M.B. dos Santos Scholz et al., J. Near Infrared Spectrosc.

22, 411–421 (2014) 411

Received: 13 October 2014 n Revised: 10 December 2014 n Accepted: 8 January 2015 n Publication: 13 January 2015

JOURNAL
OF
NEAR
INFRARED
SPECTROSCOPY

Application of near infrared spectroscopy

for green coffee biochemical phenotyping
Maria Brígida dos Santos Scholz,a,* Cíntia Sorane Good Kitzberger,a Luiz Filipe Protasio Pereira,b
Fabrice Davrieux,c David Pot,d Pierre Charmetantd and Thierry Leroyd
a
IAPAR—Instituto Agronômico do Paraná, Departamento de Fisiologia Vegetal, Londrina, Brazil. E-mail: mbscholz@iapar.br
b
Embrapa Café, Londrina, Paraná, Brazil
c
Cirad-UMR Qualisud, F-34398 Montpellier, France
d
Cirad-UMR AGAP, F-34398 Montpellier, France

Accessions resulting from surveys in Ethiopia (the centre of origin of Arabica coffee) can be used as a source of genetic variability in
breeding coffee plants. They may contain some genes of interest for coffee breeding, specifically in relation to beverage quality. Near
infrared (NIR) spectroscopy was used to develop models for predicting the major coffee constituents related to quality beverage (pro-
teins, caffeine, lipids, chlorogenic acids, phenolic compounds, total sugars and sucrose). We selected coffee samples listed in a data-
base containing data of chemical contents from samples of traditional and modern cultivars and of Ethiopian accessions to construct
models to predict these compounds. Spectra were collected between 1100 nm and 2500 nm, and mathematical pretreatments were
applied. The number of samples for the calibration step for each compound was set so as to be representative of distribution values.
Cross-validation was performed on the total set of samples to select the optimal number of terms for the prediction models of each
component. The prediction models were developed employing a modified partial least-squares regression. The total set of samples for
each component was divided randomly into two subsets: one for developing the prediction model and the other to evaluate the predicted
values. The best prediction models obtained were for chlorogenic acids (r2 = 0.94, RPD = 4.16), proteins (r2 = 0.94, RPD = 4.09) and caf-
feine (r2 = 0.92, RPD = 4.16). Models for lipids and phenolic compounds were not as accurate (r2 = 0.87, RPD = 2.77 and r2 = 0.86, RPD = 2.62,
respectively), while models for sucrose (r2 = 0.84, RPD = 2.44) and total sugars (r2 = 0.85, RPD = 2.55) were even less accurate. All these
models can be used for identifying coffee lines with more desirable traits in breeding programmes. The models were effective in dis-
criminating Ethiopian coffee accessions from modern cultivars of coffee. Additionally, the NIR technique will make it possible to analyse
a large number of samples in breeding programmes and may be used as a high-throughput analysis for green coffee phenotyping.

Keywords: green coffee components, NIR spectroscopy, Ethiopian coffee accessions, coffee cultivars, high-throughput phenotyping

Introduction
In the global economy, coffee is a major commercialised agri-
cultural product, the production of which involves thousands
of people around the world. In 2010, there were approximately
26 million farmers and coffee workers in over 50 producing
countries engaged in coffee production.1 Coffee production
in the 2013–2014 season was 145 million 60 kg bags of coffee.
Brazil is the largest producer and exporter of coffee followed
by Colombia and Vietnam. Many producing countries are also
large consumers of coffee, such as Brazil, which is currently
the second largest consumer of this beverage.2
Coffee is consumed mainly for the stimulating effect of
caffeine, but the aroma and flavor play a fundamental role
in making coffee one of the most appreciated and consumed
beverages in the world.3

