1
Impac o ozen s o age ime on he ola ile p o ile o whea b ead c umb
Joana Pico a*, Ma io M. Ma ínez b,c, José Be nal a, Manuel Gómez c
a I.U.Cinquima, Analy ical Chemis y G oup, Uni e si y o Valladolid, Paseo de Belén
S ee 7, E-47011 Valladolid, Spain.
b P esen add ess: Whis le Cen e o Ca bohyd a e Resea ch, Depa men o Food
Science, Pu due Uni e si y, 745 Ag icul u al Mall D i e, Wes La aye e, IN 47906,
USA.
c Food Technology A ea, E.T.S. Ingenie ías Ag a ias, Uni e si y o Valladolid, Mad id
A enue 57, E- 34071 Palencia, Spain.
*Co esponding au ho : Joana Pico
e-mail: joana.pico@u a.es
Abs ac
The eezing o whea b ead be o e a oma analyses is a common p ac ice in o de o
p ese e loss o he ola ile p o ile. Howe e , he impac o he ozen s o age ime on
he a oma p o ile has no been s udied. Fo his pu pose, he ola ile p o iles o whea
b ead ozen o 1, 2 and 4 weeks we e analysed employing sol en ex ac ion and s a ic
headspace me hoologies wi h GC/MS. The esul s e ealed ha he eezing was
e ec i e o p e en he loss o ola iles du ing he i s week. Howe e , a e wo
weeks, he e was an inc ease o ola ile compounds, p obably gene a ed by chemical
eac ions. Thus, a maximum o one week o ozen s o age was ecommended when
using he sol en ex ac ion me hodology. When using he s a ic headspace me hod, he
samples should be analysed on he same day as p epa a ion, since he ex ac ion was
su p isingly inc eased due o he s a ch e og ada ion ha occu ed du ing eezing.
Key wo ds: whea b ead a oma; ozen s o age ime; sol en ex ac ion; SHS-GC/MS;
s a ch e og ada ion.
Abb e ia ions: Ano a (analysis o a iance); FU ( a inog aph uni s); GC/MS (gas
ch oma og aphy-mass spec ome y); RH ( ela i e humidi y); SHS (s a ic headspace);
SIM (selec ed ion moni o ing).
2
1. In oduc ion
The a oma o b ead is one o he main cha ac e is ics pe cei ed by consume s. The
mo e a ac i e he a oma is, he mo e likely he b ead will be consumed. Thus, he
de elopmen o new ecipes ha imp o e he b ead a oma as well as he quali y con ol
o he b ead a oma i sel a e key ac o s o ensu e consume accep abili y. The e o e,
accu a e analy ical me hods a e essen ial o measu e he a oma o b ead. In his con ex ,
he eezing o whea b ead samples, in o de o p ese e he ola ile compounds, is
usually equi ed p io o chemical analyses due o logis ic ques ions o shipping o
p oduc ion on a di e en day han he analyses. Nume ous s udies epo ed he
“ eezing o he b ead sample un il he a oma analysis” wi hou checking i he ola ile
p o ile e en changed a eezing empe a u es (Bianchi, Ca e i, Chia a o, Musci, &
Vi adini, 2008; Luning, Roozen, Moës , & Pos humus, 1991; Pa aske opoulou,
Ch ysan hou, & Kou idou, 2012). Thus, i is decisi e o ensu e ha he con en o
ola ile compounds emains almos unchanged du ing eezing o achie e eliable
esul s in a oma esea ch. The p esen li e a u e conce ning he e olu ion o he ola ile
compounds du ing s o age has been ocused on he changes o he a oma p o ile a
oom empe a u e (Chia a o, Vi adini, Musci, Bianchi, & Cu i, 2008; Jensen, Oes dal,
Skibs ed, La sen, & Thybo, 2011a; Jensen, Oes dal, Skibs ed, & Thybo, 2011b; La ou,
Mexis, Badeka, & Kon ominas, 2010; Plessas e al., 2008, 2011). The ex ension o
shel -li e is one o he bigges challenges o he baking indus y oday, since he sho
shel -li e o b ead has caused conside able economic losses annually (Plessas e al.,
2011). Howe e , as o ou knowledge, he e is no li e a u e conce ning he e olu ion o
ola ile compounds du ing eezing.
The e o e, he aim o he p esen s udy was o in es iga e he ozen s o age ime
sui able o p ese e he ola ile p o ile o whea b ead samples, in o de o achie e
eliable a oma analyses. Fo his pu pose, b ead samples ozen o one, wo and ou
3
weeks we e analysed using a s a ic headspace me hodology o he e y ola ile
compounds and a sol en ex ac ion me hodology o he es o common ola ile
compounds s udied in whea b ead, bo h wi h GC/MS.
