Research Article

Received: 22 March 2023 Revised: 25 September 2023 Accepted article published: 27 September 2023 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.13016

Integrating new variables into a framework to

support cacao denomination of origin: a case
study in Southwest Colombia
Carlos E González-Orozco,a* Mario Porcel,a,b Roxana Yockteng,c,d
Alejandro Caro-Quintero,c,e Caren Rodriguez-Medina,f,g
Margareth Santander,c,g Martha Zuluaga,c Mauricio Soto,c
Jader Rodriguez Cortina,c Fabrice Eric Vaillantf
and Sebastian Escobar Parraf,g*

Abstract
BACKGROUND: Cocoa quality plays a pivotal role in establishing denominations of origin, with genotypes, geography, climate
and soil conditions being key variables. However, these factors have not been comprehensively explored in defining cacao
denominations of origin. The present study addresses this gap by laying the foundation for cacao denomination of origin, focus-
ing on the Buenaventura region on Colombia's Pacific coast. Our goal is to provide a holistic understanding of the elements under-
pinning cacao denomination of origin, emphasizing Buenaventura's unique cocoa quality and geographical significance.
RESULTS: Through the Buenaventura case, we propose a robust framework applicable to other cacao-producing regions, elevat-
ing the recognition and value of cacao denomination of origin. Our framework encompasses geography, agronomy, genetics,
microbial diversity, pests and diseases and cocoa quality. In a pioneering move, we propose a cacao denomination of origin in
Colombia, specifically examining Bajo Calima, Sabaletas and Cisneros within Buenaventura region. Buenaventura stands out
for its cocoa quality, characterized by fruity flavors attributed to the rich biodiversity of the lowland rainforest.
CONCLUSION: Our analysis indicates specific geographical indicators for each of the study zones, with Buenaventura identified
as a region with natural characteristics to produce fine flavour cocoa products. Each zone exhibited a high differentiation and
diversity of cacao cultivars. Buenaventura has the potential to be designated as a future denomination of origin for cacao from
the Pacific region of Colombia, characterized by its unique fruity-aroma chocolates. Our framework is adaptable to other cacao-
producing regions, facilitating the establishment of denominations of origin within the cocoa industry and agriculture.
© 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of
Chemical Industry.
Supporting information may be found in the online version of this article.

Keywords: Buenaventura; chocolate; cocoa flavour-aroma; rainforest

* Correspondence to: C E González-Orozco, Corporación Colombiana de Investigación Agropecuaria – Agrosavia. Centro de Investigación La Libertad- Km 14 vía
Villavicencio-Puerto López, Meta, Colombia. E-mail: cegonzalez@agrosavia.co; or S Escobar, E-mail: sescobar@agrosavia.co

a Corporación Colombiana de Investigación Agropecuaria – Agrosavia, Centro de Investigación La Libertad, km 14 via Puerto Lopez, VILLAVICENCIO, Meta, Colombia

b Instituto de Investigación y Formación Agraria, Pesquera, Alimentaria y de la Producción Ecológica (IFAPA), Málaga, Spain

c Corporación Colombiana de Investigación Agropecuaria – Agrosavia, Centro de Investigación Tibaitatá, vía a Mosquera, Bogotá, Colombia

d Departamento de Biología, Facultad de Ciencias, Universidad Nacional de Colombia sede Bogotá, Ciudad Universitaria, Bogotá, Colombia

e Corporación Colombiana de Investigación Agropecuaria – Agrosavia, Centro de Investigación La Selva, via Rionegro - Las Palmas, Sector Llano Grande, Rionegro,
Colombia

f Muséum National d'Histoire Naturelle, UMR-CNRS 7205, Paris, France

g Corporación Colombiana de Investigación Agropecuaria – Agrosavia, Centro de Investigación Palmira, Valle del Cauca, Colombia
1

