Variation in Glucosinolates and Anthocyanins of Radish (Raphanus sativus L.) with Cultivar and Growth Period = 무(Raphanus sativus L.)의 품종 및 생육시기 별 글루코시놀레이트 및 안토시아닌류 특성 변화

다국어 초록 (Multilingual Abstract)

Kimchi produced with radishes cultivated in late spring has been reported to exhibit a stronger pungency compared to that prepared using radishes grown in autumn, which has been identified as a major challenge in the kimchi manufacturing industry. The biochemical mechanisms responsible for seasonal variations in radish pungency have not been thoroughly investigated. In this study, three radish (Raphanus sativus L.) cultivars, characterized by different pungency levels, were cultivated during spring and autumn. The contents of favor (pungency and sweetness)-related metabolites were analyzed to determine the factors responsible for the seasonal dependency of favor. The concentrations of glucoraphasatin-a key pungency-related metabolite-and various polar metabolites were analyzed. Although previous studies have demonstrated a correlation between glucoraphasatin content and radish pungency, this study suggests that the concentrations of glucoraphasatin and its hydrolysis product (raphasatin) do not fully account for the seasonally mediated variation in pungency among the cultivars. The seasonal variation in radish pungency may be attributed to the ratio of raphasatin content to the total sweetness of sugars [Raphasatin/ (fructose content x 2.0) + (glucose content x 0.6) + (sucrose content x 1.0)]. The levels of raphasatin and soluble sugars in white radish roots were influenced by differences in temperature and humidity between spring and autumn. The ratio of raphasatin content to total sweetness of sugars was found to vary by cultivar and season, suggesting its potential use as an indicator compound for evaluating pungency in radish. Furthermore, seasonal variation in metabolite profiles was analyzed to identify seasonal biomarkers and to understand insights into the physiological and biochemical changes associated with seasonal conditions. 1. Introduction Radish (Raphanus sativus L., 2n = 18), a key root crop in the Brassicaceae family, has long been cultivated across various regions including China, India, Japan, Korea, Europe, and the Americas (Carlson et al., 1985). In Korea, diverse F1 hybrid varieties have been bred to align with different seasonal cultivation systems (spring, summer, autumn, and winter) and consumer preference (big root, small root; Altari, Yeolmu, and processing for pickling) (Lee and Park, 2017). Due to the seasonal and cultivar-based variation, achieving standardization in radish is challenging. Therefore, the taste of radish-based kimchi varies depending on the properties of the radishes used (Ki-Don, 2000). Although radish exhibits substantial morphological variation, its glucosinolate profile is relatively simple and dominated by a few major compounds (Nishio and Kitashiba, 2017). Kimchi, a traditional Korean fermented dish with a historical background of over a millennium (Lee, 1991), continues to be a staple food, with approximately 467,000 tons produced in South Korea in 2018 (Han, 2020). Kimchi is primarily made by fermenting ingredients such as radish or kimchi cabbage, a process facilitated by lactic acid bacteria (Jang et al., 2015). Glucosinolates (sulfur-containing secondary metabolites) are widely present across species within the Brassica genus (Sadowski and Kole, 2016). These compounds are characterized into three major types based on the amino acid from which they originate: aliphatic glucosinolates (from methionine), aromatic glucosinolates (from phenylalanine or tryptophan), and indolic glucosinolates (from tryptophan) (Giamoustaris