Food Research International 60 (2014) 59–65

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Food Research International

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Verification of fresh grass feeding, pasture grazing and organic farming

by FTIR spectroscopy analysis of bovine milk
Edoardo Capuano a,⁎, Jan Rademaker b, Harrie van den Bijgaart b, Saskia M. van Ruth a
a
RIKILT, Institute of Food Safety, Wageningen University and Research Centre, P.O. Box 230, 6700 AE Wageningen, The Netherlands
b
Qlip N.V., Postbus 119, 7200 AC Zutphen, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: In the present study, a total of 116 tank milk samples were collected from 30 farms located in The Netherlands
Received 12 August 2013 and analysed by Fourier-transform infrared (FTIR) spectroscopy. Samples were collected in April, May and
Received in revised form 11 December 2013 June 2011 and in February 2012. The samples differed in the time spent by the cows on pasture, presence/absence
Accepted 20 December 2013
of fresh grass in the daily ration and the farming system (organic/biodynamic or conventional). Classification
Available online 28 December 2013
models based on partial least square discriminant analysis (PLS-DA) of FTIR spectra were developed for the pre-
Keywords:
diction of fresh grass feeding, pasture grazing and organic farming. The PLS-DA model discriminated between
Milk milk from cows that had fresh grass in the daily ration and milk from cows that had not fresh grass with sensi-
Authentication tivity and specificity values of 88% and 83% in external validation and all the samples from cows that had no
FTIR spectroscopy fresh grass collected in spring were correctly classified. The PLS-DA model developed for the authentication of
Grass feeding pasture grazing showed comparable accuracy when the whole sample set is considered but was less accurate
Pasture on the spring samples (75% of samples from cows indoors in spring correctly classified). Discrimination of organic
Chemometrics and conventional milk was also accomplished with acceptable accuracy with % correct classification of 80% and
PLS-DA
94% respectively in external validation. The results suggest that milk FTIR spectra contain valuable information
on cows' diet that can be used for authentication purposes.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction verification of cheese geographical origin (Boscaini, van Ruth, Biasioli,

Chemometrics can be defined as the science of extracting chemical
information from data by using mathematical and statistical tools.
Chemometrics has proven to be particularly suited for the verification
of certain food quality attributes or the detection of adulterations
which could not be attained by the analysis of one single food property
but requires the generation of multivariate dataset, i.e. based on the
analysis of more than one sample property. The application of chemo-
metric modelling aims at detecting specific patterns or fingerprints in
a multivariate dataset that can be used to infer about the genuine nature
of a food sample.
A large number of scientific studies have been published over the
last years wherein chemometrics has been applied to data generated
from a variety of analytical methodologies (chromatography, spectros-
copy, mass spectrometry, calorimetry, wet chemistry) for the authenti-
cation of dairy products. Many studies have focused on cheese.
Chemometric modelling has been applied, for instance, for the
⁎ Corresponding author at: RIKILT, Institute of Food Safety, Wageningen UR, P.O. Box
230, 6700 AE Wageningen, The Netherlands. Tel.: +31 317 480260; fax: +31 317 417717.
E-mail address: edoardo.capuano@wur.nl (E. Capuano).
Gasperi, & Mark, 2003; Galle et al., 2011; Karoui, Bosset, Mazerolles,
Kulmyrzaev, & Dufour, 2005; Pillonel, Ampuero, Tabacchi, & Bosset,
2003) or ripening time (Frau, Massanet, Rossello', Simal, & Canellas,
1997). Fluid milk has been also extensively investigated. Chemometrics
has been applied for the discrimination of the animal species (Andreotti,
Trivellone, Lamanna, Di Luccia, & Motta, 2000; Nicolaou, Xu, &
Goodacre, 2011; Smiddy, Huppertz, & van Ruth, 2012), for the determi-
nation of milk composition and origin (Ntakatsane, Yang, Lin, Liu, &
Zhou, 2011), for the discrimination between raw and processed milk
(Dufour & Riaublanc, 1997) and for the detection of milk adulteration
(Souza et al., 2011).
Further examples of application of chemometric modelling to dairy
products other than cheese and fluid milk may be given: discrimination
of low-fat and full-fat yoghurt (Cruz et al., 2013), verification of purity of
butter (Koca, KocaoglufVurma, Harper, & Rodriguez-Saona, 2010; Lipp,
1996) and detection of adulterations in milk powders (Borin, Ferrao,
Mello, Althmann Maretto, & Poppi, 2006; Maraboli, Cattaneo, Maria, &
Giangiacomo, 2002).
Consumers are nowadays increasingly interested in information
about the origin of their foods including information on dairy cows'
diet, housing and herd management system. That is because specific
feeding regimen and herd management practices such as fresh grass
feeding or pasture grazing are linked to superior organoleptic and

