Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate

FRIN-05218; No of Pages 11 Food Research International xxx (2014) xxx–xxx

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Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate Michael Crafack a, Hanna Keul a, Carl Emil Eskildsen a, Mikael A. Petersen a, Sofie Saerens b, Andreas Blennow c, Mathias Skovmand-Larsen a, Jan H. Swiegers b, Gert B. Petersen d, Hanne Heimdal d, Dennis S. Nielsen a,⁎ a

Department of Food Science, University of Copenhagen, Denmark Chr. Hansen A/S, Hørsholm, Denmark Department of Plant and Environmental Sciences, University of Copenhagen, Denmark d Toms Confectionary Group A/S, Ballerup, Denmark b c

a r t i c l e

i n f o

Article history: Received 18 December 2013 Received in revised form 13 April 2014 Accepted 16 April 2014 Available online xxxx Keywords: Starter culture Pichia kluyveri Kluyveromyces marxianus Cocoa fermentation technique Volatile aroma compounds Sensory profiling

a b s t r a c t The sensory quality of chocolate is widely determined by the qualitative and quantitative composition of volatile compounds resulting from microbial metabolism during fermentation, and Maillard reactions taking place during drying, roasting and conching. The influence of applying mixed starter cultures on the formation of flavour precursors, composition of volatile aroma compounds and sensory profile was investigated in cocoa inoculated with cultures encompassing a highly aromatic strain of Pichia kluyveri or a pectinolytic strain of Kluyveromyces marxianus, and compared to commercially fermented heap and tray cocoa. Although only minor differences in the concentration of free amino acids and reducing sugars was measured, identification and quantification by dynamic headspace gas chromatography–mass spectrometry (HS/GC–MS) revealed pronounced differences in the composition of volatiles in roasted cocoa liquors and finished chocolates. 19 of the 56 volatile compounds identified in the chocolates were found in significantly higher amounts in the tray fermented sample, whilst significantly higher amounts of 2-methoxyphenol was measured in the two inoculated chocolates. The P. kluyveri inoculated chocolate was characterized by a significantly higher concentration of phenylacetaldehyde and the K. marxianus inoculated chocolate by significantly higher amounts of benzyl alcohol, phenethyl alcohol, benzyl acetate and phenethyl acetate compared to a spontaneously fermented control. Sensory profiling described the heap and tray fermented chocolates as sweet with cocoa and caramel flavours, whilst the inoculated chocolates were characterized as fruity, acid and bitter with berry, yoghurt and balsamic flavours. The choice of fermentation technique had the greatest overall impact on the volatile aroma and sensory profile, but whilst the application of starter cultures did affect the volatile aroma profile, differences were too small to significantly change consumer perception of the chocolates as compared to a spontaneously fermented control. © 2014 Elsevier Ltd. All rights reserved.

Introduction The unique flavour characteristic of chocolate is determined by the genetically inherited flavour potential of the cocoa bean variety, the methods by which the primary processing steps of fermentation and drying are conducted, and to the conditions applied during grinding, roasting and conching in the chocolate manufacture (Afoakwa, Paterson, Fowler, & Ryan, 2008; Owusu, Petersen, & Heimdal, 2011). More than 600 volatile compounds are reported to make up the complex mixture that characterizes chocolate aroma including aldehydes,

⁎ Corresponding author at: Department of Food Science, Section for Food Microbiology, University of Copenhagen, Rolighedsvej 30, 1958 Frederiksberg C, Denmark. Tel.: +45 35333287. E-mail address: [email protected] (D.S. Nielsen).

pyrazines, acids, alcohols, esters, ketones, furans, pyrroles, phenols, terpenes and terpene alcohols (Counet & Callemien, 2002; Ziegleder, 2009). There is consensus in the literature that fermentation is the single most important factor influencing the flavour quality of cocoa and chocolate. A properly conducted fermentation ensures liberation of flavour precursors vital for the formation of aroma compounds, but also imparts a ‘fermentative flavour’ derived from the acids, alcohols, esters and ketones excreted by yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation (Afoakwa et al., 2008; Lima, Almeida, Nout, & Zwietering, 2011; Lopez & Dimick, 1995; Rohan, 1964; Schwan & Wheals, 2004; Thompson, Miller, & Lopez, 2001). Fermentation normally occurs spontaneously through inoculation of the fruit pulp with microorganisms naturally present on the surface of the cocoa pods, hands of workers, tools, fermentation vessels and insects (Jespersen, Nielsen, Hønholt, & Jakobsen, 2005; Ostovar &

http://dx.doi.org/10.1016/j.foodres.2014.04.032 0963-9969/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article as: Crafack, M., et al., Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.04.032

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Keeney, 1973). The initial phase of cocoa fermentation is dominated by yeasts and LAB which thrive in the sugar rich, acidic and anaerobic environment. Yeasts convert pulp sugars into ethanol and excrete pulp degrading enzymes causing liquid known as “sweatings” to drain away, whereby air is allowed to enter the fermenting mass. Simultaneously, citric acid assimilating heterofermentative LAB cause a slight increase in pulp pH, which together with the increased oxygen tension favours growth of aerobic AAB. Ethanol initially formed by the yeasts is oxidized into acetic acid by AAB in an exothermic process causing a temperature increase in the fermenting mass reaching 45 °C or more (Camu et al., 2007; Nielsen et al., 2007; Schwan, Rose, & Board, 1995). Ethanol and acetic acid diffuse into the beans where they, together with the elevated temperature, are responsible for killing the bean germ, cell wall breakdown and liberation of endogenous enzymes important for the hydrolysis of globulin storage proteins into free amino acids, peptides and the inversion of sucrose into glucose and fructose (Biehl, Brunner, Passern, Quesnel, & Adomako, 1985; Hansen, Del Olmo, & Burri, 1998; Quesnel, 1965; Thompson et al., 2001; Voigt, Heinrichs, Voigt, & Biehl, 1994). The formation of hydrophobic free amino acids and hydrophilic peptides is reported to be dependent on the successive action of an aspartic endoprotease and a carboxypeptidase responsible for the specific cleavage of vicilin storage proteins in the cocoa bean cotyledons (Kratzer et al., 2009; Voigt et al., 1994). These enzymatic reactions are highly influenced by the degree of acidification during fermentation, as in vitro studies by Biehl et al. (1985) show that strong cotyledon acidification (pH 4.0–4.5) causes an unspecific proteolysis of all proteins present in the cocoa cotyledon yielding a cocoa with low flavour potential, whereas a moderate acidification (pH 5.0–5.5) causes specific proteolysis of the storage proteins yielding cocoa with a higher flavour potential. The accumulation of free amino acids, peptides and reducing sugars forms the basis of Maillard reactions taking place during subsequent drying, roasting and conching. Maillard reactions include dehydration, cyclization, fragmentation and condensation reactions initiated by the formation of imines from a reaction of free amino groups with carbonyl groups of reducing sugars (Belitz, Grosch, & Schieberle, 2004). Intermediate products of the Maillard reaction include a range of aroma-active compounds such as pyrazines, furans, aldehydes, ketones, pyrroles and aldols, many of which are of prime importance for the aroma profile of cocoa and chocolate. Volatile aromatic aldehydes are predominantly formed by Strecker degradation of the hydrophobic amino acids alanine, valine, leucine, isoleucine and phenylalanine yielding their respective aldehydes acetaldehyde, 2-methylpropanal, 3-methylbutanal, 2methylbutanal and phenylacetaldehyde (Afoakwa et al., 2008; Belitz et al., 2004; Ziegleder, 2009). Furthermore, these aldehydes can be partly converted into their corresponding alcohols, acids and esters. Strecker degradation also yields aminoketones which are regarded as important precursors of the formation of pyrazines (Belitz et al., 2004; Perez-Locas & Yaylayan, 2010), a group of compounds considered essential for chocolate aroma (Counet & Callemien, 2002; Owusu et al., 2011). Whilst many Maillard products impart aromas described as earthy, roasted, baked and nutty, the alcohols and esters formed mainly by yeasts during the initial phase of fermentation are often described as fruity, flowery and sweet, making these compounds very desirable for balancing the heavy roast flavours. Using dynamic headspace GC–MS and GC–olfactometry, Owusu et al. (2011) showed that cocoa fermented using the tray fermentation technique, as described by Allison and Rohan (1958), yielded chocolates with a different aroma profile compared to chocolates produced from conventional heap fermented cocoa. Some of the key aroma compounds distinguishing the tray chocolates from heap chocolates were identified as 1,3/2,3-butanediol and benzyl acetate, both compounds which are reported to be products of microbial fermentation (Janssens, Pooter, Schamp, & Vandamme, 1992; Verstrepen et al., 2003), indicating that secondary metabolites of microbial origin could be important for the distinct aroma profiles of certain cocoas (Aculey et al., 2010).

