foods

Article
Development and Characterization of a Pilot-Scale
Model Cocoa Fermentation System Suitable for
Studying the Impact of Fermentation on Putative
Bioactive Compounds and Bioactivity of Cocoa
Kathryn C. Racine 1 , Andrew H. Lee 1 , Brian D. Wiersema 1 , Haibo Huang 1 ,
Joshua D. Lambert 2 , Amanda C. Stewart 1, * and Andrew P. Neilson 1, *,†
1 Department of Food Science and Technology, Virginia Polytechnic Institute and State University, Blacksburg,
VA 24060, USA; racinekc@vt.edu (K.C.R.); andhlee@vt.edu (A.H.L.); wiersema@vt.edu (B.D.W.);
huang151@vt.edu (H.H.)
2 Department of Food Science, Pennsylvania State University, University Park, PA 16801, USA;
jdl134@psu.edu
* Correspondence: amanda.stewart@vt.edu (A.C.S.); aneilso@ncsu.edu (A.P.N.); Tel.: +1-540-231-0868 (A.C.S.);
+1-705-250-5495 (A.P.N.)
† Current affiliation: Plants for Human Health Institute, Department of Food, Bioprocessing and Nutrition
Science, North Carolina Research Campus, 600 Laureate Way, Kannapolis, NC 28081, USA.




Received: 26 February 2019; Accepted: 15 March 2019; Published: 19 March 2019


Abstract: Cocoa is a concentrated source of dietary flavanols—putative bioactive compounds

associated with health benefits. It is known that fermentation and roasting reduce levels of native
flavonoids in cocoa, and it is generally thought that this loss translates to reduced bioactivity.
However, the mechanisms of these losses are poorly understood, and little data exist to support this
paradigm that flavonoid loss results in reduced health benefits. To further facilitate large-scale studies
of the impact of fermentation on cocoa flavanols, a controlled laboratory fermentation model system
was increased in scale to a large (pilot) scale system. Raw cocoa beans (15 kg) were fermented in 16 L
of a simulated pulp media in duplicate for 168 h. The temperature of the fermentation was increased
from 25–55 ◦ C at a rate of 5 ◦ C/24 h. As expected, total polyphenols and flavanol levels decreased as
fermentation progressed (a loss of 18.3% total polyphenols and 14.4% loss of total flavanols during
fermentation) but some increases were observed in the final timepoints (120–168 h). Fermentation
substrates, metabolites and putative cocoa bioactive compounds were monitored and found to follow
typical trends for on-farm cocoa heap fermentations. For example, sucrose levels in pulp declined
from >40 mg/mL to undetectable at 96 h. This model system provides a controlled environment
for further investigation into the potential for optimizing fermentation parameters to enhance the
flavanol composition and the potential health benefits of the resultant cocoa beans.

Keywords: Theobroma cacao; simulated pulp media; polyphenols; fermentation; procyanidin; catechin

1. Introduction
Recently, cocoa (Theobroma cacao) and its putative bioactive compounds (particularly flavonoids)
have been associated with various health benefits, including positive effects on cardiovascular, metabolic
and endocrine diseases [1]. There is interest among health researchers, scientists, cocoa suppliers
and manufacturers alike in tailoring the processing of cocoa to produce products with maximum
health benefits. Three main groups of flavonoids exist within cocoas beans: proanthocyanidins
(oligomeric and polymeric flavanols) constitute approximately 58% of the total phenolic content,

Foods 2019, 8, 102; doi:10.3390/foods8030102 www.mdpi.com/journal/foods

Foods 2019, 8, 102 2 of 20

followed by catechins (monomeric flavanols, ~37%) and anthocyanins (~4%) [2]. It is known that
oxidation, condensation and other reactions that take place during cocoa fermentation and roasting
reduce levels of native flavonoids, warranting investigation into how these reactions ultimately impact
cocoa’s health benefits [3–8]. The widely-accepted assumption is that preservation of native flavonoids
is critical for retaining bioactivity [9]. However, reactions (oxidation, epimerization, condensation,
etc.) during processing may generate compounds with novel activities, potentially preserving or even
enhancing health benefits [2,10–13] despite flavonoid loss. Recent findings by Ryan et al. [11] contradict
the widely-accepted assumption that loss of native cocoa flavonoids corresponds with reduced activity
in some cases. In their study, lower concentrations of flavonoids and total polyphenols in fermented
cocoa products were not found to be associated with reduced bioactivity in in vitro digestive enzyme
inhibition assays. These findings indicate the potential for optimization of processing factors such as
fermentation and roasting to maximize the health benefits of cocoa.
Commercial cocoa fermentation is conducted on or in close proximity to the farm of origin in large
heaps on the ground or in wooden boxes covered with banana leaves. Tremendous variability exists
among on-farm heap fermentations, as differences in environmental microbiota, climate, substrate
and fermentation methods all play key roles in microbial ecology and activity. These conditions are
poorly documented and beans with differing fermentation histories are commingled into large batches,
making post hoc evaluation of the impact of fermentation on bioactivity essentially impossible [8,12,14].
Furthermore, sourcing fermented beans for scientific research is difficult, and complex supply chains
have left researchers with uncertainty on cultivar, processing parameters and heap consistency.
Sourcing intact cocoa pods is also logistically challenging. With growing interest in the potential for
optimization of processing to influence composition and subsequent bioactivity of cocoa, a controlled
model pilot-scale fermentation system is needed. Employing dried, unfermented beans and simulated
pulp media eliminates the needs for fresh unopened pods, a major limitation for cocoa fermentation
research in regions distant from cultivation. Such a system would provide the ability to control all
aspects of fermentation using the exact same starting beans, eliminating confounding variables when
sourcing is not under full control of the investigators.
Some preliminary work has been done to explore controlling cocoa fermentation for research
purposes (both for characterizing fermentation processes as well as modifying outcomes). Several
laboratory bench-scale model fermentations have been conducted in a variety of vessels, including
plastic [4,15] and stainless-steel [16], to examine the microbial influence and overall impact of starter
cultures on cocoa fermentation. These model fermentations generally use pulp and beans from
freshly harvested pods—which is not practical for frequent large-scale use in cocoa fermentation
research conducted in non-tropical regions—and use inoculated and ambient pulp mediums [6,17–21].
Our group recently developed a large laboratory bench-scale fermentation using simulated pulp media
and dried unfermented cocoa beans as starting material [22]. To our knowledge, ours was the first
model of this scale. Along with other laboratory bench-scale model fermentation systems, this model
can be used to evaluate the impact of controlled fermentation on putative bioactive compounds in cocoa.
Fermentation can cause anywhere from 0–70% loss of total polyphenols, and following fermentation
the beans are dried and roasted, causing an additional 15–40% loss [2,3,23–31]. Nonetheless, recent
findings have indicated that lower concentrations of specific cocoa flavonoids and total polyphenols in
a given product are not always associated with decreased bioactivity [11].
Additional studies employing a combination of analytical, in vitro and in vivo approaches are
needed to advance the understanding of how specific cocoa flavonoid losses during cocoa processing
affect the bioactivities of cocoa. Studies are then needed to optimize processing, including fermentation,
to maximize the desirable health benefits of cocoa. However, to employ techniques that go beyond
analytical characterization of the fermented product, such as in vivo bioactivity studies, larger amounts
of experimentally fermented cocoa would be required than can be reasonably produced using existing
laboratory bench-scale model systems (pilot scale: tens of kgs of fermented beans or more, instead of
bench scale: hundreds of grams). Hence, our objective was to develop and characterize a pilot-scale
Foods 2019, 8, 102 3 of 20

model cocoa fermentation system suitable for studying the impact of fermentation on putative bioactive
compounds. Dried, unfermented beans and a simulated pulp media and ambient microorganisms
were used due to the limitations associated with sourcing and transporting fresh whole cocoa pods.
Specifically, our hypothesis was that broad microbial and chemical changes similar to those generally
observed in heap fermentations could be replicated in non-tropical regions by using a pilot-scale
model system designed to simulate the conditions occurring in the middle of a well-turned cocoa heap,
and that this system could be used to generate sufficient amounts of fermented cocoa beans for further
research on the effect processing has on the potential health benefits of cocoa.

