folia biologica et geologica 57/3, 11–18, ljubljana 2016
CONCENTRATION OF PROTEINS, BETA-GLUCANS, TOTAL 
PHENOLS AND ANTIOXIDANT CAPACITy OF SLOVENIAN 
SAMPLES OF BARLEy
VSEBNOST PROTEINOV, BETA-GLUKANOV, SKUPNIH 
FENOLOV IN ANTIOKSIDATIVNA VREDNOST SLOVENSKIH 
VZORCEV JEČMENA
Giovanni Bonafacciaa, Nicolò Merendinob, Francesco Bonafacciab, Romina Molinarib, 
Vincenzo Gallia, Igor Pravstc, Vida Škrabanjad, Zlata Luthard, Aleksandra Golobd & Mateja 
Germd *
Received October 27, 2016; accepted December 31, 2016.
Delo je prispelo 27. oktobra 2016, sprejeto 31. decembra 2016. 
 a Food and Nutrition Research Center (CRA - NUT), Via Ardeatina 546, 00178 Roma, Italy.
 b Department of Ecological and Biological Sciences (DEB), Laboratory of Cellular and Molecular Nutrition, University of Tus-
cia, Largo dell’Università snc, 01100 Viterbo, Italy. 
 c Nutrition Institute, Tržaška 40, SI-1000 Ljubljana, Slovenia.
 d Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia.
 * e-mail: Mateja.Germ@bf.uni-lj.si
abStRact
concentration of proteins, beta-glucans, total phenols 
and antioxidant capacity of Slovenian samples of barley
Four Slovenian barley samples were analyzed for the 
content of the ash, proteins, β-glucans and total polyphe-
nols; and for the antioxidant capacity by two methods. Anti-
oxidant capacity was determined by the ferric reducing anti-
oxidant power (FRAP) activity and by the oxygen radical 
absorbance capacity (ORAC) assay. For the comparison, four 
foreign barley commercial samples were analyzed, three of 
German origin and one from Italy. Slovenian samples had 
equal or lower content of ash, proteins and β-glucans in 
comparison to foreign samples. A Slovenian barley sample 
from Šalovci, Prekmurje had the highest content of 
β-glucans, namely 5.73% in dry weight (DW). The results 
obtained by both methods of the determination of antioxi-
dant capacity showed significant correlation with the total 
polyphenols content.
Key words: Hordeum, barley, beta-glucans, phenols
iZVleČeK
Vsebnost proteinov, beta-glukanov, skupnih fenolov in 
antioksidativna vrednost slovenskih vzorcev ječmena
Štiri vzorce ječmena iz Slovenije smo analizirali na vs-
ebnost pepela, beljakovin, β-glukanov in skupnih fenolov. S 
pomočjo dveh metod smo tudi ugotavljali antioksidativno 
zmogljivost. Antioksidantivna zmogljivost je bila določena z 
zmanjšanjem antioksidativne sposobnosti železa (metoda 
FRAP) in s sposobnostjo absorbance kisikovega radikala 
(metoda ORAC). Za primerjavo smo analizirali štiri komer-
cialne vzorce tujega ječmena, tri nemške in enega iz Italije. 
Slovenski vzorci so imeli enako ali nižjo vsebnost pepela, 
beljakovin in β-glukanov v primerjavi s tujimi. Vendar je 
slovenski vzorec ječmena iz Šalovcev (Prekmurje) vseboval 
najvišjo vsebnost β-glukanov, in sicer 5,73% suhe teže (DW). 
Rezultati obeh metod določanja antioksidativne kapacitete 
so bili primerljivi in so pokazali pomembno povezavo z vs-
ebnostjo celokupnih fenolov.
