J Food Sci Technol (May 2021) 58(5):1819–1828

https://doi.org/10.1007/s13197-020-04693-y

ORIGINAL ARTICLE

Lime juice and enzymes in clean label pan bread: baking quality
and preservative effect
Michele Scarton1 • José Ricardo Crepaldi Ganancio1 • Matheus Henrique Mariz de
Avelar1 • Maria Teresa Pedrosa Silva Clerici1 • Caroline Joy Steel1

Revised: 30 July 2020 / Accepted: 4 August 2020 / Published online: 17 August 2020
Ó Association of Food Scientists & Technologists (India) 2020

Abstract Clean label breadmaking is a challenge for the Introduction

bakery industry, considering the removal of additives,
especially preservatives. Through dough acidification and In bread products, apart from wheat flour, salt, yeast,
the use of enzymes, shelf-life of breads may be extended in shortening and other optional ingredients (eggs, sugar,
terms of sensory quality and preservation against micro- dehydrated fruits, nuts etc.), it is common practice to use
biological deterioration. This study aimed to evaluate the additives. Additives are natural or artificial substances that
effect of lime juice (0, 10.2 or 20.4 g.100 g-1, flour basis, are added to food products for purposes of conservation or
f.b.) on technological characteristics and preservation of modification of their chemical or sensory properties (Gioia
clean label pan breads, with and without enzymes (0.0015 et al. 2017). Among additive categories used in bakery
and 0.0050 g.100 g-1 of phospholipase and fungal xyla- products, oxidants, emulsifiers and preservatives stand out
nase, f.b., respectively). The technological parameters of (Saltmarsh and Insall 2013).
dough (high-speed mixing time, temperature, pH before Among the current demands for the breadmaking
and after proofing) and pan bread (specific volume, firm- industry are consumer desires for more healthy, authentic,
ness, crust and crumb color, aw, pH and mold occurrence) and natural products (Queiroz and Nabeshima 2014). To
were evaluated. Doughs with lime juice required a longer fulfill these trends, the bakery industry is making invest-
mixing time, and produced breads with lower volume, ments in clean label versions of their products. Clean label
firmer crumb, lighter crust and lower mold occurrence, products are those without synthetic ingredients or addi-
when compared to the market standard. Pan breads with tives, aiming to attend the demand for a simpler and more
10.2 g.100 g-1 of lime juice and enzymes showed a higher consumer-friendly ingredient lists (Asioli et al. 2017).
volume and softer crumb, compared to those with only One of the clean label alternatives that can be used in
lime juice. Thus, the joint use of lime juice and enzymes substitution of many additives in bakery products are
may be suitable for clean label pan breads, maintaining a enzymes, that are obtained from plants, animals or
soft crumb and longer shelf life without additives. microorganisms and added to food for technological pur-
poses. In breadmaking processes, enzymes are added to act
Keywords Acidification
Phospholipase
Xylanase
during the mixing, proofing or baking steps, but are inac-
Additives
Shelf-life
Molds tivated at some point in the oven. For this reason, they are
considered processing aids and, in many countries, do not
need to be declared on the product label (Nascimento et al.
2018).
In breadmaking, the most common enzyme categories
& Caroline Joy Steel used are: (1) fungal alpha-amylases, which randomly
steel@unicamp.br hydrolyze starch into lower molecular weight polysaccha-
1 rides, or even into more simple sugars, that will be fer-
Department of Food Technology, School of Food
Engineering, University of Campinas, Campinas, São Paulo, mented by the yeast with the production of carbon dioxide,
Brazil contributing to bread volume; (2) maltogenic amylases,

