Mechanism of Browning Occurring during the Processing of Semi-dried Persimmons

Transcription

1 J. Japan. Soc. Hort. Sci. 78 (1): Available online at JSHS 2009 Mechanism of Browning Occurring during the Processing of Semi-dried Persimmons Hisashi Yamada 1 *, Tomomi Ando 1, Kenta Tsutani 1 **, Shoji Amano 1 and Yoshihiko Yamamoto 2 1 Faculty of Agriculture, Ehime University, Matsuyama , Japan 2 Toyoengei Agriculture Co-operative Association, Saijo , Japan To investigate the mechanism of browning occurring during the processing of semi-dried Atago persimmon (Diospyros kaki Thunb.) fruit, the changes in several components related to enzymatic and non-enzymatic browning were determined. The browning of semi-dried persimmons developed when the water content of fruit decreased to about 50% or less, and hand massage (HM) treatment promoted both the drying process and development of browning. The polyphenol content as a substrate of polyphenol oxidase (PPO) markedly decreased during the first 7 days of drying before the occurrence of browning. PPO activity gradually decreased and was lower in dry + HM than dry treatment alone during drying. The ascorbic acid (AsA) content decreased and HM accelerated the reduction by 7 days of drying. Dehydroascorbic acid (DHA) also decreased with stagnation in the mid-stage during 14 days of drying and was lower in dry + HM than dry treatment alone during the latter period of drying. Furfural, a non-oxidative degradation product of AsA, started to increase from 4 or 10 days of drying in dry + HM or dry treatment, respectively. Reducing sugars, fructose and glucose, markedly increased for the first 4 days with a concomitant decrease in the sucrose content, and were higher in dry + HM than dry treatment. The content of amino acids gradually decreased, and no difference was observed between treatments. Hydroxymethylfurfural (HMF), as an intermediate product from fructose to brown pigments, decreased during drying and was lower in dry + HM than dry treatment alone. These results suggest that the oxidative and nonoxidative degradation of AsA would contribute markedly to the browning of semi-dried persimmon fruit, and enzymatic browning by PPO and the Maillard reaction between amino acids and reducing sugars might not play an important role. Key Words: browning, degradation of ascorbic acid, Maillard reaction, polyphenol oxidase, semi-dried persimmon. Introduction Atago persimmon originated in the Shuso district of Ehime Prefecture in Japan and has long been grown there. Since the cultivar belongs to the pollination constant astringent (PCA) type, it has usually been marketed after the deastringency of fresh fruit by ethanol and/or CO 2 treatment. Recently, growers there started to produce semi-dried persimmons, called Anpo-gaki in Japan, using unfavorable Atago fruit for the fresh fruit market. However, browning occurring during the drying process decreased the market value of products Received; April 28, Accepted; July 10, * Corresponding author ( hyamada@agr.ehime-u.ac.jp). ** Present address: Graduate School of Natural Science and Technology, Okayama University, Okayama , Japan. because semi-dried persimmon is characterized by a softer texture and lighter reddish orange color compared to fully-dried fruit. Many studies on the discoloration of fruit and juice have suggested the involvement of enzymatic and nonenzymatic reactions (Handwerk and Coleman, 1988; Kurata, 1976; Murata and Homma, 1998). Polyphenol oxidase (PPO) is one of the most important enzymes involved in the enzymatic browning of food, and a close relationship between the browning potential and PPO activity and/or polyphenol content was found in several fruits (Brandelli and Lopes, 2005; Ding et al., 1998; Murata et al., 1995a, 1995b; Nguyen et al., 2003). On the other hand, the oxidative and non-oxidative degradation of ascorbic acid (Kurata, 1976; Li et al., 1989; Shinoda et al., 2005) and the Maillard reaction (Bolin and Steele, 1987; Caglarirmak, 2006; Sanz et al., 124

