International Journal of

Environmental Research
and Public Health

Article
Comparative Quality Assessment of Five Bread Wheat and Five
Barley Cultivars Grown in Romania
Elena Moros, an 1 , Ana Andreea Secareanu 2 , Adina Magdalena Musuc 3, * , Magdalena Mititelu 1, * ,
Ana Corina Ionit, ă 1 , Emma Adriana Ozon 2, *, Ionut, Daniel Raducan 4 , Andreea Ioana Rusu 4 ,
Adriana Maria Dărăban 4 and Oana Karampelas 2

1 Department of Clinical Laboratory and Food Safety, Faculty of Pharmacy, “Carol Davila” University of
Medicine and Pharmacy, 6 Traian Vuia Street, 020945 Bucharest, Romania
2 Department of Pharmaceutical Technology and Biopharmacy, Faculty of Pharmacy, “Carol Davila” University
of Medicine and Pharmacy, 6 Traian Vuia Street, 020945 Bucharest, Romania
3 “Ilie Murgulescu” Institute of Physical Chemistry, 202 Spl. Independentei, 060021 Bucharest, Romania
4 Faculty of Pharmacy, “Vasile Goldis, ” Western University of Arad, 86 Liviu Rebreanu Street,
310045 Arad, Romania
* Correspondence: [email protected] (A.M.M.); [email protected] (M.M.);
[email protected] (E.A.O.)

Abstract: Cereals whole grains contain vitamins, phytochemicals, antioxidants, resistant starch, and
minerals with potential benefits to human health. The consumption of whole grains is correlated
with a lowered risk of the most important chronic diseases, including type II diabetes, cardiovascular
Citation: Moros, an, E.; diseases, and some cancers. This study aimed to characterize and evaluate the content of five
Secareanu, A.A.; Musuc, A.M.; cultivars of wheat (Triticum aestivum L.) and five cultivars of barley (Hordeum vulgare L.) obtained
Mititelu, M.; Ionit, ă, A.C.; Ozon, E.A.;
by conventional plant breeding using crossing and selection methods. The novelty and the purpose
Raducan, I.D.; Rusu, A.I.;
of this research was to quantitatively and qualitatively analyze these ten cultivars from Romania
Dărăban, A.M.; Karampelas, O.
and to show the importance of, and the changes produced by, crossing and selection methods when
Comparative Quality Assessment of
these are aimed at the physiological or morphological development of the cultivars. Studies based
Five Bread Wheat and Five Barley
Cultivars Grown in Romania. Int. J.
on gluten dosing; spectrophotometry using Bradford, fructan and protein dosing; Kjeldahl protein
Environ. Res. Public Health 2022, 19, dosing; GC-MS/MS-protein and amino acid dosing; and identification of protein fractions using
11114. https://doi.org/10.3390/ polyacrylamide gel electrophoretic method were conducted. This study demonstrates the possibility
ijerph191711114 of developing future cultivars using conventional methods of improvement to modify the content
and composition of nutrients to increase their health benefits.
Academic Editors: Giuseppe
Valacchi, Antonella De Donno and
Keywords: Triticum aestivum L.; Hordeum vulgare L.; conventional plant breeding; nutritional knowledge;
Francesca Serio
human health
Received: 26 July 2022
Accepted: 1 September 2022
Published: 5 September 2022

Publisher’s Note: MDPI stays neutral

1. Introduction
with regard to jurisdictional claims in Consumption of wheat-based products is associated with a reduced risk of develop-
published maps and institutional affil- ing chronic diseases [1,2]. Recent studies regarding the primary potential health benefits
iations. of wheat-based products show that an increased consumption of wheat fiber and cereal
whole grains improves intestinal transit, reduces obesity risks and cardiovascular dis-
eases, prevents type-II diabetes and decreases the chances of developing some forms of
cancer [3–6]. Carbohydrate fermentation in the colon promotes the development of the
Copyright: © 2022 by the authors.
intestinal flora and improves the production of beneficial compounds such as short-chain
Licensee MDPI, Basel, Switzerland.
fatty acids [7]. Additionally, wheat seeds contain polyphenols, carotenoids, ascorbic acid,
This article is an open access article
vitamin E and phytosterols. These bioactive chemicals provide various biological activities,
distributed under the terms and
such as antioxidant, antidiabetic, antiinflammatory and antithrombotic properties [8,9].
conditions of the Creative Commons
Barley (Hordeum vulgare L.) is one of the oldest crops cultivated in the world with a natural
Attribution (CC BY) license (https://
tolerance and a highly adapted capacity [10]. Its use over time as a food source has also
creativecommons.org/licenses/by/
4.0/).
carried it through the malt, beer, and distillery manufacturing industries [11]. Barley seeds

Int. J. Environ. Res. Public Health 2022, 19, 11114. https://doi.org/10.3390/ijerph191711114 https://www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2022, 19, 11114 2 of 18

contain little fat, carbohydrates (mainly starch), and enough protein to provide the body
with the necessary amino acids, minerals, vitamin E, polyphenolic compounds and soluble
or insoluble fiber [12]. The endosperm is rich in B-glucans and plays an important role
in the regulation of serum cholesterol and blood sugar, supporting the proper function
of the cardiovascular system and controlling diabetes [13]. The high fiber content gives
the effect of satiety, having a positive impact on weight control, but also on the transit of
food in the colon [11]. In comparison with barley, wheat (Triticum aestivum L.) is one of the
most widespread and important cereal crops, being a major source of feed for livestock and
industry, and for nutrition [12].
In the last decade, great efforts have been made to find strategies that improve human
public health. These have focused mainly on nutritional knowledge and information
regarding dietary components [14–19]. In 2015, the most cultivated plants obtained by
conventional plant breeding were soybeans (83% of crops), cotton (75% of crops), corn (29%
of crops) and canola, a hybrid of rapeseed (24% of crops) [20].
In Romania, the modification of plant crops by crossing and selection methods is an
important process in which higher hybrid varieties can be obtained. For this study, five
cultivars of wheat (Triticum aestivum L.) and five cultivars of barley (Hordeum vulgare L.)
obtained by crossing and selection methods of improvement, were purchased from the
National Agricultural Research and Development Institute Fundulea. The selection of these
cultivars was preferred due to their high consumption and their large cultivated areas—
with Romania occupying sixth place at for these at the European level. Simultaneously,
quantitative changes in the content of some nutrients in cereals can be correlated with
certain diets in order to correct deficiencies or adjuvants for treating certain pathologies.
Improving the nutritional quality of wheat varieties plays an important role in the
bakery industry. The ability to make quality dough depends on both the gluten content
(the majority protein component of the wheat grain) and also on the content of fermentable
carbohydrates (small molecule carbohydrates). The nutritional value of wheat flour is also
provided by the content of essential amino acids, minerals, vitamins and fibers. Increasing
fiber content is important for the diets of diabetic, obese, or constipated people. Increasing
the gluten content improves the baking properties [21,22], but restricts the consumption by
people with gluten allergies or intolerance [23].
Enhancing the nutritional qualities of barley varieties plays an important role in the
beer industry because barley is the raw material for obtaining malt. In the technological
process of obtaining beer, the quality of the raw materials is essential for obtaining a
nutritious final product with special organoleptic characteristics [24,25].
Both types of cereal cultivars that are the subject of this study (wheat and barley culti-
vars) represent important raw materials for obtaining food products with a large market
worldwide. Considering the current interest in finding ways to increase the nutritional
value of food products, this work characterizes, from a qualitative viewpoint, some im-
proved cereal cultivars that are provided from raw materials that are themselves rich in
nutrients and that have special technological qualities for the food and beer industries.
The novelty of the study consists in the characterization of new, improved cereal culti-
vars, especially in terms of the important nutrients designed for the quality of the final
products derived from them—the bakery products obtained from the processing of wheat
flour and the beer varieties obtained from malt. Both types of cereal cultivars represent
important raw materials for food products with increased consumption among the pop-
ulation: bakery products and alcoholic beverages such as beer. These findings will help
in filling the knowledge gap for the wheat and barley cultivars produced by crossing and
selection methods.