0963-9969/$ – see front matter © 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2013.12.024
60 E. Capuano et al. / Food Research International 60 (2014) 59–65
nutritional quality attributes of milk and dairy products (Elgersma,
Tamminga, & Ellen, 2006). Furthermore those specific management sys-
tems are perceived by large groups of consumers as more natural,
healthy and respectful of animal welfare. Therefore such products
would gain an added value compared to conventional counterparts
and might therefore be fraudulently mislabelled. In The Netherlands,
for instance, a quality label has been recently introduced for milk
and dairy products that are produced from cows on pasture at
least 4 months per year, 6 h per day: “Weidemelk”, which is the
Dutch for “pasture milk” (Elgersma, 2012). The European regula-
tion for organic farming also requires that the animals are given
an adequate access to an outdoors area and that at least 60% of
the dry matter in the daily ration of herbivores should consist of
roughage (Commission regulation (EC) No 889/2008). In addition
to the paper-trail and inspection based systems of certification of
product traceability, reliable analytical methodologies may help
the various stakeholders in verifying that the claimed quality attributes
are fully met.
In an earlier research we already found that milk fatty acid (FA) pro-
file can be proficiently used for the authentication of fresh grass feeding
at a level of the amount of fresh grass as low as 30% of the daily ration.
PLS-DA binary classification models based on algorithm appear to be
highly successful, with sensitivity and specificity values close to 100%
(Capuano et al., submitted for publication). However, the proposed an-
alytical methodology is based on gas chromatography with flame
ionisation detection analysis which involves labour intensive and time
consuming fat extraction and analysis and is therefore relatively expen-
sive. For the routine analysis of dairy samples for authentication pur-
poses, cheap, fast, non-destructive and user friendly technologies
would be more valuable. Infrared (IR) spectroscopy is a suitable candi-
date for that. The IR regions of the electromagnetic spectrum that are
relevant to food analysis are the mid IR (MIR, 4000–400 cm−1) region
and the near IR region (NIR, 14,000–4000 cm−1). Recently, it has been
shown that NIR spectroscopy has a potential for the discrimination be-
tween raw milk from cows on pasture and milk from stabled cows
(Coppa et al., 2012). Discrimination (95% of correct classification in
cross-validation) between milk from cows on pasture from stabled
cows was reported based on the spectral interval from 25,000 cm− 1
to 4000 cm−1. MIR spectroscopy is applied for the quantitative analysis
of major milk components since decades and is extensively employed
for routine compositional quality control analysis in the dairy industry
and in milk production registration of individual cows. Applications of
MIR spectroscopy in milk include the prediction of milk coagulation
properties (Cecchinato, De Marchi, Gallo, Bittante, & Cainier, 2009; De
Marchi, dal Zotto, Cassandro, & Bittante, 2007), protein composition
(Rutten, Bovenhuis, Heck, & van Arendonk, 2010; Sorensen, Lund, &
Jull, 2003) and fat composition (Rutten, Bovenhuis, Hettinga, van
Valenberg, & van Arendonk, 2009). A notable advancement in IR spec-
troscopy technology is represented by Fourier transform infrared
(FTIR) spectroscopy. FTIR differs from standard IR technology because
it employs the interferometric modulation of radiation to measure mul-
tiple frequencies simultaneously. The resulting interferogram is then
converted to the original spectrum using complex algorithms. The
major advantage of FTIR over dispersive-based instrumentations are:
improved sensitivity due to higher signal to noise ratio, improved
speed of analysis, higher energy throughput and superior accuracy
(Rodriguez-Saona & Allendorf, 2011).
Despite these advantages, to our knowledge only one study has in-
vestigated the potential of FTIR spectroscopy for milk authentication
purposes. Maamouri et al. (2008) have used FTIR spectroscopy to mon-
itor the quality of milk from Sicilo-Sarde ewes and to discriminate be-
