LWT - Food Science and Technology 77 (2017)

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LWT - Food Science and Technology

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Modulation of the volatile and non-volatile

f lespro
of coffee
fermented with Yarrowia lipolyticaI.
: Green coffee

Liang Wei Lee a, Geng Yu Tay a, Mun Wai Cheong b, Philip Curran c, Bin Yu d, Shao Quan Liu a, e,
*

a
Food Science and Technology Programme, Department of Chemistry, National University of Singapore, S14 Level 5, Science Drive 2, 117542 Singapore
b Kerry Ingredients Asia Pacifc, 8 Biomedical Grove, #02-01/04 Neuros, 138665 Singapore
c
Firmenich Asia Pte Ltd., 10 Tuas West Road, 638377 Singapore
d Silesia Flavours South East Asia Pte Ltd., 41 Science Park Road, The Gemini, Science Park II, 117610 Singapore
e National University of Singapore (Suzhou) Research Institute, No. 377 Linquan Street, Suzhou Industrial Park, Suzhou,
Jiangsu, 215123 China

articleinfo abstract

Article history: Yeast starter cultures have been used in the fermentation of different coffee substrates to modulate the volatile and aroma
Received 10 June 2016 profles of coffees with the exception of green coffee beans. This two-part study aimed to modulate the volatile pro fles of
Received in revised form roasted coffee via the fermentation of green coffee beans with a nonconventional aerobic yeast Yarrowia lipolytica. The
27 October 2016 objective of part I of this study was to evaluate the effects of Y. lipolytica fermentation on the volatile and non-volatile pro fles
Accepted 18 November 2016 Available of green coffee beans. After fermentation, sugars concentration decreased by 1.2-fold while the amino and phenolic acids
online 21 November 2016
concentrations decreased by 1.3-fold. The decrease in phenolic acid concentration could account for the 1.5-fold increase in
the volatile phenols levels resulting from phenolic acid catabolism. The degradation of Lphenylalanine via the Ehrlich pathway
Keywords: by the yeast led to a 1.9-fold increase in 2-phenylethanol levels, whereas the decreases in alkanes, acids and aldehydes levels
Green coffee beans were attributed to the hydrophobic substrate metabolizing pathways that were unique to Y. lipolytica. Hence, this work showed
Solid-state fermentation that Y. lipolytica fermentation of green coffee beans resulted in the modi fcation of the aroma precursors and volatile
Yarrowia lipolytica composition of green coffees.
Aroma precursors
2016 Elsevier Ltd. All rights reserved.
Volatile compounds

