INFLUENCE OF VARIOUS BIOTIC AND ABIOTIC FACTORS …shodhganga.inflibnet.ac.in/bitstream/10603/69216/7... · · 2016-01-08FACTORS ON THE SECONDARY METABOLITES PRODUCTION IN COFFEE

Chapter 4

INFLUENCE OF VARIOUS INFLUENCE OF VARIOUS INFLUENCE OF VARIOUS INFLUENCE OF VARIOUS

BIOTIC AND ABIOTIC AND ABIOTIC AND ABIOTIC AND ABIOTIC BIOTIC BIOTIC BIOTIC

FACTORS ON THE FACTORS ON THE FACTORS ON THE FACTORS ON THE

SECONDARY SECONDARY SECONDARY SECONDARY

METABOLITES METABOLITES METABOLITES METABOLITES

PRODUCTION IN COFFEE PRODUCTION IN COFFEE PRODUCTION IN COFFEE PRODUCTION IN COFFEE

BEANSBEANSBEANSBEANS

Influence of biotic and abiotic factors on secondary metabolites of coffee beans

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4.1. Summary

Plant growth and development is affected by various abiotic factors that include

topography, soil, and climatic factors. Similarly biotic factors, determine the extent in

which the genetic factor is expressed in the plant. Abiotic elements methyl jasmonate

(MJ) and salicylic acid (SA) followed by biotic stress factors such as mycelial extracts of

Rhizopus oligosporus and Aspergillus niger have shown significant augmentation of

major secondary metabolites of green beans of robsuta coffee plants that were subjected

to floral spray. Up to 42 % caffeine, 39 % theobromine, 46 % trigonelline along with 32

% cafestol and kahweol improvement were evident under respective elicitor treatments.

Over all, surge in respective metabolites content depends on elicitor stress type and

concentration. The elicitors MJ and SA are found to be efficient at 1 to 5 µM

concentration in augmenting all the metabolites compared to R. oligopsorus and A. niger

mycelial extracts spray at 0.5 -2.0 % (w/v). To check the influence of altitude

environment on Coffee beans metabolite profile, fruits were collected from C. canephora

plants grown at different elevations to find out the influence of altitude on various

metabolites of coffee beans. Caffeine content was found always more at all stages of

coffee fruit ontogeny compared to the plants grown at low elevations. Maximum caffeine

content of 1.868 ± 0.149 g. 100g-1

dry weight was evident in stage V seeds (9 months

DAF) of low altitude collected samples. There was a 32% reduction in caffeine content

of beans from plants grown at high elevations. Both nicotinic acid and trigonelline

contents were always higher at all stages of coffee fruit ontogeny from plants grown at

higher elevations. Maximum nicotinic acid and trigonelline content of 5.164 ± 0.131

mg.100g-1

and 975.8 ± 7.24 mg.100g-1

respectively was evident in stage V seeds (9


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months DAF) of high altitude collected samples.The levels of free diterpenes cafestol

and kahweol were found to be more in beans collected from low elevations (0.12% of

cafestol and 0.045% kahweol) than that of high elevation grown plants (0.042% of

cafestol and 0.014% of kahweol). Apart from these, some major cafestol and kahweol

esters viz., cafestol linoleate, cafestol oleate, cafestol palmitate, cafestol stearate,

kahweol linoleate, kahweol oleate and kahweol palmitate were identified. This, stress

mediated augmentation of secondary metabolites of coffee would have a wider scope for

studying the expression pattern of major biosynthetic pathway genes of caffeine,

trigonelline, and diterpenes in coffee, under selected elicitor treatment, which would be

helpful to develop a metabolomics and proteomics approach for improving the quality of

the beans.

4.2. Introduction

Secondary metabolites production in plants is influenced by various extrinsic and

intrinsic factors (Ramakrishna and Ravishankar, 2011). Extrinsic factors such as

environmental and biotic factors role on plant growth and important metabolites

production was investigated under both in vitro and ex vitro conditions. The biotic factors

such as fungi, bacteria and algae are reported to be good in eliciting production of wide

range of chemical compounds such as alkaloids, flavonoids, phenyl propanoid

intermediates (Prasad et al., 2006), as shown by using cell suspension cultures or root

cultures (reviewed by Ramakrishna and Ravishankar, 2011).Recent studies have

demonstrated the efficiency of this elicitor mediated approach to augment food value

metabolites such as annatto pigment in Bixa orellana (Mahendranath et al., 2011;


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Giridhar and Parimalan, 2010), phenyl propanoid intermediates and capsaicin in Chilli

(Gururaj et al., 2012).

Coffee is rich in bioactive secondary metabolites such as caffeine, trigonelline,

nicotinic acid, and diterpenes cafestol and kahweol. Caffeine (1,3,7 trimethyl xanthine) is

one of the major secondary metabolite in coffee and some other plants are reported to

combat physical and biotic stress factors such as pathogens and predators (Nathanson,

1984; Kim et al., 2011). Coffee also contains trigonelline which is considered as the

second abundant alkaloid compound and it is thermally converted to nicotinic acid and

some flavour compounds during roasting (Taguchi, 1988). In addition it is considered

important for taste and nutrition (Adrian and Fragne, 1991). Other important components

that are associated with lipid fraction of coffee are diterpenes- cafestol (caf) and kahweol

(kah) which are unique to coffee, and they are mainly pentacyclic diterpene alcohols

based on kaurene skeleton (Urgert et al., 1995) and only a small portion is present in the

free-form, and the remain is in esterified form.

High levels of caffeine produced by coffee seedlings exhibit allelopathy there by

inhibit the germination of other seeds in the vicinity of the growing plants (Peneva,

2007). Elicitation is an induction process which results in increased activities of enzymes

of caffeine biosynthesis. The production of the purine alkaloid caffeine was shown to be

stimulated by stressors such as high light intensity and high NaCl concentration (Peters et

al., 2004). Moreover, exogenous calcium influences caffeine alkaloids production in C.

arabica and C. canephora (Ramakrishna et al., 2011). Though sporadic reports are

available on biotic elicitors influence on in vitro cultures of Coffea for caffeine

production, no such reports are available on Coffea grown ex vitro and also during the


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ontogeny of fruits. Such studies pertaining to trigonelline, cafestol and kahweol

production in green beans of C. canephora are lacking. From coffee quality point of

view metabolite profiles of coffee beans is having paramount importance in global coffee

market, hence understanding the influence of various extraneous factors on bean

metabolite profiles of Coffea standing crop in field is warranted as it facilitate to

understand the biochemical profiling and their biosynthetic pathways. Moreover, various

microbes incidence on coffee berries is reported (Velmourougane et al., 2011; Velazquez

et al., 2011) which may influence the quality of bean.

The altitude at which coffee is grown plays a major role in determining the

quality of the bean, because there is less oxygen, so coffees grown at higher altitudes

take longer to mature than plants grown at lower altitudes. This allows the flavours to

develop more fully and produces beans that are delicate and flavourful (Clifford, 1985).

