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Urea determination in milk by a differential pHtechnique

Massimo Luzzana, Raffaella Giardino

To cite this version:Massimo Luzzana, Raffaella Giardino. Urea determination in milk by a differential pH technique. LeLait, INRA Editions, 1999, 79 (2), pp.261-267. �hal-00929648�

Lait (1999) 79, 261-267© Inra!EIsevier, Paris

261

Original article

Urea determination in milkby a differential pH technique

Massimo Luzzana-", Raffaella Giardino"

a Dipartimento di scienze e tecnologie biomediche, Università degli Studi di Milano, Milan, Italyb CNR Centro Studi Latte, via Celoria 2, Milan, Italy

(Received 9 September 1997; accepted 2 November 1998)

Abstract - A differential pH method for the routine determination of urea in milk is presented.The measure is based upon a single enzymatic reaction, the urea hydrolysis by urease, which causesa pH variation directly proportional to the urea content in the milk sample. No sample pretreat-ment is required. Repeatability expressed as a coefficient of variation was 0.85 % at a level of416 mg-urea-L -1. Recovery of added urea averaged 99.4 %. The preservatives Bronopol and sodiumazide at a ten times higher level than recommended did not affect the results. Comparison with the man-ual enzymatic French reference method showed a very good agreement. The method is very simple,accurate and rapid. © InralElsevier, Paris

urea 1 differential pH 1 milk 1 enzymatic assay

Résumé - Dosage de l'urée dans le lait avec la technique de pHmétrie différentielle. Uneméthode de pHmétrie différentielle pour la détermination de l'urée dans le lait est présentée. Lamesure est basée sur une seule réaction enzymatique, l'hydrolyse de l'urée par l'enzyme urease, quiprovoque une variation de pH directement proportionnelle au niveau de l'urée dans l'échantillon delait. Aucun prétraitement de l'échantillon n'est nécessaire. Le coefficient de variation de la répétabilitévaut 0,85 % au niveau de 416 rng-urée-L -1. La détermination de l'urée ajoutée était en moyenne de99,4 %. L'addition de conservateurs tels que le Bronopol ou l'azide de sodium à une concentrationdix fois supérieure à la quantité conseillée n'avait aucune influence sur les résultats. La comparaisonavec la méthode manuelle enzymatique française de référence donne une très bonne corrélation. Laméthode est très simple, précise et rapide. © Inra!Elsevier, Paris

urée 1 pHmétrie différentielle liait 1 essai enzymatique

* Correspondence and reprints.

262 M. Luzzana, R. Giardino

1. INTRODUCTION

Ureic nitrogen in milk is derived essen-tially from the ureic nitrogen in blood; sev-eral reports have demonstrated a close cor-relation between urea levels in blood and inmilk [3, 12]. Measuring the milk urea andthe blood urea contents is approximately thesame, but since milk is more easily avail-able, it is a very suitable medium for theanalysis of urea.

In mammals, urea represents the moleculeused for excretion of ammonia; it is synthe-sized in the liver from the ammonia, which,in free form, is extremely toxic for the organ-ism. Inruminants, the ammonia reaching theliver has two origins: exogenous, absorbedthrough the rumen' s walls, and endogenous,originated from the amino acid of the ani-mal organism. Its concentration in blood ishighly affected by dietary factors, especiallythe amount of protein ingested and the pro-tein/energy ratio [4, 8]. Consequently, deter-mination of the urea in milk provides impor-tant information about the cows' feedingpractices.

Several authors have also demonstrated agood correlation between the urea content inmilk and the health problems of cows [6,7], heat stability of milk proteins [2, 5] andcheese characteristics and yield [9-11, 13].

Regular determinations of urea in milkfrom a large number of cows can be helpfulin several ways; however, for routine anal-ysis, simple, rapid, accurate and inexpen-sive methods are required.

