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1、Polyphenol oxidase activity in tea, apple and potatoMao YuLinCollege of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, ChinaCorrespondence: Words: 1278; Figures: 4Abstractl Background Polyphenol oxidase exists in most of plants, changes plants colors, and thus decreases food qual
2、ity. Therefore, finding the main factors that impacts the PPO is benefit to store food from plants.l Methods The content of enzyme in tea leaves, apple fruits, and potato tubers was measured by Folin- phenol method. The main influence factor of PPO activity was determined by orthogonal method. l Res
3、ults The protein yields of the crude enzymes extracted from tea, apple, and potato were 44.318 mg/g, 38.626 mg/g, and 52.114 mg/g, respectively. Tea leaves showed the highest PPO activity at 8 mM catechol, 20C, and pH 6.5. Apple fruits showed the highest PPO activity at 8 mM catechol, 20C, and pH 5.
4、 Potato tubers showed the highest PPO activity at 20 mM catechol, 30C, and pH 6.l Conclusion The activator and inhibitor are the main factors that are in control of PPO.Temperature is the second important factor and is easy to control. Therefore, we should inhibit PPO activity by controlling storing
5、 temperature. Tea and potato could be stored at 40, and apple should be stored below 20 C.Key words: Polyphenol oxidase; Orthogonal method; Activator and inhibitor; TemperatureIntroductionPolyphenol oxidase (PPO) catalyzes polyphenols to quinones. PPO widely exists in various plants, and plays roles
6、 in plant growth and development, and plant adaptability to the environment. Importantly, PPO activity results in brown reactions in plant tissues, and affect the color and quality of the food derived from plants (Kong et al., 2011). Tea, apple, potato are common food and easily available, and conta
7、in abundant PPO. A recent study compared PPO activities in fresh tea, apple and potato (L.J.Song et al., 2009). This study used the same methods to extract crude PPO enzymes and measure PPO activities and used an orthogonal method to find the main factor controlling PPO activities in tea, apple and
8、potato.Materials and MethodsPlant materials Leaves of green tea grown at ChangBai Mountain were harvested in March. Local apple fruits and potato tubers were purchased from market. Crude enzyme extractionApple fruits and potato tubers were peeled and cut into small pieces; tea leaves were cut into s
9、mall pieces; 150 g of each material were homogenized in a NaF buffer (pH 7) with a ZZ homogenizer (HanNuo, Shanghai, China). After adding 150 ml 0.1 M NaCl, the homogenates were filtered with 4 layers of gauze. Fifty milliliters of the filtrate were centrifuged at 4000 rpm for 10 min at 4C. The supe
10、rnatant was mixed with 50 ml ethanol and put at 4C for 30 min, and then centrifuged at 4000 rpm for 10 min at 4C. The precipitation was dissolved in a phosphate buffer (pH 6.8) and used as the crude PPO solution.Protein determination by Folin-phenol method Protein was determined by the Folin-phenol
11、method as the protocol described by the manufacturer of the Folin-phenol regents (Sangon Biotech, Shanghai, China).Standard protein solutions were prepared from a 500 g.ml-1 invertase protein solution. After adding 10 l, 20 l, 40 l, 60 l, 80 l, and 100 l of the 500 g.ml-1 protein solution in tubes,
12、distilled water was added to make the solution volume to 1.0 ml; 1.0 ml distilled water was the blank control. The blank and standard protein solutions (5, 10, 20, 40, and 50 g.ml-1) were mixed with 5.0 ml Folin-phenol regent A and put at room temperature for 25 min, and then mixed with 0.5 ml Folin
13、-phenol regent B and put at room temperature for 20 min. The optical density of the reaction solutions were measured at 500 nm with an ultraviolet spectrometer (Shanghai Yuanxi Instruments Co. Ltd, Shanghai，China).The protein concentrations of the crude PPO solutions were then determined by the Foli
14、n-phenol method. Each solution consists of three replicates; each replicate was measured three times.Determination of factors controlling PPO activity using orthogonal methodPPO activities were measured by the catechol oxidation method (L. J. Song et al., 2009). The absorbance of the oxidation produ
