International Food Research Journal 31(3): 681 - 695 (June 2024)

Journal homepage: http://www.ifrj.upm.edu.my

Effect of commercial lipase incorporation

on technological properties of bread
1
Santos, A. C., 1*Morais, R. A., 1Martins, G. A. S., 2Carvalho, E. E. N.,
2
Souza, A. R. M., 2Miguel, K. O. R. and 1,2Damiani, C.
1
Department of Food Science and Technology, Federal University of Tocantins, 77001-090, Palmas, Brazil
2
Department of Food Engineering, Federal University of Goiás, 74690-900, Goiânia, Brazil

Article history Abstract

Received: Additives are used to improve bread quality; for this reason, lipase stands out, as it
25 June 2023 simulates the effect of emulsifiers. The present work aimed to verify the effect of
Received in revised form: commercial lipase addition on the physical and technological characteristics of the bread.
21 March 2024 The first stage used 33 factorial design namely three enzymes, five concentrations, and
Accepted: three fermentation times. The second stage used 2 3 factorial design, and stored over 28
1 April 2024 days, with two formulations (control and with enzyme) and three storage temperatures.
The PCA identified the variables with the most significance in PC1: specific volume,
Keywords volume, and variables related to texture and expansion. In the second stage (shelf life), the
additive, PCA revealed that the bread samples with enzymes in the formulation differed from the
enzymes, control samples. Based on the circle of correlations, enzyme samples were distinguished
texture, by lower chewiness, gumminess, and hardness. Microscopic analysis showed that the
microstructure, starch molecules were more uniform in the first 14 days. However, the starch molecules
principal component analysis lost their conformational structure afterwards. Therefore, the incorporation of lipase
improved the bread’s technological parameters such as volume, texture, and structure.

DOI
https://doi.org/10.47836/ifrj.31.3.13 © All Rights Reserved

Introduction the interest of the population, producers, and

researchers. In this sense, many enzymes are suitable
Worldwide, bread is considered essential for for the bakery industry, especially amylases,
human nutrition, providing macronutrients and cellulases, proteases, xylanases, and lipases (Dahiya
micronutrients such as dietary fibres and vitamins et al., 2020; Haghighat‐Kharazi et al., 2020; Both et
(Lockyer and Spiro, 2020). Traditionally, bread is al., 2021; Taddia and Tubio, 2022; Wu et al., 2022).
produced from wheat flour, water, salt, and yeast. The functional and technological
However, due to the need to produce different types understanding of lipases has been continuously
of bread with different functionalities, and the discussed. However, they are mainly based on the
increase in mechanisation in the bakery industry, knowledge of the effects of lipids in baking (Dahiya
other components can be added to improve the et al., 2020). Lipases positively influence the physical
characteristics of the dough during processing, and characteristics of bread, particularly the specific
consequently, the quality of the final product. These volume, acting mainly on the lipid fraction,
additives include fats, sugars, emulsifiers, oxidising consequently increasing the number of molecules
agents, and enzymes (Dahiya et al., 2020). For this with emulsifying properties (Wang et al., 2018; Melis
purpose, the bakery industry has been considering the et al., 2019). As a result of the increase in bread
incorporation of commercial enzymes to improve the volume, a more uniform, and therefore, softer crumb
physical-chemical, thermal, and rheological structure is obtained; they also modify the handling
properties of doughs in general. Bearing in mind that properties, increasing the stability and strength of the
many of the existing chemical additives can harm dough (Dahiya et al., 2020).
health, the use of compounds generally recognized as Lipase catalyses triglycerides into mono- or
safe (GRAS) in bakery products is a topic that arouses diglycerin, glycerol, and free fatty acids. Although
______
*Corresponding author.
Email: [email protected]
682 Santos, A. C., et al./IFRJ 31(3): 681 - 695

