The Chemistry Behind Chocolate Production

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Review The Chemistry behind Chocolate Production

Veronika Barišić , Mirela Kopjar , Antun Jozinović , Ivana Flanjak , Đurđica Aˇckar* , Borislav Miliˇcević,Drago Šubarić,Stela Jokić and Jurislav Babić

Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek, Franje Kuhaˇca20, 31000 Osijek, Croatia * Correspondence: [email protected]

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 (Figure1) 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 Molecules 2019, 24, 3163 3 of 13

Molecules 2019, 24, x FOR PEER REVIEW 3 of 13 from tryptophan is spontaneous, or whether endogenous enzymes or enzymes of microﬂora during fermentationinvolved. Since are involved.biogenic amines Since biogenic are known amines to be are th knowne most abundant to be the most in fermented abundant foods, in fermented it is most foods,likely it that is most fermentation likely that of fermentation cocoa beans of and cocoa microflo beansra and involved microﬂora in the involved process in are the the process most arerelevant the mostin tryptamine relevant in synthesis. tryptamine synthesis.

FigureFigure 1. 1.Possible Possible transformation transformation of of tryptophan tryptophan during during fermentation fermentation of of cocoa cocoa beans. beans.

HydrophobicHydrophobic oligopeptides oligopeptides that that are are formed formed from from hydrophobic hydrophobi aminoc amino acids acids mentioned mentioned above above becomebecome substrates substrates for for serine serine carboxy-(exo) carboxy-(exo) peptidase. peptidase. Hydrophilic Hydrophili oligopeptidesc oligopeptides and and hydrophobic hydrophobic freefree amino amino acids acids (chocolate(chocolate aromaaroma precursors) precursors) are are obta obtainedined by by the the activity activity of carboxypeptidase of carboxypeptidase from fromhydrophobic hydrophobic oligopeptides oligopeptides [16]. [16 ].During During fermen fermentation,tation, precursor precursor compoundscompounds derivedderived fromfrom free free aminoamino acids acids are are released. released. 3-methylbutanol,3-methylbutanol, phenylacetaldehyde, 2-methyl-3-(methyldithio)furan, 2-methyl-3-(methyldithio)furan, 2- 2-ethyl-3,5-dimethyl,ethyl-3,5-dimethyl, and 2,3-diethyl-5-methylpyrazine2,3-diethyl-5-methylpyrazine are amongamong thethe mostmost importantimportant [5[5].]. Also, Also, decarboxylationdecarboxylation of of amino amino acidsacids duringduring fermentationfermentation results in in formation formation of of biogenic biogenic amines amines (2- (2-phenylethylamine,phenylethylamine, 2-methylpropylamine, andand 2-2- andand 3-methylbutylamine). 3-methylbutylamine). These These components components are are alreadyalready present present in in unfermented unfermented beans, beans, but but during during fermentation, fermentation, decarboxylases decarboxylases lead lead to an to increase an increase in thein content the content of bioactive of bioactive amines amines [8,10 [8,10].].

2.2.2.2. Roasting Roasting SinceSince roasting roasting is is conducted conducted at at high high temperatures, temperatures, precursors, precursors, which which were were obtained obtained by by protein protein degradationdegradation during during fermentation, fermentation, are are subjected subjected to to chemical chemical reactions. reactions. The The most most important important is is the the MaillardMaillard reaction reaction between between thethe ca carbonylrbonyl group group of of reducing reducing sugars sugars and and the theamino amino group group from fromamino aminoacids acidsor proteins. or proteins. Through Through these reactions, these reactions, cocoa obtains cocoa obtainsits chocolate its chocolate specific specificaroma and aroma flavor and [4]. flavorThe [4condensation]. The condensation product product that results that results from from these these reactions reactions is is transformed byby Amadori Amadori rearrangementrearrangement to to 1-deoxy-2-ketosyl 1-deoxy-2-ketosyl [2 ,[2,4].4]. Roasting Roasting is is more more eff effectiveective in in the the generation generation of of amines amines and and aldehydesaldehydes than than fermentation. fermentation. Strecker-type Strecker-type reactions reactions form form aldehydes aldehydes in roasted in roasted beans frombeans amino from acidsamino (Figureacids 2(Figure)[10,17] 2). In [10,17]. addition, In Oraczaddition, and NebesnyOracz and [18 Nebesny] reported [18] that reported at higher roastingthat at higher temperatures roasting (135temperatures and 150 ◦C), (135 Maillard and 150 reactions °C), Maillard advance reactions to produce advance melanoidins, to produce high-molecular melanoidins, weight high-molecular (HMW) compoundsweight (HMW) that confer compounds the specific that confer brown the color specific and texture brown to color cocoa and beans. texture They to distinguishcocoa beans. three They typesdistinguish of melanoidins: three types “(1) of melanoidins: polymers consisting “(1) polymers of repeating consisting furans of repeating or pyrrole furans units or pyrrole linked viaunits polycondensationlinked via polycondensation reactions; (2) carbohydrate reactions; skeletons(2) carbohydrate built mainly skeletons from sugar built degradation mainly from products sugar polymerizeddegradation through products aldol-type polymerized condensation through reactionsaldol-type and condensation possibly linked reactions by nitrogenous and possibly residues; linked andby (3)nitrogenous protein-like residues; structures and arising(3) protein-like from crosslinks structures between arising proteinsfrom crosslinks and low-molecular between proteins weight and (LMW)low-molecular compounds weight formed (LMW) in the compounds advanced formed stages ofin Maillardthe advanced reactions”. stages Theyof Maillard also imply reactions”. that, sinceThey the also carbohydrate imply that, content since the in cocoacarbohydrate beans is conten relativelyt in lowcocoa (unlike beans co isff eerelatively beans, for low example), (unlike coffee it is morebeans, likely for thatexample), products it is of more lipid like oxidation,ly that products rather than of lipid sugars, oxidation, are involved rather in than reactions sugars, with are proteins involved toin form reactions HMW with melanoidins. proteins to However, form HMW this linkmelanoidins. is not well However, established this by link the is results not well of their established research by andthe should results be of furthertheir research investigated. and should be further investigated.

