Research Article Received: 9 April 2014
Revised: 20 June 2014
Accepted article published: 18 July 2014
Published online in Wiley Online Library: 15 August 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6831
A comparative study of aroma-active compounds between dark and milk chocolate: relationship to sensory perception Jianbin Liu,a Mengya Liu,a Congcong He,a Huanlu Song,a* Jia Guo,b Ye Wang,b Haiying Yangb and Xiaoxia Sub Abstract BACKGROUND: The most important aroma-active compounds of two types of chocolate and cocoa liquor used for their production were analysed by gas chromatography–olfactometry–mass spectrometry (GC-O-MS) and aroma extract dilution analysis (AEDA). Furthermore, the relationship between odorants and sensory perception of chocolate was measured by quantitative analysis, sensory evaluation and correlation analysis. In addition, some chemicals were added to the original dark or milk chocolate to validate their roles in the aroma property of chocolate. RESULTS: A total of 32 major aroma-active compounds were identified in the chocolate with the flavour dilution factors of 27–729 by AEDA, including seven aldehydes, six pyrazines, three pyrroles, four carboxylic acids, four lactones, two alcohols, two ketones, one ester, one pyrone, one furan and one sulfur-containing compound. Further quantitative analysis showed that dark chocolate had higher contents of pyrazine, pyrrole, carboxylic acids, alcohols and Strecker aldehydes, whereas the concentration of lactones, esters, long chain aldehydes and ketones were higher in the milk type. CONCLUSION: Differences in volatile composition and descriptive flavour attributes between the dark and milk chocolate were observed. The relationship between aroma-active compounds and sensory perception in the chocolate was verified. © 2014 Society of Chemical Industry Keywords: chocolate; gas chromatography–olfactrometry–mass spectrometry (GC-O-MS); solvent-assisted flavour evaporation (SAFE); aroma extract dilution analysis (AEDA); sensory perception
INTRODUCTION
1362
Chocolate is an important food appreciated by consumers all over the world. The complex matrix of semi-solid chocolate is composed of sugar, cocoa liquor (mass), cocoa butter, lecithin and dairy products.1 Chocolate has been classified based on its formula, particularly, two basic commodity forms of chocolate, i.e. the dark and milk, have many differences in their ingredients and sensory properties, which highly influenced the eating behaviour of consumers.2 Milk powder is one of the most important food additives for the manufacture of milk chocolate, which can greatly change the physical properties of chocolate as well as organoleptic properties.3 – 5 The addition of dairy products may increase the milky and caramel-like aroma of milk chocolate.6 On the other hand, the presence of milk fat induced a significant decrease in the release volatiles.7 Together with different changing patterns of conching, the typical flavour of each type of chocolate was formed.8 Many investigations have aimed to clarify the aroma-active compounds of cocoa products in the early stage. More than 40 years ago, research was performed to reveal the contribution of Strecker aldehydes as well as pyrazine to the cocoa-like odour of roasted cocoa beans.9,10 Later studies evaluated the key aroma-active compounds of cocoa liquor, milk chocolate, dark chocolate and cocoa powder, respectively.11 – 13 These authors have identified 2-methylpropanal, 3(or 2)-methylbutanal, phenylacetaldehyde, J Sci Food Agric 2015; 95: 1362–1372
2-ethyl-2(or 3),5-dimethylpyrazine, 2-acetyl-1-pyrroline, 2,3dimethylpyrazine, trimethylpyrazine, tetramethylpyrazine, 3,5(or 2)-diethyl-2(or 5)-methylpyrazine, 3-methylbutanoic acid and 4-hydroxy-2,5-dimethyl-3(2H)-furanone (FuraneolTM ) as the important compounds to the overall flavour of cocoa products. Particularly, 1-octen-3-one, dimethyl trisulfide, (E,E)-2,4nonadienal, (E,E)-2,4-decadienal, 5-methyl-(E)-2-hepten-4-one and 𝛾-decalactone were important contributors to the aroma of milk chocolate but did not show much contribution to the flavour of cocoa liquor.11 The impact of various storage conditions on the flavour quality of dark chocolate was also investigated.14 However, few systematic studies have been conducted to evaluate the relative importance of flavour compounds in the different types of chocolate. The relationship between these odorants and odour property of different types of chocolate is also unclear.
