designed using a 23 factorial arrange­ ment (2´2´4): (2) CaCl2 at 2 levels (0 and 4%), (2) 1­ MCP at 2 levels (0 and 10 μg l­1), and (4) storage peri­ od at four levels (0, 2, 4, and 6 days). Statistical procedures Analyses were carried out with analysis of vari­ ance (ANOVA) using the Sisvar Statistical Program (version 5.3) (Ferreira, 2011). Treatments that showed a significant effect were subjected to multi­ ple comparisons of the mean using the Tukey test at a 5% probability level. 3. Results Results from ANOVA showed that CaCl2 promoted significant effect on firmness and soluble solids of the ‘Roxo­de­vlinhos’ ripe figs, that storage periods had significant influence on soluble solids, titratable acidi­ ty, weight loss, and maturation index, whereas 1­ MCP only affected significantly the maturation index (Table 1). The exclusive interaction that showed a sig­ nificant effect was CaCl2 x 1­MCP on the fig ratio. As expected, storage influenced the quality parameters evaluated in the ripe figs, except in terms of firm­ ness. There was no significant effect of CaCl2 and 1­ MCP on all parameters evaluated in this present work after storage of 6 days (Table S1). Adv. Hort. Sci., 2022 36(2): 89­96 92 Except for effect of the interaction between CaCl2 x 1­MCP on the maturation index of figs, we could also observe from ANOVA that there was no signifi­ cant effect of interactions between the factors on parameters evaluated in this present work (Table 1). When ripe figs were post­harvest treated with CaCl2, they demonstrated more firmness than untreated figs (3.37 lb and 1.91 lb, respectively) (Table 2). CaCl2 postharvest treatment also influ­ enced TSS on ripe figs, where untreated fruits showed a higher value (8.70%) compared to the treated figs (7.98%). Figs stored for 4 (8.74%) and 6 days (8.81%) showed higher TSS than non­stored fruits (7.47%) (Table 3). The titratable acidity was higher after 6 days of storage (2.58%), when compared with no storage (1.98%) and 2 days of storage (2.07%) (Table 3). The weight loss of ripe figs was significantly Table 1 ­ Analysis of variance (ANOVA) for parameters evaluated on storage of ripe figs treated with calcium chloride (CaCl2) and 1­ methylcyclopropene (1­MCP) CC= Calcium Chloride; MCP= Methylcyclopropene; SP= Storage Periods; CV= Coefficient of Variation; FI= Firmness; SS= Soluble Solids; TA = Titratable Acidity; WL= Weight Loss; MI= Maturation Index; MS= Mean Square; F= F test; ** Significant at P≤0.01; * Significant at P<0.05. NS = not significant. Table 2 ­ Effects of calcium chloride (CaCl2) treatments on quality parameters of ripe figs stored over different periods * Standard deviation. Values followed by the different lowercase letter in a column are significantly different from each other (P<0.05). Table 3 ­ Effects of storage periods on quality parameters of ripe figs * Standard deviation. Values followed by the same lowercase letter in a column are not significantly different from each other (P<0.05). Source of Variation df FI (Lb) SS (°Brix) TA (%) WL (%) MI (Ratio) MS F MS F MS F MS F MS F CC 1 25.37 17.06 ** 6.31 9.84 ** 0.02 0.09 NS 102.11 0.36 NS 404.26 1.70 NS MCP 1 2.34 1.57 NS 0.08 0.13 NS 0.64 3.15 NS 28.29 0.11 NS 1029.53 4.33 * SP 3 3.94 2.65 NS 4.57 7.14 ** 1.04 5.09 ** 97.99 104.25 ** 765.64 3.22 * CC´MCP 1 0.07 0.05 NS 0.61 0.95 NS 0.81 3.97 NS 2.53 1.69 NS 987.36 4.15 * CC´SP 3 0.48 0.33 NS 1.01 1.57 NS 0.18 0.86 NS 0.04 0.14 NS 9.85 0.01 NS MCP´SP 3 1.39 0.93 NS 0.86 1.35 NS 0.09 0.44 NS 0.09 0.05 NS 342.21 1.44 NS CC´MCP´SP 3 3.90 2.62 NS 0.58 0.91 NS 0.46 2.27 NS 0.33 0.39 NS 84.59 0.36 NS CV (%) 46.2 9.6 19.9 13.2 22.2 Fruit parameter CaCl2 Firmness (Lb) Soluble Solids (%) Untreated 1.91 ± 1.34* b 8.70 ± 1.00 a Treated 3.37 ± 1.29 a 7.98 ± 0.91 b Coefficient of Variation (%) 46.2 9.6 Storage (days) Fruit parameters Soluble solids (%) Titratable acidity (%) Weight loss (%) Maturation index 0 7.47 ± 0.99* b 1.98 ± 0.35 b 0 ± 0.00 d 69.22 ± 12.56 ab 2 8.35 ± 0.68 ab 2.58 ± 0.73 b 7.67 ± 0.86 c 62.22 ± 16.49 b 4 8.74 ± 0.82 a 2.07 ± 0.47 ab 13.67 ± 2.84 b 80.47 ± 21.62 a 6 8.81 ± 1.02 a 2.46 ± 0.22 a 18.75 ± 3.96 a 65.23 ± 11.54 ab Coefficient of Variation (%) 9.6 19.9 27.3 22.2 Tofanelli et al. ‐ Application of CaCl2 and 1‐MCP for ripe fig quality 93 enhanced as storage duration increased, as expected. The greatest weight loss was 18.75% after 6 days of cold storage. The maturation index (TSS/TA ratio) was the para­ meter that showed a significant interaction effect (Table 1). When ripe figs were treated with CaCl2 after with 1­MCP, they showed a higher ratio (75.55) than those treated only with CaCl2 (57.22) (Table 4). 