Plant Science 219–220 (2014) 19–25
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Lipase activity and antioxidant capacity in coffee (Coffea arabica L.) seeds during germination Sonia Patui a , Luisa Clincon a , Carlo Peresson a , Marco Zancani a , Lanfranco Conte b , Lorenzo Del Terra c , Luciano Navarini c , Angelo Vianello a , Enrico Braidot a,∗ a
Department of Agricultural and Environmental Sciences, Unit of Plant Biology, University of Udine, via delle Scienze 91, 33100 Udine, Italy Department of Food Science, University of Udine, via Sondrio 2/A, 33100 Udine, Italy c Illycaffè s.p.a, via Flavia 110, 34147 Trieste Italy b
a r t i c l e
i n f o
Article history: Received 30 October 2013 Received in revised form 19 December 2013 Accepted 25 December 2013 Available online 3 January 2014 Keywords: Free fatty acids Germination Coffee seed Lipase Oxidative stress Parchment
a b s t r a c t In this paper, lipase activity was characterized in coffee (Coffea arabica L.) seeds to determine its involvement in lipid degradation during germination. The lipase activity, evaluated by a colorimetric method, was already present before imbibition of seeds and was further induced during the germination process. The activity showed a biphasic behaviour, which was similar in seeds either with or without endocarp (parchment), even though the phenomenon showed a delay in the former. The enzymatic activity was inhibited by tetrahydrolipstatin (THL), a selective and irreversible inhibitor of lipases, and by a polyclonal antibody raised against purified alkaline lipase from castor bean. The immunochemical analysis evidenced a protein of ca. 60 kDa, cross-reacting with an anti-lipase antibody, in coffee samples obtained from seeds of both types. Gas chromatographic analyses of free fatty acid (FFA) content confirmed the differences shown in the lipolytic activity of the samples with or without parchment, since FFA levels increased more rapidly in samples without parchment. Finally, the analyses of the antioxidant capacity showed that the presence of parchment was crucial for lowering the oxidation of the lipophylic fraction, being the seeds with parchment less prone to oxidation processes. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Coffee is an economically relevant commodity, internationally traded as green coffee [1]. The Coffea genus contains around 100 species, being C. arabica and C. canephora the most important ones for commercial purposes. The coffee plant is an evergreen shrub or small tree, native to the forests in the Ethiopian mountains. Its white and bisexual flowers form a short inflorescence called glomerule. The fruit is classified as a drupe, consisting of a smooth skin or exocarp, a soft yellowish pulp or mesocarp, and a greyish-green fibrous endocarp surrounding the seeds, which are commonly called coffee beans. The exocarp becomes transiently yellow and then red at the final stage of development.
Abbreviations: FFA, free fatty acids; TAGs, triacylglycerols; ROS, reactive oxygen species; DAI, days after imbibition; DGGMR, 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6 -methylresorufin)-ester; THL, tetrahydrolipstatin; MAGs, monoacylglycerols; DAGs, diacylglycerols. ∗ Corresponding author. Tel.: +39 0432 558792; fax: +39 0432 558784. E-mail addresses: [email protected] (S. Patui), [email protected] (L. Clincon), [email protected] (C. Peresson), [email protected] (M. Zancani), [email protected] (L. Conte), [email protected] (L. Del Terra), [email protected] (L. Navarini), [email protected] (A. Vianello), [email protected] (E. Braidot). 0168-9452/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2013.12.014
The mesocarp is rich in reducing sugars, sucrose and water. The endocarp (also called parchment, or “pergaminho”) is a hard and lignified tissue, protecting coffee seeds against digestive enzymes from the gut of frugivorous animals [2]. Coffee seeds represent vital organs, in which various metabolic reactions take place during post-harvest processing, e.g. germination-related metabolism and stress metabolism [3]. Coffee seeds are classified as intermediate seeds [4], able to withstand considerable drying in comparison to recalcitrant seeds. Such seeds cannot tolerate extreme water loss as in the case of orthodox ones [5]. Indeed, coffee beans can tolerate desiccation down to a water content of about 7–12%, although further drying leads to rapid loss in viability [3,4]. Similarly, the coffee embryo is very sensitive to low temperature, and it is heavily damaged when seeds are kept to temperature lower than 25 ◦ C. In particular, it is demonstrated that the lower the storage temperature, the more critical the seed water content becomes [6]. Seed storage for medium periods at 25 ◦ C is possible if environment relative humidity is maintained around 50%, while for conservation at freezing temperatures, a lower content in coffee seed moisture and hermetic conditions are required [7]. Both intermediate and recalcitrant seeds are characterized by the lack of dormancy and partial desiccation, features that are associated to an appreciable metabolism, which makes the seeds ready
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to germinate. In coffee seeds, the germination-related processes have been monitored by the expression of enzymes related to germination, like isocitrate lyase, and by -tubulin accumulation, as markers of cell cycle activity [8]. The extent and time-course of this metabolism depends strongly on post-harvest processing; indeed, germination-related reactions are activated earlier during “wet” processes in comparison to “dry” ones [3,8]. Another aspect of coffee processing is represented by the unusual maintenance of the parchment in coffee seeds after “wet” processes. In this case, green coffee beans exhibit high viability for longer periods, if compared to the hulled beans obtained by “typical wet” and “dry” processes, where the parchment is usually removed [9]. In addition, this maintenance of viability seems to be positively correlated to the preservation of green coffee quality during prolonged storage [9]. Indeed, the active metabolism of green coffee seeds could have a negative impact on the quality features of commercial beans through the deterioration of well-known precursors of coffee flavour, such as free amino acids and free sugars, which could lead to a poor final cup quality [9,10]. During green coffee storage, the “off-flavours” are also produced by the oxidation processes acting on the lipid fraction [9,11]. Oxidative stress, lipid hydrolysis, phospholipid loss and decrease in concentrations of two crucial antioxidant compounds, such as glutathione and ascorbate, are involved in the ageing of coffee seeds stored at 20 ◦ C and at intermediate relative humidity (81%) [5]. Therefore, the control of oxidative processes is an essential aspect, not only for storage but also for germination of coffee seeds, since the embryo viability could be affected by the uncontrolled release of free fatty acids (FFA). The lipid fraction in green C. arabica seeds represents a significant part of dry matter, ranging from 13 to 17%, consisting mainly of triacylglycerols (TAGs) [11], which are responsible for the major aroma in roasted beans [12]. Since the lipid fraction is a typical substrate for degradation reactions, the content of FFA is positively correlated to increases of temperature, oxygen content and moisture, during storage for 18 months [11]. The role of lipid hydrolysis in the behaviour of coffee seeds during storage has been further confirmed by the negative correlation observed between coffee seed viability and FFA content [13]. Nevertheless, it is not yet clear how environmental conditions could affect lipid metabolism during storage and the initial phases of germination. As a consequence of the features described above, the storage behaviour of coffee beans depends on the result of various and complex factors, acting at different times. Considering the crucial role of lipid metabolism during both seed germination and post-harvest processing, these aspects appear to be scarcely investigated [14]. In particular, to our knowledge, lipase activity in coffee seeds has been only preliminarily identified. Lipases are ubiquitous enzymes belonging to the class of serine hydrolases (triacylglycerol acylhydrolases EC 3.1.1.3) with a Ser-His-Asp triad in their active site. They mainly catalyze the hydrolysis of ester bonds in monoacylglycerol, diacyglycerol and TAGs into FFA and glycerol at the oil/water interface. In plants, these enzymes play a relevant role in the mobilization of reserves (stored as TAGs) during oilseed germination [15,16]. A further aspect involved in both germination and storage is the control of the levels of reactive oxygen species (ROS). As recently proposed, low levels of ROS are present in ungerminated dormant seeds, while during germination ROS act as signalling molecules leading to seed dormancy release. Nevertheless, the homeostasis of ROS should be strictly controlled since their overproduction leads to oxidative damage in both germinating and stored seeds [17]. In this work lipase activity was examined in both ungerminated and germinating C. arabica L. seeds, either in presence or absence of the parchment. In addition, the antioxidant properties
