Accuracy of Fourier transform infrared spectrometry

Prévia do material em texto

<p>Accuracy of Fourier transform infrared spectrometry</p><p>in determination of casein in dairy cows’ milk</p><p>Lambert K Sørensen*, Mads Lund and Bent Juul</p><p>Steins Laboratorium, Ladelundvej 85, 6650 Brørup, Denmark</p><p>Received 23 April 2002 and accepted for publication 20 November 2002</p><p>For practical purposes the casein content of milk is often estimated from the crude protein</p><p>content using a general conversion factor. This is done even though it is known that several</p><p>factors can influence the percentage of casein nitrogen in the total nitrogen (casein number).</p><p>The uncontrolled variation in casein number affects optimization of cheese production because</p><p>casein is a limiting factor for cheese yield. Fourier transform infrared spectrometry has recently</p><p>been introduced for casein determination. Using this technique we obtained a standard error of</p><p>prediction (SEP) of 0.033% for casein concentrations in the range 2.1–4.0%. The true prediction</p><p>error was estimated as 0.025%. The correlation between casein numbers obtained by reference</p><p>analysis and infrared spectrometry could be expressed by an R2 of 0.73 and an SEP of 0.89 for</p><p>casein numbers in the range 70.7–81.0.</p><p>Keywords: Casein, casein number, milk, Fourier transform infrared spectrometry.</p><p>Milk crude protein (CP), defined as total Kjeldahl nitrogen</p><p>multiplied by a conversion factor, typically 6.38, can be</p><p>classified into three main fractions: a casein fraction</p><p>(y78% of CP), a whey protein fraction (y17% of CP) and</p><p>a fraction of non-protein compounds containing nitrogen</p><p>(y5% of CP) (Erasmus, 2001). The main milk components</p><p>contributing to cheese yield are fat and casein, with casein</p><p>as the significant and limiting factor. Other milk proteins</p><p>are normally not concentrated in the cheese curd to any</p><p>significant degree. To increase cheese yield it is necessary</p><p>to focus on the casein fraction rather than on the CP or</p><p>true protein fractions. Several studies show that casein/</p><p>protein ratio is not fixed, but may be influenced by genetic</p><p>and physiological factors. Milk containing the BB pheno-</p><p>type of b-lactoglobulin and/or k-casein has a higher casein/</p><p>protein ratio (Rahali & Ménard, 1991; Coulon et al. 1998;</p><p>Mackle et al. 1999). Some breeds may give higher casein/</p><p>protein ratios than others (Blake et al. 1980; Malossini et al.</p><p>1996) which may be correlated to the presence of variants</p><p>B of b-lactoglobulin and/or k-casein (Coulon et al. 1998).</p><p>It is known that casein/protein ratio is decreased when the</p><p>content of somatic cells in milk exceeds 100 000–200 000</p><p>cells/ml (Barbano et al. 1991; Ballou et al. 1995; Coulon</p><p>et al. 1998); the ratio also decreases slightly with parity</p><p>(Coulon et al. 1998). Reports on the effect of stage of</p><p>lactation are variable but a low casein/protein ratio gen-</p><p>erally occurs immediately after calving (Ostersen et al.</p><p>1997; Coulon et al. 1998). In contrast, dietary factors seem</p><p>to have little effect on the casein/true protein ratio except</p><p>in more extreme feeding conditions (Laurent et al. 1992;</p><p>Coulon et al. 1995; Malossini et al. 1996; Coulon et al.</p><p>1998). However, this area deserves to be more fully</p><p>investigated.</p><p>A major constraint to optimization of casein and cheese</p><p>production is the lack of rapid, inexpensive and accurate</p><p>analytical methods for casein determination. It has not yet</p><p>been possible to perform systematic analysis of the casein</p><p>content in milk from herds and single cows. Rapid and</p><p>inexpensive methods have only been available for determi-</p><p>nation of total protein content but not for more defined</p><p>protein fractions because, until recently, high through-put</p><p>infrared (IR) spectrometers had been based on a technology</p><p>using a few fixed optical filters. Although direct casein</p><p>measurements are not possible with these filter instruments,</p><p>they have been used for indirect casein determination</p><p>based on the difference in CP determined in milk and the</p><p>whey obtained from acid precipitation (Barbano & Della-</p><p>valle, 1987) or in milk and the whey obtained from treat-</p><p>ment with rennet (Karman et al. 1987; Taha & Puhan, 1992).