ISSN: 0967-0335 © IM Publications LLP 2014

doi: 10.1255/jnirs.1134 All rights reserved
 412 Green Coffee Biochemical Phenotyping
During roasting, the reactions between various constituents
of green beans produce nearly 1000 compounds (volatile and
nonvolatile), but approximately 30 compounds are responsible
for the main impression of the coffee aroma.4 To identify the
role of these compounds in beverage quality, the relationship
between chemical compounds in green beans and beverage
quality has been built over the years.5
The compounds in green beans, such as caffeine, proteins,
lipids, phenolic compounds and sugars, are among the main
precursors of the typical aroma and flavor of roasted coffee.6
Caffeine was the first compound studied in coffee, and the
content varies greatly within a single species owing to the
environmental and agronomic conditions.7 The protein content
of coffee beans also varied greatly and is influenced by envi-
ronmental conditions, fertilisation practices and the cultivar.8,9
The presence of lipids is associated with aroma retention and
foaming in the beverage, and their accumulation depends on
several factors, particularly the species and cultivars. Beans
of the same cultivar of coffee grown in different environmental
conditions show lipid contents ranging from 10.65 g 100 g–1 to
13.16 g 100 g–1.9 However, a wider range (10.69–16.75 g 100 g–1)
was found by measuring different canephora cultivars.10
Sugars are important precursors of the aromatic compounds
generated during roasting. Sucrose, fructose and glucose are
the major sugars found in coffee, and an increase in their
concentrations has been associated with maturation stage of
the beans. The sucrose level increases as the beans develop,
and the glucose and fructose levels decrease at the end of the
cycle.11
The concentrations of phenolic compounds, especially chlo-
rogenic acids, are inversely associated with bean ripening. The
immature beans contain large amounts of such compounds,
and their presence in the beverage creates a high acidity and
astringency.12
Despite the range of compositions observed for the chem-
ical compounds, the genetic diversity in coffee varieties is
very narrow. Basically, with rare exceptions, the cultivars of C.
arabica plantations in Central and South America are derived
from Typica and Bourbon cultivars.13 This narrow origin has
limited the search for polymorphic markers associated with
the formation of the main compounds in the coffee bean.14
Ethiopia is considered the centre of origin of Arabica
coffee.13 Accessions resulting from surveys in Ethiopia can
be used as a source of genetic variability. They may contain
some genes of interest for coffee breeding related mainly
to beverage quality. One hundred and thirty-two samples of
accessions were collected in Ethiopia, and planted and main-
tained in the experimental fields of the Agronomic Institute of
Paraná, Brazil (IAPAR) in 1976. The phenotypic and genotypic
characterisation of this population is the starting point for
studies of linkage disequilibrium mapping, or mapping by
association, current tools that are used to accelerate genetic
gain in breeding programmes.
Near infrared (NIR) spectroscopy is a technique that makes
it possible to evaluate a large number of phenotypes in a short
time. This technique has many advantages compared with
traditional techniques: it is fast and inexpensive and requires
only small quantities of samples without the use of chem-
ical reagents. Furthermore, samples can be analysed in their
natural state or in small preparations, and several compounds
can be measured simultaneously.15,16
In the area of plant breeding, the composition and quality
of new lines must be evaluated for at least three years to be
confirmed, generating a large number of determinations. NIR
has been widely used in this area to quantify the oil, protein and
total and free fatty acids content in sunflower lines17, to assess
the seed quality of various species18,19 and to identify intro-
gression between different coffee species.20 Prediction models
were developed to determine caffeine and21,22 proteins,23 and
more recently, this technique was also shown to be able to
