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Optimization and Kinetic Modeling of Honey

Fermentation for Laboratory and Pilot-Scale Mead
Production

Marta Cuenca, Amaury Blanco, Marta Quicazán & Carlos Zuluaga-

Domínguez

To cite this article: Marta Cuenca, Amaury Blanco, Marta Quicazán & Carlos Zuluaga-
Domínguez (2021): Optimization and Kinetic Modeling of Honey Fermentation for Laboratory
and Pilot-Scale Mead Production, Journal of the American Society of Brewing Chemists, DOI:
10.1080/03610470.2021.1966590

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Journal of the American Society of Brewing Chemists
https://doi.org/10.1080/03610470.2021.1966590

Optimization and Kinetic Modeling of Honey Fermentation for Laboratory

and Pilot-Scale Mead Production
Marta Cuencaa, Amaury Blancob, Marta Quicazánb and Carlos Zuluaga-Domínguezc
a
Facultad de Ingeniería – Programa de Ingeniería Química, Universidad de Cartagena, Cartagena, Colombia; bInstituto de Ciencia y
Tecnología de Alimentos, Universidad Nacional de Colombia, Bogotá DC, Colombia; cFacultad de Ciencias Agrarias – Departamento de
Desarrollo rural y Agroalimentario, Universidad Nacional de Colombia, Bogotá DC, Colombia

ABSTRACT KEYWORDS
Honey is usually employed for the elaboration of mead, an alcoholic beverage obtained through Assimilable nitrogen; bee-pollen;
fermentation with yeast. However, its production in many places is incipient and still has such honey-must; mead;
drawbacks as long process times, undesired fermentation, and sensory quality problems. In this Saccharomyces cerevisiae
research, a three-level factorial design was used to evaluate the effect of the initial honey
monosaccharide concentration (glucose and fructose) and yeast assimilable nitrogen content on
the alcoholic fermentation yield at a laboratory scale. Fermentation tests were conducted at 25 °C
for 20 days using Saccharomyces cerevisiae subsp. bayanus and bee-pollen as a nutrient source.
Residual sugars, assimilable nitrogen, glycerol, ethanol, and ethanol/sugar yield were calculated.
Optimization by Response Surface Methodology showed that maximal predicted yield (46.1%)
was obtained when sugar concentration and yeast assimilable nitrogen levels were 22% and
123 mg/L, respectively. Once the best conditions were determined, pilot-scale fermentations were
performed, and then, logistic and Gompertz models were adjusted. The results were consistent
with the results found on the laboratory scale, which suggest fermentable sugars and yeast
assimilable nitrogen content can vary between 21 to 25% and 120 to 150 mg/L, respectively, to
obtain a physical, chemical, and sensory acceptable beverage.

Introduction risk of delayed or arrested processes,[6] and improving the

Mead is a traditional alcoholic product, with 8 to 18%v/v
of ethanol, obtained by the alcoholic fermentation of
diluted honey using Saccharomyces cerevisiae.[1] This bev-
erage is important for beekeepers since it is considered a
potential alternative for adding value to honey and gener-
ating economic development and competitiveness. However,
in most countries, production volumes are very low and
its production still shows some drawbacks as long or
incomplete fermentations, unreached appropriate ethanol
content, and sensory quality problems, mainly due to high
osmotic stress and/or deficient nitrogen content in the
honey-must.[2]
Honey is a natural raw material with a high sugar (glu-
cose and fructose) concentration, but it lacks other nutri-
ents, such as nitrogen,[3] essential for yeast growth during
alcoholic fermentation. Carbohydrates are the most import-
ant nutrient for the growth and metabolism of yeast cells
since they are the main source of structural carbon and
energy. Yeast assimilable nitrogen (YAN) includes ammo-
nium ions, α-amino acids (except proline), some proteins,
small peptides, and nucleic acids.[4] YAN availability has
shown to be related to an increase in fermentation rate
and the production of volatile compounds responsible for
odors and aromas,[5] reducing fermentation length and the
sensory characteristics of mead.[7] Several studies have men-
tioned nitrogen additions such as ammonium salts, vita-
mins, amino acids, and commercial nutritive mixes to
optimize honey must composition.[7,8] In winemaking, a
minimum YAN concentration is recommended to avoid
problems during fermentation, and it can vary between 120
to 150 mg/L.[9] It has also been demonstrated that bee-pollen,
the main source of proteins, amino acids, lipids, minerals,
and vitamins for bees, improves fermentation kinetics, and
sensory properties when it is used as a nitrogen and nutri-
ent supplement in honey alcoholic fermentations.[10]
The optimization of both variables, sugar and YAN con-
tent, represents a critical stage in mead production.
Response Surface Methodology (RSM) is one of the most
common tools for optimization because it does not only
consider interactions between multiple factors but also
requires fewer experimental tests. RSM has been used for
modeling and optimization of alcoholic fermentation pro-
cesses [11,12] including honey fermentation. [13] This work
aimed to optimize the initial content of sugar and YAN of
honey-must to shorten the time for complete fermentation
and to maximize the ethanol/sugar yield. Optimization was
performed at the laboratory scale and then applied at pilot
scale production.

