WAYS OF PREPARING STANDARDS FOR CALIBRATI ON OF INDIRECT METHODS OF DETERMINATION OF UREA CONCENTRATION IN MILK

The results of recent experiments have confirmed that urea concentrati on (UC) in milk in combination with protein contents is an indicator o f balanced protein-energy nutrition of cows. Instruments based on an i ndirect principle of measurement of urea concentration in milk that ar e highly efficient and automated are used for this purpose. Similarly like all indirect methods, these procedures require to prepare calibra tion standards for calibration while reference procedures are used. Th is paper deals with repeatability, accuracy and correctness of some pr ocedures of determining urea concentration in milk. Some ways of prepa ring milk urea calibration standards have also been included. Fig. I d ocuments natural variability of urea concentration in individual milk of cows from bucket samples. Two photometric methods were used to dete rmine urea concentration in milk: staining reactions with para-dimethy laminobenzaldehyde (FME) and with diacetylmonoxime (FMD). Four sets of milk urea calibration standards (A to D, Tab. I) were prepared: A = r andom selection of bulk milk samples, B = bulk milk sample with differ entiated constant artificial addition of urea at five steps 1.17 mmol/ l at each step, C = bulk milk sample with gradually increasing artific ial addition of urea at six steps from 0.83 mmol/l (sample no. 2) to 1 3.33 mmol/l (sample no. 7), D bulk milk sample with zero urea concentr ation and with gradually increasing artificial addition of urea at six steps-from 0.83 to 13.33 mmol/l. Enzymatic urea hydrolysis after addi tion of urease to milk was used to decrease the original natural conce ntration (5.70 mmol/l) to zero urea concentration (0.33 mmol/l). A hyd rolysis process was controlled photometrically. Residual urease was th ermally inactivated before artificial additions of urea. Tab. Ii shows the results of measurement repeatability. Overall repeatability was 3 .25% for FME and 4.73% for FMD. Repeatability of FME method was better in this trial, in general repeatability of both methods of urea conce ntration determination was good. Tab. III shows the basic data on UC d etermination by the two photometric methods in calibration sets A to D while Tab. IV indicates the significance of average differences betwe en the groups. The differences were statistically insignificant with o ne exception (P > 0.05). Such results document good agreement and accu racy of the used methods FME and FMD and the methods of artificial add itions of urea to milk substrates for the preparation of sets of milk urea calibration standards (CS). Tab. V and Figs. 2 to 4 and 7 to 11 s ummarize the interrelationships between the used methods of UC adjustm ent in CSs. Correlation coefficients between single groups UC within C S sets (Tab. V) were from 0.959 to 0.999 (P < 0.01). Changes in UC due to artificial additions of urea to CSs contributed to a response in t he results of photometric methods by 92.20 to 99.99%. In the trial, th e relation of the FME method to the method of artificial urea addition s was closer than that to the FMD method (Tabs. IV and V). It can also be pointed in general to very good agreement and closeness of the int errelationships between the used photometric and urea addition methods applied to determine UC in milk. Fig. 5 shows the process of the prep aration of zero UC (set D). The residual UC 0.33 mmol/l was taken as z ero. Residual ureolysis was checked by measuring the conductivity of b uffer solution with urea addition before and after addition of treated milk, and it was not demonstrated (Fig. 6). An expressive reduction i n UC was determined photometrically after a longer storage of CS (set D, 5 days, 4 degrees C). The reason was probably residual ureolysis. H ypothetically it could also be reconstitution of the beforehand therma lly inactivated urease. The results of this study do not however docum ent it. it suggests a necessity of prolonged maintainability of CSs, w hich can be provided by the previously used procedure but the proper h ydrolysis of urea is not based on urease addition to milk but it takes place on a column with cellulose-fixed insoluble urease. This procedu re also makes it possible to reduce the thermal denaturation stress of CSs during preparation (set D), which is a necessary step as well. An other way is to replace pasteurization before hydrolysis by NaN3 chemi cal preservation of CSs. The used analytical and preparation methods t urned out appropriate as to their repeatability, accuracy and result c orrectness, therefore they can be recommended as potential unification procedures for calibration of indirect methods of milk urea concentra tion determination.