</p><p>However, this methodology is tedious and not suitable for</p><p>routine analysis of milk.</p><p>Technological developments have now made full spec-</p><p>trum spectrometers available for routine analysis of milk*For correspondence; e-mail : lks@steins.dk</p><p>Journal of Dairy Research (2003) 70 445–452. f Proprietors of Journal of Dairy Research 2003 445</p><p>DOI: 10.1017/S0022029903006435 Printed in the United Kingdom</p><p>(Andersen, 2002). The spectral information provided by</p><p>these instruments makes it possible to obtain more detailed</p><p>information on milk composition compared with fixed-</p><p>filter instruments. Despite the increased data information,</p><p>rapid measurements can still be obtained, especially when</p><p>the technique is based on interferometry rather than on</p><p>dispersive spectrometry (Agnet, 1998). The objective of the</p><p>present study was to investigate the possibility of direct</p><p>casein measurement in herd milk by high through-put</p><p>Fourier Transform infrared (FT-IR) spectrometry in order</p><p>to obtain a more precise estimate of casein content than</p><p>calculation from CP content.</p><p>Materials and Methods</p><p>Experimental design</p><p>Test samples collected for subsequent payment to milk</p><p>producers in Denmark were used as the pool for selection</p><p>of test samples. These samples covering Danish Holstein,</p><p>Red Danish Breed and Jersey breeds were analysed for</p><p>fat and protein by FT-IR spectrometry. Casein content was</p><p>also measured by the FT-IR technique, but only for internal</p><p>information. During a period of 7 months, August 2001–</p><p>March 2002, five to six test samples were selected weekly</p><p>or at intervals of a few weeks using a stratified sampling</p><p>procedure. A total of 86 test samples was selected. The</p><p>samples selected each week were taken on a single day</p><p>from the pool of incoming laboratory samples. At least one</p><p>sample was taken from each category listed in Table 1.</p><p>The test samples were not preserved.</p><p>Three control materials were included in each test series.</p><p>The first control material consisted of UHT milk. Several</p><p>units were taken as an unbroken series from a production</p><p>plant and stored at 3–5 8C until use. A new unit was used</p><p>in each run of test samples. Two batches of control units</p><p>were used in succession during the study. The other two</p><p>control materials were reference samples (check samples)</p><p>used for routine calibration control of IR instruments. Fat</p><p>levels in these samples were 5.8–6.3% and 3.9–4.3%,</p><p>while protein levels were 3.9–4.2% and 2.5–3.5%. Check</p><p>samples were preserved with 0.02% 2-bromo-2-nitro-1,3-</p><p>propanediol (Bronopol) and stored at 3–5 8C until use. New</p><p>check samples were produced every week. Check samples</p><p>were 10–12 d old when used in the study. The effect of</p><p>Bronopol preservation of milk samples was investigated at</p><p>levels of 200 and 800 mg/l. Bulk milk from six different</p><p>herds was divided into six non-preserved subsamples, six</p><p>subsamples preserved at low level, and six subsamples</p><p>preserved at high level. The six sets of subsamples were</p><p>analysed on different days.</p><p>IR spectrometry</p><p>A Milkoscan FT6000 FT-IR spectrometer (Foss, Hillerød,</p><p>Denmark) was used in the study. The same instrument</p><p>was used for routine analysis of farm milk. The test and</p><p>control samples were heated to 41±2 8C for 10–15 min</p><p>before measurement. Three replicate measurements were</p><p>performed on each sample. Data acquisition was per-</p><p>formed in the wave number range 1000–3000 cm–1. A</p><p>partial least square (PLS) calibration equation (casein bulk</p><p>measurement using a dedicated spectrum calibration ver-</p><p>sion 1.2.1) for bulk milk was supplied by Foss. This cali-</p><p>bration was used without modification and with the same</p><p>slope and intercept values during the entire test period.