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FigureFigure 2. Strecker-typeStrecker-type reaction reaction for formation of al aldehydesdehydes during roasting of cocoa beans.

2.3. Other Other Processes Processes Casein is one of the most important proteins in milk. It It can can be be present present in in milk milk chocolate chocolate and form an interface between solid and and fatty components, components, mean meaninging that that casein casein can can act act as as an an emulsifier emulsifier [2,3]. [2,3]. During conching,conching, caramelization caramelization of of lactose lactose in milkin milk chocolates chocolates and Maillard and Maillard reactions reactions with milk with proteins milk proteinsoccur [19 occur]. Conching [19]. Conching does not does change not the change amino the acid amino content acidof content chocolate of chocolate [20]. [20]. 3. Carbohydrates 3. Carbohydrates 3.1. Fermentation 3.1. Fermentation Unfermented cocoa beans contain 2–4% of low molecular weight carbohydrates and their derivatives,Unfermented mostly cocoa sucrose beans (90% contain of total 2–4% sugars), of lo glucose,w molecular fructose, weight galactose, carbohydrates sorbose, arabinose,and their derivatives,xylose, mannitol, mostly and sucrose inositol. (90% Polysaccharides of total sugars), (starch glucose, (4–6%), fructose, pectins, galactos cellulosee, (2–3%)sorbose, etc.) arabinose, are also xylose,present mannitol, in beans at and a proportion inositol. Polysaccharides of approximately (starch 12%. (4–6%), Redgwell pectins, and Hansencellulose [21 (2–3%)] reported etc.) thatare also cell presentwall polysaccharides in beans at a proportion (CWP) include of approximately cellulose (28% 12%. of Redgwell CWP), pectic and Hansen polysaccharides [21] reported (containing that cell wallgalactose polysaccharides as the predominant (CWP) include side-chain cellulose sugar (28% (9% of of CWP), CWP) pectic and containingpolysaccharides arabinose (containing as the galactosepredominant as side-chainthe predominant sugar (52% side-chain of CWP)), sugar hemicellulosic (9% of CWP) polysaccharides and containing (xyloglucan arabinose (8% of as CWP), the predominantand gallactoglucomannan side-chain sugar (3% of (52% CWP)). of CWP)), They also hemicellulosic reported that fermentationpolysaccharides does (xyloglucan not influence (8% CWP. of CWP),Sucrose and is the gallactoglucomannan main subject of reactions (3% duringof CWP)). fermentation. They also Sugars reported from that pulp fermentation are converted does to acids not influenceduring fermentation. CWP. Sucrose These is the acids main move subject into the of beansreactions and during lower their fermentation. pH, which Sugars leads to from breakdown pulp are of convertedstorage cells. to acids There during are two fermentation. types of storage These cells acids in the move cotyledon: into the polyphenolic beans and lower cells, their which pH, consist which of leadsa large to vacuole breakdown that is filledof storage with polyphenols cells. There and are alkaloids; two types and of lipid-protein storage cells cells, in which the havecotyledon: small polyphenoliclipid and protein cells, vacuoles which consist tightly of packed a large insidevacuole the that cytoplasm. is filled with Yeasts polyphenols present during and alkaloids; fermentation and lipid-proteinconvert the present cells, which sugars have to alcohol, small lipid which and benefits protein developmentvacuoles tightly of lacticpacked acid inside bacteria the cytoplasm. [7,8,12,22]. YeastsThe alcohol present is converted during tofermentation acetic acid under convert aerobic the conditions present andsugars the activityto alcohol, of acetic which acid bacteria.benefits developmentSucrose isof hydrolyzed lactic acid bacteria into fructose [7,8,12,22]. and glucose The alcohol (reducing is converted sugars) dueto acetic to invertase acid under activity aerobic [8]. conditionsMegías-Pé rezand et the al. activity [23] found of acetic seyllo-inositol acid bacteria. (trace—504.9 µg/g in unfermented and 15.5–491.9 µg/g in fermentedSucrose is bean), hydrolyzed 1-kestose into (36.1–133.5 fructose andµg/ gglucose in unfermented (reducing andsugars) traces—115.5 due to invertaseµg/g in activity fermented [8]. Megías-Pérezbean), and 6-kestose et al. [23] (no found reported seyllo-inositol values) for (trace—504.9 the first time. μg/g The in contentunfermented of these and compounds 15.5–491.9 μ wasg/g 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 in3.2. the Drying later processes of chocolate production.

