Chlorogenic acid content swap during fruit maturation in Coffea pseudozanguebariae

Plant Science 165 (2003) 1355–1361

Chlorogenic acid content swap during fruit maturation in Coffea pseudozanguebariae Qualitative comparison with leaves Claire Bertrand, Michel Noirot∗ , Sylvie Doulbeau, Alexandre de Kochko, Serge Hamon, Claudine Campa Centre IRD of Montpellier, BP 64501, 34394 Montpellier Cedex 5, France Received 26 March 2003; received in revised form 22 July 2003; accepted 22 July 2003

Abstract Chlorogenic acids (CGA) are products of phenylpropanoid metabolism, i.e. one branch of the phenolic pathway. A wild species, Coffea pseudozanguebariae, native of East Africa, is a caffeine-free species with low CGA content (1.2% dmb in green beans). It is also used as a gene donor to improve C. canephora cup taste quality. In the current study, contents of the different CGA isomers were observed during the development in fruits and leaves. In both organs, CGA content decreased strongly during the growth and feruloylquinic acids (FQA) constituted most CGA. In fruits, a critical step was emphasised at the growth end, beyond which caffeoylquinic acid content (CQA) drastically increased. Previous results on beans suggest that the qualitative change concerned seeds and not pulp. The breeding implications and potential for further studies are discussed. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Coffea; Chlorogenic acids; Fruit maturation; Leaf development

1. Introduction CGA are products of phenylpropanoid metabolism, one branch of the phenolic pathway [1]. CGA sensu stricto (CGAs.s.) include only depsides of quinic acid with caffeic acid, i.e. caffeoylquinic acids (CQA) and dicaffeoylquinic acids (diCQA), but also, to a lesser extent, other hydroxycinnamoyl conjugates, such as ferulic or p-coumaric acid derivatives. In green coffee beans, 98% of CGA belong to three classes, i.e. CQA, diCQA and FQA (feruloylquinic acids) [2,3]. Each class includes three isomers according to the acylating residue positions [4] (Fig. 1). CGA are common in many plants including Coffea species. Two coffee tree species are of worldwide importance: Coffea arabica and C. canephora (commonly known as Robusta), but C. arabica coffee—with its lower bitterness and better flavour—is preferred by consumers. Aroma and taste are produced during roasting as a result of Maillard and Stecker’s reactions and thermal degradation of precursors ∗

Corresponding author. E-mail address: [email protected] (M. Noirot).

present in green beans [5]. This is how the chlorogenic acids (CGA) increase bitterness after their degradation into phenol derivatives [6]. The difference in CGA contents in C. arabica and C. canephora—4.1% dry matter basis (dmb) versus 11.3% dmb, respectively [7]—is a major element explaining flavour differences between these two coffees [4,8,9]. A wild species, C. pseudozanguebariae, native of East Africa, has no caffeine and low CGA content (1.2% dmb) in green beans [10,11]. It can be used as a gene donor to improve C. canephora cup taste quality [11,12]. Evaluation and comparison of CGA contents in leaves and fruits during their growth are the first steps before analysing the expression of the genes controlling CGA biosynthesis, accumulation or degradation. From an organogenesis standpoint, leaves and fruits show some similarities, at least at youngest stages. Their differentiation over time, may be accompanied by changes both in total CGA content and isomers proportions, due to tissue specific gene expression. To date, time-course variations of CGA contents have not been surveyed in leaves and fruits of Coffea species. To determine critical steps for CGA content variation in fruits have to be emphasised to understand modification of gene expression

0168-9452/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2003.07.002

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R1

R2 feruloyl moiety

caffeoyl moiety

R1

5-CQA

R1

4,5-diCQA R1

R2

5-FQA

Fig. 1. Structure of CGA. Three main classes of CGA exists in coffee beans: CQA, diCQA and FQA depending on the type and the number of moieties. Within each class, three isomers are observed depending on the position of moieties on the quinic acid. Examples concern 5-CQA, 4,5-diCQA and 5-FQA.

over time. It is also important to observe qualitative changes between organs. In the current study, CGA isomer contents were evaluated in C. pseudozanguebariae fruits and leaves during their development. Within each organ, a quantitative time-course analysis was carried out. A qualitative comparison (presence/absence of some isomers; trend change) was also made between fruits and leaves. Hypotheses on CGA biosynthesis pathways are put forward on the basis of relationships between isomers.

