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Carotenoids as flavour precursors in coffee
W.L.P. Bredie and M.A. Petersen (Editors) Flavour Science: Recent Advances and Trends 9 2006 Elsevier B.V. All rights reserved.
379
C a r o t e n o i d s as flavour p r e c u r s o r s in coffee A n d r e a s D e g e n h a r d t a, M a r t i n P r e i n i n g e r b and F r a n k Ullrich a
aKrafi Foods R&D Inc., Bayerwaldstr. 8, D-8173 7 Munich, Germany," bKrafi Foods, 801 Waukegan Rd., Glenview, IL 60025, USA
ABSTRACT Carotenoids and carotenoid-derived products were identified in green coffee beans of different origins. After extraction with acetone, the carotenoids, lutein and zeaxanthin, were identified by HPLC-Diode Array Detection. Glycosidically bound ionols were identified as carotenoid-derived products which are known to be formed by oxidative degradation of lutein and zeaxanthin [1 ]. These glycosides were analysed by absorption on XAD-1180 resin followed by enzymatic hydrolysis of the eluted extract with [3-glucosidase, and by GC-MS analysis of the released aglycones [2]. The impact of carotenoids on aroma formation was investigated by using a model roasting system. For this purpose, green coffee beans were depleted of their naturally occurring potential aroma precursors, and subsequently enriched with [3-carotene, a carotenoid similar in structure to lutein and zeaxanthin. Under coffee roasting conditions, [~-carotene yielded the aroma compound, [3-ionone, as its major degradation product [3]. Furthermore, the importance of 3-oxo-a-ionol as an aroma precursor was investigated in the model system. 1. I N T R O D U C T I O N Carotenoids is a class of natural compounds which has been extensively studied due to their nutritional importance. Carotenoids are also valued as pigments for use in food products. Some powerful aroma compounds are derived from carotenoids and have been thoroughly investigated in tea, wine and fruits [3]. 13-Damascenone is among the very few carotenoid-derived potent aroma compounds in coffee [4-5], and has very low flavour thresholds in the low ppb to ppt range. Carotenoids have been found in green coffee beans [4,6], and their relevance as precursors for [~-damascenone has been mentioned in the literature. However, no clear identification and quantification of carotenoids in green coffee has been reported so far. Therefore, we quantified carotenoids and conducted model studies to evaluate their relevance as flavour
380 precursors in coffee. This study on glycosidically bound flavour precursor compounds adds to the knowledge on glycoconjugated progenitors in coffee [7].
2. MATERIALS AND METHODS
2.1. Analysis of carotenoids Ground green coffee (50 g) was extracted with acetone (2 x 200 ml; 30 rain each). The extract was filtered and evaporated nearly to dryness. The carotenoids were extracted into diethyl ether (50 ml). The extract was saponified with methanolic potassium hydroxide (30 g/l; 10 ml) at room temperature for 10 min under constant shaking. The solution was washed with brine (10% NaCI) until pH 7 was reached. The ether was removed in vacuo, the residue taken up in acetone, and completed to 5 ml. The solution was filtered through hydrophobic filters (Sartorius Minisart SRP25, 0.45 lain), and 10 lal injected into an HPLC/Diode Array Detector system (Agilent 1100 Series). Chromatography was performed on an RP18 column (Luna; 5 lam mesh; 250 mm x 4.6 mm ID) with the eluents, acetonitrile/water (9/1, v/v; solvent A) and ethyl acetate (solvent B). A linear elution gradient (0-60% B in 16 rain, 60-100% B in 16-36 rain) was applied at 1 ml/min flow rate. Photometric detection was performed at 440 nm. 2.2. Analysis of carotenoid degradation products Green coffee beans (about 200 g) were deactivated with ethanol (100 ml) and water (120 ml) for 24 h at room temperature. After evaporation of the solvents, drying and grinding, the residue (100 g) was defatted with n-hexane (300 ml).The defatted beans were extracted with methanol (3 x 250 ml) at room temperature. The methanol was evaporated, the residue taken up in water (150 ml), and cleaned up on a small XAD1180 column (40 g resin). The column was washed with water (500 ml), eluted with methanol (about 500 mi), and the eluate evaporated to dryness (yield about 800 mg). An aliquot of the residue (400 rag) was dissolved in phosphate buffer (pH 5.0, 50 ml) and extracted with pentane/ether (1/1, v/v; 3 x 40 ml) to remove free volatile aroma compounds. After evaporation of residual organic solvent, the aqueous solution was hydrolysed with