© 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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INTRODUCTION
Geographical indications (GI) and denomination of origin (DO) are
widely used concepts to determine the geographical and commer-
cial status of food production.1 According to the World Intellectual
Property Organization,2 a GI is a sign used on goods with a specific
geographical origin and possessing qualities or a reputation
because of that origin. DO, on the other hand, refers to food prod-
ucts specific to a particular region or town, conveying a special
quality or characteristic of the designated area (European Union
Commission). Although the guidelines for GI and DO are globally
accepted. standardized scientific frameworks to support their appli-
cation to emerging products in developing economies are lacking.
Consequently, evidence-based frameworks are needed to provide
rigor in defining or understanding GI and DO. The present study
presents a new approach to define and support the establishment
of cacao GI and DO in an understudied tropical region. We propose
a science-based framework to support GI and DO establishment,
using the cacao-producing Pacific region of Buenaventura in
Colombia as a case study. Here, we will refer to the cacao tree as
‘cacao’ and derived products made from cocoa beans as ‘cocoa’.
GI and DO are effective tools for differentiating foods and agro-
nomical products.3 Global guidelines on GI regulations have been
developed by FAO to strengthen capacities at various levels of the
food industry. Even within Europe, each country develops its own
regulations. In 2017, France published a booklet with user guide-
lines for GI and DO,4 providing clarity on rules for licensing DOs. In
Latin America, the commission of the Andean community created
a policy document on industrial property called Decision
486, which is related to patents of genetic resources. However, it
does not offer a detailed guideline on DO requirements specifi-
cally; instead, it states that registration is a matter for each coun-
try. Consequently, regional legislations are unclear, complicating
the application process for GIs or DOs. This lack of clarity leads
to misinformation and a poor understanding of the commercial
value of GIs and DOs as tools. Colombia, for example, is no excep-
tion. The Colombian Commerce and Industry institution (CCI), the
government entity responsible for issuing DOs, provides just four
bullet points outlining the requirements for applying for a certifi-
cate of denomination of origin. This list is clearly insufficient for
the extensive task at hand in the cocoa industry.
Native tropical cacao trees and their cultivated cousins are signif-
icant sources of cocoa aromas.5 However, they are not utilized as fre-
quently as expected for proposing DOs and GIs. Several examples of
cacao DOs or GIs exist in South America, such as Cacao Chuau in
Venezuela,6 Cacao Arriba in Ecuador,7,8 Cacao Grijalva in Mexico9
and Cacao Tomé-açu in Brazil.10 The Cacao Arriba region in
Ecuador shares similar latitudes and climates with the Buenaventura
region. In Ecuador, the provinces of Los Rios and Guayas have a long
history as cacao-producing areas since the colonial period. In 2011,
the Ecuadorian Institute of Intellectual Property granted a DO for
Ecuador's fine aroma cacao, including the Cacao Arriba region on
the Pacific Coast. However, the DO for Ecuador is applied under a
broad scheme, and more specificity is needed in delimitating DO
regions. For example, it would be an improvement if Ecuador could
derive from the National cacao a set of specific DO´s delimitated
based on new surveys or methodologies. In the case of Venezuela,
the Cacao Chuau in Venezuela's Aragua state covers a specific geo-
graphic valley along a river that leads to the Caribbean Ocean.
Validating DOs over time is a constant challenge for these
regions because Chuau's DO is still under scrutiny, and finalizing
its designation remains an ongoing task. Despite a few examples
2
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CE González-Orozco et al.
of Cacao DOs in the tropical region of South America, a lack of sci-
entific rigor in the processes involved in supporting DO assign-
ments is a common characteristic of all the mentioned cacao
cases. The documents supporting the request for the DO license
often consist of four sections: (1) a legal framework; (2) a descrip-
tion of the geographic region; (3) quality characteristics of the
product and its marketplace; and (4) communities and cacao asso-
ciativity or trading networks. Adopting a more evidence-based
and focused approach would greatly benefit intellectual property
and legal processes in the domain of food product qualifications.
Colombia lags behind in the realm of cacao DOs. However, it
holds the potential to become a future stronghold for cacao in
the region as a result of its unique geographical conditions and
diverse environments, encompassing 54 cacao regions in
Colombia.11 As mentioned before,12 achieving a comprehensive
system to improve GI and DOs legislation in Colombia requires
significant inter-institutional coordination between the govern-
ment, communities and private industry. Indeed, over the last
decade, the CCI has only granted 19.13 There are some examples
of DOs in the south American coffee industry including
Colombia and Honduras.14,15 However, the lack of scientific
advances in cacao DOs and GIs in Colombia is counterproductive,
considering the country's abundant agrobiodiversity, and its sta-
tus as a centre of cacao origin, accompanied by cultural richness.
Together, these factors present numerous opportunities in the
food industry. Despite these natural advantages, Colombia has
yet to issue any cacao DOs or GIs. As mentioned in Section 3.3
of the CCI technical manual, one of the weaknesses is limited sci-
entific research on the topic, compounded by a lack of qualified
professionals in the field. However, the situation is currently evolv-
ing in Colombia. Recent cacao studies provide new evidence to
advance the creation of GIs for cacao in the country, such as pro-
posing a cacao regionalization.11 This regionalization is the first of
its kind and serves as the scientific foundation for defining the
separation of geographical cacao regions, which is useful because
it goes beyond political boundaries. It represents a novel
approach because the territory's geography drives the proposed
classification the river systems at different levels (i.e. catchment,
sub-catchment), with microclimate based on the soil temperature
and macroclimate data being used to define the cacao regions.
This stands in contrast to existing cacao crop classifications, which
rely on agronomical aptitude and departmental classification.16
The knowledge of cacao bean flavor quality serves as an exam-
ple to illustrate the differentiation of cacao origins.17 Similar to
Venezuela and Ecuador, Colombia also produces fine-flavored
cocoa. Certain Colombian regions have gained worldwide recog-
nition for their high-quality cocoa, solely based on organoleptic
characteristics. However, there is a lack of scientific data support-
ing the holistic evaluation of Colombian cacao beans. Neverthe-
less, recent studies have demonstrated the unique sensory
qualities of Colombian cacao.18,19 Cocoa beans from the Arauca
region in eastern Colombia have twice been awarded the title of
best cacao in the world at the ‘International Cacao Awards’ held
during the ‘Salon du Chocolat’ in Paris. This recognition was also
achieved by cacao beans from Tumaco region, situated in south-
western Colombia geographically close to the Buenaventura
region. Additionally, other studies emphasized the quality poten-
tial of the FEAR 5 cacao cultivar developed in the Arauca region,
known by its fruity and nutty aromas.19 In parallel, more work
has been carried out on developing a fermentation methodology
for cacao cultivated in the pacific region,18 aiming to enable cacao
farmers to consistently produce fine flavored cacao beans. These
J Sci Food Agric 2023

Cacao denomination of origin
are examples of cultivated cacao, but Colombia is home to many
unexplored materials of wild cacao species,20 with numerous wild
genotypes from the Amazon still waiting to be discovered and uti-
lized in determining future designations of origin.
To date, no comprehensive characterization has been con-
ducted to define or classify cocoa quality based on its genetic
diversity, regional or local climate, soil characteristics, landscape
features, sensorial and biochemical indicators, and productivity
aspects. This holds true not only for Colombia, but also for other
cacao-producing countries in South America. Consequently, the
series of steps and variables that our framework contains repre-
sent a significant advancement in the field of cacao DOs and
GIs, particularly for unexplored geographic areas within the trop-
ical Pacific region of Colombia. The present study demonstrates
the feasibility of establishing cacao DOs and GIs by employing a
multidisciplinary and robust scientific approach that lays the
foundation for supporting cacao DOs through a series of indepen-
dent steps.
MATERIALS AND METHODS
Study region and sampling zones
The Buenaventura region is located in the department of Valle del
Cauca, situated in the southwestern part of Colombia along the
Pacific Ocean (Fig. 1). Buenaventura is part of the Biogeographical
Chocó region.21 The region is encompassed by tropical rainforests
that experiences some of the highest precipitation levels on
earth.22 The exceptionally high rainfall is a result of the conver-
gence of vast amounts of air rising from the ocean, combined with
the intricate and rugged topography of the area, as well as the
intense evapotranspiration occurring within the tropical rainfor-
est. Within the Buenaventura region, three specific cacao sam-
pling zones were selected, each representing a distinct cacao
cropping system.
The average climate conditions for each study zone are pre-
sented in Table 1. Zone 1 (Cisneros) is situated closer to the foot-
hills of the western Andean mountain range at an elevation of
515 m a.s.l. It experiences the highest amount of solar radiation
and notable temperature variations between the maximum and
minimum values during the day and night. Zone 2 (Bajo Calima),
located on the lowlands closer to the Pacific Ocean at approxi-
mately 40 m a.s.l, is characterized as the rainiest and most humid
among all three zones. Lastly, zone 3 (Sabaletas) is positioned fur-
ther south on the marine plains of the coastal areas, with an eleva-
tion of 15 m a.s.l. This zone exhibits the highest average
maximum temperature and is the warmest among the three.
The soils found in the cacao plantations of the study region are
primarily Inceptisols and Entisols. These soils are characteristic of
the area and are highly acidic, with pH levels from 5.0 to 5.4. Addi-
tionally, they occasionally show significant limitations as a result
of aluminium toxicity. Lime application and fertilization practices
are infrequent. The dominant soil textures in the region are loam
and clay loam, and the organic matter content varies, typically
ranging from moderate to high levels.
Summary of the proposed framework
Our framework is not a mere variable-based methodology but
comprises a series of steps to establish cacao denominations of
origin in tropical regions. Cocoa quality, including other unique
traits, is vital for such denominations. Our proposal integrates
these variables, contributing significantly to the concept,
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although we do not claim to devise a comprehensive analytical
methodology.
The proposed bottom-up framework comprises two inter-
connected cycles (green and blue cycles in Fig. 2). The first step
is a regionalization mapping, which involves scaling from a
national to regional level using climate and soil indicators specific
to each geographic area (green cycle). Once the region of interest
is identified, an agronomical assessment is conducted to charac-
terize the cacao cropping system, including the evaluation of
pests and diseases present in the region. Moving forward, the
blue cycle focuses on quantifying product typicity variables of
cacao at both the individual farm and clusters of farms levels. This
cycle involves conducting a genetic identity analysis, a detailed
typification of cocoa quality, and a survey of microorganism diver-
sity, all of which are crucial in defining the sensorial characteristics
of chocolate. All these steps are interconnected and collectively
constitute the fundaments and key variables in the proposed
framework that are required to achieve a well-established denom-
ination of origin for cacao.
Geography
Regionalization of GIs
A biogeographical regionalization approach was employed to
identify distinct cacao regions in Colombia, which were subse-
quently designated as GIs.11 To conduct the regionalization, a
dataset consisting of 4974 cacao farm occurrences of cacao across
Colombia, along with macroclimate variables,23 and microclimate
variables,24 were utilized as inputs. The macroclimate WorldClim
v.2 dataset23 comprises seven variables, including precipitation,
maximum temperature, minimum temperature, mean tempera-
ture, relative humidity, wind speed and solar radiation, with a spa-
tial resolution of 1 km2 and a soil depth of 0–5 cm. The
microclimate analysis employed the SBIO dataset,24 which con-
tains eleven soil temperature variables. The macroclimate analysis
enabled an understanding of the climate variations between dif-
ferent geographic regions, whereas the microclimate analysis
focused on determining specific microclimate conditions based
on soil properties.
Mean climate and soil temperature values for each cacao farm
occurrence were extracted from the WorldClim2 climate raster
and the SBIO soil temperature raster. Principal component analy-
sis (PCA) was performed using the extracted climate data from
WoldClim2 and SBIO rasters, employing R, version 3.6.3
(R Foundation, Vienna, Austria) and the ‘vegan’ package. The
PCA axis 1 results were then mapped onto the climate and soil
temperature values using a geospatial application.25 This applica-
tion facilitated the generation of climate and soil temperature
maps for all cacao producing regions in Colombia, based on the
principal components. These results serve as GIs, reflecting spe-
cific geographical origins, and were utilized to identify the cacao
GI for Buenaventura at the national level. Lastly, a geospatial
local-level PCA analysis was independently conducted for the
three sampling zones using the same methodology, aiming to
characterize the finer differences in local climate and soils
between the three zones. This local analysis can be considered
as a fine-scale identification of GIs.
Agronomic baseline
A technical diagnosis of cacao farmers was conducted using a par-
ticipatory methodology, where farmers provided descriptive
information regarding their agronomic practices and farming sys-
tems. In total, 12 cacao farms for the three zones were visited, and
3
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Figure 1. Map of Colombia with the location of the Buenaventura study region on the Pacific coast and the three sampling zones.

