J Sci Food Agric 1997, 73, 39È45

Could the Dumas Method Replace the KjeldahlDigestion for Nitrogen and Crude ProteinDeterminations 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, USAb Department of Horticulture, 101 Funchess Hall, Auburn University, AL 36849-5408, USAc 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 crudeprotein (CP), has led to wider use of the Dumas method in place of the tradi-tional Kjeldahl methods. Although Kjeldahl N (KN) and Dumas N (DN) rep-resent di†erent N fractions, published studies on infant formula, animal feed andmeat products have indicated that DN could replace KN with little practicalimpact on the reliability of the N values obtained. This study was conducted toestablish whether DN determination could replace that of KN in a broaderrange of foods for CP calculation. Statistical analysis was performed on in-houseassayed KN and DN values together with published KN and DN values forselected food products. In the range 0É05È6É8% N, KN may be estimated fromDN with the equation : KN\ 1É00(P:0Õ01)] DN [ 0É09(P/0Õ50) (n \ 101,R2\ 0É98, P-regression \ 0É01). Because N levels in individual groups of fooddid not span the entire range of N contents, KN : DN ratios were calculated foreach food group. KN : DN ratios di†ered signiÐcantly (R2\ 0É25, P\ 0É01)from group to group. Ratios of 1É01 for dairy, 1É00 for oilseeds, 0É99 for feed, 0É98for infant formulas, 0É95 for cereals, 0É94 for meats, 0É89 for vegetables, 0É80 forÐsh and 0É73 for fruits were valid for the estimation of KN and CP using DNdata. CP was independently calculated as CP1\ H ] KN orCP2\ H ] KN : DN] DN, where H is the nitrogen to protein conversionfactor for the food group. Mean di†erences between CP1 and CP2 values were0% for dairy, oilseeds, feed, infant formulas and baby foods, cereals, meat andmeat products, vegetables and vegetable products and fruit, and 1% for Ðsh.These results suggest that DN may replace KN for the determination of N andCP 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 nutrientsof the Nutrition Labeling and Education Act of 1990and is usually estimated by multiplying nitrogen (N)content by a nitrogen to protein conversion factor. Inmost foods, amino-N accounts for approximately 16%

* This paper was presented at the 1995 annual meeting of theInstitute of Food Technologists 3È7 June, 1995 in Anaheim,CA, USA.” To whom correspondence should be addressed at : Depart-ment of Nutrition and Food Science, 328 Spidle Hall, AuburnUniversity, AL 36849, USA.

of the protein weight. Hence, the nitrogen to proteinconversion factor is usually 6É25, but speciÐc foods havesmaller (cereals) or higher (milk) conversion factorsdepending on the proportions of N in their proteins.

Direct methods for protein determination includebiuret, Lowry, bicinchoninic acid (BCA), ultraviolet(UV) absorption (280 nm), dye binding, Bradford,ninhydrin and turbidimetry. These methods are basedon properties of speciÐc proteins, speciÐc amino acidresidues in proteins or peptide bonds present in proteinsor peptides and involve extraction, isolation and some-times puriÐcation (Chang 1994 ; Pomeranz and Meloan1994). These methods are often used in biochemical

39J Sci Food Agric 0022-5142/97/$09.00 1997 SCI. Printed in Great Britain(

40 A H Simonne et al

investigations but they are completely impractical forroutine food analysis because foods contain a complexmixture of proteins (Chang 1994 ; Pomeranz andMeloan, 1994). Moreover, only dye binding methods(official methods 967É12 and 975É17) are approved fordirect determination of protein in milk (AOAC 1995).