2. Materials and Methods

2.1. Chemicals and Standards

Citric acid, yeast extract, malt extract, calcium lactate pentahydride, tween 80, sodium hydroxide,
magnesium sulfate heptahydride, manganese sulfate monohydride, sucrose, glucose, fructose,
peptone, calcium carbonate and agar were obtained from Thermo Fisher Scientific (Waltham,
MA, USA). Cycloheximide was obtained from MP Biomedicals, LLC (Solon, OH, USA) and
oxytetracycline dihydrate was obtained from Acros Organics (Springfield Township, NJ, USA). Lactic
acid, Folin–Ciocalteu reagent, 4-dimethylaminocinnamaldehyde (DMAC), (±)-catechin, (–)-epicatechin
and procyanidin B2 (PCB2) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Procyanidin C1
(PCC1) and cinnamtannin A2 (CinA2) were obtained from Planta Analytica (New Milford, CT, USA).
Solvents were ACS grade or higher.

2.2. Pilot-Scale Fermentation Model

Raw unfermented Criollo cocoa beans (18 kg) (Natural Zing LLC, Mount Airy, MD, USA), sourced
from Ecuador, were rehydrated in 66.4 × 44.3 × 34.3 cm plastic fermentation boxes (Polypropylene,
Sterilite, Townsend, MA, USA) by submersion in approximately 20 L of distilled, deionized (DI) water
for 24 h. The final moisture content of the beans after rehydration was 39.7% (IR-120 Moisture Analyzer,
Denver Instrument, Bohemia, NY, USA), close to the lower end of the typical moisture range (40–60%)
for fresh beans [32,33]. Rehydrated beans were then drained and 15 kg were mixed with 16 L of
simulated pulp media, prepared as described by Lee et al. [22] with minor modifications. Simulated
pulp media were obtained by mixing 3 separate solutions (solutions A, B and C). Solution A consisted
of citric acid (10 g/L), yeast extract (5 g/L), peptone (5 g/L), calcium lactate pentahydride (1 g/L)
and tween 80 (1 mL/L); the pH was adjusted to 3.6 using 1 N NaOH and then volume corrected to a
total of 9.6 L before autoclaving (121 ◦ C, 15 min) to ensure sterility of the medium. Solution B was a
4.8-L sugar solution, with 1.6 L each of sucrose (83.3 g/L), glucose (133.3 g/L) and fructose (150 g/L).
Each sugar solution was autoclaved and cooled prior to fermentation. Solution C was prepared the
day of fermentation and consisted of magnesium sulfate heptahydride (1 g/L) and manganese sulfate
monohydride (0.4 g/L) for a total of 1.6 L. Boxes were loosely covered with their plastic lids and
placed inside a pre-heated (25 ◦ C) incubator (Forma 29 cu ft Reach-In Incubator, Model No. 3950,
Thermo Fisher Scientific, Waltham, MA, USA). Scale-up from the bench scale was required to produce
sufficient material for experiments evaluating the impact of fermentation on cocoa bioactivity using
in vivo models. Two factors determined the scale of the pilot system: (1) the amount of fermented
product required for animal studies using the fermented material as substrate, and (2) the size of the
largest incubator available in our pilot plant. Taking these two factors into account, the batch size was
increased by over 10-fold from a 1.2-kg batch of rehydrated beans at the bench scale to a 15-kg batch
at the pilot scale. The bench-scale system was under constant agitation in a shaker/incubator, which
was not feasible in this pilot system. To maintain maximum dissolved oxygen (DO) possible in a static
system, fermentation vessel size and shape were selected to maximize the surface area to volume ratio,
and a stirring regime was implemented wherein the contents of the vessel were well-mixed twice daily.
Through preliminary work, this mixing regime, in combination with mixing due to gas evolution
Foods 2019, 8, 102 4 of 20

during fermentation, proved sufficient to maintain a well-mixed condition in the fermentation vessel
and to support the microbial succession required for fermentation.
The fermentation was performed in duplicate, simultaneously under identical conditions
(2 replicate boxes each employing the conditions described above: 15 kg rehydrated beans and
16 L simulated pulp per box) using ambient microorganisms (i.e., no inoculation) and took place
over a period of 168 h (representing the upper end of the spectrum of reported heap fermentation
times that occur on-farm). In the future, fermentation time can be varied to obtain different extents of
fermentation. The incubator set point was raised 5 ◦ C per 24 h to a final temperature of 55 ◦ C in order to
mimic temperature progressions seen in heap fermentation [3,8,34,35]. Beans were manually agitated
for 3 min every 12 h to ensure that the simulated pulp media were aerated [14,34–36]. The agitation
step was critical to ensure the expected succession of the microbial communities due to our model’s
inability to introduce oxygen by draining pulp away, as typically happens during heap fermentations
when the pulp is liquefied and the heap is manually turned. Pulp and bean samples were collected
every 24 h throughout the fermentation. Pulp dissolved oxygen (DO) and pH values were monitored
using benchtop meters (Orion DO Probe 083005MD; Orion Versa Star Pro pH meter; Thermo). After
168 h of fermentation, the beans were drained to remove the remaining pulp media, rinsed with water
and spread evenly onto baking sheets. Beans were oven dried (Rational, Landsberg am Lech, Germany;
Blodgett, Burlington, VT, USA) at 45–65 ◦ C until the moisture content fell below 8%. These conditions
mimicked typical commercial drying protocols [3,34,36,37]. After drying, beans were thoroughly
mixed together and stored at 4 ◦ C.

2.3. Microbial Enumeration

Enumeration methods and selective media were based on the protocols of Nielsen et al. [34]
and Ho et al. [38] with minor modifications. Collected pulp media (5 mL) from each time point was
diluted with 45 mL sterile 0.1% peptone water, and 1 mL of the resulting mixture was then diluted
10-fold. Aliquots (0.1 mL) were spread inoculated to nutrient agar appropriate for the growth of yeast,
lactic acid bacteria (LAB) and acetic acid bacteria (AAB). Yeast cultures were spread on YM media
(3 g/L yeast extract, 3 g/L malt extract, 3 g/L peptone, 10 g/L glucose, 20 g/L agar) with 100 mg/L
oxytetracycline and incubated at 37 ◦ C. LAB were cultured anaerobically by a BD GasPAK EZ gas
generating system (Franklin Lakes, NJ, USA) on de Man–Rogosa–Sharpe (MRS) agar (Sigma) with
400 mg/L cycloheximide at 37 ◦ C, and AAB were cultured on GYC media (50 g/L glucose, 10 g/L
yeast extract, 30 g/L calcium carbonate, 20 g/L agar, pH = 5.6) with 400 mg/L cycloheximide by
incubating at 25 ◦ C. Bacterial enumeration was performed in duplicate on analytical replicates for each
fermentation box and time-point. Colonies were counted and presented as log colony-forming units
(CFU)/mL.

