244 1. Introduction Haskap (Japanese blue honeysuckle; Lonicera caeru- lea L. var. emphyllocalyx Nakai), a deciduous berry shrub, growing to 1.5-2.0 m tall, bearing a type of blue-berried honeysuckle about 1 cm in diameter, has recently been listed as one of four promising, emerging berry crops with commercial potential despite its current status of low eco- nomic importance (Hummer et al., 2012). Therefore, these crops have attracted the attention of world agriculturalists and breeders even if the history of their agricultural han- dling is still brief (Hummer et al., 2012). Blue-berried hon- eysuckles, including haskap, are native throughout the cool temperate Northern Hemisphere; haskap is cultivated in Hokkaido, the coldest local region in Japan. Haskap berry is now known as the earliest fresh fruit harvested in Hokkaido prefecture, Japan, starting in late June (Fu et al., 2011). In the last two decades, an American research team has conducted a survey on the adaptability of various blue honeysuckle plants to the northwestern United States. They have considered qualities from several geographic sources including botanical varieties edulis, kamtchatica, altaica, and boczkarnikovae from Russia, edulis and bocz- kamikovae from northeast China, and emphyllocalyx from Hokkaido, Japan, and concluded that the Japanese vari- ety emphyllocalyx (Haskap) has superior adaptability in Oregon (Thompson, 2006; Thompson and Barney, 2007). Therefore, a breeding program utilizing this variety was initiated in 2003 in the states of Oregon and also Idaho, aiming for outstanding selections in hopes of identifying superior individuals to release as cultivars as the basis for a new berry industry in the United States. To date, a research team at Hokkaido University has surveyed the ploidy level and geographical distribution of wild haskap, based on the flow cytometric analysis reveal- ing the presence of DNA diploid and DNA tetraploid plants sampled in Japan (Miyashita et al., 2011). Accordingly, chromosomal analysis confirmed that diploid and tetraploid plants showed 2n=2x = 18 and 2n = 4x = 36, respectively. The DNA diploid populations were found only in lowland mires, Betsukai, Bekanbeushi, Kushiro and Kiritappu locat- ed in eastern Hokkaido prefecture. On the other hand, DNA tetraploid populations were widely distributed in most areas in Hokkaido prefecture, and also in mainland Japan. In fact, commercial cultivars of haskap have been se- lected only from wild plants and thus, fruit traits and other agricultural characteristics have been largely limited until recently (Miyashita and Hoshino, 2010). Breeding and plant biotechnological attempts to obtain novel haskap cultivars have followed, resulting in enhanced yield and quality. Around a decade ago, selection of haskap wild lines show- ing notable edible qualities and some horticultural char- Attempt for postharvest ripening of immature fruits of Haskap (Lonicera caerulea L. var. emphyllocalyx Nakai), an emerging fruit in Northern Japan Y. Yamamoto1, Y. Hoshino2, H. Masago3, T. Kawano1,3,4,* 1 Graduate School of Environmental Engineering, The University of Kitakyushu, Kitakyushu, Japan. 2 Field Science Center for Northern Biosphere, Hokkaido University, Sapporo, Japan. 3 LINV Kitakyushu Research Center (LINV@Kitakyushu), Kitakyushu, Japan. 4 Université Paris Diderot, Sorbonne Paris Cité, Paris 7 Interdisciplinary Energy Research Institute (PIERI), Paris, France. Key words: agricultural mechanization, fruit softening, postharvest ripening. Abstract: Haskap or Japanese blue honeysuckle (Lonicera caerulea L. var. emphyllocalyx Nakai) is a deciduous shrub berry crop, which is recently listed as one of promisingly emerging berry crops. In the present short survey, an attempt for postharvest ripening of immature fruits of haskap was testified by examining the changes in fruit hardness, peel color, pigment synthesis and sugar-acid balance during storage at 5, 10, 15 or 20°C. Softening and coloring were shown to be induced during postharvest storage, especially at 20°C. The extent of maturation was largely enhanced by longer storage period. It is conclusive that haskap berries can be harvested at premature stage if postharvest maturation was allowed during storage and/or transportation to the markets. Adv. Hort. Sci., 2014 28(4): 244-249 Corresponding author: kawanotom@kitakyu-u.ac.jp Received for publication 14 October 2014 Accepted for publication 20 November 2014 Short note 245 acteristics were performed for a further breeding program (Takada et al., 2003). Accordingly, interspecies crosses be- tween L. caerulea var. emphyllocalyx and Lonicera gracili- pes var. glabra Miquel were examined to increase genetic variability of L. caerulea