You are on page 1of 12

Food Chemistry 444 (2024) 138608

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Fermentation of coffee fruit with sequential inoculation of


Lactiplantibacillus plantarum and Saccharomyces cerevisiae: Effects on volatile
composition and sensory characteristics
Mariane Helena Sances Rabelo a, *, Flávio Meira Borém a, Ana Paula de Carvalho Alves a,
Rodrigo Soares Pieroni c, Claudia Mendes Santos a, Makoto Nakajima b, Ryosuke Sugino b
a
Departamento de Engenharia Agrícola, Universidade Federal de Lavras, POB 3037, 37.200-000, Lavras, MG, Brazil
b
Suntory Company, Japan
c
Associação dos Cafeicultores da Canastra, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Mixed starter cultures of lactic acid bacteria and yeasts used in the production of fermented foods, including
Coffee fermentation coffee, can improve the sensory quality and food safety. The objective of this study was to evaluate the effects of
Sequential inoculation fermentation of coffee with inoculation of Lactiplantibacillus plantarum followed by Saccharomyces cerevisiae and
Mixed starter culture
the effects of fermentation time on the aroma and flavor of the coffee beverage and on the volatile composition of
the roasted coffee beans. The coffee was fermented for 48 h or 96 h after inoculation of Lactiplantibacillus
plantarum followed by inoculation of Saccharomyces cerevisiae or the respective controls. The aroma and flavor of
the coffee beverage fermented with sequential inoculation showed complexity, with a predominance of fruity
and fermented sensory notes. Forty-seven volatile compounds were identified. In addition, the sequentially
inoculated coffees had greater formation of volatiles and led to greater perception of fruity and fermented flavor
and aroma.

1. Introduction predominantly yeast (Silva et al., 2008).


Spontaneous food fermentation processes occur through naturally
Bacteria, yeasts, and fungi are naturally present in coffee, and established mixed cultures. Starter and nonstarter microorganisms
colonization by these microorganisms occurs throughout the entire contribute to the physicochemical and sensory characteristics of the
production chain, while the fruit is still on the coffee plant up to the final product, especially in nonsterile processes (Evangelista et al., 2015;
storage of beans. In the postharvest stages, coffee fruit serves as a sub­ Lu et al., 2018; Yunita & Dodd, 2018; Zhang et al., 2019b). However,
strate for the development of these microorganisms, supplying them microorganisms in a consortium can interact and affect each other’s
with carbon and nitrogen (Junqueira et al., 2019). In addition, chemical metabolism. This interaction between microorganisms can be positive,
and physical changes occur in the fruit, and microbial groups or species neutral, or negative, and it occurs through physical contact of signaling
that can affect the final quality of the coffee beverage through their molecules or through changes in the physicochemical properties of the
metabolic activity can be selected (Haile & Kang, 2019). environment (Sieuwerts et al., 2008).
Studies on microbial succession during the spontaneous fermenta­ García et al., (2017,2018) used a sequential inoculation in which
tion and drying process of natural coffees have identified that bacteria lactic acid bacteria (LAB) was inoculated in second place dairy sub­
and yeasts predominate at different times (Haile & Kang, 2019). At the strates as a means of preventing competitive exclusion of the sensitive
beginning of the fermentation and drying process, when the fruit had Pseudomonas taetrolens by antimicrobial compounds produced by LAB.
approximately 69 % water content and 0.95 water activity, bacteria Sequential inoculation has been used in the production of various fer­
represented 96.3 % of all the microorganisms isolated. When the fruit mented products, such as wine, cider, and dairy and nondairy fermented
water activity reaches values between 0.82 and 0.85 as a result of the beverages (García et al., 2017,2018; Lu, Putra & Liu, 2018), but it has
drying process, the microorganisms that were present were not yet been used for fermentation of coffee fruit. In a sequential

* Corresponding author.
E-mail address: marianerabelo1@hotmail.com (M. Helena Sances Rabelo).

https://doi.org/10.1016/j.foodchem.2024.138608
Received 20 June 2023; Received in revised form 25 January 2024; Accepted 26 January 2024
Available online 28 January 2024
0308-8146/© 2024 Elsevier Ltd. All rights reserved.
M. Helena Sances Rabelo et al. Food Chemistry 444 (2024) 138608

Fig. 1. Sensory descriptors of aroma and flavor used in the sensory evaluation. Note: Figure .
adapted from Spencer et al., 2016

2
M. Helena Sances Rabelo et al. Food Chemistry 444 (2024) 138608

fermentation system, different stages can be implemented in which company (ML Prime/Lallemand, Blagnac, France). The yeast used in this
specific operational conditions are applied. That way, the processes can study was a strain of the Saccharomyces cerevisiae species isolated and
be optimized, and conditions that are more favorable for each micro­ marketed in lyophilized form by the company Lallemand (Lalvin
organism can be adopted. EC1118/Lallemand, Blagnac, France).
In the development of dairy and nondairy fermented beverages The LAB was activated by rehydration with water at a ratio of twenty
involving LAB, the introduction of yeast in a second stage resulted in times the weight of the lyophilized bacteria at a water temperature of
greater survival of the LAB, due to the symbiotic relationship that arose approximately 25 ◦ C, following the manufacturer’s instructions. The
(García et al., 2017,2018; Lu, Putra & Liu, 2018). Thus, sequential yeast was also activated with water, but at the ration of ten times the
inoculation can allow the use of new microbial consortia, avoiding weight of the lyophilized yeast, and the water temperature was
possible amensalistic interactions. approximately 36 ◦ C.The inoculations were performed directly on the
In this respect, a key parameter in fermentations with sequential coffee fruit at the concentration of 107 colony-forming units per gram.
inoculation is the interval between the first and second inoculations All procedures were carried out according to the manufacturer’s
(Hranilovic et al., 2020). The interval between inoculations can affect instructions.
the development and metabolic activity of the first microorganism and,
consequently the concentration of substrates, the physicochemical 2.2. Experimental procedure
conditions of the medium, and the products and other metabolites that
affect the second microorganism. This interval can create an environ­ This study was conducted in the Serra da Canastra region in the
ment with more favorable conditions for inoculation and development municipality of Vargem Bonita, Minas Gerais, Brazil. Ripe Coffea arabica
of the second culture. Freire et al. (2017) clearly showed such an fruit of the Catuaí Vermelho variety (50 Kg for each treatment) were
interaction, in which the yeast population in the co-culture fermentation processed by the dry method, and fermentations were induced in closed
was favored by the reduction in pH to 4.0–4.5 due to the metabolic polypropylene bioreactors (induced anaerobic fermentation). The
activity of LAB. average temperature during fermentation was 21 ◦ C (minimum tem­
Yeasts and LAB have been used in recent studies as pure starter perature of 17 ◦ C and maximum of 26 ◦ C). Two factors were studied in
cultures in the postharvest processing of coffee to shorten the fermen­ the coffee fruit fermentation process: sequential inoculation of starter
tation time and modulate the chemical and sensory characteristics of cultures of Lactiplantibacillus plantarum and Saccharomyces cerevisiae
coffee beans (Bressani et al., 2018; Carvalho Neto et al., 2020; Evan­ (LPSC) and total fermentation time. Based on the study of microbial
gelista et al., 2014; Ribeiro et al., 2017; Wang et al., 2019). The mi­ succession in coffee fruit conducted by Silva et al. (2008), it was
croorganisms that have been studied most intensively include the yeast established that LAB would be inoculated first, and yeast would be
Saccharomyces cerevisiae and the LAB Lactiplantibacillus plantarum, which inoculated 24 h later.
show adaptability to the stress factors in postharvest processing, effi­ The sequential inoculation factor was composed of four levels that
cient consumption of the sugars in coffee fruit pulp, and adequate pro­ corresponded to the presence or absence of the starter cultures. The
duction of organic acids and volatile compounds (Cassimiro et al., factor of total fermentation time of the coffee fruit was evaluated at two
2022). levels, 48 h and 96 h after the beginning of the fruit fermentation pro­
Although the use of pure starter cultures in processing coffee fruit cess. The two levels were chosen after preliminary tests and reference to
offers advantages, recent studies show that mixed starter cultures of industry standards. Thus, the experiment had a 4 × 2 factorial design
bacteria and yeasts used in the production of fermented foods, including (four levels of the sequential inoculation factor and two levels of the
coffee fruit, can provide improved sensory quality and the promote total time factor), for a total of eight treatments, performed in triplicate.
production of toxin-free foods (Garcia et al., 2019; Vale et al., 2019; Specifically, the treatments performed were (i) L. plantarum inoculation
Cassimiro et al., 2022). followed by S. cerevisiae (LPSC), with 48 h of fermentation; (ii)
Co-inoculation with yeast and LAB has potential in forming aromatic L. plantarum inoculation followed by S. cerevisiae (LPSC), with 96 h of
compounds desirable for the production of specialty coffees (Vale et al., fermentation; (iii) L. plantarum inoculation alone (LP), with 48 h of
2019). Cassimiro et al. (2022) investigated the co-cultivation of the fermentation; (iv) L. plantarum inoculation alone (LP), with 96 h of
L. plantarum CCMA1065 and S. cerevisiae CCMA0543 strains in the fermentation; (v) S. cerevisiae inoculation alone (SC), with 48 h of
fermentation of wet processing coffees and observed an improvement in fermentation; (vi) S. cerevisiae inoculation alone(SC), with 96 h of
sensory quality. There are numerous reports in the literature indicating fermentation; (vii) control, with 48 h of fermentation; and (viii) control,
that the use of mixed cultures offers advantages. However, the use of with 96 h of fermentation. As the fifth and sixth treatments represented
mixed cultures employimg the sequential inoculation technique for the control of L. plantarum inoculation, inoculation of S. cerevisiae was
production of high quality coffees has not yet been investigated. performed only 24 h after the beginning of the fermentation process.
The aim of the present study was to carry out a coffee fermentation
process through sequential inoculation using Lactiplantibacillus planta­ 2.3. Sensory evaluation
rum followed by Saccharomyces cerevisiae in an attempt to improve
sensory quality. A further aim was, to evaluate the volatile composition The sensory evaluation was performed by a sensory panel composed
of roasted beans and the sensory characteristics (aroma and flavor) of of five trained cuppers with international Q-grader coffee certification.
the coffee beverage to better understand the effect of fermentation using The five trained cuppers were men, from 35 to 50 years of age, and
sequential inoculation and the effect of sequential inoculation on total professionals in the coffee industry. All sample preparation and roasting
fermentation time. procedures followed the recommendations of the Specialty Coffee As­
sociation (SCA) Protocols - Cupping Specialty Coffee (Lingle, 2011).
2. Materials and methods In the first step of sensory evaluation, a survey was performed of the
sensory descriptors that best described the aroma and flavor charac­
2.1. Starter cultures teristics of all the fermented coffee samples. With the results of the
survey, two lists of sensory descriptors were generated, one for aroma
Two starter cultures were used for the fermentation process of coffee and another for flavor. Based on the Coffee Taster’s Flavor Wheel
fruit with sequential inoculation: a LAB and a yeast of the genus (Spencer et al., 2016), the sensory descriptors listed were grouped into
Saccharomyces. nine classes, and each class was named with a term that best described
The LAB used in this study was a strain of the Lactiplantibacillus the class (Fig. 1). The nine classes were Fruity, Fermented, Herb-like,
plantarum species marketed in lyophilized form by the Lallemand Defects, Spices, Nutty, Cocoa, Sweet and Floral. The aroma descriptors

