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Improvement of techniques for analysis of membrane lipids in coffee

This study focuses on improving techniques for analyzing membrane lipids in coffee seeds, specifically in regards to solid phase extraction, high-performance liquid chromatography, and gas chromatography. The findings will be applicable to other plant materials as well.

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Improvement of techniques for analysis of membrane lipids in coffee

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  1. Improvement of techniques for analysis of membrane lipids in coffee Andréina Laffargue IRD Montpellier (P4)

  2. Background • During a one week visit to P1 at Leuven on september 2003 : • Phospholipid (PL), free fatty acid (FFA), and sterol content and composition of seeds of 9 coffee species were analysed. • It was concluded that the techniques employed present several limitations when applied to coffee seeds, due mainly to their high content in triacylglycerols and their small amount of PL. • The following techniques needed improvement • Solid phase extraction (SPE) • High performance liquid chromatography (HPLC) of PL • Gas chromatography (GC) of sterols • GC of fatty acids

  3. Lipid separation using SPEMethodology • Seven SPE methods published in plant physiology • and technology journals were tested. • Fraction purity was checked using thin layer chromatography : • Diethylether/Methyl alcohol/Acetic acid (90/1/1) • Fraction composition was determined using HPLC

  4. NH2 / Elution method 2 NH2 / Elution method 1 Lipid separation using SPE Effect of the elution method Recovery (%) Whatever the phase used, the elution method has a major effect on PL recovery percentages

  5. Lipid separation using SPE Phase effect : example of 3 different phases Phase NH2 PhaseSi Phase C8 Selectivity and different recovery percentages were observed Some Si phase cartridges (the most employed in plant physiology) underestimated the PC content All phospholipids were recovered with C8

  6. Phospholipids PL composition using HPLC Fatty acid composition GC after TLC purification Free fatty acids TAG pollution NH2 cartridge 2g 150 mg coffee lipid extract Elution : 16 ml Chloroform/MeOH (2/1) 16 ml Diethylether/AcAc (2%) 16 ml Methyl alcohol FFA fraction GC analysis Purety = 100% FFA Selected methods for lipid fractioning C8 cartridge 2g 150 mg coffee lipid extract Elution : 16 ml Methyl alcohol 20 ml Chloroform/MeOH(2/1) 20 ml Chloroform PL fraction

  7. HPLC analysis of PL • Five detectors tested from Eurosep, Waters, Sedere, Polymer labs, and Alltech • Three columns tested : one Silica (Restek) and two Diol (Supelco and Merck) • Phase modifiers: TEA and Acetic acid Chromachem (EUROSEP) Good repeatability The best detection limit Lichrospher diol (MERCK) TR stability, sharp peaks TEA : phase damage Acetic acid : TR stabilisation

  8. HPLC analysis of PL Temperature influence on retention times

  9. HPLC analysis of PLStandard curves Beckman HPLC system Column : Lichrospher 100 Diol 5µ (25 cm*4,6 mm i.d), Merck Column temperature : 30°C ELSD : Chromachem, Eurosep TNeb = 35°C, TEvap = 45°C, P = 1,5 bar Binary gradient : A = Hexan/Isop/AcAc (82/18/1,3) B = Isop/Water/AcAc (86/14/1,3) Flow rate : 1 ml/min

  10. Standards 160 140 120 100 80 60 40 20 0 PC PE LPE LPC Detector response (mV) PI PS PG 0 10 20 30 40 Time (min) PS + PG PI Coffee 200 100 0 PC Detector response (mV) PE LPE LPC 0 10 20 30 40

  11. Seven transmethylation methods were compared • FAMEs were analysed by the usual GC method Fatty acids composition (%) FA BF3/MeOH 45 min ISO Standard 14:0 0.2 0.1 16:0 37.8 33.7 18:0 18:1 6.9 7.3 6.8 7.6 18:2 41.7 46.2 18:3 20:0 20:1 22:0 1.3 2.9 0.3 0.8 1.4 2.5 0.3 0.6 Fatty acids composition Lipid extraction Saponification KOH 0,5M, 90°C, 5min BF3/MeOH 14%, 90°C, 3min Hexane extraction of FAMEs GC injection GC separation of FAME. Coffee sample

  12. Silylation On-column injection Sterol identification and quantification Lipid extract (+ I.S) sitosterol I.S Saponification KOH 2M, 90°C, 60min stigmasterol Unsaponifiable matter campesterol TLC purification Ether extraction of sterol-containing band GC separation of sterols. Coffee sample GC analysis of Sterols

  13. Application to CRYMCEPT studies

  14. WP4 main results– interspecific variability for seed desiccation tolerance

  15. Principal component analysis: WP3+WP4 data Desiccationtolerant : high PC, high PI, high di+oligo. Desiccationsensitive: high PE high PA WP4 main results – interspecific variability for seed desiccation tolerance PE:PC ratio chosen as potential ‘simple’ marker

  16. WP4 main results – interspecific variability for seed desiccation tolerance In the dry state, PE form non-lamellar(Hexagnal II) phase = irreversible damage PC form gel lamellar phase = no damage

  17. WP4 main results – loss of cryo-ability during storage Cryo-ability 1. Storage 2. Desiccation Lyso-PE 3. Cooling The rapid loss of cryo-ability during storage of seeds in the Laboratory, after harvest and beforecryopreservation,is associated with phospholipidde-esterification = irreversible lossof membrane integrity 4. Rewarming Lyso-PI 5. Rehydration

  18. Conclusions • Methods for analyzing all lipid components are now available for coffee species • They will be employed for Crymcept studies during the following months • Some methods are certainly applicable to other plant materials

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