Ključne besede: Hordeum, ječmen, beta-glukani, fenoli
CONCENTRATION OF PROTEINS, BETA-GLUCANS, TOTAL PHENOLS AND ANTIOXIDANT CAPACITY
12 folia biologica et geologica 57/3 – 2016
Interest in growing barley, a crop cultivated by human-
kind for a long time, has become more intense in recent 
years in Slovenia, Europe and elsewhere, due to the nu-
tritional value of this ancient crop. Barley was culti-
vated for centuries for human food, but the areas of 
growing of barley declined in the middle of the 
20th century. However high fibre foods are recently 
preferred in human nutrition. It should be noted that 
the cultivation of barley was for many years particu-
larly focused on achieving high dry matter content, 
high starch, and low content of dietary fibre. This is 
because barley has been used mostly for either animal 
feed or for production of barley malt. Cultivars with 
higher levels of dietary fibre, particularly of β-glucans, 
are therefore now gaining importance for use in human 
nutrition and some of these are being introduced to 
Southern and Central Europe, including Slovenia. 
Identification of suitable cultivars high in β-glucan 
content, evolution of milling and dry separation tech-
niques and using the β-glucan-enriched fractions as an 
ingredient in bakery products are subjects for further 
needed research. The methods developed will allow 
obtaining new, nutritionally valuable barley products 
with high potential for use in foods.
In recent months it was a quick development of re-
search and published papers on barley β-glucans. 
Shaik et al. (2016) reported that in amylose-only 
starch chemotypes it is showed significant reduction in 
starch accumulation with the re-direction to protein 
and β-glucan accumulation. Ho et al. (2016) newly re-
ported a systematic review of the effects of different 
high molecular weight β-glucans on cholesterol and 
cardiovascular disease risk reduction.
Moza & Gujral (2016) reported that in India 
high altitude barley cultivars (between 1200 m and 
3500 m above sea level) contain higher levels of total 
β-glucans in comparison to the barley cultivars grown 
in plains (97-126 m altitude). Cultivars grown in higher 
altitudes may find better utilization in nutraceutical 
foods. Han et al. (2016), Zhu et al. (2016) and Dimi-
troff et al. (2016) suggested how, understanding ge-
netic background, barley β-glucans based functional 
food may be developed for the management of obesity. 
Belobrajdic et al. (2016) suggest a wholegrain barley 
high molecular weight β-glucan to lover food intake.
After the separation in barley after milling to ten 
milling fractions, the highest β-glucan content was de-
termined in the bran fraction (Wiege et al. 2016). It 
was a strong positive correlation between β-glucan and 
protein content. Fractions containing high level of 
β-glucan have as well high concentration of protein 
and ash (Wiege et al. 2016).
Croatian barley samples were screened for total 
high molecular weight β-glucans content (Krstano-
vić et al. 2016), but such kind of research was not yet 
performed in Slovenia.
1 INTRODUCTION
2 MATERIALS AND METHODS
2.1 Samples
Barley samples analysed are presented in Table 1.
table 1: Description of studied barley samples
Preglednica 1: opis preučevanih vzorcev ječmena
Sample Description
A Winter barley sample from Šentjernej, Dolenjska, Slovenia
B Winter barley sample from Trbonje, Dravograd, Slovenia
C Winter barley sample from Šalovci, Prekmurje, Slovenia
D Winter barley sample from Murska Sobota, Prekmurje, Slovenia
E Winter barley sample from Nienstad, Germany
F Winter barley sample from Germany, cultivated in Jable, Slovenia
G Naked barley sample from Asheberg, Germany
H Winter barley sample from Lazio, Italy
Samples A-D are samples of barley grain, each of 
barley domestic cultivars was grown for years by farm-
ers in different parts of Slovenia. The origin of barley 
cultivars is not known. Samples E-F are commercial 
barley samples available in Slovenia, originating, as far 
as it is known, in Germany or Italy.
CONCENTRATION OF PROTEINS, BETA-GLUCANS, TOTAL PHENOLS AND ANTIOXIDANT CAPACITY
13folia biologica et geologica 57/3 – 2016
2.2 Methods
2.2.1 Determination of moisture, ash, proteins and 
β-glucans 
Constituents of barley samples were determined using 
the standard methods.
For the determination of moisture: Method ICC 
No. 109/1, was used.
In short: This method can be taken as the standard 
for the development of methods which are specifically 
suited to the practical determination of the moisture 
content of wheat, rice (hulled paddy), barley, maize or 
whole maize meal, millet, rye and oats, as grains, 
ground grains, semolina and flour. It is not to be used 
for the settlement of commercial disputes. Measure-
ment of moisture loss when the material, ground if 
necessary without change of moisture content, is 
equilibrated in an anhydrous atmosphere at a tempera-
ture between 45 and 50 °C and at a pressure of 1.3 ... 2.7 
KPa (10 ... 20 mm Hg).