3
M. Helena Sances Rabelo et al. Food Chemistry 444 (2024) 138608

Fig. 2. Multiple factor analysis for evaluation of beverage aroma (A, B and C) and beverage flavor (D, E and F) of coffees under treatments of fermentation with
sequential inoculation and controls treatments. Legend: FR - fruity, FL - floral, FE - fermented, SP - spices, CH - cocoa, SW - sweet, NU - nutty.

4
M. Helena Sances Rabelo et al. Food Chemistry 444 (2024) 138608

were red fruits, dried fruits, yellow fruits, tropicals fruits, citric fruits, samples evaluated (Rabelo et al., 2020).
coffee pulp, milk notes, winey, alcohol, herb-like, vinegar, earthy,
woody, tabacco, spices, peanuts, nut and almond, milk chocolate, dark 3. Results and discussion
chocolate, caramel, brown sugar, caramelized sugar, honey, black tea,
floral. Flavor descriptors were red fruits, dried fruits, yellow fruits, 3.1. Sensory characterization
tropical fruits, orange, Sicilian lemon, lemon, coffee pulp, milk notes,
winey, alcohol, herb-like, vinegar, earthy, woody, tobacco, spices, The use of a mixed starter culture instead of a pure starter culture is a
peanuts, nut and almond, milk chocolate, dark chocolate, caramel, common practice in wine production for the purpose of providing
brown sugar, sugar cane, sucrose, caramelized sugar, honey, black tea, greater complexity to the sensory characteristics of wine (Cañas et al.,
orange flower, coffee flower, and jasmine. 2015; Gobbi et al., 2013). Fermentations with mixed starter cultures not
In the second step, each sample was evaluated using the descriptive only offer several advantages over conventional fermentations with pure
“check all that apply” (CATA) method (Adams et al., 2007). The CATA starter cultures, but also more closely resemble what spontaneously
sensory evaluation questionnaire consisted of the list of sensory de­ occurs in nature, where consortia of bacteria and yeast are naturally
scriptors generated in the first step. The cuppers were asked to taste the established (Zhang et al., 2019b). Microorganisms in consortia can
samples and answer the CATA questionnaire, indicating the class of interact and affect each other’s metabolism (Sieuwerts et al., 2008).
descriptors that best described the aroma and flavor of each sample. The Thus, the sequential inoculation technique in mixed culture fermenta­
number of items selected was not limited; the cuppers could select as tions was developed to enable new microbial consortia and avoid
many classes of sensory descriptors as they wished. In addition, the possible negative interactions (Garcia et al., 2008, 2019).The research
cuppers could select the sensory descriptors within the assigned class, developed here was a pioneering study in the use of the sequential
thus providing a more specific description. inoculation technique for fermentation of coffee fruit for production of
The sensory analysis procedure was approved by the Ethics Com­ specialty natural coffees with complex aroma and flavor characteristics.
mittee on Human Research of the Universidade Federal de Lavras – Flavor is a sensory characteristic that can be defined as a “complex
UFLA, registered under number 40641620.8.0000.5148 of the ethical combination of the olfactory, gustatory, and trigeminal sensations
consideration certificate (CAAE). perceived during tasting” that “may be influenced by tactile, thermal,
painful, and/or kinesthetic effects” (International Standards Organiza­
2.4. Analysis of volatile composition tion - ISO 5492, 1992, 2008). The gustatory sense, through the tastebuds
distributed on the surface of the tongue, is responsible for perception of
The roasted coffee samples were ground in an IKA 11A mill, and for the basic tastes, which are only five: sweet, bitter, salty, sour, and
each sample, 2 g was placed in a hermetically sealed vial for later umami. In contrast, the sense of smell, through stimulation of the ol­
analysis of its volatile composition, according to the method of Rabelo factory epithelium, can distinguish numerous volatile compounds that
et al. (2020). The volatile compounds were extracted using the static give rise to the aromas of foods and beverages. Thus, it is the aromas
headspace of the gas chromatograph/mass spectrometer (GCMS) model perceived by the sense of smell that actually contribute much more in­
QP − 2010 SE (Shimadzu Corporation, Kyoto, Japan) equipped with an formation to the flavor experience (Spence, 2016; Spence, 2015). Aroma
NST- 100 column (30 m × 0.25 mm × 0.25 μm) with a Carbowax®-like is therefore the most important component of coffee beverage flavor.
polyethylene glycol phase. For a better understanding of multifactorial analysis, it should be
Data analysis and compound identification were performed using the noted that the results are presented in three different graphs. The first
GCMS solution software (version 4.4, Shimadzu Corporation, Kyoto, graph (Fig. 2 – A and D) is the inertia of the group and presents differ­
Japan) and the NIST NIST/EPA/NIH 2014 database. Chemical identifi­ entiation in characterization of the sensory descriptors according to
cation was performed by comparing the mass spectra against the data­ fermentation time. It is represented by two centroids, one for 48 h of
base. The results are expressed as the relative percentage area fermentation and the other for 96 h of fermentation. When the centroids
corresponding to the peak area of each identified compound out of the are close or overlapping, there is no significant difference in the sensory
total area of the chromatogram Rabelo et al. (2020). analysis between fermentation times. Distance between the centroids
indicates that the fermentation time affected the sensory description.
2.5. Statistical analysis The second graph (Fig. 2 – B and E) presented is the correspondence
graph. This graph shows the interrelationship between sequential
2.5.1. Sensory analysis inoculation and total fermentation time. The centroids represent
Multiple factor analysis was applied to the data obtained from the sequential inoculation, and the vectors symbolize total fermentation
CATA questionnaire for the aroma and flavor characteristics. The data time. The position of the centroids represents the similarity or difference
correspond to the frequency that each class of descriptors was used to among the sensory descriptors. Centroids in the same quadrant indicate
describe each treatment and the frequency was determined by counting that their descriptors are similar; centroids in different quadrants indi­
the number of cuppers who used the class, multiplied by the number of cate difference in their descriptors. Vectors representing total fermen­
treatment replications. Classes with a frequency lower than 20 % were tation time are presented for each centroid. The direction and modulus
excluded from the multiple factor analysis. The analyses were performed of the vectors show the effect of the total fermentation time on sensory
as described by Ossani et al. (2017) with R software Version 3.6.3, description. When the vectors are in opposite directions, they indicate
package MVar (R Development Core Team, 2017). differentiation between the sensory descriptors. The modulus of the
vectors indicates magnitude; the larger the modulus, the greater the
2.5.2. Volatile compounds differentiation between fermentation times.
The relative percentage areas of the volatile compounds found in the The third and final graph (Fig. 2 – C and F) is the correspondence
treatments were subjected to analysis of variance, and when significant circle. Using this graph, the main sensory descriptors for each treatment
differences were obtained by the F-test, the Scott-Knot test was applied. can be listed. Sensory descriptors are represented by vectors. The red
These analyses were performed using the SISVAR statistical software vectors represent the sensory descriptors for 48 h of fermentation, and
(SISVAR, version 5.3). To better understand the relationship between the green vectors represent the sensory descriptors for 96 h of fermen­
the treatments and the volatile composition, principal component tation. The list of the main sensory descriptors for each treatment was
analysis was performed using the CHEMOFACE statistical software obtained by associating the position of the centroid (graph 2, Fig. 2 – B
(CHEMOFACE, version 1.64). An m × n matrix was constructed, with and E) and the position of the vectors of the sensory descriptors for each
the relative areas of the 47 chromatographic peaks identified in the 8 total fermentation time.

