Journal of Food Engineering 289 (2021) 110182

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Journal of Food Engineering

journal homepage: http://www.elsevier.com/locate/jfoodeng

Development and optimization of beer containing malted and non-malted

substitutes using quality by design (QbD) approach
~o a, Joa
Simone Tessarini Esteva ~o Batista de Almeida e Silva b, Felipe Rebello Lourenço c, *
a
Departamento de Química Fundamental, Instituto de Química, Universidade de S~ ao Paulo, Av. Prof. Lineu Prestes, 748 – Bloco 1, CEP: 05508-000, S~ ao Paulo, SP,
Brazil
b
Departamento de Biotecnologia. Escola de Engenharia de Lorena, Universidade de S~ ao Paulo, Estrada Municipal do Campinho, s/n – Planta Piloto de Bebidas, CEP:
12602-810, Lorena, SP, Brazil
c
Departamento de Farm�acia, Faculdade de Ci^encias Farmac^euticas, Universidade de S~
ao Paulo, Av. Prof. Lineu Prestes, 580 – Bloco 15, CEP: 05508-000, S~
ao Paulo, SP,
Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Quality by Design (QbD) approach has been widely used in the pharmaceutical field, but its application in the
Beer development of food products is recent. The objective of this work was to apply QbD in the development of Ale-
Barley malt substitute type beer, with up to 45% of barley malt substitutes, with alcohol content of 5.50 � 0.55% v/v and a good
Mixture design
sensory evaluation. A simplex-centroid mixture design was used, in which five cereals were tested: maize, oat
Quality by design
malt, rice, rye malt, and sorghum. In the screening design, rice, maize, and oat malt were selected. The opti­
mization design allowed the definition of the design space to meet critical quality attributes. The standard (100%
barley malt) and optimized (55% barley malt, 42% oat malt, and 3% maize) formulations had similar physi­
cochemical, fermentative, and sensory parameters. The implementation of QbD helps improve quality and reduce
quality deviations.

1. Introduction (QTPP), which identifies the critical quality attributes (CQAs) of a

Quality by Design (QbD) approach is more than 25 years old and is
relatively well established in industries whose products must comply
with quality, safety, and performance or efficacy requirements. The
notion of quality as a science was born in the 1980s and 1990s with the
work of Juran and Deming and has a relationship with the
manufacturing processes created by two major global companies:
Motorola’s Six Sigma and Toyota Lean Manufacturing (Menezes; Flo ^res;
Gouveia, 2014).
In the pharmaceutical industry, the notions of QbD first appeared in
the 21st century and in documents from the Food and Drug Adminis­
tration (FDA) from the International Council for The Harmonisation of
Technical Requirements for product registration Pharmacists for Human
Use (ICH), which provides recommendations and guidelines on the
subject (Food and Drug Administration, 2009). In the food area, both in
industries and research, these methodologies are not yet well
disseminated.
Because it is a systematic scientific-based approach, according to Yu
(2014), QbD includes some elements: a quality target product profile
product; product design and understanding, including identification of
critical material attributes (CMAs); process design and understanding,
including identifying critical process parameters (CPPs), critical mate­
rial attributes, and critical process parameters of critical quality attri­
butes; a control strategy that includes specifications for product
components and controls for each step of the manufacturing process;
process capability; and continuous improvement. QbD tools and studies
include prior knowledge, risk assessment, mechanistic models, data
analysis, process analytical technology (PAT), and design of experiments
(DoE).
Traditionally, the development and optimization of food products
were carried out by analyzing one factor at a time (univariate approach)
(Politis, 2017; Candioti et al., 2014). One of the factors is changed
within an appropriate range or level, while the others are kept constant.
In addition to requiring a large number of experiments, the univariate
approach cannot evaluate the existence of interaction between factors,
which can lead to inadequate development and optimization (Politis,
2017). DoE is a tool in which experiments are planned according to a
pre-established matrix, interactions between factors are evaluated, and

* Corresponding author.
E-mail address: feliperl@usp.br (F.R. Lourenço).