For the determination of ash: Method ICC No. 
104, was used.
In short: Samples were ground and placed into 
crucibles, and crucibles placed into a muffle furnace. 
The ashing was carried out at 900 °C, and is completed 
when the cool residue was white or nearly white. As the 
ash quantity has to be related to dry matter, the mois-
ture content of the test sample has to be determined 
separately.
For the determination of proteins: Method ICC 
105/2. Kjeldhal Method was used.
In short: The organic matter of the sample is oxi-
dized with concentrated sulfuric acid in the presence 
of a catalyst: the product of the reaction (NH4)2SO4 is 
treated by alkali; free ammonia is distilled and titrated.
For the determination of β-glucans: Method ICC 
No.166 was used.
In short: β-D-glucan is determined using highly 
purified lichenase and β-D-glucosidase. β-D-glucan is 
specifically hydrolyzed by lichenase to oligosaccha-
rides, which are quantitatively cleaved to glucose by 
β-glucosidase. Glucose is measured using glucose oxi-
dase - peroxides - buffer mixture. Method is rapid pro-
cedure for direct, quantitative measurement of (1->3) 
(1->4)- β-D-glucan (β-D-using highly purified li-
chenase and β-glucosidase). β-D-glucan is specifically 
hydrolyzed by lichenase to oligosaccharides, which are 
then quantitatively cleaved to glucose by β-glucosidase. 
Glucose is measured using glucose oxidase-peroxi-
dase-buffer mixture.
2.2.2 Extraction method
In this study, 80% methanol extracts from barley were 
used for the determination of total phenolic content 
and antioxidant property. Barley samples (1 g) were 
extracted with 6 ml acidified methanol (HCl/metha-
nol/water, 1:80:10, v/v/v) at room temperature (25 °C) 
for 2 h using orbital shaker. The mixture was centri-
fuged at 3000g for 10 min. The supernatant was used 
for determination of total phenolic content and anti-
oxidant capacity. Several studies have shown that 80% 
methanol is an effective solvent in extracting phenolic 
and other polar substances from cereals (Lahouar et 
al. 2014).
2.2.3 Total phenolic content determination
Total phenols (TP) were determined using a modifica-
tion of the Folin–Ciocalteu method method described 
by Singleton & Rossi (1965). Briefly, the assay was 
conducted by mixing 4 mL of deionized water, 0.25 mL 
of extracts (see ‘Extract preparations’ in this section), 
0.25 mL of Folin–Ciocalteu reagent, and 0.5 mL of 
Na2CO3. After 30 min at room temperature, the absor-
bance of the mixture was measured at 725 nm. A stan-
dard curve was prepared with gallic acid. The final 
results were expressed as mg of gallic acid equivalents 
(GAE) per g of dry weight (DW). All of the analyses 
were conducted in triplicate and the results report the 
sum of bound and soluble phenolic compounds.
2.2.4 Determination of ferric reducing antioxidant 
power (FRAP) activity
A ferric reducing antioxidant power (FRAP) assay was 
performed according to the method described by Ben-
zie & Strain (1999), which was adapted for 96-well 
plates and an automatic reader (Infinite 2000, Tecan, 
Salzburg, Austria). The method is based on the reduc-
tion of the Fe3+-2,4,6-tripyridyl-s-triazine (TPTZ) 
complex to its ferrous form at a low pH. Briefly, 160 mL 
of FRAP assay solution (consisting of 20 mM ferric 
chloride solution, 10 mM TPTZ solution, and 0.3 M 
acetate buffer at pH 3.6) was prepared daily, mixed 
with 10 mL of the sample, standard, or blank, and dis-
pensed into each well of a 96-well plate. The absorb-
ance was measured at 595 nm at 37 °C after 30 min of 
incubation. All of the analyses were conducted in trip-
licate. The final results are expressed as µmol Fe2+ 
equivalents per g of the DW of the samples, and the 
results were obtained using a standard curve with dif-
ferent concentrations of ferrous sulfate heptahydrate 
(FeSO4 x 7H2O).
