Research Article

Received: 4 November 2017 Revised: 9 May 2018 Accepted article published: 13 June 2018 Published online in Wiley Online Library: 3 August 2018

(wileyonlinelibrary.com) DOI 10.1002/jsfa.9202

Potential of lactic acid bacteria to modulate

coffee volatiles and effect of glucose
supplementation: fermentation of green coffee
beans and impact of coffee roasting
Chenhui Wang,a Jingcan Sun,b Benjamin Lassabliere,b Bin Yu,b
Feifei Zhao,c Fangju Zhao,c Ying Chenc and Shao Quan Liua,d*

Abstract
BACKGROUND: Coffee flavor can be significantly influenced by microbial activities in spontaneous fermentation of coffee
cherries. The potential of lactic acid bacteria for flavor modulation through controlled fermentation of green coffee beans has
not been explored.

RESULTS: Fermentation by Lactobacillus rhamnosus HN001 with and without 1% w/w glucose supplementation led to modifi-
cation of flavor-related constituents in green coffee beans, which translated into modulation of coffee volatiles upon roasting.
The lactic acid bacteria consumed almost all glucose and fructose, leaving sucrose behind. Amino acids and malic, citric, and
succinic acids were partially catabolized. Glucose supplementation enhanced lactic acid production but repressed acetic acid
formation. After roasting at 235 ∘ C for 9 min, 12 min, and 15 min, the levels of furfurals in glucose-supplemented-fermented
coffee were 10.5-, 2.7-, and 1.1-fold higher than those in the controls (nonsupplemented-unfermented coffee); furthermore, the
levels of pyrazines in the controls were 11.9-, 10.1-, and 6.5-fold higher than those in the treated coffee. Glucose-supplemented
fermentation yielded roasted coffee with stronger caramelic and burnt characteristics but weaker nutty notes. In roasted
non-supplemented-fermented coffee, volatile production was generally reduced, resulting in a milder overall aroma.

CONCLUSION: Lactic acid fermentation of green coffee beans is a new strategy for coffee flavor modulation, creating novel aroma
characteristics.
© 2018 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: coffee; lactic acid fermentation; Lactobacillus rhamnosus; volatile precursors; coffee flavor

INTRODUCTION attracted much research interest. Inoculation of selected starter

The flavor of coffee is a decisive consumer parameter and signif-
icantly influenced by the on-farm processing to release the cof-
fee green beans from the cherries. On-farm processing is mainly
conducted through wet, dry, and semi-dry methods.1 The wet
method features the submerged spontaneous fermentation of
the de-pulped cherries to degrade the mucilage, while the dry
method usually involves sun-drying of the whole coffee cherries
and mechanical separation of the dried green beans. In semi-dry
processing, de-pulped cherries are sun-dried and milled to release
the green beans.2
Microbial activities during on-farm processing are thought to
play an important role in the development of coffee flavor.1 Proper
mucilage fermentation in wet processing modulates the con-
stituents in green coffee beans and characterizes the final product
with preferable flavor.3 Involving a wide range of naturally occur-
ring microorganisms, including yeast, bacteria, and fungi,4–7 the
spontaneous fermentation suffers from the lack of controllability
cultures to modify the natural microbiota in mucilage fermen-
tation has been introduced to enhance the consistency of the
process and, moreover, to enhance or impart certain sensorial
attributes. The yeast Pichia fermentans and the lactic acid bac-
terium (LAB) Lactobacillus plantarum seem to be suitable starter
cultures for such purposes.8,9 Furthermore, fermentation of whole
∗ Correspondence to: SQ Liu, Department of Chemistry, Food Science and
Technology Programme, National University of Singapore, Science Drive 3,
Singapore, 117543, Singapore. E-mail: chmlsq@nus.edu.sg
a Department of Chemistry, Food Science and Technology Programme, National
University of Singapore, Singapore, Singapore
b Mane SEA Pte Ltd, Singapore
c Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd,
Shanghai, China
409

and consistency.1 The need for improvement of the processing has d National University of Singapore (Suzhou) Research Institute, Suzhou, China

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 www.soci.org
coffee cherries with selected fermentation microorganisms has
also been reported.10
Apart from modulating microbiota during mucilage or cherry
fermentation, attempts have been made regarding the direct
application of fully controlled microbial fermentation on green
coffee beans.1,11 In comparison with the spontaneous fermenta-
tion of de-pulped coffee cherries in the wet on-farm processing,
such a process features fully controlled fermentation of green
coffee beans with the inoculated starter cultures. As the starter
cultures are not restricted to those isolated from spontaneous
coffee fermentation, microorganisms traditionally used in other
food fermentations can be applied because of their availability,
unique metabolic functions, and ease of handling.11–13 The fun-
gus Rhizopus oligosporus and the yeasts Yarrowia lipolytica and
Saccharomyces cerevisiae have been shown to successfully modify
roasted coffee aroma attributes through the alteration of aroma
precursors and generation of volatile metabolites in the green cof-
fee beans.11–15 Still, there remains a need to expand the pool of
suitable fermenting microorganisms for such a process. With the
ability to significantly modify various flavor-related constituents,
including carbohydrates, organic acids, amino acids, and phenolic
acids,16 LABs play an important role in spontaneous coffee fermen-
tation and have been used as starter cultures for fermentation of
the de-pulped coffee cherries.4,5,7,9 Although LABs have been pro-
posed as suitable starter cultures for the modification of the con-
stituents in green coffee beans, such an application with specific
LAB strains has not been documented.11
As a member of LABs, Lactobacillus rhamnosus is a faculta-
tive heterofermenter and has been demonstrated as a suitable
bio-producer of lactic acid and diacetyl.17–19 Fermentations of
plant products by this Lactobacillus species have also been fre-
quently reported, and its robustness has been demonstrated in
fermentations of cereals, legumes, soymilk, and fruit juices.20–22
Thus, L. rhamnosus has the potential to alter various flavor-related
constituents in green coffee beans, which may lead to the modifi-
cation of final coffee flavor after roasting. Originally isolated from
a dairy source, the probiotic strain L. rhamnosus HN001 has been
proven to be safe for human consumption and commercialized as
a dietary supplement.23,24
In this study, we present the modulation of coffee flavor through
L. rhamnosus HN001 fermentation of green coffee beans with
and without glucose supplementation. The effects of fermenta-
tion on the flavor-related constituents in the green coffee beans
were studied. Following the roasting of the treated green coffee
beans, detailed analyses were conducted to reveal the impact of
L. rhamnosus fermentation on the volatile profiles of roasted coffee
beans.
MATERIALS AND METHODS
Green coffee beans, microorganism, and chemicals
Green coffee beans (Coffea arabica) from Indonesia were sup-
plied by Yong-In Traders Pte Ltd (Singapore). L. rhamnosus HN001
was obtained from Danisco A/S (Copenhagen, Denmark) in
freeze-dried form.
High-performance liquid chromatography (HPLC)-grade ace-
tonitrile was procured from Tedia (Ohio, USA); HPLC-grade
methanol, ACS-grade ethanol, acetone, and petroleum ether
(boiling point 35–60 ∘ C), and glycerol were from Merck (Darm-
stadt, Germany); acetic acid was from RCI Labscan (Bangkok,
Thailand); and sulfuric acid was from VWR (Pennsylvania, USA).
410
Glucose, fructose, sucrose, quinic, lactic, citric, caffeic, ferulic,
wileyonlinelibrary.com/jsfa © 2018 Society of Chemical Industry
C Wang et al.
p-coumaric, and chlorogenic (5-caffeoylquinic) acids, and tryp-
tophan were procured from Sigma-Aldrich (Missouri, USA); malic
and succinic acids were from Fluka Analytical (Steinheim, Austria);
amino acid standard H and phosphate-buffered saline (PBS) were
from Thermo Scientific (Illinois, USA). AccQ-Tag Ultra Chemistry
Kit, consisting of AccQ-Fluor reagent kit and AccQ-Tag eluent A
concentrate, was bought from Waters (Dublin, Ireland). Satu-
rated alkane standards (C7 –C40 ) were purchased from Supelco,
Sigma-Aldrich (Barcelona, Spain). Bacteriological peptone and
de Man–Rogosa–Sharpe (MRS) broth and agar were purchased
from Oxoid (Hamsphire, UK).
Inoculum preparation
A pure stock culture was prepared by incubating the freeze-dried
L. rhamnosus HN001 in MRS broth at 37 ∘ C for 24 h and stored in
glycerol (15% v/v) at −80 ∘ C. To prepare an inoculum, a thawed
stock culture was inoculated into MRS broth and subcultured
at 37 ∘ C for 24 h twice. L. rhamnosus cells were harvested by
centrifuging and washed twice with 100 mmol L−1 PBS (20 mL
each). Subsequently, the pellet was resuspended in 100 mmol L−1
PBS (20 mL) to obtain the inoculum.
Fermentation of green coffee beans
Submerged fermentation of green coffee beans (with an origi-
nal moisture content of around 7%) using L. rhamnosus HN001
was conducted in triplicate with and without 1% w/w glucose
supplementation. The treatments of green coffee beans included:
control without supplementation (G-CN), fermentation without
supplementation (G-FN), control with supplementation (G-CS),
and fermentation with supplementation (G-FS). For all the treat-
ments, including two fermentations and two controls, the mix-
ture of green coffee beans (1 kg) and deionized water (2.2 kg) was
heat treated together by boiling for 30 min and cooling to room
temperature. For glucose-supplemented fermentation and con-
trol, the heat-treated mixture of green coffee beans and water
was supplemented with 100 mL of filter-sterilized glucose solu-
tion (33% w/v) to obtain a final glucose concentration of 1% w/w.
For nonsupplemented fermentation and control, 100 mL of steril-
ized water was added. The fermented media were inoculated with
the inoculum described earlier to obtain an initial cell population
of approximately 7.5 log CFU mL−1 in the media. For the control
media, 100 mmol L−1 PBS (20 mL) was added. Incubation was con-
ducted at 37 ∘ C for 72 h. Samples (5 mL of liquor and 10 g of green
coffee beans) were collected from the fermentation media at 0, 12,
24, 48, and 72 h. The pH of the fermentation liquor was measured
with a pH meter, model FEP20 (Metrohm, Herisau, Switzerland).
The moisture content of green coffee beans stabilized at around
54% after the heat treatment and throughout the fermentation.
The moisture measurements were conducted with 5 g green cof-
fee beans sampled at each time point using a moisture analyzer
(MOC-120H, Shimadzu, Kyoto, Japan). After the incubation of 72 h,
the remaining liquid was drained off, and the green coffee beans
were dried in an oven at 45 ∘ C until the moisture content reached
approximately 7%. The dried green coffee beans were ground
and sieved (aperture diameter: 426 μm) prior to volatile and non-
volatile analyses. The grounds and remaining green coffee beans
were stored at −30 ∘ C.
Enumeration of L. rhamnosus HN001
Samples containing 5 mL of liquor and 5 g of green coffee
beans from fermentation media were shaken well and the liquor
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was diluted serially with 0.1% w/v peptone solution and then
spread-plated on MRS agar. Plates were incubated at 37 ∘ C for
48 h.