</p><p>The calibration was based on 86 natural samples from</p><p>New Zealand, Germany, Holland and Denmark covering</p><p>seasonal variations.</p><p>Reference analyses</p><p>Reference analyses were performed within 1 h of measure-</p><p>ment by FT-IR spectrometry to prevent uncontrolled effects</p><p>such as casein degradation by native milk proteinases</p><p>(primarily plasmin) and</p><p>microbial proteinases (Barbano,</p><p>2000). Four subsamples of test and control samples were</p><p>precipitated for casein determination by a modification of</p><p>the IDF Standard 29 (International Dairy Federation, 1964)</p><p>using direct determination of casein nitrogen. A 5.0-ml</p><p>volume of sample heated to 41±2 8C for y10 min was</p><p>transferred to a digestion tube for nitrogen determination</p><p>(International Dairy Federation, 1993) and 70 ml water at</p><p>41±2 8C was added. The weight of the sample was re-</p><p>corded. The sample was swirled with 1.00 ml acetic acid</p><p>(10% v/v) and left to stand at 38±1 8C for 10 min. The</p><p>mixture was then swirled with 1.00 ml sodium acetate</p><p>solution (1 M), cooled to 5–10 8C and left to stand until the</p><p>precipitate had settled. The mixture was filtered through</p><p>a no. 1 filter paper (Whatman International, Maidstone,</p><p>UK). The tube was rinsed with 30 ml buffer solution pre-</p><p>pared by diluting a mixture of 10 ml acetic acid (10% v/v)</p><p>and 10 ml sodium acetate solution (1 M) to 1 l with water.</p><p>The buffer rinse was filtered through the filter paper con-</p><p>taining the precipitate. The rinse step was repeated. The</p><p>Table 1. Experimental design for sample selection. Samples</p><p>were selected evenly among categories based on FT-IR results</p><p>Category Casein content Casein number</p><p>1 low low</p><p>2 low high</p><p>3 high low</p><p>4 high high</p><p>Table 2. Chemical composition of test samples</p><p>Minimum Maximum Mean± SD</p><p>Fat, % 3.36 8.02 4.90±1.05</p><p>CP, % 2.94 5.03 3.67±0.46</p><p>Casein, % 2.08 3.96 2.87±0.40</p><p>Casein number 70.7 81.0 77.9±1.68</p><p>446 LK Sørensen and others</p><p>filter paper with precipitate was then folded and trans-</p><p>ferred to the digestion tube. Nitrogen determination was</p><p>performed by a Kjeldahl method on a Kjeltec 2400 in-</p><p>strument (Foss, Hillerød, Denmark) after block-digestion</p><p>under IDF Standard 20B (International Dairy Federation,</p><p>1993). The four subsamples were analysed in two different</p><p>series to reduce the reproducibility error of the final result.</p><p>Statistical analysis</p><p>The standard error of prediction (SEP) was computed from:</p><p>SEP=[S(yi –xi –bias)2=(N–1)]0�5, i=1, … ,N</p><p>where xi=Result obtained by reference method on sample</p><p>i. yi=Result obtained by routine method on sample i.</p><p>Bias=S(yi –xi)=N, i=1, … ,N</p><p>N=Total number of samples in the test.</p><p>The root mean square prediction error (RMSEP) was com-</p><p>puted from:</p><p>RMSEP=[S(yi –xi)</p><p>2=N]0�5, i=1, … ,N</p><p>The RMSEP expresses the average error that can be ex-</p><p>pected in future predictions.</p><p>The true accuracy of FT-IR spectrometry (SEPtrue) was</p><p>estimated from (SEP2–SDt</p><p>2)0</p><p>.5, where SDt is the total SD of</p><p>the final reference results used for calibration (Sørensen,</p><p>2002). SDt was calculated as the day-to-day standard</p><p>Table 3. Effect of preservation with Bronopol on protein and</p><p>casein results obtained by FT-IR spectrometry</p><p>Values are means±standard deviation of mean for n=30</p><p>200 mg Bronopol/l</p><p>(preserved–non-preserved)</p><p>800 mg Bronopol/l</p><p>(preserved–non-preserved)</p><p>Crude</p><p>protein</p><p>0.0007±0.0008 0.0036±0.0008</p><p>Casein 0.0027±0.0009 0.0117±0.0008</p><p>Table 4. Effect of NPN fractions on protein and casein</p><p>calibrations</p><p>Fortification