var. emphyllocalyx (Miyashita and Hoshino, 2010). Furthermore, Miyashita et al. (2009) re- ported the regeneration of haskap plantlets from endosperm culture of selected lines to enhance the breeding strategy. Although haskap is one of most promising berry crops in Hokkaido, the amount of cultivation and supply to the market is largely limited due to the attention required to handle soft ripe berries. In Japan’s small-scale orchards, farmers carefully harvest the haskap berries by hand so as to avoid losing any juice through damage of the deli- cate peel (Fu et al., 2011). To date, conventional agricul- tural machinery has failed to be introduced for the har- vest. There could be two distinct approaches applicable to improve harvesting efficiency: (1) development of novel machinery or devices for automated or semi-automated harvesting with maximal care from aids such as sensors and actuators; and (2) development of novel working al- gorithms to simplify the overall processes so that conven- tional agricultural machines or equipment can be readily introduced, as recently proposed for other horticultural crops (Kawano et al., 2012). Some examples of recent approaches can be found in a series of studies conducted by Fu et al. (2011). After testing various combinations of separating, collecting and cleaning methods, they concluded that the harvesting rate for haskap went from 1.45 kg/h (conventional hand pick- ing) to a maximum of 10.36 kg/h. The present study aims to contribute to the documenta- tion, designing a novel harvesting strategy based on the rip- ening physiology of haskap berries, which could be readily automated with minimal efforts. Mature haskap berries are easily damaged by mechanical operations during harvest, selection and transportation. Therefore, mature haskap ber- ries are hard to handle by conventional harvesting machines and those harvested in the Hokkaido region bear transporta- tion with difficulty over great distances to markets in large cities in mainland Japan. To avoid these problems or diffi- culties which could be attributed to fruit maturity, it is tempt- ing to propose harvesting immature green berries which are less sensitive to mechanical stresses, to then be followed by postharvest forced maturation during storage or transporta- tion. In this brief survey, we report an attempt at posthar- vest ripening of immature fruits of haskap by examining the changes in fruit hardness, peel color, pigment synthesis and sugar-acid balance during storage at different temperatures. 2. Materials and Methods Plant materials Mature and immature haskap berries were picked from two-year-old shrubs cultivated at the Experiment Farm at Hokkaido University. The berries, harvested by hand, were immediately packed for transportation under temperature- controlled conditions (exposure to heat or cold was avoided), and used for the experiments within two days after harvest. Color measurements As described elsewhere (Kawano and Shimokawa, 2003), changes in color of the fruit peel were monitored using a handy CIELAB color reader (CR-13, Konica Minolta Sens- ing Inc., Osaka, Japan) by measuring the a* and b* values of CIE (Commission International de l’Eclairge) 1976 L*a*b* color space units (CIELAB system), at 0, 1, 3, and 7 days of storage. As the a* value corresponds to a red-green scale (red, positive; green, negative) and the b* value corresponds to a yellow-blue scale (yellow, positive; blue, negative) (Kawano, 2013), we assumed that the a* and b* values represent de- greening due to chlorophyll loss and blue color development due to the synthesis of anthocyanin, respectively. Hardness Changes in fruit hardness during storage were deter- mined using a universal fruit hardness meter (max., 1 kg; model KM, Fujiwara Scientific Co. Ltd, Tokyo). Storage As the studied species are native to a cold region, we tested the effect of a low to moderate range of tempera- ture, namely, 5, 10, 15 and 20°C, by keeping the samples in temperature-controllable cool incubators (model CN-25C, Mitsubishi Electronic Engineering Co. Ltd., Tokyo, Japan). Sugar and acid contents Freshly squeezed berry sap was used for determination of sugar content using a pocket sugar meter (model PAL-1, ATAGO Co. Ltd., Tokyo, Japan) and organic acid content using a portable amperometric acid sensor (model FS-101N, ATAGO Co. Ltd., Tokyo, Japan). For determination of sugar and organic acid, 0.5 mL and 0.1 mL of berry sap were used, respectively. Quantification of organic acids is based on the voltammetric reduction of 3,5-di-tert butyl 1,2-benzoqui- none (in quinone reagent mixture provided by ATAGO Co. Ltd.) in the presence of acids (Kotani et al., 2008). Pigment analysis Anthocyanins were extracted from 0.1 g fresh weight of homogenates in 20 ml of 50% (w/v) acetic acid kept at 23°C for 24 h. Then, 1 ml of crude extract was sub- jected to centrifugation at 10,000 rpm (8217 x g) at room temperature for 10 min and the resultant supernatant was used for optical reading at 550 nm using a spectrophotom- eter (U-3310, Hitachi, Tokyo). Total anthocyanin content (expressed as of cyanidine-3-glucoside) was estimated ac- cording to Matsuzoe et al. (2006). 