Prediction and suppression of internal blue discoloration in roots of daikon, the Japanese radish (Raphanus sativus L.)

Abstract The internal blue discoloration of edible daikon roots often occurs on day 3 after harvest during storage at 20°C and is a serious problem. This study reports a rapid and simple method for predicting discoloration at harvest and proposes two methods for suppressing the discoloration of roots that are at discoloration risk. The soaking of freshly harvested roots in aqueous hydrogen peroxide resulted in immediate blue discoloration. The correlation between discoloration after storage at 20°C and discoloration after soaking in hydrogen peroxide was positive. Discoloration using hydrogen peroxide at harvest is a useful way of predicting discoloration risk. The storage of roots at 10°C in air or at 20°C in an atmosphere containing 1% (v/v) molecular oxygen resulted in no discoloration for at least 8 days. These storage conditions can guarantee no discoloration for the distribution after harvest.


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TERANISHI ANd NAGATA therefore a result of oxidative stress within daikon roots, leading to the oxidation of 4-hydroxyglucobrassicin and the production of blue-colored molecules (Teranishi, Nagata, & Masuda, 2016).
4-Hydroxyglucobrassicin also works as an antioxidative compound, akin to ascorbic acid, and the blue discoloration is a realtime indicator of extreme oxidative stress in the roots. Although 4-hydroxyglucobrassicin, which was first discovered in cabbage seed and rapeseed in 1982 (Truscott, Burke, & Minchinton, 1982), is generally contained in the seeds, sprouts, and roots of Brassica plants (Agerbirk & Olsen, 2012), this was the first observation that 4-hydroxyglucobrassicin generates blue-colored compounds in plants. 4-Hydroxyglucobrassicin has been known to be unstable and is converted to a blue-colored compound(s) (Sang & Truscott, 1984).
The aim of the current study was to develop simple and rapid methods for predicting internal blue discoloration in daikon roots at the time of harvest and methods to suppress discoloration during storage based on the molecular mechanism underlying the blue discoloration. If the prediction of blue discoloration indicates a high discoloration risk, the daikon lots need to be stored under suitable conditions to avoid the discoloration. The likelihood of the blue discoloration depends on the cultivar of daikon and the conditions of cultivation (Ikeshita et al., 2010). Thus, a method to predict the risk of discoloration for each lot and discoloration-suppressing storage conditions for each lot is required.

| Plant materials and cultivation
A total of 29 cultivars of daikon were used in the current study.

| Chemicals
Chemicals were purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan. The concentration of hydrogen peroxide was determined using the standard titration with KI and Na 2 S 2 O 3 .

| Storage experiments
Freshly harvested roots of 29 cultivars of daikon (n = 5) were washed with water and stored at 20°C and approximately 80% relative humidity for 4 days in open air and in the dark using the MLR-350HT growth cabinet (Sanyo Electric Co., Ltd., Osaka, Japan). The roots were vertically cut along the middle, and the degree of discoloration of the root sections was visually assessed for the intensity, but not the area, of blue discoloration and was expressed as four levels from 0 to 3 in order of increasing discoloration as shown in Figure 3. The results are expressed as the mean of the samples from five roots.
For the temperature storage study, freshly harvested roots of Hukuhomare daikon cultivated in 2017 were washed with water and stored at 10, 12.5, 15, 17.5, or 20°C at approximately 80% relative humidity in the dark in MLR-350HT growth cabinet, and the roots were vertically cut along the middle on day 2, 3, 4, 6, or 8. The degree of discoloration was visually assessed in the manner described above.
Alternatively, roots were stored at 20°C in soil containing 12% (w/w) water. The roots were vertically cut along the middle on day 2, 3, 4, 6, or 8, and the degree of discoloration was visually assessed in the manner described above.