of germinating coffee seeds were analyzed, aiming at identifying possible correlations between the antioxidant protection and the lipid degradation processes. 2. Materials and methods 2.1. Plant material and seed germination Coffee seeds (C. arabica L.) harvested in Colombia were provided by illycaffè spa, Trieste, Italy, as a single bulk, stored in polypropylene plastic bags for a maximum of two months at room temperature. The seeds were provided with the parchment and then were divided into two lots: the first had the parchment manually removed (− parchment), while the second still conserved the endocarp (+ parchment). The seeds were kept in water for 7 days at 28 ◦ C in the dark. After imbibition, the seeds were sown in perlite at 28 ◦ C in the dark and daily watered. At each sampling day (0, 3, 7, 10, 12, 15, 18, 21, 24, 28 days after imbibition, DAI), about 40 ungerminated or germinating seeds were collected, the parchment was manually removed when present, and the seeds were finally frozen in liquid nitrogen and stored at −80 ◦ C. The experimental design consisted of three independent replicates for all the considered variables. The replicates were performed using the same seed lot for three progressive sowing within two months. 2.2. Acetone powder preparation Ten g of frozen coffee beans was ground by a blender (Ika Werke, Staufen, Germany) to obtain a fine powder. Then, the powder was stirred for 4 h at 4 ◦ C in 50 ml of chilling acetone (−20 ◦ C), and subsequently centrifuged at 1900 × g (SS-34 rotor, Sorvall) for 15 min. The pellet was dried under nitrogen, resuspended in 50 mM TrisHCl (pH 7.5), 1 mM EDTA and 0.4 M sucrose, homogenized with an Ultra-Turrax (Ika Werke, Staufen, Germany), and finally centrifuged at 11,900 × g (SS-34 rotor) for 20 min. The supernatant was filtered through cotton gauze and stored at −80 ◦ C. This preparation was used to evaluate the lipase activity, protein content, and to perform SDS-PAGE and Western blotting. 2.3. Assay of lipase activity Lipase activity was assayed by a colorimetric method, using a kit from Randox (Lipase, Crumlin, UK). The assay was based on the hydrolysis of a specific substrate (1,2-O-dilauryl-racglycero-3-glutaric acid-(6 -methylresorufin)-ester) (DGGMR): this chromogenic compound is cleaved by the catalytic action of lipases into 1,2-o-dilauryl-rac-glycerol and glutaric acid-(6-methyl resorufin) ester, an unstable intermediate. The latter decomposes spontaneously in alkaline solution to form glutaric acid and methylresorufin, a coloured compound that was determined photometrically by a Multilabel plate reader at 570 nm (Wallac 1420, PerkinElmer Waltham, MA, USA). The reaction mixture was composed by 5 l (approx. 7.5 g protein) of extract, 155 l of buffer and the reactions were started by the addition of 40 l of DGGMR. The analysis was carried out for 2 h at 30 ◦ C. The enzymatic activity was expressed as nmol methylresorufin mg−1 protein min−1 , using an extinction coefficient ε = 54 × 103 M−1 cm−1 at 570 nm. The analysis of lipase activity inhibition was performed using tetrahydrolipstatin (THL) and an anti-lipase antibody (Ab) raised against the lipase of castor bean (Ricinus communis L.) (Agrisera, Cernusco sul Naviglio, Milan, Italy). THL (up to 300 M) and antilipase Ab (up to 0.6 ng) were added to the reaction mixture in the assay and incubated for 30 min at 30 ◦ C.
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2.4. Antioxidant capacity assay
2.7. Protein determination
The antioxidant capacity of germinating coffee seeds was evaluated by crocin kinetic competition test, as described by Tubaro and coworkers [18]. This test was performed on the hydrophobic compounds extracted with 1,4-dioxane from 1 g of ground material obtained from germinating coffee seeds at different sampling days. Then, the samples were centrifuged at 7600 × g (SS-34 rotor) for 20 min and the supernatant was collected. Peroxy radicals were generated in situ by diazo compound decomposition. The bleaching of crocin, monitored at 443 nm, was directly correlated to radical production and occurred with a constant speed. When part of the peroxyl radicals was quenched by other antioxidants, the bleaching rate was lower, and was correlated to the concentration of the antioxidants present in the sample. The ratio between the bleaching speed of crocin, with or without other antioxidants, yielded the whole antioxidant capacity of the sample. The slope of a known antioxidant, such as ␣-tocopherol, was calculated as reference. The results were expressed as milliequivalents (meq) of ␣-tocopherol g−1 fresh weight.
Protein content of the samples was determined according to Bradford [20], using bovine serum albumin as a standard. The dye kit was from Sigma–Aldrich. The increase of absorbance due to dye-protein interaction was evaluated at 595 nm using Lambda 15 Perkin Elmer spectrophotometer. 2.8. Statistics The statistical analyses were performed using Statistica software 10 (Statsoft Inc., USA). The presented data are means ± S.D. Two-way ANOVA analysis has been performed on lipase activity, antioxidant capacity and FFA content, considering time and presence/absence of parchment as factors. Significant differences (p ≤ 0.05) between means were determined for each variable by post hoc Fisher LSD test to rank the experimental groups. 3. Results 3.1. Characterization of lipase activity in coffee seeds
2.5. SDS-PAGE and Western blotting Proteins from acetone powder preparation (about 20 g) were separated by 10% (w/v) SDS-PAGE, under reducing conditions and the gels were stained with Coomassie Brilliant Blue R-250. Immunoblotting was performed according to standard techniques, using the anti-lipase Ab at 1:20,000 dilution. The immune reaction was detected with an antirabbit IgG alkaline phosphatase-conjugated secondary Ab (1:2500 dilution), followed by the addition of the substrate 5-bromo-4-chloro3indolyl phosphate/nitroblue tetrazolium, buffered by substrate tablets (Sigma–Aldrich, Milan, Italy). Computer-assisted densitometric analysis of the Western blot was performed by Quantity One Software (Bio-Rad, Hercules, CA, USA). 2.6. Gas chromatography analysis of lipid extracts Two grams of frozen coffee beans (0, 7, 12, 21, 28 DAI) was ground by a blender (Ika Werke, Staufen, Germany) to obtain a fine powder. The powder was stirred for 2 h at 4 ◦ C in 10 ml of hexane-ether (1:1, v/v) and the organic phase was dried with anhydrous sodium sulphate (0.1 g ml−1 ) and finally filtered with Whatman paper (Maidstone, England). The solvent was removed under nitrogen to recover the extracted oil. About 100 mg of oil was added to 1 ml of internal standard solution containing 1 mg methyl heptadecanoate and 1 mg triheptadecanoin. Then 20–30 l of solution was dried under nitrogen stream and solubilized in 200 l of silylating reagent (a mixture of pyridine, bistrimethylsilyltrifluoroacetamide/trimethylchlorosilane, 1:1, v/v), kept at room temperature for 20 min in a tightly closed vial. Finally, it was dried by a soft nitrogen flow and resuspended in 2 ml of n-heptane. Analysis was performed within 6 h. One l was injected for GC analyses, performed using a Thermo Fished MEGA 5300 capillary gas chromatography system with cold on column injection port with flame ionization detection and a fused silica column (3 m × 0.32 mm i.d., coated with SPB5 stationary phase (df 0.1 m) Supelco, Bellefonte, Pennsylvania, USA – Sigma–Aldrich, Milan, Italy). The analyses were carried out in programme temperature mode from 80 ◦ C to 300 ◦ C (at 30 ◦ C min−1 ) and then from 300 ◦ C to 360 ◦ C (at 5 ◦ C min−1 ) and hold at 360 ◦ C for 10 min. The carrier gas was helium (flow rate 1.5 ml min−1 ), auxiliary gases: hydrogen (flow rate: 25 ml min−1 ), air (flow rate: 300 ml min−1 ) [19]. Compounds were identified by comparison with commercial standards.