Roasting of green coffee beans

Roasting of coffee beans (120 g), including G-CN, G-FN, and G-FS,
was conducted in triplicate with a Genecafe batch-top roaster,
mode CR-100 (Genesis, Ansan, Korea), at 235 ∘ C for 9, 12, and
15 min, representing light, medium, and dark roasts respectively.
The roasting temperature was selected according to the manu-
facturer’s instruction and previous trials. The roasted coffees from
G-CN, G-FN, and G-FS were denoted as R-CN, R-FN, and R-FS
respectively. The three roast times were denoted as 9, 12, and 15.
The G-CS was not roasted to avoid the interference from the added
glucose, and R-CN served as the control for both R-FN and R-FS.
The roasted coffee beans were ground and sieved (aperture diam-
eter: 426 μm) for further analyses. The roasted coffee beans and
grounds were stored at −30 ∘ C. Roast degrees were assessed with
L* (lightness) values that were measured with ground-roasted cof-
fees using a Konica Minolta CM-3500d spectrophotometer with a
D65 illuminant (Osaka, Japan).

Quantification of carbohydrates, phenolic acids, organic

acids, and amino acids in green and roasted coffee beans
Extraction of nonvolatiles
Nonvolatile extractions were conducted following the methods
described by Lee et al. with modifications.14 Grounds of green cof-
fee beans (72 h) and grounds of roasted coffees were defatted with
petroleum ether in a Soxhlet extractor, mode SOX 416 (Garhardt,
Königswinter, Germany), and stored at −30 ∘ C prior to extraction.
Carbohydrates were extracted separately by shaking the defatted
coffee ground (2 g) in aqueous ethanol (80% v/v, 3 × 20 mL). Phe-
nolic acids were extracted by shaking the defatted coffee ground
(2 g) in a mixture of methanol, acetone, and water (7 : 7 : 6 v/v/v,
4 × 20 mL) for 30 min. The extracts of carbohydrates and phenolic
acids were evaporated to dryness and reconstituted with deion-
ized water (2 mL) and aqueous methanol (80% v/v, 2 mL) respec-
tively. Organic and amino acids were co-extracted by shaking the
coffee ground (2 g) in deionized water (10 mL) for 30 min, and the Figure 1. Growth of Lactobacillus rhamnosus HN001 (a) and the change
extract was obtained by centrifugation. of pH in the media (b) during fermentations. G, green coffee beans;
CN, control without glucose supplementation; FN, fermented without
glucose supplementation; FS, fermented with glucose supplementation;
HPLC analyses CS, control with glucose supplementation; Cell counts and pH are the
The extracts from green (72 h) and roasted coffee beans, together means of triplicate fermentations (n = 3), with error bars representing the
with the liquors from the fermentation media (72 h), were filtered standard deviation of the mean. Data points with an asterisk indicate
through 0.2 𝜇m regenerated cellulose membranes (Sartorius, Got- statistical differences between fermented green coffee beans with and
without supplementation (P < 0.05).
tingen, Germany) before analyses. HPLC analyses of carbohydrates
(fructose, glucose, and sucrose), phenolic acids (chlorogenic, caf-
feic, ferulic, and p-coumaric acids), organic acids (citric, lactic, (100%), as described by Cheong et al.25 Derivatization and HPLC
acetic, malic, quinic, and succinic acids), and amino acids were analysis of amino acids were performed according to the method
performed using a Shimadzu Prominence system (Kyoto, Japan). provided by the manufacturer.26 An evaporative light-scattering
Carbohydrates were separated using a Zorbax carbohydrate col- detector was used for analyses of carbohydrates, and a photodi-
umn (150 mm × 4.6 mm) coupled with a Zorbax NH2 guard col- ode array detector was used for analyses of phenolic, organic, and
umn (12.5 mm × 4.6 mm; Agilent, Santa Clara, CA, USA) at 30 ∘ C amino acids, at 320 nm, 210 nm, and 248 nm respectively. Identifi-
with acetonitrile (80% v/v, flow rate 1 mL min−1 ) as the mobile cation and quantification of the nonvolatiles were achieved using
phase. Organic acids were resolved with a Supelcogel C-610 H col- authentic standards with external calibration curves constructed.
umn (Supelco, Bellefonte, PA, USA) at 40 ∘ C with aqueous sulfu-
ric acid (0.1%, v/v, flow rate 0.4 mL min−1 ) as the mobile phase. Volatile analysis with headspace solid-phase microextraction
Phenolic acids were separated in a Zorbax Eclipse C18 column gas chromatography–mass spectrometry/flame ionization
(150 mm × 4.6 mm) coupled with a Zorbax C18 guard column detection
(12.5 mm × 4.6 mm) at 40 ∘ C with gradient elution of a binary The coffee grounds (2 g) were sealed into a headspace vial
(10 mL) for the volatile extraction at 60 ∘ C for 30 min with a
411

mobile phase of aqueous acetic acid (0.1% v/v) and methanol

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 www.soci.org C Wang et al.

Table 1. Effects of Lactobacillus rhamnosus fermentation with and without glucose supplementation for 72 h on sugars, organic
acids, and phenolic acids in green coffee beans and 12-min-roasted coffees
Concentration (×103 mg kg−1 dry matter) Concentration (×103 mg kg−1 dry
in green coffee beans matter) in 12-min roasted coffees