Arabica beans grow at altitudes between 600 and 1,800 metres above sea level and take

six to nine months to mature. Robusta plants grow at low altitudes (sea level to 600

meters), more cold and moisture-tolerant and disease-resistant. Variation in the content

of trigonelline, nicotinic acid and diterpenes in coffee beans is mainly based on

geographical distribution of Coffea species (Lercker et al., 1995) and the altitude at

which coffee is grown may play a major role in determining metabolites profiles (Decazy

et al., 2003) and also quality of the bean (Muschler, 2001). As there are no reports

available about the influence of elevation (altitudinal) variations on Coffee bean

metabolite profiles from plants grown in India such studies are warranted.

A study has been proposed to investigate the influence of abiotic elicitors

(salicylic acid and methyl jasmonate) and biotic elicitors (Aspergillus niger and Rhizopus


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oligosporus) on caffeine alkaloids, trigonelline and antinutritioal diterpenes cafestol and

kahweol production in green beans of C. canephora under elicitor stress. Apart from this,

analysis of major metabolites profile during the ontogeny of C. canephora fruit

development in samples collected from plants grown at different altitudes was carried

out.

4.3. Material and methods

4.3.1. Preparation of elicitors

Biotic elicitors were prepared using two fungal cultures viz., Aspergillus niger

and Rhizopus oligosporus that were procured from microbial culture facility of Food

Microbiology Department of CFTRI, Mysore. Fresh cultures of A. niger and R.

oligosporus were made on PDA slants and incubated for 7 days. Then the spores of the

respective fungi were used to prepare spore suspension in 0.1 % sodium lauryl sulphate

(w/v) and diluted with sterile distilled water under aseptic conditions to obtain a spore

density of ~ 2.5 X 106 spore mL

-1. Later the same was inoculated into the 40 ml of PDA

broth contained in 150 ml Erlenmeyer conical flasks and the cultures were incubated in

dark for 10days. After culture growth, the cultures were autoclaved and the mycelium

was separated from the culture broth by filtration and its fresh weight was recorded.

Aqueous extract of mycelium mat was made by homogenizing in mortar and pestle using

neutralized sand. The extract was filtered through Whatman no. 1 filter paper. The filtrate

was made up to the known concentration and kept as the stock solution from which the

individual fungal mycelia extracts at a working concentration of 0.5, 1.0 and 2% % w/v

(wet weight of fungal mycelium in 100 ml of distilled water) were prepared in sterile

water and used for elicitation experiment.


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Figure 4.1. A pictorial depiction of work component under objective 2 of the study


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Abiotic elicitors viz. salicylic acid (SA) and methyl jasmonate (MJ) purchased

from (Sigma, Bangalore) were dissolved in distilled H2O and diluted to obtain three

concentrations (1.0, 2.5 and 5.0 µM) for the study.

4.3.2. Elicitor application

Five years old plants of Coffea caenphora CxR were used for small-scale field

study to find out influence of elicitors on annatto pigment on standing crop through

elicitor mediated approach. Different plants were selected for spraying various elicitors

in our study. All these plants were under cultivation in an area of 50 x40 ft at Plant Cell

Biotechnology Department, of this institute, with a spacing of 4 x 4 ft between each

plant. The prepared biotic (A. niger, R. oligosporus) and abiotic elicitors methyl

jasmonates (MJ) and salicylic acid (SA) were sprayed to the flowers during anthesis

(between 9-11 Hrs). The ripened red fruit from plants subjected to respective treatments

were harvested at maturity (after 9 months), and used for extraction of respective

metabolites and their analysis.

4.3.3. Extraction and analysis of caffeine and theobromine

Fruit pulp of harvested fruits was removed and beans were taken out and dried to

attain ~12 % of moisture content. To determine caffeine, 5g of fresh tissue (of green

beans) was extracted using 80% ethanol using mortar and pestle, the resultant slurry was

homogenized using neutralized sand, followed by centrifugation for 10min at 8000 rpm

and supernatant was collected after centrifugation. The extract was flash evaporated to

dryness and dissolved to 1ml of 80% ethanol prior to estimation of caffeine and

theobromine by HPLC (Ashoor et al., 1983) as explained in chapter 3 material and

methods 3.3.3. The compounds were identified by their retention times, chromatographic


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comparisons with authentic caffeine and theobromine standard (Sigma, Bangalore), and

their UV spectra. Quantification was based on the external standard method.

4.3.4. Extraction and analysis of trigonelline and nicotinic acid

Extraction and HPLC analysis of trigonelline and nicotinic acid were carried out

as explained earlier in chapter 3 material and methods section.

4.3.5. Extraction and analysis of diterpenes cafestol and kahweol.

Known quantity ground coffee sample was weighed (5 g of beans/500 mg of

pericarp) separately for extraction of each sample for free forms of diterpenes (cafestol

and kahweol). The extraction and analysis of free forms of cafestol and kahweol was

performed as explained under materials and methods section of Chapter 3.

4.3.6. Influence of altitude on metabolite profile of coffee beans

Fruits of C. canephora P. ex Fr. CxR variety at different stages of development

were collected from plantations grown at different elevations near Mudigere of

Chikamagalur District of Karnataka during February 2009. Ripened fruits from plants

grown at high (3700 ft MSL), medium (3300 ft MSL) and low altitudes (3000 ft MSL)

were collected from different places viz., Devaramane, Guthi (Javali) and Tripura

respectively (Lat 13° 7' 60N, Long 75° 37' 60E). Ten random plants were selected at each

of the said altitude, and at least 250 g of fresh fruits were collected for caffeine

trigonelline and nicotinic acid profiles and for diterpenes around 500 g of fresh fruits

were collected from these plants. These samples were immediately taken into sterile

polythene bags with small perforations and used for experiment within 24 hours.


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To determine caffeine, trigonelline, nicotinic acid, cafestol and kahweol contents,

fruits of five different stages were harvested i.e. Stage I (3 months DAF), Stage II (5

months DAF), Stage III (7 months DAF), Stage IV (8 months DAF) and Stage V (9

months DAF). These stages roughly represent pinhead stage immediately after dormancy

period, rapid expansion stage, pericarp growth stage, efficient endosperm formation stage

and lastly dry matter accumulation stage to harvesting stage respectively (Koshiro et al.,

2006). Respective fruit tissues (whole fruit/pericarp/beans) of five different stages were

used for metabolites analysis. The green beans were allowed to dry to attain ~12%

moisture content. All the green beans were then grounded finely. The extraction and

analysis of caffeine, trigonelline, nicotinic acid, was carried out as explained in materials

and methods section of chapter 3.

4.3.7. Extraction and separation of both free form of cafestol and kahweol and their

methyl esters by GPC and SPE

The coffee oil obtained after soxhlet extraction as explained earlier in Chapter

3, was dissolved in dichloromethane and membrane filtered in a 5 ml measuring flask

and the flask was made up to the mark with dichloromethane. Approximately 250 µL

solution was subjected to Gel Permeation Chromatography (GPC) on Bio-Beads SX3

(BioRad, India) with dichloromethane as the mobile phase at a flow rate of 4ml/min. The

fraction with the diterpenes esters was concentrated in a rotary evaporator and solvent

residues were removed in a nitrogen steam. The diterepene esters were then separated by

solid phase extraction (SPE) on the silica gel column with n-hexane (5ml) and washed

the column with n-hexane:ethylacetate (2ml: 96+4,v/v) for elution of the 16-O-Methyl

cafestol esters, with 2 ml n-hexane:ethylacetate (9:1) for cafestol/kahweol esters, with


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4ml n-hexane:ethylacetate (8:2 v/v) for free diterpenes (Kurzrock and Speer, 2001).