2 J. Japan. Soc. Hort. Sci. 78 (1): ) have also been suggested to play important roles in the discoloration of many foods including dried fruit as non-enzymatic browning. In our preliminary observations, browning started to occur at a water content of about 50% or less, suggesting excessive drying to be a key factor (Yamada et al., 2005). Hand massage (HM) of fruit, which is well-known to promote the drying process and is often used in producing full-dried persimmons, also accelerated both the decrease in the water content and browning development (Yamada et al., 2005). However, no information is available on the mechanism of browning during the drying of persimmon fruit. Therefore, we determined the changes in PPO activity and polyphenol content as an enzymatic reaction and the content of some components related to non-enzymatic reactions, such as ascorbic acid (AsA), dehydroascorbic acid (DHA), furfural, amino acids, sugars, and hydroxymethylfurfural (HMF) during the processing of semi-dried Atago persimmons. We also compared the changes in these compounds between the treatments with and without HM to clarify the involvement of each reaction in browning development. Materials and Methods Experiment for enzymatic browning in 2005 Atago persimmons (Diospyros kaki Thunb.) were harvested in early December from a commercial orchard in the Shuso district of Ehime Prefecture, Japan and then transferred to the laboratory at Ehime University, Matsuyama. Forty-five fruit of a uniform size and color were selected and randomly divided into 9 groups with 5 replicate fruit. Fruit preparation and drying procedures followed the commercial method adopted at Toyoengei Agriculture Co-operative Association at that time. Fruit were peeled with a knife, and soaked in a solution containing 1% sodium sulfite and 1.3% citric acid for 30 min to inhibit the oxidation of the fruit surface. Then, fruit were hung on strings at 30 C and 30 45% RH. Half of the drying fruit were massaged by hand at 3 days of drying to impose 2 treatments with different levels of water content and browning, as described above. Sampling was conducted at 3- or 4-day intervals during 13 days of drying, and we determined the extent of browning, the levels of water, polyphenols, and proteins, and the activity of PPO. The degree of browning was assessed using a visual rating scale (0 = none to 4 = severe). The water content was determined after drying the diced flesh in an oven at 80 C for 24 h. The total polyphenol content was analyzed with 80% methanolic extracts using the Folin- Denis method (Swain and Hillis, 1959). PPO was extracted using the polyethylene glycol (PEG)-acetone method of Badran and Jones (1965) and Zheng et al. (2005) with some modifications. One or five grams of persimmon flesh were homogenized in the extraction solution containing 15 ml of 0.1 M NaH 2 PO 4 /Na 2 HPO 4 buffer (ph 6.5) and 1.7 ml of 10% PEG. The homogenate was mixed with 30 ml of cold acetone ( 20 C) and centrifuged at 7000 rpm for 10 min at 10 C. The precipitate was dissolved in 10 ml of extraction buffer containing 0.2 g of insoluble polyvinylpyrrolidone at 4 C for 1 h and then centrifuged at rpm for 10 min at 4 C. The supernatant was purified with a 0.45 μm membrane filter and a Sephadex G-25 column (Amersham Biosciences Ltd., Sweden) and used as a crude PPO solution. PPO activity was assayed using a modification of the procedure described by Ding et al. (1998). A decrease in absorbance of 0.01 per min at 325 nm at 30 C after adding chlorogenic acid and crude enzyme solution to McIlvaine buffer (ph 4) was defined as 1 unit of PPO activity. The protein content of the crude enzyme solution was measured according to the Bradford method (Bradford, 1976). Experiment for non-enzymatic browning in 2006 and 2007 Atago fruit were harvested at the end of November 2006 and in early December 2007 from an orchard of Ehime University Farm. The preparation and treatment of fruit were almost the same as those in Measurements were conducted of the chemical components related to the non-enzymatic browning reactions, such as AsA, DHA, sugars, and amino acids in 2006 and furfural and HMF in AsA and DHA were determined with 5% metaphosphoric acid extracts using the hydrazine method (Tsujimura, 1982). Sugars were extracted with 80% ethanol