2. Ma e ials and me hods
2.1. Ma e ials
S ong whea lou (11.73% and 11.20% w/w o mois u e and p o ein con en s,
espec i ely) om Ha ine a Cas ellana (Medina del Campo, Valladolid, Spain), asco bic
acid om Sigma Ald ich (Gillingham, UK), Saccha omyces ce e isiae (Sa -ins an
yeas ) om Lesa e (Lille, F ance), sal om Yba a (Se illa, Spain) as well as ap
wa e we e used o make he b ead samples. To check he e en ion ime and he mass
spec a o he main ola ile compounds, he 38 analy ical s anda ds lis ed in Table S1
we e pu chased om Sigma Ald ich (Gillingham, UK).
2.2. Me hods
2.2.1. B ead making and s o age condi ions
The ollowing ing edien s, as % on whea lou basis, we e u ilized: sal (1.8%), ins an
yeas (1%), asco bic acid (0.01%) and wa e (52.7%, calcula ed o ob ain 500
Fa inog aph Uni s, FU). The dough was made wi h 1500 g (± 0.05 g) o lou and he
amoun o wa e was adjus ed o an a e age mois u e con en o 12%. The ing edien s
we e mixed using a Ki chen-Aid P o essional mixe (KPM5, Ki chenAid, S . Joseph,
Michigan, USA) o 15 min a speed 2. Six pieces o dough o 500 g each we e ounded
and le o e men a ion o 90 min in a chambe a 30°C wi h 75% RH. La e , he
pieces o e men ed dough we e baked a 180°C o 40 min and le o 30 min o each
oom empe a u e. One piece o b ead was analysed as eshly p epa ed sample (day 0),
as a baseline o compa ison o e ime. Fi s , he esh b ead was cu in o slices o 5 cm
long and hen he c umb was sepa a ed 1 cm om he c us , o a oid con amina ion o
4
he c umb wi h c us ola ile compounds. Then, he c umb was ozen wi h liquid
ni ogen and g ound in an Ika g inde model M20 (S au en, Ge many) o 10 seconds.
Finally, 50 g o he powde was submi ed o ola ile compounds analyses (sub-sec ion
2.2.2). The c umb o ano he piece o b ead was sepa a ed om he c us in he same
way, g ound and ozen wi h liquid ni ogen, as was epo ed by s udies ha eeze he
c umb sepa a ed om he c us un il hei analysis (Pa aske opoulou e al., 2012;
Bianchi e al., 2008). The c umb powde was sepa a ed in o h ee aluminum packe s
placed in polye hylene bags and ozen a -21°C o one, wo and ou weeks,
espec i ely, un il hei ola ile compounds we e analysed. Finally, ano he piece o
b ead was aken as a con ol sample o he e olu ion o he ola ile compounds o e
ime a oom empe a u e, in o de o compa e he changes du ing eezing wi h
con en ional oom s o age. I was s o ed in a labo a o y o en, w apped in aluminum
oil, a a con olled empe a u e o 22°C. This b ead was s o ed wi h he c us in o de o
p o ec he ola ile compounds om d ama ic losses ha could p e en he analysis o
changes due o he na u al aging o b ead. A e one week, he c umb was sepa a ed
om he c us , ozen wi h liquid ni ogen and g inded and inally submi ed o ola ile
compounds analyses (as was explained o he esh b ead). Expe imen s we e made
wi h a piece o b ead s o ed o wo weeks a oom empe a u e, bu he ola ile
compounds analysis was no accomplished because he b ead was comple ely s ale. All
he samples we e hawed o 30 min be o e he a oma analyses we e conduc ed. The
whole expe imen was conduc ed in duplica e (n=2).
2.2.2. Vola ile compounds analyses: Sol en ex ac ion, S a ic Headspace & GC/MS
The esh sample, he con ol sample (s o ed one week a oom empe a u e) as well as
he h ee ozen samples (one, wo and ou weeks) we e analysed ollowing he sol en
ex ac ion me hodology o he analysis o ola ile compounds in whea b ead c umb
de eloped by ou esea ch g oup (Pico, Nozal, Gómez & Be nal, 2016). This is
5
conside ed a sui able me hod o examine he possible changes in he ola ile compounds
o ozen c umb since he epo ed limi s o de ec ion ha e been lowe han 35 µg Kg-1.
