molecules

Review
The Chemistry behind Chocolate Production
Veronika Barišić , Mirela Kopjar , Antun Jozinović , Ivana Flanjak , Đurđica Ačkar * ,
Borislav Miličević, Drago Šubarić, Stela Jokić and Jurislav Babić
Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek, Franje Kuhača 20,
31000 Osijek, Croatia
* Correspondence: dackar@ptfos.hr




Received: 2 July 2019; Accepted: 29 August 2019; Published: 30 August 2019


Abstract: Chocolate production is a complex process during which numerous chemical reactions
occur. The most important processes, involving most of the reactions important for development of the
proper chocolate flavor, are fermentation, drying and roasting of cocoa bean, and chocolate conching.
During fermentation, formation of important precursors occurs, which are essential for further
chemical reactions in the following processes of chocolate production. Roasting is one of the most
important processes due to the occurrence of Maillard’s reactions, during which aroma compounds
are formed. In this paper, we have reviewed the most important chemical reactions that occur with
proteins, carbohydrates, lipids, and polyphenols. Additionally, we present other components that
may be naturally present or form during the production process, such as methylxanthines, aldehydes,
esters, ketones, pyrazines, acids, and alcohols.

Keywords: chocolate; cocoa beans; Maillard reactions; polyphenols; pyrazines

1. Introduction
Chocolate is a product widely consumed by all generations. It is rich in fat, proteins, carbohydrates,
polyphenols and other bioactive compounds [1]. Cocoa beans are main ingredient for production of
chocolate. The chocolate production process consists of fermentation, drying, roasting, grinding of
cocoa beans, mixing of all ingredients (cocoa mass, sugar, cocoa butter, emulsifiers, aroma, and milk
components if needed), conching, and tempering. Major chemical reactions occur during fermentation,
drying, roasting of cocoa beans, and conching of chocolate mass. These reactions are the most important
for flavor and aroma development [2].
Fermentation of cocoa beans is a process in which growth of yeasts and bacteria occur in the
pulp and it is conducted at cocoa plantations as a part of the cocoa beans’ production. In this stage,
breakdown of sugars and mucilage occurs. It consists of three phases:

(1) The first phase is dominated by anaerobic yeasts and lasts for 24–36 h. In this phase, low content
of oxygen and low pH (<4) are present.
(2) The second phase is dominated by lactic acid bacteria. They are present from the beginning but
become active between 48–96 h.
(3) Third phase is dominated by acetic acid bacteria when aeration increases. During this last
phase, an exothermic reaction (conversion of alcohol into acetic acid) occurs, responsible for
a temperature rise (50 ◦ C or higher).

These phases of cocoa bean fermentation are described in detail in Beckett et al. [3]. Fermentation
is the key stage for the production of precursors for development of proper chocolate aroma [3–5].
Drying of cocoa beans is conducted after fermentation to lower the moisture content and complete
oxidative processes induced during fermentation of cocoa beans. After drying, cocoa beans have 6–8%

Molecules 2019, 24, 3163; doi:10.3390/molecules24173163 www.mdpi.com/journal/molecules

Molecules 2019, 24, 3163 2 of 13

moisture. Low moisture content prevents mold growth and makes the beans more stable for transport
and storage [2,3].
Roasting of cocoa bean is usually conducted in a chocolate factory as a first stage of chocolate
production. It is a high temperature process, usually conducted at temperatures between 120 and
140 ◦ C, which is important for the occurrence of Maillard reactions. Roasting reduces contents of
undesirable components, produces chocolate-specific aroma and flavor, and decontaminates the
cocoa beans. In this phase, all precursors formed in the previous phases react and form numerous
compounds [2,3,5].
Grinding and mixing of all chocolate ingredients are very important for achievement of the right
particle size of all ingredients. The main ingredients used in chocolate production are cocoa liquor
(obtained by grinding cocoa beans), cocoa butter (obtained by pressing cocoa liquor), sugar, and milk
(in the case of milk chocolate) [3].
Conching is a mixing and heating treatment that is conducted to produce liquid chocolate (all solid
particles are coated with fat), evaporate volatile acids, achieve a proper viscosity, remove excess
moisture, and develop a desirable color [5–7].
Tempering is a process by which to obtain a stable product. Tempering is conducted thermally
and results in stable and consistently sized crystals of cocoa butter which then affect growth of a stable
crystalline network during cooling [3].
This paper reviews the major chemical reactions that occur during the chocolate production
process and gives insights into the latest understandings of the most important components of cocoa
beans and chocolate.

2. Proteins

2.1. Fermentation
Proteins make 10–15% of the dry weight of unfermented cocoa beans. The most important are
albumin (water soluble), prolamin (alcohol soluble), globulin (vicilin-type, salt soluble), and glutein
(soluble in dilute acids and alkali) [8,9]. Fermentation is a process important for supplying precursors
for further reactions. The low pH that develops during fermentation activates the endogenous enzymes
carboxypeptidase and aspartic endoprotease. Carboxypeptidase and aspartic endoprotease are most
active at pH 3.8 to 5.8 [8,10,11]. These enzymes hydrolyze proteins and, during fermentation, there is
an increase in free amino acids and oligopeptides. The highest activity of these enzymes occurs shortly
after bean death (24–48 h) [8,12,13]. Unfermented beans have a lower content of free amino acids
then fermented beans. The ratio of acidic/hydrophobic/basic/other free amino acids in unfermented
beans depends mostly on the origin and type of the beans. Regardless of the aforementioned ratio,
during fermentation, the content of hydrophobic free amino acids increases and the content of acidic
amino acids decreases. Hydrophobic amino acids, especially leucine, alanine, and phenylalanine,
released during fermentation are responsible for development of Strecker aldehydes and pyrazines [8,13].
This was confirmed by the research of Adeyeye et al. [4] who discovered that fermented beans have
a higher content of crude protein when compared with unfermented beans. They showed that
phenylalanine and leucine are the predominant amino acids after fermentation [10]. Free tryptophan
(which can be present as protein-bound as well), is transformed during fermentation into biogenic
amines (5-hydroxytryptamine and tryptamine) [9]. Although the exact mechanism of tryptamine
formation in cocoa beans has not been revealed, Oracz and Nebesny [14] proposed a mechanism of its
enzymatic formation from tryptophan (Figure 1) and do Carmo Brito et al. [15] reported that the level
of tryptamine decreases in the first four days of fermentation, only to slowly increase to virtually the
same level by the sixth day. The presence of small amounts of tryptamine in raw cocoa beans has also
been reported, as well as differences regarding variety (due to different composition of the bean) and
origin (due to differences in cultivation conditions and microflora involved in the fermentation process)
of cocoa beans [14]. Therefore, it is yet to be revealed whether the process of tryptamine synthesis
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from tryptophan is spontaneous, or whether endogenous enzymes or enzymes of microflora during
fermentation
involved. Sinceare involved. Since biogenic
biogenic amines amines
are known to beare known
the to be the most
most abundant abundantfoods,
in fermented in fermented
it is most
foods,
likelyit that
is most likely that of
fermentation fermentation
cocoa beansofand
cocoa beans and
microflora microflora
involved involved
in the processinare
thethe
process
most are the
relevant
most relevant in tryptamine
in tryptamine synthesis. synthesis.

