Comparison of Different Types of NIR Instruments in Ability
to Measure β-Glucan Content in Naked Barley
J. Schmidt,1,2 S. Gergely,3 R. Schönlechner,1 H. Grausgruber,4 S. Tömösközi,3 A. Salgó,3 and E. Berghofer1
ABSTRACT
Importance of β-glucan in human nutrition is mirrored in numerous
approval applications registering β-glucan containing products as health
beneficial products in accordance with forthcoming EU Health Claims
Regulation. In comparison to other cereals, barley contains considerable
amounts of β-glucan. Naked barley is of particular interest because it
circumvents the costs and loss of beneficial substances related to dehusk-
ing. In this study, the potential of near-infrared spectroscopy as an accu-
rate, fast and economic method of determination of β-glucan in naked
barley was appraised. Four different near-infrared instruments were used
to analyze 107 barley samples, in both whole grain and milled form.
Importantly, both black and purple pericarp samples, which are of addi-
β-Glucan is a large, linear nonstarch polysaccharide built on
mixed linkage β(1→3)/(1→4)-D-glucose units localized in large
amounts in the endosperm cell wall of oats (Avena sativa) and
barley (Hordeum vulgare). It initially drew attention and was la-
beled as problematic for causing a decrease in extract yields and
filtration problems in brewery, wine, and fruit juice industries
(Bamforth 1982) as well as problems in broiler chicken feedings
where barley leads to remarkable reduction in weight gain and
hygienic problems (von Wettstein et al 2000). These problems can
be attributed to high viscosity caused by high molecular weight
and high molecular asymmetry.
On the other hand, β-glucan is predicted to become an impor-
tant supplement in human nutrition for proven cholesterol-
lowering effect (Delaney et al 2003; Yang et al 2003; Behall et al
2004) and augured positive effect on diabetes (Behall et al 2006),
several immune diseases (Estrada et al 1997), and suppression of
cancer development (Murphy et al 2004; Modak et al 2005). In-
deed, in accordance with a forthcoming EU Health Claims Regu-
lation, a number of entities filed an approval procedure toward
registering β-glucan-containing products as health beneficial
products.
To accommodate brewery and animal breeders demand for bar-
ley low in β-glucan as well as nutritionists requirements for the
optimal usage of the positive nutritional effects ascribed to β-
glucan, optimal breeding of barley is essential. Breeding supervi-
sion and entrance control of resultant barley cultivars in relevant
industries is therefore necessary and as such requires an adequate
quantification method for β-glucan. Frequently used methods for
the detection of β-glucan including the Calcoflour method, EBC
method 4.16.3 offered by NovaBiotec, the enzyme assay after
McCleary, HPLC, HPAEC-PAD, MALDI-MS, and mid-IR suffer
from one or more drawbacks including low sensitivity, long de-
termination times, destructiveness, and high costs related to the
methods. Hence, the need for an accurate, fast, nondestructive,
1 Department of Food Science and Technology, University of Natural Resources
and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
2 Corresponding author. E-mail: julia.schmidt@boku.ac.at
3 Department of Applied Biotechnology and Food Science, Budapest University of
Technology and Economics, Műegyetem rkp. 3, 1111 Budapest, Hungary.
4 Dept. Applied Plant Sciences and Plant Biotechnology, University of Natural
Resources and Applied Life Sciences, Gregor Mendel Strasse 33, 1180 Vienna,
Austria.
doi:10.1094 / CCHEM-86-4-0398
© 2009 AACC International, Inc.
398 CEREAL CHEMISTRY
Cereal Chem. 86(4):398–404
tional nutritional interest due to high anthocyanin content, and waxy
samples, which show an extraordinary high β-glucan content could be
analyzed within the same calibration set as the normal samples. All tested
dispersive near-infrared reflection instruments showed suitability for
supervision of breeding experiments and β-glucan monitoring in food
industries (R2 > 0.78). Common, industrially used near-infrared transmis-
sion instruments also provided reasonable results, although only suitable
for rough selection according to β-glucan levels. On the other hand, the
Fourier transform near-infrared reflection instrument was able to perform
analytical analyses (R2 = 0.96–0.98).
and economic method with minimal requirements related to sam-
ple preparation is obvious.