The purpose of our work has been to opti-mize a new method to determine the ureacontent in milk with an analytical appara-tus which fulfils the following requirements:the instrument is smal1 and easy to use, nosample pretreatment is necessary, the anal-ysis rate and stability reagents are high, andit is accurate and precise.

2. MATERIALS AND METRODS

2.1. Principle

The addition of urease to a urea-containingsolution promotes the following reaction:

Reaction (1) is rate limited by the amount ofurease added. Its half-time varies between 20 to2 s (10-100 U'-rnl,"! of urease) and producesammonia, whieh, at pH 6.7 immediately hydro-lyzes, thus releasing hydroxyl ions. Carbon diox-ide also hydrolyzes, with a half-time of about30 s, thus partially neutralizing the OH- pro-duced by step (2):

2NH3 + 2HzÜ H 2NH4 + + 20H- (2)

COz + H20 H HC03- + W (3)

2.2. Instrumentation

The change in pH produced by the urea' sreaction in a properly buffered system is me a-sured by a commercially available differentialpH analyzer, CL 10 Micro (kindly supplied byEurochem, S.r.!, Ardea, Rome, Italy). The appa-ratus (figure 1) is essentially composed of a mea-surement block thermostatic stabilized between30 and 37 "C, containing a mixing chamber, twocapillary glass electrodes and five peristalticpumps. The analyser is controlled by a personalcomputer running program 'pH 4000', with areal-time graphie display of a differential pH sig-nal.

2.3. Method

Twenty ul, of milk are added to the mixingchamber containing 970 ul, of buffer solution; ahalf volume of milk and buffer solution is aspi-rated in both the electrodes and the pH differ-ence between the electrodes is measured. Four-teen ul, of enzyme are then automatically addedto the milk and buffer solution still remaining inthe mixing chamber and the solution is aspiratedin only one electrode. The new pH difference ismonitored.

Endogenous ammonia in milk does not inter-fere with the differential pH measurement,because both electrodes contain the same dilutedmilk sample.

P5

c

Urea determination in milk 263

A P

EL!

lTIllE

1 1 1

G

EL2"?1 it

1M

_~le. • ._'

; .it<

•wFigure 1. Diagram of the differential pH apparatus. A: DifferentiaI amplifier; B: buffer solution;C: mixing chamber; D: display; El and E2: glass capillary electrodes; EL: electronics; ES: enzymeor substrate solution; G: ground; K: keyboard; M: magnetic stirrer; P: printer; PI to P5: peristalticpumps; S: micropipette for injection of sample; W: waste.Figure 1. Diagramme du pHmètre différentiel: A amplificateur différentiel; B solution tampon;C chambre de mélange; D display ; El et E2 électrodes capillaires en verre; EL électronique; ES solu-tion de l'enzyme ou du substrat; G terre; K clavier; M agitateur magnétique; P imprimante; PI àP5 pompes péristaltiques; S micropipette pour l'injection de l'échantillon; W déchets.

2.4. Materials

The buffer solution used had the followingcomposition: 8 mmol-L:' NaH2P04, 12 mmol-L:'Na2HP04, 100 mmol-Lr! KCI, 1 g.L-1 NaN),10 mg-L:" acetazolamide, 2 mmol-L"! Mg?",2 g-L -1 Triton x 100, 1 g-L -1 Brij 35 (supplierFluka), 2 % v/v LM1 (supplier Kartell), pH 6.7(at 25 oC).

Urease (E.C. 3.5.1.5.) Iyophilized, suppliedby Genzyme, was diluted in an aqueous solutionof glycerol 50 % v/v to the final concentrationof 1 800 V·mL-I.

Aqueous urea standard solutions were pre-pared by diluting a stock solution of 100 g·L-1urea in KCI 100 mmol-Lr! and 1 g·L-1 NaN).

Ali of the milk sampI es used were raw wholemilk samples or raw whole milk samples addedto Bronopol (2-bromo-2-nitropropane-1 ,3-diol)

either individual or taken From farm tanks. ThesampI es were stored at 4 "C and analyzed within1 or 2 d, if not mixed with Bronopol, or within1 week if mixed with Bronopol.

