Rind Color Development in Satsuma Mandarin Fruits Treated by Low-intensity Red Light-emitting Diode (LED) Irradiation

Abstract

Here, we investigated the effect of irradiation by a low-intensity red light-emitting diode (LED) (photon flux 12 µmol•m⁻²•s⁻¹) on rind color development in Satsuma mandarins after harvesting at two different periods. In the early harvest fruit, the a* value for Satsuma mandarin peel treated by red LED irradiation was 2.7 and 2.4 times higher than that for the dark treatment fruit at 4 and 8 days irradiation, respectively. In the commercial harvest fruit, the a* value of the peel treated by red LED irradiation was 1.2 and 1.4 times higher than for dark treatment at 4 and 8 days irradiation, respectively. Additionally, we examined the influence of red LED irradiation on the internal fruit quality of Satsuma mandarins from the two harvest periods. Low-intensity red LED irradiation did not affect fruit quality. We elucidated the effect of intermittent red LED irradiation on the change in a* value of mandarin fruits. These results indicated that treatment with low-intensity red LED irradiation is sufficient to develop a degree of rind color in mandarins without affecting the internal fruit quality.

Introduction

The peel color of Satsuma mandarin fruits is an important factor in customer satisfaction. The delay of color development in harvested fruits has resulted in reduced market value. In recent years, there have been problems with delayed coloration of Satsuma mandarins because of unstable weather conditions, including high temperatures in early fall because of global warming and sharp drops in temperature before harvesting. There are advantages to the early shipment and sale of Satsuma mandarins. In recent years, fruits with a low level of coloration despite the degree of flesh maturation have been shipped; this type of fruit development occurred because high fall temperatures caused the flesh to ripen while some green coloration remained in the peel. Satsuma mandarin production is faced with challenges due to global warming (Sugiura and Yokozawa, 2004; Sugiura et al., 2007). One impact of global warming on fruit cultivation is the occurrence of rind puffing. Therefore, it has become increasingly common for growers to treat fruit with gibberellic acid and prohydrojasmon mixtures (hereafter, GP solutions) three months before harvesting, as a method to reduce rind puffing (Makita and Yamaga, 2006; Nakatani et al., 2014). However, treatment with GP solutions is problematic, as gibberellin delays coloration (Porat et al., 2001), and interested parties are keen to find a method that simultaneously solves issues related to both rind puffing and coloration delay. The ability to improve coloration through some type of fruit processing would increase its marketability. In Japanese citrus fruit production areas, mulching the ground with light-reflective, non-woven fabric sheets during cultivation has been embraced as a means of improving coloration and increasing sugar content (Morinaga et al., 2004; Muramatsu et al., 2005). In addition, in the past there have been many studies on methods of improving fruit coloration during post-harvest storage. High-temperature pretreatment, in which the fruit is maintained at 15 – 20°C for a week, has been brought into practical use (Murata and Yamawaki, 1992). However, this treatment involves the extended use of heaters (at least 8 days), and improved technology is being sought to reduce costs. In addition, several studies have been conducted over the years on promoting coloration by treating fruit with ethylene (Kitagawa et al., 1971; Kitagawa, 1973; Houck et al., 1978; Mayuoni and Porat, 2011). Ethylene is used for quality improvement, including promotion of fruit ripening and decomposition of chlorophyll in citrus fruits. However, a drawback of ethylene treatment in citrus fruit is that if the fruit is immature and the ethylene concentration is too high, damage, such as browning of the peel and calyx, can occur. Reduction in the fruit's storage-life because of ethylene is also a concern.