5
M. Helena Sances Rabelo et al. Food Chemistry 444 (2024) 138608

Fig. 3. Principal component analysis of volatile compounds present in coffee beans fermented with sequential or simple inoculation (LPSC, LP, SC, and control) at
different total fermentation times (48 h and 96 h). Legend: 1:butanal; 2: 2-methylbutanal; 3: 3-methylbutanal; 4: 2,2-dimethylpropanal; 5: acetic acid; 6: propionic
acid; 7: 4-hydroxybutanoic acid; 8: 3-methylbutanoic acid; 9: ethyl acetate; 10: propanoic acid ethyl ester; 11: 2-propenoic acid ethyl ester; 12: 2-methylfuran; 13:
2,5-dimethylfuran; 14: 2-vinylfuran; 15: furfuryl methylether; 16: furfural; 17: furfuryl formate; 18: furfuryl acetate; 19: 5-methyl-furfural; 20: furfuryl alcohol; 21:
thiophene; 22: thiazole; 23: 2,3-pentanedione; 24: 3,4-hexanedione; 25: 4-hydroxy-3-hexanone; 26: 2-hydroxy-3-pentanone; 27: 1-hydroxy-2-butanone; 28: 1-(2-
furanyl)-ethanone; 29: pyrazine; 30: methylpyrazine; 31: 2,5-dimethylpyrazine; 32: 2,6-dimethylpyrazine; 33: ethylpyrazine; 34: 2,3-dimethylpyrazine; 35: 2-ethyl-
6-methylpyrazine; 36: 2-ethyl-3-methylpyrazine; 37: pyridine; 38: 1-acetyl-1,4-dihydropyridine; 39: 1-methylpyrrole; 40: 1-methylpyrrole-2-carboxaldehyde; 41: 2-
methyl-3-buten-2-ol; 42: 1-pentanol; 43: 3-methyl-3-buten-1-ol; 44: 3-methylphenol; 45: 5-nonylamine; 46: 4-ethylamino-n-butylamine; 47: methyl-urea.

The predominant aromas of the coffee beverage that underwent the processes that use coffee mucilage as a substrate for the production of
fermentation process with LPSC were from the fruity, fermented, and volatile compounds that underlie these aromas (Zhang et al., 2019a;
floral classes at both fermentation times; and their distribution of Zhang et al., 2019b). Studies conducted in different coffee-producing
importance differed from those under the other treatments, as observed regions in Brazil used pure starter cultures of LAB strains and Saccha­
in the multiple factor analysis depicted in Fig. 2 – A, B and C. The results romyces cerevisiae strains for the fermentation of coffee fruit, and the
of the treatments with different fermentation times differed from each sensory profile of the coffees was mostly described as fruity (Evangelista
other, as visible in the first graph (Fig. 2A). The centroid that represents et al., 2014; Pereira et al., 2016). However, in this study, the coffees
sequential inoculation (LPSC) is isolated in the negative part of the first from the LP and SC treatments, which consisted of inoculation of pure
coordinate (Fig. 2B), and the centroids of the LP, SC, and control starter cultures, showed a predominance of the cocoa (48 h), nutty (96
treatments are located in the positive part of the first coordinate, which h), and sweet (96 h) classes in the LP treatment and nutty (48 h) and
explained 57.88 % of the data variability. spices (96 h) in the SC treatment. Thus, the multiple factor analysis
The sensory descriptors that best characterized the aroma of the indicated that the joint action of LPSC in the fermentation of coffee fruit
coffees that underwent fermentation for 48 h and 96 h with sequential potentiated the fruity aromas, diversifying the aromatic notes within
inoculation (LPSC) were red fruits, yellow fruits, tropical fruits, floral, this class and incorporating aromas characteristic of fermented products
winey, and alcohol. Although the “sweet” sensory descriptor class was described as winey and alcohol.
not directly related to LPSC in the multiple factor analysis, it should be Total fermentation time also had an effect on aroma, especially in the
noted that 60 % of the sensory panel assigned brown sugar scores to this LP, SC, and control groups. The main classes of aromas that differed with
treatment. The coffee from the control treatment was also described by fermentation time were cocoa and sweet.
the majority of the sensory panel as having predominant brown sugar In the evaluation of flavor, the centroid of each treatment was ar­
notes at both fermentation times. Sweet aromatic notes are more related ranged in a quadrant of the multiple factor analysis, as observed in
to coffee characteristics and not to the action of microorganisms (Cas­ Fig. 2E. The first coordinate, which explained 43.22 % of the variability
simiro et al., 2022). of the data, was responsible for discriminating the treatments containing
The fruity aromas present in coffee and its beverage are most often the starter culture Saccharomyces cerevisiae. The second coordinate,
derived from the action of microorganisms, i.e., from fermentation which explained 32.21 % of the variability of the data, discriminated the

6
M. Helena Sances Rabelo et al. Food Chemistry 444 (2024) 138608

Table 1
Effect of sequential inoculation on the mean relative areas of the volatile compounds of coffees fermented with different total fermentation times. Analysis of variance
and breakdown of the interaction of the sequential inoculation and total fermentation time factors, with the inoculation treatment (LPSC, LP, SC, and control) broken
down within each total fermentation time (48 h and 96 h).
Volatile Compound Similarity 48 h 96 h
Index
LPSC LP SC Control LPSC LP SC Control