One method with the potential to meet these requirements and
even allows for simultaneous quantification of other cereal quality
determining components, is near-infrared spectroscopy. Intro-
duced for food analysis purposes in the 1970’s by NIR pioneers
Williams and Norris, this technique is now widespread in this
field. Specifically, several calibrations for different constituents
have been established in cereal analyses. Recently, feasible cali-
brations for main components like moisture, protein, starch, and
fat have been employed with success. In Europe, milling and
malting companies use cheap NIT instruments for entrance con-
trol estimating moisture and protein. One instrument frequently
employed in these instances is the Foss Infratec 1241 grain ana-
lyzer, thus this machine was also included in this study. Besides
those well-established applications already under further devel-
opment for fast inline sorting methods (Pasikatan and Dowell
2004), approaches have been made in establishing food safety
monitoring of grain in terms of detection of parasites (Baker et al
1999) and quantification of mycotoxins (Börjesson et al 2007)
using NIT/NIR technology. In addition, functional properties de-
termining the end product yield such as malting quality (Ratcliffe
and Panozzo 1999) have also been assessed by NIRS. The in-
creasing popularity of functional food is also reflected in trials to
adapt NIRS and NITS for quantification of soluble and insoluble
dietary fiber in cereal food products (Kays and Barton 2002).
The need to establish a method for fast and economic determi-
nation of β-glucan in barley as well as the fact that NIRS may be
suitable for that purpose was a subject of previous studies. For
example, Blakeney and Flinn (2005) tested NIRS for quantifica-
tion of β-glucan (among other nonstarch polysaccharides [NSP])
in barleys, wheats, triticales, oats, sorghums, maize, lupins, field
peas, chickpeas, and faba beans. Although the calibration parame-
ters appeared to be acceptable, a plot of β-glucan predicted versus
β-glucan determined enzymatically revealed apparent splitting of
samples in two groups: 1) high (>2.5%) in β-glucan (mainly bar-
ley) and 2) low (<1%) in β-glucan (other cereals and fruits) which
classifies this heterogeneous sample set as invalid. Furthermore,
the number of barley samples (26) was clearly too small for use as
calibration basis and the relatively small range of 2.5–5.2% was
insufficient to draw any conclusions regarding β-glucan amounts
in different barley cultivars. Indeed, this study intended to acquire
a broad overview regarding the suitability of NIRS for the quanti-
fication of several single NSP and not on determination of β-
glucan in barley in detail.
 In contrast, de Sa and Palmer (2006) concentrated on the detec-
tion of β-glucan in barley in detail. The objective of their study
was to detect the changes in β-glucan content in hulled brewing
barley in the course of malting process. A calibration was estab-
lished, yielding R2 = 0.59, which may be due to the small amount
and narrow amount range of β-glucan present in the sample set
(0–1%). In validation, for samples from the beginning of germi-
nation (high in β-glucan) β-glucan could be roughly estimated
whereas for samples taken later on (when β-glucan is partially
degraded) it was impossible to quantify β-glucan. This observa-
tion indicates that NIRS analysis of β-glucan levels in barley
samples requires samples richer in β-glucan than those used by de
Sa and Palmer.
Indeed, a relatively early work of Szczodrak et al (1992) did in-
clude waxy barley samples, resulting in a broader amount range
of 3–9.5%. The Technicon 500 InfraAlyzer was used to perform
this analysis. As in all previously mentioned studies, only a few
naked barley samples were used (out of total 84 flour samples).
Using best fit of wavelength search, the analysis at 2234, 2374
and 2500 nm yielded reasonable R2 = 0.85 but with an unaccept-
able SEE (standard error of estimate) of 0.68%. These results are
not surprising, considering that the authors used the reference β-
glucan determination method described by Ahluwalia and Ellis
(1984). This reference method normally yields an accuracy of
±0.25%, a result considerable higher than the ±0.1% charac-
teristic for currently used AACC McCleary Method.
Recently, the detection of β-glucan in barley selected for fuel
production by different NIR instruments was the subject of two
reports (Sohn et al 2007, 2008).
