LWT - Food Science and Technology xxx (2018) xxx-xxx

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LWT - Food Science and Technology

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Influence of acerola pulp concentration on mead production by Saccharomyces
cerevisiae AWRI 796
Thaíse Souza Amorima⁠ , Solimar de Brito Lopesb⁠ , Jose Ailton Conceição Bispob⁠ ,

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Carlos Francisco Sampaio Bonafec⁠ , Giovani Brandão Mafra de Carvalhob⁠ , Ernesto Acosta Martínezb⁠ ,⁠ ∗⁠
a
Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana (UEFS), Av. Transnordestina s/n, Bairro Novo Horizonte, 44036-900, Feira de Santana, BA, Brazil
b
Departamento de Tecnologia, Universidade Estadual de Feira de Santana (UEFS), Av. Transnordestina s/n, Bairro Novo Horizonte, 44036-900, Feira de Santana, BA, Brazil
c
Departamento de Bioquímica e Biologia Tecidual, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Rua Monteiro Lobato, 255, 13083-862, Campinas, SP, Brazil

ARTICLE INFO ABSTRACT

Keywords:
Acerola

D In this work the influence of acerola (Malpighiae marginata DC) pulp, at concentrations of 0, 10, 15, 25 and
30%, on mead production by Saccharomyces cerevisiae AWRI 796 was evaluated. A novel approach based on
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Honey cell growth to obtain high accuracy fits for the data and to estimate optimization parameters such as Gibbs free
Mead energy was used. Fermentation was done using 107⁠ cells/mL at pH 5.0 and 30 °C for up to 288 h. The addition
Saccharomyces cerevisiae
of increasing concentrations of acerola pulp progressively enhanced the cell growth, with the highest cell con-
centration reached being ∼2.09 × 108⁠ cells/mL. Under these conditions, the lowest Gibbs free energy of growth
was −4.81 kJ/mol after fermentation for 288 h at a pulp concentration of ∼18%. The velocity of substrate con-
sumption was −14 × 10− ⁠ 3
A.U./h whereas the velocity of ethanol formation was 17 × 10−
⁠ 3
A.U.⁠ /h with ethanol
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production of 15.2% v/v or 120.1 g/L.

anaerobic conditions (Knauf & Kraus, 2006; Lima, Basso, & Amorim,
1. Introduction 2001).
Mead production is still largely an empirical and homemade process.
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Mead is perhaps the oldest alcoholic beverage and is produced from
honey. Mead contains many nutritional elements required by the body
and has favorable effects on digestion, metabolism and the treatment
of anemia and chronic diseases of the gastrointestinal tract (Gupta &
Sharma, 2009). Honey, a natural food produced by honey bees from
flower nectar or plant secretions, contains a complex mixture of carbo-
hydrates (mainly glucose and fructose), organic acids, lactones, amino
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In the last 15 years, there have been attempts to improve the process
of mead production, such as the use of a better fermentation agent
(Fernandes, Locatelli, & Sacartazzini, 2009; Pereira, Mendes-Ferreira,
Oliveira, Estevinho, & Mendes-Faia, 2013, 2009), the use of a wort
formulation with salts (Fabian, 1935; Ferraz, 2015; Pereira,
Mendes-Ferreira, Estevinho, & Mendes-Faia, 2015; Steinkraus & Morse,
1966), and the use of mixtures of honey with fruits and vegetables such

acids, minerals, vitamins, enzymes, pollen, wax and pigments (Lemos,
Santos, & Santos, 2010). This composition accounts for the widespread
use of honey to sweeten food and improve the palatability of medicines.
Honey is generally fermented using the yeast Saccharomyces cere-
visiae, the most important microorganism in alcohol fermentation be-
cause of its high capacity for fermentation, its high tolerance to ethanol
and other inhibitors formed during the pre-treatment of raw materi-
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as banana, berry, soya, grape, black rice, apple, coconut milk (Balogu
& Towobola, 2017; Ferraz, 2015; Gupta & Sharma, 2009; Koguchi,
Saigusa, & Teramoto, 2009), organic acids (Mendes-Ferreira et al., 2010;
Sroka & Tuszyński, 2007) and pollen (Roldán, van Muiswinkel, Lasanta,
Palacios, & Caro, 2011). Some investigations have also sought to im-
prove the quality of honey used (Kempka & Mantovani, 2013; Koguchi
et al., 2009), in addition to attempts to improve the fermentation

als and during fermentation, and its capacity for rapid growth under process (Czabaj, Kawa-Rygielska, Kucharska, & Kliks, 2017; Gomes,
2010; Gomes et al., 2013; Iglesias et al., 2014; Morse & Steinkraus,
1971; Navrátil, Šturdík, & Gemeiner, 2001; Pereira,

