Changes in Biochemical and Volatile Flavor Compounds of Shine Muscat at Different Ripening Stages
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applied sciences Article Changes in Biochemical and Volatile Flavor Compounds of Shine Muscat at Different Ripening Stages Kyeong-Ok Choi , Dong Hoon Lee, Seo Jun Park, Dongjun Im, Youn Young Hur and Su Jin Kim * Fruit Research Division, National Institute of Horticultural and Herbal Science, Wanju 55365, Korea; [email protected] (K.-O.C.); [email protected] (D.H.L.); [email protected] (S.J.P.); [email protected] (D.I.); [email protected] (Y.Y.H.) * Correspondence: [email protected]; Tel./Fax: +82-63-238-6750 Received: 27 July 2020; Accepted: 13 August 2020; Published: 14 August 2020 Abstract: Changes in the biochemistry and flavor of Shine Muscat grapes at different ripening stages (RS) were analyzed to identify factors affecting these characteristics. The yellowness index values were 45.1, 49.4, and 50.2 in the ripening stage 1 (RS1), ripening stage 2 (RS2), and ripening stage 3 (RS3) groups, respectively, representing the different ripening stages. The yellowness of the grape berries tended to increase with ripening due to the gradual breakdown of chlorophylls and the evolution of carotenoids. The total content of monoterpenes, on the other hand, was approximately two-fold higher at RS3 than RS1 and RS2. Moreover, linalool was the most abundant compound contributing to the total content of monoterpenes. The highest correlation was observed between the linalool content and ◦Brix/acid ratio (r = 0.9981), followed by the monoterpene content and ◦Brix/acid ratio (r = 0.9933). These findings indicate that changes in the contents of linalool and its oxidized forms may be used as a quality index and an indicator of the timing of harvest for Shine Muscat grapes. Keywords: linalool; ripening; Shine Muscat; volatile flavor compounds; ◦Brix/acid ratio 1. Introduction The composition and distribution of volatile flavor compounds are important characteristics that contribute to the quality of grape berries and wine. The flavors of grape berries and wine can be characterized by complex families of volatile compounds, including terpenes, esters, C6-aldehydes and alcohols, and methoxypyrazines, which contribute to fruity, floral, green leafy, and green capsicum flavors, respectively [1]. The development of flavor compounds in grape berries is likely to be affected by various factors, including grape cultivars, growing conditions, and climate [2]. The flavor intensity and aromatic profiles of grape berries are primarily affected by the concentrations and odor thresholds of volatile flavor compounds and their interactions with other compounds [1]. It has been reported that grape berries develop in two successive growth stages: berry formation and ripening [3]. The first stage is characterized by the formation of berry and seed embryos, followed by rapid cell division. During the second stages, berries become softer and start to change color. Furthermore, various secondary metabolites, including volatile aroma compounds, are developed in the berries during this stage [4]. Thus, the changes in biochemical and volatile flavor compounds of grape berries during the second stage are more closely related to consumer’s sensory perception than those of the first growth stage. For this reason, the degree of ripening is a crucial factor influencing the development and distribution of volatile compounds, and is closely related to consumer’s preferences for grape berries. In recent years, a new grape cultivar called Shine Muscat (Vitis labruscana Baily Vitis vinifera L.), × developed in Japan in the late 1980s and commercialized in the early 2000s [5], has been gaining Appl. Sci. 2020, 10, 5661; doi:10.3390/app10165661 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 5661 2 of 12 attention as a table grape in Korea. This cultivar has attracted consumer attention due to its unique taste, namely, a strong muscat flavor with excellent sweetness and not much sourness, despite its high price. The muscat flavor of Shine Muscat is the most important attribute in determining consumer preferences. Monoterpene compounds are associated with the distinctive taste and flavor of Muscat grape varieties [6]. In particular, terpenic alcohols, including linalool, geraniol, nerol, citronellol, and α-terpineol, are known to be the major aroma compounds in Muscat grape varieties [1], and their chemical structures are represented in Figure1. Figure 1. Chemical structures of the major aroma compounds in Muscat grape varieties. Thus, the development and alteration of flavor compounds in Muscat grape varieties during berry ripening has been widely investigated [1,7]. Wilson et al. [6] investigated the alterations in volatile monoterpene compounds of Muscat of Alexandria during berry ripening and they found that only the concentration of free linalool increased with ripening in parallel to its glycoside, and Ribereau-Gayon et al. [8] compared the concentrations of monoterpene compounds among various Muscat grape varieties, such