Food Chemistry 139 (2013) 3134

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Food Chemistry
o!rnal home"a#e$ %%%&elsevier&com'locate'(oodchem
Short comm!nication
)l!ms ()r!n!s domestica *&) are a #ood so!rce o( yeasts "rod!cin# or#anic acids
o( ind!strial interest (rom #lycerol
)a!la +arc,a-Fraile a.1. *!,s /& Silva b. Sal!d S0nche1-20r3!e1 c. 4ncarna Vel013!e1 a. /a5l /ivas a.
a
De"artamento de 2icrobiolo#,a y +en6tica. 7niversidad de Salamanca. 38008 Salamanca. S"ain
/497:2;4'*aborat<rio de Farmaco#nosia. De"artamento de 9!,mica. Fac!ldade de Farm0cia. 7niversidade do )orto. /& =or#e Viterbo Ferreira. 22>. 40?0-313 )orto. )ort!#al
c:nstit!to de /ec!rsos @at!rales y A#robiolo#,a de Salamanca. :/@ASA-CS:C. 3800> Salamanca. S"ain
b
a r t i c l e i n ( o a b s t r a c t
;he "rod!ction o( or#anic acids (rom several yeasts isolated (rom mat!re "l!ms on media containin#
#lycerol as carbon so!rce %as analysed by B)*C-7V& ;he yeasts isolated %ere identiCed by se3!encin#
the ?&>S internal transcribed s"acer as )ichia (ermentans. DicEerhamomyces anomal!s and Candida oleo-
"hila& ;he or#anic acid "roCles o( these strains com"rise acetic. citric. s!ccinic and malic acids that 3!al-
itatively and 3!antitatively vary bet%een di((erent s"ecies as %ell as amon# strains (rom the same
s"ecies& ;he "rod!ction (rom #lycerol o( s!ccinic. acetic. citric. malic and oFalic acids (rom C& oleo"hila
and D& anomal!s. and that o( s!ccinic. oFalic and acetic acids by )& (ermentans is re"orted (or the Crst time
in this %orE. as is the "rod!ction o( oFalic acid (rom #lycerol in yeasts& G!r res!lts also sho%ed that
mat!re (r!its can be a #ood so!rce o( ne% yeasts able to metabolise #lycerol. "rod!cin# di((erent or#anic
acids %ith ind!strial and biotechnolo#ical interest&
H 2013 4lsevier *td& All ri#hts reserved&
Article history$
/eceived 2? Se"tember 2012
/eceived in revised (orm 18 December 2012
Acce"ted 2> December 2012
Available online 1I =an!ary 2013
Jey%ords$
Gr#anic acids
Keasts
+lycerol
)r!n!s
)l!ms
1& :ntrod!ction
Gr#anic acids are nat!ral com"o!nds in (r!its and ve#etables&
;hese com"o!nds have a stron# inL!ence on the or#anole"tic
"ro"erties o( (r!its and ve#etables. "artic!larly as re#ards Lavo!r.
colo!r and aroma& ;hey are also !sed eFtensively as (ood additives
in the man!(act!re o( bevera#es. (r!it and ve#etable drinEs and
!ices& ;he main acids !sed to enhance bevera#es are citric. malic
and tartaric as acid!lants and ascorbic as an antioFidant (Scherer
et al&. 2012)&
+lycerol is a by-"rod!ct o( biodiesel "rod!ction "rocesses that
can be !sed (or di((erent "!r"oses (Kan#. Banna. M S!n. 2012)&
;he eF"ansion o( the biodiesel "rod!ction sector has increased
the amo!nt o( cr!de #lycerol obtained ann!ally. %hich has also
res!lted in a red!ction o( the "rice o( reCned #lycerol (Kan#
et al&. 2012)& Altho!#h cr!de #lycerol may be !sed to obtain several
ind!strial "rod!cts (Kan# et al&. 2012). the red!ced "rice o( reCned
#lycerol can also (acilitate its !se in ind!strial "rocesses& For
eFam"le. the "rod!ction o( or#anic acids. !sed in (ood. "harmace!-
tical and chemical-related ind!stries. constit!tes an interestin#
%ay to !se #lycerol as a ra% material (or bioconversion by yeasts
Corres"ondin# a!thor& Address$ De"artamento de 2icrobiolo#,a y +en6tica.