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which hydrolyze gelatinized starch and reduce bread stal- acid was determined according to the 2,6-dichloropheno-
ing rates, keeping the softness of breads for longer periods; lindophenol titrimetric method no. 967.21 of the Associa-
(3) hemicellulases, such as xylanases, which hydrolyze or tion of Official Analytical Chemists (AOAC 2006). The
solubilize non-starch polysaccharides (NSPS), releasing lime juice presented 8.4 8Brix and 16.37 mg.100 mL-1 of
water, relaxing the dough and enhancing its machinability ascorbic acid. It was immediately frozen and used for
and proofing tolerance, also contributing to improve bread breadmaking maximum 24 h after its extraction.
volume; and (4) phospholipases, which improve dough Type 1 wheat flour, enriched with iron and folic acid
structural properties by producing potent in situ emulsifiers (Renata, Pastifı́cio Selmi, Sumaré, SP, Brazil), was kindly
derived from wheat flour phospholipids; all of them have donated by the manufacturer. The market standard for-
important effects on final bread quality (Gioia et al. 2017). mulation used the following additives: sodium stearoyl-2-
However, it is difficult to substitute traditional preser- lactylate emulsifier (Du Pont, Pirapozinho, SP, Brazil);
vatives. Preservatives are used in breads for shelf-life ascorbic acid oxidant (Granotec, São José dos Pinhais, PR,
extension, since breads usually have a high-water activity Brazil); and calcium propionate mold inhibitor (Mix Ali-
(aw [ 0.90) in comparison to other bakery products such as mentos, São Bernardo do Campo, SP, Brazil).
biscuits, for example, and pH (5.0–6.2) that permits its The enzymes used in this study were fungal alpha-
spoilage by molds, mainly from Aspergillus and Penicil- amylase VeronÒ M4, maltogenic alpha-amylase VeronÒ
lium strains (Sluimer 2005). MAC, phospholipase VeronÒ Hyperbake-T and fungal
Some studies have reported the utilization of natural xylanase VeronÒ 191S, all manufactured by AB Enzymes
ingredients as alternatives to substitute preservatives for (Darmstadt, Germany).
clean label bakery products. As an example, the addition of
ingredients containing organic acids for dough acidifica-
Methods
tion, as in studies using vinegar as acetic acid source
(Williams and Pullen 2007), resulted in longer preservation
Wheat flour characterization
of bakery products by reduction of mold growth during
storage.
Wheat flour was characterized according to the American
Lemons and limes, besides having ascorbic acid, also
Association of Cereal Chemists International (AACCI
have a high content of citric acid (Penniston et al. 2008),
2010) methods for its wet and dry gluten contents and
that is a weak organic acid safe for consumption and
gluten index (method 38-12.02); diastatic activity in a
widely used as a natural preservative and acidifying agent
Falling Number equipment (Perten Instruments, Hägersten,
in foods and beverages (EFSA 2015). Differently than
Sweden) (method 56-81.03); and farinographic and exten-
observed for other fruits, limes present an increase in
sographic properties (methods 54-21.01 and 54-10.01,
acidity and a decrease in pH as their ripening progresses,
respectively). For the determination of farinographic
with citric acid content between 70 and 80 g.100 g-1 of the
properties, a Brabender farinograph model 810130 (Duis-
soluble solids of the juice (Ramana et al. 1981).
burg, Germany) was used, and the parameters obtained
Therefore, this study aimed to evaluate the feasibility of
were: water absorption (ABS, in g.100 g-1), dough
the production of clean label pan breads using lime juice
development time (DDT, in min), stability (STA, in min)
and enzymes, assessing their effects on technological and
and mixing tolerance index (MTI, in BU). The extenso-
preservation characteristics of pan breads.
graphic properties were evaluated in a Brabender exten-
sograph model 860703 (Duisburg, Germany), and the
parameters obtained were: resistance to extension (R, in
Material and methods
BU), extensibility (E, in mm) and ratio number (R/E), at
135 min.
Material

For the obtainment of lime juice, fruits of Tahiti lime Bread formulation
(Citrus latifolia Tanaka), acquired in a supermarket in the
city of Campinas, SP, Brazil, were washed, cut in halves Six different pan bread formulations were tested in this
and submitted to mechanical extraction in a domestic juice study and are presented in Table 1. All formulations con-
extractor Arno Citrus Power 70 W (Groupe SEB, São tained wheat flour, sugar (6 g.100 g-1 flour basis, f.b.),
Paulo, SP, Brazil). The juice was sieved for the removal of instant dry yeast (2 g.100 g-1 f.b.), salt (1.5 g.100 g-1
fibers and seeds and analyzed for its soluble solids content f.b.), canola oil (1 g.100 g-1 f.b.), maltogenic a-amylase
in an automatic refractometer (Reichert Technologies, (0.0060 g.100 g-1 f.b.) and fungal a-amylase
Depew, NY, USA). The total titratable acidity in ascorbic (0.0020 g.100 g-1 f.b.).

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Table 1 Tested pan bread

Ingredients Pan breads
formulations (g.100 g-1 flour
basis) T1 T2 T3 T4 T5 T6

Wheat flour 100 100 100 100 100 100

a a b a b b
Water
Sugar 6 6 6 6 6 6
Dry instant yeast 2 2 2 2 2 2
Salt 1.5 1.5 1.5 1.5 1.5 1.5
Canola oil 1 1 1 1 1 1
Fungal a-amylase 0.0020 0.0020 0.0020 0.0020 0.0020 0.0020
Maltogenic a-amylase 0.0060 0.0060 0.0060 0.0060 0.0060 0.0060
Ascorbic acid 0.005 0 0 0 0 0
Sodium stearoyl lactylate 0.43 0 0 0 0 0
Calcium propionate 0.20 0 0 0 0 0
Lime juice 0 0 20 0 20 10
Phospholipase 0 0 0 0.0015 0.0015 0.0015
Fungal xylanase 0 0 0 0.0050 0.0050 0.0050
CL clean label, LJ lime juice, P phospholipase, X fungal xylanase. T1 (market standard); T2 (CL); T3
(CL ? 20 g.100-1 g LJ); T4 (CL? P ? X); T5 (CL ? P ? X ? 20 g.100 g-1 LJ); T6
(CL ? P ? X ? 10 g.100 g-1 LJ)
a
Amount adjusted considering wheat flour water absorption determined in the farinograph
b
Amount adjusted considering wheat flour water absorption determined in the farinograph and lime juice
soluble solids content