2.4. Cut Test

The cut test is the standard assessment of post-fermentation bean quality and suitability to move
forward in processing [3,39]. A reduced sample-size version of the cut test was performed as follows:
6 beans from each 24-h sampling were cut through lengthwise and each half examined for color and
quality defects. Although this test is not very applicable to low-anthocyanin beans like Criollo, a purple
interior is indicative that the fermentation ended prematurely, while a brown interior is indicative of a
successful fermentation [3,40].

2.5. Fermentation Index

Fermentation index (FI) monitors the color change within the bean cotyledon during fermentation.
This color change is due to the decreasing anthocyanin content as beans progress through fermentation.
FI was measured based on the method of Romero-Cortes et al. [41] with minor modifications. Five
to seven randomly selected cocoa beans from each time point were frozen with liquid nitrogen and
ground to a fine powder. A 50-mg sample of the resulting powder was weighed and mixed with 5 mL
Foods 2019, 8, 102 5 of 20

MeOH:HCl (97:3 v/v). Samples were extracted at 4 ◦ C for 16–18 h on a rotating shaker, centrifuged
for 5 min at 3500× g, and the supernatant was collected. Absorbance was measured using a BioTek
Synergy 2 plate reader (BioTek, Winooski, VT, USA) on a 96-well plate (Corning Inc., Corning, NY, USA)
at 460 and 530 nm. These wavelengths were chosen to express structural properties and distributions
through fermentation, as 530 nm is a general λmax for anthocyanin spectra and 460 nm reflects the
glycoside distribution [42]. FI was determined based on the ratio of the absorbance at 460 nm compared
to that of 520 nm.

2.6. Bean pH
Approximately 5–7 cocoa beans collected at each time point were frozen with liquid nitrogen,
the shells were removed and the nibs were ground. Ground nibs (5 g) were collected and mixed with
100 mL hot water (90 ◦ C) and stirred for 30 s. The cocoa water solution (25 mL) was then filtered
through Whatman #4 filter paper and collected for pH analysis. It is important to note that this
procedure was not for quantifying the actual pH of the cocoa bean itself, but rather to measure the
acidity derived when bean acids are diluted into water; it is useful for comparison between the pH of
solutions produced by beans at different time points.

2.7. High-Performance Liquid Chromatography (HPLC) Analysis of Fermentation Metabolites

2.7.1. Pulp Media Sample Preparation

Pulp media samples were diluted 10-fold with distilled water, vortexed and centrifuged for 5 min
at 5000× g. Next, 1 mL of supernatant was removed and filtered through a 0.45-µM polyvinylidene
difluoride (PVDF) membrane filter (Thermo Fisher Scientific, Waltham, MA, USA) into vials.

2.7.2. Bean Sample Preparation

Approximately 5–7 cocoa beans were peeled so that the nibs were exposed. Next, 5 g of nib were
weighed and added to distilled water at a 10× dilution. The bean water solution was homogenized at
high speed for 2 min in a blender (Waring Products, Calhoun, GA, USA). The blended mixture was
then centrifuged for 5 min at 5000× g and 1 mL supernatant was removed and filtered into vials.

2.7.3. Analysis
Bean and pulp samples were analyzed by HPLC on an Agilent HPLC 1260 Infinity Series (Agilent
Technologies, Santa Clara, CA, USA) using an Aminex HPX-87H column (300 × 7.8 mm, 50 ◦ C)
(Bio-Rad Laboratories, Hercules, CA, USA) and a refractive index (RI) detector (35 ◦ C). A 0.005 M
H2 SO4 isocratic mobile phase at a flow rate of 0.6 mL/min was used for analyte separation. The sample
injection volume was 5 µL. Triplicate analytical replicates were prepared and analyzed from each
fermentation time point. A standard curve was prepared with a range from 0.5 to 5.0 g/L. Sugars
(sucrose, glucose, fructose), ethanol, glycerol and organic acids (acetic acid, lactic acid, succinic acid,
citric acid) were quantified.

2.8. Polyphenol Extraction and Quantification

Whole cocoa beans (40 g) were frozen with liquid nitrogen and ground into a fine powder. To defat,
the powder was mixed with 150 mL hexane and sonicated for 10 min at 22 ◦ C. The mixture was then
centrifuged for 5 min at 5000× g, the supernatant was discarded and then the process was repeated.
Once defatted, the powder was allowed to dry at room temperature. Once dry, the powder was
mixed with 150 mL extraction solution (70:28:2 acetone, water, acetic acid v/v/v), sonicated for 10 min
at 22 ◦ C and centrifuged for 5 min at 5000× g. The supernatant was collected, and this procedure
was repeated three times for a total volume of 450 mL. All collected supernatant was pooled and
placed under vacuum on a rotary evaporator at 40 ◦ C until all acetone had evaporated. The resulting
extract was freeze dried for 72 h and the yield was calculated. Total phenolic content (all polyphenols,
Foods 2019, 8, 102 6 of 20

including flavanols as well as other flavonoids and non-flavonoid phenolics) of both the nib and
shell was determined by the Folin–Ciocalteu colorimetric assay and total flavanols (only catechins
and proanthocyanidins) measured by the 4-dimethylaminocinnamaldehyde (DMAC) colorimetric
assay, as previously described by Dorenkott et al. [10]. These values were expressed in mg gallic-acid
equivalents (mg GAE)/g bean and mg PCB2/g bean, respectively.

2.9. Individual Polyphenol Analysis by Reversed Phase UPLC-MS

Monomeric catechins and low molecular weight procyanidins were measured by UPLC-MS
on an Acquity H-Class UPLC-QDa Mass Detector (Waters Corporation, Milford, MA, USA). Cocoa
extract (CE) was diluted with 0.1% formic acid (v/v) in water and 0.1% formic acid in acetonitrile
(95:5 v/v), to a final concentration of 0.1 mg/mL, filtered (13 mm diameter syringe filters, 0.22 µm nylon
membrane with propylene housing, Microsolv, Leland, NC, USA) into vials, and held at 10 ◦ C. Samples
were analyzed on an Acquity HSS T3 column (2.1 × 100 mm, 1.8 µm particle size) in combination
with an Acquity HSS T3 VanGuard pre-column (2.1 × 5 mm column, 1.7 µm particle size) at 43 ◦ C.
Binary gradient elution was performed using 0.1% formic acid (v/v) in water (Phase A) and 0.1%
formic acid (v/v) in acetonitrile (Phase B). The solvent flow rate was 0.6 mL/min and the linear
gradient elution was as follows: 95% A (0–0.5 min), 65% A (6.5 min), 20% A (7.5–8.6 min) and 95%
A (8.7–10.5 min). Samples were held at 4 ◦ C and the injection volume was 10 µL. (–)-electrospray
ionization (ESI) together with mass spectrometry (MS) was used to analyze the UPLC eluent. The
ionization settings were as follows: (–) mode, 0.8 kV capillary voltage, 15 V cone voltage and 600 ◦ C
probe temperature. Triplicate analytical replicates were prepared and analyzed from each fermentation
time point. Authentic standards of (±)-catechin, (–)-epicatechin, PCB2, PCC1 and CinA2 were utilized,
and settings for selected ion response (SIR) monitoring of each compound are listed in Table 1. Data
were collected using Empower 3 software (Milford, MA, USA).