1. Introduction 0023-6438/ 2016 Elsevier Ltd. All rights reserved.

Coffee aroma is one of the main drivers of rising global

coffee consumption over the past 50 years. This has led to
strong interest in coffee aroma modulation, with a shift in
focus towards the optimization of post-harvest processes and
treatment of coffee substrates in recent years. The yeast
fermentation of different coffee substrates has been
documented to induce coffee aroma modulation. Aqueous
coffee extracts fermented by specifc yeast strains were found
to possess fruity and foral notes as a consequence of the
conversion of 2-, 3-methylbutanal to their corresponding
alcohols and thiols to their respective thioacetates during
fermentation (Duboc & Milo, 2003).
* Corresponding author. Food Science and Technology Programme, Department
Chemistry, National University of Singapore, S14 Level 5, Science Drive 2, 117542 Singapore.
E-mail address: chmLsq@nus.edu.sg (S.Q. Liu).
http://dx.doi.org/10.1016/j.lwt.2016.11.047
On the other hand, the use of indigenous yeast starter cultures
like Pichia fermentans in coffee fermentation during post-harvest
wet processing gave rise to coffees with novel and distinctive
aroma profles (de Melo Pereira et al., 2015). The primary role of
fermentation in dry, semi-dry and wet processing of coffee
cherries is to facilitate mucilage degradation by indigenous
microorganisms consisting of pectinolytic yeasts (Masoud, Cesar,
Jespersen, & Jakobsen, 2004). However, the reliance on naturally
occurring microfora in coffee cherries for fermentation in coffee
processing gave rise to inconsistency and uncontrollability issues
which affected the corresponding coffee aroma profle. Therefore,
the shiftof towards the use of yeast starter cultures in coffee
fermentation not only provides greater control and consistency, it
also allows the selection of yeast species with desirable features
 L.W. Lee et al. / LWT - Food Science and Technology 77 (2017) 225e232
such as exhibiting strong pectinolytic activity and volatile
metabolites production.
A review of the literature showed that the utilization of green
coffee beans as substrates for biotransformation/fermentation
processes to promote coffee aroma modulation has not been
explored. While Li, Li, and Li, (2010) have outlined a method for
the solid-state fermentation of green coffee beans using
macrofungi such as Antrodia camphorata and Hericium
erinaceum, quantitative and qualitative data regarding the impacts
of fermentation on coffee aroma were not reported in their study.
Therefore, with increasing evidence showing that the microbial
fermentation of other coffee matrices can promote coffee aroma
modulation (Lee, Cheong, Curran, Yu, & Liu, 2015), extending
the process to green coffee beans presents a potentially feasible
method of producing coffees with novel and desirable aroma
profles. Previous studies have shown that the fermentation of
green coffee beans with a food grade mold Rhizopus oligosporus
signifcantly modifed the volatile profle and aroma precursors
of green coffee beans which corresponded to signifcant changes
in the volatile and aroma profles of roasted coffees (Lee,
Cheong, Curran, Yu, & Liu, 2016a, 2016b).
Compared to molds, yeasts exhibit different metabolic
processes and secrete different types of extracellular enzymes
during fermentation. This suggested that fermenting green coffee
beans with suitable yeast species would modify the aroma
precursors and volatile profles of green coffee beans differently
and thereby, promote different extent of coffee aroma modulation
after roasting.
The dairy yeasts Yarrowia lipolytica and Geotrichum
candidum have been found to exhibit strong proteolytic and
lipolytic activities which infuenced the organoleptic and textural
characteristics of dairy products like cheeses (Boutrou &
Gueguen, 2005; Lanciotti, Vannini, Lopez, Gobbetti, &
Guerzoni, 2005). The strong lipolytic activity of Y. lipolytica
would account for the generation of aroma compounds such as
methyl ketones and free fatty acids in addition to volatile sulfur
compounds like dimethyl disulfde and dimethyl trisulfde that
were also detected after fermentation (Srensen, Gori, Petersen,
Jespersen, & Arneborg, 2011). In addition, Y. lipolytica has also
been associated with organic acids (citric, succinic and a-
ketoglutaric acids) and lactones (g-decalactone) production under
suitable fermentation conditions (Harzevili, 2014). Y. lipolytica
also possesses unique metabolic pathways such as hydrophobic
substrate (n-alkanes and fatty acids) metabolism (Fickers et al.,
2005).
Therefore, the objective of this two-part study was to conduct
solid-state fermentation (SSF) of green coffee beans using Y.
lipolytica, with the aim of altering the composition of aroma
precursors and production of volatile metabolites during
fermentation, hence modulating the volatile profles of roasted
coffees. In part I, the effects of Y. lipolytica fermentation were
evaluated by characterizing the volatile and non-volatile profles
of green coffee beans after fermentation. The corresponding
effects of Y. lipolytica fermentation after roasting will be
evaluated in Part II. 2. Materials and methods
2.1. Green coffee beans, yeast strain and chemicals
Green coffee beans (Coffea arabica) from the Toraja region of