3.2. DryingDuring 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 sugarsDuring [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 sugars3.3. Roasting [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 dehydration of cocoa of sugars.beans leads During to caramelization,caramelization of molecular the present degradation sugars. Caramelization of sugars leads occurs 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

Molecules 2019, 24, 3163 5 of 13 Molecules 2019, 24, x FOR PEER REVIEW 5 of 13 developmentdevelopment of of aroma aroma compounds. compounds. Also,Also, duringduring roasting,roasting, dehydrationdehydration ofof sugarssugars can can occur occur which which producesproduces furfural furfural and and its its derivatives derivatives (Figure (Figure3)[ 3)2 ,[2,17].17].

FigureFigure 3. 3.Dehydration Dehydration of of sugars. sugars.

FinishedFinished chocolate chocolate contains contains about about 50% 50% sugar, sugar, which which is is mainly mainly added added sucrose, sucrose, but but it it can can also also be be lactoselactose (in (in milk milk chocolate). chocolate). In In the the last last few few years, years, fructose, fructose, sorbitol, sorbitol, polydextrose, polydextrose, etc. etc. have have been been added added inin chocolates chocolates [ 2[2].]. AfterAfter chocolate chocolate production,production, sugar bloom bloom on on the the chocolate chocolate surface surface may may appear. appear. This This happens happens due dueto high to high humidity humidity when when moisture moisture condenses condenses on on th thee surface surface of of chocolate. Sugar fromfrom chocolatechocolate isis extractedextracted out out on on the the surface surface and and recrystallizes recrystallizes after after the the water water is is removed removed [12 [12].]. Noor-SoNoor-Soffalinaﬀalina et et al. al. [24 [24]] found found that that cocoa cocoa beans bean withs with higher higher polyphenol polyphenol content content have lower have sugarlower content.sugar content. This phenomenon This phenomenon is most likelyis most due likely to binding due to of binding carbohydrates of carbohydrates to polyphenols. to polyphenols. Hydrogen bondingHydrogen of these bonding components of these is created components between is hydroxyl created groups between and throughhydroxyl hydrophilic groups and interactions. through Tanninshydrophilic are more interactions. likely to Tannins bond with are more carbohydrates likely to bond because with they carbohydrates are rich in closelybecause set they hydroxyl are rich groupsin closely [25 ].set hydroxyl groups [25].

4.4. Lipids Lipids CocoaCocoa beans beans containcontain 50–58%50–58% fats,fats, 97–98% of which which are are triacylglycerols triacylglycerols (TAGs). (TAGs). TAGs TAGs consist consist of of24.1–27.1% 24.1–27.1% palmitic palmitic acid, acid, 32.9–37.6% 32.9–37.6% stearic stearic acid, acid, and and 32.7–37.6% 32.7–37.6% oleic oleic acid, acid, and and low low amounts amounts of oflinoleic linoleic acid acid (2.3–3.7%). (2.3–3.7%). Fatty Fatty acid acid 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]. [There8,26]. Thereare differences are differences in cocoa in cocoa butter butter considering considering origin origin and andbean beantype. type. Softer Softer cocoa cocoa butter butter has hasa higher a higher content content of of1-palmitoyl-2-3-dioleoyl-glycerol 1-palmitoyl-2-3-dioleoyl-glycerol (POO) (POO) and and 1- 1-stearoyl-2-3-dioleoyl-glycerolstearoyl-2-3-dioleoyl-glycerol (SOO),(SOO), while while harder harder cocoa cocoa butter butter has has increased increased content content of saturated of saturated fatty acidsfatty[ 26acids]. Sirbu [26]. et Sirbu al. [26 ,et27 ]al. found [26,27] that found only unfermented that only unfermented beans contain beans TAG containingcontain TAG hydroxyl-allyl containing fattyhydroxyl-allyl acid substituent fatty byacid which substituent they can by be which differentiated they can from be differentiated fermented beans. from They fermented named beans. this newly They identifiednamed this component newly cacaoicidentified acid component and stated that cacaoic during acid fermentation, and stated degradation that during or transformation fermentation, ofdegradation this component or transformation may occur. Future of this research component is needed may to occur. further Future explore research this new is component needed to andfurther its fateexplore during this fermentation. new component and its fate during fermentation. FatsFats which which are are rich rich in symmetricalin symmetrical monounsaturated monounsaturated triglycerides, triglycerides, such such as cocoa as cocoa butter, butter, show show a high a degreehigh degree of polymorphism of polymorphism [3]. Regardless[3]. Regardless of theof th TAGe TAG composition, composition, cocoa cocoa butter butter can can exist exist in in six six γ α β β β β polymorphs:polymorphs: γ,, α,, β’(III),’(III), β’(IV),’(IV), β(V),(V), andand β(VI)(VI) whichwhich havehave meltingmelting pointspoints ofof 17.3,17.3, 23.3,23.3, 25.5,25.5, 27.5,27.5, 33.8,33.8, and and 36.3, 36.3, respectively, respectively, and and can can transform transform from from one one to to another, another, depending depending on on temperature temperature and and β time.time. The The most most desirable desirable form form in in chocolate chocolate is is(V) β(V) which which melts melts at 29–31.5at 29–31.5◦C. °C. This This form form is obtained is obtained by aby properly a properly conducted conducted tempering tempering process. process. Fat Fat bloom bloom is is one one of of main main problems problems in in finished finished chocolate. chocolate. β β InIn well-tempered well-tempered chocolates, chocolates, it it can can occur occur from from transition transition of of β(V)(V) to to more more stable stable β(VI)(VI) form form [ 3[3,28].,28]. MilkMilk fat fat is is present present in milkin milk chocolates chocolates and and consists consists of 98% of TAGs,98% TAGs, 2% phosphoglycerides, 2% phosphoglycerides, and traces and oftraces diacylglycerides of diacylglycerides and sterols and [8 ].sterols In milk [8]. chocolates, In milk chocolates, conjugated conjugated linoleic acid linoleic (CLA) isacid also (CLA) detected is also in 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