CONCENTRATION OF PROTEINS, BETA-GLUCANS, TOTAL PHENOLS AND ANTIOXIDANT CAPACITY
14 folia biologica et geologica 57/3 – 2016
2.2.5 The oxygen radical absorbance capacity (ORAC) 
assay
The oxygen radical absorbance capacity (ORAC) was 
determined using the hydroxyl radical antioxidant ca-
pacity (HORAC) assay kit (Oxford Biomedical Re-
search, Oxford USA) according to the manufacturer’s 
instructions. The final ORAC values were expressed as 
gallic acid equivalents and determined according to 
the standard curve. All of the analyses were conducted 
in triplicate.
3. RESULTS AND DISCUSSION
Slovenian barley samples were analysed and compared 
with the foreign ones. Results of the ash (i.e. total con-
tent of mineral elements), protein and β-glucans con-
centration are presented in Table 2.
table 2: concentration of ash, protein and β-glucans in barley samples, mean values and standard deviations 
(DW=dry weight).
Preglednica 2: Koncentracija pepela, beljakovin in β-glukanov v vzorcih ječmena, povprečne vrednosti in 
standardni odkloni (DW=sušina).
Sample Moisture % Ash % DW Protein % DW β-glucans % DW
A 10.9 ± 0.3 1.83 ± 0.08 8.7 ± 0.1 4.56 ± 0.09
B 11.0 ± 0.5 1.67 ± 0.10 8.8 ± 0.2 4.06 ± 0.07
C 11.0 ± 0.3 1.30 ± 0.11 8.2 ± 0.6 5.73 ± 0.11
D 11.3 ± 0.2 1.66 ± 0.04 8.2 ± 0.5 4.19 ± 0.06
E 10.9 ± 0.4 1.72 ± 0.07 9.7 ± 0.5 5.36  ± 0.08
F 10.7 ± 0.3 1.95 ± 0.09 9.8 ± 0.2 4.76 ± 0.04
G 10.3 ± 0.5 2.25 ± 0.06 11.1 ± 0.7 5.19 ± 0.06
H 11.4 ± 0.3 2.42 ± 0.03 9.6 ± 0.4 3.73 ± 0.10
All analyzed samples show a high protein content 
(average 9.3% DW), with values  ranging from 8.2% 
DW to 11.1% DW. The sample G had a highest value. 
From the point of view of β-glucans the sample D 
(Šalovci, Slovenia) is the most interesting because it 
had highest β-glucans concentration. 
Correlation coefficient between ash and proteins 
in grain among all samples analysed was 0.74; correla-
tion coefficient between ash and ß-glucans was -0.46; 
correlation coefficient between proteins and ß-glucans 
was 0.15. We used Pearson correlation coefficient. 
Thus the correlation between ash and proteins is sig-
nificant (p<0.5) and positive, correlation between ash 
and β-glucans was significant (p<0.5) and negative. 
There was no significant correlation between the 
amount of proteins and β-glucans in studied barley 
samples. Barley has a distinct advantage over some 
other grains in that β-glucan soluble fiber is found 
throughout the entire barley kernel. In some other 
grains, the fiber is only found in the outer bran layer. If 
these grains are processed, the fiber can be easily lost 
(Wiege et al. 2016). 
The all samples analysed showed average values  of 
β-glucans 4.69% DW, with a maximum value of 5.73% 
DW and a minimum value of 3.73% DW. 
The ash values were with a minimum of 1.3% DW, 
and a maximum of 2.42% DW. 
To better understand the relationship between the 
antioxidant activity and total phenolic content, the 
TPC of the sample extracts were determined using the 
Folin–Ciocalteu phenol reagent. The results are ex-
pressed as mg of gallic acid equivalents per gram of dry 
mater, and are presented in Table 3. Barley samples 
with high amount of phenolics also showed high anti-
oxidant activity (FRAP and ORAC assay). 
The total antioxidant capacity was in linear re-
gression with the total phenolic content in barley sam-
ples (R2 = 0.9948 and 0.8192 for the results of FRAP 
and ORAC assays, respectively) (Fig. 1).
There were differences in the antioxidant activities 
by ORAC and FRAP, and in content of total polyphe-
nols of investigated barley samples.  