Each sample was analysed in duplica e (n=2).In o de o e alua e he changes o he
e y ola ile compounds, s a ic headspace analyses o e hyl ace a e and e hyl alcohol
we e pe o med, which elu ed wi h he sol en in he lipases me hod. Thus, 1 g (± 0.050
g) o each sample was placed in a 20 mL ial and sealed wi h a sep um cap. The
samples we e hen ex ac ed o 90 min a 90°C, wi hou agi a ion, in a S a ic
Headspace au osample 7694 om Hewle Packa d (Palo Al o, Cali o nia, USA). The
loop and ans e line empe a u es we e 100°C and 105°C, espec i ely. The ca ie gas
employed was helium, supplied by Ca bu os Me álicos (Ba celona, Spain), wi h a
ca ie gas p essu e o 23 psi. The ial p essu iza ion was 14 psi o 0.2 min. The loop
illing ime was 0.2 min, he equilib a ion loop ime was 0.05 min and he injec ion ime
was 1 min. Each sample was analysed in duplica e (n=2). GC-MS condi ions o he
sol en ex ac ion me hodology a e he same ha hose p e iously desc ibed (Pico,
Nozal, Gómez & Be nal, 2016). Speci ying he g adien condi ions, o he sol en
ex ac ion me hodology he empe a u e anged om 45°C (1.5 min) o 100°C (0 min)
a 7°C/min, a e which he empe a u e was inc eased o 114°C (3 min) a 6°C/min, and
hen o 136°C (0 min) a 1.5°C/min. Finally, he empe a u e was aised o 245°C a
85°C/min. This empe a u e was held o 25 min in o de o elu e he hyd olysed a
(glyce ol and ee a y acids). Fo s a ic headspace (SHS) analyses, he empe a u e
anged om 45°C (1.5 min) o 100°C (0 min) a 7°C/min, and a e wa ds he
empe a u e was inc eased o 114°C (6.7 min) a 1°C/min. Analyses we e pe o med in
Selec ed Ion Moni o ing (SIM) mode and he 38 ola ile compounds we e iden i ied
and con i med by compa ison o hei e en ion imes and mass spec a ( a ge and
quali ie ions) wi h s anda ds (Table S1) and wi h he Mass Spec a Lib a y (Wiley 7N
edi ion).

6
2.2.3. Da a analysis.
The One-way Analysis o Va iance (ANOVA) o he peak a eas (n=4, each b ead
p epa ed in duplica e and analysed in duplica e) was compu ed by he so wa e
S a g aphics Cen u ion e sion XVII (S a poin Technologies, Wa en on, Vi ginia)
wi h s a is ical signi icance se a p < 0.05.
3. Resul s and discussion
3.1. E olu ion o he ola ile compounds du ing s o age a oom empe a u e
A o al o 38 ola ile compounds epo ed as main ola ile compounds in esh whea
b ead (Bi ch, Pe e sen, & Hansen, 2014; Chia a o e al., 2008; Jensen e al., 2011a;
La ou e al., 2010; Makhoul e al., 2015; Plessas e al., 2008., 2011) we e selec ed o
examine he e olu ion du ing oom empe a u e and ozen s o ages (Table S1). The
esul s o he 38 selec ed ola ile compounds o he esh sample and he con ol
sample s o ed one week a e summa ised in Table 1. Nea ly all o he ola ile
compounds, dis ega ding he boiling poin , pola i y o unc ional g oup, dec eased a e
one week o s o age a oom empe a u e. Only o 2,3-bu anedione, 1-pen anol and 1,3-
bu anediol he e we e no signi ican di e ences be ween he esh sample and he s o ed
sample, al hough hey we e p esen in low amoun . This gene al endency o ola ile
compounds o dec ease a e a ew days o s o age a oom empe a u e has been
commonly epo ed (Chia a o e al., 2008; Jensen e al., 2011b; Plessas e al., 2011).
These changes ha e been a ibu ed o e apo a ion, s aling o b ead o oxida ion
eac ions, al hough hey ha e no been explained. Chia a o e al. (2008) ound in hei
s udy ha he ola ile compounds in he whea b ead c umb dec eased be ween 1.5 and
3 imes a e 8 days o s o age a 25°C, which is in conco dance wi h he a e age
dec ease o 2.75 imes (39% o losses) o ou s udy. E hyl ace a e as well as e hyl
alcohol also showed la ge dec eases o 65 and 70%, espec i ely, which can mainly be
7
explained by hei low boiling poin s. This dec ease in he concen a ion o e hyl
alcohol has been also epo ed by Plessas e al. (2008, 2011). Howe e , he e a e some
con o e sies wi h e hanol, since La ou e al. (2010) epo ed an inc ease a e ou days
in he concen a ion o e hanol, al hough no explana ion was gi en o his ou come.