Figure Possible
1. 1.
Figure transformation
Possible ofof
transformation tryptophan during
tryptophan fermentation
during of of
fermentation cocoa beans.
cocoa beans.

Hydrophobic
Hydrophobic oligopeptides
oligopeptidesthatthatare formed
are formed from hydrophobic
from hydrophobic amino
amino acids mentioned
acids mentionedabove
above
become
become substrates
substrates forfor
serine carboxy-(exo)
serine carboxy-(exo) peptidase.
peptidase.Hydrophilic
Hydrophilic oligopeptides
oligopeptides and hydrophobic
and hydrophobic
free
freeamino
aminoacids
acids(chocolate
(chocolate aroma precursors) are
aroma precursors) areobtained
obtainedbybythe
the activity
activity of carboxypeptidase
of carboxypeptidase from
from
hydrophobic oligopeptides [16]. During fermentation, precursor compounds derived
hydrophobic oligopeptides [16]. During fermentation, precursor compounds derivedfrom
fromfree
free
amino
aminoacids
acidsare
arereleased.
released.3-methylbutanol,
3-methylbutanol, phenylacetaldehyde, 2-methyl-3-(methyldithio)furan,2-
phenylacetaldehyde, 2-methyl-3-(methyldithio)furan,
2-ethyl-3,5-dimethyl,
ethyl-3,5-dimethyl, and and 2,3-diethyl-5-methylpyrazine
2,3-diethyl-5-methylpyrazine are are among
among the the most
mostimportant
important[5].
[5].Also,
Also,
decarboxylation
decarboxylationofofamino aminoacids
acids during
during fermentation results in
fermentation results in formation
formation ofofbiogenic
biogenicamines
amines(2-
(2-phenylethylamine, 2-methylpropylamine, and
phenylethylamine, 2-methylpropylamine, and 2-
2-and
and3-methylbutylamine).
3-methylbutylamine).These Thesecomponents
componentsare are
already
already present in unfermented beans, but during fermentation, decarboxylases leadantoincrease
present in unfermented beans, but during fermentation, decarboxylases lead to in
an increase
the
incontent of bioactive
the content amines
of bioactive [8,10].
amines [8,10].

2.2. Roasting
2.2. Roasting
Since
Sinceroasting
roasting is is
conducted
conducted at at
high
high temperatures,
temperatures, precursors,
precursors, which
which were
were obtained
obtained byby protein
protein
degradation
degradation during fermentation, are subjected to chemical reactions. The most important isthe
during fermentation, are subjected to chemical reactions. The most important is the
Maillard
Maillardreaction
reactionbetween
between the carbonyl group
the carbonyl groupofofreducing
reducing sugars
sugars andandthe the
aminoamino
groupgroup
from from
amino
amino
acids acids or proteins.
or proteins. Through Through these reactions,
these reactions, cocoa obtains
cocoa obtains its chocolate
its chocolate specific specific
aroma and aromaflavorand [4].
flavor
The [4]. The condensation
condensation productproduct that results
that results fromfrom thesethese reactionsisis transformed
reactions transformed by byAmadori
Amadori
rearrangement
rearrangement toto
1-deoxy-2-ketosyl
1-deoxy-2-ketosyl [2,4]. Roasting
[2,4]. Roasting is is
more
more effective
effective inin
thethegeneration
generation ofof
amines
amines andand
aldehydes than fermentation. Strecker-type reactions form aldehydes in roasted
aldehydes than fermentation. Strecker-type reactions form aldehydes in roasted beans from amino beans from amino acids
(Figure
acids 2) [10,17].2)In[10,17].
(Figure addition, In Oracz and Nebesny
addition, Oracz and [18]Nebesny
reported[18]that reported
at higher roasting temperatures
that at higher roasting
(135 and 150 ◦ C), Maillard reactions advance to produce melanoidins, high-molecular weight (HMW)
temperatures (135 and 150 °C), Maillard reactions advance to produce melanoidins, high-molecular
compounds
weight (HMW) that confer the specific
compounds brownthe
that confer color and texture
specific browntocolor
cocoa beans.
and They
texture to distinguish
cocoa beans. three
They
types
distinguish three types of melanoidins: “(1) polymers consisting of repeating furans or pyrrole via
of melanoidins: “(1) polymers consisting of repeating furans or pyrrole units linked units
polycondensation reactions; (2) carbohydrate skeletons built mainly
linked via polycondensation reactions; (2) carbohydrate skeletons built mainly from sugar from sugar degradation products
polymerized
degradationthroughproducts aldol-type
polymerizedcondensation
through reactions
aldol-type and possibly linked
condensation by nitrogenous
reactions and possibly residues;
linked
and (3) protein-like structures arising from crosslinks between proteins
by nitrogenous residues; and (3) protein-like structures arising from crosslinks between proteins and low-molecular weight and
(LMW) compounds formed in the advanced stages of Maillard reactions”.
low-molecular weight (LMW) compounds formed in the advanced stages of Maillard reactions”. They also imply that,
since
They thealso
carbohydrate
imply that,content
since the in cocoa beans is content
carbohydrate relatively inlow (unlike
cocoa beanscoffee beans, for
is relatively example),
low it is
(unlike coffee
more likely that products of lipid oxidation, rather than sugars, are involved in
beans, for example), it is more likely that products of lipid oxidation, rather than sugars, are involvedreactions with proteins
toin
form HMWwith
reactions melanoidins.
proteins to However,
form HMW this link is not wellHowever,
melanoidins. established bylink
this the is
results of their
not well researchby
established
and should be further investigated.
the results of their research and should be further investigated.
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Figure Strecker-type reaction

Figure 2. Strecker-type reaction for formation of aldehydes during roasting of cocoa beans.