emulsin (almond 13-glucosidase; 75 rag, about 7 units) at 37 ~ for 72 h. After addition of 100 IaL n-tridecane in ether (0.01m) as internal standard, the liberated aglycones were extracted with pentane/ether (1/1, v/v; 3 x 40 ml). The organic extract was washed with hydrochloric acid (0. IN; 2 x 40 ml), concentrated on a small Kuderna-Danish column to about 0.5 ml, and 1 pl analysed by GC-FID (HP 5890 Series II) and GC-MS (Agilent 5973, Gerstel CIS 3 injector) using a DB-WAX capillary (Agilent; 60 m x 0.32 mm ID x 0.25 ~tm FD). The GC oven was ramped from 40 ~ to 240 ~ at a 5 ~ heating rate. 2.3. Model roasting trials In order to remove naturally occurring potential aroma precursors, green Robusta coffee beans were extracted with water at 80 ~ The extraction was considered complete when a coffee brew from the extracted, dried, and roasted beans almost lost its typical
381 coffee flavour, and instead tasted cereal/popcorn-like. These depleted beans were infused with 13-carotene and 3-oxo-a-ionol, respectively. For that purpose, [3-carotene or 3-oxo-a-ionol (10 mg each) were dissolved in ethanol (about 5 ml), diluted with water (100 ml), and infused into the depleted beans (150 g) at 80 ~ for 2 h. The infused beans were dried and roasted (Neuhaus Neotec Signum roaster; 260 ~ 180 s). 2.4. Isolation of aroma compounds from model roasting trials The volatile compounds were extracted from roast and ground (R&G) coffee (100 g) for 2 h with pentane/ether (1/1, v/v; 50 ml) using a conventional Likens-Nickerson apparatus for simultaneous-distillation-extraction (SDE). The extract was concentrated to about 2 ml on a Kuderna-Danish column, and dried over anhydrous sodium sulfate. The dried concentrate was analysed under similar GC conditions as described in section 2.2.
3. RESULTS
3.1. Identification of carotenoids in green coffee The carotenoids, lutein and zeaxanthin, were identified in green coffee beans by HPLCDAD analysis with photometric detection at visible wavelength. An external standard method for quantitation of carotenoids in green coffee was developed, involving alkaline saponification of bound forms. Green Robusta coffee beans contained a higher carotenoid level (sum of lutein and zeaxanthin) of 1.5 mg/kg versus Kenyan, Brazilian and Colombian Arabica with 0.2 to 0.7 mg/kg. 3.2. Identification of carotenoid-derived flavour precursors Carotenoids are labile compounds. By oxidative degradation, primary cleavage products are formed which undergo subsequent enzymatic modification [3]. These are usually glycosidically bound, non-volatile compounds, and can be cleaved by 13-glucosidase to release the respective aglycones. The aglycones can be potent aroma compounds or their volatile precursors [11]. Two glycosidically bound ionols (3-oxo-Gt-ionol and 3oxo-7,8-dihydro-a-ionol) were identified in green coffee after enzymatic release from the isolated glycosidic mixture [2]. The presence of 3-oxo-a-ionol in green coffee was verified with a synthesised reference compound [8], whereas 3-oxo-7,8-dihydro-a-ionol was tentatively identified by its retention index and mass spectrum. These two ionols are known lutein- and zeaxanthin-derived aroma precursors in many foods [3,10]. 3.3. Model roasting trials The role of carotenoids as potential precursors of coffee flavour compounds was investigated in model roasting trials. Green coffee beans were depleted of their flavour precursors by extraction with hot water and were then used as a reaction matrix for added aroma precursors. [~-Carotene, which has not been detected in green coffee, was added as a model compound in these roasting trials. The aroma volatiles were extracted using a Likens-Nickerson-type apparatus. The known carotenoid degradation product,
382 13-ionone, was identified in the enriched and roasted beans by GC-MS with the help of an authentic reference compound. This finding confirms that carotenoids are degraded to aroma compounds during coffee roasting, and that carotenoids are a source of potential aroma for roasted coffee. Furthermore, synthesised 3-oxo-ct-ionol [8] was infused into the depleted beans, and the bean matrix roasted. Four newly formed megastigmatrienone isomers were identified in the roasted coffee by GC-MS after SDE, utilising literature mass spectra as references [9]. A generation pathway for the megastigmatrienone isomers via 3-oxo-Gt-ionol has been proposed [ 12]. 4. D I S C U S S I O N A N D C O N C L U S I O N