Aldehyde
1 Butanal* 95,8 ± 0,4 0,84 ± 0,70 ± 0,92 ± 0,79 ± 0,74 ± 0,66 ± 0,56 ± 0,60 ±
0,07a 0,11a 0,09a 0,07a 0,15a 0,03a 0,03a 0,01a
2 2-Methylbutanal* 97,0 ± 0,0 5,62 ± 5,91 ± 6,17 ± 6,86 ± 5,45 ± 5,94 ± 4,81 ± 5,04 ±
0,31a 0,17a 0,66a 0,73a 0,84a 0,39a 0,38a 0,33a
3 3-Methylbutanal 96,2 ± 0,4 1,96 ± 2,03 ± 2,05 ± 2,10 ± 1,82 ± 2,02 ± 1,62 ± 1,68 ±
0,15 0,04 0,10 0,29 0,22 0,06 0,09 0,23
4 2,2-Dimethylpropanal* 90,5 ± 1,0 0,26 ± 0,21 ± 0,24 ± 0,22 ± 0,15 ± 0,23 ± 0,18 ± 0,20 ±
0,03a 0,02a 0,04a 0,05a 0,03a 0,04a 0,01a 0,03a
Acid
5 Acetic acid* 98,2 ± 0,4 5,31 ± 6,95 ± 6,04 ± 5,74 ± 6,59 ± 5,82 ± 4,79 ± 6,51 ±
0,47a 0,55a 0,38a 0,16a 0,97a 0,19a 0,44a 0,93a
6 Propionic acid* 89,9 ± 3,0 0,33 ± 0,50 ± 0,44 ± 0,42 ± 0,41 ± 0,29 ± 0,25 ± 0,31 ±
0,07b 0,02a 0,08a 0,02a 0,04a 0,07b 0,01b 0,04b
7 4-Hydroxybutanoic acid 94,2 ± 1,4 0,26 ± 0,27 ± 0,28 ± 0,20 ± 0,21 ± 0,24 ± 0,25 ± 0,27 ±
0,03 0,06 0,14 0,08 0,09 0,06 0,03 0,02
8 3-Methyl-butanoic acid* 94,2 ± 1,4 0,28 ± 0,34 ± 0,28 ± 0,34 ± 0,31 ± 0,20 ± 0,16 ± 0,21 ±
0,02b 0,03a 0,04b 0,03a 0,00a 0,04b 0,02b 0,02b
Ester
9 Ethyl acetate* 83,2 ± 0,4 8,16 ± 2,99 ± 10,23 ± 3,41 ± 20,89 ± 20,55 ± 30,57 ± 27,43 ±
0,61b 0,44c 0,99a 0,90c 0,96c 1,32c 1,98a 1,23b
10 Propanoic acid, ethyl ester* 97,9 ± 1,5 0,33 ± 0,10 ± 0,23 ± 0,08 ± 0,43 ± 0,35 ± 0,44 ± 0,30 ±
0,01a 0,00c 0,04b 0,02c 0,02a 0,05b 0,01a 0,05b
11 2-Propenoic acid, ethyl 95,6 ± 1,3 0,08 ± 0,00 ± 0,05 ± 0,00 ± 0,08 ± 0,06 ± 0,08 ± 0,06 ±
ester* 0,01a 0,00c 0,01b 0,00c 0,03a 0,01b 0,01a 0,01b
Furan
12 2-Methylfuran 99,0 ± 0,0 9,19 ± 8,57 ± 8,73 ± 9,30 ± 7,04 ± 8,30 ± 7,42 ± 7,39 ±
0,77 0,72 0,72 0,24 0,41 0,99 0,32 0,57
13 2,5-Dimethylfuran 91,7 ± 2,6 0,51 ± 0,33 ± 0,43 ± 0,42 ± 0,33 ± 0,44 ± 0,37 ± 0,39 ±
0,02 0,09 0,01 0,08 0,13 0,06 0,13 0,07
14 2-Vinylfuran 93,8 ± 2,0 0,20 ± 0,22 ± 0,20 ± 0,22 ± 0,14 ± 0,16 ± 0,14 ± 0,14 ±
0,04 0,03 0,02 0,05 0,04 0,03 0,04 0,02
15 Furfurylmethyl ether 92,7 ± 1,1 0,12 ± 0,13 ± 0,11 ± 0,11 ± 0,07 ± 0,10 ± 0,09 ± 0,09 ±
0,02 0,01 0,00 0,03 0,01 0,01 0,01 0,00
16 Furfural* 98,6 ± 2,0 6,40 ± 9,82 ± 7,16 ± 8,50 ± 6,26 ± 5,03 ± 3,66 ± 4,41 ±
0,40c 0,54a 0,92c 0,42b 0,75a 0,26b 0,31b 0,37b
17 Furfuryl formate 93,8 ± 1,3 0,32 ± 0,33 ± 0,30 ± 0,26 ± 0,22 ± 0,29 ± 0,18 ± 0,22 ±
0,05 0,01 0,09 0,09 0,07 0,05 0,03 0,04
18 Furfuryl acetate* 93,7 ± 1,5 1,67 ± 1,12 ± 1,21 ± 0,99 ± 1,17 ± 1,42 ± 1,46 ± 1,46 ±
0,07a 0,27b 0,24b 0,16b 0,12a 0,14a 0,02a 0,07a
19 5-Methyl-furfural* 97,9 ± 0,3 2,98 ± 3,55 ± 2,95 ± 3,65 ± 2,51 ± 2,32 ± 2,07 ± 2,27 ±
0,13b 0,07a 0,05b 0,39a 0,14a 0,11a 0,02a 0,06a
20 Furfuryl alcohol 98,0 ± 0,0 15,79 ± 17,19 ± 15,10 ± 16,92 ± 12,98 ± 12,59 ± 11,19 ± 12,29 ±
0,68 0,39 1,02 0,68 0,89 0,47 0,60 0,90
Thiophene
21 Thiophene* 87,0 ± 1,9 0,17 ± 0,11 ± 0,14 ± 0,13 ± 0,10 ± 0,13 ± 0,11 ± 0,10 ±
0,01a 0,01b 0,01b 0,01b 0,02a 0,01a 0,01a 0,01a
Thiazole
22 Thiazole 79,8 ± 2,4 0,06 ± 0,05 ± 0,06 ± 0,06 ± 0,06 ± 0,04 ± 0,04 ± 0,04 ±
0,00 0,00 0,01 0,02 0,01 0,01 0,01 0,01
Ketone
23 2,3-Pentanedione* 92,6 ± 1,1 5,13 ± 5,09 ± 4,95 ± 5,55 ± 4,59 ± 4,34 ± 3,79 ± 3,59 ±
0,12a 0,18b 0,44b 0,33b 0,30a 0,20a 0,13b 0,17b
24 3,4-Hexanedione 97,9 ± 0,5 0,19 ± 0,18 ± 0,16 ± 0,20 ± 0,14 ± 0,15 ± 0,12 ± 0,13 ±
0,02 0,03 0,03 0,01 0,02 0,01 0,01 0,01
25 4-Hydroxy-3-hexanone* 90,5 ± 0,7 0,22 ± 0,28 ± 0,20 ± 0,26 ± 0,18 ± 0,17 ± 0,16 ± 0,15 ±
0,01b 0,03a 0,03b 0,03a 0,01a 0,02a 0,01a 0,02a
26 2-Hydroxy-3-pentanone* 82,7 ± 4,5 0,15 ± 0,18 ± 0,11 ± 0,16 ± 0,14 ± 0,10 ± 0,11 ± 0,11 ±
0,02a 0,01a 0,01b 0,02a 0,03a 0,00a 0,02a 0,04a
27 1-Hydroxy-2-butanone* 96,4 ± 1,9 0,91 ± 1,15 ± 0,78 ± 0,94 ± 0,70 ± 0,65 ± 0,55 ± 0,56 ±
0,04b 0,13a 0,02c 0,08b 0,04a 0,10a 0,11a 0,04a
28 1-(2-furanyl)-ethanone* 95,5 ± 0,9 0,28 ± 0,43 ± 0,36 ± 0,51 ± 0,22 ± 0,25 ± 0,21 ± 0,23 ±
0,06b 0,08a 0,04b 0,12a 0,02a 0,07a 0,04a 0,03a
Pyrazine
29 Pyrazine 98,3 ± 0,4 1,78 ± 1,78 ± 1,71 ± 1,84 ± 1,43 ± 1,44 ± 1,32 ± 1,23 ±
0,11 0,06 0,16 0,11 0,06 0,13 0,12 0,06
30 Methylpyrazine * 97,3 ± 0,6 8,59 ± 9,86 ± 8,81 ± 10,34 ± 7,49 ± 7,21 ± 6,13 ± 6,20 ±
0,43b 0,42a 0,39b 0,46a 0,15a 0,36a 0,76b 0,17b
31 2,5-Dimethylpyrazine 97,5 ± 0,5 0,55 ± 0,69 ± 0,59 ± 0,67 ± 0,49 ± 0,45 ± 0,43 ± 0,45 ±
0,05 0,03 0,12 0,08 0,04 0,04 0,06 0,01
(continued on next page)

7
M. Helena Sances Rabelo et al. Food Chemistry 444 (2024) 138608

Table 1 (continued )
Volatile Compound Similarity 48 h 96 h
Index
LPSC LP SC Control LPSC LP SC Control

32 2,6-Dimethylpyrazine* 98,0 ± 0,2 0,82 ± 1,02 ± 0,70 ± 1,03 ± 0,62 ± 0,69 ± 0,55 ± 0,56 ±
0,04b 0,08a 0,06c 0,04a 0,02a 0,03a 0,06b 0,05b
33 Ethylpyrazine 96,9 ± 0,5 0,89 ± 1,02 ± 0,88 ± 0,98 ± 0,69 ± 0,73 ± 0,70 ± 0,71 ±
0,07 0,05 0,10 0,04 0,15 0,11 0,09 0,04
34 2,3-Dimethylpyrazine* 94,7 ± 1,1 0,20 ± 0,26 ± 0,18 ± 0,23 ± 0,21 ± 0,17 ± 0,19 ± 0,21 ±
0,02b 0,02a 0,03b 0,05a 0,03a 0,01a 0,03a 0,01a
35 2-Ethyl-6-methylpyrazine* 91,6 ± 4,9 0,07 ± 0,10 ± 0,08 ± 0,09 ± 0,06 ± 0,05 ± 0,03 ± 0,04 ±
0,01b 0,00a 0,01a 0,01a 0,01a 0,01b 0,02b 0,00b
36 2-Ethyl-3-methylpyrazine 87,8 ± 1,3 0,19 ± 0,21 ± 0,20 ± 0,15 ± 0,22 ± 0,15 ± 0,19 ± 0,13 ±
0,05 0,08 0,06 0,03 0,03 0,02 0,03 0,02
Pyridine
37 Pyridine * 98,7 ± 0,5 12,03 ± 8,78 ± 9,70 ± 8,81 ± 8,36 ± 10,21 ± 9,44 ± 8,90 ±
0,19a 0,31b 1,05b 0,27b 0,90a 0,89a 0,84a 0,21a
38 1-Acetyl-1,4- 82,8 ± 2,1 0,14 ± 0,13 ± 0,10 ± 0,09 ± 0,11 ± 0,09 ± 0,10 ± 0,08 ±
dihydropyridine 0,02 0,01 0,02 0,01 0,02 0,03 0,02 0,01
Pyrrole
39 1-Methylpyrrole * 96,1 ± 0,6 1,28 ± 0,82 ± 1,07 ± 0,99 ± 0,89 ± 1,04 ± 0,92 ± 0,77 ±
0,06a 0,13c 0,04b 0,11b 0,03a 0,05a 0,13a 0,10a
40 1-Methylpyrrole-2- 93,9 ± 1,2 0,12 ± 0,12 ± 0,12 ± 0,11 ± 0,08 ± 0,10 ± 0,09 ± 0,10 ±
carboxaldehyde 0,02 0,01 0,02 0,04 0,04 0,01 0,01 0,01
Alcohol
41 2-Methyl-3-Buten-2-ol* 91,6 ± 4,8 0,20 ± 0,17 ± 0,22 ± 0,26 ± 0,06 ± 0,22 ± 0,08 ± 0,20 ±
0,01b 0,02b 0,02a 0,02a 0,04b 0,03a 0,01b 0,02a
42 1-Pentanol* 96,1 ± 1,0 0,30 ± 0,14 ± 0,26 ± 0,10 ± 0,70 ± 0,34 ± 0,56 ± 0,25 ±
0,03a 0,02b 0,09a 0,03b 0,05a 0,02c 0,11b 0,05c
43 3- Methyl-3-Buten-1-ol 93,6 ± 1,6 0,06 ± 0,08 ± 0,05 ± 0,07 ± 0,04 ± 0,06 ± 0,04 ± 0,05 ±
0,01 0,00 0,00 0,03 0,01 0,01 0,00 0,01
Phenol
44 3-Methylphenol* 89,8 ± 0,7 0,44 ± 0,41 ± 0,41 ± 0,45 ± 0,34 ± 0,37 ± 0,30 ± 0,28 ±
0,04a 0,03a 0,04a 0,03a 0,03a 0,04a 0,02b 0,03b
Amine
45 5-Nonylamine 93,4 ± 0,5 2,32 ± 2,59 ± 2,46 ± 2,55 ± 2,07 ± 1,86 ± 1,84 ± 1,87 ±
0,05 0,11 0,17 0,27 0,22 0,06 0,08 0,07
46 4-Ethylamino-n-butylamine 83,3 ± 0,8 0,55 ± 0,52 ± 0,44 ± 0,38 ± 0,42 ± 0,59 ± 0,44 ± 0,40 ±
0,04 0,10 0,05 0,09 0,02 0,05 0,01 0,05
Amide
47 Methyl-urea* 92,7 ± 0,5 1,76 ± 2,60 ± 2,11 ± 2,52 ± 1,82 ± 1,38 ± 1,25 ± 1,39 ±
0,11c 0,20a 0,13b 0,20a 0,36a 0,06b 0,03b 0,17b