2. Materials and Methods

2.1. Materials
Five wheat cultivars (Triticum aestivum L.) and five barley cultivars (Hordeum vulgare L.)
obtained by crossing and selection methods were used in this study. The cereal cultivars
Int. J. Environ. Res. Public Health 2022, 19, 11114 3 of 18

were purchased from the National Agricultural Research and Development Institute Fun-
dulea (Calarasi County, Romania). The Lugol’s reagent and bovine serum albumin (BSA
lyophilized powder fraction V ≥ 98%) were purchased from Sigma Aldrich (Steinheim,
Germany). The chemicals were used as received.
The characteristics of the analyzed cereals obtained from the National Agricultural
Research and Development Institute [26] are represented in Table 1.

Table 1. The morphological and physiological characteristics of the wheat cultivars (Triticum aestivum L.)
and barley cultivars (Hordeum vulgare L.) [26].

Cereal Cultivars Morphological Characteristics Physiological Characteristics Cereal Quality

The wheat cultivars
It has a semi-erect plant twig, in the twinning
phase. The leaf has a semi-bent bearing after It is an early cultivar type
the flowering phase. The average height of with a good resistance to fall,
the plant is about 85–95 cm. The to winter, the drought and the
It is characterized by hard
grain-bearing tip part of the stem is white, heat and has also a good
gluten. It has average
Glosa aristate, cylindrical in shape and it has a resistance to sprouting. It has
protein content and bread
medium density. The grains are of medium a medium resistance to brown
volume.
size, elongated in shape. The color is red, rust, and it is resistant to
and, under normal cultivation conditions, powdery mildew and current
they have a mass of 42–43 g/1000 grains and yellow rust strains;
a mass of 76–82 kg/hL;
It has a semi-erect plant bush in the twinning
phase. The leaf has a semi-erect bearing after
It is an early cultivar type
the flowering phase. The average height of It proved to have good
with a medium resistance to
the plant is 85–95 cm. The grain-bearing tip milling and baking indices.
falling. It is resistant to winter,
part of the stem is white, fusiform, large and In general, this cultivar is
Dropia drought and heat. This
of medium density. The grains are of characterized by a good
cultivar is medium sensitive
medium size, elongated in shape, have a red consistency of bakery
to brown rust, yellow rust and
color and, under normal growing conditions, quality.
powdery mildew;
a mass of 41–46 g/1000 grains and a mass of
76–80 kg/hL;
It has a semi-erect plant bush, in the twinning
phase; the leaf has a semi-erect bearing after
flowering. The leaves are medium in length It has good resistance to
and width, and they are covered with a light falling, wintering, drought
It is distinguished by very
waxy layer. The average height of the plant is and heat. It is resistant to
good bakery quality, being
80–95 cm. The spike is white, of medium brown rust and powdery
Pitar from this point of view
density, aristate, pyramidal in shape, of mildew and medium resistant
superior to the Glosa
medium length and with a semi-nuanced to Septoria and yellow rust. It
cultivars.
position at maturity. The grains are has a medium level of
medium-sized, well-filled, elongated, and resistance to ear Fusariosis.
red in color. Mass of 1000 grains: 42–44 g.
Hectoliter mass: 79–82 kg/hL;
The cultivar is early type
It has a semi-erect plant bush in the twinning (with a vegetation period It has a good baking quality,
phase. The leaves are medium in length and similar to the control cultivars being from this viewpoint
width, and they are covered with an intense Dropia and Glosa), with very similar to the Glosa cultivar.
waxy layer and the average height of the good resistance to falling, It achieves on average
plant is between 75 and 92 cm, being shorter good resistance to wintering, production an increase of
Pajura than the control cultivars Dropia and Glosa drought and heat. Pajura 5–8% compared to the Glosa
by 5–10 cm. The grains are of medium size, wheat cultivar is resistant to cultivars, in the same
elongated shape, red in color and have, brown rust and powdery technological conditions, the
under normal cultivation conditions, a mass mildew and medium resistant increases being higher in
of 40–44 g/1000 grains and a hectoliter mass to Septoria and yellow rust, favorable conditions of
of 77–80 kg/hL; and it has a medium level of attack of foliar diseases.
resistance to fusariosis;
Int. J. Environ. Res. Public Health 2022, 19, 11114 4 of 18

Table 1. Cont.

Cereal Cultivars Morphological Characteristics Physiological Characteristics Cereal Quality

It has a semi-lying to lying plant bush, in the
twinning phase. The leaves have a semi-bent
position after the flowering phase. The leaves
Early cultivars, with a
are medium in length and width, and they
vegetation period similar to
are covered with a not too intense waxy layer Tested both on laboratory
the control cultivars Dropia,
in the second part of the grain filling period. microplates and in the pilot
with good resistance to fall,
The average height of the plant is between 95 phase, the Litera cultivars
winter, drought and heat. It is
and 105 cm, being the same height as the proved to have good baking
Litera resistant to brown rust and
control cultivars Dropia. The spike is white, characteristics. The quality
medium resistant to current
semi-dense, aristate, pyramidal in shape and indices of these cultivars are
breeds of yellow rust and
with a semi-nuanced position at maturity. quite close to those of the
powdery mildew. It has a
The grains are of medium size, elongated in Dropia cultivars.
medium level of resistance to
shape, have a red color and have, under
fusariosis.
normal cultivation conditions, a mass of 1000
grains of 42–45 g and a hectoliter mass of
77–81 kg/hL;
The barley cultivars
Dana The reference cultivars
Typical semi-early autumn cultivars, with a
good twinning capacity, medium to high
waist, with medium length spike, yellow It has superior resistance to
It achieved a 17% increase in
color and long edges; the mass of 1000 grains winter, fall and foliar
production compared with
varied between 40 and 45 g, the average (environment resistant to
Simbol the control cultivars Dana
protein content was 10.5–12.5%, the average brown reticular staining of
under the same
starch content was 62.0% (qualitative barley leaves
technological conditions
parameters depend on the technology (Pyrenophora teres f. teres);
applied but also on the conditions climate
change).
Typical semi-early autumn cultivars, with
It has superior resistance to The assortment level (I + II)
good twinning capacity, medium size, with
winter, fall and leaf, can reach a maximum of
the spike of medium length, yellow color and
environmental resistant to the 94.5% (qualitative
Ametist long edges; The mass of 1000 grains varied
brown reticular staining of parameters depend on the
between 44 and 52 g, the average protein
barley leaves applied technology but also
content is 9.9–12.3%, the average starch
(Pyrenophora teres f. teres); on the climatic conditions)
content is 57.9–64.4%
It is a genotype of autumn barley, with a
The cultivars are semi-early,
growing period equal to that of the control
uniform, with superior
cultivars Dana. It has a good twinning
resistance to wintering, fall, Qualitative parameters
capacity, medium to high waist, with
foliar and ear diseases depend on the applied
Onix medium length spike, and long
(environment resistant to technology, but also on the
anthocyanin-colored edges. The mass of 1000
brown reticular staining of climatic conditions.
grains had values between 46.0–50.0 g, the
barley leaves-
protein content was 10.4–11.9%, and the
Pyrenophora teres f. teres)
starch content was 62.3%.
The shape is intermediate, with a medium to Semi-early cultivars, with Its basic characteristic is the
Smarald high frequency of plants, with a curved flag good resistance to wintering, high productivity and the
leaf. Plant height is medium. falling and foliar diseases. quality of the grains.