Because our main interest lies in the introduction of barley
products into human nutrition (within the scope of our project) for
which dehulling is uneconomic (in contrast to breweries where
husks serve as a mean to optimize filtration), naked barley sam-
ples were selected for this study. Importantly, waxy barley sam-
ples that have been proposed to contain higher amounts of β-
glucan than other barley cultivars (Bhatty 1999; Zheng et al 2000)
were also analyzed. In addition, the study also included dark bar-
ley cultivars rich in anthocyanin, which is assumed to play a role
in immune response and neutralization of free radicals (Sieben-
handl et al 2007). It should be noted that these naked dark barley
samples retain anthocyanin which is commonly but unintention-
ally removed in the dehulling process of hulled dark barleys,
stressing another advantage of naked barleys in human nutrition
and food processing. In summary, the objectives of this study
were to verify whether it is possible to quantify β-glucan in naked
barley using near infrared spectroscopy; whether there are some
limitations regarding special sample groups (dark pericarp, waxy)
and application form (whole grain vs. whole meal); and which
technologies and instruments should be optimally used (NIT, dis-
persive NIR, FT-NIR).
MATERIALS AND METHODS
Grain Samples
The data set comprised 107 barley samples representing differ-
ent barley cultivars and special breeding lines out of which 106
samples were naked whereas one cultivar belonged to the hulled
group. To obtain robust calibrations and to evaluate the influences
of sample-associated characteristics on the calibration qualifica-
tion, a relatively diverse data set was provided in terms of peri-
carp color, origin, and crop year, as well as breeding system. In
general, samples grown at different locations distributed all over
the world (Czech Republic, Germany, Syria, Italy, Sweden, Can-
ada, United States, Australia, and Japan) from three crop years
(2004-2006) and from 20 different breeders who used organic or
conventional breeding systems were analysed. The samples in-
cluded six black, one black and purple, three purple, and five
waxy lines.
All samples were freed from loosely adhered husks by a labora-
tory trasher and cleaned manually to get rid of disturbing plant
and ground material, insects, and broken kernels (relevant for
whole kernel measurements). An aliquot of each sample meant
for chemical analyses as well as for establishing calibrations for
whole meal was milled to a particle size of 0.5 mm using a Teca-
tor Cyclotec 1093 sample mill. Grinding took place immediately
before further sample processing and analysis. Throughout the
experiments, whole grain samples were stored in sealed plastic
bags at 4°C.
Chemical Analyses
Protein content was assessed by method after Dumas combus-
tion method (FP-528, Leco, St Joseph, MI). β-Glucan quan-
tification was measured according to the McCleary method using
an enzyme kit for mixed linkage β-glucan (Megazyme, K-BGLU
04/06). Specifically, the assay of mixed-linkage β-glucan in oat
and barley flour samples, streamlined method (AACC Approved
Method 32-23; AOAC Method 995.16; ICC Standard Method No.
168) was employed. Each of these measurements was performed
in duplicate and mean data were used in various calibrations.
NIRS Instrumentation
Four different instruments that were selected after three differ-
ent measuring principles (NIT, dispersive NIR, and FT-NIR),
were used to collect the spectra.
Transmission measurements were recorded on a grain analyzer
(Tecator 1241, Foss Analytical AB, Höganäs, Sweden) in the
wavelength range (λ) of 850–1048 nm at 2-nm intervals yielding
an output of 100 data points. A tungsten halogen lamp served as
the light source; detection was conducted by a SiO2 detector. The
optical pathlength was 18 mm in whole grain samples, accepting
the recommended value for wheat and barley of the manufacturer.
Flour module and sample cups with a 3-mm pathlength were used
for whole meal samples after a pathlength optimization step
where 3- and 6-mm optical pathlength was compared checking
the absorbance range of spectra.
The collection of reflectance spectra was performed (Foss NIR-
systems model 6500, serial number 2327) equipped with a sample
transport module (STM) and model 6500 (serial number 7997)
using a rapid content analyzing (RCA) system containing fiber
optics. They both operated in λ 400–2498 nm scanning at 2-nm
intervals yielding an output of 1050 data points. An SiO2 detector
was used for the λ 400–1098 nm range, whereas a PbS detector
was used for the λ 1100–2498 nm range. For transmission meas-
urements, a tungsten halogen lamp was used as the light source.
Standard sample cups equipped with threaded back were used.