the application of lipase in bread is a popular area of sodium chloride), which was in the form of dry
research in recent years, as well as other enzymes and granules, was mixed with the other ingredients; first
emulsifiers that can help improve the characteristics the dry ones followed by the wet ones. Next, the
of bakery products, it is worth emphasising that the dough was homogenised in an automatic
concentration and number of enzymes added to bread homogeniser (Master Bread NPF-53 Mondial 2970-
reflect on the improvement of rheofermentative 01) for 10 min (a time defined by preliminary tests
characteristics, water-holding capacity, and crumb guaranteeing the shortest time and a homogeneous
structure (Huang et al., 2020). They also have the dough), and left to rest for another 10 min. Then, the
advantage of improving the processing of the dough, dough was divided into three parts of approximately
as well as the general quality of the bread, increasing 220 g, shaped into an ellipse, and placed in an oven
the stability of the dough, resistance to extension and (Fanem®, model 515A) at 30°C with a relative
hardness, decreasing the viscosity of the dough, humidity of 70% for the fermentation stage (1 h, 1 h
increasing the specific volume, improving the 30 min, and 2 h 30 min). After this period, the doughs
softness and structure of the crumb, and delaying were baked in an electric oven (Layr®) preheated at
retrogradation during storage (Melis et al., 2019). In 150°C for 23 min. After cooking and cooling, the
this way, lipases make it possible to replace bread was packed in low-density polyethylene plastic
conventional emulsifiers, partially or fully in bakery bags, and stored at 25°C until further analysis.
products, promoting an improvement in the
technological performance of bread in general. Until Experimental design for bread-making
now, the use of lipases as bread improvers has been In the first stage, the tests were conducted
little explored. In this context, the main objective of following the 3 × 5 × 3 factorial scheme, considering
the present work was to apply commercial lipases, three types of enzymes (Lipopan F®, Lipopan
and evaluate their effects on the technological XTRA®, and Lipopan Prime®), five enzyme
characteristics of loaf bread during storage. concentrations (0.75, 1.5, 2.0, 2.25, 3.0, and 3.75
KLU g-1), and three fermentation times (1 h, 1 h 30
Materials and methods min, and 2 h 30 min), and the treatments were
compared with the control (without enzyme). Five
Ingredients and reagents repetitions were used for assessing the texture profile
Dona Benta® Type 1 wheat flour, responses. Three repetitions were used for assessing
Fleischmann® biological yeast, commercial the expansion index, dough fermentation, specific
®
granulated sugar, Cisne refined salt, and ice water volume, colorimetric, number of alveoli, area,
were used. All ingredients were purchased from local perimeter, and circularity.
businesses in Goiânia, Brazil. The basic parameters
of the flour used were moisture (14.59 g 100 g-1), wet Shelf life
gluten (28 g 100 g-1), ash (0.48 g 100 g-1), colour For the evaluation of shelf life, a 2 × 3
parameter concerning brightness (92.64), and water experimental design was adopted over 28 days with
absorption (57.50%). The enzymatic preparations evaluation every seven days (0, 7, 14, 21, and 28
(Lipopan F®, Lipopan XTRA®, and Lipopan Prime®) days), with two types of formulations (control and
used as source of lipases were donated by the with enzyme), and at three different storage
company Novozymes Latin América Ltd., and based temperatures (15, 25, and 35°C, stored in a biological
on the manufacturer's information, they have oxygen demand (B.O.D.) incubator to maintain stable
enzymatic activities of 25 KLU g-1 (25 kg lipase units temperatures). Shelf-life data were subjected to
for each 1 g of enzyme), 7.2 KLU g-1 (7.2 kg lipase analysis of variance, and then, the test of mean
units for each 1 g of enzyme), and 10 KLU g-1 (10 kg comparison to compare the storage times of the
lipase units for each 1 g of enzyme), respectively. treatments. A comparison was also made between the
means of the treatments, performing a multivariate
Bread preparation analysis using the principal component analysis
All ingredients were weighed on an analytical (PCA) to identify patterns of dispersion and similarity
scale (Marte®, model Ay220) to prepare the bread. between the treatments based on the response
Then, the enzymatic mixture (lipase, wheat, and variables.
 Santos, A. C., et al./IFRJ 31(3): 681 - 695 683

Dough analysis cohesiveness, gumminess, chewiness, and resilience).

Expansion rate and specific volume The analysis was performed in quintuplicate at a
The expansion index and specific volume were temperature of 25°C.
determined according to Mudgil et al. (2016). First,
the bread expansion rate (IE) was calculated using Eq. Colorimetric analysis
1. Then, the quotient established the specific volume The colorimetry of the bread crumb was
between the volume (cm3) and the dough (g) of each determined using Colorquest® Colorimeter, a
sample supplied, with results expressed in cm3 g-1 standard of the International Commission on
(Eq. 2): Illumination (CIE), calibrated with a standard white
plate, following the manufacturer's instructions.
(𝑑𝑝+ℎ𝑝)
𝐸𝑥𝑝𝑎𝑛𝑠𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 (𝐼𝐸) = (𝑑𝑚=+ℎ𝑚) (Eq. 1) Colour measurements were expressed numerically
2 using the coordinates L*, a*, and b*, where L* is
2

brightness, a* is the red-to-green coordinate, and b*

𝑉
𝑉𝐸 = 𝑚 (Eq. 2) is the yellow-to-blue coordinate. According to
Minolta, the chromaticity value, C*, and hue angle,
where, dp and hp = diameter and height of bread after or H°, are called the CIELAB colour system. Colour
baking (cm); dm and hm = diameter and height of measurements were expressed numerically using the
moulded doughs (cm); VE = specific volume (cm3 g- chromaticity coordinates or C* and the hue angle or
1
); V = volume (cm3); and m = dough (g). H°. The determinations of all colorimetric
coordinates were performed in triplicates.
Number of alveoli and perimeter
The bread crumb structures were evaluated Chemical analysis
using digital images according to Juárez et al. (2008). The moisture content was determined by the
The images were obtained by digitalisation at a gravimetric test, and expressed in g by 100 g of
resolution of 550 DPI in an HP ScanJet 2400 scanner sample (g 100 g-1). The water activity was determined
in the central area of the crumb with a resolution of at an average temperature of 25°C in the Aqualab®
900 × 900 pixels. The images were analysed using the equipment model (Decagon Devices, Inc. Pullman
ImageJ® 1.47v software (National Institute of Health, Washington). Determination of hydrogen ion
USA). These, in turn, were saved as files in JPEG potential (pH) was performed using a digital
format, and cropped for a field of view of 900 × 900 potentiometer. The titratable acidity was determined
mm. Finally, colour images were converted to 8-bit by titration, and the results expressed in g of citrus
grayscale, and the Otsu thresholding algorithm was acid per 100 g sample (g 100 g-1). All methodologies
applied. From this, it was possible to obtain the values were performed in triplicate, and followed the
of the number of alveoli and the perimeter. protocols AOAC (2012).