2. Material and methods 2.1. Plant material C. pseudozanguebariae fruits were harvested at the CNRA Station of Divo (Cˆote-d’Ivoire) on field-grown trees. Fructification time is defined as the time from flowering to bean maturity. In C. pseudozanguebariae, fructification time is 9–10 weeks. Fruits were harvested at four maturity levels, corresponding to 40, 60, 80 and 100% of the fructification time (stages 1, 2, 3 and 4, respectively). C. pseudozanguebariae leaves were sampled on trees maintained in a greenhouse under a controlled environment (30 ◦ C, 90% RH) at the IRD Centre, Montpellier (France). Three leaf maturation stages were defined. At stage 1, leaves

were cut off at the tip of branches and were <40 mm long. At stage 2, leaves were at their maximal size and located on non-ligneous branches (green internode). At stage 3, leaves were located on ligneous branches (brown internode). All samples were frozen in liquid nitrogen immediately after collecting and then stored at −80 ◦ C until CGA extraction. 2.2. CGA extraction, purification and determination CGA extraction and purification were carried out using a fast and accurate method [13]. Each sample was crushed in a grinder (Ika Labortechnik, Germany) and about 50 mg of powder was extracted in a 50 ml Falcon tube with 50 ml of 0.5% sodium bisulfite in a methanol–water (70/30; v/v) solution. The Falcon tubes were shaken overnight at 4 ◦ C in darkness on a stirring plate at 125 rpm. Three extractions were obtained per stage in leaves and four in fruits. The organic extracts were directly treated with Carrez reagents to precipitate colloidal material [11]. Solutions were then filtered through a 0.2 ␮m filter and directly analysed by high pressure liquid chromatography (HPLC). All extracts were separately processed for HPLC analysis. HPLC was performed with a Waters 510 apparatus using a C18 pre-column and a LiChrospher® 100 RP-18 column (5 ␮m, 250 mm × 4 mm, Merck). The mobile phase consisted of phosphoric acid (2 mM, pH 2.7) in MeOH and

C. Bertrand et al. / Plant Science 165 (2003) 1355–1361

was delivered over a gradient at a flow rate of 0.8 ml/min, according to Ky et al. [13]. Samples (50 ␮l) were analysed at room temperature at 325 nm with a photodiode-array detector (Waters). The extract processing order was fully randomised for HPLC analysis. Every 10 extracts, a 5-CQA standard (ref. D11.080-9, Aldrich-Chimie) was used at 50 mg/l to verify the stability of the HPLC measurements. Every 40 extracts, the HPLC column was washed overnight with a solution of pure MeOH at a flow rate of 0.2 ml/min. CGA were identified and characterised on the basis of their chromatograms (at 280 and 325 nm), retention times and UV spectra [15] and compared to Rakotomalala’s results [14]. Quantification was achieved by peak-area measurement and comparison with the standard 5-CQA. A calibration curve was plotted for each injected volume (10 and 50 ␮l) using three replicate points at 1, 5, 10, 25, 50, 100 and 150 mg/l. CGA isomer content was expressed in percentage of dry matter basis (% dmb). Leaf dry matter was evaluated in triplicate by desiccation of about 100 mg of powder in an oven (15 h, 100 ◦ C). Only one sample per stage was evaluated in fruits. 2.3. Variables CGA are presented according to the IUPAC [15] numbering system. Abbreviations proposed by Clifford [4,16] were adopted. The primary variables are: 3-CQA: 3-caffeoylquinic acid; 4-CQA: 4-caffeoylquinic acid; 5-CQA: 5-caffeoylquinic acid; 3,4-diCQA: 3,4dicaffeoylquinic acid; 3,5-diCQA: 3,5-dicaffeoylquinic acid; 4,5-diCQA: 4,5-dicaffeoylquinic acid; 3-FQA: 3-feruloylquinic acid; 4-FQA: 4-feruloylquinic acid; 5-FQA: 5-feruloylquinic acid. All of these variables were expressed in percentage of dry matter. Secondary variables were defined on the basis of primary variables: • • • • •

CQA = 3-CQA + 4-CQA + 5-CQA; DiCQA = 3,4-diCQA + 3,5-diCQA + 4,5-diCQA; FQA = 3-FQA + 4-FQA + 5-FQA; CGAs.s. = CQA +DiCQA; and CGA = CGAs.s. + FQA.