Table 1. MS settings for individual polyphenol analysis by reverse-phase (RP)-UPLC-MS.

a b
Compound tR [M–H]–
(min) (m/z)
(±)-catechin 2.946 288.95
(–)-epicatechin 3.625 289.01
PCB2 3.366 576.84
PCC1 3.904 864.85
CinA2 4.063 1153.19
a Retention time b QDA detector uses singly charged parent ions for selected ion response (SIR) monitoring.

2.10. HILIC UPLC-MS/MS

Monomeric flavanols and procyanidins were analyzed by hydrophilic interaction liquid
chromatography (HILIC) UPLC-MS/MS as previously described [43]. A Waters Acquity H-class
UPLC equipped with an Acquity Torus DIOL column (2.1 × 100 mm, 1.7 µL, 45 ◦ C) and Torus DIOL
VanGuard Pre-column (2.1 × 5 mm, 1.7 µL) were used for analysis. Gradient elution was performed
with 2% acetic acid in acetonitrile (phase A) and 3% water and 2% acetic acid in methanol (phase B).
Solvent flow rate was 0.8 mL/min and elution was carried out as followed: 100% A (0 min), 55% A
(5.7 min), 5% A (6.0 min) and 100% A (6.7–9.0 min). The UPLC eluent was analyzed by (−)- mode
ESI coupled to tandem mass spectrometry (MS/MS) on a Waters Acquity triple quadrupole (TQD).
Aqueous ammonium formate (0.04 M, 5 µL/min) was added to the eluent flow stream post-column
to enhance ionization. Ionization settings were as follows: (−) mode, capillary and cone voltages:
−4.5 kV and 60.0 V, extractor voltage: 1.0 V, source and desolvation temperatures: 150 ◦ C and 500 ◦ C.
N2 was used for the cone and desolvation gas at 50 and 1000 L/h, respectively. For MS/MS, Ar was
the collision gas at 0.1 mL/min. Parent and signature daughter ions were subjected to multi-reaction
Foods 2019, 8, 102 7 of 20

monitoring (MRM) with a mass span of 0.2 Da and 1.0 sec of inter-channel delays and inter-scan times.
Calibration curves for standards DP 1-9 (Planta Analytica, New Milford, CT, USA) were prepared and
analyzed with dilutions ranging from 6.93 × 10−7 – 0.091 mg/mL. MRM settings for each compound
are listed in Table 2. MassLynx software (version 4.1, Waters) was used to acquire data.

Table 2. Tandem MS/MS settings for multi-reaction monitoring (MRM) detection of monomeric-
decameric flavanols.

a
tR MW [M–H]– b Daughter Ion
Compound
(min) (g mol−1 ) (m/z) (m/z)
Monomer 0.61 290.27 289.03 245.06
Epigallocatechin 0.74 458.37 305.04 124.98
Dimer 2.03 578.52 577.14 425.10
Trimer 3.05 866.77 865.22 287.07
Tetramer 3.73 1155.02 576.40 125.02
Pentamer 4.26 1443.28 720.41 125.02
Hexamer 4.66 1731.53 864.52 125.02
Heptamer 5.00 2017.81 1008.40 125.17
Octamer 5.28 2308.03 1152.58 125.17
Nonamer 5.53 2596.54 864.12 125.17
Decamer 5.75 2884.54 960.18 125.17
a Retention time. b All MRMs used singly charged parent ions except for pentamer, hexamer, heptamer and octamer,

which are double-charged ([M–2H]2− ), and nonamer and decamer, which are triple-charged ([M–3H]3− ).

2.11. Data Analysis and Statistics

The fermentation was performed in duplicate, simultaneously under identical conditions. Analyses
were performed on samples from each replicate; analytical replicates were averaged together to create
a composite value for each fermentation replicate. Data from distinct time points were analyzed by
one-way ANOVA to determine overall significance. If significant differences were detected, Tukey’s
HSD post-hoc test was then used to compare all time point means. Significance was defined as p < 0.05.
Analyses were performed using GraphPad Prism 7.03 (GraphPad, La Jolla, CA, USA).

3. Results

3.1. pH, DO, FI, Cut Test

As shown in Figure 1A, the mean pH of the simulated pulp media started at approximately 3.6,
increased to a maximum value at 72 h and slightly declined to a final mean of 4.6. The solution made
from the nib had a mean pH of 5.4, and the pH declined throughout fermentation to a final mean of
4.6. Initial DO levels were high due to the fresh mixing of simulated pulp and beans but decreased
rapidly over the first 24 h followed by consistent levels for the remainder of the fermentation. It has
been determined that cocoa mass is adequately fermented when FI measurements are ≥1 [41] and,
as shown in Figure 1B, FI values began at 0.872 ± 0.065 and values ≥1 were initially achieved between
48–72 h. Figure 1C shows the cut test of beans selected at each time point. There were a variety of
colors over the span of the fermentation, from light/dark brown to purple.
Foods 2019, 8, 102 8 of 20
Foods 2019, 8, x FOR PEER REVIEW 8 of 20

A
C

Figure
Figure 1. 1. (A)
(A) pH
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measurements for for both
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simulated pulp
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points. Dissolved Dissolved
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oxygen (DO) measurements within the simulated pulp media are expressed in
measurements within the simulated pulp media are expressed in mg/L. (B) Fermentation index (FI) mg/L. (B) Fermentation
index (FI) expressed
expressed as a ratio asof aabsorbance
ratio of absorbance
at 460 and at 460
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replicates. Significance
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3.2. Microbial
3.2. Microbial Enumeration
Enumeration and
and Fermentation
Fermentation Products
Products
Microbial population
Microbial population changes
changes are are shown
shown in in Figure
Figure 2.
2. Yeasts
Yeasts proliferated
proliferated early
early (Figure
(Figure 2A)
2A) with
with
a 6-log increase over the first 48 h followed by a decline over the remainder of
a 6-log increase over the first 48 h followed by a decline over the remainder of fermentation, ending fermentation, ending
with no
with no measurable
measurable colonies.
colonies. LAB LAB (Figure
(Figure 2B) 2B) presented
presented aa similar
similar butbut less
less dramatic
dramatic trend
trend with
with
approximately a 7-log increase over the first 72 h, followed by a moderate decline.
approximately a 7-log increase over the first 72 h, followed by a moderate decline. AAB (Figure 2C) AAB (Figure 2C)
levels fluctuated before peaking at 72–96 h and then exhibiting a similar
levels fluctuated before peaking at 72–96 h and then exhibiting a similar decline as LAB. decline as LAB.
Concentrationof
Concentration offermentation
fermentationsubstrates
substratesand andmetabolites
metabolites inin simulated
simulated pulp
pulp media
media areare shown
shown in
in Figure 3. During the first 48–72 h of fermentation, sugar and
Figure 3. During the first 48–72 h of fermentation, sugar and citric acid concentrations dropped citric acid concentrations
dropped significantly
significantly and remainedand remained
close to zero closefortothe
zero for the remainder
remainder of the fermentation.
of the fermentation. Contrarily,Contrarily,
ethanol,
ethanol, glycerol and acetic acid remained relatively constant for the first
glycerol and acetic acid remained relatively constant for the first 24–48 h of fermentation before 24–48 h of fermentation
before demonstrating
demonstrating a dramatic
a dramatic increase
increase in simulated
in simulated pulpmedia
pulp mediaconcentrations.
concentrations. Succinic
Succinic acid
acid
concentrations remained
concentrations remained constant
constant for for the
the first
first 48
48 hh followed
followed byby aa 3-fold
3-fold increase
increase in in simulated
simulated pulp
pulp
media. Lastly, lactic acid was the only metabolite that showed a consistent
media. Lastly, lactic acid was the only metabolite that showed a consistent increase throughout the increase throughout the
entire fermentation, increasing almost 6-fold by the end
entire fermentation, increasing almost 6-fold by the end of the 168 h. of the 168 h.
Foods 2019, 8, 102 9 of 20
Foods 2019, 8, x FOR PEER REVIEW 9 of 20