level Measured content†</p><p>NPN compound</p><p>used for</p><p>fortification</p><p>Pure</p><p>compound</p><p>(mg/l)</p><p>Protein</p><p>equivalent</p><p>(Nr6.38)</p><p>(%)</p><p>Protein</p><p>(%)</p><p>Casein</p><p>(%)</p><p>No addition 0 0 3.36 2.50</p><p>Urea 200 0.06 3.34 2.49</p><p>400 0.12 3.33 2.49</p><p>800 0.24 3.33 2.49</p><p>Hippuric acid 200 0.01 3.37 2.50</p><p>400 0.02 3.38 2.51</p><p>800 0.04 3.38 2.51</p><p>Creatine 200 0.05 3.36 2.50</p><p>400 0.09 3.37 2.50</p><p>800 0.19 3.38 2.49</p><p>† Mean of triplicate analyses</p><p>0·1</p><p>0·08</p><p>0·04</p><p>0·02</p><p>0</p><p>–0·1</p><p>0·06</p><p>–0·02</p><p>–0·04</p><p>–0·06</p><p>–0·08</p><p>0 5 10 15 20 25 30 35</p><p>Weeks from project start</p><p>FT</p><p>-I</p><p>R</p><p>c</p><p>as</p><p>ei</p><p>n</p><p>%</p><p>(</p><p>ac</p><p>tu</p><p>al</p><p>-m</p><p>ea</p><p>n</p><p>r</p><p>es</p><p>u</p><p>lt</p><p>)</p><p>Fig. 1. Reproducibility of FT-IR spectrometric method determined on UHT milk. The batches used for week periods 0–6 and 11–30</p><p>were different. FT-IR results were corrected for the mean result in both periods.</p><p>Determination of casein 447</p><p>deviation of the final reference values. A test for outliers</p><p>(Wernimont, 1990) was conducted prior to calculation of</p><p>SDt to obtain homogeneous data sets and reliable results.</p><p>The repeatability SD, SDr (i.e., the variability of independent</p><p>analytical results obtained by the same operator, using the</p><p>same apparatus under the same conditions on the same</p><p>test sample and in a short interval of time) was calculated</p><p>in accordance with ISO standard 5725-2 (International Or-</p><p>ganization for Standardization, 1994). The intra-laboratory</p><p>reproducibility standard deviation SDR,intra (i.e., the varia-</p><p>bility of independent analytical results obtained on the</p><p>same test sample in the same laboratory on different days)</p><p>was calculated by the same principle used for determi-</p><p>nation of reproducibility (International Organization for</p><p>Standardization, 1994).</p><p>Results and Discussion</p><p>Sampling plan</p><p>Test samples were selected by a stratified sampling pro-</p><p>cedure in order to obtain a relatively flat distribution</p><p>across the entire range of variation for casein concen-</p><p>tration and casein nitrogen/total nitrogen percent (casein</p><p>number) in normal milk. This design was applied to obtain</p><p>sufficient variation for a conclusion of general validity.</p><p>Test samples were selected over a half-year period to in-</p><p>clude variation of uncontrolled factors affecting milk com-</p><p>position and long-term use of the instrument. The variation</p><p>ranges for main milk components are listed in Table 2.</p><p>Cell count was 100 000–800 000 cells/ml.</p><p>Casein determination by reference method</p><p>Casein determination was based on the Rowland principle,</p><p>defining casein as proteins precipitating at pH 4.6 (Row-</p><p>land, 1938). This principle of casein determination can be</p><p>applied in practice in either an indirect or a direct way. In</p><p>the indirect method, the casein content is calculated from</p><p>the difference in nitrogen contents of the milk and the</p><p>whey obtained from casein precipitation. In the direct</p><p>method, the casein content is calculated from the nitrogen</p><p>content of the precipitate. A preliminary study in our lab-</p><p>oratory showed slightly better precision by the direct</p><p>0·1</p><p>0·08</p><p>0·04</p><p>0·02</p><p>0</p><p>–0·1</p><p>0·06</p><p>–0·02</p><p>–0·04</p><p>–0·06</p><p>–0·08</p><p>0 5 10 15 20 25 30 35</p><p>Weeks from project start</p><p>C</p><p>as</p><p>ei</p><p>n</p><p>%</p><p>(</p><p>FT</p><p>-I</p><p>R</p><p>-r</p><p>ef</p><p>er</p><p>en</p><p>ce</p><p>)</p><p>Fig. 2. Difference between FT-IR and reference methods on test samples ($), UHT milk (m) and check samples (X) measured during</p><p>the test period. Results on test samples are mean results obtained on 5 or 6 different samples. Results obtained on UHT milk were</p><p>corrected for the average mean differences of 0.31% and 0.40% for the two batches.