2.2. The Milling Process

The cereal seeds were ground using FOSS Cyclotec 1093 Mill laboratory equipment to
achieve a fineness and uniform size of the particles. A Mettler Toledo XS205 DualRange
analytical balance was used for the weighing of the sample.
Int. J. Environ. Res. Public Health 2022, 19, 11114 5 of 18

2.3. Gluten Detection Method from Wheat

We moistened 10 g flour with 5 mL of purified water, it was kneaded to a non-sticky
consistency and was then left to stand for 20 min. Then, it was washed for 30 min with
purified water to remove the starch. The elimination of starch was complete when the
residual water did not change the color of a drop of Lugol’s reagent. The gluten wet mass
was weighed. The sample was then crushed and placed in a heat-resistant vessel in the oven
at 105 ◦ C and 760 mmHg, for two hours. After that, the dry mass of gluten was weighed.

2.4. Wheat Protein Content Using the Bradford Assay

The Bradford assay [27] is based on an absorbance shift of the dye Coomassie Brilliant
Blue (CBB) G-250, by the binding of protein molecules. The absorbance of the dye–protein
complex and the efficiency depend on the amino acid composition of the protein, and it
has a maximum absorption at a wavelength of 595 nm.
An amount of 0.010 g Coomassie Brilliant Blue G was dissolved in 5 mL ethanol and
10 mL phosphoric acid. Then, it was brought to a final volume of 100 mL with distilled
water. Before use, it was refrigerated and filtered. Tris-glycine buffer at pH = 8.3 was
obtained as follows: 0.600 g tris (hydroxymethyl) aminomethane and 2.88 g glycine is
dissolved in purified water and then diluted to 100 mL with the same solvent. Before use, it
was made a 1:10 dilution with distilled water as follows: 20 mL tris-glycine buffer prepared
above is brought to a final volume of 200 mL with distilled water. Bovine serum albumin
(BSA) stock solution (1 mg/mL) was prepared as follows: 0.025 g BSA was put into a 25 mL
volumetric flask. An amount of 10 mL tris-glycine buffer diluted at pH = 8.3 (or any other
solvent used to dissolve the unknown sample) was added. The solution was stirred until
the substance was solubilized and it was brought to a level of 25 mL with Tris-glycine at
pH = 8.3 buffer. The test solution was prepared by weighing 50 mg wheat and 50 g barley.
The samples were prepared in duplicate. An amount of 3 mL tris-glycine buffer was added
over the first row of samples, and 3 mL distilled water over the second row. For efficient
dispersion, the samples are placed on the sonicate bath for 5 min at 4 ◦ C. Then, the samples
were centrifuged for 30 min on ice, at 4000 rpm. The samples were kept in a refrigerator.
The calibration curves were obtained using the dilutions shown in Table 2, which are made
in ten Eppendorf vials of 1 mL volume.

Table 2. The used concentrations for the calibration curves.

BSA Concentration BSA Volume (1 mg/mL) Tris-Glycine/Distilled Water

0.05 5 µL 95 µL
0.1 10 µL 90 µL
0.2 20 µL 80 µL
0.4 40 µL 60 µL
0.6 60 µL 40 µL
Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 6 of 19
The obtained calibration curves in tris-glycine and distilled water are given in Figure 1a,b.

Figure 1. The calibration curves in (a) tris-glycine and (b) distilled water.
Figure 1. The calibration curves in (a) tris-glycine and (b) distilled water.

2.5. Total Nitrogen by the Kjeldahl Method [28]

The determination of total nitrogen (Nt) in a product of vegetable or animal origin
can be done only after its mineralization by wetting. Wet mineralization, by boiling the
Int. J. Environ. Res. Public Health 2022, 19, 11114 6 of 18

2.5. Total Nitrogen by the Kjeldahl Method [28]

The determination of total nitrogen (Nt ) in a product of vegetable or animal origin
can be done only after its mineralization by wetting. Wet mineralization, by boiling the
product in concentrated sulfuric acid and in the presence of an oxidizing agent (perchloric
acid, hydrogen peroxide or solid catalyst) releases nitrogen from the organic compounds
present in the material under analysis and its fixation in the form of ammonium borate.
Thus, transformed nitrogen can be determined either by a volumetric method (Kjeldahl
method) or using a spectrophotometric method (the Nessler reaction). To determine Nt
from ammonium borate (mineralization product), the ammonia presented in the solution is
released by using sodium hydroxide and then it was distilled. In the obtained distillate,
nitrogen was determined volumetrically using the titration method with a solution of
hydrochloric acid of known concentration and a known factor in the presence of the Groack
indicator (mixture of methyl red and methylene blue). The Nt from the sample is calculated
from the amount of hydrochloric acid used in the titration.
0.5 g Glosa wheat and 0.5 g Amethyst barley cultivars were weighted. Also, 4 g
disintegrant mixture consisting of 5 g of copper sulfate, 100 g potassium sulfate and 2.5 g
selenium was weighted. The samples were placed in a Kjeldahl balloon. An amount of
10 mL concentrated sulfuric acid was added to the above mixture, and the beaker was
connected to a digestion apparatus. The temperature was settled at 440 ◦ C and it was kept
in the flask for 30 min. At the end of the digestive stage, the contents of the Kjeldahl flask
were quantitatively brought into the distillation flask by multiple washes with distilled
water. By connecting the flask to the distillation plant, a 50 mL sodium hydroxide (30% wt.)
was introduced into the solution. It was brought to half with distilled water. An amount
of 50 mL 4% boric acid and 2–3 drops of Groack indicator solution (a mixture of methyl
red and methylene blue indicator) were brought into the collection flask. The mixture was
refluxed for 20–30 min or until the volume in the distillation flask reached 100 mL. Nitrogen
was massively distilled in the first 5–6 min from the beginning of reflux. The contents of
the distillation flask were titrated with a 0.1 N hydrochloric acid solution of known factor
(0.9740), the volume of used acid was noted, and the Nt content of the sample and the total
proteins using the conversion factor of 6.25 were calculated.

2.6. GC-MS/MS Analysis of Amino Acids Derived from Proteins

An amount of 100 mg wheat and 100 mg barley cultivars were weighed using the
analytical balance into centrifuge tubes. The samples were made in duplicate. Half of the
samples were brought to 5 mL with tris-glycine buffer and half with distilled water. The
samples were centrifugated for 30 min, on ice, at 4000 rpm.
A 600 µL sample of 20 mg/mL was taken from each test tube and was then brought
into derivatization vials and supplemented with 720 µL concentrated hydrochloric acid.
The vials were stapled and were left in the oven for 8 h at 110 ◦ C for protein hydrolysis
process. Then, 250 µL was transferred to other vials and dried at 40–50 ◦ C. After drying, the
samples were brought with 250 µL acetonitrile, and were derivatized with 250 µL BSTFA
and they were sealed.
The vials were introduced in the stirring module of the Gas Chromatograph coupled
with the Quadrupol Triple Mass Spectrometer for one hour at a maximum temperature
of 200 ◦ C and a stationary one of 105 ◦ C; afterward, the contents were transferred into
injection vials and left for two hours. A standard solution of marine fish extract (Biotehnos
S.A., Otopeni, Romania) in concentrations of 25 µL, 50 µL, 100 µL, 200 µL, and 400 µL was
used to draw the calibration curve.