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amountsMolecules 2019 between, 24, x FOR 0.07 PEER and REVIEW 0.18%. The origin of CLA in milk chocolate is probably from milk, where6 of it13 is recognized as group of positional and geometrical isomers of 18-carbon conjugated dienoic acid. CLAdienoic is known acid. CLA for anticarcinogenicis known for anticarcinogenic activity [29–31 activi]. Milkty fat[29–31]. is known Milk forfat itsis known inhibition for ofits fatinhibition bloom. Itof lowers fat bloom. solid It fat lowers content solid and fat slows content down and degree slows of down cocoa degree butter of crystallization. cocoa butter crystallization. Milk fat is β’ stable Milk andfat is delays β’ stable transition and delays of β(V) transition to β(VI) formof β(V) of cocoato β(VI) butter form because of cocoa of itsbutter crystal because size, betterof its consistencycrystal size, ofbetter crystals, consistency and smaller of crystals, distance and between smaller unit distance cells [between32]. unit cells [32].

5.5. PolyphenolsPolyphenols ItIt isis wellwell knownknown thatthat cocoa cocoa and and chocolate chocolate are are rich rich in in polyphenols. polyphenols. UnfermentedUnfermented cocoacocoa beansbeans containcontain 12–18% 12–18% polyphenols polyphenols of of the the dry dry weight weight of of beans beans on on average average [33 [33].]. Cocoa Cocoa polyphenols polyphenols consist consist of approximatelyof approximately 37% 37% ﬂavan-3-ols, flavan-3-ols, 4% 4% anthocyanins, anthocyanins, and and 58% 58% proanthocyanidins. proanthocyanidins. These These compounds compounds areare storedstored in polyphenolic polyphenolic cells cells in in unfermented unfermented beans. beans. In this In thisform, form, they theyconfer confer a white a whiteto deep to purple deep purplecolor to color unfermented to unfermented beans. Catechins beans. Catechins make up make approximately up approximately 29–38% 29–38%of total polyphenols. of total polyphenols. In cocoa In cocoa and chocolate, they are represented by ( )-epicatechin, (+)-catechin, (+)-gallocatechin, and chocolate, they are represented by (−)-epicatechin,− (+)-catechin, (+)-gallocatechin, and (−)- and ( )-epigallocatechin (Figure4). ( )-epicatechin makes up up to 35% of total polyphenols. They are epigallocatechin− (Figure 4). (−)-epicatechin− makes up up to 35% of total polyphenols. They are essentialessential forfor beanbean ﬂavorflavor and and color color development development [ 5[5].].

FigureFigure 4.4. StructureStructure ofof mainmain ﬂavan-3-olsflavan-3-ols presentpresent inin cocoacocoa andand chocolate.chocolate.