https://doi.org/10.1016/j.jfoodeng.2020.110182
Received 7 January 2020; Received in revised form 25 April 2020; Accepted 1 June 2020
Available online 8 June 2020
0260-8774/© 2020 Elsevier Ltd. All rights reserved.
S.T. Estev~
ao et al.
the entire set of experiments can provide more information using a
smaller number of experiments compared to the univariate approach
(Leng et al., 2017).
DoE is designed from a structured and organized method to deter­
mine the relationships between input factors (xi - independent variables)
that affect one or more output responses (y - dependent variables), by
establishing mathematical models (y ¼ f (xi)). In the DoE approach, the
input factors studied are systematically varied to determine their effects
on output responses, which allows determination of the most important
input factors, identification of the configuration of input factors leading
to optimized output responses, and the elucidation of interactions be­
tween input factors. To provide better understanding, experimental
projects can be divided into two types: screening designs and optimi­
zation designs (Fukuda et al., 2018; Politis, 2017; Candioti et al., 2014).
To design and develop a robust food product that has the desired
QTPP, development should consider the physical, chemical, biological,
and sensory properties of food. The experimental design can be con­
structed through factorial designs or, depending on the type of food and
the objectives established, through a mixture design.
When mixed variables are involved in an optimization, the result
depends on the proportion of these components, and their levels cannot
be varied without taking into account the other components. Thus,
mixtures are systems whose properties depend on the relative pro­
portions of their components and not their concentrations. There are no
restrictions on physical state, so components can be solid, liquid, or
gaseous, provided that system properties are defined by their proportion
in the mixture. Thus, the amount of each component of the system
should be treated as a variable (mixing variable), which is not inde­
pendent of the others, because the sum of the proportions of its com­
ponents must be constant (Cornell, 1990).
Thus, mixture design consists of adjusting a polynomial mathemat­
ical model to a response surface obtained according to a specific
experimental design, known as mixture design. This name is used to
differentiate it from the factorial design employed in modeling process
variables. The mixture design to be employed will depend on the
complexity of the mathematical model that is desired to adjust and the
number of components of the mixture (Coscione et al. 2005).
Simplex-centroid design is a type of mixture design which employs
binary and tertiary mixture of the components. The number of points of
this project depends on the number of components of the mixture
(Nunes, 1998). Although the simplex-centroid design is a simple appli­
cation, it cannot test for lack of fit of the model (Cornell, 1973). This
issue can be solved by including replicas of the central point (Cornell,
1990).
According to Yu (2014), mixture designs fulfilling the role of DoE can
help identify ideal conditions, CMAs, CPPs, and finally design space.
Thus, the main objectives of applying the QbD approach in the food area
are to develop a robust product that can deliver the target quality profile
of the desired product during its lifetime, increase the capacity of the
process, reduce the variability of the food manufacturing process, and
incorporate quality into the product. In this work, QbD was applied to
develop beer formulations produced with malted and non-malted
cereals.
Beer is the drink resulting from fermentation, by the brewing yeast,
of a malt barley or malt extract previously submitted to an additional
cooking process of hops or hop extract. Part of malted barley or malt
extract may be replaced by brewer substitute (Brasil, 2019). In Brazil,
the use of substitutes may not exceed forty-five percent of the primitive
extract (Brasil, 2019; Brasil, 2009; Brasil, 2001). Barley malt substitutes
are considered other cereals suitable for human consumption, malt or
not, as well as starches and sugars.
With the premise of scientifically comparing beer produced with
barley malt and its substitutes, the objective of this work was to apply
the QbD methodology to beer production. Mixture design of experi­
ments were adopted aiming to observe the effects of the presence of non-
malted and malted cereal substitutes on physicochemical and sensory
2
Journal of Food Engineering 289 (2021) 110182
characteristics of beers. QbD approached allowed definition of an opti­
mized formulation that complies with the parameters established in the
QTPP.
2. Material and methods
To apply Quality by design (QbD) in the development of beer, the
quality target product profile (QTPP) was established. The critical
quality attributes (CQAs), the critical materials attributes (CMAs), and
the critical parameters of the process (CPPs) were identified. In addition,
screening and optimization designs of experiments (DoE) were
performed.
A simplex-centroid mixture design was adopted. The varied and
studied components were the proportions of the brewers: maize, oat
malt, rice, rye malt, and sorghum. Mixture design occurred in two steps:
the screening design (SD) and optimization design (OD).
2.1. Screening design
The control consisted of a formulation with 100% barley malt with
four replicas: F0I, F0II, F0III, and F0IV. In the other formulations, barley
malt was fixed at a concentration of 55% and the substitutes were varied
in proportions between zero and 45%. The concentrations of hops and
yeasts were fixed in all formulations.
The central point formulation (F1) was composed a mixture of barley
malt (55%), sorghum (9%), rye malt (9%), maize (9%), rice (9%), or oat
malt (9%). At the vertex points, 45% of the barley malt was substitute by
sorghum (F2), rye malt (F3), maize (F4), rice (F5), or oat malt (F6).
2.2. Optimization design
The three barley malt substitutes selected in SD were submitted to
the optimization design (OD) in which a new mixture design was per­
formed. The formulations of the vertex points of the selected substitutes
had been performed in the SD (F4, F5, and F6). The double mixture
formulations (F7, F8, and F9) were manufactured with two substitutes
ranging in a ratio of 22.5%. The formulations of axial points (F10, F11,
and F12) were composed of one of the substitutes in the proportion of
30% and the others at 7.5%. A central point (F13) was also held in which
each substitute was used together in the proportion of 15%, which was
manufactured in four replicas (F13I, F13II, F13III, and F13IV). Fig. 1 il­
lustrates the distribution of each formulation in the study space.
2.3. Manufacturing of formulations
The manufacturing of each formulation was performed according to
the following steps: mashing (barley malt is added to water and starch is
saccharified in smaller sugars by the enzymes present in malt); boiling
for 1 h (addition of hops in the brewer must for the development of
bitterness and aromas); inoculation of the yeast Saccharomyces cerevisiae
