molecules

Article
Chemical and Biological Characterization of Green and
Processed Coffee Beans from Coffea arabica Varieties
Javier Gallardo-Ignacio 1 , Anislada Santibáñez 2 , Octavio Oropeza-Mariano 3 , Ricardo Salazar 4 ,
Rosa Mariana Montiel-Ruiz 2 , Sandra Cabrera-Hilerio 5 , Manasés Gonzáles-Cortazar 2 ,
Francisco Cruz-Sosa 1, * and Pilar Nicasio-Torres 2, *

1 Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa,

Av. Ferrocarril de San Rafael Atlixco No. 186, Col. Leyes de Reforma 1ª Sección, Iztapalapa,
Mexico City 09310, Mexico; gajarig-07mx@hotmail.com
2 Centro de Investigación Biomédica del Sur, Instituto Mexicano del Seguro Social (CIBIS-IMSS),
Argentina No. 1 Col Centro, Xochitepec 62790, Mexico; anisszg@gmail.com (A.S.);
montielrmariana@gmail.com (R.M.M.-R.); gmanases@hotmail.com (M.G.-C.)
3 Cafeticultores Mephaa de la Montaña, Paraje Montero, Malinaltepec 41500, Mexico; ooropezam05@gmail.com
4 Consejo Nacional de Humanidades, Ciencia y Tecnología (CONAHCyT), CONACYT, Laboratorio de
Bromatología y Tecnología de Alimentos Universidad Autónoma de Guerrero, Av. Lázaro Cárdenas S/N,
Chilpancingo de los Bravo 39086, Mexico; rsalazarlo@conacyt.mx
5 Laboratorio de Bromatología, Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla,
Av. San Claudio S/N Ciudad Universitaria, Puebla 72000, Mexico; sandra.cabrera@correo.buap.mx
* Correspondence: cuhp@xanum.uam.mx (F.C.-S.); pisaliva@yahoo.com.mx (P.N.-T.)

Abstract: Coffee is one of the most consumed beverages in the world; its production is based
mainly on varieties of the Coffea arabica species. Mexico stands out for its specialty and organic
coffee. In Guerrero, the production is done by small indigenous community cooperatives that
Citation: Gallardo-Ignacio, J.; market their product as raw material. Official Mexico Standards stipulate the requirements for its
Santibáñez, A.; Oropeza-Mariano, O.; commercialization within the national territory. In this work, the physical, chemical, and biological
Salazar, R.; Montiel-Ruiz, R.M.; characterizations of green, medium, and dark roasted beans from C. arabica varieties were carried out.
Cabrera-Hilerio, S.; Gonzáles-Cortazar, Analysis by HPLC showed higher chlorogenic acid (55 mg/g) and caffeine (1.8 mg/g) contents in
M.; Cruz-Sosa, F.; Nicasio-Torres, P.
the green beans of the Bourbon and Oro Azteca varieties. The caffeine (3.88 mg/g) and melanoidin
Chemical and Biological
(97 and 29 mg/g) contents increased according to the level of roasting; a dissimilar effect was found
Characterization of Green and
in the chlorogenic acid content (14.5 mg/g). The adequate nutritional content and the sensory
Processed Coffee Beans from Coffea
evaluation allowed the classification of dark-roasted coffee as premium coffee (84.25 points) and
arabica Varieties. Molecules 2023, 28,
4685. https://doi.org/10.3390/
medium-roasted coffee as specialty coffee (86.25 points). The roasted coffees presented antioxidant
molecules28124685 activity without cytotoxic effects; the presence of CGA and caffeine supports the beneficial effects of
drinking coffee. The results obtained will serve as a basis for making decisions on improvements to
Academic Editors: Patricia Morales,
the coffees analyzed.
Virginia Fernández-Ruiz and
Maria Ciudad-Mulero
Keywords: antioxidant; C. arabica beans; chlorogenic acid; caffeine; cytotoxicity; melanoidins
Received: 12 May 2023
Revised: 6 June 2023
Accepted: 8 June 2023
Published: 10 June 2023 1. Introduction
Coffee commercialization is mainly based on Coffea arabica, which accounts for 70% of
global production. The cultivation of C. arabica occurs mainly in Latin American countries
Copyright: © 2023 by the authors.
such as Colombia, Honduras, Peru, and Mexico, with a production of 13.8, 5.6, 4.45, and
Licensee MDPI, Basel, Switzerland. 3.7 million bags of 60 kg, respectively [1]. Peru and Mexico are recognized for their organic
This article is an open access article and high-altitude coffee production. In Mexico, 3.33% of total coffee production is organic,
distributed under the terms and and 28,000 t are principally exported to the European Union [2].
conditions of the Creative Commons In Mexico, there are more than 600 cooperatives dedicated to coffee cultivation whose
Attribution (CC BY) license (https:// producers have obtained certifications such as USDA Organic, Fair Trade, Shade Grown,
creativecommons.org/licenses/by/ Rainforest Alliance, and Small Producer to achieve new markets and offer their products [3].
4.0/). Mexican coffee is cultivated in four main regions: the Gulf of Mexico, the Soconusco, the

Molecules 2023, 28, 4685. https://doi.org/10.3390/molecules28124685 https://www.mdpi.com/journal/molecules

Molecules 2023, 28, 4685 2 of 17

north-central part of Chiapas, and the Pacific Ocean slope; the latter includes Colima,
Guerrero, Jalisco, Nayarit, and Oaxaca [4]. Chiapas State produces 41% of Mexican coffee.
Of the total coffee production, 70% is exported to the United States, European Union, Japan,
Cuba, and Canada as green coffee (85%), soluble coffee (12%), and roasted coffee (3%); the
rest (30%) is for national sale [5].
In Guerrero, C. arabica varieties are cultivated in Costa Grande, Costa Chica, and the
Región de la Montaña. The coffee cultivated in the “Región de la Montaña” is characterized
by growing under shade, also known as “benefit”, and it is cultivated traditionally by
people from the Mixtec and Tlapaneco ethnic groups, grouped in cooperatives [6]. The
coffee produced by these cooperatives is sold to intermediary companies such as Asociación
Rural de Interés Colectivo de R.L. (ARIC), CAFECO Agroindustrial del Pacífico S.A. de
C.V., and the Unión of Ejidos y Comunidades Luz de La Montaña, A.C. [7]. More than 60%
of the production is marketed as green coffee to Nestlé S.A. Company; a small amount is
exported to Europe by the Network of Sustainable Self-Managed Farmers S.C (RASA); and
the remaining ≈30% is destined for coffee shops highlighting Starbucks as the main buyer,
self-service, and convenience stores (Oxxo, 7 Eleven, among others), as well as for local
consumption [8].
Some producers have organized themselves to give added value to their coffee by
looking for methods that improve the quality of their products and marketing; one of them
is the Cooperative Cafeticultores Mephaa de La Montaña. This cooperative currently pro-
duces small batches of commercial and specialty coffee from mixtures of C. arabica varieties,
Typica, Bourbon, and Oro Azteca, which are marketed at regional and national levels. The
cooperative sells green and roasted coffee to coffee shops and roasters in the country (70%)
such as Buzz Café, Bombilla Errante, Sonata tostadores, and Comercializadora Golmex de
México S.A de C.V.; the rest is locally sold as roasted and ground coffee.
In Mexico, there are Official Mexico Norms such as NMX-F-013-SCFI-2010 and PROY-
NOM-255-SE-2021 that establish physical, chemical, and nutritional specifications to market
roasted and ground coffee within the national market. The Cooperative Cafeticultores
Mephaa de “La Montaña” coffee production is carried out on a small scale and organically.
To expand its market, it is necessary to carry out studies that contribute to improving yields
and cost reduction [9,10].
The main compounds related to the cup quality of the coffee drink, which gives
it astringency and flavor, are chlorogenic acid (CGA), caffeine, caffeic acid, ferulic acid,
vanillic acid, cinnamic acid, trigonelline, and volatile compounds such as furans, pyridines,
pyrazines, and pyrroles [11,12].
The major compounds present in the coffee are CGA, caffeic, ferulic, catechin, epicate-
chin, and anthocyanin phenolic compounds; caffeine and trigonelline alkaloid compounds;
and those that form during roasting, such as melanoidins and acrylamide (Figure 1) [11,12].
The Official Mexican Norms specify the caffeine and acrylamide levels required to market
roasted and ground coffee. Furthermore, these compounds have important biological
activities such as antioxidants, caffeine as a neurostimulator, CGA as an anti-inflammatory,
and glucose and lipid metabolism [13–15].
The Cooperative Cafeticultores Mephaa de “La Montaña” has implemented a physical
analysis of green beans as a first step in improving their commercial coffees. To address
this point, in the present study, it was proposed to determine the nutritional and chemical
specifications of green, medium, and dark roasted coffee beans that are currently marketed
in addition to the antioxidant effect, as well as the cup quality of coffee drinks obtained
from these commercial coffees.
Molecules 2023,
Molecules 28,28,
2023, x FOR
4685 PEER REVIEW 3 17
3 of of 17

