Food Microbiology 101 (2022) 103896

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Bioprospecting of indigenous yeasts involved in cocoa fermentation using

sensory and chemical strategies for selecting a starter inoculum
Marcelina María Mendoza Salazar a, *, Olga Lucia Martínez Álvarez b,
Maurem Paola Ardila Castañeda b, Pilar Ximena Lizarazo Medina a
a
Grupo de Investigación en Ecología Microbiana y Bioprospección, Facultad de Ciencias Exactas y Naturales, Universidad de Antioquia, Calle 67 No. 53 - 108, 050010,
Medellín, Antioquia, Colombia
b
Grupo de Investigación en Ciencia Sensorial, Facultad de Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia, Calle 67 No. 53 - 108, 050010, Medellín,
Antioquia, Colombia

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

Keywords: Cocoa fermentation is the key and most relevant process in the synthesis of aroma and flavor precursor molecules
Cocoa in dry beans or raw material for producing chocolate. Because this process occurs in an uncontrolled manner, the
Fermentation chemical and sensory quality of beans can vary and be negatively affected. One of the strategies for the stan­
Sensory
dardization and improvement of the sensory quality of chocolate is the introduction of microbial starter cultures.
Starter culture
Hanseniaspora thailandica
Among these, yeasts involved in fermentation have been studied because of their pectinolytic and metabolic
Pichia kluyveri potential in the production of volatile compounds. This study was aimed at isolating and characterizing, both
sensory and chemically, yeasts involved in cocoa fermentation that could be used as starter cultures from two
agro-ecological regions for the cultivation of cocoa in Colombia. The microbiological analyses identified 22
species represented mostly by Saccharomyces cerevisiae, Wickerhamomyces anomalus and Pichia sp. The pre­
liminary sensory analysis of eight of these species showed that Hanseniaspora thailandica and Pichia kluyveri
presented sensory profiles characterized by high intensity levels of fruity notes, which could be ascribed to the
production of ethyl acetate, isoamyl acetate, and 2-phenylethyl acetate.

1. Introduction
The sensory characteristics of chocolate are determined by fer­
menting cocoa beans because aroma and flavor precursor molecules are
synthesized during this process with microbial volatile compounds that
influence the sensory profile of beans (Batista et al., 2016; Ho et al,
2014, 2018). Spontaneous cocoa fermentation is characterized by a
microbial succession initially directed by yeasts, which synthesize
ethanol from the fermentation of carbohydrates present in the mucilage,
and lactic acid bacteria (LAB), which produce lactic acid, consume citric
acid, and reduce fructose into mannitol. At the end of the process, an
increase in the abundance of acetic acid bacteria (AAB) is observed,
these produce acetic acid from ethanol oxidation. The penetration of this
acid, together with the increase in temperature triggered by the
oxidation of ethanol, induces the death of the embryo and the synthesis
of aroma and flavor precursor compounds in the cotyledons (De Vuyst
and Leroy, 2020; Schwan and Wheals, 2004).
Because cocoa fermentation occurs in spontaneous or an uncon­
trolled manner, the chemical and sensory quality of beans varies from
location to location and can be compromised because of defects in the
process. One of the strategies for the standardization and improvement
of the sensory quality of chocolate is the introduction of microbial
starter cultures (Figueroa-Hernández et al., 2019). Starter cultures
composed of yeasts (Batista et al., 2015; Leal et al., 2008; Meersman
et al., 2016; Mota-Gutierrez et al., 2018; Ramos et al., 2014; Visintin
et al., 2017), bacteria (Kresnowati et al., 2013; Lefeber et al., 2012), or
both microbial groups (Crafack et al., 2013; Lefeber et al., 2012; Mag­
alhães da Veiga Moreira et al., 2017; Sandhya et al., 2016; Schwan,

Abbreviations: LAB, lactic acid bacteria; AAB, acetic acid bacteria; SDA, Sabouraud dextrose agar; MEA, malt extract agar; PSM-yeast, cocoa pulp simulation
medium for yeast; GC-MS, gas chromatography-mass spectrometry; HS-SPME, headspace solid-phase microextraction; PCA, principal component analysis; NIST,
National Institute of Standards and Technology; VOC, volatile organic compound.
* Corresponding author.
E-mail addresses: marcelina.mendoza@udea.edu.co (M.M. Mendoza Salazar), olga.martinez@udea.edu.co (O.L. Martínez Álvarez), maurem.ardila@udea.edu.co
(M.P. Ardila Castañeda), pilar.lizarazo@udea.edu.co (P.X. Lizarazo Medina).