These extracts were dried in a nitrogen stream and redissolved in 1ml mobile phase

(acetonitrile: isopropanol 70:30 v/v) and were used for chromatographic analysis. The

sample injection volume was 20µl. As standards of cafestol and kahweol palmitate are

not available the same were synthesized for reference standards purpose as suggested

earlier (Kurzrock and Speer, 2001). In brief it is: The mixture of cafestol and kahweol

standards ~ 60 mg was taken in 55:45 ratio (m/m) into round-bottom flask (100 mL)

and dissolved in benzene and pyridine (2mL;3+1, v/v). Palmitoyl chloride (~ 120 mg)

dissolved in benzene (0.5mL) and dichloromethane (3mL) were added to the mixture,

followed by its stirring for one hour and then allowed to stand overnight in the dark. The

reaction was stopped by addition of water (20mL) and then concentrated to about 10 mL

on flash evaporator, and the residue was transferred to a 250mL separating funnel. To

this 10% Na2CO3 solution (60mL) was added and the aqueous solution was extracted

with tert-butyl methyl ether (2+100mL) in order to remove the free fatty acid. The

combined ether phases were dried for 15 min over Na2SO4 (3g) filtered concentrated to

about 5mL in rotary evaporator. To this added 25 mL acetone and concentrated again.

The solvent residues were removed in the N2 stream. The diterpenes esters and the free

diterpenes were separated off by means of solid-phase extraction (SPE) on a silica-gel

column as explained above for different fractions from samples.

4.3.8. Free diterpenes analysis by HPLC

Free diterpenes fraction that obtained after GPC and SPE fractionation, was

subjected to HPLC analysis (Waters Alliance 2695 HPLC, USA) by using Nucleosil 120-

3, C18, 250/4 column (Macherey – Nagel, Gmbh, Germany) to quantify cafestol and


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kahweol (Kurzrock and Speer, 2001). The UV detector was set to 230 nm for cafestol

and 290 nm for kahweol. Eluent solvent system used was acetonitrile: isopropanol (70:30

v/v) with a flow rate of 0.6 ml/min. Peaks were identified by comparing with the

retention time of reference standards (Cafestol and Kahweol from Sigma, Bangalore) and

by spiking.

4.3.9. LC-MS analysis of diterpene esters

Analysis was performed (Kurzrock and Speer, 2001) on Waters Alliance 2695

HPLC, equipped with an auto sampler and coupled with a Waters 2696 photodiode array

detector, using Nucleosil 120-3, C18, 250/4 column (Macherey – Nagel, Gmbh,

Germany) with UV detection in the range of 200-350 nm (optimal is 230 nm for cafestol

esters and 290 for kahweol esters). The mobile phase used was acetonitrile: isopropanol

(70:30 v/v) and flow rate of 0.6 ml/min for 30 min. Mass spectrum was acquired in a Q-

TOF Ultima mass spectrometer (Waters Corporation, Micro Mass, and Manchester, UK).

The adopted ionization mode was of positive type, APCI-temperature was 3500C, gas

temperature (nitrogen) was 3300C, nebulizer gas pressure was 60 psi, and flow rate of the

dry gas is 4.0 mL/min. The ionization energy (CID voltage) was 20V or 50 V as

suggested earlier (Kurzrock and Speer, 2001). The confirmation of the occurrence of

compounds is based on the molecular ions in the MS spectra as well as with the literature

(Kurzrock and Speer, 2001). To get clear spectra for free forms of cafestol and kahweol

low CID (<20v) was used.


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4.3.10. Statistical Analysis

The entire experiment was conducted in a randomized block design with five

replicates. For each treatment fifty flowers (bunches) were sprayed. Five fruits of

respective tagged flowers were taken for analysis of caffeine, theobromine, trigonelline,

nicotinic acid, free diterpenes cafestol and kahweol content for each and every treatment.

Regression Values from all five replicate determinations of each sample were averaged

and represented as means with standard deviations. Data were analysed statistically by

the SPSS 17.0 software by one-way ANOVA. Similarly, for studying influence of

altitude on metabolites profile of developing fruits of C. canephora, five coffee bean

samples, out of the ten samples each that were randomly collected from coffee plants

grown at higher, medium and lower elevations were extracted and analysed for caffeine

trigonelline, nicotinic acid, cafestol and kahweol. Values from all five replicate

determinations of each sample were averaged and represented as means with standard

deviations. Data were analysed statistically by the SPSS 17.0 software by Two-way

ANOVA and homogenous subsets were determined to separate the mean values of the

different altitudes and developmental stages.

4.4. Results and Discussion

The increase in respective secondary metabolites of coffee beans was evident

under elicitor treatments which vary with the elicitor and its concentration. Both biotic

and abiotic elicitors responded in a similar way, however, the number of folds increase in

metabolites was better in abiotic elicitors. The purine alkaloid caffeine content was

maximum (2900 ± 244 mg.100g-1

) at 2.5 µM MJ which was 42% more than that of


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control. Even (Fig 4.2) under 2.5 µM SA treatment, 39% increase in caffeine content was

noticed. On par with caffeine content, theobromine content too increased at respective

elicitor treatments and maximum production under 2.5 µM SA spray was noticed which

~ 38% was more to control. When biotic elicitors (Fig.4.3) R. oligosporus and A.niger

mycelial extracts were sprayed at 1% concentration as elicitors to flowers, there was a

maximum enhancement of 20 % and 18 % for theobromine, 25 % and 21% for caffeine

respectively was noticed in beans of harvested fruits, which was less than that of abiotic

elicitors SA and MJ (Fig. 4.2).

The content of pyrimidine alkaloid trigonelline and its precursor nicotinic acid too

were influenced by respective elicitors (Fig 4.4). Trigonelline production was maximum

at 1.0 µM MJ. Though there was an increase in nicotinic acid content it was in the range

of 4-5 mg.100 g-1

dry wt., compared to control (3.78 ±1.17 mg.100g-1

). At higher

concentrations of SA (5 µM) and MJ (5µM) there was significant decrease in caffeine,

theobromine, trigonelline content and also to moderately in case of nicotinic acid (Fig

4.5).


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Figure 4.2. Caffeine and theobromine profile of harvested beans of C.canephora

plants (values are mean ± SD of five analyses; significant at p< 0.05) under

abiotic stress.

Figure 4.3. Caffeine and theobromine profile of harvested beans of C.canephora

plants under biotic stress (values are mean ± SD of five analyses; significant at

p< 0.05; RO: R. oligosporus; AS: A. niger).

Figure 4.4. Trigonelline and nicotinic acid profiles of harvested beans of

C.canephora plants under abiotic stress (values are mean ± SD of five analyses;

significant at p< 0.05).


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Figure 4.5. Trigonelline and nicotinic acid profiles of harvested beans of

C.canephora plants under biotic stress (values are mean ± SD of five analyses;

significant at p< 0.05; RO: R. oligosporus; AS: A. niger).