Ace oin was he only ola ile compound ha showed signi ican di e ences wi h an
inc ease in he peak a ea a e one week o s o age a oom empe a u e, which is in
conco dance wi h he wo k o Jensen e al. (2011a). Ace oin is mainly o med om he
glycolysis o py u ic acid in e men a ion (Ma ínez-Anaya, 1996) by he yeas
(Capozzi e al., 2016) and i can also be gene a ed by Mailla d eac ions du ing baking
(Poino e al., 2010). Howe e , Mailla d p ocesses ypically do no occu du ing s o age
as a consequence o he mode a e empe a u es. Ne e heless, Bi ch, Pe e sen, &
Hansen (2013) epo ed ha ace oin can be o med om he deca boxyla ion o 2-
ace olac a e, which could en a i ely p oceed a oom empe a u e. 2-e hyl-1-hexanol, 2-
me hyl-1-p opanol and 5-me hyl-2- u aldehyde p esen ed losses o a ound 90% in he
peak a ea o he esh b ead, which was su p ising o 2-e hyl-1-hexanol and 5-me hyl-
2- u aldehyde ega ding hei high boiling poin s. 2-me hyl-1-bu anol and 3-me hyl-1-
bu anol also p esen ed high losses, a ound 60%. Coinciden ally, hose we e i e o he
se en ola ile compounds ha con ained a me hyl/e hyl adical g oup ha could lead o
some o m o s e ic hind ance, making he in e ac ion di icul be ween he ola ile
compounds molecules and he b ead ma ix. The in e ac ions be ween he ola ile
compounds and s a ch ha e been a ibu ed mainly o he amo phous ac ion o s a ch,
he amylose ac ion (A isene , Le Bail, Voilley, & Cayo , 2002). This in e ac ion is
based on he gene a ion o complexes, he ola ile compound being he ligand ha
induces he o ma ion o amylose single helices, no mally o six glucoses pe u n
(Rappenecke & Zugenmaie , 1981). As a consequence, he me hyl/e hyl adical o 2-
e hyl-1-hexanol, 2-me hyl-1-p opanol, 5-me hyl-2- u aldehyde, 2-me hyl-1-bu anol and
8
3-me hyl-1-bu anol could ha e he po en ial o lead o high s e ic hind ances ha
complica ed hei access o he amylose helix and made he in e ac ion di icul be ween
he hyd oxyl g oups o he ola ile compound and he amylose o s a ch. Al hough 3-
me hylbu anoic acid and 2-me hylbu anoic acid also con ained he me hyl adical, he
high pola i y o he acidic g oup could ha e he abili y o e aining hem in he c umb
ma ix ia linkages o he s a ch by hyd ogen bonds (Le Bail, Biais, Pozo-Bayón, &
Cayo , 2004). In he case o 1-p opanol, he low boiling poin jus i ied a loss o 56%.
Howe e , he losses highe han 50% o R-(+)-limonene, u u al, e hyl oc anoa e,
phenylace aldehyde and 2,4-(E,E)-decadienal could be explained by a combina ion o
s e ic hind ance oge he wi h a low pola i y ha could o ce he ola ile compounds o
be easily eleased om he ma ix. The o he 25 ola ile compounds p esen ed losses
lowe han 25% ha we e pe ec ly explained by he s o age ime.
3.2. Changes in he ola ile p o ile du ing ozen s o age
3.2.1. E olu ion o he ola ile compounds o e he ou weeks o eezing
The esul s o he 38 selec ed ola ile compounds o he samples ozen o one, wo
and ou weeks a e p o ided in Table 2. Only 1-p opanol and 4- inylguaiacol showed
no signi ican di e ences, as hey emained almos cons an du ing he eezing
expe imen . Rega ding he i s week o eezing, o e all he e was a dec ease in
concen a ion o he ola ile compounds, wi h an a e age loss o 32% o he ola ile
compounds using he sol en ex ac ion me hodology and 21% and 19% o e hyl
ace a e and e hyl alcohol, espec i ely. Ne e heless, in compa ison o he con ol
sample, he eezing achie ed an a e age p ese a ion o 34% a e one week.
Fu he mo e, del ing in o mo e de ail, in he sample s o ed a oom empe a u e, 2-
me hyl-1-p opanol, 1-p opanol, 3-me hyl-1-bu anol, e hyl alcohol, 2-me hyl-1-bu anol,
e hyl ace a e and hexanal expe ienced losses ha we e 79%, 56%, 54%, 51%, 50%,
44% and 17% highe , espec i ely, han he ozen sample a e one week. This inding
9
could be a ibu ed o hei high ola ili y. Fu u al and 2,4-(E,E)-decadienal also
showed losses ha we e 25% and 73% highe in he oom empe a u e sample han in
he ozen one, bu hey do no p esen low boiling poin s. 2,3-bu anedione was he only
ola ile compound ha was be e p ese ed a oom empe a u e han du ing eezing,
which was e y su p ising aking in o accoun ha 2,3-bu anedione p esen s he lowes
boiling poin o he s udied ola ile compounds. None heless, 2,3-bu anedione can be
gene a ed om he oxida i e deca boxyla ion o 2-ace olac a e (Bi ch e al., 2013), a
eac ion ha hypo he ically is mo e spon aneous a oom empe a u e. Al e na i ely,
ace oin showed he same beha iou du ing eezing as a oom empe a u e and
inc eased a e one week o s o age bu in a lesse deg ee. This can also be heo e ically
a ibu ed o he possible decele a ion o he deca boxyla ion o 2-ace olac a e upon
eezing. Fo he o he 27 ola ile compounds he e we e sligh di e ences be ween
s o age a oom empe a u e and eezing, wi h he di e ences lowe han 15%
a ibu able o he luc ua ions o he GC/MS ins umen in he measu emen be ween
di e en days (in e day epea abili y). Thus, he c us o he con ol sample seemed o
ac as an e icien p o ec o o hese 27 ola ile compounds ha di e ed minimally om
he ozen samples.
