Zinabu Hailu, Basha Mekonnen, IJFNR, 2019; 3:33

Research Article IJFNR (2019) 3:33

International Journal of Food and Nutrition Research

(ISSN:2572-8784)

Optimization of Local Wort and Fermented Beer from Barley as

Substitute Raw Material for Ethanol Production Using Response
Surface Methodology
Zinabu Hailu, Basha Mekonnen

Chemical Engineering Department, Adigrat University

ABSTRACT

The research work was aimed to find the exact optimized *Correspondence to Author:
operating temperature, time, pH and substrate which is important Zinabu Hailu, Basha Mekonnen
for the production of wort and fermented beer in both mashing Chemical Engineering Department,
and fermentation processes. Here, the barley was used as Adigrat University
a basic source of substrate and enzymes. To determine the
optimum operating temperature, pH, time and substrate under How to cite this article:
mashing and fermentation processes, Central Composite Zinabu Hailu, Basha Mekonnen.
Experimental Design (CCD) was used. The results showed that, Optimization of Local Wort and Fer-
the maximum % malt extracts (92.36 %), fermentable sugar mented Beer from Barley as Substi-
(10.53 oBx) were observed at 70oC, 120 min., pH value of 4.5 tute Raw Material for Ethanol Pro-
and 30 gm substrate source addition. After optimizing the wort, duction Using Response Surface
the fermentation experiment was conducted accordingly the Methodology. International Journal
combination which was given by design expert software. The of Food and Nutrition Research,
maximum degree of attenuation value (86 %) was observed at 2019; 3:33.
20oC, 96 hr, and pH value of 4.4 and 75 gm substrate source
added. Therefore, good barley type and optimum condition for
mashing and fermentation process were found to be significant
effect for high wort, and distillery beer. eSciPub LLC, Houston, TX USA.
Website: https://escipub.com/
Key words: Mashing, At tenuation, Fermentable sugar,
Optimization, Wort

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 Zinabu Hailu, Basha Mekonnen, IJFNR, 2019; 3:33