In order to identify the technological and preservative Pan bread manufacturing process
effects of lime juice and enzymes in pan breads, the for-
mulations tested were: T1, representing a common for- Pan breads were manufactured in 1.5 kg flour batches,
mulation of Brazilian commercial pan bread, considered as using the modified straight dough method. The dry ingre-
‘‘market standard’’, with the addition of 0.0050 g.100 g-1 dients (flour, sugar, yeast, salt, enzymes and additives)
f.b. of ascorbic acid oxidant, 0.43 g.100 g-1 f.b. of sodium were homogenized at slow speed (1st speed) for 1 min, in a
stearoyl-2-lactylate (SSL) emulsifier and 0.20 g.100 g-1 VMI SPI 11 spiral mixer (VMI Pétrins et Mélangeurs,
f.b. of calcium propionate preservative; T2, as the control Montaigu, France). After homogenization, cold water (at
clean label formulation, without additives, enzymes or approximately 4 °C) was added according to wheat flour
lime juice; T3, clean label formulation without additives or water absorption (55 g.100 g-1 f.b.), obtained from the
enzymes, with the addition of 20.4 g.100 g-1 f.b. of lime farinographic analysis. For the formulations containing
juice; T4, clean label formulation without additives or lime lime juice, the total amount of water remained the same,
juice, with the addition of enzymes phospholipase and with water additions (26.8 g.100 g-1 f.b. in T3 and T5, and
fungal xylanase; T5, clean label formulation without 40.9 g.100 g-1 f.b. in T6) considering the water present in
additives, with the addition of enzymes phospholipase and lime juice, discounting its soluble solids content. Further
fungal xylanase, and 20.4 g.100 g-1 f.b. of lime juice; and mixing was carried out during 4 min at slow speed. After
T6, clean label formulation with the addition of enzymes this period, the mixing speed was increased to 2nd speed.
phospholipase and fungal xylanase, and 10.2 g.100 g-1 f.b. The canola oil was added after complete dough hydration
of lime juice. (approximately after 8 min of mixing at 2nd speed). Mix-
Lime juice quantities were defined based on the ascorbic ing continued until complete dough development and this
acid content of the lime juice and also taking into account time was monitored.
preliminary tests, where quantities of up to 30 g.100 g-1 After mixing, dough was divided into 200 g portions,
were tested, and an adverse effect on taste was noted. Due which were rounded in an automatic rounder (Prática,
to these factors, we set a maximum quantity of lime juice. Pouso Alegre, MG, Brazil) and left to rest for 10 min under
The number of replicates produced was 8 pan breads for a plastic film, to avoid dehydration of the dough surface.
each formulation. After resting, dough portions were molded in an automatic

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molder (Prática, Pouso Alegre, MG, Brazil), put in greased Pan bread pH
pans (height 4.8 cm 9 width 20.2 cm 9 depth 9.0 cm)
and taken to a proofing chamber (Klimaquip, Prática, Pan bread pH was measured (10 g bread crumb was sus-
Pouso Alegre, MG, Brazil), adjusted to 35 °C and pended in 100 mL distilled water), according to method
[ 80 g.100-1 g relative humidity (RH). The proofing time 02-52.01 (AACCI 2010), using a pH-meter model 300 M
of each dough formulation was evaluated by pressing a (Analyser, São Paulo, SP, Brazil), in triplicate, after 1, 5
fingertip on the dough surface until the perception of a and 8 days of storage.
lower resistance of the fermented dough to pressure. Fer-
mented doughs were baked in an electric forced convection Pan bread crumb firmness
oven (Prática, Pouso Alegre, MG, Brazil), at 175 °C, for
12 min. Baked breads were removed from the pans, cooled Pan bread crumb firmness was evaluated according to
for 2 h, sliced and packaged in polyethylene bags, closed method 74-09.01 (AACCI 2010), in six replicates, using a
with twist ties, and stored at room temperature texture analyzer model TA-XT2i (Stable Micro Systems,
(25 ± 1 °C). During breadmaking and bread handling, Surrey, UK), with a 25 kg load cell, P36/R probe and
good manufacturing practices and hygiene procedures were software developed by the manufacturer. Two central slices
observed in order to avoid product contamination, espe- of pan bread (1.2 cm thick each) were overlaid and sub-
cially after baking. mitted to compression by the probe. Maximum compres-
sion force represented crumb firmness, registered in gf.
Parameters set in the texture analyzer were compression
Dough pH before and after proofing
force: 20 g; pre-test speed 1.0 mm/s; test speed: 1.7 mm/s;
post-test speed: 3.0 mm/s; and sample compression:
The pH of dough was measured immediately after mixing
40 g.100 g-1.
and after proofing. The analysis was made in triplicate,
using 10 g of dough suspended in 100 mL distilled water,
Evaluation of pan bread preservation
according to method 02-52.01 (AACCI 2010), using a pH-
meter model 300 M (Analyser, São Paulo, SP, Brazil).
The indication of pan bread microbiological deterioration
was evaluated by visual inspection of the presence or
Specific volume
absence of mold colonies on the surface, with photographic
registration. From the total of 8 loaves, the number with
Pan bread loaf volume was evaluated by millet displace-
visible mold colonies was quantified from the 1st to the 8th
ment, according to method 10-05.01 (AACCI 2010), and
day of storage of breads.
weight was measured in a semi-analytical balance. Specific
volume was calculated by the ratio between loaf volume
Statistical analysis
and weight (cm3.g-1). The analysis was made in duplicate,
after crumb center temperature reached room temperature
Results for pH, color, specific volume, firmness and aw are
(25 °C, measured with a thermometer).
expressed as means ± standard deviations. Analysis of
variance (one-way ANOVA) and Tukey’s test were carried
Pan bread crust and crumb color out for comparison between means (p \ 0.05), using the
software SISVAR, version 5.6 (Ferreira 2000).
Pan bread crust and crumb color was evaluated in tripli-
cate, using a colorimeter model LabScan XE (HunterLab,
Reston, VA, USA) with 108 observation angle and D65 Results and discussion
illuminant. The parameters obtained were L* (lightness),
a* (green to red) and b* (blue to yellow). Wheat flour characterization

