Food Chemistry 153 (2014) 81–86
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Characterisation of volatile compounds in a smoke flavouring from rice husk Jorge A. Pino ⇑ Food Industry Research Institute, Carretera al Guatao km 3½, P.O. Box 19200, Havana, Cuba
a r t i c l e
i n f o
Article history: Received 4 October 2013 Received in revised form 4 December 2013 Accepted 9 December 2013 Available online 15 December 2013 Keywords: Smoke flavouring Rice husk Volatiles Aroma extract dilution analysis
a b s t r a c t An aqueous smoke flavouring from rice husk was obtained on a laboratory scale. The volatile compounds were isolated by simultaneous steam distillation–solvent extraction and its identification and quantitative composition was studied by GC–MS and GC–FID. A total of 93 compounds were isolated and 86 of them were positively identified. Major compounds (more than 5% GC area) were 2-furfural, phenol, 2-methoxyphenol, 4-ethyl-2-methoxyphenol, and 2,6-dimethoxyphenol. Application of aroma extract dilution analysis on the volatile fraction revealed that 2-methoxyphenol, 4-methyl-2-methoxyphenol, 2,6-dimethoxyphenol, 2-furfural, 2-acetylfuran, 3-methyl-1,2-cyclopentanedione, acetic acid, 5-methyl-2-furfural, 4-(2-propenyl)-2-methoxyphenol, 4-methyl-2,6-dimethoxyphenol, phenol, 2,6dimethylphenol, 4-ethyl-2-methoxyphenol, 2-methylphenol were the most odour-active compounds. Ó 2014 Published by Elsevier Ltd.
1. Introduction Liquid smoke flavourings, manufactured from wood pyrolysates are reported to offer many advantages over traditional smoking in a kiln, namely ease of application, speed and product uniformity. A large literature exists on the composition of smokes and liquid smoke preparations and about the sensory properties of various smoke fractions and isolated compounds (Cadwallader, 1996; Chen & Maga, 1993; Goma, Gray, Rabie, López Bote, & Booren, 1993; Guillén & Ibargoitia, 1999; Guillén & Manzanos, 1996, 1997, 2002; Guillén, Manzanos, & Ibargoitia, 2001; Guillén, Manzanos, & Zabala, 1995; Kostyra & Baryłko-Pikielna, 2006; Vichi et al., 2007; Wittkowski, Baltes, & Jennings, 1990; Wittkowski, Töth, & Baltes, 1981; Zandersons et al., 2009), but our knowledge is still incomplete. Although the main method for obtaining volatile compounds related to ‘‘smokiness’’ is the thermal pyrolysis of cellulose, hemicellulose and lignin (Cadwallader, 2007), no research has been done on the use of cellulosic materials, such as agricultural residues and by-products, in place of wood. Rice husk is one of the most important, due to its production volume and more than 25 possible uses of this by-product have been proposed (González, Alvira, & González, 1986), but to this moment the attention of researchers has not been focused on its thermal pyrolysis to produce smoke flavouring. This in spite of the fact that the cellulose-hemicellulose-lignin ratio in rice husk is 3:1:1 (González,
⇑ Tel.: +53 7 2793956. E-mail address: [email protected] 0308-8146/$ - see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.foodchem.2013.12.041
Alvira, & González, 1987), similar to 2:1:1 of wood (Gilbert & Knowles, 1975). A smoke flavouring manufactured by pyrolysis of rice husk, highly appreciated for its sensory characteristics, has been developed. This paper reports the characterisation of the volatile compounds from this product and the identification of potent odorants in the aroma extract by application of aroma extract dilution analysis. 2. Materials and methods 2.1. Chemicals Standards of chemicals were purchased from Sigma–Aldrich (St. Louis, MO) and Fluka (Buchs, Switzerland), and some were supplied by Dallant (Barcelona, Spain). An n-alkane solution (C8– C32) was purchased from Sigma–Aldrich. Anhydrous sodium sulfate, absolute ethanol, n-pentane and diethyl ether were purchased from Merck (Darmstadt, Germany); the solvents were redistilled and checked for purity. 2.2. Sample Liquid smoke flavouring was prepared by a dry distillation of rice husk from a Cuban commercial rice variety. The laboratory smoke producer consisted of a steel cylindrical tube (35 6 cm i.d.) externally heated by a stove and connected to a 15-cm water-cooled condenser with a glass wool filter plug to catch tar aerosol. The following parameters were used: 350 g of rice husk