Figure 2. Enumeration of (A) yeast, (B) lactic acid bacteria (LAB) and (C) acetic acid bacteria (AAB) in
Figure 2. Enumeration of (A) yeast, (B) lactic acid bacteria (LAB) and (C) acetic acid bacteria (AAB) in
simulated pulp media, expressed in log colony-forming units (CFU)/mL. Values are presented as the
simulated pulp media, expressed in log colony-forming units (CFU)/mL. Values are presented as the
mean ± SEM of fermentation replicates. Significance between time points was determined by one-way
mean ± SEM of fermentation replicates. Significance between time points was determined by one-way
ANOVA and Tukey’s HSD post-hoc test (p < 0.05). Time points with different letters are significantly
ANOVA and Tukey’s HSD post-hoc test (p < 0.05). Time points with different letters are significantly
different within values.
different within values.

Concentrations
Concentrations of of fermentation
fermentation substrates
substrates and
and metabolites
metabolites from
from thethe beans
beans are are shown
shown in Figure
in Figure 4.
4. Fructose, glucose and citric acid concentrations rose significantly over the first
Fructose, glucose and citric acid concentrations rose significantly over the first 48 h before peaking48 h before peaking
andthen
and then quickly
quickly decreasing.Succinic,
decreasing. Succinic,lactic
lacticand
andacetic
aceticacid
acidfluctuated
fluctuated before
before reaching
reaching maximum
maximum
concentrations at 120 h, followed by decreasing levels for the final 48 h of fermentation. Sucrose is is
concentrations at 120 h, followed by decreasing levels for the final 48 h of fermentation. Sucrose thethe
only
only compound
compound thatthat showed
showed a quick
a quick and sharp
and sharp decrease
decrease in concentration.
in concentration. After
After a 96-h a 96-hsucrose
decline, decline,
sucrose concentrations ended at undetectable levels. Bean alcohol trends were
concentrations ended at undetectable levels. Bean alcohol trends were like those of the simulated pulp like those of the
simulated pulp media, with ethanol and glycerol remaining constant for 24 h
media, with ethanol and glycerol remaining constant for 24 h before demonstrating a sharp increase.before demonstrating
a sharpconcentrations
Glycerol increase. Glycerol concentrations
fluctuated more than fluctuated more than
those of ethanol, peakingthose of hethanol,
at 96 compared peaking at 96 h
to ethanol’s
compared to ethanol’s
48 h maximum concentration. 48 h maximum concentration.
Foods 2019, 8, 102 10 of 20
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3. (A–I) Concentration
Figure 3. Concentration of fermentation
fermentation substrates and metabolites
metabolites inin simulated
simulated pulp
pulp media
media
across
across 168
168 h.
h. Values
Values are
are presented
presentedasasthe mean±
themean SEM of
± SEM of fermentation
fermentation replicates. Significance between
between
time
time points
pointswaswasdetermined
determined bybyone-way
one-way ANOVA
ANOVA andand
Tukey’s HSDHSD
Tukey’s post-hoc test (ptest
post-hoc < 0.05).
(p < Time
0.05).points
Time
with different letters are significantly different within values.
points with different letters are significantly different within values.

3.3. Total Polyphenol and Flavanol Content

3.5. Total Polyphenol and Flavanol Content
Mean total polyphenol levels were initially 38.0 mg gallic acid equivalents (GAE)/g bean and
Mean total polyphenol levels were initially 38.0 mg gallic acid equivalents (GAE)/g bean and
after 168 h finalized at 31.1 mg GAE/g bean, for a total loss of 18.3%. Yet after 120 h, there was a
after 168 h finalized at 31.1 mg GAE/g bean, for a total loss of 18.3%. Yet after 120 h, there was a more
more significant net polyphenol loss, quantified at 47.1% loss from initial levels. This difference can be
significant net polyphenol loss, quantified at 47.1% loss from initial levels. This difference can be
attributed to the 35.3% apparent polyphenol gain in the final 48 h, 120–168 h (Figure 5A). When looking
attributed to the 35.3% apparent polyphenol gain in the final 48 h, 120–168 h (Figure 5A). When
at total flavanol concentrations in Figure 5B, a similar trend can be seen in the last day of fermentation.
looking at total flavanol concentrations in Figure 5B, a similar trend can be seen in the last day of
A 14.4% loss in flavanol concentration is accounted for when looking at initial and final hours (0 and
fermentation. A 14.4% loss in flavanol concentration is accounted for when looking at initial and final
168 h), whereas a 59.3% loss is seen between 0 and 144 h, with this difference being attributed to the
hours (0 and 168 h), whereas a 59.3% loss is seen between 0 and 144 h, with this difference being
apparent 52.5% gain in total flavanol concentration between the final 24 h (Figure 5B).
attributed to the apparent 52.5% gain in total flavanol concentration between the final 24 h (Figure
5B). Individual Polyphenol Analysis
3.4.
Concentrations
3.6. Individual of (±Analysis
Polyphenol )-catechin, (–)-epicatechin, PCB2, PCC1 and CinA2 are shown in Figure 5C–G.
(–)-Epicatechin levels decreased the most, with 66.2% of initial concentrations lost between 0 h and
Concentrations of (±)-catechin, (–)-epicatechin, PCB2, PCC1 and CinA2 are shown in Figure 5C–
168 h. CinA2 concentrations fell by 61.3%, followed by PCC1 at 51.8%, catechin at 51.3% and PCB2
G. (–)-Epicatechin levels decreased the most, with 66.2% of initial concentrations lost between 0 h and
at 38.0%. Each individual compound mirrored the trend seen in total polyphenol concentrations in
168 h. CinA2 concentrations fell by 61.3%, followed by PCC1 at 51.8%, catechin at 51.3% and PCB2 at
the final 48 h (120–168 h). The most significant loss in all compounds was within the first 48–72 h,
38.0%. Each individual compound mirrored the trend seen in total polyphenol concentrations in the
followed by fluctuating values until the final 48 h (120–168 h), where all compounds then increased.
final 48 h (120–168 h). The most significant loss in all compounds was within the first 48–72 h,
Concentrations of individual procyanidins analyzed by degree of polymerization (DP) from
followed by fluctuating values until the final 48 h (120–168 h), where all compounds then increased.
monomer through decamer mirrored trends seen previously in that significant losses occurred in
Concentrations of individual procyanidins analyzed by degree of polymerization (DP) from
the first 72 h, followed by an increase in concentrations of all compounds from 120–168 h. Dimer
monomer through decamer mirrored trends seen previously in that significant losses occurred in the
first 72 h, followed by an increase in concentrations of all compounds from 120–168 h. Dimer
concentration (Figure 6B) increased over 2-fold from 120–168 h, returning to initial (0 h) concentration
Foods 2019, 8, 102 11 of 20

Foods 2019, 8, x FOR PEER REVIEW 11 of 20

concentration (Figure 6B) increased over 2-fold from 120–168 h, returning to initial (0 h) concentration
levels
levels by
by the
the end
end of
of fermentation.
fermentation. As
As expected,
expected, monomers
monomers (Figure
(Figure 6A)
6A) had
had the
the greatest
greatest loss
loss with
with an
an
80%
80% decline
decline in
in native
native compounds
compounds from
from 0–120
0–120 h.
h. All
All other
other compounds
compounds had had losses
losses between
between 42–63%
42–63% inin
the first 120 h of fermentation, followed by increases in the final 48 h (120–168
the first 120 h of fermentation, followed by increases in the final 48 h (120–168 h).h).