Nonsupplemented Supplemented Nonsupplemented Supplemented

Compound G-CN G-FN G-CS G-FS R-CN12 R-FN12 R-FS12

Soluble saccharides
Fructose 0.141 ± 0.011aB ND 0.136 ± 0.004a tr 0.884 ± 0.005aA 0.810 ± 0.021b ND
Glucose 0.293 ± 0.006bB ND 9.98 ± 0.70a 0.182 ± 0.013c 0.497 ± 0.024aA 0.349 ± 0.024b ND
Sucrose 25.5 ± 0.4aA 23.7 ± 2.2aA 26.6 ± 0.2a 24.2 ± 3.0aA 9.04 ± 0.30bB 14.5 ± 1.03aB 8.01 ± 7.76bB
Total 25.8 ± 0.5bA 23.7 ± 2.2bA 36.8 ± 0.9a 27.9 ± 6.3bA 10.4 ± 0.31bB 15.6 ± 1.02aB 8.01 ± 7.76cB
Organic acids
Lactic acid 1.07 ± 0.09bA 6.45 ± 0.12aA 1.07 ± 0.09b 29.1 ± 0.3bA 0.508 ± 0.061cB 6.30 ± 0.50bA 24.3 ± 0.58aB
Acetic acid ND 2.21 ± 0.06B ND tr ND 4.14 ± 0.41A ND
Citric acid 7.75 ± 0.38aA tr 7.85 ± 0.35a 5.13 ± 0.03bA 5.13 ± 0.12aB tr 2.21 ± 0.03bB
Malic acid 2.89 ± 0.16aA 0.713 ± 0.023bA 2.69 ± 0.12a 0.614 ± 0.005bA 3.30 ± 0.11aA 0.324 ± 0.039cB 2.13 ± 0.07bB
Succinic acid 1.16 ± 0.05aB 0.765 ± 0.023cB 1.24 ± 0.01a 0.901 ± 0.055bB 4.07 ± 0.41bA 4.59 ± 0.23bA 6.69 ± 0.36aA
Quinic acid 2.38 ± 0.04aB 1.70 ± 0.06bB 2.29 ± 0.09a 2.08 ± 0.01bB 10.1 ± 0.15aA 7.05 ± 0.74bA 5.66 ± 0.07cA
Total 15.4 ± 0.7bB 12.0 ± 0.2cB 15.3 ± 0.4b 37.9 ± 0.3aA 23.1 ± 0.32bA 22.5 ± 2.22bA 40.1 ± 0.88aA
Phenolic acids
Chlorogenic acid (5-CQA) 32.2 ± 0.9aA 27.0 ± 0.9bA 30.9 ± 1.7a 28.7 ± 0.5bA 11.4 ± 0.6aB 10.6 ± 1.1aB 11.3 ± 0.6aB
Caffeic acid 0.175 ± 0.006bA 0.207 ± 0.002aA 0.176 ± 0.004b 0.218 ± 0.004aA 0.0171 ± 0.0006aB 0.0148 ± 0.0011abB 0.0143 ± 0.0003bB
Ferulic acid 0.0524 ± 0.0005b 0.0582 ± 0.0012a 0.0522 ± 0.0002b 0.0604 ± 0.0013a trace trace trace
p-Coumaric acid ND ND ND ND ND ND ND
Total 32.4 ± 0.9aA 27.3 ± 0.9bA 31.1 ± 1.7a 31.0 ± 0.5aA 11.4 ± 0.6aB 10.6 ± 1.1aB 11.3 ± 0.6aB

G, green coffee beans; R, roasted coffees; CN, control without glucose supplementation; FN, fermented without glucose supplementation; CS, control with glucose
supplementation; FS, fermented with glucose supplementation; G-CS was not roasted to prevent the interference from the added glucose; only 12-min-roasted coffees
were analyzed for nonvolatiles as the 12 min roast time led to the most differentiated volatiles profile among the coffees roasted to three different degrees. Values are
expressed as the mean plus/minus standard deviation of triplicate fermentations (n = 3). Mean values in each row with different lower-case letters (a–c) indicate statistical
differences among the green coffee beans or the roasted coffees with different treatments. Mean values in each row with different upper-case letters (A, B) indicate statistical
differences between the green coffee bean and the roasted coffee with the same treatment. ND, not detected; tr, concentration below limit of quantitation.

Carboxen/polydimethylsiloxane fiber (85 𝜇m film thickness;
Supelco, Bellefonte, PA, USA). Volatile analysis was conducted with
a gas chromatograph (7890 N, Agilent Technologies, Palo Alto, CA,
USA) equipped with a DB-FFAP column (60 m × 0.25 mm, 0.25 𝜇m
film thickness), and coupled to a flame ionization detector (FID)
and inert mass spectrometer (mode 5975). The injector was oper-
ated in the splitless mode at 250 ∘ C. The flow rate of the carrier
gas (helium) was maintained at 1.2 mL min−1 . The oven temper-
ature was programmed from 50 ∘ C (5 min) to 230 ∘ C (30 min) at
5 ∘ C min−1 . The mass spectrometry (MS) analysis was performed
in the full scan mode, recording electron ionization mass spec-
tra (m/z 40–500). The FID was operated at 250 ∘ C. The volatile
analyses of all the samples were conducted in one continuous
session with the same solid-phase microextraction (SPME) fiber
for a better consistency. Identification of volatiles was done by
matching their linear retention indices in relation to the mixture
of n-alkanes (C7 –C40 ) standards to the literature values and mass
spectral comparison with the NIST library. The FID peak areas were
used for the semi-quantification of the volatiles.
Evaluation of aroma profiles of the 12-min-roasted coffees
Because the medium roast (12 min) best differentiated the volatile
profiles of the three coffees, aroma profiles of the 12-min-roasted
coffees were analyzed at the Wilmar (Shanghai) Biotechnology
Research and Development Center, China. The panel consisted of
seven experienced panelists (four females and three males, age
412
the aroma attributes – including nutty, roasted, caramelic, burnt,
smoky, and acidic, which were selected due to their distinctiveness
in the roasted coffee samples. The scale used was of 0–15 (0.5
increments), with ‘0’ indicating ‘not detected’ and ‘15’ indicating
‘strongest intensity’. The coffee grounds (10 g) from the three
12-min-roasted coffees were prepared just before the evaluation
and each was served in a 30 mL brown glass sniff bottle. The
evaluation was conducted in triplicate in three separate sessions.
Statistical analysis
The analysis of variance (P < 0.05), Tukey’s test, and principal
component analysis (PCA) were performed with XLSTAT 19.02
(Addinsoft, New York, NY, USA).
RESULTS
Growth of L. rhamnosus and changes of pH
Preliminary experiments showed that the growth of L. rhamnosus
HN001 was inhibited in solid-state fermentation of soaked
green coffee beans (data not presented), suggesting such
conditions were unsuitable. Thus, submerged fermentation of
green coffee beans was conducted. An overall increase of about
2 log CFU mL−1 within 24 h was achieved in both nonsupple-
mented and glucose-supplemented fermentations, and the
LAB populations maintained afterwards (Fig. 1(a)). The 1% w/w
glucose supplementation led to significantly faster growth, par-

20–40 years). Quantitative descriptive analysis was performed on ticularly in the first 12 h (P < 0.05). In the unfermented controls,

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Figure 2. Effects of Lactobacillus rhamnosus fermentation with and without glucose supplementation for 72 h on amino acids in the green coffee beans.
G, green coffee beans; CN, control without glucose supplementation; FN, fermented without glucose supplementation; FS, fermented with glucose
supplementation; CS, control with glucose supplementation. Values of amino acid contents are the means of triplicate fermentations (n = 3), with error
bars representing the standard deviation of the mean. Columns with an asterisk indicate statistical differences between fermented and control green
coffee beans within the supplemented or nonsupplemented groups (P < 0.05). With concentrations lower than the limit of quantification in all samples,
changes of methionine are presented in the supplementary material (Table S2).