and Mithen, 1996; Nishio and Kitashiba, 2017). Among them, aliphatic glucosinolates are the most prevalent in Brassica crops. Their accumulation is influenced by various factors, including environmental conditions, abiotic stresses, and genetic background (Nishio and Kitashiba, 2017). The dominant glucosinolate is 4-methylthio-3-butenyl glucosinolate (glucoraphasatin) in radish roots, also referred to as dehydroerucin, which belongs to the aliphatic group and is derived from methionine. Glucoraphasatin accounts for over 90% of the total glucosinolate content in Japanese and Chinese radish varieties (Ishida et al., 2012). The concentration of glucoraphasatin in radish roots has been reported to vary depending on the cultivar (Yi et al., 2016). Raphasatin (4-methylthio-3-butenyl isothiocyanate) was identified as the pungency compound derived from radish roots (Kim and Rhee, 1986). Radish is known to have very low levels of epithiospecifier protein (ESP) activity, which allows for highly efficient conversion of glucoraphasatin into raphasatin by endogenous myrosinase (Ku et al., 2015; Nakamura et al., 2008; Wang et al., 2017). As a result, the pungency of radish is largely determined by its glucoraphasatin content. In addition, variations in raphasatin concentration may arise due to differences in myrosinase activity (Liu et al., 2021). The activity of myrosinase in radish can be influenced by environmental and seasonal factors, which may affect the conversion of glucosinolates into bioactive compounds (Charron and Sams, 2004; Charron et al., 2005; Rosa and Rodrigues, 1998; Wei et al., 2011). Therefore, accurate quantification of raphasatin is essential for understanding the variation in radish pungency. Glucosinolates contribute to plant defense mechanisms by deterring a range of biological threats, including fungi, nematodes, herbivores, and competing weeds (Rosa and Rodrigues, 2001). The enzymatic hydrolysis of glucosinolates into isothiocyanates upon tissue disruption contributes to the pungency taste, bitterness, and sulfurous aroma typical of Brassica species. (Padilla et al., 2007; Sadowski and Kole, 2016). These findings indicate that raphasatin levels in radish can be influenced by biotic and abiotic factors. Seasonal changes in glucosinolate accumulation have been documented across various Brassica crops, including radish, turnip, cabbage, and oilseed rape (Cartea et al., 2008; del Carmen Martínez-Ballesta et al., 2013; Sarwar & Kirkegaard, 1998; Schreiner et al., 2002; Zhang et al., 2008). A positive correlation between soil temperature and glucosinolate concentration has been observed in Brassica oleracea (Charron and Sams, 2004). In addition, drought-induced water stress has been shown to enhance glucosinolate levels in several Brassica species (Sánchez-Pujante et al., 2017). Sugars contribute not only to the characteristic sweetness of radish but also provide critical sources of carbon and energy and actively regulate plant growth and metabolic pathways by functioning as signaling molecules in the plant's life cycle (Merillon and Ramawat, 2017). In radish, high temperatures have been shown to accelerate the breakdown of sucrose into glucose and fructose (Hayata, 1986). However, optimal growth conditions (22/18℃) significantly enhance sugar accumulation in the hypocotyl, coinciding with rapid root thickening (Hayata, 1986). The perception of sweetness in radish is closely linked to the concentrations of glucose and fructose (Beck et al., 2014). Altari radish kimchi, known as Chonggak kimchi, is popular in Korea. Raphasatin-induced pungency is commonly identified as a key factor contributing to negative consumer perceptions of Altari radish kimchi. In