Removal of astringency in persimmon fruits (Diospyros kaki) subjected to different freezing temperature treatments (2024)

The effects of two different freezing temperatures (− 20°C and − 80°C) on the astringency trait of persimmon fruits during 15 to 60days of storage were investigated. The levels of soluble and insoluble tannins, proanthocyanidins and other physicochemical characteristics were evaluated. Storage at − 20°C and − 80°C temperatures up to 60days has been found to be an effective method to remove astringency of persimmon fruits. Proanthocyanidin concentration was negligible at both temperatures during storage. Total soluble solid contents were decreased as 3.34 from 4.59 (mg/g DW) whereas, insoluble tannin contents were increased as 20.30 from 16.45 (mg/g DW) by freezing temperatures treatment during storage. Comparatively, higher soluble tannin content 11.68 (mg/g DW) and lower insoluble tannin content 10.02 (mg/g DW) was observed in control (day 0). Therefore, the astringency of persimmon fruits incubated at − 20°C and − 80°C was markedly reduced and after 15 up to 60days of storage, the astringent taste virtually disappeared. The proanthocyanidin contents were decreased as 0.02 from 0.52 (mg/g DW) at − 20°C storage and 0.17 from 0.47 (mg/g DW) at − 80°C storage, in comparison with the control 2.65 (mg/g DW). The moreover, along with the removal of astringency, other physicochemical parameters including color, pH, moisture content, total soluble solid, and sensory attributes were also conserved on freezing at both the temperatures. These findings suggest that freezing temperature treatments aid the removal of astringency from persimmon fruits which could be used in different food preparations or as supplements.

Materials

Persimmon fruits (Diospyros kaki L. cv. Daebong) were purchased from a Korean farm. Analytical grade chemicals, such as gallic acid, (−) -catechin (C), Folin–Denis’ reagent (F–D), and 4-(dimethylamino) cinnamaldehyde (DMAC), were procured from Sigma Chemicals (St. Louis, MO, USA). Sodium carbonate was obtained from Daejung Chemicals and Metals (Siheung, Gyeonggi, South Korea). All other solvents used were of analytical grade.

Treatment of persimmon fruits at freezing temperatures

Fruits were selected on the basis of skin color, and uniformity of weight, size, and shape. The fruits were then divided into groups and packaged using an airtight Ziploc bag for storage at − 20°C and − 80°C for 15- to 60-day time intervals. The day 0 samples comprised the control group, and the ripened non-astringent persimmon (RNAP) fruits comprised the standard group.

Thawing of persimmon fruits

The frozen persimmon fruits were thawed at room temperature (25°C) for 24h. After 24h thawing, the internal temperature of the fruits was determined as 0–4°C, which was taken by inserting the thermometer to the center of the fruits.

Sampling

For conducting further experiments, samples were prepared as follows: persimmon fruits (10 fruits in each group) were peeled, and flesh from each fruit was processed using a blender (FM-681C, Hanil, South Korea).

Soluble tannin content

Determination of soluble tannin content

One hundred microliters of the extract were mixed with 9.3mL of distilled water, and 300 µL of F–D reagent was added. After 3min, 300 µL of saturated Na₂CO₃ was added. The mixture was left for 1h in the dark at room temperature, and the absorbance was measured at 760nm using a spectrophotometer (Optizen 2120 UV, Mecasys Co., South Korea). Gallic acid solutions, in the specific concentration range, were used to construct a calibration curve. The soluble tannin content was expressed as milligrams of gallic acid per gram fresh weight (mg/g DW).

Insoluble tannin content

Determination of insoluble tannin content

The extract was filtered through Whatman no. 1 filter paper, and used for the determination of insoluble tannin content. One hundred microliters of the extract were mixed with 9.3mL of distilled water, and 300 µL of F–D reagent was added to the mixture. After 3min, 300 µL of 7.5% Na₂CO₃ was added. The solution was kept at room temperature for 1h in the dark, and the absorbance was measured at 760nm. The total insoluble tannin content was expressed as milligram of gallic acid per gram DW based on a gallic acid standard curve (mg/g DW).

Total proanthocyanidin content

Determination of proanthocyanidin content

Twenty microliters of the sample were mixed with 2380 µL of methanol, and 100 µL of freshly prepared DMAC reagent (1:1 (v/v) 6N H₂SO₄ and 2% DMAC (w/v) in methanol). The mixture was then allowed to equilibrate for 20min, and the absorbance of the colored complex was measured at 640nm. Total proanthocyanidin contents were calculated as catechin equivalents, based on a catechin standard curve, and expressed as milligram of catechin per gram DW (mg/g DW).