Sulawesi, Indonesia were used in this study. The freeze-dried
yeast cultures were purchased from the respective suppliers:
food-grade Y. lipolytica NCYC 2904 from National Collection of
Yeast Cultures (NCYC) (Norwich, UK) and G. candidum Geo 3A
from Christian Hansen (Hrsholm, Denmark).
226
The following reference standards were purchased from the
respective suppliers: glucose, fructose, sucrose, quinic, lactic,
citric, caffeic, ferulic, chlorogenic, and p-coumaric acids from
SigmaAldrich (Missouri, USA); malic, a-ketoglutaric, sinapic
and succinic acids from Fluka Analytical (Steinheim, Austria);
amino acid standards from Thermo Scientifc (Illinois, USA); and
saturated alkane standards (C7 e C40) from Supelco, Sigma-
Aldrich (Barcelona, Spain). AccQ-Fluor reagent kit and AccQ-
Tag eluent A concentrate for amino acid derivatization were
obtained from Waters (Dublin, Ireland).
HPLC-grade solvents methanol and acetonitrile were
obtained from Merck (Darmstadt, Germany) and Tedia (Ohio,
USA), respectively, while ACS-grade solvents ethanol,
acetone and petroleum ether (35 Ce60 C) were purchased
from Merck (Darmstadt, Germany). Acetic and sulfuric acids
were obtained from RCI Labscan Limited (Bangkok,
Thailand) and VWR (Pennsylvania, USA), respectively.
2.2. SSF of green coffee beans
Pure cultures were prepared by propagating a freeze-dried
Y. lipolytica culture in sterile yeast malt (YM) broth (0.5% w/v
bacteriological peptone, 0.3% w/v yeast extract, 0.3% malt
extract and 1% w/v dextrose) adjusted to pH 5.0 with 1 M
hydrochloric acid at 30 C for 48 h under aerobic conditions
and stored in 15% glycerol at 80 C prior to use. Pre-cultures,
with cell counts of approximately 10 7 CFU ml1, were prepared
by further sub-culturing twice (5% v/v) under similar media
and conditions. Y. lipolytica cells were obtained via
centrifugation at 8000 g, washed twice and resuspended in
equivolumes of 100 mM phosphate buffer saline
(PBS) solution.
Green coffee beans (800 g) were soaked in deionized water
at 4 C for 24 h, steamed at 80 C for 40 min and cooled to room
temperature under sterile conditions prior to fermentation.
Triplicate batches of heat-treated green coffee beans were
inoculated with Y. lipolytica at an inoculum size of
approximately 105 CFU g1 of green coffee beans and incubated
at 30 C for 4 days. The fermented green coffee beans were
then steamed at 100 C for 10 min, oven-dried to a moisture
content of 7% and stored at 20 C. The unfermented green
coffee beans were subjected to the same treatment steps, with
the exception of the inoculation of Y. lipolytica starter culture.
Both green coffee bean samples were thawed thoroughly to
room temperature for 90 min prior to subsequent analyses.
2.3. Volatiles analysis
Volatile compounds were analyzed via headspace-solid
phase microextraction e gas chromatography-mass
spectrometry/fame ionization detector (HS-SPME-GC-
MS/FID). Green coffee beans were ground (Krups GVX2,
Germany) and the coffee grounds (2 g) were extracted in a
screw-top headspace vial at 90 C for 30 min, with constant
agitation at 250 rpm using a SPME fber (carboxen/
poly(dimethylsiloxane)) (Supelco, Sigma-Aldrich, Barcelona,
Spain).
As described by Lee, Toh, Yu, Curran, and Liu (2013), GC-
MS/FID analysis of volatiles in green coffee beans was
conducted with Agilent 7890N GC coupled to 5975 inert MS
and FID. Volatiles from the SPME fber were thermally
desorbed at 250 C and resolved with a fused silica capillary
column (60 m 0.25 mm x 0.25 mm DBFFAP, Agilent
Technologies, Woodbridge, USA) coated with a 0.25 mm flm
of nitroterephthalic acid modifed with polyethylene glycol.
 L.W. Lee et al. / LWT - Food Science and Technology 77 (2017) 225e232
Identifcation of volatile compounds was achieved via mass
spectral comparison with Wiley database and linear retention
indices (LRI), derived from C7 e C40 alkane standards analyzed
under similar conditions while FID peak areas were used for
semiquantifcation.
2.4. Extraction of sugars, phenolic, organic and amino acids
Prior to extraction, green coffee grounds were defatted
with petroleum ether (35 Ce60 C) (FOSS Soxtec 2050 auto
fat extraction system, Hillerd, Denmark), freeze-dried (VirTis
AdVantage, Genevac, SP Scientifc, Ipswich, UK) and stored
in a desiccator pending non-volatiles extraction. The
extractions of sugars, phenolic, organic and amino acids from
the defatted green coffee grounds were conducted in triplicate.
For sugar extraction (Maness, 2010), green coffee grounds
(5 g) were sonicated with 80% (v/v) aqueous ethanol (4 50
ml) for 30 min. The extracts were evaporated to dryness
(Eyela rotary vacuum evaporator N-1200 series, China) and
reconstituted with deionized water (5 ml) before storing at 30
C prior to analysis. For phenolic acids extraction (Cheong et
al., 2013), green coffee grounds (4 g) were extracted using a
ternary solvent system consisting of methanol, acetone and
water (7:7:6 ratio, 6 40 ml) for 30 min. The extracts were
evaporated to dryness at 40 C and reconstituted with 80%
methanol (5 ml) before storing at 30 C prior to HPLC analysis.
Organic and amino acids were co-extracted from green coffee
grounds (2 g) with deionized water (10 ml) for 30 min at 200
rpm (Heidolph Rotamax 120, Schwabach, Germany). The