It has been shown that carotenoids in citrus fruits are increased by light emitting diode (LED) light irradiation. Ma et al. (2013) reported that red light irradiation (at 660 nm) was effective in accumulating carotenoids in the flavedo of Satsuma mandarin. LEDs are increasingly being used in plant cultivation (Yorio et al., 2001; Hidaka et al., 2013) and in the prevention of disease and insect damage (Kudo et al., 2011). However, besides the study mentioned above, there are few research examples of their use in the storage of Satsuma mandarins. Given the cost of installation, it is necessary to conduct research at practical, low-intensity irradiation levels and to investigate how coloration benefits sales. In this study, we investigated the effect of low-intensity red LED light irradiation on the coloration of Satsuma mandarin fruit after harvesting. In addition, it has been reported that when immature fruit with low levels of carotenoids in the flavedo is treated with ethylene, chlorophyll decomposes but the flavedo only yellows (Kitagawa and Tarutani, 1973). Thus, in the present study, we examined color changes in the fruits after harvesting at two different periods.

Materials and Methods

Red LED irradiation of Satsuma mandarin fruits    Satsuma mandarin (Citrus unshiu Marc.) cultivar ‘Aoshima unshu’ (a late-maturing cultivar; harvest period late-November to mid-December) was harvested from an orchard at Shizuoka Prefectural Agriculture and Forestry Research Institute Fruit Tree Research Center on November 14 (early harvest) and November 25 (commercial harvest), 2013. The fruits were continuously irradiated with a red LED with an emission peak of 660 nm at a photon flux of 12 µmol•m⁻²•s⁻¹ (Yamato Industrial Co., Ltd., Shizuoka, Japan) (Fig. 1) for 8 days. The photon flux was measured using an illuminance spectrophotometer (CL-500A; Konica Minolta, Inc., Tokyo, Japan). The fruits were placed 30 cm from the irradiation source (Fig. 2). Fruits that were not irradiated were used as controls. The fruits were kept at 13°C and 85% – 90% relative humidity for 8 days. Fifteen fruits constituted a single replicate and each treatment was repeated four times. In each experiment, different fruits samples were chosen randomly.

Fig. 1.

Relative light-emitting diode (LED) power distribution.

Fig. 2.

Experimental setup of red LED irradiation for Satsuma mandarin fruits.

Rind color analysis    Rind color was measured at a single point between the pedicel and the lateral part of the fruit using a color meter (TC-1500SX; Tokyo Denshoku, Tokyo, Japan). CIELAB (L* = lightness, a* = cyan/magenta hue component, b* = yellow/blue hue component) were measured, and chroma (C*) = [(a*² + b*²)^1/2] values were calculated. The degree of rind color (coloration) was assessed on a scale of 1 (green) to 10 (full coloring), discriminated by visual observation, which is the system used in packing houses.

Internal quality characteristics    Soluble solids concentration (SSC) and titratable acidity (TA) were determined using freshly extracted juice from irradiated and non-irradiated fruits after 8 days of treatment. Each harvest involved a random sampling from multiple trees. Five fruits constituted a single replicate and each treatment was repeated four times. In each experiment, different fruit samples were chosen randomly. SSC was determined with a digital refractometer (DBX-55A; Atago, Tokyo, Japan). TA was determined by titrating 5 mL of the juice to pH 8.1 with 0.156 mol L⁻¹ NaOH. Acidity was expressed as g citric acid per 100 mL juice.

Effect of intermittent red LED irradiation on the change in a* value of Satsuma mandarins    Satsuma mandarin (C. unshiu Marc.) cultivar ‘Takabayashi wase’ (an early-maturing cultivar; harvest period September to October) was harvested from an orchard at Shizuoka Prefectural Agriculture and Forestry Research Institute Fruit Tree Research Center on October 2, 2013. The fruits for LED treatment were continuously irradiated with a red LED at a photon flux of 12 µmol•m⁻²•s⁻¹. The fruit for LED/dark treatment was irradiated with a red LED at the same photon flux in cycles of 12 hours of red LED light followed by 12 hours of dark. The fruits were placed 30 cm from the irradiation source. Fruits that were not irradiated were used as controls. The fruits were kept at 13°C and 85% – 90% relative humidity for 8 days. At days 5 and 8, the a* values of the rinds were measured as described above. A change in the a* value (Δa*) for the fruits was calculated according to the formula:   

Fifteen fruits constituted a single replicate and each treatment was repeated three times. In each experiment, different fruit samples were chosen randomly.