2.4. Statistical analysis All results were analysed using the Statistica software package. A one-way ANOVA with fixed effect was used to compare maturity levels in leaves and fruits. When the F test was significant, a Newman and Keuls-test was carried out to compare means. Standard deviations were computed from the residual mean square, the latter being the best estimate (unbiased and accurate) of the residual variance. Linear regression was also performed to highlight relationships between some CGA isomers.

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A clustering analysis with Ward’s criteria and Euclidian distance as parameters was applied to CGA data in order to pinpoint clusters and similarities between stages and organs.

3. Results The first results concerned time-course modifications in the percentage of dry matter in leaves and fruits. In leaves, dry matter increased from 19 to 34% throughout development (Table 1). In fruits, dry matter increased from 16 to 29% between stages 1 and 4 (Table 2). 3.1. Changes in CGA content During fruit ripening, the highest CGA content was observed in stage 1 (11.6% dmb) (Table 1). CGA content decreased linearly up to stage 3 and no difference was recorded between stages 3 and 4. FQA was the main CGA throughout ripening, representing 94% of CGA in young fruit and 63% at stage 4. FQA content was 10.9% dmb at stage 1 and decreased linearly up to 2.1% dmb at stage 3, to reach 1.8% dmb at stage 4. In CGAs.s., the proportion of CQA. ranged from 76% at stage 1 to 100% at stages 3 and 4. In leaves, as in fruit, CGA content was the highest (13.5% dmb) at stage 1 and FQA was still the main class of CGA irrespective of the stage (Table 2). As in fruit, 1: contents drastically decreased from stages 1 to 2 for all CGA classes, particularly diCQA and FQA; 2: a relative enrichment in CGAs.s. was observed from stages 2 to 3; and 3: the proportion of CQA in CGAs.s. increased. The three isomers were not always detected in each CGA class. One isomer in CQA and FQA classes (3-CQA and 3-FQA) was never observed in leaves or fruits at any developmental stage. As 4-CQA was poorly accumulated, 5-CQA appeared to be the only CQA-class isomer stored and its level represented the entire CQA content. Concerning the FQA class, 5-FQA was the main isomer detected in leaves and fruits, while 4-FQA was encountered at all stages, except at stage 3 in fruits. There was a close linear relationship between 5-FQA and 4-FQA in fruits from stages 1 to 3 (Fig. 2). Stage 4 was markedly out of line with the regression trend, clearly indicating that a qualitative change occurred between the two last stages. This relationship was also recorded in leaves from stages 1 to 3. Concerning diCQA isomers, marked changes were observed during leaf development and fruit maturation. Only the isomer 3,5-diCQA was always detected in leaves, while it disappeared at stage 2 in fruits. The presence of 3,4-diCQA was only recorded in leaves and fruits at stage 1. The 4,5-diCQA isomer was not detected in leaves at stage 3 or in fruits. A relationship between 3,5-diCQA and 4,5-diCQA was noted in leaves but not in fruits. 4,5-diCQA accumulation was observed when 3,5-diCQA content was higher than 0.035% (Fig. 3).

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Table 1 CGA contents in fruit of C. pseudozanguebariae Trait

Stage 1

Stage 2

Stage 3

Stage 4

F test

P

S.D.

Seeda

CQA DiCQA CGAs.s. FQA CGA %CQA %CGAs.s. %Dry matter

0.51a 0.16b 0.68b 10.9d 11.6c 75.7a 5.9a 16.4

0.61b 0.02a 0.62b 5.89c 6.51b 97.5c 9.6b 21.7

0.48a 0.00a 0.48a 2.04b 2.51a 100c 19.4c 25.8

1.08c 0.00a 1.08c 1.83a 2.91a 100c 37.2d 29.4

162 82 126 201 179 76 568 –

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 –

0.043 0.018 0.045 0.60 0.63 2.70 1.17 –

1.40 0.04 1.44 0.08 1.51 97.2 95.4 –

Absolute contents (CQA, diCQA, CGAs.s., FQA and CGA) are expressed as a percentage of dry matter basis (dmb). Relative contents (%CQA and %CGAs.s.) are also expressed in percentage, but representing CQA/CGAs.s. and CGAs.s./CGA, respectively. The sixth and seventh columns give ANOVA results (degrees of freedom are d.f. 1 = 3 and d.f. 2 = 12). Newman and Keuls test results are indicated with on-line letters. The eighth column gives the standard deviation estimated from the ANOVA residual mean square. a Seed results were published in Ky et al. [11].

