Distribution of Polyphenol Oxidase in Flour Millstreams of Canadian Common Wheat Classes Milled to Three Extraction Rates

Cereal Chem. 70(l):51 -55 Polyphenol oxidase (PPO) levels were determined on individual and for the different wheats at similar cumulative flour yields. Less than 10% pooled millstreams of five cultivars representative of five different classes of the total PPO activity was present in cumulative flour streams of Canadian wheat. The wheats were milled on a pilot mill to extraction corresponding to 70% extraction, after which the amount of the enzyme rates of approximately 75 (conventional), 80, and 85%. Enzyme activity rapidly increased. Slightly more PPO activity appeared in the lower ash in individual streams ranged widely but increased with increasing bran streams if the wheats were milled to a higher extraction using this mill contamination in the millstreams. With the exception of the soft spring flow. PPO activity was linearly correlated with ash content (up to 2.0% wheat cultivar, PPO levels, as a percentage of total activity, were similar ash) and flour grade color figure (up to 5 units). Polyphenol oxidase (PPO) has been implicated in enzymatic browning reactions in whole wheat flours used for making chapati (Abrol et al 1971, Singh and Sheoran 1972). The enzyme is located 'Paper 683 of the Canadian Grain Commission, Grain Research Laboratory, 1404303 Main Street, Winnipeg, Manitoba R3C 3G8, Canada. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. American Association of Cereal Chemists, Inc., 1993. mainly in the bran of milled wheat and is not readily extractable with common buffers (Marsh and Galliard 1986). A number of studies have elaborated on the chemical and physical properties of the enzyme, as well as its multiplicity (Tikoo et al 1973; Taneja and Sachar 1974; Kruger 1976; Interesse et al 1980, 1981, 1983). Wheat enzyme levels are dependent on wheat class (Lamkin et al 1981). A number of end products are commonly prepared from flours milled to higher extraction rates in which the presence of PPO Vol. 70, No. 1, 1993 51 may play a role in affecting product color. These include Chinese steamed bread (Dexter et al 1984) and Middle East flat breads (Faridi 1988). Color in certain end products, such as Cantonese noodles, is extremely important and may be influenced by this enzyme, even at lower flour milling extraction rates (Miskelly 1984; Kruger et al, in press). The purpose of this study, therefore, was to determine levels of the enzyme that are found in wheat flour millstreams and to ascertain the effects on these levels of increasing flour extraction rate. Five cultivars of Canadian wheat representative of the common wheat classes grown in western Canada were milled to three extraction rates. MATERIALS AND METHODS Wheat Samples The wheats employed in this study (Table I) were obtained as certified seed. The samples were grown within the same year TABLE I Properties of Wheats Used in These Studies Falling Wheat Number Moisture Protein' Ash' Wheat Class Variety (sec) (%) (%) (%) Canada Western Red Spring Katepwa 405 11.8 15.7 1.75 Canada Utility Glenlea 385 11.5 13.1 1.69 Canada Western Red Winter Norstar 375 11.9 12.0 1.18 Canada Prairie Spring HY320 335 12.0 11.4 1.54 Canada Western Soft White Spring Fielder 380 11.8 9.0 1.36 a13.5% moisture basis. TABLE II Flour Yield and Ash of Straight-Grade Flours Milled to Extraction Rates of Approximately 75, 80, and 85%' 75% 80% 85% Variety Yield Ash Yield Ash Yield Ash Katepwa 75.7 0.54 79.8 0.66 84.1 0.99 Glenlea 73.9 0.59 79.0 0.70 83.7 1.00 Norstar 77.5 0.42 80.1 0.48 84.2 0.66 HY320 75.0 0.57 78.6 0.66 82.9 0.80 Fielder 71.7 0.43 77.6 0.53 83.2 0.70 AAll values are percents. but were harvested from different sites across western Canada. As shown in Table I, the wheats ranged in protein from 9.0 to 15.7%. The Falling Number indicated that all wheats were free of sprout damage. Wheat Milling Wheats were milled on the Grain Research Laboratory's pilot mill as described by Black (1980). The mill has a capacity in excess of 300 kg/hr. The wheats were cleaned and tempered as described by Black (1980). Temper moisture ranged from 14 to 16.5% depending on wheat hardness and flour extraction rate desired. Wheats were milled by a typical North American milling that gives a flour extraction rate near 75% (Black 1980) and by two higher extraction milling procedures that give flour extraction rates of about 80 (Dexter et al 1987) and 85% (Dexter et al 1984). Flour yields and resulting ash values of straight-grade flours of the five cultivars at the different extraction rates are shown in Table II. PPO Assay Analyses of PPO were carried out using a YSI model 5300 biological oxygen monitor (Yellow Spring Instrument Co., Yellow Spring, OH) per Marsh and Galliard (1986) with slight modifications. Temperature of the assay was raised to 370C, and the assay medium consisted of 4 ml of air-saturated 0.01M McIlvaine's buffer, pH 6.8 (Kruger 1976). Ground wheat or flour was added to the buffer, and the suspension monitored for 5 min to establish endogenous oxygen consumption. Freshly prepared substrate, 0.1 ml of 0.8M catechol, was added and the oxygen consumption monitored for 3-5 min. All values were corrected for substrate autooxidation. Activity was linear with added sample in the range of 10-200 mg. Results are expressed as nanomoles of 02 consumed per minute per gram at 37°C. Analyses were performed in triplicate. Flour Color Color was obtained using a Simon series IV (Henry Simon, Stockport, UK) flour color grader, which gives the relative reflectance (with filter no. 58) of a sample. Results are reported as Kent-Jones color units: the lower the number, the less bran contamination in the flour