Statistical analysis    The data are expressed as mean values. Statistical analysis was performed using the statistical functions in Microsoft Office Excel 2007 for Windows (Microsoft Corporation, Redmond, WA, USA). Differences between means were evaluated by a Student's t-test or Tukey's multiple range test.

Results and Discussion

The changes in L*, a*, b*, and Chroma (C*) values with red LED irradiation in C. unshiu Marc. ‘Aoshima unshu’ at early and commercial harvest times are shown in Fig. 3. In the early harvest fruits, the a* value of the mandarin peel treated by red LED irradiation was 2.7 and 2.4 times higher than with the dark treatment at 4 and 8 days of irradiation, respectively. Similarly, the L*, b*, and C* values for the LED-irradiated fruits were significantly higher compared with those for the dark treatment fruits. In the commercial harvest fruit, the a* value of the peel treated by red LED irradiation was 1.2 and 1.4 times higher than with the dark treatment at 4 and 8 days of irradiation, respectively. The C* values for the LED-irradiated fruits were significantly higher than for the dark treatment fruits, as with the a* value. However, in the commercial harvest, the L* and b* values for the irradiated fruits were not significantly different compared with those for the dark treatment fruits. Changes in L* and b* values in the fruits differed between the harvest periods and with red LED irradiation. Kon et al. (1987) indicated that the L* and b* values in citrus fruit peels over a season increase rapidly when the peel changes color from green to yellow. L* and b* values might be affected by chlorophyll decomposition of the surface peel. In the current study, we did not measure chlorophyll content; hereafter, we need to evaluate the relationship between red light irradiation and chlorophyll decomposition.

Fig. 3.

The effect of red light emitting diode irradiation on changes in L*, a*, b* and Chroma (C*) values in C. unshiu Marc. ‘Aoshima unshu’ harvested at early (a) and commercial (b) harvests. Vertical bars indicate standard error (SE) (n = 4). ^z **, * and n.s.: significant difference at p < 0.01, 0.05 and no significant difference by t-test, respectively.

The change in rind color with red LED irradiation in C. unshiu Marc. ‘Aoshima unshu’ from the early and commercial harvests is illustrated in Fig. 4. In the early harvest fruit, the degree of rind color of the fruits irradiated by red LED at days 4 and 8 was significantly increased from that of the dark treatment fruits. The degree of rind color after dark treatment for 8 days was 7.2, while following LED treatment it was 8.2. Similarly, even at commercial harvest, the LED-irradiated fruits had a significantly increased degree of rind color when compared with the dark treatment fruits. Ma et al. (2013) found that irradiation with red light at a photon flux of 50 µmol•m⁻²•s⁻¹ for 6 days was effective in enhancing the carotenoid content and the expression of carotenoid metabolism-related genes in Satsuma mandarins. They also reported that irradiation with blue light at a photon flux of 50 µmol•m⁻²•s⁻¹ enhanced the expression of these carotenogenic genes at 3 days of irradiation, but the expression of these genes was not affected at 6 days. Our results suggest that treatment with low-intensity red LED irradiation is sufficient to develop the degree of rind color of mandarin fruits. This method of irradiation would also enhance the content of carotenoids as in previous studies (Ma et al., 2013; Ma et al., 2015); thus, further investigation of carotenoid content is required.

Fig. 4.

The effect of LED irradiation on the degree of rind color in C. unshiu Marc. ‘Aoshima unshu’ harvested in two harvest periods. Vertical bars indicate standard error (SE) (n = 4). ^z **, * : significant difference at P < 0.01, 0.05 by t-test, respectively.

The influence of red LED irradiation on the internal fruit quality of Satsuma mandarins at early and commercial harvests is shown in Tables 1 and 2, respectively. In the present study, irradiation with red LED at a photon flux of 12 µmol•m⁻²•s⁻¹ did not influence the fruit quality characteristics (SSC, TA, SSC/TA, and moisture loss). We observed that the content of β-cryptoxanthin in the flavedo was increased by red LED irradiation, but the content in the pulp was not increased (data not shown). On the other hand, Kaewsuksaeng et al. (2011) reported that UV-B treatment induces a gradual increase in citric acid formation and suppresses the increase of sugar content in limes (Citrus latifolia Tan.) during storage. Our data indicate that the influence of red LED irradiation on citric acid and sugar contents appears to be small, unlike the influence of UV-B irradiation.