*ab& 210. 4diCcio De"artamental de Niolo#,a. 38008 Salamanca. S"ain& ;el&$ O34 923
294?00F1919P (aF$ O34 923 294I11&
4-mail address$ ra!lr#Q!sal&es (/& /ivas)&
1)resent address$ 2olec!lar 2icrobiolo#y De"artment. =ohn :nnes Centre. Colney
*ane. @or%ich @/4 87B. 7J&
030>->14I'R - see (ront matter H 2013 4lsevier *td& All ri#hts reserved&
htt"$''dF&doi&or#'10&101I'&(oodchem&2012&12&043
(*evinson. J!rt1man. M J!o. 2008P )a"aniEolao!. 2!ni#lia. Cheva-
lot. A##elis. M 2arc. 2002)& ;he "rod!ction o( some or#anic acids.
s!ch as citric acid. by #lycerol (ermentation has been re"orted
(or some yeasts. s!ch as Karro%ia. Candida. and /hodotor!la ()a"a-
niEolao! et al&. 2002P /oehr. J!biceE. M Jom,eE. 1993)& @everthe-
less. the ca"ability o( most yeasts to "rod!ce or#anic acids o(
ind!strial interest (rom #lycerol remains !nEno%n&
Since (r!its and !ices %ith hi#h s!#ar content are a #ood so!rce
o( yeasts (DeaE M Ne!chat. 1993P ;o!rnas. Beeres. M N!r#ess. 200IP
;o!rnas M Jatso!das. 200?). in the "resent %orE %e eF"lore
mat!re "l!ms as a so!rce o( yeasts able to #ro% in #lycerol as a
sin#le carbon so!rce& Altho!#h. d!e to their hi#h s!#ar content
and %ater activity. "l!ms are ade3!ate (or yeast #ro%th there
are (e% data abo!t the yeasts "resent in these (r!its beca!se only
st!dies (oc!sin# on ethanol-"rod!cin# yeasts have been "er(ormed
S(Satora M ;!s1ynsEi. 200?. 2010)& ;he or#anic acid "roCles (rom
yeasts isolated (rom "l!ms able to metabolise #lycerol %ere
analysed by B)*C-7V&
2& 2aterial and methods
2&1& Keast isolation
Keasts %ere isolated (rom mat!re commercial "l!m (r!its o(
variety TTCla!diaUU. (or %hich several "l!ms %ere transversely
c!t into "ieces& :n order to avoid eFternal contamination. the
s!r(ace o( the "ieces %as disin(ected by a 1-min treatment %ith
32 )& +arc,a-Fraile et al& ' Food Chemistry 139 (2013) 3134
a 0&01 #'l ;%een->0 a3!eo!s sol!tion. (ollo%ed by a 10-min treat-
ment %ith a sol!tion o( ho!sehold bleach (10 #'l active chlorine)&
A(ter bein# rinsed in sterile %ater. 10 # o( the "l!m (ra#ments %ere
s!s"ended in 100 ml sterile distilled %ater and shaEen vi#oro!sly
(or (e% min!tes& Serial dil!tions o( the "l!m sam"le s!s"ension
%ere made in sterile distilled %ater& An ali3!ot o( 0&1 ml o( each
dil!tion %as inoc!lated on K@N-+*K (8 #'l yeast nitro#en base.