CONTACT Carlos Zuluaga-Domínguez cmzuluagad@unal.edu.co

Supplemental data for this article is available online at https://doi.org/10.1080/09585192.2021.1967422.
© 2021 American Society of Brewing Chemists, Inc.
2 M. CUENCA ET AL.
Experimental
Materials
Multifloral honey from Apis mellifera bees from the munic-
ipality of Rivera (Huila, Colombia, 2°46’42” N 75°14’35” W)
and dry bee-pollen from the municipality of Viracachá
(Boyacá, Colombia, 5°26’04” N 73°17’53” W) were used.
Both products were stored at 4 °C for a period not exceeding
six months. The yeast Saccharomyces cerevisiae subsp.
bayanus (Uvaferm BC®, Lallemand Inc, Canada) was selected
and kept in its original sealed packaging at 4 °C until use.
Characterization of honey and bee pollen
Honey was characterized according to the content of total
soluble solids, pH and free acidity, ash, and moisture,
according to AOAC methodologies. Bee-pollen was charac-
terized by measuring the moisture content, pH, acidity, total
protein, assimilable nitrogen, lipids, and ash, based on bib-
liographic reports and AOAC methodologies. Methods are
further described in the supplementary material.
Determination of yeast assimilable nitrogen (YAN)
content
YAN was measured based on the Sorensen formaldehyde
titration method, as described by Baroň.[14] Briefly, 50 mL
of sample was placed into a Falcon tube and the pH was
adjusted to a value of 8.0 by adding 1 N sodium hydroxide.
Then, 10 mL of neutralized formaldehyde (pH = 8.0) was
added. The sample was shaken and after one min it was
titrated with 0.0862 N sodium hydroxide to reach a stable
pH value around 8.0. The volume of sodium hydroxide
required was recorded and the content of YAN was calcu-
lated as indicated in Equation 1.
 mg N  vol NaOH ( mL ) *0.0862 N*14*1000
YAN  =
 L  sample vol (mL)
(1)
Inoculum preparation and yeast cells count
In all assays, inoculums were prepared according to man-
ufacturer directions: briefly, 0.4 g/L of lyophilized yeast was
fully dissolved and hydrated in enriched pasteurized honey
must at 37–40 °C under gentle agitation.
The monitoring of cell growth during the alcoholic fer-
mentation was conducted by counting colony-forming units
by an immersion plate method. For the initial yeast count,
0.4 g/L of lyophilized yeast was suspended in 50 mL of sterile
water at 39 °C for 20 min with gentle shaking. Successive
dilutions of the inoculum or honey-must sample (10−1 to
10−10) in peptone water (NaCl 0.85%, peptone 0.1%) were
performed. One mL of each dilution was spread onto YPD
agar media and incubated at 25 °C for 7 days in duplicates.
The inoculum yeast count was 1.6 × 106 CFU/mL.
Factorial design for micro-fermentation assay (0.2 L)
The effect of the initial concentration of sugars (glucose
and fructose) and YAN was evaluated according to a com-
plete factorial design 32. The factors and levels evaluated
were the content of fermentable sugars (22, 27, and 32%
w/w) and YAN (65, 130, and 195 mg/L, corresponding to
2.3, 4.6 and 7.0% w/w of pollen). Every treatment was pre-
pared by diluting with drinking water the amounts of honey
and bee pollen as required for the experimental design. The
initial pH of the fermentation media, which ranged between
4.2–4.3, was not adjusted as recommended by Hernández
et  al.[15] The mead was pasteurized at 65 °C for 20 min,
quickly cooled in a cold-water bath and then inoculated
with a yeast culture suspension prepared as explained later.
The treatment samples were incubated at 25 °C for 20 days.
All tests were carried out in triplicate.
Laboratory scale fermentations (3 L)
In this phase, the monitoring of the alcoholic fermentation
of honey was carried out in triplicate under optimal con-
ditions for the initial concentration of sugars (glucose and
fructose) and YAN as determined previously. The reactor
consisted of 316 stainless steel with a stirring system and
a Rushton turbine impeller with four flat blades, deflectors,
connections for sampling and gas exhaust, a thermocouple
well, heating blanket, and glass electrode for pH measure-
ments. During the alcoholic fermentation, samples were
taken periodically for 11 days and the monitoring and char-
acterization of mead was performed as explained later.
Fermentation at pilot-scale batches (100 L)
The third phase of experimentation was carried out in a
pilot plant, using a kettle with a thermal jacket, gas heating
and an electric stirring systems, and a Rushton impeller
with four flat blades, a conical bottom fermenter, and two
storage tanks. All equipment was 316 stainless steel.
Pasteurization was carried out at 65 °C for 20 min and cooled
by circulating cold water through the jacket of the kettle
until it reached 25 °C. Alcoholic fermentation was performed
at room conditions (temperature: 18–28 °C, average relative
humidity: 66%), and it was periodically monitored for
14 days, with sampling every 24 h. The fermentation was
performed in triplicate.
Monitoring and characterization of mead
The content of total soluble solids was measured by refrac-
tometry according to the OIV-MA-AS2-02 method. The pH
was monitored by potentiometry according to the
OIV-MA-AS313-15 method and volatile acidity (g acetic
acid/L) was determined according to the OIV-MA-AS313-02
method (Organization Internationale de la Vigne et du Vin,
2015). The determination of glucose, fructose, glycerol, and
ethanol was performed according to the AOAC 977.20 and
AOAC 979.23 (2012) using a high-performance liquid

