Astringency reduction using ethanol-associated to the storing under refrigeration at 5oC promotes physiological and structural alterations in ‘Giombo’ persimmons

This study aimed to study the effect of astringency reduction using ethanol and application of 1-MCP in ‘Giombo’ persimmons, storage in cold chamber at 5oC and to assess its physiological and structural alterations. After the deastringency treatment using ethanol, and the application of 1-methylcyclopropene (1-MCP), the fruits were stored at 5oC and 90% RH during 60 days and shelf life (SL) for 5 days at 22oC. The dosage of 1.70 ml/kg-1 ethanol during 12 hours was efficient for the astringency removal in ‘Giombo’ persimmons. However, deastringency using ethanol reduces pulp firmness and 1-MCP preserves this characteristic both in detannized fruits and in controls. The increase of soluble pectins and solubilization did not foster the polymerization of the soluble tannins both in control and detannized fruits. ‘Giombo’ persimmons displayed a severe firmness reduction, increase of soluble pectins, and degradation of the parenchyma cells, gelling of the pulp, increase of the production of ethanol and acetaldehyde after 60 days of storing + 5 days SL, inclusively in control fruits, and those treated with 1-MCP. These results suggest, cultivar ‘Giombo’ is susceptible to damages at 5oC and that further studies are required using different temperatures to improve the storage.


Introduction
The production of persimmons has grown in the last years because of the excellent acceptance of this fruit in the market. Today Brazil ranks as the fourth largest producer of persimmons in the world, with a production of 158,241 tons (Faostat, 2015).
The variety 'Giombo' belongs to the group of pollination variant (PVA), has a clear pulp, and astringency as the fruit develops for parthenocarpy (Campo-Dall'orto et al., 1996).
From the commercial point of view, this variety has several advantages, because of its late harvest. Even the same, requires an astringency removal before of the consumption of the fruits (Neuwald et al., 2009).
The astringency in persimmons is derived from the tannins accumulated in specialized cells of the mesocarp during the development of the fruits (Tessmer et al., 2014). These tannins are called condensed tannins or proanthocyanidins.
They are classified as phenolic oligomers derived from the polymerization of flavan-3-ols units (Akagi et al., 2011;Akagi et al., 2009). 'Giombo' persimmons are commercialized and consumed after deastringency. This procedure is performed by the use of ethanol or the exposition to high CO 2 concentrations Salvador et al., 2007). Ethanol promotes the increase of the activity of the enzyme alcohol dehydrogenase, and the following production of acetaldehyde, which polymerizes the soluble tannins and minimizes the astringency ).
The treatments with ethanol and the following production of acetaldehyde may affect the cellular structure, promote the degradation of the cell wall and affect the taste (Chung et al., 2015). The consequence of the is the acceleration of the reduction of pulp firmness, which shortens the shelf life of the fruits (Antoniolli et al., 2000).
In addition to the deastringency, it is necessary to provide the extension of the period of preservation of the fruits and preserve their quality Soluble tannins content were assessed by spectrophotometry, using the Follin-Ciocalteau (50%) reagent, according to the Taira (1995) technique. The extract for soluble tannins was prepared with 1 g of minced pulp and centrifuged adding 80% methanol, filtered, and adjusted with distilled water until 100 ml. From this solution, a 1 ml aliquot was withdrawn. To this aliquot was added the Follin-Ciocalteau (50%), reagent and 1 ml of a supersaturated solution of sodium carbonate, and 7.5ml of distilled water.
Reading was performed in a Biochrom Libra S22 Spectrophotometer, with absorbance at 725 nm.
The results were expressed as g 100 g -1 .
Histochemical test with hydrochloric vanillin

Pectins
The extract for total and soluble pectins was prepared using 2 g of frozen fruit pulp, and its determination was performed according to Bitter & Muir (1962). According to this technique is used 1 ml of the extract, 3.6 ml of H 2 SO 4 solution, containing 0.0125M of sodium borate and 60 µl of 3-phenylphenol (90%). The readings were performed in a spectrophotometer at 520 nm wavelength and compared to a standard curve of the galacturonic acid. The results were expressed as mg 100 g -1 .