Figure 4. (A–I) Concentration of fermentation substrates and metabolites in cocoa beans across 168 h.
Values are
Values arepresented
presentedas asthe
themean
mean± ± SEM of fermentation replicates. Significance between time points
points
determined by one-way
was determined one-way ANOVA
ANOVA and and Tukey’s
Tukey’s HSD
HSD post-hoc
post-hoc test
test (p
(p << 0.05).
0.05). Time points
points with
with
different letters
different letters are
are significantly
significantly different
differentvalues.
values.
Foods 2019, 8, 102 12 of 20
Foods 2019, 8, x FOR PEER REVIEW 12 of 20

Figure 5.
Figure 5. (A)
(A) Concentration
Concentration of of total
total polyphenols
polyphenols over over the
the 168-h
168-h fermentation,
fermentation, as as quantified
quantified by
by the
the
Folin–Ciocalteu colorimetric assay, expressed in mg GAE/g cocoa bean. (B) Concentration
Folin–Ciocalteu colorimetric assay, expressed in mg GAE/g cocoa bean. (B) Concentration of total of total
flavanols over
flavanols overthethe 168-h
168-h fermentation,
fermentation, as quantified
as quantified by the 4-dimethylaminocinnamaldehyde
by the 4-dimethylaminocinnamaldehyde (DMAC)
(DMAC) colorimetric
colorimetric assay,in expressed
assay, expressed mg PCB2/ginbean. mg (C–G)
PCB2/g bean. (C–G)
Individual Individual
polyphenol polyphenol
concentrations (C,
concentrations
EC, PCB2, PCC1, (C,CinA2)
EC, PCB2, PCC1,
over the 168-hCinA2) over theas
fermentation, 168-h fermentation,
quantified as quantified
by reversed by reversed
phase UPLC-MS, and
phase UPLC-MS,
expressed as mg/gand expressed
cocoa as mg/g
bean. Values are cocoa
presentedbean.asValues
the meanare ±presented as the mean replicates.
SEM of fermentation ± SEM of
Significance between time points was determined by one-way ANOVA and Tukey’s HSD post-hoc and
fermentation replicates. Significance between time points was determined by one-way ANOVA test
Tukey’s
(p < 0.05).HSD
Timepost-hoc testdifferent
points with (p < 0.05). Time
letters arepoints with different
significantly differentletters
withinare significantly different
values.
within values.
Foods 2019, 8, 102 13 of 20
Foods 2019, 8, x FOR PEER REVIEW 13 of 20

Figure6.6.(A–J)
Figure (A–J)Individual
Individual polyphenol
polyphenol concentrations
concentrations by mean degree of polymerization (mDP) over
the 168-h
the 168-hfermentation
fermentation asas quantified
quantified by
byHILIC
HILICUPLC-MS/MS
UPLC-MS/MS and expressed as mg/g mg/g cocoa
cocoa bean.
bean.
Valuesare
Values arepresented
presentedasasthe mean±± SEM of fermentation replicates. Significance between time points
themean
wasdetermined
was determined byby one-way
one-way ANOVA
ANOVA and and Tukey’s
Tukey’s HSD
HSD post-hoc
post-hoc test
test (p
(p << 0.05).
0.05). Time points with
differentletters
different lettersare
aresignificantly
significantlydifferent
differentwithin
withinvalues.
values.

4.4.Discussion
Discussion
The
The primary
primary goal goal ofof this
this work
work waswas to
to develop
develop and and characterize
characterize aa pilot-scale
pilot-scale model cocoa
model cocoa
fermentation system suitable for conducting cocoa fermentation research in the
fermentation system suitable for conducting cocoa fermentation research in the absence of fresh cocoa absence of fresh cocoa
pods,
pods, and
and capable
capable of of producing
producingsufficient
sufficientquantities
quantities(tens
(tens ofof
kg)kg)
of of material
material for for further
further evaluating
evaluating the
the
impact of cocoa fermentation on putative bioactive compounds and cocoa bioactivity in in in
impact of cocoa fermentation on putative bioactive compounds and cocoa bioactivity in vitro
vitro and
and in vivo
in vivo experiments.
experiments. OurOur model
model was
was notnotdesigned
designedtotophysically
physicallymimicmimic aa scaled-down
scaled-down heap heap
fermentation, but rather to serve as a model system suitable for the study of
fermentation, but rather to serve as a model system suitable for the study of heap fermentation usingheap fermentation using
(1)
(1)dried,
dried,unfermented
unfermentedbeans beansand and aa simulated
simulated liquid
liquid pulp
pulp media
media asas the
the starting material, (2)
starting material, (2) ambient
ambient
microbiota
microbiotaand and(3)(3)regular
regularstirring.
stirring. The
The goal
goal was
was toto achieve
achieve similar
similar chemical
chemical changes
changes asas observed
observed in in
on-farm
on-farm heap fermentations by putting beans in similar conditions to those found at the center of aa
heap fermentations by putting beans in similar conditions to those found at the center of
well-turned
well-turnedheap.heap.InIn cocoa
cocoa fermentations
fermentations conducted on farms,
conducted highlyhighly
on farms, variablevariable
conditions exist between
conditions exist
regions,
betweencountries, farms and
regions, countries, even
farms andbetween specificspecific
even between heaps, heaps,
as temperature
as temperatureand environment
and environment play
important roles in fermentation [44]. Using separate boxes under identical
play important roles in fermentation [44]. Using separate boxes under identical conditions providedconditions provided some
insight into theinto
some insight amount of variability
the amount to expect
of variability in this in
to expect controlled model.model.
this controlled Variability within within
Variability our model
our
was generally
model very minor
was generally very andminor
the metabolic
and the profiles
metabolic were similarwere
profiles to those seento
similar in those
heap fermentations.
seen in heap
Although our model
fermentations. Althoughwas consistent,
our model thewas
replicate fermentations
consistent, were conducted
the replicate fermentations simultaneously in the
were conducted
same incubator. in
simultaneously Further
the samework is needed
incubator. to determine
Further work isthe consistency
needed between
to determine thebatches conducted
consistency between at
different times and in
batches conducted at different incubators.
times and in different incubators.
At
Atan
aninitial
initial pH
pH of of 3.6,
3.6, the
the pulp
pulp media
media created
created aa favorable
favorable environment for yeasts to proliferate
within
withinthe
thefirst
first48
48hh[12].
[12]. Citric
Citric acid was then metabolized by LAB and and ethanol
ethanol production
production continued,
continued,
increasing
increasingthe thepHpH andandencouraging
encouraging thethe
growth of LAB.
growth As lactic
of LAB. and acetic
As lactic acids dominated
and acetic the system,
acids dominated the
pH declined
system, pH (72–168
declinedh)(72–168
and bean h) cotyledon
and bean was penetrated
cotyledon to initiate bean
was penetrated death, bean
to initiate wheredeath,
endogenous
where
endogenous biochemical reactions began the formation of the characteristic chocolate flavor, and pH
concluded at approximately 4.6 [45]. In heap fermentations, turning patterns are a primary factor in
Foods 2019, 8, 102 14 of 20