extract was decanted after centrifugation (Sigma 3e18 K
centrifuge, Osterodeam Harz, Germany) at 20 380 g for 30
min and stored at 30 C before HPLC analysis.
2.5. Non-volatile analysis
The respective coffee extracts were fltered through a 0.20-
mm regenerated cellulose (RC) membrane (Sartorius,
Gottingen, Germany) prior to HPLC analysis. The HPLC
analyses of sugar and organic acids were carried out as
described by Lee et al. (2013). Sugars were resolved using a
Zorbax carbohydrate column (150 4.6 mm; Agilent, Santa
Clara, CA, USA) attached to a Zorbax SB-Aq guard column
via isocratic elution with 80% (v/v) acetonitrile and detected
with an evaporative light scattering detector (ELSD)
(Shimadzu, Kyoto, Japan). They were identifed using
reference standards (sucrose, fructose and glucose) and
quantifed with the respective calibration curves in the range
of 50 mg/L to 5000 mg/L. Organic acids were resolved using
Supelcogel C-610 H column (Supelco, Bellefonte, PA, USA)
via isocratic elution with 0.1% (v/v) sulfuric acid and detected
using a photodiode array (PDA) detector at 210 nm. The
organic acids were subsequently identifed with reference
standards (citric, lactic, malic, quinic and succinic acids).
Quantifcation was carried out with calibration curves in the
range of 20 mg/L to 10 000 mg/L.
Phenolic acids were resolved with a Zorbax Eclipse C18
column (4.6 150 mm, 5 mm; Agilent Technologies,
California, USA) attached to a C18 guard column via gradient
elution with a binary mobile phase system consisting of 1%
(v/v) acetic acid and methanol neat followed by detection with
a PDA detector at 320 nm (Cheong et al., 2013). Phenolic
acids were identifed with reference standards (chlorogenic,
caffeic, p-coumaric, ferulic and sinapic acids) and quantifed
with their respective calibration curves. The calibration curve
for chlorogenic acid was in the range of 5e700 mg/L while the
227
calibration curves for other phenolic acids were in the range of
2e280 mg/L.
Amino acids, as described by Waters (1993), were
derivatized with AccQ-Fluor reagent kits and subsequently
resolved using a Waters AccQ-Tag Nova-Pak C18 column
(150 3.9 mm; Waters, Dublin, Ireland) via gradient elution
with a binary mobile phase system consisting of 9.1% (v/v)
AccQ-Tag Eluent A concentrate and 60% (v/v) aqueous
acetonitrile. Identifcation and quantifcation were carried out
with reference amino acids standards and their respective
calibration curves in the range of 2 mMe50 mM.
2.6. Statistical analysis
Statistical signifcance in the differences arising from the
quantitative data obtained was evaluated with the analysis of
variance (ANOVA) at p < 0.05 (IBM, Illinois, USA).
3. Results and discussion
3.1. Screening of yeast strains for green coffee bean fermentation
G. candidum is a mold-like yeast which has been found to
exhibit a large extent of lipolytic activity and volatile production
(Mdaini, Gargouri, Hammami, Monser, & Hamdi, 2006).
Therefore, both G. candidum and Y. lipolytica were initially
screened on their suitability for green coffee bean fermentation
based on the extent of modulation of the volatile profle of green
coffee beans induced. The fermentation of green coffee beans
with G. candidum was conducted in the same manner as Y.
lipolytica fermentation described in Section 2.2. The volatile
profles of green coffee beans fermented by Y. lipolytica for 4 and
5 days and G. candidum for 4 and 7 days were evaluated via HS-
SPME-GC-MS/FID described in Section 2.3 (results not shown).
Whilst G. candidum fermentation mainly resulted in the
production of fusel alcohols and volatile phenols from amino and
phenolic acids metabolism respectively, changes in the aldehyde,
alkane, ketone and volatile acid levels were observed from Y.
lipolytica fermentation, in addition to the aforementioned
changes. Consequently, Y. lipolytica was selected on the basis of
its extensive, complex and unique metabolic processes that
occurred during fermentation.
3.2. Effects of Y. lipolytica fermentation on volatile profles of
green coffee beans
The volatile profle of green coffee beans was found to have a
signifcant impact on the aroma quality of roasted coffee (Lee &
Shibamoto, 2002). These volatile compounds were either formed
naturally in green coffee beans or during post-harvest processing
such as mucilage removal, drying and storage (Gonzalez-Rios et
al., 2007). A comparison of the volatile profles of fermented and
unfermented green coffee beans revealed 105 compounds
consisting of acids, alcohols, aldehydes, alkanes, alkenes,
aromatics, esters, furans, furanones, ketones, lactones, phenols,
pyrazines, pyridines, pyrroles, sulfur-containing compounds and
terpenes. Their respective GC-FID peak areas are represented in
Table 1. Despite the lack of signifcant difference in total volatile
levels after fermentation, changes in the levels of the respective
classes of compounds were observed.
One notable observation was the decrease in volatile acids,
alkanes and aldehydes levels which was attributed to decreases in
the levels of compounds within the respective classes such as
hexanoic, nonanoic acids, dodecane, tridecane, 3-methylbutanal
and 2-phenylethanal. Such observations could be explained by the
228 L.W. Lee et al. / LWT - Food Science and Technology 77 (2017) 225e232