The results from the present model studies prompted us to search for the occurrence of megastigmatrienones in roasted coffees. However, in R&G coffee no megastigmatrienones were detected, although they were found in green coffee. Therefore, megastigmatrienones are potentially newly identified green coffee constituents. Since glycoconjugates are labile products, which undergo cleavage and rearrangement reactions in their free forms, the content of megastigmatrienones might be altered by the sample preparation step [13]. The aroma relevance of megastigmatrienones, and their role as precursors for other aroma compounds, needs to be clarified, requiring further model reactions as well as GC-olfactometry studies. The presented data will help increasing the knowledge of flavour precursors in coffee, and elucidating further generation pathways of aroma compounds via glycosides. References 1. R. Teranishi, G.R. Takeoka and M. GOntert (eds.), Flavour precursors - thermal and enzymatic conversions, ACS symposium series 490, Washington, DC, USA (1992) 75. 2. P. Winterhalter and R.I~. Rousefl'(eds.), Carotenoid-derived aroma compounds, ACS symposium series 802, Washington, DC, USA (2002) 20. 3. P. Winterhalter and R.L. Rouseff(eds.), Carotenoid-derived aroma compounds, ACS symposium series 802, Washington, DC, USA (2002) 1. 4. T.H. Parliment, M.J. Morello and R.J. McGorrin (eds.), Thermally generated flavours Maillard, microwave, and extrusion processes, ACS symposium series 543, Washington, DC, USA (1994) 206. 5. I. Blank, A. Sen and W. Grosch, Z. Lebensmittel Untersuch. Forsch., 195 (1992) 239. 6. C. Yeretzian, A. Jordan, R. Badoud and W. Lindinger, Eur. Food Res. Technol., 214 (2002) 92. 7. B. Weckerle, G. Toth and P. Schreier, Eur. Food Res. Technol., 216 (2003) 6. 8. A.J. Aasen, B. Kimland and C.R. Enzell, Acta Chem. Scand., 27 (1973) 2107. 9. A.J. Aasen, B. Kimland, S.-O. Almqvist and C.R. Enzeli, Acta Chem. Scand., 26 (1972) 2573. 10. C.R. Strauss, B. Wilson and P.J. Williams, Phytochem., 26 (1987) 1995. 11. C. Enzell, Pure Appl. Chem., 57 (1985) 693. 12. P. Winterhalter and R.L. Rouseff(eds.), Carotenoid-derived aroma compounds, ACS Symposium Series 802, Washington, DC, USA (2002) 131. 13. P. Winterhalter and P. Schreier, J. Agric. Food Chem., 36 (1988) 1251.
379
C a r o t e n o i d s as flavour p r e c u r s o r s in coffee A n d r e a s D e g e n h a r d t a, M a r t i n P r e i n i n g e r b and F r a n k Ullrich a
aKrafi Foods R&D Inc., Bayerwaldstr. 8, D-8173 7 Munich, Germany," bKrafi Foods, 801 Waukegan Rd., Glenview, IL 60025, USA
ABSTRACT Carotenoids and carotenoid-derived products were identified in green coffee beans of different origins. After extraction with acetone, the carotenoids, lutein and zeaxanthin, were identified by HPLC-Diode Array Detection. Glycosidically bound ionols were identified as carotenoid-derived products which are known to be formed by oxidative degradation of lutein and zeaxanthin [1 ]. These glycosides were analysed by absorption on XAD-1180 resin followed by enzymatic hydrolysis of the eluted extract with [3-glucosidase, and by GC-MS analysis of the released aglycones [2]. The impact of carotenoids on aroma formation was investigated by using a model roasting system. For this purpose, green coffee beans were depleted of their naturally occurring potential aroma precursors, and subsequently enriched with [3-carotene, a carotenoid similar in structure to lutein and zeaxanthin. Under coffee roasting conditions, [~-carotene yielded the aroma compound, [3-ionone, as its major degradation product [3]. Furthermore, the importance of 3-oxo-a-ionol as an aroma precursor was investigated in the model system. 1. I N T R O D U C T I O N Carotenoids is a class of natural compounds which has been extensively studied due to their nutritional importance. Carotenoids are also valued as pigments for use in food products. Some powerful aroma compounds are derived from carotenoids and have been thoroughly investigated in tea, wine and fruits [3]. 13-Damascenone is among the very few carotenoid-derived potent aroma compounds in coffee [4-5], and has very low flavour thresholds in the low ppb to ppt range. Carotenoids have been found in green coffee beans [4,6], and their relevance as precursors for [~-damascenone has been mentioned in the literature. However, no clear identification and quantification of carotenoids in green coffee has been reported so far. Therefore, we quantified carotenoids and conducted model studies to evaluate their relevance as flavour
380 precursors in coffee. This study on glycosidically bound flavour precursor compounds adds to the knowledge on glycoconjugated progenitors in coffee [7].