Anthocyaninsincocoaarerepresentedbycyanidin-3-Anthocyanins in cocoa are represented by cyanidin-3-α-l-arabinosideandcyanidin-3-α-L-arabinoside andβ -dcyanidin-3--galactoside[β-7D].- Procyanidinsgalactoside [7]. are Procyanidins represented by are dimers, represented trimers, by and dimers, oligomers trimers, of flavan-3,4-diols.and oligomers of Epicatechin flavan-3,4-diols. is the mainEpicatechin subunit, is linked the main by 4–8 subunit, or 4–6 linked bonds [by5,7 4–8]. Procyanidins or 4–6 bonds can [5,7]. exist Procyanidins in A or B form can [29 exist,34]. in All A these or B compoundsform [29,34]. contribute All these tocompounds astringency contribute and bitterness to astringency specific of and unfermented bitterness cocoaspecific [1 ].of unfermented cocoa [1]. 5.1. Fermentation 5.1. FermentationPolyphenolic cells are destructed during fermentation and polyphenols exude from the cells. DuringPolyphenolic fermentation, cells aerobic are destructed oxidation during of polyphenols fermentation occurs. and polyphenols This reaction exude is carried from the out cells. by polyphenolDuring fermentation, oxidase (Figure aerobic5), which oxidation is released of polyphenols because of cotyledon occurs. changesThis reaction a ffected is bycarried fermentation. out by Thepolyphenol results of oxidase this process (Figure are reduced5), which bitterness is released and astringencybecause of andcotyledon increased changes brown coloraffected [5, 35by]. Duringfermentation. oxidation, The polyphenolsresults of this react process with are proteins reduced and bitterness are converted and astringency into insoluble and forms increased [8]. brown colorCatechin [5,35]. During content oxidation, is mainly polyphenols decreased react during wi fermentationth proteins and (more are converted than 90% into of initialinsoluble content). forms It is found that ( )-epicatechin is significantly decreased [7]. During fermentation, procyanidin A2 [8]. − epimerizesCatechin to procyanidin content is mainly F2. F1 decreased and F2 procyanidins during fermentation are specific (more to fermented than 90% cocoa of initial beans content). [35]. It is foundHydrolysis that (− of)-epicatechin glycosides is (mainly significantly anthocyanins), decreased nonenzymatic [7]. During fermentation, oligomerization procyanidin of catechin, A2 andepimerizes transfer to of procyanidin proanthocyanidins F2. F1 and into F2 a procyani more complexdins are form specific lead to to fermented brightening cocoa of thebeans cotyledon. [35]. SinceHydrolysis the decrease of of glycosides anthocyanin (mainly content anthocyanins), during fermentation nonenzymatic is significant, oligomerization it is considered of catechin, as a good and fermentationtransfer of proanthocyanidins index [7,36,37]. into a more complex form lead to brightening of the cotyledon. Since the decrease of anthocyanin content during fermentation is significant, it is considered as a good fermentation index [7,36,37].

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Figure 5. Oxidation of phenols by polyphenol oxidase. FigureFigure 5. 5.Oxidation Oxidation of of phenols phenols by by polyphenol polyphenol oxidase. oxidase. 5.2.5.2. DryingDrying 5.2. Drying DryingDrying alsoalso decreasesdecreases thethe contentcontent ofof polyphenolspolyphenols inin beans.beans. ThisThis reductionreduction isis catalyzedcatalyzed byby Drying also decreases the content of polyphenols in beans. This reduction is catalyzed by polyphenolpolyphenol oxidase, oxidase, after after which, which, new new aroma aroma compounds compounds arise arise alongside alongside the formation the formation of brown of brown color, polyphenol oxidase, after which, new aroma compounds arise alongside the formation of brown andcolor, bitterness and 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. RoastingRoasting 5.3. Roasting AsAs previouslypreviously mentioned,mentioned, roastingroasting isis conductedconducted atat highhigh temperaturestemperatures andand thusthus signiﬁcantlysignificantly As previously mentioned, roasting is conducted at high temperatures and thus significantly aaffectsﬀects the the content content and and composition composition of thermolabileof thermolabile molecules molecules such such as polyphenols. as polyphenols. Reduction Reduction of total of affects the content and composition of thermolabile molecules such as polyphenols. Reduction of polyphenoltotal polyphenol content content during during roasting roasting is observed is observed in numerous in numerous studies studies [8,38,39 [8,38,39].]. Aprotosoaie Aprotosoaie et al. [7 et] total polyphenol content during roasting is observed in numerous studies [8,38,39]. Aprotosoaie et observedal. [7] observed that the that decrease the decrease of high molecularof high molecular weight proanthocyanidinsweight proanthocyanidins is more pronounced.is more pronounced. During al. [7] observed that the decrease of high molecular weight proanthocyanidins is more pronounced. roasting,During roasting, the ratio the of epicatechinratio of epicatechin/catechin/catechin is also changing.is also changing. Ioannone Ioannone et al. [et40 al.] concluded [40] concluded that thethat During roasting, the ratio of epicatechin/catechin is also changing. Ioannone et al. [40] concluded that epicatechinthe epicatechin/catechin/catechin ratio changedratio changed during duri roastingng roasting and that and the contentthat the of (content)-epicatechin of (−)-epicatechin decreased. the epicatechin/catechin ratio changed during roasting and that the content− of (−)-epicatechin Stanleydecreased. et al. Stanley [41] also et observedal. [41] also a decrease observed of a ( decrease)-epicatechin of (−)-epicatechin but they found but that they it found is subjected that it to is decreased. Stanley et al. [41] also observed a −decrease of (−)-epicatechin but they found that it is epimerizationsubjected to epimerization and that the content and that of the ( )-catechin content of was (−)-catechin increased was (Figure increased6). Also, (Figure high temperatures 6). Also, high subjected to epimerization and that the− content of (−)-catechin was increased (Figure 6). Also, high cantemperatures induce (+ )-catechincan induce epimerization (+)-catechin to epimeriz (+)-epicatechination to [ 42(+)-epicatechin]. The content [42]. of procyanidin The content B2 isof temperatures can induce (+)-catechin epimerization to (+)-epicatechin [42]. The content of alsoprocyanidin decreased B2 during is also roasting, decreased while procyanidinduring roasting, B1 is increasedwhile procyanidin [39]. Proanthocyanidins B1 is increased are also[39]. procyanidin B2 is also decreased during roasting, while procyanidin B1 is increased [39]. subjectedProanthocyanidins to epimerization, are also so subjected it is noted to that epimerization, after roasting, so theit is content noted that of large after proanthocyanidins roasting, the content is Proanthocyanidins are also subjected to epimerization, so it is noted that after roasting, the content increasedof large proanthocyanidins [38,41]. During roasting, is increased a decrease [38,41]. of ( )-epicatechinDuring roasting, and procyanidina decrease of B2 (− and)-epicatechin an increase and of of large proanthocyanidins is increased [38,41].− During roasting, a decrease of (−)-epicatechin and (+procyanidin)-catechin and B2 and procyanidin an increase B1 wasof (+)-catech observedin [ 39and,41 procyanidin]. B1 was observed [39,41]. procyanidin B2 and an increase of (+)-catechin and procyanidin B1 was observed [39,41].