To investigate the influence of starter cultures and fermentation techniques on the chemical and sensory qualities of cocoa liquors and chocolate, we here report the impact of two defined mixed starter cultures, encompassing a highly aromatic strain of Pichia kluyveri and a pectinolytic strain of Kluyveromyces marxianus, on the formation of flavour precursors, composition of volatile compounds, and on the sensory profile of finished chocolate. Results are compared to commercial heap and tray fermented cocoa. Materials and methods Cocoa samples Five types of Ghanaian Forastero cocoa (mixed hybrid varieties) including three cocoas from an experimental fermentation setup and two commercial heap and tray fermented cocoas obtained from Toms Confectionary Group (Ballerup, Denmark) were analysed. Spontaneous and inoculated experimental fermentations were conducted at the Cocoa Research Institute of Ghana (CRIG) in a small scale tray setup using two defined mixed starter cultures, as described in detail by Crafack et al. (2013). In brief, inoculation cultures were composed of Lactobacillus fermentum L18 and Acetobacter pasteurianus A149, previously isolated from spontaneous Ghanaian cocoa fermentations by Nielsen et al. (2007), in combination with either a highly aromatic commercially available strain of P. kluyveri (Viniflora® FrootZen™, Chr. Hansen A/S, Hørsholm, Denmark) or a pectinolytic K. marxianus strain KM16-6, previously isolated from spontaneous Ghanaian cocoa fermentations by Jespersen et al. (2005). Fresh cocoa beans were either inoculated to bacterial cell densities of 5 × 105 cells/g and yeast cell densities of 1 × 106 cells/g or fermented spontaneously in modified plastic trays, each containing 20 kg of wet beans. Three trays were stacked on top of each other for a total volume of 60 kg per fermentation and the beans were fermented for 120 h before being sun dried for 10 days on bamboo mats. In the following, the two cultures will be referred to as P. kluyveri and K. marxianus although they are mixed cultures encompassing strains of Lb. fermentum and A. pasteurianus as well. Analysis of metabolites and flavour precursors Contents of sugars, organic acids and pH were measured in the dried beans following the protocol described by Crafack et al. (2013). Concentrations of free amino acids were determined by GC–MS in un-roasted defatted cocoa powders to assess the degree of protein hydrolysis. To produce defatted cocoa powder, 500 g of cocoa nibs were ground into cocoa liquor using a melangeur (Spectral 10, Santha Inc., Hickory, NC, USA). Fat was removed from the liquor by repeated extraction in a Soxhlet (Soxtec System HT 1043, Tecator A.B., Hoganas, Sweden) using petroleum ether (bp 40–70 °C, Sigma-Aldrich, St. Louis, MO, USA) as solvent. Samples were extracted twice by boiling for 45 min followed by a 90 min rinsing step. Amino acids were extracted for 3 h at room temperature by mixing 0.5 g of defatted powder with 7.5 ml sodium citrate solution [20 g/l sodium citrate dihydrate (Sigma), pH adjusted to 2.2 with 37% HCl (Sigma)] as described by De Brito et al. (2000). Following 10 min centrifugation at 5000 × g, the supernatant was filtered through a 0.22 μm syringe filter and frozen until further analysis. Samples and standards of pure amino acids were prepared for GC–MS analysis by derivatization with methyl chloroformate (MCF). In brief, 250 μl of sample was mixed with 50 μl internal standard (1 mM norvaline), 200 μl catalyst [32% methanol/8% pyridine/60% water (v/v)] and 25 μl 1% MCF (derivatization agent). When gas development occurred, the amino acids were extracted by the addition of 500 μl 1% (v/v) MCF in chloroform. After vigorous mixing, the phases were separated by centrifugation at 500 rpm for 10 min and the supernatant was used for GC–MS. An injector split ratio of 1:15 was applied and the amino acids were separated on a GC system (G6890N, Agilent Technologies) equipped with a DB-XLB column (15 m × 0.25 mm × 0.25 μm,

Please cite this article as: Crafack, M., et al., Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.04.032

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Agilent Technologies) operating with He as carrier gas at a constant head pressure of 50 kPa resulting in a flow rate of app. 1.8 ml/min. The column oven temperature was increased from 110 °C to 350 °C at a rate of 30 °C/min and a mass spectrometric detector (5973 MSD, Agilent Technologies) used for selected ion monitoring (SIM) of the target ions 88 (glycine), 102 (alanine), 115 (threonine), 128 (proline), 130 (tryptophan), 130 (valine), 142 (lysine), 144 (leucine), 144 (isoleucine), 147 (methionine), 162 (phenylalanine), 174 (glutamate), and 236 (tyrosine). Quantifications were based on calibration curves which were calculated using five points in the standard concentration range (0.1–100 ppm). The analyses were performed in duplicates. Production of cocoa liquors and chocolate Following the protocol of Owusu et al. (2011), chocolates with 66% (w/w) of cocoa solids were produced. The cocoa beans were cracked and de-shelled using pilot scale cracking and winnowing machines (Westrup A/S, Denmark) before being roasted at 106 °C for 60 min in a continuous pilot scale Lehmann roaster (F.B. Lehmann GmbH, Aalen, Germany) according to the roasting conditions employed during commercial production of tray fermented chocolates at Toms Confectionary Group. Cocoa liquor was produced from the cooled roasted nibs in a laboratory melangeur (Spectral 10, Santha Inc., Hickory, NC, USA). 4 kg of chocolate was produced from each sample by mixing 63% cocoa liquor with 37% of sugar (beet root) before refining the mixture on a three roll refiner (WDL/H300, F.B. Lehmann GmbH) with a hydraulic pressure of 100 bar. The refined chocolate mass was conched for 6 h at 80 °C in a small scale conche (Frisse ELK0005, Bühler GmbH, Switzerland) with the addition of 12.5% cocoa butter and 0.05% rapeseed lecithin (as emulsifier) 20 min before end of conching. The final chocolate was tempered manually in a metal pot using cold and warm water baths under continuous stirring. To melt all fat crystals, the chocolate was initially heated above 40 °C, then cooled to 26 °C and finally heated to 28–29 °C before being moulded into ~ 4 g pieces. Particle size distribution of the final chocolates was measured by laser light scattering using a CILAS 990 L Granulometer (CILAS S.A., Orléans, France) to ensure a d90-value below 20 μm (i.e. 90% of the particles in the liquor are finer than 20 μm). Dynamic headspace GC–MS analysis of volatiles from cocoa liquor and chocolate The volatile aroma profiles of un-roasted cocoa liquors, roasted cocoa liquors and chocolates were analysed in duplicate using a combination of dynamic headspace sampling and gas chromatography–mass spectrometry (GC–MS) following the protocol of Owusu et al. (2011). To collect volatiles from chocolate, 20 g of sample was placed in a 500 ml glass flask which was closed with a purge head and placed on a magnetic stirrer in a water bath at 50 °C. After 20 min of equilibration, the headspace was purged with nitrogen at a flow-rate of 200 ml/min for 60 min. Volatile compounds were collected on Tenax-TA traps each containing 250 mg of Tenax-TA with a mesh size of 60/80 and a density of 0.37 g/ml (Buchem bv, Apeldoorn, The Netherlands). Volatiles from un-roasted and roasted cocoa liquors were collected as described above, but at a reduced flow-rate of 50 ml/min for 15 min to avoid overloading the traps with acetic acid. Primary desorption of volatiles was performed by heating the traps to 250 °C and applying a 60 ml/min flow of carrier gas (H2) for 15 min in an automatic thermal desorption unit (ATD 400, Perkin Elmer, Norwalk, USA). The stripped volatiles were trapped on a cold Tenax-TA trap containing 30 mg Tenax at 5 °C before being flash heated to 300 °C and desorbed for 4 min. Using an outlet split ratio of 1:10, the volatiles were transferred through a heated transfer line (225 °C) to a GC (7890A, Agilent Technologies) equipped with a polar Agilent DBWax column (30 m × 0.25 mm × 0.50 μm) operating with H2 as carrier gas at a constant column pressure of 16.5 kPa resulting in an initial flow rate of app. 1.2 ml/min. Column oven temperature was initially held at

