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J Sci Food Agric 1997, 73, 3945

Could the Dumas Method Replace the Kjeldahl Digestion for Nitrogen and Crude Protein Determinations in Foods?*
A H Simonne,a E H Simonne,b R R Eitenmiller,a H A Millsc and C P Cresman IIIa
a Department of Food Science and Technology, University of Georgia, Athens, GA 30602, USA b Department of Horticulture, 101 Funchess Hall, Auburn University, AL 36849-5408, USA c Department of Horticulture, University of Georgia, Athens, GA 30602, USA
(Received 19 January 1996 ; revised version received 25 May 1996 ; accepted 9 August 1996)

Abstract : Increased demand for determinations of nitrogen (N), and hence crude protein (CP), has led to wider use of the Dumas method in place of the traditional Kjeldahl methods. Although Kjeldahl N (KN) and Dumas N (DN) represent dierent N fractions, published studies on infant formula, animal feed and meat products have indicated that DN could replace KN with little practical impact on the reliability of the N values obtained. This study was conducted to establish whether DN determination could replace that of KN in a broader range of foods for CP calculation. Statistical analysis was performed on in-house assayed KN and DN values together with published KN and DN values for selected food products. In the range 00568% N, KN may be estimated from DN with the equation : KN \ 100(P:001) ] DN [ 009(P/050) (n \ 101, R2 \ 098, P-regression \ 001). Because N levels in individual groups of food did not span the entire range of N contents, KN : DN ratios were calculated for each food group. KN : DN ratios diered signicantly (R2 \ 025, P \ 001) from group to group. Ratios of 101 for dairy, 100 for oilseeds, 099 for feed, 098 for infant formulas, 095 for cereals, 094 for meats, 089 for vegetables, 080 for sh and 073 for fruits were valid for the estimation of KN and CP using DN data. CP was independently calculated as CP1 \ H ] KN or CP2 \ H ] KN : DN ] DN, where H is the nitrogen to protein conversion factor for the food group. Mean dierences between CP1 and CP2 values were 0% for dairy, oilseeds, feed, infant formulas and baby foods, cereals, meat and meat products, vegetables and vegetable products and fruit, and 1% for sh. These results suggest that DN may replace KN for the determination of N and CP in selected food groups when appropriate coefficients are used. Key words : Kjeldahl nitrogen, Dumas nitrogen, crude protein.

INTRODUCTION Crude protein (CP) is among the mandatory nutrients of the Nutrition Labeling and Education Act of 1990 and is usually estimated by multiplying nitrogen (N) content by a nitrogen to protein conversion factor. In most foods, amino-N accounts for approximately 16%
* This paper was presented at the 1995 annual meeting of the Institute of Food Technologists 37 June, 1995 in Anaheim, CA, USA. To whom correspondence should be addressed at : Department of Nutrition and Food Science, 328 Spidle Hall, Auburn University, AL 36849, USA.

of the protein weight. Hence, the nitrogen to protein conversion factor is usually 625, but specic foods have smaller (cereals) or higher (milk) conversion factors depending on the proportions of N in their proteins. Direct methods for protein determination include biuret, Lowry, bicinchoninic acid (BCA), ultraviolet (UV) absorption (280 nm), dye binding, Bradford, ninhydrin and turbidimetry. These methods are based on properties of specic proteins, specic amino acid residues in proteins or peptide bonds present in proteins or peptides and involve extraction, isolation and sometimes purication (Chang 1994 ; Pomeranz and Meloan 1994). These methods are often used in biochemical 39