Official methods for N determinations in foodsinclude modiÐcations of the Kjeldahl method (981É10,955É04, 977É02, 978É04, 920É152, 920É87, 945É18B, 950É36,930É33, 930É29, 928É08, 977É14, 950É48 and 935É39 with aHg catalyst ; and 991É20, 991É22 and 991É23 with a Cucatalyst) (Sullivan and Carpenter 1993). AOAC OfficialMethod 984É13 “Protein (Crude) in Animal FeedÏ alsoutilises a Cu catalyst (AOAC 1995). The Kjeldahlmethod consists of (a) a digestion step where N is con-verted into ammonium and (b) an analytical(NH4`),step where is quantiÐed by titrimetry, colorimetryNH4`or using an ion-speciÐc electrode. This method assumesthat N recovered during digestion is mainly amino-Nfrom proteins (total organic N) and that the contribu-tion of inorganic N (nitrate, nitrite, ammonium) orother organic N (nucleotides, nucleic acids) is negligible.Presently, methods used for determining the totalprotein (referred to as crude protein) content of foodsinvolve (a) determination of N, and (b) calculationwhere the N value is converted to protein by a food-speciÐc nitrogen to protein conversion factor, which willbe refer to as “HÏ throughout this manuscript.

The main advantage of Kjeldahl methods is its widelyestablished use in CP estimation. However, Kjeldahlmethods require wet chemistry, the handling of concen-trated sulphuric acid and a heavy-metal catalyst. Inconsequence, for routine analysis the Kjeldahl methodis a tedious and time-consuming procedure requiringdisposal of hazardous wastes. For these reasons, ana-lytical laboratories tend to adopt automated and easy-to-use equipment (such as the LECO FP-428, LECOCHN 600 or Carlo Erba NA-1500) for routine, unat-tended N determination. These instruments use theDumas method which consists of (a) converting all theforms of N into gaseous nitrogen oxides by com-(NO

x)

plete combustion in an induction furnace, (b) reducingthe gases to and (c) quantifying by thermalNO

xN2 N2

conductivity (Sweeney and Rexroad 1987 ; Jones 1991).Thus, for the Dumas method (also referred to as the

combustion method) wet chemistry is not involved andtime for analysis is reduced to approximately 6 min persample, furthermore liquid, semi-solid or solid samplescan be analysed. Moreover, its accuracy and repeatabil-ity may be superior to that of the Kjeldahl method(Schmitter and Rihs 1989).

Because of its nature, Dumas N (DN) is a truemeasure of total N. In food products (Minagawa et al1984) and in plants (Sweeney and Rexroad 1987 ;Simonne et al 1994), DN and Kjeldahl N (KN) recov-ered di†erent fractions of N (Fig 1). During the Kjeldahldigestion, reducing conditions caused by the release offree carbon favour the conversion of nitrate to NH4`and losses of nitrate as (Bradstreet 1960). Therefore,N2KN may include some non-amino N from nitrates,nitrites, nucleotides or nucleic acids (Nelson andSommers 1980 ; Pace et al 1982 ; du Preez and Bate1989a,b ; Goyal and Hafez 1990 ; Barbano et al 1991).The reduction of non-amino N during Kjeldahldigestion depends on the chemical form of the non-amino N and sample matrix. Hence, the recovery ofnon-amino N is non-quantitative. The combustionmethod was approved for CP determination in animalfeed (Sweeney 1989), meat and meat products (King-Brink and Sebranek 1993), and cereal grains and oil-seeds (Bicsak 1993). An in-depth study was also con-ducted with infants foods (Bellomonte et al 1987) andtwo 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 evaluatingthe e†ect of analytical method in each food group isvery thorough, it is slow and expensive. In addition, it issometimes difficult to classify food products into asingle category. For example, a food designated forinfant food and containing turkey is classiÐed as ainfant food, while a meat or blood meal designated foranimal consumption was part of a study on animal feed.Matrix di†erences between these two samples may notbe di†erent enough to justify the distinction implied bythe two categories. Another limitation to this classi-Ðcation is that compound foods may belong to two dif-ferent food groups. For instance, soups containing milk,chicken and fruits may be regarded as a modiÐed dairyproduct 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 sizeof the cells is not proportional to the mean sample content. Levels of free ammonium and nitrite are negligible in(NH4`) (NO2~)

plants because of their toxicity (from Simonne et al 1994).