Figure
Figure1.1.Chemical
Chemicalstructures
structures of main compounds
of main compoundsidentified
identifiedininthe
thebeans
beansofof Coffea
Coffea arabica:
arabica: (a)(a) 3-O-
3-O-
caffeoylquinic
caffeoylquinic acid (CGA); (b) Caffeine; (c) Epicatechin; (d) Catechin; (e) Ferulic acid; (f) Trigonelline; (f)
acid (CGA); (b) Caffeine; (c) Epicatechin; (d) Catechin; (e) Ferulic acid;
Trigonelline; (g) Acrylamide.
(g) Acrylamide.

2. Results and Discussion

The Cooperative Cafeticultores Mephaa de “La Montaña” has implemented a
physicalTheanalysis of green
organic coffee beans
of the Stateasofa Guerrero
first stepisinknown
improving
for its their commercial
natural dry process, coffees.
which To
address
preservesthis
thepoint,
pulp. in
Inthe presentdestudy,
La Región it was in
la Montaña, proposed
the State to
of determine the nutritional
Guerrero, coffee is producedand
by people
chemical of the Mixtecofand
specifications Tlapaneca
green, medium, ethnic
andgroups, whose coffee
dark roasted crops are mainly
beans that the
are Typica,
currently
Bourbon,in
marketed Caturra,
addition and
to Mundo Novo varieties
the antioxidant effect, as C. arabica,
of well as the although hybrid
cup quality varieties
of coffee drinks
are being promoted such as
obtained from these commercial coffees.Colombia, Costa Rica 95, Oro Azteca, and Sarchimor. The
Cooperative Cafeticultores Mephaa de “La Montaña” cut the ripe cherries and processed
2.them in an
Results andartisanal way; cherries are dehydrated under the sun until they obtain dry
Discussion
fruits with a humidity of 11–12%, and the shell is removed to obtain the green coffee beans
(GC)The organic
[13]. coffee
The green of the
beans areState of Guerrero
principally is known
marketed for its
on a small natural
scale dry process,
as mixtures which
of Typica,
preserves
Bourbon, andthe Oro
pulp. In Lavarieties,
Azteca Regiónbased
de laonMontaña,
their colorinand
thesize.
State of Guerrero, coffee is
produced by people of the Mixtec and Tlapaneca ethnic groups,
The next step is to determine the nutritional and chemical analyses whose crops are mainly
of green and
the Typica, Bourbon, Caturra, and Mundo Novo varieties of C. arabica,
ground roasted beans that are currently marketed, in addition to the antioxidant effect, although hybrid
as
varieties
well as thearecup
being promoted
quality such
of coffee as Colombia, Costa Rica 95, Oro Azteca, and Sarchimor.
drinks.
The Cooperative Cafeticultores Mephaa de “La Montaña” cut the ripe cherries and
2.1. Nutritional
processed themComposition
in an artisanal way; cherries are dehydrated under the sun until they
obtainThe
drybromatological analysis ofofgreen
fruits with a humidity 11–12%,and and
processed beans
the shell in coffee allows
is removed for the
to obtain knowl-
green
edge of
coffee the quality
beans and nutritional
(GC) [13]. The green composition of commercial
beans are principally coffees.on
marketed Thea protein and as
small scale
carbohydrate
mixtures content
of Typica, were similar
Bourbon, andbetween
Oro Aztecatoasted beans and
varieties, theon
based mixture (GCM)and
their color of Typica,
size.
Bourbon, and Oro Azteca (Table 1); the fat content was higher in the dark
The next step is to determine the nutritional and chemical analyses of green roast coffee (DCR). and
The higher
ground humidity
roasted beanscontent
that arewas found in
currently GCM, which
marketed, in decreasestoasthe
addition theantioxidant
roasting tempera-
effect, as
ture is increased (MRC 180 ◦ C and DCR 210 ◦ C), while the ashes generated are similar in
well as the cup quality of coffee drinks.
GCM and DRC samples. The humidity (<6.0%) and ash (<6.5%) contents in the medium
roasting coffee (MRC) and DCR commercial coffee beans are within the range established
2.1. Nutritional Composition
by the Official Mexican Norm for roasted and ground coffee, NMX-F-013-SCFI-2010. The
The bromatological
humidity of green beans isanalysis
inferior of green
to the rangeand processed
specified beans the
to prevent in growth
coffee of allows
fungifor
knowledge
and bacteriaof(10–12%)
the quality
andand nutritional
preserve composition
physical propertiesof
andcommercial
nutritionalcoffees.
content.The protein
Instead,
and
onlycarbohydrate content
the fat content of DCRwere similar
beans as anbetween toasted(8–18%)
etheric extract beans and the mixture
achieves (GCM) of
the parameter
stipulated
Typica, by the norm.
Bourbon, TheAzteca
and Oro roasted(Table
coffee 1);
beans
thedarken, and the
fat content released
was higheroils givedark
in the themroast
a
shiny appearance.
coffee (DCR). The higher humidity content was found in GCM, which decreases as the
roasting temperature is increased (MRC 180 °C and DCR 210 °C), while the ashes
generated are similar in GCM and DRC samples. The humidity (<6.0%) and ash (<6.5%)
contents in the medium roasting coffee (MRC) and DCR commercial coffee beans are
within the range established by the Official Mexican Norm for roasted and ground coffee,
 NMX-F-013-SCFI-2010. The humidity of green beans is inferior to the range specified to
prevent the growth of fungi and bacteria (10–12%) and preserve physical properties and
nutritional content. Instead, only the fat content of DCR beans as an etheric extract (8–
Molecules 2023, 28, 4685 18%) achieves the parameter stipulated by the norm. The roasted coffee beans darken, and4 of 17
the released oils give them a shiny appearance.