Lipase activity was evaluated in coffee seeds without parchment at 12 DAI, just before radicle protrusion. The activity was detected at increasing concentration of DGGMR, ranging from 0 to 100 M (Fig. 1). The initial rate values fitted the Michaelis–Menten equation, whose kinetic parameters were: Vmax = 0.23 ± 0.01 mol min−1 mg−1 protein and KM = 14.18 ± 2.79 M. This lipase was extremely sensitive to the specific lipase inhibitor THL, which reacts with a serine present in the active site [21], reaching approx. 67% inhibition at 300 M (Fig. 2). Furthermore, the activity was inhibited by the polyclonal anti-lipase Ab, in a concentration-dependent manner (Fig. 3). The inhibition followed the equation y = y0 + a × e(−b×x) and showed a maximum inhibition of approx. 20% in the presence of 0.6 ng Ab with respect to the control. 3.2. Parchment influence on germination, antioxidant capacity and lipase activity The developmental stages of coffee seeds during germination are exemplified in Fig. 4, panel A. Coffee seeds sown without parchment exhibited a visible radicle protrusion at 7 DAI, indicating the embryo growth inside of the endosperm. The radicle emission appeared at about 15 DAI and in the following days the radicle elongation was clearly evident. On the contrary, in coffee seeds with
Fig. 1. Characterization of lipase activity in coffee seeds at 12 DAI. The initial rate of lipase activity on DGGMR concentration fitted (r2 = 0.97) the equation V = Vmax [DGGMR]/(KM [DGGMR]). Data (n = 3) are means ± S.D.
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Residual lipase activity (%)
120 100 80 60 40 20 0 0
10
100
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THL ( M) Fig. 2. Inhibition by THL of lipase activity in coffee seeds at 12 DAI. The initial rate was expressed as percentage with respect to the control. Data (n = 3) are means ± S.D.
parchment the radicle emission was strongly delayed, occurring approximately at 28 DAI. During germination, lipase activity was measured in coffee seeds with or without parchment up to 28 DAI (Fig. 4, panel B). This activity was very similar in seeds with or without parchment at the first three sampling days (0, 4 and 8 DAI), and the values were not significantly different. From 10 DAI, the enzymatic activity showed a different pattern: coffee seeds with parchment were characterized by a slow continuous increase of lipase activity, with the highest value detected at 28 DAI. Conversely, coffee seeds without parchment showed a peak of lipase activity at 12 DAI, followed by a decrease. This pattern was supported by the immunoblotting assays performed using the anti-lipase Ab. In coffee seeds, either without (Fig. 5, panel A) or with (panel B) parchment, the anti-lipase Ab cross-reacted with a protein of approx. 60 kDa. The intensity of this cross-reaction, obtained from the densitometric analysis of the immunoblot membrane, appeared different during the germination stages in both seed lots (Fig. 5, panel C). In coffee seeds without parchment, the lipase increased during the first sampling days, exhibiting its maximum at 15 DAI, to decrease hereafter. A different pattern was observed in coffee seeds with parchment, where the lipase continued to increase after 15 DAI,
Fig. 4. Influence of parchment on lipase activity in germinating coffee seeds. Panel A: morphological development of germinating coffee seeds with (+) and without (−) parchment. Panel B: lipase activity in coffee seeds with () or without () parchment. Data (n = 3) are means of the initial rate of lipase activity ± S.D.
reaching its highest level at 28 DAI. The cross-reactivity of coffee seed lipase with the anti-lipase Ab was, therefore, correlated with the enzymatic activity shown in Fig. 4, panel B. Aiming at describing the degree of oxidative status of coffee seeds with or without parchment, the antioxidant capacity of coffee beans was measured during germination at 0, 7, 12, 21, 28 DAI (Fig. 6). The pattern exhibited a trend resembling that described for lipase activity. At 0 and 7 DAI, total antioxidant capacity was very similar for seeds with or without parchment. During the following germination steps, the lipophilic antioxidant activity in the seeds without parchment showed a peak at 12 DAI, while decreasing later. In the seeds with parchment the maximum was delayed, showing the highest total antioxidant capacity at 21 DAI. 3.3. Gas chromatographic analysis of lipid extracts Lipid profile throughout germination was characterized in coffee oil. An example of a typical chromatogram of the pattern composition (FFA, monoacylglycerols, MAGs, diacylglycerols, DAGs, and TAGs) is shown in Fig. 7 panel A. In coffee seeds with parchment, the accumulation of FFA increased steadily throughout the period of germination (Fig. 7, Panel B). On the contrary, coffee seeds without parchment showed a maximum level of FFA at 14 DAI that decreased afterwards until 28 DAI. The analysis of TAGs, DAGs and MAGs did not exhibit significant differences in coffee seeds, with or without parchment, and throughout germination (results not shown). 4. Discussion
Fig. 3. Inhibition by anti-lipase Ab on lipase activity in coffee seeds at 12 DAI. The initial rate was expressed as percentage with respect to the control (without antilipase Ab). Data (n = 3) are means ± S.D. Data fitted (r2 = 0.95) to an exponential decay function, two parameter equation y = y0 + a × e(−b×x) , where the parameters found were: y0 = 79.9 ± 1.2, the residual relative lipase activity, a = 20.8 ± 2.4, the fraction of relative lipase activity that is amenable to inhibition by the antibody; b = 61.8 ± 17.1, the rate of decline of relative lipase activity with increasing concentrations of the antibody.
Lipid bodies in oilseeds are mainly composed of TAGs and in coffee seeds they are generally located in the endosperm [11]. Uncontrolled TAGs metabolism during storage could lead to impairment of germination in propagation material and also to production of undesired compounds responsible for decrease in final cup quality [9]. The release of FFA could, indeed, trigger either degenerative
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Fig. 7. Analyses of FFA in coffee seeds at different DAI. Panel A: GC/FID chromatogram of FFA, MAGs, DAGs and TAGs in coffee beans; Panel B: FFA content in coffee seeds either with (black columns) or without (white columns) parchment.
-1
Antioxidant capacity (meq -tocopherol g )
Fig. 5. Immunoblot analyses of lipase from coffee seeds at different DAI. Western blots of proteins (approx. 20 g) obtained from coffee seeds without (Panel A) or with (Panel B) parchment. The values represent the apparent molecular mass of molecular standards. Panel C: densitometric analysis of immunoblot. Data (n = 3) are mean values ± SD, calculated from three independent experiments. Columns represent the spot density from coffee seeds either with (black) or without (white) parchment.