US-05 (Fermentis, France); fermentation at 20 � 1 � C for six days;
maturation at 2 � 1 � C for thirty days; carbonation at 20 � 1 � C for seven
days; and filling steps. The formulations were not filtered.
The barley malt substitutes generally entered production along with
barley malt. Rice and sorghum, which are difficult to saccharification,
underwent a pre-preparation stage, which required cooking each cereal
soaked in mineral water (1:4) until the absorption of all water. In the
case of sorghum, after cooking, the mixture was cooled to between 60
and 70 � C, the enzyme thermostable alpha-amylase Brautec-α (Prozyn
Biosolution, Brazil) was added to 0.3%, and the mixture kept at this
temperature range for about an hour. Maize syrup did not require any
pre-preparation and was added to the formulation during boiling step.
Beer formulations were manufactured at the Beverage Pilot Plant of
the Escola de Engenharia de Lorena at the Universidade de Sa ~o Paulo
(EEL-USP) and in the Faculdade de Ci^ encias Farmac^euticas at the Uni­
versidade de Sa ~o Paulo (FCF-USP) according to Good Manufacturing
S.T. Estev~
ao et al.
Fig. 1. Mixture design of experiments that defines the proportions of maize,
rice, and oat malt as barley malt substitutes in formulations F4 to F13.
Practices (GMP) for food described in RDC 216 (Brasil, 2004) and in
random order to obtain statistical independence from errors.
2.4. Physicochemical analyses
The analyzed responses were alcohol content (% v/v), density (g/
cm3), original extract (% w/w), real extract (% w/w), apparent extract
(% w/w), calories (kcal/100 mL), real degree of fermentation (%, RDF
¼ real extract – original extract/original extract), and apparent degree
of fermentation (%, ADF ¼ apparent extract – original extract/original
extract), in triplicate, were obtained using a beer analyzer equipment
(Alex 500 model, Anton Paar, Brazil). Based on the values of original,
real, and apparent extracts, and according to Medeiros et al. (2018), real
and apparent degrees of attenuation were calculated. Color analyses,
according to EBC (2005), and pH analyses were also performed, in
triplicate, for all formulations. Additionally, the efficiency of the
mashing step process (%) was calculated for the formulations of the SD,
based on the masses of components used and the final amount of ob­
tained for each formulation.
2.5. Sensory analysis
Sensory analysis was based on affective tests, using a hedonic scale of
9 points, adapted according to the Likert scale (Likert, 1932), in which
the tasters (untrained, but consume beer products regularly) were able
to choose the option that best expressed their opinion about the samples
for the attributes: color, aroma, and flavor. The scale had at its extremes
the terms “I really liked” (9) and “I really disliked” (1). The results of the
sensory analysis were calculated in a total sensory score, the possible
interval to be obtained in each formulation is 3–27 points. The sensory
analysis was performed by 109 tasters with a minimum age of 18 years,
who provided a grade from 1 to 9 for color, aroma, and flavor for each
tasted formulation. The sensory responses of each formulation were
compared using a repeated ANOVA measures. Sensory evaluations were
approved by the Research Ethics Committee of the Faculdade de Ci^ en­
cias Farmac^ euticas da Universidade de Sa ~o Paulo.
3
Journal of Food Engineering 289 (2021) 110182
2.6. Fermentative and kinetic parameters and sensory analysis used to
characterize the optimized formulation and compare with control
formulation
Inferences about the proportions of the barley malt substitutes were
addressed by the regression model. Response surface analyses were
performed to obtain the optimized formulation (F14). Optimized (F14)
and control (F0) formulations were submitted to the same character­
ization analyses performed for the beers of the OD. In addition,
fermentative and kinetic parameters were calculated, such as extract
masses, the conversion factor of extract into alcohol, the real and
apparent degrees of attenuation, as well as the efficiency of the con­
version of extract into alcohol. The formulations were also submitted to
a 5-point hedonic scale purchase intention test, taking in their extremes
the terms “certainly buy” (5) and “certainly wouldn’t buy” (1). The
purchase intention test was performed by 109 tasters with a minimum
age of 18 years old.
2.7. Statistical analyses
The results of mixture design were subject to analysis of variance
(ANOVA) and regression analysis. The results of characterization ana­
lyses and sensory analysis were analyzed by the ANOVA tests and Tukey
tests, at 5% significance. All statistical analyses were performed using
Minitab™ 18 software (Minitab Inc., USA). Principal component anal­
ysis (PCA) was also performed using Minitab™ 18. The charts and tables
were prepared in Microsoft Office Excel 2010), Microsoft Office Word
2010, Minitab™ 18, and GraphPad Prisma 6.
3. Results and discussion
Implementation of QbD approach was initiated by defining the
quality target product profile (QTPP). This definition contributes to the
development of the product design and concept. The QTPP defined was
an Ale beer, with up to 45% of barley malt substitutes, with an alcohol
content of 5.50 � 0.55% v/v and well evaluated in sensory analysis.
Sensory analysis was measured by means of the hedonic scale, in which
a well-evaluated beer received, at least 6 out of 9 possible points (which
means “liked slightly”) for each of the following attributes: color, aroma,
and flavor (totaling, at least, 18 points from a total of 27 possible points).
The values of alcohol content and sensory analysis that comprise the
QTPP were defined a priori, before manufacturing the beer formulations.
The reactions that lead to the development of color also contribute to
the formation of aromas and flavors. The pH of the must is related to the
transformation of hops during boiling, affecting the extraction of alpha
acids (Olec, 2019) and consequently the production of iso-α-acids,
which is related to the bitterness of the final product, a parameter that
can contribute to acceptance or rejection of the taste. For these reasons,
color and pH were defined as critical quality attributes (CQAs) directly
related to the sensory part of the QTPP. Thus, the defined CQAs were the
results of sensory analysis, alcohol content, color (measured according
to EBC), and pH analyses.
The critical materials attributes (CMAs) were the proportions of
barley malt and its substitutes in the final products. The optimized
proportions of barley and its substitutes was defined using mixture
design of experiment. Moreover, time and temperature of mashing,
fermentation, maturation, and carbonation stages are potentially critical
process parameters (CPPs). Because these parameters were kept con­
stant for all formulations, we assumed that no CCPs were identified and
studied in the work.
The efficiency of the mashing process in the screening design (SD)
was not defined as a critical attribute but as a performance parameter,
which was evaluated because it was related to the proper control of
CPPs.
S.T. Estev~
ao et al. Journal of Food Engineering 289 (2021) 110182