The mean values within the same row and at the same total fermentation time (48 h and 96 h) followed by different superscript letters in lowercase differ significantly
(p < 0.05) by the Scott-Knott test.

treatments containing the Lactiplantibacillus plantarum culture. 3.2. Volatile profile


The total fermentation time factor showed greater variation in the
flavor evaluation than in the aroma evaluation. Fruity flavors were more More than 800 volatile compounds have been found in green and
prevalent in the coffee beverage whose fruit had fermented for 96 h with roasted coffee beans (Farah et al., 2006). Two factors contribute to the
sequential inoculation (LPSC). The main sensory descriptor of this class formation of volatile compounds in coffee beans: compounds inherent to
was red fruits, as occurred for aroma. Zhang et al. (2019b) observed that the bean itself and microbial metabolites resulting from fermentation
the fermentation time had a greater impact on the sensory characteris­ processes (Yeretzian et al., 2002). Yeasts produce many low-molecular-
tics of coffee beverages than the type of processing when comparing weight volatile compounds during the fermentation of mucilage, such as
pulped coffee and coffee with total removal of mucilage. esters, higher alcohols, aldehydes, ketones, and terpenoids. Among
The LP coffee fruit had a sweet flavor at 48 h of fermentation and these volatile compounds, the most abundant groups were ethyl and
floral and nutty flavors at 96 h of fermentation. In the SC coffee acetate esters (Pereira et al., 2019). Recent studies have been conducted
beverage, the predominant flavors were cocoa and floral at 48 h of to select ester-producing yeasts, such as Candida parapsilosis, Pichia fer­
fermentation. The control had nutty and spices flavors after 48 h of mentans, P. guilliermondii, Saccharomyces cerevisiae, Torulaspora del­
fermentation, and cocoa and sweet flavors after 96 h of fermentation. brueckii, and Yarrowialipolytica, for use as starter cultures in the
Zhang et al. (2019b) observed that lactic acid bacteria played a role fermentation of coffee fruit and to increase the levels of these com­
in protecting the ecosystem from unwanted microorganisms, such as pounds in the beans (Bressani et al., 2018; Evangelista et al., 2014; Lee
Enterobacteria and Clostridia. The ecosystem was protected from the et al., 2017; Pereira et al., 2014; Pereira et al., 2015; Silva et al., 2013).
production of compounds that cause unpleasant aromas and flavors so The compounds n-butyl acetate, ethyl acetate, isoamyl acetate, propyl
that a desirable profile of aroma and flavor precursors could develop. In acetate, and ethyl hexanoate contributed to the development of fruity
addition to contributing to desirable aroma and flavor characteristics in sensory notes (Evangelista et al., 2014; Pereira et al., 2015).
coffee beverages, LAB starter cultures that grow in consortium with Forty-seven compounds were identified in this study, including al­
yeasts secrete compounds that cause the suppression of toxin-producing dehydes (4), acids (6), an ester (1), furans (9), a thiophene (1), a thiazole
filamentous fungi, such as Aspergillus ochraceus (Masswe & Lifa, 2010). (1), ketones (6), pyrazines (8), pyrroles (2), alcohols (3), a phenol (1),
The sequential inoculation technique used in the fermentation of amines (2), and an amide (1). To explain the chemical characteristics
coffee fruit contributes fruity and fermented aromas and flavors to the and clustering of the samples, the volatile composition was analyzed by
beverage of specialty natural coffee. The main sensory descriptors of the principal component analysis (Fig. 3). The first three principal compo­
natural coffee beverage fermented with LPSC were red fruits and winey. nents together explained 86.18 % of the total variability in the data. The

8
M. Helena Sances Rabelo et al. Food Chemistry 444 (2024) 138608

Table 2
Effect of total fermentation time on the mean relative areas of the volatile compounds of coffees fermented with different sequential inoculation treatments. Analysis of
variance and determination of the interaction between inoculation type and total fermentation time with the total fermentation time (48 h and 96 h) within each
inoculation treatment (LPSC, LP, SC, and control).
Volatile Compound Similarity LPSC LP SC Control
Index
48 h 96 h 48 h 96 h 48 h 96 h 48 h 96 h

Aldehyde
1 Butanal* 95,8 ± 0,4 0,84 ± 0,74 ± 0,70 ± 0,66 ± 0,92 ± 0,56 ± 0,79 ± 0,60 ±
0,07a 0,15a 0,11a 0,03a 0,09a 0,03b 0,07a 0,01b
2 2-Methylbutanal* 97,0 ± 0,0 5,62 ± 5,45 ± 5,91 ± 5,94 ± 6,17 ± 4,81 ± 6,86 ± 5,04 ±
0,31a 0,84a 0,17a 0,39a 0,66a 0,38b 0,73a 0,33b
3 3-Methylbutanal 96,2 ± 0,4 1,96 ± 1,82 ± 2,03 ± 2,02 ± 2,05 ± 1,62 ± 2,10 ± 1,68 ±
0,15 0,22 0,04 0,06 0,10 0,09 0,29 0,23
4 2,2-Dimethylpropanal* 90,5 ± 1,0 0,26 ± 0,15 ± 0,21 ± 0,23 ± 0,24 ± 0,18 ± 0,22 ± 0,20 ±
0,03a 0,03b 0,02a 0,04a 0,04a 0,01b 0,05a 0,03a
Acid
5 Acetic acid* 98,2 ± 0,4 5,31 ± 6,59 ± 6,95 ± 5,82 ± 6,04 ± 4,79 ± 5,74 ± 6,51 ±
0,47a 0,97a 0,55a 0,19a 0,38a 0,44a 0,16a 0,93a
6 Propionic acid* 89,9 ± 3,0 0,33 ± 0,41 ± 0,50 ± 0,29 ± 0,44 ± 0,25 ± 0,42 ± 0,31 ±
0,07a 0,04a 0,02a 0,07b 0,08a 0,01b 0,02a 0,04b
7 4-Hydroxybutanoic acid 94,2 ± 1,4 0,26 ± 0,21 ± 0,27 ± 0,24 ± 0,28 ± 0,25 ± 0,20 ± 0,27 ±
0,03 0,09 0,06 0,06 0,14 0,03 0,08 0,02
8 3-Methyl-butanoic acid* 94,2 ± 1,4 0,28 ± 0,31 ± 0,34 ± 0,20 ± 0,28 ± 0,16 ± 0,34 ± 0,21 ±
0,02a 0,00a 0,03a 0,04b 0,04a 0,02b 0,03a 0,02b
Ester
9 Ethyl acetate* 83,2 ± 0,4 8,16 ± 20,89 ± 2,99 ± 20,55 ± 10,23 ± 30,57 ± 3,41 ± 27,43 ±
0,61b 0,96a 0,44b 1,32a 0,99b 1,98a 0,90b 1,23a
10 Propanoic acid, ethyl 97,9 ± 1,5 0,33 ± 0,43 ± 0,10 ± 0,35 ± 0,23 ± 0,44 ± 0,08 ± 0,30 ±
ester* 0,01b 0,02a 0,00b 0,05a 0,04b 0,01a 0,02b 0,05a
11 2-Propenoic acid, ethyl 95,6 ± 1,3 0,08 ± 0,08 ± 0,00 ± 0,06 ± 0,05 ± 0,08 ± 0,00 ± 0,06 ±
ester* 0,01a 0,03a 0,00b 0,01a 0,01b 0,01a 0,00b 0,01a
Furan
12 2-Methylfuran 99,0 ± 0,0 9,19 ± 7,04 ± 8,57 ± 8,30 ± 8,73 ± 7,42 ± 9,30 ± 7,39 ±
0,77 0,41 0,72 0,99 0,72 0,32 0,24 0,57
13 2,5-Dimethylfuran 91,7 ± 2,6 0,51 ± 0,33 ± 0,33 ± 0,44 ± 0,43 ± 0,37 ± 0,42 ± 0,39 ±
0,02 0,13 0,09 0,06 0,01 0,13 0,08 0,07
14 2-Vinylfuran 93,8 ± 2,0 0,20 ± 0,14 ± 0,22 ± 0,16 ± 0,20 ± 0,14 ± 0,22 ± 0,14 ±
0,04 0,04 0,03 0,03 0,02 0,04 0,05 0,02
15 Furfurylmethyl ether 92,7 ± 1,1 0,12 ± 0,07 ± 0,13 ± 0,10 ± 0,11 ± 0,09 ± 0,11 ± 0,09 ±
0,02 0,01 0,01 0,01 0,00 0,01 0,03 0,00
16 Furfural* 98,6 ± 2,0 6,40 ± 6,26 ± 9,82 ± 5,03 ± 7,16 ± 3,66 ± 8,50 ± 4,41 ±
0,40a 0,75a 0,54a 0,26b 0,92a 0,31b 0,42a 0,37b
17 Furfuryl formate 93,8 ± 1,3 0,32 ± 0,22 ± 0,33 ± 0,29 ± 0,30 ± 0,18 ± 0,26 ± 0,22 ±
0,05 0,07 0,01 0,05 0,09 0,03 0,09 0,04
18 Furfuryl acetate* 93,7 ± 1,5 1,67 ± 1,17 ± 1,12 ± 1,42 ± 1,21 ± 1,46 ± 0,99 ± 1,46 ±
0,07a 0,12b 0,27b 0,14a 0,24b 0,02a 0,16b 0,07a
19 5-Methyl-furfural* 97,9 ± 0,3 2,98 ± 2,51 ± 3,55 ± 2,32 ± 2,95 ± 2,07 ± 3,65 ± 2,27 ±
0,13a 0,14b 0,07a 0,11b 0,05a 0,02b 0,39a 0,06b
20 Furfuryl alcohol 98,0 ± 0,0 15,79 ± 12,98 ± 17,19 ± 12,59 ± 15,10 ± 11,19 ± 16,92 ± 12,29 ±
0,68 0,89 0,39 0,47 1,02 0,60 0,68 0,90
Thiophene
21 Thiophene* 87,0 ± 1,9 0,17 ± 0,10 ± 0,11 ± 0,13 ± 0,14 ± 0,11 ± 0,13 ± 0,10 ±
0,01a 0,02b 0,01a 0,01a 0,01a 0,01b 0,01a 0,01b
Thiazole
22 Thiazole 79,8 ± 2,4 0,06 ± 0,06 ± 0,05 ± 0,04 ± 0,06 ± 0,04 ± 0,06 ± 0,04 ±
0,00 0,01 0,00 0,01 0,01 0,01 0,02 0,01
Ketone
23 2,3-Pentanedione* 92,6 ± 1,1 5,13 ± 4,59 ± 5,09 ± 4,34 ± 4,95 ± 3,79 ± 5,55 ± 3,59 ±
0,12a 0,30b 0,18a 0,20b 0,44a 0,13b 0,33a 0,17b
24 3,4-Hexanedione 97,9 ± 0,5 0,19 ± 0,14 ± 0,18 ± 0,15 ± 0,16 ± 0,12 ± 0,20 ± 0,13 ±
0,02 0,02 0,03 0,01 0,03 0,01 0,01 0,01
25 4-Hydroxy-3-hexanone* 90,5 ± 0,7 0,22 ± 0,18 ± 0,28 ± 0,17 ± 0,20 ± 0,16 ± 0,26 ± 0,15 ±
0,01a 0,01b 0,03a 0,02b 0,03a 0,01a 0,03a 0,02b
26 2-Hydroxy-3-pentanone* 82,7 ± 4,5 0,15 ± 0,14 ± 0,18 ± 0,10 ± 0,11 ± 0,11 ± 0,16 ± 0,11 ±
0,02a 0,03a 0,01a 0,00b 0,01a 0,02a 0,02a 0,04b
27 1-Hydroxy-2-butanone* 96,4 ± 1,9 0,91 ± 0,70 ± 1,15 ± 0,65 ± 0,78 ± 0,55 ± 0,94 ± 0,56 ±
0,04a 0,04b 0,13a 0,10b 0,02a 0,11b 0,08a 0,04b
28 1-(2-furanyl)-ethanone* 95,5 ± 0,9 0,28 ± 0,22 ± 0,43 ± 0,25 ± 0,36 ± 0,21 ± 0,51 ± 0,23 ±
0,06a 0,02a 0,08a 0,07 0,04a 0,04a 0,12a 0,03b
Pyrazine
29 Pyrazine 98,3 ± 0,4 1,78 ± 1,43 ± 1,78 ± 1,44 ± 1,71 ± 1,32 ± 1,84 ± 1,23 ±
0,11 0,06 0,06 0,13 0,16 0,12 0,11 0,06
30 Methylpyrazine * 97,3 ± 0,6 8,59 ± 7,49 ± 9,86 ± 7,21 ± 8,81 ± 6,13 ± 10,34 ± 6,20 ±
0,43a 0,15b 0,42a 0,36b 0,39a 0,76b 0,46a 0,17b
31 2,5-Dimethylpyrazine 97,5 ± 0,5 0,55 ± 0,49 ± 0,69 ± 0,45 ± 0,59 ± 0,43 ± 0,67 ± 0,45 ±
0,05 0,04 0,03 0,04 0,12 0,06 0,08 0,01
(continued on next page)