Table
Table1.1.Nutritional
Nutritionalcomposition
compositionofof
commercial coffee
commercial with
coffee Coffea
with arabica
Coffea varieties
arabica in the
varieties mixture.
in the mixture.
Content in Percentage (%)
Coffee Content in Percentage (%)
Coffee Humidity Ash Fats Proteins Carbohydrates
Humidity Ash Fats Proteins Carbohydrates
GCM 8.48 ± 0.13 ** 4.54 ± 0.06 ** 5.09 ± 0.89 12.34 ± 0.29 69.56 ± 1.06
GCM
MRC 8.48 ±
4.23 0.13 **
± 0.14 * 3.84 ±
4.54 0.06 ** 6.48
± 0.11 5.09 ± 0.89
± 0.34 12.34
* 13.04 ± 0.29 72.41
± 0.28 69.56 ± 1.06
± 1.26
MRC 4.23 ± 0.14 * 3.84 ± 0.11 6.48 ± 0.34 * 13.04 ± 0.28 72.41 ± 1.26
DRC
DRC
3.59 ± 0.12 4.44
3.59 ± 0.12
± 0.08 ** 8.15 ± 0.63 ** 13.01
4.44 ± 0.08 ** 8.15 ± 0.63 **
± 0.38
13.01 ± 0.38
70.81 ± 1.11
70.81 ± 1.11
Values are mean ± standard deviation (n = 3). According to the ANOVA and Tukey’s test, the means
Values are mean ± standard deviation (n = 3). According to the ANOVA and Tukey’s test, the means with
with * and
* and ** were ** were significantly
significantly different.
different. Humidity
Humidity F = 1251.75
F = 1251.75 p ≤ p0.0001,
≤ 0.0001,
TukeyTukey 0.05 = 0.33; Ash F =
0.05 = 0.33; Ash F = 62.89
62.89 p ≤ 0.0001,
p ≤ 0.0001, TukeyTukey 0.05 =Fats
0.05 = 0.20; 0.20;
F =Fats F =p 16.26,
16.26, p ≤Tukey
≤ 0.0001, 0.0001,
0.05Tukey
= 1.65;0.05 = 1.65;F Proteins
Proteins = 4.56 p >F0.05,
= 4.56 p >0.05
Tukey 0.05,
= 0.80;
Carbohydrates
Tukey = 7.55 p ≤ 0.0001;
0.05 = 0.80;FCarbohydrates F Tukey p ≤=0.0001;
= 7.550.05 2.25. Tukey0.05 = 2.25.

InInthe
theC.C.arabica
arabicabeans
beanswith
withlight (176
light (176 ◦ C),
°C), medium
medium(204 ◦ C),
°C),
(204 and dark
and (232
dark °C)◦roasts,
(232 C) roasts,
the
thehumidity
humiditycontent
contentwas
wasreduced
reduced with
withthethe
temperature
temperatureincrease, while
increase, protein
while (16%)
protein andand
(16%)
fat
fat(16.2%)
(16.2%)contents
contents increased
increased in in
thethe
dark
darkroast. Ashes
roast. and and
Ashes sugar contents
sugar werewere
contents similar (2
similar
(2 ◦ Brix)
°Brix) in the
in three roasts
the three [16].[16].
roasts

2.2.Chemical
2.2. ChemicalAnalysis
Analysis
The High-Performance
The High-Performance Liquid Liquid Chromatography
Chromatography (HPLC) (HPLC)analysis
analysisshowed
showed that com-
that
pounds of greater
compounds predominance
of greater predominance identified in the
identified infusions
in the of greens
infusions andand
of greens processed beans
processed
were were
beans CGACGA(λ = 330
(λ = nm) and and
330 nm) caffeine (λ =(λ
caffeine 280 nm),
= 280 with
nm), a retention
with time
a retention ofof
time 8.51 min
8.51 minand
8.888.88
and min, respectively
min, respectively (Figure
(Figure2).
2).Minority compoundsaround
Minority compounds aroundCGA CGAand and caffeine
caffeine were
were
detected at these long waves; one of them was caffeic acid with a retention time of 8.3 min. of
detected at these long waves; one of them was caffeic acid with a retention time
8.3 min.
These These compounds
compounds are relatedare to
related to the
the cup cup quality
quality of theofcoffee
the coffee drink,
drink, which
which gives
gives it it
astringencyand
astringency andflavor
flavor[15,17].
[15,17].

Figure 2. Chromatograms of HPLC at λ = 330 nm of (a) CGA standard (25 µg/mL) and infusions
of GCM (125 µg/mL), MCR, and DCR (500 µg/mL); (b) at λ = 280 nm of caffeine (CAF) standard
(10 µg/mL) and infusions of GCM (125 µg/mL), MCR, and DCR (500 µg/mL).

The 1 H and 13 C nuclear magnetic resonance (NMR) analysis of the compound isolated
from the MRC infusion validated that it corresponds to CGA; chemical displacements
Molecules 2023, 28, 4685 5 of 17

and coupling constants (Table 2) correspond to those reported in the literature for this
compound [18].

Table 2. 1 H (400 MHz) and 13 C NMR (100 MHz) Spectroscopic Data of Chlorogenic Acid (MeOH-d4 ,
δ, ppm, J/Hz).

Data of [18]
C Atom δ 1 H-Experimental δ 13 C-Experimental δ 1H δ 13 C
1 70.24 71.06
2a 2.09 (m)
39.44 2.21 (m) 37.65
b 1.92 (dd,12.1, 12.4 Hz)
3 5.23 (ddd, 5.1,5.5, 10.2 Hz) 71.29 5.17 71.06
4 3.62 (d, br, 10.1 Hz) 72.97 4.89 68.48
5 4.12 (s, br) 71.91 4.77 73.90
6a 1.98 (m)
37.21 1.84 36.66
b 1.98 (m)
10 126.03 126.05
20 7.10 (d,1.4 Hz) 115.06 7.00 114.99
30 146.07 148.80
40 148.80 145.71
50 6.82 (d,8.1 Hz) 116.25 6.98 116.20
60 7.03 (dd,1.5, 8.1 Hz) 121.68 7.00 114.99
70 7.52 (d,15.8 Hz) 145.18 7.42 145.71
80 6.30 (d,15.9 Hz) 115.20 6.15 114.99
90 166.74 166.18
COOH 175.88 175.38

Among the green beans of C. arabica varieties analyzed (Table 3), the Typica variety has
the lowest contents of CGA (36.81 mg/g) and caffeine (1.16 mg/g). The GCM is composed
of a greater proportion of the beans of the Typica species (Typica-Bourbon-Oro Azteca,
40–30–30%), and the content of both compounds in the GCM is close to those detected in
this variety. It has been reported that in C. arabica green coffee, the CGA content ranges
between 52 and 76 mg/g [19]. The CGA content in the Bourbon and Oro Azteca varieties is
within this range.

Table 3. Contents of CGA, caffeine, and melanoidins in green and processed beans of Coffea arabica
varieties.

Coffee Beans CGA Caffeine Melanoidins

Unclarified Clarified
mg/g Coffee Kmix Lg−1 cm−1
GCM 30.81 ± 2.22 0.87± 0.09 15.41 ± 1.15 2.04 ± 0.88 0.07
Bourbon-GC 55.75 ± 2.31 ** 1.78 ± 0.12 ** - - -
Oro Azteca-GC 54.63 ± 2.43 ** 1.77 ± 0.15 ** - - -
Typica-GC 36.81 ± 0.10 1.16 ± 0.18 - - -
MRC 30.26 ± 0.45 ** 2.52 ± 0.17 * 85.51 ± 5.99 * 18.95 ± 1.9 ** 1.586
DRC 14.52 ± 0.65 3.88 ± 0.23 ** 96.79 ± 3.44 ** 29.06 ± 7.7 ** 1.614
Values are mean ± standard deviation (n = 9). According to the ANOVA and Tukey’s test, the means with
** were significantly different. CGA F = 328.13 p ≤ 0.0001, Tukey0.05 = 2.66; caffeine F = 101.83 p ≤ 0.0001,
Tukey0.05 = 0.17 in mg/g of coffee in green beans; CGA F = 415.72 p ≤ 0.0001, Tukey0.05 = 1.60; caffeine F = 678.07
p ≤ 0.0001, Tukey0.05 = 0.20 in mg/g of coffee in processed beans. For melanoidins, values are mean ± standard
deviation (n = 3). According to the ANOVA and Tukey’s test, the means with * and ** were significantly different.
Unclarified melanoidins F = 1423.09 p ≤ 0.0001, Tukey0.05 = 4.055 in mg/g of coffee, and clarified melanoidins
F = 26.45 p ≤ 0.0001, Tukey0.05 = 11.516 in mg/g of coffee.