∗
Correspondence to: Huanlu Song, Laboratory of Molecular Sensory Science, Beijing Technology and Business University, Beijing 100048, China. E-mail: [email protected]
a Laboratory of Molecular Sensory Science, Beijing Technology and Business University, Beijing 100048, China b Nutrition and Health Research Institute of China Oil & Foodstuffs Corporation (COFCO), Beijing 100020, China
www.soci.org
© 2014 Society of Chemical Industry
Aroma-active compounds in chocolate
www.soci.org
The objective of this study was to investigate the differences in volatile composition and descriptive flavour attributes between dark and milk chocolate. Furthermore, the relationship between odorants and the sensory perception of each types of chocolate was attempted to determine by sensory evaluation, clustering analysis and correlation analysis. Finally, some chemicals were added to the original dark or milk chocolate to validate their roles in the aroma property of chocolate.
and vanillin. Dark chocolate samples (designated as DC1, DC2, DC3 for three batches, 5 days between batches) and milk chocolate samples (designated as MC1, MC2, MC3 for three batches, 5 days between batches) were provided by the Nutrition and Health Research Institute of China Oil & Foodstuffs Corporation (Beijing, China). Cocoa liquor samples (designated as CL1, CL2, CL3 for three parallel samples, variety is forastero) used for their production were supplied by Barry Callebaut (Klang, Malaysia). All the chocolate samples were stored at 10 ∘ C, relative humidity ≤ 5.5% for about 1 month before analysis.
MATERIALS AND METHODS Sample and chemicals Chocolates The ingredients of commercial dark chocolate were 47% sucrose, 10% cocoa butter, 42.6% cocoa liquor, 0.4% lecithin as emulsifier and moderate vanillin as flavour enhancer. The commercial milk chocolate formulation used in this study was 47% sucrose, 15.6% milk powder, 15% cocoa liquor, 22% cocoa butter, 0.4% lecithin
Chemicals and reference odorants Dichloromethane (AR), sodium bicarbonate (AR), anhydrous sodium sulfate (AR) and hydrochloric acid (AR) were purchased from Beijing Chemical Reagents Company (Beijing, China). The reference compounds 1, 2, 4–6, 8–17, 19, 21–25, 27–29 and 31 listed in Table 1, n-alkanes (C7 –C30 ) (chromatographic reagent) and 2-methyl-3-heptanone were purchased from Sigma–Aldrich
Table 1. Calibration data used for quantification of selected aroma-active components of chocolate by SAFE–gas chromatography–mass spectroscopy Nr 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Compound
CAS no.
2-Methylpropanal 3-Methylbutanal Hexanal 2,5-Dimethylpyrazine 2-Acetyl-1-pyrroline Dimethyl trisulfide 2-Nonanone 2-Ethyl-6-methylpyrazine Nonanal Trimethylpyrazine 3-Ethyl-2,5-dimethylpyrazine Tetramethylpyrazine Acetic acid 3,5-Diethyl-2-methylpyrazine Benzaldehyde Linalool 2-Methylpropanoic acid 5-Methyl-1H-pyrrole-2-carboxaldehyde Benzeneacetaldehyde 2-Furan methanol 3-Methylbutanoic acid 2-Phenethyl acetate Hexanoic acid Phenylethanol 𝛾-Octalactone Maltol 2-Acetylpyrrole 4-Hydroxy-2,5-dimethyl-3(2H)-furanone 𝛾-Decalactone 𝛿-Decalactone Vanillin
78-84-2 590-86-3 66-25-1 123-32-0 85213-22-5 3658-80-8 821-55-6 13925-03-6 124-19-6 14667-55-1 13360-65-1 1124-11-4 64-19-7 18138-05-1 100-52-7 78-70-6 79-31-2 1192-79-6 122-78-1 98-00-0 503-74-2 103-45-7 142-62-1 60-12-8 104-50-7 118-71-8 1072-83-9 3658-77-3 706-14-9 705-86-2 121-33-5
m/z 72 86 100 108 111 126 142 122 142 122 136 136 60 150 106 154 88 109 120 98 102 164 116 122 142 126 109 128 170 170 152
Slope 53 637 839 73 911 288 10 445 250 36 572 649 2 320 016 3 567 353 9 419 696 19 553 356 35 891 998 17 663 628 158 756 23 110 844 4 993 595 9 270 488 9 141 053 311 244 5 806 347 1 403 949 9 673 341 51 403 493 2 640 771 15 563 057 5 482 296 22 222 657 21 453 630 15 791 631 56 303 948 14 336 573 1 394 150 115 726 111 747 450
Y-intercept −5 707 329 −4 875 376 783 375 −266 981 1 780 023 284 319 486 500 −1 730 063 2 638 126 57 492 10 185 2 164 424 −422 978 −253 146 −540 203 47 819 −214 078 −38 085 −773 939 −564 669 104 577 1 009 613 −508 486 212 712 −135 415 235 085 −4 781 953 −5 362 677 −19 876 −15 072 6 596 916
R2 0.99 0.97 0.99 0.99 0.99 0.99 0.99 0.98 0.99 0.99 0.99 0.98 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.98
J Sci Food Agric 2015; 95: 1362–1372
© 2014 Society of Chemical Industry
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m/z is the mass ion used for identification of compounds. R2 is the coefficient of determination of regression curve. SAFE, solvent-assisted flavour evaporation.