As depic ed in Figu e 1, he o al con en o alcohols, acids and ke ones ollowed he
same gene al endency du ing eezing, wi h a dec ease in he o al a ea du ing he i s
week, an inc ease du ing he second week and a inal dec ease leading up o he ou h
week. Rega ding he SHS-GC/MS analyses (Table 2), e hyl ace a e also showed a
dec ease du ing he i s week bu hen a cons an inc ease leading up o he ou h
week. Fo e hanol, he e also was a dec ease in he i s week bu a g ea inc ease in he
second week (much highe han he esh sample) and a sligh dec ease leading up o he
ou h week. This indica es ha he key di e ences be ween he e olu ions o he
16
Plessas, S., Alexopoulos, A., Beka o ou, A., Man zou ani, I., Kou inas, A. A., &
Bezi zoglou, E. (2011). Examina ion o eshness deg ada ion o sou dough b ead
made wi h ke i h ough moni o ing he a oma ola ile composi ion du ing s o age.
Food Chemis y, 124(2), 627–633.
Plessas, S., Beka o ou, A., Gallanagh, J., Nigam, P., Kou inas, A. A., & Psa ianos, C.
(2008). E olu ion o a oma ola iles du ing s o age o sou dough b eads made by
mixed cul u es o Kluy e omyces ma xianus and Lac obacillus delb ueckii ssp.
bulga icus o Lac obacillus hel e icus. Food Chemis y, 107, 883–889.
Poino , P., A isene , G., G ua-P iol, J., Fillonneau, C., Le-Bail, A., & P os , C. (2010).
In luence o inulin on b ead: Kine ics and physico-chemical indica o s o he
o ma ion o ola ile compounds du ing baking. Food Chemis y, 119(4), 1474–
1484.
Rappenecke , G., & Zugenmaie , P. (1981). De ailed e inemen o he c ys al s uc u e
o Vh amylose. Ca bohyd a e Resea ch, 89(1), 11-19.
Ronda, F., Caballe o, P. A., Quilez, J. & Roos, Y. H. (2011). S aling o ozen pa ly
and ully baked b eads. S udy o he combined e ec o amylopec in
ec ys alliza ion and wa e con en on b ead i mness. Jou nal o Ce eal Science,
53, 97 – 103.
Ronda, F., & Roos, Y. H. (2011). S aling o esh and ozen glu en- ee b ead. Jou nal
o Ce eal Science, 53, 340 – 346.

17
Table 1. Peak a ea o he a ge ions (x 106) o he 38 ola ile compounds s udied in he c umb
o he esh b ead and he b ead s o ed o one week a 22°C. S anda d de ia ions (SD) a e
gi en a e ± (n=4). Di e en le e s in he same ow indica e signi ican di e ences in One-
way Ano a (95% signi icance le el).