When the green beans are subjected to the roasting process, the CGA content is reduced
by the effects of temperature and time exposure. CGA is hydrolyzed into the molecules of
Molecules 2023, 28, 4685 6 of 17

simple phenols that compose it, caffeic acid and quinic acid [17]. In the GCM and MRC
beans, the CGA content was similar, while in the DRC beans roasted at 210 ◦ C, the CGA
content was reduced by 53% in comparison to the unroasted beans (Table 3).
Studies carried out at different levels of roasting show that the content of CGA in the
beans is correlated with the temperature and the time of roasting, finding greater content
in a light roast (186.5 ◦ C, 7:15 min) of 11.24 mg/g of coffee; when the roasting time is
prolonged to a dark roast (186.5 ◦ C, 14:02 min), the content is reduced by 70% [20].
Similarly, the Typica variety has a low content of caffeine (1.16 mg/g) compared with
the other two varieties; therefore, the GCM presents a low level (0.87 mg/g) of caffeine
(Table 3). The caffeine content determined in green beans increased in the roasted beans as
the level of roasting increased (Table 3). Darkly roasted beans presented a higher caffeine
content; when the green beans are roasted, they lose moisture, increase in size, and become
porous, allowing a better caffeine extraction [21]. Nevertheless, this content is minor
compared with that established for the commercialization of roasted coffee; according
to the Mexican Norm (NMX-F-013-SCFI-2010), the acceptable caffeine content range is
between 10 and 20 mg/g for roasted coffee.
The CGA and caffeine content are related to the variety of coffee, growing conditions,
and degree of ripeness of the fruits. In a study conducted on coffees from Veracruz, Nayarit,
Oaxaca, and Chiapas states of Mexico, it was found that the level of caffeine ranges between
2.9 and 7.0 mg/g of roasted coffee [22]. The caffeine content determined in the processed
coffee from Guerrero State (Región de la Montaña) and reported in this study is between
these ranges.
In Typica variety coffee beans from plants grown in two different places, the caffeine
content in light roasting was 4.19 mg/g and 5.01 mg/g; when the level of roasting was
increased to a dark level, the content presented an increase (5.18 mg/g and 6.12 mg/g),
a similar effect that was reflected in this study [21]. Other studies on C. arabica have not
reported variations in its content at different levels of roasting (11.9–13 mg/g), even using
temperatures between 194 and 217 ◦ C, showing a thermostable behavior of caffeine [23].
In other studies, it has been reported that in C. arabica beans from Brazil, the highest
content of caffeine was present in light (6.42 mg/g) and medium (5.77 mg/g) roasts,
compared with dark roast (2.63 mg/g). A similar effect was reported in the Typica and
Bourbon varieties of coffee beans; the highest content was presented in light roasting
(14.59 mg/g), followed by medium and dark (5.57 mg/g). The caffeine content in green
beans of Sidama (16.4 mg/g), Yirgacheffe (15.72 mg/g), and Harar (15.03 mg/g) varieties
was reduced to 7.96, 8.87, and 4.52 mg/g, respectively, after the beans were subjected to a
dark roasting process [20,24].

2.3. Melanoidins
During roasting practice, the phenolic compounds can be degraded by Maillard and
Strecker reactions, or they can be followed by the formation of new compounds such as
melanoidins, acrylamides, and hydroxymethylfurfural [25,26]. Melanoidins give the beans
a brown pigment, flavor, and color, and they are associated with antioxidant activity that
is enhanced by simple phenolic compounds such as caffeic, ferulic, and chlorogenic acids
binding to their structure [17].
In this study, two procedures were performed to determine the melanoidin content of
the coffee infusions. In unclarified solutions, the melanoidins are present in the GCM, and
the content more than doubled after beans were roasted in the DRC (~2.87 fold). The coffee
beans presented a dark brown coloration (Table 2). Carrez I and II solutions are used to
precipitate proteins and remove turbidity and micelles, reducing interference at the time of
reading (λ = 420 nm). In the clarified samples, the melanoidin content was lower compared
with those not clarified, but with similar behavior in their content; it was related to the
increase in the level of roasting. The samples subjected to the clarification process lost color
and presented the formation of a precipitate, which may be influencing the elimination of
compounds of high molecular weight. The contents of melanoidins determined are lower
Molecules 2023, 28, 4685 7 of 17

than those reported in the literature (200–250 mg/g) [27,28]. Other authors report that the
highest melanoidin content was determined in soluble coffee obtained from C. canephora
(676 mg/g) and C. arabica varieties (peaberry, known as caracolillo in Spanish, 305 mg/g)
roasted with sugar [28].

2.4. Sensory Evaluation

An important evaluation for commercial coffees is the cup quality of coffee drinks; this
value depends on the physical and organoleptic properties of the beverage, such as flavor,
aroma, acidity, body, and balance. These properties are associated with the content of their
chemical compounds (Figure 1), mainly CGA, caffeine, caffeic acid, ferulic acid, vanillic acid,
and cinnamic acid, among others [17]. In this work, CGA and caffeine contents were analyzed.
The GCM infusions had a pH of 5.6, the MRC infusions had a pH of 4.74, and the
DRC infusions had a pH of 6.15. The MRC infusions had a higher acidity correlated with
the CGA content (Table 3), as reported [16]. The sensory attributes established by the
Mexican Norm PROY-NOM-255-SE-2021 in fragrance/aroma, flavor, acidity, balance, and
taster score must present a score ≥ 8.0; in residual flavor and body, a score ≥ 7.5; and
in uniformity, cup cleanliness, and sweetness, a score of 10. Natural coffees or honeyed
coffees of specialty are those that present a total rating of 85 to 87.75 points according to the
Mexican Norm.
The sensory analysis of the infusions prepared from MRC achieved a score of 86.25,
and it was considered a natural specialty coffee. The points obtained in each parameter
are within the values considered by the Norm (Table 4). In natural coffee or dry coffee, it
is common to obtain characteristic fruity aromas and flavors due to the preservation of
the peel and pulp of the fruit [29], such as those determined in this study, which present
aromas of tropical fruits and flavors of white wine, grape, and honey.

Table 4. Sensory profile of commercial coffee blend of Coffea arabica varieties.

Coffee Beans MRC DRC

Aroma 8.00 ± 0.16 * 7.75 ± 0.20
Taste 7.75 ± 0.29 7.75 ± 0.29
Aftertaste 8.00 ± 0.20 8.00 ± 0.20
Acidity 8.00 ± 0.61 8.00 ± 0.13
Body 8.25 ± 0.20 * 8.00 ± 0.13
Balance 8.00 ± 0.29 * 7.25 ± 0.29
Uniformity 10 ± 0 10 ± 0
Clean cup 10 ± 0 10 ± 0
Sweetness 10 ± 0 10 ± 0
Taster score 8.25 ± 0.29 * 7.5 ± 0.41
Total Score 86.25 84.25
Values are mean ± standard deviation (n = 4). According to the Student’s t-test (p ≤ 0.05), the means with * were
significantly different.