chromatograph (Jasco Inc., Japan) equipped with a PU-2031
pump, CO-2065 oven, RI-2031 PLUS refractive index detec-
tor, and a 10 µm Sugar-Pak I column, 6.5 mm x 300 mm
(Waters Inc., USA). The assimilable nitrogen content was
determined by the Sorensen method, as described by
Baroň.[14] Sensory analysis of the final mead was also carried
out (see Supplementary Material).
Statistical analysis and mathematical modeling
Response surface methodology (RSM) was used as a tool
to optimize the conditions for mead production. The depen-
dent variable was ethanol/sugars yield Ye/s calculated with
Equation 2, as an indicator of the effectiveness of sugar
conversion to ethanol.
xE
Ye /s =
∆x S
(2)
Where XE is the final ethanol concentration in g/L and
ΔXS is the total sugar (glucose and fructose) consumption.
Meanwhile, the kinetic modeling was carried out using
Gompertz and logistic models. The Gompertz equation con-
sists of a double exponential function with three fitting
parameters describing an asymmetric sigmoidal curve
(Equation 3).
−k (t −c )
−e
y = a *e
(3)
Where a is the maximum potential value of the response
variable y when the independent variable t tends to infinity,
c is the inflection point of the curve, and k is a factor
strictly related to the slope of the curve. On the other hand,
the logistic model has a generalized form as presented in
Equation 4.[16]
dX  X 
= µmax X  1 −  these was total (see Table 1). The best treatments were found
dt  X max  (4)
Where X is the biomass concentration at time t, μmax is
the maximum specific growth rate, and Xmax is the maxi-
mum cell concentration.
In addition, the three and four-parameter logistic regression
models were employed. In the three-parameter logistic model
(Equation 5), x is the independent variable, y is the response
variable, A is the maximum value of the response variable,
d is the inflection point of the curve (where changes of direc-
tion), and p is a factor related to the slope of the curve.
A concentration of sugars at the lowest YAN levels (T00 and
y =
− p ( x −d )
1+e (5)
Journal of the American Society of Brewing Chemists 3
In the four-parameter logistic model (Equation 6), A1 is
the value of y when x approaches zero, and A2 is the value
of y when x approaches infinity.
A1 − A2
y = + A2
p
x 
1+ 
d  (6)
Statistical analysis was performed using SAS® 9.4 TS Level
1M1 software (SAS Inc., USA) by validating the assumptions
of normality and homoscedasticity through the Bartlett and
Levene tests, an analysis of variance (ANOVA) of several
factors, and a multiple comparison test of the treatments
using the Tukey test. All statistical tests were performed
with a significance level of 5%. The modeling of the kinetic
data and the validation of their significance was performed
using the OriginPro 2017 software (OriginLab, USA).
Results and discussion
Characterization of honey and bee-pollen
The honey had adequate physicochemical conditions that
allowed its use as raw material to produce mead, given its
high concentration of total soluble solids, mainly glucose
and fructose. Among the results obtained for the physico-
chemical characterization of bee pollen an important frac-
tion of fermentable sugars (32% w/w) stands out and a
high protein content coherent to what is reported in the
literature.[17] A ratio of YAN was found that corresponds
to 6% of the total nitrogen content present in bee-pollen.
The complete composition of honey and bee-pollen used
in the study is found in the supplementary material.
Micro-fermentation assay (0.2 L)
In all of the tests carried out at concentrations of 22% and
27% sugars (fructose/glucose of 1.1), the consumption of
at the lowest level of initial sugar concentration due to
higher yields. The opposite happened with the tests at 32%
sugar content, which presented a residual sugar content that
represented 12% of the initial sugar content, mainly fructose,
and high fructose/glucose ratios ranging between 5.4–8.6,
which indicated a preference for glucose uptake under con-
ditions of high sugar content. This situation is undesirable
in the production of mead as it increases the risk of con-
tamination of the mead and/or re-fermentation in the bottle,
as well as altering the organoleptic profile.
In general terms, the assimilable nitrogen consumption
by the yeast was low. The highest assimilable nitrogen con-
sumptions were found in the tests with the lowest initial
T01), together with the T12 test, suggesting a greater inabil-
ity to assimilate these compounds at higher concentrations
4 M. CUENCA ET AL.