and the brighter the flour color (KentJones et al 1950). Ash Content Ash content was determined by using AACC method 08-01 .TABLE III Polyphenol Oxidase Levels' in Canadian Wheats Milled to Extraction Rates of 75 (A), 80 (B), and 85% (C)b Katepwa Glenlea Norstar Fielder HY320 Streamc A B C A B C A B C A B C A B C BI 125 141 201 299 366 398 52 106 149 174 93 191 93 115 106 B2 76 94 80 189 201 266 33 18 68 114 111 119 56 54 72 B3 60 98 82 133 138 60 41 62 93 77 197 56 63 86 129 B4 277 498 1,410 339 716 116 366 647 796 348 494 987 386 860 1,072 B5 3,239 4,060 1,061 1,274 1,831 S1 13 21 32 25 36 51 15 57 39 20 20 12 40 53 34 S2 0 4 17 12 11 12 7 11 19 12 4 8 16 18 8 M1 12 29 25 22 28 22 13 31 33 16 19 8 39 21 17 M2 24 45 52 45 41 57 14 29 43 21 28 30 46 9 58 M3 39 51 337 55 77 150 32 51 409 35 52 246 53 92 485 M4 63 259 196 66 23 116 55 291 207 52 168 111 101 323 196 M5 133 441 710 127 680 834 141 1,071 573 133 482 544 175 945 515 M6 172 548 585 122 716 627 141 1,014 318 156 479 265 205 907 724 M7 2,754 2,786 1,273 1,963 2,295 M8 4,195 3,900 2,070 3,423 3,237 M9 3,868 4,378 3,502 3,662 4,537 SD 690 915 3,773 1,401 1,328 7,960 445 1,008 1,990 689 1,083 2,896 669 1,305 2,547 BF 697 785 5,143 366 1,130 3,980 323 371 684 737 716 1,393 360 662 1,604 aPolyphenol oxidase levels in whole wheat samples were 1,266, 1,769, 1,011, 864, and 1,369 for Katepwa, Glenlea, Norstar, Fielder, and HY320, Alo~ioy I-usviu r nnnmoe 1U-e ga e iue 52 CEREAL CHEMISTRY respectively. All values are in nanomoles Of012 per gram per minute. bAverage coefficient of variation for sample analysis = 4.68%. BI1-B5 = first through fifth breaks, Sl-S2 = first and second sizings, Ml-M9 = first through ninth middlings, SD = shorts duster, BF = bran flour. (AACC 1983) on a 4-g sample in a silica dish incinerated overnight at 6000 C. Statistical Analyses Regression analyses were performed using the SAS version 6.03 (SAS Institute Inc., Cary, NC) software on an IBM-compatible personal computer. RESULTS AND DISCUSSION PPO Levels in Individual Millstreams and Pooled Flours PPO levels on a per gram basis in individual flour streams of Canadian wheat milled to an extraction rate of approximately 75% and to two higher extraction rates of approximately 80 and 85% are shown in Table III. In general, the relative order of increasing enzymic activity in the streams were the sizings, followed by the first through third middling flours interdispersed with break flours, followed by the remaining middling flours, the bran flours, and finally the shorts duster flours. As expected, the bran flours and shorts duster contained notably higher levels than the other flour streams, coincident with the expected anatomical location of the enzyme in the bran (Marsh and Galliard 1986). In general, enzyme activity in a particular flour stream increased on a per gram basis from the 75-80% extraction rate. Direct comparison with the 85% extraction millstreams was not possible because of the separation of additional streams. The cumulative PPO activity as a percentage of total activity versus the cumulative flour yield on the basis of increasing ash content is shown in Figure 1. It is interesting to note that at a particular extraction rate, all cultivars, with the exception of the soft white spring wheat Fielder, behaved quite similarly in terms of percent PPO activity present at a particular cumulative flour yield. The percent PPO ending up in the flour is fairly low. Thus, at the conventional extraction rate (75%), the percentages of total PPO activity found at 50, 60, and 70% cumulative flour yields were approximately 3, 4, and 5%, respectively, of total wheat PPO activity. Even at the highest extraction rate (approximately 85%), the percentages of total PPO activity found at 50, 60, and 70% cumulative flour yields were only around 4, 6, and 7%, respectively. The results indicate, however, that if wheat is being milled to a higher-than-conventional flour extraction rate for a particular end product, the quality of lower-ash-bulked flour streams, i.e., top patent, may suffer with respect to PPO activity. It is recognized, however, that in a commercial milling operation, the mill flow for an elevated extraction rate can be extended to produce a very fancy patent flour. Under such conditions, PPO levels may be only minimally influenced. Although the increase in total PPO in top patent flours with increased extraction was observed to be minimal compared with the total PPO present in the grain, it is conceivable that products in which color is highly important, i.e., Oriental white salted noodles, could be affected. Relationship Between PPO Activity and Ash or Color The anatomical location of the enzyme PPO in the bran suggests that there should be fairly strong relationships between PPO activity and either ash or flour color values. In general, overall relationships were poor when all flour streams were considered. As shown in Figure 2, however, fairly linear correlations were found between PPO activity and ash for the individual flour streams from the wheats milled to different extractions up to an ash value of about 2%, with Pearson correlation coefficients (P < 0.001) of 0.98 for Katepwa, 0.96 for Glenlea and Norstar, and 0.94 for Fielder and HY320. Similarly, the relationship between PPO activity and flour color of individual flour streams for the five cultivars (Fig. 3) were fairly linear up to a Kent-Jones flour color value of approximately 5 units, with correlation coefficients (P< 0.001) of 0.93 (Katepwa), 0.88 (HY320), 0.82 (Glenlea and Fielder), and 0.78 (Norstar). McCallum and Walker (1990) have also reported a high correlation (r = 0.977) between o-diphenol oxidase and flour color with 30 millstreams from New Zealand wheat cultivars.