8 #'l #lycerol and 20 #'l a#ar) containin# ?00 m#'l o( chloram"hen-
icol. and %as inc!bated (or 4I days at 22 VC& A(ter that. 12 isolated
colonies %ere selected (rom the "lates accordin# to sha"e. colo!r.
s!r(ace (eat!res and (re3!encies& ;he yeast c!lt!res %ere "re-
served on Sabo!ra!d a#ar "lates at 4 VC and s!b-c!lt!red every
I months. or Ee"t at W>0 VC in Sabo!ra!d broth (GFoid. Nasin#-
stoEe. 7J) s!""lemented %ith sterile #lycerol (in "ro"ortion 1$1)&
2&2& :dentiCcation o( the strains isolated
;he molec!lar identiCcation o( the strains %as based on
analysis o( their ?&>S internal transcribed s"acer (?&>-:;S)
se3!ences& +enomic D@A %as eFtracted (rom cells collected (rom
2-day-old Sabo!rea!d a#ar c!lt!res !sin# a commercial Eit
(/ed4Ftract-@-Am" )lant )C/P Si#maAldrich. St& *o!is. 2G)
accordin# to the man!(act!rerUs instr!ctions& ;he ?&>S-:;S
(ra#ments %ere am"liCed !sin# the Am"li;a3 +old rea#ent Eit
(A""lied Niosystems :nc&. Foster City. CA) and the "rimers :;S1
(?0 -;CC;CC+C;;A;;+A;A;+C-30 ) and :;S4 (?0 -++AA+;AAAA+;+C
;AACAA++-30 )& Am"liCcation %as "ro#rammed as (ollo%s$ initial
denat!ration at 9? VC (or 2 min. 3? cycles %ith denat!ration at
94 VC (or 1 min. annealin# at ?4 VC (or 1 min and D@A synthesis
at 82 VC (or 1 minP a(ter Cnal eFtension at 82 VC (or ? min& Ali3!ots
o( 10 ll o( am"liCed "rod!cts %ere se"arated electro"horetically
on 1X (%'v) a#arose #el in ;risborate4D;A (;N4) b!((er at a con-
stant volta#e o( I V'cm (or I0 min& ;he bands %ere stained %ith
ethidi!m bromide and "hoto#ra"hed !nder transill!minated 7V
li#ht& ;he am"liCed D@A (ra#ments %ere "!riCed (rom a#arose
#el a(ter electro"horesis !sin# the Eit 9:A3!icE (9ia#en. Bilden.
+ermany). and se3!enced in an AN:388 se3!encer (A""lied Niosys-
tems) !sin# a Ni#Dye terminator v3&0& ;he 3!ality o( the se3!ences
obtained %as analysed by means o( the se3!encin# reaction chro-
mato#rams and vis!alised %ith Chromas 1&4? so(t%are& ;he se-
3!ences obtained %ere com"ared %ith all available se3!ences in
the +enNanE database !sin# the N*AS;@ "ro#ram (Altsch!l. +ish.
2iller. 2yers. M *i"man. 1990)&
2&3& Gr#anic acids identiCcation
;he or#anic acids %ere identiCed by com"arison o( their
retention times %ith those o( standards and they %ere 3!antiCed
by !sin# an eFternal standard calibration& ;he standards (oFalic.
cis-aconitic. citric. ascorbic. malic. 3!inic. (!maric. Eeto#l!taric. s!c-
cinic. shiEimic. lactic. acetic. "yr!vic. malonic and tartaric acids)
%ere (rom Si#maAldrich and (rom 4FtrasynthYse (+enay. France)&
2ethanol and hydrochloric acid %ere obtained (rom 2ercE (Darms-
tadt. +ermany) and s!l"h!ric acid (rom )ronalab (*isbon. )ort!#al)&
;he %ater %as treated in a 2illi-9 %ater "!riCcation system&
;ri"licated yeast c!lt!res in ? ml K@N-+*K broth %ere inc!-
bated %itho!t shaEin# at 2I VC (or 1? days. and then centri(!#ed
at 9000# (or ? min& ;he s!"ernatants %ere Cltered. eva"orated to
dryness !nder red!ced "ress!re (40 VC) and redissolved in s!l"h!-
ric acid (0&01 @ Z ? m2). Cltered %ith );F4 membrane (0&4? lm)
and 20 ll %ere analysed by B)*C'7V& ;he sam"les corres"onded
to a miFt!re o( the three re"licates& 4ach sam"le %as (!rther
divided into three ali3!ots and analysed se"arately&
B)*C analysis %as carried o!t as "revio!sly re"orted (;aveira
et al&. 2010)& ;he se"aration o( or#anic acids %as "er(ormed by
analytical B)*C. !sin# an ion eFcl!sion @!cleo#el[ :on 300 GA
(300 mm \ 8&8 mm) col!mn (2acherey@a#el. D]ren. +ermany).