3.1. Screening design

Product development according to QbD approach, adopting the

screening design (SD), provided the responses presented in Table 1.
The efficiency of the mashing process was determinant for the choice
of the first barley malt substitute selected in the screening step. The
calculation of the efficiency of the mashing process considered the pre-
preparation stage of rice and sorghum, which justifies that the formu­
lations containing these barley malt substitutes (F5 and F2, respectively)
were the least efficient. On the other hand, the formulations containing
maize as barley malt substitute (F4 and F1) presented the greatest effi­
ciency, since the addition of maize is in the form of syrup during the
boiling stage of the manufacturing process, dispensing with the need for
saccharification. Thus, maize was the first substitute selected for the
optimization design.
The results of the sensory analysis in Table 1, in the form of a total
sensory note, indicate that F6 was the formulation that presented the
best sensory evaluation, and for this reason, oat malt was the second
barley malt substitute selected in the SD.
The selection of the third substitute was through the principal
component analysis (PCA), in which the various variables are trans­
formed into a few new components that are able to explain much of the
variability, according to Fig. 2. The PCA can explain, in total, 89.0% of
variability with only two components (67.2% explained by PC1 and
21.8% by PC2).
The formulations at the right side of the chart (CP1 < 0) have higher
alcohol content, have greater real and apparent degrees of fermentation,
are less dense, and have lower apparent extract in relation to the for­
mulations at the left side (CP1 > 0). Thus, PC1 was strongly related to
the fermentation process. The formulations at the lower portion of the
chart (CP2 < 0) have lighter color (EBC) and higher values than the real
and original extracts, besides being more caloric than the formulations
at the upper portion (CP2 > 0). Thus, PC2 was strongly related to the
composition of the formulations.
The biplot chart obtained from PCA indicates the formulation with
45% rice (F5) is the one that appears closest to the formulation with 9%
of each substitute tested in screening (F1). In other words, F5 presented
similar characteristics to F1, which demonstrates that rice was the
determining substitute for the characteristics of the mix. Thus, it was
determined that rice would be the third cereal selected in the SD to
compose as a substitute the OD formulations.
The results of all analyses of the SD formulations allowed the choice
of the three barley malt substitutes that best met the established QTPP,
namely: maize, rice, and oat malt. These substitutes were adopted in
optimization design, considering the high efficiency of the mashing
process, the sensory analysis results, and the similar characteristics of
the formulation as the control formulation (100% barley malt formu­
lation F0).
Fig. 2. Principal component analysis for the screening design (SD) for the
physicochemical characteristics (color, pH, density, alcohol content, calories,
AE – apparent extract, RE – real extract, OE – original extract, ADF – apparent
degree of fermentation, and RDF – real degree of fermentation) of formulations
F1 to F6 (n ¼ 3 for each formulation). (For interpretation of the references to
color in this figure legend, the reader is referred to the Web version of
this article.)
3.2. Optimization design
The formulations of the optimization design (OD) were manufac­
tured and the CQAs analyzed. According to Rosa and Afonso (2015), the
pH in the brewing process acts directly in the regulation of enzymatic
activity. The enzymes involved in the saccharification of the must,
especially amylases, can act at different levels of pH, but the optimal pH
range for the activity of these enzymes is between 5.2 and 5.5 (Gon­
çalves, 2006). The pH is also related, also according to Rosa and Afonso
(2015), with the solubilization of astringent components, with color
variation and coagulation of the protein components of the must. The pH
analyses results are in Fig. 3.
Several statistically significant differences can be observed between
the final pH of the formulations. The definition of a pH range intended
for the final product of each formulation was not one of the aspects of
quality target product profile because, according to Briggs et al. (2004),
the fermentation process tends to establish an adequate pH, as it usually
falls with the secretion of carbonic acid.
Brazilian legislation does not define an ideal pH range for ready-
made beer, but according to Lindstrom and Hansen (2014), beer usu­
ally has pH between 4.1 and 4.5. According to Rosa and Afonso (2015),
the pH of beer should be around 4.0, and the pH of 4.5 is considered very
high, which increases the biological susceptibility of the product. Thus,
the pH presented in the formulations of the optimization design (be­
tween 4.05 and 4.16) were adequate.
The results of the alcohol content allowed the elaboration of the
graphs presented in Fig. 4.
The formulations F8 and F10 presented a statistically significant
difference in alcohol content. Since F8 and F10 have identical

