Reliability of Five Methods for Protein Determination in Barley and Malt1 Y. FOMERANZ, R. B. MOORE, and F. S. LAI, U.S. Grain Marketing Research Center, Agricultural Research Service, U.S. Department of Agriculture, Manhattan, KS 66502 Protein was determined in 44 barley and 49 malt samples by the Kjeldahl binding capacity of proteins can be used to estimate the protein method. The data were used to establish a regression line of Kjeldahl protein contents of agricultural products and food (20), including barley and protein contents as estimated by biuret, dye-binding, alkaline (11,16). The empirical alkaline-distillation method has been distillation, and infrared reflectance (on two commercial instruments) developed for determination of protein in wheat and barley (17). methods. Protein contents of additional samples (76 of barley, 72 of malt) The biuret method, presumably, has been revised to eliminate were determined by the Kjeldahl method (actual Kjeldahl protein). Protein contents of those were estimated by each of the five methods and were interferences from barley aleurone components (7) and to adapt it converted to the Kjeldahl nitrogen by use of the regression lines (predicted to simple and routine testing (8,10,13). Finally, investigations Kjeldahl nitrogen). Reliability of the five methods was evaluated by conducted by K. H. Norris and coworkers at the Instrumentation comparison of predicted with actual Kjeldahl protein of those additional Research Laboratory, Agricultural Research Service, U.S. samples. Six-rowed barleys contained, on the average, more protein than Department of Agriculture have led to the development of an two-rowed barleys; varietal ranges in protein contents were smaller than instrument which uses infrared reflectance to measure the moisture, location ranges; differences between barley and malt protein were small. protein, and oil contents of oilseeds and cereal grains. Since 1971, Protein contents affected particle size of ground barley more than that of two firms (Dickey-john, Inc. and Neotec Instruments, Inc.) have ground malt; ground high-protein barleys contained more large particles been marketing commercial infrared reflectance instruments than the low-protein barleys. Malting reduced the particle size difference (19,21). between high- and low-protein samples and increased amounts of fine particles. Correlation coefficients for linear regression between protein We compared the reliability of protein determination in barley determined by the Kjeldahl and any of the other methods for calibration and malt by the Kjeldahl, biuret, dye-binding, alkaline distillation, were significant at the 99% level and were generally above 0.97. The and infrared reflectance procedures. Theoretically, the factors that Kjeldahl method was the most precise and the biuret method the least affect reliability are: specificity (absence of interfering substances), precise. The biuret and dye-binding methods gave best agreement (for accuracy (degree of conformity of a measurement to a standard or averages of two determinations) with the Kjeldahl method, infrared true value), precision (agreement among repeated measurements of methods were intermediate, and alkaline distillation was the poorest. the same parameter), and sensitivity (the smallest detectable Key words: Alkiline distillation, Biuret, Dye binding, Infrared composition difference between two samples). In practice, reflectance, Kjeldahl, Protein methods. however, the main criterion for reliability of a protein assay is comparison with the Kjeldahl determination. rotein content of malting barley is important because it affects malting, brewing, and their end products (2,3,4,9,12,18). P MATERIALS AND METHODS Kjeldahl-N determination is the commonly accepted method of protein estimation in barley and malt. The main objections to that Barley and Malt procedure are the corrosive reagents and the time, facilities, and The barleys (120 samples) and malts (121 samples) used in this personnel it requires. Consequently, rapid and simple tests have