in con!nction %ith a col!mn heatin# device at 30 VC& 4l!tion
%as carried o!t at a solvent Lo% rate o( 0&2 ml'min. isocratically.
%ith s!l"h!ric acid (0&01 @) as the mobile "hase& Detection %as
"er(ormed %ith a +ilson 7V detector set at 214 nm& Gr#anic acids
3!antiCcation %as achieved by the absorbance recorded in the
chromato#rams relative to eFternal standardsP "eaEs %ere inte-
#rated !sin# a de(a!lt baseline constr!ction techni3!e& ;his "roce-
d!re %as "er(ormed in tri"licate&
2&4& Statistical analysis
)rinci"al com"onent analysis ()CA) %as carried o!t !sin#
^*S;A; 2010 so(t%are& ;he )CA method sho%s the similarities
bet%een or#anic acids and yeasts strains& ;he "roection o(
chemical variables. #ro!"ed by or#anic acids classes "rod!ced
d!rin# inc!bation time and by all yeast strains !sed into the "lane
com"osed by the "rinci"al aFes F1 and F2 contained >I&?1X o( the
total variance&
3& /es!lts and disc!ssion
3&1& :dentiCcation o( strains isolated (rom "l!ms
:n order to select "ossible #lycerol-trans(ormin# yeasts %e !sed
a selective medi!m containin# #lycerol as the only carbon so!rce
and a(ter I days o( inoc!lation 12 yeast strains %ere isolated on
K@N-+*K a#ar "lates (see ;able 1)& All strains #re% on a li3!id
medi!m containin# #lycerol as the only carbon so!rce. "rod!cin#
acids d!rin# #ro%th. detected by a decrease in "B 4I days a(ter
inoc!lation at 2> VC&
;he se3!encin# o( the inter#enic (ra#ment located bet%een the
1>S and 2>S ribosomal #enes (?&>S-:;S) is c!rrently one o( the most
reliable methods (or yeast identiCcation (J!rt1man. Fell. M
NoeEho!t. 2011b)& ;here(ore %e se3!enced this (ra#ment in the
yeasts isolated and a(ter the se3!ence analysis (;able 1) the strains
C:/01. C:/03. C:/0?. C:/08. C:/0>. C:/09 and C:/11 %ere identiCed
as o( DicEerhamomyces anomal!s since they have 100X identity
%ith res"ect to its ty"e strain (CNS ?8?9;)& ;his s"ecies %as
"revio!sly named )ichia anomala bein# reclassiCed by J!rt1man.