82
J.A. Pino / Food Chemistry 153 (2014) 81–86
(8% moisture content), generation temperature 300 °C, and smouldering period 3 h. The resulting aqueous condensate was capped and permitted to equilibrate at room temperature for three days before analysis. The aqueous liquid smoke obtained was filtered through a paper filter to eliminate the oily phase. The liquid smoke flavouring was brown in colour, and its odour has been described as strong smoky. Three replicates of the procedure were made. 2.3. Isolation of volatile compounds The choice of isolation method and solvent was based on published information (Guillén & Manzanos, 2002; Zandersons et al., 2009). A sample of 50 mL of the liquid smoke flavouring was extracted three times with dichloromethane (30 + 20 + 20 mL). An internal standard (2-isopropyl-5-methylphenol, 2 mg, dissolved in ethanol) was previously added to the sample. The extracts were combined and dried over anhydrous sodium sulfate, and carefully concentrated to 0.9 mL in a Kuderna-Danish evaporator with a Vigreux column (12 1 cm i.d.) and then to 0.3 mL with a gentle nitrogen stream. Extractions were made in triplicate. 2.4. GC–FID analysis Analyses were conducted on a Hewlett–Packard 6890N series II (Agilent, Santa Clara, CA) equipped with DB-Wax (30 m 0.25 mm, 0.25 lm film thickness; J & W Scientific, Folsom, CA), DB-5ms (30 m 0.25 mm, 0.25 lm film thickness; J & W Scientific) and HP-1 (30 m 0.25 mm, 0.25 lm film thickness; Agilent) capillary columns, working with the following temperature programme and conditions for all columns: 50 °C for 2 min, ramp of 4 °C min 1 up to 250 °C; injector and detector temperatures 250 °C; carrier gas helium (1 mL min 1); detector FID; injections 1 lL in split mode with 1:10 ratio. Quantitative determination of odour-active volatiles exhibiting FD higher than 32, was done by internal standard method in the DB-5ms column. For that purpose, 2-isopropyl-5-methylphenol was added as internal standard, together with a defined mixture of the respective analytes, to 50 mL of distilled water, and processed by using the same procedure presented above in Section 2.3. Calibration curves were constructing using a series of solutions of varying nominal concentrations containing each analyte, where the slope was assumed as the response factor. An identical amount of internal standard was added to each solution and the corresponding chromatograms obtained (IOFI, 2011). All data were obtained in triplicate. 2.5. GC–MS analysis Analyses were performed on a Hewlett–Packard 6890N series II (Agilent) gas chromatograph equipped with a HP-5973N massselective detector and with a DB-5ms column (30 m 0.25 mm, 0.25 lm film thickness; J & W Scientific). The temperature programme and carrier gas flow rate were the same as for GC–FID. EI-MS, electron energy, 70 eV; ion source and interface temperature, 250 °C. The acquisition was performed in scan mode (mass range m/z 35–400). Compounds were identified by their linear retention indices, by their mass spectra, by comparing their mass spectra with those in libraries (NIST 05, Wiley 6, NBS 75k, Adams 2001 and in-house Flavorlib) and with those in the literature and, in most cases, by using standards. All standard compounds used, from Aldrich, Fluka and Merck, are asterisked in Table 1. 2.6. Aroma profile analysis Aroma profile analyses were performed by a trained sensory panel consisting of five trained panellists. The following aroma
