International Journal of Biological Macromolecules 94 (2017) 663668

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

The impact of germination time on the some selected parameters

through malting process
Vahid Farzaneh a, , Alireza Ghodsvali b , Hamid Bakhshabadi c , Zahra Zare d ,
Isabel S. Carvalho a
a
MeditBio, Faculty of Sciences and Technology, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
b
Department of Agricultural Engineering, Golestan Agricultural and Resources Research Center, Iran
c
Young Researchers and Elites club, Gorgan Branch, Islamic Azad University, Gorgan, Iran
d
Young Researchers and Elites club, Shahre Qods Branch, Islamic Azad University, Shahre Qods, Iran

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

Article history: In the present study, the impacts of germination time on the enzymes activity attributed in malting and
Received 3 July 2016 some polysaccharides contents of the malt prepared from the Joseph barley variety have been screened
Accepted 17 October 2016 using a completely random design with three levels of germination time(3, 5 and 7 days). The archived
Available online 18 October 2016
outcomes revealed that the highest quantity of starch has been observed in the malt resulted from 3 days
germination, and an enhancement in the germination period from 3 to 7 days decreased the quantity of
Keywords:
available starch. An enhancement in the germination period presented a reduction in the beta-glucan
Germination time
quantity in the malting seeds. The malt produced 7 days after germination had the highest enzymatic
Enzymatic activity
Malting
activity(253 U. kg1 ). The comparison of data average using Duncan test showed that the minimum and
maximum value of -Amylase enzyme activity and diastatic power were recorded in the malts produced 3
and 7 days after germination, respectively. Increasing in the germination time led to a reduction in malting
efciency, however the efciency of the hot water extraction showed enhancement. The outcomes of
the correlation between the studied parameters showed that the beta-glucan and starch quantities are
negatively affected by the activities of beta-Glucanase and -Amylase.
2016 Elsevier B.V. All rights reserved.

1. Introduction cantly enhanced in Iran [1]. Malting is a combined biotechnological

Economic development of developing countries with similar cli-
mates and agricultural conditions depends on the expanding of
agricultural industries as a main parameter in availability of job
vacancies. A solution to attract foreign currency income for the
upcoming years could be restriction of exiting of valuable foreign
currency out of borders via reducing food products import. In this
regard, the role of food processing industries is certainly undeni-
able. Barley with the scientic name of Hordeum Vulgar L., is among
the agricultural and strategic products supported commercially
regarding the recent agricultural policies and is considered as one
of the important forage plants. Recently, researchers and interna-
tional research centers pay considerable notice to this plant and its
malting procedures. In the resent years, this issue has been signi-
Corresponding author at: Food Science Laboratory, Faculty of Sciences and
Technology-University of Algarve, Campus Gambelas, Building 8, 8005-139 Faro,
Portugal. Tel. +351 9690 18 099.
E-mail address: Farzaneh1364@gmail.com (V. Farzaneh).
approach involving of steeping, germination and drying of the ger-
minated seeds in controlled conditions of different temperatures
and humidities [2]. The major purpose of germination is synthesiz-
ing of the hydrolytic enzymes to decompose the cells wall, protein
and endosperm starch compounds leading to a desirable enhance-
ment of brittleness and fragility of the barley grains [3]. Usually,
within the germination leg, the relative humidity of the air passing
through the grains bed is almost 100% and the temperatures range
are between 12 and 19 C depending on the barley variety applied in
malting. Ref. [4] reported that an enhancement in the germination
time will lead to an increase in the amount of albumin and globulin
contents; therefore, the amount of available soluble proteins gets
increased in the obtained extract. Ref. [5] examined the physico-
chemical characteristics of the oat grains during germination and
stated that during germination the activities of enzymes including
-Amylase, beta-Amylase and beta-Glucanase are increased, more-
over the starch content is reduced from 60% to 20%, on the other
hand the quantity of reducing and soluble sugars got increased, also
the amount of protein showed an enhancement, but the amount of
lysine got reduced at the end of the germination phase. Finally,

http://dx.doi.org/10.1016/j.ijbiomac.2016.10.052
0141-8130/ 2016 Elsevier B.V. All rights reserved.