biochemical reactions began the formation of the characteristic chocolate flavor, and pH concluded at
approximately 4.6 [45]. In heap fermentations, turning patterns are a primary factor in pH variability.
On average, heaps that are turned at least twice progress from pH 3.9–4.6 [8,12,14,34]. Our system
was stirred every 12 h to mimic conditions in the center of a well-mixed heap, and our observed
pH values align with previously reported values. DO values stabilized after 24 h as the lag phase
of yeast metabolism ended. Likely due to a lack of monitoring equipment, there are no published
data regarding DO progression in heap fermentations. Moving forward, aeration in the model could
be increased to elevate DO. It would be worthwhile to monitor DO in various on-farm fermentation
systems to determine accurate values for modeling a given system. Additionally, comparison of
our measured FI values with reported values provide evidence that sufficient fermentation can be
achieved under these pilot-scale conditions. Anthocyanins and catechin monomers polymerize during
fermentation, rapidly disappearing from the bean cotyledon [2,24]. This can also be observed in the
cut test. In traditional cut tests of ≥300 beans, bean color should uniformly progress from purple to
brown throughout the fermentation, showing the effect of polyphenol oxidase and other reactions that
reduce the appearance of color within the bean. Criollo beans have low anthocyanin levels and as
such the cut test and FI for these beans does not follow the pattern typically seen with other cultivars
such as Trinitario and Forastero [46–50]. However, it is important to include these tests in reference
to typical cocoa fermentation quality checks, and future work is needed using this model with other
cocoa cultivars.
Yeasts proliferate during the early stages of fermentation, consuming available sugars and
converting them to ethanol and carbon dioxide. Sucrose concentrations exhibited a decline in both
pulp media and bean, while pulp glucose and fructose concentrations had a 24-h lag period, and bean
concentrations of these sugars increased in the beginning hours, likely due to diffusion from the
pulp media into the bean. The early decrease in sucrose is likely due to yeast-derived invertase that
hydrolyze sucrose into glucose and fructose during the lag period, before the yeasts begin to consume
these sugars. Although sugar concentrations vary between replicates, these trends are similar to
those seen in traditional heaps [8,16,51]. Ultimately, yeasts consumed all available substrates for
growth, inducing inhibition of their own activity, and LAB began to dominate the system with citric
acid degradation, subsequently increasing pH and creating an optimal environment for bacterial
growth [52]. AAB then thrived in this newly aerobic, less-acidic environment (48–72 h), facilitating the
oxidation of ethanol to acetic acid and, further, to carbon dioxide and water, ultimately resulting in
bean death. Although the reactions that occur within the bean itself are not well understood, reported
consumption and production of metabolites during industrial cocoa fermentation follow a similar
pattern to that of the simulated pulp media in the model system described in this study.
During the first days of fermentation, polyphenols are oxidized via polyphenol oxidase and
condense into high molecular weight tannins and other complex compounds. These reactions occur as
polyphenols, such as (–)-epicatechin, diffuse out of the bean cotyledon and into the media, subsequently
aligning with bean death [9,24]. Criollo beans have been reported to have approximately two-thirds
of the total polyphenol content of Forastero and Trinitario varieties, yet other studies have indicated
that Criollo beans have high levels of procyanidins with no significant difference in total polyphenol
content between the three main cultivars [40,53,54]. Total phenolic content of Criollo beans is often
not thoroughly analyzed, but values of 40–50 mg GAE/g have been reported, aligning with the data
presented in this study (Figure 5A) [53]. The most dramatic decrease in phenolic concentrations took
place in the first 72 h (Figures 5 and 6), confirming this model system’s activity and succession of
bean death, as well as representing the quick decline of polyphenol compounds seen in Criollo bean
fermentation [40]. Additionally, the observed loss of polyphenols from raw to fermented is comparable
to that of heap fermentations. The average loss of total polyphenol content previously reported is
40–60%, which is within the range of loss for this study [3,9,17,23,46,55,56]. Payne et al. [55] found
that heap fermentation resulted in an 86–94% decrease in (–)-epicatechin and an 83–89% decrease in
(±)-catechin levels. Similarly, Kim and Keeney [23] demonstrated that (–)-epicatechin levels declined
Foods 2019, 8, x FOR PEER REVIEW 15 of 20

concluded
Foods at 120 h, and it cannot be determined if the trend seen in the final 48 h of our model system
2019, 8, 102 15 of 20
would correspond with observations from that study. The quantification of high molecular weight
procyanidins (trimer-decamer) over the course of fermentation (Figure 6) is the first of these values
77–91% during in
to be reported fermentation,
the literature,withas bean originare
standards andoften
variety
notplaying a key role
commercially in rate and
available. Thistotal
datadecline.
shows
Furthermore, when examining polyphenols based on degree
the elevated presence of very large molecular weight compounds (Figure 6I–K) throughout of polymerization, Kealey et al. [30]
reported
fermentationlosses andof 61% (monomer),
highlights 54% (dimer),
the research gap on 60% (trimer)
these and 68%in
compounds (tetramer).
cocoa, with Although
most of these
the
values align with our data, that fermentation concluded at 120 h,
literature focusing on monomeric compound quantification, specifically (–)-epicatechin and (±)- and it cannot be determined if
the trend
catechin. seen in the final 48 h of our model system would correspond with observations from that
study. The quantification
Elevated temperatures of high
of the molecular
fermentationweightsystemprocyanidins
may also (trimer-decamer)
be responsible over forthe course
phenolic
of fermentation (Figure 6) is the first of these values to be reported
degradation, with further polymerization of these smaller monomers and procyanidins into larger in the literature, as standards
are
more often not commercially
complex compounds [55]. available.
TheseThiscould data
form shows the elevated
complex compounds presence
with of veryresponses
higher large molecularin our
weight compounds (Figure 6I–K) throughout fermentation and
assays, but further investigation into the relationship between fermentation temperature highlights the research gap on these and
compounds
polyphenol loss in cocoa, with most
is warranted of the
as this literature
theory has not focusing on monomeric
been adequately compound
investigated. Figure quantification,
7 shows the
specifically (–)-epicatechin
visual appearance and (±)-catechin.
of the polyphenol-rich cocoa extract used for the quantification of total/individual
Elevated temperatures
polyphenols and total flavanols. of Atthe144fermentation
h, a change insystem maycan
the extract also be responsible
be noted in the colorfor andphenolic
texture.
degradation, with further polymerization of these smaller monomers
Because the four different analyses (Folin, DMAC, UPLC-MS, UPLC-MS/MS) were performed and procyanidins into larger at
more complex compounds [55]. These could form complex compounds
different times and produced similar results in terms of time-course trends, it is unlikely that the with higher responses in
our assays,
phenolic but further
increase in theinvestigation
final hours into is due thetorelationship
human error between
duringfermentation
analysis. Antemperature
error in extract and
polyphenol
preparationloss is warranted
is possible but alsoas this theoryashas
unlikely, thenot been adequately
appearance investigated.
of the cocoa extracts Figure
change7inshows the
multiple
visual appearance of the polyphenol-rich cocoa extract used for the quantification
instances. Further investigation into a wider range of compounds is warranted to determine the cause of total/individual
polyphenols
of this phenolic andincrease,
total flavanols. At 144 h, with
as it is inconsistent a change in the extract
any previously can be
reported noted
data andin the is
there color and
a lack of
texture. Because the four different analyses (Folin, DMAC, UPLC-MS,
thorough data quantifying these individual compounds across varying degrees of fermentation. TheUPLC-MS/MS) were performed
at different by
mechanism times
which andthese
produced
late-stagesimilar
changesresults in terms
occurred of time-course
warrants further study.trends,It isitimportant
is unlikely that
to note
the
thatphenolic increase
the specific beansinused
the final
for thehours
curtistest
due(Figure
to human 1C) error
were during
not theanalysis.
same actual An errorbeansinused extract
for
preparation is possible but also unlikely, as the appearance of the cocoa
extraction and other assays, and furthermore that extraction isolates and concentrates components extracts change in multiple
instances.
that cannotFurther
alwaysinvestigation into a wider
be visibly observed range of compounds is warranted to determine the cause
in beans.
of this phenolicpolyphenol
Although increase, asloss it is may
inconsistent with any
have negative previously on
implications reported data and
the overall health there is a lack
benefits of
of thorough data quantifying these individual compounds across varying
cocoa, this relationship is still poorly understood. It is important to understand that these losses degrees of fermentation.
The mechanism
correspond withby thewhich these late-stage
development changes
of positive flavoroccurred warrants
profiles, further must
and a balance study.be It found
is important
between to
note that the specific
optimization beans used
of polyphenol for the
content for curt
health testoutcomes
(Figure 1C)andwere not the same
for acceptable actual
flavor [3].beans used for
Furthermore,
extraction and other assays, and furthermore that extraction isolates and
the chemistry of these losses, and the structures and bioactivity of the subsequent products, remain concentrates components
that
to becannot always be visibly observed in beans.
elucidated.