2.5. Reagents stability

Three preservatives, sodium azide, sodiummerthiolate and Bronopol, were evaluated at dif-ferent concentrations in relation to their influ-ence on the urease activity.

The stability as say was carried out at refrig-erated and room temperature, on different lotsof the reagents, added to sodium azide orBronopol, while monitoring the followingparameters: pH and buffer power for the buffersolution with sodium azide 1g·L-1 and the buffersolution with Bronopol 1 g-L-1; pH and urea con-

264 M. Luzzana, R. Giardino

tent for the standard solution with sodium azide1 g-L-1 and the standard solution with Bronopol1 g·L-I; pH and enzyme activity for the ureasesolution.

Ali the reagents being tested were analyzedonce a week for a period of 6 months.

2.6. Reference method

Milk samples were analyzed using the Frenchreference method, NFV04-217-Afnor [1], a man-ual enzymatic determination. Urea is hydrolyzedby the enzyme urease to give ammonium ionsand carbonate. The ammonium ions formed thenact as substrate for the enzyme glutamate dehy-drogenase:

œ-ketoglutarate + NH/ + NADH-7 glutamate + NAD+ + HzÜ (4)

The decrease in absorbance at 340 nm fol-lowing the oxidation of NADH is monitoredspectrophotometrically. Milk protein and fat haveto be precipitated and filtrated before the analy-sis and the decrease of absorbance due to theendogenous ammonia in the milk has to be sub-tracted from the total absorbance variationobtained.

3.RESULTS

The linearity between pH signal and ureaconcentration, in aqueous standard solutionwith a urea content of up to 5 000 mg-L-1, ispresented infigure 2. The regression of theresults of differential pH method (f) on theurea content (X) is Y = 31.5 X - 8.8, and thecorrelation coefficient (R) = 0.999.

3.1. Repeatability

The repeatability of the method was eval-uated by analyzing ten times three whole milksamples at three different levels of urea con-tent-respectively, 140,223 and 416 mg-L:':the coefficients of variation were 1.50, 0.86and 0.85 %, respectively. The results are pre-sented in table J.

3.2. Recovery

The recovery was evaluated by addingdifferent known amounts of an aqueous urea

160-,--------------------:/'1

i 120E<1

:::E:Q. 80iii

] 40

"Cl

y = 31.5 X - 8.8R = 0.999

1000 50002000 3000 4000

urea standard (mq.L")

Figure 2. Method linearity with aqueous urea standards (500, 1 000, 2 000, 3 000, 4 000 and5 000 mg-L").Figure 2. Linéarité de la méthode avec standards aqueux d'urée (500, 1 000, 2 000, 3 000, 4 000,5 000 mg-urée-L''').

Urea determination in milk

solution (100 g·L-I) to a whole raw milksample with endogenous urea concentrationof 247 mg-L:'.

The average recovery was 99.4 %;regression of measured urea minus endoge-nous urea content on urea added was:y = 0.99 X + 1.2, R = 0.999. The results arepresented in table II.

3.3. Accuracy

The accuracy of the differential pHmethod was evaluated by comparison witha manu al enzymatic method. The results ofthis method comparison, carried out in dou-ble on 47 whole milk samples, preservedwith Bronopol, containing 80 to 500 mg-L:'of urea, are presented infigure 3.

Calculation of the regression of the resultsof differential pH method on the results ofenzymatic manual method yields the fol-lowing equation: Y = 0.968 X + 12.191,R =0.997.

The mean value of the 94 determinationswas 300.9 mg-Ir" for the manual enzymaticmethod and 303.6 mg-L-1 for the differentialpH method. The mean difference and thestandard deviation of the differencesbetween the two methods were, respectively,-2.7 and 8.5 mg-L:'.