2.3. Other
2.3. Other Processes
Processes
Casein is
Casein is one
one of
of the
the most
most important
important proteins
proteins in milk. It
in milk. It can
can be
be present
present in
in milk
milk chocolate
chocolate and
and form
form
an interface
an interface between
between solid
solid and
and fatty
fatty components,
components, meaning
meaning that
that casein
casein can
can act
act as
as an
an emulsifier
emulsifier [2,3].
[2,3].
During conching, caramelization of lactose in milk chocolates and Maillard reactions
During conching, caramelization of lactose in milk chocolates and Maillard reactions with milkwith milk proteins
occur [19].
proteins Conching
occur does not does
[19]. Conching changenotthe amino
change acid
the content
amino acidof chocolate
content [20].
of chocolate [20].
3. Carbohydrates
3. Carbohydrates
3.1. Fermentation
3.1. Fermentation
Unfermented cocoa beans contain 2–4% of low molecular weight carbohydrates and their
Unfermented
derivatives, mostlycocoa
sucrosebeans
(90%contain
of total2–4% of low
sugars), molecular
glucose, weight
fructose, carbohydrates
galactose, and their
sorbose, arabinose,
derivatives, mostly
xylose, mannitol, andsucrose
inositol.(90% of total sugars),
Polysaccharides glucose,
(starch (4–6%), fructose,
pectins,galactose, sorbose,
cellulose (2–3%) arabinose,
etc.) are also
xylose, mannitol, and inositol. Polysaccharides (starch (4–6%), pectins, cellulose
present in beans at a proportion of approximately 12%. Redgwell and Hansen [21] reported that (2–3%) etc.) are also
cell
present in beans at a proportion of approximately 12%. Redgwell and Hansen
wall polysaccharides (CWP) include cellulose (28% of CWP), pectic polysaccharides (containing [21] reported that cell
wall polysaccharides
galactose (CWP) include
as the predominant cellulose
side-chain sugar (28%(9%ofofCWP),
CWP)pectic polysaccharides
and containing (containing
arabinose as the
galactose
predominant side-chain sugar (52% of CWP)), hemicellulosic polysaccharides (xyloglucan (8% ofas
as the predominant side-chain sugar (9% of CWP) and containing arabinose the
CWP),
predominant side-chain sugar
and gallactoglucomannan (3% of(52% of CWP)),
CWP)). They also hemicellulosic polysaccharides
reported that fermentation does(xyloglucan
not influence (8%
CWP.of
CWP), and gallactoglucomannan (3% of CWP)). They also reported that fermentation
Sucrose is the main subject of reactions during fermentation. Sugars from pulp are converted to acids does not
influence CWP. Sucrose
during fermentation. is the
These acidsmain
movesubject of beans
into the reactionsand during fermentation.
lower their pH, which Sugars
leads tofrom pulp are
breakdown of
converted to acids during fermentation. These acids move into the beans and lower
storage cells. There are two types of storage cells in the cotyledon: polyphenolic cells, which consist of their pH, which
leads
a largeto breakdown
vacuole of storage
that is filled cells. Thereand
with polyphenols arealkaloids;
two types and of storage cells
lipid-protein cells,inwhich
the have
cotyledon:
small
polyphenolic cells, which consist of a large vacuole that is filled with polyphenols
lipid and protein vacuoles tightly packed inside the cytoplasm. Yeasts present during fermentation and alkaloids; and
lipid-protein cells, which
convert the present sugars have small lipid
to alcohol, whichandbenefits
proteindevelopment
vacuoles tightly packed
of lactic inside
acid the cytoplasm.
bacteria [7,8,12,22].
Yeasts present during fermentation convert the present sugars to alcohol,
The alcohol is converted to acetic acid under aerobic conditions and the activity of acetic acid bacteria. which benefits
development
Sucrose isofhydrolyzed
lactic acid bacteria [7,8,12,22].
into fructose The alcohol
and glucose (reducingis converted to acetic
sugars) due acid under
to invertase aerobic
activity [8].
conditions and the activity of acetic acid bacteria.
Megías-Pérez et al. [23] found seyllo-inositol (trace—504.9 µg/g in unfermented and 15.5–491.9 µg/g
Sucrose isbean),
in fermented hydrolyzed
1-kestose into fructose and
(36.1–133.5 µg/gglucose (reducing and
in unfermented sugars) due to invertase
traces—115.5 µg/g inactivity
fermented [8].
Megías-Pérez et al. [23] found seyllo-inositol (trace—504.9 μg/g in unfermented and 15.5–491.9 μg/g
bean), and 6-kestose (no reported values) for the first time. The content of these compounds was
in fermented bean), 1-kestose (36.1–133.5 μg/g in unfermented and traces—115.5 μg/g in fermented
related to the origin of the cocoa beans. They also reported presence of galactinol in unfermented beans
bean), and 6-kestose (no reported values) for the first time. The content of these compounds was
(traces—1970.4 µg/g). Since they were unable to detect galactinol in fermented beans, they related this
related to the origin of the cocoa beans. They also reported presence of galactinol in unfermented
compound to the fermentation status of the cocoa beans.
beans (traces—1970.4 μg/g). Since they were unable to detect galactinol in fermented beans, they
Reducing sugars obtained during fermentation are the main compounds of aroma development
related this compound to the fermentation status of the cocoa beans.
in the later processes of chocolate production.
Reducing sugars obtained during fermentation are the main compounds of aroma development
in the later processes of chocolate production.
3.2. Drying
During drying, reducing sugars obtained during fermentation react with other components of
3.2. Drying
cocoa beans. These reactions are actually Maillard reactions between free amino acids and reducing
During
sugars [16]. drying, reducing sugars obtained during fermentation react with other components of
cocoa beans. These reactions are actually Maillard reactions between free amino acids and reducing
3.3. Roasting
sugars [16].
Roasting of cocoa beans leads to caramelization of the present sugars. Caramelization occurs
3.3. Roasting
during heating of carbohydrates (especially sucrose and reducing sugars), where high temperature
causesRoasting of cocoa
dehydration beans leads
of sugars. to caramelization,
During caramelization of the present
molecular sugars. Caramelization
degradation of sugars leadsoccurs
to the
during heating of carbohydrates (especially sucrose and reducing sugars), where high temperature
causes dehydration of sugars. During caramelization, molecular degradation of sugars leads to the
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development
developmentofofaroma
aromacompounds.
compounds.Also,
Also,during
duringroasting,
roasting,dehydration
dehydrationof
ofsugars
sugarscan
canoccur
occurwhich
which
produces furfural and its derivatives (Figure 3) [2,17].
produces furfural and its derivatives (Figure 3) [2,17].

Figure3.3.Dehydration
Figure Dehydrationofofsugars.
sugars.

Finished
Finishedchocolate
chocolatecontains
containsabout
about50%50%sugar,
sugar,which
whichisismainly
mainlyadded
addedsucrose,
sucrose,butbutititcan
canalso
alsobe
be
lactose
lactose(in
(inmilk
milkchocolate).
chocolate).InInthe thelast
lastfew
fewyears,
years,fructose,
fructose,sorbitol,
sorbitol,polydextrose,
polydextrose,etc.
etc.have
havebeen
beenadded
added
ininchocolates
chocolates[2].[2].
After
Afterchocolate
chocolate production, sugar bloom
production, sugar bloomon onthe
thechocolate
chocolatesurface
surface may
may appear.
appear. This
This happens
happens due
due to high
to high humidity
humidity when
when moisturecondenses
moisture condenseson onthe
thesurface
surface of
of chocolate.
chocolate. Sugar
Sugar from
from chocolate
chocolateisis
extracted
extractedoutoutononthethesurface
surfaceandandrecrystallizes
recrystallizesafter
afterthe
thewater
waterisisremoved
removed[12].
[12].
Noor-Soffalina et al. [24] found that cocoa beans with higher
Noor-Soffalina et al. [24] found that cocoa beans with higher polyphenol polyphenol content have lower
content have sugar
lower
content. This phenomenon
sugar content. This phenomenon is most likely duelikely
is most to binding
due toofbinding
carbohydrates to polyphenols.
of carbohydrates Hydrogen
to polyphenols.
bonding
Hydrogen of these components
bonding of theseis created betweenishydroxyl
components createdgroups
between and through
hydroxylhydrophilic
groups and interactions.
through
Tannins are more
hydrophilic likely toTannins
interactions. bond with carbohydrates
are more because
likely to bond with they are rich in closely
carbohydrates becausesettheyhydroxyl
are rich
groups [25].
in closely set hydroxyl groups [25].