664 V. Farzaneh et al. / International Journal of Biological Macromolecules 94 (2017) 663668
the quantity of Phytic acid presented a reduction from 0.35% to
0.11%. Beta-Glucan is a water-soluble non-starch polysaccharide
which is the main component of the polysaccharides detected in
the endosperm cell walls of barley and has been made of a non-
branched chains containing beta-d-glucopyranose with bonds of
(1 3) and (1 4) [6]. Alterations in the quantity of beta-Glucan
and the activity of beta-Glucanase in germination and malting pro-
cess are presenting substantial impacts in malting, since both of
the abovementioned parameters are associated with the efciency
and quality of the produced malt. Analysis of the endosperm cell
walls and the subsequent changes in the beta-Glucan structures
during the malting process is signicantly associated with the beta-
Glucanase activity [7]. Beta-Glucanase is often synthesized in the
Aleurone and Scutellum of the germinated grains and remains
hidden in the endosperm cells. Therefore, during the malting pro-
cess, the beta-Glucan content decreases signicantly as a result
of enhancement in beta-Glucanase activity. Therefore, developed
malting process depends on the less amounts of beta-glucan con-
tents in the grain and higher quantities of beta-Glucanase enzyme
in the obtained malt [7]. Beta-Glucanase produced during the malt-
ing process hydrolyses the cell walls into soluble beta-Dextrin
with low molecular weight; it is almost thermolabile and rapidly
inactivated during the extraction phase above 50 C temperature.
However, liquefying of beta-Glucan from the intact cell walls is
continued and leads to the accumulation of soluble beta-Glucan
molecules in the extract [8]. Temperature, humidity and period of
the germination are the key factors on the nal characteristics of
the produced malts. High temperatures accelerate the germination
process but decrease the activity of beta-Glucanase and protease
enzymes. Moreover, a rapid increase is observed in the amount of
-Amylase enzyme which is decreased before the completion of
the endosperm amendment [9]. In fact, extraction by hot water
shows an enhancement in the quantity of dissolved solids in the
sweet extract obtained from the malt or other materials added to
the malting medium during the malting process in a small-scale,
there is a direct relationship between the extraction efciency of
the cold water extract and changes in dissolved solids quantity
within the germination process [10]. Ref. [11] stated that remaining
of beta-Glucan increases the viscosity at the end of the extraction
phase and decreases the ltration rate. It might be presented that
decomposition and reduction of beta-Glucan quantity is attributed
to increased beta-Glucanase activities, the beta-Glucan quantity in
grain is greatly reduced after the accomplishment of 50% of malting
process. The activity of all the decomposing factors of barley malt
starch including alpha and beta-amylase, alpha-Glucosidase, lim-
ited dextrinase and the maltase are called diastatic power which is
a substantial parameter in assessing of the activity of decompos-
ing agents of starch in malting industry. Alpha and beta-amylase
together are creating more than 99% of the total diastatic activity
of the malt [1]. The aim of this study is to examine the impacts of
germination period on the activity of the enzymes and important
polysaccharides of the barley malt grains.
2. Materials and methods
2.1. Materials
Barley seeds applied in this study, variety Joseph has been pro-
vided from the Agricultural Research Center of Golestan-Gorgan
and chemical materials such as toluene, sulfuric acid, sodium
hydroxide, copper sulfate, methylene blue, zinc acetate, ammo-
nia and Fehling solutions A and B were provided in analytical
grade from Merck company (Germany); kits for the determina-
tion of polysaccharides starch, beta-Glucan, and enzyme including
beta-Glucanase and alpha-Amylase activities have been obtained
from the Megazyme Company (Ireland). Equipments used in the
present study are as the following: germinator machine (Tabai
Espec Corp-Japan), laboratory sieve, milling machine (Huddinge
14105-Sweden), desiccator, laboratory oven (Memert-Germany),
pycnometer, spectrophotometer (Novaspec II) digital scale (Gec
Avery-England) and centrifuge (Model: HERMLE Z323K-Germany).