after the microwave irradiation of flesh tissue to inhibit sucrose degradation during extraction by invertase (Zheng and Sugiura, 1990). The sugar composition was analyzed with a gas chromatograph (GC-14B, Shimadzu Co., Japan) equipped with a flame ionization detector and a silicone column after silylating the ethanolic extracts with a trimethylsilylating reagent (Yamada et al., 1994). Total amino acids were determined with the same ethanolic extracts using the ninhydrin method (Chachin, 1981). Furfural and HMF were determined with 50% ethanol extracts using the high-performance liquid chromatograph (HPLC) method described by Li et al. (1988). The ethanolic extracts were passed through a membrane filter (0.45 μm), and 10 μl of the filtrates was injected into a HPLC reverse-phase column (TSK-gel ODS-80TM, mm i.d., Tosoh Co., Japan) at 40 C. Tetrahydrofuran (THF)-water (0.3: 99.7, v/v) was used isocratically at 1.0 ml min 1. The effluent was monitored at 280 nm with a UV detector (L-4000, Hitachi Co., Japan). All data are expressed on a fresh weight basis before drying to eliminate the concentrating effect of drying. Results and Discussion Experiment for enzymatic browning in 2005 HM accelerated the rate of drying, and about a 50% water content was achieved at 7 and 10 days of drying

3 126 H. Yamada, T. Ando, K. Tsutani, S. Amano and Y. Yamamoto on dry + HM and dry treatment alone, respectively (Fig. 1). Browning started to occur at about this water content in each treatment, consistent with our previous observations (Yamada et al., 2005). These results suggest that growers should take care not to decrease the water content to less than 50% to produce semi-dried persimmons of a high quality. Browning occurred in the inner flesh of drying fruit and not directly on the surface, indicating that the soaking treatment with sulfite solution could inhibit the oxidation of the peeled fruit surface just as sulfur fumigation often used in producing dried persimmons. PPO widely exists in plants and oxidizes polyphenols to their corresponding quinines; then, these quinones are polymerized with other quinones and amines to form brown pigments (Ding et al., 1998; Murata and Homma, 1998). Many studies with fresh or cut fruit and juice showed the relationships between browning and the polyphenol content and/or PPO activity (Brandelli and Lopes, 2005; Ding et al., 1998; Murata at al., 1995a, 1995b; Nguyen et al., 2003; Sannomaru et al., 1998). However, there has been no report that associated PPO and its substrate with browning during drying persimmon fruit, although the polyphenol or tannin content was often determined in relation to the removal of astringency (Ishii and Yamanishi, 1982; Taira et al., 1988). In the present study, the polyphenol content decreased rapidly to less than 1 mg g 1 FW for the first 7 days of drying, and HM promoted the decreasing rate during this period (Fig. 2). Since no astringency was tasted at 7 days of drying, the rapid decrease of the polyphenol content would be due to insolubilization by acetaldehyde accumulation (Taira et al., 1988). Polyphenol remained at a lower level thereafter and no difference was found between the treatments, indicating that the substrate of PPO was reduced to a very low level before the start of browning development. PPO activity per g fresh weight or mg protein decreased in both treatments and was lower in dry + HM than dry treatment alone throughout the drying period (Fig. 3A, 3B). These results suggest that the enzymatic reaction by PPO and polyphenol might not play an important role in browning during the drying Fig. 2. Changes in polyphenol content during drying Atago persimmons at 30 C in Vertical bars indicate SE (n = 5). HM, hand massage treatment at 3 days of drying. Fig. 1. Changes in water content (circles) and degree of browning (triangles) during drying Atago persimmons at 30 C in Vertical bars indicate SE (n = 5). HM, hand massage treatment at 3 days of drying. Fig. 3. Changes in PPO activity per g fresh weight (A) and mg protein (B) during drying Atago persimmons at 30 C in Vertical bars indicate SE (n = 5). HM, hand massage treatment at 3 days of drying.