4. Discussion and Conclusions CaCl2 has been widely used for food conservation, mainly because it can maintain firmness during the shelf life of vegetables, especially in fruits immersed in calcium chloride solution after harvest (Li et al., 2014). Calcium plays an important role in maintaining the structure of the cell wall, since its cation binds to pectins producing calcium pectates that structure the cell wall, thereby inhibiting enzymes such as poly­ galacturonase and polymethylesterase. Irfan et al. (2013) recorded the lowest sugar con­ tent when figs were treated with CaCl2 at 4%, as reported before when we showed that figs treated with CaCl2 obtained lower sugar content against untreated figs. These authors concluded that this likely occurred because calcium caused a delay in fruit ripening through the inhibition of certain events such as starch hydrolysis and formation of sugars, a response to the high amounts of calcium deposition in the fruit cell wall of the treated figs. These authors also mentioned that components such as pectic sub­ stances, calmodulin, and hemicellulose polysaccha­ rides are the most likely binding sites for calcium, preserving the texture and stability of the treated fruit and promoting higher fruit resistance by main­ taining high calcium concentration in the cytosol, as well as less free sugars released during storage. Storage having no influence on firmness likely relates to the figs being harvested at a relatively advanced ripening stage, when they showed at least a 50% change in skin color, from green to reddish brown at stage 6. In Brazil, the late season to harvest ripe figs for table takes place from late March to early April, when they usually get better prices in the mar­ ket. Thereby, it would be interesting whether grow­ ers had a technique that was able to extend shelf life of ripe figs in order to add value to their product. Thus, at this harvest time, figs already displayed pre­ harvest firmness reduction; therefore, the collected data of firmness from harvest to last storage period did not show a significant difference. According to Freiman et al. (2012), postharvest treatment on figs at the maturation stage can be ineffective for their quality conservation or may have a slight effect on softening, promoting its retardation, especially when figs are collected at an advanced stage. These authors mentioned that marketable ripe figs are defined as commercially mature when they have changing skin coloration from 20 to 70% of their surface. Tofanelli et al. (2018) also showed TSS increasing during storage in ‘Roxo­de­Valinhos’ figs collected at stage 5 (Freiman et al., 2012) and treated with 1­ MCP. According to those authors, this TSS increase likely occurs either due to the hydrolysis of several polysaccharides, such as pectins, starch, and other oligosaccharides in the cell wall, which become part of the cellular juice when they are solubilized in the aqueous phase, or due to starch accumulation during fruit maturation, which is degraded into sugars by the action of enzymes such as α­amylase, β­amylase, and starch phosphorylase, consequently increasing the TSS concentration. Although the acidity of figs tends to decrease dur­ ing the storage period (Irfan et al., 2013; Song et al., 2019), it has been observed that in the initial storage time, acidity tends to increase for a short period in the first few days. Gözlekçi et al. (2008) evaluated the effect of 1­MCP on the quality of figs stored dur­ ing three different periods (5, 10, and 15 days) and Table 4 ­ Effects of calcium chloride (CaCl2) and 1­methylcyclopropene (1­MCP) treatments on the ratio (%) of ripe figs * Standard deviation. Values followed by the same uppercase letter in a column and lowercase letter in a row are not significantly diffe­ rent from each other (P<0.05). CaCl2 1­MCP Untreated Treated Untreated 72.09 ± 11.65* aA 72.28 ± 18.67 aA Treated 57.22 ± 11.10 bB 75.55 ± 20.28 aA Coefficient of Variation (%) 22.2 94 Adv. Hort. Sci., 2022 36(2): 89­96 observed that fruit acidity slightly increased until the first storage period. In addition, Álvarez­Herrera et al. (2016) studied the effect of 1­MCP on the posthar­ vest conservation of pitahaya (Selenicereus megalan‐ thus) during seven storage periods (4, 8, 12, 16, 20, 24, and 28 days), and discovered an increase in acidi­ ty in the treated pitahayas until the second storage period (8 days). In the present work, the last evaluat­ ed storage period was 6 days; therefore, it was expected that the acidity of ripe figs would increase during the entire experimental period. Fruit transpiration and several physiological disor­ ders