The score of DRC coffee (84.25) was lower than that required to be considered a natural
specialty coffee, but it is within the natural coffees named premium (80 to 84.75 points)
according to the Norm, and attributes such as aroma, flavor, body, and balance presented
the lowest score. In this drink, a floral and fruity aroma with date, grape, and red apple
flavors was detected, along with tartaric citrus acidity, a juicy-silky body, and medium-high
sweetness. The sensory analysis performed on the MRC beans is within the considered
range of specialty coffees; a lower flavor was reported (7.75) and a greater body (8.25); in
the case of DRC, beans showed a lower aroma (7.75), a lower balance (7.25), as well as a
smaller body than in MRC beans.
In roasted coffee, it is very common to find mixtures of C. arabica varieties identifying
different organoleptic characteristics influenced by the method to which the cherries are
subjected to obtain the green beans. Other aspects that must be considered are the altitude at
which the crops are located and their degree of maturity. The commercial coffees analyzed in
this study were carefully collected by hand, considering the uniformity of ripe cherries, from
Molecules 2023, 28, 4685 8 of 17

C. arabica plant varieties grown from 1900 m.a.s.l. under the shade of other native trees of the
place; furthermore, during drying, cherries were placed on beds to avoid contact with the soil
and the growth of contaminating microorganisms. In addition, selected and sorted grains are
stored in moisture-controlled spaces to preserve the quality of the green beans.
C. arabica beans from Brazil processed as natural coffee (87.8 points) presented better
attributes in aroma, flavor, acidity, and body than honeyed coffee (83.8 points), possibly
due to fermentation carried out by the microorganisms present in the fruit [30]. Likewise,
natural coffee (dry, 80), wet (85), and semi-dry (86) with the fermentation of C. arabica
variety Colombia presented similar attributes such as medium fruity body, medium fresh
acidity, and chocolate and caramel flavors. Sensory attributes are related to geographic
conditions, climate, altitude, and crop field practices [31].

2.5. Biological Activities

2.5.1. Antioxidant Effect
The coffee beverage has antioxidant activity related to the content of chemical com-
pounds such as caffeine, CGA, and melanoidins [17]. The antioxidant activity of processed
coffee infusions was determined by the equivalents of Trolox and CGA related to the
inhibitory effect of the radicals DPPH, ABTS, and FRAP (Table 5). In the tests carried out,
the MRC extract showed a greater capacity for radical scavenging compared with the DRC
extract. The CGA content in the infusion of MRC was related to the greater inhibitory
activity, likely due to the low degradation of phenolic compounds involved in antioxidant
processes. Furthermore, DRC presented a higher content of melanoidin, although its activ-
ity was lower than that presented by MRC. Finally, in the case of caffeine, it acts indirectly
in the antioxidant process by increasing levels of glutathione [32]. Other molecules related
to the main effect of coffee beverages are ferulic acid, caffeic acid, vanillic acid, guaiacol,
epicatechin, catechin, and anthocyanins, which can unravel various mechanisms for the
elimination or inhibition of free radicals [11,12,33].

Table 5. Antioxidant activity of commercial coffee with Coffea arabica varieties in the mixture.

DPPH ABTS FRAP

Sample
eq CGA eq Trolox eq CGA eq Trolox eq CGA eq Trolox eq FeSO4
MRC 1.60 ± 0.27 * 52.74 ± 4.84 * 16.09 ± 0.33 * 14.39 ± 1.16 16.22 ± 1.04 * 14.59 ± 2.35 * 54.68 ± 1.46 *
DRC 1.12 ± 0.37 42.52 ± 1.91 12.49 ± 0.46 12.15 ± 0.49 8.82 ± 0.94 6.38 ± 1.40 33.30 ± 0.63
Values are mean ± standard deviation (n = 3). According to the Student’s t-test (p ≤ 0.05), the means with * were
significantly different.

The roasted beans of Colombia, Typica, and Bourbon varieties presented a similar
effect to that reported in this study; the greatest antioxidant activity was found in the light
and medium roasts through the tests carried out with DPPH and ABTS [20,31]. The high
content of phenolic compounds provides greater radical inhibitory activity in beans with
light roasting of the Cataui variety [34].
The concentration needed to inhibit 50% of DPPH and ABTS radicals depends on the
content of phenolic compounds present, such as CGA. According to the tests performed, the
MRC extract (higher CGA content, Table 3) presents a greater antioxidant effect (Table 5),
and a lower concentration is required to achieve IC50 compared with DRC (Table 6). In
C. arabica, the content of CGA was higher in green beans than in roasted beans, so less
content of the green extract was required to achieve IC50 using the DPPH test [35].
Molecules 2023, 28, 4685 9 of 17

Table 6. Inhibitory concentration (IC50 ) of commercial coffee with Coffea arabica mixture varieties.

IC50
Assay MRC DRC MRC DRC CGA Trolox
mg/mL Extract µg/mL CGA Content µg/mL Standard
DPPH 2.22 ± 0.08 2.59 ± 0.05 * 56.92 ± 1.90 66.20 ± 1.46 * 28.18 ± 0.83 91.88 ± 3.75 *
ABTS 0.38 ± 0.02 0.49 ± 0.02 * 9.69 ± 0.35 12.67 ± 0.44 * 6.51 ± 0.16 * 6.29 ± 0.03
Values are mean ± standard deviation (n = 3). According to the Student’s t-test (p ≤ 0.05), the means with * were
significantly different.