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40 °C for 10 min followed by an increase to 240 °C (at a rate of 8 °C/ min) were it was held for 5 min. Mass-to-charge ratios between 15 and 300 m/z were scanned using a Triple Axis mass spectrometric detector (5975C VL MSD, Agilent Technologies) operating with an electron ionization mode at 70 eV. Linear retention indices relative to a mixture of n-alkanes were calculated according to Kovats (KI). Volatile compounds were tentatively identified by probability based matching of their mass spectra with those obtained from a commercial database (Wiley275.L, HP product no. G1035A) and by matching the KI of the compounds with literature values reported on equivalent columns. Peak areas were used for relative quantification of well identified compounds using the MSD Chemstation software (version E.02.00, Agilent Technologies). One-way analysis of variance (ANOVA) was conducted to compare peak areas of the individual compounds identified by GC–MS. Compounds showing significant variance (p b 0.05) were subjected to a post-hoc Tukey's HSD (Honestly Significant Difference) test to identify significantly different samples. Principal component analysis of GC–MS peak areas Principal component analysis (PCA) was performed on the peak areas of selected aroma compounds using Matlab version R2012b (8.0.0.783, MathWorks Inc., Natrick, MA, USA) and PLS_Toolbox version 7.0.2 (Eigenvector Research Inc., Manson, WA, USA). Data were meancentred by subtracting the average value of each variable and scaled to have unit variance for each variable (auto-scaling). This was done prior to PCA modelling to give each aroma compound the same weight to influence the PCA model (Wold, Esbensen, & Geladi, 1987). Sensory analysis of chocolates Sensory analysis was performed to elucidate whether the sensory profiles of chocolates produced from the three experimental and the two commercial fermentations differed. A descriptive analysis was performed according to the CATA method (Check-All-That-Apply) as described by Ares, Deliza, Barreiro, Giménez, and Gámbaro (2010) and Valentin, Chollet, Lelièvre, and Abdi (2012) using a panel of 24 semi-trained judges at the Department of Sensory Science, University of Copenhagen. The panellists were familiar with sensory analysis in general, but not specifically trained in evaluating chocolates. To generate attributes for use in the CATA questionnaire, a focus group of 9 people were asked to describe the flavours perceived when tasting the different chocolates. Following attribute generation, 25 descriptors were selected in plenum to best describe the taste, flavour and mouth feel of the chocolates. Two tasting sessions were conducted allowing the panellists to taste the chocolates in duplicate. The recognition of an attribute by a panellist was marked by ticking the corresponding box in the questionnaire. The samples were blind labelled and the order of presentation completely randomised as recommended by Valentin et al. (2012). The results of the questionnaires were converted to binominal data, where ‘1’ indicates the recognition of an attribute whilst ‘0’ marks that the attribute was not ticked by the panellist. Statistical analyses including a Friedman's test and a post-hoc paired Wilcoxon rank sum test were performed to check for significant differences in the CATA profiles using the software IBM SPSS Statistics vers. 19. The same semi-trained panel was used for conducting Triangle tests on the three experimental chocolates. During the test each participant obtained three triangle tests to cover all combinations of the three samples. The order of presentation as well as the order of combination was completely randomised. To investigate whether samples were perceived as significantly different, the number of correct answers was assessed and the statistical tables of Meilgaard, Civille, and Carr (2007) were consulted.

Please cite this article as: Crafack, M., et al., Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.04.032

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A hedonic preference test was conducted on the three experimental chocolates with a total of 121 consumers. The participants were asked to rank the three chocolates by assigning numbers from 1 to 3 to indicate which samples they preferred most, second most and least. A Friedman's test was performed as recommended by Lawless and Heymann (2010) in order to evaluate preferential differences between the samples. Results and discussion Flavour precursor and organic acid content of the fermented cocoas The experimentally fermented cocoas contained 2–2.5 times more acetic acid (8.4–9.1 mg/g) than the conventional heap and tray fermented cocoas (3.7–4.4 mg/g, Table 1). Consequently, the pH of the experimental cocoas (pH 4.9–5.0) was 0.4–0.5 units lower compared to the conventional cocoas (pH 5.3–5.4). The comparably high concentration of acetic acid in the experimental samples can most probably be ascribed to sub-optimal drainage in the small plastic trays used for fermentation. Lactic acid concentrations in the range of 1.2–1.7 mg/g were measured with the highest amounts being found in the heap and tray fermented cocoas. In small amounts, lactic acid gives a clean freshness to the chocolate, however due to its non-volatile nature, high concentrations can be detrimental to cocoa quality since excess acidity cannot be removed by evaporation during conching (Thompson et al., 2001; Wood & Lass, 1985). Only minor differences in the total concentration of flavour precursors were measured in the five cocoas. Whilst sucrose is the major carbohydrate found in unfermented cocoa, fermented and dried cocoa contains substantial amounts of reducing sugars formed by enzymatic inversion of sucrose into glucose and fructose during fermentation (Hansen et al., 1998; Thompson et al., 2001). With exception of the experimental control containing 19.3 mg/g of reducing sugars, all other

samples contained between 11.9 and 14.2 mg/g. As reported by Reineccius, Andersen, Kavanagh, and Keeney (1972) and Hashim, Selamat, Muhammad, and Ali (1998) fructose was the most abundant sugar found in the fermented and dried cocoas. The concentration of free amino acids ranged from 9.80 to 11.37 mg/ g of fat-free cocoa, with the heap and tray cocoas containing the lowest amounts. Although the total content of free amino acids is indicative of the overall degree of protein hydrolysis, it is not necessarily a good indicator of high flavour potential (Biehl et al., 1985). More important is the accumulation of hydrophobic amino acids which readily undergo Strecker degradation during drying and roasting to yield aromatic Strecker aldehydes and aminoketones which are considered important precursors in the formation of pyrazines (Perez-Locas & Yaylayan, 2010). Hydrophobic amino acids were found to compose 68–73% of the total amount of amino acids with leucine, phenylalanine and alanine being the most abundant. The three experimental cocoas contained slightly higher concentrations of hydrophobic amino acids (8.1– 8.2 mg/g) compared to the heap and tray cocoas (7.0–7.4 mg/g), indicating either a stronger proteolysis in the experimental cocoas or that initial Maillard reactions taking place during drying were further progressed in the heap and tray cocoas. Composition of volatiles in un-roasted and roasted cocoa liquors Using dynamic headspace GC–MS, a total of 46 volatile compounds were identified in the un-roasted cocoa liquors, whilst 63 compounds were found in the roasted liquors (Table 2). To visualize the impact of roasting on the volatile aroma profile, a two component PCA model was calculated based on the GC–MS data from the un-roasted and roasted liquors. With 68.8% explained variation, the score plot in Fig. 1 shows a clear separation of the un-roasted and the roasted samples along PC1. Un-roasted samples cluster on the negative axis, whilst the roasted samples cluster along the positive axis. Separation of the

Table 1 pH and dry weight contents (mg/g) of organic acids, sugars and free amino acids in cocoa inoculated with Pichia kluyveri, Kluyveromyces marxianus or spontaneously fermented (control) in an experimental small scale tray setup or from commercial heap and tray fermentations. Values represent averages of biological and technical replicates (n = 2 × 2) with SD (±). P. kluyveri K. marxianus