An FT-NIR spectroscopy instrument Matrix I (Bruker Optics,
Billerica, MA) using a white light source and an InGaAs detector
was used. Reflectance was measured with a resolution of 4 cm–1
in an operating range of λ 800–2780 nm (12492–3598 cm–1).
Analyzing Procedure
Whole meal was analyzed on all instruments, whereas grain
was analyzed on all instruments except the Matrix I. All spectral
analyses on Foss NIRSystems instruments as well as meal analy-
ses using Foss Infratec 1241 grain analyzer were conducted in
duplicate with independent sample aliquots in independent sam-
ple holders. Additionally, each of those two sample aliquots was
scanned 32 times. The 32 scans were averaged for each sampling
individually to produce two single spectral data files for each
sample.
In contrast, whole grain spectra collection from the Foss In-
fratec 1241 grain analyzer was conducted twice with five repacks
between scans, with each pack functioning as an independent
subsample. Analogous to the first scheme, the average of each
five analyses was used for calibration. In Matrix I, within-sample
differences were addressed by rotating the sample cup by 180°.
Vol. 86, No. 4, 2009 399
Data Evaluation
Extensive statistic quality evaluation of the reference data was
performed using software (v.7.1, Statsoft, Tulsa, OK) to uncover
potential outliers as well as time-dependent trends occurring over
the measuring period that would declare reference data as invalid.
Furthermore, statistic reference data analyses were applied to
identify potential subpopulations whose presence could indicate
the requirement of another discrete calibration set.
As for reference data, spectral data quality was also evaluated by
visually inspecting the raw spectra such as 2nd derivative and
performing principal component analyses (PCA) for identification
of outliers and examination of the difference between cultivars
and potential subpopulations as well as potential differences be-
tween instruments. Detailed evaluation of spectral data provided
the basis for choosing concrete calibration parameters.
Finally, calibration was performed (WinISI II 1.50, Infrasoft In-
ternational LLC, Port Matilda, PA) for a set of auspicious settings
using partial least square (PLS) algorithm with 10 cross-
validation groups. PLS regression methods were used for all spec-
tra collecting NIT, dispersive NIR, and FT-NIR instruments. PLS
reduces the dimension of the wavelength space from the number
of wavelength points originally stored to a handful of orthogonal
factors, while simultaneously incorporating the influence of a
dependent variable. The maximum number of factors (fmax) of 15
was chosen considering the size of sample population. Outlier
detection and elimination in the course of calibration process took
place by judging the T outliers (actual vs. predicted), the PLS H
outliers (distance from the spectral mean), and the X outliers
(spectra that are poorly modeled). The recommended default val-
ues (T = 2.5, H = 10.0, and X = 10.0) were used to evaluate PLS
calibrations.
Obtained calibrations were assessed, compared, and discussed
on basis of R2, standard error of cross validation (SECV) and re-
sidual predictive deviation (RPD, ratio standard deviation of ref-
erence data/SECV) considering the number of factors (f) needed
and outliers detected.
RESULTS AND DISCUSSION
Within the chosen sample set mean β-glucan content were
4.46% (related to fresh weight). Importantly, values were rela-
tively high and distributed over the relatively wide range (3.33–
7.41%) thereby indicating a better suitability for establishing a
robust calibration equation than the low levels in combination
with the small range of ≈0–1% achieved in a recent study on β-
glucan (de Sa and Palmer 2006). Neither time-dependent trends
nor any unexplainable outliers could be identified. Furthermore,
this study clearly strengthens findings of Bhatty (1999) and Zheng
et al (2000) who stated that waxy barley has extraordinary high β-
glucan levels. Tested waxy samples contained higher amounts of β-
glucan when compared with normal, black, and purple samples.
As shown in Fig. 1, the difference between waxy and normal and
black samples is statistically significant.
The λ 1100–2200 nm suggested a calibration basis for all NIR
instruments for whole grain as well as whole meal application
because it appeared to be the least common denominator due to a
remarkable influence of sample color in lower wavelength regions
for all instruments and types of application as well as a high
background noise in higher wavelength regions for whole grain
measurements by RCA. In at λ 1100–2200 nm, spectra attained
by all three instruments in both whole grain and whole meal form
were expected to be comparable in later steps. In addition, for
whole meal analyses, λ 1100–2500 nm was also used as a basis
for calibrating for ground samples. In the NIT instrument, maxi-
mal range λ 850–1050 nm was used.