1.4 f
1.2 1.0
de
e
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cd abc bc
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ab
ab
a a
0.4 0.2 0.0 0
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12
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DAI Fig. 6. Lipophilic antioxidant capacity in coffee seeds at different DAI. Antioxidant capacity was measured in coffee seeds either with (black columns) or without (white columns) parchment. Data (n = 3) are means ± S.D.
processes, resulting in lipid oxidation/peroxidation, or fuelling high-energy demanding processes, e.g. respiration and sugar synthesis for embryo growth [22]. Even though lipase in coffee seeds represents a crucial enzyme during storage and germination, few studies have dealt with this aspect [14]. Therefore, this paper represents, to our knowledge, the first characterization of lipases from coffee seeds. The lipase activity in coffee seeds, evaluated with a colorimetric method, was characterized by kinetics parameters (KM and Vmax , Fig. 1), that were similar to those reported for lipases from Barbados nut (Jatropha curcas L.), whose seeds are classified as recalcitrants [23], but different to those found in orthodox seeds such as sunflower, French peanut (Panchira aquatica), as well as in seedlings of wheat, oat [24], rice [25] and rape [26]. The higher affinity for the substrates exhibited by coffee lipase suggests that in such seeds, containing a high amount of stored lipids, the lipolytic activities could play a regulative role during the first steps of germination. This lipase activity was further characterized in germinating coffee seeds by the inhibition by THL (Fig. 2), a selective and irreversible inhibitor of lipases, and by a polyclonal antibody raised against purified alkaline lipase from castor (Ricinus communis L.) beans (Fig. 3). This antibody also cross-reacted with a protein of approx. 60 kDa (Fig. 5), a value similar to that of the acid lipase RcOBL1, isolated and characterized in oil body membranes from castor bean [27]. The lipase activity was present in coffee seeds before imbibition and further induced by the germination process. During this phase, it followed a biphasic trend, which was similar in seeds either with or without parchment (Fig. 4, panel B), even though the phenomenon showed a delay in the former. In coffee seeds without
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parchment, a sharp second peak was observed after 12 DAI. On the contrary, the lipase activity gradually increased in coffee with parchment, in parallel with the delay in germination, while the other samples exhibited a precocious germination. A similar trend was also observed by the Western blot analysis of total coffee bean proteins, where the detection of lipases was possible since the very first DAI. Afterwards, it decreased at around 8 DAI, to rise again after about 12 up to 28 DAI in parchment coffee seeds (Fig. 5). This biphasic activity could possibly be attributable to ex novo synthesis and/or to different lipase isoenzymes, similarly to what was recently found in rice [25]. In this species, lipase-I and lipase-II have been reported to be involved in lipolytic activity. In particular, lipase-I is responsible for lipid metabolism for root and shoot growth, while lipase II is active during germination [25]. Therefore, it is possible to speculate that lipase activity in coffee would be exerted first by enzymes already present in the beans and, subsequently, during the second phase, by ex novo synthesized lipases induced by the germination process. The delay in activation of germination and root development could be explained by the presence of parchment, that slowed down metabolic activities by restricting gas exchanges. According to the observations reported above, the analysis of antioxidant capacity indicated that parchment was crucial for preventing the oxidation of the lipophilic fraction, being the seeds with parchment less prone to oxidation (Fig. 7). Indeed, the parchment could act as a barrier to oxygen fluxes towards the embryo/endosperm, thus limiting the production of ROS [28]. This environment would allow the germinating seeds to control ROS levels within the “oxidative window” for germination, as proposed by Bailly and coworkers [17]. Oxidative signals, mainly represented by ROS, are involved in seed germination, but their concentration must be adequate to allow the completion of this process [17,29,30]. Nevertheless, ROS excess is detrimental and a delicate balance is needed to prevent the activation of a high oxidative metabolism, which is a harmful feature, causing a rapid decline of embryo viability [31]. The release of FFA in coffe seeds, as expected, increased in both samples, but it reached a very high value earlier in coffee seeds without parchment, when compared to those with parchment (Fig. 7, panel B). This result would depend on the initial higher lipase activity observed until 12 DAI (Fig. 4, panel B). Further research will be useful to clarify if the early accumulation of high amount of FFA at 12 DAI would prevent seeds to metabolize all FFA by -oxidation. This would instead lead to lipid oxidation, thus explaining the depletion of antioxidant capacity in seeds without parchment (Fig. 7). On the other hand, this hypothesis is consistent with what observed in seeds still protected by parchment where a slower but steady release of FFA would lead to a lower consumption of the antioxidant capacity. In conclusion, our study suggests that coffee beans possess lipase activity that was already present during storage and increased during germination. The lack of quiescence and ABA insensitivity of intermediate coffee seeds require a more strict metabolic control of the degradation processes, to provide new organic skeletons for the development of the seedling. In particular, a fine tuning is necessary to avoid the uncontrolled release of ROS, especially in seeds exhibiting high lipid content in the endosperm. Therefore, we propose that in coffee seeds the regulation of the gas exchange exerted by the parchment would be considered similar to what demonstrated in holm oak seed, where the use of plastic films limiting the gaseous exchange prolonged the dormant-like state and consequently enhanced seed viability [31]. In particular, the decrease of oxygen permeation due to the parchment in coffee seeds could lead to positive effects during storage and germination, e.g. through the decrease of both degeneration products and oxidative processes. It is reasonable to suggest that parchment exerts a regulative role during germination, allowing the well-ordered
progress of all the different stages. Besides its restriction to gas exchange, we speculate that the polyphenol content of parchment, in particular chlorogenic acid, might inhibit lipase activity. Such inhibition would be useful to limit the amount of FFA initially released, allowing the physiological development of germination in coffee that, as intermediate seeds, is characterized by a partially activated metabolic machinery. Hence, our results supply a physiological explanation to field experience and to technological practices, which indicate that the presence of parchment leads to prolonged maintenance of seed viability and preservation of green coffee quality with respect to hulled beans [9]. To better understand the physiology of lipid metabolism in coffee seeds during germination, a crucial aspect to study will be the characterization of free fatty acid release in relation with the oxidative damage. Further research could also open new scenarios to clarify the modulation of lipase activities and the possible positive effects due to the control of oxygen permeability in green coffee during storage.
Acknowledgements We are grateful to Dr. Alessio Colussi (formerly Head of green coffee department illycaffè) for the critical reading of the manuscript. This work was supported by European Regional Development Funds, Cross-Border Cooperation Italy-Slovenia Programme 2007–2013 (TRANS2CARE and AGROTUR projects). We are also grateful to prof. Franco Tubaro (University of Udine) for the critical reading of the manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2013. 12.014.
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S. Patui et al. / Plant Science 219–220 (2014) 19–25 [14] T. Kurzrock, I. Kolling-Speer, K. Speer, Effects of controlled storage on the lipid fraction of green arabica coffee beans, in: 20’eme Colloque Scientifique International sur le Cafe´ı, Bangalore, India, 2005, pp. 161–168. [15] F. Beisson, A. Tiss, C. Rivière, R. Verger, Methods for lipase detection and assay: a critical review, Eur. J. Lipid Sci. Technol. 102 (2000) 133–153. [16] I.A. Graham, Seed storage oil mobilization, Ann. Rev. Plant Biol. 59 (2008) 115–142. [17] C. Bailly, H. El-Maarouf-Bouteau, F. Corbineau, From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology, C. R. Biol. 331 (2008) 806–814. [18] F. Tubaro, E. Micossi, F. Ursini, The antioxidant capacity of complex mixtures by kinetic analysis of crocin bleaching inhibition, J. Am. Oil Chem. Soc. 73 (1996) 173–179. [19] I.T.C.f.F.a. Oils, NGD method C 77., Norme Grassi e Derivati. Stazione sperimentale per le industrie degli oli e dei grassi. (1976). [20] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [21] P. Hadvary, H. Lengsfeld, H. Wolfer, Inhibition of pancreatic lipase invitro by the covalent inhibitor tetrahydrolipstatin, Biochem. J. 256 (1988) 357–361. [22] A.L. Quettier, P.J. Eastmond, Storage oil hydrolysis during early seedling growth, Plant Physiol. Biochem. 47 (2009) 485–490.