https://doi.org/10.1016/j.fm.2021.103896
Received 5 May 2021; Received in revised form 11 July 2021; Accepted 1 September 2021
Available online 3 September 2021
0740-0020/© 2021 Elsevier Ltd. All rights reserved.
M.M. Mendoza Salazar et al.
1998) have been evaluated. However, Pereira et al. (2016) reported that
the addition of a yeast strain is a necessary requirement to overcome the
variability in the final product quality because great diversity is
observed during the fermentation process among locations. Another
reason to use yeast starter cultures is their ability to produce volatile
compounds with a positive effect on the sensory quality of cocoa
(Meersman et al, 2015, 2016; Mota-Gutierrez et al., 2019), as well as
mucilage depectinization and carbohydrate consumption (Batista et al,
2015, 2016; Ramos et al., 2014).
Some authors propose for the establishment of microorganisms as
starter cultures the selection of the most abundant species of the
fermentation process (Lima et al., 2021). However, we consider that this
selection requires developing a systematic and interdisciplinary
approach through the application of sequential tests to progressively
reduce the number of candidates according to its performance in favor of
the chemical and sensory quality of the product. Vinicius De Melo Per­
eira et al. (2020), indicates criteria for the selection of food-associated
starter cultures that involves the assessment of growth under stressful
environmental conditions, the identification of strains that produce
metabolites of interest, and the evaluation of technological parameters
such as sensory characterization to finally select the isolates that present
the highest number of functional properties.
Different methodologies have been used to select starter cultures in
cocoa (de Melo Pereira et al., 2012; Lefeber et al., 2010; Meersman et al.,
2016, 2015; Saerens and Swiegers, 2013) and to establish their contri­
bution to the volatilome of fermented cocoa beans (Koné et al., 2016).
However, none of them has used sensory analysis as a strategy to
determine their potential in the production of volatile compounds.
In this study we propose the selection of a yeast starter culture based
on chemical and sensory characterization. Based on these criteria, select
the yeast with the potential to synthesize volatile compounds to improve
the sensory profile of cocoa.
2. Materials and methods
2.1. Spontaneous fermentation
Cocoa fermentation was studied using the same parameters in farms
in two locations in agroecological regions for the cultivation of cocoa at
Magdalena Medio and Urabá subregions. The cocoa clones CCN-51, FEC-
2, FLE-2, and ICS-1 were used in all locations. Fruits were harvested
separately, stored for 36 h, and then opened using a machete to carry out
the shelling. The mass of beans was placed in the three central boxes of
an oak wood fermenter; 0.70 m length, 0.65 m height, and 0.65 m width,
each. A total of 109.7 kg were placed in each box in identical proportions
for each subregion, namely 85.9% CCN-51, 7.9% FEC-2, 3.2% FLE-2,
and 3% ICS-1.
The mass of beans was covered with leaves of bijao (Calathea lutea)
and fique bags, and held in stillness. This was considered the starting
time of fermentation. After 48 h, the first mixing or turning of the beans
was carried out, and then repeated every 24 h.
2.2. Microbiological analysis
To analyze the composition of the culturable yeast community,
separate samples of mass of fermenting beans were collected from the
three boxes, at the starting time and every 24 h until the end of
fermentation. For the analysis, 25 g of cocoa beans from each sample
were suspended in 225 mL of 0.1% peptone solution and 3 drops of
Tween 80. Samples were ground in a Smasher homogenizer (Bio­
merieux) at low speed for 10 min. Serial 1:10 dilutions were made and
100 μl aliquots were plated in triplicate on Sabouraud 4% dextrose agar
(SDA) (Merck) and malt extract agar (MEA) (Merck) supplemented with
0.05 mg/mL gentamicin. Plates were incubated at 25 ◦ C for 48 h.
Morphotypes were determined in these cultures and one colony of each
was randomly selected. All isolates were cryopreserved at − 80 ◦ C in
2
Food Microbiology 101 (2022) 103896
Sabouraud 2% dextrose broth with 30% glycerol.
2.3. Identification of microorganisms
2.3.1. DNA extraction
Yeast DNA was extracted using the rapid lysis method. Briefly, a
colony grown for 15 h was suspended in 50 μl deionized water and
exposed to 95 ◦ C for 5 min.
2.3.2. Amplification of molecular markers
The ITS marker was used for taxonomic classification of yeasts using
the universal primers ITS1 (5′ -TCCGTAGGTGAACCTGCGG-3′ ) and ITS4
(5′ - TCCTCCGCTTATTGATATGC-3′ ) (White et al., 1990). The amplifi­
cation reactions were performed in 25 μl as follows: 12.5 μl 2X Master
Mix (Thermo Scientific), 0.5 μM of each primer, 0.4 mg/mL bovine
serum albumin, and 3–5 μl yeast lysate. The PCR program comprised an
initial denaturation step at 95 ◦ C for 3 min, followed by 35 cycles of
amplification at 94 ◦ C for 1 min, 53 ◦ C for 50 s, and 72 ◦ C for 1 min, and
a final elongation step at 72 ◦ C for 10 min in a SimpliAmp thermal cycler
(Applied Biosystems) (Chen et al., 2011).
The taxonomic classification was verified by amplifying and
sequencing the ACT1 gene and the D1/D2 region of the 26 S ribosomal
subunit. For the first marker, the following primer combinations were
