Introduction: Coffee is consumed by over 800 million people worldwide, who appreciate it for its taste, aroma, and health benefits. The flavor of coffee is closely linked to its quality, and the lipids it contains play a significant role in determining this quality. Lipids constitute approximately 10–20% of the total chemical compounds in Coffea arabica L. and Coffea canephora, which together account for about 70% of global coffee production (1).
The primary fatty acids in coffee oil are linoleic acid, averaging 44.1%, and palmitic acid, averaging 34%. Minor fatty acids include myristic, palmitoleic, eicosenoic, and behenic acids, each contributing less than 1%, while linoleic and arachidic acids are present in amounts ranging from 1.5% to 3% (2).
Green coffee lacks a distinctive taste or aroma. The desirable flavor develops during roasting, which alters the coffee’s color and flavor, enhancing consumer acceptance. Roasting also induces significant changes in coffee’s chemical composition.
Three main reactions occur simultaneously during roasting: the Maillard reaction, lipid oxidation, and sugar decomposition. The Maillard reaction is a nonenzymatic reaction between amines and carbonyl compounds, particularly reducing sugars (3). It is responsible for the cooked, dried, or stored food characteristics, influencing color, flavor, and nutritional value through the formation of stable and sometimes mutagenic compounds. In green coffee, carbohydrates and amino acids are the primary precursors contributing to the characteristic aroma formed during roasting. Standard roasting procedures range from 240°C for 6 minutes to 270°C for 3 minutes, though industrial roasting often uses higher temperatures. These conditions not only develop color and aroma but can also produce undesirable compounds such as acrylamide (4).
Key lipids in coffee include the diterpenes kahweol and cafestol, which are important for both aroma and potential health benefits. The fatty acid composition of green coffee depends on factors such as bean species and growing conditions (5,6). Previous studies on oxidized oils by Damanik & Murkovic (2017, 2018) provided a foundation for toxicological studies on oxidized coffee oils (7,8). Aliphatic aldehydes ranging from hexanal to decanal, including decenal, have been identified using LC-MS/MS and quantified as DNPH derivatives. Research on coffee oil fatty acids and secondary oxidation products is essential to determine optimal roasting conditions.
Materials and Methods
Reagents and solvents
Arabica coffee beans were obtained from Trieste, Italy. Acetonitrile (Chem Lab, NV, Belgium) was of gradient-grade purity. Boron trifluoride in methanol was sourced from Fluka (Buchs, Switzerland). Sodium hydroxide was purchased from Sigma Aldrich. 2,4-Dinitrophenylhydrazine (2,4-DNPH) was obtained from Sigma Aldrich (St. Louis, USA), hydrochloric acid (37%) from Merck (Darmstadt, Germany), and all other solvents (methanol, ACN, acetone) were HPLC grade from Merck (Darmstadt, Germany). Acetic acid was purchased from Roth (Karlsruhe, Germany), and β-carotene and α-tocopherol were obtained from Sigma Aldrich (St. Louis, USA).
Fatty acid analysis: saponification and methylation
Twenty mg of coffee oil in a Pyrex tube was hydrolyzed with 6 mL of 0.5 M NaOH in methanol at 80°C for 1 hour. After cooling to room temperature, free fatty acids (FFAs) were methylated with 6 mL of BF3 in methanol at 80°C for 15 minutes. Water was added, and the resulting fatty acid methyl esters (FAMEs) were extracted with 10 mL of heptane.
Liquid chromatography (UV and ELSD) for fatty acids
Fatty acids in Arabica coffee oil were analyzed using an Agilent 1100 series HPLC system equipped with a quaternary pump, vacuum degasser, autosampler, and variable wavelength UV detector. Separation was performed on an AccQ-Tag column (60Å, 4 µm, 3.9 × 150 mm) using 95% acetonitrile in water as the mobile phase.
