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Polyphenol Oxidase in Wheat Grain: Whole Kernel and Bran Assays for Total and Soluble Activity

¹Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164-6394. ²USDA-ARS Western Wheat Quality Laboratory, Washington State University, Pullman, WA 99164-6394. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. ³USDA-ARS Biosciences Research Laboratory, 1605 Albrecht Blvd., Fargo, ND 58105-5674. ⁴Correspondong author. Phone: +1.509.335.4062. Fax: +1.509.335.8573. E-mail: morrisc@wsu.edu

Color is a key quality trait of wheat products, and polyphenol oxidase (PPO) is implicated as playing a significant role in darkening and discoloration. In this study, total and soluble PPO activities were characterized in whole kernel assays and bran extracts. In whole kernel assays similar to AACC Approved Method 22–85, four wheat cultivars were ranked the same for both total and soluble (leached) PPO activity with L-DOPA (diphenol) as the substrate. Total kernel PPO activity was much greater than soluble PPO activity in three hexaploid wheat cultivars, indicating that insoluble PPO was the major contributor to kernel PPO measurements. Tyrosine (monophenol) was an excellent PPO substrate in kernel assays as expected but had no activity as a substrate for soluble PPO. However, soluble PPO activity with tyrosine was activated by the addition of the diphenols chlorogenic acid and caffeic acid. When PPO was assayed in homogenized bran, 89–95% of total PPO activity remained insoluble, associated with the bran particles. The kernel assay detected <2% of PPO measured in an equivalent amount of homogenized bran. However, total PPO activity was 2-fold higher in Klasic than in ID377s, both when measured in the kernel assay and in homogenized bran, indicating that the kernel assay was an accurate predictor of relative total extracted PPO activity in these two cultivars. Adding detergents (0.1% SDS plus 0.2% NP-40) to the bran extraction buffer increased both soluble and insoluble PPO activity. Results indicate that relative PPO activities among wheat cultivars are similar in whole kernel and kernel leachate assays, and that the predominant insoluble fraction of PPO, which is relatively uncharacterized, may be largely responsible for wheat product discoloration.