∗ Corresponding author.
Email address: ernesto.amartinez@yahoo.com.br (E.A. Martínez)

https://doi.org/10.1016/j.lwt.2018.07.009
Received 5 October 2017; Received in revised form 21 March 2018; Accepted 7 July 2018
Available online xxx
0023-6438/ © 2018.
T.S. Amorim et al.
Mendes-Ferreira, Oliveira, Estevinho, & Mendes-Faia, 2014) as well as
the quality (Ukpabi, 2006) and sensory parameters (Gomes et al., 2015;
Rivaldi, Silva, Coelho, Tomazi, & Maciel, 2009; Roldán et al., 2011;
Vidrih & Hribar, 2007) of the final product.
Honey-based drinks have little body and are very sweet, so fruits in
the form of juices, pulps or pieces can be added to contribute to acidity,
and growth factors can be used to enhance the fermentation and senso-
rial characteristics (Gupta & Sharma, 2009; Koguchi et al., 2009). Fruits
and their pulps have been highly recommended because of their rich-
ness in carbohydrates, fibers, minerals, vitamin C, carotenoids, pheno-
lic substances and sulfuric substances, and also because of their antiox-
idant action that helps to maintain a balance between production and
elimination of reactive oxygen species and other related compounds,
thereby attenuating free radical-induced damage to cells (Maia, Sousa,
& Lima, 2007). Acerola pulp, which is very appreciated for its flavor
and color (Johnson, 2003; Pino & Marbot, 2001), has a high level of
ascorbic acid (1000–4500 mg/100 g of fruit) and a total phenolic con-
tent of 835 ± 32 mg and 449 ± 10 mg/100 g for aqueous and hydroalco-
holic extracts, respectively. These levels of ascorbic acid and phenolic
compounds indicate a high antioxidant capacity (Vieira, Sousa, & Lima,
2011). In this work, we examined the influence of acerola pulp concen-
tration on mead production by S. cerevisiae AWRI 796.
2. Materials and methods
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2.1. Raw material
Wild flower honey was acquired through the Beekeeping Cooper-
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ative of Ribeira do Pombal (COOARP) located in the city of Ribeira
do Pombal, BA, Brazil. Commercial acerola pulp [4.0 ± 0.2% reducing
sugars, 96.0 ± 0.2% non-reducing sugars, 685.00 ± 49.81 mg vitamin C/
100 g, 8.69 ± 0.45 o⁠ Brix, pH 3.23 and 0.61 ± 0.09% proteins, assayed as
previously described by Bastos, Martinez, and Souza (2016)] was pur-
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chased in markets at Feira de Santana city, BA, Brazil.
2.2. Yeast propagation
The experiments were done with S. cerevisiae strain AWRI 796, a
commercial yeast from Mauri Yeast (Burns Philp, Sydney, Australia)
that was obtained as freeze-dried cultures from the Australian Wine Re-
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search Institute culture collection (Adelaide, Australia). A 30 °Brix blend
containing distilled water and honey was prepared and autoclaved at
120 °C for 15 min in a 125 mL Erlenmeyer flask containing 50 mL of
medium. For all experiments, a starter culture was prepared by adding
0.2 g of lyophilized yeast (S. cerevisiae AWRI 796) to a shaker (Tecnal
TE-420) followed by incubation at 30 °C and 150 rpm for 24–48 h. The
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initial appropriate amount of inoculum (at least 108⁠ cells/mL) was de-
termined by counting in a Neubauer chamber (Cadena-Herrera et al.,
2015).
2.3. Preparation and treatment of must
All fermentations were done in triplicate using a system that con-
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sisted of 500 mL flasks containing 250 mL of wort mixture and fitted
with an airlock valve used to release CO2⁠ produced during fermenta-
tion. This CO2⁠ displaced the air and served as a blanket, allowing the
yeast to grow under anaerobic conditions. Water supplemented with
yeast extract (5 g/L), malt extract (5 g/L), peptone (10 g/L), magne-
sium chloride (0.05 g/L), ammonium sulfate (0.3 g/L) and dibasic am-