15、cts was measured at 420 nm. The relative unit (U) of the enzyme activity is defined as the enzyme quantity needed for 0.001of absorbance change per minute (Qiao et al., 2007).An orthogonal experiment was designed (Table 1) to investigate the major factors determining the PPO activities. Four factors
16、 1) substrate concentration (s1, s2, s3), 2) temperature (20C, 30C, 40C), 3) buffer pH (5.5, 6.5, 7.5), and 4) activator (SDS) and inhibitor (Na2SO3) were investigated. ResultsProtein determination The optical density values at 500 nm of the standard protein solutions were correlated with the protei
17、n concentrations (R2 = 0.99) and resulted in a linear calibration curve: y= 0.0066x+ 0.0184(x represents protein concentration, y represents OD500 value) (Fig. 1). The protein yields of the crude enzymes extracted from tea, apple, and potato were 44.318 mg/g, 38.626 mg/g, and 52.114 mg/g, respective
18、ly.PPO activities and determination factorsTea leaves, apple fruits, and potato tubers showed different PPO activities at the same condition and different changes of PPO activities at different conditions. Tea leaves showed the highest PPO activity at 8 mM catechol, 20C, and pH 6.5. Apple fruits sho
19、wed the highest PPO activity at 8 mM catechol, 20C, and pH 5. Potato tubers showed the highest PPO activity at 20 mM catechol, 30C, and pH 6. The PPO activator SDS greatly increased the PPO activities in tea leaves, apple fruits, and potato tubers whereas the PPO inhibitor Na2SO3 greatly inhibited t
20、he PPO activities. Temperature also showed a strong influence on the PPO activities.DiscussionThis study shows that tea leaves, apple fruits, and potato tubers have different PPO activities at the same condition and different changes of PPO activities at different conditions. These results are simil
21、ar to those shown by a previous study (J. L. Song et al., 2009). This study also shows that activator SDS and inhibitor Na2SO3 have the greatest impact on the PPO activities, and temperature has a high impact on the PPO activities.PPO activity leads to the browning of plant tissues and thus decrease
22、s the quality of foods from plants (Kong et al., 2011). To reduce PPO activity for plant food storage, PPO inhibitor, concentrations of the substrate catechol, and pH are difficult to control. In contrast, temperature is easy to control. Therefore, temperature control is useful and practical for low
23、ering PPO activities of plant food. Based on this study and previous studies ( Meng et al., 2006; J.L.Song et al., 2009), tea and potato can be stored at 40C while apple below 20 C to inhibit PPO activities during storage.AcknowledgementsI thank Prof. Wenjun Guan (College of Life Sciences, Zhejiang
24、University) for advices, Xia Zheng and Canyuan Xu (College of Agriculture & Biotechnology, Zhejiang University) for assistance.ReferencesKong J-H, Sun Q-L, Tu Y-F. 2011. Enzyme properties research of polyphenol oxidase and application progress.Chinas wild plant resources 30(4): 13-17Meng Y, Li G, Cu
25、i Y. 2006. The research of the extraction and purification conditions of the potato polyphenol oxidase on the effects of the active. Chemical ad Biological Engineering 23(10): 47-49.Song J-L, Zhao S-X, Zhang L-M, Guo S-W, Cui J-Y. 2009. Fresh tea, apple and potato polyphenol oxidase activity. Modern
26、 Food Science and Technology 25(11): 60-64.Song L-J, Tang G-F, Zhao Q-Y, Qiao M-W. 2009. The extraction of Fuji apple polyphenol oxidase and characteristic research. Zhejiang Agricultural Science (4): 459-462.Table 1. Orthogonal design for catechol oxidation solution (3 ml)GroupNo.PB buffer（1.0 ml）A
27、ctivatorinhibitor(1.0 ml)Catechol solution(0.12 M)Temperature12pH7.5 distilled water 0.5 ml20 4pH6.5 10 % SDS0.2 ml20 9pH5.5 Na2SO30.8 ml20 21pH5.5 10 % SDS0.5 ml 30 6pH7.5 Na2SO30.2 ml30 8pH6.5 distilled water0.8 ml30 33pH6.5 Na2SO3 0.5 ml 40 5pH5.5 distilled water0.2 ml40 7pH7.5 10 % SDS0.8 ml40 Fig. 1. Protein standard curve Fig. 2. Activities of tea polyphenol oxidase in the orthogonal experimentFig. 3. Activities of apple polyphenol oxidase in the orthogonal experimentFig. 4. Activities of potato polyphenol oxidase in the orthogonal experiment- 12 -