as Muscat of Alexandria, Muscat de Frontignan, Muscat Saint-Vallier, Italia, and Muscat Hamburg, and they found that linalool and geraniol were the most important aroma compounds in Muscat grape varieties. In addition, Bordiga et al. [9] evaluated the alteration of flavor compounds in Muscat-based wines (Asti Spumante and Moscato d’Asti), and they showed the changes of linalool, β-damascenone, ethyl hexanoate, and ethyl octanoate levels in both two Muscat-based wines. However, to the best of our knowledge, the flavor compound profiles of Shine Muscat grapes during ripening have not yet been extensively investigated. Although several relevant studies have been conducted in China and Japan [10–12], no such studies have been performed in Korea. Increases in consumer demand for Shine Muscat grapes and higher prices have led many grape growers to harvest and market immature berries, thereby decreasing the quality of Shine Muscat grape and thus consumer demand. The objective of this study was to analyze changes in the biochemistry and flavor in Shine Muscat grapes at different ripening stages to identify factors that affect the flavor characteristics of this cultivar. Grape berries were classified into three groups depending on their color in order to analyze the sugar content, titratable acidity and pigments at different ripening stages. Furthermore, the volatile flavor compound profiles were evaluated by SPME/GC/MS. 2. Materials and Methods 2.1. Reagents Analytical standard grade 1-dodecanol, 1-hexanol, hexanal, benzaldehyde, geraniol, linalool, nerol, terpinen-4-ol, α-phellandrene, α-terpineol, β-myrcene, β-ocimene, 4-nonanol, and C8–C20 Appl. Sci. 2020, 10, 5661 3 of 12 alkane standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical reagent grade. 2.2. Grape Samples Shine Muscat grape cultivated in the orchards at the National Institute of Horticultural and Herbal Science (Ansung, Korea) were used to evaluate the volatile aroma compounds. Grape berries were harvested at 85, 95, and 105 days after full bloom (DAFB) on late September 2019 and classified into three groups depending on their DAFB and color: ripening stage 1 (RS1, 85 DAFB), ripening stage 2 (RS2, 95 DAFB), and ripening stage 3 (RS3, 105 DAFB) (Figure2), where the numbers indicate the degree of ripening. All grape samples were stored at 70 C prior to analysis. − ◦ Figure 2. Visual appearances of Shine Muscat berries at different ripening stages: RS1, ripening stage 1; RS2, ripening stage 2; and RS3, ripening stage 3. The increasing numbers behind the abbreviation RS indicate the degree of ripening. 2.3. Sugar Content, Acidity, Color, and Pigments The total sugar content in the extracts of the grape berries was measured using a digital refractometer (Palete Digital Refractometer PR-32 Alpha; ATAGO Co. Ltd., Tokyo, Japan). Twenty berries from each group were crushed to collect free flowing juice. After filtering the juice, the total soluble solid content was measured at the constant temperature of 2 ◦C. The total sugar content was expressed as ◦Bx. The titratable acidity was determined using an automatic titrator (TitroLine Easy; SI Analytics GmbH, Mainz, Germany). Briefly, 5 mL juice was mixed with 20 mL distilled water. Then, 0.1 M NaOH standard solution was titrated until the pH of the sample solution reached 8.2. The titratable acidity was expressed as tartaric acid equivalent and calculated using the following Equation: mL NaOH 0.1 Mw. tartaric acid Titratable acidity (%) = × × (1) S 10 × The ◦Brix/acid ratio is known to be a better attribute to describe the consumers acceptability, such as sweetness, sourness, and flavor attributes, as compared with individual ◦Brix or acidity value [13]. Thus, the ◦Brix/acid ratio was calculated by dividing the ◦Brix value of the grape juice samples by the titratable acidity (◦Brix/% acidity). For color measurement, the color values L, a, and b of twenty randomly selected berries were measured using a colorimeter (DP-400; Konica Minolta, Tokyo, Japan). Standard illuminant C was used as a reference. The hunter color indices L, a, and b were converted into their corresponding X, Y, and Y values. The yellowness index (YI) of the berries was calculated using the equation below [14]. YI = 100(1.28X 1.06Z)/Y (2) − Appl. Sci. 2020, 10, 5661 4 of 12 The concentrations of pigments, including chlorophyll a and b (Chla and Chlb, respectively) and total carotenoids (x+c) (Cx+c), were estimated by spectrophotometry [15]. For the extraction of pigments, 0.15 g of grape skin was added to 1.5 mL of 0.1% CaCO3 in acetone solution, vortexed, and centrifuged at 4500 rpm for 5 min. The supernatant was isolated from the sediment. Residual pigments were further extracted using fresh extraction solution. The resulting supernatants were merged and filtered using a 0.2 µm membrane filter. The absorbances of the extracted pigment samples were recorded at 661.6 nm, 644.8 nm, and 470 nm on a microplate reader (Multiscan GO; Thermo Fisher Scientific, Waltham, MA, USA). The whole experimental process was performed under dim light to prevent pigment loss.