tween milk samples from ewes that have been fed soybean meal or
scotch bean. The aim of the present study was therefore to investigate
whether FTIR spectroscopy combined with chemometrics is an accurate
and valid technique for the authentication of cows' milk with respect to
grass feeding, pasture grazing and organic farming.
2. Material and methods
2.1. Study design
A total of 116 tank milk samples were collected from 30 different
farms located in the Gelderland region (6 farms) and in the Friesland/
Groningen region (24 farms) in The Netherlands. The sampling was
conducted in the framework of a larger project in which other method-
ologies were tested for the authentication of fresh grass feeding, pasture
grazing and organic farming. With few exceptions, each farm provided 4
milk samples, one in each of the following month: in April, May and
June 2011 and in February 2012. Questionnaires and interviews were
used to collect information on farm management and cow diet from
the week of sampling. The grass intake with cut fresh grass fed indoor
was estimated from the acreage of cut land and weight of fed fresh
grass. The grass intake via grazing was estimated by the farmer through
their experience from the animal energy needs (based on milk produc-
tion) minus what was fed next to the grassland. The milk samples were
collected from the tank by using 30-mL plastic screw-top containers
after adequate stirring of the tank. The milk samples were cooled
(4 °C) immediately after sampling and frozen (− 18 °C) within 6 h
after sampling. Samples were categorized in five groups: Milk samples
from cows at least 19 h outdoors on pasture on a daily basis at the
time of sampling (group P2); Milk samples from cows 6–9 h outdoors
on pasture on a daily basis at the time of sampling (group P1); Milk
samples from cows indoors with fresh grass in the diet (group GI);
Milk samples collected in spring from cows indoors with no fresh
grass in the diet (group NG); Milk samples collected in winter (group
W). In groups P2, P1 and GI, the estimated amount of fresh grass in
the cows' daily ration varied from 36% to 94%. All the milk samples of
group W were from cows indoors all the time with no fresh grass in
the daily ration. Two certified organic farms, three certified biodynamic
farms and one farm converting to organic farming were included in the
sample set. In Table 1 an overview of the study design is provided.
2.2. FTIR analysis
The content of fat, protein, lactose, urea, total unsaturated FAs as
well as the milk freezing point depression (FPD) were measured using
MilkoScan FT 6000 equipment (Foss, Hillerod, Denmark) with the man-
ufacturer supplied basic calibration models according to manufacturer's
recommended procedures (Foss, Hillerod, Denmark) at Qlip N.V.
(Zutphen, The Netherlands) on 10-mL milk subsamples in conformity
with ISO FDIS 9622|IDF 141: 2013. The applying reference methods
were ISO 1211|IDF 1 (fat), ISO 8968-1|IDF 20-1 (total protein), ISO |IDF
198 (lactose), ISO 14637|IDF 195 (urea), ISO 15884|IDF 182 and ISO
15885|IDF 184 (total unsaturated FAs) and ISO 5764|IDF 108 (FPD).
FTIR absorption spectra were separately recorded for all 116 milk sam-
ples. They consisted of 1060 infrared frequencies (wavenumbers) ranging
from 925 to 5008 cm−1.
2.3. Statistical analysis
First, the normality of the distribution of fat, protein, lactose, urea,
total unsaturated FAs and FPD within the groups P2, P1, GI, NG and
W was checked by means of a Shapiro–Wilk test for normality
(P b 0.05). Since the distributions appeared to be non-normal in the
groups, one Kruskal–Wallis 1-way ANOVA test for group comparisons
was performed among the groups. If the Kruskal–Wallis test result
was significant (P b 0.05), pairwise comparisons between groups
were performed by means of a Mann–Whitney U test (P b 0.05 was
considered significant). SPSS v19.0 (IBM Statistics Inc., Armonk, NY)
was used to perform all those tests. Principal Component Analysis
(PCA) of the FTIR spectra was performed to screen the multivariate
dataset for outliers and to explore the presence of any grouping in the
data (Berrueta, Alonso-Salces, & Héberger, 2007). Partial least squares-
 E. Capuano et al. / Food Research International 60 (2014) 59–65 61

