The purpose of this paper is to present a detailed account of the precalibration procedures developed and implemented by the USDA Federal Milk Market Administrators (FMMA) for evaluating mid-infrared (MIR) milk analyzers. Mid-infrared analyzers specifically designed for milk testing provide a rapid and cost-effective means for determining milk composition for payment and dairy herd improvement programs. These instruments determine the fat, protein, and lactose content of milk, and enable the calculation of total solids, solids-not-fat, and other solids. All MIR analyzers are secondary testing instruments that require calibration by chemical reference methods. Precalibration is the process of assuring that the instrument is in good working order (mechanically and electrically) and that the readings before calibration are stable and optimized. The main components of precalibration are evaluation of flow system integrity, homogenization efficiency, water repeatability, zero shift, linearity, primary slope, milk repeatability, purging efficiency, and establishment of intercorrection factors. These are described in detail and apply to both filter-based and Fourier transform infrared instruments operating using classical primary and reference wavelengths. Under the USDA FMMA Precalibration Evaluation Program, the precalibration procedures were applied longitudinally over time using a wide variety of instruments and instrument models. Instruments in this program were maintained to pass the criteria for all precalibration procedures. All instruments used similar primary wavelengths to measure fat, protein, and lactose but there were differences in reference wavelength selection. Intercorrection factors were consistent over time within all instruments and similar among groups of instruments using similar primary and reference wavelengths. However, the magnitude and sign of the intercorrection factors were significantly affected by the choice of reference wavelengths.
Our first objective was to optimize center wavelengths and bandwidths for virtual filters used in a Fourier transform mid-infrared (MIR) milk analyzer. Optimization was accomplished by adjusting center wavelengths and bandwidths to minimize the size of intercorrection factors. Once optimized, the virtual filters were defined as follows: fat B sample, 3.508 microm (2,851 cm(-1)), and bandwidth of 0.032 microm (26 cm(-1)); fat B reference, 3.556 microm (2812 cm(-1)), and bandwidth of 0.030 microm (24 cm(-1)); lactose sample, 9.542 microm (1,048 cm(-1)), and bandwidth of 0.092 microm (20 cm(-1)); lactose reference, 7.734 microm (1,293 cm(-1)), and bandwidth of 0.084 microm (14 cm(-1)); protein sample, 6.489 microm (1,541 cm(-1)), and bandwidth of 0.085 microm (20 cm(-1)); protein reference, 6.707 microm (1491 cm(-1)), and bandwidth of 0.054 microm (12 cm(-1)); fat A sample, 5.721 microm (1,748 cm(-1)), and bandwidth of 0.052 microm (16 cm(-1)); fat A reference, 5.583 microm (1,791 cm(-1)), and bandwidth of 0.050 microm (16 cm(-1)). The bandwidth and its proximity to areas of intense water absorption had the largest effect on the intercorrection factors. The second objective was to quantify the impact of fatty acid chain length and unsaturation on fat B and fat A MIR measurements. Increasing the chain length increased the difference (i.e., MIR minus reference) between MIR prediction and reference chemistry by 0.0429% and by -0.0566% fat per unit of increase in carbon number per 1% change in fat, for fat B and fat A, respectively. Increasing the unsaturation decreased the difference (i.e., MIR minus reference) between MIR prediction of fat and chemistry for fat B by -0.4021% and increased fat A by 0.0291% fat per unit of increase in double bonds per 1% change in fat concentration.
Mid-infrared (MIR) milk analyzers are traditionally calibrated using sets of preserved raw individual producer milk samples. The goal of this study was to determine if the use of sets of preserved pasteurized modified milks improved calibration performance of MIR milk analyzers compared with calibration sets of producer milks. The preserved pasteurized modified milk sets exhibited more consistent day-to-day and set-to-set calibration slope and intercept values for all components compared with the preserved raw producer milk calibration sets. Pasteurized modified milk calibration samples achieved smaller confidence interval (CI) around the regression line (i.e., calibration uncertainty). Use of modified milk calibration sets with a larger component range, more even distribution of component concentrations within the ranges, and the lower correlation of fat and protein concentrations than producer milk calibration sets produced a smaller 95% CI for the regression line due to the elimination of moderate and high leverage samples. The CI for the producer calibration sets were about 2 to 12 times greater than the CI for the modified milk calibration sets, depending on the component. Modified milk calibration samples have the potential to produce MIR milk analyzer calibrations that will perform better in validation checks than producer milk-based calibrations by reducing the mean difference and standard deviation of the difference between instrument values and reference chemistry.
Currently, the reference procedure for determination of the "protein" content of milk is based on measurement of the total nitrogen content of milk by the Kjeldahl method (AOAC method, 920.105). About 6% of the total nitrogen content of milk Is nonprotein nitrogen. Therefore, total nitrogen multiplied by the conversion factor 6.38 overestimates the true protein content of milk on average by about 6%. In the present study, new direct and Indirect methods were developed for measurement of the true protein content of whole milk by Kjeldahl nitrogen determination. Both new methods are sample preparation procedures used to fractionate the nitrogen-containing compounds In milk prior to measurement of the nitrogen content of these fractions by Kjeldahl analysis. The collaborative study consisted of 9 pairs of blind duplicate milk samples that were analyzed for total nitrogen, nonprotein nitrogen, and protein nitrogen by each of 10 laboratories. Both methods for true protein measurement (direct and Indirect) gave acceptable statistical performance characteristics and good agreement between methods. The new direct method requires about half the laboratory analysis work of the indirect method (i.e., total minus nonprotein nitrogen). The methods have been adopted official first action by AOAC as (1) a new method for nonprotein nitrogen determination in milk, (2) a new method (direct) for determination of protein nitrogen content of milk, and {3) an alternative method (indirect) for determination of protein nitrogen content of milk.
The classic method for determination of milk casein is based on precipitation of casein at pH 4.6. Precipitated milk casein is removed by filtration and the nitrogen content of either the precipitate (direct casein method) or filtrate (noncasein nitrogen; NCN) is determined by Kjeldahl analysis. For the indirect casein method, milk total nitrogen (TN; Method 991.20) is also determined and casein is calculated as TN minus NCN. Ten laboratories tested 9 pairs of blind duplicate raw milk materials with a casein range of 2.42- 3.05℅ by both the direct and indirect casein methods. Statistical performance expressed in protein equivalents (nitrogen ⨯ 6.38) with invalid and outlier data removed was as follows: NCN method (wt%), mean = 0.762, sr = 0.010, SR = 0.016, repeatability relative standard deviation (RSDr) = 1.287℅, reproducibility relative standard deviation (RSDR) = 2.146%; indirect casein method (wt℅), mean = 2.585, repeatability = 0.015, reproducibility = 0.022, RSDr = 0.560℅, RSDR = 0.841; direct casein method (wt℅), mean = 2.575, sr = 0.015, sR = 0.025, RSDr = 0.597℅, RSDR = 0.988℅. Method performance was acceptable and comparable to similar Kjeldahl methods for determining nitrogen content of milk (Methods 991.20, 991.21,991.22, 991.23). The direct casein, indirect casein, and noncasein nitrogen methods have been adopted by AOAC INTERNATIONAL.
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