Table 1
Physicochemical responses, efficiency values of the mashing and sensory analysis results (n ¼ 3) for the formulations of the screening design (SD).
Variables F0I F1 F2 F3 F4 F5 F6

Alcohol (% v/v) 5.72 6.06 5.91 5.11 6.14 6.04 5.20

Density (g/cm3) 1.0061 1.0068 1.0047 1.0091 1.0055 1.0070 1.0083
Real Extract (% w/w) 4.1 4.4 3.8 4.6 4.1 4.5 4.5
Apparent Extract (% w/w) 2.0 2.3 1.7 2.8 1.9 2.3 2.6
Original Extract (% w/w) 12.7 13.5 12.8 12.4 13.4 13.5 12.3
Calories (Kcal) 46.0 49.0 46.0 44.6 48.3 49.0 44.5
RDF (%) 69.5 69.3 71.7 63.7 71.1 69.0 65.3
ADF (%) 84.2 83.9 86.9 77.1 86.1 83.4 79.0
pH 4.21 4.30 4.04 3.99 3.84 4.07 4.30
Color (EBC) 6.1 4.5 5.4 6.6 3.4 3.1 5.6
Efficiency (%) 74.54 80.09 63.98 75.54 89.36 72.04 78.53
Total Sensory 18.43 17.43 18.59 20.00 19.00 20.29 21.71

Legend: F0I: control; F1: mix; F2: 45% sorghum; F3: 45% rye malt; F4: 45% maize; F5: 45% rice; and F6: 45% oat malt. RDF ¼ real degree of fermentation and ADF ¼
apparent degree of fermentation.

4
S.T. Estev~
ao et al.
Fig. 3. Results of pH values (n ¼ 3) for the formulations F0 and F7 to F13
manufactured as part of the optimization design (OD).
compositions, the difference in alcohol content could be due to the
random variability of the temperature during the fermentation process.
According to the mixture contour chart, formulations with higher pro­
portions of maize in their composition contained higher alcohol content,
while those with higher concentrations of oat malt were those with the
alcohol content close to the intended target values (5.50 � 0.55% v/v).
The color analyses results (EBC) of the formulations are illustrated in
Fig. 5.
The control formulation (F0) had with the highest level of color in
Fig. 4. Results of alcohol content (n ¼ 3) and response surface counter plot for the formulations F0 and F7 to F13 manufactured as part of the optimization
design (OD).
Fig. 5. Results of color (EBC) (n ¼ 3) and response surface counter plot for the formulations F0 and F7 to F13 manufactured as part of the optimization design (OD).
5
Journal of Food Engineering 289 (2021) 110182
the EBC scale. This may be due to the use of only malted cereal, since the
malt color is mainly affected by toasting (Maillard reactions between
sugars and amino acids) and caramelization (sugar decomposition)
during malting process. The oat malt was the only one of the three
substitute that malted and it also contributed to an increase in color level
on the EBC scale in the formulations in which it was used in greater
proportion, such as in F8 and F12; the only formulation composed only
of non-malted substitutes, F7, was the one with the lightest color.
The F8 and F9 formulations that were manufactured with 22.5% oat
malt are statistically different, because the development of color does
not depend solely on the malting of ingredients. F8 was also composed of
22.5% maize and F9 by 22.5% rice. The difference in the development of
the color of both occurs by the interactions between the different sub­
stitutes, since the types of sugars formed during the saccharification of
the must is one of the factors that influence the Maillard reaction that
occurs during the boiling phase. Another factor that can influence the
Maillard reaction is the pH of the must. The higher pH during
manufacturing process, the greater the color of the formulation (Nas­
cimento et al., 2015). However, this interactive effect between the two
CQAs (pH and color) was not relevant when the pH values were within
the specifications for all tested formulations. Thus, the color of the inal
product was mainly affected by the amount of barley malt and its
substitutes.
Sensory analyses were also performed in the OD. These results are
showed in Fig. 6.
There is no statistically significant difference between formulations
(p-value > 0.05), but the presence of maize in the composition in the
formulations was preferred by the tasters. F10, with 30% maize,
S.T. Estev~
ao et al. Journal of Food Engineering 289 (2021) 110182