used: CA1 (5′ - GCCGGTGACGACGCTCCAAGAGCTG-3′ )/CA5r (5′ -
GTGAACAATGGATGGACCAGATTCGTC-3′ ), CA21 (5′ - ATTGA­
TAACGGTTCCGGTATGTG-3′ )/CA22r (5′ - TCGTCGTATTCTTGCTTT­
GAGATCCAC-3′ ), and Act1-f (5′ - CTCGTGCTGTCTTCCCATCT-3′ )/
CA22r (Cadez et al., 2006; Daniel and Meyer, 2003). The PCR reactions
were performed as previously described. For the amplification, a
touch-down program of 20 denaturation cycles at 94 ◦ C for 5 min in the
first cycle and 30 s in the remaining 19 cycles, annealing at 60 ◦ C for 30 s
(reducing 0.5 ◦ C per cycle) and extension at 72 ◦ C for 1 min, followed by
15 cycles of denaturation at 94 ◦ C for 30 s, annealing at 50 ◦ C for 30 s
and final extension at 72 ◦ C for 10 min was used (Daniel and Meyer,
2003). For the second marker, the primers NL-1
(5′ -GCATATCAATAAGCGGAGGAAAAG-3′ ) and NL-4 (5′ -
GGTCCGTGTTTCAAGACGG-3′ ) were used. The amplification program
comprised an initial denaturation step at 94 ◦ C for 5 min, 30 cycles of
amplification at 94 ◦ C for 1.5 min, 53 ◦ C for 30 s, and 72 ◦ C for 1.5 min
and a final elongation step at 72 ◦ C for 10 min (Crafack et al., 2013).
The PCR products were analyzed by electrophoresis using 1%
agarose gel in TBE buffer, at 80 V for 30 min. The amplicons were pu­
rified and sequenced by Macrogen Inc. (Seoul, South Korea).
2.3.3. Sequence editing and classification
Sequences in .ab1 format were edited using the package sanger­
analyseR (Lanfear, 2015) in R software version 3.5.1 (R Core Team,
2018) with a Phred Score of 40 or above. Classification was performed
using the Ribosomal Database Project (RDP) classifier (Wang et al.,
2007) (80% bootstrap threshold) and BLAST (Basic Local Alignment
Search Tool).
2.4. Selection of microorganisms as starter inoculum
The physiology of all identified species had been previously reviewed
in scientific publications in terms of volatile compound production and
health safety. Based on this information eight species were selected and
sensory analyzed in a cocoa pulp simulation medium for yeast (PSM-
yeast) containing 17 g/l fructose, 25 g/l glucose, 10 g/l citric acid, 5 g/l
yeast extract, 5 g/l soy peptone, and 20% fresh cocoa pulp (de Melo
Pereira et al., 2012). Each species was grown separately in 100 mL
Sabouraud broth at 30 ◦ C for 24 h without shaking to standardize the
inoculum concentration. Cellular biomass (cells/mL) was estimated by
viability staining using 0.01% methylene blue and counting in a Neu­
bauer chamber. The estimated volume to obtain a final concentration of
106 cells/mL was centrifuged at 6000×g for 10 min. The pellet was
M.M. Mendoza Salazar et al.
inoculated in 100 mL of PSM yeast and incubated at 30 ◦ C for 24 h
without shaking. These cultures were used for sensory analyses and
volatile compound profiles.
2.4.1. Sensory analysis of cocoa simulation cultures
Every 24 h, 1 mL aliquots were collected from the cultures and
sensory evaluated by a panel of three experts using the descriptive
method of olfactory profile. The panelists were trained in the recogni­
tion of volatile compounds of cocoa fruit quality and defects, and of
fermenting and drying beans. Moreover, the experts used the cocoa ol­
factory kit designed by the Grupo de Investigación en Ciencia Sensorial
of the Universidad de Antioquia- Colombia, together with Casa del Sur S.
A.S, supplemented with reference substances under NTC 4503:2011-ISO
5496:2006. Samples were analyzed in terms of odor descriptors (acid,
alcohol, sweet, floral, fruity, herbal, yeast, and vegetable) and overall
quality, using an ordinal scale from 0 to 10, where 0 means absence, 1 to
2 low intensity, 3 to 5 medium intensity, 6 to 8 high intensity, and 9 to
10 very high intensity.
2.4.2. Volatile compound profile of selected yeasts
The volatile compound profile produced by yeasts growing in cocoa
simulation broth was determined every 24 h for 120 h. VOCs composi­
tion was analyzed by headspace solid-phase microextraction (HS-
SPME), followed by gas chromatography-mass spectrometry (GC-MS) by
the Grupo de Investigación en Ingeniería de Alimentos GRIAL of the
Corporación Universitaria Lasallista – Colombia. Briefly, 1 mL of a 40%
saturated NaCl solution was added to 5 mL of each sample and vortexed
at 3000 rpm for 10 s. Samples were stored at 8 ◦ C for 12 h before
compound extraction. The microextraction was performed using a
polydimethylsiloxane-divinylbenzene (PDMS/DVB) fiber activated at
250 ◦ C for 30 min and conditioned at 48.6 ◦ C for 15 min. Then, it was
exposed to the headspace at 10 mm depth with initial shaking for 2 min,
followed by static incubation at 48.6 ◦ C until the end of the 50 min cycle.
The chromatographic separation and spectrometric detection started
with the desorption of the concentrated volatile compounds on the fiber
at the injection port at 250 ◦ C for 10 min. A 1000 μl sample was sepa­
rated in the splitless mode on a TRACE TR-WaxMS GC column (60 m ×
0.25 mm I.D., 0.25 μm film thickness) (Thermo Scientific, USA). The
temperature at the transfer line and the injector was 250 ◦ C. Helium was
used as the carrier gas for elution at a flow rate of 1 mL/min with a
temperature ramp of 40 ◦ C for 5 min, increased up to 180 ◦ C at a rate of
4 ◦ C min− 1, then up to 260 ◦ C at a rate of 10 ◦ C min− 1, and a final hold
time of 2 min. Detection in the mass spectrometer started with ioniza­