Aldehyde analysis in oxidized coffee oil
Forty grams of green coffee were roasted at 140°C for 11 minutes, with samples taken at 3, 6, 9, and 11 minutes. Roasted beans were cooled, ground, and extracted using a Soxhlet apparatus for 5 hours with petroleum benzene.
Derivatization with 2,4-DNPH
To 1 mL of oxidized oil, 4 mL of acetonitrile and 3 mL of 2,4-DNPH reagent (3.48 mg/mL) were added. The mixture was kept in the dark for 1 hour, followed by extraction with 2 mL ethyl acetate and phase separation with 1 g KCl. The organic layer was analyzed directly by HPLC.
HPLC-MS conditions for aldehyde identification
DNPH derivatives of carbonyl compounds were analyzed using reversed-phase HPLC (Kinetex EVO C18, 150 × 3 mm) with a gradient elution from methanol (45%), water (30%), and acetonitrile (25%) to methanol (6%), water (4%), and acetonitrile (90%) over 15 minutes. Absorbance was measured at 400 nm. Mass detection was performed with a QTRAP 2000 in APCI mode at 250°C, 4000 V capillary voltage, and 150 V fragment potential.
Results and Discussion
Fatty acids in coffee oil
Various solvent combinations were tested for optimal separation of fatty acids in coffee oil using ELSD-HPLC: (A) 100% acetonitrile, (B) acetonitrile:water 95:5%, and (C) acetonitrile:methanol:water 47.5:47.5:5%. Combination B provided the best separation. Saponification with BF3-methanol was used to quantify fatty acids, followed by isocratic elution of FAMEs on a C18 column with ELSD detection. Gradient RP-HPLC was employed to analyze fatty acids in roasted coffee. The classical saponification-methylation method produced FAMEs from glycerolipids and sterol esters. A gradient of acetonitrile and water with 0.1 M formic acid was used to improve peak shape and provide protons for LC-MS analysis of roasted coffee FAMEs (Figure 1).
Figure 1 : Fatty acids in coffee oil profile with/without BF3
The main fatty acids content in coffee lipid extracts of arabica coffee determined with LC/MS. Four fatty acids were are considered : Palmitic acids ( C16:0),oleic acid (C18:1), linoleic acid (C18:2) and stearic acid (C18:0) (Figure 2a; 2b; 2c; and 2d).
Figure 2. Identification fatty acids in coffee oil with LC/MS
Fatty acids are combined in more complex molecules such as acylglycerols, cholesterol esters, waxes, and glycosphingolipids — these products obtained by saponification (inorganic or basic organic solution) or acidic hydrolysis and then derivatized. FAME may also be obtained directly by transesterification (alcoholysis or methanolysis) of the fatty acid-containing lipids.
An alternative analysis of fatty acids which used the reversed-phase HPLC has done. It’s convenient for the analysis of non-volatiles acylglycerols, which need to derivatize before the analysis. This method is more common to encounter the fatty acids, i.e., C14 to C22 of fatty acids. The clear separation of saponifiable and unsaponifiable material required continuing steps, which is extraction, hydrolysis also methylation of coffee oil. Also, it is recommended for tiny samples to avoid any loss of fatty acids during analysis.
Carbonyls formation during the coffee roasting
Arabica coffee roasted in a Probat roaster 1 Z at ca. 140 °C for 11 minutes to obtain a roasted coffee comparable to standard quality. Nonvolatile lipid secondary oxidation product of roasting coffee oils was analyzed using MS coupled with a reversed phase-HPLC. Most of the LC methods available for other food matrices based on the detection of stable carbonyl derivatives. Therefore, the carbonyl compound in triacylglycerol is volatile. It is preferable to derivatize them into a nonvolatile stable form. One of the most popular methods for the determination of individual secondary oxidation product in edible oils is the reaction of 2,4- dinitrophenyl hydrazine (9).