Control

Heap

Tray

pH, cotyledon

5.0 ± 0.0

4.9 ± 0.0

5.0 ± 0.0

5.3 ± 0.0

5.4 ± 0.0

Organic acids Acetic acid Lactic acid

8.5 ± 0.5 1.3 ± 0.2

9.1 ± 0.3 1.2 ± 0.2

8.4 ± 0.5 1.4 ± 0.3

4.4 ± 0.2 1.6 ± 0.2

3.7 ± 0.1 1.7 ± 0.2

Sugars Glucose Fructose Sucrose

4.5 ± 0.3 9.7 ± 0.7 3.1 ± 0.1

4.6 ± 0.3 9.5 ± 0.4 2.8 ± 0.3

6.4 ± 0.5 12.9 ± 0.9 4.3 ± 1.0

2.9 ± 0.5 9.0 ± 0.3 5.4 ± 0.6

3.2 ± 0.3 10.6 ± 0.7 4.9 ± 0.4

11.4 ± 0.6

11.4 ± 0.3

11.1 ± 0.4

10.9 ± 1.3

9.8 ± 0.7

1.3 1.0 0.56 2.3 1.9 1.1

1.4 0.95 0.54 2.3 2.0 1.1

1.3 1.0 0.56 2.3 1.9 1.0

1.0 1.1 0.57 1.9 1.7 1.2

Free amino acidsa Total Hydrophobic Alanine Valine Isoleucine Leucine Phenylalanine Tyrosine Acidic Glutamate Histidine Others Glycine Threonine Proline Methionine Lysine Tryptophan

± ± ± ± ± ±

0.1 0.0 0.02 0.1 0.1 0.2

± ± ± ± ± ±

0.0 0.03 0.02 0.1 0.1 0.1

± ± ± ± ± ±

0.0 0.0 0.03 0.1 0.1 0.0

± ± ± ± ± ±

0.2 0.1 0.03 0.1 0.2 0.4

1.2 1.0 0.52 1.7 1.5 1.1

± ± ± ± ± ±

0.1 0.1 0.03 0.1 0.1 0.5

0.90 ± 0.05 0.17 ± 0.06

1.0 ± 0.1 0.16 ± 0.03

0.87 ± 0.10 0.14 ± 0.02

1.3 ± 0.2 0.16 ± 0.06

0.97 ± 0.04 0.12 ± 0.03

0.28 0.22 0.48 0.22 0.78 0.15

0.28 0.21 0.47 0.22 0.71 0.14

0.29 0.23 0.50 0.23 0.63 0.13

0.19 0.22 0.75 0.07 0.71 0.14

0.20 0.19 0.70 0.05 0.56 0.13

± ± ± ± ± ±

0.01 0.01 0.02 0.01 0.33 0.04

± ± ± ± ± ±

0.01 0.01 0.02 0.02 0.14 0.00

± ± ± ± ± ±

0.01 0.01 0.02 0.01 0.05 0.01

± ± ± ± ± ±

0.04 0.02 0.11 0.01 0.26 0.03

± ± ± ± ± ±

0.01 0.01 0.02 0.00 0.19 0.05

Abbreviations: P.: Pichia, K.: Kluyveromyces. a Content (mg/g) of defatted cocoa powder.

Please cite this article as: Crafack, M., et al., Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.04.032

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Table 2 Volatiles identified in un-roasted and roasted liquors of cocoa inoculated with Pichia kluyveri (P), Kluyveromyces marxianus (K) or spontaneously fermented (C) in an experimental small scale tray setup or from commercial heap (H) and tray (T) fermentations. Means represent averages of GC–MS peak areas including biological and technical replicates (n = 2 × 2). Means with different superscripts are significantly different within each of the two groups (un-roasted and roasted), whilst * indicates a significant difference between the un-roasted and roasted sample (Tukey's HSD, p b 0.05). Compound1

KI

Mean GC–MS peak area × 105 Un-roasted liquor

Roasted liquor

P

K

C

H

T

P

K

C

H

T

Acids Propanoic acid 2-Methyl propanoic acid 3-Methyl butanoic acid

1548 1580 1682

187 3431 1408ab

179 3262 972a

203 4051 1504ab

123 2659 1409ab

125 3407 1980b

– 4487 2278

– 4126 2031

– 4102 2212

– 3795 2626⁎

– 3678 2943

Aldehydes 2-Methylpropanal 2-Methylbutanal 3-Methylbutanal Hexanal Heptanal Nonanal Benzaldehyde Phenylacetaldehyde 2-Phenyl-2-butenal 5-Methyl-2-phenyl-2-hexenal

825 920 924 1087 1194 1407 1539 1660 1950 2077

2193 3401 4072a 22a – 41 233a 18a – –

1449 1936 2390abc 17a – 25 174a 10a – –

1823 2713 3397ab 30a – – 207ab 24a – –

2377 2244 1319c 102b – 30 382b 22a – –

2888 3419 2144bc 31a – 16 582c 42a – –

2338a 3856 6691 – – – 290a 363⁎

2947ab⁎ 4602⁎ 7315⁎

3703b⁎ 5889⁎ 7220⁎

9 1.0

– – – 271a 210 8 1.1

2485a 4488 7199⁎ – – – 264a 182 11 1.3

145a 13 36 655b⁎ 117 14 0.6

3489b 5501 7879⁎ 45 – 33 705b 140 13 1.9

Alcohols 2-Propanol 2-Methyl-3-buten-2-ol Isobutyl alcohol 2-Pentanol Isoamyl alcohol 1-Pentanol 2-Heptanol 2,3-Butanediol 1,3-Butanediol 1-Phenylethanol Benzyl alcohol Phenethyl alcohol

937 1048 1109 1146 1229 1278 1347 1557 1591 1832 1894 1929

217 402a 50 1510 857 60 105a – – – 6 178

152 325a 24 987 682 34 98a – – – 8 178

451 932ab 33 1339 767 47 97a – – – 6 138

474 648ab 37 2006 433 31 270b – – – 8 207

296 1349b 46 3862 699 34 323b – – – 10 198

– 55a – – 359ab 50ab 153a 5647 4993ab 16 15 534⁎

– 130a – – 338ab 63ab 178a 3369 1127a 13 10 433⁎

– 93a⁎ – – 121a 34a 131a 5749 8662b 21 15⁎ 468⁎

– 961b 267 2793 862b 85ab 569b⁎ 4939 4262ab 22 18⁎ 602⁎

– 727b 182 1074 443ab 119b⁎ 490c 5239 5113ab 27 19⁎ 567⁎

Esters Methyl acetate Ethyl acetate Ethyl isobutyrate Isobutyl acetate Methyl 3-methylbutanoate Ethyl 2-methylbutyrate 2-Pentyl acetate Isoamyl acetate Benzyl acetate Ethylphenyl acetate Phenethyl acetate Ethyl cinnamate

838 903 973 1019 1023 1055 1080 1142 1749 1807 1838 2127

2145 571ab – 335ab 9a 7a 287 1845 4 50ab 273ab –

1523 706a – 230ab 6a 5a 244 1369 4 45ab 270ab –

1988 678a – 385a 15a 10a 326 1574 5 64a 237ab –

1607 189bc – 163b 54b 26b 369 1588 6 49ab 312a –

1759 82c – 212ab 61b 41c 568 1917 4 35b 182b –

2633 136ab⁎ – 510 – – 690a 1274ab 6ab 62ab 357 0.4

2749 204a⁎ – 664 – – 847a 1553ab 5a 54ab 333 0.5

2449 222a⁎ – 712 – – 704a 886a 7ab 80a 315 0.6

2851 155ab 103 997⁎ 108a 25 2320b⁎ 3612b 10b 65ab 419 0.9

2653 31b 62 550 57 18 1821ab⁎ 2412ab 7ab 48b 255 0.8

Ketones 2-Butanone 2-Pentanone 2,3-Butanedione 2-Heptanone Acetoin 2,3-Octanedione 2-Nonanone 1-Phenyl ethanone

911 985 987 1192 1305 1344 1402 1671

203 – 2637a 86a 4111 – 20a 92ab

148 – 1587b 66a 3136 – 19a 73a

151 – 2828a 67a 6446 – 16a 121bc

195 – 668c 197b 3432 – 113b 118abc

251 – 926bc 261c 3327 – 102b 154c

382 – 4735a⁎ 81a 1409 2a 24a 134ab

390 – 4768a⁎ 109a 998 9a 31a 121a

385 – 5412a⁎ 44a 78⁎ – – 163ab

534⁎ – – 447b⁎ 2935 36b 188b 229b⁎

435 3248 3464b⁎ 251ab 3168 – 149a 233b

Furans 2-Methyltetrahydrofuran 2-Ethyl-5-methylfuran 2-Pentylfuran

884 1032 1249

56a 91a –

30a 46ab –

62ab 86a –

94b 32b –

225c 79ab –

22a – 8a

38a – 12ab

35a – 6a

167b – 44c

161b – 25b

Pyrazines Methylpyrazine 2,5-Dimethylpyrazine 2,6-Dimethylpyrazine Ethylpyrazine 2,3-Dimethylpyrazine 2,3,5-Trimethylpyrazine 2,3-Dimethyl-5-ethylpyrazine 2,3,5,6-Tetramethylpyrazine 2,3,5-Trimethyl-6-ethylpyrazine