J Sci Food Agric 0022-5142/97/$09.00 ( 1997 SCI. Printed in Great Britain

40 investigations but they are completely impractical for routine food analysis because foods contain a complex mixture of proteins (Chang 1994 ; Pomeranz and Meloan, 1994). Moreover, only dye binding methods (official methods 96712 and 97517) are approved for direct determination of protein in milk (AOAC 1995). Official methods for N determinations in foods include modications of the Kjeldahl method (98110, 95504, 97702, 97804, 920152, 92087, 94518B, 95036, 93033, 93029, 92808, 97714, 95048 and 93539 with a Hg catalyst ; and 99120, 99122 and 99123 with a Cu catalyst) (Sullivan and Carpenter 1993). AOAC Official Method 98413 Protein (Crude) in Animal Feed also utilises a Cu catalyst (AOAC 1995). The Kjeldahl method consists of (a) a digestion step where N is converted into ammonium (NH`), and (b) an analytical 4 step where NH` is quantied by titrimetry, colorimetry 4 or using an ion-specic electrode. This method assumes that N recovered during digestion is mainly amino-N from proteins (total organic N) and that the contribution of inorganic N (nitrate, nitrite, ammonium) or other organic N (nucleotides, nucleic acids) is negligible. Presently, methods used for determining the total protein (referred to as crude protein) content of foods involve (a) determination of N, and (b) calculation where the N value is converted to protein by a foodspecic nitrogen to protein conversion factor, which will be refer to as H throughout this manuscript. The main advantage of Kjeldahl methods is its widely established use in CP estimation. However, Kjeldahl methods require wet chemistry, the handling of concentrated sulphuric acid and a heavy-metal catalyst. In consequence, for routine analysis the Kjeldahl method is a tedious and time-consuming procedure requiring disposal of hazardous wastes. For these reasons, analytical laboratories tend to adopt automated and easyto-use equipment (such as the LECO FP-428, LECO CHN 600 or Carlo Erba NA-1500) for routine, unattended N determination. These instruments use the Dumas method which consists of (a) converting all the forms of N into gaseous nitrogen oxides (NO ) by comx plete combustion in an induction furnace, (b) reducing the NO gases to N and (c) quantifying N by thermal x 2 2 conductivity (Sweeney and Rexroad 1987 ; Jones 1991). Thus, for the Dumas method (also referred to as the

A H Simonne et al combustion method) wet chemistry is not involved and time for analysis is reduced to approximately 6 min per sample, furthermore liquid, semi-solid or solid samples can be analysed. Moreover, its accuracy and repeatability may be superior to that of the Kjeldahl method (Schmitter and Rihs 1989). Because of its nature, Dumas N (DN) is a true measure of total N. In food products (Minagawa et al 1984) and in plants (Sweeney and Rexroad 1987 ; Simonne et al 1994), DN and Kjeldahl N (KN) recovered dierent fractions of N (Fig 1). During the Kjeldahl digestion, reducing conditions caused by the release of free carbon favour the conversion of nitrate to NH` 4 and losses of nitrate as N (Bradstreet 1960). Therefore, 2 KN may include some non-amino N from nitrates, nitrites, nucleotides or nucleic acids (Nelson and Sommers 1980 ; Pace et al 1982 ; du Preez and Bate 1989a,b ; Goyal and Hafez 1990 ; Barbano et al 1991). The reduction of non-amino N during Kjeldahl digestion depends on the chemical form of the nonamino N and sample matrix. Hence, the recovery of non-amino N is non-quantitative. The combustion method was approved for CP determination in animal feed (Sweeney 1989), meat and meat products (KingBrink and Sebranek 1993), and cereal grains and oilseeds (Bicsak 1993). An in-depth study was also conducted with infants foods (Bellomonte et al 1987) and two collaborative studies carried out on dairy products (Bradley 1995) ; one on fruit and vegetable products (Huang et al 1995) is currently being prepared. Although the approach of systematically evaluating the eect of analytical method in each food group is very thorough, it is slow and expensive. In addition, it is sometimes difficult to classify food products into a single category. For example, a food designated for infant food and containing turkey is classied as a infant food, while a meat or blood meal designated for animal consumption was part of a study on animal feed. Matrix dierences between these two samples may not be dierent enough to justify the distinction implied by the two categories. Another limitation to this classication is that compound foods may belong to two different food groups. For instance, soups containing milk, chicken and fruits may be regarded as a modied dairy product or infant formula. Therefore, the present study

Fig 1. Expected recovery of main nitrogen fractions in plant tissues for selected analytical methods for N determination. The size of the cells is not proportional to the mean sample content. Levels of free ammonium (NH`) and nitrite (NO~) are negligible in 4 2 plants because of their toxicity (from Simonne et al 1994).