</p><p>Table 5. Mean values for casein concentration and casein</p><p>number obtained on UHT milk by FT-IR spectrometry and the</p><p>reference method</p><p>Fat (%) Casein (%) Casein number</p><p>Batch no Reference Reference FT-IR Reference FT-IR</p><p>1 3.22 3.03 2.63 88 76</p><p>2 3.06 2.81 2.50 86 76</p><p>Table 6. Effect of heat treatment of raw milk on casein</p><p>concentration and casein number obtained by FT-IR</p><p>spectrometry and the reference method</p><p>Fat (%) Casein (%) Casein number</p><p>Treatment Reference Reference FT-IR Reference FT-IR</p><p>None 3.26 2.76 2.75 76 76</p><p>Heated to 80 8C 3.26 3.18 2.74 88 76</p><p>448 LK Sørensen and others</p><p>method, and it was therefore decided to use that method</p><p>throughout the study.</p><p>Precision of reference analyses</p><p>The final precision of the reference method was calcu-</p><p>lated using all of the test and control samples analysed</p><p>during the study. Before calculation, the four single results</p><p>obtained for each sample were investigated for outliers</p><p>using a homogeneity test (Wernimont, 1990). Detected</p><p>outliers (1.1% of all results) were removed before calcu-</p><p>lations. SDr was then calculated to be 0.016%. The ex-</p><p>panded repeatability SD (i.e., the variation between two</p><p>analyses when precipitation is performed in the same</p><p>series within a short time interval but nitrogen determi-</p><p>nation is performed in two independent series) was 0.021%.</p><p>SDR,intra was calculated from results for the UHT milk as no</p><p>significant systematic trend in measured casein concen-</p><p>tration was observed during the test period. SDR,intra was</p><p>0.029%,</p><p>which agreed with the expected level estimated</p><p>from SDr. SDt describing the day-to-day variation of final</p><p>reference results was 0.022%.</p><p>Calibration of the FT-IR instrument</p><p>The calibration model was provided by the instrument</p><p>manufacturer. A PLS regression algorithm was used to</p><p>correlate spectral data with reference data. Multivariate</p><p>methods such as PLS are often superior to classical tech-</p><p>niques when many more or less inter-correlated variables</p><p>have to be considered. Using these methods, it is possible to</p><p>project data to a space of independent variables (compo-</p><p>nents), which makes it easier to extract hidden information</p><p>and to build rugged calibration models. Detailed infor-</p><p>mation on the PLS equation for determination of casein</p><p>was proprietary. However, some information has been re-</p><p>leased: (a) wavelength regions carrying significant infor-</p><p>mation related to reference data were pre-selected before</p><p>calibration, and (b) the model included absorption data</p><p>from amide bonds at y1530 cm–1, bands at y1450 cm–1</p><p>and the traditional bands for fat (1750, 2830 cm–1) and</p><p>lactose (1100–1200 cm–1).</p><p>Repeatability of FT-IR analyses</p><p>SDr for each series was calculated from the triple FT-IR</p><p>measurements on test samples. No outliers were detected</p><p>and no systematic trends during the test period were ob-</p><p>served. The pooled SDr was then calculated as 0.006%.</p><p>Reproducibility of FT-IR analyses</p><p>Week-to-week stability of the FT-IR measurements was</p><p>evaluated using the results obtained on UHT milk. No</p><p>marked systematic trends were observed during the test</p><p>period (Fig. 1). SDR,intra was 0.016%, which was signifi-</p><p>cantly better than for the reference analyses. The precision</p><p>was also determined on the check samples used for routine</p><p>calibration control. The check samples were analysed</p><p>every day at the start and the end of a series of samples</p><p>and with a 3-h time interval between them. A series was</p><p>typically equivalent to 8–10 h of uninterrupted analysis.</p><p>Average within-day SD was 0.012% and average between-</p><p>day SD for one-week periods was 0.015%.