Generally NIT/NIR spectra are pretreated to eliminate un-
wanted variability due to path length or particle size variations
before developing calibrations. There is also more than one driv-
400 CEREAL CHEMISTRY
Fig. 1. Box-Whisker plot visualizing β-glucan contents separated according
to sample characteristics
ing force for working with derivative spectra. This pretreatment
can deconvolute overlapping peaks and has scatter correction
effects. The raw spectra were transformed into 2nd derivative
using 2-2-2-1, 2-5-5-1, and 2-10-10-1 in NIT, dispersive NIR, and
FT-NIR spectra, respectively, where the first digit stands for the
number of the derivative, the second digit for the gap over which
the derivative is calculated, the third digit for the number of data
points in a running average or smoothing, and the fourth digit for
the second smoothing. Band sharpness such as the positive side-
effect of eliminating the baseline shift between instruments used
(esp. FOSS NIRSystems), which in turn implies the chance of
mathematical correction, led to the use of 2nd derivative in all
cases. Because 2nd derivative of spectra obtained by Matrix I had
an enhanced background noise in λ < 1700 nm, increased smooth-
ing (2-10-10-1) was employed only for this instrument in attempt
to correct for this problem.
In general, the waxy hulled sample was excluded because spec-
tra strongly deviated from those of naked barley in both shape and
absorbance, whereas for grain samples, its spectrum showed much
lower absorbance in the whole range while for meal it showed
slightly lower absorbance at λ 1100–1300, in agreement with
Czuchajowska et al (1992). In contrast, naked waxy samples
qualified to remain within the calibration set. In detail, in whole
grain analyses, waxy sample spectra were distributed randomly
within the main stream of normal samples, while in whole meal
analyses they formed a distinct area directly adjacent to the main
stream in spectra as well as in PCA. The influence of the dark
pericarp color on spectral data seemed to be considerable. Inter-
estingly, dark samples grouped themselves as separate subpopula-
tions despite using only the typical NIR region with all NIR
instruments and irrespective of application form (whole grain and
whole meal) used. To maintain the chance of analyzing dark sam-
ples of interest due to extraordinary high anthocyanin contents
and to determine the extent of loss in calibration qualification in
dark cultivars, calibration was performed using all samples (ex-
cept waxy hulled) and only normal plus waxy samples.
Finally, based on perceptions mentioned above, most promising
settings were conveyed into the calibration process; these results
are listed in Table I for normal and waxy samples only and analo-
gous for all samples (except waxy hulled) in Table II. Because the
spectra of black and black and purple samples attained by NIT
referred to opaque materials using the given instrument settlings
(18 mm optical pathlength), these samples were also excluded
from all samples calibration set. Tables also contain information
about the PLS regression method: application form of samples,
the respective instruments, wavelength regions, extent of smooth-
ing (2-X-X-1 used), number of spectra, and statistic of calibration
 TABLE I
Selected PLS Models of β-Glucan Calibrations of Normal and Waxy Samples
Application Form
Whole Grain Whole Meal
NIR NIR NIR NIR NIR NIR
Instrumenta (RCA) (STM) NIT (RCA) (RCA) (STM) (STM) NIT FT-NIR FT-NIR FT-NIR FT-NIR
Wavelength range (nm) 1100- 1100- 850- 1100- 1100- 1100- 1100- 850- 1100- 1100- 1100- 1100-
2200 2200 1050 2200 2500 2200 2500 1050 2200 2500 2200 2500
Math treatment 2-5-5-1 2-5-5-1 2-2-2-1 2-5-5-1 2-5-5-1 2-5-5-1 2-5-5-1 2-2-2-1 2-5-5-1 2-5-5-1 2-10-10-1 2-10-10-1
All spectra 192 192 96 192 192 192 192 192 192 192 192 192
Outlier spectra 16 7 4 9 1 7 7 4 6 7 10 17
Outlier percentage (%) 8.3 3.6 4.2 4.7 0.5 3.6 3.6 2.1 3.1 3.6 5.2 8.9
Calibration spectra 176 185 92 183 191 185 185 188 186 185 182 175
Minimum (%)b 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33