Statistical analysis
The experimental design was completely randomized with five repetitions of five fruits each treatment. Data were submitted to the analysis of variance (ANOVA). Means were compared by the Tukey test (P < 0.05) using the program SASM-Agri.

Results and Discussion
Astringency and structure of the tannins during refrigerated storage The dosage of 1.70 ml kg -1 of ethanol during 12 hours was sufficient for the astringency removal in 'Giombo' persimmons, in agreement with  and Monteiro et al. (2012).
We observed a significant reduction of the soluble tannins of 0.085 g 100g -1 in the fruits treated with ethanol and 0.185 g 100g -1 in the fruits treated with ethanol and 1-MCP after the treatments + 1 day at 22ºC and before the entrance in the AR.
During the period of AR at 5ºC, the fruits with the astringency reduced, both those treated with ethanol and ethanol + 1-MCP had their soluble tannins content cut to about 0.050 g 100g -1 and astringency index note 1.0 at 30 and 60 days of storage + 5 days of SL ( Figures 1A and B).
In the control fruits, even the significant reduction of the soluble tannins content and astringency index at 30, 60 days in storage + 5 days SL, the results were not yet acceptable for human consumption (Figures 1A and B). The    Figure 2D), probably because of the low solubility of the pectins ( Figure 3B). After 60 days at 5ºC + 5 days SL, the firmness reduction in these treatments was drastic, reaching values as low as 10 N. Figure 2E shows the disruption of the parenchyma cells which coincides with firmness reduction as a consequence of the astringency reduction. Decreasing levels of firmness were also described by Antoniolli et al. (2000) in 'Giombo' persimmons treated with ethanol at 3.85 ml kg -1 during 24, 36 or 48 hours and stored during ten days at 25ºC.
Firmness was higher in the control fruits and those fruits treated with 1-MCP for 30 days at 5ºC + 5 days SL. In these samples firmness was respectively 20.7 and 29.7 N. At 60 days + 5 days SL, also happened the firmness reduction below At the end of the 60 days storage period we observed a significant increase of soluble pectins ( Figure 3B) in deastringent fruit, either treated or not with 1-MCP, associated with a reduction of the firmness ( Figure 3A).
At 30 days at 5ºC + 5 days of SL at 22 ºC, deastringent fruit and deastringent fruit + 1-MCP displayed ethanol (0.60 and 0.67 µg g -1 ) and acetaldehyde (0.020 and 0.025 µg g -1 ) ( Figures 4A and B) content which could be sufficient for the complete astringency reduction (Figures 1A and   B). In the control fruit, and in those treated only with 1-MCP, ethanol and acetaldehyde content kept low during this period ( Figures 4A and B).
At 60 days at 5ºC + 5 days SL at 22 ºC we observed an increase in the ethanol and acetaldehyde production in all the treatments ( Figures 4A and B). The increase in the production of these volatile compounds shall be associated to the exposition to high temperatures, and the susceptibility of this cultivar to cold storage damages (Besada et al., 2014;Salvador et al., 2008).
The astringency reduced fruits and the astringency reduced + 1-MCP, our results suggest that the increase in the ethanol and acetaldehyde production occurred after the withdrawal of the fruits from storage and at 22ºC + 5 days of SL at 22 ºC.
In the control fruits and those only treated with 1-MCP we also observed a significant increase of ethanol and acetaldehyde, probably after the withdrawal from AR at 60 days of storage, and the transfer to the storage at 22ºC. These high volatiles levels were not adequate to reduce the soluble tannins (0246 g 100g -1 and 0,466 g 100g -1 ) and the astringency (notes 3 and 4), respectively.  of Mogi das Cruzes (SP-Brazil) who kindly offered the products that have been used in this study.