3. Results and Discussion Appearance of berries before and after the storage Prior to and after storage, the color of berry peels was compared with that of mature samples with the naked eye 246 (Fig. 1). While the peel of mature berries were apparently highly pigmented with a deep blue color, the immature berry samples lacked the blue pigmentation but were green due to the remaining chlorophylls. After a week of storage at 20°C, the peel of immature berry samples was slightly colored blue (Fig. 1 bottom). These observations suggest that postharvest ripening of haskap berries could likely be achieved under controlled conditions. Softening and coloring The progress in postharvest maturation in haskap ber- ries kept under various temperature was scored by the changes in mechanical hardness (Fig. 2) and peel color (Fig. 3). The results suggest that the softening of berries is affected by the storage temperature. At time 0, the differ- ence in hardness between mature and immature samples was significantly large. The hardness of the immature samples kept at 20°C was gradually lowered and finally attained a level comparable to naturally-matured berry samples (Fig. 2). Similarly, coloring was enhanced in the samples stored at higher temperature. Peel color was non-destructively determined by a colorimeter and the changes in peel color were expressed as values in CIELAB system (Fig. 3). At 5°C, neither lightness (L* values), greenness (negative a* values), nor blueness/yellowness (b* values) showed sig- nificant change during seven days of storage. In contrast, under higher temperatures, all coloring values were altered with time, suggesting that chlorophyll degradation and an- thocyanin biosynthesis were induced. Loss of chlorophylls (de-greening) is one key feature of fruit ripening found in various fruits with ethylene-producing climacteric (Kawano Fig. 1 - Images of typical haskap berries at three different stages of maturation. (a) Mature berries. (b) Immature berries prior to storage. (c) Semi-colored berries obtained after storing the im- mature samples for 1 week at 20°C. Fig. 2 - Changes in mechanical hardness of haskap berries during sto- rage under various temperatures. Vertical bars on the graph in- dicate se (n=4). Fig. 3 - Colorimetric changes in peel color in haskap berries during storage under various temperatures. Peel color was determined by colorimeter and expressed as values in CIELAB system, namely L* (a), a* (b), and b* (c). Vertical bars on the graph indicate se (n=4). 247 and Shimokawa, 1994; Kawano et al., 1999; Kawano and Shimokawa, 2004, 2005 a) and non-climacteric natures (Kawano and Shimokawa, 2003, 2005 b). Thus, further in- vestigation of the de-greening mechanism is encouraged. Anthocyanin content As the decrease in b* value in the peel color (Fig. 3 c) reflects the increase in blue pigments, the changes in anthocyanin content during postharvest storage were monitored (Fig. 4). The mature samples were rich in an- thocyanin (Fig. 4 a-i) and immature samples contained no detectable anthocyanin (Fig. 4 a-ii). Among the samples which were subjected to postharvest maturation, only the samples kept at 20°C showed signs of induced pigmenta- tion (Fig. 4 a-vi). Similarly to the colorimetrically determined blueness (Fig. 3), the blue pigmentation in the samples stored at 20°C was shown to be linearly increased with time (Fig. 4). Based on the observed tendency, we can further expect that enhanced pigmentation can be manifested by longer storage. Sugar/acid contents It is well known that both the absolute values and bal- ance in Brix index and acidity in the berry sap largely de- termine the consumption quality of fresh haskap berries (Takada et al., 2003). During postharvest maturation un- der various temperatures, no significant increase in sugar content in the immature berries was observed (Fig. 5 a). Instead, compared to green immature samples examined prior to storage, there was significant decrease in acidity over the seven days of storage in most samples kept at dif- ferent temperatures (Fig. 5 b), thus contributing, although slightly, to the increase in sugar/acid ratio (Fig. 5 c). In the end, significance between the sugar acid ratio in ma- ture samples and that in stored samples was lost, suggest- ing that sugar/acid