tion at the source at 230 ◦ C, the quadrupole temperature was 150 ◦ C.
The detection mode was set to scan every 0.2 s in a mass range of 30–500
amu.
2.5. Data analysis
Composition and richness of the yeast community was established as
per the taxonomic determination and the number of isolated species.
To determine the most important sensory attributes between the
different treatments, the sensory analysis data of the yeast cultures in
simulation broth for the selection of species was analyzed by principal
component analysis (PCA) using the packages FactoMineR (Le et al.,
2008) and factoextra (Kassambara and Mundt, 2017) in R version 3.5.1.
Trend charts were made to describe the overall sensory impression
scores. The data to compare sensory profiles between isolates of the
same species was analyzed using radial and trend graphs.
To establish the volatile compound profile, the chromatographic
peak integration was performed using the Cobra software, and the NIST
(National Institute of Standards and Technology) spectra database was
used for identification applying the RMatch option. Data were expressed
as the relative area of each peak with respect to the total areas of all
compounds identified in the sample. Compounds with the largest rela­
tive areas were compared between treatments by analysis of variance,
3
Food Microbiology 101 (2022) 103896
followed by Tukey’s multiple comparisons test using the functions of the
stats package in the R software version 3.5.1.
3. Results and discussion
3.1. Microbiological analysis
Forty-five cocoa bean samples were collected and analyzed from a
fermentation of 168 h or 132 h in the Magdalena Medio and Urabá re­
gions, respectively.
A total of 247 yeast isolates were obtained, 144 from the fermenta­
tion in Magdalena Medio and 103 from Urabá. Twenty-two species were
recorded, of which seven were exclusively reported in Magdalena
Medio, seven exclusively in Urabá, and eight in both locations. Yeast
diversity was higher at the beginning of fermentation (0 h–72 h) than at
the end (96 h–168 h) in the Magdalena Medio subregion. In the Urabá
subregion, the number of different species was highest at 0 h of
fermentation, decreased after 24 h, and then increased between 48 h and
96 h of fermentation. In the two subregions, ~70% of the morphotypes
were classified into the genera Saccharomyces, Wickerhamomyces, and
Pichia, and were represented by the species S. cerevisiae, W. anomalus,
and Pichia sp., which are frequently reported in cocoa fermentation
(Chagas Junior et al., 2020; Koné et al., 2016; Ooi et al., 2020; Papal­
exandratou et al., 2013). Moreover, species belonging to Candida,
Debaromyces, Hanseniaspora, Kodamaea, Schwanniomyces, Trigonopsis,
and Zygosaccharomyces genera were recorded (Table 1).
3.2. Sensory characterization of species with potential for the production
of volatile compounds
To evaluate the aromatic potential of the isolated yeast and select a
microbial inoculum to promote the sensory quality of fermented beans,
the species were sensory analyzed for their performance in a cocoa
simulation matrix. Firstly, those species reported in the literature as
showing the potential to produce volatile compounds without any bio­
logical risk were selected among the species obtained. Issatchenkia ori­
entalis, S. cerevisiae, W. anomalus, and a representative of the genus
Hanseniaspora were selected based on their ability to synthesize ethyl
acetate and 2-phenylethyl acetate (Koné et al., 2016; Moreira et al.,
2005; Rojas et al., 2001). The genera Pichia spp. and Candida spp. were
selected for their potential to primarily produce isoamyl acetate (Buzzini
et al., 2003a, 2003b). The species Clavispora lusitaniae (Krcmery et al.,
1999), Kodamaea ohmeri (Diallo et al., 2019), Meyerozyma guilliermondii
(Pfaller et al., 2006), and Schwanniomyces etchellsii (Relich et al., 2016)
were discarded because they have been reported to cause disease in
humans, similar to Trigonopsis cantareli, which was associated with
sensory defects in wine fermentation (Portugal et al., 2015). Eight spe­
cies identified as Candida oleophila, Hanseniaspora thailandica, I. ori­
entalis, Pichia deserticola/Candida ethanolica, P. kluyveri, Pichia sp.
(classified as Pichia manshurica by sequencing of actin gene and D1/D2
region) S. cerevisiae, and W. anomalus were evaluated by sensory
analysis.
A PCA was made based on the results obtained during the 120 h of
the yeast sensory analysis. The two principal components accounted for
78.8% of total variance (Fig. 1). The descriptors sweet, fruity, and
alcohol were the variables with the highest contribution to the first
component; however, herbal notes were the primary contributor to the
second component. This analysis indicated that most yeasts produced
important sensory changes after 24 h of growth, except for the
P. deserticola/C. ethanolica isolate that showed similar results to those of
the culture medium without inoculation. P. manshurica presented herbal
notes. C. oleophila was characterized by acid descriptors. H. thailandica,
I. orientalis, S. cerevisiae, P. kluyveri, and W. anomalus showed alcohol,
sweet, floral, fruity, and yeast notes (Fig. 1), which could be attributed
to the ethanol produced by fermentation of sugars in the culture medium
and the presence of volatile compounds product of the amino acid
M.M. Mendoza Salazar et al. Food Microbiology 101 (2022) 103896