Table 1. Average climate conditions for the three sampling zones

Maximum Mean Minimum Vapor Wind

Diurnal temp Solar radiation temperature temperature temperature Rainfall pressure speed
Zones range (°C) (kJ m−2 day−1) (°C) (°C) (°C) (mm) (kPa) (m s−1)

1. Cisneros 8.74 16.310 29.06 24.70 20.32 2686 2.52 1.05

2. Bajo Calima 7.65 15.766 30.15 26.33 22.45 6262 2.88 1.31
3. Sabaletas 8.25 16.123 30.54 26.37 22.24 4169 2.86 1.21

Note: Underlined values are the maximum averages of the three zones for each climate variable.

interviews were conducted with 32 farmers. Some of these
farmers were members of cacao grower associations active in
the study area, including ACABC (association of farmers and cacao
growers of Bajo Calima), APCC (Cisneros growers association) and
Sabaletas Community Council. The cacao plantations in the study
region contained genetic materials of cacao related to FQ-1,
FEAR5, F61, SUI-83, SUI-62, GS, TSH-565, ICS-95, CCN-51, FCH-8,
FQ-1 and FEE-2.
Pests and diseases
The infestation level of the cacao pest species and the incidence
of fruit diseases were determined through a participatory
4
approach that involved close collaboration between farmers and
researchers. In total, 11 plantations were selected, with three
located in zone 1, three in zone 2 and five in zone 3. The sampling
took place between the 6th and 10th of October 2018, during the
peak cacao harvest period of the year. In each plantation, 10 trees
were randomly chosen by following a zigzag transect pattern that
aimed to cover the entire field. The path and distance of the tran-
sect were approximately determined beforehand during an
inspection of the plantation, with guidance from the farmer.
For each sampled tree, we conducted a comprehensive assess-
ment of cacao fruit damage. Specifically, we recorded the damage
caused by different categories of pests, including:

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Cacao denomination of origin
Figure 2. Flow diagram of the proposed framework.
(1) cacao mirids (Hemiptera: Miridae),
(2) sucking insects (other than mirids), that included treehoppers
and thorn hoppers (Hemiptera: Membracidae) as well as scale
insects (Hemiptera: Coccidae) and mealybugs (Hemiptera:
Pseudococcidae),
(3) pod borers (Lepidoptera),
(4) geometer moths (Lepidoptera: Geometridae), and
(5) rodents and other mammalians.
To identify cacao mirid damage, we looked for the characteristic
dark, round spots on the pod surface26 (see Supporting informa-
tion, Fig. S1A). Damage caused by other pod-sucking insects
was differentiated from mirid damage by its black to brown scar-
ring, irregular shape and often connected scars, indicating the
previous presence of clustered mealybug or membracid colonies
(see Supporting information, Fig. S1B). These scars sometimes had
yellowish–green halos, which were not observed around mirid
spots. To confirm this type of damage, we removed mealybug
and membracid colonies from the pod surfaces to examine the
scarring caused by their feeding activity. Because of their similar
appearance, this type of damage was grouped into a single cate-
gory and could not be distinguished without the presence of the
hemipteran colony.
Pod borer damage was characterized by black entry spots
accompanied by humid frass buildup in their vicinity, often with
multiple spots per pod. We checked these spots for tunneling
activity and the presence of larvae. Geometrid damage (see Sup-
porting information, Fig. S1C) was identified by cherelles or pods
with gnawed scars, resulting in wide depressions or holes on the
smooth surface (the latter likely caused by late instar larvae). Dam-
age to cherelles and young fruits often led to misshapen fruits as
they matured (see Supporting information, Fig. S1C). Geometrids
were observed on leaves, fruits and leaf surfaces, where they con-
sumed the plant material.
Finally, rodents created large holes in the pods to access and
consume the pulp and seeds (see Supporting information,
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Fig. S1E). When available, insect pests associated with the dam-
aged pods were collected for taxonomic identification.
The primary disease issues affecting cacao, including (1) the
frosty pod rot (FPR), caused by Moniliophthora roreri (Cif. & Par.);
(2) the black-pod rot (BPR), caused by Phytophthora spp.; and
(3) witches' broom disease (WBD), caused by Moniliophthora per-
niciosa, were identified, although not all were surveyed in the pre-
sent study.
To determine the causal agent responsible for each disease,
plant tissue samples were collected from infected plants. Dis-
eased pods were carefully removed, including healthy tissues
extending approximately 4 cm beyond the lesions. These samples
were then placed in plastic bags and transported to the laboratory
for further analysis.
In the laboratory, the samples underwent surface sterilization
using a 2% sodium hypochlorite solution for 15 min. Subse-
quently, they were treated with 70% ethanol for 1 min and rinsed
three times with sterile water. The plant tissue was dried and then
sectioned into six segments measuring 0.5 cm each. These seg-
ments were directly placed on Potato Dextrose Agar (PDA; Oxoid,
Basingstoke, UK) supplemented with 0.1% Triton 100X (Sigma-
Aldrich, St Louis, MO, USA) and 0.3% chloramphenicol to prevent
bacterial contamination. The plates were incubated at 25 ± 2 °C
under 24 h of light for 7 days. Any fungal growth observed was
sub-cultured onto fresh PDA or water agar (1% w/v) until pure col-
onies were obtained.
To compare infestation levels across the different study zones,
we employed generalized mixed effect models (GLMM) with a
Poisson error distribution and a log-link, utilizing the ‘lmer4’
package. The experimental unit was defined as the individual
tree, with the number of pods displaying signs of pest and dis-
ease damage compared to the total number of pods per tree.
This ratio was used as an offset variable.27 The sampling zone
was included as an explanatory variable, and the plantation
was treated as a normally distributed random effect to account
for any spatial autocorrelation among trees within the same
5
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transect. Pairwise post-hoc comparisons between zones were
conducted using Tukey's tests on the model estimations. Models
were assessed for overdispersion, and the Pearson residuals ver-
sus the fitted values were visually inspected to validate the
models.
Genetics
Plant material and DNA extraction
Leaf samples were collected from the three study zones to obtain
genotypic information on the cultivated cacao trees in the region.
Leaves were sampled from ten farms in zone 1, six farms in zone
2 and 10 farms in zone 3. DNA extraction from the leaves and subse-
quent genotyping was performed via LGC Biosearch Technologies
(Teddington, UK). KASP genotyping assays were utilized to identify
single nucleotide polymorphisms (SNPs) at specific loci of the cacao
genome, which had been previously used in various studies.28–32 Bi-
allelic discrimination was achieved by competitively binding two
allele-specific forward primers, each having a unique tail sequence
that corresponded with two universal fluorescence resonant energy
transfer cassettes. One cassette was labeled with FAM dye, whereas
the other was labeled with HEX dye.
Phylogenetic analysis
Out of the initial 96-SNP panel selected for studying genetic diversity,
87 SNP markers were utilized to align the new dataset generated in
the present study with the dataset published previously.31 The align-
ment of the SNP dataset was performed using ClustalX, version
1.83,33 and visually confirmed using Geneious, version 8.1.7.34 After
concatenating the data, a phylogenetic tree was reconstructed using
the maximum likelihood method with PhyML, version 3.0,35 imple-
mented in a bioinformatics platform (http://www.atgc-montpellier.
fr/phyml). The phylogenetic analysis involved 100 bootstrap repli-
cates, and gap sites were treated as missing data. The species Theo-
broma grandiflorum (Wild Ex. Spreng. Schum) was used as an
outgroup in the phylogenetic tree.
To assess possible significant genetic differences between the study
zones, a one-way permutational multivariate analysis of variance
(PERMANOVA) was conducted, and the data was visualized through
PCA using Past, version 4.1.36 The PCA analysis was based on the
sum of SNP differences between samples. Furthermore, the ancestry
of each genotype was analyzed using the intersected SNPs represent-
ing the 10 recognized cacao genetic groups.37 A maximum likelihood
analysis was performed using Admixture, version 1.3,38 in a supervised
mode, where the reference genetic groups were specified.
Quality
Typification
One post-harvest transformation site for each zone was con-
ducted to evaluate the quality potential of cacao cultivated.
Figure 3 illustrates the stages involved in the process. The assess-
ment of quality potential involved studying the flavour profiles of
chocolate (or cocoa) and analyzing the cocoa metabolome after
post-harvest processing. The entire process was carried out at a
single site in each region, utilizing the same fermentation meth-
odology and equipment.
Postharvest transformation process
Harvesting involved selecting a representative sample of cacao
cultivars from each of the 32 farms in each study zone. Healthy
and fully ripe cacao pods were carefully harvested one day prior
to the fermentation process and then transported to the central
postharvest transformation site.
6
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CE González-Orozco et al.
The fermentation and drying processes were conducted as fol-
lows: Cacao pods were carefully opened using sharp knives, and
the seeds were manually extracted and mixed to create an initial
mass comprising all the collected cacao cultivars. From this
initial mass, 50 kg of seeds were extracted and placed into six sep-
arate compartments within a wooden fermentation box (Fig. 3),
allowing for six repetitions of the process. The fermentation fol-
lowed a standard method widely employed by cacao producers
in Colombia.39 Initially, the cacao mass underwent an anaerobic
stage lasting 48 h without any mixing. Subsequently, mixing
and aeration of the cacao seeds were performed every 12 h for
a total duration of 120 h. Throughout the fermentation process,
the temperature of the fermentation mass was monitored at the
central point of each compartment using a thermocouple (model
735-1; Titisee-Neustadt, Germany). Finally, the cacao samples
intended for sensory and metabolomics analysis were dried in a
forced convection oven at 50 °C, utilizing hot air.
During the sampling process, 300 g of cacao beans were
extracted from the central portion of the cacao seeds mass in each
of the six compartments of the fermentation box. These beans
were specifically designated for sensory analyses. Additionally,
50 g of cacao seeds were extracted for untargeted metabolomics
analyses.
Quality analysis
For the descriptive sensory analysis and evaluation of chocolate
flavour profiles, chocolate bars containing 67% (w/w) cacao solids
were produced. The laboratory of sensory analysis at CIRAD
(Centre de Coopération Internationale en Recherche Agronomi-
que pour le Développement, Paris, France) employed a standard-
ized protocol.18 The descriptive sensory analysis was conducted
using the consensus profile method outlined by the International
Organization for Standardization (ISO).40
The blind tasting sessions involved 8 trained judges who evalu-
ated the chocolate samples in duplicate. Two evaluation sessions
took place. In the initial session, the judges agreed upon the sen-
sory attributes to be assessed. Subsequently, each judge individu-
ally evaluated all the chocolate samples, assigning scores on a
scale from 0 to 10 for the predetermined sensory attributes. Cron-
bach's alpha coefficient was used to assess the reproducibility of
the judges’ evaluations.
To confirm the assumptions of normality and homogeneity of
variance, Shapiro–Wilk and Levene's tests were conducted.
Because these assumptions were not met, a non-parametric test,
specifically the Kruskal–Wallis test, was employed to determine
significant differences among the chocolate samples from the
various geographic zones. A Bonferroni correction was applied
to post-hoc test pairwise comparison zones.
Untargeted metabolomic analysis: the chemical fingerprint of
fermented and dried cacao beans
Extraction, instrumental analysis, and data processing procedures
followed stablished methodologies.19,41 Initially, all samples were
ground and defatted. An extraction solution composed of ace-
tone water:formic acid (70:29.5:0.5) was employed, and ultrasoni-
cation was utilized during the extraction process.
For instrumental analysis, an ultra-performance liquid
chromatography-electrospray ionization (UPLC-ESI)/quadrupole-
time-of-flight-mass spectrometry (MS) system (Acquity; Waters
Inc., Milford, MA, USA) was employed. Chromatographic separa-
tion was achieved using a UPLC CSH C18 column (1.7 μm particle
size, 2.1 mm × 100 mm; Waters Inc.) coupled with a pre-column
J Sci Food Agric 2023