pH values remained stable around the initial level of 6.0, while
significant acidification occurred in both fermentations (P < 0.05)
(Fig. 1(b)). Faster acidification and significantly lower pH values
were observed in glucose-supplemented fermentation, compared
with nonsupplemented fermentation (P < 0.05).
Changes of nonvolatiles in green coffee beans
In the green coffee beans, sucrose was the predominant soluble
carbohydrate, followed by glucose and fructose, but the latter two
were present in significantly lower amounts (P < 0.05) (Table 1).
Glucose and fructose were both depleted in G-FN. With regard
to G-FS, fructose was reduced to trace level and 98% of glucose
was consumed, leaving a residual hexose level lower than that in
the G-CN (P < 0.05). In contrast, sucrose was not utilized in both
fermentations.
Organic acids in the green coffee beans were significantly
altered by the fermentations (P < 0.05) (Table 1). In the unfer-
mented green coffee beans, the organic acid with the highest
concentration was citric acid, followed by malic, quinic, succinic,
and lactic acids, while acetic acid was not detected with HPLC.
LABs activities produced primarily lactic acid, followed by acetic
acid. Addition of glucose resulted in a significant increase in lactic
acid generation by 5.2-fold, which was 22.6 mg g−1 dry matter of
green coffee beans, relative to G-FN. Citric acid was reduced to
trace levels in G-FN, while 65% of citric acid remained in G-FS.
Malic, succinic, and quinic acids were also significantly reduced
(P < 0.05). For phenolic acids, the chlorogenic acid 5-caffeoylquinic
acid (5-CQA) exists in two to three orders of magnitude higher
concentration than the free phenolic acids, namely caffeic acid and
ferulic acid. Degradation of 5-CQA and generation of caffeic acid
took place during fermentations of green coffee beans. However,
the increase in caffeic acid was not sufficient to stoichiometrically
balance the loss of 5-CQA.
All the 18 amino acids and pairs were detected in the unfer-
mented green coffee beans (Fig. 2). Serine–asparagine and
histidine–glutamine are presented together, as the co-elution
of these two pairs of amino acids occurred in the HPLC analy-
were significantly reduced in both fermentations, and several
amino acids (i.e., leucine, isoleucine, and methionine) were
reduced to levels lower than the limit of detection (Table S1).
Glucose-supplemented fermentation generally led to higher
amino acid reduction than nonsupplemented fermentation did
(P < 0.05).
Changes of volatiles in green coffee beans
The volatiles associated with the metabolic activities during
the fermentations of coffee green beans by L. rhamnosus are
shown in Table 2. The major volatile generated was acetic acid,
with lower production of diacetyl, acetoin, 3-methylbutanoic
acid, and 2-phenylethanol. The fermentations generated lim-
ited types of volatiles, but significantly increased the levels of
diacetyl (25–50%), acetoin (7- to 10-fold), and acetic acid (four- to
eightfold). The SPME gas chromatography analysis revealed the
presence of acetic acid in all green coffees, with the highest con-
tent in G-FN (P < 0.05). In contrast, HPLC analysis of nonvolatiles
detected acetic acid in G-FN only (Table 1), which could be due to
the loss of volatiles in the defatting process prior to the isolation
of organic acids. Therefore, SPME gas chromatography would be
more sensitive for acetic acid analysis.
Change of volatiles in the roasted coffees
For each type of green coffee beans, a longer roast time led to
a darker color that indicated a deeper roast degree (P < 0.05;
Table S4). R-FS was significantly darker than R-CN and R-FN
(P < 0.05) for each roast time.
Volatile profiles of R-CN, R-FN, and R-FS coffees roasted to three
degrees are compared in Table 3. In total, 4 acids, 3 aldehydes,
7 ketones, 13 furans, 18 pyrazines, 3 pyridines, 5 pyrroles, and 3
phenols were identified. For each roast degree, the total amounts
of volatiles in the roasted coffees were significantly impacted
by L. rhamnosus fermentations (P < 0.05), with more noticeable
effects being observed from the fermentation with glucose sup-
413

sis adopted in this study.26 Most of the amino acids analyzed plementation. For all three roasts, the 12 min (medium) roast

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 www.soci.org C Wang et al.

Table 2. Changes in key volatiles associated with Lactobacillus rhamnosus fermentation with and without glucose supplementation for 72 h in green
coffee beans

FID peak area (means × 106 ± SD)

Nonsupplemented Supplemented
Compounds LRI (DB-FFAP) G-CN G-FN G-CS G-FS Identification

Diacetyl 99946 12.37 ± 0.34c 18.90 ± 0.70a 12.68 ± 1.50c 15.85 ± 1.01b MS
Acetoin 1298 7.83 ± 0.23c 56.10 ± 3.45b 7.89 ± 1.09c 79.00 ± 5.28a MS, LRI46
Acetic acid 1448 21.98 ± 1.38c 175.60 ± 6.68a 20.02 ± 1.17c 94.97 ± 2.87b MS, LRI46–49
3-Methylbutanoic acid 1643 21.18 ± 0.45c 32.69 ± 0.99b 21.62 ± 0.55c 42.38 ± 1.99a MS, LRI47,49
2-Phenylethanol 1995 0.27 ± 0.03c 1.73 ± 1.05a 0.19 ± 0.02c 0.71 ± 0.75b MS, LRI48

LRI, linear retention index. FID, flame ionization detector; SD, standard deviation; G, green coffee beans; CN, control without glucose supplementation;
FN, fermented without glucose supplementation; CS, control with glucose supplementation; FS, fermented with glucose supplementation; MS, mass
spectrometry.
Values are expressed as the mean plus/minus standard deviation of triplicate fermentations (n = 3). Mean values in each row with different letters are
significantly different (P < 0.05).

yielded the highest total amount of volatiles, compared to the
9 min (light) and 15 min (dark) roasts (P < 0.05).
Furans detected in the roasted coffees consisted mostly of fur-
fural, 5-methylfurfural, and furanmethanol. Total furan levels were
significantly raised in R-FS compared with R-CN (P < 0.05), which
was contributed to by the elevation in amounts of furfural and
5-methylfurfural in R-FS. The quantities of furfural in R-FS9, R-FS12,
and R-FS15 were 10.2-, 2.7-, and 1.9-fold higher than those in the
respective controls, with similar trends found for 5-methylfurfural.
Both furfural and 5-methylfurfural increased continuously in R-CN,
but degraded in R-FS as roasting was intensified, which accounted
for the diminishing difference between the amounts of the total
furfurals in R-CN and R-FS from 9 to 15 min. The other abundant
furan, furanmethanol, was lower in R-FS than R-CN for all the roasts
(P < 0.05). The furans contents in R-FN were generally lower than in
R-CN (P < 0.05). Similar to furans, the pyrroles contents were gen-
erally elevated in R-FS, but slightly reduced in R-FN, compared with
the respective R-CN.
Pyrazines belong to another predominant category among
volatiles in roasted coffees. Most of the pyrazines detected were
methyl- and/or ethyl-substituted. In contrast with furans, the lev-
els of pyrazines in R-CN were 11.9-, 10.1-, and 6.5-fold higher than
those in the respective R-FS after roasting for 9, 12, and 15 min.
Eight pyrazines with relatively low contents were not detected in
R-FS. For R-FN, the pyrazines contents were generally comparable
to those in the respective R-CN. Similar to pyrazines, the level of
pyridines was significantly lowered in R-FS (P < 0.05), but not so in
R-FN, compared with the respective R-CN.
The volatile phenols detected in the roasted coffees consisted
primarily of guaiacol and 4-vinyl guaiacol. The two guaiacols were
found at higher levels in one or both of the fermented coffees
compared with the respective R-CN (P < 0.05). The most abundant
aldehyde detected in the roasted coffees was 3-methylbutanal, the
amount of which was highest in R-CN, followed by R-FN and R-FS.
Among the five volatile metabolites detected in L. rhamnosus
fermented green coffee beans, acetic acid, 3-methylbutanoic acid,
and diacetyl were present in the roasted coffees. The three volatiles
were all reduced by roasting, with acetic acid being retained to a
higher extent. The acetic acid level in R-FN was significantly higher
than in R-FS in all roasts (P < 0.05). As roasting was prolonged
from 9 to 12 min, an increase in acetic acid was found in R-CN,
and an increase in diacetyl was found in all three roasted coffees
414
PCA of volatiles in roasted coffees
PCA was conducted with the volatile profiles of various treated
coffees and controls after roasting (Fig. 3). The PCA analysis was
performed with the semi-quantitative data of each volatile cate-
gory and the major volatiles with maximum FID peak areas larger
than 2.5 × 107 (Table 3). Principal component (PC)1 and PC2 are
responsible for 74.70% and 23.41% of the total variance respec-
tively. Total furans and total pyrazines are highly responsible for
the positive loadings of PC1, and the negative loading is mainly
attributed to the variables with lower impact. For PC2, total furans
and total pyrazines contribute to the positive and negative load-
ings respectively. R-FS coffees are tightly clustered on the positive
values on PC2 and clearly differentiated from R-CN coffees. In con-
trast, alternating R-FN and R-CN are placed on the negative values
of PC2, along an arc-shaped line in the order of roast time. The loca-
tions of the three 12-min-roasted coffees are the most scattered.
Evaluation of aroma profiles of the 12-min-roasted coffees
Figure 4 illustrates the aroma profiles of roasted R-FN12, R-FS12,
and R-CN12. In general, the three coffees were characterized with
burnt, caramelic, roasted, and smoky notes, with low levels of a
nutty note and various extents of an acidic note. A strong acidic
odor was detected in R-FN12. Noticeable differences between
the aroma profiles of R-FN12 and R-FS12 were observed. The
aroma profile of R-FN12 resembled that of R-CN12, with weaker
burnt and caramelic notes detected in R-FN12. Compared with
the R-CN12 and R-FN12, R-FS12 was characterized by stronger
caramelic, burnt, and smoky notes, together with a slightly weaker
nutty note.
Nonvolatile profiles in roasted coffees
Total nonvolatiles detected in the green coffees were reduced after
roasting for 12 min (Table 1). The predominant soluble carbohy-
drate in the roasted coffee was sucrose, the level of which was two
orders of magnitude higher than fructose and glucose (P < 0.05).
Reduction of total soluble carbohydrates from 71% w/w (in R-FS12)
to 34% w/w (in R-FN12) during roasting was mainly contributed by
the sucrose degradation. The hexose contents were exhausted in
R-FS12 but increased in R-CN12 and R-FN12 (P < 0.05). The major-
ity of amino acids were depleted during roasting (Table S1). The