of substrates. These maximum values ​​were between 13–23%
of the initial concentration of YAN, which correspond to
only 22% and 18% of the minimum level recommended in
the literature for wine production. In other treatments, the
assimilable nitrogen consumption was very low (tests T11
and T20) and even null (test T22).
These results indicate an excessive supplementation of
assimilable nitrogen, even at the lowest level evaluated. This
fact may be associated with other essential compounds and
nutrients provided by bee-pollen and important for yeasts,
such as minerals, amino acids, organic acids, salts, which
can enhance their metabolism, implying less nitrogen con-
sumption. It should also be noted that the nitrogen require-
ment is a characteristic that depends on the yeast strain
used and therefore is a parameter that must be determined
for each microorganism. In this case, this is typically a
strain having a low nitrogen requirement, corroborated with
the results obtained.
All meads presented high values ​​for ethanol content,
above 11% v/v, which represents a favorable result since this
minimizes the risk of contamination by acetic bacteria and
increases the conservation time in the bottle. The highest
yields were obtained for the tests with the lowest level of
sugars for all YAN concentrations, that is, T00, T01, and
T02, and significantly higher for the T01 test, which also
presented the highest alcohol content. On the contrary,
although the consumption of sugars had been carried out
in total in the tests T10, T11, and T12 and high values ​​had
been reached for the alcoholic degree, the conversion of the
sugars present was not efficient. This result suggests that
part of the substrate could be used for maintenance activities
or the production of secondary metabolites due to condi-
tions of osmotic stress in the fermentation honey-must (high
concentration of sugars and nitrogen, turbidity due to the
effect of adding pollen). This result was even more evident
in tests T20, T21, and T22, where the level of sugars in the
medium was higher. Similarly, it was observed that although
these meads had a high ethanol content, the production of
glycerol, a metabolite associated with conditions of osmotic
stress,[18] was significantly higher.
Glycerol is an important sensory compound in wine since
it contributes to softening sensations in the mouth and also
provides a sweet taste.[19] The glycerol content in wines
usually varies between 3–15 g/L, with an average value of
around 7 g/L.[20,21] All of the meads obtained in this phase
presented glycerol contents within this range.
It was found that as the initial concentration of sugars
in the honey-must increased, the nitrogen consumption
decreased, and the concentration of total residual sugars
rose. This finding was related to stimulation in glycerol
production as previously discussed. The ethanol content did
not vary markedly, again suggesting that under conditions
of osmotic stress, yeast cells opt for glycerol production.
The response surface obtained (see Figure 1a) presented
a turning point for the yield for which, within the range
of experimentation evaluated, a minimum condition was
observed towards higher values of​​ the concentration of sug-
ars and low values of
​​ the YAN content. This correlated with
the yields shown in Table 1, where the tests with 27% and
32% of initial sugar showed significantly lower results.
On the other hand, a region was found between the
intermediate levels of the YAN content and the lower levels
of the sugar concentration, in which, the yield exhibited an
increasing behavior until reaching a maximum within the
experimental range examined. The contour graph (see Figure
1b) showed that the highest yields for alcoholic fermentation
of honey were in the range between 20 and 24% of initial
sugars and between 100 and 140 mg/L of assimilable nitrogen
by the yeast.
A regression analysis was performed that allowed for an
evaluation of the model parameters for yield, including the
linear and quadratic components of the factors with a sig-
nificant effect on the response variable. The model was
found to be statistically significant, with an acceptable
Figure 1. Results for micro-fermentation assay a) Response surface
for the influence of the initial sugar content and YAN on the yield.
b) Contour lines for the micro-fermentation of honey.

Table 1. Final concentrations of sugars (glucose and fructose), YAN, ethanol, glycerol, and yield of alcoholic fermentations for
micro-fermentation assay.
Factor levels Experimental results
Test Xsugar XYAN Sugars (g/L) YAN (mg/L) Ethanol (%) Glycerol (g/L) YE/S (g/g)
T00 22 65 0 49.8 ± 2.6i 13.2 ± 0.4bc 8.5 ± 0.4d 0.447 ± 0.014b
T01 22 130 0 108.9 ± 6.1f 15.8 ± 0.8ª 12.2 ± 0.4b 0.518 ± 0.026ª
T02 22 195 0 183.0 ± 4.8b 12.9 ± 0.1c 9.7 ± 0.4 cd 0.408 ± 0.003bc
T10 27 65 0 57.9 ± 3.9h 14.5 ± 0.3ªb 10.6 ± 0.1c 0.399 ± 0.009c
T11 27 130 0 128.2 ± 4.3d 12.9 ± 1.0c 9.3 ± 1.0 cd 0.344 ± 0.025d
T12 27 195 0 169.0 ± 3.0c 15.4 ± 0.6ª 12.2 ± 0.4b 0.400 ± 0.016c
T20 32 65 47.7 ± 4.9 63.4 ± 0.0g 14.7 ± 0.5ªb 13.7 ± 0.6ª 0.393 ± 0.011c
T21 32 130 39.9 ± 2.8 116.2 ± 2.6e 15.2 ± 0.1ª 14.3 ± 0.3ª 0.386 ± 0.003 cd
T22 32 195 39.4 ± 3.3 195.0 ± 0.0a 15.7 ± 0.4ª 14.8 ± 0.3ª 0.387 ± 0.012 cd
Different letters in the same column indicate significant differences among treatments.
 Journal of the American Society of Brewing Chemists 5