CONCENTRATION OF PROTEINS, BETA-GLUCANS, TOTAL PHENOLS AND ANTIOXIDANT CAPACITY
15folia biologica et geologica 57/3 – 2016
table 3: the total phenolic contents (tP) and antioxidant capacity (FRaP and oRac method) of barley sam-
ples. Data are reported to mean (n=3) ± standard error. Values in the same column not sharing a common 
letter differ significantly at P≤0.05. test aNoVa. X  mg gae/g dried weight (DW). y µmol Fe2+ equivalent/g 
DW. z gae mM/g DW. gae: gallic acid equivalent; DW: dry weight
Preglednica 3: Vsebnost skupnih fenolov (tP) in antioksidantivna zmogljivost (metodi FRaP in oRac) pri 
različnih vzorcih ječmena. Podatki so povprečje (n = 3) ± standardna napaka. Vrednosti v istem stolpcu, ki 
nimajo iste črke se med seboj značilno razlikujejo P ≤ 0.05. test aNoVa. X  mg gae/g suhe teže (DW). y 
µmol Fe2+ ekvivalent/g DW. z gae mM/g DW. gae: ekvivalent galne kisline; DW: suha teža
Sample TPCx FRAPy ORACz
A 2.8 ± 0.2b 227.0 ± 1.5b 21.0 ± 0.1b
B 2.0 ± 0.3b 180.2 ± 2.9c 21.0 ± 0.3b
C 1.5 ± 0.2c 149.2 ± 1.6d 17.1 ± 0.2c
D 2.4 ± 0.3b 202.7 ± 0.2b 20.6 ± 0.4b
E 3.5 ± 0.5a 273.0 ± 0.9a 23.2 ± 0.9a
F 2.1 ± 0.2b 184.2 ± 1.3c 21.1 ± 0.2b
G 3.8 ± 0.4a 286.6 ± 2.1a 25.7 ± 0.2a
H 3.0 ± 0.1 a 231.0 ± 2.2 b 24.1 ± 0.1 a
Figure 1: Linear regression between total phenolic content and the total antioxidant capacity (FRAP method - a ferric reducing 
antioxidant power) of barley samples. GAE: gallic acid equivalent; DW: dry weight
Slika 1: Linearna regresija med skupno vsebnostjo fenolov in skupno antioksidativno kapaciteto vzorcev ječmena (metoda FRAP 
- zmanjšanje antioksidativne moči železa). GAE: ekvivalent galne kisline; DW: suha teža
CONCENTRATION OF PROTEINS, BETA-GLUCANS, TOTAL PHENOLS AND ANTIOXIDANT CAPACITY
16 folia biologica et geologica 57/3 – 2016
The Slovenian barley sample from Šalovci, Prek-
murje had the highest content of β-glucans among the 
studied Slovenian and foreign samples, namely 5.73% 
Zanimanje za pridelovanje ječmena, stare poljščine, 
se je zadnje čase povečalo v Sloveniji, Evropi in drugod, 
predvsem zaradi njegove hranilne vrednosti. Ječmen 
gojijo za prehrano ljudi že stoletja, vendar so se območja 
pridelave zmanjšala zlasti v sredini 20. stoletja. V pre-
hrani ljudi so zadnje čase zelo zaželena živila, kot je je-
čmen, z visoko vsebnostjo vlaknin. Žlahtnjenje in pri-
delovanje ječmena sta bili vrsto let osredotočeni pred-
vsem na doseganje visokih pridelkov sušine, veliko 
škroba in nizko vsebnost prehranskih vlaknin, ker so ga 
večinoma uporabljali bodisi za živalsko krmo ali za pro-
izvodnjo ječmenovega sladu. Sorte, ki vsebujejo več pre-
hranskih vlaknin, še posebej β-glukanov, so v novejšem 
času vedno bolj pomembne za uporabo v prehrani ljudi. 