Cacao denomination of origin
Figure 3. Scheme of the methodology developed to analyze the quality potential of the cacao cultivated in the selected farms from Sabaletas, Cisneros
and Bajo Calima.
VanGuard CSH C18 (1.7 μm particle size, 2.1 mm × 5 mm; Waters
Inc.). A gradient elution of water and acetonitrile (solvent B) was
utilized. ESI-MS analyses were conducted in positive ionization
mode, employing a full scan range of 100 to 1500 Da in continu-
ous mode. To ensure quality control, two pooled samples were
measured after every 10 samples.
Data processing involved the use MassLynx, version 3.1 SCN
639 (Waters Inc.) and Progenesis QI software (Waters Inc.) to select
and align chromatographic peaks. The effect of batch-related drift
on the quality control baseline was corrected using Metabodrift.42
Data normalization was performed using the LOESS (i.e. locally
reweighted scatterplot smoothing) method (⊍ = 0.5). Finally, the
data were transformed using a dilution factor that accounts for
the mass of the cacao powder and the mass of the sample upon
resuspension.
To integrate the extensive metabolomic data and conduct mul-
tivariate statistical analysis, a PCA was employed to visualize the
data patterns and trends. Subsequently, a PERMANOVA was con-
ducted to identify significant differences in the metabolomes of
the fermented and dried beans between the three zones. Addi-
tionally, partial least square discriminant analysis (PLS-DA) was
utilized to evaluate the relationship between the metabolomes
and the different zones, as well as to assess the predictive ability
of the classification model.
Microorganisms (bacteria)
The bacterial cells associated with the cacao bean mass were sep-
arated from the samples using the protocol previously
described.43 Briefly, frozen beans (20 g) were homogenized with
J Sci Food Agric 2023 © 2023 The Authors.
Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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www.soci.org
0.85 g per 100 mL of NaCl in 250-mL Erlenmeyer flasks using a
rotatory shaker (Thermo Scientific MaxQ 4000; Invitrogen, Burling-
ton, ON, Canada) at 150 rpm for 30 min. The resulting fluid was
decanted and filtered through sterile gauze. The free-pulp solu-
tion was then centrifuged at 3220 × g at 12°C for 20 min
(Hermle, Z32HK; Labortechnik GmbH, Wehingen, Germany) and
the pellet obtained was resuspended in 10 mL of sterile saline
solution. DNA extraction from this solution was performed using
an UltraClean Microbial DNA isolation kit (MoBio, Carlsbad, CA,
USA). The concentration and purity of the extracted DNA were
quantified using a NanoDrop 1000 Spectrophotometer (Thermo
Fisher Scientific, DE, USA) and stored at −20 °C.
The bacterial communities associated with the cacao bean mass
were analyzed by sequencing the hypervariable V4 region of the
16S rRNA gene. Bacterial libraries were prepared following
the protocol previously described44 which involved a two-step
PCR procedure. In the first PCR, modified primers 515F-806R,45
with a linker region in the 50 end were used. In the second PCR,
barcodes, as well as the Illumina i5 and i7 regions, were added
to the amplicons and followed a specific protocol.46 The libraries
were sequenced using an Illumina MiSeq with the kit V2
(Illumina, San Diego, CA, USA) (2 × 250 bp).
Bioinformatic analysis was conducted to taxonomically identify
the composition of the bacterial communities. First, the samples
were demultiplexed using an in-house script. The quality of the
sequences was assessed using FastQC v0.11.7 (http://www.
bioinformatics.babraham.ac.uk/projects/fastqc). Adapters and
linker sequences were removed from the partial sequences of
the 16S rRNA gene using Cutadapt, version 1.12.47 Low-quality
7
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www.soci.org
sequences, with an average Phred score < 20 and a read
length < 200 nucleotides, were identified and removed from
the datasets using Trimmomatic, version 0.38.48 The Qiime 2
pipeline v_2018 (https://forum.qiime2.org/t/qiime-2-2018-8-
release-is-now-live/5860) was employed for the analysis of the
16S rRNA gene amplicon libraries. The Dada2 (https://qiime2.org)
algorithm was used to denoize the sequences and generate
amplicon sequence variants (ASVs). The pair-end sequences were
inputted into Dada2 with a truncation length of 200 bp. ASVs that
were present in fewer than two samples or had less than four
sequences in all the samples in the feature table were excluded
from further analyses. The taxonomic assignment of the ASVs
was performed using the Silva Database, version 132.49
RESULTS AND DISCUSSION
Regionalization of GIs
The GI for the Buenaventura study region was identified as part of
the cacao southwest region, specifically the 4C sub-region and
central Calima province number 43 in the department of Valle
del Cauca.11 The climate and soil temperature conditions in this
province are characteristic of the lowland rainforests in the Pacific
region. However, the GI for zones 2 and 3 differs from that
observed for zone 1 as a result of their lower elevation. Soil tem-
perature seasonality is lower in zones 2 and 3 compared to zone 1.
The local level geospatial PCA analysis demonstrated that each
sampling zone possesses specific climate characteristics (Fig. 4).
Consequently, the study zones have distinct climate GIs, as indi-
cated in Table 1, which are further supported by the national level
regionalization. Solar radiation and daily temperature range
emerge as the primary driving variables in zone 1, suggesting
fewer extreme conditions compared to the other zones. Con-
versely, zone 2 is strongly influenced by high levels of rainfall
and humidity. Temperature exhibits the most significant effect
on zone 3.
Figure 4. Local level climatic zones determined by a geospatial analysis
of climate mapped as a principal component analysis of axis 1 (% explained
variance by the principal component).
8
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Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
10970010, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jsfa.13016 by Cochrane Colombia, Wiley Online Library on [19/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
CE González-Orozco et al.
Agronomic baseline
The types of cacaos found in the whole study region were FQ-1,
FEAR5 (Trinitario), F61, SUI-83, SUI-62, GS, EET-72, TSH-565
(Trinitario), ICS-95, CCN-51, FCH-8, FQ-1 and FEE-2. In zones
3 and 2, the main cacao cultivars grown were ICS 95 (Trinitario x
Criollo) and CCN 51, which were planted in the context of a rural
development project aimed at encouraging cacao cultivation.
Most farms in the region were small-scale, ranging from 1 to
4 ha in size. The average age of the cacao plantations was approx-
imately 9 years, but most farmers did not maintain records of the
exact number of cacao trees on their farms. Excessive shading was
observed in certain farms in zones 2 and 3, likely as a result of the
efforts to mitigate the effects of high rainfall. This shading can
affect cacao production. Diseases such as witch's broom, frosty
pod rot and black-pod rot also require intensive agronomic man-
agement to mitigate their impact on the plantations. The three
study zones featured diversified farming systems, with a focus
not only on cash crops, but also on other native crops for domes-
tic consumption. The most common were peach palm (Bactris
gasipaes Kunth), coconut (Cocos nucifera L.), cassava (Manihot
esculenta Crantz), citrus fruits (Citrus spp.), borojó (Borojoa patinoi
Cuatrecasas) and Chinese potato (Coleus parviflorus L.). Some
farmers also mentioned the presence of timber trees such as
guino (Carapa guianensis Aubl.) and sajo (Campnosperma pana-
mense Standl.).
Pests and diseases
In total, 1358 cacao pods were assessed for pest and disease dam-
age. Regarding pests, rodent-damaged pods were only recorded
in a single plantation in zone 2, with two pods (1.5% damage)
so they were not statistically analyzed. In the overall study region
of Buenaventura, geometrid and sucking insect damage (exclud-
ing cacao mirids) were the most commonly observed on cacao
pods, accounting for 19.2 ± 1.9% and 17.9 ± 1.9% of the pods,
respectively (mean ± SE). Pod borers and signs of cacao mirid
feeding activity were much less frequent, present in only 3.5
± 0.8% and 3.4 ± 0.3% of the pods, respectively.
Zone 1 had higher infestation levels of cacao mirids and pod
borers compared to the other two zones (Fig. 5A,D). No pods dam-
aged by these pests, nor the insects themselves, were observed in
zone 2. However, zone 2 recorded the highest levels of fruits dam-
aged by geometrids compared to the other zones (Fig. 5B). In
these plantations, a large number of geometrid larvae were
observed feeding on cacao foliage (M. Porcel, personal observa-
tion). Zone 2 also had a high number of pods with signs of sucking
activity, presumably from membracids. Zone 3 showed high activ-
ity of geometrids and membracids (Fig. 5B,C), whereas a moder-
ate number of pods damaged by cacao mirids and pod borers
were observed (Fig. 5A,D).
Pest damage was similar between zones 2 and 3 but differed sig-
nificantly from zone 1, which showed higher damage caused by
Monalonion spp. and cacao pod borers, which are well-established
pest species in the country.50 These differences in pest damage
could be attributed to the climatic conditions determined by altitude
(Fig. 4). Previous studies on cacao pests in other regions of the coun-
try have primarily focused on inland cacao plantations that have cli-
matic conditions more similar to zone 1. Therefore, the high levels of
geometrid and sucking insect damage (mainly caused by the mem-
bracid Horiola picta Coquebert) could be associated with the high
humidity and temperatures found in these zones. Under such condi-
tions, there may be a higher turnover of cacao damage, particularly
in the case of geometrids, which can cause severe deformations to
J Sci Food Agric 2023
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Cacao denomination of origin www.soci.org