The free form of cafestol and kahweol profiles were less influenced by elicitor

treatments (Fig 4.6) compared to other metabolites of the study viz., caffeine,

theobromine, trigonelline and nicotinic acid. There was 12, 26 and 18% increase in

cafestol content under lower to high concentration of SA treatments compared to

control (156 ± 4.43 mg.100g-1

). But under 1.0, 2.5, 5.0 µM MJ spray, the increase in

cafestol content was more to SA treatments, and it was 17, 32 and 22%. Similarly,

both R. oligosporus and A. niger triggered moderate enhancement of cafestol

compared to abiotic elicitors and it was 4%, 14% and 10% for R. oligosporus and 6,

15 and 18% for A. niger respectively compared to control (Fig.4.7). A similar trend

was evident for kahweol content enhancement with maximum kahweol production at

2.5 µM SA treatment.


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Figure 4.6. Cafestol and kahweol profiles of harvested beans of C.canephora

plants under abiotic stress (values are mean ± SD of five analyses; significant at

p< 0.05)

Figure 4.7. Cafestol and kahweol profiles of harvested beans of C.canephora

plants under abiotic stress (values are mean ± SD of five analyses; significant at

p< 0.05; RO: R. oligosporus; AS: A. niger).

The significant achievement in this study is the augmentation of major

secondary metabolites under ex vitro conditions through floral spray of respective

biotic or abiotic elicitors which is a novel method with reference to natural

compounds of any category. The bioactive metabolites from beans of coffee that


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investigated in the present study are compounds that not only are connected with

important traits of the plant itself, with reference to coffee quality, resistance to pests

and diseases, but also proved to be functionally important at physiological level in

consumers (Fredholm et al., 1999; Van Dam et al., 2006). Elicitors of both microbial

origin and abiotic elicitors are known for triggering varied responses in plants

including augmented levels of secondary metabolites for value addition (reviewed by

Ramakrishna and Ravishankar, 2011), which upon contact with higher plant cells,

trigger the increased production of phytoalexins (Giridhar and Komor, 2002), and

other defense related compounds (Robbins et al., 1985; Eilert et al., 1984; Eilert et

al., 1986; Flores and Curtis, 1992; Sim et al., 1994; Singh 1999). Also they trigger

pigments (Mahendranath et al., 2011) and isoflavones (Saini et al., 2013). Similarly

the role of biotic elicitors on increased production of alkaloid in Brugmansia candida

was reported (Pitta-Alvarez et al., 2000).

In general plants produce jasmonic acid and methyl jasmonate in response to

biotic and abiotic stresses, which accumulates in the damaged parts of the plant. The

same MJ is also demonstrated as best abiotic elicitor. Treatment with jasmonates can

elicit the accumulation of several classes of alkaloids (Zabetakis et al., 1999),

phenolics (Lee et al., 1997) activation of phenylalanine ammonia-lyase, accumulation

of scopoletin and scopolin in tobacco (Sharan et al., 1998), betacyanin synthesis in

Portuluca (Bhuiyan and Adachi, 2003) and capsaicin in Capsicum (Prasad et al.,

2006). Moreover, a mechanism by which MJ induced-gene expression involved in

plant secondary metabolites biosynthesis at molecular level was demonstrated

http://en.wikipedia.org/wiki/Jasmonic_acid


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(Suzuki et al., 2005). But such reports are scanty with reference to augmentation of

coffee metabolites especially trigonelline, diterpenes cafestol and kahweol. Hence,

the present study is first of its kind, wherein, floral application of MJ could increase

the major secondary metabolites content in robusta coffee beans as discussed. In the

present study along with caffeine, its precursor theobromine was also analysed.

Through it is not necessary for theobromine profiles in this study, the data pertains to

this theobromine further substantiates caffeine content in beans. Moreover extraction

and analysis is same as for caffeine.

Similarly salicylic acid is known as essential component of the plant resistance

to pathogens and plays a part in the plant response to adverse environmental

conditions. Recent studies have demonstrated about the importance of SA in

isoprenoids (precursors of carotenoids pathway) production and accumulation

(Czerpak et al., 2003; Moharekar et al., 2003; Çag et al., 2009), alkaloids production

in Stemona curtisii hairy root cultures (Chotikdachanarong et al., 2011) and

trigonelline production in Trigonella foenum-graceum cell cultures (Mathur and

Yadav, 2011) .

The production of caffeine was shown to be stimulated by stress factors such as

high light intensity and high NaCl concentration (Peters et al, 1985). Exogenous

calcium too influences alkaloids in C. arabica (Ramakrishna et al., 2011). In the

present study, biotic elicitors A. niger and R. oligosporus at 0.1% (w/v) enhanced ~

15% of cafestol and kahweol which is more to control. But studies on elicitor

mediated enhancement of trigonelline, and diterpenes cafestol and kahweol are


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lacking and for the first time the same is reported in the present study. Under elicitor

stress, the activity of the N-methyltransferases will be enhanced that leads to

enhanced production of caffeine. The levels of caffeine in developing seeds are

important as there are no reports about the actual role of this in developing fruits

unlike polyamines (Sridevi et al., 2009). Caffeine that initially synthesized in

pericarp tissues subsequently shifts to endosperm of seed and it accumulates apart

from its own caffeine content. Once caffeine synthesis stops in pericarp in ripened

fruits, caffeine content rather stabilizes in matured seeds of harvested fruits

(Baumann and Wanner, 1972). So the actual elicitor response triggers during the

initial developmental stage and it possibly lasts till fruit maturity stage.

Nicotinic acid being the precursor for trigonelline production, the trigonelline

data in the present study at all elicitor treatments was in direct relation to nicotinic

acid levels. As C. canephora fruit development requires 8 to 9 months there is a

scope for variation in bioactive content in beans during ontogeny of fruit as they get

exposed to various microbes and pathogens. Though trigonelline is normally

synthesized in almost all parts of coffee plant (Zheng et al., 2004), its accumulation is

high in young tissues (Zheng and Ashihara, 2004).

Generally the diterpenes cafestol and kahweol are associated with creamy fat

part of the coffee brew. Though there are no reports on direct or indirect influence of

elicitor stress on lipid content and diterpenes of beans, the increase in cafestol and

kahweol content may be due to the higher activity of gibberellic acid pathway

enzymes wherein the entekaurene is the precursor for both cafestol and kahweol (De

Roos et al., 2006). A part from elicitor stress, other associated factors such as


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climate, temperature, and availability of light and water during the ripening stage of

fruit play crucial role on metabolite profiles of beans (Clifford, 1985; Decazy et al,.

2003; Bertrand et al., 2006).

The data obtained in the present study clearly showed the potential of elicitor

mediated augmentation of major bioactive metabolites of coffee which is important

from quality aspect and also have a wider scope for studying the expression pattern of

major biosynthetic pathway genes of caffeine, trigonelline, and diterpenes in coffee,

which would be helpful to develop a metabolomics and proteomics approach for

deciphering the quality aspects of coffee.

4.4.1. Influence of altitude on caffeine content

There were significant variations in caffeine levels of green beans (dry wt.

basis) of fruits collected from plants grown at different altitudes. The caffeine levels

were positively influenced by altitude variation, with 18.5, 1.5 and 1.25 mg g-1

dry

wt. at low, middle and high altitude samples respectively (Fig 4.8). Similarly there

was significant variation observed in caffeine levels during the ontogeny of coffee

bean development (Fig 4.8). In stage I samples (3 months DAF) i.e. immediately

after dormant period, wherein very small fruits appear, the caffeine content was

0.381 ± 0.047 to 0.287± 0.009 g.100g-1

. Maximum content of caffeine was detected

in harvested beans (Stage V, 9 M DAF). The proportion of increase in caffeine

content was more from the stage II (5M DAF) to stage III (7M DAF).