contrast, in Japan, the strong pungency of raw and processed radish products-such as grated Oroshi-has been cited as a factor contributing to lower consumer preference (Nakamura et al., 2008). While fermentation under acidic conditions reduces radish pungency by degrading myrosinase and glucosinolates (Kim and Rhee, 1993), kimchi prepared from radishes grown in spring or summer-particularly those harvested in late spring-tends to exhibit a stronger pungency compared to kimchi made from autumn-harvested radishes. The glucoraphasatin content in radish roots have been reported to be influenced by genotype (Charron and Sams, 2004). However, limited research has investigated how seasonal variation affects raphasatin-the key compound responsible for radish pungency-as well as other taste-related metabolites. Thus, the present study analyzed glucosinolates, their hydrolysis products, and additional flavor-associated compounds in three radish cultivars cultivated during two distinct growing seasons (spring and autumn) in Korea (Coogan et al., 2001). In the current study, we employed a metabolomics-based approach to identify candidate metabolites that could explain the differences in pungency among the radish cultivars.

국문 초록 (Abstract)

무 (Raphanus sativus L.)는 유럽 및 아시아 지역에서 널리 소비되는 초본 식물로서, 다양한 기능성 성분을 함유하고 있어 식품 소재 및 건강기능식품으로의 활용 가능성이 높다. 최근 소비자들의 건강 기능성 식품에 대한 관심이 증가함에 따라, 무에 포함된 기능성 성분인 글루코시놀레이트와 안토시아닌에 대한 관심이 증가하고 있다. 그러나 백색무와 자색무에 함유된 기능성 물질은 품종, 계절, 생육 환경. 가공 및 저장 조건 등 다양한 요인에 의한 함량 및 조성 변화에 대한 체계적인 연구가 미흡한 실정이다. 따라서, 본 연구는 백색무와 자색무의 주요 기능성 성분인 글루코시놀레이트와 안토시아닌을 중심으로, 환경 변화가 대사체 함량 및 조성에 미치는 영향을 다양한 크로마토그래프 기법을 이용하여 분석하고자 하였다. 또한, 자색무 품종을 중심으로 품종, 생육 시기, 가공 및 저장에 따른 품질 변화 및 기능성 성분의 변화를 파악하고, 자색무에 적합한 재배, 가공, 저장 방법 확립에 이바지하고자 하였다. 백색무 3품종 (‘대양1호’, ‘452949’, ‘250704’)을 대상으로 봄과 가을의 재배 환경 차이가 글루코시놀레이트, 그 가수 분해 산물인 라파사틴 및 유리당 함량에 미치는 영향을 분석하였다. 글루코라파사틴과 라파사틴의 함량은 계절에 따른 뚜렷한 차이를 보였으며, 특히 봄철의 상대적으로 높은 온도 조건에 의해 myrosinase 효소 활성이 활성화되어 라파사틴의 축적이 촉진되었다. 또한, 라파사틴과 단맛 비율은 무의 아린맛 정도와 밀접한 상관관계를 나타냈으며, 이는 글루코시놀레이트 대사 및 당 축적이 온도 조건에 민감하게 조절됨을 시사한다. 이러한 결과는 라파사틴/단맛비율이 무의 관능적품질을 예측할 수 있는 실용적 지표로 활용 가능성을 시사하였다. 자색무 3 품종(정운무, 스위트베이비, 보라킹)을 대상으로 한 LC-MS 기반 대사체 분석 결과, 안토시아닌을 포함한 페놀성 화합물, 플라보노이드, 글루코시놀레이트 등의 기능성 성분의 함량 및 조성이 품종 및 부위(뿌리·잎)에 따라 유의한 차이를 보였다. 특히 보라킹은 다른 품종의 자색무에 비해 다수 및 다량의 cyanidin glycoside가 검출되었으며, 두 개 이상의 페놀산이 결합되어 있는 구조적 특징이 관찰되었다. 이를 통해 국내에서 재배되고 있는 자색무 품종의 성분학적 차이에 대한 이해도를 높힐 수 있었으며, 국내 자색무 품종에 적합한 재배 방법 확립 및 고도화에 기초 자료로 활용 가능성을 시사하였다. 자색무 ‘보라킹’을 대상으로 생육기간이 지남에 따라 LC-MS 대사체 분석 결과, 자색무에 함유된 글루코라파사틴 함량이 유의하게 감소하였으며, 생육 80일에 cyanidin glycoside 함량이 급격하게 증가하였다. 또한, 생육 40-70일 자색무에는 caffeic acid와 coumaric acid가 결합되어 있는 cyanidin glycoside가 주로 검출되었지만, 생육 80일에는 coumaric acid와 ferulic acid가 결합되어 있는 개별 안토시아닌류의 함량이 급격하게 증가하였다. 이러한 생육 시기에 따른 cyanidin glycoside의 구조적 변화는 생육 후기의 노화 및 환경 등에 스트레스에 의한 cyanidin glycoside의 구조적 안정성을 높이기 위한 변화로 판단된다. 자색무 무청의 경우 생육 40일에 cyanidin glycoside와 일부 kaempferol glycoside의 함량이 다량 함유되어 있으나, 생육 후기로 갈수록 유의하게 감소하였다. 이를 통해 자색무 ‘보라킹’을 대상으로 하여 생육 단계 구별 및 수확 시기 선정 등의 재배 기술 고도화에 기초 자료로 활용하고자 하였다. 저장 기간에 따른 초절임 자색무의 품질 분석 결과, 초절임 자색무의 적색도가 저장 기간이 지만에 따라 유의하게 감소하였다. 이러한 초절임 자색무의 색도 변화는 안토시아닌 함량과 유의한 상관관계를 나타내는 것으로 확인되었다. LC-MS 대사체 분석 결과, 5종의 cyanidin glycoside가 검출되었으며, cyanidin-3-O-feruloylsophoroside-5-O-malonylglucoside와 cyanidin-3-O-diferuloylsophoroside-5-O-malonylglucoside가 주된 안토시아닌임을 확인되었다. 이를 통해 초절임 자색무의 품질 지표 성분으로써 cyanidin glycoside류의 활용 가능성을 시사하였으며, 자색무에 적합한 저장 및 가공 방법 확립에 기초 자료로 활용하고자 하였다. 본 연구는 무의 기능성 성분에 영향을 미치는 유전적, 생리적, 환경적 요인을 규명함으로써, 기능성 성분 및 품질 유지에 기여할 수 있는 기초 자료를 제공하고자 하였다. 특히, 글루코시놀레이트 및 안토시아닌의 함량 및 조성 변화에 대한 통합적 분석을 통해, 무의 품질과 기능성 특성을 이해하기 위한 성분학적 접근법을 제시하였다. 이를 통해 자색무를 포함한 다양한 무 품종의 고기능성 식품 소재 개발뿐만 아니라, 저장 및 가공 방법 확립 및 고도화를 위한 기초 자료로 활용될 것으로 기대된다.