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[23] R. Staubmann, I. Ncube, G.M. Gubitz, W. Steiner, J.S. Read, Esterase and lipase activity in Jatropha curcas L-seeds, J. Biotechnol. 75 (1999) 117–126. [24] M. Barros, L.F. Fleuri, G.A. Macedo, Seed lipases: sources, applications and properties – a review, Braz. J. Chem. Eng. 27 (2010) 15–29. [25] K.R. Vijayakumar, L.R. Gowda, Temporal expression profiling of lipase during germination and rice caryopsis development, Plant Physiol. Biochem. 57 (2012) 245–253. [26] H. Belguith, S. Fattouch, T. Jridi, J. Ben Hamida, Immunopurification of a rape (Brassica napus L.) seedling lipase, Afr. J. Biochem. Res. 3 (2009) 356–365. [27] P.J. Eastmond, Cloning and characterization of the acid lipase from castor beans, J. Biol. Chem. 279 (2004) 45540–45545. [28] S.R. Mousavi, M.M.A. Rezaei, A general overview on seed dormancy and methods of breaking it, Adv. Environ. Biol. 5 (2011) 3333–3337. [29] I. Kranner, F.V. Minibayeva, R.P. Beckett, C.E. Seal, What is stress? Concepts, definitions and applications in seed science, New Phytol. 188 (2010) 655–673. [30] S. Pukacka, M. Malec, E. Ratajczak, ROS production and antioxidative system activity in embryonic axes of Quercus robur seeds under different desiccation rate conditions, Acta Physiol. Plant. 33 (2011) 2219–2227. [31] S. Pasquini, M. Mizzau, E. Petrussa, E. Braidot, S. Patui, F. Gorian, M. Lambardi, A. Vianello, Seed storage in polyethylene bags of a recalcitrant species (Quercus ilex): analysis of some bio-energetic and oxidative parameters, Acta Physiol. Plant. 34 (2012) 1963–1974.
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Lipase activity and antioxidant capacity in coffee (Coffea arabica L.) seeds during germination Sonia Patui a , Luisa Clincon a , Carlo Peresson a , Marco Zancani a , Lanfranco Conte b , Lorenzo Del Terra c , Luciano Navarini c , Angelo Vianello a , Enrico Braidot a,∗ a
Department of Agricultural and Environmental Sciences, Unit of Plant Biology, University of Udine, via delle Scienze 91, 33100 Udine, Italy Department of Food Science, University of Udine, via Sondrio 2/A, 33100 Udine, Italy c Illycaffè s.p.a, via Flavia 110, 34147 Trieste Italy b
a r t i c l e
i n f o
Article history: Received 30 October 2013 Received in revised form 19 December 2013 Accepted 25 December 2013 Available online 3 January 2014 Keywords: Free fatty acids Germination Coffee seed Lipase Oxidative stress Parchment
a b s t r a c t In this paper, lipase activity was characterized in coffee (Coffea arabica L.) seeds to determine its involvement in lipid degradation during germination. The lipase activity, evaluated by a colorimetric method, was already present before imbibition of seeds and was further induced during the germination process. The activity showed a biphasic behaviour, which was similar in seeds either with or without endocarp (parchment), even though the phenomenon showed a delay in the former. The enzymatic activity was inhibited by tetrahydrolipstatin (THL), a selective and irreversible inhibitor of lipases, and by a polyclonal antibody raised against purified alkaline lipase from castor bean. The immunochemical analysis evidenced a protein of ca. 60 kDa, cross-reacting with an anti-lipase antibody, in coffee samples obtained from seeds of both types. Gas chromatographic analyses of free fatty acid (FFA) content confirmed the differences shown in the lipolytic activity of the samples with or without parchment, since FFA levels increased more rapidly in samples without parchment. Finally, the analyses of the antioxidant capacity showed that the presence of parchment was crucial for lowering the oxidation of the lipophylic fraction, being the seeds with parchment less prone to oxidation processes. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Coffee is an economically relevant commodity, internationally traded as green coffee [1]. The Coffea genus contains around 100 species, being C. arabica and C. canephora the most important ones for commercial purposes. The coffee plant is an evergreen shrub or small tree, native to the forests in the Ethiopian mountains. Its white and bisexual flowers form a short inflorescence called glomerule. The fruit is classified as a drupe, consisting of a smooth skin or exocarp, a soft yellowish pulp or mesocarp, and a greyish-green fibrous endocarp surrounding the seeds, which are commonly called coffee beans. The exocarp becomes transiently yellow and then red at the final stage of development.
Abbreviations: FFA, free fatty acids; TAGs, triacylglycerols; ROS, reactive oxygen species; DAI, days after imbibition; DGGMR, 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6 -methylresorufin)-ester; THL, tetrahydrolipstatin; MAGs, monoacylglycerols; DAGs, diacylglycerols. ∗ Corresponding author. Tel.: +39 0432 558792; fax: +39 0432 558784. E-mail addresses: [email protected] (S. Patui), [email protected] (L. Clincon), [email protected] (C. Peresson), [email protected] (M. Zancani), [email protected] (L. Conte), [email protected] (L. Del Terra), [email protected] (L. Navarini), [email protected] (A. Vianello), [email protected] (E. Braidot). 0168-9452/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2013.12.014
The mesocarp is rich in reducing sugars, sucrose and water. The endocarp (also called parchment, or “pergaminho”) is a hard and lignified tissue, protecting coffee seeds against digestive enzymes from the gut of frugivorous animals [2]. Coffee seeds represent vital organs, in which various metabolic reactions take place during post-harvest processing, e.g. germination-related metabolism and stress metabolism [3]. Coffee seeds are classified as intermediate seeds [4], able to withstand considerable drying in comparison to recalcitrant seeds. Such seeds cannot tolerate extreme water loss as in the case of orthodox ones [5]. Indeed, coffee beans can tolerate desiccation down to a water content of about 7–12%, although further drying leads to rapid loss in viability [3,4]. Similarly, the coffee embryo is very sensitive to low temperature, and it is heavily damaged when seeds are kept to temperature lower than 25 ◦ C. In particular, it is demonstrated that the lower the storage temperature, the more critical the seed water content becomes [6]. Seed storage for medium periods at 25 ◦ C is possible if environment relative humidity is maintained around 50%, while for conservation at freezing temperatures, a lower content in coffee seed moisture and hermetic conditions are required [7]. Both intermediate and recalcitrant seeds are characterized by the lack of dormancy and partial desiccation, features that are associated to an appreciable metabolism, which makes the seeds ready
