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HANUŠ, O., GENČUROVÁ, V., KOPEC, T., YONG, T., JANŮ, L.: Validation of modifi ed milk reference sample in terms of its suitability for infra-red analysis calibration via evaluation of physical properties. Acta univ. agric. et silvic. Mendel. Brun., 2010, LVIII, No. 2, pp. 101–110 Routine milk analyses using the effi cient indirect infra-red method are important for the milk food chain quality. The reliability of the results depends on the calibration quality. It is important to use a relevant set of reference calibration samples (RCSs). RCSs with right range of values can be prepared using various methods. This paper was aimed to balance the impacts of dilution for decrease of main components in RCSs because of minimal change of matrix interference eff ects. Cow milk samples (MSs) were diluted (4/1) using distilled water, NaCl solution and a solution with specifi c composition (SC; because of disturbance in the balance of the milk matrix (NaCl 1.145; KCl 0.849; K2HPO4 1.8463; citric acid 1.7; urea 0.3 g / l)) for reduction in main milk components. Fat (F), crude protein (CP), lactose (L), milk freezing point (MFP), osmolality (OS) and electrical conductivity (EC) were measured in all (original as well as modifi ed) MSs. The lowest MFP and OS were in the original milk −0.5559 °C and 274.5 mOsmol/kg. The MFP was increased to −0.4369 °C and osmolality decreased to 217.83 mOsmol/ kg by the addition of water. The MFP was decreased (−0.4903 °C) and returned to original milk value by the addition of NaCl solutin. MFP was −0.4788 °C due to SC addition. The decrease was less than for NaCl. The ability of other SC components (K2HPO4, KCl, citric acid and urea) to MFP decrease is less than for NaCl solution. EC was highest for NaCl set 4.69 mS / cm, EC for SC was 4.48 mS / cm (P < 0.001). The original MSs set showed EC 4.27 mS / cm. The SC was the nearest to original MSs in terms of total mineral composition. ECs for both modifi cations diff ered (P < 0.001) from original MSs. The procedure is applicable for balance of interference eff ects of milk matrix because of relevant calibration. milk, interference eff ect, composition, milk freezing point, osmolality, electrical conductivity The results of routine milk analysis are important for improving the genetics of dairy cattle and ensuring the quality of the milk food chain. Modern methods include effi cient indirect methods as infra-red (IR) analysis. This procedure is usual ly performed in mid IR or as near IR variant. Over the years a number of authors (Sjaunja, 1984 a, b, c; Sjaunja et al., 1984; Sjaunja and Andersson, 1985; Hanuš et al., 1992, 1995 a, 2002; Tsenkova et al., 2000; Kukačková et al., 2000; Jankovská and Šustová, 2003; Kráčmar et al., 2004; Bijgaart, 2006; Broutin, 2006; Šustová et al., 2006, 2007) have investigated calibration procedures for various IR methods. This was done to measure various milk components using various indirect IR methods for result reliability and stability. The reliability of the results depends on the quality of the indirect method of calibration. It is important to use a set of reference calibration samples (RCSs) with the necessary range of values for the relevant calibration. RCSs can be prepared using various methods of modifi cations. These should avoid damaging the natural milk composition and changing the interference eff ects of basic matrix for calibration accuracy. The additions (milk fat, protein, 102 O. Hanuš, V. Genčurová, T. Kopec, T. Yong, L. Janů urea) or withdrawals (milk fat) of components or mechanical stress of milk samples (free fatty acids) are commonly used etc. (Hanuš et al., 1995 b, 1997, 2003, 2008 a, b; Hering et al., 2008). The addition of water or other solutions is used to achieve lower values of milk components. The required row of milk composition values in RCSs is achieved by modifi cations for IR analysis calibration purposes. Milk samples (MSs) used in the regular calibration of analytical instruments which work on basis of IR absorption spectrophotometry are seldom purposely modifi ed to cover the range of investigated components found in the real fi eld. This is why distilled water is added in selected MSs of the calibration set owing to decrease in fat, protein and lactose contents. Of course, such addition can disturb the whole milk composition (milk matrix) to a certain extent in particular in terms of soluble mineral composition. The mineral matters or salts of some inorganic and organic acids in milk can be con side red as agent of interference eff ects for measurement via IR analyzer (Hanuš