descriptors (Ojeda, Bárcenas, Pérez-Elortondo, Albisu, & Guillén, 2002), represented by the compounds given in parentheses, were chosen for sensory evaluation, and their intensities were ranked on a five-point scale (steps of 0.5) from 0 (not perceivable) to 5 (strongly perceivable): smoky (2,6-dimethoxyphenol), burnt (4methyl-2,6-dimethylphenol), sweet (2-methyl-2-cyclopenten-1one); caramel (3-methyl-1,2-cyclopentanedione), acid (acetic acid), phenolic (2,6-dimethylphenol), pungent (phenol), spicy [4(1-propenyl)-2-methoxyphenol], and woody [4-(2-propenyl)-2methoxyphenol]. The judgments of the panellists were averaged. 2.7. Gas chromatography–olfactometry analysis (GC–O) GC–O analyses were performed with a Hewlett–Packard 6890N series II gas chromatograph (Agilent) equipped with an FID, using two capillary columns, DB-Wax and DB-5ms (each 30 m 0.25 mm, 0.25 lm film thickness; J & W Scientific). Analytical conditions were the same as for the GC–FID analyses. The end of the capillary column was connected to a deactivated Y-shaped glass splitter dividing the effluent into two equal parts, which were transferred via two deactivated fused silica capillaries (50 cm 0.25 mm) to a sniffing port and an FID. The sniffing port, mounted on a detector base of the GC, consisted of a cylindrically shaped aluminium device with a bevelled top and a central drilled hole housing the capillary. Nitrogen (30 mL min 1) was used as make-up gas. The injection volume was 1 lL. During a GC–O run, the nose of the panellist was placed closely above the top of the sniffing port and the odour of the effluent was evaluated. If an odour was recognised, the retention time was marked in the chromatogram, and the odour quality was assigned. The sniffing port temperature was approximately 250 °C. The GC–O analyses were performed by three experienced assessors. Each one had a minimum of 20 h of previous GC–O experience and they had also previously taken part in the barramundi sensory descriptive analysis training and had actively contributed to the development of the descriptive sensory vocabulary. 2.8. Aroma extract dilution analysis (AEDA) The aroma extract was stepwise diluted to obtain dilutions of 1:1, 1:2, 1:4, 1:8, 1:16, 1:1024 of the original solutions (Schieberle, 1995). Each dilution was submitted to GC–O, using DB-Wax and DB-5ms capillary columns. Analytical conditions were the same as for GC–FID analyses. The odour-active compounds were located in the chromatograms, and each odorant detected was assigned an FD factor representing the highest dilution at which the odorant was detectable. The FD factors obtained by three assessors were averaged.
3. Results and discussion An important challenge in the analysis of food aroma compounds is that the method used for aroma isolation is appropriate. To address this problem, the entire volatiles from the smoke flavouring, isolated by liquid–liquid extraction, were evaluated by the sensory panel by smelling a drop of the organic extract on a strip of filter paper as done by perfumers. After evaporation of the solvent, all five panellists agreed that the distillate evoked the characteristic odour of the original smoke preparation. Furthermore, the aroma profiles of the smoke flavouring and that of the organic extract were determined (Fig. 1). Both aroma profiles showed a good agreement apart from some differences in the smoky, sweet, and spicy odour notes, which were perceived a bit more intensely in the smoke flavouring. These data suggest that the key
83
J.A. Pino / Food Chemistry 153 (2014) 81–86 Table 1 Volatile compounds identified in a smoke flavouring from rice husk. Compound
RI1
RI2
RI3
Relative amount (%)a
Acids Acetic acid* Propanoic acid* 2-Methylpropanoic acid* Butanoic acid* Pentanoic acid* Hexanoic acid* Heptanoic acid* Octanoic acid* Decanoic acid*
605 716 723 781 861 983 1064 1184 1359
600 743 765 820 911 984 1097 1179 1380
1435 1522 1584 1644 1728 1840 – 2051 2264