Moisture content determination

Moisture content was determined by the AOAC (1980) method, and was calculated using the following formula:

where X = Weight of sample before drying; Y = Weight of sample after drying.

Determination of color, pH, total soluble solids (°Brix), and titratable acidity (TA)

Hunter color values (L, a, and b) were determined for the persimmon fruits using a colorimeter (Chroma meter CR-400, Konica Mintola, Osaka, Japan). The color values were expressed as L (whiteness or brightness/darkness), a (redness/greenness), and b (yellowness/blueness). pH values were measured using a pH meter (EcoMet P25, Guro-gu, Korea). Total soluble solids (°Brix) contents were measured using a refractometer (HI 96801, Hanna Instruments; RI, Woonsocket, USA).

Titratable acidity of persimmon fruits was determined by the titrimetric method proposed by Islam et al. (2013). Phenolphthalein indicator (2–3 drops) was added to 10mL of the diluted sample. The solution was then titrated with 0.1N NaOH until a permanent pink color appeared. The volume of NaOH solution required for titration was recorded. Titratable acidity was calculated by the following formula:

Sensory evaluation

The intensity of astringency for persimmon fruits was evaluated by sensory panelists, based on the sensations before and after chewing (Taira et al. 1998). The scores were defined as follows: 0, no astringency; 1, almost non-astringent; 2, slightly astringent; 3, rather astringent; 4, strongly astringent. Evaluations were carried out at intervals of several minutes.

Statistical analysis

All experiments were performed thrice, and the values of ten replications are represented as mean ± standard deviation (SD). Data were subjected to analysis of variance (ANOVA), and multiple comparisons between means were determined by Duncan’s multiple range tests with the significance level set at p < 0.05, using the SAS 9.1.3 software (North Carolina, Cary, USA). Independent t test was used to determine whether there were significant differences in soluble tannin, insoluble tannin, and proanthocyanidin contents between the fruits stored at the two different freezing temperatures. An analysis of variance (ANOVA) was performed on data of soluble and insoluble tannin and proanthocyanidin contents to test the statistical significance of the main factors (freezing temperatures and days of storage). All analyses were performed using SAS 9.1.3 software (SAS Institute, Cary, NC, USA). Principal component analysis (PCA) analysis was performed using the factor analysis and data mining with R (FactoMineR) package (Le et al. 2008).

Effects of freezing temperatures treatment on soluble and insoluble tannin content

The astringency of persimmon fruits can be removed by the insolubilization of soluble tannins, which give rise to the astringent taste. Treatment at both freezing temperatures significantly reduced the soluble tannin concentration, as well as increased the insoluble tannin contents (Fig.1a, b). The significant difference (p < 0.05) were not observed between the fruits stored at the two different freezing temperatures for all the storage times. Statistically, no significant variances were observed from 15 to 60days of storage, except in the case of 15-day storage at − 20°C. Soluble tannin contents were decreased as 3.52 from 4.59 (mg/g DW) for − 20°C storage and 3.34 from 4.18 (mg/g DW) for − 80°C storage, whereas, more than three times higher contents 11.68 (mg/g DW) were determined in control (day-0) samples. Importantly, the soluble tannin contents of samples treated with freezing temperatures were in ranges similar to that of the RNAP fruits 3.43 (mg/g DW).

As a consequence, insolubilization of soluble tannin was observed after incubation at freezing temperatures. The insoluble tannin content in both the samples treated with freezing temperatures and the RNAP samples was almost two times higher than that in the control. The increased insoluble tannin contents were 18.18 from 16.45 (mg/g DW) at − 20°C storage and 20.30 from 18.41 (mg/g DW) at − 80°C storage, compared to that of control 10.02 (mg/g DW). In contrast, the insoluble tannin contents of the RNAP samples were 16.67 (mg/g DW). Our results indicate that in the absence of gas treatment, the freezing conditions, by themselves, are capable of polymerizing the soluble tannin to insoluble tannin, and reducing astringency. The polymerization of soluble tannins could be due to the effects of the freezing and thawing treatments. In a study reported by Taira et al. (1998), after the CO₂ treatment, small pieces of persimmon fruits (D. Kaki Thunb. Cv. Hiratanenashi) were stored using rapid and slow freezing, and it was found that slow freezing removed astringency better than rapid freezing.

According to the structural characteristics of astringent and non-astringent persimmon fruits, astringent cultivars cell walls are weaker than those of the non-astringent variety. Likewise, weak cell walls could be severely damaged by freezing. Soluble tannins from the damaged tannin cells would be readily released from their vacuoles, but as they are frozen, they could not bind or adhere to cell component fragments. Therefore, during the thawing period, thawed soluble tannins may bind loosely to the cell wall fragments and cell membranes, thus becoming insoluble (Gottreich and Blumenfeld 1991).