2.5.2. Cytotoxic Activity

Coffee, being a food product with high demand around the world, must be free of
harmful chemical compounds and biological agents. It promotes the formation of toxic
compounds in coffee beans when exposed to high temperatures, such as acrylamide and
hydroxymethylfurfural, which are considered carcinogenic or genotoxic [26,36]. Based
on the IC50 determined for DRC (216.26 ± 27.7 µg/mL) and MRC (234.63 ± 29.6 µg/mL)
infusions on the growth of the 3T3-L1 fibroblast cell line at 48 h of exposition, it was
determined that the infusions did not present cytotoxic effects. A plant extract has been
determined to be cytotoxic when it presents IC50 values <100 µg/mL [37]. Studies on
coffee extracts have reported a cytotoxic effect on prostate cancer cell lines DU145 and
PC3, without showing toxic effects in macrophage cell lines (RAW 2647), hepatocytes
(AML-12), or normal CCD-18Co fibroblast cells [34]. The commercial samples (MRC
and DRC) analyzed did not show toxic effects, likely due to the low content of harmful
compounds in these coffees.
The acrylamide content is regulated by each country; in the European Union, the
permissible limit for acrylamide content is 400 µg/kg for roasted coffee and 800 µg/kg for
soluble coffee. Studies reported variable amounts of acrylamide, but it has been shown that
in dark coffees or with prolonged levels of roasting, the acrylamide content may be higher
than in medium or light coffees [25,38].
Several studies have shown that the content of chemical compounds is influenced by
the processing method of beans and infusion preparation [39]. In the infusion preparation,
it is essential to consider the pressure, temperature, and contact time of the beans with
the water; within the populations, the amount of coffee used can vary; for example, for
filtered coffees in Europe, the United States, and Canada, 7 g per 100 mL of water is usually
used, in Brazil 10 g, and in Italy 20 g [40]. In Mexico, it is common for coffee preparation to
use one or two tablespoons per coffee cup for Turkish coffee or drip coffee. In this study,
infusions were prepared with 6.6 g of coffee (equivalent to one tablespoon) per 100 mL of
water by extraction using a French press due to its practicality.
On average, people usually ingest two to four cups (≈150 mL per cup) of coffee
per day. Moderate coffee drinkers consume per day between 200 and 400 mg of caffeine
and between 200 and 500 mg of CGA per cup [13,41]. Studies have shown that coffee
consumption of four cups per day can benefit health by reducing the risk of Parkinson’s
disease, Alzheimer’s disease, and type 2 diabetes mellitus [42]. Infusions of MCR using
9.9 g of coffee in 150 mL of water would contain 25.0 mg of caffeine and 299.5 mg of CGA,
while in DRC infusions the content would be 38.4 mg of caffeine and 143.7 mg of CGA per
cup. Coffee beverage consumption of MCR or DCR coffee can benefit people’s health.
The antioxidant properties of coffee infusions are mainly attributed to CGA and caf-
feine, although other compounds that influence antioxidant properties, such as epicatechin,
catechin, and anthocyanins, have also been reported. Oxidative stress is a trigger for chronic
degenerative diseases such as arthritis, diabetes mellitus, and cancer. Important pharmaco-
logical effects such as anti-inflammatory, regulation of glucose and lipid metabolisms, and
anticancer have been reported for coffee beverages and CGA [13,15,40,43]. Caffeine exerts
physiological effects associated with the functions of the central nervous system [44].
 caffeine,and
epicatechin, catechin, although other compounds
anthocyanins, that reported.
have also been influence Oxidative
antioxidantstress
properties,
is a such
epicatechin, catechin, and anthocyanins, have also been reported. Oxidative
trigger for chronic degenerative diseases such as arthritis, diabetes mellitus, and cancer. stress i
trigger for chronic degenerative diseases such as arthritis, diabetes
Important pharmacological effects such as anti-inflammatory, regulation of glucose and mellitus, and canc
Important
lipid metabolisms, pharmacological
and anticancer effectsreported
have been such as anti-inflammatory,
for coffee beverages regulation
and CGA of glucose a
Molecules 2023, 28, 4685 lipid metabolisms,
[13,15,40,43]. Caffeine and anticancer
exerts physiological effectshave been reported
associated with theforfunctions
coffee beverages
of the
10 of 17 and CG
central nervous[13,15,40,43].
system [44]. Caffeine exerts physiological effects associated with the functions of t
central nervous system [44].
3.
3. Materials
Materialsandand Methods
Methods and Methods
3. Materials
3.1. Biological Material
3.1. Biological Material
3.1. Biological Material
The
The cherries
cherriesof of Coffea
Coffeaarabica
arabicavarieties
varietiesTypica,
Typica, Bourbon,
Bourbon, and
and Oro
Oro Azteca
Azteca employed
employed in in
The cherries of Coffea arabica varieties Typica, Bourbon, and Oro Azteca employed
the
thepresent
presentstudy
study were
were harvested
harvested in
inthe
the2020–2021
2020–2021 and
and 2021–2022
2021–2022 cycles
cycles in
inthe
the plantations
plantations
the present study were harvested in the 2020–2021 and 2021–2022 cycles in the plantatio
of
ofthe
theCooperative
Cooperative Cafeticultores Mephaa “Region dedeLaLaMontaña”, at the localities of La
of theCafeticultores Mephaa
Cooperative Cafeticultores “Region
Mephaa Montaña”,
“Region at the
de La Montaña”, localities of
at the localities of
Soledad
La SoledadandandParaje
ParajeMontero,
Montero, municipality
municipality ofofMalinaltepec
Malinaltepec (longitude:
(longitude: 98.704167
98.704167 and
and
Soledad and Paraje Montero, municipality of Malinaltepec (longitude: 98.704167 a
latitude:
latitude: 17.164167),
17.164167), Guerrero,Mexico
Guerrero, Mexico(Figure
(Figure3).3). Coffee
Coffee cherries
cherries of each variety were
latitude: 17.164167), Guerrero, Mexico (Figure 3). of each
Coffee variety
cherrieswere dried
of each variety w
dried on drying
on drying bedsdriedbeds
under under
the the
sun sun
at roomat room temperature
temperature for for
15 15
days; days;
after after
that
on drying beds under the sun at room temperature for 15 days; after that
time, time,
the the
husk
that time, t
husk was removed
was removed to towas
obtain
husk obtain
the the GC.
GC.
removed to obtain the GC.

Figure Figureof
Figure3.3.Photographs
Photographs 3.the
of Photographs
thevarieties of thearabica
varietiesCoffea
Coffea varieties
arabica(a)Coffea
(a) arabica
Typica,
Typica,(b) (a) Typica,and
(b)Bourbon,
Bourbon, (b) (c)
and Bourbon,
(c)Oro and (c)
OroAzteca
Azteca Oro Azteca gr
grew
grew
in the Paraje Montero locality municipality of Malinaltepec at 1980 m.a.s.l.
in
inthe
theParaje
ParajeMontero
Montero locality
locality municipality
municipality ofof Malinaltepec
Malinaltepec atat 1980
1980 m.a.s.l.
m.a.s.l.

Subsequently, the Subsequently,

the GC
GC of of the the GC of
the Typica,
Typica, the Typica,
Bourbon, andBourbon,
Oro Aztecaandvarieties
Azteca Oro Azteca
were varieties
mixedwere mix
Subsequently, Bourbon, and varieties were mixed
(GCM) in a ratio of 40–30–30%. The mixture of coffee beans was processed in
(GCM) in
(GCM) in aa ratio
ratio of
of 40–30–30%.
40–30–30%. The The mixture
mixture of of coffee
coffee beans
beans was
was processed
processed in 100MEX®® a 100ME
in aa 100MEX
brand industrial roaster with a steel rotating cylinder to reach an Agtron Gourmet Be
brand industrial
brand industrialroaster
roaster with
with aa steel
steel rotating
rotating cylinder
cylinder to
to reach
reach anan Agtron
Agtron Gourmet
Gourmet Bean Bean
roasting level of 45 (180 °C per 15 min) to obtain a medium roast coffee (MRC) and
◦ C per
roasting level
roasting level ofof 45
45 (180
(180 °C 15 min)
per 15 min) to
to obtain
obtain aa medium
medium roast
roast coffee
coffee (MRC)
(MRC) and and an
an
Agtron Gourmet Bean roasting ◦level of 35 (210 °C per 15 min) for a dark roast coff
Agtron Gourmet
Agtron Gourmet Bean
Bean roasting
roasting level of of
level 35 35
(210(210C per
°C 15 min)
per 15 for a for
min) dark
a roast
dark coffee
roast (DRC).
coffee
(DRC). The roasted beans are transferred onto plates and allowed to cool at roo
The roasted
(DRC). beans arebeans
The roasted transferred onto plates
are transferred andplates
onto allowed andto allowed
cool at room temperature
to cool at room
temperature (Figure 4).
(Figure 4).
temperature (Figure 4).

Figure 4. Processed cherries and beans of C. arabica (a) Drying of cherries of different varieties
(GCM); (b) Mixture of green beans of different varieties; (c) Medium-roasted coffee beans (MRC);
(d) Dark-roasted coffee beans (DRC).

3.2. Bromatological Analysis

The analysis of the nutritional components of GCM, MRC, and DRC beans was
performed based on the standard methods of the American Cereal Chemical Association
(AACC International) [45]. The methods employed were moisture (44–15.02), protein
(46–16.01), fat (30–25.01), and ash content values (08–01.01). Moisture (%) was determined
by the weight difference of the grain before and after drying in a forced-air oven (VWR,
DHG-9070A)® (Beijing, China) for 5 min. The ash content (%) was obtained from the
Molecules 2023, 28, 4685 11 of 17

difference in weight of the waste generated by the samples placed in a porcelain pot and
incinerated in a muffle at 550 ◦ C for 4 h. The fats of the grains were extracted by Soxhlet
with petroleum ether, the solvent was evaporated, and the extracted residue was kiln-dried
at 100 ◦ C, weighed, and expressed as % ethereal extract (crude fat). The protein content
(%) was determined by the quantification of total free nitrogen by the modified Kjeldahl
method. Carbohydrate content was calculated by subtracting the sum of the percentages of
moisture, lipids, protein, and ash from 100%.
The values of humidity, ash, proteins, lipids, and carbohydrates obtained in the
GCM, MRC, and DRC were expressed as an average of three replicates and their standard
deviation (SD). Each variable was analyzed with a simple ANOVA and a Tukey post-test
with a confidence level of 95% (p < 0.05) using the SAS System for Windows 9.1 software
(Statistical software, SAS Institute, Inc., Cary, NC, USA).