4.4.Lipids
Lipids
Cocoa
Cocoabeans
beanscontain
contain 50–58%
50–58% fats, fats, 97–98%
97–98% of of which
which are are triacylglycerols
triacylglycerols(TAGs).
(TAGs).TAGs TAGsconsist
consistof
of24.1–27.1%
24.1–27.1%palmitic
palmiticacid,acid,32.9–37.6%
32.9–37.6%stearicstearicacid,
acid,and
and32.7–37.6%
32.7–37.6%oleicoleicacid,
acid,and
andlow lowamounts
amountsof
oflinoleic
linoleic acid
acid (2.3–3.7%).
(2.3–3.7%). Fatty Fatty
acidacid composition
composition depends
depends on origin,
on origin, variety variety of beans,
of beans, growing growing
season,
season, and method
and method of cultivation
of cultivation [8,26].[8,26].
There There are differences
are differences in cocoa
in cocoa butterbutter considering
considering origin origin
and andbean
bean
type.type.
SofterSofter
cocoa cocoa
butterbutter
has has a higher
a higher content
content of of 1-palmitoyl-2-3-dioleoyl-glycerol
1-palmitoyl-2-3-dioleoyl-glycerol (POO)
(POO) andand1-
1-stearoyl-2-3-dioleoyl-glycerol
stearoyl-2-3-dioleoyl-glycerol (SOO), (SOO),while
whileharder
harder cocoa
cocoabutter hashas
butter increased content
increased of saturated
content fatty
of saturated
acids [26]. Sirbu et al. [26,27] found that only unfermented beans contain
fatty acids [26]. Sirbu et al. [26,27] found that only unfermented beans contain TAG containing TAG containing hydroxyl-allyl
fatty acid substituent
hydroxyl-allyl fatty byacidwhich they canby
substituent bewhich
differentiated
they canfrom fermented beans.
be differentiated fromThey namedbeans.
fermented this newlyThey
identified component cacaoic acid and stated that during fermentation,
named this newly identified component cacaoic acid and stated that during fermentation, degradation or transformation
ofdegradation
this component may occur. Future
or transformation research
of this is needed
component may to occur.
furtherFuture
exploreresearch
this newiscomponent
needed to and its
further
fate during fermentation.
explore this new component and its fate during fermentation.
Fats
Fatswhich
which are
arerich in symmetrical
rich in symmetrical monounsaturated
monounsaturated triglycerides, suchsuch
triglycerides, as cocoa butter,
as cocoa show show
butter, a higha
degree
high degree of polymorphism [3]. Regardless of the TAG composition, cocoa butter can existininsix
of polymorphism [3]. Regardless of the TAG composition, cocoa butter can exist six
polymorphs: γ, α, β’(III), β’(IV), β(V), and β(VI) which have melting points
polymorphs: γ, α, β’(III), β’(IV), β(V), and β(VI) which have melting points of 17.3, 23.3, 25.5, 27.5, of 17.3, 23.3, 25.5, 27.5,
33.8,
33.8,and
and36.3,
36.3,respectively,
respectively,and andcan cantransform
transformfrom fromoneonetotoanother,
another,depending
dependingon ontemperature
temperatureand and
time. The most desirable form in chocolate is β(V) which melts at 29–31.5 ◦ C. This form is obtained by
time. The most desirable form in chocolate is β(V) which melts at 29–31.5 °C. This form is obtained
abyproperly
a properlyconducted
conducted tempering
tempering process.
process.FatFatbloom
bloom is is
one
oneofofmain
mainproblems
problemsininfinished
finishedchocolate.
chocolate.
InInwell-tempered
well-tempered chocolates, it can occur from transition of β(V) to more stable β(VI)form
chocolates, it can occur from transition of β(V) to more stable β(VI) form[3,28].
[3,28].
Milk fat is present in milk chocolates and consists of 98% TAGs, 2%
Milk fat is present in milk chocolates and consists of 98% TAGs, 2% phosphoglycerides, phosphoglycerides, and traces
and
oftraces
diacylglycerides and sterols
of diacylglycerides and [8]. In milk
sterols [8].chocolates, conjugatedconjugated
In milk chocolates, linoleic acidlinoleic
(CLA) isacidalso(CLA)
detected in
is also
detected in amounts between 0.07 and 0.18%. The origin of CLA in milk chocolate is probably from
milk, where it is recognized as group of positional and geometrical isomers of 18-carbon conjugated
Molecules 2019, 24, 3163 6 of 13

amounts between
Molecules 2019, 0.07PEER
24, x FOR andREVIEW
0.18%. The origin of CLA in milk chocolate is probably from milk, where 6 of it
13
is recognized as group of positional and geometrical isomers of 18-carbon conjugated dienoic acid.
CLA
dienoicis known
acid. CLAfor anticarcinogenic activity [29–31].
is known for anticarcinogenic Milk fat
activity is known
[29–31]. Milkforfat its inhibition
is known for of
itsfat bloom.
inhibition
Itoflowers solidItfatlowers
fat bloom. content andfat
solid slows down
content anddegree
slowsofdown
cocoadegree
butter of
crystallization. Milk fat is β’ stable
cocoa butter crystallization. Milk
fat is β’ stable and delays transition of β(V) to β(VI) form of cocoa butter because of its crystal size,
and delays transition of β(V) to β(VI) form of cocoa butter because of its crystal size, better consistency
better
of consistency
crystals, and smallerof crystals,
distanceand smallerunit
between distance between unit cells [32].
cells [32].