110

Figure 4.8. Caffeine profiles in beans of C.canephora CxR variety during ontogeny

of fruit (values are mean ± SD of five analyses); Different alphabet letters indicate

the statistical significant difference within the different developmental stages (p<

0.05), and different symbols (α, β and γ) represent statistical significant difference

within the altitude (p< 0.05).

In seeds of stage III fruit there was 14% and 20% increase in caffeine content in

samples from medium and high altitudes compared to low altitude samples. A

progressive increase in caffeine content of stage V seeds was evident from low altitude

collected samples (1.868 ± 0.149 mg.100g-l) and there was 32% increase in high altitudes

samples. To validate the efficiency of extraction procedure one sample was extracted six

times and the coefficient of variation was 1.95%. Similarly a linear relationship between


111

Figure 4.9. HPLC chromatogram of caffeine a) standard (RT 13.56 min); b)

samples from low altitude (RT 13.49 min); c) samples from middle altitude (RT

13.58 min) and d) samples from high altitude (RT 13.47 min).


112

Figure 4.10. Regression coefficients for effect of elevation on a) variation in caffeine

content in stage V (9 months DAF) beans of C. canephora fruit, b) Variation in

average caffeine content during ontogeny of C. canephora fruit.

caffeine concentration and UV absorbance (270 nm) was perceived, wherein the linearity

was managed over the concentration range of 10-100 ppm and the correlation coefficient

for the caffeine standard curve was 0.9999. The retention time of caffeine was 13.56 min

(Fig 4.9) with a relative standard deviation of RSD = 0.54%.The method of detection was

0.030 ppm and the precision was 1.95% at 50 ppm caffeine concentration. The spiked

recoveries of caffeine were 1.02% for tested coffee bean extract. The variation in

retention time for caffeine of test samples was insignificant. Caffeine concentration at

advanced stages of fruit development decreased significantly with increasing elevation

(Fig 4.8). The coefficient of correlation (R2) was recorded as 0.5938 at stage V beans

(Fig 4.10a). Similarly the average caffeine content of all stages also decreased and

inversely proportional to elevation, wherein the coefficient of correlation (R2) was

recorded as 0.8553 (Fig 4.10b).

In the present study, there was significant variation in caffeine content in

developing seeds during ontogeny of C. canephora fruits. A decreasing trend in caffeine


113

content of pericarp was evident when the fruit becomes mature and the rate of reduction

was more (58%) between stage IV to stage V fruits. The trend was same in samples

collected from plants grown at different altitudes. In C. canephora in general the fruit

development requires 8-9 months or even up to 10 months and it is to some extent

asynchronous and could be responsible for variation in major secondary metabolites in

beans during ontogeny of coffee fruit. However, a tendency for synchrony was observed

during the later stages of maturation when a significantly high proportion of fruits

entered the largest sized ripe ‘cherry’ stage as opined by De Castro et al., (2005).

Caffeine was well documented from over 63 plant species worldwide (Neigishi et al.,

1998; Jin et al., 2005; Ali et al., 2012) and also from different Coffea species (Ashihara

et al., 1998; Zheng et al., 2002; Koyama et al., 2003). Caffeine is normally synthesized

in almost all parts of coffee plant, especially in young tissues such as leaves, even in

floral parts (Baumann 2006), and highly concentrated initially in pericarp Baumann

(2006). Our results too indicate that though caffeine was found in significant amounts in

pericarp at initial growth stages of fruit, its accumulation was less in beans at maturation

stage of fruit, which was supported by earlier studies. The high levels of caffeine in

harvesting stage beans (Stage V) is mainly due to acquisition from the perisperm, import

from the pericarp (Baumann and Wanner, 1972) and also due to high activity of S-

adenosine methionine (SAM) dependent N-methyl transferases involved in caffeine

biosynthesis in developing endosperm (Mazzafera, 1994; Mizuno et al., 2003). Apart

from these metabolism aspects of caffeine in developing fruits of Coffea, catabolism of

caffeine too influence the caffeine content of beans. The ratio between biosynthesis and


114

biodegradation controls the difference in caffeine levels during ontogeny especially at

fruit ripening stage as reported in C. arabica and C. dewevrei (Lopes and Monaco 1979).

High-grown coffee beans usually have a high density than low-grown beans.

Longer maturation times and increased bean sizes are usually observed for plants grown

at high altitude or in shade conditions (Guyot et al., 1996; Muschler, 2001; Vaast et al.,

2006). In C. arabica the effects of shade on the development and sugar metabolism was

investigated and also showed that shade grown plants produce less caffeine (Geromel et

al., 2008). Similarly, post-harvest processing conditions and brewing methods too

influence the metabolic profile of beans (Sridevi et al., 2011).Though caffeine production

takes place in pericarp of young fruits, the same will decline in pericarp of ripened fruits

(Zheng et al., 2004). Similarly the caffeine content in seeds during fruit development was

varied and there was a progression in caffeine content with the advancement of age of

fruit. The increase in caffeine content was more significant from fruit development stage

III to stage V. Though caffeine biosynthesis actively takes place during leaf let

emergence (Frischknecht et al., 1986), also the same takes place in developing fruit, the

pericarp and perisperm (Keller et al., 1972). Mazzafera and Goncalves (1999) reported

that, absolute amounts of caffeine parallel the dry weight of bean, with more or less

constant caffeine content during maturation, nearing 1% on a dry matter basis at the time

of harvest. The increase in caffeine content during stage III (6 months) to stage IV (8

months) in our study is further supported by similar observations in C. arabica (Keller et

al., 1972). The levels of caffeine in developing seeds are important though we don’t have

any information about the actual role of this in developing fruits unlike polyamines

(Sridevi et al., 2009). Most of the caffeine that synthesized in pericarp shifts to


115

endosperm of seed where, it accumulates apart from its own caffeine content, and once

further caffeine synthesis stops in pericarp in ripened fruits, caffeine content rather

stabilizes in matured seeds of harvested fruits (Baumann and Wanner, 1972). This study

indicates that the altitudes at which coffee plants grow had influence on caffeine content.

4.4.2. Influence of altitude on trigonelline and nicotinic acid content

Significant variations in trigonelline (Trig) and a nicotinic acid (NA) level of

green beans of fruits collected from plants grown at different altitudes was noticed. The

Trig levels were positively influenced by altitude variation, with a maximum of

(975.8±7.24 mg.100g-l) in beans of high altitude samples, which was 20% and 25% more

than that of medium and lower altitudes respectively (Fig 4.11). (Fig 4.12).

Figure 4.11. Trigonelline (mg.100g-1

) profile in beans of C.canephoara CxR variety

during ontogeny of fruit (values are mean ± SD of five analyses); Different alphabet

letters indicate the statistical significant difference within the different

developmental stages (p< 0.05), and different symbols (α, β and γ) represent

statistical significant difference within the altitude (p< 0.05).