adjusted coefficient of determination (R2 = 0.8973). The −6 2 2 −4 2

components of the evaluated factors (A: sugars; N: assimi-
lable nitrogen), the interactions with significant effect, the
test statistic, and the estimators of the model parameters
are presented in Table 2.
From the results shown in Table 2, the regression model
for the yield of alcoholic fermentation (Equation 7) was
developed from the initial conditions of concentration of
sugars (glucose and fructose) and nitrogen assimilated
by yeast.
Ye = −3.30 × 10−1 − 1.04 × 10−7 x2A x2N
a
+2.70 × 10−5 x2A x N + 6.34 × 10−6 x A x2N
−9.00 × 10−4 x2A − 9.38 × 10−5 x2N
−1.65 × 10−3 x A x N + 5.23 × 10−2 x A + 2.4
44 × 10−2 x N
(7)
Where x A represents the concentration of fermentable
sugars (glucose and fructose, % w/w), x N represents the
concentration of YAN (mg/L), and Y (e/s) represents the
ethanol/sugar yield of the alcoholic fermentation (g/g).
From the regression model, the optimal conditions were
established for the evaluated factors that would maximize
the fermentation yield: initial concentration of sugars
(glucose and fructose): 22% w/w, initial concentration of
YAN: 123 mg/L, and maximum estimated ethanol/sugar
yield: 0.461.
Similarly, regression models were obtained to estimate
the total residual sugar content (g/L) (Equation 8) (R2 =
0.9887), the final concentration of YAN (mg/L) (Equation
9) (R2 = 0.9934), the ethanol content (% v/v) (Equation 10)
(R2 = 0.8082) and the glycerol content (g/L) (Equation 11)
(R2 = 0.9508) as a function of the initial concentrations of
sugars (% w/w) and assimilable nitrogen (mg/L).
−3
2
Sugar content = 81.21 + 0.28 x A − 6.40 × 10 x A xN
−10.18 x A + 0.15 xN
−5 2 2 −3 2
YAN f = 51.50 + 3.71 × 10 x A x N − 6.44 × 10 x A x N −
−3 2 −1 2 −2 2
1.85 × 10 x A x N + 1.64 × 10 x A + 2.28 × 10 x N
−1
+3.23 × 10 x A x N − 6.57 x A − 3.01x N
(9)
Ethanol = −33.82 − 3.66 × 10 x A x N + 9.50 × 10 x A x N
−4 2 −2 2 −3 2
+2.19 × 10 x A x N − 5.15 × 10 x A − 3.19 × 10 x N
−2 −1
−5.70 × 10 x A x N + 3.21x A + 8.29 × 10 x N
(10)
−6 2 2 −4 2
Glycerol = −34.15 − 3.84 × 10 x A x N + 9.98 × 10 x A x N
−4 2 −2 2 −3 2
+2.31 × 10 x A x N − 3.40 × 10 x A − 3.38 × 10 x N
−2 −1
−6.02 × 10 x A x N + 2.59x A + 8.90 × 10 x N
(11)
Laboratory scale fermentations (3 L)
Table 3 summarizes the final characteristics of the mead
obtained. The assimilable nitrogen consumption (59.5 mg/L)
represented 51% of the initial YAN content, more than
double the maximum found in the previous phase, suggest-
ing better assimilation of the cells to the optimized condi-
tions of the medium. It was also demonstrated that the
extraction of YAN from bee-pollen in the production of
mead is proportional to the alcoholic degree,[22] as well as
being related to lower production of volatile acidity.[23]
The pH of the honey-must decreased rapidly from 4.22
to 3.77, reaching its lowest value and stabilizing at around
100 h of fermentation in the bioreactor. This behavior is
due to the production of organic acids in the first days of
fermentation, mainly succinic and acetic, and to the low
buffering capacity of the honey-musts.[24] The rapid variation
in acidity can have an antiseptic effect, helping to control
the growth of other microorganisms, but it can also extend
the lag phase of the yeast, reducing growth speed or even
causing yeast death.[25]
Volatile acidity, made up mainly of acetic and formic
acid, is a very important quality parameter for wines as it
is associated with contamination by acetic bacteria and is
considered to be a sensory defect, therefore, it must be kept
(8)
at low concentration levels.[26] Acetic acid can react with
ethanol to produce ethyl acetate, the most important ester
found in wines, which in low concentrations adds complex-
ity to the wine, but when it exceeds 0.15 g/L it can generate
unpleasant acetone odors.[21] The mead obtained in this
phase presented a very low volatile acidity (0.14 ± 0.01 g/L),
lower than that reported by various studies and similar
experimental conditions: 0.24 g/L, [27] 0.54 g/L, [8] 0.51–
0.84 g/L,[28] and 0.56 g/L.[29] This same value was found below

