Influence of flavan-3-ol oxidation polymerization on the browning of table grapes during storage

Abstract:【Objective】The objective of the study is to explain the effects of oxidative polymerization of flavan-3-ol on the browning of'Ma Nai'table grapes during storage. Flavan-3-ols are an important class of polyphenols found in grapes, which are described as hydrogen-donating antioxidants. The oxidation of flavan-3-ols can generate the corresponding quinone compounds. However, the polymerization of flavan-3-ols (i.e., procyanidin) can also be obtained because the transformation is an oxidative intramolecular reaction. Grape is one of the main fruits in China which often has the problem of color deterioration, and can be partially attributed to the oxidative polymerization of flavan-3-ol. In addition, the storage of table grapes is very important for the development of the grape industry. Therefore, the relationship between flavan-3-ol oxidative polymerization and the browning of table grapes is investigated to reveal the biochemical mechanism of the color deterioration and to provide knowledge for the storage of table grapes.【Methods】The experimental materials were'Ma Nai'grapes from Zhuolu county, Hebei, which were packaged in food wraps with a humidity of 80%-90% and stored at (0.5±0.5) ℃ in a refrigerator. Sampling days were at 0 d, 40 d and 90 d after storage. On the sampling day, 3 berries from each cluster were randomly collected, then 30 berries were sampled from the collected berries for analysis. Grape skins were peeled from the berries, freeze-dried using a vacuum freeze drier and grinded inliquid nitrogen. The individual flavan-3-ols were extracted from the dry powder of the grape skins; then the extractions were analyzed using the high-performance liquid chromatography (HPLC) method. The model reaction systems were constructed at different pH values: 3.0, 4.0, 5.0, 6.0, 7.0. For each pH value, 10 µL tyrosinase and 80 µL catechin (EN) , 10 µL citric acid buffer solution and 80 µL catechin (E01) , 10 µL inactivated tyrosinase and 80 µL catechin (E02) , 10 µL tyrosinase and 80 µL methanol (S0) , 10 µL inactivated tyrosinase and 80 µL methanol (S0 (ia) ) were dissolved in a 110 µL citric acid buffer solutions, respectively. The solutions were maintained at 30 ℃ for 1 h, and the reactions were terminated by adding 200 µL ethyl acetate. After terminating the reactions, the solutions were dried rapidly with a stream of dry nitrogen at 30 ℃, then dissolved in 200 μL of methanol for analysis using the HPLC method.【Results】The model reaction systems, which were constructed by using catechin and activated tyrosinase, generated colored products. However, the color of other model reaction systems remained unchanged. The color intensity increased with the pH value, which indicated that the resulting products were strongly related to pH. At pH 3.0, the tyrosinase catalyzed the conversion of catechin to a colorless B-type procyanidin. Moreover, another three isomers of B-type procyanidin were detected both at pH4.0 and pH 5.0. Except for that, another two yellow A-type procyanidins were identified at pH 5.0. In the range pH 6.0-7.0, two yellow A-type procyanidins were identified while none of the colorless B-type procyanidins were found. The peak area of A-type procyanidins was observed at pH 5.0 which was significantly smaller than that at pH 6.0 and pH 7.0. The higher pH value contributed to the formation of yellow A-type procyanidins, which was in accordance with the observed higher color intensity. Thus, the formation of yellow A-type procyanidins was directly related with the browning. During the storage, the browning degree of'Ma Nai'grapes increased, and even displayed unacceptable appearance after 90 d storage. However, A-type procyanidins were not detected in'Ma Nai'grape skins during all the storage stages. On the contrary, four colorless B-type procyanidins were detected during all the storage stages, two of which were the same as that detected in the model reaction systems. Therefore, we supposed that the colorless B-type procyanidins might be acting as a substrate for further oxidation. Their oxidative products might be responsible for the observed browning of table grapes. Meanwhile, the transformation of colorless B-type procyanidin into yellow A-type procyanidin was also likely to happen.【Conclusion】In other words, the tyrosinase could catalyze the conversion of catechin into procyanidin dimers. The different kinds of resulting procyanidin dimers were strongly related to their pH values. At a low pH, colorless B-type procyanidins were formed, while yellow A-type procyanidins were formed at a high pH.When the individual flavan-3-ols of'Ma Nai'grape skins were detected, we found the colorless B-type procyanidins instead of the yellow A-type procyanidins. Thus, we concluded that the polymerization of flavan-3-ols was driven by tyrosinase. On the one hand, the polymerized product of yellow A-type procyanidins directly accounted for the browning. On the other hand, another polymerized product of colorless B-type procyanidins might be acting as a substrate for further oxidation, which was indirectly responsible for the browning. The illuminated relationship between the oxidative polymerization of the flavan-3-ol and browning allows for a better understanding of the browning of table grapes during storage.