116

Similarly there was significant variation observed in both Trig and NA during the

ontogeny of coffee bean development (Fig 4.11, 4.12). The levels of NA too were

positively influenced by altitude variation, with a maximum of (5.16 mg 100 g-l) in

beans of high altitude samples. In stage I samples (3 months DAF) i.e. immediately after

dormant period, wherein tiny fruits appear, the NA and Trig contents were (0.686 ±

0.078 mg.100g-l) to 1.25 ± 0.068 mg.100g

-1) and (20.86 ± 3.23 to 32.4 ± 4.75 mg.100 g

-l)

respectively at low to high altitudes and in subsequent stages of bean development there

was a gradient increase in both NA and Trig content and it reached to (3.92 ± 0.127 to

5.164 ± 0.131 mg.100g-l) and (734.8 ± 7.39 to 975 ± 7.24 mg.100 g

-l) at harvesting

stage of samples collected from low and high altitudes respectively (Fig. 4.11, 4.12).

Trigonelline concentration at all stages of fruit development increased significantly with

increasing elevation (Fig 4.11). The regression was significant (y=0.1739x-217.17), with

high coefficient of determination as R2 = 0.9774. Similarly, NA concentration too

increased significantly with increasing elevation, wherein regression was y=0.0015x-

2.6174 with coefficient of determination as R2

= 0.8526 which is slightly less than that of

trigonelline (Fig 4.13).

In stage V fully ripened fruit’s bean Trig concentration regression too was

significant (y=0.3509x-336.53) with high coefficient of determination as R2 =0.9434

which is almost same to R2 values of average trigonelline content of at all stages of

ontogeny. Even for NA of stage V fruit beans (Fig. 4.13), the coefficient of determination

(R2

= 0.8729) was similar to that of ontogeny stages (R2 =

0.8526). So in the present

study, the way altitude (quantitative variable) affects the secondary metabolite Trig and


117

also its precursor nicotinic acid was interpreted through linear correlation. A glance at

NA profile during ontogeny shows that, there was significant variation at stage II fruit,

stage III, IV and V seed of medium altitude (mean = 2.35, 3.964, 4.108, 4.634; standard

deviation = 0.367, 0.149, 0.291, 0.268 respectively), compared to low and high altitudes,

which indicates the influence medium altitude was prominent for nicotinic acid. Similar

observations were noticed for trigonelline too.

Figure 4.12.Nicotinic acid mg.100g-1

dry wt) profiles in beans of C.canephoara CxR

variety during ontogeny of fruit (values are mean ± SD of five analyses); Different

alphabet letters indicate the statistical significant difference within the different

developmental stages (p< 0.05), and different symbols (α, β and γ) represent

statistical significant difference within the altitude (p<0.05).


118

Figure 4.13. Regression coefficients for effect of elevation on a) variation in trigonelline

content during ontogeny of C. canephora fruit, b) variation in nicotinic acid content during

ontogeny of C. canephora fruit, c) variation in stage V seed trigonelline content, d)

variation in stage V seed nicotinic acid content


119

Figure 4.14. HPLC chromatogram of nicotinic acid (RT 1.990) and trigonelline (RT 2.663)

in seed extracts of C. canephora fruit grown at different altitudes; a) standards b) Lower

altitude sample c) medium altitude sample d) higher altitude sample


120

In the present study, the extraction and HPLC analysis of both Trig and NA

facilitated to obtain the data significantly (p < 0.05). To validate the efficiency of extraction

procedure one sample was extracted three times and the coefficient of variation was 1.54%

and 1.12% for Trigonelline (Trig) and nicotinic acid (NA). Similarly a linear relationship

between Trig / NA concentration and UV absorbance was perceived, wherein the linearity

was managed over the concentration range of (0.15-400 µg mL and 0.10-400 µg mL) and

the correlation coefficient for the Trig/NA standard curve invariably exceeded 0.999. In

HPLC the retention time (RT) for NA and Trig were 1.990 and 2.663 respectively (Figure

4.14). In case of seed sample extracts another major peak was evident at 4.54 RT. This peak

is nothing but caffeine of seed extracts and data of caffeine is not quantified as it requires

different extraction method and also absorption maxima (274 nm). In fact caffeine was

quantified separately as explained in material and methods of this chapter.

In the present study, there was significant variation in Trig and NA content in

developing seeds during ontogeny of C. canephora fruits. The pyridine alkaloid Trig

accumulates in seeds, however its biosynthetic activity is reported to be higher in the

pericarp (Koshiro et al., 2006). Nicotinic acid being the precursor for Trig production,

the Trig data in the present study at all stages of ontogeny of fruit was in direct relation to

NA levels. A decreasing trend in Trig content of pericarp was evident when the fruit

becomes mature and the rate of reduction was more from stage III to stage V fruits. The

trend was almost same in samples collected from plants grown at different altitudes. In

stage III fruit pericarp more trigonelline was detected compared to earlier stage of

growth. In C. canephora in general the fruit development requires 8 to 9 months and it is

to some extent asynchronous. This may be one of the reasons for variation in bioactive


121

content in beans during ontogeny of fruit. However, a tendency for synchrony was

observed during the later stages of maturation when a significantly higher proportion of

fruits entered the largest sized ripe ‘cherry’ stage as opined (De Castro et al., 2005).

Trigonelline was well documented from various plants (Poulton, 1981; Barz, 1979; 1985)

and trigonelline is normally synthesized in almost all parts of coffee plant (Zheng et al.,

2004), and its accumulation is higher in young tissues as reported by Zheng et al (2004).

Zheng et al (2004) also reported trigonelline activity in endosperm stage. Our results

indicate that though Trig was found in significant amounts in pericarp at initial growth

stages of fruit, its accumulation was more evident in beans at maturation stage of fruit,

which was supported by earlier studies of (Zheng et al., 2004 ) in C. arabica, wherein,

high net biosynthetic activity of Trig was demonstrated in dry matter accumulation stage

of seeds i.e. stage III - IV, which subsequently decreases markedly, however, Trig

transportation takes place from pericarp to seeds. A similar observation for caffeine too

was showed (Baumann and Wanner, 1972) for C. arabica.

Longer maturation duration and increased bean sizes are usually observed for

plants grown at high altitude or in shade conditions (Muschler, 2001; Guyot et al., 1996).