9
M. Helena Sances Rabelo et al. Food Chemistry 444 (2024) 138608

Table 2 (continued )
Volatile Compound Similarity LPSC LP SC Control
Index
48 h 96 h 48 h 96 h 48 h 96 h 48 h 96 h

32 2,6-Dimethylpyrazine* 98,0 ± 0,2 0,82 ± 0,62 ± 1,02 ± 0,69 ± 0,70 ± 0,55 ± 1,03 ± 0,56 ±
0,04a 0,02b 0,08a 0,03b 0,06a 0,06b 0,04a 0,05b
33 Ethylpyrazine 96,9 ± 0,5 0,89 ± 0,69 ± 1,02 ± 0,73 ± 0,88 ± 0,70 ± 0,98 ± 0,71 ±
0,07 0,15 0,05 0,11 0,10 0,09 0,04 0,04
34 2,3-Dimethylpyrazine* 94,7 ± 1,1 0,20 ± 0,21 ± 0,26 ± 0,17 ± 0,18 ± 0,19 ± 0,23 ± 0,21 ±
0,02a 0,03a 0,02a 0,01b 0,03a 0,03a 0,05a 0,01a
35 2-Ethyl-6- 91,6 ± 4,9 0,07 ± 0,06 ± 0,10 ± 0,05 ± 0,08 ± 0,03 ± 0,09 ± 0,04 ±
methylpyrazine* 0,01a 0,01a 0,00a 0,01b 0,01a 0,02b 0,01a 0,00b
36 2-Ethyl-3-methylpyrazine 87,8 ± 1,3 0,19 ± 0,22 ± 0,21 ± 0,15 ± 0,20 ± 0,19 ± 0,15 ± 0,13 ±
0,05 0,03 0,08 0,02 0,06 0,03 0,03 0,02
Pyridine
37 Pyridine * 98,7 ± 0,5 12,03 ± 8,36 ± 8,78 ± 10,21 ± 9,70 ± 9,44 ± 8,81 ± 8,90 ±
0,19a 0,90b 0,31a 0,89a 1,05a 0,84a 0,27a 0,21a
38 1-Acetyl-1,4- 82,8 ± 2,1 0,14 ± 0,11 ± 0,13 ± 0,09 ± 0,10 ± 0,10 ± 0,09 ± 0,08 ±
dihydropyridine 0,02 0,02 0,01 0,03 0,02 0,02 0,01 0,01
Pyrrole
39 1-Methylpyrrole * 96,1 ± 0,6 1,28 ± 0,89 ± 0,82 ± 1,04 ± 1,07 ± 0,92 ± 0,99 ± 0,77 ±
0,06a 0,03b 0,13b 0,05a 0,04a 0,13a 0,11a 0,10b
40 1-Methylpyrrole-2- 93,9 ± 1,2 0,12 ± 0,08 ± 0,12 ± 0,10 ± 0,12 ± 0,09 ± 0,11 ± 0,10 ±
carboxaldehyde 0,02 0,04 0,01 0,01 0,02 0,01 0,04 0,01
Alcohol
41 2-Methyl-3-Buten-2-ol* 91,6 ± 4,8 0,20 ± 0,06 ± 0,17 ± 0,22 ± 0,22 ± 0,08 ± 0,26 ± 0,20 ±
0,01a 0,04b 0,02b 0,03a 0,02a 0,01b 0,02a 0,02b
42 1-Pentanol* 96,1 ± 1,0 0,30 ± 0,70 ± 0,14 ± 0,34 ± 0,26 ± 0,56 ± 0,10 ± 0,25 ±
0,03b 0,05a 0,02b 0,02a 0,09b 0,11a 0,03b 0,05a
43 3- Methyl-3-Buten-1-ol 93,6 ± 1,6 0,06 ± 0,04 ± 0,08 ± 0,06 ± 0,05 ± 0,04 ± 0,07 ± 0,05 ±
0,01 0,01 0,00 0,01 0,00 0,00 0,03 0,01
Phenol
44 3-Methylphenol* 89,8 ± 0,7 0,44 ± 0,34 ± 0,41 ± 0,37 ± 0,41 ± 0,30 ± 0,45 ± 0,28 ±
0,04a 0,03b 0,03a 0,04a 0,04a 0,02b 0,03a 0,03b
Amine
45 5-Nonylamine 93,4 ± 0,5 2,32 ± 2,07 ± 2,59 ± 1,86 ± 2,46 ± 1,84 ± 2,55 ± 1,87 ±
0,05 0,22 0,11 0,06 0,17 0,08 0,27 0,07
46 4-Ethylamino-n- 83,3 ± 0,8 0,55 ± 0,42 ± 0,52 ± 0,59 ± 0,44 ± 0,44 ± 0,38 ± 0,40 ±
butylamine 0,04 0,02 0,10 0,05 0,05 0,01 0,09 0,05
Amide
47 Methyl-urea* 92,7 ± 0,5 1,76 ± 1,82 ± 2,60 ± 1,38 ± 2,11 ± 1,25 ± 2,52 ± 1,39 ±
0,11b 0,36a 0,20a 0,06b 0,13a 0,03b 0,20a 0,17b

The mean values within the same row and the same treatment (LPSC, LP, SC, and control) followed by different superscript letters in lowercase differ significantly (p <
0.05) by the Scott-Knott test.