(P < 0.05). lactic acid content in R-FS12 was 17% lower than that in G-FS,

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 Table 3. Effects of Lactobacillus rhamnosus fermentation with and without glucose supplementation for 72 h on volatile profiles of coffees roasted for 9, 12, and 15 min
FID peak area (means × 106 ± SD)

9 min 12 min 15 min

LRI
Compound (DB-FFAP) R-CN9 R-FN9 R-FS9 R-CN12 R-FN12 R-FS12 R-CN15 R-FN15 R-FS15 Identification

Acids

J Sci Food Agric 2019; 99: 409–420

Acetic acid 1447 38.45 ± 0.62cB 117.06 ± 2.76aA 52.25 ± 0.60bB 47.58 ± 0.07cA 104.34 ± 4.83aB 52.15 ± 1.20bB 45.67 ± 2.57cA 85.70 ± 2.10aC 56.12 ± 3.65bB MS, LRI46,47,3,48,49
3-Methylbutanoic acid 1643 27.36 ± 1.44aA 23.76 ± 0.88bA 9.31 ± 0.60cB 14.48 ± 1.95aB 14.01 ± 0.97aB 6.51 ± 0.18bA 8.62 ± 0.24aC 7.85 ± 0.49aC ND MS, LRI47,49
3-Methyl-2-butenoic acid 1783 5.72 ± 0.12abA 5.37 ± 0.21aA 5.12 ± 0.02bA 5.96 ± 0.23aA 0.54 ± 0.01cB 4.75 ± 0.02bB 5.66 ± 0.04aA ND 4.70 ± 0.13bB MS, LRI47,3
Hexanoic acid 1828 4.38 ± 0.06bC 4.46 ± 0.37bC 7.75 ± 0.05aC 7.98 ± 0.34bB 7.52 ± 0.15bB 12.1 ± 0.01aB 13.74 ± 0.22bA 12.0 ± 0.12bA 15.54 ± 0.08aA MS, LRI49
Total acids 75.92 ± 1.90bA 150.67 ± 4.21aA 74.44 ± 1.06bB 76.01 ± 1.91bA 126.4 ± 3.86aB 75.57 ± 1.36bB 73.70 ± 2.39cA 105.62 ± 2.52aC 82.62 ± 0.55bA

Aldehydes
3-Methylbutanal 91646 15.74 ± 0.67aA 7.76 ± 0.29bA 3.83 ± 0.19cB 10.01 ± 0.58aB 6.77 ± 0.24bB 5.13 ± 0.42cA 7.11 ± 0.36aC 6.90 ± 0.40aB ND MS
Hexanal 1091 9.17 ± 0.55aA 4.72 ± 0.23bA 2.15 ± 0.10cA 3.81 ± 0.56aB 2.63 ± 0.21bB 1.65 ± 0.20cB 2.51 ± 0.08aC ND ND MS, LRI46,50,51
3-Methyl-2-butenal 1189 5.91 ± 0.17aA 4.00 ± 0.18bA ND 2.15 ± 0.23aB 1.52 ± 0.07bB ND ND ND ND MS, LRI46,48
Total aldehydes 30.83 ± 1.28aA 16.49 ± 0.69bA 5.99 ± 0.29cB 15.98 ± 1.38aB 10.93 ± 0.43bB 6.79 ± 0.63cA 7.11 ± 0.36aC 9.414 ± 0.48aC ND

Ketones
Diacetyl 99946 3.56 ± 0.13aB 1.79 ± 0.05cB 2.42 ± 0.09bB 3.79 ± 0.12aAB 2.92 ± 0.09bA 3.20 ± 0.29abA 4.41 ± 0.51aA 3.28 ± 0.26aA 3.80 ± 0.21aA MS
2-Hexanone 1065 12.44 ± 1.12aA 6.76 ± 0.37bB 6.49 ± 0.56bA 13.02 ± 1.21aA 9.592 ± 1.03bA 7.06 ± 0.92bA 9.19 ± 1.05aA 6.47 ± 0.23cB 7.32 ± 0.34bA MS, LRI46
1-Hydroxy-2-propanone 1321 23.50 ± 0.52aB 16.13 ± 0.20cC 19.38 ± 0.23bA 28.49 ± 0.72aA 23.87 ± 0.84bA 16.26 ± 0.48cB 18.77 ± 1.06aC 17.49 ± 0.14aB 10.62 ± 0.10bB MS, LRI50,47,3,48,49
1-Acetyloxy-2-propanone 1473 8.38 ± 0.38aC 6.44 ± 0.02bC 4.94 ± 0.06cC 16.51 ± 1.10aB 15.48 ± 0.87aB 11.11 ± 0.59bB 24.75 ± 2.31aA 20.85 ± 1.09abA 18.99 ± 0.35bA MS, LRI47–49
2-Methyl-3-pentanone 1528 2.62 ± 0.17aB 1.83 ± 0.05cB 2.01 ± 0.07bC 7.92 ± 0.52aA 7.17 ± 0.27aA 4.08 ± 0.06bB 8.78 ± 0.25aA 6.69 ± 0.29bA 6.49 ± 0.08bA MS, LRI49

© 2018 Society of Chemical Industry

4-Cyclopentene-1,3-dione 1581 1.57 ± 0.08bC 1.01 ± 0.05cB 4.26 ± 0.14aA 2.68 ± 0.14aB 1.83 ± 0.05bA 2.94 ± 0.07aB 3.81 ± 0.04aA 2.93 ± 0.10bA 2.55 ± 0.14cB MS, LRI3
Butyrolactone 1624 11.24 ± 0.52aB 9.17 ± 0.50bC 6.91 ± 0.17cC 12.30 ± 0.36aB 11.16 ± 0.26bB 9.58 ± 0.06cB 17.55 ± 0.76aA 14.51 ± 0.63bA 14.59 ± 0.16bA MS, LRI46,50,47,3,49
Total ketones 63.34 ± 1.40aB 43.16 ± 0.57cB 46.45 ± 0.46bC 84.73 ± 2.76aA 72.06 ± 1.70bA 54.25 ± 1.61cB 87.31 ± 3.46aA 72.25 ± 2.06bA 64.41 ± 0.87cA
Furans
2-Pentylfuran 1232 6.96 ± 0.09aA 6.74 ± 0.42aA 5.03 ± 0.16bA 4.96 ± 0.07aB 4.55 ± 0.21aB 3.14 ± 0.13bB 3.11 ± 0.28aC 2.99 ± 0.08aC 2.80 ± 0.22aB MS, LRI46,49
Potential of lactic acid bacteria to modulate coffee volatiles and effect of glucose supplementation

Furfuryl methyl ester 1243 ND ND ND 1.05 ± 0.01aB 0.88 ± 0.05bB 0.95 ± 0.05abB 1.81 ± 0.22abA 1.43 ± 0.16bA 1.95 ± 0.15aA MS, LRI46,3,51,48
2-Methyltetrahydrofuran- 1270 1.91 ± 0.13bA 1.16 ± 0.02cA 2.72 ± 0.08aB 1.06 ± 0.01bB 0.93 ± 0.02cB 3.62 ± 0.26aA 1.06 ± 0.12bB 0.86 ± 0.02bC 3.89 ± 0.29aA MS, LRI46,47,3
3-one
Furfural 1479 47.26 ± 3.13bC 23.09 ± 0.86cB 483.10 ± 2.23aA 157.5 ± 1.13bA 92.17 ± 1.12cA 422.30 ± 5.72aB 128.20 ± 3.62bB 91.07 ± 3.41cA 245.35 ± 11.7aC MS, LRI46,50,47,3,51,48,49
Furfuryl formate 1503 1.05 ± 0.08cC 2.35 ± 0.07aC 1.40 ± 0.01bC 3.89 ± 0.05bB 4.39 ± 0.24aB 2.68 ± 0.16cB 6.76 ± 0.61aA 6.72 ± 0.32aA 4.94 ± 0.05bA MS, LRI46,50,3,48,49
2-Acetylfuran 1515 6.18 ± 0.42bC 4.65 ± 0.09cC 18.46 ± 0.17aC 15.71 ± 0.72bB 11.97 ± 0.40cB 22.16 ± 0.63aB 19.77 ± 1.15bA 14.21 ± 0.46bA 28.24 ± 0.57aA MS, LRI46,47,51,48,49
Furfuryl acetate 1533 4.00 ± 0.12cC 7.22 ± 0.28aC 5.68 ± 0.06bC 13.63 ± 0.08bB 24.78 ± 3.25aB 9.90 ± 0.29cB 26.85 ± 2.10bA 42.27 ± 0.64aA 23.16 ± 0.21bA MS, LRI50,47,3,51,48,49
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5-Methylfurfural 1571 10.20 ± 0.74bC 5.72 ± 0.19cC 115.2 ± 0.82aB 74.58 ± 3.03bB 49.36 ± 1.53cB 206.10 ± 6.33aA 127.14 ± 2.47bA 88.96 ± 3.75cA 213.40 ± 5.10aA MS, LRI46,50,47,3,51,48,49
2-Furfurylfuran 1590 0.58 ± 0.05abC 0.50 ± 0.00bC 0.71 ± 0.04aC 1.54 ± 0.01aB 1.62 ± 0.04aB 1.12 ± 0.03bB 3.02 ± 0.08bA 2.89 ± 0.16bA 3.39 ± 0.12aA MS, LRI50,51,48