A positive relation between altitude and taster preferences of Arabica coffee from

different territory of Costa Rica was reported (Avelino et al., 2005). The increase in

trigonelline content of robusta beans from high altitude in the present study was further

supported by earlier report of Avelino et al (2005). Though the slope exposure where in,

the light or shade availability too have some influence on Arabica coffee quality as

opined by Avelino et al (2005) in the present study, altitude influence only was

investigated as it mainly influence biochemical profile of robusta beans (Guyot et al.,


122

1996). Apart from this, in the present investigation, the range of altitude difference from

where samples were collected was 3000-3700 ft and this mesoclimate conditions

influence was found to be significant on both Trig and NA content of beans. In contrast

to the study of Avelino et al (2005) negative relation between elevation and triogonelline

content was found by Bertrand et al (2006), but this study is based on Near-Infrared

Spectra (NIR) of some Arabica hybrids involving Sudanese-Ethiopian origins with

traditional varieties. Such variations in metabolic profiles trend is a common

phenomenon as it is indirectly influenced by some environmental and coffee agroforestry

systems (Vaast et al., 2006). Similarly, roasting conditions and brewing methods too

influence the metabolic profile of beans (Sridevi et al., 2011). Unlike other metabolites

of Coffee, Trig is important from post-harvest processing aspects that imparts quality to

Coffee. Because Trig alkaloids gives rise to many flavour (aroma) compounds, like

alkyl-pyradines and pyrroles (De Maria et al., 1994). In view of this, the levels of Trig in

developing seeds are important though we don’t have any information about the actual

role of this in developing fruits unlike polyamines (Sridevi et al., 2009). Shimizu and

Mazzafera (2000) showed the role of trigonelline during inhibition and germination of

coffee seeds. Though Trig production takes place in pericarp of young fruits, the same

will decline in pericarp of ripened fruits (Koshiro et al., 2006). However, our data

suggests that trigonelline accumulation takes place in seeds from stage III to stage V

fruits. This was in concurrence with earlier studies (Zheng et al., 2004; Koshiro et al.,

2006). All these substantiate the trend in Trig content and also its precursor nicotinic acid

content in the present study. Moreover the content of Trig in harvested beans is on par

with its trend in C. canephora as reported earlier which would be in the range of (0.75 to


123

1.24%) as reviewed by De Castro and Marracccini (2006). Certainly, the altitudes at

which coffee plants grow had influence on Trig and NA content.

4.4.3. Influence of altitude on free diterpenes content

Free diterpenes levels of beans in fruits collected from high elevation grown

coffee plants were less (cafestol 420 ±9.5 µg.g-1

and kahweol 140 ±4.5 µg.g-1

) compared

to samples collected from low elevation (cafestol 1200 ±18.4 µg.g-1

and kahweol 450

±7.5 µg.g-1

) which were analysed by HPLC (Fig. 4.15, 4.16, 4.17). Though cafestol was

detected right from the early stage of fruit development, kahweol was not found to be at

detectable levels till stage III of fruit development. Similarly, cafestol was not detected in

pericarp of stage III fruits. In case of stage IV fruits, both cafestol and kahweol were

detected at significant levels but in the subsequent stages both these diterepenes were

reduced drastically (Fig. 4.15, 4.16). This indicates that stage IV of fruit development is

the most crucial stage, because at this stage both diterpenes were found in detectable

levels. This trend was more or less same as in case of other metabolites such as caffeine

and trigonelline.


124

Figure 4.15. Cafestol profiles (µg.g-1

dry weight) during ontogeny of C.canephoara

CxR variety fruit (values are mean ± SD of five analyses); Different alphabet letters

indicate the statistical significant between the altitude (p< 0.05).

Figure 4.16. Kahweol profiles (µg.g-1

dry weight) during ontogeny of C.canephoara

CxR variety fruit (values are mean ± SD of five analyses); Different alphabet letters

indicate the statistical significant between the altitude (p< 0.05). In stage I & II

fruits, stage III & IV (pericarp) kahweol not detected


125

Figure 4.17. HPLC analysis of kahweol (peak 1-RT 7.85) and cafestol (peak 2-RT

12.92): a) Standards, b) high altitude, c) medium altitude, d) low altitude.


126

Figure 4.18. Regression coefficients for effect of elevation on a) Variation in cafestol

content in C. canephora b) variation in kahweol content in C. canephora

This HPLC analysis of free diterpenes facilitated to obtain the data significantly

(p<0.05). To validate the efficiency of extraction procedure one sample was extracted

thrice and the coefficient of variation was 1.15 %. The linear relationship between

cafestol / kahweol concentration and UV absorbance was perceived, wherein, the

linearity was managed over the concentration range of 10 µg -150 µg mL-l. In HPLC the

retention time (RT) for kahweol and cafestol were 7.85 and 12.92 (Figure 4.17).

Unlike the method of Campanha et al (2010) that followed in Chapter 3, some

changes were made to HPLC analysis of free form of cafestol and kahweol in fractions

that obtained through GPC and SPE as explained in materials and methods part of this

Chapter 4. In place of Nucleosil 100-5 C-18 column of size 5μ X 250mm (HPLC

Trennsaule, - Macherey-Nagel Gmbh & Co. KG., Duren, Germany), another column i.e.

Nucleosil 120-3, C18, 250/4 column (Macherey – Nagel, Gmbh, Germany) was used

(Kurzock and Speer, 2001) to detect cafestol and kahweol at 230 nm and 290 nm by

using acetonitrile: isopropanol (70:30) in place of acetonitrile: water: glacial acetic acid


127

(70:29:1) as shown in Chapter 3. This modification was inevitable to get good separation

and apt resolution of cafestol and kahweol peaks in column purified fractions. Due to this

there was a slight shift in RT of kahweol to 7.85 min and cafestol to 12.92 min instead

7.967 & 15.950 min respectively as described in Chapter 3. As absorption maximum was

at 230 nm and 290 nm for kahweol and cafestol standards the same was for samples

analysis under the specified conditions. There was 1% improvement in sensitivity for

detection of standards of cafestol and kahweol in both the methods. Due to this, the

method of Kurzrock and Speer (2001) as explained in this chapter was only used for

quantification of cafestol and kahweol.

Regression coefficients for effect of elevation on variation in cafestol and

kahweol content in C. canephora were studied (Fig. 4.18). Apart from the free form of

diterpenes, the levels of caf and kah esters were found in beans collected from at all

elevations grown plants. Among the caf and kah esters the major esters are cafestol

linoleate, cafestol oleate, cafestol palmitate, cafestol stearate, kahweol linoleate, kahweol

oleate and kahweol palmitate (Figure 4.19). Three kahweol esters and four cafestol esters

were identified by MS (Figure 4.20). Though their presence in green harvested beans was

not reported so far, (Kurzrock and Speer, 2001) identified five kahweol esters along with

cafestol esters in roasted Arabica coffee beans. Similarly Pettitt (1987) investigated the

diterpene esters of Robusta coffee and also Arabica coffee by means of HPLC. Some

reports suggest cafestol and kahweol esters separation on silver nitrate impregnated silica

gel, however, but complete separation of these esters is difficult hence, LC/MS equipped

with a DAD was used in this study as advocated by (Kurzrock and Speer, 2001). In our

study low CID values (20V) were found to be good enough to achieve effective


128

fragmentation of cafestol palmitate wherein, the fragment ion [M+H-H2O]+ was detected.