hydrophobic substrate metabolizing pathways that are unique to
Y. lipolytica. During fermentation, alkanes and aldehydes act as
fatty acids precursors which undergo hydroxylation and oxidation
reactions upon assimilation into the cells (Fickers et al., 2005).
The fatty acids generated, along with those that were assimilated
from the fermentation matrix, could either undergo further
peroxisomal b-oxidation to yield acetyl-CoA or be re-esterifed
into triglycerides for storage within the cells (Harzevili, 2014). In
the former, the acetyl CoA generated could subsequently enter the
tricarboxylic acid (TCA) cycle for ATP production via oxidative
Table 1
Effects of Y. lipolytica fermentation on the volatile profle of green coffee beans.
Compounds
phosphorylation or glyoxylate cycle for gluconeogenesis (Fickers
et al., 2005).
The increase in the total alcohols level observed after
fermentation was mainly attributed to the increase in the levels of
2phenylethanol. This was consistent with studies showing
2phenylethanol production by Y. lipolytica from the metabolism
of L-phenylalanine via the Ehrlich pathway (Celinska, Kubiak,
Bialas, Dziadas, & Grajek, 2013). Furthermore, the increase in g-
butyrolactone levels after fermentation could be explained by the
presence of associated peroxisomal b-oxidation enzymes like
acyl-CoA
4
LRI FID peak area (x10
) Identif cation