3.4 ± 0.3 0.1 ± 0.01
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Characterisation of volatile compounds in a smoke flavouring from rice husk Jorge A. Pino ⇑ Food Industry Research Institute, Carretera al Guatao km 3½, P.O. Box 19200, Havana, Cuba
a r t i c l e
i n f o
Article history: Received 4 October 2013 Received in revised form 4 December 2013 Accepted 9 December 2013 Available online 15 December 2013 Keywords: Smoke flavouring Rice husk Volatiles Aroma extract dilution analysis
a b s t r a c t An aqueous smoke flavouring from rice husk was obtained on a laboratory scale. The volatile compounds were isolated by simultaneous steam distillation–solvent extraction and its identification and quantitative composition was studied by GC–MS and GC–FID. A total of 93 compounds were isolated and 86 of them were positively identified. Major compounds (more than 5% GC area) were 2-furfural, phenol, 2-methoxyphenol, 4-ethyl-2-methoxyphenol, and 2,6-dimethoxyphenol. Application of aroma extract dilution analysis on the volatile fraction revealed that 2-methoxyphenol, 4-methyl-2-methoxyphenol, 2,6-dimethoxyphenol, 2-furfural, 2-acetylfuran, 3-methyl-1,2-cyclopentanedione, acetic acid, 5-methyl-2-furfural, 4-(2-propenyl)-2-methoxyphenol, 4-methyl-2,6-dimethoxyphenol, phenol, 2,6dimethylphenol, 4-ethyl-2-methoxyphenol, 2-methylphenol were the most odour-active compounds. Ó 2014 Published by Elsevier Ltd.
1. Introduction Liquid smoke flavourings, manufactured from wood pyrolysates are reported to offer many advantages over traditional smoking in a kiln, namely ease of application, speed and product uniformity. A large literature exists on the composition of smokes and liquid smoke preparations and about the sensory properties of various smoke fractions and isolated compounds (Cadwallader, 1996; Chen & Maga, 1993; Goma, Gray, Rabie, López Bote, & Booren, 1993; Guillén & Ibargoitia, 1999; Guillén & Manzanos, 1996, 1997, 2002; Guillén, Manzanos, & Ibargoitia, 2001; Guillén, Manzanos, & Zabala, 1995; Kostyra & Baryłko-Pikielna, 2006; Vichi et al., 2007; Wittkowski, Baltes, & Jennings, 1990; Wittkowski, Töth, & Baltes, 1981; Zandersons et al., 2009), but our knowledge is still incomplete. Although the main method for obtaining volatile compounds related to ‘‘smokiness’’ is the thermal pyrolysis of cellulose, hemicellulose and lignin (Cadwallader, 2007), no research has been done on the use of cellulosic materials, such as agricultural residues and by-products, in place of wood. Rice husk is one of the most important, due to its production volume and more than 25 possible uses of this by-product have been proposed (González, Alvira, & González, 1986), but to this moment the attention of researchers has not been focused on its thermal pyrolysis to produce smoke flavouring. This in spite of the fact that the cellulose-hemicellulose-lignin ratio in rice husk is 3:1:1 (González,
⇑ Tel.: +53 7 2793956. E-mail address: [email protected] 0308-8146/$ - see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.foodchem.2013.12.041
Alvira, & González, 1987), similar to 2:1:1 of wood (Gilbert & Knowles, 1975). A smoke flavouring manufactured by pyrolysis of rice husk, highly appreciated for its sensory characteristics, has been developed. This paper reports the characterisation of the volatile compounds from this product and the identification of potent odorants in the aroma extract by application of aroma extract dilution analysis. 2. Materials and methods 2.1. Chemicals Standards of chemicals were purchased from Sigma–Aldrich (St. Louis, MO) and Fluka (Buchs, Switzerland), and some were supplied by Dallant (Barcelona, Spain). An n-alkane solution (C8– C32) was purchased from Sigma–Aldrich. Anhydrous sodium sulfate, absolute ethanol, n-pentane and diethyl ether were purchased from Merck (Darmstadt, Germany); the solvents were redistilled and checked for purity. 2.2. Sample Liquid smoke flavouring was prepared by a dry distillation of rice husk from a Cuban commercial rice variety. The laboratory smoke producer consisted of a steel cylindrical tube (35 6 cm i.d.) externally heated by a stove and connected to a 15-cm water-cooled condenser with a glass wool filter plug to catch tar aerosol. The following parameters were used: 350 g of rice husk