Analysis of bread during shelf life Scanning electron microscopy (SEM)

Texture profile The samples were initially prepared by
The texture profile of the bread was determined removing the crumb portion, followed by drying at
by the instrumental method in a benchtop 75°C for 6 h. After this period, they were placed in a
texturometer (Texture Analyzer, TA-XT Plus, beaker, and ethyl ether was added until the samples
Surrey, England). Ten slices of bread were cut 15 mm were covered and stirred in four rotations for 50 min.
thick for each treatment, and the external slices on Next, they were taken to an oven at the same
both sides were discarded. For sample compression, temperature until complete evaporation of the
an aluminium probe with a diameter of 35 mm and solvent. Next, the bread was subjected to scanning
the following parameters were used: test velocity, 2.0 electron microscopy (JSM 6610 model, Jeol, Tokyo,
mm s-1; pre-test velocity, 5.0 mm s-1; post-test Japan), equipped with EDS (Thermo Scientific NSS
velocity, 5.0 mm s-1; compression rate, 40% Spectral Imaging).
deformation; and an interval of 5 s between
compression cycles, according to Carr and Tadini Differential scanning calorimetry (DSC)
(2003). Each slice of bread was compressed twice to The stored bread's enthalpy of retrogradation
obtain texture parameters (hardness, elasticity, (△H) was analysed using a Q100 differential
684 Santos, A. C., et al./IFRJ 31(3): 681 - 695

scanning calorimeter (DSC, TA Instruments, New 30 min of fermentation); B 2.25 and B 3.0 at time 2
Castle, DE, USA). The samples (4 mg) were weighed, (1 h 30 min of fermentation), B 3.75 at time 1 (1 h of
mixed with deionised water (1:3), and sealed in a fermentation), C 2.25 and C 3.0 at time 2 (1 h 30 min
DSC aluminium pan. Samples were considered and fermentation), and C 3.75 at time 3 (2 h 30 min of
packed in a DSC tray without loss of weight or fermentation) stood out concomitantly for higher
moisture, followed by heating from 10 to 120°C at a rates of specific volume, volume, resilience,
heating rate of 10°C min-1 (Bosmans et al., 2013). elasticity, cohesiveness, and expansion. The
multivariate technique allows for selecting treatments
Statistical analysis by simultaneously viewing the variables. These
A factorial analysis of variance was performed treatments were superior in proceeding with a more
to identify the significance of factors and their detailed univariate analysis, and looking for
interactions. The data were then submitted to a significant differences between their means.
multivariate analysis using the principal component The data presented in Table 1 show very subtle
analysis (PCA) via a covariance matrix to identify differences between the chosen treatments based on
treatments with an ideal profile. With the treatments the multivariate analysis of principal components
identified, the Tukey’s test was applied to compare (PCA). However, treatment C 3.75 was eliminated
the means (p < 0.05). Finally, statistical analyses were since it presented a value greater than p < 0.05 of
performed in the R software (R Development Core hardness. According to Serventi et al. (2016), both the
Team 2021). sensory and instrumental analyses correlated very
well in the description of bread volume, crumb
Results and discussion hardness, and pore size; so, it was decided to discard
it since greater hardness would compromise
Experimental design for bread-making technological parameters. Therefore, the analysis of
First, principal component analysis was principal components significantly correlated with
performed to understand the data set better, and the following variables: hue angle, hardness,
reduce the multivariate dimensionality, thus looking gumminess, chewability, expansion, and
for correlated treatments (Figure 1) (Morais et al., cohesiveness values. Sample C 3.75 presented the
2024). The first component (PC1) corresponded to best results compared to the others, which was
49.32% of the total data variation, and the second positive for loaf bread since low values indicate that
component (PC2) was 19.32%, totalling 68.64% of the bread would disintegrate more quickly.
the variance. The variables with the greatest weight in Furthermore, statistical averages (p < 0.05) were
PC1 were hardness, gumminess, chewiness, and hue found in several tests for the other parameters. In
angle, and variables related to texture and expansion. addition to analysing the standards, the PCA was used
Among the response variables, the microstructure and again, and it inferred that B 3.75 behaved better,
colorimetric data (number of alveoli, perimeter, area, considering the objectives of the studies.
circularity, and hue angle) least explained the Based on the correlations found, the samples
variation of data in PC1 (Figure 1B). The variables with enzymes were distinguished by having less
with the most significant weight in PC1 distributed chewiness, gumminess, and hardness, indicatives of a
the scores horizontally. This indicated that the Time softer bread. The results agreed with Giannone et al.
1 scores on the left side of the graph (dotted circle) (2016), in which, when applying lipase and amylase
had higher hardness, gumminess, and chewiness in loaf bread, they observed that during the entire
indexes, and lower volume indexes (Figure 1A). The storage period, the samples containing the enzyme
desirable characteristics of loaf bread are those with were markedly softer than the control, demonstrating
greater volume and lower levels of chewiness, the effectiveness in delaying retrogradation in all
gumminess, and hardness. stages of enzymatic formulations assessed.
The correlation circle showed negative Furthermore, the results showed that the tested
correlations between variables related to bread enzymes promoted positive effects in wheat bread,
volume and chewiness, gumminess, and hardness, and showed synergistic interactions in preventing
making it possible to identify treatments with aging, showing a more marked effect in delaying
desirable characteristics. It was possible to observe staling and chewiness during storage.
that the treatments A 2.25 and A 3.75 at time 3 (2 h
 B
A
Santos, A. C., et al./IFRJ 31(3): 681 - 695

Figure 1. Principal component analysis (PCA) and scatter plot (PC1 vs. PC2) of variables studied as control sample
(without enzyme) and enzymatic sample at times studied with projection in factorial plane. In graph B, A: Lipopan F ®;
B: Lipopan XTRA®; C: Lipopan Prime®.
685
 686