1286 1346 1350 1354 1365 1425 1481 1495 1534

– – – – 17ab 34ab – 294a –

– – – – 7a 19a – 379ab –

– – – – 13a 34ab – 241a –

– – – – 37b 75b – 731b –

– – – – 79c 120c – 624ab –

49 65 53 28ab 163⁎ 379⁎

50 58 37 51a 136⁎ 307⁎

40 69 61 3b 122⁎ 444⁎

53 54 45 23ab 158⁎ 428⁎

60 57 40 13ab 177⁎ 505⁎

53a 2554⁎ 25

44ab 1908⁎ 19

62a 2716⁎ 30

29b 2411⁎ 19

31b 2760⁎ 21

(continued on next page)

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M. Crafack et al. / Food Research International xxx (2014) xxx–xxx

Table 2 (continued) Compound1

KI

Mean GC–MS peak area × 105 Un-roasted liquor

Roasted liquor

P

K

C

H

T

P

K

C

H

T

1179 1202 1253

41a 16ab 10a

42a 12a 15a

59a 22abc 24a

146b 27bc 112b

194c 30c 131b

59a 12 23a

51a 18 20a

73a 14 10a

239b 25 166b

260b 30 184b

Terpene alcohols Linalool oxide Linalool Epoxylinalool

1488 1561 1780

– 19a –

– 20a –

– 29a –

– 58b –

– 59b –

25ab 29a 5a

23a 22a 4a

29ab 41ab 6a

43ab 74bc –

50b 91c 11b

Others Dihydro-2(3H)-furanone 2-Methoxyphenol 2-Acetylpyrrole

1649 1878 1983

44a 10c –

67a 7a –

45a 4ab –

187b 2b –

105ab 4b –

132 17a 65

78 13ab 46

130 8ab 84

172 4b 60

162 5b 72

Terpenes Myrcene Limonene Ocimene

1

Compounds identified by probability based matching of mass spectra and Kovats retention index (KI).

experimentally fermented samples from the commercial samples was possible along PC2 with the former clustering along the negative axis, whilst the latter clustered along the positive axis. Separation of experimental and commercial samples was most pronounced in the roasted liquors, indicating a greater difference in the quality and/or quantity of the volatiles developed after roasting. The loading plot in Fig. 1 shows that the majority of the volatile compounds identified have positive loadings on PC1 (clustering to the right in the plot), indicating that the roasted liquors in general are characterized by higher concentrations of volatiles. From the average GC–MS peak areas presented in Table 2, it is seen that roasting had a pronounced impact on the composition and relative concentration of the volatiles identified in the liquors. Whilst the total concentration of aldehydes, alcohols and pyrazines increased remarkably upon roasting, the concentration of acids, ketones, terpenes and terpene alcohols changed only slightly or stayed at the same level. Pronounced increases of the Strecker aldehydes 2-methylpropanal, 2methylbutanal, 3-methylbutanal and phenylacetaldehyde were observed during roasting, whilst 2-phenyl-2-butenal and 5-methyl-2phenyl-2-hexenal, not present in the un-roasted liquors, were identified in the roasted liquors. 2-Phenyl-2-butenal and 5-methyl-2-phenyl-2hexenal are formed through aldol condensation of phenylacetaldehyde with acetaldehyde and 2-methylpropanal (Ziegleder, 2009), and

especially the latter is reported as being a key constituent of chocolate aroma (Bonvehí, 2005; Counet & Callemien, 2002; Owusu et al., 2011). The most abundant alcohols identified in the roasted liquors were 1,3- and 2,3-butanediol, both of which were not identified in the unroasted liquors. Although normally considered as fermentation products, Owusu et al. (2011) measured increasing concentrations of these two alcohols during prolonged conching, probably caused by the reduction of 2,3-butanedione and acetoin. A significant increase of benzyl and phenethyl alcohol was observed in the majority of the liquors upon roasting. These two alcohols are known products of yeast metabolism (Delfini, Gaia, Bardi, & Mariscalco, 1991; Nykänen, 1986) but can also be formed by conversion of benzaldehyde and phenylacetaldehyde (Ziegleder, 2009). Roasting reduced the content of ethyl acetate in all liquors, whilst the concentration of methyl acetate, isobutyl acetate, 2-pentyl acetate, benzyl acetate, ethylphenyl acetate and phenethyl acetate increased. Furthermore, ethyl cinnamate was formed as a result of roasting. Generally the highest ester concentrations were measured in the heap and tray fermented cocoas. Whilst methyl 3-methylbutanoate and ethyl 2-methylbutyrate disappeared from the experimentally fermented liquors upon roasting, they were still present in the roasted heap and tray samples. Likewise, isoamyl acetate concentrations decreased in the

Fig. 1. Principal component analysis (PCA) of GC–MS peak areas showing the effect of roasting on the composition of volatiles identified in un-roasted and roasted liquors of cocoa inoculated with Pichia kluyveri (P), Kluyveromyces marxianus (K), spontaneously fermented (C) or from commercial heap (H) and tray (T) fermentations. The score plot includes biological replicates of fermentations (1, 2) and technical replicates of GC–MS analysis.

Please cite this article as: Crafack, M., et al., Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.04.032

M. Crafack et al. / Food Research International xxx (2014) xxx–xxx

three experimentally fermented liquors, but increased in the heap and tray samples. Furthermore the increase of 2-pentyl acetate upon roasting was significantly (p b 0.05) greater in the heap and tray liquors. The comparably high concentration of acetic acid and low pH measured in the experimental cocoas is thought to have a negative influence on the formation of esters and their stability during roasting. Acetoin and 2,3-butanedione (diacetyl) were the major ketones identified in the roasted liquors, but whilst the concentration of acetoin decreased upon roasting, significant increases in the concentration of diacetyl was observed. Diacetyl is a well-known secondary metabolite of yeasts and bacteria and imparts a buttery aroma to many foodstuffs. Roasting also increased the contents of 2-butanone and 1-phenyl ethanone (acetophenone), the latter being derived from phenylalanine. Contents of the terpenes myrcene and ocimene increased slightly upon roasting, whilst the concentration of limonene stayed at a stable level. Roasting caused a 2–3 fold increase in the concentration of terpene alcohols, with the heap and tray fermented liquors containing the highest amounts. Whilst un-roasted liquors solely contained linalool, roasted liquors contained a mixture of linalool and its derivatives linalool oxide and epoxylinalool. Linalool is generally considered a product of biosynthesis and is found in its glycosidically bound form in the fruit pulp and in the cocoa bean cotyledons. During fermentation and drying the glycosidic bonds are believed to be hydrolysed by the combined

7

action of enzymes, acids and heat. De novo synthesis of linalool by Saccharomyces cerevisiae has however also been reported (Carrau et al., 2005). Although roasting is reported to decrease the concentration of linalool (Ziegleder, 1990), a slight increase was observed in the present study. Linalool and its derivatives are often described as having a floral aroma and are considered key aroma components found in high concentrations in “noble-flavour” cocoas like Criollo and Arriba (Bonvehí, 2005; Counet & Callemien, 2002; Owusu et al., 2011; Ziegleder, 1990). The group of compounds undergoing the biggest changes as a result of roasting were the pyrazines. Pyrazines are nitrogen-containing heterocyclic intermediates of the Maillard reaction and are predominantly formed through Strecker degradation reactions between amino acids and α-dicarbonyls (Perez-Locas & Yaylayan, 2010). Besides the 2,3-dimethyl-, 2,3,5-trimethyl- and 2,3,5,6-tetramethylpyrazines identified in the un-roasted liquors, six new pyrazines were formed as a result of roasting. Significant increases of 2,3-dimethyl-, 2,3,5-trimethyl- and 2,3,5,6-tetramethylpyrazine were observed upon roasting with the latter being the most abundant pyrazine. Several authors report the accumulation of pyrazines during fermentation. Whilst Reineccius, Keeney, and Weissberger (1972) argue that tetramethylpyrazine might be formed during fermentation through thermally initiated reactions or microbial synthesis, Hashim and Chaveron (1994) showed the presence of 2,3-dimethyl-, trimethyl- and tetramethylpyrazine after 1 day of

Fig. 2. Principal component analysis (PCA) of GC–MS peak areas showing the effect of fermentation technique (A) and the use of starter cultures (B) on the composition of volatiles identified in chocolate inoculated with Pichia kluyveri (P), Kluyveromyces marxianus (K), spontaneously fermented (C) or from commercial heap (H) and tray (T) fermentations. Score plots include biological replicates of fermentations (1, 2) and technical replicates of GC–MS analysis.