wileyonlinelibrary.com/jsfa
www.soci.org (St Louis, MO, USA). Other authentic odorants – 3, 7, 18, 20, 26 and 30 – were from Huihai Scientific Instruments Co., Ltd (Beijing, China). Isolation of volatiles Solution extraction was applied to achieve the aroma extract. A total of 50 g chocolate powder was mixed with 100 mL freshly distilled dichloromethane and further homogenised for 2 h. 2-Methyl-3-heptanone (1 μL, 1.632 g L−1 in n-pentane) was added before homogenising as internal standard. The extract obtained was introduced into a solvent-assisted flavour evaporation (SAFE) system (Deutsche Forschungsanstalt für Lebensmittelchemie, Freising, Germany), which consisted of a high-vacuum pump, diffusion pump, receiving tube and waste tube operating at high vacuum (10−2 to 10−3 Pa) and very low temperature (−196 ∘ C) to trap volatile substances. Volatiles from the organic phase were distilled at 50 ∘ C as previously described.15 The condensation part in the liquid nitrogen cooled flask was washed three times with 40 mL (0.5 mol L−1 ) aqueous solution of sodium bicarbonate, followed by 40 mL of saturated sodium chloride solution two times to remove acidic volatile compounds. The bottom layer (dichloromethane), containing the neutral basic volatiles, was dried over anhydrous sodium sulfate overnight and concentrated in a Vigreux column (50 cm × 1 cm internal diameter) to about 200 μL . The upper layer (aqueous phase), containing the acidic volatiles, was acidified with hydrochloric acid (2 mol L−1 ) to pH 2.5 and then extracted three times with 30 mL dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and concentrated to about 200 μL by a Vigreux column. Gas chromatography–olfactometry–mass spectrometry All the volatiles were analysed by GC-MS (7890A-7000B; Agilent Technologies Inc., Santa Clara, CA, USA), which was equipped with an olfactory detector port (Sniffer 9000; Brechbühler, Schlieren, Switzerland). After injection, separations by GC were performed on a polar DB-Wax column (30 m × 0.32 mm × 0.25 m; J & W Scientific, Folsom, CA, USA) or a non-polar DB-5 column (30 m × 0.32 mm × 0.25 m; J & W Scientific), ultra-high purity helium was used as the carrier gas at 1.2 mL min−1 . The GC oven program was 35 ∘ C held 2 min and ramped at 6 ∘ C min−1 to 230 ∘ C, then held at this temperature for 20 min. The effluent from the capillary column was split 1:1, v/v, between the mass spectrometry detector (MSD) and the olfactory detector port (Sniffer 9000; Brechbühler. The GC-MS transfer line temperature was 240 ∘ C (DB-Wax, DB-5). Electron-impact liquor spectra were generated at 70 eV, with a m/z scan range from 40 to 300. The MS source temperature was 230 ∘ C. Three experienced panellists were recruited to perform sniffing test on GC-O. To prevent drying of the nasal mucosa, humidified air was supplied to the sniff port at 30 mL min−1 .
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Aroma extract dilution analysis The average flavour dilution (FD) factor of every volatile fraction was determined at the sniffing port according to the AEDA procedure analysis. For example, serial dilutions were prepared from the initial acidic or neutral-basic fractions at a ratio of 1:3 in dichloromethane. Aliquots were then analysed by GC-O. GC-O was performed by three experienced panellists, the FD factor of one compound was determined at one dilution (1, 3, 9, 27 and so on) in which none of the panellist could detect the smell of
wileyonlinelibrary.com/jsfa
J Liu et al.