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415
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Table 3. Continued
FID peak area (means × 106 ± SD)

9 min 12 min 15 min

LRI
Compound (DB-FFAP) R-CN9 R-FN9 R-FS9 R-CN12 R-FN12 R-FS12 R-CN15 R-FN15 R-FS15 Identification

5-Methyl-2-acetylfuran 1602 0.88 ± 0.01cC 0.78 ± 0.03bC 2.61 ± 0.02aC 1.37 ± 0.05bB 1.20 ± 0.02cB 4.95 ± 0.06aB 2.93 ± 0.02bA 2.22 ± 0.09cA 7.13 ± 0.11aA MS, LRI46
Furanmethanol 1667 124.79 ± 1.60aB 110.06 ± 3.19bB 75.10 ± 2.34cB 197.17 ± 8.36aA 181.95 ± 4.12bA 78.51 ± 1.82cB 208.92 ± 8.02aA 185.04 ± 9.19bA 98.64 ± 0.49cA MS, LRI46,50,47,3,51,48,49

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3,4-Dimethyl-2,5-furandione 1738 0.66 ± 0.05C ND ND 1.25 ± 0.07aB 1.20 ± 0.05aB 1.12 ± 0.02aB 2.32 ± 0.04aA 1.91 ± 0.09bA 2.47 ± 0.20aA MS, LRI46,3
2(5H)Furanone 1763 3.76 ± 0.24bC 3.71 ± 0.11bC 5.04 ± 0.11aA 5.31 ± 0.06aB 5.22 ± 0.20aB 5.55 ± 0.13aA 6.65 ± 0.16aA 5.46 ± 0.09bA 5.25 ± 0.07bA MS, LRI47
Total furans 208.26 ± 5.80bC 166.01 ± 1.84cC 715.87 ± 3.56aB 479.18 ± 7.33bB 380.28 ± 9.45cB 762.27 ± 12.1aA 538.61 ± 15.0bA 446.10 ± 16.3cB 640.73 ± 18.0aC
Pyrazines
Pyrazine 1223 8.90 ± 0.73aB 6.59 ± 0.32bB 1.18 ± 0.03cC 10.2 ± 0.02aA 10.5 ± 0.27aA 1.63 ± 0.07bB 10.3 ± 0.63aA 10.5 ± 0.16aA 2.76 ± 0.23bA MS, LRI46,50,47,3,51,48,49
2-Methylpyrazine 1276 189.11 ± 8.47aA 142.46 ± 2.10bB 12.52 ± 0.47cB 166.75 ± 12.4aB 175.86 ± 9.60aA 12.70 ± 0.75bB 120.93 ± 11.4aC 122.36 ± 2.45aC 15.09 ± 0.34bA MS, LRI46,50,47,3,51,48,49
2,5-Dimethylpyrazine 1333 109.42 ± 2.21aA 88.21 ± 1.74bA 4.92 ± 0.17cA 88.12 ± 5.93aB 86.37 ± 4.19aA 4.18 ± 0.13bA 56.71 ± 3.54aC 54.53 ± 0.78aB 4.55 ± 0.12bA MS, LRI46,50,47,3,51,48,49
2,6-Dimethylpyrazine 1338 85.30 ± 3.38aA 70.74 ± 2.21bB 6.74 ± 0.30cB 86.51 ± 7.11aA 92.44 ± 4.55aA 6.71 ± 0.26bB 59.00 ± 4.08aB 61.16 ± 0.72aC 7.40 ± 0.22bA MS, LRI46,50,47,3,51,48,49
2-Ethylpyrazine 1342 31.12 ± 0.97aA 25.99 ± 0.50bB 4.14 ± 0.09cB 30.37 ± 2.59aA 33.00 ± 2.05aA 4.29 ± 0.11bB 20.74 ± 1.43aB 22.17 ± 0.23aC 4.91 ± 0.17bA MS, LRI46,50,47,3,51,48,49
2,3-Dimethylpyrazine 1359 16.47 ± 0.99aA 16.85 ± 0.60aB 1.98 ± 0.07bB 15.46 ± 1.62aA 18.92 ± 0.86aA 2.18 ± 0.04bB 12.19 ± 0.89aB 13.80 ± 0.18aC 2.81 ± 0.05bA MS, LRI46,50,47,3,51,48,49
2-Ethyl-6-methylpyrazine 1392 25.96 ± 0.94aB 23.84 ± 0.72aC 5.54 ± 0.08bB 31.70 ± 0.72bA 35.16 ± 0.99aA 6.55 ± 0.04cA 25.74 ± 1.01aB 27.45 ± 0.10aB 6.29 ± 0.83bA MS, LRI46,50,47,3,51,48,49
2-Ethyl-5-methylpyrazine 1398 19.66 ± 0.46aA 18.22 ± 0.41bB 2.25 ± 0.04cC 19.23 ± 0.40aA 20.11 ± 0.72aA 2.51 ± 0.03bB 14.13 ± 0.30aB 14.76 ± 0.17aC 3.13 ± 0.11bA MS, LRI46,47,3,51,48,49
2,3,5-Trimethylpyrazine 1415 26.70 ± 2.45aA 30.53 ± 1.00aB 2.90 ± 0.11bC 28.77 ± 0.91bA 35.83 ± 0.65aA 3.47 ± 0.03cB 24.60 ± 0.71aB 29.44 ± 0.46bB 4.27 ± 0.07cA MS, LRI47–49
2-Propylpyrazine 1426 0.95 ± 0.05aC 0.85 ± 0.06aB ND 1.67 ± 0.01aB 1.67 ± 0.11aA ND 1.82 ± 0.08aA 1.88 ± 0.01aA ND MS, LRI51,48
2,5-Diethylpyrazine 1441 1.72 ± 0.06C ND ND 2.41 ± 0.01aA 1.95 ± 0.14bB ND 2.18 ± 0.03bB 2.44 ± 0.02aA ND MS, LRI46,3,51,48,49
2-Ethyl-3,5-dimethylpyrazine 1454 14.79 ± 0.16aA 14.79 ± 0.19aB ND 15.79 ± 0.87aA 15.71 ± 0.21aA ND 14.26 ± 0.68aA 15.15 ± 1.08aAB ND MS, LRI51,48,49
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2,3-Diethylpyrazine 1469 3.65 ± 0.26bB 4.40 ± 0.04aC 0.82 ± 0.02cC 4.44 ± 0.01bA 5.69 ± 0.05aA 1.04 ± 0.00cB 4.44 ± 0.13bA 5.37 ± 0.06aB 1.26 ± 0.01cA MS
5-Vinyl-2-methylpyrazine 1494 0.58 ± 0.01aC 0.52 ± 0.01aC ND 1.05 ± 0.02aB 0.86 ± 0.02bB 0.85 ± 0.02bB 2.55 ± 0.21bA 2.21 ± 0.11bA 3.00 ± 0.02aA MS, LRI46,3,48,49
3,5-Diethyl-2-methylpyrazine 1499 2.65 ± 0.12aB 0.35 ± 0.00bC ND 3.06 ± 0.09aA 2.85 ± 0.02bB ND 3.21 ± 0.05aA 3.05 ± 0.01bA ND MS, LRI