Figure 7.7.Polyphenol-rich
Figure Polyphenol-rich cocoa
cocoa extracts
extracts (CE) prepared
(CE) prepared at each
at each point point throughout
throughout the 168-h
the 168-h fermentation.
fermentation. See Section 2.8 for methodology. It is important to note the change in color
See Section 2.8 for methodology. It is important to note the change in color and texture towardsand texture
the
towards the final hours of
final hours of fermentation. fermentation.

Although polyphenol loss may have negative implications on the overall health benefits of cocoa,
this relationship is still poorly understood. It is important to understand that these losses correspond
Foods 2019, 8, 102 16 of 20

with the development of positive flavor profiles, and a balance must be found between optimization of
polyphenol content for health outcomes and for acceptable flavor [3]. Furthermore, the chemistry of
these losses, and the structures and bioactivity of the subsequent products, remain to be elucidated.
Interest in controlled fermentation systems has emerged as a strategy to experimentally manipulate
cocoa fermentation on various scales in regions where cocoa does not grow. Controlled fermentations
using a model system offer reliable, reproducible methods to understand the microbial and biochemical
reactions that occur. In heap fermentations, beans and pulp are removed directly from the pod, heaped
on the ground or in wooden boxes and covered with banana leaves. Traditional systems, while
effective for cocoa production, offer little ability to implement experimental conditions or controls
to enable research on the impact of environment, materials and fermentation management practices on
outcomes of fermentation. In a controlled setting, factors such as temperature, bulk density, relative
humidity, oxygenation, microbial inoculation and other influences can be regulated and manipulated,
furthering scientific understanding of the complex interactions occurring during cocoa fermentation.
Additionally, previously published fermentation-like model systems range in capacity from 25 beans
to 1.2 kg rehydrated bean weight [19,22,57]. With the capacity of our model to ferment approximately
30 kg of rehydrated cocoa beans simultaneously, this novel pilot-scale model can produce relatively
large quantities of fermented beans in a controlled setting. Production of a larger quantity of material
under controlled experimental fermentation conditions will enable further study of the bioactivity of
the resulting cocoa, using animal feeding experiments, for example. Furthermore, this pilot-scale model
fermentation can be conducted using food-grade inputs, making it applicable to the production of
substrates for human clinical trials as well, where large amounts of material are required.
It is critical to note that the system developed in this study is intended to serve as a model designed
to replicate the chemical outcomes of cocoa fermentation, but not to physically duplicate the heap
fermentation process on a smaller scale. As such, our model aims to produce conditions mimicking the
center of a well-turned heap in simulated pulp media. Our data indicate that this system is a reliable
and controlled pilot-scale fermentation model, which replicates the outcomes of heap fermentation
with acceptable fidelity. It is important to note that cocoa fermentation varies significantly. Our model
system was not tested in the present study to mimic all possible combinations of turning, aeration,
temperature gradients or lengths of fermentation. However, the model we describe here can be used
to study such variations in a controlled environment. We are currently expanding upon this study
by introducing variables such as cool vs. hot fermentations to generate cocoas with distinct chemical
profiles for evaluating the impact of composition on health benefits. Although our results show promise
moving forward, this model is not without limitations. The beans used were commercially available
and of largely unknown origin. Although the beans were food-grade and declared to be dried and
unfermented, drying temperature and duration, as well as the conditions between harvest and drying,
are unknown. Moving forward, we will address this limitation by obtaining dried unfermented beans
from suppliers with more knowledge of the supply chain. The impact of using dried, unfermented
beans on the fidelity of our model, compared with fermentation using fresh beans, will need to be
evaluated by using a single batch of beans and conducting fermentations using fresh beans, as well as
by drying and then employing our model. For example, early flavanol degradation by endogenous
polyphenol oxidase may be impacted. Also, aeration could be further optimized to mimic conditions at
the center of a well-mixed heap or box fermentation, or at least to gain an understanding of the effects
of aeration on the chemical changes observed. Acquiring fresh cocoa pods for use in laboratories
distant from production regions without spoilage has proven to be expensive and ineffective.
To summarize the novelty of the present work, several key advances have been made. First,
by using dried, unfermented beans and a simulated pulp medium instead of relying on fresh cocoa
pods, this model system will allow cocoa fermentation research to progress anywhere in the world
regardless of location, climate or season. This greatly expands the ability of researchers distant
from cocoa cultivation to control and manipulate cocoa fermentation for research purposes. Second,
the increased scale of this model system (from previous controlled lab fermentations using tens or
Foods 2019, 8, 102 17 of 20

hundreds of grams of beans to the present model employing 2 × 15 kg beans) will facilitate production
of large amounts of custom cocoas designed specifically for animal or human clinical studies. Both of
these advances will result in expanded scope of cocoa fermentation research. Thus, the development
and characterization of this pilot-scale model system represents a promising new and controlled
method for expanding upon cocoa fermentation research.

Author Contributions: K.C.R. collected the data, interpreted the results, and wrote the original manuscript. A.H.L.
contributed to formal data analysis and interpretation of the results. B.D.W. provided conceptualization and
investigation for the pilot fermentation model system. H.H. contributed to the methodology and interpretation
of HPLC analysis. J.D.L. conceptualized the study design, reviewed and edited the manuscript. A.C.S.
conceptualized the study design, provided supervision for the fermentation model system, reviewed and edited
the manuscript. A.P.N. conceptualized the study design, provided supervision and assisted with formal analysis,
reviewed and edited the manuscript.
Funding: This research was funded by USDA AFRI Grant 2017-67017-26783. Funding for this work was also
provided, in part, by the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute
of Food and Agriculture, US Department of Agriculture.
Acknowledgments: The authors would like the acknowledge Qing Jin and Nicholas Poe for their assistance with
HPLC set-up and analysis.
Conflicts of Interest: The authors declare no conflict of interest.

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