T he Dumas method for nitrogen and crude protein determinations 41

TABLE 1Samples, their categories and their N contents (% sample as is)

Category Sample names and Mean Mean Kjeldahl Referencedescription Kjeldahl Dumas N : Dumas

N N N Ratio

Cereals Gold medal all-purpose Ñour 1É680 1É98 0É85 In-houseGold medal bread Ñour 2É290 2É52 0É91 In-houseLong grain rice 1É320 1É36 0É97 In-housePillsbury all-purpose Ñour 1É960 2É12 0É92 In-housePillsbury bread Ñour 2É130 2É32 0É92 Bicsak (1993)Barley 2É030 2É05 0É99 Bicsak (1993)Corn 1É410 1É43 0É99 Bicsak (1993)Sorghum 1É400 1É42 0É99 Bicsak (1993)Wheat 1 2É360 2É37 1É00 Bicsak (1993)Wheat 2 3É010 3É04 0É99 Bicsak (1993)

Chemicals EDTA 9É570 9É58 1É00 Sweeney and Rexroad (1987)Ammonium sulphate 1 21É40 21É80 0É98 Minagawa et al (1984)Ammonium sulphate 2 21É20 21É60 0É98 Minagawa et al (1984)Histidine 20É40 20É00 1É02 Minagawa et al (1984)Lysine HCl 15É36 15É35 1É00 Sweeney and Rexroad (1987)Lysine HCl 15É36 15É27 1É01 Bicsak (1993)Nicotinic acid 10É65 11É32 0É94 Bicsak (1993)Thiourea 37É40 36É90 1É01 Minagawa et al (1984)Tryptophan 13É68 13É70 1É00 Sweeney and Rexroad (1987)Valine 12É10 12É10 1É00 Minagawa et al (1984)

Dairy products Chocolate milkshake 0É560 0É56 1É01 In-houseCottage cheese 1 1É820 2É01 0É90 In-houseCottage cheese 2 2É070 1É97 1É05 In-houseLow-moisture Kroger

mozzarella cheese 4É090 3É64 1É12 In-houseSkim-milk 1 0É500 0É48 1É05 In-house1% milk 1 0É510 0É49 1É06 In-houseSkim-milk 2 0É500 0É50 1É01 In-house1% milk 2 0É500 0É49 1É02 In-houseSargento Mozzarella 4É050 4É53 0É89 In-houseDry milk 5É540 5É58 0É99 Sweeney and Rexroad (1987)

Animal feeds Alfalfa pellets 2É760 2É77 1É00 Sweeney and Rexroad (1987)Blood meal 13É04 13É06 1É00 Sweeney and Rexroad (1987)Broiler Ðnisher 3É420 3É42 1É00 Sweeney and Rexroad (1987)Cattle concentrate 6É640 6É69 0É99 Sweeney and Rexroad (1987)Feather meal 13É56 13É61 1É00 Sweeney and Rexroad (1987)High nitrate grass 2É430 2É62 0É93 Sweeney and Rexroad (1987)Hog feed 3É380 3É39 1É00 Sweeney and Rexroad (1987)Meat meal 8É730 8É75 1É00 Sweeney and Rexroad (1987)Soya protein concentrate 13É98 14É02 1É00 Sweeney and Rexroad (1987)Soya bean meal 7É980 8É00 1É00 Sweeney and Rexroad (1987)

Fish Bumble bee tuna in oil 1 3É370 4É66 0É72 In-houseBumble bee tuna in oil 2 4É520 5É62 0É80 In-houseBumble been tuna in water 4É310 5É65 0É76 In-houseFlounder 3É000 2É85 1É05 In-houseFlat Ðsh (uncooked) 2É610 2É80 0É93 In-houseStar kist tuna in oil 1 3É730 4É73 0É79 In-houseStar kist tuna in oil 2 3É780 5É18 0É73 In-houseWhite tuna in oil 4É700 6É57 0É72 In-houseWhite tuna in water 3É810 5É80 0É66 In-house

Fruit Guava 0É170 0É67 0É25 In-houseNectarines (with skin) 0É190 0É22 0É83 In-housePeaches 1 0É140 0É16 0É89 In-housePeaches 2 0É200 0É21 0É91 In-housePears 0É070 0É07 0É88 In-housePlum 1 0É120 0É13 0É96 In-house