predict cafestol and kahweol in green coffee beans.24
The quantification of these compounds in green coffee
by NIR was used to evaluate the influence of altitude and
cultivar on the biochemical composition of Arabic hybrids5
as well as differentiation between Arabica non-introgressed
and C. canephora derivate genotypes.20 Furthermore, NIR
was employed to evaluate the quality of Arabica and Robusta
coffees from different geographical locations25 and the quality
of roasted coffee.26
The objective of this study was to evaluate the potential of the
NIR technique for quantifying the main components of green
coffee (caffeine, chlorogenic acids, proteins, lipids, phenolic
compounds, total sugars and sucrose) and for discriminating
cultivars from different origins (Ethiopian coffee accessions,
traditional and modern cultivars).
Materials and methods
Samples
To construct prediction models of each of the proposed constit-
uents, representative sampling of the coffee was conducted.
Thus, a database containing data on chemical contents from
coffee samples collected and analysed during various seasons
in the coffee region of Paraná State was created. In the 2004
season, samples of one cultivar (by IAPAR 59), cultivated under
different fertiliser levels, were collected from various agri-
cultural properties. Samples were collected from Ethiopian
coffee accessions at IAPAR for two seasons (2006 and 2007).
Modern coffee cultivars developed by IAPAR (Iapar 59, IPR 97,
IPR 98, IPR 99, IPR 100, IPR 101, IPR 102, IPR 103, IPR 104,
IPR 105, IPR 106, IPR 107 and IPR 108) were collected for
three seasons (2008, 2009 and 2010) in experimental plots
in the coffee region of Paraná State. Finally, to complete the
database, traditional and modern coffee cultivars (Iapar 59,
IPR 99, Catuaí, Mundo Novo, Tupi, Obatã, and Bourbon) were
harvested on coffee farms in the region Norte Pioneiro of
Paraná State in the 2009 and 2010 seasons.
To ensure that the maximum range of values was covered
for the constituents of green coffee, specific subsets were
separated manually from the total set of samples. This action
resulted in a variable number of samples for each constituent
 M.B. dos Santos Scholz et al., J. Near Infrared Spectrosc. 22, 411–421 (2014) 413
model. The variability of the samples in the data sets (calibra-
tion and validation) was evaluated for each group by the coef-
ficient of variation, standard deviation and range values of the
contents.
In order to verify the applicability of the developed prediction
models, another group of samples was analysed. This group
was formed by traditional cultivars [Bourbon (BA10), Catuaí
(Ctai)], modern cultivars (by IAPAR 59, IPR 97, IPR 98, IPR 99,
IPR 100, IPR 101, IPR 102, IPR 103, IPR 104, IPR 105, IPR 106,
IPR 107 and IPR 108) and coffee accessions from the collec-
tion of Ethiopian coffees (Eastern side of the Rift Valley: E007
and E237, Western side: E041, E044, E047, E087, E123a, E123b,
E331, E338, E383, E464, E511 and E516). These accessions
were planted in experimental fields at IAPAR, and the samples
were collected for two seasons (2010 and 2011). The chemical
composition of these samples was determined by applying the
prediction model developed here.
Sample preparation
Ripe coffee beans were collected manually, washed and then
dried in the sun until a moisture content of 12–12.5% was
reached. The husk and parchment were removed mechani-
cally in portions of approximately 300 g. The green coffee beans
were dipped in liquid nitrogen and ground (0.5 mm particles) in
a disk mill (Perten 3600, Kungens Kurva, Sweden). The ground
samples were frozen at –20°C for further chemical analysis
and spectral measurements.
Biochemical analyses
To quantify the caffeine content, the ground coffee was added
to water and MgO, and heated to the boiling point for 30
min. After filtering, an aliquot of chloroform was evaporated
completely and added to the boiling water. After cooling, the
absorbance was read at 273 nm, and the concentration was
calculated from a standard curve for caffeine. The total sugars
(reducing and non-reducing) were extracted with water, which
was maintained at 80°C, for 30 min. After cooling, the sample