According to the t-test the mean resultsfrom the two methods are not significantlydifferent from each other. The coefficients ofvariation of the repeatability between thedouble determinations were 0.97 % for thedifferential pH method and 1.36 % for themanual enzymatic method.

3.4. Reagents stability

The activityof a ureasesolution1800 UmL-1

was measured in the buffer solution added todifferent concentrations of preservatives.The results are shown in table III. Bronopolhas no inhibitory effect, sodium azideslightly inhibits the urease activity at theconcentration of 2 % and sodium merthiolate

265

Table I.Repeatability on three raw whole milksamples with different urea content (resultsexpressed in mg-L:'),Tableau I.Répétabilité sur trois échantillons delait cru avec différents niveaux d'urée (résultatsen mg-L:').

Milk 1 Milk3Milk2

Averagecrn_1

CV%

141142139141140141142136143138

1402.111.50

226222222220223225224223221225

2231.910.86

412420419414416421417419411413

4163.550.85

Table II. Recovery on raw milk added with 100,200,400,600 and 800 mg-urea-L:'.Tableau Il. Détermination de l'urée ajoutée danslait cru, 100,200,400,600,800 mg-urée-L".

x 1 x 2 Average Rec. %

Milk 0 (mg-Ir")+ 100+200+400+600+ 800

248346446643839

1041

246346450645848

1032

247346448644844

1037

99.0100.599.399.598.8

has a strong inhibitory effect only at con-centration five times higher than those nor-mally used. However, we decided to excludesodium merthiolate from the stability assay.

No modification of any buffer and stan-dard solutions was observed after 6 monthsof storage.

The activity of the urease solutions atrefrigerated temperature was unmodifiedafter 6 months of storage, while the pHdecreased of approximately 0.002 pH.

266 M. Luzzana, R. Giardino

600 ~--------------------------.

y = 0.968X + 12.2n =47R = 0.997

400

200

Table III. Influence of different preservativesin phosphate buffer on the urease activity.Tableau III. Influence de différents conserva-teurs en tampon phosphate sur l'activité del'uréase.

Preservative Activity(U·mL-I)

Concentration(%)

0.1 1820Bronopol 0.5 1 760

2.0 1800

0.1 1790Sodium azide 0.5 1 810

2.0 1570

0.1 1800Sodium merthiolate 0.5 1 120

2.0 490

The urease solutions at room tempera-ture after 17 weeks of storage graduallystarted loosing activity until the average lossof 20 % after 6 months. A graduai acidifi-cation was observed, probably caused bythe protein denaturation.

o+-----,.------,----,..----,----~--~o 100 200 300 400 500 600

enzymatic method (mg.L·1)

Figure 3. Correlation between the manual enzymatic method (standard Afnor V04-217) and thedifferential pH method.Figure 3. Corrélation entre la méthode manuelle enzymatique (standard Afnor V04-217) et laméthode de pHmétrie différentielle.

3.5. Influence of preservatives

The influence of preservatives was eval-uated by adding increasing amounts of anaqueous solution (5 %) of Bronopol (2-bromo-2-nitropropane-I,3 diol) and an aqueoussolution (5 %) of sodium azide to three dif-ferent raw milk samples. The resultsobtained (table IV) do not show any differ-ence, within the experimental error, forunpreserved and preserved samples, for ailthe Bronopol and sodium azide concentra-tions tested.

4. DISCUSSION

The method presented here has a num-ber of definite advantages as compared tothe other existing methods for the determi-nation of milk urea: the system usesuntreated whole milk and no interference iscaused by fat, fat drop lets or by other sus-pended material.

The method derives its accuracy fromthe high specificity of the single enzymatic

Urea determination in milk

Table IV. Influence of Bronopol and sodiumazide at four concentration levels (0.02, 0.05, 0.1and 0.2 %) on three different raw milk sam pies(results expressed in mg-L").Tableau IV. Influence de Bronopol et azide desodium à quatre niveaux de concentration (0,02,0,05, 0,1 et 0,2%) sur trois échantillons de laitcru.