Table 1
Taxonomic classification of yeasts isolated every 24 h from the spontaneous cocoa bean box fermentation in the Magdalena Medio and Urabá subregions.
Species Magdalena Medio Urabá

0 24 48 72 96 120 144 168 Total 0 24 48 72 96 120 132 Total

Candida oleophila * 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
Candida railenensis ** 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 2
Candida sp. * 6 1 0 0 0 0 0 0 7 0 0 0 0 0 0 0 0
Candida zemplinina ** 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 2
Clavispora lusitaniae * 0 0 1 2 0 0 0 0 3 0 0 0 0 0 0 0 0
Debaryomyces prosopidis/hansenii ** 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 3
Hanseniaspora occidentalis * 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
Hanseniaspora opuntiae 2 1 0 0 0 0 0 0 3 1 0 0 0 0 0 0 1
Hanseniaspora thailandica ** 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 2
Hanseniaspora uvarum * 1 2 1 0 0 0 0 0 4 0 0 0 0 0 0 0 0
Issatchenkia orientalis 0 2 2 2 0 0 0 1 7 3 0 4 0 0 0 2 9
Kodamaea ohmeri ** 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1
Meyerozyma guilliermondii ** 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 2
Pichia deserticola/Candida ethanolica * 0 1 0 5 2 1 0 4 13 0 0 0 0 0 0 0 0
Pichia kluyveri 2 0 0 0 0 0 0 0 2 4 0 0 0 0 0 0 4
Pichia sp. 0 2 0 4 3 2 1 5 17 1 0 0 2 0 8 10 20
Saccharomyces cerevisiae 6 8 10 6 8 6 5 3 52 13 12 3 8 1 1 0 38
Saccharomycodes ludwigii 0 0 1 0 0 0 0 0 1 0 0 0 1 0 0 0 1
Schwanniomyces etchellsii 0 0 0 0 0 2 0 0 2 0 0 0 0 0 2 0 2
Trigonopsis cantarellii * 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0
Wickerhamomyces anomalus 16 5 6 3 0 0 0 0 30 6 0 7 0 1 1 0 15
Zygosaccharomyces bailii/parabailii ** 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1

* Taxa detected exclusively in Magdalena Medio.

** Taxa detected exclusively in Urabá.

Fig. 1. PCA with sensory assessments of C. oleophila, H. thailandica, I. orientalis, P. deserticola/C. ethanolica, P. kluyveri, P. manshurica, S. cerevisiae, and W. anomalus
cultures, and control (PSM-Yeast medium without microorganisms) from 0 h to 120 h.

metabolism (higher alcohols) and esterification reactions (esters)
(Loviso and Libkind, 2018; Pires et al., 2014).
As per the overall sensory impression determined by the panel of
experts, H. thailandica and P. kluyveri were the most outstanding yeast
species (Fig. 2). H. thailandica has been reported as the dominant species
in the initial stage of cocoa fermentation in Malaysia (Meersman et al,
2013, 2016) and Ghana (Crafack et al., 2013). Moreover, it has been
used as a starter culture and has shown a positive effect on the antiox­
idant content of cocoa beans (Ooi et al., 2020). No studies were reported
on the production of volatile compounds in this species, but on related
taxa such as Hanseniaspora uvarum, Hanseniaspora guilliermondii, Han­
seniaspora osmophila (Rojas et al., 2001), and Hanseniaspora vineae. The