In its mass spectrum the fragment ions m/z 281 and 147 were found. However, for

kahweol esters high CID value (50V) was used which yielded more number of

characteristic fragment ions such as [M+H]+-ion and [M+H-H2O]

+. In this mass

spectrum, one fragment ion m/z 279 was obtained due to separation of the fatty acid and

of the second OH-group as well as the fragment ion m/z 131, which are in concurrence

with observations of (Kurzrock and Speer, 2001). Apart from mass spectra, the retention

times were taken into account, as most of the cafestol and kahweol esters have the same

molar masses, but different retention values. Accordingly, the RT and MS values were

compared with earlier reports (Kurzrock and Speer, 2001), which indicates the presence

of cafestol and kahweol esters (Figure 4.19). The [M+H]+ and [M+H-H2O]

+ values are

575 & 557 (kahweol linolenate), 553 & 535 (cafestol linolenate), 579 & 559 (cafestol

linoleate), 581 & 563 (cafestol oleate), 555 & 537 (cafestol palmitate),583 & 565

(cafestol stearate), 611 & 539 (cafestol arachidate), 639 & 621 (cafestol behenate), 579 &

561 (kahweol oleate), 553 & 535 (kahweol palmitate), 569 & 551 (cafestol

heptadecanoate) and 637 & 619 (kahweol behenate) respectively. Also presented mass

spectrum of cafestol and kahweol fractions that eluted from HPLC (Figure 4.20). In

general, the free form of diterpenes are in less quantity (< 200 mg/kg d.m.) as reported

earlier (Kaufman and Hamasagar, 1962; Kurzock and Speer, 2001), but their levels vary

depending on the coffee species and geographical and agronomic conditions (Speer and

Kolling-Speer, 2006).

In coffee trade, elevation at which plantations are grown is a vital indicator of

quality as it influences biochemical composition of the coffee beans (Decazy et al., 2003)


129

and also influenced by genetic factors (Montanon et al., 1998). Various factors such as

temperature, light, shaded conditions, and water at different altitudes had influence on

growth of fruits (Guyot et al., 2006, Decazy et al., 2003). Effect of elevation on Arabica

bean chemical composition with reference to caffeine, trigonelline, chlorogenic acids and

sucrose was investigated (Avelino et al., 2005; Bertrand et al., 2006). Especially CGAs

and fat concentrations are positively influenced up to low altitude, but fat concentrations

decrease at highest altitudes (Bertrand et al., 2006). Higher leaf to fruit ratios at higher

altitudes leads to augmented carbohydrates supply to berries and increased bean fat

synthesis (Vaast et al., 2006). Being part of overall fat component of beans, the

antinutritional diterpenes cafestol and kahweol levels too are effected and the decrease in

their levels at higher altitudes in the present study is in agreement with fat profile trend as

reported (Bertrand et al., 2006). In the present investigation, samples were collected

from robusta coffee plantations at three different altitudes, where in, shade cultivation

was in practice as a part of Coffee-agro forest systems. This will help organic matter

availability and nutrient cycling that positively influence the growth of coffee plants

which will contribute to some extent the coffee quality (Leroy et al., 2006). Generally the

diterpenes caf and kah are associated with creamy fat part of the coffee brew. Certainly

there would be a change in their profile with a shift in elevation for respective

plantations. In the present study, we analysed both free and esterified forms of caf and

kah. While extracting samples for diterpenes through saponification method, only free

diterpenes are recovered, because of the presence of fatty acids, soaps are formed which

generate an emulsion lowing caf and kah recovery (Silva et al., 2012). In view of this to


130

find out esterified form of diterpenes we relied on GPC method as reported earlier

(Kurzrock and Speer, 2001).

Both caf and kah are mainly found in coffee oil and are esterified with fatty acids

(Kurzrock and Speer, 2001). Apart from the major diterpenes cafestol and kahweol,

additionally 16-O-mehtyl cafestol (16-OMC) and 16-O-methylkahweol (16-OMK) were

reported in robusta beans (Speer and Mishnick-Lubbecke, 1989; Speer Montog, 1989;

Speer et al., 1991b; Kolling-Speer et al., 2001), which may aid as marker to find robusta

in arabica blends, in view of their stability even at roasting temperatures. As both these

16-OMC and 16-OMK are in trace amounts both these things were not considered for

analysis in our study. In robusta the free diterpenes are in the range of 1.1 – 3.5% (Speer

and Kolling-Speer, 2006) along with 14% of diterpene esters (Duraan et al., 2011),

which are together considered as total diterpenes. In our study, we got ~0.12% of free

form of cafestol and 0.045% of kahweol respectively in beans of robusta that collected

from low altitude grown plants. So the remaining part of coffee oil would be contributed

by diterpene esters as reported earlier (Speer and Kolling-Speer, 2006), wherein again the

C16, C18, C18:1, C18:2, C20 and C22 are the major esters which are reported to be in the

range of 0.22% to 0.76% on dry weight basis in robusta beans, which corresponds to

0.12% to 0.42% of cafestol which is also the primary diterpene component of coffee

(Speer and Kolling-Speer, 2006). Similarly, kahweol esters too represent more or less

similar levels of cafestol esters. In view of this, in our study it is envisaged that up to

0.22% of these cafestol esters would present in robusta beans.


131

Figure 4.19. HPLC chromatogram of diterpene esters in low altitude robusta coffee

beans. 1- kahweol linolenate (RT 7.49); 2- cafestol linolenate (RT 7.72), 8- cafestol

linoleate (RT 9.02), 3- cafestol oleate (RT 11.18), 4 - cafestol palmitate (RT 11.65), 5

- cafestol stearate (RT 15.18), 6 - cafestol arachidate (RT 19.85), 7- cafestol behenate

(RT 26.02). 9- kahweol oleate (RT 11.02), 10- cafestol heptadecanoate (RT 13.23),

11- kahweol behenate (RT 25.42).

Among the esterified forms of caf and kah, cafestol linoleate, kahweol linolete,

cafestol palmitate, cafestol stearate, kahweol linoleate, kahweol oleate and kahweol

palmitate are the major esterified forms (based on peak resolution and area) followed by

other minor forms viz., cafestol arachidate, cafestol behenate, cafestol heptadecanoate

and kahweol behenate, which were further supported by earlier report on diterpene esters

in coffee bean (Kurzrock and Speer, 2001). The main purpose of the present study was to

find out the influence of altitudinal conditions on cafestol and kahweol profiles in coffee

beans. Along with the free forms of diterpenes, esters of cafestol and kahweol also

increased from the high to low altitudes. Though the levels appears to be more, the same


132

may not go into coffee brew, because, there will be a drift in diterpenes profiles, once

green beans are subjected to roasting (Urgert et al., 1995b; Sridevi et al., 2011 Silva et

al., 2012).


133

Figure 4.20. Mass spectrum of a) cafestol palmitate, b) kahweol oleate, c) kahweol

palmitate, d) cafestol linoleate, e) cafestol f) kahweol


134

It is known that the levels of caf and kah are significantly low in Arabica beans

compared to Robusta beans. The reason for this is attributed to high altitude cultivation

of Arabica (Campanha et al., 2010). Apart from elevation at which coffee plants grow,

other associated factors such as climate, temperature, and availability of light and water

during the ripening stage of fruit play crucial role metabolite profiles of beans (Clifford,

1985; Decazy et al., 2003; Bertrand et al., 2006).Especially high concentration of

chlorogenic acids, trigonelline and sucrose in sun-grown (low elevation) coffee plant’s

beans attributed to its increased bitterness and astringency of the brew (Muchler, 2001).

Another reason for the high quantity of diterpenes could be due to accumulation of these

diterpenes during ontogeny of fruit (Dias et al., 2010) as in case of polyamines (Sridevi

et al., 2009).

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INFLUENCE OF VARIOUS BIOTIC AND ABIOTIC FACTORS …shodhganga.inflibnet.ac.in/bitstream/10603/69216/7... · · 2016-01-08FACTORS ON THE SECONDARY METABOLITES PRODUCTION IN COFFEE