In the dairy sector, there has been a long run debate about an appropr
iate basis of calibration for milk protein content of routine indirect
instrumental methods (IM) calibrated according to reference methods.
Using traditional methods, milk protein content in per cent is given i
n terms of the content of so called crude protein, i.e. total nitrogen
(according to Kjeldahl method) x 6.38. Indirect methods are usually c
alibrated for these values. In France, the content of milk true protei
n, that means the content of protein nitrogen (according to Kjeldahl m
ethod) x 6.38, has been used as a calibration basis for a longer time.
Optimizing discussion about the aspects of suitability, accuracy and
correctness as well as about the advantages and difficulties of these
different approaches can be encountered in literature. The objective o
f the present paper is to discuss theoretical and practical problems:
analyses of milk nitrogen matters; relationships of some methodical pr
ocedures used for analyses of milk nitrogen matters; interrelationship
s between milk nitrogen matters. Within a year, calibration of a milk
infraanalyzer Milko-Scan 133 B (MSc, Foss Electric, Denmark) was done
eleven times for the content of crude protein (CP) that was determined
by Kjeldahl method (KM) on a Kjeltec instrument (Tecator AB, Sweden).
The patterns and results of these calibrations (Tab. I) were very goo
d with respect to generally respected requirements. At the same time,
288 individual samples of cow's milk from the middle part of lactation
were analyzed in the middle of intercalibration intervals. The cows c
ame from three herds of two breeds: Czech Pied and Black-Pied Lowland
breed. The cows were at the second and higher lactations. Kjeldahl met
hod, calculations and photometric method were used for the particular
analyses in order to quantify the separate nitrogen fractions of milk
according to a scheme shown in Tab. II and/or III. Absolute and relati
ve proportions of nitrogen (N) fractions in milk are summarized in Tab
. II. The absolute values of the levels of most N fractions were lower
in comparison with those established in previous studies, but they we
re comparable, in absolute and relative terms, with the levels determi
ned for the Holstein breed (Cerbulis and Farrell, 1975). Protein nitro
gen equalled on average 96.3% of total N. The share of casein N in pro
tein N was 82.6%. Urea nitrogen made 72% of nonprotein nitrogen (NN).
The per cent of nonprotein N is important for calibrations of indirect
methods (so called instrumental methods IM). In this case, it amounte
d to 3.7% of total N. The difference between the determination of CP b
y reference (RM), i.e. Kjeldahl method (KM) and MSc was insignificant
(p > 0.05, the difference 0.006+/-0.081%). Figs. 1, 2 and 3 show the r
elationships between CP and TP contents determined by various procedur
es. Theoretically it is more appropriate to calibrate the infraanalyze
rs for the content of true protein (TP) in milk according to KM. But a
pplying a practical procedurs, there was no closer relationship betwee
n infraanalyzer readings and TP content according to KM in comparison
with CP content according to KM: r = 0.947 > 0.937 (Figs. 1 and 2). Ma
nual analytical inaccuracies at KM method when determining TP content
in curd (direct) may be a reason. Determination of TP content by Kjeld
ahl method is more labor- and time-consuming than the determination of
CP content. These inaccuracies may overlap the theoretico-practical a
nd expected effect of natural variability of NN on the agreement of in
fraanalysis and reference method (RM = KM). Although the better agreem
ent of IM x RM was not achieved empirically, further evaluations show
(Figs. 4, 5 and 7) that an improvement should be achieved theoreticall
y. It is possible that determination of NN content (by KM) in filtrate
that decrease the risk of inaccuracies, could help to improve empiric
ally the IM x RM relationship. TP content for the purposes of IM calib
ration could be calculated as the difference: total N (by KM) minus NN
(by KM), i.e. indirect. The relationships between the content of NN a
nd/or urea (U) and the difference between the content of crude protein
determined by infraanalysis and Kjeldahl method (CP, MSc - CP, KM) we
re as follows: r = -0.38 and r = -0.33 (p < 0.001), resp., Figs 4 and
5, resp. A relationship between U and difference CP, MSc - TP, KM was
insignificant (Fig. 7). These three evaluations (Figs. 4, 5 and 7) con
firm the theoretical assumptions for improvement of the IM x RM relati
onship at IM calibration for TP content by KM. On the other hand, the
theoretical assumptions were not confirmed by evaluation in Fig. 6, wh
ich can also result from the methodical procedure of protein N determi
nation by KM in filter curd. Eg. Grappin et al. (1980), Grappin (1992)
and Grappin and Lefier (1993) did not confirm in several cases, but m
ostly they did the better agreement between IM and RM at IM calibratio
n for TP content by KM. Therefore this procedure has been used for abo
ut 20 years in the only country - in France. Barbano and Lynch (1990,
1992) also recommend this procedure in theoretical discussions. Discus
sion of dairy specialists about this problem is going on, each system
(calibration for CP or TP) has its pros and cons. But the reason for T
P to be accepted as a calibration basis for IM seem to more serious. T
he relationships between the particular nitrogen matters are shown in
Tab. III. The highest correlation was found between crude protein and
true protein: r=0.96. All important correlation coefficients are posit
ive, except one. coefficient. The relationship between U and NN conten
t was relatively close: r=0.45. In order to decide what calibration ba
sis for IM should be accepted in future, it is important to know the r
elationships between nitrogen matters of milk and methods of their det
ermination. So this paper could contribute to the knowledge of this pr
oblem.