Table 1. Treatments giving best behaviour in analysing principal components (PCA) of produced breads.
Hardness Elasticity Cohesiveness Gumminess Chewability Dough
Treatment Resilience
(N) (m) (N) (N) (N) volume
b a ab bc bc a
A 2.25 (T3) 384.90 ± 36.86 0.95 ± 0.01 0.76 ± 0.00 275.422 ± 19.35 259.28 ± 23.51 0.38 ± 0.00 32.25 ± 2.25a
b a bc bc bc bc
A 3.75 (T3) 390.32 ± 16.06 0.95 ± 0.00 0.75 ± 0.00 287.88 ± 12.15 269.78 ± 11.41 0.33 ± 0.00 29.93 ± 0.11ab
B 2.25 (T2) 394.74 ± 6.10b 0.95 ± 0.01a 0.76 ± 0.01ab 295.444 ± 15.27bc 281.16 ± 17.07bc 0.33 ± 0.01bc 30.00 ± 0.50ab
b a d c c c
B 3.00 (T2) 346.60 ± 11.28 0.95 ± 0.02 0.73 ± 0.00 251.72 ± 11.32 241.34 ± 9.74 0.30 ± 0.02 31.10 ± 1.10a
B 3.75 (T1) 344.64 ± 9.06b 0.96 ± 0.00a 0.77 ± 0.00a 309.42 ± 37.47ab 300.64 ± 26.20ab 0.35 ± 0.00 ab 27.73 ± 0.75b
b a ab c c bc
C 2.25 (T2) 333.54 ± 24.45 0.94 ± 0.00 0.76 ± 0.00 252.24 ± 24.90 246.85 ± 26.20 0.33 ± 0.02 30.00 ± 1.00ab
C 3.00 (T2) 374.98 ± 21.56b 0.97 ± 0.01a 0.75 ± 0.00c 278.33 ± 12.66bc 275.10 ± 8.92bc 0.33 ± 0.01bc 30.50 ± 0.50ab
a a bc a b
C 3.75 (T3) 560.20 ± 21.56 0.95 ± 0.01 0.75 ± 0.00 354.14 ± 35.37ª 332.99 ± 31.93 0.34 ± 0.01 30.73 ± 0.25a
Specific volume Expansion Number of
Treatment H° Perimeters Circularity
(cm3 g-1) (cm3 g-1) alveoli
A 2.25 (T3) 4.02 ± 0.18ab 1.49 ± 0.07b 95.50 ± 0.13bc 751 ± 87.15a 6.88 ± 0.58b 0.79 ± 0.01a
b a b a b
A 3.75 (T3) 3.88 ± 0.07 1.87 ± 0.11 96.47 ± 0.38 672 ± 9.29 6.11 ± 0.23 0.80 ± 0.01a
B 2.25 (T2) 3.88 ± 0.13b 1.36 ± 0.11b 94.82 ± 0.46c 707 ± 28.00a 6.49 ± 0.32b 0.79 ± 0.02a
b b bc a b
B 3.00 (T2) 3.77 ± 0.08 1.48 ± 0.04 95.44 ± 0.60 774 ± 85.49 6.45 ± 0.40 0.79 ± 0.01a
B 3.75 (T1) 3.93 ± 0.06ab 1.44 ± 0.03b 96.27 ± 0.34b 742 ± 51.73a 6.16 ± 0.04b 0.79 ± 0.00a
a b b a a
Santos, A. C., et al./IFRJ 31(3): 681 - 695

C 2.25 (T2) 4.24 ± 0.13 1.48 ± 0.03 96.20 ± 0.25 623 ± 29.70 8.19 ± 0.38 0.77 ± 0.01a
C 3.00 (T2) 3.90 ± 0.14b 1.46 ± 0.04b 95.85 ± 0.42bc 726 ± 63.22a 6.47 ± 0.47b 0.77 ± 0.00a
b b a a b
C 3.75 (T3) 3.79 ± 0.05 1.46 ± 0.03 98.55 ± 0.74 759 ± 77.09 5.94 ± 0.15 0.79 ± 0.00a
Means followed by different lowercase superscripts in the same column differ statistically using the Tukey’s test (p < 0.05). T1: 1 h of
fermentation; T2: 1 h and 30 min of fermentation; T3: 2 h and 30 min of fermentation; A: Lipopan F®; B: Lipopan XTRA®; and C: Lipopan
Prime®. Enzyme concentrations are expressed in KLU g-1: 2.25, 3.00, 3.75.
 Santos, A. C., et al./IFRJ 31(3): 681 - 695 687