monium phosphate (0.05 g/L) was autoclaved at 110 °C for 10 min. Fur-
ther addition of honey resulted in final sugar concentrations of 50.9 g/L
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LWT - Food Science and Technology xxx (2018) xxx-xxx
for sucrose, 99.9 g/L for glucose and 118.9 g/L for fructose. Different
quantities of acerola pulp were then added to obtain final pulp concen-
trations of 10, 15, 20, 25 and 30%. Each wort sample was adjusted to
30 °Brix by adding honey and the pH of the wort was adjusted to pH
5.0 using calcium carbonate or lactic acid. A yeast inoculum (108⁠ cells/
mL) was added to each assay to yield an initial cell concentration of
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∼107⁠ cells/mL in must. During alcohol fermentation, the flasks were
incubated at 30 °C in a biochemical oxygen demand (BOD) incubator
(model Q315M25, Quimis greenhouse incubator, Diadema, SP, Brazil)
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without shaking. The total length of fermentation was 288 h.
2.4. Monitoring of fermentation
At the end of each 24-h period, samples were obtained aseptically to
assess the fermentation and growth parameters. Cells were counted with
a syringe-type system and cell viability was assessed using methylene
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blue in a Neubauer chamber. The concentration of soluble solids (o⁠ Brix)
was determined with a digital portable refractometer (Reichert Tecnal
AR-200, Piracicaba, SP, Brazil). The alcohol content (% v/v) was mea-
sured with a DDM 2911 bench densitometer from Rudolph Analytical
Research (De Melo, Araújo, Bispo, Del Bianchi, & De Carvalho, 2017).
2.5. Thermodynamic analysis
The results were analyzed thermodynamically essentially as de-
scribed elsewhere (Bispo, Bonafe, Santana, & Santos, 2015, 2012). Some
modifications were made to the previous procedure to allow the in-
clusion of more than two species states and the occurrence of reaction
rates involving distinct stoichiometric coefficients between reagents and
products. In this case, the experimental results and 3D surface plots
were fitted using Eq. (2) of Bispo et al. (2015) described below. Eq. (2)
was applied to n equal to the number of experimental points for yeast
cell growth (y) and time (x).
(1)
Considering the process of yeast duplication during growth, the equi-
librium constant should be expressed as:
(2)
where L is the initial concentration of yeast species prior to division
while L*⁠ corresponds to the yeast generated by division. Note that
the superscript (*) is used here only to differentiate the starting yeast
species from those arising from duplication. In addition, Eq. (2) gives
only a simplified reaction equation without the presence of other reac-
tants, e.g., substrate, and reaction products such as ethanol and carbonic
gas. However, the process described below allows independent charac-
terization of these other compounds.
Thus, considering the process described by relationship (2) and the
extent of process parameter (xL⁠ ) (Bispo et al., 2015), the concentration
(cL⁠ ) and (cL⁠ *) of each species at time zero (t0⁠ ) and any time (t) can be
described as:
(3)
(4)
whereas at time (t), the relationship would be
(5)
T.S. Amorim et al. LWT - Food Science and Technology xxx (2018) xxx-xxx

(6) figures, there was good agreement between the theoretical curves and
the experimental data, with random residual distribution and correla-
where corresponds to the initial yeast concentration. tion coefficients (R2⁠ ) >0.9 (data not shown). The use of a surface plot
As previously described [eq. (1) in Bispo et al. (2015)], from Eqs. (4) (Fig. 1c) allowed better visualization of the relationship between cell
and (5) the Gibbs free energy of yeast growth/duplication related to eq. growth and the pulp concentration and duration of fermentation. The
(1) can be defined as: influence of these parameters can be evaluated by analyzing the contour