Please cite this article as: Crafack, M., et al., Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.04.032

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M. Crafack et al. / Food Research International xxx (2014) xxx–xxx

Table 3 Volatile compounds identified in chocolates produced from cocoa inoculated with Pichia kluyveri, Kluyveromyces marxianus or spontaneously fermented (control) in an experimental small scale tray setup or from commercial heap and tray fermentations. Means represent averages of GC–MS peak areas including biological and technical replicates (n = 2 × 2). Means with different superscripts are significantly different (Tukey's HSD, p b 0.05). Compound1

KI

Odour description^

Mean GC–MS peak area × 105 P. kluyveri

K. marxianus

Control

Heap

Tray

Acids 2-Methyl propanoic acid 3-Methyl butanoic acid

1580 1682

831a 739a

438a 785a

562a 837a

841a 1364a

3440b 3009b

Rancid1 Sweaty1,2

Aldehydes 2-Methylpropanal 2-Methylbutanal 3-Methylbutanal Pentanal Hexanal Heptanal Octanal 2-Heptenal Nonanal Benzaldehyde Phenylacetaldehyde 2-Phenyl-2-butenal 5-Methyl-2-phenyl-2-hexenal

825 920 924 986 1087 1194 1311 1343 1407 1539 1660 1950 2077

87a 428a 1711a 537a 147a 28b 20 11 245a 240a 795a 61 10

84a 296a 1230a 675ab 151a 29b 22 13 238a 263a 552b 64 7

84a 439a 1806ab 997bc 144a 34ab 24 17 339ab 292a 561b 74 7

115ab 788ab 2448ab 1058c 325b 44a 25 5 459b 387a 564b 79 7

200b 1444b 3673b 1014c 147a 31b 17 24 342ab 664b 652ab 80 14

Malty1, chocolate3 Malty1, chocolate3, cocoa2 Malty1, chocolate3, cocoa2 Pungent, bitter almond4 Green5 Oily, fatty4 Orange peel2, oily, fatty, soapy4 Green, fermented2 Tallowy, soapy–fruity4 Bitter almond, grass2 Honey1,3, green2, flowery2,3 Flowery2, cocoa2,3, roasted3, rum3 Sweet2, roasted cocoa2

Alcohols 2-Pentanol Isoamyl alcohol 1-Pentanol 2,3-Butanediol 1-Octanol 1,3-Butanediol 1-Phenylethanol Benzyl alcohol Phenethyl alcohol

1146 1229 1278 1557 1574 1591 1832 1894 1929

31a 31a 228a 4026a 57a 5229a 42a 47ab 2147a

25a 24a 329ab 3942a 52a 4978a 49a 63ac 2661b

31a 25a 486bc 3933a 75a 5387a 53ab 43b 1750a

112a 56a 571c 3684a 116b 3170b 46a 50ab 1829a

349b 123b 520c 5452b 74a 5139a 68b 77c 2147a

Green6 Harsh, nail polish7 Flowery, sweet2 Sweet, flowery2 Sharp, fatty, waxy, citrus6 Sweet, flowery2 Honey, floral6 Sweet, fruity2 Flowery1–3

Esters Ethyl acetate Isoamyl acetate Ethyl decanoate Benzyl acetate Ethylphenyl acetate Phenethyl acetate Ethyl dodecanoate Ethyl cinnamate

903 1142 1654 1749 1807 1838 1857 2127

2 101a 25a 19a 328ac 2048ab 21 6

3 68a 27a 32b 376ab 2396a 21 7

2 94a 28a 21a 449b 1894b 21 10

2 91a 32ab 23ab 260cd 1881b 13 10

2 223b 38b 19a 214d 1285c 20 9

Nail polish, fruity7 Banana, pear7 Pear, grape6 Flowery, rose2,6 Flowery, rose2 Honey, flowery2,6 Fruity, floral6 Sweet, cinnamon-like5

Ketones 2-Heptanone Acetoin 2,3-Octanedione 2-Nonanone

1192 1305 1344 1402

31ab 3381a 9a 32a

27ab 1979b 11ab 38a

24a 2748ab 19b 30a

42b 766c 41c 122b

65c 2296b 50c 154c

Fruity, green2 Buttery, cream8 Earthy, mushroom2 Flowery, fatty4

Pyrazines Methylpyrazine 2,5-Dimethylpyrazine 2,6-Dimethylpyrazine Ethylpyrazine 2,3-Dimethylpyrazine 2,3,5-Trimethylpyrazine 2,3,5,6-Tetramethylpyrazine 2,3-Dimethyl-5-ethylpyrazine 2,3,5-Trimethyl-6-ethylpyrazine

1286 1346 1350 1354 1365 1425 1495 1481 1534

27a 22a 7a 7a 41a 195a 3087a 77a 78a

16a 15a 5a 5a 30a 205a 3247a 80a 83a

23a 24a 8a 7a 39a 229a 3148a 93a 98a

44a 23a 14a 11a 36a 148a 2515b 31b 40b

167b 110b 58b 47b 150b 446b 3244a 58c 61c

Cocoa2, green2,3, hazelnut2 Popcorn2 Nutty, coffee, green6 Peanut-butter, musty, nutty6 Caramel6, cocoa6, sweet2, baked2 Cocoa3,6, fried potato2 Chocolate6, mocha3, roasted3 Cocoa, chocolate8 Candy, sweet8

Terpenes Myrcene Limonene 1,8-Cineole Ocimene

1179 1202 1214 1253

20a 567 11 9a

19a 471 9 14ab

19a 309 10 16ab

61ab 1655 8 35b

89b 858 9 86c

Terpene alcohols Linalool oxide Linalool Epoxylinalool

1488 1561 1780

27a 63a 28a

36a 80a 33a

34a 98ab 32a

36a 133b 36ab

69b 189c 49b

Sweet2, flowery2 Rose2, flowery4 Spicy, liquorice2

Others 2-Pentylfuran Dihydro-2(3H)-furanone 2-Methoxyphenol 2-Acetylpyrrole

1249 1649 1878 1983

77a 117ab 38a 266ab

77a 94a 45a 242ab

79a 106ab 15c 321ac

145b 191b 2b 200b

81a 308c 5b 349c

Musty, green2 Sweet2 Spicy, smoky1 Hazelnut3

Herbaceous, metallic4 Citrus-like4 Spicy, camphor-like4

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M. Crafack et al. / Food Research International xxx (2014) xxx–xxx

fermentation. Following 3 days of fermentation they furthermore identified methyl-, 2,5-dimethyl- and 2,6-dimethylpyrazine whilst observing a rapid increase in the concentration of all six pyrazines. Although the formation of pyrazines is most often described in relation to heated food systems, biosynthesis of pyrazines by Bacillus subtilis has been reported by Zak, Ostovar, and Keeney (1972) who suggest that condensation reactions between acetoin, 2,3-butanediol or pyruvaldehyde with amino acids or ammonia could lead to the formation of methyl-substituted pyrazines. Other volatiles identified include 2-ethyl-5-methylfuran, present only in the un-roasted liquors, together with 2-methyltetrahydrofuran and dihydro-2(3H)-furanone, identified in both un-roasted and roasted liquors. 2-Pentylfuran and 2-acetylpyrrole were found exclusively in the roasted liquors. Another compound identified in both un-roasted and roasted liquors was 2-methoxy phenol (guaiacol), a compound derived from microbial synthesis or thermal degradation of ferulic acid (Belitz et al., 2004).