FigureFigure 6. 6.Epimerization Epimerization of of ( (−)-epicatechin)-epicatechin to to ( (−)-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 waterIn and the short-chain initial stage fatty of conching, acids. It volatile has been polyphen establishedols are that lost the due content to evaporation, of volatile polyphenolstogether with water and short-chain fatty acids. It has been established that the content of volatile polyphenols is iswater reduced and byshort-chain 80% in this fatty process acids. [ 5It]. has During been conching, established oxidation that the ofcontent tannins of occursvolatile as polyphenols well as color is reduced by 80% in this process [5]. During conching, oxidation of tannins occurs as well as color changereduced [20 by]. Some80% in studies this process showed [5]. a During decrease conching, of procyanidin oxidation B2 of during tannins conching, occurs as but well there as wascolor change [20]. Some studies showed a decrease of procyanidin B2 during conching, but there was an anchange increase [20]. of Some epicatechin studies and showed catechin a decrease content [of34 ].procyanidin Also, Dimick B2 etduring al. [43 conching,] concluded but that there oxidation was an increase of epicatechin and catechin content [34]. Also, Dimick et al. [43] concluded that oxidation of ofincrease tannins of and epicatechin some enzymatic and catechin mechanisms content (during[34]. Also, tanning Dimick process) et al. [43] influence concluded polyphenol that oxidation content of tannins and some enzymatic mechanisms (during tanning process) influence polyphenol content aftertannins conching. and some Complexes enzymatic between mechanisms polyphenols, (during amino tanning acids, process) peptides, influence and proteins polyphenol are formedcontent after conching. Complexes between polyphenols, amino acids, peptides, and proteins are formed duringafter conching. this process. Complexes This is onebetween of the polyphenols, reasons why amino conching acids, aff peptides,ects chocolate and proteins flavor and are reduces formed during this process. This is one of the reasons why conching affects chocolate flavor and reduces astringencyduring this [5process.]. However, This Becketis one etof al.the [3 reasons] state that why reactions conching during affects conching chocolate are flavor not extensive and reduces and astringency [5]. However, Becket et al. [3] state that reactions during conching are not extensive and asastringency important [5]. as redistribution However, Becket of aroma et al. compounds[3] state that betweenreactions chocolate during conching particles—in are not the extensive initial stage and as important as redistribution of aroma compounds between chocolate particles—in the initial 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 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 redistributes6. Other Components them between sugar, cocoa, and fat, making sugar an aroma-carrier as well. 6.6.1. Other Methylxanthines Components 6.1. Methylxanthines Methylxanthines are alkaloids (theobromine and caffeine) that have stimulatory effects on the 6.1. MethylxanthinesMethylxanthines 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. Cocoa is the main source of theobromine but has a lower content of caffeine thenMethylxanthines coffee and tea. The are reported alkaloids values (theobromine for theo andbromine caffeine) and thatcaffeine have content stimulatory in nonfat effects cocoa on bean the then coffee and tea. The reported values for theobromine and caffeine content in nonfat cocoa bean centralsolids nervousare about system. 4% and Cocoa 0.2% isof the dry main weight, source respectively of theobromine [3,33]. but Contents has a lower can be content influenced of ca ffbyeine the solids are about 4% and 0.2% of dry weight, respectively [3,33]. Contents can be influenced by the thenfermentation coffee and process tea. The and reported depend values on forbean theobromine type [1,44]. and Unfermented caffeine content cocoa in beans nonfat often cocoa contain bean fermentation process and depend on bean type [1,44]. Unfermented cocoa beans often contain solidstheobromine are about bound 4% and to tannins. 0.2% of During dry weight, fermentati respectivelyon, acetic [3 ,33acid]. Contentshydrolyzes can theobromine–tannin be influenced by theobromine bound to tannins. During fermentation, acetic acid hydrolyzes theobromine–tannin thebonds fermentation and part processof theobromine and depend is released. on bean typeFree [theobromine1,44]. Unfermented then diffuses cocoa beansinto the often cocoa contain shell bonds and part of theobromine is released. Free theobromine then diffuses into the cocoa shell theobromine[15,45,46]. bound to tannins. During fermentation, acetic acid hydrolyzes theobromine–tannin bonds and[15,45,46]. part of theobromine is released. Free theobromine then diffuses into the cocoa shell [15,45,46]. 6.2. Aldehydes 6.2.6.2. Aldehydes Fermentation and drying of cocoa beans produce low concentrations of aldehydes. In further FermentationFermentation andand dryingdrying ofof cocoacocoa beansbeans produceproduce lowlow concentrationsconcentrations ofof aldehydes.aldehydes. InIn furtherfurther processes of chocolate production, they are important not only for flavor development, but also for processesprocesses ofof chocolatechocolate production,production, theythey areare importantimportant notnot onlyonly forfor flavorflavor development,development, butbut alsoalso forfor further reactions and formation of pyrazines [7,47]. furtherfurther reactionsreactions andand formationformation ofof pyrazinespyrazines [[7,47].7,47]. Three aldehydes present in chocolate have strong chocolate aroma: 2-methylpropanal, 2- ThreeThree aldehydesaldehydes present in in chocolate chocolate have have strong strong chocolate chocolate aroma: aroma: 2-methylpropanal, 2-methylpropanal, 2- methylbutanal, and 3-methylbutanal [48]. They are produced during cocoa bean roasting from amino 2-methylbutanal,methylbutanal, and and 3-methylbutanal 3-methylbutanal [48]. [48]. They They are are produced produced during during cocoa cocoa bean roasting from aminoamino acids, which determine their structure. In chocolate, aldehydes formed by Strecker degradation are acids,acids, which determine their their structure. structure. In In chocolat chocolate,e, aldehydes aldehydes formed formed by byStrecker Strecker degradation degradation are also present [5]. Counet et al. [20] reported these were: 2-methylpropanal (derived from valine), 3- arealso alsopresent present [5]. Counet [5]. Counet et al. [20] et al. reported [20] reported these were: these 2-methylpropanal were: 2-methylpropanal (derived from (derived valine), from 3- methylbutanal (from leucine), 2-methylbutanal (from isoleucine), phenylacetaldehyde (from valine),methylbutanal 3-methylbutanal (from leucine), (from leucine),2-methylbutanal 2-methylbutanal (from isoleucine), (from isoleucine), phenylacetaldehyde phenylacetaldehyde (from phenylalanine), and 3-(methylthio)propionaldehyde (from methionine). (fromphenylalanine), phenylalanine), and 3-(methylthio)propionaldehyde and 3-(methylthio)propionaldehyde (from (from methionine). methionine). During conching, some aldehydes are lost due to chemical reactions and evaporation (e.g., DuringDuring conching, some some aldehyde aldehydess are are lost lost due due to chemical to chemical reactions reactions and andevaporation evaporation (e.g., concentration of 2-methylpropanal, 2-methylbutanal, and 3-methylbutanal is reduced) and other (e.g.,concentration concentration of 2-methylpropanal, of 2-methylpropanal, 2-methylbutanal, 2-methylbutanal, and 3-methylbutanal and 3-methylbutanal is reduced) is reduced) and other and aldehydes increase through aldol condensation (e.g., 2-phenyl-5-methyl-2-hexenal (Figure 7) otheraldehydes aldehydes increase increase through through aldol aldol condensation condensation (e.g., (e.g., 2-phenyl-5-methy 2-phenyl-5-methyl-2-hexenall-2-hexenal (Figure (Figure 77)) formation from phenylacetaldehyde and 3-methylbutanal followed by dehydration) [5,20]. formationformation fromfrom phenylacetaldehydephenylacetaldehyde andand 3-methylbutanal3-methylbutanal followedfollowed byby dehydration)dehydration) [5[5,20].,20].