/obnett. and Nasehoar-)o%ers (200>) based on m!lti#ene "hylo-
#enetic analysis& ;he strains C:/02. C:/04. C:/0I and C:/14 %ere
identiCed as Candida oleo"hila %ith 100X identity %ith res"ect to
the ty"e strain o( this s"ecies (CNS 2219;)& Finally. the strain
C:/10 %as identiCed as )ichia (ermentans. since its ?&>S-:;S
(ra#ment sho%ed 100X identity %ith the ty"e strain o( this s"ecies
(CNS 1>8I;)&
@one o( these endo"hytic "l!m yeasts %ere (o!nd by Satora and
S;!s1ynsEi (200?. 2010). %ho identiCed A!reobasidi!m s"& in (resh
bl!e "l!ms and re"orted that yeasts res"onsible (or (ermentation
in "l!ms %ere Saccharomyces cerevisiae. JloecEera a"ic!lata and
Candida "!lcherrima& ;his can be d!e to the di((erent "l!m varieties
!sed. the di((erent ecosystems. the di((erent isolation methods or
the !se in o!r %orE o( a rare carbon so!rce. #lycerol. instead o(
the common one. #l!cose& ;his is the Crst re"ort abo!t the "res-
ence o( )& (ermentans and C& oleo"hila in mat!re "l!ms& D& anomal!s
((ormerly Bansen!la anomala) has been "revio!sly (o!nd in "l!msP
ho%ever. this Cndin# %as re"orted by Satora and ;!s1ynsEi in an
!nre(ereed so!rce (htt"$''%%%&ar&EraEo%&"l't1'EtCmt'"!bliEace'
"sa_d02&"d()&
3&2& )rod!ction o( or#anic acids by the isolated strains
Altho!#h the "rod!ction (rom #lycerol o( some or#anic acids
has been re"orted (or a (e% yeasts (Chat1i(ra#Eo! et al&. 2011P
)& +arc,a-Fraile et al& ' Food Chemistry 139 (2013) 3134
;able 1
Gr#anic acids com"osition o( yeasts "rod!ced in c!lt!re medi!m containin# #lycerol as carbon so!rce (#'l)&
Keast strain
C:/01
C:/02
C:/03
C:/04
C:/0?
C:/0I
C:/08
C:/0>
C:/09
C:/10
C:/11
C:/14
:dentiCcation by ?&>S-:;S se3!encin#
DicEerhamomyces
Candida oleo"hila
DicEerhamomyces
Candida oleo"hila
DicEerhamomyces
Candida oleo"hila
DicEerhamomyces
DicEerhamomyces
DicEerhamomyces
)ichia (ermentans
DicEerhamomyces
Candida oleo"hila
anomal!s
anomal!s
anomal!s
anomal!s
anomal!s
anomal!s
anomal!s
GFalic acid
nd
nd
nd
nd
nd
nd
nd
nd
nd
0&I3 ` 0&03
nd
0&3I ` 0&03
Citric acid
nd
1&34 ` 0&0?
0&91 ` 0&023&01
3&009 ` 0&31
nd
nd
nd
nd
0&4? ` 0&0?
nd
0&?0 ` 0&0?
0&?1 ` 0&01
S!ccinic acid
0&0? ` 0&01
0&0> ` 0&01
0&0? ` 0&01
nd
0&2? ` 0&08
0&18 ` 0&08
0&13 ` 0&02
0&12 ` 0&01
0&0? ` 0&01
1&23 ` 0&0?
0&03 ` 0&01
nd
2alic acid
nd
nd
nd
nd
>&48 ` 0&09
1&09 ` 0&0?
0&>1 ` 0&1I
1&I> ` 0&0I
nd
nd
nd
nd
Acetic acid
0&?8 ` 0&01
1&08 ` 0&0?
1&4> ` 0&11
nd
nd
nd
nd
nd
0&82 ` 0&08
2?&88 ` 0&80
0&>8 ` 0&09
4&12 ` 0&3?
33
nd. not detected& Val!es are eF"ressed as mean ` standard deviation o( three assays&
)a"aniEolao! et al&. 2002P /osenber#. 2iEova. M JristoCEova. 1999P
;ain# M ;ain#. 2008). to o!r Eno%led#e the "rod!ction o( other
or#anic acids %ith "otential ind!strial interest by yeasts #ro%in#
%ith #lycerol as carbon so!rce has not been analysed in detail
be(ore&
;he B)*C-7V analysis allo%ed the identiCcation o( Cve or#anic
acids in o!r isolates$ oFalic. citric. s!ccinic. malic and acetic acids
(;able 1)& Strains (rom di((erent s"ecies have di((erent or#anic acid
"roCles& )CA analysis (Fi#& 1) sho%s that C:/10 ()& (ermentans) is
"roected into the "lane (ormed by the F1 "ositive aFis. d!e to its
hi#h concentration o( oFalic. s!ccinic and acetic acids& :n contrast.