9

Sweet 6 Caramel

Sour

5 4

Balsamic

Bitter

3 2 1 0

Banana

Cocoa

Berry

Fruity

Yoghurt

Melts quickly

Effect of fermentation technique on the composition of chocolate volatiles

Flat A total of 56 volatile compounds were identified in chocolates produced from the experimentally and commercially fermented cocoas. In general, the 6 hour conching process at 80 °C had a notable influence on the composition of volatiles as compared to the roasted liquors. Unfortunately, quantitative comparisons between the relative concentrations of volatiles in the roasted liquors and finished chocolates were impossible, as different flow rates and purge times were applied during collection of the volatiles in order to avoid column damage by excess acetic acid from the un-conched samples. Interestingly though, the composition of volatiles was qualitatively the same in all samples, hence no compounds were found to be unique for a certain chocolate. Whilst the composition of acids, terpene alcohols and pyrazines remained unchanged upon conching, three new aldehydes (octanal, 2heptenal and pentanal), two esters (ethyl decanoate and ethyl dodecanoate), one alcohol (1-octanol) and one terpene (1,8-cineole) were detected in the chocolates. Major changes occurred within the group of esters where methyl-, isobutyl- and 2-pentyl acetate were lost along with methyl 3-methylbutanoate, ethyl 2-methylbutyrate and ethyl isobutyrate. Furthermore, four ketones (2-butanone, 1phenyl ethanone, 2,3-butanedione and 2-pentanone), three alcohols (2-heptanol, 2-methyl-3-buten-2-ol and 2-propanol) and two furans (2-ethyl-5-methylfuran and 2-methyltetrahydrofuran) were absent in the chocolates. The effect of fermentation technique on the volatile aroma profile was visualized by calculating a two component PCA model based on GC–MS data from the experimentally and commercially fermented chocolates. The score plot presented in Fig. 2A explains 67.9% of the total variation between the 20 samples. From the score plot it is seen that all experimental samples, both inoculated and spontaneously fermented, cluster together on the negative axis of PC1 and positive axis of PC2. The two commercial samples are both placed along the positive axis of PC1, but are separated on PC2 with the tray fermented samples generally clustering on the positive axis, whilst the heap fermented samples cluster on the negative axis. The choice of fermentation technique therefore seems to be a major discriminating factor influencing

Fig. 3. Sensory profiles of chocolate produced from cocoa inoculated with Pichia kluyveri (□), Kluyveromyces marxianus (◊) or spontaneously fermented (○) in an experimental small scale tray setup or from commercial heap (♦) and tray (●) fermentations. Profiles are based on CATA (check-all-that-apply) analysis showing descriptors judged significantly different (p b 0.01) by Wilcoxon rank sum test.

the aroma profile of the chocolates. The loading plot in Fig. 2A shows that the majority of the volatile compounds are grouped in the right part of the plot along the positive PC1 axis, indicating that the tray fermented samples have the highest overall concentration of volatiles. This observation is confirmed by the average GC–MS peak areas presented in Table 3, showing that the highest concentration of 32 volatile compounds is measured in the tray samples, 19 of which are found in significantly (p b 0.05) higher concentrations compared to the experimental and heap fermented samples. Whilst many of these compounds have desirable attributes described as cocoa, nutty, fruity and flowery, the tray samples also contain app. four times as much 2methylpropanoic acid and 3-methylbutanoic acid which are described as rancid and sweaty (Table 3). 2-Methylpropanoic acid and 3methylbutanoic acid are short chain fatty acids derived from oxidative deamination and decarboxylation of their parent amino acids valine and leucine. Growth of Bacillus ssp. during the late stages of fermentation has been associated with the production of short chain fatty acids, however they can also be liberated through degradation of Strecker aldehydes (Lopez & Quesnel, 1973; Zak et al., 1972; Ziegleder, 2009). As these two acids were not found in significantly higher quantities in the un-roasted and roasted tray liquors (Table 2), it is assumed that they are not products of microbial synthesis but are liberated during the conching process as a result of the degradation of Strecker aldehydes, indicating a higher chemical reactivity in the tray sample. The composition of volatiles in the heap fermented chocolate was quantitatively closer to the experimental chocolates than to the tray chocolate. 9 compounds were found in highest concentrations in the heap chocolate, with hexanal and 2-pentylfuran being significantly

Notes to Table 3: Abbreviations: P.: Pichia, K.: Kluyveromyces. ⁎Compounds identified by probability based matching of mass spectra and Kovats retention index (KI). ^ Odour description from literature. 1 Frauendorfer and Schieberle (2006). 2 Owusu (2010) and Owusu et al. (2011). 3 Counet and Callemien (2002). 4 Belitz et al. (2004). 5 Schnermann and Schieberle (1997). 6 Bonvehí (2005). 7 Swiegers et al. (2005). 8 Afoakwa et al. (2009).

Please cite this article as: Crafack, M., et al., Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.04.032

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higher. Hexanal and 2-pentylfuran are described as having “green” and “green, musty” odours, respectively (Table 3). The small scale tray system used for the experimental fermentations yielded chocolates which were distinguishable from the commercial heap and tray cocoas by significantly higher concentrations of 2methoxyphenol, 2,3-dimethyl-5-ethylpyrazine, and 2,3,5-trimethyl-6ethylpyrazine and significantly lower concentrations of 2-nonanone and 2,3-octanedione. The two ethylpyrazines are reported to impart cocoa, chocolate and candy aromas whilst 2,3-octanedione imparts an earthy, mushroom smell and 2-methoxyphenol a burnt, smoky odour (Table 3). Although only minor differences in the contents of flavour precursors were observed in the five cocoas before roasting (Table 1), significant differences in the quantitative composition of volatiles were measured in the roasted liquors and finished chocolates. The seemingly greater reactivity of the flavour precursors in the heap and tray chocolates is most likely attributed to the higher pH measured in these two samples compared to the experimental cocoas. As shown by Biehl et al. (1985), strong cotyledon acidification (pH 4.0–4.5) causes an unspecific proteolysis of all proteins present in the cocoa cotyledon yielding cocoa with low flavour potential, whereas a moderate acidification (pH 5.0–5.5) causes specific proteolysis of the storage proteins yielding cocoa with a higher flavour potential. Furthermore the initiation of the Maillard reaction is known to be favoured by alkaline pH at which the amine groups of free amino acids and peptides will be negatively charged (when pH is above their isoelectric point), thus increasing their reactivity with the electropositive carbonyl group of reducing sugars (Perez-Locas & Yaylayan, 2010). It therefore seems plausible that the 0.4–0.5 pH unit difference observed between the experimental and commercial samples is the main cause of the quantitative variation in the composition of volatiles. Effect of starter cultures on the composition of chocolate volatiles To evaluate the isolated effect of the starter cultures, a two component PCA model was calculated on a reduced data set by excluding data from the commercial heap and tray fermented chocolates. The score plot shown in Fig. 2B explains 55.8% of the variation between the 12 experimental samples. From the score plot it appears that the biological duplicates of the spontaneous control fermentations (C1 and C2) are quite distinct. As described by Crafack et al. (2013), control fermentation C2 was performed using freshly harvested cocoa pods, whereas all other fermentations were conducted using cocoa pods which had been stored for 3–4 days before fermentation. Microbial and physico-chemical data showed that fermentation C2 was app. 12 h delayed compared to fermentation C1 — a delay which apparently had pronounced effect on the volatile profile of the chocolate. Whilst the inoculated samples were difficult to separate from the control (C1) on PC1 (Fig. 2B), they generally cluster on the negative axis of PC2, whereas the control is situated on the positive axis of PC2. Samples inoculated with P. kluyveri are found with slightly lower score values on PC1 than samples inoculated with K. marxianus, indicating that only minor differences exist between the two inoculated samples. In general, both inoculated chocolates contain significantly (p b 0.05) more 2-methoxyphenol compared to the spontaneous control, whilst K. marxianus inoculated chocolates contain significantly higher amounts of benzyl alcohol, phenethyl alcohol, benzyl acetate, phenethyl acetate and P. kluyveri inoculated chocolates significantly more phenylacetaldehyde than the control. Furthermore, the control contains significantly more pentanal, 1-pentanol, ethylphenyl acetate and 2,3octanedione than P. kluyveri inoculated samples (Table 3). Although the volatile profile of the two inoculated samples is very similar, the P. kluyveri inoculated chocolates contain significantly higher concentrations of phenylacetaldehyde and acetoin, whilst K. marxianus inoculated chocolates contain significantly higher concentrations of phenethyl alcohol and benzyl acetate (Table 3). Interestingly, these