4
M.M. Mendoza Salazar et al.
Fig. 2. Overall impression to different fermentation time of sensory quality of yeast species cultures.
latter has been extensively studied in grape fermentation because it
produces high amounts of acetate esters such as 2-phenylethyl acetate
and ethyl acetate, which increase fruity aromas in wine (Martin et al.,
2018). P. kluyveri has been frequently isolated from cocoa fermentation
but at low abundance (de Melo Pereira et al., 2013; Jespersen et al.,
2005; Moreira et al., 2013; Nielsen et al., 2005). It has been reported to
produce isobutyl acetate, which is associated with apple and banana
scents (Rodriguez-Campos et al., 2011); isoamyl acetate, with charac­
teristic aromas of banana (Aprotosoaie et al., 2016); phenyl methyl ac­
etate, described with sweet, honey, and jasmine odors (Aprotosoaie
et al., 2016); and isoamyl propionate, associated with fruity notes
(Lyumugabe et al., 2018; Buzzini et al., 2003a).
3.2.1. Sensory characterization of species belonging to the genus
Hanseniaspora
Because genus Hanseniaspora has been reported as dominant in cocoa
fermentations and in this research were isolated five species, was made a
Fig. 3. Olfactory sensory profile of the different Hanseniaspora species cultures at 0 h (a), 24 h (b), 48 h (c), 72 h (d), and 96 h (e).
5
Food Microbiology 101 (2022) 103896
sensory characterization of these. The individual sensory analysis of the
five isolates showed profile differences over time, especially in the
period between 0 h and 24 h, when the initial vegetable scent associated
with the culture medium was replaced by fruity, herbal, and yeast notes
at 24 h (Fig. 3a and b). Hanseniaspora occidentalis and H. uvarum pre­
sented similar profiles at 24 h, standing out for their fruity and yeast
notes. Hanseniaspora opuntiae isolates from Magdalena Medio showed
fruity notes and H. opuntiae isolates from Urabá presented yeast and
herbal notes. In contrast, H. thailandica profile was characterized by a
higher number of sensory descriptors with high intensity of herbal and
yeast notes (Fig. 3b). Later, at 48 h, 72 h, and 96 h, trends were similar
among treatments (Fig. 3c, d, and 3e). At 48 h, the profiles were char­
acterized by descriptors of higher intensity, among which the fruity,
floral, sweet, and acid ones stood out. At this time point, fruity pineapple
scents with high intensity of alcohol, fruity, and sweet notes were
perceived in H. opuntiae and H. thailandica cultures. Sulfur, rotten, and
overripe pineapple notes (associated with defects) were detected in
M.M. Mendoza Salazar et al. Food Microbiology 101 (2022) 103896

H. occidentalis and H. uvarum, with yeast, vegetable, and acid charac­ chosen to proceed with the inoculum selection.
teristics standing out (Fig. 3c). At 72 h, the notes primarily perceived
were alcohol, fruity, acid, and yeast, and the intensity decreased for
3.3. Profile of volatile compounds of H. thailandica and P. kluyveri
most descriptors (Fig. 3d). Moreover, at this time point, over-fermented
fruit attributes were detected in H. uvarum cultures, and unpleasant
To further explain the sensory characteristics attributed to the cul­
scents (sewer, leachate, and sulfur) in H. occidentalis. However, acid and
tures inoculated with H. thailandica or P. kluyveri, their volatile com­
acetone notes were perceived in H. opuntiae and H. thailandica cultures.
pounds and corresponding relative areas were determined in the
At 96 h, all profiles were very similar, characterized by alcohol, fruity,
fermentation of a cocoa simulation broth every 24 h for 120 h using GC-
and sweet notes (Fig. 3e).
MS. A total of 45 volatile compounds were detected and classified as
The dynamics of these profiles was the result of yeast metabolism
acids (6), alcohols (11), aldehydes (2), ketones (1), esters (20), hetero­
because in the control (culture medium) only vegetable scents were
cycles (1), and terpenes (4). Out of these, 32 have been reported in cocoa
detected. Alcohol, fruity, floral, and sweet notes could be explained by
and 26 have been sensory described. Alcohols, esters, and terpenes have
the ethanol produced through alcoholic fermentation of sugars. Fruity,
been described as having pleasant attributes with sweet, floral, fruity,
floral, and sweet notes could be due to higher alcohols and esters, which