T he Dumas method for nitrogen and crude protein determinations


TABLE 1 Samples, their categories and their N contents (% sample as is) Category Sample names and description Mean Kjeldahl N 1680 2290 1320 1960 2130 2030 1410 1400 2360 3010 9570 2140 2120 2040 1536 1536 1065 3740 1368 1210 0560 1820 2070 4090 0500 0510 0500 0500 4050 5540 2760 1304 3420 6640 1356 2430 3380 8730 1398 7980 3370 4520 4310 3000 2610 3730 3780 4700 3810 0170 0190 0140 0200 0070 0120 Mean Dumas N 198 252 136 212 232 205 143 142 237 304 958 2180 2160 2000 1535 1527 1132 3690 1370 1210 056 201 197 364 048 049 050 049 453 558 277 1306 342 669 1361 262 339 875 1402 800 466 562 565 285 280 473 518 657 580 067 022 016 021 007 013 Kjeldahl N : Dumas N Ratio 085 091 097 092 092 099 099 099 100 099 100 098 098 102 100 101 094 101 100 100 101 090 105 112 105 106 101 102 089 099 100 100 100 099 100 093 100 100 100 100 072 080 076 105 093 079 073 072 066 025 083 089 091 088 096 Reference

41

Cereals

Chemicals

Dairy products

Animal feeds

Fish

Fruit

Gold medal all-purpose our Gold medal bread our Long grain rice Pillsbury all-purpose our Pillsbury bread our Barley Corn Sorghum Wheat 1 Wheat 2 EDTA Ammonium sulphate 1 Ammonium sulphate 2 Histidine Lysine HCl Lysine HCl Nicotinic acid Thiourea Tryptophan Valine Chocolate milkshake Cottage cheese 1 Cottage cheese 2 Low-moisture Kroger mozzarella cheese Skim-milk 1 1% milk 1 Skim-milk 2 1% milk 2 Sargento Mozzarella Dry milk Alfalfa pellets Blood meal Broiler nisher Cattle concentrate Feather meal High nitrate grass Hog feed Meat meal Soya protein concentrate Soya bean meal Bumble bee tuna in oil 1 Bumble bee tuna in oil 2 Bumble been tuna in water Flounder Flat sh (uncooked) Star kist tuna in oil 1 Star kist tuna in oil 2 White tuna in oil White tuna in water Guava Nectarines (with skin) Peaches 1 Peaches 2 Pears Plum 1

In-house In-house In-house In-house Bicsak (1993) Bicsak (1993) Bicsak (1993) Bicsak (1993) Bicsak (1993) Bicsak (1993) Sweeney and Rexroad (1987) Minagawa et al (1984) Minagawa et al (1984) Minagawa et al (1984) Sweeney and Rexroad (1987) Bicsak (1993) Bicsak (1993) Minagawa et al (1984) Sweeney and Rexroad (1987) Minagawa et al (1984) In-house In-house In-house In-house In-house In-house In-house In-house In-house Sweeney and Sweeney and Sweeney and Sweeney and Sweeney and Sweeney and Sweeney and Sweeney and Sweeney and Sweeney and Sweeney and In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house

Rexroad Rexroad Rexroad Rexroad Rexroad Rexroad Rexroad Rexroad Rexroad Rexroad Rexroad

(1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987)

42
TABLE 1 Continued Category Sample names and description Mean Kjeldahl N 0120 0010 1560 1650 1810 1360 1770 1010 1640 1640 2480 2850 2500 1830 2160 2000 2560 7420 2180 1190 1270 1920 9260 1320 2050 1620 0130 0110 1750 3330 3710 5640 6540 2970 0830 0260 0410 0410 0180 0070 0060 0230 0240 0110 0910 0280 0560 0210 0180 0110 0220 Mean Dumas N 013 008 159 167 187 139 175 103 167 169 252 292 262 192 220 208 259 757 223 121 132 199 946 132 208 175 013 013 184 334 373 564 657 297 085 027 047 048 012 011 007 023 018 016 105 035 049 064 019 025 019 Kjeldahl N : Dumas N Ratio 093 015 098 099 097 098 101 098 098 097 098 098 095 095 098 096 099 098 098 098 096 096 098 100 099 092 101 086 095 100 099 100 100 100 098 097 086 085 140 064 082 097 133 068 087 078 113 033 095 042 120