Introduction hardness and physical and chemical grain

properties.
The distillery mashing process involves the
conversion of milled malted barley into a Malting is a controlled germination process
fermentable extract or wort [1]. The mashing consisting of steeping or hydration of grains (to
temperature, PH and Mash time are extremely ensure good absorption of water by the grain
important in the enzymatic degradation of the from 12% to at least 45% of moisture)[14].
milled malted barley and is essential to attain Fundamentally, the aim of malting is to unmask
the best wort composition[2]. Heat labile starch granules from the surrounding cell walls
enzymes can be quickly denatured if the and protein matrix so that fermentable sugars
temperature is too high, [2] and at lower can be optimally released from starch during the
temperatures starch gelatinization may not be mashing process [14]. Due to this Enzymes are
fully achieved. Although the mashing process synthesized or activated in the aleurone and
is a simple and straight- forward procedure, scutellar cells by the action of embryonic
it includes a complicated system of chemical gibberellin activated signal transduction
and biochemical reactions [8] Starch granules pathways and are secreted into the starchy
become hydrated and soluble in the mash endosperm [15, 16]. Thus, the main objective of
liquor and are made available to the malted the research work was to determine the optimum
barley enzymes. Any insoluble polymers such local wort and fermented beer from barley as
as cell-wall glucans still associated with the substitute raw material for ethanol production.
insoluble milled malted barley particles may Materials and Methods
be attacked by enzymes during the mashing Raw Materials
process, thus heterogeneous mixture of
The food barely type was purchased from
enzymes and substrate molecules are found
Gayent wereda south Gonder and packed
within the wort. These may contribute to the
through plastic bag and transported to Bahirdar
yield of soluble sugars available to the
institute of Technology, Bahirdar University (BiT-
fermentation.[2]
BDU).The purchased barely used as source of
Barley is used for a wide range of traditional malt with 10.4 % moisture content, 11.4 %
[9] and novel end uses [10]. Barley is fed to protein content, 96 % germination capacity and
animals as a significant part of the diet of cattle, energy, thousand kernels weight, and 2.5mm
pigs, and poultry. Malting barley varieties are sieve size.
usually soft, whereas non-malting varieties are
Supplementary Substrate
hard. According [11] report, significant
Preparation/Adjuncts
relationships between hardness of barley grain
as assessed using the particle size index and hot For this research, corn grain was selected as
water extract of malt as well as the malt quality source of supplementary substrate. The raw corn
index of barley malt. was collected from the local market and cleaned
in order to remove metal, dirt, cobs, etc. This corn
A large number of parameters have been
grain sample was prepared by pre-cooking the
proposed to define malting quality, it is also a
corn sample until changed its color to slightly
fact that the texture of the endosperm influences
dark. Then, the cooked corn ground to a
the malt modification process by affecting water
diameter of 0.5 mm particle size. The ground
uptake and consequently enzyme synthesis and
meal was used as a source of additional
movement within the endosperm [12, 13]
substrate source on both mashing and
studied the variation and correlation between
fermentation processes.
chemical and physical characteristics of barley
samples including kernel hardness, but found Preparation of kilned malt
only a low correlation between kernel Two raw barley samples of 5 kg (2.5 mm Size)
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 Zinabu Hailu, Basha Mekonnen, IJFNR, 2019; 3:33
were malted at (Faculty of Chemical and Food points and 6 center points, totally 30 number of
Engineering (FCFE) Research Grade runs for each step experiment. The independent
Laboratory, Bit-BDU. The malt was produced by variables or factors studied for the first step
applying the pure traditional malting procedure:- experiment were temperature (X1: 40-70 oC),
First, the malting procedure consists of steeping pH (X2: 4.5-6.5), residence time (X3:120 -240
of the raw barley at room temperature (an min), and supplementary substrate added
average atmospheric temperature was 20.5 oC) (X4:15-30 gm) and for the second step
for 2.5 days to achieve an out-of- steep moisture experiment were temperature (X‟1:20-40
of at least 42-45% and the moisture content oC ),pH (X‟2: 4.0-4.8 ), substrate concentration
was calculated at each steeping day (at day1, (X‟3:50-100 gm) and residence time (X‟4:48-
day 2 and day 2.5). 96 hour). The response variables from the
Second, the germination phase was lasted for 5 first step experiments were: % extract content
days at room temperature (an average (Y1),and dissolved sugar content (Y2). The
atmospheric temperature was 20.5 oC) in a flow second step experiment has two responses
of fully humidified air. which are percent alcohol by volume (Y‟1) and
Third step of malting process was kilning, it was Apparent Attenuation Limit (AAL).
conducted by exposing the sample to sunlight Malt Analysis
for five days to produce malt around 4% Malt samples were analyzed according to [7] in
moisture. Gonder Malt Factory (Gonder, Ethiopia).Sieve
Milling condition and Sieving analysis test, moisture content, fine-coarse extract,
All the malt samples were milled using disk mill. friability, color, PH, thousand kernels,
The desired particle size for this study was 0.2 Glassiness ( wholly unmodified grain), partly
mm sieve size. Sieving analysis of the milled unmodified grain, odor, appearance, and wort
malt was performed in order to achieve the viscosity was analyzed.
desired particle size distribution. The sieve sizes Mashing condition
used were 0.2mm, 0.5mm, 0.75mm, 1mm, Wort was produced in a small-scale by
1.5mm and 2 mm. All the required samples of temperature type mashing. The mash
milled malt were sieved to have 0.2 mm size. temperature, pH, mash time and amount of
Experimental Design supplementary substrate conditions were taken
This research was carried out to optimize according to the design expert version 7 output
dependent process variables in two steps of data in the experimental design section 2.4. After
experiments i.e at mashing and fermentation unit conducting each combination of experiment the
operations. Response Surface mash products were filtered with filter paper and
Methodology/central composite design (CCD) the spent grain was separated from the
was used to identify the relationship between the supernatant. The following Filtrate wort
response functions and the process variables, properties such as: % extract, soluble protein,
as well as to determine those conditions that sugar content or fermentability, wort viscosity
optimize the malt enzymes activity by were analyzed according to [7].
measuring the response variables from the two Determination of % Extract Level
step experiments. The research has four factors The extract level of the samples was determined
for each step experiment, then the experiment using the density/specific gravity to degree plato
was designed according to; 24 factorial points, correlation. The density/specific gravity of the
2*4 axial points and 6 center points. So the wort samples were measured using Hydrometer.
RSM/CCD contain 16 factorial points, 8 axial The density/specific gravity to degree plato data
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 Zinabu Hailu, Basha Mekonnen, IJFNR, 2019; 3:33
were used to calculate the amounts of dry weight specific gravity using a hydrometer as shown in
base. They the percentage of malt extract fig.2.1 (b), the specific gravity data were used
present in the filtrates was calculated [7]. to determine the sugar content in degree plato
Determination of fermentable Sugar (oP). The sugar content in degree brix (oBx)
Wort dissolved sugar content was determined by was estimated directly from the digital
using AR200 Digital Hand-Held Refractometer. refractometer as shown in Figure 2.1(a).
It was also estimated by measurement of