was clarified with Carrez reagent, and the reducing sugars
were determined using the Somogyi–Nelson assay.27 To deter-
mine the total sugars, an aliquot of the clarified solution was
hydrolysed before determining the reducing sugars. The sugar
content was quantified using a standard curve prepared with
glucose. The amount of sucrose was calculated by subtracting
the amount of reducing sugars and the total sugar value. The
protein content was determined by the Kjeldahl method. 28
After digestion of the organic matter with sulfuric acid, the
protein concentration was obtained by multiplying the value
of the nitrogen by a factor of 6.25. The lipids were solvent-
extracted (petroleum ether) over 16 h with heating (45–50°C).
The phenolic compounds (PC) were determined in the aqueous
extract with the Folin–Ciocalteau reagent, and the concen-
tration of these compounds was obtained from a standard
curve prepared with gallic acid.29 The total chlorogenic acids
were extracted with isopropyl alcohol, and after a reaction
with potassium periodate, the absorbance was measured at
530 nm. A standard curve was prepared with 5-CQA.30
All the biochemical determinations were performed in
duplicate, all reagents were suitable for chemical analysis,
and all results were expressed on a dry basis.
NIR spectrum acquisition
NIR spectra were collected using a spectrphotometer (model
6500, Foss NIRSystems, Silver Spring, MD) equipped with a
reflectance detector. Approximately 6 g of ground coffee was
placed in a rectangular cell, compressed gently with a metal
spatula and closed with a cardboard lid. The ISIscan software
package (Foss, Silver Spring, Maryland-USA) was used to
control the spectrophotometer, collect the spectra, import and
analyse the data. Although spectra were collected between
400 nm and 2500 nm, only the region between 1100 nm and
2500 nm was used to develop prediction models. Spectra were
collected between 1100 nm and 2498 nm at 2 nm intervals, and
saved as the average of 32 scans. Scans were stored as log
(1/R), where R was the reflectance at each wavelength. All
calculations were performed in WinISI 4.5 software (Infrasoft
International, Port Matilda, PA, USA).
The noise in the spectra caused by light scattering was
corrected mathematically by applying standard normal variate
and detrend corrections.15,31,32 Then, a second derivative was
calculated on five points of the spectrum, and additional
smoothing was performed using the Savitzky–Golay poly-
nomial method with a gap of five points to reduce the peak
overlap, eliminate the baseline shift and preserve the essen-
tial features of the data in the spectrum.15,31
Prior to developing any of the calibration, outlier samples in
the population were detected using the Mahalanobis distance
(H) from a principal-component analysis (PCA) of corrected
spectra. The Mahalanobis distance of each spectrum with
respect to the average spectrum was calculated.18 Samples
with an H statistics value greater than 3 units from the mean
spectrum were defined as outliers and removed when calibra-
tions were developed.15,31,32
NIR calibration development
The calibration model was developed using the regression
algorithm of modified partial least squares (mPLS),19 and a
cross-validation was performed to select the optimal number
of terms for the prediction models of each component and
avoid over-fitting.33 For cross-validation, the sample set was
divided and analysed automatically using WinISI 4.5 software.
During this procedure, 25% of the samples were randomly
assigned as validation samples, and the calibration model
was developed with the remaining 75%. This procedure was
repeated four times, and the average standard error of cross-
validation (SECV) was calculated. The optimal number is
defined as number terms beyond which there is no significant
decrease in the SECV.22,31
After this step, the total sample set was divided randomly
using the WinISI software into two parts: a subset containing
about 75% of the samples was used to construct the calibra-
tion model, and another subset (about 25% of the samples)
was used as the validation set.
 414 Green Coffee Biochemical Phenotyping