Table 1
Summary of the study design. Characteristics and composition of the different groups.

Group Hours on pasture per day Min–max (average) % fresh grass per day Samples collected in each montha Totala

April May June February

GI 0 38–71 (54) 4 4 4 0 12
P1 6–9 36–74 (51) 12 (5) 11 (1) 8 0 31 (6)
P2 N19 52–94 (76) 4 (1) 9 (5) 12 (6) 0 25 (12)
NG 0 0 7 6 6 0 19
W 0 0 0 0 0 29 (5) 29 (5)
Totala 27 (6) 30 (6) 30 (6) 29 (5) 116 (23)
a
In parenthesis, the number of samples from organic/biodynamic farms.

discriminant analysis (PLS-DA) was performed to develop binary classi-
fication models to verify the cows' feeding regime (fresh grass feeding
vs no fresh grass feeding), the housing management system (indoors
vs pasture) and the farming management system (organic vs conven-
tional). In a PLS-DA model, the dimensionality of the dataset is reduced
by calculating new variables (latent components or factors) from the
combination of the original variables in order to find the maximum cor-
relation between the new variables and the class variable and therefore
to obtain the maximum separation among the target classes. To develop
and validate the PLS-DA classification models, the sample set was divid-
ed into a training set, consisting of a random selection of 75% of the sam-
ples from each of the class (class GRASS or NO GRASS, INDOORS or
PASTURE, ORGANIC or CONVENTIONAL, see also Section 3.3. for the
composition of the classes from the original 5 groups) and a validation
set consisting of the remaining 25% of samples from each class. The
training set of samples was used to develop the models. Various data
pre-processing techniques, including mean-centering, autoscaling
(scaling to unit variance), Pareto scaling (scaling to square root of the
variance), first and second derivatives, smoothing and orthogonal signal
correction (OSC) and a combination of them were tested. The models
were internally validated by leave 10% out cross-validation and exter-
nally validated on the samples of the validation set. For each model,
the optimal number of factors was selected for each class which gave
the least standard error of cross validation (SECV). For each binary clas-
sification, the best performing model was selected which gave the
highest % of correct predictions in external validation. The PLS1 algo-
rithm described in Manne (1989) was used for the development of
PLS-DA classification models. A tie-breaker criterion was used to classify
the samples in either of the two classes: the sample is considered to
better fit the class with the Y prediction N 0.5. The first derivative, the
second derivatives and the smoothing transforms were based on a
Savitzky–Golay polynomial filter (Savitzky & Golay, 1964) modified as
suggested by Gorry (1990). A convolution is applied to independent
variables in a window containing a central data point and n points on ei-
ther side. A weighted second-order polynomial is fit to the data window
and the centre point is replaced by the fitted value. Different weighting
coefficients are applied for the three transforms. OSC pre-processing
removes the variables that contribute little to the correlation with the
target class before the regression is performed. PCA and PLS-DA were per-
formed in Pirouette 4.5 (Infometrix, Seattle, USA).
3. Results and discussion
3.1. Physical-chemical analysis
The content in fat, protein, lactose, urea, unsaturated FAs, and FPD
values of milk samples were directly measured by the MilkoScan
based on absorbance in specific MIR regions. One of the samples showed
abnormal values for fat (9.6%), protein (2.95%), unsaturated FAs (2.92 g/
100 g) and FPD (−489 m °C(H)). This sample was therefore excluded
from further statistical analysis. The results for groups P2, P1, GI, NG
and W are reported in Table 2. From Table 2 it appears that the main dif-
ference among the groups concerns the milk fat content which is signif-
icantly higher in the winter samples compared to milk from cows on
pasture and milk from cows stabled without fresh grass in the diet but
no differences were found between milk from stabled cows and cows
on pasture in spring. A significantly higher fat content in stabled cows
compared to grazing cows is reported, for instance, by White et al.
(2001) and Morales-Almaraz et al. (2010) but the opposite is reported
by Slots et al. (2008). The effect of grass feeding and pasture grazing
on FA profile has been repeatedly reported. The inclusion of fresh
grass in the ration is known to increase the level of unsaturation of
milk fat (Couvreur, Hurtaud, Lopez, Delaby, & Peyraud, 2006;
Dewhurst, Shingfield, Lee, & Scollan, 2006; Ellis et al., 2006). In our
study the unsaturated FA distribution was significantly different only
between group NG and group P1. Lactose distribution was significantly
different only between groups P2 and NG whereas the protein distribu-
tion was significantly different only between groups NG and W. The dis-
tribution of the FPD values was not statistically different among the 5
groups even though a higher average value was found in milk from
cows on continuous grazing. The FPD of genuine milk depends on
many factors including cow breed, lactation stage, diet and year
(Elisses & Zee, 1980; Elschner, Jacobi, Buchberger, & Grun, 1997). A
higher FPD during the grazing period has already been reported
(Henno, Ots, Joudu, Kaart, & Kart, 2008). All in all, with the exception
of the fat content in winter milk and the unsaturated FAs content in

Table 2
Physical–chemical properties of milk samples analysed by FTIR in groups GI (milk from cows fed grass indoors), P1 (milk from cows on pasture 6–9 h per day), P2 (milk from cows on
pasture N19 h per day), NG (milk from cows with no fresh grass collected in spring) and W (samples collected in winter). Values are means ± SD.