5.5. Polyphenols
Polyphenols
ItItisiswell
wellknown
knownthat
thatcocoa
cocoaand andchocolate
chocolateare
arerich
richininpolyphenols.
polyphenols.Unfermented
Unfermented cocoa
cocoabeans
beans
contain 12–18% polyphenols of the dry weight of beans on average [33]. Cocoa polyphenols
contain 12–18% polyphenols of the dry weight of beans on average [33]. Cocoa polyphenols consist consist of
approximately 37% flavan-3-ols, 4% anthocyanins, and 58% proanthocyanidins.
of approximately 37% flavan-3-ols, 4% anthocyanins, and 58% proanthocyanidins. These compounds These compounds
are
are stored
stored in in polyphenolic
polyphenoliccells
cellsininunfermented
unfermented beans.
beans. In this
In this form,form,
they they
confer confer a white
a white to deeptopurple
deep
purple color to unfermented beans. Catechins make up approximately 29–38% of
color to unfermented beans. Catechins make up approximately 29–38% of total polyphenols. In cocoatotal polyphenols.
In
andcocoa and chocolate,
chocolate, they arethey are represented
represented by (−)-epicatechin,
by (−)-epicatechin, (+)-catechin,
(+)-catechin, (+)-gallocatechin,
(+)-gallocatechin, and (−)-
and (−)-epigallocatechin
epigallocatechin (Figure(Figure 4). (−)-epicatechin
4). (−)-epicatechin makesmakes up up
up up to 35%
to 35% ofoftotal
totalpolyphenols.
polyphenols. They
They are
are
essential for bean flavor and color development
essential for bean flavor and color development [5]. [5].

Figure4.4. Structure
Figure Structureof
ofmain
mainflavan-3-ols
flavan-3-olspresent
presentin
incocoa
cocoaand
andchocolate.
chocolate.

Anthocyanins
Anthocyanins in cocoa are represented
in cocoa by cyanidin-3-α-l-arabinoside
are represented and cyanidin-3-β-d-galactoside
by cyanidin-3-α-L-arabinoside and cyanidin-3-β-[7]. D-
Procyanidins are represented by dimers, trimers, and oligomers of flavan-3,4-diols. Epicatechin
galactoside [7]. Procyanidins are represented by dimers, trimers, and oligomers of flavan-3,4-diols. is the
main subunit,islinked
Epicatechin by 4–8
the main or 4–6 linked
subunit, bonds [5,7].
by 4–8Procyanidins
or 4–6 bonds can[5,7].
existProcyanidins
in A or B form [29,34].
can AllAthese
exist in or B
compounds contribute to astringency and bitterness specific of unfermented cocoa [1].
form [29,34]. All these compounds contribute to astringency and bitterness specific of unfermented
cocoa [1].
5.1. Fermentation
5.1. Fermentation
Polyphenolic cells are destructed during fermentation and polyphenols exude from the cells.
During fermentation,
Polyphenolic cellsaerobic oxidation
are destructed of polyphenols
during fermentation occurs. This reaction
and polyphenols is carried
exude from the outcells.
by
polyphenol oxidase (Figure 5), which is released because of cotyledon changes affected
During fermentation, aerobic oxidation of polyphenols occurs. This reaction is carried out by by fermentation.
The results ofoxidase
polyphenol this process are reduced
(Figure 5), which bitterness and astringency
is released because ofand increased
cotyledon brown color
changes [5,35].
affected by
During oxidation, polyphenols react with proteins and are converted into insoluble
fermentation. The results of this process are reduced bitterness and astringency and increased brown forms [8].
colorCatechin content
[5,35]. During is mainly
oxidation, decreased react
polyphenols during fermentation
with proteins and (more than 90%into
are converted of initial content).
insoluble forms
It[8].
is found that (−)-epicatechin is significantly decreased [7]. During fermentation, procyanidin A2
epimerizes
Catechinto procyanidin F2. F1decreased
content is mainly and F2 procyanidins are specific(more
during fermentation to fermented
than 90%cocoa beans
of initial [35]. It
content).
Hydrolysis
is found of glycosides is(mainly
that (−)-epicatechin anthocyanins),
significantly decreasednonenzymatic oligomerization
[7]. During fermentation, of catechin,
procyanidin A2
and transfer of proanthocyanidins into a more complex form lead to brightening
epimerizes to procyanidin F2. F1 and F2 procyanidins are specific to fermented cocoa beans of the cotyledon.
[35].
Since Hydrolysis
the decreaseofofglycosides
anthocyanin content
(mainly during fermentation
anthocyanins), is significant,
nonenzymatic it is considered
oligomerization as a good
of catechin, and
fermentation index [7,36,37]. into a more complex form lead to brightening of the cotyledon. Since
transfer of proanthocyanidins
the decrease of anthocyanin content during fermentation is significant, it is considered as a good
fermentation index [7,36,37].
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Figure5.5.Oxidation
Oxidation of phenolsbyby polyphenoloxidase.
oxidase.
Figure 5. Oxidationofofphenols
Figure phenols bypolyphenol
polyphenol oxidase.

5.2.Drying
5.2. Drying
5.2. Drying
Drying also
Drying also decreases
decreases the
the content
content of
of polyphenols
polyphenols in in beans.
beans. This
This reduction
reduction isis catalyzed
catalyzed by
by
Drying also decreases the content of polyphenols in beans. This reduction is catalyzed by
polyphenoloxidase,
polyphenol oxidase,after
after which,
which, new new aroma
aroma compounds
compounds arisearise alongside
alongside the formation
the formation of brownof brown
color,
polyphenol oxidase, after which, new aroma compounds arise alongside the formation of brown
color,
and and bitterness
bitterness and astringency
and astringency are reduced
are reduced [7,16]. [7,16].
color, and bitterness and astringency are reduced [7,16].
5.3.
5.3.Roasting
Roasting
5.3. Roasting
As
Aspreviously
previouslymentioned,
mentioned,roasting
roastingisisconducted
conductedatathigh hightemperatures
temperaturesand andthusthussignificantly
significantly
As previously mentioned, roasting is conducted at high temperatures and thus significantly
affects the content and composition of thermolabile molecules such as polyphenols.
affects the content and composition of thermolabile molecules such as polyphenols. Reduction Reduction of totalof
affects the content and composition of thermolabile molecules such as polyphenols. Reduction of
polyphenol content
total polyphenol duringduring
content roasting is observed
roasting in numerous
is observed studiesstudies
in numerous [8,38,39]. Aprotosoaie
[8,38,39]. et al. [7]et
Aprotosoaie
total polyphenol content during roasting is observed in numerous studies [8,38,39]. Aprotosoaie et
observed that the decrease of high molecular weight proanthocyanidins is more
al. [7] observed that the decrease of high molecular weight proanthocyanidins is more pronounced. pronounced. During
al. [7] observed that the decrease of high molecular weight proanthocyanidins is more pronounced.
roasting, the ratiothe
During roasting, of epicatechin/catechin is also changing.
ratio of epicatechin/catechin Ioannone
is also changing. et al. [40]
Ioannone et al.concluded that the
[40] concluded that
During roasting, the ratio of epicatechin/catechin is also changing. Ioannone et al. [40] concluded that
epicatechin/catechin ratio changed during roasting and that the content of
the epicatechin/catechin ratio changed during roasting and that the content of (−)-epicatechin (−)-epicatechin decreased.
the epicatechin/catechin ratio changed during roasting and that the content of (−)-epicatechin
Stanley
decreased.et al.Stanley
[41] also
et observed a decrease
al. [41] also observedofa(−)-epicatechin but they found
decrease of (−)-epicatechin butthat
theyit found
is subjected
that ittois
decreased. Stanley et al. [41] also observed a decrease of (−)-epicatechin but they found that it is
epimerization and that the content
subjected to epimerization and that of the
(−)-catechin
content of was increased was
(−)-catechin (Figure 6). Also,
increased high temperatures
(Figure 6). Also, high
subjected to epimerization and that the content of (−)-catechin was increased (Figure 6). Also, high
can induce (+)-catechin epimerization to (+)-epicatechin [42]. The
temperatures can induce (+)-catechin epimerization to (+)-epicatechin [42]. The content content of procyanidin B2 isof
temperatures can induce (+)-catechin epimerization to (+)-epicatechin [42]. The content of
also
procyanidin B2 is also decreased during roasting, while procyanidin B1 is increased also
decreased during roasting, while procyanidin B1 is increased [39]. Proanthocyanidins are [39].
procyanidin B2 is also decreased during roasting, while procyanidin B1 is increased [39].
subjected to epimerization,
Proanthocyanidins are alsososubjected
it is notedtothat after roasting,
epimerization, so the
it iscontent
noted thatof large
afterproanthocyanidins
roasting, the content is
Proanthocyanidins are also subjected to epimerization, so it is noted that after roasting, the content
increased [38,41]. During roasting, a decrease of (−)-epicatechin and procyanidin
of large proanthocyanidins is increased [38,41]. During roasting, a decrease of (−)-epicatechin and B2 and an increase of
of large proanthocyanidins is increased [38,41]. During roasting, a decrease of (−)-epicatechin and
(+)-catechin
procyanidinand procyanidin
B2 and B1 was
an increase observed [39,41].
of (+)-catechin and procyanidin B1 was observed [39,41].
procyanidin B2 and an increase of (+)-catechin and procyanidin B1 was observed [39,41].