The calibration statistics included the following parameters:

standard deviation (SD), coefficient of determination (R2) and
standard error of calibration (SEC). The Student (t) test was
used to identify t-outlier samples during calibration develop-
ment in order to identify samples that could not be predicted
by the model. In this case, outlier detection was based on
the standardised residuals ( = error/SECV), and the limit for
acceptance was t ≤ 2.5.15,18
In the validation step, the following statistical parameters
were used to evaluate the models: SD, r2 and standard error
of validation (SEP), bias, slope and ratio of performance devia-
tion (RPD).13,34 This later parameter is the ratio of the SD of
the external validation set to SEP (SD/SEP), and it is valuable
when evaluating the capability of the prediction of the model.
In contrast to the SEP and SECV, the RPD is a dimensionless
parameter and aids comparisons between models. Williams
and Sobering35 suggest that values between 5 and 10 are suit-
able for estimating absolute contents, and values in the range
from 2.5 to 4.9 are satisfactory for comparison purposes and
screening in breeding programmes.

Other statistical analysis

PCA was conducted using the program XLSTAT (Addinsoft,
2008).36

Results and discussion

NIR spectral analysis
Figure 1(a) represents a typical spectrum of green coffee
beans: many overlaps in the vibrations of chemical bonds
result in large bands or poor peak resolution. Using the second
derivative and smoothing techniques solved these problems
and produced a spectrum with defined peaks [Figure 1(b)].
Then, specific chemical bonds can be linked to these peaks.
The spectrum for ground green coffee beans contained
various peaks between 1100 nm and 2500 nm. The main
absorption bands are observed at 1208 nm related to C–H
second overtone stretching. These bands are associated with
carbohydrate (e.g. sucrose) or lipids and amino acids (proteins)
and caffeine. The region near 1400 nm contains the first over-
tones of C–H and O–H that could be attributed to water and
other compounds of green coffee. The aromatic bonds can be
found at around 1500 nm, corresponding to the first overtone
of aromatic structures C–H, which is assumed to be related in
PC and chlorogenic acids (CGA). On the other hand, the bands
situated between 1700 nm and 1800 nm are linked to the first
overtone of C–H corresponding to lipids, carbohydrates and
caffeine. In the region 1800–2000 nm important bands related
to water, carboxylic acids and carboxylates or amides are
found. Next to this region (2016 nm), the 2 × O–H deforma-
tion and C–O deformation, associated with various chemical
compounds, can be found.
Signals between 2200 nm and 2300 nm are associated with
the O–H (water) bonds and C–C vibrations, and peaks above
2300 nm correspond to combinations of tones between C–H
Figure 1. Typical spectrum of green coffee in the wavelength
range of 1100–2500 nm: (a) raw spectrum (log 1/R) and
(b) second-derivative spectrum.
from CH2 of lipids. In the last region of the NIRS spectra (2300–
2480 nm) C–H combinations related mainly to carbohydrates
are also observed. Information on the functional group in the
spectrum was obtained from WinISI software and previously
published articles.26,37,38
These chemical bonds are found in several compounds of
green coffee; thus, it was possible to establish the relation
between these peaks and the concentration of the compounds
to construct a prediction model.
NIR calibration
The numbers of samples and terms of the model and statis-
tical description of samples employed in the cross-validation
step for the components of green coffee are presented in
Table 1.
The number of samples used to build the prediction model
for each compound was variable. These samples were chosen
at random within a database so as to cover broad ranges
for each component (Table 1). Different numbers of samples
were used because the quantity of components arises from
different metabolic pathways, and thus it is not usual to have
the appropriate concentration ranges of each component in
the same group of samples.
 M.B. dos Santos Scholz et al., J. Near Infrared Spectrosc. 22, 411–421 (2014) 415

Table 1. Number of samples (N), number of terms (T), mean and

range values (g 100 g–1) for the cross-validation analysis.

Constituents N T Mean Range SD

Proteins 309 10 13.81  9.62–18.34 1.87
Caffeine 361 11  1.98 0.66–1.57 0.18
Lipids 254  7 13.30 10.15–16.69 1.26
TS 355 10  8.26  5.01–10.94 1.47
Sucrose 358 12  7.96  4.72–10.58 1.37
CGA 427 10  8.39  3.92–13.71 2.85
PC 381  9  4.90  3.68–6.86 0.70

TS: total sugars. CGA: chlorogenic acids. PC: phenolic compounds.

The optimum number of terms defined in the cross-valida-

tion step ranged from seven for the lipids PLS model to 12 for
the sucrose model (Table 1).
All constituents showed a large variability of values (Table 1).
This distribution of the concentrations was sufficient for the
purposes of calibration and validation, and suggests wide
applicability for each of the developed models.39 Biotic and
abiotic stress, different conditions of harvesting and drying,
and interspecific crosses may modify the content of these
compounds in green coffee.7,20,24 For studies of coffee biochem-
ical phenotypes, samples with an extremely low or extremely
high values of chemical composition should be carefully
reviewed.40 Therefore, values outside the limits described in
the literature were included in this study to ensure wide appli-
cability in the developed models.
A summary of all parameters for the predicting models is
presented in Table 2 and Figures 2–8. The prediction models
were formed using a variable number of PLS terms (seven
and 10) that did not exceed those established in the cross-
validation (Table 1).
Proteins and caffeine
When coffee is cultivated using various agronomic practices
(fertilisation, irrigation) or grown under different climatic
Figure 2. NIR-predicted values versus laboratory measure-
ments of proteins in green coffee beans.
conditions, the contents of compounds such as caffeine and
proteins are modified. In our study, the range of caffeine and
proteins contents in the calibration models was large, covering
almost all situations that could occur in a general study of
coffee. Studies involving low caffeine values occur frequently
because obtaining cultivars with low levels of caffeine is one of
the objectives of breeding coffee.41,42
The calibration model developed for the prediction of
proteins and caffeine produced coefficients of determination
equal to 0.97 and 0.83, respectively (Table 2).
The comparison of the laboratory values and NIR predicted
contents in the group of validation samples (Figures 2 and
3) resulted in coefficients of determination of 0.94 and 0.92
for protein and caffeine, respectively (Table 3). The ranges
of different values in the groups of calibration and valida-
tion for caffeine may have caused the highest value of R2 in
the validation group of caffeine (Table 3). It is well known