42 A H Simonne et al

TABLE 1 Continued

Category Sample names and Mean Mean Kjeldahl Referencedescription Kjeldahl Dumas N : Dumas

N N N Ratio

Fruit Plum 2 0É120 0É13 0É93 In-houseSapodilla 0É010 0É08 0É15 In-house

Baby foods and Beef 1É560 1É59 0É98 Bellomonte et al (1987)infant formula Beef and ham 1É650 1É67 0É99 Bellomonte et al (1987)

Biscuits 1 1É810 1É87 0É97 Bellomonte et al (1987)Biscuits 2 1É360 1É39 0É98 Bellomonte et al (1987)Chicken 1É770 1É75 1É01 Bellomonte et al (1987)Chicken carrots and potatoes 1É010 1É03 0É98 Bellomonte et al (1987)Creme of cereal 1É640 1É67 0É98 Bellomonte et al (1987)Creme of rice 1É640 1É69 0É97 Bellomonte et al (1987)Formula 1 2É480 2É52 0É98 Bellomonte et al (1987)Formula 2 2É850 2É92 0É98 Bellomonte et al (1987)Formula 3 2É500 2É62 0É95 Bellomonte et al (1987)Formula 4 1É830 1É92 0É95 Bellomonte et al (1987)Formula 5 2É160 2É20 0É98 Bellomonte et al (1987)Formula 6 2É000 2É08 0É96 Bellomonte et al (1987)Formula 7 2É560 2É59 0É99 Bellomonte et al (1987)Ham and eggs 7É420 7É57 0É98 Bellomonte et al (1987)Milk soup with cereal and fruits 2É180 2É23 0É98 Bellomonte et al (1987)Milk soup with cereal and apples 1É190 1É21 0É98 Bellomonte et al (1987)Semolina 1É270 1É32 0É96 Bellomonte et al (1987)Turkey 1É920 1É99 0É96 Bellomonte et al (1987)Veal 9É260 9É46 0É98 Bellomonte et al (1987)Veal and brain 1É320 1É32 1É00 Bellomonte et al (1987)Wheat Ñour with milk and oat 2É050 2É08 0É99 Bellomonte et al (1987)

Meats and meat Eckrich bologna 1É620 1É75 0É92 In-houseproducts Heinz beef gravy (in jar) 0É130 0É13 1É01 In-house

Heinz chicken gravy 0É110 0É13 0É86 In-houseOscar myer bologna 1É750 1É84 0É95 In-house

Oilseeds Canola 1 3É330 3É34 1É00 Bicsak (1993)Canola 2 3É710 3É73 0É99 Bicsak (1993)Soya bean 1 5É640 5É64 1É00 Bicsak (1993)Soya bean 2 6É540 6É57 1É00 Bicsak (1993)SunÑower 2É970 2É97 1É00 Bicsak (1993)

Vegetables Bush red kidney beans (canned) 0É830 0É85 0É98 In-houseand vegetable Campbell tomato soup 0É260 0É27 0É97 In-houseproducts Cooked broccoli 1 0É410 0É47 0É86 In-house

Cooked broccoli 2 0É410 0É48 0É85 In-houseCooked cabbage 0É180 0É12 1É40 In-houseCucumber with skin 0É070 0É11 0É64 In-houseCucumber without skin 0É060 0É07 0É82 In-houseHeinz catsup 0É230 0É23 0É97 In-houseHunts catsup 0É240 0É18 1É33 In-houseIceberg lettuce 0É110 0É16 0É68 In-houseKroger red kidney beans (canned) 0É910 1É05 0É87 In-houseKroger tomato soup 0É280 0É35 0É78 In-houseRaw broccoli 1 0É560 0É49 1É13 In-houseRaw broccoli 2 0É210 0É64 0É33 In-houseRaw cabbage 0É180 0É19 0É95 In-houseTomato (raw with skin) 1 0É110 0É25 0É42 In-houseTomato (raw with skin) 2 0É220 0É19 1É20 In-house