Figure 5. Proportion of cacao pods (mean ± SE) with feeding damage caused by (A) cacao mirids, (B) geometrid larvae, (C) sucking insects (other than
mirids) and (D) lepidopteran pod borers and incidence per pod of (E) frosty pod rot disease, (F) black pod rot disease and (G) witches' broom disease.
Different letters indicate statistically significant differences (GLMM: Tukey's test, P < 0.100). No letter is assigned when the percentage is zero.

pod and consume entire cherelles. The cacao mirid species identified
in the study region was Monalonion dissimulatum Dist, which is the
most common species associated with cacao in the neotropics.51
All the pod borers identified belonged to the Ecdytolopha genus,
whereas species of the Carmenta genus were not found in the pre-
sent study.
As part of the participatory research approach, it was observed that,
although farmers were able to identify unhealthy cacao pods, they
struggled to determine the specific type of pest or disease affecting
the plants. There was significant variation in the way farmers managed
unhealthy plants in the field. Some farmers mentioned following man-
agement methods recommended by agrochemical dealers, whereas
others relied on traditional approaches for disease management such
as sanitary harvesting. Additionally, important agronomic practices
such as pruning and removing cacao pods affected by pathogens
were not commonly practiced in the region, leading to a continuous
presence of diseased pods and facilitating the spread of pathogens.
Cacao growers expressed that the implementation of more rigorous
agronomic practices increased production costs, thereby reducing
profitability. However, a few cases of farmers with better management
practices were also observed.
FPR was identified as the most significant disease affecting the
cacao production system (Fig. 5E). In mature pods (2.5–
3.5 months old), brown spots with irregular edges were observed.
The severity of the disease varied, but many infected pods were in
the sporulation phase, which is critical for the optimal spread of
spores by the pathogen. This finding indicates that infected pods
attached to the trees were not effectively removed. In total, 33 iso-
lates were obtained from three study zones. Macroscopic charac-
teristics confirmed that the isolates collected corresponded to
Moniliophthora spp. for FPR and WBD. However, it was not possi-
ble to isolate Phytophthora spp. from cacao pods affected by BPR.
Previous reports have already highlighted that FPR disease is one
of the main factors limiting yields in tropical America.52-53 It is twice
as destructive as BPR and more challenging and dangerous to con-
trol compared to WBD. This poses a serious challenge for cacao
growers in the region, emphasizing the urgency to develop new
cacao materials that are resistant to FPR.
Genetics
Phylogenetic analysis
The maximum likelihood phylogenetic analysis resulted in a tree
with low bootstrap values, indicating insufficient support for the tree
nodes. The samples from Buenaventura were distributed across dif-
ferent branches, suggesting that they likely have diverse genetic
backgrounds and belong to distinct cacao genetic groups (Fig. 6).
Farms in zone 1 exhibited materials related to those from the
Colombian Cacao Federation (Fedecacao), including FQ-1 and
FEAR5, as well as Colombian hybrid commercial materials such as
F61, SUI-83, SUI-62 and GS. One tree in zone 1 was potentially asso-
ciated with Ecuadorian cacao EET-72. Materials from zone 2 showed
close relationships with Ecuadorian materials (EET), the Trinidad
commercial material TSH-565 and materials from Fedecacao. In zone
3, the materials were related to ICS-95 and CCN-51. Other cultivated
materials demonstrated stronger connections with Fedecacao, such
as FCH-8, FQ-1, and FEE-2, whereas one material was linked to cacaos
from Tumaco (Colombian Pacific coast region). The PCA results
(Fig. 7) indicated that the samples did not form distinct clusters
based on their zones, and the PERMANOVA test confirmed no signif-
icant genetic differentiation between the zones (P = 0.19).
In the supervised mode of population structure analysis, we
were able to determine the ancestry of the samples from Buena-
ventura (Fig. 7). Our findings suggest that the materials found in
this region are likely the product of admixture. All the genotypes
9

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Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
 10970010, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jsfa.13016 by Cochrane Colombia, Wiley Online Library on [19/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
www.soci.org CE González-Orozco et al.

