The Effect Of Green-making Process On The Activity And Enzymatic Properties Of β-glucosidase In Oolong Tea

Oolong tea is well-known for its refreshing flavor, and fine manipulation of green tea leaves (green-making) is a key process to help forming its unique aroma. Thus, it is of great significance to study activity and enzymatic properties of β-glucosidase which is closely related to the aroma in the green-making process. This thesis studies the effects of three different fixed conditions on the activity of β-glucosidase to determine the best condition. By taking Tie Guanyin and Jin Guanyin as the research material and setting three different green-making temperatures (17℃、22℃、27℃), this research studied dynamic change of β-glucosidase’s activity in the green-making process and under the three corresponding temperatures. In addition, enzymatic properties have also been studied to explore change rule of β-glucosidase and further a brief analysis of the change mechanism will be described.The results show:The best method of study on the sample green tea leaves’ β-glucosidase’s activity and enzymatic properties is:Use the stainless steel scissor to cut two fresh tea leaves, then wrap them with aluminum foil (3g per package), the third step is to process those wrapped leaves with liquid nitrogen for3-5min and last put the processed leaves into the refrigerator with the temperature being-30℃for two weeks.Under the temperature of17℃,activity change of Tie Guanyin’s β-glucosidase demonstrates the waving upward trend, showing"drop,rise,drop,rise" in the green-making process, whereas activity change of Jin Guanyin’s β-glucosidase represents evenly waving downward trend, showing"drop,rise,drop". Under the same temperature, compared with static treatment,average activity values of Tie Guanyin and Jin Guanyin’s P-glucosidase are lower in the green-making process than that under the static treatment. Even though there is difference on the enzymatic activity of Tie Guanyin between those two different treatments, this difference is not that significant. On the contrary, there is significant difference on the enzymatic activity of Jin Guanyin between the two different treatments and the difference becomes greatly significant before the phase of the fifth shake. Under the temperature of22℃, enzymatic activity of Tie Guanyin demonstrates the waving change,which is "drop,rise,drop" in the green-making process, while that of Jin Guanyin shows the bimodal changing trend, which is"rise,drop,rise,drop.rise". Under the same temperature being22℃, average activity value of Tie Guanyin’s β-glucosidase is apparently1.31times in the green-making process of that under the static treatment, whereas that of Jin Guanyin’s β-glucosidase is obviously1.30times under the static treatment of that in the green-making process. It is found that although difference exists on the enzymatic activity of Tie Guanyin between the green-making process and static treatment, yet the difference is not that significant. In contrast, there is difference on the enzymatic activity of Jin Guanyin between the two different treatments above, but there is significant difference before the third shake, the forth shake and the fifth shake, in particular,the difference is greatly significant before the phase of the fifth shake. Under the condition of27℃, enzymatic activity of Tie Guanyin demonstrates the change from "drop" to "rise" in the green-making process, while that of Jin Guanyin represents the changing trend,which is "drop,rise,drop". When under the same temperature (27℃), it is proved that average enzymatic activity values of Tie Guanyin and Jin Guanyin are both lower in the green-making process than that under the static treatment. In addition, in terms of difference, no significant difference exists on enzymatic activity of Tie Guanyin under those two treatments while significant difference exists on that of Jin Guanyin, especially before the phase of second shake, third shake, forth shake and fifth shake.Under three different temperatures,the orders of average enzymatic activity values of Tie Guanyin and Jin Guanyin in every step of green-making process are respectively:With regards to activity of Tie Guanyin’s β-glucosidase under three different temperatures (17℃、22℃、27℃) in the green-making process, before the phases of the first shake and the second shake, there is significant difference and even greatly significant on the enzymatic activity between17℃and other two temperatures(22℃,27℃) but no significant difference exists between22℃and27℃; before the phase of the third shake, there is significant difference on the activity between27℃and the rest two temperatures and this difference is extremely obvious; before the forth shake phase significant difference exists between17℃and22℃，while the difference between27℃and the other two temperatures (17℃,22℃) is not significant.Take Tie Guanyin and Jin Guanyin under three different green-making temperatures (17℃、22℃、27℃) as the materials to extract β-glucosidase and study its enzymatic properties. The results are:When Tie Guanyin and Jin Guanyin are under17℃, even though there are some differences on β-glucosidase’s response in certain stages of the green-making process, in general the temperature maintains47℃both in the green-making process and under the static treatment. When the temperature is22℃, the optimum reaction temperature of Tie Guanyin’s β-glucosidase under the static treatment is47℃.When the temperature reaches51℃,β-glucosidase represents higher enzymatic activity and its enzymatic reaction can be described by a M-shaped curve. In contrast, its enzymatic reaction in the green-making process can be shown by an-shaped curve and the enzymatic activity becomes relatively high in the range of47℃-51℃.The optimum reaction temperature is51℃before the second shake. With respect to Jin Guanyin, the optimum reaction temperature of its β-glucosidase under the static treatment is49℃before the forth shake while in the green-making process,β-glucosidase’s reaction to the temperature is complicated, with the optimum reaction temperature being40℃before the second shake and forth shake,40℃and47℃before the third shake and47℃before the fifth shake and after the green-making period. When the temperature is27℃, the optimum reaction temperature of Tie Guanyin’s β-glucosidase in the green-making process is47℃and the optimum reaction temperature is also47℃under the static treatment in every phases except the phase after green-making process with the temperature being In terms of Jin Guanyin’s0-glucosidase, the optimum reaction temperature is47℃under the static treatment in every phases except the phase before the fifth shake with the temperature being40℃.β-glucosidase’s reaction curve to the temperature demonstrates the same tendency both in the green-making process and under the static treatment with the enzymatic activity becoming relatively high in the scope of40℃-51℃.The results also show that in spite of the subtle difference on the optimum Ph of β-glucosidase between Tie Guanyin and Jin Guanyin in every step of green-making process, all β-glucosidase is acidic protein with the optimum PH in the range of5.0-6.0. The study on Km value of Tie Guanyin and Jin Guanyin’s β-glucosidase under the two different treatments concludes:when the temperature is17℃, Km value represents obvious falling tendency before the third and forth shake under the static treatment,indicating affinity between β-glucosidase and pNPG gradually increased whereas Km value is rising in the green-making process,which means affinity between β-glucosidase and pNPG is decreasing, when the temperature is22℃, Km value of Tie Guanyin’s β-glucosidase is rising sharply in the phases of the second,third and forth shake, meaning the affinity between β-glucosidase and pNPG weakened while the Km value has the upward tendency in the green-making process except in the phase of the second shake, signifying that the affinity is decreasing gradually. When the temperature is27℃, Km values of Tie Guanyin’s β-glucosidase in the green-making process and under the static treatment have little difference with the values being slightly lower in the green-making process than that under the static treatment. Besides, Km values represents the upward tendency after the second shake in the two different conditions above, indicating affinity is evenly decreasing between β-glucosidase and pNPG under the high temperature(27℃). With respect to Jin Guanyin, even though there are fine distinction on the Km value under three different temperatures and different treatments, Km values tend to be same in general. Overall, Km value under static treatment is lower than that in the green-making process, which means affinity between β-glucosidase and pNPG is stronger under static treatment than that in the green-making process. By comparing Km values of Tie Guanyin’s and Jin Guanyin’s β-glucosidase, it is found there is significant difference and generally speaking, Km value of Tie Guanyin’s β-glucosidase is higher than that of Jin Guanyin’s β-glucosidase,signifying affinity between β-glucosidase and pNPG is weaker in Tie Guanyin than that in Jin Guanyin.