Table 3.  Physicochemical characteristics of the elaborated mead

and comparison with predicted values in laboratory scale
Table 2. Estimators of the regression model parameters obtained
fermentations.
during the micro-fermentation assay.
Predicted
Model components P-value Model estimators Parameter Experimental values values Error (%)
Intercept <0.0001 0.40937 pH 3.77 ± 0.02 –
AL <0.0001 −0.03411 YAN (mg/L) 59.5 ± 2.0 103.3 73.7
AC <0.0001 0.01396 Ethanol (%v/v) 15.1 ± 0.2 14.2 5.8
A LN C <0.0001 0.01569 Glycerol (g/L) 6.6 ± 0.6 10.1 53.4
ACNC <0.0001 −0.01094 Volatile acidity 0.14 ± 0.01 –
The letters L and C indicate the linear and quadratic components of the (g/L)
factors. Yield (g/g) 0.507 ± 0.005 0.461 9.1
6 M. CUENCA ET AL.
the odor threshold for the detection of acetic acid in wines
− 0.2 g/L,[21] nevertheless, this defect was not observed
during the sensory evaluation. The results obtained for the
ethanol content confirmed the tolerance to high concentra-
tions in this yeast strain. At a sensory level, a high ethanol
content negatively impacts the appreciation of the other
sensory characteristics and the general rating of the mead,
as it is related to a thermal sensation in the mouth that
can be unpleasant for the consumer, especially when body
and balance related to acidity and sweetness are uncontrolled
(see supplementary material).
Additionally, the consumption of sugars (glucose and
fructose) was complete, with an observation of a simulta-
neous consumption of both monosaccharides, which relates
to the fructophilic capacity of the yeast used. It was con-
firmed that the alcoholic fermentation time was around
160 h, enough time for all of the sugars to be consumed
and this was considered the endpoint of the process. In this
sense, a decrease in soluble solids was
​​ observed. This result
was much lower than the fermentation times reported by
previous researches: 216–360 h,[15] > 216 h[30] and 192 h.[10]
These authors found that concentrations of YAN greater
than 120 mg/L, using bee-pollen in the alcoholic fermenta-
tion of honey-musts, produced an increase in fermentation
speed, achieving times around 240 h and exceptionally 48 h
when the honey-must was supplemented with 168 mg/L of
YAN. Therefore, it is confirmed that the addition of
bee-pollen decreased the alcoholic fermentation time of
honey under the conditions evaluated in this study.
The production of ethanol and glycerol occurred as the
sugars were consumed, without an evident association with
the consumption of YAN. After 160 h, the ethanol and glyc-
erol reached their maximum concentration. The values ​​for
the ethanol content (15.1 ± 0.2% v/v) and the yield of the
alcoholic fermentation (0.507 ± 0.005 g/g) were very similar
to those obtained in the first experimental phase for the
T01 treatment, which was carried out under a similar assim-
ilable nitrogen concentration condition. This result shows
consistency of the dynamics of alcoholic fermentation, at
least for these parameters. On the contrary, the glycerol
content (6.6 ± 0.6 g/L) was lower than the values ​​obtained
in all of the tests of the first experimental phase, which,
together with the higher consumption of assimilable nitro-
gen, indicates a lower exposure of yeasts to stress conditions.
However, this value is in agreement with the alcoholic fer-
mentation of honey found in the literature under similar
experimental conditions: 5.4 − 9.4 g/L.[31,32]
Compared with the T01 test, the glycerol content obtained
was 46% lower; although it is within the usual range
reported for wines (5 − 12 g/L).[33] It was also evidenced that
the yeast cells rapidly consumed the assimilable nitrogen
present in the honey-must in the first 90 h of fermentation.
Similar behavior was found during the alcoholic fermenta-
tion of a synthetic medium of glucose and fructose supple-
mented with ammonium sulfate, in which nitrogen was
depleted by yeast cells in five days.[34] Nitrogen available at
the beginning of fermentation is used by cells to increase
their number through the synthesis of amino acids, proteins,
and nucleic acids, meanwhile, another part is stored in
vacuoles as cellular reserves for the synthesis of new proteins
during the stationary growth phase.[35]
Although the adaptation and reproduction of yeast are
largely influenced by the conditions of the medium, the
growth obtained was similar to that reported for other mead
production processes.[31,36] The maximum yeast cell count
reached in this study (2.98 × 107 ± 0.07 × 107 CFU/mL) was
similar to the reported by Mendes-Ferreira et  al.[28] for fer-
mentations of honey must at soluble solids of 22 g/100g with
YAN concentrations between 35 and 259 mg/L. These results
can be compared although the initial cell count was not the
same (105 vs. 106 CFU/mL), taking into account what was
reported by Pereira et  al.,[36] in which the alcoholic fermen-
tation of honey-must at different cell concentrations showed
minimal differences in growth kinetics, as well as no sig-
nificant differences were found in the evaluated parameters.
It was observed that the values predicted by the regres-
sion models presented big differences. Only the models
for the ethanol content and the yield of alcoholic fermen-
tation presented acceptable estimates with errors lower
than 10%, therefore, it is recommended to use these regres-
sion models as tools to predict the probable ethanol con-
tent based on the initial level of sugars and assimilable
nitrogen in the honey-must for the alcoholic fermentation
of honey at 25 °C. The ethanol/sugar yield was high and
similar to the stoichiometric value (0.511), suggesting a
high efficiency in the transformation of sugars (glucose
and fructose) into ethanol, confirmed by the high alcohol
content obtained.
It was possible to obtain a model with a high degree of
adjustment of the experimental results for cell growth, pre-
dicting the lag, exponential, and stationary phases of the
microorganism.
Then, the behavior of the specific growth rate (μ/h) was
graphed as a function of the concentration of total sugars
in the fermentation honey-must (S, g/L), to which the
3-parameter logistic (3 P) (Equation 11) and Gompertz
(Equation 12) models were adjusted.
0.0741
µ=
−0.0518*(S −147.6 )
1+e
(12)
−0.0259(S −140.1 )
−e
µ = 0.0885e (13)
The evaluated models showed a high degree of fit of the
experimental data see (Figure 2). However, in values around
the initial concentration of sugars in the fermentation
honey-must (235.3 g/L), the adjusted models showed notable
differences. The 3 P logistic model predicts a more precise
behavior in this area, according to which the growth rate
of yeast is almost constant when the concentration of sugars
is high.[37] Despite this, for most of the alcoholic fermen-
tation, both models showed adequate fit.