CHOCOLATE_4

CRICF_37
CRICF_30
CRICF_11
CRICF_26
CRICF_46
CRICF_31
CRICF_50
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Figure 6. Maximum likelihood phylogenetic tree showing the relationships of the samples from Buenaventura (green, red and blue labels) with
AGROSAVIA cacao germplasm.

exhibit mixed ancestry from the Amelonado and Criollo genetic study, an integrated approach combining untargeted metabolomics
groups, with the majority also displaying ancestry from the Iquitos and multivariate statistics was employed to analyze the chemical
and Contamana genetic groups. Only two materials, one from profiles that define the unique characteristics of cocoa products from
zone 2 and one from zone 3, showed apparent ancestry different agroecological regions in Buenaventura.
from the Nacional genetic group. These results indicate that the After processing the raw files, approximately 4400 features were
cultivated genotypes in these farms have a hybrid origin, combin- detected within the mass range of 100–1500 Da in positive mode.
ing genetic traits from different cacao genetic groups. The unsupervised PCA revealed distinct clustering of metabolo-
mic fingerprints among the regions (Fig. 8A). The majority of the
Quality analysis variance was explained by the first two components (Fig. 8B), with
Untargeted metabolomic analysis to define a chemical notable differentiation observed between zone 1 and zones 2 and
fingerprint of cacao 3, as confirmed by the significant differences identified through
Metabolomics serves as a valuable tool for evaluating the impact of the PERMANOVA analysis (P < 0.001). Each metabolome repre-
various factors, including geographical origin and postharvest pro- sents a unique chemical fingerprint of cacao from this specific
10

cessing stages, on the biochemistry of cacao beans. In the present area of Colombia, as validated by the adjusted PLS-DA model.

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Cacao denomination of origin www.soci.org

1.00
Proportion of ancestry

Genetic groups
Amelonado
0.75
Criollo
Purus
0.50 Iquitos
Nacional
0.25 Parinari
Guiana
0.00 Nanay
Bajo_Calima_1

Cisneros_1

Sabaleta_ML1

Sabaleta_P1

Amel_1

Criollo_1

GU-1

IMC-1

LCTEEN_1

NA_1

Nacional_1

PA_1

Purus_1

SCA_1
Sabaleta_M
Bajo_Calima_2

Cisneros_2

Sabaleta_P2

Amel_2

Criollo_2

GU-2

IMC-2

LCTEEN_2

NA_2

Nacional_2

PA_2

Purus_2

SCA_2
Bajo_Calima_4

Cisneros_4

Sabaleta_ML2

Amel_4

Nacional_4

PA_4
Cisneros_6

Sabaleta_ML4

Sabaleta_P4

Criollo_4

GU-4

IMC-4

LCTEEN_4

NA_4

Purus_4

SCA_4
Bajo_Calima_3

Bajo_Calima_6

Cisneros_3

Amel_3

Nacional_3

PA_3

Purus_3

SCA_3
Sabaleta_ML3

Sabaleta_P3

Criollo_3

GU-3

IMC-3

LCTEEN_3

NA_3
Bajo_Calima_5

Cisneros_5

Amel_5

Criollo_5

GU-5

IMC-5

LCTEEN_5

NA_5

Nacional_5

PA_5

Purus_5

SCA_5
Sabaleta_Pr
Contamana
Curaray

Buenaventura's materials

Figure 7. PCA of the population structure of the Theobrom cacao materials using single nucleotide polymorphisms (SNPs). Colors in each bar correspond
to the probability of belonging to a reference genetic group. The pure clusters on the right side correspond to the reference samples.

Figure 8. (A) PCA score plot of the metabolomic fingerprints of the fer-
mented and dried cacao samples from each zone; (B) Scree plot of the var-
iance percentage explained by each component. The first two PCs
explained 45.4% of the total variance.

The model demonstrated high predictive power, with a cross-

validation using the leave one out algorithm (LOOCV) yielding a
predicted residual sum of squares (Q2) of 0.884. Moreover, the
individual component R2 indicated robust model fitting, explain-
ing 0.962 of the variance.
These chemical fingerprints consist of low molecular weight
metabolites known as chemical flavor precursors, which play a Figure 9. Dynamics of temperature (red line) and pH (blue line) during
crucial role in the non-enzymatic browning reactions during sub- the cacao fermentation process in the three zones: (A) zone 1, (B) zone
sequent stages, such as roasting (see Supporting information, 2 and (C) zone 3.
Fig. S2). Optimal conditions of temperature, time and water activ-
ity facilitate the interaction of these metabolites in Maillard reac- associated with distinct sensory descriptors of cacao, including
fine, bulk or undesirable flavors. Additionally, non-volatile
11

tions, leading to the generation of volatile aroma compounds

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www.soci.org CE González-Orozco et al.

(A) (B) (C)

Figure 10. Bacterial succession during cacao bean fermentation. The relative composition of the bacterial communities throughout the cacao fermen-
tation process is shown for (A) zone 1, (B) zone 2 and (C) zone.

Acidity
Global quality 7 Vegetal
6 Sensory Bajo
Cisneros Sabaletas
5 attribute calima
Spices Grilled
4 Fine flavor
3 Fresh Fruits ab ab c
Florality 2
Animal
Nuts a a a
1 Floral a a a
0 Spices a a a
Nuttiness
Global quality ab ab c
Bitterness
Basic flavor
Sweet a a a
Astringency Sour a a a
Fruitiness
Bitter a a a
Cocoa taste Cocoa aroma
Astringent a b c
Sweetness Cocoa taste a a a
Bajo calima Sabaletas Cisneros

Basic flavors atributes Fine flavors atributes

Figure 11. Sensory attributes of chocolates produced with cacao beans from zone 1 (Cisneros), zone 2 (Bajo Calima) and zone 3 (Sabaletas). Different
letters indicate significant differences (ANOVA, Tukey's test, P < 0.050).

compounds such as phenolic compounds and methylxanthines
(theobromine, caffeine and theophylline) contribute to defining
the flavor profile and overall quality of cacao.
Fermentation variables play a crucial role in the cacao seed's
mass and heat transfer processes, which are closely tied to the
biochemical changes occurring within the seed. These changes
12
that contribute to the formation of flavor precursor metabolites.
To monitor these processes, important physicochemical variables
such as internal cacao mass temperature and internal pH of the
cacao beans were measured throughout the fermentation pro-
cess (Fig. 9).
Overall, an increase in temperature was observed in all regions,

involve a combination of enzymatic and non-enzymatic reactions albeit with slight variations in the trend. Zone 1 exhibited a