Considering the correlation circle of the hardness with the use of the enzyme was significantly
variables, it can be suggested that the acidity, area, lower (p < 0.05) than the control. This result
volume, and the number of alveoli were directly corroborated that observed in the multivariate
correlated with bread that had commercial enzymes analysis. These changes could be attributed to the
in their composition, that is, breads that had in their more excellent gas retention by the dough treated
composition enzymes tended to show higher values with lipase, since according to Dahiya et al. (2020),
of acidity, volume, and number of alveoli when the addition of lipases leads to an increase in bread
compared to control treatments. This indicated that volume, resulting in an improved and highly uniform
adding Lipopan to the formulations brought positive crumb, and therefore, greater firmness. This also
effects. This was mainly related to generated lipids, corroborated Serventi et al. (2016) who, when
which could indirectly stabilise gas cells in the dough. evaluating the addition of different enzymes to
This is an important characteristic, as the gas improve the sensory quality of bread, found that
retention capacity of the dough is one of the formulations with lipase incorporation (Lipopan
characteristics, if not the most important, in bread- XTRA®) showed an increase in the hardness. In this
making, as it is associated with crumb structure and sense, Soares et al. (2017) stated that sucrose
high specific volume (Gerits et al., 2014). increases the glass transition temperature Tg,
Based on PCA results, it can be suggested that consequently reducing the retrogradation of starch,
the variables were directly correlated with the bread making the bread harder over the days of storage.
with commercial enzymes in their composition The elasticity indicated a decrease in its values
compared to the controls. The data showed very over the storage periods, both for the control bread
subtle differences between the chosen treatments, and those with the addition of enzyme (Lipopan
which were based on multivariate analysis (PCA). XTRA®). However, in samples with enzyme, only
However, C 3.75 was initially eliminated as it those stored at room temperature (35 ± 2°C) and at
presented a hardness value greater than p < 0.05. On 25°C differed statistically (p < 0.05). It was also
the other hand, B 3.75 presented higher cohesiveness observed that the mean of elasticity did not present a
values, which is positive for loaf bread, since very significant difference (p > 0.05) between the control
low values indicate that the bread would disintegrate bread and those with lipase, taking into account the
more easily. Regarding the other parameters, same storage conditions. Therefore, it was impossible
statistically equal means (p < 0.05) were found in to predict lipase's effect on texture. These results
several tests. Therefore, B 3.75 showed better agreed with those found by Becker et al. (2009) who
behaviour, taking into account the objectives of the found that adding different enzymes intensified the
study, and was therefore chosen for the subsequent distending properties of the dough (extensibility), and
stages (B 3.75, Lipopan XTRA®, 1 h of fermentation, minimised the elasticity of the bread. Furthermore,
and enzyme concentrations expressed in 2.25 KLU g- this decrease in the elasticity value was inversely
1
). This result indicated that adding Lipopan XTRA® proportional to the hardness value found in the bread
to the formulations brought positive effects. This analysed during storage; bread with higher hardness
enzyme was then used to estimate shelf life for 28 values tended to show a decreasing behaviour for the
days at two different temperatures (25 and 35°C). elasticity during storage (Esteller, 2014).
At 28 days, bread with enzyme addition,
Shelf life regardless of storage temperature, had statistically
Texture profile lower cohesiveness (p < 0.05) than the days before it.
The TPA results, obtained through double- It was also observed that the overall average did not
cycle compressions at 40% depth, showed the present a significant difference (p > 0.05),
structural alterations that affected the bread samples considering the control treatment and enzyme
added with Lipopan XTRA® and in the control tests addition at the same temperature. This parameter
during storage. refers to the deformation force before failure. It is
Evaluating each analysed variable separately, understood that higher values of this attribute indicate
after 21 days of storage, a significant increase in the bread with greater freshness. This behaviour can be
hardness was observed in the formulations with explained by the fact that during the kneading process
enzyme submitted to refrigeration at 15 and 25°C. (beating), water is incorporated into the flour, and
Furthermore, it was observed that the overall mean gluten develops. Disulphide bridges and ionic bonds
688 Santos, A. C., et al./IFRJ 31(3): 681 - 695

(addition of salt) maintain cohesiveness, and ensure The treatments with the addition of Lipopan XTRA®
the retention of volatiles during baking. However, (3.75 KLU) showed lower resilience values (p < 0.05)
during the storage period, changes in these bonds within the same temperature, except under 15°C, in
(water migration, starch crystallisation, fat which the means of the control and with the addition
hydrolysis) lead to a gradual disarrangement of the of enzyme were statistically equal. For this parameter,
structure, which consequently tends to decrease the higher values indicate the bread's quality. Therefore,
cohesiveness values (Esteller, 2014). Lipopan XTRA® appeared ineffective under the
The gumminess of the bread samples showed a study's conditions.
trend similar to hardness. The gumminess increased
gradually and significantly over the days of storage, Colorimetric analysis
regardless of the temperature at which the bread was Colour evaluation may indicate failures during
stored compared to the control. However, the processing, which can be perceived by the crust
treatments that used enzymes, regardless of storage colour being too light or too dark. Table 2 shows the
temperature, obtained lower overall averages (p < chromaticity and hue angle values for the bread of
0.05). In the loaf bread prepared here, it was verified control treatment and those with the addition of
that the application of Lipopan XTRA® (3.75 KLU) enzyme (Lipopan XTRA®), where the results showed
could reduce the gumminess. The results agreed with a tendency for the values to decrease in different
Serventi et al. (2016) who, when studying the proportions, depending on the storage conditions, that
addition of enzymes to improve the sensory quality of is, it made the crumb colour lighter, with a more
wheat-cassava compound bread, found that the opaque colour, tending more towards cream.
addition of lipase (Lipopan XTRA®) promoted a However, when analysing the control bread
reduction in crumb gumminess, and associated this (without the addition of enzyme), it was possible to
behaviour with increased volume. In this sense, the verify that they presented the opposite behaviour; that
decrease in gumminess levels can be explained due to is, their chromaticity tended to increase over the days
the decrease in the content of soluble solids present in of storage. As for the overall average, it was found
the samples (López and Goldner, 2015). that only the bread with enzyme (Lipopan XTRA®) at
After 21 days, as occurred for hardness, there 25°C obtained a lower value (p < 0.05) compared to
was a significant increase in chewability (p < 0.05) in the control within the same storage conditions.
the formulations with the addition of the enzyme Almeida et al. (2013) found a similar behaviour when
subjected to study temperatures of 15 and 25°C. It studying dietary fibre sources in frozen pre-baked
was also observed that the tests with the incorporation bread and their influence on technological quality.
of Lipopan XTRA® (3.75 KLU) presented lower Therefore, it can be inferred that the control
overall averages (p < 0.05) for chewiness under the bread (without the addition of enzyme) tended to have
three storage conditions analysed. This result a colour tending to red/brown at the end of storage, as
confirmed what was observed in the analysis of they showed an increasing behaviour (from 11.02 on
principal components. Therefore, the application of day 0 to 13.85 on day 28 at room temperature). On
commercial lipase was promising in reducing the the other hand, the bread added with the enzyme
chewiness of the breadcrumbs studied. The same (Lipopan XTRA®) at the end of storage tended to
behaviour was verified in the study by Barret et al. become more yellow and lighter, as their behaviour
(2002) when producing bread with oxidising gums, shows decreasing results over the days of storage. In
stored at temperatures of 4, 21, and 38°C and stored this sense, Dube et al. (2020) stated that crumb colour
for 0, 2, 6, and 12 weeks. The authors found an is related to the type of flour and the structure of the
increase in hardness and chewiness values, and a crumb's air cell, affecting how light reflects on a piece
decrease in elasticity and cohesiveness during storage of bread. Emulsifiers, e.g., lipases, change the
compared to a control without additives. structure of the crumb, and make the air cells smaller
Bread resilience decreased with storage time and more evenly distributed, reducing the crumb's
regardless of treatment. These results could be blackness.
attributed to water loss during storage, and water When evaluating the hue angle (Table 2), it
molecules' migration to the crust. These agreed with was observed that the bread stored at room
De La Hera et al. (2014), finding that resilience temperature presented the same decreasing trend over
measures were affected by the water content in bread. the days of storage. However, all the samples were
 Santos, A. C., et al./IFRJ 31(3): 681 - 695 689