C:/0? (D& anomal!s) is "roected into the "lanes (ormed by the F1
ne#ative aFis and F2 "ositive aFis. d!e to its hi#h levels o( malic
acid& C:/04 (C& oleo"hila) is "roected into the ne#ative F1 and F2
aFesP this strain is characterised by hi#her "rod!ction o( citric acid&
@one o( the other yeasts %ere associated %ith any "artic!lar acid&
Bo%ever all strains. even those (rom the same s"ecies. have
3!antitative di((erences in the or#anic acids "rod!ced (;able 1)&
S!ccinic acid %as "rod!ced by most o( the isolates in amo!nts
ran#in# (rom 0&03 to 1&23 #'l. %ith the maFim!m amo!nt "rod!ced
by )& (ermentans C:/10& Seven strains. C:/01. C:/02. C:/03. C:/09.
C:/10. C:/11 and C:/14 "rod!ced acetic acidP )& (ermentans C:/10
%as the strain %hich "rod!ced the hi#hest amo!nt o( this acid
(2?&> #'l)& Citric acid %as "rod!ced by ?0X o( the strains isolated
in this st!dy$ C:/02. C:/03. C:/04. C:/09. C:/11 and C:/14. %ithin
the ran#e 0&4?3&01 #'l& ;he hi#hest amo!nts o( this acid %ere
"rod!ced by t%o strains (rom C& oleo"hila. C:/02 and C:/04& 2alic
acid "rod!ction %as relatively lo% (or all strains. %ith a maFim!m
val!e o( >&48 #'l in strain C:/0?& Finally only t%o strains. )& (ermen-
tans C/10 and C& oleo"hila C/14. "rod!ced oFalic acid in amo!nts o(
I&32 and 0&3I #'l. res"ectively&
All the or#anic acids "rod!ced by the "l!m yeast isolates have
ind!strial a""lications and may be "rod!ced by microor#anisms
b!t they are !s!ally "rod!ced (rom carbon so!rces di((erent to
#lycerol (Sa!er. )orro. 2attanovich. M Nrand!ardi. 200>)& For
eFam"le. s!ccinic acid is "rod!ced by S& cerevisiae in media con-
tainin# #l!cose (AriEa%a et al&. 1999) and by Karro%ia li"olytica
in media %ith ethanol (Jam1olova et al&. 2009)& ;he acetic acid "ro-
d!ction in media containin# #l!cose and ethanol is %ell Eno%n in
DeEEera (Narnett. )ayne. M Karro%. 1991P J!rt1man. Fell. M NoeEh-
o!t. 2011a) and the "rod!ction o( citric acid (rom K& ly"olytica in
media containin# #l!cose and s!crose has been re"orted (Sa!er
et al&. 200>)& Bo%ever (rom #lycerol only the "rod!ction o( citric
and acetic acids has been re"orted (or K& ly"olytica (Chat1i(ra#Eo!
et al&. 2011P )a"aniEolao! et al&. 2002) and that o( malic acid (rom
#lycerol (or Di"odasc!s and ay#osaccharomyces ro!Fii (/osenber#
et al&. 1999P ;ain# M ;ain#. 2008)& Altho!#h the "rod!ction o( oFalic
acid thro!#h microbial conversion o( #lycerol has been re"orted (or
(!n#i (2!siab M /ymo%ic1. 2009). this is the Crst re"ort on its
Fi#& 1& )CA o( all or#anic acids "rod!ced by yeast strains$ "roection o( or#anic acids
in (A) (variables$ acetic. citric. malic. oFalic and s!ccinic) and yeast strains in (N)
(variables$ C:/01. C:/02. C:/03. C:/04. C:/0?. C:/0I. C:/08. C:/0>. C:/09. C:/10.