| Blue discoloration using hydrogen peroxide
Freshly harvested roots of 29 cultivars of daikon (n = 5) were washed with water, vertically cut along the middle, and soaked in 1% (w/w) aqueous hydrogen peroxide solution at 20°C. The intensity (but not the area) of the resulting color change was visually assessed after 10 min of soaking the roots in 1% aqueous hydrogen peroxide solution, and each sample was assigned a value from 0 to 4 in order of increasing discoloration, as shown in Figure 4. The results are expressed as the mean value of samples from five roots.2.4.1 | Hy droxyglucobrassicin analysis Xylem sections (5.0 g) at 50 mm from the root tip of Hukuhomare stored at 10°C in air, at 20°C in air, at 20°C in the gas mixture, O 2 :CO 2 :N 2 = 1:1:98 (v), and at 20°C in soil containing 12% (w/w) water were immediately immersed in methanol (5 ml) and frozen at −80°C, and the mixtures were homogenized in an ice bath. The homogenates were filtered through absorbent cotton. The resulting filtrates were centrifuged at 10,000 g at 0°C for 10 min, and the supernatants were filtered through 0.45μm membrane filters (Millex-HP; Merck Millipore Ltd., Carrigtwohill, Co. Cork, Ireland). The resultant filtrates were stored at −80°C until further use. 4-Hydroxyglucobrassicin in the filtrates was analyzed using a JASCO Gulliver HPLC system equipped with a photodiode array (PDA) detector MD-910 (JASCO Corp., Tokyo, Japan), and a Cosmosil 5C18-PAQ column  . The F I G U R E 3 Index for the three levels 0-3 of discoloration after storage of freshly harvested daikon roots at 20°C and approximately 80% relative humidity for 4 days in the dark. The blue discoloration eventually changes to brown (level 3) F I G U R E 4 Index for the levels 0-4 of discoloration after treatment of freshly harvested daikon roots with 1% aqueous hydrogen peroxide at 20°C for 10 min content in the sections is reported as the mean ± SD of samples from five roots and is expressed as nanomoles per gram of wet weight.

| RE SULTS AND D ISCUSS I ON
The Hukuhomare daikon cultivar has been known to suffer from internal blue discoloration in Japan. We investigated the degree of the discoloration in 29 daikon cultivar roots, including Hukuhomare, on the 4th day of storage at 20°C. The resulting blue color intensities were scored on a visual analogue scale as shown in Figure 3, and the scores are reported in Table 1. Hukuhomare, RA-305, NR-515, MRX-120, and Aozumari-2 scored the highest for internal blue discoloration. The degree of discoloration of both Hukuhomare daikon cultivated at the Kanagawa Agricultural Center and at the Ishikawa Sand Dune Agricultural Research Center was the same. Taibyousoubutori, Yuuto, Gensuke, Hakusyu, and Huyunourasoubutori daikon produced no discoloration. The internal blue discoloration has been known to turn brown with time in the same region of the root . We present an example to brown discoloration in Figure 3, although no cultivars developed brown discoloration after 4 days in this study.
Recently, we discovered that soaking fresh Hukuhomare daikon root sections in aqueous hydrogen peroxide solution induces artificial blue discoloration ). In the current study, the artificial blue discoloration of Hukuhomare daikon root Soaking in a 1% solution produced a high degree discoloration that can be readily distinguished by visual assessment, whereas at 0.01%, the color change is too low for reliable detection (Supporting information Figure S1). Thus, in this study, soaking in 1% aqueous hydrogen peroxide solution at 20°C for 10 min was employed for the visual assessment. Samples of the same 29 fresh cultivars of daikon were treated with 1% hydrogen peroxide for comparison with the internal blue discoloration produced after storage at 20°C for 4 days. The visual evaluation of blue discoloration on the 29 cultivars, using the color index in Figure 4, is summarized in Table 1. The Hukuhomare cultivar produced the highest degree of blue discoloration, and Taibyousoubutori produced the lowest. Yuuto, Gensuke, Hakusyu, and Huyunourasoubutori daikon, which produced no internal blue discoloration during storage at 20°C for 4 days, generated only slight blue discoloration after treatment with 1% hydrogen peroxide.
The relationship between discoloration by treatment with 1% aqueous hydrogen peroxide and in storage at 20°C for 4 days is shown in Figure 5. There was a positive correlation between the two datasets (R 2 = 0.79), indicating that discoloration with hydrogen peroxide facilitates a visual prediction of internal blue discoloration. If the degree of blue discoloration after hydrogen peroxide treatment of freshly harvested roots was above 2, then internal blue discoloration was likely to occur upon storage at 20°C, and a farmer would be advised to store the roots under conditions that suppress the discoloration. This prediction method using 1% aqueous hydrogen peroxide would be convenient for farmers to use for visual assessment because of the low concentration of hydrogen peroxide required, the minimal costs involved, and the simple and rapid operation of the method that requires no technical expertise or analytical equipment.
In Japan, edible daikon roots are sold at supermarkets within a week of harvest. Thus, when the prediction test for internal blue discoloration using hydrogen peroxide by sampling inspection at the time of harvest indicates a high risk of discolouration, suitable measures must be taken to prevent discolouration in those daikon lots for at least 1 week.
The internal blue discolouration occurs by chemical oxidation of 4-hydroxyglucobrassicin under the oxidative stress in daikon roots . We speculated that oxidative stress could be suppressed by lowering the storage temperature. The cost of temperature control, however, should be as low as possible to maintain economic viability. We therefore sought to determine the highest storage temperature that would permit storage of roots for 1 week with no discolouration. For this purpose, we used Hukuhomare daikon, which produced the highest degree of internal blue discolouration during storage at 20°C. We stored roots for up to 8 days at five different temperatures and determined internal discolouration levels at different times ( Figure 6). No discolouration occurred after 8 days of storage at 10°C, whereas slight internal blue discolouration occurred on day 8 at 12.5°C. Thus, storage at 10°C is effective in suppressing the discolouration for a week while minimizing cost.
F I G U R E 5 Relationship between levels of blue discoloration after treatment with 1% hydrogen peroxide and of internal discoloration after storage at 20°C for 4 days. Data presented are mean values ± SD of samples from five roots F I G U R E 6 Effects of temperature and molecular oxygen concentration at the time of storage on internal discoloration. Data presented are mean values ± SD of samples from five roots. When stored at 10°C in air or at 20°C in the mixed gas O 2 :CO 2 :N 2 = 1:1:98 (v), no internal blue discoloration occurred for 8 days The 4-hydroxyglucobrassicin content, which was analyzed using an HPLC-PDA-MS system (Supporting information Figure S2), in the roots stored at 10°C did not change significantly (Figure 7a), and the 4-hydroxyglucobrassicin content at 8 days of 10°C storage was similar to that at 2 days at 20°C, which was before the blue discoloration occurred (Figure 7b). When 4-hydroxyglucobrassicin contents were similar, the blue discoloration from 4-hydroxyglucobarssicin could be used as a real-time indicator of extreme oxidative stress.
Therefore, these results indicate that the suppression of blue discoloration at 10°C was not due to the 4-hydroxyglucobrassicin content but because of the suppression of oxidative stress.
The effect of molecular oxygen concentration on the onset of blue discoloration was also investigated ( These results indicate that it is only when daikon roots are directly exposed to air (i.e., at harvest) that oxidative stress begins and the development of blue discoloration ultimately results.