목차 (Table of Contents)

• ABSTRACT 1
• Chapter 1. General introduction 4
• 1. Radish (Raphanus sativus L.) 4
• 2. Glucosinolate 5
• 3. Anthocyanin 6
• 4. Study objectives 7
• 5. Reference 10
• Chapter 2. Seasonal Effects of Glucosinolate and Sugar Content Determine the Pungency Radishes (Raphanus sativus L.) 16
• Abstract 16
• 1. Introduction 17
• 2. Materials and Methods 20
• 2.1. Cultivation and Growing Conditions 20
• 2.2. Quantification of Glucosinolate 21
• 2.3. Glucosinolate Hydrolysis Products Measurement 22
• 2.4. Water-Soluble Primary Metabolite and Sugar Contents 23
• 2.5. Raphasatin Content/Total Sweetness Value from Sugars Ratio 23
• 2.6. Statistical Analysis 24
• 3. Result and Discussion 25
• 3.1. Growing Environmental Factors 25
• 3.2. Quantification of Glucosinolate 25
• 3.3. Glucosinolate Hydrolysis Products 27
• 3.4. Water-Soluble Primary Metabolite Changes with Season 28
• 3.5. Sweetness Value Based on Major Sugar Contents 29
• 3.6. Ratio between Raphasatin and Total Sweetness Values from Sugars 31
• 4. Conclusion 32
• 5. Reference 43
• Chapter 3. Comparison of metabolites and antioxidative activity in leaves and roots of purple radish cultivars 51
• Abstract 51
• 1. Introduction 52
• 2. Materials and methods 54
• 2.1. Sample preparation 54
• 2.2. Total phenolic content and total flavonoid content 54
• 2.3. Total anthocyanin content 55
• 2.4. Determination of glucosinolate content 55
• 2.5. Non-Volatile Metabolite Extraction and LC-MS Analysis 56
• 2.6. Measurement of ABTS Radical Scavenging Activity and Ferric Reducing Antioxidant Power (FRAP) 57
• 2.7. Statistical analysis 58
• 3. Result and Discussion 58
• 3.1. Total Phenolic and Flavonoid Contents 58
• 3.2. Total Anthocyanin Contents 60
• 3.3. Glucosinolate content 61
• 3.4. Characterization of Non-Volatile Metabolites in Radish using LC-MS 63
• 3.4.1. Purple Radish Leaf 63
• 3.4.2. Purple Radish Root 64
• 3.5. ABTS+ Radical Scavenging Activity and Ferric Reducing Antioxidant Power (FRAP) 66
• 4. Conclusion 68
• 5. Reference. 78
• Chapter 4. Changes on Non-Volatile Metabolites of Purple Radish Root and Leaf during the Cultivated Periods by LC-ESI-QToF-MS Analysis 85
• Abstract 85
• 1. Introduction 86
• 2. Materials and Methods 88
• 2.1. Sample preparation 88
• 2.2. Growing Degree Day 88
• 2.3. Purple radish growth parameter 89
• 2.4. Determination of the total flavonoid content (TFC) 89
• 2.5. Determination of the total anthocyanin content (TAC) 90
• 2.6. Quantification of Glucosinolate 90
• 2.7. Analysis of non-volatile metabolites by liquid chromatography quadrupole time-of- flight mass spectrometry (LC-ESI-QToF-MS) 91
• 2.8. Statistical analysis 92
• 3. Result and Discussion 93
• 3.1. Purple radish environmental condition, growth parameters (width, length, and weight), and physicochemical properties (color, and hardness) during the cultivated period. 93
• 3.2. Total flavonoid content and total anthocyanin content in purple radish during the cultivated period 95
• 3.3. Glucosinolate contents in purple radish during the cultivated period 97
• 3.4. Metabolite of purple radish during the cultivated period 98
• 3.4.1. Purple radish root 98
• 3.4.2. Purple radish leaf 102
• 4. Conclusion 104
• 5. Reference. 123
• Chapter 5. Changes in Quality and Metabolites of Pickled Purple Radish During Storage 134
• Abstract 134
• 1. Introduction 135
• 2. Materials and methods 137
• 2.1. Sample preparation 137
• 2.2. Determination of the Hunter color, hardness, and pH 138
• 2.3. Determination of total phenolic (TPC) and total flavonoid (TFC)contents 138
• 2.4. Determination of total anthocyanin content (TAC) 139
• 2.5. Determination of free sugar content using gas chromatography-mass spectrometry (GC-MS). 139
• 2.6. Analysis of non-volatile metabolites by liquid chromatography quadrupole time- of-flight mass spectrometry (LC-ESI-QToF-MS) 140
• 2.7. Determination of the ABTS+ radical-scavenging activity and ferric reducing antioxidant power (FRAP) 141
• 2.8. Statistical analysis 142
• 3. Result and Discussion 143
• 3.1. Changes in the physicochemical properties (color, hardness, and pH) of pickled purple radish during storage period 143
• 3.2. Changes in the TPC, TFC, and TAC of pickled purple radish during storage 145
• 3.3. Change in the free sugar (fructose, glucose, and sucrose) content of pickled purple radish during storage 147
• 3.4. Metabolites of pickled purple radish during storage 148
• 3.5. Changes in the ABTS+ radical-scavenging activity and FRAP of pickled purple adish during storage 151
• 4. Conclusion 153
• 5. Reference 163
• Overall conclusion 175
• 국문 초록 179