C:/11 and C:/14) into the "lane com"osed by the "rinci"al aFes F1 and F2 (>I&?1X)&
;he val!es are mean o( three eF"eriments&
"rod!ction in yeasts& Also. this is the Crst record abo!t the "rod!c-
tion (rom #lycerol o( s!ccinic. acetic. citric. malic and oFalic acid
(rom C& oleo"hila and D& anomal!s. and the "rod!ction o( s!ccinic.
oFalic and acetic acid by )& (ermentans&
34 )& +arc,a-Fraile et al& ' Food Chemistry 139 (2013) 3134
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and 2icrobial ;echnolo#y. 41. 29229?&
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oFalic acid "rod!ction by As"er#ill!s ni#er& :n +& A##elis (4d&). 2icrobial
conversions o( ra% #lycerol (""& 3140)& @e% KorE$ @ova Science )!blishers :nc&&
)a"aniEolao!. )&. 2!ni#lia. *&. Chevalot. +&. A##elis. +&. M 2arc. :& (2002)& Karro%ia
li"olytica as a "otential "rod!cer o( citric acid (rom ra% #lycerol& =o!rnal o(
A""lied 2icrobiolo#y. 92. 838844&
/oehr. 2&. J!biceE. C& )&. M Jom,eE. =& (1993)& Citric acid& :n B& C& /ehm M +& /eed
(4ds&)& Niotechnolo#y (vol& I. ""& 3>>39?)& +ermany$ Verla#s#esellscha(t&
/osenber#. 2&. 2iEova. B&. M JristoCEova. *& (1999)& Formation o( *-malic acid by
yeasts o( the #en!s Di"odasc!s& *etters in A""lied 2icrobiolo#y. 29. 221223&
SSatora. )&. M ;!s1ynsEi. ;& (200?)& Niodiversity o( yeasts d!rin# "l!m De#ierEa
a%yEla s"ontaneo!s (ermentation& Food ;echnolo#y and Niotechnolo#y. 43.
2882>2&
SSatora. )&. M ;!s1ynsEi. ;& (2010)& :nL!ence o( indi#eno!s yeasts on the (ermentation
and volatile "roCle o( "l!m brandies& Food 2icrobiolo#y. 28. 41>424&
SSatora. )&. M ;!s1ynsEi. ;& (sd)& G#<lna charaEterystyEa %ybranych s1c1e"<%
dro1d1y i "l6sni %yi1olo%anych 1 sad! sli%o%e#o& 7/* htt"$''
%%%&ar&EraEo%&"l't1'EtCmt'"!bliEace'"sa_d02&"d(& Accessed 14&02&11&
Sa!er. 2&. )orro. D&. 2attanovich. D&. M Nrand!ardi. )& (200>)& 2icrobial "rod!ction
o( or#anic acids$ 4F"andin# the marEets& ;rends in Niotechnolo#y. 2I. 10010>&
Scherer. /&. /ybEa. A& C& )&. Nall!s. C& A&. 2einhart. A& D&. Filho. =& ;&. M +odoy. B& ;&
(2012)& Validation o( a B)*C method (or sim!ltaneo!s determination o( main
or#anic acids in (r!its and !ices& Food Chemistry. 13?. 1401?4&
;ain#. G&. M ;ain#. J& (2008)& )rod!ction o( malic and s!ccinic acids by s!#ar-
tolerant yeast ay#osaccharomyces ro!Fii& 4!ro"ean Food /esearch and ;echnolo#y.