Parameter GI P1 P2 NG W

Fat (%) 4.17 ± 0.16ab 4.15 ± 0.19 a 3.94 ± 0.31 a 4.10 ± 0.24a 4.66 ± 1.00b
Proteins (%) 3.45 ± 0.19ab 3.46 ± 0.11ab 3.52 ± 0.19ab 3.42 ± 0.09a 3.54 ± 0.17b
Lactose (%) 4.52 ± 0.08ab 4.51 ± 0.08ab 4.50 ± 0.08a 4.56 ± 0.07b 4.50 ± 0.12ab
Urea (mg/100 g) 20.0 ± 7.2 21.6 ± 6.6 20.3 ± 9.4 21.1 ± 4.4 20.6 ± 4.0
Unsaturated fatty acids (g/100 g) 1.29 ± 0.10ab 1.35 ± 0.11 a 1.27 ± 0.11ab 1.18 ± 0.08b 1.32 ± 0.34ab
Freezing point depression (−m °C(H)) 518 ± 5 518 ± 6 516 ± 9 518 ± 6 519 ± 7
a,b,x,y
Different superscripts in a row indicate significant differences between distributions (Kruskal–Wallis test for group comparisons, P ≤ 0.05 and Mann–Whitney U test for pairwise
comparisons, P b 0.05).
62 E. Capuano et al. / Food Research International 60 (2014) 59–65
spring milk from stabled cows few remarkable differences among the 5
groups appear from Table 2.
3.2. Exploring the FTIR data: raw spectra and PCA
As a first step, a PCA was carried out on the whole dataset using the 6
physical-chemical properties described in Section 3.1. as variables. The
PCA scores plot did not highlight any natural clustering of the milk sam-
ples between the 5 groups (data not shown). Additional to the physical–
chemical parameters, the information contained in the whole FTIR spec-
tra themselves was explored. In Fig. 1, the FTIR spectra of a milk sample
of group P2 is shown as an example. The absorption bands observed in
the MIR region are associated with the fundamental vibrations of func-
tional groups of the molecules. For quantification purposes, the absorp-
tion bands of the MIR spectrum occurring at approximately 1175 cm−1
(corresponding to transitions in the vibrational energy of the triglycer-
ide ester linkage C\O), 1750 cm− 1 (carbonyl group of FAs), and in
the region 2800–3000 cm− 1 (FAs acyl chain C\H) are commonly
used to quantify fat (Dupuy, Duponchel, Huvenne, Sombret, &
Legrand, 1996; Safar, Bertrand, Roberts, Devaux, & Genot, 1994; Yang
& Irudayaraj, 2000) while the bands at 1653 cm−1 (amide I) and
1567 cm−1 (amide II) are used for the quantification of proteins and
to obtain information on protein secondary structure (Dufour, Robert,
Renard, & Llamas, 1998; McQueen, Wilson, Kinnunen, & Jensen, 1995;
Van de Voort, Sedaman, Emo, & Ismail, 1992). Vibrations arising from
C\O and C\H stretching produce bands in the region between
1100 and 1000 cm−1 which give information on sugar molecules
(Hashimoto & Kameoka, 2008). Water significantly absorbs in the MIR
region and can interfere with the quantification of other major milk
components. Major bands are present at 3920, 3490, 3280 cm− 1
(O\H stretching vibrations), and 1645 cm−1 (resulting from water
bending vibrations), the exact location depending on the specific inter-
actions of water molecules with solutes (Lin & Brown, 1992) and tem-
perature (Libnau, Kvalheim, Christy, & Toft, 1994). In our milk
samples, strong absorption from water molecules was apparent be-
tween 1620 and 1670 cm−1 and between 3000 and 3600 cm−1. The re-
gion between 3000 cm−1 and 3600 cm−1 was eliminated for further
analysis whereas the region between 1620 and 1670 cm− 1 was
retained because information on protein composition is embedded in
it. The regions ranging from 1800 cm−1 to 2800 cm−1 as well as the re-
gion between 4000 cm− 1 and 5000 cm−1 was eliminated prior to
further modelling because it does not contain valuable information on
milk composition.
Few evident differences among the 5 groups of Table 1 could be
highlighted by comparing the FTIR raw spectra. Chemometric treatment
of the MIR spectra combined is usually required to exploit the multivar-
iate information contained in these spectra and to reveal subtle differ-
ences that might exist between different types of samples (Woodcock,
Downey, & O'Donnell, 2008). In the raw spectra, it was apparent that
one sample was very different from the bulk of the data showing a
Fig. 1. FTIR raw spectra of a milk sample from cows on pasture N19 h per day.
very atypical profile at different wavelengths. This sample was the
same which showed an unusual proximate composition (see
Section 3.1.) This sample was thus excluded from further chemometric
modelling. PCA was then conducted with the MIR spectral data (900–
3000 cm−1 with the exclusion of the region 1800–2800 cm−1 of the re-
maining 115 samples) to reveal natural clustering of the samples and to
detect outliers. Several data pre-processing methods were tested. In
Fig. 2 the PCA scores plot of the pre-processed (autoscaling, smoothing
and second derivative) FTIR spectra is depicted, whereas Fig. 3 shows
the eigenvectors corresponding to the first two PCs. The PCA scores
plot revealed that the milk samples collected in winter are clearly dis-
tinct from the milk samples collected in spring even though some over-
lapping occurs. Milk from cows with no fresh grass in the diet collected
in spring are clearly distinct from the winter samples and almost