found close to the 90° axis, confirming the tendency dietary fibre in frozen pre-baked bread and their
towards a more yellowish colour, a typical colour influence on functional and technological quality,
tone of breadcrumbs. Similar results were reported by obtaining results very similar to those reported in the
Almeida et al. (2013) when verifying new sources of present work about the tonality of the crumb.

Table 2. Means of chroma and hue angle of breads stored at three temperatures over 28 days.
Chroma (C)
Ambient (35°C) 15°C 25°C
Day
Control Enzyme bread Control Enzyme bread Control Enzyme bread
b a
0 11.02 ± 0.47 12.78 ± 2.24 - - - -
7 11.99 ± 1.07b 10.14 ± 0.07ab 11.92 ± 2.55a 10.73 ± 0.17a 11.61 ± 1.12a 11.50 ± 0.56a
14 12.05 ± 0.02b 10.48 ± 0.77ab 11.20 ± 0.40a 10.19 ± 0.67a 11.46 ± 0.85a 11.10 ± 0.42ab
21 11.98 ± 0.62b 9.14 ± 0.57b 11.7 ± 0.60a 9.99 ± 1.27a 11.39 ± 0.59a 10.30 ± 1.27ab
28 13.85 ± 0.27a 12.56 ± 0.27a 12.09 ± 0.60a 10.48 ± 0.74a 12.12 ± 0.54a 9.87 ± 0.78b
A ABC AB C AB
Mean 12.17 11.29 11.73 10.34 11.64 10.69BC
Hue angle (H°)
Day Control Enzyme bread Control Enzyme bread Control Enzyme bread
a a
0 95.21 ± 0.37 94.16 ± 1.18 - - - -
a ab a ab a
7 94.68 ± 1.07 95.45 ± 0.32 94.73 ± 0.79 95.38 ± 0.08 94.93 ± 0.55 94.69 ± 0.63a
a ab a b a
14 93.79 ± 0.0.2 93.96 ± 0.19 94.45 ± 0.41 94.36 ± 0.27 95.35 ± 0.07 94.52 ± 0.21a
21 93.64 ± 0.62a 94.5 ± 0.35a 94.97 ± 0.32a 95.95 ± 0.08a 94.71 ± 0.63ª 94.33 ± 0.16a
28 93.07 ± 0.27a 92.94 ± 0.05a 94.13 ± 0.06a 95.17 ± 0.36ab 95.41 ± 0.61a 94.23 ± 0.46a
Mean 94.08B 94.21B 94.57AB 95.22A 94.45AB 95.10A
Means followed by different lowercase superscripts in the same column differ statistically by Tukey's test
(p < 0.05). Means followed by different uppercase superscripts in the same row differ statistically by
Tukey's test (p < 0.05).

Chemical analysis increased starch crystallinity, and crumb firmness

Water activity is one of the most critical factors (Dahiya et al., 2020). However, a different behaviour
in the food industry. It quantifies the water available was observed in tests at room temperature, in which
for the growth of microorganisms,a and the the means were higher than in the first days of
biochemical and technological reactions that can alter storage, and decreased at the end of shelf life (p <
food quality. Results ranged from 0.89 to 0.94 aw for 0.05). This phenomenon could have been attributed to
control treatments and 0.91 to 0.95 aw for treatments the storage temperature (35 ± 2°C) which favoured
with lipase incorporation (3.75 KLU). An increase (p the evaporation of water molecules. Following the
< 0.05) was observed from the twenty-first day of legislation in force in the country (Brazil, 2000), the
storage onwards for treatments with the addition of humidity of loaf bread must have a maximum of 38%;
enzyme at a temperature of 15°C, whose average was therefore, all treatments were within the humidity
0.94 aw. In general, there were no significant parameters prescribed by the legislation in force.
differences between the control and the formulations As for the pH, the doughs with the addition of
with added enzyme (Lipopan XTRA®) at any of the Lipopan XTRA® (3.75 KLU) showed lower overall
temperatures evaluated (p > 0.05). pH means (p < 0.05) when compared to the control
The moisture content tended to increase in all samples under the same temperature conditions, but
treatments kept under refrigeration (25 and 15°C) still within the normal range for loaves of bread.
over storage. However, after seven days, a lower According to Debonne et al. (2020), the pH value is
moisture content (p < 0.05) was observed. This directly correlated with the ingredients present in the
behaviour during bread storage is directly related to bread formulation. So, most studies conducted with
bread staling, caused by changes in starch and water bread mention the pH reduction as responsible for
migration. Because of this, bread freshness loss technological and nutritional changes, and bread
occurs due to increased moisture in the crust, conservation. In the evaluation of the titratable
690 Santos, A. C., et al./IFRJ 31(3): 681 - 695