2.2. Methods
2.2.1. Preparing of the malt samples
After cleaning, sifting and winnowing of the grains manually
using a sieve, they were separately moved to the soaking pro-
cess for 2448 h to reach to the nal moisture content of 4246%
(the water temperature and hardness have been set on about 20 C
and 250 ppm respectively). Then, the obtained soaked grains were
weighed into three equal parts and transferred and kept into ger-
minator machine for the designed duration of 3, 5 and 7 days for
germination; the germinator temperature was set in the range
1720 C. Finally, the samples were dehydrated at 5565 C for
2448 h and then their rootlets were separated by frequent siev-
ing and abrasion followed by milling, the extraction was performed
using the temperature timing [12].
2.2.2. Measuring the starch
Determination of the starch quantity has been performed using
the commercial kits regarding the released methods by AACC
Method 1176 and the Eq. (1) and its amount was reported as
percentage [12,13].
F
Starch% = A F FV O.9 (1)
V
Where A = absorbance (reaction) read against the reagent blank,
100(g of Dglucose)
F = absorbance for 100g of glucose , FV = nal volume, W = the weight in
milligrams (as is basis) of the our analysed. It should be noted
that the wave length of 510 nm in spectrophotometric approach
has been used in this research.
2.2.3. Determination of the barley beta-Glucan content
According to EBC method No.3, 11, and 1, 0.5 g of malt grains
our was precisely weighed with 7% moisture content and moved
into polypropylene tubes, then 1 mL of 50% aqueous ethanol (W/W)
was added to disperse the samples. Then, 5 mL of sodium phos-
phate buffer (20 mM with pH = 6.5) was added to the tube and
vortexed vigorously. After that, the tubes containing the samples
were placed in a boiling water bath for about 2 min, then the tubes
were removed from the bath and vortexed again, afterward they
were moved in the bath for more 3 min. The tubes were cool down
to 40 C, then 0.2 mL of Lichenase was added to the tube and the
tubes have been diluted to 30 mL using distilled water followed by
vortexing. The tubes were centrifuged with 1000 g for 10 min and
0.1 mL of sodium acetate buffer (50 mM with pH = 4) was added to
one of the tubes (blank) against 0.1 mL of beta-Glucosidase added to
two other tubes (samples). Then, the tubes were incubated at 40 C
for 15 min. In the next stage, 3 mL of GOPOD reagent was added to
each tube and incubated at 40 C for 20 min. After changes in color,
samples absorption was read at 510 nm. The beta-Glucan value has
been calculated by Eq. (2) [12,13].
F
Beta Glucan (%) = A 27 (2)
W
Where, A is demonstrating the difference in absorption at 510 nm
between the samples and control, F expresses the conversion factor
of uptake to Glucose and W indicates the dry weight of our sample.
 V. Farzaneh et al. / International Journal of Biological Macromolecules 94 (2017) 663668
2.2.4. Determining of beta-Glucanase enzyme activity
Measuring of beta-Glucanase enzyme was performed using the
commercial kits using Eq. (3); the enzymatic activity is expressed
as (U. Kg1 ) [12,13]
Y = MX + C
In Eq. (3), M = 630, C = 4 and X presents the absorbance value of the
test solution at 590 nm.
2.2.5. Determining the activity amount of alpha-amylase enzyme
Measuring the activity of alpha-amylase enzyme was deter-
mined using the commercial kits of Megazyme Company of Ireland
using Eq. (4):
Y = E400 376
In Eq. (4), E400 presents the difference of absorbance at 400 nm
between the our and control samples and Y indicates the activity
value of Alpha-Amylase enzyme in unites per gram of malt our
(U. g1 ) where each unite (U) indicates the activity leading to the
release of a micromole of the restored sugar (substrate) per minute
in the dened temperature and pH [12,13].
2.2.6. Determination of malt diastatic power
The malt diastatic power (DP) has been detected using Fehlings
solution as described formerly [14] and was presented as a Linter
( L). Fischer chemical starch was applied to prepare starch solution
with 2% concentration for this determination. An infusion of the
obtained malt was prepared. 3 mL of the prepared infusion has been
shifted into 100 mL of 2% buffer starch solution in a 200 mL ask.