82
J.A. Pino / Food Chemistry 153 (2014) 81–86
(8% moisture content), generation temperature 300 °C, and smouldering period 3 h. The resulting aqueous condensate was capped and permitted to equilibrate at room temperature for three days before analysis. The aqueous liquid smoke obtained was filtered through a paper filter to eliminate the oily phase. The liquid smoke flavouring was brown in colour, and its odour has been described as strong smoky. Three replicates of the procedure were made. 2.3. Isolation of volatile compounds The choice of isolation method and solvent was based on published information (Guillén & Manzanos, 2002; Zandersons et al., 2009). A sample of 50 mL of the liquid smoke flavouring was extracted three times with dichloromethane (30 + 20 + 20 mL). An internal standard (2-isopropyl-5-methylphenol, 2 mg, dissolved in ethanol) was previously added to the sample. The extracts were combined and dried over anhydrous sodium sulfate, and carefully concentrated to 0.9 mL in a Kuderna-Danish evaporator with a Vigreux column (12 1 cm i.d.) and then to 0.3 mL with a gentle nitrogen stream. Extractions were made in triplicate. 2.4. GC–FID analysis Analyses were conducted on a Hewlett–Packard 6890N series II (Agilent, Santa Clara, CA) equipped with DB-Wax (30 m 0.25 mm, 0.25 lm film thickness; J & W Scientific, Folsom, CA), DB-5ms (30 m 0.25 mm, 0.25 lm film thickness; J & W Scientific) and HP-1 (30 m 0.25 mm, 0.25 lm film thickness; Agilent) capillary columns, working with the following temperature programme and conditions for all columns: 50 °C for 2 min, ramp of 4 °C min 1 up to 250 °C; injector and detector temperatures 250 °C; carrier gas helium (1 mL min 1); detector FID; injections 1 lL in split mode with 1:10 ratio. Quantitative determination of odour-active volatiles exhibiting FD higher than 32, was done by internal standard method in the DB-5ms column. For that purpose, 2-isopropyl-5-methylphenol was added as internal standard, together with a defined mixture of the respective analytes, to 50 mL of distilled water, and processed by using the same procedure presented above in Section 2.3. Calibration curves were constructing using a series of solutions of varying nominal concentrations containing each analyte, where the slope was assumed as the response factor. An identical amount of internal standard was added to each solution and the corresponding chromatograms obtained (IOFI, 2011). All data were obtained in triplicate. 2.5. GC–MS analysis Analyses were performed on a Hewlett–Packard 6890N series II (Agilent) gas chromatograph equipped with a HP-5973N massselective detector and with a DB-5ms column (30 m 0.25 mm, 0.25 lm film thickness; J & W Scientific). The temperature programme and carrier gas flow rate were the same as for GC–FID. EI-MS, electron energy, 70 eV; ion source and interface temperature, 250 °C. The acquisition was performed in scan mode (mass range m/z 35–400). Compounds were identified by their linear retention indices, by their mass spectra, by comparing their mass spectra with those in libraries (NIST 05, Wiley 6, NBS 75k, Adams 2001 and in-house Flavorlib) and with those in the literature and, in most cases, by using standards. All standard compounds used, from Aldrich, Fluka and Merck, are asterisked in Table 1. 2.6. Aroma profile analysis Aroma profile analyses were performed by a trained sensory panel consisting of five trained panellists. The following aroma