Figure6.6.Epimerization
Figure Epimerizationofof(−)-epicatechin
(−)-epicatechintoto(−)-catechin.
(−)-catechin.
Figure 6. Epimerization of (−)-epicatechin to (−)-catechin.
5.4. Conching
5.4. Conching
5.4. Conching
In the initial stage of conching, volatile polyphenols are lost due to evaporation, together with
In the initial stage of conching, volatile polyphenols are lost due to evaporation, together with
water Inandtheshort-chain
initial stagefatty
of conching,
acids. It volatile
has beenpolyphenols
establishedarethatlost
thedue to evaporation,
content together with
of volatile polyphenols
water and short-chain fatty acids. It has been established that the content of volatile polyphenols is
is reduced by 80% in this process [5]. During conching, oxidation of tannins occurs as well as coloris
water and short-chain fatty acids. It has been established that the content of volatile polyphenols
reduced by 80% in this process [5]. During conching, oxidation of tannins occurs as well as color
reduced
change by Some
[20]. 80% instudies
this process
showed [5].a During
decrease conching, oxidation
of procyanidin B2 of tannins
during occurs as
conching, butwell as was
there color
change [20]. Some studies showed a decrease of procyanidin B2 during conching, but there was an
change
an [20].
increase of Some studies
epicatechin andshowed a decrease
catechin of procyanidin
content [34]. Also, Dimick B2 et
during
al. [43]conching,
concluded but there
that was an
oxidation
increase of epicatechin and catechin content [34]. Also, Dimick et al. [43] concluded that oxidation of
of tannins and some enzymatic mechanisms (during tanning process) influence polyphenol contentof
increase of epicatechin and catechin content [34]. Also, Dimick et al. [43] concluded that oxidation
tannins and some enzymatic mechanisms (during tanning process) influence polyphenol content
tannins
after and some
conching. enzymatic
Complexes mechanisms
between (duringamino
polyphenols, tanning process)
acids, influence
peptides, polyphenol
and proteins content
are formed
after conching. Complexes between polyphenols, amino acids, peptides, and proteins are formed
after conching. Complexes between polyphenols, amino acids, peptides, and
during this process. This is one of the reasons why conching affects chocolate flavor and reduces proteins are formed
during this process. This is one of the reasons why conching affects chocolate flavor and reduces
during this process. This is one of the reasons why conching affects chocolate flavor
astringency [5]. However, Becket et al. [3] state that reactions during conching are not extensive and and reduces
astringency [5]. However, Becket et al. [3] state that reactions during conching are not extensive and
asastringency [5].redistribution
However, Becket et al. compounds
[3] state that reactionschocolate
during conching are not extensive and
asimportant
importantas as redistributionofofaroma
aroma compoundsbetween between chocolateparticles—in
particles—inthe theinitial
initialstage
stage
as important as redistribution of aroma compounds between chocolate particles—in the initial stage
(dry phase), aromatic compounds are contained in cocoa particles; however, mixing and shearing
(dry phase), aromatic compounds are contained in cocoa particles; however, mixing and shearing
redistributes them between sugar, cocoa, and fat, making sugar an aroma-carrier as well.
redistributes them between sugar, cocoa, and fat, making sugar an aroma-carrier as well.
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Molecules 2019, 24, x FOR PEER REVIEW 8 of 13
(dry phase), aromatic compounds are contained in cocoa particles; however, mixing and shearing
6. Other Components
redistributes them between sugar, cocoa, and fat, making sugar an aroma-carrier as well.
6. Other Components
6.6.1.
Other Components
Methylxanthines
6.1. Methylxanthines
Methylxanthines are alkaloids (theobromine and caffeine) that have stimulatory effects on the
6.1. Methylxanthines
Methylxanthines are alkaloids (theobromine and caffeine) that have stimulatory effects on the
central nervous system. Cocoa is the main source of theobromine but has a lower content of caffeine
central nervous system. areCocoa is the(theobromine
main source of theobromine buthave
has astimulatory
lower content of caffeine
thenMethylxanthines
coffee and tea. The alkaloids
reported and
values for theobromine caffeine)
and that
caffeine effects
content in nonfat cocoaon bean
the
then
centralcoffee and
nervous tea. The
system. reported
Cocoa is values
the main for theobromine
source of and
theobromine caffeine
but content
has a lowerin nonfat
content cocoa
of bean
caffeine
solids are about 4% and 0.2% of dry weight, respectively [3,33]. Contents can be influenced by the
solids
then are about
coffee 4%The
andprocess
tea. andreported
0.2% of dry weight,theobromine
values respectively [3,33]. Contents can benonfat
influenced
cocoaby the
fermentation and depend on for
bean type [1,44].and caffeine
Unfermented content
cocoainbeans often bean
contain
fermentation
solids process
are about 4% and and depend on
0.2% of During bean
dry weight, type [1,44].
respectively Unfermented
[3,33]. cocoa beans often contain
theobromine bound to tannins. fermentation, acetic acid Contents
hydrolyzescantheobromine–tannin
be influenced by
theobromine
the fermentationbound to tannins.
process and During
depend on fermentation,
bean type acetic
[1,44]. acid hydrolyzes
Unfermented cocoa theobromine–tannin
beans often contain
bonds and part of theobromine is released. Free theobromine then diffuses into the cocoa shell
bonds and
theobromine part
boundof theobromine
to tannins. is
During released. Free
fermentation, theobromine
acetic acid then
hydrolyzesdiffuses into the
theobromine–tannincocoa shell
bonds
[15,45,46].
[15,45,46].
and part of theobromine is released. Free theobromine then diffuses into the cocoa shell [15,45,46].
6.2. Aldehydes
6.2. Aldehydes
6.2. Aldehydes
Fermentation and drying of cocoa beans produce low concentrations of aldehydes. In further
Fermentation and
Fermentation and drying
drying ofof cocoa
cocoa beans
beans produce
produce low lowconcentrations
concentrations of ofaldehydes.
aldehydes. In
In further
further
processes of chocolate production, they are important not only for flavor development, but also for
processes of
processes of chocolate
chocolate production,
production, they
they are
are important
important notnot only
only for
for flavor
flavor development,
development, but
but also
also for
for
further reactions and formation of pyrazines [7,47].
further reactions and formation of pyrazines
further reactions and formation of pyrazines [7,47]. [7,47].
Three aldehydes present in chocolate have strong chocolate aroma: 2-methylpropanal, 2-
Three aldehydes
Three aldehydes present in in chocolate
chocolate have
have strong
strong chocolate
chocolatearoma:
aroma:2-methylpropanal,
2-methylpropanal, 2-
methylbutanal, and 3-methylbutanal [48]. They are produced during cocoa bean roasting from amino
methylbutanal, and
2-methylbutanal, and3-methylbutanal
3-methylbutanal[48]. [48].They
Theyareareproduced
produced during
during cocoa
cocoa bean roasting from
from amino
amino
acids, which determine their structure. In chocolate, aldehydes formed by Strecker degradation are
acids, which determine
acids, determine theirtheir structure.
structure.InInchocolate,
chocolate, aldehydes
aldehydes formed
formed by by
Strecker degradation
Strecker degradation are
also present [5]. Counet et al. [20] reported these were: 2-methylpropanal (derived from valine), 3-
alsoalso
are present [5]. Counet
present et al. [20]
[5]. Counet et al.reported these were:
[20] reported these2-methylpropanal
were: 2-methylpropanal(derived from valine),
(derived from3-
methylbutanal (from leucine), 2-methylbutanal (from isoleucine), phenylacetaldehyde (from
methylbutanal
valine), (from leucine),
3-methylbutanal 2-methylbutanal
(from leucine), (from isoleucine),
2-methylbutanal phenylacetaldehyde
(from isoleucine), (from
phenylacetaldehyde
phenylalanine), and 3-(methylthio)propionaldehyde (from methionine).
phenylalanine),
(from and 3-(methylthio)propionaldehyde
phenylalanine), and 3-(methylthio)propionaldehyde (from(from
methionine).
methionine).
During conching, some aldehydes are lost due to chemical reactions and evaporation (e.g.,
During conching,
During conching, some somealdehydes
aldehydesare arelost duedue
lost to chemical
to chemical reactions and and
reactions evaporation (e.g.,
evaporation
concentration of 2-methylpropanal, 2-methylbutanal, and 3-methylbutanal is reduced) and other
concentration of 2-methylpropanal, 2-methylbutanal, and 3-methylbutanal
(e.g., concentration of 2-methylpropanal, 2-methylbutanal, and 3-methylbutanal is reduced) and is reduced) and other
aldehydes increase through aldol condensation (e.g., 2-phenyl-5-methyl-2-hexenal (Figure 7)
aldehydes
other increase
aldehydes through
increase throughaldol condensation
aldol condensation (e.g.,
(e.g.,2-phenyl-5-methyl-2-hexenal
2-phenyl-5-methyl-2-hexenal(Figure (Figure 7)
formation from phenylacetaldehyde and 3-methylbutanal followed by dehydration) [5,20].
formation from
formation fromphenylacetaldehyde
phenylacetaldehydeand and3-methylbutanal
3-methylbutanalfollowed
followedby bydehydration)
dehydration)[5,20].
[5,20].