LEAVES: y=0.0207(x-0.983); r=0.998 FRUITS: y=0.0133(x-2.101); r=0.993

4-FQA content (%dmb)

0.24

0.18

Stage 4 Stage 1

0.12

Stage 2

0.06

Stage 3

0.00 0

2

4

6

8

10

12

14

5-FQA content (%dmb) Fig. 2. Linear regression between 5-FQA and 4-FQA contents in leaves and fruits. Stage 4 in fruit is markedly out of line with the regression trend, clearly indicating that a qualitative change occurred between stage 3 and 4 in fruits.

3.2. General comparison Table 2 CGA contents in leaves of C. pseudozanguebariae Trait

Stage 1

Stage 2

Stage 3

F test

P

S.D.

CQA diCQA CGAs.s. FQA CGA %CQA %CGAs.s. %Dry matter

0.78b 0.21c 0.99c 12.5c 13.5c 78.7a 7.3a 18.6a

0.58a 0.06b 0.64a 3.75b 4.40b 90.6b 14.7b 34.0b

0.62a 0.04a 0.66a 2.08a 2.74c 94.9c 24.1c 33.7b

11.4 269 50 2197 2029 73 79 43

0.009 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

0.053 0.010 0.047 0.21 0.22 1.65 1.33 2.32

Absolute contents (CQA, diCQA, CGAs.s., FQA and CGA) are expressed as a percentage of dry matter basis (dmb). Relative contents (%CQA and %CGAs.s.) are also expressed in %, but representing CQA/CGAs.s. and CGAs.s./CGA, respectively. The fifth and sixth columns give ANOVA results (degrees of freedom are d.f. 1 = 2 and d.f. 2 = 6). Newman and Keuls test results are indicated with on-line letters. The seventh column gives the standard deviation estimated from the ANOVA residual mean square.

The results of a clustering analysis using CGA, FQA and diCQA as variables are given in Fig. 4. At the highest level, two clusters G1 and G2 were observed: G1 included leaves and fruits at stage 1, whereas G2 pooled other stages. Young fruits were more similar to young leaves in terms of CGA content as compared to other organs and stages. At an intermediate clustering level, G2 can be split into G2-1 and G2-2. G2-1 corresponded to maturity stage 2, again highlighting the similarity between leaves and fruit for CGA content during the first stages of development, while G2-2 included stages 3 and 4. At the lowest clustering level, leaves and fruits can be separated within stages 1 and 2, giving G1-1 (leaf stage 1), G1-2 (fruit stage 1), G2-1-1 (leaf stage 2), and G2-1-2 (fruit stage 2) clusters. Mature fruits (stage 4) can also be clearly defined (G2-2-2). In contrast, at maturity stage 3, fruits cannot be easily distinguished from leaves (G2-2-1).

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0.040

4,5-diCQA content (%dmb)

0.035 0.030 2

r = 0.953

0.025 0.020 0.015 0.010 0.005 0.000 0.02

0.03

0.04 0.05

0.06

0.07

0.08

0.09

0.10

0.11

3,5-diCQA content (% dmb) Fig. 3. Linear relationship between 3,5-diCQA and 4,5-diCQA contents in leaves: Y = 0.611X + 0.022.

4. Discussion Our results revealed three main points that will be discussed, 1: similarities in CGA content patterns in growing leaves and ripening fruits, 2: relationships between isomers, and 3: qualitative biosynthesis changes occurring in seeds between stages 3 and 4. In addition, the results are discussed in terms of breeding. 4.1. CGA content similarities in growing leaves and ripening fruits As fruit and leaf development took place under different conditions (natural conditions in Cˆote-d’Ivoire versus greenLeaf Sta ge 1 Leaf Sta ge 1 Fruit Sta ge 1 Leaf Sta ge 1 Fruit Sta ge 1 Fruit Sta ge 1 Fruit Sta ge 1 Leaf Sta ge 2 Leaf Sta ge 2 Leaf Sta ge 2 Fruit Sta ge 2 Fruit Sta ge 2 Fruit Sta ge 2 Fruit Sta ge 2 Leaf Sta ge 3 Leaf Sta ge 3 Leaf Sta ge 3 Fruit Sta ge 3 Fruit Sta ge 3 Fruit Sta ge 3 Fruit Sta ge 3 Fruit Sta ge 4 Fruit Sta ge 4 Fruit Sta ge 4 Fruit Sta ge 4