The wheat flour used presented diastatic activity of

Pan bread crust and crumb water activity 470 ± 18 s. Wet and dry gluten contents, and gluten index
were 24.7 ± 0.4 g.100 g-1, 9.2 ± 0.0 g.100 g-1, and
Water activity (aw) of pan bread crust and crumb was 95.7 ± 2.9, respectively. In the farinograph, the results
measured during the storage period (days 1, 5 and 8) using obtained were: water absorption of 58.1 g.100 g-1, dough
an Aqualab Series 2 apparatus (Decagon Devices, Pullman, development time of 16.4 min, stability of 26 min and
WA, USA), at 25 °C, and the measurements were con- mixing tolerance index of 12.5 BU, indicating a strong
ducted in triplicate. flour. In the extensograph, wheat flour presented the

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following results: 737 ± 27 BU, 113 ± 4 mm and gluten suspensions caused structural modifications and a
6.5 ± 0.1 for resistance to extension (R), extensibility decrease of the exposure of free-thiol groups, that are
(E) and ratio number, respectively, at 135 min. According important for disulfide bond formation. In the study of Su
to these parameters, the wheat flour presented suit- et al. (2019), on the use of organic acids in bread formu-
able characteristics for breadmaking, exhibiting a strong lations, the authors found that the addition of citric acid
flour profile (Pizzinatto 1997). caused an increase in dough stability, but also led to a
The amount of water used in the formulation of pan decrease of mixing tolerance. Thus, pH is a key factor in
breads is generally lower than the water absorption of flour bread dough performance and limits the increase of the
indicated by the farinograph analysis, and in the present quantity of added lime juice in pan breads, once in the
study it was considered 55 g.100 g-1 f.b., in order to mixing step the hydration and chemical interactions for the
obtain proper machinability. formation of the gluten network must adequately occur.
All doughs produced were maintained below 29 °C until
Process parameters the end of the mixing time, to avoid the beginning of yeast
activity before the dough molding step. Proofing time was
Process parameters high-speed mixing time, final dough 120 min. For straight-dough processes using S. cerevisiae
temperature and proofing time, besides dough pH before yeast, it is usual to have a proofing time range from 60 to
and after proofing, are shown in Table 2. 190 min (Cauvain and Young 2012a).
It was observed that pan breads containing lime juice In studies using sourdough, a prolonged proofing time is
required a longer high-speed mixing time to reach optimum necessary for dough acidification and production of com-
dough development. Independent of enzyme dosage level, pounds related to aroma and flavor derived from natural
it was observed that the higher the lime juice content, the microorganism metabolism. It is important to note that,
longer the time needed for dough to reach its optimum differently, dough acidification by lime juice occurs
development point (11 min for T6, prepared with instantly, and also impairs yeast activity (Su et al. 2019).
10.2 g.100 g-1 f.b. of lime juice, compared to 14 min for
T3 and T5, with 20.4 g.100 g-1 f.b. of lime juice). Dough pH
Therefore, the increase in acidity provoked by citric and
ascorbic acids present in lime juice, leading to a pH \ 5, Dough pH was measured before and after proofing, and
interfered in dough formation, retarding the optimal these values are shown in Table 2. Dough pH before
development of the gluten network. proofing showed that formulations without lime juice (T1,
Bagagli et al. (2014) reported that the variation of pH T2 and T4) presented no significant difference among each
had a significant effect on dough rheology. Garg et al. other, whilst formulations with lime juice (T3, T5 and T6)
(2019) noted that an acidic pH between 2 and 3 in wheat presented greater pH reduction in relation to those without