Figure 7. Structure of 2-phenyl-5methyl-2-hexenal.

7. Structure
Figure 7.
Figure Structure of
of 2-phenyl-5methyl-2-hexenal.
2-phenyl-5methyl-2-hexenal.

6.3.Esters
6.3. Esters
6.3. Esters
Estersare
Esters arecompounds
compoundspresent
presentininunfermented
unfermentedand androasted
roastedbeans,
beans,but
butcan
canalso
alsoresult
resultfrom
from
Esters are compounds present in unfermented and roasted beans, but can also result from
fermentation process (yeast metabolism). One of compounds formed during
fermentation process (yeast metabolism). One of compounds formed during fermentation is ethylfermentation is ethyl
fermentation process (yeast metabolism). One of compounds formed during fermentation is ethyl
acetate,which
acetate, which
is aisproduct
a product of esterification
of esterification from from
ethanolethanol and acid
and acetic acetic acid 8).
(Figure (Figure
Other8). Other
esters esters
present
acetate, which is a product of esterification from ethanol and acetic acid (Figure 8). Other esters
present
during during production
production are isobutylare isobutyl
acetate, acetate,
isoamyl isoamyl
acetate, acetate,
phenylethyl phenylethyl
acetate, acetate, methyl
methyl isopentanoate,
present during production are isobutyl acetate, isoamyl acetate, phenylethyl acetate, methyl
isopentanoate,
and and methyl
methyl isovalerate [7,49].isovalerate [7,49].
isopentanoate, and methyl isovalerate [7,49].

Figure8.8.Production
Figure Productionofofethyl
ethylacetate
acetateduring
duringfermentation.
fermentation.
Figure 8. Production of ethyl acetate during fermentation.
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Theaerobic
The aerobicphase
phaseofoffermentation
fermentationisisthe
themost
mostimportant
importantfor
forsynthesis
synthesisofofesters
estersfrom
fromalcohols.
alcohols.
Estersare
Esters areone
oneofofthethemost
mostimportant
importantvolatile
volatilecompounds
compoundsbecause
becausethey
theyare
areassociated
associatedwith
withfruity
fruity
flavors [49,50]; however, in chocolate they have secondary role in aroma development,
flavors [49,50]; however, in chocolate they have secondary role in aroma development, after aldehydes after
aldehydes
and pyrazinesand pyrazines [3].
[3].

6.4.
6.4.Ketones
Ketones
Ketones
Ketones present
present in chocolate are
in chocolate are 2-heptanone,
2-heptanone,2-pentanone,
2-pentanone, 2-nonanone,
2-nonanone, acetophenone,
acetophenone, and
and acetoin.
acetoin. These
These compounds
compounds areare favorablefor
favorable forcocoa
cocoaflavor
flavorandand quality.
quality. Ketones
Ketones are
areproduced
producedduring
during
fermentation,
fermentation,and andRodriguez-Campos
Rodriguez-Camposetetal.al.[47]
[47]reported
reportedthat
thatthe
thelonger
longerthe
thefermentation,
fermentation,the
thehigher
higher
the
thecontent
contentofofketones.
ketones.Acetoin
Acetoinisisthe
thedominant
dominantketone
ketoneininfermented,
fermented,dried,
dried,roasted
roastedbeans
beansand
andcocoa
cocoa
liquor and it is produced by alcoholic fermentation [7,51,52].
liquor and it is produced by alcoholic fermentation [7,51,52].