2.7. Protein Analysis by Polyacrylamide Gel Electrophoresis

The protein fingerprint of the samples is based on the separation of the polypeptide
bands according to the molecular weight and after the migration into the polyacrylamide
gel. The highlighting of the bands occurs by staining with reagents, which bind stoichio-
metrically, non-specifically, to the protein. The evaluation of their molecular weight was
Int. J. Environ. Res. Public Health 2022, 19, 11114 7 of 18

performed by comparison with a protein marker (20–220 kDa). The general principle of the
method is described in the European Pharmacopoeia [29].
Reagents used: (A)—Samples from Tris-glycine buffer and distilled water previously
obtained; (B)—Sample solubilization solution: 2.5 mL concentration gel buffer, 2.0 mL
glycerol, 4 mL 10% SDS, 0.5 mL Coomassie Brilliant Blue G-250 0.1%, 1 mL purified
water. To 950 µL solution for solubilization was added 50 mL samples; (C)—Acrylamide-
bisacrylamide solution: 29.2 g acrylamide and 0.8 g N0 N0 —bis-methylene—acrylamide
dissolved in 100 mL purified water; (D)—10% SDS solution: 10 g SDS is dissolved in 90 mL
purified water with stirring and brought to a final volume of 100 mL with the same solvent;
(E)—Gel buffer for separation: 18.15 g 1.5 M Tris-Hcl, pH = 8.8 Trizma base® dissolved
in 80 mL purified water. The pH was adjusted to 8.8 with 6 N HCl and it was brought to
100 mL with the same solvent; (F)—Concentration gel buffer: 6 g Tris-HCl (0.5 M), pH = 6.8
Trizma base® was dissolved in 60 mL purified water. The pH to 6.8 was adjusted with 6 N
HCl and brought to 100 mL with the same solvent; (G)—10% ammonium persulfate (APS):
0.1 g APS was dissolved in 1 mL purified water; (H)—TEMED: Tetramethylenediamine;
the migration buffer pH = 8.3: it was weighed exactly 3.03 g Trizma base® , 14.400 g Glycine,
10 mL 10% SDS and it was brought to a final volume of 1000 mL. The pH was not adjusted.
(I)—Fixation solution: Trichloroacetic acid (TCA) 12%; (J)—Staining solution: This was
dissolved 0.18 g Coomassie Brilliant Blue G-250® in 45 mL methanol, 45 mL purified water
and 10 mL acetic acid; (K)—Discoloration solution: methanol: acetic acid: purified water
in a ratio of 40:10:50 (v/v); (L)—Discoloration stop solution: 2% acetic acid in purified
water; (M)—Tris-glycine buffer pH 8.3: This was a precisely weighed 0.6 g Trizma base®
and 2.88 g Glycine which were dissolved in purified water. It was brought to 100 mL with
the same solvent. Procedure: The test solution is prepared from 60 µL sample solution (A)
and 60 µL sample solubilization solution (B). It was stirred and kept in the water bath at
95 ◦ C for 5 min.
The presence of proteins was highlighted by the appearance of distinct blue horizontal
bands, the size of the molecular masses was assessed by comparison with the marker bands.
The migration distances for the constituent polypeptides from the marker (from the starting
line of the separation gel to the center of the band) and the total migration distance (from
the starting line of the separation gel to the marked level of the dye front) were measured
using the software Chemidoc. The graph was obtained by plotting log (Mw ) as a function
of Rf , where: Mw is the molecular mass of each protein in the marker mixture and Rf is the
individual protein band migration distance/total migration front distance. Due to the low
intensity of the bands obtained by staining with Coomasie Brilliant Blue, it was opted for
a double marking with Thermo Scientific Pierce Silver Stain® according to the following
steps: The polyacrylamide gel was washed with water twice for 5 min and then the water
was removed; A EtOH:Acetic Acid:Water = 3:1:6 was used twice for 15 min for the solution
used for fixation; this was washed with 10% ethanol solution twice for 5 min, with water
twice for 5 min and then the water was removed; the gel was immersed in a mixture of
25 mL water and 50 µL Silver Stain Sensitizer® for 1 min; it was then washed with water
twice for 1 min and the water was then removed; 25 mL water and 250 µL Silver Stain
Enhancer® were immersed in the mixture for 5 min; it was washed with water twice for
20 s and the water was removed; 25 mL Silver Stain Developer® and 250 µL Silver Stain
Enhancer® , were mixed for 30 s until the strips became visible; the reaction was stopped
with a solution of 5% acetic acid.

2.8. Dosing of Fructan Fibers Using the Seliwanoff Method

The Seliwanoff method is based on the differentiation between ketosis and aldoses. At
high temperatures, ketoses are hydrolyzed faster than aldoses, with the breakdown of the
carbohydrate cycle and with the formation of furfural. This reacts in an acidic medium with
the resorcinol solution resulting in a red color that can be measured spectrophotometrically
at a wavelength of 520 nm.
 until the strips became visible; the reaction was stopped with a solution of 5% acetic acid.

2.8. Dosing of Fructan Fibers Using the Seliwanoff Method

The Seliwanoff method is based on the differentiation between ketosis and aldoses.
At high temperatures, ketoses are hydrolyzed faster than aldoses, with the breakdown of
Int. J. Environ. Res. Public Health 2022, 19, 11114 8 of 18
the carbohydrate cycle and with the formation of furfural. This reacts in an acidic medium
with the resorcinol solution resulting in a red color that can be measured
spectrophotometrically at a wavelength of 520 nm.
Fructose
Fructose stock
stock solution
solution ofof 11mg/mL,
mg/mL, 1% 1% resorcinol
resorcinol solution
solution (1(1 gg resorcinol,
resorcinol, 0.25
0.25 gg
thiourea,
thiourea, 100 mL glacial acetic acid) and 5:1 hydrochloric acid solution was prepared.Nine
100 mL glacial acetic acid) and 5:1 hydrochloric acid solution was prepared. Nine
successive
successivedilutions
dilutionswere
wereprepared
prepared from thethe
from stock solution
stock for drawing
solution for drawing the calibration line
the calibration
by taking 25 µL, 50 µL, 75 µL, 100 µL, 125 µL, 200 µL, 300 µL, 400 µL, 500
line by taking 25 µL, 50 µL, 75 µL, 100 µL, 125 µL, 200 µL, 300 µL, 400 µL, 500 µL. The µL. The standard
solutions
standardand 500 µLand
solutions sample
500 were treatedwere
µL sample with treated
0.5 mL distilled
with 0.5 water, 0.5 mL water,
mL distilled 1% resorcinol
0.5 mL
solution and 3.5 mL hydrochloric acid solution. The tubes were heated
1% resorcinol solution and 3.5 mL hydrochloric acid solution. The tubes were heated in a water bathinata
80 ◦ C for 10 min, then they were cooled. They were then read on the Perkin Elmer Life and
water bath at 80 °C for 10 min, then they were cooled. They were then read on the Perkin
Analytical
Elmer LifeScience LAMBDA
and Analytical 25 molecular
Science LAMBDA absorption spectrophotometer
25 molecular at 520 nm in a
absorption spectrophotometer
maximum of 30 min. The calibration curve is presented in Figure 2.
at 520 nm in a maximum of 30 min. The calibration curve is presented in Figure 2.

Figure2.2.The
Figure Thecalibration
calibrationcurve
curvefor
fordosing
dosingof
offructans.
fructans.

All analyses
All analyses were
were effectuated
effectuated in
in triplicate,
triplicate, and
and the
the data
data were
were registered
registered as
as means
means
values±
values ± standard
standard deviation
deviation (SD).
(SD).