completely separated from milk from cows under continuous grazing
whereas a more extensive overlapping is apparent with milk from
cows on pasture only 6–9 h. A slight difference appeared between the
two groups of cows on pasture whereas the difference between the
milk from cows fed fresh grass indoors and the pasture groups seems
very limited. The PCA scores plot also revealed that one sample belong-
ing to group P2 always laid very far from the sample cloud and exhibited
a Mahalanobis distance far beyond the 95% probability threshold value
calculated from the PCA models irrespective of the pre-processing tech-
nique applied. This sample also exhibited a residual variance significant-
ly larger than PCA model variance (F test, P b 0.05). This sample was
therefore considered an outlier and excluded from further modelling.
3.3. Binary classification models: PLS-DA
The aim of the present study was to investigate the possibility of
using FTIR spectra for the authentication of fresh grass feeding, pasture
grazing and organic farming. We used PLS-DA for building three binary
classification models. Binary classification models aim at predicting the
actual class membership of a new unknown sample. In our case model 1
aimed at predicting whether the cows have been fed fresh grass or not,
model 2 aimed at predicting whether the cows have been grazing on
pasture or not, whereas model 3 aimed at predicting whether milk
was from organic/biodynamic or conventional farms.
For model 1 the groups P2, P1 and GI were pooled in the class GRASS,
whereas the groups NG and W were pooled in the class NO GRASS. The
whole sample set was constituted by 114 raw milk samples, after the
elimination of two outliers as described in Section 3.2. The most satisfac-
torily results in terms of number of misclassified samples in external
validation was obtained by a PLS-DA model after autoscaling, smooth-
ing and 2nd derivative transformation of the raw data and the applica-
tion of one OSC component (Table 3). The smoothing and the 2nd
Fig. 2. PCA scores plot of the FTIR data (autoscaling, smoothing and second derivative).
Variance explained by each factor is provided in parentheses. In the circle the outlier sam-
ple that was excluded from further modelling.
 E. Capuano et al. / Food Research International 60 (2014) 59–65
Fig. 3. Eigenvectors of the first two PCs for the PCA model based on the FTIR data
(autoscaling, smoothing and second derivative). The % of explained variance is provided
in parenthesis.
derivative transformation were calculated including 25 points on either
side of the centre point of the window. In general, the performance of
the PLS-DA models was found to be dependent on the width of the win-
dow (number of data points) used for the computation of the smooth-
ing and the 2nd derivative. The sensitivity (% of corrected
classification in class GRASS) and specificity (% of correct classification
in class NO GRASS) of the model was very high in internal validation
with approximately 88% of the samples of the class GRASS and 100%
of the samples of the class NO GRASS correctly assigned to their actual
classes. In external validation, the sensitivity was ≈88% and the speci-
ficity was ≈83%. The two samples from class NO GRASS that were incor-
rectly predicted as GRASS in external validation were collected in
February whereas the two samples from class GRASS incorrectly pre-
dicted as NO GRASS were collected in spring. That means that all the
samples of the class NO GRASS collected in spring were correctly classi-
fied by the model. Indeed, for authentication purposes, the ability to ac-
curately predict whether the cows have been fed grass or not is by far
more important on samples collected during the grazing period than
on samples collected during the winter indoor period when fresh
grass is not available. In Fig. 4, the regression vectors of class GRASS
for the PLS-DA model described above is shown (the regression vector
of class NO GRASS is not shown since it is the mirror-image of that of
class GRASS). The regression vector can be regarded as the weighted
sum of the loadings included in the model. It gives therefore informa-
tion on the importance of each single variable for the model prediction.
Variables that have lower scores in the regression vector contribute
less in the prediction. From the regression vector it appears that
bands around 1175 cm − 1 , 1215 cm − 1 , 1260 cm − 1 , 1375 cm − 1 ,
1500 cm−1, 1550 cm−1, 1750 cm−1 and the region 2800–3000 cm−1
are important for the prediction. The last regions and the bands around
1175 cm−1 and 1750 cm−1 are attributed to the absorption by milk fat
and that would confirm what emerged from the results reported in
Table 2. The region of 1500–900 cm−1 is called the fingerprint region
and refers to C\O and C\C stretching modes. The bands around
1199–1474 cm−1 are due to the bending modes of O\C\H, C\C\H
and C\O\H bonds. In comparison with the regions corresponding to
the absorption of fats, the regions corresponding to the absorption of