</p><p>Effect of preservation with Bronopol</p><p>Because Bronopol was added to check samples, its spec-</p><p>trometric determination of protein and casein was investi-</p><p>gated. The results showed a negligible effect on protein but</p><p>a small effect on casein when milk was preserved with</p><p>200 mg Bronopol/l (Table 3). Results from this study were</p><p>used to correct the casein results obtained by FT-IR on</p><p>check samples.</p><p>Effect of NPN</p><p>The effect of different NPN fractions on the CP and casein</p><p>calibrations was investigated by spiking samples with pure</p><p>solid standards. Samples were spiked with urea, hippuric</p><p>acid and creatine to levels of 200, 400 and 800 mg/l. Urea</p><p>is the main single compound responsible for the NPN frac-</p><p>tion in milk. The study showed that CP and casein de-</p><p>terminations were insensitive to variations in urea content</p><p>(Table 4). The same was true for hippuric acid and cre-</p><p>atine, where the levels investigated were much higher than</p><p>normally found in milk.</p><p>4·0</p><p>3·5</p><p>3·0</p><p>2·5</p><p>2·0</p><p>2·0 2·5 3·0 3·5 4·0</p><p>Casein % (FT-IR)</p><p>C</p><p>as</p><p>ei</p><p>n</p><p>%</p><p>(</p><p>R</p><p>ef</p><p>er</p><p>en</p><p>ce</p><p>)</p><p>Fig. 3. Correlation between FT-IR and reference method in</p><p>determination of casein content. Casein% (reference)=0.987r</p><p>casein% (FT-IR)+0.048. SE of slope and intercept were 0.009%</p><p>and 0.026% respectively.</p><p>Determination of casein 449</p><p>Effect of heat denaturation of whey proteins</p><p>The results obtained for UHT milk clearly demonstrated</p><p>that the reference method does not give reliable results</p><p>when whey proteins are heat denatured (Table 5). On the</p><p>other hand the results indicate that the FT-IR calibration for</p><p>casein is insensitive to denatured whey proteins because</p><p>the measured casein value of 76 was close to the mean</p><p>result obtained on raw milk samples. The effect of heat</p><p>denaturation was investigated further by heating milk</p><p>samples to 80 8C. Results confirmed that denaturation of</p><p>whey proteins did not influence CP and casein contents</p><p>measured by FT-IR (Table 6).</p><p>Stability of FT-IR calibration</p><p>Figure 2 shows the mean difference between FT-IR and</p><p>reference results obtained in each series of test and control</p><p>samples. Results obtained on UHT milk were corrected</p><p>for the average mean differences of 0.31% and 0.40% for</p><p>the two batches to bring the results within the same scale</p><p>as the test samples. Some fluctuation in the mean differ-</p><p>ence of test sample results was seen from week to week.</p><p>Generally, the variation could be explained by the com-</p><p>bined reproducibility error of reference and FT-IR data.</p><p>Because the UHT milk and the check samples, which were</p><p>10–12 d older than the test samples, showed the same</p><p>variation profiles as the test samples, it was concluded that</p><p>the accuracy of the PLS calibration was not influenced</p><p>significantly by seasonal or other uncontrolled changes in</p><p>sample composition during the test period.</p><p>Prediction of casein content</p><p>Plots comparing reference and FT-IR results for casein are</p><p>shown in Fig. 3. Because no outliers were identified, all</p><p>results were used in the statistical analysis. The measured</p><p>accuracy of the spectrometric method could be described</p><p>by an SEP of 0.033%. The corresponding RMSEP was</p><p>0.035%. The residuals were slightly dependent on fat</p><p>content (Fig. 4). Standard errors calculated in this way do</p><p>not give a precise estimate of the true accuracy of the</p><p>0·20</p><p>0·16</p><p>0·12</p><p>0·08</p><p>0·04</p><p>0</p><p>–0·04</p><p>–0·08</p><p>–0·12</p><p>–0·16</p><p>–0·20</p><p>R</p><p>es</p><p>id</p><p>u</p><p>al</p><p>s</p><p>o</p><p>f</p><p>ca</p><p>se</p><p>in</p><p>%</p><p>3 4 5 6 7 8 9</p><p>Fat % (FT-IR)</p><p>Fig. 4. Residual plot of casein content (reference – FT-IR) as a function of fat content. Residual= –0.0060rfat%+0.041. SE of slope</p><p>and intercept were 0.0034% and 0.017% respectively.