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to germinate. In coffee seeds, the germination-related processes have been monitored by the expression of enzymes related to germination, like isocitrate lyase, and by -tubulin accumulation, as markers of cell cycle activity [8]. The extent and time-course of this metabolism depends strongly on post-harvest processing; indeed, germination-related reactions are activated earlier during “wet” processes in comparison to “dry” ones [3,8]. Another aspect of coffee processing is represented by the unusual maintenance of the parchment in coffee seeds after “wet” processes. In this case, green coffee beans exhibit high viability for longer periods, if compared to the hulled beans obtained by “typical wet” and “dry” processes, where the parchment is usually removed [9]. In addition, this maintenance of viability seems to be positively correlated to the preservation of green coffee quality during prolonged storage [9]. Indeed, the active metabolism of green coffee seeds could have a negative impact on the quality features of commercial beans through the deterioration of well-known precursors of coffee flavour, such as free amino acids and free sugars, which could lead to a poor final cup quality [9,10]. During green coffee storage, the “off-flavours” are also produced by the oxidation processes acting on the lipid fraction [9,11]. Oxidative stress, lipid hydrolysis, phospholipid loss and decrease in concentrations of two crucial antioxidant compounds, such as glutathione and ascorbate, are involved in the ageing of coffee seeds stored at 20 ◦ C and at intermediate relative humidity (81%) [5]. Therefore, the control of oxidative processes is an essential aspect, not only for storage but also for germination of coffee seeds, since the embryo viability could be affected by the uncontrolled release of free fatty acids (FFA). The lipid fraction in green C. arabica seeds represents a significant part of dry matter, ranging from 13 to 17%, consisting mainly of triacylglycerols (TAGs) [11], which are responsible for the major aroma in roasted beans [12]. Since the lipid fraction is a typical substrate for degradation reactions, the content of FFA is positively correlated to increases of temperature, oxygen content and moisture, during storage for 18 months [11]. The role of lipid hydrolysis in the behaviour of coffee seeds during storage has been further confirmed by the negative correlation observed between coffee seed viability and FFA content [13]. Nevertheless, it is not yet clear how environmental conditions could affect lipid metabolism during storage and the initial phases of germination. As a consequence of the features described above, the storage behaviour of coffee beans depends on the result of various and complex factors, acting at different times. Considering the crucial role of lipid metabolism during both seed germination and post-harvest processing, these aspects appear to be scarcely investigated [14]. In particular, to our knowledge, lipase activity in coffee seeds has been only preliminarily identified. Lipases are ubiquitous enzymes belonging to the class of serine hydrolases (triacylglycerol acylhydrolases EC 3.1.1.3) with a Ser-His-Asp triad in their active site. They mainly catalyze the hydrolysis of ester bonds in monoacylglycerol, diacyglycerol and TAGs into FFA and glycerol at the oil/water interface. In plants, these enzymes play a relevant role in the mobilization of reserves (stored as TAGs) during oilseed germination [15,16]. A further aspect involved in both germination and storage is the control of the levels of reactive oxygen species (ROS). As recently proposed, low levels of ROS are present in ungerminated dormant seeds, while during germination ROS act as signalling molecules leading to seed dormancy release. Nevertheless, the homeostasis of ROS should be strictly controlled since their overproduction leads to oxidative damage in both germinating and stored seeds [17]. In this work lipase activity was examined in both ungerminated and germinating C. arabica L. seeds, either in presence or absence of the parchment. In addition, the antioxidant properties
of germinating coffee seeds were analyzed, aiming at identifying possible correlations between the antioxidant protection and the lipid degradation processes. 2. Materials and methods 2.1. Plant material and seed germination Coffee seeds (C. arabica L.) harvested in Colombia were provided by illycaffè spa, Trieste, Italy, as a single bulk, stored in polypropylene plastic bags for a maximum of two months at room temperature. The seeds were provided with the parchment and then were divided into two lots: the first had the parchment manually removed (− parchment), while the second still conserved the endocarp (+ parchment). The seeds were kept in water for 7 days at 28 ◦ C in the dark. After imbibition, the seeds were sown in perlite at 28 ◦ C in the dark and daily watered. At each sampling day (0, 3, 7, 10, 12, 15, 18, 21, 24, 28 days after imbibition, DAI), about 40 ungerminated or germinating seeds were collected, the parchment was manually removed when present, and the seeds were finally frozen in liquid nitrogen and stored at −80 ◦ C. The experimental design consisted of three independent replicates for all the considered variables. The replicates were performed using the same seed lot for three progressive sowing within two months. 2.2. Acetone powder preparation Ten g of frozen coffee beans was ground by a blender (Ika Werke, Staufen, Germany) to obtain a fine powder. Then, the powder was stirred for 4 h at 4 ◦ C in 50 ml of chilling acetone (−20 ◦ C), and subsequently centrifuged at 1900 × g (SS-34 rotor, Sorvall) for 15 min. The pellet was dried under nitrogen, resuspended in 50 mM TrisHCl (pH 7.5), 1 mM EDTA and 0.4 M sucrose, homogenized with an Ultra-Turrax (Ika Werke, Staufen, Germany), and finally centrifuged at 11,900 × g (SS-34 rotor) for 20 min. The supernatant was filtered through cotton gauze and stored at −80 ◦ C. This preparation was used to evaluate the lipase activity, protein content, and to perform SDS-PAGE and Western blotting. 2.3. Assay of lipase activity Lipase activity was assayed by a colorimetric method, using a kit from Randox (Lipase, Crumlin, UK). The assay was based on the hydrolysis of a specific substrate (1,2-O-dilauryl-racglycero-3-glutaric acid-(6 -methylresorufin)-ester) (DGGMR): this chromogenic compound is cleaved by the catalytic action of lipases into 1,2-o-dilauryl-rac-glycerol and glutaric acid-(6-methyl resorufin) ester, an unstable intermediate. The latter decomposes spontaneously in alkaline solution to form glutaric acid and methylresorufin, a coloured compound that was determined photometrically by a Multilabel plate reader at 570 nm (Wallac 1420, PerkinElmer Waltham, MA, USA). The reaction mixture was composed by 5 l (approx. 7.5 g protein) of extract, 155 l of buffer and the reactions were started by the addition of 40 l of DGGMR. The analysis was carried out for 2 h at 30 ◦ C. The enzymatic activity was expressed as nmol methylresorufin mg−1 protein min−1 , using an extinction coefficient ε = 54 × 103 M−1 cm−1 at 570 nm. The analysis of lipase activity inhibition was performed using tetrahydrolipstatin (THL) and an anti-lipase antibody (Ab) raised against the lipase of castor bean (Ricinus communis L.) (Agrisera, Cernusco sul Naviglio, Milan, Italy). THL (up to 300 M) and antilipase Ab (up to 0.6 ng) were added to the reaction mixture in the assay and incubated for 30 min at 30 ◦ C.
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2.4. Antioxidant capacity assay
2.7. Protein determination
The antioxidant capacity of germinating coffee seeds was evaluated by crocin kinetic competition test, as described by Tubaro and coworkers [18]. This test was performed on the hydrophobic compounds extracted with 1,4-dioxane from 1 g of ground material obtained from germinating coffee seeds at different sampling days. Then, the samples were centrifuged at 7600 × g (SS-34 rotor) for 20 min and the supernatant was collected. Peroxy radicals were generated in situ by diazo compound decomposition. The bleaching of crocin, monitored at 443 nm, was directly correlated to radical production and occurred with a constant speed. When part of the peroxyl radicals was quenched by other antioxidants, the bleaching rate was lower, and was correlated to the concentration of the antioxidants present in the sample. The ratio between the bleaching speed of crocin, with or without other antioxidants, yielded the whole antioxidant capacity of the sample. The slope of a known antioxidant, such as ␣-tocopherol, was calculated as reference. The results were expressed as milliequivalents (meq) of ␣-tocopherol g−1 fresh weight.