The mixture was mingled well and preserved at room temperature
for 1 h from the time that sample was added. Finally, 15 mL of 0.1 M
NaOH solution was poured to stop the reaction and total solution
has been diluted to 200 mL with deionized water. Into a 150 mL
narrow-necked boiling ask, 5 mL of mixed Fehling solutions A and
B were also sprinkled. Titration was modied by adjoining from a
burette containing the digested starch solution to the ask with
1 mL of the nal end point. The contents of the ask were thor-
oughly stirred and boiled for 2 min and then 3 driblets of methylene
blue indicator were added to carry out the titration. The end point
was achieved when the methylene blue was changed to reddish
color in appearance. The blank was prepared by titration of the
undiluted 2% starch solution against 1 mL of mixed Fehlings solu-
tion A and 2 mL Fehlings solution B using methylene blue indicator
as described previously.
The diastatic power has been computed using the Eq. (5):
DiastaticPower = (2000 200/XY XS)
Where:
X = No.ofmLofmaltextract
Y = No. of mL of converted starch to 5 mL of the Fehlings solution
S = Titerforstarchblank
2.2.7. Malting yield
Malting efciency was calculated regarding the obtained data
by digital balance (with the precision of 0.01 g) using the Eq. (6):
 
Maltingyield (%) = Mm /Ms 100
Where, Mm presents the weight of the obtained nal dehydrated
malt and Ms indicates the weight of the applied seeds in this study.
The nal outcome value is between 0 and 1.
665
2.2.8. Determination of the efciency of hot water extract
After extracting by temperature timing method, specic weight
of extract was determined using the pycnometer, then with ref-
erence to plateau table, extract Brix was detected and Finally, the
efciency percentage of hot water extract was determined using
(3) the Eq. (7) [11].
(800 + M) P
E= (7)
100 P
E, M and P, are respectively equivalent to the percentage of hot
water extract based on dry matter, moisture percentage in malta
and total soluble solids in 100 g of extract obtained through plateau
table.
(4) 2.2.9. Statistical analysis
Data analysis has been performed using a completely random-
ized design with three replications. SAS software was used for data
analysis, and comparing of the means of the studied parameters
has been performed using post hoc Duncans test at the condence
level of 5%. The graphs have been drawn using Microsoft ofce Excel
software version 2010.
3. Results and discussion
3.1. Starch amount
Data analysis showed that the treatment of germination period
had signicant impacts (P < 0.01) on the starch quantity of malting
grains. According to the achieved outcomes, the maximum amount
of starch was observed in the malt obtained from 3 days after ger-
mination and an enhancement in the germination period from 3 to
7 days led to a decrease in starch quantity (Fig. 1a). It seems that
with enhancement in the germination period from 3 to 7 days, the
respiration rate and as a result starch consumption in the grains
has been gradually increased due to an increase in consumption of
nutritional compounds for Acrospires and Radicle growth, and also
by increasing the hydraulic enzymes activities particularly alpha
and beta-amylase which break the starch molecular contents into
small molecules including Maltose, Dextrin and Glucose, the starch
quantity gets decreased considerably. The results of this section
conrmed the results obtained by [15].
3.2. Beta-Glucan amount
The results of analysis of variance presented that the impacts
of germination period treatment on the malt Beta-Glucan was sig-
(5) nicant (P < 0.01). Samples with 7 days of germination treatment
compared to the samples with 3 and 5 days germination treat-
ments, presented beta-Glucan amount of the malt about 103.23%
and 17.33%, respectively. In other words, increasing of the germi-
nation period led to a signicant reduction of beta-glucan quantity
of the malt grains (Fig. 1b). It seems that increasing of the activity
of beta-Glucanase enzyme has led to a decrease and decomposi-
tion of beta-Glucan contents to smaller molecules [16]. Ref. [17]
studied the impacts of germination periods (2 to 6 days) on the
beta-Glucan quantity of oat and barley malt seeds and presented
that increasing of the germination length results in more decompo-
sition of the soluble edible bers particularly beta-Glucan [17]. The
results obtained by [18] on the impacts of malting process on the
activity of beta-Glucanase enzyme in the barley grain have shown
that the maximum alterations in the quantity of beta-Glucan and
(6)
beta-Glucanase enzyme occur at the germination time [18]. During
the germination, germ is grown and reached to half to two-third of
the grain length; the growing fetus excretes Gibberellic acid. This
fact leads to an increase in the amount of beta-Glucanase enzyme in
666 V. Farzaneh et al. / International Journal of Biological Macromolecules 94 (2017) 663668
Fig. 1. The effect of germination time on the starch content (a), Beta-Glucan amount (b), beta-Glucanase enzyme activity (c), alpha-amylase enzyme activity (d).
barely about 500600 folds as much and leads to the decomposed
beta-Glucan and additional polysaccharides of cell wall of the seed.