samples were categorized into two groups according to the total volatile compounds detected during the fermentation processes. Ac­
fermentation time factor. This distinction was mainly due to the larger cording to Evangelista et al. (2014), esters at high concentrations in
number of volatile compounds isolated from coffee beans fermented for coffee beans may confer a flavor described as “over-fermented” to the
48 h. beverage, which is an undesirable characteristic for specialty coffees.
Principal component 1, which explained 60.76 % of the variability in The analysis of variance showed that the interaction between the
the data, was responsible for grouping the treatments according to the inoculation factors and total fermentation time was significant for the
total fermentation time factor. All treatments had the same number of compounds butanal, 2-methylbutanal, 2,2-dimethylpropanal, propanoic
chromatographic peaks, that is, the same number of volatile compounds. acid ethyl ester, 2-propenoic acid ethyl ester, acetic acid, propionic acid,
However, the treatments that fermented for 48 h showed peaks with a 3-methyl-butanoic acid, ethyl acetate, furfural, furfuryl acetate, 5-
greater relative area than the treatments that fermented for 96 h. In the methyl-furfural, thiophene, 2,3-pentanedione, 4-hydroxy-3-hexanone,
principal component analysis (Fig. 3), this difference allowed discrimi­ 2-hydroxy-3-pentanone, 1-hydroxy-2-butanone, 1-(2-furanyl)-etha­
nation of the treatments LPSC48h, LP48h, SC48h, and Control48h, none, methylpyrazine, 2,6-dimethylpyrazine, 2,3-dimethylpyrazine, 2-
which clustered on the same side of the graph. This result may be related ethyl-6-methylpyrazine, pyridine, 1-methylpyrrole, 2 -methyl-3-buten-
to the degradation and consumption of these compounds by microor­ 2-ol, 1-pentanol, 3-methyl-3-buten-1-ol, and methyl-urea.
ganisms due to the long fermentation time of the coffee fruit. The vol­ Upon comparing the inoculations and the control within the same
atile compounds most related to the LPSC48h treatment were 2,2- total fermentation time (Table 1; breakdown of the inoculation factor
dimethylpropanal, thiophene, 1-methylpyrrole, 2,5-dimethylfuran, and within the total fermentation time factor), the compounds furfuryl ac­
pyridine. The SC96h coffees were related to the compound ethyl acetate, etate, thiophene, pyridine, and 1-methylpyrrole showed peaks with
an ester with fruity sensory characteristics (Pereira et al., 2019) that is greater relative area in the coffee fruit fermented with sequential inoc­
widely found in coffees fermented by this yeast species (Evangelista ulation for 48 h (LPSC48h). When comparing the fermentation times
et al., 2014). Ethyl acetate was the volatile compound with the highest within each inoculation process (Table 2 breakdown of the total
relative peak area among all compounds found in this study (Table 1), fermentation time factor within the inoculation factor), all the com­
especially in coffees fermented for 96 h. This ester was also identified in pounds showed peaks with greater relative areas in the LPSC48h coffees,
the treatments with sequential inoculation (LPSC48h and LPSC96h), but with the exception of the propionic acid compound. Propionic acid
the relative area of its peak was smaller in those groups. Vale et al. showed a peak with a greater relative area at 48 h in both processes with
(2019) studied the use of mixed cultures in coffee fermentation and inoculation of pure cultures (LP and SC).
observed that ethyl acetate and ethanol compounds were the main The furfuryl acetate compound has been found in studies of coffee

10
M. Helena Sances Rabelo et al. Food Chemistry 444 (2024) 138608

fermentation with pure Saccharomyces cerevisiae cultures (Evangelista different inoculation methods. LWT –. Food Science and Technology, 92, 212–219.
https://doi.org/10.1016/j.lwt.2018.02.029
et al., 2014). This compound is characterized by sweet, fruity, and ba­
Cañas, P. M. I., Romero, E. G., Pérez-Martín, F., Seseña, S., & Palop, M. L. (2015).
nana aromas (Pereira et al., 2019). In sensory evaluation, fruity notes Sequential inoculation versus co-inoculation in Cabernet Franc wine fermentation.
were assigned to treatment LPSC48h, in agreement with the literature. Food Science and Technology International, 21(3), 203–212. https://doi-org.ez26.perio
In that treatment, the furfuryl acetate compound had the highest relative dicos.capes.gov.br/10.1177/1082013214524585.
Carvalho Neto, D. P. De, Pereira, G. V. De M., Finco, A. M. O., Rodrigues, C., De Carvalho,
peak area. A description of the aroma through reference to each volatile J. C., & Soccol, C. R. (2020). Microbiological, physicochemical and sensory studies of
compound identified in this study can be found in the Supplementary coffee beans fermentation conducted in a yeast bioreactor model. Food Biotechnology,
Material in Table 2. 34(2), 172–192.
Cassimiro, D. M. de J., Batista, N. N., Fonseca, H. C., Naves, J. A. O., Dias, D. R., &
Schwan, R. F. (2022). Coinoculation of lactic acid bacteria and yeasts increases the
4. Conclusion quality of wet fermented Arabica coffee. International Journal of Food Microbiology,
369. 10.1016/j.ijfoodmicro.2022.109627.
Evangelista, S. R., Miguel, M. G. C. P., Silva, C. F., Pinheiro, A. C. M., & Schwan, R. F.
The aroma and flavor of the coffee beverage whose fruit was fer­ (2015). Microbiological diversity associated with the spontaneous wet method of
mented with LPSC showed desirable sensory notes required of specialty coffee fermentation. International Journal of Food Microbiology, 210, 102–112.
natural coffees. The main sensory descriptors of aroma and flavor were https://doi.org/10.1016/j.ijfoodmicro.2015.06.008
Evangelista, S. R., Silva, C. F., Miguel, M. G. P. D. C, Cordeiro, C. d. S., Pinheiro, A. C. M.,
from the fruity and fermented classes. The coffee beans fermented with & Duarte, W. F., et al. (2014). Improvement of coffee beverage quality by using
sequential inoculation for 48 h showed volatile compounds with peaks selected yeasts strains during the fermentation in dry process. Food Research
of greater relative areas than the beans fermented for 96 h. The main International, 61, 183–195. 10.1016/j.foodres.2013.11.033.
Farah, A., Monteiro, A. C., Calado, V., Franca, A. S., & Trugo, L. C. (2006). Correlation
volatile compounds related to this treatment were furfuryl acetate and between cup quality and chemical attributes of Brazilian coffee. Food Chemistry, 98
ethyl acetate. Thus, this study shows the viability of the sequential (2), 373–380. https://doi.org/10.1016/j.foodchem.2005.07.032
inoculation technique for the formation of volatile compounds and the Freire, A. L., Ramos, C. L., da Costa Souza, P. N., Cardoso, M. G. B., & Schwan, R. F.
(2017). Nondairy beverage produced by controlled fermentation with potential
production of specialty natural coffees with complex sensory probiotic starter cultures of lactic acid bacteria and yeast. International Journal of
characteristics. Food Microbiology, 248, 39–46. https://doi.org/10.1016/j.ijfoodmicro.2017.02.011
García, C., Bautista, L., Rendueles, M., & Díaz, M. (2018). A new synbiotic dairy food
containing lactobionic acid and Lactobacillus casei. International Journal of Dairy
CRediT authorship contribution statement Technology, 72(1), 47–56. https://doi.org/10.1111/1471-0307.12558
García, C., Rendueles, M., & Díaz, M. (2017). Synbiotic fermentation for the co-
Mariane Helena Sances Rabelo: Writing – review & editing, production of lactic and lactobionic acids from residual dairy whey. Biotechnology
Progress, 33(5), 1250–1256. https://doi.org/10.1002/btpr.2507
Writing – original draft, Validation, Software, Methodology, Formal García, C., Rendueles, M., & Díaz, M. (2019). Liquid-phase food fermentations with
analysis, Data curation, Conceptualization. Flávio Meira Borém: Proj­ microbial consortia involving lactic acid bacteria: A review. Food Research
ect administration, Funding acquisition. Ana Paula de Carvalho Alves: International, 119, 207–220. https://doi.org/10.1016/j.foodres.2019.01.043
Gobbi, M., Comitini, F., Domizio, P., Romani, C., Lencioni, L., Mannazzu, I., & Ciani, M.
. Rodrigo Soares Pieroni: Resources, Funding acquisition, Formal (2013). Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and
analysis. Claudia Mendes Santos: Writing – review & editing. Makoto sequential co-fermentation: A strategy to enhance acidity and improve the overall
Nakajima: Resources, Funding acquisition. Ryosuke Sugino: Re­ quality of wine. Food Microbiology, 33(2), 271–281. https://doi.org/10.1016/j.
fm.2012.10.004
sources, Funding acquisition.
Haile, M., & Kang, W. H. (2019). The Role of Microbes in Coffee Fermentation and Their
Impact on Coffee Quality. Journal of Food Quality, 2019, 1–6. https://doi.org/
10.1155/2019/4836709
Declaration of competing interest Hranilovic, A., Gambetta, J. M., Jeffery, D. W., Grbin, P. R., & Jiranek, V. (2020). Lower-
alcohol wines produced by Metschnikowia pulcherrima and Saccharomyces
cerevisiae co-fermentations: The effect of sequential inoculation timing. International
The authors declare that they have no known competing financial
Journal of Food Microbiology, 329(16), Article 108651. https://doi.org/10.1016/j.
interests or personal relationships that could have appeared to influence ijfoodmicro.2020.108651
the work reported in this paper. Junqueira, A. C. O., Pereira, G. V. M., Medina, J. D. C., Alvear, M. C. R., Rosero, R.,
Carvalho Neto, D. P., Enriquez, H. G., & Soccol, C. R. (2019). First description of
bacterial and fungal communities in Colombian coffee beans fermentation analysed
Data availability using Illumina-based amplicon sequencing. Scientific reports, 9(1), 1–10. https://doi.
org/10.1038/s41598-019-45002-8
The data that has been used is confidential. Lee, L. W., Tay, G. Y., Cheong, M. W., Curran, P., Yu, B., & Liu, S. Q. (2017). Modulation
of the volatile and non-volatile profiles of coffee fermented with Yarrowiali polytica:
I. Green coffee. LWT –. Food Science and Technology, 77, 225–232. https://doi.org/
Acknowledgments 10.1016/j.lwt.2016.11.047
Lingle, T. R. (2011) The Coffee Cuppeŕs Handbook: Systematic Guide to the Sensory
Evaluation of Coffeés Flavor. (2011 Specialty Coffee Association of America, Ed.) (4,
The authors are grateful to the company Suntory Beverage & Food revisad ed.).
Limited- Japan, the Associação dos Cafeicultores da Canastra, CNPq - Lu, Y., Putra, S. D., & Liu, S. Q. (2018). A novel non-dairy beverage from durian pulp
fermented with selected probiotics and yeast. International Journal of Food
Conselho Nacional de Desenvolvimento Científico e Tecnológico,
Microbiology, 265, 1–8. https://doi.org/10.1016/j.ijfoodmicro.2017.10.030
FAPEMIG - Fundação de Amparo para Pesquisa do Estado de Minas Lu, Z. M., Wang, Z. M., Zhang, X. J., Mao, J., Shi, J. S., & Xu, Z. H. (2018). Microbial
Gerais, CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível ecology of cereal vinegar fermentation: Insights for driving the ecosystem function.
Superior, and INCT/Café - Instituto Nacional de Ciência e Tecnologia do Current Opinion in Biotechnology, 49, 88–93. https://doi.org/10.1016/j.
copbio.2017.07.006
Café. Massawe, G. A., & Lifa, S. J. (2010). Yeasts and lactic acid bacteria coffee fermentation
starter cultures. International Journal of Postharvest Technology and Innovation, 2(1),
41–82. https://doi.org/10.1504/IJPTI.2010.038187
Appendix A. Supplementary material
Pereira, G. V. M., Carvalho Neto, D. P., Magalhaes, A. L., Vasquez, Z. S.,
Medeiros, A. B. P., Vandenberghe, L. P. S., & Soccol, C. R. (2019). Exploring the
Supplementary data to this article can be found online at https://doi. impacts of postharvest processing on the aroma formation of coffee beans - A review.
Food Chemistry, 272, 441–452. https://doi.org/10.1016/j.foodchem.2018.08.061
org/10.1016/j.foodchem.2024.138608.
Pereira, G. V. M., Carvalho Neto, D. P., Medeiros, A. B. P., Soccol, V. T., Neto, E.,
Woiciechowski, A. L., & Soccol, C. R. (2016). Potential of lactic acid bacteria to
References improve the fermentation and quality of coffee during on-farm processing.
International Journal of Food Science Technology, 51(7), 1689–1695. https://doi.org/
10.1111/ijfs.13142
Adams, J., Williams, A., Lancaster, B., & Foley, M. (2007). Advantages and uses of check
Pereira, G. V. M., Neto, E., Soccol, V. T., Medeiros, A. B. P., Woiciechowski, A. L., &
all-that-apply response compared to traditional scaling of attributes for salty snacks.
Soccol, C. R. (2015). Conducting starter culture-controlled fermentations of coffee
In 7th Pangborn Sensory Science Symposium, 12–16 August, Minneapolis, MN, USA.
beans during on-farm wet processing: Growth, metabolic analyses and sensorial
Bressani, A. P. P., Martinez, S. J., Evangelista, S. D., & Schwan, R. F. (2018).
Characteristics of fermented coffee inoculated with yeast starter cultures using