| CON CLUS IONS
The internal blue discoloration of daikon roots during storage at approximately 20°C after harvest has recently been recognized as a serious problem for farmers. Therefore, a method for predicting F I G U R E 7 Contents of 4-hydroxyglucobrassicin in xylem sections obtained at 50 mm from the root tip of Hukuhomare daikon stored at (a) 10°C in air, at 20°C in a mixed gas of O 2 :CO 2 :N 2 = 1:1:98 (v), at 20°C in soil, or at (b) 20°C in air. Data are presented as the mean values ± SD of samples from five roots. When stored at 20°C in air, internal blue discoloration occurred on day 3, and the 4-hydroxyglucobrassicin content was not measured after that discoloration at the time of harvest and suitable storage measures for daikon lots at high discoloration risk is both needed. We found that artificial blue discoloration induced by soaking daikon sections in aqueous hydrogen peroxide solution can predict the risk of internal blue discoloration during storage at 20°C. This simple visual assessment method would be practical and useful, particularly when used in conjunction with the storage methods we developed. The storage of high-risk daikon lots at 10°C produced no discoloration after 8 days, and lowering the concentration of molecular oxygen to 1% (v/v) also prevented discoloration onset for 8 days at 20°C.
4-Hydroxyglucobrassicin content did not change significantly for 8 days under these storage conditions, indicating that oxidative stress was suppressed by these conditions. These storage methods can help prevent discoloration for a week after harvest.

ACK N OWLED G EM ENTS
We thank Daisuke Masuda and Yoshihiko Masuda for cultivat-

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.