4 J. Japan. Soc. Hort. Sci. 78 (1): of persimmons, although there is a possibility that a part of the browning is caused by the reaction. Experiment for non-enzymatic browning in 2006 and 2007 Since the results regarding the water content and browning in 2007 were almost the same as those in 2006, only the data in 2006 are presented in Figure 4 as representative. Similar changes in the water content and degree of browning to those in 2005 were obtained in 2006, although the rate of drying was slightly higher in 2006 due to the smaller size of fruit used. HM treatment also promoted the drying process and browning development. AsA is an antioxidant but it contributes to the browning of foods because it is easily decomposed by oxidative and non-oxidative pathways (Handwerk and Coleman, 1988; Kurata, 1976; Shinoda et al., 2005). In the presence of oxygen, AsA is degraded primarily to DHA, which is further degraded to reductones via some intermediate products (Shinoda, 2005). On the other hand, furfural is produced through the non-oxidative degradation of AsA and often used as an indicator of the browning of citrus juice (Li et al., 1989; Shinoda et al., 2005). In this study, the AsA content decreased during drying and HM accelerated the degradation reaction by 7 days of drying (Fig. 5A). DHA also gradually decreased and it was lower in dry + HM than dry treatment alone during the late period of drying (Fig. 5B). The absence of a difference in the DHA content between the treatments during the early stage of drying and slower rate of decrease from 4 to 7 or 10 days of drying in dry + HM or dry treatment, respectively, might reflect the active supply of DHA by the oxidation of AsA during the decomposition of DHA to reductones. Furfural was not detected in the flesh of fresh persimmon fruit, but then showed an increasing presence after 4 or 10 days of drying in dry + HM or dry treatment, respectively, and the changes were related to browning (Figs. 4 and 6). A few days delay in the furfural increase compared to browning in dry treatment suggests that furfural had not accumulated to a detectable level by 10 Fig. 5. Changes in the content of AsA (A) and DHA (B) during drying Atago persimmons at 30 C in Vertical bars indicate SE (n = 5). HM, hand massage treatment at 3 days of drying. Fig. 4. Changes in water content (circles) and degree of browning (triangles) during drying Atago persimmons at 30 C in Vertical bars indicate SE (n = 5). HM, hand massage treatment at 3 days of drying. Fig. 6. Changes in furfural content during drying Atago persimmons at 30 C in Vertical bars indicate SE (n = 5). HM, hand massage treatment at 3 days of drying.

5 128 H. Yamada, T. Ando, K. Tsutani, S. Amano and Y. Yamamoto days of drying when only a slight degree of browning at 0.7 and 0.5 was observed in 2006 (Fig. 4) and 2007, respectively. The results suggest that both the oxidative and non-oxidative degradation pathways of AsA function and might be involved in the browning of semi-dried persimmons, as with citrus juice (Li et al., 1989; Shinoda et al., 2005). The importance of AsA may be supported by the observation in our preliminary experiment that the addition of AsA to the sodium sulfite solution instead of citric acid induced browning on the fruit surface (unpublished data). Another important non-enzymatic reaction in food browning is the Maillard reaction between reducing sugars and free amino groups of amino acids, peptides, and proteins (Sanz et al., 2001). HMF is derived from fructose and considered an important intermediate and indicator of the browning of citrus juice (Berry and Tatum, 1965; Kanner et al., 1982; Kato and Sakurai, 1964; Ohta et al., 1983) and raisins (Caglarirmak, 2006). However, there are some reports that provide negative data regarding the role of HMF in browning (Li et al., 1989; Shinoda et al., 2005). In the current study, fructose and glucose markedly increased during the first 4 days of drying, accompanied by a concomitant decrease of sucrose and total sugars (Fig. 7). Treatment of dry + HM showed a higher fructose and glucose and lower sucrose content than that of dry alone (Fig. 7A, B, C). These changes in sugar compositions would be induced by the greater activity of invertase in persimmon flesh (Tsuji and Komiyama, 1988; Zheng and Sugiura, 1990). Free amino acids tend to decrease, and no difference was observed between the treatments throughout the drying period (Fig. 8), suggesting almost no contribution of the Maillard reaction between reducing sugars and amino acids to the browning of semi-dried persimmons. HMF decreased during the 14 days of drying and HM treatment promoted the reduction (Fig. 9). The changes in fructose and HMF indicated that HMF would be broken down during drying without any supply via fructose decomposition. These results suggest that the Maillard reaction including the pathway through HMF would not be closely involved in browning development in the processing of semi-dried persimmons. In conclusion, the browning of semi-dried Atago persimmon fruit was related to the excessive drying to a water content of 50% or less. The main factors involved in browning would be the oxidative and non-oxidative degradation pathways of AsA. The enzymatic reaction Fig. 7. Changes in the content of fructose (A), glucose (B), sucrose (C), and total sugars (D) during drying Atago persimmons at 30 C in Vertical bars indicate SE (n = 5). HM, hand massage treatment at 3 days of drying.