A H Simonne et al

Reference

Fruit Baby foods and infant formula

Meats and meat products

Oilseeds

Vegetables and vegetable products

Plum 2 Sapodilla Beef Beef and ham Biscuits 1 Biscuits 2 Chicken Chicken carrots and potatoes Creme of cereal Creme of rice Formula 1 Formula 2 Formula 3 Formula 4 Formula 5 Formula 6 Formula 7 Ham and eggs Milk soup with cereal and fruits Milk soup with cereal and apples Semolina Turkey Veal Veal and brain Wheat our with milk and oat Eckrich bologna Heinz beef gravy (in jar) Heinz chicken gravy Oscar myer bologna Canola 1 Canola 2 Soya bean 1 Soya bean 2 Sunower Bush red kidney beans (canned) Campbell tomato soup Cooked broccoli 1 Cooked broccoli 2 Cooked cabbage Cucumber with skin Cucumber without skin Heinz catsup Hunts catsup Iceberg lettuce Kroger red kidney beans (canned) Kroger tomato soup Raw broccoli 1 Raw broccoli 2 Raw cabbage Tomato (raw with skin) 1 Tomato (raw with skin) 2

In-house In-house Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et Bellomonte et In-house In-house In-house In-house Bicsak (1993) Bicsak (1993) Bicsak (1993) Bicsak (1993) Bicsak (1993) In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house In-house

al al al al al al al al al al al al al al al al al al al al al al al

(1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987) (1987)

T he Dumas method for nitrogen and crude protein determinations


TABLE 2 Inuence of food group on lowest, highest and mean KN : DN ratio Food type Number of observations 10 5 10 10 23 10 4 17 9 8 L owest ratio 089 099 094 093 095 085 086 033 066 015 Highest ratio 112 100 102 100 101 100 101 140 105 096 Mean ratioa 101 100 099 099 098 095 094 089 080 073 a a a a a a a a bc c Standard deviation 007 000 002 002 001 005 006 028 012 033

43

Dairy products Oilseeds Chemicals Animal feed Infant formulas and baby foods Cereal Meats and meat products Vegetables and vegetable products Fish Fruit

a Mean followed by dierent letters are signicantly dierent according to Duncans multiple range test (alpha \ 5%).

aimed to (1) compare the Kjeldahl and Dumas methods over several food types and (2) evaluate the eect of replacing KN by DN on the calculation of CP.

MATERIALS AND METHODS Food samples from grocery stores of the Athens (GA, US) area and from the Georgia School Lunch Program monitoring study were selected to cover a wide range of matrices and expected N values. Published data from comparative studies using food and chemical samples were also included (Table 1). Kjeldahl N and DN were determined in duplicate on in-house samples. The mean of duplicates was used as the estimate of N content. KN was determined with a macro-Kjeldahl apparatus using copper sulphate as a catalyst by Method 98413 (AOAC 1995). For DN determination, 20 g of fresh sample was weighed in a tin foil and placed in a

drying oven at 70C for 12 h. Samples were pre-dried so that dierences in sample moisture would not inuence the results. After cooling at room temperature, samples were analysed (FP-428, Leco Corp, St Joseph, MI, USA). KN and DN were expressed as g N per 100 g of the sample before drying (%N). CP was independently calculated as CP1 \ H ] KN or CP2 \ H ] mean KN : DN ratio ] DN. CP was also calculated with DN and the single lowest (CP3) and highest (CP4) calculated KN : DN ratios. Dierences between CP1 and CP2 were used to evaluate the eect of N from dierent analytical method on CP. CP3 and CP4 provided extreme CP estimates. Nitrogen to protein conversion factors (H) of 57 for the cereal group, 638 for the milk group and 625 for the vegetable, fruit and meat groups were used to estimate CP. Regression analysis was performed on in-house and compiled values. For each food type, KN : DN ratios were calculated and dierences were evaluated by ANOVA (SAS 1987). Coefficients of variation (CV) were

TABLE 3 Predicted crude protein value using Kjeldahl N or Dumas N data Food type Nitrogen to protein conversion factors (H) 570 638 625 625 625 625 625 625 625 Crude protein estimatesa (g per 100 g as is) CP1 Cereal Dairy products Animal Feed Fish Fruit Infant formulas and baby foods Meat and meat products Vegetables and vegetable products Oilseeds 11 13 47 23 1 15 6 2 28 CP2 11 13 47 24 1 15 6 2 28 CP3 10 11 44 20 0 15 5 1 26 CP4 12 14 48 32 1 16 6 3 28

a CP1, Kjeldahl N ] H ; CP2, Dumas N ] Mean KN : DN ratio ] H ; CP3, Dumas N ] Lowest KN : DN ratio ] H ; CP4, Dumas N ] Highest KN : DN ratio ] H.