2.9.
2.9. Measurements
Measurements
The
Theabsorbances
absorbancesof of
thethe
samples werewere
samples obtained usingusing
obtained a Perkin Elmer Life
a Perkin andLife
Elmer Analyti-
and
cal Science Molecular
Analytical Absorption
Science Molecular Spectrophotometer
Absorption LAMBDA 25,
Spectrophotometer at a λ = 595
LAMBDA nm.
25, at a For
λ = the
595
total
nm. nitrogen determination
For the total by the Kjeldahlby
nitrogen determination method, a Digesdahl
the Kjeldahl 23130–21
method, digestion
a Digesdahl appa-
23130–21
ratus was used.
digestion A Thermo
apparatus Scientific
was used. TSQ 8000
A Thermo EVO gas
Scientific TSQchromatograph
8000 EVO gaswas used under
chromatograph
the following conditions: Column, Thermo Scientific TraceGOLD TG-5SilMS,
was used under the following conditions: Column, Thermo Scientific TraceGOLD L = 30.00TG-
m,
D = 0.25 mm;
5SilMS, The used
L = 30.00 m, D carrier gas, He;
= 0.25 mm; The Flow
used rate, 1 mL/min;
carrier Analysis
gas, He; Flow rate,time, 46.667 Analysis
1 mL/min; min; De-
tection with Total
time, 46.667 min;Ion Chromatogram
Detection with Total(ICT),
Ion 40–600 M/z; Software,
Chromatogram (ICT),Chromeleon
40–600 M/z; 7.Software,
Protein
analysis by polyacrylamide gel electrophoresis was performed using the Bio-Rad PowerPac
Basic Mini electrophoresis system. A Perkin Elmer Life and Analytical Science LAMBDA
25 molecular absorption spectrophotometer was used for dosing of fructan fibers at 520 nm.

3. Results
Qualitative Assessment of Wheat Cultivars and Barley Cultivars
Due to the increasing use of gluten-free products for different food applications [30],
it is necessary to achieve deep characterization of the content of gluten in the studied
cultivars and to determine whether differences in composition are dependent from the
cereal cultivars type. The results of gluten detection from wheat are presented in Table 3.
Int. J. Environ. Res. Public Health 2022, 19, 11114 9 of 18

Table 3. Measured contents of gluten in wheat cultivars (means values ± standard deviation).

The Wheat Cultivars Initial Mass (g) Wet Mass (g) Dry Mass (g) Gluten Concentration (%)
Glosa 10.01 ± 0.02 3.67 ± 0.03 1.32 ± 0.02 13.22 ± 0.12
Dropia 10.00 ± 0.12 4.52 ± 0.05 1.65 ± 0.06 16.46 ± 0.16
Pitar 10.07 ± 0.06 5.31 ± 0.02 1.96 ± 0.11 19.60 ± 0.20
Pajura 10.01 ± 0.04 4.74 ± 0.04 1.63 ± 0.09 16.32 ± 0.13
Litera 10.01 ± 0.08 4.18 ± 0.06 1.56 ± 0.08 15.60 ± 0.11

According to the gluten concentrations in the flour, they can be divided into: (i) low
gluten flour (7–8%); (ii) usual flour (8–12%); (iii) flour with high gluten content (12–13%);
(iv) gluten-free flour [31]. The dosage of gluten from the five wheat samples, as can be seen
in Table 3, highlighted the presence of gluten quantities greater than 13%.
The protein concentrations of the five selected wheat cultivars and five barley cultivars
were determined using the extraction from tris-glycine buffer and distilled water. The
results are presented in Tables 4 and 5.

Table 4. Measured contents of the extracted proteins concentration in tris-glycine buffer (means
values ± standard deviation).

Sample Concentration Sample Mass Protein Concentration

Sample Used Volume (µL) Absorbance
(µg/mL) (mg) (%)
Wheat cultivars
Pajura 50 0.275 185.9 50.0 3.72 ± 0.14
Litera 50 0.285 192.8 53.2 3.62 ± 0.03
Pitar 50 0.305 206.1 50.5 4.08 ± 0.06
Dropia 50 0.313 211.7 51.4 4.12 ± 0.07
Glosa 50 0.293 198.3 52.6 3.77 ± 0.05
Barley cultivars
Dana 50 0.237 159.5 50.5 3.16 ± 0.03
Simbol 50 0.208 139.7 53.6 2.61 ± 0.02
Ametist 50 0.251 169.2 54.0 3.13 ± 0.06
Onix 50 0.243 163.7 52.6 3.11 ± 0.14
Smarald 50 0.189 126.6 54.4 2.33 ± 0.11

Table 5. Measured contents of the extracted proteins concentration in distilled water (means values
± standard deviation).

Sample Concentration Sample Mass Protein Concentration

Sample Used Volume (µL) Absorbance
(µg/mL) (mg) (%)
Wheat cultivars
Pajura 50 0.114 82.19 ± 0.22 53.2 1.55 ± 0.14
Litera 50 0.117 84.29 ± 0.16 56.4 1.49 ± 0.06
Pitar 50 0.126 90.44 ± 0.23 54.6 1.66 ± 0.14
Dropia 50 0.194 137.90 ± 0.32 57.9 2.38 ± 0.08
Glosa 50 0.107 76.95 ± 0.12 56.7 1.36 ± 0.12
Barley cultivars
Dana 50 0.149 106.90 ± 0.18 55.6 0.96 ± 0.11
Simbol 50 0.134 96.16 ± 0.12 55.8 0.86 ± 0.12
Ametist 50 0.145 103.60 ± 0.22 54.2 0.96 ± 0.12
Onix 50 0.158 112.90 ± 0.24 56.4 1.00 ± 0.08
Smarald 50 0.159 113.70 ± 0.20 52.6 1.08 ± 0.13
Int. J. Environ. Res. Public Health 2022, 19, 11114 10 of 18

The experimental results regarding the concentration of proteins for Glosa wheat and
Ametist barley cultivars obtained using the Kjeldahl method are shown in Table 6.

Table 6. Measured contents of the protein concentrations obtained using the Kjeldahl method (means
values ± standard deviation).

Cereal Cultivars Mass (g) HCl (mL) Nt (%) Proteins (%)

Glosa wheat 0.52 ± 0.02 7.8 2.04 ± 0.04 12.80 ± 0.06
Ametist barley 0.50 ± 0.02 5.9 1.59 ± 0.02 9.99 ± 0.04

Considered as a reference method for the dosing of proteins from plant and ani-
mal products, the Kjeldahl method was further used to extrapolate the results obtained
previously using the Bradford method. The results are presented in Table 7.

Table 7. The results obtained using the extrapolation method (means values ± standard deviation).

Proteins by Bradford Calculated

Cereal Cultivars Total Proteins (%)
(%) Conversion Factor
Wheat cultivars
Pajura 1.54 ± 0.04 9.48 14.59 ± 0.11
Litera 1.49 ± 0.13 9.48 14.12 ± 0.22
Pitar 1.65 ± 0.05 9.48 15.64 ± 0.06
Dropia 2.38 ± 0.08 9.48 22.56 ± 0.08
Glosa 1.35 ± 0.12 9.48 12.79 ± 0.02
Barley cultivars
Dana 0.96 ± 0.04 10.51 10.08 ± 0.06
Simbol 0.86 ± 0.02 10.51 9.03 ± 0.04
Ametist 0.95 ± 0.11 10.51 9.98 ± 0.15
Onix 1.00 ± 0.14 10.51 10.51 ± 0.06
Smarald 1.08 ± 0.06 10.51 11.35 ± 0.08

The experimental results, obtained after extrapolation from Table 7, indicated an

increase or a very high content in the case of wheat samples of proteins. This increase in
protein concentration can be justified by the increase in gluten content.
The calculated amino acid concentrations from studied cultivars based on peak areas
obtained using Chromeleon 7 software from GS-MS/MS analysis are represented in Table 8.