Maximum (%)b 6.84 7.41 7.41 7.24 7.41 7.24 7.41 7.41 6.32 7.24 7.24 7.24
Range (%)b 3.51 4.08 4.08 3.91 4.08 3.91 4.08 4.08 2.99 3.91 3.91 3.91
Mean (%)b 4.399 4.417 4.455 4.426 4.462 4.427 4.455 4.460 4.370 4.403 4.438 4.432
SD (%)b,c 0.639 0.702 0.784 0.715 0.782 0.720 0.784 0.783 0.620 0.703 0.729 0.728
d
f 12 14 9 13 8 12 8 10 8 9 14 15
e
R2 0.837 0.848 0.786 0.871 0.783 0.866 0.861 0.735 0.952 0.976 0.976 0.989
SECV (%)f 0.384 0.385 0.452 0.370 0.474 0.324 0.339 0.460 0.328 0.279 0.260 0.183
RPDg 1.66 1.82 1.73 1.93 1.65 2.22 2.31 1.70 1.89 2.52 2.80 3.98
a NIR (STM), FOSS NIRSystems model 6500 equipped with sample transport module; NIR (RCA), FOSS NIRSystems model 6500 equipped with rapid content
analyzer; NIT, FOSS Tecator 1241 grain analyzer; FT-NIR, BRUKER Matrix I.
b β-Glucan content (as is) of calibration spectra population.
c Standard deviation.
d Number of PLS factors.
e Squared correlation coefficient.
f Standard error of cross-validation.
g Residual predictive deviation (SD/SECV).

Fig. 2. Second derivative spectra of FOSS NIRSystems 6500 + STM for whole kernels (A) and for whole meal (B) in λ = 1100–2200 nm.

samples regarding β-glucan content. The well-established calibra-
tion quality parameters R2, SECV, and RPD as well as the dis-
qualification criteria number of factors (f) and outlier percentage
(Williams 2001) were calculated. Importantly, during the outlier
elimination procedure, neither dark samples nor waxy samples
were pushed out of the calibration set in above-average number
for any model; this allowed us to study their influence on calibra-
tion quality.
Overall, models that include dark samples are not of lesser
quality and only with marginal differences in comparison to re-
gressions based only on normal and waxy samples.
The results for grain analyses consistently appeared less prom-
ising than those acquired through whole meal application (shown
in the spectroscopic background for Foss NIRSystems 6500 +
STM in Fig. 2). On one hand, this is reflected in the ineligible
high number of factors of 14 for four out of six models; on the
other hand, in the relatively high number of outliers of 8.3 and
8.5% for the remaining two models. Apparent better suitability of
whole meal application could be attributed to lower absorbance
variance in spectra, which in turn probably is due to high influ-
ence of surface consistency on absorption. Nevertheless, despite
the relatively high amount of outliers (8.3%) and RPD = 1.66,
employing RCA in grain application could be considered as a fair
model for screening whole kernels (f = 12; R2 = 0.84; RPD =
1.66) with limitation to normal and waxy samples only (Fig. 3).
To evade sample limitation in whole kernel measurements, STM
can be used alternatively, in which case another factor becomes
limiting (R2 = 0.78). Such rough quantification of β-glucan in
whole kernels is important because it would meet the demand for
nondestructive single kernel inline screening (for β-glucan, but
also for other parameters like protein content) combined with
subsequent sorting of kernels. Using this approach, problems
arising in currently used solutions, which involve detailed analy-
sis of a small (but not necessary representative) sample aliquot,
would not occur. Single-kernel sample injection systems are al-
ready on the market (e.g., Perten instruments); these instruments,
although continuously being improved, still require moderate
improvements in sample processing speed.
Comparing whole meal analyses with respect to instrument
qualification clearly demonstrates best fitness of models con-
structed by use of mathematically pretreated (2-5-5-1) spectra
attained by the Matrix I FT-instrument (especially at λ = 1100-

Vol. 86, No. 4, 2009 401

2500 nm), whose superiority is reflected in all considered pa-
rameters such as f = 8–9, R2 ≈ 0.96–0.98, RPD = 2.33–2.52, and
SECV ≈ 0.28–0.30% (number of outliers 3.6–4.2%). The superi-
ority of the Matrix I FT-instrument is corroborated by homogene-
ity in PCA that is far higher than for other instruments.