Figure 2.1: (a) Sugar content determination in oBx by Refractometer, (b) Sugar content
determination from SG in ⁰P

Determination of Wort Viscosity fermentation (determination of density for

Wort viscosity was determined According to [7] sample after fermentation) and the data/values
using free ball viscometer as described in were used to determine the degree of
section 3.8.3 and the viscosity was determined. attenuation.
Distillery Fermentation Condition Calculating attenuation: Attenuation refers to
the percentage of original extract (the level of
The fermentation process was conducted by
fermentable sugar) found in the wort sample that
taking the wort samples with high % malt extract
has been converted by the fermentation
and sugar content from the mashing condition.
process. It can be calculated as:
The controlling factors were according to the
(𝑂𝐺−𝐹𝐺)
design expert software output data which was 𝐴𝑡𝑡𝑒𝑛𝑢𝑎𝑡𝑖𝑜𝑛 = ∗ 100 %. (1)
𝑂𝐺−1.0
described in section 2.4.
Where: OG is the Original Gravity of the wort,
Determination of Apparent Attenuation Limit FG is Final Gravity of the beer/ sample after
(AAL) fermentation process
Fermentability /Apparent Attenuation Limit Data Analysis
(AAL) is the malt, wort and beer evaluating
RSM is a tool, which was applied t o
parameter that describe the ability of yeast to
designed the mathematical relationship to
turn sweet (sugar) in wort into alcohol. It is also
link the controllable parameters to the
used to measure the performance of starch
experimental responses so as to explore the
converting enzymes (degree of hydrolyzing) and
effect of parameters on responses. In the thesis
the amount of sugar level present in the wort.
work, Design Expert software, Response
The density/specific gravity of wort and beer Surface Methodology (RSM) and central
samples was determined by Anton paar DMA composite design (CCD) was used to analyze
4500M density meter. Samples was taken from the data.
the clear wort and injected to the density meter.
Result and Discussion
This experiment was used for the samples after
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 Zinabu Hailu, Basha Mekonnen, IJFNR, 2019; 3:33
Effect of Mashing Parameters on Wort which was fermentation results, discussed by 3-
Quality D plot response surface graphical
The discussion part was based on interaction representation, and also based on the combined
effect of factors on the responses of mashing effect of factors (temperature, pH, time and
experiment using 3-D plot of response surface amount of substrate source added) on the
graphical representation. The second one, responses of fermentation experiment.