proteins and of lactose seem to contribute less to the PLS-DA models.
For model 2, the groups P1 and P2 were merged together in the class
PASTURE whereas the groups GI, NG and W were merged together in
the class INDOORS. The best performing PLS-DA model was obtained
after Pareto scaling of the data, smoothing and 2nd derivative transfor-
mation of the raw data and the application of one OSC component. The
smoothing and the 2nd derivative were calculated including 25 points
on either side of the centre point of the window. The results were
63
very satisfactory in internal validation with an average % of correct pre-
diction of 93% and 95% for class PASTURE and class INDOORS respective-
ly, whereas in external validation 91% of milk samples from class
PASTURE and 81% of milk samples from class INDOORS were correctly
classified in their actual class (Table 3). Out of the three samples
misclassified in class INDOORS, two were collected in spring and one
in winter which reduces to 75% the specificity of the model on spring
samples. The lower specificity of model 2 compared to model 1 was ex-
pected and related to the higher misclassification rate of milk from cows
fed grass indoors compared to milk from stabled cows without fresh
grass. Indeed the two samples of class INDOORS misclassified in internal
validation and one of the samples misclassified in external validation
belonged to group GI. The PLS-DA regression vector showed high scores
(in absolute value) for bands around 1640 cm−1 (positive score) and
around 1585 cm− 1 and 1695 cm− 1 (negative scores) (data not
shown) and smaller peaks around 1020 cm−1, 1380 cm−1 and in the
region 2800–3000 cm−1. The unambiguous assignment of the first
three bands to specific vibrations is difficult but they are related to pro-
tein content, protein secondary structure and interactions with water
and other solutes as well as amino acid side chains, i.e. carboxylic
group of aspartic acid. Water also strongly absorbs in the region
1600–1650 cm−1. The structure of the PLS-DA regression vector was
very different from that of the PLS-DA model developed for the verifica-
tion of grass feeding even though the two pairs of classes (GRASS/NO
GRASS and PASTURE/INDOORS) are very similar. This behaviour de-
pends on the different data pre-processing applied in the two models.
For model 3, since major differences were highlighted between win-
ter and spring samples, only the latter ones were used to develop the bi-
nary classification model. A classification model based on winter milk
was not developed because of the few number of samples available
(29). The milk samples from the farms converting to organic farming
were included in the class ORGANIC. A total of 87 samples were includ-
ed in the model with 69 samples in the class CONVENTIONAL and 18 in
the class ORGANIC. The most accurate model was obtained after mean-
centering, smoothing (25 points) and 2nd derivative transformation of
the raw spectra and application of 1 OSC component and in external
validation gave 94% of correct classification in class CONVENTIONAL
and 80% in the class ORGANIC (Table 3). The PLS-DA regression
vectors gave very high scores (in absolute values) for bands around
1640 cm− 1 (positive score) and around 1592 cm− 1 and 1695 cm− 1
(negative scores) (data not shown). The scores associated to these
bands were 1–2 orders of magnitude higher than any other wave-
lengths which implies they are by far most important for the discrimina-
tion between organic and conventional milk samples.
3.4. FTIR vs FA profiling
In a previous part of this study, using the same set of samples as that
used here, we found that it is also possible to highly satisfactorily au-
thenticate fresh grass feeding by means of FA fingerprinting (Capuano
et al., submitted for publication). In fact, the results achieved using FA
profiling were better, in terms of sensitivity and specificity than those
achieved by FTIR reported in the present paper (≈100% in external val-
idation). However, the analytical procedure required to obtain the FA
profile is relatively time consuming, labour intensive, requires expen-
sive instruments and trained personnel and makes use of chemicals.
FTIR on the other hand is a more rapid, high-throughput technique, it
is non-destructive, does not require specialized personnel, is environ-
mental friendly since it does not require solvents and chemicals and
does not produce waste and may also be applied in situ. In addition,
FTIR is a common and widespread technique in the dairy routine QA
laboratories, being widely applied to dairy quality control, milk pay-
ment and herd improvement purposes. Based on those practical as-
pects, FTIR might be used as screening tool to verify the fresh grass
feeding of cows. The samples that are claimed from cows that have
been fed fresh grass but predicted by the FTIR analysis otherwise can
64 E. Capuano et al. / Food Research International 60 (2014) 59–65