Table 2. Number of samples (N), number of terms (T), mean values (g 100 g–1), range values (g 100 g–1) standard error of laboratory (SEL) and
statistical parameters for the calibration models (SD, SEC, R2).

Constituents N T Mean Range SEL SD SEC R2

Proteins 254 10 13.82  9.62–18.34 0.35 1.85 0.32 0.97
Caffeine 305 10  1.19 0.69–1.57 0.05 0.18 0.07 0.83
Lipids 195  7 13.27 10.15–16.69 0.40 1.25 0.46 0.86
TS 288 10  8.24  5.01–10.81 0.25 1.448 0.45 0.90
Sucrose 287 10  7.97  4.72–10.51 0.25 1.37 0.44 0.90
CGA 368 10  8.41  3.93–13.71 0.30 2.86 0.73 0.93
PC 315  9  4.88 3.68–6.86 0.30 0.71 0.22 0.91
TS: total sugars. CGA: chlorogenic acids. PC: phenolic compounds. SD: standard deviation. R2: coefficient of determination. SEC: standard error of
calibration­.
 416 Green Coffee Biochemical Phenotyping
Figure 3. NIR-predicted values versus laboratory measure-
ments of caffeine in green coffee beans.
that the range of values in the validation group affects the
value of the coefficient of determination.18,43 Thus, in this case,
other parameters such as SEP and RPD would be better indi-
cators of fitting of model.18 The SEP values for caffeine and
proteins are near the values of SEC, indicating good adjust-
ment models for these compounds, and they are similar to
respective standard error of laboratory (SEL) values (Table
2). Published models developed for caffeinein green Arabica
Figure 4. NIR-predicted values versus laboratory measure-
ments of CGA in green coffee beans.
Figure 5. NIR-predicted values versus laboratory measure-
ments of PC in green coffee beans.
coffee beans grown in different geographical locations found
SEC and R2 values of 0.07 and 0.88, respectively.22 On the other
hand, models for caffeine employing Brazilian coffees showed
a better performance (SEC = 0.02, R2 = 0.95) than models for
protein (SEC = 0.064, R2 = 0.69).21,23
Furthermore, the models developed in the present study for
protein and caffeine yield RPD values equal to 4.09 and 3.32,
Figure 6. NIR-predicted values versus laboratory measure-
ments of lipids in green coffee beans.
 M.B. dos Santos Scholz et al., J. Near Infrared Spectrosc. 22, 411–421 (2014) 417

Figure 7. NIR-predicted values versus laboratory measure- Figure 8. NIR-predicted values versus laboratory measure-
ments of total sugar in green coffee beans. ments of sucrose in green coffee beans.

respectively; these values were similar to those indicated for
comparison purposes.
CGA and PC
The contents of CGA and PC in the green coffee beans depend
primarily on climatic conditions and the degree of ripeness.12
The calibration models for CGA and PC showed a high preci-
sion for NIR determinations. The values of SEL and SEP for
the PC model were also very close (Tables 2 and 3). CGA model
showed SEP values slightly higher than the respective SEL
(Tables 2 and 3). The validation models of CGA and PC showed
r2 values of 0.94 and 0.86, respectively (Table 3, Figures 4 and
5). The RPD values of 4.16 and 2.65 for CGA and PC, respec-
tively, indicate that both models can be used to screen samples
for these compounds in a coffee-plant breeding programme.
Although these compounds have similar chemical structures,
the prediction model of CGA was better than the PC model. The
specificity of the chemical reaction in the laboratory determi-
nation probably contributed to a more accurate model for CGA.
Total sugars, sucrose and lipids
The contents of total sugars, sucrose and lipids in green coffee
were strongly influenced by the stage of maturity.
The calibration models for lipids (Figure 6), total sugars
(Figure 7) and sucrose (Figure 8) showed R2 values of 0.86,
0.90 and 0.90, respectively, and SEC values of 0.46, 0.45 and
0.44, respectively (Table 2). However, in the sample set used to
validate the models of TS and sucrose, the r2 values of these
compounds showed a slight decrease relative to calibration
values (Table 3).
The RPD values for validation models for the lipids, total
sugars and sucrose (2.77, 2.55 and 2.44, respectively)