Table 8. Measured contents of the amino acid concentrations (means values ± standard deviation).

Concentration of Compound (%)

Wheat Cultivars Barley Cultivars
Compound Pajura Litera Pitar Dropia Glosa Dana Simbol Ametist Onix Smarald
L-Valine 0.19 ± 0.08 0.15 ± 0.06 0.25 ± 0.04 0.14 ± 0.02 0.31 ± 0.03 0.07 ± 0.11 0.06 ± 0.31 0.09 ± 0.02 0.04 ± 0.14 0.06 ± 0.31
Glicine 17.54 ± 0.11 18.09 ± 0.32 21.42 ± 0.04 18.06 ± 0.02 25.81 ± 0.08 22.28 ± 0.04 24.99 ± 0.06 26.96 ± 0.16 13.93 ± 0.33 25.25 ± 0.26
L-Leucine 0.12 ± 0.07 0.09 ± 0.05 0.13 ± 0.02 0.08 ± 0.08 0.16 ± 0.13 0.08 ± 0.02 0.06 ± 0.01 0.10 ± 0.04 0.04 ± 0.03 0.07 ± 0.11
L-Isoleucine 0.13 ± 0.04 0.07 ± 0.01 0.12 ± 0.03 0.07 ± 0.01 0.17 ± 0.04 0.07 ± 0.03 0.06 ± 0.04 0.11± 0.08 - 0.06 ± 0.03
L-Serine 0.08 ± 0.01 0.08 ± 0.01 0.10 ± 0.02 0.07 ± 0.01 0.13 ± 0.02 0.06 ± 0.01 0.05 ± 0.01 0.07 ± 0.04 0.03 ± 0.01 0.05 ± 0.11
L-Threonine 0.06 ± 0.01 0.05 ± 0.01 0.07 ± 0.01 0.05 ± 0.01 0.18 ± 0.04 0.08 ± 0.01 0.05 ± 0.01 0.11 ± 0.02 0.03 ± 0.01 0.05 ± 0.01
L-Proline - 0.03 ± 0.01 0.19 ± 0.02 − 0.38 ± 0.06 0.27 ± 0.04 - 0.65 ± 0.06 - 0.17 ± 0.12
L-Aspartic
0.17 ± 0.12 0.18 ± 0.14 0.24 ± 0.13 0.12 ± 0.11 0.31 ± 0.24 0.16 ± 0.22 0.14 ± 0.12 0.23 ± 0.14 0.06 ± 0.01 0.17 ± 0.14
acid
L-Glutamic
1.24 ± 0.08 1.01 ± 0.12 1.45 ± 0.23 0.69 ± 0.03 1.95 ± 0.04 0.56 ± 0.07 0.43 ± 0.02 1.05 ± 0.06 0.15 ± 0.01 0.54 ± 0.14
acid
DL-
0.29 ± 0.17 0.22 ± 0.32 0.29 ± 0.15 0.11± 0.11 0.53 ± 0.03 0.11 ± 0.08 0.25 ± 0.02 0.23 ± 0.12 0.02 ± 0.21 0.14 ± 0.26
Phenylalanine
Tyrosine 0.04 ± 0.04 0.02 ± 0.08 0.04 ± 0.06 - 0.07 ± 0.01 - - 0.04 ± 0.11 - 0.03 ± 0.21

The results of protein analysis by polyacrylamide gel electrophoresis are shown in

photographs from Figures 3 and 4.
 L-Proline - 0.03 ± 0.01 0.19 ± 0.02 − 0.38 ± 0.06 0.27 ± 0.04 - 0.65 ± 0.06 - 0.17 ± 0.12
L-Aspartic acid 0.17 ± 0.12 0.18 ± 0.14 0.24 ± 0.13 0.12 ± 0.11 0.31 ± 0.24 0.16 ± 0.22 0.14 ± 0.12 0.23 ± 0.14 0.06 ± 0.01 0.17 ± 0.14
L-Glutamic acid 1.24 ± 0.08 1.01 ± 0.12 1.45 ± 0.23 0.69 ± 0.03 1.95 ± 0.04 0.56 ± 0.07 0.43 ± 0.02 1.05 ± 0.06 0.15 ± 0.01 0.54 ± 0.14
DL-Phenylalanine 0.29 ± 0.17 0.22 ± 0.32 0.29 ± 0.15 0.11± 0.11 0.53 ± 0.03 0.11 ± 0.08 0.25 ± 0.02 0.23 ± 0.12 0.02 ± 0.21 0.14 ± 0.26
Tyrosine 0.04 ± 0.04 0.02 ± 0.08 0.04 ± 0.06 - 0.07 ± 0.01 - - 0.04 ± 0.11 - 0.03 ± 0.21

Int. J. Environ. Res. Public Health 2022, 19, 11114 11 of 18

The results of protein analysis by polyacrylamide gel electrophoresis are shown in
photographs from Figures 3 and 4.

Int. J. Environ. Res. Public Health 2022,Figure 3. Polyacrylamide Gel

Gel Staining
Staining Steps Using Coomassie
Coomassie Brilliant
Brilliant Blue
Blue G-250
G-250®® and Thermo
19, x FOR
Figure 3. PEER REVIEW
Polyacrylamide Steps Using 12 ofThermo
and 19
Scientific Pierce Silver Stain®® Dye.
Scientific Pierce Silver Stain Dye.

Figure 4. Polyacrylamide gels after migration of colored samples with Coomassie Brilliant Blue G-
Figure 4. Polyacrylamide gels after migration of colored samples with Coomassie Brilliant Blue
250® and Thermo Scientific Pierce Silver Stain® (M-Thermo molecular marker (kDa); 1—Simbol in
G-250® and Thermo Scientific Pierce Silver Stain® (M-Thermo molecular marker (kDa); 1—Simbol in
tris-glycine; 2—Simbol in distilled water; 3—Pitar in tris-glycine; 4—Pitar in distilled water; 5—
tris-glycine; 2—Simbol 6—Pajura
Pajura in tris-glycine; in distilledinwater; 3—Pitar
distilled in 7—Onix
water; tris-glycine; 4—Pitar in distilled
in tris-glycine; 8—Onixwater; 5—Pajura
in distilled
in tris-glycine; 6—Pajura in distilled water; 7—Onix in tris-glycine; 8—Onix
water; 9—Ametist in tris-glycine; 10—Ametist in distilled water; 11—Litera in tris-glycine; 12—in distilled water;
Litera in distilled water; 13—Smarald in tris-glycine; 14—Smarald in distilled water; 15—Dropia
9—Ametist in tris-glycine; 10—Ametist in distilled water; 11—Litera in tris-glycine; 12—Litera in in
tris-glycine;
distilled 16—Dropia
water; in distilled
13—Smarald water; 17—Glosa
in tris-glycine; 14—Smaraldin tris-glycine;
in distilled 18—Glosa in distilled
water; 15—Dropia water;
in tris-glycine;
19—Dana in tris-glycine; 20—Dana in distilled water.).
16—Dropia in distilled water; 17—Glosa in tris-glycine; 18—Glosa in distilled water; 19—Dana in
tris-glycine; 20—Dana in distilled water.).
The strips from the two sample gels were scanned and were identified using the
Chemidoc program. Subsequently, it was decided to process the data corresponding to
the double-labeled gels, stained with Thermo Scientific Pierce Silver Stain reagent.
Comparing the samples extracted in tris-glycine buffer with those extracted in water, a
high extraction yield can be observed in the case of the buffer solution. The bands have
Int. J. Environ. Res. Public Health 2022, 19, 11114 12 of 18

The strips from the two sample gels were scanned and were identified using the
Chemidoc program. Subsequently, it was decided to process the data corresponding to the
double-labeled gels, stained with Thermo Scientific Pierce Silver Stain reagent. Comparing
the samples extracted in tris-glycine buffer with those extracted in water, a high extraction
yield can be observed in the case of the buffer solution. The bands have been associated
with proteins type based on literature data. In the case of both wheat and barley cultivars,
important proteins have been successfully identified and included in their mass domains.
The results are represented in Tables 9 and 10.