6 J. Japan. Soc. Hort. Sci. 78 (1): Fig. 8. Changes in total free amino acid content during drying Atago persimmons at 30 C in Vertical bars indicate SE (n = 5). HM, hand massage treatment at 3 days of drying. Fig. 9. Changes in HMF content during drying Atago persimmons at 30 C in Vertical bars indicate SE (n = 5). HM, hand massage treatment at 3 days of drying. via PPO and the non-enzymatic Maillard reaction might play a minor role in browning during the processing of semi-dried persimmons. Literature Cited Badran, A. M. and D. E. Jones Polyethylene glycols tannins interaction in extracting enzymes. Nature 206: Berry, R. E. and J. H. Tatum Hydroxymethylfurfural in stored foam-mat orange powders. J. Agric. Food Chem. 13: Bolin, H. R. and R. J. Steele Nonenzymatic browning in dried apples during storage. J. Food Sci. 52: Bradford, M. M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: Brandelli, A. and C. H. G. L. Lopes Polyphenoloxidase activity, browning potential and phenolic content of peaches during postharvest ripening. J. Food Biochem. 29: Caglarirmak, N Ochratoxin A, hydroxymethylfurfural and vitamin C levels of sun-dried grapes and sultanas. J. Food Proc. Pres. 30: Chachin, K Amino-san. p In: S. Nakagawa, Y. Ohsawa, Y. Sakanishi, T. Iwata and R. Takahashi (eds.). Engeigaku-jikken-jisshuu (In Japanese). Yokendo, Tokyo. Ding, C. K., K. Chachin, Y. Ueda and R. Mochioka Changes in polyphenol concentrations and polyphenol oxidase activity of loquat (Eriobotrya japonica Lindl.) fruits in relation to browning. J. Japan. Soc. Hort. Sci. 67: Handwerk, R. L. and R. L. Coleman Approaches to the citrus browning problem. A review. J. Agric. Food Chem. 36: Ishii, Y. and T. Yamanishi The changes of soluble tannin and free sugars of astringent persimmon in the process of sun drying. Nippon Shokuhin Kogyo Gakkaishi 29: (In Japanese with English abstract). Kanner, J., J. Fishbein, P. Shalom, S. Harel and I. Ben-Gera Storage stability of orange juice concentrate packaged aseptically. J. Food Sci. 47: Kato, H. and Y. Sakurai Studies on browning mechanisms of fruit juice products. Part III. Mechanism of browning in citric acid-sucrose system. Nippon Nogeikagaku Kaishi 38: (In Japanese). Kurata, T Mechanism of degradation and discoloration reaction of L-ascorbic acid. Nippon Nogeikagaku Kaishi 50: (In Japanese). Li, Z. F., M. Sawamura and H. Kusunose Rapid determination of furfural and 5-hydroxymethylfurfural in processed citrus juices by HPLC. Agric. Biol. Chem. 52: Li, Z. F., M. Sawamura and H. Kusunose Role of furfural and 5-hydroxymethylfurfural in browning of yuzu juice. Nippon Shokuhin Kogyo Gakkaishi 36: (In Japanese with English abstract). Murata, M. and S. Homma Recent progress in polyphenol oxidase and control of enzymatic browning. Nippon Shokuhin Kagaku Kogaku Kaishi 45: (In Japanese). Murata, M., I. Noda and S. Homma. 