V zadnjih mesecih je bil hiter razvoj raziskav in 
objavljanja člankov o ječmenovih β-glukanih. Shaik 
Figure 2: Linear regression between total phenolic content and the total antioxidant capacity (ORAC method - the oxygen 
radical absorbance capacity) of barley samples. GAE: gallic acid equivalent; DW: dry weight
Slika 2: Linearna regresija med skupno vsebnostjo fenolov in skupno antioksidativno kapaciteto vzorcev ječmena (ORAC 
metoda - absorbanca kisikovega radikala). GAE: ekvivalent galne kisline; DW: suha teža
4 CONCLUSIONS
in dry weight. The results obtained by both methods of 
the determination of antioxidant capacity were in li-
near relation with the total phenols content. 
5 POVZETEK
in sod. (2016) so poročali, da je genotip ječmena z ami-
loznim škrobom pokazal pomembno zmanjšanje kopi-
čenja škroba s preusmerjanjem sinteze na beljakovine 
in akumulacijo β-glukanov.
Štiri vzorce ječmena iz Slovenije smo analizirali na 
vsebnost pepela, beljakovin, β-glukanov in skupnih fe-
nolov ter za antioksidativno kapaciteto z dvema meto-
dama. Antioksidativna zmogljivost je bila določena z 
zmanjšanjem antioksidativne moči železa (metoda 
FRAP) in s sposobnostjo absorbcije kisikovega radikala 
(metoda ORAC). Za primerjavo smo analizirali štiri 
tuje komercialne vzorce ječmena, tri nemškega izvora 
in enega iz Italije. Slovenski vzorci so imeli enako ali 
nižjo vsebnost pepela, beljakovin in β-glukanov v pri-
merjavi s tujimi. Vendar je slovenski vzorec ječmena iz 
Šalovcev (Prekmurje) imel najvišjo vsebnost β-glukanov 
CONCENTRATION OF PROTEINS, BETA-GLUCANS, TOTAL PHENOLS AND ANTIOXIDANT CAPACITY
17folia biologica et geologica 57/3 – 2016
in sicer 5,73% suhe teže (DW). Vzorci imajo povprečne 
vrednosti β-glukanov 4,69% suhe teže (DW), z najvišjo 
vrednostjo 5,73% suhe teže (DW) in minimalno vre-
dnostjo 3,73% suhe teže (DW). Rezultati obeh metod 
določanja antioksidativne kapacitete so pokazali po-
membno korelacijo s celotno vsebnostjo polifenolov.
Za boljše razumevanje razmerja med antioksida-
tivnim delovanjem in skupno vsebnostjo fenolov so 
bili analizirani ekstrakti vzorcev za TPC z uporabo Fo-
lin-Ciocalteu-jevega fenolnega reagenta. Rezultati so 
bili izraženi kot mg ekvivalentov galne kisline na gram 
suhe teže in so predstavljeni v razpredelnici 2. Raziska-
ni vzorci ječmena E, G, H vsebujejo najvišjo vrednost 
TPC, medtem ko je bila najnižja vrednost v vzorcu C 
(razpredelnica 2, prvi stolpec). Na splošno je ječmen z 
veliko količino fenolov pokazal tudi visoko antioksida-
tivno aktivnost (po metodah FRAP in ORAC). Skupna 
antioksidativna zmogljivost je bila tudi linearno pove-
zana s skupno vsebnostjo fenolov v vzorcih ječmena 
(R2 = 0,9948 in 0,8192 za rezultate iz FRAP in ORAC 
metod) (sl. 1). Rezultati so pokazali, da so različni 
vzorci imeli različne antioksidativne sposobnosti.
ACKNOWLEDGEMENT
This study was financially supported in part by a grant 
from the Agriculture Ministry of Italy (MIPAF) AL-
IMED Project (cod 7110/730303/10) and by the Slove-
nian Research Agency, through programmes P3-0395 
“Nutrition and Public Health”, and P1 – 0212 “Biology 
of Plants” and projects “young researchers” [grant 
number 34326], and L4-7552, and co-financed by Eu-
ropean Community under project No. 26220220180: 
Building Research Centre “AgroBioTech”. Ministry of 
Agriculture, Forestry and Food, Republic of Slovenia, 
financed Slovenian Plant Gene Bank (Slovenska rast-
linska genska banka) at Biotechnical Faculty, PP 
142910. Ms. Lucija Glorija Jelen Krivonog is acknowl-
edged to perform formating of the paper.
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