study are listed in Table I, and were supplied by the Barley and Malt been tried as substitutes. Laboratory, ARS, USDA, Madison, Wis. The barleys were plant The three main procedures for protein determination on the basis breeders' samples; the malts were prepared on a laboratory scale as of nitrogen content are the Kjeldahl, the Dumas, and the neutron described by Dickson and Burkhart (5). As expected, average activation methods. The principles, limitations, and reliability of protein contents between the barleys and malts (Tables II and III) those tests have been reviewed in detail elsewhere (15). The specific differed little. Protein for six-rowed barleys and malts was higher methods used in this study are summarized briefly here. The dye- than for two-rowed varieties. For 1974, overall average was 14.7% for six-rowed and 12.6% for two-rowed barleys. The protein ranges 1 Mention of firm names or trade products does not constitute endorsement by the for individual samples were 10.4—21.5% for barley and 10.3—20.3% U.S. Department of Agriculture over others of a similar nature not mentioned. for malt. TABLE I Description of Barleys and Malts Used in Study Year Varieties Locations 1974 2-rowed Firlbecks 111, Shabet, Vanguard, Larker' Aberdeen, Idaho, Twin Falls, Idaho, Klages, ID 181053, WA 766467, Fairfield, Mont., Pullman, Wash., WA 765267, Hannchen, MT 548448 Laramie, Wyo. 1973 6-rowed Barbless, Manchuria, Trail, Larker, Fargo, N. Dak., Langdon, N. Dak., Conquest, Bonanza, Minn. M-16 Brookings, S. Dak. (Manken), ND 1156 1974 6-rowed Barbless, Manchuria, Trail, Larker East Lansing, Mich., Langdon, N. Dak., Conquest, Bonanza, Prilar, Crookston, Minn., Manken, Minn. M-18, ND 231 Carrington, N. Dak., Brookings, S. Dak. JA 6-rowed barley. 86 No. 2 ASBC JOURNAL 87 TABLE II Range, Mean, and Coefficient of Variation (CV) of Varietal Kjeldahl-Protein Means for Barley and Malt Barley Malt Type and Variety Range Mean CV% Range Mean cv% 1974 2-rowed Firlbecks III 10.8-14.9 12.9 14.3 10.7-15.1 12.9 15.0 Shabet 11.3-14.7 12.9 11.4 11.1-14.7 12.9 12.4 Vanguard 10.8-13.9" 12.4' 10.9' 10.6-14.0' 12.2" 12.0° Larker" 11.9-15.6 14.2 10.3 11.8-16.5 14.0 14.0 Klages 11.2-14.3 12.6 10.4 10.9-14.4 12.6 11.1 ID 181053 10.7-13.8 12.1 12.6 10.5-14.1 12.1 14.4 WA 766467 10.6-13.7 12.0 11.6 10.4-13.4 11.9 11.4 WA 765267 10.4-13.4 11.9 10.9 10.3-13.2 11.7 11.1 Hannchen 11.6-14.9 13.7 11.0 11.1-14.6 13.4 12.9 MT 548448 11.1-14.6' 12.5" 12.3" 11.3-14.6' 12.4' 12.5° 1973 6-rowed Barbless 15.5-16.8 16.2 4.14 15.4-16.9 16.2 4.69 Manchuria 14.4-15.0 14.7 2.17 15.1-15.4 15.2 1.06 Trail 13.8-14.4 14.1 2.20 13.4-14.8 14.3 5.22 Larker 14.5-14.9 14.7 1.50 14.5-15.2 14.9 2.48 Conquest 14.0-14.4 14.2 1.32 14.2-14.3 14.3 0.49 Bonanza 14.1-14.9 14.5 2.98 14.3-15.6 15.0 4.48 M-16 14.3-14.5 14.4 0.659 14.3-14.6 14.5 1.26 ND-1156 14.4-15.1 14.8 2.43 15.0-15.4 15.2 1.33 1974 6-rowed Barbless 13.2-18.4 16.0 13.0 13.1-18.7' 15.8C 14.9° Manchuria 12.6-17.1 15.3 12.4 12.7-17.2 15.3 11.9 Trail 11.5-19.1 14.9 20.2 .4-18.1 14.8 18.8 Larker 11. 4- 1 8.2 14.1 17.0 .2-17.0 14.7 15.2 Conquest 12.2-18.3 15.4 15.6 .9-17.2 15.0 20.0 Bonanza 11.4-17.7 14.7 16.4 .1-16.6 14.4 15.6 Prilar 14.1-17.3" 15.6d 9.50" .6-18.0 15.1 16.3 Manken 11.6-18.6 15.2 17.9 .8-17.6 15.0 14.9 M-18 14.0-17.4 15.6 10.8 .8-17.0 14.5 13.4 ND-231 12.6-21.5" 16.3d 20. ld 12.5-20.3 16.1 18.3 'Missing from Laramie, Wyo. "A 6-rowed barley. 'Missing from Brookings, S. Dak. JMissing from East Lansing, Mich. TABLE III Range, Mean, and Coefficient of Variation (CV) of Location Kjeldahl-Protein Means for Barley and Malt Barley Malt Type and Location Range Mean cv% Range Mean CV% 1974 2-rowed Aberdeen, Idaho 13.4-15.2 14.3 4.41 13.2-15.2 14.2 5.06 Twin Falls, Idaho 10.4-12.0 11.2 5.27 10.4-12.0 11.0 5.19 Fairfield, Mont. 10.7-14.9 12.4 11.10 10.5-14.1 12.3 11.90 Pullman, Wash. 10.9-12.7 11.8 5.42 10.7-12.4 11.7 5.06 Laramie, Wyo. 12.9-15.6° 14.1a 6.38° 12.8-16.5' 14.3° 7.69° 1973 6-rowed Fargo, N. Dak. 14.0-16.8 14.7 6.11 13.4-15.6 14.8 4.89 Langdon, N. Dak. 13.8-15.5 14.7 3.33 14.3-16.3 15.0 4.27 Brookings, S.