et al., 1992). There are above all chlorides, sodium, potassium, citric acid, urea, free fatty acids etc. (Kerkhof Mogot et al., 1982; Sjaunja, 1984 c). The dilution of milk with distilled water alone leads to decrease in the fat, protein and lactose contents and a change in concentration of minerals and other soluble matter at the same time. This can cause a change in interference eff ects in measured MS and consequently also error measurement in the IR analysis. The calibrated instrument automatically compensates mean interference effects on the results of measurement in the current (average) MSs. However, this could be diff erent in modifi ed milk. MS was diluted using NaCl solution at a concentration that was approximately equal to the total milk mineralization, instead of plain water to avoid possible unfavourable eff ects such as change of normal interference eff ect in milk matrix. However, the NaCl solution could cause diff erent interference eff ect to the above mentioned whole specifi c composition (SC; chlorides, sodium, potassium, citric acid and urea). For this reason the SC water solution which approaches the modifi ed MS to fi nal current matrix composition a er its addition to milk as much as possible, was created. This leads to desired decrease in fat, protein and lactose concentrations in modifi ed MS. The other composition (minerals, organic acids) should approach the composition of original milk. The change of interference eff ects for IR analysis should be minimal. For instance the measurable signals and in this way also possible interference eff ects of minority milk components such as urea and citric acid on infrared milk analysis are marked in results more papers (Grappin, 1987; Hanuš et al., 1995 b, 2001, 2008 b, 2009 a, b; Hering et al., 2008). The measurement results of milk composition by the indirect method of IR spectrophotometry can be infl uenced by interference eff ects. Average intereference eff ects are automatically compensated by the technical solution of the method. These interference eff ects are caused by mutual interactions of milk components in simultaneous measurement. The changes in interference eff ects can be caused by milk components which have been manipulated. These eff ects are known but missing is detailed information about their size or dependence on quantity of manipulated components. The hypothesis tested was that it was possible to increase the calibration quality of indirect analytical method (IR analysis) for reliability of measurement of concrete milk components via compensation of possible negative interference eff ect caused by modi fi ca tion changes in milk matrix during the preparation of RCSs. This could be successfully done by verifying targeted method for modifi cation of milk RCSs in terms of composition. Hence the goal of this study was to verify and balance the impact of specifi c dilution for reduction in main milk components in RCSs, which are intended for IR analysis calibration, into milk matrix via manipulation in the soluble composition of cow milk. MATERIAL AND METHODS Experimental sample preparation Cow milk RCS was prepared with targeted decreased fat, protein and lactose content for calibration of IR analyzer. The milk dilution was carried out using: distilled water; NaCl solution; solution of other matter composition (SC). This was based on the rationale of approximating the percentage of main mineral and other minority non-protein nitrogen matter and organic acids in milk as much as possible (Tab. I). The replacement of distilled water by NaCl solution was made to balance the decrease in mineral matter caused due to sample dilution. The total mineral concentration approximated the original concentration a er sample treatment although the composition was a little diff erent. The effort to minimize the interference eff ect changes and maximize the analytical improvement of method led consequently to proposed SC which should correspond to original milk as much as possible. A row of six MSs was prepared with original fat content from 3.61 to 6.94%. This row was marked as set 1 (original set). Three further sets (Tab. II) were prepared from this original row as follows: – set 2 (water): six MSs of original set were diluted by distilled water in the ratio milk/water 4/1; – set 3 (NaCl): six MSs of set 1 was diluted by NaCl solution (4 g / l I: Specifi c composition (SC) used for preparation of reference milk sample (MS) set 4 Component Mass [g/l]

Validation of modified milk reference sample in terms of its suitability for infra-red analysis calibration via evaluation of physical properties