Table 3.1: Main Experimental Result for mashing process (on wort Optimization)

Run No. Factors Resposes

Temperature Time pH S.S.A. % Extract Sugar Protein Viscosity

o (min.) (gm) (%) Content content (cp)
( C) o (g/l)
( Bx
1 70.00 240.00 5.50 22.50 68.35 8.1 0.794 4.1
2 55.00 240.00 6.50 15.00 59.76 7.1 0.879 2.7
3 70.00 180.00 5.50 22.50 66.25 7.9 1.734 3.9
4 55.00 120.00 4.50 15.00 56.08 6.4 1.65 2.9
5 70.00 180.00 6.50 30.00 76.93 8.84 2.188 3
6 55.00 180.00 6.50 15.00 68.35 7.86 1.144 2.4
7 40.00 240.00 4.50 30.00 83.33 9.57 1.988 1.3
8 40.00 240.00 6.50 15.00 76.93 8.84 1.063 1.9
9 55.00 240.00 6.50 22.50 72.64 8.35 1.64 2.58
10 70.00 120.00 5.50 22.50 81.23 9.32 1.406 3.2
11 55.00 180.00 4.50 30.00 83.33 9.57 1.216 2.2
12 70.00 240.00 6.50 15.00 61.95 7.12 0.881 3.8
13 40.00 120.00 4.50 30.00 66.24 7.61 2.138 1.5
14 55.00 120.00 6.50 30.00 76.79 8.84 0.653 2.1
15 40.00 180.00 5.50 22.50 74.83 8.6 1.766 1.9
16 70.00 240.00 4.50 30.00 85.44 9.8 2.719 4
17 40.00 240.00 4.50 15.00 76.79 8.84 1.263 1.3
18 70.00 120.00 4.50 30.00 92.36 10.53 1.734 4.1
19 40.00 240.00 6.50 30.00 89.73 10.30 0.891 2.3
20 40.00 120.00 6.50 15.00 44.51 5.12 0.969 2
21 55.00 180.00 5.50 22.50 71.1 8.4 1.066 3.18
22 40.00 120.00 6.50 30.00 61.95 7.12 0.847 1.5
23 55.00 180.00 5.50 15.00 59.76 6.87 2.578 2.65
24 55.00 240.00 5.50 30.00 79.03 8.84 1.016 3.05
25 55.00 180.00 4.50 22.50 78.86 9.08 1.606 2.5
26 40.00 120.00 4.50 15.00 57.57 6.62 1.175 1.8
27 55.00 120.00 5.50 22.50 68.35 7.86 1.9531 2.63
28 70.00 120.00 6.50 15.00 66.24 7.61 1.61 3.4
29 70.00 240.00 4.50 15.00 76.93 8.84 1.053 3.7
30 40.00 180.00 6.50 15.00 66.24 7.61 0.628 2.15

Table 3.1 presents the main experimental result similar amount % malt extract of 83.33 % and
for mashing process. In this table run number 7 sugar content of 9.57oBx, but different values for
and 11 were conducted at different time and protein and viscosity.
temperature combination, and there were give
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 Zinabu Hailu, Basha Mekonnen, IJFNR, 2019; 3:33
This is due to diastatic and proteyoltic enzymes was 70°C. This higher temperature indicated the
are work optimally at different conditions. thermos-stability of malt enzymes and working
Diastatic enzymes are used to break down / at greater than 50°C without deactivating. As
hydrolysis starch material into simple sugar at a shown in figure 3.1 (a) after 120 min the activity
temperature range of 45 -70 oC .This means that of enzymes was decreased drastically and
diastatic enzymes are not functional below or enzyme was completely inactivated when
above the given range. Similarly proteyoltic heated further of 70°C. Thus, the results
enzymes are used to breakdown proteins and concluded that the malt enzyme is moderately
works optimally below temperature of 40 oC. temperature stable. The % malt extract is one of
the most important quality parameter. It
It was observed (Figure 3.1 (a)) that, the enzyme
measures the soluble materials from the malt
exhibited its maximum activity at 120 min of
when hydrolysis enzymes in the malt acted
reaction time. [8] Reported that enzyme
optimally.
obtained from Bacillus sp. AB68 was active in a
broad temperature range between 20 and 90°C, A pH ranges from 4.5-6.5 was used to study the
with an optimum of 50°C. But in case of malt effect of pH on diastatic enzyme activity (Figure
enzymes the optimum temperature observed 3.1 (b)) and optimum pH was found at 4.5.