Table 9. Identification of proteins from the colored sample strips using Thermo Scientific Pierce Silver
Stain® (Wheat cultivars).

Mass Types of the Wheat Cultivars in Tris-glycine Wheat Cultivars in Distilled Water
(kDa) Proteins Pajura Litera Pitar Dropia Glosa Pajura Litera Pitar Dropia Glosa
112.8 X X X X X
108.1
101.4 X X X X X X
HMW
96.2 glutenin X X
87.0 X X X X X X X X
79.9
77.0 X X X X X
72.6 X X X X X X X X X X
Ω-gliadin,
63.9 LMW X X X X X X X X
glutenin
60.0 X X X X X X X
51.7 X X X X X
γ-gliadin,
47.0 LMW X
glutenin
45.0 X X X X X X X X
42.2 X
40.5 α-gliadin, X X X X X X
34.5 LMW X X X X X X X X X X
glutenin
31.5 X X X X X X X
28.5
25.8 X X X X X
22.0 X X X X X X X X
19.0 X
14.9 X X X X X X X X X

Table 10. Identification of proteins from the colored sample strips using Thermo Scientific Pierce
Silver Stain® (Barley cultivars).

Mass Types of the Barley Cultivars in Tris-glycine Barley Cultivars in Distilled Water
(kDa) Proteins Dana Simbol Ametist Onix Smarald Dana Simbol Ametist Onix Smarald
112.8 X X
108.1 X
101.4 X X
96.2 D-hordein X X
Int. J. Environ. Res. Public Health 2022, 19, 11114 13 of 18

Table 10. Cont.

Mass Types of the Barley Cultivars in Tris-glycine Barley Cultivars in Distilled Water
(kDa) Proteins Dana Simbol Ametist Onix Smarald Dana Simbol Ametist Onix Smarald
87.0 X X X X X X X
79.9 X X X
77.0 X X
72.6 X X X X X X X X X
63.9 C-hordein X X X X X
60.0 X
51.7 X X X X X
47.0 X X X X
45.0 X X X X
42.2 X X X
40.5 X X X X
34.5 B-hordein X X X X X X X X X
31.5 X X X X
28.5 X X X
25.8 X
Albumin
22.0 X X X X X X X X
19.0 X X
Globulin
14.9 X X X X X X

The results of fructan dosage are shown in Table 11.

Table 11. Fructan dosage in cereal cultivars (means values ± standard deviation).

Sample Concentration Mass Sample

Sample Used Volume (µL) Absorbance Fructose (%)
(µg/mL) (mg)
Wheat cultivars
Pajura in Tris-glycine 500 0.229 9730.0 53.2 1.83 ± 0.14
Pajura in distilled water 500 0.274 1119.0 53.2 2.10 ± 0.16
Litera Tris-glycine 500 0.184 8190.0 56.4 1.45 ± 0.21
Litera in distilled water 500 0.162 7450.0 56.4 1.32 ± 0.22
Dropia Tris-glycine 500 0.233 9816.7 57.9 1.69 ± 0.13
Dropia in distilled water 500 0.331 1313.3 57.9 2.27 ± 0.24
Pitar Tris-glycine 500 0.188 8323.3 54.6 1.52 ± 0.13
Pitar in distilled water 500 0.105 5576.7 54.6 1.02 ± 0.12
Glosa Tris-glycine 500 0.171 7753.3 56.7 1.37 ± 0.16
Glosa in distilled water 500 0.172 7793.3 56.7 1.37 ± 0.08
Smarald Tris-glycine 500 0.216 9260 52.6 1.76 ± 0.07
Smarald in distilled water 500 0.081 4780.0 54.2 0.88 ± 0.12
Ametist Tris-glycine 500 0.102 5473.3 54.2 1.01 ± 0.04
Ametist in distilled water 500 0.122 6143.3 54.2 1.13 ± 0.05
Barley cultivars
Dana Tris-glycine 500 0.181 8113.3 55.6 1.46 ± 0.04
Dana in distilled water 500 0.202 8796. 7 55.6 1.58 ± 0.05
Simbol Tris-glycine 500 0.152 7130.0 55.8 1.28 ± 0.22
Simbol in distilled water 500 0.207 8950.0 55.8 1.60 ± 0.05
Onix Tris-glycine 500 0.209 9060.0 56.4 1.61 ± 0.07
Onix in distilled water 500 0.235 9890.0 56.4 1.75 ± 0.16
Int. J. Environ. Res. Public Health 2022, 19, 11114 14 of 18

It can be seen that, in most cases, the extraction in distilled water had a better yield.

4. Discussions
Gluten remains an essential protein for the food and bakery industry. Given that this
quantitative analysis was performed on raw material, the impact of crossing and selection
methods of improvement on the gluten content of the wheat samples was demonstrated.
The literature supports the fact that the proteins (gluten) from wheat, which are associated
with the elasticity and extensibility of the bakery and related products, play an important
role in human nutrition [32]. Gluten represents one of the major protein components of
wheat (~80% from the total protein endosperm) in which gliadins and glutenins represent
the most important parts. These two types of gluten determine its quality by their inter-
action and variation in composition [33–35]. The highest increase in the gluten content
(Table 3) was observed in the case of Pitar wheat, where the amount of 19.60 ± 0.20% is 6.5%
higher than in the case of commercial flour with a high gluten content. In the case of the
other cultivars, the increase was not so pronounced. An increase of 3.4%in the maximum
gluten content was observed in the case of the Dropia cultivars (16.46 ± 0.16%).
The literature data report that the quality of the final products is affected by the flour
protein content. The protein is represented by the different proportions of low-molecular-
weight glutenin subunit (LMW-GS), high-molecular-weight glutenin subunit (HMW-GS),
albumins, α-, γ-, and ω-gliadins, and globulins [36–39]. According to the Department of
Agriculture of the United States of America (USDA), the protein content in wheat flour
is between 6.67 and 11.8%, and the protein content in barley is between 9 and 10% [40].
The results obtained using the Bradford assay (Tables 4 and 5) were below the lower limit.
This is probably due to the inefficiency of protein extraction using a single solvent (tris-
glycine buffer or distilled water) and the structural differences between plant and animal
proteins used as standard Bovine Serum Albumin (BSA) in this dosage. Subsequently, for
developing a future quantitative extraction method, the total nitrogen (Nt ) was dosed using
the Kjeldahl method.
The results of the GC-MS/MS analysis (Table 8) showed quantitative and qualitative
differences between different cultivars from the same species. For example, the amino
acid L-proline was not identified in the Pajura and Dropia wheat cultivars, but occurs
in the other three, and Tyrosine is absent only in the case of Dropia wheat. From a
quantitative perspective, an increased content of L-Glutamic acid (0.68–1.94%) and DL-
Phenylalanine (0.1–0.53%) can be observed in all wheat samples. In the case of other
amino acids, no significant differences were observed between cultivars or important
concentrations. However, the Glosa wheat cultivars proved to be clearly superior in terms
of the concentration of each amino acid.
Qualitative differences in barley cultivars showed a lack of L-Isoleucine in Onix, L-
Proline in Symbol and Onix and Tyrosine in Dana, Symbol and Onix. The glutamic acid
content was increased compared with other amino acids present in the sample (0.14–1.04%).
The concentrations of the other amino acids were compared, but there were no major
differences. No quantitatively superior cereal cultivars were found. The plant-based
proteins have relatively low essential amino acid contents when compared with animal-
based and human proteins. This assumption was demonstrated by comparing the amino
acids content of various plants and animal-based protein sources [41,42].
According to the literature, in the case of wheat, non-gluten proteins represent 20–25%
of the total mass of proteins, most of which are monomeric [43]. Albumins and globulins
represent about 14% and 22%, respectively. Molecular weights are generally of 25 kDa,
although there are significant proportions between 60 and 70 kDa. From the nutritional
perspective, these proteins have a balanced intake of amino acids (high content of lysine,
tryptophan, methionine). Wheat gluten can be separated into three broad groups: sulfur-
rich proteins of about 50 kDa (α-, β-, γ-gliadins and B-, C-glutenins), low-sulfur proteins
of the same mass (ω-gliadins and D-glutenins) and high mass proteins of about 100 kDa.
Glutenins and gliadin make up 60–90% of all wheat proteins and tend to be rich in as-
Int. J. Environ. Res. Public Health 2022, 19, 11114 15 of 18