T he Dumas method for nitrogen and crude protein determinations 43

TABLE 2InÑuence of food group on lowest, highest and mean KN :DN ratio

Food type Number of L owest Highest Mean Standardobservations ratio ratio ratioa deviation

Dairy products 10 0É89 1É12 1É01 a 0É07Oilseeds 5 0É99 1É00 1É00 a 0É00Chemicals 10 0É94 1É02 0É99 a 0É02Animal feed 10 0É93 1É00 0É99 a 0É02Infant formulas and baby foods 23 0É95 1É01 0É98 a 0É01Cereal 10 0É85 1É00 0É95 a 0É05Meats and meat products 4 0É86 1É01 0É94 a 0É06Vegetables and vegetable products 17 0É33 1É40 0É89 a 0É28Fish 9 0É66 1É05 0É80 bc 0É12Fruit 8 0É15 0É96 0É73 c 0É33

a Mean followed by di†erent letters are signiÐcantly di†erent according to DuncanÏs multiple rangetest (alpha \ 5%).

aimed to (1) compare the Kjeldahl and Dumas methodsover several food types and (2) evaluate the e†ect ofreplacing 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 Programmonitoring study were selected to cover a wide range ofmatrices and expected N values. Published data fromcomparative studies using food and chemical sampleswere also included (Table 1). Kjeldahl N and DN weredetermined in duplicate on in-house samples. The meanof duplicates was used as the estimate of N content.

KN was determined with a macro-Kjeldahl appar-atus using copper sulphate as a catalyst by Method984É13 (AOAC 1995). For DN determination, 2É0 g offresh sample was weighed in a tin foil and placed in a

drying oven at 70¡C for 12 h. Samples were pre-dried sothat di†erences in sample moisture would not inÑuencethe results. After cooling at room temperature, sampleswere analysed (FP-428, Leco Corp, St Joseph, MI,USA). KN and DN were expressed as g N per 100 g ofthe sample before drying (%N).

CP was independently calculated as CP1\ H ] KNor CP2\ H ] mean KN : DN ratio] DN. CP wasalso calculated with DN and the single lowest (CP3)and highest (CP4) calculated KN : DN ratios. Di†er-ences between CP1 and CP2 were used to evaluate thee†ect of N from di†erent analytical method on CP. CP3and CP4 provided extreme CP estimates. Nitrogen toprotein conversion factors (H) of 5É7 for the cerealgroup, 6É38 for the milk group and 6É25 for the veget-able, fruit and meat groups were used to estimate CP.

Regression analysis was performed on in-house andcompiled values. For each food type, KN : DN ratioswere calculated and di†erences were evaluated byANOVA (SAS 1987). Coefficients of variation (CV) were

TABLE 3Predicted crude protein value using Kjeldahl N or Dumas N data

Food type Nitrogen to protein conversion Crude protein estimatesa (g per 100 g as is)factors (H)

CP1 CP2 CP3 CP4

Cereal 5É70 11 11 10 12Dairy products 6É38 13 13 11 14Animal Feed 6É25 47 47 44 48Fish 6É25 23 24 20 32Fruit 6É25 1 1 0 1Infant formulas and baby foods 6É25 15 15 15 16Meat and meat products 6É25 6 6 5 6Vegetables and vegetable products 6É25 2 2 1 3Oilseeds 6É25 28 28 26 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 A H Simonne et al

calculated as group standard deviation divided by thegroup mean multiplied by 100. Published results onchemical samples were statistically analysed separatelyfrom food samples.

RESULTS AND DISCUSSION

Chemical samples

The mean KN : DN ratio calculated from publisheddata on chemical samples was 0É99 (CV\ 2%) andranged between 0É94 and 1É02 (n \ 10 observations).Mean KN : DN ratios were 1É00 for valine, 0É98 forNBS N-1 and NBS N-2 ammonium sulphate(Minagawa et al 1984), 0É93 for EDTA and 1É00 fortryptophan and lysine HCl (Sweeney and Rexroad1987), 0É94 for nicotinic acid (Bicsak 1993), 0É99 fornicotinic acid and 1É00 for lysine HCl (King-Brink andSebranek 1993), 1É01 for thiourea, 1É02 for histidinehydrochloride (Minagawa et al 1984) and 1É01 for lysineHCl (Bicsak 1993). In these chemicals, N was involvedin ÈCÈNÈ (EDTA), HÈNÈ (ammonium sulphate, lysine,thiourea, valine) or heterocyclic (tryptophan, histidine,nicotinic acid) linkages. Because KN : DN ratios wereclose to 1É00, N recovery by either method can be con-sidered identical.