the compound in the olfactory detecting port. Each panellist has been received at least two years of weekly GC-O training. Compounds with higher FD factor are considered to be more important. Identification of the compounds The preliminary identification of volatile compounds was based on the NIST 2.0 mass spectra libraries installed in the GC-MS. Reconfirmation was performed by comparison of the mass spectrum (MS), retention index (RI) and odour descriptions with authentic standards as described previously.16 Quantification of major aroma-active compounds Quantitative data was obtained by calculating standard curve of each major aroma-active compound. To achieve this, a serial dilution (1:10, five dilutions) containing all 31 authentic standards was constructed, and an aliquot (1 μL) of these solutions were injected into the GC-MS system at the same analysis condition, respectively. The concentration of components was proposed by using the regression curve obtained from a series of peak area of standard compounds. Calibration data was listed in Table 1. Sensory evaluation Fifteen trained panellists (seven males and eight females, aged 20–39 years) were recruited from Laboratory of Molecular Sensory Science, Beijing Technology and Business University and Nutrition and Health Research Institute of COFCO by verifying the lexicon in
Table 2. Flavour attributes selected for sensory evaluation Attribute Malty Nutty
Milky
Characteristic
Relevant aroma model
Typical flavour of malt extract Delicate aroma of indistinguishable nuts without roast
The aroma of malt extract Mixed raw nuts powder (hazelnut, walnut, peanut, and sunflower seeds) Fresh milk
The flavour of newly boiled milk Sour/harsh The flavour of distilled white vinegar with a little pungent aroma Fruity(fresh) Desirable odour of fresh fruit; it is closer to orange than to apple Sweet/honey-like The smell that is reminiscent of the aroma of honey Floral The aroma associated with a mixture of freshly cut flowers Roasty The aroma associated with popcorn or roasted peanut Caramel Typical flavour of roasted sugar
A 10% vinegar (acetic acid) solution
New cut orange peel or apple peel
Honey dissolved in warm water Newly cut Jasminum sambac Roasted peanut and popcorn Roasted sugar
All the attributes were generated as a result of discussions between the panellists, with guidance from the references.
© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 1362–1372
Aroma-active compounds in chocolate
www.soci.org
the technique of descriptive analysis. All samples were prepared, served and evaluated under appropriate conditions. To train the sensory panel to recognise the aroma character of chocolate, daily training sessions were conducted for 3 months. For example, the panellists were trained to evaluate the aroma of following an aroma model: five different commercial dark/milk chocolates for the overall aroma profile of chocolate, malt extract for malty aroma, mixed raw nuts powder for nutty aroma, fresh milk for milky aroma, 10% vinegar (acetic acid) solution for sour/harsh aroma, newly cut orange peel or apple peel for fruity (fresh) aroma, honey solution for honey-like/sweet aroma, newly cut Jasminum sambac for floral aroma, roasted peanut for roasty aroma and roasted sugar for caramel aroma. These items were generated by the panel with the guidance of reference compounds (Table 2).17
Sensory evaluation was performed by the method recently described with a little revolution.18 The detail was as follows: after introducing 5 g of chocolate or cocoa liquor powder into the mouth for 15 s followed by chewing 10 times, deep expiration was performed several times to obtain the aroma, evaluation was asked on a scale from 0 (not detectable) to 5 (strongly detectable) with 10-point category scales for each attribute. Each panellist was asked to take a rest for sensory recovery between each set of three different samples. Random Arabic numerals were given to the three types of samples; all the evaluations were performed under a dim light to mask the colour differences between samples. All nine samples were evaluated in one-time evaluation; three independent sessions were included. Differences of the same result between sessions were not more than two category scales.
Table 3. Predominant odorants identified in dark chocolate (DC), milk chocolate (MC) and cocoa liquor (CL) by aroma extract dilution analysis Flavour dilution factorc
RI Nr
Compound name
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
2-Methylpropanal 3-Methylbutanal Hexanal 1-Octen-3-one 2,5-Dimethylpyrazine 2-Acetyl-1-pyrroline Dimethyl trisulfide 2-Ethyl-6-methylpyrazine 2-Nonanone Nonanal Trimethylpyrazine 3-Ethyl-2,5-dimethylpyrazine Tetramethylpyrazine Acetic acid 3,5-Diethyl-2-methylpyrazine Benzaldehyde Linalool 2-Methylpropanoic acid 5-Methyl-1H-pyrrole-2-carboxaldehyde Phenylacetaldehyde 2-Furanmethanol 3-Methylbutanoic acid 2-Phenethyl acetate Hexanoic acid Phenylethanol 𝛾-Octalactone Maltol 2-Acetylpyrrole 4-Hydroxy-2,5-dimethyl-3(2H)-furanone 𝛾-Decalactone 𝛿-Decalactone Vanillin