Protein content of the samples was determined according to Bradford [20], using bovine serum albumin as a standard. The dye kit was from Sigma–Aldrich. The increase of absorbance due to dye-protein interaction was evaluated at 595 nm using Lambda 15 Perkin Elmer spectrophotometer. 2.8. Statistics The statistical analyses were performed using Statistica software 10 (Statsoft Inc., USA). The presented data are means ± S.D. Two-way ANOVA analysis has been performed on lipase activity, antioxidant capacity and FFA content, considering time and presence/absence of parchment as factors. Significant differences (p ≤ 0.05) between means were determined for each variable by post hoc Fisher LSD test to rank the experimental groups. 3. Results 3.1. Characterization of lipase activity in coffee seeds
2.5. SDS-PAGE and Western blotting Proteins from acetone powder preparation (about 20 g) were separated by 10% (w/v) SDS-PAGE, under reducing conditions and the gels were stained with Coomassie Brilliant Blue R-250. Immunoblotting was performed according to standard techniques, using the anti-lipase Ab at 1:20,000 dilution. The immune reaction was detected with an antirabbit IgG alkaline phosphatase-conjugated secondary Ab (1:2500 dilution), followed by the addition of the substrate 5-bromo-4-chloro3indolyl phosphate/nitroblue tetrazolium, buffered by substrate tablets (Sigma–Aldrich, Milan, Italy). Computer-assisted densitometric analysis of the Western blot was performed by Quantity One Software (Bio-Rad, Hercules, CA, USA). 2.6. Gas chromatography analysis of lipid extracts Two grams of frozen coffee beans (0, 7, 12, 21, 28 DAI) was ground by a blender (Ika Werke, Staufen, Germany) to obtain a fine powder. The powder was stirred for 2 h at 4 ◦ C in 10 ml of hexane-ether (1:1, v/v) and the organic phase was dried with anhydrous sodium sulphate (0.1 g ml−1 ) and finally filtered with Whatman paper (Maidstone, England). The solvent was removed under nitrogen to recover the extracted oil. About 100 mg of oil was added to 1 ml of internal standard solution containing 1 mg methyl heptadecanoate and 1 mg triheptadecanoin. Then 20–30 l of solution was dried under nitrogen stream and solubilized in 200 l of silylating reagent (a mixture of pyridine, bistrimethylsilyltrifluoroacetamide/trimethylchlorosilane, 1:1, v/v), kept at room temperature for 20 min in a tightly closed vial. Finally, it was dried by a soft nitrogen flow and resuspended in 2 ml of n-heptane. Analysis was performed within 6 h. One l was injected for GC analyses, performed using a Thermo Fished MEGA 5300 capillary gas chromatography system with cold on column injection port with flame ionization detection and a fused silica column (3 m × 0.32 mm i.d., coated with SPB5 stationary phase (df 0.1 m) Supelco, Bellefonte, Pennsylvania, USA – Sigma–Aldrich, Milan, Italy). The analyses were carried out in programme temperature mode from 80 ◦ C to 300 ◦ C (at 30 ◦ C min−1 ) and then from 300 ◦ C to 360 ◦ C (at 5 ◦ C min−1 ) and hold at 360 ◦ C for 10 min. The carrier gas was helium (flow rate 1.5 ml min−1 ), auxiliary gases: hydrogen (flow rate: 25 ml min−1 ), air (flow rate: 300 ml min−1 ) [19]. Compounds were identified by comparison with commercial standards.
Lipase activity was evaluated in coffee seeds without parchment at 12 DAI, just before radicle protrusion. The activity was detected at increasing concentration of DGGMR, ranging from 0 to 100 M (Fig. 1). The initial rate values fitted the Michaelis–Menten equation, whose kinetic parameters were: Vmax = 0.23 ± 0.01 mol min−1 mg−1 protein and KM = 14.18 ± 2.79 M. This lipase was extremely sensitive to the specific lipase inhibitor THL, which reacts with a serine present in the active site [21], reaching approx. 67% inhibition at 300 M (Fig. 2). Furthermore, the activity was inhibited by the polyclonal anti-lipase Ab, in a concentration-dependent manner (Fig. 3). The inhibition followed the equation y = y0 + a × e(−b×x) and showed a maximum inhibition of approx. 20% in the presence of 0.6 ng Ab with respect to the control. 3.2. Parchment influence on germination, antioxidant capacity and lipase activity The developmental stages of coffee seeds during germination are exemplified in Fig. 4, panel A. Coffee seeds sown without parchment exhibited a visible radicle protrusion at 7 DAI, indicating the embryo growth inside of the endosperm. The radicle emission appeared at about 15 DAI and in the following days the radicle elongation was clearly evident. On the contrary, in coffee seeds with
Fig. 1. Characterization of lipase activity in coffee seeds at 12 DAI. The initial rate of lipase activity on DGGMR concentration fitted (r2 = 0.97) the equation V = Vmax [DGGMR]/(KM [DGGMR]). Data (n = 3) are means ± S.D.
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Residual lipase activity (%)
120 100 80 60 40 20 0 0
10
100
200
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THL ( M) Fig. 2. Inhibition by THL of lipase activity in coffee seeds at 12 DAI. The initial rate was expressed as percentage with respect to the control. Data (n = 3) are means ± S.D.
parchment the radicle emission was strongly delayed, occurring approximately at 28 DAI. During germination, lipase activity was measured in coffee seeds with or without parchment up to 28 DAI (Fig. 4, panel B). This activity was very similar in seeds with or without parchment at the first three sampling days (0, 4 and 8 DAI), and the values were not significantly different. From 10 DAI, the enzymatic activity showed a different pattern: coffee seeds with parchment were characterized by a slow continuous increase of lipase activity, with the highest value detected at 28 DAI. Conversely, coffee seeds without parchment showed a peak of lipase activity at 12 DAI, followed by a decrease. This pattern was supported by the immunoblotting assays performed using the anti-lipase Ab. In coffee seeds, either without (Fig. 5, panel A) or with (panel B) parchment, the anti-lipase Ab cross-reacted with a protein of approx. 60 kDa. The intensity of this cross-reaction, obtained from the densitometric analysis of the immunoblot membrane, appeared different during the germination stages in both seed lots (Fig. 5, panel C). In coffee seeds without parchment, the lipase increased during the first sampling days, exhibiting its maximum at 15 DAI, to decrease hereafter. A different pattern was observed in coffee seeds with parchment, where the lipase continued to increase after 15 DAI,
Fig. 4. Influence of parchment on lipase activity in germinating coffee seeds. Panel A: morphological development of germinating coffee seeds with (+) and without (−) parchment. Panel B: lipase activity in coffee seeds with () or without () parchment. Data (n = 3) are means of the initial rate of lipase activity ± S.D.
reaching its highest level at 28 DAI. The cross-reactivity of coffee seed lipase with the anti-lipase Ab was, therefore, correlated with the enzymatic activity shown in Fig. 4, panel B. Aiming at describing the degree of oxidative status of coffee seeds with or without parchment, the antioxidant capacity of coffee beans was measured during germination at 0, 7, 12, 21, 28 DAI (Fig. 6). The pattern exhibited a trend resembling that described for lipase activity. At 0 and 7 DAI, total antioxidant capacity was very similar for seeds with or without parchment. During the following germination steps, the lipophilic antioxidant activity in the seeds without parchment showed a peak at 12 DAI, while decreasing later. In the seeds with parchment the maximum was delayed, showing the highest total antioxidant capacity at 21 DAI. 3.3. Gas chromatographic analysis of lipid extracts Lipid profile throughout germination was characterized in coffee oil. An example of a typical chromatogram of the pattern composition (FFA, monoacylglycerols, MAGs, diacylglycerols, DAGs, and TAGs) is shown in Fig. 7 panel A. In coffee seeds with parchment, the accumulation of FFA increased steadily throughout the period of germination (Fig. 7, Panel B). On the contrary, coffee seeds without parchment showed a maximum level of FFA at 14 DAI that decreased afterwards until 28 DAI. The analysis of TAGs, DAGs and MAGs did not exhibit significant differences in coffee seeds, with or without parchment, and throughout germination (results not shown). 4. Discussion
Fig. 3. Inhibition by anti-lipase Ab on lipase activity in coffee seeds at 12 DAI. The initial rate was expressed as percentage with respect to the control (without antilipase Ab). Data (n = 3) are means ± S.D. Data fitted (r2 = 0.95) to an exponential decay function, two parameter equation y = y0 + a × e(−b×x) , where the parameters found were: y0 = 79.9 ± 1.2, the residual relative lipase activity, a = 20.8 ± 2.4, the fraction of relative lipase activity that is amenable to inhibition by the antibody; b = 61.8 ± 17.1, the rate of decline of relative lipase activity with increasing concentrations of the antibody.