Fig. 6. Results of sensorial analysis (n ¼ 3) and response surface counter plot for the formulations F0 and F7 to F13 manufactured as part of the optimization
design (OD).

received the best evaluation, and F9, without maize in its composition,
had the worst. The good evaluation in the SD formulation with only oat
malt as barley malt substitute (F6) did not remain in the results of the
OD. The lack of statistical significance may be explained due to high
variability of sensory responses among the tasters, particularly for
screening and optimization designs, which required the tasters to test a
large number of formulations (up to 13 formulations). Despite the lack
of statistical significance, several formulations provided sensory re­
sponses above the minimal value defined in QTPP (minimal of 18 points
out of 27 possible points).
The mixture contour plot for the sensory response confirms that
maize formulations were better evaluated by tasters, as well as formu­
lations with higher proportions of rice, the two non-malted substitutes
used in OD.
Each mixture contour plot contributed suggestions. (a) The mixture
contour plot for alcohol content (Fig. 4) suggests that for the optimized
formulation to achieve the alcohol content close to the intended, which
is 5.50 � 0.55% v/v, it should be manufactured with a high proportion
of oat malt and with low proportions of the other substitutes. (b) The
mixture contour plot for color (Fig. 5) suggests the use of malt substitute
for a greater level of color on the EBC scale and use of non-malted to
lower level. This information is relevant to demonstrate that color Fig. 7. Overlapped counter plot obtained for the surface count plot of alcohol
attribute can be measured an related as part of the QTPP. (c) The content (blue lines), color (red lines), and sensory analysis (green lines) that
mixture contour plot for sensory analysis (Fig. 6) suggests that for an define the design space region (white region) and optimized formulation (F14).
(For interpretation of the references to color in this figure legend, the reader is
optimal sensory score (of 24 total points or more, including at least 6
referred to the Web version of this article.)
points in each category – color, aroma, and flavor), the optimized
formulation should contain higher concentrations of non-malted
substitutes. content (according to the contour plot of Fig. 4) resulted in a design
All the results obtained with the manufacture of the SD and OD space (white region) presented in Fig. 7. Formulations adopted within
allowed the construction of the overlapped contour plot (Fig. 7) for the the design space region ensure the requirements defined in QTPP.
development of the final optimized formulation (F14), in which the Alternatively, optimized formulation can be defined using a mathe­
results of the alcoholic content, color, and sensory analysis are simul­ matical method, based on desirability functions. The combined desir­
taneously optimized. The pH determination was not considered during ability value (D) was calculated by the square root of the product of the
the optimization step because all tested formulations provided pH values individual desirability values of alcohol content (dAlcohol) and sensory
within the specifications defined in QTPP. The optimization parameters analysis (dSensorial). D value ranges from 0 to 1, and the closer to 1, the
consider alcohol content values between 5 and 6% v/v (as defined in the better the results complies with the CQAs. If one of the parameters do
QTPP), color on the EBC scale of 6 (range between 5 and 7), and sensory not complies with CQAs requirements (d ¼ 0), then D ¼ 0, that is, this
analysis with a total evaluation greater than 18 (as defined in the combination is outside the design space region. The combined desir­
QQTP). Although the color was not considered in the QTPP, the color ability value (D) for the optimized formulation was found to be 0.55,
range around 6 was adopted from the mean results obtained with the OD which indicates that all CQAs were simultaneously achieved.
formulations. Although the design space region considers the use of rice as barley
Fig. 7 establishes the design space region, which defines the pro­ malt substitute, it was not included in the optimized formulation, since it
portions of barley malt substitutes that the optimized formulation would require the pre-preparation stage, without presenting significant
should have to comply with the CQAs. Although the formulations con­ improvement in compliance with the CQAs.
taining maize and rice are promising for sensory analysis (according to The design space region (Fig. 7) and the combined desirability
the contour plot of Fig. 6), the overlap of this parameter with the alcohol function helped to define the final optimized formulation (F14), which
contains 55% barley malt, 42% oat malt, and 3% maize.