Effects of treatment with freezing temperatures on total proanthocyanidin contents

Proanthocyanidins are the secondary metabolites formed after the condensation of flavon-3-ol units, and are responsible for flavor and the astringent taste in some fruits and vegetables during the early development stages (Dixon et al. 2005). Persimmon fruits contain condensed tannins, particularly proanthocyanidin, which is responsible for their astringent taste. When compared to the control, the total proanthocyanidin contents of persimmon fruits significantly decreased after treatment with both the freezing temperatures (Fig.1c). No significant differences (p < 0.05) were determined in total proanthocyanidin contents between − 20 and − 80°C storage treatment. Proanthocyanidin contents decreased to a very negligible content as 0.02 from 0.52 (mg/g DW) at − 20°C storage and 0.17 from 0.47 (mg/g DW) at − 80°C storage. The proanthocyanidin contents in RNAP sample was 0.04 (mg/g DW). The control group had very high proanthocyanidin content 2.65 (mg/g DW). ANOVA analysis is performed to observe the significant effects of temperature treatments on soluble tannin, insoluble tannin and proanthocyanidin contents (Supplementary Table1). The effects of different storage days on proanthocyanidin content are highly significant (***p < 0.001) than others (Supplementary Table1). However, the results obtained by our study gives clear understanding that without the application of gas (CO₂), conventional freezing treatments alone is capable to reduce the specific astringency that occurs due to condensed tannins (proanthocyanidin). These condensed tannins could be released from damaged tannin cells; predictably, after thawing, the adherence of the frozen condensed tannins to the cell wall fragments makes them insoluble and renders the fruit non-astringent (Taira and Ono 1997).

Effects of the treatment with freezing temperature on physicochemical characteristics

Color is considered as an important indicator that can directly affect the appearance and consumer preference of fruits. The color on the outside (with peel) and inside (after cutting into halves) of the persimmon fruits after the storage at freezing temperatures are demonstrated in Table1. In comparison with the control, the outside and inside color (L, a, b) values of persimmon fruits decreased after storage at both the freezing temperatures, and these values were close to those of the RNAP samples. These might be the effects of the freezing temperatures. The color properties were still retained after long time storage conditions (Fig.3). For outside (with skin) and inside (cutting into halves) color values, significantly higher (p < 0.05) L, a and b values were obtained from control fruits rather than that of freezing temperature treatment fruits. Whereas, no significant differences (p < 0.05) were determined among both freezing temperatures treatment on inside (cutting into halves) color values. Nevertheless, significant differences (p < 0.05) were observed for outside (with skin) color values between both temperatures treatment. A similar finding was described by Arnal and Del Río (2004), which indicated that cold storage can maintain initial color for a longer time than other storage conditions.

Compared to the control sample, pH values increased in case of storage at both freezing temperatures (Fig.2a). The increased pH values were due to the decreasing TA values during the incubation at the freezing temperatures. pH and TA were found to be inversely related.

The different freezing temperatures also exerted significant effects on the TSS (°Brix) contents (Fig.2b). Compared to the control, TSS contents significantly increased in case of both freezing temperatures, and they were similar to those of the RNAP samples. During storage, the ripening of fruits accumulates higher contents of sugar as well as TSS which results in sweet taste than that observed before freezing. Compared to the control, titratable acidity decreased significantly after storage at both freezing temperatures, and these values were almost similar to those for the RNAP samples (Fig.2c). According to a study described by Khan et al. (2007), the reduction of TA after storage is mainly due to the degradation of biochemical constituents (citric acid, malic acid) of the un-ripened fruits throughout the respiration, which leads to ripening. Another reason is thought to be the conversion of starch to sugar results in reduced acidity of most fruits (Jha et al. 2012).

Moisture content has important effects on the impact of weight loss of fruits during storage. The moisture content of the freezing sample was slightly higher than that of the control sample (Fig.2d). A similar result by Khan et al. (2007), has shown that a slight increase in the moisture content of persimmon fruits was observed after storage (Fig.3).