Smoothing led to obviously overfitted models with unacceptable
high factor numbers and relatively high number of outliers.
Out of the two dispersive NIR instruments, STM in comparison
with RCA, did slightly better in both regions studied (number of
outliers 1.4–3.6%; f = 8-12; R2 ≈ 0.85–0.86; RPD = 2.09–2.31;
SECV ≈ 0.32–0.38%). In addition, RCA qualification parameters
(outlier% = 0.5-4.7; f = 8–13; R2 = 0.78–0.87; RPD = 1.65–1.93;
SECV = 0.37–0.47%) showed higher calibration-to-calibration
cultivar than those of STM. The common, industrially used NIT
instrument FOSS Tecator 1241 grain analyzer appeared to be
inferior to all other instruments tested (R2 ≈ 0.74–0.76, RPD =
1.70, and SECV = 0.43–0.46%); f and SECV resulted in values
comparable to other instruments used in this study.
Recently, Sohn et al (2007, 2008) analyzed a number of differ-
ent barley samples using FT-NIR and dispersive NIR spectros-
copy. Because their aim was to identify the samples suitable for
bioethanol production using the above mentioned methods, the
barley samples rich in β-glucan (i.e., waxy barley samples) were
not included in their study. Indeed, one of the requirements for
bioethanol production is a low β-glucan content. In comparison to
our study, Sohn et al used fewer and partly different instruments.

TABLE II
Selected PLS Models of β-Glucan Calibrations of All Samples (Except Waxy Hulled)
Application Form
Whole Grain Whole Meal
NIR NIR NIR NIR NIR NIR
Instrumenta (RCA) (STM) NIT (RCA) (RCA) (STM) (STM) NIT FT-NIR FT-NIR FT-NIR FT-NIR
Wavelength range (nm) 1100- 1100- 850- 1100- 1100- 1100- 1100- 850- 1100- 1100- 1100- 1100-
2200 2200 1050 2200 2500 2200 2500 1050 2200 2500 2200 2500
Math treatment 2-5-5-1 2-5-5-1 2-2-2-1 2-5-5-1 2-5-5-1 2-5-5-1 2-5-5-1 2-2-2-1 2-5-5-1 2-5-5-1 2-10-10-1 2-10-10-1
All spectra 212 212 99 212 212 212 212 198 212 212 212 212
Outlier spectra 12 18 6 9 2 7 3 8 20 9 16 40
Outlier percentage (%) 5.7 8.5 6.1 4.2 0.9 3.3 1.4 4.0 9.4 4.2 7.5 18.9
Calibration spectra 200 194 93 203 210 205 209 190 192 203 196 172
Minimum (%)b 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33
Maximum (%)b 7.41 5.85 7.24 7.24 7.41 7.24 7.41 7.24 5.85 7.24 7.24 7.41
Range (%)b 4.08 2.52 3.91 3.91 4.08 3.91 4.08 3.91 2.52 3.91 3.91 4.08
Mean (%)b 4.453 4.349 4.419 4.410 4.457 4.441 4.461 4.433 4.356 4.417 4.448 4.522
SD (%)b,c 0.745 0.605 0.723 0.729 0.786 0.732 0.786 0.732 0.602 0.695 0.741 0.807
d
f 14 13 9 13 9 11 11 11 9 8 15 15
e
R2 0.870 0.778 0.770 0.843 0.782 0.846 0.847 0.756 0.972 0.959 0.978 0.994
SECV (%)f 0.428 0.356 0.438 0.394 0.468 0.333 0.376 0.430 0.257 0.298 0.261 0.147
RPDg 1.74 1.70 1.65 1.85 1.68 2.20 2.09 1.70 2.34 2.33 2.84 5.49
a NIR (STM), FOSS NIRSystems model 6500 equipped with sample transport module; NIR (RCA), FOSS NIRSystems model 6500 equipped with rapid content
analyzer; NIT, FOSS Tecator 1241 grain analyzer; FT-NIR, BRUKER Matrix I.
b β-Glucan content (as is) of calibration spectra population.
c Standard deviation.
d Number of PLS factors.
e Squared correlation coefficient.
f Standard error of cross-validation.
g Residual predictive deviation (SD/SECV).