ratio was amended by the postharvest maturation process employed. Texture, taste, flavor, and attractive components Softening of the berries is one of the important factors determining the texture of haskap berries. As described above, the mechanical hardness of berries was signifi- cantly lowered during postharvest incubation at 20°C and attained a level comparable to naturally-ripened samples (Fig. 2). As shown in figure 5 a, the postharvest maturation approach was barely successful for enhancing the sweet- ness increase, although sweetness and sourness represent- ed by the contents of sugar and organic acids are major factors determining the tastes of berries (Takada et al., 2003). However, due to the induced decrease in total acid Fig. 4 - Changes in anthocyanin content in haskap berries during sto- rage under various temperatures. (a) Typical spectral profiles of extracts from different samples. (i) Mature berries. (ii) Im- mature berries prior to storage. (iii, iv, v, vi) Immature berries subjected to one- week-long storage under 5, 10, 15, 20°C, re- spectively. (b) Increase in anthocyanin content in haskap ber- ries during storage under various temperatures. (i) Comparison of anthocyanin content between mature samples and immature samples subjected to storage. (ii) Data in (i) were enlarged for ease of comparison among the samples stored under different temperatures. Vertical bars in (b) indicate se (n=4). Fig. 5 - Changes in sugar and acid contents in haskap berries during storage under various temperatures. (a) Sugar content (per- centage by weight). (b) Organic acid content (percentage by weight). (c) Sugar/acid ratio are compared. Vertical bars on the graph indicate se (n=4). 248 level (with limited extent, Fig. 5 b), the sweetness/acid- ity balance might be enhanced reaching a level relatively close to the level of naturally-matured samples (sugar/acid ratio in Fig. 5 c). A preliminary organo-lip test performed by laboratory members is in support of the data on sugar/ acid balance, but it is still early to provide any conclusion from such a limited survey. One of major uses of haskap in Hokkaido is in bak- ery goods as sour taste accents and as color-attractive top- pings. Therefore, rather than sweetness, production of pig- ments, chiefly anthocyanin, is of more importance from this point of view. It is notable that haskap is considered a new berry crop with high antioxidant capacity due to its wealth in anthocyanins and related substances: anti-oxidative scores for haskap varieties were the highest among commercial fruits examined through multiple methods such as ferric reducing antioxidant power (FRAP) assay, oxygen radi- cal absorbance capacity (ORAC) assay, the 1,1-diphe- nyl-2- picrylhydrazyl (DPPH) free radical scavenging as- say, the aluminum chloride colorimetric method and the Folin-Ciocalteu method (Rupasinghe et al., 2012). More recently, Takahashi et al. (2014) examined the effects of dietary intake of anthocyanin-rich phenolic phytochemi- cal (containing 13.2% anthocyanin) purified from haskap fruit on postprandial serum triglyceride and blood glucose levels in rats, concluding that a decrease in postprandial blood lipids and blood glucose by short or long-term has- kap phytochemical ingestion is due to anthocyanin and other polyphenols contained in the haskap phytochemical. Reports on the antioxidant capacity of haskap and other health-related studies regarding haskap-derived pigments have been reviewed elsewhere (Celli et al., 2014). In addition to pigments, the flavors or aromas of haskap berries are of commercial importance and have recently gained attention from food industries. For instance, haskap residues after juice extraction have been used preliminary as natural flavoring for teas (Sakamoto et al., 2012). Thus, the impact of postharvest maturation of haskap berries on the production of flavors and aromas should be document- ed in future studies. 4. Conclusions Two key parameters of fruit maturation, namely soft- ening and coloring, were significantly amended by post- harvest storage at moderate temperature and the extent of maturation is likely to be enhanced by a longer stor- age period. As enhanced coloring represents an increase in anthocyanin content, valued for its antioxidant action, the postharvest maturation approaches presented here may contribute to the market quality of this crop. However, the sugar content in the berry sap could not be altered during storage and the change in sap acidity induced during stor- age was limited. In conclusion, haskap can be harvested at premature stage if postharvest maturation is allowed dur- ing storage and/or transportation to the markets. 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