224. 343348&
;aveira. 2&. Silva. *& /&. Vale-Silva. *& A&. )into. 4&. Valentaco. )&. Ferreres. F&. et al&
(2010)& *yco"ersicon esc!lent!m seeds$ An ind!strial by"rod!ct as an
antimicrobial a#ent& =o!rnal o( A#ric!lt!ral and Food Chemistry. ?>. 9?299?3I&
;o!rnas. V& B&. Beeres. =&. M N!r#ess. *& (200I)& 2o!lds and yeasts in (r!it salads and
(r!it !ices& Food 2icrobiolo#y. 23. I>4I>>&
;o!rnas. V& B&. M Jatso!das. 4& (200?)& 2o!ld and yeast Lora in (resh berries. #ra"es
and citr!s (r!its& :nternational =o!rnal o( Food 2icrobiolo#y. 10?. 1118&
Kan#. F&. Banna. 2& A&. M S!n. /& (2012)& Val!e-added !ses (or cr!de #lycerol A
by"rod!ct o( biodiesel "rod!ction& Niotechnolo#y (or Nio(!els. ?. 13&
From the res!lts o( this st!dy %e can concl!de that$ (i) "l!ms
are a #ood so!rce o( yeasts able to !se #lycerol as a carbon so!rceP
(ii) this is the Crst re"ort abo!t the "resence o( C& oleo"hila and )&
(ermentans in "l!msP (iii) all strains isolated (rom these s"ecies
in the "resent %orE are able to "rod!ce several or#anic acids o(
ind!strial interest !sin# #lycerol as the carbon so!rceP (iv) this is
the Crst re"ort abo!t the "rod!ction o( s!ccinic. acetic. citric. malic
and oFalic acid (rom C& oleo"hila and D& anomal!s. and the
"rod!ction o( s!ccinic. oFalic and acetic acid by )& (ermentans in
media containin# #lycerol as carbon so!rce and (v) this is the Crst
re"ort abo!t the "rod!ction o( oFalic acid (rom #lycerol by yeasts&
AcEno%led#ements
;hanEs are eFtended to )a!la Andrade (or her hel" %ith the
man!scri"t& )rimary s!""ort %as "rovided by a +rant (rom the
7niversity o( Salamanca (7SA*200>N2-10)& Additional s!""ort
%as "rovided by #rants (rom 2:C:@@ and =CK*& )+F is #rate(!l to
the S"anish +overnment (or Cnancin# her "ostdoctoral contract&
/e(erences
Altsch!l. S& F&. +ish. D&. 2iller. D&. 2yers. 4& 2&. M *i"man. D& =& (1990)& Nasic local
ali#nment search tool& =o!rnal o( 2olec!lar Niolo#y. 21?. 403410&
AriEa%a. K&. J!royana#i. ;&. ShimosaEa. 2&. 2!rats!baEi. B&. 4nomoto. J&. Jodaira.
/&. et al& (1999)& 4((ect o( #ene disr!"tions o( the ;CA cycle on "rod!ction o(
s!ccinic acid in Saccharomyces cerevisiae& =o!rnal o( Nioscience and
Nioen#ineerin#. >8. 2>3I&
Narnett. =& A&. )ayne. /& D&. M Karro%. D& (1991)& Keasts$ Characteristics and
identiCcation (2nd ed&)& Cambrid#e$ Cambrid#e 7niversity )ress& Cha"ter I&
Chat1i(ra#Eo!. A&. 2aEri. A&. NelEa. A&. Nello!. S&. 2avro!. 2&. 2astorido!. 2&. et al&
(2011)& Niotechnolo#ical conversions o( biodiesel derived %aste #lycerol by
yeast and (!n#al s"ecies& 4ner#y. 3I. 1098110>&
DeaE. ;&. M Ne!chat. *& /& (1993)& Keasts associated %ith (r!it !ice concentrates&
=o!rnal o( Food )rotection. ?I. 8888>2&
Jam1olova. S& V&. K!s!"ova. A& :&. VinoE!rova. @& +&. Fedotcheva. @& :&. Jondrashova.
2& @&. Fino#enova. ;& V&. et al& (2009)& Chemically assisted microbial "rod!ction
o( s!ccinic acid by the yeast Karro%ia li"olytica #ro%n on ethanol& A""lied
2icrobiolo#y and Niotechnolo#y. >3. 10281034&
J!rt1man. C& )&. Fell. =& D&. M NoeEho!t. ;& (2011a)& +ene se3!ence analyses and
other D@A-based methods (or yeast s"ecies reco#nition& :n C& )& J!rt1man. =& D&