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Cacao denomination of origin
significant upward trend starting at 24 h, whereas, in zones 2 and
3, the temperature increase began after 48 and 72 h, respectively.
Beyond the 72-h mark, a consistent progressive temperature rise
was observed in all regions, eventually reaching approximately
45°C. This temperature increase is attributed to the exothermic
reactions caused by acetic acid bacteria thriving in the aerobic
conditions generated by the aeration of the fermentation mass.
A concurrent sharp decrease in pH was observed throughout the
fermentation process across all zones. The pH levels ranged from
6.5 to 4.5. These pH conditions in all regions facilitate the optimal
functioning of enzymatic processes and biochemical changes
within the cacao seeds. Enzymes such as proteases, invertases
and polyphenol oxidases effectively act on proteins, carbohydrates
and phenolic compounds at the ideal temperature and pH, leading
to the generation of lower molecular weight compounds known as
flavor precursor metabolites in cacao. These metabolites include
free amino acids, peptides and reducing sugars, all of which con-
tribute to the development of unique flavors in cacao.
Microorganisms
Bacterial diversity
Cacao bean fermentation is a vital process that plays a key role in
the development of chocolate (or cocoa) flavor by generating
essential chemical precursors. During fermentation, both bacteria
and fungi undergo physical and biochemical transformations of
the bean structures. This transformation follows a predictable
microbial succession, involving yeast and various functional
groups of bacteria. Specifically, the bacterial succession starts with
organisms related to the Enterobacteriaceae (ENT group), which
then transition to lactic acid bacteria (LAB group) and finally to
acetic acid bacteria (AAB group).54 The dynamics of microbial
communities involved in cacao bean fermentation can now be
assessed using high-throughput sequencing of phylogenetic
markers, a method known as metataxonomy. This approach over-
comes the limitations of traditional cultivation-based methods
and provides a more detailed understanding of the microbial
composition involved in the fermentation process.55
The fermentation processes in the three study zones were
examined using 16S rRNA metataxonomy (Fig. 10). Although all
three microbial groups were observed in all fermentations, there
were differences in the fine-level composition, such as genus
and species, as well as relative abundance, between the fermenta-
tions. For example, Tatumella was present during the initial fer-
mentation steps in zones 1 and 3 but not in zone 2, whereas
Pantoea was only found in zone 3. Another unexpected difference
was the presence of Pseudomonas in zone 1, a bacterial taxo-
nomic group that is not typically found in fermentations. Perhaps
the most notable difference between the fermentations was the
atypical microbial succession observed in the fermentation from
zone 1, where it transitioned directly from ENT to AAB, bypassing
the LAB group. This faster transition to AAB might be responsible
for the rapid increase in fermentation temperature and drop in pH
observed at 48 h (Fig. 10) because the production of acetic acid by
AAB is an exergonic reaction. This accelerated transition may
impact the transformation of the pulp and beans during fermen-
tation and could explain the differences in the metabolomics pro-
file observed between zone 1 and the other two locations.
Although recent studies have not found a clear relationship
between the absence of lactic acid bacteria (LAB) in cacao fermen-
tation and distinct characteristics in the organoleptic profile of
chocolate, it is still possible that LAB play a role in the fine transfor-
mation of pulp and contribute to the development of sweet and
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distinct cacao notes. LAB has been shown to transform citric acid
during the initial stages of fermentation, preventing its transfer
into the beans, and also reduces fructose to mannitol, which
may diffuse into the beans.56-57 Recent evidence has demon-
strated the accumulation of mannitol during the fermentation
process. These transformations can lead to additional processes
that contribute to the cacao flavor, beyond those caused by acetic
acid alone.58
Sensory profile of chocolate and special quality
Chocolates made from the cacao beans of the three sampling
zones exhibited distinct sensory attributes, including fresh fruits,
floral notes, spices and nuts (Fig. 11). These unique attributes clas-
sify the cacao from this region as a fine flavor product, according
to the International Cocoa Organization.59 Producers who are able
to obtain higher quality cacao beans have greater opportunities
to access different market segments and achieve better profitabil-
ity in their sales.39,54
Although there were differences in climate and genetic compo-
sition of the plantations, as well as variations within each fermen-
tation batch per zone, we did not find significant differences in
cacao bean quality among the zones. Some minor variations
in the intensity of certain attributes, such as fresh fruits, overall
quality and astringency, were observed (Fig. 11); however, there
were no distinct patterns in the types of attributes that formed
the sensory profiles of each region.
Thus, there is no direct correlation between climate and the
cacao varieties cultivated in these regions that determine the pro-
duction of fine flavor cacao beans. Buenaventura demonstrates a
combination of natural characteristics that contribute to the pro-
duction of high-quality cacao products, with a unique and spe-
cialty quality associated with fruity-aroma chocolates.
CONCLUSIONS
Colombia boasts a rich diversity of cultivated cacao throughout
the country, which is preserved in ex situ collections such as the
national germplasm collection conserved at AGROSAVIA research
stations.31 This genetic reservoir plays a crucial role in the devel-
opment of high-quality cacao in the country but, to enhance this
diversity, methodologies for DOs need to be implemented.
In the present study, we propose an evidence-based framework
for supporting establishing DOs of cacao. Our analysis indicates spe-
cific GIs for each of the three sampling zones, with Buenaventura
identified as a region with natural characteristics to produce fine fla-
vour cocoa products. Each zone exhibited a high differentiation and
diversity of cacao cultivars. Buenaventura has the potential to be des-
ignated as a future DO for cacao from the Pacific region of Colombia,
characterized by its unique fruity-aroma chocolates. However, each
study zone exhibited specific metabolomic characteristics influ-
enced by the unique climatic conditions of each zone. Additionally,
distinct pest species and genetic footprints were identified across
the zones. Overall, this proposed framework highlights the potential
for supporting GIs and DOs in cacao. The integration of scientific
expertise and community collaboration was vital in achieving a com-
prehensive understanding of the cacao quality and characteristics
specific to Buenaventura.
AUTHOR CONTRIBUTIONS
CEG-O and SE conceived the idea. CEG-O, SE, MP, RY, CR, MS, MZ,
13
AC-Q, MS and JR collected, cleaned and analyzed the data. CEG-O
wileyonlinelibrary.com/jsfa

www.soci.org
and SE led the writing and revision of the manuscript. All authors
contributed critically to the drafts and approved the final version
11
of the manuscript submitted for publication.
12
ACKNOWLEDGMENTS
We thank the Corporación Colombiana de Investigación Agrope-
cuaria (AGROSAVIA) for providing funding. We gratefully acknowl-
13
edge the generous assistance provided by cacao farmers
associations of Buenaventura: Asociación de Productores Campe-
sinos de Cisneros, Asociación de Agricultores y Cacaoteros de Bajo
Calima, and Asociación de Productores y Transformadores de
14
Zacarías-Sabaletas. We also thank the University of the Andes,
especially Professor Bart Van Hoof, Maite Rosales, Juanita Duque
and Jesús Riascos, as well as the researchers from AGROSAVIA,
Darwin Martínez, Eliseo Polanco and Rafael Novoa. Bancoldex 15
and the Buenaventura Chamber of Commerce are acknowledged
for contributing resources for the development of this research 16
project.
CONFLICTS OF INTEREST 17
The authors declare that they have no conflicts of interest.
18
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from
the corresponding author upon reasonable request. 19
20
SUPPORTING INFORMATION
Supporting information may be found in the online version of this
21
article.
22
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