Table 2 Process parameters for pan bread manufacturing

Pan breads High-speed mixing Dough final Proofing time Dough pH before Dough pH after
time (min) temperature (°C) (min) proofing proofing

T1 (market standard) 6.5 25 106 5.51 ± 0.02a, A

5.20 ± 0.00a, B

a, A a, B
T2 (CL) 5 25.8 106 5.60 ± 0.10 5.20 ± 0.01
T3 (CL ? 20.4 g.100 g-1 LJ) 14 25.8 106 3.69 ± 0.01d, A
3.60 ± 0.07c, A

T4 (CL? P ? X) 5 25.9 108 5.55 ± 0.03a, A

5.18 ± 0.02a, B

c, A c, B
T5 14 27 104 3.94 ± 0.00 3.69 ± 0.01
(CL ? P ? X ? 20.4 g.100 g-1
LJ)
T6 11 27 98 4.16 ± 0.00b, A
4.11 ± 0.03b, B

(CL ? P ? X ? 10.2 g.100 g-1

LJ)
CL clean label, LJ lime juice, P phospholipase, X fungal xylanase
For pH: means ± standard deviations; Different lowercase letters in the same column indicate significant differences (p \ 0.05); Different
uppercase letters in the same row indicate significant differences (p \ 0.05)

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lime juice, increasing with the amount of lime juice added Water activity (aw) of crust and crumb
(20.4 or 10.2 g.100-1 g of lime juice).
The pH after proofing of the dough of pan bread for- As shown in Table 3, aw values above 0.9 were observed
mulations without lime juice (T1, T2 and T4) presented a for all pan breads. Different to other bakery products, the
reduction due to the generation of organic acids by yeast crust and the crumb of breads are highly susceptible to the
activity. According to Jayaram et al. (2013), in a straight- development of molds, due to their aw greater than 0.7, as
dough fermentation, the succinic acid produced by the indicated by Damodaran et al. (2008). In biscuits, for
yeast (S. cerevisiae) metabolism is the major contributor to example, that have high quantities of added fat and sugar,
acidification of the dough, determining its pH. low quantities of added water, and high baking tempera-
Wheat proteins plays an important role as buffering tures for small portions, the average aw is below 0.4,
substances of dough pH during fermentation, due to the explaining their stability against mold development (Čer-
presence of acid and basic regions in their molecules. The venka et al. 2006). An increase in crust aw was verified for
weak basic groups can absorb H? ions and, therefore, pH most pan breads, as observed in Table 3. This could be due
changes after fermentation are not as drastic as they could to the retrogradation of amylose and amylopectin, and the
be, considering the amount of acids formed (Sluimer consequent release and migration of water molecules from
2005). The pH of conventional dough after fermentation the crumb to the crust (Cauvain and Young 2012a).
remains between 4.7 and 5 (Cauvain and Young 2012b). In However, crumb aw did not present great changes during
doughs with lime juice (T3, T5 and T6), the direct effect of the storage period, which could be a positive quality
acid addition caused the reduction of pH to values between parameter as, according to Cauvain and Young (2012b),
3.5 and 4. These values are similar to sourdough, where the the higher the aw in the crumb, the fresher the bread is
acidification due to the bacterial synthesis of acids such as perceived by the consumer.
acetic and lactic acid can lead to pH values between 3.5
and 4 (Nogueira et al. 2015). Color of crust and crumb
Among the enzymes used in breadmaking, fungal
xylanase stands out due to its capacity to release water Regarding the instrumental color of bread crust and crumb,
from arabinoxylans, influencing dough viscosity and, thus, with values described in Table 4, for crust lightness (L*)
its machinability. This enzyme improves dough extensi- there was no significant difference between the breads
bility, that contributes to bread volume increase, due to an without addition of lime juice (T2 and T4) and the market
enhancement of dough extension (Gioia et al. 2017). Due standard formulation (T1). The addition of lime juice in
to its optimal pH between 3.5 and 4 (Damodaran et al. T3, T5 and T6 breads promoted the increase of the
2008), fungal xylanase could be a suitable option to obtain parameter L*, indicating that the crusts obtained were
softer and finer crumb in the manufacturing of pan bread lighter in color.
using lime juice, keeping good technological quality. The golden and dark color of breads comes from both
the caramelization reaction between sugars formed during
Bread pH during storage the fermentation process in the initial stages of baking, and
the Maillard reaction between amino acids and reducing
Bread pH was measured during the storage period, and is sugars, which also plays an essential role in the develop-
shown in Table 3. A slight increase in pH was observed ment of the aroma of breads (Cauvain and Young 2012b).
during the storage period. Volatile acids may be eliminated Among the three routes in the final stage of the Maillard
during baking, cooling and storage (Sluimer 2005), leading reaction, the 2,3-enolization route of the Amadori products
to these changes. Citric acid is classified as a non-volatile is favored at neutral pH, promoting darker colors (Brião
acid (Penniston et al. 2008), which can also be verified by et al. 2011). Doughs elaborated with the addition of lime
the maintenance of the pH of the pan breads with lime juice presented lower pH before and after proofing (range
juice, even after baking, that can also impact the sensory from 3.6 to 4.2), which may have consequently reduced the
characteristics of these breads. formation of products from the Maillard reaction, yielding
As well as the texture and color modifications of pan lighter breads in relation those without lime juice in their
breads observed in this study, it is also expected that lime formulations.
juice cause other sensory modifications, namely a charac- In relation to the chromaticity coordinate b* of the crust,
teristic citric aroma and sour taste, as can be observed in the breads evaluated did not present significant differences
different food products with lime juice, such as sweets, pies among them, all showing a tendency to a yellow shade
and salads. These modifications could be a limit for the after baking, which is an important aspect for acceptability.
quantities of lime juice added, as we noted in preliminary According to Sabanis et al. (2009), due to the presence of
tests. 70 g.100 g-1 reducing sugars in concentrated raisin juice,