© 2018 Society of Chemical Industry

2-Methoxy-6-methylpyrazine 1523 0.80 ± 0.03bC 0.73 ± 0.03bC 1.54 ± 0.02aC 2.29 ± 0.03bB 1.82 ± 0.10cB 3.06 ± 0.06aB 3.81 ± 0.16bA 2.65 ± 0.09cA 4.64 ± 0.06aA MS
Acetylpyrazine 1618 3.20 ± 0.20aB 2.74 ± 0.14aB 1.46 ± 0.05bB 6.44 ± 0.20aA 6.46 ± 0.13aA 2.20 ± 0.07bA 6.83 ± 0.07aA 6.77 ± 0.12aA ND MS, LRI46,51,49
2-Acetyl-3-methylpyrazine 1692 3.78 ± 0.09aC 3.74 ± 0.13aC ND 5.63 ± 0.39bB 6.72 ± 0.07aB ND 7.05 ± 0.57aA 8.74 ± 0.02bA ND MS, LRI46,49
Total pyrazines 544.51 ± 16.3aA 451.64 ± 9.92bB 46.04 ± 1.29cC 520.04 ± 30.02aA 552.09 ± 24.4aA 51.45 ± 1.55bB 389.43 ± 23.9aB 403.51 ± 3.84aC 60.17 ± 1.82bA
Pyridines
Pyridine (Pd1) 1200 15.09 ± 0.94aC 13.79 ± 0.47aC 4.20 ± 0.11bC 21.56 ± 0.48bB 25.22 ± 1.15aB 8.27 ± 0.34cB 40.23 ± 4.67aA 39.70 ± 2.41aA 21.57 ± 0.27bA MS, LRI46,50,47,3,51,48,49
2-Acetylpyridine 1593 0.82 ± 0.03aC 0.80 ± 0.02aC 0.84 ± 0.01aC 1.76 ± 0.10aB 1.77 ± 0.01aB 1.46 ± 0.02bB 4.91 ± 0.15aA 4.01 ± 0.08bA 2.02 ± 0.01cA MS
1-Acetyl-4(H)pyridine 1712 3.08 ± 0.19aB 2.72 ± 0.05aB ND 4.95 ± 0.16aA 4.50 ± 0.11bA 2.33 ± 0.02cB 5.16 ± 0.32aA 4.71 ± 0.09aA 3.07 ± 0.02bA MS, LRI50
Total pyridines 19.00 ± 0.85aC 17.31 ± 0.50aC 5.04 ± 0.11bC 28.28 ± 0.20bB 31.50 ± 1.18aB 12.07 ± 0.32cB 50.31 ± 5.05aA 48.44 ± 2.47aA 26.67 ± 0.23bA
Pyrroles
Pyrrole 1518 4.79 ± 0.17aA 3.45 ± 0.06bA ND 2.91 ± 0.05bC 3.17 ± 0.07aA ND 3.02 ± 0.13aB 2.77 ± 0.02bB ND MS, LRI46,50,3,51,48,49
2-Formyl-1-methylpyrrole 1611 2.92 ± 0.20bC 2.78 ± 0.08bC 5.49 ± 0.08aC 8.03 ± 0.37cB 9.05 ± 0.28bB 9.97 ± 0.08aB 15.25 ± 0.61aA 16.04 ± 0.37aA 13.8 ± 0.19bA MS, LRI50,47,3,51,48,49
Furfurylpyrrole 1823 0.54 ± 0.03bC 0.47 ± 0.00bC 0.69 ± 0.01a 0.79 ± 0.01bB 0.84 ± 0.02aB ND 1.49 ± 0.04aA 1.25 ± 0.04bA ND MS, LRI46,51,49
2-Acetylpyrrole 1976 3.40 ± 0.30bC 2.99 ± 0.05bC 5.70 ± 0.05aC 8.48 ± 0.23bB 7.20 ± 0.54cB 12.30 ± 0.23aB 19.53 ± 0.18bA 17.26 ± 0.08cA 27.50 ± 1.23aA MS, LRI50,3,51,48,49
2-Formylpyrrole 2033 2.97 ± 0.22bC 2.73 ± 0.10bC 14.65 ± 0.30aB 10.75 ± 0.65bB 9.43 ± 0.56bB 27.17 ± 0.91aA 19.14 ± 1.11bA 17.28 ± 0.61cA 27.13 ± 1.18aA MS, LRI50,47,3,51,48,49
Total pyrroles 14.64 ± 0.62bC 12.45 ± 0.16cC 26.56 ± 0.27aC 31.00 ± 1.47bB 29.72 ± 0.81bB 50.16 ± 1.57aB 58.46 ± 0.47bA 54.62 ± 1.17cA 71.27 ± 1.94aA

J Sci Food Agric 2019; 99: 409–420

C Wang et al.
Potential of lactic acid bacteria to modulate coffee volatiles and effect of glucose supplementation www.soci.org

whereas lactic acid content in R-FN12 was not significantly dif-

LRI, linear retention index; FID, flame ionization detector; SD, standard deviation; R, roasted coffees; CN, control without glucose supplementation; FN, fermented without glucose supplementation; FS, fermented with glucose

Values of FID peak areas are expressed as the mean plus/minus standard deviation of triplicate fermentations (n = 3). Mean values in each row with different lower-case letters (a–c) indicate statistical differences among the three
MS, LRI46,50,47,3,51,48,49
ferent from that in G-FN. Furthermore, the 12 min roasting led to

Identification

MS, LRI47,3,51,48,49
increases in succinic and quinic acids, as well as reductions in citric,
malic, and phenolic acids (P < 0.05).

MS, LRI47,3,48
DISCUSSION

1209.62 ± 41.41 1158.92 ± 18.13 954.18 ± 21.51bB

11.93 ± 0.44bA
5.94 ± 0.13bA

3.27 ± 0.24bB
2.71 ± 0.08aA
The L. rhamnosus fermentations resulted in significant modula-

coffees roasted for the same time (P < 0.05). Mean values in the same row with different upper-case letters (A–C) indicate statistical differences among the same coffee roasted for different times (P < 0.05).
R-FS15

tions of flavor-related substrates in coffee green beans. The pro-

duction of the primary metabolic product, lactic acid, could
be associated with several pathways. As a facultative hetero-

aB
13.15 ± 0.21aA fermenter, L. rhamnosus metabolized hexoses, such as glucose
6.28 ± 0.10aA
2.74 ± 0.07aA
4.11 ± 0.03aA

and fructose detected in the fermentation matrix, through the

R-FN15
15 min

Embden–Meyerhof–Parnas pathway to generate mainly lactic

acid.19 The catabolism of citric acid by L. rhamnosus involves
the conversion of citric acid via oxaloacetate – thereby liberating
acetate – into pyruvate that could be reduced to lactic acid.17,18
aA
10.96 ± 0.31bA
4.00 ± 0.30abA
2.02 ± 0.06bA
4.93 ± 0.04cA

L. rhamnosus HN001 could also perform malolactic fermentation,

R-CN15

in which malic acid is converted into lactic acid and carbon diox-
ide in an equal molar ratio.27 The lower production of acetic acid
during the fermentation of green coffee beans was in agreement
with L. rhamnosus fermentations in other media.28 As citric acid is
961.51 ± 21.6 914.05 ± 5.08 965.60 ± 4.65 1242.90 ± 41.47 1243.12 ± 29.66 1033.92 ± 16.10 bA
4.93 ± 0.07aB
1.56 ± 0.04aB
3.61 ± 0.14aB
10.1 ± 0.12aB

converted into pyruvate through oxaloacetate as just mentioned,

R-FS12

acetic acid is released in an equimolar ratio.18 The existence of lac-

FID peak area (means × 106 ± SD)

tic and acetic acids in the nonfermented green coffee beans is con-
sistent with previous reports,14,15 which could be generated during
semi-dry processing of green coffee beans.29
aA
4.17 ± 0.04bB

3.17 ± 0.12bB
1.66 ± 0.07aB

9.00 ± 0.12aB

Phenolic acids in green coffee beans exist essentially in esteri-

R-FN12
12 min

fication with quinic acid to form chlorogenic acids, among which

5-CQA is the predominant species.30 By performing metabolisms
of phenolic acids, L. rhamnosus HN001 detoxifies the antimicrobial
constituents in the fermentation of green coffee beans.31
aA
3.39 ± 0.43abB
1.06 ± 0.07bB

7.64 ± 0.70bB
3.19 ± 0.20cB

The volatiles production during the fermentations was associ-

R-CN12

ated with citric acid and amino acid catabolisms. Besides lactic
acid, part of the pyruvate generated from citric acid degradation
could be channeled into four-carbon volatiles, leading to sig-
G-CS was not roasted to prevent the interference from the added glucose in volatile formation.

nificantly higher levels of diacetyl and acetoin in the fermented

aA
4.06 ± 0.11aA
4.54 ± 0.03aC
0.90 ± 0.02aC

4.55 ± 0.42cC 5.82 ± 0.12bC 9.51 ± 0.17aB

green beans.18 Transamination of leucine and phenylalanine

R-FS9

could account for the production of 3-methylbutanoic acid and

2-phenylethanol respectively.32 Nevertheless, the metabolic activ-
ities during the L. rhamnosus fermentation of green coffee beans
bB
1.70 ± 0.09bC

3.32 ± 0.08bB
0.79 ± 0.06aC

contributed limited types of volatiles.