DB-wax 820 921 1041 1245 1312 1338 1369 1377 1381 1383 1394 1435 1464 1469 1481 1510 1525 1542 1606 1628 1634 1639 1791 1835 1910 1920 1944 1956 2033 2148 2197 2258
DB-5
Odour propertyb
DC
MC
CL
Revised: 20 June 2014
Accepted article published: 18 July 2014
Published online in Wiley Online Library: 15 August 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6831
A comparative study of aroma-active compounds between dark and milk chocolate: relationship to sensory perception Jianbin Liu,a Mengya Liu,a Congcong He,a Huanlu Song,a* Jia Guo,b Ye Wang,b Haiying Yangb and Xiaoxia Sub Abstract BACKGROUND: The most important aroma-active compounds of two types of chocolate and cocoa liquor used for their production were analysed by gas chromatography–olfactometry–mass spectrometry (GC-O-MS) and aroma extract dilution analysis (AEDA). Furthermore, the relationship between odorants and sensory perception of chocolate was measured by quantitative analysis, sensory evaluation and correlation analysis. In addition, some chemicals were added to the original dark or milk chocolate to validate their roles in the aroma property of chocolate. RESULTS: A total of 32 major aroma-active compounds were identified in the chocolate with the flavour dilution factors of 27–729 by AEDA, including seven aldehydes, six pyrazines, three pyrroles, four carboxylic acids, four lactones, two alcohols, two ketones, one ester, one pyrone, one furan and one sulfur-containing compound. Further quantitative analysis showed that dark chocolate had higher contents of pyrazine, pyrrole, carboxylic acids, alcohols and Strecker aldehydes, whereas the concentration of lactones, esters, long chain aldehydes and ketones were higher in the milk type. CONCLUSION: Differences in volatile composition and descriptive flavour attributes between the dark and milk chocolate were observed. The relationship between aroma-active compounds and sensory perception in the chocolate was verified. © 2014 Society of Chemical Industry Keywords: chocolate; gas chromatography–olfactrometry–mass spectrometry (GC-O-MS); solvent-assisted flavour evaporation (SAFE); aroma extract dilution analysis (AEDA); sensory perception
INTRODUCTION
1362
Chocolate is an important food appreciated by consumers all over the world. The complex matrix of semi-solid chocolate is composed of sugar, cocoa liquor (mass), cocoa butter, lecithin and dairy products.1 Chocolate has been classified based on its formula, particularly, two basic commodity forms of chocolate, i.e. the dark and milk, have many differences in their ingredients and sensory properties, which highly influenced the eating behaviour of consumers.2 Milk powder is one of the most important food additives for the manufacture of milk chocolate, which can greatly change the physical properties of chocolate as well as organoleptic properties.3 – 5 The addition of dairy products may increase the milky and caramel-like aroma of milk chocolate.6 On the other hand, the presence of milk fat induced a significant decrease in the release volatiles.7 Together with different changing patterns of conching, the typical flavour of each type of chocolate was formed.8 Many investigations have aimed to clarify the aroma-active compounds of cocoa products in the early stage. More than 40 years ago, research was performed to reveal the contribution of Strecker aldehydes as well as pyrazine to the cocoa-like odour of roasted cocoa beans.9,10 Later studies evaluated the key aroma-active compounds of cocoa liquor, milk chocolate, dark chocolate and cocoa powder, respectively.11 – 13 These authors have identified 2-methylpropanal, 3(or 2)-methylbutanal, phenylacetaldehyde, J Sci Food Agric 2015; 95: 1362–1372
2-ethyl-2(or 3),5-dimethylpyrazine, 2-acetyl-1-pyrroline, 2,3dimethylpyrazine, trimethylpyrazine, tetramethylpyrazine, 3,5(or 2)-diethyl-2(or 5)-methylpyrazine, 3-methylbutanoic acid and 4-hydroxy-2,5-dimethyl-3(2H)-furanone (FuraneolTM ) as the important compounds to the overall flavour of cocoa products. Particularly, 1-octen-3-one, dimethyl trisulfide, (E,E)-2,4nonadienal, (E,E)-2,4-decadienal, 5-methyl-(E)-2-hepten-4-one and 𝛾-decalactone were important contributors to the aroma of milk chocolate but did not show much contribution to the flavour of cocoa liquor.11 The impact of various storage conditions on the flavour quality of dark chocolate was also investigated.14 However, few systematic studies have been conducted to evaluate the relative importance of flavour compounds in the different types of chocolate. The relationship between these odorants and odour property of different types of chocolate is also unclear.