Figure 3.1 (a-c): 3-D % malt extract graphs for two varying parameters

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 Zinabu Hailu, Basha Mekonnen, IJFNR, 2019; 3:33
Higher enzyme activity was observed in the with pH and temperature with S.S.A. interaction
acidic pH range, but when the pH range parameters, we can observe that % malt extract
proceeds to alkaline, the activity was lower. The was minimum. This is due to, during enzyme
effect of temperature on enzyme activity was substrate reaction, the initial reaction velocity
assayed at different temperatures ranging from gradually increases with increasing both
40-70°Cat optimum pH, time and S.S.C. The substrate concentration and temperature.
results showed that enzyme activity was Increasing temperature increases the kenetic
increased with temperature and it showed energy that molecules possess.
highest enzymes activity at temperature 70°C Fig. 3.2 (a) describe the combined effect of
(Figure 3.1 (a & b)). At this temperature the temperature and time on the fermentable sugar
highest % malt extract value of 92.36 % was content, with pH and S.S.C. held constant at its
observed. Above 70°C temperature enzyme lower and upper value. The color key on the
activity was also decreased. For determination green, yellow and red surface becomes “hot‟ at
of optimum reaction time, enzyme assay was higher response levels, this means that when the
carried out at different reaction time ranging from interaction factors (temperature and time)
120 –240 minutes at constant temperature pH increase the sugar content also increase to a
and S.S.C. The relation between temperature higher value and the maximum fermentable
with respect to substrate source is presented in sugar was observed at the right edge of x-axis
figure 3.1 (c). The maximum response value is with the value 10.53.
observed at maximal of temperature and
maximal of S.S.A. At maximal of the temperature

Figure 3.2 (a-b): 3-D plot of sugar content graphs for two varying parameters

This is due to the starch hydrolyzing enzymes increasing the red color key. It showed that the
was worked at the optimum value of 70 oC and maximum value was recorded at 70 oC, and 4.5,
time of 120 min. Fig.3.2 (b) shows the temperature and pH respectively. In the other
interaction effect of temperature and pH hand the sugar content was decreased as the
keeping the actual factors constant at (pH, 4.5 pH increased, but is observed increase as
and S.S.A., 30 gm). In this case this fig. temperature increase.
presented a plot of sugar content as a function 3.2 Effect of Fermentation Parameters on
of temperature and pH. As clearly shown the distilled beverage quality
fermentable sugar content was increase as
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 Zinabu Hailu, Basha Mekonnen, IJFNR, 2019; 3:33
Since the research work is originated from the (Yeast strain) as shown in table 3.2. In order to
Ethiopian indigenous ethanol (“Areki”) optimize the distillery beer in the fermentation
production process, the optimized wort (i.e wort with
Technology, the fermentation process was done maximum % extract and fermentable sugar
without the presence of any external bio-catalyst content) was taken from the previous mashing
experiment.

Table 3.2: Result of degree of attenuation for distillery beer in fermentation process

Run Temperatur eTime PH S.S.S OG FG AAL (%)