2. MATERIALS AND METHODS
2.1. Analysis of carotenoids Ground green coffee (50 g) was extracted with acetone (2 x 200 ml; 30 rain each). The extract was filtered and evaporated nearly to dryness. The carotenoids were extracted into diethyl ether (50 ml). The extract was saponified with methanolic potassium hydroxide (30 g/l; 10 ml) at room temperature for 10 min under constant shaking. The solution was washed with brine (10% NaCI) until pH 7 was reached. The ether was removed in vacuo, the residue taken up in acetone, and completed to 5 ml. The solution was filtered through hydrophobic filters (Sartorius Minisart SRP25, 0.45 lain), and 10 lal injected into an HPLC/Diode Array Detector system (Agilent 1100 Series). Chromatography was performed on an RP18 column (Luna; 5 lam mesh; 250 mm x 4.6 mm ID) with the eluents, acetonitrile/water (9/1, v/v; solvent A) and ethyl acetate (solvent B). A linear elution gradient (0-60% B in 16 rain, 60-100% B in 16-36 rain) was applied at 1 ml/min flow rate. Photometric detection was performed at 440 nm. 2.2. Analysis of carotenoid degradation products Green coffee beans (about 200 g) were deactivated with ethanol (100 ml) and water (120 ml) for 24 h at room temperature. After evaporation of the solvents, drying and grinding, the residue (100 g) was defatted with n-hexane (300 ml).The defatted beans were extracted with methanol (3 x 250 ml) at room temperature. The methanol was evaporated, the residue taken up in water (150 ml), and cleaned up on a small XAD1180 column (40 g resin). The column was washed with water (500 ml), eluted with methanol (about 500 mi), and the eluate evaporated to dryness (yield about 800 mg). An aliquot of the residue (400 rag) was dissolved in phosphate buffer (pH 5.0, 50 ml) and extracted with pentane/ether (1/1, v/v; 3 x 40 ml) to remove free volatile aroma compounds. After evaporation of residual organic solvent, the aqueous solution was hydrolysed with emulsin (almond 13-glucosidase; 75 rag, about 7 units) at 37 ~ for 72 h. After addition of 100 IaL n-tridecane in ether (0.01m) as internal standard, the liberated aglycones were extracted with pentane/ether (1/1, v/v; 3 x 40 ml). The organic extract was washed with hydrochloric acid (0. IN; 2 x 40 ml), concentrated on a small Kuderna-Danish column to about 0.5 ml, and 1 pl analysed by GC-FID (HP 5890 Series II) and GC-MS (Agilent 5973, Gerstel CIS 3 injector) using a DB-WAX capillary (Agilent; 60 m x 0.32 mm ID x 0.25 ~tm FD). The GC oven was ramped from 40 ~ to 240 ~ at a 5 ~ heating rate. 2.3. Model roasting trials In order to remove naturally occurring potential aroma precursors, green Robusta coffee beans were extracted with water at 80 ~ The extraction was considered complete when a coffee brew from the extracted, dried, and roasted beans almost lost its typical
381 coffee flavour, and instead tasted cereal/popcorn-like. These depleted beans were infused with 13-carotene and 3-oxo-a-ionol, respectively. For that purpose, [3-carotene or 3-oxo-a-ionol (10 mg each) were dissolved in ethanol (about 5 ml), diluted with water (100 ml), and infused into the depleted beans (150 g) at 80 ~ for 2 h. The infused beans were dried and roasted (Neuhaus Neotec Signum roaster; 260 ~ 180 s). 2.4. Isolation of aroma compounds from model roasting trials The volatile compounds were extracted from roast and ground (R&G) coffee (100 g) for 2 h with pentane/ether (1/1, v/v; 50 ml) using a conventional Likens-Nickerson apparatus for simultaneous-distillation-extraction (SDE). The extract was concentrated to about 2 ml on a Kuderna-Danish column, and dried over anhydrous sodium sulfate. The dried concentrate was analysed under similar GC conditions as described in section 2.2.