house at Montpellier, respectively), we did not statistically compare absolute CGA contents in leaves and fruits. Indeed, a comparison of CGA contents in leaves (13.5%) and fruit (11.7%) at stage 1 would obviously have no biological relevance. In both organs CGA content was maximal at the youngest stages. This resemblance between the two organs may be explained by the sporophytic origin of most tissues forming the fruit at the beginning of its growth: during the first half of the fructification period, the perisperm dominates the interlocular space [17]. By contrast, in endosperm, i.e. in tissue with non-sporophytic origin, CGA accumulation is observed during seed growth in C. canephora, C. liberica and C. arabica [18].

G1-1

G1 G1-2 G2-1-1

G2-1

G2-1-2

G2

G2-2-1

G2-1

G2-2-2

Fig. 4. Clustering tree showing similarities between CGA profiles observed in C. pseudozanguebariae fruits and leaves at different stages of maturity. There were four replicates per stage-organ combination.

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This could explain the distinctive feature of stage 4 in fruits. A second similarity concerns the substantial decrease observed during development in both organs. Two hypotheses, at least, could explain such a decrease, 1: as measured in growing C. arabica leaves [19], the decrease in CGA may be concomitant to an increase in the amount of cell wall-bound polymers (consumption process). Indeed, CGA are also considered to be storage forms of cinnamic acid derivatives that can be used for lignification [20,21]; and 2: the growing period was also characterised by a marked increase in dry matter in both organs. Consequently, the decrease in CGA content could result from the dry matter increase, with the CGA content remaining constant (dilution process). The dilution and consumption processes could occur simultaneously. Finally, the proportion of FQA in CGA is preponderant from stage 1 to 3. At stage 1, FQA represented over 92% of CGA content and the proportion decreased slightly during development. Inversely, the share of CGAs.s. in CGA increased and this cannot be explained by the dry matter increase. It indicates that relationships between biochemical pathways leading to CGAs.s. and FQA are modified during development, associated with the different roles of these compounds in different tissues. The situation is both similar and contrasted in tomato. The FQA/CQA ratio changes during fruit development in coffee, whereas in tomato it is minimal in young fruits and maximal in mature fruits [22]. The close similarities between leaves and fruits, despite their different environmental growing conditions, is unanticipated. We could have expected that the different external conditions would have increased developmental differences between organs rather than favouring similarities. This strongly suggests that the observed similarities between organs (stage 1 > stage 2 > stage 3) were qualitatively independent of external variations. Some minor differences were noted between leaves and fruits concerning diCQA isomers. The 4,5-diCQA was not detected in fruits as early as stage 1 and some isomer concentrations were at the detection limit for the method used. We cannot overlook the possibility that an environmental origin might account for the difference. This could be the focus of a future investigation. 4.2. Relationships between isomers There was a linear relationship between 4-FQA and 5-FQA, which has been noted in mature seeds of Coffea liberica Dewevrei, even though the 5-FQA content range was narrower (0.18–0.7%) [11]. There was a relationship between 4- and 5-FQA in both organs, at least up to stage 3. The straight line suggests an equilibrium between 5-FQA and 4-FQA contents at each stage of organ development. In fruits as in leaves, the 4-FQA isomer appeared only when the 5-FQA content was over a