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 Table 3 pH and water activity (aw) of pan breads during storage
Pan breads pH during storage Crust aw during storage Crumb aw during storage
1st day 5th day 8th day 1st day 5th day 8th day 1st day 5th day 8th day
ns, A ns, A ns, NS
T1 (market standard) 5.33 ± 0.03a, C 5.46 ± 0.01a, A 5.38 ± 0.02a, B 0.92 ± 0.00d, B 0.94 ± 0.00 0.95 ± 0.01 0.95 ± 0.00b, NS 0.95 ± 0.00ab, NS 0.96 ± 0.00
ns, B ns, A ns, A
T2 (CL) 5.23 ± 0.01b, B 5.38 ± 0.01b, A 5.39 ± 0.02a, A 0.94 ± 0.00b, C 0.94 ± 0.00 0.95 ± 0.00 0.96 ± 0.00a, A 0.95 ± 0.00bc, B 0.96 ± 0.00
ns, A ns, AB ns, AB
T3 (CL ? 20.4 g.100 g-1 LJ) 3.69 ± 0.01d, B 3.80 ± 0.04e, A 3.86 ± 0.03d, A 0.94 ± 0.00b, B 0.95 ± 0.00 0.95 ± 0.00 0.96 ± 0.00a, A 0.96 ± 0.00a, B 0.96 ± 0,00
ns, B ns, B ns, AB
T4 (CL? P ? X) 5.21 ± 0.01b, C 5.32 ± 0.01c, A 5.26 ± 0.02b, B 0.96 ± 0.00a, A 0.94 ± 0.00 0.95 ± 0.00 0.96 ± 0.00a, A 0.95 ± 0.00d, B 0.95 ± 0.00
ns, A ns, A ns, A
T5 (CL ? P ? X ? 20.4 g.100 g-1 3.66 ± 0.01d, B 3.81 ± 0.02e, A 3.80 ± 0.01e, A 0.94 ± 0,00c, B 0.94 ± 0.00 0.95 ± 0.00 0.96 ± 0.00ab, A 0.95 ± 0.00ab, B 0.96 ± 0.00
LJ)
B A A B ns, A ns, A NS cd, NS ns, NS
T6 (CL ? P ? X ? 10.2 g.100 g-1 4.14 ± 0.01c, 4.30 ± 0.02d, 4.27 ± 0.02c, 0.94 ± 0.00c, 0.94 ± 0.00 0.94 ± 0.00 0.95 ± 0.00b, 0.95 ± 0.00 0.95 ± 0.00
LJ)

CL clean label, LJ lime juice, P phospholipase, X fungal xylanase

Means ± standard deviations; Different lowercase letters in the same column indicate significant differences (p \ 0.05); different uppercase letters in the same row indicate significant
differences (p \ 0.05), during the storage period (1, 5 or 8 days). ns or NS: non-significant
J Food Sci Technol (May 2021) 58(5):1819–1828

