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1、- 1 -1Polyphenol oxidase activity in tea, apple and potato2Mao YuLin3College of Agriculture Figures: 4- 2 -6Abstract7Background Polyphenol oxidase exists in most of plants, changes plants colors, 8and thus decreases food quality. Therefore, finding the main factors that impacts 9the PPO is benefit t
2、o store food from plants.10Methods The content of enzyme in tea leaves, apple fruits, and potato tubers was 11measured by Folin- phenol method. The main influence factor of PPO activity 12was determined by orthogonal method. 13Results The protein yields of the crude enzymes extracted from tea, apple
3、, and 14potato were 44.318 mg/g, 38.626 mg/g, and 52.114 mg/g, respectively. Tea 15leaves showed the highest PPO activity at 8 mM catechol, 20C, and pH 6.5. 16Apple fruits showed the highest PPO activity at 8 mM catechol, 20C, and pH 5. 17Potato tubers showed the highest PPO activity at 20 mM catech
4、ol, 30C, and pH 186.19Conclusion The activator and inhibitor are the main factors that are in control of 20PPO.Temperature is the second important factor and is easy to control. Therefore, 21we should inhibit PPO activity by controlling storing temperature. Tea and potato 22could be stored at 40, an
5、d apple should be stored below 20 C.23Key words: Polyphenol oxidase; Orthogonal method; Activator and inhibitor; 24Temperature- 3 -26Introduction27Polyphenol oxidase (PPO) catalyzes polyphenols to quinones. PPO widely exists in 28various plants, and plays roles in plant growth and development, and p
6、lant 29adaptability to the environment. Importantly, PPO activity results in brown reactions 30in plant tissues, and affect the color and quality of the food derived from plants (Kong 31et al., 2011). Tea, apple, potato are common food and easily available, and contain 32abundant PPO. A recent study
7、 compared PPO activities in fresh tea, apple and potato 33(L.J.Song et al., 2009). This study used the same methods to extract crude PPO 34enzymes and measure PPO activities and used an orthogonal method to find the main 35factor controlling PPO activities in tea, apple and potato.36Materials and Me
8、thods37Plant materials38 Leaves of green tea grown at ChangBai Mountain were harvested in March. 39Local apple fruits and potato tubers were purchased from market. 40Crude enzyme extraction41Apple fruits and potato tubers were peeled and cut into small pieces; tea leaves 42were cut into small pieces
9、; 150 g of each material were homogenized in a NaF buffer 43(pH 7) with a ZZ homogenizer (HanNuo, Shanghai, China). After adding 150 ml 0.1 44M NaCl, the homogenates were filtered with 4 layers of gauze. Fifty milliliters of the 45filtrate were centrifuged at 4000 rpm for 10 min at 4C. The supernata
10、nt was mixed 46with 50 ml ethanol and put at 4C for 30 min, and then centrifuged at 4000 rpm for 10 47min at 4C. The precipitation was dissolved in a phosphate buffer (pH 6.8) and used 48as the crude PPO solution.- 4 -49Protein determination by Folin-phenol method 50Protein was determined by the Fol
11、in-phenol method as the protocol described by 51the manufacturer of the Folin-phenol regents (Sangon Biotech, Shanghai, China).52Standard protein solutions were prepared from a 500 g.ml-1 invertase protein 53solution. After adding 10 l, 20 l, 40 l, 60 l, 80 l, and 100 l of the 500 g.ml-1 54protein s
12、olution in tubes, distilled water was added to make the solution volume to 1.0 55ml; 1.0 ml distilled water was the blank control. The blank and standard protein 56solutions (5, 10, 20, 40, and 50 g.ml-1) were mixed with 5.0 ml Folin-phenol regent 57A and put at room temperature for 25 min, and then
13、 mixed with 0.5 ml Folin-phenol 58regent B and put at room temperature for 20 min. The optical density of the reaction 59solutions were measured at 500 nm with an ultraviolet spectrometer (Shanghai Yuanxi 60Instruments Co. Ltd, Shanghai，China).61The protein concentrations of the crude PPO solutions
14、were then determined by 62the Folin-phenol method. Each solution consists of three replicates; each replicate was 63measured three times.64Determination of factors controlling PPO activity using orthogonal method65PPO activities were measured by the catechol oxidation method (L. J. Song et al., 6620
15、09). The absorbance of the oxidation products was measured at 420 nm. The 67relative unit (U) of the enzyme activity is defined as the enzyme quantity needed for 680.001of absorbance change per minute (Qiao et al., 2007).69An orthogonal experiment was designed (Table 1) to investigate the major 70fa
16、ctors determining the PPO activities. Four factors 1) substrate concentration (s1, s2, - 5 -71s3), 2) temperature (20C, 30C, 40C), 3) buffer pH (5.5, 6.5, 7.5), and 4) activator 72(SDS) and inhibitor (Na2SO3) were investigated.73 74Results75Protein determination 76The optical density values at 500 nm of the standard protein solutions were 77correlated with the protein concentrations (R2 = 0.99) and resulted in a linear 78calibration curve: y= 0.0066x+ 0.0184