Fig. 3. Scatterplot of FOSS NIRSystems 6500 + RCA (A) and FOSS NIRSystems 6500 + STM (B) for normal and waxy whole kernel samples.

402 CEREAL CHEMISTRY

Specifically, for whole meal analysis, Sohn et al (2007) employed
Foss NIRSystems 6500 and Bruker Vector22/N; for whole grain
analysis, Sohn et al (2008) used a Foss XDS rapid content ana-
lyzer. For NIRSystems 6500, which is the only instrument used
both in this study and in Sohn et al (2007, 2008), we were able,
regardless of waxy and dark samples used in our studies, to gen-
erate clearly better calibrations for both whole grain and whole
meal sample sets. Sohn et al (2007, 2008) results were R2 = 0.67,
RMSECV = 0.42%, RPD = 1.58 for ground barley and R2 =
0.47, RMSECV = 0.5%, RPD = 1.43 for whole kernels, respec-
tively). This can most probably be attributed to sample set; as
obvious from occurring subpopulations in spectra and PCA as
published in Sohn et al (2007, 2008), these authors have used
considerably different samples, specifically, hulled barley and
malt together with naked barley as one calibration set, whereas
we focused on naked barley samples alone. The selection of bio-
ethanol-relevant Doyce cultivar grown at numerous different
places and other samples grown at a single location probably had
a further influence on calibration quality.
Importantly, our analysis included the commonly industrially
used instrument Foss Tecator 1241 grain analyzer, to show
whether instrumentation currently used in food quality assurance
can be directly used for β-glucan analysis of barley samples. This
instrument yields relatively little information in a narrow wave-
length range (100 data points at λ = 850–1050) and was expected
to be unsuitable for analysis of β-glucan, which is supposed to be
indicated at higher wavelengths (Czuchajowska et al 1992).
Nonetheless, a calibration of moderate quality was acquired for
both whole meal and whole grain samples, albeit the dark barley
samples had to be excluded from the sample set (as discussed
above). This surprising result indicates not only that sufficient
information on β-glucan can be collected in the narrow range of
850–1050, but that common instrumentation can easily be em-
ployed in selection according to β-glucan levels.
Interestingly, this finding might be of further relevance consid-
ering different ranges employed by Sohn et al (2007, 2008) in
their comparison of different instruments (780–2500 nm used for
FT instrument and 1100–2500 nm used for dispersive instrumen-
tation).
Nonetheless, the analyses performed by Sohn et al (2007, 2008)
in general support our findings, primarily in that the correlation
between NIR spectra and β-glucan content exists, but also in that
milled samples yield better results than whole kernel samples and
that FT instrumentation combined with 2nd derivation analysis
yields best results.
CONCLUSIONS
Several near-infrared spectroscopy calibrations could be estab-
lished that are able to estimate β-glucan levels in normal, waxy,
and black and purple naked barley. Accuracy met the require-
ments of two processes that considerably depend on β-glucan
levels in barley: monitoring of breeding and classification of bar-
ley deliveries to entrance control into groups high containing and
low β-glucan. FT-NIRS instruments, such as both dispersive NIRS
instruments tested, were capable of simultaneous determination of
waxy, purple and black samples, whereas the common industrial
transmittance measuring instrument operating at λ = 850–1050
nm was suitable only for recording of normal, waxy, and purple
samples but not for recording black samples. In general, whole
meal analyses seemed to be slightly superior compared with
whole grain application. The best results, indicating that the in-
strument was suitable for analytical studies, were attained with
the FT instrument. In comparison, the two tested dispersive NIRS
instruments yielded results of slightly reduced quality, whereas
the NIT instrument appeared at least suitable for the β-glucan
determination in barley, albeit still suitable for analysis of non-
black barley samples.
ACKNOWLEDGMENTS
We wish to thank Vollmann for granting us access to Bruker Matrix I
instrument. A part of results described in this study was attained with
financial support of WTZ Österreich/Ungarn, ÖAD (project ID 19/2005).
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[Received March 26, 2008. Accepted April 12, 2009.]

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