Table 3. Number of samples (N), number of terms (T), mean values (g 100 g–1), range values (g 100 g–1) and statistics parameters for the
validation analysis

Constituents N SD Mean Range SEP r2 RPD

Proteins 56 1.70 13.86 9.94–16.90 0.42 0.94 4.09
Caffeine 65 0.19 1.20 0.76–1.63 0.06 0.92 3.32
Lipids 57 1.28 13.32 10.38–15.66 0.46 0.87 2.77
TS 62 1.35 8.34 5.47–10.71 0.53 0.85 2.55
Sucrose 62 1.30 8.04 5.28–10.37 0.53 0.84 2.44
CGA 55 2.97 8.42 4.28–12.89 0.72 0.94 4.16
PC 64 0.69 4.87 3.79–6.37 0.26 0.86 2.62
TS: total sugars. CGA: chlorogenic acids. PC: phenolic compounds. r 2: coefficient of determination. SEL: standard error of laboratory. SEP: standard error of
prediction. RPD: ratio of performance deviation.
 418 Green Coffee Biochemical Phenotyping

indicated  that these models can be used for comparison absorption bands relative to C–H + CH combinations (2284 and
purposes. 2356) Models for determining cereal and roasted coffee lipids
had high coefficients in a similar spectrum bands.26,48
Principal mPLS loadings for the formation of The prediction models for total sugars showed similar sets
models of the constituents of green coffee beans of bands with high coefficients to those for sucrose, because
MPLS loadings (coefficients) were analysed for each constit- sucrose is the main sugar of green coffee beans,6,7 and to
uent of green coffee beans. The first factor in the mPLS model other sugars present (glucose, fructose), as these have chem-
proteins was mainly associated with absorption bands of ical structures very similar to sucrose.
chemical bonds relevant to protein structure. High coeffi- The prediction models showed settings that allowed its
cient values are found in the region of 1212 nm for the second application in breeding coffee. The possibility of phenotyping
overtone of the C–H stretching bands from the –CH2 group, at the genotypes for these compounds in coffee will contribute to
1388 nm associated with the first overtone bands of the C–H substantial research advances in functional genomics.
combinations from CH3 and at 1452 nm owing to the first over-
tone of N–H and O–H. The coefficients with high values found Composition variability of Ethiopian
in the region between 1708 nm and 1764 nm were attributed accessions­and cultivars
to absorption bands of the first overtone of C–H. Furthermore, Biochemical phenotyping is one of the tools that can be used
absorptions at 1972 nm were related to N–H and at 1978 nm by breeders for genetic mapping (quantitative trait loci) and
to asymmetric N–H stretching and amide II, respectively. In for association studies. Therefore, chemical compounds
addition, significant coefficients attributed to the absorption related to quality beverages were determined by the predic-
arising from the structure of proteins, N–H combinations tion models developed in this study. A greater variability in
(2060 nm and 2076 nm), CH + CC combination bands (2300 nm) the biochemical composition was observed in the Ethiopian
and C–H + CC combinations bands from CH, CH2 and CH3 accessions than in traditional and modern cultivars [Figure
groups (2276 nm and 2348 nm), were found. Similar bands 9(a, b)]. This behaviour­in the traditional and modern cultivars
were associated with models for predicting protein in oilseeds can be attributed mainly to the narrow genetic base from
and press cakes of rosehips hips, hazelnut and soy.43 which these cultivars originated. In comparison, Ethiopian
Many absorption bands that contributed to the prediction accessions showed higher contents for all constituents of
model of protein (1212 nm, 1388 nm, 1708 nm, 1760 nm and coffee, with the exception of total sugars and sucrose contents
1920 nm) were also present in the model of caffeine. However, [Figure 9(b)].
in the caffeine model, the contribution of bands at 2276 nm Usually, the constituents analysed in the present study play
owing to the absorption by N–H and O–H bands combinations a fundamental role in the description and discrimination of
was noted. Models built for caffeine were formed by similar
bands.39,44
The model for prediction of chlorogenic acid showed high
coefficients in bands similar to those in models of caffeine and