3.3. Chemical Analyses

3.3.1. Infusion Preparation
The beans of Typica-GC, Bourbon-GC, Oro Azteca-GC, GCM, MRC, and DRC were
ground in an MCT-750 100MEX® (Veracruz, Mexico) grinder with a particle size of 1.0 mm.
Infusions of each coffee were prepared in the laboratory in triplicate at room temperature
with 13.2 g of ground coffee beans in 200 mL of boiling water to 98 ◦ C in a French press
for 5 min. The infusions were filtered, and the pH was measured with a potentiometer
Oakton pH 510; subsequently, the infusions were concentrated at reduced pressure in
a rotatory evaporator (Heidolph Laborota 4000). Then, the extracts were freeze-dried
(Heto Drywinner DW3), and the powders were stored in amber glass containers at room
temperature.

3.3.2. Chlorogenic Acid and Caffeine Quantification

The concentrations of chlorogenic acid (CGA) and caffeine (CAF) in the Typica-GC,
Bourbon-GC, Oro Azteca-GC, GCM, MRC, and DCR infusions were determined by High-
Performance Liquid Chromatography (HPLC) in Waters equipment consisting of a sepa-
ration module (Waters 2695) and a photodiode detector (Waters 2696). For this purpose,
10 µL of each infusion at concentrations of 0.25, 0.5, and 1.0 mg/mL were injected and
eluted through an RP-18 column (250 × 4.6 mm, 5 µm, SUPELCO Discovery® , Merck,
Darmstadt, Germany) with a flow of 0.9 mL/min in a gradient system of 30 min based
on water HPLC grade (VWR, Mississauga, ON, Canada) with 0.5% trifluoroacetic acid
(Sigma-Aldrich, Saint Louis, MO, USA) (A) and acetonitrile-HPLC (Merck, Germany) (B).
Gradient flow starts at A-100%, with gradient changes to 95% in 2 min, 70% in 2 min, 50%
in 17 min, 20% in 3 min, 0% A and 100% B in 3 min, and finally system return to initial
conditions (A-100%) in 3 min. Data were processed with the Empower Pro 3.0 software
(Waters, Milford, MA, USA), and the chromatograms were obtained at wavelengths (λ)
of 330 nm for CGA and at λ = 280 nm for caffeine. The identification of both compounds
was determined by comparison of their retention time (RT) and absorption spectra. The
CGA and caffeine concentrations were calculated according to external standards for CGA
(3-(3,4-Dihydroxycinnamoyl) quinic acid; ≥95% purity, Sigma-Aldrich) and caffeine (≥99%
purity, Sigma-Aldrich). The calibration curves of CGA and caffeine were constructed using
a lineal square model y = mx + b with the Microsoft Office Excel 365 Software (Microsoft®
Excel V.16.70) with correlation coefficients ≥0.9995; for CGA, it was a regression equation
of y = 11,702x + 19,276 with an R2 = 0.9982, and for caffeine, y = 87,483x + 38,786 with an
R2 = 0.9993.
The CGA and CAF contents were expressed in mg/g of coffee beans as the mean of
nine analyses and their standard deviation (SD). The GC contents of the varieties, GCM,
MRC, and DRC infusions, were compared with an ANOVA and a Tukey post-test with a
95% confidence level (p < 0.05).
Molecules 2023, 28, 4685 12 of 17

3.3.3. Chemical Fractionation of MRC Infusion

The MRC extract (2 g) was fractionated by open column chromatography (2.7 cm
diameter × 44 cm high), packed with 20 g of RP-18 silica gel (Supelco, Germany), and
eluted with a gradient system of H2 O: CH3 CN. Aliquots of 10 mL were collected with an
initial system of 100% H2 O (1–10) and polarity changes from CH3 CN at 5% (11–15), CH3 CN
at 50% (16–17), and CH3 OH at 100% (18). The fractions were analyzed by reverse-phase-
thin layer chromatography with an elution system of 90:10 H2 O:CH3 CN and displayed
in a UV lamp (UVP UVGL-58) at λ = 254 nm and λ = 365 nm. Aliquots with a similar
chromatographic profile (13–15) were grouped and diluted in dimethyl sulfoxide to be
analyzed by NMR of 1 H and 13 C spectra at 100 MHz, 2-dimensional (2-D) correlated
spectroscopy (COSY), heteronuclear simple quantum coherence (HSQC), and heteronuclear
multiple bond coherence (HMBC) at 400 MHz on Varian INOVA-400 equipment.

3.4. Melanoidins
The melanoidin content was analyzed in infusions of GCM, MRC, and DRC by
two different procedures:
(1) Serial dilutions (2.0–0.0625 mg/mL) were prepared from a solution of 10 mg/mL of
each infusion, and the absorbance of each concentration was measured at
λmax = 420 nm in a UV-VIS spectrophotometer (Genesys 20-Thermo Scientific, Waltham,
MA, USA). The melanoidin content in the infusions was determined by the Lambert-
Beer formula: C = A/cb, where C is concentration, A absorbance, b cell length (1 cm),
and c extinction coefficient (1.1289 L/g cm) [28,46].
(2) Extracts from 2 g of each infusion were dissolved in 20 mL distilled water, and they
were filtrated through Acrodiscs Pall® (0.45 µm). The calibration curves for each coffee
were built from dilutions with absorbances between 1.0 and 0.01. For melanoidin
determination, 1 mL of the filtrate was diluted with water (1/5, v/v), and 1 mL of
Carrez I and II solutions were added (Sigma-Aldrich). The solution was homoge-
nized and completed to a volume of 10 mL. Then, each sample was centrifuged at
4000 rpm for 5 min, and the clarified samples were filtered through Acrodiscs
Pall® (Pall Port Washington, NY, USA) (0.20 µm). The corresponding readings for
melanoidins were carried out to obtain the content and the specific extinction coeffi-
cient (Kmix ) determined by Lambert-Beer’s law.
The melanoidin content values in the GCM, MRC, and DRC were expressed as the
mean of three analyses and their SD, compared with an ANOVA and a Tukey post-test with
a 95% confidence level (p < 0.05).