acidity, the control samples presented a variation of provide alteration between the components (Sharma
1.30 - 1.05 g 100 g-1, whereas the samples with the and Gujral, 2019).
addition of lipase (Lipopan XTRA®) presented an The micrographs were separated into control
acidity of 1.39 - 1.00 g 100 g-1. Until the end of bread (without enzyme addition) and bread with
storage, there was an increase in titratable acidity enzyme addition (Lipopan XTRA®) over 28 days of
values in all samples, reflecting the drop observed in storage to obtain more precise and cohesive results
pH values. In general, among the samples, those (Figure 2). It could be inferred that the results
added with lipase showed significantly (p < 0.05) obtained for the micrographs of the control samples
higher acidity than the control samples. The intense ratified the results of the physical, chemical, and
acidifying activity of yeasts is mainly responsible for technological analyses since at room temperature (35
this pronounced acidity. In addition, the presence of ± 2°C), on the fourteenth day, damaged zones (DZ)
lactic acid bacteria, responsible for producing organic began to appear, a fact mainly stemming from the
acids, also promotes this behaviour (Sharma and decrease in water activity, which consequently
Gujral, 2019). increased the hardness, making the bread more brittle
and with easily identifiable damaged areas. On the
Scanning electron microscopy (SEM) other hand, the opposite behaviour was observed for
In Figure 2, the micrographs of the bread stored temperatures of 15 and 25°C, where humidity and
at time 0 are shown, being categorised as a control water activity showed a slight increase, thus ensuring
sample and with the addition of the enzyme lipase a more homogeneous and less crumbly bread (<
(Lipopan XTRA®), obtained at 500× magnification. hardness).
It consisted of water and wheat fractions, such as By evaluating the micrographs obtained from
starch, protein, and bran. All bread samples contained the bread with the addition of the enzyme lipase
small and large starch granules of spherical and (Lipopan XTRA®) (Figure 2), it was possible to
lenticular shapes distributed over the protein matrix. notice a change in the conformation of the starch
This observation was similar to reports by Aponte et molecules, where visually they were different over
al. (2014) on baked bread during the first day of the days of storage; the same behaviour could also be
storage. observed in the control bread samples. According to
Still, on the micrographs at time 0, it was Deckardt et al. (2014), when evaluating the effects of
possible to observe that the starch had oval, spherical, applying organic acids in starch granules, the authors
or polygonal shape, as previously reported in the showed that their addition led to the modification of
literature, without any cavities or fissures, with a the starch granules. Furthermore, they found that
smooth surface (Deckardt et al., 2014; Liu et al., changes in starch granules underwent slight surface
2015). By analysing the two micrographs (A and B), deformations with increasing acidity. This same
no significant differences were observed between the behaviour could be observed in the micrographs of
control bread and those with the addition of enzyme, the present work; in the first fourteen days of storage,
with a clear division of starch molecules and protein the starch molecules were uniform, smooth, circular,
networks and their cavities. As it was time 0, no and defined. However, over the days of storage, we
damaged zones were found since the physical and noticed that such molecules began to lose their
chemical variables such as humidity, water activity, structural shape, becoming less circular and more
pH, acidity, and the technological variables such as oval, or even losing their conformational structure
hardness and cohesiveness had not undergone completely.
changes that would justify the appearance of damaged The scanning electron microscopy data also
zones that would compromise the integrity of the showed significant modifications on the surface of the
sample. Damaged areas indicate deformation of the starch granules between the fourteenth and twenty-
gluten network and consequent release of small starch eighth days. Severe changes and deformation in the
granules. However, this aspect may be related to appearance of the surface, especially on the twenty-
sample preparation. Depending on the preparation eighth day at temperatures of 25 and 15°C, are also
method, there may be changes in its morphology. visible in Figure 2, in line with the higher acidity
Therefore, the sample must not undergo excessive values for this bread, which would contribute to the
preparation, which can distort the structure and conformational derangement of the structure. At the
 Santos, A. C., et al./IFRJ 31(3): 681 - 695 691

Figure 2. Micrographs of control bread (A) and enzymatic bread (B), with 500× magnification at 14, 21,
and 28 days of storage, at temperatures 15, 25, and 35°C ± 2. Arrows show granules of starch (G), cavities
(C), protein networks (N), and damaged areas (ZD).

end of storage, the starch granules appeared trapped fresh bread. This behaviour, as mentioned, could have
inside the compact protein matrix, especially those been due to increased crumb firmness (hardness and
subjected to 25 and 35°C (Figure 2). This visually gumminess) and decreased elasticity (elasticity and
detected interaction between starch granules and cohesiveness) for all breads during storage. Similar
matrix could have decreased the enzymatic behaviour was observed by Hayta and Hendek Ertop
hydrolysis of starch, and increased the amount of (2017) when studying bread stored for ten days. This
resistant starch in the final product (Deckardt et al., change in the microstructure of the stored bread also
2014; Hayta and Hendek Ertop, 2017). indicated that the starch-starch interaction resulted
At the end of storage, the starch-protein matrix from retrogradation, and not from the starch-gluten
that formed the microstructure of stored bread was interaction.
more discontinuous, and appeared weaker than in
692 Santos, A. C., et al./IFRJ 31(3): 681 - 695