of figs, such as skin side cracking and ostiole­end cracking/splitting occur during storage, and these could promote increasing levels of TA and TSS, as well as the maintenance of several organic acids dur­ ing the storage period (Byeon and Lee, 2020). In our study, 1­MCP treatments only affected on maturation index (ratio). Cantín et al. (2020) also did not obtain improvement on shelf life of ‘Cuello Dama Negro’ dark­skin commercial figs treated with 1­MCP at 1 μL l ­1 and according to these authors the advance maturity stage of the figs at the moment of harvest could be promoted a high increase in ethyl­ ene production even under 1­MCP treatment. They also highlighted that, although the best moment to harvest fresh­market figs is variable according to each cultivar or variety, it is usually done as early as possible. In our work, studied figs were harvested at maturation stage 6 (Fig. 1B), thus an early harvest (Fig. 1C) could be promoted different results, what we would release here as a challenge for future stud­ ies, as well as evaluate how these figs harvested on early maturation stage would be taken up by the market. However, figs may be a climacteric or non­ climacteric fruit due to their maturation shows rapid changes of compositional features that are typical of climacteric fruit, whereas figs are not capable to keep ripening after harvested (D’Aquino et al., 2015). Thus, that is a good question when would be the bet­ ter time to harvest ripe figs for post treatment in order to enlarge their shelf life as well as promote their quality conservation. Fig fruits are highly perishable products in which physiological events such as transpiration, respira­ tion, and degradation occur quickly (Gözlekçi et al., 2008; Ozkaya et al., 2014; Byeon and Lee, 2020). It is interesting to consider the unique morphological structure of figs, as they have a thin skin over the entire fruit with a distal end orifice called the ostiole where water loss through evaporation occurs by exu­ dation of a syrupy liquid (D’Aquino et al., 2003; Freiman et al., 2012). CaCl2 combined with 1­MCP applied on posthar­ vest ripe figs likely promoted sugar content accumu­ lation and was not favorable to acidity oscillation of fruits during storage. Some structural effects on figs have been observed when they are treated with calcium, such as mechanical strength improvement and increased resistance of fruit tissue due to absorbed calcium in the apoplast primarily complexing with the cell wall, where the plasma membrane works as a cementing material. Calcium is also involved in the maintenance of the cell wall structure by interacting with pectic acid and forming calcium pectate (Irfan et al., 2013). Thus, in the present study, CaCl2 application likely promoted these events in figs, resulting in sugar con­ tent accumulation and acidity stabilization, conse­ quently enhancing the maturation index (ratio). In addition, when 1­MCP was applied after calci­ um on the ripe figs, the MI ratios increased, poten­ tially due to ethylene inhibition, decreased fruit res­ piration, and glucose and fructose stabilization during cold storage (Ozkaya et al., 2014; Song et al., 2019). Overall, the results showed that a 4% CaCl2 solu­ tion was capable of promoting higher firmness and sugar content stabilization, whereas when CaCl2 was applied in combination with 1­MCP at 10 μg l­1, the maturation index increased. These results reinforce Fig. 1 ­ Figs of Roxo­de­Valinhos harvested at different stages of ripening. A ­ Advanced maturation; B ­ Maturation at 50% change skin color; C ­ Maturation at 25% change skin color, D ­ Maturation at 10% change skin color. Tofanelli et al. ‐ Application of CaCl2 and 1‐MCP for ripe fig quality 95 that calcium really may maintain more stable the structure of the cell wall, seeing that when it was combined with 1­MCP there was no effect on firm­ ness. An explanation in this case, may be because humidity into the container increased softly due to 1­ MCP solution applied indoor, what could be sufficient to promote moisture saturation from CaCl2 reducing its absorbing capacity. The present work encourages the development of further investigation in order to verify the effects of both CaCl2 and 1­MCP applied at numerous different concentrations on quality conservation of ‘Roxo­de­ Valinhos’ ripe figs harvested at earlier maturation stage and during a longer storage period than stud­ ied here. In conclusion, this study showed the effect of cal­ cium chloride and 1­methylcyclopropene on con­ serve quality of ‘Roxo­de­Valinhos’ ripe figs to market for table. 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