Table 4 CIELab color parameters (L*, a* and b*) for crust and crumb, specific volume and firmness of pan breads during storage
Pan breads Crust color Crumb color Specific volume Firmness during storage (N)
(cm3.g-1)
L* a* b* L* a* b* 1st day 5th day 8th day
ns B A AB
T1 (market standard) 66.19 ± 4.21ab 15.18 ± 1.74abc 36.66 ± 1.49 86.01 ± 0.19a 0.61 ± 0.15bc 18.39 ± 0.36d 4.66 ± 0.07a 3.00 ± 0.17e, 6.45 ± 0.49c, 5.76 ± 0.11f,
ns B A AB
T2 (CL) 55.90 ± 2,30c 18.44 ± 0.27a 36.98 ± 0.86 80.95 ± 0.22d 0.90 ± 0.10a 20.90 ± 0.11b 4.08 ± 0.11b 6.66 ± 0.17c, 13.79 ± 0.51bc, 12.50 ± 0.06d,
-1 a c ns c a a d a, B a, A a, B
T3 (CL ? 20.4 g.100 g LJ) 70.71 ± 1.45 13.06 ± 0.83 38.17 ± 1.34 82.03 ± 0.36 1.03 ± 0.06 22.78 ± 0.26 2.75 ± 0.01 14.23 ± 0.03 26.49 ± 0.35 29.30 ± 0.05
ns B A A
T4 (CL? P ? X) 59.09 ± 3.39bc 17.47 ± 1.06ab 38.12 ± 0.49 80.03 ± 0.51e 0.82 ± 0.07ab 20.83 ± 0.51b 4.30 ± 0.20ab 4.65 ± 0.07d, 10.10 ± 0.32bc, 9.17 ± 0.06e,
ns cd C B A
T5 70.94 ± 2.83a 12.42 ± 2.27c 38.80 ± 1.60 81.18 ± 0.25 1.04 ± 0.13a 22.66 ± 0.20a 3.07 ± 0.06d 8.87 ± 0.08b, 16.94 ± 0.10b, 23.24 ± 0.05b,
(CL ? P ? X ? 20.4 g.100 g-1
LJ)
ns C B A
T6 69.29 ± 2.98a 14.24 ± 1.91bc 39.53 ± 1.00 84.24 ± 0.23b 0.52 ± 0.08c 20.01 ± 0.12c 3.50 ± 0.04c 6.76 ± 0.06c, 13.68 ± 0.18bc, 16.51 ± 0.04c,
(CL ? P ? X ? 10.2 g.100 g-1
LJ)

CL clean label, LJ lime juice, P phospholipase, X fungal xylanase, L* lightness, a* positive values (red) and negative values (green), b* positive values (yellow) and negative values (blue)
Means ± standard deviations; Different lowercase letters in the same column indicate significant differences (p \ 0.05); Different uppercase letters in the same row indicate significant
differences (p \ 0.05), during the storage period (1, 5 or 8 days); ns: non-significant
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(20.4 g.100 g-1 f.b. of lime juice) and T6 (10.2 g.100 g-1
f.b. of lime juice) on the same day of shelf-life showed
higher values of firmness for the former, indicating that the
higher the lime juice dosage, the denser the crumb.

Bread specific volume

The results for the specific volume of the loaves are shown in
Table 4. It was observed that the loaves containing
20.4 g.100 g-1 of lime juice (T3 and T5) presented the
lowest specific volumes and did not show significant dif-
ferences between each other, but differed from the other pan
breads. They were followed by the loaves containing
10.2 g.100 g-1 of lime juice (T6), that presented the second
lowest specific volumes. This effect may mainly be due to the
inhibitory effect of acidity (dough pH shown in Table 2) on
yeast fermentation, since the ideal pH for this process stage is
between 5 and 5.5 (Cauvain and Young 2012a).
It was also observed that not only lime juice addition
caused a specific volume reduction, as in the other clean
label breads (T2 and T4), the specific volume was also
lower when compared to the market standard (T1). This
Fig. 1 Pan breads visual aspects. a Top view of loaves after produc- effect may be mainly due to the lack of oxidants and
tion—T1 to T6 (1st day of storage), b Sliced loaves after production—
T1 to T6 (1st day of storage), c Pan breads at the end of shelf-life—T1 emulsifiers in T2 and T4, as these ingredients were present
to T6 (8th day of storage). T1 (market standard); T2 (CL); T3 in T1, and there was no significant pH difference among
(CL ? 20.4 g.100 g-1 LJ); T4 (CL ? P ? X); T5 (CL ? P ? X ? them.
20.4 g.100 g-1 LJ); T6 (CL ? P ? X ? 10.2 g.100 g-1 LJ). CL clean
label, LJ lime juice, P phospholipase, X fungal xylanase (color
figure online) Crumb firmness during storage

there was an enhancement of the Maillard reaction during The results obtained for crumb firmness of pan breads are
baking, leading to a darker color of breads. A similar result shown in Table 4. Breads from the market standard for-
was obtained by Lasekan et al. (2011) in their study of mulation (T1) presented the lowest crumb firmness on all
breads with the addition of pineapple juice. In the case of of the shelf-life days evaluated. Breads from formulation
lime juice, the lower content of reducing sugars, when T6, which presented phospholipase and fungal xylanase
compared to other fruits, led to a lower rate of the Maillard enzymes, associated with 10.2 g.100 g-1 f.b. of lime juice,
reaction in bread crusts. maintained similar crumb softness behavior to formulation
All formulations tested presented lower crumb lightness T2 (clean label, without enzymes or lime juice) during the
(L*) than the market standard, which may be related to the storage period. The firmest loaves were those from T3
color of lime juice and to the volume reduction provoked (clean label with 20.4 g.100 g-1 f.b. of lime juice and no
by its addition. The removal of additives, as well as the enzymes) and T5 (clean label with 20.4 g.100 g-1 f.b. of
addition of lime juice and enzymes in pan breads con- lime juice with phospholipase and fungal xylanase
tributed to increase the chromaticity coordinates a* and b* enzymes). These were also the formulations with the
of the crumb, highlighting reddish and yellowish shades, lowest specific volumes. Thus, texture can also be a con-
which may be due to carotenoids and chlorophyll, that are sequence of a lower volume and a denser crumb. Despite
the colored compounds of lime juice (Sandhu et al. 2006). the addition of phospholipase and fungal xylanase to obtain
As shown in Table 4, the yellowish shade provided by the a well-developed dough, the best results in terms of volume
added lime juice, allied to the effect of a higher density and softness were not produced, due to a poor gas pro-
crumb, contributed to the darker aspect of the crumb, as duction by the yeast, probably partially injured by the
suggested by the comparison of chromaticity coordinate a* abrupt pH reduction caused by the higher dosage of lime
for T3 and T5, which received 20.4 g.100 g-1 f.b. of lime juice. When yeast fermentation occurs adequately, bread
juice, and T6, whose lime juice dosage was 10.2 g.100-1g crumb resembles a sponge, also increasing volume (Slui-
f.b. Also, the comparison of firmness for T3 and T5 mer 2005).