Preservative Milk 1 Milk 2 Milk 3

Unpreserved 267 351 217Bronopol 0.02 % 263 352 221Bronopol 0.05 % 270 354 219Bronopol 0.1 % 265 352 220Bronopol 0.2 % 269 353 218Sodium azide 0.02 % 269 355 220Sodium azide 0.05 % 264 351 219Sodium azide 0.1 % 266 '351 221Sodium azide 0.2 % 265 356 220

reaction, with no interference of the mostcommon preservatives used in milk. ThepH signal obtained is directly proportion alonly to the urea content in the milk sample.Any spurious pH drift eventually present,due to aspecific side reactions, is auto mati-cali y eliminated by the continuous blankcompensation.

The apparatus is easy to use and thechemical reagents show good stability.

The method allows the analysis of 60 milksamples per hour.

The routine work flow for the methodcomparison study was eight samples in dou-ble per day, analyzed in parallel with bothmethods. With the manual enzymaticmethod alone, it required approximately 4 h.

ACKNOWLEDGEMENTS

The authors want to thank F. Perrotta ofEurochem for pro viding the differential pH ana-lyzer CL 10 Micro and R. Lodi of C.N .R. CentroStudi Latte.

267

REFERENCES

[1] Anonymous, Lait et produits laitiers. Détermi-nation de la teneur en ammoniac et urée.Méthode enzymatique, norme Afnor V04-217,1992.

[2] Banks W., Clapperton J.L., Muir 0.0.,Powell A.K., Sweetsur A.W.M., The effect ofdietary-induced changes in milk urea levels onthe heat stability of milk, J. Sei. Food. Agric.35 (1984} 165-169.

[31 Bertoni G., Valutazione degli alimenti e dellostato metabolico dei ruminanti, Cappa V.,Bertoni G. (Eds.), AIA, Roma, 1985.

[4] Carlsson J., Pehrson B., The influence of thedietary balance between energy and protein onmilk urea concentration. Experimental trialsassessed by two different protein evaluation sys-tems, Acta Vet. Scand. 35 (I994) 193-201.

[5] Dalgleish D.G., Pouliot Y., Paquin P., Studies onthe heat stability of milk. II. Association anddissociation of particles and effect of added urea,J. Dairy Res. 54 (1987} 39-45.

[6] Duby R.T., Tritschler J.P., Prange R.W., AbdulWahid ET., Effect of dietary crude protein onurea in fluids of the reproductive tract of ewesand dairy cows, J. Anim. Sei. 59 (I986) 339(abstr).

[7] Ferguson J.O., Chalupa W., Impact of proteinnutrition on reproduction in dairy cows, J. DairySci. 72 (1989} 746-753.

[8] Lewis O., Blood urea concentration in relationto protein utilisation in the ruminant, J. Agric.Sei. 48 (I987) 438-445.

[9] Mariani P., Bonatti P., Sandri S., Contenuto diurea, pH, acidità titolabile e caratteristiche dicoagulazione dei latte di singoli allevamenti,Ind. Latte 28 (1992} 139-149.

[ID] Mietton B., Preparazione dei latte per l'industrialattiero-casearia, Latte 14 (I989) 742-748.

[II] MilIetJ., Incidence de la teneur en urée du lait etde la standardisation du pH du lait sur la qualitéd'une pâte pressée cuite: comté, Process 1039(I989) 56-62.

[12] Oltner R., Wiktorsson H., Urea concentrationin milk and blood as influenced by feeding vary-ing amounts of protein and energy to dairy cows,Livest. Prod. Sci. ID (I983) 457-462.

[13] Pecorari M., Mariani M.S., Calzolari M.G.,Tedeschi G., Il contenuto di urea nellatte: vari-azioni e rapporti con i parametri tecnologici,Sci. Tee, Latt. Casearia 44 (1993} 144-151.