An inability of the yeast to maintain a high growth rate
throughout alcoholic fermentation was observed, a phenom-
enon that may be associated with stress conditions due to
environmental conditions. From the two adjusted models,
maximum specific growth of 0.0885 h−1 and 0.0741 h−1 were
obtained. The latter value was taken as the most appropriate
for the maximum growth rate of yeast during the alcoholic
fermentation of honey, using bee-pollen as an assimilable
nitrogen source and sugars (glucose and fructose) at levels
of 120 mg/L and 235 g/L, respectively. This value is close to
reported in the literature for mead production processes:
0.045 h−1,[29] 0.16–0.18 h−1,[38] 0.19–0.21 h−1.[39]
Statistical analyses were carried out on the significance
of these models and the parameter estimators, finding that
the two models adjusted for the yeast kinetics were signif-
icant, as well as the estimated parameters. Furthermore, the
adjusted coefficients of determination (R2) for each model
were above 0.999, and the sums of the squared residuals
were of the order of 10−10 for the Gompertz and 3-parameter
logistic models, indicating a low discrepancy between the
values ​​predicted by the models and the experimental ones.
The kinetics of glucose consumption (Glu, g/L) and fruc-
tose (Fru, g/L) were also modeled using the 3 P logistic
model (R2glu = 0.9994, R2fru = 0.9986), assimilable nitrogen
consumption by yeast (YAN, mg/L), and ethanol production
(Eth, % v/v) by the logistic model of 4 P (R2YAN = 1, R2eta
= 0.9904) and glycerol production (Gly, g/L) by the
Gompertz model (R2gly = 1), whose results are presented in
Figure 3.
Fermentation at pilot-scale batches (100 L)
The 3 P logistic models were adjusted for glucose and fruc-
tose consumption (R2glu = 0.9999, R2fru = 0.9997) and the
4 P logistic models for ethanol and glycerol production (R2eth
= 0.9416, R2gly = 0.9807), which are presented in Figure 4.
The pH decreased more rapidly until reaching a value
of 3.94 in the first 50 h of fermentation, approximately in
Figure 2. Laboratory scale fermentation. Kinetic adjustment of the
specific cell growth rate as a function of time for the alcoholic
fermentation of honey at 25 °C using Saccharomyces cerevisiae
subsp. bayanus.
Journal of the American Society of Brewing Chemists 7
half the time required in the laboratory tests, indicating
more accelerated kinetics.
The kinetics for assimilable nitrogen consumption showed
similar behavior to that obtained at a laboratory scale, where
YAN was rapidly consumed by yeast in the first 24 h until
reaching a concentration around 60 mg/L and maintained
until the end of the fermentation, confirming an excessive
nitrogen supplementation of approximately 50%. It was
found that the kinetic model for YAN consumption adjusts
satisfactorily to the results at pilot scale since the analysis
of the differences between the values predicted
​​ by the mod-
els and the experimental results yielded a value of the root
mean square error RMSE of 4.2 mg/L and an average esti-
mation error of 6.2%.
The fermentation at the pilot-scale showed a more accel-
erated consumption of sugars, which became slower towards
the end of the monitoring. Glucose was consumed in
approximately 20 h less than in the 3 L scale tests, while
fructose, on the other hand, took longer, showing less assim-
ilation of this carbohydrate by the yeast. Compared to the
laboratory-scale, total soluble content stabilized at 10.5 g/100 g
after 140 h of fermentation, and there were no marked dif-
ferences in the variation profiles.
Since the kinetic models obtained in the previous phase
presented a high correlation, they were used to analyze the
results of the fermentation at the pilot scale. In this sense,
considerable differences were found between the kinetic pro-
files between tests. The RMSE values for the glucose and
fructose consumption kinetics were 14.7 g/L and 16.4 g/L, from
which mean estimation errors of the model of 37.3% and
44.5%, respectively, were obtained. The errors of the estimates
for the initial concentrations of glucose and fructose were
acceptable, 3% and 8%, respectively. In consequence, the dif-
ferences were due to modifications in the activity of the yeast
and not to the goodness of fit of models. This fact is related
to different scales used for experimentation. Originally this
yeast was isolated from grapes. It can be adapted to other
substrates, especially in small scale fermentations. In the case
of a larger fermenter, probably it would be necessary to per-
form more inoculum steps than those we have used.
Despite the high rate of sugar consumption, the fermen-
tation exhibited a slower start for ethanol production in the
first 24 h compared to the laboratory process, where the
concentration was more than 250% less than the predicted
values. The RMSE, in this case, was 3.5% v/v, which cor-
responded to an average overestimation of the model of
34% to the final value of the ethanol content.
Similar to the 3 L bioreactor, the highest amount of eth-
anol was produced in the first 90 h of fermentation when
the yeast cells showed accelerated growth. During this
period, fermentation showed a recovery in ethanol produc-
tion, although the concentration was not higher at any time
throughout the process.
In the case of glycerol, the adjusted model also showed
large deviations in the predicted value, which reached
higher levels at the pilot-scale throughout the process.
Unlike ethanol production kinetics, the glycerol content
increased rapidly during the first 48 h of fermentation,
8 M. CUENCA ET AL.