Differential scanning calorimetry (DSC) being subjected to different temperatures and during
The results obtained through differential 28 days of storage, substantial fluctuations in their
scanning calorimetry (DSC) analysis are enthalpy temperatures were not observed (Table 3). It was also
variation (ΔH), heat capacity of materials, and noted that the enthalpy of retrogradation (ΔH) values,
temperature of thermal events (Table 3). From the as observed in hardness, increased with storage time.
thermograms obtained in this analysis, it was possible The increase in ΔH indicates the presence of less
to determine the T0 (initial temperature of the starch amorphous and more crystalline starch regions (Ou et
retrogradation process), Tp (peak temperature), and al., 2022). This change does not result from a single
ΔH1 (enthalpy of retrogradation). It can be seen that effect but includes the reorganisation of polymers
starch retrogradation started at a temperature of within the amorphous region, loss of moisture
138.60°C (T0). The other analysed times were below content, water content distribution between the
173.00°C since starch is mainly composed of amorphous and crystalline zones, and the
amylopectin, which, according to Zobel et al. (1988), macroscopic structure of the crumb (Ozkoc et al.,
is one of the components responsible for the 2009). Similar behaviour was observed by Ma et al.
crystalline part of the granule; this compound being (2022) when evaluating the effects of sugars on the
one of the main factors in the process of starch retrogradation of starch gels during storage, who
recrystallisation or retrogradation. observed that the enthalpy of fusion (ΔH) of all
It was observed that the retrogradation process samples increased significantly with increasing
reached its maximum point when the peak storage time.
temperature was measured (Tp), showing that, despite

Table 3. Differential scanning calorimetry (DSC) results of breads stored at three temperatures over 28 days.
Control Enzyme
Time T0 Tp Tp - T0 ΔH1 T0 Tp Tp - T0 ΔH1
-1
(°C) (°C) (°C) (J g ) (°C) (°C) (°C) (J g-1)
gB fA cA bA eA eA dB
0 138.60 147.90 9.30 812 139.60 147.90 8.3 251mB
Ambient

7 121.50kB 122.20mB 0.70kA 625gA 126.00jA 126.70lA 0.70hA 593kB

14 148.50dB 157.30cB 8.80dA 628fA 160.90aA 164.60aA 3.70eB 627iB
21 149.40cA 150.50eA 1.10iB 389kB 138.10hB 147.70fB 9.60cA 653fA
iB kA bA lB fA jB iB
28 130.70 144.40 13.70 384 138.70 139.30 0.60 691eA
0 138.60gB 147.90fA 9.30cA 812bA 139.60eA 147.90eA 8.3dB 251mB
7 137.80hB 146.00gB 8.20eA 231mB 148.50bA 149.50cA 1.00gB 706bA
15°C

14 155.10bA 157.50bA 2.40gA 605hB 138.60gB 139.20kB 0.60iB 730aA

21 124.00jB 143.40lA 19.40aA 743cA 141.70dA 142.80iB 1.10fB 626jB
fA iB jB jB iB bA bA
28 144.30 145.10 0.80 481 134.70 151.70 17.00 636hA
0 138.60gB 147.90fA 9.30cA 812bA 139.60eA 147.90eA 8.3dB 251mB
7 144.30fA 144.90jA 0.60lB 697dB 123.30kB 144.20hA 20.90aA 700dA
25°C

14 173.00aA 175.70aA 2.70fA 3465aA 148.50bB 149.30dB 0.80hB 701cB

bA dA hA eA cB gB gB
21 155.10 157.00 1.90 664 144.00 145.00 1.00 637gB
28 144.70eA 145.40hA 0.70kA 547iA 119.60lB 120.20mB 0.60iA 498lB
Means followed by different lowercase superscripts in the same column differ statistically by Tukey's test
(p < 0.05). Means followed by different uppercase superscripts in the same row differ statistically by t-test
(p < 0.05).

Conclusion most significant weight in PC1 were hardness,

gumminess, specific volume, and chewiness, and
Among the formulations proposed by the variables related to texture and expansion, since they
experimental design, all showed slight differences are technological parameters. The means of hardness,
between the chosen treatments based on the gumminess, and chewiness were significantly lower
multivariate analysis (PCA). The variables with the (p < 0.05) with the use of enzymes in the formulation
 Santos, A. C., et al./IFRJ 31(3): 681 - 695 693

at all storage temperatures. The hardness, elasticity, application: Dough properties and bread
cohesiveness, gumminess, chewiness, and resilience quality. Journal of Food Science and
showed significant differences (p < 0.05) between the Technology 58: 3902-3912.
samples during storage. Despite the impairment of Brazil. 2000. Resolution - RDC No. 90, of October
some variables during storage, incorporating lipase 18, 2000 - Technical regulations for fixing the
(Lipopan XTRA®) was promising for further studies identity and quality of bread. Brazil: Brazilian
and subsequent commercialisation. In this sense, Health Surveillance Agency.
further research should be conducted to verify the Carr, L. G. and Tadini, C. C. 2003. Influence of yeast
sensory acceptance profile, and application in fibre- and vegetable shortening on physical and
rich products, such as products enriched with textural parameters of frozen part baked French
vegetable extracts, ensuring that such bread becomes bread. LWT - Food Science and Technology
technologically and economically viable. 36: 609-614.
Dahiya, S., Bajaj, B. K., Kumar, A., Tiwari, S. K. and
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