</p><p>85</p><p>80</p><p>75</p><p>70</p><p>65</p><p>65 70 75 80 85</p><p>Casein number (FT-IR)</p><p>C</p><p>as</p><p>ei</p><p>n</p><p>n</p><p>u</p><p>m</p><p>b</p><p>er</p><p>(R</p><p>ef</p><p>er</p><p>en</p><p>ce</p><p>)</p><p>Fig. 5. Correlation between FT-IR and reference method in</p><p>determination of casein number in milk containing 5.5% fat (#). The 5.5% fat limit was used to</p><p>discriminate between Danish Holstein and Danish Red Breed as</p><p>a group and Jersey. Casein number (reference)=0.930rcasein</p><p>number (FT-IR)+5.73. SE of slope and intercept were 0.052 and</p><p>4.06 respectively.</p><p>450 LK Sørensen and others</p><p>spectrometric method as, in addition to the spectrometric</p><p>precision term and the spectrometric trueness term related</p><p>to the individual sample, they also contain the precision</p><p>term from the reference results (Martens & Næs, 1989;</p><p>Faber & Kowalski, 1997; Sørensen, 2002). When SEP was</p><p>corrected for an SDt of 0.022%, an estimate of the true</p><p>prediction error of 0.025% was obtained.</p><p>When milk samples were spiked with lyophilized bovine</p><p>b-lactoglobulins and a-lactalbumins (obtained from Sigma,</p><p>St. Louis, MO, USA) CP and casein contents as determined</p><p>by FT-IR were both increased by the fortification. This in-</p><p>dicates that a casein calibration based solely on natural</p><p>milk samples does not give a specific calibration but prob-</p><p>ably will model some inner relations present in natural</p><p>milk.</p><p>Prediction of casein number</p><p>The predictive ability of the FT-IR method could theoreti-</p><p>cally be due to the close relationship between CP and</p><p>casein contents in milk rather than to a more direct</p><p>measurement of casein. To investigate this in more detail,</p><p>the prediction of reference casein separately from FT-IR CP</p><p>and FT-IR casein was compared. Prediction of reference</p><p>casein from FT-IR CP was characterized by a SE of 0.043%</p><p>when data were modelled by linear regression. In com-</p><p>parison, prediction of reference casein from FT-IR casein</p><p>was characterized by a SE of 0.033%. Thus, prediction of</p><p>reference casein from FT-IR casein was significantly more</p><p>precise than prediction from CP content. It was not poss-</p><p>ible to estimate the precision of casein calculation from</p><p>FT-IR true protein results because a true protein calibration</p><p>was not available. However, a significantly better pre-</p><p>cision would not be expected because even in the case of</p><p>CP determinations, the instrument only responds spectro-</p><p>scopically to true protein and not significantly to NPN</p><p>(Table 4). The difference</p><p>between FT-IR-measured CP and</p><p>true protein is mainly a level difference equal to the mean</p><p>level of NPN in milk.</p><p>The predictive ability of FT-IR was also investigated by</p><p>comparing casein numbers based on reference and FT-IR</p><p>casein results. A clear correlation was observed with a</p><p>symmetrical distribution around the diagonal when plotted</p><p>(Fig. 5). If the casein content determined by FT-IR spec-</p><p>troscopy was essentially a linear function of the CP con-</p><p>tent, then a random or vertical distribution of results, rather</p><p>than a diagonal distribution, would be expected. A linear</p><p>regression model explained 73% of the variation in refer-</p><p>ence casein numbers on the basis of FT-IR-determined</p><p>casein numbers. Measured SEP was 0.89 for casein num-</p><p>bers in the range 70.7–81.0. The corresponding RMSEP</p><p>was 1.0%. If SEP was corrected for a SDt of 0.52 calculated</p><p>from results obtained on the UHT milk, an estimated true</p><p>prediction error of 0.72% was obtained.</p><p>The breed origin of the selected test samples was not</p><p>known. Consequently, it was not possible to divide the</p><p>milk samples exactly into Danish Holstein, Red Danish</p><p>Breed and Jersey populations. Within the geographic con-</p><p>straints, however, milk samples containing >5.5% fat are</p><p>generally considered to come from Jerseys. These samples</p><p>showed a higher average casein number than samples</p><p>containing</p>