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plots in relation to the cell concentration axis. Fig. 1d shows four regions
(7) of high cell growth (∼2 × 108⁠ cells/mL) after 48 h of fermentation at a
pulp concentration of 17%, after 168 h of fermentation at a pulp concen-

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where R is the gas constant and T is the absolute temperature (K). tration of ∼24%, after 216 h of fermentation at a pulp concentration of
Thus, the degree of growth for an initial yeast cell concentration (cL0 ∼10% and after 288 h of fermentation at a pulp concentration of ∼18%.
According to Pereira et al. (2013), the maximum cell biomass and the
) is given by:
maximum number of CFUs (S. cerevisiae Lalvin QA23 and ICV D47) were
obtained at a pitching rate (yeast net growth) of 108⁠ CFUs/mL. Pereira et
(8) al. (2015), subsequently examined effect of supplementing honey-must
with minerals and/or vitamins on the growth of yeast strains QA23 and

Introducing Eq. (8) into Eq. (7) yields the following relationship that
directly correlates the degree of growth (∝G) with the Gibbs free energy
of growth/duplication:
(9)
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ICV D47. The growth behavior of strain QA23 was similar in all fermen-
tations and the population almost reached 108⁠ CFUs/mL after 48 h. Inde-
pendently of the honey-must composition, the stationary phase of strain
ICV D47 started after 48 h of fermentation and after the population had
reached 7–8 x 107⁠ CFUs/mL; the number of CFUs/mL was slightly lower
in fermentations with vitamins and salts + vitamins. For both strains
and in all fermentations the population remained constant between 48 h

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and 168 h, and then decreased slightly after 288 h (Pereira et al., 2015).
Gomes et al. (2015) observed that in sweet and dry mead fermenta-
tions there was a lag phase of ∼10 h followed by an exponential phase
This equation shows that the energy change of reaction is obtained of ∼45 h duration.
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from the degree of growth, the initial yeast concentration and the spec- Fig. 1e shows the cell concentration as a function of pulp concen-
ified stoichiometry of reaction. Analogously, considering now the con- tration (0, 5, 10, 15, 20, 25 and 30%) at distinct times of fermenta-
version of substrate (glucose/fructose) into ethanol, the same procedure tion. The cell growth seen here agreed with the steady progressive mi-
should be applied. Thus, crobial growth (from 1.0 × 106⁠ cells/mL to 1.6 × 108⁠ cells/mL) observed
(10) during 60 days of fermentation of a honey-coconut milk blend using S.
cerevisiae (Balogu & Towobola, 2017). Cell growth from 105⁠ cells/mL to
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which yields 1.5–2.8 × 107⁠ cells/mL was also seen during honey fermentation supple-
mented with nitrogen and organic acids (Mendes-Ferreira et al., 2010).
According to Pereira, Dias, Andrade, Ramalhosa, and Estevinho (2009),
all yeast strains showed the same behavior at 10% (v/v) ethanol, al-
(11) though there was a decrease in cell viability and none of the strains
grew at ethanol concentrations of 15–20% (v/v).
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where the subscript R in ΔGR⁠ and αR⁠ are related, respectively, to the
Gibbs free energy and degree of reaction during ethanol formation, and
s0 is the initial substrate concentration.
In addition, the differentiation of the degree of reaction with respect
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Fig. 1f shows the degree of growth when the data were normalized
from 0 to 1. From these results and Eq. (9) it was possible to calcu-
late the Gibbs free energy of growth/duplication (Fig. 1g). Cell growth
was spontaneous under all conditions studied, with values ranging from
∼3.2 to 4.8 kJ/mol. Comparison of Fig. 1d,h indicated that the fermen-
tation time and pulp concentration that yielded the highest cell growth

to time, which can be determined based on cell growth, substrate deple-
tion and ethanol formation, yields
(12)
Eq. (12) provides νG⁠ , νS⁠ and νP⁠ , additional important parameters ob-
tained from the experimental data that are directly related to the veloc-
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(∼2.09 × 108⁠ cells/mL) were those that yielded the lowest Gibbs free en-
ergy of growth (−4.81 kJ/mol). For 1.84 × 108⁠ cells/mL, the process was
also spontaneous, with a free Gibbs energy of growth of −4.51 kJ/mol.
The surface profile for the velocity of growth during fermentation
can be obtained from Eq. (12) and is shown in Fig. 2a. Fig. 2b and c
shows that for 1.84 × 108⁠ cells/mL a higher growth rate, corresponding
to 0.0315 A.U./h, was obtained after ∼48 h of fermentation at a pulp