∗
Correspondence to: Huanlu Song, Laboratory of Molecular Sensory Science, Beijing Technology and Business University, Beijing 100048, China. E-mail: [email protected]
a Laboratory of Molecular Sensory Science, Beijing Technology and Business University, Beijing 100048, China b Nutrition and Health Research Institute of China Oil & Foodstuffs Corporation (COFCO), Beijing 100020, China
www.soci.org
© 2014 Society of Chemical Industry
Aroma-active compounds in chocolate
www.soci.org
The objective of this study was to investigate the differences in volatile composition and descriptive flavour attributes between dark and milk chocolate. Furthermore, the relationship between odorants and the sensory perception of each types of chocolate was attempted to determine by sensory evaluation, clustering analysis and correlation analysis. Finally, some chemicals were added to the original dark or milk chocolate to validate their roles in the aroma property of chocolate.
and vanillin. Dark chocolate samples (designated as DC1, DC2, DC3 for three batches, 5 days between batches) and milk chocolate samples (designated as MC1, MC2, MC3 for three batches, 5 days between batches) were provided by the Nutrition and Health Research Institute of China Oil & Foodstuffs Corporation (Beijing, China). Cocoa liquor samples (designated as CL1, CL2, CL3 for three parallel samples, variety is forastero) used for their production were supplied by Barry Callebaut (Klang, Malaysia). All the chocolate samples were stored at 10 ∘ C, relative humidity ≤ 5.5% for about 1 month before analysis.
MATERIALS AND METHODS Sample and chemicals Chocolates The ingredients of commercial dark chocolate were 47% sucrose, 10% cocoa butter, 42.6% cocoa liquor, 0.4% lecithin as emulsifier and moderate vanillin as flavour enhancer. The commercial milk chocolate formulation used in this study was 47% sucrose, 15.6% milk powder, 15% cocoa liquor, 22% cocoa butter, 0.4% lecithin
Chemicals and reference odorants Dichloromethane (AR), sodium bicarbonate (AR), anhydrous sodium sulfate (AR) and hydrochloric acid (AR) were purchased from Beijing Chemical Reagents Company (Beijing, China). The reference compounds 1, 2, 4–6, 8–17, 19, 21–25, 27–29 and 31 listed in Table 1, n-alkanes (C7 –C30 ) (chromatographic reagent) and 2-methyl-3-heptanone were purchased from Sigma–Aldrich
Table 1. Calibration data used for quantification of selected aroma-active components of chocolate by SAFE–gas chromatography–mass spectroscopy Nr 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Compound
CAS no.
2-Methylpropanal 3-Methylbutanal Hexanal 2,5-Dimethylpyrazine 2-Acetyl-1-pyrroline Dimethyl trisulfide 2-Nonanone 2-Ethyl-6-methylpyrazine Nonanal Trimethylpyrazine 3-Ethyl-2,5-dimethylpyrazine Tetramethylpyrazine Acetic acid 3,5-Diethyl-2-methylpyrazine Benzaldehyde Linalool 2-Methylpropanoic acid 5-Methyl-1H-pyrrole-2-carboxaldehyde Benzeneacetaldehyde 2-Furan methanol 3-Methylbutanoic acid 2-Phenethyl acetate Hexanoic acid Phenylethanol 𝛾-Octalactone Maltol 2-Acetylpyrrole 4-Hydroxy-2,5-dimethyl-3(2H)-furanone 𝛾-Decalactone 𝛿-Decalactone Vanillin
78-84-2 590-86-3 66-25-1 123-32-0 85213-22-5 3658-80-8 821-55-6 13925-03-6 124-19-6 14667-55-1 13360-65-1 1124-11-4 64-19-7 18138-05-1 100-52-7 78-70-6 79-31-2 1192-79-6 122-78-1 98-00-0 503-74-2 103-45-7 142-62-1 60-12-8 104-50-7 118-71-8 1072-83-9 3658-77-3 706-14-9 705-86-2 121-33-5
m/z 72 86 100 108 111 126 142 122 142 122 136 136 60 150 106 154 88 109 120 98 102 164 116 122 142 126 109 128 170 170 152
Slope 53 637 839 73 911 288 10 445 250 36 572 649 2 320 016 3 567 353 9 419 696 19 553 356 35 891 998 17 663 628 158 756 23 110 844 4 993 595 9 270 488 9 141 053 311 244 5 806 347 1 403 949 9 673 341 51 403 493 2 640 771 15 563 057 5 482 296 22 222 657 21 453 630 15 791 631 56 303 948 14 336 573 1 394 150 115 726 111 747 450
Y-intercept −5 707 329 −4 875 376 783 375 −266 981 1 780 023 284 319 486 500 −1 730 063 2 638 126 57 492 10 185 2 164 424 −422 978 −253 146 −540 203 47 819 −214 078 −38 085 −773 939 −564 669 104 577 1 009 613 −508 486 212 712 −135 415 235 085 −4 781 953 −5 362 677 −19 876 −15 072 6 596 916
R2 0.99 0.97 0.99 0.99 0.99 0.99 0.99 0.98 0.99 0.99 0.99 0.98 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.98
J Sci Food Agric 2015; 95: 1362–1372
© 2014 Society of Chemical Industry
1363
m/z is the mass ion used for identification of compounds. R2 is the coefficient of determination of regression curve. SAFE, solvent-assisted flavour evaporation.