Vola ile compounds
F esh
1 week
% losses b
p- alue
E hanol a
39.332 b ± 4.720
11.651 a ± 1.305
70
0.0026
E hyl ace a e a
6.345 b ± 0.318
2.243 a ± 0.067
65
0.0012
2,3-Bu anedione
3.354 a ± 0.323
2.320 a ± 0.157
31
0.0553
1-P opanol
1.179 b ± 0.065
0.516 a ± 0.021
56
0.0053
2-Me hyl-1-p opanol
6.715 b ± 0.102
0.882 a ± 0.020
87
0.0002
Hexanal
12.671 b ± 0.129
7.705 a ± 0.032
39
0.0004
3-Pen en-2-ol
4.069 b ± 0.103
3.140 a ± 0.028
23
0.0066
2-Me hyl-1-bu anol
4.942 b ± 0.032
1.841 a ± 0.011
63
0.0001
3-Me hyl-1-bu anol
9.970 b ± 0.095
3.983 a ± 0.056
60
0.0002
1-Pen anol
1.224 a ± 0.019
1.186 a ± 0.014
3
0.1502
Ace oin
7.808 a ± 0.183
12.309 b ± 0.268
-58
0.0026
R-(+)-Limonene
0.019 b ± 0.002
0.0085 a ± 0.0004
55
0.0114
2-Oc anone
0.348 b ± 0.017
0.193 a ± 0.001
44
0.0058
1-Hexanol
2.539 b ± 0.034
1.646 a ± 0.018
35
0.0009
Ace ic acid
403.680 b ± 1.859
344.030 a ± 5.336
15
0.0045
Fu u al
1.817 b ± 0.025
0.470 a ± 0.004
74
0.0002
Me hional
0.0066 b ± 0.0003
0.0046 a ± 0.0002
31
0.0198
1-Oc en-3-ol
0.196 b ± 0.002
0.113 a ± 0.006
42
0.0031
Nonanal
0.863 b ± 0.007
0.604 a ± 0.025
30
0.0050
2,3-bu anediol
203.014 b ± 4.621
162.075 a ± 7.192
20
0.0211
2-E hyl-1-hexanol
0.444 b ± 0.003
0.030 a ± 0.001
93
0.0000
Isobu y ic acid
20.648 b ± 0.139
15.903 a ± 0.743
23
0.0125
Benzaldehyde
0.136 b ± 0.004
0.094 a ± 0.002
31
0.0057
1,2-P opanediol
12.571 b ± 0.194
10.569 a ± 0.491
16
0.0331
E hyl oc anoa e
0.259 b ± 0.008
0.132 a ± 0.001
49
0.0021
5-Me hyl-2- u aldehyde
1.041 b ± 0.011
0.133 a ± 0.003
87
0.0001
Bu y ic acid
4.651 b ± 0.022
3.596 a ± 0.183
23
0.0149
Bu y olac one
1.177 b ± 0.025
0.825 a ± 0.015
30
0.0034
2-(E)-Nonenal
1.751 b ± 0.006
1.395 a ± 0.080
20
0.0244
3-Me hylbu anoic acid
5.054 b ± 0.007
3.960 a ± 0.121
22
0.0061
2-Me hylbu anoic acid
1.568 b ± 0.002
1.156 a ± 0.044
26
0.0058
Phenylace aldehyde
0.059 b ± 0.000
0.022 a ± 0.002
62
0.0014
1,3-Bu anediol
0.848 a ± 0.020
0.733 a ± 0.072
14
0.1627
Hexanoic acid
18.920 b ± 0.019
14.855 a ± 0.777
21
0.0178
Benzyl alcohol
1.782 b ± 0.041
1.494 a ± 0.039
16
0.0186
Phenyle hyl alcohol
13.132 b ± 0.303
11.589 a ± 0.111
12
0.0212
2,4-(E,E)-Decadienal
0.206 b ± 0.001
0.056 a ± 0.003
73
0.0002
4-Vinylguaiacol
7.876 b ± 0.048
5.965 a ± 0.154
24
0.0036
a E hyl alcohol and e hyl ace a e we e analysed by SHS-GC/MS. The o he 36 ola ile
compounds we e analysed by sol en ex ac ion and GC/MS.
b % losses calcula ed compa ed o he esh sample. Nega i e alues imply ha he peak a ea
o he a ge ion inc eased compa ed o he esh c umb.
18
Table 2. Peak a ea o he a ge ions (x 106) o he 38 ola ile compounds s udied in he c umb o he esh b ead and he b ead ozen o one, wo and ou
weeks a -21°C. S anda d de ia ions (SD) a e gi en a e ± (n=4). Di e en le e s in he same ow indica e signi ican di e ences in One-way Ano a (95%
signi icance le el).