ities of growth, substrate depletion and product formation, respectively.
3. Results and discussion
Fig. 1a shows the experimental results (symbols) and curve fits
(lines) obtained with eq. (1) and Fig. 1b shows the respective percent-
age residuals determined from the fitted curves. As indicated in these
concentration of ∼17%. These figures also indicate regions of negative
growth that reflect greater cell death relative to cell division.
Application of the analytical approach described above to the eval-
uation of soluble solids in solution (S.S., °Brix) is shown in Fig. 3a
and b. The optimal conditions should contemplate an economy in the
most expensive component in mead fermentation, namely honey. Fig.
3c,d,e shows that such optimization is reached at 1.84 × 108⁠ cell/mL
after 168 h of fermentation at a pulp concentration of ∼24% and at
∼2.09 × 108⁠ cells/mL after 288 h of fermentation at a pulp concentration

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Fig. 1. A) Cell concentration [X] (symbols) as a function of fermentation time (h) and pulp concentration (%). Lines were fitted using the function described in Eq. (1). b) Residual plot
for the data fitted to the lines in panel a. c) Surface plot and d) contour plot of cell concentration (S. cerevisiae AWRI 796) as a function of fermentation time (h) and pulp concentration
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(%). e) Cell concentration [X] as a function of pulp concentration (%) in each fermentation time (h). f) Surface plot of the degree of growth as a function of fermentation time (h) and pulp
concentration (%). g) Surface plot and h) contour plot of the Gibbs free energy of growth ( Joule/mol) as a function of fermentation time (h) and pulp concentration (%).

of ∼18%. These optimal conditions represent an economy in the use of ∼1.7 × 10−
⁠ 2 A.U./h. Based on the stoichiometry of the reaction as 1:2 (1

honey (Fig. 3d and e). Fig. 3f–h corroborates these results since the °Brix
decreased over time and the negative velocity of reaction reached at
∼40 h indicated high substrate consumption and ethanol formation (Fig.
3h).
Fig. 4 shows a similar analysis for substrate consumption and
ethanol production. Fig. 4c,f shows a velocity of substrate consumption
of −1.4 × 10−⁠ 2
A.U./h, whereas the velocity of ethanol formation was
glucose => 2 ethanol), these data suggest that ∼39% of the substrate
was used to produce other cell compounds.
Substrate consumption was 49.1% and ethanol production was
14.7% (v/v; 116.2 g/L) in the absence of acerola pulp (Figs. 3a and
5a-d). The addition of 10–30% acerola pulp enhanced substrate con-
sumption (55–58%) and increased ethanol production (15.2–16.6%,
v/v, or

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Fig. 2. A) Surface plot of the velocity of growth (vG, expressed in absorbance units (A.U.)/h) as a function of fermentation time (h) and pulp concentration (%). b) Contour plot and c)
surface plot of the velocity of growth (VG⁠ ) as a function of fermentation time (h) and pulp concentration (%).
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120–127 g/L) after 288 h of fermentation (Figs. 3a and 5a-d). Similar
values for substrate consumption (58.5%, 54.5% and 42.9%) and lower
ethanol production (9 g/L, 8 g/L and 6.5 g/L) were obtained during the
fermentation of mead (16 o⁠ Brix) with honeydew, wild and angico honey,
respectively, using S. cerevisiae at pH 5.45 and 36 °C for 168 h (Kempka
& Mantovani, 2013).
Multiflorous and honeydew diluted in water (36 o⁠ Brix) incubated
with Saccharomyces bayanus and S. cerevisiae resulted in a substrate con-
sumption of 49.7–59.6% and ethanol content of 11.98–16.53% (v/v)
at the end of mead production (720 h) (Czabaj et al., 2017). In con
trast, higher substrate consumption (88.5%) and greater ethanol pro-
duction (8%, v/v) were reported by Ilha et al. (2008) for 21 o⁠ Brix af-
ter 84 h at pH 4.5 and 25 °C. Gomes et al. (2015) also observed high
rates of sugar consumption (80%) and low ethanol production (7.54
and 13.54%) with S. cerevisiae (pH 3.5, 35 °C) after sweet and dry
mead fermentations for 79 h and 198 h, respectively. Similar high sub-
strate consumption (82%) and ethanol production of 10.03–10.33% (v/
v) and 9.7–10.37% (v/v) were obtained in fermentations with S. cere-
visiae Lalvin QA23 and ICV D47, respectively (Pereira et al., 2013).