11
M. Helena Sances Rabelo et al. Food Chemistry 444 (2024) 138608

effects. Food Research International, 75, 348–356. https://doi.org/10.1016/j. Spencer, M., Sage, E., Velez, M., & Guinard, J. X. (2016). Using Single Free Sorting and
foodres.2015.06.027 Multivariate Exploratory Methods to Design a New Coffee Taster’s Flavor Wheel.
Pereira, G. V. M., Soccol, V. T., Pandey, A., Medeiros, A. B. P., Andrade Lara, J. M. R., Journal of Food Science, 81(12), S2997–S3005. https://doi.org/10.1111/1750-
Gollo, A. L., & Soccol, C. R. (2014). Isolation, selection and evaluation of yeasts for 841.13555
use in fermentation of coffee beans by the wet process. International Journal of Food Vale, A. S., Pereira, V. M. P., Carvalho Neto, D. P., Rodrigues, C., Pagnoncelli, M. G. B., &
Microbiology, 188, 60–66. https://doi.org/10.1016/j.ijfoodmicro.2014.07.008 Soccol, C. R. (2019). Effect of Co-Inoculation with Pichia fermentans and Pediococcus
Rabelo, M. H. S., Borém, F. M., Lima, R. R. de, Alves, A. P. de C., Pinheiro, A. C. M., acidilactici on Metabolite Produced During Fermentation and Volatile Composition of
Ribeiro, D. E., Santos, C. M., Pereira, R. G. F. A. (2020). Impacts of Quaker beans Coffee Beans. Fermentation., 5(67), 1–17. https://doi.org/10.3390/
over sensory characteristics and volatile composition of specialty natural coffees. fermentation5030067
Food Chemistry, 342(16), 128304. 10.1016/j.foodchem.2020.128304. Wang, C., Sun, J., Lassabliere, B., Yu, B., Zhao, F., Zhao, F., Chen, Y., & Liu, S. Q. (2019).
Ribeiro, L. S., Pedrozo, M. G. C. P., Evangelista, S. R., Martins, P. M. M., van Mullem, J., Potential of lactic acid bacteria to modulate coffee volatiles and effect of glucose
Belizario, M. H., & Schwan, R. F. (2017). Behavior of yeast inoculated during semi- supplementation: Fermentation of green coffee beans and impact of coffee roasting.
dry coffee fermentation and the effect on chemical and sensorial properties of the Journal of The Science of Food and Agriculture, 99(1), 409–420. https://doi.org/
final beverage. Food Research International, 92, 26–32. https://doi.org/10.1016/j. 10.1002/jsfa.9202
foodres.2016.12.011 Yeretzian, C., Jordan, A., Badoud, R., & Lindinger, W. (2002). From the green bean to the
Sieuwerts, S., de Bok, F. A. M., Hugenholtz, J., & van Hylckama Vlieg, J. E. (2008). cup of the coffee: Investigating coffee roasting by on-line monitoring of volatiles.
Unraveling microbial interactions in food fermentations: From classical to genomics European Food Research and Technology, 214(2), 92–104. https://doi.org/10.1007/
approaches. Applied and Environmental Microbiology, 74(16), 4997–5007. https://doi. s00217-001-0424-7
org/10.1128/AEM.00113-08 Yunita, D., & Dodd, C. E. R. (2018). Microbial community dynamics of a blue-veined raw
Silva, C. F., Batista, L. R., Abreu, L. M., Dias, E. S., & Schwan, R. F. (2008). Succession of milk cheese from the United Kingdom. Journal of Dairy Science, 101(6), 4923–4935.
bacterial and fungal communities during natural coffee (Coffea arabica) https://doi.org/10.3168/jds.2017-14104
fermentation. Food Microbiology, 25(8), 951–957. https://doi.org/10.1016/j. Zhang, S. J., De Bruyn, F., Pothakos, V., Contreras, G. F., Cai, Z., Moccand, C., Weckx, S.,
fm.2008.07.003 & De Vuyst, L. (2019a). Influence of Various Processing Parameters on the Microbial
Silva, C. F., Vilela, D. M., Cordeiro, C. S., Duarte, W. F., Dias, D. R., & Schwan, R. F. Community Dynamics, Metabolomic Profiles, and Cup Quality During Wet Coffee
(2013). Evaluation of a potential starter culture for enhance quality of coffee Processing. Frontiers in Microbiology, 10, 2621. https://doi.org/10.3389/
fermentation. World Journal of Microbiology and Biotechnology, 29(2), 235–247. fmicb.2019.02621
https://doi.org/10.1007/s11274-012-1175-2 Zhang, S. J., De Bruyn, F., Pothakos, V., Torres, J., Falconi, C., Moccand, C., Weckx, S., &
Spence, C. (2015). Just how much of what we taste derives from the sense of smell? De Vuyst, L. (2019b). Following coffee production from cherries to cup:
Flavour, 4, 30. https://doi.org/10.1186/s13411-015-0040-2 Microbiological and metabolomic analysis of wet processing of Coffea arabica.
Spence C. (2016) The neuroscience of flavor. In: Piqueras-Fiszman B, Spence C, eds. Applied and Environmental Microbiology, 85(6), 1–22. https://doi.org/10.1128/
Multisensory Flavor Perception: From Fundamental Neuroscience through to the AEM.02635-18
Marketplace. Oxford, UK: Elsevier; 2016:235e248.

12

You might also like