Vola ile compounds
F esh
1 week
%
losses b
2 weeks
%
losses b
4 weeks
%
losses b
p- alue
E hyl ace a e a
6.345 c ± 0.189
5.000 a ± 0.186
21
5.220 ab ± 0.175
18
5.415 b ± 0.143
15
0.0035
E hanol a
39.332 b ± 3.456
32.000 a ± 3.001
19
44.614 c ± 4.065
-13
41.821 bc ± 4.013
-6
0.0025
2,3-Bu anedione
3.354 c ± 0.323
1.606 b ± 0.024
52
0.953 a ± 0.010
72
nd c
100
0.0001
1-P opanol
1.179 a ± 0.065
1.381 a ± 0.018
-17
1.415 a ± 0.058
-20
1.425 a ± 0.130
-21
0.0960
2-Me hyl-1-p opanol
6.715 c ± 0.102
6.187 b ± 0.056
8
6.788 c ± 0.010
-1
4.284 a ± 0.034
36
0.0000
Hexanal
12.671 d ± 0.129
9.938 c ± 0.051
22
9.019 b ± 0.188
29
4.526 a ± 0.209
64
0.0000
3-Pen en-2-ol
4.069 c ± 0.103
3.526 b ± 0.047
13
3.952 c ± 0.102
3
0.056 a ± 0.001
99
0.0000
2-Me hyl-1-bu anol
4.942 c ± 0.032
4.507 b ± 0.018
9
4.859 c ± 0.003
2
3.130 a ± 0.183
37
0.0001
3-Me hyl-1-bu anol
9.970 c ± 0.095
8.998 b ± 0.058
10
8.951 b ± 0.036
10
5.774 a ± 0.009
42
0.0000
1-Pen anol
1.224 d ± 0.019
1.060 c ± 0.015
13
0.586 b ± 0.009
52
0.314 a ± 0.010
74
0.0000
Ace oin
7.808 a ± 0.183
9.435 b ± 0.064
-21
12.868 c ± 0.161
-65
7.226 a ± 0.379
7
0.0001
R-(+)-Limonene
0.019 c ± 0.002
0.008 b ± 0.001
55
0.0017 a ± 0.0001
91
nd c
100
0.0001
2-Oc anone
0.348 c ± 0.017
0.2363 b ± 0.0003
32
0.251 b ± 0.005
28
0.130 a ± 0.002
63
0.0001
1-Hexanol
2.539 c ± 0.034
1.912 b ± 0.035
25
2.034 b ± 0.083
20
0.597 a ± 0.006
76
0.0000
Ace ic acid
403.680 d ± 1.859
301.126 c ± 3.202
25
319.549 b ± 9.703
21
177.242 a ± 2.381
56
0.0000
Fu u al
1.817 d ± 0.025
0.926 c ± 0.005
49
0.755 b ± 0.001
58
0.426 a ± 0.007
77
0.0000
Me hional
0.0066 d ± 0.0003
0.0042 c ± 0.0002
37
0.0026 b ± 0.0001
61
0.00118 a ± 0.00003
82
0.0001
1-Oc en-3-ol
0.196 c ± 0.002
0.127 b ± 0.002
35
0.131 b ± 0.011
33
0.073 a ± 0.006
63
0.0002
Nonanal
0.863 d ± 0.007
0.504 c ± 0.006
42
0.294 b ± 0.007
66
0.112 a ± 0.004
87
0.0000
2,3-bu anediol
203.014 c ± 4.621
150.509 b ± 1.506
26
208.044 c ± 0.271
-2
113.890 a ± 3.010
44
0.0000
a E hyl alcohol and e hyl ace a e we e analysed by SHS-GC/MS. The o he 36 ola ile compounds we e analysed by sol en ex ac ion and GC/MS.
b % losses calcula ed compa ed o he esh sample. Nega i e alues imply ha he peak a ea o he a ge ion inc eased compa ed o he esh c umb.
c nd = no de ec ed.
19
Table 2. (con inued).
Vola ile compounds
F esh
1 week
% losses b
2 weeks
%
losses b
4 weeks
%
losses b
p- alue
2-E hyl-1-hexanol
0.444 b ± 0.003
0.025 a ± 0.006
94
nd c
100
nd c
100
0.0000
Isobu y ic acid
20.648 d ± 0.139
13.145 b ± 0.316
36
14.232 c ± 0.551
31
7.769 a ± 0.410
62
0.0000
Benzaldehyde
0.136 d ± 0.004
0.119 c ± 0.004
12
0.105 b ± 0.001
23
0.079 a ± 0.004
42
0.0004
1,2-P opanediol
12.571 d ± 0.194
8.196 c ± 0.099
35
6.226 b ± 0.322
50
3.649 a ± 0.025
71
0.0000
E hyl oc anoa e
0.259 b ± 0.008
0.131 a ± 0.006
49
nd c
100
nd c
100
0.0000
5-Me hyl-2- u aldehyde
1.041 d ± 0.011
0.3741 c ± 0.004
64
0.3018 b ± 0.0002
71
0.155 a ± 0.003
85
0.0000
Bu y ic acid
4.651 b ± 0.022
3.271 a ± 0.028
30
5.314 c ± 0.299
-14
3.197 a ± 0.107
31
0.0004
Bu y olac one
1.177 d ± 0.025
0.801 c ± 0.011
32
0.624 b ± 0.008
47
0.348 a ± 0.014
70
0.0000
2-(E)-Nonenal
1.751 d ± 0.006
1.111 c ± 0.002
37
0.847 b ± 0.019
52