and wine notes; however, acids have been described as having un­
are synthesized during the metabolism of amino acids and lipids through
pleasant notes, such as butter, rancid, cheese, sweaty, sour, and astrin­
the Ehrlich pathway, and of alcohols through the action of alcohol
gent (supplementary material).
acetyltransferase, respectively (Pires et al., 2014). The over-fermented
There were 55.6% of VOCs detected in both the microbial cultures
fruity notes in H. opuntiae and H. thailandica cultures could be caused
and the control (sterile culture medium without inoculation), and 31.1%
by overproduction of esters because of the high concentration of glucose
were only present in the treatments with H. thailandica and P. kluyveri,
and fructose in the medium, while acid notes could be attributed to the
including ethanol. Moreover, VOCs exclusive to each treatment were
carboxylic acids synthesized from the hydrolysis of esters (Loviso and
observed. H. thailandica culture presented four unique VOCs, namely
Libkind, 2018). Sulfur notes in H. occidentalis and H. uvarum, could be
acetic acid at 96 h and 120 h, 4-methyl-2-hexanol at 72 h and 120 h, and
attributed to the production of sulfuric compounds, which have been
the esters ethyl dodecanoate and ethyl tetradecanoate detected from 72
reported in these two species (Moreira et al., 2005).
h to 144 h. In P. kluyveri, only 1-(R)-phenyl ethyl acetate was detected
One of the determining factors in ester biosynthesis is the yeast
between 0 h and 24 h.
species used. Variations in aroma and flavor may be attributed to
The analysis of the dynamics of all VOCs over time allowed the
interspecific differences associated with the mechanisms that regulate
identification of five compounds characterized by their constant pres­
the expression of genes responsible for ester synthesis and the activity of
ence and high percentage of relative area, namely, 2-phenylethyl ace­
the involved enzymes (Loviso and Libkind, 2018). The genotypic and
tate, 3-methylbutyl acetate or isoamyl acetate, ethyl acetate, pentan-2-yl
metabolic diversity of certain species of Hanseniaspora has been studied
acetate or 2-pentanol acetate (between 0 h and 120 h), and ethanol
in the fermentation of must because the presence of these species at the
(between 24 h and 120 h) (Fig. 5). The first three compounds have been
beginning of the process has been associated with the production of
considered as markers of sensory quality in cocoa fermentation
fruity aromas in wine. Among the genetic differences, the number of
(Meersman et al., 2016; Mota-Gutierrez et al., 2019); consequently, their
copies of the genes coding for alcohol dehydrogenases varies from four
biosynthesis has been used as a criterion to select starter cultures (Sae­
to eight depending on the species. This could influence the fermentation
rens and Swiegers, 2013).
capacity and the production of higher alcohols (Martin et al., 2018).
Ethyl acetate, which is associated with fruity descriptors such as
Moreover, differences in the production of 2-phenylethanol, 2-phenyl­
pineapple, showed high relative area values between 0 h and 96 h in
ethyl acetate (Rojas et al., 2001), and ethyl acetate (Moreira et al., 2005)
H. thailandica, and tended to decrease over time. Conversely, 2-phenyl­
between H. guilliermondii and H. uvarum have been reported.
ethyl acetate, with sensory notes of rose, honey, tobacco, and flowers,
Based on the overall sensory impression analysis, H. opuntiae (iso­
was detected between 0 h and 96 h with the highest relative area in
lated from Magdalena Medio) and H. thailandica were rated with the
P. kluyveri culture. Isoamyl acetate, whose main attribute is banana
highest scores during most of the measurements (Fig. 4). According to
scent, was detected similarly at 0 h, 72 h and 96 h (p > 0.05) in the yeast
additional observations by the panel of experts, characteristic aromas of
treatments and the culture medium. However, in the fermentation with
goldenberry (Physalis peruviana L.) and sweet corn were detected in the
P. kluyveri, the estimated relative area was higher at 24 h, 48 h, and 120
fermentation of H. thailandica. Moreover, this was the only treatment
h when compared to control (Table 2).
that did not present undesirable odors. Consequently, this specie was
The acetate esters reported in the highest proportion (i.e., 2-