3.3. Activity amount of beta-Glucanase
Some researchers [19] reported that the germination period is
effective on the enhancement of enzyme quantity. Analyzing of
variance of the data obtained for the enzymatic activity within the
different germination times expressed that there is a signicant
difference between the impacts of different treatments (P < 0.01).
The comparison of the achieved results of the treatments (Fig. 1c)
showed that a 7-day germination had the maximum enzymatic
activity (253 U. kg1 ) which was 73.46% rather than the minimum
enzymatic activity obtained from a 3-day germination, and increas-
ing in the germination period led to the increase in the activity
amount of Beta-Glucanase enzyme. The results obtained of this
study were in solid agreement of the results obtained by [20,18]
who stated that with an enhancement in the germination period,
the activity of the beta-Glucanase enzyme gets increased. Before
malting, the amount of beta-Glucanase enzyme activity in barley
is less and then it increases signicantly during malting process.
Within malting germination causes some alterations in the seed
endosperm through D-polymerization of beta-Glucan. In brewery
industry, a large amount of beta-Glucan leads to less damages in the
seed cell wall during the process and continuing that, it prevents
the distribution of enzymes in the seeds during the germination
leg causing some difculties including reduction of the efciency
of the malt extract, increasing of the yeast viscosity, hardening of
the ltration operation as well as darkening in the resulted syrup
[6].
3.4. Activity amount of alpha-Amylase
Analysis of variance (ANOVA) of data achieved for alpha-
amylase activity with the germination period indicated that there is
a signicant difference between the effect of treatments (P < 0.01).
Comparing of the means using Duncan post hoc test showed that the
maximum and minimum activity values of alpha-amylase enzyme
were related to the malt obtained from 3-day and 7-day germina-
tion, respectively (Fig. 1d). Scientists [19] have reported that the
germination and soaking periods are effective on the boosting of
enzyme amount. Highly active enzyme is generally required for the
conversion of starch into oligosaccharides. According to the nd-
ings of [21] the activity of amylase increases with enhancement in
germination time and the maximum value has been achieved on
the sixth day of germination and started reduction on the seventh
day of germination, that researcher also reported that fat content
of the seeds decreases while the germination time increases, the
lower values were observed at the seventh day of germination [21].
3.5. Diastatic power
The results obtained from the analysis of variance showed
that the treatment of germination period has a signicant effect
(P < 0.01) on the malt diastatic power. Comparing of means of the
obtained data using Duncan post hoc test showed a statistically
signicant effect on the malt diastatic power in different treat-
ments of germination times. The maximum diastatic power of malt
was observed with the average ( IOB 115) in the 7-day germina-
tion, and the minimum value has been observed with the mean of
( IOB 74.66) in the 3-day germination (Fig. 2a). The cause for the
increase of malt diastatic power with increasing in the germina-
tion period might be attributed to the increase in alpha-amylase
enzyme activity. In a research, the impact of germination period
(5, 7 and 9 days) on the amount of rice malt diastatic power has
been studied and the results showed that with an increase in the
germination period, the amount of malt diastatic activity value has
been signicantly increased. Also, the results of that study have
shown a signicant negative correlation between the malt diastatic
power and the starch content. The activity of all of the decompos-
 V. Farzaneh et al. / International Journal of Biological Macromolecules 94 (2017) 663668
Fig. 2. The effect of germination time on the diastatic power (a), malting yield (b), hot water extract (c).
ing factors of barley malt starch including alpha and beta-amylase,
alpha-Glucosidase, limited dextrinase and the maltase is called the
diastatic power which as an important quality factor is used for
assessing of the activity of decomposing enzymes of starch and
additional polysaccharides such as beta-Glucan in malting indus-
try. Alpha and beta-amylase are included in more than 99% of malt
total diastatic activity [1].
3.6. Malting yield
The obtained results showed that the treatment of germina-
tion period had signicant effect (P < 0.01) on the malting yield.