3.5. Sensory Assessment

Infusions in cups of MRC or DRC ground coffee (MCT-750 100MEX® grinder (100MEX
Veracruz, Mexico) with a particle size of 1.0 mm) were prepared with 8.25 g in 150 mL
of distilled water at 93 ◦ C in a simple infusion. The sensory evaluation of the coffee
in cup was carried out by four tasters certified by the Mexican Association of Specialty
Coffees and Cafeterias, AC (AMCCE, abbreviations in Spanish), following the cupping
protocol of the Specialty Coffees of America Association (SCAA) [47]. The parameters
of the SCAA are based on a reference of 100 points; a score of 0 to 10 points was given
to each organoleptic characteristic of aroma/fragrance, flavor, residual flavor, sweetness,
acidity, body, uniformity, balance, clean cup, and taster. The point total sum was the result
of the sensory analysis; scores ≥ 80 are considered very good coffees and are classified as
specialty coffees.
Molecules 2023, 28, 4685 13 of 17

3.6. Biological Analyses

3.6.1. Antioxidant Activity Assays
Radical Scavenging of 2,2-Diphenyl-1-picrylhydrazyl (DPPH)
In each well of microplates of 96 wells, 175 µL of DPPH (1 mg/mL of methanol)
solution was added, followed by 25 µL of CGA (3.0–100 µg/mL), Trolox (6-Methoxy-
2,5,7,8-tetramethylchromane-2-carboxylic acid) (3.0–100 µg/mL), MRC (0.1–5.0 mg/mL),
or DRC (0.1–5.0 mg/mL) solutions. The absorbance was measured at the beginning of the
reaction (A0 ), and the plates were stored protected from light for 30 min. After this period,
the absorbance was measured (A1 ) again with an ELISA lector (Perkin-Elmer Lambda
40 UV/Vis) to λ = 515 nm. The inhibition percentage was calculated with the equation:

( A0 − A1 )
 
% inhibition = × 100
A0

The CGA, Trolox, MRC, and DRC mean inhibitory concentrations (IC50 ) of scavenging
DPPH radicals were determined based on the curves generated by recording the inhibition
percentages of the reaction against the concentration. In addition, the absorbance values of
the samples were compared with the graphed curves of the inhibition percentage against
the concentration of the standards and reported in GCA or Trolox equivalents (eq CGA and
eq Trolox) [25].
The IC50 and GCA, or Trolox equivalents, were expressed as the mean of three repli-
cates and their SD. Each variable of MRC and DRC was compared by a Student’s t-test
with a p < 0.05.

ABTS (2,20 -Azinobis (3-Ethylbenzothiazoline-6 sulfonic Acid) Radical

The 7 mM ABTS radical solution was prepared with persulfate of potassium 140 µM
in the dark under agitation for 12 to 16 h; after, the solution was diluted with methanol
until it had an absorbance value of 0.7 at λ = 734 nm. In each well, 230 µL of ABTS solution
and 20 µL of CGA (0.3–1.3 µg/mL), Trolox (0.3–1.3 µg/mL), MRC (0.1–5.0 mg/mL), or
DCR (5.0–0.1 mg/mL) were added. After a 30 sec stirring, the absorbance was determined
at λ = 734 nm. The antioxidant activity was expressed by inhibition percentage, IC50, and
CGA or Trolox equivalents, as previously described.
The IC50 and GCA, or Trolox equivalents, were expressed as the mean of three repli-
cates and their SD. Each variable of MRC and DRC was compared by a Student’s t-test
with a p ≤ 0.05 value.

Ferric Reducing/Antioxidant Power (FRAP)

The FRAP solution was produced with 1 mL of 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine
(TPTZ) solution and 1 mL 20 mM ferric chloride (FeCl3 . 6H2 O) solution in 10 mL of 300 mM
sodium acetate buffer (CH3 COONa) to pH 3.6. The FRAP solution was prepared freshly
and kept at 37 ◦ C. To each well, 175 µL of FRAP was added, along with 50 µL of CGA
(13–0.3 µg/mL), Trolox (0.3–1.3 µg/mL), MRC (0.1–5.0 mg/mL), or DCR (0.1–5.0 mg/mL).
The absorbance was measured at λ = 595 nm after 30 s of the reaction.
The IC50 and GCA, Trolox, or ferric sulfate equivalents were expressed as the mean of
three replicates and their SD. Each variable of MRC and DRC was compared by a Student’s
t-test with a p ≤ 0.05 value.

3.6.2. Cytotoxic Evaluation

The 3T3-L1 (ATCC® CL-173) cell line was used for cytotoxic assays; this culture is a
mouse embryonic fibroblast cell line obtained from IN VITRO S.A. The 3T3-L1 fibroblasts
are committed to differentiation into adipocytes. The cells were cultivated in Dubelco’s
Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, L-glutamine
(1 mL × 100), antibiotic 3X (1 mix100), non-essential amino acids (1 mL × 100), and sodium
Molecules 2023, 28, 4685 14 of 17

bicarbonate (3 mL × 100). Cellular cultures were incubated at 37 ◦ C in a humidified

atmosphere with 5% CO2 .
For the cytotoxic assay, the 3T3-L1 cells were cultivated in plates of 96 wells with a
cellular density of 3 × 104 cells per well. After 24 h of plate attachment, the non-adherent
cells were eliminated and subsequently treated with DMSO 1% as the negative control,
different concentrations of MRC and DRC infusions (15.6–500 µg/mL), and paclitaxel as the
positive control (0.6–20 µg/mL). The plates were incubated for 24 and 48 h at the conditions
above described.
After each incubation time, the medium was discarded, and 80 µL of DMEM medium
and 20 µL of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT, 5 mg/mL
in PBS) were added to each well and incubated for 4 h at 37 ◦ C to allow the formation of
formazan crystals. The supernatant was removed, 100 µL of isopropanol was added, and
the plates were incubated at room temperature with stirring for 15 min until the formazan
crystals were dissolved. The optical density of the solution was measured with a microplate
spectrophotometer at λ = 490 nm.
The cell viability percentage was determined by the following equation:

(negative control OD − tested sample OD)

 
Cell viability(%) = × 100
negative contro OD

The mean inhibitory concentration (IC50 ) for each infusion was calculated with a
dose-response curve regression analysis.

4. Conclusions
The artisanal coffees showed variable chemical contents related to the roasting process
to which they were subjected. The nutritional composition of MRC and DRC beans is
according to normative requirements. Both infusions presented variations in sensory
attributes, allowing the classification of the MRC as specialty coffee and the DRC coffee as
premium. It is well known that coffee consumption is related to its flavor and aroma; both
coffees had aromas of tropical fruits and flavors of white wine, grape, and honey.
The conditions of cultivation, the proportions of the C. arabica varieties in the coffee
bean mixture, the roosting processing of the beans, and the infusion preparation are
parameters related to the CGA and caffeine contents. CGA content was reduced by the
effect of roasting, while caffeine and melanoidins were increased, although below the
suggested levels by the norm. The commercial samples analyzed showed antioxidant
activity without presenting toxic effects; the content of harmful compounds in these coffees
may be at low concentrations.
The results obtained in this experimental work will be the basis for making modifica-
tions to improve the manufacturing processes of these commercial coffees, comply with the
standards for green and roasted coffee dealing by the Mexican Standard, and preserve their
chemical composition and biological potential.

Author Contributions: Conceptualization, F.C.-S. and P.N.-T.; methodology, J.G.-I., A.S., R.S., R.M.M.-R.,
S.C.-H., M.G.-C. and O.O.-M.; formal analysis, J.G.-I., A.S. and P.N.-T.; writing—original draft preparation,
J.G-I., A.S., P.N-T. and F.C-S; writing—review and editing, J.G-I., A.S., R.S., R.M.M.-R., S.C.-H., M.G.-C.,
O.O.-M., F.C.-S. and P.N.-T.; supervision, F.C.-S. and P.N.-T.; project administration, F.C.-S. and P.N.-T. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated during this study are included in this published article.
Molecules 2023, 28, 4685 15 of 17

Acknowledgments: The first author (J.G.-I.) acknowledges that this work was supported by Consejo
Nacional de Ciencia y Tecnología of Mexico (CONACYT-Mexico), for the Basic Grant 745198 for his
Doctoral studies at the Biotechnology Doctoral Program of Universidad Autónoma Metropolitana-
Iztapalapa. To CIBIS-IMSS for providing its infrastructure to carry out the experimental work. To the
Cooperativa Cafeticultores Mephaa de la Región de la Montaña for providing the biological material
for this study.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not applicable.

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