descriptors (Ojeda, Bárcenas, Pérez-Elortondo, Albisu, & Guillén, 2002), represented by the compounds given in parentheses, were chosen for sensory evaluation, and their intensities were ranked on a five-point scale (steps of 0.5) from 0 (not perceivable) to 5 (strongly perceivable): smoky (2,6-dimethoxyphenol), burnt (4methyl-2,6-dimethylphenol), sweet (2-methyl-2-cyclopenten-1one); caramel (3-methyl-1,2-cyclopentanedione), acid (acetic acid), phenolic (2,6-dimethylphenol), pungent (phenol), spicy [4(1-propenyl)-2-methoxyphenol], and woody [4-(2-propenyl)-2methoxyphenol]. The judgments of the panellists were averaged. 2.7. Gas chromatography–olfactometry analysis (GC–O) GC–O analyses were performed with a Hewlett–Packard 6890N series II gas chromatograph (Agilent) equipped with an FID, using two capillary columns, DB-Wax and DB-5ms (each 30 m 0.25 mm, 0.25 lm film thickness; J & W Scientific). Analytical conditions were the same as for the GC–FID analyses. The end of the capillary column was connected to a deactivated Y-shaped glass splitter dividing the effluent into two equal parts, which were transferred via two deactivated fused silica capillaries (50 cm 0.25 mm) to a sniffing port and an FID. The sniffing port, mounted on a detector base of the GC, consisted of a cylindrically shaped aluminium device with a bevelled top and a central drilled hole housing the capillary. Nitrogen (30 mL min 1) was used as make-up gas. The injection volume was 1 lL. During a GC–O run, the nose of the panellist was placed closely above the top of the sniffing port and the odour of the effluent was evaluated. If an odour was recognised, the retention time was marked in the chromatogram, and the odour quality was assigned. The sniffing port temperature was approximately 250 °C. The GC–O analyses were performed by three experienced assessors. Each one had a minimum of 20 h of previous GC–O experience and they had also previously taken part in the barramundi sensory descriptive analysis training and had actively contributed to the development of the descriptive sensory vocabulary. 2.8. Aroma extract dilution analysis (AEDA) The aroma extract was stepwise diluted to obtain dilutions of 1:1, 1:2, 1:4, 1:8, 1:16, 1:1024 of the original solutions (Schieberle, 1995). Each dilution was submitted to GC–O, using DB-Wax and DB-5ms capillary columns. Analytical conditions were the same as for GC–FID analyses. The odour-active compounds were located in the chromatograms, and each odorant detected was assigned an FD factor representing the highest dilution at which the odorant was detectable. The FD factors obtained by three assessors were averaged.
3. Results and discussion An important challenge in the analysis of food aroma compounds is that the method used for aroma isolation is appropriate. To address this problem, the entire volatiles from the smoke flavouring, isolated by liquid–liquid extraction, were evaluated by the sensory panel by smelling a drop of the organic extract on a strip of filter paper as done by perfumers. After evaporation of the solvent, all five panellists agreed that the distillate evoked the characteristic odour of the original smoke preparation. Furthermore, the aroma profiles of the smoke flavouring and that of the organic extract were determined (Fig. 1). Both aroma profiles showed a good agreement apart from some differences in the smoky, sweet, and spicy odour notes, which were perceived a bit more intensely in the smoke flavouring. These data suggest that the key
83
J.A. Pino / Food Chemistry 153 (2014) 81–86 Table 1 Volatile compounds identified in a smoke flavouring from rice husk. Compound
RI1
RI2
RI3
Relative amount (%)a
Acids Acetic acid* Propanoic acid* 2-Methylpropanoic acid* Butanoic acid* Pentanoic acid* Hexanoic acid* Heptanoic acid* Octanoic acid* Decanoic acid*
605 716 723 781 861 983 1064 1184 1359
600 743 765 820 911 984 1097 1179 1380
1435 1522 1584 1644 1728 1840 – 2051 2264
3.4 ± 0.3 0.1 ± 0.01