44 calculated as group standard deviation divided by the group mean multiplied by 100. Published results on chemical samples were statistically analysed separately from food samples.

A H Simonne et al vegetable and fruit samples and to the low level of N. However, this dierence may not be of practical importance since N levels in fruits and vegetables are usually low (\1% N). The high CV observed for the sh food group may be attributed to the relatively high non amino nitrogenous compounds in sh.

RESULTS AND DISCUSSION Chemical samples The mean KN : DN ratio calculated from published data on chemical samples was 099 (CV \ 2%) and ranged between 094 and 102 (n \ 10 observations). Mean KN : DN ratios were 100 for valine, 098 for NBS N-1 and NBS N-2 ammonium sulphate (Minagawa et al 1984), 093 for EDTA and 100 for tryptophan and lysine HCl (Sweeney and Rexroad 1987), 094 for nicotinic acid (Bicsak 1993), 099 for nicotinic acid and 100 for lysine HCl (King-Brink and Sebranek 1993), 101 for thiourea, 102 for histidine hydrochloride (Minagawa et al 1984) and 101 for lysine HCl (Bicsak 1993). In these chemicals, N was involved in CN (EDTA), HN (ammonium sulphate, lysine, thiourea, valine) or heterocyclic (tryptophan, histidine, nicotinic acid) linkages. Because KN : DN ratios were close to 100, N recovery by either method can be considered identical. Food samples Regression analysis on in-house samples (n \ 52 observations, R2 \ 096, P-regression \ 001) over the 0166% N range and all data combined (n \ 101 observations, R2 \ 098, P-regression \ 001) showed a close relationship between KN and DN. For all data, KN may be estimated from DN with KN \ 100(P:001) ] DN [ 009(P:050) in the 005 68% N range. Because dierent food matrices were included and because N levels within food types did not span the entire N range, KN : DN ratios were calculated for each food type. KN : DN ratios were signicantly (P \ 001 ; R2 \ 025) aected by food type (Table 2). KN may be estimated from DN using the appropriate corrective ratio. The numerical value of KN : DN ratios suggest that for dairy, feed, infant formula, cereal and meat, DN may replace KN without adjustment. However, adjustments of 089, 080 and 073 should be used for vegetable, sh and fruit samples, respectively. For the dairy, feed, infant formula, cereal and meat types, CV of the mean KN : DN ratios ranged between 1 and 5%. Values of 15, 31 and 45% were observed for the sh, vegetable and fruit types, respectively. The highest ratio found was 140 for cooked cabbage (sample no 85). This variation in mean KN : DN ratios may be attributed to dierences in nitrate contents in Crude protein Mean CP1 and CP2 values were [2, 0, 0, 1, 0, 0 and 0% and mean CP4 and CP3 values were 7, 3, 4, 12, 1, 1 and 2 for the cereal, dairy, feed, sh, fruit, infant formula, meat and vegetable food type, respectively (Table 3). These results suggest that dierences between CP estimates using KN or DN are of little practical importance in CP calculation.

CONCLUSIONS Dumas N could replace Kjeldahl N in food analysis when the numerical dierences between KN and DN are small (KN : DN ratios [ 090) without practical eect on CP calculation (CP1 and CP2 \ 2%). When the KN : DN \ 090, replacing KN by DN without adjustment will result in an overestimation of N and CP. However, DN may be used along with the appropriate KN : DN ratio. This adjustment may be easily included in computer programs that handle nutrient data. KN : DN ratios for the fruit and vegetable groups suggested the need for adjustment. However, expected N levels are low in these food groups ; the error introduced by replacing KN with DN will be of little practical importance. For complex foods which are difficult to classify in a single food group, CP may be estimated by adding the weighed partial values of each component. Partial values may be calculated with the appropriate KN : DN ratio and DN. CP may also be estimated by a t-all KN : DN ratio. The mean KN : DN ratio calculated in this study was 093. However, a larger database would provide a better estimate of the t-all ratio. DN would be less appropriate for CP calculations in cases such as shelf-life studies where changes of proteinor amino-N to other non-amino N compounds occur.

ACKNOWLEDGEMENTS The authors wish to thank Mrs Wonda Rogers (Food Science and Technology Department, University of Georgia) and Mr Mark Couvillon (Micro Macro International) for their technical assistance.

T he Dumas method for nitrogen and crude protein determinations REFERENCES


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