Sensory evaluation of persimmon fruits after treatment with freezing temperatures

Removal of astringency by treatment with freezing temperatures was evaluated by sensory scores. In case of both temperatures, the sensory evaluation scores were almost similar (Fig.4). Sensations after the initial tasting of samples were not astringent in case of both the freezing temperatures; these values were similar to those of RNAP fruits. However, the control sample was strongly astringent. This sensory evaluation score was consistent with the results of the evaluation of soluble tannin, insoluble tannin, and proanthocyanidin contents. The astringency perceived by sensory evaluation was reduced due to the conversion of soluble tannins to insoluble tannins, as well as the reduction of the proanthocyanidin content after storage at freezing temperatures. After storing the fruits at both temperatures for 15 and 30days, the sensory scores after chewing revealed slight astringency levels. Slightly high contents of soluble tannin could be responsible for this astringency trait after chewing. However, the sensory evaluation also exhibited very good scores for astringency removal using freezing treatment.

Principal component analysis (PCA)

Principal component analysis (PCA) was performed to observe any possible clusters during treatment with the two different freezing temperatures for astringency removal from persimmon fruits. PCA was applied in order to evaluate the data of soluble tannin, insoluble tannin, and proanthocyanidin contents, TSS, TA, pH, moisture content, color properties (outside and inside), and sensory evaluations of persimmon fruits at all temperatures and times (Fig.5). The cumulative contribution of the first and the second principle components was 90.2%. Principal component 1 (PC1) accounted for up to 83.2% of total variance, and principal component 2 (PC2) accounted for up to 7.0%. Soluble tannin (0.99), insoluble tannin (− 0.98), and proanthocyanidins (0.98) contents, TSS (0.99), TA (0.94), pH (− 0.82), color values with peel (L, 0.93); (a, 0.92); (b, 0.97), inside color value (L, 0.98); (a, 0.94); (b, 0.97), and the sensory evaluation before (0.83) and after chewing sensation (0.95) contributed greatly to PC1. In contrast, moisture content (0.99) contributed to PC2. Notably, all samples after storage at freezing temperatures, and the RNAP samples were clustered into one group, as they had a strong negative correlation with PC1, whereas, the control positively correlated with PC1. This analysis indicates that compared to that of the untreated control fruits, astringency of persimmon fruits was effectively reduced by freezing temperature treatment.

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Conflict of interest

The authors declare no conflict of interest.