four compounds are all related to one common precursor amino acid — phenylalanine. Although known as secondary metabolites of yeasts, it seems that the accumulation of these four compounds is accelerated by roasting and conching (Tables 2 and 3), indicating that they are products of Maillard reactions rather than being products of microbial metabolism. Sensory evaluation of chocolates To correlate findings of the aroma analysis with the perceived quality of the chocolates, three sensory analyses were conducted using a semi-trained panel of judges. Initially, a consumer based sensory profiling of the chocolates was conducted using the CATA methodology. The major advantage of CATA being that it is a cost-effective alternative to the traditional generic descriptive analysis as the method does not require extensive training of a dedicated panel of judges (Meilgaard et al., 2007). The disadvantage is however that the method is less thorough compared to a generic descriptive analysis. The results presented in Fig. 3 show the 12 descriptors judged by the sensory panel as being significantly (p b 0.01) different amongst the five types of chocolate. In general, chocolates from the commercial heap and tray fermentations were described as being sweet with cocoa and caramel flavours whilst the descriptors “acid” and “bitter” were used less often to describe these two samples. Both were characterized as quick melting and flat flavoured compared to the experimental chocolates, which were described as acidic and bitter tasting with fruity, berry, yoghurt and balsamic flavours. Despite the uncertainties associated with linking individual volatile compounds to specific sensory attributes in a complex flavour matrix as chocolate, the cocoa flavour perceived by the tasters in the tray chocolate correlates well with the high concentrations of Strecker aldehydes and pyrazines measured by GC–MS (Table 3). The acidity perceived in the experimental chocolates correlates to the high concentrations of acetic acid measured in the fermented and dried cocoa (Table 1). More judges described the experimental chocolates as being fruity which corresponds well with the P. kluyveri inoculated chocolate having the highest concentration of phenylacetaldehyde and 1,3-butanediol, the K. marxianus inoculated chocolate having the highest concentration of phenethyl alcohol, benzyl acetate and phenethyl acetate and the spontaneous controls having the highest concentration of ethylphenyl acetate. Surprisingly, the descriptor “fruity” was less often used to describe the tray chocolate although GC–MS revealed significantly higher concentrations of fruity and floral smelling compounds like 2,3-butanediol, isoamyl acetate, 2heptanone, linalool and linalool oxide. The descriptor “banana” was used significantly more often to describe the heap chocolate although the concentration of the banana-smelling ester isoamyl acetate was significantly higher in the tray chocolate. The three experimental chocolates were subjected to a hedonic preference test to determine whether certain chocolates were preferred over others by a total of 121 consumers. Since app. one third of all consumers preferred one of each of the three samples, no preferential differences were observed. The result strongly indicated that the samples were indistinguishable to the consumers, but does not exclude the possibility of the consumers simply having different preferences. To clarify whether consumers are able to differentiate the chocolates, a triangle test was performed using 22 semi-trained panellists. Non-significant differences between the P. kluyveri inoculated chocolate and the spontaneous control was proven with α = 0.4 as 9 judges were able to distinguish the two chocolates, whilst the K. marxianus inoculated chocolate was distinguished from the control with α = 0.2 and 10 correct answers from the judges. No difference was observed between the two inoculated chocolates as only 7 judges were able to distinguish the two. The triangle tests therefore proved that it was difficult for consumers to distinguish the inoculated from the spontaneous chocolates. Results of the sensory analyses show a clear differentiation between the chocolates produced from commercial heap and tray fermented

Please cite this article as: Crafack, M., et al., Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.04.032

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cocoa, and the experimental chocolates produced from cocoa fermented in a small scale tray setup. A good correlation between the volatile aroma profile discussed in the Effect of fermentation technique on the composition of chocolate volatiles section and the sensory perception of the chocolates seems to exist. Conclusion The results obtained in the present study indicate that the choice of fermentation technique has a more profound impact on the volatile aroma and sensory profile of chocolate than does the use of starter cultures. Whilst the application of starter cultures did change the aroma profile of the resulting chocolate as determined by GC–MS, the differences observed were too small to significantly change consumer perception of the chocolates. Arguably, using a higher inoculation density and a fermentation setup with better drainage properties could accentuate the impact of the cultures investigated. Acknowledgements This research was funded by the Danish Agency for Science, Technology and Innovation and Toms Confectionary Group A/S (10-084295). The author is grateful for the help of Louise Olsen (chocolate production), Julie Ogstrup and Bodil Allesen-Holm (sensory analysis), Abdelrhani Mourhrib (aroma analysis) and Henriette Erichsen (HPLC). References Aculey, P. C., Snitkjaer, P., Owusu, M., Bassompiere, M., Takrama, J., Nørgaard, L., et al. (2010). Ghanaian cocoa bean fermentation characterized by spectroscopic and chromatographic methods and chemometrics. Journal of Food Science, 75(6), 300–307. Afoakwa, E. O., Paterson, A., Fowler, M., & Ryan, A. (2009). Matrix effects on flavour volatiles release in dark chocolates varying in particle size distribution and fat content using GC–mass spectrometry and GC–olfactometry. Food Chemistry, 113(1), 208–215. Afoakwa, E. O., Paterson, A., Fowler, M., & Ryan, A. (2008). Flavor formation and character in cocoa and chocolate: A critical review. Critical Reviews in Food Science and Nutrition, 48(9), 840–857. Allison, H. W. S., & Rohan, T. A. (1958). A new approach to the fermentation of West African Amelonado Cocoa. Tropical Agriculture (St. Augustine), 35, 279–288. Ares, G., Deliza, R., Barreiro, C., Giménez, A., & Gámbaro, A. (2010). Comparison of two sensory profiling techniques based on consumer perception. Food Quality and Preference, 21(4), 417–426. Belitz, H. -D., Grosch, W., & Schieberle, P. (2004). Food chemistry (3rd ed.) Berlin– Heidelberg, Germany: Springer-Verlag. Biehl, B., Brunner, E., Passern, D., Quesnel, V., & Adomako, D. (1985). Acidification, proteolysis and flavour potential in fermenting cocoa beans. Journal of the Science of Food and Agriculture, 36(7), 583–598. Bonvehí, J. S. (2005). Investigation of aromatic compounds in roasted cocoa powder. European Food Research and Technology, 221(1–2), 19–29. Camu, N., De Winter, T., Verbrugghe, K., Cleenwerck, I., Vandamme, P., Takrama, J. S., et al. (2007). Dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in Ghana. Applied and Environmental Microbiology, 73(6), 1809–1824. Carrau, F. M., Medina, K., Boido, E., Farina, L., Gaggero, C., Dellacassa, E., et al. (2005). De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts. FEMS Microbiology Letters, 243(1), 107–115. Counet, C., & Callemien, D. (2002). Use of gas chromatography–olfactometry to identify key odorant compounds in dark chocolate. Comparison of samples before and after conching. Journal of Agricultural and Food Chemistry, 50(8), 2385–2391. Crafack, M., Mikkelsen, M. B., Saerens, S., Knudsen, M., Blennow, A., Lowor, S., et al. (2013). Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation. International Journal of Food Microbiology, 167, 103–116. De Brito, E. S., García, N. H. P., Gallão, M., Cortelazzo, A. L., Fevereiro, P.S., & Braga, M. R. (2000). Structural and chemical changes in cocoa (Theobroma cacao L) during fermentation, drying and roasting. Journal of the Science of Food and Agriculture, 81(2), 281–288. Delfini, C., Gaia, P., Bardi, L., & Mariscalco, G. (1991). Production of benzaldehyde, benzyl alcohol and benzoic acid by yeasts and Botrytis cinerea isolated from grape musts and wines. Vitis, 30, 253–263. Frauendorfer, F., & Schieberle, P. (2006). Identification of the key aroma compounds in cocoa powder based on molecular sensory correlations. Journal of Agricultural and Food Chemistry, 54(15), 5521–5529. Hansen, C. E., Del Olmo, M., & Burri, C. (1998). Enzyme activities in cocoa beans during fermentation. Journal of the Science of Food and Agriculture, 77(2), 273–281.

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Please cite this article as: Crafack, M., et al., Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.04.032