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Fig. 3. A) Soluble solid (S.S.; symbols) content of mead as a function of fermentation time (h) and pulp concentration (%). Lines were fitted using the function described in Eq. (1). b)
Residual plot for the data fitted to the lines in panel a. c) Surface plot and d) contour plot of soluble solids (S.S.) as a function of fermentation time (h) and pulp concentration (%). e)
Soluble solid (S.S.; symbols) content as a function of pulp concentration (%) in each fermentation time (h). f) Surface plot of the degree of reaction as a function of fermentation time (h)
and pulp concentration (%). g) Surface plot and h) contour plot of the Gibbs free energy of reaction (ΔGR⁠ , Joule/mol) as a function of fermentation time (h) and pulp concentration (%).

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Fig. 4. A) Surface plot of the velocity of substrate consumption (VB⁠ rix) as a function of fermentation time (h) and pulp concentration (%). b) Contour plot and c) surface plot of the pulp
concentration (%) as a function of velocity of substrate consumption (VB⁠ rix) and fermentation time (h). d) Surface plot of the velocity of ethanol production (VP⁠ ) as a function of fermenta-
tion time (h) and pulp concentration (%). e) Contour plot and f) surface plot of pulp concentration (%) as a function of the velocity of ethanol production (VP⁠ ) and fermentation time (h).
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Fig. 5. A) Ethanol production [P] as a function of fermentation time (h) and pulp concentration (%). Lines were fitted using the function described in Eq. (1). b) Residual plot for the data
fitted to the lines in panel a. c) Surface plot and d) contour plot of ethanol production as a function of fermentation time (h) and pulp concentration (%).

Lower ethanol production (10.7–11.4%) was reported by
Mendes-Ferreira et al. (2010) for honey fermentation after nitrogen
supplementation and the addition of organic acids under the follow-
ing initial conditions: 22 o⁠ Brix, pH 3.50–4.72, 105⁠ cells/mL of S. cere-
visiae UCD522
and 600 h of fermentation. A mead with an alcohol content of 10–17.2%
and a substrate consumption of 60% after nine days was obtained
from honey using commercial S. cerevisiae (20 g/L) at an initial pH
of 3.55. Ethanol production of 12.7–15.0% was reported by Ukpabi

7
T.S. Amorim et al.
(2006) for mead obtained with cassava (Manihot esculenta) floral honey
under farm conditions. Vidrih and Hribar (2007), who studied the
fermentation of chestnut, lime and honeydew meads, and Gupta and
Sharma (2009), who studied home-brewed and commercial meads
made from soya honey, reported ethanol production of 14.2% and
4.6–11.8 (v/v), respectively.
As shown here, S. cerevisiae AWRI 976 grew at ethanol concentra-
tions up to 16.6% (v/v). This finding suggests that acerola pulp may in-
crease the tolerance of yeast to ethanol.
4. Conclusion
The addition of acerola pulp during mead fermentation enhanced
cell growth (greater cell concentration) and increased substrate con-
sumption and ethanol production. This is the first study to examine the
influence of fruit pulp concentration on mead production and indicates
that acerola pulp could be a useful substitute for honey, thereby reduc-
ing the cost of mead production. A complementary sensorial analysis of
mead produced with acerola pulp is required to demonstrate its suitabil-
ity as a substitute for honey.
Conflicts of interest
The authors declare no conflict of interest.
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Acknowledgments
The authors thank the Beekeeping Cooperative of Ribeira do Pombal
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(COOARP), Ribeira do Pombal, BA, Brazil for providing the honey and
Dr. Stephen Hyslop for editing the English of the manuscript. This work
was supported by Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP), Brazil.
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