Lipid bodies in oilseeds are mainly composed of TAGs and in coffee seeds they are generally located in the endosperm [11]. Uncontrolled TAGs metabolism during storage could lead to impairment of germination in propagation material and also to production of undesired compounds responsible for decrease in final cup quality [9]. The release of FFA could, indeed, trigger either degenerative
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Fig. 7. Analyses of FFA in coffee seeds at different DAI. Panel A: GC/FID chromatogram of FFA, MAGs, DAGs and TAGs in coffee beans; Panel B: FFA content in coffee seeds either with (black columns) or without (white columns) parchment.
-1
Antioxidant capacity (meq -tocopherol g )
Fig. 5. Immunoblot analyses of lipase from coffee seeds at different DAI. Western blots of proteins (approx. 20 g) obtained from coffee seeds without (Panel A) or with (Panel B) parchment. The values represent the apparent molecular mass of molecular standards. Panel C: densitometric analysis of immunoblot. Data (n = 3) are mean values ± SD, calculated from three independent experiments. Columns represent the spot density from coffee seeds either with (black) or without (white) parchment.
1.4 f
1.2 1.0
de
e
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cd abc bc
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ab
ab
a a
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12
16
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DAI Fig. 6. Lipophilic antioxidant capacity in coffee seeds at different DAI. Antioxidant capacity was measured in coffee seeds either with (black columns) or without (white columns) parchment. Data (n = 3) are means ± S.D.
processes, resulting in lipid oxidation/peroxidation, or fuelling high-energy demanding processes, e.g. respiration and sugar synthesis for embryo growth [22]. Even though lipase in coffee seeds represents a crucial enzyme during storage and germination, few studies have dealt with this aspect [14]. Therefore, this paper represents, to our knowledge, the first characterization of lipases from coffee seeds. The lipase activity in coffee seeds, evaluated with a colorimetric method, was characterized by kinetics parameters (KM and Vmax , Fig. 1), that were similar to those reported for lipases from Barbados nut (Jatropha curcas L.), whose seeds are classified as recalcitrants [23], but different to those found in orthodox seeds such as sunflower, French peanut (Panchira aquatica), as well as in seedlings of wheat, oat [24], rice [25] and rape [26]. The higher affinity for the substrates exhibited by coffee lipase suggests that in such seeds, containing a high amount of stored lipids, the lipolytic activities could play a regulative role during the first steps of germination. This lipase activity was further characterized in germinating coffee seeds by the inhibition by THL (Fig. 2), a selective and irreversible inhibitor of lipases, and by a polyclonal antibody raised against purified alkaline lipase from castor (Ricinus communis L.) beans (Fig. 3). This antibody also cross-reacted with a protein of approx. 60 kDa (Fig. 5), a value similar to that of the acid lipase RcOBL1, isolated and characterized in oil body membranes from castor bean [27]. The lipase activity was present in coffee seeds before imbibition and further induced by the germination process. During this phase, it followed a biphasic trend, which was similar in seeds either with or without parchment (Fig. 4, panel B), even though the phenomenon showed a delay in the former. In coffee seeds without
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parchment, a sharp second peak was observed after 12 DAI. On the contrary, the lipase activity gradually increased in coffee with parchment, in parallel with the delay in germination, while the other samples exhibited a precocious germination. A similar trend was also observed by the Western blot analysis of total coffee bean proteins, where the detection of lipases was possible since the very first DAI. Afterwards, it decreased at around 8 DAI, to rise again after about 12 up to 28 DAI in parchment coffee seeds (Fig. 5). This biphasic activity could possibly be attributable to ex novo synthesis and/or to different lipase isoenzymes, similarly to what was recently found in rice [25]. In this species, lipase-I and lipase-II have been reported to be involved in lipolytic activity. In particular, lipase-I is responsible for lipid metabolism for root and shoot growth, while lipase II is active during germination [25]. Therefore, it is possible to speculate that lipase activity in coffee would be exerted first by enzymes already present in the beans and, subsequently, during the second phase, by ex novo synthesized lipases induced by the germination process. The delay in activation of germination and root development could be explained by the presence of parchment, that slowed down metabolic activities by restricting gas exchanges. According to the observations reported above, the analysis of antioxidant capacity indicated that parchment was crucial for preventing the oxidation of the lipophilic fraction, being the seeds with parchment less prone to oxidation (Fig. 7). Indeed, the parchment could act as a barrier to oxygen fluxes towards the embryo/endosperm, thus limiting the production of ROS [28]. This environment would allow the germinating seeds to control ROS levels within the “oxidative window” for germination, as proposed by Bailly and coworkers [17]. Oxidative signals, mainly represented by ROS, are involved in seed germination, but their concentration must be adequate to allow the completion of this process [17,29,30]. Nevertheless, ROS excess is detrimental and a delicate balance is needed to prevent the activation of a high oxidative metabolism, which is a harmful feature, causing a rapid decline of embryo viability [31]. The release of FFA in coffe seeds, as expected, increased in both samples, but it reached a very high value earlier in coffee seeds without parchment, when compared to those with parchment (Fig. 7, panel B). This result would depend on the initial higher lipase activity observed until 12 DAI (Fig. 4, panel B). Further research will be useful to clarify if the early accumulation of high amount of FFA at 12 DAI would prevent seeds to metabolize all FFA by -oxidation. This would instead lead to lipid oxidation, thus explaining the depletion of antioxidant capacity in seeds without parchment (Fig. 7). On the other hand, this hypothesis is consistent with what observed in seeds still protected by parchment where a slower but steady release of FFA would lead to a lower consumption of the antioxidant capacity. In conclusion, our study suggests that coffee beans possess lipase activity that was already present during storage and increased during germination. The lack of quiescence and ABA insensitivity of intermediate coffee seeds require a more strict metabolic control of the degradation processes, to provide new organic skeletons for the development of the seedling. In particular, a fine tuning is necessary to avoid the uncontrolled release of ROS, especially in seeds exhibiting high lipid content in the endosperm. Therefore, we propose that in coffee seeds the regulation of the gas exchange exerted by the parchment would be considered similar to what demonstrated in holm oak seed, where the use of plastic films limiting the gaseous exchange prolonged the dormant-like state and consequently enhanced seed viability [31]. In particular, the decrease of oxygen permeation due to the parchment in coffee seeds could lead to positive effects during storage and germination, e.g. through the decrease of both degeneration products and oxidative processes. It is reasonable to suggest that parchment exerts a regulative role during germination, allowing the well-ordered
progress of all the different stages. Besides its restriction to gas exchange, we speculate that the polyphenol content of parchment, in particular chlorogenic acid, might inhibit lipase activity. Such inhibition would be useful to limit the amount of FFA initially released, allowing the physiological development of germination in coffee that, as intermediate seeds, is characterized by a partially activated metabolic machinery. Hence, our results supply a physiological explanation to field experience and to technological practices, which indicate that the presence of parchment leads to prolonged maintenance of seed viability and preservation of green coffee quality with respect to hulled beans [9]. To better understand the physiology of lipid metabolism in coffee seeds during germination, a crucial aspect to study will be the characterization of free fatty acid release in relation with the oxidative damage. Further research could also open new scenarios to clarify the modulation of lipase activities and the possible positive effects due to the control of oxygen permeability in green coffee during storage.
Acknowledgements We are grateful to Dr. Alessio Colussi (formerly Head of green coffee department illycaffè) for the critical reading of the manuscript. This work was supported by European Regional Development Funds, Cross-Border Cooperation Italy-Slovenia Programme 2007–2013 (TRANS2CARE and AGROTUR projects). We are also grateful to prof. Franco Tubaro (University of Udine) for the critical reading of the manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2013. 12.014.
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