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• Akagi T, Ikegami A, Yonemori K. DkMyb2 wound-induced transcription factor of persimmon (Diospyros kaki Thunb.), contributes to proanthocyanidin regulation. Planta. 2010;232:1045–1059. doi:10.1007/s00425-010-1241-7. [PubMed] [CrossRef] [Google Scholar]
• AOAC . Official methods of analysis. 14. Washington, DC: Association Official Analytical Chemists; 1980. [Google Scholar]
• Arnal L, Del Río MA. Effect of cold storage and removal astringency on quality of persimmon fruit (Diospyros kaki, L.) cv. Rojo Brillante. Food Sci Technol Int. 2004;10:179–185. doi:10.1177/1082013204044824. [CrossRef] [Google Scholar]
• Baltacioğlu H, Artik N. Study of postharvest changes in the chemical composition of persimmon by HPLC. Turk J Agri For. 2013;37:568–574. doi:10.3906/tar-1210-21. [CrossRef] [Google Scholar]
• Barea-Álvarez M, Delgado-Andrade C, Haro A, Olalla M, Seiquer I, Rufián-Henares JA. Subtropical fruits grown in Spain and elsewhere: a comparison of mineral profiles. J Food Compost Anal. 2016;48:34–40. doi:10.1016/j.jfca.2016.02.001. [CrossRef] [Google Scholar]
• Ben-Arie R, Sonego L. Temperature affects astringency removal and recurrence in persimmon. J Food Sci. 1993;58:1397–1400. doi:10.1111/j.1365-2621.1993.tb06191.x. [CrossRef] [Google Scholar]
• Besada C, Sanchez G, Salvador A, Granell A. Volatile compounds associated to the loss of astringency in persimmon fruit revealed by untargeted GC–MS analysis. Metabolomics. 2013;9:157–172. doi:10.1007/s11306-012-0436-2. [CrossRef] [Google Scholar]
• Bubba MD, Giordani E, Pippucci L, Cincinelli A, Checchini L, Galvan P. Changes in tannins, ascorbic acid and sugar content in astringent persimmons during on-tree growth and ripening and in response to different postharvest treatments. J Food Comp Anal. 2009;22:668–677. doi:10.1016/j.jfca.2009.02.015. [CrossRef] [Google Scholar]
• Butt MS, Sultan MT, Aziz M, Naz A, Ahmed W, Kumar N, Imran M. Persimmon (Diospyros kaki) fruit: hidden phytochemicals and health claims. EXCLI J. 2015;14:542–561. doi:10.17179/excli2015-159. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Dixon RA, Xie DY, Sharma SB. Proanthocyanidins—a final frontier in flavonoid research? New Phytol. 2005;165:9–28. doi:10.1111/j.1469-8137.2004.01217.x. [PubMed] [CrossRef] [Google Scholar]
• Giordani E, Doumett S, Nin S, Bubba MD. Selected primary and secondary metabolites in fresh persimmon (Diospyros kaki Thunb.): a review of analytical methods and current knowledge of fruit composition and health benefits. Food Res Int. 2011;44:1752–1767. doi:10.1016/j.foodres.2011.01.036. [CrossRef] [Google Scholar]
• Gottreich M, Blumenfeld A. Light microscopic observation of tannin cell walls in persimmon fruit. J Hortic Sci. 1991;66:731–736. doi:10.1080/0022189.1991.11216205. [CrossRef] [Google Scholar]
• Islam MK, Khan MZH, Sarkar MAR, Absar N, Sarkar SK. Changes in acidity, TSS, and sugar content at different storage periods of the postharvest mango (Mangifera indica L.) influenced by bavistin DF. Intl J Food Sci. 2013;8:1–8. doi:10.1155/2013/939385. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Islam MT, Lee BR, Par SH, La VH, Bae DW, Kim TH. Cultivar variation in hormonal balance is a significant determinant of disease susceptibility to Xanthom*onas campestris pv. campestris in Brassica napus. Front Plant Sci. 2017;8:1–12. doi:10.3389/fpls.2017.02121. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Jha SN, Rai DR, Shrama R. Physico-chemical quality parameters and overall quality index of apple during storage. J Food Sci Technol. 2012;49:594–600. doi:10.1007/s13197-011-0415-z. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Jiménez-Sánchez C, Lozano-Sánchez J, Marti N, Saura D, Valero M, Segura-Carretero A, Fernández-Gutiérrez A. Characterization of polyphenols, sugars, and other polar compounds in persimmon juices produced under different technologies and their assessment in terms of compositional variations. Food Chem. 2015;182:282–291. doi:10.1016/j.foodchem.2015.03.008. [PubMed] [CrossRef] [Google Scholar]
• Kader AA, Yahia EM. Postharvest biology of tropical and subtropical fruits. In: Yahia EM, editor. Postharvest biology and technology of tropical and subtropical fruits. Abington: Woodhead Publishing Limited; 2011. pp. 79–111. [Google Scholar]
• Khan D, Khan AR, Bibi S, Ali S, Khalil IA. Storage stability of persimmon fruits (diospyros kaki) stored in different packaging materials. J Agric Biol Sci. 2007;2:1–4. doi:10.3844/ajabssp.2007.1.14. [CrossRef] [Google Scholar]
• Le S, Josse J, Husson F. FactoMineR: an R package for multivariate analysis. J Stat Softw. 2008;25:1–18. doi:10.18637/jss.v025.j01. [CrossRef] [Google Scholar]
• Lee YA, Cho EJ, Yokozawa T. Effects of proanthocyanidin preparations on hyperlipidemia and other biomarkers in mouse model of type 2 diabetes. J Agric Food Chem. 2008;56:7781–7789. doi:10.1021/jf800639m. [PubMed] [CrossRef] [Google Scholar]
• Prior RL, Fan E, Ji H, Howell A, Nio C, Payne MJ, Reed J. Multi-laboratory validation of a standard method for quantifying proanthocyanidins in cranberry powders. J Sci Food Agric. 2010;90:1473–1478. doi:10.1002/jsfa.3966. [PubMed] [CrossRef] [Google Scholar]
• Taira S. Astringency in persimmon. In: Linskens HF, Jackson JF, editors. Fruit analysis: modern methods of plant analysis. Berlin: Springer; 1996. pp. 97–110. [Google Scholar]
• Taira S, Ono M. Reduction of astringency in persimmon caused by adhesion of tannins to cell wall fragments. Acta Hortic. 1997;436:235–241. doi:10.17660/ActaHortic.1997.436.25. [CrossRef] [Google Scholar]
• Taira S, Ono M, Otsuki M. Effects of freezing rate on astringency reduction in persimmon during and after thawing. Postharvest Bio Technol. 1998;14:317–324. doi:10.1016/S0925-5214(98)00057-X. [CrossRef] [Google Scholar]
• Veberic R, Jurhar J, Mikulic-Petkovsek M, Stampar F, Schmitzer V. Comparative study of primary and secondary metabolites in 11 cultivars of persimmon fruit (Diospyros kaki L.) Food Chem. 2010;119:477–483. doi:10.1016/j.foodchem.2009.06.044. [CrossRef] [Google Scholar]