Hydrazine, such as 2,4- dinitrophenylhydrazine (DNPH), selectively react with aldehydes and ketones to form stable hydrazones. The aromatic hydrazines react with carbonyls under acidic condition, formed insoluble hydrazone derivatives. Total carbonyl content can be determined in oxidized lipids by the reaction with 2,4-dinitrophenyl- hydrazine — the colored hydrazone (2,4-DNPH) due to its high reactivity, selectivity, and stability.During 3 minutes of roasting, the oxidation product is low. Started from 6 minutes the forming of carbonyls are increasing . The highest number and concentration of the carbonyls can be observed more than 9 minutes of roasting. The secondary oxidation product with low molecular weight included carbonyls can be identified by an Orbi- trap LC-MS which has high sensitivity and the possibility (Figure 3).
Figure 3 : The profile of secondary oxidation products in coffee oil during roasting at 0,3,6,9, 11 (fully roasted)min
The secondary oxidation product with low molecular weight included carbonyls can be identified by an Orbitrap LC-MS which has high sensitivity and the possibility. From the orbitrap’s data of the fully roasted, we can identify that is formed two kinds of carbonyls compounds which the molecular mass is 102 and 108 (Figure 4).
Figure 4 : LC/MS analysis of coffee oil
| Full ms2 (-H+) Molecule Fragment Proposed Structure 281 282 101 Hydroxypentanal 287 288 107 Heptatrienal |
Conclusions
An alternative analysis of fatty acids using RP-HPLC was performed. The primary fatty acids in Arabica coffee—palmitic, stearic, oleic, and linoleic acids—were analyzed. The separation of saponifiable and unsaponifiable materials was achieved through subsequent processing steps. The procedure involved extraction, hydrolysis, and methylation of coffee oil.
MS coupled with reverse phase HPLC was employed to determine secondary oxidation products (carbonyls), which typically occur at low concentrations. The derivatization method produced stable hydrazone derivatives. Among the carbonyl compounds analyzed were hydroxypentanal and heptatrienal, with concentrations of 57.45 μmol/mL and 224.80 μmol/mL, respectively.
Conflict of interest
The authors declare that they have no conflict of interest.
References
- Illy, A.; Viani, R. Espresso Coffee: The Science of Quality; Academic Press: London, 2005.
- Oliveira, L.S.; Franca, A.S.; Mendonça, J.C.F.; Junior, M.C.B. Proximate composition and fatty acid profile of green and roasted defective coffee beans. LWT – Food Sci. Technol. 2006, 39, 235–239.
- Kawamura, S. Seventy years of the Maillard reaction. ACS Symposium Series, American Chemical Society, 1983, 215, 3–18.
- Murkovic, M.; Derler, K. Analysis of amino acids and carbohydrates in green coffee. J. Biochem. Biophys. Methods 2006, 69, 25–32.
- Alves, M.R.; Casal, S.; Oliveira, M.B.P.P.; Ferreira, M.A. Contribution of fatty acid profile obtained by high-resolution GC/chemometric techniques to the authenticity of green and roasted coffee varieties. J. Am. Oil Chem. Soc. 2003, 80, 511–517.
- Villareal, D.; Laffargue, A.; Posada, H.; Bertrand, B. Genotypic and environmental effects on coffee (Coffea arabica L.) bean fatty acid profile: Impact on variety and origin chemometric determination. J. Agric. Food Chem. 2009, 57, 11321–11327.
- Damanik, M.; Murkovic, M. Formation of potentially toxic carbonyls during oxidation of triolein in the presence of alimentary antioxidants. Monatsh. Chem. 2017, 148, 2031–2035.
- Damanik, M.; Murkovic, M. The stability of palm oils during heating in a Rancimat. Eur. Food Res. Technol. 2018, 244, 1293–1299.
- Decker, E.A.; Elias, R.J.; McClements, D.J. Oxidation in Foods and Beverages and Antioxidant Applications; Woodhead Publishing: Philadelphia, USA, 2010; p. 202.