Impact of frozen storage time on the volatile profile of wheat bread crumb

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).