6.5.
6.5.Pyrazines
Pyrazines
Pyrazines
Pyrazinesare areheterocyclic
heterocyclic volatile compoundsthat
volatile compounds thatgive
giveimportant
important cocoa
cocoa flavor
flavor andand aroma,
aroma, and
and were recognized in 1967 when seven alkyl-substituted pyrazines where
were recognized in 1967 when seven alkyl-substituted pyrazines where identified in chocolate aroma: identified in
chocolate aroma: methylpyrazine;
methylpyrazine; 2,3-dimethylpyrazine;
2,3-dimethylpyrazine; 2-ethyl-5-methylpyrazine;
2-ethyl-5-methylpyrazine; trimethylpyrazine;
trimethylpyrazine; 2,5-dimethyl-
2,5-dimethyl-3-ethylpyrazine; 2,6-dimethyl-3-ethylpyrazine; and
3-ethylpyrazine; 2,6-dimethyl-3-ethylpyrazine; and tetramethylpyrazine [53]. tetramethylpyrazine [53].
Tetramethylpyrazine
Tetramethylpyrazineand andtrimethylpyrazine
trimethylpyrazineare arethe
themost
mostimportant
importantfor fordevelopment
developmentofofnutty nutty
and grassy flavor. Most pyrazines are produced during roasting by Maillard reactions
and grassy flavor. Most pyrazines are produced during roasting by Maillard reactions and Strecker and Strecker
degradation
degradation(Figure
(Figure9).9).The
Thecontent
contentofofthese
thesecompounds
compoundsdependsdependson onorigin
originofofthe
thecocoa
cocoabeans
beansand and
the
theduration
durationand andtemperature
temperatureof of roasting. It is
roasting. It is known
known thatthat Ghanaian
Ghanaianbeans beanshave
havehigher
highercontent
contentof
ofpyrazines
pyrazinesafterafterroasting,
roasting,mostly
mostlybecause
because of
of longer
longer fermentation
fermentation [3].[3]. The
The content
content andandratio
ratioofofthe
the
above-mentioned
above-mentioned pyrazines can be good indicators of roasting degree [7,47] becauseincreased
pyrazines can be good indicators of roasting degree [7,47] because applying applying
time and temperature
increased improves production
time and temperature of pyrazines
improves production of[3]. There are
pyrazines [3].two more
There are ways of pyrazine
two more ways of
pyrazine production: Maillard reactions during drying of beans and as a metabolic product ofsubtilis
production: Maillard reactions during drying of beans and as a metabolic product of Bacillus Bacillus
during
subtilisfermentation [7,54]. [7,54].
during fermentation

Figure9.9.Production
Figure Productionofofpyrazine.
pyrazine.

6.6.
6.6.Acids
Acids
Acids
Acidsarearefermentation
fermentationproducts
productswhich
whichare areproduced
producedmainly
mainlyfrom
fromsugars.
sugars.Predominantly,
Predominantly,acetic
acetic
and
andlactic acids
lactic are are
acids formed. TheyThey
formed. reduce the pHthe
reduce to approximately 4.5–5.5, enabling
pH to approximately 4.5–5.5, enzymatic reactions
enabling enzymatic
necessary
reactions in this stepinofthis
necessary cocoa
stepbean processing.
of cocoa SubsequentSubsequent
bean processing. fermentation, drying, and
fermentation, roasting
drying, and
significantly
roasting significantly reduce the content of volatile acids, which is one of prerequisites of
reduce the content of volatile acids, which is one of prerequisites for development for
the pleasant aroma of the final products [3]. If cocoa beans are dried too quickly,
development of the pleasant aroma of the final products [3]. If cocoa beans are dried too quickly, volatile acids are
trapped
volatilewithin
acids arethetrapped
bean, which
withinisthenot desirable,
bean, which since
is not high acid content
desirable, since highleads
acidtocontent
off-flavors
leadscocoa
to off-
and
flavors cocoa and chocolate [7,55]. Low content can be connected to loss of proper flavor ofNormally,
chocolate [7,55]. Low content can be connected to loss of proper flavor of chocolate. chocolate.
milk chocolate
Normally, milk has lower acid
chocolate hascontent thancontent
lower acid dark chocolate
than dark[56].
chocolate [56].

6.7. Alcohols
Alcohols are present in cocoa and chocolate after fermentation of beans. They are produced
mainly by yeasts but some can be formed by combined activity of bacteria and yeast. 2-
Molecules 2019, 24, 3163 10 of 13

6.7. Alcohols
Alcohols are present in cocoa and chocolate after fermentation of beans. They are produced mainly
by yeasts but some can be formed by combined activity of bacteria and yeast. 2-phenethylethanol
and 2,3-butanediol are dominant during all fermentation stages [49,56]. Alcohols are involved in
esterification reactions, predominantly with acetic acid, to produce acetates [3].

7. Conclusions
The chemistry behind chocolate production is very complex due to large number of involved
compounds and processes. The most intensive changes occur during fermentation and roasting of
cocoa bean, and during conching of chocolate. These processes must be carefully controlled due to
the influence of chemical reactions and their products on chocolate’s flavor and aroma. The main
flavor compounds in chocolate are polyphenols, present in raw cocoa bean and going through various
forms during production, and pyrazines formed during production, followed by aldehydes, ketones,
and esters. Some mechanisms of reactions during cocoa bean and chocolate processing have been
revealed and are well-established; however, as new analytical techniques emerge, new compounds
are being identified (e.g., seyllo-inositol, 1-kestose and 6-kestose, cacaoic acid) and their involvement
in reactions during cocoa bean and chocolate processing and their influence on overall chocolate
properties (flavor, aroma, color) are yet to be revealed. Furthermore, as analytical instruments are more
and more sensitive in detection and quantification of already identified compounds, new knowledge is
expected in this area as well. All this knowledge will contribute not only to science, but to cocoa bean
and chocolate producers as well, who will be able to select varieties and target processes to increase
contents of desirable compounds and decrease contents of less desirable ones, as well as to optimize
ratios of different compounds, which is important for overall quality.

Author Contributions: All authors contributed to the conceptualization and implementation of the research
study; Writing—original draft preparation, V.B., M.K., I.F., Đ.A. and S.J.; Writing—review and editing, A.J., B.M.,
D.Š. and J.B.
Funding: This work has been supported in part by Croatian Science Foundation under the project “Application of
cocoa husk in production of chocolate and chocolate-like products” (UIP 2017-05-8709).
Conflicts of Interest: The authors declare no conflict of interest.

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