Increasing in the germination time led to a decrease in the malting
yield (Fig. 2b). A decrease in malting efciency with an increase
of germination time might be attributed to more respiration of
the cell of the seeds and the reduction of stored starch weight. A
group of researchers studied the impact of different germination
times on the drop of malting scale in the millet malt and found
out that with increasing of the germination time the malting drop
experiences growing process. They stated that the dry weight loss
during the germination is due to the increase in metabolic activ-
ity and malting carbohydrates decomposition [22]. The research
results on the rice malt in 2007 showed that by increasing the ger-
mination period from 5 to 9 days, the malting yield had a decline
process, and the malting drop experienced a growing process. In
the present study, the maximum malting yield has been observed
in the malt obtained from a 5-day germination (92.81%), and its
minimum value has been observed in the malt obtained from a 9-
day germination. These researchers have considered the chemical
changes, decomposing a part of the material with high molecular
weight in the starch endosperm and decreasing the dry material
percentage of the seed during the malting process as the rea-
sons of increasing in the malting drop and decreasing the malting
yield. These researchers have also reported that the malting drop is
occurred due to the metabolic activity and separating the rootlets;
and increasing of the germination period [11].
3.7. Hot water extraction efciency
Data analysis have shown that germination period had a sig-
nicant effect (P < 0.01) on the efciency of hot water extraction.
Table 1
Correlation coefcient detected between different determined parameters in this study.
Parameter Starch quantity -Gluconase
Starch amount 1
-Gluconase 0.993 1
-Glucan 0.969 0.940
-Amylase 0.897 0.934
Malting yield 0.976 0.986
Hot water extract 0.982 0.965
Diastatic index 0.953 0.978
667
Comparing of the data averages using Duncan test showed (Fig. 2c)
that increasing of the germination period led to an increase in
the efciency of hot water extraction. Increasing the hot water
extraction efciency might be due to the higher activity of the beta-
Glucanase enzyme and lower amount of the Beta-glucan. Increasing
in the germination period will increase the efciency of extrac-
tion. In fact, extraction with hot water represents the quantity of
dissolved solids in the extract obtained from the malt or other mate-
rials added to malt our during the extraction process on a small
scale. Small samples of malt or other milled materials are ground
and squashed in different ways and soaked under standard condi-
tions of time, temperature, in warm distilled water. At that point,
they are extended to a certain volume and are ltered, then the
extract specic gravity is measured at a constant temperature and
over this measurement the extraction amount resulted from the
malt or other soaked samples has been calculated [10,23]. Extrac-
tion efciency of the hot water is one of the most important quality
factors of malting which is economically substantial due to it rep-
resents the extraction obtained from a certain amount of malt [25].
3.8. Examining the interactive effects of studied parameters
As it is observed in Table 1, the beta-Glucan and starch amounts
are negatively correlated with the activity of beta-Glucanase and
alpha-Amylase enzyme. On the other hand, the higher the enzyme
activities the lower polysaccharides amount in the seeds and as
a result the higher the extraction efciency of hot water. Some
researchers have studied the interactive effects of some parame-
ters of malt seeds on each other. For instance, [24] showed that
there is a signicant positive correlation coefcient between the
total malt protein and the total dissolved protein.
4. Conclusions
Increasing of the germination time results in the reduction
of starch quantity, beta-Glucan as well as malting efciency, but
the amount of beta-Glucanase and alpha-amylase enzymes, the
diastatic power alongside of the hot water extract efciency have
been got increased. As it could be noticed, enhancement in the
activity of enzymes and further decomposing factors of polysaccha-
-Glucan -Amylase Malting yield Hot water extract Diastatic index
1
0.765 1
0.909 0.953 1
0.989 0.815 0.935 1
0.854 0.987 0.982 0.895 1
668 V. Farzaneh et al. / International Journal of Biological Macromolecules 94 (2017) 663668
rides in malt seeds might be resulted in an increase of extraction
efciency.
Acknowledgments
This work is a part of the PhDstudies of student Vahid Farzaneh.
Acknowledgement is given to Erasmus Mundus, program SALAM
(2013) for nancial support and food science lab (FSL) of MeditBio,
Faculty of Sciences and Technology, University of the Algarve for
facilities provided during this study. We greatly appriciate Golestan
Agricultural and Natural Resourses Research Center, Gorgan, Iran
for collaboration in this research.
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