threshold. This suggest that 4-FQA is very likely produced by an enzymatic reaction. Our experimental conditions did not enable us to compare regression slopes. A linear relationship was noted in leaves when comparing 3,5-diCQA and 4,5-diCQA levels. In this relationship, there was a threshold under which 4,5-diCQA would disappear. This underlines that 4,5-diCQA would be formed from 3,5-diCQA, as already suggested [11,23] by trans-esterification. The absence of 4,5-diCQA in fruits after stage 1 again reflects a difference between organs in terms of gene expression and/or enzyme activity. However, this could result from the different environmental conditions of development for leaves and fruits (greenhouse versus tropical field). 4.3. CGA biosynthesis changes in fruits just before maturity The main difference between stage 3 and 4 in fruits concerned the increased proportion of CGAs.s. among total CGA content (Table 2). This resulted from a slow decline in FQA content and a substantial CQA content increase. When seed CGA composition was taken into account along with Ky’s data [11], the greatest difference between fruits and seeds concerned FQA, which is residual in seeds. This means that FQA in pulp would be clearly higher than 2%, whereas CGA metabolism in seeds would be completely oriented towards CQA accumulation. The second difference between stages 3 and 4 involved the sharp increase in the proportion of 4-FQA in FQA. These results indicate the presence of a critical stage when fruits become mature, with some expression changes in seeds. Such differentiation should also be discussed in terms of fitness. What would be the advantage of a CGA modification in seeds for the species? This could be related to the mode of seed dispersal. Indeed, Coffea species have different fruit colours. Preferential dispersal of dark fruits and red fruits could be due to nocturnal animals like bats and diurnal animals like monkeys, respectively. The selective advantage appears when the pulp is eaten, whereas seed is rejected [24]. In wild C. canephora populations present in Kibale national park, the pulp of ripe fruits is eaten by black and white colobus and redtail monkeys, whereas most seeds are still undamaged on the ground [25]. Seeds could be rejected by bats and monkeys on the basis of different biochemical criteria. Such effects on animal behaviour could explain between-species differences in the biochemical composition of seeds, but also within-species differences between pulp and seeds. 4.4. Consequences on breeding C. pseudozanguebariae can be used as a gene donor to improve C. canephora cup quality. Its main favourable traits are the absence of caffeine, a low CGA content, high trigonelline and sucrose contents [7,11,12,26]. For CGA, evidence of

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a critical stage differentiating pulp and seed in C. pseudozanguebariae highlights new potential focuses of investigation at biochemical and molecular levels. In particular, the expression of enzymes implicated in the CGA biosynthesis pathway, e.g. phenylalanine ammonialyase (PAL), caffeoyl CoA O-methyltransferase (CCoAOMT), hydroxycinnamoyl CoA: quinate hydroxycinnamoyl transferases (CQT), CoA ligase (4CL) or cytochrome P450 enzymes, such as C4 H or C3 H, should now be investigated. Studies will be undertaken to obtain specific cDNA libraries from stages 3 to 4 in order to develop EST and co-localize candidate-genes with QTLs. Such markers will then be used to assist selection in successive backcross generations. References [1] J.B. Harborne, Plant Phenolics, in: Bell, Charlwood (Eds.), Encyclopedia of Plant Physiology, New series, vol. 8, Springer, Berlin, 1980, pp. 329–402. [2] M.N. Clifford, P.S. Staniforth, A critical comparison of six spectrophotometric methods for measuring chlorogenic acids in green coffee bean, Proc. Int. Congr. ASIC 8 (1977) 109–113. [3] H. Morishita, H. Iwahashi, R. Kido, Chlorogenic acids composition of green and roasted coffee beans, Bull. Fac. Edu. Wakayama Univ. Nat. Sci. 38 (1989) 33–39. [4] M.N. Clifford, Chlorogenic Acids, in: Clarke, Macrae (Eds.), Coffee I Chemistry, Elsevier, London, 1985, pp. 153–202. [5] C.A.B. De Maria, L.C. Trugo, R.F.A. Moreira, C.C. Werneck, Composition of green coffee fractions and their contribution to the volatile profile formed during roasting, Food Chem. 50 (1994) 141– 145. [6] V. Leloup, A. Louvrier, R. Liardon, Degradation mechanisms of chlorogenic acids during roasting, Proc. Int. Congr. ASIC 16 (1995) 192–198. [7] C.L. Ky, J. Louarn, S. Dussert, B. Guyot, S. Hamon, M. Noirot, Caffeine, trigonelline, chlorogenic acids and sucrose diversity in wild Coffea arabica L. and C. canephora P. accessions, Food Chem. 75 (2001) 223–230. [8] B. Guyot, E. Petnga, J.C. Vincent, Analyse qualitative d’un café Coffea canephora var. Robusta en fonction de la maturité. Part I. Evolution des caractéristiques physiques, chimiques et organoleptiques, Café Cacao Thé 32 (1988) 229–242. [9] B. Guyot, D. Gueule, J.C. Manez, J.J. Perriot, J. Giron, L. Villain, Influence de l’altitude et de l’ombrage sur la qualité des cafés arabicas, Plant. Rech. Dével. 3 (1996) 272–280. [10] F. Anthony, M.N. Clifford, M. Noirot, Biochemical diversity in the genus Coffea L.: chlorogenic acids, caffeine and mozambioside contents, Genet. Res. Crop Evol. 40 (1993) 61–70.

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