Figure 3. Experimental kinetics and models adjusted during the alcoholic fermentation of honey at laboratory scale for a) the consumption
of glucose and fructose, b) the consumption of assimilable nitrogen, c) the production of ethanol, and d) the production of glycerol.

Figure 4. Experimental kinetics and models adjusted during alcoholic fermentation of honey at pilot scale for a) glucose and fructose
consumption, b) ethanol production, and c) glycerol production.

reaching values up to 60% higher compared to the esti-
mates. Subsequently, the production rate decreased, and
this difference remained around 30%. The RMSE calculated
was 2.6 g/L, which corresponded to an average underesti-
mation of 30% over the final glycerol content.
The physicochemical characterization of the mead obtained
at the pilot scale showed the following final characteristics:
pH: 3.94, YAN: 62.7 mg/L, ethanol: 12.4%v/v, glycerol: 8.5 g/L,
volatile acidity: 0.27 g/L, and yield: 0.413 g/g. Those values
were within the normal ranges reported for this product. In
comparison with the meads obtained at the laboratory scale,
considerable differences were observed except for the pH
and the residual assimilable nitrogen content.
Lower ethanol content was found and, therefore, lower
ethanol/sugar yield, unlike the glycerol content and volatile
acidity that were higher compared to laboratory fermenta-
tion. Despite the increase in volatile acidity, this value was
lower than the normal maximum limit found in wines
(1.2 g/L), which provides sensory complexity to the mead.[40]
The differences found in the kinetics at the laboratory
and pilot-scale fermentation may be related to variations in
yeast behavior due to the difficulty in promoting adequate
agitation during sample collection and cell sedimentation.[29]
The variation of the environmental temperature (18 − 30 °C)
during the pilot-scale fermentation, was another determining
factor for these differences. Glycerol production, glucose,
and fructose consumption, and specific growth rate in alco-
holic fermentation using Saccharomyces cerevisiae have been
reported to increase as temperature increases, with an opti-
mum around 30 °C,[41,42] while ethanol production and eth-
anol/sugar yield decreased.[43,44]
Conclusion
The best conditions for the production of mead were a sugar
concentration between 22 and 24% w/w and assimilable nitro-
gen between 65–130 mg/L. The addition of 50 g/L of pollen
exerted a positive effect on the kinetics of alcoholic fermen-
tation of honey-must at 22°Bx and 25 °C, leading to a 17–30%
decrease in fermentation time compared to other studies.
The production of mead under the operating conditions used
in this study is useful to obtain products of high sensory
quality, with few odor and aroma defects and an attractive
descriptive profile. The logistic and Gompertz regression
models are alternatives to successfully model the kinetic
behavior of the alcoholic fermentation of honey for moni-
toring and control purposes, not only for the laboratory but
also at the pilot-scale.
Acknowledgments
The authors thank the beekeepers’ associations Apisred, Asoapiboy,
and Apiario Los Cerezos, as well as the Institute of Food Science and
Technology (ICTA) and the Colleges of Agricultural Sciences and
Engineering of Universidad Nacional de Colombia.
Disclosure statement
The authors declare not having any conflict of interest.
Journal of the American Society of Brewing Chemists 9
Funding
This research was funded by the Colombian Ministry of Science,
Technology, and Innovation (Minciencias), through the projects
“Development of a production model of fermented honey beverages
as a strategy to generate value in the characteristic field of beekeeping
in Colombia” (Code 1101-586-35750) and “Development of fermented
beverages of native red fruits of Colombia and evaluation of their
bioactive and sensory characteristics” (Resolution No. 046 of 2013 of
the Institute of Food Science and Technology).
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