% correctly classified in CLASS 2c

81% (13/16)
83% (10/12)

80% (4/5)
% correctly classified in CLASS 1b

91% (12/13)
88% (15/17)

94% (16/17)

Fig. 4. Regression vector for the class GRASS in the PLS-DA model (autoscaled data,
smoothing, second derivative and one OSC component).

then be subsequently confirmed by FA profiling. From this standpoint it

RMSECV

is remarkable that the FTIR model developed herein exhibits an espe-

0.206
0.266
0.221

cially good specificity for the spring samples, which means that the per-
centage of misclassifications is very low for the samples from cows that
have not been fed fresh grass during spring. This would reduce in prac-
0.825
0.718
0.697
r2a

tice the rate of mislabelling detection failure. On the other hand, the di-
rect authentication of pasture grazing is not achievable with acceptable
Explained variance %

accuracy by FTIR combined with chemometrics on samples collected in

spring (the same was found with FA profiling), but the verification of
fresh grass feeding might be the starting point for the authentication
of pasture grazing provided that additional biomarkers are considered.
86
29

90

4. Conclusions
No. of factors

In this feasibility study we showed that FTIR spectra of raw, herd bulk
milk contain valuable information on cows' diet that can be used for the
6
3

authentication of cow feeding regime and herd management system.

Mean-centering, smoothing (25 points), 2nd derivative (25 points) and 1 OSC

PLS-DA classification models developed to predict whether cows have

Pareto scaling, smoothing (25 points), 2nd derivative (25 points) and 1 OSC

been fed fresh grass exhibited satisfactory accuracy. An analogous binary

Autoscaling, smoothing (25 points), 2nd derivative (25 points) and 1 OSC

model developed to predict whether cows have been on pasture or not

Class 2 is NO GRASS for model 1, INDOORS for model 2 and ORGANIC/BIODYNAMIC for model 3.

showed comparable accuracy even though the model specificity reduced

when applied on spring samples only. The discrimination between
Description of the optimal PLS-DA models and their performance during external validation.

organic and conventional milk collected in spring seems also feasible by

Class 1 is GRASS for model 1, PASTURE for model 2 and CONVENTIONAL for model 3.

FTIR but the relatively few samples in the class ORGANIC would call
for more cautious conclusions and validation with a larger and more
balanced dataset. The results reported in this study suggest that FTIR
spectroscopy might be used as a screening technique for the authentica-
tion of fresh grass feeding before more accurate but time-consuming
techniques are applied (e.g. FA profiling) for confirmatory purposes.
However, the validation of the proposed model with a truly independent
validation set is mandatory to support these conclusions.
RMSECV = Root Mean Square Error of Cross Validation.
Data pre-processing

Acknowledgements

The authors gratefully acknowledge the Dutch Ministry of Economic

Affairs and Innovation for financial support, Grishja van der Veer for the
OSC = orthogonal signal correction.

fruitful discussion on the modelling and Nisha Shetty for reading the
Obtained in cross validation.

manuscript. They also acknowledge Anjo Elgerma and the farmers for
collecting and providing the milk samples.
Model 3: Organic farming
Model 2: Pasture feeding
Model 1: Fresh grass

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