wileyonlinelibrary.com/jsfa
www.soci.org (St Louis, MO, USA). Other authentic odorants – 3, 7, 18, 20, 26 and 30 – were from Huihai Scientific Instruments Co., Ltd (Beijing, China). Isolation of volatiles Solution extraction was applied to achieve the aroma extract. A total of 50 g chocolate powder was mixed with 100 mL freshly distilled dichloromethane and further homogenised for 2 h. 2-Methyl-3-heptanone (1 μL, 1.632 g L−1 in n-pentane) was added before homogenising as internal standard. The extract obtained was introduced into a solvent-assisted flavour evaporation (SAFE) system (Deutsche Forschungsanstalt für Lebensmittelchemie, Freising, Germany), which consisted of a high-vacuum pump, diffusion pump, receiving tube and waste tube operating at high vacuum (10−2 to 10−3 Pa) and very low temperature (−196 ∘ C) to trap volatile substances. Volatiles from the organic phase were distilled at 50 ∘ C as previously described.15 The condensation part in the liquid nitrogen cooled flask was washed three times with 40 mL (0.5 mol L−1 ) aqueous solution of sodium bicarbonate, followed by 40 mL of saturated sodium chloride solution two times to remove acidic volatile compounds. The bottom layer (dichloromethane), containing the neutral basic volatiles, was dried over anhydrous sodium sulfate overnight and concentrated in a Vigreux column (50 cm × 1 cm internal diameter) to about 200 μL . The upper layer (aqueous phase), containing the acidic volatiles, was acidified with hydrochloric acid (2 mol L−1 ) to pH 2.5 and then extracted three times with 30 mL dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and concentrated to about 200 μL by a Vigreux column. Gas chromatography–olfactometry–mass spectrometry All the volatiles were analysed by GC-MS (7890A-7000B; Agilent Technologies Inc., Santa Clara, CA, USA), which was equipped with an olfactory detector port (Sniffer 9000; Brechbühler, Schlieren, Switzerland). After injection, separations by GC were performed on a polar DB-Wax column (30 m × 0.32 mm × 0.25 m; J & W Scientific, Folsom, CA, USA) or a non-polar DB-5 column (30 m × 0.32 mm × 0.25 m; J & W Scientific), ultra-high purity helium was used as the carrier gas at 1.2 mL min−1 . The GC oven program was 35 ∘ C held 2 min and ramped at 6 ∘ C min−1 to 230 ∘ C, then held at this temperature for 20 min. The effluent from the capillary column was split 1:1, v/v, between the mass spectrometry detector (MSD) and the olfactory detector port (Sniffer 9000; Brechbühler. The GC-MS transfer line temperature was 240 ∘ C (DB-Wax, DB-5). Electron-impact liquor spectra were generated at 70 eV, with a m/z scan range from 40 to 300. The MS source temperature was 230 ∘ C. Three experienced panellists were recruited to perform sniffing test on GC-O. To prevent drying of the nasal mucosa, humidified air was supplied to the sniff port at 30 mL min−1 .
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Aroma extract dilution analysis The average flavour dilution (FD) factor of every volatile fraction was determined at the sniffing port according to the AEDA procedure analysis. For example, serial dilutions were prepared from the initial acidic or neutral-basic fractions at a ratio of 1:3 in dichloromethane. Aliquots were then analysed by GC-O. GC-O was performed by three experienced panellists, the FD factor of one compound was determined at one dilution (1, 3, 9, 27 and so on) in which none of the panellist could detect the smell of
wileyonlinelibrary.com/jsfa
J Liu et al.