6
S.T. Estev~
ao et al. Journal of Food Engineering 289 (2021) 110182

In addition to the composition of final formulation, the quality and
compositional parameters of raw materials may affect the final charac­
teristics of the products. All raw materials used in this work fulfilled the
quality and compositional parameters defined in literature. However,
these issues were not considered in this work because all formulations
were manufactured using the same raw materials (varying only the
proportions of the raw materials in the final formulation).
3.3. Characterization of optimized formulation and comparison with
control formulation
Yeasts are the agents of alcoholic fermentation, and the substrate is
the extract obtained in the previous stages of the manufacturing process.
This extract is classified as original extract (EO). For growth and
reproduction, yeasts preferably use sugars, to the detriment of other
carbon sources present in the original extract. According to Bento and
Almeida (2006), the formation and concentration of products during
fermentation depend mainly on the metabolic routes that the yeast uses.
The product formed after fermentation is also called extract. If the
alcohol content is considered, this extract is classified as apparent
extract (As). On the other hand, if the measure does not suffer alcohol
influence this extract is called real extract (ER). The fermentation
response variables monitored over time were the alcohol content and the
real extract. The fermentation kinetics of the formulations were
analyzed approximately every 12 h for 5 days, according to Fig. 8. The
initial time showed in the real extract curve refers to the original extract.
The fermentation stage was halted when the results of alcohol content
and real extract were already constants.
In both formulations, the alcohol and real extract contents, which are
inversely proportional, were constants after about 48 h of fermentation.
At the end of the process, the degree of attenuation could be measured.
Attenuation, according to Costa (2017), is the measurement of how
complete the fermentation of the must was, that is, how much of the
sugars present in the extract were consumed during fermentation. Re­
sidual sugars are present in beer as part of the complex set that generates
the characteristic flavor and aroma of this product. The real (ATR) and
apparent (ATA) degrees of attenuation were calculated based on the
results of the original, real, and apparent extracts. These results have the
same meaning as the real (RDF) and apparent (ADF) degrees of
fermentation. The difference lies in the fact that the RDF and ADF are
responses from the beer analyzer of samples only from the end of
fermentation. Despite the greater accuracy that the results of ATR and
ATA offer by analyzing the beginning and end of the fermentation pro­
cess, throughout this experimental project, the RDF was used as the
fermentation evaluation parameter. These and other fermentative pa­
rameters, which could be measured or calculated, are in Table 2.
During fermentation, yeasts grow and reproduce forming a yeast
cream that was separated from beer so that the beer can enter the
maturation process; thus, the volume available for maturation is lower
than that of the beginning of fermentation.
For a laboratory scale process, the efficiency of 93.4% for F0IV and
87.8% for F14 was considered good results. Comparing with values in
the literature, the real degrees of fermentation values of 64.1% (F0IV)
and 63.0% (F14) were higher than those obtained by Andrade (2007),
which was 61.7%, and Curi (2006), which were 59.7% and 62.5%. The
real degrees of attenuation values of 65.6% (F0IV) and 65.7% (F14) were
higher than the 62.7% obtained by Cerri (2012). For the conversion
factor of extract into alcohol, the values of 0.452 g/g (F0IV) and 0.425
g/g (F14) were lower than 0.475 g/g of the laboratory scale formulation
obtained by Castro (2014). These authors also used barley malt sub­
stitutes in their beer formulations. The factors for conversion of extract
into alcohol obtained were also lower than the theoretical factor of
0.4839 g/g (Balling factor). The lower conversion factor of extract into
alcohol in relation to the Balling factor indicates less efficiency in the
fermentation process. Greater control of this parameter could have been

Table 2
Fermentative and kinetic parameters for control (F0IV) and optimized (F14)
formulations (n ¼ 3).
Parameters F0IV F14

di (g/cm3) 1.0500 1.0497

df (g/cm3) 1.0093 1.0094
Vi (mL) 9.000 9.500
Vf (mL) 6.500 6.500
OE(% w/w) 13.50 13.43
RE(% w/w) 4.65 4.61
AE (% w/w) 2.80 2.86
A(% w/w) 4.00 3.75
mEi (g) 1.275.7 1.339.3
mEf (g) 305.1 302.5
YA=E (g/g) 0.452 0.425
RDF (%) 64.1 63.0
ADF (%) 77.5 76.2
ATR (%) 65.6 65.7
ATA (%) 79.3 78.7
EfA (%) 93.4 87.8

Legend: di : initial density, df : final density, Vi : initial volume, Vf : final volume,

Fig. 8. Fermentation kinetics of the control (F0IV) and optimized (F14) for­
mulations (n ¼ 3).
OE: original extract, RE: real extract, AE: apparent extract, A: alcohol content,
mEi : initial mass extract, mEf : final mass extract, YA=E : conversion factor of extract
in alcohol, RDF: real degree of fermentation, ADF: apparent degree of fermen­
tation, ATR : real degree of attenuation, ATA : apparent degree of attenuation, and
EfA : conversion efficiency of extract in alcohol.