Fig. 4. Overall impression to different fermentation time of sensory quality of Hanseniaspora species cultures.

6
M.M. Mendoza Salazar et al. Food Microbiology 101 (2022) 103896

Fig. 5. Volatile compounds detected with higher percentage of relative area during 120 h of analysis. Ht: H. thailandica, Pk: P. kluyveri and Cm: culture medium.

ethyl alcohol (the 2-phenylethyl acetate precursor) was larger at 0 h,

Table 2
96 h, and 120 h in the fermentation with H. thailandica, suggesting
Comparison of the relative area of three volatile compounds detected in high
differences in the alcohol acetyl transferase activity of and/or in regu­
proportion. Values denote mean ± standard deviation.
latory mechanisms. The synthesis of 2-phenylethyl acetate among spe­
Volatile Time Control H. thailandica P. kluyveri
cies of the genus Hanseniaspora is a particular characteristic of H. vineae,
compound (h)
which may be attributed to the presence of six sequences with alcohol
2-phenylethyl 0 0.00 ± 0.00 0.65 ± 0.92 (a) 23.05 ± 3.37 acetyl transferase domains in its genome. These species are more effi­
acetate (a) (b)
cient in the production of ethyl acetate (Giorello et al., 2019), which is
24 0.00 ± 0.00 1.50 ± 0.01 (a) 30.00 ± 1.51
(a) (b) synthesized from ethanol and acetyl-CoA. It has been reported at levels
48 0.00 ± 0.00 1.94 ± 0.01 (a) 29.50 ± 1.10 between 0 and 18.45 μg/kg in fresh cocoa, and between 0 and 22.82
(a) (b) μg/kg in fermented cocoa, and may persist up to chocolate (Mota-Gu­
72 0.00 ± 0.00 1.86 ± 0.11 (a) 30.11 ± 0.28
tierrez et al., 2019). Ethyl acetate was detected between 0 h and 96 h
(a) (b)
96 0.00 ± 0.00 1.89 ± 0.01 (a) 31.45 ± 0.67
with a larger relative area in H. thailandica than in P. kluyveri, and tended
(a) (b) to decrease over time. Ethyl acetate synthesis possibly occur as a
Ethyl acetate 0 3.47 ± 4.91 42.89 ± 6.22 (b) 2.46 ± 0.69 mechanism for ethanol detoxification in H. thailandica because this
(a) (a) compound was observed with a large relative area in this culture.
24 0.00 ± 0.00 23.98 ± 1.48 (b) 2.24 ± 0.49
However, the observed decrease may be attributed to the action of es­
(a) (a)
48 4.79 ± 2.13 21.35 ± 1.08 (b) 2.66 ± 0.12 terases that hydrolyze the compound and lead to the synthesis of acetic
(a) (a) acid (Lilly et al., 2000), which was only detected in H. thailandica
72 6.52 ± 0.06 19.34 ± 0.51 (b) 3.19 ± 0.09 culture.
(a) (c)
Isoamyl acetate is synthesized from isoamyl alcohol and acetyl-CoA,
96 8.71 ± 1.50 18.61 ± 0.73 (b) 3.20 ± 0.10
(a) (c)
and has been reported at higher concentration in fresh cocoa (0–56.5
120 9.52 ± 2.16 16.31 ± 3.19 (a) 3.05 ± 0.09 μg/kg) than in fermented cocoa (0–17.65 μg/kg) (Mota-Gutierrez et al.,
(a, b) (b) 2019). In this study, a larger relative area was observed in P. kluyveri
Isoamyl acetate 0 6.07 ± 0.56 23.53 ± 18.27 31.08 ± 4.79 culture compared to the control at 24 h, 48 h, and 120 h, while in
(a) (a) (a)
H. thailandica the relative area was different from the control and similar
24 6.85 ± 1.44 41.76 ± 2.98 (b) 53.82 ± 0.54
(a) (b) to P. kluyveri only at 24 h. Isoamyl alcohol, the isoamyl acetate pre­
48 22.03 ± 7.53 39.19 ± 4.79 (a, 49.98 ± 2.99 cursor, was not detected in any treatment. P. kluyveri has been reported
(a) b) (b) to have a great potential to produce isoamyl acetate, and it has been
72 26.36 ± 8.80 35.70 ± 4.82 (a) 47.04 ± 2.36 studied and patented since it was observed that its use as a starter cul­
(a) (a)
96 27.09 ± 5.26 33.64 ± 5.67 (a) 44.54 ± 2.23
ture in cocoa fermentation increased the concentration of this com­
(a) (a) pound in beans (Saerens and Swiegers, 2013). These yeast species
120 27.84 ± 4.22 36.71 ± 2.59 (a, 43.04 ± 2.40 presented helped in the production of volatiles and notes with a high
(a) b) (b) sensory value, and are therefore considered to have bioprospective po­
tential to be used as inoculums in cocoa fermentations.
phenylethyl acetate, ethyl acetate, and isoamyl acetate) have been re­
ported as products of yeast metabolism. Once synthesized within a cell, 4. Conclusion
they can diffuse across the cell membrane because of their lipophilic
nature and be released into the fermentation medium (Loviso and Lib­ In this study, the sensory and chemical potential for producing vol­
kind, 2018). The alcohol acetyl transferase transfers the acetyl group atile compounds of interest was determined in yeasts isolated from
from acetyl-CoA to 2-phenylethanol producing 2-phenylethyl acetate, spontaneous fermentation processes through sensory and chromato­
which has been reported in raw and fermented beans at concentrations graphic analyses of cocoa simulation cultures. Based on our results,
ranging between 0.03 and 0.12 μg/kg, and between 0.73 and 1.69 H. thailandica and P. kluyveri presented sensory profiles characterized by
μg/kg, respectively (Mota-Gutierrez et al., 2019). This compound pre­ high intensity in fruity notes. This could be attributed to the production
sented a larger relative area in P. kluyveri culture than in H. thailandica of acetate esters such as ethyl acetate, 2-phenylethyl acetate, and iso­
culture between 0 and 96 h. However, the estimated area for 2-phenyl amyl acetate. Thence, these species are promising starter inoculums to

7
M.M. Mendoza Salazar et al.
improve the sensory characteristics of fermented beans. However,
further studies are needed to determine their pectinolytic or stress
tolerance potential, which are also important for them to be considered
as starter cultures for the cocoa fermentation process.
Acknowledgements
Authors express their gratitude to Secretary of Agriculture and Rural
Development from Government of Antioquia and to General System of
Royalties of the Nation for the funding of the research project
4600003895 “Implementation of Models to Guarantee the Quality of the
Cocoa Bean in Post-Harvest: Sustainable Strategies Belong to Interna­
tional Trade.” Gratitude to Corporation of Tropical Pathologies and to
Faculty of Exact and Natural Sciences from Universidad de Antioquia for
providing human, financial and physical resources from the institution
for the development of the project. To The National Cacao Federation of
Colombia for the technical support in the field. To Vicerrectoría de
Investigación from Universidad de Antioquia for the support in the
translation of the research paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fm.2021.103896.
Funding
This work was supported by General System of Royalties of the
Nation, Secretary of Agriculture and Rural Development from Govern­
ment of Antioquia and University of Antioquia.
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