the compound in the olfactory detecting port. Each panellist has been received at least two years of weekly GC-O training. Compounds with higher FD factor are considered to be more important. Identification of the compounds The preliminary identification of volatile compounds was based on the NIST 2.0 mass spectra libraries installed in the GC-MS. Reconfirmation was performed by comparison of the mass spectrum (MS), retention index (RI) and odour descriptions with authentic standards as described previously.16 Quantification of major aroma-active compounds Quantitative data was obtained by calculating standard curve of each major aroma-active compound. To achieve this, a serial dilution (1:10, five dilutions) containing all 31 authentic standards was constructed, and an aliquot (1 μL) of these solutions were injected into the GC-MS system at the same analysis condition, respectively. The concentration of components was proposed by using the regression curve obtained from a series of peak area of standard compounds. Calibration data was listed in Table 1. Sensory evaluation Fifteen trained panellists (seven males and eight females, aged 20–39 years) were recruited from Laboratory of Molecular Sensory Science, Beijing Technology and Business University and Nutrition and Health Research Institute of COFCO by verifying the lexicon in
Table 2. Flavour attributes selected for sensory evaluation Attribute Malty Nutty
Milky
Characteristic
Relevant aroma model
Typical flavour of malt extract Delicate aroma of indistinguishable nuts without roast
The aroma of malt extract Mixed raw nuts powder (hazelnut, walnut, peanut, and sunflower seeds) Fresh milk
The flavour of newly boiled milk Sour/harsh The flavour of distilled white vinegar with a little pungent aroma Fruity(fresh) Desirable odour of fresh fruit; it is closer to orange than to apple Sweet/honey-like The smell that is reminiscent of the aroma of honey Floral The aroma associated with a mixture of freshly cut flowers Roasty The aroma associated with popcorn or roasted peanut Caramel Typical flavour of roasted sugar
A 10% vinegar (acetic acid) solution
New cut orange peel or apple peel
Honey dissolved in warm water Newly cut Jasminum sambac Roasted peanut and popcorn Roasted sugar
All the attributes were generated as a result of discussions between the panellists, with guidance from the references.
© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 1362–1372
Aroma-active compounds in chocolate
www.soci.org
the technique of descriptive analysis. All samples were prepared, served and evaluated under appropriate conditions. To train the sensory panel to recognise the aroma character of chocolate, daily training sessions were conducted for 3 months. For example, the panellists were trained to evaluate the aroma of following an aroma model: five different commercial dark/milk chocolates for the overall aroma profile of chocolate, malt extract for malty aroma, mixed raw nuts powder for nutty aroma, fresh milk for milky aroma, 10% vinegar (acetic acid) solution for sour/harsh aroma, newly cut orange peel or apple peel for fruity (fresh) aroma, honey solution for honey-like/sweet aroma, newly cut Jasminum sambac for floral aroma, roasted peanut for roasty aroma and roasted sugar for caramel aroma. These items were generated by the panel with the guidance of reference compounds (Table 2).17
Sensory evaluation was performed by the method recently described with a little revolution.18 The detail was as follows: after introducing 5 g of chocolate or cocoa liquor powder into the mouth for 15 s followed by chewing 10 times, deep expiration was performed several times to obtain the aroma, evaluation was asked on a scale from 0 (not detectable) to 5 (strongly detectable) with 10-point category scales for each attribute. Each panellist was asked to take a rest for sensory recovery between each set of three different samples. Random Arabic numerals were given to the three types of samples; all the evaluations were performed under a dim light to mask the colour differences between samples. All nine samples were evaluated in one-time evaluation; three independent sessions were included. Differences of the same result between sessions were not more than two category scales.
Table 3. Predominant odorants identified in dark chocolate (DC), milk chocolate (MC) and cocoa liquor (CL) by aroma extract dilution analysis Flavour dilution factorc
RI Nr
Compound name
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
2-Methylpropanal 3-Methylbutanal Hexanal 1-Octen-3-one 2,5-Dimethylpyrazine 2-Acetyl-1-pyrroline Dimethyl trisulfide 2-Ethyl-6-methylpyrazine 2-Nonanone Nonanal Trimethylpyrazine 3-Ethyl-2,5-dimethylpyrazine Tetramethylpyrazine Acetic acid 3,5-Diethyl-2-methylpyrazine Benzaldehyde Linalool 2-Methylpropanoic acid 5-Methyl-1H-pyrrole-2-carboxaldehyde Phenylacetaldehyde 2-Furanmethanol 3-Methylbutanoic acid 2-Phenethyl acetate Hexanoic acid Phenylethanol 𝛾-Octalactone Maltol 2-Acetylpyrrole 4-Hydroxy-2,5-dimethyl-3(2H)-furanone 𝛾-Decalactone 𝛿-Decalactone Vanillin
DB-wax 820 921 1041 1245 1312 1338 1369 1377 1381 1383 1394 1435 1464 1469 1481 1510 1525 1542 1606 1628 1634 1639 1791 1835 1910 1920 1944 1956 2033 2148 2197 2258
DB-5
Odour propertyb
DC
MC
CL