FFAP Ref. Unfermented Fermented

Acids
Acetic acid 1452 1452 583 92a 476 189a MS, LRIa
Isobutyric acid 1566 4 2a 17 4b MS
Pentanoic acid 1667 1725 278 102a 134 45a MS, LRIc
3-methyl-2-butenoic acid 1799 34 14a 44 16a MS
Hexanoic acid 1843 1848 149 13a 47 13b MS, LRIa
Heptanoic acid 1950 18 10 e MS
Octanoic acid 2057 5 1a 4 1a MS
Nonanoic acid 2164 121 10a 73 19b MS
Benzoic acid 2451 2430 107 7a 47 5b MS, LRIa
Total acids 1299 63a 843 168b
Alcohols
1-phenylmethanol 1895 1874 64 5a 33 5b MS, LRIa
2-phenylethanolI 1936 1903 387 24a 723 80b MS, LRIa,b
Total alcohols 451 21a 756 81b
Aldehydes
3-methylbutanalI e 137 28a 64 11b MS
HexanalI 1086 1087 88 51a 29 4a MS, LRIa,b,c
Octanal 1286 e 64 33 MS
NonanalI 1393 1391 61 8a 20 2b MS, LRIa
trans-2-octenalI 1435 43 15a 30 4a MS
BenzaldehydeI 1540 1514 270 72a 217 26a MS, LRIa
2-phenylacetaldehydeI 1656 582 71a 280 16b MS
Dodecanal 1712 32 11a 25 13a MS
trans,trans-2,4-decadienalI 1823 18 2a 10 1b MS
VanillinI 2599 2564 19 5a 12 4a MS, LRIb,c
Total aldehydes 1249 220a 750 11b
Alkanes
Dodecane 1193 16 1 e MS
Tridecane 1296 35 5 e MS
Tetradecane 1397 79 17a 92 11a MS
Pentadecane 1498 58 8a 32 14b MS
Hexadecane 1599 11 2a 23 13a MS
Total alkanes 200 19a 147 31b
Alkenes
1-acetyl-2-methylcyclopentene 1250 e 13 6 MS
cis,trans-1,3,5-undecatriene 1387 e 92 MS
2,5-dimethyl-1,4-hexadiene 1518 e 19 4 MS
Total alkenes e 42 5
Aromatics
TolueneI 1044 1031 51 15a 38 4a MS, LRIa
EthylbenzeneI 1121 1125 14 4a 14 5a MS, LRIa
1,3-dimethylbenzene 1133 1132 6 1a 7 3a MS, LRIa
1,2-dimethylbenzene 1138 23 8a 8 3b MS
1-methyl-3-ethylbenzeneI 1218 e 30 MS
StyreneI 1258 302 72a 346 154a MS
2-phenylpropene 1331 e 72 10 MS
1,2-dimethyl-4-ethylbenzene 1364 e 84 MS
1,2-dimethoxybenzene 1733 16 8a 13 5a MS
Total aromatics 412 95a 509 142a
Esters
Hexyl 2-methyl butyrate 1422 e 12 7 MS
Hexyl 3-methylbutanonate 1441 26 5a 26 3a MS
Methyl salicylateI 1795 90 42a 13 5b MS
Methyl palmitate 2215 123 41a 176 5a MS
Ethyl linoleate 2494 e 42 MS
Total esters 240 75a 231 11a
Furans
2-pentylfuranI 1222 1229 171 74a 114 21a MS, LRIa
 L.W. Lee et al. / LWT - Food Science and Technology 77 (2017) 225e232 229
FurfuralI 1475 1457 685 147a 300 70b MS, LRIa
2-acetylfuranI 1518 34 3 e MS
5-methyl-2-furfuralI 1589 1565 116 26a 17 4b MS, LRIa
Furfuryl alcoholI 1669 1664 168 26a 72 23b MS, LRIa
5-(hydroxymethyl)furfural 2529 2505 17 6a 7 2b MS, LRIa
Total furans 1192 273a 511 82b
Furanones
2(5H)-furanone 1779 13 4a 16 6a MS
5-methyl-2(5H)-furanone 1919 8 2a 3 1b MS
Total furanones 21 5a 19 6a
Ketones
2,3-butanedioneI 125 11a 362 83b MS
AcetoinI 1290 1282 220 72a 955 242b MS, LRIa
2,3-octanedione 1323 20 9 e MS
Table 1 (continued )
4
Compounds LRI FID peak area (x10
) Identif cation