o (Hr) (gm)
( C)
1 40.00 96.00 4.00 75 1.0546 1.028 49
2 30.00 96.00 4.40 75 1.055 1.0175 68
3 40.00 72.00 4.40 75 1.0557 1.0218 60
4 20.00 96.00 4.80 75 1.055 1.008 86
5 30.00 96.00 4.40 50 1.0549 1.0185 66
6 30.00 72.00 4.40 75 1.055 1.016 71
7 20.00 48.00 4.80 100 1.054 1.016 70
8 30.00 72.00 3.60 50 1.055 1.0178 67
9 40.00 96.00 4.00 100 1.055 1.025 54
10 20.00 48.00 4.80 75 1.0548 1.015 73
11 20.00 48.00 4.00 50 1..055 1.0155 72
12 30.00 72.00 4.40 100 1.0551 1.0186 66
13 40.00 72.00 4.80 100 1.055 1.0258 52
14 20.00 72.00 4.00 100 1.0545 1.014 74
15 30.00 48.00 4.40 50 1.055 1.021 62
16 40.00 48.00 4.80 50 1.0551 1.0268 51
17 20.00 72.00 4.00 50 1.055 1.013 76
18 30.00 48.00 5.20 100 1.055 1.020 64
19 40.00 48.00 4.80 100 1.055 1.0248 55
20 20.00 72.00 4.80 75 1.0548 1.012 78
21 40.00 96.00 4.80 50 1.0549 1.026 53
22 30.00 96.00 4.40 100 1.0556 1.0195 64
23 30.00 48.00 4.40 75 1.0548 1.0175 68
24 40.00 72.00 4.40 75 1.0551 1.022 60
25 20.00 96.00 4.00 100 1.055 1.0115 79
26 40.00 48.00 4.00 50 1.055 1.024 56
27 20.00 96.00 4.40 50 1.0545 1.010 82
28 20.00 96.00 4.40 75 1.055 1.0085 85
29 40.00 72.00 4.00 50 1.0551 1.0245 56
30 30.00 48.00 4.40 75 1.055 1.018 67

With Saccharomyces it has long been known enzymes are affected by the growth
that the rate of alcohol production increases temperature [5].
with temperature up to 40 oC, [3]. [6] and [4] The optimized mash bill from the mashing
showed an increase in ethyl acetate production experiment was used as fermentation feed in
with increases in incubation temperature. There order to optimize the distillery beer by controlling
are many reports that the syntheses of four factors as described in section 2.5. The
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 Zinabu Hailu, Basha Mekonnen, IJFNR, 2019; 3:33
interaction and single effect of controlling used as a catalyst for the fermentation process
variables on degree of attenuation is presented was very high at 20 oC, and lower at higher
in fig. 3.2 (a-c).
temperature of 40 oC. This result also indicated
The maximal AAL, which is 86 % was observed
that the unknown yeast strain was
at minimum fermentation temperature and
breakdown/convert much of the fermentable
maximum fermentation time. The metabolic
activity of the unspecific yeast strain which, is sugar to alcohol and carbon dioxide at 20 oC.

Figure 3.2 (a-c):3-D plot of effect of time, temperature, pH and S.S.A. on AAL (%)

This indicated that the rate of fermentable sugar response levels, yellow in the ‟70‟s and red
consumption by unspecified yeast and the above 80 for AAL. The effect of single factor
production of ethanol (EtOH) and carbon (temperature and time) can also discussed from
dioxide (CO2) increased as the fermentation fig.3.2 (a). The degree of attenuation (AAL) was
temperature decreased and fermentation time reducing as increase of fermentation
increased. As indicated by the color key in figs. temperature and increase as the fermentation
3.2(a), the surface becomes ‘’hot‟ at higher time increase. This indicated that the
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 Zinabu Hailu, Basha Mekonnen, IJFNR, 2019; 3:33
fermentation yeast strain (unspecified) was respectively.
more active at minimal temperature and Acknowledgement
maximal time. The indigenous ethanol (“areki”)
We would like to address our special thanks to
production technology is operating at
those persons who were participated in all
atmospheric (average 23.5 oC) temperature and aspects of the research work. Next, we also
at maximum fermentation time (four day). Fig. would like to forward our great gratitude to
3.2(b) described the impact of temperature and University of Adigrat for its fund that was
pH on fermentability. The interaction effect of provided during the research work. We
temperature and pH on degree of attenuation is appreciate the University for the continuous
similar with the effect of temperature and time Support and giving great attention in research
which is presented in this figure. As observed in and community servicing aspect in all collages.
this figure 3.2(b) the AAL become increases as Conflict interest
the pH increase and the temperature decreases.
There is no conflict interest among the authors
The third interaction which is presented in fig.
3.2 (c) is the effect temperature and SSC on Data Availability statement
AAL. The fermentability recorded at the No data used to support the findings of this
interaction effect of temperature and SSC was study.
increased as both temperature and SSC References
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