1995a. Enzymatic browning of apples on the market: Relationship between browning, polyphenol content, and polyphenol oxidase. Nippon Shokuhin Kagaku Kogaku Kaishi 42: Murata, M., M. Tsurutani, M. Tomita, S. Homma and K. Kaneko. 1995b. Relationship between apple ripening and browning: Changes in polyphenol content and polyphenol oxidase. J. Agric. Food Chem. 43: Nguyen, T. B. T., S. Ketsa and W. G. van Doorn Relationship between browning and the activities of polyphenol oxidase and phenylalanine ammonia lyase in banana peel during low temperature storage. Postharvest Biol. Technol. 30: Ohta, H., K. Yoshida, K. Hyakudome, H. Aoyagi, M. Okabe and W. Susukida Effects of headspace, pasteurization condition and storage temperature on quality of canned satsuma mandarin juice during storage. Nippon Shokuhin Kogyo Gakkaishi 30: (In Japanese with English abstract). Sannomaru, Y., O. Katayama, Y. Kashimura and K. Kaneko Effects of polyphenol content and polyphenoloxidase activity on browning reaction of apple fruits. Nippon Shokuhin Kagaku Kogaku Kaishi 45: (In Japanese with English

7 130 H. Yamada, T. Ando, K. Tsutani, S. Amano and Y. Yamamoto abstract). Sanz, M. L., M. D. del Castillo, N. Corzo and A. Olano Formation of amadori compounds in dehydrated fruits. J. Agric. Food Chem. 49: Shinoda, Y., H. Komura, S. Homma and M. Murata Browning of model orange juice solution: factors affecting the formation of decomposition products. Biosci. Biotechnol. Biochem. 69: Swain, T. and W. E. Hillis The phenolic constituents of Prunus domestica. I. The quantitative analysis of phenolic constituents. J. Sci. Food Agr. 10: Taira, S., Y. Kimura and S. Watanabe Effect of harvest maturity on removal of astringency during sun drying of Japanese persimmon (Diospyros kaki Thunb.) Hiratanenashi fruit after peeling. Nippon Shokuhin Kogyo Gakkaishi 35: (In Japanese with English abstract). Tsuji, M. and Y. Komiyama Influences of the sulfur amount on quality, chemical components and alcohol dehydrogenase and invertase activities in sulfur fumigated astringent persimmon fruit during sun-drying. Nippon Shokuhin Kogyo Gakkaishi 35: (In Japanese with English abstract). Tsujimura, M Vitamin C-no-teiryo. p In: T. Obara, T. Suzuki and H. Iwao (eds.). Shokuhin-bunsekihandbook (In Japanese). Kenpakusha, Tokyo. Yamada, H., H. Ohmura, C. Arai and M. Terui Effect of preharvest fruit temperature on ripening, sugars, and watercore occurrence in apples. J. Amer. Soc. Hort. Sci. 119: Yamada, H., R. Hondo, S. Amano and Y. Yamamoto Study on browning in semi-dried fruit of Atago persimmons. Abst. Chugoku-Shikoku Br., Japan. Soc. Hort. Sci. 44: 11 (In Japanese). Zheng, Q. L., A. Nakatsuka and H. Itamura Extraction and characterization of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase from wounded persimmon fruit. J. Japan. Soc. Hort. Sci. 74: Zheng, G. H. and A. Sugiura Changes in sugar composition in relation to invertase activity in the growth and ripening of persimmon (Diospyros kaki) fruits. J. Japan. Soc. Hort. Sci. 59: (In Japanese with English abstract).