FFAP Ref. Unfermented Fermented

3-octen-2-oneI 1410 e 72 MS
1-(acetyloxy)-2-propanone 1468 101 20a 47 10b MS
2-methyl-3-pentanone 1531 24 3 e MS
1-(acetyloxy)-2-butanone 1536 59 31 e MS
3,5-octadien-2-oneI 1581 13 3a 9 1a MS
6,10,14-trimethyl-2-pentadecanone 2127 30 4a 29 3a MS
Total ketones 592 62a 1409 322b
Lactones g-butyrolactoneI
1648 1607 119 40a 167 7a MS, LRIa
g-hexanolactone 1719 18 7a 11 6a MS
g-nonanolactoneI 2047 100 12a 69 10b MS
Total lactones 237 49a 246 19a
Phenols
Guaiacol 1871 1859 7 1a 6 1a MS, LRIa
PhenolI 2013 1998 94 10a 104 19a MS, LRIa
4-ethylguaiacol 2040 2015 6 2a 12 1b MS, LRIc
4-methylphenolI 2091 e 18 10 MS
3-methylphenolI 2099 14 3a 19 8a MS
4-vinylguaiacolI 2211 2185 560 66a 845 78b MS, LRIa,b,c
2,4-di-tert-butylphenol 2307 9 1a 8 2a MS
4-vinylphenolI 2407 28 2a 70 18b MS
Total phenols 717 58a 1082 73b
Pyrazines
Pyrazine 1222 17 6 e MS
2-methylpyrazineI 1275 842 209a 361 126b MS
2,5-dimethylpyrazineI 1332 82 5a 41 12b MS
2,6-dimethylpyrazineI 1337 149 31a 88 28a MS
2-ethylpyrazineI 1339 217 30a 13 1b MS
2,3-dimethylpyrazine 1359 25 15a 6 2b MS
2-ethyl-6-methylpyrazineI 1390 12 6 e MS
2,3,5-trimethylpyrazineI 1414 7 2a 6 1a MS
2-methoxy-3-isobutylpyrazineI 1524 1511 34 13a 34 9a MS, LRIb,c
2-acetylpyrazineI 1646 83 41 e MS
Total pyrazines 1467 257a 549 169b
Pyridines
PyridineI 1307 1185 7 4a 6 1a MS, LRIa
2-methoxypyridine 1574 e 13 6 MS
2-acetylpyridineI 1619 20 7a 18 7a MS
Total pyridines 27 9a 36 11a
Pyrroles
3-methylpyrrole 1224 e 36 12 MS
2-methylpyrrole 1557 e 82 MS
1-furfurylpyrrole 1835 42 e MS
2-acetylpyrrole 1985 1970 29 2a 27 3a MS, LRIa
2-formylpyrrole 2043 9 1a 12 4a MS
2-pyrrolidinone 2067 5 1a 7 1a MS
IndoleI 2468 43 2a 34 5b MS
Total pyrroles 90 6a 125 17b
Sulfurous compounds
2-acetyl-4-methylthiazole 1707 12 3 e MS
Benzothiazole 1983 5 2a 4 0a MS
Total sulfurous compounds 17 5a 4 0b
Terpenes
Myrcene 1151 e 92 MS
Limonene 1186 13 1a 278 72b MS
g-terpinene 1234 e 85 32 MS
b-damascenoneI 1826 1800 12 2a 15 3a MS, LRIb
6,10-dimethyl-5,9-undecadien-2-oneI 1856 e 91 MS
230 L.W. Lee et al. / LWT - Food Science and Technology 77 (2017) 225e232
Total terpenes 26 3a 396 98b
Others
Protoanemonine 1602 19 12a 32 4a MS
Methylethylmaleimide 2280 11 2 e MS
2,3-dihydro-3,5-dihydroxy-6-methyl-(4H)-pyran-4-one 2285 16 4a 10 2a MS
Caffeine e 1766 262a 1704 207a MS
Total peak area 10 046 560a 9399 435a
I
Compounds collated by Holscher and Steinhart (1995). Mean values (n 3) with different letters (a e b) in the same row indicate statistical differences between
unfermented and fermented green coffee bean samples at 5% signifcance level (p < 0.05). Identifcation method: MS mass spectrum, LRI linear retention
indices from literature values. LRI a refers to values obtained from Gonzalez-Rios et al. (2007); LRIb refers to values obtained from Scheidig, Czerny, and Schieberle
(2007); LRIc refers to values obtained from Czerny and Grosch (2000).
The letters a, b, and c (in superscript) were included to indicate the linear retention indices obtained from 3 different journal articles that were used for
volatile compounds identifcation.
oxidase that were responsible for g-decalactone production by Y. The signifcant decrease in total sugars concentration after
lipolytica (Aguedo et al., 2005). These enzymes helped catalyze fermentation was mainly attributed to the decrease in sucrose
the conversion of methyl ricinoleate to g-decalactone via b- concentration, which was a consequence of sucrose metabolism
oxidation and intra-esterifcation steps (Aguedo et al., 2005). that is unique to the strain of Y. lipolytica (NCYC 2904) used in
Therefore, g-butyrolactone could be synthesized from hydroxy this study (National Collection of Yeast Cultures, 1999). While
the fructose concentration remained relatively unchanged, one
fatty acids that were present in green coffee beans via a similar
interesting observation was the unexpected increase in glucose
fashion. In addition, g-butyrolactone has also been found to be
concentration after fermentation. This was in spite of the
generated from glutamic acid in other yeast species like S.
preferential metabolism of glucose over fructose in the glycolytic
fermentati (Wurz, Kepner, & Webb, 1988).
pathway during aerobic respiration of Y. lipolytica (Forster,
Another notable observation was the increase in total volatile
Aurich, Mauersberger, & Barth, 2007). A likely explanation could
phenol levels after fermentation that was attributed mainly to the
be the higher extent of gluconeogenesis via the entry of fatty
increase in the levels of 4-ethylguaiacol, 4-vinylguaiacol and
acid-derived acetyl CoA into the glyoxylate cycle (Fickers et al.,
4vinylphenol. Studies have found that other yeast species such as
2005) compared to glucose metabolism. This would be consistent
Saccharomyces spp. and Brettanomyces spp. could generate
with the decrease in the total alkanes, aldehydes and fatty acids
volatile phenolic compounds from hydroxycinnamic acid
levels via the hydrophobic substrate metabolizing pathways in Y.
precursors like ferulic and p-coumaric acids (Vanbeneden, Gils,
lipolytica as
Delvaux, & Delvaux, 2008). 4-vinylphenol can be generated Table 2
from the decarboxylation of p-coumaric acid, whereas successive Effects of Y. lipolytica fermentation on sugars, organic and phenolic acids of
decarboxylation and reduction of ferulic acid result in the green coffee beans.
generation of 4vinylguaiacol and then 4-ethylguaiacol. Compounds Unfermented Fermented
The levels of 2,3-butanedione and acetoin tripled and Sugars, mg/g dw.
quadrupled, respectively after fermentation. This could be Fructose 0.68 0.06a 0.65 0.05a
explained by the generation of the above compounds from Glucose 0.93 0.10a 1.34 0.09b
Sucrose 26.16 1.64a 21.14 2.59b
intermediates along the valine biosynthesis pathway in yeasts
Total 27.77 1.77a 23.12 2.50b
(Krogerus & Gibson, 2013). The decarboxylation of a- Organic acids, mg/g dw.
acetolactate generated 2,3butanedione which could be Citric acid 7.57 0.22a 7.05 0.20b
subsequently reduced by the yeast cells to form acetoin (Krogerus Lactic acid 2.04 0.10a 2.59 0.82a
& Gibson, 2013). Malic acid 3.81 0.09a 3.77 0.26a
Quinic acid 3.26 0.27a 3.17 0.05a
It was also noted that there were signifcant increases in the
Succinic acid 4.15 0.05a 3.09 0.30b
levels of limonene and g-terpinene which could be attributed to Total 20.83 0.62a 19.66 0.85a
terpenes formation by Y. lipolytica. The de novo biosynthesis of Phenolic acids, mg/g dw.
terpenes by S. cerevisiae (Carrau et al., 2005) and the hydrolysis Chlorogenic acid 15.85 1.88a 11.97 0.16b
of non-volatile terpene glycosides by Saccharomyces and Caffeic acid 0.14 0.01a 0.11 0.01b
p-coumaric acid 0.01 0.00a 0.01 0.00b
nonSaccharomyces yeast species such as Debaryomyces hansenii
Ferulic acid 0.03 0.00a 0.02 0.00b
and Hanseniaspora uvarum (Fernandez-Gonzalez, Di Stefano, & Total 16.03 1.89a 12.11 0.16b
Briones, 2003) have been reported in wine fermentation by
differences at 5% signiMean values (n 3) with different letters (a fcance level
various studies.
(p < 0.05).e b) in the same row indicate statistical e : not detectable
(concentration
3.3. Effects of Y. lipolytica fermentation on non-volatile profles
below LOD); Trace: LOD < concentration < LOQ.
of green coffee beans