Figure 7. Structure of 2-phenyl-5methyl-2-hexenal. FigureFigure 7.7. StructureStructure ofof 2-phenyl-5methyl-2-hexenal.2-phenyl-5methyl-2-hexenal. 6.3.6.3. Esters Esters 6.3. Esters EstersEsters are are compounds compounds present present in in unfermented unfermented and and roasted roasted beans, beans, but but can can also also result result from from Esters are compounds present in unfermented and roasted beans, but can also result from fermentationfermentation process process (yeast (yeast metabolism). metabolism). One One of of compounds compounds formed formed during during fermentation fermentation is is ethyl ethyl fermentation process (yeast metabolism). One of compounds formed during fermentation is ethyl acetate,acetate, which which is ais product a product of esteriﬁcation of esterification from from ethanol ethanol and acetic and acidacetic (Figure acid 8(Figure). Other 8). esters Other present esters acetate, which is a product of esterification from ethanol and acetic acid (Figure 8). Other esters duringpresent production during production are isobutyl are acetate, isobutyl isoamyl acetate, acetate, isoamyl phenylethyl acetate, acetate, phenylethyl methyl isopentanoate,acetate, methyl present during production are isobutyl acetate, isoamyl acetate, phenylethyl acetate, methyl andisopentanoate, methyl isovalerate and methyl [7,49 ].isovalerate [7,49]. isopentanoate, and methyl isovalerate [7,49].