3. RESULTS
3.1. Identification of carotenoids in green coffee The carotenoids, lutein and zeaxanthin, were identified in green coffee beans by HPLCDAD analysis with photometric detection at visible wavelength. An external standard method for quantitation of carotenoids in green coffee was developed, involving alkaline saponification of bound forms. Green Robusta coffee beans contained a higher carotenoid level (sum of lutein and zeaxanthin) of 1.5 mg/kg versus Kenyan, Brazilian and Colombian Arabica with 0.2 to 0.7 mg/kg. 3.2. Identification of carotenoid-derived flavour precursors Carotenoids are labile compounds. By oxidative degradation, primary cleavage products are formed which undergo subsequent enzymatic modification [3]. These are usually glycosidically bound, non-volatile compounds, and can be cleaved by 13-glucosidase to release the respective aglycones. The aglycones can be potent aroma compounds or their volatile precursors [11]. Two glycosidically bound ionols (3-oxo-Gt-ionol and 3oxo-7,8-dihydro-a-ionol) were identified in green coffee after enzymatic release from the isolated glycosidic mixture [2]. The presence of 3-oxo-a-ionol in green coffee was verified with a synthesised reference compound [8], whereas 3-oxo-7,8-dihydro-a-ionol was tentatively identified by its retention index and mass spectrum. These two ionols are known lutein- and zeaxanthin-derived aroma precursors in many foods [3,10]. 3.3. Model roasting trials The role of carotenoids as potential precursors of coffee flavour compounds was investigated in model roasting trials. Green coffee beans were depleted of their flavour precursors by extraction with hot water and were then used as a reaction matrix for added aroma precursors. [~-Carotene, which has not been detected in green coffee, was added as a model compound in these roasting trials. The aroma volatiles were extracted using a Likens-Nickerson-type apparatus. The known carotenoid degradation product,
382 13-ionone, was identified in the enriched and roasted beans by GC-MS with the help of an authentic reference compound. This finding confirms that carotenoids are degraded to aroma compounds during coffee roasting, and that carotenoids are a source of potential aroma for roasted coffee. Furthermore, synthesised 3-oxo-ct-ionol [8] was infused into the depleted beans, and the bean matrix roasted. Four newly formed megastigmatrienone isomers were identified in the roasted coffee by GC-MS after SDE, utilising literature mass spectra as references [9]. A generation pathway for the megastigmatrienone isomers via 3-oxo-Gt-ionol has been proposed [ 12]. 4. D I S C U S S I O N A N D C O N C L U S I O N
The results from the present model studies prompted us to search for the occurrence of megastigmatrienones in roasted coffees. However, in R&G coffee no megastigmatrienones were detected, although they were found in green coffee. Therefore, megastigmatrienones are potentially newly identified green coffee constituents. Since glycoconjugates are labile products, which undergo cleavage and rearrangement reactions in their free forms, the content of megastigmatrienones might be altered by the sample preparation step [13]. The aroma relevance of megastigmatrienones, and their role as precursors for other aroma compounds, needs to be clarified, requiring further model reactions as well as GC-olfactometry studies. The presented data will help increasing the knowledge of flavour precursors in coffee, and elucidating further generation pathways of aroma compounds via glycosides. References 1. R. Teranishi, G.R. Takeoka and M. GOntert (eds.), Flavour precursors - thermal and enzymatic conversions, ACS symposium series 490, Washington, DC, USA (1992) 75. 2. P. Winterhalter and R.I~. Rousefl'(eds.), Carotenoid-derived aroma compounds, ACS symposium series 802, Washington, DC, USA (2002) 20. 3. P. Winterhalter and R.L. Rouseff(eds.), Carotenoid-derived aroma compounds, ACS symposium series 802, Washington, DC, USA (2002) 1. 4. T.H. Parliment, M.J. Morello and R.J. McGorrin (eds.), Thermally generated flavours Maillard, microwave, and extrusion processes, ACS symposium series 543, Washington, DC, USA (1994) 206. 5. I. Blank, A. Sen and W. Grosch, Z. Lebensmittel Untersuch. Forsch., 195 (1992) 239. 6. C. Yeretzian, A. Jordan, R. Badoud and W. Lindinger, Eur. Food Res. Technol., 214 (2002) 92. 7. B. Weckerle, G. Toth and P. Schreier, Eur. Food Res. Technol., 216 (2003) 6. 8. A.J. Aasen, B. Kimland and C.R. Enzell, Acta Chem. Scand., 27 (1973) 2107. 9. A.J. Aasen, B. Kimland, S.-O. Almqvist and C.R. Enzeli, Acta Chem. Scand., 26 (1972) 2573. 10. C.R. Strauss, B. Wilson and P.J. Williams, Phytochem., 26 (1987) 1995. 11. C. Enzell, Pure Appl. Chem., 57 (1985) 693. 12. P. Winterhalter and R.L. Rouseff(eds.), Carotenoid-derived aroma compounds, ACS Symposium Series 802, Washington, DC, USA (2002) 131. 13. P. Winterhalter and P. Schreier, J. Agric. Food Chem., 36 (1988) 1251.