protein. However, the band at 2348 nm attributed to C–H + C–C
bands from combinations CH, CH2 and CH3 made a greater
contribution to the CGA and protein models than the caffeine
model. Perhaps some chemical bonds in caffeine were
unavailable because this compound was present as a caffeine
chlorogenate complex in green coffee beans.45,46
The PC evaluated in this study were those containing
many polymerised aromatic rings and reacted with the Folin–
Ciocalteau reagent.12,29,47 The highest coefficients for the
prediction model were found at 1388 nm and 1452 nm (first
overtone of aromatic structures OH), 2060 nm and 2116 nm
(first overtone of C = O and OH bands combinations) and those
in the regions 2300–2480 nm (CH, CH + C–H, C–H + CC and
combinations). Compounds of olive oil determined with the
same reagent also showed similar bands (1332–1470 nm and
1834–2175 nm), which were used to form the model for the
prediction of these compounds.47
The highest coefficients for the prediction model of lipids
were found: at 1212 nm (second overtone of OH), 1388 nm
(2 × CH stretching) and 1740 nm (first CH stretch). High coef-
Figure 9. (a) Coefficient of variation and (b) mean values of
ficients were also associated with the first overtone of C = O chemical compounds for Ethiopian accessions and cultivars.
and OH band combinations of various fatty acids (2052 nm) and
 M.B. dos Santos Scholz et al., J. Near Infrared Spectrosc. 22, 411–421 (2014) 419
Figure 10. PCA: plot of chemical contents and coffee cultivars/
accessions in the space formed by PC1 and PC2.
coffee cultivars. PCA has been frequently used to associate
the chemical composition of coffee with the geographical
origin,9,49 physicochemical and sensory traits50 and botanical
origin.10,51–53 Here, we apply this technique to discriminate
Ethiopian accessions and traditional/modern cultivars.
PCA applied to compounds predicted through the NIR
showed that the first two PCA axes accounted for 75.22% and
15.11% of the total variability. Proteins, caffeine, lipids, total
sugars, sucrose and PC contributed the most to first principal
component. The CGA content had a similar correlation with
both principal components.
PC1 components showed a clear separation between
Ethiopian accessions and modern cultivars. The Ethiopian
accessions, to the right of the biplot (Figure 10), are associated
with high values of caffeine, lipids, CGA and PC. Total sugar
and sucrose are found in greatest concentrations in the tradi-
tional and modern cultivars. It is probable that the selection
process has given priority to lines with higher levels of sucrose
in relation to taste.
The compounds predicted with these models were effective
for characterising and separating the biochemical composi-
tion of coffee from traditional and modern cultivars, and from
Ethiopia. Therefore, it was possible to identify biochemical
features in these cultivars and accessions of interest for future
crosses.
Conclusions
This study has demonstrated the effectiveness of near infrared
spectroscopy in the biochemical characterisation of the most
important constituents of green coffee (caffeine, proteins,
lipids, total sugars, sucrose, CGA and PC). Prediction models
were proposed for these compounds and appropriately
adjusted.
Furthermore, this study also showed the capacity to
separate coffee cultivars based on the biochemical compo-
sition determined by NIR. Furthermore, the description of
biochemical characteristics and a multivariate analysis made
it possible to identify which genotypes are appropriate for
desired characteristics.
Knowledge of the concentration of the various constituents
of coffee from a single spectrum will pave the way for studies
that are currently limited by human and financial resources.
Our study demonstrated how the NIR technique can be used
to improve biochemical phenotyping in breeding programmes.
Acknowledgements
Thanks to Capes (Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior-Brazil), to Agropolis Foundation
France for their support to the joint Cirad–UEL–IAPAR Project
No. 1002-02 and to Foss Company (now Metrohm NIRSystem)
for 3 months of the WINISI 4.5 software licence.
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