FigureFigure 8. 8.Production Production of of ethyl ethyl acetate acetate during during fermentation. fermentation. Figure 8. Production of ethyl acetate during fermentation.

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TheThe aerobic aerobic phase phase of of fermentation fermentation is is the the most most important important for for synthesis synthesis of of esters esters from from alcohols. alcohols. EstersEsters are are one one of of the the most most important important volatile volatile compounds compounds because because they they are are associated associated with with fruity fruity ﬂavorsflavors [49 [49,50];,50]; however, however, in chocolate in chocolate they havethey secondaryhave secondary role in aroma role in development, aroma development, after aldehydes after andaldehydes pyrazines and [3 pyrazines]. [3].

6.4.6.4. Ketones Ketones KetonesKetones presentpresent inin chocolate are are 2-heptanone, 2-heptanone, 2-pentanone, 2-pentanone, 2-nonano 2-nonanone,ne, acetophenone, acetophenone, and andacetoin. acetoin. These These compounds compounds are are favorable favorable for for cocoa cocoa fl ﬂavoravor and and quality. quality. Ketones areare producedproduced during during fermentation,fermentation, and and Rodriguez-Campos Rodriguez-Campos et et al. al. [47 [47]] reported reported that that the the longer longer the the fermentation, fermentation, the the higher higher thethe content content of of ketones. ketones. Acetoin Acetoin is is the the dominant dominant ketone ketone in in fermented, fermented, dried, dried, roasted roasted beans beans and and cocoa cocoa liquorliquor and and it it is is produced produced by by alcoholic alcoholic fermentation fermentation [7 [7,51,52].,51,52].

6.5.6.5. Pyrazines Pyrazines PyrazinesPyrazines are are heterocyclicheterocyclic volatile compounds compounds that that give give important important cocoa cocoa flavor flavor and and aroma, aroma, and andwere were recognized recognized in 1967 in when 1967 seven when alkyl-substitu seven alkyl-substitutedted pyrazines where pyrazines identified where in chocolate identified aroma: in chocolatemethylpyrazine; aroma: 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;3-ethylpyrazine; 2,6-dimethyl-3-ethylpyrazine; 2,6-dimethyl-3-ethylpyrazine; and tetramethylpyrazine and tetramethylpyrazine [53]. [53]. TetramethylpyrazineTetramethylpyrazine and and trimethylpyrazine trimethylpyrazine are are the the most most important important for for development development of of nutty nutty andand grassy grassy flavor. flavor. Most Most pyrazines pyrazines are are produced produced during during roasting roasting by by Maillard Maillard reactions reactions and and Strecker Strecker degradationdegradation (Figure (Figure9 ).9). The The content content of of these these compounds compounds depends depends on on origin origin of of the the cocoa cocoa beans beans and and thethe duration duration and and temperature temperature ofof roasting.roasting. It It is is known known that that Ghanaian Ghanaian beans beans have have higher higher content content of ofpyrazines pyrazines after after roasting, roasting, mostly mostly because because of of longer fermentation [[3].3]. TheThe contentcontent andand ratioratio of of the the above-mentionedabove-mentioned pyrazines pyrazines can can be good be good indicators indicators of roasting of roasting degree[ 7degree,47] because [7,47] applying because increased applying timeincreased and temperature time and temperature improves production improves production of pyrazines of [pyrazines3]. There are[3]. twoThere more are waystwo more of pyrazine ways of production:pyrazine production: Maillard reactions Maillard duringreactions drying during of beansdrying and of beans as a metabolic and as a metabolic product ofproductBacillus of subtilis Bacillus duringsubtilis fermentation during fermentation [7,54]. [7,54].

FigureFigure 9. 9.Production Production of of pyrazine. pyrazine.

6.6.6.6. Acids Acids AcidsAcids are are fermentation fermentation products products which which are are produced produced mainly mainly from from sugars. sugars. Predominantly, Predominantly, acetic acetic andand lactic lactic acids acids are are formed. formed. They They reduce reduce the pH the to approximatelypH to approximately 4.5–5.5, enabling4.5–5.5, enzymaticenabling enzymatic reactions necessaryreactions innecessary this step in of this cocoa step bean of cocoa processing. bean processing. Subsequent Subsequent fermentation, fermentation, drying, and drying, roasting and significantlyroasting significantly reduce the reduce content the of volatile content acids, of volatile which isacids, one ofwhich prerequisites is one forof developmentprerequisites offor thedevelopment pleasant aroma of the of pleasant the final aroma products of the [3]. final If cocoa prod beansucts [3]. are If dried cocoa too beans quickly, are volatiledried too acids quickly, are trappedvolatile withinacids are the trapped bean, which within is the not bean, desirable, which sinceis not highdesirable, acid contentsince high leads acid to content off-flavors leads cocoa to off- andflavors chocolate cocoa [and7,55 chocolate]. Low content [7,55]. can Low be content connected can be to connected loss of proper to loss flavor of prop of chocolate.er flavor of Normally, chocolate. milkNormally, chocolate milk has chocolate lower acid has content lower acid than content 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-

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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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