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Fig. 2 Evaluation of pan breads preservation. T1 (market standard, T6 (CL ? P ? X ? 10.2 g.100 g-1 LJ, green). CL clean label, LJ
purple); T2 (CL, yellow); T3 (CL ? 20.4 g.100 g-1 LJ, gray); T4 lime juice, P phospholipase, X fungal xylanase. Total of eight
(CL ? P ? X, red); T5 (CL ? P ? X ? 20.4 g.100 g-1 LJ, blue); replicates of pan breads for each formulation (color figure online)

Breads with less lime juice (10.2 g.100 g-1 f.b.) and the was expected as these breads did not have calcium propi-
presence of phospholipase and fungal xylanase enzymes onate or lime juice. It also showed that lime juice, in the
maintained a softer crumb during storage, suggesting that proposed concentrations, influenced shelf-life prolonga-
the dough produced with these two enzymes was well- tion, since pan breads with higher amounts of added juice
developed and was able to retain the gas produced by the (T3 and T5) presented the lowest quantities of apparent
yeast. Softness along the shelf-life period is an important molds until the 8th day of storage. That could be due to pH
quality characteristic for the consumer and is also associ- reduction caused by acidification. Similar results of shelf-
ated with freshness of breads (Cauvain and Young 2012a). life extension were found by Scarnato et al. (2017) for
sourdough breads with the addition of transglutaminase and
Mold development during storage without preservatives, where no visible molds were
observed until 7 days of storage at room temperature
Considering the perception of visible spoilage as a (24 ± 2 °C), and this was related to the presence of acids
parameter that leads consumers to discard pan breads, we produced by microorganisms during the fermentation pro-
considered the presence of any visible mold colonies as a cess and consequent pH reduction.
quality parameter. The molds observed on the pan breads
formed fuzzy colonies, with a rounded shape, cottony
appearance, and green, yellow and black coloration, as can Conclusion
be observed in Fig. 1.
According to Legan (1993), the most common bread In this study, we have shown that the addition of lime juice
spoilage molds are Penicillium spp., and also Aspergillus to pan bread formulations promoted an increase of the
spp., mainly in tropical countries. These molds are light period of conservation of these breads related to acidifi-
colored, white, grey, or blue. Another mold, Rhizopus cation. However, lime juice promoted modifications in
stolonifer, known as ‘‘bread mold’’, visually presents black dough characteristics and yeast activity that reflected in the
colored sporangiophores (reproductive structures). The baking quality of the pan breads, resulting in volume
growth of R. stolonifer is reduced at pH below 3 and totally reduction, loss of softness and modification of crumb and
inhibited only at pH below 2.5 (Amiri et al. 2011). Thus, crust color. The association of lime juice and enzymes was
we still observed its characteristic spores on pan breads, important to obtain breads with volume, softness and
even with the addition of lime juice and consequent pH conservation closer to the control formulation (market
reduction. standard), even after storage, without the use of additives.
The results obtained for the shelf-life of pan breads are Among the enzymes used, we highlight fungal xylanase,
shown in Fig. 2. Pan breads T2 and T4, both without which has its effect potentiated in the pH range of the
the addition of lime juice, presented apparent molds in all dough with lime juice addition. Based on our results, we
breads before the others, after 5 days of storage. This result strongly recommend the continuity of these studies for

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more detailed sensory and microbiological analysis of the Food additives. Intechopen, London, pp 147–166. https://doi.org/
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Acknowledgements The authors are thankful to the National metabolites in fermenting wheat straight-dough reveals succinic
Council for Scientific and Technological Development (CNPq) for the acid as pH-determining factor. Food Chem 136:301–308. https://
doctoral scholarship granted to Michele Scarton (Process doi.org/10.1016/j.foodchem.2012.08.039
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for the donation of the raw materials used in this study (wheat flour microstructure, staling and textural properties of wheat bread.
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