Food samples

Regression analysis on in-house samples (n \ 52observations, R2\ 0É96, P-regression \ 0É01) over the0É1È6É6% N range and all data combined (n \ 101observations, R2\ 0É98, P-regression \ 0É01) showed aclose relationship between KN and DN. For all data,KN may be estimated from DN withKN\ 1É00(P:0Õ01) ] DN[ 0É09(P:0Õ50) in the 0É05È6É8% N range. Because di†erent food matrices wereincluded and because N levels within food types did notspan the entire N range, KN : DN ratios were calcu-lated for each food type.

KN : DN ratios were signiÐcantly (P\ 0É01 ;R2\ 0É25) a†ected by food type (Table 2). KN may beestimated from DN using the appropriate correctiveratio. The numerical value of KN : DN ratios suggestthat for dairy, feed, infant formula, cereal and meat, DNmay replace KN without adjustment. However, adjust-ments of 0É89, 0É80 and 0É73 should be used for veget-able, Ðsh and fruit samples, respectively.

For the dairy, feed, infant formula, cereal and meattypes, CV of the mean KN : DN ratios ranged between1 and 5%. Values of 15, 31 and 45% were observed forthe Ðsh, vegetable and fruit types, respectively. Thehighest ratio found was 1É40 for cooked cabbage(sample no 85). This variation in mean KN : DN ratiosmay be attributed to di†erences in nitrate contents in

vegetable and fruit samples and to the low level of N.However, this di†erence may not be of practical impor-tance since N levels in fruits and vegetables are usuallylow (\1% N). The high CV observed for the Ðsh foodgroup may be attributed to the relatively high nonamino nitrogenous compounds in Ðsh.

Crude protein

Mean CP1 and CP2 values were [2, 0, 0, 1, 0, 0 and0% and mean CP4 and CP3 values were 7, 3, 4, 12, 1, 1and 2 for the cereal, dairy, feed, Ðsh, fruit, infantformula, meat and vegetable food type, respectively(Table 3). These results suggest that di†erences betweenCP estimates using KN or DN are of little practicalimportance in CP calculation.

CONCLUSIONS

Dumas N could replace Kjeldahl N in food analysiswhen the numerical di†erences between KN and DNare small (KN : DN ratios [ 0É90) without practicale†ect on CP calculation (CP1 and CP2\ 2%). Whenthe KN : DN \ 0É90, replacing KN by DN withoutadjustment will result in an overestimation of N andCP. However, DN may be used along with the appro-priate KN : DN ratio. This adjustment may be easilyincluded in computer programs that handle nutrientdata.

KN : DN ratios for the fruit and vegetable groupssuggested the need for adjustment. However, expectedN levels are low in these food groups ; the error intro-duced by replacing KN with DN will be of little practi-cal importance.

For complex foods which are difficult to classify in asingle food group, CP may be estimated by adding theweighed partial values of each component. Partialvalues may be calculated with the appropriate KN : DNratio and DN. CP may also be estimated by a Ðt-allKN : DN ratio. The mean KN : DN ratio calculated inthis study was 0É93. However, a larger database wouldprovide a better estimate of the Ðt-all ratio.

DN would be less appropriate for CP calculations incases such as shelf-life studies where changes of protein-or amino-N to other non-amino N compounds occur.

ACKNOWLEDGEMENTS

The authors wish to thank Mrs Wonda Rogers (FoodScience and Technology Department, University ofGeorgia) and Mr Mark Couvillon (Micro MacroInternational) for their technical assistance.

T he Dumas method for nitrogen and crude protein determinations 45

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