The non-volatile compounds present in green coffee beans are Table 3

important aroma precursors which play a major role in coffee Effects of Y. lipolytica fermentation on the amino acids of green coffee beans.
aroma development during roasting and in turn, have a strong Compounds Unfermented Fermented
infuence on the corresponding coffee aroma. Therefore, by Amino acids, mg/kg dw.
characterizing the non-volatile profles of green coffees, the Asp 586.70 33.69a 482.40 71.51a
effects of Y. lipolytica fermentation on the composition of aroma Ser Asn 631.75 27.10a 368.82 53.43b
Glu 550.10 56.33a 549.39 61.64a
precursors can be evaluated while changes to the volatile profle
Gly 198.19 13.96a 147.25 1.57b
of green coffees can be accounted for. Tables 2 and 3 show the His Gln Trace Trace
effects of Y. lipolytica fermentation on the sugars, organic, NH3 92.43 21.35a 241.52 22.13b
phenolic and amino acids profles of green coffee beans. Arg 177.31 14.90a 105.81 3.87b
Thr 46.56 5.22 Trace
 L.W. Lee et al. / LWT - Food Science and Technology 77 (2017) 225e232
Ala 322.77 33.44a
Pro 195.59 21.96a
Cys 59.40 2.60a
Tyr Trace
Val 280.96 1.55a
Met e e
Lys 134.27 21.67
Ile Trace
Leu 66.16 13.47
Phe 119.22 12.17
Trp Trace
Total 3738.29 118.87a
differences at 5% signiMean values (n 3) with different letters (a fcance level
(p < 0.05).e b) in the same row indicate statistical e : not detectable
(concentration
below LOD); Trace: LOD < concentration < LOQ.
illustrated in section 3.2. The relative stable concentration of
fructose suggested that the lower extent of fructose utilization
by Y. lipolytica (compared to glucose) might have negated the
increase in concentration resulting from sucrose hydrolysis.
The signifcant decrease in citric and succinic acids
concentration after fermentation was consistent with studies
documenting the utilization of these acids by Y. lipolytica as
carbon sources (Rodrigues & Pais, 2000). These acids may be
metabolized via the TCA cycle during aerobic respiration or
the glyoxylate cycle in gluconeogenesis. Although there have
been some studies reporting the production of citric (Levinson,
Kurtzman, & Kuo, 2007) and succinic acids (Kamzolova et
al., 2009) by Y. lipolytica, these occurred in media of high
carbon/nitrogen ratio which was uncharacteristic of green
coffee beans. The presence of proteins, free amino acids and
nitrogenous compounds like trigonelline contributes to the
high nitrogen content of green coffee beans. Therefore, the net
decrease in the concentrations of the above acids would be
expected since the extent of acid metabolism was likely to be
higher than production.
The signifcant decrease in the concentration of total
phenolic compounds after fermentation pointed to the
metabolism of these compounds by Y. lipolytica. This was
supported by studies showing that Y. lipolytica fermentation
resulted in a 70% decrease in total phenolic compounds during
the bio-treatment of olive mill wastewater (Lopes et al., 2009).
Even though the mechanisms leading to the degradation of the
respective phenolic compounds by Y. lipolytica have not been
elucidated, they have been documented in other yeast species
like Brettanomyces yeasts during wine fermentation. During
fermentation, ferulic and p-coumaric acids are decarboxylated
to form 4-vinylguaiacol and 4-vinylphenol which are
subsequently reduced to 4-ethyguaiacol and 4ethylphenol
respectively (Vanbeneden et al., 2008). Such metabolic
pathways would not only account for the decrease in the
concentrations of ferulic and p-coumaric acids, but also
explain the increase in the levels of volatile phenols detected
in the headspace of fermented green coffee beans (Section
3.2).
The detection of cinnamoyl esterase in other yeast species
like S. cerevisiae (Coghe, Benoot, Delvaux, Vanderhaegen, &
Delvaux, 2004) would explain the decrease in chlorogenic
acid concentration after Y. lipolytica fermentation. However,
chlorogenic acid hydrolysis did not correspond to an increase
in the concentrations of the hydrolytic products, caffeic and
quinic acids. This could be explained by the higher rate at
which they could be catabolized.
The signifcant decrease in total free amino acids
concentration was indicative of amino acids catabolism by Y.
231
160.57 12.75 lipolytica. This could be correlated with the increase in
152.25 6.98 ammonia concentration following fermentation since
59.79 2.13
ammonia is liberated from the oxidative deamination of amino
Trace
267.66 2.35
acids. The corresponding a-keto acids can be subsequently
converted into fusel alcohols via the Ehrlich pathway. This
Trace would thus account for the decrease in Lphenylalanine
Trace concentration and increase in 2-phenylethanol level.
Trace Furthermore, the conversion of pyruvate and other TCA
Trace
intermediates, formed from the deamination of gluconeogenic
Trace
2980.98 208.74 amino acids like alanine, into glucose via the glyoxylate cycle
has been observed in S. cerevisiae (Meyer et al., 2005).
Therefore, amino acid metabolism by Y. lipolytica could partly
account for the increase in glucose concentration after
fermentation. Despite the decrease in total amino acids
concentration after fermentation, some extent of proteolysis
should still be expected as Y. lipolytica has been reported to
secrete large amounts of proteases (Madzak, Gaillardin, &
Beckerich, 2004). However, the net decrease in the free amino
acid concentration could be explained by the higher extent of
amino acid catabolism compared to proteolysis.
The decrease in the concentration of amino acids observed after
Y. lipolytica fermentation was in contrast to that observed
after R. oligosporus fermentation (Lee et al., 2016a). The
higher extent of protein hydrolysis during R. oligosporus
fermentation led to the signifcant increase in the
concentrations of alanine, proline, phenylalanine, aspartic and
glutamic acids (Lee et al., 2016a). Amino acids participate in
Maillard reaction during coffee roasting which is responsible
for the generation of potent classes of odorants such as
pyrazines, furanones and thiols. Therefore, the contrasting
impacts of R. oligosporus and Y. lipolytica fermentations on
the amino acid profle of green coffee beans could correspond
to variations in the volatile profles of the respective roasted
coffees (Lee, Cheong, Curran, Yu, & Liu, 2016b; Part II of
this study).
4. Conclusion
This study presents a novel method of coffee aroma modulation
via the fermentation of green coffee beans with Y. lipolytica,
which resulted in signifcant changes to the volatile and non-
volatile profles of green coffee beans. The changes were
correlated and explained with metabolic pathways in Y. lipolytica
or other yeasts. The decreases in the levels of acids, alkanes and
aldehydes provided evidence of hydrophobic substrate
metabolism while the increase in 2-phenylethanol levels coupled
with the decrease in L-phenylalanine concentration corresponded
to amino acid metabolism via the Ehrlich pathway shown in Y.
lipolytica. The effects of fermentation on the volatile and non-
volatile profles of roasted coffees will be evaluated in a separate
study (Part II).
Acknowledgement
This research was partially supported by a grant from
Firmenich Asia Pte Ltd (R-143-000-650-592).
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