7
S.T. Estev~
ao et al. Journal of Food Engineering 289 (2021) 110182

considered one of the CQAs of this experimental project, which would Table 3
allow greater success in obtaining the alcohol content intended in the Physicochemical, sensory analysis and purchase intention test results (n ¼ 3) for
formulations. control (F0IV) and optimized (F14) formulations.
However, according to Fermentis (2017), an 81% apparent attenu­ Variables F0IV F14 p-value
ation (ATA) value is expected after the proper use of US-05 yeasts, which Alcohol (% v/v) 5.93 (0.12) 5.73 (0.12) 0.1108
were used in this study. The values of 79.3% and 78.7% for control and Density (g/cm3) 1.0072 (0.0070) 1.0077 (0.0070) 0.9345
optimized formulations, respectively, were close to that indicated by the pH 4.35 (0.02) 4.33 (0.02) 0.1778
yeast strain manufacturer, which demonstrates that the fermentative Color (EBC) 6.7 (0.2) 4.9 (0.2) 0.0006
Calories (Kcal) 48.6 (1.5) 47.6 (1.5) 0.4741
process was well controlled, and that the final product had an adequate
Sensory
sweetness (residual sugars). Color 6.5 (1.6) 6.2 (1.7) 0.1870
The finalized F0IV and F14 formulations, the results of alcohol con­ Aroma 6.7 (1.7) 6.2 (1.7) 0.0055
tent determinations, density, pH, color, and calories, as well as the re­ Flavor 6.1 (2.2) 5.9 (2.2) 0.3802
sults of sensory analysis and purchase intention test for finished Total 19.4 (4.2) 18.3 (4.4) 0.0604
Purchase intention test 3.3 (1.2) 3.1 (1.3) 0.2491
products are presented in Table 3.
Among the physicochemical determinations, the formulations only Results are expressed as mean values (standard deviation).
presented a statistically significant difference in color analysis. This
difference was because the optimized formulation has 3% of non-malted selection of target customers, and understanding their needs. However,
maize syrup and oat malt are lighter than barley malt. The color vari­ the methodology can also be applied to improve existing products, to
ation of oat malt ranges from 2 to 4 EBC, while the barley malt used in add new manufacturing lines, to reduce cost, and even to reposition the
this experimental design ranges from 4 to 7 EBC. company in the market.
The pH values of both formulations were suitable and according to
expected, which, according to Rosa and Afonso (2015), is around pH 4.0. 4. Conclusion
The optimized formulation exhibited an alcohol content of 5.73 �
0.12% v/v, reaching the desired content in the QTPP. The lack of sta­ The Quality by Design approach was applied, from beginning to end,
tistically significant difference in alcohol between F14 and F0IV (5.93 � for the development of beer in this experimental project. The results
0.12% v/v) was because the optimized formulation contains 97% of indicate that industry and scientific research can promote innovation in
malted cereals (55% barley malt and 42% oat malt), thus the sugars the creation of new food products in a well-structured way using this
formed from the saccharification of starch were similar and fermenta­ methodology, contributing to the improvement of product quality and to
tion also occurred in a similar way. This fact was corroborated by the the reduction of quality deviations.
statistical similarity between the real degrees of fermentation (RDF) of
both formulations (Table 2). Declaration of competing interest
For sensory attributes color and flavor, the control and optimized
formulations did not present averages with statistically significant dif­ The authors declare that they have no known competing financial
ferences (p-value > 0.05). Only the aroma attribute of the control interests or personal relationships that could have appeared to influence
formulation was considered superior to the optimized formulation (p- the work reported in this paper.
value ¼ 0.0055). The value of 18 total sensory grade points for the
optimized formulation was the minimum score defined in the QTPP, CRediT authorship contribution statement
thus the optimized formulation obtained sensorially scores within the
pre-established requirements. There was no statistically significant dif­ Simone Tessarini Esteva ~o: Formal analysis, Investigation, Meth­
ference between control and optimized formulations for the total sen­ odology, Validation, Writing - original draft. Joa
~o Batista de Almeida e
sory values (p-value > 0.05). Silva: Investigation, Methodology, Resources, Supervision, Writing -
For the purchase intention test, the formulations received, on review & editing. Felipe Rebello Lourenço: Conceptualization, Fund­
average, the grade 3, which expressed the opinion “perhaps bought, ing acquisition, Project administration, Resources, Supervision, Writing
maybe did not buy”. These values did not present statistically significant - review & editing.
difference between them (p-value > 0.05).
As the optimized formulation was constructed throughout the
Acknowledgments
experimental project, it was expected that in addition to meeting the
attributes stipulated in the QTPP that the sensory evaluation of the F14
We thank the Programa de Po �s-Graduaça ~o Interunidades em Bio­
would be higher than the final result. Although Brazil is the third largest
tecnologia, Faculdade de Ci^encias Farmac^euticas and the Escola de
producer of beer in the world, the participation of craft beers in the
Engenharia de Lorena da Universidade de Sa ~o Paulo for offering the
Brazilian market is still negligible, with less than 1% of the total (Ayer,
necessary structure for the development of this work. Anton Paar
2017). The formulations produced in this experimental project fall into
gratefully loaned his Alex 500 beer analyzer.
craft beer and even with the increase in the availability of this product
on the market in recent years, the flavors and aromas differentiated from
Appendix A. Supplementary data
this type of beer are only beginning to be appreciated. The Brazilian
brewing market currently employs maize as a widely used substitute
Supplementary data to this article can be found online at https://doi.
(Mardegan et al., 2013) and the tasters demonstrated a tendency to
org/10.1016/j.jfoodeng.2020.110182.
appreciate more what they already know, since the formulations with
predominantly maize in the composition of the OD presented better
results in sensory analysis than the formulations produced with pre­ References
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