R-FN9
9 min

Glucose supplementation significantly influenced the

L. rhamnosus fermentation of coffee green beans. The enhanced
lactic acid production in the glucose-supplemented fermentation
aA

contributed to a significantly larger reduction in pH. However, the

supplementation; MS, mass spectrometry; ND, not detected.
0.38 ± 0.01bC
3.12 ± 0.31bB
1.04 ± 0.10cC

additional lactic acid generated could not be fully accounted for

R-CN9

by 1% w/w glucose supplementation and metabolism of citric and

malic acids in the glucose-supplemented fermentation. The stim-
ulating effect of glucose on the propagation of L. rhamnosus in the
(DB-FFAP)

early stage was also demonstrated in the fermentation of other

1861
2006
2205
LRI

plant materials.20 Afterwards, decoupling of L. rhamnosus growth

and acid formation took place in the glucose-supplemented
fermentation, where the propagation was slowed down to a
Table 3. Continued

larger extent possibly due to its lower pH in the first 12 h, while

carbohydrate metabolism by enzymatic systems continued to
generate organic acids and further reduced pH.33 The decoupling
4-Vinylguaiacol

Total peak area

did not occur in nonsupplemented fermentation due to the lower

Total phenols
Compound

carbohydrate content. Furthermore, the glucose supplementation

Guaiacol
Phenols

lowered the production of acetic acid through the suppression of

Phenol

citric acid catabolism, as glucose was the preferred carbon and

417

energy source compared with citrate.28,34

J Sci Food Agric 2019; 99: 409–420 © 2018 Society of Chemical Industry wileyonlinelibrary.com/jsfa
 www.soci.org C Wang et al.

Figure 3. Biplot of principal component analysis for major volatiles in the coffees roasted for 9, 12, and 15 min. R, roasted coffees; CN, control
without glucose supplementation; FN, fermented without glucose supplementation; FS, fermented with glucose supplementation. Major volatile
compounds selected as variables are: Ac1, acetic acid; Ac2, 3-methylbutanoic acid; Ke1, 1-hydroxy-2-propanone; Ke2, 1-acetyloxy-2-propanone;
Fu1, furfural; Fu2, 2-acetylfuran; Fu3, furfuryl acetate; Fu4, 5-methylfurfural; Fu5, furanmethanol; Pz1, 2-methylpyrazine; Pz2, 2,5-dimethylpyrazine;
Pz3, 2,6-dimethylpyrazine; Pz4, 2-ethylpyrazine; Pz5, 2-ethyl-6-methylpyrazine; Pz6, 2,3,5-trimethylpyrazine; Pd1, pyridine; Pr1, 2-acetylpyrrole; Pr2,
2-formylpyrrole.

Besides the green coffee beans, the liquors sampled at the
end of the submerged fermentations were also analyzed for the
nonvolatiles (Tables S2 and S3). The relative concentrations and
changes of nonvolatiles in the liquors showed similar trends to
those in the respective green coffee beans, suggesting substance
exchange between the two phases. Diffusion of green coffee beans
substances into liquor could provide substrates for microbial activ-
ities. The metabolites that were generated infiltrated back, con-
tributing to the modification of the constituents in the green cof-
fee beans.
During roasting, the Maillard reaction, caramelization, and other
pyrolytic reactions involving nonvolatile precursors, such as car-
bohydrates and amino acids, generate a wide range of volatiles
that are responsible for the aroma of roasted coffees.35 Furans
detected in the roasted coffees consisted mostly of furfurals, which
are formed through the 3-deoxyosone route in a Maillard reac-
tion and a similar pathway in caramelization. In coffee roasting,
the carbohydrates that are more accessible for pyrolytic reac-
tions would be hexoses, including glucose and fructose, both in
418
3-deoxyosone route in the Maillard reaction leads to the produc-
tion of 5-hydroxymethylfurfural, which is degraded into furfural
and 5-methylfurfural.36–39 Further decomposition of furfural yields
other furan derivatives.38 In the presence of ammonia or other
amino compounds, the 3-deoxyosone route in the Maillard reac-
tion also generates pyrroles.40
Pyrazines and pyridines are formed through the 1-deoxyosone
route in the Maillard reaction. Breakdown of 1-deoxyosone gener-
ates dicarbonyls, which react with amino acids to yield aminoke-
tones in Strecker degradation. The subsequent condensation of
aminoketones generates pyrazines.39 Strecker degradation also
converts amino acids into corresponding Strecker aldehydes, but
such conversion of proline and hydroxyproline yields pyridines.35
The production of 3-methylbutanal could be attributed to the
Strecker degradation of leucine.39 The lower residual levels of
leucine in fermented green coffee beans could be associated with
lower 3-methylbutanal production in these coffees after roasting.
In addition, guaiacol and 4-vinylguaiacol are both formed
from degradation of ferulic acid.41 The Maillard reac-

the free form and from sucrose hydrolysis. With the hexoses, the tion contributed to acetic acid and diacetyl contents,39

wileyonlinelibrary.com/jsfa © 2018 Society of Chemical Industry J Sci Food Agric 2019; 99: 409–420
Potential of lactic acid bacteria to modulate coffee volatiles and effect of glucose supplementation
Figure 4. Aroma profile of the coffee beans roasted for 12 min. R, roasted
coffees; CN, control without glucose supplementation; FN, fermented
without glucose.
resulting in the elevation of the two volatiles during
roasting.
The modification of flavor-related constituents in green coffee
beans by L. rhamnosus fermentation influenced the characteristics
of the roasted coffees. The formation pathways for furfurals in
caramelization and the Maillard reaction through 3-deoxyosone
were both enhanced under higher acidity.39,42 The faster color for-
mation in R-FS could also be attributed to its more acidic condition
that promoted the browning process from caramelization.42 Con-
versely, the moderate pH reduction in R-FN might not be sufficient
to shift the Maillard reaction toward the 3-deoxyosone pathway,
and the lower amounts of available hexoses further suppressed
the production of furans and pyrroles in R-FN. The relatively lower
acidity in R-FN and R-CN shifted the Maillard reaction toward
the 1-deoxyosone route and enhanced pyrazine and pyridine
formations.39 Despite the lower total amino acid content in G-FN,
pyrazine production in R-FN might be compensated by other
factors, such as the higher level of aspartic acid, which was shown
to be particularly important in Strecker degradation.43 Thus, to
achieve a more noticeable modulation on the coffee volatiles
with L. rhamnosus fermentation of green coffee beans, parameter
optimization (e.g., glucose supplementation) would be required
to obtain more salient changes in acidity and composition of
volatile precursors.
The medium roast could reflect the effect of L. rhamnosus fer-
mentation better than the light and dark roasts, as medium roast
is typically more suitable to reflect the on-farm processing, origin,
and variety of the green coffee beans.44 The characteristic aroma of
the three 12-min-roasted coffees correlated well with their volatile
profiles. The major volatiles that were responsible for the aroma
characteristics of the roasted coffees included aromatic furfurals,
pyrazines, guaiacols, and acetic acid. The resemblance between
the aromas of R-FN12 and the control could be accounted for by
the similarity between the volatile profiles of the two roasted cof-
fees, while the weaker burnt and caramelic notes in R-FN12 could
be attributed to the lower contents of furfurals.6 The strong acidic
odor detected in R-FN12 was caused by a high content of acetic
acid. The higher contents of furfurals and guaiacols and lower lev-
els of substituted pyrazines in R-FS12 could account for its stronger
caramelic, burnt, and smoky notes, together with a slightly weaker
nutty note.6,41
J Sci Food Agric 2019; 99: 409–420 © 2018 Society of Chemical Industry
www.soci.org
For the nonvolatiles in the 12-min-roasted coffees, the increases
in fructose and glucose could have resulted from sucrose
hydrolysis.41 Nevertheless, pyrolytic degradation of soluble
carbohydrates still played a major role during roasting. The defat-
ting process prior to nonvolatile extraction might liberate bound
sucrose, leading to an overestimation of the sucrose content in the
roasted beans. The elevations in quinic acid and succinic acid lev-
els in the 12-min-roasted coffees was associated with the thermal
degradation of chlorogenic acids and citric acid respectively.45
CONCLUSION
This study explored the potential to modulate roasted coffee
flavor through L. rhamnosus fermentation of green coffee beans
with and without glucose supplementation. The changes of
volatile precursors and production of organic acids in green
coffee beans translated into modifications in volatile and aroma
profiles of the roasted coffees, which was most prominent in the
medium-roasted coffees. The glucose-supplemented fermenta-
tion resulted in roasted coffees with stronger caramelic and burnt
characteristics but a weaker nutty note. In contrast, the effect of
the nonsupplemented fermentation was mainly reflected in a
milder overall aroma. Furthermore, with higher contents of car-
boxylic acids, the LABs-fermented coffees would be expected to
exhibit a noticeable acidic attribute that distinguishes them from
other coffees. The lactic acid fermentation of green coffee beans is
therefore a promising strategy for the creation of specialty coffees
with novel characteristics.
ACKNOWLEDGEMENTS
We would like to thank the panelists, Wang Youfei, Wu Pinpin,
Mei Fangyi, and Wang Xu from Wilmar (R&D) Center, for their
participation in the evaluation of aroma profile.
SUPPORTING INFORMATION
Supporting information may be found in the online version of this
article.
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