Table 1. Influence of red LED irradiation on fruit quality in Satsuma mandarin (C. unshiu Marc.‘Aoshima unshu’) at early harvest.

Treatment	Average fruit weight (g)	SSC (°Brix)	TA (g·100 mL−1 as citric acid)	SSC/TA	Moisture loss (%)
LED	109 ± 3	9.7 ± 0.1	0.86 ± 0.02	11.3 ± 0.4	3.4 ± 0.1
Dark	113 ± 6	9.8 ± 0.1	0.88 ± 0.02	11.2 ± 0.4	2.8 ± 0.3
Significancez	−	n.s.	n.s.	n.s.	n.s.

Data are expressed as the means ± SE (n = 4).

^z  n.s.: no significant difference by t-test. -: not analyzed.

Table 2. Influence of red LED irradiation on fruit quality in Satsuma mandarin (C. unshiu Marc.‘Aoshima unshu’) at commercial harvest.

Treatment	Average fruit weight (g)	SSC (°Brix)	TA (g·100 mL−1 as citric acid)	SSC/TA	Moisture loss (%)
LED	135 ± 5	10.1 ± 0.4	0.71 ± 0.03	14.4 ± 0.9	2.5 ± 0.1
Dark	124 ± 4	9.6 ± 0.3	0.72 ± 0.02	13.3 ± 0.7	1.8 ± 0.3
Significancez	−	n.s.	n.s.	n.s.	n.s.

Data are expressed as the means ± SE (n = 4).

^z  n.s.: no significant difference by t-test. -: not analyzed.

The effect of intermittent red LED irradiation on the change in the a* value in Satsuma mandarins is presented in Fig. 5. The fruits, which were irradiated by intermittent red LED for 8 days had significantly increased a* values relative to the dark treatment fruits, as did those with continuous red LED irradiation. On the other hand, there were no significant changes in L* and b* with intermittent red LED irradiation (data not shown). The circadian clock plays various roles in the regulation of plant responses to environmental stress and plant growth (Blasing et al., 2005; Liao and Burns, 2010). Simkin et al. (2004) showed that the circadian rhythm controls the carotenoid cleavage dioxygenase in petunia flowers and leaves. The circadian rhythm may affect the expression of carotenoid metabolism-related genes in the flavedo. From these results, it might not be necessary to irradiate continuously with a red LED, provided there is intermittent red LED irradiation (12 h LED/12 h dark).

Fig. 5.

The effect of intermittent red LED irradiation on the change in a* value in C. unshiu Marc. ‘Takabayashi wase’. Vertical bars indicate standard error (SE) (n = 3). Different letters indicate significant differences between the treatments; P < 0.05 by Tukey's multiple range test.

To the best of our knowledge, this is the first report showing that low-intensity red LED irradiation with a photon flux of 12 µmol•m⁻²•s⁻¹ develops the rind coloration of C. unshiu Marc. ‘Aoshima unshu’ without affecting the internal fruit quality. However, the study did not allow us to evaluate the detailed mechanism, such as the biosynthetic pathway, of carotenoid accumulation due to low-intensity or intermittent red LED irradiation in citrus fruit. Further studies are required to evaluate the relationship between the photon flux of red light and carotenoid synthesis during the ripening process using biochemical and molecular approaches. This technique of rind color control by visible light irradiation could then be combined with other methods, such as temperature and ethylene. Ma et al. (2015) reported that a combination of ethylene treatment and red LED irradiation increased the expression of carotenoid metabolism-related genes and carotenoid accumulation in the flavedo of C. unshiu to a greater extent than ethylene treatment alone. Further research is required to develop alternative methods of postharvest management to enhance market value. To enable the practical application of combined red LED and other techniques in storage, export, and sale environments, it is necessary to develop a method of uniform LED light irradiation.

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