paragine, glutamine, arginine or proline, but poor in nutritionally important amino acids
such as lysine, tryptophan or methionine [44].
Proteins separated from barley cultivars can be classified into several fractions: al-
bumin, globulin, prolamine (hordein) and gluteline. These represent 8–15% of the seed
mass, of which 3–5% are albumin and 10–20% globulin. The main components are hordein
and glutelin, representing 40–45% of the total mass. According to the literature, three
protein bands were highlighted: BPI-1 (albumin and globulin fraction with masses less
than 20 and between 30 and 70 kDa), and BPI-2 and BPI-3 (C-, D-, B- hordein with masses
between 29 and 55 kDa) [45]. Previous studies have shown that fructans, soluble fiber,
are good for human health [46]. Fructans are non-reducing polysaccharides composed of
fructose units and terminated with a glucose molecule, which have different degrees of
polymerization [46]. In wheat, fructans have a value of 0.7–2.9%, and in barley 0.9–4.5%.
This concentration is high enough to regulate intestinal transit and acts as a probiotic in the
intestine. However, in some cases it can cause symptoms such as abdominal pain, flatu-
lence, diarrhea, constipation, and intestinal cramps, both in patients with irritable bowel
syndrome and in non-celiac patients [47]. Experimentally obtained concentrations ranged
from 1.01 ± 0.04 to 2.27 ± 0.24% fructose for wheat cultivars and from 1.28 ± 0.22% to
1.75 ± 0.16% for barley cultivars. Comparing the results obtained after the fructose dosing
process, it was observed that some cultivars of the same species have double saccharide
concentrations compared to others, examples being wheat Dropia in the first case, and
barley Onix cultivars in the second one.
All the above analyzed cultivars have concentrations consistent with the data found in
the literature, but the differences that appear compared to them can be based on the used
breeding techniques. Thus, future studies can be performed to improve or decrease the
fructan content of the desired cultivars.
Bread and bakery products are food products that occupy a high percentage in the
food preferences of Romanians, as evidenced by recent studies conducted both during the
pandemic and before [48,49]. As a result, the development of cereal cultivars with good
baking power is an important aspect of the bakery industry. This involves an increase in the
amount of gluten and in fermentable carbohydrates. Moreover, the increase in the amount
of protein and especially in the amino acid content improves the nutritional value of bakery
products. Moreover, fructan enrichment can have positive effects on bakery products, due
to the increase in prebiotic content, fructan behaving as a fiber with beneficial effects on
the intestinal microbiome and at the same time affecting the stimulation of the intestinal
transit, thus helping to detoxify the body [50].
Several scientific studies have demonstrated the importance of using improved cul-
tivars of wheat in obtaining bakery products with superior digestibility and special
aroma [51–53].
Nevertheless, we must also consider numerous restrictive aspects, the varieties that
are enriched in gluten and fructan are not recommended for the manufacture of bakery
products for people who have an intolerance to them or celiac disease.
If wheat cultivars obtained with improved characteristics for the bakery industry are
a valuable raw material for obtaining quality products, then barley cultivars with higher
concentrations of valuable nutrients are important raw materials for the beer industry.
Numerous studies carried out on different varieties of beer have demonstrated that the
organoleptic properties of beer and its nutritional value are strongly influenced by the
quality of the raw materials and the characteristics of the technological process. In this
sense, the use of improved cultivars of barley leads to the obtaining of quality malt that,
through their content of carbohydrates, vitamins, mineral salts, fibers, and proteins with
increased biological value, helps to create nutritional, aromatic drinks with optimal alcohol
content [54–56].
In addition to the process of obtaining improved cultivars, the qualities of the cultiva-
tion areas must be considered because environmental pollution or the practice of intensive
Int. J. Environ. Res. Public Health 2022, 19, 11114 16 of 18

agriculture can lead to the accumulation of toxic substances in the cereal grains, affecting
the quality of the raw material and the safety of consumption [57–60].

5. Conclusions
In conclusion, this study has shown the quantity and quality differences in terms of gluten,
protein, amino acids and sugar parameters of both wheat cultivars of Triticum aestivum L. and
barley cultivars Hordeum vulgare L., respectively. Multiple studies have been conducted
based on conventional dosing methods (gluten dosing), spectrophotometric (Bradford and
protein dosing), digestion (Kjeldahl protein dosing), gas chromatography (GC- MS amino
acid from protein dosing), or qualitative methods (identification of protein fractions by
polyacrylamide gel electrophoretic method). Large differences in gluten concentration,
protein, amino acids and carbohydrates were noted for both cultivars. These types of
studies regarding the new wheat and barley cultivars are the maximum exploitation of
interventions based on crossing and selection methods, beyond those which acquired a
better yield. Changing the nutritional profile by improving and dosing the constituent
nutrients can lead to the emergence of special diets for patients over time, based on their
needs or deficiencies. Obtaining wheat and barley cultivars with high concentrations of
valuable nutrients (especially protein and fructan) represents a success and improvement
for both the bakery and beer industries because the use of fortified raw materials does
not rise to the level of quality of improved natural products. The greatest applicability
for the bakery and beer industries was determined for all of the studied wheat and barley
cultivars due to the high values of their nutritional constituents, which may contribute to
increased interest in this cultivar by bakery and beer producers. This study highlighted
the production of improved cereal cultivars, with increased content of the main nutrients
involved in the quality of food products derived from them. The organoleptic characteristics
and nutritional value of bakery products and beer varieties obtained from these cultivars
will be the future scope of the study.

Author Contributions: Conceptualization, E.M., A.A.S. and M.M.; methodology, A.C.I., E.M. and
I.D.R.; formal analysis, A.I.R., A.M.D., O.K. and A.A.S.; investigation, E.M., A.I.R., A.M.D., O.K.,
A.A.S. and M.M.; data curation, M.M., A.M.M. and E.A.O.; writing—original draft preparation,
A.M.M., M.M. and E.A.O.; writing—review and editing, A.M.M., M.M. and E.A.O. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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