0.402 a ± 0.008
77
0.0000
3-Me hylbu anoic acid
5.054 c ± 0.007
3.664 a ± 0.011
28
5.949 d ± 0.046
-18
3.775 b ± 0.058
25
0.0000
2-Me hylbu anoic acid
1.568 b ± 0.002
1.041 a ± 0.008
34
1.586 b ± 0.049
-1
1.041 a ± 0.037
34
0.0001
Phenylace aldehyde
0.05909 c ± 0.00002
0.021 b ± 0.001
65
0.0113 a ± 0.0002
81
nd c
100
0.0000
1,3-Bu anediol
0.848 d ± 0.020
0.719 c ± 0.022
15
0.600 b ± 0.033
29
0.211 a ± 0.001
75
0.0000
Hexanoic acid
18.920 b ± 0.019
14.661 a ± 0.173
23
14.717 a ± 0.250
22
14.277 a ± 0.211
25
0.0000
Benzyl alcohol
1.782 b ± 0.041
1.418 a ± 0.027
20
2.404 c ± 0.089
-35
1.438 a ± 0.015
19
0.0001
Phenyle hyl alcohol
13.132 b ± 0.303
11.280 a ± 0.101
14
20.252 d ± 0.140
-54
17.401 c ± 0.348
-33
0.0000
2,4-(E,E)-Decadienal
0.206 a ± 0.001
0.255 b ± 0.009
-24
0.263 b ± 0.010
-28
0.204 a ± 0.001
1
0.0017
4-Vinylguaiacol
7.876 a ± 0.048
6.301 a ± 0.039
20
6.528 a ± 0.040
17
6.395 a ± 0.345
19
0.1598
b % losses calcula ed compa ed o he esh sample. Nega i e alues imply ha he peak a ea o he a ge ion inc eased compa ed o he esh c umb.
c nd = no de ec ed.
20
Table S1. Vola ile compounds s udied in he esh sample, he sample s o ed o one week a
22°C as well as he samples ozen o one, wo and ou weeks, in o de o elu ion (R ,
e en ion ime). Ta ge (T) and quali ie (Q1, Q2, +Q) ions employed o each compound a e
gi en in he able.
Vola ile compounds
R
T
Q1
Q2
Q+
E hanol a
5.824
31
45
46
29
E hyl ace a e a
5.831
43
61
70
29
2,3-Bu anedione
6.647
43
31
86
15
1-P opanol
7.267
31
42
59
60
2-Me hyl-1-p opanol
8.432
43
41
74
55
Hexanal
9.623
56
44
72
82
3-Pen en-2-ol
9.909
71
43
53
86
2-Me hyl-1-bu anol
10.883
55
70
41
57
3-Me hyl-1-bu anol
10.919
57
41
70
29
1-Pen anol
12.115
42
55
70
91
Ace oin
12.081
45
88
27
15
R-(+)-Limonene
14.920
68
93
79
107
2-Oc anone
14.922
58
71
85
128
1-Hexanol
14.870
56
41
42
55
Ace ic acid
14.474
45
60
15
29
Fu u al
17.594
96
39
29
67
Me hional
18.182
48
104
76
61
1-Oc en-3-ol
18.994
57
72
43
85
Nonanal
19.769
57
41
70
98
2,3-bu anediol
20.642
45
57
29
75
2-E hyl-1-hexanol
21.159
57
41
70
83
Isobu y ic acid
21.446
43
41
73
27
Benzaldehyde
21.907
106
105
77
51
1,2-P opanediol
22.528
45
43
61
29
E hyl oc anoa e
22.823
88
101
127
57
5-Me hyl-2- u aldehyde
23.579
110
109
53
81
Bu y ic acid
24.854
60
73
42
27
Bu y olac one
25.533
42
28
86
56
2-(E)-Nonenal
26.491
70
55
41
83
3-Me hylbu anoic acid
27.929
60
43
87
39
2-Me hylbu anoic acid
27.985
57
74
87
41
Phenylace aldehyde
28.320
91
120
92
65
1,3-Bu anediol
32.366
43
45
57
72
Hexanoic acid
40.674
60
73
87
41
Benzyl alcohol
41.605
79
108
91
51
Phenyle hyl alcohol
42.424
91
122
65
77
2,4-(E,E)-Decadienal
43.101
81
67
95
152
4-Vinylguaiacol
45.617
150
135
107
77
a E hyl alcohol and e hyl ace a e we e analysed by SHS-GC/MS. The o he 36 ola ile
compounds we e analysed by sol en ex ac ion and GC/MS.
21
Fig. 1. E olu ion o he main g oups o ola ile compounds in he whea c umb s o ed
o one week a oom empe a u e (black lines) and in he whea c umb ozen o
one, wo and ou weeks (g ey lines). The esul s a e he sum o he peak a eas o he
ke ones (con inuous line, x 106), aldehydes (discon inuous line, x 106), alcohols
(sc a ch-doubly spo ed line, x 107) and acids (spo ed line, x 107).